Progress in Brain Research Volume 26A Correlative Neurosciences: Fundamental Mechanisms

PROGRESS I N BRAIN RESEARCH VOLUME 21A CORRELATIVE NEUROSCIENCES PART A: F U N D A M E N T A L MECHANISMS

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

M. T. Chang E. De Robertis

J. C. Eccles J. D. French

H. H y d h J. Ari8ns Kappers S. A. Sarkisov J. P,Schad6

F. 0. Schmitt

Kiel Shanghai Buenos Aires Canberra Los Angeles

Giiteborg Amsterdam Moscow Amsterdam Brookline (Mass.)

T. Tokizane

Tokyo

H. Waeisch

New York

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH VOLUME 21A

CORRELATIVE NEUROSCIENCES PART A :

F ~ N ~ A M E N T AMECHANISMS L EDITED BY T. TOKIZANE Institute of Brain Research, University of Tokyo, Tokyo (Japan) AND

J. P. SCHADI? Netherlands Central Institute for Brain Research, Anisterdam (The NetherlanrJs)

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List of Contributors

T. ABE,Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan).

H. AKIMOTO, Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Tokyo (Japan). T. BAN, Department of Anatomy, Osaka University Medical School, Osaka (Japan). T. FURUKAWA, Department of Physiology, Osaka University Medical School, Osaka (Japan).

K. HAMA,Department of Anatomy, School of Medicine, Hiroshima University, Hiroshima (Japan). T. HUKUHARA, Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

M. ITO,Department of Physiology, Osaka University Medical School, Osaka (Japan). M. KATO,Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Tokyo (Japan). Y. KATSUKI,Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan). E. KAWANA, Department of Neuroanatomy, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan). H. KUMAGAI, Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan). M. KUROKAWA, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan). T. KUSAMA, Department of Neuroanatomy, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

H. MANNEN, Anatomical-PhysiologicalSection, Institute of the Deaf, Tokyo Medical and Dental University, Tokyo (Japan). K. MIYAMOTO, Department of Physiology, Osaka University Medical School, Osaka (Japan).

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LIST OF C O N T R I B U T O R S

K. MOTOKAWA, Department of Physiology and Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan). H. NAKAHAMA, Department of Physiology, Keio University School of Medicine. Tokyo (Japan).

H. NARUSE, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan).

S. NISHIOKA, Department of Physiology, Keio University School of Medicine, Tokyo (Japan). K. OTANI,Department of Anatomy, School of Medicine, Chiba University, Chiba (Japan). T. OTSUKA,Department of Physiology, Keio University School of Medicine, Tokyo (Japan).

Y. SAITO,Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo Tokyo (Japan). F. SAKAI,Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan). A. SAKUMA, Department of Pharmacology, Institute of Cardiovascular Diseases, Tokyo Medical and Dental University, Tokyo (Japan). N. SHIMIZU,Department of Neuroanatomy, Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan). M. SHIMOKOCHI, Department of Physiology, Osaka University Medical School, Osaka (Japan). H. SUZUKI,Department of Physiology and Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan).

Y. TSUKADA, Department of Physiology, Keio University School of Medicine, Tokyo (Japan). N. YOSHII,Department of Physiology, Osaka University Medical School, Osaka (Japan).

Other volumes in this series:

Volume 1 : Brain ~echanisms Specific und aspecific Mechanisms of Sensory Motor ~ntegrut~on Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2: Nerve, Bruin and Memory Models Edited by Norbert Wiener? and J. P. Schadt

Volume 3: The Rhinencephalon and Related Structures Edited by W.Bargmann and J. P. Schadi:

Volume 4: Growth and Maturation of the Brain Edited by D. P. Purpura and J. P. Schadk

Volume 5 : Lectures on the Diencephalon Edited by W. Bargmann and J. P. Schade

Volume 6: Topics in Basic Neurology Edited by W . Bargmann and J. P. Schadt Volume 7: Slow Electrical Processes in the Brain by N . A. Aladjalova

Volume 8: Blogenic Amhes Edited by Harold E. Himwich and Williamina A, Himwich

Volume 9: The Developing Brain Edited by Williamina A. Himwich and Harold E. Himwich

Volume 10: The Structure and Function ofthe Epiphysis Cerebri Edited by 1. Ariens Kappers and J. P. Schadi:

Volume 11 : Organization of the Spinal Cord Edited hy J . C. Eccles and J. P. Schade

Volume 12: Physiology of Spinal Neurons Edited by J. C. Eccles and J. P. Schadi:

Volume 13: Mechanisms of Neural Regeneration Edited b y M. Singer and J. P. Schadt

VlII

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schad6

Volume 15 : Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea

Volume 16 : Horizons in Neuropsychopharmacology Edited by Williamina A. Himwich and J. P. Schad6

Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener1 and J. P. Schadk

Volume 18 : Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. Schadk

Volume 19: Experimental Epilepsy by A. Kreindler

Volume 20: Pharmacology and Physiology of the Reticular Formation Edited by A. V. Valdman

Volume 21B : Correlative Neurosciences Part B: Clinical Studies Edited by T. Tokizanc and J. P. Schad6

Volume 22: Brain Reflexes Edited by E. A. Asratyan

Volume 23 : Sensory Mechanisms Edited by Y . Zotterman Volume 24: Carbon Monoxide Poisoning Edited by H.Bow and I. McA. Ledingham Volume 25: The cerebellum Edited by C. A. Fox and R. S. Snider Volume 26 : Developmental Neurology Edited by C . G. Bernhard

Volume 21 : Structure and Function of the Limbic System Edited by W. Ross Adey and T.Tokizane

1x

Preface

Medical and biological sciences in Japan have a long history. As far back as 562 AD medical books were introduced from China, initiating a long period of fruitful medical education and practice. An important era of scientific interest in the structure and function of the nervous system began in 19 11 with the publication by Prof. Shiro Tashiro on the carbon dioxide production of nerve fibers. Prof. Genichi Kato announced in 1920 his famous theory of non-decremental nerve conduction and presented all the evidence at the International Physiological Conference in 1926. His research was a major breakthrough in the physiology of single nerve fibers. He had a profound influence on the development of physiology in Japan and directing interest toward neurophysiology. From that time on the majority of Japanese scientists have been engaged in research in the brain sciences. The present volume is the first of a set of two, containing reviews and surveys of brain research in the majot Japanese laboratories and institutes. It particularly reflects the progress of Japanese research in the basic and clinical neurological sciences. Part A covers important fields such as: neural regulations of autonomic functions, basic mechanisms of vision and hearing, histochemistry and submicroscopy of synapses and dendrites, enzymatic and metabolic parameters of behavior and convulsive states. Part B will deal with clinical neurological studies and the relationship of neuroanatomy, neurophysiology and neurochemistry to the clinical sciences. It is a rare occasion that one acquires an overall view of the research activities of a large country in such an important field of the medical sciences. We trust this volume will provide a means of evaluating the level of brain research in Japan. The Editors.

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Con tents

List of contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX The septo-preoptico-hypothalamicsystem and its autonomic function T. Ban (Osaka, Japan). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Synaptic interaction at the Mauthner cell of goldfish T. Furukawa (Osaka, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Neural mechanism of hearing in cats and monkeys Y.Katsuki (Tokyo, Japan). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Relationship between activity of respiratory center and EEG H. Kumagai, F. Sakai, A. Sakuma and T. Hukuhara (Tokyo, Japan) . . . . . . . . . . 98 Metabolic studies on ep mouse, a special strain with convulsive predisposition M. Kurokawa, H. Naruse and M. Kato (Tokyo, Japan) . . . . . . . . . . . . . . . . 112 Contribution to the morphological study of dendritic arborization in the brain stem H. Mannen (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Central mechanism of vision 163 K. Motokawa and H. Suzuki (Sendai, Japan). . . . . . . . . . . . . . . . . . . . . Excitation and inhibition in ventrobasal thalamic neurons before and after cutaneous input deprivation H. Nakahama, S. Nishioka and T. Otsuka (Tokyo, Japan) . . . . . . . . . . . . . . . 180 Histochemical studies of the brain with reference to glucose metabolism N. Shimizu and T. Abe (Osaka, Japan) . . . . . . . . . . . . . . . . . . . . . . . 197 Studies on the neural basis of behavior by continuous frequency analysis of EEG N. Yoshii, M. Shimokochi, K. Miyamoto and M. Ito (Osaka, Japan) . . . . . . . . . . 217 Studies on fine structure and function of synapses K. Hama (Hiroshima, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Amino acid metabolism and its relation to brain functions Y.Tsukada (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 Projections of the motor, somatic sensory, auditory and visual cortices in cats T. Kusama, K. Otani and E. Kawana (Chiba, Japan) . . . . . . . . . . . . . . . . . 292 Synchronizing and desynchronizing iduences and their interactions on cortical and thalamic neurons H. Akimoto and Y.Saito (Tokyo, Japan) . . . . . . . . . . . . . . . . . . . . . . 323 Author index. 352 Subject index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

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The Septo-Preoptico-Hypothalamic System and its Autonomic Function TADAYASU BAN Deparrtiient of Anaroniy, Osaka University Medical School, Osaka (Japan)

THREE Z O N E S I N THE H Y P O T H A L A M U S

In 1935, Hasegawa reported that the body temperature rose after needle (0.1-0.2 mm in diameter) puncture in Griinthal’s (1929) b cell-group of the hypothalamus in guineapigs. On the other hand, Megawa reported in 1940 that needle puncture of Griinthal’s a and c cell-groups of the hypothalamus and the lateral part of the midbrain tegmentum in guinea-pigs showed a fall in body temperature. The b cell-group also showed increases in blood sugar (Shimizu, 1941) and in number of leucocytes (Satani, 1943) with increased mononuclear leucocytes after needle puncture in rabbits, although in the a and c cell-groups blood sugar (Shimizu, 1941) and leucocytes (Satani, 1943) decreased. In these cases, the coagulation time of the blood was shortened and the sedimentation rate was raised by the puncture of the b cell-group, although the coagulation time was prolonged and the sedimentation rate was lowered by the puncture of the a and c cell-groups (Iwakura, 1944; Kurotsu et al., 1943). Electrical stimulation of the cell-groups mentioned above showed almost the same results as shown by needle puncture. These results prompted Kurotsu and his associates (1947) to propose the hypothesis that the wall of the third ventricle in the hypothalamus was physiologically divided into three zones medio-laterally, namely, a-parasympathetic, b-sympathetic and c-parasympathetic zones respectively. The a-parasympathetic zone corresponds to the hypothalamic periventricular stratum (Simidu, 1942) and the medial mamillary nucleus, and the c-parasympathetic zone to the lateral hypothalamic area (the lateral hypothalamic nucleus). The b-sympathetic zone corresponds to the medial hypothalamic area including the anterior, supraoptic, paraventricular, dorsomedial, ventromedial, posterior and the lateral mamillary nuclei, but the stimulation of the anteromedial part of the paraventricular nucleus near the periventricular stratum decreased the blood sugar level (Shimizu, 1941). The b and c zones are separated from each other by the fornix (Fig. l). STIMULATION A N D DESTRUCTION EXPERIMENTS

O F THE H Y P O T H A L A M U S

( I ) Circulatory system Generally speaking, the blood pressure (Ban et al., 1949, 1951a, 1953; Kurotsu et al., Rrfirenccs p . 39-43

T. B A N

Fig. 1. Frontal sections of the septa1region (SEP) and the preoptic and hypothalamic areas are shown from left to right. ACA, anterior limb of the anterior commissure; AH, anterior hypothalamic nucleus; ARC, arcuate nucleus; CA, anterior commissure; CAU, caudate nucleus; CC, corpus callosum; CI, internal capsule; CHOP, optic chiasm; COMH, commissura fornicis; CORA, Ammon's horn; DM, dorsomedial hypothalamic nucleus; DSM, supramamillary decussation ; F, fornix; FM, fasciculus retroflexus; HYP, hypophysis; LH, lateral hypothalamic nucleus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; MT, mamillothalamic tract; PC, cerebral peduncle; PCA, posterior limb of the anterior commissure; PH, posterior hypothalamic nucleus; PMD, dorsal premamillary nucleus; PMV, ventral premamillary nucleus; POL, lateral preoptic area; POM, medial preoptic area; PV, paraventricular hypothalamic nucleus; SCH, suprachiasmatic nucleus; SM, supramamillary nucleus; SOP, supraoptic nucleus; SPVH, hypothalamic periventricular stratum; SPVP, preoptic periventricular stratum; STM, stria medullaris; STT, stria terminalis; SUB, subthalamic nucleus; TOL, lateral olfactory tract; TOP, optic tract; VL, lateral ventricle; VM, ventromedial hypothalamic nucleus; VIII, third ventricle.

19%) was increased by electrical stimulation of the nuclei in the medial hypothalamic area, but it was decreased after a longer latent period by stimulation with low frequency and voltage. This decrease could not be prevented by administration of atropine, and it was slightly accelerated by administration of Imidalin. The blood pressure was decreased (Kurotsu et al., 1954c) by electrical stimulation with low frequency and, after bilateral adrenalectomies, was increased by the same stimulation. However, even in normal rabbits, the same stimulation produced an increase in blood sugar, inhibition of gastric motility and a decrease in renal volume. In hypophysectomized, thyroidectomized or adrenalectomized rabbits, the latent period was about 1.0 sec, which was similar to that in normal rabbits (Ban et al., 1953). The pressor response obtained by the stimulation was pronounced in bilaterally adrenalectomized rabbits. In hypophysectomized rabbits, pressor response was rapid and the secondary rise of blood pressure became apparent as the stimulation was repeated. This secondary rise was not modified by extirpation of the thyroid gland, but disappeared after extirpation of the suprarenal glands. Even when all three glands were extirpated, the blood pressure still increased after medial hypothalamic stimulation (Ban et al., 1953). On the other hand, blood pressure was decreased by electrical stimulation of the lateral hypothalamic area as well as the periventricular stratum in normal rabbits (Ban e l al., 1949, 1951a, 1953; Kurotsu er al., 1954~). On strong stimulation, the blood pres-

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

3

sure sometimes increased and then decreased. When the basic level was markedly lowered by extirpation of the adrenal glands, stimulation of the lateral hypothalamic area did not produce a fall but a small rise of the pressure. However, when the basic level was elevated again by intravenous injection of physiological saline solution, the same stimulation decreased the blood pressure (Ban et at., 1953). These results suggest that the effect of nervous stimuli is subject to the internal environment of animals. The electrocardiographic changes (Morimoto, 1951 ; Yuasa et a]., 1957) during medial hypothalamic stimulation under ether or chloralose anesthesia in rabbits were as follow. The RR intervals were shortened after a latent period of 0.5-1.0 sec. The RQ and QT intervals were also shortened and the P wave increased by the stimulation (Fig. 2). Lateral hypothalamic stimulation markedly prolonged the RR intervals after a latent period of 0.4-0.8 sec. The PQ and QT intervals were also prolonged and the P wave was decreased. At the same time, sinus bradycardia, sinoauricular block or auriculoventricular block was observed. Sometimes auriculoventricular or ventricular automatism was recognized (Fig. 2). These reactions induced by the stimulation of the lateral hypothalamic nucleus were suppressed by bilateral vagotomies, but sometimes slight temporary prolongation of RR intervals could be observed 4-10 sec after the beginning of the stimulation in bilaterally vagotomized rabbits, which might be caused humorally. Effects of stimulation of the periventricular stratum on the electrocardiogram were almost the same to those mentioned above. According to Iwakura (1944), an increase in fibrinogen and thrombin was demonstrated with a decrease in the coagulation time of blood after medial hypothalamic stimulation. At the same time, the sedimentation rate was accelerated (Iwakura, 1944) and the total amount of protein, albumin and globulin, especially y-globulin, in serum increased (Morimoto, 1950). An increase in aspartic acid in serum was also demonstrated (Tazuke, 1951). On the other hand, after lateral hypothalamic stimulation, the coagulation time was prolonged and the sedimentation rate was retarded (Iwakura, 1944), and the total amount of protein, albumin and globulin in serum was gradually reduced (Morimoto, 1950). Kotake asserted in 1930 that the method for estimating the serum-iodometric titration value was the most suitable for ascertaining the state of intermediate metabolism of protein. Tazuke (Kurotsu et a]., 3954d), using this method, reported that thevalue was rapidly increased by 40-90 % after medial hypothalamic stimulation, and stated that this increase was due to an increase in the ether-insoluble material and not to an ether-soluble one such as a-ketonic acid. From the results of these experiments, it is concluded that the medial hypothalamic area can accelerate protein metabolism and the lateral hypothalamic area as well as the periventricular stratum suppress it. The total nonprotein nitrogen in blood also increased up to 30% after medial hypothalamic stimulation (Kurotsu et at., 1954d). The total nonprotein nitrogen and albumin in blood are closely related to renal function, which will be discussed later. At any rate, albuminuria was observed until 3 days after medial hypothalamic stimulation in rabbits, even in anesthetized rabbits (Ban et at., 1951a). An increase in blood sugar after medial hypothalamic stimulation has been mentioned above (Shimizu, 1941), but even when the hypophysis, thyroid and adrenal glands had been ReJermcrr p. 39-43

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T. B A N

all extirpated, an increase in blood sugar occurred on stimulation (Kurotsu et al., 1953~).This fact is very interesting for studying liver metabolism. The changes in the total cholesterol and lipid phosphorus in blood and total lipid in serum induced by the stimulation of the ventromedial hypothalamic nucleus were

2

3

Fig. 2. 1 shows shortening of PR, PQ and QT and increase of P induced by the stimulation of the nucleus hypothalamicus posterior under ether anesthesia. 2 shows shortening of RR and PQ and increase of P induced by the stimulation of the nucleus hypothalamicus ventromedialis under chloralose anesthesia. 3 shows the ventricular automatism induced by the stimulation of the nucleus hypothalamicuslateralis under chloralose anesthesia.

SEPTO-PREOPTICO-HY POTHALAMIC SYSTEM

5

measured by means of Bloor’s and Fiske-Subbarow’s methods and the phenol turbidity method of Kunkel and the results were as follow (Inoueetal., 1954).Totalcholester01 decreased in all 15 rabbits, lipid phosphorus decreased in 8, increased in 5 and remained unchanged in 2. Total lipid decreased slightly in 7 rabbits and remained unchanged in 5. After lateral hypothalamic stimulation, total cholesterol remained almost unchanged, but slightly increased (8 mg/dl) in 3 out of 10 rabbits. Lipid phosphorus increased in 1 1 out of 15 rabbits, remained unchanged in 3 and decreased in 1. Total lipid in serum remained unchanged in 7 out of 12 rabbits, while it increased in 5 (Inoue et al., 1954). As to the histamine content in total blood (Kurotsu et al., 1955a; Tane et al., 1958) measured by Code’s method, medial hypothalamic stimulation was inclined to lower the blood histamine, but an increase was observed in rabbits that died after the stimulation. In bilaterally adrenalectomized rabbits, the same stimulation caused an increase in blood histamine as shown in thyroidectomized rabbits, but the histamine content tended to decrease on stimulation when adrenocortical extract (Interenin) was satisfactorily administered to the adrenalectomized rabbits. In hypophysectomized rabbits, a decrease in blood histamine was observed on the same stimulation. On the other hand, lateral hypothalamic stimulation produced an increase in blood histamine in all normal rabbits, whereas the same stimulation showed a decrease of blood histamine content in adrenalectomized or thyroidectomized rabbits. In hypophysectomized rabbits, the same stimulation showed an increase in the same manner as in normal rabbits. Regarding the changes (Ban et al., 1951b) of K+ and Ca2+in total blood induced by the hypothalamic stimulation measured by Kramer-Tisdall’s method, K+ increased while Ca++ decreased slightly on ventromedial hypothalamic stimulation. On lateral hypothalamic stimulation, K+ decreased while Ca2+ was apt to increase. According to Okamoto and Oda (1952) mobilization of lymph from the lymph gland was accelerated by medial hypothalamic stimulation : the lymphocyte count in the efferent lymphatic vessels was increased and the related lymph gland was reduced in size by the stimulation. On the other hand, they (Okamoto and Oda, 1952) reported that production of lymph in the lymph gland was accelerated by lateral hypothalamic stimulation, because the lymphocyte count in the efferent lymphatic vessels remained almost unchanged and the related lymph gland was enlarged. (11) Cerebrospina1,fluidand choroid plexus (Kurotsu et al., 19536)

The cerebrospinal fluid pressure was markedly elevated up to 200 mm HzO in a glass tube (1.5 mm in diameter) immediately after the ventromedial hypothalamic nucleus was stimulated. In the course of repetition of the stimulation, a marked antagonistic action occurred between the sympathetic and parasympathetic systems. The stimulation resulted in positive globulin reaction and proportionate increases i n cell count, total protein and sugar contents. Portal permeability from blood into cerebrospinal fluid was increased by the stimulation. At the same time, the vitamin C content was noticeably lowered and the epithelial layer cells of the choroid plexus

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T. B A N

seemed to indicate enhancement of their secretion cytologically. After repetition of the ventromedial hypothalamic stimulation, hydrocephalus internus could often be observed. On the other hand, a decrease in cerebrospinal fluid pressure was observed down to -100 mm HzO when the lateral hypothalamic nucleus was stimulated. No changes occurred in permeability from blood to cerebrospinal fluid, in vitamin C or sugar contents. Epithelial layer cells of the choroid plexus showed features which made it seem that secretory function was at rest cytologically. (III) Eye and intraorbital glands

When the medial hypothalamic area was stimulated in rabbits, exophthalmos and mydriasis were observed (Ban et al., 1951a, b). At the same time, the intraocular pressure rose markedly (Nagai et al., 1951), even when the common carotid artery was ligated. This rise in pressure was believed to be due first to the contraction of Miiller’s muscles (Nagai, 1951) and then to an increase in blood pressure. The total protein content in the aqueous humor (Nagai and Ito, 1951) and the permeability from blood to aqueous humor (Nagai and Morimoto, 1952) were also increased by the same stimulation. According to histochemical tests, glycogen in the retina decreased during the stimulation and then increased after the stimulation (Matsumoto and Ishino, 1957). The lacrimal gland and Harder’s gland showed features of intracellular production of secretion on stimulation (Kurotsu et al., 1956b). On the other hand, when the lateral hypothalamic nucleus was stimulated, enophthalmos and miosis were observed (Ban et al., 1951a, b). At the same time, the intraocular pressure fell slightly after the drop in the blood pressure, even when the common carotid artery was ligated. Thus the fall in the intraocular pressure was presumed to be due partly to a decrease in blood pressure and partly to extension of Miiller’s muscles as well as pupillary constriction (Nagai et al., 1951). Glycogen in the retina seemed to be increased, according to histochemical examination (Matsumoto and Ishino, 1957). After lateral hypothalamic stimulation, the lacrimal and Harder’s glands showed features of secretion cytologically (Kurotsu et al., 1956b). ( I V ) Digestive system

In 1943, Fujita (1943; Fujita and Amano, 1943) in our laboratory reported that lateral hypothalamic stimulation in rabbits produced stomach bleeding which was prevented by bilateral vagotomies or administration of atropin before the stimulation. In coeliac gangliectomized rabbits, marked stomach bleeding or ulcer (Fig. 3) occurred after the same stimulation. These phenomena were presumed to be produced by rupture of the blood capillaries due to the high pressure of arterial blood caused by venal constriction induced by muscular contraction of the gastric body. Lateral hypothalamic stimulation increased the intragastrointestinal pressure and motility, and produced hemorrhage in the gastric mucosa (Kurotsu et al., 1951c, 1952~).The impulse from the lateral hypothalamic nucleus to the stomach and small intestine was transmitted chiefly through the vagi, but the rectum had no relation with the vagi and

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

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coeliac ganglia, because their extirpation did not modify the responses of the rectum to lateral hypothalamicstimulation (Kurotsu el a/., 1951c, 1952~).The same stimulation increased intraesophageal pressure (Kurotsu et a/., 1953a), but it decreased the motilities of the cardia and pylorus (Takeda and Ito, 1951). According to Fujita (1943; Fujita and Amano, 1943), the stimulation ofthe medial hypothalamic area in rabbits produced small dotted bleeding in the stomach in 50%

Fig. 3. Stomach ulcer induced by lateral hypothalamic stimulation in the coeliac gangliectomized rabbit (Kurotsu e/ a/., 1951~).

which was prevented by extirpation of the coeliac ganglia but not influenced by bilateral vagotomies or administration of atropine. Medial hypothalamic stimulation decreased the intragastrointestinal pressure and obliterated their motilities completely through both coeliac ganglia (Kurotsu et a/., 195lc, 1952~).Intraesophageal pressure also showed a slight fall (Kurotsu et af., 1953a) but the cardiac and pyloric motilities were increased by the same stimulation (Takeda and Ito, 1951). We sometimes observed minor bleeding or ulcers in the cardia or pylorusafter medial hypothalamic stimulation. The complete obliteration of the rectal motility induced by the same stimulation had no relation with the coeliac ganglia. The sexual cycle in female rabbits markedly affected all responses to the hypothalamic stimulation especially in the gastrointestinal system as well as genital organs (Kurotsu et a/., 1952b). The alveolar cells of the parotid and submandibular glands in rabbits (Kurotsu et a/., 1951b), and the chief and parietal cells of the fundus gland (Amano, 1947) and the surface epithelium cells in cats (Kurotsu eta/., 1954a), as well as the duodenal gland cells (Kurotsu et al., 1958a) and the acinus cells of the pancreas (Kurotsu, 1954) in Rr/i,renrrs p 39-43

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T. B A N

rabbits, after lateral hypothalamic stimulation, all had features observable cytologically in which they seemed to discharge their intracellular contents to the ducts, whereas after medial hypothalamic stimulation, they showed features in which they seemed to produce secretory substances in the cells. The epithelium cells of the submandibular duct discharged supranuclear vacuoles to the duct and large vacuoles along the basic membrane to the intercellular space outside the duct after medial hypothalamic stimulation. The former was taken to be the sympathetic salivary fluid and the latter to be an endocrine substance of the salivary gland. On the other hand, the surface epithelium cells of the stomach also showed features in which they discharged the contents to the lamina propria after lateral hypothalamic stimulation. This is likely to be an endocrine function of the gastric mucous membrane. ( V ) Genital organs and ejection of milk

The electrical stimulation of the medial hypothalamic area, medial preoptic area or the midbrain central gray substance produced ovulation in mature rabbits (Kurotsu et a/., 1950). In rabbits whose ovarial nerve or internal carotid nerves, including the superior cervical ganglia, were extirpated, or whose ovary was autotransplanted in the anterior chamber of the eye, follicular hematomata were also produced by the stimulation. In pregnant or pseudopregnant rabbits as well as hypophysectomized rabbits (Kurotsu et al., 1952a), ovulation could not be observed after the same stimulation. From these results we conclude that the gonadotropic stimulus in the hypothalamus was transmitted to the anterior lobe of the pituitary gland through the pituitary stalk. On the other hand, the lateral hypothalamic stimulation inhibited ovulation induced by medial hypothalamic stimulation, but it could not prevent ovulation produced by the injection of urine of pregnant women (Kurotsu eta/., 1950). The motility and tone of the uterus were increased by medial hypothalamic stimulation, but these reactions varied according to the sexual cycle (Kurotsu ef a/., 1952b). Three days after castration, spontaneous motility and reactions of the uterus to the hypothalamic stimulation disappeared, but they reappeared on administration of the follicular hormone. Spontaneous motility of the uterus and its reactions to sympathetic stimulation became evident in accord with disappearance of the corpora luteal function in pregnant or pseudopregnant rabbits. The tone of the uterus was increased, while the frequency and amplitude of the uterine motility were decreased by the lateral hypothalamic stimulation in normal mature rabbits (Kurotsu et a/., 1952b). Regarding the influence of the hypothalamus upon pregnancy in the rabbit (Tsutsui et al., 1957), ventromedial hypothalamic stimulation at the last stage of pregnancy often caused delivery, but lateral hypothalamic stimulation had no effect on the delivery or the puerperium. The gestation was prolonged by bilateral destruction of the medial hypothalamic areas during pregnancy. After bilateral destruction of the lateral hypothalamic areas at various stages of pregnancy, different changes were found as follow. Destruction on the seventh day of pregnancy caused abortion without placentation. Destruction on the 14th day of pregnancy produced necrotized uterine contents which

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

9

were absorbed or discharged later and promoted atrophy of the corpus luteum gravidarum. Destruction on the 25th day of pregnancy caused premature labor. However, even with this destruction of the lateral hypothalamic nuclei pregnancy safely could be maintained by administration of more than 40 mg of progesterone, but not by administration of follicular hormone. Medial hypothalamic stimulation in rabbits on the 3rd postpartum day increased the ejection of milk (Shimizu et al., 1956; Ban et al., 1958), to the maximum value of 38 mm3 in a glass cannula of 0.8 mm in diameter inserted in a teat duct, which was almost equal to the value induced by 100 mU of oxytocin. The same stimuldtion could not produce any ejection of milk in hypophysectomized rabbits, but it showed a vigorous ejection in thyroidectomized rabbits. It is probable that the medial hypothalamic stimulation induces milk ejection by the posterior pituitary hormone via the hypothalamohypophysial tract. Stimulation of the lateral hypothalamic nucleus or the periventricular stratum did not increase milk ejection. Bilateral destruction of the ventromedial hypothalamic nuclei of rabbits at postpartum caused reduction of the mammary gland cells as early as the 4th day after the destruction and often the sucklings died. Even though they could continue to live, their growth was not satisfactory. In these cases, milk secretion could be maintained by administration of more than 5 R.U. of the anterior pituitary hormone (Hypophorin) after the bilateral destruction of the medial hypothalamic areas. On the other hand, bilateral destruction of the lateral hypothalamic nuclei maintained milk secretion well and all sucklings showed satisfactory growth. Histological changes in the testis and prostate in mature rabbits induced by ventromedial hypothalamic stimulation were as follow (Nakamura et al., 1962). In the seminiferous tubules, marked dilatation of the lumen, discharge of spermium and reduction of fat granules were observed, while in the interstitial cells, diminution of the cell body, disappearance of vacuoles and reduction of fat granules were observed. At the same time, the prostate showed marked secretory activity similar to that in apocrine glands. Accordingly, Leydig’s interstitial cell as well as the prostate were presumed to secrete on medial hypothalamic stimulation. On the other hand, lateral hypothalamic stimulation induced contraction of the lumen, acceleration of spermatogenesis and increase of fat granules in the seminiferous tubules, while in the interstitial cells, swelling of the cell body and increase of vacuoles and fat granules were observed after the stimulation. In the prostate also fat granules were increased. ( V I ) Neurosecretion

In 1940, Kurotsu and Kondo reported the seasonal changes of neurosecretion, an increase in summer and a decrease in winter in the hypothalamus of the toad. In rabbits, some neurosecretory granules were seen which were transmitted partly to the intracellular spaces of the pars tuberalis and the frontal part of the pars distalis via primary capillaries or the perivascular spaces or the hypophysial portal system, and partly to the intercellular spaces in the caudal part of the pars distalis via the posteRcVc.rmcrs p . 39-43

10

T. B A N

rior and intermediate lobes from the hypothalamus (Okada et at., 1955) (Fig. 4). These observations may be related to the hypothalamic control of the anterior lobe. We also observed morphological changes which made it seem likely that the neurosecretory material was released into the hypothalamic and hypophysial blood vessels, and partly into the third ventricle, by the ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation its outflow was suppressed and it was

- - - .c

,

-- PI

PD Fig. 4. Hypothalamohypophysialneurosecretory pathways in the rabbit hypophysis(sagittal section). HS, hypophysial stalk; NR, posterior lobe; PD, anterior lobe; PI, intermediate lobe; PT, pars

tuberalis; a, b and c, descending course of neurosecretory granules to the anterior lobe.

retained in the axons (Shimazu et at., 1954). By irradiating rat heads with X-rays, neurosecretory granules in the hypothalamus and hypophysis were increased in one or two days (Tanimura, 1957). During pregnancy, parturition and post-partum periods in rabbits, neurosecretory material showed some changes as follow (Tanimura et at., 1960). Early in the pregnancy the supraoptic and paraventricular nuclei contained many vacuoles and comparatively few granules. At mid-pregnancy, granules increased markedly in the nuclei, infundibular area and neurohypophysis. Granules and droplets also invaded the intercellular spaces of the pars intermedia. Immediately before parturition neurosecretory granules decreased rapidly, and Herring-bodies of the neurohypophysis became vacuolated and irregularly shaped. This decrease in neurosecretory material continued to the 7th day post-partum. In rabbits which were allowed to suckle their young, neurosecretory granules in the hypothalamohypophysial system tended to increase from the 7th day.

SE PTO- PR EO PTI C O - H Y POTH A L A M I C SYSTEM

11

( VII) Urinary system

Ventromedial hypothalamic stimulation in normal rabbits anesthetized with urethane showed a marked diminution in renal volume recorded by an oncometer, followed by a decreasing number of urine drops, and then marked dilatation of the kidney followed almost simultaneously by an increase in urine drops. The same stimulation in bilaterally splanchnicotomized, hypophysectomized or bilaterally adrenalectomized rabbits showed a marked decrease in renal volume, but it recovered without exceeding the initial renal volume (Hirahara et al., 1953). On the other hand, lateral hypothalamic stimulatjon in normal rabbits showed an increase in renal volume followed by an increasing number of urine drops and then reduction of the renal volume with diminution of urine drops. In biIaterally splanchnicotomized, hypophysectomized or bilaterally adrenalectomized rabbits, the renal volume was increased by the stimulation and recovered to the initial volume after the stimulation without any rebound response. The number of urine drops in the former 2 groups was almost normal, but in the adrenalectomized rabbits, no urine drop was observed in the course of our experiments (Hirahara ef al., 1953). The histological changes in the kidney after hypothalamic stimulation were as follow. During the ventromedial hypothalamic stimulation, the majority of the renal corpuscles and the intracapsular spaces became smaller, and the permeability of the blood vessels decreased simultaneously. Consequently the filtration activity was diminished. At the same time, the proximal convolution cells showed changes in their fine structures, in which the cells were presumed to absorb the filtrate from the lumina during the stimulation. During lateral hypothalamic stimulation, the renal corpuscles became much larger, and the intracapsular spaces dilated strikingly up to 18 /.I in diameter. The glomerular capillaries also dilated from 9 to I 1 p in diameter. These features were taken to indicate promoted glomerular filtration, while the proximal convolution cells showed changes in their finer structures, in which thecells were presumed to discharge the absorbed substance into the blood vessels (Kurotsu et al., 1954b). These results show that the changes in the renal volume took place in parallel with the changes in dimensions of the renal corpuscles and the inner diameter of the uriniferous tubules. In bilaterally adrenalectomized rabbits (Kurotsu et al., 1955b), the renal corpuscles seemed to decrease in size slightly during ventromedial hypothalamic stimulation, and then they gradually enlarged after the stimulation; whereas during the lateral hypothalamic stimulation they enlarged with dilated intracapsular spaces, and after the stimulation they gradually returned to their initial size. The proximal convolution cells always showed features which suggested that they absorbed the filtrate and then discharged it to the blood stream. It was also probable in these adrenalectomized rabbits that the changes in the renal volume were mainly due to changes in size of the renal corpuscles and the other blood vessels. The anuria following bilateral adrenalectomy, which continued even at the stage of the hypothalamic stimulation, was thought to be mainly due to the intensive fall of the general blood pressure and the absorption of the proximal convolution cells. Rrfprenres p . 39-43

12

T. B A N

According to Yokoyama (Yokoyama et al., 1960) who studied urinary bladder responses to the electrical stimulation of the hypothalamus in male mature rabbits anesthetized with small doses of urethane (0.5-0.7 g per kg in body weight), the stimulation of the medial hypothalamic area or the mamillary peduncle produced relaxation response only or relaxation response after an initial contraction, whereas stimulation of the lateral hypothalamic area, mamillotegmental tract or the periventricular stratum produced a prompt, vigorous and sustained contraction as well as miosis and somatic urinary movement. Stimulation of the boundary of the three zones showed almost biphasic responses. (VIII) Respiratory system

In 1951,Ban et al. (1951a) reported hemorrhage of the lung induced by ventromedial hypothalamic stimulation in rabbits (Fig. 5 ) . Accordingly the effects of hypothalamic stimulation on the lung were studied histologically in rabbits (Kurotsu et al., 1956a).

Fig. 5. Hemorrhage of the lung induced by the stimulation of the ventromedial hypothalamic nucleus in the rabbit.

After ventromedial hypothalamic stimulation, the alveolar lumina enlarged, walls thinned and capillaries contracted. In 96 % of all cases, many scattered hemorrhages occurred at the beginning of the stimulation. This hemorrhage was due to rupture of the capillaries by an increase of blood pressure. Immediately after the stimulation, bronchial and bronchiolar dilatations were observed. Goblet cells of the bronchi and bronchioles were also distended, mitochondria increased in number, and then vacuoles began to appear. Forty min after the stimulation, vacuoles began to be discharged. On the other hand, after lateral hypothalamic stimulation, narrowing of the alveolar

SEPTO- PR EOPTI C O - H Y P O T H A LAM I C SYSTEM

13

lumina, thickening and loosening of the walls and dilatation of the capillaries were observed. Sometimes, leucocytes and emigrated cells were found to be more numerous in the alveolar sacs. Pulmonary hemorrhage occurred in 31 % in gross solitary form. This hemorrhage was believed to occur through an increase in permeability of blood vessels. Pulmonary edema accompanied by congestion was seen in the bleeding area. Atelectasis was observed in 40 %. Two out of 8 cases showed pneumonia-like features. The bronchi constricted into asteroid shape, and the bronchial lumina were covered with mucous secretion. Goblet cells were constricted and mucous secretion was observed in both apocrine and ecrine types. Shinoda studied the types of respiratory reactions induced by the hypothalamic stimulation (Shinoda et al., 1958). Electrical stimulation of the various nuclei of the medial hypothalamic area caused respiratory acceleration and also marked levelshifting towards inspiration. At the same time, enlargement of the alveoli was perceived histologically (Kurotsu et al., 1956a). Strong stimulation caused various types of panting with periodical gasping. Stimulation, if repeated, caused marked continuous acceleration in respiratory activity. The effect was greatest after stimulation of the ventromedial hypothalamic nucleus. On the other hand, electrical stimulation of the lateral hypothalamic nucleus and the periventricular stratum caused level-shifting towards expiration. On the whole, weak stimulation gave slight and gradual decrease of respiratory activity (generally, decrease in frequency and amplitude), slightly stronger stimulation caused paroxysmal hyperpnea with preponderance of expiration, and a strong one produced panting attended by marked inhibition of inspiration, also maintaining the shift towards expiration. This panting was smaller in amplitude and shallow, extremely rapid and convulsive in respiration, with gasping hardly intermingled. During these types of respiration, deflation of the alveoli of lungs was also perceived histologically (Kurotsu et al., I956a). Stimulation, if repeated, induced continuous decrease in respiratory activity. This decrease, different from the one seen in the non-narcosis, non-stimulation and untreated, soon (1 5-20 min later) reached the same degree as in natural sleep, but had no such peculiarity of respiratory waves as was seen in natural sleep. On stimulation of the periventricular stratum, the only peculiarity was that respiration was often made to stop by strong stimulation (Shinoda et at., 1958). ( I X ) Gaseous metabolism

The apparatus used was Knipping Gas Metabolimeter produced by Ei-Ken Co. combined with Saeki’s Respiration Chamber for animals. Mature male rabbits weighing about 2.5 kg were used. Experimental results were as follow (Ban el al., 1955). Gaseous metabolism was increased markedly by ventromedial hypothalamic stimulation, whereas lateral hypothalamic stimulation showed a decrease in gaseous metabolism or a slight increase directly after the stimulation followed by a decrease soon after. Choralose or urethane anesthesia produced a slight increase in gaseous metabolism, while lsoamytal(5,5-isoamylethylbarbit~ric acid) anesthesia markedly inhibited the increase in gaseous metabolism induced by ventromedial hypothalamic stimuReferences p . 3 W 3

14

T. B A N

lation. TEAB (tetraethylammoniumbromide) administered to rabbits showed almost the same change in gaseous metabolism as seen in non-anesthetized rabbits. Gaseous metabolism was increased by ventromedial hypothalamic stimulation even in thyroidectomized, unilaterally adrenalectomized or hypophysectomized rabbits, but the increase was less than that in non-operated rabbits. The increase in gaseous metabolism produced by the same stimulation in bilaterally adrenalectomized rabbits was much less than that of healthy rabbits. Bilateral adrenalectomy caused a marked decrease in gaseous metabolism : therefore it was difficult to determine whether the decrease was due to bilateral adrenalectomy only, or partly due to the lateral hypothalamic stimulation.

( X ) Endocrine glands and some other glands According to histological and cytological studies on some gland cells induced by the hypothalamic stimulation, ventromedial hypothalamic stimulation in rabbits caused swelling of the cell body by vacuolization, whereas lateral hypothalamic stimulation induced shrinkage of the cell body, and the intercellular space became much dilated in the medulla of the suprarenal gland (Kurotsu, 1954; Ishida, 1944). The thyroid follicular cells also showed an increase in vacuole and became taller, and the size of the follicle became smaller due to discharge of its contents on ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation, the cells became lower again due to gradual discharge of the vacuole contents to the follicular lumen, and the lumen became larger (Kurotsu, 1954; Fujita, 1947). The cytological changes of the anterior lobe of the hypophysis in rabbits studied by Heidenhain, Mallory and Gomori methods were as follow (Okada, 1954). Lateral hypothalamic stimulation caused accumulation of the secretion in the intercellular spaces. Dilatation of the capillaries, dark cytoplasm and obliteration of the fine structure of the cells were observed. The cell bodies shrunk and the intercellular space dilated markedly, while the ventromedial hypothalamic stimulation showed constriction of the capillaries and swelling of the cell body due to an increase in vacuoles and mitochondria. Concomitantly, the intercellular spaces were reduced markedly. In the pancreatic islets (Kurotsu et al., 1957), an increase in the number of cells was induced, and the cell body became larger by vacuole formation after ventromedial hypothalamic stimulation, whereas lateral hypothalamic stimulation caused a decrease in the number of b cells, and the cell body became smaller by discharging its content. Even in bilaterally adrenalectomized rabbits, ventromedial hypothalamic stimulation induced an increase in the number of @ cells whereas lateral hypothalamic stimulation caused a decrease. The cytological changes in the gland of the mucous membrane of the maxillary sinus induced by hypothalamic stimulation were as follow (Kato, 1958). After ventromedial hypothalamic stimulation, the serous gland cells as well as the mucous gland cells showed secretory production, even in bilaterally adrenalectomized rabbits, whereas lateral hypothalamic stimulation produced a secretory discharge, even in bilaterally adrenalectomized rabbits. Argentaffine cells (Kubo, 1960) in the epithelium of the digestive tube decreased in number on ventro-

-

-

S E P T 0 P R E 0 P T 1C 0 H Y P O T H A L A M I C

S Y ST E M

15

medial hypothalamic stimulation. This result was presumed to be due to a decrease in argentaffinity by liquefaction of the granules.

( X I ) Liver metabolism Yamada ( I 950) reported that the bile capillaries were markedly enlarged with secretory fluid, and in the liver cells granules containing iron decreased after stimulation of the ventromedial hypothalamic nucleus. On the other hand the bile capillaries remained narrow and granules containing iron increased after lateral hypothalamic stimulation. Yamada concluded that the medial hypothalamic area might stimulate bile secretion by the liver cells, and the lateral hypothalamic nucleus might produce the secretion and expel the secretory fluid from the liver by narrowing the bile capillaries in fasted rabbits. After ventromedial hypothalamic stimulation, the acid phosphatase reaction increased in the liver cells and the alkaline phosphatase reaction increased markedly in the bile capillaries (Kurotsu et al., 1951a). The latter is believed to have some relationship to the increase of the secretory fluid in the dilated bile capillaries (Yamada, 1950) in rabbits. The gallbladder contracted, the folds of the mucous membrane increased and the secretion of its cells was cytologically promoted by ventromedial hypothalamic stimulation, whereas after lateral hypothalamic stimulation in the rabbit, the gallbladder became bigger, the folds of mucous membrane decreased and the ordinary epithelium cells appeared cytologically to absorb water (Matsui et a/., 1961). In higher animals, certain hepatic enzymes that metabolize amino acids have been shown to be controlled by the hypothalamus (Shimazu, 1962, 1964a,b). Electrical stimulation of the ventromedial hypothalamic nucleus (20 sec stimulation, every 5 min for 18-20 h) in rabbits, resulted in about an 8-fold increase in activity of tryptophan pyrrolase in the liver homogenate; and lateral hypothalamic stimulation by the same method caused about a 5-fold increase in this enzyme activity. Knox and Auerbach (1955) found the activity of this enzyme was increased by administration of cortisone, and we found an increase of about 43 % of corticosteroid in blood after ventromedial hypothalamic stimulation in rabbits. Studies were made to determine whether the effect of the hypothalamic stimulation on tryptophan pyrrolase was secondary by causing an increase in adrenal activity. Even in bilaterally adrenalectomized rabbits, a 6-fold increase in enzyme activity over the control was recorded after stimulation of either the ventromedial hypothalamic nucleus or the lateral hypothalamic nucleus. These results indicate that the increase in the level of tryptophan pyrrolase observed after electrical stimulation of the hypothalamus is not a secondary effect of increased adrenal activity, but rather a primary effect of hypothalamic activity. To analyze in detail the induction of tryptophan pyrrolase by hypothalamic stimulation, the total amount of apoenzyme and the level of holoenzyme were measured differentially. The activity of tryptophan pyrrolase was assayed on the cell sap fraction of liver homogenate in the presence or absence of excess cofactor. Rat liver microsomes were used as a cofactor preparation. Electrical stimulation of either the sympathetic or parasympathetic zone of the hypothalamus caused a marked elevation in Refirenrer p. 39-43

16

T. B A N

the total amount of apoenzyme. The level of holoenzyme was markedly increased after stimulation of the sympathetic zone (the medial hypothalamic area), but was only slightly increased after stimulation of the parasympathetic zone (the lateral hypothalamic area). Thus, the ratio of holoenzyme to apoenzyme was changed from 1/3 in normal rabbits to 1/2 and 1/5, respectively, in rabbits stimulated in the sympathetic zone and the parasympathetic zone. The activities of tyrosine transaminase and alanine transaminase (Shimazu, 1964a) were likewise elevated about 2- to 3-fold after electrical stimulation of the sympathetic zone. But stimulation of the parasympathetic zone had no influence on these transarninases. Serine dehydratase was affected by stimulation neither of the sympathetic zone, nor the parasympathetic zone.

(XU) Mal$ormation The influence of electrical stimulation or destruction of the mother’s hypothalamus on development of her fetus was studied in rabbits, and the results were as follow (Takakusu et al., 1962). Acute stimulation caused abnormalities chiefly of the central nervous system such as infoldings of the brain and a flexed spinal cord. Besides these, a few cases of herniation of the heart and hypodactyly were found. Malformation of the face was observed in one case whose mother’s ventromedial hypothalamic nucleus had been chronically stimulated during the middle stage of pregnancy. In one case, whose mother’s fornix was destroyed unilaterally, syndactyly and oligodactyly were obtained. A microcephaly (Fig. 6) and herniation of the midbrain were produced by destruction of a large part of the hypothalamus and a part of the thalamus. It was suggested that abnormal proliferation by inhibition of differentiation

Fig. 6. Microcephaly induced by destruction of the mother’s hypothalamus in the rabbit.

SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

17

potency might be a factor in the genesis of malformation in the central nervous system. The mother’s parasympathetic zone of the hypothalamus was thought to be important for the development of the trabeculae in the adrenal gland and thymus, and the sympathetic zone of the hypothalamus was believed to be necessary for their differentiation into parenchymal elements. The effects of electrical stimulation of the hypothalamus on the placenta and uterine vessels were studied in rabbits, and the following results were obtained (Takakusu et al., 1964). Stimulation of the ventromedial hypothalamic nucleus, which belongs to the sympathetic zone, produced dilatation of the uterine vein, narrowing of the villi, withdrawal of fetal blood from the villous capillaries, widening of the maternal blood spaces in the labyrinth, some fragmentation of the syncytium, and bleeding in the intermediate layer. Stimulation of the lateral hypothalamic nucleus, which is the c-parasympathetic zone, induced little change in the uterine vessels, but resulted in contact of the syncytial layers, withdrawal of the maternal blood from the labyrinth and widening of the villi. Accordingly, it was suggested that inhibition of oxygen and nutriment transport from the maternal blood to the fetal blood produced by changes in the placental circulation induced by the hypothalamic stimulation might be one of the causal mechanisms of malformations induced by maternal hypothalamic stimulation or destruction. The specific changes in the placenta mentioned above are similar to the initial feature of the placental change in the toxemia in pregnancy. ( X I I I ) Waking, sleep and emotional behavior

In 1951. it was reported (Ban et al., 1951b) that electrical stimulation of the ventromedial hypothalamic nucleus in rabbits elicited the rage reaction, with which ovulation (Kurotsu et al., 1950) occurred in mature female rabbits, while repeated stimulations of the lateral hypothalamic nucleus (c-parasympathetic zone) induced sleep, and that bilateral destruction of the medial hypothalamic areas (b-sympathetic zone) could produce a state of predominance or super-predominance of the parasympathetic tonus, while bilateral destruction of the c-parasympathetic zones could produce a state of predominance or super-predominance of the sympathetic tonus (Fig. 7). Accordingly, it was thought that sleep could be induced by the state of balance in which the level of the parasympathetic tonus became a little higher than that of the sympathetic after they had conflicted with one another, and that waking was the state of balance in which the level of the sympathetic tonus was a little higher than that of the parasympathetic. Further, it was reported (Ban et al., 1951b) that rage or excitation might be produced in the state of super-dominance of the sympathetic, while lethargy or narcolepsy might be induced in the state of super-dominance of the parasympathetic tonus. These changes could be produced with hypothalamic emotion. It is considered that rage or excitation developing from the waking state belongs to the positive emotional behavior, and that sleep developing from the waking state belongs to the negative emotional behavior (Ban, 1964a). Electroencephalographic changes (Ishizuka et al., 1954) in the hypothalamus in oestrus, anoestrus, pregnant and nonpregnant rabbits also supported these hypotheses on the balance mechanisms. References p. 39-43

18

T. B A N

Fig. 7. Left above: rage reaction induced by stimulation of the ventromedial hypothalamic nucleus, and left below: sedate state induced by bilateral destruction of the same nuclei. Right above: excitatory state induced by bilateral destruction of the lateral hypothalamic nuclei, and right below: sedate state induced by bilateral destruction of the ventromedial hypothalamic nuclei.

Recently, Sano (1962) succeeded in obtaining most marked sedative effects in patients with violent behavior by his postero-medial hypothalamotomy, namely by bilateral destruction of our b-sympathetic zones; and he also demonstrated our aparasympathetic, b-sympathetic and c-parasympathetic zones in the hypothalamus of patients by electrical stimulation before his hypothalamotomy. The sites of electrodes were decided with the help of X-ray photographs. The EEG changes after the posteromedial hypothalamotomy were almost the same as those in the hypothalamotomized rabbits which had been described by Sawyer et al. (1961). It is interesting that our results with rabbits or cats are very similar to Sano’s results with man. ( X I V ) Conditioned reflex induced by hypothalamic stimulation

We produced a conditioned reflex in the pupil, respiration, gastric motility and the general condition of rabbits by using electrical stimulation of the hypothalamus as unconditioned stimulus and sound as conditioned stimulus (Ban and Shinoda, 1956).

19

SEPTO-PREOPTICO-HY POTHA L A M l C SYSTEM

Two forms of the conditioned reflex, namely the sympathetic conditioned reflex and the parasympathetic conditioned reflex, were constructed by using separate reinforcement of electrical stimulation of the ventromedial hypothalamic nucleus or the lateral hypothalamic nucleus, each as the unconditioned stimulus. The reinforcement was given 20 times or so a day at intervals of 5 min. In the sympathetic conditioned reflex, mydriasis, exophthalmos, acceleration of respiration and inhibition of the stomach cs

r. Fronto parietal ~

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Fig. 8. (a) Conditioned EEG response in synipathetic conditioned reflex. CS, conditioned stiniulation (2 c/s). 5th day of the experiment. (b) Conditioned EEG response in parasympathetic conditioned reflex. The gain from the lateral hypothalamic nucleus alone is recorded as well as a quarter of the other three, for its response is extremely peculiar high voltage slow waves. 5th day of the experiment. (c) The same animal that showed the responsive change of (b) showed differential inhibition under the conditioned stimulation of a different rhythm (I c/s). Re/>rmrcs p . 39-43

~

~

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20

T. B A N

movement (not easily produced) were induced by sound from the 3rd or 4th day of the reinforcement. On the 7th or 8th day the response became maximal. In the parasympathetic conditioned reflex, miosis (not easily produced), enophthalmos, depression of respiration and acceleration of stomach movements were all observed to be induced by sound. When the sympathetic conditioned reflex was gradually built up, sham-rage could be induced by such a weak stimulation as could not ordinarily induce the sham-rage, if it had only been applied to the medial hypothalamic area as an unconditioned stimulation that was adopted in the course of reinforcement. Another remarkable observation was that, if any kind of unconditioned reflex was combined in the course of reinforcement, there occurred a special change in the size of the effect of the conditioned reflex of the organ influenced by that unconditioned reflex. For example, mydriasis was more easily produced by reinforcement in a dark room than in a lighted room. Consequently it may be accepted that the hypothalamus has a dynamic adaptability in its functioning. In conditioned EEG responses (Ban and Shinoda, 1960) which were established by electrical stimulation of the hypothalamus in rabbits as unconditioned stimulation, frequency-specific slow waves appeared conspicuously in the hypothalamus. In the sympathetic conditioning, the frequency-specificharmonized slow waves carried superimposed high frequency fast waves and the voltage might be slightly reduced (generalized desynchronization). In the parasympathetic conditioning, on the other hand, high voltage slow waves of 200-300 pV appeared without fast wave activity. Both sympathetic and parasympathetic conditioning established generalization, differentiation and extinction which were confirmed in the EEG responses (Fig. 8). An increase in the blood sugar level and the leucocyte count was observed in the sympathetic conditioned reflex which was formed by using the electrical stimulation of the ventromedial hypothalamic nucleus as unconditioned stimulus (Ban and Shinoda, 1961). On the other hand, a decrease in the blood sugar level and the leucocyte count was observed in the parasympathetic conditioned reflex which was built up through the electrical stimulation of the lateral hypothalamic nucleus as unconditioned stimulus. From these results, we made it clear that the hypothalamic conditioning was also possible in the interoceptive reaction. F U N C T I O N O F THE P R E O P T I C A N D S E P T A L AREAS

The preoptic area belongs to the telencephalon and is closely related to the hypothalamus morphologically (Ban, 1963, 1964b) and functionally. The boundary between the hypothalamus and the preoptic area is not distinct. The preoptic area is divided into 3 cell groups, i.e., the lateral preoptic area, the medial preoptic area and the preoptic periventricular stratum which is in contact with the ependymal layer of the third ventricle (Fig. 1). But their boundaries are not clear. The lateral preoptic area, including the nucleus preopticus magnocellularis, is occupied chiefly by the medial forebrain bundle and the interstitial nuclei of the bundle scattering in this area. The medial

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

21

preoptic area is divided into the pars ventralis and pars dorsalis of the nucleus preopticus principalis. Rostrally the preoptic area continues to the septal region. The stria terminalis originating in the amygdala and partly in the periamygdaloid cortex enters the lateral preoptic area and the medial hypothalamic area from dorsal side (Ban and Omukai, 1959), and these connections were certified physiologically by Yuasa et al. ( 1959). Kurotsu et al. (1950), Kurotsu et al. (1958b), Sakai et af. (1958), Shinoda et al. ( 1958), Ban et a/. (1958) and Yokoyama et a/. (1 960) reported influences of the electrical stimulation of the preoptic and septal areas on ovulation, blood pressure, gastric motility, respiratory movement, milk ejection and urinary bladder response in rabbits. According to these experimental results, the septal region, the preoptic periventric-

Fig. 9. Septo-preoptico-hypothalamicsystem (SPH system) of the rabbit brain. Horizontal section through the septal region, preoptic area and hypothalamus. Area parasympathica A consisting of the septal region (SEP), preoptic periventricular stratum (SPVPI, hypothalamic periventricular stratum (SPVH) and medial niamillary nucleus (MM), and area parasympathica C consisting of the septal region, lateral preoptic area (APL) and lateral hypothalamic area (AHL) are marked by oblique lines. Areas A and C unite in the septal region. The medial preoptic area (APM), medial hypothalamic area (AHM) and lateral mamillary nucleus (ML) belong t o area sympathica B. AH, anterior hypothalamic nucleus; AHIP, anterior continuation of the hippocampus; BOLF, bulbus olfactorius; CAU, caudate nucleus; CE, external capsule; CI, internal capsule; DM, dorsomedial hypothalamic nucleus; F, fornix; HIP, hippocampus; PH, posterior hypothalamic nucleus; PC, cerebral peduncle; PCMS, precommissural portion of the septum; PRM, premamillary nucleus; PUT, putamen; VM, ventromedial hypothalamic nucleus. Rt:firmc,rs p. 3Y-43

22

T. B A N

ular stratum and the lateral preoptic area, including the medial forebrain bundle, showed parasympathetic reactions, and the medial preoptic area showed sympathetic reactions. In accord with these findings, such preopticareas belonging to the telenceph-

Fig. 10. Schematic summary of the courses and terminations of the medial forebrain bundle (MFB) and A-group of fibers. The small black squares show the site of the lesion. (A), A-group of fibers: ACA, anterior limb of the anterior commissure; AD, anterodorsal thalamic nucleus; AH, anterior hypothalamic nucleus; AHIP, anterior continuation of the hippocampus; AHM, medial hypothalamic area; AM, anteromedial thalamic nucleus; APL, lateral preoptic area; APM, medial preoptic area; AV, anteroventral thalamic nucleus; BOLF, olfactory bulb; (C), C-group of fibers; CC, corpus callosum: D, nucleus of Darkschewitsch; EW, nucleus of Edinger-Westphal; HIP, hippocampus; HL, lateral habenular nucleus; H M , medial habenular nucleus; IS, interstitial nucleus of Cajal; LH, lateral hypothalamic nucleus; LT, lateral thalamic nucleus; MD, mediodorsal thalamic nucleus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; OA(P), pars posterior of the anterior olfactory nucleus; PCMS, precommissural portion of the septum; PT, pretectal nucleus; PTAE, parataenial nucleus; PVA, anterior paraventricular nucleus; RT, thalamic reticular nucleus; SGC, central gray substance; SH, septohippocampal nucleus; SPL. lateral septal nucleus; SPM, medial septal nucleus; SPV, preoptic and hypothalamic periventricular stratum; STLL, stria longitudinalis lateralis; STLM, stria longitudinalis medialis; STM, stria medullaris; TD, dorsal tegmental nucleus of Gudden; TOL, lateral olfactory tract; TUBO, olfactory tubercle; 111, oculomotor nucleus; IV, trochlear nucleus; VI, abducens nucleus.

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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alon and locating rostrally to the hypothalamus are thought to be continuations of 3 zones of the hypothalamus. Namely, the medial preoptic area is a continuation of the medial hypothalamic area (b-sympathetic zone), and the lateral preoptic area is a continuation of the lateral hypothalamic area (c-parasympathetic zone). The preoptic periventricular stratum is a continuation of the hypothalamic periventricular stratum (a-parasympathetic zone). And rostrally, the preoptic periventricular stratum and the lateral preoptic area are thought to be united with each other at the septal region. From the functional point of view, the septal, preoptic and hypothalamic areas can be united into one system named the septo-preoptico-hypothdamic system or the SPHsystem (Ban, 1963, 1964b), which can be divided into 3 areas longitudinally, namely ( I ) area parasympathica A or area A consisting of the septal region and the preoptic and hypothalamic periventricular layers, (2) area symparhica B or area B consisting of the medial preoptic area and the medial hypothalamic area, and (3) area parasympathica C or area C consisting of the septal region, the lateral preoptic area and the lateral hypothalamic area (Fig. 9). FIBER C O N N E C T I O N S IN T H E SEPTO-PREOPTICO-HYPOTHALAMIC S Y S T E M

( I ) A-group of Jibers (tractus hypothalamicus periventricularis)

In our Marchi’s sections a few fine fibers from the lateral part of the septum pellucidum occupy the medial part of the diagonal band of Broca, proceed caudad in the periventricular stratum of the third ventricle wall, decrease in number and terminate in the subependymal layer of the rostral part of the cerebral aqueduct. On the way, some fibers enter the pars medianus of the medial mamillary nucleus. These fine fibers belong to our A-group of Jibers. A few fine fibers, occupying the medial part of the tract, originate in the medial forebrain bundle which belongs to our C-group offibers and terminate in the subependymal layer of the cerebral aqueduct bilaterally (Fig. 10). So a part of the C-group of fibers joins the A-group of fibers at the rostral border of the midbrain. Megawa (1960) recognized parasympathetic reactions by electrical stimulation of the subependymal layer of the midbrain central gray substance at the level of the superior colliculus. Ascending fibers of our A-group of fibers proceed in the periventricular stratum of the third ventricle wall to the level of the anterior hypothalamus, and on the way, ramify to the pars medianus of the medial mamillary nucleus. Masai et al. (1953) demonstrated fine degenerated fibers from the lesion in the cortical areas 6 and 8 to the subependymal layer. These fibers are also included in our A-group of fibers: they are shown in Fig. 11. ( I / ) B-group offibers

The dorsal longitudinal fasciculus, which originates in the medial hypothalamic area and descends through the central gray substance, corresponds to the dorsales LangsReferenres p. 39-43

T. B A N

24

nucl. nucl. camp zona

infund. \

corp.

Fig. 1 1 . Frontal sections of cat brain of which cortical areas 6 and 8 were destroyed. Degenerated fibers are recognized in the hypothalamic periventricular stratum (above) and subependymal layer of the central gray substance (below). These fibers belong to our A-group of fibers.

biindel of Schiitz (1 891), which was found in the brain of progressive paralysis. Gurdjian (1927) called these fibers the periventricular system of fibers, and divided the system into the hypothalamic and the thalamic divisions. He reported that the hypothalamic division originated in the ventral premamillary nucleus, posterior hypothalamic nucleus, ventromedial hypothalamic nucleus and the posterior hypothalamic periventricular nucleus; on the other hand the thalamic division was closely related to some cells near the nucleus reuniens. Fibers of both divisions could be traced through the central gray substance to the tectum and medulla oblongata. Ascending and descending fibers of the fasciculus obtained by us (Zyo et al., 1962) by the Marchi technique are shown in Figs. 12 and 13. ( a ) Descending fibers of the hypothalamic component. Fibers originating in the ventromedial hypothalamic nucleus, dorsomedial hypothalamic nucleus, posterior hypothalamic nucleus and the dorsal premamillary nucleus, which all belong to the medial hypothalamic area, run dorsocaudad through the third ventricle wall to the central gray substance of the midbrain. The fibers terminate in the central gray substance at the level of the superior colliculus, and partly in the interstitial nucleus of Cajal. If lesion exists dorsally to the supramamillary decussation, degenerated fibers being traced dorsocaudad to the central gray substance reach dorsally to the tegmental nucleus of Gudden decreasing in number, and in part, terminate in the pars dorsalis of this nucleus. The fiber-group sends the sympathetic impulses from the medial hypothalamic area (b-sympathetic zone) to the lower autonomic centers. Therefore, we call this fiber-group B-group of fibers.

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Some descending fibers from the ventromedial and the dorsomedial hypothalamic nuclei are traced to the central gray substance contralaterally through the supramamillary decussation and terminate dorsally to the oculomotor nucleus (Fig. 13). In all our experiments no degenerated fibers were traced to the oculomotor nucleus, nucleus of Edinger-Westphal, nucleus of Darkschewitsch or the trochlear nucleus.

Fig. 12. Schematic summary of courses and terminations of the dorsal longitudinal fasciculus (FLD). The small black round points show site of lesion. AH, anterior hypothalamic nucleus; AL, nucleus dorsalis vagi; AMB, nucleus ambiguus; CA. anterior commissure; CC, corpus callosum; CHOP, optic chiasm; COLI, inferior colliculus; COLS, superior colliculus; CMT, medial central nucleus; CP, posterior commissure; D, nucleus of Darkschewitsch; DM, dorsomedial hypothalamic nucleus; EW, nucleus of Edinger-Westphal; FLD,dorsal longitudinal fasciculus; HIP, hippocampus; HL, lateral habenularnucleus; HM, medial hypothalamic nucleus; IC, nucleus intercalatus Staderini; IS, interstitialnucleusof Cajal; IP, interpeduncular nucleus; LAM, nucleus laminaris (pars anterior); LI, nucleus of locus incertus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; PH, posterior hypothalamic nucleus; PMD, dorsal premamillary nucleus; PMV, ventral premamillary nucleus; PRH, nucleus prepositus hypoglossi; PVA, anterior paraventricular nucleus; PVP, posterior paraventricular nucleus; RE, nucleus reuniens; RVM, subnucleus reticularis ventralis medullae oblongatae; SG, nucleus supragenualis; SM, supramamillary nucleus; SOL, nucleus tractus solitarii; STPV, hypothalamic periventricular stratum; TD, dorsal tegmental nucleus of Gudden; VM, ventromedial hypothalamic nucleus; 111, oculomotor nucleus; IV, trochlear nucleus; VI, abducens nucleus; VII, facial nucleus; VIIIL, VIIIM and VIIIS, lateral, medial and superior vestibular nuclei ; XII, hypoglossal nucleus.

The dorsal longitudinal fasciculus in which central gray substance was destroyed at the level of the superior colliculus, proceeds caudad ventrally to the cerebral aqueduct as well as the fourth ventricle, and sends fibers to the pars dorsalis of the dorsal tegmental nucleus of Gudden, the nucleus of locus incertus and the nucleus supragenualis (Meessen and Olszewski, 1949). Some fibers of the fasciculus terminate in the medial and lateral vestibular nuclei (Figs. 12 and 13). The dorsal tegmental nucleus of Gudden is divided into two parts, namely the small celled pars dorsalis and the large celled pars ventralis. According to our findings on their fiber connections, the pars dorsalis is connected with the dorsal longitudinal Rt:ferenres p. 39-43

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T. B A N

Fig. 13. Horizontal aspect of the B-group of fibers including the dorsal longitudinal fasciculus (FLD), hypothalamicotegmentaltract (THT), hypothalamiconigraltract (THN)and tegmentohypothalamic tract (TTH). Abbreviations are same as in Fig. 12. VESI, VESL, VESM and VESS, inferior, lateral, medial and superior vestibular nuclei; XDI nucleus dorsalis vagi. TTML shows the lateral tegmentomamillary tract.

fasciculus and the pars ventralis receives fibers of the mamillotegmental tracts and the mamillary peduncle (Ban and Zyo, 1963) (Fig. 14). Degenerated fibers, destroyed in the medial part of the gray substance of the fourth ventricle floor at the level of the facial genu, descend in the gray substance to the level of the nucleus intercalatus (Staderini). On the way, they terminate in the medial vestibular nucleus, nucleus prepositus hypoglossi and nucleus intercalatus. A few fibers entering and penetrating the medial vestibular nucleus terminate in the lateral vestibular nucleus. If the lesion exists in the gray substance of the fourth ventricle floor at the level of the nucleus intercalatus, degenerated fibers terminate in the hypoglossal nucleus and the nucleus intercalatus, and partly in the nucleus alaris (nucleus dorsalis vagi). The majority of degenerated fibers descend laterally to the central canal, and then ventromediad to the subnucleus reticularis ventralis (Meessen and Olszewski, 1949) of the medulla oblongata and the first cervical cord (Figs. 12 and 13). In other cases (Matano et al., 1964) degenerated fibers originating in each part of the vestibular nuclei proceed to the gray substance of the fourth ventricle floor, join the dorsal longitudinal fasciculus and descend laterally to the central canal to reach the rostra1 end of the cervical cord. On the way, some of them terminate in the nucleus prepositus hypoglossi and the nucleus intercalatus (Fig. 15).

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27

3 Fig. 14. Schematic summary of the courses and terminations of the mamillary peduncle (PM) and mamillotegmental tracts observed in our experiments. I , tr. tegmentomamillaris intermedius; 2, tr. tegmentomamillaris medialis; 3, tr. tegmentomamillaris lateralis; 3', tr. tegmentohypothalamicus; 4, tr. hypothalamicotegmentalis; 4, tr. hypothalamiconigralis; 5, tr. mamillotegmentalis lateralis: 6, tr. mamillotegmentalis medialis; 8, tr. tegmentopeduncularis. AHL, lateral hypothalamic area; AHM, medial hypothalamic area; F, fornix; FLM, medial longitudinal fasciculus; FM, fasciculus retroflexus; IP, interpeduncular nucleus; LM, medial lemniscus; ML, lateral mamillary nucleus; MM, medial mamillary nucleus; MT, mamillothalamic tract; PC, cerebral peduncle; PM, mamillary peduncle; PYR,pyramidal tract ;SGC, central gray substance; SM, supramamillary nucleus; SN, substantia nigra; SPVH, hypothalamic periventricular stratum; TD, dorsal tegmental nucleus; TV, ventral tegmental nucleus; 111, oculomotor nucleus; IV, trochlear nucleus.

Rcferenrrs p . 39-43

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T. B A N

Fig. 15. Schematic summary of the courses and terminations of the vestibular component of rhe medial longitudinal fasciculus (FLM) observed in our experiments. The small black round points show the site of the lesion. CHOP, optic chiasm; CNTS, superior central nucleus; CP, posterior commissure; D, nucleus of Darkschewitsch; DSOD, dorsal supraoptic decussation; EP, entopeduncular nucleus; FLD, dorsal longitudinal fasciculus; FUNA, anterior funiculus of the spinal cord; IC, nucleus intercalatus Staderini; IS, interstitial nucleus of Cajal; LH, lateral hypothalamic nucleus; PRH, nucleus prepositus hypoglossi; PRT, pretectal nucleus; RET, reticular nucleus; SGC, central gray substance; SUB, subthalamic nucleus; TEG, tegmentum; VESI, inferior vestibular nucleus; VESL, lateral vestibular nucleus; VESM,medial vestibular nucleus; VESS, superior vestibular nucleus; VS, vestibulospinal tract; VT, ventral thalamic nucleus; ZI, zona incerta; 111, oculomotor nucleus; IV,trochlear nucleus; VI, abducens nucleus.

From these descriptions, it may be concluded that the descending pathway of the dorsal longitudinal fasciculus from the medial hypothalamic area to the caudal end of the medulla oblongata or the rostra1 end of the cervical cord, consists of a t least 3 neurons, and some of the fibers originating in the vestibular nuclei join the fasciculus. ( 6 ) Descendingfibers of the thalamic component. According to Glees and Wall (1946) the fasciculus receives fibers from the medial dorsal nucleus and the centrum medianum of the thalamus. In our cases which were destroyed in midline part of the

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thalamus including the nucleus reuniens and the medial central nucleus (Figs. 12 and 13), degenerated fibers proceed dorsad, occupy the posterior paraventricular nucleus, shift caudad ventrally to the habenula and join the hypothalamic component at the rostral level of the posterior commissure. These fibers occupy the medial part of the B-group of fibers in the midbrain central gray substance and disappear at the level of the oculomotor nucleus. ( c ) Ascending fibers of the thalamic component. The thalamic component runs rostrad ventrally to the habenular nucleus, sends a few fibers to the region ventral and ventrolateral to the lateral habenular nucleus and enters the posterior paraventricular nucleus (Fig. 12). Some fibers of this component terminate in the posterior paraventricular nucleus, and residual fibers proceed ventrad and terminate in the nucleus reuniens, the central medial nucleus and Niimi’s nucleus laminaris (pars anterior). ( d ) Ascendingfibers of the hypothalamic component. Ascending fibers of the hypothalamic component from the midbrain central gray substance terminate most numerously in the posterior nucleus, but also in the dorsal premamillary nucleus, the dorsomedial nucleus and the ventromedial nucleus. A part of these fibers disperses and disappears in the dorsal area to the dorsomedial hypothalamic nucleus. After a lesion has been made in the medial part of the fourth ventricle floor at the level of the facial genu, ascending degenerated fibers can be traced rostrad through the gray substance of the fourth ventricle floor and the midbrain central gray substance as far as the level of the trochlear nucleus. On the way, some fibers enter the nucleus of the locus incertus as well as the pars dorsalis of the dorsal tegmental nucleus of Gudden (Figs. 12 and 13). But ascending fibers of the medial longitudinal fasciculus enter the trochlear nucleus as well as the oculomotor nucleus and the central gray substance dorsal to these nuclei as shown in Fig. 15; hence more rostrally degenerated fibers in the central gray substance include 2 components of the dorsal and medial longitudinal fasciculi. After destruction of gray substance of the fourth ventricle floor at the level of the nucleus intercalatus (Staderini), ascending degenerated fibers are traced in the gray substance of the fourth ventricle floor to the level of the ventral tegmental nucleus. On the way, some fibers enter the nucleus prepositus hypoglossi, the medial vestibular nucleus and the nucleus tractus solitarii, and a few fibers terminate in the lateral and superior vestibular nuclei. More rostrally, some fibers enter the nucleus (supragenualis, the abducens nucleus and the nucleus of locus incertus (Figs. 12 and 13). In other experiments (Matano et al., 1964) (Fig. 1 9 , ascending degenerated fibers originating in the lesions in the superior, medial, lateral and inferior vestibular nuclei could be traced to the rostral level of the pons via the gray substance of the fourth ventricle floor. It is therefore concluded that the ascending hypothalamic component consists of at least 3 neurons and has almost the same connections and courses as shown by the descending fibers. On the other hand (Fig. 15), the ascending fibers in the medial longitudinal fasciculus that originate in the vestibular nuclei penetrate the trochlear and oculomotor nuclei and terminate in the central gray substance dorsally to these nuclei. These Refcsrences p. 39-43

30

T. B A N

fibers may join the dorsal longitudinal fasciculus. The fibers that originate in the vestibular nuclei and mix with the dorsal longitudinal fasciculus may send impulses to the medial hypothalamic area to produce sympathetic responses. The vestibular nucleus receives fibers from the fastigial nucleus of the cerebellum, namely via the fastigiobulbar tract, and sends fibers to the motor nerve nuclei of the eye via the medial longitudinal fasciculus. And some fibers in the medial longitudinal fasciculus could be traced in the Ganser’s commissure (DSOD in Fig. 15) and partly

-

r i g h t truncus

l e f t truncus

sgmpa t h i cue

sympa t h i c u s

before stimul.

stimulation

-

before stimul. 7

etimul a t ion

after s t imul after

. W&Mh v

Left hemisection on t h e l e v e l of t h e decuaaatio nervorum trochlearium before stimul.

before e t imul

W

. r

8 t imulation

stimulation

after stimul. 60 c/a

after 8 t imul

I z z . *. n 1 . 1 n h A 6 1.1

50 FV

.

Fig. 16. Stimulation of right nucleus hypothalamicus ventromedialis.

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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in the lateral hypothalamic nucleus on the opposite side (Zyo and Ban, 1963) (LH in Fig. 15). Regarding nystagmus and deviation of the eye which are sometimes recorded by the electrical stimulation of the hypothalamus, it may be that the impulses enter the vestibular nuceus via the dorsal longitudinal fasciculus and reach the motor nerve nuclei of the eye through the medial longitudinal fasciculus, or that the impulses enter the abducens nucleus directly via the dorsal longitudinal fasciculus, or that the impulses pass through the hypothalamo-cerebello-vestibularpathway and the medial longitudinal fasciculus to the motor nerve nuclei of the eye. After the ventromedial and dorsomedial hypothalamic nuclei had been destroyed, a small number of fibers was traced to the contralateral side of the central gray substance through supramamillary decussation. But no other decussation of fibers descending or ascending in the central gray substance ventral to the cerebral aqueduct and in tee gray substance of the fourth ventricle floor could be demonstrated. Yuasa (1959) reported that the efferent discharges of both cervical sympathetic trunks were increased by unilateral electrical stimulation of the medial hypothalamic area, and the efferent discharges of the trunk on the operated side were not increased by hemisection at the level of the trochlear or trigeminal nucleus (Fig. 16). These results accord with the results mentioned above. The dorsal longitudinal fasciculus decussates partially at the supramamillary decussation and passes through the central gray substance and the gray substance of the fourth ventricle floor without any further decussation to the cervical cord. When the lateral part of the ventromedial and dorsomedial hypothalamic nuclei was destroyed, some fibers of the descending degenerated fibers were traced through the dorsal part of the mamillary peduncle to the ventral part of the tegmentum. Their course is similar to that of the tractus hypothalamico-tegmentalis of Bodian (1940’1 or the fasciculus hypothalamico-tegmentalis of Shimizu (1948). Some of the fibers enter the caudal part of the homolateral substantia nigra (Fig. 13). We call this group of fibers the tractus hypothalamico-nigralis (Zyo et at., 1962; Ban and Zyo, 1963). The hypothalamico-tegmental tract is also included in our B-group of fibers. (III) C-group of fibers It has been recognized that the medial forebrain bundle originates in the olfactory tubercle and the septal region, and that it reaches the midbrain tegmentum via the lateral preoptic and hypothalamic areas. It seems to correspond to ‘das basale Riechbundel’ of Wallenberg (1901). (a) Descendingfibers. According to our Marchi preparations of albino rats (Ban and Zyo, 1962) and rabbits (Zyo et al., 1963), the course and the termination of the medial forebrain bundle are summarized as shown in Figs. 10 and 17. The fiber group running mediad from the olfactory tubercle, and the fiber group forming the diagonal band of Broca from the septal nuclei enter the medial forebrain bundle and terminate in the rostra1 part of the midbrain tegmentum bilaterally. That is, the medial forebrain bundle is divided into the tubercular and septal groups. Fibers originating in the anterior olfactory nucleus, the olfactory tubercle, and the precommissural portion References p. 39-43

32

T. B A N

Fig. 17. Schematic summary of the courses and terminations of the medial forebrain bundle (MFB) and mamillothalamic tract (MT) observed in our experiments. Abbreviations are the same as shown in Fig. 10. AQ, cerebral aqueduct; CA, anterior commissure; CHOP,optic chiasm; COLI, inferior colliculus; COLS, superior colliculus; CORA, Arnmon’s horn; CP, posterior commissure; IP, interpeduncular nucleus; MT, mamillothalamic tract; PRA, preamygdaloid cortex; TV, ventral tegmental nucleus.

of the septum are followed in the medial forebrain bundle to the midbrain tegmentum. A small number of fibers originating in the olfactory bulb passes through the anterior limb of the anterior commissure as well as the lateral olfactory tract and enters the lateral preoptic area, and a few fibers reach the rostral end of the lateral hypothalamic nucleus. The medial forebrain bundle descends in the lateral preoptic and hypothalamic areas and ramifies to these nuclei. On the way, the bundle which originates in the regions more rostral than the preoptic area does not enter the mamillary body. When our lesions are located in the lateral preoptic area or the lateral hypothalamic area, the medial forebrain bundle sends numerous fibers to the medial mamillary nucleus and some to the lateral mamillary nucleus. Descending fibers in the medial forebrain bundle originating in the septum pellucidum, the olfactory tubercle, and the lateral preoptic area enter the stria medullaris (STM in Figs. 10 and 17) through the inferior thalamic peduncle and reach the habenula. These fibers terminate chiefly in the lateral habenular nucleus but partly in the medial habenular nucleus. On the way, some of the fibers from the lateral preoptic area terminate in the anteroventral nucleus (AV), anterodorsal nucleus (AD), mediodorsal nucleus (MD), parataenial nucleus (PTAE), reticular nucleus (RT) jand anterior paraventricular nucleus (PVA) of the thalamus as shown in Figs. 10 and 17. Fibersprighating in the small lesion of the septum pellucidum terminate in the anteromedial nucleus, the anterodorsal nucleus and the parataenial nucleus (Figs. 10

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and 17). Regarding the fibers traced to the habenula via the stria medullaris, we (Ban and Zyo, 1962) followed fibers to the medial forebrain bundle from the olfactory tubercle, and traced fibers originating in the septum pellucidum to the stria medullaris via the fornix system (Fig. 18). Fibers traced to the medial forebrain bundle from the rostrolateral part of the septum pellucidum enter the stria medullaris via the inferior thalamic peduncle and terminate in the habenula.

Fig. 18. Schematic summary of the courses and terminations of the fornix system recorded in our experiments. The small squares show the site of the lesion. AHIP, anterior continuation of the hippocampus; APM, medial preoptic area; CA, anterior commissure; CC, corpus callosum; CHOP, optic chiasm; CM, mamillary body; COMH, commissura fornicis; DSM, supramamillary decussation; F, fornix column; F contr, contralateral fornix column; FS, superior fornix ; H, habenula; HIP, hippocampus; IP, interpeduncular nucleus; ML, lateral mamillary nucleus; MM(L), lateral part of the medial mamillary nucleus; NDB, bed nucleus of the diagonal band of Broca; NSF, fimbrial septal nucleus; NST, triangular septal nucleus; NSTT, interstitial nucleus of the stria terminalis; PCMS, precommissural portion of the septum; SH, septohippocampal nucleus; SPL, lateral septal nucleus; SPM, medial septal nucleus; STM, stria medullaris.

As Wallenberg (1901), Krieg (1932) and Nauta (1958) reported, we also found that descending fibers in the dorsal part of the medial forebrain bundle ran dorsad along the third ventricle wall and entered the central gray substance. Some of them enter the central gray substance on the opposite side via the supramamillary decussation. In the midbrain central gray substance, these fibers scatter and disappear in the subnucleus lateralis of Olszewski and Baxter. The lateral group disperses ventrolaterad in the dorsal tegmental area of the rostra1 midbrain (Fig. 19). When the caudal portion of the lateral hypothalamic nucleus has been destroyed some fibers on the operated side enter the interstitial nucleus 01 Cajal, the nucleus of Darkschewitsch, the nucleus of Edinger-Westphal and the oculomotor nucleus on the same side. Some fibers, after crossing via the supramamillary decussation, also reach the interstitial nucleus of Cajal, but do not reach the other nuclei (Fig. 10). The medial forebrain bundle is called the C-group offibers, because it is in close relationship Riferenus p. 39-43

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T. B A N

A0

R SN pc

PM

Fig. 19. Degenerated fibers descending from the lesion existing dorsally to the supramamillary decussation are divided into 3 groups: A-group of fibers in the subependymal layer, B-group of fibers in the subnucleus medialis of the central gray substance (fasciculus longitudinalis dorsalis) and Cgroup of fibers belonging to the medial forebrain bundle in the subnucleus lateralis. AQ, cerebral aqueduct; COLS, superior colliculus; FM, fasciculus retroflexus of Meynert ; GM, medial pniculate body; IS, interstitial nucleus of Cajal ; MTEG, medial mamillotegmental tract; PC, cerebral peduncle; PM. mamillary peduncle; R, red nucleus; SGC, central gray substance; SN, substantia nigra.

with our c-parasympathetic zone. Evidently the fibers in the subnucleus lateralis of the midbrain central gray substance also belong to the C-group of fibers. The medial forebrain bundle was generally traced to the tegmentum at the level of the interpeduncular nucleus. But when the caudal portion of the lateral hypothalamic nucleus had been destroyed, degenerated fibers were traced to the ventromedial area of the pontile tegmentum at the rostra1 level to the superior olivary nucleus. These fibers pass through dorsolaterally to the interpeduncular nucleus and dorsally to the medial part of the medial lemniscus. These fibers take almost the same course although their direction is reversible, as the ascending fibers observed in other cases which originate in the lesions of the dorsal pontile tegmentum run ventrorostrad t o mingle with the medial forebrain bundle as shown in Figs. 10 and 17. Our descending fiber-bundle is situated dorsally to the posterior hypothalamo-tegmental tract of Crosby and Woodburne (1951), descends in the ventromedial part of the tegmentum, decreases in number and disappears at the level of the caudal part of the pons, but its terminations are not yet clear (Fig. 17). This fiber-bundle connects the caudal part of the lateral hypothalamic nucleus with the ventromedial part of the pontile tegmentum. So this fiber-bundle may send impulses from the lateral hypothalamic nucleus to the metencephalon. ( b ) Ascendingfibers. Guillery (1957) divided the ascending fibers in rats into 2 groups : the hypothalamo-septa1 group and the mesencephalo-septa1 group. The hypothalamo-septa1 group which originates in the lateral hypothalamic area, terminates mostly in the lateral septal nucleus and partly in the bed nucleus of the anterior commissure and dorsal part of the nucleus accumbens, and the mesencephalo-septa1 group which originates in the midbrain, terminates principally in the medial septal nucleus and partly in the dorsal fornix, the supracallosal striae, the Ammon’s horn and the subiculum. In our Marchi sections (Zyo et al., 1963) of rabbits, ascending fibers of the medial forebrain bundle were traced as follows (Figs. 10 and 17). Ascending fibers from the lateral hypothalamic area run rostrad in the lateral hypothalamic and preoptic areas,

SEPTO-PREOPTICO-HYPOTHALAMIC SYSTEM

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decrease in number and reach’the septum pellucidum. The ventral group enters the olfactory tubercle and partly reaches the rostra1 part of the tubercle. In the septal region, fibers ascending in the lateral hypothalamic and preoptic areas proceed to the diagonal band of Broca. The more thickly myelinated numerous fibers in the medial part terminate in the medial septal nucleus. And thinly myelinated fibers in the lateral part terminate in the lateral septal nucleus. A few fibers enter the nucleus septohippocampalis, and in part, enter the nucleus septohippocampalis of the opposite side after crossing in contact with the corpus callosum (Fig. 20, d and c). Some fibers are traced rostrad to the precommissural portion of the septum. These fibers in the precommissural portion of the septum are divided into 5 groups (Fig. 20b):

d STLM

STLL SPM

U

SPL

Fig. 20. Ascending degenerated fibers originating in the lesion of the rostrolateral part of the lateral preoptic area. ACA, anterior limb of the anterior commissure; AHIP, anterior continuation of the hippocampus; CAU, caudate nucleus; CC, corpus callosum; CI, internal capsule; OA(P), pars posterior of the anterior olfactory nucleus; PCA, posterior limb of the anterior commissure; PCMS. precommissural portion of the septum; SH, septohippocampal nucleus; SPL, lateral septal nucleus; SPM, medial septal nucleus; STLL, stria longitudinalis lateralis; STLM, stria longitudinalis medialis; TOL, lateral olfactory tract; TUBO, olfactory tubercle; VL, lateral ventricle. References p. 39-43

36

T. B A N

Fig. 21. Schematic summary of the courses and terminations of the supraoptic decussations recorded

in our experiments. AQ, cerebral aqueduct; CI, internal capsule; CM, mamillary body; COLI, inferior colliculus; COLS, superior colliculus; EP, entopeduncular nucleus ; F, fornix ; FLM. medial lonpitudinal

fasciculus; FM, fasciculus retroflexus; GL, lateral geniculate body; GM, medial geniculate body; GP, globus pallidus; IP, interpeduncular nucleus; LH, lateral hypothalamic nucleus; LM,medial lemniscus; MT, mamillothalamic tract; PC, cerebral peduncle; PYR, pyramidal tract; RT, thalamic reticular nucleus; SGC, central gray substance; SN, substantia nigra; SOP, supraoptic nucleus; SUB,subthalamic nucleus; TD, dorsal tegmental nucleus; TROP, optic tract; VIII, third ventricle; ZI, zona incerta; 111, oculomotor nucleus; IV, trochlear nucleus.

(I) fibers terminating in the cortex of the medial wall of the precommissural portion of the septum, (2) fibers proceeding dorsad vertically and terminating in the dorsal part of the medial surface of the cortex, (3) fibers terminating in theanteriorcontinuation of the hippocampus, (4) fibers traced to the supracallosal striae, and (5) fibers proceeding rostrad medially to the lateral ventricle and the anterior limb of the anterior commissure and terminating in the pars posterior of the anterior olfactory nucleus (Fig. 20a). The fibers of group ( 4 ) turn dorsad passing rostrally to thegenu corporis callosi and are traced to the striae longitudinales medialis and lateralis at

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the dorsal side of the corpus callosum (Fig. 20, d and c). The fibers in the stria longitudinalis medialis disperse in the cingulum, and partly project dorsad to the dorsal part of the medial surface of the cortex at the level of the rostral part of the septum to the rostral end of Ammon’s horn. The fibers in the stria longitudinalis lateralis disappear in the indusium griseum at the level of the rostral part of the septum. Some fibers of the ascending medial forebrain bundle are followed to the stria medullaris through the inferior thalamic peduncle and terminate in the habenula (Figs. 10 and 17). The ventrolateral group of fibers in the medial forebrain bundle forms the ventral supraoptic decussation (Meynert’s and Gudden’s commissures) at the rostral part of the hypothalamus (Fig. 21). In a case of lesion in the dorsal tegmental area just lateral to the medial longitudinal fasciculus at the level of the dorsal tegmental nucleus of Gudden (Fig. 21), fibers proceeding ventrorostrad in the medial part of the tegmentum and fibers going ventrad along the abducens root and running dorsally to the medial part of the medial lemniscus are united into one group at the level of the inferior colliculus and are traced rostrad dorso-laterally to the interpeduncular nucleus. This fiber-group passes through the region dorsal to the mamillary peduncle and medial to the medial lemniscus and enters the ventrolateral part of the lateral hypothalamic nucleus. These fibers decrease in number terminating in the lateral hypothalamic nucleus and reach the rostral part of this nucleus. A few fibers terminate in the lateral preoptic area. A small number of fibers on the medial group of this fiber-bundle traverses the midline dorsally to the mamillary body, proceeds rostrad in the ventral part of the lateral hypothalamic area and terminates in the nucleus at the level of the ventromedial hypothalamic nucleus (Figs. 10 and 17). Hence it seems that some fibers of the medial forebrain bundle originating in the pontile tegmentum pass through the ventromedial part of the midbrain tegmentum and reach the lateral hypothalamic nucleus (partly on the opposite side) and the lateral preoptic area on the same side. This course is almost the same as the course of the descending medial forebrain bundle from the caudal part of the lateral hypothalamic nucleus, though their directions are reversed. As mentioned above, both descending and ascending fibers compose the medial forebrain bundle and they take almost the same course. These fibers connect the following areas : rhinencephalon, septal area, habenula, hippocampus, thalamus, lateral preoptic and hypothalamic areas and midbrain as well as pontile tegmentum. According to our experiments, it appears that the medial forebrain bundle is closely related to the olfactory or feeding reflexes and t o the parasympathetic responses. This bundleis thought to be the most important main route of the parasympathetic centers. SUMMARY

According to our histological and experimental studies on the central autonomic nervous system, the preoptic area and the septal region are closely related to the hypothalamus from the viewpoints of their functions and fiber connections. The medial preoptic area, which is located rostrally to the medial hypothalamic area (bsympathetic zone), shows sympathetic reactions. The preoptic periventricular stratum Rrfircvwrr p . 39-43

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continues to the hypothalamic periventricular stratum (a-parasympathetic zone) and the lateral preoptic area to the lateral hypothalamic area (c-parasympathetic zone), and they all react parasympathetically. The septum pellucidum, which is situated rostrally to the preoptic area shows, on the whole, parasympathetic responses. Therefore, it is considered that the a- and c-parasympathetic zones of the hypothalamus continue rostrad via the preoptic periventricular stratum and the lateral preoptic area respectively to be united at the septal region (Fig. 9). The A-group of fibers, or the tractus hypothalamicus periventricularis, originating in the septum pellucidum is traced through the periventricular stratum of the third ventricle wall to reach the subependymal layer of the midbrain aqueduct, and ramifies on the way to the medial mamillary nucleus. The tractus corticohypothalamicus periventricularis originating in the cortical areas 6 and 8 to reach the periventricular stratum of the third ventricle wall and the periaqueductal gray stratum is also included in the A-group of fibers. The afferent fibers from the midbrain subependymal layer to the septum are also included in the A-group of fibers. The dorsal longitudinal fasciculus, the hypothalamicotegmental tract with the hypothalamiconigral tract and the tegmentohypothalamic tract (Ban and Zyo, 1963) are included in the B-group of fibers which connects the medial hypothalamic area with other autonomic centers of the brain stem. The dorsal longitudinal fasciculus runs through the central gray substance and the gray substance of the fourth ventricle floor between the hypothalamus and the spinal cord. This fasciculus consists at least of 3 neurons between the hypothalamus and the first cervical cord, and contains both efferent and afferent fibers. Afferent fibers ascend almost the same course as the efferent fibers. The hypothalamicotegmental tract descends caudad to the midbrain tegmentum, and the tegmentohypothalamic tract ascends from the midbrain and pontile tegmentum to the hypothalamus. The sympathetic impulses descend to or ascend from the lower centers through the B-group of fibers, especially via the dorsal longitudinal fasciculus. But some sympathetic impulses can be conducted to the midbrain tegmentum via the hypothalamicotegmental tract. Therefore, these fiber bundles should be considered first when the sympathetic responses of the brain stem are discussed. The C-group of fibers participating in the parasympathetic reactions is the medial forebrain bundle. This bundle includes ascending and descending fibers and connects the lateral preoptic and hypothalamic areas with the rhinencephalon, the septal region, the midbrain and the pontile tegmenta. Some fibers enter the central gray substance of the midbrain and mingle in the A-group of fibers. These fibers are also included in the A-group of fibers. The septal region and the preoptic and hypothalamic areas can be united into one system named the septo-preoptico-hypothalamic system or the SPH-system, based on our studies of the stimulation and destruction experiments and the fiber connections. In conclusion, the septo-preoptico-hypothalamic system is divided into 3 areas: (1) area parasympathica A or area A consisting of the septal region, the preoptic periventricular stratum and the hypothalamic periventricular stratum; (2) area sympathica B or area B consisting of the medial preoptic area and the medial hypothalamic

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area; and (3) area parasympathica C or area C consisting of the septa1 region, the lateral preoptic area and the lateral hypothalamic area.

ACKNOWLEDGEMENT

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Synaptic Interaction at the Mauthner Cell of Goldfish TARO FURUKAWA Department of Physiology, Osaka City University Medical School, Osaka (Japan)

INTRODUCTION

In the spinal cord of teleost fishes a pair of large myelinated nerve fibers runs down closely parallel with each other on the ventral side of the central canal. These fibers constitute a remarkable structure in the fish’s spinal cord, since their diameter can be as large as 50 p. The Mauthner cells, located one on each side in the medulla, are neurons that give rise to these large axons. Because of their big size and peculiar shape, the Mauthner cells have attracted the interest of histologists from early times (Beccari, 1907). Studies of Bartelmez (1915) on catfish and the later work of Bodian (1937, 1952) on goldfish furnished a picture of the structure of the cell with synaptic endings that cover its surface. Especially interesting from the physiological point of view is the fact that synaptic endings of different types are distributed as groups on different parts of the cell’s surface. Furthermore there are types of synaptic endings that are not commonly found in other neurons. Electrophysiological studies disclosed many different kinds of synaptic mechanism that operate on this single neuron. It has been well established that transmission at synapses is ordinarily mediated chemically. In the M-cell, however, there exist excitatory as well as inhibitory synapses that are operated electrically. These electrical synapses coexist with a more common type of synapse where transmission is made chemically. Another interesting fact is that we can distinguish five inhibitory processes each having different sites of action on the Mauthner cell system. Descriptions of these various types of synaptic activity and their possible role in synaptic integration in the Mauthner cell will constitute the main topics of the present communication. Descriptions of some basic properties of the Mauthner cell are based on two papers by Furshpan and the author (Furshpan and Furukawa, 1962; Furukawa and Furshpan, 1963), and descriptions of the excitatory electrical transmission are based on a recent report by Furshpan (1964). The second half of the article is mostly concerned with experiments carried out in the author’s laboratory. METHODS

Anatomy. Fig. lA, taken from Bodian (1952), shows the peculiar shape of the M-cell with various types of synaptic ending that cover the cell’s surface. Of particular

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interest are the large myelinated club endings on the lateral dendrite (see next Section) and the helicoidal feltwork of fine fibers (sp.) that is embedded in the so-called axon cap (Bartelmez, 1915; Bodian, 1952). The axon cap is an approximately spherical structure of glial and nervous elements surrounding the axon of the M-cell from its axon hillock origin to the beginning of its myelination (broken line of Fig. 1A). The axon of each M-cell then expands to about 40 u , in diameter, decussates with its fellow and runs down the spinal cord (Fig. I B). In the spinal cord many fine collaterals come out from the axon. They make contact with motor neurons in the spinal cord with special axo-axonal synapses (Bodian, 1952). This structural feature suggests that M-cells are concerned with synchronous activities of tail muscles. In fact, it has been A

Club endings A

Lateral dendrite

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Cerebellum

Fig. 1. Structure of the goldfish Mauthner cell. (A) Taken from Bodian (1952). A semischematic drawing projected on a transverse plane. Midline is t o the left, dorsal above. Note particularly the thick dendrites and the several types of presynaptic endings. VIII (Xed), the unmyelinated club endings; sp. = fibers which spiral around the axon neck; d = fine dendrites. (B)A schematic drawing of the location of the M-cells within the medulla as seen from above. The cerebellum is shown retracted forward to expose the subjacent medulla. The cells are more than 1 mm beneath the surface of the medulla. Note the decussation of the M-axons.

observed that a single activity of the M-cell evokes a fairly strong tail movement toward the opposite side, i.e. to the same side as the M-axon (Diamond and Furshpan, unpublished observation). Experimental procedures. Experiments were performed upon common Japanese goldfish (Carassius auratus L.), measuring about 12 cm from nose to the tail tip. They were immobilized by intramuscular injection of Flaxedil (ca. 1 pg/g of body weight) and held in position in a fish chamber. The fish were kept alive during the Huferences p. 69/70

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experiment by flowing aerated and dechlorinated tap water through the mouth piece, Because M-cells are located more than 1 mm below the medullar surface, the first problem in working with these cells is to determine their position by blind exploration with the microelectrode. This can be done by taking advantage of the fact that the M-cell, when activated antidromically, generates an extracellular field of action potential around it. By far the most predominant extracellular action potential of the M-cell is a negative potential that can be recorded when the microelectrode approaches within 200p or so of the axon hillock of the cell. The amplitude of this negative spike can be as large as 40mV when the electrode is placed very close to the axon hillock (upper trace of Fig. 2A). In this way it is not difficult to bring the tip of the microelectrode to within 20-3Op from the axon hillock of the M-cell. It is also not difficult to impale the soma or even the lateral dendrite a t various distances from the axon hillock, because these structures are known to extend toward the lateroposterior direction from the axon hillock. Also the axon can be impaled at various points along its course. Although the positional relationship was used mostly as a basis for locating various parts of the cell with the microelectrode, it was also possible to tell from responses recorded whether the structure entered by the electrode was the soma or the axon. For example, the antidromic action potential of the soma is small (30-40 mV) and it is further reduced in size when evoked during the late phase of collateral inhibition (Fig. 5A). On the other hand, when the electrode impales the axon a larger action potential is recorded (60-90 mV) and it is not reduced in size during inhibition (Furukawa and Furshpan, 1963). Stimulation of the M-cell. It is very simple t o activate M-cells antidromically. M-axons can be excited more or less selectively by simply applying electrical pulses to the unopened spinal cord. Presumably M-axons have a much lower threshold than most of other fibers in the spinal cord. Usually two M-axons are excited together, but very often one or the other M-axon is excited selectively when stimulus parameters are finely adjusted. Usually two pairs of stimulating electrodes were embedded close to the unopened vertebral column. This arrangement made it possible to deliver a pair of stimuli at different intensities. Direct stimulation of the M-cell was made by passing a depolarizing current pulse through the intracellular electrode. A bridge circuit was used so that the same electrode served for potential recording and current passing (Araki and Otani, 1955). Orthodromic stimulus to the M-cell was applied t o the 8th nerve root through a very fine bipolar electrode. Activity in the 8th nerve was also evoked by a sound stimulus: a tone pip was applied through a loudspeaker. Action potentials of the M-cell. Action potentials of the M-cell are unique, so that some preliminary account of them may be useful. Fig. 2A shows potential changes produced by antidromic stimulation of the M-cell. In the upper trace, recorded extracellularly from the vicinity of the axon hillock, spike potential appears as a negative deflection of a verp large size. In the lower trace, recorded intracellularly from the soma, the action potential appears as a positive spike. This spike potential, about 40 mV, is much smaller than usually observed in other neurons. Two spikes in Fig. 2A make an almost complete mirror image. These special relationships between

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8

OUTSIDE

MEMBRANE

INSIDE

Fig. 2. (A) Potentials recorded from the M-cell following a single spinal cord shock. Upper trace, extracellular potential recorded from the vicinity of the axon hillock; lower trace, intracellular potential recorded from the soma of the same M-cell. The arrow marks the EHP. Positivity upwards in this and the following oscillographic records. Scales in this applies also to C. (B) A circuit diagram illustrating recording conditions. The left side of the diagram pertains to cell parts which were active during the action potential (axon hillock region); the right side to the inactive region. VI = extracellular recording of action potential; VZ = intracellular recording; E = e.m.f. during the spike; R I = active membrane resistance; RZ = convergence resistance; R3 = tissue resistance around the soma and the dendrite; R.I = membrane resistance of the inactive part of the cell. (C) Block by the EHP of antidromic spike. Extracellular recording; 1 = the EHP produced by the conditioning stimulus; 2 = testing antidromic spike; 3 = 1 and 2, the spike is absent. (D)A schematic diagram illustrating the origin of the EHP. Electromotive forces that are oriented around the initial axon segment give rise to the electrical inhibition.

extra- and intracellularly recorded spikes may be attributable to the fact that the area of M cell membrane that takes part actively in spike generation is limited to a small part in the vicinity of the axon hillock and also to the fact that the membrane time constant of the cell is very short (Furshpan and Furukawa, 1962). In Fig. 2B, E and R1 represent the e.m.f. of action potential and active membrane resistance of the axon hillock region respectively. The action current leaves the cell across the inactive part of the cell membrane (R4), then flows through extracellular space (R3 and Rz) to converge to a small active membrane located in the vicinity of the M-cell axon hillock. Therefore, the intracellular spike potential (VZ)represents the potential drop produced by the action current in the membrane resistance of the soma and the dendrite (R4), while the negative extracellular spike (Vl) represents the potential drop in convergence resistance of the tissue (Rz). R4, when measured as an input References p. 69/70

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resistance of the cell, takes a value of about 105 ohm. The value of Rz is about the same as that of R4. but R3 is much smaller. EXCITATORY RESPONSES

Agerent input to the M-cell. M-cells receive an abundant synaptic input from fibers of the 8th nerve. According to Bartelmez (1915) this direct 8th nerve supply distinguishes the Mauthner neuron from the other cells of the nucleus motorius tegmenti. Both ipsilateral and contralateral 8th nerve fibers innervate the cell but end on different regions of it. The large myelinated club endings (Fig. 1A) are the endings of ipsilateral 8th nerve fibers and are restricted to the distal portion of the lateral dendrite. The proximal portion of the dendrite receives end-bulbs of collaterals from contralateral 8th nerve fibers. The cell body, near the axon hillock, also receives collaterals from contralateral 8th nerve fibers (unmyelinated club endings in Fig. 1A). The ventral dendrite apparently does not receive a direct input from the 8th nerve but is supplied with end-bulbs of axons from the ventral acoustic nucleus and thus has an indirect 8th-nerve input; and the axon cap receives numerous collaterals of secondary 8th nerve fibers. Fiber connections as described above are mostly taken from Bartelmez (191 5) and Bodian (1952). They stimulate our imagination about the role each fiber connection plays in the functional organization of the M-cell system. Although in principle it should be possible to elucidate the function of each fiber group by stimulating it separately and observing the effects thereby produced on the M-cell, our knowledge in this direction is still very incomplete. We know, however, that stimulation of the ipsilateral and contralateral 8th nerves has different effects on the M-cell. Only stimulation of the ipsilateral root normally gives rise to firing of the cell, whereas contralateral stimulation could suppress this firing (Furshpan and Furukawa, 1962; also see Retzlaff, 1957). The origin and properties of large myelinated fibers that terminate on the distal part of the M-cell lateral dendrite will be described. Sound receptor organ and the Mauthner cell. The work of Von Frisch and his school on sound perception in a series of bony fishes has shown that hearing is localized in the pars inferior of the labyrinth which consists of sacculus and lagena, while the pars superior, which consists of utriculus and three semicircular canals, is concerned with equilibrium function (Von Frisch, 1936). Some bony fishes, called Ostariophysi, have notably better hearing ability than other fishes. In this group of fish, in which goldfish is included, the air bladder is connected with the anterior part of the sacculus by means of a chain of three Weberian ossicles which may serve as sound conductors linking the air bladder, acting as a resonator, with the sound receptors in the saccular macula (Fig. 3A). The saccular otolith, the sagitta, has striking wing-like extensions and lies delicately suspended over the saccular macula, while the lagenal otolith is a very massive structure. These findings indicate that the sacculus is the main hearing organ in this group of fishes, though the lagena was also found to be sensitive to sound stimuli. Histological studies showed that there exist two distinct groups of fibers in afferent

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nerves from the saccular macula (Fig. 3B). Large myelinated fibers (diameter 10-15 p ) are distributed over the anterior part of the saccular macula, while small fibers (diameter 5 p), also myelinated, are distributed over a larger area toward the posterior part of the macula. There is a short strip in between the two innervated areas, where fibers terminate only sparsely. Afferent fibers from the lagenal macula are largely similar to those distributed over the posterior part of the sacculus. Action potentials of these fibers were recorded with a microelectrode a t certain points before they enter the medulla. Responses to sound were markedly different between two groups of fibers from the sacculus. Large fibers had their optimal frequency at about 600 c/s; they did not show any spontaneous activity and adapted very rapidly to sound stimuli. On the other hand, optimal frequency of the small fibers from the sacculus lay at about 200 c/s; they usually showed spontaneous activity and adapted only slowly to sound. Small fibers were more sensitive to sound than large fibers. A further difference was found in the relationship between sound frequency and discharge rate in nerve fibers. Some of the large fibers (about half of the total) discharged impulses at a rate twice as frequent as the sound (see Fig. 9A): that is, they fired at a rate of 1000/sec when stimulated by a sound at 500 c/s. But this type of response was not found in the small fiber group. All other large fibers and all small fibers followed the sound with the same frequency of discharge (Fig. 9B). A close examination disclosed that fibers of the latter group were divided into two by the phase of the sound to which they responded. About half the fibers were found to fire in response to the arrival of a certain phase of the sound, e.g. the compression phase, while the rest of the fibers responded at the phase 180" from the former, i.e. at the rarefaction phase. Now turning back to the M-cell again, the existence of a special group of large fibers in the nerve from the sacculus seems to suggest that fibers that terminate with club endings on the lateral dendrite would be a direct extension of these large fibers. This hypothesis was confirmed by recording potentials intracellularly from the 8th nerve fibers in the close vicinity of the lateral dendrite and also from the lateral dendrite itself. It must be mentioned here that the morphology of the M-cell is somewhat different among different species of fish. Otsuka (1962, 1964), who made an extensive survey of the morphology of M-cells in various species of bony fish of non-Ostariophysi type, found only a small number of club endings on the lateral dendrite of M-cells. Catfish (Arneiurus) and goldfish (Curussius uuratus), in both of which large myelinated club endings were found on the distal part of the lateral dendrite, belong to the Ostariophysi. Further studies are needed to clarify the species difference. Since we now know the response pattern of different fiber groups of the auditory nerve, it should be possible to follow their course in the brain by taking these response patterns as an indicator. But trials in this direction are only just beginning. We do not even know definitely the destination of the small fiber group of the saccular nerve. We are sure now that large fibers from the sacculus are connected with the lateral dendrite-of the M-cell with an-electrical synapse (Furshpan, 1964). But electrical stimulation of the 8th nerve produces, besides a short-latency electric EPSP, a large RrScriwrs p. 69/70

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EPSP in the soma of the M-cell that seems to be evoked, a.s judged from the latency and its time course, by the activity of a chemically transmitted synapse. It would therefore be an attractive explanation that fibers of the small fiber group of the saccular nerve end directly on the soma of the M-cell with an ordinary chemical synapse. But the potential change evoked in the soma of the M-cell in response to a sound that gives a maximal response to the small fiber group was disappointingly small. These findings seem to indicate that small fibers of the 8th nerve are connected with the M-cell only indirectly (Fig. 10). Excitatory electrical transmission. Since first described by Bartelmez (1915 ) the large club endings on the lateral dendrites have been known as a special type of synaptic terminal. Here the myelin sheath extends right into the ending and terminates only a few microns from the Mauthner cell surface. Therefore the unmyelinated axon does not extend appreciably at the endings. The synaptic junctions ofthis ending are constructed as an apposition membrane with no intervening material (Bodian,

,, ShL Fig. 3A. For legend see next page.

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Fig. 3. (A) Connection between swimbladder and labyrinth by the Weberian ossicles in Ostariophysi. Black = Weberian ossicles; Sch. = swimbladder; S.i. = sinus impar (perilymphatic space); C.tr. = canalis transversus (endolymphatic space); S. = sacculus; L. = lagena; U. = utriculus; H. r- brain (posterior part being removed); I. = incus; St. :stapes; M. = malleus. (From Von Frisch, 1936). (B) A transverse section of the nerve from the sacculus, fixed with osmic acid. Large fibers that innervate the anterior part of the saccular macula occupy the left half of the bundle, while small fibers that come from the posterior part occupy the right half. A cross section of the saccular macula appears in the lower left corner.

1952). Electron microscopic study by Robertson et a/. (1963) has disclosed that the synaptic membrane complex in cross section shows segments of closure of the synaptic cleft for 0.2 to 0.5 ,u long. These alternate with desmosome-like regions of about the same length in which the gap widens to 150 A. These findings are interpreted as suggestive of electrical transmission in the club endings. Furshpan (1964), who carried out an electrophysiological study quite independently of the electron microscopic study mentioned above, has elucidated detailed processes of transmission at this synapse. As mentioned above, the EPSP evoked in the M-cell in response to a stimulus to the ipsilateral 8th nerve is often constituted of a deflection that starts with a latency of about 0.6 msec. Besides this, however, a very early intracellular potential change is observed (Furshpan and Furukawa, 1962). Furshpan (1964) demonstrated that this early potential change is an EPSP transmitted electrically. The early EPSP appears with a latency of about 0.1 msec, and its over-all duration is only 1.5 to 2 msec, the rising phase being complete in 0.2 to 0.3 msec. lntracellular recordings made at a number of positions along the M-cell disclosed that this short latency EPSP was maximal in the distal half of the lateral dendrite (up to 50 mV), but declined steeply toward the cell body. This regional difference in the size of the early EPSP clearly References p . 69/70

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indicates that the origin of the potential must be located in the distal part of the lateral dendrite. When the EPSP remained above the threshold after having been reduced in size during electrotonic conduction from its site of origin toward the soma, an orthodromic spike was set up from the initial axon segment. I N H I B I T O R Y RESPONSES

Inhibitory input to the M-cell. The 8th nerve on the opposite side constitutes an inhibitory input to the M-cell. As mentioned above, it can easily be demonstrated that stimulation of the ispilateral 8th nerve gives rise to firing of the M-cell, whereas contralateral stimulation can suppress this firing. Moreover, the inhibition is mediated not only chemically but also electrically (Furukawa and Furshpan, 1963). Perhaps reciprocal actions of two 8th nerves are most important in considering the functional organization of the system. However the effect of contralateral stimulation is not purely inhibitory, but partly excitatory to the M-cell, and this prevents further elucidation of the detailed mechanism. Since this confused state seems to be attributable more or less to a crudeness of our technique in stimulating the nerve, it is hoped that clarification would ensue with an improved method. But there is an indication that the reciprocal action of an 8th nerve volley on M-cells, if it exists at all, would be 8 very subtle one. Although we tend to suppose that the 8th nerve on the side closer to the sound source would be stimulated earlier or more intensely than the opposite one, there is some doubt whether this is so in goldfish. As shown in Fig. 3A, the goldfish has only one swimbladder; the Weberian apparatus, which exists as a pair, transmits sound from the swimbladder to the perilymphatic space which is common to both sides; sound is then transmitted to one common endolymphatic space and finally to the sacculus on each side. These structural features of the fish's sound conducting svstem seem to suggest that the activity of the 8th nerve evoked by a sound would be almost identical on both sides. In accord witl' this reasoning is the fact that fishes lack the ability of localizing a sound source accurately (Lowenstein, 1957). Another inhibitory input to the M-cell is its own axon collaterals. Collateral inhibition of M-cells is well developed, and it can be evoked easily and without any mixture of excitatory action. Therefore our analysis of inhibitory actions to be described below was mostly concerned with this type of inhibition. Collateral inhibition of M-cells. Fig. 2A shows potential changes produced by antidromic stimulation of the M-cell. In the upper tract, recorded extracellularly from the vicinity of the axon hillock, an M-cell spike appears as a very large negative deflection. In the lower trace, recorded intracellularly from the soma, the antidromic spike appears as a positive deflection. Now the extracellular spike potential in the upper trace is followed by a positive wave of about 1 msec duration (see arrow). This extracellular positive wave is of maximal size (10-15 mV) in the region of the axon hillock and there is only a very small intracellular potential change that corresponds to this extracellular positivity. This means that most of the extracellular potential is actually impressed on the membrane of the axon hillock as a hyper-

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polarization : hence the potential is called extrinsic hyperpolarizing potential (EHP) (Furukawa and Furshpan, 1963). Fig. 2D is a schematic diagram illustrating the orientation of electromotive forces that give rise to the EHP around the axon hillock of the M-cell. The origin of the EHP is attributable to the activity of nerve fibers that converge to the axon hillock. In the lower trace of Fig. 2A the spike potential is followed by a slow depolarization. This is an inhibitory postsynaptic potential (IPSP) that appeared in a depolarizing direction. In the M-cell the equilibrium potential of the IPSP lies very close to the resting membrane potential, hence the IPSP is not normally recorded as a potential change. In Fig. 2A the IPSP was turned into a depolarization because of a migration of C1 ions into the cell from the microelectrode (Furukawa and Furshpan, 1963; Asada, 1963). Both the EHP and IPSP are evoked through the activity of M-cell axon collaterals. So far as it has been tested with antidromic or direct stimulation of the M-cell, collateral inhibition appears in association with the EHP and IPSP. These points will be described below. Collateral inhibition of M-cells is characterized by the fact that the effects are evoked, so to speak, in all-or-none manner. A single stimulation of M-cells evoked full-sized EHP and IPSP. Not only antidromic excitation, but orthodromic or direct excitation of the M-cell were almost equally effective in producing the EHP and IPSP. Moreover, the EHP and IPSP appeared on both M-cells even when only one of them was activated. Therefore, it was possible to study the inhibitory effects without being disturbed by previous activity if the inhibition were evoked by stimulating the contralateral M-cell only. Another interesting property of collateral inhibition of M-cells is that it is very easily fatigued. The EHP and IPSP appeared in their full size only when evoked at a rate of once per 2 sec, or less. If M-cells were stimulated at a rate of S/sec, for instance, there was no trace of the EHP or the IPSP. Generally, inhibitory effects are assessed by measuring how activities of the target structure are suppressed by inhibition. Upon testing the inhibition of M-cells in this way, we found that different phases of inhibition showed up depending on how the test-stimulus was applied. Three different tests were used : (1) antidromic stimulation; (2) direct stimulation; and (3) orthodromic stimulation. Only the effects of postsynaptic inhibition were detected by tests 1 and 2, whereas inhibitory actions on presynaptic elements could be detected, in addition to postsynaptic inhibition, by test 3. Tests with antidromic stimulation. Both conditioning and testing stimuli were delivered to the spinal cord. It was then found that the EHP and IPSP had quite different effects on the testing antidromic spike (Furukawa and Furshpan, 1963). The testing antidromic spike that was made to arrive near the peak of the EHP was often blocked and could not invade the axon hillock of the M-cell. In the experiment of Fig. 2C, the microelectrode was positioned in the neighborhood of the M-cell axon hillock. The conditioning spinal cord shock was adjusted to obtain the EHP in the absence of the spike, as occurred when only the opposite M-cell fired (CI). C2 shows the testing spike fired by itself. In C3, where the testing stimulus was preceded by the conditioning, the spike is absent. By recording the potential not only extracellularly but also intracellularly from the soma or from the axon, it was confirmed Rcfrrmcvs p. 69/70

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that the block of antidromic impulse during the EHP took place within the axon hillock. A block by the EHP was not always observed, however, because the occurrence of a block depends on the balance between the safety margin of antidromic invasion and the EHP size. Moreover, the all-or-nothing nature of a block made it difficult to analyze the result quantitatively. This difficulty was avoided by applying an artificial electrotonus to the axon hillock region of the M-cell. A microelectrode was positioned with its tip in the axon cap; a recording microelectrode was also inserted into the cap or into the M-cell itself, to monitor the spike. It was then found that the EHP could be imitated by relatively small currents applied by an extracellular electrode. The blocking of the spike took place at the same level of extracellular potential whether brought about by the EHP or by externally applied current pulses. It was also found that critical depolarization needed for an orthodromic firing of the M-cell was elevated during the EHP by exactly the same amount as the size of the EHP. These findings indicate that the EHP suppresses the excitability of the M-cell by an electrotonus extracellularly applied to the initial axon segment (Fig. 2D). A suppression of excitability during the EHP was demonstrated very clearly also with a direct stimulation of the M-cell as will be described later. The effect of the IPSP on the antidromic spike was quite different from that of the EHP. The antidromic spike was not blocked, but it was reduced in size when it arrived during the IPSP. The reduction was observed only with intracellularly recorded spikes (lower trace in Fig. 5A). Antidromic spike size was reduced during postsynaptic inhibition due to the special recording conditions in the M-cell. In the diagram of Fig. 2B, the spike potential recorded inside the soma (VZ)represents a potential drop produced by the action current across the input resistance of the cell (R4).Therefore, spike amplitude is reduced when the input resistance of the cell is decreased by the shunting effect of inhibitory postsynaptic action. The same reasoning explains why the extracellularly recorded spike is not reduced during the IPSP. On the contrary a reduction in the input resistance should be followed by an increase in the action current, and hence an increase in the extracellular spike amplitude. Such an increase was actually observed (Furukawa and Furshpan, 1963). Although the inhibitory synapses here concerned are distributed not only over the soma of the cell but on the base of the lateral dendrite, the reduction of antidromic spike size reflects more sensitively the conductance change that takes place across the soma membrane than that which occurs across the dendritic membrane. On the other hand, the EPSP that originates in the lateral dendrite receive a stronger suppression from the dendritic inhibition (see later). Tests with direct stimulation of the M-cell. The excitability change of the M-cell during inhibition was measured by stimulating the cell directly with cathodal pulses applied through an intracellular electrode (Fukami et al., 1965). This method of testing made it possible to compare the intensity of inhibitory action of the EHP quantitatively with that of the IPSP. Such a comparison was not possible with antidromic testings, for the effect of the EHP and the IPSP on antidromic spike is of a different nature. Nor was it possible with orthodromic testings. There are a few

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i i i; b io ;2 1’4 1’6 ;i3 r n s u Fig. 4. Excitability change of the M-cell during collateral inhibition as tested with direct stimulation. I direct stimulation of the M-cell through a K-citrate-filled microelectrode; I1 : similar to I, but a KCI-filled microelectrode was used. A and B - extra- and intracellular potential changes produced by conditioning spinal cord shock; C = amplitude of test antidromic spike (per cenf of the control), plotted against intervals between conditioning shock and peak time of antidromic spikes; D = excitability, i.e. reciprocals of threshold current strength. Pulses are shown as solid rectangles the length of which represents the pulse duration. (From Fukami el al., 1965). drawbacks in using orthodromic stimulation for testing the postsynaptic inhibition in the M-cell. ( I ) Results of orthodromic stimulation are modified greatly by an inhibitory mechanism acting also on the presynaptic side (see next section). (2) Excitatory inflow to the M-cell produced by an orthodromic stimulus is not correlated linearly with stimulus strength. This makes it difficult to express the excitability change in terms of change in the threshold. Direct stimulation with brief depolarizing pulses applied through the intracellular microelectrode is apparently free from these obstacles. The test pulse was delivered at various intervals after the conditioning spinal cord shock. The latter fired the M-cell antidromically thus giving rise to collateral inhibition. The excitability of the M-cell is then expressed by the reciprocal of the threshold needed to make the cell fire. Fig. 4 shows results of experiments carried out in this way. In the experiment represented in Fig. 4 I, an electrode filled with 2 M Kcitrate was used for intracellular recording and stimulation. Record A and B show respectively extra- and intracellular potential changes produced by the conditioning spinal cord shock. Since the strength of the conditioning spinal cord shock was so adjusted as to activate only the M-cell on the opposite side, no antidromic spike appeared in records A and B. The positive deflection in A represents the EHPI the References p . 69/70

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small positive notch recorded in B being its counterpart in the intracellular record. Although no IPSP appeared in the record (B) as a potential change, this by no means indicates the absence of the postsynaptic inhibitory action: it only means that the reversal potential of the IPSP is at about the same level as the resting potential. Fig. 4 I-C shows the antidromic test-spike height (expressed as per cent of the control) against intervals between the conditioning shock and the peak of each antidromic test-spike. In I-D the excitability (reciprocal of the current strength required for threshold stimulation of the M-cell) is plotted against time interval after the conditioning antidromic shock. The test-current pulses are shown as solid rectangles the length of which represents the pulse duration. As shown in Fig. 4 I-D, the excitability change of the M-cell took place with a rather complicated time course; there came first a brief depression of excitability having its peak at an interval a little less than 2 msec, then followed a long lasting suppression with peaks at about 2.7 msec and 3.8 msec of intervals. Two factors may contribute to the change in the excitability as measured with this method of direct stimulation: i.e. changes in the transmembrane potential and in the input resistance of the cell. The first peak of decreased excitability is clearly due to the EHP which hyperpolarized the membrane because it took place before any change in the input resistance of the M-cell started. On the other hand, the third peak of suppression seems to be attributable to a decrease in the input resistance of the cell, because the EHP was completed by then and also because there was no change in the inside potential of the M-cell as shown in B. This is also supported by the observation that the excitability was lowered approximately by the same amount as the reduction in the test-spike height. The curve drawn with a dotted line in Fig. 4 I-D represents the part of the lowered excitability attributable to a shunting effect of the inhibitory postsynaptic action. The difference between the continuous and dotted lines, having a time course very similar to the EHP, represents the suppression attributable to the EHP. In working with a microelectrode filled with 3 M KCI, it was usually observed that the IPSP became positive in sign with a lapse of time after the insertion of the electrode. This depolarizing IPSP grew for a while and eventually reached a steady level. An attempt was made to measure the excitability of the M-cell during such a depolarizing IPSP. Fig. 4 I1 shows an example of such an experiment. From the top downwards are shown the antidromic responses recorded extracellularly (A) and intracellularly (B), the antidromic test-spike height (C) and the excitability of the cell measured with direct stimulation (D). The solid rectangles plotted in Fig. 4 11-D are the reciprocals of the threshold current strengths expressed in per cent of the control. The initial brief dip in the excitability curve seen in D corresponds in its time course to the first peak of the E H P (record A). Therefore, the initial dip must be attributed to an increase in the transmembrane potential caused by the EHP.The suppression was then followed by an increase in the excitability. The transition took place very rapidly; the excitability reached its maximum (165 % of the control) within a few tenths of a millisecond. Thereafter it gradually returned to its original level, the en-

S Y N A P T I C INTERACTION OF MAUTHNER CELL

57

hancement of the excitability taking place with a time course similar to that of the depolarizing IPSP. This finding, taken together with the fact that no enhancement was observed when a K-citrate-filled electrode was used, strongly suggests that the increased excitability observed in this instance is attributable to the presence of a depolarizing IPSP. These results with direct stimulation of the M-cell showed that, although its duration was brief, the suppression during the EHP was very marked, being comparable to that during the IPSP, and that excitability changes during the IPSP depended on changes in the membrane conductance as well as in the membrane potential. bome simple formulae were advanced which describe excitability change in terms of conductance and potential changes, and they were found to coincide fairly well with the experimental observations (Fukami et al., 1965). Tests with orthodromic stimulation. So far as the results of antidromic and direct stimulation are concerned, collateral inhibition of M-cells can be fully accounted for as the sum of the effects of the EHP and the IPSP. This is no longer true, however, when the inhibition is tested by orthodromic stimulation. The effect of orthodromic stimulation was found to be suppressed very strongly by an inhibition different in its time course from either the EHP or the IPSP. This additional inhibition will provisionally be called the third type of inhibition. Existence of this third type of inhibition can easily be shown by comparing the time course of inhibitory effects on antidromic spikes and on orthodromic responses. But a much clearer demonstration was obtained after the action of procaine because it abolishes the EHP and the IPSP while conserving the third type of inhibition (Furukawa et al., 1963, 1964). In Fig. 5, potentials were recorded extracellularly from the axon hillock region (upper traces) as well as intracellularly from the soma of the same M-cell (lower traces). Although our analysis was based in the main upon a study of the intracellular records, the extracellular records also supplied useful information. A spinal cord shock was delivered as a conditioning stimulus at the beginning of each sweep, and this stimulus fired the M-cell antidromically. Following this, after various intervals, a test shock was delivered to the spinal cord again in A and B (antidromic test), and to the ipsilateral 8th nerve in C and D (orthodromic test). Control responses to the 8th nerve stimulus are shown to the left in C and D. As shown, stimulation of the 8th nerve gave rise to positive deflection in both upper and lower traces. The intracellularly-produced positive potential is the EPSP. The extracellular positivity in response to 8th nerve stimulation is a potential named by us ‘extracellular orthodromic response (EOR)’. Presumably this potential is produced by the activity of the 8th nerve fibers that converge to the axon hillock. In the lower traces of Fig. 5A and C, one can see the depolarizing IPSP. Both orthodromic and antidromic test responses were reduced in size during the IPSP. The antidromic test spikes showed a maximum reduction near the peak of the IPSP, but the inhibition of the EPSP became very intense suddenly at about the peak of the IPSP and was maintained thereafter. It is to be noted that a marked reduction of the EOR started at about the same time as the reduction of the EPSP. Fig. 5B and D show records 50 min after intramuscular injection of procaine Referenres p . 69/70

T. F U R U K A W A

58 antidromic rest

orthodromic test

Fig. 5. Acomparison of the effects of a preceding antidromic stimulation on the EPSP, EOR, and antidromic spike. Upper traces, extracellular potentials recorded from the vicinity of the axon hillock of the M-cell; lower traces, intracellular potentials recorded from the soma (A-D) and from the lateral dendrite (E). An antidromic stimulus to the M-cell was delivered at the start of each sweep. Testing antidromic stimuli (A and B) or orthodromic stimuli (C and D) were delivered with varied intervals after the conditioning antidromic stimulus. Shown to the left in C and D are control responses to an orthodromic stimulation (i.e. EOR, upper trace; EPSP, lower trace). A and C, before injection; B and D, 50 min and E, 120 min after i.m. injection of procaine (0.3 mg/g of body weight). (From Furukawa et a/., 1964).

(0.3 mg/g of body weight). The effect of the drug in this instance advanced slowly for about 40 min after the injection until it became stationary, the state shown in B and D being maintained almost indefinitely. As shown in these records, the EHP and the IPSP were removed by procaine. There remained a small IPSP which was evoked with a much longer latency than the IPSP in A and C. The size of the antidromic spike was reduced slightly with approximately the same time course as this late IPSP. Despite these rather drastic reductions in the size of the IPSP and its effect on the antidromic spike size, the inhibitory effect as tested by orthodromic stimulus remained almost unchanged as shown in Fig. 5D. The results of this experiment are plotted in Fig. 6 I. The change in amplitude of the antidromic spike (filled circles), the EPSP (open circles), and the EOR (crosses) is plotted against the interval between the conditioning shock and the peak of each

SYNAPTIC INTERACTION O F MAUTHNER CELL

1

0

1

2

% loo

4

1

I

6

8

59

I

10msec

--*TI spike

40

20 0

2

4

6

10 msec

Fig. 6 (I) Plots of results shown in Fig. 5A-D. Amplitudes of the antidromic spike, EPSP, and EOR relative to amplitudes of control responses are plotted against intervals between conditioning stimuli and the peak of each response. A = before injection of procaine; B = 50 min after injection. Note that early reduction of the antidromic spike and EPSP is absent in (B). (From Furukawa et a/., 1964). (11) A comparison of the effect of a preceding antidrornic stimulation on antidrornic spike size. A = recorded inside the soma; B = recorded inside the lateral dendrite of the same M-cell.

test response. In A (before the injection of procaine) the reduction of antidromic spike size started with a latency of 3 msec, whereas the reduction of the EOR started at about 5 msec. The reduction of the EPSP, though it started at about the same time as the reduction of the antidromic spike, became much more intense thereafter in coincidence with the start of the EOR suppression. After the injection of procaine early reduction of the antidromic spike and the EPSP were abolished as shown in B, and all the inhibitory effects started at about the same time with a delay of about 5 msec. The reduction of antidromic spike size was much weaker in B, whereas the reduction of the EOR and the EPSP was maintained almost unchanged. These findings indicate the presence of an inhibition whose effects appear predominantly on the EPSP and EOR. At first we thought that suppression of the EPSP was mostly attributable, as suggested by the suppression of the EOR, to an inhibitory action on the 8th nerve. We also observed that other signs of 8th nerve activity were suppressed during the inhibition. Hence the presence of an inhibitory mechanism similar to presynaptic inhibition in the cat spinal cord (Frank and Fuortes, 1957; Eccles et al., 1961. 1962a, b) or in the crayfish neuromuscular junction (Dudel and Kuffler, 1961: Dudel, 1963) was postulated (Furukawa et al., 1963). We know now, however, that a substantial part of the third type of inhibition can be attributed t o the postsynaptic inhibition that takes placdin the lateral dendrite. Dendritic inhibition. There are grounds for believing that a t least a part of the third type of inhibition is attributable to the postsynaptic inhibition that takes place in the lateral dendrite. According to this interpretation, the discrepancy such as observed in Fig. 5C between the time course of the somatic JPSP and the reduction in the size of theEPSPcan be attributed to the fact that theEPSPis reduced in size by an increasRcfcrencrs p. 69/70

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ed conductance taking place in the soma as well as in the dendritic membrane, whereas the IPSP recorded from the soma represents only the conductance change taking place in the somatic membrane. In the experiment of Fig. 5 , the recording electrode was withdrawn from the soma about 2 h after the injection of procaine and reinserted into the lateral dendrite of the same M-cell at a point about 250 p from the axon hillock (resting potential, 75 mV). A large depolarizing IPSP was then observed in response to an antidromic stimulus (Fig. 5E). The latency and time course of this IPSP coincide well with those of the third type of inhibition. Since this IPSP was recorded from the dendrite at the time when no IPSP was recorded from the soma, it must have its origin in the dendrite. The amplitude of the antidromic spike in the lower trace of E is unduly small (cf. Fig. 6 11-B). The electrical connection between the soma and dendrite seemed to have been impaired in this experiment while we were trying to put the electrode into the lateral dendrite. Usually, antidromic spike in the dendrite at 250-300 p from the axon hillock was 112 to 1/3 as large as the spike in the soma as shown in Fig. 6 11-B. There is also a difference in the spike duration, but it is relatively small. Fig. 6 I1 illustrates another instance in which potentials recorded from the soma (A) were compared with those from the lateral dendrite (B). It can be seen that the dendritic IPSP in this instance also started with a longer latency than the IPSP in the soma, and was maintained for a longer period. An interesting aspect in these observations is that the depolarizing IPSP in each record predominantly reflects only the permeability change taking place in the part of the cell impaled by the electrode. As described before, an elevated intracellular concentration of CI ions that had migrated from the electrode is responsible for the depolarizing IPSP. The observations mentioned above indicate, however, that an increase of the intracellular concentration occurs only in the neighborhood of the electrode: namely, when the electrode impales the soma, for instance, the concentration of C1 ions would be increased in the soma but would remain largely unchanged in the lateral dendrite. Perhaps a rapid leakage from the interior of the cell in comparison with the diffusion velocity along the long axis of the cell would be responsible for this localized change in the CI ion concentration. In the soma, as shown in Fig. 6 11-A, the reduction in the antidromic spike occurs approximately with the same time course as the IPSP. We have already discussed this (see Fig. 41I-B, C and Fig. 5A). But such a parallelism between the timecourse of the IPSP and the reduction in the size of the antidromic spike cannot be seen in Fig. 6 11-B where the potential was recorded from the dendrite. Test-spikes in this record are reduced in size well before the start of the dendritic IPSP. That is, the amplitude of antidromic test-spikes was reduced, when recorded from the dendrite, not only during the dendritic IPSP but also during the somatic IPSP. Another finding which characterizes the dendritic inhibition is the presence of a spontaneous activity. A vigorous spontaneous inhibitory activity was almost always found in the lateral dendrite. The activity often occurred in a form of bursts that came at an irregular interval without any noticeable rhythm. The amplitude not only

61

SYNAPTIC INTERACTION O F MAUTHNER CELL

of the antidromic spike but of the EPSP was markedly reduced (up to 112) during such a burst of activity of dendritic inhibition. This resulted in an irregular fluctuation in the size of the antidromic spike and the EPSP in the dendrite. Model experiment on the dendritic inhibition. In order to confirm the conclusion in the preceding section, it would be desirable to carry out a systematic exploration. A suitable experiment would require that intracellular potentials be recorded at various points along the soma and the lateral dendrite, and that the effects of somatic and dendritic inhibitions be compared at each point. Actually, this experiment is not easy because: (1) chances are large that the dendrite is damaged by repeated insertions of the electrode; and (2) it is usually not possible to evoke either somatic or dendritic inhibition in isolation. In view of these difficulties an experiment on a model neuron was planned. We started by modifying the drawing of the M-cell by Bodian(Fig. IA). The schematized neuron shown in Fig. 7A was drawn as a stretched structure and was made larger than the original drawing by a factor of 10/7 in order to compensate for the shrinkage. It is constituted of 24 sections, each having a length of 50 ,u and a width that matches the average width of the corresponding part in the original drawing. Longitudinal and membrane resistances of each section were then calculated. In making these calculations, specific resistances of the membrane and the axoplasma A

0

ventral dendrite

-<

soma

soma

RI 12 RI 13

>-

RI 14

lateral dendrite

t loterol dendrite

R I 15

R i 16 Ri 17

+

Ri 18 Ri 19

Fig. 7. (A) A schematized M-cell consisting of 24 sections of equal length (50 p). (B) Circuit diapram of a model M-cell designed for elucidating the difference between the effects of somatic and dendritic

inhibitions. All resistanceswere scaled down to one hundredth of the original value. Rm 1-11 = 4.5 kn; Rm 12 and Rm I5 = 10 k n ; Rrn 13 and Rm 14 = 6 klR; Rm 16 and Rm 19 = 17 kR; Rm 17 and Rm 18 = I9 k a ;Rm 2&24 = 4.2kR; Ri 12 and Ri I5 = 0.4kR; Ri 13 and Ri 14 = 0.1 kR; Ri 16 and Ri 19 = 1 k a ; Ri 17 and Ri 18 = 1.2 Rm 12-Rm 15 and Rm 17-Rm 19 may be switched down to 1/2, 1/3. and 1/4 of the values in the above to simulate the shunting effect of inhibitory postsynaptic action. See text for details.

a.

References p. 69/70

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T. F U R U K A W A

were taken as 53 Q/cm2 and 63 Q/cm2 respectively. The results of these calculations were used in making an equivalent circuit of the M-cell that is constituted of 24 sections of resistance network. The input resistance (at the soma) of this equivalent circuit was 1.1 x 105 Q and was very close to values actually observed on the M-cell ( I .2 x 105 Q, Furshpan and Furukawa, 1962). This in turn legitimated the use of specific resistance values mentioned above. The model neuron actually constructed with electrical resistors and rotary switches consisted of 10 sections as shown in Fig. 7B. In this figure, Rm 12-Rm 19 and Ri 12Ri 19 represent respectively the membrane resistance and the longitudinal resistance of each section that bears the same number in Fig. 7A. The input resistance of the ventral dendrite viewed from section 12 is represented by Rm 1-1 1, and that of the lateral dendrite viewed from section 19 by Rm 20-24. All resistances used in this actual model were scaled down to one hundredth of the original values (see legend of Fig. 7). Membrane capacitance was not taken into consideration in the present model. Because the membrane time constant of the M-cell is very short (about 0.5 msec), omission of the capacitative component should not produce any serious error. In order to simulate an increased membrane conductance during the somatic and dendritic inhibition, provisions were made that values of Rm 12-Rm 15 and Rm 17-Rm 19 may be reduced to 1/2,1/3 and 1/4 of their normal values by changing the positions of rotary switches. Measurements on this model neuron were made as follows. Brief electrical pulses were injected through a lo5 Q resistor at either of two points: in order to simulate an antidromic spike it was injected at the point marked 0 p, whereas to simulate the EPSP generated in the distal part of the lateral dendrite it was injected at the point marked 300 p. In either event potentials were measured at 7 points along the network as shown in the figure. Results of such measurement are summarized in Fig. 8. In this figure, potential sizes are plotted against the distance from the center of the soma as abscissae. Electrical pulses were injected into the soma in A and C and into the dendrite in B and D (see arrows). Therefore curves in A and C show decrements in the antidromic spike in spreading electrotonically from the soma toward the lateral dendrite, while B and D represent electrotonic decrement of the EPSP in the reversed direction. Open circles plot the decrement under a normal condition, while filled circles, crosses, and small dots plot decrements observed when membrane resistances were decreased to '12, and of the normal value in the region indicated by a thick base line. Therefore, A and B correspond to the somatic inhibition, and C and D to dendritic inhibition. Results shown in Fig. 8 can be summarized as follows. (1) Dendritic inhibition, as shown in C, has only a negligible effect on the antidromic spike recorded in the soma. (2) The EPSP recorded in the dendrite receives only a slight influence from the somatic inhibition. (3) The antidromic spike, recorded either from the soma or from the dendrite, is reduced in size by the somatic inhibition. (4) The EPSP, recorded either from the soma or from the dendrite, is reduced in size by the dendritic inhibition. Thus it is clear that situations observed in the M-cell under various conditions were fairly faithfully reproduced on the model e x p e r i m ~ ~ t .

SYNAPTIC INTERACTION OF MAUTHNER CELL

63

0 10 -

0

I

.O

r

P

100

D 10

08 -

08

06 -

06

-

0.4

04

02

-

2 200 =P 0

100

Fig. 8. Results of the model experiment. Potentials recorded on the model are plotted against the distance from the axon hillock. Current pulses were injected at 0 ,LA in A and C, and at 300 p in R and D (see arrows), hence the former simulates electrotonic decrement of the antidromic spike from the soma to the dendrite, whereas the latter simulates electrotonic decrement of EPSP in the reversed direction. Open circles, control; filled circles, crosses, and small dots, effects of various intensities of somatic inhibition (A and B) and dendritic inhibition (C and D).

Frank and Fuortes (1957) produced convincing evidence of spinal presynaptic inhibition, evidence that was thereafter greatly developed by Eccles and others (Eccles, 1964). But Frank (1959) proposed an alternative explanation to his finding. He argued that if an inhibitory action were exerted far out on the dendrite of the motor neuron, the EPSP depression could occur without any trace of the inhibitory influence itself as detected by a microelectrode in the motor neuron soma. He designated the phenomenon ‘remote dendritic inhibition’. It has been shown above that the dendritic inhibition in the M-cell produces the kind of inhibition expected by him. Inhibition at the receptor site. Afferent discharges in the 8th nerve fibers were found to be suppressed during collateral inhibition of M-cells. Fig. 9A and B show two instances in which unit discharges were recorded from large fibers that innervate the sacculus (CJ Fig. 3B). In either instance, a stimulus sound of about 600 c/s was used. I t was recorded with a dynamic microphone placed close to the animal, and was displayed on the oscilloscope. In A, the upper trace shows afferent impulses that are set up at a rate exactly twice that of the sound, monitored in the lower trace. About half of the large fibers in the sacculus showed this type of response. B1 shows impulses that were set up at the same rate as the sound and B2 shows that the discharge was absent when the sound was preceded by a conditioning antidromic shock to M-cells. References p. 69/70

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Fig. 9. Unitary action potentials of 8th nerve fibers. (A) Spikes set up at twice the frequency of the sound; lower trace, stimulus sound (about 600 c/s) recorded with a microphone placed beside the animal. (B) 1 = spikes set up at the same rate as the sound (4); 2 = suppression of spike discharge during collateral inhibition of M-cells; 3 = monitor of the M-cell spike.

In this instance, a second microelectrode was placed in the vicinity of the axon hillock to monitor the action potential in the M-cell (B3). A suppression such as shown in B2 was observed only when the spinal cord shock was strong enough to fire the M-cell antidromically. Small ripples appear in B2. A similar suppression was observed in small fibers. Judged from its latency and duration, it seems appropriate to regard the inhibition at the receptor site as a part of the third type of inhibition. An inhibitory action of the olivo-cochlear bundle on sensory fiber terminations and on the hair cells of the organ of Corti is well known (Galambos, 1956).The presence of a similar efferent control mechanism in the fish’s ear was anticipated, for endings possessing properties of efferent fibers have been demonstrated in the vestibular neuroepithelium of fishes (Lowenstein et al., 1964; Flock, 1964). Our experiments showed that this was indeed so. Efects o j strychnine on collateral inhibition of M-cells. One of the remarkable effects of strychnine at a very low concentration is its ability to block postsynaptic inhibition in the vertebrate central nervous system (Eccles, 1964). Although some

SYNAPTIC INTERACTION OF MAUTHNER CELL

65

exceptions to this generalization have been reported (e.g. Andersen et al., 1963), it is still an interesting drug to be tested on M-cells. When a small amount of strychnine (2-5 pg/g of body weight) was administered intramuscularly into the tail of a goldfish, responses of the M-cell to ipsilateral 8th nerve stimulation were augmented. The time course of the EPSP became much more prolonged, and a repetitive firing of the M-cell occurred superimposed on such a sustained depolarization. Periodic firing of the cell then ensued during progressive action of the drug. A block of inhibition occurred along with these changes. A test of inhibition was made in a way similar to that employed in testing the effect of procaine (see Fig. 5 and 6 I). It was found that the inhibitory effects as detected by antidromic stimulation (reduction of antidromic spike size) and those as detected by orthodromic stimulation (reduction of the EPSP and EOR) were removed with approximately the same time course after an injection of strychnine: namely strychnine removed the IPSP and the third type of inhibition in parallel. On the other hand the EHP was less susceptible to the effect of strychnine than the two other types of inhibition (Furukawa et al., 1964). As to the mechanism of action of strychnine, it is postulated in analogy with the action of curare at the cholinergic junction that the drug would block postsynaptic inhibition by combining with the receptor in competition with the inhibitory transmitter substance (Eccles. 1964). It is therefore suggested that the IPSP and the third type of inhibition in M-cells may be mediated by chemical mechanisms similar to those involved in postsynaptic inhibition in cat spinal motor neurons. It is also explicable from the same reasoning that the EHP, and hence the electrical inhibition, are more resistant to the action of strychnine. DISCUSSION

We have described various types of synaptic action that impinge on the Mauthner cell. There exist excitatory and inhibitory synapses that are based on electrical mechanisms in addition to those based on more conventional chemical mechanisms. To add to the complexity of the system these different types of synapses all have different sites of action. As to the excitatory synapses, electrical synapses are on the distal part of the lateral dendrite, while chemical synapses cover wider areas of the cell (cf. Diamond, 1963). Inhibitory synaptic action is mure complicated, for we know that it takes place at 5 different loci : ( I ) electrical inhibition that takes place at the axon hillock; (2) chemical postsynaptic inhibition of the soma (somatic inhibition); (3) chemical postsynaptic inhibition of the lateral dendrite (dendritic inhibition); (4) inhibition of 8th nerve activity taking place at the receptor site; (5) inhibition of 8th nerve activity taking place inside the medulla. Inhibitions of ( I ) and (2) suppress the activity of the M-cell as a whole, while inhibitions of (3)-(5) specifically suppress the excitatory input from the 8th nerve, leaving other excitatory inputs unobliterated. Fig. I0 shows a hypothetical diagram of neuronal organization subserving collateral inhibition of M-cells. The inhibitory effect is exerted upon the M-cell and upon Referenres p. 69/70

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66 blocked by fat iauc

.

Vest ibular

Sacculus halr cell

Iefr M-axon

Fig. 10. Supposed neuronal organization for collateral inhibition in the M-cell. E = excitatory junction; I = inhibitory junction. Two kinds of 8th nerve fibers, and 4 kinds of interneurons are shown. Three possible terminations (a-c) of N4 are indicated. It is assumed that, besides releasing chemical transmitter substances from their terminals, Nz axons and secondary 8th nerve fibers enter into some close relationship with the axon cap and subserve the generation of the EHP and EOR respectively. See text for further details.

the 8th nerve. There are two kinds of fibers in the 8th nerve: large fibers terminate directly on the distal part of the lateral dendrite of the M-cell with club endings, and small fibers are distributed, after being relayed in the vestibular nucleus, over the axon hillock and the soma. On the peripheral side these 8th nerve fibers are connected with hair cells of the saccular macula. In Fig. 10, the large fiber is joined with the hair cell by a chalice. Tt was drawn by analogy from the structure in the vestibular sensory epithelia of mammals such as guinea-pig, rat and cat (Wersall, 1960). No detailed morphological study has been made on saccular macula of the Ostariophysi. We assume that collateral inhibition is mediated by four interneurons, N1 to N4. The role of generating the EHP and postsynaptic inhibition of the M-cell soma is, for convenience, attributed to a common inhibitory neuron Nz. The axon of N2 is assumed to reach the axon hillock region and enter into some close relation 'with the axon cap before ending on the soma of the M-cell. N4 is another inhibitory neuron to which is allocated the role of exerting the third type of inhibition. Three possible sites of termination for the axonal branches of N4 are drawn in the diagram. Branch a, that terminates on the base of the lateral dendrite, is associated with the dendritic inhibition. Branch b, that terminates on the nerve chalice and hair cell, is associated with the inhibition at the receptor site. Branch c ends on the soma of the secondary 8,h nerve cell. As indicated by the suppression of the €OR (Fig. 5C), some part of the third type of inhibition in the M-cell must be attributed to an inhibition acting on the presynaptic element. There is a possibility that a mechanism similar t o presynaptic inhibition in the spinal cord (Eccles, 1964) or in crayfish neuromuscular junction (Dudel, 1963)

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67

is involved in the third type of inhibition. But we know that this third type of inhibition is totally blocked by strychnine. This clearly indicates that inhibition of the presynaptic element of M-cells is different in nature from presynaptic inhibition in the cat spinal cord. There are at least two possibilities. It might be supposed that the activity of the 8th nerve fibers is suppressed by the inhibitory synapse of axo-axonal type that exerts its action through a mechanism similar to the usual postsynaptic inhibition. Another possibility is that presynaptic inhibition of M-cells might simply be an inhibition in the vestibular nucleus where many of the 8th nerve fibers synapse. Fig. 10 was drawn according to the latter view. Therefore activity of non-synapsing 8th nerve fibers does not receive an inhibitory effect except a t the receptor site. N3 is interposed between N1 and N4 in order to explain the longer latency of inhibition exerted by N4. Finally, N1 is the interneuron which receives fibers from M-cell axon collaterals on both sides. Now the effects of some drugs, such as strychnine and procaine, and of repetitive stimulation may be explained by allocating different susceptibility t o each synapse. Strychnine would block the postsynaptic inhibitory action at synapses marked I in the diagram. On the other hand, the action of procaine is most conveniently explained by a block at the E-synapse between N1 and N2. The E-synapse between N1 and N3 and that between N3 and N4, however, are more resistant to the action of procaine. The fatiguability is shared by all three types of collateral inhibition of the M-cell. Therefore, this property is best attributed to the E-synapse between axon collaterals and N1. Possible functional meaning of various types of synaptic activity in the M-cell. We have succeeded in elucidating to a certain extent various types of synaptic activity in the M-cell. It now remains to discuss the possible functional meaning of this complicated system. By microelectrode recording from Mauthner axons of Protopterm (lungfish), Wilson (1959) showed that a spike was obtained only as a startle response elicited by a severe jar to the aquarium. This was followed by a sudden flip of the tail. I t seems then that the Mauthner cell system functions as an escape mechanism. Certain structural features of the M-cell system seem t o aim at a very fast response which is required for the escape. To begin with the input side, myelinated fibers of a specially large size (I 0-1 5 p, see Fig. 3B) are employed to connect the hair cells in the anterior part of the sacculus to the lateral dendrite of the M-cell. The distance connected by these fibers is very short; in fishes such as those used in the present study it is perhaps not more than 5 mm. This corresponds to a conduction time of only a few tenths of a millisecond if a velocity of 20-30 m/sec is assumed (Tasaki, 1953). Moreover, as mentioned in the results, impulses in these fibers are set up with a short delay of 0.5 msec or so after the arrival of sound. Some of the fibers can respond to both compression and rarefaction phases of the sound : this prevents an additional delay from occurring due to the phase relation of the sound (Fig. 9A). On the output side, Mauthner’s large myelinated axon (d,40 p ) conducts impulses at a velocity as fast as 80-100 m/sec (Furshpan and Furukawa, 1962). Impulses are then transmitted to spinal motor fibers via special axo-axonal synapses (Bodian, 1952). From these considerations it would be reasonable to assume that the electrical excitatory synapses R ~ f e r e n c ~p. s 69/70

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that interpose between 8th nerve fibers and the Mauthner’s dendrite may aim at a transmission with a minimal synaptic delay. We know now that a difference of a tenth of a millisecond counts in this system. It may then be supposed that the electrical inhibition of M-cells has its meaning in the fact that it can be effected without any synaptic delay. An inhibition must act rapidly in order to be in time because of a very fast excitatory action. It is to be noted in this connection that the EHP can be evoked not only by collateral inhibition but also by stimulation of the contralateral 8th nerve. We cannot fully appreciate the functional meaning of collateral inhibition of the M-cell. One may suppose, however, that its function will be to limit the duration of activity of M-cells that were initiated as a startle response. It is not difficult t o suppose that a startle response consists of a disorganized activity of M-cells. Hence it would be profitable for the animal should it stop immediately after a few initial actions. It is interesting in this connection that the duration of the third type of inhibition is much longer than inhibitions that act on the axon hillock and the soma. This means that the acoustic input that causes a startle response is obliterated for a little while even after the M-cell becomes responsive again to excitatory volleys that impinge on the soma or on the ventral dendrite. Then the excitatory influences that are associated with an organized activity of the M-cell would be transmitted on the soma and on the ventral dendrite. Such orderly activities of M-cells would not be suppressed by collateral inhibition because of a fatigue in the neuron chain (Fig. 10). SUMMARY

1. Results of neurophysiological studies on the goldfish Mauthner cell are described. Emphasis was placed on description of various types of excitatory and inhibitory synaptic actions that impinge on this fish neuron. Since synapses of different structure are distributed on different parts of the M-cell, it presents a suitable material for elucidating the function of synapses in relation to their structure and spacial location in the cell. Another interesting aspect with the synapses in the Mauthner cell is the presence of excitatory and inhibitory synapses that are mediated electrically besides those mediated chemically. 2. Large direct 8th nerve fibers terminate on the distal part of the lateral dendrite with club endings. Excitatory action at this synapse is transmitted electrically. The origin of these large fibers was traced to the anterior part of the saccular macula. 3. Collateral inhibition is very well developed in the M-cell. Inhibitory effects were tested by delivering antidromic and orthodromic stimuli to the cell, by stimulating the cell directly with electrical currents, by delivering sound stimuli to the animal, and finally by observing the drug effects. Such analyses led us to conclude that the inhibition comprises 5 different inhibitory processes, which include electrical inhibition at the axon cap, chemical inhibition that takes place separately at the soma and at the lateral dendrite, inhibition at the sound receptor, and so forth. 4. Dendritic inhibition is perhaps a new type of inhibition. It suppresses only the

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excitatory actions that impinge on the dendrite on which the inhibition acts, while excitatory actions that impinge on the soma or other dendrites suffer almost no effect. 5. Neuronal organization subserving these various types of synaptic activities and their possible functional significance are discussed. REFERENCES ANDERSEN, P., ECCLES, J. C., LBYNING,Y., AND VOORHOEVE, P. E., (1963); Strychnine-resistant inhibition in the brain. Nature (Lond.), 200, 843-845. ARAKI,T., AND OTANI,T., (1955); Response of single motoneurons to direct stimulation in toad’s spinal cord. J. Neurophysiol., 18,472-485. ASADA,Y.,(1963); Effects of intracellularly injected anions on the Mauthner cells of goldfish. Jap. J. Physiol., 13, 583-598. BARTELMEZ, G. W., (1915); Mauthner’s cell and nucleus motorius tegmenti. J. comp. Neurol., 25, 87-128. BECCARI,N., (1907); Ricerche sulle cellule e fibre del Mauthner e sulle lor0 conessioni in Pesci ed Anfibii. Arch. ital. Anar. Etnbriol., 6, 660-705. BODIAN,D., (1937); The structure of the vertebrate synapse. A study of the axon endings on Mauthner’s cell and neighboring centers in the goldfish,. J. con~p.Neurol., 68, 117-159. ~ D I A N D., , (1952); Introductory survey of neurons. Cold Spr. Harb. Symp. quanf. Biol., 17, 1-13. DIAMOND, J., (1963); Variation in the sensitivity to GABA of different regions of the Mauthner neurone. Nature (Lond.), 199, 773-775. DUDEL, J., (1963); Presynaptic inhibition of the excitatory nerve terminal in the neuromuscular junction of the Crayfish. Pflugers Arch. ges. Physiol., 277, 537-557. DUDEL,J., AND KUFFLER,S. W., (1961); Presynaptic inhibition at the crayfish neuromuscular junction. J. Physiol. (Lond.), 155, 543-563. ECCLES,J. C., (1964); The Physiology of Synapses. Berlin, Springer-Verlag. ECCLES,J. C., ECCLES,R. M., AND MAGNI,F., (1961); Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volleys. J. Physiol. (Lond.) , 159, 147-1 66. ECCLES,J. C.,MAGNI,F., AND WILLIS,W. D., (1962a); Depolarization of central terminals of Group I afferent fibres from muscle. J. Physiol. (Lond.), 160, 62-93. ECCLES,J. C.,SCHMIDT,R. F., A N D WILLIS,W. D., (1962b); Presynaptic inhibition of the spinal monosynaptic reflex pathway. J. Physiol. (Lond.), 161, 282-297. FLOCK, A., (1964); Structure of the macula utriculi with special reference to directional interplay of sensory responses as revealed by morphological polarization. J . Cell Biol., 22, 413-431. FRANK,K.,(1959); Basic mechanisms of synaptic transmission in the central nervous system. I R E Trans. med. Elecrr., ME-6, 85-88. FRANK,K.,AND FUORTES,M. G. F., (1957); Presynaptic and postsynaptic inhibition of monosynaptic reflexes. Fed. Proc., 16, 39-40. FUKAMI, Y., FURUKAWA, T., AND ASADA,Y., (1965); Excitability changes of the Mauthner cell during collateral inhibition. J . gen. Physiol., 48, 581-600. FURSHPAN, E. J., (1964); ‘Electrical Transmission’ at an excitatory synapse in a vertebrate brain. Science, 144, 878-880. FURSHPAN, E. J., AND FURUKAWA, T., (1962); Intracellular and extracellular responses of the several regions of the Mauthner cell of the goldfish. J. Neurophysiol., 25, 732-771. T., FUKAMI, Y., AND ASADA,Y.,(1963); A third type of inhibition in the Mauthnercell FURUKAWA, of goldfish. J. Neurophysiol., 26, 759-774. FURUKAWA, T., FUKAMI, Y., A N D ASADA,Y., (1964); Effects of strychnine and procaine on collateral inhibition of the Mauthner cell of goldfish. Jap. J. Physiol., 14, 386399. FURUKAWA. T., AND FURSHPAN, E. J., (1963); Two inhibitory mechanisms in the Mauthner neurons of goldfish. J. Neurophysiol., 26, 140-176. GALAMBOS, R., (1956); Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea. J. Neurophysiol., 19, 424-437. O . ,(1957); The acoustico-lateral system. The Physiology ofFishes, Vol. 2. M. E. Brown, LOWENSTEIN, Editor. New York, Academic Press (pp. 155-186). LOWENSTEIN, 0.. OSBORNE, M. P., A N D WERSALL, J., (1964); Structure and innervation of the sensory

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epithelia of the labyrinth in the Thornback ray (Raja clavata). Proc. roy. SOC. B, 160, 1-12. OTSUKA,N., (1962); Histologisch-entwicklungsgeschichtliche Untersuchungen an Mauthnerschen Zellen von Fischen. Z . Zellforsch., S8,33-50. OTSUKA. N., (1964); Weitere vergleichend-anato:nische Untersuchungen an Mauthnerschen Zellen von Fischen. Z . Zelvorsch., 62, 61-71. RETZLAFF, E., (1957); A mechanism for excitation and inhibition of the Mauthner's cells in teleosts. A histological and neurophysiological study. J. comp. Neurol., 107, 209-225. ROBERTSON, J. D., BODENHEIMER, T. S., AND STAGE, D. E., (1963); The ultrastructure of Mauthner cell synapses and nodes in goldfish brains. J. Cell Biol., 19, 159-199. TASAKI, I., (1953); Nervous Transmission. Springfield, Thomas. VON FRISCH,K., (1936); Uber den Gehorsinn der Fische. Biol. Rev., 11, 210-246. WERSALL, J., (1960); Electron micrographic studies of vestibular hair cell innervation. Neural Mechanisms of the Auditory and Vestibular Systems. G . L. Rasmussen and W. Windle, Editors. Springfield, Thomas (pp. 247-257). WILSON,D. M., (1959); Function of giant Mauthner's neurons in the lungfish. Science, 129, 841.

71

Neural Mechanism of Hearing in Cats and Monkeys YASUJI KATSUKI Department of Physiology, Tokyo Medical and Dental University, Tokyo (Japan)

I. INTRODUCTION

When the endocochlear mechanism of hearing was disclosed by Von Bektsy (1943) and later confirmed by Tasaki et a/. (1952), further information on the neural mechanism of hearing was desired, because both Von Helmholtz’s (peripheral) theory ( I 862) and Rutherford’s (central) theory (1 886) were shown not to be completely valid. Just at that time the superfine capillary microelectrode technique enabled us to explore the unitary activity of nerve cell in the brain of higher animals. The author therefore planned to study the functional organization of nuclei at different levels in the classical auditory system step by step, by single neuron analysis. The single neuron analysis in that field had already been performed at the cochlear nuclei and other regions by Galambos and his collaborators (Galambos, 1952; Galambos et a/., 1943, 1952, 1959; Rose and Galambos, 1952; Rose et a/., 1959). Tasaki (1954) also succeeded in recording the response of primary auditory neurons to sound stimulation in guinea-pigs. The present author mainly used cats as experimental animals; under light anaesthesia with nembutal various regions of the skull were opened to expose a part of brain which it was desired to study. For some experiments monkeys were used. Guinea-pigs were used only for the study of microphonic potential. The electronic apparatus for recording the responses of neurons to sound stimulation included a cathode follower preamplifier and a high-gain main RC or DC amplifier. Most records were photographed on a running film through a conventional oscilloscope, e.g. Tektronix 502, while an automatically driven sound producing apparatus was working. For the determination of thresholds of a neuron for sounds repetitive photographs were taken once per 1 or 2 sec. The sounds used were mostly short ones, tone bursts with ditrerent durations, different frequencies ranging from 30 to 20,000 c/s or higher and different intensities, 0 dB being 80 dB above the average human threshold (0.0002 dyne per sq cm) except where otherwise indicated. Long continuous pure tones were also used. When the effect of interaction of two or more sounds was studied, a pure tone was mostly used as a background sound. In order to finish successive experiments of measuring thresholds of a neuron for sounds with various frequencies within as short a time as possible, an automatic sound producing apparatus was designed, using a rotatory switch driven by a motor, because the recording time of responses from one and the same neuron was limited. Rr/rrmr.cr p. 94- 97

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The animal was usually put in a sound-proof room, where the temperature was regulated at around 28" and the sound stimuli were usually delivered to it in a free field, though the sound was, if necessary, sent separately to each ear through headphones. Since all the experiments conducted by our group during several years have been performed under the same physical conditions, all results shown below are comparable with each other, except where otherwise indicated. II. M E C H A N I S M O N T H E C O C H L E A R N E R V E

( 1 ) Coding in the cochlea

As described above the movement of the basilar membrane in the cochlea observed by Von BCkisy (1943) was a traveling wave elicited by a sound wave, by which Corti's organ on the membrane is vibrated. It has generally been accepted that the deformation of hairs on the top of hair cells caused by the hair cell movement produces the cochlear microphonic (CM) potentials. Recent electron microscopical studies by several authors (Engstrom et al., 1962; Flock et al., 1962) suggest a characteristic orderly arrangement of two kinds of hairs, many stereocilia and a single kinocilium on the outer hair cell, though this situation has not yet been clarified on the inner hair cell of the cochlea. The exact mechanism of the CM production in the cochlea remains obscure. The mechanism of the initiation of impulses at the ending of the cochlear nerve fiber which makes contact with the base of the hair cell, is so far not clear; there are two hypotheses, one is electrical and the other chemical. According to the former hypothesis the electric current flow due to the change of membrane potential of the hair cell initiates impulses at the dendritic ending of the primary cochlear neuron, whereas the latter hypothesis proposed by electron microscopists (Engstrom and Wersall, 1958; Schuknecht et a/., 1959; Smith and Sjostrand, 1961) insists on a chemical transmission between the hair cell and nerve endings where they make synaptic contact. The electron microphotographs of hair cells represent the structure which is similar to the ordinary synapse observed in neurons of lower as well as higher animals. They also distinguish two different types of dendritic endings, small and large. The former contains vesicles sparsely whereas in the latter they are distributed densely. Furthermore at the region of hair cell that faces the large ending there is often found a thickening of the cell membrane with a synaptic ribbon. In large endings synaptic vesicles are seen to be concentrated close to the cleft having a width of several hundred A. Histochemical studies of the endings have also been made on guinea-pigs. Acetylcholinesterase has been found distributed at the nerve endings, especially at large endings whereas at small endings little or none has been found (Schuknecht et al., 1959). By cutting Rasmussen's bundle (Kimura and Wersall, 1962) which is thought to be efferent, at the facial colliculi in the pons, it was confirmed that most of the large endings were the terminals of the crossed olivo-cochlear bundle and efferent in nature. Other large endings which did not degenerate at all are considered the ter-

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minals of the homolateral fibers coming from the superior olivary complex, or of fibers of some other origins. After section of the auditory nerve at the internal auditory meatus most small nerve endings showed degenerative changes which indicates that the small endings are afferent terminals. Low contents of vesicles in small nerve endings do not oppose this conclusion. From these experimental results it may be reasonably predicted that acetylcholine is a chemical transmitter released from the large endings of efferent fibers. There is, however, another finding contradictory to that prediction. Desmedt and Monaco (1962) discovered that the intravenous injection of strychnine opposed the effect of crossed and uncrossed efferents. It is generally thought that strychnine has antagonistic action to inhibitory substances and not to acetylcholine in other parts of the nervous system. The present author with his collaborators (Tanaka, 1964) tried to obtain a conclusive answer to this problem: they designed an experiment to deliver acetylcholine directly arld close to the synaptic region by the electrophoretic method. The guinea-pigs were preliminarily so operated upon that the cochlear microphonics (CM) and the neural component N1 were recorded by means of vestibulo-tympana1 leads from the basal turn of the cochlea. According to Tasaki’s method, nichrome wire electrodes were inserted into both vestibular and tympana1 scalae through tiny holes and another into the scala media, namely the former two electrodes were in the perilymph and the latter in the endolymphatic space. The electrode in the endolymph was used for the electrophoresis. A capillary microelectrode with 2 p tip diameter, filled with various ionic solutions was inserted into the cochlea through the transparent thin membrane of the round window. The electrode was further advanced through the basilar membrane while the DC potential between the capillary electrode and the perilymph was being measured. The position of the tip of the electrode was invisible, so the negative resting potential obtained was taken as an indication of the tip position of the electrode in the organ of Corti. Ionized chemicals contained in the capillary were administered in the vicinity of the hair cells by means of a 10-6 A current in the square wave form of 500 msec in duration applied at a frequency of 1 per sec for 8 to 10 min. The application of inorganic ions such as sodium, potassium and chlorine had no effect on the CM and N1 response. Fig. 1 indicates that the CM and N1 were not influenced by applied potassium ions in 8 x 10-6 A current. In contrast with the results for inorganic ions, administration of acetylcholine (ACh) or prostigmin elicited marked changes in the cochlear response. The CM decreased abruptly in amplitude during or shortly after ACh administration using the same anodic current as used for the potassium or sodium ions. Other drugs have been similarly tested but with negative results. Application of GABA, which is known as a blocking agent on the synapses in the cat brain, produced no effect. Administration of strychnine showed no consistent change in the CM and N1 responses, though Desmedt and Monaco reported its inhibitory effect on the olivo-cochlear efferent inhibition. Local application of adrenaline and atropine had no effect on the cochlear responses. The effect of ACh on the nerve response was examined by unitary responses of the primary auditory neurons. The firing rate of fibers with characteristic frequencies R<*Ji,rcnrrs p. 94-97

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4 kc

1It

I I kr

10

-

I I kc

Fig. 1. Effect of combined application of potassium (left) and acetylcholine (right) on cochlear and nerve responses in guinea-pig. The cochlear microphonics (CM) and neural responses N I were recorded from the basal turn of the cochlea of the guinea-pig. Potassium ions were applied with a current of 8 x lo-@A from 0 to 10 min, and then acetylcholine from 83 to 93 min. Abscissa, time in min. Ordinate, amplitude of the CM (open circles show results obtained with sound at 4,7 and I I kc/s)and the N I response (closed circles) in millivolts. Records above are CM (upper beam) and N I (lower beam) responses obtained at different times shown on the abscissa.

Fig. 2. Effect of acetylcholine applied tocurarized 'organ of Corti' in guinea-pig. DTC (D-tubocurarine) was applied with a current of 4 x lo-@A from 0 to 10 min, and acetylcholine with 8 x A from 85 to 95 min. Cochlear microphonics to 4,7 and 1 1 kc/s pure tone stimuli (open circles), N I (closed circles). Records above are CM (upper beam) and N1 (lower beam) responses obtained at different times shown on the abscissa.

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above 5000 c/s began to decrease first during the administration of ACh. This effect extended gradually toward the low frequency range, probably due to the diffusion of the ACh to the cochlear apex along the basilar membrane. The action of ACh was found to be blocked by curare. The application of D-tubocurarine in a current of 4 x A caused a gradual reversible decrease of the CM and N I responses. When ACh was applied during this recovery the cochlear responses were only very slightly depressed even with twice the amount of ionic current used with D-tubocurarine. It is significant, as compared with the change of CM and N1 responses to the ACh administration only, that ACh fails to diminish cochlear responses after administration of D-tubocurarine (Fig. 2). Intravenous delivery of I to 5 mg/kg dihydro-/I-erythroidine (DHE), enough to relax the respiratory muscles, did not influence the ACh effect on the cochlear responses. It has been reported that electrical stimulation of the crossed olivo-cochlear bundle inhibits neural response in the cochlea and augments the CM (Desmedt and Monaco, 1961 ; Fex, 1959). This inhibitory effect is suppressed by intravenous injection of strychnine as described above. However, when D-tubocurarine was applied to the hair cell region by the present method the amplitude of the N1 response was still reduced by electrical shocks to the efferent fibers in the floor of the 4th ventricle after removing the cerebellar vermis by suction (Fig. 3). Previous reports by other authors c - 15 Control

5 t r yc hnine

LITC

1 mV

Fig. 3. Comparison of the effects of strychnine (middle) and D-tubocurarine (right) on olivo-cochlear inhibition (2). N1 responses to 40 dB click sound and cochlear microphonics to 40 dB 3.5 kc/s pure tone recorded from the left round window in cat. 2, electrical stimulation on the crossed olivocochlear bundle at the 4th ventricle floor with square waveO.l msecat 300/sec. Strychnine nitrate was A. 1 , before, and 3, applied intravenously and D-tubocurarine ionophoretically with 8 x 2 sec after the electrical stimulation.

have not recognized the effect of the administration of ACh in the cochlea. In our experiments only ACh and anticholinesterase applied iontophoretically to the hair cell region were found to be effective in reducing the CM and N1. This result is considered reasonable in relation to histochemical findings by Schuknecht et al. (1959) and Wersall (Hilding and Wersill, 1962), showing that the organ of Corti has intense cholinesterase activity. Rrfermces p . 94-97

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It is well known that ACh depolarizes the membrane potential of the postsynaptic ending. No sign of excitatory effect has, nevertheless, so far been found in the responses of the unitary primary auditory neuron to sound stimulation. It is therefore supposed that a part of the hair cell membrane is sensitive to ACh, since anticholinesterase acts like ACh to decrease the CM, and the ACh effect is prevented by curare. The ineffectiveness of systemic DHE injection on the effect of ACh upon the cochlear responses suggests a different blood barrier to DHE in the cochlea, but so far there is no evidence of this. A suppressive effect of strychnine on the inhibitory action of both crossed and uncrossed efferent nerve bundles has been reported. But our experimental results did not show any marked influence of D-tubocurarine delivered iontophoretically into the cochlea over the inhibition of the NI response elicited by electrical stimulation of the crossed olivo-cochlear bundle. From these results the conclusion may be reached that the efferent nerve fibers to the cochlea are not cholinergic, though ACh markedly depresses the activity of hair cells. Further studies on the intracellular potential of the hair cells may shed light on this important problem, though it remains so far unsuccessful. ( 2 ) Nature of primary auditory neurons As the primary auditory neurons are deeply buried in the petrosal bone, studies on the nature of the primary neurons are difficult. Tasaki (1954) however succeeded in recording their responses to sound stimulation in guinea-pig more than 10 years later than Galambos and Davis’ success (1943) in that of the upper level in the cat. Kiang et al. (1962) recorded the primary neuron responses to sound stimulation in the cat. Both animals are anatomically inconvenient (Galambos and Davis, 1948): in the cochlear nerve which runs from the cochlea to the medulla the nerve cells migrate from the ventral cochlear nucleus so that it is not easy to decide if the responses of a single neuron, recorded by means of a microelectrode from the cochlear nerve, come from the primary or the secondary neurons. One possible method is to measure the latency of the response. Meanwhile, Katsuki et (11. (1961) discovered that the auditory nerves of the monkey differ from the above two animals, and careful study of the region between the auditory meatus and the medulla oblongata showed that migrated cells of the cochlear nucleus were only very rare. This discovery enabled them to examine the nature of the primary auditory neuron very easily. The monkeys they used were M. irus, M..fuscata, M . cyclopis and M . mulatta. The results obtained from the cats and the monkeys showed little difference. Both groups recorded the responses of primary neurons to sound stimulation, studying the latencies, the response pattern, the thresholds for sounds with different frequencies, the responsive frequency range, the characteristic frequency (CF) and so on. Thus details of the nature of the primary auditory neurons became clear. Previous results obtained with guinea-pigs differ in certain respects from those with the latter two animals. This might result from difficulty of experimenting on the small animal. It is true that very many cochlear fibers produced spontaneous discharges, while many other fibers did not.

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20

10

n

50

0 spmt

100

dis level

Fig. 4. Distribution of the spontaneous firing rate of the primary auditory neurons in cats. Abscissa and ordinate indicate the firing rate and number of neurons, respectively. Two distinct peaks were obtained.

The spontaneous discharges, when present, appeared to be highly irregular. Their frequency varied over a wide range. The average rates of discharges were divided into two groups. A histogram of frequency occurrence concerning the average rates shows a bimodal distribution (Fig. 4): a group with firing rates below IO/sec, and another group with frequencies of about 50/sec although with considerable spread around the mean frequency. However, these average frequencies were not found to be clearly correlated with the threshold measured at its characteristic frequency (CF) (Nomoto et al., 1964). The latencies of the responses of a neuron were different to sounds with different frequencies and intensities. The latencies of the responses to the same sound were also different from neuron to neuron. However, when the latencies of responses of a neuron to strong enough sounds at their C F were plotted against the frequency of the sound, a tendency was clearly observed; as shown in Fig. 5 , the higher the CF of

I

5.0 mSec

L

o 02

05

10 2 0

50

10

20

kc

Fig. 5 . Latency of response of primary cochlear neuron to sound stimulation in the monkey. Abscissa: frequency of sound which represents the characteristic frequency of the neuron. Ordinate: latency of neuron in milliseconds. Rrferenccn p . 94-97

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neuron, the shorter the latency of response (Katsuki et al., 1961). This result is consistent with the mode of the traveling wave on the basilar membrane in the cochlea. According to the conception generally accepted concerning the sound frequency analysis in the cochlea, the traveling wave on the basilar membrane caused by low frequency sound reaches the apex while that caused by high frequency only remains a t the base of the cochlea (Von BBkCsy, 1943). The different latencies of responses of neurons can therefore be explained by different lengths of nerve fibers through which impulses have to pass. When thresholds of many neurons for sounds at their C F were measured, it was found that they could be divided, on a statistical basis, into two groups, one with high threshold, the other with a low threshold as in Fig. 6 (Katsuki et al., 1962). It is commonly thought that the inner hair cell has a higher threshold than does the outer one, because the former is located at the edge of the basilar membrane while the latter is placed at the middle part of the membrane where the amplitude of vibration is larger. The division into two groups of neuronal thresholds can be reasonably

Fig. 6. Distribution of the thresholds in the primary neurons of the monkey. Three groups (a, b, c) are classified by their characteristic frequencies. In groups (b) and (c) two subgroups are found. The abscissa represents the threshold in dB and the ordinate the number of neurons with that threshold. (a) N = 116, m = -52.0 dB, y = f 15.5 dB; (b) N = 22, rn = -25.0 dB, y = & 6.7 dB and N = 92, m = -68.5 dB, y = f 14.5 dB; (c) N = 14, rn = -21.0 dB, y = & 5.5 dB and N = 117, rn = -63.0 dB, y = f 12.0 dB.

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concluded, we believe, from the observation that the fibers of high and low thresholds innervate the inner and outer hair cells respectively. It is also reported that the outer hair cells are innervated by nerve fibers of two types, the external spiral fibers and the radial fibers (Fernandez, 1951 ; Lorente de N6, 1933a,b). A fiber of the former type branches many times along its course and makes connections with many outer hair cells, whereas the radial fibers connect with only one or two hair cells. Such facts suggest some differences in the discharge patterns of nerve fibers which are thought to innervate the outer hair cells. However, up to now the functional difference between the radial and spiral fibers is not clearly understood. Through a model experiment the difference of two kinds of fibers was described by Bergeijk (1961). His model consists of an electrical analog of the basilar membrane and electronic nerve models, named neuromimes (Harmon, 1961). Van Bergeijk predicted that, when all terminal arbors of a spiral fiber are excited to the maximum extent by the sound stimulus at the CF, there is a more gentle slope of the rate of increase of impulse frequency with increase of sound intensity than is the case when sounds of other frequencies than the CF, are delivered. His explanation i s that if each terminal arbor discharges a t high rate in the fiber with many convergent arbors, such as an external spiral fiber, there is a high probability of collision at the convergent junctions of the arbors, so that the final output of the fiber is not a simple sum of the output of each single arbor. If the discharge rate is not high on the other hand, no such high probability of collision appears. For such reasons the impulse frequency does not increase rapidly with increase of the sound intensity, when the fiber with many convergent arbors is stimulated by the sound of the CF of that neuron. On the other hand, when only a part of the terminal arbor is excited by means of sounds with frequencies other than the CF, the slope of h e intensity-frequency function becomes steeper than it is at the CF, because of the diminished number of collision sites (Fig. 7). In contrast to that, in the case of fibers without arborization when the intensity of sound is increased the impulse 'mp4ec 7

1

!

dB -80 -60 - 4 0 -x) 0

300

-80 -60 -40 - 2 0

0

Fig. 7. Crossed ramp type (left) and parallel ramp type (right). in the right figure, the curves plotting the firing frequency against the relative intensity of sound are similar in shape for sounds of different frequencies. The left curves vary with the frequency of sound stimuli and show the firing frequency increasing gradually at the characteristic frequency (1.3 kc). Numbers on this diagram indicate frequency (kc) of sound. Rrferences p. 94-97

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frequency may increase in a similar way independent of the frequency of the stimulus sound. The former type is called the crossed ramp type and the latter the parallel ramp type. The fiber of the crossed ramp type with low threshold may correspond to the external spiral fiber, and the fiber of the parallel ramp type with low threshold to the external radial fiber. In fact the experimental results (Nomoto et at., 1964) show many fibers of an indistinct crossed ramp type, an intermediate type, which might be ascribed to fibers with a small number of arborizations. The high-threshold neurons nearly always belonged to the parallel ramp type. They may well correspond to the internal radial fibers. The internal spiral fibers have been thought to be the efferent fibers since Lorente de N6 (3933b). It may be reasonable to conjecture that these three groups of fibers play different roles in signaling information about sound. At the present time no one can tell exactly which group of fibers may carry information about pitch and which group about intensity of sound. From our experimental results which do not show any contradiction to the hypothesis of a pitch-loudness coordinate system advocated by Von BCkCsy (1959, 1960), it may be concluded that the radial fibers are mainly responsible for providing information useful in the discrimination of frequency. (3) Ejerent inhibition at the primary neuron In early days of the study of primary neurons no inhibitory phenomena were found (Tasaki, 1954) and this fact was even thought t o be characteristic of the primary neurons. During the course of time it has been confirmed that there are two kinds of inhibitions; one is the efferent inhibition (Desmedt and Monaco, 1961; Fex, 1962) and the other the inhibition due to the neural interaction among the primary neurons although the mechanism of the latter is not exactly understood. Inhibition under efferent control was first proposed by Rasmussen’s histological studies (Rasmussen, 1953). The efferent system is called the crossed and uncrossed olivo-cochlear fibers. The former originate from an area medial to the accessory olive and rise through the brain stem to the floor of the 4th ventricle and, after crossing, join the vestibular nerve. The latter also originate from the S-shaped olivary segment, and then join the crossed fibers which go with the vestibular nerve. The number of fibers was counted as 500 in the crossed bundle, while a fourth of that number occur in the uncrossed bundle. The cochlear and the vestibular nerve are connected by Oort’s anastomosis, through which the olivo-cochlear bundle reaches the cochlea. The electrical stimulation of the crossed olivo -cochlear bundle was reported to reduce the action potentials at the round window first by Galambos (1956) and later by Fex (1962). Fex confirmed Galambos’s discovery and further found the augmentation of the cochlear microphonics (CM) (Desmedt and Monaco, 1961 ; Fex, 1959). The reduction of the central auditory responses was also reported (Desmedt, 1962; Ruben and Sekula, 1960). Fex studied the nature of nerve fibers in Rasmussen’s bundle by recording the single nerve fiber responses at Oort’s anastomosis. While stimulating Rasmussen’s bundle, he recorded the cochlear nerve fiber responses to sounds with various frequencies and intensities and elucidated the effects of efferent fibers on the afferent fibers.

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Latencies of this effect were found to be 5-40 msec, but only one fifth had latencies below 10 msec, so the latencies of this action are rather long. The function of this efferent was to inhibit resting as well as evoked activity by sound, and therefore the auditory efferent and afferent fibers together form a closed feedback loop within the auditory system. The gating mechanism at the input which lets through the sensory input of highest significance and suppresses that of secondary importance was not confirmed by Fex’s results. The self-regulating system found in the middle ear muscles and the pupil reflex in the eye is also analogous. ( 4 ) Inhibition under nonefferent control Besides the inhibition controlled by efferent fibers, inhibition has been found in the primary auditory neurons of various animals. In frog the auditory nerve is composed of both simple and complex units (Frishkopf and Goldstein, 1963). Simple units respond to high frequency sound over 1000 c/s and are not always spontaneously active while the complex units respond to rather low frequency sound and vibration. Such complex units are spontaneously active and can be inhibited by sounds. This inhibition is graded, depending upon the intensity of the inhibitory stimulus. The latency of this sort of inhibition is very short, only a few milliseconds, and not altered by deep anesthesia or by total section of the 8th nerve. Tt is therefore clear that this is not under efferent control. But the problem remains, whether the action is at the hair-cell level and whether or not a neural interaction is concerned. General opinion is that the lateral interaction of a well-developed neural network may cause an inhibition. The same authors (1963) observed a similar inhibition in first order neurons of the bat. Here too, the latency is very short and the possibility of efferent control appears to be excluded. In the cat Rupert et al. (1963) recently observed the inhibition of spontaneous discharge of the primary neuron by sound. The latencies of the inhibition were of two kinds, some of them were very short while others were of 20-30 msec. The latter are considered to be effected by efferent fibers. These authors thought that the mechanism of short latencies might be the lateral inhibition such as seen in the eye of the horseshoe crab. Fex who studied the inhibition under efferent control, tried experimentally to exclude the effect of the crossed and uncrossed olivo-cochlear bundle. After cutting both bundles he still found inhibition by stimulation with two sounds. This observation confirms the existence of nonefferent inhibition. As described above, the cochlear nerve of the monkey is composed entirely of primary neurons (Katsuki et al., 1961). By the use of this nerve the present author with his coworkers observed the on-off responses of neurons at a higher frequency range than the C F of that neuron (Katsuki et al., 1962). These authors also found (Nomoto et al., 1964) ;nhibitory responses when the C F of the neuron was somewhat over 1000 c/s. When a weak continuous sound with frequency in the neighborhood of the C F of the neuron was used as a primary tone, and a tone burst with a strong intensity was delivered as a second tone, the response to the primary tone was partially or completely suppressed. The relation between the frequency and intensity of sounds was measured, and the so-called inhibitory area was obtained in contrast to the Refirenrrs p.194-97

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"/w

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Fig. 8. Inhibitory areas of two neurons. Closed circles represent excitatory response areas in response to a pure tone. When the sound within regions enclosed by star-lines is applied, the response to a continuous tone as shown by the cross is completely depressed. Note inhibitory areas at both sides of the characteristic frequency.

response area of the neuron as shown in Fig. 8. The inhibitory area was obtained at one or both sides of its CF. On the higher frequency side the inhibitory area is outside the response area of the neuron at the border of cut-off frequency. At the lower frequency side the inhibitory area is rather wide and the shape of this area is irregular. The meaning of this sort of inhibition is not as yet understood. However, the inhibition of this type has often been observed at the higher level in the auditory tract, e.g. a t the level of cochlear nuclei or at the trapezoid body, as will be described later. For such reason it is thought that this type of inhibition may play an important role in frequency analysis which cannot be done accurately in the cochlea. The latency of this inhibition is very brief, shorter than a few milliseconds. Therefore, the latency was measured as in Fig. 9. At first the responses of neurons t o a single tone burst with a fixed frequency were repeated more than 100 times and the frequency of occurrence was plotted on the figure. When a continuous tone with a different frequency is delivered as background and then tone bursts are given additively, the

Fig. 9. Latency of inhibition evoked by two-sound stimuli. White blocks represent responses to tone bursts of 0.4 kc/s. As shown by shaded blocks, responses to continuous tones of 4.7 kc/s are inhibited by application of the tone bursts. Blocks indicate the total number of impulses obtained from 64 successions.

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response to the tone burst is suppressed as indicated by the shaded blocks. Through this short latency the possibility of inhibition under efferent control may be excluded. There is also other evidence to support this hypothesis. As already mentioned, Desmedt and Monaco (1961, 1962) and Desmedt and La Grutta (1963) reported that the inhibition under efferent control was inhibited by intravenous injection of strychnine. On electrical stimulation of the crossed and uncrossed fibers, no effect could be seen on the responses after the strychnine injection. The mechanism of this type of inhibition is not clear yet. An experiment was attempted to ascertain whether or not the inhibition could be lateral. A three sound experiment was tried in an effort to get disinhibition. But the attempts were unsuccessful. When complete inhibition was produced by two sounds a third sound was delivered. Many experiments were tried and in about one-half of them the phenomenon of apparent disinhibition was observed. However, this phenomenon was an artifact. The interruption of the inhibition was indeed the response of two sounds (the second and the third) which occurred irrespective of the presence or absence of the first tone (Fig. 10). It is therefore thought that such responses, like apparent disinhibition, were independent of disinhibition. Direct and unequivocal evidence of disinhibition has not so far been obtained. As discussed above, there is no direct evidence of lateral inhibition, but recent electron microscopic investigations often show complicated nerve nets beneath hair

Fig. 10. Effect of third tones upon inhibition evoked by two-sound stimuli. In each record, the upper beam shows the unit response and the lower represents electrical signals for generating sounds. Numbers represent sound frequencies (kc). Spike discharges responding t o a continuous tone of 6.0 kc, 3 dB, are suppressed by tone bursts of 8.0 kc, 80 dB, or 9.9 kc, 70 dB. Since tone bursts of 9.9 kc and 12 kc, 70 dB have a rapidly rising phase, responses t o clicks appear. In E, responses are shown t o 8.0 and 9.9 kc tones outside the response area. Time scale, 50 msec. K&rc*nws

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cells and also nerve terminals in touch with other nerve fibers. The author is therefore inclined to think that the responsible mechanism may not be in the mechanical interaction in the middle ear or in the hair cells but in the neural process. 111. N E U R A L MECHANISM A T RELAY S T A T I O N S

( I ) Auditory neurons in the medulla, midhrain and thalamus The classical auditory tract in the brain is well defined. The main relay stations are ventral and dorsal cochlear nuclei (Galambos and Davis, 1943; Tasaki and Davis, 1955), trapezoid body, superior olivary nucleus (Galambos et al., 1959), lateral lemniscus, inferior colliculus (Erulkar, 1959; Thurlow et al., 1951) and medial genicd a t e body (Gross and Thurlow, 1951). All ascending fibers from the cochlea cross at certain levels and finally reach the contralateral medial geniculate body. In contrast to other sensory pathways, e.g. the visual or somatosensory pathways, the auditory one has many relay stations and many neurons connect with other neurons at the higher levels. This unique organization must have a special meaning, because other sensory pathways have only one or two relay-stations from the periphery to the thalamus. The present author wanted first to understand the meaning of this complicated organization. It was considered that the best way of solving this problem would be to secure exact knowledge of the nature of a single neuron at each level. With this object recordings of responses of single neurons were tried at various nuclei (Katsuki et al., 1958, 1959a,b; Katsuki, 1961). During the course of experiments for 5-6 years the experimental conditions were always the same, and recordings of responses of neuron to sound stimulation were continued from the periphery to the cortex. This is an important feature of the experiments. The auditory tract in the brain is situated rather superficially, so that, after exposiiig various regions of the brain, the nuclei to be investigated were easily accessible to microelectrodes, and almost all auditory nuclei could be investigated. The results obtained from various parts in the brain have already been reported in a monograph ‘Electrical Activity of Single Cells’ edited by the present author in 1960 and ‘Sensory Communication’ edited by W . A. Rosenblith in 1961. Here only the conclusions will be described in order to give an outline of the story. More details may be found in original papers that appeared in 1958 and 1959 (Katsuki et al., 1958, 1959a,b) and in the two books described above.

( 2 ) Funneling mechanism at relay stations As already elucidated by Von Bekesy the analysis of complex sounds is made partly in the cochlea in a particular way; additional analysis is believed to be made while the impulses of each auditory neuron in response to sounds are ascending to the cortex. This analysis is thought to be completed at the medial geniculate body because the narrowest response area of the neuron is found at this region (Katsuki et al., 1958, 1959a). The function of cortical neurons will be discussed later. The wide response areas at the periphery are reduced in area, but not in sensitivity, step by step at the synapses of each nuclei along the auditory pathway (Fig. 1 1). The mechanism for

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Fig. 1 1 A. Response areas for 7 primary auditory neurons in monkeys. Abscissa and ordinate show the frequency and intensity of sounds, respectively. A few units in the low frequency range represent flat response areas in contrast t o ordinary units which have sharp cut-off frequencies.

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Fig. I 1 B. Four response areas obtained from the medial geniculate body. In comparison with those in A extremely restricted areas are noticeable, though the CFs of these neurons are rather high. In this region some broad areas were also obtained which are not shown.

the gradual narrowing of response areas at the upper levels of the brain is considered to be the inhibitory interaction of neurons (Katsuki et al., 1959b). As already described in the section on the primary neurons, inhibitory interactions of the neuronal activity were very often observed (Nomoto el al., 1964; Rupert rt al., 1963). In 1944 Galambos observed the suppression of spontaneous discharges of neurons in the cochlear nucleus. Inhibitory interaction by two sounds can be observed very easily at the higher order neuron. More than half of the neurons encountered showed this type of interaction. In such neurons inhibitory response areas were measured at one side or both sides of the C F of a neuron as already described. At the higher frequency side of the CF the inhibitory area appears just adjacent to the upper edge of the response area, that is, to the cut-off frequency. In contrast thc shape of the inhibitory area at the lower frequency side is irregular, sometimes large and sometimes small. The response area of neurons at the upper level gradually becomcs narrower due to the inhibitory effects from both sides of the C F (Katsuki et a/., 1959b). This Rcfermrer p. 94-97

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neural mechanism seems to correspond t o the neural funneling action which has been advocated by Von BCkCsy. He reached this idea through his model experiment on the skin. The present author proposes that this mechanism may be called the spatial funneling action. Similar funneling mechanisms were observed by other authors in the somatosensory (Mountcastle et al., 1957; Mountcastle, 1957; Powell and Mountcastle, 1959) and visual pathways (Hubel and Wiesel, 1962) in the central nervous system. The sigmoid relation between the number of impulses per unit time and the intensity of sound could be followed up to the geniculate body, but not to the cortex (Katsuki, 1961). The response pattern of the neuron was quite brief at the cortex and markedly different from that at other regions. However, the response pattern of the neuron was not uniform at the higher levels. Depending upon the intensity of sound, varieties of inhibitory discharge patterns can be observed. Analysis of such discharge patterns was recently performed by Hind et al. (1963) and Rose et al. (1963). The inhibitory phenomena mainly come from binaural interaction and the complicated neural network system is conjectured. The degree of interaction may depend upon small differences in timing of impulse transport and arrival under the various stimulating conditions. Most recordings from the auditory neurons were done extracellularly by the present author. lntracellular recording of the membrane potential of an auditory neuron is not impossible but not easy (Katsuki, 1962), perhaps because the neuron is so small. Recently Nelson and Erulkar (1963) succeeded in recording it intracellularly at the inferior colliculus and the medial geniculate body : their results confirmed that all events already observed at auditory nuclei could be caused by synaptic excitation (EPSP) and inhibition (IPSP). In other words the synaptic mechanism in the auditory tract is not different from those already observed in other parts of the central nervous system (Eccles, 1964), e.g. the motor neuron in the spinal cord or large nerve cells in the cortex. These results have led the present author to conclude that the discrimination of the frequency and intensity of sounds is made at the medial geniculate body i n the thalamus, and the integration of the component sounds which are once analyzed may be made a t the cortex. The experiments performed by other authors through the use of conditioned reflexes after destruction of the auditory cortex also in part support this conclusion (Butler e? a/., 1957; Diamond and Neff, 1957; Diamond ef al., 1962). IV. I N T E G R A T I O N A T T H E A U D I T O R Y C O R T E X

( I ) Cortical auditory areas Since two auditory cortical centers, auditory areas 1 and 2 were well defined by Woolsey, many studies have been performed on the cortical auditory system, and he has recently summarized all data obtained for cats (Woolsey, 1960, 1961). According to him cortical auditory response mechanisms are organized in a much more complex way than that required by the concept of dual areas. In the central auditory region there are four complete representations for the cochlea, AI, A2, Ep and the suprasyl-

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vian fringe. The connections with the thalamus are thought to come from the pars principalis of the MGB at the first and the last one whereas at the other two the connections are said to be sustaining. Outside this auditory central region there are the insular area and the third auditory area. In the former the connection with the thalamus is obscure and in A3 connections are made with the posterior nuclear group. The latencies of responses are short (8 to 10 msec). In suprasylvian and anterior lateral gyri (association area) and in the precentral motor area there are response areas with a I5 msec latency. These areas can also be activated by visual and somatic stimulation. The thalamic connections, in part at least, are from the pulvinar. The region giving late responses with 100 msec latencies occupies the second visual area (V2). The activation pathway is unknown. The insular area is called A4. Neurons in A4 are found to be activated by acoustic as well as photic stirnulation. Desmedt found the inhibitory effect on the responses to sounds at the cochlear nuclei by faradic stimulation on A4. He traced the corticofugal tract to the cochlear nucleus and suggested that a loop circuit exists from the cochlear nucleus to the cortex and then back to the cochlear nucleus. This corticofugal pathway is, he says, probably responsible for gating in acoustic afferent relay (Desmedt, 1962), though the neuronal investigations still fail to prove it. A completely different hypothesis has been proposed by Hernindez-Pe6n et al. (1957). They assume that the origin of gating is in the brain stem reticular formation, while the Belgian school take it for granted that the control is in the cortex. Further studies may settle this controversy. According to recent studies in our laboratory, facilitatory and inhibitory corticofugal effects were found ipsilaterally and contralaterally, to lower centers along the auditory tract not only from A4 but also from Al and A2. The feedback system in the brain must be very delicate and complicated (Katsuki, 1964). Berman (1961a, b) found overlap of somatic and auditory cortical response fields in the anterior ectosylvian gyrus of the cat. Interaction was encountered most markedly in the region oftransition between S2 and A2. Carrerasand Anderson (1963) studied the nature of neurons in S2, the anterior ectosylvian gyrus, and found multimodal neurons, mechano-auditory and auditory-nociceptive units. In this region there were many neurons responsive to cutaneous or deep tissue stimulation and also auditoryvibratory stimulation. Some of these neurons were facilitatorily and others inhibitorily interactive. All neurons with dual modality to auditory and noxious stimuli had mutually excitatory effects and no inhibitory ones. Hotta and Kameda (1963), our coworkers, reported the interaction of auditory and skin afferent responses on the thalamic neurons. Such neurons were found in the suprageniculate, the lateral posterior, and the medial geniculate nuclei. The effect of somato-auditory interaction was always an inhibitory one. Berman’s (1961a,b) and Carreras and Anderson’s (1963) results may have some relationship with Hotta’s results. Murata and Kameda (1963) in our laboratory, examined the difference in the activities of neurons of the cat during sleep and wakefulness. They investigated the rate of spontaneous firing and found that the mean rate of impulses of auditory Rcferencrs p . 94-97

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neurons decreased with the progress of sleep, whereas arousal by natural sonic stimulation caused an increase in mean rate in most cortical neurons. Fluctuations of sensitivity of single cortical neurons in A l to sound stimuli were larger during arousal than during sleep, but statistical probit analysis showed that the thresholds defined as 50% response were not markedly different in the two states. ( 2 ) Functional organizotion of neurons Functional organization of neurons in the visual cortex and in the somato-sensory cortex was studied very extensively by Hubel and Wiesel(1962, 1963), and by Mouiitcastle and his collaborators (Mountcastle et al., 1957; Mountcastle, 1957; Powell and Mountcastle, 1959). By contrast the functional organization in the auditory cortices A1 and A2 has not been investigated to the same extent. There have been reports by Erulkar et al. (1956); Katsuki et al. (1959a); and Hind (1953, 1960). Their results became more detailed step by step, and except in minor detail most results obtained by these authors showed no marked differences from one another. In the visual cortex (Hubel and Wiesel, 1963) and the somato-sensory cortex (Mountcastle, 1957; Powell and Mountcastle, 1959) the vertically located neurons have common response properties, by which the neurons can be subdivided into many vertical columns extending from the cortical surface to the white matter. Both the somato sensory and the visual area have their own criteria for subdivision, the former determined by the same sensory modality and the latter by the axis orientation of the visual field. In the auditory area similar experiments were performed by Oonishi and Katsuki (1964). Responses to tone bursts and contiiiuous tones were studied on the vertically ocated neurons by advancing a microelectrode, a capillary type or sometimes a UNIT DEPTH LATENCY RESPONSE AREA

Fig. 12. Response areas and latencies of auditory neurons obtained in a single penetration. The number of units, depth And latency are shown on the left and the response areas on the right. The abscissa indicates the frequency, and the ordinate the intensity of the sound.

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tungsten type, vertically against the surface of the auditory cortex while the sound stimulus was being delivered to the animal.

( 3 ) Columnar structirre at the cortex Various types of response areas of neurons were encountered during a single penetration. Not all types were new, but some of them had already been found in previous reports by other authors, namely flat and broad, multi-peak, sharp-peak, high-threshold irregular and low-threshold broad types (Fig. 12). Among them the multi-peak type seemed suggestive for obtaining a clue from the viewpoint of functional architecture and frequency integration at the auditory cortex (Fig. 13). At first sight neurons of this type were believed to converge to fibers projected directly from the medial geniculate body, because their response areas and discharge patterns were like the combined form of several geniculate cells. The number of peaks in a response area differs from neuron to neuron, but the width of frequency range between one peak and a neighboring peak was often from one to two octaves. The 0.6

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Fig. 13. A response area of a cortical neuron is shown with a solid line. Each column represents sets of responses to tone bursts of the same frequency shown at the top and with different intensities shown on the left side. Three peaks (0.8, 2, 8 kc/s) in the response area are seen where the response pattern is repetitive and continuous. At other frequencies the response patterns are on or off or on-off. TABLE I T Y P E O F R E S P O N S E A R E A A N D ITS L A T E N C Y

Type of response area

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first step in the frequency integration may, therefore, occur in the auditory cortex at several frequency bands with one or two octaves. These experimental results (Table I) were not enough to determine whether the neurons of the multi-peak type were located in the 4th layer of the cortex, although they certainly seemed to be located in deeper layers than those of the flat broad type. The mean latency of the multi-peak type neuron was shorter than that of the flat type, but longer than that of the sharp-peak type. From the results described above it may be conjectured that the next step in the frequency integration consists in convergence from neurons of the multi-peak type to a neuron of the flat type by the intercortical synaptic relay. Further, there are the common peak frequencies among neurons encountered by a single vertical penetration into the cortex, or among neurons obtained successively in a certain distance by a slightly slanting electrode penetration. Hind (1960) also reported similar observations. In the present experiments most penetrations showed common frequencies of peaks in either all or a part of the neurons encountered. Hubel and Wiesel (1962, 1963) illustrated the integrative mechanism at the visual cortex by the elaborate scheme of convergence of receptive fields from simple cells to a complex cell. The simple cells converge directly from the lateral geniculate cells and the complex cells converge from the simple cells and these simple and complex cells are included together in the same vertical column. In the auditory area too, a similar functional organization is conceivable. The multi-peak type cells receive innervation from the afferent fibers or the sharp-peak tyoe cells, and the flat type ones from the multi-peak type cells. The response properties of the flat type cell to two-sound stimuli were different from both those of multi-peak type cells and sharp-peak type cells. The relation of response properties between the flat type and the multi-peak type cells can be compared with those between the complex type and the simple type cells in the visual cortex. As for the tonotopic localization, a regular spatial arrangement of the best frequency of neuron was not always observed. Some columns had their best frequency far from those of the adjacent columns, although a regular arrangement in a statistical sense may be recognizable (Hind, 1953; Tunturi, 1950, 1955). From these two considerations it may be concluded that the auditory cortex can be divided functionally into many columns, which are determined by the frequency peak or the best frequency of a neuron, and in a column the mechanism of frequency integration involved is the convergence from deep cellular layers to the superficial layer, and also from the neighboring column to that column itself. A scheme for a possible mechanism at the auditory cortex is illustrated in Fig. 14 in which the frequency integration and the inhibitory mechanism are shown. The cortical cells of D, E, F belong to the same column which have three common peak frequencies. The afferent impulses from the medial geniculate fibers A, B, C ascend into the cortex and terminate i n the cell of the multi-peak D and further up to the cortical surface vertically through the intercortical relay system. During their course impulses from cells (G, H) in the neighboring column can come in. The response area of a superficial cortical cell may thus become flat like that of F.

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/ I \ Fig. 14. The scheme of a possible mechanism of integration and inhibition in the auditory cortica neurons. Three medial geniculate (A, B, C ) and six cortical (D, E, F, G, H, 1) cells and their connec tions are shown on the left. I is an inhibitory neuron. The small solid circle means an inhibitory synapse and small open circles excitatory synapses. Geniculate cells converging on D, G and H cell belong to an adjacent column. They send fibers to E and F cells respectively. On the right each response area and its response pattern are illustrated. A chain of solid circles indicates a slowly adapted response. As illustrated in the figure the response area becomes broader and broader a t the superficial layer while the response pattern is changing.

Fig. 15. lntracellular recording from a neuron at the auditory cortex of the cat in response to tone bursts. The upper beam in each paired figure is a tone burst with frequency indicated on the left side. The lower beam represents an intracellular slow and spike potential. Calibration, 10 mV, and 100 msec. References p . 94-97

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( 4 ) Inhibition and facilitation of response at the cortex Inhibition and facilitation of response can often be seen at the auditory cortex. Their possible mechanisms may be as follows. The recurrent inhibition has been reported by several authors at the cerebral motor area (Morrel, 1959; Suzuki and Tsukahara, 1963) and the hippocampus (Kandel et al., 1961). As the present author has already reported, such an inhibitory mechanism like the recurrent inhibition may also be conceived in the auditory cortex from the response pattern of on-off neurons. All flat type neurons exhibit an on-off pattern, i.e. rapid adaptation, whereas 33 % ofthe neurons of the multi-peak type show slow adaptation. From the fact that intracellular potential recordings of on-off type cells showed a long lasting hyperpolarization following a small depolarization (Katsuki el al., 1962) it may be inferred that the flattype cell with phasic response pattern discharges repetitively, but is inhibited by interneurons around the cells (Fig. 15). Histological studies on the auditory cortical cells performed by Mannen have also shown many long recurrent fibers which run toward the cortical surface along the apical dendrite of each cell itself (Fig. 16). Facilitation of response was often observed on both the multi-peak and sharp-peak

Fig. 16. Long axon collaterals obtained from neurons at the auditory cortex A1 (depth loo0 p ) of cat. ax: axon which is markedly thinner than dendrites. (By courtesy of H. Mannen.)

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type cells by the use of two sound stimuli but never on the flat type. The facilitatory effects on the responses of cortical cells were also dependent upon the combination of the frequency and intensity of a tone burst and a continuous tone, and the manner of combination appeared to be different from neuron to neuron. , Nelson and Erulkar (1963) recently reported that the intracellular potential of nerve cells at the inferior colliculus and the medial geniculate body, both the membrane potential and the postsynaptic potential, were altered by different frequencies of the stimulus sound. Therefore, in the auditory cortex very intricate connection of the inhibitory and excitatory fibers with the neuron of the multi-peak type and of the sharp-peak type is highly probable. Such facilitatory effects on stimulation with two or more sounds may play a role in discriminating complex sounds. The multipeak type neurons are sensitive to tonal stimuli which have several frequency bands simultaneously, and moreover the responses at the peak frequencies are facilitated or inhibited from time to time by complex sounds. Each neuron also shows its specific response pattern. Such a mechanism is certainly the basis of the discrimination of complex sounds at the auditory cortex. These observations are well in agreement with the findings of Butler et a/. (1957), Diamond and Neff (1957) and Diamond et al. (1962) that, after bilateral ablation of the auditory cortex, through the use of conditioned reflexes severe deficit was found in the capacity of the experimental animal to make a discrimination of the change in temporal pattern of tones. Temporal change of the frequency and the intensity are characteristics of natural sounds. The author is of the opinion that on-off response of neurons is indeed the most important property of cortical neurons and advocates that this kind of mechanism of the temporal pattern of discharge may be called the temporal funneling activity of auditory neurons. V. C O N C L U S I O N

The investigations of all the complicated neural mechanisms at different levels along the auditory tract in the brain represented painstaking work, and the author’s group has reached the following conclusions. The frequency analysis of sound, which is one of the principal characteristics of auditory sensation, is performed in part in the cochlea and completed while the information is ascending very many (about 30,000) fibers from the cochlea to the medial geniculate body in the thalamus. On the way to the thalamic nucleus, at several relay stations the analyses are accomplished by interneuronal facilitatory and inhibitory interaction. In this process the spatial funneling mechanism is considered to be the most important factor. The auditory cortex, particularly A l , is the site of integration of simple sounds which have already been analyzed at the thalamic level. There the temporal pattern of information is altered by specific neuronal networks, namely by the temporal funneling mechanism. By such cortical neural mechanisms, natural sounds having ceaselessly changing frequencies and intensities can be perceived. The neural mechanism in the auditory system can be said to be basically similar to kferenres p. 94-97

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the visual or somato-sensory system. A principal difference may be the change of temporal pattern of information at the cortex. The author believes that further studies on the auditory mechanism necessitate the more intimate cooperation of neurophysiologists not only with morphologists but also biochemists and behavioral scientists. SUMMARY

This article comprises three parts: the mechanism on the cochlear nerve; the mechanism at relay stations; and the integration at the auditory cortex. Single neuron analysis has been used for the studies. (1) The first part deals with coding in the cochlea, the properties of the primary auditory neurons, and both efferent and nonefferent inhibition. The synaptic coding mechanism between hair cells and cochlear nerve endings, and the specific action of acetylcholine on microphonic potentials is discussed. Through recording impulses from the primary auditory neurons, differences in the nature of neurons innervated at the inner and outer hair cells were studied. The inhibitory phenomenon at the periphery due t o efferent influences was already known, but a different type of inhibition has now been found. The results obtained from the cochlear nerve of monkeys are described. (2) The mechanism at relay stations in the medulla, the midbrain and the thalamus and the spatial funneling activities of neurons along the classical auditory tracts are discussed. (3) The integrative mechanism at the auditory cortex has been studied. The columnar structure of neurons was found to be concerned in the functional organization in the auditory cortex. The inhibition and facilitation of responses observed there are also discussed. The author stresses the temporal funneling mechanism of the responses a t the auditory cortex. A scheme of integration is introduced.

ACKNOWLEDGEMENT

Experimental results described in this article were obtained in collaboration with Drs. T. Hotta, T. Watanabe, Y. Kanno, N. Suga, M. Nomoto, K. Kameda, Y. Tanaka, S. Oonishi, T. Miyoshi and K. Yanagisawa and Mr. K. Ogura. The author wishes to express his sincere thanks to Prof. H. Mannen who collaborated with them in the histological studies.

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CARRERAS, M., AND ANDERSON,S. A., (1963); Functional properties of neurons of the anterior ectosylvian gyrus of the cat. J. Neurophysiol., 26, 100-126. DESMEDT, J. E., (1962); Auditory-evoked potentials from cochlea to cortex as influenced by activation of the efferent olivo-cochlear bundle. J . Acousfical SOC.Amer., 34, 1478-1496. DESMEDT, J . E., AND LAGRUTTA, V., (1963); Function of the uncrossed efferent olivo-cochlear fibres in the cat. Nature ( L o n d ) , 200, 472-474. DESMEDT, J. E., AND MONACO,P., (1961); Mode of action of the efferent olivo-cochlear bundle on the inner ear. Nature (Lond.), 192, 1263-1265. DESMEDT, J. E., AND MONACO,P., (1962); The pharmacology of a centrifugal inhibitory pathway in the cats acoustic system. Proc. Firs? int. Pharmacol. Meef., 8, 183-188. DIAMOND, I. T., GOLDBERG, J. M., AND NEFF, W. D., (1962); Tonal discrimination after ablation of auditory cortex. J. Neurophysiol., 25, 223-235. DIAMOND, I. T., AND NEFF,W. D., (1957); Ablation of temporal cortex and discrimination of auditory patterns. J. Neurophysiol., 20, 300-315. ECCLES, J. C., (1964); Inhibitory pathways in central nervous system of vertebrate. The Physiology of Synapses. Berlin, Springer-Verlag (pp. 202-215). ENGSTROM, H., ADES,H. W., AND HAWKINS JR., J. E., (1962); Structure and functions of the sensory hairs of the inner ear. J . Acousfical SOC.Amer., 34, 1 3 5 6 1 363. ENGSTROM,H., AND WERSALL, J., (1958); The ultrastructural organization of the organ of Corti and of the vestibular sensory epithelia. Exp. Cell Res., Suppl. 5,460-492. ERULKAR, S. D . , (1959); The response of single units of the inferior colliculus of the cat to acoustic stimulation. Proc. roy. Soc. Med., 150, 336-355. ERULKAR,S . D . , ROSE,J. E., AND DAVIS, P. W., (1956); Single unit activity in the auditory cortex of the cat. Bull. Johns Hopk. Hosp., 99, 55-86. FERNANDEZ, C., (1951); The innervation of the cochlea (guinea-pig). Laryngoscope (Sf. Louis), 61, 1152-1172. FEX,J., (1959); Augmentation of cochlear microphonics by stimulation of efferent fibers to the cochlea. Acfa ofo-laryng. (Stockh.), 50, 540-541. FEX,J., (1962); Auditory activity in centrifugal and centripetal cochlear fibres in cat. Acra physiol. scand.,Suppl. 55, 189, 1-68. FLOCK,A., KIMURA, R., LUNDQUIST, P.-G., AND WERSALL, J., (1962); Morphological basis of directional sensitivity of the outer hair cells in the organ of Corti. J. AcousticalSoc. Amer., 34,1351-1 355. JR..M. H., (1963); Responses to acoustic stimuli fromsingle units FRISHKOPF, L. S., AND GOLDSTEIN, in the eighth nerve of the bullfrog. J. Acoustical SOC.Amer., 35, 1219-1228. GALAMBOS, R., (1944); Inhibition of activity in single auditory nerve fibers by acoustic stimulation. J. Neurophysiol., 7, 287-304. GALAMBOS, R., (1952); Microelectrode studies on medial geniculate body of cat. 111. Response to pure tones. J. Neurophysiol., 15, 381-400. GALAMBOS, R., (1956); Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea. J . Neurophysiol., 19, 424-437. R., AND DAVIS, H., (1943); The response of single auditory nerve fibers to acoustic stimuGALAMBOS, lation. J . Neurophysiol., 6, 39-58. GALAMBOS, R., AND DAVIS, H., (1948); Action potentials from single auditory nerve fibers? Science, 108, 513. GALAMBOS, R., ROSE,J. E.. BROMILEY, R. B., AND HUGHES, J. R., (1952); Microelectrode studies on medial geniculate body of cat. IT. Response to clicks. J. Neurophysiol., 15, 359-380. R., SCHWARTZKOPFF, J., AND RUPERT, A., (1959); Microelectrode study of superior olivary GALAMBOS, nuclei. Amer. J . Physiol., 197, 527-536. GROSS,N . B., AND THURLOW, W. R., (1951); Microelectrode studies of neural auditory activity of cat. TI. J . Neurophysiol., 14, 409-423. HARMON, L. D., (1961); Properties and functions of an artificial neuron. Kybernetik, 1, 89-101. HERNANDEZ-PE~N, R., JOUVET, M., AND SCHERRER, H., (1957); Auditory potentials at cochlear nucleus during acoustic habituation. Acla neurol. laf.-amer.. 3 , 144-1 56. HILALI, S . , AND WHITFIELD, I. C., (1953); Responses of the trapezoid body to acoustic stimulation with pure tones. J. Physiol., 122, 158-171. HILDING, D., AND WERSALL, J., (1962); Cholinesterase and its relation to the nerve endings in the inner ear. Acta oto-laryng. (Sfockh.), 55, 205-21 7. HIND,J. E., (1953); An electrophysiological determination of tonotopic organization in auditory cortex of cat. J. Neurophysiol., 16, 415489.

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HIND,J. E., (1960); Unit activity in the auditory cortex. Neural Mechanisms of the Auditory and Vestibular Systems. G . L. Rasmussen and W. F. Wingle, Editors. Springfield, Thomas (pp. 201-210). HIND,J. E., GOLDBERG, J. M., GREENWOOD, D. D., AND ROSE,J. E., (1963); Some discharge characteristics of single neurons in the inferior colliculus of the cat. J. Neurophysiol., 26, 321-341. HOTTA, T.,AND KAMEDA, K., (1963); Interactions between somatic and visual or auditory responses in the thalamus of the cat. Exp. Neurol., 8, 1-13. HUBEL, D. H., AND WIESEL,T. N., (1962); Receptive fields, biocular interaction and functional architecture in the cat's visual cortex. J. Physiol., 160, 106-154. HUBEL, D. H.,AND WiEsEL, T. N., (1963); Shape and arrangement of columns in cat's striate cortex. J. Physiol.. 165, 559-568. KANDEL, E. R., SPENCER, W. A., AND BRINLEY, F. J., (1961); Electrophysiology of hippocampal neurons. J. Neurophysiol., 24, 225-242. KATSUKI, Y., (1960); Neural mechanism of hearing in cats and insects. Electrical Activity of Single Cells. Y. Katsuki, Editor. Tokyo, Igaku Shoin (pp. 53-76). KATSUKI, Y., (1961); Neural mechanism of auditory sensation in cats. Sensory Conmumication. W. A. Rosenblith, Editor. M. I. T. Press and New York, John Wiley (pp. 561-583). KATSUKI, Y., (1962); Pitch discrimination in the higher level of the brain. Int. Audio/., I, 53-61. KATSUKI, Y., (1964); Integrative organization in the thalamic and cortical auditory centers. Symp. on the Thalamus. Columbia University Press. KATSUKI, Y., KANNO, Y., SUGA,N., A N D MANNEN, H., (1961); Primary auditory neurons of monkey, Jap. J. Physiol., 1 1 , 678-683. KATSUKI, Y., SUGA,N., AND KANNO,Y., (1962); Neural mechanism of the peripheral and central auditory system in monkeys. J. Acoustical SOC.Amer., 34, 1396-1410. T.,(1958); Electric responses of auditory KATSUKI, Y., SUMI,T., UCHIYAMA, H., AND WATANABE, neurons in cat to sound stimulation. J. Neurophysiol., 21, 569-588. Y.,WATANABE, T., AND MARUYAMA. N., (1959a); Activity of auditory neurons in upper KATSUKI, levels of the brain of cat. J . Neurophysiol.. 22, 343-359. KATSUKI, Y., WATANABE, T., AND SUGA,N., (1959b); Interaction of auditory neurons in response to two sounds stimuli in cat. J . Neurophysiol., 22,603-623. T., THOMAS, E. C., AND CLARK, L. F., (1962); Stimulus coding in the KIANG,N.-Y. S., WATANABE, cat's auditory nerve. Ann. Otol. (St. Louis), 71, 1009-1026. KIMURA, R., AND WERSALL, J., (1962); Terminations of the olivo-cochlear bundle in relation to the outer hair cells of the organ of Corti in guinea pig. Acta oto-laryng. (Stockh.), 55, 1 1-32. DE Nb, R., (1933a); Anatomy of the eighth nerve. The central projection of the nerve endings LORENTE of the internal ear. Laryngoscope (St. Louis), 43, 1-38. LORENTE DE Nb, R., (1933b); Anatomy of the eighth nerve. Laryngoscope ( S t . Louis), 43, 1-38 and 43, 327-350. MANNEN, H., Structure of cortical auditory neuron Personal communication. MORREL, R. M., (1959); Recurrent inhibition in cerebral cortex. Nature (Lond.), 183, 979-980. MOUNTCASTLE, V. B., (1957); Modality and topographic properties of single neurons of cat's somatic sensory cortex. J. Neurophysiol., 20, 408434. MOUNTCASTLE, V. B., DAVIS,P. W.,AND BERMAN, A. L., (1957); Response properties of neurons of cat's somatic sensory cortex to peripheral stimuli. J. Neurophysiol., 20, 374-407. K., (1963); The activity of single cortical neurons of unrestrained cats MURATA, K., AND KAMEDA, during sleep and wakefulness. Arch. iral. Biol., 101, 306-331. S. D., (1963); Synaptic mechanism of excitation and inhibition in the NELSON, P. G., AND ERULKAR, central auditory pathway. J. Neurophysiol., 26,908-923. NOMOTO, M., SUGA, N., AND KATSUKI,Y.,(1964); Discharge pattern and inhibition of primary auditory nerve fibers in the monkey. J.-Neurophysiol., 27, 768-787. S., AND KATSUKI, Y., (1964); Functional organization and integrative mechanism in the OONISHI, auditory cortex of cats. In preparation. T., AND MOUNTCASTLE, V.,(1959); Some aspects of the functional organization of the cortex POWELL, of the postcentral gyrus of the monkey. Bull. Johns Hopk. Hosp., 3, 108-131. RASMUSSEN, G.'L., (I 953); Further observations of the efferent cochlear bundle. J. comp. Neurol., 99, 61-74.

ROSE,J. E., AND GALAMBOS, R., (1952); Microelectrode studies on medial geniculate body of cat. 1. J . Neurophysiol., 15, 343-358. ROSE,J. E., GALAMBOS, R., A N D HUGHES, J. R., (1959); Microelectrode studies of the cochlear nuclei of the cat. Bull. Johns Hopk. Hosp., 104,211-251.

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ROSE,J. E., GREENWOOD, D. D., GOLDBERG, J. M., A N D HIND,J. E., (1963); Some discharge characteristics of single neurons in the inferior colliculus of the cat. J . Neurophysiol., 26,294320, RUBEN,R. J., AND SEKULA, J., (1960); Inhibition of central auditory response. Science, 131, p. 163. RUPERT, A., MOUSHEGIAN, G., A N D GALAMBOS, R., (1963); Unit response to sound from auditory nerve of the cat. J. Neurophysiol., 26,449465. RUTHERFORD, W., (1886); A new theory of hearing. J. Anat. Physiol., 21, 166-168. SCHUKNECHT, H., CHURCHILL, J., AND DORAN,R., (1959); The localization of acetylcholinesterase in the cochlea. Arch. Otolaryng., 69, 549-559. SMITH,C. A., (1961); Innervation pattern of the cochlea. Ann. Otol. (St. Louis), 70, 504-527. SMITH,C. A., A N D SJOSTRAND, S., (1961); Structure of the nerve endings on the external hair cells of the guinea-pig cochlea as studied by serial sections. J. Ultrastruct. Res., 5 , 523-556. SUZUKI,H., AND TSUKAHARA, Y., (1963); Recurrent inhibition of the Betz cell. Jap. J. Physiol., 13, 386-398.

TANAKA, Y.. (1964); Effects of drugs on hair cells in the cochlea. In preparation. I., (1954); Nerve impulses in individual auditory nerve fibers of guinea-pig. J. Neurophysiol., TASAKI, 17, 97-122.

TASAKI, I., AND DAVIS,H., (1955); Electric responses of individual nerve elements in cochlear nucleus to sound stimulation. J. Neurophysiol., 18, 151-158. I., DAVIS, H., A N D LEGOUIX, J. P., (1952); The space-time pattern of the cochlear microphonic, TASAKI, as recorded by differential electrodes. J. Acousrical SOC.Amer., 24,502-519. THURLOW, W. R., GROSS,N. B., KEMP,E. H., A N D LOWY,K., (1951); Microelectrode studies of neural auditory activity of cat. I. Inferior colliculus. J . Neurophysiol., 14, 289-304. TUNTURI, A. R., (1950); Physiological determination of the arrangement of the afferent connections to the middle ectosylvian auditory-area in the dog. Amer. J . fhysiol.. 162, 489-502. TUNTURI, A. R., (1955); Effect of lesions of the auditory and adjacent cortex on conditioned reflexes. Amer. J. Physiol., 181, 225-229. VAN BERGEIJK, W. A., (1961); Analog of the external spiral innervation of the cochlea. Kybernetik, I , 102-107. VON BBKLsY,G . , (1943); Uber die Resonanzkurve und die Abklingzeit der verschiedenen Stellen der Schneckentrenwand. Akustische Z., 8, 66-76. VON BBKLsY,G., (1959); Neural funneling along the skin and between the inner and outer hair cells of the cochlea. J. AcousticalSoc. Amer., 31,1236-1249. VON-BBKBsY,G., (I 960); Neural Mechanisms of the Auditory and Vestibular System. G . L. Rasmussen and W. F. Windle, Editors. Springfield, Thomas (pp. 3-20). H., ( 1862); Die Lehre von den Tonempfindungen, Braunschweig. VON’HELMHOLTZ, WOOLSEY, C. N., (1960); Organization of cortical auditory system: A review and a synthesis. Neural Mechanisms of the Auditory and Vestibular Systems. G. L. Rasmussen and W. F. Windle, Editors. Springfield, Thomas (pp. 165-180). WOOLSEY, C. N., ( I 961); Organization of cortical auditory system. Sensory Communication. W. A. Rosenblith, Editor. M. 1. T. Press and New York, John Wiley (pp. 235-258).

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Relationship between Activity of Respiratory Center and EEG HIROSHI K U M A G A I . F U M I N O R I S A K A I , A K I R A S A K U M A * AND TAKEHIKO HUKUHARA Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo (Japan)

Gibbs et al. (1940, 1950) reported that an alteration in the pattern of the electroencephalogram (EEG) was induced by changes in respiration, and that the EEG pattern of the neocortex shifted toward the drowsy pattern (slow and high voltage waves) during hyperventilation, and conversely toward the arousal pattern (fast and low voltage waves) during hypoventilation. Recent striking progress in the physiology of the central nervous system has aroused renewed interest in this field. The study of the mechanism involved in this alteration in the EEG pattern might contribute to a clarification of the functional connection between the brain stem and higher centers. Thus, in order to elucidate this mechanism, several experiments were undertaken in our department (Sakai el al., 1962; Otsuka et al., 1962, 1963). The EEG, electrical activity of the phrenic nerve and blood pressure were recorded simultaneously in cats immobilized with D-tubocurarine chloride under artificial respiration. The EEGs were recorded using bipolar electrodes from the anterior sigmoid gyrus, suprasylvian gyrus, caudate, preoptic area, amygdala, lateral hypothalamic nucleus, thalamic centre median, hippocampus and midbrain reticular formation. The rate of respiration was 10 to 20/min, and the ventilating volume was 50 to 800 ml/ kg/min. In Fig. 1, the effects of ventilation upon the EEG pattern are shown: 1, hyperventilation, 2, normal ventilation, and 3, hypoventilation. An increase in ventilation due to changing the rate or the tidal volume caused the phrenic nerve volley to decrease in amplitude and the blood pressure to become slightly lower. The EEG of the neocortex shifted toward slow and high voltage waves, and that of the hippocampus toward low voltage waves. On the other hand, a decrease in ventilation produced an increase in amplitude of the phrenic nerve volley, often with elevation of blood pressure.TheEEG pattern of the neocortex became high frequency-low voltage waves, and that of the hippocampus synchronized high voltage waves. No remarkable changes were observed in the EEG recordings of structures other than the neocortex and hippocampus. This finding in EEG of the neocortex is the same as that reported by Gibbs (1950). However, the alteration of the EEG was observed not only in the

* Present address: Department of Pharmacology, Institute of Cardiovascular Diseases, Tokyo Medical and Dental University, Tokyo (Japan).

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neocortex but also in the hippocampus, and the level of the EEG activities of these structures seemed always to be parallel with the activity of the phrenic nerve volley. In Fig. 2, the individual relationship between ventilation volume and EEG activity of four cats is shown. The activity of the EEG was scored on a blind basis by workers who did not know the degree of ventilation. The highest score 5 designated a typical arousal pattern of the EEG (fast and low voltage wave in the neocortex, synchronized high voltage wave in the hippocampus). Then the score decreased in a graded fashion 4, 3, . . . as the pattern became drowsy (slow and high voltage wave in the neocortex, fast and low voltage wave in the hippocampus). The changes of electrical activity of the neocortex and the hippocampus were not always observed simultaneously as reported by Tokizane et al. (1960). However, in every case the scores of both activities (neocortex and hippocampus) were added and the mean score was calculated. The score thus obtained was plotted against the corresponding volume of ventilation. As shown in Fig. 2, a uniform relationship exists between score and

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ventilation volume and an individual difference in the relationship is seen. There were no particular differences between the effects of changing the ventilation by the rate and by the tidal volume except upon the frequency of phrenic nerve volley. This relationship between EEG activity and ventilation volume, although individual difference exists, suggests that one can expect to obtain a highly uniform EEG pattern in an

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experiment, if the ventilation volume is kept under control with the aid of a recording of phrenic nerve discharge. In the next part of this series of experiments, the effect of cessation of artificial respiration of EEG pattern was analyzed as an extreme case of hypoventilation, in order to elucidate the mechanism inducing EEG arousal reaction under hypoventilation. On cessation of respiration, the phrenic nerve discharge increased rapidly in amplitude, the EEG tracing changed within 1 to 2 min into an arousal pattern, and the blood pressure rose by 40 to 60 mm Hg. To minimize the effect of blood pressure on the EEG pattern, which might induce the arousal reaction (Bonvallet et ul., 1954), bilaterally adrenalectomized cats were used. However, the effects of cessation of respiration were the same as those in the intact animals, except that the blood pressure rose by 10 to 20 mm Hg and sometimes not at all. This result indicates that the arousal reaction of EEG induced by the cessation of respiration is probably not caused by an elevation of blood pressure. The pretrigeminal transection (Batini et at., 1958, 1959; Hirao, 1962) of the brain stem was made in order to separate the higher structures of the central nervous system from the brain stem. After the transection, the effects of respiration arrest on the EEG were slight (Fig. 3), although the phrenic volley markedly increased in duration as well as in amplitude, and the blood pressure was elevated. One might argue that the higher structure from which EEG was recorded might lose its reactivity after transection. Therefore, before and after transection a determination was made of the threshold voltages of electrical stimulation of the midbrain reticular formation necessary to induce an arousal reaction in the neocortex. The values were almost the same, suggesting that the reactivity of the non-specific reticular activating system was not affected by transection. In order to exclude the effects of ascending impulses on the reticular formation, the spinal cord was transected at the level of C&7, or the carotid body was denervated. In all cases cessation of artificial respiration caused an arousal reaction in the EEG pattern of the neocortex and hippocampus. These results are summarized in Table I. In this table it may be noticed that the EEG pattern changed in parallel with the activity of the phrenic nerve volley, except after brain stem transection. TABLE I SUMMARY O F T H E E F F E C T S O F T H E C E S S A T I O N O F A R T I F I C I A L R E S P I R A T I O N O N T H E E E G A N D P H R E N I C VOLLEY U N D E R V A R I O U S C O N D I T I O N S

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A considerable amount of work on the effects of respiration upon the EEG has been accumulated (Wolff and Lennox, 1930; Gibbs et al., 1935, 1940; Gellhorn, 1953; Dell and Bonvallet, 1954; Bonvallet et al., 1955). In CO2-inhalation experiments Bonvallet (1955) observed that an alteration in the EEG parallels changes in the activity of the phrenic discharge and that this alteration disappeared after the brain stem was transected at a high level. He concluded that the arousal pattern of the EEG observed was induced by C02 which stimulated the brain stem reticular formation. Gibbs et al. (1940) reported that COZmay directly activate the higher centers. Gellhorn (1953) also stated that C02 activates the reticular formation of the brain stem. Besides the action of C02, we must consider the effects of hypoxia on the pattern of EEG in analyzing the present results, because respiration was stopped temporarily. Hypoxia produces an arousal pattern of the EEG (Bonvallet et a/., 1955). Bonvallet concluded that the reticular formation is stimulated by the activation of the carotid body on the basis of failure to observe an arousal pattern after denervation of the carotid body. In our experiments no changes were induced by denervation of the carotid body, Apparently in our experiments C02 ,but not hypoxia, played an important role in the activation of the EEG pattern. In agreement with Bonvallet, we concluded that the reticular formation of the brain stem might be activated by C02, and this activation in turn induced the changes in activity of the neocortex. The critica! point suggested by the present experiment is that the alteration of the EEG pattern may be caused not by general activation of the reticular formation but by an activation of the area in the reticular formation most sensitive to COZ.This localized activation might propagate to the general structures of the reticular formation and finally project on to the neocortex and hippocampus. Lobeline injected intravenously caused increased respiratory activity as judged by increased phrenic nerve discharge and a concomitant arousal response in EEG pattern. When the carotid body had been removed, however, the pattern of the EEG as well as that of the phrenic volley were not affected by lobeline to any significant degree. This observation suggests the possibility that the activity of the respiratory center modifies the activity of the central nervous system. The vasomotor center is another structure sensitive to C02 in the brain stem. We could not entirely neglect the vasomotor center as a part of the mechanism affecting the EEG, although changes in blood pressure did not play any principal role under the conditions of the present experiments as already described. The alteration in the EEG pattern caused by changes in ventilation, particularly by hypoventilation, may largely depend upon changes in the activity level of the respiratory centers, or the structures most closely related to and more directly affected by the state of ventilation. In the following experiments, the relationship between the activity of the respiratory center and the higher centers was further investigated. During the study of alteration in the EEG pattern produced by changes in ventilation, a spontaneous EEG fluctuation was noted (Fig. 4). The EEG fluctuated at regular intervals in about half of this series of experiments, particularly in the later part of an experiment, predominantly in the tracings from hippocampus and neocortex. This periodic change was

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found more frequently in the hippocampus than neocortices, and in the neocortices was more marked in the anterior sigmoid gyrus than the suprasylvian gyrus. Excepting these two areas, the midbrain reticular formation showed a periodical fluctuation in only one animal out of 60 (Fig. 5). In the hippocampus, synchronized waves were periodically repeated on a background of desynchronized waves. In the neocortex higher frequency waves than background activity were observed during the period of synchronization in the hippocampus. The periodicity of the spontaneous fluctuation of Re/urencrs p. 110/111

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the EEG pattern exhibited a close correspondence with the periodicity of the phrenic volley, the rate of artificial respiration and the fluctuation of blood pressure. When both vagi were cut, the interval between phrenic nerve volleys became more or less prolonged. Corresponding to this alteration, the interval between the periodic changes in EEG pattern was prolonged simultaneously, whereas the pattern of blood pressure fluctuation remained almost unchanged so long as the rate of ventilation was kept unchanged. When artificial respiration was temporarily stopped, the pattern of periodic EEG activities changed in parallel with the change in the pattern of phrenic discharges (Fig. 6), although the blood pressure rose gradually. The periodicity of the EEG change always paralleled the periodicity of the phrenic nerve volley. A

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Fig. 7. Periodic activity produced by phencyclidine. Periodic high-voltage waves produced by the topical application of phencyclidine to the anterior sigmoid gyrus. A = control; B = application to the left gyrus; C = application to the right gyrus. Bilaterally vagotornized between B and C. Notice close relationship between the phrenic volley and the periodic EEG activity. PN = phrenic nerve; rAs and IAs = right and left anterior sigrnoid gyri. (Reproduced from Otsuka et al., 1962). References p. 110/111

PN

c

I)

k

A

AS d ) , A & v - - - - J

&?---Wk--W+W~~

ss w

-

4

-

Irl)

w

*

I

I

L

0

m

A/-yPl

e

-1

lsec

-

5qN

Fig. 8. Correspondence between the phrenic volley and strychnine spike induced by topical application of strychnine to the anterior sigmoid gyrus. (See Fig. 1 for abbreviations).

r .

C

I P

n

L

2

= 2 1 sec Fig. 9. Twin beam oscillographic records. 1 = action potentials of respiratory neuron. Upper tracing: inspiratory neuron in medulla: lower tracing: expiratory neuron in pons. 2 = upper tracing: fluctuation in EEG of neocortex; lower tracing: action potentials of inspiratory neuron in medulla.

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107

However, the phrenic volley and the fluctuation of the EEG did not appear in phase. The characteristic waves which were recorded periodically on the background activity of EEG came at about I to 2 sec after the onset of the phrenic volley, as shown in Figs. 4, 5 and 6. It is interesting that this fluctuation was often found in the later stages of an experiment, and no fluctuation was usually observed in cases showing an arousal EEG pattern. Further experiments were performed in order to determine the conditions necessary to induce the periodic fluctuation in the EEG. During the experiments on phencyclidine HCI (Sernyl, Parke Davis & Co: Chen et al., 1959; Luby et at., 1959) on the central nervous system we made an interesting observation in connection with the characteristic periodic EEG change. When a piece of filter paper soaked in 1 % solution of the drug was topically applied to the cortical surface close to the recording electrodes, high voltage waves were periodically recorded in the localized area of application (Fig. 7). This type of localized response was most frequently observed in the anterior sigmoid gyrus. In all experiments in which both vagi were cut, or artificial respiration was temporarily stopped, this periodic discharge paralleled the phrenic volley. If phencyclidine was topically applied t o the cat’s cortex which had shown slight spontaneous periodic fluctuation, the fluctuation became more and more marked. Sometimes, however, topical application of phencyclidine failed to produce periodic change in the neocortex. On these occasions the interval between phrenic volleys was usually very short, and when the interval between phrenic volleys was prolonged by changing the tidal volume or the rate of artificial respiration, the EEG exhibited the periodic fluctuation. When the interval between periodic waves is too short, the individual periodic waves may overlap. Spontaneous periodic changes in the EEG were not usually observed in the early stage of an experimental course. In this stage the activity level of the EEG was usually high. However, when a small dose of pentobarbital, 0.5 mg/kg, was injected intravenously so that the activity of EEG was slightly depressed, it was easy to observe the periodic change. During high reactivity of the neocortex, individual periodic waves might overlap and fluctuation might less likely be recorded. These considerations suggest that the activity level of the neocortex influences the appearance of the fluctuation. When the activity of the neocortex is maintained at a slightly depressed level by some means, for example hyperventilation or application of a depressant, it is easier to observe the periodic change in EEG. The activity of the neocortex might be lowered by phencyclidine, and thus the fluctuation in EEG pattern would be revealed. When strychnine was applied topically to the neocortex a typical train of strychnine-spikes was recorded from the area. Sometimes the grouping of spikes was recorded as a periodic change coincident with the periodicity of the phrenic volley (Fig. 8). This finding supports the hypothesis that the EEG is under the influence of the periodic activity of the respiratory center. On these grounds, although the periodic changes were not observed in all animals, it is likely that the EEG activity is affected periodically by the respiratory center. References p. 110/111

108

H. K U M A G A I

et al.

The most intimate relationship between the activity of the neocortical EEG and the respiratory center is clearly demonstrated. We observed a long latent period between the onset of periodic EEG change and that of the phrenic volley. Thus, it is difficult to believe that this periodic change in the EEG is propagated by a direct ascending projection of the respiratory activity through the reticular formation. To acquire further information on this problem, another series of experiments was done. When the brain stem was transected (pretrigeminal midpontine transection), the periodic change in the EEG disappeared completely. This result confirmed the view that the periodicity of the EEG fluctuation is under the influence of respiratory activity. The recording of periodic changes in the reticular formation of the midbrain is worthy of notice (Fig. 5). The periodic changes in the neocortex and in the midbrain reticular formation start at almost the same time. Thus a possible explanation is that the activity of the respiratory center may be propagated to the reticular formation with a delay of 1 to 2 sec and then projected to the neocortex through the nonspecificactivating system. It may also be possible that some structure exists which spontaneously fires with the same periodicity as the phrenic volley but with a different phase. The phrenic volley reflects only the state of inspiratory activity. Not only inspiratory discharge but other types of periodic discharges were recorded in the brain stem with an extracellular microelectrode in cats (Hellner and Von Baumgarten, 1961 ; Gernandt and Thulin, 1952; Salmoiraghi, 1962). Some of them were observed to be periodic and to correspond to the respiratory activity (Amoroso et al., 1951 ; Von Baumgarten, 1956; Dirken and Woldring, 1951; Haber et al., 1957; Hukuhara et al., 1954). The periodic change in the EEG might possess a closer relationship with some of these activities than with the phrenic volley. In our department the periodic discharges related to respiratory activity were recorded down in the medulla oblongata as well as up in the pons. The cat was curarized, artificially ventilated and vagotomized. A steel microelectrode with a tip diameter of 5 to 10 p was inserted into the brain stem in order to record unit discharges. Besides these discharges, the EEG of the neocortex and the phrenic volley were simultaneously recorded. Four types of periodic discharges closely related to the phrenic volley were recorded: (a) a discharge in the phase of inspiration; (b) a discharge in the phase of expiration; (c) a discharge commencing during inspiration and ceasing during expiration, and (d) a discharge commencing during expiration and ceasing during inspiration (Fig. 9). The two types of discharges (c, d) are probably the same as the phase-spanning discharges reported by Cohen and Wang (1959). All these types of discharges were still recorded after transection of the brain stem at a higher level, after transection of the spinal cord at CS-C~,or after excluding the sinus nerve reflex. Therefore, these discharges are most likely derived from the neurons closely related to respiratory activity (Von Euler and Soderberg, 1952; Salmoiraghi and Von Baumgarten, 1961). Fig. 10 shows the distribution of the locations in the medulla and pons from which the four types of discharges were recorded. The discharge of phase-spanning type was observed more often in the pons than in the medulla.

EEG

A N D RESPIRATORY ACTIVITY

8

109

I

I

Fig. 10. Distribution of the locations from which the unit discharges were recorded in pons and medulla. 0 = inspiratory; x = expiratory; A = phase spanning; Broken line = transected level.

While the periodic discharges in the medulla oblongata and the pons were being recorded simultaneously, the brain stem was transected between the medulla and pons. After transection no periodic discharge in the pons was recorded, whereas the periodic discharge was still observed in the medulla (Kumagai et al., 1964). The periodic EEG fluctuation of the neocortex did not occur in the same phase as did the phrenic volley. There was a delay of 1 to 2 sec which was found also in the midbrain reticular formation. We recorded four types of respiratory discharges in the reticular formation of the pons as described above. In many experiments the fluctuation in EEG most closely coincided with that of the type c discharge among four discharges. Therefore, it may also be possible that the fluctuation in the EEG is induced by the direct ascending projection of the discharges (type c). Recently Bonvallet and Allen (1963) and Bonvallet and Bloch (1961) reported that in the medulla oblongata there is an inhibitory system for the neocortical activity. Depending on the level of transection, the EEG activity of the neocortex is differently affected by brain stem transection. The functional relationship between the medulla oblongata and pons or midbrain must be investigated more intensively in connection with respiratory activity. SUMMARY

The EEG, electrical activity of the phrenic nerve and blood pressure were recorded simultaneously in cats immobilized with D-tubocurarine chloride under artificial respiration. The alteration in EEG pattern caused by changes in ventilation paralleled the changes in the electrical activity of the phrenic nerve and in the blood pressure. However, the results of experiments in adrenalectomized or brain stem transected cats suggest that the changes in the EEG may largely depend upon the activity level of the respiratory center. Sometimes the EEG in the neocortex as well as in hippocampus fluctuated at regular intervals, and the periodicity of these changes coincided with the periodicity of phrenic nerve volley. When the activity of the neocortex was maintained at a slightly depressed level by hyperventilation or application of a depressant, it became easier to observe the periodic change in EEG. These results References p . - l l O / l l I

I10

H. KUMACAl

et d.

indicate that the EEG is always under the influence of the activity of the respiratory center and these effects would be revealed by changing the experimental conditions. The various types of respiratory neuronal discharges were recorded with microelectrodes in the brain stem and the relationships between EEG and the unit discharges are discussed. REFERENCES AMOROSO,E. C., BAINBRIDGE, J. G., BELL,F. R.,LAWN,A. N., AND ROSENBERG, H., (1951); Central respiratory spike potential. Nature, 167, 603-604. B A T I N I , ~MORUZZI, ., G . ,PALESTINI, M., R o w , G . F., AND ZANCHEITI, A., (1958); Persistent patterns of wakefulness in the pretrigeminal midpontine preparation. Science, 128, 3&32. BATINI,C., MORUZZI,G., PALESTINI, M., R o w , G. F., AND ZANCHEITI, A., (1959); Effects of complete pontine transections on the sleep-wakefulness rhythm: The midpontine pretrigeminal preparation. Arch. ital. Biol., 97, 1-12. BONVALLET, M., AND ALLEN,M. B., (1963); Prolonged spontaneous and evoked reticular activations following discrete bulbar lesions. Electroenceph. elin. Neurophysiol., 15, 969-988. BONVALLET, M., AND BLOCH,V., (1961); Bulbar control of cortical arousal. Science, 133,1133-1 134. BONVALLET, M., DELL,P., ET HIEBEL, G., (1954); Tonus sympathique et activitk klectrique corticale. Electroenceph. elin. Neurophysiol., 6, 119-144. BONVALLET, M., HUGELIN, A., ET DELL,P., (1955); Sensibilitk cornpark du systbme rkticulk activiteur ascendant et du centre respiratoire aux gaz de sang et ii I’adrknaline. J . Physiol. (Paris), 47, 651-654. CHEN,G., ENSOR,C. R.,RUSSELL, D., AND BOHNER, B., (1959); The pharmacology of I-(1-phenyl cyclohexyl) piperidine HCI. J. Pharmacol. exp. Ther., 127, 241-250. COHEN,M. J., AND WANG,S. C., (1959); Respiratory neuronal activity in pons of cat. J. Neurophysiol., 22, 33-50. DELL,P., ET BONVALLET, M., (1954); ContrBle direct et rkflexe de I’activitk du systkme rkticulk activiteur ascendant du tronc ckrkbral par I’oxygbne et le gaz carbonique du sang. C. R. SOC.Biol. (Paris), 148,855-858. DIRKEN, M. N. J., AND WOLDRING, S., (1951); Unit activity in bulbar respiratory center. J. Neurophysiol., 14, 21 1-225. GELLHORN, E., (1953); On the physiological action of carbon dioxide on cortex and hypothalamus. Electroenceph. clin. Neurophysiol., 5 , 401-41 3. GERNANDT, B. E., AND THULIN,C. A.. (1952); Vestibular connections of the brain stem. Amer. J. Physiol., 171, 121-127. GIBES,F. A., AND GIBES,E. L., (1950); Atlas of~lectroencephal~graph~. Vol. 1. Cambridge, Mass., Addison-Wesley (p. 70). GIBES,F. A., GIBES,E. L., AND LENNOX,W. G., (1935); Changes in human cerebral blood flow consequent on alterations in blood gases. Amer. J. Physiol., 111, 557-563. GIBES,F. A., WILLIAMS, D., AND GIBES,E. L., (1940); Modification of the cortical frequency spectrum by changes in COa, blood sugar and Oa. J. Neurophysiol., 3,49-58. HABER, E., KOHN,K.W., NGAI,S . H., HOLADAY, D. A., AND WANG,S . C., (1957); Localization of spontaneous respiratory neuronal activities in the medulla oblongata of the cat. A new location of the expiratory center. Amer. J. Physiol., 190, 350-355. HELLNER, K., UND VON BAUMGARTEN, R.,(1961); Uber ein Endigungsgebiet afferenter, kardiovascularer Fasern des Nervus vagus im Rautenhirn der Katze. Pfliigers Arch. ges. Physiol., 273, 223-234. HIRAO,T., (1962); Technical improvement on brain stem transection of cat. Kilakanto Zgaku, 12, 41-48. HUKUHARA, T., NAKAYAMA. S., AND OKADA, H., (1954); Action potentials in the normal respiratory centers and its centrifugal pathways in the medulla oblongata and spinal cord. Jap. J. Physiol., 4,145-153. KUMAGAI, H., SAKAI,F., HUKUHARA, T., SAJI,Y.,KUMADAKI, N., KOJIMA, H., AND TAMAKI, H., (1964); The relation of periodic change of EEG to activity of respiratory neurone in lower brain stem of cats. Proc. XZth Ann. Meet. Jap. EEG Soc.,In press.

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LUBY,E. D., COHEN,9. D., RoseNeAuM, G., GoTTLIEB,J. s.,AND KELLEY, R., (1959); Study of a new schizophrenomimetic drug - Sernyl. Arch. Neurol. Psychiaf., 81, 363-369. OTSUKA, Y.,SAKAI,F., SAKUMA, A., SAJI,Y.,NAKANISHI, S., AND SAWABE, T., (1962); Central effects of phencyclidine hydrochloride (Sernyl). Jap. J. Pharmacol., 12, 109-1 10. OTSUKA, Y.,SAKAI,F., SAKUMA, A., SAJI,Y.,NAKANISHI, S . , UND SAWABE, T., (1963); Periodische Veranderungen des Elektrencephalogramms. Jap. J . Pharmacol., 13, 253-258. SAKAI, F., SAKUMA, A., OTSUKA, Y.,SAJI,Y.,UND KUMAGAI, H., (1962); Wirkung der Hypo- und Hyperventilation auf das Elektrencephalogramm. Naunyn-Schmiedeberg’s Arch. exp. Path. Phar-

mak., 244, 145-152. SALMOIRAGHI, G . C., (1962); ‘Cardiovascular’ neurones in brain stem of cat. J . Neurophysiol., 25, 182-198.

SALMOIRAGHI, G. C., AND VON BAUMOARTEN, R., (1961); Intracellular potentials from respiratory neurones in brain stem of cat and mechanism of rhythmic respiration. J. Neurophysiol., 24, 203218.

TOKIZANE, T., KAWAMURA, H., AND IMAMURA, G . , (1960); Hypothalamic activation upon electrical activities of paleo- and archicortices. Neurologia med.-chir., 2, 63-76. VON BAUMGARTEN, R.,( 1956); Koordinationsformen einzelner Ganglienzellen der rhombencephalen Atemzentren. Pjlugers Arch. ges. Physiol., 262, 573-594. VON EULER, C., AND SODERBERG, U., (1952); Medullary, chemosensitive receptors. J . Physiol., 118, 545-554.

WOLFF,H. G., A N D LENNOX, W. G., (1930); Cerebral circulation. XII. The effect on pial vessels of variations in the oxygen and carbon dioxide content of the blood. Arch. Neurol. Psychiaf., 23, 1097-1120.

112

Metabolic Studies on ep Mouse, a Special Strain with Convulsive Predisposition MASANORI KUROKAWA, HIROSHI N A R U S E

AND

MAKOTO KATO

Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan) and Department of Neuropsychiarry, Faculty of Medicine, University of Tokyo, Tokyo (Japan)

An inbred strain of convulsive mouse was discovered in 1954 by Imaizumi in the course of his study on spontaneous hydrocephalus in mice. The strain was named ep mouse because of the apparent similarities in convulsive seizures to those of human epileptic patients. Since convulsions in the ep strain are produced in all adult animals of both sexes by means of postural stimulation unbalancing them, and the convulsive characteristic appears to be hereditary as a Mendelian dominant (Imaizumi et al., 1959), it is expected that ep mouse will give a great opportunity for metabolic studies of convulsive predisposition, which is not to be examined in cases of electrically and chemically induced convulsions. Exogenous factors known to cause convulsive disorders, such as infections and dietary deficiencies have been specifically eliminated (Imaizumi et al., 1959; Yabe, 1959). One of the further advantages in using the ep mouse for experimental studies was that the mice did not die from the convulsions. This is in sharp contrast to electrical or chemical convulsions, and also to audiogenic strains, of which it has been reported that 30% of the mice subjected to excessive acoustic stimulation died after convulsions, apparently from respiratory paralysis (Suter et al., 1958). CONVULSIVE SEIZURES I N

ep

MOUSE

Nature of stimulation Successive and alternating movements causing adult mice to lose their postural equilibrium were the effective stimuli which induced convulsions in the strain (Yabe, 1959; Naruse et al., 1960). Of the different stimuli which were examined, the simplest and the most effective was to throw the mouse up in the air 15 to 20 cm several times. With this method, the limbs were stimulated when they came into contact with the plate which supported the mouse. But this afferent stimulation was not likely to have induced convulsions, since vertical swinging of a mouse with the limbs loosely fixed to a plate was also effective in inducing convulsions. Similarly, to-and-fro horizontal swinging, pendulum movement and alternating rotation also proved effective. Continuous rotation in one direction never caused convulsions, indicating that the alternating character of stimulation was essential.

METABOLIC STUDIES ON

ep

MOUSE

113

In the majority of mice, seesaw stimulation was used routinely, as this was the most easily standardized procedure and the least muscular movements were observed during this form of stimulation. The mouse was placed with its corporeal axis in the central axis of a plate; the plate was then subjected to a seesaw motion about the axis, at an angle of inclination of 15-20', with a frequency of 20-25 cycles per 10 sec (Fig. 1). The minimal period of seesawing needed to provoke seizures varied with different mice from 5 to 35 sec. It appeared however that the threshold period of stimulation was nearly constant for a particular mouse, provided that the animal was taken care of in a well-controlled animal house and that the seesaw stimulation was given at regular intervals, i.e. once or twice a week.

Fig. 1 . Seesaw stimulation. Angle of inclination: 15-20". Frequency: 20-25 cycles per 10 sec

Ep mouse did not respond to photic and/or acoustic stimulation. Seizure threshold in ep mouse in response to chemical stimulation was found lowered in comparison with that in non-convulsive strains of mice. Thus in ep mouse, 40 mg/kg of pentamethylenetetrazole was enough to produce generalized convulsions after intramuscular injection, while 60 mg/kg and 70 mg/kg were required for non-convulsive gpc and dd mice respectively. Similarly, the threshold for electrical stimulation (50 c/sec A.C., 0.2 sec, corneal electrode) in ep mouse, 6.1 mA, was significantly lower than in dd mouse where it was 7.3 mA (Takahashi et al., 1962; Kariya, 1962). The course of convulsions A typical seizure begins with prodromal signs of very short duration, most commonly sharp squeaks and catatonic posture (Fig. 2a). During the catatonic stage the animal stands perfectly still with its tail erect, and the postural control is still well maintained. One to 3 sec later, there is a characteristic 'breast-stroke swimming' motion or 'running fit' followed by convulsions, and the postural control is lost. Convulsions, at first clonic in nature, start in the hind limbs, but rapidly pass into a generalized tonus-clonus. Maximal convulsion is characterized by generalized tonus with ventro-flexion of the neck (Fig. 2b), which is followed by the stage of generalized tonus with dorso-flexion of the neck (Fig. 2c). During the generalized convulsive stage, Rpfurences p . 1291130

1 I4

M. K U R O K A W A , H. N A R U S E A N D M. K A T O

a

b

C

Fig. 2. Convulsive seizures in ep mouse. (a) Catatonic posture; (b) Generalized tonus with ventroflexion of the neck; (c) Generalized tonus with dorso-flexion of the neck.

massive salivation and urinary incontinence are usually seen. Cessation of convulsive activity is followed by a stage of post-ictal depression, characterized by inertness and stupor, or less commonly by excitement with extreme irritability. In 22 animals, the time from the first squeak to the complete cessation of convulsive movement was 16 & 3 (S.D.) sec. There is a definite refractory period before the second seesaw stimulus is effective, but its duration showed fairly wide individual variation. Of 20 animals tested, 6 had a refractory period of 30 min, while 6 would not convulse even after 40 min.

Changes in seizure threshold Postural stimulation could never induce convulsive seizures in animals younger than 6-7 weeks. In this strain the reproductive function was generally attained in the course of the 7th week after birth, which is suggestive of the influence of hormonal factors in the occurrence of seizures. Stimulation was usually given once a week from the beginning of the 5th week after birth, and from the 5th to 7th-8th week, seizures generally took an abortive form, most commonly squeaks and catatonic posture, oc-

METABOLIC STUDIES ON

ep M O U S E

115

casionally simple muscular twitchings. If, however, the animals were first stimulated when they were older than 10-1 1 weeks, some responded with typical seizures, indicating that previous stimulation was not absolutely essential for the genesis of convulsions; a particular neurological constitution at a definite stage of development might be the critical factor. Convulsions in this strain did not seem to decrease with age; e.g. they could be produced as easily in a 60-week-old mouse. As already mentioned, there were no sex differences in the occurrence of convulsions. Pregnancy did not appear to affect the seizure threshold. It seemed that the occurrence of seizures could be influenced by weather conditions such as atmospheric pressure; thus on days of low atmospheric pressure, more stimulation was generally needed to produce convulsions than on days of high atmospheric pressure. Sodium phenobarbitone was given intraperitoneally and seesaw stimulation was applied 40 min after the injection. In 7 of 10 mice tested in this way, seizures were completely preveiited with 10 mg/kg phenobarbitone; with 30 mg/kg, prevention was complete in all of the 10 animals tested. Sodium diphenylhydantoin appeared to be less effective than sodium phenobarbitone, but this was not conclusive, since the diphenylhydantoin solution was used at ranges more alkaline than the phenobarbitone solution. L-Glutamic acid, L-glutamine, L-asparagine, y-aminobutyric acid and y-amino-p-hydroxybutyric acid did not affect the seizure susceptibility of the ep mice. In hybrid (F1) between the convulsive ep and non-convulsive gpc mice, it was observed that the seesaw stimulation was effective in producing convulsions only after the 30th week after birth, in spite of stimulation given at one week intervals from the 5th week onward; this retardation of the first appearance of convulsive seizures in response to postural stimulation was equally observed in hybrid F1 which came from either paternal or maternal ep mouse (Hirayama and Kurokawa, unpublished). Another approach has been made by Utena and his coworkers in which they demonstrated that the seizure threshold in the ep mouse could be raised by rearing the animal in a revolving cage. Thus when an ep mouse had been reared in a specially designed revolving cage for 7 to 14 days, and then transferred to an ordinary cage, the mouse did not convulse for approximately 14 days thereafter, in spite of the maximal postural stimuli given (Utena et a / . , 1961; Yuasa, 1961). They claimed that their results were in agreement with the statement of Lennox that activity would antagonize convulsions, and also with a known fact that in audiogenic seizures, activity appeared to protect the animal from convulsions. M A T E R I A L A N D METHODS

E.yperimental ariimals Ep mice were convulsed once a week by applying seesaw stimulation. Mice not so stimulated, and so not convulsed at all, and designated ep(o), were used as controls. As additional controls, mice of non-convulsive gpc, BL-57, and dd strains were used. Unless otherwise indicated, the animals used were adults of both sexes, 15-25 weeks old, and weighed between 25 and 30 g. References p. 129/130

1 I6

M. K U R O K A W A , H. N A R U S E A N D M. K A T O

Experimental arrangements Littermates of ep mice usually numbering 8-10 were divided into two groups: ep and ep(o). Ep mice were frozen in liquid nitrogen (or decapitated when indicated) at stages of rest, of preconvulsion, of generalized tonus with ventro-flexion of the neck, and of generalized tonus with dorso-flexion of the neck. Values in the refractory period were those obtained in animals fixed between 10 and 15 min after the cessation of convulsive movements. In the indicated experiments mice were placed in a desiccator in which the air was replaced by 99.98 % nitrogen. After transient excitement due to anoxia, the mouse lost its postural control in about 30 sec and went into coma after 40 sec. Convulsions were never elicited during anoxia lasting up to 120 sec. Determinations were made in brains fixed between 40 and 50 sec of anoxia. Mice of the non-convulsive strain were convulsed by intramuscular injection of pentamethylenetetrazole (100 mg/kg) which generally produced the first tonus-clonus within 2-3 min. Control mice were anaesthetized by intraperitoneal injection of sodium pentobarbitone (30 mg/kg), and when ciliar and corneal reflexes were lost, decapitated or frozen. In anaesthetized animals, intraperitoneal injection of at least 300 mg/kg pentamethylenetetrazole was needed to provoke a typical convulsion. In experiments in which eserine sulphate (1 mg/kg, intraperitoneally) was tested the animal was decapitated 5-10 min later, at the stage of generalized shiver but without convulsive manifestations. Other conditions are given in respective tables. Tissues The frozen brain was rapidly dissected out without allowing it to thaw, and finely powdered in a steel crusher containing liquid nitrogen. The details of the homogenization and fractionation procedures have been described elsewhere (Kurokawa et al., 1963); briefly, the 10% (w/v) homogenate in 0.32 M sucrose, with or without eserine, was centrifuged at 1000 g for 10 min, the residue resuspended in fresh medium and again centrifuged. The supernatant and washing were centrifuged at 22,500 g for 20 min and the residue was again washed once. Combined supernatants from these centrifugings were centrifuged at 105,000 g for 60 min. The letters H, N, Mt, Ms and S respectively were used to designate the original homogenate, nuclear-debris (1000 g), crude mitochondria1 (22,500 g), microsomal(lO5,OOOg) and supernatant fractions. In cases indicated, the Ms and S fractions were not separated, but taken as a composite Ms-S fraction. Tissue slices 0.3-0.4 mm in thickness were cut from the cerebral cortex with a blade and a recessed guide. Chemical methods Acetylcholine activity (ACh) was extracted from the tissue in eserine-containing frog Ringer solution at pH 3-4, with boiling, and assayed at room temperature using the eserinized rectus abdominis muscle of the frog (Kato, 1959; Kurokawaet ul., 1963). The non-specific effect of brain tissue extracts on the contraction of the rectus was eliminated by the addition of extracts freed from ACh. Also, interference of 0.32 M

METABOLIC STUDIES ON

ep

1 I7

MOUSE

sucrose with the bioassay was minimized (Kurokawa e t a / . , 1963). Synthesis of acetyl-CoA was determined in terms of ferrihydroxamate formation (Kurokawa et al., 1961) and choline acetyltransferase activity (ChA) in terms of ACh formation under conditions where the formation of acetyl-CoA was not ratelimiting (Kurokawa et d., 1961). Cholinesterase activity (ChE) was measured manometrically with acetylcholine or acetyl-/l-methylcholine as substrates (Kato, 1959; Yabe, 1959; Kurokawa, 1963). The free ammonia content of the brain was determined by micro-diffusion analysis, correction being made for ammonia derived from hydrolysis of glutamine (Naruse, 1959; Naruse e t a / . , 1960). Glutamine, glutamic acid and y-aminobutyric acid were separated and determined by the method of Berl and Waelsch (1958) with minor modifications (Naruse et al., 1960). Systematic separation of free amino acids in the brain wascarried out according to the method of Spackman, Moore and Stein (1938). Acid-soluble nucleotides were separated and determined as described by Hurlbert et al. (1954), but with some modifications (Hirayama, 1963). Creatine phosphate was determined according to the method of Takahashi (1955). LEVELS O F SOME B R A I N C O N S T I T U E N T S I N THE R E S T I N G A N I M A L

There were some strain differences in the levels of some brain constituents (Table I). Thus the level of ACh and of y-aminobutyric acid in ep mouse was about 50 % higher than that in controls, regardless of whether or not convulsions had been previously produced by seesaw stimulation. In contrast, ammonia, glutamic acid, glutamine and aspartic acid levels were lower in the ep mouse than in the controls. The level of free amino acids exclusive of the above-mentioned did not show any detectable strain differences (Table 11). Again, the levels of adenine nucleotides and creatine phosphate TABLE I SOME B R A I N C O N S T I T U E N T S I N D I F F E R E N T S T R A I N S O F MICE

eP

Acetylcholine Ammonia Glutamine Glutamic acid Aspartic acid y-Aminobutyric acid ATP ATP ADP AMP Creatine phosphate Inorganic phosphate NAD

+

+

15.8 0.12 2.2 2.6 2.4 3.3 1.48 2.09 2.62 6.66 0.23

Controls

eplControls

9.7-10.7 0.20-0.21 2.8-3.3 9.8-1 1 .O 2.9-3.1 2.2-2.4 1S O

1.55 0.59 0.72 0.83 0.80 1.44 0.99 0.97 1.04 1.10 1.15

2.16 2.5 1

6.07 0.20

Determinations were carried out in the brain exclusive o f cerebellum, frozen and powdered in liquid nitrogen. Acetylcholine mpmoles/g powder; others pmoles/g powder. Control strains: dd, gpc, and BL-57. (Naruse et al., 1960; Hirayarna, 1963; and unpublished data). Rpferenres p. I29jl3U

118

M. K U R O K A W A , H. N A R U S E A N D M. KATO

T A B L E I1 THE F R E E AMINO A C I D L E V E L IN A D U L T MOUSE BRAIN

@rnoles/g powder) BL-57

Strains ~-

Glycerophosphoethanolamine Phosphoethanolamine Taurine Proline

X1' Glycine Alanine Glutathione Valine Methionine lsoleucine Leucine Phenylalanine Lysine Histidine Arginine N-Acetyl-L-aspartic acid

0.49 1.40 8.1 0. I 0.13 0.89 0.39 0.79 0.12 0.08 0.10 0.14 0.21 0.04 0.03 0.04 4.5

0.44 1.35 7.6 0. I 0.99 0.40 0.74 0.10 0.08 0.10 0.20 0.23 0.04 0.04 0.03 4.2

0.48 1.60 9.4 0. I 0.16 1.02 0.47 0.85 0.12 0.08 0.10 0.20 0.23 0.04 0.03 0.03 4.8

0.43 1.65 7.8 0.1 0.09 0.81 0.45 0.69 0.10 0.09 0.12 0.14 0.18

6.5

* Leucine equivalents. (Naruse et al., 1962). did not differ between strains. The amount of extracellular water calculated on the basis of chloride space was 2.76 and 1.80 ml/g dry weight in ep and control dd mice respectively, but total water, total potassium ion and total sodium ion did not differ between the two strains (Hirano and Shimizu, 1962). C H A N G E S I N THE LEVEL OF SOME B R A I N C O N S T I T U E N T S D U R I N G SEIZURES

A marked rise in brain ammonia was induced in ep mice by applying seesaw stimulation, the level increasing to 180, 250 and 470% above the resting control with stimulation during 8, 20 and 24 sec respectively (Table 111). The muscular activity accompanying seesaw stimulation was unlikely to have caused the secondary increase in brain ammonia; in the control mice, the same or even much longer stimulation did not cause any change in the brain ammonia. The increase in brain ammonia of ep mice with postural stimulation closely paralleled that of control mice which were chemically convulsed. Mice injected with pentamethylenetetrazole or with 3-methyl-3-ethylglutarimideshowed markedly increased brain ammonia, both at the preconvulsive and at the convulsive stage, in accord with observations reported by Richter and Dawson (1948) in rats given picrotoxin. In anoxic coma induced by pure nitrogen, brain ammonia again markedly increased, in spite of the fact that convulsions were never elicited under these conditions (Table 111; cf. Richter and Dawson, 1948).

METABOLIC STUDIES ON

ep

1 I9

MOUSE

T A B L E 111 A M M O N I A A N D GLUTAMINE I N ADULT MOUSE B R A I N

UNDER VARIOUS CONDITIONS

(Naruse et al., 1960) Strains

Clutamine (pmoleslg)

Resting

0.13

2.50

eP

Resting Seesaw stimulation, 8 sec, preconvulsive Seesaw stimulation, 20 sec, preconvulsive Seesaw stimulation, 24-32 sec, convulsive

0.1 I

2.44

0.20

2.33

0.28

2.46

0.52

2.53

Resting Seesaw stimulation, 8 sec, no changes Seesaw stimulation, 240 sec, no changes PMT* injection, preconvulsive PMT injection, convulsions MEG** injection, preconvulsive MEG injection, convulsions Anoxia in pure nitrogen, comatose Water injection, no changes

0.21

3.26

0.18

3.39

0.18 0.41 0.49 0.36 0.53

3.18

dd

* PMT

Ammonia (woleslg)

ep(o)

gpc

** MEG

Treatment and remarks

= =

Resting Seesaw stimulation, 8 sec, no changes Seesaw stimulation, 240 sec, no changes PMT injection, preconvulsive

3.42

0.50 0.20

0.20 0.18

3.67 3.25

0.17 0.41

3.25

Pentamethylenetetrazole (80 mg/kg), intramuscular. Methylethylglutarimide(37 mg/kg), intramuscular.

There was a definite fall in brain ACh at both preconvulsive and convulsive stages in ep mouse (Kato, 1959; Kurokawa, 1963), the detail of which will be described later. Convulsive seizures in ep mouse in response to seesaw stimulation were also accompanied by a fall in creatine phosphate in the brain (Table IV). In general, this was in accord with the change in creatine phosphate which had been repeatedly shown in chemically or electrically induced convulsions. In contrast to the definite changes in ammonia, ACh and creatine phosphate in the ep mouse brain during seizures, no appreciable change occurred in the levels of glutamine, glutamic acid, aspartic acid and y-aminobutyric acid (Naruse et al., 1960). This was at variance with previous reports which demonstrated changes in brain amino acid content occurring during convulsions associated with either metabolic inhibitors (Dawson, 1953; Killam and Bain, 1957; Kamrin and Kamrin, 1961) or with longer duration of preconvulsive period (Berl et: al., 1959). References p. 129/130

120

M. K U R O K A W A , H. N A R U S E A N D M. KATO

TABLE IV CREATINE PHOSPHATE A N D INORGANIC PHOSPHATE I N ADULT MOUSE B R A I N D U R I N G V A R I O U S STATES O F C O N V U L S I V E A C T I V I T Y

(Hirayama, 1963) Strains

Resting Seesaw stimulation 30 sec, no changes

2.55 (3)

5.50 (3)

2.20 (3)

6.36 (3)

eP

Resting Immediately after convulsions TVF*

2.62 (3) 1.75 (3) 1.21 (3)

6.66 (3) 7.13 (3) 6.85 (3)

gpc

Resting PMT**, first convulsions PMT, final tonus immediately before death

2.51 (5) 1.13 (5)

6.07 (5) 8.60 (5)

2.85 (3)

5.90 (3)

ep(o)

*

Treatment and remarks

TVF = Tonus with ventro-flexionof the neck (seetext). PMT = Pentamethylenetetrazole(100 mg/kg). intramuscular.

S T U D I E S O N THE A C E T Y L C H O L I N E SYSTEM

Levels in situ, formation and hydrolysis of acetylcholine Fig. 3 shows the increase in brain ACh during postnatal development; higher values in the ep mouse were shown to exist during all the developmental stages. As already mentioned, higher levels of ACh in the ep mouse were observed regardless of whether or not seesaw stimulations had been previously given, and this would eliminate the possibility that this strain difference is a secondary change due to previous convulsions. The rate of formation of ACh activity in brain slices respiring in the glucosecontaining media is shown in Table V. With a potassium concentration of 6.5 mM in

-

. 15

a

-

f 2 610 U

-

f

f-

7I

4

I

I

I

I

I

I

I

I

5 6 7 8 9 1 0 1 1 12 weeks

Fig. 3. Developmental changes in mouse brain acetylcholine.

M E T A B O L I C S T U D I E S O N ep M O U S E

121

TABLE V ACETYLCHOLINE FORMATION I N THE CEREBRAL CORTEX OF A D U L T MOUSE

Unit: mymoles ACh formed/g tissue/60 min (38") Potassium concentration (mM)

ACh in slices

ACh in media

Total

eP

6.5 33

70.5 20.0

13.8 77.6

84.3 97.6

No)

6.5 33

60.0 18.2

12.3 78.0

72.3 96.2

6.5

35.5 12.6

9.4 53.1

44.9 65.7

Strain

&?PC

33 (Kato. 1959; Naruse et al., 1960).

V 0

1

I

I

I

1

I

10

20

30

40

50

60

min

Fig. 4. Formation of acetyl-CoA in mouse brain.

min With acetate.CoA and ATP

With. acetyl-CoA

Fig. 5 . Choline acetyltransferase activity in mouse brain (Kurokawa et a/., 1961, modified). Refrrenres p . 129/130

122

M. K U R O K A W A , H. N A R U S E A N D M. KATO

the test media, the rate of formation of ACh in tissues was found to be significantly higher in the ep mouse than in the control strain. The rate was slightly lower in the ep(o) than in the ep mice, but this difference was not significant. With an increased potassium concentration of 33 mM, the increase in ‘free’ ACh in the test media during incubation was larger in the ep than in the control mice; again no significant difference was found between the two series of ep mice. The rate of synthesis of acetyl-CoA was determined in crude extracts of acetonedried powder obtained from the mouse brain. Fig. 4 shows that the activity of the enzyme in the convulsive ep and control gpc mice does not differ (Kurokawa et al., 1961). The cboline acetyltransferase activity in ep mouse brain was higher by approximately 50% than that in control gpc strain (Fig. 5), which appears to furnish an explanation for the higher value of ACh in situ. The higher choline acetyltransferase

Ijm/

‘?“(I N

8 800

L

3

l

,

,

,

6

7

0

9

,

l

,

1 0 11

12

weeks

Fig. 6. Developmental changes in mouse brain cholinesteraseactivity. Substrate 5 m M acetylcholine.

12

14

wesks

16 , , 4 7

O ,’r

500 -

L 5 10

0

times

Number 01 previous ConvuIsIons

Fig. 7. Changes in cholinesterase activity in the ep mouse brain, apparently due to previous convulsion. Substrate 30 mM, acetyl-/hnethylcholinc.

METABOLIC STUDIES ON

ep

MOUSE

123

activity in ep mice was found, in accord with ACh itself, irrespective whether or not the animals had been previously convulsed, although a little higher value was invariably obtained in ep series in comparison with ep(o) series. Cholinesterase activity in the mouse brain 5-6 weeks after birth was shown to be approximately 60-70% of the adult level reached in the 10th-12th week. The esterase activity in ep mice tended to be higher than that in gpc mice, but the difference was not significant (Fig. 6). An apparent decrease in esterase activity was observed in the ep mice convulsed once a week by postural stimulation (Fig. 7). Since in ep(o) mice no changes in activity occurred until up to the 67th week after birth, it appears that the decrease in the esterase activity in ep mice is an after-effect of previous convulsions. The subcellular distribution of acetylcholine The level of ACh in the brains of resting ep mice was found to be approximately 50% higher than that of the gpc mice (Table VI), a result which agrees with findings T A B L E VI T H E S U B C E L L U L A R D I S T R I B U T I O N O F A C E T Y L C H O L I N E I N MOUSE B R A I N

(Kurokawa et al., 1963) Fractions*

H

N

Mt Ms S

Recovery *For the description of fractions, see

mpmoles ACh/g brain

Ratio eplgpc eP

RPC

20.4 1.1 12.1 2.2 3.5 93 %

13.2 0.9 7.7 1.7 2.5 96 %

1.54 1.22 1.58 1.30 1.40

-

MATERIAL AND METHODS.

in mice frozen in liquid nitrogen (see Table I). The high level is practically attributable to an increase in the Mt fraction, which contains about 60% of total ACh in both ep and gpc mice. In agreement with earlier workers, all the free ACh present in the original homogenate is recovered as the S fraction, while the bound ACh can be accounted for as the sum of activities in the N, Mt and Ms fractions (cf. Hebb and Whittaker, 1958). When the brain was homogenized in 0.032 A4 sucrose containing no eserine, and kept at 0 4 , the level of ACh in the tissue decreased as a function of time until about 90-120 min after homogenization, and then remained fairly constant at 5.8-5.9 mpmoleslg brain (Kurokawa et al., 1963). The portion thus remainingcan be regarded as the stable fraction of the bound ACh and will be so designated, taking Whittaker’s (1959) original use of the term in a wide sense. Similarly the portion of ACh which is releasable in the hypotonic medium and hydrolyzed in the absence of an anti-cholinesterase, will be called the labile fraction of the bound ACh. Refwences p . 129l130

124

M. K U R O K A W A , H. N A R U S E A N D M. KATO

TABLE V I I T H E S U B C E L L U L A R D I S T R I B U T I O N OF ACETYLCHOLINE I N ESERINE-FREE H Y P O T O N I C B R A I N HOMOGENATES

(Kurokawa et al., 1963) mpmoles ACh/g brain Ratio eplgpc

Fractions

H N Mt

Ms S

Recovery

eP

gPc

5.9 0.1 3.3 2.2 0 104%

6.0 0.8 3.0 1.9 0 95 %

0.98 0.88 1.10 1.16

-

The subcellular distribution of ACh in the eserine-free hypotonic brain suspension is shown in Table VII. It can be seen that the distribution of stable ACh estimated in this way does not significantly differ between the strains of mice examined, which suggests that the higher level of ACh in the ep mouse is exclusively due to the increase in the labile fraction. A similar hypotonic shock was applied to the Mt fraction which had been isolated In the eserine-free 0.32 M sucrose. The amount of ACh in the Mt fraction in terms of iabile and stable forms is shown in Table VIII which demonstrates that the increase TABLE V I I I A M O U N T S OSMOTICALLY LABILE A N D STABLE ACETYLCHOLINE I N THE C R U D E M I T O C H O N D R I A L F R A C T I O N FROM MOUSE B R A I N

(Kurokawa et al., 1963) Strains

eP epfo) 8PC

BL-57

mpnoles AChlg brain Total Labile Stable

Ratio LabilelS'able

8.8 8.3 4.3 2.9

2.66 2.08 1.26 0.15

12.1 12.3 7.7 6.8

3.3 4.0 3.4 3.9

in ACh in the ep mouse brain is exclusively due to that of the labile form which is more than doubled in comparison with the level in control strains. In contrast, the level of stable ACh is fairly constant in the various strains. No significant difference is found between the ep and ep(o) mice as t o the composition of particulate ACh.

Changes in acetylcholine under various conditions Table IX shows the changes in the level of ACh in the ep mice under various states of convulsive activity. At the maximal stage of convulsive activity, i.e. tonus with ventro-flexion of the neck, there was the maximal loss of ACh which amounted to

M E T A B O L I C S T U D I E S ON

ep M O U S E

125

TAB L E IX LEVELS OF B R A I N ACETYLCHOLINE

I N ep M I C E U N D E R V A R I O U S

STATES O F C O N V U L S I V E A C T I V I T Y

Determinations were made in animals frozen in liquid nitrogen. Values are the mean of 3-6 experiments (Kato, 1959; Kurokawa 1963) ACh

Conditions and remarks

eP

ep(o)

(mwoleslg)

Resting Seesaw stimulation 8 sec, preconvulsive Immediately after the beginning of convulsions Tonus with ventro-flexion of the neck Tonus with dorso-flexion of the neck 60 sec after the restoration of posture 90 sec after the restoration of posture 120 sec after the restoration of posture 150 sec after the restoration of posture 180 sec after the restoration of posture Seesaw stimulation 40 sec, during refractory period Resting Seesaw stimulation, 8 sec Seesaw stimulation, 40 sec

15.8 12.4 9.6 5.9 8.3 9.6 15.0

17.8 18.6 20. I 13.8 15.2

13.4 12.8

9.9 m,umoles/g tissue. It may be pointed out that this amount is twice as much as the fall of ACh that has been observed in ordinary animals convulsed chemically or electrically (4.Richter and Crossland, 1949; Takahashi et al., 1961; Kato, 1959); this implies that in the ep mouse, the amount of brain ACh which is susceptible to convulsive stimulation is twice that in the control strains. It should also be noted that a transient rise of ACh above the resting level is invariably observed during the T ABLE X C H A N G E S O F L A B I L E A N D S T A B L E A C E T Y L C H O L I N E I N R E L A T I O N TO ep C O N V U L S I O N S

(Kurokawa et al., 1963) mpmoles AChlg brain

Mt fraction, separated in eserine-0.32 M sucrose, with subsequent suspension in eserine-0.032 M sucrose. Homogenate, suspended in eserine-free 0.032 M sucrose Brain tissue, frozen and pulverised in liquid nitrogen

ACh

At rest

TVF

Labile

8.8

4.0

Stable

3.3

4.0

Stable

5.7

Total

15.8

TDF

4.0

RP-60 sec RP-90 sec

4.1

6.7

4.5

5.0

4.3

5.7

5.9

7.4

-

5.9

8.3

9.6

15.0

TVF = generalized tonus with ventro-flexion of the neck; TDF = generalized tonus with dorsoflexion of the neck; RP-x sec = x sec after the restoration of posture. For further details see MATERIAL AND METHODS. For labile and stable ACh, see text. Refersncrr p . I29/I30

126

M. K U R O K A W A . H. N A R U S E A N D M. KATO

recovery process after the cessation of convulsive activity. Similar observations have also been made in the case of electrically induced convulsions (Takahashi ef al., 1961). Seesaw stimulation applied to ep mice during the refractory period as well as to epro) mice did neither cause convulsions nor a fall in ACh, During and after the convulsions brought on in ep mice by applying seesaw stimulation, changes in the levels of labile and stable ACh were observed (Table X).The fall in ACh during ep convulsions is mainly attributable to loss of ACh from the Mt fraction, and occurs without apparent change in the N, Ms and S fractions. The fall in ACh in the Mt fraction is due to a preferential loss of the labile portion; in contrast, stable ACh did not decrease during convulsive activity. In order to obtain more information on the changes in the subcellular distribution of ACh, non-convulsive mice convulsed or anaesthetized chemically were examined (Table XI). During convulsionscaused by pentamethylenetetrazolea fall in brain ACh TABLE XI CHANGES I N THE LEVEL O F BRAIN ACETYLCHOLINE l N D U C E D I N g p C MICE BY D R U G S

(Kurokawa et al., 1963) Drugs

None

C ~ n d i ~ i 5 ~ s At resr

Fractions H N Mt

labile stable MS-S

bound free Brain. frozen, pulverized in liquid nitrogen

PMT

PB

PB 4- PMT

Eserine

15.0 0.9 8.8 4.8 4.0 5.3 1.4 3.9

21.6 I .O 10.4

6.9

-

anaesthesia

13.2 0.9 7.1 4.3 3.4 4.4 I .7 2.7

22.0 0.8

10.7

16.4

13.9 9.3 4.6

6.9 1.4 5.5

Figures represent mpmoles AChfg brain. PB Other details in MATERIAL AND METHODS.

6.2

6.0 4.4 10.2 0.8 9.4

= Pento~rbitone;PMT = Pen~amethylenetetrazole.

was only found in animals frozen in liquid nitrogen and this was in accord with the results of earlier workers (cf. Richter and Crossland, 1949). An increase in brain ACh in pentobarbitone-anaesthetized animals, and its decrease due to subsequent application of pentamethylenetetrazole were mainly associated with changes in labile ACh in the Mt fraction, although the stable ACh in the Mt fraction and the free ACh in the Ms-S fraction had increased to a smaller extent. This was in contrast with the finding that an increase in the brain ACh in the eserine-treated animal was practically entirely due to free ACh in the Ms-S fraction.

METABOLIC STUDIES ON

ep

127

MOUSE

In view of the fact that the fall in ACh in the decapitated brain is detected only in relation to ep mice convulsions and to chemically induced convulsions in anaesthetized mice and is not observed in animals convulsed without anaesthesia (Table XI; CJ Elliott er a/., 1950), it seems probable that the high level of labile ACh obtaining before convulsions is an essential prerequisite for demonstrating a fall in ACh during convulsions in animals later decapitated. Fig. 8 shows a compartmentation of ACh in the brain. Dark portions on the left side of the diagram indicate the amount of ACh remaining on hypotonic shock in vitro or at the maximal stage of convulsions. Light portion on the right side indicates the fraction of ACh which is apt to change in response to hypotonic shock in vitro or to convulsive activity and to anaesthesia. Although the identity of the stable ACh in vitro and that in vivo is not directly evidenced, the correspondence in amount between these two fractions strongly suggests that the fall in ACh which occurs in the brains that are later frozen is due to the loss of labile ACh. This implies that there is a preferential and almost complete loss of labile ACh in response to such an excessive 0

2

4

I

l

l

7

8

ED Homogenized

lp

l?

14

16

mpmoles AChlg braln 18 2p 2,2

Mt'

5

Homogenlzed (Hypotonic) Frozen 105.000gx60mln -105.000~x60mln

GPC Goc Homogenized (Isotonic -Esr) Homogenized (Hypotonlc)

Mt

1

s

M t ' 22,500gr20min (Lablle portlon) S Supernatant

Frozen

I

Homo enlzed ( PB) Homogenlzed ( PB* PMT) Homogenized (ESR 1

s

J

S PB PMT PBePMT ESR TVF

Pentobarbitone 3 0 m g l kg I p Pentamethylenetetrazole lOOmgl kg I m PMT 3 0 0 m g l k g l p ,subsequently to P B Eserlne l m g l k g l p Tonus-clonus wlth ventro-flexlon 01 the neck

Fig. 8. Compartmentation of acetylcholine in the brain.

neural activity as a convulsion. These findings would indicate that the labile and stable ACh, although defined on the basis of in vitro procedures, have some correspondence with the state of binding of ACh in the brain in situ. There is a possibility that the labile and stable ACh may represent a difference in its state of existence in the particle matrix (Whittaker, 1959), or they may have some morphological significance, i.e. indicate differences in size, degree of maturity and so on of the ACh-containingor binding organelles, which is an additional possibility suggested earlier by Hebb and Whittaker (1958). Rcfcrences p. 129/130

128

M. K U R O K A W A , H. N A R U S E A N D M. K A T O

Other approaches to the ep mouse Histological examination of the brain tissue using the Nissl, haematoxylin-eosin and benzidine-Pickworth staining techniques failed to show defects which could be considered a morphological basis for, or result of, convulsive disorder in ep mouse. Also, electron-microscopic observations failed to discover pathological findings in ep mouse brain; however, extensive studies have not been carried out. Chromosomes of ep mouse examined on skin biopsy 3 days after birth did not show any abnormality in terms of shape, size and number (2n = 44)and showed a good pairing (Inouye and Tsuboi, unpublished). EEG recorded during preconvulsive and convulsive periods of ep mouse showed well-defined seizure patterns, but an attempt to discover abnormalities in the resting animal has been unsuccessful (Utena et al., 1961). Effects on the ep mouse convulsions of unilateral and bilateral destruction or stimulation of the labyrinth have been examined in some animals, with the results not fully conclusive (Yuasa, 1961; Kurokawa, Naruse and Kato, unpublished). Characteristics of behaviour were recorded in mice bred in a specially designed circular runway, and behavioral patterns of the animal were formulated according to the criteria of overall motor activity, the mean and maximal velocity of running, responsiveness as a whole and responsiveness towards individual stimuli such as visual and auditory, and an aberration in behaviour of the ep mice has been described (Utena and Hirao, 1964). SUMMARY

Aspects of brain metabolism in a special strain of convulsive mouse (ep strain) are reviewed. Generalized tonus-clonus was induced in the ep mouse by applying gentle stimulation which caused loss of postural equilibrium of the animal. Factors affecting the seizure threshold are described. Strain differences in the brain constituents so far observed involved raised levels of acetylcholine and of y-aminobutyric acid, and lowered levels of ammonia, glutamic acid, glutamine and aspartic acid in the ep mouse brain in comparison with control strains. In accord with the findings previously reported in chemically or electrically induced convulsions, there was a rise in brain ammonia and fall in creatine phosphate as well as in acetylcholine accompanying ep convulsions. The level of acetylcholine in the ep brain as a whole, was higher by approximately 50 % than in control strains, in determinations with both frozen and decapitated animals. This was practically attributable to an increase in osmotically labile fraction of the particle bound acetylcholine which was found more than doubled in comparison with that of control strains. In contrast, the level of osmotically stable fraction of the particle-bound acetylcholine was fairly constant between strains. Based on the studies on changes in subcellular distribution of acetylcholine both in convulsed and in anaesthetized animals, the inference was drawn that the labile fraction of acetylcholine was responsible for the decrease and increase in acetylcholine accompanying convulsions and anaesthesia respectivelv. The choline acetyltransferase activity in the ep mouse

M E T A B O L I C S T U D I E S O N ep M O U S E

I29

brain was higher by about 50% than in the control mice, which finding appeared to furnish an explanation for the raised level of acetylcholine in situ, although the relationship between the compartmentation of acetylcholine and that of choline acetyltransferase remains to be understood. Acetylcholine formation in the sliced brain tissue was also higher in the ep mouse, in both low and high potassium media. Cholinesterase activity in the cerebral cortex from the non-convulsed ep mouse was slightly enhanced in comparison with the control mice, but this could be reduced to three-fourths by convulsing the animal once a week by postural stimulation. A possibility of metabolic approach to the problem of convulsive predispositon, and an availability of ep mouse in the study of the regulation mechanism of acetylcholine metabolism in brain is indicated. Some morphological, electrophysiological and behavioral approaches to the ep strain are briefly referred to.

REFERENCES

D. P., GIRADO, M., AND WAELSCH, H., (1959); Amino acidmetabolism in epilepBERL,S., PURPURA. togenic and nonepileptogenic lesions of the neocortex (cat). J. Neurochem., 4, 31 1-317. DAWSON,R. M. C., (1953); Cerebral amino acids in fluoroacetate-poisoned, anesthetized and hypoglycemic rats. Biochim. Biophys. A d a , 11, 548-552. ELLIOTT, K. A. C., SWANK, R. L., AND HENDERSON, N., (1950); Effects of anesthetics and convulsants on acetylcholine content of brain. Amer. J. Physiol., 162, 469474. HEBB,C. 0.. AND WHITTAKER, V. P., (1958); Tntracelhlar distribution of acetylcholine and choline acetylase. J . Physioi., 142, 187-196. HIRAYAMA, K., (1963); Metabolism of acid-soluble nucleotides and other phosphorus compounds in the brain with special reference to convulsive activity. Seishin-Shinkeigaku Zasshi (Psychiat. Neurol. jap.,) 65, 842-855. HIRANO, S., AND SHIMIZU, T., (1962); Electrolyte distribution in brain tissues from ep mice, and from mice chronically intoxicated with methamphetamine. Shinkei-Kenkyu No Shinpo (Recent Advan. Res. nerv. Syst.), 6, 651-653. HURLBERT, R. B., SCHMITZ, H., BRUMM, A., AND POTTER,V. R., (1954); Nucleotide metabolism 11. Chromatographic separation of acid-soluble nucleotides. J. biol. Chem., 209, 23-39. IMAIZUMI,K., ITO, S., KUTSUKAKE, G., TAKIZAWA, T., FUJIWARA, K., AND TUTIKAWA, K., (1959); The epilepsy-like abnormalities in a strain of mouse. Jikken-Dobutsu (Bull. exp. Animals), 8 , 6 1 0 . KAMRIN, R. P., AND KAMRIN, A. A., (1961); The effects of pyridoxine antagonists and other convulsive agents on amino acid concentrations of the mouse brain. J . Neurochem., 6, 219-225. KARIYA, T., (1962); Neurochemical studies on convulsions. (Seishin-Shinkeigaku Zasshi) Psychiat. Neurol. jap., 64, 707-720. KATO,M., (1959); Acetylcholine metabolism in brain with reference to a convulsivestrain of mouse. Seishin-Shinkeigaku Zasshi (Psychiat. Neurol. jap.), 61, 1691-1700. KILLAM, K. F., AND BAIN,J. A., (1957); Convulsant hydrazides I. In vifro and in vivo inhibition of vitamin B6 enzymes by convulsant hydrazides. J. Pharmacol. exp. Therap., 119,255-262. KUROKAWA, M., (1963); Acetylcholine metabolism in the ep strain of mouse. Shinkei-Kenkyu No Shinpo (Recent Advan. Res. nerv. Sysf.), 7 , 519-528. KUROKAWA, M., KATO,M., AND MACHIYAMA, Y., (1961); Choline acetylase activity in a convulsive strain of mouse. Biochim. Biophys. Acfa, 50, 385-386. KUROKAWA, M., MACHIYAMA, Y., AND KATO,M., (1963); Distribution of acetylcholine in the brain during various states of activity. J. Neurochem., 10, 341-348. NARUSE, H., (1959); Ammonia and amino acid metabolism in brain with reference to a convulsive strain of mouse. Seishin-ShinkeiEaku Zasshi (Psychiat. Neurol. jap.)., 61, 1701-1710. NARUSE, H., KATO,M., KUROKAWA, M., HABA,R., AND YABE,T., (1960); Metabolic defects in a convulsive strain of mouse. J. Neurochem., 5 , 359-369. NARUSE, H., KUROKAWA, M., AND AKIMOTO, H., (1962); Some metabolic aspects of convulsions. No To Shinkei (Brain and Nerve), 16,463-470.

130

M. K U R O K A W A , H. N A R U S E A N D M. K A T O

KICHTER,D., AND CROSSLAND, J., (1949); Variation in acetylcholine content of the brain with physiological state. Amer. J. Physiol., 159, 247-255. RICHTER, D., AND DAWSON, R. M. C.. (1948); The ammonia and glutamine content of the brain. J. biol. Chem., 176, 1199-1210. D. H., STEIN, W. H., AND MOORE, S., (1958); Chromatography of amino acids on sulSPACKMAN, phonated polystyrene resin, Analyr. Chem., 30, 1185-1 190. C., KLINGMAN, W. O., LACY,0. W., Booos, D.. MARKS, R.,AND COPLINGER, C. B., (1958); SUTER, Sound-induced seizures in animals. Neurology, 8, Suppl. 1, 117-120. TAKAHASHI, T.. (1955); Determination of inorganic phosphate and of creatine phosphate in the tissue. Seikagaku (J.jap. biochem. SOC.),26,690-698. R.,KARIYA.T., KOBAYASHI, K., TORU,M., KOBAYASHI, T., AND NASU, T.,(1962); TAKAHASHI, A study on the recovery process of brain dysfunction induced by repetition of electrical stimulation and by anoxia. Shinkei-Kenkyu No Shinpo (Recent. Advan. Res. nerv. Sysf.),6,637-643. TAKAHASHI, R., NASU,T., TAMURA, T., AND KARIYA. T., (1961); Relationship of ammonia and acetylcholine levels to brain excitability. J. Neurochem., 7, 103-1 12. UTENA.H., AND HIRAO,T., (1964); Formulation of normal and aberrant behavior chains. No To Shinkei (Brain and Nerve), 16. 31-39. UTENA, H., TAKANO, S., YUASA, S., SHIMIZU, T., KATO,T., AND FUNATOCAWA, S., (1961); Behavioral abnormalities in animals and metabolic changes in the brain. 11. Does activity antagonize convulsion? No To Shinkei (Brain and Nerve), 13, 692-695. WHITTAKER, V. P., (1959); The isolation and characterization of acetylcholine containing particles from brain. Biochem. J., 72,696706. YABE,T., (1959); Studies on the convulsive seizures in the ep strain of mouse. Seishin-Shinkeigaku Zasshi (Psychiat. Neurol. jap.), 61, 1683-1690. YUASA, S., (1961); Factors influencing seizure threshold in mice. Correlation between the function and metabolic changes in the ep mouse brain. Seishin-Shikeigaku Zasshi (Psychiar. Neurol. jap.), 63, 1167-1177.

131

Contribution to the Morphological Study of Dendritic Arborization in the Brain Stem HAJIME M A N N E N Anatomical-Physiological Section, Institute of the Deaf, Tokyo Medical and Dental University, Tokyo (Japan)

Since Cajal (1889, 1891, 1892, 1897), it is generally agreed that the axon mainly serves as a conductor of nerve impulses, which may also pass through the soma or the dendrites, that the soma and the dendrites serve as receptors of impulses coming from various sources. Recently, neuroanatomists have become interested in the study of axonal connexions between the gray substances by means of the methods introduced by Glees, Nauta and other authors on the one hand, and in the electron microscopic and histochemical study of the nervous tissue on the other. In contrast, relatively few studies have been carried out concerning the dendrites. The quantitative studies of Sholl (1953, 1955, 1956) on the cortex neuron, Aitken and Bridger (1962) on the motoneuron and Mannen (1965) on the vestibular elements demonstrated that the dendrites constitute more than 80% of the neuronal surface. Consequently, the dendrites are the dominant part of receptive surface of neurons. Indeed, many synaptic contacts can be found on these structures by light and electron microscopy. According to the electron microscopic investigations of Gray (1959a,b,c, 1961), de Lorenzo (1961), Hamlyn (1962) and Blackstad and Flood (1963), functional synapses can be classified into two types: type I which is found mainly on the dendrites and type I1 principally on the soma surface. On the basis of these findings, Eccles (1964) suggested that, from a functional point of view, type I would be excitatory and type 11 inhibitory. In addition to the axo-dendritic contact, there are other modalities of contact between neuronal elements : dendro-dendritic, dendrosomatic, somato-somatic and axo-axonic, though their physiological significance still remains obscure. Estable (1 961). enlarging Cajal’s argument, maintained that the axon would be receptor and conductor in the cone of origin, receptor, conductor and effector in the parasynapses, and effector in the axo-dendritic and axo-somatic synapses. The dendrites would be receptor, conductor and effector in the dendrodendritic synapses and only receptor and conductor in the axo-dendritic and axosomatic synapses. In this sense, not only morphologically but also physiologically, it is very important to make clear the exact dendritic pattern of various parts of the central nervous system and to push forward the quantitative analysis of the individual neuron with special regard to dendritic arborization. Although this kind of study has been carried References p. 160-162

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out by several authors, much remains still to be elucidated, especially at the level of the brain stem. For about 10 years, I have been systematically studying these subjects in human and cat brains, In this paper, the results obtained until now will be reviewed. In order to observe the dendrites, the Golgi method-and its modifications provide, at least at present, the most satisfactory materials. Among these, the Golgi-Cox method is used routinely in this study, because it stains the dendrites more constantly than the others. All materials have been hardened in celloidin and cut in 250 p serial sections. I. ‘ C L O S E D N U C L E U S ’ A N D ‘OPEN N U C L E U S ’

Among the many published papers on the cytoarchitecture of the brain stem in mammals including man, noteworthy are those of Jacobsohn (1909), Winkler and Potter ( I9 I 1,1914), Foix and Nicolesco (1 923, Marburg (1927), Gage1 and Bodechtel(1930), Stern (1936), Riley (1943), Krieg (1953), Meessen and Olszewski (1949) and Olszewski and Baxter (1954). All these results are based on observations obtained with Nissl method which enable us to distinguish different gray matter according t o some properties of the soma, for example form, volume, colorability and population density. The boundary of the gray substance shown by Nissl method is fairly clear, as in the case of Weigert method. The dendrites, however, escape detection by these methods. Therefore, the classification of nerve cells in studies using these methods has been possible only 011 the basis of the morphology of the cell soma. Some attempts have been made to classify the nerve cells on the basis of their dendritic patterns. Cajal (1909, 1911) mentioned that the majority of neurons ramify their dendrites in the interior of their own nucleus, but some of them can step over the boundary of the nucleus as outlined in Nissl- or Weigert-stained preparations. He called these two kinds of dendrites ‘intrafocaux’ and ‘extrafocaux’ respectively. Lorente de N6 (1927, 1933a4, 1947, 1953) analysed the dendritic patterns of various parts of the brain, having special regard for these extrafocal dendrites. The Scheibels, Walberg and Brodal(1956) classified the cells of inferior olivary nucleus, according to the dendritic pattern into two types: one tjpe with a highly ramified, densely packed dendritic arbor, the other with longer and less branching dendrites. They considered the latter to be the more primitive type. In a previous paper (1960), I classified the nerve cells of the brain stem, according to the dendritic arborization, roughly into two types : multipolar and star-shaped. The multipolar cell has generally rectilinear dendrites. The cells with conglomerated dendrites are a modification of this type. They are found in certain nuclei, for example in the external cuneate nucleus of Monakow, the inferior olivary nucleus and the pontine nucleus. The star-shaped cell has ordinarily short dendrites which ramify in the neighbourhood of the soma. These dendrites are more slender than those of the multipolar type and usually do not conglomerate. At the level of the brain stem reticular formation, the star-shaped neurons are found exclusively in certain places, particularly in the precerebellar nuclei. In addition, several similar elements may be observed dispersed in the raphe. The star-shaped cells are also concentrated in the medial or ventromedial half of the

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reticular formation. The multipolar cell has generally extrafocal dendrites while the star-shaped cell does not have them. The neurons with conglomerated dendrites mentioned above correspond to the cells with highly ramified dendrites described by the Scheibels, Walberg and Brodal and the neurons of multipolar type to those with long dendrites. Recently, Ramon-Moliner (1962) also classified nerve cells on the basis of their dendritic patterns. According to Ramon-Moliner, the radiate or generalized type can be considered to be a prototype from which other patterns, wavy, tufted, atypical and intermediate types may be derived. The cell with conglomerated dendrites and the star-shaped cell mentioned above correspond to the ‘tufted or wavy dendritic pattern’ of Ramon-Moliner. Among these various features of dendrites described by several authors, the extrafocal dendrites have to be considered as the most important element from the anatomical and physiological points of view. Recently, accurate stimulation or destruction of the regions which seem to be related with certain functions have been made possible by the progress of neurophysiological technics. The points which respond to the electric stirnulation or destruction are generally indicated in these cases on the simple diagrams of brain sections stained only by Nissl or Weigert methods. As above mentioned, the boundary of gray substances outlined by these methods is fairly clear. However, if the extrafocal dendrites are taken in account such demarcation of the gray substance is not at all certain. Hence, in order to bring the physiological data thus obtained and the histologically established localisation of the lesion into proper correlation, it is necessary to get more precise and systematical information on the extent of dendritic arborization especially concerning extrafocal dendrites. The precise mapping of the dendritic arborization of a whole section has been carried out at various levels of the brain stem as follows. Photomicrographs of a given section were taken with a low power enlargement of microscope (objective 10 x ocular 5 ) successively from one field to the next. They were then juxtaposed according to order to form a panoramic montage. While observing the section with middle or high power enlargement, the course of a given dendrite could be followed precisely and outlined upon tracing paper placed over the montage. Fig. 1 which has been prepared in this way shows well the dendritic pattern of lower level of the pontine tegmentum in cat. As seen clearly in this figure the facial nucleus, which appears in this section only its rostra1 pole, the superior olivary nucleus and the nucleus of trapezoid body show a clearcut boundary. The vestibular and spinal trigeminal nuclei are not so clearly demarcated, because their extrafocal dendrites intermingle with those of neighbowing reticular formation. In reticular formation, it is impossible to observe clearly the demarcation of the medial and lateral parts owing to the intermingling of their dendrites. It is thus possible to classify the gray substances into two groups: nuclei which have the extrafocal dendrites, ‘open nuclei’ and those which do not, ‘closed nuclei’. The dendrites of the open nuclei overstep the limits of the nucleus and penetrate the neighbourinq region. Thus, the actual territory of the nucleus is more extensive than that shown by the Nisslor Weigert methods, whereas most of the open nuclei are invaded by the dendrites of Rrferenrrr p . 160 162

134

H. M A N N E N

Fig. 1 . For legend see next page.

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135

neighbouring gray substances. As shown clearly in Fig. 2, these two gray substances thus cover a common field and overlap each other with their dendrites. On the other hand, the closed nucleus may be invaded by the dendrites of the neighbouring open nucleus while the former do not project their dendrites in the latter. From this point of view, most of the sensitive nuclei of the brain stem, the precerebellar nuclei, the cerebellar nuclei and the magnocellular red nucleus belong to the category of closed nuclei with several exceptions, for example the spinal trigeminal nucleus and the superior colliculus. Lateral reticular nucleus, one of the precerebellar nuclei, belongs to a great extent to the category of closed nuclei, but, at its rostra1 level, to the group of open nuclei. The motor nuclei of the cranial nerves are open nuclei except the facial and motor trigeminal nuclei: these two nuclei are really almost closed although they are open to the spinal trigeminal nucleus. The substantia nigra is also a closed nucleus. Finally, the reticular formation belongs to a vast open system which contains several closed nuclei. Indeed, at this level, it is possible to distinguish several cellular masses of which the dendrites interwine each other with a great complexity, for example the gigantocellular nucleus, the nucleus of raphe and the reticular parvicellular nucleus. Furthermore, the cells of raphe send their dendrites simultaneously into the ipsilateral and contralateral reticular formation. The dendrites of the gray matter often infiltrate into the white substances, for example in

open

open

closed

open

Fig. 2. Diagram showing the interrelationship between two open nuclei and between open and closed nucleus. The dotted regions represent the overlapping parts covered with the dendrites of two neighbouring nuclei. The stimulation or destruction of these regions may influence simultaneously two neighbouring nuclei.

Fig. 1. Dendritic pattern of lower pontine tegmenlum drawn by the tracing of serial photomicrography of a Golgi-Cox-stained section of kitten. cr = restiform body; ctr = trapezoid body; flm = medial longitudinal fascicle; fr = reticular formation; ncov = ventral cochlear nucleus; nctr = nucleus of trapezoid body; nf = facial nucleus; nos = superior olivary nucleus; nsptr = spinal trigeminal nucleus; nvm = medial vestibular nucleus; py = pyramidal tract. Rrferrnrrs p . 160-162

136

H. MANNEN

the pyramidal fascicle, the medial lemniscus, the lateral lemniscus and the medial longitudinal fascicle. In case of a closed nucleus, the localization of a lesion can be marked precisely with Nissl- or Weigert-stained preparations. As shown in Fig. 2, it is not the same for the open nuclei or for the closed nucleus lying adjacent to the open nucleus. When the lesion is localized in the middle of the nucleus, there is no problem. But, if the lesion lies outside the nucleus without any trace of lesion in the nucleus itself but in a territory covered by the extrafocal dendrites, the one who studies only with Nissl or Weigert methods comes probably to the erroneous conclusion that the neurons of the nucleus have not been influenced by the lesion. Similarly, electrical stimulation could influence much more neurons than expected in Nissl- or Weigert-stained materials, since at one point come together many dendrites of which the somas are situated in various distances from this point. Consequently, the stimulation of a common point situated between two open nuclei may have the effect of a simultaneous excitation of both nuclei. From these considerations, it is necessary to consider not only the physical diffusion of the stimulus, which may vary with the type of electrode used and the intensity of current, but also the morphological differences, which can modify considerably the number and the type of neurons excited. Many authors, for example Brodal (l957), the Scheibels (1958), Valverde (1962) and Leontovich and Zhukova (1963), have demonstrated that the reticular formation is not uniform in its morphological organization. At the level of the medulla oblongata, the medial region sends a great deal of fibers in a rostral or caudal direction. Brodal considers the medial region to be the effective part, and the lateral region of the reticular formation to be the sensitive or associative part. However, the limit between these different regions is not always clear and the anatomical data demonstrate us that there are many possible interactions between them. The dendrites of these two regions interwine in such a complicated way that they are quite difficult to demarcate sharply and these two regions overlap in part. In the medial part, the gigantocellular nucleus, the paramedian nucleus, the ventral nucleus of raphe etc. can be distinguished, but in reality these structures overlap each other in some places and their limits are almost impossible to define precisely. According to a calculation of the Scheibels, there are about 41 25 nerve cells in a territory occupied by the soma and the dendrites of only one element of the gigantocellular nucleus. The results of Pitts, Magoun and Ranson (1939) and Pitts (1940) and the others obtained by the stimulation of the medulla oblongata indicate that the medial, rostral and dorsal parts of the gigantocellular nucleus may be considered to be the expiratory center while the parvicellular part can be considered to be the principal center of the inhibition of inspiration. The limit between these two structures is not clear. Furthermore, it is noteworthy that, as shown in the figures of Pitts, the expiratory and inspiratory points are found in the hypoglossal nucleus, the nucleus prepositus hypoglossi, the spinal trigeminal nucleus, the lateral reticular nucleus, the inferior olivary nucleus etc. These structures, except the inferior olivary nucleus, are open nuclei. Moreover, they are penetrated by the dendrites of elements of the reticular

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formation. The inferior olivary nucleus is also invaded in some places by the dendrites of reticular elements, those of cells of lateral reticular nucleus of raphe. Consequently, the effectsof stimulation of these structures would benecessarily complicated by effects due to the excitation of reticular formation itself. Moreover, unilateral stimulation of the region near the raphe has to excite not only homolateral elements, but also heterolateral elements. Thus, excitation can be produced directly through the dendritic commissure described by Van Gehuchten (1897). Experimental anatomical studies may be seriously influenced by theseconsiderations. Selective silver impregnation of synaptic endings by Rasmussen (1957) shows that the soma and the dendrites are covered with a number of boutons terminaux and the methods of Marchi, Glees or Nauta enable us to follow the course of the nervous pathway to its termination, taking advantage of myelin shcath or axon degeneration. Even if such degeneration is found in certain nuclei, it cannot be concluded that the pathway terminates in these nuclei. Actually in case of a nucleus or a territory between two open nuclei, it is possible that the terminal contacts of this pathway occur not only with cells of the nucleus through axo-somatic junctions, but also through axo-dendritic junctions with dendrites of neighbouring elements which penetrate the interior of the nucleus. As for a pathway which terminates near the raphe, it is also possible that the contact may occur with the cells of opposite side by means of the dendritic commissures. Compact white substances contain many dendrites issuing from the neighbouring cells. Therefore, the stimulation of such white substance may produce not only excitation of their fibers but also those of neighbouring nerve cells, an etrect which is analogous to that of the direct stimulation of the gray substance. Finally, the different modality of dendritic arborization may be considered. Our data suggest that the extent of arborization and the degree of overlapping of gray substances may vary considerably. The results of stimulation may be also variable in different animals even when the stimulations werecarried out on just the same point under the same conditions due to morphological and individual differences of dendritic arborization microscopic order. Consequently, I am perfectly of the same opinion with Lorente de NO (1933d) when he wrote on the subject of the cytoarchitectonic study of the Ammon’s horn: ‘The authors who are not accustomed to work with the silver or mercury chromate methods of Golgi, will perhaps think that the foregoing division of the Ammon’s horn cells is artificial and too extreme. The opposite would be true. The above classification is still too incomplete. In spite of the efforts of Golgi, Sala, Schaffer, Lugaro, Kolliker, and chiefly of Cajal, and of my modest work, the present description of the Ammon’s horn cannot be considered more than a preliminary communication. Every one of the cell types mentioned here is perfectly justified. When two cells have similar axonal apparatus, but dendrites which are distributed differently, they belong to two different types, because they receive different impulses. When two cells have similar dendrites, but different ramified axons, they again belong to different types, because they transmit their impulses in a different manner. All of the types described here have different dendrites and different axons’. It is exactly the same at the level of the brain stem. Rrferenccr p. 160-162

138

H. MANNEN

11. MEASUREMENT O F NEURON SIZE WITH SPECIAL REGARD TO

DENDRI'rIC

ARBORIZATION

Since the soma and the dendrites constitute the receptive surface of the neuron, it is quite important to know the actual volume and surface area. However, the quantitative study of brain stem nerve elements has not yet been published, although the cortex neurons and the motoneurons have been frequently examined, for example by Bok (1959), Sholl(l953,1955,1956), Fox and Barnard (1957), Ramon-Moliner (1961), Aitken and Bridger (1962).

Fig. 3. Example of a cell of the magnocellular nucleus (cell 1 in Table 11) drawn on a serial photo for the quantitative analysis. The length and the thickness (in circle) of dendritic branches are indicated. The concentric circles separated at the interval of 50 ,u are traced round the cell body. The arrows indicate the cut ends of dendrites connected by means of a new method described in Section 111 of this paper.

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139

Parallel t o the topoqraphical study of dendritic pattern described in Section I of this paper, I have carried out a quantitative study of individual neurons of the brain stem. About two hundred cells of the vestibular nuclei and the bulbar and pontine reticular formation in kitten have been examined; in both regions, the results were almost the same. As the results obtained in vestibular elements have been already published, the results obtained chiefly in reticular formation cells will be related here. For the quantitative study of individual nerve cell photomicrographic montage was also prepared for each cell, as done in the topographical study of dendritic pattern, but in this case with high power magnification (objective 20 x ocular lo), in order to distinguish the details as much as possible. The tracing of the course of each dendrite was carried out, the photomicrographic montage being covered with a tracing paper. The result for a given cell was in outline projected on a plane (Fig. 3). Then the number, the length and the diameter of each branch was measured for calculation of various parameters which will be related later. All these measurements have been carried out with Golgi-Cox-stained serial sections 250 p thick. The mean values of various components of the vestibular elements and the reticular formation cells are summarized in Table I. All of the cells which are measured in the reticular formation belong to the multipolar cell type. The star-shaped cells which are found in the ventral nucleus of raphe, precerebellar nucleus etc. were ruled out, because they are not considered as proper reticular elements. The neurons of the vestibular nuclei and the reticular formation have been classified in three groups according to the projected area of the neuron soma alone: large, middle and small, as done generally in Nissl-stained preparations. The large cell has a soma with a projccted area of more than 3000 p2, the middle-sized cell of from 1000 to 2000 p2 and the small of less than 1000 p2. ( a ) Number of principal dendrites and number of dendritic branches There are on the average 9 principal dendrites for large cell, 6 for middle-sized cell and 5 for small cell. The principal dendrites ramify generally five times and at irregular intervals. The ramifications are principally dichotomies. The diameter of principal dendrites is very variable, at maximum of 22 p. The dendrites are divided in the majority of cases into branches of equal diameter. The surface of principal dendrites is generally smooth whereas they have the spines after the first branching. The number of dendritic branches including the principal dendrites have been calculated. In the reticular formation, the large cell has on the average 61 dendritic branches, the middle-sized cell 31 and the small cell 20. The numbers are almost the same in the vestibular nuclei. As shown in Fig. 4a, there is a simple relationship between the dimension of the soma and the number of dendritic branches in the reticular formation cells and in the vestibular elements. (b) Letigih of dendrites The length which is obtained by means of tracing photomicrographic montage is an apparent length projected on a plane. The actual length of dendrites could be calculated, taking into account the apparent length and the difference of depth in the sections Rrferences p. 160-162

H. M A N N E N

140

....

80

:8000-

c .-

' . .'

91

0

5Ol

-m-

6000-

E 4000. -

+ m 3000-

2o

I-0

t

2000.

1000

3000

Projected

5000

7000

. . ' . . .. ... ... . . . .

..

,:b

'

. ...... *.:

v

1000

area of the soma(/*)

5000

3000

7000

Projected area of the soma&*)

(a)

(b)

.

. ,. - .. ... - .. *:

.*

*

5.;

,

Projected area 01 the (C)

somay

:-.* ..:. . ,.a

Projected area of the

somap')

(d)

Fig. 4. Correlation diagrams for the relationship between some characteristicsof dendritic branches and the dimension of soma. (a) number of dendriticbranches; (b) total length of dendrites; (c) surface area and volume of dendrites; (d) the longest dendrite. s = small cell; m = middle-sized cell; 1 = large cell.

between two extremities of each dendritic branch. However, the length of several dendrites, sectioned by the microtome, could not be measured precisely and the total length given here was the minimal value. A more precise estimation of total length will be discussed in the next section of this paper. It seems likely that there may be no parallelism between the dimension of the soma and the length of the dendrites in the reticular formation cells as well as in the vestibular cells. Even if large cells very frequently have long dendrites, middle-sized or small cells can also possess long and thick dendrites. Besides, the longest dendrites

D E N D R I T I C A R B O R I Z A T I O N I N THE B R A I N STEM

141

recorded in this study in case of reticular elements was 855 p for large cell, 881 p for middle-sized and 492 p for the small cell. The average lengths of dendrites for these three groups of neurons were respectively 615, 558 and 436 p. As shown in Fig. 4d, it seems likely that there is no clear correlation between the dendrite length and the projected area of the soma. On the other hand, there is an obvious correlation between the dimension of the soma and the total length of the dendrites (Fig. 4b). The largest value of the total length of dendrites recorded was over 9010 p for the large cell, 5760 p for the middlesized and 2790 p for the small cell. As seen in Table I, the averages of the length of dendrites as well as their total length were remarkably larger in the reticular elements than in the vestibular elements. ( c ) Surface and volume of dendrites The surface and the volume of dendrites were calculated after having measured precisely the diameter (D) and the real length of each branch (L), considering them as smooth surfaced cylinder and neglecting the spines. The formulae for calculating the surface and the volume are respectively 7c

D L and

3G

- D2L

4

As shown in Fig. 4c, the surface and the volume of dendrites become larger in

proportion to the dimension of the soma. The largest values of surface and volume attain respectively 68,600 p2 and 78,700 p3 for the large cell, 37,900 p2 and 35,700 p3 for the middle-sized and 12,590 p2 and 7300 p3 for the small cell. It is evident that, at the bulbar and pontine reticular formation as well as at the level of the vestibular nuclei, the larger the nerve cell soma, the greater the dendritic surface and volume. The dispersion of the surface and volume of dendrites in different distances from the soma was calculated, using concentric circles at the interval of 50 p as shown in Fig. 3. The histograms of these data show more than 90% of the surface and volume in the circle of 600 p of diameter whatever the cell size (Fig. 5). I have a impression that the dendrites of the vestibular nuclei concentrate in the neighbourhood of soma more than those of the reticular formation. ( d ) Surface and volume of the soma The measurement of the surface and the volume of the soma seems to be one of the most difficult questions of neuroanatomy, because the soma of nerve cell has a very irregular form. Therefore, until now, one was obliged in almost all cases t o calculate their values approximately from measurement of diameters. In this study, three diameters were estimated : two transverse, minimum (A) and maximum (C) and a third, perpendicular to the plane of section, depth of which was evaluated from the difference of level of the microscopic focus. In the majority of cases, the value of minimum transverse diameter is close to the depth of the soma. When the latter exceeds the former considerably, an average was taken. The soma was considered an Rrfcrcncrs p. 140-162

H. M A N N E N

I42

mt

62.7 44.7

Large cell

60 40 20

100

200

300

400

600

500

700

Distance from t h e soma (A)

-5

Middle-sized cell

I 83

(A)

Distance from t h e soma

t

678

573

Srriall cell

60 40 20 10

05 100

200

300

400

600

500

700

(p)

Distance f r o m the soma

Fig. 5. Histograms showing the distribution of surface area and the volume of dendrites in terms of their distance from the soma.

ellipsoid or a sphere when the three diameters are almost equal. The surface area and volume were calculated from the formulae 2 n (a2

ac2 +4 -

)

a arc cos C

and 4 -

3

n a2c respectively

c

=

C -, A < C 2

2

B

TABLE I

2 I . ,

A V E R A G E OF V A L U E S OF V A R I O U S C H A R A C T E R I S T I C S I N D I F F E R E N T C E L L T Y P E S OF T H E P O N T I N E A N D B U L B A R R E T I C U L A R F O R M A T I O N A N D VESTIBULAR NUCLEI IN KITTEN

Numbers of cells used in each structure indicated in parentheses. Large cell ( ~ 3 0 0 0} I 2 ) r . f.

(28)

vest. nuclei ( 26)

Middle-sized cell (3000-2000 r. f . (37)

}I?)

vest. nuclei (32)

Small cell

(

1000 ~

S

J

r . f.

vest. nuclei

(23)

(27)

W

0

Projected surface of soma (y2) Number of principal dendrites Number of dendritic branches Total length of dendrites ( 1 6 ) The longest dendrite 01) Surface of dendrites (Sd) (pa) Spiny surface (Ssp) (y2) Volume of dendrites (Vd) (pa) Surface of soma (Ss) (112) Volume of soma (Vs) ( p 3 ) Sd/% VdfVs SspfSsm (Ssm smooth surface)

4000*730 9 f 2 61 f 15 6550 f 1200 615 + 96 42,000 -f 9800 32,920 + 8710 36,700 f 13,800 15,200 f 3270 163,480 f 51.000 2.8 $. 0.7 0.2 0.1 1.4 f 0.4

+

4246& 1100 9 f 3 48 5 18 4240 f 1120 404 -f 74 31,860 & 10,400 22,190 & 7590 31,250 -f 15,700 15,300 & 3600 161,800 & 74,660 2.1 & 0.6 0.2 i 0.2 0.9 -f 0.2

176Of 360 6 1 2 35 f 16 4 0 5 0 i 1600 558 $. 150 23,100 i 8300 19,400 ;t. 6570 16,600 i 8500 6630 ir 1830 47,700 -i 20,650 3.8 i 0.8 0.4 It 0.1 2.0 f 0.5

770 i 121 1870 i 610 5 i 2 7 1 2 20 & 3 37 + 14 2970 i 951 2100 & 780 436 & 120 385 f 103 10,060 f 4070 20,120 f 7950 8660 f 3520 14,740 f 5980 15,490 i 8800 5800 & 3750 3000 i 620 6650 i 2360 46,770 24,600 13,500 5 5550 3.4 f 1.1 3.1 i 0.9 0.4 0.2 0.4 f 0.1 1.9 & 0.6 1.2 i 0.3

+

+

621 i- 240 5 k 2 16 i 6 1370 i 580 317 i 147 6800 i 3960 5330 i- 3070 3530 i 2740 2100 i 960 8490 i 5550 3.1 f 1.0 0.4 40.3 1.6 & 0.7

H. M A N N E N

144

The maximal values of the surface arid volume of the soma obtained on the reticular elements were respectively 24,500 pz and 293,500 p3 for the large cell, 10,400 ,u2and 95,100 p3 for the middle-sized and 5000 p2 and 33,500 pa for the small cell. These values greatly exceed those obtained in the vestibular nuclei. However, the average of the surface area and volume for corresponding cell sizes are approximately equal in both structures. As shown in Table I, the surface area of the dendrites is almost three times as large as that of the soma (Sd/Ss) for the large and small cells and three and a half times for the middle-sized cell. There is no great difference in the proportion between the volume of dendrites and that of soma (Vd~Vs)for three kinds of cells. These results are almost the same as those obtained in vestibular elements. The majority of dendrites ischaracterised,in Golgi-stained materials, by the presence o f spines. However, the principal dendrites as well as the soma lack these spines. Thus, it is possible to divide the surface of neurons into two parts: smooth surface and spiny surface. They have been measured separately and the ratio between them calculated. As shown in Fig. 6 and Table I, this ratio is quite variable for the small

40

-

3.0

-

.

e

"*******

2J3-

a**

* * 1.0

.

- 0 .

-

0

. .*

** .*

*.

.

a 8

0

* * a

(S)

.

0 .

.

(MI I

1

.

(L) I

1

I

I

Projected area of the soma g21

Fig. 6. Correlation between the dimension of the soma and the relation between the spiny surface (Ssp) and the smooth surface (Ssrn).

cells. In the vestibular nuclei, in cells with larger projected area, the spiny surface tends to equal the smooth surface while, in the reticular formation, the spiny surface exceeds also the smooth surface even for the middle-sized and large cells. I t is noteworthy here that, according to Eccles, the synapse type I, found mainly on the dendrites, would be excitatory while the synapse type 11, found chiefly on the soma, inhibitory. In any way, much more analytical works must be carried out in order to confirm this conception. Aitken and Bridger (1962) demonstrated that, at the level of the anterior horn in

D E N D R l T l C A R B O R I Z A T I O N I N T H E B R A I N STEM

I45

cat, dendrites constitute about 80% of the neuronal surface, a value close to ours. However, these authors reported that the largest dendritic surface area was 76,270 ,u2 and about half of the receptive surface of dendrites can be found over 300 ,u of distance from the soma, which is quite different from our findings. This may be due to the difference of the nervous structure examined or of the preparations used. Aitken and Bridger performed the experiments on adult cats whereas 1 used kittens. I l l . N E W M E T H O D FOR M E A S U R I N G O F T H E V O L U M E A N D S U R F A C E A R E A O F T H E N E U R O N S O M A A N D FOR F O L L O W I N G U P T H E T O T A L D E N D R I T I C C O U R S E IN SUCCESSIVE S E R I A L S E C T I O N S

I have previously pointed out that the measurement of the actual value of the surface and the volume of the neuron in toto including the dendrites is one of the most difficult questions for a neuroanatomist. I t could be said that the values obtained until now by various methods are in almost all cases rough approximations. Concerning the soma the reconstruction method appears to be the most appropriate for determining the true shape of the soma and its actual dimensions. This method was previously employed by several investigators, for example Lhermitte and Kraus (1925). Kraus and Weil (1926), Weil (1927), Haggar and Barr (1950), Micklewright et a/. (1953). Recently, SchadC and Van Harreveld (1961) studied the motoneurons and Gihr (1962, 1963, 1964) concentrated on cortical and thalamic neurons. In these cases the reconstructions were made principally by means of Nissl-stained serial sections and in some cases by means of reduced silver methods. However, as the reconstruction method is really laborious, the estimation of the soma size has been based principally on measurements ot the major and minor diameters of the soma in the optical section through the nucleolus, with no consideration of the irregular shape of soma, leaving the decision as to which diameters should be chosen to the observer. Therefore, the values thus calculated may be quite variable. On the other hand, a number of equations were proposed for determining the soma volume from measurements of axis thus obtained. Using as a basis the actual values which are obtained by the reconstruction method with Nissl-stained materials, Schade and Van Harreveld (1961) tested a number of equations in search of a satisfactory way of determining the cell body volume of neurons in such a way and the equation 1.04 x 1/6 n ab d a b gave the best approximation (a and b are major and minor axis). The measurement of axis is, however, afTected by the thickness of sections. According to Treff (1963), the smaller the cell and the thicker the section, the more accurate the measurement of the mean diameter. I f the size of the cell is large in relation to the thickness of the section and the cell is irregular in shape, the measured size will be smaller than the real size. Gihr (1963, 1964) demonstrated by means of the three dimensional reconstruction with Nissl-stained serial sections that the true shape of the cell cannot be established from its contour in one section alone. Furthermore, the shrinkage factor is considerably larger in Nissl-stained materials in comparison to other methods. According to Ramon-Moliner (1961), the linear shrinkage factor in Rejerencwr p . 160-162

146

H. M A N N E N

Nissl-stained paraffin sections is 0.645, whereas the factors for the Golgi-Cox and original Golgi-stained celloidin sections are 0.845 and 0.809 respectively. Unfortunately, with Nissl-stained materials, it is impossible to view the soma and its dendrites it? toto. In this means, the Golgi method provided at present the most satisfactory material available for the quantitative study of individual neurons. Compared with other staining methods it has the advantage of simultaneously defining the neuron soma and its dendrites in the same section. For this reason, the quantitative details of the dendrites published by various authors, for example Bok (1959), Van der Loos (1960), Sholl (1933, 1955, 1956) and Ramon-Moliner (1961) in the cerebral cortex, Aitken and Bridger (1962) in the anterior horn cell, Fox and Barnard (1957) in Purkinje cell and present author (1965) in the vestibular nuclei, have been based essentially on the measurements in Golgi-stained materials. In these cases, the measurements are carried out in one section alone containing the cell soma. The results thus obtained show the m;nimal value of dendrites, since some of them owing to their vast spread are inevitably cut short by the microtome. Therefore, the measurement of the dendrites is greatly affected by the thickness of sections used for this purpose. It can be easily assumed that the thinner a section used for such measurement, the more rapidly the percentage of these escaping dendrites increases and that the data would vary considerably from case to case. As far as I know, no attempt has been carried out to follow the entire dendrite in Golgi-stained serial sections. By means of the present cytoarchitectonic analysis of the brain stem in kitten with the Golgi-Cox method, it has been found that a combination of reflecting illumination shows a clear relief of the soma surface and its relationship with the dendrites. With this method of illumination, it is possible to overcome by two above-mentioned obstacles and to follow the total course of dendrites which have been cut short by the serial sections. In addition, by using a method for the photogrammetrical representation of the neuron soma it is possible to obtain a good approximate value, no matter how irregular the shape may be. Through these procedures, the total neuron size including the dendrites can be accurately estimated. In order to achieve this aim, it was necessary to modify some minor details of histological technics and to prepare some suitable apparatus. As will be described later, it was also necessary to invert the sections in order to observe both sides. Thin coverslips were used for mounting the serial sections, instead of ordinary slides. The section thus mounted is covered with Canada-balsam and put in a special frame which prevents the balsam from touching the microscope stage when the section is turned over. A special illuminator was prepared to provide reflecting illumination (Fig. 7). I t consists of an ordinary microscope lamp and a microscopic condensor to which a glass rod, about 10 cm long, was fitted, its tip being just at the focus of the condensor. A great advantage of this apparatus is that it supplies a light intensity sufficient for high power observation without any risk of overheating the specimen. The sections used for this study have been the same which are used for the topographical and quantitative study already described in the previous two chapters; their thickness is of 250 p. The neuronal elements stained by Golgi-Cox method are

I47

D E N D R l T l C A K R O R I Z A T I O N I N T H E B R A I N STEM

E

b Fig. 7. Photograph and diagrammatic sketch of the apparatus for microscopic stereophotography a microscopic lamp; b microscopic condensor; c glass rod; d = camera; e = microscope; f long focus lens; g specimen; h mechanical stage; i seesaw-like inclining stage; j = substage lamp. ~

~

seen, by means of ordinary substage illumination, only in silhouette against a clear background (Fig. 8 4 . Therefore, it is impossible to observe directly the real profile of the soma and the spatial interrelationship of the soma and the dendrites, especially when the dendritic branches issuing from the soma are hidden by its shadow. In such a case, they cannot be easily distinguished from those issuing from the other neurons and passing by the soma only accidentally. If these branches are mistakenly grouped with those issuing from the soma, there is the risk of miscalculation. However, a combination of the reflecting illumination and ordinary substage illumination shows these features obviously (Fig. 8b). In this case, the nerve cell soma and the dendrites are seen in three dimensicnal vision, revealing clearly their spatial relationship. They appear as a brilliant silvery or sometimes a glossy brown, depending on the light intensity, due to the reflection of light by the granules of mercury plating the cell surface. As shown in Fig. 9a and b the relief of the nerve cell soma is very irregular and varies from cell to cell. They may roughly correspond to simple geometrical figures such as spheres, ovals, cylinders or discs. But sometimes this is not the case, A hollow, protuberance or cliff can be seen on the surface of soma. There are also cases where one side of the soma is very flat while the other side is considerably swollen. The interrelationship of dendritic branches between them or with the soma, even when they protrude from the soma perpendicularly to the plane of the section and are normally hidden by the shadow of the soma, can be seen very clearly with this method of illumination. The dendro-dendritic or dendro-somatic contacts, the issue of the axon or dendrites from the soma and the dendritic spines appear much clearer with this method of illumination than with ordinary substage illumination. Rtfcvnccs p . 160-162

148

H. M A N N E N

Fig. 8. Photomicrographs of a large cell of the magnocellular nucleus taken with the ordinary substage illumination (a) and with the combined illumination (b): (a) shows only its silhouette whereas (b) reveals the real profile of soma and the spatial interrelationship of the soma and the dendrites. Kitten, Golgi-Cox stain.

D E N D R I T I C A R B O R I Z A T I O N I N T H E B R A I N STEM

149

Fig. 9. Photomicrographs of two cells of the pontine reticular formation taken with the combined illumination. Both cells have very irregular shape. Kitten, Golgi-Cox stain. Hrferences p. 160-162

H. M A N N E N

150

a

I

Fig. 10. Diagrammatic sketch showing the procedure of following up the dendritic course in serial sectioned sections. (a) a cell with a vast spread of dendritic arborization sectioned by microtome in several sections. (b) plotting of cut ends of dendrites on ruled tracing papers by means of serial photos of confronting planes of neighbouring sections.

(a) A method-forfollowing up the total dendritic course in successive section As shown diagrammatically in Fig. 10a, the Golgi-stained neuron is usually cut into several sections by the microtome due to the remarkable spread of its dendrites. As mentioned above, the estimation of dendrites was hitherto limited to those appearing in the plane containing the soma, since there were no methods for following the dendritic branches spread over successive serial sections. Now, if the confronting planes of successive serial sections are brought face to face in as precise a topographical relationship as they were before the microtome cutting, it is then possible to reunite the cut ends of the dendrites with each other and reconstruct the dendritic arborization in whole. It was found that for this purpose reflecting illumination is quite favorable for visualizing clearly the cut ends on the surface of the section.

D E N D R I T I C A R B O R I Z A T I O N IN T H E B R A I N STEM

151

Under reflecting illumination the following procedures were carried out : the serial photomicrographs of one entire section were made under low-power (10 x 5 ) and successively from one field to the other while focusing on the surface of the section. They were then juxtaposed in order. The photos thus obtained give a general pattern of cut ends in this side of section where they look like stars in the night-sky (Fig. I I). Serial photos of the confronting side of the neighbouring section were made in the same way. The photos were covered with ruled tracing papers. I t is then possible to plot the positions of the cut ends of dendrites of these two sections (Fig. lob). As shown in Fig. 1 I , the cut ends in the two sections correspond exactly. By repetition of this procedure it is possible, though quite laborious, to trace the total spread of dendrites of an individual neuron in successive serial sections and to measure their dimensions in the same manner described in the previous chapter. The summing up of the volume and surface area of soma and dendrites obtained in the section containing the soma and those of dendrites followed up in serial sections give us their total values of individual neuron. With this method, the measurement of length, surface area and volume of dendrites in foto has been carried out, until now, on 5 large cells of the magnocellular nucleus of the pontine and bulbar reticular formation. Though the results thus obtained will be published in full later, they are shown in Table I in this paper. The mean values of the total length, total area and total volume are respectively 9200 p, 66,400 p2 and 70,060 p3. The ratio of the dendrites which are contained in the plane of the soma to the whole of them varies considerably from cell to cell in case of 250 p serial sections thick: 55-93 % depending upon their length. In other words, an average of 25% of the dendrites run off into neighbouring sections. According to the Scheibels, the majority of the large reticular cores extend their dendrites perpendicularly to the axe of the brain stem. In such a case, for example cell 2, 4 and 5, more than 80% of the volume and surface area can be contained in a section. However, when the dendrites radiate in all directions, for example cell 1 and 3, the values may be variable, depending on the thickness or the direction of the cutting section used for the measurement.

(6) A pliotogrammetric represenfalion of nerve cell soma A pair of st~reophotogramsare required for photogrammetric analysis. Contrary to the ordinary photogrammetry, there are certain difficulties in this case, principally due to the character of microscopic lense of which the angle of view and the depth of focus are remarkably limited. As the camera is fixed in microscopic photography, contrary to the common photography, it is necessary to slide the object. But, if it is located in order to obtain the parallax necessary for the stereography, the object escapes the view-field. For this reason, the convergent stereographic method was found to be most appropriate for our purposes. A seesaw-like inclining stage (Fig. 7) was constructed and fitted in place of the ordinary microscope stage. This stage could be inclined 6 degrees to the left or right of the vertical axis of the lens. For extending the focus depth a long focus lens (Leitz Um 32/0.30) was used as the objective instead of the ordinary lens with an HKW 10 x eyepiece (Nikon). Reference.? p. 160-162

152

H. M A N N E N

Fig. 1 1 . Photomicrographs of two confronting planes of neighbouring sections showing the exact correspondence of each cut end of dendrites (with arrows). A and B = Panoramic montage. a and b = Enlargement of small sections from A and B.

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The stereophotography has been carried out as follows. After inclining the stage to one side at the prescribed angle of 6 degrees by a bar, the specimen is rigidly mounted on the mechanical stage attached to the inclining stage. The nerve cell to be examined is mounted in the center of the field with a certain point of the center of the view finder and then photographed under the combined illumination method. Subsequently, the specimen is replaced with a specially designed stage micrometer in order to calibrate the photograph by means of a double exposure. The calibration marks are ruled on a silver-plated glass, as shown in Fig. 12a and b. Each side of the square is 150 u , long and its middle portion is divided into units

C Fig. 12. Contour lines (c) drawn on a large neuron soma (cell 3) in the pontine reticular formation of a kitten with a pair of stereophotograms (one side) (a, b). The broken lines show the limits between the soma and the dendrites. Refercwes p . 160-162

H. M A N N E N

154

(one division 10 p). The center of this square is adjusted upon the cross of the view finder. This is done in order to fix precisely the spatial relationship between the image of the already exposed nerve cell and that of the micrometer. Next, after turning the stage down to the other side, the same operations are carried out. In this way, a pair of stereophotograms of a nerve cell viewed from one side can be obtained (Fig. 12c). Then the specimen is turned over to get an equivalent pair of stereophotographs of the opposite of soma surface in the same manner (Fig. 13c). Wild’s stereophotograph A 7 was used in order to draw the contour lines on the soma surface with these pairs of photos. A piece of thin tin foil is used as a scale for depth. Its thickness was determined with Sip’s universal measuring microscope (27.8 p

C Fig. 13. Contour lines (c) of the opposite side of the same soma with a pair of stereophotograms(a, b). The results of measurement of this cell (cell 3) are shown in Table 11.

D E N D R l T l C A R B O R l Z A T l O N I N T H E B R A I N STEM

I55

in this case). It was then covered with Canada-balsam for the sake of uniformity with the histological sections with respect to refraction, and then photographed under the same conditions as the neuron. With the stereophotograms thus obtained, it is possible to regulate the depth for photogrammetry. The contour lines of both sides of the soma are seen in Figs. 12 and 13c. The contour interval in thiscase is2.5 p. Certain objects found outside of the soma, for example sediment granules, axons which are passing by, were choosen and their levels taken photogrammetrically from the both sides in order to determine the contact plane of both sides of the cell soma. The contact plane of both sides corresponds to the plane of level 0 in these cases. The measurements of the volume and the surface area of the cell body were carried out according to following formulae thought to give the best approximative values. It is impossible to determine precisely the places where the dendrites join the soma; the limits selected in these cases as shown by the broken lines in Figs. 12-14. The formula used to calculate the volume is C A x H where A is the area surrounded by a contour line, and H is the contour interval. These areas are measured with a planimeter. The values of the total soma volume thus calculated are shown in Table I I . The formula for calculating the surface area is

x

LI

+ L2 2

where LI is the length of a contour line measured by means of a curvimeter; L2 is the length of a curve drawn parallel to the former at the distance po, which is the mean value of the measured distances between two neighbouring contour lines; and el is the length of the slope calculated from p,, and the contour interval. The values thus obtained are also shown in Table 11. Using the data thus obtained it is possible to compare the volume and the area of the soma with those values for the dendrites, which were obtained by the method mentioned above. The ratioofthedendritic volume to the soma volume and the ratio of the dendritic area to the soma area are seen on average in these cases, 5.6 and 0.6 respectively. It can be seen from these results that the dendritic area may form more than 80% of the total neuron area, greater than the value shown in the previous chapter which has been calculated in one section alone. Now, it is of interest to compare the volumes thus obtained with those calculated by other formulas. According to SchadC and Van Harreveld (1961), the equation I .04 x 1/6 n ab t/ ab (a non-rotational ellipsoid in which the third axis is u/ ab) gave the best approximative value. The simple application of this formula to our cases gives the values shown in Table 11. These are in all cases quite larger than the values estimated by means of photogrammetry. Such divergence is a result of the difference in determining the major and minor axis in these cases. In Nissl-stained sections the major and minor axis through the nucleus of the soma are used in the equation cited above. However, as already mentioned, it is impossible, in case of Golgi-Coxstained material, to see through the interior of the soma due to the plating of the surface. For this reason one is obliged to choose as major and minor axis the maxRrB~rc.nci*Pp. 160- 162

H. M A N N E N

156

.-

Y \

Fig. 14. Contour lines of both sides of the cells I , 2, 4 and 5 shown in Table 11.

T A B L E I1

-

VALUES O F THE V A R I O U S CHARACTERISTICS O F T H E SOMA A N D D E N D R I T E S O B T A I N E D B Y A N E W METHOD I N COMPARISON W I T H THOSE O B T A I N E D A C C O R D I N G TO THE OTHER FORMULAE

c

The photogrammetrical representation of these cells are shown in Figs. 12, 13 and 14.

G,

p

0 A

h "8

I

Cell

4

5

Means

2

3

10,600 69,700 61,300

10,100 74,500 76,400

7100 61,400 73,600

8500 69,100 76,700

9200 66,640 70,060

8200

5600

6600

7500

6780

Total length of dendrites (Dtl) (p) Total area of dendrites (Dta) (p?) Total volume of dendrites (Dtv)( p 3 ) Length of dendrites included in the section which contains the soma (D1) (p) Area of dendrites included in the section which contains the soma @a) (p2) Volume of dendrites included in the section which contains the soma (D,) ( p 3 ) Di/Dti (%) Da/Dta (%) Dv/Dtv (%) Area of soma surface obtained by photogrammetry (Sa) (p2) Volume of soma obtained by photogrammetry (S,) ( p 3 ) Total surface area Sa Dta ( p 2 ) Total volume Sv Dtv ( p 3 ) DdSa DtvlSv

51,300

57,900

52,800

59,300

65,900

57,440

49,700 62 75 80 12,300 105,000 80,800 167,300 5.6 0.6

54,200 77 83 88 12,200 95,800 81,900 157,100 5.7 0.6

59,500 55 70 78 12,900 102,300 87,400 178,700 5.8 0.7

64,500 93 96 87 15,400 112,000 76,800 185,600 4.0 0.7

75,300 88 95 98 15,100 134,000 84,200 210,700 4.6 0.6

60,640 75 84 86 13,580 109,820 80,220 179,880 5.1 0.6

Major diameter (a) and minor diameter (b) Soma volume calculated according to f x2z (x = b/2, z Soma volume calculated according to 1.04 x 1/6 IC a b

90 x 55 142,430 188,000

95 x 72 150,470 308,000

82 x 52 116,100 152,000

124 x 98 265,380 729,000

110 x 66 250,360 337,000

184,948 342,800

+

+

=

a/2) ab

9700 68,500 62,300

a

rn

2

m

0 *r

N

m

!a

rn

r

158 H. M A N N E N

Fig. 15. Reconstruction model of cell 3 constructed according to the contour lines. (a) side view; (b) lateral view; (c) view of opposite side.

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imal diameter of the soma and the minimal diameter which is perpendicular to the former without any consideration of the disposition of nucleolus. The values thus calibrated are in almost all cases larger than those measured through the nucleolus. In our cases, as clearly shown in Table 11, the volume and area of the soma, considered to approximate a rotatory ellipsoid, are nearer to the values obtained by photogrammetry. In comparing the values obtained according to the formulas mentioned above with those obtained by means of photogrammetry, it seems clear that in former case they vary considerably from case to case whereas in latter case they concentrate within a narrow range. As concerns the various characteristics of dendrites, their values obtained by means of the method described in this paper are also very close to each other. Consequently, as far as our results show, it may be assumed that the cells which belong to the same type in certain region of the brain may have nearly the same volume and the same area, no matter how irregular their shape may be. The reconstruction models of entire soma were constructed by following the contour lines of the soma surface by stereoautography (Fig. 15). Thus, it is possible to observe the neuron soma from all directions. SUMMARY

This paper concerns the topographical and quantitative analysis of the dendritic pattern of the brain stem. Section I deals with the classification of the gray substance: ‘open nucleus’ which has extrafocal dendrites and ‘closed nucleus’ which does not have them. Even the closed nucleus may be frequently invaded by the dendrites of neighbouring cells. Concerning the open nucleus, the extent of territory covered by the totality of extrafocal and intrafocal dendrites is larger than in Nissl- or Weigert-stained preparations. An intensive exchange of dendrites is found between two neighbouring open nuclei ; they have, therefore, a common ground and overlap each other. It is thus possible to obtain, by the stimulation of one cf them, a simultaneous response of both structures. Consequently, the effect of stimulation might be much more complicated than imagined. Besides, the cells which are found near the raphe send their dendrites to the other side; the stimulation or destruction of one side can provoke, therefore, the immediate excitation cf the other side by means of dendritic commissures. Consequently, the notions ‘closed nucleus’ and ‘open nucleus’ as well as ‘partial overlapping of gray substance’ would be important not only from the morphological but also from the physiological standpoint. In section I1 the quantitative analysis of dendritic arborization of reticular formation cells has been compared with those of vestibular nuclei. The number of dendritic branches, the total length of dendrites as well as the surface area and the volume of dendrites are nearly proportional to the dimension of neuron soma. The surface area of dendrites is about two or three times greater than that of soma. However, in contrast the volume of the dendrites is about the third of that of the soma. More than 90% of the surface and of the volume of dendrites are distributed within 300 p References p. 160-162

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H. M A N N E N

from the soma. The spiny surface and the smooth surface are almost equal in vestibular elements except in small cells while the spiny surface is larger than the smooth surface in reticular formation cells in all cell types. Section 111 deals with a new method for measurement of the volume and surface area of the neuron soma and for following up the total dendritic course in successive serial sections. During the course of a histological analysis of the brain stem using Golgi-Cox-stained materials, it was found that the application of reflecting illumination provides a clear relief of the soma surface including its relationship with the dendrites. Utilizing this method it was possible to follow the course of serially sectioned dendrites and to represent faithfully the shape of the soma photogrammetrically. This has provided a means for precisely estimating the dimensions of the neuron. The values of the surface area and volume of dendrites and soma obtained by means of this new method fall within a narrow range while those obtained by other methods vary considerably from case t o case. Although based on only 3 cells from the gigantocellular nucleus of bulbar and pontine reticular formation, these results suggest that the cells which belong to the same type in certain region of the nervous system may have nearly the same volume and the same area, no matter how irregular their shape may be.

REFERENCES AITKEN, J. T., AND BRIDGER, J. T., (1962); Neuron size and neuron population density in the lumbosacral region of the cat's spinal cord. J. Anat., 95, 38-53. BLACKSTAD, T. W., AND FLOOD,P. R., (1963); Ultrastructure of hippocampal axo-somatic synapses. Nature (Lond.), 198, 542-543. BOK,S. T., (1959); Histonomy ofthe Cerebral Cortex. Amsterdam, Elsevier. BRODAL, A., (1957); The reticular formation of the brain stem. Anatomical Aspects and Functional Correlations. Edinburgh and London, Oliver and Boyd. CAJAL, S . R A M ~Y, N(1889); Conexi6n general de 10s elementos nerviosos. Med. prcict., 1-9. CAJAL, S. R A M ~Y, N(1891); Significaci6n fisiologica de las expansiones protoplasmaticas y nerviosas de las celulas de la sustancia gris. Rev. Cienc. mPd. (Barcelona), 22, 23, 1-15. CAJAL,S. R A M ~Y, N(1892); Nuevo concept0 de la histologia de 10s centros nerviosos. Rev. Cienc. mPd. (Barcelona), 16, 20, 22, 23, 1-68. CAJAL,S . R A M ~Y, N(1897); Leyes de la morfologia y dynamism0 de las celulas nerviosas. Rev. trimestral micrografica, 1, 1-25. CAJAL,S . R A M ~ YN, (1909, 1911); Histologie du SysrLme Nerveux de I'Homme et des VertPbrPs. Tome I et 11. Paris, Maloine. DE LORENZO, A. J., (1961); Electron microscopy of the cerebral cortex. I. The ultrastructure and histochemistry of synaptic junctions. Bull. Johns Hopk. Hosp., 108, 258-279. ECCLES, J. C., (1964); The Physiology of Synapses. Berlin, Heidelberg, Springer-Verlag. ESTABLE, C., (1961); Considerations on the histological bases of neurophysiology. A. Fessard, R. W. Gerard and J. Konorski, Editors. Brain Mechanisms and Learning. Oxford, Blackwell (pp. 309-334). Forx, CH., ET NICOLESCO, J., (1925); Les Noyaux Gris Centraux et la RPgion MPsencPphalo-SousOptique. Paris, Masson. Fox, C. A.. AND BARNARD, J. W., (1957); A quantitative study of the Purkinje cell dendritic branchlets and their relationship to afferent fibres. J . Anat., 91, 299-313. GAGEL, O., UND BODECHTEL, G., (1930); Die Topik und feinere Histologie des Ganglienzellgruppen in der Medulla oblongata und im Ponsgebiet mit einem kurzen Hinweis auf die Gliaverhaltnisse und die Histopathologie. Z . Anat. Entwick1.-Cesch., 91, 130-250. GIHR,M., (1962); Methode zur Rekonstruktion von Nervenzellen. J. Hirnforsch., 5,7-22.

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GIHR,M., (1963); Rekonstruktion von Nervenzellen. Separatum aus dem ersten internationalen Kongress fur Stereologie, 37, 1-1 3. GIHR,M., (1964); Die Zellformen des Nucleus medialis dorsalis thalami der Menschen. Progress in Brain Research, Vol. 5, Lectures on the Diencephalon. W. Bargmann and J. P. Schadk, Editors. Amsterdam, Elsevier (pp. 74-87). GRAY,E. G., (1959a); Electron microscopy of dendrites and axons of the cerebral cortex. J. Physiol., 145, 25-26. GRAY,E. G . , (1959b); Axosomatic and axodendritic synapses of the cerebral cortex: an electron microscopy study. J . Anat., 93, 420-433. GRAY,E. G., (1959~);Electron microscopy of synaptic contacts on spines of dendrites of the cerebral cortex. Nature (Lond.), 183, 1592-1 593. GRAY, E. G., (1961); The granule cells, mossy synapses and Purkinje spine synapses of the cerebellum: Light and electron microscope observations. J. Anat., 95, 345-356. HAGGAR, R. A., AND BARR,M. L., (1950); Quantitative data on the size of synaptic end-bulbs in the cat's spinal cord with a note on the preparation of cell models. J. comp. Neurol,, 93, 17-35. HAMLYN, L. H., (1962); The fine structure of the mossy fibre endings in the hippocampus of the rabbit. J. Anat., 96, 112-120. JACOBSOHN, L., (1909); Uber die Kerne des menschlichen Hirnstammes. Abhandl. Konigl. Preussischen Akad. (Ges. ) Wissenschaflen. Physisch-mathematische Klasse Berlin (pp. 1-70). KRAUS,W. M., AND WEIL,A., (1926); Human adult and embryo anterior horn cell. Arch. Neurol. Psychiat., 15, 686-701. KRIEG,W. J. S.,(1953); Functional Neuroanalomy. New York and Toronto, Blakiston. LEONTOVICH, T. A.. AND ZHUKOVA, G. P., (1963); The specificity of the neuronalstructureand topography of the reticular formation in the brain and spinal cord of Carnivora. J. comp. Neurol., 121, 347-379. J., AND KRAUS, W. M., (1925); On the form of the anterior horn cells. Anat. Rec., 31, LHERMITTE, 123-129. LORENTE DE N6, R., (1927); Untersuchungen iiber die Anatomie und die Physiologie des Ohrlabyrinthes und des Nervus octavus. Mschr. Ohrenheilk., 61, 857-897, 1066-1 135, 1152-1 190, 13001357. DE N6, R.,(1933a); Vestibulo-ocular reflex arc. Arch. Neurol. Psychiat., 30,245-291. LORENTE DE N6, R., (1933b); Anatomy of the eighth nerve. The central projection of the nerve LORENTE endings of the internal ear. Laryngoscope (St. Louis), 43, 1-38. LORENTE DE NO, R., (1933~);Anatomy of the eighth nerve. 111. General plan of structure of the primary cochlear nuclei. Laryngoscope (Lond.), 43, 327-350. LORENTE DE N6, R., (1933d); Studies on the structure of the cerebral cortex. 11. Continuation of the study of the Ammonic system. J. Psychol. Neurol. (Lpz), 46, 113-177. LORENTE DE N6, R.,(1947); Action potential of the motoneurons of the hypoglossus nucleus. J. cell. comp. Physiol., 29, 207-287. DE N6, R., (1953); Conduction of impulses in the neurons of the oculomotor nucleus. LORENTE Cyba Found. Symp. The Spinal Cord. G . E. W. Wolstenholme, Editor. London, Churchill (pp. 132-179). MANNEN, H., (1960); Noyau fermt et noyau ouvert. Arch. ital. Biol., 98, 333-350. MANNEN, H.,(1964a); Sttrtomttrie de la cellule nerveuse. Acta anat. nipponica, 39, 98 (abstract). MANNEN, H., (1 964b); Photogrammetric representation of Golgi-stained nerve cell. Preliminary Report. Proc. Jap. Acad., 40, 582-587. H., (1965); Arborizations dendritiques dans le noyau vestibulaire du chat. Etude topoMANNEN, graphique et quantitative. Arch. ftal. Biol., 103, 197. O., (1 927) ; Mikroskopisch-topographischer Atlas des menschlichen Zentralnervensystems. MARBURG, Leipzig und Wien, Franz Deuticke. MEESSEN, H., UND OLSZEWSKI, J., (1949); Zytoarchitektonischer Atlas des Rautenhirns des Kanincbens. Basel, Karger Verlag. MICKLEWRIGHT, H. L., KURNICK, N. B., AND HODES,R., (1953); The determination of cell volume. Exp. Cell Res., 4, 151-158. J., AND BAXTER, D., (1954); The Cytoarchitecture of the Human Brain Stem. New York OLSZEWSKI, and Basel, Karger. PITTS,R. F., (1940); The respiratory center and its descending pathways. J . comp. Neurol., 72,605625.

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P r m , R. F., MAGOUN, H. W., AND RANSON, S. W., (1939); Localisation of the medullary respiratory centers in the cat. Amer. J. Physiol., 126, 678-688. RAMON-MOLINER, E., (1961); The histology of the postcruciate gyrus in the cat. 1. Quantitative studies. J. comp. Neurol., 117, 43-62. RAMON-MOLINER, E., (1962); An attempt at classifying nerve cells on the basis of their dendritic patterns. J. comp. Neurol., 119, 21 1-227. RASMUSSEN, G. L., (1957); Selective silver impregnation of synaptic endings. New Research Techniques of Neuroanatomy. W. F. Windle, Editor. Springfield, Thomas (pp. 27-39). RILEY,H. A., (1943); An Atlas of the Basal Ganglia, Brain Stem and Spinal Cord. Baltimore, Williams and Wilkins. SCHADB, J., AND VAN HARREVELD, A., (1961); Volume distribution of moto- and interneurons in the peroneus-tibialis neuron pool of the cat. J. comp. Neurol., 117, 387-398. SCHEIBEL, M. E., AND SCHEIBEL, A. B., (1958); Structural substrates for integrative patterns in the brain stem reticular core. In Henry Ford Hospital international symposium. Reticular Formation of the Brain. H. H. Jasper, L. P. Proctor, R. S. Knighton, W. C. Noskay and R. T. Costello, Editors. New York, Brown (pp. 31-55). SCHEIBEL, M. E., SCHEIBEL, A. B., WALBERG, F., AND BRODAL, A., (1956); A real distribution of axonal and dendritic patterns in inferior olive. J. comp. Neurol., 106, 21-49. SHOLL,D. A,, (1953); Dendritic organization in the neurons of the visual and motor cortices of the cat. J. Anat., 87, 387-406. SHOLL,D. A., (1955); The organization of the visual and motor cortices of the cat. J. Anat., 89, 3346.

SHOLL,D. A., (1956); The Organization of the Cerebral Cortex. London, Methuen; New York, John Wiley. STERN,K., (1936); Der Zellaufbau des menschlichen Mittelhirns. Z. ges. Neurol. Psychiat., 154, 521-598.

WEIL,A., (1927); The form of the anterior horn cells of vertebrates. Arch. Neurol. Psychiat., 17, 783-793.

TREFF,W. M., (1963); Einfluss der Schnittdicke auf die messbaren Grossen bei Nervenzellen. Separatum aus dem ersten internationalen Kongress fur Stereologie, 18, 1-1 2. VALVERDE, F.. (1962); Reticular formation of the albino rat's brain stem. Cytoarchitecture and corticofugal connections. J. comp. Neurol., 119, 25-54. VANDER Loos, H.,(1960); On dendro-dendritic junctions in the cerebral cortex. Structure and Function of the Cerebral Cortex. D. B. Tower and J. P. SchadC, Editors. Amsterdam, Elsevier (PP. 36-42). A., (1897); Anatomie du S y s t h e Nerveux de I'Homme. Louvain, UystpruystVANGEHUCHTEN, DeeudonnC. A., (1911); An Anatomical Guide to Experimental Researches on the WINKLER.C., AND POTTER, Rabbit's Brain. Amsterdam, W . Versluys. WINKLER, C., AND POTTER.A., (1914); An Anatomical Guide to Experimental Researches on the Cat's Brain. Amsterdam, W. Versluys.

163

Central Mechanism of Vision KOITI MOTOKAWA A N D H l S A O S U Z U K I Department of Physiology and Institute of Brain Diseases, Tohoku University School of Medicine, Sendai (Japan)

The present survey is concerned with two subjects which have been investigated in our laboratory. In the first part, we shall discuss the transfer of the visual message at the dorsal nucleus of the lateral geniculate body (LGD); and in the second, modulation of color information at the central visual system. Articles which do not seem to be related directly to the present discussion are omitted. A rather detailed review on the conduction of information along the visual pathways has been published elsewhere by Motokawa (1963). THE LATERAL G E N I C U L A T E BODY

Diference in discharge pattern bet ween pre- and postgeniculate neurons

The lateral geniculate body is a prominent and well-defined thalamic nucleus with several distinct cell layers. Phylogenetically this nucleus has been developed with the process of the encephalization of the visual function (Marquis, 1934). In fishes and Amphibia there are no connections between the optic nerve and the cerebral cortex. Therefore, ablation of the cortex does not give rise to any impairment of vision. The optic tectum, which is a comparable organ to the superior colliculus of higher mammals, contributes to vision, and the small lateral geniculate nucleus receives a few incoming fibers from the retina. No geniculo-striate fibers are present. In reptiles and birds, the geniculo-striate system becomes apparent, though the optic tectum is still the main visual center. Only a small number of axons of the lateral geniculate neurons go to the forebrain. At the level of rabbits, cats and dogs, the size of the geniculostriate system increases greatly, while that of the optic tectum decreases considerably. Thus, ablation of the striate cortex results in disappearance of their object vision, but the perception of brightness is preserved. In the monkey and man, the encephalization becomes so complete that the geniculo-striate system plays an important role in the visual function. The process of phylogenetic development of the geniculo-striate system may lead us to the notion that the LGD is a switchboard for branching out a new geniculo-striate system from the optic-nerve-colliculus channel and connecting the retina to the striate cortex. Preservation of the sense of brightness in rabbits, cats Reforrrces p . 1781179

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and dogs indicates that this switching-over may not be complete, but the mesencephalic structure shares some role in vision. This interpretation may be supported by the anatomical fact that some fibers of the optic nerve show a bifurcation, sending one branch to the LGD then to the visual cortex and the other to the superior colliculus (Barris, 1935). It appears from these observations that one of the important functions of the LGD is a branching station. However, there is some evidence suggesting that visual impulses from the optic nerve are modified considerably in transmission through the geniculate synapses. According to O’Leary (1940), in the LGD of the cat each principal cell makes synaptic contacts with the terminals of several different optic nerve fibers. From the convergence type of the synaptic organization, one may expect that the genicdate neuron would operate as a ‘logical decision element’, which generates an output signal when the algebraic sum of its input signals becomes equal to or greater than a certain threshold. In other words, a geniculate neuron can generate a conducting impulse toward the cerebral cortex only when more than a certain definite number of impulses of the optic nerve fibers arrive at the neuron in sufficiently close succession. Since several impulses from the optic nerve fiber are necessary to generate one impulse in the geniculate neuron in this model, it may be expected that there is some dropping out of the impulses in transmission through the geniculate synapses. The validity of this concept may easily be tested by comparing the discharge pattern of the optic tract fibers (input to the geniculate synapse) with that of the optic radiations (output ofthe synapse).

Fig. 1. Discharge patterns obtained from single optic tract fibers (left column) and from single optic radiation fibers (right column) under dark adaptation. Upper row = on units; middle = on-off units; lower = off units. The discharge rate was decidedly low in optic radiation units as compared with that of optic tract units. Time marker 100 msec. Upward swing of lower beam in each record denotes period of illumination. (From Suzuki et al., 1960.)

In Fig. 1 is shown an example of unit discharges of the optic tract and optic radiation of the cat immobilized and locally anesthetized (Suzuki et al., 1960). When the eye of the animal was illuminated by diffuse light, on responses, on-off responses or off responses were recorded in both optic tract fibers and optic radiations (top, middle

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and bottom rows respectively). In any type of response impulses the discharge began with relatively high frequency and declined more or less rapidly. Although such a rapidly declining discharge rate is not constant, mere inspection of the records reveals that the discharge rate is decidedly low in the postgeniculate neurons (right column) as compared with that in the pregeniculate (left column). Since the discharge rate depends upon the intensity of illumination, all records shown in Fig. 1 were taken under one and the same stimulus condition. The discharge rate in the pre- and postgeniculate neurons was expressed more quantitatively by the average frequency of response which was determined by measuring the number of impulses in 300 or 600 msec from the onset or cessation of illumination (Suzuki et al., 1960). Moreover, the maximum frequency of theunit discharge was determined by using illumination of various intensities. The maximum discharge rate of the optic tract unit thus obtained was usually as high as 100 per sec, whereas the highest rate of radiation fibers was about 30-40 per sec. Therefore it can be inferred that some dropping out of the impulses occurs in the geniculate transmission. The notion that the discharge rate of the postgeniculate neurons is much lower than that of pregeniculate neurons holds good in the spontaneous discharges of both units. Most pregeniculate units of the OF. and off types showed spontaneous discharges of 30-40 per sec, while the rate of discharge in the radiation fibers was only about 10-20 per sec. Therefore, the above-mentioned results indicate that the principle of the logical decision element may be applied to the geniculate synapses so far as the discharge rate is concerned. The applicability of the logical decision principle to the geniculate synapses can be also tested by comparing the spectral sensitivity curves of the pregeniculate and postgeniculate units. As mentioned above, rapid succession of the impulse generation in the presynaptic fiber should be required before the generation of one impulse in the postsynaptic neuron. Therefore, the postgeniculate units may not respond to the light stimulus until the stimulus becomes strong enough to generate impulses in the optic nerve fibers in sufficientlyclose succession. It may be expected that the sensitivity of the optic nerve fiber to the light stimulus is higher than that of the radiation fiber. If this is true, the geniculate synapse makes some barrier for controlling the passage of visual information along the pathway to the striate cortex. Therefore, the spectral sensitivity curves were compared between the optic tract and optic radiation units (Suzuki et a/., 1960). At the pregeniculate level, the on, off and on-off type units gave spectral sensitivity curves with a maximum at 480-500 mp under dark adaptation. As to the spectral sensitivity distribution similar results were obtained with optic radiation fibers and even with cortical neurons; in these neurons the peak of the spectral sensitivity curve was also around 500 mp. However, as can be seen in Figs. 2 and 3, the sensitivity level of the postgeniculate neurons was quite different from the pregeniculate neurons: the pregeniculate neuron responded to a light stimulus of relative intensity as low as 0.716 at 500 rnp, while the postgeniculate unit could be activated only by light stimuli stronger than 0.78. This great difference in threshold intensity suggests that visual information undergoes some screening at the geniculate synapses. Glees (1941) described the existence of some divergence of the optic nerve terminaR1fircwcc.s p. 1781179

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0.7

‘

0.7

0.7

0.7’*

0.7”

0.720

Fig. 2. Spectral sensitivity curve of optic tract unit (on-off unit). Ordinate, wavelength. Wh stands for white light stimulus. Monochromatic lights of equal energy were obtained by the use of interference filters and suitable neutral filters. Abscissa, relative intensities as expressed by transmission factors of neutral filters. (From Suzuki cf a/., 1960.)

tion in the geniculate body. Each optic nerve fiber terminates in an arborization related to several geniculate cells. If it is assumed that there is a great divergence of the optic nerve fiber at the level of the LGD, the receptive field of the postgeniculate neuron may be greatly different from that of the presynaptic neuron in shape or extent. In Fig. 4, the receptive fields of an off unit of the optic radiation are illustrated. These records were obtained by an automatic scanning device which is described in detail by Suzuki et al. (1960). Under dark adaptation no definite receptive field could be mapped owing to spontaneous background discharges. When spontaneous discharges were reduced by background illumination, the receptive field could be mapped as an assembly of light spots, which was roughly circular in shape. As the intensity of adapting light was increased, the receptive field was reduced progressively in size. Similar experiments were made with presynaptic neurons, and it was found that no essential difference existed between the receptive fields of optic nerve fibers and those of radiation fibers. A similar result was reported by Hubel and Wiesel (1961). This physiological finding might support the anatomical observation that one optic nerve fiber terminates in a relatively restricted region so as to be confined to a single cell layer and adjoining interlaminar margin (O’Leary, 1940). All the experimental observations mentioned above could be explained by the assumption of the LGD as a monosynaptic system operating under the principle of the logical decision element. However, during the course of experiments on the

167

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07'

07

'

0.7'

0.7

'*

Fig. 3. Spectral sensitivity curve of optic radiation unit (on-off unit) obtained by same stimulus condition as in Fig. 2. The sensitivity of the optic radiation unit is much lower thari that of the optic tract unit. (From Suzuki ef a!., 1960.)

spectral response curves of the postgeniculate neurons, we encountered a phenomenon which could not be explained by the above-mentioned principle of the logical decision element (Suzuki e / a/., 1960; Okuda et a/., 1962). The spectral response curve of a single unit was constructed by plotting the average frequency of impulses in response to colored lights of equal energy against wavelength. At the postgeniculate neurons such curves obtained with lights of strong intensity were of complicated form (Fig. 5 ) . The 2c

0

Fig. 4. Receptive fields of the optic radiation unit (on-off unit). Relative intensity of adapting light is given above each receptive field. As the intensity of adapting light was increased, the receptive field was reduced progressively in size. (From Suzuki er al., 1960.) Ri./;rianr(s.s

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Fig. 5. Spectral response curves of single optic radiation fibers (on units). The number of impulses (ordinate) to each spectral light was found by counting the impulse number durinp an illumination period of 0.5 sec and dark period of 0.5 sec following cessation of illumination. Note two maxima of on response in C. [From Okuda et al., 1962.)

average frequency of impulses was low a t about 500 mp and two maxima appeared over the ranges of 420-480 and 520-600 mp. These two maxima disappeared when the intensity of the colored lights was reduced, and instead a maximum appeared at about 500 mp. The occurrence of two separated peaks in the spectral response curve has never been observed at the pregeniculate neurons. In the optic tract a spectral response curve obtained with strong colored lights showed either a broad maximum at about 500 mp or a plateau extending over a very wide range of wavelengths except at the red end of the spectrum at which the curves fell abruptly. When the intensity of colored lights was reduced to a certain level, the curve fell off towards both ends of the spectrum, giving a peak at about 500 mp. Thus, this curve resembled the spectral sensitivity curve obtained from a postgeniculate unit. Since the complex shape of the spectral response curves becomes manifest only in the postgeniculate neuron, it may be said that the complex pattern might be the result of modification taking place in the transmission through the geniculate synapses. Reticular influence upon geniculate transmission

Histologically the LGD is a far from simple structure. The existence of a large number of short-axon cells, each of which has synaptic relation with more than a dozen principal cells (O'Leary, 1940), suggests that some modification of the incoming visual impulses may take place in the LGD. Furthermore, many investigators have demonstrated the presence of fiber terminals of non-optic origin in the LGD. Glees (1941) found that normal terminal rings apparently remained after section of one optic nerve suggesting an extraretinal source to the LGD. Polyak (1957) also mentioned fibers differing in terminal arborization from those of retinal origin, probably from the cortex. Szenthgothai (1963) and Colonnier and Guillery (1964) also described the fiber terminals of non-optic origin in the LGD.

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There are many phenomena complementary to these anatomical observations that support the existence of a number of terminals of non-retinal origin (Long, 1959; Dumont and Dell, 1960; Bremer, 1961). The evoked potential, brain wave and even unit activities in the striate cortex were changed when an animal’s attention was attracted or its reticular activating system was stimulated. More directly, the evoked potential of the LGD was greatly modified by stimulation of the reticular formation. However, the stimulus parameters were so complicated in most experiments that no definite conclusion could be obtained from these results. Therefore, a single electric shock to the brain stem reticular formation was used as a conditioning stimulus for simplifying the experimental condition, and the mass responses of the LGD to electric stimulation of the optic tract were recorded in the cat without general anesthesia (Suzuki and Taira, 1961). The response obtained in the LGD consisted of two components, that is, t l and r l which represent pre- and postsynaptic components respectively (Bishop and McLeod, 1953). A conditioning shock was applied to the mesencephalic reticular formation 100 msec before the test shock which was applied to the optic tract. The conditioning shock clearly enhanced the postsynaptic component rl whereas the presynaptic component tl remained unaltered or was reduced slightly (Fig. 6). The time course of the effect of the reticular stimula-

II

Fig. 6. Effect of reticular stimulation on geniculate evoked potential to optic stimulus. C = Contro responses. Others are preceded by single shock stimuli to mesencephalic reticular formation by intervals indicated in numerals in msec. Time marker 1 msec. Conditioning stimulation of reticular formation produced augmentation of the second component (postsynaptic) of the evoked potential without significant alteration of the first component (presynaptic). (From Suzuki ar?d Taira, 1961 .)

tion on the geniculate evoked response was examined by altering the interval between the conditioning and test stimuli systematically. The postgeniculate component of the response evoked 30 msec after the conditioning stimulus to the reticular formation was clearly enhanced above that of the control. Enhancement ofthe second component became more marked as the interval between the conditioning and test stimuli was increased. The maximal increase occurred between 70 and 90 msec after the conditioning shock, and the facilitation continued over 500 msec. The maximal increase in amplitude produced by a reticular shock was about 30% in the animal with the intact central nervous system. In the ‘enctphale isolt’ cat, however, the increase of Rrfrrcnrrs p . 1781179

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the postgeniculate component exceeded the control value by 100%. The conditioning shock to the reticular formation alone did not evoke any detectable response in almost all experiments. Besides the mesencephalic reticular formation, many subcortical structures were found to exert a facilitatory influence upon the LGD (Okuda, 1962). The bulbar reticular formation gave a facilitatory effect as great as the mesencephalic reticular formation. Stimulation of the thalamic reticular nuclei - N. centrum medianum and N. centralis lateralis - was also effective (Fig. 7). Moreover, zona incerta and Forel's H

Fig. 7. Schematic representation of loci, stimulation of which exerted facilitatoryIand inhibitory effects upon LGD response. Dots represent facilitatory, and crosses inhibitory, effects. Ch = Chiasrna opticurn; GL = Corpus geniculatum laterale; CL = N. centralis lateralis; CM = N. centrum medianum; R-F = Formatio reticularis; VA = N. ventralis anterior. (From Okuda. 1962.)

fields, which are postulated to be anatomical continuations of the brain stem reticular formation, were found to be facilitatory structures. The only structure eliciting an inhibitory action was N. ventralis anterior, though this effect was not striking. The thalamic relay nuclei, including N. ventralis postero-medialis, N. ventralis lateralis, N. anterior ventralis and corpus geniculatum mediale, were entirely ineffective, and the association nuclei such as N. lateralis dorsalis, N. medialis dorsalis and pulvinar were also ineffective. By recording the unit activity of the postgeniculate neurons, it was further substantiated that this facilitatory effect of the geniculate mass response was due to an increase in number of the firing neurons at the postgeniculate level (Suzuki and Taira, 1961). Radiation units responded with a relatively fixed and short latency to a single shock and discharged only once to the supraliminal stimulus to the optic tract. When the stimulus to the optic tract was weakened gradually from a supra-threshold to the threshold level, the radiation unit did not respond to successive stimuli each time but only some of them fired the radiation unit (see column A in Fig. 8). At such an intensity level of the test stimulus the radiation unit became capable of responding to each stimulus when the conditioning reticular stimulus preceded the test stimulus (column B). The above-mentioned phenomenon was studied more quantitatively using the firing index which was originally devised by Lloyd and McIntyre (1955) to describe the reflex

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Fig. 8. Responses of a single neuron of optic radiation fiber to 10 successive threshold tract stimuli. A and C give control responses. B gives responses conditioned by reticular stimulation. Reticular stimuli precede optic tract stimuli by 100 msec. Time marker 1 msec. Reticular stimulation elicits great facilitatory effect on geniculate neurons. (From Suzuki and Taira, 1961.)

behavior of a motoneuron pool. In the present experiment the firing index was defined as n/10 times 100% when n responses were obtained to 10 successive stimuli. The intensity of stimuli to the optic tract was adjusted to a certain definite value so that the firing index of a given radiation unit was scores per cent. Under such a stimulus condition of the optic tract a large subliminal fringe will remain in a geniculate neuron pool, and stimulation of the reticular formation may greatly modify the firing index of the unit. The temporal pattern of the firing index following reticular stimulation was found to be different from one radiation unit to another. Usually, units received a combined influence of facilitation and inhibition in temporal pattern from the reticular formation, although facilitation was predominant in most units. This suggests that the number of neurons responding to tract stimuli will increase following reticular stimulation, and the increase may result in the augmentation of the postsynaptic component of the geniculate evoked potential. These reticular effects on the geniculate transmission can also be demonstrated by using discharge patterns of unit activities in the optic radiation and the LGD (Hubel, 1960; Arden and Soderberg, 1961 ; Taira and Okuda, 1962; Ogawa, 1963). The pathway from the retina usually showed background activity, both optic tract and optic radiation units firing spontaneously. The firing rate of these units, as many workers have observed, changed appreciably depending upon the animal’s waking state. In the cat with the EEG of the non-alerted state, radiation units discharged at relatively low frequency of 10-20/sec in both on and off type units. When the EEG activation pattern was induced either by reticular stimulation or natural stimuli, the discharge rate was raised. On the contrary, when the drowsy state was induced by intravenous References p . 1781179

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injection of a barbiturate, there appeared a reduction in the discharge rate of radiation units as well as a tendency to clustered firing patterns accompanying the appearance of the high voltage and slow activity or spindle bursts in the EEG (Taira and Okuda, 1962).The clustered firing patterns were first observed by Hubel (1960) in the geniculate neurons of the unrestrained cat. The responsiveness of radiation units to visual stimuli also changes with alteration in the animal’s waking state (Taira and Okuda, 1962). As the EEG showed the alert pattern, the responsiveness of radiation units was improved. In the alerted state produced by either reticular stimulation or natural stimuli, off units showed stronger inhibition of the spontaneous discharge and intense and long-lasting off discharge to a light stimulus, whereas in the non-alerted state these units gave a response consisting of weak suppression of the spontaneous discharge followed by an off discharge to the light stimulus. On units in the optic radiation increased the discharge rate during illumination as well as in the dark when the alerted state was induced by reticular stimulation. Thus, the response of on optic radiation units to illumination became less distinguishable from the background discharge in the alerted state because of the raised background discharge. It seems as if the signal to noise ratio, S/N, was lower in the alerted state than in the relaxed state. In other words it seems as if the alertness would be unfavorable for sensory perception. However, this argument is not always acceptable as will be shown by quantitative treatment of the data. The spike number of response was treated quantitatively as follows: 10 trials were made for each condition and the mean number of response impulses and standard deviation (S.D.) were determined; where the number of response impulses means the number of spikes during an illumination period of 0.5 sec minus the number of spikes occurring in the 0.5 sec dark period immediately prior to the illumination. Table I shows the mean numbers TABLE I EFFECT O F NO NS P E CI F I C STIMULI O N D I S C H A R G E OF LGD N E U R O N S I N R ES P ON S E TO P H O T I C STIMULUS

Number of response impulses over 0.5 sec. (From Taira and Okuda, 1962.) __~_

~.

Type o f discharge

Non alert (based on EEG)

Alert (based on EEG)

Type o f stimulus

Mean

S.D.(%)

Mean

S.D. 1%)

On

10.1 18.6 16.5 24.2 8.0

33.6 16.7 24.8 14.0 41.5

11.7 15.1 14.1 29.2 11.0

22.2 7.3 17.7 10.6 23.6

R.F. R.F. R.F. R.F. Auditory

Off Off Off Off Off

10.3 8.6 10.1 8.0 9.6

25.2 25.6 33.6 36.2 36.4

14.3 15.0 14.6 12.5 15.8

12.6 9.3 13.0 20.0 10.8

R.F. R.F. R.F. R.F. Olfactory

On On On On

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of response impulses and % S.D. of 5 randomly sampled on units in the non-alerted and alerted states. In most on units the mean number of response impulses didnot show any definite tendency to increase under reticular activation, and in some units it even decreased slightly. Unlike the mean number of response impulses, the S.D. definitely decreased in the alerted state. As to off units, the number of response impulses was defined as the number of spikes occurring in 0.5 sec after the termination of illumination minus the number of spikes occurring during the illumination period of 0.5 sec. In off units, too, a definite reduction of fluctuation in the number of response impulses was regularly observed in the alerted state. The reduction in fluctuation signifies heightened stability of responsiveness of the radiation units in the alerted state or increased reliability of information. The optic tract units did not show significant change in either response or background discharge as the EEG was altered from a non-alerted to an alerted pattern by reticular stimulation or natural stimuli. Therefore, the stabilizing effect of alerting stimuli observed above may be attributable to the synaptic action in the LGD. As mentioned above, reticular stimulation seems to reduce the subliminal fringe in the geniculate neuron pool. Since the existence of the subliminal fringe may be considered to be one of the reasons for fluctuation of the response, the reduction of the fringe may result in the stability of the response. The mechanism of corticifugal influence upon geniculate transmission

Contrasting with the prevailing facilitatory effect of the subcortical structures on the geniculate transmission, stimulation of the striate and parastriate areas gives rise to an inhibitory effect on the geniculate transmission (WidCn and Ajmone Marsan, 1960; Ajmone Marsan and Morillo, 1961). Kwak (1964) demonstrated that the cortical effect on the geniculate transmission is inhibitory when mass responses in the LGD are taken as an index. This effect was confirmed, and its mechanism was explored in our laboratory. The evoked response of the LGD t o ipsilateral optic tract stimulation consists of t and r which represent pre- and postsynaptic components respectively. A conditioning shock was applied to the striate cortex through a silver ball electrode 100 msec prior to the test shock to the optic tract. The conditioning shock clearly

Fig. 9. Effect of cortical stimulation on geniculate evoked potential to optic tract stimulus. A and C give control responses. In B, a conditioning shock was applied to the lateral gyrus 100 msec prior to a test shock to the optic tract. Theconditioninpshockclearlydecreasedthe postsynapticcomponent. (From Kwak, 1964.) References p . 1781179

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decreased the postsynaptic component r , but no conspicuous change could be observed on the presynaptic component t (Fig. 9). This depression of the postsynaptic component reached a maximum at about 70-80 msec after the conditioning stimulus, and continued over 500 msec. Stimulation of the contralateral visual cortex was also found effective though the ipsilateral cortex was usually more effective. Therefore, a possible pathway via the corpus callosum from the contralateral visual cortex could be postulated to produce the depressive effect on the geniculate synapses (Ajmone Marsan and Morillo, 1961). This effectiveness of the contralateral stimulation on the postsynaptic component of the geniculate response and the long-lasting time course of depression may indicate that the depression is a true inhibitory effect of the efferent pathway from the cortical neurons. The postexcitatory depression of the geniculate neurons (for example, the after-hyperpolarization of the geniculate neurons following the antidromic activation in response to cortical stimulation) would not contaminate the above-mentioned effect so long as the contralateral cortex is stimulated. When the experiments on the effect of the visual cortex stimulation on the geniculate evoked potential were performed, slight depression was sometimes observed in the presynaptic component of the geniculate evoked potential as well (Iwama, 1964; Suzuki and Kato, 1964). This depression of the presynaptic component was preserved even after electrical coagulation of both optic discs. Thereforea possible efferent effect on retinal elements may be ruled out. The depression of the presynaptic component became dominant when repetitive stimulation was applied to the visual cortex as the conditioning stimulus. Simultaneously with the depression of the presynaptic component, it was found that the excitability of the afferent terminals of the optic nerve fibers in the LGD was enhanced by stimulation of the visual cortex (Fig. 10). The excitability of the afferent terminals of the optic nerve fibers was tested by the procedure of Wall and Johnson (1958):a single shock was applied to the LGD to stimulate the terminals of the optic nerve fibers and the antidromic volley evoked by the exci-

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Fie. 10. Effect of cortical stirnulation (train of 5 pulses at 300/sec) on excitability in optic tract terminals. Excitability of terminals was tested by amplitude of antidrornic response in optic tract evoked by stimulation of LGD. C = control responses. The response consisted of first and second components which correspond to large and small fibers in optic tract respectively. Numerals on the other two records denote intervals between conditioning cortical and test stimuli in msec. Horizontal bar, 1 msec. Vertical bar, 500 ,uV. Both first and second components increased in amplitude after cortical stimulation.

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tation of the terminals was recorded in the optic tract. The increase in the excitability induced by the conditioning cortical stimulation could be observed as the increased amplitude of the antidromic response. The enhancement of the excitability of the afferent terminals started immediately after the cortical stimulation, reached a maximum at about 50 msec after the conditioning stimulus and waned to the control value during 100 msec. This increased excitability which accompanies the depression of the transmission may be comparable to the presynaptic inhibition observed in the spinal motoneuron (Eccles et al., 1961) and the cuneate nucleus (Andersen et al., 1964). Axo-axonal synapses in the LGD of the monkey are described by Colonnier and Guillery (1964). Such pre- and postsynaptic inhibition at the lateral geniculate body would represent a powerful negative feedback comparable to the pupillomotor system in the visual pathway. THE VISUAL CORTEX

Cortical responses to colored lights It is well established that the fundamental mechanism of color reception certainly lies at the retinal level. However, there remains the possibility that further elaboration of color information is performed in the geniculate body and visual cortex. Motokawa et a / . (1962) made experiments on color vision by recording the spike discharge of single units from the visual cortex in monkeys. Spikes of radiation fibers and cortical neurons were distinguished by their spike shape and discharge rate which is much higher in radiation fibers than in cortical neurons. The macular region and its surroundings were illuminated with colored lights of equal energy. The stimulus size was 0.5 degree i n visual angle, and its duration 0.5 sec. The stimulus spot was projected on to the center of the receptive field of each unit. At least two types of unit could be distinguished with respect to response patterns to various colored lights: the first type was commonly encountered and maintained the same discharge pattern, either on, off or on-off, throughout the whole range of the spectrum; the second type changed its discharge pattern according to wavelength. An example of the first type, an on unit in this case, is illustrated in Fig. 1 1 : a sensitivity maximum was found at about 480 mp and a submaximum at about 610 mp. A variety of discharge pattern was observed in this type; some units had a single peak, but others showed one or two submaxima besides their main peak. In some of these units the relative heights of the dominant peak and submaxima remained unaltered over a wide range of intensities of illumination, but in others, especially those having a submaximum around 500 mp, this peak at 500 mp became progressively dominant as the stimulus intensity was reduced. Many off units responded to a narrow band of the spectrum in much the same way as the on units illustrated in Fig. 11. There were many cortical neurons which responded to a very wide range of the spectrum, showing no dominant maximum of responsiveness. The fact that the spectral response curves of most cortical units have submaxima besides their dominant peak indicates that R
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color-specific channels are not entirely independent of each other. Two examples of the second type of unit are shown in Fig. 12. The unit illustrated in A responded with on-discharge to blue-green light, but with off-discharge to red light. The example B had the inverse response pattern with respect to the discharge type. In any unit, the complementary relation such as red-on and blue-green-off, or vice versa could be obtained; when the spatial organization within the receptivezfield

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0.5sec Fig. 13. Spectral sensitivity curve of cortical neuron of monkey. See explanation to Fig. 11. (From Motokawa et a/., 1962.) was studied in such units, a similar complementary relation was found between the center and periphery of the receptive field. Thus, it is apparent that these units contribute to color contrast. Some units showed a consistent maximum at about 490 mp, as exemplified in Fig. 13. This unit was an on-off type, but units of on or off types were found to have a sensitivity maximum at about 490 mp too. Judging from the spectral property, they are probably scotopic units. The wavelength dependence of cortical neurons mentioned above generally resernbles the result obtained by De Valois (1960) in the LGD neurons of the monkey and that obtained by Hubel and Wiesel(l960) at the ganglion cell level of the spider monkey. Lennox-Buchthal (1962) obtained a different result on the behavior of cortical neurons to colored lights. This may, however, be due to the difference in the stimulus conditions. SUMMARY

The mechanism of screening of the visual information at the lateral geniculate body was the main object of the present study. An electric test stimulus was applied to the optic tract, and a conditioning one to various points of the central nervous system. The postsynaptic component of the mass response thus evoked at the dorsal nucleus of the lateral geniculate body was increased in amplitude by a conditioning stimulus Referenws p . 1781179

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applied to the ascending brain stem reticular formation, but decreased by a conditioning stimulus applied to the visual cortex. The interval between conditioning and test stimuli most favorable to these effects was 80-100 msec. The presynaptic component of the same mass response suffered almost no change by the reticular conditioning stimulus. Therefore the reticular influence mentioned above may be regarded as facilitation of synaptic transmission. On the contrary, the cortical conditioning stimulus, especially a repetitive one, was found to be effective in reducing the presynaptic component of the mass response as well, and this effect was identified as presynaptic inhibition. Similar experiments were carried out with single unit discharges from geniculate cells. Further experiments with single units were carried out on the visual cortex of the monkey, and three types of units were distinguished. Units of the first type responded with pure on or off discharge to the whole range of the equal-energy spectrum, although the spectral part corresponding to the response maximum varied from unit to unit. The three spectral regions, red, green and blue tended to evoke maximal responses. In units of the second type one region of the spectrum caused on-discharge and another caused off-discharge, or vice versa. In units of the third type no clear-cut wavelength dependence could be observed. The response maximum of most units was found in the neighborhood of 500 mp when tested with lights of sufficiently low intensities, and this observation suggests that both systems, cone and rod, converge into a single cortical cell. REFERENCES AJMONEMARSAN, C., AND MORILLO, A., (1961); Cortical control and callosal mechanisms in the visual system of cat. EleLtroenceph. clin. Neurophysiol., 13, 553-563. ANDERSEN,P., ECCLES, J. C., SCHMIDT, R. F., AND YOKOTA, T., (1964); Depolarization of presynaptic fibers in the cuneate nucleus. J. Neurophysiol., 27. 92-106. ARDEN, G.B., AND S~DERBERG, U., (1961j; The transfer of optic information through the lateral geniculate body of rabbit. Semory Communication. W. A. Rosenblith, Editor. Cambridge, M.1.T Press. BARRIS, R. W., (1935); Disposition of fibers of retinal origin in the lateral geniculate body. Arch. Ophthal., 14.61-70. BISHOP,P. O., AND MCLEOD.J. G., (1953); Nature of potentials associated with synaptic transmission in lateral geniculate of cat. J . Nercrophysiol., 17. 387-414. BREMER, F.. (1961); Neurogenic factors influencing the evoked potentials of the cerebral cortex. Sensory Communication. W. A. Rosenblith, Editor. Cambridge, M.I.T.Press. COLONNIER, M.,AND GUILLERY, R.w., (1964); Synaptic organization in the lateral geniculate nucleus of the monkey. Z. Zelljorsch., 62, 333-355. DE VALOIS,R. L., (1960;; Color vision mechanisms in the monkey. J. gen. Physiol., 43, Suppl. 2 1 15-1 28. DUMONT, S., ET DELL,P., (1960); Facilitation r6ticulaire des mtchanismes visuelles corticaux. Electroenceph. clin. Neurophysiol., 12, 169-796. ECCLES,J. C., ECCLES,R. M.,AND MAGNI,F., (1961); Central inhibitory action attributable to presynaptic depolarization produced by muscle afferent volley. J. Physiol., 159, 147-1 66. GLEES,^., (1941); The termination of optic fibres in the lateral geniculate body of the cat. J . Anat., 75,434-440. HUBEL, D. H., (1960); Single-unit activity in lateral pniciilate body and optic tract of unrestrained cats. J. Physiol., 150, 91-104.

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HUBEL,D. H., AND WIESEL, T. N., (1960); Receptive fields of optic nerve fibres in the spider monkey. J. Physiol., 154, 572-580. HUBEL,D. H., AND WIESEL, T. N., (1961); Integrative action in the cat's lateral geniculate body. J. Physiol., 155,385-398. ~ W A M A ,K., (1964); Personal communication. KWAK,R., (1964); Effect of cortical stimulation on synaptic transmission in lateral geniculate body of the cat. Tohoku J . exp. Med., In the press. LENNOX-BUCHTHAL, M. A., (1962); Single units in monkey, Cercorebus iorquutus utys, cortex with narrow spectral responsiveness. Vision Res., 2, 1-15. LLOYD,D. P. c.,AND MCINTYRE, A. K.,(1955); Monosynaptic reflex responses of individual motoneuroas. J. gen. Physiol., 38, 771-787. LONG,R. G., (1959); Modification sensory mechanisms by subcortical structures. J. Nerrrophy.riol., 22,412427.

MARQUIS, D. G . , (1934); Effects of removal of the visual cortex in mammals with observations on the retention of light discrimination in dogs. Res. Publ. Ass. nerv. men/. Dis., 13, 558 -592. MOTOKAWA, K., (1963); Mechanisms for the transfer of information a!ong the visual pathways. In/. Rev. Neurobiol., 5, 121-181. MOTOKAWA, K., TAIRA,N., A N D OKUDA,J., (1962); Spectral responses of single units in the primate visual cortex. Tohoku J. exp. Med., 78, 320-337. OGAWA,T., (1963); Midbrain reticular influences upon single neurons in lateral geniculate nucleus. Science, 139, 343-344. OKUDA,J., (1962); Subcortical structures controlling lateral geniculate transmission. Tohoku J. exp. Med., 76,350-364. OKUDA,J., TAIRA,N., AND MOTOKAWA, K., (1962); Spectral response curves of postgeniculate neurons in the cat. Tohoku J. exp. Med., 78, 147-157. OLEARY,J. L., (1940); A structural analysis of the lateral geniculate nucleus of the cat. J. conip. Neurol., 73,405430. POLYAK, S . , (1957); The Ver/ebrufe Visual Sysrem. Chicago, University of Chicago Press. SUZUKI, H., AND KATO,E., (1964); T o be published. SUZUKI,H., AND TAIRA,N., (1961); Effect of reticular stimulation upon synaptic transmission in cat's lateral geniculate body. Jup. J. Physiol., 11, 641-655. SUZUKI, H., TAIRA, N., AND MOTOKAWA, K., (1960); Spectral response curves and receptive fields of pre- and postgeniculate fibres of the cat. Tohoku J. exp. Med., 71,401-415. SZENT~GOTHAI, J., (1963); The structure of the synapse in the lateral geniculate body. Ac/a unuf. (Busel), 55, 166-185. TAIRA,N., AND OKUDA,J., (1962); Se,isory transmission in visual pathway in various arousal states of cat. Tohoku J. exp. Med., 78,76-97. WALL,P. D., A N D JOHNSON, A. R., (1958); Changes associated with post-tetanic potentiation of a monosynaptic reflex. J. Neurophysiol., 21, 148-158. WIDEN,L., AND AJMONE MARSAN, C., (1960); Effects of corticopetal and corticifugal impulses upon single elements of the dorsolateral geniculate nucleus. Exp. Neurol., 2, 468-502.

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Ex itation nd Inhibition in Ventrobasal Thalamic Neurons before and after Cutaneous Input Deprivation HIROSHI N A K A H A M A , S H I N K O N I S H I O K A A N D T O S H I R O O T S U K A Department of Physiology, Keio University School of Medicine, Tokyo (Japan)

Spontaneous and background activity has long been of interest to students of the nervous system. This activity was studied by Arden and Soderberg (1961) in lateral geniculate neurons of rabbits before and after retinal blockade, and in cats by Levick and Williams (1964). An investigation has been undertaken in the authors’ laboratory in the hope ofgaining some understanding of the neural mechanisms ofthe spontaneous background activity which is recorded from ventrobasal thalamic neurons of cats, both before and after the removal of cutaneous input. The results recorded in, this paper are preliminary. Details and further developments will appear elsewhere. In the present study, thalamic neurons activated by stimulation of the skin have been classified according to whether (cz) they were activated by movement of hairs within the related cutaneous receptive fields, or (6) they were not activated by hair movements, but responded to touching the skin surface between the hairs. A new observation made in the study, which we believe t o be of importance, is that following blockade of the nerve fibers innervating a previously determined peripheral receptive field, the new receptive field sometimes appeared continuously with the excitatory field previously blocked and in the direction to the proximal side of the body. METHODS

Adult cats weighing between 3.0 and 4.0 kg were used. Anaesthesia was induced with ether. During this time the trachea was cannulated, a venous cannula was inserted in the femoral vein, all operative wounds were carefully closed, and the cat’s head was fixed in the stereotaxic instrument. After no more than one hour of ether, pentobarbital sodium was given intravenously. During this stage the skull was trephined directly over the. ventrobasal nucleus of the thalamus, a lucite chamber mounted with dental cement, the dura removed and bleeding into the chamber carefully stopped. Care was taken to insure that the animal was always surgically anaesthetized, although the anaesthesia was kept as light as possible during the recording. To prevent reflex movements, gallamine triethiodide was given, and respiration was maintained by means of a pump. The intermittent recovery from the neuromuscular blocking agent permitted control of the anaesthetic state, and additional injections of pentobarbital

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sodium were given if necessary, usually in doses of 3-7 mg. The animal was kept warm by a radiating lamp and a rectal temperature was [maintained at 37-38". Attention was paid to keeping the cutaneous temperature normal. Intravenous fluids were given at a rate of 15-20 ml/h throughout the experimental period, lasting up to 15 h. No observations were made on the animals until 3-4 h after the cessation of ether administration. A tungsten microelectrode was employed for extracellular recording from single units. The terminal 7-8 mm of a piece of tungsten wire(diameter 1 0 0 ~were ) immersed in 2 M sodium hydroxide (NaOH) solution, and an alternating current was passed between the wire and a nearby carbon rod, using an e.m.f. of 6.3 V. The wire was dissolved only when the current flowed, and in this way the final stages were easily monitored by microscopic examination. The prepared wire was then carefully inserted into a glass pipette (tip outer diameter 3-5 p) under the microscope, and sealed, for fixation, by a gas burner at the root of the pipette. Finally, the wire outside the tip of the pipette was electrically sharpened under the microscope to less than 1 p, both in length and diameter. In order to reduce pulsations due to respiration and heart beat, a modification of the 'closed head' technique was used (Amassian, 1961; Amassian et al., 1959). The microelectrode tip was brought under stereotaxic control to a position just above the cortex and inside the rigid chamber. In order to make a water- and airtight seal around the glass pipette, high melting point (60 to 65") paraffin wax was melted and poured into the chamber over a I cm depth of Ringer solution. After cooling of the paraffin wax the electrode was inserted vertically through the intact cerebral cortex. In this way it was often possible to observe the same unit for several hours. The recording equipment was conventional, and consisted of a cathode follower probe close to the recording electrode, preamplifier, amplifiers, cathode-ray oscilloscope, loud-speaker, camera, and tape-recorder. Recording was made through resistance-capacity linked with the amplifier, the time constant being suitably adjusted. The observations described were made upon cells whose electrical signs were initially negative spike potentials relative to the earth, although some did show an initial small positive deflection. Cells displaying initially positive potentials showed signs of damage, and were abandoned. To determine the site and size of the receptive fields and the response properties of individual cells, physiological stimuli were offered such as bending hairs with an air-puff or with small brushes, touching the skin lightly with a glass rod as well as pressing it with a wooden stick; the elevated skin was stimulated with strong stimuli such as pressure; joints were rotated by hand; limbs were squeezed. When the receptive field of the cell under examination extended so that it touched the stereotaxic instrument, it could be freed from the instrument by lifting a limb with a block or by suspending a limb. When this was impossible, the observation of the cell was terminated. The outline of the receptive field was drawn on the skin surface with Indian ink, and subsequently measured and reproduced on graph paper. The receptive fields drawn were photographed after the experiment, if necessary. In some experiments cutaneous sensory input from the receptive field was blocked by subcutaneous inRrfcrcncrr p . 196

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jection of 0.5 % procaine solution. After the observations with this procedure of procaine application were finished usually no further unit was studied in the same animal so as to rule out the observativon of abnormal cell activities. At the end of the experiment the animal was killed by an intravenous injection of an overdose of pentobarbital sodium. Reference pipettes were inserted into each hemisphere with the microdrive. The head was then removed and placed in a 10% formaldehyde solution. After fixation the block of the brain was embedded in celloidin and sections were cut serially at 30 p, in the frontal plane. The sections were stained with 1 thionin. The approximate position of each penetration was determined from photographs. RESULTS

( I ) Excitatory peripheral receptive fields of thalamic neurons

When the discharges of a single neuron were isolated, the peripheral field activating it was located by mechanical stimulation of the body surface, and the sensory modality determined as described by Nakahama et al. (in preparation). The modalities of hairy skin were subgrouped into 2 categories in this experiment: hair and touch. It is well known that hair receptors respond easily to movement of the hair without deformation of the skin; they are also driven by weak puffs of air. When a sustained pressure is applied to such a receptive field, a short train of impulse discharges is elicited only upon the application and removal of the stimulus. In addition to these rapidly adapting units, moderately adapting hair units were also observed. Touch units show a very slowly-adapting response during constant stimulation. In response to this type of stimulus the discharges are graded by the degree of pressure used. Touch units d o not show variation in their activity patterns, nor are they activated by soft puffs of air applied to the skin. After the determination of the modality type, the fur was carefully clipped, and the receptive field outlined with a small glass rod. Of the 134 neurons whose receptive fields lay in the hairy skin, 94 were classified as hair, 32 as touch, and 8 could not be classified. It was difficult to find a pressure unit in hairy skin which was activated only by pressure on the skin. Of the 32 touch neurons in the ventrobasal nuclei of the thalamus, 9 were related to receptive fields limited to 1-5 discrete spots; the distance between these spots was generally less than 5 mm (figs. 3,9). Twenty-three neurons were related to continuous fields. A constant mechanical deformation of the skin within the receptive field for a ‘touch’ neuron evoked a high frequency discharge which was maintained during steady stimulation. In response to this type of stimulus the discharge was graded by the degree of pressure used. Touch neurons showed no variation in their activity patterns from phasic to slowly-adapting responses. A very high frequency of discharge (approximately 200/sec) was obtained by the rapid movement of a smooth glass rod across the touch spot or by the rapidly appiied vertical movement of a stimulating probe. This phenomenon was not observed for hair units. Six ventrobasal thalamic units were activated by both air-puff and light mechanical

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stimulation applied to the hairy skin, and by light mechanical or pressure stimulation applied to the toe pads or plantar cushions of the feet. The receptive field was continuous along the boundary between hairy and non-hairy skin. Following the de-

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privation of either the hairy or non-hairy cutaneous input, the stimulus applied t o the remainder of the field still activated these neurons. These findings support the view that convergence from hairy and non-hairy skin exists for ventrobasal thalamic neurons. The area of the receptive field in hairy skin for each of the 83 hair units and the area of the 23 touch units is shown in Fig. 1 by dots and crosses, respectively. Eight hair References p. 196

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units showing peripheral receptive areas of more than 100 cm2 and 9 touch units related to the spot-like field are not included in this graph. Sixteen hair units which showed wide peripheral receptive fields of over 31 cm2 were eliminated from the statistical calculations, since they will be treated in another group, similar to the cortical units of Brooks et al. (1961a, b). A fairly good correlation exists between the size of the field of hair units and its distance from the tip of the forelimb (r = 0.58). A good correlation was also found for touch units (r = 0.71). This corresponds well with the data of Mountcastle (1957) and Mountcastle and Powell (1959) regarding neurons of the somatic sensory cortex of cats and monkeys. It is clear from Fig. 1 that the size of the receptive field of hair units is more than 6 times as large as that of the touch units. Almost the same results were obtained for the hindlimb, although the number of units was too small to treat statistically. To compare the size of the receptive field of hair units in the ventrobasal thalamic nuclei with that in the first order afferents, a dashed line in Fig. 1 indicates the mean values of the sizes of the fields of first order fibers innervating hairs, recorded by Nakahama et al. (in preparation). The size of the field is more than 30 times larger for the ventrobasal thalamic neurons than for the first order afferents. ( 2 ) Inhibition

(a) Surround inhibition. For ventrobasal thalamic neurons activated from cutaneous fields, spontaneous or evoked firing in response to sensory stimulation was sometimes reduced by stimulation of the skin. The inhibitory field was continuous with the excitatory field: surrounding it either partially or completely. This is in agreement with the results obtained in the somatic sensory area of the cerebral cortex by Mountcastle (1957, 1961), Mountcastle and Powell (1959), and Anderson (1962). They classify this type of inhibition as surround or afferent inhibition. An example is given in Fig. 2. A unit was activated by light mechanical stimulation to the excitatory receptive field of skin, but not by bending hairs with an air-puff (A). The modality of this unit was classified as touch. The spontaneous discharges of the unit were reduced by light mechanical stimulntion applied to the skin surrounding the excitatory field (B). The area of skin whose stimulation caused inhibition of the spontaneous discharges covered a large area of the arm, completely surrounding the excitatory field. A similar inhibitory phenomenon was observed even after deprivation of cutaneous input from the excitatory field (C). After the cutaneous input from the inhibitory field was blocked by subcutaneous injection of procaine solution, no inhibition was caused by stimulation applied to the inhibitory field (D). This apparently indicates that the inhibitory effect was derived from the skin. ( b ) Deep to skin inhibition. This type of inhibition was described in the cat motor cortex in front of the post-cruciate dimple, by Brooks et al. (1961a, b), described by them as ‘cross-modality inhibition’, and defined as ‘cessation of response to one type of stimulus caused by simultaneous application of another type’. In the present study this type of inhibition was found also for ventrobasal thalamic neurons. Joint movement or pressure on deep structures inhibited either spontaneous discharges, or those

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Fig. 2. Surround inhibition in the ventrobasal nucleus of the thalamus. A unit was activated by light mechanical stimulation applied t o the receptive field of the contralateral forepaw (E), as indicated in the figurine drawing. The discharge rate was reduced by a puff of air or mechanical stimulus within a much larger surrounding area (I). A and B, graphs showing the impulse frequency of the unit when the stimuli were applied t o the excitatory and the inhibitory receptive fields, respectively. C and D, the discharge rate with the application of stimuli to the inhibitory field after procaine solution was injected subcutaneously into the excitatory and the inhibitory fields, respectively. Ordinate, number of discharges per sec; abscissa, time in sec. The solid and the dotted bars at the bottom of each graph indicate the approximate time and duration of the mechanical and the air-puff stimulation respectively. The shaded area indicates the spontaneous discharge rate of the unit without any stimulation.

evoked by cutaneous stimulation. The inhibitory areas were usually located near the excitatory ones. An example is given in Fig. 3. The modality of the neuron was touch and the receptive field consisted of 4 touch spots located on the preaxial forearm near the elbow. An initial high frequency response, reaching about 170 impulses/sec, was caused by the application of steady pressure to one of the discrete touch spots, followed by a slow decline to a more or less steady rate of discharge. With release of the pressure the discharge rate dropped at once to approximately zero, and then returned slowly to its spontaneous rate over a period of 10-20 sec (A, B). The stronger the stimulus intensity, the longer the duration of the poststimulus depression. The rate of spontaneous discharge was reduced by squeezing deep tissues under the touch spots after the deprivation of cutaneous input from the peripheral area covering 4 touch spots (C), and no poststimulus depression was observed, indicating that the poststimulus depression is not due to deep structures. No effect was obtained with the application of any cutaneous stimulus. References p. 196

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Time (sec ) Fig. 3. For legend see p. 187. Hair

T -186

T-140

T -63

Touch

I

I T-115

Fig. 4. For legend see p. 187.

VD

E X C I T A T I O N A N D I N H I B I T I O N IN

NEURONS

Before

I87

/

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-

Mechanical stimulus E

40. C

0

-

10

20

30

40

50

60

Time(sec

OO

lo

26

-.30 Time(sec )

Fig. 5. Showing mixed type of both surround and deep to skin inhibition. A unit could be activated with the movement of hair without deformation of the skin by a puff of air applied to the contralateral forepaw (E), as indicated in the figurine drawing. The unit could be inhibited either by air-puff stimulus within a much larger surrounding area ( I ) or mechanical stimulus applied to deep structures located under the excitatory receptive field (X). A and B, graphs indicating the impulse frequency of the unit when the stimuli were applied to the excitatory and the surround inhibitory receptive field respectively. C and D, the discharge rate with the application of stimuli to the excitatory and the surround inhibitory field, respectively, after subcutaneous injection of procaine solution to the excitatory field.

Fig. 3. Figure showing deep to skin inhibition. A unit could be driven by light mechanical stimulation applied to each of 4 discrete spots of the contralateral preaxial forearm, as indicated in the drawing. A and B, graphs showing the rate of discharge with the application of constant pressure to spots A and B, indicated in the drawing, respectively. C, response frequencies with the application of squeezing stimulus to the small area covering touch spots where procaine solution was subcutaneously injected. Fig. 4. Spatial relation of the excitatory and inhibitory receptive field for 6 units. The modality of the excitatory field was hair in three cases (upper drawings), and touch in the other three (lower drawings). The excitatory field is indicated by black. The rate of discharge was reduced with the application of pressure to deep tissue around a joint, as indicated by crosses in the drawings, or with joint rotation of which the direction is shown by arrows. Rtfirencc,\ p. 190

188

H . N A K A H A M A , S. N l S H l O K A A N D T. O T S U K A

The topographical relation of the excitatory and inhibitory receptive field was mapped, using physiological stimuli. Several examples are indicated in Fig. 4. Usually the inhibitory field was located in the joint tissue under or near the excitatory field. The location of the inhibitory field was easily determined after the sensory block, since the effects produced by cutaneous stimulation were then eliminated, and only that of deep tissue origin remained. For example, for unit T-140 the inhibitory effect was induced by movement of the wrist joint, or by squeezing the wrist. A mixed type of both surround and deep to skin inhibition was observed in some ventrobasal thalamic neurons. An example is illustrated in Fig. 5 . The modality of the receptive field was hair. The excitatory field was found in the contralateral forepaw. The neuron was activated by the movement of hair without deformation of the skin by air-puff and mechanical stimulation, and the poststimulus depression was observed (A). The spontaneous discharges of the unit were reduced by mechanical or airpuff stimulation applied to the skin surrounding the excitatory field (B). After the cutaneous input from the excitatory field was blocked by subcutaneous injection of procaine solution, air-puff stimulation of the excitatory field had no effect, although mechanical stimulation applied to deep tissue under the excitatory field still reduced the rate of spontaneous discharge (C). Surround inhibition was observed even after the deprivation of cutaneous excitatory input. ( 3 ) Appearance of new receptive field

In general, the size or shape of the receptive field did not change with time during the course of long observations, nor during repeated stimulation, as already shown by Poggio and Mountcastle (1963). However, the fields often changed after deprivation of cutaneous sensory input by subcutaneous injections of procaine solution. An example is indicated in Fig. 6. A unit was activated by bending hairs with an air-puff applied to the peripheral receptive field of the preaxial side of the forearm. In this test the field was found to be discontinuous, and each of the two parts were separate, although the modality was the same. In only one out of 59 units was a discontinuous field observed in a cell. Less than 2 min after the deprivation of cutaneous input for one of them, the unit could be activated by air-puff stimulation applied to other fields where no response could be evoked before the deprivation. The field was continuous with the deprived one and partially surrounded it. This new excitatory receptive field did not disappear for some hours during the course of the experiment, even after the recovery of the deprived field from local anaesthesia of procaine solution. Similar results were obtained in other units which were activated by air-puff or light mechanical stimulation of the receptive field. Some examples of the results are given in Fig. 7. The modality of the new receptive field was skin, and not deep. The new receptive field often appeared in the proximal side of the body, continuously with the deprived field. Fig. 8 shows that the new excitatory receptive field from the tip of the contralateral hindleg to the hip appeared after the deprivation of cutaneous input from the excitatory field of the contralateral ankle. The excitatory field was also observed in the

EXCITATION A N D INHIBITION IN

VD

I89

NEURONS

ankle of the ipsilateral hindleg. The modality of these fields was hair in all cases and the fields remained for 2 h during the experiments. Since the ipsilateral side was not tested before the sensory block, it is difficult to determine whether the ipsilateral

17' after

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1 sec. Fig. 6. Oscillograph records showing the appearance of the new receptive field with cutaneous input deprivation. The unit could be activated by air-puff stimulation of the two separated receptive fields in the preaxial side of the contralateral forearm, as indicated in the figurine drawing. Air-puff stimuli were applied to the areas shown by the dots on the figurine drawing. Records 1, 2, 3, and 4 are from samples taken from the spots indicated on the drawing before and after subcutaneous injection of procaine solution into the whole receptive field covering spot 1. Area (3) indicates the receptive field newly-appearing covering spot 3. References p . 196

190

H. N A K A H A M A , S. N l S H l O K A A N D T. O T S U K A

Hair

Fig. 7. A representative sample of newly-appearing receptive fields (dots) of units after subcutaneous injection of procaine solution to the excitatory field (black).

B -Air-puff

Time (sec )

Time ( s e c )

Fig. 8. Showing the wide appearance of the new receptive field (dots) following subcutaneous procaine injection to the excitatory field (black) of the ankle of the contralateral hindleg. Graphs A and B indicate response frequencies when air-puff stimuli were applied to points A and B of the receptive fields, respectively, as shown in the figurine drawing, 50 min after procaine injection.

E X C I T A T I O N A N D I N H I B I T I O N IN

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.Before

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Time ( sec i Fig. 9. Appearance of the new receptive field (dots) after subcutaneous procaine injection to the small area covering the excitatory touch spots (.) of the contralateral preaxial forearm. A, graph indicating the rate of discharge when constant pressure was applied to one of the touch spots. B, the response frequencies when a puff of air was applied to the inhibitory receptive field. C,response frequencies with the application of air-puff stimulation to the inhibitory field after procaine injection. D, discharge rate with the application of light mechanical stimulus to the receptive field newly appearing on procaine injection, as indicated in the figurine drawing.

field appeared after the deprivation. This point must be investigated. This was the only cell out of 59 units in which ipsilateral projection was found. The new excitatory receptive field was also observed in the touch unit surrounded by the inhibitory field, as shown in Fig. 9.The unit was driven by gentle mechaiiical stimulation of two touch spots of the contralateral preaxial forearm, but not by airpuff stimulation. Application of steady pressure to one of the touch spots caused a rapid-onset transient response which reached about 120 impulses/sec. Thereafter the response decreased to a more or less steady rate of discharge of 55 impulses/sec. With release of pressure the rate of discharge almost dropped to zero. Thereafter the discharge frequency slowly returned to its spontaneous rate over a period of 30 sec (A). The spontaneous discharges were reduced by application of an air-puff to the surrounding inhibitory field (B). This inhibitory effect was also observed even after the deprivation of cutaneous input from the area covering 2 touch spots (C). In addition to this phenomenon, the new excitatory receptive field was observed after the deprivation (D). Gentle mechanical stimulus applied to this field was more Refwences p. 196

192

H. N A K A H A M A , S. N l S H l O K A A N D T. O T S U K A

effective in driving the unit than was an air-puff stimulus. Air-puff stimulation to the inhibitory field produced a cessation of the evoked responses caused by stimulation of the new receptive field.

(4) Reversal of responses Reversal of responses from inhibitory to excitatory following the deprivation of cutaneous inpu. from the excitatory receptive field is illustrated in Fig. 10. A unit was driven in a slow, adapting way by means of light mechanical stimulation of the excitatory receptive field of the contralateral forepaw, and its spontaneous discharge was reduced by a puff of air or a mechanical stimulation within the surrounding inhibitory area (A). The discharges of the touch unit were evoked by mechanical stimulation applied to the excitatory field. Interpolation of air-puff stimulation within the inhibitory field induced abrupt inhibition of discharge, in spite of the persisting excitatory stimulus. After the removal of the inhibitory stimulus response to the continuing excitation persisted (B). Less than 1 min after the deprivation of cutaneous input from the excitatory field, the rate of the spontaneous discharge of the unit was increased with the application of air-puff or mechanical stimulation of the surrounding area which was the inhibitory field before the sensory block.

( 5 ) Classification of cell types The classification of neurons in the inhibitory receptive field, the appearance of a new receptive field, the mixed effect of both, or reversal of response from inhibitory to excitatory are listed in Table I. Although no systematic study was made of the TABLE I CLASSIFICATION OF CELL TYPES

No. of cells examined

Surround

Deep to skin

New receptive field

36

7

5

4

0

non-spot 19

7

Modality

Hair

( spot

Type of inhibition

Mixed

Inhibition

4

ype

Excitat ion

9

1

0

2

0

2

0

5

4

2

1

Touch

occurrence of these types, a detailed study was made of at least 59 units which were examined by the deprivation of cutaneous input from the excitatory receptive field of the contralateral limbs. The results obtained from these 59 units are given in Table 1. Hair units showing a wide receptive field have been omitted from this Table. Surround

E X C I T A T I O N A N D I N H I B I T I O N IN

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60

0

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50

20

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30

40

Time (se c )

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40

Time ( s e c )

Fig. 10. Reversal of responses from inhibitory to excitatory. Surround inhibition was observed before subcutaneous injection of procaine solution, as indicated in the drawing. A, graph showing the response frequencies when constant pressure was applied to the excitatory receptive field, air-puff stimulus to the inhibitory field, and squeezing stimulus to the contralateral forepaw covering the excitatory and inhibitory fields, successively. B, the rate of discharges when an air-puff stimulus was applied to the inhibitory field during the mechanical stimulus to the excitatory field. C . response frequencies caused by air-puff stimulus to the inhibitory field and squeezing the paw, successively. For details, see text.

and deep to skin inhibition was less in hair units than in touch units. The mixed type of both the inhibitory receptive field and the appearance of the new receptive field was also less in hair units than in touch units. This seems to suggest that touch units behave more subtly and delicately than do hair units. DISCUSSION

The results reported in this paper are of two-fold interest: firstly, the peripheral receptive field of the newly-appearing ventrobasal thalamic neurons following the deprivation of cutaneous input from the excitatory receptive field, and secondly the receptive area of some touch units was limited to 1-5 discrete spots and the distance between these spots was generally less than 5 mm. It was shown by Poggio and Mountcastle (1963) that ventrobasal thalamic neurons of the unanaesthetized chronically denervated head of monkeys did not show changes in the size or the shape of the peripheral receptive field during repeated and prolonged References p. 196

194

H. N A K A H A M A , S. N I S H I O K A A N D T. O T S U K A

stimulation of the field for several hours. The findings in the present report support this view. However, ventrobasal thalamic neurons at first related t o receptive fields, over a given area of skin, and responsive to one form of stimulation, following cutaneous input deprivation, were activated by another kind of manipulation applied to a larger cutaneous area. This new receptive field was observed in 16 out of 59 units almost immediately after the sensory deprivation. Accordingly, it is conceivable that this is a common phenomenon. The new receptive field does not seem to be due to the injury effect of adjacent neurons, since it appeared only after the sensory block, and the appearance occurred very quickly. If the appearance had occurred when the adjacent neuron died, it might have occurred without sensory deprivation. This, however, was not observed in this study. The possibility that the appearance could be caused by the release of an inhibitory effect from the excitatory receptive field following the sensory block seems plausible. If so, the new receptive field should disappear with the restoration of the excitability of the deprived receptive field. However, it was observed for some hours, even after the previously blocked excitatory field had regained its excitability. Therefore it seems unlikely that this phenomenon is induced only by a release from inhibition. Plastic changes such as habituation might be produced after the occurrence of the release effect. Otherwise the phenomenon may be regarded as only a change in excitability, so that the subliminal fringe can now excite. A sensory block to the excitatory receptive field might be associated with a sensitization process of the surrounding field. An odd observation is that the response of the ventrobasal thalamic unit reversed from inhibitory to excitatory almost immediately after the deprivation of cutaneous input from the excitatory field. The neural mechanism of the reversal of the response might be the same as that of the appearance of the new receptive field. The pattern and the rate of the spontaneous or background activity is changed after the sensory block (manuscript in preparation). Whether the appearance and the reversal are related to the spontaneous or background activity is under investigation. Several touch units observed in the ventrobasal thalamic neuron received projections from a receptive area limited to discrete spots, which bears on the findings of Maruhashi et al. (1952), Hunt and McIntyre (1960), Iggo (1963), and Nakahama et al. (in preparation) who indicated that certain first order afferents ended in such touch spots. It has been shown by Iggo (1963), and Nakahamaetal. (in preparation) that a very high frequency of discharge is induced in the first order afferents by the quick movement of a smooth glass rod across the touch spot. Similar results were obtained for the third order elements of the system. This strongly supports the view of Mountcastle (1961), and Poggio and Mountcastle (1963) that the lemniscal system is one of precise anatomical connections, having the capacities for transmitting rapid and rapidly changing neural activity. The properties of touch units of the ventrobasal thalamic nuclei differ from those of hair units. The sizes of the receptive fields of touch units are much smaller than are those of hair units. Touch units showed very slowly adapting responses during constant pressure, and the rate of discharge was graded by the degree of pressure used, while hair units showed rapidly or moderately adapting responses. Touch units be-

EXCITATION A N D INHIBITION IN

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195

haved in more complex ways than did hair units before and after the cutaneous input deprivation (Table 1). It is well known, since the finding of surround inhibition in cortical neurons by Mountcastle, that its functional significance is to preserve contrast, which serves to promote sensory localization, two-point discrimination and pattern and contour recognition. The functional meaning of deep to skin inhibition is not likely to be different from that of surround inhibition, since natural stimuli usually consist of mixed modalities, as already noted by Brooks et al. (1961b). In this study, surround, deep to skin inhibition and a mixed type of both were observed in the ventrobasal thalamic neurons. Mountcastle (1 957), and Mountcastle and Powell (1959) presented examples of neurons of the somatic sensory cortex of cats and monkeys in which, upon removal of steady pressure on the skin, the rate of discharge dropped at once to zero, and returned to its spontaneous and background rate over a period of a few seconds. This poststimulus depression was also observed in touch units of the ventrobasal thalamic complex i n this study, and in first order touch afferents, by Mountcastle and Werner (personal communication). They showed quantitatively that the duration of the poststimulus depression is a function of stimulus intensity. The convergence of hairy and non-hairy skin at the thalamic level was observed in this study, although it was not found at the first order afferents (Nakahama et al., in preparation). It is likely that the convergence occurs at a higher level than the second order afferents. SUMMARY

A study was made of excitation and inhibition of neurons in the ventrobasal nuclei of the thalamus in lightly anaesthetized cats. Modalities tested were hair-bending, touch, pressure, joint movement, and squeezing. The receptive fields for some touch units were limited to 1-5 discrete spots, whereas those of other touch units and of all hair units were continuous, and not limited to spots. In the latter units direct measurements of the size of the peripheral receptive fields were made, although hair units showing wide receptive fields of over 31 cm2 were not included in the statistical calculations. The size of the area of hair and touch units increased steadily from limb tip toward shoulder. The mean value of the size of the field of hair units is more than 30 times larger for the ventrobasal thalamic neurons than for the first order afferents, and is over 6 times larger in the thalamic neurons than in the neurons of touch units. In some single ventrobasal neurons, convergence from hairy and non-hairy skin was found within the continuous receptive field. The excitatory cutareous receptive field could be surrounded by the inhibitory cutaneous field of thalamic neurons (surround inhibition). Similarly, the inhibitory field could be located in the joint tissues under or near the excitatory cutaneous field (deep to skin inhibition). A mixed type of both surround and deep to skin inhibition was also detected. After subcutaneous injection of procaine solution into the excitatory receptive field, a new receptive field often appeared, and the reversal of responses from inhibitory to excitatory was caused in one example.

196

H . N A K A H A M A , S. N I S H I O K A A N D T. O T S U K A ACKNOWLEDGEMENTS

The experiment on sensory block was originally suggested by Dr. Vernon B. Mountcastle at the Johns Hopkins University, in whose laboratory one of the authors (Nakahama) learned the technique of natural stimuli and thalamic microelectrode recording during his stay in Baltimore as Fellow of the Rockefeller Foundation in 1961-1962. The authors wish to express their gratitude to Dr. Vernon B. Mountcastle not only for his criticism, but also for his help in writing this paper. This work was supported by grants from the Rockefeller Foundation (GA MNS 6370), and from the Education Ministry of Japan. REFERENCES V. E., (1961); Microelectrode studies of the cerebral cortex. tnt. Rev. Neurobiol., 3,67-136. AMASSIAN, AMASSIAN, V. E., BERLIN, L., MACY,J., JR., AND WALLER, H. J., (1959); Simultaneous recording of the activities of several individual cortical neurons. Trans. N . Y. Acad. Sci., 21, 395-405. ANDERSSON, S. A., (1962); Projection of different spinal pathways to the second somatic sensory area in cat. Acta physiol. scand., 56, Suppl. 194, 1-74. ARDEN,G. B., AND S~DERBERG, U., (1961); The transfer of optic information through the lateral geniculate body of the rabbit. Sensory Communication. W. A. Rosenblith, Editor. New York, London, John Wiley and Sons (p. 521). BROOKS, V. B., RUDOMIN, P., AND SLAYMAN, C. L., (1961a); Sensory activation of neurons in the cat's cerebral cortex. J. Neurophysiol., 24, 286-301. V. B., RUDOMIN, P., AND SLAYMAN, C. L., (1961b); Peripheral receptive fields of neurons BROOKS, in the cat's cerebral cortex. J. Neurophysiol., 24, 302-325. A. K., (1960); Properties of cutaneous touch receptors in cat. J. HUNT,c. c . , AND MCINTYRE, Physiol. (Lond.), 153, 88-98. IGGO,A., (1963); New specific sensory structures in hairy skin. Acta neuroveg. (Wien), 24, 175-180. LEVICK, W. R., AND WILLIAMS, W. O., (1964); Maintained activity of lateral geniculate neurones in darkness. J. Physiol. (Lond.), 170, 582-597. MARUHASHI, J., MIZUGUCHI, K., AND TASAKI, I., (1952); 1. Action currents in single afferent nerve fibres elicited by stimulation of the skin of the toad and the cat. J. Physiol. (Lond.), 117, 129-151. MOUNTCASTLE, V. B., (1957); Modality and topographic properties of single neurons of cat's somatic sensory cortex. J. Neurophysiol., 20, 408-434. MOUNTCASTLE, V. B., (1961); Some functional properties of the somatic afferent system. Sensory Communication. W. A. Rosenblith, Editor. New York, London, John Wiley and Sons (p. 403). MOUNTCASTLE, V. B., AND POWELL, T. P. S., (1959); Neural mechanisms subserving cutaneous sensibility, with special reference to the role of afferent inhibition in sensory perception and discrimination. Bull. Johns Hopk. Hosp., 105, 201-232. NAKAHAMA, H., AIKAWA, S., AND ARAI,Y., (1965); Modality and topographic organization of the first order afferents. In preparation. POGGIO,G. F. ,ANDMOUNTCASTLE, V. B., (1960); A study of the functional contributions of the lemniscal and spinothalamic systems to somatic sensibility. Central nervous mechanisms in pain. Bull. Johns Hopk. Hosp., 106, 266-316. POGGIO, G. F., AND MOUNTCASTLE, V. B., (1963); The functional properties of ventrobasal thalamic neurons studied in unanesthetized monkeys. J. Neurophysiol., 26, 775-806.

197

Histochemical Studies of the Brain with Reference to Glucose Metabolism NOBUO SHIMIZU

AND

TOMIYA ABE

Department of Neuroanatomy. Institute of Higher Nervous Activity, Osaka University Medical School, Osaka (Japan)

INTRODUCTION

It has been well established that the energy necessary for the functional actibity ofthe brain is supplied exclusively by the aerobic breakdown of glucose (Himwich, 1951; McIlwain, 1955; Elliott et al., 1955; Richter, 1957). This is performed through the Embden-Meyerhof pathway followed by Krebs’ TCA-cycle (Fig. I), which effectively produces energy-rich phosphate bonds. ATP thus produced is utilized for the transport of ions, for the synthesis of acetylcholine and for other processes connected with the nervous activity of the brain. Moreover, the brain has been shown tocontainthe Warburg-Dickens pathway (hexose monophosphate, HMP, shunt) (Dickens and Glock, 1951 ; Glock and McLean, 1954), the significance of which has not been fully clarified (Coxon, 1957). TPNH

TPN

A 1 1

6- Phosphogluconolactone,

IHgBhxyOn;e-mOnOphoSphateI

Glu,cose Glucose -6- P

G6PD

i----+Nucleic

acid

I

pathway Fructose-1.6-dip

i

I m DpN4

3- Phosphoglyceraldehyde

DPNH

Dihydroxyacetone-P Triose phosphate dehydrogenase

1.3- Diphosphoglycerate

j

Pyr u,vat e =Lactate

LR

i Fig. 1. Metabolic pathway of glucose. Refcrenrcs p. 2151216

I98

N. S H I M I Z U A N D T. ABE

The location and intensity of various enzymes related to the carbohydrate metabolism have been studied with histochemical methods by several investigators. Thus, histochemical studies of succinic dehydrogenase (SD) have made possible the characteristic mapping of this enzyme activity in mammalian brains (Shimizu et al., 1957a,b; Ortmann, 1957; Friede, 1959b,c, 1960, 1961a,b; Friede and Fleming, 1962), and offered morphological evidence on the extent and intensity of the functional activity in the various regions of the brain. Further, several studies concerning the distribution of enzymes related to glycolysis and HMP-shunt have been reported (Thomas and Pearse, 1961 ;Felgenhauer and Stammler, 1962; Friede and Fleming, 1963; Abe et at., 1963). However, it seems necessary to perform experiments on the interrelation of the 3 metabolic pathways of glucose by histochemical means. We recently found (1964) a new histochemical method for demonstrating aldolase (ALD), an important member of the Embden-Meyerhof pathway. Accordingly we chose ALD, glucose-6-phosphate dehydrogenase (G6PD) and SD as representative members of the Embden-Meyerhof pathway, HMP-shunt and TCA-cycle respectively, and compared their distributions in the brain of adult and developing rats, in order to obtain deeper insight into the functional and metabolic significance of these 3 pathways. MATERIAL A N D METHODS

The materials used consisted of the brains of 5 adult male rats. For the study on the postnatal changes the brains were removed from two rats of both sexes at each of the following ages: 2, 5,7, 10 days and 2,3,4 weeks. All the animals were killed by decapitation or cutting the carotid arteries without anaesthesia, followed by prompt excision of the brain. Fresh frozen serial sections 30 ,u thick were cut in the cryostat using a sliding microtome. The sections mounted on slides were allowed to dry at room temperature for 2 to 3 h, and subjected to the histochemical methods for ALD, SD, and G6PD, and complementarily to the method for lactic dehydrogenase (LD). For demonstration of ALD the dried sections were treated according to our newly reported method (Abe and Shimizu, 1964).The sections were fixed in 80% cold ethanol (0'4") for 20 min and again allowed to dry at room temperature for 30 min. These sections were incubated in the following mixture: 10 mlO.02 M sodium fructose-l,6diphosphate, 5 mg DPN, 10 mg nitro-BT, 10 ml 0.05 M arsenate-HCl buffer, pH 7.6; at 37" for 30 min. For SD the dried sections were incubated in a mixture composed of 5 ml 0.2 M sodium succinate, 5 m10.2 M phosphate buffer, pH 7.6, 10 mg nitro-BT and 10 ml distilled water according to Nachlas et al. (1957) for 20 min at 37". For location of G6PD the sections were incubated for 30 min in a mixture (10 mlO.006 M sodium glucose-6-phosphate, 5 mg TPN, 10 mg nitro-BT, 2 ml2/3 M BaC12, 10 ml 0.05 M veronal buffer, pH 7.4) prepared according to Nachlas et al. (1958b). LD was demonstrated according to Nachlas et al. (1958a) by incubating the sections for 60 min in a mixture composed of 4 m10.5 M sodium lactate, 13 mlO.1 M phosphate buffer, pH 7.4, 5 mg DPN, 5 mg nitro-BT and 3 ml distilled water. After incubation in each mixture, the sections were briefly rinsed in distilled water,

HISTOCHEMISTRY OF THE B R A I N

199

fixed in 10% neutral formalin for 1 to 12 h, dehydrated through graded alcohol solutions, cleared in xylol and mounted in Canada balsam. OBSERVATIONS

The distribution of SD, which was previously studied with the Seligman-Rutenberg method modified by Rosa and Velardo using neotetrazolium (Shimizu and Morikawa, 1957a) was confirmed by the present observation. Microscopic comparison of the location of SD with that of ALD showed a nearly identical distribution of both enzymes in the rat brain, though a slight difference in staining intensity was observed under the present staining conditions, SD appearing slightly stronger than ALD (Figs. 2 and 3). Accordingly we will describe the location of both enzymes at the same time. Both enzymes were mainly demonstrated in the gray matter appearing as blue formazan granules. The gray matter showed the characteristic variation in the different regions of the brain with respect to the enzyme activity in the neuropil and perikaryon (Figs. 6A, 10A and 1 IC). The white matter showed slight activity in the glial elements and blood capillary walls between the nerve fiber bundles, while the nerve fibers were entirely negative (Figs. 6A and I IC). As the histochemical distribution of G6PD of the whole brain of the rat has already been reported by Abe e t a / . (1963), the present description will be limited to enough to permit a comparison with ALD and SD. G6PD was seen both in the gray and white matters (Figs. 6B, 10B and 12C). In general, nerve cell bodies, glial cells and blood

Fig. 2. Nearly the same distribution of ALD (A) and SD (B) was seen in the bulbus olfactorius, bulbus olfactorius accessorius and nucleus olfactorius anterior. Both activities are very strong in the lamina glomerulosa and lamina gelatinosa (synaptic area), while they are less active in the cell layers. x 23. Refir1.rmrr.y p. 215/216

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N. S H I M I Z U A N D T. A E E

Fig. 3. Similar distribution of ALD (A) and SD (B) was demonstrated in the frontal section through the striatum, nucleus accumbens septi and tuberculum olfactorium, which are strongly reactive for bothenzymes. x 7.8.

capillary walls showed a moderate to strong activity, while the neuropil was very weak in activity. Nerve fibers of the white matter showed slight activity probably corresponding to the axis cylinders. However, the stria terminalis characteristically demonstrated an intense activity probably due to its densely packed parallel-running blood capillaries and the contained neuroglial cells. LD showed a faint general activity in the adult rat brain under the present conditions of staining, and a slight activity could usually be observed in the molecular layer of both cerebral and cerebellar cortices, and faint staining in the nerve cell bodies in various portions of the brain. Meninges and blood vessels

Of the cerebral meninges, the arachnoid membrane was slightly positive for SD and ALD, but strongly positive for G6PD. The intracerebral blood capillary walls exhibited only a weak staining for SD and ALD, while they showed strong activity for G6PD (Fig. 10B). Ventricular system

The choroid plexuses exhibited an intense activity for SD, ALD and G6PD in the choroid epithelium (Figs. 4 and 5). The ependymal cells were slightly to moderately

HISTOCHEMISTRY O F T H E B R A I N

20 1

Fig. 4. The distribution of ALD (A) and G6PD (B) in the frontal section through the psalterium lentiforni nucleus and rostra1 end of the hypothalamus. The supraoptic crest is negative for ALD (A), while it is reactive for G6PD (B). x 7.8.

positive for SD and ALD, but less reactive for G6PD. The medial margin of the caudate nucleus corresponding to the subependymal cell plate was characteristically negative or weak for SD and ALD, whereas G6PD revealed a rather strong reaction. Paraventricular structures, such as the supraoptic crest, subfornical and subcommissural organs, pineal body and area postrema were generally slightly reactive or negative for SD and ALD (Figs. 4A and 8A). However, G6PD was characteristically strong in the parenchymal cells of these structures (Figs. 4B and 8B). Neocortex

The gray matter of the neocortex showed the architectonic layers with respect to the enzyme distribution. Both SD and ALD were similarly demonstrated most markedly in layers I, Ill and IV, where the activity mainly occurred in the neuropil (Figs. 3A, B and 1 IC). In the deeper layers (V and VI) the general activity was rather weak, and slightly reacting nerve cells could be noticed. I n G6PD strongly staining neurons were demonstrated in all layers (excepting layer 11). where the neuropil reacted rather weakly (Fig. 12C). Layers I and 11 showed relatively slight activity. Solitary hyperactive cells first reported by Thomas and Pearse (1961) were found throughout the cerebral cortex and basal ganglia. References p . ZIJIZ16

202

N. S H l M l Z U A N D T. A B E

Rhinencephalon

( I ) Bulbus olfactorius The strongest activity for SD and ALD (Figs. 2A and B) was demonstrated in the lamina glomerulosa and lamina gelatinosa (molecular layer). Slight activity was shown in the laminae granularis interna and externa, and lamina cellularum mitralium. The lamina fibrosa and lamina medullaris showed faint activity in the glial cells and vascular walls.

Fig. 5. The distribution of ALD (A) and G6PD (B) of the hippocampus, basal ganglia and diencephalon. The paraventricular and supraoptic nuclei of the hypothalamus, stratum lucidum of the hippocampus and stratum granulosum of the g y m dentatus are very intense for G6PD (B), while they are weak for ALD (A). x 7.8.

The G6PD activity was most prominent in the lamina cellularum mitralium and laminae granularis interna and externa, while a slight reaction was seen in the laminae glomerulosa and gelatinosa and a moderate reaction in the lamina fibrosa. The lamina medullaris showed a moderate activity in the axis cylinder and a strong reaction in the glial cells between the nerve fibers. Similar location and intensity were also demonstrated in ALD and SD in the bulbus olfactorius accessorius (Figs. 2A and B): a slight activity for both enzymes was observed in the lamina granulosa (Cajal) and lamina cellularum, and a strong one in the lamina glomerulosa. The hyperactivity for G6PD was present in the lamina granulosa and rather less activity in the lamina cellularum (see Fig. 1B by Abe et al., 1963), and a weak one in the lamina glomerulosa.

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( 2 ) Tuberculum olfactorium Both activities of S D and ALD were similarly most intense in the plexiform layer, and slight and moderate in the pyramidal and polymorph layers (Fig. 3A and B). The islands of Calleja showed slight activity. G6PD was most active in the nerve cell bodies of the pyramidal layer. Both the plexiform and polymorph layers were moderately reactive. Characteristic hyperactivity for this enzyme was encountered in the solidly packed granular cells of the islands of Calleja, which has already been reported by Felgenhauer and Stammler (1962) and Abe et al. (1963). ( 3 ) Pyriform lobe The pyriform lobe presented an enzyme pattern similar to the tuberculum olfactorium. S D and ALD were demonstrated most strongly in the plexiform layer, slightly or moderately in both the pyramidal and polymorph (lamina multiformis) layers (Fig. 4A). G6PD was seen strongly in the nerve cells in the pyramidal layer, and was slight in the plexiform layer. The nerve cells rather less closely packed in the third layer were moderately reactive. ( 4 ) Hippocampus and gyrus dentatus The enzyme activity of both SD and ALD was shown most intensely in the stratum oriens and stratum moleculare of the hippocampus and the stratum moleculare of the gyrus dentatus (Figs. 5A and 6A). It was moderate or slight in the stratum lucidum of the hippocampus and the stratum granulosum of the gyms dentatus. Intermediate activity was seen in the stratum radiatum of the hippocampus and the stratum multiforme of the gyrus dentatus. G6PD was most active in the pyramidal cells and their processes of the hippocampus and in the granule cells of the stratum granulosum and the scattered large neurons of the stratum multiforme of the gyrus dentatus (Figs. 5B and 6B). The neuropil of each layer of both the hippocampus and gyrus dentatus reacted moderately.

Basal ganglia The caudate nucleus and putamen showed the most intense activity for both SD and ALD in the brain (Figs. 3A, B and 4A). The strong staining seemed to be attributable to that of the neuropils, and nerve cell bodies could hardly be perceived. The globus pallidus showed slight activity (Fig. 4A) and revealed dissemination of heavily stained nerve cells. A moderate activity of both enzymes was encountered in the amygdaloid nuclei, where the lateral nucleus (pars posterior) was somewhat more reactive than other nuclei (Fig. 5A). G6PD was demonstrated strongly in the nerve cell bodies as well as glial elements, and slightly to moderately in the neuropil of the striatum (Fig. 4B). In the globus pallidus the reaction was generally moderate and rather active nerve cells were disseminated. I n the amygdaloid nuclei the cortical nucleus showed a prominent reaction, while other nuclei reacted less strongly, the lateral nucleus (pars posterior) showing nerve cells of medium-size moderately stained (Fig. 5B). ReJercnces p. 215/216

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Fig. 6. Higher magnification of the location of SD (A) and G6PD (B) of the hippocampus. SD activity is intense in the stratum oriens and stratum moleculare, and rather weak in the stratum lucidum, while G6PD reaction is most intense in the pyramidal cells and their processes, and weak in the synaptic regions. x 120.

Diencephalon

In the thalamic nuclei the greatest activity for S D and ALD occurred in the anterior dorsal nucleus and reticular nucleus. Intermediate activity was shown in the ventral, anterior ventral, posterior and habenular nuclei, and medial and lateral geniculate bodies, slighter activity being in the medial and lateral nuclei and midline nuclear group (Fig. 5A). The subthalamus, especially the corpus Luysi, reacted strongly. The hypothalamus, except for the deeply stained mammillary body, generally exhibited slighter activity than the thalamus. The medial hypothalamus showed a moderate reaction, while the periventricular gray and the lateral hypothalamus reacted very weakly. Characteristically slight activity for both enzymes was given in the paraventricular and supraoptic nuclei (Fig. 5A). In the G6PD, stronger reaction was observed in the anterior ventral nucleus and corpus Luysi, rather less activity in the midline nuclear group, where the neuropil as well as nerve cell body reacted moderately. The ventral nucleus showed a strong reaction in the perikaryon, and weakest reaction in the neuropil (Fig. 5B). In the hypothalamus the enzyme activity was shown moderately to strongly in the medial and lateral hypothalamus, where the activity was present in both the neuropil and peri-

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Fig. 7. The distribution of ALD (A) and G6PD (B) of the midbrain. ALD is strong in the interpeduncular nucleus and in the nucleus of the oculomotor nerve, while G6PD is weak in the above nuclei. The stratum griseum superficiale of the optic tectum shows moderate to intense activity for both enzymes. x 7.8.

karyon. The neurosecretory nuclei showed characteristically prominent activity in the constituent nerve cell bodies (Fig. 5B). In the mammillary body the activity was strongest in the pars lateralis of the medial nucleus.

Mesencephalon S D and ALD similarly showed the most intense activity in the stratum griseum superficiale and interpeduncular nucleus, where constituent nerve cell bodies were hardly discernible (Fig. 7A). Moderate to strong reaction was observed in the nucleus interstitialis of Cajal, nucleus of the oculomotor nerve, substantia nigra, red nucleus, and nuclei of the inferior colliculus and lateral 1emniscus.Rather weak activity was seen in the central gray matter, strata griseum mediale and profundum. G6PD showed a characteristic hyperactivity in the disseminated nerve cells in the nucleus lateralis profundus mesencephali. Moderate activity occurred in the stratum griseum superficiale, interpeduncular nucleus, pars compacta of the substantia nigra and nucleus raphe dorsalis (Fig. 7B). In the red nucleus and nucleus of the oculomotor nerve the constituent nerve cell bodies showed moderate activity, and slight or weak activity in the neuropil. Pons Moderate to strong activity for both SD and ALD was shown in the dorsal and Rrferrnces p . 215/216

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Fig. 8. The location of ALD (A) and G6PD (B) of the cerebellum and the pons. Strong ALD reaction is seen in the motor and principal sensory nuclei and the nucleus of the mesencephalic root of the trigeminal nerve, the ve.itral cochlear nucleus, the superior olivary nucleus and the nucleus ofthe trapezoidbody. Moderate G6PD reaction is mainlyseen in thenervecell bodies in theabovestated nuclei, except for hyperactive cells in the nucleus laterodorsalis tegmenti. The pineal body seen between the inferior colliculi presents strong activity for G6PD, whereas it is weak for ALD. x 10.

ventral tegmental nuclei, nuclei pontis, and superior olivary nucleus (Fig. 8A). Similar activity occurred in the motor and sensory nuclei of the cranial nerves (Fig. 8A): motor and principal sensory nuclei of the 5th nerve, nucleus of the facial nerve, ventral and dorsal cochlear nuclei, and medial vestibular nucleus. In these nuclei the constituent nerve cells and surrounding neuropils reacted to a similar extent, and the nucleus of the mesencephalic root (5th nerve), the lateral vestibular nucleus and the nucleus of the trapezoid body (subnucleus medialis) were characteristic in showing deeply stained neurons and a lack or paucity of reacting neuropil. Rather weak reaction was seen in the locus caeruleus and central gray matter. In the G6PD preparation, hyperactive cells were disseminated in the nucleus laterodorsalis tegmenti (rostroventral part), while the locus caeruleus (caudodorsal part of the nucleus laterodorsalis tegmenti) showed moderate activity. Strong to moderate staining was observed in the nerve cell bodies of most of the nuclei (motor, sensory, coordination) ofthis region, while a weak reaction was found in the neuropil (Fig. 8B).

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Fig. 9. Distribution of ALD ( A ) and G6PD (B)of the medulla oblongata and cerebellum. Intense A L D activity is in the nucleus of the hypoglossal nerve, the inferior olivary nucleus, the gracilis and cuneate nuclei, and the nucleus of the spinal tract of the 5th nerve. G 6 P D is demonstrated strongly in the ala cinerea and inferior olivary nucleus, and is very marked in the cerebellar cortex. x 10.

Medulla

Strong reactions for SD and A L D were demonstrated in the nucleus of the hypoglossal nerve, lateral reticular nucleus, inferior olivary nucleus, nucleus of the descending tract of the 5th nerve, gracilis and cuneate nuclei (Fig. 9A), where the perikaryon and neuropil reacted at the same intensity (Fig. 10A). However the ala cinerea (dorsal motor nucleus and sensory nucleus of the vagus and nucleus of the solitary tract) reacted weakly (Figs. 9A and 10A). G6PD was shown strongly in the area postrema, ala cinerea and inferior olivary nucleus. In the dorsal nucleus of the vagus nerve both perikaryon and neuropil reacted markedly. Other nuclei showed moderate activity in the constituent neurons without activity in the neuropil (Figs. 9B and IOB). Cerebellum

The cerebellar cortex showed a strong activity for SD and A L D in the molecular layer, and in the glomeruli of the granular layer, moderate staining in the Purkinje cells (Figs. 8A and 9A). G6PD was most intense in the molecular layer, moderate in the Purkinje cells and in the granular layer (Figs. 8B and 9B).

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Fig. 10. Higher magnification of distribution of ALD (A) and G6PD (B) in the dorsal nucleus of the vagus nerve (upper third of the figure) and in the nucleus of the hypoglossal nerve (lower twothirds of the figure). ALD is stronger in the nucleus of the hypoglossal nerve (both nerve cell bodies and neuropil are reactive) than in the dorsal nucleus of the vagus. Nearly the reverse activity is seen in G6PD (B). The dorsal nucleus of the vagus shows G6PD in both the perikaryon and neuropil, while the hypoglossal nucleus shows the activity mainly in the nerve cell bodies. x 170.

In the cerebellar nuclei the constituent nerve cells strongly reacted for both SD and ALD with rather weak activity of the neuropil. G6PD showed weak to moderate activity of the constituent neurons. Postnatal changes of SD and ALD

As stated above, SD in the adult rat brain showed a similar enzyme location to ALD, though the staining intensity was somewhat stronger in the former than the latter. Both enzymes in the developing brains also showed similar relations to those in adult brains. Therefore, we shall describe mainly the distribution of SD. At 2 days after birth the activity of both enzymes was generally weak in the telencephalon and diencephalon, except for moderately reactive choroid plexuses. Very weak activity of the nerve cell bodies was demonstrated in the neocortex, hippocampus and striatum. However, the midbrain, pons and medulla showed moderate activities in the nucleus of the oculomotor nerve, the dorsal tegmental nucleus, the motor nucleus and nuclei of the mesencephalic root and the spinal tract of the 5th nerve, and nuclei of the facial and hypoglossal nerves. At 1 week after birth (Fig. 1 I A ) slight activity mainly present in the nerve cell

Fig. I I . Postnatal changes of SD in the cingular area (upper part of the figure) (the right hand border is the molecular layer) and adjacent corpus callosum (lower part of the figure). A (1 week) demonstrates weakly staining nerve cell bodies. and B (2 weeks) moderately t o strongly reactive nerve cell bodies and moderately active neuropil. A t 4 weeks (C) the neuropil and molecular layer are very reactive, nerve cell bodies being hardly discernible. The white matter is weakly stained in the glial cells at each age. x 180. Ruferences p . 215/216

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bodies was revealed in the cerebral cortex, hippocampus (stratum lucidum) and gyrus dentatus (stratum granulare). In the bulbus olfactorius the laminae glomerulosa and gelatinosa showed some activity. A slight activity was also demonstrable in the habenula, and the ventral nucleus of the thalamus and putamen. In the midbrain, pons and medulla strong to moderate activity was noticed in the various nuclei. In the cerebellum only the Purkinje cells showed a moderate reaction. At 2 weeks after birth (Fig. 11B) SD showed an evident increase in activity. The neocortex and putamen showed moderate activity in the perikaryon and similar activity in the neuropil. In the hippocampus and gyrus dentatus, the molecular layers showed an evident increase in activity and the stratum oriens a slighter one. The habenula and nucleus ventralis of the thalamus increased their activity both in the nerve cell bodies and neuropils. The nucleus reticularis showed a n intense activity in the perikaryon. In the midbrain the nucleus of the oculomotor nerve and red nucleus showed a prominent activity, moderate activity being in the nucleus of the lateral lemniscus, though less activity was found in the optic tectum, substantia nigra, corpus geniculatum mediale, and interpeduncular nucleus. Most of the motor and sensory nuclei in the pons and medulla showed a strong activity comparable to that in the adult. The cerebellar cortex demonstrated the strong activity in the Purkinje cells and a lesser activity in the remaining layers. At 3 weeks the enzyme activity nearly reached the adult level in most places, except for the neocortex, some thalamic nuclei (lateral and medial), optic tectum, nucleut pontis and cerebellar cortex, where the molecular layers or neuropils were somewhas less reactive than the adult. At 4 weeks (Fig. 1 IC) the distribution and intensity of both enzymes were similar to those of the adult brain. This observation on the postnatal changes of histochemical distribution of SD was generally similar to those reported by Morikawa (1958) and Friede (1959a), and also corresponded well to the biochemical data of Hamburg and Flexner (1957).

Postnatal changes of G6PD At 2 days after birth moderate t o strong activity.for G6PD was demonstrated both in the gray and white matters of the whole-brain. The outer layers of the cerebral cortex showed moderate activity in the celi.bodies of the nervous elements. The white matter of the corpus callosum and centrum iemiovale exhibited 7trong activity in the glial cells. In the hippocampus the pyramidal cells showed a moderate staining. In the hypothalamus moderate activity was observed .in the paraventricular and supraoptic nuclei. In the midbrain, pons and medulla.diffuse moderate staining was demonstrated, and moderately staining nerve cell bodies could be discernible in the nuclei of the oculomotor, vagus and hypoglossal nerves. In the cerebellum only the Purkinje cells showed moderate activity. Paraventricular structures, such as the subfornical and subcommissural organs, pineal body and area postrema showed a moderate activity. At 1 week after birth (Fig. 12A) moderate to strong reaction was observed similarly in the gray and white matters. Outer layers of the neocortex and hippocampus

Fig. 12. Postnatal changes of G6PD in the cingular area and adjacent corpus callosum. At 1 week (A) weakly reactive nerve cell bodies in the gray matter and strongly reactive glial cells with cytoplasmic ramifications in the white matter are seen. B (2 weeks) shows strongly reactive nerve cell bodies as well as moderately to strongly staining neuropil and molecular layer. In C (4 weeks) nerve cell bodies of the cerebral cortex are clearly revealed due to the decrease in activity in the neuropil. The white matter contains reactive glial cells, which show decrease in size. x 180. Re//ermrrs p. 2151216

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showed a moderate activity in the nerve cell bodies. The white matter showed strong activity in the glial cells and their processes (Fig. 12A). In the diencephalon the midline nuclear group of the thalamus and paraventricular nucleus of the hypothalamus showed a slight staining. The midbrain, pons and medulla showed diffuse moderate activity. Characteristic hyperactive cells were seen in the nucleus laterodorsalis tegmenti, and moderate staining were in the locus caeruleus, motor nucleus and nucleus of the mesencephalic root of the trigeminal nerve and nuclei of the vagus and hypoglossal nerves. At 2 weeks after birth (Fig. 12B) an evident activity was observed in the gray matter and a characteristic strong activity in the glial cells and their processes within the white matter. The cerebral cortex, putamen and amygdala showed strong activity mainly within the nerve cell bodies. Glial cells present in the corpus callosum exhibited strong staining. Hyperactive solitary cells made their first appearance in the cerebral cortex and striatum. Strong staining for G6PD was demonstrated in the nerve cell layers of the hippocampus and gyrus dentatus. In the diencephalon the ventral and medial nuclei of the thalamus and lateral habenular nucleus showed some activity both in the nerve cell bodies and neuropil. The hypothalamus showed strong staining in the nucleus ventromedialis. Hyperactive cells were demonstrated in the nucleus laterodorsalis tegmenti of the midbrain and pons. In the cerebellum the Purkinje cells, the inner half of the molecular layer and the glial elements of the white matter showed strong staining, while the granular layer showed a slight staining. By 3 to 4 weeks (Fig. 12C) the activity seemed slightly to decrease in some gray matters, whereas the decrease occurred more markedly in the neuropil than in the nerve cell body. On the other hand the activity of the white matter appeared to be confined to the glial cell bodies, which decreased in size but reacted intensely. For example the ventral thalamic nucleus evidently gave a decreased activity. The pons and medulla generally showed rather decreased activity, while the molecular layer of the cerebellum exhibited very intense staining. LD generally showed a similar distribution and intensity to G6PD up to 10 days after birth, except for some differences. L D activity was weak in the white matter, but stronger in the matrix layer around the lateral ventricle, external granular layer of the cerebellum, ependyma and choroid plexus. From 2 weeks on the staining of the brain stem became curiously slighter for reasons unknown. DISCUSSION

ALD is an important member of the Embden-Meyerhof pathway and might constitute the rate-limiting step in this pathway in the brain (Buell et al., 1958). Although our ALD method demonstrates ALD with concomitant triose phosphate dehydrogenase and DPN-diaphorase, it gives clear and constant staining and fairly good correspondence with biochemical data (Sibley and Lehninger, 1949). Upon applying this method to the rat brain and other tissues we found that the distribution of ALD surprisingly coincides with that of SD. These histochemical data seem to indicate that the Embden-Meyerhof path might be intimately correlated or connected with the

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TCA-cycle in order efficiently to yield ATP. Parallelism between glycolytic and respiratory activities has been demonstrated by the biochemical studies of the developing as well as the adult brain (Himwich. 1951 : Mcllwain, 1955). According to Hamburg and Flexner (1957) ALD and SD in the cerebral cortex of the rat showed a parallel rapid increase from 10 to 25 days after birth. The histochemical location of ALD and SD generally showed prominent activity in the molecular layers or neuropils, which are rich i n synaptic contacts, and rather weak activity in the nerve cell bodies with some exceptions. Microanalytical data by Buell et a/. (1958) showed that the molecular and dendritic layers are far more active for enzymes of the Embden-Meyerhof path than the cell body layers in the cerebellum and Ammon's horn. These observations support the concept that the energy gained through the Embden-Meyerhof path and the TCA-cycle might be mainly used for the nervous activity in the synaptic areas of the brain. The above consideration is also substantiated by the observations in the developing brain, which showed later appearance and increase of both enzyme activities in the molecular layer or neuropil from 2 to 4 weeks after birth following initial weak activity up to 2 weeks. This finding also indicates that formation and growth of the brain structures in neonatal stages might not be directly concerned in the Embden-Meyerhof path plus the TCA-cycle. G6PD showed several characteristic locations in the brain. First, this enzyme occurred strongly i n the paravcntricular structures, such as the supraoptic crest, subfornical and subcommissural organs, pineal body and area postrema. Secondly, the enzyme activity was demonstrable very strongly in certain nuclei, such as the lamina granulosa of the bulbus olfactorius accessorius, islands of Calleja, paraventricular and supraoptic nuclei of the hypothalamus, nucleus laterodorsalis tegmenti and hyperactive solitary cells in the neocortex and striatum. I n theabove nuclei as well as moderately staining regions, the activity was gencrally stronger in the nerve cell bodies than the neuropil, this being different from SD and ALD. Thirdly, the enzyme activity was observed in the glial cells between the fiber bundles of the white matter. The functional significance of HMP-shunt in the brain has not been fully established. Reduced TPN produced by the action of G6PD is thought to be a hydrogen donor in the reductive synthesis of biologically important substances (cf. Klingenberg and Bucher, 1960). The neuroglial cells (especially oligodendrocytes), which showed especially strong G6PD in the developing white matter, might produce lipids of the myelin sheaths through reduced TPN. We made several histochemical observations on paraventricular structures: heavy deposition of glycogen (Shimizu and Kumamoto, 1952). weak or low activity of succinic dehydrogenase and cytochrome oxidase (Shimizu ef a/.. 1957a.b), strong monoamine oxidase activity (Shimizu ef a/., 1959; Hashimoto et a/., 1962). and an abundance of catecholamine containing granules (Shimizu and Ishii, 1964). Similar histochemical characteristics were also seen in the visceral portions of the brain, such as the hypothalamus and ala cinerea. From these findings the structures might be presumed to be principally concerned in the active catecholamine metabolism. G6PD might then play some role in hydroxylation of phenylalanine (Kaufman. 1957) or in formation and maintenance of a reduced cellular milieu (Hotta, 1962; etc.). RQ/rrmr.es p. 115/216

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Moreover, HMP-shunt was shown to play an important part in the nucleic acid, and therefore protein, formation (Beaconsfield and Reading, 1964). G6PD activity was generally localized in the nerve cell bodies in contrast to the low activity in the neuropil, with some exceptions in the hypothalamus and dorsal nucleus of the vagus nerve. Most nerve cells in cytoarchitectonic layers or nuclei showed moderate activity, which were somewhat stronger in the young developing brain than the adult as shown in the above stated observation. Reading (1964) recently reported similar results that the relative importance of the HMP-shunt declines with increasing age and development in the normal retina. These observations make us suppose the enzyme to be involved in nucleic acid, and therefore protein, synthesis. .' LD activity in the adult rat brain was curiously very weak under the present conditions of staining, while it was very intense in the neonatal brain as described above. These observations make us suppose that LD might take a special position in the glycolysis entirely different from ALD, showing some similarity to G6PD at least in the neonatal brain. Therefore it seems unreasonable to consider LD as an indicator of the Embden-Meyerhof pathway. Actually, it has been shown that oxidative metabolism of glucose in the brain is performed by the mechanism that does not involve lactate as an intermediary (Jowett and Quastel, 1937). Accordingly, it seems likely that the main path for energy production i n the brain is the Embden-Meyerhof pathway followed by the TCA-cycle; and that HMP-shunt, without relation to the energy yield, plays a part in the formation and maintenance of the structures, besides special activities in some restricted structures. SUMMARY

( I ) In the brain of adult as well as developing rats, localization of ALD, SD and G6PD was histochemically investigated and compared. The significance of these enzymes in the function and metabolism of the brain was discussed. (2) ALD and SD showed nearly the same distribution. Strong activity was demonstrated in the neuropil as well as in the perikaryon of the gray matter. Shortly after birth both enzymes showed very low activity followed by marked increase in activity especially in the neuropil from 2 to 4 weeks after birth. These observations suggest that both enzymes might be intimately related to the nervous function by the energy-rich phosphate bonds, which are yielded through the EmbdenMeyerhof path plus Krebs' TCA cycle. (3) G6PD showed a location different from ALD and SD. It was generally weak in the neuropil, and strong in the perikaryon and neuroglial cells i n the white matter. Moreover, characteristically strong activity was seen in the paraventricular structures, neurosecretory nuclei, lamina granulosa of the bulbus olfactorius accessorius, islands of Calleja, nucleus laterodorsalis tegmenti and stria terminalis. Directly after birth strong activity was present in both the gray and white matters, while with advancing age the activity in the neuropil of most portions decreased rather markedly, and that in the white matter seemed to decrease slightly. From these observations the enzyme, namely the hexose monophosphate shunt, is thought to participate in the formation

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and maintenance of structures of the brain, besides particular functions in some restricted structures. REFERENCES

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MORIKAWA,N., (1958); Histochemical study of succinic dehydrogenase in the brain. 11. On the alteration of succinic dehydrogenase from late fetal life to adult in the rat brain. Arch. histol. jap., 14. 165-184. NACHLAS,M. M., Tsou, K.-C.. SOUZA,E. D., CHENG, c.-s.,AND SELIGMAN, A. M., (1957); Cytochemical demonstration of succinic dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J. Histochem. Cytochem., 5, 420-436. NACHLAS, M. M., WALKER, D. G . , AND SELIGMAN, A. M., (1958a); A histochemical method for the demonstration of diphosphopyridine nucleotide diaphorase. J. biophys. biochem. Cyfol., 4, 29-38. NACHLAS, M. M., WALKER, D. G., AND SELIGMAN, A. M., (1958b); The histochemical localization of triphosphopyridine nucleotide diaphorase. J. biophys. biochem. Cytol., 4, 467-474. ORTMANN, R., (1957); h e r Succinodehydrogenase im olfactorischen System. Acra attar., 30,542-565. READING, H. W., (1964); Activity of the hexose monophosphate shunt in the normal and dystrophic retina. Nature, 203, 491-492. RICHTER,D., Metabolism of the Nervous System. London, Pergamon Press. SHIMIZU,N., AND ISHII,S., (1964); Electron microscopic observation of catecholamine-containing granules in the hypothalamus and area postrema and their changes following reserpine injection. Arch. histol. jap., 24,489497. SHIMIZU, N., AND KUMAMOTO, T., (1952); Histochemical studies on the glycogen of the mammalian brain. Anat. Rec., 114,419498. SHIMIZU, N., AND MORIKAWA, N., (1957a); Histochemical studies of succinic dehydrogenase of the brain of mice, rats, guinea-pigs and rabbits. J. Histochem. Cytochem., 5, 334-345. SHIMIZU, N., MORIKAWA, N., AND ISHII,Y., (1957b); Histochemical studies of succinic dehydrogenase and cytochrome oxidase of the rabbit brain, with special reference to the results in the paraventricular structures. J . comp. Neurol., 108, 1-21. SHIMIZU, N., MORIKAWA, N., AND OKADA,M., (1959); Histochemical studies of monoamine oxidase of the brain of rodents. Z. Zelvorsch., 49, 389400. SIBLEY, J. A., AND LEHNINGER, A. L., (1949); Determination of aldolase in animal tissues. J. hiol. Chem., 177, 859-872. THOMAS, E., AND PEARSE, A. G. E., (1961); The fine localization of dehydrogenases in the nervous system. Histochemie, 2, 266-282.

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Studies on the Neural Basis of Behavior by Continuous Frequency Analysis of EEG N A O S A R U R O YOSHII. M I N O R U SHIMOKOCHI, K E N S A K U M I Y A M O T O AND MUNEYUKI IT0 Department of Phvsiology, Osaka University Medical School, Osaka (Japan)

In April and May 1956, one of the authors (N.Y.) worked with the continuous frequency analyzer in Dr. Storm van Leeuwen’s laboratory in Leyden. The equipment recorded the EEG component for each frequency band on a photographic film in black and white (Storm van Leeuwen, 1961). After N.Y. returned to Japan the next year, he asked the San’ei Sokki Co. to make a frequency analyzer to show the instantaneous value of each frequency band with a pen-writing EEG machine. With the support of our Ministry of Education and foreign Foundations all the apparatus was given to us, and we have published some results on the experiment of conditioning the frequency specific waves in animals (Yoshii et al., 1962a,b, 1963, 1965). I n this paper we present the results, by using the continuous frequency analyzer, of (i) EEG changes in ‘switch-off behavior’ (escape from the brain-stem stimulation by lever-pressing) in rats, and of ( i i ) EEG changes in free behavior, recent memory test and the detour experiment in dogs, using the radio telemeter and telestimulator. (I)

ELECTROENCEPHALOGRAPHIC STUDIES ON THE ‘SWITCH-OF BEHAVIOR’

IN RATS I N D U C E D B Y STIMULATION OF BRAIN-STEM STRUCTURE

A number of electroencephalographic studies has been reported that aimed to clarify the neural mechanisms that control conditioned behavior. In some experiments intracerebral stimulation was used as the conditioned stimulus, as the indifferent tracer stimulus to follow the input, or to influence the conditioned response. Olds and his co-workers showed by ingenious self-stimulation and escape behavior experiments that the positive or negative points, electrical stimulation of which served as reward or punishment, were located in the rat brain (Olds and Milner, 1954; Olds, 1958, 1962; Olds and Peretz, 1960). Nakao (1962) reported changes in electrical activities of the hippocampus during ‘switch-off behavior’ of the cat to turn off the electrical current given to the antero-medial hypothalamus, and the effect of spreading of hippocampal after-discharges upon this learned behavior was also observed. References p. 2491250

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et at.

In this experiment, EEG changes were observed by continuous frequency analysis just before the lever-pressing to escape from electrical stimulation delivered to various parts of the brain. Methods

Forty male and female rats, weighing 150-250 g, were used. Each animal was anesthetized (intraperitoneal injection of amobarbital), and fixed on a stereotaxic instrument, cortical and subcortical electrodes being implanted through the small pores of the exposed skull. The electrodes were connected with a miniature tube socket which was fixed on the skull with dental cement. Deep stimulation and/or recording were obtained through the electrodes consisting of a pair of stainless steel wires, 0.2 mm in diameter and insulated to within 0.5 mm of the tip, the interpolar distance being 0.5 mm. The tiny machine screws implanted on the skull served as the surface (cortical) electrodes. The reference electrode was screwed 10 mm in front of Bregma on the midline of the skull. For stimulation, rectangular pulses were obtained with an electronic stimulator (Nihon Koden Co. Ltd.), the parameters varying according to the stimulation purpose. Animals were trained in a wooden box, 18 x 30 x 25 cm, with a lever on one side of the wall. The circuit of electrical stimulation was arranged to be turned off by lever-pressing. This experimental box was placed in a soundproof, electrically shielded room. EEG’s were recorded on paper with an 8-channel electroencephalograph (San’ei Sokki Co. Ltd.), and simultaneously put on an electromagnetic tape with an 8-channel tape-recorder (San’ed Sokki Co. Ltd.). EEG traces in the tape were subjected to continuous frequency analysis through a 23-band (1-30c/sec) frequency analyzer (San’ei Sokki Co. Ltd). The sites of the electrodes were verified histologically after the experiment. Results ( A ) Learning of ‘switch-off behavior’ Square pulse waves were used for brain stimulation in this experiment. Taking the results of the continuous frequency analysis of routine EEGs into account, the frequency mostly used was at 9.5 c/sec, the background component of which had been proved relatively small. A pulse duration of 0.5 msec was used. The experiment began with delivery of the brain stimulation at 1 V, which was switched off by the operator when the animal did not turn it off by pressing the lever within 2 min. Ten to twenty trials were carried out at the same intensity of stimulation, then it was raised successively by 1-V steps. In the course of the stepwise increase in intensity of the brain stimulation, the animal showed searching behavior and pressed the lever by chance at a certain voltage. After lever pressing the animal released the lever, and the electrical stimulation was no longer delivered for 2 min. As the trials with sufficient voltage to induce the searching behavior were repeated with a constant interval, the latency (the time from the onset of stimulation t o the lever-pressing) gradually became shorter (Fig. 1). When the intensity was raised after constant latency was obtained a t a certain

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I

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Fig. 1. Learning curves from 2 rats. For Rat 323 stimulation was applied to the lateral hypothalamus, and for Rat 329 to the pretectal area. Each learning curve was obtained by averaging latency curves of the first 3 (for Rat 323) or 4 (for Rat 329) sessions of training, one session consisting of 9 or 10 trials a day. Abscissa: successive trial number. Ordinate: latency in min. 0-0 Rat 323 L. LHA ST. 6 V, 1 msec, 9.5 counts/sec; 0-0 Rat 329 L. PRT ST. 8 V, 0.5 msec, 9.5 counts/sec.

Sec

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Fig. 2. Latency or ‘switch-off behavior’ related to stre.igth of stimulation. In this case (stimulation of medial geniculate body) the ‘switch-off behavior’ was not induced below 5 V. Abscissa: intensity of stimulation in V. Ordinate: latency in sec. Rat 314, LGM ST. 0.5 msec, 9.5 counts/sec.

stimulation intensity, the latency was further shortened (Fig. 2). The learning-positive points which motivated the learning of ‘switch-off behavior’ were scattered in the brain-stem structures without any special pattern of localization except in the midRc,/iwnrrs p. 249/250

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brain region. In the midbrain the learning-negative points were clustered in the tectum and in the dorso-medial part of the midbrain reticular formation, and the learningpositive points formed a group in the ventro-lateral part of the tegmentum (Fig. 3).

LEARNING: POSITIVE 0 LEARNING :NEGATIVE

Fig. 3. Distribution of learning-positive and learning-negative points in the brain stem. With stimulation of learning-positive points the ‘switch-off behavior’ could be induced. There was no definite localization except in the midbrain region (AP 5, AP 6.5) where the learning-negative points were found in the tectum and the dorsomedial part of the reticular formation, and the learning-positive points, more ventrolaterally. (Brain map after Fifkovi and MarSala in Elecfrophysiological Methods in Biological Research, 1962.)

( B ) EEG changes correlated with ‘switch-off behavior’ The cortical and subcortical EEG’s were obtained during ‘switch-off behavior’ and they were examined in detail afterwards by continuous frequency analysis. Fig. 4 shows the EEG record of one of these trials, in which the ‘switch-off behavior’ was induced by left medial geniculate stimulation ( 5 V, 0.5 msec, 9.5 c/sec). The vertical line on the left indicates the onset of the brain stimulation, and a t the right vertical line the animal turned off the current by lever-pressing. In the right fronto-temporal lead in Fig. 4, the responses driven by the stimulation at 9.5 clsec showed a marked increase in amplitude just before the lever-pressing. At that time the hippocampal synchronization pattern increased its frequency to 7-8 clsec in the hippocampus. And at the same time the propagation of the hippocampal synchronization pattern was observed in the right temporo-occipital and right occipito-frontal leads. The results

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of the frequency analysis of the right fronto-temporal EEG showed that the frequency component at 9-1 0 c/sec (corresponding to the stimulation frequency), which at the onset of stimulation underwent a transient increase and then retained a rela-

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tively low amplitude during the period of stimulation, was eventually markedly augmented just before the lever-pressing (Fig. 5A). On the other hand, this component (9-10 c/sec) declined in the hippocampus, and the 7-8 c/sec component (corresponding to the frequency of hippocampal synchronization) became dominant just before the lever-pressing (Fig. 5D). Fig. 6 shows another example in which ‘switch-off behavior’ was induced by stimulation of the midbrain reticular formation (6 V, 0.5 msec, 9.5 clsec). The cortical EEG’s were monopolarly recorded from the symmetrical points of both frontal cortices. From the results of frequency analysis, it was observed that thecomponent at 9.5 c/sec driven by the reticular stimulation showed a marked increasein amplitude just before the lever-pressing in the frontal Lead on both sides (Fig. 7, A and C), and the EEG component of the stimulation frequency (9.5 c/sec) in the hippocampal lead was replaced by an enhancement of the component at 7-8 c/sec due to the hippocampal arousal pattern at that time (Fig. 7E). Where the hippocampal activities were maximally driven by stimulation with the frequency at 9.5 c/sec, the component of the driven waves at the stimulation frequency was sometimes suppressed in its amplitude just before lever-pressing, only to be replaced by an increment of the component of proper frequency of hippocampal synchronization at 7-8 c/sec. After the brain stimulation was over the animal released the lever voluntarily. Just before this lever-releasing the hippocampal synchronization reappeared at about 7-8 c/sec (Fig. 8). References p . 249/2SO

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Fig. 5A-D. Data of frequency analysis of the EEG's in Fig. 4. A. right fronto-temporal cortical lead; B, right temporo-occipital cortical lead; C,right occipito-frontal cortical lead; D, right dorsal hippocampal lead. Just before lever-pressing the 9-10 c/sec component (.corresponding to 9.5 counts/sec stimulation) showed a marked increase in amplitude in the right fronto-temporal cortical lead, and the component of hippocampal arousal pattern (7-8 c/sec component) increased in the right temporo-occipital and occipito-frontal cortices and the dorsal hippocampus.

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Fig. 6. Rat 403 (stimulation of left midbrain reticular formation). Movement of floor ofexperimental box (Move) represented body movement accompanying lever-pressing. Electrical changes preceding the ‘switch-off behavior’ were barely recognized in this routine EEG record. Rat 403, L. MBRF ST. 0.5 msec, 6 V, 9.5 countslsec.

( C ) EEG’s correlated with voluntary lever-pressing After the repetition of the ‘switch-off behavior’ the animal showed spontaneous lever-pressing even in intertrial intervals in which the electrical stimulation was not applied. The EEG changes at that time were comparable to those of ‘switch-off behavior’ motivated by brain stimulation. As far as the hippocampal activity was concerned, the EEG changes in the driven (by brain stimulation) and the spontaneous (voluntary) lever-pressing resembled each other in that the frequency of hippocampal synchronization was gradually increased just before the behavior performance. In the traces other than that of the hippocampus there was no noticeable similarity between the driven and the spontaneous lever-pressings (Fig. 9). ( D ) ‘Switch-off behavior’ to high frequency stimulation As it was observed that the ‘switch-off behavior’ was characterized by a marked change in the hippocampal synchronization, the behavior of hippocampal EEG was investigated in detail in the ‘switch-off behavior’ induced by high frequency stimulation at 100 c/sec with 0.1 msec pulse to the lateral hypothalamus (Fig. lo), the frequency being beyond the limit of our frequency analysis. The hippocampal synchronization, much facilitated up to 11-12 c/sec at the beginning of the high frequency stimulation, showed a decrease in its main frequency just before the lever-pressing (Fig. IOA). When the stimulation intensity was raised to 9 V (Fig. IOC), the frequency of the hippocampal synchronization reached 14-16 c/sec. Fig. I I shows the relationship between the main frequency of the hippocampal synchronization and the intensity of stimulation. Weak stimulation (1-2 V) induced the ‘switch-off behavior’ Refrrcnws p. 249/250

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Fig. 7A-E. Data of frequency analysis of the EEG’s in Fig. 6. The 9-10 c/sec (stimulation frequency) component increased in frontal cortical E E G s o n both sides (A and C)just before ‘switch-off behavior’. At the same time an increase in amplitude of the 7-8 c/sec (frequency of hippocampal synchronization) component was observed in right hippocampus (E!. Rat 403 L. MBRF ST. 0.5 msec, 6 V, 9.5 counts/sec.

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Fig. 8. EEG’s and the data of frequency analysis correlated with lever-releasingmovement. Jncrease in the 7-8 c/sec component in temporo-occipital cortical, occipito-frontal cortical and dorsal hippocampal leads corresponded to appearance of the hippocampal arousal pattern.

with the hippocampal synchronization at 7-8 c/sec, the strong stimulation producing a rapid increase in synchronization higher than 12 c/sec; nevertheless, the animal completed the ‘switch-off behavior’ successfully. (E) Combination of indifferent low frequency ‘tracer’ stimulation with high frequency ‘driver’ stimulation After the ‘switch-off behavior’ was established with high frequency (100 c/sec) stimulation, low frequency (1 1.5 c/sec) stimulation (‘tracer’ stimulation) was applied through one of the electrodes other than the one used for ‘driver’ stimulation. The indifferent stimulus (‘tracer’) (the stimulus producing no apparent changes in behavior) was applied before and during high frequency ‘driver’ stimulation. Because of their low frequency, the electrical responses to the ‘tracer’ stimuli were easily distinguished from those to the ‘driver’ ;the frequency of the latter was far beyond the range of our frequency analysis. In order to ascertain the distribution of the response to the ‘tracer’ in the brain just before the ‘switch-off behavior’ induced by high frequency stimulation (‘driver’), the EEG component corresponding to the low frequency ‘tracer’ stimulation was followed by continuous frequency analysis. As shown in Fig. 13 (the record of frequency analysis of each EEG in Fig. 12), the EEG component (at 1 I .5 c/sec) due to the ‘tracer’ stimulation given to the midbrain reticular formation, along with its harmonic (22-24 c/sec) component, was decreased in temporo-occipital, occipito-frontal and hippocampal EEG’s when the ‘switch-off behavior’ with hypothalamic ‘driver’ stimulation appeared. Combination of the hypothalamic ‘tracer’ stimulation and the ‘driver’ stimulation of the midbrain reticular formation showed that the ‘tracer’ was suppressed in the hippocampus in correlation with the ‘switch-off behavior’ (Figs. 14 and 15). References p . 249l250

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Fig. 9A, B. EEG's and the data of frequency analysis correlated with spontaneous lever-pressing were compared to those of 'switch-off behavior' induced by electrical stimulation. Rat 303 spontaneously pressed the lever twice between 2 successive trials driven by reticular stimulation. A, there was no similarity between fronto-temporal cortical EEG of driven lever-pressing and that of spontaneous lever-pressing. B, in the hippocampal lead, an EEG change similar to that of driven lever-pressing was observed in spontaneous lever-pressing; appearance of hippocampal synchronization was followed by a decreascin its frequency. Rat 303, L. MBRF ST.0.5 msec, 7 V, 9.5 countslsec.

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Fig. 1 0 A X . Right dorsal hippocampal EEG's and the data of frequency analysis correlated with 'switch-off behavior' induced by high frequency (100 counts/sec) stimulation of the left lateral hypothalamus. For each tracing the left vertical line indicates the onset of stimulation, and the rignt vertical line the lever-pressing. Strength of stimulation: 5 V in A ; 6 V in B; 9 V in C. A, the hippocampal synchronization facilitated up to 11-12 c/sec a t the beginning of 5 V stimulation showed a decrease in frequency during the stimulation, but it returned to 11-12 c/sec, until it was suppressed just before performance of the 'switch-off behavior'. B and C. when the stimulation intensity was raised. frequency of the hippocampal synchronization reached 14-16 cisec. Rat 326, L. LHA ST. 0.1 msec, 100 counts/sec.

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Fig. 14. EEG’s in the course of combination of ‘driver’ (high frequency stimulation of the midbrain reticular formation) and ‘tracer’ (low frequency stimulation of the anterior hypothalamic area). Rat 413, L. AHA ST. 0.1 msec, 7 V, 11.5 counts/sec; L. MBRF ST. 0.1 msec, 1.8 V, l00counts/sec.

Contrary to these results, when the ‘tracer’ was given to the hippocampus the EEG component at the same frequency as that of ‘tracer’ stimulation (especially the component of its harmonics) was increased in the occipital EEG’s, while the EEG component at the hippocampal arousal theta frequency was increased presumably due to propagation from the hippocampal and related structures (Figs. 16 and 17). These results are summarized in Table 1. Refcrencrr p. 2491250

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Fig. 16. EEG’s in the course of combination of ‘driver’ (high frequency stimulation of the midbrain reticular formation) with preceding ‘tracer’ (low frequency stimulation of the dorsal hippocampus). Rat 412, R. DHPC ST.0.1 msec, 3 V, 11.5 counts/sec; L. M B R F ST.0.1 msec, 1.5 V, I 0 0 counts/sec.

DISCUSSION

( A ) Distribution of the learning-positive points of the ‘switch-of behavior’ From the self-stimulation experiment, in which the rat could stimulate himself in the brain by pedal-pressing, and the escape behavior experiment, Olds and his co-workers (1954, 1958, 1960, 1962) showed with a brain map that the motivationally positive points (self-stimulation) and the motivationally negative points (escape behavior) were widely distributed in the rat brain. As for the area of pure negative

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reinforcement (escape behavior) appearing on one half of the transverse coronal plane, Olds (1962) reported that in the midbrain it appeared to form a full circle surrounding the reticular activating system, and in the thalamus it formed a U-shaped distribution. The stimulation points which induced the ‘switch-off behavior’ learning in our experiment are assumed to be the points of the motivationally negative effects in Olds’s terminology. However, the threshold voltage sufficient to induce searching

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Fig. 17A-D. Data of frequency analysis of the EEG’s in Fig. 16. Prior to the lever-pressing the 22-24 c/sec component (second harmonic of 1 I .5 c/sec tracer) along with the frequency component 8-9 c/sec of the hippocampal arousal was increased in the temporo-occipital and the occipitofrontal cortices.

behavior was adopted to obtain a learned ‘switch-off behavior’ in our experiments. The ‘switch-off behavior’ was then performed with a relatively long latency, which was enough to follow the EEG changes in detail just before the lever pressing. As shown in Fig. 3, the distribution in the brain of the learning-positive points did not quite coincidewith Olds’s findings, perhaps owing to the fact that our stimulation points were more concerned with the searching behavior. The stimulation points which failed to induce the ‘switch-off behavior’ might be the points where the motivationally negative (or escape) effect could not be produced, or involuntary or jerky movements were induced, when the intense stimulation was delivered. Rrferenrrr p . 249/250

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( B ) BEG changes correlated to the ‘switch-off behavior’ The effectsof the intracerebral stimulation, which produced the ‘switch-off behavior’ as the peripheral manifestation, were investigated in the EEG recording. The results of this experiment were as follows, (a) The frequency component correponding to the brain stimulation was increased in the frontal cortex just before the lever-pressing. (6) As for the hippocampal EEG, the fiequency component corresponding to the brain stimulation, which had maintained a considerable amplitude from the onset of the stimulation, was replaced by an increase in the EEG component at 7-8 c/sec corresponding to the hippocampal arousal theta wave in correlation with the appearance of the “witch-off behavior’. Sometimes,however, the hippocampal electrical activity was maximally driven, and thus the component at the stimulation frequency (9.5 c/sec) underwent a noticeable increase. Even in these cases, it was replaced by an increase in the component at lower frequency band (7-8 c/sec) just before lever-pressing movements. Moreover, the frontal EEG also displayed a continuous maximal driving from the onset of the stimulation, and no further increase in amplitude of this component was noted before the ‘switch-off behavior’. The changes of the temporo-occipital and occipito-frontal EEG’s correlated with the ‘switch-off behavior’ resembled those of the hippo~mpalEEG. It is reported in the next part of this paper that the hippocampal synchronization was propagated to the occipital cortex, especially in the hippocampal arousal stage. Yoshii and his co-workers (1 958) observed the hippocampal synchronization appearing in the course of positive, negative, differential and trace conditioned reflexes of alimentary and defensive reactions. It is also noted in the next part of this paper that the hippocampal arousal pattern appeared in the instrumental conditioning and in the spontaneous appearance of conditioned behavior. Adey et al, (1960) and Holmes and Adey (1960) reported that the hippocampal arousal pattern appeared in correlation with the goal-directed movement in the EEG‘s of alimentary conditioned behavior, and they described the phase relation of this theta rhythm in the entorhinal cortex and various parts of the hippocampus. Nakao (1962) carried out an EEG study of ‘switch-off behavior’ motivated by hypothalamic stimulation, and concluded that there was a relationship between the intensity of stimulation and the frequency of hippocampal arousal waves during stimulation. These findings were supported in our experiments. ( C ) On the spontaneous lever-pressing

On repetition of trials of the ‘switch-off behavior’, lever-pressing appeared in the intertrial intervals without any electrical stimulation. When the EEG changes correlated with the ‘switch-off behavior’ driven by the brain-stem stimulation were compared to those of the spontaneous lever-pressing, a similarity was found in the hippocampus; namely, appearance of hippocampal synchronization pattern just before the lever-pressing followed by a gradual decrease in its frequency. This might suggest that as far as the hippocampus was concerned, neural process similar to that in the ‘switch-off behavior’ developed in the performance of the spontaneous lever-pressing. Gradually, the occurrence of spontaneous lever-

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pressing became more frequent, and eventually the animal would not release the lever. In this state, a long lasting process of intense tonic arousal in the hippocampus presumably kept the animal on the lever for a long time. ( D ) On the EEG changes in the hippocampus correlated with the ‘switch-off behavior’ driven by Irigh.frequency stimulation Nakao ( 1 962) reported that the frequency of the hippocampal synchronization pattern in the ‘switch-off behavior’ induced by hypothalamic stimulation was increased as the stimulation intensity was raised. Sailer and Stumpf (1957) noted that there was a linear relationship between the frequency of the hippocampal arousal pattern, which was induced by midbrain reticular formation, and the logarithm of the stimulation intensity in volts. In our experiment, similar results were obtained; the hippocampal synchronization which appeared at 7-8 c/sec by 1-2 V hypothalamic stimulation was facilitated i n its frequency up to 15 c/sec, when the intensitywasraised to as high as 10 V. However, the animal suffered from postural disturbances such as tumbling when the hippocampal synchronization frequency exceeded 12 c/sec. Even i n these situations the animal completed the lever-pressing successfully irrespective of the postural disturbances. The hippocampal synchronization, which was facilitated as mentioned above, inevitably underwent a decrease in frequency just before the ‘switch-off behavior’ took place. It was noticeable that, when the animal performed the lever-pressing from a resting state, the frequency of the hippocampal arousal pattern increased by 2-3 c/sec in correlation with the behavior performance, but when the ‘switch-off behavior’ appeared in the extreme facilitation of hippocampal synchronization (in high frequency stimulation of the brain-stem structure), the hippocampal synchronization decreased its frequency. This might suggest that the hippocampal synchronized waves play some controlling role in the learned behavior.

( E ) On distribution of the indiferent ‘tracer’ correlated with the ‘switch-ofl behavior’ induced by high frequency stimulation The occipital cortical and/or hippocampal responses evoked by indifferent low frequency (‘tracer’) stimulation of the midbrain reticular formation or of the hypothalamus were suppressed with the ‘switch-off behavior’ induced by high frequency (‘driver’) stimulation, the occipital cortical responses evoked by hippocampal ‘tracer’ stimulation being facilitated. Therefore, impulses of the ‘driver’ from the midbrain reticular formation, or from the hypothalamus presumably invaded the hippocampus, and consequently indifferent impulses of the ‘tracer’ from other regions to the hippocampus seemed to be occluded just before the ‘switch-off behavior’. On the other hand, the facilitated responses to the hippocampal ‘tracer’ in the occipital cortex at that time might suggest that a pathway of ‘tracer’ from the hippocampus to the occipital cortex was opened just before the lever-pressing performance. Moreover, the hippocampal synchronization invaded the occipital cortex where the indifferent ‘tracer’ impulses from the hippocampus were intensified, suggesting that the invasion of the hippocampal synchronization pattern inhibited the activities of the cortical area, which was not responsible for the appearance of ‘switch-off behavior’. RiV./rrc.nri*r p. 249/250

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SUMMARY

( 1 ) The responses in the central nervous system evoked by intracerebral electrical

stimulation to drive the ‘switch-off behavior’ were followed by EEG frequency analysis. (2) The learning-positive points, stimulation of which induced the ‘switch-off behavior’, and the learning-negative points, stimulation of which failed to produce the ‘switch-off behavior’, were found in the brain stem. As far as the midbrain region was concerned the learning-positive points were scattered in the lateral part of the reticular formation, the learning-negative ones being clustered in the tectum and in the dorso-medial part of the reticular formation. (3) Just before the ‘switch-off behavior’ appeared, the driven responses of the frontal sensorimotor area were facilitated and those of the hippocampus were suppressed, although the appropriate hippocampal arousal pattern was induced, showing a frequency 2-3 c/sec higher than usual. This pattern of EEG response was obtained when the behavior-driving stimuli were given to the midbrain reticular formation, the hypothalamus, the thalamic specific nucleus, etc. (4) Spontaneous lever-pressing occurred with the appearance of a hippocampal arousal pattern similar to that observed in the learned ‘switch-off behavior’, suggesting that the neural process underlying the spontaneous (voluntary) lever-pressing reproduced the EEG pattern of the conditioned lever-pressing as far as the hippocampus was concerned. (5) Indifferent impulses from the brain stem to the hippocampus were found to be shut out prior to the ‘switch-off behavior’. A pathway from the hippocampus to the occipital cortex seemed to be open for indifferent impulses and for impulses of the hippocampal synchronization of the arousal system. The significance of spreading hippocampal synchronization was suggested to suppress the local activities not responsible for the behavior.

(11) ELECTROENCEPHALOGRAPHIC STUDIES ON THE FREE BEHAVIOR, RECENT MEMORY TEST AND DETOUR EXPERIMENT, USING RADIO TELEMETER AND TELESTIMULATOR

Numerous types of investigation have been performed t o clarify the neural basis involved in the natural, conditioned or learned behavior of experimental animals (Delgado, 1964). As an approach to the study of the nervous mechanisms Yoshii and his co-workers (1958) have undertaken experiments to observe the electrical activities of the brain in the course of classical and instrumental conditioning of animals. However, it was difficult to record the electrical activities of freely moving subjects. Recently, it became possible not only to record the EEG’s but electrically to stimulate the deep structures of the brain by the use of a wireless transmission method. With a radio telemetering and telestimulating apparatus the neural mechanism has been studied by recording the EEG’s of the hippocampus and neocortex, during natural behavior and psychological tests at the various conscious levels and in activated sleep. The results are reported in this paper.

CONTINUOUS FREQUENCY ANALYSIS OF

EEG

23 5

METHODS

Four dogs with electrodes implanted in the skull and subcortical structures were used. The animal, having a part of the telemetering and telestimulating apparatus (weight, 550 g) on its back, to which it had been habituated beforehand, was allowed to move freely in the experimental room. The apparatus on its back was composed of 2 or 4 channel EEG sending and 1 channel receiving machines. The EEG’s were recorded on the paper of a 4 channel ink-writing machine and also simultaneously fed to a 2 channel tape-recorder for later frequency analysis (from 1-30 c/sec). The equipment used in this experiment was made by the San’ei Sokki Co. Ltd.

RESULTS

( A ) Electrical activities during natural behavior

Voluntary movements and the related EEG’s are described in the following. The cortical EEG was not much changed when the animal was awake. ( I ) Orienting reflex. A tonic head turning was elicited upon foot stepping or human voices outside the experimental room. At this time, synchronized waves (5-6 c/sec) of high amplitude appeared in the hippocampus with a decrease in the intermediate

Fig. 18. EEG’s of fronto-temporal cortex and hippocampus during walking and food taking. A, Walking to the fish put before the dog; hippocampal synchronization was induced. B, Seizing the fish suppressed the hippocampal synchronization. C, Masticatory waves with suppression of proper synchronization in the hippocampus. D, Licking. E, Seizing more food. F, Eating.

fast waves. On the other hand, a phasic head turning was induced upon strong sound or hand-claps, accompanied by desynchronization in the hippocampus. ( 2 ) Walking. A short run of the synchronization (5-6 c/sec) was produced in the hippocampus when the dog walked about in the experimental room (Figs. 18 and 19). (3) Fuod taking. When the dog seized food with his teeth, the hippocampal synchronization disappeared and was replaced by the desynchronization (Figs. 18 and 19). Refwencrs p . 2491250

N. Y O S H I I et al.

236 c ycles/sec

20-22

------k+

4-5 5-6 6-7 7-8 8-9 9 -10 10-11 11 -12 12-13 13-14 14 16 16-18

-

18-20 20-22 22-24 24-27 27- 30

EEG(Hipp)

Fig. 19A, B. Continuous frequency analysis of the EEG's in Fig. 18. A, Fronto-temporal EEG. B, Hippocampal EEG. Phasic arousal pattern is seen in A-B-C and D-E, and tonic arousal pattern in C-D and E-F.

With masticatory movements synchronized waves of about 5 clsec sporadically reappeared in the hippocampus. (4) Searchingmovements. The hippocampdl synchronization at 5.5 clsec was clearly seen when the dog was sniffing on the floor or at some object, or trying to escape from the experimental room. (5) Defecation and urination. Prior to excretion performance, the dog walked about

CONTINUOUS FREQUENCY ANALYSIS OF

EEG

237

with sniffing and took up the habitual posture of defecation or urination. The hippocampal synchronization disappeared from the start of taking the posture to the end of the excretion. After the end of excretion synchronized waves were reproduced in the hippocampus (Fig. 20). ( 6 ) Unpleasant stimulus. When the animal barked or feared some object, putting the tail between legs, the hippocampal synchronization was not induced. On application of an unpleasant stimulus, e.g. the ‘Matatabi’ (cat-nap) smell, when he turned DEFECATION F-T

A

H

m

URINATION

Cycles/sec 1015202530354 -

I5 20 25 30 35 40 5

6 - 7

7 8 9 10

- 8

- 9 -10 - 1 1 I I -12 12 - 1 3 13 -14 14 -16 16 - 1 8 18 - 2 0 2 0 -22 22 -24 24 - 2 7 2 7 -30 EEG

.

.-

. . .

.

Fig. 20A-C. A, EEG’s of fronto-temporalcortexand hippocampus before, during and after defecation (A) and urination (B). The hippocampal synchronization appeared in the searching behavior and was suppressed during the excretion period. ((3Data of frequency analysis of the hippocampal EEG in the case of urination. Note the tonic pattern of hippocampal EEG. ReJurences p . 249/250

23 8

N. Y O S H I I

A

et al.

B

Fig. 21 A, R. EEG’s and the data of frequency analysis on giving up an idea. A, Frequency analysis of the fronto-temporal EEG. B, Frequency analysis of the hippocampal EEG. At A in the routine EEG, food was put in the dish which the dog was unable to approach, because a short string restrained him. From A to B and C to D the dog tried and failed to approach the food, and EEG showed the phasic arousal which changed to the tonic arousal at B and D.

away his face, or when the dog could not reach the food and gave up the idea of taking it (Fig. 21), the synchronization became obscure in the hippocampus. (7) Tense state. One dog was able to put his foreleg on the experimenter’s palm on the verbal instruction, ‘ote’ (‘give me your foreleg’). Giving the foreleg, the animal was in the tense state. At this state the cortical and hippocampal EEG’s were not much changed, but the hippocampal synchronization was suppressed (Fig. 22). Short summary. The hippocampal synchronization was induced by orienting or searching behavior with attention, but the desynchronization, by startle or fear activities. The synchronization became obscure when the dog turned his face away from unpleasant objects or lost his interest. During the biting of food with the teeth, defecation or urination, the hippocampal synchronization was replaced by low voltage

CONTINUOUS FREQUENCY ANALYSIS OF

EEG

239

fast waves. The term ‘tonic arousal’ will be used for the EEG pattern of cortical and hippocampal desynchronization, and ‘phasic arousal’ for that of cortical desynchronization with hippocampal synchronization.

F-T

25 3

3

-xx_

35--.-

A

B

Fig. ZZA, B. Changes of EEG’s when the dog placed his foreleg twice on the palm of the experimenter with the instruction ‘0.e’. A, Frequency analysis of the hippocampal EEG. B, Frequency analysis of the fronto-temporal EEG. During the performance of giving the foreleg to the experimenter tonic

arousal waq witnessed.

( B ) EEG’s during sleep (I) In the drowsy state the synchronization at 3-3.5 c/sec became obscure and 11-13 c/sec waves were gradually increased in the hippocampus. Shortly after the occurrence of these changes in the hippocampus, irregular large slow waves appeared in the cortex. (2) The cortical activities were gradually changed to high amplitude irregular slow waves, and the animal fell asleep. When spindle waves and irregular large slow waves were dominant in the cortex, the hippocampal synchronization decreased in appearance and finally almost disappeared. (3) From the stage of spindles and large slow waves, the animaJ often awoke spontaneously or on receiving a weak external stimulus, e.g., faint step sounds. At this moment, the 11-13 c/sec components in the hippocampus abruptly disappeared References p. 2491250

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al.

A

E

Fig. 23A, B. Continuous frequency analysis of hippocampus (A) and fronto-temporal cortex (B). and integrated values of each 10 sec,showing long period fluctuation of hippocampal synchronization during activated sleep. E, eye movements; F and H, fore- or hindleg twitchings. The hippocampal synchronization appeared about 70 sec before behaviorally marked activated sleep, and irradiated to the cortical EEG.

and were replaced by slow wave components at 2-3 c/sec. At the same time the cortical slow waves disappeared and low voltage fast waves appeared. (4) Several tens of seconds before the cortical EEG changed into the desynchronization pattern of activated sleep from the large slow wave pattern, the hippocampal synchronization was recorded by the frequency analyzer, increasing its frequency up to 5-7 c/sec in the hippocampus, and corresponding to this, in the fronto-temporal lead (Fig. 23). The hippocampal synchronization was facilitated just before the twitching of the mouth angle and/or rapid eye movements were produced. During activated sleep the frequency of hippocampal synchronization reached 7 c/sec. The most regular synchronization with the highest amplitude in the hippocampal EEG was accompanied by general twitchings; when one or two local twitchings were produced, no peculiar enhancement of the hippocampal synchronization was observed. It was noted from the integrated value of each frequency band component that the amplitude of the main component in the hippocampal EEG fluctuated by about a 1-min period (Fig. 23). By behavioral arousal from activated sleep, whether it was elicited spontaneously or by electrical stimulation of the brain-stem reticular formation, the hippocampal synchronization gradually decreased in frequency and then became irregular with an

CONTINUOUS FREQUENCY ANALYSIS OF

EEG

24 1

increase of fast wave components in the cortex. Much similarity was found between those fronto-temporal EEG’s in the alert state and in the activated sleep state, but close investigation showed that fast wave components were dominant in the former state while the 6-7 c/sec component was conspicuous in the latter. Short summary. During activated sleep the frequency of hippocampal synchronization varied from 3.5 c/sec to 7 c/sec. Humoral mechanisms were suggested to be involved in activated sleep because of the long period fluctuation of the amplitude of the main component in the hippocampal synchronization. The lower frequency of hippocampal synchronization appeared in the initial stage of activated sleep. The frequency increased up to 7 c/sec during the generalized twitching of 4 limbs and body muscles with rapid eye movements; a corresponding increase was barely observable during the local twitchings of foreleg, hindleg or face muscles. The theta wave component in the cortex showed an increase or decrease concomitant with those in the hippocampus. It is assumed that the hippocampal synchronization spread to the cortex during the activated sleep stage. Fast waves of more than 20 c/sec were less in the hippocampus during activated sleep when compared with those in the resting awake state.

Fig. 24. Hippocampal and fronto-temporal EEG’s in correct (upper pair) and incorrect (lower pair) performance of recent memory test.

(C) EEG’s during psychological tests ( I ) Recent memory test. To examine the electrical activities of the brain during the

recent memory test, the animal, having the telemetering equipment on his back, was put into the cage, through the window of which he was able to watch two dishes. After the experimenter put food into one dish, a screen was drawn so that the animal could not see the food. Ten sec later the door of the cage was opened. From the preliminary test, changing the interval between drawing the screen and opening the door, it was confirmed that the limit of the recent memory was 10 sec for the dog. The results of frequency analysis of EEG’s (Figs. 24,25 and 26) recorded in each period of stimulation (putting the food into the dish), image (remembering the food in the dish) and performance (walking to the dish) are summarized in Table 11, in which correct and incorrect performances are compared. When the animal noticed the mistake and changed his direction, the phasic arousal pattern of EEG’s changed to the tonic one (Figs. 24 and 26). ( 2 ) Insight behavior in the detour experiment. Two obstructions were placed and a string of a certain length from the animal’s neck was fastened to a hook: the animal Rcfermces p. 2491250

N. Y O S H I I e t a l .

242

4-5 5-6 6-7 7-8 8- 9 9-10 10-11 11 -12 12 13 13 14 14 16 16-18 18-20 20-22

A

-

_h

2-2.5-

25-3 - .

-

3 35 :. 35-4 4-5 5-6 6-7 7- 8 8- 9 9 -10 10-11 11-12 12-13 13 14 14 -16 16-18 18-20 20-22 rc 22 24 24 27 27-30

I

I

I

I

I

I

I

I

-

-

1

~-

-

EEGWir

.

.

Fig. 25A, B. Data of frequency analysis of the EEG's in the correct performance of the recent memory test in Fig. 24. A, Fronto-temporal lead. B, Hippocampal lead.

could reach the food put before him only through the detour, as the string restrained him from walking directly to the dish. The insight behavior of the dog was observed electroencephalographically.As soon as the food was put in the dish the dog started to walk about (A-B) and after several seconds he noticed the detour (C-D) and stepped backward (D-E-F-G) to reach the food (Figs. 27 and 28). Hippocampal synchronized waves of 4-5 c/sec appeared with the start of walking and lasted until the arrival at the food. The component of 11-1 2 c/sec was increased in the hippocampus when the animal started. The component of 9-10 c/sec was induced in the cortex during the walking about. The important changes of EEG at the moment of detour or turning back were suppression of the hippocampal synchronous waves and increase of the fast wave components in the cortical and hippocampal EEG's showing the tonic arousal pattern.

EEG

CONTINUOUS FREQUENCY ANALYSIS OF

243

A

2-2.525-3

I, -*ux

3-35

I

-

9-10 10-11 11-12 12 -13 .-

..

14-16

--+I

I

-

B

Fig. 26A, R. Data of frequency analysis of the EEG's in the incorrect performance of the receilt memory test in Fig. 24. A, Fronto-temporal lead. B, Hippocampal lead.

Short summary. I n the recent memory test the cortical and hippocampal EEG's were continuously analyzed and compared for each period of stimulation, image and behavior performance. Characteristics of the EEG's in the correct performance were the tonic arousal pattern in the stimulation period, increase of the 16 c/sec component i n the cortical and hippocampal leads in the later image period, which disappeared in the next period, and the synchronized waves of 6-7 c/sec in the hippocampus spread to the cortex in the performance period. The difference between correct and incorrect performances was observed in the hippocampal EEG, as shown in Table 11. I n the incorrect performance, no marked change occurred in the stimulation period, but synchronization at 4.5 c/sec in the image period and its increase up to 6 c/sec in the performance period occurred with an increase in components of 11.5 c/sec and 16 c/sec. The reason for the incorrect performance was suggested from the results to Ri./i,rrnr.c,$p. 2491250

244

N. Y O S H I I et

al.

TABLE I1 EEG

Behavior

Stimulation period

Correct

8-9 c/sec incr.

16 c/sec incr.

Incorrect

fast waves 9-10 c/sec incr. fast waves

fast waves 16 c/sec incr. fast waves

Fast waves

8-10 c/sec incr. 16 c/sec incr. 4.5 c/sec incr.

Cortex

Correct Hippocampus Incorrect

None

Image period

Performance period 6 c/sec incr. 16 c/sec incr. 6 c/sec incr. 16 c/sec decr.

Turning point 5-9 c/sec remain.

fast waves

6-7 c/sec incr.

-

16 c/sec decr. 6, 11.5, 16

12 c/sec remain.

c/sec incr.

fast waves

be due to the lack of tonic arousal in the stimulation period or to the carelessness to the stimulus. When the dog noticed the incorrect way and changed his direction of walking, the hippocampal synchronization was suppressed to induce the tonic arousal pattern. With the next step in the new direction hippocampal synchronization reappeared. In the detour experiment, it was observed that when the animal tried to walk directly to the food the hippocampal synchronization increased in frequency and amplitude, and when the animal found the detour to the food the 12 c/sec component in the cortex and the synchronized waves in the hippocampus were suppressed, changing to the tonic arousal pattern.

wFig. 27. EEG’s in the detour experiment. A, A fish was put into the dish (G). A-B-C, The dog walked about but failed to reach it. C-D, He found the detour. D-E-F, Walked backward to approach the food. G, Reached and seized the food. After C and G a tonic arousal pattern was presented.

CONTINUOUS FREQUENCY ANALYSIS OF

EEG

245

7- 8 0 0 -10

8-

10-11 11-12 12 -13 13 -14 14 16

B

-

16 -18 18-20 20-22 22-24

24-27 27-30 EEG(Hipp0)

A

0

C

D

E

F

G

Fig. 28A, B. Data of frequency analysis of the EEG’s in Fig. 27. A, Fronto-temporal lead. B,

Hippocampal lead.

( D ) EEG’s and behavior changes elicited by telestimulation of some brain-stem Structures To get more detailed difference in the cortical EEG’s in between the correct and incorrect performances, we used the ‘tracer method’, i.e. we recorded the EEG responses to brain-stem stimulation, which had no apparent effect on the animal behavior except EEG response. Before presenting thc results we will describe some experiments on the telcstimulation of the geniculate bodies. ( I ) Medial geniculate body. A tonic contralateral head turning was observed by electrical stimulation of thc medial geniculate body (80 clsec, 8 V, 2 mscc pulse) when the dog was at rcst, while walking was arrested when the animal was walking. Low ReJrrmcrc p . 249!250

TABLE 111 Correct performance Stimulation period Indiff. st.

EEG

VPL

Hippo. synFwrda- 2nd 3rd ciwonimental harmonic harmonic zation

t

F P

0 0

0 0

0

0

T Hippo

0

+

0 0

f

0

0

t

F P

0

0 0 0

0 0 0

0 0 0

f

0

0 0 0

0 0 0 0

T Hippo

F P

RF

Image period

T Hippo

J.

0 0

+t

0 0

c

Hippo. synFundo2nd 3rd chronimental harmonic harmonic zarion

c

0

0

0

0 0 0 0

0 0

0

0

t

0 0

4

0 0

0 0

0

t t t

0

0

t

+

0

t

c

t

+

0

0

Performance period

+

0

+

c

J

+J-

++ 0

$ 0 0

0 0

0

J-+

0

0

t o + o t o

t o

0

0 0

0

tt

t

0

t o

0 0

tt

+

+ t +c

4 0

0

0

0

+

tt

Incorrect performance Stimulation period

Image period

0 0 0 0

0 0 0 0

(tt) (t) 0 0

0 0 0

o o

t t

0 0

t

0 0 0 0

0 0

0

0

0

0

0 0 0 0

t

t

c

0 VPL 0 Hippo 0

0 0

0 0 0

0 0 0

Hippo

t

F

P T

RF

P T

t

t t

e

J-

0

0

o

tt

t

0 0

0 0

t t t

t

t

0

0

(tt) t

Performance period

0 0

0 0 0 0

J

c c t

0 0 0

(tt)

0

4

0

4

o

0

0 0

t

0

(tt) t 0

+

4

0 0

tt t

+c 0

t

0

4

t tt

o

t

0

4

0 0 0

0 0 0 0

o

0 0

0

0

0

tt

0

t t

c

0

0 0 0 0

$4 $4

0

0

o

+

c

tt

$4

0 0 0 0

o o

+c t

0 0 0 0

o

t t

0

4

c

4

z-

=

Turning point

0

0

2:

3

Hippo. Hippo. Hippo. Hippo. ~ d i f f . Fundo- 2nd 3rd syn- F u h 2nd 3rd syn- F u h 2nd 3rd syn- Funda- 2nd 3rd synst. EEG mental harmonic harmonic chroni- mental harmonic harmonic chroni- mental harmonic harmonic chroni- mental harmonic harmonic chronization zation zation zation

F P

h,

t

tt

0

tt

+

0 0 0 0

0 0 0

mental

Hippo.syn3rd ciwoniharmonic harmonic zation 2nd

++ t o + +t

J-

0 0

t t 0 t

Fu&-

t

0

c

0 0

t t

c t

t

++

0 0

J.

+c + ++ +

0

C O N T l N U O U S F R E Q U E N C Y A N A L Y S I S OF

EEG

247

voltage fast waves increased i n the cortex, and the synchronized waves in the hippocampus. (2) Lateral geniculate body. Stimulation of the lateral geniculate body (80 c/sec, 7 V, 2 msec pulse) induced head turning to the contralateral side. Additionally, pricking up of ears and stopping of tail wagging were occasionally observed. Fast waves were increased in the frontal cortex when the animal pricked up his ears, and sharp waves temporarily appeared during the arrest of the tail wagging. Hippocampal synchronization was also induced. ( 3 ) Recent memory test during the stimulation oj' brain-stem structures. The same recent memory test as described above was performed during the stimulation of brain-stem structure (nucleus ventralis postero-lateralis, nucleus ventralis anterior, midbrain reticular formation). The purpose of this experiment was to see the difference in cortical response between correct and incorrect performances in the same animal. The experiment is not completed, so that Table 111 is preliminarily presented in this paper, and shows one sample of our 3 tested animals. Short summary. If we notice the 2 or 3 step difference (0 and ff, 4 and f or 4 and tt) the following summary will be accepted. The important difference is that in the case of the incorrect performance the indifferent impulses sent from the nucleus ventralis postero-lateralis produce marked response in the frontal cortex during the image period, which continues to the performance one. The conspicuous changes at the turning point from the incorrect way to the correct one were seen in the stimulation of the midbrain reticular formation, in which the temporal and hippocampal responses were increased at the turning point with the tonic arousal pattern in the hippocampus.

Discussion ( A ) Phasic and tonic arousals

Sharpless and Jasper (1956) used the term of phasic and tonic arousal reactions in their experiment on the habituation of auditory stimulus. We obtained both states in the freely moving dog with the characteristic EEG's, and used the same terms of phasic and tonic arousals, respectively. Our meaning is quite different; we emphasize that the hippocampus plays the leading role though we know the important role of the thalamic reticular system in the phasic arousal. The phasic arousal pattern of EEG was observed in the behavior of attention or searching, and just before the occurrence of voluntary movements, while the tonic arousal was observed in the alert and tense state of the posture of excretion (defecation, urination) or of placing a foreleg on the experimenter's palm. ( B ) Cortical invasion of the hippocampal synchronization during activated sleep From the results of continuous frequency analysis it was observed that several tens of seconds before entering the behaviorally activated sleep stage (rapid eye movements, limb and body muscle twitchings during sleep) the EEG component of4.5 c/sec appeared in the hippocampus, and the frequency increased up to 7.0 c/sec at the time RuJi,rcnws p . 249/250

248

N. Y O S H I I e f a l .

when generalized twitchings were induced during activated sleep. When the animal woke, the hippocampal synchronization pattern was replaced by a tonic arousal one. Corresponding to these, the same frequency component as the hippocampal synchronization increased and decreased in the cortex, perhaps owing to the spread from the hippocampus to the cortex with the latency of several seconds. The amplitude of the main frequency component of the hippocampal EEG fluctuated over a 1-min period, suggesting the contribution of humoral mechanisms in the activated sleep. ( C ) EEG during behavior performance When the animal started to go to the food in the recent memory test, the phasic arousal pattern of hippocampal synchronization with cortical desynchronization was induced, but when he changed his direction from the incorrect way to the correct one the EEG pattern changed to the tonic one. It seemed not due to the cessation of walking because the same cessation was observed without changing the hippocampal synchronization. The important mechanisms of changing the behavior performance with a new idea seemed to suppress the hippocampal synchronization. ( D ) Cause of incorrect performance in the recent memory test Comparing the results of the continuous frequency analysis of EEG’s in correct and incorrect performance of the recent memory test, it was seen that in incorrect behavior the tonic arousal pattern was not seen during the stimulation period. Another experiment with the indifferent ‘tracer’ stimulus, which is not yet completed, showed that the indifferent impulses sent from the nucleus ventralis posterolateralis easily invaded the frontal cortex during the image stage. The former suggested lack of attention to the stimulus, and the latter a lowered tonus of the activating system to keep the recent memory circuits. ( E ) Role of midbrain reticular formation in turning the behavior direction From our following experiment we concluded that the midbrain reticular formation played the important role of changing the behavior direction. Hayashi and Yoshii (to be published) made an experiment to separate the synchronization system from the fast wave system in the hippocampus. The synchronization system was activated by the stimulation of the hypothalamus, while the fast wave system, by that of the midbrain reticular formation. From the recording site, it was easy to produce fast waves in the ventral hippocampus and synchronized waves in the dorsal hippocampus. The synchronized waves appeared in the dorsal hippocampus with a weak stimulus and the fast ones with a strong stimulus. The desynchronization system was most susceptible to habituation because fast waves were induced at the early stage of the stimulation, and replaced by the synchronous waves; the synchronization system was much more resistant to habituation. The synchronization system was less resistant to hypothermia than the fast wave system (Table IV). One of the factors which change the behavior performance from one direction to another is suggested to be a hypothalamusmidbrain mechanism to the hippocampus. The reticular formation, with its diffuse influences and its capacity to modify conduction along sensory and moter pathways,

C O N T I N U O U S FREQUENCY A N A L Y S I S OF

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249

T A B L E IV Fast wave system

Stimulation site Recording site Appeared in Threshold Resistance to hypothermia

RF Ventral Early stage High Strong

Synchronization system

Hypothalamus Dorsal Late stage

Low Weak

may serve to control the natural and learned behaviors. It also provides the mechanism for changing the behavior performance, regulating the hippocampal activities in the background.

Summary (I) Two different states in the freely moving dog were observed from the E E G s of the neocortex and hippocampus recorded with a radio telemeter. They were named phasic and tonic arousals respectively. (2) When the dog started to perform an act, the phasic arousal of hippocampal synchronization with cortical desynchronization appeared in the EEG's. At the time when the animal changed the way with a new idea, a tonic arousal pattern temporarily appeared to suppress the hippocampal synchronization. (3) From the frequency analysis of EEG it was suggested that the causes of incorrect performance in the recent memory test were carelessness to the test stimulus in the stimulation period and invasion of indifferent impulses to the frontal cortex in the image period, disturbing the recent memory circuits. (4) Several tens of seconds before entering the behaviorally activated sleep stage (rapid eye movements, muscle twitchings) the hippocampal synchronization was recorded by the continuous frequency analyzer in the initial low frequency portion. It developed up to 7 c/sec during activated sleep and spread to the cortex with a latency of several seconds. (5) The characteristics of the synchronization and fast wave systems in the hippocampus and the hypothalamus-midbrain mechanism to control their background activities are discussed. ACKNOWLEDGEMENT

Our investigations have been supported by grants from the Ministry of Education, the Rockefeller Foundation (GA MNS 6039) and the Foundations Fund For Research in Psychiatry (FFRP Grant 61-233). REFERENCES ADEY,W. R.. DUNLOP, C. W., AND HENDRIX, C. E., (1960); Hippocampal slow waves; distribution and phase relations in the course of approach learning. Arch. Neurol. (Chic.), 3, 74-90. J. M. R., (1964); Free behavior and brain stimulation. Znt. Rev. Neurobiol., 6, 349-449. DELGADO,

250

N. Y O S H I I e t a / .

FIFKOVA,E., AND MARSALA, J., (1962); Stereotaxic atlases for the cat, rabbit and rat. Electrophysiological Methods in Biological Research. J. BureS, M. Petriih and J . Zacher, Editors. Prague, Publ. House Czechoslov. Acad. Sci. (p. 426-467). HOLMES, J. E., AND ADEY,W. R., (1960); The electrical activity of the entorhinal cortex during conditioned behavior. Amer. J. Physiol., 199, 741-744. NAKAO,H., (1962); The spread of hippocampal after-discharges and the performance of switch-off behavior motivated by hypothalamic stimulation in cats. Folia psychiat. neurol. jap., 16, 168-1 80. OLDS,J., (1958); Selective effects of drives and drugs on ‘reward’ systems of the brain. Neurological Basis of Behavior. G . E. Wolstenholme and C. M. O’Connor, Editors, Boston, Little, Brown & Company (p. 124-148). OLDS,J., (1962); Hypothalamic substrates of reward. Physiol. Rev., 42, 554-604. OLDS,J., AND MILNER,P., (1954); Positive reinforcement produced by electrical stimulation of septa1 area and other regions of rat brain. J . comp. physiol. Psyrhol., 47. 419-427. OLDS,J., AND PERETZ,B., (1960); A motivational analysis of reticular activating system. Electroenceph. clin. Neurophysiol., 12, 44-454. SAILER, S., AND STUMPF, CH., (1957); Beeinflussbarkeit der rhinencephalon Tatigkeit des Kaninchens. Nariyn-Schniiedeberg’s Arch. exp. Path. Pharmak., 231, 63-77. SHARPLESS, S., AND JASPER, H., (1956); Habituation of the arousal reaction. Brain, 79, 655-680. STORMVAN LEEUWEN, W., (1961); Comparison of EEG data obtained with frequency analysis and with correlation methods. Computer Techniques in EEG Analysis. M. A. B. Brazier, Editor. Electroenceph. clin . Neurophysiol., Suppl . 20, 37-40. YOSHII, N., MATSUMOTO, J., MAENO, S., HASEGAWA, Y., YAMAGUCHI, Y., SHIMOKOCHI, M., HORI,Y., AND YAMAZAKI, H., (1958); Conditioned reflex and electroencephalography. Med. J. Osaka Univ., 9, 353-375. YOSHII, N., MIYAMOTO, K., AND SHIMOKOCHI, M., (1965); Electrophysiological studies on the conditioning of frequency specific waves. Med. J. Osaka Univ., 15, 321-344. YosHi1, N . , StiIMoKocHi, M., AND MIYAMOTO, K., (1962a); Studies on ‘memory tracer’ with conditioni‘ig technique. 11. Conditioning by intermittent photic stimulation in the rabbit. Med. J . Osaka Univ., 13, 21-36. YOSHII, N.. AND YAMAGUCHI, Y., (1962b); Studies on ‘memory tracer’ with conditioning technique. I. Conditioning by electrical stimulation of the brainstem in the dog. Med. J. Osaka Univ., 13, 1-19. YOSHII,N., YAMAGUCHI, Y., SHIMOKOCHI, M., AND MIYAMOTO, K., (1963); Studies on ‘memory tracer’ with conditioning technique. 111. Electroencephalographic relationships between sleep and acute neurosis. Med. J . Osaka Univ.. 14, 99-123.

25 I

Studies on Fine Structure and Function of Synapses KIYOSHI H A M A Departnient of Anafoniy, School of Medicine, Hiroshitna University, Hiroshima (Japan)

There are, in principle, two basically different modes of synaptic transmission, electrical and chemical. The so-called segmental septum of the giant fibre in the earthworm or crayfish is an electrical two-way synapse. The giant to motor giant synapse of the crayfish and squid giant synapse both have the same morphological features, but their operating mechanism is considered to be different. The former is electrical rectifier synapse (Furshpan and Potter, 1959) and the latter is chemical (Hagiwara and Tasaki, 1958). The earthworm giant fibres are partitioned by oblique septa occurring at intervals, one to each body segment. The septum under a high power electron microscope is found to consist of two plasma membranes of adjacent giant axons (Figs. 1 and 2). The space between two plasma membranes is constant and narrow, being less than 100 A. Vesicles of regular size are found closely associated on both surfaces of the sept um. Thus the contact surface ofthis septum is morphologically symmetrical (Hama, 1959). The giant to motor giant synapse of the crayfish has been reported to be an electrical one-way synapse (Figs. 3 and 4). At the area of contact all sheath structures, including Schwann cells, disappear, and the plasma membranes of the synaptic processes and giant axoil become closely associated with each other. Vesicles and tubular structures are found closely associated with the contact area both in the pre- and post-fibres (Hama, 1961). Synaptic membranes do not show specialization such as increasing electron density or thickening. The synaptic cleft is narrow and constant, being about 100 A in width. No structural basis for rectifier function of this synapse has been observed. The squid giant synapse has been considered to be a chemical synapse, and many small svnaptic processes of the postsynaptic fibre form synaptic contact with the prefibre. The contact surface of the synapse consists of two opposing plasma membranes which are separated by a constant space of about 200-300 A in width. Synaptic vesicles are found in the pre-fibre. The synaptic cleft is occupied by slightly electrondense material. An electron-dense layer similar to the ‘inter-cellular contact layer’ of desmosome can frequently be found midway between the two synaptic membranes (Hama, 1962). In summary, synaptic membranes of electrical synapses do not show morphological References p. 252

252

K. H A M A

specialization such as increasing electron density or thickening. The synaptic cleft is narrow, being less than 100 8, in width, and does not show specialization. On the other hand, in chemical synapses, the synaptic cleft is wide - more than 200 8, and shows specialization such as an intercellular contact layer. The synaptic membrane shows increasing electron density, and electron-dense materials are found to be closely associated especially with the postsynaptic membrane. In the central nervous system of the rat, various atypical synapses’are found (Figs. 5-8). Vesicles are sometimes found in the soma or dendrite closely associated with the synaptic membrane. Dense materials are accumulated in the terminal cytoplasm which also contains synaptic vesicles. The morphological features suggest that the direction of transmission at these synapses is soma to axon or dendrite to axon (Figs. 9-1 1). Close apposition of plasma membranes of adjacent dendrites, suggesting the existence of electrical connection between the neurons, is also found i n [he central nervous system of the rat (Figs. 12-16). The functional significance of these atypical synapses is yet unknown.

SUMMARY

The fine structure of synapses which have different functional mechanisms has been studied with the electron microscope to elucidate the correlation between the tine structure and function of the synapses. The most significant fine structural differences between the chemically and electrically transmitting synapses are found in the width of the synaptic cleft and the distribution of synaptic vesicles. The electrical transmission is invariably associated with the narrow cleft. A small number of vesicles is irregularly distributed both in pre- and postsynaptic processes. They are absent from the electrical synapse. Various types of atypical synapse in the central nervous system of the rat were observed. From the morphological basis, the direction of transmission at these synapses is considered to be dendro-dendritic, dendro-axonic, soma-axonic and axoaxonic. The functional significance of these atypical synapses is not yet clearly understood. REFERENCES

E. J., AND POTTER,D. D., (1959); Transmission at the giant motor synapses of the FURSHPAN, crayfish. J. Physiol. (Lond.), 145, 289-325. HAGIWARA, S., AND TASAKI, I., (1958); A study on the mechanism of impulse transmission across the giant synapse of the squid. J . Physiol. (Lond.), 143, 114-137. HAMA,K., (1959); Some observations on the fine structure of the giant nerve fibers of the earthworm, Eisenia foetida. J. biophys. biochem. Cytol., 6 , 61-66. HAMA,K., (1961); Some observations on the fine structure of the giant fibers of the crayfishes (Cambarus virilus and Cambarus clarkii) with special reference to the submicroscopic organization of the synapses. Anat. Rec., 141, 275-293. HAMA, K., (1962); Some observations on the fine structure of the giant synapse in the stellate ganglion of the squid, Doryteuphis Bleekeri. Z . Zellforsch., 56, 437-444.

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253

Fig. I . A high power electron micrograph of a synapse between the synaptic process (p) and the second-order giant fibre (as). The opposing plasma membranes of adjacent nerve units are separated by a narrow space of 20 nip which is filled with slightly electron-dense material. Both the pre- and the postsynaptic membranes are thicker and more electron dense than the other part of the axon Schwann interface (f). Small vesicles (v) of 50-70 mp in diameter are accumulated in the second-order giant fibre i.e. presynaptic fibre, near the synaptic membrane. The cytoplasm of synaptic process (p) displays a fine granular appearance. x 110,000.

254

K. H A M A

Fig. 2. A high power electron micrograph of a portion of a segmental septum of the earthworm giant fibre showing the close apposition of two axon membranes (sm) of adjacent nerve units (gx). Vesicles of regular size (150-200 A) are lined up on both surfaces of the septum at regular intervals (100 A). Note that the contact of this septum is morphologically symmetrical. x 160,OOO.

ELECTRON M I C R O G R A P H S OF SYNAPSES

255

Fig. 3. An electron micrograph of a part of the synapse between the giant fibre (gx) and the synaptic process (p) of the motor giant fibre of the crayfish. The relationship between synaptic membranes (sm), axon membrane (am), Schwann cell surface membrane (sp) and Schwann cell (s) can clearly beseen. x 161.000.

256

K. H A M A

Fig. 4. A high power electron micrograph of a part of the synapse between the lateral giant fibre and the synaptic process (p) of the motor giant fibre of the crayfish. Two synaptic membranes (sm) lie close to each other with a constant gap, 100 8, in width. Vesicles of 400-600 8, in diameter are seen closely associated with both synaptic membranes. No structural difference which suggests the functional polarity at this synapse can be detected. x 164,000. (gx)

E L E C T R O N M I C R O G R A P H S OF S Y N A P S E S

257

Fig. 5. An electron micrograph showing type I synapse on the large dendrites (D,D’). The terminal (T) makes synaptic contact with two dendrites (arrow). The synaptic cleft is about 300 8, wide. The apposing synaptic membranes show a remarkable increase in electron density. (From the spinal cord of the rat.) x 57,000.

258

K. H A M A

Fip. 6. An electron micrograph showing a type 2 synapse (T) on the soma (S). The appositional membranes exhibit localized dense regions (arrows). Clusters of synaptic vesicles are accumulated at these locations. x 41,000.

ELECTRON M I C R O G R A P H S O F S Y N A P S E S

259

Fig. 7. An example of the synapse (arrow) with subsynaptic sac. The terminal (T) contains many synaptic vesicles and mitochondria. The accumulation of rough surfaced endoplasmic reticulum (N) corresponds to the Nissl body in light microscopy. (From the spinal cord of the rat.) x 39,000.

260

K. H A M A

Fig. 8. An electron micrograph showing a second type nerve terminal (T)on the receptor cell (R) of the lateral line organ of the Japanese sea eel. The subsynaptic sac (arrow) is found closely associated with the synaptic membrane of the receptor cell. The second type nerve terminal is considered to be inhibitory in nature. A similarity may be seen between this and the one illustrated in Fig. 6. x 158,000.

ELECTRON M I C R O G R A P H S O F S Y N A P S E S

26 1

Fig. 9. A n electron micrograph showing an axon terminal (T) on the dendrite (D). The terminal contains a cluster of synaptic vesicles, although they are not closely associated with the synaptic membrane. Synaptic vesicles are also found on the dendrite and they are associated with the synaptic membrane. An accuniulation of electron-dense material is visible in the cytoplasm of the nerve terminal close to the synaptic membrane. From these morphological features the direction of transmission at this synapse is considered to be dendro-axonic. x 24,000.

262

K. H A M A

Fig. 10. An example of soma (S) to axon (T) synapse. Synaptic vesicles appear on the soma closely associated with the synaptic membrane. x 39,000.

ELECTRON M I C R O G R A P H S OF S Y N A P S E S

263

Fig. I I . An electron micrograph showing an axon-axon synapse. An axon (T) makes synaptic contact with another axon (T') which also contains a cluster of vesicles. x 51,000.

264

K. H A M A

Fig. 13.

Fig. 12. Fig. 12. An electron micrograph showing close apposition (arrow) of plasma membranes of adjacent dendrites (D,D).x 75,000. Fig. 13. This picture shows a high power electron micrograph of the close contact area which is marked by the arrow in Fig. 12. A five-layered complex membrane is seen. The overall thickness of the membrane complex is about 150 A. x 173,000.

ELECTRON M I C R O G R A P H S O F SYNAPSES

265

Fig. 14. Three dendrites (D, D’, D”) run parallel to each other. The contact surfaces display specialization over long distances. The gaps between the two plasma membranes are constant and narrow, especially at the lower left corner (arrow). A small amount of dense material is accumulatde on the plasma membranes of the contact area. X 28,000.

266

K. H A M A

Fig. 15. The accumulation of dense material is conspicuous. The appearance resembles that of the terminal bar or zona adherens. However the overall thickness of the two membranes is smaller, less than 200 A. x 46,000.

E L E C T R O N M I C R O G R A P H S OF S Y N A P S E S

267

Fig. 16. A high power electron micrograph showing the contact area between adjacent dendrites. A live layered compound membrane structure is visible. The overall thickness of the layers i s about 180 A. x 177,000.

268

Amino Acid Metabolism and its Relation to Brain Functions YASUZO TSUKADA Department of Physiology, Keio University School of Medicine, Tokyo (Japan)

Amino acid metabolism in the central nervous system has been well investigated. As shown in Table I, the distributions of free amino acids such as glutamic, aspartic, yaminobutyric and N-acetylaspartic acids in various species of vertebrates were similar (Tsukada et a/., 1964). The free amino acid content in the brain showed a special pattern compared to that in other tissues. It is believed that the metabolism of glutamic acid and related substances plays a role in regulating brain functions. The relationship between amino acid metabolism and brain functions will be discussed elsewhere. CHANGES I N AMMONIA A N D GLUTAMINE CONTENTS I N RAT BRAIN I N D U C E D BY STIMULATION

(Tsukada et al., 1958, 1962)

It is well known that ammonia is formed explosively in the brain after electric shocks andafter death. This effect is specific to brain tissue and is not observed in other tissues. Glutamine is believed to participate in the detoxication of ammonia in the brain. The ammonia level in the brain has been regarded as a good biochemical indicator giving the state of physiological activity of the brain in vivo as reported by several investigators (Richter and Dawson, 1948; Vladim!rova, 1954; Vrba, 1956). The contents of ammonia and glutamine in living rat brain can be estimated accurately in various states of cerebral activity with the following method. Fig. I shows the experimental set-up in a dark and quiet room, the left apparatus being used for the active defence reflex and the right one for the avoidance reflex. A rat was put into the experimental chamber, and an electric shock of 30-50 V AC was applied to the paws through the grid as an unconditioned stimulus, and the light from a 60 W electric lamp was used as a conditioned stimulus. To fix the rat brain instantly, an electric magnet was switched off; the animal then dropped into liquid nitrogen and was frozen. The frozen brain was rapidlychiselled out, ground to powder in the frozen state, and homogenized in 10% trichloroacetic acid. Determinations of ammonia and glutamine were carried out on weighed samples in accordance with Conway’s microdiffusion method. The mean value of 0.36 ,umoles/g was obtained for the ammonia content of rat

TABLE I* FREE AMINO ACID CONTENT OF BRAIN TISSUE

Species

Aspartic acid

Glutamic acid

y-Aminobutyric N-Acetylaspartic acid acid

4.74 f 1.67(3 3.56 f 1.30(6)

9.92 f 0.14(3) 10.1 2.50(8)

3.94 f 0.74(3) 2.72 f 0.92(8)

6.53 f 1.18(3) 4.59 (2)

4.09 f 0.51(3) 5.26 f 0.84(4)

9.70 f 1.54(3) 13.9 f 0.20(4)

2.95 f 0.29(3) 6.36 f 1.49(4)

4.63 f 1.10(3) 5.63 k 0.63(3)

1.65 f 0.27(4) 2.76 f 0.23(3)

8.63 f 0.70(4) 7.68 f 0.57(3)

1.85 f 0.46(4) 2.84 f 0.11(3)

< 0.1

(3)

2.86 f 0.67(3) 2.73 f 1.35(3)

8.01 1.28(3) 6.64 f 1.05(3)

3.08 f 0.63(3) 3.27 f 1.17(3)

4.85

2.23 -f 0.76(7) 2.78 f0.22(7) 4.73 f 1.47(6)

9.08 f 1.80(7) 6.83 1.13(7) 18.1 f 1.56(6)

3.46 f0.66(3)

* 18.8 * 1.94(3)

Canisfamiliaris Cavia cobaya Rattus norvegicus albus Gallus domesticus Aves CIemmys Reptilia (japonica) Bufo vulgaris Amphibia Xesurus Pisces scalprum Osteichthyes Girella punctata Parapristipoma trilineatum Sphaeroides sp. Chondrichthyes Dasybayus akajei Heterodontus japonicus

Mammalia

10.0 f 3.12(3) 10.2 f 2.41(3) 28.5 f 2.58(5)

16.1 f 0.59(3) 16.7 f 1.69(3) 17.5 f 4.25(5)

Mollusca Sepia esculenta Nerve ganglion Optic tract Nerve fibre

Cephalopoda

~~

~

Ammonia

Glutatnine

~

~

~~

@moles/g; mean & S.D.)

~

From Tsukada et al. (1958).

+

*

I

tl 0.36 f 0.07*(14)

3.15 f 0.69*(14)

(2)

0.69 f 0.18(3)

4.38 f 0.22(3)

2.75 f 0.70(7) 2.63 f0.83(7) 5.16 f 2.16(6)

5.33 f 0.53(3) 5.02 =k 1.07(4) 5.85 f 1.58(3)

0.78 f 0.32(3) 0.31 f 0.15(3) 1.54 f 0.33(4)

6.03 (2) 3.83 f 0.74(3) 8.74 f 0.22(4)

3.64 f 0.74(3)

7.93 f 1.08(3)

1.89 f 0.31(3)

6.50 f 0.69(3)

< 0.1 < 0.1 < 0.1

< 0.3 < 0.3 < 0.3

(6) (3) (3)

(6) (6) (6)

W

w

tz

0

1:

Y. T S U K A D A

270 Unavoida b l t

voidable

P

Llcluld Nitrogen

Fig. 1. Experimental apparatus for conditioned reflex.

A M I N O A C I D METABOLISM A N D B R A I N F U N C T I O N

27 I

brain in the resting state. Electrical stimulation for 5 sec which caused an active defence reflex resulted a significant increase in the ammonia in the brain, and a mean of 0.45 pmoleslg was obtained as shown in Fig. 2. With continuous stimulation for longer than 5 sec. i.e., 30,60, I20 sec or of much longer duration, no further increase in ammonia level was observed. In these experiments, the content of glutamine did not change significantly. I 0 05 5

1

04

-

')' k

I

1: I

I

m

h

I'

03

t, m

t

02 -

'

i

C

0

E

+

01

Q

No of cf

"

14

ex e xp.

1

1

1

4

15

Fig. 2. Ammonia

-A-

Lo

0,

.4 0 0

E

x

:

4

4

4

30 60 Sec

Duration of

v

;

m

$

I

-3

0

-2

c

I

c

2

:

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5

"' 5

+

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5

11

120

30

5

5

-

0

-

.P<001

..P=

stimulation

002

and glutaniine ----0---contents in rat brain after electric shock.

When the stimulation was applied to the rat for 1 sec, the ammonia content in the brain did not increase. However, 4 sec later, after I-sec electrical stimulation the ammonia content in the brain was increased to the same extent as after 5-sec stimulation, as shown in Fig. 3. This shows that it will need the time of 5 sec to detect an increase in the ammonia in the brain after the stimulation. And an increase in the ammonia in the brain produced by the second stimulation was also suppressed during

m

t Lo

a

4 Sec

3 0 Mln

6

'"

(2nd S t l m ) Time a t t e r

1st

'p
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Fig. 3. Etfect of I-sec stimulation on ammonia and glutarnine contents in rat brain. References p . 291

Y. T S U K A D A

272

30 min after 1 sec stimulation. It is suggested that even 1 sec stimulation is enough to change the ammonia metabolism in living brain instead of 5 sec. On the other hand, when the rat was stimulated continuously for 30-60 min with electric current, the glutamine content increased significantly. though the ammonia level stayed constant at the resting level. With this kind of stimulation, the rat showed a peculiar posture during stimulation to minimize the effect of electric shock which might correspond to a strong defensive inhibition. In these experiments, 14C-incorporationinto glutamine and other amino acids from [14C]glucose injected intraperitoneally into the rat was determined. As shown in Table 11, glutamine synthesis in terms of the carbon skeleton was also accelerated with conTABLE I1 DISTRIBUTION O F l 4 c - L A B E L L E D AMINO ACIDS I N R A T B R A I N

25 pC [U-C]glucose was injected into the rat intraperitoneally. Amino acids were separated by

column chromatography of Moore and Stein. Rest

Amino acid

Content (pmoles/g)

Glutamine Glutamic acid Aspartic acid y-Aminobutyric acid

*

3.07 8.62 3.03 1.91

Stimulated* 1 4 C - s ~ .act.

444 597 314 290

Content (pmolestg)

1 4 C - s ~ .act.

3.93 8.62 3.12 1.93

582 508 350 279

Electric stimulation was applied through paws of rat for 60 min continuously. Stlm&tion

Stimulation

-,--"+I,

formed

(theoretical )

3.005

t

OdO Glutamine formed

--*--

GIU-NH, (theoretical)

Fig. 4. Schematic representation for ammonia metabolism in brain produced by stimulation.

A M I N O AC'IU M E T A B O L I S M A N D B R A I N F U N C T I O N

273

tinuous stimulation. This showed that a net synthesis of glutamine in the brain took place under continuous stimulation. These experiments suggest the following conclusions. Ammonia way formed in living brain explosively during stimulation. Five sec later, the glutamine synthetic system began to work. Thus the content of ammonia after 5 sec stayed at the resting level for several min. When stimulation was continued over 30 min, the glutamine accumulated to a significant value in the brain owing to its synthesis. These processes might relate to inhibitory mechanisms in the brain. A schematic representation for ammonia metabolism in the brain is illustrated in Fig. 4. The next experiment was carried out to investigate the effect of the second 5-sec electrical stimulation on the ammonia level in the rat brain. As shown in Fig. 5, the

Sec Min Time after 1 st. stimulation

Fig. 5. After-effect of first electrical stimulation. * = The values of ammonia and glutamine were determined just before application of the second electric shock; ** = P < 0.05; *** P < 0.01.

second 5-sec electrical stimulation applied at any moment within 120 min after the first one, did not induce an increase in the ammonia content in an unconditioned animal. When the second stimulation was applied at 15 sec after the first one, the content of ammonia which was still high from the first stimulation, tended to decrease rapidly. However, 150 min after the first stimulation, an increase in ammonia induced by electric shock was observed just as in the unconditioned one. It appears that a transient increase in ammonia in the brain caused by a stimulation induced, during two hours, an activation of the glutamine synthetic system which is thought to be a main route of detoxication of ammonia in the brain. Evidently the stimulation produces an after-effect on the chemical process for a long period of time. This might be correlated with the memory process. Experiments were carried out using conditioned rats along similar lines. When the lighting of a 60 W lamp for 5 sec as a conditioned stimulus was applied to the reinforced rat instead of electrical stimulation, the results were similar to those with electrical stimulation, as shown in Table 111. This Table indicates that the conditioned stimuli gave the same effect on the changes in ammonia in rat brain as that of electric RcYri C ' N C ~ Sp. 291

214

Y. T S U K A D A

T A B L E 111 AFTER- EFFECT O F C O N D l T I O N @ D S T I M U L U S O N AMMONIA A N D G L U T A M I N E CONTENTS I N RAT BRAIN

Condition

Resting state 5-sec cond. stim. 60-sec cond. stim. Elect. stim. 60 min after cond. stim. Twice cond. stim. with 60-min interval Three times cond. stim. with 60-min interval Elect. stim. 10 min after lighting (unconditioned)

Exprs.

NHs (,umoles/g) Glutamine (pmoles/g) (mean f s.D.) (mean f s.D.)

8 8 4

0.35 f 0.03 0.42 f 0.03* 0.35 f 0.03

3.41 f 0.38 3.19 & 0.41 3.25 f 0.28

5

0.41 f 0.02*

3.51 f 0.39

I

0.39

3.13 f 0.44

6

0.35 f 0.35

3.18

9

0.43 f 0.03*

3.40 f 0.41

0.05**

0.25

* P < 0.01.

** P < 0.05. shock. But the effect of the conditioned stimuli became weaker according to the extinction phenomenon in conditioned animals. In the reinforced rat, however, the second stimulation was already effective 60 min after the first stimulation. This shows that the after-effect caused by the first stimulation has been shortened by the repeated stimulations or learning. When the rat was conditioned by the light for avoidance reflex instead of defence reflex, the ammonia response induced by the second stimulation appeared 30 rnin after the first stimulation. This suggests that the chemical processes of activation of the glutamine synthetic sys-

Fig. 6. Effect of second stimulation on the changes of ammonia and glutamine in brain under various conditioning processes.

AMINO A C I D METABOLISM A N D B R A I N F U N C T I O N

275

TABLE IV EFFECTS OF AMMONIUM CHLORIDE INJECTION O N AMMONIA A N D Q L U T A M I N E CONTENTS I N R A T B R A I N

Injection NHaCl20 mg/kg NH&l 20 mg/kg Ha0 0.2 ml

shock

-

+ +

Ex~ts.

NH3 (pmoleslg) (mean f s.D.)

Glutamine (ptnoleslg) (mean f s.D.)

0.35 & 0.09 0.36 f 0.06 0.47 f 0.08*

3.34 i-0.24 3.31 & 0.24 3.83 &- 0.51

6 6 11

* P < 0.01. TABLE V EFFECTS OF ELECTRICAL STIMULATION O N AMMONIA A N D G L U T A M I N E C O N T E N T S OF R A T BRAIN INJECTED WITH NH4CL REPEATEDLY

Condition

Resting state 60 rnin after 1st stim.

2nd Stim. 60 min after 1st stim. 2nd Stim. 60 rnin after 1st stim.on rats inj. HzO repeatedly

Expts.

NH3 (pmoleslg) (mean f S.D.)

Clutamine (pmoleslg) (mean f S.D.)

14 6 11

0.37 f 0.06 0.37 f 0.05 0.46 f 0.10"

3.40 & 0.55 3.35 f 0.28 3.80 f 0.14

7

0.41 f 0.06

3.40 f 0.33

* P < 0.02. tem in the brain rapidly return to the normal state after repeated stimulation. Also the changes in chemical processes would be different from the procedure of learning (Fig. 6). Ammonium chloride (20 mg/kg of body weight) was injected into the rat: this dose did not by itself change the brain ammonia or glutamine levels. Then, 60 min later, electrical stimulation for 5 sec did not increase the content of ammonia. However, after injection of distilled water into the animal as a control, an increase in ammonia was induced by the electrical stimulation as usual (Table IV). When ammonium salt (5 mgN/kg body weight) was injected into the rat once aday for 10 days repeatedly, ammonia response induced by the second electrical stimulation was observed 60 min after the first stimulation (Table V). From these experiments, it is concluded that the electrical stimulation has an equivalent effect to that of the injection of ammonium salt, that is a slight increase in ammonia in the brain has the effect of accelerating the glutamine synthesis.

in V i V O et a/., 1962)

STUDIES O N AMMONIA METABOLISM I N R A T BRAIN I S O T O P E S (Tsukada

USING 1 5 N A N D 1 4 C

When ammonium salt was injected into the rat, the glutamine content in the brain increased corresponding to the amount of ammonium salt injected. On the other hand, References p. 291

Y. T S U K A D A

276

TABLE VI EFFECT O F THE A D M I N I S T R A T I O N O F AMMONIUM SALTS O N T H E AMMONIA A N D G L U T A M I N E CONTENTS O F R A T B R A I N 1 h A F T E R INJECTION

Dose

Expts.

Ammonia (pmoleslg) (mean =tS.D.)

Glutatnine (pmoleslg) (mean f S.D.)

NHdCI, 200 mg/kg NHICI, 50 mg/kg NH4CI, 20 mg/kg None

9 9 6 11

0.44 f 0.10 0.47 f 0.06 0.35 f 0.09 0.38 f 0.04

5.57 1.31 4.09 f 0.14 3.34 f 0.24 3.83 & 0.39

the content of ammonia in the brain did not reach the value of 0.50 pmoles/g while the rat was alive (Table VI). The time course of the change of glutamine content in the brain was also checked. When 80 mg N/kg ammonium glucuronate was injected intraperitoneally, the glutamine content in the brain increased 15 min after the injection, and stayed high for 2 h, but the content of glutamic acid in the brain did not change significantly. Evidently glutamic acid in the brain is supplied continuously from glucose (Fig. 7).

,

Injection

(80mg N/kg)

-m 20 0

. 0

\

\

Y)

0, -

0

0 T

0

b

i 10.0

E P

U

u ._

E

2-

(3

Fig. 7. Effect of ammonia injection on amino acid content in rat brain.

In order to study the dynamic equilibrium concerned with amino acid metabolism including ammonia and glutamine in the brain, 15N-ammonium glucuronate (96 atom % excess) and [14C]glucose (uniformly labelled) (25 pc) were injected simultaneously into the rat intraperitoneally. Sixty min after the injection, the animal was killed, and amino acids in the brain and liver tissues were measured. Amino acid separation was carried out by column chromatography in accordance with the Moore, Spackman and Stein method (Moore et al., 1958). A part of the purified amino acid was measured quantitatively by ninhydrin colour development, and

277

A M I N O A C I D METABOLISM A N D B R A I N F U N C T I O N

14C-radioactivities were detected by an automatic gas flow counter. The rest of each amino acid separated was digested by the micro-Kjeldahl method, and the 15N content was measured by a mass-spectrometer according to a modified micromethod of Rittenberg (Hirano et al., 1962). Amido-N in glutamine was detected in a macro-Conway diffusion dish by halfsaturated KOH. Alkaline media were neutralized by perchloric acid and then hydrolysed by acid to form glutamic acid again. Glutamic acid formed was separated by column chromatography and digested by the micro-Kjeldahl method. 15N in both amido- and amino-N of glutamine was measured as mentioned above. The results are summarized in Table VII. TABLE VII 14C A N D 15N

INCORPORATION INTO A M I N O ACIDS I N B R A I N A N D L I V E R TISSUES 30 MIN AFTER INJECTION OF ‘5NH3 AND 14c-tiLUCOSE

Brain Condition Amino acid

NH3 Glutamine Amido-N a-Amino-N Glutamic acid Aspartic acid GABA Urea N-Acetyl asp. Taurine Protein amido-N Protein-N

Liver

15N-Ammoniainjected Content 14Csp.act.lSNex.% hmoleslg) 0.39 5.75

11.0 3.10 1.63 4.12 4.50 2.70 40. I

20.9 42 1

474 360 260

17.5 4.63 1.96 1.84 1.44 2.71

O.l> 1.09 0.12 0.06

._

lSN-Ammonia injected

Content I4C sp. act. hdeslg) 0.65 0.95

3.14 1.oo 0 4.50 0 1.10

15NEx.

%

133

282 296

7.78 2.63 3.08 3.25 11.8 1.06 0.38 0.13

In brain tissue, 70% of the total counts of 14Cin the acid-soluble fraction was found in the free amino acid group, and specific activities of l4C in glutamic acid, glutamine, aspartic acid and GABA gave higher values of this order. On the contrary only 30 % of the total count of 14C was found in free amino acids in liver tissue, and the rest of the radioactivity was found in intermediates of carbohydrate metabolism. On the other hand, the highest value of 15N atom % excess among amino acids of brain was amidoN of glutamine, and l5N in a-amino-N of glutamine was much higher than that of glutamic acid within 60 min under the condition of abnormally high ammonia level in the body. Similar results have been reported on perfusing cat brain using an enormous amount l5N-ammonium salt by Waelsch et al. (Berl et al., 1962; Waelsch, 1961), and it was assumed that active glutamine synthesis appears only in a specialized compartment in brain tissue. However, in our experimental conditions “%labelling into glutamine was not higher than that of glutamic acid; also when 15N-ammonia was Referenres p . 291

Y. T S U K A D A

278

given in a small dose, the a-amino-N in glutamic acid was much higher than that of glutamine. The physiological significance of these characteristic phenomena in brain tissue is still obscure. 15N-incorporation into amino acids in the brain, such as glutamic acid, aspartic acid and GABA, was almost of the same order as that of 14C-incorporation. In liver tissue, the highest incorporation of 15N was found in urea instead of glutamine. 15N in other amino acids was much less than that of brain. TABLE V I I I 15N I N C O R P O R A T I O N (atom eXeSS %) I N T O AMINO A C I D S Numbers in parentheses indicate 14C-radioactivity(counts/min)

T I M E COURSE OF

I N BRAIN

Time Amino acid 30 min .

60 mitt

6h

(582) 14.5 2.89 2.83 (609)

1.51 1.89

~ _ _ _ _ _

Glutamine Amido-N a-Amino-N Clutamic acid

23.2 4.54 2.60

In the brain tissue, the glutamine synthetic system mainly contributed to the ammonia binding mechanism as a detoxication process. The 15N content in amino-N of glutamine decreased rapidly, and 1 hour after ammonia injection, heavy nitrogen in amino-N of both glutamine and glutamic acid reached almost the same level. Six hours later, 15N in glutamic acid was much higher than that of glutamine (Table VIII). Evidently ammonia and gIutamine metabolism in the brain tissue must be highly specific in comparison with other tissues, and regulate the functional state of the brain in vivo.

Centrifuged

S U P - 2 0 min

Sucrose Fraction

t N )

[ Myelin1 Mitoihondrial Layer (ME) Fraction (Mw) [Nerve Microsomat Fraction (Ms)

Supernatant

(S)

Fig. 8. Separation of s u ~ l l u l a runits of guinea-pig brain cortex,

A M I N O A C I D METABOLISM A N D B R A I N F U N C T I O N

279

b

1

C

d

e

f Fig. 9. Electron micrographs of subcellular units of guinea-pig brain cortex. a mitochondria (Mlo); c and d -- nerve endings (MR);e = vesicle fractions (LM); f R d i v r n ( ( ’ \ p . 291

=

myelin (X); b = microsomes (Ms).

280

Y. T S U K A D A

G L U T A M I C A C I D METABOLISM O N S U B C E L L U L A R U N I T S I N B R A I N T I S S U E

(Uyemura et al., 1963) Metabolic compartments of glutamic acid in subcellular units of guinea-pig brain cortex were analysed. The brain cortex was homogenized in 10 volumes of 0.32 M sucrose by a Teflon homogenizer in the cold. Differential and sucrose density gradient centrifugations (Mandel et al., 1962; De Robertis et al., 1962) were carried out to separate particulate fractions as shown in Fig. 8. Nuclei, mitochondria, nerve endings, microsomes and supernatant fractions were collected. The pellet of each fraction was embedded in Epon resin and electronmicrographs were taken to check the purity of each fraction. Electronmicroscopic patterns were fairly satisfactory (Fig. 9a-f). The chemical composition of each fraction, such as total nitrogen, RNA, DNA and lipid-P was determined (Table IX). TABLE IX TOTAL

N, RNA, DNA

AND

LIPID-PD I S T R I B U T I O N

IN SUBCELLULAR UNITS OF GUINEAP I G B R A I N CORTEX

Distribution ( %) Whole homogenate*

Total N RNA DNA Lipid-P

17.0 1.7 0.5 1.o

N

X

Mio

ME

LM

MS

S

6.0 5.4 97.7 2.5

4.7 3.9 0.5 7.8

14.6 10.9 1.8 30.1

13.0 8.2

11.8 14.4

10.3 35.8

32.0 21.4

22.5

20.4

13.0

3.7

-

-

-

-

mg/g wet wt. original tissue.

The distribution of inorganic ions in each fraction was determined. Na and C1 ions were found mostly in the supernatant, and 25% of K ions were bound on particles. 50 % of the total Ca ions were found as a bound form associated with the mitochondrial fraction, and 40 % of the total Mg ions were found on small vesicle fractions and microsomes. Mg ions in microsomes were contributed mainly by ribosomes. HowTABLE X ELECTROLYTE D I S T R I B U T I O N I N S U B C E L L U L A R U N I T S O F B R A I N CORTEX

Distribution (%)

Whole homogenate Ion

P CI Na

K

Ca

Mg

Umoleslg wet wt.)

N

63.1 34.2 41.5 85.0 1.77 6.55

3.1 0.2 1.2 0.7 7.9 6.3

X

(Mt)

Mio

ME

LM

MS

S

6.0 4.2 1.8

22.5 9.8 6.5 10.8 54.5 13.6

12.5 5.1 2.5 3.0 22.7 3.8

6.3 3.1 3.0 6.0 22.8 9.5

16.0 1.2 6.0 9.6 8.6 21.7

15.5 0.5 9.6 4.0 8.4 20.2

31.5 83.8 81.0 74.0 12.0 30.2

0.3 8.6 5.4

A M I N O A C I D METABOLISM A N D B R A I N FUNCTION

28 1

ever, the differences in ionic distribution in particulates might be due to differences in the participation of phospholipids (Table X). The enzyme distribution was examined on each fraction. The enzymes concerned TABLE XI ENZYME D I S T R I B U T I O N I N S U B C E L L U L A R U N I T S O F G U I N E A - P I G B R A I N CORTEX

Distribution (%)

Whole hornogenate

Succinic dehydrogenase Glutamic dehydrogenase G lutaminase Glutamine synthetase Glutamotransferase Glutamic decarboxylase GABA transaminase GOT GPT Cholinesterase ATPase Acetylcholine

N

(Mt)

Mlo

M0

LM

Ms

S

3.1

85.3

48.0

28.5

6.7

1.6

-

1.6* 40.0*

2.0 5.6

85.6 70.0

55.0 46.0

20.0 12.7

3.0 15.2

1.0 7.6

1.8

3.5*

3.6

28.2

14.1

9.3

4.6

2.9

58.2

7.3*

2.3

26.0

13.1

8.9

7.4

3.2

60.3

1.8*

1.0

39.3

18.1

14.9

4.0

1.8

45.0

2.1 * 60.0* 11.5. 40.0* 59.8' 2.10**

1.4 1.0 4.2 2.9 3.4 4.8

40.3 37.2 26.0 9.6

23.0 16.0 12.5 3.0 23.8 11.0

11.0 14.9 9.4 5.0 20.4 21.4

5.5 11.4 6.8 15.2 22.3 13.9

0.5 1.6 5.2 11.3 10.2 11.1

55.0 33.3 62.5 61.0 12.9 7.2

240.0.

51.0 52.2

* pmoles/rng N/h.

** yg/g wet wt. original tissue. T A B L E XI1 AMINO A C I D D I S T R I B U T I O N I N S U B C E L L U L A R U N I T S OF G U I N E A - P I G

Free amino acids (Total) Aspartic acid Glutarnic acid Alanine glutarnine GABA N-Acetylaspartic acid

+

References p. 291

B R A I N CORTEX

Distribution (%)

Whole hornogenate (pmoleslg)

N

Mi0

M0

LM

Ms

S

25.0 3.50

1.2 0

5.3 4.6

5.8 5.3

6.2 7.6

2.5 1.5

72.0 76.0

9.15

0

2.4

2.8

4.8

0.5

85.0

6.50 2.50

0.3 0.3

4.5 7.5

3.6 8.6

3.5 9.5

1.4 0.4

83.0 65.0

4.60

0

17.5

21.0

3.7

0

51.0

282

Y. T S U K A D A

with glutamate metabolism in the brain tissue, such as glutamic dehydrogenase, glutamate-a-ketoglutarate transaminase, glutamic decarboxylase, glutaminase, glutamic synthetase and glutamotransferase were located mainly on mitochondria and in the supernatant. Glutamic dehydrogenase and glutaminase were strictly attached to the mitochondria1 fraction. Glutamine synthetase, glutamotransferase, glutamic decarboxylase, and transaminase were also found in the supernatant in considerable proportion. From these results, it might be difficult to assume a specific compartment for glutamine synthesis in the intracellular matrix (Table XI). Amino acid distributions in these fractions were also determined. Glutamic acid, aspartic acid and glutamine were mainly in the supernatant portion. On the other hand, GABA and N-acetylaspartic acid were found partly in nerve ending fractions (Table XII). These latter amino acids might possibly be candidates for the transmitter substance in the synaptic regions.

NEUROCHEMICAL S T U D I E S O N EXPERIMENTAL P H E N Y L K E T O N U R I A I N RATS A N D M O N K E Y S (Tsukada,

1963; Tsukada et al., 1963)

In order to clarify the relationship between amino acid metabolism and brain functions from a different angle, a neurochemical investigation on the metabolic disorder of phenylalanine was attempted. Experimental phenylketonuric rats and monkeys were prepared by high L-phenylalanine feeding from infancy (Auerbach et al., 1958; Waisman et al., 1960; Lyman, 1963). The mother rat was fed on a 2 % L-phenylalanine and L-tyrosine diet from 10 days after parturition, and suckling rats were grown on mother’s milk rich in amino acids. Suckling rats were weaned at the 23rd day after birth and then were fed on a diet containing 3 % L-phenylalanine and L-tyrosine. The excretion of homogentisic acid, phenylalanine, tyrosine and phenylpyruvic acid in the urine was determined on both mother and weanling rats. Homogentisic acid excretion in the urine was marked and fluctuated periodically over 2-week intervals in both mother and weanlings. Phenylpyruvic acid excretion was only found in weanlings on the 30th day after birth, but never found in the mother rat. Phenylpyruvic acid excretion of weanlings also fluctuated; and it is interesting that each peak of homogentisic acid and phenylpyruvic acid in the urine was observed in different phases (Fig. 10). Changes in the level of serum phenylalanine and tyrosine, and the enzyme activities which took part in phenylalanine metabolism in liver tissue, were determined on weanlings in various stages of high amino acid feeding. The results are shown in Fig. 1 1. The serum tyrosine level was much higher than the phenylalanine level (Table XllI). The change in serum tyrosine level was just parallel with that of the excretion of homogentisic acid in the urine. On the contrary the change of serum phenylalanine level was coincident with the excretion of phenylpyruvic acid.

283

AMINO A C I D METABOLISM A N D B R A I N F U N C T i O N

-1

30001

2000j; 1000

A

0

aJ E

/1

C 0 3

z: 200

B = :

5

10.0 5

s_oi U

c

,

,

I

0

E

%

3

wpry

pmoles/g - I iver I h

,umoles/g- liver/ h

.-2 50 I'

C

L +

Phe hydrOxyldSe

-

1

'.'.

Y 0

rn urine x&j

I1 2r

200.

'

10

.5 loo

n o I

I%

'

-0 a

Fig. 11. 3 % L-phenylalanine plus 3 % L-tyrosine feeding in the rat. References p. 291

2

:L

0

284

Y. T S U K A D A

TABLE XI11 PHENYLALANINE AND TYROSINE I N R A T SERUM

Serum phenylalanine was determined fluorometrically. Serum tyrosine was determined chemically as a nitrosonaphthol derivative. Diet Basic 3 % L-Tyr 3 % L-Phe

Phenylalanine (pmoleslml)

Tyrosine (pmoleslml)

0.07 0.40 0.78

0.05 0.90 2.70

+ 3 % L-Tyr

Phenylalanine hydroxylase activity in the liver declined gradually and tyrosine-aketoglutarate transaminase activity decreased temporarily at the phase when phenylpyruvic acid excretion was observed in the urine, since this enzyme was inhibited by the metabolites of phenylalanine. In general, phenylalanine and tyrosine transaminase activities in the liver tended to increase owing to the induction of these enzymes (Knox and Goswami, 1960). p-Hydroxyphenylpyruvate oxidase activity did not change significantly (Table XIV). TABLE XIV E N Z Y M E ACTIVITIES I N LIVER TISSUE FROM RATS G I V E N A M I N O ACIDS

Diet

Basic 3 % L-Tyr 3 % L-Phe

+ 3 % L-Tyr

Phe-hydroxylase+

Phe-a-ketoglutarate transaminase* *

Phe-pyr transaminase* *

Tyr-a-ketoglutarate transaminase* *

1.02 0.43 0.48

60.1 10.8 90.5

66.5 108.0 112.0

37.0 86.0 50.5

p-HPPA oxidase* *

6.96 8.85 7.92

Activities are indicated as the amounts of

* tyrosine. * * phenylpyruvic acid ** * p-hydroxyphenylpyruvic acid (p-HPPA) formation (pmoleslg liver/h). **** p-Hydroxyphenylpyruvic acid oxidase activities are indicated as the amounts

of p-hydroxy-

phenylpyruvic acid consumption (pmoleslg liverlh).

The mechanism of fluctuation of urinary excretion of homogentisic acid and phenylpyruvic acid might be explained by the changes in amino acid level in serum and of enzyme activities concerned with phenylalanine metabolism in the liver as an autocontrol system. The contents of amino acids in the brain such as glutamic acid, aspartic acid, glutamine, GABA and phenylalanine did not change significantly compared with their normal values. Only the tyrosine content clearly increased. These results showed that rats fed on 3 % L-phenylalanine and L-tyrosine would be more likely to have tyrosinosis than phenylalaninaemia, for even phenylketone was excreted in the urine.

285

A M I N O ACID METABOLISM AND BRAIN FUNCTION

An attempt was made to produce much higher levels of serum phenylalanine in the animals. Weanling rats, which were already given high amino acid milk, were fed on a diet containing 7 % L-phenylalanine. Phenylpyruvic acid excretion appeared rapidly, and its amount tended to increase. On the other hand homogentisic acid excretion was lower than in the rats fed on 3 % L-phenylalanine and L-tyrosine (Fig. 12). The serum phenylalanine level showed a much higher level than serum tyrosine (1 st phase).

?i

"t -

:100

It ,,I I

$5.

50

0

'

F

I

u

I

._ L

1 s t Phase

i 2nd Phase

-

2

U .

r u

.-C

3 rd Phase

L

i

c

.D

W

. . _ " " k--

-7%

0 L-Phe --+Normal+7%

Fig. 12. 7 % L-phenylalanine feeding in the rat. 0 -0 gentisic acid.

L-Phe--( =

phenylpyruvic acid; 0. . . . 0 = homo-

However, the glutamic acid, aspartic acid, glutamine and tyrosine contents in the brain did not change significantly. But the GABA content in the brain was markedly low, and the phenylalanine content increased 5 times over that of the control. An extraordinarily high concentration of phenylalanine in the brain during development might have an effect on the activity of glutamic decarboxylase. However, phenylalanine and its metabolites did not inhibit glutamic decarboxylase in an experiment in vitro. Woolley (Woolley and van der Hoeven, 1964) reported that the content of serotonin in the brain decreased in phenylketonuric mice. It is assumed that phenylalanine inhibits the formation of amine from amino acid. T A B L E XV SERUM P H E N Y L A L A N I N E A N D TYROSINE CONTENTS IN VARIOUS

P H A S E S F R O M R A T S G I V E N 7 % L-PHENYLALANINE Serum phenylalanine was determined fluorometrically. Serum tyrosine was determined chemically as a nitrosonaphthol derivative.

Phase

Control 1 2 3 References p. 291

Phenylalanine (pmolesiml)

0.07 2.30 0.49 5.10

Tyrosine (pmoles/ml)

0.05 0.66 0.18 0.26

Y.

286

TSUKADA

When a rat was fed on a normal diet with the omission of high phenylalanine at the 30th day after amino acid feeding, phenylpyruvic acid excretion promptly stopped, and homogentisic acid excretion was found only in one day in enormous amounts. At this moment, the serum tyrosine level decreased to a subnormal range. During 10 days feeding on a normal diet (2nd phase), serum phenylalanine and tyrosine decreased to 0.49 pmoles/ml and 0.18 pmoleslml respectively. But these values were still higher than normal (Table XV). When the 7 % L-phenylalanine diet was restored (3rd phase), homogentisic acid and phenylpyruic acid excretion appeared again, and the serum phenylalanine level went up much more than before (Table XVI). T A B L E XVI A M I N O A C I D C O N T E N T S I N B R A I N T I S S U E I N V A R I O U S P H A S E S FROM R A T S G I V E N L - P H E N Y LA LAN1 N E

7%

The values are pnoles/g wet weight tissue and determined by paper chromatography. Phase

ASP

Glu- NHa

Glu

GABA

Phe

TYV

Control

3.75 2.75 4.00 3.52

4.43 3.43 5.03 3.70

10.0

2.65 1.45 2.66 1.75

0.10 1.21 0.13 0.80

0.08 0.52 0.22 0.26

1 2 3

8.43 11.6 8.93

In the liver, phenylalanine hydroxylase activity markedly decreased at the 1st phase and it recovered to 50% of the normal control at the 2nd phase. However, transaminase and oxidase activities were not changed much (Table XVII). The GABA content in the brain also recovered in the 2nd phase and then decreased again in the 3rd phase. To check the intellectual development of these rats, a light-dark discrimination learning test was performed for 10 days using the T-maze as shown in Fig. 13. Through T A B L E XVII E N Z Y M E A C T I V I T I E S I N LIVER T I S S U E I N V A R I O U S P H A S E S FROM R A T S G I V E N

7 % L-PHENYL-

ALANINE

Phase

Phehydroxylase*

Control

2.00 0.41 1.17 1.09

1 2 3

Phe-a-ketog/utarate IranSaminase* * 62.5 79.0 62.8 66.2

Phe-PYr transaminase* *

Tyr-a-ketoglutarate transaminase* * *

p-HPPA oxidase. *

47.6 71.7 64.0 55.5

27.6 30.8 25.8 29.8

6.57 7.86 7.41 6.56

Activities are indicated as the amounts of tyrosine

* phenylpyruvicacid ** and p-hydroxyphenylpyruvicacid * * formation (pmoleslg liver/h). p-Hydroxyphenylpyruvic acid oxidase * *** activities are indicated as the amounts ofp-hydroxyphenylpyruvicacid consumption (pmoleslg 1iver/h).

287

A M I N O ACID METABOLISM A N D BRAIN FUNCTION

lGoal I Light

I ight

Light

Start

Fig. 13. T-maze for rat.

I I

I

30

f

lo( TIME (start to goal)

20

L ul

u E

n

2

10

0

5

Fig. 14. Light-dark discrimination learning by T-maze. 3”/, Phe Tyr feeding: O-ii

+

5

10

0-0 =

10 Days = 7 % Phe feeding; control.

A-A

=

a light-light-dark course, a rat can reach the goal freely. The learning performance was carried out 10 times per day by driven rats. The number of errors and the time consumed from start to goal were recorded during every performance. The score is indicated in Fig. 14. The rat fed on the 3 L-phenylalanine and L-tyrosine diet behaved almost normally, and the learning abilities of these rats were the same as that of the control rats. On the contrary, the rats fed on the 7 % L-phenylalanine diet seemed to be retarded with regard to the number of errors. Rrf‘rences

p . 291

Y. T S U K A D A

288

Along similar lines, infantile male monkeys (Macaca fuscata yakuii), 5 months old, were fed on water and biscuit. The drinking water contained 2 % L-phenylalanine. The daily intake of L-phenylalanine was approximately 2 g/kg body weight. Phenylpyruvic acid excretion was found every day, and the plasma phenylalanine level was elevated to that of a phenylketonuric patient. But the plasma tyrosine level increased only to a small extent, and homogentisic acid in the urine was scarcely observed as shown in Fig. 15.

.

0 50. m

I

0

Fig. 15. High phenylalanine feeding in the monkey. T AB LE X V l l l A M I N O A C I D C O N T E N T S I N B R A I N T I S S U E FROM A M O N K E Y O N A H I G H P H E N Y L A L A N I N E DIET

The values are ymoleslg tissue and determined by paper chromatography. ~

Diet

Control High Phe

ASP

Glu

GABA

5.15 6.00

2.30 I .37

GIu-NHz

I .26

I .23 I .08

1.42

T AB LE X I X E N Z Y M E A C T I V I T I E S I N L I V E R T I S S U E FROM A M O N K E Y O N A H I G H P H E N Y L A L A N I N E D I E T -

Diet

Control High Phe

xylase+ 1.78 0.34

Phe a-ketoTyr-a-kerop-HPPA glutorate trans- Phe-Pyr trans- glutarate transoxidase * * * * aminase* * aminase* * * 52.0 63.7

12.6 16.0

21.6 26.6

204.0 216.0

Enzyme activities are indicated as the amounts of

* tyrosine ** phenylpyruvic acid *** p-hydroxyphenylpyruvic acid

formation (pmoleslg liver/h)

* *** p-Hydroxyphenylpyruvic acid oxidase activities are indicated phenylpyruvic acid consumption (pmoleslg liverlh).

as the amounts of p-hydroxy-

289

A M I N O A C I D METABOLISM A N D B R A I N F U N C T I O N

After 7-month feeding on high phenylalanine, when phenylalanine was omitted from the water, phenylpyruvic acid excretion in the urine ceased. The pattern of phenylalanine tolerance test in the monkey was very close to that in the human (Anderson et a/., 1962). It seems more likely model of phenylketonuria in the case of phenylketonuric monkey which was produced experimentally. The enzyme activities in liver tissue and the contents of amino acids in the brain were also determined on biopsy materials. Phenylalanine hydroxylase activity of the phenylketonuric monkey was reduced to 20% of the normal, and the GABA content in the brain was very low (Tables XVllI and XIX). I t is suggested that a decrease in the content of GABA in the brain might play a role in mental retardation.

lector

E Recorder

FRONl

JlEW

BACK V I E W

Fig. 16. Learning test instrument for the monkey.

The intellectual development of monkeys was tested by light-dark discrimination learning or 0-X figure discrimination learning using a Skinner type test instrument shown in Fig. 16. When the right sign was given through the screen, the monkey was able to obtain food by pulling out the bar. The learning performance was carried out in 100 trials per day on a driven monkey. The score is shown in Fig. 17. In light-dark discrimination, a normal monkey learned almost 100% within 3 days, though a phenylketonuric monkey learned only 70 %. In 0-X discrimination learning, a dramatic difference was found between normal and phenylketonuric monkeys, that is, a normal monkey learned 90 % in 7 days, while a phenylketonuric monkey learned nothing on such a higher learning test. This finding indicates that the intellectual development of the phenylketonuric monkey was highly retarded. The somatic development of the phenylketonuric monkey was almost normal in terms of body weight, and he had a good appetite throughout the experiment. On EEG examination, nothing of interest was found. The behavioral changes of the retarded monkey were examined now a t the stage which is returned to normal feeding. References p . 291

290

Y. T S U K A D A

c

50

(51

0

0

1 4 5

Day

Fig. 17. Discrimination learning for monkey.

0-

Day 0 =

high L-Phe feeding monkey; (1-0

=

control.

These attempts contribute to knowledge on the first step of understanding of the relationship between amino acid metabolism and brain activities. Through our better comprehension of the brain function from the biochemical point of view, it is now possible to plan experiments in a variety of animals that will give us important knowledge for application to human beings. SUMMARY

(I) Ammonia levels in the rat brain determined by the freezing technique changed rapidly at the various states of brain activity which were produced by electric shocks or conditioned stimuli. It is concluded that ammonia level in the brain could be a good biochemical indicator for the function of the brain. (2) Ammonia metabolism in the brain in vivo was analysed biochemically using 15Nand W-isotopes. 1SN-Ammonia was incorporated most powerfully into amido nitrogen in glutamine and also l5N appeared in amino nitrogen in glutamic acid, glutamine, aspartic acid and y-aminobutyric acid in the brain. 1%-Radioactivities which came from [14C]U-glucose were also found in glutamic acid, glutamine, aspartic acid and y-aminobutyric acid. These isotopic patterns were characterized only in the brain tissues. (3) The distribution of enzymes concerned with glutamic acid metabolism and amino acid contents was determined on the subcellular units of the brain tissues which were fractionated by differential centrifugation (nuclei, mitochondria, nerve endings, microsomes and supernatant). Glutamic acid was metabolized in localized metabolic pools in each subcellular fraction. ( 4 ) Experimental phenylketonuric rats and monkeys were produced dietetically,

AMINO A C I D METABOLISM A N D BRAIN F U N C T I O N

29 1

and the neurochemical changes in the brain were studied. GABA contents in the brain of phenylketonuric rat and monkey significantly decreased compared with that of the control animals. Phenylpyruvic acid was excreted in the urine; and phenylalanine hydroxylase activity in the liver wassuppressed markedly. The intellectual development of these animals was tested using a T-maze or a Skinner type test instrument. The phenylketonuric animals produced by high amino acid feeding from infancy were considerably retarded in mental development.

REFERENCES ANDERSON, J. A., GRAVEN, H., ERTEL,R., AND FISH,R., (1962); Identification of heterozygotes with phenylketonurics on basis of blood tyrosine responses. J . Petfiat., 61, 603-609. AUERBACH,H., WAISMAN, H. A., AND WYCKOFF, L. B., (1958); Phenylketonuria in the rat treated with decreased temporal discrimination learning. Nature, 27, 871-872. BERL,S., TAKAGAKI, G . ,CLARKE, D. D., AND WAELSCH, H., (1962); Metabolic compartments in vivo; a ~ m o n i aand glutamic acid metabolism in brain and liver. J. biol. Chem., 237, 2562-2569. DEROBERTIS, E., DEIRALDI,A. P., DE LORES ARNAIZ, G . R., AND SALGANICOFF, L., (1962); Cholinergic and non-cholinergic nerve endings in rat brain. 1. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase. J . Neurochem., 9, 23-35. HIRANO, s., UYEMURA, K., TSUKADA, Y.,AND KODAIRA, K., (1962); Studies on quantitative separation of amino acid labeled by I4C- and lSN-isotopes. Sei Kagaku (Japanese), 34,63-68. KNOX,W. E., AND GOSWAMI, M. N. D., (1960); The mechanism of p-hydroxyphenylpyruvate accumulation in guinea pigs fed tyrosine. J . biol. Chem., 235,2662-2666. LYMAN, F. L., (1963); ~henylketonu~ja. S p r i n ~ e l d(Ill.), C.C. Thomas. MANDEL,P., BOROKOWSKI, T., HARTH, S., AND MARDELL, R., (1962); Incorporation of Psz in ribonucleic acid of subcellular fractions of various regions of the rat central nervous system. J. Neurochem., 8, 126138. MOORE,S., SPACKMAN, D. H., AND STEIN,W. H., (1958); Chromatog~phyof amino acids on sulfonated polystyrene resin; an improved system. Anal. Chem., 30, 1185-1 190. RICHTER, D., AND DAWSON, R. M. C., (1948); The ammonia and glutamine content of the brain. J . biol. Chem., 176, t 199-1210. TSUKADA, Y., (1963); Biochemical studies on phenylketonuria. Nho to Shinkei(Jupanese), 16,747-751. TSUKADA, Y . , HIRANO, S., AND MATSUTANI,T., (1963); Neurochemical studies on experimental phenylketonuria. Shinkei Kenkyu no Shinpo (Japanese), 7, 721-725. TSUKADA, Y., HIRANO, S., UYEMURA, K., AND NAGATA, Y., (1962); Studies on ammonia, glutamine and amino acid metabolism in rat brain in vivo accompanied with behavioral changes. Shinkei Kenkyu no Shinpo (Japanese), 6, 631-636. TSUKADA, Y., TAKAGAKI, G., HIRANO, S., AND SUGIMOTO, S., (1958); Changes in the ammonia and glutamine content of the rat brain induced by electric shock. J. ~eurochem.,2, 295-303. TSUKADA, Y.,UYEMURA, K., HIRANO, S., AM) NAGATA, Y., (1964); Distribution of aminoacids in the brain in different species. ComparafiveNeurochemisfry.D. Richter, Editor. London, Pergamon Press. (p.179-183). K., TIDA, Y . ,AND TSUKADA, Y.,(19~3);Biochemi~I studies on subcellular units of guinea UYEMURA, pig brain cortex. Shinkei Kenkyu no Shinpo (Japanese), 7 , 763-771. VLADIMIROVA, E. A., (1954); Ammonia formation in rat cerebral hemisphere induced by conditioned stimuli. Dokl. Akad. Nauk SSSR (Russian), 95, 905-908. VRBA,R., (1956); On the participation of the glutamic acid-glutamine system in metabolic processes in the rat brain during physical exercise. J . Neurochem., 1, 12-1 7. WAELSCH, H., (1961); Compartmentalized biosynthetic reactions in the central nervous system. ~egion ~e/iroche~?isfr.v. ~~ S. S . Kety and J. Elkes, Editors. London, Pergamon Press. (p. 57-64). WAISMAN, H. A,, WANC,H. L., PALMER, G., AND HARLOW, H. F., (1960); Phenylketonuria in infant monkeys. Na/we, 188, 1124-1 125. WOOLLEY, D. W., AND VAN DER HOEVEN, TH.,(1964); Serotonin deficiency in infancy as one cause of a mental defect in p~enylk~tonuria. Science, 144, 883-884.

Projections of the Motor, Somatic Sensory, Auditory and Visual Cortices in Cats T O S H I O KUSAMA,, K A T S U M I OTANI**

AND

ETSURO KAWANAI

Department of Neuroanatomy, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo (Japan) * and Department of Anatomy, School of Medicine, Chiba University, Chiba (Japan) * *

The Nauta method allows us to observe fiber connections of the central nervous system which are insufficiently demonstrated with the Marchi method. With our collaborators we studied, using the Nauta-Gygax or its modified method, projections of the motor, somatic sensory, auditory and visual areas in the lateral surface of the cortex of cats. Most of our findings already have been published, but mainly in Japanese. In this paper we summarize our studies. In description we placed stress on preterminal degenerating fibers, and mostly omitted passing fibers, because space did not permit it. TABLE I A B B R E V I A T I O N S I N TEXT, T A B L E A N D F I G U R E S

AEct AL Ant. AS AS1 ASI, A ASI, P ASm ASm, L ASm, M ASup Bu Caud. Cent. Cerv. CI CL CM

co Co, A Co, Mid co, P D. D.-I. D.-m.

Anterior ectosylvian gyrus Anterior lateral gyrus Anterior Anterior sigmoid gyrus Lateral part of AS Anterior part of AS1 Posterior part of AS1 Medial part of AS Lateral part of ASm Medial part of ASm Anterior suprasylvian gyrus Cuneate nucleus (Burdach) Caudal Central Cervical Capsula interna Nucl. centralis lateralis Nucl. centrum medianum Coronal gyrus Anterior part of Co Middle part of Co Posterior part of Co Dorsal Dorso-lateral Dorso-medial

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293

T A B L E I (continued) Ect EN Except. ( * ) or G0 GL GM GP IC Intermed. L LD Lgd Lgv LP M MD MEct

Mg MSup NCM PEct PED PL Post. PS PSI PSI, A PSI, P PSm PSm, A PSm, P PSup PUT R RE Ret. form. sc Spin. SPV SUP TO VA VL VM VP VPL VPLl VPLm VPM ZI

(A)

Ectosylvian gyrus Nucl. entopeduncularis Except for dorso-lateral corner Nucl. gracilis (Goll) Lateral geniculate body Medial geniculate body Globus pallidus Inferior colliculus In termediate Lateral gyrus Nucl. lateralis dorsalis thalami Pars dorsalis of lateral geniculate body Pars ventralis of lateral geniculate body Nucl. lateralis posterior thalami Nucl. cuneatus accessorius (Monakow) Nucl. medialis dorsalis Middle ectosylvian gyrus Medial geniculate body Middle suprasylvian gyrus Nucl. centralis medialis Posterior ectosylvian gyrus Pedunculus cerebralis Posterior lateral gyrus Posterior Posterior sigmoid gyrus Lateral part of PS Anterior part of PSI Posterior part of PSI Medial part of PS Anterior part of PSm Posterior part of PSm Posterior suprasylvian gyrus Putamen Nucl. reticularis thalami Nucl. reuniens Reticular formation Superior colliculus Spinal Spinal trigeniinal nucleus Suprasylvian gyrus Tractus opticus Nucleus ventralis anterior thalami Nucl. ventralis lateralis thalami Nucl. ventralis medialis thalami Nucl. ventralis posterior thalami Nucl. ventralis postero-lateralis thalami Pars lateralis of VPL Pars medialis of VPL Nucl. ventralis postero-medialis thalami Zona incerta

P R O J E C T I O N S O F T H E MOTOR A N D S O M A T I C S E N S O R Y C O R T I C E S

A number of morphological studies on projections from the sigmoid and coronal

gyri to the thalamus have been performed with the Marchi method (Riese, 1924; References p . 319-322

294

T. K U S A M A , K. O T A N l A N D E. K A W A N A

t’

JASPER

CI

.I.

ww

KAWASA

FR. 11.0

F R. 10.0

FR. 9.0

FR. 8.0

Fig. I. Photomicrographs and diagrams showing classification of the thalamic nuclei. In the diagrams, classification of Kawana is shown on the right side in comparison with the atlas of Jasper and Ajmone-Marsan (1960) on the left side. Abbreviations in photographs: A = VPLl; B = VPLm; C = VPM; D = VL; L = lateral fiber bundle; M = medial fiber bundle.

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295

Poljak, 1927, 1928; Biemond, 1930; Le Gros Clark, 1932; Le Gros Clark and Boggen, 1935; Mettler, 1935a, b; Kariya, 1936; Hirasawa and Kariya, 1936; Sakuma, 1937; Uesugi, 1937; Peele, 1942; Gobbel and Liles, 1945). This method, however, does not seem to be so suitable for observing their fine organization. On the other hand, studies with the Nauta method have not yet been carried out in detail (Auer, 1956; Kusama el a/., 1960; Akiba, 1960; Niimi et a/., 1963).

A

B

Fig. 2. Diagrams showing the cortical areas which project the somatic cortico-sensoneuronal fibers of the lower body (A) and the upper body (B). Projections of the anterior and posterior parts of these cortical areas are distinguished by horizontal and vertical hatchings, respectively. Diagrams 5 and 6 represent the upper and lower spinal cord, respectively. Terms of the nuclei in the figure are shown in Fig. 3B. For appearance’s sake, protxtion of the posterior part on the left hemisphere is plotted in the thalamus on the right side and in the medulla oblongata and spinal cord on the left side. to the relay nuclei of the somatic sensory pathways

Studies on projections of the sigmoid and coronal gyri to the dorsal column nuclei, trigeminal terminal nuclei, the lateral medullary reticular formation and the spinal cord with silver impregnation methods including the Nauta method, have already been performed in considerable numbers (Brodal et al., 1956; Chambers and Liu, 1957; Walberg. 1957; Szentagothai and Rajkovits, 1958; Kuypers, 1958a, b, 1960, 1962; Kodaira, 1960; Kusama el al., 1960; Niimi et al., 1963; Nyberg-Hansen and References p. 319-322

296

T. KUSAMA, K. O T A N I A N D E. K A W A N A

Brodal, 1963). Their findings, however, are not in complete harmony and, furthermore, findings remain which have not yet been reported. We (Kawana, 1961, 1963a, b; Kawana and Kusama, 1962, 1964) produced small lesions in the sigmoid and coronal gyri (the first motor and somatic sensory areas, M I and S I) unilaterally in 5 cats, and bilaterally in 14 cats, to observe the topical organization of the corticothalamic tract in its origin and termination, in comparison

A

n

Fig. 3. Diagrams in A show the cortical areas which project the cortico-sensoneuronal fibers to the relay nuclei of the somatic sensory pathways of the head. Projections of the middle and posterior parts of Co are distinguished by horizontal and vertical hatchings, respectively. Projection of the posterior part of Co to the central part of the upper spinal cord is omitted to simplify the diagrams. Diagrams in B show the head area of M I and its projection (horizontal hatchings) and the upper body area of M I and its projection (vertical hatchings). Projection of the anterior parts of Co and AS1 to the intermediate zone of the upper spinal cord is omitted to simplify the diagrams. For appearance's sake, projections of the posterior part of Co and the anterior parts of Co and AS1 on the left hemispheres are plotted in the thalamus on the right side and the medulla oblongata on the left side.

with that of projections of these gyri to the dorsal column nuclei, the trigeminal terminal nuclei, the lateral medullary reticular formation, and the spinal cord. In our cases to the thalamic nuclei were exclusively ipsilateral to lesions, except for

P R O J E C T I O N S FROM C A T C O R T E X

297

those to the nucl. centrum medianum (CM) and the inferior part of the lateral central nucleus (CL) which received, concomitantly, a small number of contralateral fibers. Contrarily, the projections to the above mentioned nuclei other than the thalamus were predominantly contralateral. These facts allowed us to producelesions bilaterally in different areas of two sides to compare projections of different cortical areas in a cat, namely under the same experimental and staining conditions. As a matter of course, we gave attention as much as possible to theexistence o f a few fibers terminating contralaterally to the principal projections in the interpretation of findings of cats with bilateral lesions. In this section, furthermore, projections of the second somatic sensory areas (S 11) observed in 7 cats with unilateral lesions will be briefly described for comparison with those of S 1. Before describing our experimental findings the extent or subdivision of the lateral (VL) and posterior (VP) ventral thalamic nuclei adopted by us will be noted (Kawana, 1963a, b). On the basis of normal myelin-sheath stain material, most of VPM anterior to the section about Fr. 9.0 in the atlas of Jasper and Ajmone-Marsan (1960) was classified as belonging to VL (Fig. 1). Thus VL in our papers is generally composed of VL and most of VPM anterior to the section about Fr. 9.0 in their atlas, while our VP consists mostly of VPM posterior to the section about Fr. 8.5 and VPL in the atlas. This classification corresponds to that of Jimenez-Castellanos ( I 949) and to our experimental data. VP in our definition was divided into three subdivisions by two well defined myelinated fiber bundles, the lateral and the medial, and their extensions. VPM and VPL are delimited by the medial fiber bundle and its extension. This kind of VPL seemed to correspond to VPL of Jimenez-Castellanos and, for the most part, to that of Jasper et a/. The myelinated fiber bundle as the border between VPM and VPL was already shown by Crouch (1934) and Rose and Mountcastle (1952,. The lateral fiber bundle and its extension divides VPL further into two subdivisions, the medial (VPLm) and the lateral (VPLI). The fibrous lamina which divides VPL in such a fashion has not yet been demonstrated. The medial part of the posterior sigmoid gyrus (PSm). The distribution pattern of preterminal degenerating fibers observed following lesions in the sigmoid and coronal gyri is easily followed in Table 11, but certain details need emphasis. The posterior sigmoid gyrus (PS) is divided by the postcruciate sulcus into two parts, the anterior and the posterior. The anterior part of PSm projects mostly to the ventromedial part of the posterior half of VPLI apart from the posterior one-seventh of VPLI (Fig. 2A; Table IIA). On the other hand, its posterior part sends massive fibers not only all over its anterior half and to the dorsolateral part of its posterior half, apart from the posterior one-seventh of VPLI, but to the dorsolateral corners of VPLm and VPM as well. Cases with lesions in the sigmoid and coronal gyri showed no definite preterminal degenerating fibers in the posterior one.seventh of VPLI, but a small number of rosary-like degenerating fibers which appeared to be passing fibers to CM. Following lesions in PSm, preterminal degenerating fibers were definitely found in the gracile rucleus except for its small dorsolateral part above the lower level of the pyramidal decussation. Below the level, however, their number so decreased that it References

p.

319-322

T A B L E I1

(+

A

PSm

+ + = remarkable; + +

Ant. half

=

moderate;

+

=

small; +$ = a few; f = few; ? = questionable; no mark

VPLI

VPLm

Most part of post. half

Ant. 213

D.-I. part

V.-m. part

Post. 117

D.4. D,-I part corner* except.

VPM

Post.

cent,v , - ~ ,1 part

negative)

=

VL

D.4. part

Cent.

V.-m.

.

P

P

D.-'.

D. P

V.-I. part

M. m t

part

f + +++ +++ +++

t

? ?

Co

? ?

P Mid A

AS1 ASm

M

Cuneate nucleus

B

Gracile nucleus

Level of I. cuneate nucleus M. part

D. part

Cent. part

Terminal nucleus of V Below I. cuneate nucleus (hilus)

L. part

M. part

Cent. part

L. part

Sensory, oral and interposed nuclei D.-m. part

V.-m. part

".-I. part

Gaud,

L. ref. form'

PS1

co

A

P

*

P

+j.

Mid

+++

fff

TTT

&

zt

I

++

+

1

++

+

+

zt

+++

A

+

+

+

+

+++ +++ +++

+j.

+ + +++

+++

+

f

Spinal cord ~

Upper spin. cord

C

PSm PSI

Lower cerv. cord

A P

P Mid A

AS1

A P

Lower spin. cord

Upper spin. cord Upper cerv. cord

+++ ++

A

co

ASm

Upper cerv. cord

+

++

M

* For abbreviations see Table I .

Lower cerv. cord

f

Lower spin. cord

Upper spin. cord

+ + ++

+++ +++ +++ +

Lower spin. cord

Upper spin. cord

+

*

0

Lower spin. cord

rn cl

? ?

+i

+++

+j.

Nucl. cornu-comm. post.

U

f

++

f

Basal part of post. horn

Intermed. zone

Cent. part of post. horn

+++

0

+j.

+4

0

+J.

+i

4 rn x

++

++

.n

300

T. KUSAMA, K . O T A N I A N D E. K A W A N A

became difficult to decide clearly whether or not their distribution was still confined to its large ventromedial part. Projections of the anterior and posterior parts of PSm showed no marked difference in their termination in the nucleus, but the former were more massive than the latter (Table IIB). PSm projected markedly to the central part (Fig. 4) of the lower spinal cord (Table I1 C). The fibers from its anterior part were more numerous than those from its posterior part. There was, however, no discernible difference in their termination. The intermediate zone of the lumbar segments received only very few fibers. Using monkeys in which the somatotopical organization of the postcentral gyrus has been well established, Kuypers (1960) has shown that projections of the postcentral gyrus to the dorsal column nuclei, the trigeminal terminal nuclei, and the spinal cord are well organized somatotopically. Thus PSm which projects to the relay nuclei of the somatic sensory pathways of the lower body can be called the lower body area of S I. This finding agrees well with the physiological study of Woolsey and Fairman (1946) and Woolsey (1958), but not with that of Dusser de Barenne (1916). According to Woolsey the center for the limbs is situated anterior to that for the trunk. It is noteworthy that the anterior and posterior parts of PSm are represented in a different area of VP (Table HA). In contrast to VP, the gracile nucleus and the central part of the posterior horn receive fibers from the anterior and posterior parts with no discernible difference in their termination. The lateral part of the posterior sigmoid gyms (PSI).The anterior part of PSI sends fibers mostly to the central part of the anterior two-thirds of VPLm, while its posterior part projects massively to its dorsolateral part apart from its dorsolateral corner (Figs. 2B, 11A and B; Table 11 A). Of cases with lesions in PSI, one with lesion extending slightly anteriorly beyond the lateral extension of the cruciate sulcus showed moderate projection to the ventromedial part of VPLm, whereas cases with lesions in AS1 showed only few or no preterminal degenerating fibers, even if the lesions included the transitional area from PSI to ASl. Thus our findings on the projections to the ventromedial part of VPLm are not yet conclusive. Preterminal degenerating fibers occurring in lesions of both the anterior and posterior parts of PSI diminished in number in the posterior one-third of VPLm and were scattered all over it, showing no discernible difference in their distribution, in contrast to its anterior two-thirds. Preterminal degenerating fibers in the cuneate nucleus observed following lesions in the sigmoid and coronal gyri are not confined to its hilus, but spread over the cuneate nucleus including its hilus, at the level of the lateral cuneate nucleus, in contrast to the report of Kuypers (1958a). Below this level, however, they are restricted to its hilus. Projection fibers of the anterior part of PSI to the cuneate nucleus show the considerably different pattern of termination from those of its posterior part (Table IIB). It is of interest that the ventrolateral part of the cuneate nucleus received no projections from the sigmoid gyrus, but from the posterior part of Co, even in small numbers. Following lesions in PSI, the central part of the posterior horn shows preterminal degenerating fibers mostly in the lower cervical cord (Table IIC). Distribution of the preterminal degenerating fibers from lesions of its anterior and posterior parts showed

P R O J E C T I O N S FROM C A T C O R T E X

30 1

no noticeable difference. The central part of the upper cervical cord received the most noticeable projection from the posterior part of Co. This cortical area projected fibers to the cuneate nucleus and VP in the number and fashion shown in Table IIA and B. In addition of these cortical areas, the posterior part of AS1 sent fibers to the central part of the lower cervical cord, the cuneate nucleus and VPLm, as shown in Table IIA-C. Its projection to the cuneate nucleus was more definite than that to the other two nuclear parts and showed the pattern of preterminal degenerating fibers largely similar to that of the anterior part of PSI. Nucl. cornu-commissuralis posterior

I

Fig. 4. Subdivision of the spinal cord. The central part of the posterior horn (C) is the area between the substantia gelatinosa and the line ‘A’ which runs from the dorsal border of the reticular formation to the ventral margin of the medial lamella of the substantia gelatinosa. The intermediate zone (I) is the area ventral to the line ‘B’ which is drawn from the ventral border of the reticular formation to the ventral margin of the posterior funiculus. The anterior horn except for its motor neuron groups is included in the intermediate zone, for it is impossible to draw a clear demarcation between them. The area between the lines ‘A’ and ‘B’ except for the reticular formation and the nucl. cornu-commissuralis posterior is the basal part of the posterior horn (B).

PSI which projects to the relay nuclei of the somatic sensory pathways of the upper bodv is regarded as the upper body area of S I, which agrees with Woolsey (1958). The term of ‘upper body’ does not include the head in this paper. It is of special Referencrs p . 319-322

302

T. K U S A M A , K. O T A N l A N D E. K A W A N A

interest that lesions of the anterior and posterior parts of PSI show the different distribution of preterminal degenerating fibers in VPLm and the cuneate nucleus, while they do not show it in the central part of the spinal cord. Extension of the upper body area of S I to the posterior part of AS1 is not so definite, and is difficult to substantiate. The marked projection of the posterior part of Co to the central part of the upper cervical cord seems to show that this area includes the neck sensory center which appears to be located around the posterior end of the coronal sulcus according to Woolsey (1958). If the neck sensory area is located in the posterior part of Co, its chief representation in VP would be a part of the dorsolateral part of VPM except for its dorsolateral corner, since projection fibers of the posterior part of Co to the VPL are few (Table IIA). The middle and posterior parts of the coronalgyrus (Co). The coronal gyrus, except for its anterior part, sent a good many fibers to VPM. Its posterior part showed marked projection to the dorsolateral part of VPM, apart from its dorsolateral corner,

Fig. 5. Diagrammatic representation of ascending degenerating fibers following lesions in the caudal nucleus of the spinal trigeminal nucleus. Crosses and dots show degenerating fibers in fiber fasciculi and preterminal degenerating fibers, respectively. Vertical hatchings show the extent of lesions.

while its middle part sent massive fibers to the central part of VPM (Fig. 3A; Table IIA). None of our cases with lesions in the sigmoid and coronal gyri indicated marked preterminal degenerating fibers in the ventromedial part of VPM. Degenerating fibers caused by lesions in the middle and posterior parts of Co showed a different distribution of preterminal degenerating fibers in most of the trigeminal terminal nuclei. The middle part projected to the large ventromedial part of the main sensory nucleus and the oral and interposed spinal trigeminal nuclei,

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303

but hardly at all to the small ventrolateral part. The posterior part sent fibers markedly to the small ventrolateral part of the above-mentioned nuclei and only few, if any, fibers to their large ventromedial part. In the caudal nucleus of the spinal trigeminal nucleus, however, these two cortical areas did not show such a different termination: following lesion in either of them, preterminal degenerating fibers were observed in all of this nucleus except for the substantia gelatinosa. Stewart and King (1963) reported that ascending fibers of the caudal nucleus of the spinal trigeminal nucleus terminated in the interposed and oral nuclei and the main trigeminal sensory nucleus in cats. However, they did not mention the terminal portion of these fibers in the nuclei. According to our findings in rabbits (Kusama), these ascending fibers formed preterminal degenerating fibers mostly in the area where the middle and posterior parts of Co projected in cats. They terminated all over the trigeminal caudal nucleus except for the substantia gelatinosa and mostly in the lateral half of the other nuclei (Fig. 5). Such a resemblance in the termination of both fiber systems is most marked in the interposed and oral nuclei. Projections of the middle part of Co did not extend caudally to the spinal cord. Fibers of the posterior part of Co to the central part of the upper spinal cord have already been discussed in the section on PSI. The middle and posterior parts of Co which project to the relay nuclei of the somatic sensory pathways of the head are designated as the head area of S I, which agrees with Woolsey (1958). The middle and posterior parts of Co appear to correspond topically to the lower face center, and the upper face and occiput center of Woolsey, respectively. Lesions of the middle and posterior parts of Co demonstrate the different distribution of preterminal degenerating fibers in VPM and most of the Crigeminal terminal nuclei. Main projection areas of these two cortical parts in the nuclei could relate somatotopically to the lower face and to the upper face and occiput, respectively. I n relation to this assumption. it is of special interest that part of the second cervical dorsal root fibers, which are mostly composed of fibers of the major occipital nerve, terminates in the small ventrolateral part of the interposed nuclei at its caudal level (a part of the possible representation of the upper face and occiput) (Fig. 1 I G). Such a termination was not found following cuts of the dorsal roots of CI, C3, C4, C7 and C8 (Imai, 1964). Furthermore, our cases with lesions in the siginoid and coronal gyri did not show preterrninal degenerating fibers in the ventrolateral part of the cuneate nucleus which was the termination area of the C2 dorsal root, unless lesions were located in the posterior part of Co. The projections of the posterior part of Co, however, are very small in number. The possible sensory representation of the neck i n the posterior part of Co and VPM has already been discussed in the section on PSI. Kuypers (1960) observed no discernible difference in distribution of preterminal degenerating fibers in the dorsal column nuclei, the trigeminal terminal nuclei and the spinal cord, when comparing cases of lesions including the whole postcentral gyrus with cases of lesions including only its posterior half. In our cases, however, projection areas of the anterior and posterior parts of PSm in VP, those of the anterior and posterior parts of PSI in VP and the cuneate nucleus, and those of the middle RcJcrmrcs p . .119-.322

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and posterior parts of Co in VP and most of the trigeminal nucleus were different from each other, although some overlapping existed. The projections to the gracile nucleus, the caudal nucleus of the spinal trigeminal nucleus and the central part of the spinal cord, however, showed no discernible topical difference in their termination. The areas of the head, the upper body and the lower body in S I presumed morphologically from the projection pattern of the somatic cortico-sensoneuronal fibers* correspond generally with those of Woolsey (1958) observed with the evoked potential method. Woolsey’s sensory centers for the hindlimb and the lower trunk appear to correspond topically to the anterior and posterior parts of PSm; the centers for the forelimb and the upper trunk, to the anterior and posterior parts of PSI; the centers for the lower face and for the upper face and occiput, to the middle and posterior parts of Co, respectively. If so, the topical difference in the main projection parts of these individual cortical areas in VP, most of the trigeminal terminal nuclei and the cuneate nucleus (Figs. 2 and 3) may suggest the somatotopical localization of these nuclei. The somatotopical organization of VP thus presumed agrees at least in principle to that of Mountcastle and Henneman (1949) with tactile stimulation. The somatotopical organization of the somatic cortico-sensoneuronal fibers observed by us in cats are finer than that shown by Kuypers (1960) in monkeys. Our subdivision of VP into VPM, VPLm and VPLl by the well-defined myelinated fiber bundles and their extensions corresponded well to our experimental findings, except for the dorsolateral corners of VPM and VPLm which seemed to belong to VPLI. The lateral part of the anterior sigmoid gyrus ( A N ) and the anterior part of the coronal gyrus. Following lesions of the anterior part of AS1 and Co, marked preterminal degenerating fibers were observed in the medial part of VL, while the posterior part of AS1 projected mostly to its ventrolateral part (Figs. 3B and 11C; Table HA). These two projection areas, however, slightly overlapped each other. The small dorsomedial region of VL just adjacent to the internal medullary lamina showed no marked preterminal degenerating fibers in our cases with lesions in the coronal and sigmoid gyri. A small number of projection fibers to VL occurred concomitantly in several parts of the coronal and sigmoid gyri in the manner shown in Table 11. The anterior parts of AS1 and Co were the chief origin of the indirect corticomotoneuronal fibers of Kuypers’ definition to the lateral medullary reticular formation (Fig. 1 1 H aiid Table IIB), which shows that these cortical areas are the head area of M 1. The same projection also occurred, in a small number, in the middle and posterior parts of Co. Although their projection pattern to VL is different from that of the head area of M I, but largely similar to that of the posterior part of AS1 (upper body area of M I), it may be that the head area of M I extends to these areas at least in minimum degree. A small number of projection fibers of the posterior and middle parts of Co, however, should be carefully evaluated, since their lesions would incidentally include part of S 11. Thus the final conclusion needs further investigation. The motor face area of Ward and Clark (1935), Garol (1942) and Woolsey (1958) corresponds to our head area, though there is some difference in extent.

* Projections of the somatic sensory cortex to the relay nuclei of the somatic sensory pathways are called the somatic cortico-sensoneuronal fibers.

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Projections to the intermediate zone of the spinal cord are the indirect corticomotoneuronal fibers to the spinal cord. They terminate mainly in its dorsolateral part. Marked preterminal degenerating fibers in the intermediate zone of the upper spinal cord were observed in cases with lesions in the posterior part of AS1 (Table IIC). In some of these cases the fibers to +he upper cervical cord were approximately the same in amount as those to the lower cervical cord, while in the others the latter were considerably less than the former. Cases with lesions in PSI did not show such degenerating fibers, unless its anterior part was included in the lesions. Their number, however, was much smaller than that of the posterior part of AS]. These findings show that the posterior part of AS1 represents the upper body area of M 1 which extends in minimum degree to the anterior part of PSI. The posterior part of AS1 is regarded as the motor area of the upper body by Ward and Clark (1935), Garol (1942) and Woolsey (1 958). The anterior part of PSI was included in the area by Ward et a/. and Garol, but not by Woolsey. Thus our morphological result corresponds approximately to these physiological studies. Marked preterminal degenerating fibers in the intermediate zone of the upper cervical cord were also observed following lesions in the anterior parts of AS1 and Co (Table IIC). It may depend upon the fact that the neck area of M I is located in or around the anterior parts of Co and AS1 and the posterior part of ASI, and that the area is included in lesions of both of these parts. Whether or not a very small number of preterminal degenerating fibers in the intermediate zone of the upper spinal cord following lesions in the posterior part of Co were caused by involving a part of S I1 is not certain. The medial part of the atiierior sigmoid gyrus (ASm). While cases with lesions extending laterally to the medial tip of the presylvian sulcus showed preterminal degenerating fibers in VL, the cuneate nucleus, and the intermediate zone of the upper spinal cord, cases with lesions restricted medially to this tip offered no discernible preterminal degenerating fibers in these nuclei (Table IIA-C). The medial part of ASm (area medial to the medial tip of the presylvian sulcus) is partially included in the motor fore- and hindlimb areas by Ward and Clark (1935), whereas i t is not regarded as the motor area by Garol (1942) or Woolsey (1958). Our results correspond with that of the last two. The lateral part of ASm (area lateral to the medial tip) is included in the motor area of the upper body by all of them. In our finding, the lateral part of ASm projects fibers to V L and the intermediate zone of the upper spinal cord in small number (Table IIA, C). Thus our result seems to coincide with their physiological studies. Projection of this cortical area to V L is, however, somewhat complicated: its pattern is not similar to that of the posterior part of AS1 (upper body area of M I), but to the anterior parts of AS1 and Co (head area of M I), whereas the lateral part of ASm sends no discernible projection to the lateral medullary reticular formation. Such a contradiction was also observed in projections of the middle part of C o : this cortical area, which projected fibers to the lateral medullary reticular formation in small number, but not to the intermediate zone, showed a projection pattern to V L similar to that of the posterior part of AS1 (upper body area of M I). The fibers to V L were, however, very small in Rc./c,irn[es p. 3IY-322

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number. The posterior part of Co, which sent fibers not only to the intermediate zone of the upper spinal cord but to the lateral reticular formation as well, gave a projection pattern similar to that of the upper body area, but not to that of the head area of M 1. Except for these small contradictions, the projection of the sigmoid and coronal gyri seems to be well organized somatotopically. As far as can be determined from our findings on the sigmoid and coronal gyri, the ventrolateral part of VL appears t o relate somatotopically mostly to the upper body and its medial part, chiefly to the head. These parts representing the upper body and the head in VL slightly overlap each other. In cats with lesions in VL and the adjoining small area of CL and LP, Kawana showed that degenerating fibers passed downward through CM to distribute preterminal degenerating fibers in the dorsolateral part of the bilateral mesencephalic tegmentum, being much more numerous ipsilaterally, and in the ipsilateral superior colliculus (Kusama, 1963b). Yanagisawa et al. (1963) demonstrated that bilateral lesions in the dorsolateral part of the midbrain tegmentum resulted in much reduction, or abolition, of diffuse or reciprocal muscle spindle responses produced by VL stimulation, and suggested descending routes of VL which activate the gamma motor system through the midbrain tegmentum. Projections to the intermediate zone of the lower spinal cord were not definitely observed in our cases with lesions in the coronal and sigmoid gyri, unless lesions incidentally extended deeply into the deep surface of the cruciate sulcus, showing that the lower body area of M 1 is not located in the surface of these gyri. This result did not correspond to morphological studies of Chambers and Liu (1957) and NybergHansen and Brodal (1963) or to physiological studies of Ward and Clark (1935) and Garol (1942), but coincided with physiological studies of Delgado and Livingston ( 1 955) and Woolsey (I 958). The anterior part of PSI is included in the motor forelimb area by Ward and Clark (1935) and Garol (1942), while the anterior part of PSm and nearly all of PSm are respectively classified by Ward et al. and by Garol as belonging to the motor hindlimb area. That the anterior part of PSI is referred to as a part of the motor forelimb area by them is understandable, because this area sends the indirect cortico-motoneuronal fibers to the upper spinal cord, at least in small number. Their result that a part or all of the motor hindlimb area is situated in PSm, however, is diflicult to understand at least from our findings on the indirect cortico-motoneuronal fibers, since this area sends only few, if any, fibers to the intermediate zone of the lower spinal cord. This cortical area is the major origin of the somatic cortico-sensoneuronal fibers to the lower spinal cord. It has been reaffirmed with the Nauta method that the trigeminal terminal nuclei, a terminal portion of the somatic cortico-sensoneuronal fibers, send fibers to the lateral medullary reticular formation and the motor nuclei of the cranial nerves (Carpenter and Hanna, 1961; Sekino, 1962; Stewart and King, 1963). In Sekino’s cases preterminal degenerating fibers in the motor nuclei of the V, VII and XIlth cranial nerves were observed following lesions not only in the lateral reticular formation but in the trigeminal terminal nuclei as well. The fibers from the former are

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more numerous than those from the latter. Thus it cannot be denied that impulses mediated by the somatic cortico-sensoneuronal fibers affect concomitantly these motor nuclei in a fashion similar to those of the indirect cortico-motoneuronal fibers via the trigeminal terminal nuclei alone or, furthermore, via the lateral reticular formation. If so, the same view can be assumed on the somatic cortico-sensoneuronal fibers to the spinal cord. A largely similar assumption has been made by Kuypers (1960) on the basis of physiological evidence in the literature. In our study, projections from the sigmoid and coronal gyri to the spinal cord in cats were classified into four groups: projections to ( I ) the central part of the posterior horn, (2) the basal part of the posterior horn, (3) the nucl. cornu-commissuralis posterior, and (4) the intermediate zone (Kawana, 1963a. b; Kawana and Kusama, 1964). Projection to the central part of the posterior horn was regarded by us as the somatic cortico-sensoneuronal fibers to the spinal cord. It is of special interest that following a dorsal root cut preterminal degenerating fibers were observed by lmai (1964) massively in the region corresponding to the central part, but only sparsely in the basal part. Fibers of the sigmoid and coronal gyri to the intermediate zone are regarded as the indirect cortico-motoneuronal fibers in the present study. Their main terminal portion in the intermediate zone is its dorsolateral part. The principal projection area of the tecto- and rubrospinal tracts in the spinal cord is nearly the same as that of the indirect cortico-motoneuronal fibers, while the interstitio-, vestibulo- and reticulospinal (lesions of the last one are in the medial medullary reticular formation) tracts terminate chiefly in the medial and central parts of the anterior horn, differently from the indirect cortico-motoneuronal fibers. In the motor cell groups only its medial group received fibers from these five tracts, and only in a very small number (Kanemitsu, 1963, unpublished). It is noteworthy that the nucl. intermedius medialis where a good many dorsal root fibers terminate (Imai, 1964) received only few fibers from these tracts and the indirect cortico-motoneuronal fibers, except for fibers from the vestibular nuclear region. Following lesions in and around the lateral vestibular nucleus, the marked network of preterminal degenerating fibers was bilaterally found in the nucl. intermedius medialis in C2 and T6, but almost none in C5. C7, L3 and L5 (Kanemitsu, unpublished). The direct cortico-motoneuronal fibers could not be definitely observed in cats. as accepted by recent literature. Kodaira (1960), however, noted that dendrites of some motor nerve cells extended to the dorsolateral part of the intermediate zone, the main terminal portion of the indirect cortico-motoneuronal fibers. In our study, the projection pattern of the sigmoid and coronal gyri to the basal part of the posterior horn was similar to that of the nucl. cornu-commissuralis posterior (Table IIC). Localization of their origin showed neither complete correspondence t o that of the indirect cortico-motoneuronal fibers nor to that of thesomatic cortico-sensoneuronal fibers. Thus Kawana (1963a, b)and Kawana and Kusama( 1964) intended to distinguish the first two projections from the last two, in contrast to Kuypers (1960) and Nyberg-Hansen and Brodal (1963). It is of interest that the basal References p. 319-322

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part received only sparse fibers from the dorsal roots, in contrast with the central part o f the posterior horn, and that the nucl. cornu-commissuralis posterior above C5 received a good many fibers from the C7 and C8 dorsal roots, but not from the CI, C2, C3, C4, T4, T8, L3 and L4 dorsal roots (Imai, 1964). Of the extrapyramidal nuclei, the following five received noticeable projections from the sigmoid and coronal gyri: the striate body, the magnocellular red nucleus, the pontine nuclei, the tegmental reticular nucleus and the lateral reticular (funicular) nucleus. In these projections, only the cortico-rubral tract is well organized somatotopically (Kusama, 1963a). According to him, PSm (lower body area of S I) projected fibers mainly to the ventrolateral part of the magnocellular red nucleus (Fig. 1 ID,) while AS1 and PSI (upper body area of M I and S I, respectively) sent fibers chiefly to its dorsomedial part. This finding appeared to him to suggest that the dorsomedial part of the nucleus related somatotopically to the upper body, and its ventrolateral part to the lower body, which agrees with the study of Pompeiano and Brodal (1957) using the modified Gudden method. Citing the study of Kanemitsu (1963) on efferent fibers of the magnocellular red nucleus, Kusama (1963a) stated that the terminal portions of these fibers in the brain stem and spinal cord coincided well to those of the indirect cortico-motoneuronal fibers, except for the direct termination of the rubrofugal fibers in the contralateral facial nucleus, especially in its dorsolateral part. In the same year, Rinvik and Walberg (1963) also reported the somatotopical arrangement of the corticorubral projection. Their study is almost identical with ours. There is, however, a discrepancy between these two studies: Kusama (1963a) recognized the somatotopically well organized, marked projection from PS (a part of the somatic sensory area) to the magnocellular red nucleus, contrary to the observation of Rinvik and Walberg (1963). To examine this discrepancy, Mabuchi (1964) studied the corticorubral tract in more detail, using Kawana’s cases with bilateral lesions in the sigmoid and coronal gyri, and obtained the result shown in Fig. 6. Since her result appears to be well shown in this figure and space does not permit a detailed description, we will describe it only briefly. By the aid of the projection pattern to the magnocellular red nucleus, PS can be divided into two parts, the anterior and the posterior. The border between them is located slightly posterior to the postcruciate sulcus. The most marked difference between projection areas of the anterior and posterior parts in the magnocellular red nucleus is recognized in the rostrocaudal direction, the former projecting mostly to the caudal two-thirds of the nucleus and the latter chiefly to its rostral half. Differences of the projection area of the anterior part of PSm from that of the anterior part of PSI will be seen in the figure. The projection area of the latter is largely similar to that of the posterior part of ASI. The anterior parts of AS1 and Co project mostly to the dorsolateral part of the rostral two-thirds of the magnocellular red nucleus. On the contrary to PS (sensory body area), the middle and posterior parts of Co (sensory head area) send only few fibers to the nucleus. ASm shows no prominent projection to the magnocellular red nucleus, unless lesions extend into the deep surface of the cruciate sulcus. Distribution of preterminal degenerating fibers in the nucleus in cases with lesions including the deep surface of the cruciate sulcus is similar to that in cases with lesions in the anterior

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part of PSm. Projection areas of the posterior parts of PSm and of PSI are not markedly different, the latter being included in the lateral part of the former. Thus Mabuchi's study reaffirmed that PS projected markedly to the magnocellular red nucleus. Jn our findings, PSm and the posterior part of PSI project no discernible

Fig. 6. Diagrams showing the origin and termination of the corticorubral tract in cats. Projection of the right hemisphere is shown on the right side and vice versa In the right cortex the deep surface of the cruciate sulcus is exposed. The exact extent of the origin of the corticorubral tract in the deep surface is not clear. Solid circles indicate the hindlimb area; open circles, the forelimb area; solid triangles, the head area; vertical hatchings, the trunk area.

indirect cortico-motoneuronal fibers. Thus it can be concluded that the first somatic sensory area also prqjects the corticorubral tract. Projection patterns of the first sensory hindlimb area (anterior part of PSm) and the first motor lower body area (deep surface of the cruciate sulcus) to the magnocellular red nucleus are, furthermore, Rcfcrmccs p . 319-322

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similar to each other. The same fact is recognized in the projections of the mostly sensory forelimb area (anterior part of PSI) and the mostly motor upper body area (posterior part of ASI). I n the head area, only the motor area sends a marked projection, on the contrary to the body area. The fact that the head area projects to the particular part of the magnocellular red nucleus corresponds well to Kanemitsu's finding (1963) that shows termination of the rubrofugal fibers in the lateral reticular formation and the facial nucleus. If the difference of the projection pattern between the anterior and posterior parts of PS corresponds to the different somatotopical organization of these cortical areas which has been demonstrated by Woolsey ( 1 958), the ventromedial part of the rostra1 half of the magnocellular red nucleus, the main projection part of the posterior part of PS, may chiefly relate somatotopically to the trunk. The somatotopical organization of the sigmoid and coronal gyri suggested by the cortico-rubrospinal fibers is as follows: part of the deep surface of the cruciate sulcus and the anterior part of PSm are the hindlimb area; the posterior part of AS1 and the anterior part of PSI, the forelimb area; the anterior parts of AS1 and Co, the head area; the posterior parts of PSm and PSI, the trunk area. It is of interest that the somatotopical organization thus assumed is largely similar to that of the motor area of Garol (1942) and that the extznt of origin of this projection in these gyri nearly coincides with area 4 of Brodmann (1906) and Gurewitsch and Chatschaturian (1928). The above-mentioned findings prompt the hypothesis that the cortico-rubrospinal tract is a bypass of the indirect cortico-motoneuronal fibers. A marked difference between these two fiber systems is that the former arises not only from M I, but also from part of S I. In projection of the cortex to the striatum, the topographical organization in both the rostrocaudal and mediolateral planes has been observed i n albino rats (Webster, 1961), rabbits (Carman eta/., 1963) and cats (Mitsuhashi and Otani, 1961 ; Mitsuhashi, 1962). According to our study (Mitsuhashi and Otani, 1961 ; Mitsuhashi, 1962). the anterior portion of the lateral surface of the cortex in cats projects to the anterior part of the homolateral striatum (Fig. IOA). The gyrus proreus, AS and PS send more numerous fibers to the caudate nucleus than to the putamen, while fibers of Co, ASup and AEct to the putamen are nearly equal in number or more numerous than those to the caudate nucleus. The middle portion of the lateral surface of the cortex sends fibers chiefly to the posterior part of the ipsilateral striatum. MSup and the anterior part of AL project mostly to the caudate nucleus, while MEct, the middle sylvian gyrus and PEct send fibers only to the putamen. The posterior portion of the lateral surface of the cortex (PSup, PL and the posterior half of AL) seems to give rise to no prominent projection to the striatum. The result of our study on projection of the sigmoid and coronal gyri to the striatum which has been carried out in 10 cats with bilateral lesions in different areas of two sides is shown in Fig. 10E and F (Nagano and Kusama, unpublished). I t is noteworthy that the medial part of ASm projects no discernible fibers to the striatum, whereas this cortical area is regarded as area 6 by Gurewitsch and Chatschaturian (1928). Projections of this cortical area to the magnocellular red nucleus and the

31 I

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89

90 .

10'0

99

98

102

I

R

r\

lesions in AEct. B diagram representing difference of projection pattern of S I to Fig. 7. A VPfrom thatof S I 1 in sagittal plane. The Ifb and mfb show the level of appearance of the lateral and medial fiber bundles, respectively. By these bundles the thalamus is divided into levels I-IV. C-F show findings in cases with lesions including all of S 1 on the left side and degenerating fibers in 7 cases with lesions in AEct in a superimposed form on the right side. Dots show degenerating fibers except for those in the thalamic fiber fasciculi indicated by crosses. Terms of the nuclei are shown in Fig. I . ~

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lateral reticular (funicular) nucleus are also negligible. Although this cortical area projects relatively numerous fibers to the pontine nuclei and the tegmental reticular nucleus, these fibers are considerably smaller in number than those of the other part of the sigmoid and coronal gyri {Kusama, 1963a). The anterior ectosylvian gyrus ( A E c t ) . To compare projections of S 11 with those of S I, Kawana produced small lesions in AEct of 7 cats (Fig. 7A). S I I projects to VL, VP, the trigeminal terminal nuclei, the lateral medullary reticular formation and the dorsal column nuclei, as mentioned previously by Otani and Hiura (1961, 1963). Findings as to whether these fibers cross or remain uncrossed are the same as those on S 1. The most marked difference between projection areas of S 1 and S I1 in VP is recognized in the rostrocaudal direction (Fig. 7B-F). The preterminal degenerating fibers following lesions of S I exhibit a tendency to decrease markedly in number i n the most caudal part of VPLI, VPLm and VPM (Fig.7B). On the contrary to S I, S II projects chiefly to the most caudal part of VPLl and VPLm (Fig. 1 1 E). I n VPM, the difference between projections of S I and S I 1 was not so pronounced as between those observzd in VPLl and VPLm, since the preterminal degenerating fibers caused by lesions in the area of S I I were more evenly distributed in the rostrocaudal direction. The difference, however, can be observed likewise in the most caudal part of VPM: preterminal degenerating fibers occurring in lesions of S I I did not decrease in number in the most caudal part of VPM, while those of S 1 showed a considerable decrease. The sensory head area of S I is located very closely to that of S I1 and lesions of the latter will easily include a part of the former. This may be the reason why such a difference was not so markedly observed in VPM as in VPL. The final conclusion needs, however, further study. The somatotopical organization in the order of the head or the upper body or the lower body appears to be present in projections of S 11 t o VP. We concluded previously that the somatotopical organization could not be observed in the somatic cortico-sensoneuronal fibers of S I in cats, when comparing projections of the sigmoid gyrus with those of the coronal gyrus (Kusama et a/., 1960; Akiba, 1960; Kodaira, 1960). It depended on the fact that lesions of the coronal gyrus included part of the area of S 11. As mentioned above, the somatotopical organization was assumed in VL by the projection pattern of S I. In our 7 cases with lesions in S I I , however, the area of preterminal degenerating fibers is restricted to the small ventrolateral part of V L and does not extend to its medial part, although these cases showed preterminal degenerating fibers in the lateral medullary reticular formation. Recently, the posterior nuclear group (Po) is emphasized in relation with S 11 (Rose and Woolsey, 1958; Poggio and Mountcastle, 1963). The above-mentioned findings, however, are on VP or VL itself. PROJECTIONS O F T H E A U D I T O R Y C O R T E X

The auditory area has been subdivided into A I, A 11, A 111, Ep I , Ep I I , the insular region and A IV by morphological and physiological studies (Woolsey and Walzl,

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1942; Rose, 1949; Rose and Woolsey, 1949; Lilly, 1951 ; Tunturi, 1945; Mickle and Ades, 1952; Per1 and Casby, 1954; Butler e t a / . , 1957; Desmedt, 1960). A few workers, using the Nauta method, have reported on projections of A I and A I I (Whitlock and

..._ Auditory

area ( A I , A I[,E P E )

---.Dorsal Trapezoid nucleus

cochlear nucleus

---Ventral cochlear nucleus

! Superior olidary nucleus

Fig. 8. Diagram showing the cortical area which projects the auditory cortico-sensoneuronal fibers. The prominent projection to Mg and Ic occurs in the hatching area corresponding to A I and the much snlaller projection, in the area of dots corresponding to A I1 and Ep 11.

Nauta, 1956) or A IV (Desmedt, 1960) or the area including all parts of the auditory cortex (Rasmussen, 1960), but the difference among the projection patterns of each subdivision is still obscure. Then small lesions were made unilaterally or bilaterally in the auditory area and in its adjacent cortex of 28 hemispheres in cats to compare the projections with one another (Otani and Hiura, 1961, 1963). Rrfcrcnrc*s p. 319-322

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Projection of the auditory area to the medial geniculate body (Mg) is ipsilateral to lesions, which agrees with Whitlock and Nauta (1956) and Rasmussen (1960). The most extensive projection to Mg occurred in A 1. After lesions of A I, degenerating fibers enter the rostral end of Mg to distribute in the dense network of preterminal degenerating fibers in the dorsolateral and ventrolateral parts of the rostral half of Mg. At the caudal level of the rostral half a moderate number of preterminal degenerating fibers were concomitantly observed in the dorsomedial region of Mg (Fig. 1 1 F). In the caudal half of Mg, degenerating fibers ran ventromedially to enter the brachium colliculi inferioris, and no discernible preterminal degeneratingfibers were observed. Projection fibers of A I 1 to Mg are much smaller in number than those of A I. In lesions of A I 1 degenerating fibers enter Mg to reach the area of a small number of preterminal degenerating fibers in the middle part of the ventral portion of the caudal half of Mg. This area overlaps at least partly the most ventromedial part of the projection area of A I . Ep I I sends fibers to Mg in a fashion largely similar to A 11. The fibers of Ep 11, however, were very small in number and could not always be found. Our cases with lesions in A 111, Ep I, the insular region and A IV demonstrated no discernible degenerating fibers in Mg. The main projection area of the auditory cortex in Mg was the principal nucleus. Whether or not this cortex sends fibers to the magnocellular nucleus of Mg cannot be conclusively decided, but, if present, their number should be very small. The most noticeable preterminal degenerating fibers in the inferior colliculus (Ic) were also observed in cases with lesions in A 1. In these cases most of the degenerating fibers reached Ic via Mg and the brachium of Ic to terminate in almost all parts of the ipsilateral Ic. The preterminal degenerating fibers, however, were not evenly distributed in Ic, most of them lying in its dorsolateral and ventrolateral parts. A small number of degenerating fibers crossed the midline through the commissure of Ic to spread mainly in the dorsal and medial parts of the caudal half of Ic contralateral to lesions. A very small number of projection fibers to Ic occurred in A 11. Most of them took a course different from those of A 1, leaving the lateral one-third of the cerebral peduncle to reach Ic through the area of nucl. paralemniscalis and the brachium of Ic. They spread mainly in the middle one-third of the ipsilateral Ic. In the contralateral Ic no discernible preterminal degenerating fibers were observed. Projection of Ep I1 to Ic shows a largely similar course and distribution to that of A 11. A 111 projected massive fibers to the intermediate and deep gray layers of the caudal part of the superior colliculus (Sc). Although these areas are located closely to Ic, A I11 appears to send no discernible fibers to Ic itself. Contrary to Rasmussen’s (1960) finding, projection ofthe auditory cortex to the dorsal nucleus of the lateral lemniscus could not be definitely observed in our cases. Projection of the auditory cortex to Mg and Ic, the relay nuclei of the auditory pathway, could be placed in the same category as the projection of the somatic sensory cortex to the relay nuclei of the somatic sensory pathways and called the auditory cortico-sensoneuronat fibers. As mentioned above, the auditory corticosensoneuronal fibers occur most noticeably in A I, corresponding to the physiological evidence that A I is the principal area of the auditory cortex. Projection fibers of

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A I I and Ep I1 to Mg and Ic are much smaller in number than those of A I . A 111, Ep I, the insular region and A IV seem to send no fibers to Mg and Ic. The auditory cortex projects to the striate body (Fig. IOA) and, at least in very small number, to the lateral part of the amygdaloid nuclear complex. Projection to the pontine nuclei occurs only in the rostrodorsal end of the auditory cortex. Projections of the area of A 111 to VL, VP, the trigeminal terminal nuclei and the dorsal column nuclei have already been discussed. PROJECTIONS O F THE V I S U A L CORTEX

Numerous workers have already studied projections of the visual cortex (Probst, 1902; Poljak, 1927-1928; Barris et a/., 1935; Mettler, 1935~;Nauta and Bucher, 1954; Beresford, 1961 ; Altman, 1962). The detailed comparative study of projections of each part of the visual cortex, however, has been carried out by few, if any, workers. Whereas projection of the visual cortex to the pars dorsalis of the lateral geniculate body (Lgd) was neglected by Barris et a/. (1935), several workers have recognized it (Probst, 1902; Nauta and Bucher, 1954; Beresford, 1961 ; Altman, 1962). According to our findings on the lateral cortical surface in cats, only V I projected to Lgd (Otani and Hirai. 1962; Otani, 1964). Following lesions in V I, the ipsilateral Lgd showed preterminal degenerating fibers in its laminae A and A I . The posterior one-third of the anterior lateral gyrus (AL) projected to the anterior part of Lgd, while the posterior lateral gyrus (PL) sent fibers to its posterior part. Since von Monakow described projection of the visual cortex to the superior colliculus (Sc) in 1889, several workers studied its terminal portion in Sc (Probst, 1902; Barris et a/., 1935; Nauta and Bucher, 1954; Altman, 1962). Their results, however, are not in harmony. In our findings the visual cortex projects mostly to the stratum griseum superficiale, and rarely t o the stratum griseum intermedium on the ipsilateral side to lesions. The projection occurs not only in V 1 but in V I1 as well. The area of V I and V I I ventral to the occipital pole sends fibers mostly to the medial one-third of Sc; the area of the occipital pole, to its middle one-third; the area of V I and V I I rostra1 to the occipital pole, to its lateral one-third. According to Barris et a/. ( I 935) and Beresford ( I 961) the visual cortex projects to the nucl. lentiformis mesencephali of the pretectal region. In our findings, however, its chief projection area in the pretectal region is the ipsilaieral pretectal nucleus, which coincides with the findings of Nauta and Bucher (1954) in the rat. Preterminal degenerating fibers in the nucleus were observed following lesions not only in V 1 but in V I I as well. On the contrary to the projection to Sc, their distribution does not show the topical difference. Probst (1902) considered projection of the visual cortex to the pulvinar as its principal one. Hence. several workers studied this point (Barris et a/., 1935; Mettler, 1935~;Nauta and Bucher, 1954; Beresford, 1961; Altman, 1962). Among their findings, however, there still seem to be some discrepancies. In our cases, projection to the pulvinar occurs ipsilaterally in PL, PSup, the posterior end of AL, the posterior half of MSup and, occasionally, in the caudodorsal angle of Ect, as shown in Fig. 10D R<./i,rm
319-322

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(Otani and Hirai, 1962; Otani, 1964). Thus the area projecting to the pulvinar does not necessarily coincide with the visual cortex (V 1 and V I I): it includes not only the caudal half of the visual cortex (Fig. 9), but also a part o f the area projecting to LP

rficiale

Fig. 9. Diagram showing the visual cortex. The hatching area projects to Lgd, nucl. pretectalis and the stratum griseurn superficiale of Sc. The area of dots projects to the last two, but not to Lgd.

(thalamic association nuclei) which is located mostly in MSup and the anterior twothirds of AL, as shown in Fig. IOC (Otani and Horie, 1964). The area projecting to the pars ventralis of the lateral geniculate body corresponds to that to the pulvinar. Projection to Lgd can be called the visual cortico-sensoneuronal fibers. Its origin is in V I.

317

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D

F projection areas to the striatum. The blackened parts show the lesions which cause Fig. 10. A preterminal degenerating fibers in the striatum, while the parts surrounded by black lines show the lesions which do not cause them. B = projection areas to the pontine nuclei. The blackened regions project to the nuclei, while the regions surrounded by black lines do not project. C = prominent projection areas to LP. D = projection area to the pulvinar. E and F show projection patterns of the sigmoid and coronal gyri to the putainen (E: and the caudate nucleus (F). Density of dots represents the number of the projection fibers occurring in the individual parts of the.e gyri. Lesions in the sigmoid and coronal gyri in diagrams A and B are different from those used by Kawana (1963a,b) and Kawana and Kusama (1964) to observe the somatic cortico-sensoneuronal fibers, ReJerenws p . 319-322

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Fig. 11. A = photomicrograph showing degenerating fibers in fiber fasciculi of VPLl and in the gray substance of VPLm in a case with lesion in PSI. Broken line indicates the site of the lateral fiber bundle. Nauta-Gygax method, x 100. B = photomicrograph from VPLm in the above-mentioned case with higher magnification. Nauta-Gygax method, x 200. C photomicrograph showing degenerating fibers in the ventrolateral part of VL in a case with lesion of ASI. Nauta-Gygax method, 7

(continued p . 319)

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SUMMARY

Studies on projections of the motor, somatic sensory, auditory and visual cortices in cats which were carried out in our laboratories by Akiba, Hirai, Hiura, Horie, Imai, Kanemitsu, Kodaira, Mabuchi, Mitsuhashi, Nagano, Sekino and the authors with the Nauta-Gygax or its modified method have been summarized. ACKNOWLEDGEMENT

The authors are grateful for the generous collaboration of our collaborators and for the untiting technical assistance of Misses T. Ogisaka, K. Kobayashi and M. Akiyama, Mrs. T. Matsui and Mr. N. Nakamura.

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(Fig. I I coti!iniied) \.’ 400. D photomicrograph showing degenerating fibers in the magnocellular red nucleus in a case with lesion in PSni. Nauta-Gygax method, x 200. E = photomicrograph showing degenerating fibers in VPLni in a case with lesion in AEct. Nauta-Gygax method, i100. F photomicrograph showing degenerating fibers in Mg in a case with lesion in A I. Nauta-Gygax method, x 100. G photomicrograph showing degenerating fibers in the spinal trigeminal nucleus after the second cervical dorsal root had been cut. Nauta-Gygax method, x 400. H photomicrograph showing degenerating fibers in the reticular formation just medial to the spinal trigeminal nucleus in a case with lesion in the anterior parts of AS1 and Co. Nauta-Gygax method, x 200. :

~

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323

Synchronizing and Desynchronizing Influences and their Interactions on Cortical and Thalamic Neurons HARUO AKIMOTO

AND

YOICHI SAITO

Department of Neuropsychiatry, Faculty of Medicine, University of Tokyo, Tokyo (Japan)

INTRODUCTION

Since Morison and Dempsey (1942a,b) first described the diffuse rhythmic cortical waves induced by low frequency repetitive stimulation of the thalamic midline structure, a great number of studies has been devoted to the nature and the physiological significance of this ‘recruiting response’. In the original studies by the above authors recruiting response showed a homologous character with spontaneous ’spindle’ waves, and the response was clearly distinguished from more localized rhythmic response induced by the stimulation of thalamic sensory nuclei, i.e., ‘augmenting response’. The recruiting response was analyzed by the use of extracellular microelectrodes by Li et al. (1956). Repetitive burst discharge was found in synchronization with low frequency repetitive stimulation of non-specific thalamic nuclei. In the motor cortex of the ‘enciphale isole’ cat, besides the typical response pattern mentioned by theabove authors. a pure inhibitory respwse was also described (Akimoto et al., 1957, 1958). No appreciable mass discharge was found in the cat’s pyramidal tract during the recruiting response in the experiments of Brookhart and Zanchetti (1 956), and purely surface negative spindle waves were far less effective in accompanying the cortical unit discharge than the positive-negative type spindle waves (Spencer and Brookhart, 1961b). Single unit recording from the medullary pyramid, however, showed the existence of a synchronized discharge of pyramidal tract cells during the recruiting response (Arduini and Whitlock, 1953; Schlag and Balvin, 1964). The change in excitability of cortical neurons during the recruiting response was also examined by interaction study with the test response to the stimulation of specific sensory pathways (Creutzfeldt and Akimoto, 1958 on the visual cortical neurons; Endo et a/., 1959; Endo, 1962 on the somato-sensory cortical neurons). The latter authors showed the initial facilitation (10-50 msec) followed by a longer inhibitory phase. Cyclic changes in the membrance potential of cortical pyramidal tract cells during recruiting and augmenting responses have recently been demonstrated by an intracellular recording technique (Lux and Klee, 1962; Li, 1963; Purpura et al., 1964a,b). After each shock of low frequency repetitive stimulation first excitatory and then inhibitory References p . 349-351

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H . A K I M O T O A N D Y. S A l T O

post-synaptic potentials (EPSPs and IPSPs) were found. The results may explain most of the previous extracellular data. A similar cyclic alteration of membrane potentials was observed in the thalamic non-specific neurons (Purpura and Cohen, 1962; Purpura and Shofer, 1963). But much remains unknown about the genesis of the recruitment of the alternating EPSPs and IPSPs and also about the difference in the characteristics of the post-synaptic potentials (PSPs) during augmenting and recruiting responses. High frequency repetitive stimulation of the midbrain reticular formation has been found to suppress the amplitude of the recruiting response (Moruzzi and Magoun, 1949). The same high frequency stimulation caused tonic augmentation of the spontaneous discharge in the majority of the sampled neurons of the cat’s association, visual and somato-motor cortices (Saito et a/., 1957, 1958; Saito, 1959; Akimoto et a/., 1960). For the single pyramidal discharge, however, a suppressive influence of reticular stimulation has been reported (Whitlock e ta /. , 1953; Calma and Arduini, 1954). The relationship between wakefulness and unit discharge level has also been investigated in the chronic preparation (Evarts, 1961, 1963, 1964). In the last report the pyramidal tract cells showed a higher mean discharge rate in wakefulness than in the slow wave stage of natural sleep. Here, also, the underlying mechanism remainsunknown for the desynchronized augmentation of spontaneous discharge in the unitary arousal response. The present report consists of three aspects of our own efforts to extend the results obtained by previous authors. It is concerned with (A) the characteristics of the sequence of PSPs observed in pyramidal tract cells of the cat’s motor cortex during augmenting and recruiting responses, (B) the mode of unitary arousal response of pyramidal tract cells and its influence upon synchronized cyclic activity during recruiting response or spindle waves, and (C) the effects of the stimulation of thalamic and mesencephalic reticular formations (ThRF and M R F respectively) on non-specific thalamic neurons. Discussion will be mainly on the dynamic aspect of the problem. METHODS

All experiments were performed on adult cats. In the experiments described in section (A), the animal was anesthetized with pentobarbitone sodium (30 mg/kg intravenously for initial dose). In the experiments of sections (B) and (C), the animal was immobilized with D-tubocurarine chloride under artificial respiration. All surgical procedures were performed under ether anesthesia in the latter experiments, and local anesthesia with procaine was carefully used to prevent painful effects of fixation points and operative wounds. Recording from the pyramidal tract cell was made at its somatodendritic portion in the motor cortex or at its descending axon in the medullary pyramid. The cells were identified by the analysis of their response t o the stimulation of the medullary pyramid or of the surface of the pericruciate cortex respectively. A portion of bulbar pyramidal tract 3 to 6 mm caudal to the lower edge of the pons was used as the site for stimulation or recording. References p. 349-351

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Recording from non-specific thalamic neurons was carried out stereotaxically by a special micromanipulator(Nakamura et al., 1961).It consists of two independent parallel micromanipulators attached on a common base. One of them carries a ‘sheath’ or ‘tube’ with stimulating electrodes at its tip, and the other carries a micropipette which goes through the tube. The location of the tip of the tube was monitored by examining the effect of stimulation or by recording the evoked potential through the electrodes at the tip. Glass capillary microelectrodes (filled with 3 M KCI or 2 M K-citrate, electrode resistance 4-10 M R for extracellular recording, 15-20 M Q for intracellular recording) were used in all experiments. Silver wire or a ball electrode placed on the surface of the pericruciate anterior sigmoid gyrus adjacent to the point of microelectrode penetration served for leading out electrocorticogram or evoked potentials simultaneously. The criteria for the identification of the units will be described in each section. The medullary pyramid was exposed by ventral approach (Chang, 1955). For the stimulation of subcortical structures concentric bipolar electrodes with 0.3 to 0.5 mm tip distance were inserted stereotaxically into ipsilateral thalamic nucleus ventralis lateralis (VL), ipsilateral nucleus centrum medianum (CM), ipsilateral nucleus ventralis anterior (VA) or ipsilateral or contralateral midbrain reticular formation (MRF). The points of subcortical stimulation were confirmed histologically after each experiment. A conventional cathode follower input circuit was used for extracellular recording. A different input circuit designed for both recording and passing current (Ito, 1960) was employed for intracellular studies. For the confirmation of IPSPs reversal of a component of PSPs from hyperpolarizing to depolarizing response was observed by passing hyperpolarizing current or by injection of CI- ions (Coombs et al., 1955; also see Uno et a/., 1963). The signals were amplified and displayed on a dual beam oscilloscope simultaneously with electrical activity from the gross electrode on the surface of the motor cortex. The general arousal level of the animal was monitored by an 8 channel ink-writing oscillograph in all experiments. RESULTS

PSPs observed in pyramidal tract cells during augmenting and recruiting responses

( A ) The characteristics of the sequence of

This section of the study principally concerns the results of further analysis of the sequence of PSPs during the augmenting and recruiting responses. Some observations on the relationship between the PSP sequence and the surface potentials are also described. Some of the results have been reported elsewhere (Uno et al., 1963). The criteria generally used to discriminate the two types of cortical response by their distributions, latencies, wave forms and modes of amplitude change were employed (Jasper, 1960). Pyramidal tract cells (hereafter called PT cells) were identified by the observation of antidromic responses for the bulbar pyramidal tract stimulation according to the criteria employed by Phillips (1956). Rrfeermrrr p . 349-351

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Fig. I . (A) Surface potential from the motor cortex evoked by lO/sec VL stimulation. N I , N2, PI and P2 were labelled at the negative and positive surface potentials resptctively. (B) Corresponding to the surface-positive potential of P1 (upper trace) a short latency EPSP (lower trace) was evoked in a PT cell by V L stimulation. (C) An action potential induced antidromically at threshold stimulation. The same cell as in B. (D) A short latency EPSP evoked in another PT cell by VL stimulation at 2, 10,40, 80 and 200/sec. Positivity is represented as the upward deflection in all records except for A and the upper traces of B in which positivity is downward. Time constant for the intracellular recording was 0.02 sec.

( 1 ) Synaptic events in PT cells during the augmenting response induced by low frequency

VL stimulatioti When an augmenting response appeared on the surface of the pericruciate cortex, a preceding depolarization and a prolonged hyperpolarization were observed on a PT cell. The rhythm of cyclic change of both potentials accorded with that of the augmenting response. For convenience of further description, each component of the augmenting response on the cortical surface was labeled as shown in Fig. 1A. Initial positive potential was labeled as P1, which was initiated with a latency of 1.5 to 2.0 msec and remained unaltered in its amplitude during repetitive stimulation even when VL was stimulated at more than 100/sec. The succeeding negative potential (Nl) was most clearly seen in the first response. The next positive and negative potentials were labeled as P2 and N2respectively which were generally believed to exhibit typical augmentation during low frequency VL stimulation (Brookhart and Zanchetti, 1956; Yoshida et al., 1964).

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In a group of PT cells single or repetitive stimulation of the VL nucleus produced an EPSP with very short latency ( I .4-2.0 msec in our samples), with a simple configuration as illustrated in Fig. 2B and D, and capable of following VL stimulation up to 200/sec (Fig. ID). The EPSP was identified as a monosynaptic EPSP by the measurement of a delay between the arrival time of a presynaptic volley and the onset of the EPSP (Yoshida, U n o and Yajima, 1965). All PT cells activated monosynaptically by VL shocks responded to antidromic stimulation with a latency less than 2.0 msec (Fig. 1C) and were found in the deep layer of the motor cortex. Apparently they were included in the ‘early firing group’ by Towe et al. (1963). I n Fig. 1 B, during repetitive stimulation of the VL nucleus the EPSP corresponded to the earliest positivity (Pl) of the surface potential, which was initiated with a latency of about 1.5 msec. The surface PI did not exhibit augmentation but appeared stable in their amplitude during repetitive VL stimulation. The threshold EPSP occasionally triggered spikes in the lower trace but the augmenting phenomenon was not found. Hence this monosynaptic EPSP would not be playing a dominant role in the augmenting process. In the other group of sampled PT cells the monosynaptic component of EPSP was not induced. EPSPs with longer latencies appeared as a dominant component in the EPSP complex. The investigation on the relationship between the organization of the EPSPs and types of PT cells is in progress and the results will be reported elsewhere (Yoshida, Uno and Yajima, in preparation). When the augmenting response developed on the cortical surface, another EPSP of later onset and prolonged duration was observed following the monosynaptic or the early EPSP mentioned above. This late EPSP showed marked growth with close parallelism to the augmentation of the surface potential, and appeared in a stable state when the augmenting response reached its stable amplitude. This late EPSP always corresponded to the second positivity (P2). Hence this late EPSP would be closely connected to the augmenting process of surface potentials.

I Fig. 2. Traces of the responses evoked by lO/sec VL stimulation recorded on a moving film were superimposed at the point of stimulation. Upper traces in A and B were the surface records, while lower traces were intracellular potential changes in a PT cell. (A) Responses from the first to the third shocks. (B) Those from the 5th and the 6th shocks. Positivity is represented as the upward deflection in the lower traces while it is shown as the downward deflection in the upper traces. Time constant for the intracellular recording was 0.2 sec. Rtferenres p . 349-351

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Fig. 2 illustrates the development of the late EPSPs with the simultaneous record from the cortical surface during the repetitive VL stimulation. The first shock (1) produced an EPSP of short latency, but not a late one, and in the cortical surface record the late positive component (P2) was not prominent. The late EPSPs with the latency of approximately 10 msec appeared in response to the 2nd shock, and grew in its amplitude in the responses to the 3rd, 4th and 5th shocks. Also the rising slopes were more abrupt in the 3rd to 5th responses. The second positivity (P2) of the surface potentials developed in parallel with the growth of the late EPSPs. After several responses, a relatively stable configuration was obtained, in which the monosynaptic and late EPSPs were followed by the long lasting IPSPs which formed the typical appearance of PSPs during augmenting response as shown in Fig. 2B (see also Fig. 4B). It should be pointed out that the surface negative potentials (N2) would not have the close connection with the synaptic events in PT cells, because the fundamental configuration of the intracellular response was not changed with or without N2 as seen in Fig. 2 8 (traces 5 and 6). The onset of the IPSP varied in the different cases. The shortest onset of the IPSP observed was less than 3 msec. The whole time course of the IPSP was as long as 100 to 200 msec. In a slower recording of the response to single VL stimulation, after the early EPSP and succeeding IPSPs, a train of alternating EPSPs and IPSPs was regularly found corresponding to the triggered spindle waves of the surface record. As shown in Fig. 3 (left column), as the stimulus strength was increased, the IPSPs became larger in its magnitude and more prolonged in its duration (for example, from 100 msec at 0.5 V, 0.1 msec shock to 150 msec at 2 V, 0.1 msec shock) and the first ‘recurring’ EPSPs or ‘rebound’ EPSPs showed a corresponding delay in its onset, At the stimulus rate of 6/sec (Fig. 3, right column) recruitment of the late EPSPs was observed. The first recurring EPSPs, however, remained at almost the same timing and configuration. With weak stimuli the successive shocks fell after the recurring waves. After strong VL

Fig. 3. (a-c) Responses of a PT cell to l/sec VL stimulation at three levels of the stimulus strength (0.5, 1 and 2 V respectively). Early EPSP, prolonged IPSPs and ‘recurring’or ‘rebound‘ EPSPs were induced successively. As the stimulus strength was increased the IPSPs became larger and more prolonged. (d-f) Responses of the same PT cell to 6/sec VL stimulation at three levels of the stimulus strength (the same as in a-c). Facilitation of late EPSPs was observed. Three sweeps with initial 6 shocks are superposed. Arrows indicate the first responses. Spikes were truncated at the upper limit of the screen. Time: 100 msec. Voltage calibration: 10 mV.

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Fig. 4. Sequential intracellular potential changes obtained fron three different PT cells, during spontaneous spindle burst (a), augmenting response (b), and recruiting response (c, d and e). CM was stimulated at 4/sec in (c) and at lO/sec in (d), in the same PT cell. The upper trace in (a) and the lower traces in (e) are the records obtained from the surface of the motor cortex near the microelectrode in which negativity is represented as the upward deflection. Positivity is shown by upward deflection in the intracellular records. Action potentials were truncated at the upper limit of the scope screen.

shocks, successive shocks were seen on the top of the first recurring waves. The result suggests the importance of the prolonged IPSPs and ‘rebound’EPSPs for the timing of the optimal synchronization. And the optimal frequency varied, to some extent, according to the stimulus parameters. ( 2 ) Synaptic events in P T cells during recruiting response Two main characteristics will be described about the synaptic potentials in PT cells during recruiting responses. Firstly, as illustrated in Fig. 4C, the sequences of prolonged EPSPs and long lasting IPSPs were also observed in the PT cell after low frequency repetitive stimulation of CM. EPSPs which were very small after the first shock grew in amplitude and duration, corresponding to the increasing process in the surface-negative potentials in a similar manner to that of the augmenting responses. The rising slope of the EPSPs was smooth and their duration was more prolonged, as compared with the late EPSPs during augmenting response. The early EPSP observed in the augmenting response was not found in recruiting response. The phase relationships between the surface recruiting wave and the EPSPs of the PT cells may be summarized as follows: the onset of the negative recruiting wave and the rising phase of the negative wave up to its negative crest roughly corresponded to the duration of the EPSPs. Secondly, certain observations suggest that the frequency of the rhythmical recurrence of EPSPs and IPSPs during the recruiting response was much more influenced References p. 349-351

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by the so-called 'intrinsic factor' than the frequency of the applied stimuli itself. As illustrated in Fig. 4C and D, an interesting phenomenon was frequently observed. As in C, repetitive stimulation of CM at 4/sec produced typical rhythmical sequences of EPSPs and IPSPs at 4/sec in the PT cell. In the same PT cell, however, the frequency of the sequences remained at almost the same 4/sec, when the frequency of the CM stimulation was raised to IO/sec (Fig. 4D). Most of the lO/sec stimuli had no effect in producing another sequence of EPSPs and IPSPs. The finding that some stimuli failed to produce any PSPs in PT cells in a similar time relationship to that in 'rebound' type facilitation means that the stimulus effect is blocked in some area in the presynaptic course of the PT cell which originates from the CM. ( B ) Effect of stimulation of mesencephalic reticular formation (MRF) on pyramidal tract cells and its influence upon the synchronized cyclic activity during recruiting response and spindle waves

In this section we describe first the results obtained by single pyramidal discharge as an indicator of PT cell excitability. In the second the difference in the effect due to C

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Fig. 5. (A-B) Patterns of mass and unitary responses in medullary pyramid evoked by single shocks applied to anterior sigmoid cortex. Upper trace, microelectrode record on the surface of medullary pyramid. Lower trace, record after insertion of microelectrode into the pyramid. Time, 1 msec. A, recorded by a blunt steel microelectrode: electrode resistance below 1 Ma. B, recorded by a fine capillary microelectrode: electrode resistance 4 Ma.In lower record of B (at 950 p from the surface of the pyramid) amplification was reduced to half that of the upper record. (C) Short refractory period in D-response. Uppermost record shows the latency (I .3 msec) in faster sweep and the others show responses to twin shocks at various intervals, stimulus strength just above the threshold. Time, 1 msec. (D) Direct (D-) and indirect (I-) responses of a single pyramidal fiber. In each column are shown 4 responses to 4 identical stimuli. Left 4 columns: stimulation of the anterior sigmoid cortex. Right 3 columns: stimulation of the posterior sigmoid cortex. At the top of each column stimulus strengths are indicated in arbitrary units (10,9, 8, 7, and 10,9 and 8 respectively). Record at a depth of 300 p beneath the surface of the pyramid. Time, 1 msec. For explanation see text.

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the background activity or arousal level is presented. In the third the effect of M R F stimulation on the specific and non-specific type responses of the PT cells will be described. Paralyzed preparations were used in this part of the studies. A part of the study has been reported elsewhere (Akimoto et al., 1960). ( I ) Single pyramidal discharge and the efect of M R F stimulation on the spontaneous and evoked unitary pyramidal discharges Single shocks applied on the surface of the pericruciate cortex evoked a series of conducting triphasic mass spike potentials (latency 0.7-0.8 msec, interspike interval I .3-1.6 msec) on the surface of the medullary pyramid (upper traces in Fig. 5A and B). A thick microelectrode picked up ‘killed end potential’ (Patton and Amassian, 1954). Insertion of a fine capillary did not distort the triphasic potential but picked up single axonal potentials which often appeared in correspondence with the field potential (Fig. 5B, lower trace). The first or short latency spike corresponding to the first mass spike (D-response of Patton and Amassian) had short and constant latency (Fig. 5D) and showed a very short refractory period (for example 0.75 msec in Fig. 5C). The latter finding indicated that the unit was activated at its axonal portion (presumably at the IS portion). This type of response is called unitary D-response hereafter according to the above authors. Another type of response is called unitary I-response which appeared without preceding D-response and showed longer and more variable latencies. Eighty-three units were identified as pyramidal units by D-response (39 units) and/ or by short latency (less than 3 msec) I-response with typical discharge patterns (44 units) together with depth measurement of the recording electrode tip under direct visual control. Latencies of the D-response were distributed between 0.53 t o 2.0 msec. For about half the total units we followed the change in evoked unitary response during one or more trials of the reticular stimulation. The change in spontaneous discharge was recorded simultaneously by the second CRT and the long recording camera. The common change in the spontaneous discharge was that of the ‘tonic augmentation type’ (Saito et al., 1956; see also Fig. 7 in this report). The latency of the effect was 20-30 msec at the minimum and more often 50 to a few hundred msec after the onset of repetitive stimulation. The effect lasted for a considerable time after cessation of the stimulation, although the length of the effect depended on the state of the animal and the stimulus parameters. Barbiturate anesthesia blocked the effect completely. After cessation of the M R F stimulation the inhibitory phase was never found, in contrast with the effect of high frequency stimulation of ThRF. Neither was there found an inhibitory effect of low frequency stimulation or single shock stimulation, although such an effect was regularly found after stimulation of ThRF. In a few units at the onset of repetitive stimulation, or during the whole period of stimulation, an arrest of spontaneous discharge was observed. During the course of the stimulation, or immediately after the end of stimulation, asimilar tonically augmented discharge pattern was resumed. Exclusively facilitatory effects were found in all units examined by evoked pyramRrferenctm p . 349-351

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Fig. 6. (A) The effect of mesencephalic (left 3 columns) and thalamic (right 3 columns) reticular stimulation on subthreshold pyramidal I-response. Single shocks on the posterior pericruciate sigmoid cortex. Upper trace, single pyramidal discharge. Lower trace, evoked potential on the surface of the anterior sigmoid cortex. In the 2nd column repetitive stimulation (lOO/sec, 0.2 msec, 4 V) was applied to the mesencephalic reticular formation and thalamic nucleus centralis lateralis respectively. In the left 3 columns test stimulus strength was about 20% higher than that in (B). Time, 1 msec. (B) The effect of single reticular shock on the threshold of unitary D-response. Shock on the pericruciate posterior sigmoid cortex elicited unitary D- and I-responses. The stimulus strength was maintained at 25 %firing level for D-response. In the 2nd column a single reticular shock was delivered at 50 msec prior to the cortical test shock. The 3rd and the 4th columns show recovery after discontinuing the conditioning reticular shock. Time, I msec. (C)The effect of reticular stimulation on the unitary I-response. Supraliminal submaximal stimulation of the posterior sigmoid cortex. First column, record before reticular stimulation. In the 2nd column repetitive stimulation (100 sec, O.Zmsec, 4 V) of the contralateral mesencephalic reticular formation was added. During reticular stimulation the 2nd spikes were in interference with reticular shock artifacts. Time, 1 msec. (D) The effect of reticular stimulation on the unitary response evoked by single VL shocks (l/sec). Upper trace, unit record from two PT cells (cortical record). Stimulus strength of VL shock was kept subthreshold for the second unit of longer latency. Lower trace, record from the surface of the motor cortex. In the first record of the 2nd column lOO/sec reticular stimulation was added. The record below was taken at 0.5 sec after cessation of reticular stimulation. The 3rd column shows recovery from the effect. Time, 1 msec.

idal discharge (on both subliminal D-response and sub- or supraliminal I-responses). The examples are shown in Fig. 6A to C. In A the effect of repetitive stimulation (100/sec, 0.2 msec) of MRF and thalamic centralis lateralis (CL)was xamined on subthreshold I-response. Both stimulations caused facilitatory influence on the Iresponse. Usually the effect of MRF stimulation was more intense and longer lasting than that of ThRF stimulation, although thalamic stimulation showed marked potentiation in its early phase of stimulation. Fig. 6B shows facilitation of threshold D-response by a single conditioning shock which preceded the test shock by 50 msec. A similar facilitatory effect of a single conditioning MRF shock was demonstrated also on I-response. For example one unit showed the following number of I-discharges per shock (average of 10 responses at each interval between the conditioning MRF and testing cortical shock): 5 msec interval, 0.1, the same firing rate with the control test shock alone; 10 msec, 0.2; 40 msec, 0.5; 60 msec, 1.0; 80 msec, 2.0; 110 msec, 2.2; 120m~ec~2.4; and 200 msec, 0.6. In Fig. 6C facilitation of the supraliminal submaximal I-response is illustrated. Before MRF stimulation (the first column) the I-response consisted of 2-3 spikes with latencies of 3.0-3.4 msec. During M R F stimulation (100/sec, 0.2 msec, 4 V) the latencies of the first spike were shortened to 2.5-2.8 msec and the numbers of spikes increased to 4 (the second spikes were masked by stimulus artifacts but could be recognized). In one PT cell which showed complete suppression of spontaneous discharge during high frequency MRF stimulation the change in evoked unitary response was

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Fig. 7. Background activity level and effect of MRF stimulation. (A) Discharge interval diagram showing the change in the effect of successive MRF stimulations. The same units as in (B). Graphs (a) to (e) show continuous discharge interval diagrams with 5 successive MRF burst stimulations. Discharge intervals with stimulus are shown by black bars below diagrams. In (f) recovery to the prestimulus state 2 min after the end of (e) is shown. (B) Samples from continuous record of extracellular PT cell discharge. MRF stimulation (100/sec, 0.1 msec, 4 V, 21 shocks) was given at the rate of 0.3/sec.Upper beam, PT cell discharge recorded extracellularly with time constant of 3 msec. Lower beam, surface record. Records 1 to 4 show the effect of the lst, the 2nd, the 4th and the 5th of successive repetitive burst stimuli.

examined simultaneously. The unit which showed spontaneousdischarge incorrespondence to a spindle wave burst and was silent in the interspindle period was completely suppressed during the whole course of high frequency M R F stimulation, while the evoked I-discharge of the unit recorded simultaneously showed marked facilitation for spontaneous pyramidal discharge in this period of silence. ( 2 ) Relation between the effect of MRF stimulation and background activity level of PT cells The effect of' M R F stimulation on the PT cells was examined at various levels of its spontaneous activity. In the examples illustrated in Fig. 7 identical burst shocks of References p. 349-351

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MRF were repeated at a low rate (0.3/sec). The records 1 to 4 in Fig. 7B show the effects of the lst, 2nd, 4th and 5th stimulations. The same is shown in Fig. 7A as a continuous plot of discharge intervals during the whole course of 5 successive stimulations. In the graph the ordinate denotes discharge intervals in msec, and the abscissa shows the number of successive discharge intervals counted from the first discharge interval which fully entered into the period of the stimulation. Before the first stimulation (a in Fig. 7A) there were irregular discharges at 10 to 2/sec. During and immediately after the stimulation the unit transiently reached a firing level of 100-30/sec and at the end of (a) it recovered to the IO/sec level. As the stimulation was repeated (b and c) the slope of the curve in recovery from the maximal effect became flatter and flatter. After the 4th stimulation (d) the firing level maintained 25/sec which lasted until the start of the 5th stimulation (e). As the background firing became more frequent in the course of the experiment the effect of the stimulation, as indicated by the difference in mean discharge intervals of pre- and post-stimulus periods, became much smaller although the absolute firing level in the period of maximal effect of the stimulation was: heightened slightly in the later stimulation. The reversal of the effect (such as the facilitatory influence to an inhibitory one) was never found. The inefficacy of the later stimulations was not due to ‘adaptation’ of the stimulated M R F to the shock itself because even the first stimulation was ineffective when the prestimulus pattern was fully ‘activated’, or when the peripheral stimulation was used to induce a similar prestimulus discharge pattern. This finding suggests that some ‘saturating level’ exists in the system for this type of activation. It is interesting to find, in the interval diagram of the fully activated state, minor alternating fluctuation of the successive intervals (see right half of post-stimulus plot in Fig. 6A) that may suggest the existence of a negative feedback mechanism to maintain a certain level of activity. Similar observations had already been made on the neurons in the association cortex (Saito et al., 1959).

(3) Tonic depolarization of PT cell membrane caused by MRF stimulation Intracellular recording revealed a sustained ‘tonic depolarization’ of PT cell membrane due to MRF stimulation. Record I of Fig. 8 shows typical tonic depolarization during and after the MRF stimulation. The horizontal line is drawn at an arbitrary level to make the depolarization clearer. The depolarization lasted long after cessation of MRF stimulation in this unit. The next record (2) shows a similar tonic depolarization which returned more quickly to the original level after the end of M R F stimulation. In this unit the membrane potential was lower than in record 1, and the stimulus caused inactivation due to excessive depolarization. Record 3 shows shorter depolarization caused by a single reticular shock (marked by a vertical bar) on the same PT cell with record 2. Record 4 illustrates the effect of repetitive stimulation (70/sec) of CM. Only this record was taken under nembutal anesthesia. During the stimulation a slight increase in the level of polarization prevented the generation of a spontaneous action potential during stimulation. After the end of stimulation typical hyperpolarization which lasted over 300 msec was followed by ‘rebound’ excitation.

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Fig. 8. Effects of high frequency stimulation of mesencephalic and thalamic reticular systems on the membrane potentials of PT cells. Intracellular record. Calibration: 50 mV for 1 and 4, 30 mV for 2 and 3. Time, upper time mark indicates 100 msec for I and 4, lower time mark indicates 100 msec for 2 and 3. Records 1 to 3 were taken from the non-anesthetized animal. Record 4 was recorded from the animal anesthetized with nembutal. ( I ) Effect of high frequency stimulation (100/sec, 0.1 msec, 3.5 V, see shock artifacts on the lower trace) of the mesencephalic reticular formation. Upper trace shows intracellular record and lower trace represents surface activity. The horizontal line was added at an arbitrary level to demonstrate ‘tonic depolarization’ caused by the stimulation. (2) Effect of mesencephalic reticular stimulation (100/sec, 0.1 msec, 3 V, horizontal line indicates duration of the stimuli). Different unit from I . Membrane potential level was lower than in 1. Horizontal line was similarly drawn at an arbitrary level. ‘Tonic depolarization’ caused depolarization inactivation of action potentials. (3) Effect of single reticular shock. Single reticular shock (0.1 msec, 9 V) is indicated by a vertical bar. The same PT cell as in 2. (4) Effect of repetitive stimulation (70/sec, 0.1 msec, 3 V seen by stimulus artifacts) of the thalamic non-specific nucleus (VA). During the stimulation a slight increase in membrane polarization was accompanied by minor noise-like fluctuations of the membrane potential which remained at subthreshold level. After termination of the stimuli further hyperpolarization lasting more than 300 msec was followed by ‘rebound’ depolarization with action potentials.

In general the difference between M R F and ThRF stimulations is not so marked in the high frequency repetitive stimulation of both systems in the non-anesthetized animal. By the use of a single shock, short burst shocks or barbiturate anesthesia, the phasic inhibitory component in the response to ThRF stimulation was recorded more clearly as in this example. ( 4 ) Efect of reticular stimulation on the specific and non-specific responses of the PT cells The effect of reticular stimulation on the early excitatory response t o single VL stimulation is illustrated in Fig. 6D. The left column shows examples of control responses in which only distant units (small spike) were activated at 3 msec after the stimulus. The first record in the second column was made during 100/sec stimulation of MRF which shows activation of the second unitary response, while the response of the:distant unit remained unchanged. The response of the second unit was also found in a record taken after cessation of the M R F stimulation with increased latency. Successive records (in the right column) showed recovery to the original state. A similar References p. 349-351

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Fig. 9. Cyclic excitability change indicated by mass response and the effect of MRF stimulation on the cyclic change. Surface record from the anterior sigmoid gyrus. (A) Cyclic change in the amplitude of test evoked response during triggered spindle. Surface record. Twin shocks were delivered at ipsilateral VA. The magnitude of the evoked potential to the second shock was plotted against the time course of the triggered spindle for conditioning stimulus. Time, 100 msec/div. (B) The effect of repetitive MRF stimulation (lOO/sec) on the cyclic excitability change during triggered spindle. (a) Enhancement of suppressed test response was found at the second inhibitory phase. 1 = control record. 2 = record during MRF stimulation. Separation time of two stimuli was 210 msec; (b) Suppression of enhanced test response evoked at the second facilitatory phase. 1 = control record. 2 = record during MRF stimulation. Separation time of two stimuli was 260 msec; (c) Graph plotted on the time course of triggered spindle shows the effect. Ordinates indicate the relative amplitude of the test response in the control record (solid circle) and in the record during MRF stimulation (open circle).

facilitation of the early response of PT cells to VL shock was observed regularly in the other units examined. The effect of MRF stimulation on the cyclic change of the cortical excitability en masse was examined by a surface electrode on the motor cortex. By simple twin shock test on ThRF a cyclic change in the amplitude of the early evoked potential of the second response is evident (Fig. 9A). There was alternating inhibition and facilitation which almost paralleled the time course of triggered spindle waves. The maximal points in facilitation in the curve were in the negative rising phase or in the positive phase just before the negative rise of spindle waves and minimal points (or maximal inhibitory points) in the curve corresponded to the falling phase of the negative waves. A similar test, made by conditioning a locus in ThRF and testing the amplitude of the evoked potential to the shock on the other site in ThRF, gave an almost identical result. The effect of MRF stimulation on the cyclic change in the second evoked response by double shocks of ThRF is illustrated in Fig. 9B. The samples of records on the left (a and b) show that both inhibitory (upper two records) and excitatory (lower two records) phases reduced the effect of the shocks. In the graph (c) on the right of the figure, solid circles indicate the relative magnitude of the second response before the MRF stimulation, and open circles show the same taken during MRF stimulation. The peak to peak amplitude of the cyclic change was strikingly reduced during the stimulation. Athough it is impossible to correlate the amplitude of the predominantly surface negative evoked potential directly with the activity of the deep-lying PTcells, it was’ob-

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Fig. 10. Effect of high frequency repetitive stimulation of MRF upon the unitary recruiting responses of two PT cells (A and B). (A) 1, Low frequency (8/sec) ipsilateral VA stimulation was given on relatively ‘activated’ background discharge. Upper trace, single PT cell discharge recorded with short time constant (3 msec). Lower trace, record from the cortical surface (anterior sigmoid gyrus). VA stirnulation is indicated on the bottom of the record; 2, Contralateral high frequency (lOO/sec) MRF stimulation was applied simultaneously with the VA stimulation. Start of the MRF stimulation is indicated by an arrow above the record; 3, High frequency VA stimulation wasgiven at 2 mm posterior to the low frequency VA stimulating electrode during the low frequency VA stimulation. Parameters of high frequency stimulation were the same as in 2. Onset of high frequency stimulation is indicated by an arrow. Increased thickness of the base line during high frequency stimulation was due to the stimulus artifacts; 4, Control 8/sec VA stimulation alone. Time mark, 0.5 sec. (B) The effect of MRF stimulation on recruiting unitary response of another PT cell. The left column shows control recruiting response evoked by 8/sec ipsilateral VA stimulation which started at the 4th sweep. In the right column repetitive stimulation of MRF (lOO/sec) was added during the whole course of recruiting response. Time, 10 msec/div.

served that excitation of extracellularly recorded PT cells induced by the test shocks in the test similar to that in Fig. 9A almost paralleled the excitability curves found by the mass response. More direct examination of the effect was made by recording the unitary recruiting response of the PT cells and testing the effect of high frequency M R F stimulation on the response. Two examples are illustrated in Fig. 10. In A of the figure low frequency (8/sec) stimulation of ipsilateral VA produced a predominantly inhibitory response on the PTcell, and single spike response was found after the development of recruiting response (1). At the foot of the spikes synchronized noise-like discharges of distant units were also observed. High frequency (100/sec) stimulation of contralateral MRF was simultaneously given in 2 which almost completely suppressed the recruiting response on the cortical surface. The inhibitory component of the unitary response was suppressed and replaced by diffuse, asynchronous (in relation to the VA stimuli) sponR*/oronser p . 349-331

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taneous discharges. Synchronized single spike responses were masked out and synchronized discharges of the distant units disappeared. In 3, high frequency stimulation (of the same parameters) was given to ipsilateral VA (2 mm posterior to the low frequency stimulating electrode). The increase in discharge rate was not so marked as in the MRF stimulation but a desynchronizing effect on the discharge pattern was clearly seen. MRF and VA stimulation alone caused a tonic augmenting type of change in the spontaneous discharge in which M R F showed a more intense effect. In the left column of Fig. 9B the same effect on a PT cell from the other experiment during recruiting response is shown. Low frequency (8lsec) VA stimulation started at the 4th sweep. The record shows that, under MRF stimulation (the right column), the spontaneous discharge increased in the sweeps before VA stimulation and the repetitive unitary recruiting response was markedly depressed to the single spike response while the timing of the unitary response remained relatively unaltered. Observations were also made on the relationship between background spontaneous discharge level and mode of activity of a PT cell during spindle waves. The significance of this unitary spindle activity seems to depend largely on the background activity level of the neuron. In the experiment of Fig. 1 1 a PT cell (antidromic response with a latency of 1.6 msec is shown in the inset figure below) showed marked inhibition or decrease in average spontaneous firing level during spindle waves as compared with high firing rate in the interspindle period (non-anesthetized preparation). Slow intravenous administration of nembutal(2 in Fig. 11) greatly depressed the background

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Fig. 1 1 . The effect of barbiturate anesthesia on the PT cell activity during spindle wave burst. An example of the extracellularly recorded unit discharge is shown in the inset figure below record 3. The antidromic response showed a latency of 1.6 msec. In the records above the unit discharge was transformed into a square pulse of 3 msec duration and recorded by an ink-writing oscillograph simultaneously with surface activity. Record 1, before injection of nembutal. Record 2, after 10 mg of nembutal/kg administered intravenously. Record 3, after 30 mg of nembutal/kg. Time mark, 5 sec. Voltage scale, 100 pV,

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firing level. The average discharge rate during spindle waves was not changed markedly at this level of anesthesia, and spindle activity of the PT cell appeared as periodic activation of the uriit on the silent baseline of the inter-spindle phase. Further addition of nembutal(3 in Fig. 5) did not change the picture but merely depressed the PT cell activity during spindle waves. The underlying mechanism of this ‘reversal of spindle effect’ will be discussed later. ( C ) Efects of stimulation of ThRF and MRF on the non-specific thalamic neurons This section deals with unitary responses of the non-specific thalamic neurons to the low and the high frequency repetitive stimulation of ThRF and MRF. All recordings were made extracellularly. A paralysed preparation similar to that in section (B) was used. Electroencephalograms showed that the state of the animal was comparable to that in section B. A part of the results has been reported elsewhere (Maekawa et al., 1961, 1962; Okuma ef al., 1964).

( 1 ) Identijication of the unit recorded and general description of the unitary recruiting response The identification of the non-specific thalamic neuron was performed with the following procedures. Two fine bipolar stimulating electrodes, which were attached to the side of the protecting sheath of the microelectrode, were located about 1-1.5 mm anterior and posterior from the recording microelectrode. These bipolar electrodes will hereafter be called ‘local electrodes’. Units were identified as non-specific thalamic neurons when the stimulating electrodes evoked the typical cortical recruiting response and the microelectrode tip in the area between two electrodes recorded the positive or positive-negative waves of recruiting type during cortical recruiting response. On stimulation of a portion of ThRF, recruiting negative-positive waves (Purpura and Cohen, 1962) were found corresponding to the cortical recruiting response. The negative component had a shorter duration and smaller amplitude compared with the slow positive component, and the waves as a whole seemed to form a mirror image of the cortical recruiting waves. Unitary response appeared as a repetitive burst discharge of 1-10 spikes of the recruiting type. The frequency of the repetition reached 100-200/sec. The discharge was found most frequently in the negative phase and the succeeding positive slope to the crest of the positive wave. The second half of the positive wave corresponded to the inhibitory phase of about 100 msec following the preceding excitatory one. When the response was less, the negative phase was not so marked, and the unit discharge increased its latency toward the later phase of the positive wave. The overall timing of the burst discharge and succeeding inhibitory phase was roughly synchronous with the unitary response of the cortex except for a shorter (5-10 msec) latency for the intense local response. During spindle waves of the cortex smaller and more irregular positive waveSwere observed in rough correspondence to the former. Burst type discharge was also found in this period (Fig. 13A). References p. 319-351

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Fig. 13. (A) When a spontaneous spindle appeared in the corticogram (lower trace), CM neuron showed a synchronized repetitive discharge (I). While cortical waves changed to a low voltage fast pattern, CM discharge showed a desynchronized dispersed pattern (11). (111). Similar correlation of CM neuron and cortical waves was also seen during low cycle stimulation of CM (IV). In 4, recruiting response was observed in the cortex, and CM neuron showed burst discharge. With high cycle CM stimulation the cortical wave turned to a low voltage fast pattern, and unitarydischargesof CM tonically increased and lasted beyond the cessation of the stimuli. (B) Responses of CM neuron to various frequencies of local stimuli. The CM neuron showed a synchronous short burst on low frequency stimulation. As the frequency of the stimuli increased the number of discharges increased during stimulation. The discharge was preceded by a suppressive period of 300-500 msec at the onset of the stimuli. After the cessation of stimuli an inhibitory period and then burst discharges were found.

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In Fig. 12 an example of unitary recruiting response is illustrated. The unit was picked up in the thalamic VA. The second and the third records of Fig. 12 were obtained by a local stimulation of 7/sec. The local anterior electrode showed the maximal effect in this situation. In the first record homolateral CM was stimulated at the same parameters. A relatively weak synchronizing effect was observed. The last record in Fig. 12 shows the effect of low frequency stimulation of M R F with the same stimulus parameters on the same unit. The stimulus caused almost no Evps nor any changes in unit discharge. ( 2 ) Excitability change in non-specijc thalamic neurons after single shock to ThRF and MRF Two types of excitability change were observed after single shocks on ThRF. Local stimulation was used to evoke a test response. In the first group the inhibitory phase lasted 30 to 100 msec after the conditioning shock. A succeeding facilitatory phase was found in 100-300 msec with a peak at 150-300 msec and the effect often lasted up to several hundred msec with more or less cyclic variation. In the second group of neurons a late facilitatory phase was observed with no marked preceding inhibitory phase. The single shocks on the M R F were accompanied by an inhibitory phase dominant at 10-60 msec after the stimulus. In a small portion of the sampled units the inhibitory phase was preceded by a facilitatory phase at 10-40 msec. Relatively early recovery (at 60-150 msec) was found in contrast with the long lasting effect of ThRF shocks (Maekawa et al., 1962). ( 3 ) Efect of repetitive stimulation of ThRF and MRF ( a ) Low ,frequency (jl-lO/sec) stimulation of ThRF. The typical response pattern of synchronized repetitive burst type has already been described (Fig. 12). In some neurons only a single discharge was evoked even at the maximal height of the recruiting wave (Fig. 14C, first record). In the other neuron only a suppression of spontaneous discharge was found during the low frequency stimulation. A non-synchronized augmentation type change was rarely found by this stimulus frequency. The difference in patterns of the response mentioned above may be explained simply by different compositions of EPSPs and IPSPs in different neurons and with different stimulus parameters. The closer the stimulating point in ipsilateral ThRF the larger the recruiting positive waves which usually accompanied more intense and prolonged activation of a neuron. ( b ) Lowfrequency stimulation of MRF. As already indicated in Fig. 12, low frequency MRF stimulation showed far less marked change in the thalamic neurons. Well-synchronized burst response was hardly ever found with this stimulating condition. When the stimulus strength was raised, an increase or decrease in the number of discharges was observed without significant synchronization with the stimulus. In 24 units that responded in either way to the standardized stimulus strength, 13 units belonged to the augmenting type and 11 units showed a decrease in firing rate during stimulation. A further 9 units remained unresponsive. Refsrences p . 349-351

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Fig. 14. (A) The activity of the MRF neuron was slightly depressed during cortical triggered spindle wave evoked by ipsilateral CL single stimulation (I). During CL low frequency (6/sec) stimulation which evoked the cortical recruiting response, this neuron decreased its discharge rate and changed to a synchronized discharge pattern with each stimulus. (9) CL high frequency stimulation markedly depressed the other MRF neuron, and the inhibitory effect continued for several hundred milliseconds after the cessation of the stimulation. (C) The response of C M neuron evoked by local stimulation at 7/sec, showed a single unitary discharge at each stimulus (I). This evoked unitary discharge was suppressed almost completely by overlapped high frequency MRF stimulation (11). The suppression of unit discharge was also observed by MRF stimulation alone (111).

( c ) Highfiequency (30-100/sec) stimulation of ThRF. The synchronized burst type response could not follow the high frequency stimulus rate (Fig. 13B, the 3rd to 4th record). At the start of the stimulation phasic inhibition of a few to several hundred msec often preceded the phase of augmentation. A similar initial suppression had been found by Purpura and Cohen (1962) with the evidence of IPSPs. After the cessation of the stimulation, augmentation of the discharge lasted for a considerable time in some units (Fig. 2A, the 5th record). The majority of the units (10 in 16 units) which showed augmented discharge during stimulation exhibited suppression of spontaneous discharge for a few to several seconds after the end of stimulation which resembled the 'post-excitatory extinction' found by Schlag and Faidherbe (1961). In 9 units similar stimulation only caused suppression of spontaneous discharge. (d) High frequency stimulation of MRF. High frequency stimulation of MRF led to a decreased discharge rate in 16 units and an increased discharge rate in 13 units. In comparison with cortical neurons it is evident that the suppressive influence is far more prominent here than in the effect of the similar stimulation upon cortical neurons. The suppression was sometimes found as phasic inhibition of (Fig. 14C) spontaneous discharge in which only a brief silent period of a few hundred msec was observed after

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the end of stimulation. For the phasic inhibitory effect the collateral inhibition by the antidromic activation of the neighboring units may be excluded. Antidromic activation by the M R F shocks was observed only in a very small fraction (less than 1 %) of the units sampled in our present experimental conditions. The rather common occurrence of a phasic inhibitory influence makes it difficult to attribute it exclusively to the collateral inhibition due to antidromic activation of the thalamic neurons. ( e ) Comparison of the ascending and descending influences between ThRF and MRF. The descending influence of ThRF on MRF neurons is briefly summarized here for the purpose of making a comparison with the ascending influence described in (d). An example of the record from MRF neurons is shown in Fig. 14A. A single shock on i psilateral CL produced an inhibitory influence during the triggered spindle waves. Low frequency (6/sec) stimulation showed primarily inhibitory response with synchronized late discharge of ‘rebound’ type. This type of response (predominantly inhibitory response with weak or moderate synchronization as the late discharge) was found in the majority of the samples responded. Only in a few units was early repetitive burst response found. Recruiting positive waves were recorded in the M R F during low frequency stirnulation of ThRF. In general the characteristics of the thalamo-reticular influence seem to be similar to intrathalamic or thalamo-cortical influences but weaker than the latter two especially in its excitatory component. Among 64 MRF units which exhibited significant change in their discharging pattern during recruiting response, 46 showed a decreased mean rate of firing and 18 showed an increase. On high frequency stimulation of ThRF, 32 MRF units showed a decrease of their firing rate and 19 showed an increased rate. ( 4 ) Influence of high frequency MRF stimulation upon unitary recruiting response of non-specijic thalamic neurons Observations were made on the non-specific thalamic neurons with experimental conditions similar to those of the interaction study in section (B). In the example of Fig. 14C, high frequency M R F stimulation showed a strong suppressive effect on the spontaneous discharge (the third record). Low frequency (7/sec) local stimulation of the CM evoked a well-synchronized single spike response together with cortical and subcortical recruiting response (the first record). High frequency MRF stimulation which markedly suppressed these recruiting waves also suppressed the synchronized unitary recruiting response (the second record). In the neurons which showed a tonic augmentation type change during and after the MRF stimulation, suppression of the synchronized unitary recruiting response was found with an increase in desynchronized discharges which invaded the inhibitory phase of the synchronized response. DISCUSSION

( 1 ) The similarity and the difference in the sequences of synaptic potentials during augmenting und recruiting responses lntracellular recording from the identified PT cells revealed the existence of rhythmRejrrrncrs p . 349-351

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ically recurring synaptic potentials synchronous with the augmenting, recruiting or spindle waves on the surface of the pericruciate cortex. These findings are mainly in accord with the findings reported by Klee and Offenloch (1964), Li (1963) and Purpura et al. (1964a,b). When postsynaptic potentials were recorded simultaneously with the cortical surface potentials, an apparent correlation between the two potentials appeared. During augmenting response, corresponding to the earliest surface-positive potential (Pl), an early EPSP was observed. The EPSP was presumed to be a monosynaptic EPSP on the large cells of the Vth layer. When the thalamic non-specific nucleus (CM) was stimulated, a similar EPSP was not induced. On the other hand, prolonged EPSPs grew successively during the development of the augmenting response which behaved in a similar manner to the surface-positive potentials (P2). From these findings it may be said that the surface-positive potentials of the augmenting response reflected, at least partially, the excitatory postsynaptic potentials induced in the large PT cells. During the course of augmentation of the cortical potentials, facilitation of the prolonged EPSPs was always observed. Growth of the prolonged EPSPs was also observed during the course of recruiting response. Facilitation of the EPSPs was a common characteristic in the development of two types of rhythmical potential. The neuronal mechanism responsible for the facilitation has not yet been characterized. In our results two facts seem to be related to the production of the synchronized EPSPs. Firstly, long lasting IPSPs with a duration of 100-200 msec were evoked by single or repetitive stimulation of VL or CM. The inhibition seems to be one of the limiting factors in the timing of the rhythmic responses. There also exists a powerful recurring inhibition within the thalamus itself, having a similar duration of 100-200 msec, as reported by Purpura and Cohen (1962) and also by Andersen and Eccles (1962). Secondly, after a single shock to VL or CM, a massive EPSP with a duration of several tens of msec followed the long lasting IPSPs mentioned above. The ‘rebound’ type EPSP which represents the first of several recurring EPSPs observed during triggered spindle waves were often more prominent than the EPSPs primarily observed after the shocks. Our results suggest that the timing of this ‘rebound’ EPSP is one of the crucial factors for producing synchronized facilitation or excitation. There is no evidence to suggest that the recurrent phenomena are of true ‘rebound’ or anodaloff excitation in the recorded PT cell itself, although some facts exist that suggest the involvement, in the thalamic relay nucleus, of a true rebound phenomenon (Andersen and Eccles, 1962). The recurrent excitation in our PT cell records seems to be not proportional to the preceding IPSPs. Moreover, low frequency repetitive activation of recurrent IPSPs by antidromic stimulation could not produce the augmenting activation. ( 2 ) Tonic depolarization of PT cell membrane and tonic augmentation of spontaneous discharge induced by MRF stimulation In the present study a tonic augmentation of a single pyramidal discharge was commonly found during and after the M R F stimulation. The results were almost in

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accord with our previous observations on the neurons in the cat’s association cortex (Saito et al., 1957) and in cat’s visual cortex (Saito et al., 1958). The results are, however, in contrast with those of Whitlock et al. (1953), and Calma and Arduini (1954) in which the spontaneous spikes disappeared for a considerable time after MRF stimulation. In our experiments reticular stimulation was given in a relatively ‘relaxed’ state of the brain with EEG spindling, and on a more desynchronized EEG in the experiments by Arduini and his collaborators, although we could not find the reversal of the effect by changing the stimulus parameters or the prestimulus arousal level (Fig. 7). The present result agrees rather with the observation made on the effect of barbiturate anesthesia by Calma and Arduini (1954) although the direction of the change is reversed between stirnulation and anesthesia. Our own results on the effect of nembutal (Fig. 11) also confirmed the result reported by Calma and Arduini (1954). In the present results tonic suppression of spontaneous discharge has been observed only in a very small fraction of the sampled PT cells. Moreover a unit that was examined by the unitary I-response showed ‘paradoxical facilitation’ (which will be discussed later) during the suppression of the spontaneous discharge. A similar suppression of spontaneous discharge has been observed in a small population in the cat’s association cortex (Saito et al., 1957; Saito, 1959) and has been reported more frequently in the natural arousal of chronic or fixed animals by Hubel (1959),Creutzfeldt and Jung (1961) and Evarts (1961). It is not difficult to envisage a fundamental difference between the freely moving active animal and the passive preparation used in the present experiments, especially in a ‘specific’ area such as the primary sensory or motor cortex. An interesting result was reported in the last two articles above mentioned that, during the natural arousal, marked dispersion among the mean discharge rates of the units was found. If this is a general mode of activity among the cortical neurons during natural arousal it could cause a noise-like activity as an ensemble average when the phase relationship is randomized. In our passive preparation, such a dispersion in the firing rate was not evident. Instead, PT cells tended to approach a certain ‘saturating level’ in the spontaneous firing at which the same reticular stimulation is almost ineffective. The maximal firing rate differed among the sampled units and according t o the state of the brain, but it seems to be 30-50 /sec for early firing PT cells in the fresh preparation (Fig. 7). The tonically augmented spontaneous discharge usually showed not only a significantly smaller mean discharge rate but also smaller variance or standard deviation in the discharge intervals (Fig. 7) which is in accord with Evart’s (1964) discharge interval histogram obtained from the chronic preparations. The other type of inhibition of spontaneous discharge by repetitive reticular shocks was found in some units (also very small in number). They showed transitory inhibition at the beginning of, or only during, repetitive stimulation. This seems t o be quite different in nature. At its longest it ceased promptly at the end of the stimulation and no tonic after-effect was observed. One possible cause of this inhibition may be the recurrent type inhibition caused by the stimulation of other PT cells via axon collaterals in MRF. Actually, in very rare instances, PT cells were found responding References p. 319-351

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Fig. 15. (A) Scheme showing the relationship between background level of depolarization and pattern of unit discharge during spindle waves. (B) Scheme illustrating the possible effects of tonic depolarization upon the firing level of the unit. In both A and B, Lc denotes the critical level of the membrane potential. For explanation see text.

antidromically to reticular shocks delivered at the appropriate electrode location. Tonic depolarization of PT cell membranes found in intracellular records can reasonably be the cause of tonically augmented spontaneous discharge induced by reticular stimulation. Depolarization inactivation, such as found in records 2 and 3 of Fig. 8, should be regarded as non-physiological because the same cells showed tonic augmentation of spontaneous discharge in their extracellular record by MRF stimulation prior to electrode penetration into the cell. ( 3 ) Tonic and rhythmic discharge pattern and excitability of the PT cell In our present results (Fig. 11) the unitary PT cell discharge during spindle burst remained relatively unchanged after abolishing tonic discharge of interspindle period of unanesthetized animals by nembutal. From the results of such differential blockade we can assume, in a schematic way, that spindle activity would be superposed on a background activity level as shown by the solid line in Fig. 15 Aa. But when the background level was very high there appeared an interaction between the two systems responsible for the tonic and spindling activity (Figs. 10 and 14C). As a result the amplitude of cyclic change in membrane potential of the PT cell might be reduced as shown in the broken line of Fig. 15Aa, and the resulting unit activity during spindle waves may not be so enhanced as expected by the summation with the depolarization. The mechanism that seems to prevent the development of hypersynchronous spindling might operate at both thalamic and cortical levels. Thus the burst discharge during spindle waves did not change, or was even enhanced, by light nembutal (b). Further administration of the drug first causes real depression of the spindle discharges (c). The tonically depolarized membrane potential level after stimulation of the MRF need not necessarily reach the critical level as shown in the right half of the intracellular record in Fig. 8, record 1. The situation is shown schematically in Fig. 15B. In (a) spindle activity was superposed on a relatively low membrane potential. After arousal stimulation the average membrane potential level was raised (b). The level

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could be either above (bl) or below (b2) the critical level (Lc). In b2 the average level in the number of spontaneous discharges was paradoxically suppressed. The exceptional pyramidal unit described in section (C) which showed facilitation of evoked Idischarge simultaneously with complete suppression of spontaneous discharge may be explained in this way. ( 4 ) The activity of the non-specific thalatnic neurons during EEG synchronization and desynchronizatioir From the results of the present experiments in the cortical and thalamic levels it is evident that during typical EEG synchronization, such as recruiting response, striking in-phase synchronization of successive cyclic sequences of excitation and inhibition is maintained between cortical and non-specific thalamic neurons. A similar synchronization of activity was also found to some extent in the M R F neurons but the latter was usually less intense and less excitatory than in thalamo-cortical synchronization. This effect on the MRF neurons may possibly have some relation to the diffuse descending inhibitory effect of the same stimuli on the spindle afferent discharges found by Hongo et al. ( I 963). It was found by Purpura and Shofer (1963) that the same non-specific thalamic neuron which showed typical alternating sequence of EPSPs and IPSPs was converted to exhibit sustained depolarization during and after high frequency stimulation of ThRF. Our extracellular observations also showed that the same unit can operate in the two modes of activity, synchronized and tonic. As to the mode of poststimulus activity in our present results, the typical after-effect of the ThRF stimulation was primary inhibition (for 100-300 msec or more after the end of high frequency T h R F stimulation) followed by recurrent burst discharge. But when the stimulus was enough to evoke a very intense response of the unit, even in low frequency stimulation, a marked tonic after-discharge lasted for a longer period (Fig. 12,II). In the figure the afterdischarge was evoked only by one of the local stimulating electrodes. This finding suggests the existence of a local activation process and capability of the thalamic neuron to produce a tonic discharge which seems in accord with the gradual repolarization after the end of stimulation observed by the above authors. A markedly asymmetric mutual interrelationship between M R F and T h R F was found. Low frequency stimulation ThRF mainly evoked a synchronized response of MRF neurons with a predominantly inhibitory character. On low frequency M R F stimulation such effect was not significantly shown. With high frequency stimulation an ascending influence (suppressive or facilitatory) was more effective than a descending influence (also suppressive or facilitatory). Our results on the latter influence are in contrast with the findings of Machne et al. (1955) that many non-specific thalamic neurons are activated by stimulation of the cephalic midbrain tegmentum. Frequent appearance of tonic or phasic type suppression is also in contrast with the results on the cortical neurons described in section (B). The possible collateral inhibition by antidromic activation of the ThRF neurons appears to be highly unlikely as an explanation because of its rather frequent occurrence together with the very rare incidence of antidromic activation of the ThRF neurons. Further analysis, however, is needed to References p. 349-351

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discriminate between them completely. In our results among 29 ThRF units that responded, 13 neurons showed a similar augmentation of spontaneous discharge to that described by Machne et al. (1955). The marked difference between the effects of MRF stimulation on the neuronal activity at the cortical and thalamic levels may support the dual ascending pathwaythalamic and extrathalamic - proposed by Starzl et al. (1951). It also suggests a difference in the neuronal organization of the two structures. Our results are not decisive on the possible thalamo-reticulo-cortical pathway (Schlag and Chaillet, 1963) for the desynchronizing cortical effect of high frequency ThRF stimulation. On high frequency stimulation of ThRF, 19 units of M R F showed an increased discharge rate, 32 showed a decreased rate, and 13 showed no marked changes. From these results the thalamo-reticular relation seems to have at least an aspect of inhibitory interaction. Our results also suggest that the process of desynchronization may act at the thalamic and cortical level. In both structures cyclic sequences of facilitation and inhibition were suppressed at the elevated excitability level in the cerebral cortex and more often at the decreased background level in ThRF. The physiological significance of the synchronized activity is not yet clear. Gating function could be supposed from the finding of the EPSPs-IPSPs sequence in the cortical and non-specific thalamic neurons during spindle waves. And the production of the synchronized EPSPs might be an effective means of intermittent gating of the biological signals in a suppressed or lowered activity level. SUMMARY

(I) In the nembutized cat intracellular recordings were made from the large PT cells in the deep layer of the motor cortex during augmenting and recruiting response. In the augmenting response two groups of EPSPs (monosynaptic and polysynaptic) are described. Only the polysynaptic EPSPs showed augmentation. The existence of monosynaptic EPSP was the most distinct characteristic of the augmenting response. Polysynaptic EPSPs were superposed on and followed by long lasting IPSPs. Recruiting response showed only similar polysynaptic EPSPs accompanied by long lasting IPSPs. (2) In the curarized cat single pyramidal discharge and extracellular PT cell discharge were used to examine the excitability change in the PT cells during MRF stimulation. Except for a few units almost all PT cells showed tonic augmentation of spontaneous discharge during and after MRF stimulation. Facilitations of the unitary ‘I-response’ and of the subthreshold unitary D-response were also observed. A similar facilitation was found in the unitary response of PT cells to the VL shocks. A single conditioning shock of MRF was followed by a long lasting facilitatory phase from 10-20 msec to several hundred msec after the shock. An inhibitory phase which regularly accompanied the ThRF shock was not found. By intracellular recording, a tonic depolarization of the PT cell membrane was found during and after repetitive stimulation of M R F or even after single MRF shock.

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Both the excitatory and inhbitory components of the PT cell response in the recruiting response or in the spindle waves were suppressed on the tonically facilitated background during repetitive stimulation of MRF. (3) In conditions similar to those in 2 above, the activity of the non-specific thalamic neurons were examined. During low frequency repetitive stimulation of ThRF (local and distant) a typical ‘unitary recruiting response’ was evoked. A rhythmic burst pattern was also observed during cortical spindle waves. Two types of excitability curve were obtained in ThRF neurons when a conditioning single ThRF shock was given. One was characterized by a diphasic alternation of inhibition (10-80 msec after conditioning) and facilitation (100-300 msec) and the other byLa prolonged facilitation. A similar test by MRF conditioning shock showed a marked inhibition (30-80 msec) with no signs of tonic facilitation. About a half of the units sampled showed suppression of spontaneous discharges during high frequency MRF stimulation. The suppressive effect abolished synchronized unitary recruiting discharge when MRF stimulation was added to the low frequency ThRF stimuli. On high frequency ThRF stimulation half of the M R F neurons sampled showed suppression of the spontaneous discharge. ( 4 ) From the results presented it is concluded that in the cortical desynchronization both the facilitatory and inhibitory components of the synchronizing influence are suppressed with a tonically facilitated background level. A similar suppression in the thalamic neurons with the suppressed or facilitated background may be an important mechanism for facilitating the cortical desynchronization. REFERENCES AKIMOTO, H., NEGISHI, K., AND TORII, H., (1957); The effect of thalamic stimulation on the activity of cortical neuron in the cats. Proc. Vlth Ann. Meet. Jap. EEG SOC.,(pp. 99-1 10). AKIMOTO, H., NEGISHI, K., TORII, H., AND ENDO,M., (1958); Reaktionen dereinzelnen Neuronen des motorischen Kortex nach elektrischen Reizung spezifischer und unspezifischer Thalamuskerne. Foliapsychiat. neurol. jap., 60,1303-1311. AKIMOTO, H., SAITO,Y.,NAKAMURA, Y.,MAEKAWA, K., AND KUROIWA, S., (1960); Effects of arousal stimuli on evoked unitary responses in cat’s sensory and motor cortices. Proc. IXth Ann. Meet. Jap. EEG SOC.,(pp. 67-71). ANDERSEN, P., A N D ECCLES, J . C., (1962); lnhibitory phasing of neuronal discharge. Nature (Lond.), 196, 645-647. ARDUINI, A., AND WHITLOCK, D. G., (1953); Spike discharges in pyramidal system during recruitment waves. J. Neurophysiol., 16,430-436. BROOKHART, J. M., AND ZANCHETTI, A., (1956); Relation between electrocortical waves and responsiveness of cortico-spinal system. Electroenceph. din. Neurophysiol., 8, 427-444. CALMA, I., AND ARDUINI, A., (1954); Spontaneous and induced activity in pyramidal units. J . Neurophysiol., 17, 321-335. CHANG,H. T., (1955); Cortical response to stimulation of medullary pyramid in rabbit. J . Neurophysiol., 18, 332-352. COOMBS, J. S., ECCLES, J . C., AND FATI-,P., (1955); The specific ionic conductances and ionic movements across the motoneuronal membrane that produce the inhibitory post-synaptic potential. J. Physiol., 130, 326-373. CREUTZFELDT, O., U N D AKIMOTO, H., (1958); Konvergenz und gegenseitige Beeinflussung von Impulsen aus der Retina und den unspezifischen Thalamus Kernen an einzelnen Neuronen des optischen Kortex. Arch. Psycliiat. Nervenkr., 196, 520-538.

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CREUTZFELDT, O., AND JUNG,R.,(1961); Neuronal discharge in the cat’s motor cortex during sleep and arousal. The Nature of Sleep. G . E. W. Wolstenholme and M, O’Connor, Editors. London, Churchill (pp. 131-170). DEMPSEY, E. W., AND MORISON, R. S., (1942); Production of rhythmically recurrent cortical potentials after localized thalamic stimulation. Amer. J . Physiol., 135,293-300. ECCLES, J. C., (1957); The Physiology of Nerve Cells. Baltimore, Johns Hopkins Press. ENDO,M., (1962); Effects of specific and non-specific afferent impulses upon neuronal activity of the somatosensory cortex in cats. Folia psychiat. neurol. jap., 16,25-61. ENDO,M., ASAI,T., IHARA, S., TORII,H., AND NEGISHI, K., (1959); Stimulation effects of specific and non-specific thalamic nuclei on the unit activity of the somatosensory cortex in the cat. Proc. Vlllth Ann. Meet. Jap. EEG SOC.,(pp. 8-11). EVARTS, E. V.,(1961); Effects of sleep and waking on activity of single units in the unrestrained cat. The Nature of Sleep. Ciba Foundation Symposium Series. G. E. W. Wolstenholme and M. OConnor, Editors. London, Churchill (pp. 171-182). EVARTS, E. V.,(1962); Activity of neurons in visual cortex of cat during sleep with low voltage fast EEG activity. J . Neurophysiol., 25, 812-816. EVARTS, E. V., (1964); Temporal patterns of discharge of pyramidal tract neurons during sleep and waking in the monkey. J. Neurophysiol., 27, 152-171. HONGO,T., KUBOTA, K., AND SHIMAZU, H., (1963); EEG spindle and depression of gamma motor activity. J. Neurophysiol., 26, 568-580. HUBEL, D. H., (1959); Single unit activity in striate cortex of unrestrained cats. J. Physiol., 147, 226-238.

ITO, M., (1964); On the microelectrode investigations of nerve cells. Iyodenshi Seitaikogaku, 2, 19-26. JASPER,H. H., (1960); Unspecific thalamocortical relations. Handbook of Physiology. Sect. I. Vol. 11. J. Field, Editor. (pp. 1307-1321). KLEE,M. R.,AND OFFENLOCH, K., (1964); Postsynaptic potentials and spike patterns during augmenting responses in cat’s motor cortex. Science, 143,488489. LI, C. L., (1963); Cortical intracellular synaptic potentials in response to thalamic stimulation. J. cell. comp. Physiol., 61, 165-179. C., AND JASPER, H. H., (1956); Laminar microelectrode analysis of cortical unLI, C. L., CULLEN, specific recruiting responses and spontaneous rhythms. J . Neurophysiol., 19, 131-143. Lux,H. D., UND KLEE,M. R.,(1962); Intracelluliire Untersuchungen uber den Einfluss hemmender Potentiale im motorischen Cortex. Arch. Psychiat. Nervenkr., 203, 648-666. MACHNE, x.,CALMA,I., AND MAGOUN,H. W., (1955); Unit activity of central cephalic brain stem in EEG arousal. J. Neurophysiol., 18, 547-558. Y.,HAYASHI, A., AND AKIMOTO, H., (1962); Arousal reaction of nonMAEKAWA, K., NAKAMURA, specific thalamic neurons. Proc. Xlth Ann. Meet. Jap. EEG SOC., (pp. 60-65). MAEKAWA, K., NAKAMURA, Y.,KUROIWA, S., HAYASHI, A., AND AKIMOTO, H., (1961); A study of recruiting response. Proc. Xth Ann. Meet. Jap. EEG SOC., (pp. 4347). MORISON, R.S.,AND DEMPSEY, E. W., (1942a); A study of thalamocortical relations. Amer. J. Physiol., 135,281-292.

MORISON, R.s.,AND DEMPSEY, E. W., (1942b); Mechanism of thalamocortical augmentation and repetition. Amer. J . Physiol., 138,297-308. G . , AND MAGOUN, H. W., (1949); Brain stem reticular formation and activation of the MORUZZI, E EG . Electroenceph . clin. Neurophysiol., 1,455-473. Y.,MAEKAWA, K., HAYASHI, A,, AND AKIMOTO, H., (1961); A new micromanipulator NAKAMURA, for the deep structures of the brain. Proc. Xth Ann. Meet. Jap. EEG Soc., (pp. 110-1 13). T., AND MARUYAMA, N., (1964); EEG and OKUMA, T., MAEKAWA, K., KAWAI,N., MITZUTANI, the thalamus. Advanc. neurol. Sci., 8, 771-798. V. E., (1954); Single- and multiple-unit analysis of cortical stage of PATTON, H. D., AND AMASSIAN, pyramidal tract activation. J . Neurophysiol., 17, 345-363. PHILLIPS, C. G., (1956); Intracellular records from Betz cells in the cat. Quart. J. exp. Physiol., 41, 58-69.

PURPURA, D. P., AND COHEN,B., (1962); Intracehlar recording from thalamic neurons during recruiting responses. J. Neurophysiol., 25, 621-635. PURPURA, D. P., AND SHOFER, R. J., (1963); Intracellular recording from thalamic neurons during reticulocortical activation. J. Neurophysiol., 26, 494-505. PURPURA, D. P., AND SHOFER,R. J., (1964a); Cortical intracellular potentials during augmenting and recruiting responses. I. Effects of injected hyperpolarizing currents on evoked membrane

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potential changes. J. Neurophysiol., 27, 117-132. PURPURA, D. P., SHOFER,R. J., AND MUSGRAVE, F. s., (3964b); Cortical intracellular potentials during augmenting and recruiting responses. 11. Patterns of synapticiactivities in pyramidal and non-pyramidal tract neurones. J . Neurophysiol., 27, 133-1 51. SAITO,Y., (1959); Single cortical unit activity during EEG arousal. Psychiai. Neurol. jap., 61, 16651682. SAITO,Y., MAEKAWA, K., TAKENAKA, s.,AND KASAMATSU, A., (1957); Single cortical unit activity during EEG arousal. Proc. Vlih Ann. Meet. Jap. EEG SOC.,(pp. 95-98). SAITO,Y., NAKAMURA, Y., MAEKAWA,K., TAKENAKA, S., KOGA,E., JIMBO,S., A N D HIRANO, G., ( I 958); Influence of arousal stimulation on photically evoked cortical unit activity. Proc. VIIth Ann. Meet. Jap. EEG SOC.,(pp. 39-42). SCHLAG, J., AND BALVIN, R., (1964); Sequence of events following synaptic and electrical excitation of pyramidal neurones of the motor cortex. J . Neurophysiol., 27, 334-365. SCHLAG, J. D., AND CHAILLET, F., (1963); Thalamic mechanisms involved in cortical desynchronization and recruiting responses. Electroenceph. clin. Neurophysiol., 15, 39-62. SCHLAG, J. D., AND FAIDHERBE, J., (1961); Recruiting responses in the brain stem reticular formation. Arch. ital. Biol., 99, 135-162. SPENCER, H. A., AND BROOKHART, J. H., (1961a); Electrical patterns of augmenting and recruiting waves in depths of sensorimotor cortex of cat. J . Neurophysiol., 24,2649. SPENCER, H. A., AND BROOKHART, J. H., (1961b); A study of spontaneous spindle waves in sensorimotor cortex of cat. J. Neurophysiol., 24,50-65. STARZL, T. E.,TAYLOR, C. W.,AND MAGOUN, H. W., (1951); Ascending conduction in reticular activating system, with special reference to the diencephalon. J. Neurophysiol., 14, 461477. Towt, A. L., PAITON,H. D., AND KENNEDY, T. T., (1963); Properties of the pyramidal system in the cat. Exp. Neurol., 8, 220-238. UNO,M., YOSHIDA, M., AND HIRANO, G.,(1963); Thalamic influences upon membrane potential of pyramidal tract cells. Proc. XIlih Ann. Meet. Jap. EEG SOC.,(pp. 61-62). WHITLOCK, D. G.,ARDUINI, A., AND MORUZZI, G., (1953); Microelectrode analysis of pyramidal system during transition from sleep to wakefulness. J . Neurophysiol., 16,414429. YOSHIDA, M., UNO, M., AND YAJIMA.K., (1965); Monosynaptic activation of PT cells during VL stimulation. In preparation for publication. H., GIVRE,A., AND NARABAYASHI, H., (1964); PhysioYOSHIDA, M., YANAGISAWA, Y., SHIMAZU, logical identification of the thalamic nucleus. Arch. Neurol., 11, 435443.

352

Author Index* Abe, T., 197-216 Ades, H. W., 72, 313 Adey, W. R., 232 Aikawa, S., 182, 184 Aitken, J. T., 131, 138, 144, 146 Ajmone Marsan, C., 173, 174, 297 Akiba, H., 295, 312 Akimoto, H., 118,323-351 Altman, J., 315 Amano, T., I , 6, 7 Amassian, V. E., 181, 331 Amoroso, E. C., 108 Andersen, P., 65, 175, 344 Anderson, J. A., 289 Anderson, S. A., 87, 184 Arai, Y.,182, 184 Araki, T., 46 Arden, G. B., 171, 180 Arduini, A., 323, 324, 345 Arizono, H., 15 Asada, Y.,45, 55, 57, 58, 65 Asai, T., 323 Auer, J., 295 Auerbach, H., 282 Auerbach, V. H., 15 Bain, J. A., 119 Bainbridge, J. G., 108 Balvin, R., 323 Ban, T., 1-43 Barnard, J. W., 138, 146 Barr, M. L., 145 Barris, R. W., 164, 315 Bartelmez, G. W., 44, 45, 48, 50 Batini, C., 101 Baxter, D., 132 Beaconsfield, P., 214 Beccari, N., 44 Bell, F. R., 108 Beresford, W. A., 315 Berl, S., 117, 119, 277 Berlin, L., 181 Berman, A. L., 86, 88 Biemond, A., 295 Bishop, P. O., 169 Blackstad, T. W., 131 Bodechtcl, G., 132 Bodenheirner, T. S., 51

Bodian, D., 31, 44.45, 48, 50, 61, 67 Boggen, R. H., 295 Boggs, D., 112 Bohner, B., 107 Bok, S. T., 138, 146 Bonvallet, M., 101, 102 Borokowsky, T., 280 Bremer, F., 169 Bridger, J. T., 131, 138, 144, 146 Brinley, F. J., 92 Brodal, A., 132, 136,293,296, 306-308 Brodmann, K., 310 Bromiley, R. B., 71 Brookhart, J. H., 323, 326 Brooks, V. B., 184, 195 Brumm, A,, 117 Bucher, T., 213 Bucher, V. M., 315 Buell, M. V., 212,213 Butler, R. A., 86, 93, 313 Cajal, S. Rambn Y.,131, 132 Calma, I., 324, 345, 347, 348 Carman, J. B., 310 Carpenter, M. B., 306 Carreras, M., 87 Casby, J. U., 313 Chaillet. F., 348 Chambers, W. W., 295, 306 Chang, H. T., 325 Chang, M. W., 212, 213 Chatschaturian, A., 310 Chen, G., 107 Cheng, C.-S., 198 Churchill, J., 72, 75 Clark, L. F., 76 Clark, S. L.,297 Clarke, D. D., 277 Cohen, B. D., 107,324, 339, 342, 344 Cohen, M. J.. 108 Collonnier, M., 168, 175 Coombs, J. S., 325 Coplinger, C.B., 112 Cowan, W. M., 310 Coxon, R. V., 197 Creutzfeldt, O., 323, 345 Crosby, E. C., 34 Crossland, J., 124, 126

Italics indicate the pages on wich the paper of the author in these proceedings in printed.

AUTHOR I N D E X

Crouch, R. L., 297 Cullen, C., 323 Davis,iH., 71, 76, 84 Davis, P. W.,86, 88 Dawson,lR. M. C., 118, 119,268 De Iraldi, A. P., 280 Delgado, J. M. R., 234, 306 Dell, P., 101, 102, 169 De Lorenzo, A. J., 131 De Lores Arnaiz, G. R., 280 Dempsey, E. W.,323 De Robertis, E., 280 Desmedt. J. E., 73, 75, 80, 83, 87. 313 De Valois, R. L., 177 Diamond, I. T., 86, 93, 313 Diamond, J., 65 Dickens, F., 197 Dirken, M. N. J., 108 Doran, R., 72, 75 Dudel, J., 59, 66 Dumont, S., 169 Dunlop, C.W.,232 Dusser de Barenne, J. G., 300 Eccles, J. C., 59, 63-66, 86, 131, 175, 325, 344 Eccles, R. M., 59, 175 Elliott, K. A. C., 127, 197 Endo, M.,323 Engstram, H., 72 Ensor, C. R., 107 Ertel, R., 289 Erulkar, S. D., 84, 86,88,93 Estable, C., 131 Evarts, E. V., 324, 345 Faidherbe, J., 342 Fairman, D.. 300 Fatt, P., 325 Felgenhauer, K., 198, 203 Fernhndez, C.,79 Fex, J., 75, 80 Fifkovh, E., 220 Fish, R., 289 Fleming, L. M., 198 Flexner, L. B., 210,213 Flock, A,, 64,72 Flood, P. R., 131 Foix, Ch., 132 Fox, C. A., 138, 146 Frank, K., 59, 63 Friede, R. L.. 198, 210 Frishkopf, L. S., 81 Fujita, K., 12, 14 Fujita, M.,1, 2, 5-7 Fujita, S., 5, 295 Fujiwara, K., I12 Fukami, Y.,54, 55, 57-59,65 Funatogawa, S., 128

Fuortes, M. G. F., 59, 63 Furshpan, E. J. 4649.51-54,57.59,62,67, Furukawa, T., 44-70 Gagel, O., 132 Galambos, R., 64,71, 76, 80, 84, 85 Garol, H. W., 304,305,306,310 Gellhorn, E., 102 Gernandt, B. E., 108 Gibbs, E. L., 98, 102 Gibbs, F. A., 98, 102 Gihr, M., 145 Girado, M.,119 Givre, A., 326 Glees, P., 28, 165, 168 Glock, G. E.. 197 Gobbel, W.G., 295 Goldberg, J. M.,86, 93 Goldstein, Jr., M.-H., 81 Goswami, M. N. D., 284 Gottlieb, J. S.. 107 Graven, H., 289 Gray, E. G., 131 Greenwood, D. D., 86 Gross, N. B., 84 Griinthal, E., 1 Guillery, R. W.,168, 175 Gurdjian, E. S., 24 Gurewitsch, M., 310 Haba, R., 112, 117, 119, 120 Haber, E., 108 Haggar, R. A., 145 Hagiwara, S., 251 Hama, K.. 251-267 Hamburg, M.,210,213 Hamlyn, L. H., 131 Hanna, G. R., 306 Harlow, H. F., 282 Harmon, L. D., 79 Hatth, S.. 280 Hasegawa, A., 1,234 Hashimoto, P. H., 198, 199, 202, 203, 213 Hawkins, Jr., J. E., 72 Hayashi, A., 325,339 Hebb, C. O., 126,127 Hellner, K., 108 Henderson, N., 127 Hendrix, C. E., 232 Henneman, E., 304 Hernhndez-Peh, R., 87 Hidaka, T., 16, 17 Hiebel, G., 101 Hilding, D., 75 Hilliard, J., 18 Himwich, H. E., 197,213 Hind, J. E.. 86. 88, 90 Hirahara, T.,11 Hirai, H.,315, 316

353 251

354

AUTHOR INDEX

Hirano, G., 324,325 Hirano, S., 118,268,269,275,277,282 Hirao, T., 101, 128 Huasawa, K., 295 Hirayama, K., 117, 120 fiura, M., 312, 313 Hodes, R., 145 Holaday, D. A., 108 Holmes, J. E., 232 Hongo, T., 247 Hori, Y., 234 Horie, T., 316 Hotta, S. S., 213 Hotta, T.,87 Hubel, D. H., 86, 88, 90, 166, 171, 177, 345 Hugelin, A.. 102 Hughes, J. R.,71 Hukuhara, T., 98-111 Hunt, C. C., 194 Hurlbert, R. B., 117 Iggo, A., 194 Ihara, S., 323 Iida, Y., 280 Imai, Y., 307, 308 Lmaizumi, K., 112 Imamura, G., 100 Ingram, W. R.,315 Inoue, K., $ 2 1 Ishida, S., 14 Ishii, S., 213 Ishii, Y.,198 Ishino, T., 6, 7 Ishizuka, N., 17 Ito, A., 5-7 Ito. M., 217-250, 325 Ito, s., 112 Iwakura, I., 1, 3 Iwama, K., 174 Jacobsohn, L., 132 Jasper, H. H., 247,297, 323, 325 Jimbo, S., 324 Jimenez-Castellanos, J., 297 Johnson, A. R.,174 Jouvet, M., 87 Jowett, M., 214 Jung, R., 345 Kameda, K., 87 Kamrin, A. A., 119 Kamrin, R. P., 119 Kandel, E. R., 92 Kanemitsu, A., 308. 310 Kaneki, S., 12, 13 Kanno, Y.,76,78 Kapphan, J. I., 212, 213 Kariya, K., 295

Kariya, T., 113, 125, 126 Kasamatsu, A., 333, 345 Kato, E., 174 Kato, J., 14 Kato, M., 112-130 Kato, T., 128 Katsuki, Y., 71-97 Kaufman, S., 213 Kawamura, H., 100 Kawana, E., 292-322 Kawashima, T., 13 Kelley, R., 107 Kemp, E. H., 84 Kennedy, T. T., 327 Kiang, N.-Y. S., 76 Killam, K. F., 119 Kimura, R.,72 King, R.B., 303, 306 Kishi, S.,295 Wee, M. R., 323,344 Klingenberg, M., 2 I 3 Klingman, W. O., 112 Knox, W. E., 15,284 Kobayashi, K., 113 Kobayashi, T., 113 Kodaira, A., 277, 295, 307, 312 Koga, E., 324 Kohn, K. W., 108 Kojima, H., 109 Kondo, H., 9 Kotake, Y., 3 Kozaki, T., 17 Kraus, W. M., 145 Krieg, W. J. S., 33, 132 Kubo, N., 14 Kubota, K., 347 Kuffler, S. W., 59 Kumadaki, N., 109 Kumagai, H., 98-111 Kumamoto, T.,213 Kurachi, K., 7, 8, 17, 21 Kuritani, T.,14 Kurnick, N. B., 145 Kuroiwa, S., 324, 331, 333, 339 Kurokawa, M., 112-130 Kurotsu, T., 1-15, 17, 21, 23 Kusama, T., 292-322 Kutsukake, G., 112 Kuypers, H. G. J. M.. 295, 300, 303, 307 Kwak, R., 173 Lacy, 0. w., 112 LaGrutta, V., 83 Lawn, A. N., 108 Legouix, J. P., 71 Le Gros Clark, W. E..295 Lehninger, A. L., 212 Lennox, W. G.. 102 Lennox-Buchthal, M. A., 177

AUTHOR INDEX

Leontovich, T. A., 136 Levick, W. R.. 180 Lhermitte, J., 145 Li, C. L., 323, 344 Liles, G. W., 295 Lilly, J. C., 313 Liu, C. N., 295, 306 Livingston, R. B., 306 L.loyd, D. P. C., 170 Long, R. G., 169 Lorente de Nb, R., 79, 80, 137 Lowenstein, O., 52, 64 Lowry, 0. H., 212,213 Lowy, K., 84 Layning, Y.,65 Luby, E. D., 107 Lundquist, P.-G., 72 Lux, H. D., 323 Lyman, F. L.. 282 Mabuchi, M.. 308 Machiyama, Y., 116, 117, 120, 121, 124, 125 Machne, X.,247, 248 Macy, Jr., J., 181 Maeda, T., 213 Maekawa, K., 324, 331, 339, 345 Maeno, S., 234 Magni, F., 59, 175 Magoun, H. W., 136, 324,347, 34R Mandel, P., 280 Mannen, H., 76. 78, 131-162 Marburg, O., 132 Mardell, R., 280 Marks, R., 112 Marquis, D. G., 163 MarSala, J., 220 Marukashi, J., 194 Maruyama, N., 84, 88 Masai, H., 2, 3, 5-7, 11-13, 17, 23 Masuda, M., 5 Matano, S., 26, 27 Matsui, M., 15, 21 Matsumoto, J., 234 Matsumoto, S., 6 Matsutani, T., 282 McIlwain, H., 197, 213 Mclntyre, A. K., 170, 194 McLean, P., 197 McLeod, J. G., 169 Meessen, H., 25, 26, I 3 2 Megawa, A., 21 Megawa, N., 1, 23 Mettler, F. A., 295 Mickle, W. A., 313 Micklewright, H. L., 145 Miki, M., 295 Milner, P., 217, 230 Misaki, Y.,11 Mitsuhashi, Y.,310

355

Miyamoto, K., 217-250 Mizuguchi, K., 194 Momose, T., 10 Monaco, P., 73, 75, 80, 83 Moore, S., 117, 272, 276 Morikawa, N., 198, 199, 210, 213 Morillo, A., 173, 174 Morimoto, A., 3, 6 Morison, R. S., 323 Morrel, R. M., 92 Moruui, G., 101, 324, 345 Motokawa, K., 163-179 Mountcastle, V. B., 86, 88, 184, 188, 193-195, 297, 304, 312

Moushegian, G., 85 Murata, K., 87 Musgrave, F. S., 323, 344 Muzuguchi, K., 194 Nachlas, M. M., 198 Nagai, M., 6 Nagata, Y., 268, 275, 277 Nakahama, H., 180-196 Nakamura, T., 9 Nakamura Y.,324, 325, 331, 339 Nakanishi, S., 98, 103-105 Nakao, H., 217,232, 233 Nakayama, S., 108 Narabayashi, H., 306, 326 Naruse, H., 112-130 Nasu, T., 113 Nauta, W. J. H., 33, 313-315 Neff, W. D., 86, 93, 313 Negishi, K., 323 Nelson, P. G., 86, 93 Ngai, S. H., 108 Nicolesco, J., 132 Niimi, K., 295 Nishioka, S., 180-196 Nomoto, M., 77, 80, 85 Nyberg-Hansen, R., 295, 306, 307 Oda, S., 5 Offenlock, K., 344 Ogawa, T., 171 Okada, H., 108 Okada, M., 10, 14,213 Okarnoto, M., 5 8 k i , T., 31, 34 Okuda, J., 167-172, 175, 176 Olds, J., 217, 230, 231 OL.eary, J. L., 164, 166, 168 Olstewski, J., 25, 26. 132 Omukai, F., 21, 24, 31 Oonishi, S., 88 Ortmann, R., 198 Osborne, M. P.. 64 Otani, K., 292-322 Otani, T., 46

356

AUTHOR I N D E X

Otsuka, N., 49 Otsuka, T.,180-196 Otsuka, Y.,98-100, 103-105 Ozaki, S., 21 Page, 1. H., 197 Palestini, M., 101 Palmer, G., 282 Patton, H. D., 327, 331 Pears, A. G. E., 198 Peele, T. L., 295 Peretz, B., 217, 230 Perl, E. R., 313 Phillips, C. G., 325 Pitts. R. F., 136 Poggio, G. F., 188, 193-195, 312 Polyak, S., 168,295, 315 Pompeiano, O., 308 Potter, A., 132 Potter, D. D., 251 Potter, V. R., 117 Powell, T. P. S., 86, 88, 184, 310 Probst, M.,315 Purpura. D. P., 119, 323, 324, 339, 342, 344 Quastel, J. H., 197. 214 Rajkovitz, K., 295 Ramon-Moliner, E., 133, 138, 145, 146 Ranson, S. W.,136, 315 Rasmussen, G. L.. 30, 137, 313, 314 Reading, H. W.,214 RetzlafF, E., 48 Richter, D., 118, 125, 126, 197, 268 Riese, W.,293 Riley, H. A., 132 Rinvik, E., 208 Roberts, N. R., 212, 213 Robertson, J. D., 51 Rose, J. E., 71,86, 88,297, 312, 313 Rosenbaum, G., 107 Rosenberg, H., 108 Rossi, G. F., 101 Ruben, R. J., 80 Rudomin, P., 184, 195 Rupert, A., 71, 84, 85 Russell, D., 107 Rutherford, W.,71 Sailer, S., 233 Saito, Y.,323-351 Saji, Y.,98-100, 103-105, 109 Sakai, A,, 2, 3, 5, 6, 12, 17, 21 Sakai, F., 98-111 Sakuma, A., 98-111 Sakuma, S., 295 Salganicoff, L., 280 Salmoiraghi, G. C., 108 Sano, K.!18

Satani. R., 1 Satani, T.,1 Sawabe, T., 98, 104, 105 Sawyer, C. H., 18 Schadk, J. P., 145, 155 Scheibel, A. B., 132, 136 Scheibel, M. E., 132, 136 Scherrer, H., 87 Schlag, J. D., 323, 342, 348 Schmidt, R. F., 59, 175 Schmitz, H., 117 Schuknecht, H., 72, 75 Schiitz, H., 24 Schwartzkopff, J., 71.84 Sekino, T., 306 Sekula, J., 80 Seligman, A. M.,198 Sharpless, S., 247 Shimazu, H., 306, 326, 347 Shimazu, K., 10 Shimazu, T., 15, 16 Shimizu, N., 1, 9,197-216 Shimizu, S.,9, 21 Shimizu, T., 3. 9, 21, 31, 118, 128 Shimokwhi, M., 217-250 Shinoda, H., 13, 16-18, 20, 21 Shinya, G., 1, 2 Shofer, R. J., 323, 324, 344 Sholl, D. A., 131, 138, 146 Sibley, J. A,, 198 Simidu, M., 1 SjUstrand, S., 72 Slayman, C. L., 184, 195 Smith, C. A., 72 Siiderberg, U., 108, 171, 180 S o w , E. D., 198 Spackman, D. H.,117,272,276 Spencer, H. A., 323 Spencer, W.A., 92 Stage, D. E., 51 Stammler, A., 198, 203 Stanl, T. E.,248 Stein, W.H., 117, 272, 276 Stern, K., 132 Stewart, W.A., 303, 306 Storm van Leeuwen. W.,217 Stumpf, Ch.. 233 Suga, N., 76,78,80,84,85 Sugimoto, S., 268,269 Sugita, N., 17 Sumi, T., 84 Suter, C., 112 Suzuki, H., 92, 163-179 Swank, R. L., 127 Szabo, T., 295 Szenthgothai, J., 163, 295 Tabayashi, C., 2 4 , 8, 17 Taira, N., 164-172, 175,176

A U T H O R INDEX

Takagaki, G., 268, 269,277 Takahashi, R., 125 Takahashi, T., 117 Takakusu, A., 16, 17 Takamura, H., 10, 12 Takano, S., 128 Takeda, M., 6-8 Takenaka, S., 324,345 Takizawa, T., 112 Tamaki, H., 109 Tamura, T., 125, 126 Tanaka, Y., 73 Tane, T., 5 Tanimura, H., 10, 21 Tasaki, I., 67, 71, 76, 80, 194, 251 Taylor, C. W., 348 Tazuke, M.. 3 Thomas, E. C., 76, 198 Thulin, C. A., 108 Thurlow, W. R., 84 Tokizane, T., 100 Torii, K., 21 3, 323 Toru, M., 113 Torvik, A., 295 Towe, A. L., 327 Treff, W. M., 145 Tsou, K. C., 198 Tsukada, Y., 268-291 Tsukahara, Y., 92 Tsutsui, H., 8, 10 Tunturi, A. R.,90, 313 Tutikawa, K., 112 Uchiyama, H., 84 Uesugi, M., 295 Uno, M., 325, 327 Utena, H., 128 Uyemura, K., 268, 275,277,280 Valverde, F., 136 Van Bergeyk, W. A., 94 Van der Hoeven, Th., 285 Van der Loos, H., 146 Van Gehuchten, A., 137 Van Harreveld, A., 145, 155 Vladimirova, E. A., 268 Von Baumgarten, R., 108 Von Bektsy, G., 71, 78, 80, 86 Von Euler, C., 108 Von Frisch, K., 48 Von Helmholtz, H., 71 Von Monakow, C., 315 Voorhoeve, P. E., 65

357

Vrba, R., 268 Waelsch, H., 117. 119, 277 Waisman, H. A., 282 Walberg, F., 132,295, 308 Walker, D. G., 198 Wall, D., 28 Wall, P. D., 174 Wallenberg, A., 33 Waller, H. J., 181 Walzl, E. M., 313 Wang, H. L., 282 Wang, S. C., 108 Ward, J. W., 304-306 Watanabe, T., 76, 84, 85, 88 Webster, K. E., 310 Weil, A., 145 Wersall, J., 64,66, 72, 75 Whitlock, D. G., 313, 314, 323, 324, 345 Whittaker, V. P., 123, 127 WidBn, L., 173 Wiesel, T.N., 86, 88, 90, 166. 177 Williams, D., 98, 102 Williams, W. O., 180 Willis, W. D., 59 Wilson, D. M., 67 Winkler, C., 132 Woldring, S., 108 Wolff, H. G., 102 Woodburne, R.T.,34 Woolley, D. W., 285 Woolsey, C. N., 86, 300, 301, 303-305, 310, 312 Wyckoff, L. B., 282 Y a k , T., 112, 175 Yajima, K., 327 Yamada, M., 15 Yamada, Y., 198, 199,202, 203 Yamaguchi, Y., 217,234 Yamazaki, H., 234 Yamagisawa, N., 306 Yanagisawa, Y., 326 Yokota, T.. 175 Yokoyama, S., 12 Yoshida, M., 325-327 Yoshii, N.. 17,217-250 Yuasa, R., 3, 21, Yuasa, S., 115, 128 Zanchetti, A., 101, 323, 326 Zhukova, G. P., 136 Zyo, K., 24, 26, 27, 31, 33, 34

3 58

Subject Index Acetylcholine action on hair cell potential, 76 administration of, 75 chemical transmitter, 73 depolarization of membrane, 76 subcellular distribution, brain, 123, 281 Acetylcholinesterase distribution, 72 N-acetyl-L-aspartic acid mouse brain, 118 Adaptation dark, 166 rapid, brain, 92 Adrenaline local application cerebral cortex, 73 Alanine mouse brain, 118 ALD (aldolase) histochemical detection, 197 postnatal changes, 208 presence in olfactory bulb, 202 Amino acids interchangeability, 268 metabolic properties, 15 specific neuronal action, 268 Ammon’s horn, 2 Ammonia changes upon neuronal activity, 268 storage properties, 267 Analyzer continuous frequency, 217 Aquaduct cerebral, 32, 34, 36 Area

cortical auditory, 86 hypothalamic, 9, 12, 21 lateral hypothalamic, 27 lateral preoptic, 2, 20, 22 medial hypothalamic, 27 medial preoptic, 2,20,22 precentral motor, 87 preoptic, 2, 19, 21,22, 37 septal, 21 somatic sensory, 295,296,297 striate and parastriate, 173 Arginine mouse brain, 118 Arousal phasic frequency spectrum, 241.247 tonic neuronal correlates, 239, 241, 247 Aspartic acid presence in brain, 268

ATPase involvement in neural activity, 197 subcellular distribution, brain, 281 Atropine local application, 73 Auditory cortex lesions, 313 projections, 21 3 Axon collaterals, 45, 52 M-cell, 46 Basal ganglia endogenous activity, 302 Behavior neural basis, EEG, 217 properties of escape, 230,231 switch-off, 217 waking, sleep and emotional patterns, 17 Blood pressure cerebral control, 98 midbrain activation, rise of, 101 minimizing effect, 101 Bradycardia nervous regulation, 3 Brain stem dendritic arborization, 131-162 localization of learning sites, 220 multipolar and star-shaped neurons, 132 pretrigeminal transection, 101 reticular formation, 87, 102 telestimulation, 245 Bufo vulgaris amino acid content, 269 Bulbus olfactorius content of ALD and SD,202 medial forebrain bundle, 22 rabbit brain, 21 Canisfamiliaris amino acid content, 269 Capillaries glomerular brain tissue, 11 Capsule external, 21 internal, 21 Cavia cobaya amino acid content, 269 cells acinus, pancreas, 7 alveolar, parotis, 7

SUBJECT INDEX

epithelial layer, choroid plexus, 5 epithelium, 7 parietal, fundus gland, 7 thyroid follicular, 14 Central nervous system amino acid metabolism, 268 atypical synapses, 252 funneling mechanisms, 86 genesis of malformation, 17 phencyclidine HCL response, 107 strychnine effect, 64 Cerebellum fastigial nucleus, 30 Cerebral cortex auditory integration, 86,92 contralateral visual, 174 inhibition and facilitation, 92 integration of component sounds, 86 neo-, EEG pattern, 98, 109 periamygdaloid, 21, 32 somato-sensory area, 88, 184 visual, 88, 175 0-wave, 241 Chiasm optic, 2,25,28, 32, 33 Cholinesterase subcellular distribution, brain, 281 Clemmys (japonica) amino acid content, 269 Cochlea ascending fibers, 84 basilar membrane, 75 blood barrier, 76 coding, 72 neural response, 75 Colliculus inferior, 25, 32,36, 163 superior, 25, 32, 34, 36, 163 Commissura anterior, 2, 22,25, 33, 35 fornicis, 2, 33 posterior, 25, 28, 35 Conditioned reflex hypothalamic stimulation, 18 Convulsion chemically induced, 1 1 2 course of, 1 1 3 electrically induced, 112 Corpus callosum connection with cortical areas, 35 dorsal longitudinal fasciculus, 25 fornix system, 33 internal capsule, 2 preoptic area, 35 Curare action of ACh. 75 Dasybayus akajei amino acid content, 269

359

Degeneration axonal, myelin pattern, 137 Dehydrogenase gIucose-6-phosphate, 197 lactic, 198 succinic, histochemical detection, 197 Dendrites intrafocal and extrafocal, 132, 133, 136 lateral, 60,62 ventral, 62 Depolarization tonic, pyramidal neuron, 334 Depression postexcitatory, 174 presynaptic component, 174 Deprivation cutaneus input, 180-196 sensory, 194 Diencephalon structural organization, 204 Disinhibition neuronal artifact, 83 DPN-diaphorase interaction with ALD, 212 EEG arousal pattern, 100, 102 artificial respiration, 101 changes after posteromedial hypothalamotomy, 18 correlated with lever-pressing, 223 cortical, 221 desynchronization, 347 effects of ventilation, 98 frequency analysis, 217-250 hippocampal arousal @-wave,232 phasic arousal pattern, 241 psychological tests, 24 relationship with respiratory center, 99-1 1 1 switch-off behavior, 217 synchronization, 347 Electrophoresis acetylcholine, 73 Encephalization visual function, 163 EPSP (Excitatory postsynaptic potential) intracellularly produced positive potential, 57 monosynaptic, 327-351 polysynaptic, 327-351 Erythroidine effect on spinal neurons, 75 somatic effects, 76 Excitation antidromic axonal, 53 direct neuronal, 53 orthodromic axonal, 53 ventrobasal thalamic neurons, 180-196 Exophthalmos drug effect, 6,19

360

SUBJECT INDEX

Fasciculus ascending and descending fibers, 24 hypothalamo-tegmentalis, 30, longitudinalis dorsalis, 25, 30 longitudinalis medialis, 27-30, 36 pars ventralis, 26 retroflexus, 2,27, 34. 36 Forebrain medial bundle (MFB),22 Fornix rabbit brain, 21

superior, 33

Frequency analysis (EEG) characteristic average, 77 combination with tracer and driver stimulation, 225 histogram, 77 integration, 90 GABA (7-aminobutyric acid) interaction with amino acids, 268 specific neuronal function, 268 subcellular distribution, brain, 281 Gallus domesticus amino acid content, 269 Geniculate body lateral, 36, 163-175.247 medial, electrical stimulation. 34, 36, 245, 247 Girella punctata amino acid content, 269 Gland adrenal, 3 duodenal, 7 endocrine, 13 eye and intraorbital, 6 fundus, parietal cells, 7 lymph, production of, 5 submandibular, 7 Globus paltidus supraoptic decussations, 36 Glucose aerobic breakdown, 197 metabolic pathway, 198 metabolism, histochemical detection, 197 6-phosphate dehydrogenase, 197, 199, 210 Glutamic acid presence in brain, 276 Glutamic decarboxylase subcellular distribution, brain, 281 Glutaminase subcellular distribution, brain, 281 Glutamine changes, induced by stimulation, 268 Glutamine synthetase subcellular distribution, brain, 281 Glutathione mouse brain, 118 Glycine mouse brain, 118

Gyrus posterior sigmoid, 297 sigmoid and coronal, 295, 302 Hearing endocochlear, 71 neural mechanism, 71-97 Heterodontus japonicus amino acid content, 269 Hippocampus anterior continuation, 21, 22, 33, 35 periodic changes, 102 Histidine mouse brain, 118 Hydrocephalus experimental production. 112 Hypophorin anterior pituitary hormone, 9 Hypophysis extirpation, 3 neurosecretory granules, 10 portal system, 9 Hypothalamus antero-medial, electrical current, 2 17 electrical stimulation, nystagmus, 30 electroencephalographic changes, 17 gonadotropic stimulus, 8 neurosecretory granules, 10 parasympathetic zone, 17 periventricular stratum, 1 stimulation, 1 Hypoxia arousal pattern of EEG, 102 Inhibition chemical, 52 chemical postsynaptic, 65 collateral, 45, 52, 63 cortical response, 92 dendritic, 59, 60,62 efferent control, 80 W I t h nerve, 65 electrical, 52 model experiment, 61 neural network, 81 neuronal interaction, 85 nonefferent control, 81 presynaptic, 59 receptive properties, 192 remote-dendritic, 63 skin, 184 somatic, 62 spinal presynaptic. 63 strychnine, 64 ventrobasal thalamic neurons, 180-196 IPSP (Inhibitory postsynaptic potential) during neuronal synchronization. 328-350 receptive field, 192 reversal potential, 56

SUBJECT I N D E X

testing with EHP, 54 Isoleucine mouse brain, 118 Junction axo-dendritic, 137 axo-somatic, 137 cholinergic, 65 neuromuscular, 59, 66 LD (lactic dehydrogenase) presence in neurons, 198 Lemniscus medial, 36 Leucine mouse brain, 118 Lobe pyriform, enzyme pattern, 203 Lysine mouse brain, 118 Mamillary body fornix system, 33 supraoptic decussations, 36 Mauthner cells sound receptor organ, 48 synaptic interaction, 44-70 Medulla oblongata, 24, 207 auditory neurons, 84 caudal end, 28 inhibitory system, neocortical activity, 109 periodic discharges, 109 subnucleus reticularis ventralis, 26 Membrane arachnoid, 200 basilar, 75, 78, 79 dendritic, 60 plasma, 25 1 somatic, 60 synaptic, 51, 251 Membrane potential hair cell, 72 intracellular recording, 86 postsynaptic ending ACh, 76 stimulus sound. 93 Meninges cerebral, 200 Mesencephalon, 205 reticular formation, 330-351 Methionine mouse brain, 118 Midbrain auditory neurons, 84 central gray substance, 33 reticular formation, behavior direction, 248 rostral, 33 Neocortex EEG pattern, 98, 109

361

gray matter structure, 199 pentobarbital response, 107 periodic changes, 102 strychnine effect, 107 Nerve fiber ascending, 22, 34 cochlear, 72, 76,79 connection in the SPH-system, 21 degenerated, 22 descending, 24,30 external spinal, 79 hypothalamo-septa1 group, 34 inhibitory and excitatory, 93 internal carotid, 8 mesencephalo-septa1 group, 34 myelination, 44, 79 olivo-cochlear, 80 ovarial, 8 phrenic, 98 radial, 79 saccular, 49 superior cervical ganglia, 8 tractus hypothalamizo-nigralis, 30 vestibular, 79 Neuron auditory, membrane potential, 86 cortical, 323-351 flat-type, 90 inhibitory interaction, 85 model, 61 motor, 45 multi-peak type, 90 multipolar, 132, 133, 139 pre- and postgeniculate, discharge pattern, 163 primary auditory, nature. 76 response latency, 77 sharp-peak type, 90 star-shaped, 132, 139 thalamic, 87, 182, 323-351 ventrobasal thalamic, 180-1 96 Neuropil enzyme activity, 199 Nucleus abducens, 22,25,28 ambiguus, 25 anterior olfactory, 22, 25 anterodorsal thalamic, 22, 32 anteromedial thalamic, 22 arcuate, 2 Cajal, 22, 25, 28, 34 caudate, 2.21, 35 centrum medianum, 170 cochlear, 76, 82 cuneatus external. 132 Darkschewitsch, 22, 25, 28 dorsal premamilfary, 2 dorsomedial hypothalamic, 2, 21, 25, 26 Edinger-Westphal, 22, 25 entopeduncular, 28, 36

362

S U B J E C T INDEX

facialis, 25 Gudden, 22,25 intercalatus, 26 interpeduncular, 25,27, 32, 33, 36 lateral habenular, 22, 25 hypothalamic, 2, 3, 6, 13, 15, 22, 28, 36 mamillary, 1, 2, 21, 22, 25, 27, 33 septal, 22, 33, 35 vestibular, 25, 28 locus incertus, 25 medial central, 25 geniculate, 170 habenular, 22 hypothalamic, 25 mamillary, 1, 2, 21, 22, 27, 33 septal, 22, 33, 35 vestibular, 25, 28 mediodorsal thalamic, 22 oculomotor, 22,25,27, 28, 36 olivar inferior, 136 olivar superior, 84, 133 parataenial, 22, 32 paraventricular hypothalamic, 2, 22 posterior hypothalamic, 2, 21 paraventricular, 25 premamillary, 21 preopticus magnocellular, 20 prepositus hypoglossal, 25, 26, 28 pretectal, 22 septohippocampal, 22, 33, 35 subthalamic, 2, 36 superior vestibular, 25, 28 suprachiasmatic, 2 supragenualis, 25.29 supramamillary, 2,25 supraoptic, 2, 36 trigeminal, 30 trochlear, 22, 25. 27, 28, 36 ventral premamillary, 2 ventromedial hypothalamic, 2, 4.25 Nystagmus recording technique, 30 Parapristipoma trilineatum amino acid content, 269 Pathway (tract) auditory, 84 corticofugal, 87

hypothalamo-cerebello-vestibular,30 sensory, 84 somato-sensory, 84,86 visual, 84, 86 Pattern arousal, 102 EEG, artificial respiration, 101 response, colored lights, 175 Peduncle cerebral, 2, 21,27, 34 mamillary, 27, 34

Pentobarbital effect on neocortex, 107 method of application, 180 Perception brightness, 163 sensory pattern, 172 sound mechanism, 48 Phencyclidine HCI effect on cerebral cortex, 107 Phenylalanine mouse brain, 118 Phosphoethanolamine mouse brain, 118 Phosphor lipid content, 4 presence in cholesterol, 4 Plexus choroid, 5, 200 epithelial layer cells, 5, 6 Pons periodic discharges, 109 structural organization, 205 Potential ACh, depolarization of membrane, 76 antidromic action, 45 cochlear microphonic, 72 excitatory postsynaptic (see EPSP) extracellular, 45,46, 52 inhibitory postsynaptic (see IPSP) intracellular, 46 negative, 45, 46 postsynaptic, 93 resting, 56 reversal, 56 Procaine cortical application, 181 Proline, mouse brain, 118 Putamen rabbit brain, 21 Rattus norvegicus albus amino acid content, 269 Reflex conditioned, hypothalamic stimulation, 18 feeding, 36 olfactory, 36 orienting, 235 parasympathetic conditioned, 19 sympathetic conditioned, 19 Respiratory center relationship with EEG changes, 98-1 11 Response arousal (EEG), 102 augmenting, 323-348 cortical, colored lights, 175 excitatory, 48-52 extracellular orthodromic, 57 facilitation cortex, 92

SUBJECT INDEX

inhibitory, 52-65, 92 specific PT cells, 335 Reticular formation ascending impulses, 101 bulbar, 170 dendritic arborization, 144 mescncephalic, 170, 33C335 midbrain, dorso-medial part, 220 structural organization, 102 Rhinencephalon structural properties, 202

Synapse axo-axonal, 45, 131 dendro-dendritic, 131, 147 dendro-somatic, 131, 147 electrical, 49 excitatory, electrical, 44,131, 144 geniculate, 164 inhibitory, electrical, 44,131, 144 ordinary, chemical, 50 somato-somatic, 131 transmission, 44, 50 Synaptic delay, 68 Synaptic interaction Mauthner cell, 44-70 System septo-preoptico-hypothalamic(SPH), 21

Seizure amino acid changes, 119 convulsive, in ep mouse, 1 12-1 15 Self-stimulation response in rat, 230 Sepia esculenta Taurine amino acid content, 269 mouse brain, 118 Septum Thalamus pellucidum, commissural fibers, 32 auditory neurons, 84 precommissural, 21. 22, 33, 35 reticular formation, 335, 336 segmental, 251 Tractus Sphaeroides auditory, 82, 84, 86 amino acid content, 269 corticothalamic, 269 Spinal cord hypothalamico-nigralis, 27, 30 anterior funiculus, 28 hypothalamico-tegmentalis,27, 30 shock, conditioning stimulus, 57 hypothalamicus periventricularis, 2 1 structural aspects, teleosts, 44 hypothalamico-hypophysial, 9 Stimulation lateral olfactory, 2, 22, 32, 35 electrical, medial geniculate body, 245 mamillotegmentalis lateralis, 27 medial hypothalamic, 7 medialis, 27 ventromedial hypothalamic, 11 mamillothalamicus, 2, 27, 32, 36 visual and somatic cortex, 87 optic electric test stimulus, 164 Stratum supraliminal stimulus, 2, 170, 269 hypothalamic periventricular, 1, 2, 21, 22, 25, pyramidal, 27, 36 27, 37 tegmento-hypothalamicus,27 preoptic periventricular. 2, 20, 21, 22 tegmento-mamillaris lateralis, 27 Stria medialis, 27 longitudinalis lateralis, 22, 35 tegmentopeduncularis, 27 longitudinalis medialis, 22, 35 vestibulospinalis, 28 medullaris, 2, 22, 33 Transaminase terminalis, 2, 33 effect of tyrosine and alanine, 16 Strychnine Tuberculum olfactorium antagonistic action, 73 enzyme pattern, pyriform lobe, 203 collateral inhibition, 64 lateral preoptic area, 35 crossed and uncrossed efferents, 73 medial forebrain bundle, 30, 35 inhibitory effect, 75, 83 D-Tubocurarine mechanism of action, 65, 67 decrease of response, 75 neocortex, 107 suppressive effect, 76 Valine Substance mouse brain, 118 central gray, 22, 27, 36 Ventricle Substantia nigra lateral, 35 mamillary peduncle, 27, 34 third, 36 supraoptic decussations, 36 Succinic dehydrogenase Xesurus scalpruin subcellular distribution, brain, 281 amino acid content, 269

363

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