Using CNS Tissue in Psychiatric Research: A Practical Guide

USING CNS TISSUE IN PSYCHIATRIC RESEARCH

USING CNS TISSUE IN PSYCHIATRIC RESEARCH A Practical Guide

Edited by Brian Dean Mental Health Research Institute of Victoria, Parkville, Australia Joel E.Kleinman and Thomas M.Hyde National Institute of Mental Health, Washington DC, USA

harwood academic publishers Australia • Canada • China • France • Germany • India • Japan • Luxembourg Malaysia • The Netherlands • Russia • Singapore • Switzerland

This edition published in the Taylor & Francis e-Library, 2004. Copyright © 1999 OPA (Overseas Publishers Association) N.V. Published by license under the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group. All rights reserved. No part of this book may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and recording, or by any information storage or retrieval system, without permission in writing from the publisher. Printed in Singapore. Amsteldijk 166 1st Floor 1079 LH Amsterdam The Netherlands

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 0-203-30427-6 Master e-book ISBN

ISBN 0-203-34387-5 (Adobe eReader Format) ISBN 90-5702-298-2 (Print Edition)

Cover artwork: Dr Evian Gordon, Untitled, digital image and oil paints, 1×1½ metre canvas. Dr Evian Gordon is a brain scientist, who extrapolates basic principles about brain function into his Brainart. His work can be viewed at the Website: www.BRAINART.com.au Cover design: Jessica Cotterell

CONTENTS Foreword Contributors 1

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The Collection of Tissue at Autopsy: Practical and Ethical Issues Deborah A.Kittell, Thomas M.Hyde, Mary M.Herman and Joel E.Kleinman Psychiatric Diagnosis After Death: The Problems of Accurate Diagnosis from Case History Review and Relative Interviews Nicholas A.Keks, Christine Hill, Kenneth Opeskin, David L.Copolov and Brian Dean

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Membrane Binding Assays: Membrane Preparation and Assay Development Yogesh Dwivedi and Ghanshyam N.Pandey 39

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The Localisation and Quantification of Molecular Changes in the Human Brain Using In Situ Radioligand Binding and Autoradiography Brian Dean, Geoffrey Pavey, Siew Yeen Chai and Frederick A.O.Mendelsohn 67

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In Situ Hybridisation Histochemistry: Application to Human Brain Tissue Richard E.Loiacono and Andrew L.Gundlach

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Immunohistochemistry Techniques Applicable for Use with Human Brain Tissue James Vickers

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The Processing and Use of Postmortem Human Brain Tissue for Electron Microscopy Rosalinda C.Roberts and Lili Kung

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Isolating Components of Human Brain: The Purification of Aβ and the Alzheimer’s Amyloid Precursor Protein Robert A.Cherny, Colin L.Masters, Konrad Beyreuther and Ashley I.Bush

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Analysis of Receptor Systems in Schizophrenia Using Tissue Obtained at Autopsy and Neuroimaging Janet Mulcrone, Brian Dean and Robert W.Kerwin

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Index

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FOREWORD

The use of human postmortem tissue to discover the CNS pathophysiology of brain diseases has become critical, if not essential, for the identification of disease mechanisms. The brain is a hidden organ, difficult to assess in life, whose function is not directly apparent from its gross structure (as, for example, is the function of the heart or the stomach) or even from its microscopic analysis (as, for example, is the pancreas). Basic knowledge of normal brain mechanisms are only now being uncovered. A burgeoning neuroscience knowledge base and new research technologies are fueling this discovery. The challenge of explicating mechanisms for diseases of the human brain is now becoming possible and will inevitably require human postmortem tissue. Moreover, this knowledge is of the utmost importance to the rational discovery of targeted treatments for brain disease. Schizophrenia, in particular, is an illness whose basic pathophysiology remains entirely unknown. Clinical symptoms are highly prominent and characteristic across races and cultures. That pathophysiology has been so unsuccessfully approached, it is hard to reconcile with the florid clinical presentation. Basic neuroscience provides plausible scenarios, even rational hypotheses, for explaining the illness. Pharmacologic studies suggest relevant neurotransmitter systems. Functional imaging studies suggest CNS regions and dynamics of dysfuction. But perhaps only postmortem tissue studies can offer the definitive identification of the pathophysiology that we so sorely need for progress in understanding the illness. But postmortem tissue analysis is burdened with several categories of methodologie problems which can obscure or confound results. Indeed, these methodologic issues may have disadvantaged previous postmortem tissue research and discouraged further progress. The contents of this volume bring these areas to the forefront and suggest techniques to optimize study results. It is more important than is initially apparent to optimize and standardize tissue collection techniques, diagnostic approaches, and assay conditions for human postmortem tissue. Variability in these areas is often high; ability to control conditions may be low. All of this may introduce more variability into a postmortem human study than is generated by the illness, thus obscuring critical vii

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outcome measures. This volume identifies important methodologic issues and provides examples of approaches. Each of the chapters focuses on a discrete methodology or aspect of the postmortem research process. The chapters represent a broad range of diverse approaches, some traditional others new. Results of such analyses are easy enough to find in the published journal literature. However, nowhere in the journal literature will one be able to identify such concentrated and complete methodologies, with informal suggestions and examples as well as the generous methodologic description. These chapters will be important for experienced investigators who wish to enter new technical areas, as well as for serious students. The contents of this volume also suggest the extensive promise of the new sensitive and selective neuroscience techniques applied to postmortem analysis. Neurochemical studies at the molecular level are illustrated. Structural analysis at the electron microscopic level, as well as at the light microscopic level, offers potential new insight. Combinations of techniques are possible with high-quality and well-characterized postmortem tissue. Moreover, the combination of in vivo techniques like brain imaging with postmortem analysis is illustrated here and demonstrates the advantage of such a dual approach. It is not hard to imagine the exponential progress possible with focused application of these techniques to schizophrenia research. Postmortem neurochemical, anatomic, and pathologic studies in schizophrenia are being rediscovered as a critical methodologic approach in the study of schizophrenia after nearly 40 years of suboptimal application. It was probably both the methodologic complexities and the lack of the critical histologic and neurochemical techniques which frustrated early postmortem scientists and made them call schizophrenia the ‘Graveyard of Neuropathology’. This volume acknowledges the rebirth of postmortem tissue schizophrenia studies and contributes to the ongoing acquisition of techniques and postmortem data to use in the study of the illness. This volume should serve to reverse long held negative attitudes, and illustrates the excitement of new technical directions. CAROLYN A.TAMMINGA Professor of Psychiatry Maryland Psychiatric Research Center University of Maryland School of Medicine Baltimore, Maryland

CONTRIBUTORS

Konrad Beyreuther Center for Molecular Biology The University of Heidelberg IM Neuenheimer Feld 282 D-6900 Heidelberg, Germany

The Psychiatric Institute 1601 West Taylor Street Chicago, IL 60612, USA Andrew L.Gundlach The University of Melbourne Clinical Pharmacology and Therapeutics Unit Department of Medicine The Austin and Repatriation Medical Centre Heidelberg, Victoria 3084, Australia

Ashley I.Bush Genetics and Aging Unit Harvard Medical School Massachusetts General Hospital Building 149, 13th Street Charlestown, MA 02129, USA

Mary M.Herman Section of Neuropathology National Institute of Mental Health Neuroscience Center National Institute of Health Intramural Research Program 4th Floor Clinical Center 9000 Rockville Pike Bethesda, MD 20892, USA

Siew Yeen Chai Howard Florey Institute of Experimental Physiology and Medicine The University of Melbourne Parkville, Victoria 3052, Australia Brian Dean Division of Molecular Schizophrenia The Mental Health Research Institute 155 Oak Street Parkville, Victoria 3052, Australia

Christine Hill Division of Molecular Schizophrenia The Mental Health Research Institute 155 Oak Street Parkville, Victoria 3052, Australia

Robert A.Cherny Department of Pathology The University of Melbourne and The Alzheimer’s Disease Research Division The Mental Health Research Institute 155 Oak Street Parkville, Victoria 3052, Australia

Thomas M.Hyde Section on Neuropathology Clinical Brain Disorders Branch National Institute of Mental Health Neuroscience Center National Institute of Health Intramural Research Program 4th Floor Clinical Center 9000 Rockville Pike Bethesda, MD 20892, USA

David L.Copolov The Mental Health Research Institute 155 Oak Street Parkville, Victoria 3052, Australia Yogesh Dwivedi Department of Psychiatry (MC 912) ix

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Richard E.Loiacono Department of Pharmacology The University of Melbourne Parkville, Victoria 3052, Australia

Colin L.Masters Department of Pathology The University of Melbourne Parkville, Victoria 3052, Australia

Nicholas A.Keks Alfred Hospital Commercial Road Prahran, Victoria 3181, Australia

Frederick A.O.Mendelsohn Howard Florey Institute of Experimental Physiology and Medicine The University of Melbourne Parkville, Victoria 3052, Australia

Robert W.Kerwin Section of Clinical Neuropharmacology Department of Psychological Medicine Institute of Psychiatry De Crespigny, Denmark Hill London SE5 8AH, UK

Janet Mulcrone Section of Clinical Neuropharmacology Department of Psychological Medicine Institute of Psychiatry De Crespigny, Denmark Hill London SE5 8AH, UK

Deborah A.Kittell Section on Neuropathology Clinical Brain Disorders Branch National Institute of Mental Health Neuroscience Center National Institute of Health Intramural Research Program 4th Floor Clinical Center 9000 Rockville Pike Bethesda, MD 20892, USA Joel E.Kleinman Section on Neuropathology Clinical Brain Disorders Branch National Institute of Mental Health Neuroscience Center National Institute of Health Intramural Research Program 4th Floor Clinical Center 9000 Rockville Pike Bethesda, MD 20892, USA Lili Kung Department of Family Practice Lutheran Medical Center 150 55th Street Brooklyn, NY 11220, USA

Kenneth Opeskin Victorian Institute of Forensic Medicine 57–83 Kavanagh Street South Bank, Victoria 3006, Australia Ghanshyam M.Pandey Department of Psychiatry (MC 912) The Psychiatric Institute 1601 West Taylor Street Chicago, IL 60612, USA Geoffrey Pavey Division of Molecular Schizophrenia The Mental Health Research Institute 155 Oak Street Parkville, Victoria 3052, Australia Rosalinda C.Roberts Maryland Psychiatric Research Center University of Maryland PO Box 21247 Catonsville, MD 21228, USA James Vickers Department of Pathology Faculty of Medicine and Pharmacy University of Tasmania GPO Box 252–29 Hobart, Tasmania 7001, Australia

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THE COLLECTION OF TISSUE AT AUTOPSY: PRACTICAL AND ETHICAL ISSUES Deborah A.Kittell, Thomas M.Hyde, Mary M.Herman and Joel E.Kleinman

An abundance of new research techniques and findings have removed postmortem schizophrenia research from ‘the graveyard of neuropathology’ (Plum, 1972). Advances in neuroimaging that have allowed for in vivo pathophysiology as well as newer techniques in neuropathology such as immunocytochemistry, autoradiography, computerized neuronal morphometrics, and in situ hybridization have fueled this revitalization. However, despite these advances, many studies suffer from critical methodological deficiencies, some of which could easily be corrected. Moreover, there are few consistent thematic approaches to this disorder. To date, there have been numerous findings in postmortem schizophrenia research, but few have been replicated. The goal of this chapter is to review the pitfalls of postmortem schizophrenia research, and offer a guide towards well-constructed research protocols employing the advanced techniques now available. One example of the value of postmortem research involves Parkinson’s disease. The discovery of reduced dopamine concentrations in the nigrostriatal pathway of deceased patients diagnosed with Parkinson’s disease (Ehringer & Hornykiewic, 1960) led to the L-dopa carbidopa replacement treatment strategy (Cotzias et al., 1967). This success, in large part, was achieved by knowing where to look in the brains of Parkinson’s disease patients, a fact made more obvious by the loss of pigment in the substantia nigra (Foix, 1921). The utilization of modern neurochemical techniques, such as assays to measure dopamine in fresh-frozen postmortem specimens, was also essential to this clinical research advance. Neuropathological studies of mental illnesses raise several issues that are not encountered in the study of most neurological diseases. A number of these problems are discussed in the first section of this chapter, Neuropsychiatric Issues. The next three sections, Neuropathological Issues, 1

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Neuroanatomical Issues, and Neurochemical Issues, cover topics that may prove relevant to all brain diseases. The last section, Ethical Issues, addresses questions that must be considered when designing any study using postmortem tissue.

NEUROPSYCHIATRIC ISSUES

Tissue collection All neuropathological studies of mental illnesses are limited by the availability of sufficient numbers of brain specimens. In the United States there are at least six proven sources of specimens and equivalent sources should exist elsewhere. These sources are the medical examiners’ offices, the Veteran’s Administration Hospital system (VAH), state psychiatric hospitals, hospices, brain banks, and direct donations from individuals affiliated with patient advocacy groups. Each of these sources has advantages and disadvantages. Medical examiners’ offices are the optimal source for the study of suicide. This source of tissue offers the advantage of specimens from younger subjects. For the study of mental illness in general, and schizophrenia in particular, this advantage may be offset by the difficulty of establishing a complete psychiatric history, making an accurate diagnosis problematic. A second complication in the use of medical examiner’s cases is substance abuse. Toxicological screening of urine, blood, or brain can be routinely employed to identify this problem. The medical examiner’s office provides two other major advantages: relatively short postmortem intervals and a source of normal controls from the same facility. This may reduce the effects of postmortem artifacts in the collection, resulting from factors such as ambient temperature, delay in refrigeration, and mode of death. A second major source of brain specimens is the VAH system. Along with medical examiners’ offices, the VAH is particularly useful for the study of alcoholism and other drug addictions. The VAH usually offers the advantage of easy access to medical records essential for an accurate diagnosis. Unfortunately, a major limitation is the large percentage of patients with dual diagnoses, such as manic-depressive illness and alcoholism. Nonpsychiatric disease controls can also be obtained in these hospitals. With the closure of a number of VAHs and the use of outside sources for veterans’ care, this resource is diminishing.

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The state psychiatric hospital offers the most promise for prospective studies of mental illness. Large numbers of schizophrenic patients who have not been placed in the community make prospective studies in the state psychiatric hospitals and, to a lesser extent, the VAH system feasible. The obvious advantage of this approach is that psychiatric diagnosis can be made with greater accuracy in living patients. An unavoidable disadvantage is that subjects studied prospectively in these sites are often of advanced age, which may be associated with superimposed dementias. Careful neuropathological examination for neurological co-morbidity such as cerebrovascular disease and Alzheimer’s disease is a necessity in this type of study. A second major problem in this setting is that normal controls must be obtained from another source. Like the VAH, state psychiatric hospitals are decreasing in number. The hospice has been used with considerable success for the study of illnesses where death is imminent, such as end-stage Alzheimer’s disease and other dementias, as well as nonpsychiatric disease controls. This approach can yield extremely short postmortem intervals. Yet, since psychiatric patients rarely die in a hospice, it is of limited value for the study of mental illnesses. Although nonpsychiatric disease controls can be obtained from this source, the patients usually have a severe debilitating systemic disease such as cancer or AIDS. Another factor in this setting is that prolonged agonal states may adversely affect the quality of harvested tissue. Patient advocacy groups, such as the Tourette’s Syndrome Association and the like, have recently organized efforts to encourage individuals with specific disorders, and their family members, to donate brains to research groups in order to advance research on a particular disease or disorder. The list of organizations that have undertaken this effort is impressive and growing every year. Particularly for less common disorders, this is an excellent way to increase the volume of available brain specimens. Unfortunately, due to many factors including the psychosocial effects of having family members with severe chronic mental illness, this has not been an especially productive source of brain tissue. For those researchers who do not have access to one of the previously mentioned sources, brain banks at UCLA and Harvard University are funded by the NIMH. Brain banks obtain tissues from any of the above sources, collecting brains in a standardized fashion. They have the disadvantages of obtaining tissues from great distances and multiple sources, limiting to some degree their abilities to satisfy the diverse needs of their users. The resourcefulness of their respective leaders, Drs Wallace Tourtelotte and Francene Benes, has allowed the banks to assist numerous researchers despite limited financial resources.

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Diagnosis after death After collection, the most important issue is diagnosis. This can be accomplished in prospective studies through patient interviews employing standardized diagnostic interviews such as the SCID (American Psychiatric Association, 1994), or after death, through medical records, family interviews, and police reports. A structured interview with the next of kin and the collection of medical records should be pursued on nonpsychiatric disease controls as well as psychiatric cases. Establishing a diagnosis after death can be a difficult process. From the NIMH experience, approximately one-third of possible schizophrenic cases cannot be confirmed by existing records or interviews or are confounded by alcohol or substance abuse. Accurate premorbid psychiatric diagnosis in suicides may be even more difficult. Both of these seem simple next to determining an accurate history on substance abusers, except for those recently in detoxification programs. For the most part, toxicological screens describe the latter. Attempts have been made to better characterize their history by the systematic use of segmentai hair analysis, which sequentially incorporates residues of many substances of abuse longitudinally along the hair shaft over several months to a year. This form of toxicological analysis has yet to become routine.

Controls The need for proper controls is a third major issue. The study of schizophrenia illustrates the variety of control cohorts that can be valuable in order to place findings in the proper scientific context. Schizophrenics collected through the medical examiners office may have died through suicide. For this reason, a non-psychotic suicide control group may be utilized to control for manner of death. The confound of prior neuroleptic treatment can be met by using neuroleptic-treated patients who are not schizophrenic, such as manic depressive patients, psychotic depressed patients, or the like. The two NIMH funded brain banks at UCLA and Harvard University frequently use Alzheimer’s and Huntington’s disease patients as controls for neuroleptic treatment. Lastly, a group of nonpsychiatric disease controls is a necessity. Each of these groups should be matched for age, gender, race, postmortem interval, and freezer storage time as much as possible. Controlling for socioeconomic status and intelligence is not feasible. Tests for toxicology of urine and blood should be performed as part of routine screening, but routine brain toxicology has yet to be employed.

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Recently, testing for HIV has become routine in our laboratory, as well as for hepatitis B if warranted by history or autopsy examination. For inclusion in a series currently under study we also employ screening for preservation of mRNA. Two final methodological considerations are worth mentioning. If one collects enough tissue to match for all the relevant variables, a computerized inventory is a necessity. Lastly, maintaining a strict double blind between the collectors and the biochemist or molecular biologist is critical in order to conduct an unbiased study.

NEUROPATHOLOGICAL ISSUES

Case selection The difficulties involved in the postmortem study of mental illness make almost every brain desirable, at least for neuropathological screening. There are notable exceptions however. Cases to avoid include those individuals with a history of mechanical ventilation for greater than 12 hours, external evidence of decomposition, and gunshot wounds to the head. A common misconception involves an arbitrarily chosen postmortem interval when deciding which brains to collect. To be certain, prolonged postmortem intervals result in the degradation of many important cellular components. However, time from death to refrigeration is probably a more meaningful variable than postmortem interval. The former measure is more difficult to obtain than postmortem interval, and as a consequence, it is rarely reported. Brain and related tissue collection should be performed in an orderly and regimented fashion. At the time of opening the thoracic cavity, cardiac blood should be obtained for HIV and hepatitis serology, toxicology, and other testing. During removal of the brain, the calvarium must be carefully sectioned to avoid saw blade marks on the cortical surface. The cerebral dura is reflected dorsally and the brain is carefully freed up, using a sharp scalpel for the cranial nerves and spinal nerve roots and a curved scissor for the tentorium cerebelli and vertebral arteries. A deep cut is made in the foramen magnum to obtain all of the medulla and as much as possible of the upper cervical cord. After the brain is completely freed, it can be gently lifted from the skull, weighed, photographed, put in a plastic bag, and covered with wet ice. Traction must be avoided to prevent tears in the cerebral peduncles and other brainstem structures. The pineal will remain with the brain if the cerebral dura and proximal tentorium are kept

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attached to the brain. The pituitary is removed by carefully breaking the posterior clinoid processes and pulling them caudally. The gland is then dissected out with the tip of a sharp scalpel blade, leaving its capsule intact. The pituitary and additional segments of the cervical cord, if not obtained with the initial brain removal, should also be placed in a plastic bag on ice. A ¾–1 in. wide by 6–8 in. long piece of scalp and attached hair are removed from the caudal aspect of the scalp incision and put in a separate bag for toxicology testing. After returning to the laboratory, the pituitary and pineal are frozen separately before beginning to section the brain. If the pineal is not accessible at this point, it can be dissected out following the rostral midbrain section. Samples of dura and vessels from the base of the brain can also be frozen at this time; these are useful as non-CNS control tissues from the same subject. The brain is kept surrounded by ice while dissections are performed. A flat section is made through the rostral most midbrain above the oculomotor nerves, to remove the entire brainstem and cerebellum, taking care not to damage the inferior medial temporal lobes (entorhinal cortex and hippocampus) in the process. After bisecting the cerebrum through the corpus callosum, each tissue slab is rapidly frozen separately on a flat surface (a glass plate surrounded by a plastic bag) in 1 to 1.5 cm coronal slabs. The freezing solution is a slurry of 50:50 isopentane and dry ice granules. As soon as possible, the tissue must be loosened from the plate and turned every few seconds to ensure uniform freezing. Before removal from the freezing solution, the tissue is tested for complete freezing by palpation. It must be rock-hard before putting into a thick-walled ziplock bag and rapidly submerging in a dry ice chest. Portions of the brain that are awaiting freezing should continue to be held in a plastic bag surrounded by wet ice. Where laterality is the principle research issue, bisection should be avoided if possible. Throughout the process of brain removal and dissection, face masks, eyeglasses or goggles, durable gloves (preferably N-Dex Nitrile gloves, which are more resistant to accidental cuts) or two pairs of latex gloves, sleeve protectors, disposable shoe covers, and an apron should be worn. After the procedure is completed, all surfaces and instruments should be cleaned with a 10% solution of freshly prepared household bleach (Clorox) by submersion for a minimum of 20 minutes. This is done to protect personnel from potential infectious organisms such as hepatitis, tuberculosis, and HIV. Coronal sections of the cerebrum are placed in pre-labeled plastic bags marked left or right and numbered sequentially (level one being the most rostral) through the most caudal level. The slabs with amygdala are also designated (usually levels 3 and 4). Coronal sections are arranged so that the second or third section begins at the rostral most tip of the temporal pole; 8–10 coronal sections are

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obtained from each side of the cerebrum. As sectioning proceeds, the neuropathologist examines each slab for pathological changes, such as atrophy, hemorrhage, trauma, glial scars, lacunar infarcts, tumors, etc. and takes sections for formalin fixation and subsequent histology from any questionable areas. Sections are also obtained in each case from the frontal, temporal, and occipital lobes, from the dorsal cerebellar vermis and in patients over age 40, from the caudal hippocampus. Separate sections from the poles of the cerebrum are frozen for HIV and toxicology testing; one sample is obtained for each test. The cerebellum is separated from the brainstem by cutting through the cerebral peduncles and is then bisected into two pieces in the horizontal plane. The vermis is bisected mid-sagitally and a section is taken from the superior vermis for histology. The brainstem is cut in sections perpendicular to its long axis, with at least two levels of midbrain, two of pons, two or three of medulla, and one or two of cervical cord. Thus, tissue will be available for more than one study at each level. The levels can be indicated on the bag as to upper, middle, and lower levels. The brainstem should not be cut through the midline, as this will destroy midline structures, such as the raphe nuclei, which may be critical to mental illness research.

Fixation Although modern approaches have employed fresh-frozen tissue, fixed tissue is still a valuable resource. Formalin-fixed specimens are essential for ruling out conditions such as cerebrovascular disease, Alzheimer’s disease, and other dementias. Moreover, this approach is especially useful for determining cell counts and the size of individual brain structures. It is important when using this approach to control for storage time, because this will influence brain shrinkage. Many studies would benefit from a fixed protocol with a predetermined duration of fixation, such as one month. After two weeks the brain is blocked and the structure(s) of interest embedded and stored in paraffin blocks for further investigations. Such blocks can be safely stored for long periods in a cool dry environment. For initial fixation and storage we use a neutral phosphate-buffered 10% formalin solution, which is changed the day after tissue acquisition and thereafter at regular intervals. The fixative should be changed regularly to prevent tissue decomposition or growth of deep-seated bacteria due to changes in the potency of the fixative with time. An alternative approach for long-term storage involves blocking and fixing the tissue in a cold 4% paraformaldehyde solution for 48

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hours, immersing the blocks in a graded sucrose series (12%, 16%, and 18%) for cryoprotection, and then storing at -30 °C in an antifreeze solution of ethylene glycol and glycerol. Lastly, electron microscopic studies for viral particles or the like may require glutaraldehyde as a primary or secondary fixative (the latter is less desirable). Formalin-fixed tissue can also be used for immunohistochemistry and in situ hybridization studies. Routine stains are hematoxylin-eosin and the Bielschowsky’s method for axons, adapted for paraffin sections. Bielschowsky s stain is the most sensitive silver stain in the latter sections for the detection of neuritic pathology (i.e. neurofibrillary tangles and neuritic plaques). Freezing Brain specimens can be frozen in a number of ways. Rapid freezing with frequent rapid turning of the tissue is a necessity to prevent ice artifacts, which can create major complications in the analysis of autoradiography and in situ hybridization studies. Rapid freezing can be accomplished by immersion of 1 to 1.5 cm thick coronal slabs into a mixture of dry ice pellets and isopentane or between metal plates in liquid nitrogen. Samples are then placed in airtight plastic bags and stored in -70 °C freezers with carbon dioxide cylinders as a backup. Neuropathological screening Ruling out concurrent Alzheimer’s disease, cerebrovascular disease and tumors is essential, especially if the cases involve the elderly. Alzheimer’s cases require sections from the frontal temporal/parietal, occipital cortex, and/or the amygdala/ hippocampal region (Khachaturian, 1985; Braak & Braak, 1991). The presence of cerebrovascular disease, hemorrhage, trauma, tumors, or other pathology is screened by inspection and confirmed by appropriate sections from suspected areas. Sections of the locus coeruleus and substantia nigra may also be necessary to rule out Parkinson’s disease and related disorders. A piece of superior cerebellar vermis is necessary to evaluate ethanol-induced atrophy and hypoxic injury. Sections of the cerebellar hemisphere with dentate nucleus and hippocampus and other cortical areas also aid in the evaluation of anoxic/hypoxic encephalopathies. A neuropathology report including a macroscopic and microscopic description is provided to the referring physician who then can make it available to the families. This is very useful information for the family and aids in their cooperation.

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Further dissection issues An alternative to the storage of brain tissue in slabs is the dissection of regions of interest from the whole brain. However, fresh tissue is difficult to accurately dissect, as it is quite soft. Frozen tissue poses several problems, including the danger that thawing in the process of dissection can lead to protein denaturation and the formation of ice crystals. In fact, great care must be taken during dissection to keep the bagged tissue slabs buried in dry ice except for the few minutes of tissue trimming. Motor driven saws are especially problematic, because their heat may denature tissues adjacent to the blade. Techniques such as autoradiography and in situ hybridization may avoid some of these problems, as they lend themselves well to 14 to 20 µm thick sections that can be cut from frozen coronal blocks by use of a cryostat. Both small structures, such as the locus coeruleus, and large structures, such as the cerebral cortex, are ideal for this approach. However very large tissue blocks are much more difficult to section and necessitate large amounts of expensive reagents, as well as a specialized cryostat. NEUROANATOMICAL ISSUES Gross neuroanatomy The first consideration in neuroanatomical localization is macroscopic. The central nervous system (CNS) is exceedingly complex, composed of many cortical and subcortical structures. Random sampling of cerebral structures is very unlikely to be productive. Mental illness research should be driven by anatomically based hypotheses. Specific regions of interest should be identified and selected on the basis of scientific evidence. This task is complicated by the subtle neuropathological basis of neuropsychiatrie disorders. Brains from patients with well-recognized psychiatric disorders have been studied for more than 100 years using traditional neuropathological techniques, with few reproducible findings. The paucity of meaningful findings reflects less about the abilities of the researchers and more about the subtlety of the abnormalities. In research, gross examination together with imaging is useful in the assessment of subtle volumetric changes in large structures, such as the hippocampus (Bogerts et al., 1985). Microscopic examination Microscopic analyses of brain tissue have proven to be more valuable than most macroscopic analyses in the investigations of psychiatric disorders.

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Recently, cytoarchitectural studies have led to a number of exciting findings in the brain of patients with schizophrenia. In the mesial temporal lobe, cytoarchitectural investigations, stimulated by data from in vivo neuroimaging studies (Suddath et al., 1989), have reported subtle abnormalities in the entorhinal cortex (Jakob & Beckmann, 1986; Arnold et al., 1991; Krimer et al., 1997a). Large structures in the brain such as the hippocampus often vary in neuronal type and arrangement regionally, in both the anterior-posterior dimension as well as the dorsal-ventral domains. This also holds true for the six-layered neocortex and the more primitive cortical regions. For example, the entorhinal cortex is characterized by clusters of neurons in layer two, in its more rostral aspects, with the gradual disappearance of these clusters caudally, as it merges with the parahippocampal gyrus (Krimer et al., 1997b). It is a necessity to study the same level of entorhinal cortex within and across patient cohorts in order to make valid comparisons between schizophrenics and normal controls. Subcortical structures also have a great deal of frequently overlooked anatomical complexity. The patch-matrix pattern of the striatum reflects a heterogeneous distribution of neurons and neurotransmitters, and differing patterns of connectivity (Semba et al., 1987). The many nuclei of the thalamus, hypothalamus, and brainstem have been subdivided into subnuclei, differing in neuronal type, neurotransmitters, and patterns of connectivity. The nucleus of the solitary tract (NTS) spanning most of the medulla illustrates these concepts. The rostral third receives gustatory input from the tongue, and the posterior two-thirds receives input from a variety of chemoreceptors and mechanoreceptors associated with abdominal and thoracic viscera. Within the visceral NTS, there are ten distinct subnuclei (Hyde & Miselis, 1992). Input from one class of pulmonary receptors project primarily to just one, the ventrolateral subnucleus, whereas gastric afferents project to at least two different subnuclei (Kalia & Mesulam, 1980; Kalia & Sullivan, 1982). Receptor distribution is also inhomogeneous in this structure, with 5-HT3 receptors restricted largely to one subnucleus (Ohuoha et al., 1994). Therefore, careful consideration of subtle neuropathological changes must take into account these levels of complexity.

Other issues In addition to anatomical heterogeneity in brain structures, there is also functional heterogeneity. For example, in many cortical and subcortical structures there is a topographic organization that corresponds to specific parts of the body or

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visual fields. In the striatum, there is a complicated yet well understood somatotopy, which should be considered in any investigations involving this structure (Young & Penney, 1982). Functional heterogeneity of brain structures is yet another reason for careful anatomical matching of specimens across subjects and cohorts. Connectivity also plays a role. Neuronal networks are important in the generation and modulation of complicated behaviors. Neuropathological analyses must account for the primary and secondary changes in the neural network disrupted in the disorder. For example, primary pathological changes in the entorhinal cortex and hippocampal formation could cause profound but secondary neurochemical changes in the frontal lobe. These secondary abnormalities may explain many of the clinical manifestations in schizophrenia such as deficits in executive function (Goldberg et al., 1991). Neuropathology studies must also consider the normal cellular constituency of a structure under investigation. There can be a selective loss of one subset of neurons within a structure, with relative preservation of other neuronal subtypes. Within the striatum there are multiple types of neurons. In Huntington’s disease, there is a primary loss of Golgi type II neurons, with a relative sparing of larger neurons (Dom et al., 1973). Clarification of the pathophysiology of Huntington’s disease is now focusing on the neurobiological characteristics of the Golgi type II neurons. These principles may be applicable to psychiatric disorders as well. When fresh brain specimens are obtained, the initial handling of the tissue will dictate how these principles of anatomic inquiry can be applied. Large structures, removed in blocks taken in a pre-selected and uniform plane of section, allow consideration of many of the concepts outlined above, including issues of precise localization and cytoarchitectural configuration. Precise localization of pathology to cortical lamina, subcortical subnuclei, and neuronal subtype requires this type of tissue. Autoradiography and in situ hybridization are best performed on tissue blocks that preserve the normal anatomy and adjacent landmarks. The immediate dissection of fresh tissue can make detailed anatomical analysis difficult if not impossible. However, immediate dissection facilitates rapid neurochemical analyses such as the characterization of receptors using membranes derived from tissue homogenates or the measurement of neurotransmitter levels. Micropunch methodology can be applied to large tissue blocks, allowing more precise anatomical localization while rapidly accessing tissue for detailed neurochemical analyses. Two other issues should be considered in human postmortem studies. The

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first issue is lateralization of function, which is important in cortical structures, and may be a consideration with subcortical structures as well. In right-handed individuals, regions within the left temporal and frontal lobes are the primary repositories of language function. Damage to analogous structures in the contralateral hemisphere produces a completely different and much more subtle set of behavioral abnormalities. Lateralization of function is often tied to handedness. Unfortunately, handedness is rarely a consideration in many postmortem studies, and should be considered when studying structures whose function is lateralized. A second important issue is that of normal inter-subject variability. There is a great deal of variation between individuals’ brain size and configuration. Any diagnosis of atrophy must consider normal variations. Another factor, which may be variable between subjects, is gyral configuration and size. Lateralization of function, handedness, and the normal amount of individual variability must be considered in any anatomical study of human brain tissue. In summary, the anatomical complexity of the CNS must be recognized in the postmortem study of human brain tissue. Uniform selection of regions of interest, chosen on the basis of precise hypotheses, are then studied using methods that take into account the macroscopic and microscopic characteristics of the structure. Consideration is given to cytoarchitectural, laminar, and/or subnuclear divisions. Finally, lateralization of function, handedness, and normal inter-subject variability cannot be ignored. Although these precepts are daunting in scope, rigorous application will improve the quality of human postmortem research into neuropsychiatric disorders, and increase the value of the findings from these investigations.

NEUROCHEMICAL ISSUES RNA preservation and integrity The detection of individual messenger RNAs (mRNA) coding for particular proteins necessary for normal neurodevelopment, function, and connectivity has recendy become possible through techniques such as in situ hybridization, Northern blot analysis, and nuclease protection assays. The quantitation and localization of gene expression in postmortem human tissue have yielded many exciting findings in neuropathological research. However there are a number of factors which must be controlled for when conducting a valid study using postmortem tissue. It is necessary to match for age, race, and gender when

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conducting a comparative, quantitative study across groups. Other variables that require consideration due to their effects on RNA preservation and integrity are postmortem interval (PMI), agonal state or pre-mortem conditions, and brain tissue pH at the time of freezing. Although the possible degradation of RNA during the postmortem interval has been considered a limitation, studies indicate that PMI has a very moderate effect on the preservation and integrity of total RNA. In situ hybridization studies have shown that mRNA is abundant in tissue with a postmortem interval of 36 hours or more. Data from Northern blot analyses provide evidence that the integrity of total RNA is preserved. The synthesis of peptides from extracted mRNA through in vitro translation experiments indicates that RNA is not only intact but is also biologically active in tissue up to 84 hours after death. However, it has been suggested that individual mRNAs may be subject to postmortem degradation. Studies have shown that the variation in postmortem stability is related to the specific mRNA species, but selective degradation according to molecular size or cell type does not occur (Barton et al., 1993; Gilmore et al., 1993). In addition, thawing of tissue after it is frozen may result in a loss of RNA. It is crucial to keep tissue samples frozen at all times prior to use. The agonal state of a patient can cause considerable alterations in gene expression prior to death, leading to significant variations in subsequent measurements of RNA. For example, seizures have been demonstrated to result in a number of changes in the quantitative distribution of several mRNAs (Saffen et al., 1988; Gall et al., 1990). Acute or chronic drug administration, substance abuse, alcoholism, dehydration, hypoxia, severe pain, etc. affect many transcripts. The agonal state is likely to produce a cascade of chemical events resulting in significant changes in patterns of gene expression such that individual mRNA species may be up-regulated, down-regulated, or unaffected. Agonal state factors may confound the identification of quantitative changes in gene expression as a result of an underlying neurological disorder. Unfortunately, the specific effects of many agonal state events are unknown. Measurements of tissue pH after death are a useful marker of pre-mortem hypoxia and acidosis (Yates et al., 1990). Tissue pH levels may also be employed as an indicator of total RNA preservation. Evidence suggests that total RNA is well preserved in tissue with pH values near 6.0 or higher. In contrast, tissue with more acidic pH values yield very little intact total RNA in extracts and no detectable mRNA in hybridization blots (Kingsbury et al., 1995). Measured routinely, tissue pH levels may provide a useful screen for RNA preservation within a collection of human brain tissue. Yet, all mRNAs are not equally sensitive to pre-mortem acidosis, and thus the effect of pH may vary depending on the

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mRNA species (Harrison et al., 1995). RNA degradation may be region-specific; therefore, each region of interest should be separately screened for RNA preservation.

Internal controls Another useful screening method for RNA preservation is the measurement of a marker mRNA or an internal control. Transcripts chosen as internal controls should be naturally abundant, a constitutively expressed gene, and found to occur at the same levels in control and experimental groups, unaffected by disease state. Two ideal internal standards for human postmortem research are actin and cyclophilin (Ponte et al., 1984; Haendler & Hofer, 1990). Unfortunately, no single mRNA is constant in all situations and all anatomical areas of interest; thus, each brain region should be screened and the appropriate internal control identified. The lack of replication in neurochemical postmortem studies can largely be attributed to inconsistencies in methodological approaches to RNA isolation and RNA analysis. Experimentally determined values of a mRNA of interest can be normalized against an internal standard. This may minimize errors in the isolation and analysis process. Human internal standard kits are available through Ambion Inc. (Austin, TX).

ETHICAL ISSUES Medico-legal-ethical issues are a major concern of postmortem research in mental illness. The most notable among these is obtaining informed consent from the legally designated next of kin for brain tissue donations. The law provides ‘competent’ adults with the ability to indicate their intent to donate by signing a legal document, a donor card. When such a donor card is lacking, the next of kin may authorize the donation of organs (Jonsen, 1989). This presents many important questions surrounding neuropsychiatric postmortem research that must be considered individually with each case. First, a donor card is rarely sufficient evidence for pathologists. In the case of a mentally ill patient, there may be questions about the validity of a donor card. In such cases, the consent of a family member is required. Consent may be obtained over the telephone if witnessed by a third party or recorded on tape. A permit for donation must be detailed and include permission to collect the brain, cranial contents, blood and

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hair samples. Second, many mentally ill patients do not have families available to authorize the donation of brain tissue. When the family is available, there can be controversies over who is legally the next of kin. Usually, the surviving spouse has primary standing, but in some cases it may be the parents, the children, or the siblings. In the absence of family consent, organs usually cannot be retrieved for research purposes. The time allotted to a family to decide upon donation is another important issue. When tissue is obtained through the medical examiner’s office, the family is called either the night before or the morning of the autopsy and a request for donation is made. This situation forces the family to rapidly arrive at a decision, which, for some, might require at least a day of deliberation and contemplation. When making a request for donation, researchers are obligated to inform the families not only of the immediate project but also that the tissues may be stored and used for research in the future by other investigators at various institutions. This is especially relevant to those investigators who are compiling brain banks. The investigator also has the responsibility of ensuring each donor’s anonymity and the privacy of any medical information that may be derived from the review of relevant records (Clayton et al., 1995). Lastly, the donation of tissues and organs for biomedical research should be presented to the family with the same reassurances as donation for transplantation. Many families find consolation through donating for transplantation. They often report that by aiding in the survival of other individuals, they feel the deceased is still alive and contributing to society. The donation of tissues and organs for research purposes may not be able to console the survivors in exactly this way. Organ tissue donation for transplantation greatly exceeds donations for medical research. This imbalance in family response to research versus transplantation could most likely be resolved through education. Overall, society has not been well educated to the public health benefits of neuropathological research nor to the dependence of this research on donation.

SUMMARY The availability of advanced neurochemical assays and neuroimaging techniques has revived interest in the ambitious field of neuropsychiatrie postmortem research. This research is unquestionably a challenging task, and numerous factors should be considered at all times. Investigators must first establish a source of brain specimens. Next, it is essential to determine the diagnosis of each case for both psychiatric subjects and nonpsychiatric

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controls. Cases having gross or microscopic neuropathological abnormalities are deleted from the sample. The collection of brain tissue should be matched for age, race, gender, and whenever possible postmortem interval. Samples are screened for RNA preservation through pH screening or the use of an internal RNA control such as cyclophilin or actin. Microscopic analysis is essential for assessing the preservation of tissue integrity. Material dissected from the brains is neuroanatomically matched. Collaboration among psychiatrists, neuroanatomists, neuropathologists, biochemists, and molecular biologists is a valuable resource. Collaboration is necessary in order to make appropriate use of the donated tissue in the postmortem study of mental illness. Despite these obstacles, the potential for significant findings and advances is great.

References American Psychiatric Association. (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th edn. American Psychiatric Association: Washington, D.C. Arnold, S.E., Hyman, B.T., Van Hoesen, G.W. and Darnasio, A.R (1991) Some cytoarchitectural abnormalities of the entorhinal cortex in schizophrenia. Arch. Gen. Psychiatry, 48, 625–632. Barton, A.J.L., Pearson, R.C.A, Najlerahim, A. and Harrison, P.J. (1993) Pre- and postmortem influences on brain RNA. J. Neurochem., 61, 1–11. Braak, H. and Braak, E. (1991) Neuropathological staging of Alzheimer-related changes. Acta Neuropathologica, 82, 239–259. Bogerts, B., Meertz, E. and Schonfeldt-Bausch, R. (1985) Basal ganglia and limbic system pathology in schizophrenia. Arch. Gen. Psychiatry, 42, 784–791. Clayton, E.W., Steinberg, K.K., Khoury, M.J., Thomson, E., Andrews, L., Kahn, M.J.E., Kopelman, L.M. and Weiss, J.O. (1995) Informed Consent for Genetic Research on Stored Tissue Samples. JAMA, 274, 1786–1792. Cotzias, G.C., Van Woert, M.H. and Schiffer, L.M. (1967) Aromatic amino acids and modification of parkinsonism. N. Engl. J. Med., 276, 374–379. Dom, R., Baro, F. and Brucher, J.M. (1973) A cytometric study of the putamen in different types of Huntington’s chorea. In Advances in Neurology, vol. 1, Huntingdon’s Chorea 1872–1972, edited by A. Barbeau, T.N. Chase and G.W. Paulson, pp. 369–385. New York: Raven Press. Ehringer, H. and Hornykiewic, O. (1960) Verteilung von noradrenalin und dopamin (3hydroxytyramine) im gehirn des menschen und ihr verhalten bei erkankungen des extrapyramidalen systems. Klin. Wschr., 38, 1236–1239. Foix, M.C. (1921) Les lesions anatomiques de la maladie de Parkinson. Rev. Neurolog., 28, 593–600. Gall, C., Lauterborn, J., Isackson, P. and White, J. (1990) Seizures, neuropeptide regulation, and mRNA expression in the hippocampus. Prog. Brain Res., 83, 371–390.

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Gilmore, J.H., Lawler, C.P., Eaton, A.M. and Mailman, R.B. (1993) Postmortem stability of dopamine D1 receptor mRNA and D1 receptors. Mol. Brain Res. 18, 290–296. Goldberg, T.E., Gold, J.M. and Braff, D.L. (1991) Neuropsychological functioning and time-linked information processing in schizophrenia. In Review of Psychiatry, vol. 10, edited by A.Tasman and S.M.Goldfmger, pp. 60–78. Washington, DC: American Psychiatric Press. Haendler, B. and Hofer, E. (1990) Characterization of the human cyclophilin gene and of related pseudogenes. Eur. J. Biochem., 190, 477–482. Harrison, P.J., Heath, P.R., Eastwood, S.L., Burnet, P.W.J., McDonald, B. and Pearson, R.C.A. (1995) The relative importance of pre-mortem acidosis and postmortem interval for human brain gene expression studies: selective mRNA vulnerability and comparison with their encoded proteins. Neuroscience Letts., 200, 151–154. Hyde, T.M. and Miselis, R.R. (1992) The subnuclear organization of the human caudal nucleus of the solitary tract. Brain Res. Bull., 29, 95–109. Jakob, H. and Beckmann, H. (1986) Prenatal developmental disturbances in the limbic allocortex in schizophrenics. J. Neural Transm., 65, 303–326. Jonsen, A.R. (1989) Organ and Tissue Retrieval and Donation: The Ethical Imperative. In Pediatric Brain Death and Organ/Tissue Retrieval, edited by H.H.Kaufman, pp. 251–257. New York: Plenum Publishing Co. Kalia, M. and Mesulam, M-M. (1980) Brainstem projections of sensory and motor components of the vagus complex in the cat. J. Comp. Neurol., 193, 435–465. Kalia, M. and Sullivan, J.M. (1982) Brainstem projections of sensory and motor components of the vagus nerve in the rat. J. Comp. Neurol., 211, 248–264. Khachaturian, Z.S. (1985) Diagnosis of Alzheimer’s Disease. Arch. Neurolog., 42, 1097– 1105. Kingsbury, A.E., Foster, O.J.F., Nisbet, A.P., Cairns, N., Bray, L., Eve, D.J., Lees, A.J. and Marsden, C.D. (1995) Tissue pH as an indicator of mRNA preservation in human postmortem brain. Mol. Brain Res., 28, 311–318. Krimer, L.S., Herman, M.M., Saunders, R.C., Boyd, J.C., Hyde, T.M., Carter, J.M., Kleinman, J.E. and Weinberger, D.R. (1997a) A qualitative and quantitative analysis of the entorhinal cortex in schizophrenia. Cerebral Cortex, 7, 732–739. Krimer, L.S., Hyde, T.M., Herman, M.M. and Saunders, R.C. (1997b) The entorhinal cortex: an examination of cyto- and myelo-architectonic organization in humans. Cerebral Cortex, 7, 722–731. Ohuoha, D.C., Knable, M.E., Wolf, S.S., Kleinman, J.E. and Hyde, T.M. (1994) 5-HT3 receptor distribution in the dorsal vagal complex of the human medulla: a quantitative autoradiographic study. Brain Res., 637, 222–226. Plum, F. (1972) Prospects for research on schizophrenia. 3. Neuropsychology. Neuropathological findings. Neurosci. Res. Prog. Bull., 10, 384–388. Ponte, P., Ng, S.Y., Engel, J., Gunning, P. and Kedes, L. (1984) Evolutionary conservation in the untranslated regions of actin mRNAs: DNA sequence of a human beta-actin cDNA. Nucl. Acids Res., 12, 1687–1696. Saffen, D.W., Cole, A.J., Worley, P.F., Christy, B.A., Ryder, K. and Barban, J.M. (1988) Convulsant induced increase in transcription factor messenger RNAs in rat brain. Proc. Natl. Acad. Sci. USA, 85, 7795–7799.

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Semba, K., Fibiger, H.C. and Vincent, S.R. (1987) Neurotransmitters in the mammalian striatum: neuronal circuits and heterogeneity. Can. J. Neurol. Sci., 14, 386–394. Suddath, R.L., Casanova, M.F., Goldberg, T.E., Daniel, D.G., Kelsoe, J.R. and Weinberger, D.R. (1989) Temporal lobe pathology in schizophrenia: a quantitative magnetic resonance imaging study. Am. J. Psychiatry, 146, 464–472. Yates, C.M., Butterworth, J.,Tennant, M.C. and Gordon, A. (1990) Enzyme activities in relation to pH and lactate in postmortem brain in Alzheimer-type and other dementias. J. Neurochem., 55, 1624–1630. Young, A.B. and Penney, J.B. (1982) Neurochemical anatomy of movement disorders. Neurol. Clinics, 2, 417–433.

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PSYCHIATRIC DIAGNOSIS AFTER DEATH: THE PROBLEMS OF ACCURATE DIAGNOSIS FROM CASE HISTORY REVIEW AND RELATIVE INTERVIEWS Nicholas A.Keks, Christine Hill, Kenneth Opeskin, David L.Copolov and Brian Dean

In addition to controlling the laboratory techniques involved in the study of postmortem brain neurobiology, it is also necessary to ensure the subjects from whom tissue is collected have an accurate psychiatric diagnosis. However, in a recent review of 60 major studies we showed that about half did not explain psychiatric diagnostic procedure and less than half specified the diagnostic criteria used (Keks et al., 1995a). There would thus seem to be a need to give more consideration to the validity of diagnosis on which neurobiological studies using postmortem tissue are based. Postmortem diagnosis poses a number of practical challenges, the most obvious being that the subject is not available for clinical evaluation of history, symptoms and signs. It is therefore necessary to rely on secondhand accounts from medical records and informants such as relatives. Theoretically such limitations could be overcome by antemortem prospective psychopathological evaluation, an approach that has proved successful in studying the neurobiology of diseases of old age. However, in the cases of illness such as schizophrenia, which have an early age of onset, the difficulties in tracing assessed patients until death limit the application of this strategy. Furthermore the samples thus obtained tend to be nonrepresentative by virtue of being from the elderly and having long and complex treatment histories. There are inherent problems in the quality of sources of information relating to postmortem diagnosis of psychiatric illness which are not amenable to correction. The reliability, comprehensiveness, completeness and organisation, as well as the clinical skill involved in creating medical records can vary 19

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drastically. There may be major differences and contradictions from one illness episode to another. Information from relatives or other sources may be of variable quality if at all available. However, there is no alternative to the acceptance of these sources of information, which can therefore introduce unreliability in postmortem investigations. On the other hand the methods and procedures through which postmortem diagnoses are made are amenable to standardisation in order to improve reliability and comprehensiveness. Elsewhere, the reliability of diagnosis has been improved through use of diagnostic criteria and structural assessments, such as the Research Diagnostic Criteria (RDC) and the related instrument, the Schedule for Affective Disorders and Schizophrenia (Spitzer et al., 1975).

Diagnostic evaluation after death In postmortem neurobiology the only published instrument known to the authors is the Diagnostic Evaluation After Death (DEAD) (Zalcman et al., 1983). However, it has only been utilised in limited circumstances (Karson et al., 1993) despite its positive characteristics. The DEAD is intended for use by a trained clinician who needs to review all available data and, if possible, to interview informants. ‘Best clinical judgement’ is used to convert non-specific case notes to symptom data so that where a patient is recorded as being ‘consistently up through the night’, sleep disturbance may be diagnosed. The absence of a symptom is recorded if the symptom is not mentioned in the notes and relevant behaviours are absent. A key strength of the DEAD is in its meticulous approach to recording treatment history, including medications and physical illness. The record distinguishes items as having been ever present, received in the last six months or received in the 24 hours before death. Evidence of alcohol and drug abuse is sought, as well as the presence of related physical complications. Specific psychopathologic syndromes (or subsyndromes) and symptoms are diagnosed. Evidence for depression, mania/hypomania, hallucinations, delusions, other psychotic symptoms, organic symptoms, and antisocial behaviour is gathered. Again the items are divided into ever present, present in last 6 months, and present in the 24 hours prior to death. The clinician using the DEAD also applies to the data the diagnostic criteria of RDC (Spitzer et al., 1975), Feighner (Feighner et al., 1972) and DSM-III (American Psychiatric Association, 1980). The diagnoses are divided into lifetime diagnosis and diagnosis at time of death. The level of certainty is rated

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as either definite or probable. The clinician is also able to make a clinical diagnosis (that is, not based on diagnostic criteria), and if all else fails, make a clinical ‘hunch’. Family history is also noted. Although the diagnostic criteria used by the DEAD have been partly superseded, and criticism may be leveled at the absence of subsyndrome diagnosis (e.g. positive symptoms, formal thought disorder, negative symptoms), the DEAD has major strengths. The DEAD has an approach to psychopathological assessment that is semi-structured and the evaluation required is clearly specified, which does improve reliability. It is also multidiagnostic. However the instrument does not appear to have been tested for reliability, and its use in actual studies has been limited. Diagnostic criteria for schizophrenia One of the major difficulties facing research into neurobiological correlates of schizophrenia is that there is no gold standard for the diagnosis of the disorder. Over 14 reputable systems are in use and differences between the systems are important. This can be readily seen on a broad-brush summary of whether or not the more common diagnostic systems include key clinical manifestations of schizophrenia (Table 2.1). A practical example of the importance of diagnostic evaluation occurred in one of our neuroendocrine studies where findings on plasma prolactin levels changed from significant to non-significant depending on the diagnostic criteria for schizophrenia (Keks et al., 1990). It has also been shown that the application of different diagnostic criteria changes the population of subjects included within diagnostic groups. When 10 diagnostic systems were used to diagnose 119 acutely psychotic patients Table 2.1 Summary of key symptoms which contribute significantly to diagnosis using common diagnostic criteria for schizophrenia

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depending on the diagnostic system used, the percentage diagnosed with schizophrenia ranged from 4% to 45% (Brockington et al., 1978). The major differences between diagnostic systems tend to be concerned with the ways in which positive, negative and affective symptoms, as well as course of illness are regarded (Table 2.2) (Fenton et al., 1981). In addition, our neuroendocrine studies revealed particularly poor concordance between Schneiderian criteria (Schneider, 1959), for the diagnosis of schizophrenia and the DSM-III, Feighner and RDC criteria. In those studies, the level of diagnostic agreement between DSM-III and Feighner was only moderate (kappa=0.433), and only slighdy better for Feighner and RDC (kappa=0.472) (Keks et al., 1990, 1992). It is likely that research studies being currently undertaken will utilise DSMIV (American Psychiatric Association, 1994) or ICD-10 (World Health Organization, 1990). The criteria for these systems are given in Tables 2.2. Results generated from these studies may not be directly comparable with older studies using DSM-III-R, DSM-III, Feighner, Schneider or RDC criteria, as there may be significant psychopathological differences in the inclusion/ exclusion criteria for the patient samples. To enable comparisons, criteria for these diagnostic systems are listed in Tables 2.3 to 2.9. We have investigated the ramifications of differences between diagnostic systems in a review of 83 subjects who were referred for autopsy with a clinical diagnosis of schizophrenia and adequate case notes were available. Subjects were diagnosed according to DSM-III-R, ICD-10, Feighner, RDC and Schneider criteria (Hill et al., 1996). In that study, 26 of the 83 subjects that had a provisional diagnosis of schizophrenia did not fulfil the criteria for schizophrenia in any of the diagnostic systems. Most of these 26 subjects were reclassified as either schizoaffective disorder or major depression on DSM-III-R criteria. In addition, 68.7% of the group met the criteria for at least one system of schizophrenia, but only 20.5% met the criteria for all 5 systems. Thus, whilst the argument that postmortem studies should include ‘schizophrenic’ patients that meet the criteria for all or at least a substantial majority of diagnostic systems (Kleinman, J., pers. comm.), such patients are relatively few in number. Nonetheless, studies on such a ‘core’ schizophrenic group could be of particular interest. Finally, the concordances for schizophrenia among the 5 systems are given in Table 2.10. It can be seen that because the concordance between certain diagnostic systems is either fair or poor, the investigator’s choice of diagnostic system may have a major effect on the extent to which sprecific neurobiological findings are reported to be present ‘in schizophrenia’.

Table 2.2 Comparison of diagnostic criteria for six diagnostic systems of schizophrenia

Table 2.3 DSM-IV diagnostic criteria for schizophrenia

Source: American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders (4th edn). Washington D.C.: American Psychiatric Association.

Table 2.4 Research criteria for ICD-10 schizophrenia

Source: World Health Organization (1993) The ICD-10 Classification of Mental Health and Behaviour Disorders: Diagnostic Critieria for Research. Geneva: World Health Organization.

Table 2.5 DSM-III-R diagnostic criteria for schizophrenia

Source: American Psychiatrie Association (1987) Diagnostic and Statistical Manual of Mental Disorders (3rd edn-rev.). Washington D.C.: American Psychiatric Association.

Table 2.6 DSM-III diagnostic criteria for a schizophrenic disorder

Source: American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders (3rd edn). Washington D.C.: American Psychiatric Association.

Table 2.7 Feighner criteria for schizophrenia

Table 2.8 Schneiderian criteria for schizophrenia

Table 2.9 Research diagnostic criteria (RDC) for schizophrenia

Source: Spitzer, R.L., Endicott, J. and Robins, E. (1975) Research Diagnostic Criteria (RDC) for a Selected Group of Functional Disorders (2nd edn). New York: New York State Psychiatric Institute.

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Table 2.10 Concordance (expressed as Kappa) for schizophrenia among five diagnostic systems

In our study, the nature of diagnostic mismatch between clinical and system diagnosis was also interesting. There was a trend towards a higher rate of diagnostic disagreement for women. The subjects not meeting criteria for schizophrenia despite a clinical diagnosis of this illness tended to have affective disorders. Contamination of a schizophrenic cohort with affective patients can have major implications for neurobiological findings. Biological abnormalities associated with affective dysfunction may differ significantly from those associated with schizophrenia. Together, these factors clearly show that diagnostic variability between studies could contribute to misleading and non-reproducible findings in neurobiological studies using brain tissue obtained postmortem (Keks et al., 1995; McGuffin et al., 1984; Hirschowitz et al., 1986). It is notable that the concordance between DSM-III-R and ICD-10 criteria was only moderate (kappa=0.59). One key difference, for example, concerns the duration of illness requirement: a minimum of 1 month for ICD-10, but at least 6 months for DSM-III-R and DSM-IV (Hiller et al., 1994). Differences reported in the American and European psychiatric literature may, in part, derive from the differences in clincial populations characterised by, respectively, DSMIV and ICD-10 criteria for particular disorders. It has been suggested that differences between diagnostic systems have little effect on the assignment of a diagnosis of schizophrenia in postmortem samples (Benes, 1988). It is indeed likely that the effect will be less marked than that seen in other contexts, such as the acute psychosis study of Brochington et al. (1978). Nevertheless our study clearly demonstrates that the selection of particular diagnostic criteria do matter in defining patient samples (Hill et al., 1996), thereby influencing the nature of the findings (e.g. neuroendocrine responses to pharmacological challenge (Keks et al., 1990), which are reported in various studies.

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In the absence of any valid reason for accepting one diagnostic system of schizophrenia over another, and while different systems are being used in different studies, it is necessary to use a multi-diagnostic strategy in neurobiological research (Kendell, 1982). The multi-diagnostic evaluation poses some novel methodological challenges, particularly because attempts at comparing samples are confounded by lack of sample independence (McGuffin et al., 1984; Keks et al., 1992). However, at a basic level the ability to approach diagnostic classification of schizophrenia flexibly has significant advantages for neurobiological research, particularly for avoiding false negative results and enhancing comparability between studies. However, such an approach requires the availability of a large number of tissue samples to ensure that there is a significant number of individuals with the required diagnosis using each diagnostic criteria. Beyond schizophrenia: Subsyndromes and symptoms There are strong arguments to the effect that the concept of schizophrenia is invalid (Bentall et al., 1988) due to poor descriptive, predictive and construct validity. Crow has picked up Griesinger’s pre-Kraepelinian notion of unitary psychosis to suggest the existence of a continuum extending from unipolar, to bipolar, to schizoaffective psychosis, to chronic schizophrenia, with increasing degrees of deficit (Crow, 1986, 1987). At the sub-syndromal level, the concepts of positive syndrome and negative syndrome have gained strong support (Andreasen & Olsen, 1982). There is now a strong likelihood that the positive subsyndrome should be further divided into two groups, one delusions and hallucinations and the other thought disorder and disorganisation (Minas et al., 1992) (Table 2.11). Finally, depression and elevation of mood frequently occur in schizophrenia, and may be associated with significant neurobiological correlates (Meltzer, 1987). Table 2.11 Postulated subsyndromes in schizophrenia

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There are major uncertainties as to whether to regard the problem in terms of state (e.g. time of death) or trait (e.g. lifetime diagnosis). Thus, the question is whether research should be commenced with schizophrenia as the starting point? Given competing definitions and doubts about validity, it may be more appropriate to study all patients with idiopathic psychoses, and subsequently to proceed with sub-categorization. Thus, it has been argued that a purely symptomatic approach could be used in neurobiological studies in which, for example, results from patients who were or were not hallucinating could be compared (Bentall et al., 1988). Investigators in postmortem neurochemistry should seriously consider adding a subsyndrome and symptom level of psychopathological evaluation in studies. Currently such approaches appear to be rarely undertaken. Another vital consideration demonstrated the utility of subsyndromal and symptom-level approaches in neuroendocrine studies (Keks et al., 1995). Prolactin response to a single low dose challenge with haloperidol was found to be most impaired in a subgroup of psychotic patients characterised by both the presence of thought disorder and the absence of mood disorder.

Diagnostic Instrument for Brain Studies (DIBS) Bearing in mind the above considerations, we have developed a novel semistructured instrument to carry out psychopathological evaluation postmortem using documentation and informants where available. Although versions for other disorders are intended, the first is intended for patients with idiopathic (i.e. not due to substances or medical conditions) psychoses. The aim of the DIBS was to formulate a standardised procedure for postmortem psychopathological assessment which enabled multidiagnostic, subsyndrome and symptom analysis while enhancing reliability over clinical diagnosis. The Diagnostic Instrument for Brain Studies (DIBS)1 has five modules. The first focuses on history, background and physical details. The second module collects data on substance abuse in a form which is intended to allow diagnoses to be made on DSM-IV and ICD-10 criteria. The third and fourth modules of the DIBS concern psychotic and affective symptomatology respectively. The items reflect the actual requirements of different diagnostic systems and subsyndromes, and thus seek highly specific information. Finally, the 1

The Diagnostic Instrument for Brain Studies can be obtained from The Mental Health Research Institute of Victoria, Locked Bag 11, Parkville, Victoria 3052, Australia.

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data gathered in the first four modules enable diagnostic classification for schizophrenia on DSM-IV, DSM-III-R, ICD-10, RDC, Schneider and Feighner criteria. Other psychoses and mood disorders are also diagnosed on DSM-IV and ICD-10 criteria (Figure 2.1). The DIBS is accompanied by a glossary which defines symptoms consistent with the needs of the different diagnostic systems, since significant differences are present. Instructions for evaluation are also given, but clinical judgement remains a critical variable in the interpretation of documentation and secondhand accounts. A key issue currently under examination is whether the process can yield any degree of reliability between different clinical raters. As with the DEAD, the variable quality of source data requires notation of diagnostic uncertainty. The rater can specify clinical level diagnosis, probable diagnosis on criteria and definite diagnosis on criteria. Conflicting data (a not uncommon occurrence) are duly recorded for a judgement to be made. In contrast to the DEAD, all symptoms are rated as either present in the three months before death, or ever present. The three-month time frame was chosen because, even though it might appear preferable to record the mental state examination immediately prior to death (e.g. within 24 hours), experience casts doubt on the validity and reliability of such assessments. Furthermore, such assessments are often not present in suicides or not recorded as a specific mental state in deaths caused by physical illness. Three months prior to death was judged by the authors to be a biologically more relevant period than the six months used in the DEAD, as state abnormalities are being sought. Clinical states are usually defined over a three-month period while six months can encompass a significant portion of illness course, as suggested by the DSM-III/IV duration criterion (American Psychiatric Association, 1980, 1987, 1994). The raters also make subsyndrome diagnoses on specified criteria for depression, mania, positive symptoms, negative symptoms and formal thought disorder (Figure 2.2). Symptom-level evaluation can be carried out in addition as required.

CONCLUSION While the neuroscientific methodology of postmortem brain studies into schizophrenia has received intensive scrutiny (Casanova & Kleinman, 1990), psychopathological evaluation needs considerably more attention. The authors recommended that in view of uncertainties concerning the boundaries, subcategorization and overall validity of schizophrenia, researchers address

Figure 2.1 DIBS diagnostic summary

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Figure 2.2 DIBS sub-syndromes

all idiopathic psychoses as the index group. Rather than a rigid adherence to a single concept of schizophrenia, multidiagnostic, subsyndromal and symptomlevel hypotheses can be pursued. False positive and negative findings may thus be minimised and reproducibility of findings should be improved. In order to improve reliability of psychopathological evaluation, standardised instruments such as the DEAD and DIBS should be utilised.

References American Psychiatric Association (1980) Diagnostic and Statistical Manual of Mental Disorders (3rd edn). Washington D.C.: American Psychiatric Association. American Psychiatric Association (1987) Diagnostic and Statistical Manual of Mental Disorders (3rd edn-rev.). Washington D.C.: American Psychiatric Association. American Psychiatric Association (1994) Diagnostic and Statistical Manual of Mental Disorders (4th edn). Washington, D.C.: American Psychiatric Association. Andreason, N.C. and Olsen, S. (1982) Negative versus positive schizophrenia: definition and validation. Arch. Gen. Psychiatry, 39, 789–794. Benes, F.M. (1988) Post-mortem structural analyses of schizophrenic brain: study designs and the interpretation of data. Psychiatr Dev, 3, 213–226. Bentall, R.P, Jackson, H.F. and Pilgrim, D. (1988) Abandoning the concept of schizophrenia: some implications for validity arguments for psychological research into psychotic phenomena. Br. J. Clin. Psychology, 27, 303–324. Brockington, I.F., Kendell, R.E. and Leff, J.P. (1978) Definitions of schizophrenia: concordance and prediction of outcome. Psychol. Med., 8, 387–398.

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Casanova, M.F., and Kleinman, J.K. (1990) The neuropathology of schizophrenia: a critical assessment of research methodologies. Biol. Psychiatry, 27, 353–362. Crow, T.J. (1986) The continuum of psychosis and its implications for the structure of the gene. Br. J. Psychiatry, 149, 419–429. Crow, T.J. (1987) Psychosis as a continuum and the virogene concept. Brit. Med. Bull, 43, 754–767. Feighner, J.P., Robins, E., Guze, S.B., Woodruff, R.A. Jr, Winokur, G. and Munoz, R. (1972) Diagnostic criteria for use in psychiatric research. Arch. Gen. Psychiatry, 26, 57–63. Fenton, W.S., Mosher, L.R. and Matthews, S.M. (1981) Diagnosis of schizophrenia: a critical review of current diagnostic systems. Schizophr. Bull, 7, 452–476. Hill, C., Keks, N., Roberts, S., Opeskin, K., Dean, B. and Copolov, D.L. (1996) Postmortem brain studies in schizophrenia: The problems of diagnosis. Am. J. Psych., 153, 533–537. Hiller, W., Dichtl, G., Hecht, H., Hundt, W. and vonZerssen, D. (1994) Testing the comparability of psychiatric diagnoses in ICD-10 and DSM-IIIR. Psychopathology, 27, 19–28. Hirschowitz, J., Zemlam, F.P., Hitzemann, R.J., Fleischmann, R.L., and Garver, D.L. (1986) Growth hormone response to apomorphine and diagnosis: a comparison of three diagnostic systems. Biol. Psychiatry, 21, 445–454. Karson, C.N., Casanova, M.F., Kleinman, J.E. and Griffin, W.S.T. (1993) Choline acetyltransferase in schizophrenia. Am. J. Psychiatry, 150, 454–459. Keks, N.A., Copolov, D.L., Kulkarni, J., Mackie, B., Singh, B.S., McGorry, P.D., Rubin, R.T., Hassett, A., McLaughlin, M.. and van Riel, R. (1990) Basal and haloperidolstimulated prolactin in neuroleptic-free men with schizophrenia defined by 11 diagnostic systems. Biol. Psychiatry, 27:1203–1215. Keks, N.A., Mckenzie, D., van Riel, R., Low, L., McGorry, P., Hill, C., Singh, B.C. and Copolov, D.L. (1992) Multidiagnostic evaluation of prolactin response to haloperidol challenge in schizophrenia: maximal blunting in Kraepelinian patients. Biol. Psychiatry, 32, 426–437. Keks, N., Hill, C., Dean, B., Opeskin, K. and Copolov, D. (1995a) Diagnosis as a confounding variable for post-mortem brain studies in schizophrenia. Schizophrenia Research, 15, 29. Keks, N.A., Copolov, D.L., McKenzie, D.P., Kulkarni, J., Hill C.,Hope, J.D. and Singh, B.S. (1995b) Basal and haloperidol-stimulated prolactin and symptoms of nonaffective psychoses in neuroleptic free men. Biol. Psychiatry, 37, 229–234. Kendell, R.E. (1982) The choice of diagnostic criteria for biological research. Arch. Gen. Psychiatry, 39, 1334–1339. Meltzer, H.Y. (1987) Psychopharmacology: The Third Generation of Progress. New York: Raven Press. McGuffin, P., Farmer, A.E., Gottesman, I.I., Murray, R.M. and Reveley, A.M. (1984) Twin concordance for operationally defined schizophrenia: confirmation of familiarity and heritability. Arch. Gen. Psychiatry, 1, 541–545. Minas, I.H., Stuart, G.W., Klimidis, S., Jackson, H., Singh B. and Copolov, D. (1992) Positive and negative symptoms in the psychoses, multidimensional scaling of SAPS and SANS items. Schizophrenia Research, 8, 143–156.

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Schneider, K. (1959) Clinical Psychopathohgy. Translated by Hamilton, M.W. London: Grune & Stratton. Spitzer, R.L., Endicott, J. and Robins, E. (1975) Research Diagnostic Criteria (RDC) for a Selected Group of Functional Disorders (2nd edn). New York: New York State Psychiatric Institute. World Health Organization (1993) The ICD-10 Classification of Mental and Behavioural Disorders: Diagnostic Criteria for Research. Geneva: World Health Organization. Zalcman, S., Endicott, J., Clayton, P. and Winokur, G. (1983) Diagnostic Evaluation After Death (DEAD). Rockville: National Institute of Mental Health.

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MEMBRANE BINDING ASSAYS: MEMBRANE PREPARATION AND ASSAY DEVELOPMENT Yogesh Dwivedi and Ghanshyam N.Pandey

In the last two decades the use of human postmortem brain tissue in neurochemical and neuropharmacological research has received wide attention. Such studies have implicated serotonergic (Stanley & Mann, 1983; Arora & Meltzer, 1986), glutamatergic (Conley & Farber, 1995), dopaminergic (Crawley et al., 1986; Parade et al., 1990), GABAergic (Benes et al., 1996), cholinergic (Leonard et al., 1996), adrenergic (Mann et al., 1986; Meana et al., 1992; Arangoetal., 1993; Ordwayetal., 1994), and benzodiazepine (Kiuchi et al., 1989) receptors in the pathophysiology of mental disorders. Prior to the widespread availability of postmortem brain tissue most studies of patients with mental disorders were based on measurement of neurotransmitters or their metabolites in cerebrospinal fluid (e.g. serotonin or its metabolite 5-hydroxyindoleacetic acid (5HIAA) (Asberg et al., 1976)), urine or plasma (e.g. norepinephrine or its metabolite 3-methoxy-4hydroxyphenylglycol (MHPG) (Agren et al., 1992; Roy et al., 1986)). Other strategies such as neuroendocrinological studies were also employed to delineate the abnormalities associated with mental disorders (e.g. growth hormone secretion in response to clonidine (Checkley et al., 1981)). Direct study of abnormalities in neurotransmitter receptors became possible only after the development of receptor binding assay techniques. This powerful technique uses radioactive ligands to study ligand-receptor interactions and hence the density of receptors and the affinity of drug binding. Earlier, studies of receptors and their involvement in the pathophysiology of mental disorders utilised peripheral tissues such as platelets, leukocytes, and lymphocytes. These studies were carried out on the premise that peripheral receptors would provide information relevant to the receptors in the brain. However, the increased accessibility of human postmortem brain samples and the use of receptor binding 39

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assays has allowed the direct assessment of abnormalities in receptors that may be associated with mental disorders. In this chapter we will describe briefly the techniques used to determine the affinity and number of receptors and quantification of receptor proteins in postmortem brain samples.

LIMITATIONS OF HUMAN POSTMORTEM BRAIN STUDIES Certain limitations exist in the study of human postmortem brain samples, and one must design experiments and analyze data carefully, particularly matching specific variables in the affected and unaffected subjects. Some important factors to consider are: (a) (b) (c) (d) (e) (f) (g) (h)

age postmortem interval tissue storage time tissue volume changes agonal state (the clinical state of patients immediately prior to dying) ethanol or drug toxicity psychologic and neurologic history cause of death

It is important that postmortem tissue from control subjects and from diseased subjects is collected under identical conditions. Control subjects need to be free from diseases associated with the central nervous system and from major medical conditions such as cardiac, pulmonary, or renal disease to be considered as suitable controls. Age and sex are two factors that can be easily matched. Age affects some neurotransmitter receptors as well as components of signal transduction systems (e.g. protein kinase C (PKC) and guanine trisphosphate (GTP) binding protein have been shown to be decreased during aging (Greenwood et al., 1995)). In a preliminary study, we observed that the number of receptors for PKC is higher in the postmortem brain of younger subjects than in older subjects. It has also been reported that there is a loss of specific pre-synaptic γ-aminobutyric acid (GABA) function in the cortex and of cholinergic markers in the striatum of older subjects (Allen et al., 1993). No differences have been reported in neurotransmitter receptors in males versus females, and there are no reports of ethnic differences in functional responsiveness of receptors. The agonal state

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and the cause of death also influence neurotransmitter receptors. Recently, we observed that benzodiazepine receptors are significantly increased in the postmortem brain of suicide victims, and that this increase is more profound in subjects who died by violent means (Pandey et al., 1997). Antipsychotic drugs, stimulants, antidepressants, and anxiolytics taken prior to death can also affect various neurochemical parameters in animals and have been shown to induce neurochemical changes in human brain tissue. For example, it has been reported that the density of dopamine-D2 like receptors is increased in schizophrenic subjects on antipsychotic drugs at death compared with those who had stopped taking medication a month or more before death (Iverson & Mackay, 1981). We have shown that alcohol affects a variety of neurotransmitter receptors in the rat brain (Pandey et al., 1993; Negro et al., 1995) and hence data from subjects who have a known history of alcohol abuse should either be eliminated from study or should be matched with subjects with no alcohol history to determine whether the variable is mediated by alcohol. Thus, before analysis of the data is attempted, it is important that the medical and pharmacological history of tissue donors should be carefully examined. Storage and postmortem delay are important variables that can influence data interpretation. Many neurochemicals are stable for several hours if the brain has not been removed from the skull and the corpse has been stored at 4 °C. Drugreceptor binding sites are quite stable in the postmortem brain and can be assessed by radioligand binding procedures. Most pertinent postmortem changes take place within 12 hours after death. After that, further changes tend to be much slower and approach a stable plateau level. In the disease state, postmortem delay is an important variable in considering neurotransmitter changes. Correlation between neurotransmitter receptor changes and the length of postmortem delay should be determined by studying neurotransmitter receptors in rats or mice at various postmortem intervals. As an example, cooling curves for brain have been established by monitoring brain temperature after death in mice (Spokes & Koch, 1978). One must also consider the differences between left and right brain areas. Although we have not found any variations in neurotransmitter receptors between left and right areas of postmortem human brain in 5HT2A receptors or PKC, there is a report showing that norepinephrine (NE) is asymmetrically distributed between the right and the left hemisphere of thalamic nuclei (Rosser et al., 1980). CRITERIA FOR RECEPTOR SITES Before describing radioreceptor ligand binding assaying techniques, there is a

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need to consider certain criteria that must be met to identify the desired receptor site. Criteria for receptor sites can be defined on the basis of these properties: (a) (b) (c) (d) (e) (f)

regional distribution stereospecificity saturability appropriate affinity ligand specificity correlation with biological response

The regional distribution of neurotransmitter receptors is a very important consideration, since some receptors are localized in certain parts of the brain whereas other areas of the brain lack these receptors. (We will further discuss regional distribution in the Receptor Binding Assay Techniques section.) Stereospecificity is important because the stereoisomers of a drug bind to receptors with different affinities and the degree of stereospecificity differs from one receptor to another and from one drug to another. Thus, the stereospecificity of the binding site should be the same as that of the biologically active receptors. Saturability is another criteria for binding assay studies. Specific binding is saturable whereas nonspecific binding is non-saturable. The affinity of a receptor for an endogenous ligand should be appropriate to the physiological concentration of the ligand. It has been shown that high affinity receptors exhibit a signal at low concentrations of the ligand, whereas low affinity receptors require higher concentrations of the ligand. Also, the receptors should have specificity for a given class of ligands, and agonists and antagonists of the same class should compete for their receptors in rank order of potency, corresponding to their biological responses as measured by clinical, behavioral, and biochemical studies. If a number of drugs with varying potencies correlate well with radiolabeled sites affinity, then this is a proper receptor to study. RECEPTOR BINDING ASSAYING TECHNIQUES Radioligand binding is a simple technique; however, designing an experiment requires an understanding of certain fundamental techniques of receptor binding assays. We will briefly discuss the theoretical basis of receptor binding assays; however, our main emphasis will be receptor binding assay techniques and membrane preparation development. In a receptor binding assay, radioactive ligands are used to selectively label the receptor, and the amount of drug (D) bound to the receptor (R) in the presence of the free concentration of the drug is

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then measured. The specific binding, i.e. the radioactive drug bound to the receptor, is separated from the nonspecific binding (the radioligand bound to components other than the receptors), and on this basis, the association and dissociation constants (the rates at which ligands bind or dissociate), the affinity of receptors for a particular ligand (KD), and the number of receptors (Bmax) can be calculated. D+R Free

D.R Bound

Before starting a receptor binding assay in tissue homogenate, it is imperative to localize the neurotransmitters in various brain regions. Certain receptors are abundant only in specific brain regions, whereas other regions contain only sparse amounts of those particular receptors, β-adrenergic receptors are present abundantly in the cerebral cortex, the cerebellum, the striatum, and the limbic forebrain; however, when β-adrenergic receptors are subdivided into the 1 and the β2 subtypes, their distribution is found to differ. β1 is relatively more predominant than β2 in the cerebral cortex and the striatum (65% versus 35%, respectively). In the cerebellum, β1 is absent, whereas in the limbic forebrain, 55% of receptors are β1 and 45% are β2 (Rugg et al., 1978). Dopamine receptors are very dense in the striatum, whereas they are poorly represented in the cerebellum. Serotonin receptors are mainly present on neurons, including those in the raphe area, as well as the striatum, the hippocampus, the hypothalamus, the septum, the amygdala, and the cerebral cortex (reviewed by Hoyer et al., 1994). It has also been reported that the binding of [3H] lysergic acid diethylamide (LSD) to 5HT2 receptors in the cerebral cortex of the monkey was about oneeighth that of binding in the frontal cortex (Snyder & Bennett, 1975). Similarly, differences in 5HT2 receptors exist between the cerebral cortex and the cerebellum in the rat brain. Relatively higher [3H]LSD binding has been observed in the cerebral cortex, the hippocampus, the caudate, and the putamen of the monkey, whereas diencephalic areas showed somewhat less binding. Other serotonergic receptors, i.e. 5HT2C, are present not only in the cortex and the hippocampus but also in the choroid plexus in high concentrations, where no other serotonin receptors exist. The regional distribution of endogenous levels of neurotransmitters and putative receptor binding sites are important study factors. It has been shown that endogenous serotonin levels do not correlate with the density of postsynaptic receptors (Snyder & Bennett, 1995). Similarly, receptor binding for GABAA or α2 adrenergic receptors (Weiss & Costa, 1968; Chasin et al., 1971)

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does not correlate with the level of endogenous neurotransmitters and their respective receptor binding sites but the number of dopamine receptors tends to parallel the dopamine concentration in the brain. Receptor binding assays In designing a radioligand binding assay five components must be considered: 1. 2. 3. 4. 5.

choice of radiolabeled ligand tissue/membrane preparation incubation conditions separation of bound and free ligands calculation of data

The concentration of most receptors in a given tissue ranges from femtomolar to picomolar. Considering these low concentrations, the choice of a radioligand is the most important factor in a receptor binding study. The ligand should be such that the ligand-receptor dissociation constant lies within the range of the receptor concentration. It should be stable under assay conditions, i.e. free of enzymatic degradation. High selectivity is another criteria for choosing a radioligand. It would be best if the radioligand only bound to one type of receptor; however, if the radioligand also binds to other receptors and if no other appropriate ligand is available, then it can be used after irreversibly blocking any other receptors to which it binds. We have used this technique to label 5HT2C receptors in the cortex and the hippocampus using [3H]mesularzine in the presence of 100 µM mianserin. Similarly, 5HT1B can be labeled with [125I]cyanopindolol in the presence of adequate concentrations of drugs that block β-adrenergic receptors, such as 30 µM isoproterenol. To label 5HT1A receptors, we employ [3H]OH DPAT as the ligand in the presence of serotonin (10 µM), which blocks 5HT2A receptor sites. In most cases, the affinity of the radioligand to the receptor ranges from 10–7 to –11 10 M. At low concentrations the ligand has a low ratio of specific to nonspecific binding whereas at higher concentrations ligand binding kinetics is slow. Another criterion for choosing a radioligand is specific activity. Compounds with a higher specific activity require a smaller amount of the radioligand, which decreases the binding to nonspecific sites. Several radioisotopes are available, for example, 14C, 3H, 32P and 125I; however, for binding assays, the choice of ligands ranges among 14C, 3H, and 125I. Due to its low specific activity, 14C (50–500 mCi/ mole) is not usually used for this purpose. The most commonly used radioisotope is tritium [3H]. Tritiation of a drug does not change the structure of the compound;

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however, there is a disadvantage: tritium-labeled compounds tend to degrade radiochemically and to produce a spectrum of radioactive byproducts. If possible, iodinated compounds are the best choice because they possess a higher specific activity (2200 Ci/mmol) and can be introduced easily into phenolic and indole rings. Since iodination requires the presence of an aromatic hydroxyl group in the molecule, however, the introduction of such a moiety into the ligand may change the biological activity of the compound. Iodinated compounds have another disadvantage, that of having a shorter half-life. Tritiumlabeled compounds are more active biologically compared to iodinated ligands. Thus, the biological activity of iodinated compounds needs to be routinely checked. The stability of radiolabeled components should also be routinely assessed because some ligands, such as [3H]5HT, are quite unstable. Another criterion for selecting a specific radioligand is its hydrophilicity. The radioligand should be hydrophilic in nature because most receptors are localized in the membranes and the hydrophobicity of the ligand may increase nonspecific binding. Both agonists and antagonists can be used as ligands. A good example is the use of agonists or antagonists to label α2 adrenergic receptors in the postmortem brain of suicide victims and in platelets of depressed patients. Most studies show no change in Bmax of α2 adrenergic receptors after antagonist binding (Pelletz & Halaris, 1988; Pandey et al., 1989; Ordway et al., 1994), whereas agonist binding does demonstrate an increase in Bmax of α2 adrenergic receptors in depressed patients (Daiguji et al., 1981; Campbell et al., 1985; Ordway et al., 1994). Membrane preparation Membrane preparation is one of the most crucial steps in the receptor binding assay, particularly in the use of human postmortem brain tissue. Tissue should be devoid of blood cells, since blood cells can interfere with the binding assay and may produce false results, particularly when cytosolic fractions are utilized to measure receptor number. Another step in membrane preparation is the separation of white matter from gray matter. It has been shown that [3H]LSD binding is 10 times higher in areas enriched with gray matter than in white matter areas (Bennett & Aghajanian, 1974). We also have observed that inclusion of white matter with gray matter has decreased the specific binding of other receptors, such as inositol 1,4,5-trisphosphate and protein kinase C, in the postmortem human brain. Membrane preparation techniques are similar for most receptors; however, preparations may vary depending upon the receptor subtype. Most receptor binding assays have been performed in crude participate membrane preparations.

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PREPARATION OF CRUDE PARTICULATE MEMBRANE 1. Homogenized tissue in 10 volumes of ice-cold 0.32 M sucrose in a glass homogenizer using 10–20 strokes. 2. Centrifuge homogenate at 1,000×g for 10 minutes at 4 °C to separate white, gray matter and nuclear fraction. 3. Collect supernatant. 4. Centrifuge supernatant at 40,000×g for 15 minutes. 5. Homogenize the pellet in a suitable washing buffer using the Polytron homogenizer. 6. Centrifuged at 48,000×g for 15 minutes. 7. Repeat 5 and 6 and suspend in the incubation buffer. Note: These washing steps usually remove any residual drug that has bound to the membrane and any endogenous substance that may interfere with the binding.

In some receptor binding assays, certain other steps are required to ensure proper binding. For example [3H]muscimol binding to GABAA receptors requires freezing at –20 °C after homogenization and then further processing. This enables the release of endogenous GABA, which may interfere with [3H]muscimol binding to GABAA receptors (Enna & Snyder, 1975). With certain other receptors, for example, 5HT2C receptors, we pre-incubate the tissue homogenate at 37°C for 10 minutes to destroy the endogenous serotonin (Pandey et al., 1993). Some receptor binding assays require freshly prepared membranes; however, in the case of a few receptors, the homogenate can be stored at –70°C for some time with only a small loss of binding capacity (Glasel et al., 1980). Certain receptors, such as PKC, are present in both membranal and cytosolic fractions and therefore binding to both membranal and cytosolic PKC should be measured. PREPARATION OF MEMBRANAL AND CYTOSOLIC FRACTION 1. Homogenize tissue into a suitable buffer. 2. Centrifuge at 1,000×g for 10 minutes. 3. Collect supernatant. 4. Centrifuge at 100,000×g for 60 minutes. 5. Collect supernatant. 6. Suspend pellet in buffer. 7. Repeated 2 and 3. 8. Collect supernatant. 9. Combine supematants (cytosolic fraction). 10. Collect pellet (membranal fraction) and suspend in a volume of incubation buffer that is dependent on the size of the pellet and on the protein concentration required to perform the binding assay.

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Note: Before processing the binding assay, a protein concentration curve must be determined to ensure that the specific binding is linear. For example, we use approximately 100 µg of protein per assay for [125I]LSD binding to 5HT2A receptors, but 20 to 50 µg of protein are enough to provide saturable binding for protein kinase C. Under some circumstances, a purified synaptic plasma membrane preparation can be used to measure receptors, for example, steroid receptors have been measured with this technique.

PREPARATION OF SYNAPTIC PLASMA MEMBRANE 1. Homogenize tissue into 10 volumes of isotonic sucrose (0.32 M) containing 10 mM CaCI2 at 4 °C. 2. Centrifuge at 1000×g for 10 minutes. 3. Collect supernatant. 4. Centrifuge at 12,000×g for 20 minutes. 5. Collect the pellet and suspend in 5 mM Tris MCI, pH=8.6. by homogenizing with a Polytron homogenizer. 6. Centrifuged at 40,000×g for 20 minutes. 7. Suspended pellet in distilled water and centrifuged at 48,000×g for 20 minutes. 8. Collect pellet and suspend in 5 mM Tris (pH=8.6) containing 1.2 M sucrose. 9. Overlay suspension into centrifuge tubes with layers of 0.9 and 0.3 M sucrose. 10. Centrifuge at 82,000×g for 2 hours. 11. The interface between 1.2 and 0.9 M sucrose is used as purified synaptic plasma membrane (Peck & Kelner, 1982).

Incubation conditions The conditions for incubation are determined by many factors, including the nature and the pH of the buffer, the assay temperature, the time of incubation, and the presence of mono- or divalent cations. Tris HCl is the most commonly used buffer for binding assays. For example, 50 mM Tris HCl buffer has been used in binding assays to determine 5HT receptor subtypes (5HT1A, 5HT2A, 5HT2C, and 5HT3) (Gozlan et al., 1983; Pazos et al., 1984; Bruinvels et al., 1991). HEPES buffer (10 mM) has been used for receptors such as 5HT4 (Grossman et al., 1993). Certain other buffers, such as citrate and Tris-acetate, have also been utilized for binding assays. Ideally, the pH of the buffer for a binding assay ranges between 7.4 to 7.7, but other pH may be better for different types of receptors. For example, maximal opiate receptor binding occurs at pH 8.0. Higher pH causes deportation of the opiate, whereas lower pH causes protonation of some moieties on the receptor. Both deportation and protonation decrease the binding of the ligand to the opiate receptors (Squires & Braestrup, 1978). [3H]Naloxone binding shows pH

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dependence, with maximum binding at pH=8.0. At pH=7.5, binding drops to 85%, at pH=7.0, binding is 65%, and at pH=6.5, binding drops to 25% (Squires & Braestrup, 1978). We have observed similar properties in [3H]inositol 1,4,5trisphosphate binding to inositol 1,4,5-trisphosphate (IP3) receptors, and found that the optimum pH for IP3 receptor binding is 8.4. At acidic pH (4.0 to 6.0), the specific binding is relatively low; it increases to 50% as pH approaches 7.0, remains stable between pH 7.0 to 8.0, and then increases further between pH 8.0 to 8.4. After that, the specific binding to IP3 receptors again decreases. Temperature affects both the receptor number and the dissociation constant. For example, in the erythrocytic system it has been shown that the number of β adrenergic receptors is decreased with increasing temperature. At 15 ° to 40 °C, 40% of the β receptors become increasingly undetectable, perhaps because of the embedd ing of receptors in the lipid bilayer as the fluidity of the membrane increases with temperature; however, this effect is reversible upon cooling (Rimon et al., 1980). Binding assays are usually carried out at temperatures such as 4 °C, 20 ° to 30 °C, or at 37 °C. Most receptor binding for serotonin receptor (5HT1A 2A, 2C, 4) assays is conducted at 25 °C, however, for IP3 receptor binding assays, we use a temperature of 4 °C. Certain receptors are more sensitive to temperature, for example, low-affinity [3H]naltrexone binding to opiate receptors is not detectable at temperatures greater than 20 °C (Squires & Braestrup, 1978); however, high-affinity sites slow down maximum binding, which is detectable between 10 ° to 30 °C. The ionic concentrations of the buffer greatly affect receptor binding assays. High concentrations of monovalent cations (Na+ or K+, 20 mM) cause significant reductions in [3H]LSD or [3H]5HT binding to brain 5HT2A receptors in membranes (Bennett & Snyder, 1975, 1976; Mallat & Hamon, 1982). Divalent cations such as Ca+2, Mn2+, and Mg2+ increase the specificity of [3H] 5HT binding to brain membranes (Bennett & Snyder, 1976; Mallat & Hamon, 1982); however, these ions reduce [3H]LSD and [3H]spiperone binding. We observed that nanomolar concentrations of Ca2+ inhibit the binding of [3H]IP3 receptors, whereas increasing the concentration of Ca2+ to the miilimolar range stimulates binding. Ca2+ also increases Bmax of 5HT2A sites (Bennett & Snyder, 1976), due to enhanced viscosity of brain membranes. The time of incubation is also a crucial variable since it affects the ability of a binding reaction to reach equilibrium. With most neurotransmitter receptors, equilibrium is reached within 60 minutes; however, certain receptors require a longer period of time, for example, 5HT 1B receptors as measured by [125I]cyanopindolol require 90 minutes of incubation time. Thus, it is necessary to establish association and dissociation constants using different incubation

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time intervals. For the IP3 receptor, we found that specific binding increases from 15 seconds to 5 minutes and remains stable up to 2 hours. The concentration of the radioligand normally should be at or below the KD of the ligand for the receptor. Usually, a selection of five or six different concentrations of the ligand below and above the KD value are needed to obtain an accurate Bmax. For example, the concentration of the ligand required for [125I]LSD binding to 5HT2A receptors in the brain is in the range of 0.25 to 3.0 nM (Pandey et al., 1995), whereas for protein kinase C, the appropriate [3H] phorbol 12, 13-dibutyrate (PDBu) ranges are between 1.5 to 30 nM (Pandey et al. 1997). [3H]Muscimol (0.5 to 50 nM) (Negro et al., 1995), [3H]GABA (10 to 40 nM) (Corda et al., 1986), and [3H]nipecotic acid (1 nM to 2 nM) (Sundman et al., 1997) have been used to measure GABAA receptors and GABA uptake sites, respectively. In general, if the affinity is very high for a given ligand (above 10–9 M), then a very low concentration of the ligand should be used. This minimizes nonspecific binding. On the other hand, if the affinity is low, then the concentration of ligand should be higher; however, in this case, nonspecific binding may also be very high. The concentration of the displacing compound usually needs to be 100 times greater than the KD values, which will provide enough displacing agent to block all of the specific binding sites. The concentrations of the ligand and the displacing agent are determined by calculating the amount of specific binding displaced by increasing concentrations of the displacing agent. In the radioligand binding assay, the concentration of the displacing agent is fixed, whereas the concentration of the radioligand is increased so that the binding of the radioligand is able to reach its plateau. Separation of free ligands from ligands bound to tissue Free ligands are separated from receptor-bound ligands using centrifugation or filtration. The centrifugation process is primarily used when KD values range from 100 to 1000 mM. In this protocol, the reaction is terminated by rapid centrifugation of the tubes at 15,000×g for 0.5 to 5 minutes. This speed is high enough to separate the tissue from the supernatant. During the centrifugation process, the tissue is constantly exposed to the radioligand and thus there is less chance of losing the bound radioactivity. After centrifugation, the supernatant is decanted and the membrane pellet is washed once with cold buffer. This method is advantageous in that separation and washing procedures occur under equilibrium conditions and it provides a true estimate of nonspecific binding. This method also has certain disadvantages as it cannot be used when:

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1. 2. 3.

assays incubation times are short; a high or saturating concentration of radioligand ligand is present; nonspecific binding is high.

Filtration is the most commonly used method to separate receptor-bound ligand from the free ligand. It is very rapid and provides reproducible results. In this procedure, the assay mixture is passed through a filter under vacuum conditions so that the ligand bound to the receptor is retained on the filter, passing the free ligand into a disposable tank. This is followed by washing 2–3 times with icecold buffer. The total procedure takes less than 30 seconds. This procedure also has certain limitations: 1.

2.

3.

4.

5.

It can only be used with ligands with a slow dissociation rate. Ligands with a high dissociation rate will give low values because some bound counts are removed during washing. Retention of small membrane fragments on the filter may be poor. For instance, GF/C filters are not adequate for some membrane suspensions. In our laboratory, we generally use the Brandel Cell Harvester (Brandel Inc.) with GF/B filter paper. Iodinated ligands can bind to the tubing in multi-tube filtration systems such as the Brandel Cell Harvester, however, the Teflon tubing can be used to prevent such binding. Non-specific binding can be high. The most common procedure for decreasing nonspecific binding is the use of 0.1% (w/v) bovine serum albumin (BSA) in the washing buffer. Another procedure is to soak the filter paper in 0.1 % polyethylenimine for a few minutes before passing the incubation mixture through the filter paper. For certain receptors localized in the cytosolic fraction (e.g. PKC), an additional procedure is required during filtration. In this method, solubilized receptors are precipitated by polyethylene glycol (12% w/v). The incubation mixture is diluted with 0.1% bovine γ-globulin and mixed, and then 200– 500 µl of polyethyleneglycol are added and left for 15 minutes at 4 °C. The incubation mixture is then passed through the filter, or it can be centrifuged.

MEASURING BOUND AND FREE RADIOACTIVITY In the filtration procedure, the filters are dried on aluminum foil and placed in plastic vials. We add 10 ml of BBS scintillation cocktail (3 liters toluene, 120 ml scintillation fluid, and 300 ml SAS solubilizing agent). Vials are kept at 4 °C

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for several hours, and then mixed and counted in the β-counter. For iodinated radioligands radioactivity can be measured in a γ-counter by placing the filters in plastic tubes. For calculation purposes, we take readings of the total counts in triplicate, using two different volumes, for example, 5 µl and 10 µd. We use non-refrigerated counters, which are approximately 55–60% efficient.

Calculation of binding data The basis of ligand receptor binding assaying is derived from the law of mass action. The simplest equation is: (1)

where D=radiolabeled drug; R=receptor. The concentration [D+R] is usually referred to as bound [B]. At equilibrium: (2)

where KD=equilibrium dissociation constant of D for R. K1 and K2 (dissociation and association rate constants, respectively) and KD are related to each other: (3)

The total number of receptors equals (Bmax) [R]+[D+R]: (4)

(5)

In a typical binding assay, the amount of receptor [D+R] is determined by adding varying concentrations of radioligands to a fixed amount of tissue until saturation of the receptor is achieved. Nonspecific binding is usually defined as the amount of radioligand bound to the receptor in the presence of the displacer. Usually, nonspecific binding is nonsaturable. The specific bound radioligand concentration is determined by subtracting the nonspecific binding from the total binding. The determination of Bmax and KD can be achieved by plotting the

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ratio of the bound/free (B/F) ligand against the concentration of the ligand [B]. This procedure produces a straight line and its interception at the bound axis provides Bmax, whereas the slope is the negative reciprocal of KD. This is detailed in Figure 3.1 for the determination of Bmax and KD for [3H]PDBu binding to membranal and cytosolic PKC in postmortem human brain. We usually analyze our binding data by Scatchard plot analysis (McPherson et al., 1985) using the EBDA program. By using Scatchard analysis, the total number of receptors can be determined without knowing the saturation concentration of the radioligand. If the result is a straight line whose slope is equal to the –1/KD in the Scatchard plot, this means that the receptors belong to a single class. The linearized form of the binding data can also be obtained using a Hill plot (log P/[I-P] versus log F, where P is the receptor occupancy). The Hill plot is generally employed when quantitation of the mode of saturation is required. Another way to visualize the data is to determine the percent inhibition of specific radioligand binding versus that of its competitor. However, if the radioligand binds to more than one type of receptor with different affinities, then the slope of the Scatchard plot changes and becomes concave upward. These possibilities can be elucidated by detailed kinetic analysis (Munson et al., 1980). For multiple binding sites, the Hill plot is more useful; however, it also has some limitations: it does not allow quantification of the sites, nor can it determine the affinity of the receptor for the radioligand. Competitive curve experiments are also useful in determining multiple binding sites. Details of the possibilities for determining multiple binding sites have been reviewed by Titeler (1989). A TYPICAL RADIORECEPTOR BINDING ASSAY To illustrate the points discussed a specific example of a radioreceptor assay is provided. This assay details the method to measure [3H]phorbol dibutyrate binding to PKC in cytosolic and membranal fractions of postmortem human brain tissue, and thus shows the procedure that allows the preparation of the fractions. DETERMINATION OF Bmax AND KD OF [3H]PHORBOL DIBUTYRATE (PDBu) BINDING TO POSTMORTEM HUMAN BRAIN Materials • • •

Glass homogenizer Polytron Centrifuges: Refrigerated centrifuge capable of operating at 100×g to 100,000×g

Figure 3.1 Saturation isotherm of [3H]PDBu binding to membranal (A) and cytosolic (B) PKC. Each point is the mean of triplicate determinations. Inset: Scatchard plot of the specific binding of [3H]PDBu. B=[3H]PDBu specifically bound (pmol/mg protein); B/F=bound over free [3H]PDBu (pmol/mg protein×nM). For this particular experiment, the binding indices in the membranal fraction were KD=3.4 nM; Bmax=9.9 pmol/mg protein, and the correlation coefficient (r)=0.99. For the cytosolic fraction (B), KD=0.8 nM, and Bmax=3.44 pmol/mg protein. The correlation coefficient (r)=0.98.

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• Brandel Cell Harvester (Brandel, Inc., 8561 Atlas Dr., Gaithersburg, MD 20877, USA) • GF/B filter (Brandel, Inc.) • Scintillation cocktail (can be obtained from Research Products Inc., 2692 Delta Lane, Elk Grove Village, IL 60007) (a) SAS Solubilizing agent (b) RPI Scintillation fluid (c) Toluene

Chemicals Tris HCI, polyethylene glycol, phenylmethylsulfonyl fluoride, phosphatidyl serine, bovine gamma globulin, phorbol-12-myristate 13-acetate, EDTA, EGTA, MnCI2, magnesium acetate, CaCI 2, bovine serum albumin. Radioligand: [3H]phorbol dibutyrate (New England Nuclear).

Methods Preparation of Membranal and Cytosolic Fractions An outline of the preparation of membranal and cytosolic fractions is given in Figure 3.2. 1. Postmortem brain tissue is homogenized in 0.32 M sucrose with 20 strokes using a glass homogenizer. 2. The homogenate is centrifuged at 1,000×g rpm for 10 minutes. 3. The supernatant is centrifuged at 40,000×g for 15 minutes. 4. The pellet is homogenized by Polytron at setting #8 for 15 seconds in 10 volumes of homogenizing buffer (50 mM Tris HCI; 2 mM ethylene glycol-bis [b-aminoethyl ether]-N,N,N’,N’-tetraacetic acid; 1 mM MnCI2; and 1 mM phenylmethylsulfonyl fluoride [PMSF]). 5. The homogenate is centrifuged at 100,000×g for 60 minutes at 4 °C. 6. This supernatant is stored (S1) and the pellet is homogenized in homogenizing buffer and centrifuged at 100,000×g for 60 minutes at 4 °C. 7. This supernatant (S2) is combined with S1 to make up the cytosolic fraction. 8. The final pellet is collected and is the membranal fraction. The pellet and the supernatant fraction are used to measure membranal and cytosolic PKC, respectively.

[3H]PDBu Binding to Membranal PKC 1. The membranal pellet obtained above is suspended in the an amount of incubation buffer (50 mM Tris HCI, pH=7.4; 1 mM CaCI2; 75 mM magnesium acetate; 0.1% BSA; 50 mg/ml of phosphatidylserine) so that the protein ranges between 20–50 µg/tube. 2. The binding assay is carried out in triplicate tubes containing incubation buffer, [3H]PDBu ranging from 0.8 to 30 nM (6 different concentrations), and 150 ml of membrane suspension, with or without 10 µM phorbol 12-myristate 13-acetate (PMA) as the displacing agent, in a total volume of 500 µl.

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3. All tubes are incubated for 30 minutes at 37 °C. 4. Bound [3H]PDBu is separated from free [3H]PDBu by the addition of 5.0 ml of washing buffer (50 mM Tris MCI, pH=7.4, 0.1% bovine serum albumin) rapidly filtered through a Whatman GF/B filter, and then washed 3 times using a Brandel cell harvester. Air-dried filters are used for liquid scintillation counting.

[3H]PDBu Binding to Cytosolic PKC 1. The binding assay for the cytosolic PKC is carried out in triplicate tubes containing incubation buffer (50 mM Tris MCI, pH=7.4; 1 mM CaCI2; 75 mM magnesium acetate; 0.1% BSA); 150 ml of the cytosolic fraction; [3H]PDBu (8.0 to 30 nM); bovine gamma globulin (100 mg/ml); and phosphatidylserine (50 mg/ml) in a total volume of 500 µl. 2. All tubes are then incubated for 30 minutes at 37 °C. 3. Proteins are precipitated by the addition of 200 µl of chilled 12% polyethylene glycol (w/v in 50 mM Tris MCI, pH=7.4: to allow complete precipitation). 4. Keep samples at 15 minutes at 4 °C. 5. Bound [3H]PDBu is separated from free [3H]PDBu according to the method described for the membranal fraction (see step 4).

Specific binding in both membranal and cytosolic fractions is defined as the difference between binding observed in the presence or in the absence of 10 µM PMA. Bmax and KD are calculated by Scatchard analysis. The protein content in membranal and cytosolic fractions is determined (Lowry et al. 1951). Scatchard plots for [3H]PDBu binding to the membranal and the cytosolic fractions are given in Figures 3.1 A and B, respectively. The saturation isotherm shows that the binding is saturable. We also observed that nonspecific binding was non-saturable. DETERMINATION OF EXPRESSED LEVELS OF PROTEIN IN HUMAN POSTMORTEM BRAIN Receptor binding studies using specific radioligands provide the number of receptors and the affinity of the ligand toward the receptor. If the receptor number is altered in any pathological condition, it is difficult to know from binding studies alone whether the alteration in receptor number is due to changes in expressed protein level of the receptor or only due to changes in receptor binding sites. Thus, it is important to determine the protein level of receptors using specific antibodies. The simplest method is the Western blot technique, in which the membrane is prepared by a procedure similar to that used to assay radioligand binding; however, certain proteins are quite labile and susceptible to proteolytic degradation. To prevent this, protease inhibitors are added to the homogenizing buffer.

Figure 3.2 Protocol for the separation of membranal and cytosolic fractions from postmortem brain tissue

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57

WESTERN BLOT 1. Homogenate containing protease inhibitors is mixed with the loading buffer and kept in boiling water for 3–4 minutes. (This is required to denature the protein.) 2. Protein samples are then separated by SDS-polyacrylamide gel electrophoresis using acrylamide gel. The percentage of acrylamide gel depends on the molecular weight of the protein to be determined and may range from 4% to 20%. The proteins are separated on the gel under an electrical field according to their molecular weights. 3. The separated proteins are then electrophoretically transferred to a nitrocellulose membrane. 4. The nonspecific binding sites of the membrane are blocked with powdered nonfat milk in Tris-base, sodium chloride and Tween 20 (TBST). 5. The nitrocellulose membranes are then incubated with primary antibodies. The dilution of the antibody and the duration of the incubation depend on the amount of protein present in a given tissue and the specificity of the antibody utilised. 6. The membranes are washed again with TBST and incubated with secondary antibody. 7. Finally, membranes are washed with TBST and exposed to film.

Two different procedures are available to detect protein. One is a radioactive procedure, wherein the secondary antibody is labeled with [125I] and the membranes are exposed to X-ray film from a few hours to 2 to 3 days. In an alternate procedure, the nitrocellulose membrane is treated with chemiluminescent reagents after incubation with the secondary antibody linked to horseradish peroxidase. The treated membranes are then exposed to film. A representative autoradiogram showing the immunolabeling of PKC isozymes in human brain is shown in Figure 3.3. Importantly, before starting the experiment, the conditions for a given protein need to be established. For example, the protein concentration curve is plotted, and the selected concentration of protein should fall within the linear range.

Figure 3.3 Immunolabeling of PKC isozymes (α, β, γ, δ, ε) in the cerebral cortex of human postmortem brain tissue. Molecular weights of PKC isozymes are: α, β, γ=80 kDa, δ=77.6 kDa, ε=90 kDa.The antibody concentrations used here for α, β, γ, δ=1:3000 for primary and 1:5000 for secondary (anti-rabbit IgG); for ε=1.5000 for primary and 1:5000. for secondary (anti-rabbit IgG).

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Also, exposure time is crucial, and membranes should be exposed to film for different time intervals. The density of the band appearing on the film can be calculated using the densitometer. To avoid inter-blot variation, a standard pool of brain samples should be simultaneously run on the gel and the optical density of the desired protein should be divided by the optical density of the band for standard brain pool samples. DETERMINATION OF EXPRESSED LEVELS OF PKC ISOZYMES IN POSTMORTEM HUMAN BRAIN TISSUE

Materials • • • • • • •

Refrigerated centrifuge Densitometer (Loats Associates, Inc., Westminister, MD, 21157, USA) Mini Protein II Cell (BioRad) Mini Protein II Transblot (BioRad) Power supply (250V, BioRad) ECL-Hyperfilm (Amersham) Chemiluminescent ECL detection reagent (Amersham)

Chemicals Tris HCI, 2-mercaptoethanol, sodium dodecylsulfate, (SDS), glycerol, bromphenol blue, acrylamide, bis-acrylamide, TEMED, glycine, Tween-20, nonfat dry milk, nonidet P-40, ammonium persulfate, methanol, leupeptin, dithiothreitol (DTT), pepstatin, apoproteinin, Triton x-100, primary antibodies (PKC α, β, γ, δ, ε, Gibco-BRL), horseradish peroxidase-linked secondary antibodies (Amersham).

Reagent Preparation Preparation of acrylamide gel 1. Prepare the 30:0.8 acrylamide stock solution take 30 g acrylamide and 0.8 g bis-acrylamide and make up the volume to 100 ml with water. 2. Filter this solution through Whatman filter paper. 3. Prepare lower gel stock solution by adding 0.4 g sodium dodecylsulfate (SDS) to 100 ml 1.5 M Tris HCI, pH 8.8. 4. Prepare stacking gel stock solution by adding 0.4g SDS to 100 ml of 0.5 MTris HCI, pH 6.8. 5. To prepare resolving gel (7.5%) take 3 ml of lower gel stock solution and add 3 ml acrylamide (30:0.8) and 6 ml distilled water. 6. Degas the solution for 3 minutes, then add 60 µl freshly prepared ammonium persulfate (10% w/v) and 6 µl TEMED. 7. Pour solution between electrophoresis plates allowing sufficient room for stacking gel and allow to set. 8. To prepare stacking gel (4.5%) add 1.5 ml acrylamide (30:0.8) and 6 ml distilled water to 2.5 ml upper gel stock solution. 9. Degas for 3 minutes and then add 30 µl of ammonium persulfate (10% w/v) and 10 µl TEMED.

MEMBRANE BINDING ASSAYS

10. Pore over set resolving gel and add comb. 11. Allow to set. 12. Remove comb.

Membrane preparation 1. Homogenize brain tissue into buffer containing 20 mM Tris HCI, pH=7.5; 2 mM EGTA, 5 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1.45 mM pepstatin, 0.2 units/ml aprotinin, and 2 mM dithiothreitol. 2. Centrifuged homogenate at 100,000×g for 60 minutes at 4 °C. 3. Collect supernatant (the cytosolic fraction). 4. Homogenized pellet into the same buffer containing 0.2% Triton X-100. 5. Place suspension on ice for 60 minutes and then centrifuged at 100,000×g for 60 minutes. 6. Collect the supernatant (the solubilized membranal fraction). 7. Both the membranal and the cytosolic fractions are used for the measurement of PKC isozymes.

Measurement of PKC isomers 1. Measure the protein content in each sample (e.g. method of Lowry et al. (1951)). 2. Mix equal volumes of protein sample and gel loading solution (50 mM Tris HCI, pH=6.8; 4% β-mercaptoethanol; 1% SDS; 40% glycerol; and bromphenol blue), boil for 3 minutes and keep on ice for 10 minutes. 3. Load samples (30 µg protein in each lane) onto 10% (w/v) acrylamide gel using the Mini Protein II gel apparatus (Biorad). A standard pooled brain sample is also run in the same gel. 4. Run gel in 25 mM Tris base, 192 mM glycine, and 0.1% (w/v) SDS buffer at 150 volts. 5. Transfer proteins electrophoretically from the gel to ECL nitrocellulose membrane (Amersham, IL) using the Mini TransBlot transfer unit at 0.150 amp constant current. 6. Wash membranes with TBST buffer (10 mM Tris base, 0.15 M NaCl, and 0.05 % (v/v) Tween 20) for 10 minutes. 7. Block membrane by incubating with 5% (w/v) powdered nonfat milk in TBST, 2 ml nonidet P-40, and 0.02% (w/v) SDS (pH=8.0). 8. Incubate membrane overnight at 4 °C with primary monoclonal antibody (antiPKC α, β, γ, δ, ε) at a dilution of 1:1000 to 1:3000 (depending on the antibody used). 9. Wash membrane with TBST and incubate with horseradish-peroxidase-linked secondary antibody (anti-rabbit IgG) for 3 to 5 hours at room temperature. 10. Wash membrane thoroughly with TBST and exposed to ECL film. 11. The optical density of the bands on the autoradiogram are quantified using the Loats Image Analysis System, and the optical density of each sample is corrected by the optical density of the pooled brain sample. The values are represented as a percent of the control.

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Before starting the experiment, the procedure is standardized using 10 to 100 µg protein. We found that the bands were linear up to a protein concentration of 50 µg. In addition, the dilution of antibodies and the duration of the exposure of nitrocellulose membranes on autoradiographic film are standardized. DATA ANALYSIS The major factors that influence postmortem brain studies are discussed in detail in the section Limitations of Human Postmortem Brain Studies. These factors should be considered before analyzing the data. In general, data can be analyzed by using a multivariate analysis of variance. Different variables, as described earlier (age, sex, postmortem delay, drug toxicity, drug exposure), should be correlated with the data obtained. If any significant associations exist (using the multiple linear regression method), they should be added as covariates in the ANOVA and used to further characterize the association between the data and the patient groups. However, if such associations disappear following adjustments for psychopathology and/or current and historical substance abuse, that would suggest that these associations are due to these factors alone, and that the differences were produced by an interaction between the patient and the psychopathology and/or substance abuse. CONCLUSION This chapter covers a variety of topics related to the assay of membrane-bound receptors in postmortem human brain samples. Because some of these receptors are also localized in the cytosolic fraction, we chose to describe protein kinase C as an example, since it exists in both cytosolic and membranal fractions. Although radioreceptor binding is a very important tool in determining the number of receptors and the affinity of the ligand for the receptor, related techniques, such as autoradiography and immunolabeling of receptors, are equally important. In the receptor ligand binding assay section, we discussed the most familiar assays used routinely in our laboratory; however, other procedures can be found in other reviews. Interpretation of data, particularly regarding postmortem human brain samples, is very difficult. For this reason, we have discussed the various factors that may influence the results and urge that caution be applied both in the experimental design and in the analysis of the results. References Agren, H. (1982) Depressive symptom patterns and urinary MHPG excretion. Psychiatry Res., 6, 185–196.

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Allen, S.J., Benton, J.S., Goodhart, M.J., Haan, E.A., Sims, N.R., Smith, C.C.T., Spillane, J.A., Bowden, D.M. and Davidson, A.N. (1983) Biochemical evidence of selective nerve cell changes in the normal aging human and rat brain. J. Neurochem., 41, 256–265. Arango, V., Ernsberger, P., Sved, A.F. and Mann, J.J. (1993) Quantitative autoradiography of α1 and α2 adrenergic receptors in the cerebral cortex of controls and suicide victims. Brain Res., 630, 271–282. Arora, R.C. and Meltzer, H.Y. (1986) Serotonergic measures in the brain of suicide victims: 5HT2 binding sites in the frontal cortex of suicide victims and control subjects. Am. J. Psychiatry, 146, 730–736. Arranz, B., Cowburn, R., Eriksson, A., Vestling, M. and Marcusson, J. (1992) Gammaaminobutyric acid-B (GABAB) binding sites in portmortem suicide brains. Biol. Psychiatry, 26, 33–36. Asberg, M., Traskman, L. and Thoren, P. (1976) 5-HIAA in cerebrospinal fluid: a biochemical suicide predictor. Arch. Gen. Psychiatry, 33, 1193–1197. Benes, F.M., Vincent, S.L., Marie, A. and Khan, Y. (1996) Upregulation of GABAA receptor binding on neurons of the prefrontal cortex in schizophrenic subjects. Neuroscience, 75, 1021–1031. Bennett, J.L. and Aghajanian, G.K. (1974) Stereospecific binding of D-LSD and physiological response of serotonergic neurons. Fed. Proc., 33, 256. Bennett, J.P. and Snyder, S.H. (1975) Stereospecific binding of D-lysergic acid diethylamide (LSD) to brain membranes: relationship to serotonin receptors. Brain Res., 94, 523–544. Bennett, J.P. and Snyder, S.H. (1976) Serotonin and lysergic acid diethylamide binding in rat brain membranes: relationship to postsynaptic serotonin receptors. Mol. Pharmacol., 12, 373–389. Bruinvels, A.T., Landwehrmeyer B., Waeber, C., Palacios J.M. and Hoyer D. (1991) Homogenous 5-HT1D recognition sites in the human substantia nigra identified with a new iodinated radioligand. Eur. J. Pharmacol., 202, 89–91. Campbell, I.C., McKenna, R.M. and Checkley, S.A. (1985) Characterization of platelet alpha 2-adrenoreceptors and measurement in control and depressed subjects. Psychiatry Res., 14, 17–31. Carlsson, A. (1988) The current status of the dopamine hypothesis of schizophrenia. Neuropsychopharmacohgy, 1, 179–186. Chasin, M., Rivkin, I., Mamrak, F., Samaniego, S.G. and Hess, S.M. (1971) α- and βadrenergic receptors as mediators of accumulation of cyclic adenosine 3', 5' monophosphate in specific areas of guinea pig brain. J. Biol. Chem., 246, 3037–3041. Checkley, S.A,Slade, A.P. and Shur, E. (1981) Growth hormone and other responses to clonidine in patients with endogenous depression. Br. J. Psychiatry, 138, 51–55. Crawley, J.C.W., Crow, T.J., Johnstone, E.G., Oldland, S.R.D., Owens, D.G.C., Poulter, M., Smith, T., Veall, N. and Zanelli, G.D. (1986) Dopamine receptors in schizophrenia studied in vivo. Lancet, 2, 224–225.

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Daiguji, M., Meltzer, H.Y., Tong, C., U’Prichard, D.C., Young, M. and Kravitz, H. (1981) α2-Adrenergic receptors in platelet membranes of depressed patients: no change in number or [3H]yohimbine affinity. Life Sci., 29, 2059–2064. Enna, S.J. and Snyder, S.H. (1975) Properties of γ-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res., 100, 81–97. Enna, S.J. and Snyder, S.H. (1977) Influences of ions, enzymes, and detergents on γaminobutyric acid-receptor binding in synaptic membranes of rat brain. Mol. Pharmacol., 13, 442–453. Farde, L., Wiesel, F.A., Stone-Elandes, S., Halldin, C., Nordstrom, A.L., Hall, H. and Sedvall, G. (1990) D2-dopamine receptors in neuroleptic naïve schizophrenic patients. Arch. Gen. Psychiatry, 47, 213–219. Glasel, J.A., Venn, R.F. and Barnard, E.A. (1980) Distribution of stereospecific opiate receptor binding activity between subcellular fractions from ovine corpus striatum. Biochem. Biophys. Res. Commun., 95, 263–268. Gozlan, S., El Mestikawy, L., Pichat, J., Glowinski, and Hamon, M. (1983) Identification of presynaptic serotonin autoreceptors using a new ligand: 3H80H-PAT. Nature, 305, 140–142. Greenwood, A.F., Powers, R.E., and Jope, R.S. (1995) Phosphoinositide hydrolysis, Gaq, phospholipase C, and protein kinase C in postmortem human brain: effects of postmortem interval, subject age, and Alzheimer’s disease. Neuroscience, 69, 125–138. Grossman, C.J., Kilpatrick, G.J. and Bunce, K.T. (1993) Development of a radioligand binding assay for 5HT4 receptors in guinea pig and rat brain. Br. J. Pharmacol., 109, 618–624. Hanada, S., Mita, T., Nishino, N. and Tanaka, C. (1987) [3H]Muscimol binding sites increased in autopsied brains of chronic schizophrenics. Life Sci., 40, 259–266. Hoyer, D., Engel, G. and Kalkman, H.O. (1985) Characterization of the 5-HT1B recognition site in rat brains: binding studies with (-) [125I] iodocyanopindolol. Eur. J. Pharmacol., 118, 1–12. Hoyer, D., Palacios, J.M. and Mengod, G. (1992) 5HT receptor distribution in the human brain: Autoradiographic studies. In Central Serotonin Receptors andPsychotropic Drugs, edited by C.A.Marsden and D.J.Heal, pp. 100–125. London: Blackwell Scientific Publications. Iversen, L.L. and Mackay, A.V.P. (1981) Brain dopamine receptor densities in schizophrenics. Lancet, ii, 149. Kiuchi, Y., Kobayashi, T., Takeuchi, J., Shimizu, H., Ogata, H. and Toru, M. (1989) Benzodiazepine receptors increase in postmortem brain of chronic schizophrenics. Eur. Arch. Psychiatr. Neurol. Sci., 239, 71–78. Leonard, S., Adams, C., Breese, C.R., Adler, L.E., Bickford, P., Byerley, W., Coon, H., Griffith, J.M., Miller, C., Myles-Worsley, M., Nagamoto, H.T., Rollins, Y., Stevens, K.E., Waldo, M. and Freedman, R. (1996) Nicotinic receptor function in schizophrenia. Schizophrenia Bull., 22, 431–445.

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Lowry, O.H., Rosebough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with folin-phenol reagent. J. Biol. Chem., 193, 265–275. Mallat, M. and Hamon, M. (1982) Ca2+-guanine nucleotide interactions in brain membranes. I. Modulation of central 5-hydroxytrypfamine receptors in the rat. J. Neurochem., 38, 151–161. Manji, H.K., Etcheberrigary, R., Chen, G. and Olds, J.L. (1983) Lithium decreases membrane-associated protein kinase C in hippocampus: selectivity for the α isozyme. J. Neurochem., 61, 2303–2310. Mann, J.J., Stanley, M., McBride, P.A. and McEwen, B.S. (1986) Increased serotonin-2 and β-adrenergic receptor binding in the frontal cortices of suicide victims. Arch. Gen. Psychiatry, 43, 945–959. McPherson, G.A. (1985) Analysis of radioligand binding experiments: a collection of computer programs for the IBM PC. J. Pharmacol. Methods, 14, 213–228. Meana, J.J., Barturen, F. and Garcia-Sevilla, A.(1992) α2-adrenoreceptors in the brain of suicide victims: increased receptor density associated with major depression. Biol. Psychiatry, 31, 471–490. Munson, P.J., and Rodband, D. (1980) Ligand: A versatile computerized approach for characterization of ligand-binding system. Anal. Biochem., 107, 220–239. Negro, M., Chinchetru, M.A., Fernandez, A. and Calvo, P. (1995) Effect of ethanol treatment on rate and equilibrium constants for [3H]muscimol binding to rat brain membranes: alteration of two affinity states of GABAA receptor. J. Neurochem., 64, 1379–1389. Olney, J.W. and Farber, N.B. (1995) Glutamate receptor dysfunction and schizophrenia. Arch. Gen. Psychiatry, 52, 988–1025. Ordway, G.A., Widdowson, P.S., Smith, K.S. and Halaris, A. (1994) Agonist binding to α2-adrenoceptors is elevated in the locus ceruleus from victims of suicide. J. Neurochem., 63, 617–624. Palacios, J.M., Mengod, G. and Hoyer, D. (1993) Brain serotonin receptor subtypes, radioligand binding assays, second messengers, ligand autoradiography, and in-situ hybridization histochemistry. In Methods in Neurosciences, edited by P.M.Conn, vol 12, pp. 238–262. New York: Academic Press Inc. Pandey, S.C., Dubey, M.P., Piano, M.R., Schwertz, D.W., Davis, J.M. and Pandey, G.N. (1993) Modulation of 5HT2C receptors and phosphoinositide signaling system by ethanol consumption in rat brain and choroid plexus. Eur. J. Pharmacol., 247, 81–88. Pandey, G.N., Dwivedi, Y., Pandey, S.C., Conley, R.C. and Tamminga, C.A. (1997) Protein kinase C in the postmortem brain of teenage suicide victims. Neuroscience Lett., 228, 111–114. Pandey, G.N., Janicak, P.G., Javaid, J.I. and Davis, J.M. (1989) Increased [3H]clonidine binding in the platelets of patients with depressive and schizophrenic disorders. Psychiatry Res., 28, 73–88.

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Pandey, S.C., Piano, M.R., Schwertz, D.W., Davis, J.M. and Pandey, G.N. (1992) Effect of ethanol administration and withdrawal on serotonin receptor subtypes and receptormediated phosphoinositide hydrolysis in rat brain. Alc. Clin. Exp. Res., 16, 1110– 1116. Pazos, A., Hoyer, A and Palacios, J.M. (1984) The binding of serotonergic ligands to the porcine choroid plexus: characterization of a new type of serotonin recognition site. Eur. J. Pharmacol., 106, 539–546. Pazos, A., Hoyer, A and Palacios, J.M. (1984) Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors. Eur. J. Pharmacol., 106, 531– 538. Peck, E.J. and Kelner, L. (1982) Receptor measurement. In Handbook of Neurochemistry, edited by A.Lajtha, pp. 53–75. New York: Plenum Press. Piletz, J.E. and Halaris, A.G. (1988) Super high affinity [3H]paraamino-clonidine binding to platelet adrenoreceptors in depression. Prog. Neuropsychopharmacol. Biol. Psychiatry. , 12, 541–553. Raymond, J.R., Hnatowich, M. Lefkowitz, R.J., and Caron, M.G. (1990) Adrenergic receptors: models for regulation of signal transduction processes. Hypertension, 15, 119–131. Rimon, G., Hanski, E. and Levitzki, A. (1980) Temperature dependence of β-receptor, adrenosine receptor and sodium fluoride-stimulated adenylate cyclase from turkey erythrocytes. Biochemistry, 19, 4451–4460. Rossor, M., Garrett, N. and Iversen, L. (1980) No evidence for lateral asymmetry of neurotransmitters in post-mortem human brain. J. Neurochem., 35, 745–745. Roy, A., Jimerson, D.C. and Pickar, D. (1986) Plasma MHGP in depressive disorders and relationship to the dexamethasone suppression test. Am. J. Psychiatry, 143, 846– 851. Rugg, E.L., Barnett, D.B. and Nahorski, S.R. (1978) Coexistence of beta1 and beta2 adrenoreceptors in mammalian lung: evidence from direct binding studies. Mol. Pharmacol., 14, 996–1005. Snyder, S.H. and Bennett, J.P. (1975) Biochemical identification of the postsynaptic serotonin receptor in mammalian brain. In Pre and Postsynaptic Receptors, edited by E.Usdin and W.E.Bunney Jr., pp. 191–205. New York: Marcel Dekker Inc. Spokes, E.G.S. and Koch, D.J. (1978) Postmortem stability of dopamine, glutamate decarboxylase and choline acetylcholine transferase in the mouse brain under conditions simulating the handling of human autopsy material. J. Neurochem., 31, 381–383. Squires, R.E and Braestrup, C. (1978) Characteristics and regional distributions of two distinct [3H]naloxone binding sites in the rat brain. J. Neurochem., 30, 231–236. Stanley, M. and Mann, J.J.(1983) Increased serotonin-2 binding sites in frontal cortex of suicide victims. Lancet, 1, 214–216.

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THE LOCALISATION AND QUANTIFICATION OF MOLECULAR CHANGES IN THE HUMAN BRAIN USING IN SITU RADIOLIGAND BINDING AND AUTORADIOGRAPHY Brian Dean, Geoffrey Pavey, Siew Yeen Chai and Frederick A.O.Mendelsohn

Early studies investigating molecular abnormalities in the brain of subjects with psychiatric illness measured levels of neurotransmitter and their metabolites in the brain or the activity of neuronal specific enzymes (Bird et al., 1979). The development of radioactive ligands that specifically bound to an increasingly diverse number of target sites then made it possible to measure other molecules in the brain, such as neurotransmitter receptors (Mackay et al., 1982) and transporters (Stanley et al., 1990). Initially such experiments were carried out using particulate membrane prepared from dissected brain regions. However, whilst this approach had the advantage of allowing extensive characterisation of molecules in brain tissue, it could not localise any change to a discrete area within specific brain regions. Unlike earlier radioactive ligand binding methodologies, in situ radioligand binding with autoradiography utilises the binding of radioactive ligands to frozen tissue sections rather than to particulate membranes. This approach has the advantage of maintaining the anatomical integrity of the brain and hence allows localisation of molecules within brain regions. Indeed, the initial studies using autoradiography were designed solely to identify the distribution of molecules in the brain (Kuhar, 1991). With the on-going development of increasingly specific radioactive ligands and the development of more sophisticated computer aided image analysis, it is now possible to localise and quantify molecules in the brain. Thus, quantitative autoradiography is now a powerful tool that is being used to investigate the biochemical changes that underlie psychiatric diseases. 67

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AUTORADIOGRAPHY USING HUMAN POSTMORTEM BRAIN Experimental protocols that utilise autoradiography to analyse the molecular make up of postmortem brain tissue rely on many of the principles that apply to methods utilising tissue homogenates. However, with in situ radioligand binding coupled with autoradiography it is essential that tissue preparation and handling preserves the architectural integrity of the brain. Tissue preparation In situ radioligand binding usually requires frozen tissue, however where localising binding to the cellular level is essential, tissue may be lightly fixed then frozen to preserve cellular cytoarchitecture (Benes et al., 1986). In addition, there are two methods by which tissue can be processed before freezing or fixation and the method used will usually be dictated by the frequency and quantity of brain tissue to be processed. Where a significant proportion of the brain is to be collected from a large number of donors it is often only feasible to store brain tissue as slices. However, if facilities are available, the storage of brain tissue blocked into specific regions prior to freezing is preferable. The major advantage of the latter process is that anatomical structures within the brain are more easily localised for dissection in the tissue before it is frozen. In addition, freezing to the centre of small blocks of tissue can be more rapid than for larger blocks, depending on the surface area to volume ratios. However, freezing brain slices will reduce the total time taken to process the tissue to ultracold temperatures. The main disadvantage of storing tissue as frozen slices is the need to repeatedly thaw slices when dissecting specific brain regions. We have developed a process by which the impact of repeatedly thawing tissue is avoided. FREEZING TISSUE SLICES 1. Slice available tissue into 0.5 to 1.0 cm slices. 2. Photograph each tissue slice prior to freezing and carefully file each photograph referenced to slice and brain. 3. Place slice on a steel tray and place on the base of an ultracold freezer. 4. After 2 hours, remove tissue slices from the freezer, place each slice into separate sequentially numbered plastic bags. 5. Reassemble slices into cutting order, place slices into a suitable container and return to ultracold storage.

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DISSECTION OF BRAIN REGIONS 1. To identify a specific brain region the photographs of the relevant brain slices from each brain should be utilised to determine the slice that contains the region of interest. 2. Remove the slice of interest from ultracold storage. 3. With the tissue at -70 °C rapidly dissect the required brain region using a standard coping saw (blade: W4, 18 teeth/25 mm). 4. Return the brain slice to cold storage. 5. Process dissected brain region quickly and return to ultracold storage. The dissected brain regions should ideally be stored in a separate facility and catalogued. This ensures that when the region is required for another experiment it can be accessed without removing the rest of that brain from ultracold storage. In addition, regions that have been blocked can be prepared for sectioning in 2– 3 minutes.

PROCESSING TISSUE BLOCKS 1. Mount tissue block on a pre-cooled cryomicrotome specimen disc using a commercial embedding medium such as OCT compound (Miles Scientific). 2. Equilibrate to the temperature of the cryomicrotome chamber before cutting (approximately 30 minutes). This minimises problems with distortion or fracturing of the tissue during sectioning. For human brain tissue the optimal cutting temperature appears to be in the range of -17 ° to -20 °C. 3. To minimise variations in anatomy, and possible regional variability in radioactive ligand binding, serially cut the required number of 15–20 µm sections. 4. Transfer the sections onto a gelatinised microscope slide (see below) using one of two strategies. 5. Thaw-mount the frozen sections onto slides kept at room temperature by carefully placing the slide against the frozen section on the microtome blade. 6. Transfer the sections onto pre-chilled slides using a soft brush and thaw mount the section by warming the back of the slide with a finger. 7. Process the sections through the experimental procedure. NB: If it is impractical to immediately process the sections, sections should be thoroughly dried. This can be achieved by placing the section in a desiccator under reduced pressure before the sections are frozen for storage. The section can then be placed in a sealed slide box containing a few capsules of desiccant (Aldrich Z16,348–1). The slide box should then be sealed into plastic bags and stored at -70 °C. This will prevent the formation of ice-crystal artifacts on the sections.

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Cleaning and gelatinising microscope slides It is important to use the best quality slides available to ensure the slides are of uniform thickness (typically 1.0–1.2 mm thick). This is critical if a combination of a tritiated radioligand and a film-based emulsion is used because the short path length of β emissions requires the sections to be in intimate contact with the emulsion. Standard (25×76 mm) microscope slides can be used with smaller human brain structures (i.e. hippocampus) but large regions (i.e. caudateputamen) may require bigger slides (50×76 mm). All slides must be free of all dirt and contamination. CLEANING AND GELATINISING MICROSCOPE SLIDES 1. 2. 3. 4.

Soak slides in a detergent solution (i.e. 5–10% DECON). Rinse thoroughly in distilled water. Dry. Prepare a 0.5% gelatin solution: Dissolve 2.5 g gelatin (Bloom 300; Sigma G 2500) into 500ml distilled water heated to 50 °C. 5. Allow the gelatin solution to cool to ~37 °C and add 0.25 g chromium potassium sulphate (Aldrich 24,336–1). 6. Briefly dip (~10 sees) slides into a 0.5% solution of gelatin at 37 °C. 7. Drain excess solution from the slide allowed to dry either at room temperature or in an incubator at 37 °C.

AUTORADIOGRAPHY: THEORETICAL AND PRACTICAL ISSUES Prior to incubating the tissue sections for autoradiography a number of parameters must be considered. These include the following. 1. 2. 3.

The availability of a suitable radioactive compound with which to label the molecule of interest. The availability of non-radioactive ligands to compete with the radioactive ligand to bind to the molecule of interest. Determining appropriate assay conditions including: (a) pre-incubation washes (b) buffer (c) incubation temperature and time (d) appropriate post-incubation wash conditions

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RADIOACTIVE LIGANDS The choice of radioactive ligand will have a significant effect on experimental protocols. The most commonly used isotopes for in situ radioligand binding with autoradiography are tritium (3H) and iodine125, but sulphur35 and phosphate32 are also employed.

Tritiated radioactive ligands For studies where anatomical resolution is paramount, tritiated radioactive ligands will produce the best image resolution because of the short path length of the β emission. However, it is important to note that although tritiated radioactive ligands will have long radioactive half-lives (approx. 12 years) radiolytic degradation of the ligand can result in an effective ‘shelf-life’ of only six months. There are two major disadvantages in using tritium: 1. 2.

Emissions are susceptible to differential quenching by the grey and white matter of the brain. Relatively low specific activities (typically 20–100 Ci/mmol) may necessitate lengthy exposure of several months but exposure times of two to six weeks are most common.

Iodinated radioactive ligands Iodinated compounds are of much higher specific activity (up to 2000 Ci/ mmol) and therefore the time in exposing the tissue sections to film will be in the order of days rather than months. In addition, quenching is much less a problem with iodine125 but the higher energy particle emissions tend to reduce the resolution of the autoradiographic image. It is also important to note that with iodine, unlike tritium, section thickness is directly related to image density. Thus particular care must be taken in achieving uniform tissue section thickness particularly if an autoradiograph is to be used for densitometry. Iodine125 can be incorporated into the compound of interest using direct oxidation reaction provided the compound contains a phenolic ring. The most common oxidizing reagents used include chloramine T (a strong oxidizing agent), iodogen and lactoperoxidase (a mild oxidising system used for unstable compounds) (McFarthing, 1992). More recently, commercial companies, such

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as Pierce, have released simplified methodologies that allow iodination of compounds in pre-coated test tubes. This could be an attractive methodology for laboratories seeking to establish ‘in house’ iodination facilities. Radioiodinated compounds generally require purification by high pressure liquid chromatography (HPLC) to ensure purity of the compound and to separate unlabelled, mono-iodinated and di-iodinated compounds. The integrity of compounds which, for example, may be susceptible to degradation to smaller peptides, can be evaluated before and after incubation using HPLC. The absence of degradation products will ensure that only the binding of intact radioligand to tissue sections is measured.

Sources of radioactive ligands Most commonly, tritiated compounds are obtained from commercial companies such as Amersham and New England Nuclear who offer a comprehensive range of radioactive ligands of a consistent quality. Custom-labelling services are also available when it is necessary to obtain a novel radioactive ligand.

The use of radioactive ligands with autoradiography The most common method used to measure the density of radioactive ligand binding in tissue slices is by single point saturation analysis. This approach relies on the concentration of the radioactive ligand being present at a sufficient level to saturate all the binding sites of interest in the tissue. In that instance, the difference between total and non-specific binding will be directly related to the density of binding sites in the tissue (Figure 4.1) (Stephenson, 1956). If not known from previous experiments, the concentration of radioactive ligand to be used in single saturation analysis must be determined by an experimental approach. This can be done using particulate membrane from the same tissue to be used in slices and determining the specific binding of radioactive ligand at multiple concentrations of the radioligand. It is possible to use the same approach using tissue sections. To accelerate this approach the tissue sections may be wiped from the slide onto a small fibre glass filter disc. In the case of tritium, the filter disc containing the tissue is placed into liquid scintillation fluid. For iodine125 the filter would be placed in a test tube in a γcounter. In both cases the amount of bound radioactive ligand on the filter, and hence on the tissue section, can be calculated. If the specific binding of a

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particular radioligand is localised to a small portion of the tissue section, tissue wipe studies or particulate membrane studies may not give an accurate measure of the signal to noise ratio. In that situation, a dose response curve using the full in situ radioligand binding and autoradiography will need to be carried out. Once the affinity (Kd) of the radioactive ligand binding is known a concentration of radioactive ligand at 3 to 4 times higher than the Kd is needed for single point analysis to ensure the majority of binding sites of interest are occupied. In that case, the density of binding sites is equal to the specific binding of the radioactive ligand per area of tissue. Non-radioactive ligand The role of the non-radioactive ligand is to compete with the radioactive ligand for, ideally, a single binding site that is common to both compounds and is the molecule of interest. In the case where the radioactive ligand has only one binding site the same ligand in a non-radioactive form can be used. However, many radioactive ligands may bind to multiple binding sites. If that is the case the molecule of interest can still be visualised and measured by carefully combining radioactive and non-radioactive compounds. The aim is to use two ligands that have only one common binding site, which is the molecule

Figure 4.1 Theoretical representation of the binding of a radioactive ligand to a specific binding site on tissue obtained at autopsy

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of interest. The density of the molecule of interest is then calculated by subtracting the binding of the radioactive drug in the presence of the nonradioactive drug (non-specific binding) from the binding of the radioactive ligand in the absence of the non-radioactive drug (total binding).

OPTIMISATION OF ASSAY CONDITIONS Pre-incubation washes Washing sections prior to incubation with a radioactive ligand is usually necessary when the affinity of the radioactive ligand is similar to that of an endogenous ligand. In that instance it is necessary to remove the residual endogenous ligand from the tissue to ensure an accurate estimation of the density of binding sites. If the ‘drop assay’ method (see below) is to be used then it is important to thoroughly dry the sections after the pre-incubation. This is necessary to ensure that when the assay medium is applied to the section there is enough surface tension between the liquid and the section to prevent the incubation medium from dispersing over the entire slide. Furthermore, before drying sections, it is advisable to dip the sections into distilled water to remove residual salts from the sections. Assay buffer An appropriate buffer to use in combination with specific radioactive ligands can often be derived from previous studies using that radioactive ligand with tissue homogenates. Buffers will normally be close to physiological pH (7.4) and contain various ions which have been found to enhance ligand/molecule interactions. For example, using paniculate membrane the affinity of [3H]mazindol binding to the dopamine transporter was shown to be sodiumdependent using paniculate membrane (Javitch et al., 1985) and subsequent autoradiographic techniques use buffers that include sodium chloride up to 300 mM (Donnan et al., 1989). By contrast, the binding of some radioactive ligands can be reduced by various ions and therefore assays using such ligands tend to use only Tris-HCl buffers. Finally, radioactive ligand binding may be pH dependent. For example, the binding of the dopamine receptor antagonist (-)sulpiride to the dopamine-D2 receptor is optimal at pH 7.5–8.0 and is strongly reduced below pH 7.0 (Presland & Strange, 1991).

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Incubation temperature The optimal incubation temperature relates to the pharmacokinetics of the radioactive ligand used in the experiment. In most cases, the association rate for radioactive ligand binding will be known and will have been used to determine the optimum incubation temperature. However, if this information is not available, it will be necessary to ensure that incubation time and temperature are sufficient to allow the binding to reach equilibrium. Non-specific binding tends to increase linearly with time and hence carrying out incubations at high temperatures, when equilibration times are shorter, is desirable. Unfortunately whilst higher incubation temperatures shorten incubation times they also severely disrupt tissue integrity. In addition, peptide radioligands are prone to degradation during incubation, even in the presence of a proteolytic enzyme inhibitor cocktail, a factor that is a greater problem the higher the incubation temperature. These considerations tend to limit the temperature at which assays may be performed. Hence most incubations seem to be carried out between 4 °C and room temperature. Post-incubation washes Often the non-specific binding to tissue sections can be significant. However, washing sections after the primary incubation will often reduce non-specific binding without any effect on specific binding. This is because non-specific binding, unlike total binding, is not temperature dependent, therefore washing the tissue sections at 4 °C should not affect specific binding of high affinity radioactive ligands. Washing steps will often involve two to four washes in icecold assay buffer followed by a rapid wash in distilled water to remove salt from the tissue section. Establishing optimal assay conditions For well-established methodologies the optimal conditions under which to perform in situ radioligand binding will have been determined and published. However, for newer radioactive ligands optimal conditions can be rapidly established by varying the parameters listed above systematically. To accelerate this process tissue sections can be exposed to the radioactive ligand under a variety of conditions. However, rather than exposing the sections to autoradiographic analysis, the sections whilst still wet should be ‘wiped’ from the slide onto a filter paper as described previously.

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PROCESSING TISSUE SECTIONS The Drop Technique 1. Remove tissue sections from ultracold storage or the cryomicrotome chamber and thoroughly dry at room temperature or in a stream of warm air (e.g. hair dryer). 2. Add the radioactive ligand or the radioactive ligand and non-radioactive ligand to the sections in a sufficient volume of buffer to ensure the entire tissue section is covered. 3. To avoid excessive evaporation of the assay medium place the slides in a humidifier chamber (see below). 4. Incubate the tissue sections with the assay medium at the appropriate temperature for the appropriate time. 5. Tip the assay medium from the sections. 6. Wash the sections an appropriate number of times in a large volume of buffer in a Coplin jar or other suitable container. 7. Rinse the sections in distilled water to remove excess ions. 8. Thoroughly dry the sections under a stream of cool air and in some circumstances dry using vacuum and desiccation. 9. In the darkroom, appose the section and a relevant radioactive calibration scales (see below) to an appropriate autoradiographic film in an autoradiographic cassette (e.g. Kodak X-Omatic, Amersham Hypercassette). 10. Expose the section and film for the time required to generate an image of the correct intensity. Storage should be where the cassettes will not be disturbed to ensure the slides and calibration scales do not move and produce blurred or ‘ghost’ images. NB: Exposure time will relate to the specific activity of the radioactive ligand and the quantity of the ligand bound to the tissue. Exposure time, which will often be derived by trial and error, needs to ensure the area of interest in the resulting autoradiographs have optical densities between 0.05 and 0.8. 11. Remove film from the cassette and develop using an appropriate developer (e.g. for Amersham Hyperfilm-[3H] recommended developers are Kodak D19, Ilford D19, DuPont GAP 30). When developing film rubber tipped forceps should be used to avoid damage to the photographic emulsion. 12. Wash developed film extensively (e.g. 10 minutes in running water), fix and dry thoroughly. 13. Analyse the image on an image analysis system–ensure that the OD reading of the section is in the linear portion of the standard calibration curve. 14. Stain the sections (e.g. cresyl violet) so that anatomical regions can be accurately identified in reference to the autoradiographic image.

The Slide Immersion Technique 1. Follow steps 1 and 2 for the drop technique. 2. Add sufficient buffer containing radioactive ligand or radioactive and nonradioactive ligand to a slide mailers so that all sections will be completely immersed.

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3. Place slides in slide mailers. 4. Incubate the tissue sections with the assay medium at the appropriate temperature for an appropriate time. 5. Remove the slides from the slide mailer. 6. Follow steps 7 to 14 for the drop technique.

Designing a humidifier chamber A humidifier chamber can be constructed from 4 mm clear acrylic measuring 10 cm wide×30 cm long×2 cm high (Figure 4.2). The base of the ‘moist box’ is covered with a strip of 1 cm thick black foam (to assist in visualising the sections on the slides) which can be moistened prior, and if necessary, during the incubation to help maintain humidity. Up to 10 slides can be accommodated inside such a humidifier chamber which is sealed with an overhanging clear acrylic lid. VISUALISATION Most commonly, autoradiography relies upon the generation of images using photographic emulsion to detect the radioactive ligand bound to the tissue sections. The film used in autoradiography consists of a single coat of emulsion, with no protective layer, on one side of a support medium. This design of film allows intimate contact between the tissue section and the emulsion

Figure 4.2 A design for a humidifier chamber that can be constructed from acrylic

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which means there is a short path for emitted radiation to travel between the section and film emulsion. Having such a short path minimises the scattering of the emitted radiation which is critical to ensure a resulting high resolution autoradiograph. Where cellular localisation of radioactive ligand binding is required, nuclear emulsions such as Amersham LM-1 or Kodak NTB, can be used. These emulsions are highly sensitised solutions of silver halide crystals which can be used dry on emulsion-coated cover slips or on tissue sections partially fixed in paraformaldehyde vapour. The major advantage of using nuclear emulsions is that the small grain size (less than 0.2 µm) allows for very high resolution images to be produced. Thus, nuclear emulsions can be used when obtaining cellular rather than regional localisation of a molecule is the prime objective. Unfortunately, these techniques are rather laborious and, with the exception of improved resolution, do not confer any major advantages over film-based autoradiography. Most recent, a non-emulsion technique for detecting radioactivity on sections has been developed which uses photo-stimulable phosphor crystals to absorb and store energy from radioactive emissions. When such crystals are subsequently stimulated by a laser this stored energy is released as luminescence which can

Figure 4.3 The binding of [3H]phorbol 12, 13 dibutyrate to Brodmann’s area 9 in the human frontal cortex in the absence (total binding) or presence (non-specific binding) of phorbol 12, 13 dibutyrate

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then be captured and digitised to form a latent image that is subjected to quantitative analysis. Importantly, latent images can be generated by opposing the tissue sections to a phosphor crystal plate for a shorter time than would be possible using photographic film. Moreover, in instruments such as the Fuji BAS 5000, the resolution with the phosphor crystal technique is in the order of 25 µm which compares favorably with that of 18 µm achieved with film-based emulsions. Whichever methodology is utilised the aim is to have the largest signal above noise as possible. Thus, the intensity of the non-specific binding image should be less than 20% of that for the total binding image (Figure 4.3).

Image calibration If the experimental design includes quantitation of the autoradiograph, the density of the image needs to be calibrated to a known concentration of radioactivity. Most laboratories find it convenient to use commercially available standards for this purpose. Calibration standards for [3H] and [125I] made from pieces of methacrylate polymer, each containing a different concentration of isotope, are available (e.g. Amersham). These standards are designed to produce a linear gradation of optical densities which are representative of exact levels of radioactivity and span the effective density range of the film used to produce the autoradiograph. In the case of tritium standards two calibration strips may be needed. One strip should be of a high enough level of radioactivity to require an exposure of 1–8 weeks, whilst the other would need to be exposed for 2–12 months. Commercial standards, such as those supplied by Amersham, may also be standardised to what is termed ‘tissue equivalents’. This is achieved by impregnating rat brain with increasing concentrations of 2-deoxy [3H] glucose to approximate the quenching effects of tritium by grey matter, which is then factored into the results obtained with the calibrated standards. Where commercial standards are not available similar standards can be made ‘in-house’. Thus, sections of the same thickness as the tissue are cut from a 5 mm diameter tissue core that has been snap frozen in isopentane at –40 °C. Six to eight sections of the tissue core are thaw-mounted onto a gelatine coated slide, equidistant apart and air-dried. A range of known iodine125 dilutions are applied to the tissue discs in a volume of 5 ml and dried. Mean protein content is determined on adjacent sections of the tissue which have been digested in 1.0 M sodium hydroxide. The radioactivity on each tissue disc is then corrected for decay to the middle of the exposure period and will enable conversion of the

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optical density of the autoradiographs to be converted to dpm or radioligand bound to unit area or, more commonly, fmol radioligand bound/mg protein. Post-exposure processing When carrying out film-based autoradiography it may become necessary to gain some impression of the precise anatomical localisation of the molecule of interest. This can be achieved by comparing the autoradiographic image with its corresponding tissue section that has been stained after exposure to the film. The most common type of stain used in such a situation would be a simple nuclear stain, such as cresyl violet. STAINING WITH CRESYL VIOLET 1. Fix each section by immersion in 10% solution of formalin in phosphate buffer (10ml commercial formalin, 0.701 g NaCl, 0.522 g KH2PO4, 0.179 gNa2HPO4 90 ml dH2O) for 30 minutes to one hour. 2. Prepare a 1.0% (w/v) solution of cresyl violet (Sigma C1791) in distilled water. This solution can be stored for several months. 3. Prepare a 0.1% solution of cresyl violet acetate in glacial acetic acid (10 ml of 1% cresyl violet+1 ml acetic acid+89 ml distilled water). 4. Rinse fixed tissue sections briefly in water. 5. Incubate rinsed sections with cresyl violet for 5–10 minutes at 37 °C. 6. Rinse sections in water. 7. Differentiate the sections by placing them in 70% ethanol and then 100% ethanol. 8. Clear the sections by placing them in 2 changes of xylene. 9. Cover section with an appropriate mounting medium such as DPX (BDH/ Merck 36029) and a coverslip.

Analysis With the ongoing development of computer technology it has become possible to utilise software that allows the quantification of autoradiographs. Typically, a system to quantify autoradiographic images consists of a light box with a stable light source, a cooled CCD camera and a computer containing appropriate software (e.g. MCID software from Image Research Inc.) Where cellular localisation of a molecule is desired the image would be viewed using an appropriate microscope with a CCD camera attached. Limitations of image capture using a CCD camera One of the most critical components of such systems is the camera which allows the image to be captured prior to digitisation into spatial elements (i.e. pixels)

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which are then stored as information in a Cartesian co-ordinate system. The spatial resolution of most medium resolution cameras is 640×480 pixels, however resolution with these cameras is often further limited by the pixel count of the frame buffer. Together these factors result in the capture of low to medium resolution images. More recently, 1024×1280 pixel cameras have become commonly available and would be the camera of choice. Importantly, with camera-based analysis systems, the overall variation in repeated analysis of density should be less than 1%. To achieve such consistency all systems will have procedures that must be followed to ensure an image is captured under optimum conditions. This will involve establishing a fixed specimen magnification at a fixed illumination intensity adjusted to a blank field of view at medium density. Quantitative autoradiography is based on the principle that an increase in the optical density of an image represents an increase in the amount of radioligand bound to the tissue. Unfortunately, the relationship between image density and ligand concentration is not strictly linear, largely due to the limitations of the emulsion itself. At high ligand concentrations photographic emulsions exhibit image saturation such that small increases in optical density represent large increases in tissue radioactivity. This problem of non-linearity can be compounded by the limited dynamic range of the camera which may cause inaccuracies at low or high densities. The sensitivity of image analysis systems is governed by the format in which the image is encoded. Thus, most systems support 8-bit precision which translates to 256 grey levels ranging from black (0) to white (255). This range of values, however, has no fixed reference point and is only useful in providing relative measures of the optical densities encountered within any given sample. The most common way of overcoming these limitations is to standardise the densitometric instrument against images produced by calibration strips as discussed previously. Such an approach allows the creation of a calibration table against which the range of densities in the images of the tissue sections can be analysed. Importantly, there is a limitation with this technique relating to the sensitivity of the system because the 8-bit CCD cameras are limited to recording 256 grey levels. For example, if a system is calibrated from 0 dpm/mg TE to 60,000 dpm/mg TE the theoretical value of each grey scale, assuming linearity, is 230 dpm/mg TE (60,000 divided by 256). In practice, experimental sensitivity would be less than this value. If high sensitivity is viewed as essential then a more sophisticated camera (such as the Princeton Instruments range) offering up to 16-bit precision should be obtained.

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Limitations of image capture using phosphor plates Significantly, the dynamic range of phosphor plates used in systems such as the Fuji BAS 5000 is significantly greater than photographic emulsions or film. In addition, the dynamic range is essentially linear which overcomes problems at high and low densities. In addition, the image is captured in a 16-bit format which translates into a grey scale of 65536 levels making the system, in the main, more sensitive to variations in image density. However, at present this is not matched by the availability of calibrations scales with smaller incremental increases in levels of radioactivity. Furthermore, it is essential that appropriate image analysis software is used with phosphoimagers. This software needs to accommodate the need to extrapolate accurately to get messages of very high or very low intensity images. However, one disadvantage of phosphor plate technology is that it involves large costs when the system is initially established. Furthermore, the ongoing costs related to maintaining such a system will relate to the care of the phosphor plates. However, such disadvantages may well be outweighed by the advantage of increased working ranges, increased sensitivity, good resolution and rapid image exposure times. SUMMARY In situ radioligand binding with quantitative autoradiography is now a wellestablished technique. In disease states, where neuroanatomical localisation of molecular change is viewed important, autoradiography can be a powerful research tool. Furthermore, advancing technology is improving image production times and is increasing image resolution making the use of autoradiography increasingly attractive. However, as with other techniques, it is important to be aware of its technical limitations. References Benes, F.M., Khan, Y., Vincent, S.L. andWickramasinghe, R. (1996) Differences in the subregional cellular distribution of GABAA receptor binding in the hippocampal formation of schizophrenic brain. Synapse, 22, 238–349. Bird, E.D., Spokes, E.G.S. and Iversen, L.L. (1979) Increased dopamine concentration in limbic areas of brain from patients dying with schizophrenia. Brain, 102, 347–360. Donnan, G.A., Kaczmarczyk, S.J., McKenzie, J.S., Kalnins, R.M., Chilco, P.J. and Mendelsohn, F.A.O. (1989) Catecholamine uptake sites in mouse brain: distribution determined by quantitative [3H]mazindol autoradiography. Brain Res., 504, 64–71.

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Javitch, J.A., Blaustein, R.O. and Snyder, S.H. (1985) [3H]mazindol binding associated with neuronal dopamine and norepinephreine uptake sites. Mol. Pharmacol., 26, 35–44. Kuhar, M.J. (1991) Perspectives on receptor autoradiography in human brain. In Receptors in the Human Nervous System, edited by F.A.O. Mendelsohn and G.Paxinos, pp. 1– 8. London: Academic Press Ltd. McFarthing, K. (1992) Selection and synthesis of receptor-specific radioligands. In Receptor– ligand Interactions: A Practical Approach, edited by E.C.Hulme, pp. 1– 18. Oxford: Oxford University Press. Mackay, A.V.P., Iversen, L.L., Rosser, M., Spokes, E., Bird, E., Arregui, A., Creese, I. and Snyder, S.H. (1982) Increased brain dopamine and dopamine receptors in schizophrenia. Arch. Gen. Psychiatr., 39, 991–997. Presland, J.P. and Strange, P.G. (1991) pH dependence of sulpiride binding to D2 dopamine receptors in bovine brain. Biochem. Pharmacol., 41, R9–R12. Stanley, M., Virgilio, J., and Gershon, S. (1982) Tritiated imipramine binding sires are decreased in the frontal cortex of suicides. Science, 216, 1337–1339. Stephenson, R.P. (1956) A modification of receptor theory. Br. J. Pharmacol., 11, 379–393.

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IN SITU HYBRIDISATION HISTOCHEMISTRY: APPLICATION TO HUMAN BRAIN TISSUE Richard E.Loiacono and Andrew L.Gundlach

The biochemical phenotype or functional properties of a given cell can be determined by its pattern of gene expression. In turn, gene expression can be identified by measuring specific mRNA species within a cell using the powerful technique of in situ hybridisation histochemistry (ISHH). This involves the use of a labelled nucleotide probe with a sequence complementary to all or part of the sequence of target mRNA within the cell, and its hybridisation to that target mRNA. Furthermore, ISHH is a histochemical technique in which cell-to-cell relationships are maintained. Thus, macroscopic or microscopic localisation of the probe-mRNA hybrids in tissue sections can be used to precisely determine the cell type and structural organisation between different cells types expressing the gene(s) of interest. ISHH can be used to characterise neurones based on their expression of receptors for neurotransmitters, neuropeptides, synthetic enzymes for non-peptide neurotransmitters, signal transduction systems and structural proteins. ISHH allows the examination of characteristics and functional state of neurones and neuronal systems in response to external physiological and pathophysiological events, or in response to developmental signals during neurogenesis. As with any technique there are methodological limitations associated with ISHH. ISHH will usually only label neuronal cell bodies or associated dendrites, as this is where the majority of mRNA and the machinery involved in mRNA translation is located. Thus, ISHH does not provide information on the axonal projections of labelled neurones or the final location of the gene product. Furthermore, as ISHH only detects target mRNA, it does not provide information concerning the post-translational processing of gene products. ISHH can be readily applied to the examination of gene expression in pathophysiological conditions and diseases where no genetically linked 85

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influences are readily apparent. In addition, in several neurological disorders where genetic linkages have been established, ISHH can assist in determining how the linked gene produces the biochemical phenotype of the disorder. Thus, ISHH has become widely used as a method for the characterisation and localisation of mRNA in brain tissue. Importantly, ISHH is completely feasible for many investigators who are not directly trained in, or equipped for, molecular biological techniques. Following its introduction ISHH was adapted and popularised in the early 1980s for use in brain and other tissues of experimental animals (e.g. Lewis et al., 1985; Young et al., 1986, 1990). More recently, the application of ISHH to human brain tissue obtained at autopsy has been described (e.g. Mengod et al., 1990) along with special requirements needed in the collection and processing of this tissue (Harrison and Pearson, 1990). The methodologies for ISHH described here are essentially those described by Wisden and Morris (1994). PRE- AND POSTMORTEM INFLUENCES ON NEURONAL mRNA In studying neuronal mRNA levels in human brain it is important to consider factors that may affect the technique of ISHH when applied to investigate human neuropsychiatrie diseases (Barton et al., 1993). Generally, cellular mRNA is very stable after death and persists in frozen tissues almost indefinitely; ribonucleases are compartmentalised and not in contact with cellular mRNA in frozen tissues. However, there are two main factors that may affect neuronal mRNA levels in unfrozen tissue: postmortem interval (PMI) and agonal state. FACTORS AFFECTING mRNA IN BRAIN TISSUE COLLECTED POSTMORTEM PMI • • • •

PMI of up to 36 hours does not appear to be associated with any substantial decline in extractable human brain mRNA. Individual mRNA species do not appear to differ in their susceptibility to the effects of PMI. There may be a modest decline in mRNA if PMI is greater than 48 hours (Barton et al., 1993). The histochemical approach of ISHH depends on good tissue morphology for meaningful results, and it is therefore important to match PMI for

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tissue from subjects with a mental illness to that for the tissue from the control subjects. For ISHH, tissue processing and treatment conditions must preserve cellular integrity.

•

Agonal states •

Factors such as age and events such as coma, hypoxia, ischaemia, pyrexia, seizures, endocrine status, hypoglycaemia, dehydration, food deprivation and drug administration have substantial effects on the expression of individual mRNAs in experimental animals. Such variables are impossible to eliminate from studies using human brain tissues. Nonetheless these variables need to be considered and controlled as carefully as possible. It may also be necessary to use internal controls, comparing disease-affected with disease-spared regions of the same brain to attempt to account for interactions between agonal states and the effects of any neuropsychiatrie or neuropathological disease on human brain mRNA levels. The number of specimens from different individuals should be high to provide sufficient statistical power to account for inherent variation amongst humans in any particular study.

•

•

PROTOCOLS FOR ISHH Generally, these procedures should be carried out under sterile conditions (sterile plastics, gloves, sterile or baked glassware, etc.) to minimise the risk of RNAase contamination of tissues and/or equipment. DNA oligonucleotides are more resistant to breakdown than RNA probes, which is the main reason for an RNAsefree system, rather than any potential loss of tissue mRNA. PREPARATION OF SLIDE-MOUNTED BRAIN SECTIONS FOR ISHH

Equipment and material requirements • • • • • • • •

Autoclave Baking oven (temperatures up to 180 °C) Chloroform Continental staining troughs and matching slide racks Cryostat Diethyl pyrocarbonate (DEPC) 100% ethanol -70 °C freezer

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•

Glass slides (washed)

•

Poly-L-lysine hydrobromide (Sigma)

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Sealable plastic tupperware®-like tubs (~ 20×30×5 cm; these will fit 6 slide boxes)

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Slide boxes (Becton-Dickenson)

Solutions •

•

Diethyl pyrocarbonate (DEPC)-treated solutions: to destroy any RNAase contamination, solutions are generally treated with DEPC (0.1% v/v) in a fumehood and solutions then autoclaved. Poly-L-lysine solution: poly-L-lysine hydrobromide (100 mg) is dissolved in 20 ml DEPC-treated H2O and stored as 1 ml aliquots at -20 °C. On the day of use, a 1 ml aliquot is thawed and diluted to 50 ml with DEPC-treated H2O, giving a 0.01% solution ready for use.

Slide preparation 1. Glass slides (up to 200) are wrapped in aluminium foil and baked at 180 °C for at least 4 hours. 2. Slides are cooled, individually dipped into a 0.01% solution of poly-L-lysine and allowed to dry (air dry or in a oven at 25 °C) standing upright in a slide rack (frosted edge down to avoid streaking on the slide surface). 3. Once dry, the slides are repacked in foil and can be stored indefinitely at -20 °C. NB: Coating slides with poly-L-lysine ensures the adhesion of the tissue section to the slide for subsequent ISHH protocols. Several other adhesive treatments are available including pre-coated slides (e.g. silane solutions, or ‘Probe-on’ slides from Fisher Scientific). The cost effectiveness of these has not been investigated in our laboratories.

Processing of brain tissue 1. ISHH is routinely done using fresh frozen brain tissue. In the case of human brain tissue, fresh tissue can be firstly cut into ~ 2 cm square blocks. 2. The blocks of frozen brain tissue are placed in isopentane that has been cooled to -70 °C in liquid N2 or on solid CO2 for approximately 15–30 seconds. NB: It is advisable not to freeze the blocks directly in liquid N2, as this may crack or shatter the tissue block. An alternative that we commonly use is to float a plastic weighing boat containing the tissue on the surface of liquid N2. 3. Once frozen the tissue blocks are wrapped in Parafilm® (American National Can Co.) and foil, prior to storage at -70 °C. NB: The stability of mRNA in this material stored in this manner is excellent.

Tissue sectioning and processing 1. On the day of sectioning frozen blocks are equilibrated to -15 ° to -20 °C in a cryostat for 30–60 minutes.

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2. Commonly, 14 µm tissue sections are cut and mounted on poly-L-lysinecoated glass slides but 5 to 20–30 µm sections may be used, depending on the resolution or type and strength of signal required. 3. The sections on the slides can either be dried in air (~ 30 minutes to 1 hour) or on a warming plate at 25 ° to 30 °C (~ 1 to 2 minutes). Once dried, sections can be stored for brief periods (overnight to a few days) at -20 °C or -70 °C, prior to further processing, but slide boxes should subsequently be equilibrated to room temperature before opening to avoid condensation on the slides. 4. Once at room temperature sections are placed in slide racks and transferred through a series of solvent washes using Continental Staining Troughs: (a) 70% v/v ethanol in DEPC-treated H2O for 2 minutes. (b) 95% v/v ethanol in DEPC-treated H2O for 2 minutes. (c) 100% ethanol for 2 minutes. (d) Chloroform for 10 minutes. (Chloroform should be used in a fumehood.) (e) 100% ethanol for 2 minutes. Sections can then be fixed in 4% w/v paraformaldehyde solution, but this is not absolutely necessary for good results (Dagerlind et al., 1992). A good signal to background ratio can be obtained by simply dehydrating sections in ethanol followed by delipidation in chloroform (e.g. Ryan & Gundlach, 1996). It is important to note that conditions of fixation are a compromise; strong fixation yields better preservation of cellular morphology, but increased cross-linking lowers the accessibility of probe to target mRNA and may increase background. Brain tissues areas such as white matter tracks have high lipid concentrations and tend to bind oligonucleotide probes non-specifically in unfixed sections, however this can be minimised by delipidating the sections using chloroform as described. 5. Processed sections can be stored indefinitely in unsealed slide boxes submerged under 100% ethanol and sealed in large plastic tubs in an appropriately vented cold room at 4 °C, NOT in refrigerators, because of the risk of solvent ignition. Sections stored in ethanol can be easily viewed, selected and regrouped without the need for freeze-thawing of frozen, slide-mounted sections (Wisden & Morris, 1994).

Design of oligonucleotide probes for detection of mRNA Oligonucleotides for ISHH are generally short (usually 20–50 nucleotides in length), single-stranded lengths of DNA complementary to the target mRNA of interest (Lewis et al., 1985; Stahl et al., 1993). This length of oligonucleotide ensures a high level of specificity of the oligonucleotide for a unique sequence on the mRNA of interest and readily penetrates fixed or unfixed tissue sections. The use of oligonucleotides for studies in human brain is assisted, but not necessarily restricted, by the availability of the human cDNA sequence for the gene of interest. If this is unavailable, oligonucleotides directed against regions

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of the sequence for a rodent or other species may, on a trial and error basis, give positive results in human tissue. Oligonucleotide probes can be readily obtained through commercial sources or possibly through a central facility available in a research institute or university. The cost of such services is low as a typical ‘research quantity’ synthesis provides 50 nmol (~ 500 µg) of oligonucleotide and, typically, 0.2–0.5 ng is sufficient to hybridise 2 to 3 sections of tissue on a standard microscope slide. A single synthesis should provide an almost indefinite supply of oligonucleotide for ISHH. Oligonucleotides are resistant to RNAases and are quite stable, and so concentrated stocks can therefore be stored frozen for long periods without significant degradation. In selecting oligonucleotide probes for targeting a specific mRNA, it is usually necessary to identify regions of the mRNA sequence that show the lowest sequence homology (<25%) between closely related members of a given gene family (e.g. different neuronal receptor or enzyme subunits; Stahl et al., 1993) in order to differentiate between such closely-related transcripts. Once this region is identified an oligonucleotide can be designed to target it. Alternatively, oligonucleotide probes can be targeted to regions of the mRNA sequence that are highly conserved, in order to be able to use the same oligonucleotide probe to detect several related target mRNAs, such as related receptor isoforms. OLIGONUCLEOTIDE PROBES FOR ISHH 1. Obtain DNA sequence of gene of interest from the original publication or a commercial database. NB: The National Centre for Biotechnology and Information runs a gene database that can be accessed through the World Wide Web at the address . A keyword search for the gene of interest is carried out using the ENTREZ search system (a submenu in the NCBI Resources Home Page) to search the database for the protein of choice. Once the protein of interest is identified the DNA sequence coding for this protein can be directly retrieved together with the original publication details. Vector NTI Viewer (Informax Inc) is a useful program that can be used in viewing and navigating the DNA sequence (it also possesses some limited tools for searching the sequence and identifying potential oligonucleotide probes). Vector NTI Viewer supports the NCBI Genbank file format and can be obtained at the internet address . Published DNA sequences are given as the coding strand of DNA. The oligonucleotide probes sequence designed to detect the transcribed mRNA are constructed to be complementary and ‘antisense’ to this sequence. 2. Locate the region of the gene with least homology to other genes, especially from closely related members of a given gene family.

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3. Manually identify candidate oligonucleotide sequences that will target this region. NB: With standard hybridisation protocols oligonucleotides of 39–48 nucleotide bases in length are generally used and the total of G and C nucleotides should be 50–60% of the oligonucleotide probes base content. Oligonucleotide probes with large numbers of AT residues will form less stable hybrids with the target mRNA, while oligonucleotides with large numbers of GC residues will form more stable hybrids and affect the ‘stringency’ and Tm (see below) in the hybridisation protocol. Many investigators prepare more than one oligonucleotide directed at nonoverlapping regions of the target mRNA. This is useful for demonstrating the specificity of the hybridisation results obtained and it also allows the amplification of the hybridisation signal, particularly when detecting lower abundance transcripts. (Another useful control for hybridisation is the use of labelled sense probes or random/ scrambled oligonucleotides to demonstrate accurately the level of nonspecific hybridisation.) 4. To ensure an oligonucleotide probe sequence is unique and does not detect sequences related to, but distinct from, the target mRNA, a search for sequence similarity is facilitated by the use of a BLAST search (basic local alignment search tool; also a submenu in the NCBI Resources Home Page). The section of the DNA sequence from which the oligonucleotide probe is derived is submitted and all the related and unrelated sequences that show some or total homology with the selected DNA sequence are identified. 5. It may be necessary to again manually redefine an appropriate oligonucleotide sequence. This last step is useful in verifying the oligonucleotide probe selection process; a BLAST search should identify with 100% identity the original DNA sequence from which the oligonucleotide probe was designed. Generally, probe sequence similarity less than 95% with a non-target mRNA (i.e. 2–3 bases different in a 39 mer) should not produce a positive hybridisation signal using the stringency conditions described in the current protocol.

Labelling oligonucleotide probes For the detection of mRNA by radioactivity, oligonucleotides are made radioactive by the enzymic addition of a radiolabelled ‘tail’ to the oligonucleotide using one of several possible radioisotopes, each with its own distinct advantages and disadvantages. Most commonly used, 35S-labelled oligonucleotide probes give good resolution equal to roughly one cell in diameter (10–15 µm). For a moderately abundant target mRNA, the use of 35S-labelled probes can give relatively fast results via short autoradiographic exposure time on X-ray film (range of 1 to 3 weeks). By contrast, exposures to photographic emulsion are quite lengthy. The half-life of this isotope is 87 days, and labelled probes should be used within a month. 35S-labelled probes are subject to oxidation; thus it is important

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to include a reducing agent (such as DTT) in all solutions to protect the sulphur from oxidation. Standard safety precautions should be used in handling 35Slabelled probes (β-particle radiation; principle energy 167 keV). 32 P-labelled oligonucleotide probes have a higher specific activity than 35Slabelled probes and, for moderately abundant target mRNA, can give very rapid results via very short autoradiographic exposure to X-ray film (typically in the range of a few days). 32P-labelled probes generally give a poorer resolution of signal (several cells in diameter; 20–30 µm); the half-life of this isotope is only 14 days and this tends to negate its higher specific activity. All the above notwithstanding, 32P-labelled probes are extremely useful in detecting low abundance target mRNA. Labelled probes should be used within the week. The high specific activity of 32P-labelled probes necessitates the use of special safety measures. Film badge dosimeters, acrylic plastic shielding screens and containment boxes (5–6 mm thick) should be used in the handling and use of labelled probes (β-particle radiation; principle energy 1710 keV). 33 P-labelled oligonucleotide probes give better resolution of signal than 32Plabelled probes, but slightly less than 35S-labelled probes (several cells in diameter; 15–20 µm). Given the higher specific activity, the use of 33P-labelled probes can give fast results with little loss of cellular resolution. Autoradiographic exposure times on X-ray film are roughly half as long as 35S-labelled probes for moderately abundant target mRNA (1 week). It is claimed by some investigators (mostly using riboprobes) that 33P- is preferable to 35S- for labelling probes (McLaughlin & Margolskee, 1993; Liu & Salpeter, 1994). It is our experience that a problem in using 33P-labelled oligonucleotide probes may be an inability of photographic emulsions to effectively detect sufficient 33P emissions to allow cellular resolution of the hybridisation signal. (In our laboratory, the exposure of sections labelled with a number of different 33P-labelled probes directed against both rare and moderately abundant mRNA species to several different brands of nuclear emulsion for up to 20 weeks have been unsuccessful in detecting hybridisation-associated silver grains.) A further disadvantage in using 33Plabelled probes is that they should be used within a week: the half-life of this isotope is only 25 days. Standard safety precautions should be used in handling 33 P-labelled probes (β-particle radiation; principle energy 248 keV). Oligonucleotides are labelled by using terminal deoxynucleotidyl transferase (TdT) and radiolabelled deoxyadenosine triphosphate (dATP). TdT catalyses the addition of radiolabelled dA residues to the 3' end of the oligonucleotide. The end result of the labelling reaction is a radioactive poly-deoxyadenylic (poly-dA) ‘tail’ at the 3'-end of the oligonucleotide; each radioactive dA residue added contributing to the overall specific activity of the oligonucleotide probe. The specific activity can

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be controlled and altered by altering the incubation time and the ratio of concentrations of radiolabelled dATP to oligonucleotide. A 30:1 molar ratio of radiolabelled dATP to oligonucleotide appears to be optimal; longer poly-dA ‘tails’ tend to show higher non-specific hybridisation, shorter poly-dA ‘tails’, labelling that require longer exposure times for adequate detection (Wisden & Morris, 1994). There are also protocols available for the non-radioactive detection of mRNA using a range of different detection systems, with alkaline phosphatase-based systems quite popular for use with oligonucleotides (Kiyama et al., 1990; Trembleau & Bloom, 1995). Such techniques are useful when very high resolution or co-localisation is required and are appropriate (using oligonucleotides) when the target mRNA is very abundant, and/or large numbers of probes are used simultaneously (Trembleau & Bloom, 1995). There is no potential reason why these protocols should not be suitable for use in human brain, but the details of these procedures will not be further discussed here. LABELLING OLIGONUCLEOTIDE PROBES

Equipment and materials • • • • • • • • • • •

• • •

•

Autoclave Dithiothreitol (DTT; Sigma/Aldrich) EDTA Fine glasswool Heated incubation block or water bath (accurate at 37 °C) Liquid scintillation counter Low speed centrifuge NaCl 1 ml plastic syringes (Terumo) 1.5 ml and 10 or 15 ml plastic centrifuge tubes Radiolabelled dATP (35S-dATP, 33P-dATP or 32P-dATP; MEN). These nucleotides are packaged in EasyTide® form, consisting of aqueous solutions buffered with Tricine-Tris pH 7.6, containing a dye and stabiliser. Sephadex® G-25 medium (Pharmacia) Sodium dodecyl phosphate (BDH) Terminal deoxynucleotidyl transferase (TdT) labelling kit (Boehringer-Mannheim). This kit contains 25 U/ml TdT, 25 mM CoCI2 and 5×reaction buffer (1 M potassium cacodylate, 125 mM Tris buffer, pH 6.6, and 1.25 mg/ml bovine serum albumin) Tris HCI/Tris base

Solutions Dithiothreitol (DTT) solution: DTT (3.09 g, Sigma/Aldrich) is dissolved in 20 ml DEPC-treated H2O and stored as 1 ml aliquots at -20 °C. On the day of use, a 1

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ml aliquot is thawed and used as required. Only refreeze once or twice before discarding.

Sephadex® spin columns Sephadex® solution 1. Add 14 ml 5.0 M NaCl, 10 ml 1.0 M Tris buffer (pH 8.0), 5 ml 0.5 M EDTA (BDH; pH 8.0), 5 ml 5% w/v sodium dodecyl phosphate (BDH) to 466 ml of DEPCtreated H2O 2. Mix well. 3. Add 20 g Sephadex® G-25 medium (Pharmacia). 4. Mix. 5. Autoclave and store at 4 °C. Spin columns are prepared just prior to oligonucleotide labelling and separation.

Single-use spin column 1. Pack a small wad of autoclaved fine glasswool into the tip of 1 ml plastic syringe body (final packed volume of ~ 0.1 ml). 2. Gradually fill the entire syringe body with the Sephadex® slurry (the solution will freely drain, leaving a packed Sephadex® column within the syringe body). 3. The spin column is finally dried by placing the syringe body in a 10 or 15 ml centrifuge tube and spinning at 2000 rpm for 1 minute at room temperature (Sambrook et al., 1989).

Method Preparing 35S-dATP- or 33P-dATP-labelled probes 1. To prepare sufficient labelled oligonucleotide probe to hybridise to 50 slides of mounted tissue sections, add to a 1.5 ml centrifuge tube on ice: (a) 1 µl of a working dilution (0.3 pmol/ml; 5 ng/ml) of oligonucleotide probe (stored at -20 °C). (b) 2.4 µl 5×Reaction Buffer (Boehringer Mannheim TdT labelling kit; stored at 20 °C; the Reaction Buffer can be repeatedly freeze-thawed without loss of activity). (c) 1 µl CoCI2 (Boehringer Mannheim TdT labelling kit; stored at -20 °C; the CoCI2 can be repeatedly freeze-thawed without loss of activity). (d) 1.5 µl 35S-dATPαS (NEG-734H ~ 1300 Ci/mmol) or 33P-dATP (NEG-612H ~ 2000 Ci/mmol; NEN). These EasyTide® formulations can be stored at 4 °C without the need for multiple freezing and thawing required for other formulations. Multiple freezing and thawing may lead to breakdown of the isotopes and ineffective labelling. (e) 5.6 µl DEPC treated H2O. (f) 1 µl TdT enzyme (Boehringer Mannheim TdT labelling kit; stored

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2. 3. 4. 5.

6. 7.

8. 9.

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at -20 °C; the TdT should be removed from the freezer and held on ice only when needed). The addition of TdT starts the reaction. Mix the reaction mixture after the addition of TdT by repeated ‘drawing-up’ and expulsion of the reaction mixture against the side of the tube using a pipette set at 50 µl. Care should be taken in avoiding the introduction of air bubbles into the reaction mix as this tends to decrease the effectiveness of the enzyme. A short pulse in a centrifuge will settle the reaction mixture contents and remove any air bubbles if they are introduced. Place the 1.5 ml centrifuge tube containing the reaction mix into a 37 °C heated incubation block or water bath for 8–15 minutes. Stop the reaction by the addition of 38 µl of DEPC treated H2O. Draw up the reaction mix (50 µl) and pipette it onto a prepared Sephadex® spin column. Place an empty 1.5 ml centrifuge tube (with the lid removed) in a 10 or 15 ml centrifuge tube. Place the Sephadex® spin column into the 10 ml centrifuge tube inserting the tip into the 1.5 ml centrifuge tube. Spin at 2000 rpm for 1 minute at room temperature. The eluate (50 µl) collected in the 1.5 ml centrifuge tube contains the labelled oligonucleotide probe; unincorporated radionucleotides remain on the column. Test a 2 µl sample for radioactive content by liquid scintillation counting. For 35 S-labelled probes the counts should be in the range of 200,000–500,000 dpm/µl. If the counts are 100,000 dpm/µl or below the specific activity of the labelled probe may be too low and the probe should not be used. If the counts are significantly above 500,000 dpm/µl the specific activity of the labelled probe may be too high and the probe should not be used. Such probes show high non-specific hybridisation, probably due to the presence of a long polydA ‘tail’. For 33P-labelled probes the counts should be in the range of 100,000– 300,000 dpm/µl. If dATPα35S is used as the radiolabelling agent, add 1 µl of 1M DTT to the eluate. The DTT protects the sulphur group from oxidation. 35 S- and 33P-labelled probes can be conveniently stored at 4 °C or -20 °C for up to a month or a week, respectively, but should probably be prepared on the day of hybridisation.

In situ hybridisation Hybridisation of the labelled probe to the target mRNA in the slide-mounted tissue is carried out under optimal conditions for DNA-mRNA annealing and for washing non-specifically bound labelled probe away. Much of this methodology is empirically derived for ISHH. The processes of annealing oligonucleotides to target mRNA that is embedded and cross-linked in tissue sections is not well understood, but nonetheless the methodology developed by Wisden and Morris (1994) and used here is guided by the theory of hybridisation kinetics based on in vitro systems.

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Hybridisation of the labelled probe to the target mRNA depends on the length of the oligonucleotide probe, its base composition (see Design of oligonucleotide probes for detection of mRNA, page 89), on the presence and concentrations of Na+ ions, agents such as formamide and dextran sulphate present in the hybridisation buffer and ultimately on the temperature and duration of hybridisation. The choice of temperature, salt concentration and formamide concentration are critical in determining the ‘stringency’ or specific annealing of the labelled probe to the target mRNA during hybridisation, as opposed to ‘non-specific binding’ or the annealing of the labelled probe to sequences related to, but distinct from, the target mRNA or binding to other cellular material within the tissue section. Generally, the exact hybridisation conditions and required ‘stringency’ are empirically derived for the specific application and methodology. However, a considerable advantage offered by the use of oligonucleotide probes is that if a similar length oligonucleotide is used, then conditions used for in situ hybridisation are not likely to be different for different probe sequences, allowing for a standardisation of hybridisation conditions across all target tissues and target mRNAs. Hybridisation buffer Na+ ion concentration increases the stability and thus the ‘stringency’ of the hybridisation reaction by neutralising repulsive forces between the negativelycharged phosphate groups in the sugar-phosphate backbone of the nucleotide (be it oligonucleotide probe or target mRNA). The rate of hybridisation of probe to the target mRNA is also increased by a high salt concentration. Formamide is a destabilising agent that disrupts hydrogen bonding of the probe to the target mRNA, thus increases the ‘stringency’ of hybridisation. Formamide also lowers the temperature at which effective hybridisation takes place, thus by carrying out the hybridisation process at lower temperatures brain tissue morphology in the slide-mounted section can be better preserved. Finally, the effective probe concentration and rate of hybridisation of probe to the target mRNA can be increased by using a volume exclusion agent such as dextran sulphate. Duration of hybridisation The duration of incubation of slide-mounted sections being hybridised is variable; the majority of the hybridisation reaction will have probably taken place within the first 6 hours following probe application. Most protocols use

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an overnight incubation for convenience; there appears to be little difference in the time course of development of specific hybridisation and non-specific probe interactions over this time period.

Hybridisation temperature Increasing the temperature at which hybridisation takes place increases the ‘stringency’ of hybridisation. The temperature of hybridisation is chosen to be sufficiently ‘stringent’ to reduce or prevent the probe from hybridising to sequences related to, but distinct from, the target mRNA or binding nonspecifically to other cellular material within the tissue section, but low enough to allow a high level of hybridisation to the target mRNA. The temperature of hybridisation also affects the integrity of the tissue; high hybridisation temperatures result in poor tissue morphology. Hybridisation is usually carried out at 15–20 °C below the ‘melt’ temperature or Tm, where Tm is the mid-point of the temperature range over which the oligonucleotide probe dissociates from the target mRNA. At the Tm, 50% of the oligonucleotide-target mRNA pairings are dissociated. The Tm, which links the length of the oligonucleotide probe, its base composition, the presence and concentrations of Na+ ion and agents such as formamide and dextran sulphate present in the hybridisation buffer medium, can be calculated using the following formula: Tm= 16.6 log (molarity of monovalent cations)+0.41 (%GC content of oligonucleotide probe)+81.5–675/(length of oligonucleotide probe)–0.65 (% formamide concentration) Typically, the Tm for synthesised oligonucleotide probes in an entirely aqueous medium (no formamide) is ~ 70–75 °C. Using the hybridisation conditions outlined here, the T m is reduced and typical incubation temperatures are 37–42 °C.

Post-hybridisation washing Following hybridisation, washing of sections is carried out to remove labelled probe that has non-specifically annealed to non-target mRNA or bound to other cellular components within the tissue section. The ‘stringency’ of washing is affected,

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controlled and optimised in much the same way as during the hybridisation conditions. Most protocols use a higher ‘stringency’ during post-hybridisation washing by carrying out the procedures at a higher temperature (55–60 °C). POST-HYBRIDISATION WASHING

Equipment and materials • • • • • • • • • • • • • •

Amberlite monobed resin MB-1 (BDH) Continental staining troughs and matching slide racks Diethyl pyrocarbonate (DEPC) Dextran sulphate (Pharmacia) 100% ethanol Formamide (BDH or Fluka1) Hybridisation oven (accurate temperatures from 35–60 °C) Incubation water bath (accurate temperatures from 35–65 °C) Kimwipes® tissues Na Citrate NaCl Parafilm® (American National Can Co.) 10 or 15 ml plastic centrifuge tubes Plastic petri-dishes or trays to hold multiple slides, face-up

Solutions Deionised formamide 1. 2. 3. 4.

Add 2.5 g of Amberlite monobed resin to 25 ml of formamide. Stir for 30–60 minutes at room temperature. Filter the formamide solution to remove ‘exhausted’ resin. Stored filtered solution at –20 °C until required.

Saline sodium citrate buffer (SSC) 1. To prepare stock solution of 20×SSC buffer (3 M NaCl, 0.3 M Na citrate, pH 7.0) dissolve 175.32 g NaCl and 88.23 g Na citrate in 1000 ml H2O. 2. Treat 20×SSC solution with DEPC (0.1% v/v). 3. Autoclave. 4. Stored at 4 °C or room temperature for up to a month (Sambrook et al., 1989).

Hybridisation buffer 1. Add 25 ml of 100% formamide (Fluka, stored at room temperature; or deionised formamide (BDH), stored at -20 °C) to 10 ml of 20×SSC buffer, 5 ml of DEPCtreated H2O and 5 g of dextran sulphate in a sterile tube. 1

As an alternative, Fluka molecular biology grade formamide can be used without prior deionisation (Wisden and Morris, 1994).

IN SITU HYBRIDISATION

2. Agitate the mixture until all of the dextran sulphate is dissolved (~ 30–60 minutes). 3. Adjust the volume to 50 ml with DEPC-treated H2O. 4. Store the solution at 4 °C (can be stored for several months). NB: This amount of hybridisation buffer is sufficient to hybridise 500 slides with multiple mounted tissue sections.

Method Once the probe has been labelled, in situ hybridisation is performed by applying the labelled probe in hybridisation buffer onto the prepared slide-mounted brain sections, incubating the slide-mounted sections for 6–18 hours to allow hybridisation to occur and then washing the sections to remove ‘non-specific binding’.

In situ hybridisation 1. Warm hybridisation oven to 42 °C. 2. Fold several tissue paper sheets (Kimwipes®) into small 2×2 cm wads. 3. Soak tissue wads in a petri-dish in a mixture of 50% formamide/4× SSC (25 ml formamide, 10 ml 20×SSC buffer and 15 ml DEPC-treated H20). 4. Remove slide-mounted sections from storage in 100% ethanol. 5. Allow the sections to dry for ~ 15–10 minutes. 6. Place dried sections face-up in a petri-dish. 7. Add 50 µl of labelled oligonucleotide probe to 5 ml of hybridisation buffer in a 10ml plastic centrifuge tube (sufficient for 50 slides). The hybridisation buffer mixture should not be stored for lengthy periods (overnight is okay) and should be discarded after use. NB: Unmixed labelled oligonucleotide probe can be stored as described above. 8. Vortex the mixture vigorously. 9. An option to aid spreading of the mixture on the slides is to warm the hybridisation buffer to ~ 42 °C in the hybridisation oven before use. Vortex the mixture vigorously once more to ensure complete mixing of the labelled oligonucleotide probe with the hybridisation buffer. 10. Pipette 70–250 µl of hybridisation buffer-oligonucleotide probe mixture onto the surface of each slide in the petri-dish(es); slowly expelling the pipette contents so that the buffer mixture is evenly distributed across the tissue(s). 11. Immediately place a previously cut-to-size Parafilm® strip over the slide, to act as a coverslip. This will help disperse the hybridisation buffer mixture over the tissue. 12. In each petri-dish containing slide-mounted sections, place a soaked wad of tissue paper (see 2 above) against the edge of the dish and replace the lid. 13. An option is to gently wrap the petri-dish with Parafilm® to help maintain the slide-mounted sections in a moist atmosphere, preventing the hybridisation buffer mixture from drying onto the slide (see below).

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14. Place the petri dish(es) in the pre-warmed hybridisation oven set at 42 °C and hybridise the slide-mounted sections overnight for 8–16 hours.

Post-hybridisation washing 1. Pre-warm the incubation water bath to 60 °C. 2. Place several continental staining troughs containing 1×SSC buffer in the 60 °C water bath and allow to equilibrate to temperature. 3. Remove the petri-dishes from the hybridisation oven and gently remove the Parafilm® coverslip from one slide and immediately place this slide in a rack in a continental staining trough filled with 1×SSC buffer at room temperature. (This removes excess hybridisation buffer mixture from the slide and continues to maintain the slide in an aqueous environment.) 4. Repeat for each slide, one at a time. NB: If the slides are allowed to dry, the hybridisation buffer mixture appears to become ‘baked-on’ resulting in a dark aura surrounding the tissue section when detecting the hybridisation signal on film. 5. Using continental staining troughs, transfer slides through a series of washes as follows: (a) 1×SSC buffer at 60 °C for 1 hour. (b) 1×SSC buffer at room temperature for 1 minute. (c) 0.1×SSC buffer at room temperature for 1 minute. (d) 70% ethanol at room temperature for 30 secconds. (e) 95% ethanol at room temperature for 30 secconds. (f) 100% ethanol at room temperature for 30 seconds. This procedure removes labelled probe that has non-specifically annealed to nontarget mRNA or bound to other cellular components within the tissue section. 6. Remove slide-mounted sections from 100% ethanol and allow to dry for ~ 5–10 minutes.

Detection of the hybridisation signal The detection of the hybridisation signal involves the apposition and exposure of the labelled slide-mounted sections to a sheet of X-ray film or similar (e.g. Hyperfilm β-max, Amersham, or Kodak Biomax MR). Subsequent development of the film results in an autoradiographic image indicating the localisation of the target mRNA within specific cell groups within the slide-mounted section. Film resolution may be sufficient to determine which area or cell type is expressing the target mRNA of interest; however, for an enhanced cellular resolution, which may be essential for very small regions of interest or closely grouped but different cell types within the tissue; slide-mounted sections may

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be directly coated with photographic emulsion and exposed and developed as outlined below to obtain an autoradiographic image. To obtain a suitable autoradiographic image, emulsion-coated slides require ~ 4–5 times the exposure time that X-ray film apposed slides do.

DETECTION OF THE HYBRIDISATION SIGNAL

Equipment and materials • • • • • • • • • • • • •

Continental staining troughs and matching slide racks D-19 developer (Kodak) Double-sided adhesive tape Sheet film development trays Fixer (Ilford rapid fixer or 30% sodium thiosulphate) Glycerol Hyperfilm β-max (Amersham), Kodak Biomax MR film or equivalent Incubation water bath Photographic emulsion (Ilford K5 or equivalent) Safe lights (darkroom lights fitted with Kodak 6B filters or similar) Slide boxes Stop bath (Kodak) X-ray cassettes

Solutions D-19 developer: Add a packet of D-19 powder to tap water according to the manufacturer’s instructions to obtain the final developer solution. D-19 developer solution should be stored in dark bottles until required. The solution can be reused several times before being discarded. D-19 developer solution is a yellow colour when freshly made up and should be discarded when the solution is a darker colour or contains a large sediment of fine, brown-black grains. Rapid fixer: Fixer solution (Ilford) and 30% sodium thiosulphate can be prepared and reused once or twice before being discarded. Photographic emulsion: Photographic emulsion is prepared on the day of use in a darkroom fitted with Kodak 6B safe light or equivalent. Pre-warm a 25 ml solution of glycerol in water (0.5% v/v) to 42 °C in an incubation water bath and add the solid emulsion chunks (Ilford) to bring the volume up to 50 ml, allowing the emulsion to melt and stirring with a wooden cherry-stick. The glycerol makes the emulsion more elastic and prevents cracking of the emulsion. The emulsion is ready for use (in 15–20 minutes) when the solution appears homogenous. The emulsion solution cannot be stored; 50 ml of emulsion is usually sufficient for 100–120 standardsized slides with 2–3 tissue sections mounted per slide to about 1 cm below the frosting.

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Method Exposure to radioactivity-sensitive film 1. Using double-sided adhesive tape, stick the dry slide-mounted sections (section side-up) onto a suitably sized piece of card and ensure that the card fits snugly in an X-ray film cassette. 2. In a darkroom fitted with Kodak 6B safe light place a sheet of Hyperfilm β-max or Kodak Biomax MR onto the sheet holding the slides. (These films are singlesided (the emulsion only coats one side of the film), thus it is important that the emulsion side is down in apposition to the tissue surface of the slides. The emulsion side can be determined by holding the film surface parallel to a safelight source. The shiny side of the film is not emulsion-coated, while the dull surface is emulsion-coated.) 3. Place the apposed film sheet and slide-card into the X-ray cassette ensuring that they fit snugly, pressing the film tightly and evenly against the slides. 4. The X-ray cassette can be stored for film exposure at room temperature in any suitable darkened spot (e.g. a desk drawer where the cassette is not likely to be knocked) for an appropriate time (usually 3 days to 3 weeks), depending on the type of isotope used to label the oligonucleotide probe (see Labelling oligonucleotide probes, page 91) and the relative abundance of the target mRNA. 5. Once exposed for a suitable period, in a darkroom under safelight the film sheet can be removed from the X-ray cassette and developed either in an automatic processor (Kodak film) or in photographic trays (Amersham film) as follows, keeping the emulsion surface of the film facing-up to avoid scratching the emulsion on the bottom of the developing tray. (a) Develop the film sheet in a tray containing D-19 developer for 5 minutes at room temperature. Ensure that the film is completely submerged for complete development, agitating the tray gently every 30 seconds. Remove the film sheet and briefly drain. (b) Place the film sheet in a tray containing Kodak stop bath at room temperature for a minute or so, completely submerging the film and agitating the tray. Remove the film and gently drain. (c) Place the film in a tray containing fixer at room temperature for 5–10 minutes, completely submerging the film and agitating the tray every minute. Remove the film sheet and gently wash the film surface with tap water directly under a tap, drain and air dry the film sheet. 9. Upon development the film should show autoradiographic images of the target mRNA localisation on the tissue sections.

Exposure to photographic nuclear emulsion 1. In a darkroom fitted with Kodak 6B safelights or equivalent, slide-mounted sections are individually dipped into photographic emulsion (ensuring that the emulsion does not contain any air bubbles) and left to dry, in complete darkness, for up to 2 hours.

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2. Place the dried, dipped-slides in light-tight slide boxes, seal these and place them at 4 °C for exposure. The appropriate time of exposure depends on the type of isotope used to label the oligonucleotide probe (see Labelling oligonucleotide probes, page 91) and the relative abundance of the target mRNA. 3. Once exposed for a suitable period, in a darkroom under safelight the dipped slides can be removed from the slide box, placed in slide racks and developed in continental slide troughs as follows: (a) Place the slide rack containing the emulsion-dipped slides in a continental slide trough containing D-19 developer at room temperature for 2 minutes. Ensure that the slides are completely submerged to ensure complete development, agitating the rack every few seconds. Remove the rack and gently drain. (b) Place the slide rack in a trough containing Kodak stop bath at room temperature for a minute, taking care to completely submerge and occasionally agitate the rack. Remove the rack and gently drain. (c) Place the slide rack in a trough containing 30 % sodium thiosulphate fixer for 2 minutes, completely submerging the slides and agitating the tray every 20–30 seconds. Remove the rack and gently drain. (d) Place the slide rack under running tap water for 5 minutes, then remove and drain. (e) Allow the dipped slides to air dry. 4. The dipped slides should be lightly stained with an appropriate histological stain (and coverslipped) to identify the cells of interest. The labelled target mRNA is represented by silver grains overlying the cells within the tissue. Visualisation of the silver grains can be carried out under darkfield or high-power brightfield microscopy. 5. Aspects of the quantitative analysis of ISHH have been previously described (e.g. O’Shea & Gundlach, 1994) and the topic is beyond the scope of this article. Generally, the analysis of film autoradiographs involves the conversion of film optical densities to concentration of isotope per unit area or mg tissue protein through the use of simultaneously-exposed, radioactive standards, while the analysis of photographic emulsion-coated sections involves ‘counting’ grains by computer-assisted methods.

Controls for ISHH An important consideration in the analysis of ISHH results is the use of appropriate controls to determine ‘background’ or ‘non-specific hybridisation or binding’ which represents the annealing of the labelled probe to sequences related to, but distinct from, the target mRNA or sequestration of the probe to other cellular material within the tissue section. Unfortunately, in ISHH, there is no (best) control to determine non-specific binding. There are, however, several controls used to validate the signal obtained from target mRNA:

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(i)

The use of RNAase treatment of slide-mounted sections; any signal obtained following this treatment represents non-mRNA labelling. (ii) Carrying out the hybridisation in the presence of a 100-fold excess of unlabelled oligonucleotide probe; any signal obtained probably represents a non-saturable, non-specific binding site. (iii) The use of multiple oligonucleotide probes each targeting a different segment of the DNA sequence of interest. The observation of a similar distribution for all oligonucleotides suggest a single target mRNA is being detected. (iv) The use of Northern blot analysis using the oligonucleotide probe under the same stringency conditions. Northern analysis should result in a single band of the appropriate molecular weight, suggesting that the signal obtained in ISHH studies is the result of the oligonucleotide annealing to a single target mRNA. (v) The use of a sense oligonucleotide probe, i.e. an oligonucleotide that has a sequence that is identical to the oligonucleotide probe chosen but runs sense rather than antisense to the DNA sequence. The sense probe has the same base sequence, thus any signal obtained with this probe probably represents hybridisation or sequestration to mRNA other than the target mRNA or other cellular components. NB: Controls (ii) and/or (iii) are routinely practiced in our laboratories.

References Barton, A.J.L., Pearson, R.C.A, Najlerahim, A. and Harrison, P.J. (1993) Pre- and postmortem influences on brain RNA. J. Neurochem., 61, 1–11. Dagerlind, Å., Friberg, K., Bean, A.J. and Hökfelt, T. (1992) Sensitive mRNA detection using unfixed tissue: Combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry, 98, 39–49. Harrison, P.J. and Pearson, R.C.A. (1990) In situ hybridization histochemistry and the study of gene expression in the human brain. Prog. Neurobiol., 34, 271–312. Kiyama, H., Emson, P.C. and Tohyama, M. (1990) Recent progress in the use of the technique of non-radioactive in situ hybridization histochemistry: new tools for molecular neurobiology. Neurosci. Res., 9, 1–21. Lewis, M.E. Sherman, T.G. and Watson, S.J. (1985) In situ hybridization histochemistry with synthetic oligonucleotides: Strategies and methods. Peptides, 6 (suppl 2), 75–87. Liu, E. and Salpeter, M.M. (1994) In situ hybridization and cytochemistry: Localization of mRNA at stained neuromuscular junctions with 33P-labeled probes. J. Histochem. Cytochem.,42, 1407–1411.

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McLaughlin, S.K. and Margolskee, R.E (1993) 33P is preferable to 35S for labeling probes used in in situ hybridization. Bio Techniques, 15, 506–511. Mengod, G., Charli, J.-L. and Palacios, J.M. (1990) The use of in situ hybridization histochemistry for the study of neuropeptide gene expression in the human brain. Cell. Mol. Neurobiol., 10, 113–126. O’Shea, R. and Gundlach, A.L. (1994) Quantitative analysis of in situ hybridization histochemistry. In In Situ Hybridization Protocols for the Brain, edited by W.Wisden and B.J.Morris, pp. 57–79. London: Academic Press. Ryan, M.C. and Gundlach, A.L. (1996) Localization of preprogalanin messenger RNA in rat brain: Identification of transcripts in a subpopulation of cerebellar Purkinje cells. Neuroscience, 70, 709–728. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edn). Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Stahl, W.L., Eakin, T.J. and Baskin, D.G. (1993) Selection of oligonucleotide probes for detection of mRNA isoforms. J. Histochem. Cytochem., 41, 1735–1740. Trembleau, A. and Bloom, F.E. (1995) Enhanced sensitivity for light and electron microscopic in situ hybridization with multiple simultaneous non-radioactive oligodeoxynucleotide probes. J. Histochem. Cytochem., 43, 829–841. Wisden, W. and Morris, B.J. (1994) In situ hybridization with synthetic oligonucleotide probes. In In Situ Hybridization Protocols for the Brain, edited by W.Wisden and B.J. Morris, pp. 9–34. London: Academic Press. Young, W.S., III (1990) In situ hybridization histochemistry. In Handbook of Chemical Neuroanatomy, Vol. 8, Analysis of Neuronal Microcircuits and Synaptic Interactions, edited by A.Björklund, T.Hökfelt, F.G.Wouterlood and A.N.van den Pol, pp. 481– 512. Amsterdam: Elsevier Science Publishers. Young, W.S., III, Bonner, T.I and Brann, M.R. (1986) Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc Natl. Acad. Sci. U.S.A., 83, 9827–9831.

6

IMMUNOHISTOCHEMISTRY TECHNIQUES APPLICABLE FOR USE WITH HUMAN BRAIN TISSUE James Vickers

Immunohistochemistry utilises antibodies to localise specific antigens in tissue. While such techniques have been widely used in studies of the nervous system in non-human species, it has only been in recent years that they have seen more extensive utilisation in investigations using human tissue. Immunohistochemistry can reveal the presence of a diverse range of markers. Thus, antibodies can be used to localise a broad range of nervous system components including neurotransmitters, neurotransmitter-related enzymes, receptor and channel subunits, cytoskeletal proteins, protein kinases, protein phosphatases, proteases, second-messenger molecules, membrane-associated proteins, calcium binding proteins, inflammatory markers and non-neuronal cell antigens. Immunocytochemical markers may be used to localise different components of various neurotransmitter systems such as the transmitters themselves, associated enzymes and receptors in the human central nervous system (CNS). Other applications include the use of particular markers to study neuronal morphology and brain cytoarchitecture, as well as the cell type-specific biochemical features that distinguish particular subclasses of neurons. These techniques are applicable for investigations of the pathology of the nervous system, where antibody markers may reveal certain hallmarks of diseases, as well as the cellular alterations associated with neurodegeneration. Immunohistochemistry is also being increasingly used to determine the structural and biochemical underpinning of psychiatric conditions such as schizophrenia. This chapter describes the fundamentals of the immunohistochemical techniques that are particularly applicable to the study of the human CNS and describes how such material is optimally prepared for such techniques. 107

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The various alternatives for visualising immunohistochemical labelling are also reviewed as well as some ‘tricks of the trade’ and trouble-shooting suggestions.

ANTIBODY PRODUCTION: GENERAL PRINCIPLES An antibody for immunohistochemical investigation is usually generated by challenging the immune system of a non-human animal with an antigen of interest. Insoluble antigens, or those combined with an adjuvant, appear to be more effective at stimulating this antibody response. Typical antigens include proteins purified from nervous system material, synthetic peptides or larger proteins generated from cell expression systems. Immunogenicity is often increased by coupling prospective antigens to larger ‘carrier’ proteins. This chapter will not detail antibody generation but rather how they are used for immunohistochemical study. However, it is important to note the differences between monoclonal and polyclonal antibodies. Polyclonal antibodies, traditionally raised in rabbits, are comprised of immunoglobulins derived from different lymphocyte cell lines stimulated by the epitopes on the one antigen. Thus, this mixture of different antibodies would bind to a number of sites on the antigen, but they may also bind to other antigens that have a similar sequence or conformational structure. Polyclonal antisera are often further purified to separate out the antibodies specific for particular proteins/ epitopes. A major drawback of the use of polyclonal antibodies for immunohistochemistry is the limited amount of sera that can be obtained from a given animal, consequently variations in the titre, stability or specificity may occur between batches and between animals. Therefore, if a particular batch obtained commercially proves to be particularly effective for immunohistochemical study, it is advisable to purchase as much of this antibody as can be used, as there is the risk that the next batch may not be as effective. To generate monoclonal antibodies, single clonal lines of lymphocytes are isolated that have a high degree of specificity for a particular domain on the antigen. To immortalise an antibody-producing cell line, lymphocytes from the spleen of animals (usually mice) challenged with a particular antigen are fused with myeloma cells. These hybridomas are then selectively screened for their specific reactivity to the antigen of interest. Culture fluid (supernatant) from these hybridomas can then be used for immunohistochemistry. In order to

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produce higher titres of the antibody, the hybridoma can be injected back into the peritoneum of mice and the ascitic fluid collected from the tumours that develop. The distinct advantage of monoclonal antibodies include their high degree of specificity for particular epitopes, as well as the relative reliability of producing as much of a given antibody as required.

ANTIBODIES FROM COMMERCIAL SOURCES A broad range of antibodies suitable for immunohistochemical studies are now available from a variety of companies. For those commencing immunohistochemistry, it is important to keep a number of factors in mind when purchasing antibodies. For example, there are usually many companies offering antibodies to the same markers in varying concentrations, volumes and working dilutions. In addition, for monoclonal antibodies, the exact same clones may be available from different companies for different prices. A further important consideration is stability—an antibody may have already degraded before purchase, or it may degrade rapidly after purchase. Importantly, an antibody that appears non-functional will require exhaustive testing before it can be concluded that it does not perform to stated specifications and either a refund or replacement obtained from the supplier. Therefore, it is important to determine which companies have stringent quality control practices for the type of antibody markers required. Prior to purchase, it is also advisable to obtain data sheets from the company that describe some of the above features, and which may also contain references to literature detailing previous characterisation and use of the antibody. Upon obtaining the antibody, it should be tested for appropriate reactivity and then partitioned into smaller aliquots which are frozen until required. Freezing and thawing of antibodies should be avoided. Antibodies should also be tested over a range of dilutions to determine their effectiveness for immunohistochemistry. A company may suggest a working dilution but the antibody may work at higher dilutions, which means they last longer. Immunohistochemistry usually requires a ‘secondary’ antibody system for visualising the binding of the primary antibody to the tissue section. There are a wide variety of conjugated secondary antibodies and relevant kits available commercially. Some of the considerations noted for the ‘primary’ antibody described above are important, although there tends to be less variability in the quality of secondary antibodies. In this respect, secondary antibody visualisation kits from Vector Laboratories and DAKO are very reliable.

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PREPARATION OF HUMAN CNS FOR IMMUNOHISTOCHEMISTRY An important consideration for immunohistochemistry on human CNS is the preservation of the epitope of interest. For most immunohistochemical markers, it is necessary to ‘fix’ tissue using aldehyde-based solutions. The great advantage of non-human brain studies is the ability to easily perfuse the animal with fixative solutions. It is also possible to perfusion-fix human brains (Halliday et al., 1990). The basilar and carotid arteries can be connected to a perfusion apparatus, and the brain perfused with a large volume of an aldehyde-based solution. However, it is more common practice, particularly with brains taken at autopsy, to immersionfix human brain tissue for long periods of time in formalin. It is important to note that over-fixation may result in interference with the localisation of many epitopes but this problem can be overcome using various ‘antigen-retrieval’ methods (Evers and Uylings, 1994; McQuaid et al., 1995). Typically this may involve the rapid boiling of tissue sections in a microwave—this can be done in distilled water alone (e.g. two periods of five minutes in a 850 W microwave on ‘high’; Vickers et al., 1994), or in buffered solutions (e.g. 0.01 M sodium citrate, pH 7.8). In this author’s experience, immunohistochemistry is optimal following fixation of blocks dissected from 1 cm thick slices of brain with phosphatebuffered 4% paraformaldehyde (see below) for 24–48 hours. This is followed by washes in a phosphate-buffered saline solution (PBS). The use of small blocks avoids the delayed fixation of inner structures that can occur with immersionfixation of the whole brain. PHOSPHATE BUFFER STOCK SOLUTIONS Solution A: Solution B:

NaH2PO42H2O Na2HPO4

31.2 g/litre 28.4 g/litre

0.1 M Phosphate-Buffered Saline Solution (PBS) To prepare 1 litre of PBS, add 9 g NaCl to 850 ml distilled water and add 10 ml of solution A and 40 ml of solution stock.

Paraformaldehyde Solution (pH 7.4–7.6) Add 40 g (4%) paraformaldehyde to 500 ml distilled water and then add 100 ml solution A plus 400 ml solution B. Stir and heat solution (60–70 °C) to dissolve paraformaldehyde—do not boil!

Another important consideration for the use of tissue obtained at autopsy for immunohistochemical studies is the length of time between death and fixation.

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The longer the period of autolysis, the more tissue degradation occurs, and the greater the possibility of alterations in epitopes which would result in inaccurate localisation of the marker of interest. Autolysis times of less than 6 hours are ideal, but some epitopes can still be reliably preserved up to 24 hours after death. As with many aspects of immunohistochemistry, it is important to test how the postmortem interval to fixation may affect labelling, and the use of perfusion-fixed non-human brain sections can often be used as a positive control for immunohistochemistry. An alternative source of material is that derived from surgical biopsy. This material is used for studies/diagnosis of tumours, and immunohistochemistry is often used to determine tumour type. In addition, the region around the tumour may be used for studying the localisation of other epitopes of interest (Vickers et al., 1995). Brain material is often removed from the cortex to treat intractable epilepsy, and such tissue has been studied for the brain changes associated with this condition (DeFelipe et al., 1994).

CHOICE OF SECTIONS Some human CNS material can be utilised as wholemounts (e.g. retina; Straznicky et al., 1992) for immunohistochemistry. Microtome sectioning of material embedded in paraffin is the most common approach for routine histology, but is often not ideal for immunohistochemistry. Sectioning of frozen brain material is favored for most immunohistochemical techniques. For this procedure, the material is ‘cryoprotected’ by impregnation with sucrose solutions. We utilise 30% sucrose in PBS—the block is suitably impregnated when it sinks in the solution. However, we have also encountered particular neuronal epitopes that are diminished or altered by tissue freezing. For example, the localisation of the subunits that comprise excitatory receptors is optimal in sections that have been cut using a vibratome (Vickers et al., 1995). Following sectioning, there are two main alternatives for processing for immunohistochemistry. Sections can be first mounted on ‘subbed’ slides (see below) and the antibody incubations and washes conducted on the section. This is most commonly done with the relatively thin (e.g. 10–15 µm) paraffin, and sometimes cryostat, sections. For these techniques, the various solutions can be dropped onto the slide. Incubations are usually done in a sealed container (humid chamber) containing a small amount of water to prevent evaporation of the antibody solution. The sections can be encircled by a ring of silicone or rubber cement on the slide—this will create a shallow well that maintains the solution

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over the sections. These slides may also be processed through staining trays or Coplin jars, but this has the disadvantage of requiring relatively large quantities of the immunoreagents. CHROME ALUM/GELATINE SUBBED SLIDES 1. Place pre-cleaned slides in racks and rinse 3×30 minutes in distilled water. 2. While wet, dip slides in chrome alum-gelatine solution for 5 minutes (Subbing solution: Add 2.5 g gelatin and 0.25 g chrome alum to 500 ml distilled water. NB: Heat to dissolve and filter solution before use.) 3. Cover slides until they have dried.

Alternatively, all incubations and washes can be conducted on sections that are loose in solutions, the so-called ‘free-floating’ method. This is usually conducted with relatively thicker (30–50 µm) freezing microtome or vibratome sections. In our laboratory, sections are placed in a covering volume of the solutions in appropriately sized tissue culture trays. Sections are transferred between wells with fine paint brushes. The main advantage of the free-floating method is the accessibility of the solution to both faces of the section and the ease of use of shaker tables for rotations of sections during incubations and washes. In addition, this approach results in superior penetration of the antibody into the section and usually less non-specific binding of antibodies and other immunoreagents. Once sections are cut for this free-floating technique, they can be kept in a storage solution of either PBS containing 0.1% sodium azide at 4 °C, or in an ‘anti-freeze’ solution, detailed below, at -20 °C. With either solution, sections should be washed (e.g. 3×10 minute washes in PBS) before commencing the immunohistochemical procedure. TISSUE STORAGE SOLUTION (-20 °C) Add 300 ml glycerol, 300 ml ethylene glycol, 300 ml distilled water, 20 ml solution A and 80 ml solution B.

IMMUNOPEROXIDASE There are a number of ways in which the binding of an antibody to a tissue section can be visualised. An antibody that binds to the epitope of interest can be conjugated to a molecule that can be viewed with a microscope. This is often referred to as the ‘direct’ method (Figure 6.1). The substance that is conjugated

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to the antibody may be an enzyme, such as horseradish peroxidase or alkaline phosphatase. These enzymes can then reacted with appropriate substrates and these reactions coloured opaque deposits that are visible using transmitted light microscopy. A typical substrate is 3,3'-diaminobenzidine (DAB). DAB is a suspected carcinogen, and it may therefore be more practical to buy this chromogen in either a tablet or liquid form rather than as a powder. An alternative to the chromogen method requires the antibody to be conjugated to a substance, that when excited with a certain wavelength of incandescent (or laser) light emits fluorescent light at a longer wavelength. The use of these fluorophores for immunohistochemistry is discussed in following sections. With the direct method of antibody visualisation, all the antibodies of interest need to be conjugated, and there will be a loss of antibody in the process of conjugation. Alternatively, the antibody bound to the tissue section (the primary antibody) can be visualised by a second antibody that has a specific affinity for the first antibody and is conjugated to a substance that can be visualised by microscopy (Figure 6.1). The main advantage of this indirect method is that a range of unconjugated primary antibodies can be utilised, and visualisation can be conducted using standard secondary antibody-conjugate systems that are easily available from commercial sources.

Figure 6.1 Methods of visualising the binding of primary antibodies to tissue preparations

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The indirect approach has been further modified to amplify the signal from the primary antibody (Hsu et al., 1981) (Figure 6.1). In this procedure, a secondary antibody is conjugated to the molecule, biotin. Biotin binds with a second molecule, avidin, with a high degree of avidity at a ratio of 4:1, respectively. Biotin conjugated to a reporter molecule (e.g. peroxidase) can be mixed with avidin in a particular ratio to form an avidin-biotin-peroxidase complex with spare binding sites on the avidin molecule. Combining this mixture with the secondary antibody conjugated to biotin results in the association of multiple peroxidase molecules with an individual bound primary antibody. Thus, this method is often described as highly ‘sensitive’, and is particularly useful for detecting epitopes that may be present in relatively small amounts. A typical immunoperoxidase procedure using the ABC (Avidin-BiotinComplex) method utilised by a Vectastain kit (Vector Laboratories) on freefloating brain sections is presented below. All steps are conducted at room temperature unless noted. Examples of immunoperoxidase labelling of different subsets of neocortical cells with particular antibody markers is shown in Figure 6.2. IMMUNOPEROXIDASE METHOD Preparation: Blocking non-specific labelling Inhibiting endogenous peroxidase activity in the tissue section. 1. Place tissues sections in a 3:1 solution of methanol and 3% hydrogen peroxide for 15 minutes. 2. Wash 3×10 minute in PBS.

Optional: Blocking non-specific binding of antibodies 1. Place section in 10% serum (from the species used for raising the secondary antibody) in PBS for 30 minutes. 2. Wash briefly in PBS following incubation. This stage blocks the binding of antibodies to non-specific sites in the section and is not usually necessary when using well-characterised primary and secondary antibodies. May be useful with unpurified polyclonal antibodies. One percent bovine serum albumin or 5% non-fat skim milk may also be added to the diluent to promote blockage of sites that are generally ‘sticky’ for protein.

ABC immunoperoxidase method The following stages utilise a detergent (Triton X-100) to increase antibody penetration. However, it is important to note that such detergents may damage particular epitopes (e.g. cell surface antigens).

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1. Incubate in primary antibody diluted to the appropriate concentration in 0.3% Triton X-100/PBS. (This stage is usually conducted overnight at 4 °C, but may be lengthened for two to three days to promote maximal binding of the antibody.) 2. Three×10 minute PBS washes. 3. Incubate in biotinylated secondary antibody at appropriate concentration (usually 1:200 or 1 drop/10 ml diluent), in 0.3% Triton X-100/PBS, for one hour. 4. Three×10 minute PBS washes. 5. Incubate in AB (avidin-biotin-peroxidase complex) solution for one hour. Make AB solution twenty minutes before use. Dilute solutions A and B to 1:100 in 0.3% TritonX-100/PBS (alternatively 2 drops of each in 10 ml of diluent). 6. Three×10 minute PBS washes. 7. Place sections in DAB solution. This can be bought in kit form (e.g. Vector Laboratories), as a solution (e.g. DAKO) or made up from tablets (e.g. Sigma). To decrease background labelling, the DAB solution can be cooled to 4 °C before use. When using the powdered form, dissolve 0.05% DAB in a 1:10 solution of PBS (low molarity is required for DAB to solubilise) and activate with 1 µl of 3% H2O2 per ml of DAB. NB: When treating sections with DAB, carefully observe tissue sections against a white background until they change colour (labelled elements will be brown). This procedure usually takes about 3 minutes. 8. Stop the reaction by placing the sections in PBS. 9. Three×10 minute PBS washes. 10. Mount sections on chrome alum/gelatine subbed slides and air dry. Dehydrate through graded alcohol’s, clear in xylene or Polyclear, and coverslip with permanent mounting media (e.g. Eukitt). NB: As the reaction product with DAB should be fairly stable, it is possible to combine immunoperoxidase labelling with other histological methods such as cresyl violet and haematoxylin and eosin staining. Counterstaining is particularly useful for providing cytoarchitectural information about the distribution of immunolabelling.

Cleaning Up 1. Combine remaining DAB with bleach and dispose of appropriately. 2. Use bleach to clean all containers that have come into contact with DAB and then wash thoroughly.

UNCOVERING EPITOPES IN TISSUE SECTIONS As discussed above, antigen-retrieval methods such as microwave pretreatment may be used to uncover epitopes obscured by over-fixation in aldehyde-based solutions. Additional methods may also be used on sections to uncover particular

Figure 6.2 Examples of immunoperoxidase labelling. A shows a subset of human neocortical pyramidal cells labelled with an antibody to neurofilament proteins. B shows a subset of non-pyramidal cells in the human neocortex labelled with an antibody to the calcium binding protein, calretinin. C shows immunoperoxidase labelling with an antibody to β-amyloid in the hippocampus of an Alzheimer’s disease sufferer. From the same case, more plaques are β-amyloid immunolabelled in a different section pretreated with formic acid (D), and these plaques show a higher degree of immunoreactivity. Scale bar for A to D is 100 µm.

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epitopes that may be present. Some of these techniques can be illustrated by considering immunohistochemical studies of Alzheimer’s disease-related pathology. This degenerative condition is characterised by the presence of certain pathological ‘hallmarks’ in the brain, including plaques, neurofibrillary tangles, dystrophic neurites and neuropil threads. Plaques are extracellular structures that are comprised of amyloid fibrils, derived from the insoluble form of the βamyloid protein (Glenner et al., 1994; Masters et al., 1995). Thus, immunohistochemistry using antibodies to β-amyloid will reveal the plaques of Alzheimer’s disease. However, it has become common practice to pre-treat brain sections with formic acid (e.g. 90% formic acid for twenty minutes followed by several PBS washes) to visualise the full spectrum of β-amyloid deposition that occurs in this disease (Kitamoto et al., 1987) (Figure 6.2). Interestingly, such techniques have also been used to determine the molecular features of the cytoskeletal changes that lead to the generation of neurofibrillary tangles, dystrophic neurites and neuropil threads. For example, formic acid pretreatment has been shown to result in the uncovering of specific neuronal cytoskeletal epitopes in neurofibrillary tangles, including particular phosphorylated epitopes on neurofilaments (Cammarata et al., 1990; Vickers et al., 1992; 1994). Thioflavine S is a histochemical fluorescent stain for both plaques and neurofibrillary pathology, but this staining is abolished by formic acid pretreatment, indicating that this method of epitope retrieval may be altering the conformational state of certain protein constituents or removing the binding sites for this histochemical stain. Enzymes such as proteases and phosphatases may also be used to modify and/ or uncover certain epitopes in human brain sections prior to immunohistochemistry. With the example of Alzheimer’s disease material, abnormal phosphorylation of cytoskeletal proteins, such as the microtubule-associated protein, tau, has been implicated in the disease process. Thus, antibodies to specific phosphorylated or dephosphorylated epitopes of tau have been extensively utilised and pretreatment of sections with enzymes such as alkaline phosphatase may be used to modify these epitopes (e.g. Iqbal et al., 1989). Other pretreatment strategies, such as autoclaving (Shin et al., 1992), have also been shown to enhance tau immunoreactivity in the pathology of Alzheimer’s disease.

IMMUNOFLUORESCENCE Immunoperoxidase, due to its simplicity, is the most appropriate immunohistochemical technique for routine light microscopy.

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Immunofluorescence offers a number of advantages over chromogen-based methods but is more complex and requires a microsope fitted with specific optics for the visualisation of the fluorescent markers. Fluorescence immunohistochemistry can provide detailed resolution of immunolabelling as the marker is observed against a completely black background. This technique also gives a more reliable indication of the relative concentration of the epitope present as it does not involve any significant ‘amplification’ of the signal, and it lacks the enzymatic step necessary for the visualisation of chromogens. Thus, this method is often more appropriate for the quantification of the intensity of immunolabelling in a tissue preparation, as long as appropriate reference controls are used and the light source is constant (e.g. Gazzaley et al., 1996). A crucial advantage of immunofluorescence is the ease with which multiple antibody markers can be simultaneously visualised in the same tissue section. Multiple immunolabelling with chromogens is possible, but can be complicated to interpret when the markers overlap. With immunofluorescence, filter combinations can be used to separate the signals from each marker. Immunofluorescence usually relies on the indirect visualisation method, with a fluorophore-conjugated secondary antibody that recognises the primary antibody bound to the tissue section. Alternatively, a biotinylated secondary antibody can be followed by avidin conjugated to a fluorophore. Many fluorophores are available with variable excitation and emission parameters. Fluorophores have a maximal wavelength peak for excitation (absorption) and emit with a longer maximal wavelength peak. Fluorescence microscopes are equipped with filter sets used to visualise specific fluorophores. These are usually comprised of an excitation filter that selects out the wavelengths of light that are in the maximal absorption range of the fluorophore, a dichroic mirror that controls the path of excitatory and reflected light based on their wavelengths, and a barrier filter that allows the light emitted by the stimulated fluorophore to pass through, but blocks out wavelengths that are extraneous to the signal of interest. A common fluorophore is fluorescein isothiocyanate (FITC), with has a maximal excitation peak of 494 nm and an emission peak of 520 nm, which means that, with the appropriate filter cubes, blue fluorescent light causes this molecule to maximally emit in the wavelength corresponding to green. Other common fluorophores used for these methods include rhodamine and Texas Red, both of which, when stimulated with green to orange wavelengths of light, emit with red fluorescence. A third fluorophore type being increasingly

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utilised in immunofluorescent studies are the coumarin-based labels which are excited by light in the UV range and emit in the blue spectra. It is therefore possible to have several antibodies visualised in the tissue section at a given time. This is achieved by the use of multiple antibody-linked fluorophores that can be distinguished with appropriate optics. In this situation, it is critical to avoid any inappropriate cross-reactivity between primary and secondary antibodies. Fluorescence immunohistochemistry and, in particular, multi-labelling variants of this procedure are commonly used for studies of the nervous system in non-human animals. One reason for the less widespread use of these techniques in investigations of human material is the relatively high amount of autofluorescence in such preparations. This mainly comes from the pigments that accumulate in lipofuscin within nerve cell bodies. The degree of lipofuscin accumulation is variable between cases but tends to be age-related. Such autofluorescent material typically appears red in filter combinations for Texas Red/rhodamine, yellow in filter sets for FITC and white in filter sets for coumarin-based fluorophores. This may not be a problem if, for example, the epitope-bound antibody is visualised with FITC, as distinction between antibody labelling (green) and autofluorescence (yellow) is possible (Vickers et al., 1992). In addition, epitopes located in regions other than the cell body, where the autofluorescent lipofuscin is present, may still be reliably distinguished with the different fluorophores. It may, however, be necessary to use colour photography for recording data (which can also be costly for publication) as black-and-white photographs will not distinguish between autofluorescence and antibody-related fluorescence, particularly if, to some degree, these are colocalised to the same tissue elements. A technique developed to abolish the autofluorescence of human brain sections (Styren et al., 1991; Vickers et al., 1992) is described below. This method is often used to eliminate autofluorescence for the purposes of staining sections with thioflavine S, the histochemical stain for Alzheimer’s disease-related pathology described above. Thioflavine S-related fluorescence appears white with UV filter optics, yellow with FITC-related filters and is not usually visualised with Texas Red/rhodamine-related filters (Vickers et al., 1992). Thus, this method may be reliably combined with other fluorophores to examine the localisation of particular epitopes in relation to the pathological structures of Alzheimers disease. We have used this method to examine the staging of the cytoskeletal changes leading to neuroflbrillary pathology (Vickers et al., 1992, 1994), as well as the relative vulnerability of different subpopulations of cortical

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neurons to the pathology of Alzheimer’s disease (Sampson et al., 1997). The major problem associated with this method is that it enhances the fragility of tissue sections, and therefore the immunohistochemical steps that follow must be conducted with great care. AUTOFLUORESCENCE QUENCHING TECHNIQUE (from Styren et al., 1991; Vickers et al., 1992) This technique can be conducted on free-floating section or sections mounted on slides. The steps 5, 6 and 7 are required for staining with thioflavine S. 1. Place sections in PBS containing 0.25% potassium permanganate for 20 minutes. (Sections will turn a deep purple/brown colour.) 2. Two×2 minute PBS washes. 3. Place material in a ‘destaining’ solution (PBS containing 1% potassium metabisulfite and 1% oxalic acid) for 2–3 minutes or until all colour has been removed from the section. Conduct destaining in a fume hood as this solution releases a noxious gas. 4. Four×5 minute PBS washes. 5. Place in a 40% ethanol/60% PBS solution containing 0.0125% thioflavine S for 3 minutes. This, and subsequent reactions, should be conducted in the dark as much as possible. Aluminium foil or a cardboard box can be used to cover the preparation. 6. Two×10 second washes in 50% ethanol/50% PBS. This is the differentiating step for thioflavine S staining. 7. Three×5 minute PBS washes. 8. Proceed to primary antibody incubation.

A standard double labelling immunofluorescence protocol, combining primary antibodies raised in mice and rabbits, and using secondary antibodies from Vector Laboratories, is presented below (see also Figures 6.3 and 6.4). After sections are exposed to fluorophore-conjugated secondary antibodies, it is important to conduct subsequent steps in the dark as much as possible. However, there should be no appreciable loss of fluorescence labelling if the material is exposed to normal light during the transferring stages and when mounting sections on slides and coverslipping. This method is designed for free-floating sections but can be easily modified for sections mounted on slides. Notably, this procedure uses a biotinylated anti-rabbit secondary antibody followed by an avidin-Texas Red conjugate. It has been this author’s experience that anti-rabbit IgG secondary antibodies conjugated to fluorophores do not produce as strong a signal as the avidin-biotinfluorophore method.

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Figure 6.3 Immunofluorescence double labelling

Figure 6.4 Double exposure showing neurofilament (green) and tau (red) immunofluorescence labelling in the human cerebral cortex. In the center, a neuron labelled for neurofilaments contains a neurofibrillary tangle labelled for tau. DOUBLE LABELLING IMMUNOFLUORESCENCE NB: All antibodies are diluted in PBS containing 0.3% Triton X-100 to enhance penetration. 1. Blocking stage as described for immunoperoxidase procedure above (Optional). 2. Incubate sections in diluent containing both antibodies overnight (usually at 4 °C). This step may be lengthened for two to three days to promote maximal binding of the antibody. 3. Three×10 minute PBS washes. 4. Incubate in a combination of horse anti-mouse IgG conjugated to FITC and goat anti-rabbit IgG conjugated to biotin in diluent for 2 hours. 5. Three×10 minute PBS washes. 6. Incubate in avidin-Texas Red conjugate for 1 hour. 7. Three×10 minute PBS washes

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COVERSLIPPING AND STORAGE OF FLUORESCENT MATERIAL After the final washes, mount sections on non-subbed glass slides, air dry and coverslip using either an aqueous or permanent mounting media. Buffered glycerol can be easily prepared as an aqueous mounting media (see below)— buffering is important to prevent fluorescence fading. Coverslips can be later sealed with nail polish, allowing the slides to be stored upright. Alternatively, a permanent mounting media (e.g. Permafluor by Immunotech) that dries may make the storage of slides more straightforward as coverslips need not be sealed. In either case, slides should be kept in the dark at 4 °C if possible. Under these conditions, fluorescence should be preserved for many weeks, although it is advisable to analyse and photograph prepared slides as soon as possible.

BUFFERED GLYCEROL (FLUORESCENCE MOUNTING MEDIA) To prepare 150 ml of mounting media, titrate 50 ml of a 0.5 M NaHCO3 (4.2 g/100 ml) solution with 0.5 M Na2CO3 (5.3 g/100 ml) (approximately 0.6 ml) until pH is 8.6. Mix with 100 ml of glycerol. Final pH should be approximately 8.4.

APPROPRIATE DOUBLE LABELLING CONTROLS For most immunohistochemical techniques, it is important to run positive and negative control preparations. The best positive control for comparing with a new primary antibody is the concurrent processing of a tissue preparation with a primary antibody raised in the same species that is known to work well. With an antibody known to bind to, for example, rat brain, it may also help to conduct any new immunolabelling on human CNS in conjunction with the equivalent material from the non-human species. A negative control, which should be conducted concurrently with all immunohistochemical investigations, involves the entire procedure with the omission of the primary antibody. This confirms that any labelling is due to the specific binding of the primary antibodies to the tissue section. For multiple labelling procedures, further controls are very important. With most commercially available secondary antibodies, there should be little cross reactivity between species such as rabbits and mice. However, with every new combination of primary and/or secondary antibodies, it is prudent to check for reactivity of the secondary antibodies with the inappropriate primary

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antibody or for reactivity between secondary antibodies. Similarly, it may also be useful to check that any avidin-based fluorophore conjugates bind to the primary or secondary antibodies, or the tissue preparation itself. Example of an appropriate antibody combination mouse+rabbit IgGs→anti-mouse IgG FITC+biotinylated anti-rabbit IgG→avidin Texas Red Double labelling control preparations rabbit IgG→anti-mouse IgG FITC mouse IgG→biotinylated anti-Rabbit IgG→avidin Texas Red That is, do the secondary antibodies bind to the inappropriate primary antibody? mouse IgG→anti-mouse IgG FITC+biotinylated anti-Rabbit→ avidin Texas Red rabbit IgG→anti-mouse IgG FITC+biotinylated anti-Rabbit→ avidin Texas Red That is, do the secondary antibodies cross-react with each other? no primary IgG (diluent only)→anti-mouse IgG FITC+ biotinylated anti-rabbit IgG→avidin Texas Red no primary IgG (diluent only)→avidin Texas Red That is, do the secondary antibodies or avidin bind to elements in the tissue preparation other than the primary antibody?

CONCLUDING REMARKS As with most techniques, there will be some degree of experimentation and further manipulation to attain a standard set of immunohistochemistry protocols for a given laboratory. The advantage for the beginner in immunohistochemistry is the availability of a broad range of primary and secondary antibodies, as well as immunoreagents in kit form, from a variety of commercial sources. In addition, competition in this field has also led to improvements in the quality of these products. While immunoperoxidase techniques are the most straightforward approach for immunohistochemistry on human nervous system material, fluorescencebased immunohistochemistry can provide exquisite cellular detail and offers

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the advantage of the simultaneous localisation of multiple markers in a given tissue section. In this respect, with optimally prepared material, it may be reasonable to expect that the advanced immunohistochemical methods being developed and applied in investigations of non-human species can be extended to studies of the human nervous system. This should not only increase the understanding of the normal organisation of the human CNS but is vital for unravelling disease mechanisms and/or the structural abnormalities that underlie neurological and psychiatric conditions. Acknowledgements Many of the techniques outlined in this chapter have their origin in the laboratories in which the author has worked, including the Flinders University of South Australia, the Mount Sinai School of Medicine (New York) and the University of Tasmania. The author is grateful to the numerous individuals in these institutions who have instructed him in this area. In particular, the author would like to thank Paul Adlard, Mark Cozens, Tracey Dickson and Carolyn King for their help in the preparation of this chapter. References Cammarata, S., Mancardi, G., and Tabaton, M. (1990) Formic acid treatment exposes hidden neurofilament and tau epitopes in abnormal cytoskeletal filaments from patients with progressive supranuclear palsy and Alzheimers disease. Neurosci. Letts, 115, 351–355. DeFelipe, J., Huntley, G.W., del-Rio, M.R., Sola, R.G. and Morrison, J.H. (1994) Microzonal decreases in the immunostaining for non-NMDA ionotropic excitatory amino acid receptor subunits GluR 2/3 and GluR 5/6/7 in the human epileptogenic neocortex. Brain Res., 657, 150–158. Evers, P. and Uylings, H.B.M. (1994) Effects of microwave pretreatment on immunocytochemical staining of vibratome sections and tissue blocks of human cerebral cortex stored in formaldehyde fixative for long periods. J. Neurosci. Methods, 55, 163–172. Gazzaley, A.H., Siegel,.SJ., Kordower, J.H., Mufson, E.J. and Morrison J.H. (1996) Circuitspecific alterations on N-Methyl-D-Aspartate receptor subunit 1 in the dentate gyrus of aged monkeys. PNAS, 93, 3121–3125. Glenner, G.G. and Wong, C.W. (1984) Alzheimers disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. B.B.R.C., 120, 885–890. Halliday, G.M., Li, Y.W., Blumbergs, P.C., Joh, T.H., Cotton, R.G.H., Howe, P.RC.,Blessing, W.W. and Geffen, L.B. (1990) Neuropathology of

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immunohistochemically identified brainstem neurons in Parkinson’s disease. Annals Neurol., 27, 373–385. Hsu, S.M., Raine, L. and Fanger, H. (1981) Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577–580. Iqbal, K., Grundke-Iqbal, I., Smith, A.J., George L, Tung, Y.C. and Zaidi, T. (1989) Identification and localization of a tau peptide to paired helical filaments of Alzheimer disease. P.N.A.S., 86, 5646–5650. Masters, C.L., Simms, G., Weinman, N A., Multhaup, G., McDonald, B.L. and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer’s disease and Down syndrome. P.N.A.S., 82, 4245–4249. McQuaid, S., McConnell, R., McMahon, J. and Herron, B. (1995) Microwave antigen retrieval for immunocytochemistry on formalin-fixed, paraffin-embedded post-mortem CNS tissue. J. Path., 176, 207–216. Sampson, V.L., Morrison, J.H. and Vickers, J.C. (1997) The cellular basis for the relative resistance of parvalbumin and calretinin immunoreactive neocortical neurons to the pathology of Alzheimer s disease. Experimental Neurol., 145, 295–302. Shin, R.-W., Iwaki, T., Kitamato, T., Sato, Y. and Tateishi, J. (1992) Massive accumulation of modified tau and severe depletion of normal tau characterise the cerebral cortex and white matter of Alzheimer’s disease. American J. Path., 140, 937–945. Straznicky, C., Vickers, J.C., Gabriel, R. and Costa, M. (1992) A neurofilament protein antibody selectively labels a large ganglion cell type in the human retina. Brain Res., 582, 123–128. Styren, G., Civin, C.W. and Rogers, J. (1991) Quenching lipofuscin autofluorescence in aged and Alzheimer’s brain: a double-label fluorescence method with thioflavin counterstain. Soc. Neurosri. Abstracts, 17, 694. Vickers, J.C., Delacourte, A. and Morrison, J.H. (1992) Progressive transformation of the cytoskeleton associated with normal aging and Alzheimer’s disease. Brain Res., 594, 273–278. Vickers, J.C., Riederer, B.M., Marugg, R.A., Buee-Scherrer, V., Buee, L., Delacourte, A. and Morrison, J.H. (1994) Alterations in neurofilament protein immunoreactivity in human hippocampal neurons related to normal aging and Alzheimer’s disease. Neurosci., 62, 1–13. Vickers, J.C., Huntley, G.W., Hof, P.R., Bederson, J., DeFelipe, J. and Morrison, J.H. (1995) Immunocytochemical localization of non-NMDA ionotropic excitatory amino acid receptor subunits in human neocortex. Brain Res., 671, 175–180.

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THE PROCESSING AND USE OF POSTMORTEM HUMAN BRAIN TISSUE FOR ELECTRON MICROSCOPY Rosalinda C.Roberts and Lili Kung

Ultrastructural studies of the adult human brain are scarce due to the difficulty in obtaining brains with short postmortem intervals (Aganova & Uranova, 1992; Roberts & Francis, 1993; Roberts et al., 1994, 1996a,b, 1997; Kung et al., 1996, 1998; Uranova, 1996). Most reports are performed in hippocampal or cortical tissue obtained during surgical resection (Babb et al., 1991; Ong & Garey, 1993; Smiley et al., 1994). Surgical samples have the advantage of no postmortem interval, but may be abnormal since they are removed adjacent to aberrant tissue, particularly epileptic foci. It is in fact possible to perform ultrastructural studies using postmortem tissue, which offers the advantage of normal control tissue, access to any part of the brain, and comparisons to diseased material. Sources of tissue for the study of human development include spontaneous or medically induced abortions and stillborn premature infants. The following chapter details methods which have been successful in our laboratory for analysis of adult and fetal human brain. The most critical detail for success in doing this research is the ability to obtain fresh brain tissue with postmortem intervals (PMI) compatible with electron microscopy. However, factors other than the PMI can affect the utility of postmortem tissue for electron microscopy, such as the agonal status of the individual and ambient temperature. In our case, we obtained adult postmortem brain tissue, collected within seven hours of death, from the Maryland Brain Collection. We obtained second trimester fetal tissue, collected within one hour of delivery, from the University of Maryland Brain and Tissue Bank for Developmental Disorders. The short PMI tissue from both developing and adult brain is suitable for a variety of qualitative and quantitative analyses. By examining the type, frequency and size of synapses in unstained tissue, it is possible to gain insight on synaptic organization, clues as to the origin of 127

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the neurons forming the synapses and in specific neurological and psychiatric disorders, whether synaptic number or efficacy may be altered. Even though postmortem tissue requires the use of glutaraldehyde in the fixative, it can be processed successfully for immunocytochemistry using a number of antibodies.

METHODS The first step is to organize a system for tissue collection, for example from a brain bank or collection, a medical examiner’s office or hospital. Once a system is in place for tissue collection, the tissue can be processed, with some noted exceptions, in a similar manner to tissue from experimental animals. Thus, a number of options are available for fixation and analysis as detailed below. The electron microscopy (EM) fixatives and embedding solutions are very standard products which, for example, can be ordered from Electron Microscopy Science (Ft. Washington, PA) or Ted Pella Inc. (Redding, CA). The buffers and table top shaker are also standard products which can be purchased from a number of scientific suppliers. Standard protocols for buffer and embedding mixtures should work with postmortem human tissue. The ultrastructural integrity of human tissue is compromised if the PMIs are much longer than seven hours (Roberts et al., 1996b). As the postmortem interval increases beyond a few hours, a number of structures deteriorate rendering quantitative analysis difficult, if not impossible. For example, membranous structures, such as pre- and postsynaptic membranes, synaptic vesicles and myelin, are lost or distorted. The postsynaptic density becomes unnaturally thick, making it difficult to determine synapse symmetry. Profiles such as axon terminals, dendrites, spines and mitochondria become swollen and/or irregular in contour, making assessments of size inaccurate. The extracellular spaces enlarge in size, making measurements of synaptic density potentially inaccurate. Extracellular space is very sensitive to PMI and may be expanded in some regions even in tissue with very short PMIs. Nevertheless, using tissue with a PMI shorter than seven hours yields good results (Figure 7.1). TISSUE FIXATION AND CUTTING 1. Immerse blocks (0.5–1.0 cm thick) of the brain in a cold solution of 4% paraformaldehyde and 1% glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.2–7.4 (4 °C) for a period of at least one week. The fixative should be kept in a brown glass bottle; paraformaldehyde may be prepared ahead of time, but

Figure 7.1 Electron micrographs from adult human neostriatum showing fields of neuropil taken from cases where the cause of death was sudden and the postmortem interval short. A: An example from a case (35-year-old male) with a postmortem interval of 3 hours; the cause of death was a motor vehicle accident. Higher magnification examples from this case are illustrated in the next three figures. B: An example of a case (32-year-old male) with a postmortem interval of six hours; the cause of death was multiple fractures from a fall. Note in both cases the structural integrity of the tissue is similar. These samples have little, if any, postmortem artifacts, such as profile swelling, expansions of extracellular space and loss of membrane integrity. Synapses (arrows), axon terminals (at), dendrites (d), spines (s), and mitochondria (*) are indicated. The scale bar=1 µm.

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the glutaraldehyde should be added immediately prior to the brain tissue. The optimum fixative in our hands is the 4% paraformaldehyde, 1% glutaraldehyde combination. In fact, for fetal tissue, which is fragile, 1% is the minimum recommended concentration for glutaraldehyde. For antibodies that are sensitive to glutaraldehyde, it will be necessary to reduce the concentration of the glutaraldehyde, perhaps to as low as 0.2%. Unfortunately, the paraformaldehyde, lysine, periodate (PLP) fixative formula of McLean and Nakane (1974), which is excellent for combined EM-immunocytochemistry in perfused animals, is not recommended for human postmortem tissue. Brain tissue fixed in PLP was never suitable for EM analysis, even when samples of the same brain gave adequate or excellent results with glutaraldehyde. (a) Fixed tissue may need special preparation prior to sectioning. If the arachnoid membrane and/or choroid plexus are present in the brain region of interest, there will be problems cutting consistent sections. Thus, they must be carefully removed from the surface of the block prior to sectioning. 2. Trim the tissue to the desired size and embed it in 5% agar (5 g agar-agar in 100ml of boiling double distilled water). 3. Cut the tissue with a vibratome at a thickness of 40 µm for the adult tissue or 100 µm thick for the fetal tissue. The fetal tissue needs to be cut substantially thicker than that of the adult, due to the fragile nature of developing tissue.

EMBEDDING UNSTAINED TISSUE SECTIONS Unstained tissue sections may be flat embedded or block embedded (2 mm plugs of tissue) using standard techniques. 1. Briefly, rinse either the sections or blocks in phosphate buffer (PB) (3 washes, 10 minutes each), immerse them in 1% osmium tetroxide for one hour, followed by 1% uranyl acetate for one hour. 2. Dehydrate in successively higher concentrations of alcohol. 3. Embed in resin. 4. Heat at 60 °C for 72 hours.

Flat embedded sections 1. Block areas of interest. 2. Glue areas of interest to beam capsules. 3. Cut semi-thick sections followed by ultra-thin sections, 70–90 nm in thickness with an ultramicrotome. 4. Examine with the electron microscope.

Immunocytochemistry Several antibodies can be successfully used in human postmortem tissue (Figure 7.4 and 7.5) (Kung et al., 1998b; Roberts et al., 1997). A partial list of these

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antibodies and parameters for use are noted in Table 7.1. Process free floating sections for immunocytochemistry. Do not use triton to increase antibody penetration or a pre-block with hydrogen peroxide (H2O2) to quench endogenous peroxidase, as these substances will diminish ultrastructural integrity. PROTOCOL Day 1 1. 2. 3. 4.

Collect the sections into cold PB. Wash three times (10 minutes each) in PB. Incubate the sections in 2% normal serum in PB for 30 minutes. Incubate the sections with primary antibody for a period of 24–48 hours. The tissue should be agitated gently on a table top shaker in a cold room during incubation with the primary antibody.

Day 2–3 For the following steps, it is preferable to incubate the tissue at room temperature on a table top shaker. React the tissue with reagents from the avidin-biotin peroxidase kit (ABC standard kit) using recommended dilutions and times (Hsu et al., 1981). 1. Wash tissue (PB, three changes, 10 minutes each). 2. Incubate sections for one hour in biotinylated secondary antibody (44 µl/10 ml PB). 3. Wash, incubate for one hour in the avidin-biotin complex (88 µl each of solutions A and B/10 ml PB).

Table 7.1 Antibodies which have been utilised with human postmortem tissue and the parameters for their use in immunocytochemistry

Animal refers to the animal in which the antibody was raised. Abbreviations: GABA, γaminobutyric acid; GFAP, glial fibrilary acidic protein; P, paraformaldehyde; G, glutaraldehyde.

a

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4. Wash [if desired, insert BLAST procedure here, see below]. 5. Incubate sections in diaminobenzidine (6 mg/10 ml PB) containing 0.03% hydrogen peroxide for 5–30 minutes to visualize the reaction product. Controls consist of eliminating the primary antibody or pre-absorbing the primary antibody but otherwise processing the tissue in an identical fashion. Control sections should not exhibit any specific staining.

Amplification of signal The necessity of using glutaraldehyde to fix human postmortem tissue may result in diminished staining for some antibodies. Thus, amplification techniques may be necessary for certain antibodies. Several types of signal amplification techniques are likely to work in postmortem tissue. A method that we have found to be particularly successful is the BLAST reaction (purchased from DuPont) (Borrow et al., 1989; Adams, 1992), which can amplify the signal an order of magnitude.

THE BLAST REACTION 1. Biotinylated tyramine is diluted 1:1000 with PB containing 0.005% H2O2. This step is inserted in the basic ABC reaction after the wash which follows the avidin-biotin step (which is shortened to 30 minutes). 2. Incubate tissue in the BLAST solution for 20 minutes. 3. Washed 3×5 minutes in PB. 4. Returned to the avidin-biotin solution for another 30 minutes and washed in PB. 5. The remainder of the incubations steps are the same as above. The BLAST reaction is particularly recommended for antibodies against tyrosine hydroxylase. 6. Incubate sections in diaminobenzidine (6 mg/10 ml PB) containing 0.03% hydrogen peroxide for 5–30 minutes to visualize the reaction product.

Quantitative analysis For the most part, the analysis of human postmortem tissue is the same as the analysis of fixed tissue from experimental animals. Thus, descriptive analyses as well as measures of synaptic density, proportion, length and cross-sectional areas of structures (such as axon terminals, dendritic shafts and spines, or mitochondria) can be performed. Some recommendations and problems are highlighted below.

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Synapse counting Photograph random fields of neuropil at 8,000–15,000 X and analyze at a total magnification of 22,500–30,000 X (Figures 7.1–7.3). The criteria for differentiating a symmetric versus an asymmetric synapse (Roberts et al., 1996b) is the thickness of the postsynaptic density: symmetric synapses exhibit a postsynaptic density less than 15 nm thick and at times a presynaptic thickening as well, whereas asymmetric synapses exhibit a prominent postsynaptic density typically between 20–50 nm thick. For synaptic density counts and measures, a final magnification of at least 28,000 X is preferable. Although lower magnifications are acceptable in well-fixed animal tissue, a little extra magnification is advised for postmortem work. At 28,000 X the postsynaptic target and the symmetry of the synapse are clear (Figures 7.1– 7.3) and can be used together to subcategorize synapses into: 1. 2. 3. 4.

asymmetric axospinous symmetric axospinous asymmetric axodendritic symmetric axodendritic

Simple profile counts versus stereology Although recent studies are moving away from simple profile counts and are utilizing stereological methods, certain questions can be answered using the traditional method. For example, when comparing the synaptic organization of the human to that of other species, which have been analyzed using simple profile counts, this same technique must be used in human material in order to yield directly comparable data. The application of stereological methods (Sterio, 1984; Geinisman et al., 1996) to the quantification of synapses in human postmortem tissue adds another level of complexity to the analyses. In regions of maximum tissue preservation, serial sections can be analyzed. However, some tissue which ordinarily would be adequate for simple profile counts or any analysis that could be performed in one or two serial sections, may not be practical for stereology. As the postmortem interval increases, the extracellular space expands, decreasing the number of synapses that can be followed in a long series of sections. Nevertheless, serial sections can be analyzed in well-preserved tissue using stereological methods. Figure 7.4 illustrates a series of four sections obtained from a tyrosine hydroxylase (TH) immunostained adult human striatum. One spine (s1) is present in all four sections, but only receives a synapse in two of these sections. Therefore, using Figure 7.4C as the counting section and Figure 7.4D as the look-up section, the

Figure 7.2 Electron micrographs from the adult human neostriatum, from the same case as shown in Figure 7.1A, showing several types of synapses (arrows). Note profile identity, vesicular morphology, symmetry of the synapses and intact membranes and mitochondria. A: A spine (s) emanating from a dendrite (d), receives an asymmetric synapse (open arrows). The postsynaptic density is perforated (arrowhead at perforation). B: A dendrite (d) is postsynaptic to an axon terminal (at) forming an asymmetric synapse (open arrow). A nearby axon terminal (at) forms a perforated asymmetric synapse (open arrows at synapse, arrowhead at perforation) with a spine (s), which it partially engulfs. C: A dendrite (d) is postsynaptic to an axon terminal (at) forming a symmetric synapse (arrows). A nearby spine (s) receives an asymmetric synapse (open arrows). Arrowheads indicate a dense core vesicle in an adjacent bouton. Mitochondria are marked with asterisks. Scale bars=0.5 µm. (Reprinted from Figure 1, Roberts et al. (1996) J. Comp. Neurol., 374, 523–534. With permission from Wiley-Liss.)

Figure 7.3 Electron micrographs of adult human striatal neuropil taken from the same case as shown in Figure 7.1A. A: An example of a spine (s) which is postsynaptic to more than one axon terminal. One terminal (at 1) is packed with round and pleomorphic vesicles (arrows) and forms an asymmetric synapse (open arrows). The other terminal (at 2) is sparsely filled with vesicles (arrows) and forms a symmetric synapse (arrows) with the same spine. Note the presynaptic density in at 2. A third terminal (at 3), containing only a few vesicles (arrows) contacts the spine but does not form a synapse in this section (*, mitochondria). B: An example of a synapse en passant formed with a spine (s). Synaptic vesicles (arrows) are clustered at the synapse (open arrow). Scale bars=0.5 µm. (Reprinted from Figure 3, Roberts et al., 1996, J. Comp. Neurol., 374, 523–534. With permission from Wiley-Liss.)

Figure 7.4 Examples of serial sections through two synaptic complexes in the adult human striatum stained for the immunocytochemical localization of tyrosine hydroxylase (TH). A myelinated axon (*) is labeled as a reference point in each micrograph. This plate demonstrates the feasibility of EMimmunocytochemistry and serial sections for stereology. While the TH labeled axon does not form a synapse in any of these sections, an example of a THlabeled synapse is shown in the next figure. A–D: TH-immunoreactive (TH-i) axon (open arrow) is apposed to two synaptic complexes (unlabeled axon terminals (at 1, at 2) and spines (S1 & S2)). TH-i axon is closely apposed to at 1 and at 2, which both appear in all four sections. (C). A large perforated synapse is formed (arrows) between at 2 and spine 2 (C, D). A synapse is formed between at 1 and spine 1 (B, C). Scale bars=0.25 µm.

Figure 7.5 Electron micrographs of calbindin immunoreactive structures in the fetal and adult striatum. A: Calbindin-i cell (n) in the fetal putamen exhibiting a notched nucleus, scant cytoplasm and a tortuous process (open arrows). B: A typical arrangement of TH-labeled profiles and a synaptic complex in the adult human striatum. A TH-i bouton (open arrow) forms a symmetric synapse (little arrow) with a spine (s), which is also postsynaptic to an unlabeled axon terminal (at) which forms an asymmetric synapse (arrow) with it. Another TH-i bouton (open arrow) is closely apposed to the axon terminal, but does not form a synapse with it. C: Calbindin-i neuronal somata (n) in the adult, exhibiting an unindented nucleus and scant cytoplasm, features of medium spiny neurons. An adjacent glial cell (g) is indicated. D: Calbindin-i process receives a synapse (arrow) from an unlabeled axon terminal (at) in the fetal putamen. E: An asymmetric synapse (arrow) in the fetal putamen is formed between an unlabeled dendrite (d) and an axon terminal (at). Two calbindin-i fibers (open arrows) are present. F: Calbindin-i spines (s) receive synapses (arrow) from nonlabeled axon terminals (at) in the adult striatum. Scale bars=2 µm (A, C), 0.5 µm (B, D–F).

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synapse formed onto spine1, which is present in 7.4C but not in 7.4D, can be counted. When counting large synapses, such as striatal perforated synapses which are greater than 1 µm in length (Roberts et al., 1996b), long ribbons of serial sections should be collected in order to obtain an accurate tally (see Geinisman et al., 1996). Analyses of fetal tissue Ultrastructural studies of various regions of the developing human brain indicate the presence of synapses in tissue from second trimester brains (Kostovic et al., 1989; Kostovic & Rakic, 1990). Many areas of the fetal brain, including the striatum, are characterized by large expansions of extracellular space, few synapses (Figure 7.5 A, D, E), growth cones and cells in various stages of maturation. Due to the scarcity of synapses encountered in some brain regions and the large areas of extracellular space, measures of synaptic density are especially challenging, though possible (Kostovic & Rakic, 1990). CONCLUDING COMMENTS Ultrastructural studies of human postmortem brain are possible and many techniques developed in animal studies can be successfully applied to human tissue. With patience, the collection of a meaningful number of subjects is feasible. Due to the problems inherent with the technique, there is a paucity of information on the synaptic organization of the human brain. In psychiatric research, where few good animal models exist which model fundamental mental deficits such as hallucinations, delusions, thought disorder, and suicidal ideation, the study of postmortem brain tissue from patients with mental illness is crucial.

Acknowledgements The authors are indebted to the personnel of the Maryland Brain Collection and the Brain and Tissue Bank for Developmental Disorders at the University of Maryland, especially Boris Lapidus and Robert Vigorito, for their special efforts in obtaining the short postmortem interval brains. Also, we thank Michelle Force, Kristoffer Crosby, and Joyce J.Kelley for excellent technical assistance and Sharon Jones for help with the preparation of the manuscript. This work was supported in part by the Scottish Rite Benevolent Foundation’s Schizophrenia

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Research Program, NMJ, USA, the Stanley Foundation, P30MH40279 and DRIF funds from the University of Maryland.

References Adams, J.C. (1992) Biotin amplification of biotin and horseradish signals in histochemical stains. J Histochem. Cytochem., 40, 1457–1463. Aganova, E.A., and Uranova, N.A. (1992) Morphometric analysis of synaptic contacts in the anterior limbic cortex in the endogenous psychoses. Neurosci. Behav. Physiol., 22, 59–65. Babb, T.L., Kupfer, W.R., Pretorius, J.K., Crandall, P.H., andLevesque, M.F. (1991) Synaptic reorganization by mossy fibers in human epileptic fascia dentata. Neuroscience, 42(2), 351–363. Borrow, M.N., Harris, T.D., Shaughnessy, K.J., and Litt, G.J. (1989) Catalysed reporter deposition, a novel method of signal amplification. Application to immunoassays. J. Immunol. Methods, 125, 279–285. Geinisman, Y., Gundersen, H.J.G., Van Der Zee, E., and West, M.J. (1996) Unbiased stereological estimation of the total number of synapses in a brain region. J. Neurocytol, 25, 805–819. Hsu, S.M., Raine, L., and Fanger, H. (1981) The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 29, 577–580. Kostovic, I. and Rakic, P. (1990) Developmental history of the transient subplate zone in the visual and somatosensory cortex of the macaque monkey and human brain. J. Comp. Neurol., 297, 441–470. Kostovic, I., Seress, L., Mrzljak, L., and Judas, M. (1989) Early onset of synapse formation in the human hippocampus: a correlation with nissl-golgi architectonics in 15- and 16.5-week-old fetuses. Neuroscience, 30, 105–116. Kung, L., Force, M., Chute, D.J., Smialek, J. and Roberts, R.C. (1996) The immunocytochemical localization of tyrosine hydroxylase in the human striatum: an ultrastructural study. Soc. Neurosci. Abstr., 22, 892. Kung, L., R.Conley, D.J.Chute, J.Smialek and R.C.Roberts (1998a) Synaptic changes in the striatum of schizophrenic cases: a controlled postmortem ultrastructural study. Synapse, 28, 125–239. Kung, L., M.Force, D.J.Chute and R.C.Roberts (1998b) Immunocytochemical localization of tyrosine hydroxylase in the human striatum: a postmortem ultrastructural study. J. Comp. Neurol., 390, 52–62. McLean, I.W. and Nakane, P.K. (1974) Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J. Histochem. Cyctochem., 22, 1077–1083. Ong, W.Y. and Garey, L.J. (1993) Ultrastructural features of biopsied temporopolar cortex (area 38) in a case of schizophrenia. Schizophr. Res., 10, 15–27.

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Roberts, R.C. and Francis, S.M.N. (1993) The synaptic organization of the human striatum in schizophrenics and controls: a quantitative ultrastructural study. Schizophr. Res., 9(2,3), 150. Roberts, R.C., Strain-Saloum, C.E., Gaither, L.A, Perretti, F.J. and Vigorito, R.D. (1994) Ultrastructural features of the developing and adult human striatum. Soc. Neurosd. Abstr., 20, 785. Roberts, R.C., Conley, R., Kung, L., Peretti, F.J. and Chute, D.J. (1996a) Reduced striatal spine size in schizophrenia: a postmortem ultrastructural study. NeuroReport, 7, 1214–1218. Roberts, R.C., Gaither, L.A., Peretti, F.J., Lapidus, B. and Chute, D.J. (1996b) The synaptic organization of the human striatum: a postmortem ultrastructural study. J. Comp. Neural., 374, 523–534. Roberts, R.C., McKim, R., Kung, L., Crosby, K. and Chute, D.J. (1997) The immunocytochemical localization of tyrosine hydroxylase in the human substantia nigra: an ultrastructural study. Schizophr. Res., 24, 41. Smiley, J.F., Levey, A.I., Ciliax, B.J., and Goldman-Rakic, P.S. (1994) D1dopamine receptor immunoreactivity in human and monkey cerebral cortex: Predominant and extrasynaptic localization in dendritic spines. Proc. Natl. Acad. Sci. USA, 91, 5720–5724. Sterio, D.C. (1984) The unbiased estimation of number and size of arbitrary particles using the dissector. J. Microsc., 134, 127–136. Uranova, N.A. (1996) Brain synaptic plasticity in schizophrenia. Vestn. Ross Akad. Med. Nauk., 4, 23–29.

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ISOLATING COMPONENTS OF HUMAN β AND BRAIN: THE PURIFICATION OF Aβ THE ALZHEIMER’S AMYLOID PRECURSOR PROTEIN Robert A Cherny, Colin L.Masters, Konrad Beyreuther and Ashley I.Bush

INTRODUCTION Human brain tissue presents a variety of challenges to those planning to extract and purify biologically active molecules. These issues include: • • • • •

the potential need to maintain close to physiological conditions, the need to prevent degradative processes associated with cell damage, the need to accommodate the solubility characteristics of the molecule of interest, brain tissue is soft and when thawed is especially yielding, white matter containing the neuronal axons is highly enriched in lipid in the form of myelin which poses a potential obstacle to extraction procedures.

Of equal importance are the safety concerns that relate to working with human brain tissue. In particular brain tissue represents a significant biohazard to the operator. The presence of viruses and prions make protection against these agents of prime concern to those designing laboratories in which human brain research will be carried out. In particular, the potential for the presence of particular infectious agents should accommodated in the design of laboratories in which human brain tissue is used. These agents include: • • •

the HIV virus, numerous forms of hepatitis, transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease and related conditions. 141

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In our laboratory we extract and purify Alzheimer’s disease-related proteins, such as the amyloid precursor protein (APP) and Aβ (also known as βA4) (Figure 8.1). While Aβ is a cleavage product of APP, the two molecules represent opposite poles in terms of size, ionic and solubility characteristics. Because of this, these proteins will serve to demonstrate the range of procedures which may be employed to extract and purify biologically significant molecules from brain tissue. Thus, while this chapter by no means exhausts the variety of procedures available to the investigator, hopefully it will serve as a guide to the perplexed and a starting point for the novice.

LABORATORY DESIGN AND SAFETY In addition to the usual care taken when processing potentially infectious material, human brain tissue presents a unique hazard to the operator in relation

Figure 8.1 A: Structural features of the Amyloid Precursor Protein. The soluble APP exists in 100–110 kD forms while the membrane-bound APP is larger at 120–130 kD. Note the heparin (HBD) and zinc binding sites and the secretase sites at which A is cleaved from the precursor. B: A is cleaved from the parent APP at several alternative sites by as yet unknown secretases resulting in variable length forms of the molecule. The predominant species are 40 or 42 residues in length (ca. 4 kD) but other variants may have significant roles in the seeding and promotion of amyloid deposition.

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to potentially transmissible diseases. The putative transmissible agent for some of these diseases, the prion, is an altered, protease-resistant form of a normal neuronal membrane protein which accumulates in the CNS. Prions are remarkably resistant to commonly used proteolytic and anti-viral agents and specific decontamination measures should be instituted. The British Advisory Committee on Dangerous Pathogens (London: HMSO) has published a handbook detailing recommended procedures for decontamination of such agents. These include autoclaving at 134 °C (not 121 °C) for 18 minutes and chemical disinfection of laboratory ware with sodium hypochlorite at 20,000 ppm for 1 hour. Any procedure that may generate aerosols must be conducted in a Class II biohazard cabinet. (NB: UV light and formalin, commonly used to decontaminate laminar flow cabinets, stabilise the prion proteins and should not be used.) Although the natural incidence of prion related diseases is low, workers in a laboratory regularly dealing with human brain tissue should anticipate coming into contact with infected specimens. In our laboratories the processing of brain tissue is conducted in a dedicated negative-pressure environment serviced by a separate air-conditioning system incorporating a removable HEPA filter. Processing of human brain tissue takes place within a Class II biohazard cabinet and all dissection equipment, including centrifuges, is restricted to work involving human brain tissue. Operators wear disposable, waterproof full-arm length gowns, safety glasses, overshoes and two pairs of latex gloves. All material is subjected to high temperature autoclaving (see above) in special hightemperature autoclave bags before leaving the confines of the laboratory. If facilities where only infrequent work is being contemplated, the minimum requirements should be a biohazard cabinet, a dedicated area which can be swabbed with sodium hypochlorite after use and suitable disposal facilities. Under these circumstances the operator should ensure that, wherever possible, all activities are conducted within the cabinet or within sealed vessels, e.g. during centrifugation. Full details should be obtained from your regional authority for regulations concerning decontamination and disposal procedures. Tissue collection and preparation Procedures for the collection of postmortem brains are covered elsewhere in this book. However, some practical suggestions for those contemplating the use of frozen tissue include the following. •

Generally the histopathological indicators of neurological conditions are manifested bilaterally, thus it can be advantageous to formalin fix one

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hemisphere for histopathological analysis whilst the other, for protein purification, is frozen at -80 °C. Tissue should be frozen in such a way as to avoid distortion, enabling the operator to distinguish major anatomical features. This is particularly the case if the person processing the tissue is not trained in neuroanatomy. To this end, to minimise such distortion we provide specially commissioned cardboard containers of a size such that the hemisphere (in a resealable zip-lock plastic bag) can be frozen without being folded upon itself. Be aware that many plastics are not resistant to extremely low temperatures and care should be taken when selecting suitable storage vessels. A dedicated -80 °C chest freezer is most suitable for long-term storage as this type is least likely to gain temperature if remaining open for lengthy periods. Storage in a standard freezer at -20 °C is not recommended as the activity of degradative enzymes will not be abolished at that temperature. Connecting the freezer to an emergency power generator is another worthwhile precaution. If the entire brain is not to be processed at one time, the hemisphere can be cut into 1 cm thick coronal slices using a modellers handsaw or a hacksaw. The slices can be separated by pieces of plastic laboratory film such as Parafilm (American National Can Co.).

Tissue frozen at -80 °C is extremely hard and we prefer to allow the brain to thaw to around -20 °C before attempting to section. Smaller sections can be collected and stored in clear plastic screw-top specimen vials. As experiments frequently require repeating, the operator is well advised to prepare multiple specimens from the anatomical regions of interest. Clear labelling, careful storage and systematic record keeping are essential to avoid repeated thawing and refreezing of tissue.

SAMPLE PREPARATION As the Alzheimer’s disease-associated proteins are manifested primarily within the neocortex we have concentrated on establishing methods for processing this portion of the brain. In our laboratory we routinely process an entire hemisphere to obtain APP in its soluble and membrane bound forms. The protocol we have developed for this purpose has been scaled up from preliminary small-scale experiments, such small-scale experiments can provide information on ideal

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buffering conditions, centrifugation speeds, and tissue to buffer ratios. Optimisation of the extraction conditions usually involves a compromise between relatively harsh conditions that would allow extraction of most protein from the tissue and less harsh conditions that will extract less protein but preserve the biological activity of the material of interest. Scaling-up presents additional challenges in the form of extraction efficiencies and logistical obstacles (Scopes, 1994). For those commencing protein purification, the following details provide information on the equipment and techniques required for such protocols. PROTEIN PURIFICATION: EQUIPMENT AND TECHNIQUES

Equipment 1. 2. 3. 4. 5.

Two pairs of long, blunt-tip forceps. Scalpel blade handle to suit no. 10 blades. Flat bladed safety razor blade. Long, flat-bladed carving knife. Blender for processing several hundred grams of tissue (e.g. Sorval Omnimixer, Dupont instruments, USA) or a smaller homogenising instrument, e.g. UltraTurrax T25 (Janke & Kunkel Labortechnik, Germany) for gram quantities. A Potter homogeniser is a serviceable alternative. 6. A deep tray filled with ice. 7. A small waste bin fitted with an autoclavable bag. This sits inside the biohazard cabinet and is used for minor waste. 8. A 10 litre vessel with autoclavable spigot containing 2 L of 10 M NaOH for large volumes of liquid waste. 9. As an option, a cooled dissection surface. This may take the form of a shallow vessel containing dry ice upon which a shallow stainless steel instrument tray is placed. 10. Autoclavable centrifuge tubes. (NB: For small samples we find it easiest to conduct the homogenisation in the same vessels used for centrifugation). 11. An electronic balance with tare facility. 12. Parafilm or aluminium foil squares 5 cm×5 cm. 13. Spray bottle filled with sodium hypochlorite for decontamination.

Homogenisation and extraction buffers The constituents of homogenisation and extraction buffers will be determined by the nature of the molecule to be extracted from brain tissue and will usually be based on a Tris or phosphate buffered saline. Whenever possible buffers should be prepared using HPLC-grade reagents and deionised water, e.g. Milli-Q

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(Millipore). Filter all solutions through a 0.22Å membrane. Keep the buffers on ice during the extraction process. HOMOGENISATION BUFFER 1. To 1800 ml distilled water add 12.11 g Tris.HCI (50 mM) and 20.50 g NaCl (175 mM). 2. Adjust to pH 8.5. 3. Add 0.29 g EDTA (5 mM) 4. Add 0.2 g sodium azide (0.01% w/v). 5. Adjust to pH 7.4. 6. Make to 2 L. 7. Filter and store at 4 °C

EXTRACTION BUFFER 1. Prepare 25% Triton X-100 in H2O (prepare this by slow stirring overnight, the concentrated detergent is very viscous). 2. Add 80 ml of 25% Triton X-100 to 920 ml of homogenisation buffer. 3. Filter and store at 4 °C. 4. Add 400 µl of a stock solution of 25 g/L phenyl, methyl, sulphonyl, fluoride (PMSF) in methanol to 1 L of buffer just prior to use. (PMSF is a protease inhibitor and has a short half life in the presence of its substrate.)

The pH and ionic strength of a buffer are critical variables in maintaining solubility and will dictate the behaviour of molecules during the purification. Extraction of membrane or vesicle bound proteins will require a detergent in the homogenisation buffer and the type and concentration of detergent will vary with the solubility properties of the protein to be purified. For example, APP can be solubilised with a dilute solution of the non-ionic detergent Triton X-100, whereas to solublise Ab we apply the powerful detergent sodium dodecyl sulphate (SDS). Homogenisation results in cellular disintegration which release of a wide variety of proteases (Table 8.1). To prevent proteolytic degradation protease inhibitors must be added to the buffers before proceeding with the extraction. Protease inhibitors can be purchased in the form of a ‘cocktail’ combining inhibitors of all the known classes of protease. Importantly, if metals are to be used in a subsequent affinity purification chelating agents such as EDTA cannot be used. In such a case the individual protease inhibitors must be purchased and made up according to the recommended concentrations. Also note that protease inhibitors have a short half-life in the presence of their substrates and must be added to the buffer at the last possible moment. Maintain the buffers at 4 °C at all times.

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CHROMATOGRAPHY SYSTEMS Manufactured by Pharmacia (Uppsala, Sweden), FPLC (Fast, Protein Liquid Chromatography) is a modular, medium pressure chromatographic system employing computer-linked pumps in combination with a variety of valves to produce solvent gradients from two buffers of differing ionic strengths. Recent software for the FPLC contains interactive guide through the often baffling process of selecting a purification strategy. The FPLC is flexible and can be used for semi-preparative and analytical level purification. The Biosys is another chromatography system produced by Beckman Instruments which is suitable for high, medium and low pressure applications including semi-preparative and preparative HPLC. This system does not require a PC controller and can be operated at 4 °C if desired. The Biologic (Biorad) is marketed as a less expensive alternative to FPLC and is again compatible with most columns and fittings. The system does not operate without a dedicated computer, however the software is user-friendly and the hardware easy to maintain. HPLC, originally ‘high pressure liquid chromatography’ now ‘high performance liquid chromatography’, is useful for very high resolution of minute quantities of mixtures and is recommended for the last step in a purification which results in less than 5 mg of product (see Scopes, 1994 for an explanation of the theoretical basis of the different chromatographic techniques). Purification processes may also include technologies such as capillary electrophoresis, but a description of such techniques is beyond the scope of this discussion as they require extremely specialist knowledge and skills. Table 8.1 Protease inhibitors that can be obtained from Boerhringer Mannheim (Germany)

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PROCESSING HUMAN BRAIN TISSUE 1. Remove the brain from -80 °C freezer. 2. Place brain in a refrigerator at 4 °C approximately 15 hours prior to dissection. (Slices and smaller specimens should be processed without preliminary thawing.) 3. Remove meninges using forceps to prevent this tough membrane from interfering with the blades of the blender if not removed. 4. Dissect grey matter from the interdigitating myelin-rich white matter. 5. For small samples: (a) Place Parafilm in weighboat. (b) Mince tissue using a safety razor blade and weigh out the desired mass. (c) Place specimen into the homogenisation vessel. 6. Wherever possible maintain tissue and equipment at 4 °C. 7. Before homogenisation weigh tissue. 8. Begin homogenisation at low speed and gradually increase speed to minimise the generation of heat and foam. NB: The volume of buffer used per gram of tissue should be determined empirically but as a guideline 1 volume of tissue to 2–3 volumes of buffer. 9. After homogenisation lipids and particulate matter are removed from the homogenate by filtering through Whatman no. 1 filter paper. 10. Transfer filtrate to centrifuge bottles.

An example of a protein purification schema is summarised in Figure 8.2 (Moir et al., 1992). The described protocol for the purification of APP was established empirically, based on known characteristics such as molecular weight, isoelectric point as well as parameters predicted from the known amino acid sequence. This information included the observation that APP contained a putative heparin binding site and amino-acid sequences that predicted the molecule would undergo hydrophobic interactions (Figure 8.1). PURIFICATION OF APP 1. Pass solubilised brain extract through an anion exchange column (Macroprep, Pharmacia) under low pressure applied using a peristaltic pump (A). 2. Elute bound proteins off the column using a 1 M Tris-buffered salt solution. 3. To remove excess salt, pass fractions containing APP through a desalting column (B). 4. Taking advantage of the high-affinity heparin binding site, the desalted fraction containing APP is then loaded, at a low rate (overnight at 4 °C), onto a heparinsepharose affinity column (C) using a high accuracy benchtop peristaltic pump (Econopump, Biorad) to maintain a stable flow rate.

Figure 8.2 Schematic diagram of a protocol for the purification of APR from human brain. (Reproduced with the kind permission of Dr Robert Moir.)

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5. Using the FPLC automated chromatography system, APR (and other heparinbinding proteins) are eluted from the column by a salt gradient generated by combining two buffers at a predetermined flow rate(D). The eluting heparinbinding proteins are detected by monitoring for absorbance at 280 nm. 6. The heparin binding proteins are collected and loaded onto an anion exchange column (Mono Q, Pharmacia) for further purification (E). 7. Bound protein is eluted using a salt gradient and 0.5 ml fractions are collected using a pre-programmed fraction collector. 8. Duplicate samples from each fraction are analysed using polyacrylamide gel electrophoresis (PAGE, Glycine, 10%) and subjected to Western transfer (see discussion). 9. APP is detected using a specific an anti-APP monoclonal antibody (22C11, Boehringer-Mannheim, Germany) in conjunction with an enzymatically-catalysed chemiluminesence reaction (Amersham ECL). 10. Silver staining of the duplicate gels provides a measure of the efficiency and selectivity of the purification process. 11. The APP-containing fractions (as determined in the preceding step) are pooled and loaded onto a column containing phenyl superose (Pharmacia) for a final purification employing hydrophobic interaction chromatography (F). 12. Fractions that make up the largest 280 nm absorbance peak are again collected and are subjected to PAGE, Western blot and immunodetection as described previously and duplicate silver stain gels confirm the absence of contaminating protein. (Fig. 8.3: Representative phenyl superose chromatogram and accompanying polyacrylaminde gels.) NB: Periodically, samples of the APP preparation undergo amino acid sequencing to establish the purity of the product.

Column buffers will vary according to the chromatographic medium used. For our Macroprep, Desalting, Mono-Q and Heparin columns we utilise: • •

Buffer A: 50 mM Tris, 5 mM EDTA, 0.02% NaN3, pH 7.4, filter. Buffer B: Buffer A plus 1 M NaCl, pH to 7.4, filter.

GEL ELECTROPHORESIS Polyacrylamide gel electrophoresis (PAGE) and FPLC are the workhorses of our protein chemistry laboratory. An excellent treatment of the subject of PAGE is to be found in the manual Gel Electrophoresis of Proteins: A Practical Approach (Hames & Rickwood, 1990), which includes a comprehensive section on the Western blot procedure. If a PAGE system is not already in use in the laboratory, the operator may choose to purchase a system which employs ready made ‘pre-cast’ gels or may choose to cast the gels in the laboratory. The advantages of the former

Figure 8.3 Top: Representative chromatogram from the final (phenyl superose) stage of the purification of APR from human brain; 0.5 ml fractions are collected across the salt gradient. Bottom: The fractions are run on duplicate SDS-PAGE gels. Upper panel: Western blot, APR detected with monoclonal antibody 22c11 and visualised using alkaline phosphataseconjugated secondary antibody. Lower panel: silver stained duplicate gel showing that APR is uncontaminated by other protein.

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are convenience and reproducibility although for some laboratories the high cost (ca, US$10 per gel) may outweigh the disadvantages. We prepare simple ‘homemade’ 10% glycine gels using the Hoefer system (Hoefer Scientific Instruments, San Francisco, Ca. USA, recently merged with Pharmacia Biotech) for qualitative APP analysis. For resolution and quantitation of the small protein/ peptide Aβ we employ pre-cast Novex 10–20% Tris-Tricine gradient gels (Novex, San Diego, Ca. USA). Biorad (Hercules, Ca. USA) manufacture a system which accepts both pre-cast and manually cast gels. Each of the systems offers advantages and disadvantages in ease of use, cost and reliability and each provides modules for Western transfer. Unfortunately, none of the systems are interchangeable so it is advisable to seek advice before committing to purchase. If resolution of proteins of similar molecular weight is required, a second separation based upon isoelectric point (pI) may be necessary. Two-dimensional electrophoresis (2D PAGE) is often required when purifying a protein for mass spectrometry or amino-acid analysis/ sequencing. Pharmacia (Uppsala, Sweden) manufactures an integrated 2D system which includes pre-cast isoelectric and molecular weight gels. A much lower cost system is produced by Biorad which requires manual gel casting. β EXTRACTION AND PURIFICATION: AN EXAMPLE OF A Aβ ‘DIFFICULT’ PROTEIN The purification and characterisation of the major component of the amyloid plaques of Alzheimer’s disease was a turning point in the elucidation of the biochemical basis of the disease (Glenner & Wong 1984, Masters et al., 1985). Protocols for extraction of the 40 amino acid polypeptide named Aβ or alternatively βA4, exploited the extreme insolubility of the crystalline cores at the centres of the plaques. Highly hydrophobic, with a strong tendency to aggregation, Aβ does not lend itself to separation techniques which involve interactions between itself and the chromatographic medium, anion exchange for example, however it is a remarkably robust molecule (a quality which most likely contributes to its persistence in the brain over decades). In addition, its small size restricts the use of desalting procedures which might otherwise be used to remove solubilising agents such as detergents. These characteristics, coupled with the extreme insolubility of the amyloid plaque cores suggested a purification strategy that employed a combination of differential solubilisation steps followed by size exclusion chromatography (gel filtration) using a resolving solvent of high stringency. Formic acid has been found to be highly effective in dissolving the aggregated Aβ although this solvent presents complications

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downstream where complicated procedures are required to remove it from the purified product. The following protocol represents an extreme example of the combination of techniques which may be required to obtain a purified specimen of the desired molecule, however each and any of these methods may be used in alone or in combination depending on the characteristics of the species of interest and the degree of purity desired. β FROM AMYLOID PLAQUES EXTRACTION AND PURIFICATION OF Aβ (from Roher et al., 1993)

1. Mince grey matter from brain (Alzheimer’s disease brains give high amounts Aβ) in a blender at 4 °C for 5 seconds in 20 volumes of homogenisation buffer (10 mM Tris-HCI pH7.5, 0.25 M sucrose, 2 mM EDTA, 200 mg/ml PMSF mg/ml leupeptin, 0.7 mg/ml pepstatin, 50 mg/ml gentamicin sulphate, 0.25 mg/ml amphotericin B). 2. Filter homogenate through stainless multiple steel mesh 700, 350, 150, 75 and 45 µm. 3. Adjust sucrose concentration to 1.2 M. 4. Centrifuge at 25,000×g for 30 minutes at 4 °C. 5. Suspend resulting pellet in 12 volumes of buffer and filter through a 45 µM mesh. 6. Adjust sucrose to 1.9 M and spin at 125,000×g for 30 minutes at 4 °C. 7. Remove solids that float to the tops of tubes, pool and wash four times with 50 mM Tris-HCI pH 8.0. 8. Add 2 mM CaCI2, collagenase CLS3 100 mg, DNAse 15 mg to 500 ml suspension and incubate at 37 °C for 14 hours with continuous shaking. 9. Centrifuge suspension at 6000×g for 30 minutes at 4 °C. 10. Wash pellets thrice with Tris-HCI pH 8.0 and add solid SDS to a final concentration of 5%. 11. Incubate at room temperature for 2 hours. 12. Precipitate the insoluble material by centrifugation at 15,000×g for 10 minutes at 20 °C. 13. Suspend the resulting pellet in 800 ml of Tris-HCI pH 8.0. 14. Add solid sucrose to a final concentration of 1.3 M and centrifuge at 50,000×g for 45 minutes at 20 °C. 15. The pellets which form above and below an intermediate supernatant are collected. 16. The bottom pellet is suspended in 18 ml of Tris-SDS buffer (50 mM Tris-HCI pH 8.1, 1% SDS). 17. The suspension is then layered onto a discontinuous sucrose density gradient composed of 2 ml steps of 2.1, 2.0, 1.7, 1.4 and 1.3 M sucrose and spun at 200,000×g for 1 hour at 20 °C. 18. The material from within the 1.4/1.7 M interface is recovered and layered over a second ladder of 2.3 ml steps consisting of 2.2, 1.8, 1.7, 1.6 and 1.5 M sucrose and spun as in 17.

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19. The amyloid cores are now recovered from the 1.4/1.7 M interface. 20. The amyloid cores are then washed by suspension in 1 ml Tris-SDS buffer. 21. The suspended amyloid cores are then centrifuged in 1.1 M sucrose at 300×g for 15 minutes at 20 °C in conical 1 ml vials. 22. The supernatants and top portions of pellets are discarded. 23. The remaining pellets are washed once with distilled water and twice with 0.1% SDS dissolved in water and spun at 1500↔g for 10 minutes at 20 °C. 24. Dissolve core preparations in 80% glass-distilled formic acid at 4 °C. 25. Incubate for 20 minutes at RT then spin at 435,000↔g for 10 minutes at 4 °C. 26. Collect clear supernatant and dialyse against 6 M guanidine HCI, 0.1 M TrisHCI, pH 8.5 using 1000 Dalton cutoff dialysis tubing. 27. A flocculent precipitate will form in the dialysis tubing which is recovered by centrifugation at 1500×g for 10 minutes at RT. 28. The resultant precipitant is dissolved in 80% formic acid. 29. The solubilised precipitant is submitted to size exclusion HPLC on a 300×10 mm Superose 12 column equilibrated in 80% formic acid at a flow rate of 0.2 ml/ minute with monitoring at 280 nm. 30. Fractions containing βA4 are identified by immunodetection (slot blot) and then concentrated by vacuum centrifugation and dialysed against 8% aqueous betaine in 0.1 M ammonium bicarbonate pH 7.8. Alternatively, high concentrations of chaotropic agents such as urea and guanidine thiocyanate are used in place of the formic acid, as in the following (from Dyrksetal., 1992). 1. Prepare the insoluble fraction as described previously. 2. Wash insoluble fraction twice with solution A (1 M urea, 1% Triton X-100, βmercaptoethanol) and once with 1 M urea under sonification. 3. Further dissolve insoluble proteins in 9 M urea under sonification. 4. Fractionate using DEAE sepharose CL-6B anion exchange column equilibrated in 9 M urea, 100 mM Tris-HCI (pH 8.0), 1 mM EDTA, 10 mM NaCl. 5. Proteins are eluted with 10–200 mM NaCl gradient. Nβ: A fragment elutes between 40 and 120 mM NaCl.

More recent investigations revealed the existence of substantial quantities of soluble Aβ species of variable length and aggregating potential co-existing with the insoluble deposits. (Harigaya et al., 1995). In our laboratory we are investigating the factors controlling the flux between soluble and insoluble forms and have established a protocol for extraction of the various forms. The soluble forms of Aβ are extracted relatively easily using buffered saline solutions or detergents, however, obtaining a pure specimen of the soluble Aβ is complicated by its propensity to aggregate at relatively low concentrations. The aggregation is believed to involve a conformational change from helix to β sheet, the latter form being insoluble and precipitating out of solution. Some extraction

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procedures employ solvents such as hexafluoroisopropanol which act to maintain alpha-helical conformation and thus solubility. Such organic solvents have the advantage of being volatile and easily driven away using vacuum centrifugation following solubilisation but these agents severely restrict the operator in the range of plasticware which can be used. Generally, only polypropylene-type plastics are compatible (e.g. ‘Eppendorf’ microfuge tubes).

Alternative techniques and general hints •

•

•

•

The ‘molecular sieving’ approach (Rohr et al. 1996). Low molecular weight Aβ is separated from larger proteins by being forced through a membrane of limiting pore size. Disposable filters such as ‘Centricon’ (Amicon, USA) are available in a range designed to exclude proteins varying from 3,000 to 100,000 kilodalton in size and may be used sequentially to trap a species of known molecular weight. These filters are also regularly used to remove undesired salts from protein preparations and to perform buffer changes whilst maintaining sample volume. (Normal desalting columns will allow passage of smaller proteins and peptides with the flowthrough.) Care must be taken as the membranes are not compatible with all reagents, particularly organic solvents. Also depending upon the circumstances, anything up to 80% of the molecule of interest may be lost through adherence to the plastic vessel components. This applies to all peptides and proteins, and the operator is advised to test a variety of plasticware for ‘stickiness’. A variation on this technique is the Centri Spin system (Princeton Separations Inc., NJ, USA) which incorporates a gel filtration matrix of known pore size (a selection of pore sizes is available) in a microfuge tube to which the sample is applied and then spun to wash out species below the molecular weight cutoff. We have found that the extraction process is enhanced several fold by freeze drying the tissue followed by homogenisation in phosphate buffered saline (PBS) containing a small quantity of metal chelator such as EDTA. The extract is passed through a superose 12 or superdex 75 size exclusion column (Pharmacia) resolved with PBS in the presence of a chelating agent and maintained at pH 8.5 to reduce aggregation. Optimisation of detection wavelength: Aβ exhibits several fold higher absorbance at 214 nm, than at the more commonly used 280 nm and although the greater sensitivity gained by detecting at this frequency is

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offset by the fact that some solvents including formic acid also absorb highly at 214 nm. (A UV monitor which can detect at variable frequencies is an expensive but worthwhile purchase.) We find that if the fractions containing Aβ are freeze dried they can be resolubilised in water without causing excessive re-aggregation. Metal chelation chromatography makes use of the affinity of many proteins for metals including copper, iron cobalt and nickel, and several purification strategies include a purification step employing a metal-bearing chelating matrix such as ‘Hi Trap’ (Pharmacia). We have also developed methods for selectively precipitating Aβ from solution by exploiting a high affinity zinc binding domain. Immunoprecipitation: Methods have been developed for the purification of Aβ in bodily fluids, e.g. urine (Ghiso et al., 1997), using immunoprecipitation. The standard protocol utilises agarose or sepharose beads conjugated to protein A which selectively bind immunoglobulins which in turn specifically bind the desired antigen (Firestone et al., 1990). We have found that Aβ binds non-specifically to Con A sepharose but have had some success when substituting iron-coated beads pre-conjugated with goat-anti-mouse immunoglobulins (Dynabeads: Dynal, Oslo, Norway) in conjunction with a specific anti-Aβ antibody. This system has an added advantage in that the bead-antigen-antibody complex is separated from the other components of the fluid by simple application of a magnet rather than being spun down as a pellet together with possible contaminants.

The development of highly specific monoclonal antibodies has been critical in establishing the success or otherwise of purification protocols for Alzheimer s-related proteins, and PAGE in conjunction with Western transfer remains an intrinsic element of the purification procedure. For Alzheimer’srelated proteins such antibodies are commercially available, 22C11 (Boehringer-Mannheim, Germany) for APP and 6e10 (Senetec, USA) for Aβ are two of the most commonly used antibodies but it must be noted that these are expensive and their purchase may represent a significant proportion of a laboratory’s budget. For reasons of cost or in the event that no antibody is commercially available, a program to generate ‘in-house’ antibodies may be required. The production of monoclonal antibodies should be tendered to a laboratory with experience in this technology but the generation of polyclonal antibodies is generally within the grasp of most biological laboratories. A polyclonal antibody may in fact prove advantageous with a

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heterogeneous protein such as Aβ. Often a purified sample of the molecule of interest is required to generate a specific antibody, leading to a ‘chicken and egg’ situation only resolved in several steps and frequently by the use of a fragment of the protein obtained by synthetic or recombinant technologies.

CONCLUSION In summary, methods have been described to recover proteins with many different physicochemical properties. Notably the vast majority of proteins found in the brain will exhibit some of the characteristics of either or both proteins. In any event, the scientist wishing to investigate a novel or less characterised protein should be clear about the quantity and purity of material required, as well as the required bio-activity after purification. In addition, any known characteristics of the protein should be investigated before setting up equipment and methodologies to purify the protein. The most expensive and complicated equipment is not required for many applications. Extensive consultation with informed scientists provides the most valuable foundation upon which to base these decisions and is highly recommended.

References Firestone, G.L. and Winguth, S.D. (1990) Immunoprecipitation of proteins. Meth. Enzymol., 182, 688–700. Ghiso, J., Calero, M., Matsubara, E., Governale, S., Chuba, J., Beavis, R., Wisniewski, T. and Frangione, B. (1997) Alzheimer’s soluble amyloid beta is a normal component of human urine. Febs Lett., 408, 105–8. Hames, B.D. and Rickwood, D. (1990) Gel Electrophoresis of Proteins: A Practical Approach (2nd edn). Oxford: Oxford University Press. Glenner, G.G. and Wong, C.W. (1984) Alzheimer’s Disease: initial report of the purification and characterisation of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun, 120, 885–890. Harigaya, Y., Shoji, M., Kawarabayashi, T., Kanai, M., Nakamura, T., Lizuka, T., Igeta, Y., Saido,T.C., Sahara, N., Mori, H. and Hirai, S. (1995) Modified amyloid β protein ending at 42 or 40 with different solubility accumulates in the brain of Alzheimer’s disease. Biochem. Biophys. Res. Commun., 211, 1015–1022. Masters, C.L., Simms, G., Weinman, N A., Multhaup, G., McDonald, B.L. and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl. Acad. Sci. USA, 82, 4285–4249.

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Moir, R.D., Martins, R.N., Small, D.H., Bush, A.I., Milward, E.A., Multhaup, G., Beyreuther, K. and Masters, C.L. (1992) Human brain βA4 amyloid protein precursor of Alzheimer’s disease: purification and partial charaaerization. J Neurochem, 59, 1490–1498. Scopes, R.K. (1994) Protein purification: Principles and Practice (3rd Edn). New York: Springer-Verlag. Rohr, A.E., O’Chaney, M.O., Kuo, Y-M., Webster, S.D., Stine, W.B., Haverkamp, L.J., Woods, A.S., Cotter, R.J., Tuohy, J.M., Krafft, G.A., Bonnell, B.S. and Emmerling, M.R. (1996) Morphology and toxicity of Aβ (1–42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer’s disease. JBC, 271, 202631–20635.

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ANALYSIS OF RECEPTOR SYSTEMS IN SCHIZOPHRENIA USING TISSUE OBTAINED AT AUTOPSY AND NEUROIMAGING Janet Mulcrone, Brian Dean and Robert W.Kerwin

The use of tissue obtained at autopsy from subjects with psychiatric disorders has slowly gained credence because they have begun to provide reproducible and robust data. Such consistent outcomes have been achieved by increasing care in controlling for confounding variables such as tissue collection, tissue preservation, donor medication and postmortem delay. More recently, studies using positron emission tomography (PET) and single photon emission computer tomography (SPECT) have also provided information on the molecular changes underlying schizophrenia. Thus, in future these two approaches to investigating molecular changes underlying psychiatric illness should provide complimentary and confirmatory data on the pathology of such illnesses. To demonstrate this point, outcomes of the study of three major neurotransmitter systems in schizophrenia, using both approaches, is reviewed.

THE STUDY OF SCHIZOPHRENIA USING TISSUE OBTAINED AT AUTOPSY Tissue obtained at autopsy has been used to examine the morphological and cytological changes in the CNS of schizophrenic subjects and has revealed characteristic cytoarchitecture resulting in the developmental theory of schizophrenia (Murray et al., 1988). In addition, by comparing receptor distribution patterns in tissue obtained at autopsy using radioligand binding characteristic changes in receptor density have been identified in CNS from schizophrenic subjects. The use of tissue obtained at autopsy has also allowed specific CNS organisation to be identified, such as the two neurochemically 159

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distinct divisions in the human nucleus accumbens which has been postulated to be equivalent to the ‘shell’ and ‘core’ of the rat (Voorn et al., 1994). More recently, a number of studies have shown that nucleic acid, particularly in RNA, extracted from CNS tissue obtained at autopsy is of sufficient quality to allow molecular analysis (Harrison et al., 1995). This has lead to the examination of gene expression at the level of mRNA using in situ hybridisation, reverse transcriptase-polymerase chain reaction (RT-PCR) and Northern analysis. These techniques can now complement studies at the protein level using radioligand binding assays, antibody detection and Western blots. Data obtained from such analyses may be correlated with cytological studies allowing more definitive conclusions to be derived. Determining the temporal and spatial expression of genes in the brain in both normal and diseased states will allow the elucidation of the complex mechanisms underlying brain function and hence ‘malfunction’. Furthermore, the use of neuroimaging techniques should allow some of the findings in tissue obtained at autopsy to be followed up in living brain. Here we will summarise some of the results obtained from postmortem and neuroimaging studies in understanding the aetiology of schizophrenia, concentrating on three of the major neurotransmitter systems that have been implicated in the disorder.

DOPAMINE SYSTEM The primary biochemical theory of schizophrenia has centred on the role of dopaminergic dysfunction in the illness (Meltzer, 1987): the dopamine hypothesis predicts that an excess of dopaminergic activity results in the psychotic symptoms characteristic of the disorder. This hypothesis mainly relies on the observations that dopamine receptor agonists can induce or worsen psychosis whilst dopamine receptor antagonists can reduce psychoses in some individuals with schizophrenia. The most enduring evidence from the study of postmortem tissue to support the dopamine hypothesis is the replicable increase in dopamine in the nucleus accumbens and caudate putamen (MacKay, 1982) as well as the left amygdala of schizophrenic patients (Reynolds, 1983, 1988). However, newer techniques including more complex molecular analysis of CNS tissue and the use of PET have failed to provide any conclusive evidence to support the dopamine hypothesis of schizophrenia. Following the demonstration that antipsychotic drugs have a high affinity for dopamine receptors, these receptors became a focus for research into the

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neuropathology of schizophrenia. More recently, cloning and sequencing studies have identified five dopamine receptors: the dopamine D1-like (DA-D1) receptors comprising of the DA-D1 and DA- D5 receptors and the dopamine D2-like receptors, consisting of DA-D2, DA-D3 and the DA-D4 receptors (Sibley & Monsma, 1992). All these receptors are G-protein coupled and exhibit heterogeneous distribution within the brain. The identification of these five distinct receptors has renewed interest in the study of dopamine receptors in tissue obtained at autopsy from schizophrenic subjects. Research on the DA-D1 family of receptors is limited. The only evidence for a role for DA-D1 receptors in schizophrenia is its localisation in the prefrontal cortex, a region strongly implicated in the pathophysiology of schizophrenia. However, existing studies do not suggest there are widespread changes in DA D1-like receptors in schizophrenia (Pimoule et al., 1985, Knable et al., 1994, 1996). Historically, in relation to the pathology of schizophrenia, most interest has centered on the dopamine DA-D2 receptors. Primarily this was because DA-D2 receptor blockade correlates with antipsychotic efficacy across a wide range of antipsychotic drugs (Creese et al., 1976). However direct evidence to support a role for DA-D2 receptors has been inconclusive. Early studies using tissue obtained at autopsy with [3H]spiperone to measure DA-D2 receptors showed an increase in DA-D2 receptor density in the basal ganglia from schizophrenic subjects (Seeman, 1985). Significantly, results on DA-D2 receptors were confounded when an elevation of DA-D2 receptors was demonstrated in rats treated with antipsychotic drugs (Clow et al., 1980). Subsequent in vivo studies using patients who were drug naive or treated with atypical antipsychotics have also failed to implicate the DA-D 2 receptor in the pathophysiology of schizophrenia (Pilowsky et al., 1992). The DA-D2 receptor has now been shown to exist as two isoforms, a long form (DA-D2L) and a short form (DA-D2S), which are thought to act via different G-proteins. These differentially spliced forms show regional selectivity for striatal and extra striatal sites. Furthermore, some atypical antipsychotics such as clozapine demonstrate a small differential binding preference to the limbic form of the DA-D2 receptor (Malmberg et al., 1993). A postmortem study by Roberts et al. (1994) showed an increase in the abundance of mRNA for both DA-D2 isoforms in the ventral orbital gyrus from schizophrenic subjects. In addition DA-D2L mRNA was significantly increased in the caudate nucleus, whilst DA-D2S mRNA was significantly increased in the inferior temporal gyrus. It is possible therefore that a subtle abnormality of certain DA D2-like receptors may be involved in schizophrenia.

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The DA-D3 receptor has a limbic distribution and binds the new atypical antipsychotics. Schmauss et al. (1993) reported a significant, selective loss of DA-D3 mRNA in the parietal and motor cortex of schizophrenic patients compared to controls. The presence of a truncated DA-D3 receptor mRNA has also been detected in the CNS tissue obtained at autopsy which has been termed D3nf mRNA. D3nf has been shown not to be reduced in CNS tissue from schizophrenic subjects. However, subsequent experiments performed by Schmauss (1996) showed that DA-D3 receptor pre-mRNA was spliced in vivo to generate both DA-D3 and D3nf mRNA in similar amounts. Analysis of the anterior cingulate cortex, obtained at autopsy from schizophrenic subjects, showed significant differences between the relative levels of DA-D3 and D3nf mRNA. The author concluded that enhanced D3nf specific splicing of DA-D 3 pre-mRNA in schizophrenia resulted in a decreased expression of DA-D3 mRNA. In addition to studies examining DA-D3 receptor mRNA levels, genetic studies at the D3 locus have implicated it as a candidate susceptibility gene for schizophrenia. An excess of homozygosity in schizophrenics for a polymorphism close to the N-terminus of the protein has been demonstrated by several groups (Mant et al., 1994). Gurevich et al. (1997) analysed DA-D3 receptor levels in postmortem tissue using quantitative autoradiography. They found a two-fold elevation in the number of DA-D3 receptors in the basal ganglia and ventral forebrain of the schizophrenic group. This group of patients had been free of antipsychotic drugs for at least one month prior to death. Examination of tissue from the patients receiving antipsychotic medication showed DA-D3 levels similar to the control group indicating that reduction detected in the drug-free group may be reversed by antipsychotic drugs. This study demonstrates the importance of complete clinical histories for the samples used in postmortem analyses and how correlation of data obtained with both clinical and methodological variables can increase the robustness of the results. The DA-D4 receptor is present at low levels in the brain, however gene expression studies have demonstrated differential expression with relatively high levels of DA-D4 gene expression being detected in the hippocampus and cortex, whilst lower levels are expressed in the basal ganglia (Mulcrone & Kerwin, 1997). The most recent and controversial results obtained from analyses of tissue obtained at autopsy has involved the DA-D4 receptor, which has a high affinity for the atypical antipsychotic drugs clozapine, olanzapine and risperidone. In studies of the putamen, Seeman et al. (1993) subtracted the binding of a DA-D2/D3 ([3H]raclopride) selective ligand from a DA-D2/D3/D4

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([3H] YM-09151–2) selective ligand to demonstrate a six-fold elevation of DAD4 receptors in the putamen from schizophrenic subjects. In a similar study, using in situ radioligand binding and quantitative autoradiography, Murray et al. (1995) demonstrated a two-fold elevation of DA-D4 receptors in the striatum and nucleus accumbens from schizophrenic subjects compared to controls. Conversely ligand binding studies by Reynolds and Mason (1995) consistently failed to provide evidence for the presence of DA-D4 receptors in the putamen of either the control or schizophrenic group. Similarly, Helmeste et al. (1996) could not detect differences in DA-D4 receptors in tissue obtained at autopsy from schizophrenic subjects. Low levels of DA-D4 receptor gene expression are present in the basal ganglia and it is proposed that the subtractive methodology utilised by Seeman et al. (1993) was in fact detecting other receptor types. Subsequent comparisons of DA-D4 receptor gene expression (by RT-PCR analysis of RNA extracted postmortem) in control and schizophrenic brain have failed to identify any significant differences in mRNA levels between the two sample groups (Roberts et al., 1996; Mulcrone & Kerwin, 1997). Thus, gene expression studies and more recendy genetic studies of the DA-D4 receptor gene (Shaikh et al., 1994) have failed to implicate a role for the DA-D4 receptor in the aetiology of schizophrenia and have not supported the initial postmortem ligand binding studies. Because of this, a major role for the DA-D4 receptor in schizophrenia aetiology looks unlikely though it still cannot be excluded as a gene of minor effect.

SEROTOMN SYSTEM A number of lines of evidence implicate a role for serotonin (5HT) in the aetiology of schizophrenia. Lysergic acid, which has a molecular structure similar to 5HT, produces psychotic symptoms. In addition, there are a number of reports of increased levels of 5-indoleacetic acid, a metabolite of 5HT, in the CSF of schizophrenic patients and elevations of 5HT in blood samples that could not be accounted for by differences in MAO activity (Owen et al., 1987). The pharmacology of the new atypical antipsychotics, which uniquely act on serotonergic function, has renewed interest in this receptor system. Fifteen serotonin receptors have now been identified and classified into groups based on gene homology (Hoyer et al., 1994), these groups being the 5HT1, 5HT2, 5HT3, 5HT4, 5HT5, 5HT6 and 5HT7 receptors. All these receptors, except

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the 5HT3 receptor, belong to a G-coupled receptor super family with 7 transmembrane domains, although the subtypes exhibit heterogeneous distributions within the brain. The 5HT3, 5HT6 and 5HT7 receptors are of theoretical importance in the aetiology of schizophrenia due to their limbic distribution and high affinity for the atypical antipsychotics. However, to date research on these subtypes is limited. The 5HT2 receptor subtypes (5HT2A, 5HT2B and 5HT2C) are all closely homologous. The 5HT2A and 5HT2C receptors are, at present, considered the main candidates in schizophrenia aetiology due to their high affinity for clozapine. The spatial distribution of the newly identified subtypes has been achieved using tissue obtained at autopsy and ligand binding whilst in situ hybridisation, Northern analysis and RT-PCR can be used to identify mRNA distribution in the same tissue. Thus, analysis of the distribution of mRNA for 5HT1A and 5HT2A receptors has been reported by Burnet et al. (1995), for 5HT4 by Reynolds et al. (1995), and for the 5HT6 by Monsma et al. (1993). Significantly, the density of 5HT2A receptors has been repeatedly shown to be decreased in cortical tissue obtained at autopsy from subjects with schizophrenia (Aurora & Meltzer, 1991; Mita et al., 1986; Burnet et al., 1996; Dean & Hayes, 1996). Thus, the frontal reduction in 5HT2A receptors is now one of the most replicable findings from postmortem studies. However, the decrease in 5HT2A receptors in the dorsolateral frontal cortex and parahippocampal gyrus was not accompanied by a change in receptor mRNA levels (Burnet et al., 1996). This would suggest that the decrease in receptor density is not simplistically related to a decrease in the rate of receptor production by neurons. These studies on serotonin receptors demonstrate that comparisons of both protein receptor and corresponding mRNA levels in the same tissue will help to elucidate the complex pathways of the brain and help to reveal the mechanisms that underlie the changes in expression of receptors in neuropsychiatrie disorders. Another reproducible finding in schizophrenia is the demonstration of a significant increase in the density of 5HT1A receptors in the prefrontal and temporal cortex from schizophrenic subjects (Hashimoto et al., 1991; Burnet et al., 1996). It has now been postulated that the imbalance in the 5HT2A/ 5HT 1A receptor ratios may contribute to impairment of cortico-cortical association pathways. Finally, one study has reported that the 5HT3 receptor is not altered in the amygdala form subjects with schizophrenia (Abi-Dargham et al., 1993). Two independent postmortem studies (Joyce et al., 1993; Lamelle et al., 1993a) have demonstrated decreased densities of 5HT transporters in the

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frontal cortex of schizophrenic subjects compared to controls, whilst other brain regions examined showed no differences. By contrast, others have reported that the density of the serotonin transporter was not changed in the frontal cortex from subjects with schizophrenia (Dean et al., 1995) but that a conformational change in the serotonin transporter is present in the hippocampus from subjects with schizophrenia (Naylo, et al., 1996). Interestingly, levels of mRNA for the serotonin transporter has been reported to be increased in the frontal cortex from subjects with schizophrenia (Hernandez & Sokolov, 1997). Thus, overall these studies strongly indicate a prefrontal reduction in 5HT innervation in schizophrenia and also suggest there may be regionally specific changes in the 5HT transporter in schizophrenia.

GLUTAMATE SYSTEM Glutamate is the most prevalent excitatory neurotransmitter in the CNS and is crucial in the early development of the brain. A number of cytological studies have implicated that schizophrenia is a disorder of neurodevelopment; as glutamate and its receptors are vital in the formation of neuronal cytoarchitecture, an abnormality of this system may be involved. The possible involvement of glutamate is supported by the observation that phencyclidine (PCP), which non competitively blocks NMDA receptors, is a potent psychotomimetic (Olney, 1989). In addition, there are reports of abnormal excitatory amino acid levels in the CSF and postmortem brain tissue from schizophrenic subjects (Kim et al., 1980), a finding that has not been consistently replicated (Perry, 1982). A study by Squires et al. (1993) reported reduced glutamate binding in the cingulate cortex and hippocampus of schizophrenic brains compared to controls which they hypothesised was due to a selective loss of glutamatergic neurons from specific brain regions in schizophrenia. In addition, a small left lateralised decrease in glutamate uptake sites in the polar temporal cortex and amygdala of schizophrenic subjects has been reported (Deakin et al., 1989). This finding was confirmed and extended by a second similar study using tissue samples from antipsychotic free subjects in which a bilateral decrease in glutamate uptake sites in the hippocampus was detected (Deakin et al., 1990). The authors proposed that schizophrenia may involve abnormal glutamatergic innervation within the temporal lobe. Thus there is evidence of an abnormality of the glutamate system in schizophrenia.

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The glutamate receptor system is complex, being comprised of the ionotropic and metabotropic receptors. The ionotropic receptors can be further subdivided into those that respond to N-methyl-D-aspartate [NMDA] and those that respond to kainic acid [KA] or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA]. NMDA, KA and AMPA are analogues of glutamate that exhibit different sensitivities and receptor binding affinities. As with certain dopaminergic and serotonergic agonists, the NMDA ion channel blocker PCP induces a psychotic state closely resembling schizophrenia in normal individuals. It elicits both the positive and negative symptoms of the disorder and the resulting psychosis is considered to resemble schizophrenia more closely than, for example, amphetamine induced psychosis. PCP and its related compounds induce their unique behavioural effects by blocking neurotransmission mediated at NMDA-type glutamate receptors, indicating that dysfunction of NMDA receptor mediated transmission may play a role in the pathophysiology of schizophrenia (Halberstadt, 1995). Significantly, an increase in NMDA receptors has been reported in the orbitofrontal cortex from subjects with schizophrenia (Simpson et al., 1992), a result suggestive of an increase in glutamate synapses in the region. This has been supported by reports of cellular abnormalities in the prefrontal area (Benes et al., 1994). Using in situ hybridisation, Akbarian et al. (1996) reported an increase in the level of NR2D mRNA (encoding a subunit of the NMDA receptor) in the prefrontal cortex of schizophrenics compared to controls. However, there are studies that have failed to find alterations in NMDA receptor levels in schizophrenic brain (Kerwin et al., 1990). Changes in non-NMDA receptors have also been reported in schizophrenia. Kerwin et al. (1990) reported a significant loss in the density of kainate receptors in the hippocampus and parahippocampal gyrus of schizophrenic patients. This result was supported by the finding of Harrison et al. (1991) that showed reduced levels of mRNA encoding for the KA/AMPA receptors in the CA3 region of the hippocampus. Nishikawa et al. (1983) measured an increase in kainate binding sites in the prefrontal cortex of schizophrenic subjects a finding that has since been replicated (Toru et al., 1988).

THE STUDY OF SCHIZOPHRENIA USING NEUROIMAGING TECHNIQUES As with studies using tissue obtained at autopsy, studies of receptors using neuroimaging techniques have mainly focused on the dopamine system. In

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particular, these studies have focused on the DA-D2 receptors, however two recent studies using PET have reported a decrease in the density of DA-D1 receptors in the prefrontal cortex (Okubo et al., 1997) and basal ganglia (Sedvall et al., 1995) in subjects with schizophrenia. Using (3-N-[11C])methylspiperone and PET, an increase in the density DA-D 2 receptors was reported in the caudate-putamen in subjects with schizophrenia (Tune et al., 1993; Wong et al., 1986). These data being in apparent agreement with data from the study of tissue obtained at autopsy from subjects with schizophrenia. Significantly, no difference in the density of DA-D2 receptors was reported using [11C]raclopride and PET (Farde et al., 1990, Hietala et al., 1994). It has been suggested that these differing results could be due to (3-N-[11C)metKylspiperone binding to DA-D2, DAD3 and DA-D4 receptors whereas [11C]raclopride binds only DA-D2 and DAD3 receptors (Seeman et al., 1993). If that was the case in living brain then the difference in results obtained using raclopride and spiperone derivatives could reflect the presence of elevated DA-D4 receptors in the brain of schizophrenic subjects. Alternatively, if changes in the affinity of [3H]raclopride binding in CNS tissue obtained at autopsy from subjects with schizophrenia (Ruiz et al., 1992; Dean et al., 1997) are present in living brain this could cause a falsely low estimate of DA-D2 receptors using [11C] raclopride and PET. This could also account for differences in results using different receptor ligands. However, studies that showed no change in the density of DA-D2 receptors in subjects with schizophrenia using PET and (3-N-[ 11 C])methylspiperone (Nordstrom et al., 1995) or [ 76 Br] bromospiperone (Martinet et al., 1990) may indicate methodological explanations may not totally explain differing results. Studies using SPECT have mainly focused on the ability of antipsychotic drugs to occupy DA-D2 receptors and whether this may provide information on the therapeutic affects of such drugs (Volk et al., 1994; Pilowsky et al., 1992). However, 123I-IBZM binding to DA-D2 receptors was not altered in schizophrenia (Pilowsky et al., 1994). Finally, the increase in 18F-fluro-DOPA uptake measured in caudate-putamen in subjects with schizophrenia using PET has lead to the suggestion that changes in pre-synaptic dopaminergic function could be important in schizophrenia (Dao-Catellana et al., 1997). At present, studies on the 5HT2A receptor using PET have focused on the occupancy of this receptor by atypical antipsychotic drugs (Farde et al., 1994, 1995). As yet there appears to be no reports on the status of components of the serotonin system in schizophrenia. However, the development of ligands that will allow the measurement of serotonin turnover (Reibring et al., 1992),

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serotonin receptors (D’haenen et al., 1992) and the serotonin transporter (Laruelle et al., 1993b) is a likely forerunner to the investigation of the serotonin system in schizophrenia.

CONCLUSION In the absence of any true animal model the examination of human brain tissue is the only option for direct biochemical investigations of schizophrenia. Recently, the studies of a polymorphism in DNA extracted from peripheral tissue (lymphocytes) suggested the 5HT2A receptor gene as a gene possibly involved in the pathology of schizophrenia (Williams et al., 1997). This result has not been supported by a study examining its presence in DNA from CNS tissue obtained at autopsy from subjects with schizophrenia (Kouzmenko et al., 1997). Significantly, the study using tissue obtained at autopsy also showed that the polymorphism in the 5HT2A receptor gene was not associated with a change in the density of that receptor in the frontal cortex. This type of study demonstrates that with continual improvements in the collection and collation of CNS tissue and controlling for known variables it will be possible to apply new molecular analysis techniques to the study of psychiatric illness. In addition, the studies reported here demonstrate how convergent, supportive evidence can be obtained from postmortem and neuroimaging studies leading to testable hypotheses.

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INDEX

affective symptoms 23 age 40 agonal state 40, 86, 127 α2-adrenergic receptors 43, 45 antibody amyloid precursor protein 150 batch variability 108 blast reaction 132 commercial sources 109, 131 immunofluorescence 117 pre-treatment 116 production 108 protein kinase C (PKC) 59 secondary antibodies 109 visualisation 112 visualisation with peroxidase 114 antidepressant drugs 41 antipsychotic drugs 41 anxiolytics 41 association constant 43, 48 autofluorescence 119 quenching 120 autoradiographic developer 76 type 76 autoradiography 68, 70, 76 avidin-biotin-complex 114, 131 β-adrenergic receptors 43, 48 basal ganglia 161 binding affinity 39, 42, 43, 49, 73 binding density 39, 43 Bipolar disorder 31, 34 blast reaction 132 brain bank 3

brain slices dissecting 6, 9, 69 tissue sections 69 brain tissue collection 2 freezing 8 British Advisory Committee on Dangerous Pathogens 143 buffered glycerol 122 calibration standards 79 CCD camera 80 cresyl violet stain 80 depression 20 delusions 20, 24, 25, 26, 27, 28, 31, 138 determination of assay/extraction buffer 47, 145 controls 122, 132 filtration 50 image calibration 79 incubation period 48 pH 47 radioligand 71 radioligand concentration 72 temperature 48, 75 diagnosis 4 Diagnostic Evaluation After Death 20, 21 Diagnostic Instrument for Brain Studies (DIBS) 32, 34, 35 dissociation constant 48, 162, 168 dopamine-D1 receptor 162, 168 dopamine-D2 receptor 41, 74, 163, 168 dopamine-D3 receptor 162, 163 175

176

INDEX

dopamine-D4 receptor 162, 163, 164, 168 dopamine-D5 receptor 162 dopamine transporter 74 double-labelling immunofluorescence 121 DSM-III 20, 22, 27 DSM-III-R 22, 23, 25, 30, 33 DSM-IV 21, 22, 23, 24, 30, 32, 33 duration of illness 24 equipment for in situ radioligand binding 79 in situ hybridisation 87 protein purification 145, 147 Western blot 58 fast protein liquid chromatography 147 Feighner 21, 22, 23, 28, 33 formal thought disorder 21, 29, 31, 33, 138 Fuji BAS 5000 82 GABAA receptors 43, 46, 49 gene expression 85 genetic linkage 86 glutamate transporter 168 glutaraldehyde 130 guanine triphosphate (GTP) 40 hallucinations 20, 24, 25, 26, 27, 28, 31, 138 high pressure liquid chromatography 72, 147 humidifier chamber 77 ICD-10 21, 22, 23, 24, 30, 32, 33 in situ hybridisation (ISSH) 85, 95 selection of controls 103 signal detection 101

non-specific binding (NSB) 43, 49, 50, 51, 74, 75 non-specific labelling 114 oligonucleotide probes design 89 radiolabelling 91, 93 osmium tetroxide 130 paraformaldehyde 89, 110, 130 phosphor plates 82 polyacrylamide gel electrophoresis 150 polyethylenimine 50 polyethylene glycol 50 positive symptoms 21, 22, 27 positron emission tomography (PET) 160, 168 postmortem interval (PMI) 40, 41, 86, 110, 127, 133 preparation of crude particulate membrane 46 gelatinised slides 70, 112 membrane and cytosol fractions 46 particular membrane 46 purified Aβ 152 purified APP 148 radioactive oligonucleotides 91, 93 soluble protein 148 synaptic plasma membrane 47 tissue sections 69, 88, 89, 128 tissue storage solutions 112 prion 143 protein kinase C (PKC) 40, 46, 49, 52, 57 protein purification extraction buffer 141 homogenisation buffer 141 protease inhibitors 146

major depression 22 mania 20

radioligand binding theory 42, 44, 51 radioisotopes 44, 71 Research Diagnostic Criteria (RDC) 20, 21, 22, 23, 33

negative symptoms 21, 23, 24, 25, 33, 167 neuropathology 10

safety issues 142 Scatchard plot 52, 55

INDEX

Schedule for Affected Disorders and Schizoaffective Disorder 22, 24, 26 schizophrenia 3, 4, 20, 22, 23, 31, 41 dopamine hypothesis 161 glutamate hypothesis 166 Schneider 21, 22, 23, 29, 33 SCID 4 selection of CCD camera 80 controls for in situ hybridisation 103 duration of hybridisation 96 hybridsation buffer 96 hybridisation temperatures 97 protein purification systems 147 radioactive probes 44, 71, 92 tissue fixation 110, 128 washing conditions 97 serotonin1B receptor 44, 48 serotonin1A receptor 44, 47, 48, 165 serotonin2A receptor 47, 48, 49, 165, 168 serotonin2c receptor 44, 45, 47, 48, 165 serotonin3 receptor 47, 165 serotonin4 receptor 47, 48 serotonin6 receptor 165

177

serotonin7 receptor 165 serotonin transporter 166 single photon emission computer tomography 160 sources of brain tissue hospice 3 medical examiners office 2 psychiatric hospitals 3 Veteran’s Administration Hospitals 2 specific binding 42 synapse counting 133 fetal tissue 138 simple profile 133 stereology 133 tissue embedding 130 tissue storage time 40 total binding 74 2D electrophoresis 153 uranyl acetate 130 Western blot 55, 58, 150, 161