- Email: [email protected]
Mapping receptors in the human brain
TINS- June 1986
284
Mat0¢
l
ors in
man brain
J. M. Palacios, A. Probst and R. Cortes A utoradiographic techniques are being increasingly used to localize receptor binding sites at the microscopic level. These techniques have recently been applied to the study of neurotransmitter receptors in human postmortem material. Receptors in these tissues appear to be resistant to postmortem changes and their study is amenable to autoradiographic procedures. Receptors for many amine, amino acid and peptide neurotransmitters, as well as other related binding sites, have been visualized and mapped in human brain. In some cases the pharmacological characteristics, and~or the anatomical distribution of these sites are different from those found in rat brain. Changes in the densities of some neurotransmitter receptors have been found in the brains of patients that had suffered from a number of neurological diseases, and in patients with localized lesions or as a consequence of drug treatment. These studies, which offer an invaluable counterpart to the rapidly developing non-invasive techniques of brain imaging, are already providing important insights into human brain receptor mechanisms. During the last two decades, the development of novel neurochemical techniques and the establishment of 'brain banks' for the systematic collection of tissues have considerably expanded our knowledge of the chemistry of the human brain 1. Many studies have been carried out involving biochemical analysis of p r e p arations obtained from hand-dissected human brain samples. However, in an organ of the anatomical and cellular complexity of the human brain, techniques that allow microscopic resolution of cells and their components possess important advantages. With the application of techniques such as histofluorescence, immunohistochemistry and autoradiography to nervous tissue, it is now possible to obtain microscopic resolution in the study of transmitter mechanisms 2. Radioligand binding techniques for the study of receptors and other recognition sites have been extensively applied to the analysis of these sites in human tissue 1. The development of autoradiographic methods of receptor visualization3, particularly the in-vitro receptor autoradiography technique of Young and Kuhar 4, now offer the possibility of visualizing and quantifying receptors within the resolution of the light microscope. Over the past few years, several laboratories have used autoradiography to investigate the properties and anatomical distributions of receptors in human postmortem tissue. In this review, we examine the goals of these studies, some of the technical problems encountered in them, the main results regarding the distribution of these sites in normal (neurologically healthy) subjects, the similarities and differences between results from human post-
mortem tissue and laboratory animals (mainly the rat), and results of the examination of material from patients who either died from neurological diseases or exhibited brain lesions upon autopsy. Two obvious questions one can ask in
the study of receptors are 'are all receptors found in laboratory animals also present in the human brain?' and 'where are they localized?'. Once we know what the distribution of receptors in the normal human brain is, we can ask 'are receptor densities or their Iocalizations changed as a result of disease or drug treatment? It would then be important to assess whether these receptor changes are primary or secondary to neuronal changes caused by the disease. For example, are they an expression of neuronal degeneration or distant trans-synaptic effect? If they are primary, could they have a diagnostic value? And since drug treatment and/or disease can affect receptor localization and density, this topic is of considerable practical and economic interest for anyone involved in the development of new psychotropic agents.
F i g . 1. Autoradiographic localization of receptors in human brain. This figure illustrates two types of preparation,~ for the localization of receptors in human brain tissue. Photographs are from autoradiograms obtained using a 3H-sensitive film sheet. Dark areas are rich in binding sites while clear areas contain low densities of receptors. (A) The distribution of muscarinic cholinergic receptors in a section of a whole hemisphere, labeled with 3H-N-methylscopolamine. This section was obtained using a large stage microtome. High densities o f receptors are observed in the co rtex and p utamen, while the globus pallidus and white matter areas are poor in receptors. (B) Distribution of muscarinic receptors in a section o f the human midbrain. This section was made with a standard microtome. High densities of receptors are localized to the superior colliculus and periaqueductal grey, while the substantia nigra exhibits lower receptor densities. (C) Illustration of the resolution of the autoradiographic method for receptor localization. High densities of serotonin receptors, labeled with 3H-LSD, are seen localized to the cell bodies oftheserotonergicneurons oftheraphd nuclei. The barin (A ) is 10 ram; in (B) is5 ram; andin ( C) is 1 ram.
t~) 1986, Elsevier Science Publishers B.V., Amsterdam 0378 - 5912/86/$02.00
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Fig. 2. Similarities and differences in the distribution o f receptors in human and rat brains. The distribution o f muscarinic cholinergic receptors in the rat (A) and human (B) h~opocampus is an example o f a receptor showing similar distribution in both species. In contrast ¢xl-adrenoceptors, labeled in rat (C) and human (D) hippocampus with the ligand 1271.BE 2452, are highly concentrated in the human hippocampUs but present only in very low densities in the rat hippocampus. Differences in the pharmacological properties o f receptors also exist between human and rat brains. (E) to (lid are photographs from autoradiograms illustrating the autoradiographic localization o f serotonin2 receptors in the rat (E and G) and human (F and H) labeled with the ligands 3H-ketanserin (E and F) or 3Hmesulergine ( G and 1t). While in the rat both ligands labeled the same population of receptors, 3H.mesulergine (tt) did not label these sites in the human cortex, indicating that serotoninz receptors are pharmacologically different in the two species. The bars in (A )-( D ) are 2 ram; in (E) and (G) are O.5 ram; and in (F) and (H) are 1 mm.
286 Methodology of receptor mapping in human brain The application of receptor autoradiographic techniques to human postmortem material has encountered on the one hand some of the problems already known from previous neurochemical studies and, on the other hand, problems specific to the use of in-vitro autoradiographic techniques. Some of the technical problems and the application of this technique have been reviewed in these pages 5'6. A clear technical problem in the application of receptor autoradiography to the human brain is due to the large size of the organ, compared to the brains of standard laboratory animals. Ideally, it would be desirable to produce microtome sections from a whole brain hemisphere so that many related anatomical structures could be observed in the same sample. Fortunately, microtomes able to produce such large sections do exist; they are often used in 'whole-body' autoradiographic studies of drug distribution and metabolism. This microtome uses a plastic, flexible tape, glued on one side, to hold the section during the cutting procedure. We have applied this technique to cut large human brain sections7 for receptor localization. An example, in which muscarinic cholinergic receptors are visualized, is shown in Fig. 1A. Unfortunately, this procedure detrimentally affects the binding properties of many receptor sites. Other groups, in particular Young, Penney and collaboratorss at the University of Michigan produce large hemispheric sections by rolling the sections onto large lantern slides. Receptor types in normal human brain In the rat, no less than 50 different neurotransmitter and drug receptors have been localized by autoradiography9. Not every receptor visualized in the rat has yet been studied in the human brain, but until now there have been no major absences of receptor types reported for human brain, although differences have been observed regarding some receptor subtype,. Table I summarizes the receptors whose presence and distribution has now been documented in human brain by autoradiography. This list includes receptors for amine, amino acid, peptide and purine neurotransmitters. Other sites associated with neurotransmitter mechanisms, such as uptake sites or ionic channels, are also listed.
T I N S - June 1986
TABLE I. Neurotransmitterreceptors and related bindingsites that have been examined by autoradiographyin the humanbrain Receptor
Subtypea
Amines Acetylcholine Noradrenaline Dopamine Serotonin Histamine
(Muscarinic,M~ and M2) (cq, a2, 131and 152) (D1 and D2) (5-HTIA,5-HTm,5-HTlo 5-HT2) (Hi)
Aminoacids Glycine y-aminobutyricacid (GABA)
(GABAA and GABAB, BenzodiazepineType I and II)
Glutamate Peptides Opiate Neurotensin Somatostatin TRH
(g, ,5, ~)
Purines Adenosine
(A0
Other binding sites Dopamineuptake Serotoninuptake Monoamineoxidase Calciumchannels Chloride channels
(Nomifensine) (Imipramine) (MPTP) (Dihydropyridine) (TBPS)
aOr drug used to characterize the bindingsite. Pharmacological characteristics In mapping receptors in human tissues, we have also been interested in the pharmacological characteristics of the binding sites being visualized. Similarities and differences have been found between human brain receptors and those of laboratory animals. For example, the two putative subtypes of the muscarinic receptor, the so-called M1 and M2, appear to be pharmacologically identical in human and rat tissues~°-12. In contrast, serotonin receptors exhibit clear pharmacological differences: the 5-HT2 receptor is recognized and labeled by some ligands (e.g., the ergot derivative mesulergine 13) in the rat brain, but not in the human brain (Fig. 2). Three subtypes of the 5-HT~ receptor are thought to exist in the rat brain~. One of these subtypes, 5-HTm, is apparently absent in the human brain. Minor differences between the two species have also been detected, e.g. a generalized decrease in the affinity of drugs for the 5-HTlc subtype in human tissue. The densities of receptors (expressed in fmol mg -~ protein) in the human brain appear to be generally lower than in the rat brain. Autoradiographic results from tissue sections are in agreement on this point with measurements in membrane preparations pro-
duced by homogenization, centrifugation, etc. For example, muscarinic receptor densities in the human caudate nucleus are only 50% of those measured in the rat caudate/putamen11A2. Glycine receptor densities in some brainstem nuclei or in the spinal cord of man, measured using 3H-strychnine as a ligand, are less than half those measured in the rat ~5. These differences probably reflect the lower density of neuronal cell bodies in humans compared to rats. R e e q a ~ di~ilmtion The distributionof receptors was also compared between human and rat brains. Muscarinic cholinergic subtypes 11"12"16 (Fig. 2), [3-adrenoceptors 17, ~2-adrenoceptorsis and glycine receptors 15 all show similar ~ b u t i o n s in rat and man. Other receptor types differ in their regional distribution. For example, opioid receptors are present at high levels in the human cerebellum and in the deepest laminae of the neocortex 19, but are absent from these areas in rat. ~-Adrenoceptors, labeled with 1251-BE-2452 or with 3H-prazosin~°, are particularly concentrated in the human hippocampal formation but scarce in rat hippocampus (Fig. 2). In some cases, e.g., opioid receptors, the difference in distribution is a reflection of differences in the distribution of
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specific receptor subtypes. In man, opioid receptors in the cerebellum and deep cortical laminae belong to the rsubtype, which appears in the rat to be present at only low levels. Differences in the distribution of receptor subtypes is probably not the only explanation for species differences in distribution because in other cases, receptor types are common to both species. Species differences between human and laboratory animals are not only of intrinsic biological interest, but are also of great importance in the development of new drugs and the assessment of their pharmacological effects in humans. These studies have revealed that the rat is not always an appropriate model for the study of receptors in man. Fortunately, the systematic search of other laboratory species has provided appropriate animal models for human receptor distribution. The cerebellar opiate receptors observed in humans are also present in the guinea-pig, and
exhibit similar pharmacology21. For serotonin receptors we have found that pig or cat brains compared adequately with humans. Finally, for some neurotransmitter systems there is information on the distribution of presynaptic markers in the human brain, allowing a comparison between these markers and neurotransmitter receptors. As in studies with laboratory animals, the mismatch problem between pre- and post-synaptic markers has been observed in the human brain. In some cases, for example with muscarinic cholinergic receptors, correlations with the distribution of presynaptic cholinergic markers have given mixed results 12'16. A good correlation has been seen between the distribution of pre- and post-synaptic markers in the brainstem, whereas there is a clear mismatch in the neocortex and in areas of the basal forebrain containing cholinergic cells that innervate the forebrain. A classical
example of high densities of receptors in an area with low levels of transmitter is the [~-adrenoceptor in the human and rat striatum. Possible hypotheses to explain this mismatch problem have recently been reviewed in TINS 22. Receptor changes in human neuropathology examined by autoradiography The development of simple quantitative autoradiographic techniques, particularly the introduction of the film method that facilitates quantification and generation of autoradiograms from large histologic specimens, has led a number of laboratories to examine receptor changes in pathological sampies°. The receptors and different diseases examined are listed in Table II. These and many other examples have already been studied with binding techniques; however the techniques of in-vitro receptor autoradiography possess a number of important advantages
Fig. 3. A utoradiographic localization o f muscarinic cholinergic receptors in the hippocampas o f patients having suffered from senile dementia. (A ) shows a tissue section stained for acid phosphatase activity to reveal the presence o f senile plaques, seen as small round dots o f high enzyme activity (arrow). In spite o f the presence o f numerous senile plaques the distribution and density o f muscarinic cholinergic receptors, shown in the autoradiogram in (B), was similar to that seen in comparable control cases (Bars = 3 mm). In other cases (C), where few senile plaques were seen but a marked neuronal loss was observed (area between the arrows in the cresyl-violet stained tissue, a parallel decrease in receptors was seen (D) (area between the arrows in the autoradiogram ). The bars in (A ) and ( B) are 3 mm; the bar in (C) is 1 mm; and the bar in (D) is 3 mm.
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YABLEIL Receptorautoradiographystudies in human pathologies Disease
Receptors examined
Receptor changes
Amyotrophic lateral sclerosis
Muscarinic cholinergic24 Benzodiazepine 24 Glycine24
Decreased in spinal cord grey matter mostly in Rexed layer IX (concomitant with cell death)
Huntington's chorea
GABA/benzodiazepines,26,31 (includingsubtypes) Muscariniceholinergics Opiates32 Glutamate29
Decreased in caudate and putamen, increasedh globus pallidus Decreased in putamen Decreased in globus pallidus and putamen Decreased in caudate and putamen
Parkinson'sdisease
Neurotensin 23 GABA/benzodiazepine Muscarinic cholinergic Opiate 31
Decreased in substantia nigra
Increased in neocortex, putamen and caudate
Senile dementia
Muscarinic cholinergic (M 1 and M2)25'27'2s Serotonergic (5-HT~ and 5-HT2) zs'27,:s GABA/benzodiazepine 2~ Glutamate 29
No change in neocortex and hippocampus 2s Decreased in hippocampus in some cases 25 Decreased in hippocampus in some cases Decreased in hippocampus in some cases Decreased in cerebral cortex
Schizophrenia
Neurotensin 33 (effects of chronic neuroleptic treatment)
Increased in substantia nigra
Numbers correspond to references.
over this biochemical procedure. The biggest advantage is the possibility of correlating both receptor and histopathological changes in the same section (Fig. 3). The specificity of the effect observed and its relationship to the disease process can be examined by comparisons with appropriate age- and sex-matched controls and with samples from other pathologies. The possibility that the pathological process may generate technical artifacts has also been considered. For example, in Parkinson's disease the neuronal loss in the substantia nigra is accompanied by gliosis. These changes in the cellular composition of the region being studied could result in modification of the local tissue absorption of [~-radiation (selfabsorption), pa.rticulady when 3Hlabeled ligands are used. Uhl et al. 23 have analysed this problem and they reported no change in self-absorption. This is nevertheless an experiment that clearly must be performed when quantitative data are generated. While the number of autoradiographic studies on receptors in pathological tissues is still very limited, a number of conclusions can already be drawn. Two general types of change have been observed. The first is receptor loss, which is directly related to neuronal loss. Decreases in receptors have been observed in the ventral horn of amyotrophic lateral sclerosis • 24 pattents , m the htppocampal formation of some patients with senile dementia 25 (Fig. 3) or in the striatum in
patients with Huntington's chorea 8. In most of these cases, a clear relationship has been established between receptor decrease and neuronal loss. While receptor changes associated with neuronal degeneration are probably not surprising, receptor autoradiography has revealed local and distant changes in receptor number in some pathologies that could not have been completely predicted. Analyses of receptor changes by autoradiography in several degenerative diseases have revealed that they can precede the actual neuronal degeneration. Such changes have been seen in early cases of Huntington's disease, where decreases in G A B A and benzodiazepine receptors in the putamen and increases of these receptors in the lateral globus pallidus were observed before cell loss and atrophy 26. In some instances receptor changes occur in areas where no evident pathology is present. For example, muscarinic receptors decrease in number in the dorsal horn of the spinal cord in patients that had suffered from amyotrophic lateral sclerosis, although there is no sign of any other pathology at that site24. In Alzheimer's disease autoradiographic studies have illustrated a number of interesting effects of this disease on neurotransmitter receptors. Surprisingly, the presence of the main neuropathologlcal lesion, namely the senile plaque, does not seem to alter receptor distribution; the neuropil in the senile plaque contains a receptor
density similar to that seen in unaffected areas 27'2s (Fig. 3). Specific receptor changes have been observed in Alzheimer's disease in the glutamatergic 29 and serotonergic systems (Pazos et al., unpublished observations), while other systems involving muscarinic choiinergic or GABA/benzodiazepine receptors are less affected25'27'28. The changes in receptors for glutamate and serotonin appear to be localized mainly in cortical areas and the hippocampus. In summary, receptor autoradiography can be applied to the study of the distribution and characteristics of drug and neurotransmitter receptors in normal and diseased human brain• These receptor maps have revealed the heterogeneous distribution of these sites in human brain, their differences and similarities with receptors in laboratory animals, and their alterations in diseases. New ligands are being developed for the labeling of important functional sites in the brain and in the future they will be applied to human tissues. This includes not only receptor sites but also ligands for enzymes, transport sites and ionic channels. Autoradiographic techniques for the localization of antigens using radioactive antibodies and the use of in-situ hybridization techniques for the localization of specific mRNAs are now being used in postmortem materials, and will continue to yield interesting results. Some of the information generated by autoradiographic techniques used to develop specific positron emis-
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9 Palacios, J. M. (1984)J. Recept. Res. 4,633-Kuhar, M. J. (1983) Ann. Neurol. 14, 8--16 644 25 Probst, A., Palacios, J. M. and Cortes, R. 10 Wastek, G. J. and Yamamura, H. I. (1978) (1984) Soc. Neurosci. Abstr. 10, 889 Mol. Pharmacol. 14, 768-780 26 Walker, F. O., Young, A. B., Penney, J. B., 11 Cort6s, R. and Palacios, J. M. (1986) Brain Dovorini-Zis, K. and Shoulson, I. (1984) Res. 362,227-238 Neurology 34, 1237-1240 12 Cortes, R., Probst, A., Tobler, H. J. and 27 Palacios, J. M. (1982) Brain Res. 243, 173Palacios, J. M. (1986) Brain Res. 362,239--253 175 13 Pazos, A., Hoyer, D. and Palacios, J. M. 28 Lang, W. and Henke, H. (1983) Brain Res. (1984) Eur. J. Pharmacol. 106, 531-538 267,271-280 14 Pazos, A. and Palaeios, J. M. (1985) Brain 29 Greenamyre, J. T., Penney, J. B., Young, Res. 346, 205--230 A . B . , D'Amato, C. J., Hicks, S.P. and Selected references 15 Probst, A., Cortes, R. and Palacios, J. M. Shoulson, I. (1985) Science 227, 1496-1499 1 Bird, E. D. and Iversen, L. L. (1982) in (1986) Neuroscience 17, 11-35 30 Phelps, M. E. and Mazziotta, J. C. (1985) Handbook of Neurochemistry (2nd edn Vol. 16 Cort~s, R., Probst, A. and Palacios, J. M. Science 228, 799-809 2), (Lajtha, A., ed.), pp. 225-251, Plenum (1984) Neuroscience 12, 1003-1023 31 Pan, H. S., Penney, J. B., Young, A. B., Press 17 Pazos, A., Probst, A. and Palacios, J. M. Arbor, A., Sboulson, I. and Eskin, T. (1985) 2 Bj6rklund , A. and H6kfelt, T., (eds.) (1984) (1985) Brain Res. 358, 324-328 Neurology Suppl. 1, 35, 175 Methods in Chemical Neuroanatomy (Hand- 18 Probst, A., Cortes, R. and Palacios, J. M. 32 Penney, J. B., Young, A. B., Walker, F. O. book of Chemical Neuroanatomy, Vol. 1), (1984) Eur. J. Pharmacol. 106, 477-488 and Shoulson, I. (1984) Neurology Suppl. 1, Elsevier 19 Maurer, R., Cort6s, R., Probst, A. and 34, 153 3 Kuhar, M. J. (1985) in Neurotransmitter Palacios, J. M. (1983) Life Sci. 33, 231-234 33 Uhl, G. R. and Kuhar, M. J. (1984) Nature Receptor Binding (2nd edn) (Yamamura, 20 Biegon, A., Rainbow, T. C., Mann, J. J. and 309, 350-352 H. I., ed.), pp. 153-176, Raven Press McEwen, B. S. (1982) Brain Res. 247, 3794 Young, W. S. and Kuhar, M. J. (1979) Nature 382 280, 393-395 21 Robson, E. E., Foote, R. W., Maurer, R. and 5 Kuhar, M. J. and Unnerstall, J. R. (1985) Kosterlitz, H. W. (1984) Neuroscience 12,621 Trends NeuroSci. 8, 49-53 22 Kuhar, M. J. (1985) TrendsNeuroSci. 8,1906 Whitehouse, P. J. (1985) Trends NeuroSci. 8, 191 J. M. Palacios and R. Cortes are at the 434-437 23 Uhl, G. R., Whitehouse, P. J., Price, D. L., Pharmaceutical Division, Sandoz Lid, CH.4002 7 Palacios, J. M., Mcier-Ruge, W. and Ulrich, J. Tourtelotte, W. W. and Kuhar, M. J. (1984) Basle, Switzerland, and A. Probst is at the (1982) Neurosci. Supp. to Vol. 7, 165 Brain Res. 308, 186-190 Department of Neuropathology, Institute of 8 Penney, J. B. and Young, A . B . (1982) 24 Whitehouse, P. J., Wamsley, J. K., Zarbin, Pathology, University of Basle, Sch6nbeinstrasse Neurology 32, 1391-1395 M. A., Price, D. L., Tourtelotte, W. W. and 40, CH-4003 Basle, Switzerland. For technical reasons we are unable to reproduce this figure in colour. See the June issue of Trends in NeuroScience~ for full colour illustration.
s i o n t o m o g r a p h y ( P E T ) p r o b e s 3° c o u l d h e l p in t h e e a r l y d i a g n o s i s o f s o m e degenerative and psychiatric diseases a n d in t h e m o n i t o r i n g o f t h e t h e r a p e u t i c effects o f d r u g s . T h i s , t o g e t h e r w i t h t h e development of improved non-invasive i m a g i n g t e c h n i q u e s for r e c e p t o r s a n d o t h e r sites, will c h a n g e t h e w a y w e t h i n k about our own brain.
284
Mat0¢
l
ors in
man brain
J. M. Palacios, A. Probst and R. Cortes A utoradiographic techniques are being increasingly used to localize receptor binding sites at the microscopic level. These techniques have recently been applied to the study of neurotransmitter receptors in human postmortem material. Receptors in these tissues appear to be resistant to postmortem changes and their study is amenable to autoradiographic procedures. Receptors for many amine, amino acid and peptide neurotransmitters, as well as other related binding sites, have been visualized and mapped in human brain. In some cases the pharmacological characteristics, and~or the anatomical distribution of these sites are different from those found in rat brain. Changes in the densities of some neurotransmitter receptors have been found in the brains of patients that had suffered from a number of neurological diseases, and in patients with localized lesions or as a consequence of drug treatment. These studies, which offer an invaluable counterpart to the rapidly developing non-invasive techniques of brain imaging, are already providing important insights into human brain receptor mechanisms. During the last two decades, the development of novel neurochemical techniques and the establishment of 'brain banks' for the systematic collection of tissues have considerably expanded our knowledge of the chemistry of the human brain 1. Many studies have been carried out involving biochemical analysis of p r e p arations obtained from hand-dissected human brain samples. However, in an organ of the anatomical and cellular complexity of the human brain, techniques that allow microscopic resolution of cells and their components possess important advantages. With the application of techniques such as histofluorescence, immunohistochemistry and autoradiography to nervous tissue, it is now possible to obtain microscopic resolution in the study of transmitter mechanisms 2. Radioligand binding techniques for the study of receptors and other recognition sites have been extensively applied to the analysis of these sites in human tissue 1. The development of autoradiographic methods of receptor visualization3, particularly the in-vitro receptor autoradiography technique of Young and Kuhar 4, now offer the possibility of visualizing and quantifying receptors within the resolution of the light microscope. Over the past few years, several laboratories have used autoradiography to investigate the properties and anatomical distributions of receptors in human postmortem tissue. In this review, we examine the goals of these studies, some of the technical problems encountered in them, the main results regarding the distribution of these sites in normal (neurologically healthy) subjects, the similarities and differences between results from human post-
mortem tissue and laboratory animals (mainly the rat), and results of the examination of material from patients who either died from neurological diseases or exhibited brain lesions upon autopsy. Two obvious questions one can ask in
the study of receptors are 'are all receptors found in laboratory animals also present in the human brain?' and 'where are they localized?'. Once we know what the distribution of receptors in the normal human brain is, we can ask 'are receptor densities or their Iocalizations changed as a result of disease or drug treatment? It would then be important to assess whether these receptor changes are primary or secondary to neuronal changes caused by the disease. For example, are they an expression of neuronal degeneration or distant trans-synaptic effect? If they are primary, could they have a diagnostic value? And since drug treatment and/or disease can affect receptor localization and density, this topic is of considerable practical and economic interest for anyone involved in the development of new psychotropic agents.
F i g . 1. Autoradiographic localization of receptors in human brain. This figure illustrates two types of preparation,~ for the localization of receptors in human brain tissue. Photographs are from autoradiograms obtained using a 3H-sensitive film sheet. Dark areas are rich in binding sites while clear areas contain low densities of receptors. (A) The distribution of muscarinic cholinergic receptors in a section of a whole hemisphere, labeled with 3H-N-methylscopolamine. This section was obtained using a large stage microtome. High densities o f receptors are observed in the co rtex and p utamen, while the globus pallidus and white matter areas are poor in receptors. (B) Distribution of muscarinic receptors in a section o f the human midbrain. This section was made with a standard microtome. High densities of receptors are localized to the superior colliculus and periaqueductal grey, while the substantia nigra exhibits lower receptor densities. (C) Illustration of the resolution of the autoradiographic method for receptor localization. High densities of serotonin receptors, labeled with 3H-LSD, are seen localized to the cell bodies oftheserotonergicneurons oftheraphd nuclei. The barin (A ) is 10 ram; in (B) is5 ram; andin ( C) is 1 ram.
t~) 1986, Elsevier Science Publishers B.V., Amsterdam 0378 - 5912/86/$02.00
TINS - June 1986
285
Fig. 2. Similarities and differences in the distribution o f receptors in human and rat brains. The distribution o f muscarinic cholinergic receptors in the rat (A) and human (B) h~opocampus is an example o f a receptor showing similar distribution in both species. In contrast ¢xl-adrenoceptors, labeled in rat (C) and human (D) hippocampus with the ligand 1271.BE 2452, are highly concentrated in the human hippocampUs but present only in very low densities in the rat hippocampus. Differences in the pharmacological properties o f receptors also exist between human and rat brains. (E) to (lid are photographs from autoradiograms illustrating the autoradiographic localization o f serotonin2 receptors in the rat (E and G) and human (F and H) labeled with the ligands 3H-ketanserin (E and F) or 3Hmesulergine ( G and 1t). While in the rat both ligands labeled the same population of receptors, 3H.mesulergine (tt) did not label these sites in the human cortex, indicating that serotoninz receptors are pharmacologically different in the two species. The bars in (A )-( D ) are 2 ram; in (E) and (G) are O.5 ram; and in (F) and (H) are 1 mm.
286 Methodology of receptor mapping in human brain The application of receptor autoradiographic techniques to human postmortem material has encountered on the one hand some of the problems already known from previous neurochemical studies and, on the other hand, problems specific to the use of in-vitro autoradiographic techniques. Some of the technical problems and the application of this technique have been reviewed in these pages 5'6. A clear technical problem in the application of receptor autoradiography to the human brain is due to the large size of the organ, compared to the brains of standard laboratory animals. Ideally, it would be desirable to produce microtome sections from a whole brain hemisphere so that many related anatomical structures could be observed in the same sample. Fortunately, microtomes able to produce such large sections do exist; they are often used in 'whole-body' autoradiographic studies of drug distribution and metabolism. This microtome uses a plastic, flexible tape, glued on one side, to hold the section during the cutting procedure. We have applied this technique to cut large human brain sections7 for receptor localization. An example, in which muscarinic cholinergic receptors are visualized, is shown in Fig. 1A. Unfortunately, this procedure detrimentally affects the binding properties of many receptor sites. Other groups, in particular Young, Penney and collaboratorss at the University of Michigan produce large hemispheric sections by rolling the sections onto large lantern slides. Receptor types in normal human brain In the rat, no less than 50 different neurotransmitter and drug receptors have been localized by autoradiography9. Not every receptor visualized in the rat has yet been studied in the human brain, but until now there have been no major absences of receptor types reported for human brain, although differences have been observed regarding some receptor subtype,. Table I summarizes the receptors whose presence and distribution has now been documented in human brain by autoradiography. This list includes receptors for amine, amino acid, peptide and purine neurotransmitters. Other sites associated with neurotransmitter mechanisms, such as uptake sites or ionic channels, are also listed.
T I N S - June 1986
TABLE I. Neurotransmitterreceptors and related bindingsites that have been examined by autoradiographyin the humanbrain Receptor
Subtypea
Amines Acetylcholine Noradrenaline Dopamine Serotonin Histamine
(Muscarinic,M~ and M2) (cq, a2, 131and 152) (D1 and D2) (5-HTIA,5-HTm,5-HTlo 5-HT2) (Hi)
Aminoacids Glycine y-aminobutyricacid (GABA)
(GABAA and GABAB, BenzodiazepineType I and II)
Glutamate Peptides Opiate Neurotensin Somatostatin TRH
(g, ,5, ~)
Purines Adenosine
(A0
Other binding sites Dopamineuptake Serotoninuptake Monoamineoxidase Calciumchannels Chloride channels
(Nomifensine) (Imipramine) (MPTP) (Dihydropyridine) (TBPS)
aOr drug used to characterize the bindingsite. Pharmacological characteristics In mapping receptors in human tissues, we have also been interested in the pharmacological characteristics of the binding sites being visualized. Similarities and differences have been found between human brain receptors and those of laboratory animals. For example, the two putative subtypes of the muscarinic receptor, the so-called M1 and M2, appear to be pharmacologically identical in human and rat tissues~°-12. In contrast, serotonin receptors exhibit clear pharmacological differences: the 5-HT2 receptor is recognized and labeled by some ligands (e.g., the ergot derivative mesulergine 13) in the rat brain, but not in the human brain (Fig. 2). Three subtypes of the 5-HT~ receptor are thought to exist in the rat brain~. One of these subtypes, 5-HTm, is apparently absent in the human brain. Minor differences between the two species have also been detected, e.g. a generalized decrease in the affinity of drugs for the 5-HTlc subtype in human tissue. The densities of receptors (expressed in fmol mg -~ protein) in the human brain appear to be generally lower than in the rat brain. Autoradiographic results from tissue sections are in agreement on this point with measurements in membrane preparations pro-
duced by homogenization, centrifugation, etc. For example, muscarinic receptor densities in the human caudate nucleus are only 50% of those measured in the rat caudate/putamen11A2. Glycine receptor densities in some brainstem nuclei or in the spinal cord of man, measured using 3H-strychnine as a ligand, are less than half those measured in the rat ~5. These differences probably reflect the lower density of neuronal cell bodies in humans compared to rats. R e e q a ~ di~ilmtion The distributionof receptors was also compared between human and rat brains. Muscarinic cholinergic subtypes 11"12"16 (Fig. 2), [3-adrenoceptors 17, ~2-adrenoceptorsis and glycine receptors 15 all show similar ~ b u t i o n s in rat and man. Other receptor types differ in their regional distribution. For example, opioid receptors are present at high levels in the human cerebellum and in the deepest laminae of the neocortex 19, but are absent from these areas in rat. ~-Adrenoceptors, labeled with 1251-BE-2452 or with 3H-prazosin~°, are particularly concentrated in the human hippocampal formation but scarce in rat hippocampus (Fig. 2). In some cases, e.g., opioid receptors, the difference in distribution is a reflection of differences in the distribution of
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specific receptor subtypes. In man, opioid receptors in the cerebellum and deep cortical laminae belong to the rsubtype, which appears in the rat to be present at only low levels. Differences in the distribution of receptor subtypes is probably not the only explanation for species differences in distribution because in other cases, receptor types are common to both species. Species differences between human and laboratory animals are not only of intrinsic biological interest, but are also of great importance in the development of new drugs and the assessment of their pharmacological effects in humans. These studies have revealed that the rat is not always an appropriate model for the study of receptors in man. Fortunately, the systematic search of other laboratory species has provided appropriate animal models for human receptor distribution. The cerebellar opiate receptors observed in humans are also present in the guinea-pig, and
exhibit similar pharmacology21. For serotonin receptors we have found that pig or cat brains compared adequately with humans. Finally, for some neurotransmitter systems there is information on the distribution of presynaptic markers in the human brain, allowing a comparison between these markers and neurotransmitter receptors. As in studies with laboratory animals, the mismatch problem between pre- and post-synaptic markers has been observed in the human brain. In some cases, for example with muscarinic cholinergic receptors, correlations with the distribution of presynaptic cholinergic markers have given mixed results 12'16. A good correlation has been seen between the distribution of pre- and post-synaptic markers in the brainstem, whereas there is a clear mismatch in the neocortex and in areas of the basal forebrain containing cholinergic cells that innervate the forebrain. A classical
example of high densities of receptors in an area with low levels of transmitter is the [~-adrenoceptor in the human and rat striatum. Possible hypotheses to explain this mismatch problem have recently been reviewed in TINS 22. Receptor changes in human neuropathology examined by autoradiography The development of simple quantitative autoradiographic techniques, particularly the introduction of the film method that facilitates quantification and generation of autoradiograms from large histologic specimens, has led a number of laboratories to examine receptor changes in pathological sampies°. The receptors and different diseases examined are listed in Table II. These and many other examples have already been studied with binding techniques; however the techniques of in-vitro receptor autoradiography possess a number of important advantages
Fig. 3. A utoradiographic localization o f muscarinic cholinergic receptors in the hippocampas o f patients having suffered from senile dementia. (A ) shows a tissue section stained for acid phosphatase activity to reveal the presence o f senile plaques, seen as small round dots o f high enzyme activity (arrow). In spite o f the presence o f numerous senile plaques the distribution and density o f muscarinic cholinergic receptors, shown in the autoradiogram in (B), was similar to that seen in comparable control cases (Bars = 3 mm). In other cases (C), where few senile plaques were seen but a marked neuronal loss was observed (area between the arrows in the cresyl-violet stained tissue, a parallel decrease in receptors was seen (D) (area between the arrows in the autoradiogram ). The bars in (A ) and ( B) are 3 mm; the bar in (C) is 1 mm; and the bar in (D) is 3 mm.
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YABLEIL Receptorautoradiographystudies in human pathologies Disease
Receptors examined
Receptor changes
Amyotrophic lateral sclerosis
Muscarinic cholinergic24 Benzodiazepine 24 Glycine24
Decreased in spinal cord grey matter mostly in Rexed layer IX (concomitant with cell death)
Huntington's chorea
GABA/benzodiazepines,26,31 (includingsubtypes) Muscariniceholinergics Opiates32 Glutamate29
Decreased in caudate and putamen, increasedh globus pallidus Decreased in putamen Decreased in globus pallidus and putamen Decreased in caudate and putamen
Parkinson'sdisease
Neurotensin 23 GABA/benzodiazepine Muscarinic cholinergic Opiate 31
Decreased in substantia nigra
Increased in neocortex, putamen and caudate
Senile dementia
Muscarinic cholinergic (M 1 and M2)25'27'2s Serotonergic (5-HT~ and 5-HT2) zs'27,:s GABA/benzodiazepine 2~ Glutamate 29
No change in neocortex and hippocampus 2s Decreased in hippocampus in some cases 25 Decreased in hippocampus in some cases Decreased in hippocampus in some cases Decreased in cerebral cortex
Schizophrenia
Neurotensin 33 (effects of chronic neuroleptic treatment)
Increased in substantia nigra
Numbers correspond to references.
over this biochemical procedure. The biggest advantage is the possibility of correlating both receptor and histopathological changes in the same section (Fig. 3). The specificity of the effect observed and its relationship to the disease process can be examined by comparisons with appropriate age- and sex-matched controls and with samples from other pathologies. The possibility that the pathological process may generate technical artifacts has also been considered. For example, in Parkinson's disease the neuronal loss in the substantia nigra is accompanied by gliosis. These changes in the cellular composition of the region being studied could result in modification of the local tissue absorption of [~-radiation (selfabsorption), pa.rticulady when 3Hlabeled ligands are used. Uhl et al. 23 have analysed this problem and they reported no change in self-absorption. This is nevertheless an experiment that clearly must be performed when quantitative data are generated. While the number of autoradiographic studies on receptors in pathological tissues is still very limited, a number of conclusions can already be drawn. Two general types of change have been observed. The first is receptor loss, which is directly related to neuronal loss. Decreases in receptors have been observed in the ventral horn of amyotrophic lateral sclerosis • 24 pattents , m the htppocampal formation of some patients with senile dementia 25 (Fig. 3) or in the striatum in
patients with Huntington's chorea 8. In most of these cases, a clear relationship has been established between receptor decrease and neuronal loss. While receptor changes associated with neuronal degeneration are probably not surprising, receptor autoradiography has revealed local and distant changes in receptor number in some pathologies that could not have been completely predicted. Analyses of receptor changes by autoradiography in several degenerative diseases have revealed that they can precede the actual neuronal degeneration. Such changes have been seen in early cases of Huntington's disease, where decreases in G A B A and benzodiazepine receptors in the putamen and increases of these receptors in the lateral globus pallidus were observed before cell loss and atrophy 26. In some instances receptor changes occur in areas where no evident pathology is present. For example, muscarinic receptors decrease in number in the dorsal horn of the spinal cord in patients that had suffered from amyotrophic lateral sclerosis, although there is no sign of any other pathology at that site24. In Alzheimer's disease autoradiographic studies have illustrated a number of interesting effects of this disease on neurotransmitter receptors. Surprisingly, the presence of the main neuropathologlcal lesion, namely the senile plaque, does not seem to alter receptor distribution; the neuropil in the senile plaque contains a receptor
density similar to that seen in unaffected areas 27'2s (Fig. 3). Specific receptor changes have been observed in Alzheimer's disease in the glutamatergic 29 and serotonergic systems (Pazos et al., unpublished observations), while other systems involving muscarinic choiinergic or GABA/benzodiazepine receptors are less affected25'27'28. The changes in receptors for glutamate and serotonin appear to be localized mainly in cortical areas and the hippocampus. In summary, receptor autoradiography can be applied to the study of the distribution and characteristics of drug and neurotransmitter receptors in normal and diseased human brain• These receptor maps have revealed the heterogeneous distribution of these sites in human brain, their differences and similarities with receptors in laboratory animals, and their alterations in diseases. New ligands are being developed for the labeling of important functional sites in the brain and in the future they will be applied to human tissues. This includes not only receptor sites but also ligands for enzymes, transport sites and ionic channels. Autoradiographic techniques for the localization of antigens using radioactive antibodies and the use of in-situ hybridization techniques for the localization of specific mRNAs are now being used in postmortem materials, and will continue to yield interesting results. Some of the information generated by autoradiographic techniques used to develop specific positron emis-
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9 Palacios, J. M. (1984)J. Recept. Res. 4,633-Kuhar, M. J. (1983) Ann. Neurol. 14, 8--16 644 25 Probst, A., Palacios, J. M. and Cortes, R. 10 Wastek, G. J. and Yamamura, H. I. (1978) (1984) Soc. Neurosci. Abstr. 10, 889 Mol. Pharmacol. 14, 768-780 26 Walker, F. O., Young, A. B., Penney, J. B., 11 Cort6s, R. and Palacios, J. M. (1986) Brain Dovorini-Zis, K. and Shoulson, I. (1984) Res. 362,227-238 Neurology 34, 1237-1240 12 Cortes, R., Probst, A., Tobler, H. J. and 27 Palacios, J. M. (1982) Brain Res. 243, 173Palacios, J. M. (1986) Brain Res. 362,239--253 175 13 Pazos, A., Hoyer, D. and Palacios, J. M. 28 Lang, W. and Henke, H. (1983) Brain Res. (1984) Eur. J. Pharmacol. 106, 531-538 267,271-280 14 Pazos, A. and Palaeios, J. M. (1985) Brain 29 Greenamyre, J. T., Penney, J. B., Young, Res. 346, 205--230 A . B . , D'Amato, C. J., Hicks, S.P. and Selected references 15 Probst, A., Cortes, R. and Palacios, J. M. Shoulson, I. (1985) Science 227, 1496-1499 1 Bird, E. D. and Iversen, L. L. (1982) in (1986) Neuroscience 17, 11-35 30 Phelps, M. E. and Mazziotta, J. C. (1985) Handbook of Neurochemistry (2nd edn Vol. 16 Cort~s, R., Probst, A. and Palacios, J. M. Science 228, 799-809 2), (Lajtha, A., ed.), pp. 225-251, Plenum (1984) Neuroscience 12, 1003-1023 31 Pan, H. S., Penney, J. B., Young, A. B., Press 17 Pazos, A., Probst, A. and Palacios, J. M. Arbor, A., Sboulson, I. and Eskin, T. (1985) 2 Bj6rklund , A. and H6kfelt, T., (eds.) (1984) (1985) Brain Res. 358, 324-328 Neurology Suppl. 1, 35, 175 Methods in Chemical Neuroanatomy (Hand- 18 Probst, A., Cortes, R. and Palacios, J. M. 32 Penney, J. B., Young, A. B., Walker, F. O. book of Chemical Neuroanatomy, Vol. 1), (1984) Eur. J. Pharmacol. 106, 477-488 and Shoulson, I. (1984) Neurology Suppl. 1, Elsevier 19 Maurer, R., Cort6s, R., Probst, A. and 34, 153 3 Kuhar, M. J. (1985) in Neurotransmitter Palacios, J. M. (1983) Life Sci. 33, 231-234 33 Uhl, G. R. and Kuhar, M. J. (1984) Nature Receptor Binding (2nd edn) (Yamamura, 20 Biegon, A., Rainbow, T. C., Mann, J. J. and 309, 350-352 H. I., ed.), pp. 153-176, Raven Press McEwen, B. S. (1982) Brain Res. 247, 3794 Young, W. S. and Kuhar, M. J. (1979) Nature 382 280, 393-395 21 Robson, E. E., Foote, R. W., Maurer, R. and 5 Kuhar, M. J. and Unnerstall, J. R. (1985) Kosterlitz, H. W. (1984) Neuroscience 12,621 Trends NeuroSci. 8, 49-53 22 Kuhar, M. J. (1985) TrendsNeuroSci. 8,1906 Whitehouse, P. J. (1985) Trends NeuroSci. 8, 191 J. M. Palacios and R. Cortes are at the 434-437 23 Uhl, G. R., Whitehouse, P. J., Price, D. L., Pharmaceutical Division, Sandoz Lid, CH.4002 7 Palacios, J. M., Mcier-Ruge, W. and Ulrich, J. Tourtelotte, W. W. and Kuhar, M. J. (1984) Basle, Switzerland, and A. Probst is at the (1982) Neurosci. Supp. to Vol. 7, 165 Brain Res. 308, 186-190 Department of Neuropathology, Institute of 8 Penney, J. B. and Young, A . B . (1982) 24 Whitehouse, P. J., Wamsley, J. K., Zarbin, Pathology, University of Basle, Sch6nbeinstrasse Neurology 32, 1391-1395 M. A., Price, D. L., Tourtelotte, W. W. and 40, CH-4003 Basle, Switzerland. For technical reasons we are unable to reproduce this figure in colour. See the June issue of Trends in NeuroScience~ for full colour illustration.
s i o n t o m o g r a p h y ( P E T ) p r o b e s 3° c o u l d h e l p in t h e e a r l y d i a g n o s i s o f s o m e degenerative and psychiatric diseases a n d in t h e m o n i t o r i n g o f t h e t h e r a p e u t i c effects o f d r u g s . T h i s , t o g e t h e r w i t h t h e development of improved non-invasive i m a g i n g t e c h n i q u e s for r e c e p t o r s a n d o t h e r sites, will c h a n g e t h e w a y w e t h i n k about our own brain.