Receptors for Amines, Amino Acids and Peptides: Biochemical Characterization and Microscopic Localization

Receptors for Amines, Amino Acids and Peptides : Biochemical Characterization and Microscopic Localization

INTRODUCTION Receptors are defined as the structures which recognize hormones and neurotransmitters and initiate their biological effects. The concept of receptor was introduced in pharmacology by Langley at the end of the last century. Later it was further developed by Ehrlich and Langley during the first decade of the 1900s (see Parascandola, 1980 for a review). Since then, the concept of receptor has formed the basis of the development of a rational pharmacology. Receptors have been rather hypothetical entities until relatively recently. The development of new biochemical and biophysical methods and their application to the study of receptors have, however, led to the establishment of the receptor as a real, molecular entity. The solubilization and purification of the nicotinic cholinergic receptor protein signaled the beginning of the era of molecular biology of receptors (Changeux, 1976). In this paper, we will briefly review two aspects of the current research in brain receptors: the biochemical characterization and the microscopic localization of receptors for amine, amino acid and peptide neurotransmitters. BIOCHEMICAL CHARACTERIZATION OF NEUROTRANSMITTER RECEPTORS From the results of the investigations on receptor mechanisms in the last 20 years, has emerged a model of what could be called the “receptor complex”. An example of this is presented schematically in Fig. I . Two major parts of the receptor complex have been studied in detail. One, the “recognition” or “binding” site is the specific portion of the receptor to which the neurotransmitter binds. This binding induces modifications in a second major component of the complex that leads to the “translation” of the message into a physiological effect. Interconnecting the recognition and translation parts are “regulatory” proteins.

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Present addresa : Sandoz Ltd., Prrclinical Research 3601604. CH-4002 Baael. Switzerland

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“I Fig. I , A model of the “receptor complex”. The different proteins of the “receptor complex” are schematically represented, embedded in the lipid bilayer of the cell membrane. “Out” indicates the extracellular space. “In” indicates the intracellular space. M, methylating enzymes; R, recognition or binding site; I, ion channel; Cy, cyclase; G , GTP binding protein. See text for details.

The recognition site

-

binding studies

It was recognized for a long time that the first step in the action of a neurotransmitter is its binding to highly selective molecules in the membrane of cells. The first attempts to label neurotransmitter receptors using radioactive ligands were hampered by the high concentration of non-specific, non-receptor related binding sites when compared with the low concentrations of specific receptor sites (in the order of picomoles per gram of tissue). Only by working with very enriched material (such as the electric organ of the torpedo) and by using irreversible ligands, was it possible to characterize the nicotinic receptor (Changeux, 1976). The problem of the separation of the small quantities of specific receptor binding from the much greater non-specific sites, when reversible ligands are used, was solved by applying to the neurotransmitter receptors methods previously developed in hormone receptor studies (Cuatrecasas, 1975). A pioneer study of neurotransmitter receptors in brain tissue was the characterization of the opiate receptor by S . H . Snyder and his collaborators (Snyder, 1975). By using ligands with a very high affinity for the receptor site (labeled with a very high specific activity) and using low concentrations of ligand, it is possible to maximize the specific binding. In addition, the use of rapid filtration techniques for the separation and washing of the membranes allows the efficient removal of the non-specifically bound ligand with minimal perturbation of the ligand specifically bound to receptors. The demonstration that the radiochemical binding was to a pharmacologically relevant receptor was performed by using several criteria. The binding was: saturable, indicating the presence of a finite number of binding sites and was stereospecijic, that is, levorotatory (-) isomers were much more potent displacing the binding of the radioactive ligand than the dextro (+) isomers, in accordance with their pharmacological properties. More importantly, a large series of opiate drugs presented affinities for the receptor site that closely paralleled their potencies in classical pharmacological tests. Finally, the regional distribution and the subcellular localization of the opiate binding indicated the association of these sites with areas relevant to the actions of opiates and with fractions containing neuronal membranes. Even when all these parameters are taken into account caution is advised when interpreting results from radioligand binding studies. There are

267 examples in the literature of high affinity, low capacity and even stereospecific binding of radioligands to non-biological materials such as talcum powder or glass filters (Snyder and Bennett, 1976). Finally, in many cases, the biological relevance of some "binding sites" and the correlation between density of binding sites and biological response is not completely understood. Radioligand binding studies provide detailed information about the affinity of drugs or neurotransmitters for a recepter but they do not give information about the "intrinsic activity" or "efficacy" of these molecules, i.e., their ability t o produce a biological response. While, in general, there is a good correlation between the results obtained in binding assays and in classical pharmacological tests both parameters are not always determined in the same tissue. In some cases both the biological response and the binding activity of a series of molecules had been determined in the same tissue. For example Creese and Snyder (1 975), found a very good correlation between the pharmacological activity and receptor binding of opiate drugs in the guinea-pig intestine. The demonstration of opiate receptors in brain was rapidly followed by the application of these binding techniques to the characterization of many different neurotransmitter receptors (for recent reviews see Y amamura et al., 1978, 1980 ; Pepeu et al., 1980). Table I summarizes the receptors for putative neurotransmitters that have been characterized, up to now, and indicates the ligands most often used in these studies. Some general properties emerge from the large quantity of receptors studied until now. TABLE 1 PUTATIVE NEUROTRANSMITTERS : RECEPTORS AND TYPICAL LIGANDS USED IN LABELING

Atninrs

Noradrenal ine Doparnine Serotonin Hi stami ne Acetylcholine

alpha beta

3H]WB4101, ['H]clonidine 'H]dihydroalprenolol 3H] spiperone 'HILSD 'H]mcpyramine 'HIQNB 3H]alpha-bungarotoxin

HI rnuscarinic nicotinic

Amino a d s

GABA Glutamic acid Glycine Peprides Opioids Substance P Neurotensin Angiotensin I1 Somatostatin VIP TRH Bombesin Camosine CCK Bradykinin

H"][

niuscimol

[ 'Hlkainic acid [ 'H1strychnine

(?),

[ 'Hjglutarnate

'Hldihydromorphine 'Hlsubstance P 3H]neurotens~n 'Hlangiotenaln I1 1?51]somatostatin-28 12511VIP 'HITRH '25I]bombesin 'Hlcarnosl ne 1251]CCK 'H]bradykinin

268 Receptors have high affinities for their ligands, the dissociation constants ( K d ) being in the order of the nanomolar range ( 10- M) ;the concentration of receptors sites in the brain is very small, in the order of picomoles per gram of tissue; and receptors have a high degree of pharmacological and steric specificity for their particular ligands. Furthermore, no major differences appear to exist between the properties of the receptors for the different chemical groups of putative neurotransmitters (i.e. receptors for peptides as opposed to amines). The application of the binding techniques to the study of receptors have also uncovered some properties of the recognition site for neurotransmitters. Two important characteristics are the multiplicity of receptors for a single neurotransmitter and the regulation of the binding by nucleotides and ions. The concept of multiple receptors for a neurotransmitter is not new. The existence of muscarinic and nicotinic receptors for acetylcholine (Dale, 1914) or alpha and beta receptors for noradrenaline (Ahlquist, 1948) have been known for many years. The application of binding techniques for the study of many neurotransmitter receptors has revealed a much greater multiplicity of binding sites (Snyder and Goodman, 1980). Some examples are presented in Table 11. Again, the receptors for the major chemical groups of neurotransmitters TABLE I1 MULTIPLE RECEPTORS FOR NEUROTRANSMITTERS Neurotrunsmiiter Amines Noradrenaline

Dopamine Serotonin Histamine Acetylcholine

Recepor beta, beta-1 and beta-2 alpha, alpha- I and alpha-2 D-1, D-2 (D-3, D-4, ?) 5 HT-I, 5 HT-2 H-1 and H-2 muscarinic (high and low affinity) nicotinic

Amino acidx

GABA Peptides Opioids

high and low affinity presynaptic (haclofen-sensitive)

mu, delta. kappa

do not seem to vary in their characteristics. Amines such as dopamine, serotonin or histamine have multiple receptors. Amino acids such as GABA (gamma-aminobutyric acid) also have more than one receptor. The opiate peptides have at least 3 different types of receptors designated mu, delta and kappa. The study of neurotransmitter receptors by binding methods has also revealed the important regulatory role played by ions and guanine nucleotides. Already in the pioneering experiments with the opiate receptor, the regulatory role of low concentrations of sodium ion was observed. Sodium ions selectively increase opiate antagonist binding while reducing agonist binding. The regulatory role of monovalent ions such as sodium, and divalent ions such as manganese, magnesium and calcium, has now been described for several receptors including alpha adrenergic, histamine-H,, muscarinic cholinergic receptors and others (see for example, U'Prichard and Snyder, 1978).

269

Another important regulatory factor is the nucleotide, guanosine triphosphate (GTP). It is postulated that GTP effects on the binding of neurotransmitter receptors reflect the linkage of these receptors to the enzyme adenylate cyclase. In general, GTP produces a decrease in the affinity of agonists for their binding sites. In recent years the mechanism of GTP regulation of hormone receptors has been studied in some detail (Rodbell, 1980). A nucleotide regulatory component (aprotein denominated GIF or N factor) situated on the inner face of the membrane, contains sites that bind GTP. It has been proposed that the binding of the hormone or neurotransmitter leads to the formation of an “active state” of the regulatory protein that then binds GTP. The neurotransmitter-receptor-regulatory protein-GTP complex couples preferentially with the catalytic subunit of adenylate cyclase to produce an active form of this enzyme. On the other side of the membrane, the formation of this macromolecular complex also alters the affinity of the ligand for the receptor site. Guanosine nucleotide regulation has now been observed in receptors for peptides (such as the opiates) and amines (for example, noradrenaline and dopamine receptors) (U’Prichard and Snyder, 1978).

Translation units : the g e n e r d o n qf second messengers How the recognition of a neurotransmitter by its receptors is translated into a cellular response, is a central question in the understanding of receptor mechanisms. Several “translation” mechanisms have been postulated. In general, the binding of the neurotransmitter to the receptor is followed by alterations in the activity of membrane-bound enzymes (such as cyclases) or in the change of ion permeability (by opening or closing of ion channels). Table 111 presents some examples of “second messenger” mechanisms. One of the best characterized “second messengers” are the cyclic nucleotides CAMPand cGMP (3‘,S‘-cyclic adenosine and 2’ ,3’-cyclic guanosine monophosphate). Neurotransmitters such as noradrenaline, dopamine, histamine (Daly, 1975) acting on specific receptors (beta, D1, H2, etc.) produce increases in the activity of adenylate cyclase leading to increases in the intracellular levels of CAMP. In other cases some neurotransmitters, such as acetylcholine acting through muscarinic receptors or histamine acting on H l-receptors, produce increases in guanylate cyclase increasing cGMP levels. Inhibition of cyclase activity can also be mediated by neurotransmitter receptors. Examples of this are noradrenaline (acting on n,-receptors) and opiates. Not all neurotransmitter receptors are, however, associated to cyclases. An important group of receptors seem to act by mobilizing calcium ion movement (Table 111) through the membrane and affecting the turnover of a minor phospholipid, phosphatidylinositol (Michell and Kirk. 1981). The stimulation of for example, alpha adrenergic, histamine-HI, or muscarinic cholinergic receptors elicits an increase in the intracellular concentration of calcium ions and (connected in some way) the increased breakdown of phosphatidylinositol. Peptide receptors like those for the vasoactive intestinal peptide, also produce a similar effect. Besides the two mechanisms described above, other “translation” mechanisms have been proposed. For example, opioid receptor stimulation can produce changes in the synthesis of gangliosides in neuroblastoma (Dawson et al., 1979). Recently, receptor-stimulated phospholipid methylation has been described by Axelrod and co-workers (Hirata et al., 1979). This mechanism has been studied in some detail in the case of beta adrenergic receptors in rat reticulocytes. Phospholipid methylation, although independent of cyclase stimulation, seems to facilitate the coupling of the beta-receptor with the cyclase. Receptor-mediated phospholipid methylation can also be implicated in the regulation of the number of sites and in phenomena such as receptor desensitization. Besides beta adrenergic receptors, at least one

270 TABLE 111 NEUROTRANSMITTER RECEPTORS AND “SECOND MESSENGERS” A . Receptors linked to cycluses : Amines Noradrenaline Dopamine Histamine Serotonin Acetylcholine Amino acids GABA Glutamate Peptides Opioids VIP CCK

bcta D-I

HI

( t . CAMP), alpha (1. CAMP) (t, CAMP)

(1, cGMP), H2 (1, CAMP)

(1. CAMP) muscarinic

(1, cGMP)

B . Receptors linked to CaZ+ m o b i l i ~ r i t i o n ~ ~ h o s p h u t i d y l ~ nresponse ~~sitol Amines Noradrenaline alpha Acetylcholine muscarinic Histamine HI Serotonin Peprides Vasopresin Substance P Bombesin Angiotensin I1 Bradykinin C. Receptors linked to phospholipid methylation. beta Noradrenaline Benzodiazepine

other receptor (the receptor for the minor tranquilizing drugs benzodiazepines) also mediates changes in phospholipid methylation (Hirata et al., 1980).

MICROSCOPIC LOCALIZATION OF NEUROTRANSMITTER RECEPTORS One question that follows the characterization of neurotransmitter receptors is where are these receptors localized? The brain is a highly organized and interconnected organ. The different neurotransmitter systems also present a very discrete and predetermined anatomical organization. The development, for instance, of histochemical techniques for the localization of catecholaminergic (Swanson and Hartman, 1975 ; Moore and Bloom, 1979) or peptidergic (Hokfelt et al., 1980)neurons in the brain have greatly improved our knowledge of the role the different neurotransmitters play in the function of the brain. Only very recently have similar techniques become available for the localization of neurotransmitter receptors (Kuhar, 198 1 ). In this section we will describe these techniques and some of the results obtained with them.

27 1

Methodology : autororlio,srriphic.locnlizritiori of receptors The ability to localize receptors by Hutoradiographic methods stems directly from the development of ligands of high affinity for these receptors and which can be radioactively labeled with a high specific activity. Also, the development of autoradiographic procedures for the localization of water-diffusible substances has been of primary importance. Basically, the method of autoradiographic localization of receptors consists of labeling these sites with a radioactive drug (administered to the living animal or introduced to brain tissues in vitro) and the subsequent contact of sections of the labeled tissue with a photographic emulsion. Because of the presence of non-receptor binding sites (the “non-specific” binding) and the reversible nature of the majority of the ligands available, many precautions need to be taken in labeling receptors for autoradiography. The characteristics of the ligands used and the conditions for labeling have been extensively reviewed (Kuhar, 1978a,b; 1981. Young et al., 1980) and will not be described here. Young and Kuhar (1979) have developed a simple method that consists of performing the binding of radioligands in vitro to tissue sections that are subsequently prepared for histological obscrvation. Under these conditions, the parameters of labeling can be rigorously controlled and the properties of the receptors assessed. The autoradiograms are then generated by opposing emulsion-coated coverslips (Young and Kuhar, 1979) or tritium-sensitive X-ray films (Palacios et al., 198 la). Using these techniques the conditions for the labeling of a large number of receptors have been defined. Subsequently, the distribution of these receptors in the brain has been studied at the light microscopic level (Table IV). One of the advantages of the autoradiographic techniques is that they are quantitative. By using the ”-sensitive film and microdensitometric procedures it is possible to determine receptor densities in very small areas of the brain. The light microscopic techniques do not have. however, enough resolution as to allow the cellular and subcellular localization of receptor sites. For example, whether a receptor is localized in neurons or glial cells, or if it is pre- or postsynaptic to a given neuron cannot be determined from these studies. In some cases by combining autoradiographic techniques with others, such as specific lesions with 6-hydroxydopamine or with kainic acid, it is possible to get more information on the cellular localization of receptors. The development of electron microscopic techniques is then a necessary improvement (Kuhar et al., 198 I ) .

Receptor localization : relationship to presynuptic intiervation and function The mapping of receptors in the brain has revealed that, as with the neurotransmitters themselves, they have a heterogeneous distribution in the central nervous system. These studies have also revealed some characteristics or general trends of receptor localization in the CNS. One of the first questions addressed by these studies is: what kind of correlation is there between the distribution of receptor sites for a neurotransmitter and that of presynaptic terminals using the transmitter? The answer that is derived from the receptor distributions studied so far, is that there is not a simple correlation between pre- and postsynaptic markers. In some cases, a remarkable complementation between receptors and terminals has been found. Examples of this are the distribution of the opioid peptides and morphine receptors in the spinal cord (Kuhar, 1978a,b) or hippocampus (Goodman et al., 1980and Gall et al., 198 1 ; see Fig. 2). In other cases, while there is a general correspondence between terminals and receptors there is not a one-to-one relationship. An example of this is the distribution of high

TABLE IV

N

4

RECEPTORS LOCALIZED IN THE RAT BRAIN BY AUTORADIOGRAPHY AND LIGANDS USED Amines Carecholamines Dopamine ([3H]spiperone) Alpha-I-noradrenaline ([3H]WB4101) Alpha-2-noradrenaline ([3H]para-aminoclonidine, [3H]clonidine) Beta-noradrenaline ( [3H]dihydroalprenolo1) Serotonin

5-HT-I ([3H]LSD, [3H]5-HT) 5-HT-2 ([3H]LSD, [3H]spiperone) Acetylcholine Muscarinic cholinergic high and low affinity ([3H]QNB. ['HINMS. ['Hlpropylbenzilylcholine) Nicotinic ([ 1251]alpha-bungarotoxin)

Histamine Histamine HI ([3H]mepyramine)

Amino acids

Peptides

GABA ([3H]muscimol) Opiates - mu and delta ([3H]dihydromorphine, [3H]naGABA related drugs, benzodiazepines (['Hlflunitraze[3H]diprenorphine, ['H]Leu-enkephalin, loxone, ['251]FK-33-824, [ 1251]D-Ak-~-Leu-enPam) glycine ([3H]strychnine) kephalin) Glutamate (['Hlkainic acid) Neurotensin ([3H]neurotensin) CCK ([ '*'I]CCK-33) Insulin ([12SI]INS)

N

273

Fig. 2. Relationship between the distribution of terminals and receptors for neurotransmitters. The distribution of receptors is illustrated by dark-field micrographs of autoradiographic emulsions, where the different densities of receptors are indicated by the concentrations of autoradiographic grains, seen as bright points. Distribution of terminals is illustrated by schematic drawings, where the densities of terminals are represented by the dotted patterns. A : the correlation between the distribution of opiate receptors (Goodman et al.. 1980) and enkephalin immunoreactivity (as described by Gall et al., 198 1 ) in rat hippocampus. B : GABA receptors and GABA terminals in rat cerebellum (from Palacios et al., 1980). C : Beta-adrenergic receptors (from Palacios and Kuhar, 1980) and catecholamine terminals (from Swanson and Hartman, 1975). See text for details. Abbreviations: sl-m, stratum lacunosum inoleculare; sp, stratum pyramidale; m, molecular layer of the dentate gyrus (in A ) ; h. hilus of the area dentata: sg, stratum granulosum; g, granule cell layer; m, iiiolecular layer (in B); p. Purkinje cell bodies (arrow in the dark field): wm, white matter; Cx, cortex; CC. corpus callowni; Cd, caudate-putamen; BST, bed nucleus of the stria terminalis; ac. anterior commisure; OT. olfactory tubercles

affinity GABA receptors in the rat cerebellum. These receptors are highly concentrated in the granule cell layer while the density of GABAergic terminals is approximately the same in both the granule and molecular layers (Palacios et al., 1980). Finally, dramatic examples of absence of a correlation between terminals and receptors have also been found. Perhaps one of the most impressive is the localization of beta-adrenergic receptors in the rat striatlim. While this area contains few, if any, noradrenergic terminals, it has one of the highest concentrations of beta-adrenergic receptors in the rat brain (Palacios and Kuhar, 1980 ; see Fig. 2). A convincing explanation for these discrepancies is still absent. Several different hypotheses have been proposed (Snyder and Bennett, 1976 ; Kuhar, 198 I ), including possible differences in the

274

Fig. 3. Autoradiographic localization of multiple binding sites for ['HILSD in the rat brain. Two types of serotonin rcccptors have been characterized by binding of [IHILSD (Peroutka and Snyder, 1979). A: the autoradiographic image, obtained with a 'H-sensitive film (LKB. ZH-Ultrofilm). of the localization of ['HILSD binding sites in a coronal section of the rat brain at the levcl of the anterior nucleus caudatus. High densities of receptors are localized in the nucleus accunibens, striaturn, olfactory tubercles and lamina 1V of the cortex. B : a section consecutive to (A) which was incubated with ['HILSD in the presence of 30 nM spiperone, a neuroleptic that inhibits the binding of LSD to serotonin-2 receptors. The image shows the localization of serotonin-] receptors. In the cortex, for example, high concentrations of serotonin-1 receptors are found in the deeper layers. C : the binding of [3H]LSD to this section was performed in the presence of 300 nM serotonin which blocks the binding to serotonin-1 receptors. The picture shows the localization of serotonin-2 receptors. In the cortex high concentrations of these receptors are found in lamina IV. D: this photomicrograph shows that ['HILSD hinds to additional receptor sites in striaturn, accumhens and olfactory tubercles. This section was incubated with ['HILSD and 100 ,uM serotonin, and binding still remains in these areas. Thus, LSD binds to dopamine receptors in these nuclei.

cellular localization of receptors, the influence of the geometry of the postsynaptic cells and others. The questions of the functionality of "non-innervated" receptors and the physiological meaning of the differences in receptor densities are important and will certainly receive much attention in the future. The problem of multiple neurotransmitter receptors has also been addressed from the anatomical point of view. Multiple receptors for several neurotransmitters have been localized. The results obtained indicate that the multiple receptors are localized to different anatomical elements and possibly represent different molecular entities. Examples are the differential localization of multiple opioid (Goodman et al., 1980), alpha adrenergic (Young andKuhar, 1980),muscarinic cholinergic (Wamsley et al., 1980) or serotonin (Palacios et al., 1981b) receptors. The localizations of multiple binding sites for ["H]LSD is illustrated in Fig. 3. One important result of the autoradiographic studies has been their contribution to the understanding of the mode of action of psychoactive drugs. The early studies with the opiate

275 receptor are an example. Atweh and Kuhar (1977a,b,c) demonstrated the presence of high concentrations of opiate receptors in areas associated with analgesia and also in regions of the brain involved in other physiological and hormonal effects that are known to be altered after opiate administration. Other examples of correlation between drug action and receptor localization are provided by recent studies of the binding of drugs like clonidine an alpha-2-adrenergic agonist (Young and Kuhar, 1980), and strychnine, a glycine antagonist (Zarbin et al., 1981). SUMMARY Receptors for amine, amino acid and pcptide neurotransmitters have now been characterized by biochemical techniques such as the high affinity binding of radioactive ligands. Different mechanisms of translation of the neurotransmitter binding into a cellular response have also been identified. These include cyclase activation, calcium ion mobilization, phospholipid methylation and others. Receptor distribution in brain has been studied by autoradiographic methods. Receptors for different neurotransmitters present a very heterogeneous localization. Some areas seem to be very rich in receptors for different neurotransmitters. In general, there is not a simple correlation between receptor and terminal distributions for a given neurotransmitter. The distribution of receptors provides information about the anatomical locus of action of neurotransmitters and drugs. No special characteristics have been observed regarding the properties or distribution of receptors for amine, amino acids or peptide neuroftxiwi tters. Thus, receptors as structural entities may be similar although the neurotransmittcsi. wlxrunces differ. Hopefully, research in the near future will involve isolation and purification 01’ rcccptors in the central nervous system so that we may better understand the physical or molecular structure which constitutes receptors for aniines, amino acids and peptides. ACKNOWLEDGEMENTS The authors wish to express their appreciation to Roberta Proctor for her artistic contributions, to Naomi Taylor for technical and photographic assistance and to Leslie Bangerter for typing the manuscript. We would also like to thaqk Dr. Michael J. Kuhar for his guidance and contributions in the field of receptor localization. REFERENCES Ahlquist, R.P. (1948) A study of the adrenotropic receptors. Amer. J. Physiol., 153: 58&600. Atweh, S. and Kuhar. M.J. (1977a) Autoradiographic localization of opiate receptors in rat brain. I. Spinal cord and lower medulla. Bruin Res., 124: 53-67. Atweh, S. and Kuhar, M.J. ( I 977b) Autoradiographic localization of opiate receptors in rat brain. 11. The brain stem. Bruin Res., 129: 1-12. ) localization of opiate receptors in rat brain. 111. The telencepAtweh, S. and Kuhar, M.J. ( 1 9 7 7 ~Autoradiographic halon. Bruin Res., 134: 393405. Changeux, J.P. ( 1976) The cholinergic receptor protein from fish electric organ. In Handbook of Psjchophurmucology, Vol. 6, L.L. lversen et al. (Eds.), Plenum, New York, NY, pp. 235-301. Creese, 1. and Snyder, S.H. (1975) Opiate receptorbinding and pharmacological activity in theguinea pig intestine../. Pharmucol. exp. Ther., 94: 205-2 19. Cuatrecasas, P. (1975) Hormone receptors - their function in cell membranes and some problems related to methodology. Ad. C y l i c . Nucleotide R e s . , 5 : 79-104. Dale. H.H. (1914) The action of certain esters and ethers ofcholine, and their relation to muscarine. J. Phurmucol. e.xp. Ther., 6 : 147-190.

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277 Yamamura, H.I.. Olsen, R . W . and Usdin. E. (Eds.) (1980) P . ~ ~ c h ~ ) p h u r m uand ~ o Biochernistrj l~)~~ of Neurorrurzsmitter Receptors, ElsevieriNorth-Holland, New York, NY. Young, W.S. 111 and Kuhar, M.J. (1979) A new method for receptor autoradiography: [3H]opioid receptors in rat brain. Bruin Rex., 179: 255-270. Young, W.S. 111 and Kuhar, M.J. (1980) Noradrenergic a , and a2 receptors: light microscopic autoradiographic localization. Proc. nut. Acczd. Sci. U.S.A.. 77: 1696-1700. Young, W.S. 111, Palacios, J.M. and Kuhar, M.J. (1980) Histochemistry of receptors. In Receptorsfor NeurotrunsmitrersundPeptideHariiioiies, G . , Pepeu, M.J. KuharandS.J. Enna(Eds.), Raven, New York. NY, pp. 51-56. Zarbin, M.A., Wamsley, J.K. and Kuhar, M.J. (198 I ) Glycine receptor: light microscopic autoradiographic localization with ’H-strychnine. J . Neurosci. , I : 532-547. DISCUSSION

V . GALLO: (1) You have shown in one of your slides that GABA increases cGMP, but it is well known that GABA in cerebellum decreases cGMP levels. (2) [3H]Kainic acid as a ligand for glutamate receptors. It seems to have a different binding receptor. J.M. PALACIOS: ( I ) Some investigators get an increase in cGMP by adding GABA to the incubation medium (Ferrendelli et al., 1974). The differences in results can depend upon the conditions, species, age of the animal, etc. However, the point of the table is to show that the different types of neurotransmitters (amines, amino acids and peptides) do in fact act through similar mechanisms with respect to the second messenger generation. (2) Yes, it seems now that the binding of the glutamate analogue kainic acid is not to a “glutamate receptor”. We should then add a question mark to the tables where KA is proposed as a glutamate ligand. D. SWAAB : You showed hot spots for diazepine receptors in lamina IV of the cortex. On which structure(s) are these receptors localized exactly ? J.M. PALAClOS: From our results we can only say that the receptors are localized to lamina IV. The resolution of these techniques is not enough to localize these receptors to specific cell populations. This can be done by combining autoradiographic studies with e.g. lesions of specific pathways or cell populations.

G . MILLIGAN : In ontogeny of cultured cell lines it is often the case that dedifferentiated and more differentiated cells possess the same number of binding sites but activation of these binding sites is repressed in the dedifferentiated state, so this may reflect production of receptor effector coupling, with synthesis of the information coupling molecule with greater states of differentiation. R.G. HILL: Where there is no evidence for function o f a receptor site, i.e. the only experiments performed have been binding ones, then we should perhaps not talk about receptors at all but restrict ourselves to “binding sites” until function has been established’? J.M. PALACIOS : ( I ) I agree. However, for many years we have called “receptors” sites whosefunctional role we do not know, for example the “spare receptors”. V . CHAN-PALAY : The fact that you have binding of ligands in autoradiography in regions of the brain that are very

well known, in layers etc., where there is no distribution of cells or terminals with the appropriate transmitter remains the most serious problem with your method. J.M. PALACIOS : I do not think this is a problem of rhr method ifse(f.Our results agree very well with those obtained by dissection and subsequent membrane binding. An examplc of localization of receptors in areas without endogenous ligand is beta receptors in the rat striatum. This has been found in membrane binding studies too. In my opinion the real problem is what the functional role of these “non-innervated” receptors is (if any?).

F. LOPES DA SILVA: Are there enkephalin binding sites in CAI ?This has been put in doubt although we know there are physiological data showing an effect of o-Ala-enkephalin on CAI ?And on the fascia dentata of the hippocampus, stratum moleculare. J.M. PALACIOS: ( 1 ) Yea. there are. ( 2 ) Yes

278 REFERENCES Ferrendelli, J . A . , Chang, M.M. and Kinscherf. D.A. ( I 974) Elevation of cyclic GMP levels in central nervous system by excitatory and inhibitory amino acids. J . Ncurochern., 22: 535-540.