Distribution of dopaminergic receptors in the primate cerebral cortex: Quantitative autoradiographic analysis using [3H]raclopride, [3H]spiperone and [3H]SCH23390

Neuroscience Vol. 40, No. 3, Pp. 657-671, 1991 Printed in Great Britain

0306-4522/91$3.00+ 0.00 Pergamon Press plc 0 1991IBRO

DISTRIBUTION OF DOPAMINERGIC RECEPTORS IN THE PRIMATE CEREBRAL CORTEX: QUANTITATIVE AUTORADIOGRAPHIC ANALYSIS USING [‘HIRACLOPRIDE, [3H]SPIPERONE AND [3H]SCH23390 M. S. Lmow,*

P. S.

D. W. GALLAGER and P. RAKIC

GOLDMAN-RAKIC,

Yale University, School of Medicine, Section of Neuroanatomy,

New Haven, CT 06510, U.S.A.

Abstract-A widespread distribution of dopamine D, receptors in the neocortex is well recognized. However, the presence of dopamine D, receptors in this structure has only recently been established [Martres er al. (1985) Eur. J. Pharmac. 118, 211-219; Lidow et al. (1989) Proc. mtn. Acad. Sci. U.S.A. fJ6, 6412-64161. In the present paper, a highly specific antagonist, [‘Hlraclopride, was used for autoradiographic determination of the distribution of D, receptors in 12 cytoarchitectonic areas of the frontal, parietal, and occipital lobes of the rhesus monkey. A low density of D,-specific [3H]raclopride binding (1.5-4.0 fmol/mg tissue) was detected in all layers of all cortical areas studied. Throughout the entire cortex, the highest density of binding was consistently found in layer V. This is a unique distribution not observed so far for any other neurotransmitter receptor subtype in monkey cerebral cortex, including D, receptor. In addition, a comparison was made of the distribution of [‘Hlraclopride and [3H]spiperone, which has been commonly used in previous attempts to label cortical D, receptors. We found marked differences in the distribution of these two radioligands. In the prefrontal cortex, the pattern of [3H]spiperone binding in the presence of ketanserin resembled the combined distribution of 5-HT,c serotoninergic and a,-adrenergic sites as well as D, receptors. Thus, [3H]raclopride provides a better estimation of the D, receptor distribution than does [3H]spiperone. The distribution of D,-specific binding of [3H]raclopride was also compared with the D,-specific binding of [3H]SCH23390 in the presence of mianserin to block labeling to 5-HT, and 5-HT,, sites. The density of D,-specific [)H]SCH23390 binding was lo-20 times higher than that of D,-specific [3H]raclopride binding throughout the cortex. The densities of both [‘Hlraclopride and [“H]SCH23390 binding sites display a rostral-caudal gradient with the highest concentrations in prefrontal and the lowest concentrations in the occipital cortex. However, the binding sites of these two ligands had different laminar distributions in all areas examined. In contrast to preferential [‘Hlraclopride binding in layer V, a bilaminar pattern of [3H]SCH23390 labeling was observed in most cytoarchitectonic areas, with the highest concentrations in supragranular layers I, II and IIIa and infragranular layers V and VI. Whereas [3H]raclopride binding was similar in all cytoarchitectonic areas, [‘H]SCH23390 exhibited some region-specific variations in the primary visual and motor cortex. The different regional and laminar distributions of D, and D, dopaminergic receptors indicates that they may subserve different aspects of dopamine function in the cerebral cortex.

An understanding of the distribution of dopamine receptors in the cerebral cortex depends on the availability of receptor subtype-specific ligands. However, most ligands have considerable problems of specificity. For example, [-‘H]SCH23390, an antagonist with high affinity for dopamine D, sites, has been used widely to evaluate the distribution of D, However, recently it has become receptors. 15-‘7~‘9-2’,49 clear that this ligand also labels 5-HTz and 5-HT,, serotonergic sites present in the cortex.6.46 Thus, the data from earlier studies employing [3H]SCH23390 *To whom correspondence

should be addressed. ( + )N,N-diethyl-N’-[(3a,4aa, lO~)-1,2,3,4,4a,10,10a-octahydro-6-hydroxy-l-propyl-3benzo-[Glquinolinyll-sulfamide; 5-HT, 5-hydroxytryptamine; SCH23390, (R)-( +)-8-chloro-2,3,4,5_tetrahydro3-methyl-5-phenyl-lH-3-benzazepin-7-01; SCH23982, (5R)-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-IH-3benzozepine 7,ol; SKF83566, ( + )7-bromo-8-hydroxy-3methyl- l-phenyl-2,3,4,5_tetrahydrolH-3-benzazepine hydrochloride; TH, tyrosine hydroxylase.

Abbreviations:

CV205-502,

should be re-evaluated. The detection of Dr receptors in the cerebral cortex has proven to be even more difficult. Indeed, the unreliability of cortical dopamine D, receptor labeling with available D,-sensitive radioligands has lead many investigators to doubt the presence of D, receptors in the cortex.‘~‘4*20~22~24~32 We recently addressed this problem and demonstrated that the D,-specific antagonist of the substituted benzamide group, [3H]raclopride, labels a population of cortical receptors with the pharmacological properties of D, sites.37 Furthermore, these D, sites were found to be widespread in the cerebral cortex of both rat and monkey.37 In the present paper, we report the results of an autoradiographic study of the distribution of D,specific [3H]raclopride binding in the monkey cerebral cortex and compare it to that of [3H]spiperone in the presence of ketanserin. The latter has been widely used in previous attempts to label cortical D, sites (for review see Ref. 47). We also compared the 657

658

M. S. LIDOW et al.

cortical localization o f D 2 sites with that o f D 1 receptors detected with [3H]SCH23390 in the presence o f mianserin, which prevents radioligand binding to 5-HT 2 and 5-HTlc serotonergic sites. In the course o f this analysis, the effect o f mianserin on the distribution [3H]SCH23390 binding was also examined. EXPERIMENTAL PROCEDURES

Tissue preparation The cerebral hemispheres of three adult rhesus monkeys (obtained from the Yale monkey colony) were prepared as described elsewhere. 38,48 Briefly, the animals were anaesthetized and perfused with ice-cold phosphate-buffered saline followed by 0.1% paraformaldehyde containing increasing concentrations of sucrose. The brains were rapidly removed, blocked and immersed in isopentane at - 4 0 ° C for 5 min before storing at -80°C. Brains were cut into 20-#mthick sections on a Bright Cryostat. Sections were mounted on acid cleaned, chrom~alum subbed slides and kept at - 8 0 ° C until assayed, usually within 2 weeks of the tissue being sectioned. Binding assay For labeling with [3H]raclopride, tissue sections were first preincubated for 20 min at room temperature in 50 mM Tris-HC1 buffer (pH 7.4) containing 150 mM NaC1. Sections were then incubated for 45 min at room temperature with 0.3 3.0 nM of radioligand in 50 mM Tris-HC1 buffer (pH 7.4) containing 150 mM NaC1 and 0.1% ascorbic acid. To determine non-specific binding, parallel sections were incubated in the presence of 1 #M (+)-butaclamol. At the end of the incubation, tissue sections were rinsed six times (60 s each) in ice-cold 50 mM Tris HC1 buffer (pH 7.4),

a

b

dipped in distilled water, dried and apposed to 3H-sensitive Ultrofilm for 8 months. Receptor binding with [3H]spiperone was conducted as described previously.38,48Tissue sections were incubated for 30 min at room temperature with 0.7q5.6 nM of radioligand in 170mM Tris-HC1 buffer (pH 7.7) containing 120mM NaCI, 5mM KC1, 2mM CaC12, 1 mM MgCI2, 0.1% ascorbic acid and 0.3 #M ketanserin. Non-specific binding was determined in the presence of 1 # M (+)-butaclamol or 1 0 # M (-)-sulpiride or 10#M dopamine. Sections were rinsed five times (60 min each) in ice-cold 170 mM Tris HC1 buffer (pH 7.7), dipped in distilled water and apposed against 3H-sensitive Ultrofilm for 2 months. Labeling of tissue sections with 1 10 nM [3H]SCH23390 was conducted for 90 min at room temperature in 50 mM Tris HC1 buffer (pH 7.4) containing 120 mM NaC1, 5 mM KC1, 2 mM CaC12, 1 mM MgC12 and 1 #M mianserin. To evaluate the effect of mianserin in the incubation buffer, some sections were incubated in mianserin-free media. Nonspecific binding was determined in the presence of 1 #M SKF83566. Sections were rinsed twice (10min each) in ice-cold 50 mM Tris-HC1 buffer (pH 7.4), dipped in distilled water and apposed to 3H-sensitive Ultrofilm for 3 months. After the films were developed, the tissue was stained with Cresyl Violet for identification of cortical layers. Tissue sections from each animal were assayed separately. For every animal, three consecutive sections were incubated with each concentration of radioactive ligand and the next two sections were used for evaluation of non-specific binding. Radioligands were obtained from New England Nuclear (Boston, MA). Other chemicals were purchased from Research Biochemicals (Natick, MA) and Sigma (St. Louis, MO).

Quantitative densitometry The autoradiographs were analysed with a computer imaging system which allows the overlay of the digitized

c

Fig. 1. Diagrams of the lateral (A) and medial (B) surfaces of the rhesus monkey cerebral hemisphere. Different patterns indicate the cortical areas where the distributions of muscarinic cholinergic receptors were examined. The cytoarchitectonic areas of prefrontal cortex (46, 9, 12 and 25) were identified according to Walker; ss the remaining areas (4, 3, 1, 2, 5, 7, 18 and 17) were identified according to Brodmann. s Line "a" shows the plane of sections presented in Fig. 2; line "b" shows the plane of sections presented in Fig. 4; and line "c" shows the plane of sections presented in Fig. 7.

Dopaminergic receptors in neocortex images of Cresyl Violet-stained sections and the corresponding autoradiograms on the computer screen in order to facilitate histological identification of specific layers on the autoradiographic images. Further, film images of sections with non-specific binding were substracted from those of adjacent sections with total binding, thus permitting the direct observation of images representing specific binding on

659

the screen. Our programs allow comparison of the optical densities of the film images of individual cortical layers with those of ‘H-standards (Amersham Corp., Arlington Heights, IL) that were apposed to the film along with the tissue sections and converts the optical densities of autoradiographs into concentrations of labeled compounds per tissue wet weight for each cortical layer. The optical

Fig. 2. Autoradiograms of the prefrontal cortex. (A) [‘H]SCH23390 labeling in the presence of mianserin (D, sites). (B) [3e]SCH23390 labeling in the absence ofmianserin (presumably Di , S-HT,, and 5-HT, sites). (C) I3HlRaclomide labeling (D, sites) and (D) I3Hlsuioerone binding in the nresence of ketanserin (presumably D2, a and 5-HT,, s&j. Roxes’mark the sites where quantitatiie measurement of radioligand distribution was made. (a) Area 46; (b) area 9; (c) area 12; (d) area 25. Arrow shows the slight increase in density of labeling in layer V corresponding with increase in [3H]raclopride binding in this layer. These and other photographs can be used only to compare the distributions of the ligands. They do not allow a comparison of the absolute densities of labeling between ligands.

660

M. S. Lmow e/ ul.

densities were between 0.08 and 0.80 (diffuse optical density) on all autoradiograms used in this study. In this range they are linearly related to tisue radioactivity on ‘H-sensitive Ultrofilm.3h We examined Walker’s areas 46, 9, 12 and 2555 in the prefrontal cortex, Brodmann’s area 48 in the precentral gyrus, Brodmann’s areas 1, 2, 3, 5 and 7 in the parietal cortex, and Brodmann’s areas 17 and 18 in the occipital cortex (Fig. 1). For the primary visual cortex (area 17) the cytoarchitectonic laminar divisions follow the criteria of Lund.4’ All other cortical areas were defined as described by WalkerSS and Brodmann.* Also, since the distribution of labeling in layer III was not homogeneous in all cortical areas, we found that an accurate description and quantification of the results required a division of layer III of most cytoarchitectonic areas into two strata: sublayer IIIaapproximately the upper one-third of the layer, and sublayer III&the lower two-thirds of the layer. Statisticalanalysis The analysis of saturation binding utilized the nonlinear curve-fitting computer programs KINETIC/EDBA/ LIGANDiLOWRY from Elsevier-BIOSOFT Co.. Cambridge, U.‘K. The analyses were based on concentrations of radioactive ligands specifically bound to tissue labeled with five different concentrations of ligands in incubating solutions. The number of data points employed for quantative analysis of saturation binding is a compromise between the desired accuracy and the availability of appropriate tissue. Five concentrations of free ligand in incubating solution is a minimal number which allows a relatively accurate estimation of B,,, and Kd for a one-site receptor model.9,39B,,,,, and Kdvalues obtained from different layers of each neocortical area were compared with the Gabriel modification of the GT2 method.s3 This method utilizes the data provided by KINETIC/EDBA/LIGAND/LOWRY and produces 95% comparison intervals for each Kd or B,,, value. K,, or Bmdxvalues with overlapping intervals are considered statistically identical; Kd or B,, with intervals which do not overlap are considered statistically different. Plots of these comparison intervals provide a comprehensive visual display of the degree of variation in Kd or B,, values between cortical layers and areas. We have previously demonstrated that sublayer IVb of area 17 and the deep strata of layer III, and layers V and VI of area 4 absorb significantly more ‘H-generated emissions than the rest of the cortex due to increased myelin content of these laminae.j6 The B,, values obtained for these layers and sublayers are underestimated and were therefore corrected.r6 RESULTS

[‘H]SCH23390

and [‘Hlraclopride

binding in the

cerebraI cortex D,-specific

labeling

of

[‘Hlraclopride

and

D,-

specific labeling of [3H]SCH23390 were observed in all layers of all cortical areas examined (Figs 2a,c, 4

and 7). We found no statistically significant changes in Kd values of either ligand between any cortical

layers or areas (Figs 3, 5, 6 and 8). The specific binding of [‘Hlraclopride constituted 4060% of the total binding and the specific binding of [3H]SCH23390, in the presence of mianserin, constituted 7&80% of the total binding. The density of [3H]SCH23390 binding was l&20 times higher than that of [‘Hlraclopride throughout the entire cortex (Figs 3, 5, 6 and 8). The prefrontal cortex had the highest overall density of both [‘Hlraclopride and [‘H]SCH23390 binding sites. In this area, the density of [‘Hlraclopride binding varied from 1.7 to 4 fmol/mg tissue (Fig. 3). In cytoarchitectonic areas 46, 9 and 12, the density of [‘H]SCH23390 binding varied from 18 to 40 fmol/mg. The binding of this ligand in area 25 was particularly high, reaching 50 fmol/mg tissue. In contrast, occipital cortex (areas 17 and 18) had the lowest density of binding for both ligands. [‘H]Raclopride binding varied from 0.6 to 2.7 fmol/mg and [3H]SCH23390 binding varied from 12 to 27 fmol/mg tissue (Fig. 8). Primary motor (area 4) and parietal cortex (areas 3, 1, 2, 5 and 7) had intermediate binding densities for both ligands (Figs 5 and 6). In these regions, [‘Hlraclopride binding was 1.4-9.5 fmol/mg tissue and [3H]SCH23390 binding was 18-38 fmol/mg tissue. The laminar distributions of [3H]SCH23390 and [‘Hlraclopride labeling were very different from each other in all cortical regions examined. The distribution of [‘Hlraclopride was the same in all cytoarchitectonic areas (Figs 2, 3, 4b, 5, 6, 7b and 8). In frontal, parietal and occipital cortex, this ligand consistently has the highest binding density in layer V. Other layers had approximately 4&50% lower density of binding sites. In contrast, [‘H]SCH23390 showed bilaminar patterns of labeling in a majority of cytoarchitectonic areas including all subdivisions of prefrontal and parietal cortex as well as prestriate region, area 18 (Figs 2a, 3, 4a, 5, 6, 7a and 8). The highest densities of labeling with this ligand were found in superficial layers I, II and IIIa and in deep layers V and VI. The middle strata of the cortex (sublayer IIIb and layer IV) were relatively poor in [‘H]SCH23390 binding sites. In parietal cortex, the density of [3H]SCH23390 binding sites in layers V and VI

Fig. 3. Histograms representing the distributions of specific [‘H]SCH23390 binding in the presence of mianserin (D, sites), specific [‘H]SCH23390 labeling in the absence of mianserin (presumably D, , 5-HT,, and 5-HT, sites), specific [3H]raclopride labeling (D2 sites) and specific [3H]spiperone binding in the presence of ketanserin (presumably D,, a and 5-HT,, sites) in area 46 of the prefrontal cortex. The data on the distribution of D, and D, receptors in areas 40, 9, 12 and 25 of monkey prefrontal cortex have already been published in Goldman-Rakic et a1.26Since the distributions of dopaminergic receptors were similar in all of these cortical areas, here we present only the data for area 46 as an example. Three histograms are presented for each Iigand. The middle histogram presents the distribution of B,, averaged for three animals + S.E.M. The Roman numerals indicate cortical layers. The histogram at the top shows B,, values with 95% comparison intervals generated by the GT2 test. The histogram below shows the distribution of Kd values with their 95% comparison intervals. If comparison intervals for any two Bm, or Kd values overlap, there are no statistically significant differences between them.

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Fig. 4. Typical labeling patterns of [‘H]SCH23390 in the presence of mianserin (A) and [‘Hlraclopride (B) in area 4 of the frontal cortex and areas 3, I , 2, 5 and 7 of parietal cortex. Boxes mark the sites where quantitative measurement of radioligand distribution was made. (a) Area 4; (b) area 3; (c) areas 1 and 2; (d) area 5; (e) area 7. Due to differential absorption of 3H-generated emission, the patterns of optical densities of autoradiographic images of area 4 do not precisely reflect the distributions of radioligands; however, the distortions are insufficient to obscure the general pattern of ligand binding.36

was slightly (2&23%) lower than that in superficial cortical strata (Figs 4a, 5 and 6). In prefrontal and prestriate cortex, both supragranular and infragranular layers contained similar densities of binding sites (Figs 3 and 8). Exceptions to the general rule of bilaminar patterning of [‘H]SCH23390 labeling were found in the primary motor (area 4) and visual (area 17) cortices (Figs 4a, 6, 7a and 8). In the primary motor cortex, high binding density was observed in superficial layers I, II and IIIa where the binding was at least twice that in the deeper layers (Figs 4a and 5). In the visual cortex, on the other hand, [3H]SCH23390 labeling took on a trilaminar pattern with highest density in superficial layers I and II, middle sublayer IVa and deep layers V and VI (Figs 7a and 8). In other cortical layers of this area the density of labeling was 40-50% lower.

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Fig. 5. Histograms representing the distribution of D,-specific binding of [‘H]SCH23390 and D,-specific binding of [3H]raclopride in areas 4, 3, 1 and 2. Conventions as in Fig. 3.

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of [3H]spiperone using (-)-sulpiride or dopamine as blanks. Another widely used blank, (+)-butaclamol, was found to displace 25-30% of [3H]spiperone binding and the non-specific binding appeared to be homogeneous. Accordingly, we used (+)-butaclamol as a blank to compare the binding patterns of [3H]raclopride and [3H]spiperone (the latter in the presence of ketanserin) in the monkey prefrontal cortex. In this cortical region, the [3H]raclopride- and [3H]spiperone-specific sites had very different densities and distributions (Figs 2c,d and 3). Thus, [3U]spiperone binding density was much higher than that of [3H]raclopride (Figs 2c,d and 3). In addition, while [3H]raclopride labeled largely cortical layer V, the highest density of [3H]spiperone binding was found in

Effect o f rnianserin on [3H]SCH23390 binding

In this study, we routinely added mianserin to the [3H]SCH23390 incubation buffer to prevent radioligand binding to 5-HT 2 and 5-HTIc sites. In order to see how this affected [3H]SCH23390 binding, some sections of the prefrontal cortex were labeled with this radioligand in the absence of mianserin. While we found no change in the non-specific binding, there was a 10-50% increase in the total and specific binding

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Dopaminergic

receptors

of [3H]SCH23390 (Fig. 3). The removal of mianserin from incubation media also produced a somewhat different pattern of cortical labeling (Figs 2a,b and 3). Thus, in areas 46, 9 and 12, the highest binding density in layers I, II, IIIa, V and VI was replaced by another pattern in which the highest density of labeling was in layers I, II, IIIa, IIIb and V (Figs 2a,b and 3). The most dramatic change was found in area 25. In the absence of mianserin, the bilaminar pattern of labeling in this area became a single band of relatively dense binding which occupied layers I-V (Figs 2a,b). DISCUSSION

Detection of dopamine D, sites: differences [3Hlraclopride and [’ Hlspiperone binding

in

Based on our recent pharmacological characterization of D, receptor sites in cortical homogenateq3’ we used a highly selective antagonist, [3Hlraclopride, for in vitro autoradiographic labeling of these sites in monkey cerebral cortex. Our previous study had indicated that, in the cortex, the specific binding is

in neocortex

665

higher than non-specific only within a limited range of [3H]raclopride concentrations,37 suggesting to us that the failure of some previous studies to autoradiographically detect this ligand in monkey neocortex32 could be related to the use of [3H]raclopride concentrations largely outside the range of relatively low non-specific binding. Indeed, in the present study, we showed that by using concentrations of [3H]raclopride within the range of 0.3-3.0 nM it is possible to detect the laminar distribution of D,-specific binding in the monkey cerebral cortex. While some binding is present in all cortical layers, the most dense labeling was in layer V in all areas studied. This finding stands in contrast to the previous reports of a homogeneous distribution of D2 sites in monkey cerebral cortex obtained with [’ H]spiperone.49 The high affinity of [‘Hlspiperone for D,-receptors and widespread use of this ligand for detection of such sites (for review see Ref. 47) has prompted us to compare [3Hlraclopride and [3Hlspiperone binding. As is customary, [3H]spiperone binding was conducted in the presence of ketanserin to prevent The specificity of binding to 5-HT, sites. 7~1120~43~47~49

Fig. 7. Typical labeling patterns of [)H]SCH23390 in the presence of mianserin (A) and [‘Hlraclopride (B) in areas 17 and 18 of occipital cortex. Boxes mark the sites where quantitative measurement of radioligand distribution was made. (a) Area 18; (b) area 17. Due to differential absorption of 3H-generated emission by sublayer IVb of area 17 the optical densities corresponding to this sublayer on autoradiograms do not represent the true concentrations of the ligandsJ6

666

M.S. LIDOWet al.

binding to D 2 receptors was further assured by the use of (-)-sulpiride or dopamine as a blank. 7,u,43,49 We found that under these conditions the nonspecific binding was very high and precluded the reliable detection of D2-specific binding. Similarly high non-specific binding (up to 90% of the total binding) has also been reported by Richfield et al., ~9 who employed the same binding conditions in their autoradiographic study of D2 sites in monkey neocortex. The specificity of the assay is further complicated by the fact that (-)-sulpiride does not have a sufficient safety margin between the concentration needed to block D 2 receptors and the one at which non-dopaminergic inhibition of [3H]spiperone binding occurs. 52(+)-Butaclamol has also often been used

as the blank. ~9'2°,3s'54Using this compound, we found that the density and pattern of specific [3H]spiperone binding in prefrontal cortex was markedly different from that of [3H]raclopride binding. While there was some increase of binding density in layer V, corresponding to that found with [3H]raclopride binding, the highest density of [3H]spiperone binding was in the superficial cortical strata. This should not be surprising, since our previous studies of cerebral cortex homogenates 37showed that, under these assay conditions, only a fraction of specific [3H]spiperone binding consists of D 2 sites displaceable with raclopride. A substantial portion of binding is to other, most likely ~t2-adrenergic and 5-HT~A serotoninergic sites. Indeed, the superficial cortical layers heavily

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Dopaminergic receptors in neocortex

labeled with [3H]spiperone are rich with c+ adrenergic and S-HT,* serotoninergic sites.26,29,3*,48 The present results lead us to conclude that [3H]spiperone is not a ligand of choice for labeling D2 receptors in the cerebral cortex. Dopamine receptor-specifk

binding of [3HlSCH23390

The radioligand, [3HlSCH23390, and its analogs, [‘*‘I]SCH23982 and rHlSKF83566, have been widely used for labeling of D, receptors in the brain (for review see Ref. 47). Based on these ligands, the laminar distribution of D, receptors has been reported in rat,7,16*18,49 cat,49 monkey26*38149 and human neocortex.‘7*‘9s20 However, the question of the laminar distribution of D, sites in all of these species is far from being resolved. This situation is most likely due to variations in the level of serotoninergic binding of these ligands. For example, Boyson et aZ.’ and Dawson et aLI6 reported the highest density of labeling in layer V of rat prefrontal cortex. However, [3H]SCH23390 (or its analog SKF83566) binding to serotoninergic sites was not blocked in either study. When Dawson et al.‘* subsequently labeled the same cortical region with [‘25IlSCH23982 (which has pharmacological properties similar to SCH23390@) in the presence of ketanserin, thus blocking 5-HT, sites, they found the highest density of binding in layers IV, V and VI; however, 5-HT,, receptors were not blocked. Finally, Richfield et a1.49used cis-flupentixol as a blank and found the highest density of D, receptors in layers V and VI. In this case, the use of cis-flupentixol as a blank allowed detection of pure D,-specific binding of [3H]SCH23390, but nonspecific binding was high, sometimes up to 75% of the total binding. In the present study, we found that the failure to block 5-HT, and 5-HT,, serotonergic sites alters the density and the distribution of the [3H]SCH23390 specific binding in the cerebral cortex. Moreover, the binding pattern of [3H]SCH23390 in the absence of mianserin (which blocks radioligand binding to both 5-HT, and 5-HT,, sites) closely resembles the combined distributions of D,, 5-HT, and 5-HT,c sites.26,30v39 Thus, the blockade of [3H]SCH23390 binding to serotoninergic sites is necessary for more accurate discrimination of D,-specific labeling in macaque cerebral cortex. Cortical distribution of D, receptors

The highest density of D,-specific [3H]raclopride binding was consistently found in layer V of frontal, parietal and occipital cortex. This distribution is interesting for several reasons. First, this is a unique distribution not expressed by any other receptor so far studied in monkey neocortex, including tl, , u2, B, and ‘fir adenergic, 5-HT, and 5-HT,, serotoninergic, M, and M, cholinergic, D, dopaminergic, GABA, and benzodiazepine receptors.26~3~~” The highest density of D, receptors in layer V was particularly

667

unexpected in primary motor cortex where all other receptor subtypes are concentrated in the upper cortical strata.31138Also unusual is the similarity in the pattern of distribution between different cortical areas. Virtually every other neurotransmitter receptor subtype displays some variation in distribution between cortical areas with sharp cytoarchitectonic differences.26,3b40,48 Layer V contains predominantly soma of pyramidal cells which project to the basal ganglia, the tecturn and the spinal cord (for review see Ref. 23) and it is reasonable to assume that the D2 receptors are largely concentrated on these soma. This suggests an involvement of the dopaminergic system in the regulation of cortical output including the direct regulation of primary motor neurons. In light of this preferential distribution, it is understandable that disturbances in the cortical dopaminergic system have such a profound effect on motor activity in monkey.‘3,50 Comparison

of [3Hlraclopride

and [3HlSCH23390

binding

The D, receptor is clearly the predominant dopaminergic receptor subtype in the primate cerebral cortex. Thus even in layer V, where D, sites are most abundant, the density of D, sites is nearly 10 times higher. Furthermore, the laminar distribution of D,specific binding of [3H]SCH23390 is very different from that of [3H]raclopride. The predominant pattern of [3H]SCH23390 binding is bilaminar with the densest binding in the most superficial cortical layers and equal or slightly less dense in the deep cortical strata. This distribution is not uncommon among neurotransmitter subtypes in monkey cerebral cortex.26.3w,48 Moreover, in contrast to [‘Hlraclopride (but similar to the majority of other receptor subtypes) this radioligand changes its distribution in primary motor and visual cortex. In the motor cortex, [‘H]SCH23390 has the highest density in superlicial cortical layers, thus conforming to the general “motor” distribution that is characteristic for the majority of receptors in this cytoarchitectonic area.38 In the primary visual cortex, [3H]SCH23390 has a trilaminar pattern. Thus, in contrast to D, receptors, the distribution of D, sites is more typical of the distribution of neurotransmitter receptors in monkey neocortex. The different laminar distribution of receptor subtypes serving the same neurotransmitter is a consistent finding in monkey cerebral cortex.26,38-40,48 The differential distribution of D, and D, receptor subtypes indicates that they may serve different functions. Indeed, recent studies indicate that cortical D, and D, receptors are involved in different aspects of cognitive functions.33v51

66X

M. S. Linow er (11.

As we already speculated, D, receptors are probably situated on soma of pyramidal cells of layer V. It has recently been found that a subset of pyramidal cells of layer V in monkey cortex are rich in cyclic AMP-regulated neuronal phosphoproteins associated with dopaminoceptive neurons containing D, receptors.5 Thus, in spite of the differences in laminar distribution and involvement in different aspects of cortical functions, it is possible that both D, and D, sites are situated on the very same pyramidal cells (for discussion see Ref. 26). The functional implication of this is that dopamine activation of different receptor subtypes on different cortical cells as well as on the same neurons can differentially influence neuronal activity and behavior. Species-specl$c d#erences A comparison of the distributions of dopaminergic receptor subtypes in rhesus monkey found in this study with those reported in the literature for other species is complicated by the fact that in the majority of studies the specificity of the binding is questionable.37 For example, the laminar distribution of D,-specific [‘Hlraclopride binding in monkey cerebral cortex is different from the rather homogeneous distribution of cortical D, sites reported in [3H]spiperone studies of rat cerebral cortex.7,4y In contrast, when the highly specific antagonist [‘251]iodosulpiride was used to label D, receptors in rat cerebral cortex,45 the highest density of binding sites was found in layer V, which is similar to that reported here for monkey. The labeling of D, receptors in human neocortex has been carried out only with [3H]spiperone or agonists such as [3H]CV205-502. ” 12,43,47 Labeling of D, sites with agonists is as problematic as with [3H]spiperone, given that the specificity of agonist binding strongly depends on assay conditions.2,‘2 Agonists are prone to bind to catechol recognition sites as well as other non-specific sites2.12 Using the above-mentioned radioligands, Camps et al.” reported the highest density of D, sites to be in the most superficial layers of human cerebral cortex. This distribution does not match that found for D, receptors in the present study of monkey neocortex. It would be informative to study D, receptors in human cerebral cortex using selective antagonists of the benzomide group, such as [3Hlraclopride or [125fliodosulpiride. These studies could show if the distribution of D, sites is conserved in evolution, or whether the unprecedented expansion and differentiation of human neocortex is accompanied also by a reorganization in the distribution of this receptor subtype. None of the studies in rat cortex7~‘6~‘8,49have reported the bilaminar pattern of D,-specific binding of [3H]SCH23390 observed in the present study of monkey cerebral cortex. In contrast, the distribution of D, receptors in the cat cerebral cortex determined with [‘H]SCH23390 and cis-flupentixol as a b1ank49 is similar to that which we found in monkey. In

comparable studies of human cerebral cortex. Cortes et 01.‘~ found the distribution of D, sites in primary motor and posterior parietal cortex similar to that found in the present study in comparable areas of monkey cerebral cortex. However, this was not the case for all cytoarchitectonic areas examined. Thus, in humans, a high density of D, has been reported in layer I of the somatosensory cortex, layers l-111, V and VI of primary visual cortexIs and layer V of Brodmann’s area 9 of prefrontal cortex.” Since the binding of D, receptors in human and monkey was done under different assay conditions, the distributions of D, receptors in these two species should be compared with caution. Nevertheless, the review of the literature indicates that the differences between D, receptor distributions in monkey and human neocortex are much smaller than those found between distributions of these sites in primates and rodents. Dopaminergic receptors and innervation While [3HlSCH23390 and [‘Hlraclopride have different laminar distributions, they both show a rostrocaudal density gradient having the highest overall concentration in the prefrontal cortex and lowest concentration in the occipital cortex. The rostrocaudal density gradient of dopaminergic receptors closely parallels the reported concentration gradient of dopamine itself.” This, however, does not correspond with area1 densities of dopaminergic innervation defined by tyrosine hydroxylase (TH) immunoreactivity35 or by [‘Hldopamine uptake,4 which display the highest density in the motor cortex and the lowest in somatosensory and visual cortex. The prefrontal and posterior parietal regions contain intermediate densities of innervation. The correspondence between the laminar distribution of dopamine receptors and dopaminergic innervation largely depends on the way the innervation is defined. The greatest difference appears to be between the distribution of dopamine receptors and innervation defined by dopamine uptake sites.3.4 In the motor cortex, these uptake sites are concentrated in layers I, IIIa and V while in the primary visual cortex they are found predominantly in layer 1. This does not correlate precisely with the distribution of dopaminergic receptors in these areas. In the prefrontal and parietal cortex, both the dopamine uptake sites and the combined distribution of dopamine receptor subtypes have a bilaminar distribution with high densities in layer I and layers V and VI. The match, however, is not perfect since a relatively high density of dopamine receptors can also be found in layers II and IIIa, which are poor in dopamine uptake sites. When the dopaminergic innervation is defined by TH immunoreactivity,3s the labeled fibers are homogeneously distributed across all layers of the motor cortex, most prominent in layer I of primary visual cortex, layers I and IV of preastriate and layers I, V and VI of somatosensory cortex. None of these match exactly the distribution of dopaminergic

Dopaminergic receptors in neocortex receptors in these regions. On the other hand, the laminar organization of the immunoreactivity in the prefrontal and posterior parietal region is very similar to that of the combined distribution of both subtypes of dopamine receptors. Multiple reasons have been offered for mismatches between distributions of receptors and their neurotransmitter-specific innervation.27v34 Although not ruling out other explanations, we feel that in many cases the mismatch is the result of failure to reliably label an entire population of receptor sites as well as deficiencies in defining neurotransmitter-specific axonal terminals. In the case of the dopaminergic innervation, both TH immunoreactivity and dopamine uptake are capable of labeling non-dopaminergic fibers, and while investigators using these methodologies made efforts to minimize unwanted labeling, the contamination of results cannot be totally ruled out (for discussion see Ref. 4). Recently, the dopaminergic innervation of monkey neocortex has been defined with monoclonal antibodies directed against dopamine itself.25.56We have found that the distribution of dopaminergic terminals labeled in this way matches precisely the

669

distribution of dopaminergic receptors in virtually all areas of the neocortex that have been examined to date. It has been suggested that the distribution of the dopaminergic innervation in the cortex correlates with the distribution of D, rather than D, receptors.‘6,49 In contrast, our results indicate that the distribution of dopaminergic terminals56 corresponds much better with the combined distribution of these two receptor subtypes. For example, in primary motor cortex, the highest density of D, receptors was found in layers I, II and IIIa, while D, receptors are largely concentrated in layer V. Thus, the combined distribution of dopaminergic receptors in this area is bilaminar with the superficial laminae having a higher receptor density than the deep laminae. The middle cortical strata have the lowest receptor densities. This is also the distribution of axonal endings labeled by dopamine-specific antibodies in this area.56

Acknowledgement-This work was supported from the U.S. Public Health Service.

by grants

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