The Journal of Neuroecience, July 1990, R?(7): 2125-2138
Overlap of Dopaminergic, Adrenergic, and Serotoninergic Receptors and Complementarity of Their Subtypes in Primate Prefrontal Cortex
P. S. Goldman-Rakic,’ M. S. Lidow,’ and D. W. Gallager*
‘Section of Neuroanatomy and *Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06510
Quantitative in vitro autoradiography was used to determine portions of the same cell. Understanding the anatomical ar- and compare the areal and laminar distribution of the major rangement of receptors within the cortical layers may aid in dopaminergic, adrenergic, and serotonergic neurotransmit- the analysis of monoaminergic modulation of higher cortical ter receptors in 4 cytoarchitectonic regions of the prefrontal function. cortex (Walker’s areas 12, 46, 9, and 25) in adult rhesus monkeys. The selective ligands, 3H-SCH-23390, 3H-raclo- The prefrontal cortex of primates is widely acknowledgedto pride, 3H-prazosin, and 3H-clonidine were used to label the play an essentialrole in the regulation of behavior by goals, D, and D, dopamine receptor subtypes and the (Y,- and (Y*- ideas,and expectations (for review, seeGoldman-Rakic, 1987). adrenergic receptors, respectively, while iZ51-iodopindolol was Modern neuroanatomical tracing techniqueshave provided un- used to detect B-adrenergic receptors. The radioligands, paralleled information about the input-output relationshipsof 3H-5-hydroxytryptamine and 3H-ketanserin labeled, respec- this area and, together with fluorescencehistochemical, auto- tively, the 5-Hi, and 5-HT, receptors. Densitometry was per- radiographic, and immunohistochemical studies,have enabled formed on all cortical layers and sublayers for each of the precise localization of its major neurotransmitters and neu- 7 ligands to allow quantitative as well as qualitative com- reactive peptides (Levitt et al., 1984; Berger et al., 1986, 1988; parison among them in each cytoarchitectonic area. Lewis et al., 1986, 1987, 1988; Schwartz and Goldman-Rakic, Although each monoamine receptor was distributed in a 1988). The distribution of tyrosine hydroxylase (TH), the rate- distinctive laminar-specific pattern that was remarkably sim- limiting enzyme in catecholamine biosynthesis is denserin the ilar from area to area, there was considerable overlap among dorsomedial and ventrolateral prefrontal areascompared with the dopaminergic, adrenergic, and serotoninergic receptors, the principal sulcalcortex (Lewis et al., 1987; Bergeret al., 1988). while subtypes of the same receptor class tended to have Further, the TH-positive arborizations in these areas have a complementary laminar profiles and different concentra- bilaminar distribution with particularly high concentrations in tions. Thus, the D, dopamine, the QI,- and a,-adrenergic, and layer I (Berger et al., 1988; Lewis et al., 1988). Noradrenergic the 5-HT, receptors were present in highest relative con- projections to prefrontal areasalso have a bilaminar distribution centration in superficial layers I, II, and llla (the “S” group). (Levitt et al., 1984), with highest concentrations in the infra- In contrast, the @,- and &,-adrenergic subtypes and the granular layers (Morrison et al., 1982; Lewis et al., 1986; Lewis 5-HT, receptor had their highest concentrations in the inter- and Morrison, 1989). In contrast, serotoninergicfibers are more mediate layers, lllb and IV (the “I” group), while the D, re- concentrated in layer IV and in deep layer III than in other ceptor was distinguished by relatively high concentrations layers (Lewis et al., 1985, 1986). The laminar profiles of some in the deep layer V compared to all other layers (the “D” of the monoaminetransmitters may be distinctive for prefrontal class). Consequently, clear laminar differences were ob- cortex sincethe distribution ofTH-immunoreactive fibers(Lew- served in the D, vs D, dopaminergic, the a- vs @-adrenergic, is et al., 1986, 1987, 1988) and of labeling due to high-affinity and the 5-HT, vs 5-HT, serotoninergic receptor subtypes in uptake of 3H-dopamine in the prefrontal cortex (Berger et al., all 4 areas examined. 1986, 1988) differ both qualitatively and quantitatively from The anatomical overlap of different monoaminergic re- that in the cingulate gyrus or motor cortex (Bergeret al., 1986, ceptors in the same cortical strata suggests that there may 1988; Lewis et al., 1986, 1987). be families of receptors linked by localization on common In contrast to the relatively broad store of information on the targets, while the complementary laminar distribution of the monoaminergic innervation in the macaque prefrontal cortex, D, vs D,, the 5-HT, vs 5-HT, and the (Y- vs @-adrenergic knowledgeabout the monoaminereceptors which are the targets receptors raises the possibility that different subtypes within of thesemodulatory neurotransmittersis more limited. Detailed a given class may have distinctive actions in cortex by virtue information on this subject would be useful in specifying more of their localization on different cells or possibly different precisely the postsynaptic targets of various classesof brain- stem projections to this part of the primate cortex, as well as for providing much needed information about the potential Received Nov. 6, 1989; revised Jan. 11, 1990; accepted Jan. 25, 1990. neural sitesof action of psychoactive drugs, including the neu- This research was supported by grants from the USPHS. Correspondence should be addressed to P. S. Goldman-Rakic, Section of Neu- roleptic medications. Accordingly, the present study employed roanatomy, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06510. in vitro autoradiographic techniques (Kuhar et al., 1986; Pala- copyright 0 1990 Society for Neuroscience 0270-6474/90/072125-14$03.00/O cios et al., 198 1) and quantitative densitometry to examine the 2126 Goldman-Rakic et al. * Monoaminergic Receptors in Prefrontal Cortex
80
60
1 2 3 4 5 Log zinterol concentrations (nM)
B / Figure 2. Inhibition of 1251-iodopindolol binding with the &-selective agonist, zinterol in layer II of area 9 in one of the monkeys. The line is the computer-generated best fit to a 2-site model. In this case, p, re- ceptors (low-affinity sites; K, = 2.0 pm) constituted 66.3% and & re- ceptors (high-affinity sites; K, = 0.04 pm) constituted 33.7% ofthe entire population of fl-adrenergic receptors.
very samecytoarchitectonic areasof the sameanimals provides an opportunity for a comprehensive view of monoamine re- ceptor allocation in prefrontal cortex. Materials and Methods Tissuepreparation. Data on receptor binding were obtained from 4 adult (2 females, 2 males) rhesus monkeys (Macaca mulatta). Prefrontal cor- tex from each animal was sectioned at 20 pm and assayed independently within 1 or 2 months of each other. The monkeys were anesthetized with Na-pentobarbital(40 mg/kg) and perfused with ice-cold PBS (pH 7.4, 1.25 liters) followed by 1 liter of 0.1% paraformaldehyde containing increasing concentrations of sucrose: 500 ml 0% sucrose; 500 ml 5% sucrose; 1 liter 10% sucrose; 500 ml 15% sucrose; 1 liter 20% sucrose. We have determined that this fixation protocol enhances tissue pres- ervation without measurably decreasing binding or altering kinetic con- stants. The brains were rapidly removed, blocked, and immersed in isopentane at -30°C for approximately 5 min before storing at -40°C until use. For autoradiography, 20 pm sections were processed according to individual assay procedures. In each case, tissue sections were cut not more than 2 weeks prior to assay, mounted on acid-cleaned chrom alum-coated slides, and stored at -80°C until the time of assay. Binding assays. The comparative analysis of the monoamine recep- tors in this study was confined to well-characterized ligands which rec- ognize monoaminergic receptor subtypes and, if possible, label the entire subtype. As the binding procedures used have been described previously Figure I. Schematic drawing showing the location of Walker’s area 9, in Rakic et al. (1988) and Lidow et al. (1989a), they are summarized 46, 12, and 25 examined in the present study. PS, principal sulcus; LO, for each ligand only briefly here and in Table 1. Dopamine D, receptors lateral orbital sulcus; MO, medial orbital sulcus; AS, arcuate sulcus; CS, were labeled with the antagonist, 3H-SCH23390 (Faim et al., 1985; cingulate sulcus; RS, rostra1 sulcus. Cytoarchitectonic areas are num- Boyson et al., 1986; Dawson et al., 1986, 1987), and D, receptors were bered according to Walker (1940). labeled with the antagonist ‘H-raclopride (Kohler and Radesater, 1986; Lidow et al., 1989d). The ‘H-SCH23390 binding assay was carried out in the presence of mianserin to prevent its binding to 5-HT, (Bischoff distribution of 7 major monoamine receptor subtypesin 4 dis- et al., 1986) and 5-HT,, sites (Nicklaus et al., 1988). ol,-Adrenergic receptors were labeled with the antagonist JH-prazosin tinct prefrontal areas of rhesusmonkeys. While (Y- and p-ad- (Rainbow and Biegon, 1983; Rakic et al., 1988); high-affinity q-adrener- renergic-specific and 5-HT, and 5-HT, serotonin-specific li- gic receptors were labeled with the partial agonist, 3H-clonidine (Young gandsand their appropriate binding conditions have been known and Kuhar, 1980; Rakic et al., 1988) and the P-adrenergic receptors for some time (seeMaterials and Methods), the recent devel- were labeled with the antagonist ‘251-iodopindolol (Rainbow et al., 1984; opment of the dopamine-specific ligands, ‘H-SCH23390 and Aoki et al., 1986; Reznikoff et al., 1986; Rakic et al., 1988). In the case of ‘2SI-iodopindolol, the use of isoproteronol as the blanking agent as- 3H-raclopride make it possibleto identify the D, and D, recep- sured that 5-HT, binding was not included in specific binding (Rainbow tors without ambiguity for the first time. Finally, comparison et al., 1984). The relative percentage of @, and p2 receptors was deter- of dopaminergic, adrenergic, and serotonergic receptors in the mined in experiments in which the ability of various concentrations of The Journal of Neuroscience, July 1990, IO(7) 2127
Figure 3. Autoradiographs of coronal sections cut through the prefrontal cortex. A, Labeling with 3H-SCH23390 in the presence of mianserin. B, Labeling with ‘H-raclopride. Abbreviations as in Figure 1. the & selective agonist zinterol (Minneman et al., 1979) to inhibit the film in this study were between 0.08 and 0.80 (diffuse optical density). specific binding of 1251-iodopindolol was measured. For this purpose, In this range, they are linearly related to variations in tissue radioactivity tissue sections were incubated for 70 min with 150 PM ‘251-iodopindolol (Geary et al., 1985). in the presence of 10 nM-100 mM zinterol. GTP, 100 mM, was added The distribution of radioligands was examined in 4 subdivisions of to the incubation buffer (Table 1) in order to reduce agonist-specific prefrontal cortex following the cytoarchitectonic map of Walker (1940): negative cooperativity. the inferior frontal gyrus or convexity that extends from the lateral Serotonin 5-HT-1 receptors were labeled with the agonist 3H-5-HT orbital sulcus to beneath the principal sulcus (Walker’s area 12); the (Pazos and Palacios, 1985; Hoyer et al., 1986; Lidow et al., 1989c), and banks and depths of the principal sulcus (area 46); the dorsomedial 5-HTz receptors were labeled with the antagonist ‘H-ketanserin (Pazos cortex lying between the principal sulcus and longitudinal fissure (area et al.. 1985: Hover et al.. 1986: Lidow et al.. 1989~). The incubation 9); and the cortex of the medial wall anterior to the genu of the corpus buffer for )H-5HT binding contained fluoxetine to prevent 3H-5-HT callosum (area 25) (Fig. 1). Comparable regions were sampled from each binding to serotonin uptake sites (Fuller, 1985) and pargyline to prevent animal, and the cytoarchitectonic area from which the reading was taken the oxidation of the ligand by monoamine oxidase present in the tissue was confirmed by analysis of Nissl-stained sections. Sections in which (Fuller, 1985). Incubation of tissue sections with 3H-ketanserin was the plane of section through the cortex was tangential rather than per- conducted in the presence of prazosin to prevent its binding to (Y, re- pendicular were excluded from analysis. ceptors (Leysen et al., 1982). In addition, our use of methysergide as Statistzcal analysis. Analysis of saturation binding was performed the blank assured that the ligand did not bind to 01~,histamine (Leysen using the nonlinear curve-fitting computer prograinS BINKIN~ and et al., 1982), or a unique ketanserin site associated with dopamine nerve FITFUNCTION, which were accessed through the NIH-sponsored PROPHET terminals (Leysen et al., 1987). computer network. The analysis was based on ligand-specific binding The binding assays of 3H-raclopride, )H-prazosin, ‘H-clonidine, and obtained with 5 concentrations of free ligand in incubating solutions. ‘H-ketanserin included preincubation to eliminate endogenous ligands We have previously established that 5 concentrations is the minimum (see Table 1). number needed to obtain a relatively accurate estimate of B,,, and Kd Quantitative densitometry. The density of receptor binding in cortical for a l-site receptor model (Lidow et al., 1989~). Twelve replications areas and layers was assessed using an image-analysis system consisting of total binding (3 replicates/animal) and 4 replications of blanks (l/ of a DAGE-MT1 series 68 video camera and video signal digitizing animal) were measured for each concentration of free ligand. B,,, and circuitry interfaced with a Digital VT100 computer. This computer- Kd values obtained from different layers of each neocortical area of each imaging system allows the overlay on a TV monitor of the digitized animal were compared with the GT2 modification of Gabriel (1978). images of cresyl violet-stained sections and corresponding autoradi- The latter is a conservative a posteriori multiple-comparison method ograms in order to histologically identify the autoradiographic image based on analysis of variance that allowed us to compare each layer of each cortical layer and sublayer. The system is also capable of sub- with each other layer within and between cortical areas. Using this traction of the film images of sections representing nondisplaceable, method, 95% confidence intervals were calculated for each Kd or B,,,, hence nonspecific, binding from film images of adjacent sections with and these values were plotted graphically on histograms in order to total binding, thus allowing direct on-screen observation of the images provide a comprehensive visual display of all possible comparisons representing specific binding. Optical densities were converted to con- within and between areas. Kd or B,,, values with overlapping confidence centration of labeled compounds per tissue wet weight for each cortical intervals are statistically identical; Kd or B,,, values in which intervals layer. The optical densities for all autoradiographs produced on Ultro- do not overlap are considered statistically different. Plots of these com- 2128 Goldman-Rakic et al. - Monoaminergic Receptors in Prefrontal Cortex
Area 46 Area 9 Area 46
25
0 I II llla lllb IV V VI
Area 12 Area 12 Area 25
0 I /I llla lllb IV V VI llla lllb IV V VI 0 I II llla lllb IV V VI
Figure 4. Histograms representing the layer-by-layer distribution of Figure 5. Histograms representing the laminar profile of D,-specific D,-suecific ‘H-SCH22390 labeling in Walker’s areas 46. 9. 12. and 25. 3H-raclopride binding in the 4 prefrontal areas. In contrast to Figure 3, Error bars represent means + SEM. Note that layers I, ‘II,’ and IIIa, as the highest concentrations of D, receptors are found in layer V. Con- well as V and VI, contain the high concentrations of the ligand in all ventions as in Figure 4. areas, while layers IIIb and IV are characterized by a relatively low density of labeling. The Gabriel coefficients for B,,, values are displayed at the top of each histogram; those for Kd values are presented at the bottomof each. In this and all subsequent histograms, Kd or B,,, value with overlapping confidence intervals do not differ significantly; only Results those with nonoverlapping intervals are statistically significant. Each radioligand had a characteristic laminar distribution in the prefrontal cortex with little variation over the 4 cytoarchitec- tonic areas examined, and the Kds for each ligand were statis- tically equivalent in all layers of all 4 areas(see Figs. 4, 5, 7, 8, parison intervals are provided in the histograms of this report (Figs. 4, 10, 11, 13, and 14). Also, nonspecific binding was low and in 5, 7, 8, 10, 11, 13, and 14) to facilitate comnarison of variation in K,Y no caseexceeded 25% of total binding. The specific labeling in or L within each cortical area as well as to identify the layers for areas 12, 46, 9, and 25 is described below both qualitatively which these variations are statistically significant. and quantitatively. Analysis of the inhibition of /3-adrenergic binding of ‘251-iodopindolol by the p-2 specific agonist, zinterol, were performed using the FITCOMP Dopamine D, receptors (3H-SCH23390). In the presenceof program, which is a part of the PROPHET computer network. Sixteen mianserin, the density of 3H-SCH23390 binding sitesin the 6 concentrations of displacing ligand were used in this particular exper- layers of the cortex rangedfrom 23 fmol/mg tissue to 47 fmol/ iment. A typical inhibition curve is presented in Figure 2. The F test, mg tissue. 3H-SCH23990-specificbinding was present in every which compares the sum of the squares of the residuals, showed a statistically significant improvement of fit from a 1-site to a 2-site model layer. However, the labeling was greatest in superficial layers I, (p < 0.05). FITCOMP provided percentages of low-affinity p, and high- II, and IIIa, only somewhatless dense in deep layers V and/or affinity 0, receptor sites and IC,, values for these sites. These nercentaaes VI and least in middle layers IIIb and IV. Also, this ligand was and the B,,, of 1251-iodopindolol for the entire population of@ receptors one of the few examined that had as high or higher density in were used to calculate the concentrations of PI and & receptor sites. layer VI compared to layer V. Little regionalvariation in binding The K,‘s for each receptor subtype were calculated by the method of density was observed in this pattern (Figs. 3A, 4), but area 25 Cheng and Prusoff (1973). The data provided by FITCOMP cannot be used to calculate standard errors of the mean or comparison intervals contained the highest density of binding sites, particularly in for K,‘s. the superficial layers. The Journal of Neuroscience, July 1990, W(7) 2129
Figure 6. Autoradiographsof prefrontalsections labeled with jH-prazosin(A) and 3H-clonidine(B). The complementarynature of the binding patternsin A andB areevident. Abbreviationsas in Figure 1.
DopamineD2 receptors(3H-raclopride). The presenceof D, re- twice that in layer VI in all 4 prefrontal areasexamined. Area ceptors in cerebral cortex has been questioned (for review, see 25 wasthe only area in which there wasany deviation from this DeKeyser et al., 1988a; Lidow et al., 1989d) and, indeed the pattern. In this area, layer II binding was lessthan that of both B,,, values for D, specific binding of 3H-raclopride were ex- adjacent layers and hence disrupted the descendingconcentra- tremely low, ranging from approximately 2 to 3.5 fmol/mg tissue tion gradient acrossthe layers. However, both the density and acrossthe different layers. Nevertheless,a clear and distinctive pattern of ol,-specific labeling was remarkably similar among pattern of labeling resulted: D, specificbinding was consistently areas9, 46, and 12. most concentrated in layer V, while the binding in all other ,&Adrenergic receptors(1251-iodopindolol). Norepinephrine p layers waslower (Figs. 3B, 5). This pattern of stratification, which receptors, as measuredby lZSI-iodopindolol binding, had ex- was amazingly uniform in all 4 prefrontal regions, was unlike tremely low concentrations, ranging from 16 to 25 fmol/mg that observed with any other ligand. tissue. Although present in all layers, this labeling was denser cu,-Adrenergicreceptors (:‘H-prazosin). The density of CY,-spe- in layers IIIa and IIIb than in deeperor more superficial layers cific binding of ‘H-prazosin sites in the cortical layers varied in all areas(Figs. 9, 10). This pattern was particularly charac- from about 50 to 120 fmol/gm tissue.In all 4 prefrontal areas, teristic of the p, subtype which had somewhat higher concen- or,-specificbinding showed a descendingpattern of concentra- trations than the pZsubtype (Figs. 11, 12). The 0, receptor also tion from layer I through layer IV and then a modest increase appearedto be lessevenly distributed acrossthe laminae than again in layers V and VI (areas46 and 12) or mainly in layer its & counterpart. V (areas9 and 25) (Figs. 6A, 7). This pattern wassimilar to that 5-HT, receptor(3H-5-HT). The concentration of specificbind- observedwith the D,-specific ligand, although the concentration ing of )H-5-HT in the prefrontal cortex was among the highest of 01,binding sitesin the deeperlayers waslower relative to that of all radioligandsexamined and had a distinctive layering pat- in superficial strata. Area 25 generally contained the highest tern in which the highest density was found in the superficial absolute binding, which was especially apparent in the supra- layers, I, II, and IIIa, the lowest in the intermediate layers IIIb granular layers. and IV and an intermediate level of binding defined the deepest a,-Adrenergic receptors(jH-clonidine). The a,-specific bind- layers V and VI (Figs. 13, 14). The absolute density of binding ing of 3H-clonidine rangedfrom 22 to 62 fmol/mg tissue (Figs. was remarkably similar from region to region, including area 6B, 8). Unlike the a), subtype, cyZreceptors generally showeda 25. For example, the B,,, values for layers I, II, and IIIa were monotonic pattern of decreasingconcentration from layer I approximately 150 fmol/mg in all 4 areasof cortex examined through layer VI with the density in layer I being more than and half that (75 fmol/mg) in layers IIIb and IV in all 4 areas. 2130 Goldman-Rakic et al. * Monoaminergic Receptors in Prefrontal Cortex
Area 46 Area 9
Area 12 Area 25 Area 12 Area 25
.s 120 120
F 100 100 F 6o 60
;i > 50 50 Jg 80 60 J 5 60 60 5 2 4030 3040
Ei 40 40 20 mE 20 20 10
0 0 cl 0
Figure 7. Histograms of layer-by-layer labeling of q-specific 3H-pra- zosin binding. Note that the ligand is most concentrated in the superficial Figure 8. Histograms of a,-specific ‘H-clonidine binding, again show- laminae and is least concentrated in layer IV in all 4 areas. Conventions ing the highest concentrations to be located in the upper layers, I, II, as in Figure 4. and IIIa in all areas. Conventions as in Figure 4.
5-HT, receptors(jH-ketanserin). In the presence of prazosin An unexpected related finding is that the peak concentrations (Table l), the density of 3H-ketanserinbinding rangedfrom 25 of the different subtypeswithin a given neurotransmitter cate- to 90 fmol/mg tissue and the laminar pattern of binding was gory were in some degree complementary. Clear differences virtually identical in all 4 prefrontal subdivisions. The highest characterized the disposition of the D, versus D, dopaminergic, concentrations were found in layers IIIa, IIIb, and IV, with lower the a- versus /3-adrenergic,and the 5-HT, versus the 5-HT, values in the surrounding layers (Figs. 13, 15). serotonergicreceptors. Finally, a striking feature of the present results is the degree of similarity among different prefrontal Discussion subregions in the laminar distribution and density of each The present analysis of 7 major monoaminergic receptor sites monoaminergicligand examined.A common profile acrossareas in the primate prefrontal cortex has revealed several findings was found for all monoamine receptors examined, although that may have implications for understanding neurochemical some receptors were more concentrated in area 25 compared interactions in normal and diseasedprimate cerebral cortex. with the other prefrontal regions.Each of thesefindings will be First, there is considerable overlap in the location of several discussedin greater detail below. dopaminergic, adrenergic, and serotoninergic receptors in the prefrontal cortex. One grouping, which will be referred to as the Overlapping laminar distribution of receptor subtypes “s” group, was formed by the D,, (Ye,CQ, and 5HT, subtypes A consistent result in the present study is the preferential lo- that were most concentrated in the superficial layers, while the calization and high concentration of the D, dopaminergic re- p,, pz, 5-HT, receptors(the “I” group) had their highestdensity ceptor, and (Y,-and oc,-adrenergicreceptors, and the 5-HT, sero- in the intermediate layers. The D, dopamine receptor subtype toninergic receptor in superficial laminae I, II, and IIIa in all (the “D” class) alone was most concentrated in a deep layer areasstudied. Similar findingshave been obtained in other areas (layer V) and evenly distributed acrossall others. The D, re- of the cortical mantle (Rakic et al., 1988; Lidow et al., 1989a). ceptor could arguably belongto this category asD, -specificbind- It is tempting to speculate that the convergence of different ing was as high or higher in layer VI than in superficial strata receptorsin common cortical strata reflects an associationwith in areas9 and 12. common cortical elements such as the spine-laden, and hence The Journal of Neuroscience, July 1990, W(7) 2131
Area 46 Area 9
Area 12 Area 25
I II llla lllb IV V VI
Figure 9. Autoradiographof 1Z51-iodopindololbinding in the prefron- tal cortex. Abbreviationsas in Figure 1. Figure 10. Histogramof b-specific1251-iodopindolol binding in the various layersof the 4 prefrontalareas. Binding is most concentrated in the intermediatelayers in all areas.Conventions as in Figure4. synapse-dense,apical dendritesof layer III and V corticocortical neurons,whose dendrites ascendvertically through the cortical layers to the upper strata. Spinesare most concentrated on these The unique distribution of the D, receptor in layer V raises apical dendrites (Globus and Scheibel, 1966, 1967; Feldman the possibility of an association with the corticostriatal and and Dowd, 1975; Parnavelas et al., 1977) and, in general,there corticotectal neurons that reside in this layer. We should note, is an exponential increase in the mean spine density with in- however, that becausethe D, receptor density is so low, every creasingdistance from the cell body along the apical dendritic other receptor examined in this study has a concentration in shaft (Valverde, 1967; seeFeldman, 1984, for review). It may layer V higher than that of D,, even though each is more con- be relevant that the spineheadsof pyramidal neurons are a centrated in superficial or intermediate layers. The considerable prominent target. of DA- or TH-positive boutons in the super- convergenceof monoaminergic receptors in the infragranular ficial layers of the macaque prefrontal cortex (Goldman-Rakic layers placesthem all in position to influence, to one degreeor et al., 1989). The superficial cortical layers in prefrontal cortex another, the output and feedback functions thought to be lo- are a prime location for integrative functions as they receive calized in theselayers. The further dissection of the functional extensive callosal and ipsilateral corticocortical projections contribution of monoaminereceptors in cortex should be aided (Goldman-Rakic and Schwartz, 1982). by detailed knowledge of their preciselocation within the cor- The P-adrenergicand 5-HT, receptorswere also clustered in tical mantel. the samelayers, in this case,the intermediate thalamorecipient layers of the cortex. It is possible that these receptors may, be Complementary distribution of receptor subtypes associatedpredominantly with the granule cell or local circuit A remarkable finding in the presentstudy is the complementary neurons in layer IV and/or with pyramidal neurons in layers distribution of subtypes within different neurotransmitter clas- IIIa&b that are the main targets of mediodorsalthalamic .(Gi- sifications. Thus, while D, receptors were concentrated in the guere and Goldman-Rakic, 1988) and corticocortical (Schwartz superficial and to a lesserdegree, the deep strata, D, receptor and Goldman-Rakic, 1984) afferents in primate prefrontal cor- siteswere most prominent in layer V. Likewise, (Y- and p-ad- tex. In addition, the I receptors could also be associatedwith renergic receptor subtypes had their highest concentration in the smooth and/or spinous surfacesof the proximal dendrites, different layers: the a-adrenergic receptors in superficial strata cell soma, and basilar dendrites of the very same pyramidal (layers I, II, and III& the P-adrenergicreceptors in intermediate neuronsthat sendtheir distal processesto the superficial layers. strata (IIIb and IV). Finally, the S-HT, and 5-HT, receptorswere 2132 Goldman-Fiakic et al. * Monoaminergic Receptors in Prefrontal Cortex
Area 46 Area 9 CALCULATED DENSITY OF p2 ADRENERGIC SITES
Area 46 Area 9
Area 2.5
Area 25
0.036 0 036 0.032 0.032 0.037 0.030 Figure II. Histograms representing the distributions of @, receptors 0 033 in prefrontal areas obtained as described in the text. Conventions as in previous histograms. Figure 12. Histograms of & receptors in prefrontal cortex. Conven- tions as in previous histograms.
concentrated in the superficial and intermediate layers, respec- tively. Our quantitative analysispermitted the further charac- few D , receptors. The widespreadand denserdistribution of D , terization that for each “pair” of receptor subtypes,one subtype receptors relative to that of the D, receptors in the primate was always more concentrated than the other; the more con- prefrontal cortex is interesting in light of our recent finding that centrated was in the superficial layers and was invariably more SCH23390 injected into area 46 of rhesusmonkeys produced concentrated even in the deeperlayer of peak concentration of an increasedlatency and decreasedaccuracy of responsein de- its counterpart. This cannot be due to differential subtraction layed-responseperformance, whereasD, antagonistswere with- since in all cases,different ligandswere usedto define subtype out effect in this region (Sawaguchiand Goldman-Rakic, 1989). specific binding. The consistency of this pattern in all 3 neu- However, in the rat, blockade of the D, receptor, but not of the rotransmitter classes-dopaminergic, adrenergic, and seroto- D, receptor, antagonizes dopamine’s inhibitory effects on the ninergic-is notable. One clear functional implication is that spontaneousactivity of prefrontal neurons(Sesack and Bunney, the different receptor subtypesare related to different neuronal 1988). The role of the dopamine receptors in cortical function compartments, either on the same cell (e.g., soma and/or den- appearsto be quite complicated and may even be different in drites vs spines)or to different cells (e.g., projection vs. inter- anesthetized and awake behaving animals or on spontaneous neuron). Different target structures may, in turn, be related to neural activity and integrated behavior; speciesdifferences also dual sourcesof neurotransmitter innervation, as has been de- need to be considered. scribedfor 5-HT, for example (Wilson et al., 1989). Our finding The high concentration of D, -specific 3H-SCH23390binding that dopamine-immunopositive boutons are widely distributed in superficial layers of the macaque cortex found in the present on the spines,dendritic shafts, and soma of the samepyramidal study in fact differs from studiesin other species.Thus, studies neuron (Goldman-Rakic et al., 1989) is compatible with a het- in rat prefrontal cortex show a high density of binding in layer erogeneousdistribution of receptor subtypes at different sites V (Boyson et al., 1986; Dawson et al., 1986) layers V and VI along the neuron and its processes. (Richfield et al., 1989) or layers IV, V, and VI (Dawson et al., 1988). It is likely that the discrepanciesin laminar profiles be- tween the studiesin rodents reflect different degreesof blocking Dopaminergic receptorsin prefrontal cortex serotoninergicbinding. )H-SCH23390 binds to both 5-HT, and Among the neurotransmitter receptorsexamined in the present 5-HT,, sites in addition to D, receptors, and failure to block study, dopamine receptors are of special interest in relation to theseadditional sitessignificantly alters the distribution of bind- the sitesof action of neuroleptic medications in the CNS. The ing by this ligand (M. S. Lidow, personal communication). recent availability of SCH23390 (Hyttel, 1982; Iorio et al., 1983) Nevertheless, none of the binding patterns observed in rat has made it possible to selectively label D, receptors, and using matches the bilaminar distribution of 3H-SCH23390 binding this compound in the present study we have found that D, found in the present study of monkey prefrontal cortex, even receptor sitesare particularly prominent in layers I, II, III (up- when all serotonergic sitesare effectively blocked, as they were per), V, and VI; only layers IV and lower layer III had relatively in one of these studies (Richfield et al., 1989). Thus, species The Journal of Neuroscience, July 1990, iO(7) 2133
Figure 13. Autoradiographs representing 3H-5-HT (A) and ‘H-ketanserin in the presence of prazosin (B) binding in prefrontal areas. Note complementary pattern of binding of the 2 receptor subtypes in all prefrontal areas shown. Abbreviations as in Figure 1. differencescannot be ruled out, particularly asa densedopamine Liskowsi and Potter, 1985; Martres et al., 1985; Boyson et al., innervation of superficial layers in prefrontal cortex appearsto 1986; Camus et al., 1986; Bouthenet et al., 1985; Palaciosand be a primate specialization (Bergeret al., 1988);such differences Pazos, 1987; Stefanini et al., 1987). In the present study of in dopamine input could explain the high concentration of D, primates, we consistently found a low density of D, receptors receptorsin the superficial layers found in this study in monkey in the prefrontal cortex, and similarly low concentrations were compared with comparable analysesin the rat. observed in our recent homogenate binding studies with the )H-SCH23390 binding in cat prefrontal cortex is bilaminar sameD,-specific antagonist(Lidow et al., 1989d).These findings as in the monkey, despite very high (up to 75%) nonspecific concur with an earlier study of rodent cortex using the D,- binding (Richfield et al., 1989). However, Dawsonet al. (1987) specific ligand 3H-iodosulpiride (Martres et al., 1985) and to- reported that ‘the highest density of 3H-SCH23390 D,-specific gether with it, they provide strong evidence that the difficulty binding (determined by using pifluxol as blank) in Brodmann’s in detecting D, receptors in the cortex is due to their low con- area 9 of human prefrontal cortex was in sublayer Va, and no centration combined with the use of nonspecific ligands. evidence of the bilaminar pattern characteristic for monkey pre- The preferential localization of D, receptors in layer V of frontal cortex was observed. Thus, a number of different results primate prefrontal cortex is in agreementwith a study in the has been obtained in several studiesof prefrontal cortex in rat, rat by Martres et al. (1985). However, the relatively homoge- cat, monkey, and human. These differencesmay reflect species neouscortical distribution of D, sitesreported in rat (Boyson, differences, particularly between rodent and primate, as dis- 1986; Richfield et al., 1989)and cat (Richfield et al., 1989)may cussedabove, but a largemeasure of the variability acrossstud- be due to the assayconditions used. The use of 3H-spiperone ies is likely due to the binding assaysused, and specifically to in the presenceof ketaserin (mianserin) using (-)sulpiride or the use of nonspecific ligands. dopamine asblanks createsvery high nonspecificbinding which D,-binding sitesare alsopresent in all layers ofthe 4 prefrontal can reach 90% of the total binding (Richfield et al., 1989), with areasexamined. This bearsemphasis because the very existence attendant problems in evaluating binding data. The high con- of D, receptorsin any cortical area,including prefrontal cortex, centration of D, receptors in layers I and II of human cortex has been questioned(see Fallon and Loughlin, 1987, for recent found with 3H-CV205-502 and 3H-spiperonein the presenceof review). The literature rangesfrom reports of the complete ab- ketanserin (Campset al., 1989) with (+)butaclomol asa blank, senceof cortical D, receptorsin cortex (e.g., Altar et al., 1985; also doesnot conform to our findings. Again, the D, specificity Bouthenet et al., 1985; Gehlert et al., 1985; Dubois et al., 1986; of this labelingpattern can be questionedbecause of the binding Kohler and Radesater, 1986; Charuchinda et al., 1987; De- of both radiolabeled compounds to a-adrenergic and 5-HT,, Keyser et al., 1988) to positive findings (Carboni et al., 1985; sites,in addition to the D, sites(Lidow et al., 1989d). Also, as 2134 Goldman-Rakic et al. * Monoaminergic Receptors in Prefrontal Cortex
SPECIFIC BINDING OF [3H]5-HT SPECIFIC BINDING OF [3H]KETANSERIN TO 5-HT, SEROTONERGIC SITES TO 5-HT, SEROTONERGIC SITES
Area 46 Area 9 Area 46 Area 9
Area 12 Area 25
Figure 15. Laminarhistograms of SHT,-specific3H-ketanserin bind- ing in the 4 prefrontal areasshow the highestdensity present in the intermediatelayers in all 4 areas.Conventions as in Figure4. Figure 14. Histogramsof 4 prefrontalareas showing the concentration of 5-HT- 1 specificbinding )H-5-HT in the variouslayers. Note the high concentrationsin layersI, II, and IIIa in all areas.Conventions as in binding is highest in layer V rather than in superficial layers Figure4. (Pazos and Palacios, 1985), a finding reminiscent of the D, receptor comparisonbetween rat and monkey describedabove. 3H-CV205-502 is an agonist, the specificity of its binding is In contrast, the highest density 5-HT,-specific binding of 3H- strongly dependent on assayconditions (Camus et al., 1986). ketanserin is consistently found in the middle cortical layers in monkey, human (Pazoset al., 1987b)and rat (Pazoset al., 1985). Adrenergic and serotoninergicbinding sites The distribution of adrenergicreceptors also appearsto exhibit Relationship to monoamine innervation speciesdifferences. For example, the highest density of a, re- The specificreceptor patterns revealed in different cortical areas ceptors in rat prefrontal cortex defined with lZSI-HEAT (Jones generally appear to correspond closely to the monoaminergic et al., 1985) is found in layers III and IV, while in monkey they innervation of the cortex, although it is not possiblefrom the are highestin the superficial strata but are abundant in all layers present results to determine whether the various receptors are except IV. The distributions of P-adrenergicreceptors in mon- located presynaptically on the afferent terminals or postsynapti- key and rat prefrontal cortex found with lZSI-iodopindololalso tally on cortical neurons.TH immunohistochemistry (Lewis et do not correspond. Thus, the rat hasa homogenousdistribution al., 1988), dopamine immunohistochemistry (Goldman-Rakic of /3 sitesacross all cortical layers (Rainbow et al., 1984), while et al., 1989; Williams et al., 1989), and autoradiography of 3H- in monkey layers III and IV have a higher receptor density than dopamine uptake (Berger et al., 1986, 1988) all reveal a basic other layers. bilaminar pattern of fibers with a high density of fibers in su- Our data on 5-HT,-specific binding of 3H-5-HT in monkey perficial strata and another band in layers V/VI in several areas are, however, in accord with findings in human prefrontal cortex of prefrontal cortex. The bilamination of the dopamine inner- (Biegon et al., 1986; Pazoset al., 1987a). In all cases,the highest vation thus correspondsto our observations on the relatively binding is found in the most superficial layers. Again, however, high concentrations of D,-binding sitesin the superficial layers there is a difference with rat prefrontal cortex, where 3H-5-HT and D, and D, sites m the deep layers of prefrontal areas as The Journal of Neuroscience, July 1990, IO(7) 2135
Table 1. Protocols of saturation autoradiographic assays
Site Cont. Blank Exposure Ligand labeled CUM) GM) Protocol Buffer Time Temn. time dopamine l-10 SKF83566 incubation 0.05M Tris- 90 min room to 3 month JA (1) HCl (pH 7.4), 120mM NaCl, 5mM KCl, 2mM rinse CaCl,, lrn~ 2 x 10 min 4°C MgCl,, 1 .@M mianserin, 0.05h1 Tris-HCl (pH 7.4) dopamine 0.3-3.0 (+)butac- preincu- 50 mM Tris- 20 min room t” 8 month Q lomol(1) bation HCl (pH 7.4) 150mM NaCl, 50mM Tris- HCl (pH 7.4), incubation 150mM NaCl, 45 min room to 5mM KCl, 2mM CaCl,, lmh4 MgCl,, 0.1% rinse ascorbic acid 6 x 1 min 4°C 50mM Tris- HCl (pH 7.4) [?H]prazosin norepi- 0.2-1.0 phenyleph- preincu- 0.17M Tris- 30 min 4°C 3.5 month nephrine rine (100) bation HCl (pH 7.1) aI incubation same buffer 70 min 4°C rinse same buffer 2 x 5 min 4°C [?H]clonidine norepi- 0.5-4.0 norepineph- preincubation 0.17M Tris- 30 min room to 3.5 month nephrine tine (100) HCl (pH 7.6), 012 5mM EDTA, 0.17M Tris- incubation HCl (pH 7.6), 60 min room to 1mM MnClz 0.1% ascorbic acid 0.17~ Tris- rinse HCl (pH 7.6) 2 x 5 min 4°C [1151]iodo- norepi- 0.025- isoproter- incubation 0.02M Tris- 70 min room to 2d pindolol nephrine 0.200 on01 ( 100) HCl (pH 7.4) P 100 pM rinse phentolamine 0.02M Tris- 3 x 20 min 4°C HCl (pH 7.4) [‘HIS-HT serotonin 2-32 serotonin incubation 0.17M Tris- 60 min room to 2.5 month 5-HT, (10) HCl (pH 7.7), 0.4mr.4 CaCl,, 0.1 Yoascorbic rinse acid, ~NM 5 x 1.0 min 4°C fluoxetine 1O~LM pargyline 0.17~ Tris-HCl (pH 7.7) serotonin 0.5-3.5 methyser- preincubation 0.05M tris-HCl (pH 30 min room to 1 month 5-HT, gide (10) 7.7), 0.1 PM prazosin incubation same buffer 0.05~ 20 min room to rinse Tris-HCl (pH 5 x 0.4 min 4°C 7.7) 2136 Goldman-Rakic et al. l Monoaminergic Receptors in Prefrontal Cortex contrasted with the intermediate laminae. Yet the correspon- they are in prefrontal cortex (Lidow et al., 1989a). In addition, dence between dopamine afferents and their putative postsyn- the muscarinic M, receptor is distributed differently in prefron- aptic targets is far from precise. For example, dopamine inner- tal cortex than in many other cortical areas(Lidow et al., 1989b). vation is reported to be densest in deep layer I (Berger et al., Thus, in general, the concept of a “prefrontal” archetype for 1988; Lewis et al., 1988), whereas no marked sublamination is rhesusmonkeys seemsvalid and in accord with other regional evident in our autoradiograms, although D, binding tends to be differencesthat have previously been documented in primary slightly higher in layer I than in II and IIIb. Neither could we and secondary visual areas (Kritzer et al., 1987; Rakic et al., find a tight relationship between the overall concentration of 1988) as well as in primary motor and somatosensorycortices dopaminergic binding sites and the overall density of the do- of rhesusmonkeys (Lidow et al., 1989a). pamine innervation revealed by TH immunohistochemistry in the cytoarchitectonically defined subdivisions of the prefrontal References cortex. Thus, Lewis et al. (1988) found the highestconcentration Altar CA, Kim H, MarshallJF (1985) Computerimaging and analysis of TH-immunoreactive fibers in Walker’s area 9, while the low- of dopamine(D-2) and serotonin(S2) binding sites in rat basalganglia est concentration of these fibers was found in area 46, with or neocortexlabeled by [3H]spiroperidol.J PharmacolExp Ther 233: intermediate concentrations in areas 12 and 25. However, with 527-538. few exceptions, the B,,, values for D, and D, receptor binding Aoki C, KaufmanD, RainbowTC (1986) The ontogenyof the laminar distributionin the visual cortex of cats,normally rearedand dark- in each layer were remarkably similar in all 4 areas. reared.Devel Brain Res27: 109-l 11. The noradrenergic innervation of prefrontal cortex, like the Amsten AFT, Goldman-RakicPS (1985) Alvha-2 adrenernicmech- dopaminergicinnervation, is also reported to be bilaminar (Lev- anismsin prefrontalcortex associatedwith cognitivedecline in aged itt et al., 1984; Lewis et al., 1988) with bands of fibers in nonhumanprimates. Science 230:1273-1276. BergerB, Trottier S, GasparP, Vemey C, Alvarez C (1986) Major superficial and deep strata and, according to a recent immu- dopamineinnervation of the corticalmotor areasin the cynomolgus nohistochemical analysis of dopamine+hydroxylasc (DBH) monkey. A radioautoradiographic study with comparative assessment staining, more concentrated in the deeper, especially layer V, of serotoninergicafferents. Neurosci Lett 72: 12l-l 27. than superficial cortical layers (Lewisand Morrison, 1989). Con- Berger B, Trottier S, Vemey C, Gaspar P, Alvarez C (1988) Regional sequently, a-adrenergic receptorsare densein superficial layers and laminar distribution of dopamine and serotonin innervation in the cynomolgus cerebral cortex. Major differences of dopamine input where the norepinephrine input is weaker, although the mod- in the granular and agranular cortices. A radioautographic study. J erate density of a), receptors present in the deeper layers does Comp Neurol 273:99-l 19. correspond to the preferential adrenergic innervation in this Bicgon A, Kargmar S, Snyder L, McEwen BS (1986) Characterization layer. The discrepancy between DBH immunohistochemistry and localization of serotonin receptors in human brain postmortem. Brain Res 363:91-98. and clonidine receptor autoradiography is interesting in light of Bischoff S, Heinrich M, Sonntag JM, Krauss J (1986) The D-l do- findingsboth in rat (UP&chard et al., 1979) and monkey (Am- pamine receptor antagonist SCH23390 also interacts potently with sten and Goldman-Rakic, 1985), indicating that most of the LYE brain serotonin (5HT-2) receptors. Eur J Pharmacol 129:367-370. receptorsin prefrontal cortex are postsynaptic. Bouthenet ML, Sales N, Schwartz JC (1985) Autoradiographic local- The 5-HT input to the prefrontal cortex has variously been ization of [3H]apomorphine binding sites in rat brain. 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