Glutamate Binding in Primate Brain

Journal of Neuroscience Research 27512-521 (1990)

Quisqualate- and NMDA-Sensitive [3H]Glutamate Binding in Primate Brain

A.B. Young, G.W. Dauth, Z. Hollingsworth, J.B. Penney, K. Kaatz, and S. Gilman Department of Neurology. University of Michigan, Ann Arbor

Excitatory amino acids (EAA) such as glutamate and Young and Fagg, 1990). Because EAA are involved in aspartate are probably the neurotransmitters of a general cellular metabolic functions, they were not orig- majority of mammalian neurons. Only a few previous inally considered to be likcl y neurotransmitter candi- studies have been concerned with the distribution of dates. Electrophysiological studies demonstrated con- the subtypes of EAA receptor binding in the primate vincingly the potency of these substances as depolarizing brain. We examined NMDA- and quisqualate-sensi- agents and eventually discerned specific subtypes of tive [3H]glutamate binding using quantitative autora- EAA receptors that responded preferentially to EAA an- diography in monkey brain (Macaca fascicularis). alogs (Dingledine et al., 1988). Coincident with the elec- The two types of binding were differentially distrib- trophysiological studies, biochemical studies indicated uted. NMDA-sensitive binding was most dense in that EAA were released from slice and synaptosome dentate gyrus of hippocampus, stratum pyramidale preparations in a calcium-dependent fashion and were of hippocampus, and outer layers of cerebral cortex. accumulated in synaptosomc preparations by a high-af- Quisqualate-sensitive binding was most dense in den- finity transport system. With these methodologies, EAA tate gyrus of hippocampus, inner and outer layers of release and uptake were found to be selectively de- cerebral cortex, and molecular layer of cerebellum. creased in the projection regions of a variety of neuronal In caudate nucleus and putamen, quisqualate- and pathways after the pathways were lesioned. Studies us- NMDA-sensitive binding sites were nearly equal in ing immunocytochemical methods to stain conjugated- density. However, in globus pallidus, substantia ni- glutamate or aspartate have also demonstrated the wide gra, and subthalamic nucleus, quisqualate-sensitive distribution of presumed glutamatergic and aspartergic binding was several-fold greater than NMDA-sensi- neurons (Otterson and Storm-Mathisen, 1987; Giuffrida tive binding. In thalamus, r3H]glutamate binding was and Rustioni, 1989; Conti et al., 1988). Initial experi- generally low for both subtypes of binding except for ments in cerebellum indicatcd that the granule cells use the anterior ventral, lateral dorsal, and pulvinar nu- glutamate as a putative neurotransmitter (Young et al., clei. In the brainstem, low levels of binding were 1974). Cortico-cortical association pathways were also found, and strikingly the red nucleus and pons, which linked to glutamate (Fonnum et al., 1981; Barbaresi et are thought to receive glutamatergic projections, had al., 1987). Subsequently, numerous pathways, including approximately 1/20 the binding observed in cerebral afferents, intrinsic neurons, and efferents of the hippo- cortex. campal formation, were found to use EAA as neurotrans- These results demonstrate that NMDA- and quis- mitters (Fagg and Foster, 1983; Fonnum, 1984; Storm- qualate-sensitive [3H]glutamate binding are observed Mathisen, 1977). Cortical inputs to subcortical regions in all regions of primate brain, but that in some regions such as thalamus, caudate/putamen, red nucleus, pons, one subtype predominates over the other. In addition, and spinal cord were shown to use EAA as probable certain areas thought to receive glutamatergic projec- neurotransmitters (Bernays et al., 1988; Christie et al., tions have low levels of both types of binding. 1986; Fosse and Fonnum, 1987; Giuffrida and Rustioni, 1988; Rouzaire-Dubois and Scarnati, 1987a,b; Young et Key words: primate, excitatory amino acid recep- al., 1981, 1983). Most of these pathways have been tors, autoradiography

Received August 13, 1990; revised September 28, 1990; accepted In the last 15 years, excitatory amino acids (EAA) September 28, 1990. such as glutamate and aspartate have been identified as Address reprint requests to Sid Gilman, M.D., Department of Neu- thc probable neurotransmitters of a large percentage of rology, University of Michigan Medical Center, 1500 E. Medical neurons in mammalian brain (Watkins et al., 1990; Ccntcr Drivc, 1914 Taubman Center, Ann Arbor. MI 48109-0316.

G 1990 Wiley-Liss, Inc. Subtypes of EAA Receptor Binding in Primate Brain 513 examined in rodents and cats, but some have been in- pM quisqualate. and 1 mM NMDA or 1 mM glutamate. vestigated in primate (Young et al., 1981 and 1983). After a 45 minute incubation at 4"C, sections were rinsed Electrophysiological studies now indicate the pres- three times with cold buffer and once in cold acetone ence of at least four subtypes of EAA receptors in brain with 2.5% glutaraldehyde (viv) and then rapidly dried. (Watkins et al., 1990; Young and Fagg, 1990). Three of Rinse times were less than 10 seconds. Sections were these receptors are linked to ion channels: the kainate, apposed to tritium-sensitive LKB Ultrofilm 'H for 3 AMPA, and N-methyl-D-aspartate (NMDA) receptors. weeks along with plastic standards from American Ra- The fourth receptor type modulates inositol triphosphate diolabeled Chemical, Inc. that had been calibrated to metabolism and is sensitive to quisqualate (as in the dpm/pg protein in our laboratory. After exposure the AMPA site) but is not particularly sensitive to other films were developed in D- 19 and analyzed for binding selective ion channel-activating AMPA or quisqualate density with computerized densitometry . agonists (Nicoletti et al., 1985; Rescasens et al.. 1987). The BRS2 quantitative imaging program from Im- Thus, quisqualate-sensitive ['H]glutamate binding rep- aging Research, Inc. (St. Catherine, Ontario, Canada) resents binding to both the AMPA and metabotropic was used to digitize the autoradiograms. The program receptors (Cha et al., 1990). Biochemical studies in ho- converted the density readings to dpmipg protein by mogenates and autoradiographic studies on tissue sec- comparison to the standards (Pan et al., 1983). The data tions have demonstrated NMDA-, quisqualate-sensitive from the BRS2 program were then converted to fmol/mg ['Hlglutamate binding sites that have pharmacological protein. properties similar to those of the physiologically defined Five to 20 individual readings were taken from NMDA and quisqualate receptors, respectively (Young each structure bilaterally and from each level at which and Fagg, 1990). the structure appeared. In cortex, readings were taken Only a limited number of studies have been per- from both the outer (1-111) and inner IV-VI) layers. The formed showing the distribution of receptor subtypes in individual readings from each structure along with iden- the primate brain (Geddes et al., 1989; Jansen et al., tifying information were incorporated into a database us- 1989). In this study, we examined the regional distribu- ing the SYSTAT statistical analysis program. All subse- tion of NMDA-sensitive and quisqualate-sensitive quent transformations, merging and averaging of data, [3H]glutamate binding in many regions of monkey brain both within and across subjects, was accomplished with as part of an ongoing series of studies of plasticity of the SYSTAT. A mean value for each structure was com- motor system in monkeys (Dauth et al., 1985; Gilman et puted from the data from duplicate sections. Specific al., 1987; Shimoyama et al., 1988). binding data were calculated as described below. The mean values of the data were then computed by structure across all section levels of the brain for each structure in MATERIALS AND METHODS each animal. The values for each structure from each Four adult Macaca ,fascicularis monkeys weighing animal were used to calculate the means and standard 2.5-3.5 kg were anesthetized with pentobarbital and errors for each structure listed in the tables (Tables I, IT). killed by an intravenous injection of saturated KCI. The The cortical outer layedinner layer (OiI) binding brain was rapidly removed and froLen in isopentane on ratios were calculated from the specific binding under dry ice. Coronal sections of 20 pm were taken serially each displacer condition, i.e., total binding minus bind- from the rostral caudate through the deep cerebellar nu- ing in the presence of 2.5 pM quisqualate and total bind- clei of the brain and assayed for [3H]glutamate binding ing minus binding in the presence of 1 mM NMDA. within 36 hours of sectioning. Two sections were taken Mean values of these data were then computed and tab- for histological analysis every 20 sections. One section ulated as described above (Table 111). was stained with cresyl violet for cell bodies, the other The specific binding for the glutamate subtypes with osmium tetroxide for myelin. Sections for ['HI was calculated from the total binding data in the follow- glutamate autoradiography were taken at the level of the ing manner: rostral caudate, anterior commissure, medial globus pallidus, subthalamic nucleus, ventral anterior thalamic nu- S(NMDA) = B(3H-g1utamate) - b(NMDA) cleus, substantia nigra, and deep cerebellar nuclei. Sec- S(quis) = B(3H-glutamate) - b(quisqua1ate) tions were given three 5 minute prewashes at 4°C in SO mM Tris-HC1 buffer containing 2.5 mM CaCI, at pH where S ( ) = specific subtype binding, B ( ) = total 7.2. Duplicate sections were then incubated with 20 nM binding, b ( ) = nonspecific binding as defined by (3Hlglutamate (specific activity 40 Ciimmol) in 50 mM binding in the presence of 1 mM NMDA (for NMDA- Tris-HC1 buffer with 2.5 mM CaCI, in the presence or sensitive binding) or 2.5 pM quisqualate (for quis- absence of 2.5 pM quisqualate, 1 mM NMDA, both 2.5 qualate-sensitive binding). With these equations, it was 514 Young et al. possible to determine specific NMDA-sensitive and quis- than the NMDA-sensitive binding; and the parvocellular qualate-sensitive glutamate binding. portion of the ventral posteromedial nucleus, which had mainly quisqualate-sensitive binding. The overall density of [3H]glutamate binding was substantially less in RESULTS other thalamic regions than in the cerebral cortex. In NMDA-sensitive and quisqualate-sensitive sites particular, very low densities of binding were found in were differentially distributed (Tables I, 11; Figs. 1, 2). the motor thalamus and the geniculate nuclei. Binding was highest for each of the subtypes in hippo- In the brainstem, binding densities were again low campus and cerebral cortex (Table I). except for the central gray zones, superior colliculus, and NMDA-sensitive binding was most dense in hip- vestibular nuclei. In the central gray, NMDA-sensitive pocampal formation and cerebral cortex. In the hippo- binding predominated. In superior colliculus , quis- campal region, NMDA-sensitive binding was dense in qualate-sensitive binding was more dense. In the granule dentate gyrus, stratum pyramidale of CAI, and entorhi- cell layer of the cerebellum, quisqualate-sensitive bind- nal cortex. In cerebral neocortex, NMDA-sensitive binding was nearly equal to NMDA-sensitive binding. In the ing was highest in the outer layers, with Brodmann areas molecular layer, however, quisqualate-sensitive binding 8, 12, and 6 having the highest binding. Inner layers was four times higher than NMDA-sensitive binding, showed variable densities depending on the region. and quisqualate-sensitive binding in the molecular layer There was an area of decreased apparent binding asso- was twice as dense as that in the granule cell layer. The ciated with the increased white matter in layer IV. A thin deep cerebellar nuclei displayed low binding densities band of higher density NMDA-sensitive binding was ob- for both subtypes. served at the border between layers IV and V. Primary motor cortex, which had no band of increased binding, showed the highest ratio of OiI binding in cerebral cor- DISCUSSION tex. Temporal association cortex (Brodmann areas 21, Several different techniques for measuring NMDA- 22, and 24), entorhinal, and insular cortex showed the and quisqualate-sensitive ['Hlglutamate binding have lowest OiI ratios (Table 111). been reported (Young and Fagg, 1990). Some methods Quisqualate-sensitive binding was most dense in are more selective than others and new assays are being dentate gyrus and stratum pyramidale of CAI in hippo- developed continually. ['HIGlutamate can be used to la- campal formation with intermediate densities in subicu- bel all the EAA receptor subtypes, although the subtypes lum and entorhinal cortex. In cerebral cortex, quis- must be identified by competition studies because gluta- qualate binding was highest in density in outer layers of mate itself does not distinguish between the sites. prefrontal cortex (Brodmann areas 45 and 46) and insular NMDA appears to interact with a homogeneous group of cortex. There was less of a band of increased binding in ['Hlglutamate binding sites whereas quisqualate inter- layer V and the 011 ratios of quisqualate-sensitive bind- acts with receptors linked to ion channels and receptors ing were lower than those for NMDA-sensitive binding. linked to inositol triphosphate metabolism (Monaghan et In motor cortex, the OiI ratios were approximately 1.3- al., 1989; Young and Fagg, 1990). Quisqualate-sensitive 2.3 (compared to 2.2-3.2 for NMDA-sensitive binding). ['Hlglutamate binding is highly chloride sensitive and is The basal ganglia showed marked regional hetero- stimulated at least four-fold in the presence of 40 mM geneity of NMDA-sensitive and quisqualate-sensitive chloride and 2.5 mM calcium chloride (Cha et al., binding (Table 11). In caudate/putamen quisqualate-sen- 1988). In the presence of calcium, chloride, and KSCN, sitive binding was somewhat higher than NMDA-sensi- the quisqualate agonist AMPA will compete for a subset tive binding. Giobus pallidus binding of both types was of quisqualate-sensitive sites, presumably those on the considerably lowcr than caudateiputamen. In external ion channel linked quisqualate receptor (Cha et al., globus pallidus quisqualate-sensitive binding was higher 1988). This so-called "metabotropic" quisqualate recep- than NMDA-sensitive binding by 2: I and in internal glo- tor is insensitive to AMPA (Nicoletti et al., 1985; Palmer bus pallidus by nearly 8: 1. Similar relationships held for et al., 1989; Rescasens et al., 1987). Quisqualate-sensi- the substantia nigra pars reticulata and subthalamic nu- tive ['H]glutamate binding probably measureb binding to cleus. both quisqualate-linked EAA subtypes, i.e., the ion In the thalamus, binding was generally only of low channel and the metabotropic receptor (Young and Fagg, density. The exceptions were the anterior ventral nu- 1990). cleus, which had a NMDA:quisqualate binding ratio of Quisqualate inhibits ['H]glutamate binding in a bi- 2:3; the pulvinar, which displayed a high density of phasic manner with a high-affinity component in the low NMDA-sensitive binding; the lateral and medial dorsal nanomolar range and a low-affinity component in the nuclei, where quisqualate-sensitive binding was denser high micromolar range (Greenamyre et al., 1985; Cha et Subtypes of EAA Receptor Binding in Primale Brain 515

TABLE I. NMDA-Sensitive and Quisqualate-Sensitive ['H]Glutamate Binding in Macaca fascicularis Cerebral Cortex" NR4DA-sensitive, Quisqualate-sensitive, Inner (I) and fmolimg protein, fmolhg protein, Brodmann area outer (0)layers" mean (SEM)~ mean (SEM)" Frontal motor 4 I 108 (26) 132 (19) 4 0 350 (64) 300 (53) 6 I 162 (49) 202 (47) 6 0 405 (58) 298 (52) Supplementary motor I 179 (52) 218 (31) Supplementary motor 0 368 (49) 300 (37) Association 8 I 257 ( 154) 203 (49) 8 0 550 (190) 307 (58) 12 I 198 (10) 249 (32) 12 0 434 (80) 325 (21) 45 I NA 290 (37) 45 0 NA 434 (70) 46 I NA 3 12 (70) 46 0 NA 392 (96) Anterior cingulate 24 I 211 (48) 247 (35) 24 0 324 (65) 302 (44) Insular cortex I 263 (33) 293 (46) Insular cortex 0 387 (43) 344 (44) Parietal sensory 1 &2 I 201 (57) 185 (39) 1 &2 0 351 (91) 246 (59) 3 I 180 (54) 164 (31) 3 0 373 (85) 258 (59) Association 5 I 176 (107) 165 (54) 5 0 346 (171) 275 (85) 7 1 IS4 (94) 182 (58) 7 0 296 (165) 264 (106) 'Temporal Auditory A1 I 151 (43) 161 (43) A1 0 301 (84) 247 (69) Association 21 1 175 (48) 202 (42) 21 0 295 (58) 265 (57) 22 I 209 (37) 254 (43) 22 0 324 (53) 294 (61) Hippocampal region Entorhinal cortex I 155 (52) 218 (46) Entorhinal cortex 0 244 (66) 270 (61) Subiculum 210 (57) 233 (38) Presubiculum 207 (81) 243 (60) Stratum moleculare, CAI 243 (56) 247 (26) Stratum pyramidale, CAI 310 (68) 301 (45) Stratum pyramidale, CA3 228 (46) 229 (27) Stratum molecular dentate gyms 382 (87) 363 (55)

*Values are the mean (SEM) of 4 monkeys. NA = not available. "Inner layers (I) were layers IV-VI, outer layers (0)were layers 1-111. bNMDA-sensitive [3H]glutamate binding was determined in 20 nM ligand in SO mM Tris-HCI buffer plus 2.5 mM CaCI,; binding in 1 mM NMDA was subtracted from total ['Hlglutamate binding. 'Quisqualate-sensitive [3H]glutamate binding was determined by subtracting binding in the presence of 2.5 FM quisqualate from total ['H]glutamate binding in 20 nM ligand, SO mM Tris-HCI buffer plus 2.5 mM CaCI,. 516 Young et al.

TABLE 11. NMDA-Sensitive and Quisqualate-Sensitive [3H1Glutamate Binding in Subcortical Regions of Macaca fascicularis* NMDA-sensitive, Quisqualate-sensitive, fmolimg protein, fmol/mg protein. Structure mean (SEM)" mean (SEM)~ Claustrum 145 (29) 170 (29) Septum 242 (36) 259 (23) Basal ganglia Caudate 160 (50) 219 (26) Caudate. ventral tail 163 (41) 196 (23) Putamen 147 (30) 216 (23) Globus pallidus, external 38 (24) 66 (10) Globus pallidus, internal 4 (10) 31 (6) Substantia nigra pars compacta 17 (14) 37 (11) Substantia nigra pars reticulata 14 (21) 48 (15) Subthalamic nucleus 18 (19) 49 (18) Thalamus Anteroventral 159 (105) 227 (9 1 j Centrum medianum. parafascicular 6 (14) 28 (20) Lateral dorsal 61 (16) 77 (42) Lateral geniculate -21 (10)' 6 (10) Medial dorsal 27 (31) 66 (19) Medial geniculate - 1 1 (-) 62 (-1 Pulvinar 529 (-) 267 (50) Ventral antcrior 22 (21) 56 (19) Ventral lateral, medial 2 (17) 44 (19) Ventral, lateral. oral 17 (30) 54 (32) Ventral posteroinferior -9 (12) I I (10) Ventral posterior 9 (12) 31 (14) Ventral posteromedial 15 (13) 39 (14) Ventral posteromedial, parvocellular 56 (27) 103 (12) Brainstem Central gray 114 (42) Superior colliculus 101 (40) Inferior colliculus 46 (22) Pontine nuclei 13 (12) Red nucleus 22 (25) Vestibular nuclei 66 (48) Cerebellum Granular layer, cerebellar cortex 205 (58) 233 (27) Molecular layer, cerebellar cortex 119 (57) 437 (63) Dentate nucleus 0 (13) 29 (11) Fastigial nucleus -5 (1) 6 (1) Interposed nucleus -12 (3) 8 (1) *Values are the mean (SEM) of data from 4 monkeys cxcept where indicated. (-j = Estimate based on 1 animal. NA = not available. aNMDA-sensitive [3H]glutamate binding was determined in 20 nM ligand in 50 mM Tris-HCI buffer plus 2.5 mM CaC12: binding in 1 mM NMDA was subtrdctcd froin total ['HH]glutamate binding. bQuisqualate-sensitive I3HJglutamate binding was determined by subtracting binding in the prcsence of 2.5 FM quisqualate from total ['Hlglutarnate binding in 20 nM ligand, 50 mM Tris-HCI buffer plus 2.5 mM CaCL,. 'Negative values occur when binding in the presence of NMDA (or quisqualate) was actually slightly higher than total binding.

al., 1988). The high-affinity quisqualate-sensitive sites qualate-sensitive sites can be blocked by 2.5 pM quis- appear biochemically to resemble quisqualate-linked ion qualate. This concentration of quisqualate has minimal channels and quisqualate-linked metabotropic receptors. effects on the low-affinity site. Quisqualate receptors can The low-affinity quisqualate-sensitive sites are probably also be selectively measured in Tris-chloride buffer with equivalent to the NMDA-sensitive sites (Greenamyre et calcium in the presence of 1 mM NMDA and 1 p,M al., 1985). Approximately 95% of thc high-affinity quis- kainate. Subtypes of EAA Receptor Binding in Primate Brain 517

Fig. 1. Computerized images of coronal sections demonstrat- Image Research, Inc., St. Catherine's, Ontario, Canada. The ing NMDA-sensitive (left) and quisqualate-sensitive (right) bars on the left indicate the density scales for the images to the binding in association (A,B) and motor (C,D) cortices of pri- right of the bar. Note that NMDA-sensitive sites are denser in mate brain. The images are subtraction images of specific bind- outer layers of motor cortex than inner cortex and no dense ing. They were obtained by subtracting from total [3Hlgluta- band is scen in layer IV of motor cortex. In area 2 1, a dense mate binding the binding remaining in either 1 mM NMDA (to band of binding is observed in layer IV. Abbreviations: 4, 6, measure NMDA-sensitive binding) or 2.5 pM quisqualate (to 2 1, and 22 refer to the respective Brodmann areas; I is insular measure quisqualate-sensitive binding). The subtractions were cortex. carried out on a computerized image processing system from

NMDA-sensitive ['Hlglutamate binding is not The assay used in this study is less selective than chloride sensitive and can be measured adequately in the some current binding assays, but selective information absencc of chloride or calcium (Greenamyre et al., 1985; was obtained by analyhg NMDA- and quisqualate-sen- Monaghan et al., 1989; Young and Fagg, 1990). Thus, sitive binding alone. In this study, however, quisqualate- NMDA-sensitive receptors can be measured in Tris-ac- sensitive [3H]glutamate binding probably represents etate with 2.5 pM quisqualate and 1 pM kainate. Alter- binding to both the quisqualate-sensitive ion channel and natively, NMDA-sensitive ['Hlglutarnate binding can be the metabotropic receptor. In addition, not all measured in the presence of calcium and chloride by ['Hlglutarnate binding in primate brain was inhibited by examining only the binding that is sensitive to a high 1 mM NMDA and 2.5 ~J-Mquisqualate; in some regions concentration of NMDA. considerable binding remained. 518 Young et al.

Fig. 2. Computerized images of NMDA-sensitive (left) and ations: P, putamen; GPE, external globus pallidus: GPI. inter- quisqualate-sensitive (right) binding in coronal sections of nal globus pallidus; E, entorhinal cortex; D, niolecular layer of basal ganglia (A,B), hippocampus (C,D), and cerebellum dentate gyrus; M1, stratum moleculare of CAI; P1, stratum (E,F). The images are subtraction images of specific binding pyramidale of CAI; P3. stratum pyramidale of CA3; M, mo- and were obtained as described in Figure I. The bars indicate lecular layer of cerebellum; G, granule cell layer of cerebel- the density scales for the images to the right of the bar. The lum. same bar applies to basal ganglia and hippocampus. Abbrevi-

Most biochemical studies of EAA receptor sub- tributed in the primatc brain. Although both binding subtypes have been carried out in rodent tissue where they types were present in each area of brain examined, in display strikingly independent regional distributions many areas one subtype predominated over the other. (Young and Fagg, 1990). Quisqualate- and NMDA-sen- NMDA receptors are gated by magnesium in a sitive [3H]glutamate binding was also differentially dis- voltage-depcndcnt fashion and thus are maximally acti- Subtypes of EAA Receptor Binding in Primate Brain 518 TABLE 111. Cortical NMDA-Sensitive and Quisqualate-Sensitive synapse on the distal dendrites of layer 111 pyramidal Binding: Outerhner Layers* cells and specific thalamocortical projections synapse Mean outeriinner ratios heavily in layer 1V (Brodal, 1981). Feedforward associ- Cortical area NMDA Quisqualate ation fibers from primary sensory cortex synapse in layer IV 4 3.24 2.27 of association cortex (Pons et a]., 1987). These feed- 6 2.50 1.47 forward fibers mediate fast synaptic transmission and Supplementary motor area 2.06 1.38 pyramidal cell excitation. Feedback pathways from other 8" 2.14 1.51 areas of cerebral cortex, however, synapse on layer 111 12 2.20 1.30 pyramidal dendrites and modulate neuronal activity 45 NA 1 .so (Pons et al., 1987). NMDA receptors are activated in an 46 NA 1.25 apparent voltage-dependent manner because of their 24 1.54 1.22 voltage-dependent blockade by magnesium. Thus, the I &2 1.75 1.32 strength of these responses is likely to be modulated by 3 2.07 1.57 the activity of other excitatory inputs. The high density 5 1.97 1.67 7 1.92 1.45 of NMDA binding in outer layers of cerebral cortex and Auditory cortex I .99 1.53 at the layer IV-V junction is consistent with the presence 21 I .69 1.31 of NMDA modulatory function in these areas of mam- 22 1.55 1.16 malian cortex (Thomson, 1986; Addae and Stone, 1986). Insular cortex I .47 1.17 In the hippocampal formation, both NMDA- and Entorhinal cortex 1.57 1.24 quisqualate-sensitive binding were present in high densities. This is in keeping with prior studies indicating that *Ratios of values in Table 1. NA = not available the entorhinal pathway to dentate gyrus and stratum py- "Brodmann areas. ramidale and moleculare of CAI is EAAergic, as are the prqjections from dentate granule cells to CA3 neurons, from CA3 neurons to CAI pyramidal cells, and from vated in situations in which other neurotransmitters de- CA1 pyramidal cells to subiculum (Fagg and Foster, polarize the neuron. The availability of selective NMDA 1983; Storm-Mathisen, 1977). This series of EAAergic receptor antagonists has made it possible to examine the pathways is thought to be important in learning and role of these receptors in normal behavior. NMDA re- memory (Collingridge and Singer, 1990). Long-term po- ceptor blockade has been associated with disruption of tentiation in hippocampal lormation is disrupted by both long-term potentiation, a model of memory formation quisqualate and NMDA antagonists. (Collingridge and Singer, 1990). The high density of The neocortical and hippocampal distribution of NMDA-sensitive [3H]glutamate binding in forebrain, es- EAA receptors is very similar to those found by Geddes pecially hippocampus and cerebral cortex, is consistent et al. (1989) who studied NMDA receptors in the baboon with the proposed role these receptors have in learning and Jansen et al. (1989) who studied NMDA, AMPA, and memory (Collingridge and Singer, 1990). NMDA and kainate binding sites in human cortex. All groups antagonists have been found to be anticonvulsants, find the same hippocampal and outer cortical binding. which is consistent with the high density of NMDA bind- Geddes et al. do not comment on the apparent zone of ing in cerebral cortex, the presumed site of action of decreased binding in layer IV. Whether this is due to a anticonvulsants (Meldrum et al., 1983; Schwarcz and species difference or a difference in the methodologies is Meldrum, 1985). not clear. In cerebral cortex, NMDA-sensitive binding was NMDA receptor antagonists affect motor function, most dense in outer layers (layers I to 111). There was an resulting in decreased muscle tone and ataxia (Green- apparent zone of decreased binding in layer IV. This amyre, 1986; Schwarcz and Meldrum, 1985). These ef- finding may be an artifact due to quenching of the tritium fects are probably due to NMDA receptor blockade in emissions by the increased white matter in this layer structures such as pons, spinal cord, and cerebellum. (Rainbow et al.. 1984; Geary and Wooten, 1985; Kuhar NMDA binding in these regions is low even though elec- and Unnerstall, 1985). Intermediate densities were ob- trophysiological and behavioral studies indicate an im- served in layers V and V1. In neocortical areas, except portant rolc for thesc receptors in motor function. Mis- for motor cortex, a thin band of NMDA-sensitive bind- matches between binding densities and presynaptic ing was observed at what appeared to be the junction neurotransmitter pathways have been noted in other sys- between layers IV and V. The differential di5tribution of tems (Kuhar, 1985). Whether some receptors are spare these binding sites may be related to specific cortical receptors in certain regions is unclear but striking mis- afferent systems. Nonspecific afferents from thalamus matches do appear to exist in the EAA system. 520 Young et al. Quisqualate receptors are thought to be responsible REFERENCES for EAA-induced fast synaptic transmission (Dingledine Addae J1, Stone TW (1986): Effects of topically applied excitatory et al., 1988; Watkins et al., 1990). Thus, the finding that amino acids on evoked potentials and single cell activity in rat most regions of brain contain high densities of quis- ccrebral cortex. Eur J Pharmacol 121:337-343. qualate binding was expected. Certain areas of brain con- Barbaresi P, Fabri M, Conti F, Manzoni T (1987): D-L3H] Aspartate tain predominantly quisqualate binding. Notably, the retrograde labelling of callosal and association neurones of somatosensory areas I and I1 of cats. 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