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The Journal of , July 1987, 7(7): 2214-2231

Regional Distribution of with Intense lmmunoreactivity for Glutamate Dehydrogenase in Rat Brain: Implications for - Interactions in Glutamate Transmission

Chiye Aoki, Teresa A. Milner, Kwan-Fu Rex Sheu,’ John P. Blass,’ and Virginia M. Pickel Department of Neurology, Cornell University Medical College, New York, New York 10021, and ‘Dementia Research Service, Burke Rehabilitation Center, White Plains, New York 10605

The principally mitochondrial enzyme glutamate dehydro- Glutamate dehydrogenase(GDH; L-glutamate : NAD oxidore- genase (GDH) exhibited low-intensity, uniform immunoreac- ductase; E.C. 1.4.1.2) is one of the major enzymes involved in tivity in and intense heterogeneous labeling of glial the interconversion of glutamate and alpha-ketoglutarateof the cells of rat brain. Simultaneous peroxidase labeling for GDH tricarboxylic acid cycle (Dennis and Clark, 1977). Thus, GDH and immunoautoradiography for glial fibrillary acidic protein might be expected to be present in mitochondria of both glia (GFAP) confirmed the astrocytic localization of the enzyme. and neuronsthroughout the brain. However, sinceglutamate is lmmunoreactivity in astrocytes, but not in neurons, required also an excitatory transmitter in selective neuronal pathways the presence of Triton X-100 as a solubilizing agent. Most (Cotman et al., 1981; Watkins and Evans, 1981; Fonnum, 1984) of the intensely labeled glial processes were localized to higher concentrations of the enzyme also might be expected in regions previously reported as containing moderate to high correspondingneurons or their associatedglia. The glial versus densities of binding sites for the excitatory amino acids, neuronal, or the dual glial and neuronal localization of GDH L-glutamate or L-aspartate, and glutamatergic fibers. These in brain has been contestedfor a number of years (seeNicklas, included several regions, such as the superficial 1984, for a review). Furthermore, the regional concentrations layers of the rostra1 neocortex, dorsal neostriatum, nucleus of GDH relative to glutamatergic pathways have not been exten- accumbens, septohippocampal nucleus, intralaminar tha- sively examined by cytochemical methods (Graham and Apri- lamic nuclei, and external capsules. However, the central son, 1969; Ryder, 1980; Wenthold, 1980; Leong and Clark, gray of the , the nuclei of the , 1984). brain stem regions projecting to the , and cranial In the present study we employed the peroxidase-antiperox- nuclei of the trigeminal and vagal nerves also exhibited in- idase(PAP) immunocytochemical method (Stemberger, 1979) tense glial labeling for GDH, even though some of these for determining the cellular and regional distributions of GDH regions are known to receive only weak glutamatergic pro- in adult rat brain. The glial versus neuronal labeling was dis- jections. A second factor determining the distribution of GDH tinguished by the dual localization of GDH and of glial fibrillary appeared to be neuronal activity, as assessed by corre- acidic protein (GFAP) (Bignami et al., 1972) using combined spondence with reported high densities of cytochrome ox- PAP and immunoautoradiographicmethods (Pickel et al., 1986). idase. We conclude that GDH enriched in glial populations Furthermore, since GDH is believed to exist in at least 2 forms, (1) exists in a subcellular compartment distinct from that of differing in susceptibilitiesto solubilization by detergents and neurons and (2) may serve as one of the enzymes involved salts (Colon et al., 1986; Lai et al., 1986), we examined the in glutamatergic transmission. Deficiencies of glial GDH and immunocytochemical localization of the enzyme under a variety the consequent cytotoxic effects of high levels of excitatory of fixation conditions, using different concentrations of deter- amino acids may contribute to a number of neurodegener- gentsin the labeling procedure. ative disorders. Materials and Methods Antisera. The antiserum to GDH was prepared by immunizing rabbits with bovine liver GDH (Sigma) that had been further purified by SDS- PAGE. This antiserum has been tested for its specificity by the detection of a single immunoreactive material on immunoblots, using GDH puri- Received Aug. 29, 1986; revised Dec. 30, 1986; acceptedJan. 22, 1987. fied from bovine liver, as well as GDH in whole homogenates of brain This study was supported by NIH Grants 5F32NS07782 (C.A.), HL18974, and (Lai et al., 1986). MH40342 (V.M.P.); by NS22952 (K.-F.R.S.), AGO3573 (J.P.B.), and the Will Rabbit PAP and rabbit anti-GFAP antiserum were generously sup- Rogers Institute (J.P.B.). V.M.P. is also supported by a career award from the plied bv the laboratories of Dr. T. H. Joh (Cornell Universitv Medical NIMH (MH00078) and NSF Grant BNS 80-239 14. We wish to thank Drs. David i?ollege, New York) and Drs. A. Bignami and D. Dahl (V. A. Medical Ruggiero, Joseph LeDoux, and Victoria Arango for their anatomical expertise; Center, MA), respectively. Donkey anti-rabbit rZSI-IgG was obtained Dr. Young-Tai Kim for providing Sepharose-conjugated antibodies; June Chan and Dr. Harriet Baker for their technical assistance;and Nancy Marmor, Hillary from Amersham, and goat anti-rabbit IgG from Miles Laboratory (Elk- Rettig, and William Feig for their assistance with the preparation of the manu- hart, IN). SCliDt. Fixation and sectioning. Thirty-two adult (180-250 gm) male rats Correspondence should be addressed to Chiye Aoki, Department ofNeurology/ were perfused for 6 min through the ascending aorta with one of 3 Neurobiology, Cornell University Medical College, 4 11 E. 69th Street,New York, fixatives. These were (1) 200 ml of 4% paraformaldehyde in 0.1 M NY 10021. phosphate buffer (pH 7.4); (2) 50 ml of 3.75% acrolein and 2% para- Copyright 0 1987 Society for Neuroscience 0270-6474/87/072214-18$02.00/O formaldehyde, followed by 150 ml of 2% parafotmaldehyde; and (3) The Journal of Neuroscience, July 1987, 7(7) 2215

4% paraformaldehyde with 0.1-0.2% glutaraldehyde. After the termi- inos and Watson (1982). In addition, identifications of some of the nation of perfusion, the brains were removed, cut into 1 mm coronal thalamic nuclei and neocortical regions were modified in accordance or sagittal blocks and further fixed in 2 or 4% paraformaldehyde for r/z with the delineation of boundaries of Jones (1985) and Peters and Jones hr. With each of the 3 fixation conditions, the 1 mm blocks were sec- (1984). Each stippling on the schematics of Fig. 6, A-J, represents lo- tioned at 30-50 pm on a Vibratome or were cryoprotected with 10% 15 processes. When the processes appeared to be regularly oriented sucrose in 0.1 M phosphate buffer, rapidly frozen in freon, then sectioned within structures, stippled lines were drawn to reflect the direction of on a sliding microtome. Both types of sections were collected in 0.1 M the labeled profiles. Tris-O.9% NaCl buffer (Tris-saline) at pH 7.6. Single-labeling with the GDH and the GFAP antisera. The fixed sec- tions were immunocytochemically labeled by a modification (Pickel, Results 198 1) of the PAP method of Stemberger (1979). Prior to the incubation of sections with the antiserum, they were incubated for ‘/2 hr with 3% Under a specific set of experimental labeling conditions, GDH goat serum-Tris-saline. This buffer, with the substitution of 3% goat was selectively localized to glial cells. These GDH-enriched glial serum with 1% goat serum, was used in all ofthe subsequent incubations cells were heterogeneously distributed in rat brain. The exper- and in the rinses between incubations. Sections were incubated with imental labeling conditions, morphological and chemical char- GDH or GFAP antiserum for a period of 12-48 hr and at dilutions acteristics of identified glial cells, and distribution of the labeled from 1:800 to 1:5000. Sections were then incubated in the goat anti- rabbit IgG at a dilution of 1:50 for 1 hr, followed by an incubation in cells are described in the following sections. the rabbit PAP at a dilution of 1:lOO for 1 hr. Following 2 rinses in Tris-saline not containing goat serum, the peroxidase was demonstrated Labeling conditions by reaction with 3’,3’-diaminobenzidine and H,O, Vibratome sections of brains fixed with paraformaldehyde alone For the purpose of enhancing the penetration of the antisera, some sections were permeabilized with 0.25% (vol/vol) Triton X-100 or (Wu or combined with low (O.l-0.2%) concentrations of glutaral- vol) saponin. In these cases, the detergent was added to all incubation dehyde showed immunocytochemical labeling with GDH anti- and wash solutions. All incubations and washes were carried out at serum. Intense immunoreactivity was seen in glial cells when room temperature. 0.2% Triton X-100 was included in all incubations (Fig. 1, A, Preadsorption of the GDH-antiserum with GDH. Bovine liver GDH C, E) (see next section for identity of cell types). Changes in the was coupled to cyanogen bromide-activated Sepharose 4B beads (Phar- macia), according to the manufacturer’s instructions. Two milliliters of fixation condition, sectioning procedure, and use of lipid sol- anti-GDH antiserum (ca. 40 pg of IgG, 40 mg of total protein) were vents markedly altered the intensity of the glial labeling for incubated with 100 ~1 of GDH coupled to Sepharose beads (100 pg of GDH. For example, omission of glutaraldehyde increased, GDH) for 1 hr at 37°C under gentle agitation. The supematant formed whereas addition of acrolein decreased, the intensity of labeling by a 1 min centrifugation at 100 x n was collected and substituted for the specific antiserum in the labeling procedure. in Vibratome sections with the use of Triton X-100 in all in- Dual labelinp for GDH and GFAP. The antisera to GDH and GFAP cubations. In brains fixed with 4% paraformaldehyde, frozen were raised in Tabbits. Thus, one antiserum had to be localized using a and sectioned on a sliding microtome, and immunostained in dilution sufficiently high to be recognized by autoradiography of 1251- the presence of T&on X-100, the peroxidase product in glia labeled secondary antibody, but not by the peroxidase method, as was was greatly reduced. The substitution of saponin for T&on described recently in detail (Pickel et al., 1986). The sections were first incubated with the GFAP antiserum at a dilution of 1:30,000, which X- 100 in the immunocytochemical labeling of paraformalde- was determined to be the highest possible dilution that allowed for hyde-fixed Vibratome sections also produced less intense la- reasonable detections of the immunoreactivity by autoradiography, but beling in glia. In the absence of Triton X- 100, similarly prepared not by peroxidase. Following a 12-16 hr period of incubation with or sections showed only a diffuse background labeling and a low without 0.25% Triton X-100, sections were incubated in a buffer con- taining a 1:50 dilution of donkey anti-rabbit Y-IgG (100 pCi/ml) for intensity of immunoreactivity in larger cells, presumed to be 2 hr. This step was followed by 10 rinses over a period of 5 hr to remove neuronal perikarya, in the and in most other regions unbound ‘251-IgG. The tissue was then incubated with the GDH anti- (Fig. 1, B, F). However, in areas such as the neocortex, the serum at a dilution of 1:2000 for 48 hr in the presence of 0.2% Triton labeling in the cytoplasm of neuronal perikarya was more in- X- 100. All dilutions and washes were in Tris-saline (pH 7.6) containing tense and pun&ate, particularly with higher concentrations (0.2%) 1% goat serum, and the entire labeling procedure was carried out at room temperature, with continuous agitation. The immunoreactivity of glutaraldehyde (Fig. 1D). Both glial and neuronal labeling for GDH was detected by the accumulation of peroxidase products, and were abolished when the primary antiserum was adsorbed with the immunoreactivity for GFAP was detected autoradiographically. For purified GDH prior to the immunocytochemical labeling (Fig. the purpose of autoradiography, after the PAP reaction sections were 1, G, H) and thus represented specific labeling for the enzyme. mounted on slides, dehydrated, then rehydrated through ethanol and dipped in Ilford L4 emulsion (at 50°C) diluted 1:1 with distilled water. Identification of glial cells These slides were air-dried, then stored with a desiccant in light-tight boxes at 4°C for 4-24 d exposure periods. The autoradiographs were In Vibratome sections of paraformaldehyde-fixed tissues im- developed at the end of the exposure period and examined with a Nikon munolabeled for GDH in the presence of Triton X- 100, intense Microphot FX using either bright-field or differential-interference con- peroxidase reaction product was detected in round or spindle- trast optics. The extent of labeling of the rabbit GFAP antiserum by the rabbit PAP was monitored by examining sections that were pro- shaped cells having a thin rim of cytoplasm, multiple long (1 O- cessed as described above in the absence of the GDH antiserum. 50 pm) branched processes, and small round or angular nuclei Other cytochemical and histochemical methods. Sections adjacent to (Fig. 1, A, C’, E). These cytological features, combined with their the ones used for immunocytochemistry were processed for cytochem- location in cellular portions of the and in fiber bundles, ical and histochemical procedures to facilitate neuroanatomical iden- tification of the immunoreactive structures. The histochemical proce- led to a tentative identification of protoplasmic and fibrous dure for the demonstration of AChE has been described previously by astrocytes (Figs. 1-6; described below in detail). Other presumed Hedreen et al. (1985). glial processes containing immunoreactivity for GDH were seen Presentation of labeling with the GDH antiserum. The immunoreac- exclusively in areas, such as layer I of the anterior cingulate tive structures were mapped using a camera lucida attachment on a cortex (ACg; Fig. 2, A, C). These straight, twiglike processes Leitz microscope. The boundaries of nuclei and fiber tracts, which were difficult to distinguish on the immunocytochemically stained sections, were lo-20 wrn in length, and clustered in tight bundles con- were delineated using both adjacent Nissl and AChE-stained sections. taining 3-5 processes. Nomenclature for the nuclei and fiber tracts was from the atlas of Pax- The GDH-labeled processes along blood vessels were in a 2216 Aoki et al. l Localization of Glutamate Dehydrogenase in Glia

Figure 1,: Cellular localization of GDH in relation to methods of tissue preparation. A-C, E, G, H, Fixation with 4% paraformaldehyde and 0.1% glutaraldehyde. D, F, Fixation with 4% paraformaldehyde and 0.2% glutaraldehyde. Micrographs in G and H show sections through the caudate putamen (CP) immunolabeled with control serum in the presence (G) and absence (IS) of Triton X-100. Micrographs in A and B show sections through the CP immunolabeled with GDH antiserum in the presence and absence of Triton. C, E, Higher magnifications of the GDH labeling in the corpus callosum (CC) and CP of sections prepared as in A. Arrows in A, C, and E show glial labeling. D, F, Higher-magnification micrographs The Journal of Neuroscience, July 1987, 7(7) 2217 similar pattern to the GFAP-immunoreactive processes, as seen . Twiglike processes containing GDH immu- at low (Fig. 2, A, B) and high (Fig. 2, C, D) magnifications of noreactivity were densely distributed throughout the anterior- the ACg. However, the GDH-labeled cells and processes were posterior extension of the stratum lacunosum-moleculare of far less numerous than those labeled with the GFAP antiserum the hippocampus (Fig. 6, E, F, just dorsal to the hippocampal in most areas examined. For example, in the ACg (Fig. 2B) and fissure). These processes were in close association with blood in the hippocampus, GFAP-immunoreactive cells were prom- vessels that coursed parallel to the hippocampal fissure. A few inent throughout all layers. In contrast, labeling with the GDH more delicately branched processes with GDH immunoreactiv- antiserum was enriched in the superficial layers in the ACg (Figs. ity were seen throughout all layers of the hippocampus and the 2A and 6, B-D) and within the stratum lacunosum-moleculare dentate gyrus (DG), and running in parallel with the in of the hippocampus (Fig. 6, E, F, just dorsal to the hippocampal the alveus. Diffuse labeling for GDH was most evident in the fissure), particularly in association with the walls of major blood stratum lacunosum-moleculare of CA1 and CA3, but was also vessels. In other examples, the GFAP-labeled processes showed detectable in the layer of CA1 . little structural resemblance to the straight, twiglike processes and associated nuclei. In the neostriatum (cau- containing immunoreactivity for GDH (Fig. 2, C vs E). date-putamen complex), GDH was localized to numerous cells The labeling pattern in the cerebellum was more dramatically with long, wispy processes (Figs. 1, A, E; 5D; 6, B-E) that were divergent. The radiating processes of glia, especially within the frequently associated with blood vessels coursing in an anterior- outer molecular layer, but also within the layer, posterior direction. Occasionally, labeled twiglike processes could were prominently labeled with the GFAP antiserum (Fig. 3B). also be identified within the dorsolateral quadrant. In contrast, labeling for GDH was more moderate and within The medial-ventral quadrant of the striatal complex, corre- the finer-caliber fibers of the molecular layer and diffuse within sponding to the accumbens nucleus (Acb; Fig. 6, A, B), the the granule cell layer (Fig. 3A). ventral pallidum (VP; Fig. 6, B-D) and nucleus basalis (B; Fig. The dorsal was one of the regions that 6D), and the more lateral endopyriform nucleus (En; Fig. 6, B- exhibited a close correspondence between the patterns of la- E) and claustrum (Cl; Fig. 6, A-D), were particularly rich in beling for GDH and GFAP. With both antisera, the labeling processes with GDH immunoreactivity. In contrast, the ‘sub- was intense within the nucleus of the (NTS) and stantia innominata (SI; Fig. 60) and the nucleus of the anterior dropped sharply at the boundary to the motor nucleus of vagus commissure (AC; Fig. 6, C, D) were only moderately labeled. (X) (Fig. 4, A, B). The processes with GDH immunoreactivity in the globus pal- In dual-labeling studies with dilutions sufficient to differen- lidus (GP) were as dense as those in the neostriatum, and were tiate the 2 rabbit antisera, GDH was shown to be colocalized clumped within the neuropil between the large fiber bundles with GFAP in subsets of cells located in the neuropil and in that coursed through the structure (Fig. 6, D, E). fiber bundles (Fig. 5). The dual labeling was detected in both Other telencephalic nuclei. The basolateral amygdaloid com- the radiating, branched processes (double arrows in Figs. 5, A, plex (BL, BLV, BM, LA, Fig. 6E) exhibited intense GDH B, D, 8) and in straight, twiglike processes (single arrow in Fig. immunoreactivity in evenly distributed, irregularly shaped cells 54). In most cases, the silver grains that developed from the and processes. In contrast, the corticomedial group (Me, ACo, lZSI-IgG bound to the GFAP antiserum were concentrated over PMCo; Fig. 6, E, F) was sparsely labeled. One exception was only a portion of the peroxidase-labeled GDH cells or processes. the central nucleus (Ce; Fig. 6E) of the corticomedial group, In every region examined, cells exhibiting only silver grains which exhibited more processes containing GDH immunoreac- (GFAP), only peroxidase (GDH immunoreactivity; arrowheads tivity. in Fig. 5, A, D) and both of the labels were found. The septohippocampal nucleus (SHi; Fig. 6B) contained an extremely dense plexus of twiglike processes with GDH im- Distribution of GDH-labeled cells and processes munoreactivity. These processes were uniformly oriented in Forebrain parallel to the fiber tracts running mediolaterally through the In the forebrain, dense as well as sparse labeling for GDH was nucleus. With the exception of external portions of the nucleus seen in different subregions of the (1) neocortex, (2) hippocam- of the diagonal band (DB), which also contained dense, twiglike pus, (3) basal ganglia, (4) other telencephalic subnuclei, (5) thal- processes with GDH immunoreactivity along the brain surface amus, (6) , as well as in (7) fiber tracts. (Fig. 6C) and dense labeling in the horizontal limb (HDB; Fig. Neocortex. Immunolabeling within layer I and along orthog- 6D), more delicate, branched processes of moderate distribu- onally oriented large blood vessels decreased in the rostrocaudal tions were seen in the related septal nuclei. direction. Specifically, the most prominent labeling was seen in . In the thalamus, numerous shaggy, branched pro- the ACg (Fig. 6, B-D), the primary olfactory cortex (PO), and cesses and cell bodies immunolabeled for GDH had features of tubercle (Tu) (Fig. 6, A-E). Less labeling was found in the pos- protoplasmic astrocytes. The most intensely labeled nuclei in terior cingulate (PCg; Fig. 6E) and entorhinal (Ent; Fig. 6fl the dorsal thalamus were the rhomboid (Rh), the central medial cortices and the (S; Fig. 6F). These processes were (CM), and the paracentral (PC) nuclei of the intralaminar group morphologically distinct from the radiating processes that ex- (Fig. 6E). In the ventral thalamus, the reticular nucleus (Rt) and tended outward from the corpus callosum (cc) in layer VI (Fig. (ZI) contained the densest collections of labeled 6, A, B). cells (Fig. 6E). The pattern of labeling in the neuropil of the c of sections prepared as in B, except for a higher concentration ofglutaraldehyde. The GDH-labeling in the cytoplasm of neurons is far more evident in the cortex (D) than in the CP (B, fl. Neuronal labeling is indicated by arrows in B, D, and F. J; Myelinated fibers. Bar: A, & G, H, 50 pm; C-F, 25 Mm. 2218 Aoki et al. l Localization of Glutamate Dehydrogenase in Glia

Figure 2. Localization of GDH and GFAP in the anterior . A, Low-magnification micrograph showing the “twiglike” processes with GDH immunoreactivity near the cortical surface and in relation to the walls (arrows) of a penetrating blood vessel. B, Low-magnification micrograph showing a corresponding area from the same brain processed in parallel for the localization of GFAP. Numerous labeled cells are seen throughout the cortical layers but are especially dense near the walls (arrows) of blood vessels. C, D, Higher-magnification micrographs of the fibrillar twig-type processes labeled with GDH and GFAP, respectively. E, Higher-magnification micrograph showing the labeling of GFAP in the more widely distributed, presumably protoplasmic, astrocytes, which generally lacked detectable GDH immunoreactivity in the cerebral cortex. Bars: A, B, 50 pm; C-E, 10 Wm. The Journal of Neuroscience, July 1987, 7(7) 2219

Figure 3. Localization of GDH and GFAP in the cerebellar cortex. A, Micrograph showing the fine veil-like processes labeled with GDH (double nrrows) that radiatethrough the molecularlayer (MOL) of the cerebellum.These processes encircle Purkinje cells (RX) (larger arrow). More dense GDH labelingof presumedglial cellsand processes is indicatedwith a large openarrow in the granulecell layer (CCL). B, Micrographshowing the localizationof GFAP in glialprocesses (double arrows) of the samecerebellar layers seen in A. ventral thalamus was broken up by the many fiber bundles of sure (aca, ac, acp; Fig. 6, A-C’), lateral olfactory tract (lo; Fig. the thalamic radiation and . The subthalamic nu- 6, A-D), dorsal fomix (Fig. 6E), fimbria (fx, fi; Fig. 6, D, E), cleus(Sb; Fig. 6E) also contained an extensive plexus of labeled optic tract (opt, oc, sox; Fig. 6, D, E), and cerebral peduncle processes.The remaining thalamic nuclei contained moderate, (cp; Fig. 6, F, G). The fomix (f, fx; Fig. 6, D, E), the stria low, or no detectable immunoreactivity for GDH. medullaris (sm; Fig. 6D), and the were Hypothalamus. In the hypothalamus, the highest density of lessdensely labeled, while the rostra1continuation of the su- processeswith GDH immunoreactivity was seenin the supra- perior , the mammillotegmental tract, and chiasmatic (Sch) and supraoptic (SO) nuclei (Fig. 60). The la- the (st; Fig. 6, D, E) had almost no detectable beled processesin thesenuclei appearedmore stippled or var- labeling. Finally, immunoreactivity for GDH was seenin pro- icosethan in other regions.More characteristicglia-like processes cesseswithin the (ml; Fig. 6, F-H) only in labeled with GDH were densely distributed in the dorsal hy- regions near . pothalamic area (DA; Fig. 6E), and were somewhat lessnu- merousin the rostra1part of the medial and the lateral preoptic Midbrain nuclei (MP and LPO; Fig. 60). As shown in Figure 6E, labeled In the midbrain, only the central gray, substantianigra, pontine processeswere rarely detected in the remaining hypothalamic nuclei, and a few nuclei in the reticular formation, along with nuclei. selective fiber tracts, had noteworthy densitiesof processesor Fiber tracts. Some of the most intensely immunoreactive cells cells with GDH immunoreactivity. and processeswere seenwithin fiber tracts in the forebrain; other In the central gray, immunoreactive processeswere seen tracts remainedclearly unlabeled.The processeswere numerous throughout the region, but the dorsal and caudal areas were in the corpus callosum and external capsule(cc, gee,ec; Fig. 6, more heavily labeled (CG, CGD, Fig. 6, F, G). The labeledcells A-G; Fig. lc), (ic; Fig. 6E), anterior commis- and processesin the substantianigra (SNC, SNR, Fig. 6fl were 2220 Aoki et al. * Localization of Glutamate Dehydrogenase in Glia

Figure 4. Localization of GDH and GFAP in the nucleusof the solitarytract. A, B, Micrographsshowing the respectivelocalizations of GDH and GFAP in glia of the rostra1nucleus of the solitarytract (NTS) andadjacent motor nucleusof the vagus(2’). With both antisera,the intensity of labelingof the -likeprocesses is muchdarker in the NTS than in the X. Dottedline demarksthe boundarybetween NTS andX. Bar: 40 pm.

almost exclusively restricted to the areasadjacent to the cerebral peduncles(cp), which werethemselves heavily labeled(Fig. 68). The pontine nuclei (Pn), the afferent Reticular nuclei that project to the cerebellum,namely, the para- within the longitudinal fibers of the pons (lfp), and the efferent median (PMn; Fig. 64 and lateral reticular (LRt; Fig. 6, Z, J) transverse fibers of the pons (tfp) all exhibited prominent la- nuclei of the medulla, as well as the reticulotegmental nucleus beling of cells and processes(Fig. 6G). Some of the processes of the pons (RtTg; Fig. 6G) (reviewed in Carpenter, 1978)con- were like those in other heavily myelinated structures, suchas tained high densitiesof cells and processeswith immunoreac- the globus pallidus, and others were of the straight, twiglike tivity for GDH. The (py; Fig. 6, H-J) and morphology. trapezoid body (tz; Fig. 6H) continued to exhibit intensely im- Among nuclear groups of the reticular formation, the cunei- munoreactive processesthrough the level of decussation.The form (Cnc Fig. 6G) and lateral dorsal tegmental (LDTg; Fig. area of the tract of the central and locus coeruleus 6G) nuclei exhibited densely labeled cells with radiating and were labeledmoderately. Of the raphe complex, only the dorsal sometimesvaricose processes,while the dorsaland ventral para- nuclei and the dorsal part of the median nuclei (DR and MR, brachial nuclei contained more modestdensities of labeledcells. Fig. 6G) exhibited detectable levels of immunoreactivity in as- Fiber tracts containing the highestdensity of immunoreactive trocyte-like cells or processes. cells and processesincluded the cerebral peduncle(cp; Fig. 6F) Intense labeling of glial processesin cranial nerves was re- and superior cerebellar peduncle (scp; Fig. 6G). Two auditory stricted to the selective subnucleiand tracts associatedwith the pathways, i.e., the (11;Fig. 6G) and the bra- trigeminal (spV, SpVO, SpVI, mV; Fig. 6, G, H), facial (VII; Fig. chium of the inferior colliculus (bit; Fig. 6G), contained a more 6H), acoustic (VIIIn; Fig. 6ZZ),and vagus (X; Fig. 6, I, .Z), in- moderate density of labeling. cluding the nucleusambiguus (Amb; Fig. 6, Z, 4.

Figure 5. Dual peroxidase and autoradiographic labeling for GDH and GFAP antisera.Micrographs show the labelingfor GDH (brownPAP reaction) with superimposed silver grains (lz51-autoradiography), revealing the localization of GFAP in the neocortex(A, B), caudateputamen (D), and perirhinal fissure area (F). Micrographs in C, E, and G show sections corresponding to A, B, D, and F that were similarly dually labeled, but with the omission of GDH antiserum. Openarrows in A-C point to pial surfaces, the singlesmall arrow in A to the dually immunoreactive twiglike processes, the doublearrows in A, B, D, and Fto the astrocyte ofthe more conventional morphology, and the arrowheadsto the GDH-immunoreactive but GFAP-nonimmunoreactive processes. The boldarrows in C, E and G point to the GFAP-immunoreactive processes. Note that these are devoid of cross-reactiverabbit PAP products.Bar: A-C, 50 pm; D-G, 25 pm. ‘I

f . . “V -: t I, I --.T- 2222 Aoki et al. * Localization of Glutamate Dehydrogenase in Glia

Figure 6. Distribution of GDH-immunoreactive processes. Schematic drawings of the left halves of coronal sections, A-J, represent GDH- immunoreactive processes, drawn with the aid of a camera lucida. Nuclei boundaries are delineated with dashed lines and identified on right hdves of the drawings with upper-case letters, while fiber tract boundaries are delineated with continuous lines and identified with lower-case letters. Further details are as described in Materials and Methods. Bar: A-H, 2.28 mm; Z, .Z, 1.14 mm (see Appendix for abbreviations). The Journal of Neuroscience, July 1987, 7(7) 2223

Figure 6. Continued. 2224 Aoki et al. - Localization of Glutamate Dehydrogenase in Glia

Figure 6. Continued. The Journal of Neuroscience, July 1987, 7(7) 2225

G

Figure 6. Continued. 2226 Aoki et al. * Localization of Glutamate Dehydrogenase in Glia

I

Figure 6. Continued. The Journal of Neuroscience, July 1987, 7(7) 2227

Of the nuclei and fiber tracts of the dorsal column, the nucleus scured. Alternatively, the antiserum used in the present study of the external cuneate (ECU; Fig. 6, Z, J) contained the highest may also have recognized a form of GDH distinct from the density of labeled processes within the brain. The cuneate nu- nuclear form characterized by Lai et al. (1986). Under the con- cleus (Cu; Fig. 6J) and its fiber tract, and the gracilis nucleus ditions of our study, the antisera may recognize primarily a (Gr; Fig. 65) and its fiber tract were also densely labeled. Triton-insoluble GDH that is more GTP-, NADH/NADPH-, The inferior olive complex (IO) was another structure where and heat-sensitive than is the soluble form, and which has been intensely GDH-immunoreactive processes were numerous. proposed to be “non-mitochondrial” (Colon et al., 1986). These processes were fasciculated and closely wrapped around fiber bundles (Fig. 6Z). Of the inferior olive complex, the medial Glial versus neuronal localization of GDH accessory olive (MAO; Fig. 6, Z, J) exhibited particularly intense The results of this study have shown that, in Triton-treated labeling for GDH in small branched processes. sections, GDH is primarily localized to cells having the mor- phological characteristics of astrocytes (Kosaka and Hama, 1986). Cerebellum In certain processes, dual labeling with GDH and GFAP further The cells and processes with GDH immunoreactivity were fairly supports the concept that many GDH-containing cells are as- evenly distributed throughout the rostrocaudal extent of the trocytes. However, certain processes showed only GFAP, while various folia ofthe cerebellar cortex (Fig. 6H). The entire granule others were immunolabeled exclusively for GDH. This would cell layer (GCL) exhibited a patchy, diffuse labeling. Processes not be unexpected, since immunoreactivity for GFAP is prin- with moderate intensity of GDH immunoreactivity were evi- cipally localized to large shafts of processes containing glial fil- dent in the molecular layer. Many labeled processes resembled aments (Kosaka and Hama, 1986) and GDH would more likely the fine varicose branches of Bergmann glia (called the “velate be distributed in fine ramifications of processes enveloping syn- astrocytes” by Palay and Chan-Palay, 1974). Labeling for GDH apses. Methodological factors such as differences in penetration was also seen in other types of glial processes (Ramon y Cajal, of antisera or more superficial detection of autoradiographic 1911) that appeared to radiate from various depths within the labeling probably also contribute to the differential detection of entire thickness of the molecular layer and were particularly GDH and GFAP. In addition, GDH may be found in a relatively evident in the floculonodular lobules. small population of astrocytes containing GFAP, and may also The , i.e., the fastigial (Fas), the inter- be detected in or . There is evidence positus (Int), and the dentate (De) (Fig. 6ZZ), contained equal strongly suggesting that oligodendrocytes (GFAP-negative, ga- and moderate densities of processes with GDH immunoreac- lactocerebroside-positive) and astrocytes (GFAP-positive, ga- tivity. Many labeled processes coursed parallel to the bundles lactocerebroside-negative) arise from a common progenitor cell of axons in the near the deep cerebellar nuclei. (Raff et al., 1983) and retain common antigenic determinants Other myelinated bundles of fibers in the cerebellum also con- even after maturation (Brenner et al., 1986). Also, the matter tained numerous delicate, GDH-immunoreactive processes ori- of whether or not microglia arise from astrocytes or from blood ented parallel to their longitudinal axes. monocytes remains unsettled (Miles and Chou, 1986; Schelper and Adrian, 1986). In light of these reports, it would be of Discussion interest to determine the ontogeny of expression of the antigenic Characterization of the antigenic GDH form of glial GDH in relation to the other marker proteins by The present detection of intense labeling of GDH in glia in means of immunocytochemical double-labeling. aldehyde-fixed sections of rat brain, using an antiserum raised While the most intense reaction for GDH was seen in glial against bovine liver GDH, supports earlier evidence for a high processes in the present study, neuronal with varying in- degree of conservation of the molecular structure of the enzyme tensities of immunoreactivity also were detected whenever Tri- among various species (Smith et al., 1975) and organs (Chee et ton X- 100 was omitted from the labeling procedure. The low- al., 1979). However, the detection of immunoreactivity was intensity cytoplasmic labeling seen in virtually all neurons is highly dependent on the procedures used for tissue preparation. probably attributable to levels of the enzyme associated with The optimal labeling seen in paraformaldehyde-fixed Vibra- the metabolic pools of glutamate. More intense neuronal la- tome sections using 0.2% Triton X-100 probably reflects the beling for GDH in certain regions, such as the cerebral cortex, conditions necessary for the antisera to reach certain intramo- could be associated with the neurotransmitter L-glutamate, as lecular antigenic sites or subcellular compartments with mini- described for glia in the next section. mal diffusion of GDH. Lai et al. (1986) have shown that GDH The difficulty in detecting neuronal labeling for GDH in the in rat brain is present in mitochondrial and nuclear fractions. presence of Triton X- 100 is probably attributable to the masking GDH in isolated mitochondria can be solubilized in 0.1% Triton of faint immunoreactivity in neurons by more intense labeling X- 100, while nuclear GDH is soluble only in elevated concen- of glia or, alternatively, to the greater solubility of neuronal trations of Triton X- 100 (0.3%) and salt (150 mM KCl). Colon GDH or its association with lipids that are extracted bv deter- et al. (1986) also found that rat brain contained at least 2 kinds gents. of GDH, differing in their susceptibilities to detergent extrac- tion. Our results are consistent with the possible existence of Distribution of GDH in relation to glutamatergic pathways multiple forms of the enzyme. It seems likely that the nuclear Detailed rostrocaudal mapping of GDH-immunoreactive glial fraction that was difficult to solubilize even in unfixed tissues cells and processes in the present study revealed a distribution was never made freely accessible to the antisera under the ex- remarkably similar to known glutamatergic pathways. In the perimental conditions that yielded optimal cytoplasmic labeling neocortex, we detected the highest densities of twiglike processes of glia in the present study. However, because of intense labeling containing GDH immunoreactivity within layer I, particularly in the cytoplasm, nuclear labeling of glia may have been ob- in the anterior pole, and also accompanying large, orthogonally 2228 Aoki et al. * Localization of Glutamate Dehydrogenase in Glia oriented blood vessels through layer III. Corresponding with matergic pathways supports recent studies on neuron-glia in- this pattern is the uptake of 3H-D-aspartate within cortical slices, teractions (reviewed in Hertz et al., 1983) in maintaining the which has been shown to be highest in layer I (Fonnum et al., compartmentalization of metabolic and neurotransmitter pools 198 1). These observations are further supported by Monaghan of L-glutamate. Besides their well-documented role in the con- and Cotman’s (1985) characterization of N-methyl-D-aspartate- trol of interstitial ion composition and volume (reviewed in sensitive glutamate receptors and the characterization by Hal- Abbott, 1986), astrocytes take up glutamate with a capacity that et al. of 3H-glutamate receptors, which indicate the presence is higher than that of neurons (Huck et al., 1984). Astrocytes, of a laminar distribution that is highest within superficial layers, then, can convert the glutamate to a-ketoglutarate and return more anteriorly than caudally. In the cerebellum, the laminar the product to the tricarboxylic acid cycle of neurons (Dennis distribution of these receptors also closely matches the GDH and Clark, 1977). Alternatively, the astrocytes may use gluta- distribution, and in the inferior colliculus, N-methyl-D-aspar- mine synthetase to convert glutamate to glutamine for its return tate-sensitive glutamate receptor binding exhibits a dorsal-to- to the amino acid pool of astrocytes and neurons (Bradford et ventral downward gradient (Monaghan and Cotman, 1985), al., 1978). The active uptake and metabolism of glutamate by much like the distribution of GDH-immunoreactive processes astrocytes are consistent with the present detection of GDH in seen within this area. On the basis of the present results, the regions where L-glutamate is actively released by terminals. demonstrated glutamate receptor binding may include both glia and neurons within receptive regions. Involvement of GDH in glutamate cytotoxicity The above and other studies have demonstrated convincingly Glutamate and its analogs, kainic and ibotenic acid are well- that the neostriatum, the accumbens nucleus, and, to a lesser known excitotoxins when present in excess amounts within glu- extent, the substantia nigra receive glutamatergic fibers from the tamate-receptive areas (Van Harreveld and Fitkova, 197 l), in- frontal cortex, while the lateral septal nucleus receives gluta- cluding the temporal lobe (Olney, 1985) and striatum (McGeer matergic fibers from the , and the amyg- and McGeer, 1976; Coyle et al., 1977). L-Glutamate also has dala from the frontal, pyriform, and entorhinal cortices (re- been implicated in neurodegenerative changes following met- viewed in Fonnum, 1984). In the present study, all of these areas abolic insults, such as hypoglycemia (Wieloch, 1985) and isch- demonstrated moderate to intense GDH immunoreactivity. emia (Rothman, 1984; Simon et al., 1984), since these damages The high densities of glial processes with GDH immuno- can be prevented by prior, central applications of antagonists reactivity were detected in the intralaminar (rhomboid, central to glutamate receptors. medial, and paracentral) and ventral (reticular nucleus and zona As noted above, the hindbrain areas with the highest con- incerta) thalamic nuclei. The question of whether these areas centrations of GDH were structures that project to the cerebel- are sites of termination or of origin remains unsettled (Streit, lum. These areas, together with the GDH-rich granule cell layer 1980; Rustioni and Cuenod, 1982). In support of the theory of the cerebellar cortex, characteristically undergo atrophy in that certain thalamic nuclei are termination sites for pathways extrapyramidal motor disorders of the olivopontocerebellar type containing L-glutamate are reports of high densities of N-meth- (Yamaguchi et al., 1982; Duvoisin et al., 1983; Plaitakis et al., yl-D-aspartate-sensitive receptors in these ventral and midline 1984). As postulated by Plaitakis et al. (1984), the high levels nuclei (Monaghan and Cotman, 1985). of GDH may be necessary for protection against glutamate cy- Hindbrain areas that receive cortical afferents (reviewed in totoxicity in areas having active glutamatergic neurotransmis- Carpenter, 1978), namely, (1) the reticular nuclei (reticuloteg- sion. Thus, these areas would be expected to be the first to show mental, lateral reticular, and paramedian nuclei), (2) the sensory neuronal degeneration following a systemic deficiency of GDH. relay nuclei (gracile, cuneate, external cuneate, sensory trigem- GDH-deficient patients exhibit “multiple-system degenera- inal, and solitary tract nuclei), (3) the inferior olive, and (4) the tion,” suggestive of alterations of the caudate putamen and glo- pontine nuclei, exhibited dense populations of GDH-immu- bus pallidus, the hippocampus and other limbic structures, the noreactive processes. In accordance with this, many corticofugal cortex, the oculomotor nerve, lateral vestibular nucleus, cere- systems (reviewed in Streit, 1984) and cerebellar projecting bellum, brain stem, frontal lobes, hypothalamic and thalamic pathways (reviewed in Ottersen and Storm-Mathisen, 1984) are areas. The present detection of high densities of GDH immu- believed to be glutamatergic. Our finding of large numbers of noreactivity in glial processes in all of these areas strongly sup- glial cells with GDH immunoreactivity in the intermediate and ports the concept that degenerative changes in specific regions caudal portions of the medial nuclei of the solitary tracts is also of the CNS may be associated with deficiencies of GDH. How- consistent with electrophysiological evidence that suggests that ever, the reports of GDH deficiency in patients with multiple L-glutamate is one of the neurotransmitters of afferent nerve systems degeneration were based on measurements of GDH fibers from arterial baroreceptors (Talman et al., 1980). activity in leukocytes and fibroblasts. It would be of interest to The brain stem reticular and sensory relay nuclei are reported confirm that the GDH activity within the CNS and, in partic- to contain only moderate densities of glutamate receptors (Hal- ular, within the brain stem, pontine, and cerebellar areas is pain et al., 1984; Monaghan and Cotman, 1985) which suggests similarly deficient in these patients. that GDH immunoreactivity may reflect factors other than the degree of glutamatergic innervation. It is interesting that these Appendix. -Abbreviations used in Figure 6 areas do exhibit intense levels of another mitochondrial enzyme, cytochrome oxidase (Claude, 1946; Wong-Riley, 1976), indic- AC, ant. commissural n. ative of high levels of tonic synaptic activity (Wong-Riley et al., ac, ant. commissure 1978, Wong-Riley 1979; Aoki et al., 1987). Conceivably, the aca, ant. commissure, ant. neuronal activity may determine the rate of turnover of gluta- Acb, accumbens n. mate released from axon terminals. ACg, ant. cingulate cortex The present localization of GDH in glia of presumed gluta- ACo, ant. cortical n. of The Journal of Neuroscience, July 1987, 7(7) 2229

acp, ant. commissure, post. Gr, gracile n. AHi, amygdalohippocampal a. GrCo, granule cell layer of cochlear n. ah, alveus of hippocampus HDB, n. of horizontal limb of diagonal band Amb, ambiguus n. HiF, hippocampal fissure AOP, ant. olfactory n., post. ZC, inf. colliculus AP, a. postrema ic, int. capsule APT, ant. pretectal n. ZCj, islands of Calleja Aq, aqueduct ZCjM, islands of Calleja, major island Arc, arcuate n. of hypothalamus icp, inf. cerebellar peduncle asc VII, ascending fibers of facial nerve IF, interfascicular n. B, cells of basal n. of Meynert ZZZn, oculomotor nerve bit, brachium of inferior colliculus In, intermediate gray layer of sup. colliculus BL, basolateral n. of amygdala Znt, interpositus n. of cerebellum BLV, basolateral n. of amygdala, vent. In Wh, intermediate white layer of BM, basomedial n. of amygdala 10, inf. olive bsc, brachium of sup. colliculus ZP, interpeduncular nucleus, cent. BST, bed n. of stria terminalis Z Vn, trochlear nerve Cl, crus 1 of ansiform lobule LA, lat. n. of amygdala CZ, crus 2 of ansiform lobule LD, lat. dorsal n. of thalamus CAI, field CA1 of Ammon’s horn of hippocampus LDTg, laterodorsal tegmental n. CA2, field CA2 of Ammon’s horn of hippocampus Ifp, longitudinal fasciculus of pons CA3, field CA3 of Ammon’s horn of hippocampus LH, lat. hypothalamic a. cc, corpus callosum LHb, lat. of Ce, central n. of amygdala Li, linear n. of medulla CG, central gray 11,lat. lemniscus cg, lo, lat. olfactory tract CGD, central gray, dorsal LPO, lat. preoptic a. CL, central lat. n. of thalamus LRt, lat. reticular n. Cl, claustrum LS, lat. septal n. CA4, central med. n. of thalamus LSD, lat. septal n., dorsal Cnf; cuneiform n. LSZ, lat. septal n., intermediate CP, caudate putamen LSII, lat. septal n., vent. cp, cerebral peduncle, basal LV, lat. ventricle Cu, cuneate n. LVe, lat. vestibular n. DA, dorsal a. of hypothalamus MAO, med. accessory olive DB, n. of diagonal band mcp, middle cerebellar peduncle DCo, dorsal cochlear n. MD, mediodorsal n. of thalamus dc/oc, dorsal / MDL, mediodorsal n. of thalamus, lat. De, dentate n. of cerebellum MdD, reticular n. of medulla, dorsal DG, dentate gyrus of hippocampus MdV, reticular n. of medulla, vent. Dk, n. of Darkschewitsch ME, DLL, dorsal n. of lat. lemniscus Me, med. n. of amygdala DR, dorsal raphe MG, med. geniculate n. of thalamus Dsc, lamina dissecans of MHb, med. habenula of epithalamus ec, ext. capsule ml, med. lemniscus ECU, ext. cuneate n. ml’ med. longitudinal fasciculus En, endopyriform n. MOL, molecular layer of cerebellar cortex Ent, entorhinal cortex MP, med. preoptic a. EP, endopeduncular n. mp, mammillary peduncle E W, Edinger-Westphal n. MR, median raphe n. f; postcommissural MS, med. septal n. Fas, fastigial n. of cerebellum mt, mammillothalamic tract $, fimbria of hippocampus mV, motor root of trigeminal nerve Fl, MVe, med. vestibular n. FrPaM, frontoparietal cortex, motor a. NTS, n. of solitary tract FrPaSS, frontoparietal cortex, somatosensory a. oc, optic chiasm FS, fundus striati Op, optic nerve layer of sup. colliculus fx, fomix opt, optic tract G, gelatinosus n. of thalamus Pas, parasubiculum gee, genu corpus callosum PBg, parabigeminal n. GCL, granule cell layer of cerebellar cortex PC, paracentral n. of thalamus GP, globus pallidus PCg, posterior cingulate cortex 2230 Aoki et al. - Localization of Glutamate Dehydrogenase in Glia

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