THE JOURNAL OF COMPARATIVE NEUROLOGY 326:82-90 (1992)

Immunocytochemical Localization of Kynurenine Aminotransferase in the Rat Striatum: A Light and Electron Microscopic Study ROSALINDA C. ROBERTS, FU DU, KATHLEEN E. MCCARTHY, ETSUO OKUNO, AND ROBERT SCHWARCZ Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, Maryland 21228 (R.C.R., F.D., K.E.M., R.S.) and Department of Biochemistry, Wakayama Medical College, Wakayama 640, Japan (E.O.)

ABSTRACT Kynurenine aminotransferase is the biosynthetic enzyme for kynurenic acid, an antagonist of excitatory amino acid receptors. Because of the possible role of kynurenic acid in basal ganglia diseases, the distribution of kynurenine aminotransferase immunoreactivity was examined in the adult rat striatum at the light and electron microscopic levels. Kynurenine aminotransferase immunoreactivity was detected in glial cells and in neurons. The preadsorption control vastly reduced or eliminated specific staining at both the light and electron microscopic levels. Kynurenine aminotransferase positive glial cells were abundant and contained a robust and homogeneous distribution of reaction product in both the nucleus and cytoplasm. The majority of neurons, both medium and large, were immunostained and exhibited granular kynurenine aminotransferase immunoreactivity in the cytoplasm of somata and proximal dendrites. At the ultrastructural level, kynurenine aminotransferase immunoreactive astrocytic processes were apparent throughout the neuropil where they often encircled capillaries and surrounded axospinous synapses. Reaction product was associated with the cytoplasmic matrix, filaments, rough endoplasmic reticulum, and the nucleus. In neurons, the majority of label occurred in round membrane-bound cytoplasmic organelles located adjacent to the Golgi apparatus, rough endoplasmic reticulum, and the cell or nuclear membranes. Cisternae and vesicles were identifiable in some of the labeled profiles. Polyribosomes and rough endoplasmic reticulum were also labeled. These data provide an anatomical basis for biochemical studies that have suggested the presence of striatal kynurenine aminotransferase in both astrocytes and neurons. o 1992 Wiley-Liss, Inc. Key words: kynurenic acid, excitotoxicity, basal ganglia, astrocytes

Kynurenic acid (KYNA), a metabolite of tryptophan (Ellinger, '04), is a normal component of the mammalian brain. Its concentration varies among brain regions and, even more strikingly, among species (Carla et al., '88; Turski et al., '88). KYNA has recently gained attention as a broad spectrum antagonist of ionotropic excitatory amino acid receptors (Perkins and Stone, '82; Stone et al., '89) and acts preferentially at the glycine site associated with the N-Methyl-D-Aspartate (NMDA) receptor complex (Kessler et al., '89). Moreover, KYNA has neuroprotective and anticonvulsant properties (German0 et al., '87; Andine et al., '88) and is especially potent at blocking the neurotoxic effects of quinolinic acid (Foster et al., '84), another endogenous metabolite of tryptophan and selective NMDA receptor agonist (Stone et al., '89). Thus, KYNA may play a role

o 1992 WILEY-LISS, INC.

in diseases which are believed to be caused by overstimulation of excitatory amino acid receptors by endogenous compounds (Schwarcz et al., '84; Meldrum and Garthwaite, '90; Do et al., '91). Kynurenine, the bioprecursor of KYNA, crosses the blood-brain barrier from the periphery (Gal and Sherman, '80; Fukui et al., '91) and enters both astrocytes, which accumulate it rapidly in a sodium-independent manner, and neurons, which possess a slow sodium-dependent kynurenine transporter (Speciale and Schwarcz, '90). Kynurenine aminotransferase (KAT), the biosynthetic enzyme of KYNA, is singularly responsible for the production of KYNA in the rat brain under physiological conditions Accepted August 6,1992

KAT LOCALIZATION IN RAT STRIATUM (Okuno et al., '91b). The enzyme has been characterized and purified to homogeneity (Okuno et al., '90, '91b), and immunocytochemical studies using anti-KAT antibodies have been performed at the light microscopic level. Thus, KAT was localized to both glial cells and neurons in several brain regions (Okuno et al., '90; Schwarcz et al., '92). In the neostriatum, agonists at excitatory amino acid receptors, in particular quinolinic acid, can produce a pattern of neurodegeneration which closely mimics that seen in the neostriatum of patients with Huntington's disease (HD) (Coyle and Schwarcz, '76; McGeer and McGeer, '76; Schwarcz et al., '83; Beal et al., '86; Roberts and DiFiglia '89). It was also shown that KYNA levels are decreased in the putamen of HD brains as compared to controls (Beal et al., '90). Because of the possible involvement of KYNA in the pathogenesis of HD, it clearly became important to examine the neuroanatomical features of KYNA biosynthesis in the normal brain. In preparation for future studies in the human brain (Okuno et al., '91a), we therefore decided to examine the immunocytochemical localization of KAT at both the light and electron microscopic level in the adult rat striatum. A preliminary report of this work has been published (Roberts et al., '91).

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mended dilutions followed by reaction with diaminobenzidine (6 mgi 10 ml PB) containing 0.03% hydrogen peroxide for 5-15 minutes. Controls consisted of 1) omitting the primary antibody, and 2) preadsorbing the specific antibody with pure rat kidney KAT (0.8 antigen per ml of the diluted antibody; Okuno et al., '90 for 60 hours).

Electron microscopy Selected sections from six brains, which exhibited typical staining at the light microscopic level, were processed for electron microscopy by standard techniques. Briefly, sections were immersed in 1%osmium tetroxide for 1 hour, stained with 1%uranyl acetate for 1 hour, dehydrated in alcohols, and embedded flat in Epon. Areas of interest from the striatum were excised, mounted on Epon blocks, thick (2.5 ym) and thin (90 nm) sectioned, and examined with an electron microscope.

Quantitative light microscopy

Two types of measures were taken from three animals which exhibited typical staining. 1)KAT-positive glial cells and neurons were counted from 40 ym thick, flat embedded sections at three locations throughout the rostrocaudal extent of the striatum: anterior, middle, and posterior. In METHODS ~ with an each rat, cells were counted at 4 0 0 magnification ocular eyepiece in four grids from each of three sections Animals (each grid was 250 x 250 pm). A total of 1,529neurons and The striata of 20 adult male rats were used in this study. 1,230 glial cells were counted. 2) Camera lucida drawings Animals were deeply anesthetized with either a mixture of plotting the number and distribution of KAT-i punctate xylazineiketamine (1mg xylazine + 5 mg ketamineilO0 g structures were generated of 10 large aspiny neurons, 2 body weight) or chloral hydrate (400 mg/kg) and perfused medium-sized aspiny neurons, and 27 medium-sized spiny through the heart with 50 ml of saline (4°C)followed by 350 neurons. In addition, the area of the cytoplasm (the area of ml of fixative (4°C)containing 4.0% paraformaldehyde, 0.03 the soma minus the area of the nucleus) was calculated M periodate, and 0.1 M lysine in 0.1 M phosphate buffer from the drawings with the aid of a Macintosh computer (PB) (pH 7.4) (McLean and Nakane, '74). Brains were and Mac-Measure software. The number of KAT-positive postfixed for 3 hours in the same fixative, and then either 1) granules was calculated per 100 km2 of cytoplasm. stored in 0.1 M phosphate-buffered saline (PBS) for 2-24 hours (PC), and sectioned with a vibratome (40 km thick), or 2) cryoprotected in 15% sucrose in PBS for 48 hours RESULTS (4°C)and sectioned on a cryostat (20-30 pm thick).

Immunocytochemistry

Light microscopy

KAT-i was present throughout the striatum in glial cells In each brain, every sixth coronal section collected from and neurons (Fig. lA,C,D). Specific staining was eliminated the rostrocaudal extent of the striatum was processed for or vastly reduced by the preadsorption control (Fig. 1B). the localization of KAT-immunoreactivity (KAT-1). The KAT-positiveglial cells and neurons appeared to be homogeanti-KAT polyclonal antibody used in this study was raised neously distributed throughout the striatum (Fig. 2A,B). in rabbit against rat kidney KAT, was partially purified by KAT-positive glial cells contained heavily deposited reacconventional methods, and was characterized by western tion product diffusely distributed throughout the cytoblot analysis (Okuno et al., '90). As described previously plasm and processes. Nuclear labeling was present in many (Du et al., '92; Schwarcz et al., '92), free-floating sections labeled glial cells and occurred only in cells that also were incubated in 2% normal goat serum in PB for 30 exhibited cytoplasmic staining. Based on morphological minutes prior to incubation in anti-KAT antibody (1:1,500) criteria, immunoreactive glial cells appeared to be astrofor 72 hours (4°C). Sections were then processed with cytes. Notably, KAT-positive processes surrounding blood reagents from an avidin-biotin kit (Vecta-stain)using recom- vessels were abundant throughout the striatum (Fig. 1A). KAT-i in neurons was punctate and was located in the cytoplasm of somata and proximal dendrites. KAT-i was present in many neurons, both medium sized and large. On Abbreviations the basis of qualitative observations, it was estimated that at least half the neuronal population was KAT-positive. HD Huntington's disease Medium-sized immunoreactive neurons were of both spiny KAT-i KAT-immunoreactivity and aspiny types, as revealed in semithin and ultrathin KYNA kynurenic acid KAT kynurenine aminotransferase sections by the presence of an unindented or indented NMDA N-methyl-D-aspartate nucleus, respectively. The majority of striatal neurons, the PB phosphate buffer medium-sized spiny neurons, had only a few labeled puncPBS phosphate-buffered saline tate structures in their scanty cytoplasm (Fig. 1D). Large RER rough endoplasmic reticulum

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Fig. 1. Photomicrographs of coronal sections (20 pm) of the normal rat striatum. A Distribution of KAT-i. KAT-i around blood vessles is indicated (arrows). Asterisk indicates location of high magnification view shown in panel 1C. Scale bar, 1,000 pm. B: Specific staining is eliminated or vastly reduced in the preadsorption control. Scale bar, 1,000 pm. C: High magnification view showing glial cells (arrowheads)

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and neurons (arrows) exhibiting KAT-i. Scale bar, 100 pm. D: Nomarski photograph of a semithin section (2.5 pm), showing two neurons (n) and two glial cells (g). Note that KAT labeling in glial cells is diffuse in the cytoplasm and nucleus, while in neurons label is punctate (solid arrows). KAT-positive glial processes are highlighted (open arrows). Scale bar, 10 km.

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DISTRIBUTION OF KAT-POSITIVE NEURONS

DISTRIBUTION OF KAT-POSITIVE GLIAL CELLS 70

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Fig. 2. A,B: Histograms illustrating the number of KAT-positive glial cells (A) and neurons (B) per 250 krn2throughout the rostrocaudal extent of the striatum. Both glial cells and neurons were distributed in

similar numbers across the rostrocaudal extent of the striatum. Standard deviations are (A) 0, 3.5, and 2.9, respectively; and (B) 0, 0.6, and 1.2, respectively.

aspiny interneurons, which have abundant cytoplasm, were heavily labeled with KAT-i and were apparent even a t low magnification (Fig. 1C). Since neuronal KAT labeling was discrete and punctate, it was possible to quantitate the number of KAT-positive structures in each cell type (Fig. 4). Medium-sized spiny neurons contained 9.1 3.4 immunoreactive profiles/2.5 km section, medium-sized aspiny neurons had 50.5 2 6.5, and large aspiny neurons contained 79 11.9 KAT-positive profiles. To determine whether the number of KAT-i profiles was related to the amount of cytoplasm, the number of KAT-positive granules/cell was compared to the area of cytoplasm, in 2.5 km thick sections. The results indicated 4.6 labeled granules per unit of cytoplasm in large neurons and 3.6 labeled granules per unit of cytoplasm in medium-sized spiny cells. This represents a 25% increase, albeit statistically insignificant, in the number of KATlabeled profiles in the large cells versus the medium-sized spiny cells. At low magnification, especially in unosmicated sections, immunoreactive glial cells appeared to be far more abundant than KAT-i neurons (Fig. 1C). However, at higher magnification, a very different picture emerged (Fig. lD), revealing a similar number of immunoreactive neurons and glial cells (Fig. 2A,B). This disparity between low and high magnification views is due, in part, to the fact that the majority of immunoreactive neurons contain only a few labeled granules per cell and their presence escapes detection at low magnification. Also, immunoreactive glial cells were far more conspicuous than KAT-positive neurons, because of a robust labeling pattern.

KAT-i was found in a membrane bound cytoplasmic organelle (Fig. 3B). Astrocytic processes surrounding capillaries were frequently KAT-positive (Fig. 3A). KAT-i glial processes dominated the neuropil (Fig. 3A,B,D) and frequently engulfed axospinous synapses (Fig. 7). KAT-i astrocytic processes sometimes were adjacent to axodendritic synapses, but very rarely encircled them (not shown). The subcellular localization of KAT-i in neurons was confined to more discrete regions of the cell than in glial cells. The granule labeling seen in neurons at the light microscopic level corresponded at the ultrastructural level to round membrane-bound organelles (Figs. 5, 6). The location of these KAT-i profiles within the neurons and the association of these profiles with other cytoplasmic organelles did not seem to vary amongst the different subtypes of neurons. These profiles were found adjacent to the cell membrane (Fig. 5D), the Golgi apparatus (Fig. 6A), the nuclear membrane (Fig. 6B), and rough endoplasmic reticulum (Fig. 6B). Although the reaction product obscured the contents of most of these profiles, cisternae and vesicles were discernible in some. Thus, at least some of these structures may be multivesicular bodies. Other profiles, labeled with colloidal gold (preliminary unpublished observations), contained a floccular matrix, only one or two vesicles and occasionally a fragment of membrane. These profiles may represent membrane-bound intracytoplasmic storage sites. KAT-i was also localized to rough endoplasmic reticulum and polyribosomes (Fig. 6B). Throughout the neuropil, KAT-positive membrane bound profiles were not apparent in neuronal profiles except for proximal dendrites. Immunoreactivity was not present in axonal profiles but occasionally was observed in distal dendrites where it was associated with microtubules and cisternae (Fig. 7).

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Electron microscopy

Not surprisingly, the subcellular distribution of KAT-i in neurons and glial cells differed at the ultrastructural level. DISCUSSION In astrocytes, KAT-i was diffusely distributed throughout The major finding of the present study is that KAT is the cytoplasm (Fig. 3A-E). Rough endoplasmic reticulum (RER) (Fig. 3C,E), smooth cisternae (Fig. 3C), filaments localized in both glial cells and neurons in the adult rat (Fig. 3B), and cytoplasmic matrix (Fig. 3B) were heavily striatum. This is consistent with biochemical studies of the labeled. In cells which exhibited stained nuclei, immunore- excitotoxin-lesioned striatum, which have indicated that activity was present on the euchromatin. Very rarely, the majority of KAT is present in glial cells, but that a

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Figure 3

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and confined to restricted intracellular compartments. In addition to the more discrete intracellular localization of KAT-i in neurons, quantitative deliberations must also take into account that the most heavily labeled neurons, the aspiny neurons, constitute only 5% of all striatal neurons (Graveland and DiFiglia, '85). Therefore, although the absolute number of KAT-positive neurons and glial cells is similar, the present morphological data are in agreement with the idea that striatal KYNA is preferentially produced in glial cells. MEDIUM SPINY LARGE ASPlNV M E D I U M ASPINV KAT-i was present in medium-sized spiny, medium-sized aspiny, and large aspiny striatal neurons. The similarity in the distribution of KAT-positive profiles suggests that KYNA is produced and stored in a similar way by the different types of neurons. However, the subtypes of neurons exhibited a striking difference in the number of Fig. 4. Camera lucida drawings from 2.5 pm thick sections showing KAT-immunoreactive profiles present. Thus, spiny neuthe typical distribution of KAT-positive punctate structures throughout the cytoplasm of the various subtypes of striatal neurons. Note the rons contained 5-10 times fewer KAT-positive structures abundance of KAT-positive structures in the aspiny neurons in compar- than aspiny interneurons. These absolute numbers may not be related to less cytoplasmic area since medium-sized ison to the medium-sized spiny cell type. Scale bar, 10 pm. spiny neurons had only 75% of the number of KAT-positive profiles found per unit of cytoplasm in large aspiny neurelatively small proportion of KAT exists in neurons (Turs- rons. These results indicate that spiny neurons may proki et al., '89). Since KAT is the only enzyme capable of duce less KYNA than aspiny neurons. Since KYNA can act synthesizing KYNA in the rat brain under physiological as a neuroprotective agent (Foster et al., '84; German0 et conditions (Okuno et al., '91b), the localization of KAT in al., '87; Andine et al., '88), the relative paucity of KAT in both glial cells and neurons indicates that KYNA is likely to spiny neurons may, in part, account for the high vulnerabilbe produced in both these cell types. Notably, the number of ity of this subset of neurons to excitotoxic insults (Schwarcz KAT-i glial cells and neurons was similar, a surprising et al., '83; Beal et al., '86; Roberts and DiFiglia, '89). finding considering that KAT-i glial cells appear to outnumThe subcellular localization of KAT in neurons was found ber KAT-i neurons in other brain regions, such as the to be discrete and unusual. Neuronal KAT located on hippocampus (Du et al., '92). The functional significance of ribosomes and RER is probably related to its synthesis the apparent difference in the ratio of KAT-positive neu- within neurons. Moreover, the high concentration of neurorons to glial cells in different regions of the rat brain is nal KAT found within membrane-bound organelles sugunknown at present. However, the reason for the discrep- gests that at least some KYNA in neurons is synthesized in ancy may be methodological since the large number of these structures. Based on similarities in morphology and striatal KAT-positive neurons found in the present study intracellular location, some of these profiles appear to be was only noticed at high power, particularly in semithin multivesicular bodies (Peters et al., '76). Kynurenine, which sections. Perhaps a similar analysis of other brain regions is slowly accumulated by neurons (Speciale and Schwarcz, would also reveal more KAT-positive neurons than previ- 'go), may be sequestered in these multivesicular bodies and ously thought to exist. then converted to KYNA. The identity of other labeled Irrespective of those methodological considerations, the membrane-bound organelles, containing a light matrix and subcellular distribution of KAT-i in neurons and glial cells a paucity of inclusions (identified in colloidal gold material), provides support for the conclusions drawn from biochemi- was more difficult to establish; these profiles may be cal experiments, i.e., that the majority (70-80%) of KAT in atypical multivesicular bodies or intracytoplasmic storage the rat striatum is contained in glial cells (Schwarcz et al., compartments. '92). Thus, KAT-i differed dramatically between glial cells, KAT-positive glial cells within the striatum were identiin which the reaction product was diffuse and abundant, fied as astrocytes based on morphological criteria at the and neurons, in which the reaction product was punctate light microscopic level and the presence of filaments at the ultrastructural level (Peters et al., '76). This finding is consistent with data obtained in the hippocampus which demonstrated a large number of cells double-labeled with Fig. 3. A-E: Electron micrographs of striatal astrocytes, showing KAT-immunoreactivity diffusely distributed throughout the cyto- both KAT and glial fibrillary acidic protein (Du et al., '92). plasm, processes, and nucleus. A KAT-positive astrocytic process In the present study, KAT-positive glial processes were surrounding a capillary wall exhibiting diffuse KAT-i (solid arrows) and particularly evident surrounding capillaries, where they are a rare membrane bound organelle (open arrow). Scale bar, 1 pm. B: in a position to convert kynurenine to KYNA as kynurenine Immunoreactive astrocyte with reaction product in the cytoplasmic crosses the blood-brain barrier (Fukui et al., '91). Notably, matrix (thick arrows), on RER (boxed area), and fibers (open arrows). Thin arrows in the neuropil highlight immunoreactive glial processes. striatal KAT-positive astrocytic processes frequently surBoxed area is shown at higher power in C. Scale bar, 5 pm. C : Glial RER rounded asymmetric axospinous synapses which are typi(solid arrows) is heavily labeled with KAT. Also, cisternae are labeled cally excitatory in nature. Since more than 80% of striatal (open arrow). Scale bar, 1 pm. D: An astrocyte exhibiting KAT-i in both synapses are asymmetric axospinous (Pasik et al., '801, are the nucleus and cytoplasm (thick arrows). KAT-i labeled glial processes largely of cortical origin (Kemp and Powell, '711, and use are abundant throughout the neuropil (thin arrows). Boxed area is glutamate as a neurotransmitter (Divac et al., '77; Kim et shown at higher magnification in E. Scale bar, 5 km. E: High magnification view of KAT label on RER (solid arrow) and in the al., '77; McGeer et al., '77), KYNA appears to be in an excellent position to modulate excitatory amino acid nucleus on euchromatin (open arrow). Scale bar, 1pm.

Fig. 5. A-E: Electron micrographs showing the distribution of KAT-positive profiles in striatal neurons. A: A medium-sized spiny neuron (N) (identified by its unindented nucleus) labeled with KAT (arrows).A KAT-positive &a1 soma ( G )is also present (arrows indicate reaction product). Scale bar, 5 pm. B: A KAT-positive (arrows) medium-sized aspiny neuron (identified by its indented nucleus). Scale bar, 5 pm. C: A higher magnification view of the boxed area in B

showing a typical KAT-positive membrane bound organelle. This particular profile is located next to the cell membrane. Scale bar, 1 pm. D: A large aspiny neuron containing many KAT-labeled profiles (arrows). *, stacked RER. Scale bar, 5 pm. E:A semitbin section (2.5 pm) of the neuron in D, showing numerous KAT-positive granules (arrows).Scale bar, 10 pm.

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Fig. 7. KAT-positive astroglial (G) processes (arrows) are often adjacent to and partially surround axospinous synapses; follow arrows around synaptic profile. The synapse, arrow in spine (S),is asymmetric and the axon terminal (AT) is probably of cortical or thalamic origin. Dendrite (d) contains lightly labeled cisternae and microtubules (arrows). Scale bar, 0.5 pm.

Fig. 6. A,B: High power views of typical KAT-positive round membrane bound profiles located in various regions throughout the cytoplasm of neurons. A: A KAT-positive sphere adjacent to the Golgi apparatus in a medium-sized aspiny neuron. Arrowheads indicate secretory vesicles between the Golgi apparatus (GI and the KATpositive structure (open arrow). N, nucleus. Scale bar, 0.5 pm. B: A KAT-labeled structure (open arrow) located between the nucleus and the RER in a large aspiny neuron. KAT-labeled RER (thick arrows) and polyribomes (thin arrows) between the nucleus and the KAT-positive profile are indicated. N, nucleus. Scale bar, 0.5 km.

Clearly, future studies will need to examine if and under what circumstances KYNA can be released from neurons, and to investigate the precise functional relationship in the striatum, between KAT-containing processes and glutamatergic afferents. Moreover, anatomical arrangements similar to those in the rat may exist in the human neostriatum and may be involved in the pathogenesis of neurodegenerative basal ganglia diseases. For example, a decrease in neostriatal KAT activity could trigger cellular events leading to excitotoxic destruction and could thus play a role in the pathogenesis of HD (cf. Introduction).

ACKNOWLEDGMENTS This work was supported by USPHS grants NS28236 and MH44211 and a fellowship from the Huntington's Disease Society of America (to F.D.). The authors wish to thank Nancy E. Flowers for excellent secretarial assistance.

LITERATURE CITED receptor function in the striatum. Taken together with the fact that KYNA can freelv enter the extracellular ComDartmerit after its production"in glial cells ( ~et d., ~ ,&I, it~ that KYNA may reach local is therefore trations at the synapse which are sufficient to inhibit glutamatergic activity.

AndinB, P., A. Lehmann, K. Ellren, E. Wennherg, I. Kjellmer, T. Nielsen, and H. Hagherg (1988)The excitatory amino acid antagonist kynurenic acid administered after hypoxic-ischemia in neonatal rats offers neuroprotec~ Neurosci. b Lett. 90:208-212. tion. Beal, M.F., N.W. Kowall, D.W. Ellison, M.F. Mazurek, K.F. Swartz, and J.B. Martin (1986) Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid. Nature 321:168-171.

90 Beal, M.F., W.R. Matson, K.J. Swartz, P.H. Gamache, and E.D. Bird (1990) Kynurenine pathway measurements in Huntington’s disease striatum: Evidence for reduced formation of kynurenic acid. J. Neurochem. 55r1327-1339. Carla, V., G. Lombardi, M. Bensi, P. Russi, G. Moneti, and F. Moroni (1988) Identification and measurement of kynurenic acid in the rat brain and other organs. Anal. Biochem. 169:89-94. Coyle, J.T., and R. Schwarcz (1976) Lesion of striatal neurons with kainic acid provides a model for Huntington’s chorea. Nature 263:244-246. Divac, I., F. Fonnum, and J. Storm-Mathisen (1977) High affinity uptake of glutamate in terminals of corticostriatal axons. Nature 266:377-378. Do, K.Q., J.M. Klancnik, B. Gahwiler, H. Perschak, H.-G. Wieser, and M. Cuenod (1991) Release of excitatory amino acids: Animal studies and epileptic foci studies in humans. In B.S. Meldrum, F. Moroni, R.P. Simon, and J.H. Woods (eds): Excitatory Amino Acids. New York: Raven Press, pp. 677-685. Du, F., W. Schmidt, E. Okuno, R. Kido, C. Kohler, and R. Schwarcz (1992) Localization of kynurenine aminotransferase immunoreactivity in the rat hippocampus. J. Comp. Neurol. 321:477487. Ellinger, A. (1904) Die Entstehung der Kynurensaure. 2. Physiol. Chem. 43:325-337. Foster, A.C., A. Vezzani, E.D. French, and R. Schwarcz (1984) Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid. Neurosci. Lett. 48:273-278. Fukui, S., R. Schwarcz, S.I. Rapoport, Y. Takada, and R. Smith (1991) Blood-brain transport of kynurenines: Implications for brain synthesis and metabolism. J. Neurochem. 5612007-2017. G&l, E.M., and A.D. Sherman (1980) Its synthesis and possible regulatory function in brain. Neurochem. Res. 5.223-239. Germano, I.M., L.H. Pitts, B.S. Meldrum, H.M. Bartkowski, and R.P. Simon (1987) Kynurenate inhibition of cell excitation decreases stroke size and deficits. Ann. Neurol. 22:730-734. Graveland, G.A., and M. DiFiglia (1985) The frequency and distribution of medium-sized neurons with indented nuclei in the primate and rodent neostriatum. Brain Res. 327:307-311. Kemp, J.M., and T.P.S. Powell (1971) The site of termination of afferent fibers in the caudate nucleus. Phil. Trans. R. Soc. Lond. 262t413-427. Kessler, M., T. Terramani, G. Lynch, and M. Baudry (1989) A glycine site associated with N-methyl-D-aspartic acid receptors: Characterization and identification of a new class of antagonists. J. Neurochem. 52:13191328. Kim, J.-S., R. Hassler, and P. Haug (1977) Effect of frontal cortex ablation on striatal glutamic acid level in rat. Brain Res. 132370-374. McGeer, E.G., and P.L. McGeer (1976) Duplication of biochemical changes of Huntington’s chorea by intrastriatal injection of glutamic or kainic acids. Nature 263:5 17-5 19. McGeer, P.L., E.G. McGeer, and U. Scberer (1977) A glutamatergic corticostriatal path? Brain Res. 128:369-373. McLean, I.W., and P.K. Nakane (1974) Periodate-lysine-paraformaldehyde fixative: A new fixative for immunoelectron microscopy. J. Histochem. Cytochem. 22: 1077-1083.

R.C. ROBERTS ET AL. Meldrum, B., and J. Garthwaite (1990) Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol. Sci. 11:379-387. Okuno, E., F. Du, T. Ishikawa, M. Tsujimoto, M. Nakamura, R. Schwarcz, and R. Kido (1990) Purification and characterization of kynureninepyruvate aminotransferase from rat kidney and brain. Brain Res. 534: 3 7 4 4 . Okuno, E., M. Nakamura, and R. Schwarcz (1991a) Two kynurenine aminotransferases in human brain. Brain Res. 542307-312. Okuno, E., W. Schmidt, A.P. Deborah, M. Nakamura, and R. Schwarcz (1991b) Measurement of rat brain kynurenine aminotransferase at physiological kynurenine concentrations. J. Neurochem. 57:533-540. Pasik, P., T. Pasik, and M. DiFiglia (1980) The internal organization of the neostriatum in mammals. In I. Divac and R.G.E. Oberg (eds): The Neostriatum. Oxford: Pergamon Press, pp. 5-36. Perkins, M.N., and T.W. Stone (1982) An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Brain Res. 247:184-187. Peters, A,, S.L. Palay, and H. de F. Webster (1976) The fine structure of the nervous system: The neurons and supporting cells. Philadelphia: W.B. Saunders C o . Roberts, R.C., and M. DiFiglia (1989) Short- and long-term survival of large neurons in the excitotoxic lesioned rat caudate nucleus: A light and electron microscopic study. Synapse 3:363-371. Roberts, R.C., F. Du, E. Okuno, and R. Schwarcz (1991) Immunohistochemical localization of kynurenine aminotransferase in the rat striatum. SOC. Neurosci. Abstr. 17253. Schwarcz, R., A.C. Foster, E.D. French, W.O. Whetsell, Jr., and C. Kohler (1984) Excitotoxic models for neurodegenerative disorders. Life Sci. 35: 19-32. Schwarcz, R., F. Du, W. Schmidt, W.A. Turski, J.B.P. Gramsbergen, E. Okuno, and R.C. Roberts (1992) Kynurenic acid: Apotential pathogen in brain disorders. In R.E. Heikkila, J.W. Langston, and A.B. Young (eds): Neurotoxins and their Potential Role in Neurodegeneration. Ann. N.Y. Acad. Sci., pp. 140-153. Schwarcz, R., W.O. Whetsell, and R.M. Mangano (1983) Quinolinic acid: An endogenous metabolite that produces axon-sparing lesions in rat brain. Science219:316-318. Speciale, C., and R. Schwarcz (1990) Uptake of kynurenine into rat brain slices. J. Neurochem. 54: 156-163. Stone, T.W., N.R. Burton, and D.A.S. Smith (1989) Electrophysiology of quinolinic acid and other kynurenines, and their potential roles in the CNS. In T.W. Stone (ed): Quinolinic Acid and the Kynurenines, Boca Raton, FL: CRC Press, pp. 114-148. Turski, W.A., J.B.P. Gramsbergen, H. Traitler, and R. Schwarcz (1989) Rat brain slices produce and liberate kynurenic acid upon exposure to L-kynurenine. J. Neurochem. 52: 1629-1636. Turski, W.A., M. Nakamura, W.P. Todd, B.K. Carpenter, W.O. Whetsell, Jr., and R. Schwarcz (1988) Identification and quantification of kynurenic acid in human brain tissue. Brain Res. 454:164-169.