THE JOURNAL OF COMPARATIVE NEUROLOGY 312:652-679 (1991)

Immunocytochemical Localization of Argininosuccinate Synthetase in the Rat Brain HIROYUKI NAKAMURA, TAKEYORI SAHEKI, HITOSHI ICHIKI, KIMIHIKO NAKATA, AND SHIRO NAKAGAWA Departments of Anatomy (H.N., K.N., S.N.) and Biochemistry (H.I., T.S.), Faculty of Medicine, Kagoshima University, Kagoshima 890, and Kagoshima Institute of Preventive Medicine, Kagoshima 892 (H.I.),Japan

ABSTRACT The neuronal distribution of argininosuccinate synthetase (ASS) was mapped in the rat brain. Argininosuccinate synthetase is one of the enzymes of the arginine metabolic pathway and catabolizes the synthesis of argininosuccinate from aspartate and citrulline. Since arginine is the precursor of nitric oxide, argininosuccinate synthetase may act as part of the nitric oxide producing pathway. Argininosuccinate is also suggested to have a messenger function in the nervous system. Therefore, the localization of ASS is of great interest. Polyclonal antisera against purified rat liver argininosuccinate synthetase revealed a characteristic distribution pattern of argininosuccinate synthetase-like immunoreactivity: (1) many neurons with strong argininosuccinate synthetase-like immunoreactivity were observed in the septal area, basal forebrain, anterior medial and premammillary nuclei of the hypothalamus, anterior and midline thalamic nuclei, dorsal endopiriform nucleus of the amygdala, basal nucleus of Meynert, subthalamic nucleus, laterodorsal tegmental nucleus, raphe nuclei, nucleus ambiguus, and the area postrema, (2) neuropile staining was dense in the septal areas, hypothalamus, area postrema, nucleus of the solitary tract, and the laminae I and I1 of the caudal subnucleus of the spinal trigeminal nucleus and the spinal dorsal horn, (3) relay nuclei of the specific sensory systems such as the dorsal lateral geniculate nucleus and the ventral nuclei of the thalamus were devoid of argininosuccinate synthetase-like immunoreactivity, (4)no staining was seen in the large white matter structures such as the internal capsule, corpus callosum, and the anterior commissure, and (5) most of the neurons stained were small or medium in size and appeared to be interneurons. The results suggest that argininosuccinate synthetase affects the widely distributed, neuromodulatory system in the brain. Key words: arginine, urea cycle, nitric oxide, immunohistochemistry

Argininosuccinatesynthetase (ASS)catalyzes the synthesis of argininosuccinate from aspartate and citrulline. As a result of this reaction, the amino residue of aspartate enters the urea cycle in the liver. The urea cycle comprises five enzymes: carbamoyl phosphate synthetase, ornithine transcarbamoylase,ASS, argininosuccinate lyase, and arginase. However, only three of these enzymes, i.e., ASS, argininosuccinate lyase, and arginase, are present in the brain (Ratner et al., '60). Argininosuccinate lyase catalyzes the cleavage of argininosuccinate to yield fumarate and arginine, and arginase in turn cleaves arginine to urea and ornithine. Thus ASS, argininosuccinate lyase, and arginase are thought to constitute the metabolic pathway for production and degradation of arginine in the brain (Ratner et al., '601.

o 1991 WILEY-LISS, INC.

Arginine is a precursor of nitric oxide, a recently identified neurotransmitter (Garthwaite et al., '88, '89; Bult et al., '90; Ross et al., '90). Nitric oxide synthase is localized in some population of neurons (Bredt et al., '90) and produce citrulline from arginine (Palmer and Moncada, '89; Bredt and Snyder, '90). Thus ASS, argininosuccinate lyase, and nitric oxide synthase may form a cyclic metabolic pathway to produce nitric oxide from aspartate (Rees et al., '89; Garthwaite, '91). Recently argininosuccinate is suggested

Accepted July 23,1991. Address reprint requests to Dr.Hiroyuki Nakamura, who is now at the Laboratory of Neuropsychology, NIMH, Bldg. 9, Rm 1E104,Bethesda, MD 20892.

653

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN Abbreviations 3v 4 4n 4v 6 7 7n 8vn 10 12

I-VI

A4 AC ACo ac acp

AD AH

AHi AM AMP0 Amb AOB

AP APt As Arc AStr AV AVPO B BIC b1c BL BLP BLV BM BMP bP bsc BST BSTIA BSTLP BSTMP BSTS BSTV cc Ce CG CI CIC cic CL

c1

Cli CM CnF co Cp

CPU cu cu

CVL DC DCIC DEn DG DLG DLL DM DMTg DP DPGi DpMe DR

third ventricle trochlear nucleus root of the trochlear nerve fourth ventricle abducens nucleus facial nucleus root of the facial nerve vestibular root of the vestibulocochlear nerve dorsal motor nucleus of vagus hypoglossal nucleus layers I-Vl of the cortex anterior amygdaloid area anterior commissural nucleus anterior cortical amygdaloid nucleus anterior commissure anterior commissure, posterior part anterodorsal thalamic nucleus anterior hypothalamic area amygdalohippocampal area anteromedial thalamic nucleus anterior medial preoptic nucleus nucleus ambiguus accessory olfactory bulb area postrema anterior pretectal nucleus aqueduct arcuate hypothalamic nucleus amygdalostriatal transition area anteroventral thalamic nucleus anteroventral preoptic nucleus basal nucleus of Meynert nucleus of the brachium of the inferior colliculus brachium of the inferior colliculus basolateral amygdaloid nucleus basolateral amygdaloid nucleus, posterior part basolateral amygdaloid nucleus, ventral part basomedial amygdaloid nucleus basomedial amygdaloid nucleus, posterior part brachium pontis brachium of the superior colliculus bed nucleus of the stria terminalis bed nucleus of the stria terminalis, intraamygdaloid division bed nucleus of the stria terminalis, lateral division, posterior Part bed nucleus of the stria terminalis, medial division,posterior Part bed nucleus of the stria terminalis, supracapsular division bed nucleus of the stria terminalis, ventral division corpus callosum central amygdaloid nucleus central gray caudal interstitial nucleus of the medial longitudinal fasciculus central nucleus of the inferior colliculus commissure of the inferior colliculus nucleus centralis lateralis thalami claustrum caudal linear nucleus of the raphe nucleus centralis medialis thalami cuneiform nucleus cortical amygdaloid nucleus cerebral peduncle caudate putamen cuneate nucleus cuneate fasciculus caudoventrolateral reticular nucleus dorsal cochlear nucleus dorsal cortex of the inferior colliculus dorsal endopiriform nucleus dentate gyrus dorsal lateral geniculate nucleus dorsal nucleus of the lateral lemniscus dorsomedial hypothalamic nucleus dorsomedial tegmental area deep layers of the superior colliculus dorsal paragigantocellular nucleus deep mesencephalic nucleus dorsal raphe nucleus

DTgP ECIC ECu EP EPI F f fi

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fr FStr ft G Gi GiA GiV G1 GP GR Gr gr

GrC Gu HDB Hi

I ic ICj icp IGL ILL IMD IMLF In InCo Int

I0 IOC IOM IP IPC IPDL IPDM IPI IPL IP1 IPR IRt La Lat LC LD LDTg LDTgV LH LHb LHbL LHbM 11 LM lo LP LPB LPGi LPLC LPMC LPO LRt LRtPC LSD LSI LV LVe mcP MCPO MD MdD MdV

dorsal tegmental nucleus, pericentral part external cortex of the inferior colliculus external cuneate nucleus entopeduncular nucleus external plexiform layer of olfactory bulb nucleus of the field of Fore1 fornix fimbria of the hippocampus fasciculus lentiformis fasciculus retroflexus fundus striati fasciculus thalamicus nucleus gelatinosus thalami gigantocellular reticular nucleus gigantocellular reticular nucleus, alpha part gigantocellular reticular nucleus, ventral part glomerular layer of the Olfactory bulb globus pallidus granular layer of the cerebellar cortex gracile nucleus gracile fasciculus granular layer of the cochlear nuclei gustatory thalamic nucleus nucleus of the horizontal limb of the diagonal band hippocampus intercalated nuclei of the amygdala internal capsule islands of Calleja inferior cerebellar peduncle intergeniculate leaflet intermediate nucleus of the lateral lemniscus intermediodorsal thalamic nucleus interstitial nucleus of the medial longitudinal fasciculus intermediate layers of the superior colliculus intercollicular nucleus interposed cerebellar nucleus inferior olivary nuclear complex inferior olive, subnucleus C of the medial nucleus inferior olive, medial nucleus interpeduncular nucleus interpeduncular nucleus, caudal subnucleus int.erpeduncular nucleus, dorsolateral subnucleus interpeduncular nucleus, dorsomedial subnucleus interpeduncular nucleus, intermediate subnucleus interpeduncular nucleus, lateral subnucleus internal plexiform layer of the olfactory bulb interpeduncular nucleus, rostral subnucleus intermediate reticular nucleus lateral amygdaloid nucleus lateral cerebellar nucleus locus ceruleus nucleus lateralis dorsalis thalami laterodorsal tegmental nucleus laterodorsal tegmental nucleus, ventral part lateral hypothalamic area lateral habenular nucleus lateral habenular nucleus, lateral part lateral habenular nucleus, medial part lateral lemniscus lateral mammillary nucleus lateral olfactory tract nucleus lateralis posterior thalami lateral parabrachial nucleus lateral paragigantocellular nucleus nucleus lateralis posterior thalami, laterocaudal part nucleus lateralis posterior thalami, mediocaudalpart lateral preoptic area lateral reticular nucleus lateral reticular nucleus, padcellular part lateral septal nucleus, dorsal part lateral septal nucleus, intermediate part lateral ventricle lateral vestibular nucleus middle cerebellar peduncle magnocellular preoptic nucleus nucleus medialis dorsalis thalami medullary reticular nucleus, dorsal part medullary reticular nucleus, ventral part (continued 1

H. NAKAMURA ET AL.

654 Abbreviations (continued) Me me5 Med MG MGD MGV MHb ML ml mlf MM MMn MnPO Mo mP MPA MPB MPO MPt

MS mt Mtr MVe MVeV NPC OlPt ON OP opt OtPt ox P P7 Pa PBg Pc PC PCRt PCRtA Pe PF PH Pir PMCo PMD PMV Pn PnC PnO PnV Po POT PPt PPTg PR Pr5 PrH PS PT PV PVA PVP PY

medial amygdaloid nucleus mesencephalic trigeminal tract medial cerebellar nucleus medial geniculate nucleus medial geniculate nucleus, dorsal part medial geniculate nucleus, ventral part medial habenular nucleus medial mammillary nucleus, lateral part medial lemniscus medial longitudinal fasciculus medial mammillary nucleus, medial part medial mammillary nucleus, median part median preoptic nucleus molecular layer of the cerebellar cortex mammillary peduncle medial preoptic area medial parabrachial nucleus medial preoptic nucleus medial pretectal nucleus medial septal nucleus mammillothalamic tract mitral cell layer medial vestibular nucleus medial vestibular nucleus, ventral part nucleus of the posterior commissure olivary pretectal nucleus olfactory nerve layer optic nerve layer of the superior colliculus optic tract optic tract nucleus of the pretectum optic chiasma Purkinje cell layer perifacial zone paraventricular hypothalamic nucleus parabigeminal nucleus paracentral thalamic nucleus posterior commissure parvicellular reticular nucleus parvicellular reticular nucleus, alpha part periventricular hypothalamic nucleus parafascicular thalamic nucleus posterior hypothalamic area piriform cortex posteromedial cortical amygdaloid nucleus premammillary nucleus, dorsal part premammillary nucleus, ventral part pontine nuclei pontine reticular nucleus, caudal part pontine reticular nucleus, oral part pontine reticular nucleus, ventral part posterior thalamic nuclear group posterior thalamic nuclear group, triangular part posterior pretectal nucleus pedunculopontine tegmental nucleus prerubral field principal sensory trigeminal nucleus nucleus prepositus hypoglossi parastriatal nucleus paratenial thalamic nucleus paraventricular thalamic nucleus paraventricular thalamic nucleus, anterior part paraventricular thalamic nucleus, posterior part pyramidal tract

to have a neuromodulator function on account of the different cellular localization of ASS and argininosuccinate lyase in the rat brain (Nakamura et al., '90) and the modulation of the increase in intracellular calcium produced by glutamate in isolated rat neurons (Nakamura et al., '91). Thus ASS is not only a part of nitric oxide synthetic cycle but also produces a possible neuromodulator, so that the localization of ASS in the brain is of great interest. In the present study we demonstrated the distribution of neurons and terminals with ASS by using rabbit antiserum

PYX Re Rh RMg Ro RPn RRF Rt RVL s5 sag Sch SCP SFi

SFO SI sm SNC SNR

so so1

Sol sol SOlC sox SP5 sp5c Sp5I Sp50 SPF SpVe SStr st STh StHy SubCD SubCV SuG SUM SuVe TC TS Tu tz VCA VEn

VL VLG

VM VMH VN VP

VPL VPM VRe VTA

X Y ZI ZID ZIV 21 zo

pyramidal decussation reuniens thalamic nucleus rhomboid thalamic nucleus nucleus raphe magnus nucleus of Roller nucleus raphe pontis retrorubral field reticular nucleus of the thalamus rostroventrolateral reticular nucleus sensory root of the trigeminal nerve nucleus sagulum suprachiasmatic nucleus superior cerebellar peduncle septofimbrial nucleus subfornical organ substantia innominata stria medullaris of the thalamus substantia nigra, pars compacta substantia nigra, pars reticularis supraoptic nucleus superior olivary complex nucleus of the solitary tract solitary tract nucleus of the solitary tract, commissural part supraoptic decussation spinal trigeminal tract spinal trigeminal nucleus, caudal part spinal trigeminal nucleus, interpolar part spinal trigeminal nucleus, oral part subparafascicular thalamic nucleus spinal vestibular nucleus substriatal area stria terminalis subthalamic nucleus striohypothalamic nucleus subceruleus nucleus, dorsal part subceruleus nucleus, ventral part superficial gray layer of the superior colliculus supramammillary nucleus superior vestibular nucleus tuber cinereum triangular septal nucleus olfactory tubercle trapezoid body ventral cochlear nucleus, anterior part ventral endopiriform nucleus nucleus ventralis lateralis thalami ventral lateral geniculate nucleus nucleus ventralis medialis thalami ventromedial hypothalamic nucleus vomeronasal nerve layer ventral pallidum nucleus ventralis posterolateralis thalami nucleus ventralis posteromedialis thalami ventral reuniens nucleus ventral tegmental area (Tsai) nucleus X nucleus Y zona incerta zona incerta, dorsal part zona incerta, ventral part zona limitans zonal layer of the superior colliculus

against purified rat liver ASS. The characteristic localization pattern of the enzyme indicates the existence of widely distributed systems connected with argininosuccinate or arginine in the brain.

MATERIALS AND METHODS Five male adult Wistar rats, weighing 150 to 350 g, were deeply anesthetized with 50 mgikg sodium pentobarbital (i.p.1 and perfused through the ascending aorta with 100 ml

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN of physiological saline followed by a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 M sodium phosphate buffer (pH 7.4) (PB) for 20 minutes at 4°C. The brains were immediately removed from the skull and immersed in a fixative containing 4% paraformaldehyde in PB for 1week at 4°C. The brains were then transferred to a cryoprotectant containing 30% sucrose and 0.02% sodium azide in PB for another week at 4°C and cut into 30- or 50-pm coronal or sagittal sections on a freezing microtome. Every fourth section was processed for ASS immunohistochemistry, Nissl staining, or Weigert hematoxylin staining.

94.0

6 7.0 4 3.0

30.0

Immunohistochemistry To demonstrate argininosuccinate synthetase-likeimmunoreactivity (ASS-LI), the sections were processed according to the avidin-biotin-peroxidase-complex (ABC) procedure (Hsu et al., ’81).Specific antiserum to the purified rat liver ASS was raised in rabbits. The enzyme purification procedure and characterization of the antisera are described elsewhere (Saheki et al., ’77, ’81; Ichiki et al., ’87; Imamura et al., ’87). The sections were washed in 0.3% hydrogen peroxide in 0.05 M potassium phosphate buffer (pH 7.6) (KPB) for 1 hour at room temperature, and then washed in KPB for 6 hours and in 0.1% bovine albumin in KPB overnight. They were incubated in the primary antiserum at a dilution of 1:4,000 or 1:8,000 in KPB containing 1% normal goat serum, 0.3%bovine albumin, 0.2% sodium azide, and 0.3%TritonX-100 for 5 days at 4°C. The sections were washed overnight and incubated in 0.5% biotinylated goat antirabbit IgG (Vector Labs) in JXPB containing 1% normal goat serum, 0.3% bovine albumin, and 0.3% Triton X-100 for 3 days at 4°C and were then washed overnight and incubated in 1%avidine-biotinylated horseradish peroxidase complex (Vector Labs) in KPB containing 1%goat serum and 0.3% bovine albumin. This step was followed by overnight washing in KPB and processing with cobaltglucose oxidase (Itoh et al., ’79). The sections were washed twice for 10 minutes in 0.1 M Tris buffer (pH 7.6) (TB) and incubated in 0.5% solution of cobalt chloride in TB for 10 minutes at room temperature. They were washed three times for 10 minutes in TB and then twice for 10 minutes in 0.1 M potassium phosphate buffer (pH 7.3). The sections were incubated in a reaction solution containing 0.05% 3,3’-diaminobenzidinetetrahydrochloride (DAB),0.2%p-Dglucose, 0.04% ammonium chloride, and 7 units of glucose oxidase (Toyobo) in 100 ml of 0.1 M potassium phosphate buffer (pH 7.3) for 1-6 hours at 4°C. The sections were finally washed, mounted on gelatinized slides, dried, cleared with xylene, and coverslipped. To test the specificity of immunostaining, a series of one in every eight sections was incubated in normal rabbit serum instead of antiserum, and another series was incubated in rabbit antiserum preabsorbed with purified ASS.

RESULTS The antiserum to ASS detected a 43 kDa protein as a single band in Western blot analysis of rat liver homogenate (Fig. 1).This band had the same molecular weight as the purified enzyme. This molecular weight is in good agreement with those seen in the literature (43-46 kDa) (O’Brien, ’79; Saheki et al., ’83, ’87). Several different antisera preparations raised on the enzyme gave the same histological results. The sections incubated in normal rabbit serum

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Fig. 1. Western blot analysis using rat liver homogenate shows specificity of the antiserum against argininosuccinate synthetase. Molecular weights ( m a , kilo Dalton) of markers are indicated with arrows.

or in rabbit antiserum preabsorbed with purified ASS were devoid of immunostaining. Figures 2 - 4 show the distribution of neurons, fibers, and terminal-like structures with ASS-LI throughout the rat brain. The cytoarchitectonic boundaries and the nomenclature are based on the atlas by Paxinos and Watson (’86). The other references for nomenclature are Carpenter (’83, for the subthalamic areas), Kanaselu and Sprague (‘74, for the pretectum), and Watts and Swanson (’87, for the ventral lateral geniculate nucleus). Immunoreactive material was observed mostly in the neurons but also seen in the glial cells scattered in some tracts. The size of the neurons with ASS-LI is indicated by comparison with the length of the long and short axes of the average of 10 distinctly stained neurons. The long axes of most neurons with ASS-LI were smaller than 20 pm. For convenience of description, we use the terms “small” when the long axis of neuron is less than 13 pm, “medium-size” when the long axis is 14-18 pm, and “large” when it is over 19 pm.

Telencephalon Neocortex. Medium-size pyramidal neurons (16 x 10 pm) with ASS-LI were observed in layer V and the superficial part of layer VI of the cerebral cortex (Fig. 5A). A few nonpyramidal neurons of varying sizes were also immunoreactive in layers TI, 111, V, and VI. Many small triangular and ellipsoid neurons (14 x 9 km) were immunoreactive in the deepest part of layer VI. Terminal-like or neuropile staining was seen prominently in the superficial half of layer I and weakly in layer V. Layer IV was almost devoid of ASS-LI. The distribution pattern was the same throughout the neocortex. Olfactory bulb. In the main olfactory bulb, small neurons with ovoid soma (10 x 7 pm) were immunoreactive to ASS in the periglomerular region (Fig. 5B). These immunoreactive neurons had thick dendrites with bush-like bifurcation and appeared to be external tufted cells (Fig. 5C). The

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Figs. 2-4. A series of drawings of coronal sections of the rat brain from rostra1 (Fig. 1A) to caudal (Fig. 4H) showing the distribution of ASS-like immunoreactive neurons (large dots), fibers (lines), and

terminal-like staining (small dots). Arrowheads in Figure 3D indicate patchy distribution of immunoreactive cells and terminal-like staining in the deep layer of external cortex of the inferior colliculus.

Fig. 5. Photomicrographs of ASS-like immunoreactive neurons in the sensorimotor cortex (A) and the main (B,C) and the accessory (D,E) olfactory bulb. (C) shows a ramification pattern of the dendrite of neurons with ASS-LI in the glomerular layer (GI) of the main olfactory bulb (B). Asterisk in (D) indicates a bundle of thick fibers ventromedial

to the bundles of fine fibers of the vomeronasal layer. (E) is a higher magnification photograph of a region indicated by angles in (D). Note the difference in calibers of ASS immunoreactive fibers. Bars are: 100 ym in (A), 50 yin in (B), 10 pm in (C), 100 ym in (D), and 30 pm in (E).

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morphological character and localization were similar to ASS-LI was seen in large neurons characteristic of the those of substance P-like immunoreactive neurons (Mac- medial nucleus. This is consistent with the sparsity of rides and Davis, '83). A few larger bipolar neurons (13 x 7 ASS-LI in the hippocampal formation. The segregation of pm) with moderate ASS-LI were seen in the lower part of large and small neurons on staining with antisera to ASS the glomerular layer. In the external plexiform layer and suggests the existence of distinct populations of large and the mitral cell layer, small neurons with ASS-LI were small neurons in the medial septal nucleus. The lateral septal nuclei were segregated into dorsal, occasionally observed. We sometimes observed a few fine fibers with ASS-LI in the internal plexiform layer and the intermediate, and ventral parts from the view point of type subependymallayer. In contrast to the less immunoreactive and density of neurons with ASS-LI (Figs. 2A, 6B). In the olfactory nerve layer and the glomeruli of the main olfac- dorsal part, we observed only a few immunoreactive neutory bulb, fine fibers of the vomeronasal nerve layer were rons and terminal-like grains. The intermediate part constrongly immunoreactive to ASS and the glomeruli of the tained many immunoreactive large neurons (19 x 11 pm) accessory olfactory bulb were rich in ASS immunoreactive and terminal-like grains. Within the intermediate part terminal-like staining (Fig. 5D). Just caudomedial to these staining was diflerent mediolaterally suggesting further fiber bundles, a bundle of thick fibers was also densely subdivision (Figs. 2A, 6B). In the ventral part, many stained (asterisk in Fig. 5D), Adjacent to these fibers we immunoreactive, small bipolar neurons (12 x 8 pm) were found medium-size, ovoid-shape neurons (13 x 9 pm) and observed. The ventral area, which was rich in immunoreactive cells, was separated ventrolaterally from the bed terminal-like immunoreactivity with ASS-LI (Fig. 5D,E). Medium-sizebipolar and multipolar neurons with ASS-LI nucleus of the stria terminalis by the zona limitans where (16 x 11 pm) were distributed in the deeper part of the only immunoreactive fibers were observed. In the lambdoid septal zone, medium-size (15 x 6 km) anterior olfactory nucleus around the intrabulbar part of the anterior commissure. A few immunoreactive fibers bipolar neurons were immunoreactive to ASS. In the were also observed here. These fibers could not be traced septofimbrial nucleus we observed medium-size (18 x 9 caudally in the anterior part of the anterior commissure; pm) multipolar neurons with ASS-LI (Figs. 2A, 6B). Large they appeared to continue rostrally to the subependymal fusiform or triangular neurons (21 x 10 pm) with moderand internal plexiform layer of the main olfactory bulb. ate immunoreactivity were observed in the triangular Thus these fibers may be axons of ASS-like immunoreactive septal nucleus. In the subfornical organ we observed many neurons in the anterior olfactory nucleus projecting to the small neurons with various intensities of ASS-LI (11 x 8 pm) and terminal-like grains. No fibers with ASS-LI were main olfactory bulb. The dorsal endopiriform nucleus is easily distinguished observed in the fornix or in the anterior commissure. Basal ganglia. In the caudate-putamen, ASS-LI was because a large number of neurons were densely stained in this area (Figs. 2,3A,B, 6E, 7A). The neurons with ASS-LI observed in large triangular or fusiform neurons (25 x 15 were medium in size and triangular and multipolar in shape pm) with a few, thick, aspinous and nonvaricose dendrites (16 x 10 pm), and had smooth dendrites expanded within and medium-size neurons (14 x 10 pm) with ovoid or and around the nucleus (Fig. 7A). The neuropile of the multipolar soma and a few, fine, aspinous, and nonvaricose nucleus was also stained densely. Between the dorsal dendrites (Figs. 2,6C). Terminal-like immunostaining was endopiriform nucleus and the immunoreactive neurons in observed in the neuropile between the nonimmunoreactive layer VI of the insular cortex, the claustrum was noted as fiber bundles. No compartmentalization was observed in an ASS-LI free area (Fig. 2A,B,C).In contrast to the dense the caudate-putamen. staining of the dorsal endopiriform nucleus, the ventral In the globus-pallidus, large triangular and fusiform endopiriform nucleus showed only moderate staining of the neurons (20 x 11 pm) were faintly immunoreactive in their neuropile and only a few medium-size multipolar neurons soma and strongly immunoreactive in their nucleus (Figs. (14 x 9 pm) were observed. In the piriform cortex, we 2B,C,D, 6D). Terminal-likegrains were distributed sparsely observed a few fusiform and triangular medium-size neu- around the immunoreactive neurons; fiber bundles within rons (15 x 8 pm) in layer 111. the nucleus were not immunoreactive. Large triangular Hippocampus. The hippocampal formation was less and multipolar neurons (26 x 12 pm) of the basal nucleus densely stained. In the subiculum and CA1, neurons in the of Meynert were strongly immunoreactive to ASS (Figs. deepest part of the polymorphic layer were immunoreactive BB,C,D, 6D). The neurons were scattered along the medial to ASS and were contiguous with neurons that had ASS-LI and ventral edge of the globus pallidus and in the lateral in layer V of the retrosplenial cortex. Only a few bipolar and part of the internal capsule. In the entopeduncular nucleus, multipolar neurons with ASS-LI were observed in the large fusiform and triangular neurons (20 x 9 pm) with molecular layer throughout the hippocampus and the subic- strongly immunoreactive nucleus and weakly immunoreaculum (Figs. 2D, 3A,B, 6A). Occasionallypyramidal neurons tive somata were observed. with ASS-LI were observed in the pyramidal layer. Very In the subthalamic nucleus, many medium-size trianguslight terminal-like staining was observed in the pyramidal lar neurons were immunoreactive to ASS (Figs. 3A, 6F). layer. We could not detect a clear difference among CA1, The nuclei of the immunoreactive neurons were very CA2, and CA3. In the dentate gyms, a few bipolar and densely stained with ASS-LI, whereas the somata were multipolar neurons were faintly immunoreactive to ASS in weakly immunoreactive. The neuropile was devoid of immuthe polymorphic layer (Figs. 2D, 3A,B, 6A). A few terminal- no staining. Amygdala and related structures. The amygdaloid nulike immunoreactive grains were seen in the deeper part of the granular layer with perikarya-like nonimmunoreactive clei were rather sparsely stained (Figs. 2, 3A, 6E). In the spots. nucleus of the lateral olfactory tract, a very few mediumSeptum. The septal nuclei showed heterogenous immu- size, fusiform neurons (14 x 10 pm) were observed. Terminostaining. In the medial nucleus, ASS-LI was observed in nal-like immunoreactivity was richly observed in this nua few small neurons (11 x 8 pm) with ovoid soma. No cleus. In the dorsal part of the anterior amygdaloid area,

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Fig. 6. Photomicrographs of coronal sections processed with antisera to ASS. (A) Hippocampal formation. Only a very few neurons with ASS-LI were seen in the dentate gyms (DG) and the hippocampus proper (Hi). Very weak terminal-like immunostaining was seen in the pyramidal layer. Bar: 500 ym. B. Septum at the level of anterior commissure. A large number of immunoreactive neurons were observed in the intermediate part of the lateral septal nucleus (LSI). Neurons with strong ASS-LI were also seen in the septofimbrial nucleus (SR).The fibers of the fornix (0 were devoid of immunostaining. Bar: 300 ym. C. Caudate-putamen. Large multipolar medium-size fusiform and small ovoid neurons were immunoreactive to ASS. Bar:

100 ym. D. Large, multipolar neurons with ASS-LI were observed in the basal nucleus of Meynert (B). A few small neurons were immunoreactive in the globus pallidus (GP). The internal capsule (ic) was devoid of immunoreactivity. Bar: 100 ym. E. Amygdala. A large number of neurons with ASS-LI were distributed in the medial amygdaloid nucleus (Me). A few immunoreactive neurons were distributed in the central (Ce) and basal-lateral (La, BL, BM) amygdaloid nuclei. The dorsal endopiriform nucleus was rich in both neurons and terminal-like grains with ASS-LI. Bar: 300 ym. F. A large number of immunoreactive neurons in the subthalamic nucleus (STh). Bar: 100 ym.

medium-size triangular or multipolar neurons (16 x 11 pm) were stained. In the ventral part of this area, small fusiform neurons (11 x 7 pm) were immunoreactive t o ASS. In the anterior cortical amygdaloid nucleus, mediumsize ovoid or triangular neurons (15 x 10 km) were stained.

Moderate density of terminal-like immunoreactivity also was seen in this area. In the posterolateral cortical amygdaloid nucleus, a few small ovoid neurons (12 x 7 pm) were seen to be immunoreactive to ASS. Sparse terminal-like staining was seen in the neuropile.

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Fig. 7. Photomicrographs of immunoreactive neurons in the dorsal endopiriform nucleus (A), medial amygdaloid nucleus (B),and the ventral pallidum (0. Scale bars: 50 km.

In the posteromedial cortical amygdaloid nucleus, medium-size triangular and multipolar neurons (15 x 8 pm) were immunoreactive to ASS. In the amygdalohippocampal transition area, small ovoid neurons (13 x 8 pm) were immunoreactive. Weak terminal-like staining also was seen in these areas. The lateral amygdaloid nucleus was almost devoid of immunoreactive neurons except for a very few small neurons (13 x 8 pm) with weak ASS-LI. In the basolateral amygdaloid nucleus, a few small triangular and multipolar neurons (13 x 8 pm) were weakly immunoreactive to ASS. Weak terminal-like immunoreactivity was seen in the neuropile. In the ventral basolateral nucleus, a few mediumsize bipolar and triangular neurons (14 x 7 +m) with ASS-LI were observed. In the basomedial nucleus, a few small neurons were faintly immunoreactive to ASS. Granular staining of the neuropile was also seen in the basolateral and basomedial nuclei. In the anterodorsal part of the medial amygdaloid nucleus, medium-size bipolar and triangular neurons (17 x 8 pm) were immunoreactive to ASS. The anteroventral part was almost free of neurons with ASS-LI and only faintly immunoreactive neurons were observed. In the posteroventral part of the medial amygdaloid nucleus, many mediumsize triangular neurons (15 x 9 pm) were seen with moderate ASS-LI (Fig. 7B). The posterodorsal part of the medial amygdaloid nucleus appeared to be subdivided further on the basis of the ASS-like immunostaining tendency; the posterodorsal part was further divided into dorsomedial,

ventromedial, and ventrolateral parts. The dorsomedial and ventromedial parts had sparser terminal-like staining and immunoreactive neurons than the ventromedial part. In the dorsomedial part, small to medium-size ovoid and spheroid neurons (14 x 9 pm) were observed, whereas in the ventromedial part, medium-size neurons (17 x 11 pm) of bipolar, triangular, or ovoid shape were observed. The ventrolateral part was rich in small ovoid neurons (13 x 9

wd.

In the central amygdaloid nucleus, a few small fusiform (9 x 6 pm) and larger multipolar (13 x 9 pm) neurons were observed (Fig. 6E). Terminal-like staining of the neuropile was slightly denser in the lateral division than the medial division. The intercalated nuclei of the amygdala were free of ASS-like immunoreactive cells. In the core of the nucleus accumbens, a few medium-size (16 x 10 pm) multipolar and small (10 x 6 pm) spheroidal neurons with ASS-LI were observed. The neuropile was faintly stained as in the caudate-putamen. In the shell of the nucleus accumbens several medium-size (17 x 10 pm) multipolar neurons and moderate density of neuropile staining were observed. In the fundus striati a few small (13 x 7 pm) bipolar and triangular neurons were observed. The neuropile was also weakly stained. In the diagonal band, a few small ovoid neurons (10 x 6 pm) and a very few medium-size multipolar neurons (17 x 11 pm) were stained. The nucleus of the horizontal limb of the diagonal band contained few immunoreactive cells. The ventral pallidum was characterized by some

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Dorsal thalamus. The anterior nuclei were rich in medium-size multipolar neurons (17 x 8 pm), which were strongly immunoreactive to ASS (Figs. 2A,B, 7 0 . In the ASS-like immunoreactive neurons. The neurons in the substriatal area, a few medium-size multipolar neurons anterodorsal nucleus were the most strongly immunoreac(16 x 11 pm) were seen with ASS-LI. The neuropile was tive to ASS, whereas the anteroventral nucleus was weakly immunoreactive. In the anterodorsal nucleus, many mestained moderately. In the magnocellular preoptic nucleus large multipolar dium-size triangular and multipolar neurons (16 x 11 pm) neurons (21 x 14 pm) with ASS-LI were observed (Fig. were strongly immunoreactive to ASS (Figs. ZC, 8B). The 2A). The neuropile contained a few terminal-like grains nuclei of the neurons were more strongly immunoreactive with ASS-LI.In the substantia innominata, several medium- than the somata. In the anteroventral nucleus, only the size bipolar or triangular neurons (18 x 8 pm) were moder- nuclei of the neurons were weakly immunoreactive to ASS ately immunoreactive to ASS (Figs. ZA,B,C). Weak immu- (Fig. 2C). The neuropile was also weakly immunoreactive. nostaining was also seen in these areas. In the anteromedial nucleus, a lot of large triangular and In the anterior divisions of the bed nucleus of the stria multipolar neurons (19 x 10 pm) with ASS-LI were obterminals (the dorsal, juxtacapsular, and ventral parts of served (Figs. ZC, 8C). The nucleus was more densely the lateral division, the intermediate and ventral divisions, stained than the soma in these neurons. A few terminal-like and the anterior part of the medial division in the atlas of grains were also stained. Paxinos and Watson ('86)), a few medium-size fusiform The nucleus ventralis lateralis, nucleus ventralis posteroneurons (14 x 7 pm) with weak immunostaining and a lateralis, and the nucleus ventralis posteromedialis were weak terminal-like staining were observed. The immunore- devoid of ASS-LI (Figs. 2D, 3A). In the nucleus ventralis active neurons were rather densely distributed in the medialis (Fig. 2D), large multipolar neurons (19 X 10 pm) anterior part of the medial division and very sparsely in the with ASS-LI were observed. A few fibrous or terminal-like dorsal, juxtacapsular, and ventral parts of the lateral grains were also seen in the neuropile. The nucleus gelatinodivision and the ventral division. Many neurons with sus thalami was free of ASS-LI. ASS-LI were observed in the posteromedial and posterolatThe nucleus lateralis dorsalis was free of neurons with era1 parts of the medial division of the bed nucleus of the ASS-LI (Figs. 2D, 3A) except for its lateral rim. In the stria terminalis. In the posteromedial part of the medial lateral rim of the ventrolateral part, some strongly immudivision, medium-size fusiform neurons (15 x 9 pm) were noreactive medium-size multipolar neurons (18 x 11 pm) densely immunoreactive to ASS with a few areas of terminal- were observed. In the medial part of the nucleus lateralis like immunoreactivity. In the posterointermediate part of posterior, some medium-size fusiform and triangular neuthe medial division, only a few medium-size fusiform and rons (17 x 10 pm) were immunoreactive to ASS, whereas oval neurons (14 x 9 pm) were weakly immunoreactive to the lateral part was devoid of immunostaining. ASS, and there was very little terminal-like staining. In the In the rostral part of the posterior complex of the posterolateral part of the medial division, medium-size thalamus situated dorsomedial to the ventrobasal complex, triangular neurons (15 x 11 pm) with moderate ASS-LI a few medium-size multipolar neurons (15 x 10 pm) were and terminal-like grains were observed. In the posterior immunoreactive to ASS (Fig. 3A). In the caudal part of the part of the lateral division, we saw a few large neurons posterior complex including the suprageniculate nucleus, (19 x 10 pm) with weak ASS-LI and moderate staining of many medium-size triangular and multipolar neurons with the neuropile. ASS-LI were observed (Figs. 3B, 8E). Terminal-like immuIn the islands of Calleja, a few medium-size fusiform nostaining was also seen in this area. In the triangular part neurons (18 x 9 pm) with ASS-LI were observed (Fig. 2A). of the posterior complex of the thalamus, small triangular A few terminal-like grains were observed among nonimmu- neurons (13 x 7 pm) were immunoreactive to ASS. noreactive spots of neuronal sizes. In the polymorph layer In the nucleus medialis dorsalis, three differently immuof the olfactory tubercle and the areas between the islands noreactive zones were observed: anteromedial and posteroof Calleja, large bipolar or triangular neurons (19 x 11pm) lateral immunoreactivity-free zones and an intermediate were immunoreactive to ASS. The profile of the immunore- immunoreactive cell rich zone. At its rostral levels (Figs. active neurons was different from that in the ventral 2D, 9A), the intermediate zone with immunoreactive neupallidum. The neuropile in the olfactory tubercle and the rons occupied the lateral part of the nucleus, and the area between the islands of Calleja was rich in terminal-like anteromedial non-immunoreactive zone was seen in the immunoreactivity . medial part. At more caudal levels the intermediate zone White matter. The white matter under the cerebral with immunoreactive neurons was situated more medially cortex, the internal capsule, and the corpus callosum are with the nonimmunoreactive posterolateral zones in the devoid of immunostaining. lateral part of the nucleus and the nonimmunoreactive anteromedial zone in the medial part (Fig. 9B). At caudal Diencephalon levels of the nucleus, the intermediate zone with stained Epithalarnus In the medial habenular nucleus, the neurons was seen in the medial two-thirds of the nucleus soma of medium-size ovoid and ellipsoid neurons (14 x 10 with the nonimmunoreactive posterolateral zone in the pm) were very weakly immunoreactive to ASS (Fig. 8A). In lateral part (Figs. 3A, 9C). The immunoreactive neurons in the medial part of the lateral habenular nucleus, medium- the intermediate zone were medium in size (18 x 11 pm) size fusiform, triangular, and multipolar neurons (15 x 8 and triangular or multipolar in shape (Fig. 10A). In the nucleus centralis medialis and nucleus centralis p m ) with strong ASS-LI were observed (Figs. 2D, 3A, 8A). Granular immunostaining was seen in the neuropile. The lateralis, a large number of medium-size triangular and lateral part of the lateral habenular nucleus was devoid of multipolar neurons (18 X 11 pm) were immunoreactive to immunostaining (Fig. 3A). In the stria medullaris thalami, ASS (Figs. 2D, 3A, 8D, 9 , 1 0 0 . In the paracentral thalamic a few fibers and a few glial cells were immunoreactive to nucleus, large fusiform neurons (20 x 8 pm) were immunoreactive to ASS (Figs. 2C,D). The parafascicular nucleus was ASS.

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Fig. 8. Photomicrographs taken from thalamus showing neurons with ASS-LI. A. Immunoreactive neurons in the habenular nuclei. Densely immunoreactive neurons and terminal-like grains were distributed in the lateral habenular nucleus (LHb) (the medial part) and a few weakly immunoreactive neurons were seen in the medial habenular nucleus (MHb). B. Abundant, densely immunoreactive neurons in the anterodorsal nucleus (AD). Only a few neurons with ASS-LI were

observed in the anteroventral nucleus (AV). C. Many densely immunoreactive neurons in the anteromedial nucleus. D. Immunoreactive neurons in the nucleus centralis lateralis (CL). E. Immunoreactive neurons and a few terminal-like grains in the posterior complex of the thalamus (Po). F. Neurons with ASS-LI in the ventral reuniens nucleus. The same scale is used from (A) to (F).Bar: 100 pm.

free of neurons with ASS-LI (Figs. 3A, 9C, D). In the fasciculus retroflexus, a small amount of fine fibrous staining was seen. A large number of neurons with ASS-LI were localized in the midline thalamic nuclei. In the paraventricular nucleus, many medium-size ovoid and triangular neurons (14 x 10 pm) with ASS-LI were observed (Figs. 2C,D, 3A, 9A,B,C, 10B). Granules with ASS-LI were also seen in the neuropile. In the paratenial thalamic nucleus, a very small

number of medium-size triangular and multipolar neurons (17 x 11 pm) were observed (Fig. 2C). The neuropile was devoid of immunostaining. In the intermediodorsal thalamic nucleus, medium-size fusiform and triangular neurons (17 x 11 pm) with ASS-LI were observed (Figs. 9A,B,C). In the interanteromedial thalamic nucleus, medium-size triangular or multipolar neurons (18 x 11 pm) were irnmunoreactive to ASS. In the rhomboid thalamic nucleus, medium-size multipolar neurons (14 x 10 pm)

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Fig. 9. Distribution of neurons with ASS-LI in the nucleus medialis dorsalis (MD) of the thalamus. Coronal sections with a 500-pm interval arranged from rostral (A) to caudal (D). The parafascicular nucleus (PF) was devoid of immunostaining. Bar: 500 pm.

were weakly immunoreactive to ASS. A few terminal-like so-called intergeniculate leaflet (Watts and Swanson, '871, grains were also seen in the neuropile. In the nucleus and we temporarily use this term here since we did not yet reuniens, medium-size triangular or multipolar neurons have appropriate nomenclature (Nakamura and Kawa(15 x 10 pm) with ASS-LI were observed. The nucleus mura, '88). Terminal-like grains with ASS-LI were also reuniens ventralis was rich in neurons with ASS-LI (Figs. seen in this area. In the dorsal part of the zona incerta, medium-size 2C,D, 8F). The neurons were ellipsoid or triangular in shape and medium in size (18 x 9 pm). Terminal-like spindle-shape neurons (17 x 7 pm) with ASS-LI were observed in its ventral edge adjacent to the ventral part grains with ASS-LI were also seen in the neuropile. The dorsal lateral geniculate nucleus was free of ASS-like (Figs. 3A, 11B). Terminal-like immunoreactivity was obimmunostaining (Figs. 3B, 11A). The ventral and magnocel- served in the dorsal part of the zona incerta. The ventral lular parts of the medial geniculate nucleus were also part of the zona incerta was devoid of immunostaining. In devoid of neurons and terminals with ASS-LI (Figs. 3B, the fasciculus lentiformis medium-size fusiform neurons 11A). In the dorsal part of the medial geniculate nucleus, a (17 x 5 pm) were stained. A small amount of fibers with few medium-size multipolar neurons (18 x 11 pm) with ASS-LI were also seen in this area. In the fasciculus ASS-LI were observed. thalamicus a few large, elongated triangular neurons (21 x 7 Ventral thalamus. In the dorsal and medial parts of the pm) were immunoreactive to ASS. A few fibers were also reticular nucleus of the thalamus, nuclei of medium-size stained. In the nucleus of the field of Forel, many large triangular and multipolar neurons were strongly immuno- multipolar neurons (26 x 11 pm) with ASS-LI were obreactive to ASS. The somata of the neurons were very served (Figs. 3A, 11C).In the rostral part of the subparafasweakly immunoreactive. Medium-size fusiform neurons cicular nucleus, a lot of large fusiform neurons (23 x 9 pm) (15 x 7 pm) were densely stained in the most lateral part of with moderate ASS-LI were observed (Figs. 3A, 11D). In the lateral magnocellular part of the ventral lateral genicu- the caudal, parvicellular part of the subparafascicular late nucleus (Figs. 3B, 11A). A few larger neurons in the nucleus, many medium-size fusiform neurons (17 X 5 pm) lateral magnocellular part were moderately immunoreac- were immunoreactive to ASS (Fig. 3B). Fibrous or terminaltive to ASS. In the medial parvicellular part of the nucleus, like staining was also seen densely in this area. small triangular neurons (12 x 5 km) with ASS-LI were Hypothalamus. In the anterior medial preoptic nuobserved. In the dorsomedial small part of the nucleus cleus, many medium-size multipolar neurons (17 x 10 pm) medium-size fusiform and ellipsoid neurons (17 x 9 pm) with ASS-LI were observed (Fig. 2A), as were terminal-like were immunoreactive to ASS. This part is identical to the grains in the neuropile. The median preoptic nucleus was

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Fig. 10. Photomicrographs of the stained neurons in the nucleus medidis dorsalis (A), paraventricular nucleus (B), and nucleus centralis medialis (Ci of the thalamus, and the optic tract nucleus of the pretectum (D).Scale bar: 50 km.

rich in weakly stained, small neurons (13 x 7 pm). In the medial preoptic nucleus, medium-size fusiform and triangular neurons (15 x 9 pm) were weakly immunoreactive to ASS (Fig. 2B). In the medial preoptic area, many mediumsize multipolar neurons (17 x 10 pm) with ASS-LI were observed (Fig. 2B). In the lateral preoptic area, a few immunoreactive neurons of similar size were also observed (Figs. 2A,B). In the striohypothalamic nucleus, a few small neurons (11 x 8 pm) with faint ASS-LI were seen. In the anterior commissural nucleus, medium-size triangular neurons (16 x 9 pm) were moderately immunoreactive to ASS. In the supraoptic nucleus, large cuboidal neurons (27 x 25 pm) with ASS-LI were seen in the dorsal part of the nucleus (Figs. 2B,C). In the suprachiasmatic nucleus, small ellipsoid neurons (13 x 7 pm) with ASS-LI were observed (Figs. 2B, 11F). Terminal-like staining was also seen in the ventral part of the nucleus. Immunoreactive fibers were observed in the supraoptic decussation. No immunoreactivity was seen in the optic decussation. In the lateroanterior hypothalamic nucleus, small neurons with weak immunoreactivity were observed. In the anterior part of anterior hypothalamic area, medium-size fusiform or ovoid neurons (16 x 8 pm) were weakly stained. In the central part of the anterior hypothalamic area, small ellipsoid neurons (13 x 8 pm) were weakly immunoreactive to ASS. In the posterior part of the anterior hypothalamic area, both medium-size triangular (18 x 9 pm) and small ellipsoid (12 x 9 pm) neurons with ASS-LI were observed. In this nucleus termi-

nal-like grains were also seen in the neuropile. In the lateral hypothalamic area, large triangular or multipolar (23 x 13 pm) and medium-size fusiform or triangular (17 x 8 pm) neurons were immunoreactive to ASS (Figs. 2C,D, 3A). In the tuber cinereum, large multipolar neurons (20 x 10 pm) with moderate ASS-LI were observed (Figs. 2C,D). Around the immunoreactive neurons, terminal-like staining of the neuropile was also seen. In the retrochiasmatic area, large multipolar neurons with strong immunoreactivity were seen (Fig. 2C). In the anterior parvicellular area of the paraventricular hypothalamic nucleus, honeycomb-like neuropile staining with nonimmunoreactive spots was observed. In the ventral part of this honeycomb structure, large multipolar neurons with weak ASS-LI were observed. In the dorsomedial cap of this nucleus, medium-size triangular and multipolar neurons with weak ASS-LI were observed. Honeycomb structure was again seen in the medial parvicellular part, suggesting that small neurons in the paraventricular nucleus were nonimmunoreactive. In the posterior magnocellular part of the nucleus, large fusiform and triangular neurons (23 x 12 pm) were immunoreactive to ASS (Fig. 11E). The neuropile of the paraventricular nucleus was rich in terminal-like immunoreactivity. In the periventricular hypothalamic nucleus, a few small ovoid neurons (12 x 9 pm) with strong ASS-LI were seen (Fig. 2C). In the diffuse part of the dorsomedial hypothalamic nucleus, medium-size fusiform or triangular neurons

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Fig. 11. Photomicrographs from the ventral thalamus (A,B,C,D) and the hypothalamus (E,F,G).A. Distribution of neurons with ASS-LI in the ventral lateral geniculate nucleus (VLG) and the intergeniculate leaflet (IGL). The medial geniculate nucleus (MG) was devoid of ASS-LI. Bar: 300 pm. B. Distribution of immunoreactive neurons in the zonaincerta and adjacent structures. Bar:200 pm. C. Immunoreactive neurons in the field of Forel. Bar: 100 pm. D. Immunoreactive neurons in the subparafascicular nucleus. Bar: 50 pm. E. Neurons and terminal-like grains with ASS-LI in the paraventricular nucleus of the hypothalamus (Pa). Bar: 50 pm. F. ASS-LI in the suprachiasmatic nucleus (Sch). Bar: 100 pm. G. Distribution of ASS-like immunoreactive neurons and terminal-like structures in the mammillary nucleus. Bar: 500 pm.

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(15 x 7 pm) with ASS-LI were observed (Fig. 2D). TerminalSuperior colliculus and related structure. In the superlike staining was also seen in the neuropile. In the compact ficial grey layers of the superior colliculus, a few small part, the granular neuropile staining had a honeycomb ellipsoid neurons (12 x 7 pm) were observed (Fig. 3C). A appearance. The ventromedial hypothalamic nucleus and few terminal-like grains were also seen in these layers. In its dorsolateral proximity were noted as an area with sparse the intermediate layers a few terminal-like grains were terminal-like immunostaining. In the dorsomedial part of distributed in a lattice-frame pattern (Figs. 3C, 12B). Large the ventromedial nucleus, medium-size fusiform and trian- fusiform and triangular neurons with moderate ASS-LI gular neurons (16 x 11 bm) with ASS-LI were observed. A were observed in the vertical frame of the lattice structure. few small fusiform and ovoid neurons (13 x 10 pm) were Medium-size ellipsoid neurons with strong ASS-LI were scattered in the other area of the nucleus. In the arcuate seen in the interlattice areas. In the deep layers of the hypothalamic nucleus, small ellipsoid neurons (13 x 9 pmj superior colliculus, medium-size fusiform neurons with were moderately stained. Terminal-like grains were also weak ASS-LI were observed. A patchy distribution of seen in the neuropile. terminal-like grains was also seen in these layers. In the In the posterior hypothalamic area, medium-size fusi- medial part of the deep layers, medium-size fusiform and form neurons (16 x 9 pm) with ASS-LI were observed. triangular neurons (17 x 11 pm) were strongly immunoreTerminal-like grains were also seen in the neuropile. In the active to ASS. This part was free from neuropile staining. submammillothalamic nucleus, medium-size multipolar In the commissural part of the superior colliculus, small neurons (18 x 14 pm) with strong immunoreactivity were ellipsoid neurons (13 x 9 pm) were with ASS-LI. In this compactly accumulated. In the dorsal part of the premam- part a few terminal-like grains were also seen. millary nucleus, a large number of medium-size ellipsoid, In the parabigeminal nucleus, faintly stained neurons fusiform, and triangular neurons (16 x 10 km) with strong were observed (Fig. 3C). In the subbrachial nucleus, meASS-LI were observed (Fig. 3A). In the ventral part of the dium-size fusiform neurons (16 x 8 pm) with ASS-LI were premammillary nucleus, ovoid and multipolar neurons observed. Terminal-like grains were also seen in the neurowere moderately immunoreactive to ASS. Terminal-like pile. grains were also seen in the neuropile. Inferior colliculus and related structures. The core of The mammillary body was easily demarcated on the basis the inferior colliculus was free of ASS-LI except for a very of immunoreactivity to ASS (Figs. 3B, 11G). In the median few, weakly immunoreactive, medium-size triangular neupart of the medial mammillary nucleus, medium-size bipo- rons (Fig. 3D). In the lateral part of the dorsal cortex, large lar and triangular neurons with strong ASS-LI were densely multipolar neurons (19 x 11 pm) were immunoreactive to packed as vertically oriented cell-column in the middle of ASS. In the medial part of the dorsal cortex, medium-size the mammillary nucleus (Fig. 11G). A few terminal-like fusiform neurons (15 x 9 pmj and terminal-like grains grains also were seen in this part. The medial part of the with ASS-LI were distributed between nonimmunoreactive medial mammillary nucleus had low ASS-like immunoreac- commissural fibers of the inferior colliculus. In the superfitivity. Weakly stained, medium-size ovoid and triangular cial layer of the external cortex of the inferior cortex, neurons (14 x 8 pm) were observed in this part. In the medium-size fusiform neurons (16 x 9 pm) with ASS-LI lateral part of the medial mammillary nucleus, small ovoid were observed (Figs. 3D, 12C). Terminal-like immunoreacand triangular neurons (13 x 8 pm) were immunoreactive tive grains were also seen in this layer. In the deep layer of to ASS. In the lateral mammillary nucleus, faintly stained, the external cortex of the inferior colliculus, a few patches medium-size multipolar neurons were seen. In the supra- of weakly immunoreactive, small ellipsoid neurons (13 x 8 mammillary nucleus, a large number of medium-size ellip- pm) were observed (arrowheads in Figs. 3D, 12C). soid neurons with ASS-LI were observed. Terminal-like In the nucleus of the brachium of the inferior colliculus, staining was also seen in this area. In the mammillotha- medium-size fusiform and triangular neurons (15 x 9 pm) lamic tract, a few fine fibers with ASS-LI were observed. No with strong ASS-LI were observed (Fig. 3C). A few immuimmunoreactive fibers were seen in the fornix. noreactive grains were seen in the neuropile. The brachium of the inferior colliculus was rich in immunoreactive fibers. Midbrain The dorsal and intermediate nuclei of the lateral lemniscus Pretectum. The anterior pretectal nucleus was almost were devoid of ASS-LI. In the ventral nucleus of the lateral free of ASS-LI (Figs. 3B, 12A). In the optic tract nucleus, lemniscus, small triangular neurons (13 x 9 pm) were large multipolar neurons (19 x 10 pm) with distinct smooth weakly immunoreactive to ASS. No terminal-like grains dendrites were observed (Figs. 10D, 12A). Terminal-like were observed in the nuclei of the lateral lemniscus. In the staining was seen in the neuropile. The olivary pretectal nucleus sagulum, medium-size ellipsoid neurons (16 x 9 nucleus was noticed as an immunoreactive cell sparse area pm) with ASS-LH were observed. The neuropile was also above the optic tract nucleus. In the posterior pretectal rich in terminal-like grains. The superior olivary complex nucleus, small ovoid and ellipsoid neurons (13 x 7 pm) was devoid of ASS-LI. Central gray. In the dorsal part of the central gray at were immunoreactive to ASS. A little granular staining of the neuropile was also seen in this area. In the medial the level of the pretectum, a few small fusiform neurons pretectal nucleus, a few small to medium-size ovoid and (12 x 7 pm) were observed, whereas in the ventral part a ellipsoid neurons (14 x 8 pm) were weakly immunoreac- few medium-size neurons were stained. Terminal-like grains tive to ASS. Terminal-like staining was also seen this area. were also seen in this area. The Darkschewitsch nucleus In the nucleus of the posterior commissure, medium-size was free of terminal-like grains and contained weakly fusiform and triangular neurons (16 x 8 pm) with ASS-LI immunoreactive small neurons. At the level of the superior colliculus, the central gray were observed. Terminal-like staining was also seen in this area. In the magnocellular nucleus of the posterior commis- was easily divided into the medial (central) part and the sure, large triangular neurons with weak ASS-LI were circumscribed (peripheral) part (Fig. 3C). The peripheral part was further divided into the dorsal, dorsolateral, observed.

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Fig. 12. Photomicrographs showing neurons and terminal-likestaining with ASS-LI from the midbrain. A. Pretectum. Bar: 500 pm. B. Lattice-frame distribution of terminals in the intermediate layer (In) of the superior colliculus. Bar: 200 pm. C. Inferior colliculus. Arrowheads indicate patchy distribution of immunoreactive neurons and terminal-

like staining in the deep layer of external cortex of the inferior colliculus. Bar: 100 pm. D. Ventral part of the central gray just dorsal to the supraoculomotor central gray. Bar: 50 pm. E.Red nucleus. Bar: 100 pm. F.Interpeduncular nucleus. Bar: 200 pm.

lateral, ventral, and supraoculomotor parts. The medial part was richer in terminal-like grains than the circumscribed part. A very few medium-size fusiform or ellipsoid neurons (14 x 8 km) with ASS-LI were observed in this area. In the dorsal part few immunoreactive grains were observed except in the medial dorsal part where the grain was more densely distributed. A few medium-size weakly immunoreactive neurons (18 x 11 pm) were observed. The dorsolateral part was rich in both neurons and terminallike grains. The neurons in this area were small (13 x 8 prn) and ovoid in shape. In the lateral part a few medium-

size weakly immunoreactive fusiform neurons (15 x 7 km) were seen. The ventral part was easily distinguished in the central gray by the abundance of neurons with strong ASS-LI (Figs. 3C, 12D). The neurons were fusiform and triangular in shape and medium in size (18 x 9 pm). Granular substance was also seen in the neuropile. At the level of the inferior colliculus, both weakly immunoreactive medium-size triangular neurons (18 x 11 pm) and strongly immunoreactive small ellipsoid neurons (11 x 8 pm) were observed in the dorsal part of the central gray (Fig. 3D). Terminal-like grains also were seen this

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area. In the lateral part of the central gray, small fusiform rons (23 x 13 pm) were observed (Fig. 3D). The neuropile neurons (13 x 7 pm) were immunoreactive to ASS. Termi- was also rich in immunoreactive grains. In the rostral linear nucleus, both large (27 x 11 pm) and medium-size nal-like grains were also seen this area. Oculomotornuclei and related structures. No immunor- fusiform neurons (16 x 8 pm) with ASS-LI were observed. eactive neurons and only a very few varicose fibers with In the caudal linear nucleus, medium-size fusiform neurons ASS-LI were observed in the somatic oculomotor and (14 x 7 pm) were immunoreactive to ASS. A large number trochlear nuclei. In the Edinger-Westphal nucleus, large of terminal-like grains also were observed between nonimfusiform and triangular neurons with moderate ASS-LI munoreactive raphe fibers. In the median raphe nucleus were observed. A few terminal-like grains were also seen in both large triangular (20 x 11 pm) and small ellipsoid this area. In the supraoculomotor central gray, a few (12 x 6 pm) neurons with ASS-LI were observed. A few medium-size fusiform neurons (15 x 7 pm) were immuno- terminal-like grains were also seen in the neuropile. In the reactive to ASS. The neuropile was devoid of immunostain- paramedian raphe small ellipsoid neurons (12 x 7 pm) ing. No immunoreactive neurons were seen in the para- were densely stained. Terminal-like grains were also seen in trochlear nucleus. Granular immunostaining was seen in this area. In the rostral subnucleus of the interpeduncular nucleus, the neuropile. In the interstitial nucleus of the medial longitudinal small ovoid and ellipsoid neurons (13 X 8 pm) were strongly fasciculus at the rostral level of the superior colliculus, immunoreactive t,o ASS (Fig. 12F). The ASS-LI neurons large triangular neurons (19 x 7 pm) and terminal-like were densely distributed in the ventral part of the subnugrains with ASS-LI were observed among the nonimmuno- cleus. Terminal-like immunostaining was seen in the neuroreactive fibers of the medial longitudinal fasciculus. In the pile. In the caudal subnucleus, a few small neurons (13 x 9 greater part of the nucleus of the medial longitudinal pm) with ASS-LI were seen. The intermediate subnucleus fasciculus, large triangular and multipolar neurons (20 x 10 was devoid of stained neurons. In the lateral subnucleus a very few medium-size ellipsoid neurons with ASS-LI were pm) were immunoreactive to ASS. Tegmentum. At the level of the pretectum, a few me- observed. A few terminal-like grains also were seen in this dium-size triangular and multipolar neurons (18 x 9 pm) subnucleus. In the dorsolateral subnucleus a large number with ASS-LI were observed in the deep mesencephalic of medium-sizeovoid neurons and terminal-like grains with nucleus (Fig. 3B). This group of neurons seemed to be ASS-LI were seen. In the dorsomedial subnucleus many distributed continuously in the dorsal part of the deep small, weakly immunoreactive neurons were observed. mesencephalic nucleus at the level of the superior collicu- Terminal-like grains were also seen in this area. In the lus. In the ventral part of the nucleus at this level, no interfascicular nucleus medium-size fusiform neurons immunoreactivity was observed. In the intercollicular nu- (15 x 8 pm) with ASS-LI were observed. Substantia nigru andrelated structures. In the substancleus, strongly immunoreactive, medium-size fusiform or tia nigra pars compacta, medium-size fusiform and triangutriangular neurons (16 x 9 pm) were observed. In the magnocellular part of the red nucleus, large lar neurons (17 x 9 pm) were seen with ASS-LI (Fig. 3B). neurons (39 x 25 pm) were immunoreactive to ASS (Fig. Terminal-like grains with ASS-LI were also observed. In 12E). No immunoreactivity was seen in the neuropile. The the pars reticulata, a few, large fusiform and triangular parvicellular part was free of ASS-like immunoreactivity. neurons (25 x 11 pm) were weakly immunoreactive to In the prerubral field, medium-size elongated triangular ASS. In the ventral tegmental area, large, triangular and neurons (18 x 6 pm) were scattered among the nonimmu- multipolar neurons (20 x 10 pm) with strong ASS-LI were noreactive superior cerebellar peduncle. In the retrorubral observed. Terminal-like immunoreactivity also was seen field large triangular neurons (20 x 8 pm) with strong richly in this area. In the paranigral nucleus both small ASS-LI were observed. Terminal-like grains also were seen (11 x 6 pm) and medium-size (17 x 8 pm) triangular in this area. In the paralemniscal nucleus, medium-size neurons with ASS-LI and terminal-like immunoreactive fusiform neurons (16 x 7 pm) with ASS-LI were observed. grains were observed. In the peripeduncular nucleus, a lot In the cuneiform nucleus medium-size fusiform neurons of medium-size fusiform neurons (17 x 7 pm) were immu(17 x 8 pm) with moderate ASS-LI were observed (Fig. noreactive to ASS. The neuropile was rich in terminal-like 3D). Terminal-likegrains were also seen in the neuropile. grains. In the pedunculopontine tegmental nucleus, large multipolar neurons (24 x 12 pm) were immunoreactive to ASS Cerebellum (Fig. 3 0 . A few immunoreactive grains also were seen in Small ovoid neurons (11 x 7 pm) with strong ASS-LI this area. In the anterior and ventral tegmental nuclei a few immunoreactive neurons were observed. A few terminal- were observed in the Purkinje cell layer and in the deep part like grains were seen in these nuclei. In the subpeduncular of the molecular layer (Figs. 4A,B, 13A). These neurons tegmental nucleus, a few large multipolar neurons (25 x 11 appeared to be the basket cells. In the superficialpart of the pm) with ASS-LI were observed. In the laterodorsal tegmen- molecular layer ovoid neurons of smaller size (9 x 7 pm) tal nucleus, large multipolar neurons (25 x 15 pm) with were strongly immunoreactive to ASS. In the granular strong ASS-LI were observed (Fig. 3D). Terminal-like layer, medium-sizeovoid neurons (15 x 11 pm) were immugrains also were seen in the neuropile. The dorsal tegmen- noreactive to ASS and appeared to be a type of Golgi cell tal nucleus was less immunoreactive to ASS and was seen (Fig. 13A). The white matter was crowded with immunoreas white spot in the central gray. In the central part, only a active fibers, which ascended into the granular layer and few immunoreactive fibers were observed. In the pericen- formed glomeruli. Glial cells were also faintly stained in the tral part, a few medium-size fusiform neurons (16 x 8 pm) white matter. were weakly immunoreactive to ASS. In the cerebellar nuclei small ovoid neurons (12 x 7 pm) Ruphe nuclei. In the dorsal raphe nucleus, a large with ASS-LI were observed (Figs. 4B, 13B). A large number number of strongly immunoreactive, large multipolar neu- of immunoreactive small ovoid neurons (13 x 9 pm) were

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN

671

Fig. 13. Photomicrographs from the cerebellum (A-C) and pons (D-HI. A. Cerebellar cortex. Bar: 100 pm. B. Lateral cerebellar nucleus. Bar: 50 pm. C. Ventral small celled part of the lateral cerebellar nucleus. Bar: 100 Fm. D. Medial vestibular nucleus. Bar: 50

pm. E. Locus ceruleus. Bar: 50 pm.F. Caudal interstitial nucleus of the medial longitudinal fasciculus. Bar: 50 pm. G. Ventral part of the gigantocellular reticular nucleus. Bar: 50 pm. H. Nucleus raphe magnus. Bar: 50 pm.

672

H. NAKAMURA ET AL.

aggregated in the ventral parvicellular part of the lateral nucleus (Fig. 13C).

Pons and medulla oblongata Pontine nucleus. In the pontine nucleus large, fusiform neurons (19 x 10 pm) were immunoreactive to ASS. The neurons were seen in the anterior, dorsomedial, and lateral part of the nucleus (Fig. 3C). A few fibers with ASS-LI were seen in the middle cerebral peduncle. Inferior olive. Neurons with ASS-LI were observed in the medial regions of the inferior olivary nuclei but were not seen in the lateral regions (Figs. 4C,D). In the medial nucleus of the inferior olive, medium-size triangular neurons (15 x 10 pm) were weakly immunoreactive to ASS. In the caudal levels of this nucleus, the medial subnucleus C was rich in stained neurons. The neuropile was stained densely. In the dorosmedial cell group of the inferior olive, small multipolar neurons (13 x 9 pm) were stained. Neurons with ASS-LI were seen in the medial part of the dorsal and principal nuclei, and terminal like staining was observed throughout the nuclei. Vestibular nuclei. In the superior vestibular nucleus, small ovoid neurons (11x 8 pm) with strong ASS-LI were observed (Fig. 4A). In the lateral vestibular nucleus, a few small ovoid neurons (13 x 9 pm) were densely stained (Fig. 4B). In the medial vestibular nucleus many large fusiform neurons (19 x 11 pm) with strong ASS-LI were observed with thick immunoreactive fibers (Figs. 4B,C, 13D).A few small ovoid neurons were immunoreactive in the ventral part of the medial vestibular nucleus and the spinal vestibular nucleus. In the nucleus Y, medium-size triangular neurons (18 x 10 pm) were immunoreactive to ASS (Fig. 4B). In the nucleus X, medium-size ellipsoid and ovoid neurons (14 x 8 pm) were stained (Fig. 4C). Cochlear nuclei. In the granular layer, cochlear nucleus fibers and swellings with ASS-LI were observed (Figs. 4A,B). A few small ellipsoid neurons (13 x 7 pm) were also seen immunoreactive in this part. A few small triangular neurons were immunoreactive in the anterior part of the ventral cochlear nucleus. The dorsal cochlear nucleus and the posterior part of ventral nucleus were free of immunostaining apart from a patchy distribution of swellings and neurons in the dorsal nucleus similar to that in the granular layer cochlear nucleus. Abducens nucleus. In the abducens nucleus, as in the other oculomotor nuclei, incoming fibers and terminal-like structures were immunoreactive to ASS. No immunoreactive neurons were seen here, either. In the paraabducens nucleus, a few small fusiform cells and terminal-like structures were stained. Parabrachial nuclei. The immunoreactivity in the lateral parabrachial nucleus varied between the different regions of the nucleus (Fig. 3D). In the central part of the lateral parabrachial nucleus, large fusiform neurons (20 x 10 pm) with strong ASS-LI were observed. In the dorsal part of the nucleus small ovoid neurons (13 x 7 pm) were seen immunoreactive to ASS. In the lateral part, a small number of medium-size fusiform neurons (14 x 8 pm) were stained. In the medial parabrachial nucleus, medium-size fusiform and triangular neurons (14 x 7 pm) with ASS-LI were observed. In the medial part of the superior cerebellar peduncle, densely stained medium-size (16 x 9 km) triangular neurons were seen among the nonimmunoreactive fibers. Terminal-like grains with ASS-LI were observed in the lateral but not in the medial

parabrachial nucleus. The Kolliker-Fuse nucleus was observed as an ASS-LI sparse area ventrolateral to the densely stained lateral parabrachial nucleus. In this nucleus a few terminal-like grains and weakly stained mediumsize multipolar neurons were observed. Locus ceruleus and related structures. In the nucleus locus ceruleus, a large number of medium-size polygonal neurons were moderately stained (Fig. 13E). In the subceruleus nucleus, large triangular and multipolar neurons (28 x 16 Fm) with ASS-LI were observed. A few terminallike grains were observed in the alpha part but not in the dorsal and ventral parts of this nucleus. In theA5 noradrenaline areas, large multipolar neurons (26 x 14 pm) were immunoreactive to ASS. The neuropile was also filled with terminal-like staining in this area. Hypoglossal nucleus. Fibers and terminal-like swellings with ASS-LI were observed in the hypoglossal nucleus. No immunoreactive neurons were seen in this nucleus as in the other somatic motor nuclei. In the nucleus Roller a few medium-size triangular neurons (14 x 9 pm) were immunoreactive to ASS. In the nucleus prepositus hypoglossi, medium-size fusiform neurons (17 x 8 pm) with strong ASS-LI were observed. Terminal-like grains also were seen in the neuropile. Nucleus o f the solitary tract and related structures. In the nucleus of the solitary tract, medium-size triangular neurons (15 x 9 pm) with moderate ASS-LI were observed (Figs. 4C,D,E, 14A,B).At rostral levels a few large triangular or multipolar neurons (22 x 12 pm) with ASS-LI were seen in the medial part (C2 adrenaline cell area) of the nucleus. Terminal-like staining was seen in the neuropile. In the commissural part of the nucleus of the solitary tract, a few small ovoid neurons (12 x 7 pm) were strongly immunoreactive. Terminal-likeboutons with strong ASS-LI were densely distributed in the commissural part of the nucleus of the solitary tract. In the dorsal motor nucleus of the vagus, large multipolar neurons (25 x 13 pm) were moderately stained. Immunoreactive fibers were also seen in the solitary tract. In the area postrema small ellipsoid neurons (11x 7 pm) and terminal-like boutons were stained. Trigerninal nuclei. In the motor trigeminal nucleus, fibrous and varicose structures were immunoreactive to ASS. In the accessory trigeminal nucleus, a few fibers and terminals with ASS-LI were observed. In the supratrigeminal nucleus only a few fibers were stained. No immunoreactive neurons were observed in these nuclei. In the paratrigeminal zone, a few fibers and terminals as well as a few medium-size fusiform neurons (16 x 9 pm) were observed. In the intertrigeminal nucleus, medium-sizetriangular and ellipsoid neurons (15 x 7 pm) with ASS-LI were observed. A few fibers and terminals also were seen in this nucleus. In the principal sensory trigeminal nucleus, no immunoreactive neurons were observed (Fig. 4A). Fibrous staining with or without varicosities was rarely seen. No large ovoid neurons of the mesencephalic trigeminal nucleus were immunoreactive to ASS; nonimmunoreactive spots were seen in the ventrolateral edge of the central gray at the levels of midbrain and in the area lateral t o the locus ceruleus at the upper pontine levels. In the oral part of the spinal trigeminal nucleus, a few medium-size fusiform neurons (16 x 8 pm) with ASS-LI were observed (Fig. 4B). Immunoreactive neurons were rarely seen in the interpolar part (Fig. 4C). A very few fibers were stained in these parts. In the caudal part of the nucleus, medium-size fusiform

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN

673

Fig. 14. Photomicrographs from the medulla oblongata (A-D) and the spinal cord (E-HI. A. Area postrema and the nucleus of the solitary tract. Bar: 300 pm. B. Nucleus of the solitary tract. Bar: 100 pm. C . Nucleus ambiguus. Bar: 50 pm. D. Lateral reticular nucleus. Bar: 100

pm. E.Laminae I and I1 of the dorsal horn of the cervical cord. Bar: 100 pm.F. Nucleus intermediolateralis of the thoracic cord. Bar: 100 pm. G. Ventral horn of the lumber cord. Bar: 100 pm. H. Ventral horn of the upper sacral cord. Bar: 100 pm.

674 neurons (15 x 9 pm) with ASS-LI were observed in the superficial laminae I and I1 (Fig. 4E). Thick immunoreactive fibers were seen in the sensory root of the trigeminal nerve and the spinal trigeminal tract (Figs. 3D, 4A-E). Superior olivary complex. The superior olivary complex was almost devoid ofASS-LI (Figs. 3D, 4A). The exceptions were in the following areas. In the trapezoid and the superior periolivary nucleus, a small amount of fibrous and terminal-like staining was observed. A few thick fibers in the trapezoid body were immunoreactive to ASS. Facial nucleus. In the facial nucleus, incoming fibers and terminal-like swellings with dense ASS-LI were observed (Fig. 4B). There were no immunoreactive neurons. Nucleus ambiguus. The nucleus ambiguus could be seen with the naked eye as a black dot on the sections, and with a microscope dense staining could be seen in the motor neurons as well as in the incoming fibers and terminal-like swellings (Figs. 4C, 14C). Dorsal column nuclei. In the gracile and cuneate nuclei a few small cells (11 x 7 pm) with ASS-LI were observed (Fig. 4E). The external cuneate nucleus was free of immunostaining. In the cuneate and gracile fasciculi, a few fibers with ASS-LI were observed. A few glial cells were also stained in these fasciculi. Reticular formation and related structures. In the nucleus raphe pontis, medium-size multipolar neurons (18 x 11 pm) with ASS-LI were observed (Fig. 3D). Dense neuropile staining was also seen here. In the nucleus raphe magnus, a large number of large multipolar neurons (25 x 13 pm) were immunoreactive (Figs. 4A,B, 13H). In the nucleus raphe obscurus, a few medium-size triangular neurons (18 x 11 pm) were stained. Fibers with varicosities with ASS-LI were also observed. In the caudal interstitial nucleus of the medial longitudinal fasciculus, large triangular neurons (23 x 14 pm) with long dendrites were densely stained (Figs. 4B,C, 13F). Terminal-like swellings were also stained here. In the nucleus raphe pallidus, small ellipsoid neurons (11 x 6 pm) were immunoreactive t o ASS. The neuropile in this nucleus was crowded with terminal-like staining. In the dorsomedialtegmental area, medium-size triangular neurons (15 x 8 pm) with ASS-LI were observed (Fig. 3D). The stained neurons were densely distributed in the dorsomedial part of this area. Terminal-like grains and varicose fibers with ASS-LI were seen in this area. A few large triangular neurons (25 x 11 pm) with ASS-LI were also observed here. The oral and caudal parts of the pontine reticular nucleus were almost free of ASS-LI (Figs. 3C,D, 4A). In the ventral part of the pontine reticular nucleus, a few medium-size fusiform neurons (16 x 7 pm) with weak ASS-LI were observed (Fig. 4A). In the gigantocellular reticular nucleus, a few large multipolar neurons (25 x 15 pm) with strong ASS-LI and a few, weakly stained, medium-size triangular neurons (16 x 10 pm) were scattered (Figs. 4B,C). Large multipolar neurons (32 x 19 pm) were observed in the alpha and ventral parts of the gigantocellular reticular nucleus (Figs. 4B,C, 13G).In the dorsal paragigantocellular nucleus, a few medium-size fusiform neurons (15 x 8 pm) with strong immunoreactivity were seen (Figs. 4B,C). In the lateral paragigantocellular nucleus, large triangular neurons (19 x 9 pm) were stained (Figs. 4B,C). Terminallike staining was also seen in this area. In the intermediate reticular nucleus large fusiform neurons (19 x 10 pm) were moderately stained at the level of the nucleus ambig-

H. NAKAMURA ET AL. uus (Fig. 4C). Giant multipolar neurons (27 x 14 pm) with ASS-LI were also scattered along the medial boundary to the gigantocellular reticular nucleus. In the parvicellular reticular nucleus, medium-size ellipsoid neurons (14 x 9 pm) with weak ASS-LI were observed (Figs. 4A,B). In the parvicellular part of the lateral reticular nucleus, medium-size triangular neurons (15 x 8 pm) with ASS-LI were seen (Figs. 4D, 14D). No immunoreactive neurons were observed in the lateral reticular nucleus. A few terminal-like grains were stained here. In the dorsal part of the medullary reticular field, a few medium-size triangular neurons (16 x 8 pm) were seen (Figs. 4D,E). The ventral part of the medullary reticular field was almost devoid of immunostaining except in fibers at the level of pyramidal decussation (Figs. 4D,E). The rostroventrolateral reticular nucleus was devoid of staining, whereas in the caudoventrolateral reticular nucleus, large elongated triangular neurons (19 x 7 pm) and terminal-like grains with ASS-LI were seen (Figs. 4C,D,E).

Spinal cord In lamina I, medium-size fusiform neurons (15 x 8 pm) with strong ASS-LI were observed (Fig. 14E). In lamina 11, small ovoid cells (11 x 7 pm) were strongly immunoreactive. Both these laminae were rich in terminal-like grains, especially lamina I and the deep part of lamina I1 (Figs. 4F,G,H, 14E). Immunoreactive fibers entering and leaving laminae I and I1 were seen to ascend and descend in the dorsolateral part of the dorsal fasciculus adjacent to Lissauer’s tract and in the fasciculus dorsolateralis. A few fibers were also stained in the radix dorsalis. In the superficial part of the lamina 111, a few small ovoid cells (9 x 7 pm) were densely stained. The intermediolateral nucleus was packed with large triangular neurons (23 x 12 pm) with strong ASS-LI at the middle and lower thoracic levels and the lower lumber and upper sacral levels (Figs. 4G, 14F).At the cervical and upper thoracic levels, large ovoid and multipolar neurons (21 x 14 pm) with moderate ASS-LI were observed in the medial part of the intermediate gray or the dorsal nucleus of Clarke. At the upper lumber levels, large fusiform neurons (24 x 12 pm) were observed in the region dorsal to the central canal. Terminal-like grains were observed in the ventral horn (Figs. 4F,G,H, 14G), especiallyin the ventrolateral group of the seventh segment of lumbar cord and the first three segments of sacral cord (Figs. 4H, 14H).A few terminal-like grains were seen in the lateral reticular part, the nucleus cornucommissuralis posterior, and the region around the central canal.

DISCUSSION We have described the localization of argininosuccinate synthetase (ASS) in the rat brain by using polyclonal rabbit antibody. The antiserum appears specific t o ASS because Western blot analysis detects a single band at 43 kDa. The immunohistochemistry showed that ASS-like immunoreactivity (ASS-LI) is predominantly distributed in neurons. Other than the neuronal staining, glial cells in some part of the white matter, such as the white matter in cerebellum, stria medullaris thalami, and the cuneate and gracile fasciculi, also showed weak immunoreactivity. Since ASS is one of the enzymes for terminal amino acid metabolism, it may not be so surprising that some glial cells have ASS. There were two kinds of neuronal staining: cytosomal and nuclear. In most of the regions we found stronger staining

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN in the soma than in the nucleus; however, we found stronger staining in the nucleus in some nuclei such as the anteromedial nucleus of the thalamus. Although we do not know the reason for this nuclear staining since ASS is a cytoplasmic enzyme, there are several possibilities that could explain it. One possibility is that the antiserum might recognize another protein having the same molecular weight and the same behavior during the purification procedure. Another possibility is that the enzyme may have a role in the nucleus or perinuclear regions. This problem remains to be solved in the future by using, for example, a monoclonal antibody against ASS, which we are preparing in our laboratory. We found a characteristic distribution of ASS in the rat brain (Table 1).Argininosuccinate synthetase is densely distributed in the autonomic regions such as the hypothalamus, periaqueductal gray, nucleus of the solitary tract, dorsal motor nucleus of vagus, and the intermediate gray of the spinal cord. The septa1 area and the basal forebrain, which may have modulatory effects for cortical functions and are suggested to have influence in memory processing, have high ASS-LI. Memory-related structures such as the hippocampus and amygdala, however, show only weak immunoreactivity. The other regions with high ASS-LI are the olfactory bulb, midline and anterior thalamic nuclei, raphe nuclei, interpeduncular nucleus, ventral tegmental area, peripeduncular nucleus, parabrachial nucleus, locus ceruleus, area postrema, superficial layers of the caudal part of spinal trigeminal nucleus, and superficial layers of the dorsal horn of spinal cord. Many ASS-LI positive neurons are also distributed in several nuclei of the indirectly activating motor system nuclei such as the red nucleus and the subthalamic nucleus. The caudate-putamen and the globus pallidus have only a moderate number of immunoreactive cells. Most of the first or second sensory relay nuclei are almost devoid of ASS-LI. Among these are the nucleus ventralis lateralis, nucleus ventralis posterolateralis, nucleus ventralis posteromedialis, dorsal lateral geniculate nucleus, medial geniculate nucleus, core of the inferior colliculus, nuclei of the lateral lemniscus, superior olivary nucleus, cochlear nuclei, and the principal sensory trigeminal nucleus. In the somatic motor nuclei, only incoming fibers and terminals are immunoreactive to ASS. These are the oculomotor nucleus, trochlear nucleus, abTABLE 1. Distribution Summary of Neurons and Terminal-like Structures Stained With the Antibody Against Argininosuccinate Svnthetase ~~

Structures

Neurons'

Neuropile'

Cortex Olfactory bulb Hippocampus Septum Basal ganglia Caudate-Putamen Globus pallidus Basal nucleus of Meynert Entopeduncular nucleus Subthalamic nucleus Amygdala Anterior amygdaloid area Anterior cortical amygdaloid nucleus Posterolateral cortical amygdaloid nucleus Posteromedial cortical amygdaloid nucleus Medial amygdaloid nucleus Central amygdaloid nucleus Lateral amygdaloid nucleus Basolateral amygdaloid nucleus Basomedial amygdaloid nucleus Intercalated nuclei of ainygdala

+ ++ +++

++ ++++

+ ++

++

+ +++ + + ++ ++ ++++-

+

-

-

+ +++

++ + + -

6 75 TABLE 1. (continued) Structures Nucleus accumbens, core Nucleus accumbens, shell Diagonal band Substriatal area Ventral pallidum Magnocellular preoptic nucleus Substantia innominata Bed nucleus of stria terminalis Anterior division Medial division Lateral division Ventral division Islands of Calleja Olfactory tubercle, polymorpb layer Medial habenular nucleus Lateral habenular nucleus, medial part Lateral habenular nucleus, lateral part Stria medullaris thalami Anterodorsal nucleus Anteroventral nucleus Anteromedial nucleus Nucleus ventralis lateralis Nucleus ventralis posterolateralis Nucleus ventralis posteromedialis Nucleus ventralis medialis Nucleus lateralis dorsalis Nucleus lateralis posterior Posterior complex ofthalamus, rostra1 Posterior complex of thalamus, posterior Nucleus medialis dorsalis Nucleus centralis lateralis Nucleus centralis medialis Paracentral thalamic nucleus Parafascicular nucleus Fasciculus retroflexus Paraventricular nucleus Paratenial nucleus Intermediodorsal thalamic nucleus Interanteromedial tbalamic nucleus Rhomboid thalamic nucleus Nucleus reuniens Nucleus reuniens ventralis Dorsal lateral geniculate nucleus Medial geniculate nucleus ventral part magnocellular part dorsal part Reticular nucleus of thalamus, dorsal Reticular nucleus of thalamus, ventral Ventral lateral geniculate nucleus lateral magnocellular part, lateral lateral magnocellular part, medial medial palvicehlar part intergeniculate leaflet Zona incerta, dorsal part Zona incerta, ventral part Fasciculus lentiformis Fasciculus thalamicus Nucleus of the field of Forel Suhparafascicular nucleus Anterior medial preoptic nucleus Anteroventral preoptic nucleus Median preoptic nucleus Medial preoptic nucleus Medial preoptic area Lateral preoptic area Striohypothalamic nucleus Anterior commissural nucleus Supraoptic nucleus Suprachiasmatic nucleus Optic decussation Supraoptic decussation Lateroanterior hypothalamic nucleus Anterior hypothalamic area Lateral hypothalamic area Tuber cinereum Retrochiasmatic area Paraventricular hypothalamic nucleus Periventricular hypothalamic nucleus Dorsomedial hypothalamic nucleus Ventromedial hypothalamic nucleus Arcuate hypothalamic nucleus Posterior hypothalamic area Submammillothalamic nucleus Premammillary nucleus Medial mammillary nucleus, median Medial mammillarv nucleus. medial

Neurons'

Neuropile'

++ +++

+ + + + +++

++

+ + + ++

+ + + + ++ +

(glial cells + -1

(fiber +)

+ +++

++

+ + +-

++-

-

+++ -

++ +++++ + ++ +++ ++ -

+++ +++

+-

-

+ +

-

++ +++ ++ ++++

-

++

++ ++ +

+++

+++

++

++

+ -

+ ++

+ + +++ +

+ +++ t++ +++ +++ + +

+ + ++ ++ t -

+ ++

+ + ++ + + ++ + +++

++ +++ +++ +++

++

+ +

+-

+ + -

-(fiber +) -(fiber + -1

+++ +++ +++ + ++ ++ + + + + + -

(fiber + + 1

++

++ + ++ ++ ++ ++ ++ + +++ ++ +++ +++ ++ + (contznued 1

H. NAKAMURA ET AL. TABLE 1 (continued) Structures Medial mammillruy nucleus, lateral Lateral mammillary nucleus Supramammillruy nucleus Mammillothalamic tract Fornix Anterior pretectal nucleus Optic tract nucleus Olivary pretectal nucleus Posterior pretectal nucleus Medial pretectal nucleus Nucleus of the posterior commissure Superior colliculus Superficial layers Intermediate layers Deep layers Commissural part Parabigeminal nucleus Suhhrachial nucleus Inferior colliculus, core Inferior colliculus, dorsal cortex Inferior colliculus, external cortex Brachium of the inferior colliculus Nucleus of the hrachium of the inferior colliculus Nucleus of lateral lemniscus, dorsal Nucleus of lateral lemniscus, intermediate Nucleus of lateral lemniscus, ventral Nucleus sagulum Superior olivary complex Central gray, pretectal level Central gray, collicular level Nucleus Darkschewitsch Supraoculomotor central grey Oculomotor nucleus Edinger-Westphal nucleus Trochlear nucleus Paratrochlear nucleus Interstitial nucleus of MLF Deep mesencephalic nucleus Intercollicular nucleus Red nucleus, magnocellular Red nucleus, panicellular Perirubral field Paralemuiscal nucleus Cuneiform nucleus Pedunculopontine tegmental nucleus Anterior tegmental nucleus Ventral tegmental nucleus Subpeduncular tegmental nucleus Laterodorsal tegmental nucleus Dorsal tegmental nucleus Central part Pericentral part Dorsal raphe nucleus Rostral linear nucleus Caudal linear nucleus Median raphe nucleus Paramedian raphe nucleus Interpeduncular nucleus, rostral Interpeduncular nucleus, caudal Interpeduncular nucleus, intermediate Interpeduncular nucleus, lateral Interpeduncular nucleus, dorsolateral Interpeduncular nucleus, dorsomedial Interfascicular nucleus Substantia nigra, pars compacta Substantia nigra, pars reticulata Ventral tegmental area Paranigral nucleus Peripeduncular nucleus Cerebellar cortex Cerebellum, white matter Cerebellar nuclei Pontine nucleus

Neurons'

+++

+

+++ -

-

++ +-

+++ + +

+ + + +

+

+

+++

+ + -

+ ++ + ++ + +++ -

++ -

+

+ + +

-

+ ++

+ ++ + + + ++ -

+

+++ + + ++ ++

+ + -

++++ ++ +++

+ +++ + +++ + (glial cells +) + +

TABLE 1 (continued) Neuropile'

Structures

+ ++

Inferior olive dorsal nucleus principal nucleus dorsomedial cell group medial nucleus Superior vestibular nucleus Lateral vestibular nucleus Medial vestibular nucleus Medial vestibular nucleus, ventral Spinal vestibular nucleus Nucleus X Nucleus Y Dorsal cochlear nucleus Cochlear nucleus, granular layer Ventral cochlear nucleus, anterior Ventral cochlear nucleus, posterior Abducens nucleus Paraabducens nucleus Parabrachial nucleus Locus ceruleus Subceruleus nucleus A5 noradrenaline area Prepositus hypoglossi Hypoglossal nucleus Nucleus Roller Nucleus of the solitary tract Dorsal motor nucleus of vagus Area postrema Motor trigeminal nucleus Principal sensory trigeminal nucleus Mesencephalic trigeminal nucleus Spinal trigeminal nucleus, oral Spinal trigeminal nucleus, interpolar Spinal trigeminal nucleus, caudal Laminae I, I1 Laminae 111, IV Facial nucleus Nucleus ambiguus Gracile nucleus Gracile fasciculus Cuneate nucleus Cuneate fasciculus External cuneate nucleus Nucleus raphe pontis Nucleus raphe magnus Nucleus raphe obscurus Caudal interstitial nucleus of MLF Nucleus raphe pallidus Dorsomedial tegmental area Pontine reticular nucleus, oral Pontine reticular nucleus, caudal Pontine reticular nucleus, ventral Gigantocellular reticular nucleus Gigantocellular reticular nucleus, alpha Gigantocellular reticular nucleus, ventral Dorsal paragigantocellular nucleus Lateral paragigantocellular nucleus Intermediate reticular nucleus Parvicellular reticular nucleus Lateral reticular nucleus Lateral reticular nucleus, parviceliular Medullary reticular field, dorsal Medullary reticular field, ventral Rostroventrolateral reticular nucleus Caudoventrolateral reticular nucleus Spinal cord Lamina I Lamina I1 Lamina 111 Lamina IV Intermediolateral nucleus Intermediomedial nucleus Dorsal nucleus of Clarke Ventral horn

(fiber +) -

+

+ + ++ + ++ + + ++

-

++ + ++ +

-

++ -

++ ++ -

+++

++++ ++ + ++ -

+ ++

+ +++ +

+ + ++

+ ++ ++ ++ ++ + ++ ++ ++ ++ +

-

+++ ++

+++ +

(fiber +)

+-

-

ducens nucleus, motor trigeminal nucleus, hypoglossal nucleus, facial nucleus, and the ventral horn of the spinal cord. The nucleus ambiguus contains a lot of ASS-LI positive motor neurons and terminals as in the dorsal motor nucleus of vagus. The white matter is almost devoid of ASS-LI. No ASS-LI is observed in the main output neurons of several structures such as larger pyramidal cells in the cerebral cortex, mitral cells in the olfactory bulb,

'Number of

Neurons'

Neurouile'

+-

+

+++

++

+-

+

++ + ++ + + ++ ++ + + -

+

+-

++ -

-

+

+

-

+ -

+ ++ ++ +

+ ++++ ++ +++ + + + ++ ++ +++

-

-

+-

-

++

+++ +-

-

+ +++ +++ + ++ ++ -

-

+

+ -

+++

+

+

+-

+ +++

(glial cells +)

+

(fibers +) -

(glial cells +)

(fibers +)

++

+ + ++ ++ +

+-

++

+ ++ ++ ++ + + -

-

++ + + + +++ +

-

+++ + -

+ ++-

+-

++ + +-

-

++

++ +++

-

++ ++ +

+++

+ +

-

++ -

++ +-

+ +++

-

++ + indicate approximate density of neurons and neuropile staining.

Purkinje cells in the cerebellar cortex, and the motor neurons in the motor nuclei. On the contrary, ASS-LI is observed in the neurons known to contact with the main output cells such as external tufted cells in the olfactory bulb and basket cells in the cerebellar cortex, and the incoming fibers with terminal-like swellings around the output neurons as seen in the somatic motor nuclei. Therefore it may be concluded that most of the ASS

ARGININOSUCCINATE SYNTHETASE IN RAT BRAIN containing neurons are not projection neurons but interneurons. The distribution of ASS-like immunoreactivity partially coincides with that of other transmitter candidates. Among these it will be most interesting to compare distribution of ASS and that of aspartate and glutamate, because aspartate is one of the substrates of ASS and aspartate and glutamate are convertible amino acids. In the cerebral cortex a large amount of layer V and VI pyramidal cells, horizontally elongated neurons in the deep border of layer VI, and a few layer I1 and I11 neurons show glutaminase-like immunoreactivity, whereas aspartate aminotransferase-like immunoreactivity is seen in nonpyramidal neurons (Donogue et al., '85; Kaneko and Mizuno, '88). The distribution pattern of ASS-LI in the cerebral cortex is rather similar to that of glutaminase-like immunoreactivity. Thus the localization of ASS is similar to that of glutamatergic neurons but not aspartergic ones. If ASS is localized in glutamatergic neurons it may regulate glutamate production by removal of aspartate, since aspartate is the common substrate for both glutaminase and ASS. In the thalamus, however, complete discrepancy is seen between the localization of glutamatergic neurons and that of ASS-LI. The monoclonal antibody against phosphate-activated glutaminase stained large neurons in all thalamic nuclei (Kaneko and Mizuno, '88), whereas antibody to ASS stained smaller neurons in restricted nuclei. The localization of ASS-LI neurons in the thalamus is rather similar to neuronal distribution of y-aminobutyric acid transaminase (Nagai et al., '83). Both ASS and y-aminobutyric acid transaminase are localized in the nucleus of the diagonal band, caudate-putamen, dorsal raphe, substantia innominata, and the reticular formation (Nagai et al., '83). It will also be interesting to compare the distribution of ASS-LI and that of cholinergic neurons, since nitric oxide may have some effects on acetylcholinereceptor (Furchgott and Zawadzki, '80; Palmer et al., '87; Moncada et al., '88; Rees et al., '89). Although nitric oxide is known as arterial smooth muscle relaxing factor produced by endothelial cells of blood vessels (Palmer et al., '87; Moncada et al., '88), we could not find any staining including terminal-like grains around blood vessels in the brain. Argininosuccinate synthetase-like immunoreactivity is located in large multipolar neurons of the basal nucleus of Meynert, which are known to be cholinergic and project into the wide areas of the cerebral cortex (Lehmann et al., '80; Armstrong et al., '83; Houser et al., '83; Mesulam et al., '83; Luiten et al., '87). The other cholinergic areas that contain ASS-LI positive neurons are the pedunculopontine tegmental nucleus and the dorsal tegmental nucleus in the midbrain. The cholinergic neurons in the somatic motor nuclei, however, do not show ASS-LI. Although we could not tell why some of the cholinergic nuclei contain ASS-LI positive neurons while others do not, this discrepancy also meets the hypothesis that ASS is distributed in the neurons modulating main input or output pathways and is not localized in the relay neurons and main output neurons. The distribution of neurons and terminations with ASS-LI has some similarity to that of substance P and serotonin. Substance P neurons in the main olfactory bulb, which are thought to be external tufted cells (Macrides and Davis, '831, resemble the ASS-LI positive cells. The distribution pattern of terminals with ASS-LI in lamina I and I1 of the spinal dorsal horn and in the spinal ventral horn is like that of substance P terminations (Ljungdahl et al., '78; Hunt,

677 '83). Large neurons in the raphe nuclei and the ventral gigantocellular reticular nucleus of the medulla oblongata that are known to be substance P positive (Ljungdahl et al., '78) also have ASS-LI. Argininosuccinate synthetase-like immunoreactivity positive neurons are distributed in several serotoninergic nuclei. These are the interpeduncular nucleus, the nucleus raphe obscurus, nucleus raphe pallidus, nucleus raphe magnus, nucleus raphe dorsalis, the oral linear nucleus, the ventral gigantocellular reticular nucleus, the area tegmentalis of Tsai, and the area postrema (Steinbusch et al., '78; Steinbusch, '81, '84; Steinbusch and Nieuwenhuys, '83). There also is terminal-like staining of ASS-LI and serotonin immunoreactivity in laminae I and I1 and the ventral horn of the spinal cord. There are two possible roles of ASS in the brain. The first possibility is that ASS and argininosuccinate lyase work at the same time to produce arginine from aspartate and citrulline. Arginine, in turn, may be used for a substrate for other metabolic pathways, for example, to produce nitric oxide (Deguchi and Yoshioka, '82; Hibbs et al., '87, '88; Palmer et al., '88; Garthwaite et al., '88, '89; Knowles et al., '89; McCall et al., '89; Palmer and Moncada, '89; Bredt and Snyder, '90). The second possibility is that ASS produces argininosuccinate, which then may be transmitted to modulate neuronal activity in the brain (Nakamura et al., '90, '91). Arginine is the substrate of nitric oxide synthase, which produces nitric oxide, a possible neurotransmitter in the brain (Deguchi and Yoshioka, '82; Hibbs et al., '87, '88; Palmer et al., '88; Garthwaite et al., '88, '89; Knowles et al., '89; McCall et al., '89; Palmer and Moncada, '89; Bredt and Snyder, '89, '90; Bult et al., '90; Garthwaite, '91). Arginine is related to nitric oxide formation in cytotoxic-activated macrophages (Hibbs et al., '87, '88; Marletta et al., '88; Tayeh and Marletta, '891, neutrophils (McCall et al., '89), vascular endothelium (Moncada et al., '88; Palmer et al., '88; Palmer and Moncada, '89), and nervous tissue (Deguchi and Yoshioka, '82; Garthwaite et al., '88, '89; Knowles et al., '89). Nitric oxide is synthesized from a terminal guanidino nitrogen atom of arginine, which is then converted to citrulline (Hibbs et al., '87, '88; Palmer et al., '88; McCall et al., '89; Palmer and Moncada, '89; Bredt and Snyder, '90). Nitric oxide is an endothelium-derived relaxing factor which activates guanylate cyclase and causes relaxation of smooth muscle (Deguchi, '77; Furchgott and Zawadzki, '80; Palmer et al., '87; Garthwaite et al., '88; Moncada et al., '88; Knowles et al., '89; Rees et al., '89). In the nervous system, nitric oxide is released by stimulation of glutamate receptors with N-methyl-D-aspartate (Garthwaite et al., '88). Nitric oxide synthesis is stimulated by kainate, which is an agonist of glutamate (Garthwaite et al., '89). Nitric oxide has a role in increase in cGMP levels produced by N-methyl-D-aspartate (Bredt and Snyder, '89). The transmitter role of nitric oxide is suggested by its release from nonadrenergic, noncholinergic autonomic nerves after electric stimulation (Bult et al., '90). Argininosuccinate synthetase as well as argininosuccinate lyase may have significant role in the production of nitric oxide since they may provide arginine for nitric oxide synthase. Recently an immunohistochemical study revealed the localization of nitric oxide synthase in the rat brain (Bredt et al., '90). Nitric oxide synthase is localized in the molecular and granule layers of the cerebellum, superior and inferior colliculi, granule layer of the olfactory bulb, superficial layer I of the cerebral cortex, dentate gyrus of the

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hippocampus, nucleus of the stria terminalis, islands of calleja, diagonal band of Broca, mammillary body, supraoptic nucleus, and the paraventricular nucleus (Bredt et al., '90). Among these we focus on basket cells in the cerebellar cortex to discuss a possible relation of ASS and nitric oxide synthesis. Nitric oxide synthase is localized in basket cells in the cerebellar cortex (Bredt et al., '90). Basket cells contain ASS, but no immunoreactivity is revealed against argininosuccinate lyase and arginase (Nakamura et al., '90). Since citrulline is the precursor of argininosuccinate, nitric oxide synthase may supply citrulline to ASS in basket cells. From basket cells argininosuccinate may be transported from basket cells to Golgi epithelial glia where argininosuccinate lyase (Nakamura et al., '90) and free arginine (Aoki et al., '91) are localized. The nitric oxide, in contrast, may be defused to Purkinje cells to activate guanylate cyclase (Nakane et al., '83; Bredt and Snyder, '89). Thus, ASS, argininosuccinate lyase, and nitric oxide synthetase may work as a cyclic metabolic pathway as suggested in cultured endothelial cells (Hecker et al., 'go), presuming that the metabolites are transported transcellularly. Argininosuccinate synthetase-like immunoreactivity is also observed in most of the areas with nitric oxide synthase. Compared to the wide distribution of ASS, however, the distribution of nitric oxide synthase is far more restricted. This suggests that only part of the distribution of ASS relates to nitric oxide synthesis and that ASS should also have at lease another role in the nervous system. Argininosuccinate, the product of ASS, is suggested to have a neuromodulator role in the brain (Nakamura et al., '90, '91). We reported that the enzymes of argininosuccinate metabolism are distributed in the different cells in the rat brain (Nakamura et al., '90). In the motor nuclei, incoming fibers and terminals are stained with antibody against ASS, whereas the motor neurons are immunoreactive to the antibodies against argininosuccinate lyase and arginase (Nakamura et al., '90). This indicates that ASS is in the presynaptic structure, whereas argininosuccinate lyase and arginase are in the postsynaptic motor neurons. Thus it is possible that argininosuccinate, the intermediate between ASS and argininosuccinate lyase, may be transsynaptically transported. We also reported that as little as M argininosuccinate decreases quisqualate type glutamate response in isolated neurons (Nakamura et al., '91). Although studies are clearly needed to establish the neuromodulator function of argininosuccinate, predominant distribution of ASS in interneurons shown in this study is in line with the neuromodulator role of argininosuccinate.

ACKNOWLEDGMENTS The authors gratefully thank Dr. Leslie G. Ungerleider and Dr. Yasuo Hasegawa for their discussions, Michelle M. Adams and Martin Gore for reviewing the English, and T. Fukushige, T. Nagayama, M. Ushikai, and K. Yamakuchi for technical assistance. Part of this study was supported by the Kodama Foundation for Medical Science Research.

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Immunocytochemical localization of argininosuccinate synthetase in the rat brain.

The neuronal distribution of argininosuccinate synthetase (ASS) was mapped in the rat brain. Argininosuccinate synthetase is one of the enzymes of the...
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