[3H]Nisoxetine binding sites in the cat brain: An autoradiographic study

Neuroscience Vol. 69, No. 1, pp. 259 270, 1995

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Pergamon

0306-4522(95)00257-X

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[3H]NISOXETINE B I N D I N G SITES IN THE CAT BRAIN: A N A U T O R A D I O G R A P H I C STUDY Y. C H A R N A Y , * t

L. LEGER,~: P. G. V A L L E T , * P. R. HOF,§ M. J O U V E T ~ a n d C. B O U R A S * § *Division de Neuropsychiatrie, I.U.P.G., 100 Av. Bel-Air, CH-1225 Ch6ne-Bourg/Gen6ve, Suisse :~D~partement de M~decine Exp6rimentale, INSERM U52, CNRS URA 1195, Facult6 de M6decine, F-69373 Lyon Cedex 08, France §Fishberg Research Center for Neurobiology, Mount Sinai School of Medicine, New York, NY 10029, U.S.A. Abstract--The binding of [3H]nisoxetine, a selective inhibitor of the high-affinity noradrenaline uptake sites, was studied on frontal frozen sections of the cat brain. The highest densities in autoradiographic signal were observed in the nucleus locus coeruleus and its ascending pathways, in the area postrema and in the dorsal motor nucleus of the vagus nerve. Other areas displaying high densities of signal include the dorsal part of the inferior olive, the pontine nuclei, the raphe nuclei, the colliculi, the periventricular and lateral areas of the hypothalamus, the suprachiasmatic nucleus, the nucleus accumbens and the olfactory bulb. A moderately high concentration of binding sites was observed in the hippocampal formation, especially in the molecular layer of Ammon's horn, in the superficial layers of the entorhinal cortex and in the indusium griseum. Binding sites were visualized in all the subdivisions of the neocortex. The highest density of binding was generally detected in the outer edge of the superficial layer I. In some cortical areas, especially in the visual cortex, labeling with a prevalent laminar distribution in the superficial layers I III and in the deep layers V VI was clearly observed. Moderate to low densities of binding sites were seen in most other areas of the brain except in the white matter, the caudate nucleus and putamen, which were devoid of labeling. Overall these findings indicate a good correlation between the distribution of [3H]nisoxetine binding sites and the noradrenergic systems. Furthermore, data suggest that in several areas, high-affinity noradrenaline reuptake mechanisms could play an important role in local interactions between the noradrenergic system and the other monoaminergic systems. Key words: feline brain, norepinephrine transporter, nisoxetine, uptake binding sites, autoradiography.

T h e n o t i o n t h a t nerve terminals in the b r a i n possess high-affinity u p t a k e m e c h a n i s m s selective for their respective n e u r o t r a n s m i t t e r s , such as m o n o a m i n e s or a m i n o acids, arose in the sixties (see Refs 33, 70). There is evidence t h a t such m e c h a n i s m s play a m a j o r role in the t e r m i n a t i o n o f n e u r o t r a n s m i s s i o n by rem o v i n g the t r a n s m i t t e r from the extracellular space after release. 33'7° Several m e m b r a n e proteins functionally devoted to high-affinity uptake m e c h a n i s m s were recently discovered by molecular cloning of c D N A s . 79 T h e proteins that share similar structural a n d mechanistic (i.e. ion requirements, t e m p e r a t u r e dependence) properties represent a special family o f carrier system, the N a + - d r i v e n n e u r o t r a n s m i t t e r transporters. 4'26'61'67'75'79 This family includes the noradrenaline (NA) transporter, 4~'52 the first biogenic a m i n e t r a n s p o r t e r to be cloned, the serotonin 9'39 a n d the d o p a m i n e 25'37 transporters. The m o n o a m i n e t r a n s p o r t e r s are the m a i n targets o f certain classes o f antidepressants a n d o t h e r tTo whom correspondence should be addressed. Abbreviations: NA, noradrenaline; TBS, Tris-buffered saline. 259

psychoactive drugs such as cocaine a n d a m p h e t amines which inhibit the re-uptake process after binding. 9'4°'4~'52'53 Thus, tritiated antidepressants such as desipramine a n d mazindol have been extensively used for labeling of N A - u p t a k e sites in m e m b r a n e h o m o g e n a t e s a n d for a u t o r a d i o g r a p h i c studies in tissue sections. However, it has been observed that these c o m p o u n d s bind to a heterogeneous p o p u l a t i o n of sites. TM It was more recently reported that nisoxetine, (_+)-7-(2-methoxyphenoxy)N - m e t h y l - b e n z e n e p r o p - a n a m i n e , a potential antidepressant drug, 53 binds with high affinity a n d selectivity to a h o m o g e n e o u s p o p u l a t i o n of sites associated with N A - u p t a k e . Nisoxetine thus represents a useful tool for m a p p i n g studies of the NAergic system, especially with regard to terminal areas. 12,72,73 The N A - c o n t a i n i n g neurons, the cell bodies o f which are localized in the b r a i n stem, are k n o w n to innervate a variety of functionally diverse b r a i n areas suggesting interactions with m a n y other n e u r o n a l systems (for review, see Ref. 48). The central NAergic systems have been reported to be involved in numerous functions, such as a u t o n o m i c functions, pain a n d

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affective states. 5'6'11'23'46,56'64,76 F u r t h e r m o r e , several electrophysiological a n d p h a r m a c o l o g i c a l studies, often p e r f o r m e d in the cat, suggest t h a t the NAergic n e u r o n s o f the dorsolateral p o n t i n e t e g m e n t u m , i.e., the locus coeruleus complex, play a role in the regulation of the sleep/wake cycle. It was observed t h a t these neurons, like the serotonergic n e u r o n s in the adjacent r a p h e nuclei, cease activity t h r o u g h o u t the paradoxical sleep period (see Refs 36, 63). Also m a n y drugs t h a t influence the NAergic n e u r o t r a n s mission affect sleep patterns, 31'59'66a n d it was reported t h a t selective inhibition o f N A re-uptake by nisoxetine dramatically reduces paradoxical sleep in cats. 6s O u r knowledge o f the a n a t o m i c a l distribution of n o r a d r e n e r g i c n e u r o n s in the cat b r a i n is mainly based on fluorescence histochemical investigations 16'35'45'78a n d some i m m u n o h i s t o c h e m i c a l studies using antisera against catecholamine-synthesizing enzymes, j7'28'42'47'51"57A l t h o u g h u p t a k e m e c h a n i s m s of tritiated N A have been d e m o n s t r a t e d in the locus coeruleus region, 38 in the cerebral cortex a n d the cerebellum, TM no i n f o r m a t i o n on o t h e r brain areas is available. Therefore, the aim of this work was to provide a detailed a u t o r a d i o g r a p h i c m a p p i n g of [3H]nisoxetine binding sites, a putative selective m a r k e r of the high affinity N A - t r a n s p o r t e r , 72 in the cat brain. Preliminary results have been presented in a b s t r a c t form. ~4

EXPERIMENTAL PROCEDURES

Preparation o f tissues Six adult cats (body weight 2 3 kg) of both sexes were used for this study. The animals were deeply anesthetized with pentobarbital and perfused transcardially with 250 ml of ice-cold Tyrode solution. The brains were quickly removed from the skulls, cut into a few blocks, and frozen with CO 2. Serial frontal sections were cut at 10,urn in a cryostat, thaw mounted on microscope slides coated with 0.1% poly-L-lysine and stored at - 2 0 ° C until used (less than a month). Additional blocks of tissues (few mm 3) from the dorsolateral pontine tegmentum area (including the locus coeruleus), the cerebellum and the frontal cortex were collected for homogenate studies. Binding assays in homogenates Binding assays were performed with minor modifications to published procedures. 73 Briefly, the tissue samples were homogenized at high speed for 10 s in 30 volumes of ice-cold 50 mM Tris buffer, pH 7.4, containing 300 mM NaCI and 5 mM KC1 (TBS) using a Tissue Tearor homogenizer (Biospect Products Inc., U.S.A.). The homogenates were centrifuged for 10 min at 30,000 g. The pellets were washed three times in the same conditions and resuspended in ice-cold TBS to reach a concentration of about I m g protein/ml. Four hundred microliters of membrane suspension in TBS (equivalent to 400/z g of protein) were incubated in triplicate with various concentrations (0.1-12nM) of [3H]nisoxetine (3.18 TBq/mmol, Amersham, Switzerland) for 4 h at 4°C (final volume, 500/zl). The samples were then diluted with 5ml of TBS and filtered through Whatman GF/C glass fiber filters pretreated with 0.05% polyethylenimine. The filters were dried and the radioactivity was measured by liquid scintillation spectrophotometry. Nonspecific binding was estimated in the presence

of 1 # M mazindol (generously donated by Sandoz Wander Pharma, Switzerland). Protein concentration was determined by the method of Bradford with bovine serum albumin as standard (Biorad Protein Assay, Glattbrugg, Switzerland). Autoradiographic procedures Incubation conditions were similar to those previously described by Tejani-Butt et al. n Prior to the autoradiographic study, slide-mounted brain sections were brought to room temperature. Sections were incubated with 0.1 nM [3H]nisoxetine in TBS for 4 h at 4°C. The nonspecific binding was determined in near-adjacent sections by including 1/zM mazindol. After incubation the sections were rinsed for three consecutive 5-min periods in ice-cold TBS, dipped in deionized water to remove salts, and rapidly dried under a stream of cool air. The sections were then apposed to 3H-Hyperfilms (Amersham, Switzerland) along with tritiated polymer standards (3H microscales, Amersham, Switzerland). Films were developed after two months exposure at 4°C. Autoradiograms were quantitatively assessed by computer-assisted microdensitometry (SAMBA, TITN Alcatel, France). The specific binding in each relevant area, was determined by substracting nonspecific binding from total binding and the optical densities were converted to fmol/mg equivalent tissue with the aid of 3H microscales. Results were not corrected for differential quenching of 3H by gray and white matter. The brain regions were delineated according to the atlases of Reinoso-Suarez 58 and Snider and Niemer, 69 after staining the sections with Cresyl Violet. RESULTS Binding in tissue homogenates Binding of [3H]nisoxetine in m e m b r a n e prepa r a t i o n s from one area of noradrenergic cell bodies (the dorsolateral t e g m e n t u m pontine area) a n d two areas c o n t a i n i n g NAergic terminals (the cerebellum a n d the frontal cortcx) was saturable and Scatchard plots were linear, as s h o w n in Fig. 1. C h a r a c t e r i z a t i o n studies o f [3H]nisoxetine" binding to rat cortical membranes have revealed a Kd value of 0.7 nM. 73 A Kd value o f 1.8 n M was also reported in m e m b r a n e s p r e p a r e d from rat whole b r a i n P ° In the present study, the K d values estimated ( a r o u n d 1.6 2.8 n M ) were slightly higher t h a n in rat species. The specific binding of [3H]nisoxetine at K d range c o n c e n t r a t i o n represented 80 9 0 % of the total binding (Fig. 1). Autoradiographic study' The a u t o r a d i o g r a p h i c distribution of [3H]nisoxetinc binding sites t h r o u g h o u t the b r a i n is depicted in Figs 2 - 4 a n d is quantitatively summarized in Table 1. Nonspecific binding, as d e m o n s t r a t e d by addition of 1 # M m a z i n d o l to the i n c u b a t i o n medium, was equal to the film b a c k g r o u n d in all regions examined (see Fig. 5). The distribution of radiolabeling is described from caudal to rostral levels of the brain, since the central noradrenergic systems have their cell bodies c o n c e n t r a t e d in the brainstem. However, the autorad i o g r a m s presented in Figs 2 a n d 3 are s h o w n in a r o s t r a l - c a u d a l sequence according to the frontal stereotaxic planes defined in the atlases used as references. 58.69

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Fig. 1. Saturation curves and Scatchard transformation (insert) of [3H]nisoxetine binding to homogenates taken from the locus coeruleus region, the cerebellum and the frontal cortex. Membranes were incubated with six concentrations of [3H]nisoxetine between 0.1 and 12nM, for 4 h at 4°C as described in Experimental Procedures. The saturation binding isotherms show specific (solid line) and nonspecific (dashed line) binding. The nonspecific binding was defined in the presence of 1 # M mazindol. The data points are the average of three separate experiments. From these data, the apparent K d were: 2.87 nM (locus coeruleus region); 1.62 nM (cerebellum) and 1.91 nM (frontal cortex).

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In the medulla oblongata, the dorsomedial area displayed a very dense autoradiographic signal. The highest density was seen medially over the area postrema (Fig. 3, PI3, 5-P13). Together with the nucleus locus coeruleus (see below), this structure displayed the highest concentration of [3H]nisoxetine binding sites in the cat brain (Table 1). The nucleus of the solitary tract, which contains the majority of the dorsomedial NAergic cell bodies (equivalent to the A2 group in the rat ~9) showed a high level of [3H]nisoxetine binding sites, from its caudal commissural part (Fig. 3, PI6) to its rostralmost level (Fig. 3, P9). The dorsal m o t o r nucleus of the vagus, where some cell bodies of the dorsomedial group are also located, showed a high level of binding (narrow, clear zone between the area postrema and hypoglossal nucleus at P13 level). A high number of binding sites was revealed in the hypoglossal nucleus. The region of the ventrolateral catecholaminergic cell group (A 1 in the rat ~9) and the lateral reticular nucleus (Fig. 3, P16 to P I I ) showed rather weak labeling, as did the inferior olive, except for the dorsal accessory division (Fig. 3, P9). A discrete zone of dense labeling, indicated by arrows in planes P13.5, PI4 and P16, and visible in all sections up to the pons, had the same location as the descending noradrenergic bundle issues from the pontine locus coeruleus cell group (see Ref.

45). The superficial part of the dorsal cochlear nucleus was also densely labeled. Finally, it should be noted that the granular cell layer in the cerebellar cortex was uniformely underlayed by a moderate labeling. In the pons, the most striking feature was the very high number of [3H]nisoxetine binding sites over the area of the locus coeruleus (Fig. 3, P5 to P1). This area contains the largest collection of NA-containing cell bodies in the cat brain and numerous NAergic fibers~ both types of neuronal elements being dispersed in the locus coeruleus itself and around the brachium conjunctivum, in the medial and lateral parabrachial nuclei. 7~The [3H]nisoxetine binding sites were most numerous around the tract of the mesencephalic trigeminal nucleus, i.e. in the locus coeruleus itself(Fig. 3, P3 to P1). As noted before, this nucleus showed the highest autoradiographic labeling together with the area postrema. Labeling was somewhat less dense in the parabrachial nuclei. The periventricular gray and ventral part of the periaqueductal gray (Fig. 3, P2 to AP0) displayed a substantial level of labeling. Interestingly enough~ the nucleus raphe dorsalis (Fig. 3, A1), where the largest number of serotonin cell bodies is grouped, 7~ gave rise to a high autoradiographic signal. The other raphe nuclei, from the raphe centralis superior in the pons to the raphe obscurus and pallidus in the medulla,

Ahbret:iations used in the figures

aeg AHA AHP AMY AN AP ars BC BP CA

anterior ectosylvian gyrus anterior hypothalamic area posterior hypothalamic area amygdala arcuate nucleus area postrema anterior rhinal sulcus brachium conjunctivum brachium pontis hippocampus (Ammon's horn fields 1~4 of the hippocampus) CAN anterior commissure CD dorsal cochlear nucleus CERL cerebellum CH optic chiasma cs cruciate sulcus DH dorsal hippocampus DMV dorsal motor nucleus of the vagus ENR entorhinal cortex FD fascia dentata GM medial geniculate body GL lateral geniculate body 1C inferior colliculus IG indusium griseum IL infralimbic cortex IO inferior olive IOD dorsal accessory nucleus of the inferior olive LC locus coeruleus LR lateral reticular nucleus ls lateral sulcus MM medial mammillary nucleus MOB main olfactory bulb NAC accumbens nucleus NC caudate nucleus NIP interpeduncular nucleus

NP NST NTS NVL P PA PAG Par PBL PBM peg prs RCS RD RM RO RP RPo SC SCN sg SN ss St STE TM VH 5m 5n 12 17 18 19 35

pontine nuclei nucleus of the stria terminalis nucleus of the solitary tract lateral vestibular nucleus pyramidal tract paraventricular thalamic nucleus periaqueductal gray parasubiculum lateral parabrachial nucleus medial parabrachial nucleus posterior ectosylvian gyrus posterior rhinal sulcus raphe centralis superior nucleus raphe dorsalis nucleus raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus raphe pontis nucleus superior colliculus suprachiasmatic nucleus sigmoid gyrus substantia nigra suprasylvian sulcus solitary tract stria terminalis tuberomammillary nucleus ventral hippocampus tract of the mesencephalic trigeminal nucleus 5th nerve hypoglossal nucleus Brodmann's area 17 Brodmann's area 18 Brodmann's area 19 Brodmann's area 35 of the perirhinal cortex

Nisoxetine binding sites in the cat brain

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A22

Fig. 2. Caption overleaf.

showed a medium to low level of binding. At the ventral surface of the pons, noticeable labeling was seen over the whole extent of the pontine nuclei. As noted for the descending noradrenergic bundles in the medulla, the ascending noradrenergic pathways, as described by Maeda et al. 45 appeared to be labeled. Indeed, a zone of discrete labeling was seen adjacent to the ventrolateral periaqueductal gray in the position of the dorsal ascending bundle (arrows in AI and A4 and dorsal arrow in A3). At A3 level, the ventral arrow points to an area of dense labeling corresponding to the position of the intermediate noradrenergic bundle.45 In the mesencephalon, two additional regions were underlined by a dense reaction, ventrally, the paramedian subdivision of the interpeduncular nucleus (Fig. 2, A2) and dorsally the superficialmost layer of the superior colliculus (Fig. 2, A2 to A3). There was a virtual absence of autoradiographic signal over the structure containing a large and very dense aggregation of dopaminergic cell bodies, namely the substantia nigra (Fig. 2, A3 to A6). The medially located

ventral tegmental area, also containing numerous dopamine cells, showed low labeling (Fig. 2, A4). In the diencephalon, all thalamic nuclei were weakly labeled, except for the small and dorsally located paraventricular nucleus which showed a rather strong reaction (Fig. 2, A9 to A l l ) . Conversely, the hypothalamus displayed an interesting pattern of labeling. On the whole, the posterior hypothalamus gave rise to a stronger reaction than the anterior hypothalamus (cf. A10 with A l l and A13 in Fig. 2). The area with the highest level of [3H]nisoxetine binding was the dorsal part of the pituitary stalk (Fig. 2, A10; Table 1). The arcuate nucleus was clearly labeled. In its rostral half, the high level of binding contrasted with the poor intensity of labeling of the rest of the hypothalamus (Fig. 2; A l l ) . Along all of its caudorostral extent (Fig. 2; A9 to A12), the lateral hypothalamic area contained an area of denser labeling in its lateralmost part, which would partially correspond to the course of the ascending NAergic fibers in the medial forebrain bundle. In the anterior hypothalamus, only

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et al.

P16 Figs 2, 3. Autoradiographic distribution of [3H]nisoxetinebinding to frontal sections of the cat brain. All sections were labeled with 0.1 nM [3H]nisoxetineand processed as described in Experimental Procedures. High densities of binding are represented by dark regions on the autoradiograms. Structures and stereotaxic coordinates, anterior (A) and posterior (P) to the interaural plane, were identified by reference to the atlas of Reinoso-Suarez?8 Scale bar = 5 mm.

the suprachiasmatic nucleus gave rise to noticeable labeling (Fig. 2, A14, A15, A17). In the basal telencephalon, the nuclei lying around the lateral branches of the anterior commissure showed conspicuous labeling: the nucleus of the stria terminalis (Fig. 2, A17) and the nucleus accumbens (Fig. 2, A19). In addition a narrow zone displaying a strong reaction lay in the position of the stria terminalis (Fig. 2, A13 to A15). In contrast, the caudate nucleus and associated structures were completely devoid of autoradiographic signal (Fig. 2, A 14 to A19). The hippocampal formation yielded labeling of low intensity, except for the outer edge of the molecular layer of Ammon's horn (Fig. 2, A3; Fig. 4, A4) and the indusium griseum (Fig. 2, A4 to AI9). A very high density of binding sites was also observed in the most superficial layer of the subicular complex (Fig. 4, A4) and at the same position, in the adjacent areas of the entorhinal cortex (Fig. 2, A3, A2; Fig. 3,

A1). Dense labeling remained visible in the perirhinal area especially around the posterior rhinal sulcus wall (Fig. 4, A4). The entire amygdala was remarkable for its uniformely low level of labeling (Fig. 2, A10 to A14). The neocortex showed labeling of medium intensity over all its extent, the superficial layer displaying the highest concentration of binding sites (see Fig. 2, A27 to A2; Fig. 3, A1 to P3; Fig. 4, A22 to P9). Although a lamination was slightly visible in several cortical areas, e.g., the anterior ectosylvian gyrus (Fig. 4, A22) and the suprasylvian gyrus belonging to the association cortex (Fig. 2, A9; Fig. 3, A1), with a lower number of binding sites over layer IV, binding sites seemed more homogeneously distributed over some others, e.g., the sigmoid and the posterior ectosylvian gyri, without a sharp delimitation between layers (Fig. 4, A22, A4). A more distinct lamination was seen caudally in the visual cortex, mainly in the splenial and suprasplenial gyri (Fig. 2,

LF

265

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P9 Fig. 4. Higher-magnification photomicrographs showing the distribution of [3H]nisoxetine binding to frontal sections through the frontal cortex (A22), the hippocampal formation (A4) and the occipital cerebral cortex (P9) of the cat brain (see legends of Figs 3 and 4). Note the high density of labeled sites over the most superficial layers of the cortex. In the hippocampus (A4) open arrows indicate the outer edge of the molecular layer in Ammon's horn where strong labeling is detected. The insert shows the dorsal hippocampal region in the corresponding section stained with Cresyl Violet. The occipital cortex (P9) shows a laminated pattern of labeling. Note the low level of binding in layer IV (open arrows). Scale bar = 1.2 mm.

A2 and A3; Fig. 3, P3 to A1). Thus, as illustrated in Fig. 4, the labeling seen in the Brodmann areas 17 and 18 presented a typical pattern of distribution with a very low density of binding sites over layer IV. Finally, the olfactory bulb also showed a clear laminar pattern of labeling with the plexiform and glomerular layers being more labeled than the rest of the bulb (Fig. 2, A27).

DISCUSSION

Nisoxetine and analogs have been reported to be potent and selective inhibitors of the high affinity uptake of noradrenaline.73'8°'81 Furthermore, [3H]nisoxetine has been shown to be a high affinity probe selective for the autoradiographic labeling of NAuptake sites in rat brain. 7z The present autoradiographic study indicates that specific binding sites for [3H]nisoxetine are demonstrable many regions of the cat brain and provide additional information

concerning the anatomy of the NAergic systems in the brain of this species.

Methodological considerations Studies in brain homogenates and in tissue slices of rat species indicated that [3H]nisoxetine binding was saturable and sodium-dependent to a single class of binding sites. Furthermore, it has been clearly shown that compounds that selectively bind to serotonin, dopamine uptake sites or to a variety of postsynaptic receptors (e.g. adrenoceptors, muscarinic and benzodiazepine receptors) are inactive at inhibiting the binding of [3H]nisoxetine at concentrations up to l ~ M (see Ref. 72). Our autoradiographic study of [3H]nisoxetine binding sites in the cat brain was performed according to the experimental procedures previously defined by Tejani-Butt.72 The [3H]nisoxetine labeling was almost completely inhibited by 1/~ M mazindol and binding studies in homogenates from various areas gave apparent Kd values of the same order of magnitude as those estimated in the

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Table 1. Regional distribution of specific [3H]nisoxetine binding in the cat brain Brain regions

fmol/mg tissue

Area postrema 214.9 _+ 9.4 Nucleus locus coeruleus 175.1 _+_7.6 Dorsal accessory nucleus of inferior olive 111.98 _+ 1.2 Suprachiasmatic nucleus 62.7 _+ 1.4 Sul~erficial layer of entorhinal cortex 50.4 _+ 1.2 Pontine nucleus 46.5 _+ 8.5 Pituitary stalk 41.4 _+0.3 Arcuate nucleus 37.7 +_ 1.0 Lateral parabrachial nucleus 36.4 _+ 7.1 Nucleus of the solitary tract 36.3 _+ 4.9 Brachium of the superior colliculus 35.4 _+ 1.3 Nucleus raphe dorsalis 35.2 + 5.1 Nucleus raphe pallidus 33.7 _+ 1.3 Interpeduncular nucleus 33.6 _+ 5.6 Paraventricular nucleus of the thalamus 32.6 _+ 1.6 Nucleus raphe centralis superior 31.9 + 1.5 Medial parabrachial nucleus 31.1 _+ 3.7 Indusium griseum 31.0 _+ 1.0 Accumbens nucleus 29.2 Jr 1.4 Hypoglossal nucleus 28.6 + 6.3 Anterior hypothalamic area 27.7 + 5.3 Tractus olfactorius 26.1 _+ 1.5 Dorsal lateral geniculate body 22.9 _+ 3.8 Periacqueductal gray 18.0 _ 1.6 Lateral mammillary nucleus 16.8 __+0.8 Frontal cortex 16.0 _+ 0.9 Cerebellum 10.4 _+ 1.2 Entorhinal cortex 10.2 + 2.3 Hippocampus CA 8.8 _+ 1.3 t

Optical densities were converted to finol/mg tissue equivalent with reference to ~H microscales (Amersham). Values are expressed as means _+ S.E.M. of three to five determinations per areas from four cats (see Experimental Procedures for further details). rat brain. 6°'73Therefore, it can reasonably be assumed that the labeling detected in the present study corresponds to high-affinity N A - u p t a k e sites as previously demonstrated in the rat. Additionally, the virtual absence of [3H]nisoxetine labeling through the dorsal striatum (i.e. the caudate nucleus and putamen), a brain area known to contain the highest densities of dopamine transporter associated with a massive dopaminergic input (for review, see Ref. 10), supports the lack of affinity of [3H]nisoxetine for the dopamine transporter. Main .findings and possible ,functional implications These findings provide the first anatomical description of [3H]nisoxetine binding sites in the cat brain. Overall, they confirm and extend the results of previous studies in the rat. 72 Thus, with few exceptions, the regional distribution of binding sites reported in the rat brain az'T2 is comparable to that observed in the cat brain. However, differences in the densities of binding could be noted in some areas such as the thalamus (e.g., the ventral posterior and the anteroventral thalamic nuclei) and the dentate gyrus. In these areas, the high levels of labeling reported in rat brain v2 were not found in cat brain. Furthermore, our autoradiographic study indicates

that in the cat brain, several areas such as the area postrema, the entorhinal cortex, the suprachiasmatic nucleus, the accumbens nucleus and the main olfactory bulb exhibit substantial amounts of binding sites. Lesions by selective neurotoxins like 6-hydroxydopamine and N-(2-chloroethyl)-N-ethyl-2bromobenzylamine, suggest that in the rat brain, [3H]nisoxetine binding sites are predominantly associated with the NAergic neuronal elements. 72 Recent in situ hybridization studies confirm that the NA-transporter m R N A is expressed almost exclusively within noradrenergic cell bodies. 44 Thus, high levels of NA-transporter m R N A were visualized in the locus coeruleus cells 22'44 and in all the other NAergic cell populations (A I - A 5 and A7) distributed over the pons and medulla oblongata. ~ The topographic distribution of the [3H]nisoxetine binding sites observed through the brain of the cat is compatible with these observations. Particularly dense labeling was seen throughout the whole locus coeruleus complex. More caudally, in the medulla oblongata, areas known to contain NAergic neurons such as the area postrema and the solitary complex, ~7'28'57 also, display a strong autoradiographic signal. Previous studies showed that the ability to accumulate exogenous catecholamine is a property of all parts of the adrenergic neuron including preterminal axon and cell soma. 33'38 Therefore, it is conceivable that [3H]nisoxetine binding sites located on the cell somata as well as on varicose NAergic processes surrounding these cell populations together contribute to the labeling seen in these areas. In contrast to the rat, 48"49"5°'71 the anatomical distribution of NAergic fil~ers and terminal endings in the cat brain have not been systematically investigated. However, biochemical analyses 32 and histochemical studies combined with lesions ~6'45have suggested that the organization of the NAergic pathways and the areas of projection remain comparable in both species. Thus, the distribution of [3H]nisoxetine binding sites observed here through the cerebral cortex, the septum, the hypothalamus, the cerebellar cortex and various areas in the brainstem could be reasonably associated with the presence of NAergic fibers and terminal endings. The distribution of the binding sites observed in the visual cortex is particularly illustrative for such a correlation. In the cat visual cortex, dopamine beta-hydroxylase-immunoreactive varicose fibers, thought to be mainly NAergic, exhibit a typical laminar distribution with high densities of immunoreactivity concentrated in layers I III and V VI, and a band of lower immunostaining in layer IV. 42 An almost comparable laminar distribution is clearly visible for the [3H]nisoxetine binding sites reported in the present study. The high concentration of binding sites more generally observed in layer I of the neocortex suggests that this layer receives a dense NAergic input, as previously reported in the rat.48,50,7~

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oo"

/

RM Fig. 5. Frontal sections of cat forebrain (A18) and hindbrain (P6) illustrating the total [3H]nisoxetine binding (left) and in adjacent sections, the nonspecific binding determined in the additional presence of 1 pM mazindol (right) (see Experimental Procedures for further details). Scale bar = 3 mm.

Of special interest is the high density of radiolabeling seen over the raphe nuclei. Anatomical studies indicate that these nuclei receive NAergic afferents (see Refs 16, 55). Electrophysiological and pharmacological studies in the raphe dorsalis and the raphe magnus of the rat brain suggest that the release of NA in such regions may influence the activity of the serotonergic cells via alpha-adrenoceptors. 1"3'24'43'62"65 The presence of high levels of [3H]nisoxetine binding sites observed in these nuclei, especially in the raphe dorsalis, stresses the possibility that high affinity NA-re-uptake mechanisms, by removing NA from the extracellular space, could play a major contribution to the local interactions between NAergic and serotonergic systems. More intriguing is the intensity of radiolabeling seen in some areas not usually considered as major noradrenergic terminal fields. Among them, the interpeduncular nucleus presents a typical pattern of labeling with a predominant concentration of binding sites in the paramedian subdivision. Although high concentrations of NA have been reported in the interpeduncular nucleus in humans, 2~ anatomical studies in rat have indicated a rather sparse catecholamine innervation through this nucleus.8'29 Another brain area where an unexpected high level of binding sites was observed, is the suprachiasmatic nucleus. Although circadian rhythms for NA have recently been described in the rat suprachiasmatic

nucleus, ~3 the catecholaminergic innervation of this nucleus seems relatively poor. 49'71 More conflicting results have been reported in the cat suprachiasmatic nucleus where very low ~5 or high 54 densities in catecholamine-containing terminals were observed. The presence of [3H]nisoxetine binding sites in the ventral part of the cat suprachiasmatic nucleus supports the presence of NAergic afferents in this area. The fact that high densities of [3H]nisoxetine radiolabeling were present in the nucleus accumbens is of particular interest. Despite the relative paucity in noradrenergic afferents reported through the striatal region,49'71 substantial levels of NA 22°'21"77 and NAuptake sites27'3° have been demonstrated in the nucleus accumbens of rodents and human. There are several lines of evidence for interactions between noradrenaline and dopamine within the nucleus accumbens, especially in modulating the hippocampal and amygdaloid inputs of the ventral striatum (see Ref. 18). Thus, our autoradiographic studies suggest that in this area, the noradrenaline levels could be regulated by a NA-re-uptake specific mechanism. CONCLUSION

This autoradiographic study demonstrates the neuroanatomical distribution of [3H]nisoxetine binding sites in the cat brain. In view of the

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selectivity o f nisoxetine for high-affinity n o r a d r e n a line uptake sites, 72 the distribution o f binding sites reported here provides novel i n f o r m a t i o n on the anatomical organization o f the noradrenergic systems in the cat brain. Further, it is suggested that in discrete areas such as the raphe nuclei, the suprachiasmatic nucleus, the accumbens nucleus, high-affinity re-uptake mechanisms could play a major role,

locally, in the interactions o f these systems with the other m o n o a m i n e r g i c systems. Acknowledgements--We would like to thank Dr S. M. Tejani-Butt for giving helpful advice and Dr M. Aebi for the gift of mazindol. We are grateful to B. Greggio and C. Huguenin for technical assistance and P. Y. Vallon for preparing micrographs. Y. C. is presently detached from the C.N.R.S., France.

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