Pharmacological characteristics and anatomical distribution of [3H]oxytocin-binding sites in the wistar rat brain studied by autoradiography

NeuroscienceVol. 20, No. 2, pp. 599-614,1987

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0306-4522/87$3.00+ 0.00 Pergamon Journals Ltd @ 1987IBRO

PHARMACOLOGICAL CHARACTERISTICS AND ANATOMICAL DISTRIBUTION OF ~3H]OXYT~IN-BINDING SITES IN THE WISTAR RAT BRAIN STUDIED BY AUTORADIOGRAPHY M. J. FREUNDMERCIER,* M. E. STOECKEL,* J. M. PALACIOS,~ A. PAZOS,~ J. M. REICHHART,~ A. PORTE*and Ph. RICHARD* *Laboratoire de Physiologie g&&ale, IJA 309 CNRS, 21 rue Descartes, F67084 Strasbourg, France; tPreclinica1 Research, Sandoz Ltd, CH4002 Base], Switzerland; and fLaboratoire de Biologie &ntrale, UA 672 CNRS, 11 rue de l’universiti F67000 Strasbourg, France AbPtnct-Oxytocin-binding sites were detected by autoradiography on rat brain sections incubated in the presence of the [3H]oxytocin. These sites were characterized pharmacologically using quantitative autoradiography. High pressure liquid chromatography controls of the incubation media indicated that labelling was due to the intact f”H]oxytocin molecule. P~~acolo~~l analysis of different locations (central amygdaloid nucleus, ventral subiculum and ventromedial hypothalamic nucleus) showed that the sites detected had a high affinity for oxytocin and also for arginine-vasopressin. In contrast, some areas known to bind vasopressin intensely, such as suprachiasmatic and lateral septum nuclei, had little or no affinity for oxytocin. Autoradio~aphs revealed [3~oxyt~in-binding sites in already known brain areas (olfactory centres, ventral subiculum, central amygdaloid nucleus, bed nucleus of the stria terminalis) albeit with more extensive labelling of some of these formations, in particular, the amygdaloid complex. In addition, specific [3H]oxytocin-binding sites were found in areas not yet reported lo bind oxytocin, such as the paraventricular thalamic and caudate nuclei. In the hypothalamus, specific binding sites were not detected in the supraoptic and paraventricular nuclei: the only structure labelled was the ventrolateral part

of the ventromedial nucleus. Discrepancies between the con~ntrations of [3~]oxyt~in-bin~ng sites, the known dist~bution of oxytocin-containing endings and electrophysioiogical data indicate that autoradiography, under our conditions, apparently only reveals some of the oxytocin receptors in the brain, Thus, in the hypothalamus, no relationship can be established between the known effect of oxytocin on oxytocinergic ma~~ll~ar neurons and detection of specific ~3H]oxyt~in-binding sites. Auto~diog~phy may reveal mainly oxytocin-binding sites in areas receiving diverse “parasynaptic” information, where oxytocin might play a modulatory role rather than exerting rapid, short-term effects of the neurotransmitter type.

In addition to oxytocinergic and vasopressinergic projections of hypothalamic ma~o~Iiular neurons of the paraventricular and supraoptic nuclei on the neurohypophysial capillaries, fibres and endings containing oxytocin (OT) or arginine-vasopressin (AVP) occur in various areas of the central nervous system, in the forebrain, the brainstem and the spinal cord (for reviews see Refs 43 and 45). Synaptic axo-dendritic contacts between axons containing AVP or OT and other neurons have been demonstrated ultrastructurally in cerebral areas.7 Thus, in addition to their hormonal role, AVP and OT are obviously involved in the control of different neuronal systems, acting either as neurotransmitters or neuromoduiators. Although the two types of fibre generally occur in the same brain areas, the ratio between axons conAddress correspondence and reprint requests to: Dr M. J. Freund-Mercier, Laboratoire de Physiologie g&&ale, LJA 309 CNRS, 21 rue Descartes, F67084 Strasbourg, France. Abbreviations: AVP, arginine-vasopressin; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; pH]OT, [-‘H]oxytocin; OT, oxytocin.

taining OT and AVP varies widely according to the location. Thus, OT~on~ining fibres, which probably all originate in the paraventricular nucleus, predominate in the brainstem and the spinal cord, while AVP-containing fibres, o~ginating not only from the paraventricular nucleus but also from several other areas (suprachiasmatic nucleus, bed nucleus of the stria terminalis, medial amygdaloid nucleus . . .), largely predominate in the forebrain.d3*dsThe predominance of AVP-containing fibres in the forebrain might explain why some of the neurohypophysial hormone’s central effects, e.g. the short-term ones on the homeostatic mechanisms and spontaneous behaviour, or long-lasting ones on learning and memory, seem to be more obviously related to AVP (for review see Ref. 29). However, OT has frequently been reported to have effects opposite,23*28and in some cases similar,~ to those of AVP. Oxytocin seems moreover to be specifically involved in certain comportments, e.g. inducing maternal hehaviour in the female rat. IdRecent data from our group have shown a specific facilitatory effect of OT on oxytocinergic neurons, enhancing hormone release in the suckled lactating rat. I6This stimulator effect, which requires

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the molecule to be intact, can also be demonstrated in vitro on isolated paravent~cular or supraoptic nuclei. Arginine-vasopressin release is not stimulated by OT, and AVP does not affect OT release or its own release.30 Any such effect of OT would imply its release from dendrites or recurrent collaterals of oxytocinergic neurons and the presence of specific OT receptors, either on these neurons themselves, or on a neuronal system projecting into the magnocellular nuclei, Auto~diographic studies of neurohypophysia1 hormone receptors in the rat brain showed a generally similar distribution of OT- and AVPbinding sites, except for a few areas (e.g. lateral septum, and nucleus of the solitary tract) which bind AVP exclusively.” As regards the magnocellular nuclei, OT- and AVP-binding sites have been reported in the supraoptic and paraventricular nuclei by some authors, 56,223 but not by others.31’.‘1,13.% In a recent report, OT-binding sites were occasionally found in the rostra1 part of the supraoptic nuclei, but not in paraventricular nucleLq Our preliminary observations did not reveal any specific OT-binding sites in either magnocellular nucleus.i5 In view of the extensive overlapping between AVP- and OT-binding sites and the discrepancies of the reported localizations of these sites, we have attempted (1) a more detailed characte~zation of OT-binding sites, and (2) a more extensive anatomical study of the localization of these sites in the

hypothalamus and other brain areas using the quantitative autoradiographic technique. EXPERIMENTAL PROCEDURES

Male (a = 12) and lactating female (n = 3) Wistar rats were used in this study. The animals were killed by decap itation and the brains were rapidly frozen in dry ice and stored at -20°C until sectioning. Twenty-pm-thick serial coronal and sagittal sections (interaural levels A 2.7 to A 9.2 mm, lateral levels 0 to 3.4 mm, according to Paxinos and Watso#) were cut on a microtome cryostat (Frigocut Reichert) and thaw-mounted on gelatin-coated slides. Controt sections of mammary glands from lactating females (12 days ~stpa~urn~ were prepared in parallel. The sections were stored at -20°C until use (4 weeks ~ximum). Binding procedure

After preincubation for 20 min at 15°C in T&buffer (Tris-HCl 170 mM, MgCl, 5 mM, bovine serum albumin (BSA) 0.1%; pH 7.4), the sections were incubated for 6Omin at 15°C in the same buffer containing 5 nM [3Hkxytocin ([‘HIaT) (34.4 or 39.4Ci/mmol: NEN, Du Pont de Nemours, Paris) alone or in the presence of various concentrations of unlabelled peptides: GT (Bachem, Bubendorf, Switzerland or CRB, Cambridge, U.K.); AVP (CR& the vasonressin antagonists dP,Y&n-Args,AVPar (Bacfiem) and h(CHr)5,Tyr(Me),Va14,AVPr’ (ge&ously supplied by Dr Su Sun Wana, California. U.S.A.): the oxytocin antagonists dP,Tyr(My),OTZ (generously i uqtlieh by Dr M. Manning, Toledo, U.S.A.) and dP,Hyk ,O’I”’ (generously supplied by Dr D. Gazis, New York, U.S.A.). The incubation was followed by two 5 min washes in cold but& and sections were then rapidly rinsed in cold distilled water and dried with a cold air-stream.

Optimal incubation conditions, i.e. [‘H]OT concentration, in~bation temperature and duration, influence of protease inhibitors: bacitracin, aprotinin, amastatin (Sigma). were defined in preliminary experiments on mammary gland sections. After incubation, these sections were wiped off the slides with Whatman GFjC filters and placed in scintillation vials with 2 ml of protosol (NEN) for 12- 14 h at 40°C. Six ml of scintillation fluid (omnifluor 8 g, Triton X-100 333 ml, toluene I I) were added for radioactivity measurement. Auroradiography

Autoradiographs were generated by apposing the labelled sections to [3H]Ultro~lm (LKB) as already described.“s The films were developed after 4 months’ exposure. For the evaluation of binding site densities, brain gray and white matter standards, containing known amounts of a non volatile [‘Hllabelled compound were exposed to the film along with the labelled sections. Films were analysed and quantified with a computerized image-analysis system, where the autoradiographic image was digitized onto a matrix of 256 x 256 picture units using a TV camera connected to a microscope. The areas to be measured were drawn on the TV monitor screen with a bight-~ns~tive pen. The optical densities of these areas were computed and transformed into receptor densities (fmoljmg protein) by comparison with the optical densities of the standards.9,H After exposure, the sections were postfixed in 4% phosphate-buffered paraformaldehyde and stained with thionine. High pressure liquid chromarography analysis

The stability of the [3H]OT in the medium during incubation was checked by high pressure liquid chromato~aphy (HPLC) analysis. One ml of incu~tion medium (untying protease inhibitors or not) was sampled before and after 3 or 6 incubation periods (60min each). The samples were boiled (90 s), cooled in ice for 30 min, centrifuged for 15 min at 1500g and stored at -20°C. Before HPLC analysis, the samples were vacuum-dried and taken up in 100 ~1 of 0.1% orthophosphoric acid in water with the standards (OT 1.167 pg; AVP 0.583 pg). They were applied to a Bakerbond Cl&NT column (4.4 x 250 mm), and eluted at 1 mi/min under the following conditions: 10 min at 15% of 0.1% orthophospho~c acid in acetonitrile (solution A), 1Omin gradient from A to B (30% of 0.1% or~o~hosphoric acid in acetonitrii), and 10 min in solution 8. The eluates were monitored at 280nm. Fractions of SOOfil were collected, 1 ml of scintillation fluid (ACS(R) Amersham) added, and the radioactivity was counted in a liquid scintillation counter. RESULTS

In the brain, high concentrations of OT-binding sites occur in limited areas, so mammary gIand sections were used to define optimal binding conditions for [3H]OT, because of their high concentration and homogeneous distribution of OT receptom. The total, specific and non-specific, [%@T binding as a function of in~~~on titne, was determined on sections incubated in the pre#nce 6fsnM [3H]OT at 15°C. Specific binding eq~~~~ was reached within #min (Fig. 1A). Cotnp&rison of binding at 15 ad 25°C showed much SpacirrG binding at 15°C (Fig. 113). AI1 irm&Wns were therefore performed for 60 min at 15°C. The stability of [“iffoT do&g the incubation procedure was verifkd by HPLC analysis of the

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Fig. 1. Time- and temperature-dependency of [‘HJOT binding to mammary gland sections. Mammary Bland sections were incubated in the presence of 5 nM [rH]OT at 15 or 25°C for various times. [WjOT binding was determined after the sections had been wiped off the slides (for details, see Experimental Procedures). Specific binding (e) was taken as the difference between total binding (m) and non-specitk binding (a) and non-specific-binding (0) measured in the presence of 10 JLM unlabelled OT. The results are averages for 4 destinations. (A) At WC, specific binding reached ~~iib~~ after 4Omin of incubation. (B) Specific [‘HjOT binding was higher when incu~tion was carried out at 15°C than at 25°C.

ligand. While in the freshly prepared medium, 84.8% of the total radioactivity co-migrated with OT, 79.0% still did so after three 60min incubation periods in the presence of brain sections (Fig. 2). No appreciable change could be detected even after 6 consecutive 60 min incubation periods; the stability of the peptide was similar in the presence and in the absence of protease inhibitors. ~~~~t~t~tiv~ ~utor~diog~uphic studies The method worked out in the rn~rna~ gland was assessed by a saturation kinetics of [3HjOT 20000

binding in a brain area with a high concentration of OT receptors (see below). Sections including the ventral subiculum were incubated under the conditions determined according to biochemical data, in the presence of increasing concentrations of [3H]OT. The apparent Kd and B_ for [‘H]OT binding were calculated densitometrically. A & of 4.95 nM and a maximal binding site concentration of 363.2 fmol/mg protein were determined in the ventral subiculum (Fig. 3). The method was therefore used for studying ph~a~lo~~l character&tics and anatomical distribution of ~3H]OT-bin~ng sites in the rat brain.

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Fig. 2. HPLC control of the incubation medium. Elution was performed at 1 ml/min, as indicated in Experimental Procedures. Percentage of solution 3 ( ..*,), absorption at 280 nm (-). radioactivity of the fractions ~///). (A) In the freshly prepared medium, 84.8% of the total activity co-migrated with OT. (B) After three 40 mitt incu~tion periods in the presence of brain sections, 73.0% of the total radioactivity still co-migrated with OT.

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determinations. A Scatchard analysis of the saturation curve is shown in the insert.

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The pharmacology of [3HpT binding was analysed by quantitative autoradiography in brain areas with high densities of binding sites: the ventral subiculum, the central amygdaloid nucleus, and the ventromedial hypothalamic nucleus (Fig. 4). The competition profiles for t3HpT binding of OT, two OT antagonists, AVP and two AVP antagonists was studied. Oxytocin displaced t3H]0T in all three locations, but the OT concentration which displaced 50% of [3H]OT in the ventral subiculum and central amygdaloid nucleus was about 10 times higher than in the Table 1. Densities of [~HJoxytocin-binding sites in the male rat brain

Area

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Septum Bed nucleus of the stria terminalis 112.3 f 23.9 (n = 5) Amygdala 263.6 + 44.5 (n = 5) Central amygdaloid nucleus 158.9 * 14.0 (a = 3) Medial amygdaloid nucleus 88.9 f 22*8 (a = 3) Basomedial amygdaloid nucleus ~ip~rnp~ 311.0 f 25.7 (tt = 5) Ventral subiculum Basal ganglia 64.9 f 14.4 (a = 4) Head of the caudate nucleus Cortex 76.0 f 2.3 (n = 3) Entorhinal cortex Thalamus 67.1 f 16.1 (a = 3) Paraventricular nucleus Hypothalamus 300.8 f 40.5 (n = 5) Ventromedial nucleus

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d(CH,)f,Tyr(lWLe),Va~~VP(A). ~%F.E’ W&&k& was %feasured by qua~t~ativc auto~~~~y. b bin&g in the presence of ~~~~ peptides is apressed as a percentage of the total binclmg measured in the absence d competing peptides. Each point is a mean of 2 measuremcnts each from 3 male rats.

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Localization of [‘Hloxytocin-binding sites in rat brain Anatomical distribution of [3H]oxytocin-binding sites in the brain of the male rat

The location of [)H]OT-binding sites was studied on autoradiographs of brain sections between the coronal interaural planes 2.7-9.2 mm and sagittal planes 0-3.4mm. The binding site densities were measured in several zones (Table 1). The precise location was established by comparing the autoradiographs with the sections stained with thionine after exposure. O[factory system. The anterior olfactory nucleus (posterior part) and the olfactory tubercle (islands of Calleja) had high densities of [3H]OT-binding sites (Fig. 5). Septum. The highest density of [‘H]OT binding was found in the bed nucleus of the stria terminalis, mainly in its medial part (Figs 5, lines b-c; 6 and 7). The lateral septum nucleus presented much weaker but still significant labelling (Figs 5 and 6). Amygdala. A high concentration of [3H]OTbinding sites occurred in the central amygdaloid nucleus but only in its posterior part; a very small area near the dorsal ridge of the nucleus was constantly labelled (Figs 7, line c; 8, line a; and 9, line c). The medial and the basomedial amygdaloid nuclei were moderately labelled (Fig. 8, lines b-c). In addition, diffuse weak specific labelling occurred throughout the amygdaloid complex (Fig. 8). Hippocampus. Specific [3H]OT-binding sites were highly concentrated in the ventral subiculum where they presented a laminated organization (Figs 9 and 11). The dorsal subiculum was less intensely labelled (Fig. 9). There was no specific labelling in the different fields of Ammon’s horn or in the dentate gyrus; non-specific labelling was always obvious in those areas (Figs 5 and 8-l 1). The taenia tecta, which are anterior hippocampal rudiments, were intensely labelled. Basal ganglia. Concentrations of specific [3H]OTbinding sites occurred in the head of the caudate nucleus (Figs 5, 6 and 9) and in the lateral and posterior parts of the caudate putamen complex (Fig. 8) which areas can be attributed to the caudate nucleus (body and tail). On the sagittal sections, labelling was also found on the ventral border of the nucleus accumbens, apparently extending over the ventral pallidum (Fig. 5). Cortex. Definite labelling was found in the entorhinal cortex (Figs 9 and 11); sagittal sections clearly showed labelling related to the external and internal layers (Fig. 9). Labelling was also found constantly in a cortical area dorsally adjacent to the rhinal fissure (Fig. 11). Thalamus. Binding sites were found in a restricted area corresponding to a midline nucleus, the paraventricular nucleus (Fig. 10). Hypothalamus. A very high concentration of [‘H]OT-binding sites was only detected in the ventrolateral subdivision of the ventromedial hypothalamic nucleus (Figs 7 and 8). Adjacent neuropil areas,

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anterior, basal and lateral to the nucleus were also labelled, but labelling never extended over the dorsomedial part of the nucleus nor towards the neighbouring arcuate nucleus (Fig. 8). No specific binding sites were detected in the hypothalamic paraventricular (Figs 6, line a and 7), supraoptic (Fig. 6, line a) or suprachiasmatic (Fig. 6, line a) nuclei. The [)H]OT binding which occurred irregularly in the supraoptic nucleus and the median eminence proved to be non-specific (Fig. 6). Weak labelling was observed in the choroid plexus, mainly in the lateral ventricles (Fig. 7). Labelling was also obvious in the meningal tissue in the interpeduncular fossa. Variations in [3H]oxytocin binding in the brain of lactating rats

The anatomical distribution of rH]OT-binding sites in male rats was the same as in lactating female rats. There was, however, an obvious difference in the density in the ventromedial hypothalamic nucleus; while specific binding was 300.8 + 40.5 fmol/mg protein in the male rat, it was only 68.9 + 13.5 fmol/mg protein in the lactating rat. No obvious difference was detected in other areas, such as the central amygdaloid nucleus, the ventral subiculum and the bed nucleus of the stria terminalis. DISCUSSION

In this autoradiographic study, specific OTbinding sites were found in locations already reported,%“,‘%” such as olfactory centres (olfactory anterior nucleus, olfactory tubercle or islands of Calleja), the central amygdaloid nucleus, the bed nucleus of the stria terminalis in the septum, the subiculum in the hippocampus.. . . Moreover, in some areas, labelling was more extensive, while labelled sites were also found in some formations not hitherto reported to bind OT (or AVP). The labelling of olfactory centres was found to extend to the entorhinal cortex. In the amygdala, OT-binding sites were not confined to the central nucleus; they were also concentrated in the medial and mediobasal nuclei and diffusely distributed throughout the amygdaloid complex. Significant labelling occurred in the paraventricular thalamic nucleus and in the area corresponding to the caudate nucleus in the striatum. In the hypothalamus, specific OT-binding sites were never detected in the paraventricular and supraoptic nuclei, in contrast with the findings of other authors.6 Occasional labelling of the supraoptic nucleus, also reported by Van Leeuwen et al.,” was not specific. Specific labelling was found in the ventromedial nucleus, but only in its ventral subdivision. This ventromedial area, which we have previously described as “an ill-defined area of the basal hypothalamus”,i5 undoubtedly belongs to the ventromedial nucleus, as also reported recently by Van Leeuwen et a1.49and by de Kloet et al.“’

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Abbreviatjo~s used in jigares A0 An: BM BST C ce ChP CPU Ent HP ICj ?E Me

anterior olfactory nucleus arcuate nucleus basomedial amygdaloid nucleus bed nucleus of the stria terminalis eaudate nucleus central amygdaloid nucleus choroid plexus caudate putamen entorhinal cortex hippocampus islands of Calleja lateral septum nucleus median eminence medial amygdaloid nucleus

Pa PV RF

Sch Sd so SV

VMH VMHDM VMHVL VP

paraventricular hypothalamic nucleus paravent~cular thalamic nucleus rhinal fissure suprachiasmatic nucleus dorsal subiculum supraoptic hypothalamic nucleus ventral subiculum ventromedial hypothalamic nucleus ventromedial hypothalamic nucleus-dorsomedial part ventromedial h~othalam~c nucleus-ventrolateral part ventral pallidum

Figs S-l 1. Distribution of [3H]oT-binding sites in the male rat brain. (A) Sections (20pm) stained with thionine after incubation in the prettence of S&I [3HJOT and a~tom~o~~c expoxure. {I!+)Autoradiographs generated by the sections of (A), showing distribution of [3H)t3T-bin&ng &es. (C) Autoradiographs from sections adjacent to sections of (A), incubated in the presence of 5 nM [%EOT and 10 PM OT, showing non-specific binding sites. Bars 2 mm. Fig. 5. Sagittal sections showing [JHJOT-binding sites in the olfactory tubercle (islands of Calleja, ICj), anterior olfactory nucleus (AO), bed nucltus of the &a terminahs @ST), head of the car&am .nuelqra (C), ventral pallidurn (VP), ventromedii hypothalamic nucleus (VMH) and lateral septum (LS). Note the intense non-ape&c labelling of the dorsal hippocampus (HP). Fig. 6. Coronal sections through the anterior hypothalamic and septal areas. Intense speoifk l&elling in the bed nucleus of the stria terminalis @ST’) and the head of the caudate nucleus (C). Week lab&lag in the lateral septum nucleus (LS). In the baaal hypothalamus, the area slightly anterior to the vent&me&al nucleus is weakly labelled (X). The label%8 of the supraoptie m&ens (So) and median emknee (ME), seen in (B), is not displaced by the OT anta@nist, as shown in (C). No labelhug is seen on the paraventricular (Pa) or suprachiaematic (SCh) nuclei. (C) i3HJOT binding in the presence of the OT antagonist dP,Tyr(Me),OT (I p M). Fig. 7. Successive coronal sections (2SOpm intervals) through the hypothalamic area containing the paraventricular nucleus (Pa) which is unlabelled in its anterior (line a), medial (line b) and c&e4 (l&e c), divisions. On the same sections, the cat&al portion of the bed nwleus of the stria terminO_is @ST), the central amygdaloid nucleus (Ce) and the anterior zone of the ventromedial nucleus (VI&) are intensely labelkd. The choroid plexuses (ChP) are weakly labelled. (C) 13Hm binding in the presence of 1 #M OT. Fig. 8. Coronal sections through the posterior hypothalamus and the amygdala. Intense label@ in the ventrolateral division of the ventromcdial nuclene (VMHVL); rnon diffuse labelllng ate&x around the nucleus but never towards its domomedial part (VMHDM) nor to the arcuate nncletts (Arc). In the amygdala, the central nucleus (Ce) ia inter&y l&eBed, the basomedial (BM) and the media1 (Me) amygdaloid nuclei are also labelled. Diffirse lab&lIing also ocettrs throughout the amygdala. Dopially to the amygdala, the lateral part of the cat&kite putamen (CPU) is also labekl. Sections in line a are from another brain than those of lines b and c. (C) [%foT binding in the presence of 1 fiM OT. Fig. 9. Sagittal sections through the hippocampal and amygdaloid formations. In the hippocampus, specific binding sites cecur in the domal and ventral subiculum (Sd,Sv), the latter being more intensely The internal and oxternal,layers of labelted, non-specific labelling is high ~~~ the hip. the entorhinal cortex (Ent) are lab&d. ‘Ike mo@t medial section (line a) shows laI%lBng of the border of the caudate nucleus (C), while in the meet lateral section (line c), the central amygdaloid nucleus (Ce) is intenseiy labelled. Fig. 10. Coronal section showing specitle l&?lling of the paraventricular nucleus of the tlwdemw (PV), of the ventral subieulum (Sv). Note the laminated Fig. 11. Coronal sections ahowin intenxe label organiaation of the binding sites. #h e eat&hi@ Otiex (Ent) and an area dorsally adjeeent to the rhimtl fl6snre (RF) an xlightIy lab&led.

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Further pha~~olo~~l cha~cte~zation of possAreas with high concentrations of OT-binding sites, such as the olfactory nuclei, the ventral sub- ible OT receptor subtypes was attempted using OT iculum, the central amygdaloid nucleus, have also and AVP antagonists. Indeed, So10ff~~ has shown characteristics for uterine been reported to have high concentrations of AVP- different pha~a~lo~~l binding sites. ‘*Ii However, other areas binding AVP and mammary gland OT receptors. In the present intensely such as the lateral septum, the nucleus of the study, the two OT antagonists tested were less potent solitary tract and the suprachiasmatic nucleus’5*1’*‘3*50than OT itself in displacing [‘H]OT, but were more have little or no affinity for OT. Our preliminary potent than the two vasopressor antagonists. Of the observations confirmed the high density of AVP- two OT antagonists, dP,Tyr(Me),OT, which inhibits binding sites, contrasting with the complete absence both the uterine and mammary gland response,* and of OT-binding sites from the suprachiasmatic nu- dP,Hy Ics,OT only affecting the uterine response,17 cleus, and showed similar labelling intensities with the former was more effective, mainly in the atnyg[3H]OT and [3H]AVP in the basal subdivision of the dala, so the receptor detected was probably closer to the mammary-gland-type of OT receptor. These obventromedial nucleus.‘5 servations diverge from electrophysiological data which suggest that the OT receptors in the Pharmacological considerations hippocampus and the brainstem* are rather of the These data raise the question of the specificity of uterine type. Our pharmacological data are as yet insufficient for more precise characterization of the the OT-binding sites revealed by autoradiography. OT receptors localized with the autoradiographic The HPLC controls of the incubation medium indicate that the lahelling, in fact resulted from technique in the different brain areas. It cannot be ruled out that the brain OT receptors may differ from binding of the intact [‘H]OT molecule and not from both types characterized on peripheral targets. any degradation fragments. The quantitative autoradiographic technique gave a [rH]OT-binding Kd in the ventral subiculum comparable to that determined The anatomical ~st~bution of OT-binding sites in hippoc~pal synaptic membrane preparation (4.95 also raises the rather puzzling question of the lack of and 1.8 nM, respectively) by Audigier and Barberis.’ precise to~graphi~l relationships between the There is, however, some discrepancy between the S_ projection areas of the fibres containing OT (or AVP) values determined with autora~o~aphic and bio(for review, see Ref. 45) and areas with high concenchemical techniques (363.2 and 16.9 fmol/mg protein, respectively). This is due to the fact that these mem- trations of OT- (or AVP-) binding sites. Thus, fibres containing either OT or AVP, although regularly branes were prepared from the whole hippocampus, while our measurements were taken only in the present, are not particularly concentrated in the ventral subiculum where [3H]OT-binding sites are olfactory centres which bind both peptides intensely. electively concentrated. The 13H]OT-binding sites In the hippocampus, immunoreactive axons are in revealed with the quantitative autoradiographic tech- fact abundant in the ventral subiculum which has a nique thus correspond well to the high-affinity bindhigh density of binding sites, but they are also widely ing sites characterized with biochemical techniques. distributed in other hippocampal zones (e.g. the Moreover, quantitative autoradiography allows a dentate gyrus, CA1 and CA3 fields) where only pharmacological approach to restricted brain areas non-specific OT binding is observed. Better cornot easy to tackle with membrane-binding tech- relations seem to exist in the lateral septum where niques. vasopressinergic axons predominate and which also The selectivity of the (3H]OT-binding sites for OT, binds mainly AVP, and in the central amygdaloid AVP and different antagonists of both peptides were nucleus which has similar concentrations of fibres thus evaluated in three areas with high concencontaining OT and AVP, and likewise binds both trations of [3H]OT-binding sites: the ventral sub- peptides. However, no particular concentration of iculum, the central amygdaloid nucleus and the OT-containing fibres was observed in the other OTventromedial hypothalamic nucleus. In these areas, binding areas of the amygdala where AVP-containing AVP and OT were equally potent in displacing axons largely predominate. Likewise, the distribution f3H]OT, which tallies well with the recent characterof the OT-containing fibres in the thalamus cannot be correlated with the concentration of OT-binding sites ization of a high-a~nity OT-binding site in hippocampus synaptic plasma membranes discriminating in the only ~ravent~cular thalamic nucleus. The very poorly between OT and AVP.’ On the same same is true for the caudate nucleus in the striatum. membrane preparation, a second site with a high Another striking example is the nucleus of the sohaffinity for 13H]AVP, but a low one for [3H]OT, was tary tract which essentially receives oxytocinergic also characterized.’ Vasopressin-binding sites de- projections and binds AVP exclusively.” Finally, the tected by autoradiography in areas with little or no ventromedial hypothalamic nucleus, with a high denaffinity for [3H]OT (e.g. lateral septum and supra- sity of OT- (and AVP-) binding sites, is completeiy chiasmatic nuclei) probably correspond to this bereft of fibres containing OT and AVP, while such fibres are concentrated and even seem to terminate second type of neurohypophysial hormone receptor.

612

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close to the dorsomedial nucleus in an area without any OT- (or AVP-) binding sites. Discrepancies between the distributions of peptidergic (and aminergic) fibres and related receptors are in fact generally obsented’s~26~33.?S.3”~38~‘~44 and different explanations for this mismatch have been proposed. 24z There is in fact physiological evidence of different chemically addressed forms of peptidergic or aminergic information which may be involved in either point-to-point (synaptic) transmission or “parasynaptic”4’ info~ation characterized by less precise spatial connections with a slower time-course and a far richer diversity of signals (see Refs 20 and 42). Neuromediators are known to diffuse from the nerve endings and reach the target cells via the extracellular spaces even at a considerable distance. Moreover, peptides such as OT and AVP are present in the cerebrospinal fluid and can thus affect different areas of the brain. These data might explain the different types of regulation triggered by neurohypophysial hormones*’ and, at least partially, the discrepancies between the densities of innervations and of binding sites. As for the physiological signi~can~ of the concentrations of specific OT-binding sites in restricted but well defined areas of the brain, it is clear that these locations (e.g. in the extensively labelled olfactory centres and amygdaloid complex) tally with experimental evidence for a behavioural role of OT. The fact that only the subiculum is labelled in the hippocampus may be highly significant since this zone is known to relay entorhinal and other cortical projections on the hypothalamus. On the other hand, an area with a high density of OT-binding sites, such as the ventromedial hypothaIamic nucleus, obviously changes according to physiological conditions: the concentration of OT-binding sites is clearly Iower in the lactating female than in the male rat and, as reported by de Kloet et al.,” the density of OTbinding sites decreases significantly in this nucleus after ovariectomy and reverts to normal after oestrogen administration. Oestrogen modulation of OT-binding sites in the ventromedial nucleus thus resembles that of uterus OT receptors.46*47 Physiological data also show OT-oestrogen interactions in the brain, for example in the case of OT-induced maternal behaviour, which only occurs in oestrogentreated female rats.14 There are, however, obvious discrepancies between the autoradiographic location of t3H]OT-binding

sites and electrophysiological observations. In the hippocampus, Miihlethaler et al.“’ demonstrated OT stimulation of neurons in areas bereft of OT-binding sites in our preparations (non-pyramidal neurons in the CA1 fields). In the dorsal hippocampus and the lateral septaf complex, about 60% of the neurons have been activated by OT injected microiontophoretically.*’ A similar discrepancy has been reported in the brainstem (dorsal motor nucleus of the vagus).’ In the hypothalamus, no labelling was found to explain why OT increases the firing of neurons in the paravent~cular hypothalamic nucleus in viva” and in vitro Iv and promotes periodic neurosecretory bursts on dxytocinergic neurons during the milk-ejection reflex.‘” This latter effect would have to involve receptors highly specific for OT: indeed, it is not triggered by AVP, even at doses 100 times the effective OT ones. Thus, these receptors obviously differ from the rabbit mammary gland receptors (AVP activity = 11-I 5% OT activity) and rat uterus receptors (AVP activity = 24% OT activity) (see refs in Denamur”). The OT receptors detected in the brain with biochemical and autoradiographic techniques are far less specific, in view of their equal affinities for AVP. CONCLUSION

Under our conditions, the autorad~ographic technique obviously reveals only some of the OT recep tors in the brain which are characterized by high and similar affinities for OT and AVP. These observations and physiological data suggest that this technique may not reveal receptors involved in rapid responses of the neurotransmitter type. The concentrations of OT-binding sites detected may correspond rather to areas involved in the modulatory effects of neurohypophysial hormones. More extensive physiological and pharmacological investigations are needed to clarify this. Ack~o~lie~ge~ents-his work was supported by grants from MRT (83.C.09231and INSERM (CRL 82-408. CRE 85-6018). We are deeply indebted to‘ Drs Manning and Su Sun Wang for generous supplies of oxytocin and vasopressin antagonists, Our grateful thanks are also due to Dr Diana Gazis for sending us the oxytocin inhibitor which was synthesized by the late Dr Roy. We are most grateftd to Dr Rosar Cortbs for her precious contribution with computer measurements. Finally, we must not forget to mentioti the excellent technical assistance from Mrs Georgette Haher, Catherine Reysz, and Betty Wahisperger.

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