220

Brain Research, 555 (1991) 220-232 © 1991 Elsevier Science Publishers B.V. 0006-8993/91/$03.50 A DONIS 000689939116837V

BRES 16837

Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study F. Loup, E. Tribollet, M. Dubois-Dauphin and J.J. Dreifuss Department of Physiology, University Medical Center, Geneva (Switzerland) (Accepted 5 March 1991) Key words: Basal nucleus of Meynert; Human tissue; Lateral septum; Oxytocin; Pharmacological characterization; Receptor autoradiography; Substantia nigra; Vasopressin

Sites which bind oxytocin and vasopressin with high affinity were detected in the brain and upper spinal cord of 12 human subjects, using in vitro light microscopic autoradiograpby. Tissue sections were incubated with tritiated vasopressin, tritiated oxytocin or an iodinated oxytocin antagonist. The ligand specificity of binding was assessed with unlabelled vasopressin or oxytocin in excess, as well as in competition experiments using synthetic structural analogues. The distribution of vasopressin binding sites differed markedly from that of oxytocin binding sites in the forebrain, while there was overlap in the brainstem. Vasopressin binding sites were detected in the dorsal part of the lateral septal nucleus, in midline nuclei and adjacent intralaminar nuclei of the thalamus, in the hilus of the dentate gyrus, the dorsolateral part of the basal amygdaloid nucleus and the brainstem. The distribution of oxytocin binding sites in the brainstem has been recently reported (Loup et al., 1989). Oxytocin binding sites were also observed in the basal nucleus of Meynert, the nucleus of the vertical limb of the diagonal band of Broca, the ventral part of the lateral septal nucleus, the preoptic/anterior hypothalamic area, the posterior hypothalamic area, and variably in the globus pallidus and ventral pallidum. The presence of oxytocin and vasopressin binding sites in limbic and autonomic areas suggests a neurotransmitter or neuromodulator role for these peptides in the human central nervous system. They may also affect cholinergic transmission in the basal forebrain and consequently play a role in Alzheimer's disease.

INTRODUCTION Oxytocin ( O T ) and arginine vasopressin (AVP) are closely related n o n a p e p t i d e s synthesized in neurons of the m a m m a l i a n brain and t h e r e u p o n released from h y p o t h a l a m o - n e u r o h y p o p h y s e a l axon terminals to act as h o r m o n e s on p e r i p h e r a l targets. Their m a j o r biological effects, uterine contraction and milk ejection for OT, as well as antidiuresis and vasopressor activity for AVP, are well d o c u m e n t e d . In the human, p e r i p h e r a l receptors for O T have been characterized in the uterus of pregnant and n o n - p r e g n a n t women 13A4'17'19'26'5°, and for A V P in

using light microscopic a u t o r a d i o g r a p h y . We have recently studied the distribution of specific high-affinity O T binding sites in the h u m a n brainstem and u p p e r spinal cord using in vitro light microscopic a u t o r a d i o g r a p h y 23. We presently r e p o r t (i) the distribution of O T binding sites in the f o r e b r a i n using two radioligands, (ii) the distribution of A V P binding sites in the forebrain, the brainstem and the u p p e r spinal cord using a tritiated ligand, and (iii) the characterization of O T and A V P binding sites assessed in competition experiments using synthetic structural analogues.

platelets 51'56, kidney and seminal vesicles 25.

MATERIALS AND METHODS

F u r t h e r m o r e , O T and A V P may act as n e u r o m o d u l a tors or neurotransmitters within the central nervous system35: (i) both are present in axons projecting to e x t r a h y p o t h a l a m i c areas 47, (ii) release can be evoked under e x p e r i m e n t a l conditions 3, (iii) effects on electrical activity of single neurons in the rat brain have been d e m o n s t r a t e d 36'4°-42, and (iv) specific high-affinity binding sites for O T and A V P have been characterized on purified m e m b r a n e s from rat brain 2 and localized in various areas of the rat brain 1z'39'53'54 and other species 7

Tissue preparation Twelve brains from human subjects were obtained at autopsy from patients who had died at the University Hospital of Geneva. Data on the postmortem interval, sex, age and cause of death of each subject are given in Table I. None of the patients had a history of neurological or psychiatric disorder, except one, whose results were nonetheless included in the study as they did not differ from the others. The brains were dissected into blocks of up to a few cubic centimeters, which were frozen in isopentane at -25 °C and stored at -80 °C. Selected areas comprised the basal forebrain, septal region, corpus striatum, thalamus, hypothalamus, hippocampal

Correspondence: J.J. Dreifuss, Department of Physiology, University Medical Center, 9 Av. de Champel, 1211 Geneva 4, Switzerland.

221 formation and entorhinal cortex, amygdaloid complex, olfactory bulb, portions of frontal and temporal cortex, cerebellar cortex in the vicinity of the dentate nucleus, brainstem and upper spinal cord. Series of 15-/~m-thick sections were cut, mounted on chromalum gelatin-coated slides, dehydrated for 2-14 h at 4 °C, then stored at -80 oC in sealed containers with Silica Gel until use.

TABLE I

Cases used for the study Case

Postmortem interval (h)

Sex

Age

Cause of death

Binding procedure

1 2 3 4 5 6 7 8 9 10 11 12

3 17 23 3 5 21 12 6 14 5 7 4

F M M M F M M M F M M F

61 64 65 81 79 71 40 67 42 69 60 78

Cardiac Pulmonary embolus Cardiac Pulmonary embolus Myocardial infarction Cardiac Cardiac Pneumonia Cardiac Acute pulmonary edema Cardiac Pulmonary embolus

Sections were preincubated for 15 min by dipping the slides into a solution containing 50 mM Tris-HCl (pH 7.4), 100 mM NaC1 and 50 g M GTP (optional), then rinsed twice for 5 min in 50 mM Tris-HCl alone. Incubation was carried out for I h in humid chamber by covering each section with 400 /~l of an incubation medium (composition: 50 mM Tris-HCl, 0.1 mM bacitracin, 5 or 10 mM MgCl2, 1 mg/ml bovine serum albumin) containing radioligand (see below) either alone or in presence of various unlabelled peptides. Following two 5-rain washes in ice-cold incubation medium and a quick rinse in distilled water, the slides were dried in a stream of cold air and put in a desiccator containing paraformaldehyde powder preheated at 80 °C for 2 h. When incubated with [3H]AVP or [3H]OT, they were placed in an X-ray cassette in contact with

LKB Ultrofilm during 3 months at 4 °C; when [125I]OTA was used as radioligand, in contact with Kodak X-Omat film for 3 days at 4 °C. Films were then developed in Kodak D 19. Adjacent series of sections were stained with Cresyl violet and Azure blue. In all experiments sections from human and rat kidneys and sections of mammary glands from lactating rats were included as controls. Essentially we used the nomenclature of Paxinos 37 and Paxinos and Watson as. The nuclei of the thalamus were identified according to Dekaban s, the septal region according to Andy and Stephan 1 and

Source ofpeptides [3H]OT (spec. act. 36.6 Ci/mmol) and [3H]AVP (spec. act. 67-70 Ci/mmol) were purchased from Dupont-New England Nuclear (Boston, MA) and purified as described elsewhere 53. The OT antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT was monoiodihated with 125I-Na in position 9 ([125I]OTA) to a spec. act. of - 2000 Ci/mmol 9'27. This antagonist as well as the synthetic structural analogues were kindly provided by Dr. M.M. Manning. The V 2 agonist dDAVP was purchased from Ferdng, Maim6, Sweden.

Fig. 1. [3H]OT binding in frontal hemisections at the level of the preoptic area. Medial is to the left and dorsal to the top; 40-year-old man. A and B are adjacent sections. A: incubation with 3 nM [3H]OT. B: incubation with 3 nM [3H]OT and 5/~M OT. The arrow points to the lateral ventricle which became virtual at congelation. Note specific binding in the ventral part of the lateral septal nucleus (LSV), the basal nucleus of Meynert (B) and the preoptic/anterior hypothalamic area (POA). The globus pallidus (GP) was faintly labelled in this case. For other abbreviations in this and following figures, see list. Bar = 5 mm.

222 others 15'22, and the basal forebrain according to Mesulam 33'34.

Localization of OT binding sites Sections were incubated with either 3 nM [3H]OT (6 subjects) or 0.04 nM [12SI]OTA (7 subjects). (In one brain, series of sections were incubated with either ligand at these concentrations.) Nonspecific binding was determined in adjacent sections by adding an excess of unlabelled OT (5 or 10 /tM for [3H]OT, 1-2 /,M for [1251]OTA) to the incubation medium.

Localization of A VP binding sites Sections were incubated with either 1.5 nM [3H]AVP alone or with 2-3 nM [3H]AVP supplemented with 5 nM OH[Thr4,GIy7]OT. Non-specific binding was evaluated on adjacent sections incubated with the same concentration of [3H]AVP with the addition of 1-10 /~M of unlabelled AVE

Pharmacological characterization of OT and A VP binding sites The specificity of [1251]OTA binding was assessed in series of sections obtained from three subjects by its displacement with unlabelled synthetic structural analogues of known selectivity (for refs., see Manning and Sawyer2S). For each series, one section was incubated with 0.04 nM [125I]OTA alone, and adjacent sections

were incubated with the same concentration of [~25I]OTA and in addition with 10, 20 or 75 nM of either OH[Thr4,GIyT]OT, or dDAVP, or desGly9d(CH2)sAVP or [PheE,OrnS]VT. The specificity of [3H]AVP binding was also evaluated. For each series, one section was incubated with [3H]AVP at 2 or 3 nM; adjacent sections were incubated with the same concentration of [3H]AVP with the addition of either 10, 20 or 100 nM of one of the 4 analogues mentioned above.

Stability of OT and AVP binding sites Since the postmortem stability of OT and AVP binding sites is unknown and the postmortem interval was variable, a pilot experiment was performed on 10 rat brains dissected either immediately after decapitation or after having been kept for 4, 8, 16 or 24 h in a cold room at 4 °C. Sections of the ventromedial hypothalamic nucleus and subiculum (which contain OT binding sites) and sections of the septum and kidney (rich in AVP binding sites) were processed in the same way as the human tissue. Autoradiograms were analyzed by densitometry and 15 measures taken from each animal were averaged. No significant reduction of binding was detected in rat brain tissue for up to 16 h. At 24 h, a 15% reduction in binding was observed.

l

Fig. 2. Distribution of binding sites for [125I]OTA in frontal hemisections through the preoptic area. Medial is to the left and dorsal to the top. C is more posterior than A; 60-year-old man. A,C: incubation with 0.04 nM [lz~I]OTA. B,D (adjacent sections): incubation with 0.04 nM [125I]OTA and 2/~M OT. Note specific binding in the basal nucleus of Meynert, the nucleus of the vertical limb of the diagonal band, the ventral part of the lateral septal nucleus, the globus pallidus, the ventral pallidum and the preoptic area. Bar = 5 ram.

223 RESULTS The distribution and density of binding in the human brain showed no apparent correlation with either sex or age. The number of cases studied to date is too small to exclude or establish any correlation with premortem condition or postmortem interval. However, our pilot study using the rat brain as a model indicates that central OT and AVP binding sites appear to be stable for at least 24 h postmortem.

Distribution of OT binding sites Specific O T binding sites were consistently observed in restricted areas of every brain examined, using either [3H]OT or [125I]OTA. Regions labelled with [3H]OT were the same as those labelled with [125I]OTA. Consequently, the results will be described according to the

labelled regions, irrespective of the ligand used, although [125I]OTA produced autoradiograms with better contrast (compare Fig. 1 with Figs. 2 and 4A,B). In the forebrain, intense binding for O T was observed in the basal nucleus of Meynert (Figs. 1, 2 and 4A,B) and in the nucleus of the vertical limb of the diagonal band of Broca (Figs. 2 and 4A,B). The basal nucleus of Meynert, extending caudally and laterally to the periamygdaloid area, was entirely labelled. Binding became less intense in the dorsal part of the nucleus of the vertical limb of the diagonal band and in the medial septal nucleus, which is in dorsal continuity with it (Figs. 2 and 4A,B). The nucleus of the horizontal limb of the diagonal band did not appear to be labelled. The globus pallidus and its subcommissural extension, the ventral pallidum, displayed variable density of OT binding (compare Fig. 1 with Figs. 2 and 4A,B). Low densities of binding sites

Fig. 3. Distribution of [nSI]OTA binding sites in frontal sections through the posterior tuberal hypothalamic region/anterior thalamus (A,B) and through the posterior hypothalamus (C,D). A is slightly anterior to C; 78-year-o|dwoman. A,C: incubation with 0.04 nM [I~I]OTA. B,D (adjacent sections): incubation with 0.04 nM [I~I]OTA and 2/zM OT. Note specificbinding in the posterior hypothalamicarea and the posterior tuberal region at the premammillary level. The rostral part of the paraventricular tlialamic nucleus is only faintly labelled. Bar = 5 mm.

224 were evidenced in the m a j o r island of Calleja in 2 out of 7 cases. High densities of O T binding sites were detected in the ventral part of the lateral septal nucleus (Figs. 1, 2 and 4 A , B ) , preferentially wedged between the fornix and the anterior commissure (Figs. 1 and 2). In frontal sections, but not in horizontal or parasagittal ones, it was

sometimes difficult to delineate this region from the adjacent medial division of the bed nucleus of the stria terminalis. Inconsistent faint labelling occurred in restricted areas of the dorsal part of the lateral septal nucleus (Fig. 4A,B). In the hypothalamus, a consistent p a t t e r n of binding

Fig. 4. Comparison of [125I]OTA and [3H]AVPbinding sites in the region of the nucleus of the diagonal band. Four frontal adjacent sections are shown which are slightly slanted, the right side being posterior to the left; 78-year-old woman. A: incubation with 0.04 nM [125I]OTA. B: incubation with 0.04 nM [12sI]OTA and 2/~M OT. C: incubation with 2 nM [3H]AVPand 5 nM OH[Thr4,Gly7]OT. D: same as C with addition of 2/~M AVP. Notice that regions which are labelled with [t2sI]OTA are not labelled with [3H]AVP; and that the latter binds to restricted areas of the dorsal part of the lateral septal nucleus. Bar = 5 mm.

225

Fig. 5. [3H]AVP binding in the dorsal part of the lateral septal nucleus. Three adjacent frontal hemisections through the septal/preoptic region show spot-like specific labelling; 61-year-old woman. A,B: incubation with 1.5 nM [3H]AVP. C: incubation with 1.5 nM [3H]AVP and 10/~M AVP. The arrow points to the lateral ventricle which became virtual at congelation. Bar = 5 ram.

Fig. 6. Distribution of [3H]AVP binding sites in frontal sections through the thalamus/posterior hypothalamus. C is more posterior than A; 78-year-old woman. A,C: incubation with 2 nM [3H]AVP and 5 nM OH[Thr4,Gly7]OT. B,D (adjacent sections): same as A,C with addition of 2/zM AVE Note specific binding in midline nuclei and adjacent rostral intralaminar nuclei of the thalamus. Bar = 5 mm.

226 was observed along the anteroposterior axis, the binding being more pronounced anteriorly and posteriorly and less so in between. There was no difference in labelling between the medial and lateral regions (Figs. 1, 2 and 4A,B). The preoptic/anterior hypothalamic area was moderately and inhomogeneously labelled (Figs. 1 and 2), the midtuberal region was only faintly or not labelled. Along the posterior tuberal region (at the premammillary level), the density of binding increased progressively to become moderate again at the level of the posterior hypothalamic area (Fig. 3). The medial mammillary nucleus was usually less labelled than the neighboring posterior hypothalamic area (Fig. 3). The supraoptic and paraventricular hypothalamic nuclei were not labelled (Figs. 1 and 2). In the thalamus, inconsistent faint labelling was discerned in the paraventricular thalamic nucleus (Fig. 3). In all areas mentioned above, the binding for OT was specific since it was prevented by excess unlabelled OT. No specific binding was observed in the accumbens and the caudate nuclei (Fig. 4A,B), the putamen, the hippocampal formation and entorhinal cortex, the amygdaloid complex, the olfactory bulb, selected areas of frontal, temporal and cerebellar cortex, and the choroid plexus. The distribution of O T binding sites in the brainstem has recently been published 23, and these sites were further characterized (see below).

Distribution of AVP binding sites The distribution of specific AVP binding sites differed

markedly from that of O T binding sites, as is shown in Fig. 4. Areas displaying AVP binding sites in the forebrain were small and circumscribed in all 12 cases studied. There was no difference between sections incubated with [3H]AVP alone or in presence of OH[Thra,GIyTIOT. Intense binding was observed in clearly delineated areas of the lateral septal nucleus, predominantly in the dorsal part. Labelled areas formed several streaks anteriorly (Fig. 4C,D), while posteriorly a single spot at the level of the fornix was often discerned (Fig. 5). The medial septal nucleus, the nucleus of the diagonal band and the basal nucleus of Meynert were not labelled (Fig. 4C,D). Inconsistent weak binding was found in the lateral and lateroventral divisions of the bed nucleus of the stria terminalis. The region of the median eminence/ arcuate nucleus was moderately labelled in 2 cases. In the thalamus, high densities of AVP binding sites were observed in midline nuclei and adjacent rostral intralaminar nuclei (Fig. 6). No labelling was detected in other thalamic nuclei. In the hippocampal formation, low densities of AVP binding sites were demonstrated in the hilus of the dentate gyrus, at the transition zone between the CA4 field of Ammon's horn and the polymorphic layer of the dentate gyrus (Fig. 7). The granular layer of the dentate gyrus was not labelled. Weak binding was detected in the dorsolateral part of the basal amygdaloid nucleus and, in 2 cases, the major island of CaUeja. The choroid plexus displayed variable labelling. Specificity of binding in the regions described above

Fig. 7. [3H]AVPbinding sites in the hilus of the dentate gyrus. A and B are adjacent sections through the hippocampal formation and entorhinal cortex of a 78-year-old woman. A: incubation with 2 nM [3H]AVP and 5 nM OH[Thr4,GIyT]OT.B: same as A with addition of 2 ~M AVP. Note specific binding in the hilus of the dentate gyrus (arrows), at the transition zone between the CA region and the polymorphic layer of the dentate gyms. Bar = 5 mm.

227 was demonstrated in the presence of excess unlabeUed AVP. No specific binding was observed in the corpus striatum, the olfactory bulb and selected areas of frontal, temporal and cerebellar cortex. In the brainstem, generally low densities of AVP binding sites were detected in all subjects (midbrain and pons from 6 cases and medulla from 12). Faint labelling was observed in the compact part of the substantia nigra. In the medulla oblongata and upper spinal cord, moderate (5 cases) or weak (7 cases) binding was observed in

the nucleus of the solitary tract/area postrema complex and the caudal part of the nucleus of the solitary tract. The gelatinous layer of the caudal spinal trigeminal nucleus and of the dorsal horn of the upper spinal cord was moderately to weakly labelled. Low densities were found in the interpolar part of the spinal trigeminal nucleus, while no binding was found in the hypoglossal nucleus. Binding mentioned in the above areas was displaced with unlabelled AVP in excess. However, essentially the same areas were also labelled with OT

Fig. 8. Characterization of [I~I]OTA binding sites in the substantia nigra in autoradiograms obtained from 6 adjacent transverse hemisections of the midbrain of a 78-year-old woman. A: incubation with 0.04 nM [~25I]OTA alone. B: incubation with 0.04 nM [125I]OTA and 1.5/~M OT. C-F: incubation with 0.04 nM [125I]OTAand either 20 nM OH[Thr4,GlyT]OT(C), or 20 nM dDAVP (D), or 20 nM desGly9d(CH2)sAVP (E), or 20 nM [Phe2,OrnS]VT (F), respectively. Bar = 5 mm.

228

D

Fig. 9. Characterization of [I~I]OTA binding sites in the medulla oblongata in 3 adjacent transverse sections cut at the level of the nucleus of the solitary tract/area postrema complex (A-C) and 3 cut through the gelatinous layer of the caudal spinal trigeminal nucleus (D-F) of a 78-year-old woman. A,D: incubation with 0.04 nM [125I]OTAalone. B,E: incubation with 0.04 nM [lz~I]OTAand 75 nM of OH[Thr4,GlyT]OT; C,F: 0.04 nM [125I]OTAand 75 nM of desGlygd(CH2)sAVP. Bar = 5 mm. ligands (see below). The medium to intense punctate-like binding observed in the locus ceruleus and the binding of moderate density in the inferior olive were not or only partially prevented in the presence of AVP in excess, and are thus deemed non-specific.

Pharmacological characterization of 0 T and A VP binding sites Competition experiments were performed using synthetic structural peptides on series of sections cut at the level of the midbrain (Fig. 8), the medulla oblongata

(Fig. 9) and the upper spinal cord. The aim of the study was to assess the ligand specificity of the binding sites. To this effect, the following synthetic analogues were used in increasing concentrations in conjunction with [~25I]OTA or [3H]AVP: (i) OH[Thr4,Gly7]OT, a selective OT agonist 24, (ii) dDAVP, a selective V 2 (antidiuretic) agonist, (iii) [Phe2,OrnS]VT, a V 1 (vasopressor) agonist, and (iv) desGIy9d(CH2)sAVP, a selective V1 (vasopressor) antagonist zS. When using [125I]OTA as ligand, the binding was markedly decreased in the presence of 20 nM of

229 OH[Thr4,GIy7]OT (Fig. 8C). The V 1 and V 2 analogues hardly affected binding of [125I]OTA at 20 nM (Fig. 8D-F) or even at 75 nM. Similarly in the medulla oblongata and the upper spinal cord, the selective OT agonist prevented binding in the nucleus of the solitary tract/area postrema complex, the spinal trigeminal nucleus and the hypoglossal nucleus (Fig. 9A-C). The same holds true for the gelatinous layer of the caudal spinal trigeminal nucleus (Fig. 9D-F), the gelatinous layer of the dorsal horn of the upper spinal cord and other more faintly labelled areas. As expected, the binding assumed to be non-specific in the inferior olive was not prevented by any of the synthetic structural analogues (Fig. 9B,C). Similar competition experiments were performed using [3H]AVP as ligand. OH[Thr4,GIy7]OT at 10, 20 or 100 nM totally or partially prevented binding of [3H]AVP. The AVP analogues did not or only partially affect binding at these concentrations. DISCUSSION Using in vitro light microscopic autoradiography, we have detected specific high-affinity binding sites for OT and AVP in the human brain and upper spinal cord. Our results are summarized as follows, in decreasing order of density. Intense binding for OT was observed in the basal nucleus of Meynert, the nucleus of the vertical limb of the diagonal band of Broca and the ventral part of the lateral septal nucleus. Moderate density of binding was detected in the preoptic/anterior hypothalamic area and the posterior hypothalamic area, variable density in the globus paUidus and ventral pallidum. Inconsistent weak binding was evidenced in restricted areas of the dorsal part of the lateral septal nucleus, the paraventricular thalamic nucleus and the major island of Calleja. In the brainstem and upper spinal cord, as we have already reported, intense binding for OT was detected in the compact part of the substantia nigra, the gelatinous layer of the caudal spinal trigeminal nucleus and of the dorsal horn of the upper spinal cord, as well as in the medio-dorsal region of the nucleus of the solitary tract. Other regions of moderate density were observed elsewhere in the nucleus of the solitary tract, in the area postrema, the spinal trigeminal nucleus and the hypoglossal nucleus 23. Using [3H]OT in 6 cases and [125I]OTA in the other 6 cases, we essentially labelled the same regions in a similar manner with both ligands. The ligand specificity of the OT binding sites determined in competition experiments indicates the presence of true OT binding sites. Intense binding for AVP was observed in restricted

areas of the dorsal part of the lateral septal nucleus, midline nuclei and adjacent rostral intralaminar nuclei of the thalamus. Low density of binding was detected in the hilus of the dentate gyrus in the CA4 region and the dorsolateral part of the basal amygdaloid nucleus. Inconsistent low density of binding was evidenced in the lateral and lateroventral divisions of the bed nucleus of the stria terminalis, the region of the median eminence/arcuate nucleus and the major island of Calleja. In the brainstem and upper spinal cord, generally weak binding was observed in the compact part of the substantia nigra, the nucleus of the solitary tract/area postrema complex, the gelatinous layer of the caudal spinal trigeminal nucleus and of the dorsal horn of the upper spinal cord and the spinal trigeminal nucleus. On membranes purified from rat hippocampus, AVP has been found to bind with high affinity to OT and AVP binding sites 2. Studies on human myometrial strips support the same view26. Thus in our experiments we attempted to minimize colabelling by using a low concentration of 1.5 nM [3H]AVP, which effectively labels AVP sites in the rodent brain 7'53 as well as in human pituitary tissues . Alternatively, we used [3H]AVP in conjunction with non-radioactive OH[Thr 4, Gly7]OT24 in order to reduce binding of radioligand to OT binding sites: this did not modify the type of binding observed. We therefore conclude that [3H]AVP binds to true AVP binding sites in the human brain. Competition studies using [3H]AVP as ligand yielded equivocal results due to the low densities of AVP binding sites in the brainstem and high densities of OT binding sites present in the same areas 23. Therefore, we cannot exclude that [3H]AVP also binds to OT binding sites in the brainstem. The presence of specific binding sites raises the question of whether these areas are ever exposed to endogenous OT and AVE Within the human central nervous system, several studies using immunocytochemistry have shown the presence of OT containing perikarya in the supraoptic and paraventricular hypothalamic nuclei and of AVP containing perikarya in these and in other hypothalamic and extrahypothalamic areas 6A°'4s'55. Some of these data have been recently confirmed by in situ hybridization32'43. The presence of OT and AVP outside of the hypothalamo-neurohypophyseal system has been reported in several studies using either immunocytochemistry4'1°'46'48 or radioimmunoassay2°,29,3°,44. The distribution of endogenous AVP in the above-mentioned studies however only partially coincides with that of AVP binding sites we found in our study. For example, while dense networks of AVP fibers 1° and high levels of endogenous AVP 20'29'44 were observed in the locus ceruleus, no specific binding for [3H]AVP was apparent in this region; similarly, endogenous AVP was evidenced in the magnocellular nuclei of the basal forebrain and

230 surrounding substantia innominata 1°'29'55, whereas we found no labelling with [3H]AVP in this region. The distribution of endogenous O T compared to that of O T binding sites also exhibits many discrepancies. Sparse O T fibers were detected in the septum 1° or substantia innominata 4 and low levels of O T in the globus paUidus3°; intense binding for O T was demonstrated in these regions in our investigation. Similar mismatches occur in the rat 52 and the hamster brain 7. They are known for other neurotransmitter systems 21 as well, and several explanations have been proposed for their existence TM. Comparison of data regarding the distribution of O T and A V P binding sites in the rat 12'39'53'54, golden hamster 7 and human brain reveal marked species differences. A most striking difference occurs in the basal nucleus of Meynert, a highly differentiated structure, reaching its maximal prominence in primates, especially in man ~6. It displayed intense O T binding in the present study, but is not labelled in those lower species studied. Conversely, no specific O T binding was evidenced in the human subiculum, entorhinal cortex and amygdala, where high densities of O T binding sites have been reported in the rat brain. Species differences between the human and rodent brain concerning AVP binding sites were less marked. Thus, A V P binding sites were present in the lateral septum in both. Although not in the same subdivisions and with the same densities, AVP binding sites were present in the human and rodent dentate gyrus, amaygdala and bed nucleus of the stria terminalis. ABBREVIATIONS ac

Acb AM AP Arc B

BST C CA3 CG cp Cu DG DMH Ent f Ge5 GP Gr ic ILa IO LG LSD LSV LV

anterior commissure accumbens nucleus anteromedial thalamic nucleus area postrema arcuate hypothalamic nucleus basal nucleus of Meynert bed nucleus of the stria terminalis caudate nucleus field of Ammon's horn central gray cerebral peduncle cuneate nucleus dentate gyrus dorsomedial hypothalamic nucleus entorhinal cortex fornix gelatinous layer of the caudal spinal trigeminal nucleus globus paUidus gracile nucleus internal capsule intralaminar thalamic nuclei inferior olive lateral geniculate nucleus lateral septal nucleus, dorsal part lateral septal nucleus, ventral part lateral ventricle

Differences between human and rodent distribution of binding sites for O T and A V P may indicate a possible reorganization of neural connections favoring more developed areas. The basal nucleus of Meynert along with the nucleus of the diagonal band of Broca form part of the basal forebrain cholinergic system and project to the cerebral cortex and the hippocampus respectively 33'34. Conceivably O T binding sites may be located on cholinergic cell bodies or dendrites and thus could modulate cholinergic transmission. Consequently O T may play a role in Alzheimer's disease, where a selective degeneration of the basal nucleus of Meynert occurs 57. Several studies have shown significant changes in the levels of O T and AVP in certain areas of the brain and in cerebrospinal fluid of Alzheimer's patients 11'29'3°'31'45'49.Moreover, O T and A V P may act in human limbic and autonomic areas, where we detected high-affinity binding sites for O T and AVP in the present study.

Acknowledgements. We wish to express our thanks to Dr. G. Pizzolato (Department of Pathology, University of Geneva, Switzerland) for providing the human postmortem tissue, Drs. M.M. Manning (Medical College of Ohio, Toledo, U.S.A.) and W.H. Sawyer (Columbia University, New York, U.S.A.) for the gift of peptides, Drs. C. Barberis, A. Schmidt and S. Jard (Centre CNRS-INSERM de Pharmacologie-Endocrinologie, Montpellier, France) for performing the iodination of the oxytocin antagonist. Ms. A. Marguerat, M. Berti and A. Cergneux provided excellent technical and secretarial assistance. This work was supported in part by Grant 31-28624.90 from the Swiss National Science Foundation. MD MM MS mt MTh opt Pa PG PH POA Pu PV py pyx S SC scp sm SNC SO Sol Sp5 VA VDB VP 3V 10 12

mediodorsal thalamic nucleus medial mammillary nucleus medial septal nucleus mammillothalamic tract midline thalamic nuclei optic tract paraventricular hypothalamic nucleus parolfactory gyrus posterior hypothalamic area preoptic area putamen paraventricular thalamic nucleus pyramidal tract pyramidal decussation subiculum superior colliculus superior cerebellar peduncle stria medullaris of the thalamus substantia nigra, compact part supraoptic nucleus nucleus of the solitary tract spinal trigeminal nucleus ventral anterior thalamic nucleus nucleus of the vertical limb of the diagonal band ventral pallidum third ventricle dorsal motor nucleus of vagus hypoglossal nucleus

231

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