Eliane Tribollet

Localization and pharmacological characterization of high affinity binding sites for vasopressin and oxytocin in the rat brain by light microscopic autoradiography

Sites which bind tritiated vasopressin (AVP) with high affinity were detected in the brain of male, adult rats, by light microscopic autoradiography. Their anatomical localization differed markedly from that of high affinity binding sites for tritiated oxytocin (OT) determined in the same animal. Co-labelling was minimized by using low concentrations of [3H]AVP and [3H]OT. Binding of the former occurred predominantly in several structures of the limbic system (septum, amygdala, bed nucleus of the stria terminalis, accumbens nucleus), in two hypothalamic nuclei (suprachiasmatic and dorsal tuber) and in the area of the nucleus of the solitary tract. Binding of OT was evidenced in the olfactory tubercle, the ventromedial hypothalamic nucleus, the central amygdaloid nucleus and the ventral hippocampus. The ligand specificity of the binding sites was assessed in competition experiments. Synthetic structural analogues were used, allowing to discriminate OT receptors (OH[Thr4,Gly7]OT) from V2 receptors (dDAVP and d[Tyr(Me)2]VDAVP), V1 receptors ([Phe2,Orn8]VT) and V1b receptors (desGly9d(CH2)5AVP). Our main conclusions are, firstly, that AVP and OT binding sites can be readily distinguished, and that there is virtually no overlap in their distribution in the rat brain. Second, we showed that the sites which bind AVP with high affinity in the brain are V1 receptors, different both from the renal V2 receptors and from the anterior pituitary V1b receptors. Our results support the conjecture that AVP and OT play a role in interneuronal communication in the brain.

Localization of high-affinity binding sites for oxytocin and vasopressin in the human brain. An autoradiographic study

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 autoradiography. 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 Alzheimers disease.

125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]OVT: a selective oxytocin receptor ligand

An oxytocic antagonist, [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid,2-O-methyltyrosine,4-threonine, 8-ornithine,9-tyrosylamide]vasotocin (d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT [corrected], was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. 125I-labelling was performed with 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl-glycoluril. Iodination resulted in an increased affinity for rat uterine oxytocin receptors. A considerably lower affinity for rat vascular V1- and renal V2-receptors was found, resulting in a highly specific oxytocin receptor ligand. 125I-labelled d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT [corrected] was demonstrated to bind selectively to one population of binding sites in rat uterus and ventral hippocampal membrane preparations. Dissociation constants ranged between 0.03 and 0.06 nM. After 3 days of exposure autoradiography revealed binding in regions known to contain oxytocin receptors as well as labelling in some new regions, while no binding was found in the lateral septum, a structure containing mainly [8-arginine]vasopressin receptors. The high specific radioactivity of 125I-labelling allowed important reductions in membrane protein amount, gain in precision of binding analysis as well as considerably lower exposure times for autoradiography.

Localization of neurones projecting to the hypothalamic paraventricular nucleus area of the rat: A horseradish peroxidase study

Abstract To detect the cell bodies of neurones which project to the area of the hypothalamic para-ventricular nucleus, 10–40 nl of a solution containing horseradish peroxidase and poly- l -ornithine were pressure-injected into one paraventricular nucleus of the rat. After 24 or 48 h, the enzyme remaining at the site of injection was detected by the diaminobenzidine procedure. Retrogradely transported horse-radish peroxidase was visualized by using o -dianisidine as the chromogen substrate. The extent and the intensity of labelling correlated with the apparent volume of the injection site. Labelled cell bodies were observed, ipsilateral to the injection, in the mediobasal hypothalamus, in the limbic system (lateral septum, posteromedial amygdala, ventral subiculum) and in several cell clusters in the brain stem (dorsal raphe nucleus, locus coeruleus, parabrachial nucleus, nucleus of the solitary tract and lateral reticular nucleus). In some animals, light labelling in the organum vasculosum laminae terminalis and in the subfornical organ was observed. No labelled neurones could be detected in the spinal cord.

Gonadal steroids regulate oxytocin receptors but not vasopressin receptors in the brain of male and female rats. An autoradiographical study.

The distribution and the amount of [3H]oxytocin binding were studied in the brain of adult rats of either sex, as well as in male and female castrates, some of which received injections of estradiol or testosterone. Intact males were treated with an aromatase inhibitor. Castration and inhibition of aromatase activity reduced, whereas estradiol and testosterone increased oxytocin binding, particularly in regions of the brain assumed to be involved in reproductive functions, such as the ventrolateral part of the hypothalamic ventromedial nucleus and the islands of Calleja and neighbouring cell groups. Binding of oxytocin to the uterus was also estrogen-dependent. In the same animals, we also studied the distribution of [3H]vasopressin binding sites present in the brain. It was similar in males and females, and was not affected by experimentally manipulating gonadal hormone levels. In immunocytochemical studies we noticed, as others had previously, that the vasopressin content of certain areas of the rat brain was affected by castration, whereas the oxytocin innervation was not. These results are discussed in relation to the possible functions of oxytocin in the brain and of the lack of correspondence between the immunocytochemical and the autoradiographic data.

Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain

The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.

Extra-hypothalamic afferent inputs to the supraoptic nucleus area of the rat as determined by retrograde and anterograde tracing techniques

To detect neuronal cell bodies whose axon projects to the hypothalamic supraoptic nucleus, small volumes (10-50 nl) of 30% horseradish peroxidase or 2% fast blue solutions were pressure-injected into the area of one supraoptic nucleus of rats. Both dorsal and ventral approaches to the nucleus were used. In animals where the injection site extended beyond the limits of the supraoptic nucleus, retrogradely labelled cell bodies were found in many areas of the brain, mainly in the septum, the nucleus of the diagonal band of Broca and ventral subiculum in the limbic system; the dorsal raphe nucleus, the locus coeruleus, the nucleus of the dorsal tegmentum, the dorsal parabrachial nucleus, the nucleus of the solitary tract and the catecholaminergic A1 region in the brain stem; in the subfornical organ and the organum vasculosum of the lamina terminalis, as well as in the median preoptic nucleus. In contrast, when the site of injection was apparently restricted to the supraoptic nucleus, labelling was only clearcut in the two circumventricular organs, the median preoptic nucleus, the nucleus of the solitary tract and the A1 region. Injections of wheat germ agglutinin coupled with horseradish peroxidase (60-80 nl of a 2.5% solution) made in the septum and in the ventral subiculum anterogradely labelled fibers coursing in an area immediately adjacent to the supraoptic nucleus but not within it. In contrast, labelling within the nucleus was found following anterograde transport of tracer deposited in the A1 region and in an area that includes the nucleus of the solitary tract. Neurones located in the perinuclear area were densely labelled by small injections into the supraoptic nucleus; they may represent a relay station for some afferent inputs to the supraoptic nucleus. These results suggest that the supraoptic nucleus is influenced by the same brain areas which project to its companion within the magnocellular system, the paraventricular nucleus.

Neurohypophyseal hormone receptor systems in brain and periphery.

Publisher Summary This chapter focuses on the similarities and differences between vasopressin or oxytocin receptors in the brain and peripheral receptors for these peptides. The chapter also discusses the transduction mechanisms activated by vasopressin and oxytocin receptors in the brain and peripheral nervous system. Vasopressin and oxytocin meet most of the criteria that are applied to assign a role as a neurotransmitter or a neuromodulator to a given compound. It is recognized that vasopressin and oxytocin are synthesized at multiple sites in the brain in neurons projecting to several areas. A calcium-dependent release of oxytocin and (or) vasopressin can be evoked by potassium or veratridine in various extrahypothalamic areas of the brain. No direct effect of vasopressin on the adenylate cyclase activity of nervous tissue can be demonstrated. Vasopressin stimulates inositol lipid breakdown in hippocampal slices and isolated sympathetic ganglia.

Oxytocin-induced analgesia and scratching are mediated by the vasopressin-1A receptor in the mouse

The neuropeptides oxytocin (OXT) and arginine vasopressin (AVP) contribute to the regulation of diverse cognitive and physiological functions including nociception. Indeed, OXT has been reported to be analgesic when administered directly into the brain, the spinal cord, or systemically. Here, we characterized the phenotype of oxytocin receptor (OTR) and vasopressin-1A receptor (V1AR) null mutant mice in a battery of pain assays. Surprisingly, OTR knock-out mice displayed a pain phenotype identical to their wild-type littermates. Moreover, systemic administration of OXT dose-dependently produced analgesia in both wild-type and OTR knock-out mice in three different assays, the radiant-heat paw withdrawal test, the von Frey test of mechanical sensitivity, and the formalin test of inflammatory nociception. In contrast, OXT-induced analgesia was completely absent in V1AR knock-out mice. In wild-type mice, OXT-induced analgesia could be fully prevented by pretreatment with a V1AR but not an OTR antagonist. Receptor binding studies demonstrated that the distribution of OXT and AVP binding sites in mouse lumbar spinal cord resembles the pattern observed in rat. AVP binding sites diffusely label the lumbar spinal cord, whereas OXT binding sites cluster in the substantia gelatinosa of the dorsal horn. In contrast, quantitative real-time reverse transcription (RT)-PCR revealed that V1AR but not OTR mRNA is abundantly expressed in mouse dorsal root ganglia, where it localizes to small- and medium-diameter cells as shown by single-cell RT-PCR. Hence, V1ARs expressed in dorsal root ganglia might represent a previously unrecognized target for the analgesic action of OXT and AVP.

Early appearance and transient expression of vasopressin receptors in the brain of rat fetus and infant. An autoradiographical and electrophysiological study

The development of vasopressin (AVP) receptors in the rat brain, spinal cord and pituitary gland was studied by in vitro light microscopic autoradiography. AVP binding sites were labeled using [3H]AVP in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the binding of [3H]AVP to oxytocin receptors was prevented by adding in the incubation medium a highly selective oxytocin agonist. Specific binding was first detected at E16 in the ventral pontine reticular formation. Many other brain areas were progressively labeled between E18 and PN5. The distribution of binding sites observed at PN5 remained unchanged until the beginning of the third postnatal week. Thereafter binding was markedly reduced or even disappeared in several areas, in particular in the facial nucleus. The adult distribution of AVP binding sites was established at the time of weaning. The properties of transient AVP binding sites in the facial nucleus were studied both by autoradiography and by electrophysiology. Non-radioactive AVP displaced [3H]AVP binding in this nucleus as efficiently as it did in the lateral septum of the adult. Single-unit extracellular recordings showed that AVP can excite facial motoneurones by interacting with receptors which are pharmacologically indistinguishable from V1 (vasopressor) type. Thus, AVP binding sites transiently expressed in the brain of fetal and infant rat probably represent functional neuronal receptors, having the same ligand selectivity and affinity than AVP binding sites present in the adult. This suggests that AVP acts not only as a neuropeptide in the adult brain but may play a significant role during maturation of the central nervous system.