Jean-Jacques Dreifuss

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.

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.

Localization of oxytocin binding sites in the human brainstem and upper spinal cord: an autoradiographic study.

Two different ligands, tritiated oxytocin and a newly synthesized and monoiodinated oxytocin antagonist, were used to reveal sites which bind oxytocin in the brainstem and upper spinal cord of 12 human subjects. Tissue sections were incubated with either ligand at a concentration close to their respective dissociation constants determined in human uterus and rat brain. Specificity of binding was assessed in presence of unlabelled oxytocin in excess. Comparable results were obtained using tritiated or iodinated ligand. Labelling was most intense in the substantia nigra pars compacta, the substantiae gelatinosae 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. Binding was also detected in the rest of the nucleus of the solitary tract and in other areas, including the oral and interpolar parts of the spinal trigeminal nucleus, the hypoglossal nucleus and the area postrema. Presence of oxytocin binding sites in regions concerned with sensory, autonomic and motor processing suggests that oxytocin could act as a neurotransmitter or neuromodulator in the human central nervous system.

Stimulatory action of oxytocin on neurones of the dorsal motor nucleus of the vagus nerve

Extracellular recordings were obtained from spontaneously active neurones located in the dorsal motor nucleus of vagus nerve ( DMX ) in slices of the rat brainstem. Oxytocin applied to the bath at concentrations of 10(-7) M or 10(-6) M excited 79% of these cells in a concentration-dependent, reversible manner. The remaining cells were unaffected. The stimulatory effect of oxytocin was reversibly antagonized by a synthetic structural analogue known to block the peripheral, endocrine effects of neurohypophysial peptides. A selective oxytocic agonist was as potent as oxytocin, whereas vasopressin exerted a much weaker effect. We therefore suggest that neurones located in DMX are endowed with receptors for oxytocin.

The role of central adrenergic receptors in the reflex release of oxytocin

Abstract The effects of the α-adrenergic receptor antagonists, phentolamine and phenoxybenzamine, and the β-antagonists, propranolol, oxprenolol and practolol on the milk-ejection reflex of the rat were studied. The mother rats, at day 8–11 or lactation, were anaesthetized with urethane (1.2–1.4 g/kg, i.p.) and litters of 9–11 hungry pups were placed on the nipples to suckle for 2–6 h. With 65% of the 425 rats studied, a regular pattern of milk ejection was observed, from the changes in pup behaviour and intramammary pressure, with milk ejection recurring at intervals of 3–15 min throughout the nursing period. Propranolol (1.0–1.5 mg/kg, i.v.) and the other β-antagonists failed to either facilitate or inhibit the suckling-induced reflex when given to animals that were already milk ejecting. When given to animals which were not milk ejecting or which had ceased to milk eject in response to the suckling stimulus, both propranolol and oxprenolol at doses as low as 30 μg/kg promoted a normal pattern of reflex milk ejection. Practolol (1.5 mg/kg, i.v.), a β-antagonist with limited access to the brain, did not facilitate the reflex when given to these refractory animals. Both the racemate and the l -isomer of propranolol were effective in promoting reflex milk ejection when given by either the intravenous or intraventricular route; the d -isomer was ineffective even at 1.5 mg/kg, i.v. The rats which had failed to milk eject when suckled had mammary glands that were as sensitive to oxytocin as other animals, and the abrupt onset of the milk-ejection reflex following propranolol was apparently not related to the lowered threshold of mammary gland sensitivity to oxytocin promoted by the β-antagonists. Slow infusions of adrenaline and isoprenaline (0.1–0.2 μg/min) failed to inhibit the milk-ejection reflex even at doses that reduced the gland sensitivity to oxytocin. The α-adrenergic antagonists had an opposite effect and produced a dose-dependent inhibition of the reflex when given to animals that were milk ejecting at regular intervals. Thus, 1 mg/kg phentolamine, i.v., increased the milk ejection interval 6-fold and 2 mg/kg increased the interval 13-fold. Both phentolamine and phenoxybenzamine caused a marked fall in blood pressure, but a similar fall in blood pressure was induced with hexamethonium (2 mg/kg, i.v.) without altering the pattern of reflex milk ejection. Two other centrally active drugs were also examined, picrotoxin (2–4 mg/kg, i.v.) and hyoscine (90 mg/kg, i.v.), but neither were found to influence the reflex. These results suggest that both α- and β-adrenergic receptors are involved in the central control of the milk-ejection reflex in the anaesthetized rat, and both are probably activated by neuronally released noradrenaline. The α-receptors would appear to form a component of the natural reflex and are excitatory. The β-receptors are external to the reflex and are inhibitory.

Contrasting effects of neurohypophysial peptides on pyramidal and non-pyramidal neurones in the rat hippocampus

Oxytocin and vasopressin increased the rate of firing of a class of presumed non-pyramidal neurones located in the CA1 area of rat hippocampal slices. This excitatory effect persisted in conditions of synaptic uncoupling. In contrast, pyramidal neurones were either unaffected by neurohypophysial peptides or showed one or several of the following effects: a decrease in firing rate in cells which were spontaneously active; a slight membrane hyperpolarization; and an increase in the rate of occurrence of spontaneous inhibitory postsynaptic potentials. We therefore propose that oxytocin and vasopressin excite directly a class of non-pyramidal inhibitory interneurones, whereas their observed effect on pyramidal neurones is indirect and inhibitory.

A review on neurosecretory granules: their contents and mechanisms of release.

The available evidence suggests that hormones and neurophysins are associated exclusively with the neurosecretory granules, each of which contains approximately 6 times 10-4 molecules of each. Hormones and carrier proteins are complexed within the granules and the complexes are densely packed. The processes that keep the intragranular space in osmotic equilibrium with the axoplasm require further study. Freeze-fracture data, as well as studies in which histochemical methods for the detection of glycoproteins were used, suggest that the intragranular aspect of the granule membrane mostly resembles the extracellular half of the plasma membrane; on the other hand, the cytoplasmic aspects of plasma and granule membrane have similar characteristics, which may be important in permitting membrane fusion to take place prior to secretion. Little is known about the molecular species involved in this interaction between granule and plasma membrane, except that calcium is a cofactor in this process. Release is triggered in vivo by propagated action potentials which cause an influx of calcium into the secretory endings. Newly formed granules, and other granules located at the periphery of the endings are preferentially released. Irrespective of the type of stimulation of secretion, release involves the diffusion into the extracellular space of granule core constituents. The best evidence so far in support of this view comes from ultrastructural studies showing images of exocytosis, as well as from biochemical studies demonstrating that hormones and carrier proteins are secreted concomitantly in a great variety of experimental or clinical conditions, without an associated release of granule membrane constituents or of enzymes of cytoplasmic origin. Recovery mechanisms following secretion require new synthesis of granule constituents and restoration of the resting internal concentrations of potassium, sodium, and calcium. Membrane surface area is restored following exocytosis by compensatory endocytosis which involves indiscriminate uptake of extracellular medium into the secretory axon terminals. While much progress has been made in research on the cellular and subcellular processes that take place in neurons which produce, store, and secrete neurohypophyseal hormones and their carrier proteins, neurophysins, many pressing questions remain to be answered. New developments, such as organ culture of supraoptic nuclei94-96 and the recent isolation of a clone of mouse hypothalamic cells capable of synthesizing vasopressin and neurophysin,97 will hopefully allow some of these problems to be solved in the future.

A role of central oxytocin in autonomic functions: its action in the motor nucleus of the vagus nerve

Neurones located in the dorsal motor nucleus of the vagus nerve were shown, in slices from the rat brainstem, to respond to oxytocin by a concentration-dependent increase in rate of firing. A newly available oxytocin antagonist suppressed the excitatory effect of oxytocin on single neurones; this antagonism was partially reversible. Further evidence that neurones located in the dorsal motor nucleus of the vagus nerve possess oxytocin receptors was obtained from in vitro light microscopical autoradiography using [125I]-labelled oxytocin antagonist. In conjunction with data by others which showed that oxytocin antagonist microinjected into the dorsal motor nucleus of the vagus nerve blocks gastric and cardiac effects caused by stimulation of the hypothalamic paraventricular nucleus, our results suggest a role for central oxytocin in autonomic efferent activity.