Ph. Richard

Release of oxytocin within the supraoptic nucleus during the milk ejection reflex in rats.

SummaryTo investigate the hypothesis that oxytocin may be released within the magnocellular nuclei in vivo, push-pull cannula perfusions were performed in anaesthetized lactating rats in one supraoptic nucleus of the hypothalamus while recording the intramammary pressure and/or the electrical activity of oxytocin cells in the contralateral supraoptic nucleus. Oxytocin content was measured in samples collected over 15 min, under various conditions: 1) with no stimulation; 2) during suckling and suckling-induced reflex milk ejections; 3) during electrical stimulation of the neurohypophysis by trains of pulses that mimicked oxytocin cell bursts; 4) under osmotic stimulation by i.p. injection of 2 ml of 1.5 M NaCl to evoke a tonic and sustained oxytocin release from the neurohypophysis. Oxytocin release within the supraoptic nucleus increased significantly during the milk ejection reflex and, to a lesser extent, during burst-like electrical stimulation of the neurohypophysis. In suckled rats, the increase started before the first reflex milk ejection occurred. There was no apparent correlation between the amount of oxytocin in the perfusates and the number of milk ejections and oxytocin cell bursts occurring during each perfusion period. The amount of oxytocin in the perfusates further increased during facilitation of the milk ejection reflex by intraventricular injections of oxytocin or its analogue, isotocin. When suckling failed to evoke the milk ejection reflex, there was no change in intra-supraoptic oxytocin release. There was also no change after osmotic stimulation. When the push-pull cannula was positioned outside the supraoptic nucleus, there was no increase in the amount of oxytocin during the three types of stimulation tested. These results provide evidence for an endogenous release of oxytocin within the magnocellular nuclei in lactating rats. It is suggested that the increase in such a release induced by suckling is likely to be a pre-requisite for the onset and the maintenance of the characteristic intermittent bursting electrical activity of oxytocin cells leading to milk ejections.

Electrical stimulations of perifused magnocellular nuclei in vitro elicit Ca2+-dependent, tetrodotoxin-insensitive release of oxytocin and vasopressin

Isolated rat paraventricular (PVN) and supraoptic (SON) nuclei were perifused in vitro and oxytocin and vasopressin releases were measured by radioimmunoassay during rest and during electrical stimulation. Stimulations at a frequency of 10 Hz (10-s bursts, every 10 s for 5 min) and an intensity of 4 mA, induced significant hormone release only with long duration pulses (10 ms). Short pulses (1 ms) applied at various frequencies (10, 20, 40 or 80 Hz) and intensities (4, 5, 10 or 20 mA) had no effect. The electrically evoked release of both hormones was not affected by tetrodotoxin (TTX), a sodium channel blocker, but was blocked in low-calcium medium or in the presence of gallopamil hydrochloride (D-600), a calcium channel blocker. These results suggest that, following electrical stimulation, oxytocin and vasopressin are released locally within the magnocellular nuclei even when blocking action potentials. The possibility of dendritic release is discussed.

Synchronization of oxytocin cells in the hypothalamic paraventricular and supraoptic nuclei in suckled rats: direct proof with paired extracellular recordings

SummaryIn suckled rats, neurosecretory bursts of two oxytocin cells located in two different magnocellular nuclei were almost simultaneous before each milk ejection. The time elapsing between the onset of two corresponding neurosecretory bursts varied in duration (from 0 to 368 ms) and in order from one pair of cells to another, from one pair of bursts to another for successive bursts of a given pair of cells and independantly of whether one of the two cells belonged to the paraventricular or supraoptic nuclei. However, the neurosecretory burst with the highest amplitude began before the other corresponding burst in most cases. Possible inter- and intranuclear synchronization mechanisms are discussed.

Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland—I. Characterization of different types of binding sites and their distribution in the long-evans strain

Oxytocin and vasopressin binding sites were localized and characterized by quantitative autoradiography on consecutive sections of Long-Evans rat forebrains and pituitary glands, incubated in the presence of 5 nM [3H]oxytocin or 5 nM [3H]vasopressin. In the forebrain, two types of neurohypophysial hormone binding sites were thus defined. (1) Oxytocin/vasopressin sites with similar nanomolar-range affinities for [3H]oxytocin and [3H]vasopressin; both tritiated peptides were displaced from these sites in the presence of 10 microM of either oxytocin or vasopressin. The main areas bearing such sites were the ventral subiculum, several nuclei of the amygdala, the ventromedial hypothalamic nucleus, the bed nucleus of the stria terminalis and the olfactory tubercle. (2) Selective vasopressin sites, binding [3H]vasopressin with nanomolar-range affinity and [3H]oxytocin with a much lower affinity; these sites were not labelled in the presence of 5 nM [3H]oxytocin, and 10 microM oxytocin displaced [3H]vasopressin binding by 80%. Such sites occurred in several thalamic nuclei, in the dopaminergic A13 cell group of the zona incerta, the suprachiasmatic nucleus, the fundus striati and the lateral septal nucleus. No selective oxytocin sites were detected. Different oxytocin and vasopressin binding characteristics were found in the hypothalamo-neurohypophysial system. In the paraventricular and supraoptic nuclei and in the pituitary neural lobe the [3H]vasopressin binding density was twice that of [3H]oxytocin; vasopressin was always more potent than oxytocin in displacing both [3H]vasopressin and [3H]oxytocin binding from those sites. Interaction of the tritiated peptides with neurophysins cannot be completely ruled out in these locations. The present data are discussed in correlation with the functional roles of the neurohypophysial peptides in the brain and the pharmacological characteristics of their receptors.

Oxytocin in the Bed Nucleus of the Stria Terminalis and Lateral Septum Facilitates Bursting of Hypothalamic Oxytocin Neurons in Suckled Rats

Several regions of the forebrain possess high densities of oxytocin (OT)‐binding sites including the bed nucleus of the stria terminalis (BST) and lateral septum (LS). In order to examine whether these regions participate in the central facilitation of the milk ejection reflex by OT, microinjections of OT (1 ng in 100 nl containing Janus Green dye) were made into the BST (13 tests) or LS (9 tests) of anaesthetized, suckled rats, while recording the electrical activity of OT neurons in the contralateral supraoptic nucleus. Histological localization of injection sites using Janus Green demonstrated that all BST injections were close to the anterior commissure, and LS injections were all located in the ventral division of the LS. Film autoradiographic visualization of OT‐binding sites (in 7 tests using [125I]OT antagonist) confirmed that the BST and LS injections were located within regions of high OT binding. Injections into both regions facilitated the milk ejection reflex by increasing either the frequency and/or amplitude of OT neuron bursts, or by triggering bursts in animals that previously had shown no milk ejection responses; the mean number of milk ejections in the 30 min before and after injection increasing from 1.6·0.5 to 3.6·0.5 for BST and from 1.5·0.6 to 3.9·0.4 for LS. The OT microinjections had a more variable effect on background activity of OT neurons, increasing firing in some cases and not in others. This facilitatory effect was similar to that induced by microinjections into the lateral ventricle, but was smaller and delayed compared to that induced by injection into the third ventricle (9 tests), possibly due to unilateral activation of target sites. The facilitatory effect was unlikely to have been due to diffusion of OT into the ventricle, since injections into control sites (striatum and thalamus) at similar distances from the ventricle (9 tests) had no facilitatory effect (number of bursts during 30 min before and after injection; 2.2·0.5 and 1.8·0.5, respectively). These data suggest that limbic structures (BST and LS) participate in the action of central OT on the pattern of milk ejections in the suckled rat.

Activation of N-methyl-D-aspartate receptors regulates basal electrical activity of oxytocin and vasopressin neurons in lactating rats

The control of suckling-induced bursting activity of oxytocin neurons and of phasic activity of vasopressin neurons by N-methyl-D-aspartate receptors was investigated in anaesthetized lactating rats. Receptor antagonist or agonist was applied in the vicinity of supraoptic neurons recorded extracellularly. The basal activity of oxytocin neurons was tonically decreased and increased by sustained application of the antagonist and agonist respectively. These effects occurred independently of the effectiveness of suckling to trigger the bursting pattern. When drugs were applied during an ongoing series of milk-ejection-related bursts, these changes were accompanied by parallel modifications in burst amplitude, but burst periodicity was unaffected. In rats failing to milk-eject, antagonist or agonist application did not facilitate the occurrence of bursts. Simultaneous recordings from oxytocin neurons in the contralateral supraoptic nucleus showed that neither their basal nor their bursting activity were affected, indicating the absence of cross-talk between nuclei during such application. The excitatory effect of N-methyl-D-aspartate differed from that induced in the same neurons by i.c.v. injection of oxytocin, which enhanced basal level of activity and burst amplitude, but also increased burst frequency. Furthermore, the distribution of interspike intervals indicated that N-methyl-D-aspartate, but not oxytocin, induced a regularization of the spike pattern. For vasopressin neurons, application of the receptor antagonist inhibited phasic activity by decreasing burst duration and increasing silences. Conversely, N-methyl-D-aspartate enhanced phasic activity, increasing both the duration of the active phases and the frequency of spikes during active phases. When applied to silent vasopressin neurons, N-methyl-D-aspartate induced a regular phasic activity. These results provide evidence that functional N-methyl-D-aspartate receptors regulate the excitability of both oxytocin and vasopressin neurons in lactating rats. These receptors play a paramount role in maintaining a certain level of basal activity which will favour appropriate discharge patterns, tonic for oxytocin neurons and phasic for vasopressin neurons. For oxytocin neurons, this sustained control by ambient glutamate influences the amplitude of bursts, but N-methyl-D-aspartate receptors are probably not involved in the generation of the bursting pattern.

Electrical activity of neurons in the ventrolateral septum and bed nuclei of the stria terminalis in suckled rats : statistical analysis gives evidence for sensitivity to oxytocin and for relation to the milk-ejection reflex

Our previous results obtained by lesioning or stimulating the ventrolateral part of the lateral septum and the bed nuclei of the stria terminalis suggested that this area is involved in the control of milk ejection pattern in rats. The present study was undertaken with the aim of testing ventrolateral part of the lateral septum-bed nuclei of the stria terminalis neurons as a putative link of the neuronal network controlling the bursting activity of oxytocin neurons in suckled lactating rats (anaesthetized with urethane). Ventrolateral part of the lateral septum-bed nuclei of the stria terminalis neurons were recorded simultaneously with hypothalamic oxytocin neurons in either the paraventricular or supraoptic nucleus in rats with (n = 26) or without (n = 29) periodic milk ejections. Analysis of their firing pattern enabled differentiation of two subgroups: type I, characterized by numerous high frequency spikes, often grouped in clusters; and type II with very few or no high frequency clusters of spikes. The probability density function of the interspike intervals of both patterns could be modelled using a mixture of two log-normal distributions, the parameters of which differed significantly. The presence of absence of milk ejections did not influence the overall mean level of activity (2.0 +/- 0.5 and 1.9 +/- 0.4 spikes/s, respectively). However, the characteristics of the type I firing pattern were affected by the presence of the milk-ejection reflex. The average level of activity was not always constant and 16/55 ventrolateral part of the lateral septum-bed nuclei of the stria terminalis neurons displayed cyclical activity (from 0.6 +/- 0.2 to 4.0 +/- 0.5 spikes/s) both in the presence (n = 8) and absence (n = 8) of the milk-ejection reflex. In five of eight neurons recorded during milk-ejection reflex, the cycles in firing were clearly correlated with the bursting of oxytocin neurons. These five neurons exhibited the type I firing pattern. The three remaining neurons and the eight neurons recorded in the absence of milk-ejection reflex displayed the type II firing pattern. Oxytocin (1-2 ng = 0.45-0.9 mU) was injected into the third ventricle (i.c.v.) in order to examine the possible involvement of ventrolateral part of the lateral septum-bed nuclei of the stria terminalis neurons in the facilitatory effect of oxytocin on the reflex.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland—II. Comparative study on the long-evans and brattleboro strains

The anatomical distribution and pharmacological characteristics of the different types of neurohypophysial hormone binding sites were compared in the forebrains and pituitary glands of Long-Evans rats and its mutant Brattleboro strain, genetically deficient in vasopressin. Quantitative autoradiography on sections incubated in the presence of 5 nM of either [3H]oxytocin or [3H]vasopressin revealed the presence of the same types of sites in the brains of both strains but noticeable variations in their densities were found in several areas. In the forebrain, oxytocin/vasopressin sites, which bind both peptides with similar high nanomolar affinities, had the same locations and densities in the ventral subiculum, in several nuclei of the amygdala, the bed nucleus of the stria terminalis and the olfactory tubercle. The density of such sites was, in contrast, lower in the ventromedial hypothalamic nucleus of the Brattleboro rat. Selective vasopressin sites which bind [3H]vasopressin with a nanomolar-range affinity and [3H]oxytocin with a much lower affinity showed more variations. They were not found in the Brattleboro rat thalamus but were highly concentrated in several thalamic nuclei in the Long-Evans rat. Conversely, their densities were higher in the dopaminergic A13 cell group of the zona incerta and the suprachiasmatic nucleus of the Brattleboro rat. Their densities were similar in the lateral septal nucleus and in the fundus striati of both strains. In the hypothalamo-neurohypophysial system, [3H]oxytocin and [3H]vasopressin binding occurred in the Long-Evans rat with characteristics different from those found in other brain areas. In the Brattleboro rat, no [3H]vasopressin binding and only low [3H]oxytocin binding, restricted to the magnocellular nuclei, were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Neurophysins, rather than Receptors, are Involved in [3H]Oxytocin and [3H]Vasopressin Binding Detected by Autoradiography in the Hypothalamo‐Neurohypophyseal System

The goal of the present experiments was to analyse the binding of oxytocin (OT) and vasopressin (VP) in the hypothalamo‐neurohypophyseal system to determine whether [3H]OT and [3H]VP binding in this system involved interaction with receptor sites or with neurophysins. Using quantitative autoradiography, several experiments were performed to compare [3H]OT‐ and [3H]VP‐binding characteristics in this system and in brain areas containing identified receptor sites. Saturation experiments indicated much lower affinity of [3H]OT and [3H]VP binding in the magnocellular nuclei and neural lobe than on brain receptors. Competition experiments using selective ligands indicated interaction with neurophysins rather than with receptors in the hypothalamo‐neurohypophyseal system. This system was never labelled in the presence of a [125I]OT antagonist, a selective OT receptor ligand. In contrast with receptors elsewhere in the brain, the magnocellular nuclei were labelled by [3H]OT and [3H]VP in the absence of MgCI2. In the pituitary neural lobe, density of binding sites was moreover obviously related to the amount of neurosecretory granules, as seen in acutely dehydrated rats. Taken together, these data strongly suggest that in the hypothalamo‐neurohypophyseal system [3H]OT and [3H]VP bind to neurophysins rather than to specific receptors.

Scopolamine impairs the ability of parturient ewes to learn to recognise their lambs.

Abstract Within a 4-h period after parturition, the ewe learns the odor of her lamb that will later allow recognition of her offspring from an alien lamb. This study investigated the involvement of the cholinergic system in this olfactory learning. At parturition and 2 h later, ewes received IM injections of saline (C group, n = 21), scopolamine methylbromide (METSCOP group, 100 μg/kg, n = 14) a peripherally acting muscarinic antagonist, a low dose of scopolamine hydrobromide (SCOP32 group, 32 μg/kg, n = 15) or a higher dose of scopolamine hydrobromide (SCOP100 group, 100 μg/kg, n = 18). Maternal behavior was observed at parturition and selective behavior was tested after 4 h of mother-young contact. No differences in maternal behavior at parturition were found between groups. By contrast, the proportion of ewes showing selectivity was significantly lower in the SCOP100 group (7/18) than in the METSCOP group (12/14, P = 0.01), SCOP32 group (12/15, P = 0.03), or C group (17/21, P = 0.01). In addition, saline-treated ewes, after having established their selective bond, received 100 μg/kg scopolamine and were again tested for selectivity 20 min later. Only one out of the 17 tested ewes failed to recognize their lambs after this treatment. These results indicate that intact central muscarinic transmission of the brain is required for the learning of individual lamb odor at parturition but not for the recall of this information.