The origin of somatostatin-containing nerve fibers innervating the hypothalamic supraoptic nucleus

Light and electron microscopic studies were performed to study the connections between somatostatin (SOS)-containing nerve terminals and vasopressin (VP)-containing neurons in the rat supraoptic nucleus (SON). SOS-positive fibers innervate the SON in both the oxytocinergic and vasopressinergic areas. Using double immunostaining symmetric synaptic contacts were visualized between SOS immunoreactive boutons and the soma of VP immunopositive neurons. Surgical transection deafferentiating the SON from all possible directions do not effect the presence of SOS immunopositive fibers. These results suggest a local origin of the SOS fibers. Somatostatin-containing perikarya can indeed be found at the dorsal border of the SON at the rostral and caudal pole of the nucleus--we suggest that these cells innervate the SON. The presence of synaptic contacts between SOS fibers and VP neurons as well as the lack of these fibers in the VP deficient Brattleboro rats indicate a role for SOS in the synthesis and/or release of vasopressin in the SON.     

The Noradrenergic Innervation of Identified Hypothalamic Magnocellular Somata and its Contribution to Lactation-Induced Synaptic Plasticity

Despite several studies showing that the rat supraoptic (SON) and paraventricular (PVN) nuclei are innervated by noradrenergic afferents, the respective contribution of these inputs to the oxytocinergic and vasopressinergic neuronal populations remains to be clearly defined. In the present study, we used the unbiased disector method to estimate the numerical density of noradrenergic varicosities on identified oxytocinergic and vasopressinergic somata in the rat SON and PVN. The analysis was carried out on semithin (1  μm) plastic sections cut from vibratome slices (50  μm) of the SON and PVN which had been double-labelled for noradrenaline (NA) and oxytocin- or vasopressin-related neurophysin. These preparations displayed many noradrenergic varicosities which electron microscopy showed to represent, in the main, synaptic boutons. Our quantitative analysis revealed that noradrenergic varicosities contacted oxytocinergic and vasopressinergic somata to a similar extent in male and female rats, under basal conditions of hormone secretion. The incidence of these axo-somatic contacts was similar in the SON and PVN. In contrast, in lactating rats, in which oxytocin secretion is enhanced, there was a significant increase in the density of noradrenergic varicosities apposed to oxytocinergic somata, in both nuclei. Our observations indicate that, in male and female rats under normal conditions, noradrenergic afferents innervate each type of neurosecretory somata, in both magnocellular nuclei, in a similar fashion. They reveal, moreover, that noradrenergic afferents participate in lactation-induced structural plasticity of synapses impinging on oxytocinergic somata.     

Electrophysiological Identification of the Functional Presynaptic Nerve Terminals on an Isolated Single Vasopressin Neurone of the Rat Supraoptic Nucleus

Release of arginine vasopressin (AVP) and oxytocin from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is under the control of glutamate‐dependent excitation and GABA‐dependent inhibition. The possible role of the synaptic terminals attached to SON neurones has been investigated using whole‐cell patch‐clamp recording in in vitro rat brain slice preparations. Recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of MNCs at the synaptic level in the SON. In the present study, excitatory glutamatergic and inhibitory GABAergic synaptic inputs were recorded from an isolated single SON neurone cultured for 12 h, using the whole‐cell patch clamp technique. Neurones expressed an AVP‐enhanced green fluorescent protein (eGFP) fusion gene in MNCs. In addition, native synaptic terminals attached to a dissociated AVP‐eGFP neurone were visualised with synaptic vesicle markers. These results suggest that the function of presynaptic nerve terminals may be evaluated directly in a single AVP‐eGFP neurone. These preparations would be helpful in future studies aiming to electrophysiologically distinguish between the functions of synaptic terminals and glial modifications in the SON neurones.     

Influence of Ovarian Steroids on the Ultrastructural Plasticity of the Adult Rat Supraoptic Nucleus Induced by Central Administration of Oxytocin

In earlier studies, we showed that continuous intracerebroventricular infusion of oxytocin, for several days, into the third ventricle of normally hydrated, non-lactating adult female rats significantly reduced glial coverage of magnocellular oxytocinergic neurons in the hypothalamus. It also induced synaptic remodelling whereby many oxytocinergic neurons became synaptically contacted by the same presynaptic terminals (shared synapses). Such changes were closely similar to those observed in the oxytocinergic system when it is physiologically activated, as during parturition and lactation. We now report that central oxytocin does not act alone to modify the ultrastructure of the magnocellular nuclei, but requires the concomitant action of sex steroids. Intracerebroventricular infusion of oxytocin was effective in inducing neuronal-glial and synaptic changes only in supraoptic nuclei of female rats undergoing a prolonged diestrus, or in castrated female rats treated during the infusion period with daily intramuscular injections of progesterone for 4 days followed by 17β-estradiol for 2 days. Infusion of oxytocin in rats with regular estrous cycles, or in castrated rats treated with progesterone or estrogen alone had no effect on the ultrastructure of the nucleus. Our observations also indicate that the action of oxytocin on the anatomy of its own neurons is very specific: only 4-threonine-oxytocin, a closely related oxytocin analogue, had an effect similar to that of oxytocin; vasopressin, 4-threonine-7-glycine oxytocin and cholecystokinin left the magnocellular nuclei structurally unaltered.     

Vasopressin and oxytocin decrease excitatory amino acid release in adult rat supraoptic nucleus

Oxytocin and vasopressin reduce the amplitude of excitatory postsynaptic responses in magnocellular neuroendocrine cells of the supraoptic nucleus (SON). To test whether synaptic glutamate release is modulated by these neuropeptides, we examined the combined effect of vasopressin and oxytocin on depolarization-induced glutamate and aspartate release from acutely dissected rat SON or fronto-parietal cortex punches. Glutamate release was stimulated with 60 mm K+ for 5-10 min and measured using ion exchange chromatography or high-performance liquid chromatography. During depolarization with high K+, extracellular glutamate levels increased, on average, to 204% of control values. In the presence of vasopressin/oxytocin, K+-stimulated glutamate and aspartate release were significantly reduced by 34% and 62%, respectively, in the SON. Treatment with the aminopeptidase inhibitor amastatin did not mimic the effects of exogenous vasopressin/oxytocin on glutamate or aspartate release, suggesting that, under the conditions tested here, amastatin treatment may produce more complex effects. The effects of exogenous neuropeptides are likely mediated by oxytocin and/or vasopressin receptors, as the oxytocin- and V1a-receptor antagonist, Manning Compound (10-100 micro m), partially reversed the effects of vasopressin/oxytocin on SON glutamate release. In contrast, in cortical punches, glutamate release was enhanced by high K+, but vasopressin/oxytocin did not significantly reduce glutamate/aspartate release, consistent with the relatively sparse distribution of vasopressin/oxytocin receptors in fronto-parietal cortex. These findings suggest that locally released oxytocin and vasopressin may autoregulate SON magnocellular neuroendocrine cell activity in part by modulating the release of excitatory amino acids from afferent terminals targeting these cells and/or from other cellular sources.     

Transient induction of c-fos in rat magnocellular hypothalamic neurons after hypophysectomy.

Using immunofluorescence histochemistry, the paraventricular and supraoptic hypothalamic nuclei of normal control and hypophysectomized rats were studied in double labelling experiments with antibodies against the protein c-fos (Fos) and against vasopressin or oxytocin in order to characterize the activated neurons chemically. Normal controls showed no expression of Fos, whereas in hypophysectomized animals an intense induction of Fos-like immunoreactivity (-LI) was observed 12 h and 24 h post hypophysectomy but not beyond this survival time. Both vasopressinergic and oxytocinergic magnocellular neurons were labelled with Fos-LI. Thus Fos-LI can be induced in magnocellular hypothalamic neurons by injury, suggesting that this protein may be involved in adaptive mechanisms following axotomy.     

Vasopressin from hypothalamic magnocellular neurons has opposite actions at the adenohypophysis and in the supraoptic nucleus on ACTH secretion

Magnocellular vasopressinergic and oxytocinergic neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei comprise the hypothalamic-neurohypophysial system, which is crucially involved in the regulation of body fluid and electrolyte homeostasis. However, still controversial is to what extent the same system influences the secretion of adrenocorticotropic hormone (ACTH) from the adenohypophysis. Therefore, we selectively stimulated magnocellular neurons of the SON of conscious male Wistar rats via retrodialysis. As expected, dialysis of the SON with hypertonic medium increased both the release of vasopressin within the SON and the secretion of vasopressin and oxytocin into the systemic blood circulation. This activation of the hypothalamic-neurohypophysial system was accompanied by a fivefold increase in plasma ACTH concentration. This effect was observed only if the tip of the microdialysis probe was within the SON. Intravenous infusion of the vasopressin V1 receptor antagonist d(CH2)5Tyr(Me)AVP significantly attenuated the effects of local osmotic stimulation of the SON on ACTH secretion. In contrast, administration of the same antagonist directly into the SON significantly enhanced the osmotically stimulated secretion of ACTH and corticosterone, primarily by delaying the restoration of the hormone secretion to prestimulation levels. We conclude from these findings that vasopressin from the hypothalamic-neurohypophysial system participates in the regulation of the hormonal stress response in a counterbalanced manner at the level of the SON and the adenohypophysis.     

Oestradiol potentiates hormone secretion and neuronal activation in response to hypertonic extracellular volume expansion in ovariectomised rats

Secretion of vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) is an essential mechanism for the maintenance of hydromineral homeostasis. Secretion of these hormones is modulated by several circulating factors, including oestradiol. However, it remains unclear how oestradiol exerts this modulation. In the present study we investigated the participation of oestradiol in the secretion of VP, OT and ANP and in activation of vasopressinergic and oxytocinergic neurones of the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus in response to extracellular volume expansion (EVE). For this purpose, ovariectomised (OVX) rats treated for 7 days with vehicle (corn oil, 0.1 ml/rat, OVX+O group) or oestradiol (oestradiol cypionate, 10 μg/kg, OVX+E group) were subjected to either isotonic (0.15 m NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 m NaCl, 2 ml/100 g b.w., i.v.) EVE. Blood samples were collected for plasma VP, OT and ANP determination. Another group of rats was subjected to cerebral perfusion, and brain sections were processed for c‐Fos‐VP and c‐Fos‐OT double‐labelling immunohistochemistry. In OVX+O rats, we observed that both isotonic and hypertonic EVE increased plasma OT and ANP concentrations, although no changes were observed in VP secretion. Oestradiol replacement did not alter hormonal secretion in response to isotonic EVE, but it increased VP secretion and potentiated plasma OT and ANP concentrations in response to hypertonic EVE. Immunohistochemical data showed that, in the OVX+O group, hypertonic EVE increased the number of c‐Fos‐OT and c‐Fos‐VP double‐labelled neurones in the PVN and SON. Oestradiol replacement did not alter neuronal activation in response to isotonic EVE, but it potentiated vasopressinergic and oxytocinergic neuronal activation in the medial magnocellular PVN (PaMM) and SON. Taken together, these results suggest that oestradiol increases the responsiveness of vasopressinergic and oxytocinergic magnocellular neurones in the PVN and SON in response to osmotic stimulation.     

Vesicle shape and amino acids in synaptic inputs to phrenic motoneurons: Do all inputs contain either glutamate or GABA?

Varicosities that made synapses or direct contacts with retrogradely labelled rat phrenic motoneurons were examined for their content of immunoreactivity for either glutamate or glutamate decarboxylase, the enzyme involved in synthesis of α-aminobutyric acid (GABA). Phrenic motoneurons were identified by retrograde tracing from the diaphragm with cholera toxin B subunit conjugated to horseradish peroxidase. Cell bodies and medium-sized to large dendrites were labelled. Preembedding immunocytochemistry identified glutamate decarboxylase-immunoreactive nerve fibres; glutamate-immunoreactive nerve terminals were identified using postembedding immunogold labelling of ultrathin sections. The presence of glutamate- or glutamate decarboxylase immunoreactivity in nerve terminals was correlated with the morphology of the synaptic vesicles. Two major classes of nerve terminals were identified. Nerve terminals with round (presumably spherical) synaptic vesicles (S terminals) comprised 55% of synapses and contacts on phrenic motoneuron somata and 58% of synapses and direct contacts with dendrites. Nerve terminals with flattened synaptic vesicles (F terminals) comprised 42% of synapses direct contacts with somata and 41% of synapses and direct contacts with dendrites. Analysis of immunogold-labelled sections showed that S terminals contained statistically higher levels of glutamate immunoreactivity than F terminals. At the light microscope level, many glutamate decarboxylase-immunoreactive nerve terminals surrounded retrogradely labelled motoneurons. Varicosities with glutamate decarboxylase immunoreactivity made 33% of all synapses and direct contacts on somata, and 33% of synapses and direct contacts with dendrites of the retrogradely labelled phrenic motoneurons. Flattened synaptic vesicles were present in those glutamate decarboxylase-immunoreactive nerve terminals in which synaptic vesicle morphology could be judged. An additional 10% of all nerve terminals were of the F type, but were not glutamate decarboxylase-immunoreactive. Three percent of terminals on somata and 1% of nerve terminals on dendrites could not be classified as S or F types. These findings suggest that more than 90% of all inputs to phrenic motoneuron cell bodies and proximal dendrites could contain either GABA or glutamate. Some of these glutamatergic and GABAergic nerve fibres undoubtedly represent the source of inspiratory drive to, or expiratory inhibition of, phrenic motoneurons. © 1996 Wiley-Liss, Inc.     

Excitatory effect of dopamine on oxytocin and vasopressin reflex releases in the rat

The involvement of dopamine in the release of oxytocin and vasopressin was investigated in lactating rats during suckling or after changes in plasma osmolality. The effects of intraventricular injections of dopamine, agonists and antagonists, were tested on electrical unit activity of oxytocinergic or vasopressinergic cells in the paraventricular nucleus, on intramammary pressure (index of oxytocin release) and diuresis (index of vasopressin release).In urethane-anaesthetized lactating suckled rats, dopamine (1 μg), apomorphine (2.5 and 5 μg) facilitated the established milk-ejection reflex, increasing the frequency and the amplitude of neurosecretory bursts of oxytocinergic cells. They also triggered the reflex in lactating rats without milk-ejections during suckling. The small doses injected were in no way such as to induce an acceleration in firing rate of oxytocinergic cells or an increase in mammary pressure.In alcohol-loaded rats, during water diuresis, dopamine (2 μg) and apomorphine (5 μg) activated the depressed vasopressinergic cells and inhibited diuresis. These facilitatory effects were progressive, reaching a maximum 10–15 min after injection.Haloperidol (5 μg) and α-flupentixol (10 μg) had an inhibitory effect on both types of neurosecretory cells in urethane-anaesthetized rats. They prevented the reflex activation of oxytocinergic cells induced by suckling and of vasopressinergic cells after a hyperosmotic stimulus (1 ml i.p 9% NaCl solution). These inhibitory effects were not of the ‘all-or-none’ type.So, we can postulate that dopamine regulates the reflex release of oxytocin and vasopressin in the hypothalamus. On the one hand, dopamine permits and controls the periodic activation of oxytocinergic cells as long as the mothers are being suckled. On the other hand, it modulates the activity of vasopressinergic cells whenever the plasma osmolality changes.     

Magnocellular neuropeptidergic neurons in hypothalamus: Increases in membrane apposition and number of specialized synapses from pregnancy to lactation

Morphological changes which have been hypothesized to accompany functional alterations in magnocellular neuropeptidergic cells (MNCs) were studied in female rats. Direct soma-somatic appositions between the MNC profiles of two nuclear groups, the supraoptic nucleus (SON) and nucleus circularis (NC) were investigated at the ultrastructural level in 4 groups of animals: virgin females, immediately pre-partum pregnant rats, post-partum and 14-day lactating animals. The percentage of SON MNC profiles in soma-somatic apposition and the amount of membrane in direct contact significantly increased over control levels by the last day of pregnancy. Further significant increases in these measures were observed in lactating rats. MNCs in NC showed steady gradual increases on these measures with significant differences from controls occurring in the post-partum group. The percentage of SON cell profiles with double synapses (i.e., presynaptic terminals making synaptic contact with two postsynaptic neurons) was significantly elevated in lactating rats (~10%) over the next highest group (~1% for post-partum rats). In NC, ~10% of the cell profiles sampled had such synapses but no differences among treatments occurred. The changes during late pregnancy suggest that close appositions may serve to enhance the metabolic activity of MNCs at a time when there is a build-up of stored oxytocin. Further increases in cell-cell contact and the addition of double synapses on possibly electrotonically coupled MNCs during lactation may serve a synchronizing function, particularly in the oxytocin cells participating in the milk ejection reflex.     

Taurine selectively modulates the secretory activity of vasopressin neurons in conscious rats.

Previous experiments have shown that a 10-min forced swimming session triggers the release of vasopressin from somata and dendrites, but not axon terminals, of neurons of the hypothalamic-neurohypophysial system. To further investigate regulatory mechanisms underlying this dissociated release, we forced male Wistar rats to swim in warm (20 degrees C) water and monitored release of the potentially inhibitory amino acids gamma amino butyric acid (GABA) and taurine into the hypothalamic supraoptic nucleus using microdialysis. Forced swimming caused a significant increase in the release of taurine (up to 350%; P < 0.05 vs. prestress release), but not GABA. To reveal the physiological significance of centrally released taurine, the specific taurine antagonist 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide was administered into the supraoptic nucleus via retrodialysis. Administration of this antagonist caused a significant increase in the release of vasopressin within the supraoptic nucleus and into the blood both under basal conditions and during stress (up to 800%; P < 0.05 vs. basal values), without affecting hypothalamic or plasma oxytocin. Local administration of the GABA(A) receptor antagonist bicuculline, in contrast, failed to influence vasopressin secretion at either time point. In a separate series of in vivo electrophysiological experiments, administration of the same dosage of the taurine antagonist into the supraoptic nucleus via microdialysis resulted in an increased electrical activity of identified vasopressinergic, but not oxytocinergic, neurons. Taken together our data demonstrate that taurine is released within the supraoptic nucleus during physical/emotional stress. Furthermore, at the level of the supraoptic nucleus, taurine inhibits not only the electrical activity of vasopressin neurons but also acts as an inhibitor of both central and peripheral vasopressin secretion during different physiological states.     

Modulation of GABAergic transmission by endogenous glutamate in the rat supraoptic nucleus

The presence of group III metabotropic glutamate receptors on GABAergic terminals in the supraoptic nucleus suggests that the level of glutamate in the extracellular space may regulate synaptic strength at inhibitory synapses. To test this hypothesis we examined the consequences of increasing ambient glutamate on GABA-mediated synaptic activity in supraoptic neurons. The concentration of the excitatory amino acid in the extracellular space was increased pharmacologically by blocking glutamate transporters. Inhibition of the astrocyte-specific GLT-1 glutamate transporter led to a reversible decrease in evoked inhibitory postsynaptic current amplitude. This modulation had a presynaptic origin as revealed by analysis of paired-pulse ratio and miniature inhibitory currents. Furthermore, blocking group III metabotropic glutamate receptors with the specific antagonist MAP4 prevented the depression of GABAergic transmission induced by glutamate transporter blockade. Thus, presynaptic metabotropic glutamate receptors located on inhibitory terminals in the supraoptic nucleus appear to sense changes in ambient glutamate and modify GABA release accordingly. However, it seems that such changes need to reach a certain magnitude because the discrete deficit in glutamate clearance which occurs in the supraoptic nucleus of lactating rats is not sufficient to modulate GABA-mediated transmission. These results suggest that ambient glutamate contributes to the modulation of synaptic efficacy not only at glutamatergic synapses but also at inhibitory GABAergic synapses.     

Selective Excitatory Amino Acid Uptake in Glutamatergic Nerve Terminals and in Glia in the Rat Striatum: Quantitative Electron Microscopic Immunocytochemistry of Exogenous D-Aspartate and Endogenous Glutamate and GABA

To characterize glutamate/aspartate uptake activity in various cellular and subcellular elements in the striatum, rat striatal slices were exposed to 10 and 50 μM exogenous D-aspartate. After fixation with glutaraldehyde/ formaldehyde the distribution of D-aspartate was analysed by postembedding immunocytochemistry and the ultrastructural distribution was compared with the distributions of endogenous glutamate and GABA. Light microscopically, D-aspartate-like immunoreactivity was localized in conspicuous dots along very weakly labelled dendritic profiles and neuron cell bodies. At the electron microscope level gold particles signalling D-aspartate occurred at highest density in nerve terminals making asymmetrical contacts with postsynaptic spines (i.e. resembling synapses of cortical afferents). Astrocytic processes also contained gold particles, but at a lower density than nerve endings. In contrast, dendritic spines were only weakly D-aspartate–positive. The difference in labelling at 10 and 50 μM D-aspartate was consistent with'high-affinity'uptake. Neighbouring sections processed with other antibodies showed that the D-aspartate labelling occurred in nerve terminals strongly immunoreactive for glutamate, rather than in terminals very weakly glutamate-immunopositive or in nerve endings immunoreactive for GABA. Glutamate labelling of perfusion-fixed striatum confirmed that terminals forming asymmetrical synaptic contacts with spines were enriched with gold particles, suggesting that these terminals use glutamate as a transmitter. This study demonstrates that high-affinity uptake sites for excitatory amino acids in the striatum are most strongly expressed on presumed glutamatergic nerve terminals and on astrocytes.     

Glucocorticoid modulation of neuronal activity and hormone secretion induced by blood volume expansion

The present study evaluated the involvement of glucocorticoid in the activation of vasopressinergic and oxytocinergic neurons of hypothalamic nuclei and plasma levels of vasopressin (AVP), oxytocin (OT), atrial natriuretic peptide (ANP) and corticosterone (CORT) in response to both isotonic and hypertonic blood volume expansion (BVE). Rats were subjected to isotonic (0.15 M NaCl, 2 ml/100 g b.w., i.v.) or hypertonic (0.30 M NaCl, 2 ml/100 g b.w., i.v.) BVE with or without pre-treatment with dexamethasone (1 mg/kg, i.p.). Results showed that isotonic BVE increased OT, ANP and CORT, and decreased AVP plasma levels. On the other hand, hypertonic BVE enhanced AVP, ANP, OT, and CORT plasma concentrations. Both hypertonic and isotonic BVE induced an increase in the number of Fos-OT double-labeled magnocellular neurons in the PVN and SON. Pre-treatment with dexamethasone reduced OT secretion, as well as Fos-OT immunoreactive neurons in response to both isotonic and hypertonic BVE. We also observed that dexamethasone pre-treatment had no effect on AVP secretion in response to hypertonic BVE, although this effect was associated with a blockade of Fos expression in the vasopressinergic magnocellular neurons in the PVN and SON. In conclusion, these data suggest that, not only the rapid OT release from storages, but also the oxytocinergic cellular activation induced by BVE are modulated by glucocorticoids. However, this pattern of response was not observed for AVP cells, suggesting that dexamethasone is not likely to influence rapid release of AVP but seems to modulate the activation of these neurons in response to hypertonic BVE.     

Visualization of local afferent inputs to magnocellular oxytocin neurons in vitro.

We recently showed that oxytocin (OT) neurons in organotypic slice cultures obtained from postnatal rat hypothalamus display complex patterns of electrical activity, similar to those of adult magnocellular OT neurons in vivo. Here we used such cultures to investigate the identity and, in particular, the origin of afferent inputs responsible for this activity. Multiple immunostaining with light and confocal microscopy showed that the somata and dendrites of oxytocinergic neurons were contacted by numerous synapses, visualized by their reaction to the synaptic markers, synaptophysin or synapsin. Many were GABAergic, displaying immunoreactivities for glutamic acid decarboxylase or gamma-aminobutyric acid (GABA); others were enriched in glutamate immunoreactivity. Such afferents presumably arose from GABA- or glutamate-immunoreactive neurons, respectively, with distinct and characteristic morphologies and topographies. A few dopaminergic boutons (tyrosine hydroxylase- or dopamine-immunopositive) impinged on OT neurons; they arose from dopamine-positive neurons located along the third ventricle. No noradrenergic profiles were detected. Despite the presence of choline acetyl-transferase (ChAT)-immunoreactive neurons, there were no cholinergic contacts. Lastly, we found oxytocinergic synapses, identified by immunoreaction for OT-related neurophysin and synapsin, contacting OT somata and dendrites. Our observations thus demonstrate that inhibitory and excitatory inputs to OT neurons derive from local intrahypothalamic GABA and glutamate neurons, in close proximity to the neurons. They also reveal that OT neurons are innervated by hypothalamic dopaminergic neurons. Finally, they confirm the existence of homotypic OT synaptic contacts which derive from local OT neurons.