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Durant la periode medievale, les trois saints martyrs Fuscien,Victoric et Gentien furent l’objet d’une veneration toute particuliere dans l’Eglise d’Amiens. Ce culte, dont le point d’ancrage a pu s...

Many neurons in the CNS display rhythmic patterns of activity to optimize excitation–secretion coupling. However, the mechanisms of rhythmogenesis are only partially understood. Magnocellular vasopressin (VP) neurons in the hypothalamus display a phasic activity that consists of alternative bursts of action potentials and silent periods. Previous observations from acute slices of adult hypothalamus suggested that VP cell rhythmicity depends on intrinsic membrane properties. However, such activityin vivois nonregenerative. Here, we studied the mechanisms of VP neuron rhythmicity in organotypic slice cultures that, unlike acute slices, preserve functional synaptic connections. Comparative analysis of phasic firing of VP neuronsin vivo, in acute slices, and in the cultures revealed that, in the latter, the activity was closely related to that observedin vivo. It was synaptically driven, essentially from glutamatergic inputs, and did not rely on intrinsic membrane properties. The glutamatergic synaptic activity was sensitive to osmotic challenges and κ-opioid receptor activation, physiological stimuli known to affect phasic activity. Together, our data thus strongly suggest that phasic activity in magnocellular VP neurons is controlled by glutamatergic synaptic inputs rather than by intrinsic properties.

During parturition and lactation, neurosecretory oxytocin (OT) neurons in the hypothalamus achieve pulsatile hormone secretion by coordinated bursts of firing that occur throughout the neuronal population. This activity is partly controlled by somatodendritic release of OT, which facilitates the onset and recurrence of synchronized bursting. To further investigate the cellular mechanisms underlying the control exerted by OT on the activity of its own neurons, we studied the effects of the peptide on membrane potential and synaptic activity in OT neurons in hypothalamic organotypic slice cultures. Bath application of low concentrations of OT (m) facilitated GABAAreceptor-mediated inhibitory transmission through a presynaptic mechanism without affecting membrane potential and excitatory glutamatergic synaptic activity. The facilitatory action of OT on GABAergic transmission was dose-dependent, starting at 25 nmand disappearing at concentrations >100 nm. As shown previously, higher concentrations of OT (>500 nm) had the opposite effect, inhibiting GABAAreceptors via a postsynaptic mechanism. Surprisingly, OT-mediated facilitation of GABAergic transmission promoted action potential firing in 40% of the neurons. Each action potential occurred at the end of the repolarizing phase of an inhibitory potential. Pharmacological dissection revealed that this firing involved the activation of low-threshold activated calcium channels. Detailed statistical analysis showed that OT-mediated firing upregulated bursting activity in OT neurons. It is thus likely to optimize OT secretion and, as a consequence, facilitate delivery and milk ejection in mammals.

Neurons, including their synapses, are generally ensheathed by fine processes of astrocytes, but this glial coverage can be altered under different physiological conditions that modify neuronal activity. Changes in synaptic connectivity accompany astrocytic transformations so that an increased number of synapses are associated with reduced astrocytic coverage of postsynaptic elements, whereas synaptic numbers are reduced on reestablishment of glial coverage. A system that exemplifies activity-dependent structural synaptic plasticity in the adult brain is the hypothalamo-neurohypophysial system, and in particular, its oxytocin component. Under strong, prolonged activation (parturition, lactation, chronic dehydration), extensive portions of somatic and dendritic surfaces of magnocellular oxytocin neurons are freed of intervening astrocytic processes and become directly juxtaposed. Concurrently, they are contacted by an increased number of inhibitory and excitatory synapses. Once stimulation is over, astrocytic processes again cover oxytocinergic surfaces and synaptic numbers return to baseline levels. Such observations indicate that glial ensheathment of neurons is of consequence to neuronal function, not only directly, for example by modifying synaptic transmission, but indirectly as well, by preparing neuronal surfaces for synapse turnover.

We analyzed the subtypes of group III metabotropic glutamate receptors (mGluRs) modulating inhibitory and excitatory transmission in the rat supraoptic nucleus. Bath application of the agonist l-AP4 at 200 microM, a concentration that activates all group III mGluR subtypes, inhibited the frequency but not the amplitude of miniature inhibitory and excitatory postsynaptic currents, indicating a presynaptic site of action. l-AP4 at low concentrations (10 microM), as well as ACPT-1 (50 microM), a specific mGluR III agonist, inhibited transmission at GABAergic and glutamatergic synapses to the same extent as 200 microM l-AP4. Because the potency of l-AP4 and ACPT-1 is much higher on mGluR4 and mGluR8 than on mGluR7, these results are consistent with the presence of high-affinity group III mGluRs regulating transmitter release in this nucleus. In agreement with these findings, DCPG (30 microM), a selective mGluR8 agonist, induced a significant depression of inhibitory and excitatory synaptic currents. Group III mGluRs such as mGluR8, because of their high affinity for glutamate, are particularly well suited to detect small changes in the concentration of this excitatory amino acid in the extracellular space. Their presence, therefore, may favor the negative feedback control exerted by glutamate on its own release as well as the intersynaptic crosstalk mediated by glutamate spillover on adjacent synapses.

Interactions between separate synaptic inputs converging on the same target appear to contribute to the fine-tuning of information processing in the central nervous system. Intersynaptic crosstalk is made possible by transmitter spillover from the synaptic cleft and its diffusion over a distance to neighboring synapses. This is the case for glutamate, which inhibits γ-aminobutyric acid (GABA)ergic transmission in several brain regions through the activation of presynaptic receptors. Such heterosynaptic modulation depends on factors that influence diffusion in the extracellular space (ECS). Because glial cells represent a physical barrier to diffusion and, in addition, are essential for glutamate uptake, we investigated the physiological contribution of the astrocytic environment of neurons to glutamate-mediated intersynaptic communication in the brain. Here we show that the reduced astrocytic coverage of magnocellular neurons occurring in the supraoptic nucleus of lactating rats facilitates diffusion in the ECS, as revealed by tortuosity and volume fraction measurements. Under these conditions, glutamate spillover, monitored through metabotropic glutamate receptor-mediated depression of GABAergic transmission, is greatly enhanced. Conversely, impeding diffusion with dextran largely prevents crosstalk between glutamatergic and GABAergic afferent inputs. Astrocytes, therefore, by hindering diffusion in the ECS, regulate intersynaptic communication between neighboring synapses and, probably, overall volume transmission in the brain.

During suckling, oxytocin (OT) neurons display a bursting electrical activity, consisting of a brief burst of action potentials which is synchronized throughout the OT neuron population and which periodically occurs just before each milk ejection in the lactating rat. To investigate the basis of such synchronization, we performed simultaneous intracellular recordings from pairs of OT neurons identified retrospectively by intracellular fluorescent labelling and immunocytochemistry in organotypic slice cultures derived from postnatal rat hypothalamus. A spontaneous bursting activity was recorded in 65% of OT neurons; the remaining showed only a slow, irregular activity. Application of OT triggered bursts in nonbursting neurons and accelerated bursting activity in spontaneously bursting cells. These cultures included rare vasopressinergic neurons showing no bursting activity and no reaction to OT. Bursts occurred simultaneously in all pairs of bursting OT neurons but, as in vivo, there were differences in burst onset, amplitude and duration. Coordination of firing was not due to electrotonic coupling because depolarizing one neuron in a pair had no effect on the membrane potential of its partner and halothane and proprionate did not desynchronize activity. On the other hand, bursting activity was superimposed on volleys of excitatory postsynaptic potentials (EPSPs) which occurred simultaneously in pairs of neurons. EPSPs, and consequently action potentials, were reversibly blocked by the non-NMDA glutamatergic receptor antagonist CNQX. Taken together, these data, obtained from organotypic cultures, strongly suggest that a local hypothalamic network governs synchronization of bursting firing in OT neurons through synchronous afferent volleys of EPSPs originating from intrahypothalamic glutamatergic inputs.

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.

Increasing evidence is establishing that adult neurons and their associated glia can undergo state-dependent changes in their morphology and in consequence, in their relationships and functional interactions. A neuronal system that illustrates this kind of neuronal-glial plasticity in an exemplary fashion is that responsible for the secretion of the neurohormone oxytocin (OT). As shown by comparative ultrastructural analysis, during physiological conditions like lactation and dehydration, which result in enhanced peripheral and central release of the peptide, astrocytic coverage of OT neurons is markedly reduced and their surfaces are left directly juxtaposed. Such reduced glial coverage is of consequence to neuronal activity since it modifies extracellular ionic homeostasis and glutamate neurotransmission. In addition, it is probably prerequisite to the synaptic remodeling that occurs concurrently, and results in an enhanced number of inhibitory (GABAergic) and excitatory (glutamatergic, noradrenergic) synapses, thus further affecting neuronal function. The neuronal-glial and synaptic changes occur rapidly, within a matter of hours, and are reversible with termination of stimulation. The adult OT system retains many juvenile molecular features that may allow such plasticity, including expression of cell adhesion molecules implicated in neuronal-glial interactions during development, like polysialylated NCAM, F3/contactin and its ligand, the matrix glycoprotein, tenascin-C. On the other hand, OT itself can induce the changes since in vivo (ventricular microinfusion) or in vitro (on acute hypothalamic slices) application leads to glial and neuronal transformations similar to those induced by physiological stimuli.

F3/contactin is a cell adhesion/recognition molecule of the immunoglobulin superfamily implicated in axonal growth. We examined its subcellular distribution and mobilization to the cell surface in oxytocin- (OT-) secreting neurons, which express it throughout life and the axons of which undergo activity-dependent remodelling. This was performed in hypothalamic organotypic slice cultures containing OT neurons with properties of adult neurosecretory cells. Immunocytochemistry and immunoblot analysis confirmed that OT neurons express high levels of F3/contactin in vitro. Light and confocal microscopy of cultures that underwent double immunofluorescence after fixation showed F3/contactin immunoreactivity throughout the cytoplasm of OT somata, dendrites and axons, and also in non-OT axons and in putative synaptic boutons which contacted OT neurons. By contrast, after treatment of live cultures with anti-F3/contactin antibodies followed by double immunofluorescence for the glycoprotein and OT, F3/contactin immunoreactivity was visible only on the surface of axons, whether or not OT-immunoreactivity was present. Because of its glycosylphosphatidyl-inositol (GPI) linkage, F3/contactin can occur in a membrane-bound or soluble form. As seen from immunocytochemistry of live cells and immunoblot analysis, treatment of cultures with a GPI-specific phospholipase C (GPI-PLC) resulted in loss of F3/contactin immunoreactivity from all cell surfaces. F3/contactin immunoreactivity reappeared on axonal surfaces within 5 h after enzyme washout. Such re-expression was accelerated by neuronal activity facilitation (by K+ depolarization or gamma-aminobutyric acid (GABA)-A receptor blockade with bicuculline) and inhibited by neuronal activity repression [by blockade of Ca2+ channels with Mn2+, Na+ channels with tetrodotoxin (TTX) or excitatory inputs with glutamate antagonists]. Our observations establish therefore that F3/contactin surface expression in hypothalamic neurons is polarized to the axons where it occurs mainly in a GPI-linked form. We also provide direct evidence that externalization of F3/contactin depends on Ca2+ entry and neuronal electrical activity. Taken together with our earlier finding that the glycoprotein is localized in neurosecretory granules, we demonstrate that F3/contactin is mobilized to the axonal surface via the activity-dependent regulated pathway, thus arriving at the correct place and time to intervene in activity-dependent remodelling of axons.

Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.

Understanding how neurons and glia sort and deliver cell adhesion molecules to their cell surface should provide important clues as to how such molecules participate in dynamic neuronal functions in the developing and adult brain. The present study examines translocation of polysialylated neural cell adhesion molecule (PSA-NCAM), a negative regulator of cell adhesion, in cells of the rat hypothalamo-neurohypophysial system in which it is expressed throughout life and which undergo morphological remodelling in response to stimulation. PSA-NCAM expression in this system does not vary markedly in relation to different conditions of regulated neurosecretion, suggesting that the glycoprotein reaches cell surfaces via the constitutive pathway. To study this more directly, we here used immunofluorescence for PSA on NCAM in live, unpermeabilized cells to monitor PSA-NCAM surface expression in organotypic slice cultures from postnatal rat hypothalami. Subsequent immunolabelling for oxytocin confirmed that the cultures included magnocellular oxytocinergic neurons displaying many properties of adult neurosecretory neurons in situ . In the cultures, immunoreaction for PSA-NCAM was visible on the surface of oxytocinergic and non-oxytocinergic axons. This reaction disappeared after exposure of the cultures to endoneuraminidase, an enzyme which specifically cleaves α-2-8-linked PSA from NCAM. PSA-NCAM reappeared on axonal surfaces 4 h after enzyme washout. Such reexpression was visibly not affected by neuronal activity inhibition (blockade of Ca 2+ channels with Mn 2+ , of Na + channels with tetrodotoxin, or of glutamate receptors with 6-cyano-7-nitroquinoxaline-2,3-dione or d -2-amino-5-phosphonopentanoic acid) or facilitation (K + depolarization or GABA-A receptor blockade with bicuculline). In contrast, PSA-NCAM surface translocation was inhibited reversibly by cooling the cultures at 20°C, a procedure which blocks constitutive secretion and which resulted in accumulation of PSA-NCAM in the cytoplasm of oxytocinergic and non-oxytocinergic neurons. This treatment also revealed PSA-NCAM in the cytoplasm of underlying astrocytes. Our observations provide direct evidence that PSA-NCAM reaches the cell surface of hypothalamic neurons and astrocytes via the constitutive pathway, independently of Ca 2+ entry and enhanced neuronal activity. Thus, PSA-NCAM in the hypothalamo-neurohypophysial system would be continuously available to permit its cells to undergo remodelling whenever the proper stimulus intervenes.

Oxytocin and vasopressin neurons, located in the supraoptic and paraventricular nuclei of the hypothalamus, send their axons to the neurohypophysis where the neurohormones are released directly into the general circulation. Hormone release depends on the electrical activity of the neurons, which in turn is regulated by different afferent inputs. During conditions that enhance oxytocin secretion (parturition, lactation, and dehydration), these afferents undergo morphological remodelling which results in an increased number of synapses contacting oxytocin neurons. The synaptic changes are reversible with cessation of stimulation. Using quantitative analyses on immunolabelled preparations, we have established that this morphological synaptic plasticity affects both inhibitory and excitatory afferent inputs to oxytocin neurons. This review describes such synaptic modifications, their functional significance, and the cellular mechanisms that may be responsible.Key words: oxytocin, vasopressin, GABA, glutamate, noradrenaline, hypothalamo-neurohypophysial system, lactation.

1Intracellular current clamp recordings were performed from identified oxytocin (OT) neurones in acute hypothalamic slices taken from lactating Wistar rats at early (5th day: LD-5) and late (21st day: LD-21) lactation. 2The basic electrophysiological properties of LD-21 OT neurones differed from those of LD-5 OT neurones: their resting membrane potential was more depolarised (-51·5 versus -54·9 mV); their action potential duration was longer (1·6 versus 1·2 ms); their hyperpolarising after-potential (HAP) following single spikes and after-hyperpolarisation (AHP) following a burst of action potentials had smaller amplitudes (-46 and -67 %, respectively); and they lacked spike frequency adaptation during a burst. 3In LD-21 neurones bath application of 17β-oestradiol (10−7 M, 6–14 min) reversibly restored all these properties to values observed in LD-5 cells. This treatment had no effect on LD-5 neurones. 4LD-21 neurones were less sensitive to kainate than LD-5 neurones. 17β-Oestradiol significantly potentiated the kainate-induced response in LD-21, but not in LD-5 neurones. 5The effects of 17β-oestradiol were presumably mediated through a non-genomic mechanism since they occurred within a few minutes of administration, and disappeared within 30–40 min of washout. They were not inhibited by tamoxifen, an antagonist of the nuclear oestrogen receptor ER-α. Lastly, cholesterol, a non-active lipophilic molecule, had no effect. 6Our observations demonstrate that, in the absence of 17β-oestradiol, the basic electrical properties and sensitivity to kainate of OT neurones become altered between early and late lactation. However, the rise in circulating levels of oestrogens during the late phase of lactation may contribute to maintain OT neurone reactivity as long as suckling continues.

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.

During lactation and parturition, magnocellular oxytocin (OT) neurons display a characteristic bursting electrical activity responsible for pulsatile OT release. We investigated this activity using hypothalamic organotypic slice cultures enriched in magnocellular OT neurons. As shown here, the neurons are functional and actively secrete amidated OT into the cultures. Intracellular recordings were made from 23 spontaneously bursting and 28 slow irregular neurons, all identified as oxytocinergic with biocytin and immunocytochemistry. The bursting electrical activity was similar to that described in vivo and was characterized by bursts of action potentials (20.1 +/- 4.3 Hz) lasting approximately 6 sec, over an irregular background activity. OT (0.1-1 microM), added to the medium, increased burst frequency, reducing interburst intervals by 70%. The peptide also triggered bursting in 27% of nonbursting neurons. These effects were mimicked by the oxytocin receptor (OTR) agonist [Thr4, Gly7]-OT and inhibited by the OTR antagonist desGly-NH2d(CH2)5[D-Tyr2,Thr4]OVT. Burst rhythmicity was independent of membrane potential. Hyperpolarization of the cells unmasked volleys of afferent EPSPs underlying the bursts, which were blocked by CNQX, an AMPA/kainate receptor antagonist. Our results reveal that OT neurons are part of a hypothalamic rhythmic network in which a glutamatergic input governs burst generation. OT neurons, in turn, exert a positive feedback on their afferent drive through the release of OT.

1. The adult hypothalamoneurohypophysial system (HNS) undergoes reversible morphological changes in response to physiological stimulation.

Glia and neurons of the hypothalamoneurohypophysial system (HNS) undergo reversible morphological changes, which are concomitant with the remodelling of afferents onto the neurons, under different conditions of neurohormone secretion. Here, we show that the adult rat HNS contains high levels of tenascin-C (TN-C), which is an extracellular matrix glycoprotein whose expression is usually associated with neuronal-glial interactions in the developing and lesioned central nervous system (CNS). By using light and electron microscopic immunocytochemical procedures, we visualized TN-C immunoreactivity in the hypothalamic supraoptic (SON) and paraventricular nuclei, where somata of the neurons are localized; in the median eminence, where their axons transit; and in the neurohypophysis, where they terminate. Hypothalamic areas adjacent to the magnocellular nuclei were devoid of immunoreactivity. Electron microscopy of the neurohypophysis showed immunolabelling of perivascular spaces, glial (pituicyte) and axonal surfaces, a type of labelling that also characterized the median eminence. In the hypothalamic nuclei, there was labelling of extracellular spaces and astrocytic surfaces. In normal animals, we detected no cytoplasmic reaction in glia somata, neurons, or endothelial cells. However, in animals treated with the intracellular transport blocker colchicine, there was intracytoplasmic labelling of all HNS glial cells, indicating a glial source for TN-C. Immunoblot analysis revealed TN-C isoforms of apparent high molecular weight (225, 240, and 260 kD) in the SON and median eminence, whereas lower MW forms (190/200 kD) predominated in the neurohypophysis. By using immunocytochemistry and immunoblot analysis, we found no visible differences in TN-C expression in relation to age, sex, or differing neurohypophysial secretion, which suggests that the expression of TN-C is a permanent feature of the HNS. J. Comp. Neurol. 379:386–398, 1997. © 1997 Wiley-Liss, Inc.

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 micron) plastic sections cut from vibratome slices (50 microns) 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.

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In order to investigate the role of central GABAB receptors in the control of the milk ejection reflex, we have examined the effects of third ventricular and bilateral supraoptic nucleus (SON) injections of a GABAB receptor agonist (baclofen) and antagonist (hydroxy-saclofen) on the milk ejection reflex in the urethane-anaesthetised rat. In addition, microdialysis studies have evaluated whether the activation of GABAB receptors in the SON is able to modulate the release of GABA and glutamate, two major neurotransmitters involved in the regulation of the milk ejection reflex. Intracerebroventricular injections of baclofen (n = 9) in doses of 10, 50 and 100 pmol inhibited the milk ejection reflex in a dose-dependent manner, without affecting the electroencephalogram or attenuating the intramammary pressure response to intravenous injection of 0.5 mU exogenous oxytocin. Hydroxy-saclofen given into the third ventricle in doses of 100 pmol (n = 2) and 500 pmol (n = 4) did not modify the pattern of the milk ejection reflex. Bilateral SON microinfusions of baclofen in doses of 80 (n = 2) and 200 pmol (n = 4) did not modify the pattern of the milk ejection reflex. In microdialysis experiments (n = 8), inclusion of baclofen into the microdialysate at a concentration of 500 microM had no effect upon basal or potassium-stimulated GABA and glutamate outflow. These results show that the activation of GABAB receptors located outside, but not within, the SON are capable of inhibiting the milk ejection reflex. In contrast to our previous findings regarding the GABAA receptor, we found no evidence for a tonic role of GABAB receptors within the neural network inducing the periodic synchronous bursting of oxytocin neurons during suckling.

Monolayer cultures from hypothalami of embryonic, fetal and postnatal rats were established to study the development of oxytocin-secreting neurons in vitro. Particular culture conditions, known to enhance the survival and growth of different types of neural cells in vitro, were used to investigate the conditions necessary for the appearance and survival of these peptide-producing cells in culture. They included increasing the concentration of potassium in the culture milieu and/or the addition of triiodothyronine (T(3) ). The use of immunocytochemical procedures with a monoclonal antibody that recognizes oxytocin-associated neurophysin (NP-OT) and polyclonal antibodies specific for oxytocin permitted us to identify the neurons. In cultures derived from embryonic (E16-E17) hypothalami, no NP-OT- or oxytocin-positive neurons were detected and their appearance could not be provoked by increasing extracellular potassium concentration or by administration of T(3) . On the other hand, in cultures obtained from fetal (E18-E19) rat hypothalami, a few neurons showed immunoreactivity for NP-OT (but not for oxytocin); the immunoreactivity was localized essentially in somata and proximal portions of neurites. When 10(8) M T(3) was included in the culture medium, there were cells showing immunoreactivity not only for NP-OT, but also for oxytocin, visible in somata and in dendritic- and axonal-like processes. In comparison, T(3) did not influence the total number of neurons developing in these cultures. Lastly, in cultures derived from young postnatal (PO-P2) rat hypothalami, NP-OT- and oxytocin-immunopositive neurons were found regularly; their appearance did not require any special pretreatment of the cultures. In all cultures, high extracellular potassium concentration (25 mM) resulted in a general improvement in the survival of neurons but did not induce the appearance of more oxytocin-immunoreactive cells. Our observations support in vivo results showing that although presumptive oxytocin-producing cells appear early in the development of the hypothalamus, their maturation, and in particular, their ability to produce oxytocin, occurs late at the time of birth. A factor that selectively enhances their differentiation, is the thyroid hormone, T(3) .

As c-fos expression is generally thought to be linked to neuronal activation, we compared Fos immunoreactivity in identified oxytocinergic and vasopressinergic neurons of female rats under various conditions known to elicit particular patterns of electrophysiological and secretory activity in these neurons. In suckled lactating animals, Fos immunoreactivity was visible only in rare oxytocinergic and vasopressinergic neurons of the paraventricular and supraoptic nuclei, even after interruption of suckling for 18-72 h. On the other hand, many Fos-positive cells were visible in the nuclei of parturient rats; they involved about 25% of supraoptic oxytocinergic elements. Even more Fos-positive elements were visible in the nuclei of lactating rats that had also undergone 24 h water deprivation or haemorrhage. This involved about 75% vasopressinergic neurons and 25% oxytocinergic neurons of the supraoptic nucleus. Fos immunoreactivity was particularly conspicuous in oxytocin neurons of the anterior commissural nucleus after haemorrhage. After water deprivation or haemorrhage, Fos-positive oxytocinergic neurons in the supraoptic nucleus were significantly more numerous in virgin rats than in lactating rats. Our observations show that suckling, although a most potent stimulus for oxytocin neuron activation and oxytocin release, is inefficient in inducing Fos synthesis in magnocellular neurons, even after a period of interruption. On the other hand, parturition, water deprivation and haemorrhage were more potent stimuli for both neurosecretory systems. However, under each type of stimulation, only part of the neuronal populations within each nucleus were Fos-positive, suggesting that different stimulus-specific pathways are involved in these regulations. In so far as electrical activity is one possible mechanism for c-fos expression, comparison of the patterns of c-fos activation with the known electrophysiological behaviour of hypothalamic magnocellular neurons suggests that Fos synthesis in these neurons is linked to the number of action potentials generated over a period of time, more than to the pattern of electrical activity, whatever the physiological impact of this pattern. Furthermore, within a group of neurons, the heterogeneity of the response in terms of Fos synthesis may be correlated to the variability of the electrophysiological response within this group.

In lactating rats, suckling elicits the milk ejection reflex which consists of an intermittent synchronous activation of hypothalamic oxytocin neurons which releases oxytocin into the bloodstream. We here investigated the electrophysiological behaviour of spinal cord neurons linked to mammary innervation in relation to suckling and the suckling-induced milk ejection reflex. Experiments were carried out on 58 urethane-anaesthetized rats, paralysed with gallamine triethiodide and artificially ventilated. Fixation of the spinal cord and laminectomy significantly slowed down the reflex, which occurred in only 27% of the animals. In these rats, 31 dorsal horn neurons at the thoraco-lumbar level were found to be excited by nipple stimulation. During suckling by a litter of at least nine pups, they displayed an irregular pattern of brief bursts of activity (peak firing rate 22.0 ± 3.2 Hz, mean ± SD) correlated to the bouts of suckling of the pups. Seventeen out of 19 cells tested by stimulation of at least 2 adjacent nipples received convergent input from different ipsilateral nipples. Out of 11 cells tested, 8 were also activated by stimulation of a contralateral nipple. Fourteen out of 30 units were recorded through at least one reflex milk ejection. Their firing rate was significantly higher than the firing rate of cells recorded in animals which failed to milk eject (4.4 Hz ± 2.8 versus 1.5 Hz ± 0.7). At the moment of the high frequency discharge of action potentials, occurring in oxytocinergic cells 10 to 15 s before each milk ejection, spinal neurons showed no systematic change in electrical activity. In contrast, the stretch reaction of the pups, which corresponds to an intense period of suckling when milk ejection has started, induced, in 12 cells, a considerable increase in electrical activity. One unit was found to be inhibited by suckling and during the stretch reaction. Ten more units, which were not activated by stimulation of the nipples but responded to stimulation of excitatory receptive fields near the last three pairs of nipples, were recorded through reflex milk ejections: 8 remained silent during reflex milk ejections but 2 were activated when the pups stimulated their excitatory receptive field. We conclude that some dorsal horn neurons, able to respond readily to the suckling movements of pups, appear to receive an ungated input from the nipples. At the time of the activation of oxytocin neurons, they present no particular pattern of activation or inhibition which could account in a simple manner for the intermittence of the high frequency discharge in oxytocinergic cells. However, in so far as these dorsal horn neurons may be part of the milk ejection reflex pathway, their activity, showing convergence and summation of input, and being facilitated in milk ejecting animals, indicates that the reflex does undergo a certain degree of processing at the spinal cord level.

In the nervous system, the neural cell adhesion molecule changes at the cell surface during development, from a form highly enriched in polysialic residues to several isoforms containing much less sialic acid, and is thought to participate in the structuring of neuronal groups and in the establishment of neuronal connections. Recent observations have indicated, however, that it may not be restricted to developing tissues since it is still present in certain adult neuronal centres which can undergo morphological reorganization. In this study, therefore, we examined systematically the distribution of polysialylated neural cell adhesion molecule immunoreactivity throughout the central nervous system of adult male and female rats, using light microscopic immunocytochemistry and immunoblot analysis with an antibody that specifically recognizes the polysialic residues of the molecule. Concomitantly, we compared this immunoreactivity to that due to all isoforms of the neural cell adhesion molecule, detected with a polyclonal serum raised against the NH2-terminal of the protein. Immunoreactivity due to the polysialylated isoform was consistently visualized in several discrete areas of the adult brain and spinal cord. An intercellular punctate immunolabelling characterized the staining in certain hypothalamic and thalamic nuclei, superficial laminae of the dorsal horn of the spinal cord, ventral portion of the dentate gyrus of the hippocampus, lateral geniculate, parabrachial and habenular nuclei, bed nucleus of the stria terminalis, mesencephalic central gray and olfactory bulb. In other areas, such as the piriform cortex, dorsal aspect of the dentate gyrus and fimbria and lamina X of the spinal cord, isolated neuronal-like cells were either completely filled with immunolabel or showed a surface reaction on their cell bodies and processes. Highly immunoreactive isolated glial-like cells were also noted within the ependymal layer of the central canal and lateral ventricles and at times in the peripheral white matter of the spinal cord. In contrast to this discrete localization, staining due to all isoforms of the neural cell adhesion molecule was widespread and diffuse throughout the brain and spinal cord. The expression of the polysialylated isoform in the supraoptic nucleus and hippocampus was confirmed by immunoblot analysis; it occurred together with weakly sialylated isoforms. No obvious differences were detected in the amount or distribution of immunoreactivity due to the polysialylated isoform in relation to the sex or age of the animals (between three and 12 months of age). Our study thus demonstrates that well-defined areas of the central nervous system of the adult rat continue to express the polysialylated isoform of the neural cell adhesion molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Poulain Dominique. Bernard Palissy : Sources du répertoire décoratif de l'atelier des Tuileries. In: Albineana, Cahiers d'Aubigné, 4, 1992. Bernard Palissy (1510-1590). L’écrivain, le réformé, le céramiste. pp. 187-200.

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 17beta-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.

The neural cell adhesion molecule, N-CAM, changes at the cell surface during development, from a highly sialylated form [polysialic acid (PSA)-linked N-CAM, PSA-N-CAM] to several isoforms containing less sialic acid. N-CAM and its polysialic acid may serve to regulate cell apposition, thus affecting a variety of cell interactions. In the nervous system, PSA-N-CAM has until now been localized in developing tissues where it is thought to participate in the structuring of neuronal groups and tissue pattern formation. It has been proposed, however, that PSA-N-CAM may also be expressed in the adult, where it may take part in plasticity and cell reshaping. In the present study, the use of immunoblot and immunocytochemical procedures with a monoclonal antibody that specifically recognizes PSA-N-CAM revealed that the adult rat hypothalamo-neurohypophysial system, which undergoes important neuronal-glial and synaptic rearrangements in response to physiological stimuli, contains high levels of PSA-N-CAM immunoreactivity. The use of a polyclonal serum reacting with all N-CAM isoforms indicated that PSA-N-CAM is expressed together with "adult" forms of N-CAM. Light and electron microscopy demonstrated the presence of PSA-N-CAM immunoreactivity in the supraoptic and paraventricular nuclei of the hypothalamus and in the neurohypophysis; the immunoreactivity was seen in dendrites, axons, and terminals and in associated astrocytes but not in neuronal somata. We propose that the continued expression of PSA-N-CAM confers to magnocellular neurons and their astrocytes the ability to reversibly change their morphology in adulthood. In addition, our observations suggest that evidence for polysialylation may serve to identify other neuronal systems capable of morphological plasticity in the adult central nervous system.

The possible role of the medial septum in the control of oxytocin release and of the milk ejection reflex induced by suckling was investigated in lactating rats by using electrical stimulations and lesions. In anaesthetized animals, brief electrical stimulation of the medial septum at 5 to 50 Hz elicited a single brief milk ejection similar to natural reflex milk ejections, whereas prolonged low frequency stimulations (5 to 10 Hz) induced a prolonged inhibition of the reflex. In acute experiments under anaesthesia, lesions of the medial septum did not impair the amplitude and pattern of reflex milk ejections. In chronic experiments, lesions of the medial septum resulted first in a loss of body weight of the mothers and a parallel reduction in growth of the litters. After a few days, the litters gained weight normally, and the pattern of milk ejections was normal. Thus, the pathways which pass through or originate from the medial septum and which are excitatory for oxytocin release appear not to be involved in the regulation of the milk ejection reflex. In view of these results and those from our previous study on the lateral septum, we conclude that the whole septum is not essential to the milk ejection reflex. However, the effects of septal stimulation suggest that the medial and lateral septum may be involved in a secondary neural circuitry that can inhibit the reflex.

The origin and function of type 2 astrocytes in the optic nerve are now well described, but there are few and controversial observations concerning their origin and functional significance in other regions of the mammalian brain. We here describe primary and high enriched secondary glial cultures obtained from postnatal (P 0 -P 6 ) rat hypothalami and cerebral cortices that included glial cells with morphological and immunocytochemical characteristics of type 2 astrocytes. The somata of such astrocytes were characteristically small and polygonal; they bore several processes with few branches. They were highly immunoreactive for glial fibrillary acidic protein and the surface antigens, A 2 B 5 and NSP 4 ; they were immunonegative for myelin basic protein and galactocerebroside. They grew on top of a continuous monolayer of much larger, flattened cells, that were glial fibrillary acidic protein-positive but A 2 B 5 - and NSP 4 -negative. In cultures derived from tissues younger than postnatal day 4, their appearance required the addition of adult (horse) serum to the culture millieu; they appeared spontaneously in cultures from older animals. Analysis of the origin of these cells, including experiments using tritiated thymidine incorporation, indicated that these astrocytes resulted from asymmetric divisions of the flat glial cells in the basal layer of the cultures. After their first appearance which varied according to the age of the source tissue, they were continuously generated, with a generation time no longer than 48 h; the life-span of individual cells was found to not exceed one week in neuron-free primary glial cultures. They displayed important process motility but did not show any significant migratory activity. The ready inducibility of glial cells showing many characteristics of type 3 astrocytes, in cultures derived from different brain areas, suggests that type 2-like astrocytes or their committed precursors are not restricted to particular neural structures, but are probably widely distributed within the mammalian brain. Their functional significance within the different brain areas remains to be determined.

Observations from different brain areas have established that the adult nervous system can undergo significant experience-related structural changes throughout life. Less familiar is the notion that morphological plasticity affects not only neurons but glial cells as well. Yet there is abundant evidence showing that astrocytes, the most numerous cells in the mammalian brain, are highly mobile. Under physiological conditions as different as reproduction, sensory stimulation, and learning, they display a remarkable structural plasticity, particularly conspicuous at the level of their lamellate distal processes that normally ensheath all portions of neurons. Distal astrocytic processes can undergo morphological changes in a matter of minutes, a remodeling that modifies the geometry and diffusion properties of the extracellular space and relationships with adjacent neuronal elements, especially synapses. Astrocytes respond to neuronal activity via ion channels, neurotransmitter receptors, and transporters on their processes; they transmit information via release of neuroactive substances. Where astrocytic processes are mobile then, astrocytic-neuronal interactions become highly dynamic, a plasticity that has important functional consequences since it modifies extracellular ionic homeostasis, neurotransmission, gliotransmission, and ultimately neuronal function at the cellular and system levels. Although a complete picture of intervening cellular mechanisms is lacking, some have been identified, notably certain permissive molecular factors common to systems capable of remodeling (cell surface and extracellular matrix adhesion molecules, cytoskeletal proteins) and molecules that appear specific to each system (neuropeptides, neurotransmitters, steroids, growth factors) that trigger or reverse the morphological changes.

The adult hypothalamo-neurohypophysial system undergoes a striking activity-dependent morphological remodelling that modifies the glial enwrapping of its magnocellular neurons. Although the functional consequences of such remodelling remain hypothetical, recent evidence has provided new insights into the repercussions of glial environment modifications on the physiology of magnocellular neurosecretory cells at the synaptic level. These studies have revealed that the reduced astrocytic coverage of magnocellular neurons occurring in the SON affects various functions in which astrocytes play key roles. These functions include uptake of neurotransmitters such as glutamate, restricting diffusion of neuroactive substances within the extracellular space and release of informative molecules known as gliotransmitters that act on neighbouring neurons to modulate synaptic transmission and excitability. Overall, our observations indicate that the neuron–glial anatomical reorganization leads to modifications of glutamatergic transmission that might be important for the physiology of the hypothalamo-neurohypophysial system.

Pre- and postembedding immunocytochemical procedures were used, together with antisera raised against oxytocin or its neurophysin, to characterize oxytocinergic pathways in the rat spinal cord, at the electron microscopic level. Pre-embedding immunoperoxidase staining performed on vibratome sections revealed oxytocin- and neurophysin-positive axonal profiles and terminals scattered predominantly in laminae I and II of the dorsal horn and in the central gray (lamina X). They were also visible, but to a lesser extent, in the intermediolateral columns, at thoracic and lumbar levels. Postembedding immunogold staining performed directly on ultrathin sections of the same areas, fixed in osmium and embedded in resin, permitted to show clearly that the oxytocinergic axons made symmetrical and asymmetrical synaptic contacts onto dendritic profiles. It also allowed subcellular localization of the neuropeptide immunoreactivities which were restricted to relatively large, electron-dense vesicles in the immunopositive terminals. Oxytocinergic terminals were never seen to participate in glomerular configurations in the superficial layers of the dorsal horn nor were immunoreactive cell bodies visible in any spinal area. Our results provide direct morphological evidence that oxytocinergic pathways make synapses in several regions of the spinal cord, thus supporting the contention that oxytocin may exert neurotransmitter/neuromodulator actions in this area of the CNS.

Activation of certain neurosecretory systems of the mammalian hypothalamus induces remodelling of the conformation of their neurons and glial cells. During stimulation of the hypothalamo-neurohypophysial system, astrocytic coverage of oxytocinergic somata and dendrites diminishes and their surfaces become extensively juxtaposed. In the neurohypophysis and median eminence, stimulation evokes a retraction of glial processes and an increase in the contact area between neurosecretory terminals and the perivascular space. These changes are reversible and glial coverage returns to normal upon cessation of stimulation. Neuronal-astrocytic rearrangements also occur in the arcuate nucleus in response to changes in sex steroid levels. The significance of such modifications is a matter of speculation. In the hypothalamic nuclei they may permit synaptic remodelling that takes place concurrently; in the neurohaemal structures they may facilitate neuropeptide release. We know little about the cellular mechanisms involved but glia and neurons of these systems express certain molecules implicated in cell-cell interactions in the developing central nervous system, such as the polysialylated isoform of the neural cell adhesion molecule; this may allow them to manifest their capacity for morphological plasticity in adulthood. The factors inducing the changes vary in the different structures: while oxytocin, in synergy with steroids, appears essential to the induction of the changes in the oxytocinergic system, oestrogen alone is critical in the arcuate nucleus; in the neurohypophysis noradrenaline appears important.

We here investigated inhibitory synapse turnover in the adult brain using the hypothalamic supraoptic nucleus where new synapses form during different physiological conditions, in particular on oxytocin neurons largely controlled by GABAergic inputs and locally released oxytocin. Patch clamp recordings and ultrastructural analysis of the nucleus in acute slices from late gestating rats showed that oxytocin and estrogen promoted rapid formation of inhibitory synapses. Thus, after 2-h exposure to a combination of oxytocin and 17-β estradiol, the frequency of miniature inhibitory postsynaptic currents was significantly enhanced. Since their amplitude and presynaptic GABA release probability were unmodified, this indicated an increased number of synapses. Electron microscopy confirmed increased densities of symmetric, putative GABAergic synapses within 2-h exposure to the peptide or steroid, effects which were reversible and oxytocin receptor mediated. Our observations thus offer direct evidence that hypothalamic GABAergic microcircuitries can undergo rapid and functional remodeling under changing neuroendocrine conditions. © 2006 Elsevier Inc. All rights reserved.

Under conditions of strong secretion of neurohypophysial hormone, such as during parturition, lactation and dehydration, the hypothalamic oxytocin-system displays a remarkable morphological plasticity such that astrocytic coverage of its neurones diminishes, their surfaces become directly juxtaposed and contacted by an increased number of synapses. A growing body of evidence indicates that these anatomical changes have an impact on glutamatergic neurotransmission in the supraoptic nucleus, and may be therefore of physiological consequence. We here evaluated the consequences of the inhibition of such plasticity on the overall activity of the oxytocin system during lactation. Remodeling was prevented by performing hypothalamic microinjections in gestating rats of endoneuraminidase, an enzyme that removes polysialic acid from the neural cell adhesion molecule. Our earlier studies established that the presence of polysialic acid is a prerequisite for remodeling of the oxytocin system in the supraoptic and paraventricular nuclei. In dams in which polysialic acid was absent in all magnocellular nuclei after bilateral endoneuraminidase injections, parturition was normal and neither the frequency nor the amplitude of suckling-induced reflex milk ejections was different from vehicle-treated dams. The weight gain of pups was also normal as was water intake by the dams. We then assessed the electrical activity of antidromically identified magnocellular neurones in the polysialic acid-free supraoptic nucleus of isoflurane-anesthetized lactating rats. Basal and bursting activity characteristic of oxytocin neurones before each reflex milk ejection was not significantly different from that recorded in the supraoptic nucleus of rats with normal levels of polysialic acid. Our results indicate that neuro-glial remodeling, despite its role on fine modulation of oxytocin neuronal activity, is not essential to parturition and lactation.

The morphology of axons and astrocytes in the neurohypophysis changes considerably during physiological stimulation, increasing neurovascular contact and facilitating neurosecretion. We here assessed the contribution of alpha2, 8-linked polysialic acid (PSA), which intervenes in axonal changes during development and covers all neurohypophysial axon and glial surfaces. Using an in vitro model, we first analyzed neurohypophysial ultrastructure under different conditions of plasticity. After 2 h incubation in hyperosmotic medium or with the beta-adrenergic agonist, isoprenaline, neurovascular contact significantly increased, due essentially to an enhanced number of terminals, and gliovascular contact decreased correspondingly. This morphology was maintained during 22 h exposure to isoprenaline and reversed 2 h after agonist washout. Removal of PSA from cell surfaces with endoneurominidase prevented stimulation-related induction and reversal of axon and glial changes but had no effect once remodeling had occurred. PSA, therefore, by promoting dynamic cell interactions, is necessary for plasticity of axons and their associated glia.

The supraoptic nucleus receives an abundant gamma-aminobutyric acid (GABA)ergic input which is inhibited by activation of various presynaptic metabotropic receptors. We here analysed the subtypes of voltage-gated Ca2+ channels intervening in the control of transmitter release at these synapses. To address this issue, we tested various specific inhibitors of Ca2+ channels on evoked inhibitory postsynaptic currents (IPSCs). Blocking N- and P-type voltage-gated Ca2+ channels with 1 micromomega-conotoxin-GVIA and 20 nmomega-agatoxin-IVA, respectively, dramatically reduced IPSC amplitude. Q- and L-type Ca2+ channels also contributed to GABAergic transmission, although to a lesser extent, as revealed by applications of 200 nmomega-agatoxin-IVA and of the dihydropyridines nifedipine (10 microm) and nimodipine (10 microm). Evoked IPSCs were insensitive to SNX-482 (300 nm), a blocker of some R-type Ca2+ channels. Analysis of selective blockade by the various antagonists suggested that multiple types of Ca2+ channels synergistically interact to trigger exocytosis at some individual GABA release sites. We next investigated whether inhibition of GABA release in response to the activation of metabotropic glutamate, GABA and adenosine receptors involved the modulation of these presynaptic Ca2+ channels. This was not the case, as the inhibitory actions of selective agonists of these receptors were unaffected by the presence of the different Ca2+ channel antagonists. This finding suggests that these metabotropic receptors modulate GABAergic transmission through a different mechanism, downstream of Ca2+ entry in the terminals, or upstream through the activation of K+ channels.

Astrocytes clear synaptically released glutamate from the extracellular space through high-affinity transporters present on their plasma membrane. By controlling the extracellular level of the main excitatory transmitter in the central nervous system, astrocytes thus contribute prominently to the regulation of overall cellular excitability and synaptic information processing. We recently investigated the influence of the glial environment on glutamatergic and GABAergic neurotransmission in the supraoptic nucleus of the rat hypothalamus under physiological conditions such as lactation that significantly reduce astrocytic coverage of its neurons. By performing electrophysiological analyses on this unique model of dynamic neuronal-glial interactions, we have been able to show that the fine astrocytic processes normally enwrapping synapses serve two important functions. First, they govern the level of activation of presynaptic metabotropic glutamate receptors on glutamatergic terminals, thereby regulating synaptic efficacy at excitatory synapses. Second, they act as a physical and functional barrier to diffusion in the extracellular space, limiting spillover of glutamate and other neuroactive substances and therefore contributing to the regulation of heterosynaptic transmission and intercellular communication.

The adult hypothalamo-neurohypophysial system (HNS) undergoes activity-dependent morphological plasticity which modifies astrocytic coverage of its oxytocinergic neurons and their synaptic inputs. Thus, during physiological conditions that enhance central and peripheral release of oxytocin (OT), adjacent somata and dendrites of OT neurons become extensively juxtaposed, without intervening astrocytic processes and receive an increased number of synapses. The morphological changes occur within a few hours and are reversible with termination of stimulation. The reduced astrocytic coverage has direct functional consequences since it modifies extracellular ionic homeostasis, synaptic transmission, and the size and geometry of the extracellular space. It also contributes indirectly to neuronal function by permitting formation of synapses on neuronal surfaces freed of astrocytic processes. Overall, such remodeling is expected to potentiate activated neuronal firing, especially in clusters of tightly packed neurons, an anatomical arrangement characterizing OT neurons. This plasticity connotes dynamic cell interactions that must bring into play cell surface and extracellular matrix adhesive proteins like those intervening in developing neuronal systems undergoing neuronal-glial and synaptogenic transformations. It is worth noting, therefore, that adult HNS neurons and glia continue to express such molecules, including polysialic acid (PSA)-enriched neural cell adhesion molecule (PSA-NCAM) and the glycoprotein, tenascin-C. PSA is a large, complex sugar on the extracellular domain of NCAM considered a negative regulator of adhesion; it occurs in large amounts on the surfaces of HNS neurons and astrocytes. Tenascin-C, on the other hand, possesses adhesive and repulsive properties; it is secreted by HNS astrocytes and occurs in extracellular spaces and on cell surfaces after interaction with appropriate ligands. These molecules have been considered permissive factors for morphological plasticity. However, because of their localization and inherent properties, they may also serve to modulate the extracellular environment and in consequence, synaptic and volume transmission in a system in which the extracellular compartment is constantly being modified.

The hypothalamic oxytocinergic system offers a remarkable model of morphological plasticity in the adult because its neurons and astrocytes undergo mutual remodelling in relation to differing physiological conditions. Among various factors involved in such plasticity, oxytocin (OT) itself appears of primary importance as its central administration resulted in morphological changes similar to those brought on by physiological stimuli. In the present study, we applied OT on acute hypothalamic slices from adult rats that included the supraoptic nucleus. Using ultrastructural morphometric analyses, we found that it induced a significant reduction of astrocytic coverage of OT neurons, leaving their surfaces directly juxtaposed, to an extent similar to that detected in vivo under conditions like lactation. These neuronal-glial changes were rapid and reversible, occurring within a few hours, and specifically mediated via OT receptors. They were potentiated by oestrogen and depended on calcium mobilization and de novo protein synthesis. Moreover, they depended on concurrent neuronal activation brought on by hyperosmotic stimulation or blockade of inhibitory GABAergic neurotransmission; they were inhibited by blockade of glutamatergic receptors. Taken together, our observations show that intrahypothalamic release of OT affects not only neuronal activation of the OT system but its morphological plasticity as well. Moreover, the activity dependence of the OT-induced changes strongly suggests that astrocytes can sense the level of activity of adjacent neurons and/or afferent input and this can subsequently act as a signal to bring on the neuronal and glial conformational changes.

During suckling, oxytocin (OT) neurons display a bursting electrical activity, consisting of a brief burst of action potentials which is synchronized throughout the OT neuron population and which periodically occurs just before each milk ejection in the lactating rat. To investigate the basis of such synchronization, we performed simultaneous intracellular recordings from pairs of OT neurons identified retrospectively by intracellular fluorescent labelling and immunocytochemistry in organotypic slice cultures derived from postnatal rat hypothalamus. A spontaneous bursting activity was recorded in 65% of OT neurons; the remaining showed only a slow, irregular activity. Application of OT triggered bursts in nonbursting neurons and accelerated bursting activity in spontaneously bursting cells. These cultures included rare vasopressinergic neurons showing no bursting activity and no reaction to OT. Bursts occurred simultaneously in all pairs of bursting OT neurons but, as in vivo, there were differences in burst onset, amplitude and duration. Coordination of firing was not due to electrotonic coupling because depolarizing one neuron in a pair had no effect on the membrane potential of its partner and halothane and proprionate did not desynchronize activity. On the other hand, bursting activity was superimposed on volleys of excitatory postsynaptic potentials (EPSPs) which occurred simultaneously in pairs of neurons. EPSPs, and consequently action potentials, were reversibly blocked by the non-NMDA glutamatergic receptor antagonist CNQX. Taken together, these data, obtained from organotypic cultures, strongly suggest that a local hypothalamic network governs synchronization of bursting firing in OT neurons through synchronous afferent volleys of EPSPs originating from intrahypothalamic glutamatergic inputs.

The mechanism by which dopamine induces or facilitates neurohypophysial hormone release is not completely understood. Because oxytocin- and vasopressin-secreting supraoptic neurons are under the control of a prominent GABAergic inhibition, we investigated the possibility that dopamine exerts its action by modulating GABA-mediated transmission. Whole cell voltage-clamp recordings of supraoptic neurons were carried out in acute hypothalamic slices to determine the action of dopamine on inhibitory postsynaptic currents. Application of dopamine caused a consistent and reversible reduction in the frequency, but not the amplitude, of miniature synaptic events, indicating that dopamine was acting presynaptically to reduce GABAergic transmission. The subtype of dopamine receptor involved in this response was characterized pharmacologically. Dopamine inhibitory action was greatly reduced by two highly selective D4 receptor antagonists L745,870 and L750,667 and to a lower extent by the antipsychotic drug clozapine but was unaffected by SCH 23390 and sulpiride, D1/D5 and D2/D3 receptor antagonists, respectively. In agreement with these results, the action of dopamine was mimicked by the potent D4 receptor agonist PD168077 but not by SKF81297 and bromocriptine, D1/D5 and D2/D3 receptor agonists, respectively. Dopamine and PD168077 also reduced the amplitude of evoked inhibitory postsynaptic currents, an effect that was accompanied by an increase in paired-pulse facilitation. These data clearly indicate that D4 receptors are located on GABA terminals in the supraoptic nucleus and that their activation reduces GABA release in the supraoptic nucleus. Therefore dopaminergic facilitation of neurohypophysial hormone release appears to result, at least in part, from disinhibition of magnocellular neurons caused by the depression of GABAergic transmission.

One of the functions of astroglial cells in the central nervous system is to clear synaptically-released glutamate from the extracellular space. This is performed thanks to specific transporters of the excitatory amino acid expressed on their surface. The way by which astrocytic glutamate uptake contributes to synaptic transmission has been investigated via numerous experimental approaches but has never been addressed under conditions where neuroglial interactions are physiologically modified. Recently, we took advantage of the neuroglial plastic properties of the hypothalamo-neurohypophysial system to examine the consequences of a physiological reduction in the astrocytic coverage of neurons on glutamatergic synaptic transmission. This experimental model has brought some insights on the physiological interactions between glial cells and neurons at the level of the synapse. In particular, it has revealed that the degree of glial coverage of neurons influences glutamate concentration at the vicinity of excitatory synapses and, as a consequence, affects the level of activation of presynaptic glutamate receptors. Astrocytes, therefore, appear to contribute to the regulation of neuronal excitability by modulating synaptic efficacy at glutamatergic nerve terminals.

Publisher Summary This chapter discusses the cell biology of oxytocin and vasopressin cells. Oxytocin (OT) and vasopressin (VP) are neuropeptides synthesized principally in the magnocellular neurons of the hypothalamus. They were the first neuropeptides whose peripheral hormonal actions were discovered, and whose source, mode of secretion, and chemical structure were characterized. The anatomy of the OT and VP cells, characterized by the compact localization of their somata and dendrites in the supraoptic nucleus (SON) and paraventricular nucleus (PVN), and the homogeneous projection of their axons to the neurohypophysis, has made it possible to determine the molecular and biochemical mechanisms of OT and VP synthesis and release into the bloodstream. The OT and VP neuronal populations, therefore, represent excellent models for the study of the state-dependent cell biology of neurosecretion.