Kainate receptor-induced retrograde inhibition of glutamatergic transmission in vasopressin neurons.

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. We here report that applications of agonists of GluK1-containing KARs in the rat supraoptic nucleus has an opposite action on glutamatergic transmission according to the phenotype of the postsynaptic neuron. Whereas glutamate release was facilitated in oxytocin (OT) neurons, it was inhibited in vasopressin (VP) cells. Interestingly, an antagonist of GluK1-containing KARs caused an inhibition of glutamate release in both OT and VP neurons, revealing the existence of tonically activated presynaptic KARs that are positively coupled to transmitter release. We thus postulated that the inhibition of glutamate release observed with exogenous applications of GluK1 agonists on VP neurons could be indirect. In agreement with this hypothesis, we first showed that functional GluK1-containing KARs were present postsynaptically on VP neurons but not on OT cells. We next showed that the inhibitory effect induced by exogenous GluK1 receptor agonist was compromised when BAPTA was added in the recording pipette to buffer intracellular Ca2+ and block the release of a putative retrograde messenger. Under these conditions, GluK1-containing KAR agonist facilitates glutamatergic transmission in VP neurons in a manner similar to that observed for OT neurons and that resulted from the activation of presynaptic GluK1 receptors. GluK1-mediated inhibition of glutamate release in VP neurons was also blocked by a κ-opioid receptor antagonist. These findings suggest that activation of postsynaptic GluK1-containing KARs on VP neurons leads to the release of dynorphin, which in turn acts on presynaptic κ-opioid receptors to inhibit glutamate release.

Distribution of osmoregulatory peptides and neuronal-glial configuration in the hypothalamic magnocellular nuclei of desert rodents.

The desert rodents Psammomys obesus and Gerbillus tarabuli live under extreme conditions and overcome food and water shortage by modes of food and fluid intake specific to each species. Using immunohistochemistry and electron microscopy, we found that the hypothalamic magnocellular nuclei, and in particular, their vasopressinergic component, is highly and similarly developed in Psammomys and Gerbillus. In comparison to other rodents, the hypothalamus in both species contains more magnocellular VP neurons that, together with oxytocin neurons, accumulate in distinct and extensive nuclei. As in dehydrated rodents, many magnocellular neurons contained both neuropeptides. A striking feature of the hypothalamic magnocellular system of Psammomys and Gerbillus was its display of ultrastructural properties related to heightened neurosecretion, namely, a significant reduction in glial coverage of neuronal somata and dendrites in the hypothalamic nuclei. There were many neuronal elements whose surfaces were directly juxtaposed and shared the same synapses. Their magnocellular nuclei also showed a high level of sialylated isoform of the Neural Cell Adhesion Molecule (PSA-NCAM) that underlies their capacity for neuronal and glial plasticity. These species thus offer striking models of structural neuronal and glial plasticity linked to natural conditions of heightened neurosecretion.

Glia-dependent switch of kainate receptor presynaptic action.

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. This effect can be switched from facilitation to inhibition by an increased concentration of KAR agonists. We here report that activation of presynaptic GluK1-containing KARs facilitates GABA release on oxytocin and vasopressin neurons in the supraoptic nucleus of the hypothalamus. Increase in ambient levels of glutamate associated with the physiological reduction of astrocytic coverage of oxytocin neurons in lactating rats switches this KAR-mediated facilitation to inhibition of GABAergic transmission. This effect was reproduced in both oxytocin and vasopressin neurons of virgin rats when glutamate transporters were blocked pharmacologically, thereby establishing that enhanced levels of extracellular glutamate induce the switch in KAR-mediated action. The facilitation of GABA release was inhibited with philanthotoxin, a Ca(2+)-permeable KAR antagonist, suggesting that this effect was associated with an ionotropic mode of action. Conversely, KAR-mediated inhibition was compromised in the presence of U73122, a phospholipase C inhibitor, in agreement with the involvement of a metabotropic pathway. We thus reveal that physiological astrocytic plasticity modifies the mode of action of presynaptic KARs, thereby inversing their coupling with GABA release.

Polysialic acid and activity-dependent synapse remodeling.

Polysialic acid (PSA) is a large carbohydrate added post-translationally to the extracellular domain of the Neural Cell Adhesion Molecule (NCAM) that influences its adhesive and other functional properties. PSA-NCAM is widely distributed in the developing nervous system where it promotes dynamic cell interactions, like those responsible for axonal growth, terminal sprouting and target innervation. Its expression becomes restricted in the adult nervous system where it is thought to contribute to various forms of neuronal and glial plasticity. We here review evidence, obtained mainly from hypothalamic neuroendocrine centers and the olfactory system, that it intervenes in structural synaptic plasticity and accompanying neuronal-glial transformations, making possible the formation and elimination of synapses that occur under particular physiological conditions. While the mechanism of action of this complex sugar is unknown, it is now clear that it is a necessary molecular component of various cell transformations, including those responsible for activity-dependent synaptic remodeling.

Neuron-glia interactions in the rat supraoptic nucleus.

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.

Activity-dependent structural and functional plasticity of astrocyte-neuron interactions.

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.

Glia-derived D-serine controls NMDA receptor activity and synaptic memory.

The NMDA receptor is a key player in excitatory transmission and synaptic plasticity in the central nervous system. Its activation requires the binding of both glutamate and a co-agonist like D-serine to its glycine site. As D-serine is released exclusively by astrocytes, we studied the physiological impact of the glial environment on NMDA receptor-dependent activity and plasticity. To this end, we took advantage of the changing astrocytic ensheathing of neurons occurring in the supraoptic nucleus during lactation. We provide direct evidence that in this hypothalamic structure the endogenous co-agonist of NMDA receptors is D-serine and not glycine. Consequently, the degree of astrocytic coverage of neurons governs the level of glycine site occupancy on the NMDA receptor, thereby affecting their availability for activation and thus the activity dependence of long-term synaptic changes. Such a contribution of astrocytes to synaptic metaplasticity fuels the emerging concept that astrocytes are dynamic partners of brain signaling.

Remodeling of astrocytes, a prerequisite for synapse turnover in the adult brain? Insights from the oxytocin system of the hypothalamus.

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.

Oxytocin and estrogen promote rapid formation of functional GABA synapses in the adult supraoptic nucleus.

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-beta 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.

Polysialic acid is required for active phases of morphological plasticity of neurosecretory axons and their glia.

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.

Glial modulation of synaptic transmission: Insights from the supraoptic nucleus of the hypothalamus.

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.

Neuronal, glial and synaptic remodeling in the adult hypothalamus: functional consequences and role of cell surface and extracellular matrix adhesion molecules.

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.

GABAA receptor-expressing astrocytes in the supraoptic nucleus lack glutamate uptake and receptor currents.

An important function of astrocytes is the clearance of excess extracellular glutamate via specific carriers whose expression has become an astrocytic marker. In the present study, we found that a large population of astrocytes in the supraoptic nucleus (SON) of the rat hypothalamus lacks glutamate uptake currents and receptor responses but expresses GABAA receptors. Patch clamp recordings in acute hypothalamic slices that included the SON showed typical astrocytic membrane currents and demonstrated that GABA, via GABAA receptor activation, triggered a conductance increase with the reversal potential close to the Cl- equilibrium potential and a decrease in resting K+ conductance. Intracellular labeling with Lucifer Yellow revealed that these cells had a radial glia-like morphology, with cell bodies lined up along the base of the brain and long processes traversing the nucleus; they were not dye-coupled. Parallel immunocytochemical labelings showed that they expressed strong GABAA receptor and glial fibrillary acidic protein (GFAP) immunoreactivities. In addition, our electrophysiological and morphological analyses revealed another population of astrocytes in this nucleus, located next to the subarachnoid space. They were less numerous than the radial type, had a round morphology and few processes, and were dye-coupled. Unlike the radial astrocytes, they showed little immunoreactivity for GABAA receptor or GFAP. Moreover, they did not respond to GABA but to glutamate, a response that was partially mimicked by aspartate, indicating glutamate transporter expression. Taken together, our observations add to growing evidence illustrating heterogeneity of astrocytes in the adult brain, a heterogeneity that reflects striking differences in form and function of astrocytic populations in regions as discrete as the SON of the hypothalamus.

Induction of rapid, activity-dependent neuronal-glial remodelling in the adult rat hypothalamus in vitro.

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.

Glutamatergic input governs periodicity and synchronization of bursting activity in oxytocin neurons in hypothalamic organotypic cultures.

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.

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.

Neuronal-glial remodeling: a structural basis for neuronal-glial interactions in the adult hypothalamus.

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.

Oxytocin-secreting neurons: A physiological model of morphological neuronal and glial plasticity in the adult hypothalamus.

Oxytocin-secreting neurons of the hypothalamoneurohypophysial system undergo reversible morphological changes whenever they are strongly stimulated. In the hypothalamus, such structural plasticity is represented by modifications in the size and shape of their somata and dendrites, in the extent to which their surfaces are covered by glia, and in the density of their synapses. In the neurohypophysis, there is a parallel reduction in glial (pituicyte) coverage of their axons together, with retraction of pituicyte processes from the perivascular basal lamina and an increase in the number and size of their terminals. These changes occur rapidly, within a few hours. On the other hand, the system returns to its prestimulated condition on arrest of stimulation at a rate that depends on the length of time it has remained activated. Such neuronal-glial changes have several functional consequences. In the hypothalamic nuclei, reduction in astrocytic coverage of oxytocinergic neurons and their synapses modifies extracellular ionic homeostasis and glutamate clearance and, therefore, their overall excitability. Since it results in extensive dendritic bundling, it may also lead to ephaptic interactions and may facilitate dendritic electrotonic coupling. A most important indirect effect may be to permit synaptic remodeling that occurs concomitantly and that results in significant increases in the number of excitatory and inhibitory synapses driving their activity. In the stimulated neurohypophysis, glial retraction results in increased levels of extracellular K+ which can enhance neurohormone release while an enlarged neurovascular contact zone may facilitate diffusion of neurohormone into the circulation. Ongoing work aims to unravel the cell mechanisms and factors underlying such plasticity and has revealed that neurons and glia of the hypothalamoneurohypophysial system continue to express juvenile molecular features associated with similar neuronglial interactions and synaptic events during development and regeneration. They include strong expression of cell surface adhesion molecules like F3/contactin and polysialylated neural cell adhesion molecule, extracellular matrix glycoproteins like tenascin C, and cytoskeletal proteins like vimentin and microtubule-associated protein 1D. Some of these molecules reach the cell surface constitutively while others follow the activity-dependent regulated pathway. We consider many of these molecular features permissive, allowing oxytocin neurons and their glia to undergo morphological remodeling throughout life, provided the proper stimulus intervenes. In the hypothalamic nuclei, one such stimulus is centrally released oxytocin; in the neurohypophysis, an adrenergic, cAMP-mediated mechanism appears responsible.