Despite the long-established presence of glutamate NMDA receptors at extrasynaptic sites (eNMDARs), their functional roles remain poorly understood. Factors influencing the concentration and time course of glutamate in the extrasynaptic space, such as the topography of the neuronal–glial microenvironment, as well as glial glutamate transporters, are expected to affect eNMDAR-mediated signalling strength. In this study, we used in vitro and in vivo electrophysiological recordings to assess the properties, functional relevance and modulation of a persistent excitatory current mediated by activation of eNMDARs in hypothalamic supraoptic nucleus (SON) neurons. We found that ambient glutamate of a non-synaptic origin activates eNMDARs to mediate a persistent excitatory current (termed tonic I(NMDA)), which tonically stimulates neuronal activity. Pharmacological blockade of GLT1 astrocyte glutamate transporters, as well as the gliotoxin α-aminodadipic acid, enhanced tonic I(NMDA) and neuronal activity, supporting an astrocyte regulation of tonic I(NMDA) strength. Dehydration, a physiological challenge known to increase SON firing activity and to induce neuroglial remodelling, including reduced neuronal ensheathment by astrocyte processes, resulted in blunted GLT1 efficacy, enhanced tonic I(NMDA) strength, and increased neuronal activity. Taken together, our studies support the view that glial modulation of tonic I(NMDA) activation contributes to regulation of SON neuronal activity, contributing in turn to neuronal homeostatic responses during a physiological challenge.

Key pointsr In hypothalamic magnocellular neurosecretory cells, activation of glutamate NMDA receptors leads to inhibition of the transient voltage-gated A-type K + current (I A ), in a Ca 2+ -and protein kinase C-dependent manner.r The negative NMDAR-I A functional coupling involves activation of extrasynaptic (e)NMDARs.The eNMDAR-I A coupling is engaged by endogenous extracellular glutamate, whose levels are controlled by astrocyte glutamate GLT1 transporters.r The eNMDAR-I A coupling is enhanced during dehydration, a condition in which astrocyte GLT1 efficiency is blunted.r The eNMDAR-I A coupling results in increased neuronal excitability and firing activity in magnocellular neurosecretory neurons.r Taken together these studies support the concept that the eNMDAR-I A coupling is a powerful mechanism by which glutamate increases magnocellular neurosecretory excitability and firing activity.Abstract Neuronal activity is controlled by a fine-tuned balance between intrinsic properties and extrinsic synaptic inputs. Moreover, neighbouring astrocytes are now recognized to influence a wide spectrum of neuronal functions. Yet, how these three key factors act in concert to modulate and fine-tune neuronal output is not well understood. Here, we show that in rat hypothalamic magnocellular neurosecretory cells (MNCs), glutamate NMDA receptors (NMDARs) are negatively coupled to the transient, voltage-gated A-type K + current (I A ). We found that activation of NMDARs by extracellular glutamate levels influenced by astrocyte glutamate transporters resulted in a significant inhibition of I A . The NMDAR-I A functional coupling resulted from activation of extrasynaptic NMDARs, was calcium-and protein kinase C-dependent, and involved enhanced steady-state, voltage-dependent inactivation of I A . The NMDAR-I A coupling diminished the latency to the first evoked spike in response to membrane depolarization and increased the total number of evoked action potentials, thus strengthening the neuronal input/output function. Finally, we found a blunted NMDA-mediated inhibition of I A in dehydrated rats. Together, our findings support a novel signalling mechanism that involves a functional coupling between extrasynaptic NMDARs and A-type K + channels, which is influenced by local astrocytes. We show this signalling complex to play an important role in modulating hypothalamic neuronal excitability, which may contribute to adaptive responses during a sustained osmotic challenge such as dehydration.

In addition to their peripheral vasoactive effects, accumulating evidence supports an important role for endothelins (ETs) in the regulation of the hypothalamic magnocellular neurosecretory system, which produces and releases the neurohormones vasopressin (VP) and oxytocin (OT). Still, the precise cellular substrates, loci and mechanisms underlying the actions of ETs on the magnocellular system are poorly understood. In the present study, we combined patch-clamp electrophysiology, confocal Ca(2+) imaging and immunohistochemistry to study the actions of ETs on supraoptic nucleus (SON) magnocellular neurosecretory neurones and astrocytes. Our studies show that ET-1 evoked rises in [Ca(2+) ](i) levels in SON astrocytes (but not neurones), an effect largely mediated by the activation of ET(B) receptors and mobilisation of thapsigargin-sensitive Ca(2+) stores. The presence of ET(B) receptors in SON astrocytes was also verified immunohistochemically. ET(B) receptor activation either increased (75%) or decreased (25%) SON firing activity, both in VP and putative OT neurones, and these effects were prevented when slices were preincubated in glutamate receptor blockers or nitric oxide synthase blockers, respectively. Moreover, ET(B) -mediated effects in SON neurones were also prevented by a gliotoxin compound, and when changes in [Ca(2+) ](i) were prevented with bath-applied BAPTA-AM or thapsigargin. Conversely, intracellular Ca(2+) chelation in the recorded SON neurones failed to block ET(B) -mediated effects. In summary, our results indicate that ET(B) receptor activation in SON astrocytes induces the mobilisation of [Ca(2+) ](i) , likely resulting in the activation of glutamate and nitric oxide signalling pathways, evoking in turn excitatory and inhibitory SON neuronal responses, respectively. Taken together, our study supports an important role for astrocytes in mediating the actions of ETs on the magnocellular neurosecretory system.

We showed that activation of extrasynaptic NMDARs (eNMDARs) by ambient glutamate under astrocytic control inhibited the voltage‐gated K+ current IA in neurosecretory vasopressin (VP) and PVN‐RVLM presympathetic neurons. Here, we aimed to address: i) whether the eNMDARs/IA coupling increases the firing activity of neurosecretory and presympathetic PVN neurons, and ii) whether this effect involves an imbalanced interaction with the opposing IT Ca2+ current. Patch clamp recordings were obtained from identified eGFP‐VP and PVN‐RVLM neurons. We found that activation of eNMDARs following blockade of astrocyte glutamate transporters (DHK 300μM) increased the firing activity of VP and PVN‐RVLM neurons in response to depolarizing pulses (~62%, P <0.05). This effect involved shorter latency to first spike, increased total number of action potentials, and duration of firing burst. These effects were prevented following IA blockade with 4AP (P>;0.2 in 4AP). In the presence of the IT channel blockade (mibefradil), the DHK excitatory effect was reduced to ~30%, P<0.05). Our studies indicate that activation of eNMDARs increases neuronal excitability of hypothalamic neurons by inhibiting IA function, an effect that is in part dependent on an enhanced activation of IT. Thus, the eNMDAR/IA coupling stands as a functionally relevant mechanism by which astrocytes influence hypothalamic neurohumoral outflow.

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