The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

In the mammalian CNS, excessive release of glutamate and overactivation of glutamate receptors are responsible for the secondary (delayed) neuronal death following neuronal injury, including ischemia, traumatic brain injury (TBI) and epilepsy. The coupling of neurons by gap junctions (electrical synapses) increases during neuronal injury. We report here that the ischemic increase in neuronal gap junction coupling is regulated by glutamate via group II metabotropic glutamate receptors (mGluR). Specifically, using electrotonic coupling, western blots and siRNA in the mouse somatosensory cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs increases background levels of neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) and inactivation of group II mGluRs prevents the ischemia-mediated increases in the coupling and Cx36 expression. We also show that the regulation is via cAMP/PKA-dependent signaling and post-transcriptional control of Cx36 expression and that other glutamate receptors are not involved in these regulatory mechanisms. Further, using the analysis of neuronal death, we show that inactivation of group II mGluRs or genetic elimination of Cx36 both dramatically reduce ischemia-mediated neuronal death in vitro and in vivo. Similar results are obtained using in vitro models of TBI and epilepsy. Our results indicate that neuronal gap junction coupling is a critical component of glutamate-dependent neuronal death. They also suggest that causal link among group II mGluR function, neuronal gap junction coupling and neuronal death has a universal character and operates in different types of neuronal injuries.

Coupling of neurons by electrical synapses (gap junctions) transiently increases in the mammalian CNS during development. We report here that the developmental increase in neuronal gap junction coupling and expression of connexin 36 (Cx36; neuronal gap junction protein) are regulated by an interplay between the activity of group II metabotropic glutamate receptors (mGluR) and GABAA receptors. Specifically, using dye coupling, electrotonic coupling, western blots and siRNA in the rat and mouse hypothalamus and cortex in vivo and in vitro, we demonstrate that activation of group II mGluRs augments, and inactivation prevents, the developmental increase in neuronal gap junction coupling and Cx36 expression. However, changes in GABAA receptor activity have the opposite effects. The regulation by group II mGluRs is via cAMP/PKA-dependent signaling and by GABAA receptors is via Ca2+/PKC-dependent signaling. Further, the receptor-mediated up-regulation of Cx36 requires a neuron-restrictive silencer element in the Cx36 gene promoter and the down-regulation involves the 3′UTR of the Cx36 mRNA, as shown using RT-qPCR and luciferase reporter activity analysis. In addition, the MTT analysis indicates that mechanisms for the developmental increase in neuronal gap junction coupling directly control the death/survival mechanisms in developing neurons. Altogether, the results suggest a multi-tiered strategy for chemical synapses in developmental regulation of electrical synapses.

Naturally occurring cell death is a universal feature of developing nervous systems that plays an essential role in determining adult brain function. Yet little is known about the decisions that select a subset of CNS neurons for survival and cause others to die. We report that postnatal day 0 NMDA receptor subunit 1 (NMDAR1) knockout mice display an Ϸ2-fold increase in cell death in the brainstem trigeminal complex (BSTC), including all four nuclei that receive somatosensory inputs from the face (principalis, oralis, interpolaris, and caudalis). Treatment with the NMDA receptor antagonist dizocilpine maleate (MK-801) for 24 h before birth also caused an increase in cell death that reached statistical significance in two of the four nuclei (oralis and interpolaris). The neonatal sensitivity to NMDA receptor hypofunction in the BSTC, and in its main thalamic target, the ventrobasal nucleus (VB), coincides with the peak of naturally occurring cell death and trigeminothalamic synaptogenesis. At embryonic day 17.5, before the onset of these events, NMDAR1 knockout does not affect cell survival in either the BSTC or the VB. Immunostaining for active caspase-3 and the neuronal marker Hu specifically confirms the presence of dying neurons in the BSTC and the VB of NMDAR1 knockout neonates. Finally, genetic deletion of Bax rescues these structures from the requirement for NMDA receptors to limit naturally occurring cell death. Taken together, the results indicate that NMDA receptors play a survival role for somatosensory relay neurons during synaptogenesis by inhibiting Bax-dependent developmental cell death.brainstem ͉ neuroprotection ͉ sensory systems ͉ trophic ͉ ventrobasal N eurons in the peripheral nervous system avoid developmental cell death by successfully competing for a limiting supply of neurotrophins from synaptic target tissues (1). The situation in the developing CNS is less clear, where the survival-promoting action of neurotrophins on central neurons is complemented, facilitated, or replaced by other forms of support (2). A strong candidate for this role is the electrical activity that is present in developing neurons and neural circuits (3-5). Most neurons, including those in the somatosensory relay nuclei, express NMDA receptors before or just after exiting the cell cycle, well before synapses are established (6-10). Eliminating NMDA receptor function dramatically increases neuronal cell death during development (11-17), and NMDA receptor hypofunction has been proposed to play a causal role in fetal alcohol syndrome and schizophrenia (18,19). However, the biological significance and the molecular mechanisms of NMDA receptor-regulated neuronal survival in the intact brain remain largely unknown.NMDA receptors are best known for their role in synaptic plasticity. In the adult brain many forms of long-term potentiation and long-term depression require NMDA receptor function (20). During development, the refinement and plasticity of nascent synapses have also been shown to be dependent on NMDA receptors (2...

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