Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission.

Loss of energy supply to neurons during stroke induces a rapid loss of membrane potential that is called the anoxic depolarization. Anoxic depolarizations result in tremendous physiological stress on the neurons because of the dysregulation of ionic fluxes and the loss of ATP to drive ion pumps that maintain electrochemical gradients. In this review, we present an overview of some of the ionotropic receptors and ion channels that are thought to contribute to the anoxic depolarization of neurons and subsequently, to cell death. The ionotropic receptors for glutamate and ATP that function as ligand-gated cation channels are critical in the death and dysfunction of neurons. Interestingly, two of these receptors (P2X7 and NMDAR) have been shown to couple to the pannexin-1 (Panx1) ion channel. We also discuss the important roles of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in responses to ischemia. The central challenge that emerges from our current understanding of the anoxic depolarization is the need to elucidate the mechanistic and temporal interrelations of these ion channels to fully appreciate their impact on neurons during stroke.

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39Prolonged neurotransmitter release following synaptic stimulation extends the 40 time window for postsynaptic neurons to respond to presynaptic activity. This can 41 enhance excitability and increase synchrony of outputs, but the prevalence of this at 42 normally highly synchronous synapses is unclear. We show that the postsynaptic 43 channel, pannexin-1 (Panx1) regulates prolonged glutamate release onto CA1 neurons. (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/263640 doi: bioRxiv preprint first posted online Feb. 11, 2018; 3 Fast synaptic transmission, in contrast to stimulation-independent ongoing 63 spontaneous transmission, is characterized by the highly synchronous release of 64 neurotransmitter in response to presynaptic activation 1 . Asynchronous 65 neurotransmitter release while less well understood, is typically stimulation-dependent 66 and lasts hundreds of milliseconds. At excitatory synapses, asynchronous glutamate 67 release can elevate firing rates in response to presynaptic stimulation 2,3 , enhancing 68 coincidence detection 4 , and possibly promote spread of neurotransmitter 1 . Aberrant 69 asynchronous release may influence neurodegeneration 5 or promote epileptogenesis 6,7 . 70At GABAergic synapses asynchronous release is likely involved in periods of prolonged 71 inhibition [8][9][10] . All synapses show spontaneous release and most are specialized for either 72 synchronous or asynchronous release, although some switch from being predominantly 73 asynchronous to synchronous during development 1,4,11 . The demonstration that a 74 typically synchronous synapse can switch between predominant modes of release would 75 greatly expand their signalling capacity during physiology and pathology. 76Asynchronous release typically involves a presynaptic Ca 2+ permeable channel that 77 is likely distal from the release machinery, resulting in more prolonged intracellular Ca 2+ 78 rises compared with voltage dependent Ca 2+ channel activity and synchronous release 1,11 . 79 A common thread for these presynaptic Ca 2+ sources is that they are regulated by 80 extracellular ligands, such as ATP activation of P2X2 at CA1-interneuron synapses 12 and 81 endovanniloids for TRPV1 afferents of the nucleus tractus solitaris (NTS) 13,14 . The 82 prevailing view is that these messengers function as either anterograde (presynaptic 83 source), retrograde (postsynaptic source), or glial sources and it follows that the 84 endocannabinoids/endovanilloids must be removed rapidly from the synapse for signal 85 termination. 86All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/263640 doi: bioRxiv preprint first posted online Feb. 11, 2018; 4 Pannexin-1 (Panx1) are ion...