Decay time in the presynaptic component action potential (fiber volley) was lengthened in the presence of OGD and chelator + ChTX.

<p>(A) Fiber volley recorded in the presence of BAPTA-AM + ChTX + OGD (solid line) and in presence of BAPTA-AM + ChTX only (dotted line). Fiber volleys were aligned by their negative peaks and superimposed to compare their amplitude and decay time. (B) Decay time of the fiber volley was increased in BAPTA-AM + ChTX + OGD condition versus BAPTA-AM + ChTX alone. It remained unchanged in control, OGD, BAPTA-AM and ChTX condition. (C) Amplitude of the fiber volley was unchanged in the presence of BAPTA-AM + ChTX and BAPTA-AM + ChTX + OGD, as well as control, OGD, BAPTA-AM and ChTX conditions. * p < 0.05, Student t-test</p

(A) Effect of calcium chelators and K channel antagonist, ChTX on fEPSP during OGD.

<p>1μM BAPTA-AM increases the amount of evoked neurotransmission remaining after 2 min (n = 7), 4 min (n = 6) and 6 min (n = 5) of ischemia relative to control (n = 7, 5, 5, respectively). Similarly, the amplitude of fEPSPs remaining after 2 min, 4 min, 6 min of OGD (n = 6) is increased after administration of 10 nM ChTX. Combining ChTX and BAPTA-AM led to almost no change in fEPSP amplitude up to 6 min of OGD (n = 6). <b>(</b>B) Effect of calcium chelators, and BK channel antagonist on recovery of fEPSP after OGD. BAPTA-AM (1 μM) decreases recovery time from 2 min (n = 7), 4 min (n = 6) and 6 min (n = 3) of <i>in vitro</i> OGD compared to control (n = 7, 5, 5, respectively). EGTA-AM (50μM) shows similar effects to BAPTA-AM (1μM) by decreasing time needed for electrophysiological recovery after 4 min of OGD (n = 6) but not after 6 min (n = 7). ChTX (10 nM) did not significantly decrease recovery time after 6 min of ischemia (n = 4) but a combination of BAPTA-AM (1 μM) and ChTX(10 nM) significantly decreased recovery time after 6 min of OGD (n = 6). <b>(</b>C) BAPTA-AM and BAPTA-AM + ChTX promote increased tissue resistance to a long ischemic episode. BAPTA-AM (1 μM) increases the amount of fEPSP remaining after 8 min of OGD and leads to full recovery after 40 min of reperfusion post ischemia (n = 3) when control tissue has surpassed the point of functional recovery (n = 4). A combination of BAPTA-AM (1 μM) and ChTX (10 nM) leads to almost no change in fEPSP amplitude after prolonged OGD and leads to almost full recovery after 40 min of reperfusion (n = 5). Data plotted as mean ± SE. *<i>p < 0</i>.<i>05</i>, ANOVA, all relative to control condition.</p

Intrinsic properties of the postsynaptic CA1 neurons did not change during transient OGD (n = 8).

<p>(A) Sample recording from a cell when constant current steps were applied via the patching electrode to quantify the spike and membrane properties from the hyperpolarizing and depolarizing voltage response. The current step protocol was used through all experiments when I-V curves were obtained. (B) 2–4 minutes of transient OGD did not cause significant changes in the transmembrane potential, threshold of firing action potential, and the size of AP. (C) 2–4 minutes of transient OGD did not cause significant changes in input resistance. * <i>p</i> < 0.05. mEPSC: miniature excitatory postsynaptic current</p

Presynaptic intracellular calcium fluorescence measurement from the CA1-stratum radiatum region using Ca Green-1.

<p>Calcium increases in the presynaptic terminal following OGD and is reduced by administration of cell- permeant calcium chelators (BAPTA-AM). (X40 N/A 0.8). A. High power images of axons locally loaded with Ca Green-1 (B) The effect of OGD on intracellular calcium in control (n = 5) and in the presence of 1 μM BAPTA-AM in presynaptic terminals (n = 6).</p

Ischemia depresses the amplitude of evoked synaptic transmission without changes in the presynaptic volley amplitude.

<p>(A). fEPSPs were recorded from the stratum radiatum in the CA1 region while the Schaffer collaterals were stimulated every 15 s. fEPEP amplitude decreased reversibly in a time-dependent manner after 2 min (n = 7), 4 min (n = 5), and 6 min (n = 5) of OGD. (B) fEPSP amplitudes produced by 2 min, 4 min, 6 min of <i>in vitro</i> ischemia relative to controls. (C) Fiber volley amplitude after 8 min of OGD (n = 6, p>0.05) Average plotted as mean ± SE. * p < 0.001: paired student t-test. OGD: Oxygen-glucose deprivation, fEPSP: field excitatory postsynaptic potentials.</p

ChTX channel blocker does not change synaptic transmission under baseline experimental conditions.

<p>(A) fEPSP amplitude did not significantly change during application of ChTX (10 μM). (B) Input/Output curve for fEPSP amplitude versus stimulation intensity for control and ChTX (10 μM) condition. (C) Paired-pulse ratios were not significantly different in the control and ChTX (10 μM) condition. (p>0.05)</p

Image_4_A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.JPEG

<p>Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.</p

Image_3_A Potential Compensatory Role of Panx3 in the VNO of a Panx1 Knock Out Mouse Model.JPEG

<p>Pannexins (Panx) are integral membrane proteins, with Panx1 being the best-characterized member of the protein family. Panx1 is implicated in sensory processing, and knockout (KO) animal models have become the primary tool to investigate the role(s) of Panx1 in sensory systems. Extending previous work from our group on primary olfaction, the expression patterns of Panxs in the vomeronasal organ (VNO), an auxiliary olfactory sense organ with a role in reproduction and social behavior, were compared. Using qRT-PCR and Immunohistochemistry (IHC), we confirmed the loss of Panx1, found similar Panx2 expression levels in both models, and a significant upregulation of Panx3 in mice with a global ablation of Panx1. Specifically, Panx3 showed upregulated expression in nerve fibers of the non-sensory epithelial layer in juvenile and adult KO mice and in the sensory layer of adults, which overlaps with Panx1 expression areas in WT populations. Since both social behavior and evoked ATP release in the VNO was not compromised in KO animals, we hypothesized that Panx3 could compensate for the loss of Panx1. This led us to compare Panx1 and Panx3 channels in vitro, demonstrating similar dye uptake and ATP release properties. Outcomes of this study strongly suggest that Panx3 may functionally compensate for the loss of Panx1 in the VNO of the olfactory system, ensuring sustained chemosensory processing. This finding extends previous reports on the upregulation of Panx3 in arterial walls and the skin of Panx1 KO mice, suggesting that roles of Panx1 warrant uncharacterized safeguarding mechanisms involving Panx3.</p