Abstract:
Synaptic plasticity is an extensively studied cellular correlate of learning and memory in which NMDARs play a starring role. One of the most interesting features of NMDARs is their ability to act as a co-incident detector. It is unique amongst neurotransmitter receptors in this respect. Co-incident detection is possible because the opening of NMDARs requires membrane depolarisation and the binding of glutamate. Opening of NMDARs also requires a co-agonist. Although the dynamic regulation of glutamate and membrane depolarization have been well studied in coincident detection, the role of the co-agonist site is unexplored. It turns out that non-neuronal glial cells, astrocytes, regulate co-agonist availability, giving them the ability to influence synaptic plasticity. The unique morphology and spatial arrangement of astrocytes at the synaptic level affords them the capacity to sample and integrate information originating from unrelated synapses, regardless of any pre-synaptic and post-synaptic commonality. As astrocytes are classically considered slow responders, their influence at the synapse is widely recognized as modulatory. The aim herein is to reconsider the potential of astrocytes to participate directly in ongoing synaptic NMDAR activity and co-incident detection.

Abstract:
Astrocytes are sensitive to ongoing neuronal/network activities and, accordingly, regulate neuronal functions (synaptic transmission, synaptic plasticity, behavior, etc.) by the context-dependent release of several gliotransmitters (e.g., glutamate, glycine, D-serine, ATP). To sense diverse input, astrocytes express a plethora of G-protein coupled receptors, which couple, via G(i/o) and G(q), to the intracellular Ca(2+) release channel IP(3)-receptor (IP(3)R). Indeed, manipulating astrocytic IP(3)R-Ca(2+) signaling is highly consequential at the network and behavioral level: Depleting IP(3)R subtype 2 (IP(3)R2) results in reduced GPCR-Ca(2+) signaling and impaired synaptic plasticity; enhancing IP(3)R-Ca(2+) signaling affects cognitive functions such as learning and memory, sleep, and mood. However, as a result of discrepancies in the literature, the role of GPCR-IP(3)R-Ca(2+) signaling, especially under physiological conditions, remains inconclusive. One primary reason for this could be that IP(3)R2 has been used to represent all astrocytic IP(3)Rs, including IP(3)R1 and IP(3)R3. Indeed, IP(3)R1 and IP(3)R3 are unique Ca(2+) channels in their own right; they have unique biophysical properties, often display distinct distribution, and are differentially regulated. As a result, they mediate different physiological roles to IP(3)R2. Thus, these additional channels promise to enrich the diversity of spatiotemporal Ca(2+) dynamics and provide unique opportunities for integrating neuronal input and modulating astrocyte-neuron communication. The current review weighs evidence supporting the existence of multiple astrocytic-IP(3)R isoforms, summarizes distinct sub-type specific properties that shape spatiotemporal Ca(2+) dynamics. We also discuss existing experimental tools and future refinements to better recapitulate the endogenous activities of each IP(3)R isoform.

Abstract:
Bidirectional communication between neurons and astrocytes shapes synaptic plasticity and behavior. D-serine is a necessary co-agonist of synaptic N-methyl-D-aspartate receptors (NMDARs), but the physiological factors regulating its impact on memory processes are scantly known. We show that astroglial CB1 receptors are key determinants of object recognition memory by determining the availability of D-serine at hippocampal synapses. Mutant mice lacking CB1 receptors from astroglial cells (GFAP-CB1-KO) displayed impaired object recognition memory and decreased in vivo and in vitro long-term potentiation (LTP) at CA3-CA1 hippocampal synapses. Activation of CB1 receptors increased intracellular astroglial Ca(2+) levels and extracellular levels of D-serine in hippocampal slices. Accordingly, GFAP-CB1-KO displayed lower occupancy of the co-agonist binding site of synaptic hippocampal NMDARs. Finally, elevation of D-serine levels fully rescued LTP and memory impairments of GFAP-CB1-KO mice. These data reveal a novel mechanism of in vivo astroglial control of memory and synaptic plasticity via the D-serine-dependent control of NMDARs.

Abstract:
KEY POINTS: Giant trypsin-containing endocytic vacuoles are formed in pancreatic acinar cells stimulated with inducers of acute pancreatitis. F-actin envelops endocytic vacuoles and regulates their properties. Endocytic vacuoles can rupture and release their content into the cytosol of acinar cells. Endocytic vacuoles can fuse with the plasma membrane of acinar cells and exocytose their content. ABSTRACT: Intrapancreatic activation of trypsinogen is an early event in and hallmark of the development of acute pancreatitis. Endocytic vacuoles, which form by disconnection and transport of large post-exocytic structures, are the only resolvable sites of the trypsin activity in live pancreatic acinar cells. In the present study, we characterized the dynamics of endocytic vacuole formation induced by physiological and pathophysiological stimuli and visualized a prominent actin coat that completely or partially surrounded endocytic vacuoles. An inducer of acute pancreatitis taurolithocholic acid 3-sulphate and supramaximal concentrations of cholecystokinin triggered the formation of giant (more than 2.5 mum in diameter) endocytic vacuoles. We discovered and characterized the intracellular rupture of endocytic vacuoles and the fusion of endocytic vacuoles with basal and apical regions of the plasma membrane. Experiments with specific protease inhibitors suggest that the rupture of endocytic vacuoles is probably not induced by trypsin or cathepsin B. Perivacuolar filamentous actin (observed on the surface of approximately 30% of endocytic vacuoles) may play a stabilizing role by preventing rupture of the vacuoles and fusion of the vacuoles with the plasma membrane. The rupture and fusion of endocytic vacuoles allow trypsin to escape the confinement of a membrane-limited organelle, gain access to intracellular and extracellular targets, and initiate autodigestion of the pancreas, comprising a crucial pathophysiological event.

Abstract:
Astrocytes regulate hippocampal synaptic plasticity by the Ca2+ dependent release of the N-methyl d-aspartate receptor (NMDAR) co-agonist d-serine. Previous evidence indicated that d-serine release would be regulated by the intracellular Ca2+ release channel IP3 receptor (IP3 R), however, genetic deletion of IP3 R2, the putative astrocytic IP3 R subtype, had no impact on synaptic plasticity or transmission. Although IP3 R2 is widely believed to be the only functional IP3 R in astrocytes, three IP3 R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3 R and the contribution of the three IP3 R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long-term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3 R blocker heparin, and rescued by exogenous d-serine, indicating that astrocytic IP3 Rs regulate d-serine release. To explore which IP3 R subtypes are functional in astrocytes, we used pharmacology and two-photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3 R2-/- and IP3 R2-/- ;3-/- ). This approach revealed that underneath IP3 R2-mediated global Ca2+ events are an overlooked class of IP3 R-mediated local events, occurring in astroglial processes. Notably, multiple IP3 Rs were recruited by high frequency stimulation of the Schaffer collaterals, a classical LTP induction protocol. Together, these findings show the dependence of LTP and gliotransmission on Ca2+ release by astrocytic IP3 Rs. GLIA 2017;65:502-513.

Abstract:
GABAergic synaptic transmission regulates brain function by establishing the appropriate excitation-inhibition (E/I) balance in neural circuits. The structure and function of GABAergic synapses are sensitive to destabilization by impinging neurotransmitters. However, signaling mechanisms that promote the restorative homeostatic stabilization of GABAergic synapses remain unknown. Here, by quantum dot single-particle tracking, we characterize a signaling pathway that promotes the stability of GABAA receptor (GABAAR) postsynaptic organization. Slow metabotropic glutamate receptor signaling activates IP3 receptor-dependent calcium release and protein kinase C to promote GABAAR clustering and GABAergic transmission. This GABAAR stabilization pathway counteracts the rapid cluster dispersion caused by glutamate-driven NMDA receptor-dependent calcium influx and calcineurin dephosphorylation, including in conditions of pathological glutamate toxicity. These findings show that glutamate activates distinct receptors and spatiotemporal patterns of calcium signaling for opposing control of GABAergic synapses.

Abstract:
Intracellular Ca2+ release is mostly mediated by inositol trisphosphate, but intracellular cyclic-ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are important messengers in many systems. Whereas cADPR generally activates type 2 ryanodine receptors (RyR2s), the NAADP-activated Ca2+ release mechanism is less clear. Using knockouts and antibodies against RyRs and Two-Pore Channels (TPCs), we have compared their relative importance for NAADP-induced Ca2+ release from two-photon permeabilized pancreatic acinar cells. In these cells, cholecystokinin-elicited Ca2+ release is mediated by NAADP. TPC2-KO reduced NAADP-induced Ca2+ release by 64%, but the combination of TPC2-KO and an antibody against TPC1, significantly reduced Ca2+ release by 86% (64% vs. 86%, p<0.0002). In RyR3-KO, NAADP-evoked Ca2+ release reduced by approximately 50% but, when combined with antibodies against RyR1, responses were 90% inhibited. Antibodies against RyR2 had practically no effect on NAADP-evoked Ca2+ release, but reduced release in response to cADPR by 55%. Antibodies to RyR1 inhibited NAADP-induced Ca2+ liberation by 81%, but only reduced cADPR responses by 30%. We conclude that full NAADP-mediated Ca2+ release requires both TPCs and RyRs. The sequence of relative importance for NAADP-elicited Ca2+ release from the all stores is RyR1>TPC2>RyR3>TPC1>>RyR2. However, when assessing NAADP-induced Ca2+ release solely from the acidic stores (granules/endosomes/lysosomes), antibodies against TPC2 and TPC1 virtually abolished the Ca2+ liberation as did antibodies against RyR1 and RyR3. Our results indicate that the primary, but very small, NAADP-elicited Ca2+ release via TPCs from endosomes/lysosomes triggers the detectable Ca2+-induced Ca2+ release via RyR1 and RyR3 occurring from the granules and the ER.

Abstract:
Metabotropic glutamate receptor (mGluR)-dependent calcium ion (Ca(2)+) signaling in astrocytic processes regulates synaptic transmission and local blood flow essential for brain function. However, because of difficulties in imaging astrocytic processes, the subcellular spatial organization of mGluR-dependent Ca(2)+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca(2)+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca(2)+ responses. Immunocytochemistry indicated that these polarized Ca(2)+ responses could be attributed to increased density of surface mGluR5 on processes relative to the soma. Single-particle tracking of surface mGluR5 dynamics revealed a membrane barrier that blocked the movement of mGluR5 between the processes and the soma. Overexpression of mGluR or expression of its carboxyl terminus enabled diffusion of mGluR5 between the soma and the processes, disrupting the polarization of mGluR5 and of mGluR-dependent Ca(2)+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca(2)+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

Abstract:
Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca(2)(+) entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP(3)Rs (InsP(3) receptors). The co-localization between Orai1 and IP(3)Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca(2)(+) store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca(2)(+) store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP(3)Rs and a basolateral pool that interacts with STIM1 following the Ca(2)(+) store depletion. Experiments on IP(3)R knockout animals demonstrated that the apical Orai1 localization does not require IP(3)Rs and that IP(3)Rs are not necessary for the activation of SOCE. However, the InsP(3)-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP(3)Rs could have an inhibitory effect on this Ca(2)(+) entry mechanism.

Abstract:
Alcohol abuse is a major global health problem, but there is still much uncertainty about the mechanisms of action. So far, the effects of ethanol on ion channels in the plasma membrane have received the most attention. We have now investigated actions on intracellular calcium channels in pancreatic acinar cells. Our aim was to discover the mechanism by which alcohol influences calcium homeostasis and thereby understand how alcohol can trigger premature intracellular trypsinogen activation, which is the initiating step for alcohol-induced pancreatitis. We used intact or two-photon permeabilized acinar cells isolated from wild-type mice or mice in which inositol trisphosphate receptors of type 2 or types 2 and 3 were knocked out. In permeabilized pancreatic acinar cells even a relatively low ethanol concentration elicited calcium release from intracellular stores and intracellular trypsinogen activation. The calcium sensor calmodulin (at a normal intracellular concentration) markedly reduced ethanol-induced calcium release and trypsinogen activation in permeabilized cells, effects prevented by the calmodulin inhibitor peptide. A calmodulin activator virtually abolished the modest ethanol effects in intact cells. Both ethanol-elicited calcium liberation and trypsinogen activation were significantly reduced in cells from type 2 inositol trisphosphate receptor knockout mice. More profound reductions were seen in cells from double inositol trisphosphate receptor (types 2 and 3) knockout mice. The inositol trisphosphate receptors, required for normal pancreatic stimulus-secretion coupling, are also responsible for the toxic ethanol action. Calmodulin protects by reducing calcium release sensitivity.