Misa Arizono

Receptor-Selective Diffusion Barrier Enhances Sensitivity of Astrocytic Processes to Metabotropic Glutamate Receptor Stimulation

An mGluR5-selective diffusion barrier enriches mGluR5 in astrocytic processes, enabling compartmentalized calcium signaling. Keeping Calcium Signals in the Processes Although astrocytes, the most numerous form of glial cell in the brain, are electrically inexcitable, their ability to release chemical messengers and respond to such messengers with propagated calcium signals allows them to participate actively in the regulation of local blood flow and of synaptic efficacy. Here, Arizono et al. expressed a genetically encoded calcium indicator in neuron-astrocyte cocultures and hippocampal slices and found that, compared to the soma, astrocyte processes showed enhanced calcium responses to stimulation of the metabotropic glutamate receptor (mGluR). The enhanced calcium response observed in processes resulted from an increased density of mGluRs, rather than from differences in the distribution or sensitivity of the calcium release machinery. Analysis of the movement of single mGluR5s revealed a membrane barrier that selectively blocked the movement of mGluR5 between astrocyte somata and their processes. Noting that various neurological disorders are associated with abnormal calcium signaling in astrocytes, the authors speculate that the existence of this barrier—and thereby of compartmentalized calcium signals—could allow individual processes to regulate associated partners (synapses or blood vessels) independently, in the absence of a somatic calcium signal. Metabotropic glutamate receptor (mGluR)–dependent calcium ion (Ca2+) 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 Ca2+ signaling is not well characterized and its regulatory mechanism remains unclear. Using genetically encoded Ca2+ indicators, we showed that despite global stimulation by an mGluR agonist, astrocyte processes intrinsically exhibited a marked enrichment of Ca2+ responses. Immunocytochemistry indicated that these polarized Ca2+ 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 Ca2+ signaling. Together, our results demonstrate an mGluR5-selective diffusion barrier between processes and soma that compartmentalized mGluR Ca2+ signaling in astrocytes and may allow control of synaptic and vascular activity in specific subcellular domains.

Gephyrin-Independent GABAAR Mobility and Clustering during Plasticity

The activity-dependent modulation of GABA-A receptor (GABAAR) clustering at synapses controls inhibitory synaptic transmission. Several lines of evidence suggest that gephyrin, an inhibitory synaptic scaffold protein, is a critical factor in the regulation of GABAAR clustering during inhibitory synaptic plasticity induced by neuronal excitation. In this study, we tested this hypothesis by studying relative gephyrin dynamics and GABAAR declustering during excitatory activity. Surprisingly, we found that gephyrin dispersal is not essential for GABAAR declustering during excitatory activity. In cultured hippocampal neurons, quantitative immunocytochemistry showed that the dispersal of synaptic GABAARs accompanied with neuronal excitation evoked by 4-aminopyridine (4AP) or N-methyl-D-aspartic acid (NMDA) precedes that of gephyrin. Single-particle tracking of quantum dot labeled-GABAARs revealed that excitation-induced enhancement of GABAAR lateral mobility also occurred before the shrinkage of gephyrin clusters. Physical inhibition of GABAAR lateral diffusion on the cell surface and inhibition of a Ca2+ dependent phosphatase, calcineurin, completely eliminated the 4AP-induced decrease in gephyrin cluster size, but not the NMDA-induced decrease in cluster size, suggesting the existence of two different mechanisms of gephyrin declustering during activity-dependent plasticity, a GABAAR-dependent regulatory mechanism and a GABAAR-independent one. Our results also indicate that GABAAR mobility and clustering after sustained excitatory activity is independent of gephyrin.

Spatiotemporal calcium dynamics in single astrocytes and its modulation by neuronal activity

Astrocytes produce a complex repertoire of Ca2+ events that coordinate their major functions. The principle of Ca2+ events integration in astrocytes, however, is unknown. Here we analyze whole Ca2+ events, which were defined as spatiotemporally interconnected transient Ca2+ increases. Using such analysis in single hippocampal astrocytes in culture and in slices we found that spreads and durations of Ca2+ events follow power law distributions, a fingerprint of scale-free systems. A mathematical model demonstrated that such Ca2+ dynamics can arise from intracellular inositol-3-phosphate diffusion. The power law exponent (α) was decreased by activation of metabotropic glutamate receptors (mGluRs) either by specific receptor agonist or by low frequency stimulation of glutamatergic fibers in hippocampal slices. Decrease in α indicated an increase in proportion of large Ca2+ events. Notably, mGluRs activation did not increase the frequency of whole Ca2+ events. This result suggests that neuronal activity does not trigger new Ca2+ events in astrocytes (detectable by our methods), but modulates the properties of existing ones. Thus, our results provide a new perspective on how astrocyte responds to neuronal activity by changing its Ca2+ dynamics, which might further affect local network by triggering release of gliotransmitters and by modulating local blood flow.

Bidirectional Control of Synaptic GABAAR Clustering by Glutamate and Calcium

Summary 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.

Astrocytic IP3Rs: Contribution to Ca2+signalling and hippocampal LTP: Astrocytic IP3Rs: Ca2+Signalling and LTP

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 (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs 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 IP3Rs. GLIA 2017;65:502–513

Astrocytic IP3Rs: Contribution to Ca2+ signalling and hippocampal LTP

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 (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs 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 IP3Rs. GLIA 2017;65:502–513

Imaging mGluR5 Dynamics in Astrocytes Using Quantum Dots

This unit describes the method that we have developed to clarify endogenous mGluR5 (etabotropic tamate eceptors 5) dynamics in astrocytes by single‐particle tracking using quantum dots (QD‐SPT). QD‐SPT has been a powerful tool to examine the contribution of neurotransmitter receptor dynamics to synaptic plasticity. Neurotransmitter receptors are also expressed in astrocytes, the most abundant form of glial cell in the brain. mGluR5s, which evoke intracellular Ca2+ signals upon receiving glutamate, contribute to the modulation of synaptic transmission efficacy and local blood flow by astrocytes. QD‐SPT has previously revealed that the regulation of the lateral diffusion of mGluR5 on the plasma membrane is important for local Ca2+ signaling in astrocytes. Determining how mGluR5 dynamics are regulated in response to neuronal input would enable a better understanding of neuron‐astrocyte communication in future studies. Curr. Protoc. Neurosci. 66:2.21.1‐2.21.18.

Astrocytic IP3 Rs: Contribution to Ca(2+) signalling and hippocampal LTP.

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 (IP3R), however, genetic deletion of IP3R2, the putative astrocytic IP3R subtype, had no impact on synaptic plasticity or transmission. Although IP3R2 is widely believed to be the only functional IP3R in astrocytes, three IP3R subtypes (1, 2, and 3) have been identified in vertebrates. Therefore, to better understand gliotransmission, we investigated the functionality of IP3R and the contribution of the three IP3R subtypes to Ca2+ signalling. As a proxy for gliotransmission, we found that long‐term potentiation (LTP) was impaired by dialyzing astrocytes with the broad IP3R blocker heparin, and rescued by exogenous d‐serine, indicating that astrocytic IP3Rs regulate d‐serine release. To explore which IP3R subtypes are functional in astrocytes, we used pharmacology and two‐photon Ca2+ imaging of hippocampal slices from transgenic mice (IP3R2−/− and IP3R2−/−;3−/−). This approach revealed that underneath IP3R2‐mediated global Ca2+ events are an overlooked class of IP3R‐mediated local events, occurring in astroglial processes. Notably, multiple IP3Rs 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 IP3Rs. GLIA 2017;65:502–513