Spatial separation of two different pathways accounting for the generation of calcium signals in astrocytes.
ABSTRACT: Astrocytes integrate and process synaptic information and exhibit calcium (Ca2+) signals in response to incoming information from neighboring synapses. The generation of Ca2+ signals is mostly attributed to Ca2+ release from internal Ca2+ stores evoked by an elevated metabotropic glutamate receptor (mGluR) activity. Different experimental results associated the generation of Ca2+ signals to the activity of the glutamate transporter (GluT). The GluT itself does not influence the intracellular Ca2+ concentration, but it indirectly activates Ca2+ entry over the membrane. A closer look into Ca2+ signaling in different astrocytic compartments revealed a spatial separation of those two pathways. Ca2+ signals in the soma are mainly generated by Ca2+ release from internal Ca2+ stores (mGluR-dependent pathway). In astrocytic compartments close to the synapse most Ca2+ signals are evoked by Ca2+ entry over the plasma membrane (GluT-dependent pathway). This assumption is supported by the finding, that the volume ratio between the internal Ca2+ store and the intracellular space decreases from the soma towards the synapse. We extended a model for mGluR-dependent Ca2+ signals in astrocytes with the GluT-dependent pathway. Additionally, we included the volume ratio between the internal Ca2+ store and the intracellular compartment into the model in order to analyze Ca2+ signals either in the soma or close to the synapse. Our model results confirm the spatial separation of the mGluR- and GluT-dependent pathways along the astrocytic process. The model allows to study the binary Ca2+ response during a block of either of both pathways. Moreover, the model contributes to a better understanding of the impact of channel densities on the interaction of both pathways and on the Ca2+ signal.
Project description:D2 dopamine receptor-mediated suppression of synaptic transmission from interneurons plays a key role in neurobiological functions across species, ranging from respiration to memory formation. In this study, we investigated the mechanisms of D2 receptor-dependent suppression using soma-soma synapse between respiratory interneuron VD4 and LPeD1 in the mollusk Lymnaea stagnalis (L. stagnalis). We studied the effects of dopamine on voltage-dependent Ca2+ current and synaptic vesicle release from the VD4. We report that dopamine inhibits voltage-dependent Ca2+ current in the VD4 by both voltage-dependent and -independent mechanisms. Dopamine also suppresses synaptic vesicle release downstream of activity-dependent Ca2+ influx. Our study demonstrated that dopamine acts through D2 receptors to inhibit interneuron synaptic transmission through both voltage-dependent Ca2+ channel-dependent and -independent pathways. Taken together, these findings expand our understanding of dopamine function and fundamental mechanisms that shape the dynamics of neural circuit.
Project description:mGluR long-term depression (mGluR-LTD) is a form of synaptic plasticity induced at excitatory synapses by metabotropic glutamate receptors (mGluRs). mGluR-LTD reduces synaptic strength and is relevant to learning and memory, autism, and sensitization to cocaine; however, the mechanism is not known. Here we show that activation of Group I mGluRs in medium spiny neurons induces trafficking of GluA2 from the endoplasmic reticulum (ER) to the synapse by enhancing GluA2 binding to essential COPII vesicle proteins, Sec23 and Sec13. GluA2 exit from the ER further depends on IP3 and Ryanodine receptor-controlled Ca2+ release as well as active translation. Synaptic insertion of GluA2 is coupled to removal of high-conducting Ca2+-permeable AMPA receptors from synapses, resulting in synaptic depression. This work demonstrates a novel mechanism in which mGluR signals release AMPA receptors rapidly from the ER and couple ER release to GluA2 synaptic insertion and GluA1 removal.
Project description:Group I metabotropic glutamate receptors (mGluRs) increase cellular levels of inositol-1,4,5-triphosphate (IP3) and thereby trigger intracellular Ca2+ release. Also, group I mGluRs are organized with members of Homer scaffold proteins into multiprotein complexes involved in postreceptor signaling. In this study, we investigated the relative importance of the IP3/Ca2+ signaling and novel Homer proteins in group I mGluR-mediated activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured rat striatal neurons. We found that selective activation of mGluR5, but not mGluR1, increased ERK1/2 phosphorylation. Whereas the IP3/Ca2+ cascade transmits a small portion of signals from mGluR5 to ERK1/2, the member of Homer family Homer1b/c forms a central signaling pathway linking mGluR5 to ERK1/2 in a Ca2+-independent manner. This was demonstrated by the findings that the mGluR5-mediated ERK1/2 phosphorylation was mostly reduced by a cell-permeable Tat-fusion peptide that selectively disrupted the interaction of mGluR5 with the Homer1b/c and by small interfering RNAs that selectively knocked down cellular levels of Homer1b/c proteins. Furthermore, ERK1/2, when only coactivated by both IP3/Ca2+- and Homer1b/c-dependent pathways, showed the ability to phosphorylate two transcription factors, Elk-1 and cAMP response element-binding protein, and thereby facilitated c-Fos expression. Together, we have identified two coordinated signaling pathways (a conventional IP3/Ca2+ vs a novel Homer pathway) that differentially mediate the mGluR5-ERK coupling in neurons. Both the Ca2+-dependent and -independent pathways are corequired to activate ERK1/2 to a level sufficient to achieve the mGluR5-dependent synapse-to-nucleus communication imperative for the transcriptional regulation.
Project description:Astrocytic Ca2+-mediated gliovascular interactions regulate the neurovascular network in situ and in vivo. However, it is difficult to measure directly both the astrocytic activity and fMRI to relate the various forms of blood-oxygen-level-dependent (BOLD) signaling to brain states under normal and pathological conditions. In this study, fMRI and GCaMP-mediated Ca2+ optical fiber recordings revealed distinct evoked astrocytic Ca2+ signals that were coupled with positive BOLD signals and intrinsic astrocytic Ca2+ signals that were coupled with negative BOLD signals. Both evoked and intrinsic astrocytic calcium signal could occur concurrently or respectively during stimulation. The intrinsic astrocytic calcium signal can be detected globally in multiple cortical sites in contrast to the evoked astrocytic calcium signal only detected at the activated cortical region. Unlike propagating Ca2+ waves in spreading depolarization/depression, the intrinsic Ca2+ spikes occurred simultaneously in both hemispheres and were initiated upon the activation of the central thalamus and midbrain reticular formation. The occurrence of the intrinsic astrocytic calcium signal is strongly coincident with an increased EEG power level of the brain resting-state fluctuation. These results demonstrate highly correlated astrocytic Ca2+ spikes with bidirectional fMRI signals based on the thalamic regulation of cortical states, depicting a brain-state dependency of both astrocytic Ca2+ and BOLD fMRI signals.
Project description:Astrocytic Ca2+ signals can be fast and local, supporting the idea that astrocytes have the ability to regulate single synapses. However, the anatomical basis of such specific signaling remains unclear, owing to difficulties in resolving the spongiform domain of astrocytes where most tripartite synapses are located. Using 3D-STED microscopy in living organotypic brain slices, we imaged the spongiform domain of astrocytes and observed a reticular meshwork of nodes and shafts that often formed loop-like structures. These anatomical features were also observed in acute hippocampal slices and in barrel cortex in vivo. The majority of dendritic spines were contacted by nodes and their sizes were correlated. FRAP experiments and Ca2+ imaging showed that nodes were biochemical compartments and Ca2+ microdomains. Mapping astrocytic Ca2+ signals onto STED images of nodes and dendritic spines showed they were associated with individual synapses. Here, we report on the nanoscale organization of astrocytes, identifying nodes as a functional astrocytic component of tripartite synapses that may enable synapse-specific communication between neurons and astrocytes.
Project description:Long-term information storage in the brain requires continual modification of the neuronal transcriptome. Synaptic inputs located hundreds of micrometers from the nucleus can regulate gene transcription, requiring high-fidelity, long-range signaling from synapses in dendrites to the nucleus in the cell soma. Here, we describe a synapse-to-nucleus signaling mechanism for the activity-dependent transcription factor NFAT. NMDA receptors activated on distal dendrites were found to initiate L-type Ca2+ channel (LTCC) spikes that quickly propagated the length of the dendrite to the soma. Surprisingly, LTCC propagation did not require voltage-gated Na+ channels or back-propagating action potentials. NFAT nuclear recruitment and transcriptional activation only occurred when LTCC spikes invaded the somatic compartment, and the degree of NFAT activation correlated with the number of somatic LTCC Ca2+ spikes. Together, these data support a model for synapse to nucleus communication where NFAT integrates somatic LTCC Ca2+ spikes to alter transcription during periods of heightened neuronal activity.
Project description:The functional role of astrocyte calcium signaling in brain information processing was intensely debated in recent decades. This interest was motivated by high resolution imaging techniques showing highly developed structure of distal astrocyte processes. Another point was the evidence of bi-directional astrocytic regulation of neuronal activity. To analyze the effects of interplay of calcium signals in processes and in soma mediating correlations between local signals and the cell-level response of the astrocyte we proposed spatially extended model of the astrocyte calcium dynamics. Specifically, we investigated how spatiotemporal properties of Ca2+ dynamics in spatially extended astrocyte model can coordinate (e.g., synchronize) networks of neurons and synapses.
Project description:Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other neuronal processes. Here, we investigate the role of ionotropic and metabotropic glutamate receptor (iGluR and mGluR, respectively) activation on the generation of Ca2+ and diacylglycerol (DAG) signals and the subsequent activation of the neuron-specific PKCgamma isoform in hippocampal neurons. By combining Ca2+ imaging with total internal reflection microscopy analysis of specific biosensors, we show that elevation of both Ca2+ and DAG is necessary for sustained translocation and activation of EGFP (enhanced green fluorescent protein)-PKCgamma. Both DAG production and PKCgamma translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca2+ but no DAG signals, whereas mGluR activation generated DAG but no Ca2+ signals. Both receptors were needed for PKCgamma activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca2+ and DAG for PKCgamma activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca2+-sensitive PLCdelta (phospholipase Cdelta) isoform. In a second important exception, mGluRs were sufficient for PKCgamma activation in neurons in which Ca2+ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCgamma provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.
Project description:Activation of metabotropic glutamate receptors (mGluRs) causes membrane hyperpolarization in midbrain dopamine neurons. This hyperpolarization results from the opening of Ca(2+)-sensitive K(+) channels, which is mediated by the release of Ca(2+) from intracellular stores. Neurotransmitter-induced mobilization of Ca(2+) is generally ascribed to the action of inositol 1,4,5-triphosphate (IP(3)) in neurons. Here we show that the mGluR-mediated Ca(2+) mobilization in dopamine neurons is caused by two intracellular second messengers: IP(3) and cyclic ADP-ribose (cADPR). Focal activation of mGluRs, attained by synaptic release of glutamate or iontophoretic application of aspartate, induced a wave of Ca(2+) that spread over a distance of approximately 50 microm through dendrites and the soma. Simultaneous inhibition of both IP(3)- and cADPR-dependent pathways with heparin and 8-NH(2)-cADPR was required to block the mGluR-induced Ca(2+) release, indicating a redundancy in the signaling mechanism. Activation of ryanodine receptors was suggested to mediate the cADPR-dependent pathway, because ruthenium red, an antagonist of ryanodine receptors, inhibited the mGluR response only when the cADPR-dependent pathway was isolated by blocking the IP(3)-dependent pathway with heparin. Finally, the mGluR-mediated hyperpolarization was shown to induce a transient pause in the spontaneous firing of dopamine neurons. These results demonstrate that an excitatory neurotransmitter glutamate uses multiple intracellular pathways to exert an inhibitory control on the excitability of dopamine neurons.
Project description:Epileptic seizures are associated with increased astrocytic Ca2+ signaling, but the fine spatiotemporal kinetics of the ictal astrocyte-neuron interplay remains elusive. By using 2-photon imaging of awake head-fixed mice with chronic hippocampal windows we demonstrate that astrocytic Ca2+ signals precede neuronal Ca2+ elevations during the initial bout of kainate-induced seizures. On average, astrocytic Ca2+ elevations preceded neuronal activity in CA1 by about 8 s. In subsequent bouts of epileptic seizures, astrocytes and neurons were activated simultaneously. The initial astrocytic Ca2+ elevation was abolished in mice lacking the type 2 inositol-1,4,5-trisphosphate-receptor (Itpr2-/-). Furthermore, we found that Itpr2-/- mice exhibited 60% less epileptiform activity compared with wild-type mice when assessed by telemetric EEG monitoring. In both genotypes we also demonstrate that spreading depression waves may play a part in seizure termination. Our findings imply a role for astrocytic Ca2+ signals in ictogenesis.