Enhanced astroglial Ca2+ signaling increases excitatory synaptic strength in the epileptic brain

The fine‐tuning of synaptic transmission by astrocyte signaling is crucial to CNS physiology. However, how exactly astroglial excitability and gliotransmission are affected in several neuropathologies, including epilepsy, remains unclear. Here, using a chronic model of temporal lobe epilepsy (TLE) in rats, we found that astrocytes from astrogliotic hippocampal slices displayed an augmented incidence of TTX‐insensitive spontaneous slow Ca²⁺ transients (STs), suggesting a hyperexcitable pattern of astroglial activity. As a consequence, elevated glutamate‐mediated gliotransmission, observed as increased slow inward current (SICs) frequency, up‐regulates the probability of neurotransmitter release in CA3‐CA1 synapses. Selective blockade of spontaneous astroglial Ca²⁺ elevations as well as the inhibition of purinergic P2Y1 or mGluR5 receptors relieves the abnormal enhancement of synaptic strength. Moreover, mGluR5 blockade eliminates any synaptic effects induced by P2Y1R inhibition alone, suggesting that the Pr modulation via mGluR occurs downstream of P2Y1R‐mediated Ca²⁺‐dependent glutamate release from astrocyte. Our findings show that elevated Ca²⁺‐dependent glutamate gliotransmission from hyperexcitable astrocytes up‐regulates excitatory neurotransmission in epileptic hippocampus, suggesting that gliotransmission should be considered as a novel functional key in a broad spectrum of neuropathological conditions. GLIA 2015;63:1507–1521     

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     

Neuron–astrocyte signaling is preserved in the aging brain

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca²⁺ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte–neuron signaling is derived from studies with young animals; however, the features of astrocyte–neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte–neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month‐old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter‐dependent intracellular Ca²⁺ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG‐induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte–neuron signaling in pathological conditions. Disruption of the astrocytic IP₃R2 mediated‐signaling, which is required for neurotransmitter‐induced astrocyte Ca²⁺ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte–neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca²⁺ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569–580     

Astrocyte‐mediated synapse remodeling in the pathological brain

Astrocytes, a major type of glia, reciprocally influence synaptic transmission and connectivity, forming the “tripartite synapses”. Astrocytic metabotropic glutamate receptor (mGluR)‐mediated Ca²⁺ waves and release of gliotransmitters or synaptogenic molecules mediate this neuron‐glia interaction in the developing brain, but this signaling has been challenged for adult brain. However, cumulative evidence has suggested that mature astrocytes exhibit re‐awakening of such immature phenotype in the pathological adult brain. This phenotypic change in astrocytes in response to injury may induce neural circuit and synapse plasticity. In this review article, we summarize astrocyte‐mediated synapse remodeling during physiological development, discuss re‐emergence of immature astrocytic signaling in adult pathological brain, and finally highlight its contribution to significant modification of synaptic connections correlating with functional progress of brain pathology.     

Intrinsic cellular and molecular properties of in vivo hippocampal synaptic plasticity are altered in the absence of key synaptic matrix molecules

Hippocampal synaptic plasticity comprises a key cellular mechanism for information storage. In the hippocampus, both long‐term potentiation (LTP) and long‐term depression (LTD) are triggered by synaptic Ca²⁺‐elevations that are typically mediated by the opening of voltage‐gated cation channels, such as N‐methyl‐d ‐aspartate receptors (NMDAR), in the postsynaptic density. The integrity of the post‐synaptic density is ensured by the extracellular matrix (ECM). Here, we explored whether synaptic plasticity is affected in adult behaving mice that lack the ECM proteins brevican, neurocan, tenascin‐C, and tenascin‐R (KO). We observed that the profiles of synaptic potentiation and depression in the dentate gyrus (DG) were profoundly altered compared to plasticity profiles in wild‐type littermates (WT). Specifically, synaptic depression was amplified in a frequency‐dependent manner and although late‐LTP (>24 hr) was expressed following strong afferent tetanization, the early component of LTP (<75 min post‐tetanization) was absent. LTP (>4 hr) elicited by weaker tetanization was equivalent in WT and KO animals. Furthermore, this latter form of LTP was NMDAR‐dependent in WT but not KO mice. Scrutiny of DG receptor expression revealed significantly lower levels of both the GluN2A and GluN2B subunits of the N‐methyl‐d ‐aspartate receptor, of the metabotropic glutamate receptor, mGlu5 and of the L‐type calcium channel, Ca_v1.3 in KO compared to WT animals. Homer 1a and of the P/Q‐type calcium channel, Ca_v1.2 were unchanged in KO mice. Taken together, findings suggest that in mice that lack multiple ECM proteins, synaptic plasticity is intact, but is fundamentally different.     

Effects of intravesicular loading of a Ca2+ chelator and depolymerization of actin fibers on neurotransmitter release in frog motor nerve terminals

Ca²⁺‐induced Ca²⁺ release (CICR) via type‐3 ryanodine receptor enhances neurotransmitter release in frog motor nerve terminals. To test a possible role of synaptic vesicle in CICR, we examined the effects of loading of EGTA, a Ca²⁺ chelator, into synaptic vesicles and depolymerization of actin fibers. Intravesicular EGTA loading via endocytosis inhibited the ryanodine sensitive enhancement of transmitter release induced by tetanic stimulation and the associated rises in intracellular‐free Ca²⁺ ([Ca²⁺]_i: Ca²⁺ transients). Latrunculin A, a depolymerizer of actin fibers, enhanced both spontaneous and stimulation‐induced transmitter release, but inhibited the enhancement of transmitter release elicited by successive tetanic stimulation. The results suggest a possibility that the activation of CICR from mobilized synaptic vesicles caused the enhancement of neurotransmitter release.     

Decoding glutamate receptor activation by the Ca2+ sensor protein hippocalcin in rat hippocampal neurons

Hippocalcin is a Ca²⁺‐binding protein that belongs to a family of neuronal Ca²⁺sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin‐YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor‐dependent depolarization and following Ca²⁺influx via voltage‐operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin‐YFP translocation in a dendritic shaft at the application site due to direct Ca²⁺influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin‐YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back‐propagating action potentials. The translocation to spines required Ca²⁺influx via synaptic NMDA receptors in which Mg²⁺ block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1–2 μm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site‐ and time‐specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.     

IP3 mobilization and diffusion determine the timing window of Ca2+ release by synaptic stimulation and a spike in rat CA1 pyramidal cells

Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP₃. Ca²⁺ release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca²⁺ waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca²⁺ release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca²⁺ release. The window, measured from the end of the train, lasted 250–500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP₃ concentration and [Ca²⁺]_i are elevated at the site of the IP₃ receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP₃ instead of synaptic stimulation, suggest that the most significant processes are the time for IP₃ to diffuse away from the site of generation and the time course of IP₃ production initiated by activation of mGluRs. IP₃ breakdown, desensitization of the IP₃ receptor, and the kinetics of IP₃ unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing‐dependent synaptic plasticity in these cells. © 2009 Wiley‐Liss, Inc.     

Mechanisms,pools, and sites of spontaneous vesicle release at synapses of rod and cone photoreceptors

Photoreceptors have depolarized resting potentials that stimulate calcium‐dependent release continuously from a large vesicle pool but neurons can also release vesicles without stimulation. We characterized the Ca²⁺ dependence, vesicle pools, and release sites involved in spontaneous release at photoreceptor ribbon synapses. In whole‐cell recordings from light‐adapted horizontal cells (HCs) of tiger salamander retina, we detected miniature excitatory post‐synaptic currents (mEPSCs) when no stimulation was applied to promote exocytosis. Blocking Ca²⁺ influx by lowering extracellular Ca²⁺, by application of Cd²⁺ and other agents reduced the frequency of mEPSCs but did not eliminate them, indicating that mEPSCs can occur independently of Ca²⁺. We also measured release presynaptically from rods and cones by examining quantal glutamate transporter anion currents. Presynaptic quantal event frequency was reduced by Cd²⁺ or by increased intracellular Ca²⁺ buffering in rods, but not in cones, that were voltage clamped at ?70 mV. By inhibiting the vesicle cycle with bafilomycin, we found the frequency of mEPSCs declined more rapidly than the amplitude of evoked excitatory post‐synaptic currents (EPSCs) suggesting a possible separation between vesicle pools in evoked and spontaneous exocytosis. We mapped sites of Ca²⁺‐independent release using total internal reflectance fluorescence (TIRF) microscopy to visualize fusion of individual vesicles loaded with dextran‐conjugated pHrodo. Spontaneous release in rods occurred more frequently at non‐ribbon sites than evoked release events. The function of Ca²⁺‐independent spontaneous release at continuously active photoreceptor synapses remains unclear, but the low frequency of spontaneous quanta limits their impact on noise.     

Osthole or imperatorin‐mediated facilitation of glutamate release is associated with a synaptic vesicle mobilization in rat hippocampal glutamatergic nerve endings

Osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, have previously been shown to facilitate depolarization‐evoked glutamate release from rat hippocampal nerve terminals by increasing voltage‐dependent Ca²⁺ entry. In this study, we further investigated whether osthole and imperatorin possess an action at the exocytotic machinery itself, downstream of a Ca²⁺ influx. Our data showed that ionomycin‐induced glutamate release and KCl‐evoked FM1‐43 release were facilitated by osthole and imperatorin, suggesting that some steps after Ca²⁺ entry are regulated by these two compounds. Consistent with this, osthole or imperatorin‐mediated facilitation of ionomycin‐induced glutamate release was occluded by cytochalasin D that inhibits actin polymerization, implying that the disassembly of cytoskeleton is involved. In addition, the facilitatory action of osthole or imperatorin on ionomycin‐induced glutamate release was attenuated by the Ca²⁺/calmodulin‐dependent kinase II (CaMKII) inhibitor KN62. Furthermore, Western blotting analysis further showed that osthole or imperatorin significantly increased ionomycin‐induced phosphorylation of CaMKII and synapsin I, the main presynaptic target of CaMKII. These results suggest, therefore, that osthole or imperatorin‐mediated facilitation of glutamate release involves modulation of downstream events controlling synaptic vesicle recruitment and exocytosis, possibly through an increase of CaMKII activation and synapsin I phosphorylation, thereby increasing synaptic vesicle availability for exocytosis. Synapse 64:390–396, 2010. © 2009 Wiley‐Liss, Inc.     

Heterosynaptic long‐term depression mediated by ATP released from astrocytes

Heterosynaptic long‐term depression (hLTD) at untetanized synapses accompanying the induction of long‐term potentiation (LTP) spatially sharpens the activity‐induced synaptic potentiation; however, the underlying mechanism remains unclear. We found that hLTD in the hippocampal CA1 region is caused by stimulation‐induced ATP release from astrocytes that suppresses transmitter release from untetanized synaptic terminals via activation of P2Y receptors. Selective stimulation of astrocytes expressing channelrhodopsin‐2, a light‐gated cation channel permeable to Ca²⁺, resulted in LTD of synapses on neighboring neurons. This synaptic modification required Ca²⁺ elevation in astrocytes and activation of P2Y receptors, but not N‐methyl‐D ‐aspartate receptors. Furthermore, blocking P2Y receptors or buffering astrocyte intracellular Ca²⁺ at a low level prevented hLTD without affecting LTP induced by SC stimulation. Thus, astrocyte activation is both necessary and sufficient for mediating hLTD accompanying LTP induction, strongly supporting the notion that astrocytes actively participate in activity‐dependent synaptic plasticity of neural circuits. © 2012 Wiley Periodicals, Inc.     

Calmodulin activity regulates group I metabotropic glutamate receptor‐mediated signal transduction and synaptic depression

Group I metabotropic glutamate receptors (mGluR), including mGluR1 and mGluR 5 (mGluR1/5), are coupled to Gq and modulate activity‐dependent synaptic plasticity. Direct activation of mGluR1/5 causes protein translation‐dependent long‐term depression (LTD). Although it has been established that intracellular Ca²⁺ and the Gq‐regulated signaling molecules are required for mGluR1/5 LTD, whether and how Ca²⁺ regulates Gq signaling and upregulation of protein expression remain unknown. Through pharmacological inhibition, we tested the function of the Ca²⁺ sensor calmodulin (CaM) in intracellular signaling triggered by the activation of mGluR1/5. CaM inhibitor N‐[4‐aminobutyl]‐5‐chloro‐2‐naphthalenesulfonamide hydrochloride (W13) suppressed the mGluR1/5‐stimulated activation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and p70‐S6 kinase 1 (S6K1) in hippocampal neurons. W13 also blocked the mGluR1/5 agonist‐induced synaptic depression in hippocampal slices and in anesthetized mice. Consistent with the function of CaM, inhibiting the downstream targets Ca²⁺/CaM‐dependent protein kinases (CaMK) blocked ERK1/2 and S6K1 activation. Furthermore, disruption of the CaM–CaMK–ERK1/2 signaling cascade suppressed the mGluR1/5‐stimulated upregulation of Arc expression. Altogether, our data suggest CaM as a new Gq signaling component for coupling Ca²⁺ and protein upregulation and regulating mGluR1/5‐mediated synaptic modification. © 2016 Wiley Periodicals, Inc.     

Modeling and measurement of vesicle pools at the cone ribbon synapse: Changes in release probability are solely responsible for voltage‐dependent changes in release

Postsynaptic responses are a product of quantal amplitude (Q), size of the releasable vesicle pool (N), and release probability (P). Voltage‐dependent changes in presynaptic Ca²⁺ entry alter postsynaptic responses primarily by changing P but have also been shown to influence N. With simultaneous whole cell recordings from cone photoreceptors and horizontal cells in tiger salamander retinal slices, we measured N and P at cone ribbon synapses by using a train of depolarizing pulses to stimulate release and deplete the pool. We developed an analytical model that calculates the total pool size contributing to release under different stimulus conditions by taking into account the prior history of release and empirically determined properties of replenishment. The model provided a formula that calculates vesicle pool size from measurements of the initial postsynaptic response and limiting rate of release evoked by a train of pulses, the fraction of release sites available for replenishment, and the time constant for replenishment. Results of the model showed that weak and strong depolarizing stimuli evoked release with differing probabilities but the same size vesicle pool. Enhancing intraterminal Ca²⁺ spread by lowering Ca²⁺ buffering or applying BayK8644 did not increase PSCs evoked with strong test steps, showing there is a fixed upper limit to pool size. Together, these results suggest that light‐evoked changes in cone membrane potential alter synaptic release solely by changing release probability. Synapse 70:1–14, 2016. © 2015 Wiley Periodicals, Inc.     

Dual regulation of Ca2+‐dependent glutamate release from astrocytes: Vesicular glutamate transporters and cytosolic glutamate levels

Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation‐induced Ca²⁺‐dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca²⁺‐dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca²⁺ increase. Inhibition of glutamine synthetase activity by L ‐methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca²⁺‐dependent glutamate release, without affecting intracellular Ca²⁺ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca²⁺‐dependent release of glutamate from astrocytes. © 2009 Wiley‐Liss, Inc.     

The inhibitory neurotransmitter GABA evokes long‐lasting Ca2+ oscillations in cortical astrocytes

Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca²⁺ response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser‐scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15‐20) and adult (P30‐60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca²⁺ oscillations. This response was mediated by GABA_B receptors and involved both G_i/o protein and inositol 1,4,5‐trisphosphate (IP₃) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABA_B receptor‐mediated Ca²⁺ elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long‐lasting Ca²⁺ oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch‐clamp recordings it was found that Ca²⁺ oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP₃R2^?/? mice, in which astrocytic GABA_B receptor‐mediated Ca²⁺ elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. GLIA 2016;64:363–373     

P2X7R‐mediated Ca2+‐independent d‐serine release via pannexin‐1 of the P2X7R‐pannexin‐1 complex in astrocytes

d ‐serine is a coagonist of N‐methyl‐d ‐aspartate (NMDA) subtype of glutamate receptor and plays a role in regulating activity‐dependent synaptic plasticity. In this study, we examined the mechanism by which extracellular ATP triggers the release of d ‐serine from astrocytes and discovered a novel Ca²⁺‐independent release mechanism mediated by P2X₇ receptors (P2X₇R). Using [³H] d ‐serine, which was loaded into astrocytes via the neutral amino acid transporter 2 (ASCT2), we observed that ATP and a potent P2X₇R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)adenosine‐5′‐triphosphate (BzATP), stimulated [³H]D‐serine release and that were abolished by P2X₇R selective antagonists and by shRNAs, whereas enhanced by removal of intracellular or extracellular Ca²⁺. The P2X₇R‐mediated d ‐serine release was inhibited by pannexin‐1 antagonists, such as carbenoxolone (CBX), probenecid (PBN), and ¹⁰Panx‐1 peptide, and shRNAs, and stimulation of P2X₇R induced P2X₇R‐pannexin‐1 complex formation. Simply incubating astrocytes in Ca²⁺/Mg²⁺‐free buffer also induced the complex formation, and that enhanced basal d ‐serine release through pannexin‐1. The P2X₇R‐mediated d ‐serine release assayed in Ca²⁺/Mg²⁺‐free buffer was enhanced as well, and that was inhibited by CBX. Treating astrocytes with general protein kinase C (PKC) inhibitors, such as chelerythrine, GF109203X, and staurosporine, but not Ca²⁺‐dependent PKC inhibitor, Gö6976, inhibited the P2X₇R‐mediated d ‐serine release. Thus, we conclude that in astrocytes, P2X₇R‐pannexin‐1 complex formation is crucial for P2X₇R‐mediated d ‐serine release through pannexin‐1 hemichannel. The release is Ca²⁺‐independent and regulates by a Ca²⁺‐independent PKC. The activated P2X₇R per se is also functioned as a permeation channel to release d ‐serine in part. This P2X₇R‐mediated d ‐serine release represents an important mechanism for activity‐dependent neuron‐glia interaction. GLIA 2015;63:877–893     

BDNF potentiates spontaneous Ca oscillations in cultured hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is thought to regulate neuronal plasticity in developing and matured neurons, although the molecular mechanisms are less well characterized. We monitored changes in the intracellular calcium (Ca²⁺) levels induced by BDNF using a fluorescence Ca²⁺ indicator (Fluo-3) by means of confocal laser microscopy in rat cultured hippocampal neurons. BDNF acutely potentiated spontaneous Ca²⁺ oscillations in dendrites and also in the soma of several neurons, although it increased intracellular Ca²⁺ in only selective proportion of resting neurons without Ca²⁺ oscillations. The potentiation was observed both in the frequency and the amplitude of Ca²⁺ oscillations, completely blocked by K-252a, and significantly reduced by 2-aminophosphonovaleric acid. These findings suggest that BDNF increases glutamate release and N-methyl-

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-aspartate (NMDA) channel-gated Ca²⁺ influx via TrkB and regulates the frequency and the amplitude of Ca²⁺ oscillations. BDNF may have the potential to modulate spontaneous Ca²⁺ oscillations to regulate neuronal plasticity in developing hippocampal neurons.     

Whisker experience‐dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex

Sensory experience‐dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience‐dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation‐induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience‐dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4–L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4–L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation‐induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker‐intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5‐trisphosphate (IP₃) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4–L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience‐dependent mGluR5‐IP₃ signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.     

Ionotropic and Metabotropic Glutamate Receptor Agonist-Induced [Ca2+]i Increase in Isolated Rat Supraoptic Neurons

In the present study, the effects of glutamate and of agonists for ionotropic and metabotropic glutamate receptors on intracellular Ca²⁺ concentration ([Ca²⁺]_i) were investigated in neurons of the rat supraoptic nucleus (SON). We used the intracellular Ca²⁺ imaging technique with fura-2, in single magnocellular neurons dissociated from the SON of rats. Glutamate (10^?6?10^?4 M) evoked a dose-dependent increase in [Ca²⁺]_i. The glutamate agonists exerted similar effects, although with some differences in the characteristics of their responses. The [Ca²⁺]_i response to NMDA was smaller than those of glutamate or the non-NMDA receptor agonists, AMPA and kainate, but was significantly enhanced by the removal of extracellular Mg²⁺. Glutamate, as well as quisqualate, an agonist for both ionotropic and metabotropic glutamate receptors, evoked a [Ca²⁺]_i increase in a Ca²⁺-free condition, suggesting Ca²⁺ release from intracellular Ca²⁺ stores. This was further evidenced by [Ca²⁺]_i increases in response to a more selective metabotropic glutamate receptor agonist, t-ACPD, in the absence of extracellular Ca²⁺. Furthermore, the quisqualate-induced Ca²⁺ release was abolished by the selective metabotropic glutamate receptor antagonist, (S)-4-carboxyphenylglycine. The results suggest that metabotropic glutamate receptors as well as non-NMDA and NMDA receptors are present in the SON neurons, and that activation of the first leads to Ca²⁺ release from intracellular Ca²⁺ stores and the activation of the latter two types induces Ca²⁺ entry. These dual mechanisms of Ca²⁺ signalling may play a role in the regulation of SON neurosecretory cells by glutamate.     

Deletion of Cav2.1(α1A) subunit of Ca2+‐channels impairs synaptic GABA and glutamate release in the mouse cerebellar cortex in cultured slices

Deletion of both alleles of the P/Q‐type Ca²⁺‐channel Ca_v2.1(α_1A) subunit gene in mouse leads to severe ataxia and early death. Using cerebellar slices obtained from 10 to 15 postnatal days mice and cultured for at least 3 weeks in vitro, we have analysed the synaptic alterations produced by genetically ablating the P/Q‐type Ca²⁺‐channels, and compared them with the effect of pharmacological inhibition of the P/Q‐ or N‐type channels on wild‐type littermate mice. Analysis of spontaneous synaptic currents recorded in Purkinje cells (PCs) indicated that the P/Q‐type channels play a prominent role at the inhibitory synapses afferent onto the PCs, with the effect of deleting Ca_v2.1(α_1A) partially compensated. At the granule cell (GC) to PC synapses, both N‐ and P/Q‐type Ca²⁺‐channels were found playing a role in glutamate exocytosis, but with no significant phenotypic compensation of the Ca_v2.1(α_1A) deletion. We also found that the P/Q‐ but not N‐type Ca²⁺‐channel is indispensable at the autaptic contacts between PCs. Tuning of the GC activity implicates both synaptic and sustained extrasynaptic γ‐aminobutyric acid (GABA) release, only the former was greatly impaired in the absence of P/Q‐type Ca²⁺‐channels. Overall, our data demonstrate that both P/Q‐ and N‐type Ca²⁺‐channels play a role in glutamate release, while the P/Q‐type is essential in GABA exocytosis in the cerebellum. Contrary to the other regions of the CNS, the effect of deleting the Ca_v2.1(α_1A) subunit is partially or not compensated at the inhibitory synapses. This may explain why cerebellar ataxia is observed at the mice lacking functional P/Q‐type channels.