Voltage-gated Calcium Influx Research Articles

Differential behaviors of calcium-induced calcium release in one dimensional dendrite by Nernst-Planck equation, cable model and pure diffusion model.

The source and dynamics of calcium is the key factor that regulates dendritic integration. Apart from the voltage-gated and ligand-gated calcium influx, an important source of calcium is from inner store of endoplasmic reticulum with a regenerative process of calcium-induced calcium release (CICR). To trigger this process, inositol 1,4,5-trisphosphate (IP3) and calcium are needed to satisfy certain requirements. The aim of our paper is to investigate how the CICR depends on the dynamics of membrane potential. We utilize one dimensional dendritic model to calculate membrane potential by Nernst-Planck Equation (NPE) and cable model and Pure Diffusion (PD) model, computational simulations are carried out to inject the calcium influx by synaptic stimulation and to predict subsequent CICR and calcium wave propagation. Our results demonstrate that CICR initiation and calcium wave propagation have much difference between electro-diffusion process of NPE and cable model. We find that cable model has lower threshold of IP3 stimulation to trigger CICR but is more difficult for calcium propagation than NPE, PD model requires even higher threshold of IP3 to initiate CICR process and calcium duration is shorter than NPE; the regenerative calcium wave propagates with faster speed in NPE than that in cable model and in PD model. Our work addresses the important role of electro-diffusion dynamics of charged ions in regulating CICR process in dendritic structure; and provides theoretical predictions for neurological process which requires sustaining calcium for downstream signaling processes.

GABABR silencing of nerve terminals.

Control of neurotransmission efficacy is central to theories of how the brain computes and stores information. Presynaptic G-protein coupled receptors (GPCRs) are critical in this problem as they locally influence synaptic strength and can operate on a wide range of time scales. Among the mechanisms by which GPCRs impact neurotransmission is by inhibiting voltage-gated calcium (Ca2+) influx in the active zone. Here, using quantitative analysis of both single bouton Ca2+ influx and exocytosis, we uncovered an unexpected non-linear relationship between the magnitude of action potential driven Ca2+ influx and the concentration of external Ca2+ ([Ca2+]e). We find that this unexpected relationship is leveraged by GPCR signaling when operating at the nominal physiological set point for [Ca2+]e, 1.2 mM, to achieve complete silencing of nerve terminals. These data imply that the information throughput in neural circuits can be readily modulated in an all-or-none fashion at the single synapse level when operating at the physiological set point.

Action potential-coupled Rho GTPase signaling drives presynaptic plasticity

In contrast to their postsynaptic counterparts, the contributions of activity-dependent cytoskeletal signaling to presynaptic plasticity remain controversial and poorly understood. To identify and evaluate these signaling pathways, we conducted a proteomic analysis of the presynaptic cytomatrix using in vivo biotin identification (iBioID). The resultant proteome was heavily enriched for actin cytoskeleton regulators, including Rac1, a Rho GTPase that activates the Arp2/3 complex to nucleate branched actin filaments. Strikingly, we find Rac1 and Arp2/3 are closely associated with synaptic vesicle membranes in adult mice. Using three independent approaches to alter presynaptic Rac1 activity (genetic knockout, spatially restricted inhibition, and temporal optogenetic manipulation), we discover that this pathway negatively regulates synaptic vesicle replenishment at both excitatory and inhibitory synapses, bidirectionally sculpting short-term synaptic depression. Finally, we use two-photon fluorescence lifetime imaging to show that presynaptic Rac1 activation is coupled to action potentials by voltage-gated calcium influx. Thus, this study uncovers a previously unrecognized mechanism of actin-regulated short-term presynaptic plasticity that is conserved across excitatory and inhibitory terminals. It also provides a new proteomic framework for better understanding presynaptic physiology, along with a blueprint of experimental strategies to isolate the presynaptic effects of ubiquitously expressed proteins.

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

Modulation of Voltage-Gated Calcium Influx by Electromagnetic Irradiation With Terahertz Gaussian Pulse

The voltage-gated calcium influxes modulated with terahertz Gaussian pulse is presented in this paper, and temperature rise is evaluated to monitor the terahertz thermal effects involved in the modulation. From the numerical results, inhibition effect on voltage-gated calcium influx because of the loss of the influx from electric drift is transparently revealed in the modulation with terahertz Gaussian pulse. And the inhibition effect becomes significant reflected by considerable reduction in voltage-gated calcium influx as the irradiated amplitude is raised intense enough. Compared to the terahertz sinusoidal wave, Gaussian pulse reduces the inhibition effect dramatically. The decrease in the increment of intracellular calcium concentration with the irradiated amplitude due to the inhibition effect is substantially relieved by the modulation of the calcium influx with terahertz Gaussian pulse. The thermal analyses reveal that the modulation is non-thermal effect. Meanwhile, to raise the intracellular calcium concentration to a same amount by means of the voltage-gated calcium influx, application of terahertz Gaussian pulse induces much less concurrent temperature rise than application of terahertz sinusoidal wave of the same amplitude that is intense enough to cause the inhibition effect. Additionally, terahertz Gaussian pulse irradiation remains the dependence of the increment of the calcium concentration on the irradiated time duration and frequency in terahertz sinusoidal wave irradiation. These numerical results lay the theoretical basis for the modulation of voltage-gated calcium fluxes with terahertz Gaussian pulse and for further potential clinical applications of terahertz Gaussian pulse.

2D+T Imaging of Calcium Signaling Microdomains in Cardiac Myocytes

Myocyte contraction is coupled with electrical depolarization via the process of excitation contraction coupling (ECC). During ECC, CM depolarization results in opening of voltage-gated Ca channels and calcium influx, which in turn triggers a larger calcium release via RyR calcium release channels on the sarcoplasmic reticulum (SR) store. Central to this process is the close apposition of the SR localized RyRs and plasma membrane voltage-gated calcium channels. This close coupling relies on membranous invaginations of the sarcolemma termed t-tubules, which ensure homogenous calcium elevations throughout the cytosol. In large mammals and in disease, t-tubules are not regularly distributed. Consequently, not all calcium release units (RyR clusters) are directly engaged by calcium influx, leading to potential inhomogeneities in cytosolic calcium. Here we optimized a multi-beam array confocal microscope to image signaling microdomains at high spatial (215 nm pixel size) and temporal resolution (125 fps) in intact pig myocytes. We fitted pixel-by-pixel the time evolution of calcium transients, extracted local key functional parameters and registered these pixel-based calcium changes with WGA stained cell membranes using a descriptor-based algorithm. When correlating functional parameters to the distance to closest membrane, we observed gradients in local calcium handling. Such differences were smaller during prolonged stimulation, suggesting a potential role for functional modulation, possibly through phosphorylation. We complemented live cells analysis with immunofluorescence staining of cells that were fixed after varying the duration of stimulation. We examined location and distribution of RyR clusters, their phosphorylation state and their distance to the closest membrane. We observed a site-specific RyR cluster density as well as the setting of local phosphorylation gradients that evolve with stimulation parameters and over time. Together, these data suggest that spatio-temporal patterns of phosphorylation contribute to the synchrony of calcium signals throughout the cell volume.

How chronic administration of benzodiazepines leads to unexplained chronic illnesses: A hypothesis

It is thought that an ill defined biochemical cascade may lead to protracted withdrawal symptoms subsequent to discontinuance of routine use of benzodiazepine class drugs and establish chronic illness in some patients. In this review, published findings are presented that support the novel concept that withdrawal from benzodiazepine class drugs can trigger elevated and sustained levels of a potent oxidant called peroxynitrite via potentiation of the L-type voltage-gated calcium channels, and in the later stages of withdrawal, via excessive N-methyl-D-aspartate receptor activity, as well. Potentiation of L-type voltage-gated calcium channels and excessive N-methyl-D-aspartate receptor activity both result in calcium influx into the cell that triggers nitric oxide synthesis. In pathophysiological conditions, such increased nitric oxide synthesis leads to peroxynitrite formation.The downstream effects of peroxynitrite formation that may occur during withdrawal ultimately lead to further peroxynitrite production in a system of overlapping vicious cycles collectively referred to as the NO/ONOO(−) cycle. Once triggered, the elements of the NO/ONOO(−) cycle perpetuate pathophysiology, perhaps including reduced GABAA receptor functioning, that may explain protracted withdrawal associated symptoms while the vicious cycle nature of the NO/ONOO(−) cycle may explain how withdrawal becomes a chronic state.Suboptimal levels of tetrahydrobiopterin may be one risk factor for the development of the protracted withdrawal syndrome as this will lead to partial nitric oxide uncoupling and resultant peroxynitrite formation. Nitric oxide uncoupling results in superoxide production as calcium-dependent nitric oxide synthases attempt to produce nitric oxide in response to L-type voltage-gated calcium channel-mediated calcium influx that is known to occur during withdrawal. The combination of nitric oxide and superoxide produced, as when partial uncoupling occurs, react together in a very rapid, diffusion limited reaction to form peroxynitrite and thereby trigger the NO/ONOO(−) cycle.The NO/ONOO(−) cycle may explain the nature of the protracted withdrawal syndrome and the related constellation of symptoms that are also common in other illnesses characterized as NO/ONOO(−) disorders such as myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia.

Synthesis and Biological Evaluations of NO-Donating Oxa- and Aza-Pentacycloundecane Derivatives as Potential Neuroprotective Candidates.

In order to utilize the neuroprotective properties of polycyclic cage compounds, and explore the NO-donating ability of nitrophenyl groups, an array of compounds was synthesized where the different nitrophenyl groups were appended on oxa and aza-bridged cage derivatives. Biological evaluations of the compounds were done for cytotoxicity, neuroprotective abilities, the inhibition of N-methyl-d-aspartate (NMDA)-mediated Ca2+ influx, the inhibition of voltage-mediated Ca2+ influx, and S-nitrosylation abilities. All of the compounds showed low toxicity. With a few exceptions, most of the compounds displayed good neuroprotection and showed inhibitory activity for NMDA-mediated and voltage-gated calcium influx, ranging from high (>70%) to low (20–39%) inhibition. In the S-nitrosylation assay, the compounds with the nitro moiety as the NO-donating group exhibited low to good nitrosylation potency compared to the positive controls. From the biological evaluation of the tested compounds, it was not possible to obtain a simple correlation that could explain the results across all of the biological study domains. This can be ascribed to the independent processes evaluated in the different assays, which reiterate that neuroprotection is a result of multifactorial biochemical mechanisms and interactions. However, these results signify the important aspects of the pentacylcoundecylamine neuroprotectants across different biological study realms.

Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion.

Gonadotrophs are basophilic cells of the anterior pituitary gland specialized to secrete gonadotropins in response to elevation in intracellular calcium concentration. These cells fire action potentials (APs) spontaneously, coupled with voltage-gated calcium influx of insufficient amplitude to trigger gonadotropin release. The spontaneous excitability of gonadotrophs reflects the expression of voltage-gated sodium, calcium, potassium, non-selective cation-conducting, and chloride channels at their plasma membrane (PM). These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the PM, as well as GABAA, nicotinic, and purinergic P2X channels gated by γ-aminobutyric acid (GABA), acetylcholine (ACh), and ATP, respectively. Activation of these channels leads to initiation or amplification of the pacemaking activity, facilitation of calcium influx, and activation of the exocytic pathway. Gonadotrophs also express calcium-conducting channels at the endoplasmic reticulum membranes gated by inositol trisphosphate and intracellular calcium. These channels are activated potently by hypothalamic gonadotropin-releasing hormone (GnRH) and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate cyclase-activating peptides, endothelins, ACh, vasopressin, and oxytocin. Activation of these channels causes oscillatory calcium release and a rapid gonadotropin release, accompanied with a shift from tonic firing of single APs to periodic bursting type of electrical activity, which accounts for a sustained calcium signaling and gonadotropin secretion. This review summarizes our current understanding of ion channels as signaling molecules in gonadotrophs, the role of GnRH and paracrine agonists in their gating, and the cross talk among channels.

GABA-induced uncoupling of GABA/benzodiazepine site interactions is mediated by increased GABAA receptor internalization and associated with a change in subunit composition

Persistent activation of GABAA receptors triggers compensatory changes in receptor function that are relevant to physiological, pathological and pharmacological conditions. Chronic treatment of cultured neurons with GABA for 48h has been shown to produce a down-regulation of receptor number and an uncoupling of GABA/benzodiazepine site interactions with a half-time of 24–25h. Down-regulation is the result of a transcriptional repression of GABAA receptor subunit genes and depends on activation of L-type voltage-gated calcium channels. The mechanism of this uncoupling is currently unknown. We have previously demonstrated that a single brief exposure of rat primary neocortical cultures to GABA for 5–10min (t½=3min) initiates a process that results in uncoupling hours later (t½=12h) without a change in receptor number. Uncoupling is contingent upon GABAA receptor activation and independent of voltage-gated calcium influx. This process is accompanied by a selective decrease in subunit mRNA levels. Here, we report that the brief GABA exposure induces a decrease in the percentage of α3-containing receptors, a receptor subtype that exhibits a high degree of coupling between GABA and benzodiazepine binding sites. Initiation of GABA-induced uncoupling is prevented by co-incubation of GABA with high concentrations of sucrose suggesting that it is dependent on a receptor internalization step. Moreover, results from immunocytochemical and biochemical experiments indicate that GABA exposure causes an increase in GABAA receptor endocytosis. Together, these data suggest that the uncoupling mechanism involves an initial increase in receptor internalization followed by activation of a signaling cascade that leads to selective changes in receptor subunit levels. These changes might result in the assembly of receptors with altered subunit compositions that display a lower degree of coupling between GABA and benzodiazepine sites. Uncoupling might represent a homeostatic mechanism that negatively regulates GABAergic transmission under physiological conditions in which synaptic GABAA receptors are transiently activated for several minutes.

CaRch1p does not functionally interact with the high‐affinity Ca2+ influx system (HACS) of Candida albicans

The plasma membrane protein CaRch1p of Candida albicans, homologous to the human solute carrier protein SLC10A7, is involved in the regulation of calcium homeostasis. C. albicans cells lacking CaRCH1 are hypersensitive to high extracellular Ca(2+) concentrations and show increased tolerance to ketoconazole (KCZ). We assume a higher basal Ca(2+) influx in the rch1/rch1 mutant strain at low extracellular Ca(2+) concentrations, which is not detrimental to C. albicans cells but may be sufficient to activate calcineurin, finally resulting in an increased tolerance to KCZ. However, at 8 µg/ml KCZ plus 3 mm Ca(2+) the rch1/rch1 mutant and the wild-type strains showed identical growth. By further increasing the Ca(2+) concentration to 30 mm, this phenotype was completely reversed and the rch1/rch1 mutant strain became extremely sensitive to 8 µg/ml KCZ, probably due to synergistic toxic effects of Ca(2+) and KCZ under these conditions. Furthermore, we aimed to clarify whether CaRch1p interacts with the Cch1p component of the voltage-gated calcium influx channel Cch1p/Mid1p in C. albicans cells. As disruption of the two alleles of CCH1 in the rch1/rch1 mutant strain did not alter its hypersensitivity to high extracellular Ca(2+) , and as this phenotype was completely abolished by low amounts of Mg(2+) in the rch1/rch1 mutant as well as in the cch1/cch1 rch1/rch1 double mutant, we conclude that CaRch1p is a functional component of the low-affinity calcium uptake system (LACS) system and does not functionally interact with Cch1p.

Deactivation of L-type Ca Current by Inhibition Controls LTP at Excitatory Synapses in the Cerebellar Nuclei

Long-term potentiation (LTP) of mossy fiber EPSCs in the cerebellar nuclei is controlled by synaptic inhibition from Purkinje neurons. EPSCs are potentiated by a sequence of excitation, inhibition, and disinhibition, raising the question of how these stimuli interact to induce plasticity. Here, we find that synaptic excitation, inhibition, and disinhibition couple to different calcium-dependent signaling pathways. In LTP induction protocols, constitutively active calcineurin can replace synaptic excitation, and constitutively active alpha-CaMKII can replace calcium influx associated with resumption of spiking upon disinhibition. Additionally, nimodipine can replace hyperpolarization, indicating that inhibition of firing decreases Ca influx through L-type Ca channels, providing a necessary signal for LTP. Together, these data suggest that potentiation develops after a calcineurin priming signal combines with an alpha-CaMKII triggering signal if and only if L-type Ca current is reduced. Thus, hyperpolarization induced by synaptic inhibition actively controls excitatory synaptic plasticity in the cerebellar nuclei.

Traumatic brain injury and the effects of diazepam, diltiazem, and MK-801 on GABA-A receptor subunit expression in rat hippocampus

BackgroundExcitatory amino acid release and subsequent biochemical cascades following traumatic brain injury (TBI) have been well documented, especially glutamate-related excitotoxicity. The effects of TBI on the essential functions of inhibitory GABA-A receptors, however, are poorly understood.MethodsWe used Western blot procedures to test whether in vivo TBI in rat altered the protein expression of hippocampal GABA-A receptor subunits α1, α2, α3, α5, β3, and γ2 at 3 h, 6 h, 24 h, and 7 days post-injuy. We then used pre-injury injections of MK-801 to block calcium influx through the NMDA receptor, diltiazem to block L-type voltage-gated calcium influx, or diazepam to enhance chloride conductance, and re-examined the protein expressions of α1, α2, α3, and γ2, all of which were altered by TBI in the first study and all of which are important constituents in benzodiazepine-sensitive GABA-A receptors.ResultsWestern blot analysis revealed no injury-induced alterations in protein expression for GABA-A receptor α2 or α5 subunits at any time point post-injury. Significant time-dependent changes in α1, α3, β3, and γ2 protein expression. The pattern of alterations to GABA-A subunits was nearly identical after diltiazem and diazepam treatment, and MK-801 normalized expression of all subunits 24 hours post-TBI.ConclusionsThese studies are the first to demonstrate that GABA-A receptor subunit expression is altered by TBI in vivo, and these alterations may be driven by calcium-mediated cascades in hippocampal neurons. Changes in GABA-A receptors in the hippocampus after TBI may have far-reaching consequences considering their essential importance in maintaining inhibitory balance and their extensive impact on neuronal function.

Effects of isoquinolonesulfonamides on action potential secretion coupling in pituitary cells.

Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high and steady prolactin release. Several intracellular proteins can mediate the action of calcium influx on prolactin secretion, including calmodulin-dependent protein kinases. Here, we studied effects of isoquinolonesulfonamides KN-62 and KN-93, calmodulin-dependent protein kinase inhibitors, and KN-92, an inactive analog, on spontaneous electrical activity, voltage-gated calcium influx, cyclic nucleotide production, and basal prolactin release. The effects of these compounds on electrical activity and calcium signaling was measured in single lactotrophs and cyclic nucleotide production and prolactin release were determined in static culture and perifusion experiments of anterior pituitary cells from postpubertal female rats. KN-62 and KN-93 blocked basal prolactin release in a dose- and time-dependent manner, suggesting that calmodulin-dependent protein kinase could mediate the coupling of electrical activity and secretion. However, a similar effect on basal prolactin release was observed on application of KN-92, which does not inhibit this kinase. KN-93 also inhibited cAMP and cGMP production, but inhibition of prolactin release was independent of the status of cyclic nucleotide production. Single cell measurements revealed abolition of spontaneous and depolarization-induced electrical activity and calcium transients in KN-92/93-treated cells, with a time course comparable to that observed in secretory studies. The results suggest that caution should be used when interpreting data from studies using isoquinolonesulfonamides to evaluate the role of calmodulin-dependent protein kinases in excitable endocrine cells, because inactive compounds exhibit comparable effects on action potential secretion coupling to those of active compounds.

Isradipine antagonizes hypobaric hypoxia induced CA1 damage and memory impairment: Complementary roles of L-type calcium channel and NMDA receptors

Hypobaric hypoxia leads to cognitive dysfunctions due to increase in intracellular calcium through ion channels. The purpose of this study was to examine the temporal contribution of L-type calcium channels and N-methyl-D-aspartate receptors (NMDARs) in mediating neuronal death in male Sprague Dawley rats exposed to hypobaric hypoxia simulating an altitude of 25,000 ft for different durations. Decreasing exogenous calcium loads by blocking voltage-gated calcium influx with isradipine (2.5 mg kg − 1 ), and its efficacy in providing neuroprotection and preventing memory impairment following hypoxic exposure was also investigated. Effect of isradipine on calcium-dependent enzymes mediating oxidative stress and apoptotic cell death was also studied. Blocking of L-type calcium channels with isradipine reduced hypoxia-induced activation of calcium dependent xanthine oxidases, monoamine oxidases, cytosolic phospholipase A 2 and cycloxygenases (COX-2) along with concomitant decrease in free radical generation and cytochrome c release. Increased expression of calpain and caspase 3 was also observed following exposure to hypobaric hypoxia along with augmented neurodegeneration and memory impairment which was adequately prevented by isradipine administration. Administration of isradipine during hypoxic exposure protected the hippocampal neurons following 3 and 7 days of exposure to hypobaric hypoxia along with improvement in spatial memory.

Expression of the SK2 calcium‐activated potassium channel is required for cholinergic function in mouse cochlear hair cells

Efferent inhibition of cochlear hair cells is mediated by 'nicotinic' cholinergic receptors functionally coupled to calcium-activated, small conductance (SK2) potassium channels. We recorded from cochlear hair cells in SK2 knockout mice to evaluate further the role of this channel in efferent function. Since cholinergic inhibitory synapses can be found on inner or outer hair cells, depending on developmental age, both cell types were studied. To determine if SK channel activity was indeed eliminated, seconds-long voltage-gated calcium influx was used to activate slowly rising and falling calcium-dependent potassium currents. These were identified as SK currents by their time course, calcium dependence and sensitivity to block by apamin in wild-type IHCs. IHCs from knockout mice had no SK current by these same criteria. Thus, the SK2 gene is solely responsible for encoding the SK channels of inner hair cells. Other aspects of hair cell excitability remained relatively unaffected. Unexpectedly, cholinergic synaptic currents were entirely absent from both inner and outer SK2-knockout hair cells. Further, direct application of ACh caused no change in membrane current, implying absent or otherwise dysfunctional ACh receptors. Immunohistology of whole-mounts using the antibody to the synaptic vesicle protein 2 (SV2) revealed a pronounced reduction of efferent innervation to outer hair cells (OHCs) in the knockout cochleas. Quantitative RT-PCR analysis, however, showed no change in the mRNA levels of alpha9 and alpha10 nicotinic ACh receptor (nAChR) genes. Thus, some aspect of translation or subsequent protein processing leads to non-functional or absent ACh receptors. These results indicate that SK2 channels are required both for expression of functional nAChRs, and for establishment and/or maintenance of efferent terminals in the cochlea.

Nerve growth factor inhibits Gd3+-sensitive calcium influx and reduces chemical anoxic neuronal death

To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca(2+)](i) was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 micromol/L MK-801 (glutamate receptor antagonist), 40 micromol/L CNQX (AMPA receptor antagonist) and 5 micromol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca(2+)](i) and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd(3+) (10 micromol/L) almost completely blocked KCN-mediated [Ca(2+)](i) elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca(2+)](i) increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 micromol/L) abolished NGF effects. It is concluded that Gd(3+)-sensitive calcium influx, which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd(3+)-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

L-type calcium channel blockade modifies anesthetic actions on aged hippocampal neurons

Previous studies in our laboratory demonstrated a reversal of anesthetic actions on aged neurons by decreasing extracellular [Ca2+] in hippocampal slices. Such maneuver indirectly attenuated Ca2+ influx, hence decreased exogenous intraneuronal Ca2+ loads during neuronal activity and consequently improved intracellular Ca2+ concentration homeostasis. Therefore, in the present study we hypothesized that decreasing exogenous Ca2+ loads by blocking voltage-gated calcium influx in aged neurons would oppose isoflurane actions. Conversely, increasing endogenous Ca2+ loads by suppressing calcium efflux during forced reversal of Na+/Ca2+ exchanger function would enhance anesthetic effects. Hippocampal slices were prepared from young (2–4 months) and old (24–26 months) Fischer 344 rats. Isoflurane depressed the evoked dendritic field excitatory postsynaptic potentials by approximately 45% in slices taken from old animals. However, application of isoflurane in addition with CoCl2 or nifedipine opposed the anesthetic actions, which then depressed the evoked dendritic field postsynaptic potentials by only 15%. Selective blockade of the N-type and P/Q-type calcium channels with ω-conotoxin GVIA and ω-conotoxin MVIIC respectively caused rapid but partial depression of synaptic transmission in slices taken from old Fischer 344 rats. However, isoflurane actions in these aged slices were not affected compared with slices perfused only with normal artificial cerebrospinal fluid. Young and aged slices were then exposed to a low sodium perfusate that forces the Na+/Ca2+ exchanger protein into a reverse mode, thus increasing intracellular Ca2+ concentration. Isoflurane actions under such conditions were profoundly potentiated in aged slices but were not altered in young hippocampi.The current results show that in aged central neurons, selectively blocking L-type calcium channels opposes anesthetic-induced depression of excitatory synaptic transmission. On the contrary, increasing calcium loads in aged neurons potentiates these actions.

Modeling synaptic transmission of the tripartite synapse

The tripartite synapse denotes the junction of a pre- and postsynaptic neuron modulated by a synaptic astrocyte. Enhanced transmission probability and frequency of the postsynaptic current-events are among the significant effects of the astrocyte on the synapse as experimentally characterized by several groups. In this paper we provide a mathematical framework for the relevant synaptic interactions between neurons and astrocytes that can account quantitatively for both the astrocytic effects on the synaptic transmission and the spontaneous postsynaptic events. Inferred from experiments, the model assumes that glutamate released by the astrocytes in response to synaptic activity regulates store-operated calcium in the presynaptic terminal. This source of calcium is distinct from voltage-gated calcium influx and accounts for the long timescale of facilitation at the synapse seen in correlation with calcium activity in the astrocytes. Our model predicts the inter-event interval distribution of spontaneous current activity mediated by a synaptic astrocyte and provides an additional insight into a novel mechanism for plasticity in which a low fidelity synapse gets transformed into a high fidelity synapse via astrocytic coupling.

ZD7288 inhibits exocytosis in an HCN-independent manner and downstream of voltage-gated calcium influx in pituitary lactotrophs

Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high prolactin release. Here we studied the role of hyperpolarization-activated cation channels in pacemaking activity, calcium signaling, and prolactin secretion in these cells. A slowly developing and hyperpolarization-activated inward current was identified but only in a fraction of lactotrophs. The current was blocked by ZD7288, a relatively specific blocker of these channels. However, the pacemaking activity increased in ZD7288-treated cells independently of the presence of this current. This in turn facilitated voltage-gated calcium influx and transiently stimulated prolactin secretion. Sustained ZD7288 application in concentrations that are commonly used to block the hyperpolarization-activated cation channels inhibited hormone release at elevated intracellular calcium concentrations. Agonist and Bay K 8644-stimulated prolactin release was also inhibited by ZD7288, indicating that this compound attenuates the exocytotic pathway downstream of calcium influx.