Postsynaptic IP3 receptor-mediated Ca2+ release modulates synaptic transmission in hippocampal neurons

Ca(2+)-dependent mechanisms are important in regulating synaptic transmission. The results herein indicate that whole-cell perfusion of inositol 1,4,5-trisphosphate receptor (IP(3)R) agonists greatly enhanced excitatory postsynaptic current (EPSC) amplitudes in postsynaptic hippocampal CA1 neurons. IP(3)R agonist-mediated increases in synaptic transmission changed during development and paralleled age-dependent increases in hippocampal type-1 IP(3)Rs. IP(3)R agonist-mediated increases in EPSC amplitudes were inhibited by postsynaptic perfusion of inhibitors of Ca(2+)/calmodulin, PKC and Ca(2+)/calmodulin-dependent protein kinase II. Postsynaptic perfusion of inhibitors of smooth endoplasmic reticulum (SER) Ca(2+)-ATPases, which deplete intracellular Ca(2+) stores, also enhanced EPSC amplitudes. Postsynaptic perfusion of the IP(3)R agonist adenophostin (AdA) during subthreshold stimulation appeared to convert silent to active synapses; synaptic transmission at these active synapses was completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Postsynaptic IP(3)R-mediated Ca(2+) release also produced a significant increase in spontaneous EPSC frequency. These results indicate that Ca(2+) release from intracellular stores play a key role in regulating the function of postsynaptic AMPARs.     

Endogenous inhibitors of InsP3-induced Ca2+ release in neuroblastoma cells

Cerebellar Purkinje neurons and neuroblastoma N1E-115 cells require 10-50 times more InsP3 to induce Ca2+ release than do a variety of non-neuronal cells (including astrocytes, hepatocytes, endothelial cells, or smooth muscle cells). Given the importance of InsP3-induced Ca2+ release for the development of synaptic plasticity in Purkinje neurons, a low InsP3 sensitivity may facilitate the integration of numerous synaptic inputs before initiating a change in synaptic strength. In the present study, attention is directed at the mechanism underlying this low InsP3 sensitivity of Ca2+ release. We show that permeabilization of neuroblastoma cells with saponin increased InsP3 sensitivity of Ca2+ release, indicating the presence of a diffusible, cytosolic inhibitor(s) of Ca2+ release. Consistent with this hypothesis, gel filtration of the neuroblastoma cytosol yielded three peaks that inhibited InsP3-induced Ca2+ release from permeabilized cells. The prominent inhibitory peak decreased the InsP3 sensitivity of Ca2+ release from permeabilized cells, did not bind 3H-InsP3, and was present in sufficient levels to account for the low InsP3 sensitivity of Ca2+ release in intact neuroblastoma cells. Purification of this prominent inhibitory fraction yielded a protein band that was identified by mass spectrometry as stress-induced phosphoprotein 1 (mSTI1). Furthermore, immunoprecipitation of mSTI1 decreased the inhibitory activity of N1E-115 cytosol, indicating that mSTI1 contributes to the inhibition of InsP3-induced Ca2+ release. Thus, the low InsP3 sensitivity of Ca2+ release in neuroblastoma cells can be explained by the presence of cytosolic inhibitors of Ca2+ release and include stress-induced phosphoprotein 1.     

Glutamate-induced increases in intracellular Ca2+ in cultured rat neocortical neurons

In neurons, changes in intracellular Ca2+ concentration ([Ca2+]i) trigger neurotransmitter release, regulate membrane excitability, affect gene expression, and govern short- and long-term forms of synaptic plasticity. Rises in cytoplasmic Ca2+ are thought to underlie the various effects of glutamatergic neurotransmitters within the central nervous system. In the present study, we applied a calcium imaging technique using a confocal laser scanning microscope to investigate the effects of excitatory amino acids on glutamate induced calcium influx in primary cultured neocortical neurons. Glutamate (5 microM) induced increases in [Ca2+]i in both the soma and dendritic processes of the cells. The increase was partially blocked by 10 microM DL-2-amino-5-phosphovaleric acid (APV), a NMDA antagonist. The reduction was higher in the dendritic process than in the cell body: the reduction was 58% in the cell body and 67% in the dendritic processes. In contrast, 5 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, had less effect on the response. We observed an 11% reduction in the dendritic processes, but no change in the cell bodies. The results demonstrated the heterogeneous distribution of APV- and CNQX-sensitive channels in primary cultured neocortical neurons. In both the cell body and dendritic processes, [Ca2+]i increase induced by low concentrations of glutamate was mainly due to the activation of NMDA receptors.     

Developmental regulation of action potential-induced Ca2+ entry in neocortical neurons.

Intracellular calcium plays a critical role in the regulation of membrane excitability, synaptic integration and synaptic plasticity. Using whole-cell recording and calcium imaging, we investigated intracellular calcium dynamics in apical dendrites of layer V pyramidal cells in the immature rat neocortex. Dendritic calcium increases induced by action potentials were only small on postnatal days 4-6 (P4-6), then underwent a gradual enhancement during the second post-natal week and were 2- to 3-fold larger on P16-18 than on P4-6. At P15-18 there were no significant differences in the calcium increases among the three subclasses of layer V pyramidal neurons: adapting regular spiking (RS), non-adapting RS and intrinsically bursting (IB) neurons. Developmental regulation of dendritic calcium dynamics may be crucial for functional maturation of the neocortex.     

Long-term potentiation of transmitter exocytosis expressed by Ca2+-induced Ca2+ release from thapsigargin-sensitive Ca2+ stores in preganglionic nerve terminals

We have studied whether Ca(2+)-induced Ca(2+) release (CICR) is involved in the mechanism of long-term potentiation (LTP) at nicotinic synapses of bullfrog sympathetic ganglia. Fast excitatory postsynaptic potentials (fast EPSPs) were recorded in a low-Ca(2+), high-Mg(2+) solution and quantal analysis was applied. The conditioning stimulation of the B-type preganglionic nerve at 20 Hz for 4 min consistently enhanced the amplitude and quantal content of fast EPSP for > 2 h, but only sometimes enhanced the quantal size. The LTP of quantal content produced by the conditioning tetanus was blocked by thapsigargin, a blocker of Ca(2+) pumps at Ca(2+) stores, applied before or after the conditioning tetanus, and by Xestospongin C, a blocker of inositoltrisphosphate (IP(3)) receptors, applied before the tetanus. It was not, however, blocked by ryanodine, a blocker and/or activator of ryanodine receptors, or by propranolol, a blocker of beta-adrenergic receptors. Thus the long-lasting activity of the preganglionic nerve at a high frequency causes the LTP of impulse-evoked transmitter release by the activation of CICR from thapsigargin-sensitive Ca(2+) stores in the nerve terminals. It is likely that a large Ca(2+) entry into the nerve terminals during tetanic activity primes ryanodine-insensitive Ca(2+) release channels for activation.     

姜黄素可阻止白细胞介素6损伤海马神经元钙离子的快速内流

The current study sought to investigate the potential protective action of curcumin against interleukin-6-induced injury in rat hippocampal neurons.The results revealed that interleukin-6 induced typical cellular injury,such as the swelling of cell bodies and increased Ca2+ concentration.After administration of curcumin,interleukin-6-induced neurons recovered to a normal state,and the fluorescence intensity of Ca2+ gradually returned to normal.These findings suggest that curcumin exerts a protective effect on hippocampal neurons of rats.In addition,our results suggest that the protective effect of curcumin involves prevention of the rapid Ca2+ influx induced by interleukin-6,which maintains Ca2+ homeostasis.     

Calpain inhibitors block Ca2+-induced suppression of neurite outgrowth in isolated hippocampal pyramidal neurons

Ca²⁺ is an important regulator of neurite elongation and growth cone movements but the mechanism(s) mediating these Ca²⁺-dependent effects is nuclear. Since cytoskeletal proteins are rapidly degraded by Ca²⁺-dependent proteinases (calpains) in vitro and in vivo, we investigated whether Ca²⁺-induced pruning or regression of neuronal processes is mediated by calpains. Isolated hippocampal pyramidal-like neurons were cultured and the ability of the membrane-permeable calpain inhibitors ethyl(+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarbamoyl]-2- oxiranecarboxylate (EST) and carbobenzoxyl-valyl-phenylalanyl-H (MDL 28170) to block the Ca²⁺ ionophore A23187-induced suppression in neurite outgrowth was investigated. Addition of 100 nM A23187 to the culture medium resulted in a retraction of dendrites without altering axonal elongation. The addition of 300 nM A23187 to the culture medium resulted in a significant decrease in the rate of axonal elongation as well as a retraction of dendritic processes. Administration of EST (5 or 20 m?M) to the culture medium completely blocked the pruning effect of 100 nM A23187 on dendrites and of 300 nM A23187 on axons, while EST alone did not significantly affect neurite outgrowth rate. MDL 28170 (20 m?M) showed the same effect as EST in preventing ionophore-induced pruning of dendrites and axons at 100 and 300 nM concentrations, respectively, of A23187. EST (20 m?M) did not block the A23187-induced rise of [Ca²⁺]_i as measured with fura-2. These results suggest that calpains play a role in Ca²⁺-induced pruning of neurites in isolated hippocampal pyramidal neurons. © 1994 Wiley-Liss, Inc.     

Neuropeptide Y modulates neurotransmitter release and Ca2+ currents in rat sensory neurons

Using 125I-labeled neuropeptide Y (NPY) and peptide YY (PYY), we demonstrated the existence of specific receptors for these peptides on rat dorsal root ganglion (DRG) cells grown in primary culture. Scatchard analysis of membrane homogenates indicated that the peptides bound to 2 populations of sites, with approximate affinities of 0.08 and 6.5 nM. Only low levels of binding were detected on sympathetic neurons cultured from the same animals or on a variety of neuronal clonal cell lines. The binding of 125I-NPY and 125I-PYY to DRG cell membranes was considerably reduced by the nonhydrolyzable analog of GTP, Gpp(NH)p. The major effect of Gpp(NH)p was to reduce the number of lower-affinity NPY binding sites without altering the number of high-affinity binding sites. NPY potently inhibited Ca2+ currents recorded under voltage clamp in rat DRG cells. Both the transient and sustained portions of the Ca2+ current were inhibited. The inhibitory effects of NPY were completely blocked following treatment of the cells with pertussis toxin. Depolarization elicited a large influx of Ca2+ into DRG neurons as assessed using fura-2-based microspectrofluorimetry. This influx of Ca2+ could be partially inhibited by NPY. Furthermore, NPY effectively inhibited the depolarization-induced release of substance P from DRG cells in vitro. Thus, NPY may be an important regulator of sensory neuron function in vivo.     

Probable Ca2+-mediated inactivation of Ca2+ currents in mammalian brain neurons

In rat hippocampal neurons, current- and single-electrode voltage-clamp analyses revealed a pronounced inactivation of probable Ca2+ currents (ICa), which was dependent on the amount of Ca2+ influx. Studies were conducted in cesium-loaded, tetrodotoxin-treated brain slice neurons in which known contaminating currents were blocked. These results therefore provide the first clear evidence that apparent Ca2+-mediated inactivation of ICa is an important mechanism with which mammalian brain neurons limit Ca2+ influx.     

Repetitive application of caffeine sensitizes caffeine-induced Ca2+ release in bullfrog sympathetic ganglion neurons

Cytosolic free calcium concentration ([Ca(2+)](i)) was recorded from cultured bullfrog sympathetic ganglion cells loaded with the Ca(2+)-indicator Fura-2 or Fura-6F. Repetitive application of caffeine at a low concentration, which either failed to produce any [Ca(2+)](i) elevation or induced a small gradual increase in [Ca(2+)](i) at first challenge, produced a drastic increase in the amplitude of Ca(2+) release (caffeine response). The caffeine response eventually reached peak amplitude and then remained constant even if caffeine application were continued. This augmentation was maintained for up to 2 h, and was achieved not only by repetitive application but also by a long exposure of caffeine. However, this augmentation was neither achieved by repetitive administration of high K(+)-solution, nor caused by inhibition of phosphodiesterase by caffeine. The repetitive or sustained application of caffeine is suggested to increase the caffeine sensitivity of the calcium release channel to calcium, thus causing the potentiation of the caffeine response.     

Signaling pathways involved in Ca2+- and Pb2+-induced vesicular catecholamine release from rat PC12 cells

Since Pb(2+) substitutes for Ca(2+) in essential steps leading to exocytosis, we have investigated whether Ca(2+) and Pb(2+) induce exocytosis through similar pathways. Vesicular catecholamine release was measured from dexamethasone-differentiated PC12 cells using carbon fiber microelectrode amperometry. Effects of drugs known to modulate PKC (PMA, staurosporine), calcineurin (cyclosporin A), calmodulin (W7), and CaM kinase II (KN-62) activity were investigated in intact and in ionomycin-permeabilized PC12 cells. Activation of PKC and inhibition of calmodulin decrease the frequency of exocytotic events evoked by high K(+) stimulation in intact cells. In addition, inhibition of calmodulin enhances the frequency of basal exocytosis from intact cells. Activation of PKC and inhibition of calcineurin enhance the frequency of basal exocytosis in intact as well as in ionomycin-permeabilized cells. Inhibition of PKC and of CaM kinase II cause no significant effects. None of the treatments has a significant effect on vesicle contents. The combined results indicate that PKC and calcineurin enhance and inhibit exocytosis through direct effects on the exocytotic machinery, whereas calmodulin and CaM kinase II exert indirect effects only. Conversely, Pb(2+)-evoked exocytosis in permeabilized cells is strongly reduced by inhibition of CaM kinase II, but is not sensitive to modulation of PKC and calcineurin activity. Inhibition of calmodulin only reduces the delay to onset of Pb(2+)-evoked exocytosis. Synaptotagmin I- and II-deficient PC12-F7 cells exhibit vesicular catecholamine release following depolarization or superfusion with Pb(2+). However, the frequency of exocytosis and the contents of vesicles released are strongly reduced as compared to PC12 cells. It is concluded that Ca(2+)-evoked exocytosis is modulated mainly by PKC and calcineurin, whereas Pb(2+)-evoked exocytosis is mainly modulated by CaM kinase II.     

TGF-β1 prevents gp120-induced impairment of Ca2+ homeostasis and rescues cortical neurons from apoptotic death

HIV-1 infection frequently induces neuronal death responsible for the development of neurological deficits associated with AIDS. Several reports suggest that gp120, the HIV-1 envelope glycoprotein, is the main candidate as mediator of the HIV-1-dependent neurotoxicity. Here we report the effect of gp120 on the survival of cortical neurons in vitro and the possible mechanisms whereby it occurs. Mature cortical neurons, cultured on a feeder layer of astrocytes, were treated with gp120 in a defined culture medium in absence of serum. The treatment with gp120 induced time-dependent neuronal damage displaying apoptotic features, as revealed by in situ labelling of DNA fragmentation. TGF-β1, a cytokine that has been previously shown to exert neuroprotective effects, prevented the cell death induced by exposure of cortical neurons to gp120. The prolonged treatment with gp120 also increased neuronal [Ca²⁺]_i, while the coincubation with TGF-β1 completely prevented the impairment of neuronal Ca²⁺ homeostasis. These data, taken together, demonstrate that gp120 induces apoptosis in cortical neurons, an effect that can be related to the impairment of Ca²⁺ homeostasis, and that TGF-β1 pretreatment reverts both the neuronal death and the alterations in neuronal [Ca²⁺]_i. J. Neurosci. Res. 49:600–607, 1997. © 1997 Wiley-Liss, Inc.     

Coupling of the IP3 receptor/Ca2+ channel with Ca2+ storage proteins chromogranins A and B in secretory granules

The secretory granules of neuroendocrine cells, which function as an inositol (1,4,5)-trisphosphate-sensitive intracellular Ca2+ store, contain both the inositol (1,4,5)-trisphosphate receptor/Ca2+ channel and the high-capacity low-affinity Ca2+ storage proteins, chromogranins A and B. Chromogranins A and B, which exist in approximately 2 mm range in the secretory granules, can bind 50-100 mol of Ca2+/mol with dissociation constants of 2-4 mm. These proteins interact directly with the inositol (1,4,5)-trisphosphate receptor/ Ca2+ channel at the intragranular pH 5.5, not only changing the conformation of the inositol (1,4,5)-trisphosphate receptor/Ca2+ channel but also modulating the channel activity. Given the homo- and heterotetrameric existence of both the inositol (1,4,5)-trisphosphate receptor/Ca2+ channel and chromogranins A and B, these tetrameric proteins appear to interact, thus controlling the intracellular Ca2+ concentration.     

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     

Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.     

Saikosaponin d causes apoptotic death of cultured neocortical neurons by increasing membrane permeability and elevating intracellular Ca2+ concentration

Saikosaponins (SSs) are a class of naturally occurring oleanane-type triterpenoid saponins found in Radix bupleuri that has been widely used in traditional Chinese medicine. As the main active principals of Radix bupleuri, SSs have been shown to suppress mouse motor activity, impair learning and memory, and decrease hippocampal neurogenesis. In the present study, we investigated the effect of five SSs (SSa, SSb1, SSb2, SSc, and SSd) on neuronal viability and the underlying mechanisms in cultured murine neocortical neurons. We demonstrate that SSa, SSb1 and SSd produce concentration-dependent apoptotic neuronal death and induce robust increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) at low micromolar concentrations with a rank order of SSd > SSa > SSb1, whereas SSb2 and SSc have no detectable effect on both neuronal survival and [Ca²⁺]_i. Mechanistically, SSd-induced elevation in [Ca²⁺]_i is the primary result of enhanced extracellular Ca²⁺ influx, which likely triggers Ca²⁺-induced Ca²⁺ release through ryanodine receptor activation, but not SERCA inhibition. SSd-induced Ca²⁺ entry occurs through a non-selective mechanism since blockers of major neuronal Ca²⁺ entry pathways, including L-type Ca²⁺ channel, NMDA receptor, AMPA receptor, Na⁺-Ca²⁺ exchanger, and TRPV1, all failed to attenuate the Ca²⁺ response to SSd. Further studies demonstrate that SSd increases calcein efflux and induces an inward current in neocortical neurons. Together, these data demonstrate that SSd elevates [Ca²⁺]_i due to its ability to increase membrane permeability, likely by forming pores in the surface of membrane, which leads to massive Ca²⁺ influx and apoptotic neuronal death in neocortical neurons.     

Somatic Ca2+ signaling in cerebellar Purkinje neurons

Activity‐driven Ca²⁺ signaling plays an important role in a number of neuronal functions, including neuronal growth, differentiation, and plasticity. Both cytosolic and nuclear Ca²⁺ has been implicated in these functions. In the current study, we investigated membrane‐to‐nucleus Ca²⁺ signaling in cerebellar Purkinje neurons in culture to gain insight into the pathways and mechanisms that can initiate nuclear Ca²⁺ signaling in this neuronal type. Purkinje neurons are known to express an abundance of Ca²⁺ signaling molecules such as voltage‐gated Ca²⁺ channels, ryanodine receptors, and IP3 receptors. Results show that membrane depolarization evoked by brief stimulation with K⁺ saline elicits a prominent Ca²⁺ signal in the cytosol and nucleus of the Purkinje neurons. Ca²⁺ influx through P/Q‐ and L‐type voltage‐gated Ca²⁺ channels and Ca²⁺‐induced Ca²⁺ release (CICR) from intracellular stores contributed to the Ca²⁺ signal, which spread from the plasma membrane to the nucleus. At strong K⁺ stimulations, the amplitude of the nuclear Ca²⁺ signal exceeded that of the cytosolic Ca²⁺ signal, suggesting the involvement of a nuclear amplification mechanism and/or differences in Ca²⁺ buffering in these two cellular compartments. An enhanced nuclear Ca²⁺ signal was more prominent for Ca²⁺ signals elicited by membrane depolarization than for Ca²⁺ signals elicited by activation of the metabotropic glutamate receptor pathway (mGluR1), which is linked to Ca²⁺ release from intracellular stores controlled by the IP3 receptor. © 2009 Wiley‐Liss, Inc.     

Spontaneous release from mossy fiber terminals inhibits Ni2+-sensitive T-type Ca2+ channels of CA3 pyramidal neurons in the rat organotypic hippocampal slice

Mossy fibers (axons arising from dentate granule cells) form large synaptic contacts exclusively onto the proximal apical dendrites of CA3 pyramidal neurons. They can generate large synaptic currents that occur in close proximity to the soma. These properties mean that active conductance in the proximal apical dendrite could have a disproportionate influence on CA3 pyramidal neuron excitability. Ni(2+)-sensitive T-type Ca(2+) channels are important modulators of dendritic excitability. Here, we use an optical approach to determine the contribution of Ni(2+) (100 microM)-sensitive Ca(2+) channels to action potential (AP) elicited Ca(2+) flux in the soma, proximal apical and distal apical dendrites. At resting membrane potentials Ni(2+)-sensitive Ca(2+) channels do not contribute to the Ca(2+) signal in the proximal apical dendrite, but do contribute in the other cell regions. Spontaneous release from mossy fiber terminals acting on 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)-sensitive postsynaptic channels underlies a tonic inhibition of Ni(2+)-sensitive channels. Chelating Zn(2+) with CaEDTA blocks CNQX-sensitive changes in Ca(2+) flux implicating a mechanistic role of this ion in T-type Ca(2+) channel block. To test if this inhibition influenced excitability, progressively larger depolarizing pulses were delivered to CA3 pyramidal neurons. CNQX significantly reduced the size of the depolarizing step required to generate APs and increased the absolute number of APs per depolarizing step. This change in AP firing was completely reversed by the addition of Ni(2+). This mechanism may reduce the impact of T-type Ca(2+) channels in a region where large synaptic events are common.     

Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca²⁺ channels are rapidly inactivated by a mechanism that is termed Ca²⁺‐dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca²⁺ release on CDI of high‐voltage‐activated Ca²⁺ channels in rat thalamocortical relay neurons by combining voltage‐clamp, Ca²⁺ imaging and immunological techniques. Double‐pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration‐dependent increase in CDI that was accompanied by an increase in the duration of Ca²⁺ transients. Inhibition of ryanodine receptors and endoplasmic Ca²⁺ pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5‐tris‐phosphate receptors by intracellular application of 2‐aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2‐aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L‐type Ca²⁺ channels was emphasized by the near‐complete block of CDI by nifedipine, an effect only surpassed when Ca²⁺ was replaced by Ba²⁺ and chelated by 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA). Trains of action potential‐like stimuli induced a strong reduction in high‐voltage‐activated Ca²⁺ current amplitude, which was significantly reduced when intracellular Ca²⁺ stores were made inoperative by thapsigargin or Ba²⁺/BAPTA. Western blotting revealed expression of L‐type Ca²⁺ channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross‐signalling between L‐type Ca²⁺ channels and ryanodine receptors that controls the amount of Ca²⁺ influx during neuronal activity.