Role of nonsynaptic GluN2B-containing NMDA receptors in excitotoxicity: Evidence that fluoxetine selectively inhibits these receptors and may have neuroprotective effects

In acute ischaemic brain injury and chronic neurodegeneration, the primary step leading to excitotoxicity and cell death is the excessive and/or prolonged activation of glutamate (Glu) receptors, followed by intracellular calcium (Ca²⁺) overload. These steps lead to several effects: a persistent depolarisation of neurons, mitochondrial dysfunction resulting in energy failure, an increased production of reactive oxygen species (ROS), an increase in the concentration of cytosolic Ca²⁺ [Ca²⁺]_i, increased mitochondrial Ca²⁺ uptake, and the activation of self-destructing enzymatic mechanisms. Antagonists for NMDA receptors (NMDARs) are expected to display neuroprotective effects, but no evidence to support this hypothesis has yet been reported. A number of clinical trials using NMDAR antagonists have failed to demonstrate neuroprotective effects, either by reducing brain injury or by preventing neurodegeneration. Recent advances in NMDAR research have provided an explanation for this phenomenon. Synaptic and extrasynaptic NMDARs are composed of different subunits (GluN2A and GluN2B) that demonstrate opposing effects. Synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs have different co-agonists: d-serine for synaptic NMDARs and glycine for extrasynaptic NMDARs. Both co-agonists are of glial origin.The mechanisms of cell destruction or cell survival in response to the activation of NMDAR receptors depend in part on [Ca²⁺]_i and the route of entry of this ion and more significantly on the subunit composition and localisation of the NMDARs. While synaptic NMDAR activation is involved in neuroprotection, the stimulation of extrasynaptic NMDARs, which are composed of GluN2B subunits, triggers cell destruction pathways and may play a key role in the neurodegeneration associated with Glu-induced excitotoxicity. In addition, it has been found that synaptic and extrasynaptic NMDA receptors have opposing effects in determining the fate of neurons. This result has led to the targeting of nonsynaptic GluN2B-containing NMDARs as promising candidates for drug research. Under hypoxic conditions, it is likely that the failure of synaptic glutamatergic transmission, the impairment of the GluN2A-activated neuroprotective cascade, and the persistent over-activation of extrasynaptic GluN2B-containing NMDARs lead to excitotoxicity. Fluoxetine, a drug widely used in clinical practice as an antidepressant, has been found to selectively block GluNR2B-containing NMDARs. Therefore, it seems to be a potential candidate for neuroprotection.     

N-(quinolin-2-ylmethyl)butane-1,4-diamine,a polyamine analogue,attenuated injury in in vitro and in vivo models of cerebral ischemia

It has been widely recognized that glutamate (Glu)-induced cytotoxicity, intracellular calcium overload and excessive free radical production are the key players in the development and progression of ischemic brain injury. Since MK-801, an antagonist of N-methyl-d-aspartate (NMDA) receptor, showed many adverse reactions that hampered its clinical applications, development of safe and effective agent for the treatment of cerebral ischemia is eagerly required. This study was to investigate the effects of N¹-(quinolin-2-ylmethyl)butane-1,4-diamine (QMA), a polyamine analogue, on the in vitro and in vivo models of cerebral ischemic damage. The results revealed that pretreatment with QMA could attenuate Glu, putrescine (Put) and oxygen-glucose deprivation (OGD)-induced cell death, lipid peroxidation as well as the elevation of reactive oxygen species (ROS) and intracellular [Ca²⁺]_i in pheochromocytoma (PC12) cells and in rat primary cortical neurons. The results also demonstrated that QMA could inhibit NMDA-mediated intracellular [Ca²⁺]_i accumulation in rat primary cortical neurons and reduce brain infarct volume in middle cerebral artery occlusion (MCAO) rats. The present report suggested that polyamines played a crucial role in the pathological processes of cerebral ischemic damage and that QMA or other novel polyamine analogues could be promising therapeutic candidates for stroke by virtue of their anti-hypoxia and antioxidation property.     

Selegiline (deprenyl) decreases calbindin-D28k expression in cortical neurons of rats socially deprived during the post-weaning period

Preclinical studies indicate that selegiline (deprenyl), frequently used in some neurodegenerative diseases, exert protective effects on central nervous system neurons of individuals exposed to social isolation (SI). Furthermore, it has been suggested that SI produces neuronal dysfunction due in part to an excessive intracellular Ca²⁺ overload. Since the main intracellular Ca²⁺ buffering mechanism involves changes in the calcium-binding protein calbindin-D28k (CB), and that CB neuronal expression can increase in response to Ca²⁺ transients, we hypothesized that chronic selegiline administration in early socially isolated animals could minimize cell CB expression as an indirect indicator of protective mechanism against Ca²⁺ overload. In the present study male rats were weaned at postnatal day 21 (P21) and randomly assigned to social deprivation (SI) or control (SC) environments for 30 days (P21–51). SI animals were further subdivided in two experimental groups: socially deprived-saline (SI-SAL) and socially isolated-selegiline (SI-SEL) for additional 30 days (P52–82). Medial frontal CB immunoreactivity (CB-ir) neurons were quantitatively and qualitatively analyzed. The results obtained indicate that neocortical cells of adult rats submitted to early SI show a significant increase in the number of CB-ir neurons per cortical field, while selegiline treatment significantly reduces this parameter.     

Curcumin ameliorates hippocampal neuron damage induced by human immunodeficiency virus-1

Our previous studies have shown that infection with the gp120 V3 loop can cause human immunodeficiency virus-1 associated neurocognitive disorders. Curcumin has been shown to improve these effects to some degree, but the precise mechanisms remain unknown. The present study analyzed the neuroprotective effect and mechanism of curcumin in relation to hippocampal neurons. Results showed that 1 nmol/L gp120 V3 loop suppressed the growth of synapses. After administration of 1 μmol/L curcumin, synaptic growth improved. Curcumin is neuroprotective against gp120 V3 loop-induced neuronal damage by inhibiting the activation of L-type calcium currents, relieving intracellular Ca2+ overload, promoting Bcl-2 expression, and inhibiting Bax activation. The effect of curcumin was identical to nimodipine, suggesting that curcumin has the same neuroprotective effects against gp120 V3 loop-induced neuronal damage.     

Regulation of voltage-gated Ca2 + currents by Ca2 +/calmodulin-dependent protein kinase II in resting sensory neurons

Calcium/calmodulin-dependent protein kinase II (CaMKII) is recognized as a key element in encoding depolarization activity of excitable cells into facilitated voltage-gated Ca^2 + channel (VGCC) function. Less is known about the participation of CaMKII in regulating VGCCs in resting cells. We examined constitutive CaMKII control of Ca^2 + currents in peripheral sensory neurons acutely isolated from dorsal root ganglia (DRGs) of adult rats. The small molecule CaMKII inhibitor KN-93 (1.0 μM) reduced depolarization-induced I_Ca by 16–30% in excess of the effects produced by the inactive homolog KN-92. The specificity of CaMKII inhibition on VGCC function was shown by the efficacy of the selective CaMKII blocking peptide autocamtide-2-related inhibitory peptide in a membrane-permeable myristoylated form, which also reduced VGCC current in resting neurons. Loss of VGCC currents is primarily due to reduced N-type current, as application of mAIP selectively reduced N-type current by approximately 30%, and prior N-type current inhibition eliminated the effect of mAIP on VGCCs, while prior block of L-type channels did not reduce the effect of mAIP on total I_Ca. T-type currents were not affected by mAIP in resting DRG neurons. Transduction of sensory neurons in vivo by DRG injection of an adeno-associated virus expressing AIP also resulted in a loss of N-type currents. Together, these findings reveal a novel molecular adaptation whereby sensory neurons retain CaMKII support of VGCCs despite remaining quiescent.     

Evaluation of the expression pattern of rAAV2/1, 2/5, 2/7, 2/8, and 2/9 serotypes with different promoters in the mouse visual cortex

This study compared the expression pattern, laminar distribution, and cell specificity of several rAAV serotypes (2/1, 2/5, 2/7, 2/8, and 2/9) injected in the primary visual cortex (V1) of adult C57Bl/6J mice. In order to obtain specific expression in certain neuron subtypes, different promoter sequences were evaluated for excitatory cell specificity: a universal cytomegalovirus (CMV) promoter, and two versions of the excitatory neuron‐specific Ca²⁺/calmodulin‐dependent kinase subunit α (CaMKIIα) promoter, CaMKIIα 0.4 and CaMKIIα 1.3. The spatial distribution as well as the cell type specificity was immunohistochemically verified. Depending on the rAAV serotype used, the transduced volume expressing reporter protein differed substantially (rAAV2/5 ? 2/7 ≈ 2/9 ≈ 2/8 ? 2/1). Excitatory neuron‐specific targeting was promoter‐dependent, with a surprising difference between the 1.3 kb and 0.4 kb CaMKIIα promoters. While CaMKIIα 1.3 and CMV carrying vectors were comparable, with 78% of the transduced neurons being excitatory for CMV and 82% for CaMKIIα 1.3, the shorter CaMKIIα 0.4 version resulted in 95% excitatory specificity. This study therefore puts forward the CaMKIIα 0.4 promoter as the best choice to target excitatory neurons with rAAVs. Together, these results can be used as an aid to select the most optimal vector system to deliver transgenes into specific rodent neocortical circuits, allowing further elucidation of their functions. J. Comp. Neurol. 523:2019–2042, 2015. © 2015 Wiley Periodicals, Inc.     

CaMKII antisense oligodeoxynucleotides protect against ischemia-induced neuronal death in the rat hippocampus

The present study was performed to investigate the effects of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) antisense oligodeoxynucleotides (ODNs) on the assembly of the CaMKII·GluR6·PSD-95 signaling module, GluR6 serine phosphorylation and c-Jun N-terminal kinase 3 (JNK3) activation. A further aim was to determine the neuroprotective mechanism of CaMKII antisense ODNs against ischemia-reperfusion (I/R)-induced neuronal death in the rat hippocampus. CaMKII antisense ODNs were intracerebroventricularly infused to inhibit CaMKII expression once daily for 3 days prior to the induction of ischemia. Transient cerebral ischemia (15 min) and reperfusion were induced by four-vessel occlusion in Sprague-Dawley rats as an animal model for transient cerebral I/R. The expression of related proteins was examined by immunoprecipitation and immunoblotting. Neuronal death in the rat hippocampus was detected by histology and histochemistry. The results indicate that CaMKII antisense ODNs inhibit several of the processes that are normally induced by cerebral I/R, including CaMKII expression, increased CaMKII·GluR6·PSD-95 signaling module assembly, GluR6 serine phosphorylation and JNK3 activation. Alternatively, CaMKII antisense ODNs also exhibit a significant neuroprotective role against cerebral I/R-induced cell death. These results provide the first evidence that CaMKII antisense ODNs can exert neuroprotective effects on cerebral I/R-induced cell death. The possible molecular mechanisms underlying this effect include 1) an inhibition of CaMKII expression and subsequent suppression of the assembly of the CaMKII·GluR6·PSD-95 signaling module, 2) GluR6 serine phosphorylation, and 3) reduced JNK3 activation.     

Calcium signaling in cultured rat oligodendrocytes

The syntax of neuronal-glial or axonal-glial interaction is frequently communicated through transient changes in internal calcium (Ca_i). We examined mechanisms for Ca_i signaling and intercellular propagation of Ca_i responses in cultured oligodendrocytes (OLGs) derived form adult spinal cord (SC), postnatal day 21 (P21) SC, and P21 brain. We found that (1) cultured OLGs exhibited a heterogeneous responese to norepinephrine, carbachol, ATP, histamine, and glutamate; (2) receptor-mediated Ca_i increases were derived from both Ca²⁺ influx and intracellular Ca²⁺ release; (3) the percentage of responders to neuroligands varied as a function of cell origin; (4) cultured OLGs exhibited a thapsigargin-sensitive, but not a caffeine-sensitive, intracellular Ca²⁺ pool; and (5) gap junctional contacts between OLGs permitted limited intercellular propagation of mechanically stimulated Ca_i responses. Receptor-mediated Ca_i signaling appears to occur not only in cultured OLGs but also in acutely dissociated OLGs. The heterogeneity in Ca_i responses as a function of cell origin may reflect the existence of OLG subsets of differences in the maturation stage of OLGs. © 1995 Wiley-Liss, Inc.     

Ca2+/calmodulin‐dependent protein kinase II and Dimethyl Sulfoxide affect the sealing frequencies of transected hippocampal neurons

Traumatic injury often results in axonal severance, initiating obligatory Wallerian degeneration of distal segments, whereas proximal segments often survive. Calcium ion (Ca²⁺) influx at severed proximal axonal ends activates pathways that can induce apoptosis. However, this same Ca²⁺‐influx also activates multiple parallel pathways that seal the plasmalemma by inducing accumulation and fusion of vesicles at the lesion site that reduce Ca²⁺‐influx and enhance survival. We examined whether various inhibitors of Ca²⁺/calmodulin‐dependent protein kinases (CaMKs), and/or dimethyl sulfoxide (DMSO), a common solvent for biologically active substances, affected the ability of a hippocampal‐derived neuronal cell line (B104 cells) to seal membrane damage following axotomy. Axolemmal sealing frequencies were assessed at different transection distances from the axon hillock and at various times after Ca²⁺‐influx (PC times) by observing whether transected cells took‐up fluorescent dyes. Inhibition of CaMKII by tatCN21 and KN‐93, but not inhibition of CaMKI and CaMKIV by STO‐609, affected axonal sealing frequencies. That is, CaMKII is a component of previously reported parallel pathways that induce membrane sealing, whereas CaMKI and CaMKIV are not involved. The effects of these CaMKII inhibitors on plasmalemmal sealing depended on their mechanism of inhibition, transection distance, and PC time. DMSO at low concentrations (90 µM–28 mM or 0.00064%–0.2% v/v) significantly increased membrane‐sealing frequencies at most PC times and transection distances, possibly by permeabilizing the plasmalemma to Ca²⁺. Inhibition of CaMKII, DMSO, PC time, and the transection distance significantly affect plasmalemmal sealing that is critical to somal survival in traumatic lesions.     

Workflow and Methods of High-Content Time-Lapse Analysis for Quantifying Intracellular Calcium Signals

Calcium ions (Ca²⁺) play a fundamental role in a variety of physiological functions in many cell types by acting as a secondary messenger. Variation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is often observed when the cell is stimulated. However, it is a challenging task to automatically quantify intracellular [Ca²⁺]_i in a population of cells. In this study, we present a workflow including specific algorithms for the automated intracellular calcium signal analysis using high-content, time-lapse cellular images. The experimental validations indicate the effectiveness of the proposed workflow and algorithms. We applied the workflow to analyze the intracellular calcium signals induced by different concentrations of H₂O₂ in the cell lines transfected by presenilin-1 (PS-1) that is known to be closely related to the familial Alzheimer’s disease (FAD). The analysis results imply an important role of mutant PS-1, but not normal human PS-1 and mutant human amyloid precursor protein (APP), in enhancing intracellular calcium signaling induced by H₂O₂. F. Li, X. Zhou and J. Zhu contributed equally to this work.     

Calpain-mediated proteolysis of microtubule-associated protein 2 (MAP-2) is inhibited by phosphorylation by cAMP-dependent protein kinase,but not by Ca2+/calmodulin-dependent protein kinase II

The effects of cAMP-dependent protein kinase (cAMP-PK) and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) phosphorylation on the calpain-mediated degradation of microtubule-associated protein 2 (MAP-2) were studied. Both cAMP-PK and CaMKII readily phosphorylated MAP-2. However, cAMP-PK phosphorylated MAP-2 to a significantly greater extent than did CaMKII (4.5 mol ³²P/mol MAP-2 and 1.4 mol ³²P/mol MAP-2, respectively). Phosphorylation of MAP-2 by cAMP-PK, but not by CaMKII, significantly inhibited the calpain-induced hydrolysis of MAP-2. These results demonstrate that the phosphorylation of sites on the MAP-2 molecule accessible to cAMP-PK, but not to CaMKII, result in increased resistance to calpain proteolysis. © 1993 Wiley-Liss, Inc.     

Neuroprotective Effect and Mechanism of Action of Tetramethylpyrazine Nitrone for Ischemic Stroke Therapy

Our previous studies demonstrated that the multifunctional agent TBN, a derivative of tetramethylpyrazine armed with a nitrone moiety, displayed high therapeutic efficacy in experimental ischemic stroke models. However, its molecular mechanisms of action underlying the neuroprotective effect need further exploration. In the present study, we found that TBN had significant activities scavenging free radicals such as ^·OH, O ₂ ^·? and ONOO^?, inhibiting Ca²⁺ overload, maintaining mitochondrial function and preventing neuronal damage in primary cortical cultures. Further, TBN was effective in reducing brain infarction and ameliorating impairment of behavioral functions in the permanent middle cerebral artery occlusion (p-MCAo) rat model. TBN down-regulated the expression of pro-apoptotic factors Bax, while up-regulated the expression of anti-apoptotic factor Bcl-2 and increased the expression of pro-survival factors including p-Akt and p-GSK3β in the peri-infarct cortex of p-MCAo rats. In addition, LY-294002 (a PI3K inhibitor) and MK2206 (an Akt inhibitor) significantly blocked the protective effect of TBN against OGD-induced death of cortical neurons. Taken together, the multifunctional mechanisms including scavenging free radicals, blocking calcium overload, maintaining mitochondrial function and activating the PI3K/Akt/p-GSK3β cell survival pathway were possibly involved in the neuroprotective effects of TBN, making it a promising clinical candidate for the treatment of ischemic stroke.     

Carnosine protects a primary cerebellar cell culture from acute NMDA toxicity

Activation of extrasynaptic NMDA receptors by high glutamate concentrations is one of the key pathogenic factors following a stroke. For this reason, the search for efficient neuroprotective agents that could reduce glutamate toxicity is a pressing need. Ca²⁺ overload in response to glutamate leads to activation of signaling cascades in the cell and the development of oxidative stress, which ultimately leads to apoptosis. Using a model system of acute excitotoxicity caused by 50 μM NMDA, which was used as a specific NMDA receptor activator, we demonstrated that during 2 hours of incubation the viability of the primary neuronal culture decreased by 30–50%. To demonstrate that the observed effect is associated not only with the Ca²⁺ influx into the cytoplasm through the activated NMDA receptors, we decreased the Ca²⁺ concentration in the medium. The lowered Ca2+ concentration, as well as its complete absence, did not affect NMDA toxicity. We tested carnosine, a naturally occurring dipeptide and promising antioxidant, as a neuroprotective agent. The addition of 2 mM carnosine prevented the decrease in cell viability caused by a 2-hour incubation with 50 μM NMDA, while it showed no effect on the viability of the cell culture in the control. Based on the results, we consider the further study of carnosine, its complexes, and analogues as neuroprotectors in cerebral ischemia promising.     

Amyotrophic Lateral Sclerosis Immunoglobulins Increase Intracellular Calcium in a Motoneuron Cell Line

A hybrid motoneuron cell line (VSC4.1) was used as a model system to study the relationship between alterations in intracellular calcium and subsequent cell death induced by immunoglobulin fractions purified from sera of patients with ALS. Using fluo-3 fluorescence imaging, immunoglobulins from 8 of 10 patients with ALS were found to induce transient increases in intracellular calcium ([Ca²⁺]_i) in differentiated VSC4.1 cells. These transient [Ca²⁺]_iincreases required extracellular calcium entry through voltage-gated calcium channels sensitive to synthetic FTX and to high concentrations (>1 μM) of ω-agatoxin IVa. The incidence of transient [Ca²⁺]_iincreases induced by ALS immunoglobulins correlated with the extent of cytotoxicity induced by the same ALS immunoglobulins in parallel cultures of VSC4.1 cells. Furthermore, manipulations which blocked transient [Ca²⁺]_iincreases (addition of synthetic FTX or ω-agatoxin IVa) also inhibited the cytotoxic effects of ALS immunoglobulins. No transient calcium increases were observed in VSC4.1 cells following addition of immunoglobulins from 7 neurologic disease control patients. However, transient [Ca²⁺]_iincreases were observed following addition of immunoglobulins from 4 of 5 patients with myasthenia gravis (MG). The [Ca²⁺]_ichanges induced by MG immunoglobulins were not blocked by s-FTX, suggesting that they result from a different mechanism than those induced by ALS immunoglobulins. These results suggest that immunoglobulins from patients with ALS can induce transient increases in intracellular calcium in a motoneuron cell line, which may represent early events in the cascade of processes leading to injury and death of susceptible cells.     

Calcium dependence of the priming,activation and inactivation of ryanodine receptors in frog motor nerve terminals

We studied the effects of varying extracellular Ca²⁺ ([Ca²⁺]_o) and Ca²⁺ channel density and intracellular loading of Ca²⁺ chelators on stimulation‐induced rises in intracellular Ca²⁺ ([Ca²⁺]_i) in frog motor nerve terminals with Ca²⁺ imaging. The slowly waxing and waning components of rises in [Ca²⁺]_i induced by repetitive tetani were suppressed by blockers of Ca²⁺ pumps of the endoplasmic reticulum (thapsigargin and cyclopiazonic acid) and a blocker of ryanodine receptors [8‐(N,N‐diethylamino)octyl 3,4,5‐trimethoxybenzoate hydrochloride] without affecting the initial quickly‐rising component, thus reflecting the priming (and then subsequent rapid activation) and inactivation phases of Ca²⁺‐induced Ca²⁺ release (CICR) from the endoplasmic reticulum. A short tetanus‐induced rise in [Ca²⁺]_i was proportional to [Ca²⁺]_o, whereas the component of CICR was non‐linearly related to [Ca²⁺]_o with saturation at 0.9 mm . The progressive blockade of Ca²⁺ channels by ω‐conotoxin GVIA caused proportional decreases in CICR and short tetanus‐induced [Ca²⁺]_i rises. Intracellular loading of BAPTA and EGTA reduced the magnitude of CICR as well as short tetanus‐induced rises in [Ca²⁺]_i with a greater effect of BAPTA than EGTA on CICR. The time to peak and the half decay time of CICR were prolonged by a low [Ca²⁺]_o or Ca²⁺ channel blocker or [Ca²⁺]_i chelators. These results suggest that ryanodine receptors sense the high [Ca²⁺]_i transient following single action potentials for triggering CICR, whereas the priming and inactivation processes of CICR sense a slower, persisting rise in [Ca²⁺]_i during and after action potential trains. A model is presented that includes CICR activation in elementary units.     

Calcium signalling in bovine adrenal chromaffin cells: Additive effects of histamine and nicotine

In a previous report, we described the ability of two secretogogues, histamine and nicotine, to stimulate additive effects on catecholamine (CA) release and synapsin II phosphorylation in bovine adrenal chromaffin cells (BACC) [Firestone and Browning (1992), J. Neurochem., 58:441–447]. We hypothesized that these results were due to the combined effects on cytosolic Ca⁺⁺ of the two distinct signalling pathways. We therefore examined the intracellular Ca⁺⁺ signals stimulated by histamine and nicotine, alone and together. In Ca⁺⁺ -deficient medium, nicotine-stimulated signals were abolished, whereas histamine-stimulated signals were maintained, demonstrating that nicotine depended entirely on Ca⁺⁺ influx for its effects. Indeed, the nicotine-stimulated signal could also be prevented using a Ca⁺⁺ channel blocker, nicardipine. Further, the observation that exposure of BACC to thapsigargin reduced histamine-stimulated Ca⁺⁺ signals verified that histamine mobilizes Ca⁺⁺ from intracellular stores. Thus, the two secretogogues mobilize Ca⁺⁺ from distinct pools. When BACC were stimulated with the two secretogogues together, the resulting Ca⁺⁺ signal was greater than that from either alone. These data are consistent with a model in which two distinct sources of Ca⁺⁺ can summate within the cell, producing a greater Ca⁺⁺ signal and, hence, a greater effect on neurotransmitter release. © 1994 Wiley-Liss, Inc.     

Calcium influx via L-type voltage-gated channels mediates the delayed,elevated increases in steady-state c-fos mRNA levels in cerebellar granule cells exposed to excitotoxic levels of glutamate

The altered kinetics of steady-state c-fos mRNA production in cultured cerebellar granule cells under excitotoxic conditions was investigated in neurons subjected to depolarising stimuli, namely, high KCl and L-glutamate (Glu), in which Ca²⁺ influx occurs by differing routes. Increases in intracellular-free calcium levels ([Ca²⁺]_i) stimulated by nontoxic or toxic levels of Glu were blocked by selective N-methyl-D-aspartate (NMDA) receptor antagonism; were blocked only partially by the L-type channel blocker, nifedipine; and were unaffected by α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptor antagonists. Glu-induced cell death was prevented only by NMDA receptor blockade. Exposure of cells to nontoxic levels of Glu resulted in a transient increase in c-fos mRNA levels, whereas an excitotoxic dose produced a delay in the appearance of c-fos mRNA but a subsequent, progressive, and sustained (>4 hr) increase. An excitotoxic dose of Glu in combination with either nifedipine or selective NMDA receptor antagonists resulted in the normal, transient increase of c-fos mRNA levels. Chronic exposure to 55 mM KCl caused no cytotoxicity, although it resulted in a delayed, elevated increase in c-fos mRNA levels that was unaffected by NMDA receptor blockade but reverted to the normal, transient profile of c-fos mRNA formation when it was coadministered with nifedipine. The KCl-induced increase in [Ca²⁺]_i levels was inhibited dramatically by nifedipine but was unaffected by any of the ionotropic Glu receptor antagonists. The results support the notion that the appearance of a delayed but elevated increase in steady-state c-fos mRNA levels following exposure to excitotoxic doses of Glu is mediated specifically by calcium influx via L-type voltage-gated channels. J. Neurosci. Res. 52:641–652, 1998. © 1998 Wiley-Liss, Inc.     

Calcium dependence of spontaneous neurotransmitter release

Spontaneous release of neurotransmitters is regulated by extracellular [Ca²⁺] and intracellular [Ca²⁺]. Curiously, some of the mechanisms of Ca²⁺ signaling at central synapses are different at excitatory and inhibitory synapses. While the stochastic activity of voltage‐activated Ca²⁺ channels triggers a majority of spontaneous release at inhibitory synapses, this is not the case at excitatory nerve terminals. Ca²⁺ release from intracellular stores regulates spontaneous release at excitatory and inhibitory terminals, as do agonists of the Ca²⁺‐sensing receptor. Molecular machinery triggering spontaneous vesicle fusion may differ from that underlying evoked release and may be one of the sources of heterogeneity in release mechanisms.