AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

The role of parvalbumin-containing interneurons in the regulation of spontaneous synchronous activity of brain neurons in culture

Subtypes of inhibitory GABAergic neurons containing Ca²⁺-binding proteins play a pivotal role in the regulation of spontaneous synchronous [Ca²⁺]_i transients in a neuronal network. In this study it is shown that: (1) the interneurons that containing Ca²⁺-binding proteins at buffer concentration can be identified by the shape of Ca²⁺-signa1 in response to depolarization or activation of ionotropic glutamate receptors; (2) Ca²⁺-binding proteins are involved in desynchronization of spontaneous Ca²⁺ transients. At low frequencies of spontaneous synchronous [Ca²⁺]_i transients (less than 0.2 Hz) neurons show quasi-synchronous pulsations. At higher frequencies, synchronization of spontaneous synchronous [Ca²⁺]_i transients occurs in all neurons; (3) it is established that several synchronous oscillations with different frequencies coexist in the network and the amplitude of their depolarizing pulse also varies. This phenomenon is apparently the mechanism that selectively directs information in separate neurons using the same network; and (4) in one population of interneurons at high frequencies of spontaneous synchronous [Ca²⁺]_i transients the inversion of Cl^– concentration gradient is observed. In this case, the inhibition of GABA(A) receptors suppresses the activity of neurons in this population and excites other neurons in the network. Thus, the GABAergic neurons that contain Ca-binding proteins show different mechanisms to regulate the synchronous neuronal activities in cultured rat hippocampal cells.     

Control of spontaneous synchronous Ca<Superscript>2+</Superscript> oscillations in hippocampal neurons by GABAergic neurons containing kainate receptors without desensitization

Previously we have shown that in culture of rat hippocampal neurons, the calcium responses of individual cells (changes of cytoplasmic free Ca²⁺ concentration in response to agonists of glutamate kainate receptors) differed in shape and amplitude (Kononov A.V., Bal’ N.V., Zinchenko V.P. 2011. Biochemistry (Moscow) Suppl. Series A: Membrane and Cell Biology. 5 (2), 162–170). In the majority of neurons, the amplitudes of calcium response were regularly distributed, although there were a small number of cells that generated the desensitization-free signals of far greater amplitudes. In these cells, the desensitization inhibitors did not increase the amplitude of calcium response. We identified these neurons and revealed their function. The agonists of kainate receptors inhibited the synchronized spontaneous Ca²⁺ oscillations, decreased the baseline calcium level in the majority of neurons, and considerably elevated it in some of them. After washout of the agonists, the oscillations were restored in all neurons only after a certain time lag determined by the period needed for calcium concentration to decrease to subbasal level in specific neurons with high calcium signal amplitude. This observation indicates the command role of these neurons in synchronizing the activity of the entire population. To identify the subtype of KA receptors in these neurons, we used especially selective agonists and showed that KA receptors of the neurons characterized with desensitization-free calcium signals of unusually great amplitude contained GluR5/GLUK1 subunits. These receptors are known to be located mostly in the presynaptic membrane, where they promote exocytosis of neurotransmitters due to elevation of the Ca²⁺ conductivity. Having marked the positions of these neurons, we fixed the preparation and stained the cells with fluorescently labeled antibodies raised against glutamate decarboxylase, an enzyme which is selectively expressed in GABAergic neurons. The experiments demonstrated that antibodies were localized only in the neurons, where the kainate receptor agonist evoked desensitization-free calcium responses of especially large amplitude. Thus, GABAergic neurons control the synchronous activity of a large number of neurons via glutamate-evoked activation of specific presynaptic kainate receptors with GluR5/GLUK1 subunits leading to desensitization-free calcium signals of especially large amplitude.     

Short-term hypoxia induces a selective death of GABAergic neurons

It is known that brief episodes of hypoxia protect neurons from death caused by global ischemia and hypoxia (hypoxic preconditioning). At the same time, brief hypoxia may cause a phenomenon of posthypoxic hyperexcitability during reoxygenation, which can lead to the death of separate neurons due to their individual differences. In this work we compare the effects of short-term hypoxia on the initiation of preconditioning and posthypoxic hyperexcitability in two populations of neurons: inhibitory GABAergic neurons and excitatory glutamatergic neurons. Preconditioning effect was evaluated according to the suppression of the NMDA-receptor activity. The phenomenon of posthypoxic hyperexcitability was estimated by the appearance of spontaneous synchronized Ca²⁺ spikes in the neuronal network during reoxygenation after each episode of hypoxia. It is shown that the preconditioning effect occurs only in glutamatergic neurons. In the GABAergic neurons the effect of preconditioning was not observed. The activity of NMDA receptors in these neurons was not suppressed but increased after each episode of hypoxia. At the moment of posthypoxic synchronous Ca²⁺-spike generation, a global increase of the cytoplasmic Ca²⁺ concentration occurred in a few of GABAergic neurons, followed by the apoptotic death of these cells. The anti-inflammatory cytokine, interleukin-10 (IL-10) prevented the development of posthypoxic hyperexcitability, inhibiting spontaneous synchronous Ca²⁺ spike, and protected GABAergic neurons from the death, restoring the preconditioning effect in them. PI3-kinase inhibitors wortmannin and LY294002 prevented the IL-10 protective effect abolishing the inhibiting effect of IL-10 on the generation of the Ca²⁺ synchronous spike. These findings point out to the leading role of GABAergic neurons in the development of posthypoxic hyperexcitability. We suggest that the reason for posthypoxic hyperexcitability in the network is a weakening of the inhibiting effect of GABAergic neurons. Activation of different signaling pathways leading to activation of PKB- and PKG-dependent phosphorylation in the neurons of this type represents a possible strategy to protect neurons from death during hypoxia.     

Calcium requirement of long-term depression and rebound potentiation in cerebellar Purkinje neurons

Cerebellar Purkinje neurons (PNs) receive two main excitatory inputs, from climbing fibers and parallel fibers, and inhibitory inputs, from GABAergic interneurons. The synapses formed by parallel fibers and by inhibitory interneurons on PNs are able to undergo long-lasting in efficacy. Thus, the excitatory parallel fiber-PN synapse undergoes long-term fibers. Synaptic inhibition can be potentiated by climbing fiber activity by a mechanism named rebound potentiation, resulting in a more powerful inhibitory effect of GABAergic interneurons. The induction of both long-term depression and rebound potentiation requires a transient elevation of the cytoplasmic calcium concentration ([Ca²⁺]_i). The [Ca²⁺]_i-transient is caused by Ca²⁺ entry through voltage-gated Ca²⁺ channels and, possibly, by release of Ca²⁺ from IP_3- and ryanodine-sensitive stores. Direct Ca²⁺ entry through synaptic AMPA receptor channels seems not to contribute significantly to the Ca²⁺ signal mediating the induction of both long-term depression and rebound potentiation.     

Functional organization of dendritic Ca2+ signals in midbrain dopamine neurons

Dendritic Ca²⁺ plays an important role not only in synaptic integration and synaptic plasticity, but also in dendritic excitability in midbrain dopamine neurons. However, the functional organization of dendritic Ca²⁺ signals in the dopamine neurons remains largely unknown. We therefore investigated dendritic Ca²⁺ signals by measuring glutamate-induced Ca²⁺ increases along the dendrites of acutely isolated midbrain dopamine neurons.Maximal doses of glutamate induced a [Ca²⁺]_c rise with similar amplitudes in proximal and distal dendritic regions of a dopamine neuron. Glutamate receptors contributed incrementally to the [Ca²⁺]_c rise according to their distance from the soma, with a reciprocal decrement in the contribution of voltage-operated Ca²⁺ channels (VOCCs). The contribution of AMPA and NMDA receptors increased with dendritic length, but that of metabotropic glutamate receptors decreased. At low doses of glutamate at which spontaneous firing was sustained, the [Ca²⁺]_c rise was higher in the distal than the proximal regions of a dendrite, possibly due to the increased spontaneous firing rate.These results indicate that functional organization of Ca²⁺ signals in the dendrites of dopamine neurons requires different combination of VOCCs and glutamate receptors according to dendritic length, and that regional Ca²⁺ rises in dendrites respond differently to applied glutamate concentration.     

Glutamatergic and GABAergic agonists increase [Ca2+]i in avian cochlear nucleus neurons

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are stimulated by glutamate, released from the auditory nerve, and GABA, released from both interneurons surrounding NM and from cells located in the superior olivary nucleus. In this study, the Ca²⁺ indicator dye Fura-2 was used to measure Ca²⁺ responses in NM stimulated by glutamate- and GABA-receptor agonists using a chicken brainstem slice preparation. Glutamatergically stimulated Ca²⁺ responses were evoked by kainic acid (KA), α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA), and N-methyl-D -aspartate (NMDA). KA- and AMPA-stimulated changes in [Ca²⁺]_i were also produced in NM neurons stimulated in the presence of nifedipine, an L-type Ca²⁺ channel blocker, suggesting that KA- and AMPA-stimulated changes in [Ca²⁺]_i were carried by Ca²⁺-permeable receptor channels. Significantly smaller changes in [Ca²⁺]_i were produced by NMDA. When neurons were stimulated in an alkaline (pH 7.8) superfusate, NMDA responses were potentiated. KA- and AMPA-stimulated responses were not affected by pH. Several agents known to stimulate metabotropic receptors in other systems were tested on NM neurons bathed in a Ca²⁺ free-EGTA–buffered media, including l -cysteine sulfinic acid (L-CSA), trans-azetidine dicarboxylic acid (t-ADA), trans-aminocyclopentanedicarboxylic acid (t-ACPD), and homobromoibotenic acid (HBI). The only agent to reliably and dose-dependently increase [Ca²⁺]_i was HBI, an analog of ibotenate. GABA also stimulated increases in [Ca²⁺]_i in NM neurons. GABA-stimulated responses were reduced by agents that block voltage-operated channels and by agents that inhibit Ca²⁺ release from intracellular stores. Whereas GABA-A receptor agonist produced increases in [Ca²⁺]_i GABA-B and GABA-C receptor agonists had no effect. There appear to be several ways for [Ca²⁺]_i to increase in NM neurons. Presumably, each route represents a means by which Ca²⁺ can alter cellular processes. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 321–337, 1998     

Excitatory Amino Acid-Mediated Cytotoxicity and Calcium Homeostasis in Cultured Neurons

Abstract: A large body of evidence suggests that disturbances of Ca²⁺ homeostasis may be a causative factor in the neurotoxicity induced by excitatory amino acids (EAAs). The route or routes by which an increase in intracellular calcium concentration ([Ca²⁺]_i) is mediated in vivo are presently not clarified. This may partly reflect the complexity of intact nervous tissue in combination with the relative unspecific action of the available “calcium antagonists,” e.g., blockers of voltage-sensitive calcium channels. By using primary cultures of cortical neurons as a model system, it has been found that all EAAs stimulate increases in [Ca²⁺]_i but via different mechanisms. By using the drug dantrolene, it has been shown that 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate (AMPA) apparently exclusively stimulates Ca²⁺ influx through agonist-operated calcium channels and voltage-operated calcium channels. Increased [Ca²⁺]_i due to exposure to kainate (KA) is for the major part caused by influx, as in the case of AMPA, but a small part of the increase in [Ca²⁺]_i may be attributed to a release of Ca²⁺ from intracellular stores. Quisqualate (QA) stimulates Ca²⁺ release from an intracellular store that is independent of Ca²⁺ influx; presumably this store is activated by inositol phosphates. The increase in [Ca²⁺]_i due to exposure to glutamate or N-methyl-d -aspartate (NMDA) may be compartmentalized into three components, one of which is related to influx and the other two to Ca²⁺ release from internal stores. Only one of the latter stores is dependent on Ca²⁺ influx with regard to release of Ca²⁺, whereas the other is activated by some other second messengers or, alternatively, directly coupled to the receptor. In muscles dantrolene is known to inhibit Ca²⁺ release from the sarcoplasmic reticulum, and also in neurons dantrolene inhibits an equivalent release from one or more hitherto unidentified internal Ca²⁺ pool(s). By using this drug it has been possible to show to what extent these Ca²⁺ stores are involved in the toxicity observed subsequent to exposure to the EAAs. It turned out that dantrolene, even under conditions allowing Ca²⁺ influx, inhibited toxicity induced by QA, NMDA, and glutamate, whereas that induced by AMPA or KA was unaffected. In combination with the findings that dantrolene inhibited release from the intracellular stores activated by QA, NMDA, and glutamate, it may be concluded that Ca²⁺ influx per se is not the primary event causing toxicity following exposure to these EAAs in these neurons. However, it may certainly be involved in the cases of toxicity induced by AMPA and KA. Finally, it should be pointed out that this model only serves as a much simplified working hypothesis and that the situation in vivo is much more complex.     

Early activation of Ca2+‐permeable AMPA receptors reduces neurite outgrowth in embryonic chick retinal neurons

Calcium entry through Ca²⁺‐permeable AMPA/kainate receptors may activate signaling cascades controlling neuronal development. Using the fluorescent Ca²⁺‐indicator Calcium Green 1‐AM we showed that the application of kainate or AMPA produced an increase of intracellular [Ca²⁺] in embryonic chick retina from day 6 (E6) onwards. This Ca²⁺ increase is due to entry through AMPA‐preferring receptors, because it was blocked by the AMPA receptor antagonist GYKI 52466 but not by the N‐methyl‐D ‐aspartic acid (NMDA) receptor antagonist AP5, the voltage‐gated Ca²⁺ channel blockers diltiazem or nifedipine, or by the substitution of Na⁺ for choline in the extracellular solution to prevent the depolarizing action of kainate and AMPA. In dissociated E8 retinal cultures, application of glutamate, kainate, or AMPA reduced the number of neurites arising from these cells. The effect of kainate was prevented by the AMPA/kainate receptor antagonist CNQX and by GYKI 52466 but not by AP5, indicating that the reduction in neurite outgrowth resulted from the activation of AMPA receptors. Blocking Ca²⁺ influx through L‐type voltage‐gated Ca²⁺ channels with diltiazem and nifedipine prevented the effect of 10–100 μM kainate but not that of 500 μM kainate. In addition, joro spider toxin‐3, a blocker of Ca²⁺‐conducting AMPA receptors, prevented the effect of all doses of kainate. Neither GABA, which is depolarizing at this age in the retina, nor the activation of metabotropic glutamate receptors with tACPD mimicked the effects of AMPA receptor activation. Calcium entry via AMPA receptor channels themselves may therefore be important in the regulation of neurite outgrowth in developing chick retinal cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 200–211, 2001     

Pharmacological and functional characterization of excitatory amino acid mediated cytotoxicity in cerebral cortical neurons

The cytotoxic action of the excitatory amino acids (EAAs) glutamate, N-methyl- D-aspartate (NMDA), quisqualate (QA), kainate (KA) and (RS)-2-amino-3(3-hydoxy-5-methylisoxazol-4-yl) propionate (AMPA) was studied in cerebral cortical neurons in culture. The pharmacological profile of these actions was characterized using the NMDA selective antagonist D-(-)-2-amino-5- phosphonopentanoate (APV) and the non-NMDA selective antagonists 6.7- dinitroquinoxaline-2,3-dione (DNQX), 2-amino-3[3-(carboxymethoxy)-5- methylisoxazol-4-yl]-propionate (AMOA) and 2-amino-3-[2-(3-hydroxy-5- methylisoxazol-4-yl)methyl-3-methyl-3-oxoisoxazolin-4-yl] propionate (AMNH). The role of intracellular Ca⁺⁺ homeostasis and cGMP production for development of EAA mediated cytotoxicity was assessed by measurements of changes in [Ca⁺⁺]i using the flourescent Ca⁺⁺ chelator Fluo-3 and in cGMP concentrations using a conventional radioimmune assay. It was found that glutamate toxicity involves both NMDA and non-NMDA receptor activation and that aberrations in Ca⁺⁺ homeostasis brought about by Ca⁺⁺ influx and/or liberation of Ca⁺⁺ from internal stores aare important for development of toxicity. The drug dantrolene which prevents release of Ca⁺⁺ from such stores can prevent toxicity induced by glutamate, NMDA and QA completely but has no effect on KA and AMPA toxicity. Changes in cGMP levels appear to play a role for development of glutamate, NMDA and KA toxicity but does not seem to be involved in that triggered by QA and AMPA.Abbreviations AMNH: (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate) - AMOA: (2-amino-3[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propinate) - AMPA: ( (RS) —2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propinate) - APV: (D-(-)-2-amino-5-phosphonopentanoate) - DNQX: (6,7-dinitroquinoxaline-2,3-dione) - KA (kinate) - QA (quisqualate)     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Role of mitochondria in kainate-induced fast Ca2+ transients in cultured spinal motor neurons

Motor neuron death in amyotrophic lateral sclerosis (ALS) has been linked to selective vulnerability towards AMPA receptor-mediated excitotoxicity. We investigated intracellular mechanisms leading to impairment of motor neuron Ca2+ homeostasis with near physiological AMPA receptor activation. Using fast solution exchange on patch-clamped cultured neurons, kainate (KA) was applied for 2s. This induced a transient increase in the cytosolic Ca2+ concentration ([Ca2+]c) for seconds. Inhibition of the mitochondrial uniporter by RU-360 abolished the decay of the Ca2+ transient and caused immediate [Ca2+]c overload. Repetitive short KA stimulation caused a slowing of the decay of the Ca2+ transient and a gradual increase in peak and baseline [Ca2+]c in motor neurons, but not in other neurons, indicating saturation of the mitochondrial buffer. Furthermore, mitochondrial density was lower in motor neurons and, in a network of neurons with physiological synaptic AMPA receptor input, RU-360 acutely induced an increase in Ca2+ transients. We conclude that motor neurons have an insufficient mitochondrial capacity to buffer large Ca2+ elevations which is partly due to a reduced mitochondrial density per volume compared to non-motor neurons. This may exert deleterious effects in motor neuron disease where mitochondrial function is thought to be compromised.     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Neurotransmitters affect neuronal development by regulating intracellular Ca²⁺ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca²⁺ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca²⁺ concentration were measured with Fura-2 fluorescence. The Ca²⁺ rise to glutamate first appeared at E6, reached a maximum at E9–10, and then declined before the appearance of synaptic structures (E12). Ca²⁺ rises to kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D -aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca²⁺, suggesting the functional expression of Ca²⁺-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca²⁺rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca²⁺ rise to high K²⁺, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca²⁺ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 113–125, 1998     

Intracellular Ca regulates spike encoding at cortical GABAergic neurons and cerebellar Purkinje cells differently

Spike encoding at GABAergic neurons plays an important role in maintaining the homeostasis of brain functions for well-organized behaviors. The rise of intracellular Ca²⁺ in GABAergic neurons causes synaptic plasticity. It is not clear how intracellular Ca²⁺ influences their spike encoding. We have investigated this issue at GFP-labeled GABAergic cortical neurons and cerebellar Purkinje cells by whole-cell recording in mouse brain slices. Our results show that an elevation of intracellular Ca²⁺ by infusing adenophostin-A lowers spike encoding at GABAergic cortical neurons and enhances encoding ability at cerebellar Purkinje cells. These differential effects of cytoplasmic Ca²⁺ on spike encoding are mechanistically associated with Ca²⁺-induced changes in the refractory periods and threshold potentials of sequential spikes, as well as with various expression ratios of CaM-KII to calcineurin in GABAergic cortical neurons and cerebellar Purkinje cells.     

Mechanisms of heterogeneity of calcium response to kainate and neuronal types in rat cortical primary culture

The dynamics of intracellular Ca²⁺ signal in response to NMDA (N-methyl-D-aspartate, 30 μM) or KA (kainite, 30 μM), its dependence on extracellular Ca²⁺ and the mechanisms of KA-triggered Ca²⁺ entry into neurons have been tested in neurons of rat cortical primary cultures. The level of intracellular free Ca²⁺ concentrations ([Ca²⁺] _i ) was evaluated on Leica SP5 MF confocal microscope using Fluo-3 fluorescent dye, which resolves changes in [Ca²⁺] _i in the micromolar range. The dynamics of [Ca²⁺] _i increase in response to NMDA and KA was different but in both cases the [Ca²⁺] _i increase required the presence of Ca²⁺ in the extracellular solution. The neuronal population was found to be heterogeneous, based on the response to KA applied together with either L-type calcium channel blocker nifedipine (3 μM) or IEM-1460 (3 μM), a blocker of Ca²⁺-permeable AMPAR (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) lacking GluR2 subunit. Experiments exhibited three types of calcium responses, characteristically belonging to interneurons (expressing Ca²⁺-permeable AMPAR), pyramidal neurons (with AMPAR containing GluR2, making them impermeable to Ca²⁺), and intermediate type of cells expressing both AMPAR types. Thus, we have demonstrated the role of AMPAR and L-type calcium channels in KA-triggered Ca²⁺ entry into neurons. The dynamics of [Ca²⁺] _i during the KA treatment was shown to depend on subunit composition of particular AMPAR subtype expressed in neurons. The data suggest that neuronal types existing in adult cortical tissue are probably presented in primary culture, too.     

Calcium signalling in cortical and thalamic neurons of rats with streptozotocin-induced diabetes

Intracellular Ca²⁺ transients were measured with the use of a Ca²⁺-sensitive fluorescent indicator, fura-2, in neocortical and thalamic neurons in brain slices from control rats and rats with uncompensated streptozotocin-induced diabetes. The transients were evoked by high-potassium (50 mM)-induced membrane depolarization. The amplitude of depolarization-induced Ca²⁺ transients demonstrated a tendency to increase under diabetic conditions, beeing more expressed in cortical neurons compared with thalamic ones. The transients in cortical neurons from diabetic animals became also more susceptible to the blocking action of nifedipine (100μM) and less sensitive to Ni²⁺ (50μM), indicating that diabetic changes affect mostly Ca²⁺ transients triggered by high-voltage activated (L-type) calcium channels. The duration of a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ changes. However, a statistically significant increase was observed in the residual elevation of intracellular Ca²⁺ measured 60 sec after termination of membrane depolarization in both cortical and thalamic neurons, indicating alterations in the mechanisms that restore the resting level of Ca²⁺ in the cytosol. It is concluded that uncomensated insulin-dependent diabetes, which according to earlier data substantially alters calcium signalling in primary sensory neurons, also affects such signalling in the neurons of higher brain structures including the thalamus and cortex.     

Kainic acid-induced neurotrophic activities in developing cortical neurons

Using primary cultured cortical neurons from embryonic rat brains, we elucidated an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainic acid (KA) receptor-mediated neuroprotective mechanism through actions of nerve growth factor (NGF) in developing neurons. Neurotoxicity of KA in early days in vitro neurons was quite low compared with the mature neurons. However, pretreatment with anti-NGF antibody or TrkA inhibitor AG-879 profoundly raised KA toxicity. Furthermore, KA stimulation resulted in an increase of TrkA expression and phosphorylation, which was blocked not only by the AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione and AG-879, but also by the phospholipase C inhibitor U73122 and the intracellular calcium chelator BAPTA. A study of polyphosphoinositide turnover showed that KA-stimulated phospholipase C (PLC) activity was directly triggered by the AMPA/KA receptor activity, but not by the activity of TrkA or other excitatory amino acid receptor subtypes. Sources of KA-increased intracellular calcium levels were contributed by both extracellular calcium influx and intracellular calcium release and were partially sensitive to guanosine 5'-O-(2-thiodiphosphate). These results indicate that in developing cortical neurons, activation of AMPA/KA receptors by KA may induce expression, followed by activation of TrkA via PLC signaling and intracellular calcium elevation and hence increase reception of NGF on KA-challenged neurons. A G protein-coupled AMPA/KA receptor may be involved in these metabotropic events for neuronal protection.     

Nifedipine-sensitive low voltage-activated calcium current in neurons of the rat laterodorsal thalamic nucleus

The kinetic and pharmacological properties of low voltage-activated (LVA) Ca²⁺ channels were studied in neurons of the laterodorsal (LD) thalamic nucleus in brain slices from 12-day-old rats. A homogeneous population of LVA Ca²⁺ channels was found in the tested neurons. LVA Ca²⁺ current evoked by a step depolarization from a holding potential more negative than −70 mV was found to be sensitive to nifedipine (K _d=2.6 (M). This current gained its maximum at −55 mV and demonstrated fast monoexponential decay with the time constant of 32.3±4.0 msec (n=15). Lanthanum (1 μM) effectively blocked LVA Ca²⁺ current, while nickel (25 μM) did not affect this current. It is concluded that the channels that, according to their pharmacological properties, provide the studied LVA Ca²⁺ current cannot be regarded as T-type Ca²⁺ channels and belong to some other type of LVA Ca²⁺ channels.     

Expression of 15 glutamate receptor subunits and various splice variants in tissue slices and single neurons of brainstem nuclei and potential functional implications

Abstract: Brainstem nuclei serve a diverse array of functions in many of which ionotropic glutamate receptors are known to be involved. However, little detailed information is available on the expression of different glutamate receptor subunits in specific nuclei. We used RT‐PCR in mice to analyze the glutamate receptor subunit composition of the pre‐Bötzinger complex, the hypoglossal nucleus, the nucleus of the solitary tract, and the inferior olive. Analyzing 15 receptor subunits and five variants, we found all four α‐amino‐3‐hydroxy‐5‐methyl‐4‐propionic acid (AMPA) and six NMDA receptor (NR) subunits as well as three of five kainate (KA) receptors (GluR5, GluR6, and KA1) to be expressed in all nuclei. However, some distinct differences were observed: The inferior olive preferentially expresses flop variants of AMPA receptors, GluR7 is more abundant in the pre‐Bötzinger complex than in the other nuclei, and NR2C is most prominent in the nucleus of the solitary tract. In single hypoglossal motoneurons and interneurons of the pre‐Bötzinger complex investigation of GluR2 editing revealed strong expression of the GluR2‐R editing variant, suggesting low Ca²⁺ permeability of AMPA receptors. Thus, Ca²⁺ ‐permeable AMPA receptors are unlikely to be the cause for the reported selective vulnerability of hypoglossal motoneurons during excitotoxic events.