Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Divalent Cation Entry in Cultured Rat Cerebellar Granule Cells Measured Using Mn2+ Quench of Fura 2 Fluorescence

In this study the rate of Mn²⁺ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca²⁺ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca²⁺ entry in isolation from effects on Ca²⁺ store release. The rate of fluorescence quench by 0.1 mM Mn²⁺ was markedly increased by 25 mM K⁺- evoked depolarization or by 200 μM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K⁺- or NMDA-evoked [Ca²⁺]_i response. The stimulatory effect of NMDA on Mn²⁺ quench was abolished by dizocilpine (10 μM), but nitrendipine (2 μM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn²⁺ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca²⁺ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t_1/2 of quench was unaffected by α-amino-hydroxyisoxazole propionic acid (200 μM) and carbamyl choline (1 mM). Neither thapsigargin (10 μM) nor dantrolene (30 μM) significantly affected the rate of quench under control or NMDA- or K⁺-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca²⁺]_i elevations evoked by these agents are due to actions on Ca²⁺ stores. However, thapsigargin elevated [Ca²⁺Ii in the presence of normal [Ca²⁺]_i, but not in nominally Ca²⁺-free medium, indicating that it evokes Ca²⁺ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn²⁺ quench or to occur via Mn²⁺-impermeant channels.     

Ca2+‐dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20–70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca²⁺ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca²⁺ transients in circadian pacemakers accompany tetrodotoxin (TTX)‐blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX‐sensitive sustained Na⁺ channels. Instead, they are predominantly driven by mibefradil‐sensitive, low‐voltage‐activated Ca²⁺ channels and DK‐AH269‐sensitive hyperpolarisation‐activated, cyclic nucleotide‐gated cation channels. Rhythmic depolarisations activate voltage‐gated Na⁺ channels and nifedipine‐sensitive high‐voltage‐activated Ca²⁺ channels. Together with Ca²⁺ rises, the depolarisations open repolarising small‐conductance but not large‐conductance Ca²⁺‐dependent K⁺ channels. In contrast, we hypothesise that P/Q‐type Ca²⁺ channels coupled to large‐conductance Ca²⁺‐dependent K⁺ channels are involved in input‐dependent activity.     

Voltage Oscillations in Xenopus Spinal Cord Neurons: Developmental Onset and Dependence on Co-activation of NMDA and 5HT Receptors

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 μM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2–5 μM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg²⁺ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT_1a receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT₁, a selective 5HT_1a receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg²⁺, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4–8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an ‘apparent’ decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg²⁺ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg²⁺ block. When the membrane potential was depolarized by ～20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg²⁺ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg²⁺ through activation of receptors with 5HT_1a-like pharmacology.     

Effect of kainate on the membrane conductance of hilar glial precursor cells recorded in the perforated-patch configuration

The effects of kainate on membrane current and membrane conductance were investigated in presumed hilar glial precursor cells of juvenile rats. The perforated-patch configuration was used also to reveal possible second-messenger effects. Kainate evoked an inward current that was accompanied by a biphasic change in membrane conductance in 69% of the cells. An initial conductance increase with a time course similar to that of the inward current was followed by a second delayed conductance increase. This second conductance was absent in whole-cell-clamp recordings, suggesting that it was mediated by a second messenger effect. Analysis of the reversal potentials of the membrane current during both phases of the kainate-induced conductance change revealed that the first conductance increase reflected the activation of AMPA receptors. Several lines of evidence suggest that the delayed second conductance increase was due to the indirect activation of Ca²⁺-dependent K⁺ channels via Ca²⁺-influx through AMPA receptors. (1) the delayed second conductance increase was blocked by Ba²⁺ and the reversal of its underlying current was significantly shifted towards E_K+, suggesting that it is due to the activation of K⁺ channels. (2) The delayed second conductance increase disappeared in a Ca²⁺-free saline buffered with BAPTA, indicating that it depended on Ca²⁺-influx. (3) Co²⁺, Cd²⁺ and nimodipine failed to block the delayed second conductance increase excluding a major contribution of voltage-dependent Ca²⁺ channels. (4) The involvement of metabotropic glutamate receptors also appeared unlikely, because the kainate-induced delayed second conductance increase could not be blocked by a depletion of the intracellular Ca²⁺ stores with the Ca²⁺-ATPase inhibitor thapsigargin, and t-ACPD exerted no effect on membrane current and conductance. We conclude that kainate activates directly AMPA receptors in presumed hilar glial precursor cells. This results in a Ca²⁺ influx that could lead indirectly to the activation of Ca²⁺-dependent K⁺ channels. GLIA 23:35–44, 1998. © 1998 Wiley-Liss, Inc.     

Calcium induced calcium release is involved in the afterhyperpolarization in one class of guinea pig sympathetic neurone

The mechanisms underlying two potassium conductances which are activated by Ca²⁺ influx during the action potential in ympathetic prevertebral neurones of guinea pigs have been investigated pharmacologically. One Ca-activated K⁺ conductance, which is present in all mammalian sympathetic postganglionic neurones, is maximal after the action potential and decays exponentially with a time constant of about 130 ms; this conductance was inhibited by apamin (50–100 nM) consistent with the involvement of SK channels. A second Ca-activated K⁺ conductance with much slower kinetics is present in a large subpopulation of coeliac neurones. This conductance was resistant to apamin but markedly inhibited by application of ryanodine (5–20 μM). suggesting that Ca²⁺ influx during the action potential triggers release of Ca²⁺ from intracellular stores which in turn activates a different class of K⁺ channel. Noradrenaline (100 μM) depressed the second K⁺ conductance selectively.     

Mechanisms and functional significance of a slow inhibitory potential in neurons of the lateral amygdala

A slow inhibitory potential (sIP) elicited upon synaptic activation in spiny, pyramidal-like cells with properties indicative of projection neurons was investigated in slices of the rat and guinea-pig lateral amygdala in vitro. The sIP succeeded the triphasic sequence of excitatory and fast/slow inhibitory postsynaptic potentials mediated via glutamate and GABA_A/B receptors, respectively, was readily evoked upon repetitive stimulation of the external capsule and appeared to terminate epileptiform burst discharges during pharmacologically reduced GABAergic influence. The sIP reversed close to the Cl^– equilibrium potential, but was not affected by altered transmembrane Cl^– gradients and not abolished by antagonists to ligand-gated Cl^– channels. Intracellular injection of QX 314 and resulting blockade of sodium spikes had no effect, whereas the Ca²⁺ chelator BAPTA blocked the sIP concomitantly with slow hyperpolarizing afterpotentials following intrinsically generated spike firing, thereby indicating the contribution of Ca²⁺-dependent mechanisms secondary to synaptic activation. During action of BAPTA and QX 314, an N-methyl-d -aspartate (NMDA) receptor-mediated potential was unmasked, which contributed to the sIP. The Ca²⁺-dependent mechanisms of the sIP involved a membrane K⁺ conductance, as was indicated by the dependence on the K⁺ gradient and the shift of the reversal potential towards the K⁺ equilibrium potential during blocked NMDA receptors. During the presence of GABA receptor antagonists, reduction of the Ca²⁺-activated K⁺ conductance through injection of BAPTA or application of dopamine induced a gradual shift of interictal-like single bursts of spikes towards the generation of re-occurring ictal-like activity. It is concluded that pyramidal-like projection cells in the AL can generate a sIP upon synaptic activation, which reflects the combined activation of an NMDA receptor-mediated cation current and a K⁺ current that is secondary to the rise in intracellular Ca²⁺ concentration resulting from the preceding depolarizing response. The sIP may play an important role in controlling excitatory activity in the amygdala, particularly in preventing the transformation of interictal-like activity towards recurrent epileptic discharges during periods of decreased GABAergic influence.     

Calcium-independent release of [H]spermine from chick retina

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [³H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [³H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca²⁺-independent manner. Inhibition of Na⁺/K⁺-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na⁺ electrochemical gradients, since nigericin and veratrine did not induce release in Na⁺ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.     

Glutamate receptors communicate with Na+/K+-ATPase in rat cerebellum granule cells

Two glutamate receptor agonists, NMDA (N-methyl-d-aspartic acid) and ACPD (cis-(1S/3R)-1-aminocyclopentane-1,3-dicarboxylic acid), induce the reactive oxygen species (ROS) production in rat cerebellum granule cells, whereas the third one, 3-HPG (3-hydroxyphenylglycine), decreases this parameter. The simultaneous presence of 3-HPG, together with NMDA or ACPD, prevents the generation of ROS by neuronal cells. A similar effect of these ligands on Na⁺/K⁺-ATPase can be demonstrated: NMDA and ACPD inhibited the enzyme activity, but 3-HPG activated Na⁺/K⁺-ATPase and prevented its inhibition by NMDA or ACPD. In terms of current classification, NMDA is an agonist of ionotropic glutamate receptors of the so-called NMDA class, whereas ACPD and 3-HPG belong to metabotropic agonists, the former primarily being an activator of metabotropic glutamate receptors (mGluRs) of groups 2 and 3, and the latter, that of mGluRs of groups 1 and 5. Thus, the data presented illustrate the existence of diverse mechanisms of the cross talk between Na⁺/K⁺-ATPase and different glutamate receptors, as well as that between glutamate receptors of different classes.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Pharmacology of nicotine-induced increase in cytosolic Ca concentrations in chick embryo ciliary ganglion cells

Chick embryo ciliary ganglion cells were acutely isolated, and the mechanism(s) underlying the increase in the cytosolic Ca²⁺ concentration ([Ca]_in) induced by high concentrations of nicotine examined using fura-2 microfluorometry. The order of potencies of nicotinic receptor agonists in increasing [Ca]_in was ACh > nicotine = dimethylphenylpiperazinium > cytisine. The nicotine-induced increase in [Ca]_in was inhibited not only by nicotinic antagonists but also by muscarinic antagonists, while the muscarine-induced [Ca]_in increase was little affected by nicotinic antagonists. The nicotine-induced [Ca]_in increase was inhibited by both L- and N-type Ca²⁺ channel blockers and potentiated by an L-type Ca²⁺ channel agonists, Bay-K-8644. Nicotine also increased the cytosolic Na⁺ concentration ([Na]_in) as measured by sodium binding benzofuranisophthalate microfluorometry, and this [Na]_in increase was inhibited by various agents which reportedly affected nicotinic receptor channels resulting in chromaffin cells. These results suggest that nicotine increased Na⁺ influx nicotinic receptor channels resulting in membrane depolarization, which in turn increased Ca²⁺ influx through voltage-dependent Ca²⁺ channels. However, nicotine still increased influxes of Ca²⁺ and Mn²⁺ in the absence of external Na⁺, suggesting that nicotinic receptor channels in these cells are permeable not only to monovalent cations but also to Ca²⁺ and Mn²⁺.     

N-methyl-d-aspartate and hypoxia induced Ca-changes in the CA1 region of the hippocampal slice

Hypoxic neuronal depolarization was accompanied by a large decrease in extracellular [Ca²⁺]. After reoxygenation, the time at which [Ca²⁺] normalized was correlated with the extent of recovery of N-methyl-d-aspartate (NMDA) and synaptic responses. There was no evidence that the NMDA receptor system was more disrupted following hypoxia than the receptors involved in synaptic transmission. The Na⁺/K⁺ pump appeared to be better able to recover from hypoxia than the NMDA responses or synaptic transmission.     

Properties of Single Calcium-activated Potassium Channels of Large Conductance in Rat Hippocampal Neurons in Culture

Patch-clamp recordings were made on rat hippocampal neurons maintained in culture. In cell-attached and excised inside-out and outside-out patches a large single-channel current was observed. This channel had a conductance of 220 and 100 pS in 140 mM [K⁺]_i/140 mM [K⁺]_o and 140 mM [K⁺]_i/3 mM [K⁺]_o respectively. From the reversal potential the channel was highly selective for K⁺, the P_K+/P_na+ ratio being 50/1. Channel activity was voltage-dependent, the open probability at 100 mM [Ca²⁺]_i increasing by e-fold for a 22 mV depolarization. It was also dependent on [Ca²⁺]_i at both resting and depolarized membrane potentials. Channel open states were best described by the sum of two exponentials with time constants that increased as the membrane potential became more positive. Channel activity was sensitive to both external (500 μM) and internal (5 mM) tetraethylammonium chloride. These data are consistent with the properties of maxi-K⁺ channels described in other preparations, and further suggest a role for maxi-channel activity in regulating neuronal excitability at the resting membrane potential. Channel activity was not altered by 8-chlorophenyl thio cAMP, concanavalin A, pH reduction or neuraminidase. In two of five patches lemakalim (BRL 38227) increased channel activity. Internal ruthenium red (10 μM) blocked the channel by shortening the duration of both open states. This change in channel gating was distinct from the ‘mode switching’ seen in two patches, where a channel switched spontaneously from normal activity typified by two open states to a mode where only short openings were represented.     

Functional identification of an outwardly rectifying pH‐ and anesthetic‐sensitive leak K+ conductance in hippocampal astrocytes

Astrocytes function as spatial K⁺ buffers by expressing a rich repertoire of K⁺ channels. Earlier studies suggest that acid‐sensitive tandem‐pore K⁺ channels, mainly TWIK‐related acid‐sensitive K⁺ (TASK) channels, mediate part of the passive astroglial membrane conductance. Here, using a combination of electrophysiology and pharmacology, we investigated the presence of TASK‐like conductance in hippocampal astrocytes of rat brain slices. Extracellular pH shifts to below 7.4 (or above 7.4) induced a prominent inward (or outward) current in astrocytes in the presence of tetrodotoxin, a Na⁺ channel blocker, and 4,4′‐diisothiocyanatostilbene‐2,2’‐disulfonate, a co‐transporter blocker. The pH‐sensitive current was insensitive to quinine, a potent blocker of tandem‐pore K⁺ channels including TWIK‐1 and TREK‐1 channels. Voltage‐clamp analysis revealed that the pH‐sensitive current exhibited weak outward rectification with a reversal potential of ?112 mV, close to the Nernst equilibrium potential for K⁺. Furthermore, the current–voltage relationship was well fitted with the Goldman–Hodgkin–Katz current equation for the classical open‐rectifier ‘leak’ K⁺ channel. The pH‐sensitive K⁺ current was potentiated by TASK channel modulators such as the volatile anesthetic isoflurane but depressed by the local anesthetic bupivacaine. However, unlike TASK channels, the pH‐sensitive current was insensitive to Ba²⁺ and quinine. Thus, the molecular identity of the pH‐sensitive leak K⁺ channel is unlikely to be attributable to TASK channels. Taken together, our results suggest a novel yet unknown leak K⁺ channel underlying the pH‐ and anesthetic‐sensitive background conductance in hippocampal astrocytes.     

Roles of somatic A-type K~+ channels in the synaptic plasticity of hippocampal neurons

In the mammalian brain, information encoding and storage have been explained by revealing the cellular and molecular mechanisms of synaptic plasticity at various levels in the central nervous system, including the hippocampus and the cerebral cortices. The modulatory mechanisms of synaptic excitability that are correlated with neuronal tasks are fundamental factors for synaptic plasticity, and they are dependent on intracellular Ca²⁺-mediated signaling. In the present review, the A-type K⁺ (I _A) channel, one of the voltage-dependent cation channels, is considered as a key player in the modulation of Ca²⁺ influx through synaptic NMDA receptors and their correlated signaling pathways. The cellular functions of I _A channels indicate that they possibly play as integral parts of synaptic and somatic complexes, completing the initiation and stabilization of memory.     

Molecular dissection of the myelinated axon

The membrane of the myelinated axon expresses a rich repertoire of physiologically active molecules: (1) Voltagesensitive NA⁺ channels are clustered at high density (～1,000/μm²) in the nodal axon membrane and are present at lower density(<25/μm²) in the internodal axon membrane under the myelin. Na⁺ channels are also present within Schwann cell processes (in peripheral nerve) and perinodal astrocyte processes (in the central nervous system) which contact the Na⁺ channel–rich axon membrane at the node. In some demyelinated fibers, the bared (formerly internodal) axon membrane reorganizes and expresses a higher-than-normal Na⁺ channel density, providing a basis for restoration of conduction. The presence of glial cell processes, adjacent to foci of Na⁺ channels in immature and demyelinated axons, suggests that glial cells participate in the clustering of Na⁺ channels in the axon membrane. (2) “Fast” K⁺ channels, sensitive to 4-aminopyridine, are present in the paranodal of internodal axon membrane under the myelin; these channels may function to prevent reexcitation following action potentials, or participate in the generation of an internodal resting potential. (3) “Slow” K⁺ channels, sensitive to tetraethylammonium, are present in the nodal axon membrane and, in lower densities, in the internodal axon membrane; their activation produces a hyperpolarizing afterpotential which modulates repetitive firing. (4) The “inward rectifier” is activated by hyperpolarization. This channel is permeable to both Na⁺ and K⁺ ions and may modulate axonal excitability or participate in ionic reuptake following activity. (5) Na⁺/K⁺-ATPase and (6) Ca²⁺-ATPase are also present in the axon membrane and function to maintain transmembrane gradients of Na⁺, K⁺, and Ca²⁺. (7) A specialized antiporter molecule, the Na⁺/Ca²⁺ exchanger, is present in myelinated axons within central nervous system white matter. Following anoxia, the Na⁺/Ca²⁺ exchanger mediates an influx of Ca²⁺ which damages the axon. The molecular organization of the myelinated axon has important pathophysiological implications. Blockade of fast K⁺ channels and Na⁺/K⁺-ATPase improves action potential conduction in some demyelinated axons, and block of the Na⁺/Ca²⁺ exchanger protects white matter axons from anoxic injury. Modification of ion channels, pumps, and exchangers in myelinated fibers may thus provide an important therapeutic approach for a number of neurological disorders.     

Glutamate release evoked by glutamate receptor agonists in cultured chick retina cells: Modulation by arachidonic acid

We studied the effect of ionotropic glutamate receptor agonists on the release of endogenous glutamate or of [³H]D -aspartate from reaggregate cultures (retinospheroids) or from monolayer cultures of chick retinal cells, respectively. Kainate increased the fluorescence ratio of the Na⁺ indicator SBFI and stimulated a dose-dependent release of glutamate in low (0.1 mM) Ca²⁺ medium, as measured using a fluorometric assay. Under the same experimental conditions, the release evoked by N-methyl-D -aspartate (NMDA; 400 μM) was about half of that evoked by the same kainate concentration; α-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid (AMPA; 400 μM) did not trigger a significant response. In the presence of 1 mM CaCl₂, all of the agonists increased the [Ca²⁺]_i, as determined with the fluorescence dye Indo-1, but the glutamate release evoked by NMDA and kainate was significantly lower than that measured in 0.1 mM CaCl₂ medium. Inhibition by Ca²⁺ of the kainate-stimulated release of glutamate was partially reversed by the phospholipase A₂ inhibitor oleiloxyethyl phosphorylcholine (OPC), suggesting that the effect was mediated by the release of arachidonic acid, which inhibits the glutamate carrier. Accordingly, kainate, NMDA, and AMPA stimulated a Ca²⁺-dependent release of [³H]arachidonic acid, and the direct addition of the exogenous fatty acid to the medium decreased the release of glutamate evoked by kainate in low (0.1 mM) CaCl₂ medium. In monolayer cultures, we showed that NMDA, kainate, and AMPA also stimulated the release of [³H]D -aspartate, but in this case release in the presence of 1 mM CaCl₂ was significantly higher than that evoked in media with no added Ca²⁺. The ranking order of efficacy for stimulation of Ca²⁺-dependent release of [³H]D -aspartate was NMDA ≪ kainate < AMPA. © 1996 Wiley-Liss, Inc.     

Neuroprotective activity of stiripentol with a possible involvement of voltage‐dependent calcium and sodium channels

A growing body of data has shown that recurrent epileptic seizures may be caused by an excessive release of the excitatory neurotransmitter glutamate in the brain. Glutamatergic overstimulation results in massive neuronal influxes of calcium and sodium through N‐methyl‐D‐aspartate (NMDA), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid, and kainic acid glutamate subtype receptors and also through voltage‐gated calcium and sodium channels. These persistent and abnormal sodium and calcium entry points have deleterious consequences (neurotoxicity) for neuronal function. The therapeutic value of an antiepileptic drug would include not only control of seizure activity but also protection of neuronal tissue. The present study examines the in vitro neuroprotective effects of stiripentol, an antiepileptic compound with γ‐aminobutyric acidergic properties, on neuronal–astroglial cultures from rat cerebral cortex exposed to oxygen–glucose deprivation (OGD) or to glutamate (40 µM for 20 min), two in vitro models of brain injury. In addition, the affinity of stiripentol for the different glutamate receptor subtypes and the interaction with the cell influx of Na⁺ and of Ca²⁺ enhanced by veratridine and NMDA, respectively, are assessed. Stiripentol (10–100 µM) included in the culture medium during OGD or with glutamate significantly increased the number of surviving neurons relative to controls. Stiripentol displayed no binding affinity for different subtypes of glutamate receptors (IC₅₀ > 100 µM) but significantly blocked the entry of Na⁺ and Ca²⁺ activated by veratridine and NMDA, respectively. These results suggest that Na⁺ and Ca²⁺ channels could contribute to the neuroprotective properties of sitiripentol. © 2015 Wiley Periodicals, Inc.     

Cystic Fibrosis: a disease of ion channels?

Cystic fibrosis is of interest to neuroscientists because it appears to be a disease of ion channels. It is not the conduction properties of ion channels that are affected, but rather their gating by chemical agonists. These conductance pathways appear to be unique to epithelial cells in which salt and water transport rates are governed by cAMP- and Ca²⁺-dependent regulatory processes.     

Calcium-Activated Potassium Channels in Rat Dissociated Ventromedial Hypothalamic Neurons

A potassium-selective channel, characterized by a single channel conductance of 160 pS was demonstrated to be present in rat freshly dispersed ventromedial hypothalamic nucleus neurons. The single channel activity was shown to be dependent, using inside-out membrane patches, upon the presence of intracellular calcium ions, with maximal sensitivity between 10^?6 and 10^?6 M[Ca²⁺], and to be modulated by membrane voltage, depolarization causing an increase in open-state probability in the presence of an activating concentration of calcium. Therefore these properties place this channel into the category of a large conductance (maxi-K⁺) calcium-activated potassium (Ca²⁺-K⁺) channel. This channel is active in cell-attached recordings from glucoreceptive cells when depolarized by glucose or tolbutamide with openings often associated with action current repolarization. These openings were shown to be abolished in the presence of extracellular Cd²⁺ and La³⁺ ions, which block calcium channels, suggesting that extracellular calcium entry upon cell depolarization is responsible for their activation. On a few occasions, a larger conductance (250 pS) Ca²⁺-K⁺ channel was observed in inside-out membrane patches isolated from ventromedial hypothalamic nucleus neurons. In contrast to the 160 pS channel, the presence of intracellularly-applied ATP caused a concentration-dependent, reversible inhibition of its open-state probability.