Ca2+-activated outward currents in neostriatal neurons

Whole-cell voltage-clamp recordings of outward currents were obtained from acutely dissociated neurons of the rat neostriatum in conditions in which inward Ca2+ current was not blocked and intracellular Ca2+ concentration was lightly buffered. Na+ currents were blocked with tetrodotoxin. In this situation, about 53 +/- 4% (mean +/- S.E.M.; n = 18) of the outward current evoked by a depolarization to 0 mV was sensitive to 400 microM Cd2+. A similar percentage was sensitive to high concentrations of intracellular chelators or to extracellular Ca2+ reduction (<500 microM); 35+/-4% (n=25) of the outward current was sensitive to 3.0 mM 4-aminopyridine. Most of the remaining current was blocked by 10 mM tetraethylammonium. The results suggest that about half of the outward current is activated by Ca2+ entry in the present conditions. The peptidic toxins charybdotoxin, iberotoxin and apamin confirmed these results, since 34 +/- 5% (n = 14), 29 5% (n= 14) and 28 +/- 6% (n=9) of the outward current was blocked by these peptides, respectively. The effects of charybdotoxin and iberotoxin added to that of apamin, but their effects largely occluded each other. There was additional Cd2+ block after the effect of any combination of toxins. Therefore, it is concluded that Ca2+-activated outward currents in neostriatal neurons comprise several components, including small and large conductance types. In addition, the present experiments demonstrate that Ca2+-activated K+ currents are a very important component of the outward current activated by depolarization in neostriatal neurons.     

Ca2+ channel currents in dorsal root ganglion neurons of P/Q-type voltage-gated Ca2+ channel mutant mouse, rolling mouse Nagoya

The role of the P/Q-type voltage-gated Ca(2+) channels (VGCCs) in release of neurotransmitters involved in nociception is not fully understood. Rolling mouse Nagoya (tg(rol)), a P/Q-type channel mutant mouse, expresses P/Q-type VGCC whose activation curve has a higher half activation potential and a smaller slope factor than the wild type channel. We previously reported that tg(rol) mice showed hypoalgesic responses to noxious stimuli. In this study, we examined the VGCC current in dorsal root ganglion (DRG) neurons by the whole-cell patch-clamp method. Both ω-agatoxin IVA (0.1 μM) and ω-conotoxin GVIA (1 μM) inhibited the VGCC current by about 40-50% in both the homozygous tg(rol) (tg(rol)/tg(rol)) and wild type (+/+) mice. The voltage-activation relationships of the total VGCC current and the ω-agatoxin IVA-sensitive component in the tg(rol)/tg(rol) mice shifted positively compared to the +/+ mice, whereas that sensitive to the ω-conotoxin GVIA was not different between the two genotypes. The time constant of activation of the VGCC current at -20 mV was longer in the tg(rol)/tg(rol) mice than in the +/+ mice. These changes in the properties of the VGCC in the tg(rol)/tg(rol) mouse may reduce the amount of the released neurotransmitters and account for the hypoalgesic responses.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons possess both "big" and "small" types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM omega-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 microM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM omega-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 microM omega-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM omega-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly. The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Ca2+ channels that activate Ca2+-dependent K+ currents in neostriatal neurons

It is demonstrated that not all voltage-gated calcium channel types expressed in neostriatal projection neurons (L, N, P, Q and R) contribute equally to the activation of calcium-dependent potassium currents. Previous work made clear that different calcium channel types contribute with a similar amount of current to whole-cell calcium current in neostriatal neurons. It has also been shown that spiny neurons posses both “big” and “small” types of calcium-dependent potassium currents and that activation of such currents relies on calcium entry through voltage-gated calcium channels. In the present work it was investigated whether all calcium channel types equally activate calcium-dependent potassium currents. Thus, the action of organic calcium channel antagonists was investigated on the calcium-activated outward current. Transient potassium currents were reduced by 4-aminopyridine and sodium currents were blocked by tetrodotoxin. It was found that neither 30 nM ω-Agatoxin-TK, a blocker of P-type channels, nor 200 nM calciseptine or 5 μM nitrendipine, blockers of L-type channels, were able to significantly reduce the outward current. In contrast, 400 nM ω-Agatoxin-TK, which at this concentration is able to block Q-type channels, and 1 μM ω-Conotoxin GVIA, a blocker of N-type channels, both reduced outward current by about 50%. These antagonists given together, or 500 nM ω-Conotoxin MVIIC, a blocker of N- and P/Q-type channels, reduced outward current by 70%. In addition, the N- and P/Q-type channel blockers preferentially reduce the afterhyperpolarization recorded intracellularly.The results show that calcium-dependent potassium channels in neostriatal neurons are preferentially activated by calcium entry through N- and Q-type channels in these conditions.     

Mitochondrial modulation of Ca2+ -induced Ca2+ -release in rat sensory neurons

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

P2Y1 receptor modulation of Ca2+-activated K+ currents in medium-sized neurons from neonatal rat striatal slices

ATP signaling to neurons and glia in the nervous system occurs via activation of both P2Y and P2X receptors. Here, we investigated the effects of P2Y(1) receptor stimulation in developing striatal medium-sized neurons using patch-clamp recordings from acute brain slices of 7- and 28-day-old rats. Application of the selective P2Y(1) receptor agonist 2-(Methylthio) ADP trisodium salt (2-MeSADP; 250 nM) increased outward K(+) currents evoked by a ramp depolarization protocol in voltage-clamp recordings. This effect was observed in 59 out of 82 cells (72%) and was blocked completely by the P2Y(1) antagonist, 2'-deoxy-N(6)-methyl adenosine 3',5'-diphosphate. The averaged 2-MeSADP-sensitive conductance was fitted by the sum of a linear conductance and a Boltzmann relation, giving one-half activation voltage of -14.2 mV and an equivalent charge of 2.91. The 2MeSADP-mediated effect was sensitive to submillimolar concentrations of tetraethylammonium (TEA; 200 μM), to 200 nM iberiotoxin and to 100 nM apamin, suggesting the involvement of both big and small potassium (BK and SK, respectively) calcium-activated channels. In current-clamp experiments, 2-MeSADP decreased depolarization-evoked action potential (AP) firing in all 26 cells investigated, and this effect was reversed by TEA and by apamin but not by iberiotoxin. We conclude that the stimulation of P2Y(1) receptors in developing striatal neurons leads to activation of calcium-activated potassium channels [I(K(Ca))] of both BK and SK subtypes, the latter responsible for decreasing the frequency of AP firing in response to current injection. Therefore, P2Y(1) signaling leading to activation of I(K(Ca)) may be important in regulating the activity of medium-sized neurons in the striatum.     

Slow degeneration of zebrafish Rohon-Beard neurons during programmed cell death.

Rohon-Beard cells are large, mechanosensory neurons located in the dorsal spinal cord of anamniote vertebrates. In most species studied to date, these cells die during development. We followed labeled Rohon-Beard cells in living zebrafish embryos and found that they degenerate slowly, over many days. During degeneration, the soma shrinks and finally disappears, and the processes become beady in appearance and finally break apart, but they do not retract. Zebrafish Rohon-Beard cells apparently fragment their DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) labeling, before undergoing degenerative morphologic changes. We also followed the development of labeled dorsal root ganglion neurons, as they are developing at the same stages that Rohon-Beard cells are degenerating. We found that, although axons of both cell types extend into similar regions, Rohon-Beard cells degenerate normally in mutants lacking dorsal root ganglia, providing evidence that interactions between the two cell types are not responsible for Rohon-Beard cell degeneration. Developmental Dynamics 229:30-41,2004.     

Blocker-resistant Ca2+ currents in rat CA1 hippocampal pyramidal neurons

Ca(2+) currents resistant to organic Ca(2+) channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca(2+) currents were isolated by combined application of N-, P/Q- and L-type Ca(2+) current antagonists (omega-conotoxin GVIA 2 microM; omega-conotoxin MVIIC 3 microM; omega-agatoxin IVA 200 nM; nifedipine 10 microM) and constituted approximately 21% of the total Ba(2+) current.The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of -76 and -17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca(2+) currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca(2+) channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni(2+) (half-maximal block at 28 microM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 microM, respectively).In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca(2+) channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming alpha subunits encoding high-threshold Ca(2+) channels, Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons.In summary, CA1 neurons display blocker-resistant Ca(2+) currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca(2+) channel messenger RNAs that give rise to R-type Ca(2+) currents in expression systems.     

Influence of Ca2+-binding proteins and the cytoskeleton on Ca2+-dependent inactivation of high-voltage activated Ca2+ currents in thalamocortical relay neurons

Ca²⁺-dependent inactivation (CDI) of high-voltage activated (HVA) Ca²⁺ channels was investigated in acutely isolated and identified thalamocortical relay neurons of the dorsal lateral geniculate nucleus (dLGN) by combining electrophysiological and immunological techniques. The influence of Ca²⁺-binding proteins, calmodulin and the cytoskeleton on CDI was monitored using double-pulse protocols (a constant post-pulse applied shortly after the end of conditioning pre-pulses of increasing magnitude). Under control conditions the degree of inactivation (34±9%) revealed a U-shaped and a sigmoid dependency of the post-pulse current amplitude on pre-pulse voltage and charge influx, respectively. In contrast to a high concentration (5.5 mM) of EGTA (31±3%), a low concentration (3 µM) of parvalbumin (20±2%) and calbindin_D28K (24±4%) significantly reduced CDI. Subtype-specific Ca²⁺ channel blockers indicated that L-type, but not N-type Ca²⁺ channels are governed by CDI and modulated by Ca²⁺-binding proteins. These results point to the possibility that activity-dependent changes in the intracellular Ca²⁺-binding capacity can influence CDI substantially. Furthermore, calmodulin antagonists (phenoxybenzamine, 22±2%; calmodulin binding domain, 17±1%) and cytoskeleton stabilizers (taxol, 23±5%; phalloidin, 15±3%) reduced CDI. Taken together, these findings indicate the concurrent occurrence of different CDI mechanisms in a specific neuronal cell type, thereby supporting an integrated model of this feedback mechanism and adding further to the elucidation of the role of HVA Ca²⁺ channels in thalamic physiology.     

Zinc reduces low-threshold Ca2+ currents of rat thalamic relay neurons

Whole-cell voltage-clamping of thalamic relay neurons was used to examine the possibility of a Zn(2+)-mediated reduction of the low-threshold transient Ca(2+) current (I(T)) of a central element in thalamocortical oscillations. We found that Zn(2+) reversibly decreased I(T) in a concentration-dependent manner (IC(50)=55 microM), mainly by reducing the number of I(T) channels available for activation. Zn(2+) did not affect the reaction kinetics, but did affect the voltage-dependence of I(T) channel gating. However, the apparent alterations in gating properties were not enough to account for the huge I(T) reduction.     

Kainate activates Ca2+-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices

Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/ kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the complex cells, characterized by voltage-gated Na⁺ and K⁺ channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K⁺ channels. In particular, the transient (inactivating) component of the outwardly rectifying K⁺ current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca²⁺. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca²⁺ increase was not a link to the K⁺ channel blockade, since the blockade occurred in the absence of the Ca²⁺ signal and, vice versa, an increase in cytosolic Ca²⁺ induced by ionomycin did not block the transient K⁺ current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.     

Presenilin 1 deficiency alters the activity of voltage-gated Ca2+ channels in cultured cortical neurons

Presenilins 1 and 2 (PS1 and PS2, respectively) play a critical role in mediating gamma-secretase cleavage of the amyloid precursor protein (APP). Numerous mutations in the presenilins are known to cause early-onset familial Alzheimer's disease (FAD). In addition, it is well established that PS1 deficiency leads to altered intracellular Ca(2+) homeostasis involving endoplasmic reticulum Ca(2+) stores. However, there has been little evidence suggesting Ca(2+) signals from extracellular sources are influenced by PS1. Here we report that the Ca(2+) currents carried by voltage-dependent Ca(2+) channels are increased in PS1-deficient cortical neurons. This increase is mediated by a significant increase in the contributions of L- and P-type Ca(2+) channels to the total voltage-mediated Ca(2+) conductance in PS1 (-/-) neurons. In addition, chelating intracellular Ca(2+) with 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) produced an increase in Ca(2+) current amplitude that was comparable to the increase caused by PS1 deficiency. In contrast to this, BAPTA had no effect on voltage-dependent Ca(2+) conductances in PS1-deficient neurons. These data suggest that PS1 deficiency may influence voltage-gated Ca(2+) channel function by means that involve intracellular Ca(2+) signaling. These findings reveal that PS1 functions at multiple levels to regulate and stabilize intracellular Ca(2+) levels that ultimately control neuronal firing behavior and influence synaptic transmission.     

Norepinephrine selectively reduces slow Ca2+- and Na+-mediated K+ currents in cat neocortical neurons

1. The effects of norepinephrine (NE) and related agonists and antagonists were examined on large neurons from layer V of cat sensorimotor cortex ("Betz cells") were examined in a brain slice preparation using intracellular recording, constant current stimulation and single microelectrode voltage clamp. 2. Application of NE (0.1-100 microM) usually caused a small depolarization from resting potential; hyperpolarizations were rare. Application of NE reversibly reduced rheobase and both the Ca2+- and Na+-dependent portions of the slow afterhyperpolarization (sAHP) that followed sustained firing evoked by constant current injection. The faster Ca2+-dependent medium afterhyperpolarization (mAHP), the fast afterhyperpolarization (fAHP), the action potential, and input resistance were unaffected. 3. The changes in excitability produced by NE application were most apparent during prolonged stimulation. The cells exhibited steady repetitive firing to currents that were formerly ineffective. The slow phase of spike frequency adaptation was reduced selectively and less habituation occurred during repeated long-lasting stimuli. The relation between firing rate and injected current became steeper if firing rate was averaged over several hundred milliseconds. 4. During voltage clamp in TTX, NE application selectively reduced the slow component of Ca2+-mediated K+ current. The faster Ca2+-mediated K+ current was unaffected, as were two voltage-dependent, transient K+ currents, the anomalous rectifier and leakage conductance measured at resting potential. Depolarizing voltage steps in the presence of Cd2+ revealed an apparent time- and voltage-dependent increase of the persistent Na+ current after NE application. The voltage-clamp results suggested ionic mechanisms for all effects seen during constant current stimulation except the depolarization from resting potential. The latter was insensitive to Cd2+ and TTX and occurred without a detectable change in membrane conductance. 5. NE application did not alter Ca2+ spikes evoked in the presence of TTX and 10 mM TEA. Inward Ca2+ currents examined during voltage clamp in TTX (with K+ currents reduced) became slightly larger after NE application. We conclude that NEs reduction of the slow Ca2+-mediated K+ current is not caused by reduction of Ca2+ influx. 6. Effects on membrane potential, rheobase, and the sAHP were mimicked by the beta-adrenergic agonist isoproterenol, but not by the alpha-adrenergic agonists clonidine or phenylephrine at higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+-dependent large conductance K+ currents in thalamocortical relay neurons of different rat strains

Mutations in genes coding for Ca²⁺ channels were found in patients with childhood absence epilepsy (CAE) indicating a contribution of Ca²⁺-dependent mechanisms to the generation of spike-wave discharges (SWD) in humans. Since the involvement of Ca²⁺ signals remains unclear, the aim of the present study was to elucidate the function of a Ca²⁺-dependent K⁺ channel (BK_Ca) under physiological conditions and in the pathophysiological state of CAE. The activation of BK_Ca channels is dependent on both voltage and intracellular Ca²⁺ concentrations. Moreover, these channels exhibit an outstandingly high level of regulatory heterogeneity that builds the basis for the influence of BK_Ca channels on different aspects of neuronal activity. Here, we analyse the contribution of BK_Ca channels to firing of thalamocortical relay neurons, and we test the hypothesis that BK_Ca channel activity affects the phenotype of a genetic rat model of CAE. We found that the activation of the β₂-adrenergic receptor/protein kinase A pathway resulted in BK_Ca channel inhibition. Furthermore, BK_Ca channels affect the number of action potentials fired in a burst and produced spike frequency adaptation during tonic activity. The latter result was confirmed by a computer modelling approach. We demonstrate that the β₂-adrenergic inhibition of BK_Ca channels prevents spike frequency adaptation and, thus, might significantly support the tonic firing mode of thalamocortical relay neurons. In addition, we show that BK_Ca channel functioning differs in epileptic WAG/Rij and thereby likely contributes to highly synchronised, epileptic network activity.