Neurotrophin-4/5 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons

Ca(2+) currents and their modulation by neurotrophin-4/5 were studied in cultured mesencephalic neurons. Tyrosine hydroxylase-positive neurons consistently had larger somas than tyrosine hydroxylase-negative neurons. Neurons with larger somas were therefore targeted for recording. In both control and neurotrophin-4/5-treated cultured neurons, isolation of Ca(2+) currents in cultured mesencephalic neurons revealed prominent low- and high-voltage-activated currents. These currents were separable based upon their voltage dependence of activation, the response to replacement of Ca(2+) with Ba(2+) and the response to Ca(2+) channel blockers. Replacement of Ca(2+) with Ba(2+) resulted in a slight reduction of low-voltage-activated currents and a significant enhancement of high-voltage-activated currents. Cd(2+) blocked a larger fraction of the high-voltage-activated current than Ni(2+). The synthetic conotoxins SNX-124 and SNX-230 selectively blocked high-voltage-activated currents. Morphological analysis of mesencephalic cultures pretreated with neurotrophin-4/5 revealed an increase in soma size and dendritic length in tyrosine hydroxylase-positive neurons. In agreement with the neurotrophin-4/5 induction of growth, neurotrophin-4/5 also increased cell capacitance in whole-cell recordings. Neurotrophin-4/5 significantly enhanced both low- and high-voltage-activated currents, but normalization for changes in capacitance revealed only a significant increase in high-voltage-activated current density.This study demonstrates the existence of low-voltage-activated and multiple classes of high-voltage-activated calcium currents in cultured mesencephalic neurons. Morphological and physiological data demonstrate that the increases in calcium currents due to neurotrophin-4/5 pretreatment are associated with somatodendritic growth, but an increase in high-voltage-activated Ca(2+) channel expression also occurred.     

High voltage-activated Ca(2+) channel currents in isolectin B(4)-positive and -negative small dorsal root ganglion neurons of rats

Voltage-gated Ca(2+) channels in the primary sensory neurons are important for neurotransmitter release and regulation of nociceptive transmission. Although multiple classes of Ca(2+) channels are expressed in the dorsal root ganglion (DRG) neurons, little is known about the difference in the specific channel subtypes among the different types of DRG neurons. In this study, we determined the possible difference in high voltage-activated Ca(2+) channel currents between isolectin B(4) (IB(4))-positive and IB(4)-negative small-sized (15-30 microm) DRG neurons. Rat DRG neurons were acutely isolated and labeled with IB(4) conjugated to a fluorescent dye. Whole-cell patch clamp recordings of barium currents flowing through calcium channels were performed on neurons with and without IB(4). The peak current density of voltage-gated Ca(2+) currents was not significantly different between IB(4)-positive and IB(4)-negative neurons. Also, both nimodipine and omega-agatoxin IVA produced similar inhibitory effects on Ca(2+) currents in these two types of neurons. However, block of N-type Ca(2+) channels with omega-conotoxin GVIA produced a significantly greater reduction of Ca(2+) currents in IB(4)-positive than IB(4)-negative neurons. Furthermore, the IB(4)-positive neurons had a significantly smaller residual Ca(2+) currents than IB(4)-negative neurons. These data suggest that a higher density of N-type Ca(2+) channels is present in IB(4)-positive than IB(4)-negative small-sized DRG neurons. This differential expression of the subtypes of high voltage-activated Ca(2+) channels may contribute to the different function of these two classes of nociceptive neurons.     

Maintenance of sympathetic tone by a nickel chloride-sensitive mechanism in the rostral ventrolateral medulla of the adult rat

In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni(2+); 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (-44+/-6%) and a marked depressor response (-35+/-7 mmHg). Selective blockade of high-voltage activated Ca(2+) channels with omega-agatoxin IVA (P/Q-type), omega-conotoxin GVIA (N-type) and nifedipine (L-type) did not decrease arterial pressure or splanchnic sympathetic nerve activity when injected separately into the rostral ventrolateral medulla, or combined with kynurenate. Injection of caesium chloride or ZD 7288, a blocker of the hyperpolarization-activated cation current, into the rostral ventrolateral medulla had no effect on arterial pressure or splanchnic sympathetic nerve activity. Bilateral microinjection of nickel chloride into the caudal ventrolateral medulla/pre-B?tzinger complex elicited small increases in splanchnic sympathetic nerve activity (+17+/-13%) and arterial pressure (+12+/-4 mmHg). These were substantially smaller than those evoked by blockade of glutamatergic receptors or high-voltage activated Ca(2+) channels in this area. Injection of kynurenate or high-voltage activated Ca(2+) channel blocker, but not Ni(2+), in this area evoked respiratory termination. The results indicate the existence of a distinct mechanism maintaining the tonic activity of rostral ventrolateral medulla presympathetic neurons that is different from that maintaining the tonic activity in the caudal ventrolateral medulla/pre-B?tzinger region. We conclude that ion channels that are sensitive to Ni(2+), but are insensitive to high-voltage activated (L, P/Q, N) Ca(2+) channel blockers, and are located postsynaptically on the presympathetic rostral ventrolateral medulla neurons are responsible for the tonic activity of the presympathetic neurons in rostral ventrolateral medulla. These channels could well be the low-voltage-activated (or T-type) Ca(2+) channels although other conductances cannot be conclusively excluded.     

Low-threshold L-type calcium channels in rat dopamine neurons

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.     

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.     

Increased Ca2+ channel currents in cerebellar Purkinje cells of the ataxic groggy rat

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.     

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.     

Calcineurin enhances L-type Ca(2+) channel activity in hippocampal neurons: increased effect with age in culture

The Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, modulates a number of key Ca(2+) signaling pathways in neurons, and has been implicated in Ca(2+)-dependent negative feedback inactivation of N-methyl-D-aspartate receptors and voltage-sensitive Ca(2+) channels. In contrast, we report here that three mechanistically disparate calcineurin inhibitors, FK-506, cyclosporin A, and the calcineurin autoinhibitory peptide, inhibited high-voltage-activated Ca(2+) channel currents by up to 40% in cultured hippocampal neurons, suggesting that calcineurin acts to enhance Ca(2+) currents. This effect occurred with Ba(2+) or Ca(2+) as charge carrier, and with or without intracellular Ca(2+) buffered by EGTA. Ca(2+)-dependent inactivation of Ca(2+) channels was not affected by FK-506. The immunosuppressant, rapamycin, and the protein phosphatase 1/2A inhibitor, okadaic acid, did not decrease Ca(2+) channel current, showing specificity for effects on calcineurin. Blockade of L-type Ca(2+) channels with nimodipine fully negated the effect of FK-506 on Ca(2+) channel current, while blockade of N-, and P-/Q-type Ca(2+) channels enhanced FK-506-mediated inhibition of the remaining L-type-enriched current. FK-506 also inhibited substantially more Ca(2+) channel current in 4-week-old vs. 2-week-old cultures, an effect paralleled by an increase in calcineurin A mRNA levels. These studies provide the first evidence that calcineurin selectively enhances L-type Ca(2+) channel activity in neurons. Moreover, this action appears to be increased concomitantly with the well-characterized increase in L-type Ca(2+) channel availability in hippocampal neurons with age-in-culture.     

Effects of spike parameters and neuromodulators on action potential waveform-induced calcium entry into pyramidal neurons

Neocortical pyramidal neurons express several different calcium channel types. Previous studies with square voltage steps have found modest biophysical differences between these calcium channel types as well as differences in their modulation by transmitters. We used acutely dissociated neocortical pyramidal neurons to test whether this diversity extends to different activation by physiological stimuli. We conclude that 1) peak amplitude, latency to peak, and the total charge entry for the Ca(2+) channel current is dependent on the shape of the mock action potential waveforms (APWs). 2) The percent contribution of the five high-voltage-activated currents to the whole cell current was not altered by using an APW as opposed to a voltage step to elicit the current. 3) The identity of the charge carrier affects the amplitude and decay of the whole cell current. With Ca(2+), there was a greater contribution of T-type current to the whole cell current. 4) Total Ba(2+) charge entry is linearly dependent on the number of spikes in the stimulating waveform and relatively insensitive to spike frequency. 5) Current decay was greatest with Ca(2+) as the charge carrier and with minimal internal chelation. 6) Voltage-dependent neurotransmitter-mediated modulations can be reversed by multiple spikes. The extent of the reversal is dependent on the number of spikes in the stimulating waveform. Thus the neuronal activity pattern can determine the effectiveness of voltage-dependent and -independent modulatory pathways in neocortical pyramidal neurons.     

Subtypes of low voltage-activated Ca2+ channels in laterodorsal thalamic neurons: possible localization and physiological roles

The macroscopic, low-voltage-activated (LVA or T-type) Ca2+ current in isolated associative (or local-circuit) neurons from the laterodorsal thalamic nucleus of 14-17-day old rats was dissected into two components ("fast" and "slow"), corresponding to the activation of two LVA channel subtypes, based on the difference in the kinetics of inactivation and recovery from inactivation. The steady-state activation and inactivation properties of the channel subtypes endowed slow channels with a substantial window current, whereas fast channels had almost no such current. Fast channels were almost 2 times more sensitive to 30 microM nifedipine (78% inhibition), 10 microM flunarizine (92% inhibition) and 1 microM La3+ (87% inhibition), but about 1.8-fold less sensitive to 100 microM Ni2+ (32% inhibition) than slow channels (40%, 52%, 46% and 56% inhibition respectively). Both channels were almost equally sensitive to 100 microM amiloride (58% and 51% inhibition of fast and slow channels respectively). Comparison of the fast and slow LVA Ca2+ current amplitudes and densities between enzymatically isolated and intact (in brain slices) neurons suggest a predominant localization of the fast channels in soma and the proximal dendrites that remain intact during isolation procedure, whereas the slow channels are more evenly distributed with some preference to the distal areas. These data, together with our previous studies, support the notion of two LVA Ca2+ channel subtypes in associative thalamic neurons and suggest a role for the slow channels in providing the constant Ca2+ influx necessary for the outgrowth of the neurites and for the fast channels in the generation of low-threshold Ca2+ spikes and bursting activity.     

Conductance-based model of the voltage-dependent generation of a plateau potential in subthalamic neurons

Because the subthalamic nucleus (STN) acts as a driving force of the basal ganglia, it is important to know how the activities of STN neurons are regulated. Previously, we have reported that a subset of STN neurons generates a plateau potential in a voltage-dependent manner. These plateau potentials can be evoked only when the cell is hyperpolarized. Here, to examine the mechanism of the voltage-dependent generation of the plateau potential in STN neurons, we constructed a conductance-based model of the plateau-generating STN neuron based on experimental observations and compared simulation results with recordings in slices. The model consists of a single compartment containing a Na(+) current, a delayed-rectifier K(+) current, an A-type K(+) current, an L-like long-lasting Ca(2+) current, a T-type Ca(2+) current, a Ca(2+)-dependent K(+) current, and a leak current. Our simulation results showed that a plateau potential in the model could be induced in a voltage-dependent manner that depended on the inactivation properties of L-like long-lasting Ca(2+) current. The model could also reproduce the generation of a plateau potential as a rebound potential after termination of hyperpolarizing current injection. In addition, we tested the stability of simulated plateau potentials against inhibitory perturbation and found that the model showed similar properties observed for the plateau potentials of STN neurons in slices. The effects of K(+) channel blockade by TEA and intracellular Ca(2+) ion chelation by BAPTA on the plateau duration were also tested in the model and were found to match experimental observations. Thus our STN neuron model could qualitatively reproduce a number of experimental observations on plateau potentials. Our results suggest that the inactivation of L-type Ca(2+) channels plays an important role in the voltage-dependent generation of the plateau potential.     

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic-clonic seizures. IC neurons are known to have Ca(2+) channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca(2+) channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca(2+) channels were activated by depolarizing pulses from a holding potential of -90 mV in 10 mV increments using 5 mM barium (Ba(2+)) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca(2+) channel currents that could be blocked by 50 microM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 microM, L-type channel blocker), omega-conotoxin GVIA (1 microM, N-type channel blocker), and omega-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) omega-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca(2+) channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 microM nickel and inactivated with tau of 15-50 ms, was considered as R-type Ca(2+) channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca(2+) channel antibodies showed that IC neurons express Ca(2+) channel alpha(1A), alpha(1B), alpha(1C), alpha(1D), and alpha(1E) subunits. We conclude that IC neurons express functionally all members of HVA Ca(2+) channels, but only a subset of these neurons appear to have developed functional LVA channels.     

Identification of two calcium currents in acutely dissociated neurons from the rat lateral geniculate nucleus

1. Intracellular recording in the in vitro slice preparation and whole-cell, patch-clamp recording of acutely dissociated neurons from the rat lateral geniculate nucleus (LGN) were combined to study the Ca currents underlying their electrical responses. In slices from young animals (postnatal days 13-16), we found that dorsal LGN neurons have responses similar to those of adult preparations, including the presence of a low-threshold Ca spike (LTS). After enzymatic isolation of LGN neurons from the same animals, the firing properties appeared well preserved, as indicated by whole-cell, current-clamp recordings from dissociated multipolar cells (presumably geniculocortical relay neurons). 2. Two types of Ca currents were identified in voltage-clamped, isolated LGN neurons on the basis of their voltage dependency, pharmacology, and selectivity properties. These two currents resemble the low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca channels found in rat sensory neurons (9). 3. The LVA current component required negative potentials (less than -80 mV) to deinactivate completely, started to activate around -60 mV and reached a plateau level around -25 mV. It peaked within 30-6 ms and decayed with a single time constant of approximately 24 ms at -20 mV. Its inactivation curve ranged from -100 to -40 mV, with a half-inactivation near -60 mV. The HVA current component could be isolated by holding the membrane potential positive to -60 mV, activated at potentials positive to -30 mV and peaked around +5 mV. The time-to-peak ranged from 30 to 6 ms in the voltage range from -30 to +35 mV and decayed very slowly with sustained depolarizing pulses (time constant ranged between 1,600 and 40 ms over the same voltage range). 4. The inactivation of LVA Ca current during depolarizing voltage steps was consistent with a voltage-dependent process. The recovery from inactivation after short (100 ms), inactivating prepulses displayed two exponential phases. The slower phase was predominant under conditions that induce large current flow through the membrane, suggesting a Ca-mediated mechanism. 5. The LVA current was preferentially blocked by 50 microM Ni2+, leaving the HVA currents almost unaltered. Fifty micromolars Cd2+, in contrast, seemed more effective in blocking the HVA component of the Ca current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biophysical characterization of rat caudal hypothalamic neurons: calcium channel contribution to excitability

Neurons in the caudal hypothalamus (CH) are responsible for the modulation of various processes including respiratory and cardiovascular output. Previous results from this and other laboratories have demonstrated in vivo that these neurons have firing rhythms matched to the respiratory and cardiovascular cycles. The goal of the present study was to characterize the biophysical properties of neurons in the CH with particular emphasis in those properties responsible for rhythmic firing behavior. Whole cell, patch-clamped CH neurons displayed a resting membrane potential of -58.0 +/- 1.1 mV and an input resistance of 319.3 +/- 16.6 MOmega when recorded in current-clamp mode in an in vitro brain slice preparation. A large proportion of these neurons displayed postinhibitory rebound (PIR) that was dependent on the duration and magnitude of hyperpolarizing current as well as the resting membrane potential of the cell. Furthermore these neurons discharged tonically in response to a depolarizing current pulse at a depolarized resting membrane potential (more positive than -65 mV) but switched to a rapid burst of firing to the same stimulus when the resting membrane potential was lowered. The PIR observed in these neurons was calcium dependent as demonstrated by the ability to block its amplitude by perfusion of Ca(2+)-free bath solution or by application of Ni(2+) (0.3-0.5 mM) or nifedipine (10 microM). These properties suggest that low-voltage-activated (LVA) calcium current is involved in the PIR and bursting firing of these CH neurons. In addition, high-voltage-activated calcium responses were detected after blockade of outward potassium current or in Ba(2+)-replacement solution. In addition, almost all of the CH neurons studied showed spike frequency adaptation that was decreased following Ca(2+) removal, indicating the involvement of Ca(2+)-dependent K(+) current (I(K,Ca)) in these cells. In conclusion, CH neurons have at least two different types of calcium currents that contribute to their excitability; the dominant current is the LVA or T-type. This LVA current appears to play a significant role in the bursting characteristics that may underlie the rhythmic firing of CH neurons.     

Signaling mechanisms of down-regulation of voltage-activated Ca2+ channels by transient receptor potential vanilloid type 1 stimulation with olvanil in primary sensory neurons

Olvanil ((N-vanillyl)-9-oleamide), a non-pungent transient receptor potential vanilloid type 1 agonist, desensitizes nociceptors and alleviates pain. But its molecular targets and signaling mechanisms are little known. Calcium influx through voltage-activated Ca(2+) channels plays an important role in neurotransmitter release and synaptic transmission. Here we determined the effect of olvanil on voltage-activated Ca(2+) channel currents and the signaling pathways in primary sensory neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated rat dorsal root ganglion neurons. Olvanil (1 microM) elicited a delayed but sustained inward current, and caused a profound inhibition (approximately 60%) of N-, P/Q-, L-, and R-type voltage-activated Ca(2+) channel current. Pretreatment with a specific transient receptor potential vanilloid type 1 antagonist or intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid abolished the inhibitory effect of olvanil on voltage-activated Ca(2+) channel current. Calmodulin antagonists (ophiobolin-A and calmodulin inhibitory peptide) largely blocked the effect of olvanil and capsaicin on voltage-activated Ca(2+) channel current. Furthermore, calcineurin (protein phosphatase 2B) inhibitors (deltamethrin and FK-506) eliminated the effect of olvanil on voltage-activated Ca(2+) channel current. Notably, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, calmodulin antagonists, and calcineurin inhibitors each alone significantly increased the amplitude of voltage-activated Ca(2+) channel current. In addition, double immunofluorescence labeling revealed that olvanil induced a rapid internalization of Ca(V)2.2 immunoreactivity from the membrane surface of dorsal root ganglion neurons. Collectively, this study suggests that stimulation of non-pungent transient receptor potential vanilloid type 1 inhibits voltage-activated Ca(2+) channels through a biochemical pathway involving intracellular Ca(2+)-calmodulin and calcineurin in nociceptive neurons. This new information is important for our understanding of the signaling mechanisms of desensitization of nociceptors by transient receptor potential vanilloid type 1 analogues and the feedback regulation of intracellular Ca(2+) and voltage-activated Ca(2+) channels in nociceptive sensory neurons.     

D2-like dopamine receptors modulate SKCa channel function in subthalamic nucleus neurons through inhibition of Cav2.2 channels

The activity patterns of subthalamic nucleus (STN) neurons are intimately related to motor function/dysfunction and modulated directly by dopaminergic neurons that degenerate in Parkinson's disease (PD). To understand how dopamine and dopamine depletion influence the activity of the STN, the functions/signaling pathways/substrates of D2-like dopamine receptors were studied using patch-clamp recording. In rat brain slices, D2-like dopamine receptor activation depolarized STN neurons, increased the frequency/irregularity of their autonomous activity, and linearized/enhanced their firing in response to current injection. Activation of D2-like receptors in acutely isolated neurons reduced transient outward currents evoked by suprathreshold voltage steps. Modulation was inhibited by a D2-like receptor antagonist and occluded by voltage-dependent Ca2+ (Cav) channel or small-conductance Ca2+-dependent K+ (SKCa) channel blockers or Ca2+-free media. Because Cav channels are targets of G(i/o)-linked receptors, actions on step- and action potential waveform-evoked Cav channel currents were studied. D2-like receptor activation reduced the conductance of Cav2.2 but not Cav1 channels. Modulation was mediated, in part, by direct binding of Gbetagamma subunits because it was attenuated by brief depolarization. D2 and/or D3 dopamine receptors may mediate modulation because a D4-selective agonist was ineffective and mRNA encoding D2 and D3 but not D4 dopamine receptors was detectable. Brain slice recordings confirmed that SKCa channel-mediated action potential afterhyperpolarization was attenuated by D2-like dopamine receptor activation. Together, these data suggest that D2-like dopamine receptors potently modulate the negative feedback control of firing that is mediated by the functional coupling of Cav2.2 and SKCa channels in STN neurons.     

Developmental regulation of calcium channel-mediated currents in retinal glial (Müller) cells

Whole cell voltage-clamp recordings of freshly isolated cells were used to study changes in the currents through voltage-gated Ca(2+) channels during the postnatal development of immature radial glial cells into Müller cells of the rabbit retina. Using Ba(2+) or Ca(2+) ions as charge carriers, currents through transient low-voltage-activated (LVA) Ca(2+) channels were recorded in cells from early postnatal stages, with an activation threshold at -60 mV and a peak current at -25 mV. To increase the amplitude of currents through Ca(2+) channels, Na(+) ions were used as the main charge carriers, and currents were recorded in divalent cation-free bath solutions. Currents through transient LVA Ca(2+) channels were found in all radial glial cells from retinae between postnatal days 2 and 37. The currents activated at potentials positive to -80 mV and displayed a maximum at -40 mV. The amplitude of LVA currents increased during the first postnatal week; after postnatal day 6, the amplitude remained virtually constant. The density of LVA currents was highest at early postnatal days (days 2-5: 13 pA/pF) and decreased to a stable, moderate level within the first three postnatal weeks (3 pA/pF). A significant expression of currents through sustained, high-voltage-activated Ca(2+) channels was found after the third postnatal week in approximately 25% of the investigated cells. The early and sole expression of transient currents at high-density may suggest that LVA Ca(2+) channels are involved in early developmental processes of rabbit Müller cells.     

Unique properties of R-type calcium currents in neocortical and neostriatal neurons

Whole cell recordings from acutely dissociated neocortical pyramidal neurons and striatal medium spiny neurons exhibited a calcium-channel current resistant to known blockers of L-, N-, and P/Q-type Ca(2+) channels. These R-type currents were characterized as high-voltage-activated (HVA) by their rapid deactivation kinetics, half-activation and half-inactivation voltages, and sensitivity to depolarized holding potentials. In both cell types, the R-type current activated at potentials relatively negative to other HVA currents in the same cell type and inactivated rapidly compared with the other HVA currents. The main difference between cell types was that R-type currents in neocortical pyramidal neurons inactivated at more negative potentials than R-type currents in medium spiny neurons. Ni(2+) sensitivity was not diagnostic for R-type currents in either cell type. Single-cell RT-PCR revealed that both cell types expressed the alpha1E mRNA, consistent with this subunit being associated with the R-type current.     

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.     

Raloxifene acutely reduces glutamate-induced intracellular calcium increase in cultured rat cortical neurons via inhibition of high-voltage-activated calcium current

There is increasing evidence indicating that estrogen replacement therapy produces neuroprotective actions but has undesirable side effects on the reproductive system. Raloxifene is a selective estrogen receptor modulator that exerts estrogen agonist action in the brain while acting as an estrogen antagonist in the reproductive system. In the present study, we investigated whether raloxifene affected the glutamate-induced calcium (Ca2+) overload in rat cultured cortical neurons. The bulk cytosolic intracellular Ca2+ level was measured by using confocal microscopy with fluorescent Ca2+ probe fluo3. Whole-cell recording technique was used to observe the effects of raloxifene on N-methyl-D-aspartate (NMDA)-evoked and voltage-activated Ca2+ currents in cultured cortical neurons. Pre-exposure of cortical neurons to raloxifene (0.5 microM-10 microM) for 3 min attenuated intracellular Ca2+ increase induced by application of glutamate (300 microM) for 1 min. The action of raloxifene was reversible after washout. ICI 182,780 and thapsigargin did not block the action of raloxifene. In whole-cell recording experiments, raloxifene (10 microM) significantly reduced the amplitude of the high-voltage-activated Ca2+ current but had no effect on NMDA-evoked Ca2+ current. The present study demonstrates that raloxifene acutely reduces glutamate-induced intracellular Ca2+ increase probably via inhibition of high-voltage-activated calcium channels.