Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.     

Differential expression of voltage-activated calcium currents in zebrafish retinal ganglion cells

We report a study on the characterization of voltage-activated calcium currents (I(Ca)) in retinal ganglion cells (RGCs) and the topographic distribution of RGCs that express different types of I(Ca) in zebrafish retinas. In acutely isolated zebrafish RGCs, both high-voltage-activated (HVA; peak activation potential +7.4 +/- 1.1 mV) and low-voltage-activated (LVA; peak activation potential -33.0 +/- 1.2 mV) I(Ca) were recorded. HVA I(Ca) were recorded in all of the tested RGCs, whereas LVA I(Ca) were recorded in approximately one-third of the tested cells. In RGCs that expressed both HVA and LVA I(Ca), the two currents were readily separated by depolarizing the cell membrane to different voltages from different holding potentials. Among RGCs that expressed LVA I(Ca), some cells expressed large LVA I(Ca) (up to 130 pA), whereas others expressed small LVA I(Ca) (approximately 20 pA). RGCs that expressed large and small LVA I(Ca) were designated as class I and class II cells, respectively, and RGCs that expressed only HVA I(Ca) were designated as class III cells. The topographic distribution of cell classes was similar in various areas of the retina. In the nasal-ventral retina, for example, class III cells outnumbered class I and class II cells by 10.8- and 2.6-fold, respectively. In the temporal and dorsal retinas, the density of class III cells slightly decreased, whereas the density of class I and class II cells increased. The differential expression of I(Ca) in RGCs may correlate with the development and function of the retina.     

Serotonin enhances a low-voltage-activated calcium current in rat spinal motoneurons

Calcium currents and the effects of 5-HT on these currents were investigated in visually identified motoneurons using whole-cell recording in the neonatal rat spinal cord slice preparation. In current-clamp recording, step depolarizations from a holding potential of about -90 mV produced a low-threshold transient depolarizing response and a high-threshold long-lasting spike. In voltage-clamp recording, low (LVA) and high (HVA) voltage-activated Ca2+ currents were recorded in response to depolarizing voltage steps. Low concentration of Cd2+ (50 microM) did not reduce the amplitude of the LVA current but markedly diminished the HVA current. Bath application of 5-HT (10-50 microM) markedly increased the amplitude of the LVA current without causing a shift in the current (I)-voltage (V) relation. In contrast, 5-HT did not appreciably affect the amplitude of the HVA current. We conclude that 5-HT specifically enhances the LVA Ca2+ current and that this effect together with the previously reported 5-HT-induced inward current (Takahashi and Berger, 1990), would facilitate the excitation of motoneurons.     

Synaptic modulation by dopamine of calcium currents in rat pars intermedia

Melanotrophs of the rat pars intermedia are innervated by dopaminergic fibers traveling through the pituitary stalk which inhibit secretion via an action on D-2 receptors. As secretion from the melanotroph has been shown to be calcium (Ca2+) dependent, it is possible that dopamine may have an action to inhibit Ca2+ currents in these cells. This possibility was tested by examining the effects of exogenously applied dopaminergic agonists or synaptically released dopamine upon Ca2+ currents recorded under single electrode voltage clamp in intact rat pars intermedia in vitro. Following blockade of sodium and potassium currents in melanotrophs, Ca2+ spikes were elicited with intracellular injection of depolarizing currents; electrical stimulation of the pituitary stalk caused an inhibition of the Ca2(+)-based action potentials which lasted for several seconds. Using single-electrode voltage-clamp techniques, we recorded inward Ca2+ currents corresponding to the T, N, and L types (see Williams et al., 1990). Stimulation of the pituitary stalk inhibited both the low- and high-threshold peak inward Ca2+ currents elicited from a holding potential of -90 mV. In contrast, when noninactivating Ca2+ currents were elicited from a holding potential of -30 mV, the currents were not altered by stalk stimulation. This pattern of inhibition of the Ca2+ currents was consistent with the preferential inhibition, by stalk stimulation, of the N and T Ca2+ currents, while sparing the L current. We observed that inhibition of Ca2+ currents due to stalk stimulation was completely reversed by bath perfusion of domperidone (1 microM), an antagonist of dopamine at the D-2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of voltage-activated calcium channels in III and XII motoneurones during development in the rat

To further our understanding of the role that voltage-activated Ca2+ channels play in the development, physiology and pathophysiology of motoneurones (MNs), we used whole-cell patch-clamp recording to compare voltage-activated Ca2+ currents in oculomotor (III) and hypoglossal (XII) MNs of neonatal [postnatal day (P)1-5] and juvenile (P14-19) rats. In contrast to III MNs that innervate extraocular muscles, XII MNs that innervate tongue muscles mature more rapidly, fire bursts of low frequency action potentials and are vulnerable to degeneration in amyotrophic lateral sclerosis. In neonates, low voltage-activated (LVA) Ca2+ current densities are similar in XII and III MNs but high voltage-activated (HVA) Ca2+ current densities are twofold higher in XII MNs. The HVA Ca2+ channel antagonists (nimodipine and nifedipine for L-type, omega-agatoxin-TK for P/Q-type and omega-conotoxin-GVIA for N-type) revealed that, while N- and P/Q-type HVA Ca2+ channels are present in both MN pools, a 3.5-fold greater P/Q-type Ca2+ current in XII MNs accounts for their greater HVA Ca2+ currents. Developmentally, LVA and HVA Ca2+ current densities decrease in III MNs but remain unchanged in XII MNs. Thus, the differences between these MN pools increase developmentally so that, in juveniles, the LVA Ca2+ current density is twofold greater and the HVA Ca2+ current density is threefold greater in XII compared with III MNs. We propose that this differential expression of LVA and HVA Ca2+ channels in XII and III MNs during development contributes to their distinct physiology and may also be a factor contributing to the greater susceptibility of XII MNs to degeneration as seen in amyotrophic lateral sclerosis.     

Separation of calcium currents in retinal ganglion cells from postnatal rat

A culture system of the postnatal rat retina was established to investigate Ca²⁺ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca²⁺ currents of RGNs and to clarify the potential use of the lanthanide Gd³⁺ as a selective Ca²⁺ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca²⁺ currents (I_Ca(LVA)) and high voltage activated Ca²⁺ currents (I_Ca(HVA)). The remaining 2/3 of RGNs in short term culture and most RGNs in long-term culture displayed only I_Ca(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of I_Ca(HVA) was irreversible blocked by the N channel antagonist ω-CgTx (5 μM). The L channel antagonist nifedipine (10 μM) eliminated about 25% of I_Ca(HVA). Thus, about 1/3 of the HVA Ca²⁺ or Ba²⁺ current remained unaffected by either ω-CgTx or nifedipine. ω-AgaTx (200 nM) completely failed to block HVA Ca²⁺ or Ba²⁺ currents in RGNs. Gd³⁺ exerted contrasting actions on LVA and HVA Ca²⁺ currents. While I_Ca(LVA) consistently increased in the presence of Gd³⁺ (0.32–3.2 μM), I_Ca(HVA) always decreased, especially when using higher concentrations of Gd³⁺ (10–32 μM). The blocking action of Gd³⁺ was not restricted to the ω-CgTx-sensitive HVA current component, but also concerned ω-CgTx- and nifedipine-resistant components. The decay of Ca²⁺ currents was accelerated in the presence of Gd³⁺. Even in RGNs lacking I_Ca(LVA), application of 3.2 μM Gd³⁺ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from −90 to 0 mV; 10 mM [Ca²⁺]₀; 26°C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd³⁺. Gd³⁺ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd³⁺ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca²⁺ currents, that resemble in some aspects T, N and L channel currents. A significant component of the HVA Ca²⁺ current was resistant to the available HVA channel blockers suggesting the presence of a pharmacologically distinct type of HVA Ca²⁺ channel type in RGNs. Our experiments also show that Gd³⁺ is not suitable for isolation of HVA subcomponents in RGNs, but it can be used to distinguish between LVA and HVA Ca²⁺ currents, as these currents reacted to Gd³⁺ in an opposite way. The purely depressive effect of this lanthanide on spontaneous synaptic activity is consistent with the assumption that in retinal neurons LVA Ca²⁺ channels are not involved in the regulation of glutamate release.     

Voltage-dependent calcium channels in ventromedial hypothalamic neurones of postnatal rats: modulation by oestradiol and phenylephrine

Oestradiol actions in the hypothalamus play an important role in reproductive behaviour. Oestradiol treatment in vivo induces α_1b-adrenoceptor mRNA and increases the density of α_1B-adrenoceptor binding in the hypothalamus. Oestradiol is also known to modulate neuronal excitability, in some cases by modulating calcium channels. We assessed the effects of phenylephrine, an α₁-adrenergic agonist, on low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium channels in ventromedial hypothalamic (VMN) neurones from vehicle- and oestradiol-treated female rats. Whole-cell and gramicidin perforated-patch recordings were obtained, with barium as the charge carrier. In the absence of phenylephrine, oestradiol treatment increased the magnitude of LVA currents compared to controls, but had no effect on HVA currents. Phenylephrine enhanced HVA currents in a significantly greater proportion of neurones from oestradiol-treated rats (76%) than from vehicle-treated (41%) rats. The L-channel blocker nifedipine abolished this oestradiol effect on phenylephrine-enhanced HVA currents. Preincubating slices with the N-type channel blocker omega-conotoxin GVIA completely blocked the phenylephrine response, suggesting that the N-type channel is essential. Phenylephrine also stimulated LVA currents in approximately two-thirds of neurones in slices from both vehicle- and oestradiol-treated rats. Our data show that oestradiol increases LVA currents in the VMN. Oestradiol also amplifies α₁-adrenergic signalling by increasing the proportion of neurones showing phenylephrine-stimulated HVA currents mediated by N- and L-type calcium channels. In this way, oestradiol may increase excitatory responses to arousing adrenergic inputs to VMN neurones governing oestradiol-dependent reproductive behaviour.     

Adenosine Inhibits L- and N-Type Calcium Channels in Pituitary Melanotrophs. Evidence for the Involvement of a G Protein in Calcium Channel Gating

It has been previously demonstrated that activation of A₁ adenosine receptors in frog melanotrophs causes inhibition of spontaneous action potential discharges and alpha-melanocyte-stimulating hormone secretion. In the present study, we have investigated the effect of adenosine on high-voltage-activated (HVA) calcium currents in cultured melanotrophs, using the whole-cell variant of the patch-clamp technique with barium as a charge carrier. Adenosine and the specific A₁ adenosine receptor agonist R-PIA (50 μM each) produced a decrease of the amplitude of the barium current, while the selective A₂ adenosine receptor agonist CGS 21680 did not affect the current. The inhibitory effect of R-PIA was observed throughout the activation range of the current, with stronger responses at more positive potentials. R-PIA inhibited both the L- and N-type components of the current, the effect on the N-component being two-fold higher than on the L-component. The inhibitory effect of R-PIA was rendered irreversible by addition of GTPyS (100 μM) to the intracellular solution. Pre-treatment of the cells with pertussis toxin (1 μg/ml; 12 h) totally abolished the effect of R-PIA on the HVA calcium channels. Conversely, addition of a high concentration of cAMP (100 μM) together with the phosphodiesterase inhibitor IBMX (100 μM) to the intracellular solution did not modify the effect of R-PIA on the current.
It is concluded that, in frog melanotrophs, adenosine induces inhibition of L- and N-calcium currents and that this effect is mediated by a pertussis toxin-sensitive G protein. Our data also indicate that the inhibitory effect of adenosine on the calcium currents is not mediated by inhibition of adenylyl cyclase.     

Developmental regulation of T‐, N‐ and L‐type calcium currents in mouse embryonic sensory neurones

We investigated the development of a low (T-type) and two high voltage-activated (N- and L-type) calcium channel currents in large diameter dorsal root ganglion neurones acutely isolated from embryonic mice using the whole-cell patch-clamp technique. The low and high voltage-activated barium currents (LVA and HVA) were identified by their distinct threshold of activation and their sensitivity to pharmacological agents, dihydropyridines and ω-conotoxin-GVIA, at embryonic day 13 (E13), E15 and E17–18, respectively, before, during and after synaptogenesis. The amplitude and density of LVA currents, measured during a –40 mV pulse from a holding potential of –100 mV, increased significantly between E13 and E15, and remained constant between E15 and E17–18. The density of global HVA current, elicited by 0 mV pulse, increased between E13 and E15/E17–18. The density of the N-type current studied by the application of ω-conotoxin-GVIA (1 μm ) increased significantly between E13 and E15/E17–18. The use of the dihydropyridine nitrendipine (1 μm ) revealed that the density of L-type current remained constant at each stage of development. Nevertheless, application of dihydropyridine Bay K 8644 (3 μm ) demonstrated a significant slowing of the deactivation tail current between embryonic days 13 and 15, which may reflect a qualitative maturation of this class of calcium channel current. The temporal relationship between the changes in calcium channel pattern and the period of target innervation suggests possible roles of T-, N- and L-type currents during developmental key events such as natural neurone death and onset of synapse formation.     

Calcium currents in acutely isolated stellate and pyramidal neurons of rat entorhinal cortex

Calcium currents were studied in morphologically identified pyramidal and stellate neurons acutely isolated from layer II/III of rat entorhinal cortex, using the whole-cell patch-clamp configuration. The peak amplitude of high-voltage activated current (HVA) measured at +10 mV was not different in both neuron populations with 0.94±0.08 nA for pyramidal and 1.03±0.08 nA for stellate cells. Stellate neurons had a larger capacitance (14.4±1.1 pF) than pyramidal neurons (9.6±0.8 pF), indicating a 50% larger cell surface. Most striking was the difference between the current density in stellate (79±8 pA/pF) versus pyramidal neurons (113±13 pA/pF). The potential of half maximal inactivation was not different: −37±2 mV (pyramidals) and −37±3 mV (stellates). Half of the cells contained a low-voltage activated calcium current (LVA) with a peak amplitude that was twice as large in stellate as in pyramidal neurons (0.21±0.04 nA resp. 0.11±0.03 nA; at −50 mV). In contrast to the HVA component, the current density of the LVA component was not different between cell types (13±3 pA/pF vs. 13±2 pA/pF). This implies that the relative abundance of LVA and HVA currents in stellate and pyramidal neurons is different which could result in different firing characteristics. The potential of half maximal LVA inactivation was −88±4 mV (pyramidals) and −85±3 mV (stellates). The slope of the voltage dependent steady state inactivation was steeper in stellate (7±1 mV) than in pyramidal cells (10±2 mV).     

Postnatal development of GABAB receptor-mediated modulation of voltage-activated Ca2+ currents in mouse brain-stem neurons.

GABA_B receptors modulate respiratory rhythm generation in adult mammals. However, little is currently known of their functional significance during postnatal development. In the present investigation, the effects of GABA_B receptor activation on voltage-activated Ca²⁺ currents were examined in rhythmically active neurons of the pre-Bötzinger complex (PBC). Both low- (LVA) and high-voltage-activated (HVA) Ca²⁺ currents were present from the first postnatal day (P1). The density of LVA Ca²⁺ currents increased during the first week, whilst the density of HVA Ca²⁺ currents increased after the first week. In the second postnatal week, the HVA Ca²⁺ currents were composed of L- (47 ± 10%) and N-type (21 ± 8%) currents plus a ‘residual’ current, whilst there were no N-type currents detectable in the first few days. The GABA_B receptor agonist baclofen (30 μm ) increased LVA Ca²⁺ currents (30 ± 11%) at P1–P3, but it decreased the currents (35 ± 11%) at P7–P15 without changing its time course. At all ages, baclofen (30 μm ) decreased the HVA Ca²⁺ currents by ≈ 54%. Threshold of baclofen effects on both LVA and HVA Ca²⁺ currents was 5 μm at P1–P3 and lower than 1 μm at P7–P15. The effect of baclofen was abolished in the presence of the GABA_B receptor antagonist CGP 55845A (50 n m ). We conclude that both LVA and HVA Ca²⁺ currents increased postnatally. The GABA_B receptor-mediated modulation of these currents undergo marked developmental changes during the first two postnatal weeks, which may contribute essentially to modulation of respiratory rhythm generation.     

Somatostatin inhibits two types of voltage-activated calcium currents in rat growth-hormone secreting cells

Somatostatin is known as the hypothalamic inhibitor of growth-hormone (GH) secretion from the pituitary gland. The present study examines the effects of somatostatin on voltage-gated calcium currents recorded from enriched populations of normal GH-secreting cells (somatotrophs). Two types of voltage-activated calcium currents were recorded from somatotrophs with the whole-cell mode of the patch-clamp technique. Somatostatin exerted a reversible blocking effect on these two types of calcium currents. These findings suggest that somatostatin can regulate intracellular calcium in somatotrophs by a direct control of calcium fluxes across the plasma membrane of these cells, thereby affecting the level of GH secretion.     

A guanine nucleotide-binding protein mediates the inhibition of voltage-dependent calcium currents by dopamine in rat lactotrophs

The present study examines the effect of dopamine (DA), known to inhibit prolactin (PRL) release, on voltage-activated calcium currents in identified rat lactotrophs. Two types of voltage-dependent Ca2+ currents were recorded using the whole-cell mode of the patch-clamp technique. Both were reversibly inhibited by DA application. The inhibitory action of DA was reduced by (i) sulpiride (D2 antagonist), (ii) preincubation of the cells with pertussis toxin (PTX), and (iii) inclusion of guanosine 5'-O-(2-thiodiphosphate) (GDP-beta-S) in the pipette solution, whereas it was potentiated by guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). This DA-induced response could not be overcome by changing the adenosine 3',5'-cyclic monophosphate level. These findings suggest that DA can inhibit Ca2+ entry through voltage-activated Ca2+ channels via a PTX-sensitive G protein(s) pathway thereby affecting PRL release from rat lactotrophs.     

GABA(B) receptor activation inhibits N- and P/Q-type calcium channels in cultured lamprey sensory neurons

In lamprey, sensory transmission from mechanosensory receptors (dorsal cells) to central neurons is presynaptically inhibited by GABA(B) receptor activation. The mechanisms underlying this effect were investigated using isolated dorsal cells, where voltage-dependent calcium currents were recorded in the whole-cell configuration. Activation of GABA(B) receptors by baclofen decreased the peak amplitude of high voltage-activated (HVA) calcium currents and slowed the activation phase. The role of G-proteins in mediating the effects of baclofen was examined. Intracellular dialysis of GTPgammaS occluded the effects of baclofen. Intracellular dialysis of GDPbetaS and preincubation in pertussis toxin both attenuated the effect of baclofen. Specific calcium channel blockers were used to study the types of HVA calcium channels involved in the GABA(B)-mediated modulation. The baclofen-induced inhibition was not affected by the L-type calcium channel antagonist nimodipine, but was partially blocked by the N-type blocker omega-conotoxin GVIA, and completely occluded by omega-conotoxin MVIIC, a blocker of both N- and P/Q-type channels. The pharmacology of dorsal cell GABA(B) receptors was studied using two agonists, baclofen and CGP 27492, and four antagonists, CGP 35348, CGP 55845, phaclofen and saclofen. The inhibition induced by either of the two agonists was blocked by CGP 55845, phaclofen and saclofen. The antagonist CGP 35348 completely blocked the inhibition of HVA calcium current induced by the agonist CGP 27492, but had no effect on baclofen-induced GABA(B) receptor activation. This study thus demonstrates that GABA(B) receptor activation in lamprey mechanosensory neurons inhibits N- and P/Q-type calcium channels in a voltage- and G-protein-dependent manner.     

Differential down-regulation of voltage-gated calcium channel currents by glutamate and BDNF in embryonic cortical neurons

In the embryonic brain, post-mitotic cortical neurons migrate from their place of origin to their final location. Various external factors such as hormones, neurotransmitters or peptides regulate their migration. To date, however, only a few studies have investigated the effects of these external factors on the electrical properties of the newly formed embryonic cortical neurons. The aim of the present study was to determine whether glutamate and brain-derived neurotrophic factor (BDNF), known to regulate neuronal cell migration, could modulate currents through voltage-gated calcium channels (ICa) in cortical neurons isolated from embryonic day 13 (E13) mouse foetuses. Whole cell recordings of ICa showed that E13 cortical cells kept 1 day in vitro expressed functional low- and high-voltage activated (LVA and HVA) Ca2+ channels of T-, L- and N-types. A 1-day glutamate treatment non-specifically inhibited LVA and HVA ICa whereas BDNF down-regulated HVA with N-type ICa being more depressed than L-type ICa. The glutamate-induced ICa inhibition was mimicked by NMDA. BDNF exerted its action by recruiting trkB receptors and SKF-96365-sensitive channels. BAPTA prevented the glutamate- and the BDNF-dependent inhibition of Ica, indicating a Ca2+-dependent mechanism of action. It is proposed that an influx of Ca2+ through NMDA receptors depresses the expression of LVA and HVA Ca2+ channels whereas a Ca2+ influx through SKF-96365-sensitive TRPC (transient receptor potential protein of C subtype) channels preferentially inhibits the expression of HVA Ca2+ channels. Glutamate and BDNF appear as potent modulators of the electrical properties of early post-mitotic neurons. By down-regulating ICa they could exert a neuroprotective action on embryonic cortical neurons.     

Selective blockade of N-type calcium channels by levetiracetam

PURPOSE: We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca2+ channels in freshly isolated CA1 hippocampal neurons of rats. METHODS: Patch-clamp recordings of HVA Ca2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca2+ channel blockers were used for separation of the channel subtypes. RESULTS: The currents were measured in controls and after application of 1-200 microM LEV. LEV irreversibly inhibited the HVA calcium current by approximately 18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca2+ channels. LEV also did not influence the rundown of the HVA Ca2+ current during experimental protocols lasting approximately 10 min. Finally, LEV at the highest concentration used (200 microM) did not influence the activity of L-, P- or Q-type Ca2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was approximately 37%. CONCLUSIONS: Our results provide evidence that LEV selectively inhibits N-type Ca2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action.     

Identified glial cells in the early postnatal mouse hippocampus display different types of Ca2+ currents

Based on their typical pattern of membrane currents, four populations of glial cells could be identified in thin brain slices of the postnatal hippocampus. In the present study, we applied the patch-clamp technique to glial cells in the hippocampal CA1 region, which are characterized by a complex pattern of different Na⁺ and K⁻ currents (“complex” cells). These cells were identified as non-neuronal cells, most likely astrocytes, by their glutamine synthetase immunoreactivity. Two types of glial Ca²⁺currents could be identified that differed in their kinetics and pharmacological properties. A low-voltage activated (LVA), fast inactivating component was activated at membrane potentials positive to −60 mV and reached maximum current amplitudes at about −20 mV. This current was sensitive to amiloride and thus displayed properties of neuronal LVA currents. The threshold potential of the second Ca²⁺ current component was at about −40 mV, and peak currents were observed at 0 mV. In contrast to the LVA component, the inactivation of these high-voltage activated (HVA) currents slowed down with increasing depolarizations. This current was sensitive to low concentrations of Cd²⁺ but was not affected by amiloride. A small fraction of the HVA currents was sensitive to nifedipine, and ω-conotoxin GVIA (ω-CgTx) was also found to reduce the glial HVA component. The study provides electrophysiological and pharmacological characterization of different types of Ca²⁺ currents in gray matter glial cells in situ. © 1996 Wiley-Liss, Inc.     

Regulation of L‐type Ca++ currents and process morphology in white matter oligodendrocyte precursor cells by golli‐myelin proteins

The golli myelin basic proteins are expressed in oligodendroglial precursor cells (OPCs) where they play a role in regulating Ca²⁺ homeostasis. During depolarization, they influence process outgrowth and migration through their action on voltage‐operated Ca²⁺ channels (VOCCs). To identify ion channels that are modulated by golli, we examined the electrophysiological properties of VOCCs in OPCs in the white matter of golli knock‐out and control mice. OPCs exhibited two distinct Ca²⁺ channels, which were distinguished by their voltage dependence and pharmacological profiles and which exhibited many of the hallmarks of LVA/T‐type and HVA/L‐type Ca²⁺ channels. The density of high‐voltage‐activated (HVA) currents was reduced in OPCs recorded in golli‐KO tissue, while low‐voltage‐activated (LVA) currents remained unaltered in these cells. These data indicate that golli exerts an exclusive influence on L‐type Ca²⁺ channels in OPCs. Oligodendrocytes (OLs) also displayed LVA and HVA currents, although the density of these currents was much reduced at this developmental stage. These currents were not altered in golli‐KO OLs showing the influence of golli on L‐type Ca²⁺ channels is restricted to a specific time‐window during the course of oligodendroglial development. The actions of golli on OPC L‐type Ca²⁺ channels were accompanied by changes in process morphology, including a reduction in process complexity and the appearance of enlarged varicosities that decorated these cellular processes. These data on L‐type Ca²⁺ channels and process development provide in situ evidence for the influence of golli on VOCCs, and offer an explanation for the hypomyelination observed in the brains of golli‐KO mice. © 2010 Wiley‐Liss, Inc.     

Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel

Only L- and N-type high voltage-activated calcium currents (HVA I_Ca) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA I_Ca, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other I_Ca, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type ω-GVIA-sensitive currents, motoneurons express two other HVA I_Ca. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide ω-agatoxin-IVA with a maximal effect at 100–200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50–100 nM concentration of ω-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA I_Ca is resistant to saturating concentrations of verapamil, ω-conotoxin GVIA and ω-agatoxin-IVA which block L, N and P-type HVAI_Ca, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the I_Ca in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.