Activation of metabotropic glutamate receptors inhibits high-voltage-gated calcium channel currents of chicken nucleus magnocellularis neurons

Using whole cell patch-clamp recordings, we pharmacologically characterized the voltage-gated Ca2+ channel (VGCC) currents of chicken nucleus magnocellularis (NM) neurons using barium as the charge carrier. NM neurons possessed both low- and high-voltage-activated Ca2+ channel currents (HVA I(Ba2+)). The N-type channel blocker (omega-conotoxin-GVIA) inhibited more than half of the total HVA I(Ba2+), whereas blockers of L- and P/Q-type channels each inhibited a small fraction of the current. Metabotropic glutamate receptor (mGluR)-mediated modulation of the HVA I(Ba2+) was examined by bath application of glutamate (100 microM), which inhibited the HVA I(Ba2+) by an average of 16%. The inhibitory effect was dose dependent and was partially blocked by omega-conotoxin-GVIA, indicating that mGluRs modulate N and other type HVA I(Ba2+). The nonspecific mGluR agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarbosylic acid (1S,3R-ACPD), mimicked the inhibitory effect of glutamate on HVA I(Ba2+). Group I-III mGluR agonists showed inhibition of the HVA current with the most potent being the group III agonist L(+)-2-amino-4-phosphonobutyric acid. 1S,3R-ACPD (200 microM) had no effect on K+ or Na+ currents. The firing properties of NM neurons were also not altered by 1S,3R-ACPD. We propose that the inhibition of VGCC currents by mGluRs limits depolarization-induced Ca2+ entry into these highly active NM neurons and regulates their Ca2+ homeostasis.     

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.     

Pharmacological and biophysical characterization of voltage-gated calcium currents in the endopiriform nucleus of the guinea pig

The endopiriform nucleus (EPN) is a well-defined structure that is located deeply in the piriform region at the border with the striatum and is characterized by dense intrinsic connections and prominent projections to piriform and limbic cortices. The EPN has been proposed to promote synchronization of large populations of neurons in the olfactory cortices via the activation of transient depolarizations possibly mediated by Ca(2+) spikes. It is known that principal cells in the EPN express both a low- and high-voltage-activated (HVA) Ca(2+) currents. We further characterized HVA conductances possibly related to Ca(2+)-spike generation in the EPN with a whole cell, patch-clamp study on neurons acutely dissociated from the EPN of the guinea pig. To study HVA currents in isolation, experiments were performed from a holding potential of -60 mV, using Ba(2+) as the permeant ion. Total Ba(2+) currents (I(Ba)) evoked by depolarizing square pulses peaked at 0/+10 mV and were completely abolished by 200 microM Cd(2+). The pharmacology of HVA I(Ba)s was analyzed by applying saturating concentrations of specific Ca(2+)-channel blockers. The L-type blocker nifedipine (10 microM; n = 11), the N-type-channel blocker omega-conotoxin GVIA (0.5 microM; n = 24), and the P/Q-type blocker omega-conotoxin MVIIC (1 microM; n = 16) abolished fractions of total I(Ba)s equal on average to 24.7 +/- 5.4%, 27.1 +/- 3.4%, and 22.2 +/- 2.4%, respectively (mean +/- SE). The simultaneous application of the three blockers reduced I(Ba) by 68.5 +/- 6.6% (n = 10). Nifedipine-sensitive currents and most N- and P/Q-type currents were slowly decaying, the average fractional persistence after 300 ms of steady depolarization being 0.77 +/- 0.02, 0.60 +/- 0.06, and 0.68 +/- 0.04, respectively. The residual, blocker-resistant (R-type) currents were consistently faster inactivating, with an average fractional persistence after 300 ms of 0.30 +/- 0.08. Fast-decaying R-type currents also displayed a more negative threshold of activation (by about 10 mV) than non-R-type HVA currents. These results demonstrate that EPN neurons express multiple pharmacological components of the HVA Ca(2+) currents and point to the existence of an R-type current with specific functional properties including fast inactivation kinetics and intermediate threshold of activation.     

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.     

Inactivation properties of human recombinant class E calcium channels

The electrophysiological and pharmacological properties of alpha(1E)-containing Ca(2+) channels were investigated by using the patch-clamp technique in the whole cell configuration, in HEK 293 cells stably expressing the human alpha(1E) together with alpha(2b) and beta(1b) accessory subunits. These channels had current-voltage (I-V) characteristics resembling those of high-voltage-activated (HVA) Ca(2+) channels (threshold at -30 mV and peak amplitude at +10 mV in 5 mM Ca(2+)). The currents activated and deactivated with a fast rate, in a time- and voltage-dependent manner. No difference was found in their relative permeability to Ca(2+) and Ba(2+). Inorganic Ca(2+) channel blockers (Cd(2+), Ni(2+)) blocked completely and potently the alpha(1E,)/alpha(2b)delta/beta(1b) mediated currents (IC(50) = 4 and 24.6 microM, respectively). alpha(1E)-mediated currents inactivated rapidly and mainly in a non-Ca(2+)-dependent manner, as evidenced by the fact that 1) decreasing extracellular Ca(2+) from 10 to 2 mM and 2) changing the intracellular concentration of the Ca(2+) chelator 1. 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), did not affect the inactivation characteristics; 3) there was no clear-cut bell-shaped relationship between test potential and inactivation, as would be expected from a Ca(2+)-dependent event. Although Ba(2+) substitution did not affect the inactivation of alpha(1E) channels, Na(+) substitution revealed a small but significant reduction in the extent and rate of inactivation, suggesting that besides the presence of dominant voltage-dependent inactivation, alpha(1E) channels are also affected by a divalent cation-dependent inactivation process. We have analyzed the Ca(2+) currents produced by a range of imposed action potential-like voltage protocols (APVPs). The amplitude and area of the current were dependent on the duration of the waveform employed and were relatively similar to those described for HVA calcium channels. However, the peak latency resembled that obtained for low-voltage-activated (LVA) calcium channels. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca(2+) current amplitude, suggesting an accumulation of inactivation likely to be calcium dependent. The human alpha(1E) gene seems to participate to a Ca(2+) channel type with biophysical and pharmacological properties partly resembling those of LVA and those of HVA channels, with inactivation characteristics more complex than previously believed.     

Biophysical and pharmacological characterization of voltage-sensitive calcium currents in neonatal rat inferior colliculus neurons

Calcium conductances have been found in neonatal inferior colliculus neurons, however the biophysical and pharmacological profiles of the underlying calcium currents have not yet been characterized. In this study, we examined which types of voltage-activated calcium currents comprise the whole-cell inward current of neonatal inferior colliculus neurons (10-22microm in diameter). On the basis of their voltage-dependence and pharmacological sensitivities, three major components of barium currents were identified. A low threshold voltage-activated current that activated around -70mV, a mid threshold voltage-activated current that activated near -50mV, and a high threshold voltage-activated current that activated around -40mV. Low and mid threshold voltage-activated currents were present in 33% and 41% of the recordings, respectively, whereas high threshold voltage-activated currents were recorded in all inferior colliculus neurons tested. Nickel chloride (50microM) and U-92032 (1microM), which both block low threshold voltage-activated currents, reduced the amplitude of low threshold voltage-activated peak currents at a test potential of -60mV by 72% and 10%, respectively. In addition, 50microM nickel chloride and 1microM U-92032 reduced the amplitude of mid threshold voltage-activated peak currents measured at -20mV by 55% and 21%, respectively. Further pharmacological analysis indicated the presence of multiple types of high threshold voltage-activated currents in neonatal inferior colliculus neurons. The dihydropyridine nimodipine (1microM), a selective L-type current antagonist, reduced the amplitude of high threshold voltage-activated peak currents by 25%. In addition, FPL 64176 (1microM), a non-dihydropyridine L-type current agonist caused a dramatic 534% increase in the amplitude of the slow sustained component of the tail current measured at -40mV. These data indicate that inferior colliculus neurons express L-type channels. omega-Conotoxin GVIA (1microM), a selective blocker of N-type current, inhibited high threshold voltage-activated peak currents by 28% indicating the presence of N-type channels. omega-Agatoxin IVA (300nM), a potent P/Q-type antagonist, reduced high threshold voltage-activated peak currents by 27%, suggesting that inferior colliculus neurons express P/Q-type channels. Concomitant application of nimodipine (1microM), omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (300nM) onto inferior colliculus neurons decreased the control high threshold voltage-activated peak currents only by 62%.Thus, inferior colliculus neurons may express at least one more type of calcium current in addition to low and mid threshold voltage-activated currents and L-type, N-type and P/Q-type high threshold currents.     

T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation

Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.     

Haemorrhage-evoked compensation and decompensation are mediated by distinct caudal midline medullary regions in the urethane-anaesthetised rat

Somatostatin is synthesized and released by aspiny interneurons of the neostriatum. This work investigates the actions of somatostatin on rat neostriatal neurons of medium size (ca. 6 pF). Somatostatin (1 microM) reduces both calcium action potentials (20 mM tetraethylammonium) by ca. 24% and calcium currents by ca. 35%, in all cells tested. This action was produced in the presence of tetrodotoxin and in dissociated cells and was blocked by cyclo(-7-aminoheptanoyl-phe-d-try-lys-O-benzyl-thr) acetate (CPP-1), a somatostatin receptor antagonist. Except for nitrendipine (5 microM), several calcium channel antagonists, 1 microM omega-conotoxin GVIA, 400 nM omega-agatoxin TK, and 1 microM omega-conotoxin MVIIC, partially occluded somatostatin action. According to the calcium channel types known to be blocked by these antagonists, P/Q-type channels appeared to be the channels mainly modulated by somatostatin, followed by N-type channels. Since these channel types generate the afterhyperpolarizing potential in spiny neurons, we investigated the action of somatostatin on this event. Somatostatin reduces the amplitude of the afterhyperpolarizing potential by ca. 39%. This action is occluded by omega-agatoxin TK and omega-conotoxin MVIIC but not by omega-conotoxin GVIA or nicardipine. Thus, the action of somatostatin on the afterhyperpolarizing potential is mainly mediated by P/Q-type calcium channels. The block of the slow afterhyperpolarizing potential made most neurons exhibit an irregular firing mode, suggesting that ion currents other than calcium may also be affected by somatostatin.We conclude that somatostatin exerts a direct postsynaptic effect on neostriatal neurons via the activation of somatostatin receptors. This action affects non-L-type calcium channels and therefore modifies the afterhyperpolarizing potential and the firing pattern. It is proposed that somatostatin and its analogues may have profound effects on the motor functions controlled by the basal ganglia.     

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.     

Characteristics of calcium currents in rat geniculate ganglion neurons

Geniculate ganglion (GG) cell bodies of chorda tympani (CT), greater superficial petrosal (GSP), and posterior auricular (PA) nerves transmit orofacial sensory information to the rostral nucleus of the solitary tract (rNST). We used whole cell recording to study the characteristics of the Ca(2+) channels in isolated Fluorogold-labeled GG neurons that innervate different peripheral receptive fields. PA neurons were significantly larger than CT and GSP neurons, and CT neurons could be further subdivided based on soma diameter. Although all GG neurons possess both low voltage-activated (LVA) "T-type" and high voltage-activated (HVA) Ca(2+) currents, CT, GSP, and PA neurons have distinctly different Ca(2+) current expression patterns. Of GG neurons that express T-type currents, the CT and GSP neurons had moderate and PA neurons had larger amplitude T-type currents. HVA Ca(2+) currents in the GG neurons were separated into several groups using specific Ca(2+) channel blockers. Sequential applications of L, N, and P/Q-type channel antagonists inhibited portions of Ca(2+) current in all CT, GSP, and PA neurons to a different extent in each neuron group. No difference was observed in the percentage of L- and N-type Ca(2+) currents reduced by the antagonists in CT, GSP, and PA neurons. Action potentials in GG neurons are followed by a Ca(2+) current initiated after depolarization (ADP) that may influence intrinsic firing patterns. These results show that based on Ca(2+) channel expression the GG contains a heterogeneous population of sensory neurons possibly related to the type of sensory information they relay to the rNST.     

Muscarinic M2 and M1 receptors reduce GABA release by Ca2+ channel modulation through activation of PI3K/Ca2+ -independent and PLC/Ca2+ -dependent PKC

We measured pharmacologically isolated GABAergic currents from layer II/III neurons of the rat auditory cortex using patch-clamp recording. Activation of muscarinic receptors by muscarine (1 microM) or oxotremorine (10 microM) decreased the amplitude of electrically evoked inhibitory postsynaptic currents to about one third of their control value. Neither miniature nor exogenously evoked GABAergic currents were altered by the presence of muscarinic agonists, indicating that the effect was spike-dependent and not mediated postsynaptically. The presence of the N- or P/Q-type Ca(2+) channel blockers omega-conotoxin GVIA (1 microM) or omega-AgaTx TK (200 nM) greatly blocked the muscarinic effect, suggesting that Ca(2+)-channels were target of the muscarinic modulation. The presence of the muscarinic M(2) receptor (M(2)R) antagonists methoctramine (5 muM) or AF-DX 116 (1 microM) blocked most of the muscarinic evoked inhibitory postsynaptic current (eIPSC) reduction, indicating that M(2)Rs were responsible for the effect, whereas the remaining component of the depression displayed M(1)R-like sensitivity. Tissue preincubation with the specific blockers of phosphatidyl-inositol-3-kinase (PI(3)K) wortmannin (200 nM), LY294002 (1 microM), or with the Ca(2+)-dependent PKC inhibitor G? 6976 (200 nM) greatly impaired the muscarinic decrease of the eIPSC amplitude, whereas the remaining component was sensitive to preincubation in the phospholipase C blocker U73122 (10 microM). We conclude that acetylcholine release enhances the excitability of the auditory cortex by decreasing the release of GABA by inhibiting axonal V-dependent Ca(2+) channels, mostly through activation of presynaptic M(2)Rs/PI(3)K/Ca(2+)-independent PKC pathway and-to a smaller extent-by the activation of M(1)/PLC/Ca(2+)-dependent PKC.     

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.     

Identification of T-type alpha1H Ca2+ channels (Ca(v)3.2) in major pelvic ganglion neurons

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.     

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.     

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.     

Characterization of voltage-gated ionic channels in cholinergic amacrine cells in the mouse retina

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.     

Mitral cell presynaptic Ca(2+) influx and synaptic transmission in frog amygdala

Dextran-conjugated Ca(2+) indicators were injected into the accessory olfactory bulb of frogs in vivo to selectively fill presynaptic terminals of mitral cells at their termination in the ipsilateral amygdala. After one to three days of uptake and transport, the forebrain hemisphere anterior to the tectum was removed and maintained in vitro for simultaneous electrophysiological and optical measurements. Ca(2+) influx into these terminals was compared to synaptic transmission between mitral cells and amygdala neurons under conditions of reduced Ca(2+) influx resulting from reduced extracellular [Ca(2+)], blockade of N- and P/Q-type channels, and application of the cholinergic agonist carbachol. Reducing extracellular [Ca(2+)] had a non-linear effect on release; release was proportional to Ca(2+) influx raised to the power of approximately 3.6, as observed at numerous other synapses. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1 microM), blocked 77% of Ca(2+) influx and 88% of the postsynaptic field potential. The P/Q-type Ca(2+) channel blocker, omega-agatoxin-IVA (200 nM), blocked 19% of Ca(2+) influx and 25% of the postsynaptic field, while the two toxins combined to block 92% of Ca(2+) influx and 97% of the postsynaptic field. The relationship between toxin blockade of Ca(2+) influx and synaptic transmission was therefore only slightly non-linear; release was proportional to Ca(2+) influx raised to the power approximately 1.4. Carbachol (100 microM) acting via muscarinic receptors had no effect on the afferent volley, but rapidly and reversibly reduced Ca(2+) influx through both N- and P/Q-type channels by 51% and postsynaptic responses by 78%, i.e. release was proportional to Ca(2+) raised to the power approximately 2.5.The weak dependence of release on changes in Ca(2+) when channel toxins block channels suggests little overlap between Ca(2+) microdomains from channels supporting release or substantial segregation of channel subtypes between terminals. The proportionately greater reduction of transmission by muscarinic receptors compared to Ca(2+) channel toxins suggests that they directly affect the release machinery in addition to reducing Ca(2+) influx.     

Pharmacological and biophysical properties of Ca2+ channels and subtype distributions in human adrenal chromaffin cells

In this study, we explored the pharmacological and biophysical properties of voltage-activated Ca(2+) channels in human chromaffin cells using the perforated-patch configuration of the patch-clamp technique. According to their pharmacological sensitivity to Ca(2+) channel blockers, cells could be sorted into two groups of similar size showing the predominance of either N- or P/Q-type Ca(2+) channels. R-type Ca(2+) channels, blocked by 77% with 20 muM Cd(2+) and not affected by 50 muM Ni(2+), were detected for the first time in human chromaffin cells. Immunocytochemical experiments revealed an even distribution of alpha (1E) Ca(2+) channels in these cells. With regard to their biophysical properties, L- and R-type channels were activated at membrane potentials that were 15-20 mV more negative than P/Q- and N-type channels. Activation time constants showed no variation with voltage for the L-type channels, decreased with increasing potentials for the R- and P/Q-type channels, and displayed a bell shape with a maximum at 0 mV for the N-type channels. R-type channels were also the most inactivated channels. We thus show here that human chromaffin cells possess all the Ca(2+) channel types described in neurons, L, N, P/Q, and R channels, but the relative contributions of N and P/Q channels differ among cells. Given that N- and P/Q-type Ca(2+) channel types can be differentially modulated, these findings suggest the possibility of cell-specific regulation in human chromaffin cells.     

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.