Dopamine modulates a voltage-gated calcium channel in rat olfactory receptor neurons

Dopamine D2 receptors exist in the soma of rat olfactory receptor neurons. Actions of dopamine on the voltage-gated Ca(2+) channels in the neurons were investigated using the perforated whole-cell voltage-clamp. In 10 mM Ba(2+) solution, rat olfactory receptor neurons displayed the inward currents elicited by the voltage ramp (167 mV/s) and depolarizing step pulses from a holding potential of -91 mV. The inward Ba(2+) currents were greatly reduced by 10 microM nifedipine (L-type Ca(2+) channel blocker). The Ba(2+) currents were inhibited by the external application of dopamine. The IC(50) for the inhibition was about 1 microM. Quinpirole (10 microM, a D2 dopamine agonist) also inhibited the Ba(2+) currents. Quinpirole did not affect the activation and inactivation kinetics of the Ba(2+) currents. The results suggest that dopamine modulates the L-type Ca(2+) channels in rat olfactory receptor neurons via the mechanism independent of voltage.     

α2-Adrenoceptors couple to inhibition of R-type calcium currents in myenteric neurons

Alpha2-adrenoceptors inhibit Ca2+ influx through voltage-gated Ca2+ channels throughout the nervous system and Ca2+ channel function is modulated following activation of some G-protein coupled receptors. We studied the specific Ca2+ channel inhibited following alpha2-adrenoceptor activation in guinea-pig small intestinal myenteric neurons. Ca2+ currents (I(Ca2+)) were studied using whole-cell patch-clamp techniques. Changes in intracellular Ca2+ (delta[Ca2+]i) in nerve cell bodies and varicosities were studied using digital imaging where Ca2+ influx was evoked by KCl (60 mmol L(-1)) depolarization. The alpha2-adrenoceptor agonist, UK 14 304 (0.01-1 micromol L(-1)) inhibited I(Ca2+) and delta[Ca2+]i; maximum inhibition of I(Ca2+) was 40%. UK 14 304 did not affect I(Ca2+) in the presence of SNX-482 or NiCl2 (R-type Ca2+ channel antagonists). UK 14 304 inhibited I(Ca2+) in the presence of nifedipine, omega-agatoxin IVA or omega-conotoxin, inhibitors of L-, P/Q- and N-type Ca2+ channels. UK 14 304 induced inhibition of I(Ca2+) was blocked by pertussis toxin pretreatment (1 microg mL(-1) for 2 h). Alpha2-adrenoceptors couple to inhibition of R-type Ca2+ channels via a pertussis toxin-sensitive pathway in myenteric neurons. R-type channels may be a target for the inhibitory actions of noradrenaline released from sympathetic nerves on to myenteric neurons.     

Isoflurane anesthesia enhances the inhibitory effects of cocaine and GBR12909 on dopamine transporter: PET studies in combination with microdialysis in the monkey brain

The genes encoding the alpha(1A), alpha(1B), alpha(1C) and alpha(1E) subunits of neuronal high voltage-gated Ca channels (HVGCCs) were separately expressed with beta(1B) and alpha(2)/delta subunits in Xenopus oocytes to determine the effects of volatile anesthetics (VAs) on currents through each specific channel. VA effects were determined on currents carried by Ba(2+) (I(Ba)) using the two electrode voltage clamp technique. Although time to peak was unaffected, both halothane (0.59 mM) and isoflurane (0.70 mM) reversibly inhibited peak I(Ba) by 25-35% and late current (at 830 ms) by 50-60%. A hyperpolarizing shift in steady-state inactivation of alpha(1E)-current was found which could contribute up to one third of observed decrease in the peak current. The rate of inactivation of I(Ba) seen with alpha(1A), alpha(1B) and alpha(1E)-type Ca channels was consistently increased by halothane and isoflurane. To more clearly quantify these effects, I(Ba) inactivation was fit by a single exponential function. The anesthetics depressed both the inactivating and non-inactivating residual components of I(Ba) and decreased the time constant of inactivation. In the case of I(Ba) through alpha(1C)-type channels, inactivation was minimal; however, the average current was inhibited by VAs. Similar inhibition of all these HVGCCs by halothane and isoflurane suggests that a common structural component may be involved. Furthermore, the inhibition of such neuronal HVGCCs in situ could alter synaptic neurotransmitter release and contribute to the anesthetic state.     

Role of L-type Ca2+ channels in neural stem/progenitor cell differentiation

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.     

Microglial Ca(2+)-activated K(+) channels are possible molecular targets for the analgesic effects of S-ketamine on neuropathic pain

Ketamine is an important analgesia clinically used for both acute and chronic pain. The acute analgesic effects of ketamine are generally believed to be mediated by the inhibition of NMDA receptors in nociceptive neurons. However, the inhibition of neuronal NMDA receptors cannot fully account for its potent analgesic effects on chronic pain because there is a significant discrepancy between their potencies. The possible effect of ketamine on spinal microglia was first examined because hyperactivation of spinal microglia after nerve injury contributes to neuropathic pain. Optically pure S-ketamine preferentially suppressed the nerve injury-induced development of tactile allodynia and hyperactivation of spinal microglia. S-Ketamine also preferentially inhibited hyperactivation of cultured microglia after treatment with lipopolysaccharide, ATP, or lysophosphatidic acid. We next focused our attention on the Ca(2+)-activated K(+) (K(Ca)) currents in microglia, which are known to induce their hyperactivation and migration. S-Ketamine suppressed both nerve injury-induced large-conductance K(Ca) (BK) currents and 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619)-induced BK currents in spinal microglia. Furthermore, the intrathecal administration of charybdotoxin, a K(Ca) channel blocker, significantly inhibited the nerve injury-induced tactile allodynia, the expression of P2X(4) receptors, and the synthesis of brain-derived neurotrophic factor in spinal microglia. In contrast, NS1619-induced tactile allodynia was completely inhibited by S-ketamine. These observations strongly suggest that S-ketamine preferentially suppresses the nerve injury-induced hyperactivation and migration of spinal microglia through the blockade of BK channels. Therefore, the preferential inhibition of microglial BK channels in addition to neuronal NMDA receptors may account for the preferential and potent analgesic effects of S-ketamine on neuropathic pain.     

Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.

Trains of action potentials cause Ca(2+)-dependent facilitation and inactivation of presynaptic P/Q-type Ca(2+) channels that can alter synaptic efficacy. A potential mechanism for these effects involves calmodulin, which associates in a Ca(2+)-dependent manner with the pore-forming alpha(1A) subunit. Here, we report that Ca(2+) and calmodulin dramatically enhance inactivation and facilitation of P/Q-type Ca(2+) channels containing the auxiliary beta(2a) subunit compared with their relatively small effects on channels with beta(1b). Tetanic stimulation causes an initial enhancement followed by a gradual decline in P/Q-type Ca(2+) currents over time. Recovery of Ca(2+) currents from facilitation and inactivation is relatively slow (30 sec to 1 min). These effects are strongly inhibited by high intracellular BAPTA, replacement of extracellular Ca(2+) with Ba(2+), and a calmodulin inhibitor peptide. The Ca(2+)/calmodulin-dependent facilitation and inactivation of P/Q-type Ca(2+) channels observed here are consistent with the behavior of presynaptic Ca(2+) channels in neurons, revealing how dual feedback regulation of P/Q-type channels by Ca(2+) and calmodulin could contribute to activity-dependent synaptic plasticity.     

Changes in calcium currents and GABAergic spontaneous activity in cultured rat hippocampal neurons after a neurotropic influenza A virus infection

In order to study mechanisms by which a neurotropic strain of influenza A virus (A/WSN/33) may affect neuronal function or cause nerve cell death, hippocampal cultures from embryonic rats were infected with this virus. Approximately 70% of the neurons in the infected cultures became immunopositive for viral antigens and showed reduced voltage-dependent Ca(2+) currents in whole-cell patch clamp recordings, but no changes in other membrane properties or in cytosolic Ca(2+) concentration were seen. These immunopositive neurons underwent apoptosis 3-4 days after infection. Ca(2+) channel inhibitors had no significant effect on neuronal survival. The immunonegative population of neurons survived, but displayed increased frequency of miniature inhibitory postsynaptic currents of gamma-amino-butyric acid origin compared with controls. The frequency of alpha-amino-hydroxy-5-methylisoxazole-4-propionic acid hydrobromide (AMPA) receptor-mediated miniature excitatory postsynaptic currents was not altered. Viral nucleoproteins, overexpressed using the Semliki Forest virus system, were localized to the dendritic spines as shown by double immunolabeling with actinin, but did not by themselves cause neuronal death or changes in synaptic transmission as measured by AMPA-mediated excitatory postsynaptic currents. Our results show that an influenza A virus infection can cause selective neurophysiological changes in hippocampal neurons and that these can persist even after the viral antigens have been cleared.     

Intracellular acidification in neurons induced by ammonium depends on KCC2 function

The Cl(-)-extruding neuron-specific K(+)-Cl(-) cotransporter KCC2, which establishes hyperpolarizing inhibition, can transport NH(4) (+) instead of K(+). It is, however, not clear whether KCC2 provides the only pathway for neuronal NH(4) (+) uptake. We therefore investigated NH(4) (+) uptake in cultured rat brain neurons. In neurons cultured for > 4 weeks, the response to NH(4)Cl applications (5 mM) consisted of an alkaline shift which reversed to an acid shift within seconds. Rebound acid shifts which followed brief applications of NH(4)Cl were blocked by furosemide (100 microM). They were rather insensitive to bumetanide (1 and 100 microM), in contrast to those induced in cultured glial cells. Rebound acid shifts persisted in the presence of 1 mM Ba(2+) and in Na(+)-free solution but were inhibited by extracellular K(+). In neurons with depolarizing GABA responses, indicating the absence of functional KCC2, applications of NH(4)Cl barely induced an acidosis. However, large rebound acid shifts occurred in neurons that had changed their GABA response from Ca(2+) increases to Ca(2+) decreases. Rebound acid shifts continued to increase even after the change in the GABA response had occurred and could be induced earlier in neurons transfected with KCC2 cDNA. We conclude that KCC2 provides the main pathway for fast neuronal NH(4) (+) uptake. Therefore, NH(4)Cl-induced rebound acid shifts can be used to indicate the development of KCC2 function. Further, the well known up-regulation of KCC2 function during development has the inevitable consequence of opening a major pathway for NH(4) (+) influx, which can be relevant under pathophysiological conditions.     

The cytosolic II-III loop of Cav2.3 provides an essential determinant for the phorbol ester-mediated stimulation of E-type Ca2+ channel activity

There is growing evidence that E-type voltage dependent Ca(2+) channels (Ca(v)2.3) are involved in triggering and controlling pivotal cellular processes like neurosecretion and long-term potentiation. The mechanism underlying a novel Ca(2+) dependent stimulation of E-type Ca(2+) channels was investigated in the context of the recent finding that influx of Ca(2+) through other voltage dependent Ca(2+) channels is necessary and sufficient to directly activate protein kinase C (PKC). With Ba(2+) as charge carrier through Ca(v)2.3 channel alpha(1) subunits expressed in HEK-293 cells, activation of PKC by low concentrations of phorbol ester augmented peak I(Ba) by approximately 60%. In addition, the non-inactivating fraction of I(Ba) was increased by more than three-fold and recovery from short-term inactivation was accelerated. The effect of phorbol ester on I(Ba) was inhibited by application of the specific PKC inhibitor bisindolylmaleimide I. With Ca(2+) as charge carrier, application of phorbol ester did not change the activity of Ca(v)2.3 currents but they were modified by the PKC inhibitor bisindolylmaleimide I. These results suggest that with Ca(2+) as charge carrier the incoming Ca(2+) can activate PKC, thereby augmenting Ca(2+) influx into the cytosol. No modulation of Ca(v)2.3 channels by PKC was observed when an arginine rich region in the II-III loop of Ca(v)2.3 was eliminated. Receptor independent stimulation of PKC and its interaction with Ca(v)2.3 channels therefore represents an important positive feedback mechanism to decode electrical signals into a variety of cellular functions.     

Gabapentin actions on Kir3 currents and N-type Ca2+ channels via GABAB receptors in hippocampal pyramidal cells

Gabapentin is a clinically effective anticonvulsant with an unclear mechanism of action. It was described as a GABA(B(1a,2)) receptor subtype-selective agonist, activating postsynaptic K(+) currents and inhibiting postsynaptic Ca(2+) channels in CA1 pyramidal cells, but without presynaptic actions. These activities appeared controversial and we therefore sought to further clarify gabapentin actions in rat hippocampal slices by characterizing K(+) currents and Ca(2+) channels targeted by gabapentin using whole-cell recording and multiphoton Ca(2+) imaging. 1) We found that gabapentin and baclofen induced inwardly rectifying K(+) currents (K(Gbp) and K(Bac), respectively), sensitive to Ba(2+) and Cs(+). 2) A constitutively active K(IR) current, independent of GABA(B) receptor activation and sensitive to Ba(2+) and Cs(+) was also present. 3) K(Gbp), K(Bac), and K(IR) currents showed some differences in sensitivity to Ba(2+) and Cs(+), indicating the possible activation of distinct Kir3 currents, independent of K(IR), by gabapentin and baclofen. 4) Gabapentin inhibition of Ca(2+) channels was abolished by omega-conotoxin GVIA, but not by omega-agatoxin IVA and nimodipine, indicating a predominant action of gabapentin on N-type Ca(2+) channels. 5) Gabapentin actions were linked to activation of pertussis toxin-sensitive G-proteins since N-ethylmaleimide (NEM) blocked K(Gbp) activation and Ca(2+) channel inhibition by gabapentin. 6) Finally, gabapentin reduced epileptiform discharges in slices via GABA(B) receptor activation. The anticonvulsant actions of gabapentin in hippocampal cells may thus involve GABA(B) receptor coupling to G-proteins and modulation of Kir3 and N-type Ca(2+) channels. Moreover, gabapentin and baclofen activation of GABA(B) receptors may couple to distinct cellular targets.     

Omega-conotoxin CVIB differentially inhibits native and recombinant N- and P/Q-type calcium channels

Omega-conotoxins are routinely used as selective inhibitors of different classes of voltage-gated calcium channels (VGCCs) in excitable cells. In the present study, we examined the potent N-type VGCC antagonist omega-conotoxin CVID and non-selective N- and P/Q-type antagonist CVIB for their ability to block native VGCCs in rat dorsal root ganglion (DRG) neurons and recombinant VGCCs expressed in Xenopus oocytes. Omega-conotoxins CVID and CVIB inhibited depolarization-activated whole-cell VGCC currents in DRG neurons with pIC50 values of 8.12 +/- 0.05 and 7.64 +/- 0.08, respectively. Inhibition of Ba2+ currents in DRG neurons by CVID (approximately 66% of total) appeared to be irreversible for > 30 min washout, whereas Ba2+ currents exhibited rapid recovery from block by CVIB (> or = 80% within 3 min). The recoverable component of the Ba2+ current inhibited by CVIB was mediated by the N-type VGCC, whereas the irreversibly blocked current (approximately 22% of total) was attributable to P/Q-type VGCCs. Omega-conotoxin CVIB reversibly inhibited Ba2+ currents mediated by N- (Ca(V)2.2) and P/Q- (Ca(V)2.1), but not R- (Ca(V)2.3) type VGCCs expressed in Xenopus oocytes. The alpha2delta1 auxiliary subunit co-expressed with Ca(V)2.2 and Ca(V)2.1 reduced the sensitivity of VGCCs to CVIB but had no effect on reversibility of block. Determination of the NMR structure of CVIB identified structural differences to CVID that may underlie differences in selectivity of these closely related conotoxins. Omega-conotoxins CVIB and CVID may be useful as antagonists of N- and P/Q-type VGCCs, particularly in sensory neurons involved in processing primary nociceptive information.     

Spontaneous neuronal activity in organotypic cultures of mouse dorsal root ganglion leads to upregulation of calcium channel expression on remote Schwann cells

It is well established that neurons regulate the properties of both central and peripheral glial cells. Some of these neuro-glial interactions are modulated by the pattern of neuronal electrical activity. In the present work, we asked whether blocking the electrical activity of dorsal root ganglion (DRG) neurons in vitro by a chronic treatment with tetrodotoxin (TTX) would modulate the expression of the T-type Ca(2+) channel by mouse Schwann cells. When recorded in their culture medium, about one-half of the DRG neurons spontaneously fired action potentials (APs). Treatment for 4 days with 1 microM TTX abolished both spontaneous and evoked APs in DRG neurons and in parallel significantly reduced the percentage of Schwann cells expressing Ca(2+) channel currents. On the fraction of Schwann cells still expressing Ca(2+) channel currents, these currents had electrophysiological parameters (mean amplitude, mean inactivation time constant, steady-state inactivation curve) similar to those of control cultures. Co-treatment for 4 days with 1 microM TTX and 2 mM CPT-cAMP, a cAMP analogue that induces the expression de novo of Ca(2+) channel currents in Schwann cells deprived of neurons, maintained the percentage of Schwann cells expressing Ca(2+) channel currents, showing that TTX does not directly affect the expression of Ca(2+) channel currents by Schwann cell. We conclude that blocking spontaneous activity of DRG neurons in vitro downregulates Ca(2+) channel expression by Schwann cells. These results strongly suggest that DRG neurons upregulate Ca(2+) channel expression by Schwann cells via the release of a diffusible factor whose secretion is dependent on electrical activity.     

5-HT inhibits N-type but not L-type Ca(2+) channels via 5-HT1A receptors in lamprey spinal neurons

5-HT is a potent modulator of locomotor activity in vertebrates. In the lamprey, 5-HT dramatically slows fictive swimming. At the neuronal level it reduces the postspike slow afterhyperpolarization (sAHP), which is due to apamin-sensitive Ca(2+)-dependent K+ channels (KCa). Indirect evidence in early experiments suggested that the sAHP reduction results from a direct action of 5-HT on KCa channels rather than an effect on the Ca(2+) entry during the action potential. In view of the characterization of different subtypes of Ca(2+) channels with very different properties, we now reinvestigate if there is a selective action of 5-HT on a Ca(2+) channel subtype in dissociated spinal neurons in culture. 5-HT reduced Ca(2+) currents from high voltage activated channels. N-type, but not L-type, Ca(2+) channel blockers abolished this 5-HT-induced reduction. It was also confirmed that 5-HT depresses Ca(2+) currents in neurons, including motoneurons, in the intact spinal cord. 8-OH-DPAT, a 5-HT1A receptor agonist, also inhibited Ca(2+) currents in dissociated neurons. After incubation in pertussis toxin, to block Gi/o proteins, 5-HT did not reduce Ca(2+) currents, further indicating that the effect is caused by an activation of 5-HT1A receptors. As N-type, but not L-type, Ca(2+) channels are known to mediate the activation of KCa channels and presynaptic transmitter release at lamprey synapses, the effects of 5-HT reported here can contribute to a reduction in both actions.     

Ca2+-Dependent Regulation of Ca2+ Currents in Rat Primary Afferent Neurons: Role of CaMKII and the Effect of Injury

Currents through voltage-gated Ca(2+) channels (I(Ca)) may be regulated by cytoplasmic Ca(2+) levels ([Ca(2+)](c)), producing Ca(2+)-dependent inactivation (CDI) or facilitation (CDF). Since I(Ca) regulates sensory neuron excitability, altered CDI or CDF could contribute to pain generation after peripheral nerve injury. We explored this by manipulating [Ca(2+)](c) while recording I(Ca) in rat sensory neurons. In uninjured neurons, elevating [Ca(2+)](c) with a conditioning prepulse (-15 mV, 2 s) inactivated I(Ca) measured during subsequent test pulses (-15 mV, 5 ms). This inactivation was Ca(2+)-dependent (CDI), since it was decreased with elimination of Ca(2+) influx by depolarization to above the I(Ca) reversal potential, with high intracellular Ca(2+) buffering (EGTA 10 mm or BAPTA 20 mm), and with substitution of Ba(2+) for extracellular Ca(2+), revealing a residual voltage-dependent inactivation. At longer latencies after conditioning (>6 s), I(Ca) recovered beyond baseline. This facilitation also proved to be Ca(2+)-dependent (CDF) using the protocols limiting cytoplasmic Ca(2+) elevation. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) blockers applied by bath (KN-93, myristoyl-AIP) or expressed selectively in the sensory neurons (AIP) reduced CDF, unlike their inactive analogues. Protein kinase C inhibition (chelerythrine) had no effect. Selective blockade of N-type Ca(2+) channels eliminated CDF, whereas L-type channel blockade had no effect. Following nerve injury, CDI was unaffected, but CDF was eliminated in axotomized neurons. Excitability of sensory neurons in intact ganglia from control animals was diminished after a similar conditioning pulse, but this regulation was eliminated by injury. These findings indicate that I(Ca) in sensory neurons is subject to both CDI and CDF, and that hyperexcitability following injury-induced loss of CDF may result from diminished CaMKII activity.     

Opposing effects of spinal nerve ligation on calcium-activated potassium currents in axotomized and adjacent mammalian primary afferent neurons

Calcium-activated potassium channels regulate AHP and excitability in neurons. Since we have previously shown that axotomy decreases I(Ca) in DRG neurons, we investigated the association between I(Ca) and K((Ca)) currents in control medium-sized (30-39 microM) neurons, as well as axotomized L5 or adjacent L4 DRG neurons from hyperalgesic rats following L5 SNL. Currents in response to AP waveform voltage commands were recorded first in Tyrode's solution and sequentially after: 1) blocking Na(+) current with NMDG and TTX; 2) addition of K((Ca)) blockers with a combination of apamin 1 microM, iberiotoxin 200 nM, and clotrimazole 500 nM; 3) blocking remaining K(+) current with the addition of 4-AP, TEA-Cl, and glibenclamide; and 4) blocking I(Ca) with cadmium. In separate experiments, currents were evoked (HP -60 mV, 200 ms square command pulses from -100 to +50 mV) while ensuring high levels of activation of I(K(Ca)) by clamping cytosolic Ca(2+) concentration with pipette solution in which Ca(2+) was buffered to 1 microM. This revealed I(K(Ca)) with components sensitive to apamin, clotrimazole and iberiotoxin. SNL decreases total I(K(Ca)) in axotomized (L5) neurons, but increases total I(K(Ca)) in adjacent (L4) DRG neurons. All I(K(Ca)) subtypes are decreased by axotomy, but iberiotoxin-sensitive and clotrimazole-sensitive current densities are increased in adjacent L4 neurons after SNL. In an additional set of experiments we found that small-sized control DRG neurons also expressed iberiotoxin-sensitive currents, which are reduced in both axotomized (L5) and adjacent (L4) neurons. CONCLUSIONS: Axotomy decreases I(K(Ca)) due to a direct effect on K((Ca)) channels. Axotomy-induced loss of I(Ca) may further potentiate current reduction. This reduction in I(K(Ca)) may contribute to elevated excitability after axotomy. Adjacent neurons (L4 after SNL) exhibit increased I(K(Ca)) current.     

Cobalt-sensitive and dihydropyridine-insensitive stimulation of dopamine release from tuberoinfundibular neurons by high extracellular concentrations of barium ions

Recently, it has been demonstrated that Ca2+ entrance into the neuronal cytoplasm can occur upon the activation of 3 different types of specific voltage-dependent channels which can be characterized according to the following criteria: (1) voltage threshold for activation; (2) tendency to inactivation; (3) bivalent cation permeability; and (4) drug sensitivity. In this study we investigated, in tuberoinfundibular dopaminergic (TIDA) hypothalamic neurons, the biochemical and pharmacological properties of Ca2+ channels, by comparing the effects of high extracellular concentrations of Ba2+ and Ca2+ ions on [3H]dopamine (DA) release from TIDA neurons. The results obtained show that extracellular Ba2+ ion concentrations dose-dependently (10-20 mM) stimulated [3H]DA release from superfused TIDA neurons and that this effect was prevented by Co2+ ions (2 mM). In addition, superfusion of TIDA neurons with a concentration of Ca2+ ions equimolar to that of Ba2+ ions (20 mM) failed to modify [3H]DA release. The fact that tetraethylammonium (10 mM), a blocker of K+ currents in excitable cells, did not mimick the stimulatory action of Ba2+ ions on [3H]DA release, seems to exclude that the effect of Ba2+ ions was dependent on the inhibition of K+ channels in TIDA neurons. The omission of Ca2+ ions from the extracellular medium did not prevent the stimulatory effect on [3H]DA release elicited by elevated concentrations of Ba2+ ions, but rather reinforced this effect. Finally, nitrendipine (50 microM) did not modify the stimulatory effect of high extracellular Ba2+ ions on [3H]DA release from TIDA neurons.     

Neurotensin inhibits background K+ channels and facilitates glutamatergic transmission in rat spinal cord dorsal horn

Neurotensin (NT) is a neuropeptide involved in the modulation of nociception. We have investigated the actions of NT on cultured postnatal rat spinal cord dorsal horn (DH) neurons. NT induced an inward current associated with a decrease in membrane conductance in 46% of the neurons and increased the frequency of glutamatergic miniature excitatory synaptic currents in 37% of the neurons. Similar effects were observed in acute slices. Both effects of NT were reproduced by the selective NTS1 agonist JMV449 and blocked by the NTS1 antagonist SR48692 and the NTS1/NTS2 antagonist SR142948A. The NTS2 agonist levocabastine had no effect. The actions of NT persisted after inactivation of G(i/o) proteins by pertussis toxin but were absent after inactivation of protein kinase C (PKC) by chelerythrine or inhibition of the MAPK (ERK1/2) pathway by PD98059. Pre- and postsynaptic effects of NT were insensitive to classical voltage- and Ca(2+) -dependent K(+) channel blockers. The K(+) conductance inhibited by NT was blocked by Ba(2+) and displayed no or little inward rectification, despite the presence of strongly rectifying Ba(2+) -sensitive K(+) conductance in these neurons. This suggested that NT blocked two-pore domain (K2P) background K(+) -channels rather than inwardly rectifying K(+) channels. Zn(2+) ions, which inhibit TRESK and TASK-3 K2P channels, decreased NT-induced current. Our results indicate that in DH neurons NT activates NTS1 receptors which, via the PKC-dependent activation of the MAPK (ERK1/2) pathway, depolarize the postsynaptic neuron and increase the synaptic release of glutamate. These actions of NT might modulate the transfer and the integration of somatosensory information in the DH.     

Neuroprotectant minocycline depresses glutamatergic neurotransmission and Ca(2+) signalling in hippocampal neurons

The mechanism of the neuroprotective action of the tetracycline antibiotic minocycline against various neuron insults is controversial. In an attempt to clarify this mechanism, we have studied here its effects on various electrophysiological parameters, Ca(2+) signalling, and glutamate release, in primary cultures of rat hippocampal neurons, and in synaptosomes. Spontaneous excitatory postsynaptic currents and action potential firing were drastically decreased by minocycline at concentrations known to afford neuroprotection. The drug also blocked whole-cell inward Na(+) currents (I(Na)) by 20%, and the whole-cell Ca(2+) current (I(Ca)) by about 30%. Minocycline inhibited glutamate-evoked elevation of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) by nearly 40%, and K(+)-evoked glutamate release from synaptosomes by 63%. Minocycline also depressed the frequency and amplitude of spontaneous excitatory postsynaptic currents, but did not affect the whole-cell inward current elicited by gamma-aminobutyric acid or glutamate. This pharmacological profile suggests that the neuroprotective effects of minocycline might be associated with the mitigation of neuronal excitability, glutamate release, and Ca(2+) overloading.     

Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3

RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca(2+) and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca(2+) dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3-/-) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca(2+) imaging to investigate a role for RC3 in neuronal Ca(2+) dynamics. Immunocytochemical characterization showed that the RC3-/- cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3-/- cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and gamma-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3-/- neurons was found to alter neuronal Ca(2+) dynamics including baseline Ca(2+) levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca(2+) oscillations generated by network synaptic activity, and Ca(2+) responses elicited by exogenous application of N-methyl-D-aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca(2+) dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca(2+) dynamics.     

Activation of protein kinase C antagonizes the opioid inhibition of calcium current in rat spinal dorsal horn neurons

Spinal dorsal horn (SDH) is one of important regions in both nociceptive transmission and antinociception. Opioid peptides produce analgesia via regulation of neurotransmitter release through modulation of voltage-dependent Ca(2+) channel (VDCC) in neuronal tissues. The modulatory effect of micro-opioid receptor (MOR) activation on VDCC was investigated in acutely isolated rat SDH neurons under the conventional whole-cell patch-clamp recording mode. The Ba(2+) current passing through VDCC was reversibly inhibited by a MOR agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin (DAMGO, 1 microM). Among 108 SDH neurons tested, VDCC of 39 neurons (36%) were inhibited by MOR activation, while other 69 neurons (64%) were not affected. The L-, N-, P/Q-, and R-type VDCC components shared 58.4+/-18.9%, 29.3+/-12.1%, 8.7+/-7.2%, and 3.4+/-4.8% of the total VDCC, respectively. Among VDCC subtypes inhibited by MOR activation, L- and N-types were 61.4+/-12.8% and 30.7+/-14.4%, respectively, while both P/Q- and R-types were 7.9+/-11.8%. A depolarizing pre-pulse increased the amplitude of VDCC and suppressed most of the inhibitory effect of MOR activation. Application of 1 microM phorbol-12-myristate-13-acetate completely antagonized the inhibitory effect of MOR activation without any alteration of basal VDCC amplitude. In contrast, the response of MOR activation was not altered by application of 4-alpha-phorbol (1 microM), 2-[3-Dimethylaminopropyl]indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X, 1 microM), forskolin (1 microM), N-(2-[p-Bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 1 microM). These results indicate that activation of MOR coupled to G-proteins inhibits VDCC, and that this G-protein-mediated inhibition is antagonized by PKC-dependent phosphorylation.