Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling.

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.

Non-quantal release of acetylcholine in guinea-pig airways: role of choline transporter.

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.

Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue.

INTRODUCTION: Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na(+) ) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. AIM: We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. MAIN OUTCOME MEASURES: PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. METHODS: Rat cavernosal strips were incubated with bretylium (3 × 10(-5) M) and contracted with phenylephrine (PE; 10(-5) M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10(-8) M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10(-6) M), a muscarinic receptor antagonist, N-type Ca(2+) channel blockers (ω-conotoxin GVIA, 10(-6) M) and sildenafil (3 × 10(-8) M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. RESULTS: Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca(2+) channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. CONCLUSION: We show that PnTx2-6 slows Na(+) channels inactivation in nitrergic neurons, allowing Ca(2+) influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction.

Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus.

The aim of this study was to investigate the effect of spider toxins on brain injury induced by oxygen deprivation and low glucose (ODLG) insult on slices of rat hippocampus. After ODLG insult cell viabilility in hippocampal slices was assessed by confocal microscopy and epifluorescence using the live/dead kit containing calcein-AM and ethidium homodimer and CA1 population spike amplitude recording during stimulation of Schaffer collateral fibers. Spider toxins Tx3-3 or Tx3-4 and conus toxins, omega-conotoxin GVIA or omega-conotoxin MVIIC are calcium channel blockers and protected against neuronal damage in slices subjected to ODLG insult. Confocal imaging of CA1 region of rat hippocampal slices subject to ischemic insult treated with Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC showed a decrease in cell death that amounted to 68 +/- 4.2%, 77 +/- 3.8%, 32 +/- 2.3%, and 46 +/- 2.9%, respectively. This neuroprotective effect of Tx3-4 was corroborated by eletrophysiological recordings of population spikes amplitudes in CA1. The neuroprotection promoted on hippocampal slices by Tx3-3 or Tx3-4 was also observed when the toxins were applied 10, 20, 30, 60, 90, or 120 min after induction of the ODLG injury. During the ischemic insult, glutamate release from slices was increased by 71% (from 7.0 +/- 0.3 nM/mg of protein control slices not subjected to ischemia to 12 +/- 0.4 nM/mg of protein in slices exposed to ischemia). Tx3-3, Tx3-4, omega-conotoxin GVIA or omega-conotoxin MVIIC inhibited the ischemia-induced increase on glutamate release by 54, 72, 60, and 70%, respectively. Thus Tx3-3 and Tx3-4 provided robust ischemic neuroprotection showing potential as a novel class of agent that exerts neuroprotection in an in vitro model of brain ischemia.

Calcium signaling pathways mediating synaptic potentiation triggered by amyotrophic lateral sclerosis IgG in motor nerve terminals.

Sporadic amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects particularly motoneurons. Several pieces of evidence suggested the involvement of autoimmune mechanisms mediated by antibodies in ALS. However, the significance of those antibodies in the disease and the underlying mechanisms are unknown. Here we showed that IgG purified from a group of sporadic ALS patients, but not familial ALS patients, specifically interact with the presynaptic membrane of motoneurons through an antigen-antibody interaction and modulated synaptic transmission. Immunoreactivity against nerve terminals showed strong correlation with synaptic modulation ability. In addition, several controls have ruled out the possibility for this synaptic modulation to be mediated through proteases or nonspecific effects. Effective IgG potentiated both spontaneous and asynchronous transmitter release. Application of pharmacological inhibitors suggested that activation of this increased release required a nonconstitutive Ca2+ influx through N-type (Cav2.2) channels and phospholipase C activity and that activation of IP3 and ryanodine receptors were necessary to both activate and sustain the increased release. Consistent with the notion that ALS is heterogeneous disorder, our results reveal that, in approximately 50% of ALS patients, motor nerve terminals constitutes a target for autoimmune response.

Effects of the blockade of high voltage-activated calcium channels on in vitro pineal melatonin synthesis.

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.

Testosterone modulates Ca(v2.2) calcium channels' functional expression at rat levator ani neuromuscular junction.

Spinal nucleus of bulbocavernosus and its target musculature, the bulbocavernosus and levator ani muscles, are sexually dimorphic, and their sexual differentiation depends on plasmatic levels of testosterone. Electrophysiological and immunocytochemical studies have demonstrated that at mammalian adult neuromuscular junctions only P/Q-type Ca2+ channels (Ca(v2.1)), mediate evoked transmitter release. Here we report that N-type Ca2+ channel (Ca(v2.2)) blocker omega-Conotoxin GVIA, as well as Ca(v2.1) blocker omega-Agatoxin IVA, significantly reduced quantal content of transmitter release by approximately 80% and approximately 70% respectively at levator ani muscle of the adult rats, indicating that neuromuscular transmission is jointly mediated by both types of channels. In these synapses, we also observed that castration and restitution of plasmatic testosterone in rats resulted in changes in the sensitivity to omega-Conotoxin GVIA. Castration induced, whereas testosterone treatment avoided, functional loss of Ca(v2.2), as mediators of transmitter release in these synapses. Strikingly, the expression and localization of alpha1B subunits, which form the pore of the Ca(v2.2) channel, were similar at control, gonadectomized and gonadectomized testosterone-treated rats, suggesting that testosterone may regulate the coupling mechanisms between Ca(v2.2) and transmitter release at the neuromuscular junctions of these sexually dimorphic motoneurons.

Differential Ca2+-dependence of transmitter release mediated by P/Q- and N-type calcium channels at neonatal rat neuromuscular junctions.

N- and P/Q-type voltage dependent calcium channels (VDCCs) mediate transmitter release at neonatal rat neuromuscular junction (NMJ). Thus the neonatal NMJ allows an examination of the coupling of different subtypes of VDCCs to the release process at a single synapse. We studied calcium dependence of transmitter release mediated by each channel by blocking with omega-conotoxin GVIA the N-type channel or with omega-agatoxin IVA the P/Q-type channel while changing the extracellular calcium concentration ([Ca2+]o). Transmitter release mediated by P/Q-type VDCCs showed steeper calcium dependence than N-type mediated release (average slope 3.6 +/- 0.09 vs. 2.6 +/- 0.03, respectively). Loading the nerve terminals with 10 microm BAPTA-AM in the extracellular solution reduced transmitter release and occluded the blocking effect of omega-conotoxin GVIA (blockade -2 +/- 9%) without affecting the action of omega-agatoxin IVA (blockade 85 +/- 4%). Both VDCC blockers were able to reduce the amount of facilitation produced by double-pulse stimulation. In these conditions facilitation was restored by increasing [Ca2+]o. The facilitation index (fi) was also reduced by loading nerve terminals with 10 microm BAPTA-AM (fi = 1.2 +/- 0.1). The control fi was 2.5 +/- 0.1. These results show that P/Q-type VDCCs were more efficiently coupled to neurotransmitter release than were N-type VDCCs at the neonatal neuromuscular junction. This difference could be accounted for by a differential location of these channels at the release site. In addition, our results indicate that space-time overlapping of calcium domains was required for facilitation.

High-affinity inhibition of glutamate release from corticostriatal synapses by omega-agatoxin TK.

To know which Ca(2+) channel type is the most important for neurotransmitter release at corticostriatal synapses of the rat, we tested Ca(2+) channel antagonists on the paired pulse ratio. omega-Agatoxin TK was the most effective Ca(2+) channel antagonist (IC(50)=127 nM; maximal effect=211% (with >1 microM) and Hill coefficient=1.2), suggesting a single site of action and a Q-type channel profile. Corresponding parameters for Cd(2+) were 13 microM, 178% and 1.2. The block of L-type Ca(2+) channels had little impact on transmission, but we also tested facilitation of L-type Ca(2+) channels. The L-type Ca(2+) channel agonist, s-(-)-1,4 dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (Bay K 8644 (5 microM)), produced a 45% reduction of the paired pulse ratio, suggesting that even if L-type channels do not participate in the release process, they may participate in its modulation.

Changes in Ca(2+) channel expression upon differentiation of SN56 cholinergic cells.

The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca(2+) channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the alpha(1H) (CaV3.2) Ca(2+) channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 microM), 37% by the N-type channel antagonist omega-conotoxin-GVIA (1 microM), and 15% by the P/Q-type channel antagonist omega-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca(2+) channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.

Modulation of N-type Ca2+ channel current kinetics by PMA in rat sympathetic neurons.

The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) has been used extensively in studies of G protein modulation of Ca2+ channels. PMA has been shown to be a powerful tool for inducing phosphorylation and interrupting G-protein-mediated signaling pathways. Here we re-examine the effects of PMA on whole-cell N-type Ca2+-channel currents in rat sympathetic neurons. We found that, along with an increase in the current amplitude previously reported by others, PMA pretreatment leads to alterations in current activation and inactivation kinetics. These alterations in current kinetics are voltage-dependent and are not reproduced by internal dialysis with the G protein inhibitor GDPbetaS. Alterations in current kinetics by PMA may therefore indicate the existence of a modulated state, presumably phosphorylated, of N-type Ca2+ channels. We propose that the increase in current amplitude is due primarily to alterations in current kinetics rather than to removal of tonic inhibition.

Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals.

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.

Seizures and neurodegeneration induced by 4-aminopyridine in rat hippocampus in vivo: role of glutamate- and GABA-mediated neurotransmission and of ion channels.

Infusion of the K(+) channel blocker 4-aminopyridine in the hippocampus induces the release of glutamate, as well as seizures and neurodegeneration. Since an imbalance between excitation and inhibition, as well as alterations of ion channels, may be involved in these effects of 4-aminopyridine, we have studied whether they are modified by drugs that block glutamatergic transmission or ion channels, or drugs that potentiate GABA-mediated transmission. The drugs were administered to anesthetized rats subjected to intrahippocampal infusion of 4-aminopyridine through microdialysis probes, with simultaneous collection of dialysis perfusates and recording of the electroencephalogram, and subsequent histological analysis. Ionotropic glutamate receptor antagonists clearly diminished the intensity of seizures and prevented the neuronal damage, but did not alter substantially the enhancement of extracellular glutamate induced by 4-aminopyridine. None of the drugs facilitating GABA-mediated transmission, including uptake blockers, GABA-transaminase inhibitors and agonists of the A-type receptor, was able to reduce the glutamate release, seizures or neuronal damage produced by 4-aminopyridine. In contrast, nipecotate, which notably increased extracellular levels of the amino acid, potentiated the intensity of seizures and the neurodegeneration. GABA(A) receptor antagonists partially reduced the extracellular accumulation of glutamate induced by 4-aminopyridine, but did not exert any protective action. Tetrodotoxin largely prevented the increase of extracellular glutamate, the electroencephalographic epileptic discharges and the neuronal death in the CA1 and CA3 hippocampal regions. Valproate and carbamazepine, also Na(+) channel blockers that possess general anticonvulsant action, failed to modify the three effects of 4-aminopyridine studied. The N-type Ca(2+) channel blocker omega-conotoxin, the K(+) channel opener diazoxide, and the non-specific ion channel blocker riluzole diminished the enhancement of extracellular glutamate and slightly protected against the neurodegeneration. However, the two former compounds did not antagonize the 4-aminopyridine-induced epileptiform discharges, and riluzole instead markedly increased the intensity and duration of the disharges. Moreover, at the highest dose tested (8mg/kg, i.p.), riluzole caused a 75% mortality of the rats. We conclude that 4-aminopyridine stimulates the release of glutamate from nerve endings and that the resultant augmented extracellular glutamate is directly related to the neurodegeneration and is involved in the generation of epileptiform discharges through the concomitant overactivation of glutamate receptors. Under these conditions, a facilitated GABA-mediated transmission may paradoxically boost neuronal hyperexcitation. Riluzole, a drug used to treat amyotrophic lateral sclerosis, seems to be toxic when combined with neuronal hyperexcitation.

Amyotrophic lateral sclerosis IgG-treated neuromuscular junctions develop sensitivity to L-type calcium channel blocker.

In order to search for early changes induced by the application of human immunoglobulin G (IgG) on motor nerve terminals, IgG from patients with amyotrophic lateral sclerosis (ALS) and control subjects was injected subcutaneously into the levator auris muscle of mice. A week or a month after the last injection, endplate potentials were recorded. No changes in quantal content of transmitter release were observed. In control and ALS IgG-treated muscles, neurotransmitter release remained sensitive to P/Q-type and insensitive to N-type voltage-sensitive calcium channel (VSCC) blockers as in untreated muscles. In contrast, IgG from 5 of 8 different ALS patients induced a significant reduction in quantal content of the evoked response after incubation with nitrendipine, indicating that a novel sensitivity to this calcium channel blocker appears in these motor nerve terminals. These results indicate that ALS IgG induces plastic changes at nerve terminals. The expression of transmitter release coupled to L-type VSCC indicate that ALS IgGs are capable of inducing plastic changes at the nerve terminals that may participate in the process leading to neuronal death.

Different Ca2+ source for slow AHP in completely adapting and repetitive firing pyramidal neurons.

Intracellular recordings in an in vitro neocortical slice preparation from immature rats were used to investigate the Ca2 source for slow afterhyperpolarization (sAHP) generation in pyramidal neurons that exhibit complete spike frequency adaptation (CA neurons). In pyramidal neurons that maintain repetitive firing for long periods of time (RF neurons), N-, P- and Q-type Ca2+ channels supply Ca2+ for sAHP generation. In CA neurons, the sAHP was reduced by only 50% by the combination of antagonists for these Ca2+ channel types and L-type channels. Ryanodine and dantrolene, blockers of Ca2(+)-induced Ca2+ release, reduced the sAHP by approximately 45% in CA neurons, but caused no reduction of the sAHP in RF neurons. Dantrolene application caused CA neurons to fire throughout a 1s suprathreshold current injection (as do RF neurons).

delta opioid receptor modulation of several voltage-dependent Ca(2+) currents in rat sensory neurons.

Endogenous enkephalins and delta opiates affect sensory function and pain sensation by inhibiting synaptic transmission in sensory circuits via delta opioid receptors (DORs). DORs have long been suspected of mediating these effects by modulating voltage-dependent Ca(2+) entry in primary sensory neurons. However, not only has this hypothesis never been validated in these cells, but in fact several previous studies have only turned up negative results. By using whole-cell current recordings, we show that the delta enkephalin analog [D-Ala(2), D-Leu(5)]-enkephalin (DADLE) inhibits, via DORs, L-, N-, P-, and Q-high voltage-activated Ca(2+) channel currents in cultured rat dorsal root ganglion (DRG) neurons. The percentage of responding cells was remarkably high (75%) within a novel subpopulation of substance P-containing neurons compared with the other cells (18-35%). DADLE (1 microM) inhibited 32% of the total barium current through calcium channels (I(Ba)). A delta (naltrindole, 1 microM), but not a mu (beta-funaltrexamine, 5 microM), antagonist prevented the DADLE response, whereas a DOR-2 subtype (deltorphin-II, 100 nM), but not a DOR-1 (DPDPE, 1 microM), agonist mimicked the response. L-, N-, P-, and Q-type currents contributed, on average, 18, 48, 14, and 16% to the total I(Ba) and 19, 50, 26, and 20% to the DADLE-sensitive current, respectively. The drug-insensitive R-type current component was not affected by the agonist. This work represents the first demonstration that DORs modulate Ca(2+) entry in sensory neurons and suggests that delta opioids could affect diverse Ca(2+)-dependent processes linked to Ca(2+) influx through different high-voltage-activated channel types.

L-type calcium channels unmasked by cell-permeant Ca2+ buffer at mouse motor nerve terminals.

The involvement of the different types of voltage-dependent calcium channels (VDCC) in both DM-BAPTA-AM-incubated and EGTA-AM-incubated mature mice levator auris neuromuscular junctions (NMJ) was studied. We evaluated the effects of omega-agatoxin IVA (omega-Aga IVA), nitrendipine and omega-conotoxin GVIA (omega-CgTX) (P/Q-, L- and N-type VDCC blockers, respectively) on perineurial calcium currents (ICa) and nerve-evoked transmitter release. The application of omega-Aga IVA (100 nM) drastically reduced perineurial ICa (>90%) and nerve-evoked transmitter release (>90% of reduction in quantal content, m) at both DM-BAPTA-AM-incubated and EGTA-AM-incubated NMJ. The L-type VDCC antagonist nitrendipine (10 microM) caused a significant reduction (23+/-9%, n=5) of perineurial ICa at DM-BAPTA-AM-incubated NMJ. In addition, after the block of P/Q-type VDCC with omega-Aga IVA (100 nM), nitrendipine reduced (>90%, n=2) the remaining perineurial ICa. Such reduction was not observed at EGTA-AM-incubated NMJ, before or after the total block of P/Q-type VDCC. Moreover, nitrendipine did not significantly reduce the quantal content of DM-BAPTA-AM-incubated NMJ. Finally, the application of omega-CgTX (5 microM) did not significantly affect perineurial ICa or nerve-evoked transmitter release at either DM-BAPTA-AM-incubated or EGTA-AM-incubated NMJ. These results show the existence of a nitrendipine-sensitive, L-type component of perineurial ICa in DM-BAPTA-AM-incubated NMJ of mature mice.

Calcium channels coupled to neurotransmitter release at neonatal rat neuromuscular junctions.

1. The effects of different calcium channel blockers (omega-agatoxin IVA (omega-Aga IVA), omega-conotoxin GVIA (omega-CgTx GVIA) and dihydropyridines) were tested on spontaneous and evoked transmitter release at embryonic and newborn rat neuromuscular junctions (NMJs). 2. The nerve-evoked transmitter release quantal content (m) was strongly reduced by the P/Q-type voltage-dependent calcium channel (VDCC) blocker omega-Aga IVA (100 nM) at newly formed endplates of embryos and 0- to 11-day-old rats, in agreement with the effect of this blocker on transmitter release at mature and reinnervating muscles. 3. omega-CgTx GVIA (1-5 microM), the N-type VDCC blocker, also caused a significant reduction in m at newly formed NMJs early in development (embryos and 0- to 4-day-old rats), while it was ineffective in more mature animals (5- to 11-day-old rats). 4. L-type channel blockers, nitrendipine (1 microM) and nifedipine (1 microM), did not significantly affect neurally evoked release at developing NMJs. However, nifedipine (10 microM) was able to increase m significantly at 0- to 4-day-old rat NMJs. 5. At developing NMJs, K+-evoked transmitter release was dependent on Ca2+ entry through VDCCs of the P/Q-type family (100 nM omega-Aga IVA reduced 70 % of the K+-evoked miniature endplate potential frequency). N- and L-type VDCC blockers did not affect this type of release. 6. We conclude that at rat neuromuscular junctions the presynaptic calcium channel types involved in transmitter release undergo developmental changes during the early postnatal period.

Extracellular potassium concentration regulates proliferation of immature cerebellar granule cells.

The present study examines the effect of depolarizing potassium concentrations on the proliferation of immature rat cerebellar neurons. Cells inoculated in serum free medium and 5 mM KCl (5 K) showed a high degree of 3H-thymidine incorporation that decreased 24-48 h after plating as differentiation began. During the first 24 h after inoculation, cells grown in high potassium (25 K), showed a 34 +/- 3% increase (mean +/- S.E.M., n = 12) in 3H-thymidine incorporation as compared with the values observed in 5 K. After 24 h in vitro, cells grown in 25 K showed 23 +/- 3% (mean +/- S.E.M., n = 3) less DNA synthesis than those inoculated in 5 K. The increase in DNA synthesis due to 25 K was blocked by MgCl2 and nifedipine, but not by omega-conotoxin GVIA, suggesting that it is mediated by a Ca2+ influx via voltage-gated calcium channels (VGCC) of the L-subtype. High potassium-induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor (PD98059, 75 microM). The number of neurons counted after 48 h in vitro in 25 K was 35-100% above of the number obtained with 5 K and this increase also was blocked by MgCl2 and nifedipine. These data support the hypothesis that depolarizing activity during neurogenesis plays a role in the modulation of cerebellar granule cells proliferation.