Effect of halothane on the release of [Ca2+]i in dorsal root ganglion neurons

We investigated the effect of the volatile anaesthetic halothane on [Ca2+]i of dorsal root ganglion neurons. Halothane was able to increase [Ca2+]i in those neurons in a dose-dependent manner and independent of extracellular calcium. However, halothane action was inhibited by BAPTA-AM, suggesting the involvement of intracellular calcium stores. Dantrolene, an inhibitor of ryanodine-sensitive calcium stores had no effect while 2-APB, an inhibitor of IP3-sensitive calcium store reduced by 78% the halothane-evoked increase on [Ca2+]i. These data suggests that halothane increased [Ca2+]i of ganglion neurons through calcium release from IP3-sensitive calcium store. One possible consequence of the halothane action is to alter presynaptic activity and signaling pathways that influence neurotransmission.     

Neurokinin 2 receptor-mediated activation of protein kinase C modulates capsaicin responses in DRG neurons from adult rats

Patch-clamp techniques and Ca2+ imaging were used to examine the interaction between neurokinins (NK) and the capsaicin(CAPS)-evoked transient receptor potential vanilloid receptor 1 (TRPV1) responses in rat dorsal root ganglia neurons. Substance P (SP; 0.2-0.5 microM) prevented the reduction of Ca2+ transients (tachyphylaxis) evoked by repeated brief applications of CAPS (0.5 microM). Currents elicited by CAPS were increased in amplitude and desensitized more slowly after administration of SP or a selective NK2 agonist, [Ala8]-neurokinin A (4-10) (NKA). Neither an NK1-selective agonist, [Sar9, Met11]-SP, nor an NK3-selective agonist, [MePhe7]-NKB, altered the CAPS currents. The effects of SP on CAPS currents were inhibited by a selective NK2 antagonist, MEN10,376, but were unaffected by the NK3 antagonist, SB 235,375. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C(PKC), also increased the amplitude and slowed the desensitization of CAPS responses. Phosphatase inhibitors, decamethrin and alpha-naphthyl acid phosphate (NAcPh), also enhanced the currents and slowed desensitization of CAPS currents. Facilitatory effects of SP, NKA and PDBu were reversed by bisindolylmaleimide, a PKC inhibitor, and gradually decreased in magnitude when the agents were administered at increasing intervals after CAPS application. The decrease was partially prevented by prior application of NAcPh. These data suggest that activation of NK2 receptors in afferent neurons leads to PKC-induced phosphorylation of TRPV1, resulting in sensitization of CAPS-evoked currents and slower desensitization. Thus, activation of NK2 autoreceptors by NKs released from the peripheral afferent terminals or by mast cells during inflammatory responses may be a mechanism that sensitizes TRPV1 channels and enhances afferent excitability.     

Cytosolic Ca2+ under high glucose with suppressed Na+/K+ pump activity in rat sensory neurons

Cytosolic Ca2+ concentration ([Ca2+]i) was measured in isolated rat dorsal root ganglion (DRG) neurons using the fluorescent Ca2+ indicator fura-2. Exposure to high (50 mM) extracellular K+ evoked a robust increase in [Ca2+]i, which was almost totally abolished by concomitant presence of nisoldipine (10 microM) and omega-conotoxin GVIA (10 microM). Whereas either high (30 mM) D-glucose alone or ouabain (100 microM) alone did not appreciably affect the high K+-induced [Ca2+]i elevation, neurons pretreated with high D-glucose together with ouabain exhibited a significantly larger [Ca2+]i response to high K+ stimulation, which was almost completely inhibited by nisoldipine and omega-conotoxin GVIA. These results suggest that a combination of high glucose and suppressed Na+/K+ pump activity potentiates the [Ca2+]i elevation stimulated by activation of the voltage-gated Ca2+ channels in rat DRG neurons.     

大鼠小直径痛觉三叉神经节神经元同时存在三磷酸肌醇敏感性钙库和阿诺碱敏感性钙库？P2X和P2Y嘌呤受体介导细胞内钙信号转导通路的变化

背景： 面部创伤或面部手术等可能损伤三叉神经系统而导致三叉神经疼痛,由于其疼痛剧烈难忍、易复发,长期以来一直为口腔临床治疗的一大难题。现有大量研究发现嘌呤类受体与三叉神经痛相关,目前对其作用机制知之甚少。 目的：探讨在小直径三叉神经节神经元中嘌呤类受体介导钙信号途径。 方法：用Fura-2荧光染料通过显微镜荧光测定技术实时检测急性分离成年大鼠小直径三叉神经节神经元的细胞内钙离子浓度。 结果：用正常外液或去除细胞外Ca2+灌流细胞,分别给予thapsigargin（1 μmol/L）,内质网钙泵ATP酶抑制剂,和咖啡因（20 mmol/L）,ryanodine受体激动剂,均能够引起细胞内游离钙离子浓度（[Ca2+]i）不同程度地升高。ATP（100 μmol/L）也能够产生类似的效应。在去除细胞外Ca2+条件下,ATP引起的[Ca2+]i升高可被thapsigargin可逆性地抑制,而不能被咖啡因抑制;然而在正常外液环境中,ATP引起的细胞内[Ca2+]i 升高不能完全地被thapsigargin抑制。 结论：在痛觉三叉神经节神经元中,嘌呤类受体介导的[Ca2+]i升高有两条途径,一种途径是通过代谢型P2Y受体作用于三磷酸肌醇敏感性钙库;另一种途径是通过离子型受体P2X受体引起外钙内流。     

Role of prostaglandin receptor subtype EP1 in prostaglandin E2-induced nociceptive transmission in the rat spinal dorsal horn

It has been indicated that prostaglandin E2 (PGE2) and the receptor for PGE2 (EP receptor) are key factors contributing to the facilitated generation of nociception. This study was designed to investigate the roles of PGE2 and EP1 receptors in the spinal cord in the nociceptive transmission, using behavioral and intracellular calcium ion concentration ([Ca2+]i) assays and in situ hybridization. Experiments were conducted on Sprague-Dawley rats. In behavioral assays, withdrawal thresholds to mechanical stimuli were evaluated using von Frey filament. The effect of an intrathecally administered selective EP1 antagonist, 6-[(2S,3S)-3-(4-chloro-2-methylphenylsulfonylaminomethyl)-bicyclo[2.2.2]octan-2-yl]-5Z-hexenoic acid (ONO-8711), on the intrathecal PGE2-induced hyperalgesia was examined. In [Ca2+]i assays, we measured [Ca2+]i in the dorsal horn of spinal cord slices and examined the effects of PGE2 and ONO-8711 perfusion on the [Ca2+]i changes. In situ hybridization using EP1 digoxigenin probe was performed on the slice sections of the lumbar spinal cord and bilateral L4 and L5 dorsal root ganglions (DRGs). Mechanical hyperalgesia was observed after intrathecal PGE2 administration. Intrathecal administration of ONO-8711 attenuated the PGE2-induced mechanical hyperalgesia in a dose- and time-dependent manner. Perfusion of ONO-8711 markedly suppressed PGE2-induced [Ca2+]i increment in laminae II-VI in dorsal horn of the spinal cord slice. Moreover, in situ hybridization revealed EP1 hybridization signals in the DRG neurons, but not in the spinal cord. The results of this study suggested that spinal PGE2 activates the EP1 receptors existing on the central terminals of primary afferents, subsequently increasing in [Ca2+]i in the spinal dorsal horn, which are involved in the mechanisms of spinal PGE2-induced nociceptive transmission.     

Prostaglandin E2-induced sensitization of bradykinin-evoked responses in rat dorsal root ganglion neurons is mediated by cAMP-dependent protein kinase A

Primary cultures of neonatal rat dorsal root ganglion (DRG) neurons were used to examine the mechanisms underlying both the direct activation and the sensitization of sensory neurons by prostanoids. Prostaglandin E2 (PGE2) elevated cytosolic calcium concentration ([Ca2+]i) in a subpopulation of small (< 19 microm) diameter, capsaicin-sensitive DRG neurons. PGE2 also stimulated substance P (SP) release from DRG cultures. In contrast to bradykinin, PGE2 did not stimulate phosphoinositidase C (PIC) and the PGE2-evoked increase in [Ca2+]i was dependent on extracellular calcium. Pre-treatment with PGE2 potentiated bradykinin-evoked increases in [Ca2+]i in small diameter neurons and increased the number of cells that responded to low concentrations of bradykinin. A similar effect was seen with prostaglandin I2 (PGI2) but not prostaglandin F2alpha (PGF2alpha). PGE2 pretreatment also potentiated bradykinin-evoked release of SP, inducing a leftward shift in the bradykinin concentration-response curve and an increase in the maximum response. PGE2 stimulated adenylyl cyclase activity in DRG cultures, at concentrations and times consistent with those required to observe both the direct and sensitizing effects of the prostanoid on [Ca2+]i responses. Furthermore, the direct and sensitizing effects of PGE2, on both [Ca2+]i responses and SP release, were mimicked by the membrane permeant cAMP analogue dibutyryl cAMP and inhibited by H89, an inhibitor of cAMP-dependent protein kinase A (PKA). These observations are consistent with the hypothesis that both direct activation and sensitization of sensory neurons by prostanoids, such as PGE2, are mediated by PKA-dependent phosphorylation mechanisms.     

Estrogen receptor-alpha mediates estradiol attenuation of ATP-induced Ca2+ signaling in mouse dorsal root ganglion neurons

A mechanism underlying gender-related differences in pain perception may be estrogen modulation of nociceptive signaling in the peripheral nervous system. In rat, dorsal root ganglion (DRG) neurons express estrogen receptors (ERs) and estrogen rapidly attenuates ATP-induced Ca2+ signaling. To determine which estrogen receptor mediates rapid actions of estrogen, we showed ERalpha and ERbeta expression in DRG neurons from wild-type (WT) female mice by RT-PCR. To study whether ERalpha or ERbeta mediates this response, we compared estradiol action mediating Ca2+ signaling in DRG neurons from WT, ERalpha knockout (ERalphaKO), and ERbetaKO mice in vitro. ATP, an algesic agent, induced [Ca2+]i transients in 48% of small DRG neurons from WT mice. 17beta-Estradiol (E2) inhibited ATP-induced intracellular Ca2+ concentration ([Ca2+]i) with an IC50 of 27 nM. The effect of E2 was rapid (5-min exposure) and stereo specific; 17alpha-estradiol had no effect. E2 action was blocked by the ER antagonist ICI 182,780 (1 microM) in WT mouse. Estradiol coupled to bovine serum albumin (E-6-BSA), which does not penetrate the plasma membrane, had the same effect as E2 did, suggesting that a membrane-associated ER mediated the response. In DRG neurons from ERbetaKO mice, E2 attenuated the ATP-induced [Ca2+]i flux as it did in WT mice, but in DRG neurons from ERalphaKO mice, E2 failed to inhibit the ATP-induced [Ca2+]i increase. These results show that mouse DRG neurons express ERs and the rapid attenuation of ATP-induced [Ca2+]i signaling is mediated by membrane-associated ERalpha.     

l-Menthol-induced [Ca2+]i increase and impulses in cultured sensory neurons

We investigated the effects of l-menthol on cultured dorsal root ganglion (DRG) cells, instead of free nerve endings of sensory fibers. Using Fura-2 microfluorimetry, we identified a few DRG neurons that showed an increase in intracellular free Ca2+ concentration ([Ca2+]i) in response to l-menthol. They made up only 10% of the neurons activated by a high K+ solution. l-Menthol induced the [Ca2+]i increase in a dose-dependent manner, with an EC50 of 37.9 microM and a Hill coefficient of 0.97. A related compound, cyclohexanol, had no effect. When extracellular Ca2+ was removed, l-menthol did not induce the [Ca2+]i increase. Whole-cell current-clamp recordings revealed that l-menthol induced depolarization (13.2 mV, receptor potential) leading to impulses. We conclude that l-menthol induced the impulses through activation of menthol receptors in a small subset of the cultured sensory neurons.     

Calcium dynamics in neurons treated with toxic and non-toxic concentrations of glutamate.

Intracellular calcium concentration ([Ca2+]i) dynamics were simultaneously monitored in multiple cultured rat neurons loaded with Fluo-3 and continuously stimulated with glutamate (GLU). Three response types were observed: 10 microM GLU caused an initial transient increase in [Ca2+]i; 20 microM a biphasic response characterized by a 150-350 s 'calcium trough' between peaks; and 40 microM an initial sustained increase in [Ca2+]i. Neurons in calcium-free medium treated with 40 microM GLU showed only an initial transient increase in [Ca2+]i, demonstrating the dependence of sustained secondary increases in [Ca2+]i on extracellular calcium sources. We observed synchronized responses of multiple neurons within a given culture well, after GLU treatment, supporting the hypothesis that sustained influx of extracellular calcium may be stimulated by depletion of intracellular calcium and/or the release of endogenous excitatory amino acids.     

Cisplatin modulates voltage gated channel currents of dorsal root ganglion neurons of rats

The anticancer drug cis-diammindichloroplatin (CDDP, cisplatin) causes severe side effects like peripheral sensitive neuropathy. The toxicity of CDDP has been linked to changes in intracellular calcium homeostasis ([Ca2+]i). Voltage activated calcium channel currents (ICa(V)) are important for the regulation of [Ca2+]i; therefore, this study was designed to examine the effect of CDDP on ICa(V) in comparison to voltage activated potassium (IK(V)) and sodium (INa(V)) channel currents using the whole cell patch clamp method on dorsal root ganglion neurons of rats. In small neurons (or=?25 microm) were less sensitive to CDDP. The peak ICa(V) was reduced by 14.1+/-2.3% and IK(V) by 12.8+/-3.4% (100 microM). The sensitivity of INa(V) in large neurons to CDDP was not different compared to small neurons. We conclude that the reduction of ICa(V) in small cells may be responsible for the neurotoxic side effects CDDP causes in sensory neurons.     

Immunocytochemical localization of somatostatin receptor sst2A in the rat spinal cord and dorsal root ganglia

Intrathecal administration of octreotide, a stable somatostatin analogue, provides pain relief in patients, and locally applied somatostatin inhibits firing of nociceptive dorsal horn neurons. In the present study, we have raised polyclonal antibodies that specifically detect the somatostatin receptor sst_2A and used these antisera for immunocytochemical localization of the receptor protein in the rat spinal cord and dorsal root ganglia. In the superficial layers of the dorsal horn, sst_2A-like immunoreactivity (Li) formed a dense network consisting of neuronal perikarya and dendrites which were often closely apposed by, but not co-contained within, somatostatin-14-immunoreactive nerve fibres and terminals. sst_2A-Li was resistant to dorsal rhizotomy and did not colocalize with either substance P or calcitonin gene-related peptide suggesting that sst_2A-Li was not located to primary afferents, but rather confined to second-order spinal neurons. The position of sst_2A-Li perikarya and dendrites in the dorsal horn appeared to be similar to those containing μ-opioid receptor-Li; however, double labelling experiments revealed no instances of coexistence of these two receptors. sst_2A-Li was also observed in the dorsal root ganglia predominantly targeted to the somatic plasmalemma of medium size neurons distinct from those expressing somatostatin-14 or δ-opioid receptors. Thus, the present results not only provide a morphological substrate for spinal octreotide analgesia but also show that somatostatin and opioids are poised to modulate nociceptive transmission by distinct anatomical systems.     

Pluronic F-127 affects the regulation of cytoplasmic Ca2+ in neuronal cells

Fura-2 is one of the most widely used cytoplasmic Ca2+ ([Ca2+]cyt) sensors. In studies using isolated dorsal root ganglion (DRG) neurons, the loading of Fura-2 AM is often facilitated by the use of pluronic F-127. In preliminary studies, we detected that the use of pluronic F-127 appeared to be affecting the depolarization-evoked [Ca2+]cyt transient in DRG neurons. To determine whether this was the case, we conducted a systematic study. Adult rat DRG neurons were cultured, and their response to 50 mM KCl was measured in sister cultured cells (isolated on the same day) that were loaded with 5 microM Fura-2AM in the absence or in the presence of 0.02% pluronic F-127. In the absence of pluronic F-127, the KCl-evoked [Ca2+]cyt transient changed with time, being wider on day 1 than on day 2 after plating. On day 2, the KCl-evoked [Ca2+]cyt transient was wider in neurons Fura-2 loaded in the presence of pluronic F-127. These results indicate that pluronic F-127 significantly alters depolarization-evoked [Ca2+]cyt transients, which may reflect alteration in regulation of [Ca2+]cyt in neuronal cells.     

Neuropeptide FF selectively attenuates the effects of nociceptin on acutely dissociated neurons of the rat dorsal raphe nucleus

Intracellular Ca2+ concentration ([Ca2+]i) was measured in neurons, acutely dissociated from the rat dorsal raphe nucleus (DRN), with the fluorescent calcium probe Fluo3. Nociceptin (300 nM) had no effect on resting [Ca2+]i but reduced the magnitude of the [Ca2+]i transient triggered by depolarization in 90% of neurons having polygonal or fusiform perikarya. In 94% of neurons with the same morphology 5-HT (30 microM) also reduced the magnitude of the [Ca2+]i transient. The selective 5-HT(1A) receptor antagonist 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-ben zamide hydrochloride (p-MPPI) (0.4 microM) strongly attenuated (by 72+/-7%, n=4) this effect. The responses to nociceptin and 5-HT were not affected by BaCl2 (100 microM). The neuropeptide FF analog [D-Tyr1, (N-Me)Phe3]NPFF (1DMe) altered neither the resting [Ca2+]i nor the [Ca2+]i transient triggered by depolarization but dose-dependently decreased the effect of nociceptin (EC50=1.8 nM, maximal reduction: 68+/-5%). 1DMe had no effect on the response to 5-HT. Another neuropeptide FF analog, exhibiting a different pharmacological activity in mice and rats, [D-Tyr1, D-Leu2, D-Phe3]NPFF (1 microM) also reduced the effect of nociceptin by 74+/-11% (n=4). Few neurons (5 out of 42), either with polygonal/fusiform or smaller ovoid cell bodies, responded to the mu-opioid receptor agonist [D-Ala2, (N-Me)Phe4, Gly-ol5]-enkephalin (DAGO) with a decrease in the depolarization-induced [Ca2+]i transient. 1DMe (100 nM) attenuated this response by 69+/-14%. These results suggest that, at the cellular level, neuropeptide FF selectively counteracts the effects of opioid receptor activation.     

HIV-1 envelope protein evokes intracellular calcium oscillations in rat hippocampal neurons

Treatment of single rat hippocampal neurons with 200 pM recombinant HIV-1 envelope glycoprotein, gp120, resulted in large increases in the intracellular free calcium concentration ([Ca2+]i) as measured with indo-1-based microfluorimetry. Three patterns of [Ca2+]i increases were observed: in one pattern the [Ca2+]i rose rapidly and transiently as a single peak, in a second pattern gp120 induced [Ca2+]i oscillations that subsided when the protein was removed, and in a third pattern the oscillations continued long after washout of gp120. Both single peak and oscillatory [Ca2+]i increases were completely blocked by the Ca2+ channel blocker nitrendipine (1 microM). The sustained oscillatory responses were also blocked completely and reversibly by the N-methyl-D-aspartate (NMDA) receptor antagonist CGS19755 (10 microM) and the Na+ channel blocker tetrodotoxin (1 microM). Complete block by antagonists of Ca2+, Na+, and NMDA-gated ion channels suggests that at least two cells are required to maintain the [Ca2+]i oscillations. We hypothesize that gp120 acts as an excitotoxin by increasing synaptic activity in the network of neurons established in primary culture.     

Neuropeptide Y actions and the distribution of Ca2+-dependent Cl- conductance in rat dorsal root ganglion neurons

Neuropeptide Y (NPY) increases the excitability of 'small', nociceptive, dorsal root ganglion (DRG) neurons. This effect, which may contribute to the etiology of 'neuropathic' pain, has been attributed to attenuation of Ca2+-sensitive K+ conductance(s) (gK,Ca) following suppression of Ca2+ entry via N-type Ca2+ channels. A problem arises with this conclusion because rat DRG neurons normally contain high intracellular Cl- and some of them express a Ca2+-dependent Cl- conductance (gCl,Ca). In this study, we find that in rat DRG neurons increasing intracellular Cl- does not attenuate the effect of 1 microM NPY because gCl,Ca is not found in 'small' DRG cells and the peptide failed to affect the gCl,Ca found in 'large' cells. Thus, the presence of gCl,Ca in a subpopulation of 'large' DRG neurons does not alter the conclusion that excitatory effects of NPY result from attenuation of gK,Ca.     

Mechanisms of orexin A-evoked changes of intracellular calcium in primary cultured cortical neurons

We have investigated the effect of orexin A on the intracellular free calcium concentration ([Ca2+]i) in primary cultured cortical neurons and explored the exact mechanisms of orexin A-evoked changes of [Ca2+]i. In the present study, changes of [Ca2+]i induced by orexin A in primary cultured cortical neurons were first detected by confocal laser scanning microscopy using Ca2+-sensitive dye fluo-4 as a novel calcium fluorescent probe. Our results showed that 1-0.1 microM orexin A induced the increase in [Ca2+]i in cortical neurons. The increase in [Ca2+]i by acute application of orexin A occurred in a dose-dependent manner. Orexin A-induced increase in [Ca2+]i was not observed under the condition of Ca2+-free Dulbecco's modified Eagle's medium. Pretreatment on the cells with 1 microM thapsigargin did not block orexin A-evoked response. These findings first illuminated the fact that orexin A-induced increase in [Ca2+]i may be mainly from extracellular calcium influx in cortical neurons.     

Transient increases in extracellular K+ produce two pharmacological distinct cytosolic Ca2+ transients

Transient increases in extracellular K+ are observed under various conditions, including repetitive neuronal firing, anoxia, ischemia and hypoglycemic coma. We studied changes in cytoplasmic Ca2+ ([Ca2+]cyt) evoked by pulses of KCl in human neuroblastoma SH-SY5Y cells and rat dorsal root ganglia (DRG) neurons at 37 degrees C. A "pulse" of KCl evoked two transient increases in [Ca2+]cyt, one upon addition of KCl (K+on) and the other upon removal of KCl (K+off). The K+on transient has been described in many cell types and is initiated by the activation of voltage-dependent Ca2+ channels followed by Ca2+-evoked Ca2+ release from intracellular Ca2+ stores. The level of KCl necessary to evoke the K+off transient depends on the type of neuron, in SH-SY5Y cells it required 100 mM KCl, in most (but not all) of dorsal root ganglia neurons it could be detected with 100-200 mM KCl and in a very few dorsal root ganglia neurons it was detectable at 20-50 mM KCl. In SH-SY5Y cells, reduction of extracellular Ca2+ inhibited the K+on more strongly than the K+off and slowed the decay of K+off. Isoflurane (1 mM) reduced the K+on)- but not the K+off-peak. However, isoflurane slowed the decay of K+off. The nonspecific cationic channel blocker La3+ (100 microM) had an effect similar to that of isoflurane. Treatment with thapsigargin (TG) at a concentration known to only deplete IP3-sensitive Ca2+ stores did not affect K+on or K+off, suggesting that Ca2+ release from the IP3-sensitive Ca2+ stores does not contribute to K+on and K+off transients and that the thapsigargin-sensitive Ca2+ ATPases do not contribute significantly to the rise or decay rates of these transients. These findings indicate that a pulse of extracellular K+ produces two distinct transient increases in [Ca2+]cyt.     

Functional NMDA and GABAA receptors in pioneer neurons of the cortical marginal zone

Transient pioneer neurons in the neocortical marginal zone generate an early corticofugal axonal projection at E12-E16 (Meyer et al. 1998). We have analysed the functional activity of glutamate and GABA receptors in such cells by measuring changes in intracellular calcium concentrations ([Ca2+]i). The activation of GABAA receptors with muscimol, as well as bath application of glutamate, lead to increases in [Ca2+]i in pioneer neurons. The stimulatory action of glutamate is mostly produced through the NMDA-type of ionotropic receptors. Metabotropic glutamate receptor activation has no effect on [Ca2+]i. Consistent with such results, immunocytochemical studies showed a prominent expression of GABAA and NMDA receptors in pioneer neurons. The activation of such receptors may be implicated in the remodelling of pioneer neurons during development.     

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.