Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons.

The effects of dihydropyridines (DHPs) normally considered to be specific for L-type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/- 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L-type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons.     

Spontaneous miniature hyperpolarizations affect threshold for action potential generation in mudpuppy cardiac neurons

Mudpuppy parasympathetic neurons exhibit spontaneous miniature hyperpolarizations (SMHs) that are generated by potassium currents, which are spontaneous miniature outward currents (SMOCs), flowing through clusters of large conductance voltage- and calcium (Ca(2+))-activated potassium (BK) channels. The underlying SMOCs are initiated by a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Perforated-patch whole cell voltage recordings were used to determine whether activation of SMHs contributed to action potential (AP) repolarization or affected the latency to AP generation. Blockade of BK channels by iberiotoxin (IBX, 100 nM) slowed AP repolarization and increased AP duration. Treatment with omega-conotoxin GVIA (3 microM) or nifedipine (10 microM) to inhibit Ca(2+) influx through N- or L-type voltage-dependent calcium channels (VDCCs), respectively, also decreased the rate of AP repolarization and increased AP duration. Elimination of CICR by treatment with either thapsigargin (1 microM) or ryanodine (10 microM) produced no significant change in AP repolarization or duration. Blockade of BK channels with IBX and inhibition of N-type VDCCs with omega-conotoxin GVIA, but not inhibition of L-type VDCCs with nifedipine, decreased the latency of AP generation. A decrease in latency to AP generation occurred with elimination of SMHs by inhibition of CICR following treatment with thapsigargin. Ryanodine treatment decreased AP latency in three of six cells. Apamin (100 nM) had no affect on AP repolarization, duration, or latency to AP generation, but did decrease the hyperpolarizing afterpotential (HAP). Inhibition of L-type VDCCs by nifedipine also decreased HAP amplitude. Inhibition of CICR by either thapsigargin or ryanodine treatment increased the number of APs generated with long depolarizing current pulses, whereas exposure to IBX or omega-conotoxin GVIA depressed excitability. We conclude that CICR, the process responsible for SMH generation, represents a unique mechanism to modulate the response to subthreshold depolarizing currents that drive the membrane potential toward the threshold for AP initiation but does not contribute to AP repolarization. Subthreshold depolarizations would not activate sufficient numbers of VDCCs to allow Ca(2+) influx to elevate [Ca(2+)](i) to the extent needed to directly activate nearby BK channels. However, the elevation in [Ca(2+)](i) is sufficient to trigger CICR from ryanodine-sensitive Ca(2+) stores. Thus CICR acts as an amplification mechanism to trigger a local elevation of [Ca(2+)](i) near a cluster of BK channels to activate these channels at negative levels of membrane potential.     

Dihydropyridine block of voltage-dependent K currents in rat dorsal root ganglion neurons

The dihydropyridines nifedipine, nimodipine and Bay K 8644 are widely used as pharmacological tools to assess the contribution of L-type voltage-gated Ca²⁺ channels to a variety of neuronal processes including synaptic transmission, excitability and second messenger signaling. These compounds are still used in neuronal preparations despite evidence from cardiac tissue and heterologous expression systems that they block several voltage-dependent K⁺ (Kv) channels. Both because these compounds have been used to assess the relative contribution of L-type Ca²⁺ channels to several different processes in dorsal root ganglion (DRG) neurons and because a relatively wide variety of Kv channels present in other neuronal populations is present in DRG neurons, we determined the extent to which dihydropyridines block Kv currents in these neurons. Standard whole cell patch clamp techniques were used to study acutely disassociated adult rat DRG neurons. All three dihydropyridines tested blocked Kv currents in DRG neurons; IC₅₀ values (concentration resulting in an inhibition that is 50% of maximum) for nifedipine and nimodipine-induced block of sustained Kv currents were 14.5 and 6.6 μM, respectively. The magnitude of sustained current block was 44±1.6%, 60±2%, and 56±2.9% with 10 μM nifedipine, nimodipine and Bay K 8644, respectively. Current block was occluded by neither 4-aminopyridine (5 mM) nor tetraethylammonium (135 mM). Dihydropyridine-induced block of Kv currents was not associated with a shift in the voltage-dependence of current activation or inactivation, the recovery from inactivation, or voltage dependent block. However, there was a small use-dependence to the dihydropyridine-induced block. Our results suggest that several types of Kv channels in DRG neurons are blocked by mechanisms distinct from those underlying block of Kv channels in cardiac myocytes. Importantly, our results suggest that if investigators wish to explore the contribution of L-type Ca²⁺ channels to neuronal function, they should consider alternative strategies for the manipulation of these channels than the use of dihydropyridines.     

Electrophysiological roles of L-type channels in different classes of guinea pig sympathetic neuron.

The electrophysiological consequences of blocking Ca(2+) entry through L-type Ca(2+) channels have been examined in phasic (Ph), tonic (T), and long-afterhyperpolarizing (LAH) neurons of intact guinea pig sympathetic ganglia isolated in vitro. Block of Ca(2+) entry with Co(2+) or Cd(2+) depolarized T and LAH neurons, reduced action potential (AP) amplitude in Ph and LAH neurons, and increased AP half-width in Ph neurons. The afterhyperpolarization (AHP) and underlying Ca(2+)-dependent K(+) conductances (gKCa1 and gKCa2) were reduced markedly in all classes. Addition of 10 microM nifedipine increased input resistance in LAH neurons, raised AP threshold in Ph and LAH neurons, and caused a small increase in AP half-width in Ph neurons. AHP amplitude and the amplitude and decay time constant of gKCa1 were reduced by nifedipine in all classes; the slower conductance, gKCa2, which underlies the prolonged AHP in LAH neurons, was reduced by 40%. Surprisingly, AHP half-width was lengthened by nifedipine in a proportion of neurons in all classes; despite this, neuron excitability was increased during a maintained depolarization. Nifedipine's effects on AHP half-width were not mimicked by 2 mM Cs(+) or 2 mM anthracene-9-carboxylic acid, a blocker of Cl(-) channels, and it did not modify transient outward currents of the A or D types. The effects of 100 microM Ni(2+) differed from those of nifedipine. Thus in Ph neurons, Ca(2+) entry through L-type channels during a single action potential contributes to activation of K(+) conductances involved in both the AP and AHP, whereas in T and LAH neurons, it acts only on gKCa1 and gKCa2. These results differ from the results in rat superior cervical ganglion neurons, in which L-type channels are selectively coupled to BK channels, and in hippocampal neurons, in which L-type channels are selectively coupled to SK channels. We conclude that the sources of Ca(2+) for activating the various Ca(2+)-activated K(+) conductances are distinct in different types of neuron.     

Differential expression of calcium channels in sympathetic and parasympathetic preganglionic inputs to neurons in paracervical ganglia of guinea-pigs

Neurons in pelvic ganglia receive nicotinic excitatory post-synaptic potentials (EPSPs) from sacral preganglionic neurons via the pelvic nerve, lumbar preganglionic neurons via the hypogastric nerve or both. We tested the effect of a range of calcium channel antagonists on EPSPs evoked in paracervical ganglia of female guinea-pigs after pelvic or hypogastric nerve stimulation. omega-Conotoxin GVIA (CTX GVIA, 100 nM) or the novel N-type calcium channel antagonist, CTX CVID (100 nM) reduced the amplitude of EPSPs evoked after pelvic nerve stimulation by 50-75% but had no effect on EPSPs evoked by hypogastric nerve stimulation. Combined addition of CTX GVIA and CTX CVID was no more effective than either antagonist alone. EPSPs evoked by stimulating either nerve trunk were not inhibited by the P/Q calcium channel antagonist, omega-agatoxin IVA (100 nM), nor the L-type calcium channel antagonist, nifedipine (30 microM). SNX 482 (300 nM), an antagonist at some R-type calcium channels, inhibited EPSPs after hypogastric nerve stimulation by 20% but had little effect on EPSPs after pelvic nerve stimulation. Amiloride (100 microM) inhibited EPSPs after stimulation of either trunk by 40%, while nickel (100 microM) was ineffective. CTX GVIA or CTX CVID (100 nM) also slowed the rate of action potential repolarization and reduced afterhyperpolarization amplitude in paracervical neurons. Thus, release of transmitter from the terminals of sacral preganglionic neurons is largely dependent on calcium influx through N-type calcium channels, although an unknown calcium channel which is resistant to selective antagonists also contributes to release. Release of transmitter from lumbar preganglionic neurons does not require calcium entry through either conventional N-type calcium channels or the variant CTX CVID-sensitive N-type calcium channel and seems to be mediated largely by a novel calcium channel.     

"Upstream" regulation of the release probability in sympathetic nerve varicosities

The results appear to support the following tentative working hypothesis. (1) Nerve impulse-induced transmitter release from sympathetic nerve varicosities is monoquantal and highly intermittent (probability range: 0-0.03). (2) Nerve impulses invade varicosities as all-or-none, Na+ channel-dependent action potentials; invasion failure may be rare. (3) The release probability is not controlled by properties (amplitude or duration) of the invading action potential or the resulting Ca2+ current, but by the availability of an as yet unidentified permissive factor. (4) The permissive factor is actively transported intra-axonally, probably in association with organelles (LDVs?). (5) The activation and/or transport of the permissive factor are controlled "upstream" of the varicosity; they depend on Ca2+ influx through channels insensitive to nifedipine (hence, not of L-type) but blocked by Cd2+ and apparently opened by slight depolarization of the resting membrane, in this respect behaving more as T- than N-type channels. (6) A high resting K+ efflux "upstream" of the varicosity restricts the availability of the permissive factor; it is the main mechanism maintaining the (economically necessary) low release probability. (7) Prejunctional agonists do not inhibit transmitter secretion by causing a conduction block or by reducing the action potential-induced Ca2+ influx into the varicosity itself, but by depressing the Ca2(+)-dependent activation and/or transport of the permissive factor; they act at least in part via receptors "upstream" of the varicosity. (8) This hypothesis for regulation of the release probability in sympathetic nerves may apply, at least in part, to other neurons as well.     

Dihydropyridines interact with calcium-independent potassium currents in embryonic mammalian sensory neurons

Early embryonic sensory neurons have two K currents resembling delayed rectifier and transient K currents of mature neurons. However, in contrast to those of adult neurons, the embryonic currents can hardly be separated either by electrophysiological or pharmacological methods, limiting their characterisation at these developmental stages. Using the whole-cell recording technique, we found that dihydropyridines (DHPs) inhibit the non-inactivating component of the Ca-independent K currents of 13-day mouse embryo dorsal-root ganglion (DRG) cells. The inhibitory effect of nicardipine began around 0.5 M and was nearly complete at 5 M while Na currents were not altered. This effect was reversible and voltage-dependent. The same results were obtained using another DHP Ca antagonist, nimodipine, whereas Bay K 8644, a DHP Ca agonist, had no effect. Kinetic properties of the DHP-insensitive K current have been described and compared with those of transient K currents found in differentiated neurons. These results suggest that both Ca and K channels have DHP sites, possibly homologous, at this developmental stage. The DHP inhibition of Ca-independent K channels provides a new tool with which to study K channels both at a molecular level and during DRG development.     

Ca(2+) channels involved in the generation of the slow afterhyperpolarization in cultured rat hippocampal pyramidal neurons

The advantages of using isolated cells have led us to develop short-term cultures of hippocampal pyramidal cells, which retain many of the properties of cells in acute preparations and in particular the ability to generate afterhyperpolarizations after a train of action potentials. Using perforated-patch recordings, both medium and slow afterhyperpolarization currents (mI(AHP) and sI(AHP), respectively) could be obtained from pyramidal cells that were cultured for 8-15 days. The sI(AHP) demonstrated the kinetics and pharmacologic characteristics reported for pyramidal cells in slices. In addition to confirming the insensitivity to 100 nM apamin and 1 mM TEA, we have shown that the sI(AHP) is also insensitive to 100 nM charybdotoxin but is inhibited by 100 microM D-tubocurarine. Concentrations of nifedipine (10 microM) and nimodipine (3 microM) that maximally inhibit L-type calcium channels reduced the sI(AHP) by 30 and 50%, respectively. However, higher concentrations of nimodipine (10 microM) abolished the sI(AHP), which can be partially explained by an effect on action potentials. Both nifedipine and nimodipine at maximal concentrations were found to reduce the HVA calcium current in freshly dissociated neurons to the same extent. The N-type calcium channel inhibitor, omega-conotoxin GVIA (100 nM), irreversibly inhibited the sI(AHP) by 37%. Together, omega-conotoxin (100 nM) and nifedipine (10 microM) inhibited the sI(AHP) by 70%. 10 microM ryanodine also reduced the sI(AHP) by 30%, suggesting a role for calcium-induced calcium release. It is concluded that activation of the sI(AHP) in cultured hippocampal pyramidal cells is mediated by a rise in intracellular calcium involving multiple pathways and not just entry via L-type calcium channels.     

Role of presynaptic L-type Ca2+ channels in GABAergic synaptic transmission in cultured hippocampal neurons

Using dual whole cell patch-clamp recordings of monosynaptic GABAergic inhibitory postsynaptic currents (IPSCs) in cultured rat hippocampal neurons, we have previously demonstrated posttetanic potentiation (PTP) of IPSCs. Tetanic stimulation of the GABAergic neuron leads to accumulation of Ca2+ in the presynaptic terminals. This enhances the probability of GABA-vesicle release for up to 1 min, which underlies PTP. In the present study, we have examined the effect of altering the probability of release on PTP of IPSCs. Baclofen (10 microM), which depresses presynaptic Ca2+ entry through N- and P/Q-type voltage-dependent Ca2+ channels (VDCCs), caused a threefold greater enhancement of PTP than did reducing [Ca2+]o to 1.2 mM, which causes a nonspecific reduction in Ca2+ entry. This finding prompted us to investigate whether presynaptic L-type VDCCs contribute to the Ca2+ accumulation in the boutons during spike activity. The L-type VDCC antagonist, nifedipine (10 microM), had no effect on single IPSCs evoked at 0.2 Hz but reduced the PTP evoked by a train of 40 Hz for 2 s by 60%. Another L-type VDCC antagonist, isradipine (5 microM), similarly inhibited PTP by 65%. Both L-type VDCC blockers also depressed IPSCs during the stimulation (i.e., they increased tetanic depression). The L-type VDCC "agonist" (-)BayK 8644 (4 microM) had no effect on PTP evoked by a train of 40 Hz for 2 s, which probably saturated the PTP process, but enhanced PTP evoked by a train of 1 s by 91%. In conclusion, the results indicate that L-type VDCCs do not participate in low-frequency synchronous transmitter release, but contribute to presynaptic Ca2+ accumulation during high-frequency activity. This helps maintain vesicle release during tetanic stimulation and also enhances the probability of transmitter release during the posttetanic period, which is manifest as PTP. Involvement of L-type channels in these processes represents a novel presynaptic regulatory mechanism at fast CNS synapses.     

Inhibitory effect of pranidipine on N-type voltage-dependent Ca2+ channels in mice

We investigated the N-type voltage-dependent calcium channel blocking action of pranidipine, a novel dihydropyridine (DHP) derivative. Pranidipine significantly suppressed KCl-induced intracellular calcium changes ([Ca(2+)](i)) in a dose-dependent fashion in dorsal root ganglion neurons. A patch-clamp investigation revealed a dose-dependent blocking effect on N-type currents. Intrathecal injection of pranidipine significantly shortened the licking time in the late phase of the formalin test, as occurs with cilnidipine and amlodipine, which act on L- and N-type channels. Conversely, nicardipine, which acts exclusively on L-type channels, had no antinociceptive effect. Our results indicate that pranidipine inhibits N-type calcium channels. Furthermore, it exerts an antinociceptive effect, which might be related to an attenuation of synaptic transmission by nociceptive neurons due to the blocking effect of pranidipine on N-type calcium channels in primary nociceptive afferent fibers.     

Dihydropyridines modulate K+-evoked amino acid and adenosine release from cerebellar neuronal cultures

Partial depolarization of primary cerebellar neuronal cultures with K+ evoked the release of aspartate, glutamate, adenosine, serine, taurine, gamma-aminobutyric acid (GABA), alanine and proline. The dihydropyridine calcium channel agonist, BAY K 8644, significantly augmented the K+-induced release of adenosine, aspartate, glutamate and GABA, but not that of serine, taurine, alanine or proline. However, in all cases the dihydropyridine antagonist nifedipine decreased this BAY K 8644-enhanced, K+-evoked efflux to below control levels. Neither BAY K 8644 nor nifedipine alone affected basal efflux levels. The phenylalkylamine calcium channel antagonist, verapamil, was ineffective in antagonizing K+-evoked amino acid release except at very high concentration (100 microM). These findings suggest that L-type Ca2+ channels are present in both excitatory (glutamatergic granule cells) and inhibitory (GABAergic stellate and basket cells) neurons in these cultures, and that they appear to be involved in regulating the release of not only neuroactive amino acids, but also some neutral amino acids and adenosine.     

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.     

L-type calcium channel blockade modifies anesthetic actions on aged hippocampal neurons

Previous studies in our laboratory demonstrated a reversal of anesthetic actions on aged neurons by decreasing extracellular [Ca(2+)] in hippocampal slices. Such maneuver indirectly attenuated Ca(2+) influx, hence decreased exogenous intraneuronal Ca(2+) loads during neuronal activity and consequently improved intracellular Ca(2+) concentration homeostasis. Therefore, in the present study we hypothesized that decreasing exogenous Ca(2+) loads by blocking voltage-gated calcium influx in aged neurons would oppose isoflurane actions. Conversely, increasing endogenous Ca(2+) loads by suppressing calcium efflux during forced reversal of Na(+)/Ca(2+) exchanger function would enhance anesthetic effects. Hippocampal slices were prepared from young (2-4 months) and old (24-26 months) Fischer 344 rats. Isoflurane depressed the evoked dendritic field excitatory postsynaptic potentials by approximately 45% in slices taken from old animals. However, application of isoflurane in addition with CoCl(2) or nifedipine opposed the anesthetic actions, which then depressed the evoked dendritic field postsynaptic potentials by only 15%. Selective blockade of the N-type and P/Q-type calcium channels with omega-conotoxin GVIA and omega-conotoxin MVIIC respectively caused rapid but partial depression of synaptic transmission in slices taken from old Fischer 344 rats. However, isoflurane actions in these aged slices were not affected compared with slices perfused only with normal artificial cerebrospinal fluid. Young and aged slices were then exposed to a low sodium perfusate that forces the Na(+)/Ca(2+) exchanger protein into a reverse mode, thus increasing intracellular Ca(2+) concentration. Isoflurane actions under such conditions were profoundly potentiated in aged slices but were not altered in young hippocampi. The current results show that in aged central neurons, selectively blocking L-type calcium channels opposes anesthetic-induced depression of excitatory synaptic transmission. On the contrary, increasing calcium loads in aged neurons potentiates these actions.     

Neurochemical evidence for the involvement of N-type calcium channels in transmitter secretion from peripheral endings of sensory nerves in guinea pigs.

In the guinea pig ureter, substance P-(SP) and calcitonin gene-related peptide-(CGRP) like immunoreactivity (LI) were depleted by systemic capsaicin pretreatment, indicating that they are entirely stored in peripheral endings of primary afferent neurons. Electrical field stimulation (20 Hz, 60 V, 0.5 ms) evoked the simultaneous release of SP- and CGRP-LI from superfused guinea pig ureters which was abolished by tetrodotoxin (0.3 microM). omega-Conotoxin (0.1 microM), a potent blocker of N-type voltage-sensitive calcium channels, reduced by 50-70% the evoked release of both peptides. These findings provide direct neurochemical evidence indicating that conotoxin-sensitive calcium channels play a role in transmitter secretion evoked by antidromic invasion of peripheral terminals of capsaicin-sensitive primary afferents.     

The effects on net fluid transport of noxious stimulation of jejunal mucosa in anaesthetized rats

A major aim of the present study was to investigate whether exposing the jejunal mucosa to a noxious stimulus induces a net fluid secretion by activating the enteric nervous system (ENS) and, if so, to what extent an axon reflex was involved. Net fluid transport was measured in vivo with a gravimetric method. The intestinal mucosa was exposed to an isotonic solution with an unphysiologically low pH (1.0). This evoked a fluid secretion, which was markedly attenuated by giving hexamethonium (nicotinic receptor antagonist) i.v. or exposing the intestinal serosa to lidocaine (local anaesthetic). Atropine (muscarinic receptor antagonist) had no effect. Luminal acid evoked a fluid secretion of the same magnitude in acutely denervated segments and in segments denervated about 3 weeks prior to the experiments. Luminal capsaicin (1.6–16 m M ) did not influence jejunal net fluid transport. A second aim of the study is to investigate the effect of nifedipine (Ca channel blocker of L-type) on the acid-induced fluid secretion. Nifedipine markedly attenuated acid-induced fluid secretion. In contrast to cholera toxin-evoked secretion, the nifedipine effect was not mediated via 5-hydroxytryptamine (5-HT) as judged by measurements of 5-HT release into the intestinal lumen and the lack of effect of granisetron (5-HT₃ receptor antagonist). It is concluded that the net fluid secretion evoked by hydrochloric acid in the small intestine is mainly mediated via an intramural reflex in the ENS. No experimental evidence was obtained for the involvement of an axon reflex. The site of action of the calcium channel blocker is tentatively discussed.     

Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.     

P物质对大鼠DRG神经元H+门控电流的调制作用

 目的：观察P物质（SP）对神经元H+门控电流的调制作用及其可能存在的机制。方法：采用全细胞膜片钳技术分别记录pH 4.0、pH 5.0、pH 6.0及共加H+ 与SP情况下,急性分离的大鼠背根神经节（DRG)神经元H+门控电流的形式。通过胞内透析技术分析SP对H+门控电流可能的调控机制。结果：H+门控电流可划分为短暂内流型（T型）、持续内向电流型（S型）、反向电流型（O型）及双相内向电流型（B型）。SP对S型和B型中持续成分的H+门控电流有明显增强作用,这种电流幅值增强达（85.53±22.93）%,约81.8%细胞的这种增强效应不能被选择性的SP受体NK1拮抗剂阻断,而非肽类的SP受体NK1拮抗剂对约75%的DRG神经元可明显阻断这种增强效应;SP对S型和B型中持续成分的H+门控电流也有明显抑制作用,抑制的幅值达（48.46±4.45）%,并且约88.9%细胞的这种抑制效应不能被SP受体NK1拮抗剂所阻断。通过胞内透析技术在细胞内液中加入GDP-β-S后不能消除SP对H+门控电流的调制作用。结论：H+门控电流有T型、S型、O型及B型4种电流类型。SP对H+门控电流有增强和抑制的双向调节作用。SP可能通过G蛋白偶联受体通路及与H+门控离子通道的某一位点直接结合来发挥对H+门控电流的双向调节调制作用。     

Dopamine modulates synaptic transmission between rat olfactory bulb neurons in culture

The glomerular layer of the olfactory bulb (OB) contains synaptic connections between olfactory sensory neurons and OB neurons as well as connections among OB neurons. A subpopulation of external tufted cells and periglomerular cells (juxtaglomerular neurons) expresses dopamine, and recent reports suggest that dopamine can inhibit olfactory sensory neuron activation of OB neurons. In this study, whole cell electrophysiological and primary culture techniques were employed to characterize the neuromodulatory properties of dopamine on glutamatergic transmission between rat OB mitral/tufted (M/T) cells and interneurons. Immunocytochemical analysis confirmed the expression of tyrosine hydroxylase, the rate-limiting enzyme for dopamine synthesis, in a subpopulation of cultured neurons. D2 receptor immunoreactivity was also observed in cultured M/T cells. Dopamine reduced spontaneous excitatory synaptic events recorded in interneurons. Although the D1 receptor agonist SKF38393 and the D2 receptor agonist bromocriptine mesylate mimicked this effect, evoked excitatory postsynaptic potentials (EPSPs) recorded from monosynaptically coupled neuron pairs were attenuated by dopamine and bromocriptine but not by SKF38393. Neither glutamate-evoked currents nor the membrane resistance of the postsynaptic interneuron were affected by dopamine. However, evoked calcium channel currents in the presynaptic M/T cell were diminished during the application of either dopamine or bromocriptine, but not SKF38393. Dopamine suppressed calcium channel currents even after nifedipine blockade of L-type channels, suggesting that inhibition of the dihydropyridine-resistant high-voltage activated calcium channels implicated in transmitter release may mediate dopamine's effects on spontaneous and evoked synaptic transmission. Together, these data suggest that dopamine inhibits excitatory neurotransmission between M/T cells and interneurons via a presynaptic mechanism.     

Hypoxic increase in nitric oxide generation of rat sensory neurons requires activation of mitochondrial complex II and voltage-gated calcium channels

Recently, we have demonstrated that sensory neurons of rat lumbar dorsal root ganglia (DRG) respond to hypoxia with an activation of endothelial nitric oxide (NO) synthase (eNOS) resulting in enhanced NO production associated with mitochondria which contributes to resistance against hypoxia. Extracellular calcium is essential to this effect. In the present study on rat DRG slices, we set out to determine what types of calcium channels operate under hypoxia, and which upstream events contribute to their activation, thereby focusing upon mitochondrial complex II. Both the metallic ions Cd2+ and Ni2+, known to inhibit voltage-gated calcium channels and T-type channels, respectively, and verapamil and nifedipine, typical blocker of L-type calcium channels completely prevented the hypoxic neuronal NO generation. Inhibition of complex II by thenoyltrifluoroacetone at the ubiquinon binding site or by 3-nitropropionic acid at the substrate binding site largely diminished hypoxic-induced NO production while having an opposite effect under normoxia. An additional blockade of voltage-gated calcium channels entirely abolished the hypoxic response. The complex II inhibitor malonate inhibited both normoxic and hypoxic NO generation. These data show that complex II activity is required for increased hypoxic NO production. Since succinate dehydrogenase activity of complex II decreased at hypoxia, as measured by histochemistry and densitometry, we propose a hypoxia-induced functional switch of complex II from succinate dehydrogenase to fumarate reductase, which subsequently leads to activation of voltage-gated calcium channels resulting in increased NO production by eNOS.