Heterogeneous calcium currents and transmitter release in cultured mouse spinal cord and dorsal root ganglion neurons.

1. Calcium currents and transmitter release were studied in cocultures of fetal mouse neurons from the ventral half of the spinal cord (VH neurons) and from dorsal root ganglion (DRG neurons). The effects of BayK 8644 and omega-conotoxin on calcium currents and transmitter release were compared. 2. The presence of low voltage-activated (LVA) calcium current in both VH and DRG neurons is variable. Some cells exhibit only high voltage-activated (HVA) currents, whereas others show both HVA and LVA currents. 3. BayK 8644 did not affect LVA currents but strongly augmented both steady and transient components of the HVA calcium conductance. 4. omega-Conotoxin GVIA reduces both transient and steady components of the HVA but does not abolish either component even after 3 h of application. 5. Calcium currents that were resistant to omega-contoxin were augmented by BayK 8644. 6. Synaptic transmission between pairs of spinal cord neurons from the ventral half of the spinal cord (VH-VH connections) or between dorsal root ganglion neurons and VH neurons (DRG-VH connections) were studied with two-cell recording and stimulation techniques. 7. In approximately 70% of VH-VH connections and 50% of DRG-VH connections, BayK 8644 or its active optical isomer failed to affect transmitter output. Substantial augmentation of the remainder of the connections could be reliably produced by the dihydropyridines. Raised calcium in the extracellular medium produced augmentation of synaptic connections in all cases. BayK 8644 produced substantial, consistent augmentation of voltage-sensitive calcium channels in both VH and DRG neurons. 8. The toxin, omega-conotoxin, produced no consistent effect on excitatory or inhibitory postsynaptic potentials (EPSPs or IPSPs) elicited in VH neurons by stimulation of nearby VH neurons. VH EPSPs elicited by stimulation of nearby DRG neurons were reduced to approximately 50% of control values after 10 min of omega-conotoxin perfusion. Spontaneous and evoked synaptic activity could be recorded in VH neurons as long as 2 h after cultures were incubated in 0.5 microM omega-conotoxin. omega-Conotoxin produced a modest reduction in HVA currents in both VH and DRG neurons. 9. BayK 8644 did not produce consistent augmentation of transmission at the frog neuromuscular junction. omega-Conotoxin produced total blockade of transmission in this preparation. 10. We conclude that neither sustained nor inactivating high-threshold voltage-sensitive (HVA) calcium channels sensitive to BayK 8644 or omega-conotoxin such as those measured in the neuronal cell bodies are responsible for action-potential-evoked transmitter release from the majority of VH neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of anaphylactic histamine release by calcium channel agonists and antagonists

Calcium antagonists have been reported to exert protective effects in hypersensitivity reactions in man and animals. However, their effect on anaphylactic histamine release is highly variable and controversial. In the present paper we evaluate the effect of calcium entry blockers and BAY K 8644 on the response to specific antigen in isolated hearts taken from actively sensitized guinea-pigs and from isolated rat and guinea-pig mast cells, actively or passively sensitized. Verapamil, diltiazem, nifedipine and prenylamine dose-dependently decreased anaphylactic histamine release in isolated actively sensitized guinea-pig mast cells. BAY K 8644 was found to be ineffective. In isolated, passively sensitized rat mast cells, verapamil showed a highly signficant inhibitory effect, while prenylamine (10^–4 M) was able to evoke a histamine releasing effect. In cardiac anaphylaxis verapamil, diltiazem, prenylamine, but not nifedipine, were active in reducing the release of histamine without modifying the antigen-induced arrhythmias and positive chronotropic and inotropic effects.     

The effects of calcium channel agonists and antagonists on the release of endogenous glutamate from cerebellar slices

The effects of compounds acting at the calcium channel on neurotransmitter release are equivocal. We report here the effects of the antagonists, verapamil, diltiazem and nifedipine; the agonists, bay K8644 and the calcium ionophore, A23187 on the release of endogenous glutamate from rat cerebellar slices. Of these compounds, only verapamil and diltiazem modified glutamate release and these were effective at relatively high concentrations (greater than 1 x 10(-5) M). It is suggested that the high-affinity binding sites found in neuronal tissue for the dihydropyridine-like compounds are not involved in neurotransmitter release.     

Calcium currents and transmitter output in cultured spinal cord and dorsal root ganglion neurons

The effects of repetitive activation upon voltage-dependent calcium currents (ICa) and transmitter release were studied in dissociated cell cultures of fetal mouse spinal cord and dorsal root ganglion. Sodium and potassium currents were suppressed with tetrodotoxin (TTX) and tetraethylammonium (TEA) ions, 4-aminopyridine (4-AP), and cesium sulfate. Calcium currents were compared under voltage clamp before and after a series of depolarizing clamp pulses in spinal cord (SC) and dorsal root ganglion (DRG) neurons. Repetitive activation resulted in an exponential decline in ICa, with the decrease in ICa being much more marked in DRG compared with SC neurons. Both voltage-dependent inactivation and inactivation related to the intracellular movement of Ca2+ appeared to be involved in the decrement in ICa with repetitive activation. A decrease in transmitter output occurred with repetitive activation in DRG neurons but not in SC neurons (either excitatory or inhibitory). DRG neuron synaptic boutons had fewer mitochondria than did the boutons of either excitatory or inhibitory of SC neurons. The decrement in both ICa and synaptic transmitter output in DRG neurons could last for prolonged periods (at least minutes) following repetitive activation. We hypothesize that this vulnerability of DRG neurons to repetitive activation may be related, at least in part, to a relative incapacity to maintain a low intracellular calcium ion concentration [Ca]i during periods of increased calcium ingress associated with excitation. Such an incapacity to buffer [Ca]i may be one mechanism leading to the inactive synapses seen in some studies in vitro and in vivo of synaptic transmission.     

Potentiating and depressant effects of metabotropic glutamate receptor agonists on high-voltage-activated calcium currents in cultured retinal ganglion neurons from postnatal mice

This study was aimed at clarifying the role of metabotropic glutamate receptors (mGluRs) in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in postnatal mouse retinal ganglion neurons (RGNs). RGNs were maintained for 1–2 weeks in vitro by adding brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) to the culture medium. In order to select these cells for electrophysiological measurements, RGNs were vitally labelled with an antibody against Thy-1.2. Voltage-activated Ca²⁺ currents [I _Ca(V)] were recorded with patch electrodes in the wholecell configuration. It was found that racemic ±-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (t-ACPD) or its active enantiomer 1S,3R-ACPD rapidly and reversibly either enhanced or depressed I _Ca(V). Quisqualate (QA), l-2-amino-4-phosphonobutyrate (l-AP4) and the endogenous transmitter glutamate induced similar effects when ionotropic glutamate receptors were blocked with d-2-amino-5-phosphonovalerate (d-APV) and 6,7-dinitroquinoxaline-2, 3-dione (DNQX). - Conotoxin GVIA (-CgTx GVIA), but not nifedipine prevented modulation of I _Ca(V) by mGluR agonists. The depression of I _Ca(V) by t-ACPD was irreversible when cells were dialysed with guanosine-5-O-(3-thiotriphosphate) (GTP[-S]). Ratio measurements of fura-2 fluorescence in Thy-1+ cells showed that neither t-ACPD, QA nor l-AP4 affected [Ca²⁺]_i by liberation of Ca²⁺ from intracellular stores. Our results suggest that cultured RGNs express mGluRs. These receptors cannot induce Ca²⁺ release from intracellular stores but regulate [Ca²⁺]_i by a fast and reversible, G-protein-mediated action on a subpopulation of voltage-activated Ca²⁺ channels.     

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (K(Ca)) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K(Ca) channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K(Ca) channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K(Ca) channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.     

Differential calcium dependence in basal and forskolin-potentiated spontaneous transmitter release in basolateral amygdala neurons

Action potential-independent transmitter release, or spontaneous release, is postulated to produce multiple postsynaptic effects (e.g., maintenance of dendritic spines and suppression of local dendritic protein synthesis). Potentiation of spontaneous release may contribute to the precise modulation of synaptic function. However, the expression mechanism underlying potentiated spontaneous release remains unclear. In this study, we investigated the involvement of extracellular and intracellular calcium in basal and potentiated spontaneous release. Miniature excitatory postsynaptic currents (mEPSCs) of the basolateral amygdala neurons in acute brain slices were recorded. Forskolin, an adenylate cyclase activator, increased mEPSC frequency, and the increase lasted at least 25 min after washout. Removal of the extracellular calcium decreased mEPSC frequency in both naïve and forskolin-treated slices. On the other hand, chelation of intracellular calcium by BAPTA-AM decreased mEPSC frequency in naïve, but not in forskolin-treated slices. A blockade of the calcium-sensing receptor (CaSR) resulted in an increase in mEPSC frequency in forskolin-treated, but not in naïve slices. These findings indicate that forskolin-induced potentiation is accompanied by changes in the mechanisms underlying Ca²⁺-dependent spontaneous release.     

Calcium-dependent potentials with different sensitivities to calcium agonists and antagonists in guinea-pig hippocampal neurons

Effects of organic Ca channel blockers, Ca channel activators and omega-conotoxin on guinea-pig hippocampal CA1 neurons in vitro preparations were studied with intracellular recording methods. Most of the Ca channel blockers, such as prenylamine, D 600, flunarizine, nifedipine, cinnarizine and nicardipine (0.2-4 microM), raised the threshold for Na-dependent spike generation and decreased the amplitude of the spike afterhyperpolarization. Verapamil (5 microM) and diltiazem (5 microM) did not significantly alter the threshold and amplitude of the Na spike. Action potentials elicited in the presence of either tetrodotoxin (0.5 microM) and tetraethylammonium (20 mM) or tetrodotoxin (0.5 microM) and Ba (1.25 mM) consisted of an initial spike component followed by a long depolarization. Both responses were abolished by addition of Co (2 mM) or Cd (0.25-0.5 mM), or by superfusion with a low Ca (0.25 mM)-high Mg(15 mM) medium, indicating that the potentials resulted from Ca entry. The Ca-dependent slow depolarization was preferentially blocked by most of the organic Ca channel blockers at approximately one-third the concentrations (0.1-2 microM) which were required to shorten the Ca spike. When the cell in a solution containing tetrodotoxin (0.5 microM), Co (2 mM) and 4-aminopyridine (2 mM) was hyperpolarized and then depolarized by passing current pulses across the membrane, a transient depolarizing hump occurred on the decay phase of the electrotonic potential. This transient depolarization was abolished by Co (2 mM), Ni (2 mM) or most of the organic Ca channel blockers (0.2-5 microM). Diltiazem (5 microM) did not significantly change these Ca-dependent potentials. The evoked excitatory postsynaptic potential was very resistant to the Ca channel blockers. Approximately 2-10 times higher concentrations (0.5-3 microM) were necessary to decrease the excitatory postsynaptic potential amplitude than to shorten the Ca spike. On the other hand, the minimal concentrations and order of potencies of the Ca channel blockers for depressing the evoked inhibitory postsynaptic potential and for elevating the threshold for Na spike generation were almost the same. Dihydropyridine Ca channel activators, such as Bay K 8644, CGP 28 392 and YC 170 at low concentrations (0.1-1 microM), decreased the Ca spike, the Ca-dependent slow depolarization and the evoked synaptic potentials, while the substances augmented the Ca-dependent transient depolarization. On the other hand, omega-conotoxin (5 microM) reversibly depressed the Ca spike and slow depolarization to the same degree, without affecting the transient depolarization and the evoked excitatory or inhibitory postsynaptic potentials.(ABSTRACT TRUNCATED AT 400 WORDS)     

吗啡对大鼠海马神经元钾、钙通道的作用

吗啡是常见的阿片类镇痛药,长时间应用可导致吗啡耐受和依赖,其作用机制十分复杂。海马中同时包含μ,κ,δ阿片受体,与吗啡耐受及依赖有一定的关系,并参与机体的痛觉调制过程,因而了解吗啡对海马神经元离子通道的作用对阐明吗啡的镇痛、耐受及依赖机制具有重要的理论及实际意义。本文报导吗啡对培养的海马神经元电压门控性钾、钙     

脂多糖对大鼠尾核神经元L型电压门控钙通道电流的影响和外源性2-AG的调制作用

目的:探讨内源性大麻素2-花生四烯酰甘油(2-AG)对脂多糖(LPS)损伤的大鼠尾核神经元L型电压门控钙通道(L-VGCC)电流的调制作用及其分子机制。方法:原代培养新生大鼠尾核神经元,分为对照组、LPS组、2-AG组、2-AG+LPS组、SR141716A(CB1受体反向激动剂)+2-AG+LPS组和AM630(CB2受体反向激动剂)+2-AG+LPS组,应用全细胞膜片钳记录2-AG对LPS损伤的大鼠尾核神经元L-VGCC电流的影响;采用Hoechst染色法观察2-AG对LPS诱导的尾核神经元损伤的影响,并用试剂盒测定尾核神经元caspase-3的活性。结果:(1)LPS能增强L-VGCC电流密度,且未影响L-VGCC激活及失活的电学特征;(2)2-AG能抑制LPS增强L-VGCC电流密度的作用;(3)LPS增强L-VGCC电流密度并非是通过CB1和CB2受体起作用的;(4)2-AG本身对尾核神经元L-VGCC电流密度、激活及失活等电流特性均不产生影响;(5)LPS诱导的尾核神经元caspase-3活性增强可被2-AG抑制,CB1受体反向激动剂SR141716A可取消2-AG的这种效应;(6)LPS可诱导尾核神经元表现出典型的凋亡特征,2-AG可使LPS诱导的核固缩细胞数目显著减少。结论:内源性大麻素2-AG可通过调节尾核神经元L-VGCC电流起抗炎作用和保护神经元的效应。     

Development of multiple calcium channel types in cultured mouse hippocampal neurons

The development of multiple calcium channel activities was studied in mouse hippocampal neurons in culture, using the patch-clamp technique. A depolarizing pulse (40-50 ms duration) from the holding potential of -80 mV to levels more depolarized than -40 mV produced a low threshold T-type current. The T-type current was observed in 52% of four days in vitro neurons. The number of neurons which expressed T-type current decreased with age of culture, so that the current was detected in only 18% of neurons after 16 days in vitro. The T-type current densities varied between 1.9 pA/pF and 3.29 pA/pF in the mean values during the period studied (4-16 days in vitro). A depolarizing pulse from -80 mV to levels more depolarized than -35 mV evoked a high threshold calcium channel current. The high threshold current density increased in the mean values from 3.9 pA/pF in four days in vitro neurons to 28 pA/pF in 16 days in vitro neurons. We have then examined the effect of nifedipine, omega-Agatoxin IVA and omega-conotoxin GVIA on the high threshold current. Nifedipine (1-5 microM) sensitive current density stayed in the range of 1.9-2.1 pA/pF during 4-16 days in vitro, while omega-Agatoxin IVA (200 nM) sensitive current density increased in the mean values from 1.54 pA/pF in four days in vitro neurons to 21.5 pA/pF in 16 days in vitro neurons. The omega-conotoxin GVIA sensitive N-type channel current was maximum at eight days in vitro (5.44 pA/pF) and it reduced progressively to reach almost half (2.46 pA/pF) in 16 days in vitro neurons. These results showed that diverse subtypes of calcium channels change in density during the early period of culture. We suggest that the temporal expression of each type of channel may be linked to the development of neural activities.     

Effects of calcium channel antagonists on the depolarization-evoked release of norepinephrine in the rabbit iris-ciliary body

The effects of several representative calcium channel antagonists on depolarization-evoked release of [3H]-norepinephrine were investigated in isolated, superfused rabbit iris-ciliary bodies. Potassium (50 mM)-evoked neurosecretion was blocked by 5 mM CoCl2 and partially inhibited by 10(-6) M nitrendipine or verapamil. Electrically-evoked neurosecretion was similarly blocked by CoCl2, but was unaffected by nitrendipine or verapamil. It is concluded from these results that sympathetic terminals in the rabbit iris-ciliary body contain dihydropyridine- and verapamil-sensitive calcium channels which contribute, under conditions of prolonged depolarization, to neurotransmitter release.     

Pentobarbital promotes bursts of gamma-aminobutyric acid-activated single channel currents in cultured mouse central neurons

The extracellular patch clamp method was used to study the influence of pentobarbital (PB) on membrane channels induced by gamma-aminobutyric acid (GABA) in cultured mouse spinal neurons. PB (200 microM) increased the probability of finding GABA-sensitive channels in the open state; it also increased the average frequency at which GABA-induced single channel currents occur, without decreasing the number of data sweeps which showed no single channel activity. These results indicate that PB potentiates GABA responses by promoting burst-like openings of the GABA-sensitive channel.     

Intracellular calcium and hormone release from nerve endings of the neurohypophysis in the presence of opioid agonists and antagonists

Summary Rat neural lobes and isolated nerve terminals from the neurohypophysis were stimulated in the presence of different opioid agonists and antagonists. The secretion of arginine vasopressin and oxytocin and rise in cytoplasmic calcium induced by depolarization were analyzed by radioimmunoassay and the fluorescent probe fura-2, respectively. The kappa-agonists dynorphin A_{1 -13} and dynorphin A_{1 -8} did not affect electrically evoked release of vasopressin, although oxytocin release was slightly reduced. U-50 488, a relatively specific kappa-receptor agonist, had no effect on the amount of vasopressin or oxytocin secreted, although it significantly reduced K⁺-evoked changes in [Ca²⁺]_i in isolated nerve endings. Two kappa-receptor antagonists, MR 2266 and diprenorphin, alone had no effect on vasopressin and oxytocin secretion from isolated nerve endings depolarized with potassium. Opioid agonists less selective for the kappa receptors, etorphin and ethylketocyclazocin, were found to inhibit the release of both vasopressin and oxytocin significantly. Naloxone, a nonselective opiate receptor antagonist, alone had no effect on vasopressin release but potentiated the electrically evoked release of oxytocin. Naloxone also could overcome the inhibitory effect of etorphin on oxytocin and vasopressin release observed after electrical stimulation of the neural lobe. A number of inconsistencies therefore exist between the effects of opioid agonists and antagonists on neuropeptide release and on the evoked changes in [Ca²⁺]_i. In view of these inconsistencies and the high concentrations of opioid agonists and antagonists necessary to modify release, we conclude that it is doubtful that opioid molecules have a physiological role in controlling neurohypophysial secretion.     

Neurotrophin-4/5 promotes dendritic outgrowth and calcium currents in cultured mesencephalic dopamine neurons

Ca(2+) currents and their modulation by neurotrophin-4/5 were studied in cultured mesencephalic neurons. Tyrosine hydroxylase-positive neurons consistently had larger somas than tyrosine hydroxylase-negative neurons. Neurons with larger somas were therefore targeted for recording. In both control and neurotrophin-4/5-treated cultured neurons, isolation of Ca(2+) currents in cultured mesencephalic neurons revealed prominent low- and high-voltage-activated currents. These currents were separable based upon their voltage dependence of activation, the response to replacement of Ca(2+) with Ba(2+) and the response to Ca(2+) channel blockers. Replacement of Ca(2+) with Ba(2+) resulted in a slight reduction of low-voltage-activated currents and a significant enhancement of high-voltage-activated currents. Cd(2+) blocked a larger fraction of the high-voltage-activated current than Ni(2+). The synthetic conotoxins SNX-124 and SNX-230 selectively blocked high-voltage-activated currents. Morphological analysis of mesencephalic cultures pretreated with neurotrophin-4/5 revealed an increase in soma size and dendritic length in tyrosine hydroxylase-positive neurons. In agreement with the neurotrophin-4/5 induction of growth, neurotrophin-4/5 also increased cell capacitance in whole-cell recordings. Neurotrophin-4/5 significantly enhanced both low- and high-voltage-activated currents, but normalization for changes in capacitance revealed only a significant increase in high-voltage-activated current density.This study demonstrates the existence of low-voltage-activated and multiple classes of high-voltage-activated calcium currents in cultured mesencephalic neurons. Morphological and physiological data demonstrate that the increases in calcium currents due to neurotrophin-4/5 pretreatment are associated with somatodendritic growth, but an increase in high-voltage-activated Ca(2+) channel expression also occurred.     

红藻氨酸对大鼠海马锥体细胞钙电流的影响

目的：应用膜片钳技术,观察红藻氨酸（KA）对大鼠海马锥体细胞ca2＋电流的影响,以研究癫痫的发病机制。方法：采用酶加机械分离法制备出生10～12d的大鼠海马锥体神经元标本,用全细胞膜片钳技术测定其生理学特性及观察KA对ca2＋电流的影响。结果：分离出的海马锥体细胞形态正常,有较长突起;用膜片钳技术证实,其保存了主要的离子通道活性。KA20μmol／L和100μmol／L的浓度均可使海马锥体细胞ca2＋电流峰值增大（n=8,P〈0．01）。结论：①大鼠海马锥体神经细胞具有明显的突起,细胞膜表面光洁、晕光好,适用于膜片钳实验研究;②KA使ca2＋内流增加,引起“Ca”超载”导致细胞毒性等一系列反应;③KA通过激活α-氨基羟甲基恶唑丙酸（AMPA）受体,诱发快速的兴奋性突触后电位（EPSP）,参与兴奋性突触传递。AMPA受体的激活可能是癫痫的发病机制之一。