Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Calcium dependency of excitatory chemical synaptic transmission in the frog cerebellum in vitro.

Chemical synaptic transmission was studied with microelectrode techniques in isolated frog cerebella maintained in vitro. Purkinje cell (PC) EPSPs, elicited by selective monosynaptic electrical stimulation of both the parallel fiber (PF) and climbing fiber (CF) inputs, could be inverted by depolarizing (outward) current injections. Evoked synaptic transmission at both junctions was reduced by lowering the extracellular concentration of calcium ions ([Ca2+]) below 2 mM. Raising [Ca2+] above 2 mM to 8 mM did not further increase synaptic transmission. Mg2+, Sr2+, and Ba2+ did not substitute for Ca2+ in the transmission process.     

Intracellular effects of QX-314 and Cs+ in hippocampal pyramidal neurons in vivo.

The effects of intracellular diffusion of the lidocaine derivative anesthetic QX-314 and of Cs+, which block Na+ and K+ conductances, respectively, were investigated in vivo in rat CA1 and CA3 pyramidal neurons to demonstrate slow Ca(2+)-related events. QX-314 loading prevented fast Na+ spikes, but slower presumably Ca2+ spikes remained. A slower and a faster duration type of QX-314-resistant spikes were observed. The former had high thresholds, while the latter was activated at moderate depolarized levels. The slower and the faster QX-314-resistant spikes fired at frequencies up to 8/s and 35/s and were 35-60 and 5-10 ms in duration, respectively. With Cs+ loading, pyramidal neurons depolarized and slow, presumably Ca2+, and fast Na+ spikes widened. Fast spikes usually showed a prominent shoulder and a slower repolarization. No differences were observed between drug effects in CA1 and CA3 neurons. In terms of their possible participation in theta rhythm genesis the slow QX-314-resistant events display the correct frequency and duration and can oscillate regeneratively.     

Intrinsic membrane potential oscillations in hippocampal neurons in vitro

Membrane potential oscillations (MPOs) of 2-10 Hz and up to 6 mV were found in almost all stable hippocampal CA1 and CA3 neurons in the in vitro slice preparation. MPOs were prominent for pyramidal cells but less pronounced in putative interneurons. MPOs were activated at threshold depolarizations that evoked a spike and the frequency of the MPOs increased with the level of depolarization. MPOs were distinct from and seemed to regulate spiking, with a spike often riding near the top of a depolarizing MPO wave. Analysis of the periodicity of the oscillations indicate that the period of MPOs did not depend on the afterhyperpolarization (AHP) following a single spike. MPOs persisted in low (0-0.1 mM) Ca2+ medium, with or without Cd2+ (0.2 mM), when synaptic transmission was blocked. Choline-substituted low-Na+ (0-26 mM) medium, 3 microM tetrodotoxin (TTX) or intracellular injection of QX-314 reduced or abolished the fast Na(+)-spike and reduced inward anomalous rectification. About 40% of CA1 neurons had no MPOs after Na+ currents were blocked, suggesting that these MPOs were Na(+)-dependent. In about 60% of the cells, a large depolarization activated Ca(2+)-dependent MPOs and slow spikes. MPOs were not critically affected by extracellular Ba2+ or Cs2+, or by 0.2 mM 4-aminopyridine, with or without 2 mM tetraethylammonium (TEA). However, in 5-10 mM TEA medium, MPOs were mostly replaced by 0.2-3 Hz spontaneous bursts of wide-duration spikes followed by large AHPs. Low Ca2+, Cd2+ medium greatly reduced the spike width but not the spike-bursts. In conclusion, each cycle of an MPO in normal medium probably consists of a depolarization phase mediated by Na+ currents, possibly mixed with Ca2+ currents activated at a higher depolarization. The repolarization/hyperpolarization phase may be mediated by Na+/Ca2+ current inactivation and partly by TEA-sensitive, possibly the delayed rectifier, K+ currents. The presence of prominent intrinsic, low-threshold MPOs in all hippocampal pyramidal neurons suggests that MPOs may play an important role in information processing in the hippocampus.     

The biphasic effect of phospholipase A2 inhibitors on axon elongation

We have analyzed changes in axon elongation rate in vitro induced by three different phospholipase A2 inhibitors, namely, bromophenacyl bromide (BPB), quinacrine and Sr2+. Spinal ganglia were obtained from 19.5-day-old rat fetuses and explanted onto polyornithine substrata. Axon length was measured at 1, 2 and 3 days in vitro (d.i.v.). We have previously shown that BPB added at 5 hr in vitro (h.i.v.) modifies the structure of growth cones and inhibits or promotes axon elongation according to its concentration. Now, we have observed that 0.5 x 10(-6) M BPB also stimulates axon elongation when added at 1 d.i.v. Culture media with different Ca2+ concentrations were used to test Sr2+ added at 1 d.i.v. In 2.3 mM Ca2+ only an inhibitory effect was observed with 8 mM Sr2+. On the other hand, in 0.3 mM Ca2+, axon growth was stimulated by 0.6-1.2 mM Sr2+ but inhibited by 6 mM Sr2+. Quinacrine, added at 5 h.i.v. was inhibitory at 10(-5) M and showed no effects at 10(-6) M. However, after washing quinacrine, normal elongation rate was recovered by those previously treated with 10(-5) M and axon growth was enhanced in those treated with 10(-6) M. Since three different phospholipase A2 inhibitors, tested in different situations, produce a similar biphasic effect on axon elongation rate, we postulate that this enzymatic activity is involved in the motility of axon growth cones.     

Ionic conductances underlying the activity of interneurons that control heartbeat in the medicinal leech

Electrical properties of interneurons that control heartbeat in the leech (HN cells) were studied using intracellular recording and stimulation in isolated ganglia bathed by salines of various ionic compositions. Substitution of Na+ ions in the bath by Tris stopped the spontaneous firing of HN cells and led to their gradual hyperpolarization by 15-20 mV. In the absence of Na+, HN neurons produced long-lasting regenerative plateau potentials with thresholds near -55 mV and peaks near -30 mV that were accompanied by an increase in membrane conductance. Elevation of Ca2+ concentration enhanced plateaus, as did replacement of Ca2+ by Ba2+. Plateaus were formed when Sr2+ replaced Ca2+, but were blocked by addition of Mg2+ or Co2+ to the bath, Co2+ being effective at lower concentrations than Mg2+. Hyperpolarization of HN neurons with injected currents revealed a time-dependent change in membrane potential, whereby initial maximum hyperpolarization was followed by a "sag" in potential towards more depolarized values. The sag showed dual voltage dependence, being diminished when HN neurons were hyperpolarized or depolarized outside the normal range of oscillation. The sag was found to depend on the presence of Na+ ions and to be blocked by Cs+ but not by Ba2+. This time-dependent change in membrane potential counters hyperpolarizations of HN neuron membrane potential and may contribute to the escape of these neurons from synaptic inhibition.     

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes.

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments (86)Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased (86)Rb efflux rate from 0.015 min(-1) to a maximal of 0. 049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba(2+) (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased (86)Rb efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin.     

Divalent cations and fast axonal transport in chemically desheathed (triton X-treated) frog sciatic nerve

We have studied the ability of divalent cations to restore to normal axonal transport (AXT) which was inhibited by deprivation of Ca2+ and/or Mg2+ ions. The epi- and perineurium of the frog sciatic nerve were damaged by a 30-s wash in Triton X-100 containing frog Ringer's. This treatment did not affect either AXT or nerve levels of Ca2+ and Mg2+, but made the ions more easily extractable with a Ca2+- and Mg2+-free Ringer's solution (CMFR). Inhibition of AXT was achieved by incubating Triton X-100-treated nerves in CMFR + EGTA for 5 h, followed by an additional incubation for 12 h in CMFR or Ringer's devoid of only Ca2+ (CFR). These treatments reduced Ca2+ and Mg2+ contents by 77% and 38% respectively. Addition of Ca2+ (1.1 mM) during the 12-h period stimulated AXT, measured as accumulation of 3H-labelled components in front of a ligature, several fold. Mg2+ could not substitute for Ca2+ but potentiated the stimulating effect of Ca2+. Addition of other divalent cations did not affect AXT (Sr2+ and Ba2+) or potentiated the inhibition caused by Ca2+-deprived medium (Mn2+ and Co2+). ATP and creatine phosphate contents were similar in nerves incubated in Ca2+-deprived medium and in Ca2+-containing Ringer's. Thus, inhibition of AXT in the former situation was not due to a decreased availability of high energy phosphates. Two calcium antagonists, D-600 and nifedipin, which are potent smooth muscle relaxants, effectively blocked AXT. The present results suggest that Ca2+ is specifically required to maintain AXT and that an analogy exists between Ca2+ regulation during smooth muscle contraction and AXT.     

Membrane properties and firing characteristics of rat cardiac neurones in vitro.

Electrophysiological characteristics of neurones in isolated cardiac ganglia from the left atrium and interatrial septum of the rat were studied with intracellular microelectrodes. At rest the neurones were characterized by a membrane potential of -52.6 +/- 0.83 mV, an input resistance of 85.6 +/- 7.6 M omega, a membrane time constant of 4.6 +/- 0.24 ms and an input capacitance of 63.1 +/- 5.25 pF. Removal of Ca2+ ions from the external solution resulted in a membrane depolarisation of 5.5 +/- 0.70 mV and an increase in input resistance of 96 +/- 52% which indicated that a substantial Ca(2+)-sensitive component contributed to resting membrane potential. A prolonged after-hyperpolarization (AHP) was recorded following a train of spikes; this was inhibited in a Ca(2+)-free solution, indicating that a Ca(2+)-sensitive component of potassium conductance contribute to it. On the basis of the duration of the AHP following a single spike, two types of neurones, I and II, were tentatively identified, having short (less than 300 ms) and long (greater than 300 ms) AHPs, respectively. Type I neurones responded to prolonged membrane depolarization with bursts of firing (Ib neurones) or multiple discharges (Im neurones). Type II neurones also responded with single spikes or multiple discharges to prolonged membrane depolarization. In some Im neurones, tonic firing was recorded which was inhibited by a hyperpolarizing current and accelerated by a depolarizing current injected through the recording microelectrode. Thus, neurones of isolated cardiac ganglia of the rat from the region studied here are heterogeneous in their electrical behaviour, suggesting the existence of functionally different groups within the ganglia.     

Depolarization of nonmyelinated fibers of the rat vagus nerve produced by activation of protein kinase C

The effect of activation of protein kinase C by phorbol esters has been studied on the nonmyelinated (C) fibers of the rat vagus nerve. Grease-gap recording at room temperature was used to monitor changes in the resting and action potentials. Effects of phorbol esters on the rate of efflux of 86Rb and 14C-guanidinium were also measured. The active isomer beta-phorbol 12,13-dibutyrate (PDBu), applied for 10 min at concentrations of 10 nM to 3 microM, caused a slowly developing depolarization, which persisted after the drug was washed out. The action potential was concomitantly reduced. These effects did not occur with the inactive isomer alpha-phorbol 12,13-didecanoate. The PDBu-induced depolarization was reduced by about 75% if Na+ was replaced by the impermeant cation N-methyl-(+)-glucamine (NMG); the residual effect was almost abolished if the nerves were presoaked in a solution containing gluconate in place of Cl-. It was concluded that increases in conductance mainly to Na+ and Cl- were responsible for the depolarization. The response was unaffected by tetrodotoxin or calcium-channel blockers. Omission of Ca2+, surprisingly, enhanced the PDBu-induced depolarization 3-5-fold; furthermore, addition of 2 mM Ca2+ following a PDBu-induced depolarization recorded in Ca2+-free solution caused a pronounced repolarization. This effect of Ca2+ occurred also with Sr2+ and Ba2+, but not with other divalent cations or with La3+. Divalent cations known to block Ca channels inhibited the repolarizing action of Ca2+. These results suggested that Ca2+ acts intracellularly, either to block Na channels opened by PDBu or to activate protein phosphatases. The PDBu-induced response in Ca2+-free solution was increased 2-fold by a reduction in pH from 7.4 to 6.5. Under normal conditions the nerve was reversibly depolarized by this pH change; after PDBu this pH sensitivity was enhanced, and depolarization occurred at a less acidic pH. PDBu caused a 3-4-fold increase in the rate of efflux of 86Rb (a marker for K+ ions) and of 14C-guanidinium (a marker for Na+ ions) from preloaded nerves. These effects, in contrast to the depolarization, were transient.(ABSTRACT TRUNCATED AT 400 WORDS)     

Forskolin potentiates the paraoxon-induced hyperexcitability in snail neurons by blocking afterhyperpolarization

One characteristic of organophosphate poisoning is the ability to increase excitability or induce epileptiform activity in nerve cells, but underlying mechanisms are not fully understood. We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppress Ca(2+) spikes and modulate the activity of snail neurons. This effect was unrelated to acetylcholinesterase (AChE) inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast J, Janahmadi M, Asgari AR, Sepehri H, Haeri-Rohani A. Paraoxon suppresses Ca(2+) spike and afterhyperpolarization in snail neurons: relevance to the hyperexcitability induction. Brain Res 2006a;1083(1):110-7]. In the present study, the interaction of paraoxon with cAMP formation on the modulation of Ca(2+) spikes and neuronal excitability was examined. Forskolin, the activators of adenylate cyclase, suppressed afterhyperpolarization (AHP) and increased the activity of snail neurons without any significant effect on the Ca(2+) spike duration. Pretreatment with forskolin, although attenuated the suppressing effect of paraoxon on the duration of Ca(2+) spikes but also potentiated the paraoxon-induced hyperexcitability by enhancing the suppressive effects of paraoxon on AHP. Our findings support the possible involvement of cAMP formation in the paraoxon-induced AHP suppression and neuronal hyperexcitability, although activation of cAMP pathway may attenuates some effects of paraoxon.     

Electrophysiological properties of neuroendocrine cells of the intact rat pars intermedia: multiple calcium currents

Intracellular recordings for current and voltage clamping were obtained from 130 neuroendocrine cells of the pars intermedia (PI) in intact pituitaries maintained in vitro. Spontaneous and evoked action potentials were blocked by TTX or by intracellular injection of a local anesthetic, QX-222. After potassium (K+) currents were blocked by tetraethylammonium (TEA), 4-aminopyridine, and intracellular cesium (Cs+), 2 distinct calcium (Ca2+) spikes were observed which were differentiated by characteristic thresholds, durations, and amplitudes. Both Ca2+ spikes were blocked by cobalt (Co2+) but were unaffected by TTX or QX-222. The low-threshold spike (LTS) had a smaller amplitude and inactivated when membrane potential was depolarized past -40 mV or when evoked at a fast rate (greater than 0.5 Hz). The high-threshold spike (HTS) typically had a larger amplitude and longer duration, was not inactivated at potentials which inactivated the LTS, and could be evoked at rates of up to 10 Hz. Single-electrode voltage-clamp analysis revealed that 3 distinct components of the Ca2+ current were present in most cells. From a negative holding potential (-90 mV), 2 separate peak inward currents were observed; a low-threshold transient current, similar to a T-type Ca2+ current, activated at -40 mV, whereas a large-amplitude inactivating current activated above -20 mV. This large inactivating Ca2+ current was significantly inactivated at a holding potential of -40 mV or by brief prepulses to positive potentials, and was similar to an N-type Ca2+ current. A sustained Ca2+ current (L-type) was observed which was not altered by different holding potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of calcium on the discharge pattern of primary and secondary endings of isolated cat muscle spindles recorded under a ramp-and-hold stretch

The impulse activity of muscle spindles isolated from the cat tenuissimus muscle was investigated under varying concentrations of external calcium (Ca(2+)). The outer capsule of the muscle spindle represents an effective diffusion barrier for Ca(2+) ions since activity changes were strong and rapid only if the capsule was partly removed from the sensory region of the receptor. The impulse activity of both primary and secondary muscle spindle endings was lowered by an increase in the external Ca(2+) concentration from 1.8 mM (normal Ringer's solution) to 2.7 mM and raised by a decrease in the Ca(2+) concentration from 1.8 to 0.9 mM. Primary endings were generally more strongly affected than secondary endings. With primary endings the firing rate changed by 23-52% when the external Ca(2+) concentration was altered by 0.9 mM. With secondary endings the discharge frequency changed by 15-24%. The afferent discharge patterns were obtained under repetitive ramp-and-hold stretches and were analyzed with regard to influences of external Ca(2+) ions on the static and dynamic components of the endings' responses. The stretch sensitivity and the adaptive response of both types of ending increased in the low Ca(2+) solution and decreased in the high Ca(2+) solution, but a specific effect on a single component of the responses to stretch was not observed. These findings indicate an overall change in excitability when the external Ca(2+) concentration was varied. The mechanical properties of the receptor were probably not affected since changes in the Ca(2+) concentration did not elicit a contraction or relaxation of the intrafusal muscle fibers. On the one hand, the observed effects can be explained according to the surface potential theory by an indirect influence of extracellular Ca(2+) ions on ion channels of the sensory nerve terminals, with Ca(2+) ions binding to negative charged sites at the endings' outer membrane. On the other hand, the results are consistent with the supposition that Ca(2+) ions act directly on ion channels of the sensory membrane of muscle spindle endings.     

Effects of denervation on Ca2+ channels in slow skeletal muscle fibers of the frog

Effects of denervation on calcium channels in slow skeletal muscle fibers in the frog (Rana pipiens) were studied using the three-microelectrode voltage-clamp technique in intact fibers. Ca2+, Ba2+, and Sr2+ currents were all significantly reduced in amplitude during the first 2 weeks after denervation. After nerve section the selectivity sequence Ba congruent with Ca > Sr was changed to Ba > Sr > Ca and the values for relative ratio increased from 1.04 to 2.65 for Ba2+ and from 0.58 to 1.20 for Sr2+ (with respect to Ca2+). Barium current saturation was more obvious in denervated fibers than in non-denervated fibers. The values obtained with the Michaelis-Menten type expression, I = Imax/(1+Kd/[Ba]e) were Kd = 2.7 mM and Imax = 20 microA/cm2 in fibers 2 weeks after nerve section compared with the values Kd = 4.4 mM and Imax = 60 microA/cm2 obtained in non-denervated fibers. Additionally, the effects of two calcium channel blockers (cobalt and nifedipine) were greater by a factor of two in denervated fibers than in non-denervated fibers. Three weeks or so after nerve section, all the biophysical properties studied began to show a tendency to recover toward the values obtained in non-denervated muscles (controls). These results suggest that calcium channels are modified or that there is a change in the types of calcium channels present in frog slow skeletal muscle fibers after denervation.     

Effect of neurotransmitters on axoplasmic transport: acetylcholine effect on superior cervical ganglion cells

The effect of acetylcholine (ACh) on particle movements along axons of cultured superior cervical ganglion cells was analyzed with a computer-assisted video-enhanced differential interference contrast microscope system. ACh suppressed the axoplasmic transport reversibly in both anterograde and retrograde directions. A muscarinic agonist, arecoline, mimicked the ACh effect, but nicotine did not. An experiment with the Ca(2+)-indicator dye, fura-2, revealed that ACh suppressed the transport without any change of intracellular Ca2+ concentration. ACh also suppressed the axoplasmic transport in Ca(2+)-free medium. Islet-activating protein (IAP), pertussis toxin, blocked the ACh effect. These results indicate that ACh activates muscarinic receptors and suppresses fast axoplasmic transport through the activation of IAP-sensitive GTP-binding protein, irrespective of Ca2+ ions.     

A hyperpolarization-activated inward current in heart interneurons of the medicinal leech

Heart interneurons (HN cells) in isolated ganglia of the medicinal leech were voltage-clamped with single microelectrodes. Hyperpolarizing voltage steps elicited a slow inward current (Ih), which underlies the characteristic depolarizing response of HN cells to injection of prolonged hyperpolarizing current pulses (Arbas and Calabrese, 1987a). The conductance underlying Ih begins to activate near -mV and is fully activated between -70 and -80 mV. The activation kinetics of Ih are slow and voltage dependent. The activation time constant (tau h) ranges from approximately 2 sec at -60 mV to near 700 msec at -100 mV. Ih persists in low Ca2+ (0.1 mM), 5 mM Mn2+ saline and exhibits a reversal potential of -21 +/- 5 mV. The reversal potential is shifted by altering [Na+]o or [K+]o but is unaffected by changes in [Cl-]o. Ih is blocked by extracellular Cs+ (1-5 mM) but not Ba2+ (5 mM) or TEA (25 mM). Low concentrations of Cs+ (100-200 microM) cause a partial block that exhibits strong voltage dependence. Temperature changes were also shown to affect Ih. Both the rate of activation and the steady-state amplitude of Ih are enhanced by temperature increases. HN cells are interconnected by inhibitory chemical synapses, and their normal electrical activity consists of bursts of action potentials separated by periods of inhibition. During the inhibitory phase of rhythmic bursting activity, HN cells hyperpolarize to a voltage range where Ih is activated. Block of Ih with extracellular Cs+ (4 mM) disrupted the normal bursting activity of HN cells. These results are consistent with the hypothesis that Ih contributes to escape from inhibitory inputs during normal bursting activity.     

Effects of Ruthenium ions on the sensory terminal discharges of the frog muscle spindle

The presence of a mixed Na+-Ca2+ spike along the sensory terminal of the frog muscle spindle was verified. When the terminal was perfused with Ringer's solution containing 0.1-0.5 mM ruthenium red (RuR), the amplitude and duration of the spike were increased, occurring as a prolonged or a long-lasting depolarization of up to 20-30 s duration following individual afferent spikes evoked spontaneously or antidromically by electrical stimulation. In an isotonic TEA solution, the amplitude and duration of the afferent spikes were increased; however, no prolonged depolarization occurred. Adding 0.2 mM RuR to the TEA solution produced the prolonged and long-lasting depolarization. All responses disappeared in the presence of 3 microM TTX or Na+-free Ringer's solution. An impedance decrease along the terminal was observed during the prolonged or long-lasting depolarization. The prolonged depolarization was blocked by the addition of Ca2+-blockers; the afferent spikes remained. In preparations preincubated with 0.1 mM RuR, increasing CaCl2 in Ringer's solution from 0.2 mM, resulted in shortening of the duration of individual spikes with prolonged depolarization and in increase in the maximum rate of rise (MRR) of the spikes. Preincubation with higher concentrations of RuR produced higher sensitivities in the modifications of the duration and MRR to the change in [Ca2+]O. The responses were retained by adding RuR or RuCl3 to Ca2+-free Ringer's solution containing 0.1-5 mM EGTA, although all responses disappeared in Ca2+-free EGTA Ringer's solution. It is concluded that the RuR-induced prolonged response is produced by an influx of Na+.     

Noradrenergic innervation of rabbit pancreatic ganglia

Sympathetic nerve stimulation indirectly regulates pancreatic endocrine and exocrine secretion, in part, through actions on the cholinergic parasympathetic innervation of the secretory tissues. Earlier work identified noradrenergic nerves in pancreatic ganglia and demonstrated the effects of exogenous norepinephrine (NE) on synaptic transmission but no quantitative studies of ganglionic NE content and release exist. Therefore, the distribution and density of catecholamine (CA)-containing nerves in rabbit pancreatic ganglia were studied using paraformaldehyde/glutaraldehyde (FAGLU) staining and HPLC analysis of CA concentrations. Neural release of [3H]NE was measured in ganglia isolated from the head/neck or body regions of the pancreas. CA-containing nerves densely innervated most ganglia (86%) from both regions, while neural and non-neural CA-containing cell bodies were rarely found. Ganglia from the head/neck region contained significantly higher concentrations of NE. Both 40 mM K+ and veratridine evoked Ca2+-dependent [3H]NE release and tetrodotoxin inhibited 80% of veratridine-stimulated release. omega-Conotoxin GVIA alone antagonized veratridine-stimulated release by 40% but the addition of nifedipine or omega-agatoxin IVA caused no further inhibition. There were no apparent regional differences in the Ca2+-dependence or toxin-sensitivity of NE release. In conclusion, ganglia throughout the rabbit pancreas receive a dense, functional noradrenergic innervation and NE release is dependent upon N- but not P/Q- or L-type voltage-dependent Ca2+ channels. These noradrenergic nerves may indirectly regulate pancreatic secretion through actions on ganglionic transmission.     

Alterations of spontaneous quantal transmitter release at the mammalian neuromuscular junction induced by divalent and trivalent ions

The present studies have demonstrated that Ca and Sr have a similar ability to support the release of spontaneous quanta of transmitter at the rat neuromuscular junction. The foreign ions Co, Pb, Zn and La have also been shown to support spontaneous release, although the time course of the increase in miniature end-plate potentials (m.e.p.p.s) caused by these ions is much slower than that caused by Ca and Sr. However, Sr, Co, Pb and Zn were unable to sustain m.e.p.p.s for prolonged periods. High extracellular K (10--15 mM) inhibited the steady state m.e.p.p. frequency in Ca and Sr concentrations above 1 mM, and also delayed the m.e.p.p. frequency increase induced by 1 mM Pb. It is suggested that K ions directly inhibit divalent ion entry into the nerve terminal.     

Halothane inhibits two components of calcium current in clonal (GH3) pituitary cells.

The effect of halothane on isolated calcium (Ca2+) current of clonal (GH3) pituitary cells was investigated using standard whole-cell clamp techniques at room temperature. Halothane (0.1-5.0 mM) reversibly reduced both the low-threshold, transient [low-voltage-activated (LVA)] component and the high-threshold [high-voltage-activated (HVA)] component of Ca2+ current. Halothane had little effect on the voltage dependence of activation or inactivation of either component of Ca2+ current. Inhibition of the peak high-threshold Ca2+ current was half-maximal at about 0.8 mM halothane, with maximal inhibition (100%) occurring with 5 mM halothane. When measured at the end of a 190-msec command step, half-maximal reduction of high-threshold current occurred at less than 0.5 mM halothane. The low-threshold transient current was less sensitive to halothane, with half-maximal inhibition of peak transient current activated at -30 mV occurring at approximately 1.3 mM. The effect of halothane on the HVA current was apparently not mediated by changes in intracellular Ca2+ concentration. The ability of halothane to inhibit Ca2+ current was unaffected by either the inclusion of the rapid Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) in the recording pipette or exposure of the cell to 10 mM caffeine. To assess the selectivity of the effect of halothane, the actions of halothane on two components of voltage-activated potassium (K+) current observed in the absence of extracellular Ca2+ and on voltage-dependent sodium (Na+) current were also examined. Halothane had no effect on the voltage-dependent, inactivating K+ current of GH3 cells at concentrations up to 1.2 mM. In contrast, the non-inactivating K+ current, though less sensitive to halothane than either Ca2+ current, was reduced by about 40% by 1.2 mM halothane at +20 mV. Peak Na+ current was also blocked by halothane, but 50% block required around 2.6 mM halothane with little effect at 1.6 mM. Reduction of Na+ current was associated with a substantial negative shift in the steady-state inactivation curve. Although the results indicate that a number of voltage-dependent ionic currents are sensitive to halothane, both components of Ca2+ current exhibit a greater sensitivity to halothane than any of three other voltage-dependent currents in GH3 cells. These results show that GH3 cell Ca2+ currents are selectively inhibited by clinically appropriate concentrations of halothane and that the reduction of Ca2+ current can account for the inhibition by halothane of TRH- or KCl-induced prolactin secretion in GH3 cells.