Effect of charybdotoxin and leiurotoxin I on potassium currents in bullfrog sympathetic ganglion and hippocampal neurons.

The effects of charybdotoxin and leiurotoxin I were examined on several classes of K+ currents in bullfrog sympathetic ganglion and hippocampal CA1 pyramidal neurons. Highly purified preparations of charybdotoxin selectively blocked a large voltage- and Ca(2+)-dependent K+ current (IC) responsible for action potential repolarization (IC50 = 6 nM) while leiurotoxin I selectively blocked a small Ca(2+)-dependent K+ conductance (IAHP) responsible for the slow afterhyperpolarization following an action potential (IC50 = 7.5 nM) in bullfrog sympathetic ganglion neurons. Neither of the toxins had significant effects on other K+ currents (M-current [IM], A-current [IA] and the delayed rectifier [IK]) present in these cells. Leiurotoxin I at a concentration of 20 nM had no detectable effect on currents in hippocampal CA1 pyramidal neurons. This lack of effect on IAHP in central neurons suggests that the channels underlying slow AHPs in those neurons are pharmacologically distinct from analogous channels in peripheral neurons.     

Measurement of intracellular Ca in the bullfrog sympathetic ganglion cells using fura-2 fluorescence

The intracellular free ([Ca²⁺]_i) of the bullfrog sympathetic ganglion cell was measured with fura-2 fluorescence under various conditions, and compared with changes in membrane potential recorded with an intracellular electrode. The [Ca²⁺]_i was 109 nM on average under the resting condition and increased by raising the extracellular K⁺, stimulating repetitively the pre- or post-ganglionic nerve, or by applying acetylcholine or muscarine. Since all these procedures depolarized the cell membrane, most of the rise in [Ca²⁺]_i could be the result of opening of voltage-dependent Ca²⁺ channels. However, Ca²⁺ entries through nicotinic acetylcholine receptor channels and the channel activated by the muscarinic acetylcholine receptor were also indicated by considering the threshold for the opening of voltage-dependent Ca²⁺ channels (for both entries) or a limited number of the cells showing the latter response.     

Rapid swelling of neurons during synaptic transmission in the bullfrog sympathetic ganglion

Neurons in the lumbar sympathetic ganglion of the bullfrog were found to respond to presynaptic stimulation with rapid mechanical changes. These changes represent swelling of the presynaptic nerve terminals, followed by swelling of the somas of the postsynaptic neurons. After complete blockade of impulse transmission with D-tubocurarine, swelling of the neurons associated with the excitatory postsynaptic potential is observed.     

Calcium entry through acetylcholine-channels can activate potassium conductance in bullfrog sympathetic neurons

Fast B neurons⁴ in bullfrog sympathetic ganglia were voltage clamped with two microelectrodes. Acetylcholine (ACh) was applied onto the soma membrane by iontophoresis. A rapid nicotinic inward current was followed by a slow muscarinic inward current. After an addition of scopolamine to Ringer solution so as to block the muscarinic current, the nicotinic inward current was found to be followed by an outward current lasting for several hundred ms. It disappeared when the preceding nicotinic inward current was blocked by (+)-tubocurarine. The ACh-induced outward current was due to calcium entry through ACh-channels and subsequent opening of potassium channels. This may indicate that a rapid excitatory transmission leads to non-synaptic autoinhibition. The interaction between the calcium-dependent potassium conductance and the muscarinic action of ACh is proposed.     

Effect of new O-superfamily conotoxin SO3 on sodium and potassium currents of cultured hippocampal neurons

The effects of a new O-superfamily conotoxin SO3 on sodium and potassium currents were examined in cultured rat hippocampal neurons using the whole-cell patch clamp technique. SO3 caused a concentration-dependent, rapidly developing and reversible inhibition of sodium currents (I(Na)). The IC(50) value for the blockage of I(Na) was calculated to be 0.49 and the Hill coefficient was 1.7. Using electrophysiological and pharmacological protocols, transient A-type potassium currents (I(A)) and delayed rectifiers potassium currents (I(K)) were isolated. SO3 caused a concentration-dependent, and reversible inhibition of I(K). The IC(50) value for the blockage of I(K) was calculated to be 1.6 and the Hill coefficient was 0.6, with no significant effect on I(A). These results indicate that SO3 can selectively inhibit neuronal sodium and potassium currents.     

Is Ca-activated potassium efflux involved in the formation of ischemic brain edema?

A possible role of Ca²⁺-activated potassium efflux in brain ischemia was studied using a rat focal cortical infarction model. Three days after ischemic insult, tissue contents of water, sodium, potassium and calcium ions were measured. Charybdotoxin, a specific inhibitor of Ca²⁺-activated potassium efflux, was found to reduce the formation of ischemic brain edema when a dosage of 0.15 mg/kg was given by i.v. 20–30 min prior to the onset of ischemic insult.     

Neuroleptics decrease calcium-activated potassium conductance in hippocampal pyramidal cells

Intracellular recordings were made from pyramidal CA1-neurones of the hippocampal slice preparation. Bath application of a wide variety of neuroleptics was found to depress the slow afterhyperpolarization, which is mediated in these neurons by a calcium-dependent potassium conductance occurring following a burst of spikes. The depression of this conductance took place in the presence of calcium spikes of normal amplitude and duration, and except in the case of trifluoperazine, without alteration in resting membrane potential or input resistance.     

Modulatory actions of ATP on membrane potentials of bullfrog sympathetic ganglion cells

Adenosine triphosphate (ATP) depolarized the membrane of bullfrog sympathetic ganglion cells by decreasing resting K+ conductance. ATP also depressed the maximum amplitude of after-hyperpolarization of action potentials. Voltage-clamp study revealed that ATP markedly suppressed the TEA-insensitive K+ current which appeared to correspond to the M-current, while it affected less significantly on the delayed rectifier K+ current. It was suggested that ATP depolarized resting membrane by suppressing resting K+ conductances, including the M-current, and also depressed the after-hyperpolarization of action potentials by suppressing both the M-current and delayed rectifier K+ current.     

Muscarinic modulation of TREK currents in mouse sympathetic superior cervical ganglion neurons

Muscarinic receptors play a key role in the control of neurotransmission in the autonomic ganglia, which has mainly been ascribed to the regulation of potassium M‐currents and voltage‐dependent calcium currents. Muscarinic agonists provoke depolarization of the membrane potential and a reduction in spike frequency adaptation in postganglionic neurons, effects that may be explained by M‐current inhibition. Here, we report the presence of a riluzole‐activated current (I_RIL) that flows through the TREK‐2 channels, and that is also inhibited by muscarinic agonists in neurons of the mouse superior cervical ganglion (mSCG). The muscarinic agonist oxotremorine‐M (Oxo‐M) inhibited the I_RIL by 50%, an effect that was abolished by pretreatment with atropine or pirenzepine, but was unaffected in the presence of himbacine. Moreover, these antagonists had similar effects on single‐channel TREK‐2 currents. I_RIL inhibition was unaffected by pretreatment with pertussis toxin. The protein kinase C blocker bisindolylmaleimide did not have an effect, and neither did the inositol triphosphate antagonist 2‐aminoethoxydiphenylborane. Nevertheless, the I_RIL was markedly attenuated by the phospholipase C (PLC) inhibitor ET‐18‐OCH3. Finally, the phosphatidylinositol‐3‐kinase/phosphatidylinositol‐4‐kinase inhibitor wortmannin strongly attenuated the I_RIL, whereas blocking phosphatidylinositol 4,5‐bisphosphate (PIP₂) depletion consistently prevented I_RIL inhibition by Oxo‐M. These results demonstrate that TREK‐2 currents in mSCG neurons are inhibited by muscarinic agonists that activate M1 muscarinic receptors, reducing PIP₂ levels via a PLC‐dependent pathway. The similarities between the signaling pathways regulating the I_RIL and the M‐current in the same neurons reflect an important role of this new pathway in the control of autonomic ganglia excitability.     

Single acetylcholine channel currents in sympathetic neurons

Single acetylcholine (ACh) channel currents were studied by the gigaohm patch-clamp technique in cultured sympathetic neurons of the bullfrog, Rana catesbeiana. Recordings were made at 22C oncell-attached and excised membrane patches. When ACh (0.5-1 μM) was present in the pipette, a single class of inward currents was observed with a chrod conductance of 30 pS and reversal potential of -2 mV. The mean channel open time was 11.6 ms at -65 mV and showed little or no voltage-dependence over the range -85 to -45 mV. These channels appear to mediate the fast nicotinic excitatory postsynaptic current.     

Forskolin reduces a transient potassium current in lobster neurons by a cAMP-independent mechanism

Forskolin decreases the transient potassium current, IA, in voltage-clamped somata of identified neurons in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The diterpene reduces the peak outward current and accelerates the rate of inactivation of IA. Forskolin has no detectable effects on two other identifiable potassium currents in these cells, IK(Ca) and IK(V). Three identified stomatogastric neuron types (PD, PY, AB) have marked amounts of IA which are affected by forskolin; three other cell types (LP, IC, VD) have little or no IA, and forskolin has no effect on their outward currents. Bath application of 8-bromo-cAMP, N,N-dibutyryl-cAMP and IBMX do not affect IA. In addition, the forskolin analog, 1,9-dideoxyforskolin, which does not activate adenylate cyclase, mimics forskolin's effects on IA. Thus, the effects of forskolin on IA are not mediated by cAMP elevation.     

Effects of berberine on potassium currents in acutely isolated CA1 pyramidal neurons of rat hippocampus

The effects of berberine, an isoquinoline alkaloid with antiarrhythmic action, on voltage-dependent potassium currents were studied in acutely isolated CA1 pyramidal neurons of rat hippocampus by using the whole-cell patch-clamp techniques. Berberine blocked transient outward potassium current (IA) and delayed rectifier potassium current (IK) in a concentration-dependent manner with EC50 of 22.94+/-4.96 microM and 10.86+/-1.06 microM, Emax of 67.47+/-4.00% and 67.14+/-1.79%, n of 0.77+/-0.08 and 0.96+/-0.07, respectively. Berberine 30 microM shifted the steady-state activation curve and inactivation curve of IA to more negative potentials, but mainly affected the inactivation kinetics. Berberine 30 microM positively shifted the steady-state activation curve of IK. These results suggested that blockades on K+ currents by berberine are preferential for IK, and contribute to its protective action against ischemic brain damage.     

Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons

The trunk neural crest of vertebrate embryos gives rise to dorsal root ganglion (DRG) sensory neurons and autonomic sympathetic neurons, among other derivatives. We have examined the development of DRG and sympathetic neurons during development in the zebrafish. We found that sensory neurons differentiate rapidly and that their overt neuronal differentiation significantly precedes that of sympathetic neurons in the trunk. Sympathetic neurons in different regions differentiate at different times. The most rostral population, which we call the cervical ganglion, differentiates several days before trunk sympathetic neurons. After undergoing overt neuronal differentiation, sympathetic neurons subsequently express the adrenergic differentiation markers dopamine beta-hydroxylase and tyrosine hydroxylase. A second population of adrenergic nonneuronal cells initially localized with cervical sympathetic neurons appears to represent adrenal chromaffin cells. In more mature fish, these cells were present in clusters within the kidneys. Individual DRG and sympathetic ganglia initially contain few neurons. However, the number of neurons in DRG and sympathetic ganglia increases continuously at least up to 4 weeks of age. Analysis of phosphohistone H3 expression and bromodeoxyuridine incorporation studies suggests that the increases in DRG and sympathetic ganglion neuronal cell number are due wholly or in part to the division of neuronal cells within the ganglia.     

Trans-resveratrol, a red wine ingredient, inhibits voltage-activated potassium currents in rat hippocampal neurons

The red wine ingredient trans-resveratrol was found to exert potent neuroprotective effects in different in vivo and in vitro models. Thus far, the mechanisms underlying the neuroprotection were attributed mainly to its antioxidant properties. The aim of this study was to investigate the actions of trans-resveratrol on voltage-gated K(+) channels, which have been implicated in neuronal apoptosis. Superfusion of trans-resveratrol reversibly inhibited both the delayed rectifier (I(K)) and fast transient K(+) current (I(A)) in rat dissociated hippocampal neurons with IC(50) values of 13.6 +/- 1.0 microM and 45.7 +/- 7.5 microM, respectively. The inhibition on I(K) had a slow onset, was neither voltage dependent nor use dependent. Trans-resveratrol (30 microM) shifted the steady-state inactivation curve of I(K) to the hyperpolarizing direction by 20 mV and slowed down its recovery from inactivation. The inhibition on I(A) was similar to that on I(K), but voltage dependent. Superfusion of trans-resveratrol (30 microM) shifted the steady-state activation curve of I(A) to the depolarizing direction by 17 mV. Intracellular application of trans-resveratrol (30 microM) was ineffective. Based on the comparable effective concentrations, the inhibition of voltage-activated K(+) currents by trans-resveratrol may contribute to its neuroprotective effects.     

The actions of 3,4-diaminopyridine in bullfrog sympathetic ganglia

The effects of 3,4-diaminopyridine (3,4-DAP) on isolated sympathetic ganglia were studied by means of intracellular and extracellular recording techniques. 3,4-DAP in micromolar concentrations caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges. Such stimulus-bound repetition (SBR) appeared to represent a presynaptic action of 3,4-DAP, as no repetitive firing could be evoked by antidromic or direct stimulation of ganglion cells. 3,4-DAP also increased the latency to the onset of the synaptic potential at concentrations paralleling those responsible for SBR. The actions of 3,4-DAP on synaptic transmission extended over a 10,000-fold concentration range, beginning with synaptic facilitation at micromolar concentrations (1-500 microM), and proceeding to depressant effects at millimolar concentrations (0.5-10 mM). The postganglionic repetitive discharges (SBR) induced by 3,4-DAP could be selectively suppressed by D-tubocurarine (D-Tc) or tetraethylammonium (TEA), at concentrations of these antagonists below those required to block transmission. 3,4-DAP had significant anti-curare effects, producing a four-fold shift of the D-Tc concentration-effect curve for transmission block. In contrast, 3,4-DAP neither antagonized nor enhanced the transmission block produced by TEA. These results are incompatible with traditional concepts of ganglionic blockade by D-Tc and TEA, and suggest the possibility of presynaptic sites of action in ganglion block.     

Calcium localization in the sympathetic ganglion of the bullfrog and effects of caffeine

The localization of Ca2+ in the bullfrog sympathetic ganglion was studied using electron microscopy with Oschman and Wall's technique. When the ganglion was incubated and processed in an extremely high Ca2+ solution (20 mM) for electron microscopy, electron-dense deposits (EDD) were found at or in the plasma membranes, subsurface cisterns and mitochondria of the postganglionic neurons. These EDD were proved to contain calcium by X-ray microprobe analysis. On the other hand, they were not significant in the preganglionic terminals except those in the synaptic vesicles. Addition of caffeine (10 mM) to the incubation media and fixatives caused a drastic decrease in number of EDD of the subsynaptic membranes and the subsurface cisterns. Caffeine also reduced, but less markedly, the size and number of EDD in mitochondria. Caffeine (10 mM) prolonged the afterhyperpolarization of an action potential, reduced the amplitude of the ACh (nicotinic) potential and induced slow rhythmic hyperpolarizations in a 20 mM Ca2+ solution. These effects of caffeine which were presumably the result of an increase in the intracellular free Ca2+ were discussed in relation to the morphological data.     

Effects of hypertonia on voltage-gated ion currents in freshly isolated hippocampal neurons, and on synaptic currents in neurons in hippocampal slices

We studied the effects of hypertonia on voltage-gated currents of freshly isolated hippocampal CA1 neurons, using open pipette whole-cell as well as gramicidin-perforated patch-clamp recording. Extracellular osmolarity (π_o) was raised by adding mannitol (50 or 100 mmol/l) to the bathing solution. Hypertonia depressed voltage-gated sodium, potassium and calcium currents in all trials. The threshold activation voltage of the currents did not change during hypertonic depression, but maximal activation of Ca²⁺ current shifted to a more negative potential, suggesting stronger depression of high- compared to low-voltage activated currents. During 30 min high π_o treatment (recorded with open pipette), the depression reached maximum in 10–15 min of exposure. The depression of the computed transient component of the K⁺ current recorded by open pipette was statistically not significant. Following hypertonic treatment recovery of the I_Na, the sustained I_K and sustained I_Ca were incomplete compared to control cells maintained in normal solution for an equal length of time. In hippocampal tissue slices hypertonia (+25, +50 and +100 mmol/l fructose) reversibly depressed excitatory postsynaptic currents (EPSCs). We conclude that the shutdown of membrane ion currents by elevated π_o is not selective, but the degree of the suppression varies among current types. Raising π_o in human patients, possibly combined with mild artificial acidosis, may be useful in the prevention and treatment of acute crises associated with excessive excitation or depolarization of neurons.     

The effects of pumiliotoxin-B on sodium currents in guinea pig hippocampal neurons

The actions of pumiliotoxin-B, extracted from the skin of the frog Dendrobates pumilio, were examined on hippocampal slices and on acutely dissociated hippocampal neurons from the adult guinea pig. Application of 0.5-1 microM pumiliotoxin-B to hippocampal slices caused spontaneous, repetitive field discharges in the CA3 subfield. In whole-cell patch-clamp recordings of isolated CA1 and CA3 neurons, 1-2 microM pumiliotoxin-B shifted the midpoint of Na+ current activation by -11.4 +/- 1.1 mV. This shift was not dependent upon prior activation of the sodium channel. Pumiliotoxin-B did not block macroscopic Na+ inactivation but did reduce the apparent voltage-dependence of inactivation such that currents decayed faster at membrane potentials more negative than -30 mV. Single-channel recordings of sodium currents from excised membrane patches indicated that pumiliotoxin-B had little or no effect on channel closings due to entry into inactivated state(s) but did increase the rate of channel closings due to reversal of channel opening. The increase in the channel closing rate was consistent with a +8.7 mV shift in voltage sensitivity. Negative shifts in activation and positive shifts in closing rates implied a negative shift in the voltage-dependence of channel opening, suggesting that pumiliotoxin-B increases the rate of Na+ channel opening and closing in cells at rest, which could result in spontaneous activity in the neurons.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Soman reversibly decreases the duration of Ca and Ba action potentials in bullfrog sympathetic neurons

Soman, (pinacoloxymethyl-phosphoryl fluoride) (0.1–10 μM) an irreversible cholinesterase inhibitor, reversibly reduced the duration of calcium (Ca²⁺)- and barium (Ba²⁺) spikes without significantly affecting spike amplitude in sympathetic postganglionic neurons of the adult bullfrog (Rana catesbeiana). The soman-induced shortening of the spike duration was not prevented by pretreatment with either (+)-tubocurarine (100 μM) or hexamethonium (100 μM) and atropine (10 μM) and was also recorded from acutely-dissociated sympathetic neurons. These results suggest that soman has a direct action to decrease calcium entry through voltage-dependent channels activated during a spike. This effect may contribute to both the decrease in the duration of the spike after-hyperpolarization (AHP) and the enhanced neuronal excitability produced by soman in these neurons.