Quantal mechanism of transmitter release during progressive depletion of the presynaptic stores at a ganglionic synapse. The action of hemicholinium-3 and thiamine deprivation.

In the present experiments we interfered with the mechanism of acetylcholine (ACh) synthesis in the rat superior cervical ganglion by impairing the supply of either the choline group (hemicholinium no. 3 [HC-3]treatment) or the acetyl group (thiamine deprivation). Under both conditions stimulation causes in the ganglion a progressive decline in ACh output associated with a depletion of transmitter tissue content. ACh release from the terminals of a single preganglionic fiber was estimated from the quantum content value of the evoked excitatory postsynaptic potentials (EPSP's) recorded intracellularly in the ganglion neuron under test. The present observations indicate that Poisson statistics describe transmitter release at either low or high release levels. Furthermore, the progressive decline in the rate of ACh output occurring during repetitive stimulation is shown to correspond to a progressive decrease in the number of transmitter quanta released per impulse and not to any modification in the size of individual quanta. Some 8,000 transmitter quanta proved to represent the presynaptic transmitter store initially present in those terminals on a neuron that are activated by stimulation of a single preganglionic fiber. Speculations are considered about synaptic efficacy and nerve connections in rat autonomic ganglia. It is suggested that six preganglionic fibers represent the mean input to a ganglion neuron.

A five-conductance model of the action potential in the rat sympathetic neurone.

The origin of the action potential in neurones has yet to be answered satisfactorily for most cells. We present here a five-conductance model of the somatic membrane of the mature and intact sympathetic neurone studied in situ in the isolated rat superior cervical ganglion under two-electrode voltage-clamp conditions. The neural membrane hosts five separate types of voltage-dependent ionic conductances, which have been isolated at 37 degrees C by using simple manipulations such as conditioning-test protocols and external ionic pharmacological treatments. The total current could be separated into two distinct inward components: (1) the sodium current, INa, and (2) the calcium current, ICa; and three outward components: (1) the delayed rectifier, IKV, (2) the transient IA, and (3) the calcium-dependent IKCa. Each current has been kinetically characterized in the framework of the Hodgkin-Huxley scheme used for the squid giant axon. Continuous mathematical functions are now available for the activation and inactivation (where present) gating mechanisms of each current which, together with the maximum conductance values measured in the experiments, allow for a satisfactory reconstruction of the individual current tracings over a wide range of membrane voltage. The results obtained are integrated in a full mathematical model which, by describing the electrical behaviour of the neurone under current-clamp conditions, leads to a quantitative understanding of the physiological firing pattern. While, as expected, the fast inward current carried by Na+ contributes to the depolarizing phase of the action potential, the spike falling phase is more complex than previous explanations. IKCa, with a minor contribution from IKV, repolarizes the neurone only under conditions of low cell internal negativity. Their role becomes less pronounced in the voltage range negative to -60 mV, where membrane repolarization allows IA to deinactivate. In the spike arising from these voltage levels the membrane repolarization is mainly sustained by IA, which proves to be the only current sufficiently fast and large enough to recharge the membrane capacitor at the speed observed during activity. Different modes of firing coexist in the same neurone and the switching from one to another is fast and governed by the membrane potential level, which makes the selection between the different voltage-dependent channel systems. The neurone thus seems to be prepared to operate within a wide voltage range; the results presented indicate the basic factors underlying the different discrete behaviours.

Biophysical properties of the silent and activated rat sympathetic neuron following denervation.

A biophysical description of the denervated rat sympathetic neuron is reported, obtained by the two-electrode voltage-clamp technique in mature intact superior cervical ganglia in vitro. At membrane potential values negative to -50 mV, the normal, quiescent neuron displays voltage-dependent K and Cl conductances; following direct or synaptic stimulation (15Hz for 10 s), the neuron moves to a new resting state characterized by increased amplitude and voltage dependence of Cl conductance. Denervation produces two main effects: 1) resting Cl conductance gradually increases while its voltage-dependence decreases; by 30 days a high-conductance resting state prevails, almost independent of membrane potential in the -50/-110 mV range; 2) the increase in amplitude and voltage-dependence of Cl conductance, produced by direct stimulation in control neurons, is less marked in denervated neurons, and is observed over an increasingly small range of membrane potentials. Thirty days after denervation, the prevailing high-conductance resting state appears virtually insensitive to changes in membrane potential and stimulation. Voltage-dependent potassium currents involved in spike electrogenesis (the delayed compound potassium current and the fast transient potassium current) exhibit an early drastic decrease in peak amplitude in the denervated neuron; the effect is largely reversed after 6 days. Remarkable changes in fast transient potassium current kinetics occur following denervation: the steady-state inactivation curve shifts by up to +15 mV toward positive potential and voltage sensitivity of inactivation removal becomes more steep. A comprehensive mathematical model of the denervated neuron is presented that fits the neuron behavior under current-clamp conditions. It confirms that neuronal excitability is tuned by the conductances (mostly chloride conductance) that control the resting membrane potential level, and by fast transient potassium current. Impairment of the latter reduces both inward threshold charge for firing and spike repolarization rate, and fast transient potassium current failure cancels the voltage dependence of both processes.

A model of signal processing at a mammalian sympathetic neurone.

A computational model has been developed for the action potential and, more generally, the electrical behaviour of the rat sympathetic neurone. The neurone is simulated as a complex system in which five voltage-dependent conductances (gNa, gCa, gKV, gA, gKCa), one Ca2+-dependent voltage-independent conductance (gAHP) and the activating synaptic conductance coexist. The individual currents are mathematically described, based on a systematic analysis obtained for the first time in a mature and intact mammalian neurone using two-electrode voltage-clamp experiments. The simulation initiates by setting the starting values of each variable and by evaluating the holding current required to maintain the imposed membrane potential level. It is then possible to simulate current injection to reproduce either the experimental direct stimulation of the neurone or the physiological activation by the synaptic current flow. The subthreshold behaviour and the spiking activity, even during long-lasting current application, can be analysed. At every time step, the program calculates the amplitude of the individual currents and the ensuing changes; it also takes into account the accompanying K+ accumulation process in the perineuronal space and changes in Ca2+ load. It is shown that the computed time course of membrane potential must be filtered, in order to reproduce the limited bandwidth of the recording instruments, if it is to be compared with experimental measurements under current-clamp conditions. The membrane potential trajectory and single current data are written in files readable by graphic software. Finally, a screen image is obtained which displays in separate graphs the membrane potential time course, the synaptic current and the six ionic current flows. The simulated action potentials are comparable to the experimental ones as concerns overshoot amplitude and rising and falling rates. Therefore, this program is potentially helpful in investigating many aspects of neurone behaviour.

Some properties of the transmitter release mechanism at the rat ganglionic synapse during potassium stimulation.

In the present investigation a study has been made using intracellular recordings in the rat superior cervical ganglion of the mode of transmitter release induced by raised external potassium ion concentration (40 mM), after acetylcholine synthesis has been blocked by hemicholinium-3. It is shown that the progressive decline in the rate of acetylcholine output from the ganglion is related to a decrease in the number of quanta being released. Furthermore, under these conditions there is no evidence for a reduction in the size of the transmitter quantum. The statistical foundations of the quantal release process at the rat ganglionic synapse have been investigated by comparing the distribution of the number of miniature EPSPs during successive constant time intervals in the tracings with the corresponding Poisson and binomial predictions. Analyses have shown that the probability for a quantum to be released is so small as to produce a binomial distribution of responses indistinquishable fro- the corresponding Poisson distribution, both at the beginning of the potassium-induced quantum discharge and when transmitter release level is low after exhaustion of acetylcholine tissue content.

Post-tetanic spontaneous spike activity in rat sympathetic neurons exposed to low potassium ion concentration.

Preganglionic tetanic stimulation (30 sec at 50/sec) of rat superior cervical ganglia, performed in the presence of reduced external potassium concentration (0-1 mM), is followed by a long-lasting postganglionic afterdischarge which fails to appear if stimulation is repeated in normal (5.6 mM) postassium solution. Intracellular recordings revealed that tetanus is followed by 15-30 mV membrane hyperpolarization when the neuron is exposed to normal concentrations of potassium. Conversely, after the ganglion is soaked in low potassium, stimulation results in long-lasting depolarization of the nerve cell with the consequent appearance of spontaneous spikes. This effect is reversed on returing to normal external potassium. Spontaneous activity also occurs after antidromic activation of the cell. It is suggested that tetanus causes sodium loading of the neuron, which leads to stimulation of an electrogenic sodium pump. If potassium is available, the membrane will hyperpolarize, whereas depolarization and pacemaker activity ensues if external potassium is removed. The electrogenic sodium pump thus endows. the rat sympathetic neuron with a mechanism which enables it excitability to be controlled.

Effectiveness of some anions in sustaining the efferent inhibition in the frog labyrinth.

Efferent inhibition in the frog labyrinth is sustained by the release of acetylcholine (ACh) which opens a Cl(-)-channel in the hair cell membrane. To investigate more closely the nature of the permeability change underlying the ACh reaction, the external Cl(-) was replaced by anions of increasing hydrated size, and to test the possible role of a Cl(-)-pump in the sensory cells, drugs were applied which are known to block active cl(-) pumping in other systems. Experiments indicate that the ACh-operated inhibitory channel of the hair cell is larger than at other inhibitory synapses (or approximately 0.7 nm), while pharmacological treatments (DNP, NaN3, acetazolamide, ammonium acetate, DIDS) fail to demonstrate any active distribution of Cl(-) across the hair cell membrane.

Storage and release of acetylcholine in the isolated superior cervical ganglion of the rat.

The storage and release of acetylcholine and choline were studied in the isolated superior cervical ganglion of the rat by a radioenzymic method. The acetylcholine and choline contents were 202.2 +/- 5.1 and 624.7 +/- 20.2 pmole/ganglion, respectively. The transmitter tissue store was unaffected during 1 h of superfusion in choline--Krebs solution, while a 20% decrease was exhibited after 2 h and then remained approximately stable. Conversely, choline content declined to 50% within 1 h and further to 37% of the original level by 4 h. About 24% of the choline assayed in the intact preparation is located in the connective sheath. Preganglionic nerve stimulation at 10--20/sec or potassium stimulation (40 mM KCl) invariably decreased the transmitter tissue stores by 25--45%; such a depletion is independent of the presence or absence of external choline. By contrast, the presence of choline proved to be a prerequisite for the efficient release of acetylcholine from eserinized ganglia during continuous 10/sex stimulation. A drastic depression in the acetylcholine release is described which is related to the time of preincubation of the ganglia with eserine prior to stimulation. Indeed, a 30 min exposure to eserine, compared with a 5 min period, resulted in a 4-fold decrease in the steady output rate. Under optimal conditions, the initial volley output at 10/sec was 1.3 X 10(-4) of the releasable transmitter pool and 1.9 X 10(-4) during the steady-state output. These results are discussed in the light of the electrophysiological knowledge of the quantal release process at the ganglionic synapse.

The nicotinic activation of the denervated sympathetic neuron of the rat.

Nicotinic responses to endogenous acetylcholine and to exogenously applied agonists have been studied in the intact or denervated rat sympathetic neuron in vitro, by using the two-microelectrode voltage-clamp technique. Preganglionic denervation resulted in progressive decrease of the synaptic current (excitatory postsynaptic current, EPSC) amplitude, which disappeared within 24 h. These effects were accompanied by changes in ion selectivity of the nicotinic channel (nAChR). The extrapolated EPSC null potential (equilibrium potential for acetylcholine action, E(Syn)) shifted from a mean value of -15.9+/-0.7 mV, in control, to -7.4+/-1.6 mV, in denervated neurons, indicating a decrease of the permeability ratio for the main components of the synaptic current (P(K)/P(Na)) from 1.56 to 1.07. The overall properties of AChRs were investigated by applying dimethylphenylpiperazinium or cytisine and by examining the effects of endogenous ACh, diffusing within the ganglion after preganglionic tetanization in the presence of neostigmine. The null potentials of these macrocurrents (equilibrium potential for dimethylphenylpiperazinium action, E(DMPP); and equilibrium potential for diffusing acetylcholine, E(ACh), respectively) were evaluated by applying voltage ramps and from current-voltage plots. In normal neurons, E(Syn) (-15.9+/-0.7 mV) was significantly different from E(DMPP) (-26.1+/-1.0) and E(ACh) (-31.1+/-3.3); following denervation, nerve-evoked currents displayed marked shifts in their null potentials (E(Syn)=-7.4+/-1.6 mV), whereas the amplitude and null potential of the agonist-evoked macrocurrents were unaffected by denervation and its duration (E(DMPP)=-26.6+/-1.2 mV). It is suggested that two populations of nicotinic receptors, synaptic and extrasynaptic, are present on the neuron surface, and that only the synaptic type displays sensitivity to denervation.

The calcium-dependent potassium conductance in rat sympathetic neurones.

1. Adult and intact sympathetic neurones of isolated rat superior cervical ganglia were subjected to a two-electrode voltage-clamp analysis at 37 degrees C in order to investigate the Ca2(+)-dependent K+ conductance. 2. At each potential a Ca2(+)-dependent K+ current, IKCa, was determined as the difference between the current that could be attributed to the voltage-dependent K+ current, IKV, following Ca2+ channel blockade by Cd2+ and the total current generated. The final IKCa curves were obtained after correcting the experimental tracings for the underlying ICa current component. 3. IKCa became detectable during commands to -30 mV. About 3.6 x 10(5) Ca2+ ions are required to enter the cell before IKCa is initiated. The current was modelled on the basis of a 0.4-0.6 ms delay followed by an exponential activation of a fast component, IKCaf, simultaneously with a much slower exponential activation, IKCas. Experiments indicate a sigmoidal activation curve for the fast conductance, gKCf, with half-maximal activation at -13.0 mV and a slope factor of 4.7 mV (for 5 mM-Ca2+ in the bath). The associated time constant, tau kcf, ranged from 0.8 to 2.0 ms. The slow conductance exhibited a similar steady-state activation curve but an activation time constant in the 48-280 ms range. The maximum mean gKC was 0.32 microS per neurone for either the fast or slow component. 4. Excess K+ ions accumulate in the perineuronal space during K+ current flow giving rise to rapidly occurring, large K+ reversal potential (EK) modifications (up to -45 mV for the largest currents). The kinetics of K+ extracellular load can be described satisfactorily by a simple exponential function (tau = 0.9-2.8 ms). The characteristics of K+ wash-out appear similar to those of accumulation. 5. The immediate effect of such an extracellular K+ build-up is to make the apparent IKCa activation kinetics faster and to reduce (up to 50%) the true value of the K+ conductance. We simulated the predictions of a K+ diffusion model and generated new functions describing the IKCa steady-state activation, activation rate and maximum conductance values which satisfactorily reconstruct the IKCa current tracings together with the K+ accumulation process near the membrane. 6. A small component of the Ca2(+)-dependent K+ current, IAHP, was observed which survived at membrane potential levels negative to -40 mV. Increasing Ca2+ influx by applying longer pulses enhanced IAHP, which on the other hand was also activated by depolarizations of short duration.(ABSTRACT TRUNCATED AT 400 WORDS)

The interactions between potassium and sodium currents in generating action potentials in the rat sympathetic neurone.

1. Membrane conductance parameters for the rat sympathetic neurone in vitro at 37 degrees C have been determined by two-electrode voltage-clamp analysis. The activation kinetics of two ionic currents, IA and IK(V), has been considered. Data for both currents are expressed in terms of Hodgkin-Huxley equations. 2. The isolated IA developed following third-order kinetics. The activation time constant, tau a, was estimated from the current time-to-peak and, for V less than or equal to -40 mV, from the IA tail current analysis upon membrane repolarization to various potentials. The maximum tau a occurred at -55 mV and varied from 0.26 to 0.82 ms in the range of potentials between -100 and +10 mV. The steady-state value of the variable a, corrected for inactivation, was evaluated in the voltage range from -60 to 0 mV; 14.4 mV are required to change a infinity e-fold. Steady-state gA was voltage dependent, increasing with depolarization to a maximum of 1.40 microS at +10 mV. 3. IK(V) was similarly analysed in isolation. The current proved to develop as a first-order process. tau n was determined by fitting a single exponential to the IK(V) rising phase and to the tail currents at the end of short depolarizing pulses. The bell-shaped voltage dependence of tau n exhibited a maximum (25.5 ms) at -30 mV, becoming minimal (1.8 ms) at -80 and +20 mV. The n infinity curve was obtained (n infinity = 0.5 at -6.54 mV; k = 8.91 mV). The mean maximum conductance, gK(V), was 0.33 microS per neurone at +10 mV. 4. Single spikes have been elicited by brief current pulses at membrane potentials from -40 to -100 mV under two-electrode current-clamp conditions in normal saline and in the presence of blockers of the ICa-IK(Ca) (Cd2+) and/or IK(V) (TEA, tetraethylammonium) systems. Spike repolarization was affected by the suppression of either current in the depolarized neurone, but was insensitive to both treatments when the spike arose from holding levels negative to -75 to -80 mV, indicating that at these membrane potentials the IA current mainly, if not exclusively, contributes to the action potential falling phase. 5. The basic features of the sympathetic neurone action potential were reconstructed by simulations based on present and previous voltage-clamp characterization of the IA, IK(V) and INa conductances.(ABSTRACT TRUNCATED AT 400 WORDS)

A quantitative description of the sodium current in the rat sympathetic neurone.

The somata of rat sympathetic neurones were voltage-clamped in vitro at 27 degrees C using separate intracellular voltage and current micro-electrodes. Na currents were isolated from other current contributions by using: Cd to block the Ca current (ICa) and the related Ca-dependent K current (IK(Ca)), and external tetraethylammonium to suppress the delayed rectifier current (IK(V) ). The holding potential was maintained at -50 mV to inactivate the fast transient K current (IA) when the IA contamination was unacceptable. Step depolarizations beyond -30 mV activated a fast, transient inward current carried by Na ions; it was suppressed by tetrodotoxin and was absent in Na-free solution. Once activated, INa declined exponentially to zero with a voltage-dependent time constant. The underlying conductance, gNa, showed a sigmoidal activation between -30 and +10 mV, with half-activation at -21.1 mV and a maximal value (mean gNa) of 4.44 microS per neurone. The steady-state inactivation level, h infinity, varied with membrane potential, ranging from complete inactivation at -30 mV to minimal inactivation at about -90 mV with a midpoint at -56.2 mV. Double-pulse experiments showed that development and removal of inactivation followed a single-exponential time course; tau h was markedly voltage-dependent and ranged from 46 ms at -50 mV to 2.5 ms at -100 mV. Besides the fast inactivation, the Na conductance showed a slow component of inactivation. The steady-state value, s infinity, was maximal at -80 mV and minimal at -40 mV. The removal of slow inactivation is a two-time-constant process, the first with a time constant in the order of hundreds of milliseconds and the second with a time constant of seconds. Slow inactivation onset appeared to be a faster process than its removal. When slow inactivation was fully removed the peak INa increased by a factor of 1.8. INa was well described by assuming it to be proportional to m3h. The temperature dependence of peak INa, tau m and tau h was studied in the temperature range 17-27 degrees C and found similar to that reported for other preparations. The Q10 of these parameters allowed the reconstruction of the INa kinetic properties at 37 degrees C.

Calcium currents in the normal adult rat sympathetic neurone.

1. The calcium currents evoked by membrane depolarization in the mature and intact rat sympathetic neurone have been studied at 37 degrees C using two-electrode voltage-clamp analysis. 2. Under conditions that eliminate Na+ and K+ currents and 5 mM-external Ca2+, inward currents were observed that activated at about -30 mV and reached maximum amplitude between 0 and +10 mV with time-to-peak values (2.7-1.9 ms) decreasing with increasing membrane depolarization. Thereafter, calcium current (ICa) decayed to a virtually zero level with maintained depolarization. Two exponentials were required to describe the total inactivation process. The faster rate (tau = 29.3-17.6 ms) is ten times the slower rate and proved to be only slightly voltage-dependent. Double-pulse experiments gave a similar time course of turn-off. 3. No steady-state inactivation was removed at holding potentials between -40 and -70 mV and indirect data suggest that all the ICa was available at -50 mV. Within the -30 to -50 mV holding potential range no significant modifications either in the final amount of ICa inactivation or in the inactivation time constant values were detected. 4. After an initial 100 ms, recovery from inactivation followed a single-exponential process with a mean time constant value of 1.54 s at -50 mV. 5. The kinetics of ICa observed in this neurone were consistent with the existence of a single class of Ca2+ channels. For times up to 20 ms, ICa is described reasonably well by a Hodgkin-Huxley c2hc scheme. The activation time constant was 0.57 ms close to threshold and 0.29 ms at +30 mV. Deactivation occurred with a similar fast time course. The steady-state value of the variable c was evaluated in the -40 to +20 mV voltage range: 9.9 mV are required to change c infinity e-fold. 6. Following previous analyses, we have formulated a mathematical model which incorporates the present ICa kinetic equations with Hodgkin-Huxley-type gating mechanisms for INa, IA and IK(V) conductances. The Ca2+ load of the neurone proved to be basically an 'off' effect and to be governed by the duration of the action potential falling phase. The model is consistent with the experimental observations indicating that Ca2+ channels probably do not have an important direct electrical function in the sympathetic neurone spike at normal membrane potential levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic analysis of incomplete current tracings according to the Hodgkin-Huxley model.

A set of equations is presented suitable for the activation analysis of fast ionic conductances according to the Hodgkin-Huxley model. Both the order and the time constant of the activation process can be evaluated. The method has been especially developed for the analysis of tracings whose rising phase is incomplete or contaminated, and is based on the observation that the peak co-ordinates of a transient current (time-to-peak and peak amplitude) retain all information regarding the current development. Compared to the least-squares method, the procedure illustrated is more simple, at least as precise, and has proved to be successful with experimental tracings in which the least-squares analysis failed.

Identification of delayed potassium and calcium currents in the rat sympathetic neurone under voltage clamp.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were studied at 37 degrees C under two-electrode voltage-clamp conditions. Membrane depolarization beyond -40 mV from holding levels between -50 and -100 mV produced a delayed outward current which exhibited no inactivation within this voltage range. The current is carried primarily by K+ ions and its instantaneous I-V relation is linear. The total outward current could be separated into two distinct components on the basis of ion-substitution experiments. A voltage-dependent component of the delayed current, termed IK(V), is activated by membrane depolarization beyond -40 mV when Ca2+ fluxes are selectively blocked by Cd2+ or in Ca2+-free solution. IK(V) develops following first-order kinetics and rises to a peak with a voltage-dependent delay (239 ms at -30 mV and 23 ms at +10 mV). GK(V) attains a saturating value of the order of 17 mS/cm2 at about +20 mV and can be described in terms of a simple Boltzmann distribution for a single gating particle with a valency equal to +2.5. A second component of the delayed outward current, termed IK(Ca), depends on Ca2+ entry for its activation and was isolated as difference current before and after block of Ca2+ movements across the membrane. IK(Ca) is larger and faster than IK(V): it is strictly related to Ca2+ influx and also depends on membrane potential depolarization. A distinct Ca2+ current, ICa, was recorded from the neurone exposed to Na+-free or tetrodotoxin solution. ICa was activated by membrane depolarization beyond -30 mV and reached a maximum value near 0 mV. Its activation agrees with fourth-order kinetics and becomes faster with increasing depolarization. The Ca2+ current developed with a voltage-dependent time to peak of 2.9-1.8 ms and thereafter completely inactivated. The relationship between ICa and IK(Ca) is discussed. The Ca2+-k+ repolarizing system is expected to be mainly associated with action potentials arising from a depolarized neurone, whereas the IA current (Belluzzi, Sacchi & Wanke, 1985) dominates the repolarization mechanism at the normal membrane potential. The effect of muscarine was examined. Muscarine (10-50 microM) produced a fall in conductance with a voltage dependence similar to that exhibited by GK(Ca) and was ineffective when removing extracellular Ca2+ or adding Cd2+. A partial suppression of ICa by muscarine is demonstrated. It is suggested that the decrease of the outward current magnitude in the presence of muscarine may be accounted for qualitatively by the reduction in ICa.

A fast transient outward current in the rat sympathetic neurone studied under voltage-clamp conditions.

Post-ganglionic neurones of the isolated rat superior cervical ganglion were voltage clamped at 37 degrees C using separate intracellular voltage and current micro-electrodes. Control experiments in current clamp suggested that the neurone is electrotonically compact, the soma and the proximal dendritic membranes being under good spatial voltage uniformity. Depolarizing voltage steps from membrane potentials near -50 mV evoked: (i) a voltage-dependent inward Na+ current, (ii) an inward Ca2+ current, (iii) a voltage-dependent outward K+ current, (iv) a Ca2+-activated K+ outward current. Depolarizations from holding potentials more negative than -60 mV elicited, besides the currents mentioned above, a fast transient outward current IA which peaked in 1-2.5 ms and then decayed to zero following an exponential time course. The IA current was shown to be primarily, if not exclusively, carried by K+. It was unaffected by removal of external Ca2+ or addition of Cd2+ and was weakly blocked by tetraethylammonium ions and partially by 4-aminopyridine. The IA current showed a linear instantaneous current-voltage relationship. Its activation ranged from -60 to 0 mV with a mid-point at -30 mV. The A conductance could be described in terms of a simple Boltzmann distribution for a single gating particle with a valency of +3. Both the development and removal of inactivation followed a single exponential time course with a voltage-dependent time constant which was large near the resting potential (42 ms at -70 mV) and small (11 ms) near -100 and -40 mV. Steady-state inactivation h infinity ranged from -100 to -50 mV, with a mid-point at -78 mV, suggesting that approximately 50% of the IA channels are available at the physiological resting potential. Action potentials elicited from various holding potentials showed maximal repolarization rates dependent on the holding potential itself. This voltage dependence was found to be in reasonably good agreement with that of h infinity curve. These data are consistent with the view that in the rat sympathetic neurone, under physiological conditions, it is the IA current rather than the delayed outward current that is responsible for the fast action potential repolarization.

Electrophysiological effects of a neurotoxin extracted from the skin of the Australian frog Pseudophryne coriacea.

1. The electrophysiological effects of a pumiliotoxin-B-like alkaloid extracted from the skin of the Australian frog Pseudophryne coriacea (PsC) have been studied in rat superior cervical ganglia at 37 degrees C. 2. PsC (50 mg/ml) elicits a broadening of the evoked compound action potential and, at rest, the appearance of spontaneous spike discharge at 10-20 Hz. Action potentials presumably originate far away from the soma, which is invaded in a typical IS-SD sequence. 3. The toxin effect is not related to any direct action on the preganglionic fibers of the sympathetic trunk, and does not involve synaptic mechanisms. 4. Two-electrode voltage-clamp experiments showed that the main properties of the major voltage-dependent ionic currents are apparently unaffected by the toxin, while the cell input resistance is considerably reduced. 5. The data are consistent with the hypothesis that PsC elicits a cationic permeability increase generating a pacemaker current in a region close to the cell soma.

Synaptic current at the rat ganglionic synapse and its interactions with the neuronal voltage-dependent currents.

The membrane current activated by fast nicotinic excitation of intact and mature rat sympathetic neurons was studied at 37 degrees C, by using the two-microelectrode voltage-clamp technique. The excitatory postsynaptic current (EPSC) was modeled as the difference between two exponentials. A fast time constant (tau2; mean value 0.57 ms), which proves to be virtually voltage-independent, governs the current rise phase and a longer time constant (tau1; range 5.2-6.8 ms in 2 mM Ca2+) describes the current decay and shows a small negative voltage dependence. A mean peak synaptic conductance of 0.58 muS per neuron is measured after activation of the whole presynaptic input in 5 mM Ca2+ external solution (0.40 muS in 2 mM Ca2+). The miniature EPSCs also rise and decay with exponential time constants very similar to those of the compound EPSC recorded at the same voltage. A mean peak conductance of 4.04 nS is estimated for the unitary event. Deconvolution procedures were employed to decompose evoked macrocurrents. It is shown that under appropriate conditions the duration of the driving function describing quantal secretion can be reduced to <1 ms. The shape of the EPSC is accurately mimicked by a complete mathematical model of the sympathetic neuron incorporating the kinetic properties of five different voltage-dependent current types, which were characterized in a previous work. We show that IA channels are opened by depolarizing voltage steps or by synaptic potentials in the subthreshold voltage range, provided that the starting holding voltage is sufficiently negative to remove IA steady-state inactivation (less than -50 mV) and the voltage trajectories are sufficiently large to enter the IA activation range (greater than -65 mV). Under current-clamp conditions, this gives rise to an additional fast component in the early phase of membrane repolarization-in response to voltage pulses-and to a consistent distortion of the excitatory postsynaptic potential (EPSP) time course around its peak-in response to the synaptic signal. When the stimulation initiates an action potential, IA is shown to significantly increase the synaptic threshold conductance (up to a factor of 2 when IA is fully deinactivated), compared with that required when IA is omitted. The voltage dependence of this effect is consistent with the IA steady-state inactivation curve. It is concluded that IA, in addition to speeding up the spike repolarization process, also shunts the excitatory drive and delays or prevents the firing of the neuron action potential.