Lobster shal: comparison with Drosophila shal and native potassium currents in identified neurons.

The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyte expression system.

Dopamine modulation of two subthreshold currents produces phase shifts in activity of an identified motoneuron.

1. The lateral pyloric (LP) neuron is a component of the 14-neuron pyloric central pattern generator in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. In the pyloric rhythm, this neuron fires rhythmic bursts of action potentials whose phasing depends on the pattern of synaptic inhibition from other network neurons and on the intrinsic postinhibitory rebound properties of the LP cell itself. Bath-applied dopamine excites the LP cell and causes its activity to be phase advanced in the pyloric motor pattern. At least part of this modulatory effect is due to dopaminergic modulation of the intrinsic rate of postinhibitory rebound in the LP cell. 2. The LP neuron was isolated from all detectable synaptic input. We measured the rate of recovery after 1-s hyperpolarizing current injections of varying amplitudes, quantifying the latency to the first spike following the hyperpolarizing prepulse and the interval between the first and second action potentials. Dopamine reduced both the first spike latency and the first interspike interval (ISI) in the isolated LP neuron. During the hyperpolarizating pre-steps, the LP cell showed a slow depolarizing sag voltage that was enhanced by dopamine. 3. We used voltage clamp to analyze dopamine modulation of subthreshold ionic currents whose activity is affected by hyperpolarizing prepulses. Dopamine modulated the transient potassium current IA by reducing its maximal conductance and shifting its voltage dependence for activation and inactivation to more depolarized voltages. This outward current is normally transiently activated after hyperpolarization of the LP cell, and delays the rate of postinhibitory rebound; by reducing IA, dopamine thus accelerates the rate of rebound of the LP neuron. 4. Dopamine also modulated the hyperpolarization-activated inward current Ih by shifting its voltage dependence for activation 20 mV in the depolarizing direction and accelerating its rate of activation. This enhanced inward current helps accelerate the rate of rebound in the LP cell after inhibition. 5. The relative roles of Ih and IA in determining the first spike latency and first ISI were explored using pharmacological blockers of Ih (Cs+) and IA [4-aminopyridine (4-AP)]. Blockade of Ih prolonged the first spike latency and first ISI, but only slightly reduced the net effect of dopamine. In the continued presence of Cs+, blockade of IA with 4-AP greatly shortened the first spike latency and first ISI. Under conditions where both Ih and IA were blocked, dopamine had no additional effect on the LP cell. 6. We used the dynamic clamp technique to further study the relative roles of IA and Ih modulation in dopamine's phase advance of the LP cell. We blocked the endogenous Ih with Cs+ and replaced it with a simulated current generated by a computer model of Ih. The neuron with simulated Ih gave curves relating the hyperpolarizing prepulse amplitude to first spike latency that were the same as in the untreated cell. Changing the computer parameters of the simulated Ih to those induced by dopamine without changing IA caused only a slight reduction in first spike latency, which was approximately 20% of the total reduction caused by dopamine in an untreated cell. Bath application of dopamine in the presence of Cs+ and simulated Ih (with control parameters) allowed us to determine the effect of altering IA but not Ih: this caused a significant reduction in first spike latency, but it was still only approximately 70% of the effect of dopamine in the untreated cell. Finally, in the continued presence of dopamine, changing the parameters of the simulated Ih to those observed with dopamine reduced the first spike latency to that seen with dopamine in the untreated cell. 7. We generated a mathematical model of the lobster LP neuron, based on the model of Buchholtz et al. for the crab LP neuron.

Dopamine modulation of transient potassium current evokes phase shifts in a central pattern generator network.

Bath application of dopamine modifies the rhythmic motor pattern generated by the 14 neuron pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. Among other effects, dopamine excites many of the pyloric constrictor (PY) neurons to fire at high frequency and phase-advances the timing of their activity in the motor pattern. These responses arise in part from direct actions of dopamine to modulate the intrinsic electrophysiological properties of the PY cells, and can be studied in synaptically isolated neurons. The rate of rebound following a hyperpolarizing prestep and the spike frequency during a subsequent depolarization are both accelerated by dopamine. Based on theoretical simulations, Hartline (1979) suggested that the rate of postinhibitory rebound in stomatogastric neurons could vary with the amount of voltage-sensitive transient potassium current (IA). Consistent with this prediction, we found that dopamine evokes a net conductance decrease in synaptically isolated PY neurons. In voltage clamp, dopamine reduces IA, specifically by reducing the amplitude of the slowly inactivating component of the current and shifting its voltage activation curve in the depolarized direction. 4-Aminopyridine, a selective blocker of IA in stomatogastric neurons, mimics and occludes the effects of dopamine on isolated PY neurons. A conductance-based mathematical model of the PY neuron shows appropriate changes in activity upon quantitative modification of the IA parameters affected by dopamine. These results demonstrate that dopamine excites and phase-advances the PY neurons in the rhythmic pyloric motor pattern at least in part by reducing the transient K+ current, IA.

Quantal transmitter release at snake twitch and tonic muscle fibres during prolonged nerve terminal depolarization.

1. Miniature endplate currents (MEPCs) were recorded in vitro from voltage-clamped twitch and tonic muscle fibres in costocutaneous muscles of the garter snake, Thamnophis. Recordings were made from fibres in a control sodium-containing solution and then during exposure to an isotonic potassium solution containing either 1.0 mM calcium and 4.2 mM magnesium or 3.6 mM calcium. The experiments were done at two levels of external calcium in order to demonstrate that the change in MEPC frequency was calcium dependent. During the initial exposure to the isotonic potassium solutions, the MEPC frequency was increased manyfold at both twitch and tonic fibres, but it declined progressively with continued exposure. MEPCs were recorded from both fibre types throughout a 20 h exposure to the isotonic potassium solution with 1 mM calcium, but no MEPCs were recorded at most twitch endplates after approximately 6 h in the isotonic potassium solution containing 3.6 mM calcium. In contrast, MEPCs were still present at tonic fibre endplates after 20 h in the isotonic potassium solution containing 3.6 mM calcium. 2. After 30 min in the isotonic potassium solution with 1 mM calcium, the MEPC amplitude recorded from both fibre types was approximately twice that in the control sodium-containing solution. At tonic endplates, the MEPC amplitude was also twofold greater in the isotonic potassium solution with 3.6 mM calcium than in sodium-containing solution. In contrast, after 30 min in the isotonic potassium solution containing 3.6 mM calcium, the MEPC amplitude at twitch endplates was similar to that in control solution. 3. In both fibre types, MEPC amplitude decreased progressively with continued exposure to the isotonic potassium solutions. The progressive decrease in MEPC amplitude was not due to a gradual decrease in postsynaptic sensitivity to acetylcholine. 4. The effects of high potassium were reversible as MEPCs were recorded at twitch fibre endplates in preparations which were returned to the control sodium-containing solution after a 20 h exposure to the isotonic potassium solution containing 3.6 mM calcium. 5. Ultrastructural examination showed that after a 6 h exposure to the isotonic potassium solutions most nerve terminals innervating twitch fibre endplates were devoid of synaptic vesicles whereas at the same time many synaptic vesicles were present in nerve terminals innervating tonic fibre endplates. Surprisingly, numerous synaptic vesicles were present in nerve terminals innervating either fibre type in muscle preparations exposed to the isotonic potassium solutions for 20 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of lanthanum at snake twitch and tonic muscle fibre endplates.

1. The effects of 1 mM lanthanum on miniature endplate current (MEPC) frequency, amplitude, and decay time course were studied in voltage-clamped twitch and tonic muscle fibres in the garter snake, Thamnophis. 2. Lanthanum produced a marked increase in MEPC frequency in both fibre types. The maximum frequency in lanthanum was greater at twitch endplates than at tonic endplates although the increase in frequency relative to control levels was as great in tonic fibres as in twitch fibres. 3. In twitch fibres continually exposure to lanthanum, the frequency of MEPCs reached a peak value and then declined progressively until, after approximately 6 h, no MEPCs were recorded. In contrast, at tonic endplates exposed to 1 mM lanthanum, MEPC frequency remained elevated above control levels for periods greater than 20 h. 4. Lanthanum decreased the mean amplitude of MEPCs, skewed the amplitude distribution and increased MEPC duration at both twitch and tonic fibre endplates. 5. Ultrastructural analysis showed that after a 15 min exposure to 1 mM lanthanum, approximately half of the boutons innervating a twitch fibre contained fewer synaptic vesicles than boutons at control endplates, whereas nerve terminals innervating tonic fibre endplates were similar in appearance to those in control preparations. At endplates on both fibres, the postsynaptic membrane was more electron dense than that of control preparations. 6. Following a 6 h exposure to lanthanum, all nerve terminals innervating twitch endplates contained only a few synaptic vesicles and numerous intracellular deposits of electron dense material. The nerve terminals innervating tonic endplates still contained many synaptic vesicles, but the number appeared to be less than that of tonic terminals in untreated preparations. 7. The results demonstrate that lanthanum stimulates spontaneous quantal transmitter release from nerve terminals innervating either twitch or tonic fibres. However, the terminals innervating twitch fibres become depleted of synaptic vesicles, whereas this does not occur as readily in nerve terminals innervating tonic fibres.

Staurosporine inhibits the extent of acetylcholine receptor recovery from carbachol-induced desensitization in snake twitch fibres.

1. The effect of the protein kinase inhibitor, staurosporine, on the extent and time course of recovery following carbachol-induced desensitization was studied in snake twitch-muscle fibres maintained in an isotonic potassium propionate solution and voltage-clamped to +30 mV. 2. Pretreatment with staurosporine (0.5 microM) decreased the extent of recovery of spontaneous miniature endplate current (m.e.p.c.) amplitudes following desensitization by a sustained application of 540 microM carbachol. Recovery was inhibited by approximately 50% without altering the time course of m.e.p.c. recovery. 3. Staurosporine also produced a concentration-dependent (10 nM to 0.5 microM) decrease in the amplitude of a second carbachol-induced current, following a wash period, as compared to the amplitude of the current produced by the initial carbachol application. Pretreatment with 0.5 microM K252a, another wide spectrum protein kinase inhibitor, also decreased the extent of recovery of the response to a second carbachol application following desensitization. 4. Staurosporine pretreatment (0.5 microM) had no effect on either the kinetics of receptor-channel gating or the initial endplate sensitivity to agonist. This was determined by comparing the amplitude of the carbachol (540 microM)-induced currents and the amplitude and decay rate of m.e.p.cs in control and staurosporine-treated fibres. 5. Staurosporine had no effect on the time course of desensitization onset produced during the initial application of 540 microM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540 microM carbachol.6. Elevation of the external calcium concentration from 1 to 10mM during the 540 microM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5 microM staurosporine.7. We suggest that phosphorylation of a population of acetylcholine receptors is required for complete recovery from desensitization, and that staurosporine inhibits the protein kinases responsible for this phosphorylation.8. We further propose that a transient increase in intracellular calcium, produced by an increase in calcium influx through agonist-activated endplate channels, stimulates additional protein kinase activity, which in turn, antagonizes the effect of staurosporine-treatment on recovery.