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 t...

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.

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.

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. 3 Staurosporine also produced a concentration-dependent (10nM to 0.5 pM) 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,UM 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 (0.5jUM) 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 (540Mm)-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 540piM carbachol or the depth of desensitization produced by the end of a 2-3 min exposure to 540pM carbachol.6 Elevation of the external calcium concentration from 1 to 10mM during the 540pM carbachol application completely antagonized the decreased extent of recovery of m.e.p.c. amplitude produced by pretreatment with 0.5JM 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.

The morphological effects of elevated potassium (K+) at the neuromuscular junction of garter snake twitch muscle fibers were examined in vitro. Elevated K+ depolarizes the motor neuron terminals and causes quantal release of acetylcholine. It has been shown in amphibians that prolonged (30-60 min) depolarization with high K+ depletes terminals of synaptic vesicles ; but causes no change in the average diameter of the vesicles. In the snake, however, we have shown previously that miniature endplate currents (MEPCs) are present at high frequency even after 1-2 hours of exposure to high K+ . This indicates that in the snake, synaptic vesicles are not totally depleted by depolarization with high K+. In this study, we examined the effect of depolarization with high K+ on the distribution and diameter of synaptic vesicles.Intercostal muscle preparations were exposed for 15 minutes to Ringer's + solution containing either a control (1.2mM) or high (70mM) concentration of K+ Preparations were fixed immediately in 1% glutaraldehyde for 15 minutes and washed with Millonig's buffer. The whole mount muscle preparations were then fixed for 1 h in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2; rinsed three times in fresh buffer and post-fixed in 2% OsO4 in the same buffer for 30 min, rinsed in fresh buffer and dehydrated in a graded ethanol series to 100% and then transferred into a 2% solution of uranyl acetate for 20 min to en-block stain the whole mounts. The preps were then transferred back to 100% ethanol to complete the dehydration and embedded in a mixture of Embed 812/Araldite 502 (Hard) between two microscope slides that had been precoated with Formen- Trennmittel Liquid Release Agent and polymerized overnight at 70°C.The whole mounts were then removed from the microscope slides and examined on a compound light microscope where the neuromuscular junctions on the twitch muscle fibers could be identified, marked and cut out of the preps. These pieces of the whole mounts were then remounted into a slot on the end of a pre-cast Embed-Araldite block with a drop of fresh resin and polymerized.