Presynaptic calcium-activated potassium channels and calcium channels at a crayfish neuromuscular junction.

1. We used a two-microelectrode current clamp to investigate various characteristics of the Ca(2+)-activated K+ conductance [gK(Ca)] and Ca2+ conductance (gCa), and transmitter release in presynaptic terminals of excitatory neuromuscular junctions in the crayfish walking leg. 2. Voltage-activated Na+ conductances (gNa) and K+ conductances [gK(v)] were blocked with tetrodotoxin and 3,4-diaminopyridine, respectively. Under these conditions, presynaptic depolarization produced by a first (conditioning) pulse admitted Ca2+ into the presynaptic terminals and activated gK(Ca), which modulated the amplitude of the depolarization produced by a second (test) pulse. The relative amount of gK(Ca) measured at the test pulse increased with increased magnitude or duration of the conditioning pulse. 3. A brief hyperpolarization immediately after a conditioning pulse substantially reduced gK(Ca). 4. gK(Ca) activation was blocked by funnel web spider toxin (a Ca2+ channel blocker) or by injection of the presynaptic terminal region with a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). Under current-clamp conditions, gK(Ca) was not blocked by charybdotoxin or iberiotoxin [specific gK(Ca) blockers]. 5. When gK(Ca) was blocked or reduced, the amplitude of the depolarizing afterpotential of action potentials was increased. When gK(v) was blocked or reduced, the duration of action potentials was increased. 6. Intracellular injection of BAPTA into the presynaptic terminal region eliminated evoked neurotransmitter release before test pulse modulation was affected, suggesting that the K(Ca) channel had a greater sensitivity (greater affinity or lower stoichiometry) for Ca2+ than did the transmitter release machinery. BAPTA reduced neurotransmitter release by 66-78%, but did not affect facilitation of neurotransmitter release. 7. When gNa, gK(v), and gK(Ca) were blocked, we detected a membrane depolarization produced by an increase in presynaptic gCa that was eliminated by 2 mM Cd2+ or 0 mM Ca2+.

Residual free calcium is not responsible for facilitation of neurotransmitter release.

An increase in internal free calcium ([Ca2+]i) in the presynaptic terminal is often assumed to directly produce facilitation of neurotransmitter release. Using a Ca(2+)-activated potassium conductance as a bioassay for free [Ca2+]i in the presynaptic terminal of the crayfish (Procambarus clarkii) opener neuromuscular junction, we now demonstrate that free [Ca2+]i has a decay time constant (tau) of 1-4 msec, whereas facilitation of neurotransmitter release has a decay tau of 7-43 msec. In addition, facilitation of neurotransmitter release can be markedly different at times when free [Ca2+]i values and presynaptic membrane voltages are equal. We conclude that free [Ca2+]i in the presynaptic terminal is not directly responsible for facilitation of neurotransmitter release. Our data suggest that facilitation results from bound Ca2+ or some long-lived consequence of bound Ca2+.

Functional degeneration of isolated central stumps of crayfish sensory axons.

In the crayfish, Procambarus clarkii, nerve 5 carries primarily sensory axons from the tail fan to the 6th abdominal ganglion where they synaptically activate interneuron A. Since the sensory neurons have their somata located at the periphery, transection of nerve 5 part way to the ganglion allowed us to examine the fate of their soma-less central stumps. Up to 3 weeks postlesion the response to stimulation of nerve 5 consisted of a brief latency spike in interneuron A, similar to that in control animals and to stimulation of the intact nerve 4. Stimulation of the lesioned nerve 5 beyond 3 weeks failed to fire interneuron A. This loss of function was correlated to loss of axons in nerve 5 deduced by comparing the numbers in the lesioned nerve 5 to its contralateral intact counterpart. The numbers are about equal in the paired nerves but rapidly decline on the lesioned side to 50% within 1 week, 20% within 3 weeks, and less than 10% in subsequent weeks. This loss affects all size classes of axons. However, in the 3 week lesioned nerve large glial infoldings subdivided some of the larger axons and single nuclei were seen in a few of the medium-sized axons. Possibly subdivision of large axons by glial infolding may introduce glial nuclei into axons.

Effects of ethanol and other drugs on excitatory and inhibitory neurotransmission in the crayfish.

1. Crayfish exposed to 434 mM ethanol (EtOH) showed signs of hyperactivity within 0.5-2 h, at which times crayfish hemolymph EtOH concentration had reached 60-90 mM. 2. A 10-min exposure to 60-90 mM EtOH reduced presynaptic inhibition of excitatory postsynaptic currents (EPSCs) at the crayfish opener neuromuscular junction (NMJ) in vitro but did not significantly alter excitatory neurotransmission. The same concentrations of EtOH did not alter other potentials or currents associated with inhibition at this synapse, such as presynaptic inhibitory potentials (PIPs), inhibitory postsynaptic potentials (IPSPs), and inhibitory postsynaptic currents (IPSCs). 3. Intermediate EtOH concentrations (120-180 mM) applied for 10 min in vitro reduced the amplitude of excitatory postsynaptic potentials (EPSPs) by decreasing the membrane resistance of opener muscle fibers and by reducing the amplitude of EPSCs. 4. High EtOH concentrations (434 mM) applied for 10 min in vitro had yet greater depressive effects on measures of postsynaptic properties described above. The time course of EPSCs was also significantly reduced. In addition, presynaptic properties such as action-potential (AP) amplitude and frequency of spontaneous release of neurotransmitter were reduced by 434 mM EtOH. 5. Presynaptic inhibition, gamma-aminobutyric acid (GABA; 250-500 microM), muscimol (50 microM), and baclofen (75 microM) all reduced the depolarizing afterpotential of APs in the excitor axon and reduced EPSPs in opener muscle fibers. GABA (500 microM) and baclofen (75 microM) significantly reduced presynaptic AP amplitudes, whereas presynaptic inhibition, GABA (250 microM), and muscimol (50 microM) had no effect on AP amplitude. Bicuculline (250-500 microM), a GABAA antagonist, did not entirely eliminate presynaptic inhibition, whereas picrotoxin (50 microM), another GABAA antagonist, completely removed presynaptic inhibition. Thus presynaptic inhibitory mechanisms may involve both GABAA and GABAB receptors on the opener excitor axon. 6. Our data suggest that the behavioral hyperactivity seen at hemolymph EtOH concentrations of 60-90 mM is not accompanied by a change in excitatory synaptic transmission observed at the opener NMJ. Rather, crayfish hyperactivity may be due to depressive effects of EtOH on inhibitory synapses in the CNS similar to the disinhibition evoked by EtOH at the opener NMJ.

Effect of temperature on long-term survival of anucleate giant axons in crayfish and goldfish.

The effect of temperature on the electrophysiology and morphology of anucleate axons was examined following severance of crayfish medial giant axons and goldfish Mauthner axons from their respective cell bodies. Although anucleate segments of each giant axon exhibited long-term survival for weeks to months at 5-25 degrees C in crayfish and 10-30 degrees C in goldfish, the two axons differed in their survival characteristics. All measures of long-term survival in crayfish medial giant axons were independent of animal holding temperature, whereas all measures in Mauthner axons were dependent on holding temperature. Medial giant axons survived for at least 90 days in crayfish maintained at 5-25 degrees C in this and previous studies. Mauthner axons survived for over 5 months in goldfish maintained at 10 degrees C but survived for 1 month at 30 degrees C. Postoperative time had different effects on many single measures of long-term survival (axonal diameter, amplitude of action or resting potentials) in medial giant axons compared to Mauthner axons. For example, resting and action potentials in crayfish medial giant axons remained remarkably constant at all holding temperatures for 0-90 postoperative days. In contrast, resting and action potentials in goldfish Mauthner axons declined abruptly in the first 10-20 postoperative days followed by a slower decline at each holding temperature. We suggest that the mechanism of long-term survival is not necessarily the same in all anucleate axons.

Early experience influences the development of bilateral asymmetry in a lobster motoneuron.

The development of functional asymmetry between a pair of homologous motoneurons of the claw closer muscles in lobsters, Homarus americanus, was studied. In juvenile lobsters, 3-5 years old, where the paired claws are highly specialized into a major (crusher) and minor (cutter) type, the fast closer excitor (FCE) motoneuron fired longer bursts of spikes in the crusher claw compared to those in its cutter counterpart. The intraburst impulse frequency was greater for the cutter FCE and its neuromuscular synapses showed greater facilitation at these high impulse frequencies compared to that of the crusher claw. However, such asymmetry in firing patterns and synaptic facilitation was absent in lobsters raised without a substrate and having paired cutter claws. In the earliest juvenile stage, synaptic facilitation was similar between the paired claws and then developed in either an asymmetric or symmetric manner depending on whether the lobsters experienced a substrate or not. In a substrate-free environment asymmetry could be produced by exercising one of the claws during development, implicating bilateral differences in the reflexive activity of the claws as a control mechanism.

Electrophysiological and behavioral effects of ethanol on crayfish.

The effects of EtOH on the crayfish Procambarus clarkii and P. simulans were examined behaviorally in vivo and electrophysiologically in vitro on pre- and postjunctional mechanisms of synaptic plasticity at opener excitor nerve-muscle junctions. Addition of 75 mM EtOH to the bath water of holding tanks produced 47 to 54 mM EtOH levels in the hemolymph (blood) within 24 hr. These hemolymph EtOH levels were maintained for weeks by daily changes of the bath water containing 75 mM EtOH. After 24 hr of exposure to 75 to 150 mM EtOH in vivo, crayfish showed behavioral signs of intoxication as measured by a significant increase in righting reflex times and a significant decrease in tail-flip escape behavior. After 2 weeks of chronic exposure to 75 mM EtOH, crayfish showed behavioral tolerance as measured by a decrease in righting time and an increase in tail-flip escape behavior to control levels. EtOH applied acutely to opener nerve-muscle preparations in vitro at 10 to 100 mM concentrations produced an increased probability of transmitter release as measured by an increased frequency of spontaneous release of transmitter quanta and an increased amplitude of facilitated synaptic potentials evoked by 10 to 40 Hz stimulation of the excitor axon. Acute application of 300 to 600 mM EtOH resulted in a decreased amplitude of facilitated synaptic potentials due primarily to a decrease in postsynaptic input resistance. These data suggest that EtOH has a concentration-dependent biphasic effect on synaptic transmission.