Crayfish Opener Muscle Research Articles

Historical view and physiology demonstration at the NMJ of the crayfish opener muscle.

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

Historical View and Physiology Demonstration at the NMJ of the Crayfish Opener Muscle

Here we present some of the key important discoveries made with the opener neuromuscular (NMJ) preparation of crustaceans and illustrate that there is still much to learn from this model preparation. In understanding the history one can appreciate why even today this NMJ still offers a rich playground to address questions regarding pre- and post-synaptic function and plasticity. The viability and ease of access to the terminal for intracellular as well as extracellular electrophysiology and imaging are significant advantages. The mechanisms behind the modulation of vesicular kinetics and fusion within the high- and low-output terminals are begging for investigation. The preparation also offers a testable model system for computational assessments and manipulations to examine key variables in theoretical models of synaptic function, for example calcium dynamics during short-term facilitation. The synaptic complexity of active zone and statistical nature of quantal release is also an open area for future investigation both experimentally and computationally.

SUPPRESSION OF AP4-ENHANCED EXCITATORY JUNCTIONAL POTENTIALS BY THE D3 STEREOISOMER OF A CARBOXY DERIVATIVE OF BUCKMINSTERFULLERENE.

Purpose In spinal cord injury (SCI), glutamate excitotoxicity can cause secondary injury. NMDA glutamate receptor blockade is a potential protective strategy following SCI. A crayfish neuromuscular junction (NMJ) model is useful for studying NMDA receptor activity. The D3 stereoisomer of the malonic acid (carboxy) derivative of buckminsterfullerene suppresses excitatory junctional potentials (EJPs) of NMJs of the opener muscle of crayfish (Procambarus clarkii and simulans) walking limbs. DL-2-amino-4-phosphonobutyric acid (AP4, a specific NMDA receptor agonist) potentiates EJPs in this preparation. This study examines whether D3 can suppress AP4-enhanced EJPs. Materials/Methods A first or second walking limb was removed, and the meropodite and propodite were dissected to expose the excitatory axon and the opener muscle, respectively. The preparation was bathed in Van Harreveld9s Solution (VH, pH 7.2 ± 0.1) for collection of pretreatment data. Following a pretreatment control, a solution of 3.75 μm AP4 in VH was applied. EJPs were recorded immediately following application and at 1 and 2 minutes, using standard intracellular electrophysiologic techniques. Short-term facilitation (10-second stimulus at 35 Hz) averaging eight tracings per second for 10 seconds was used to record EJPs. AP4/VH was then replaced with 3.75 μm AP4 and 25 μm D3 in VH. EJPs were recorded immediately and at 1, 2, 3, and 4 minutes. Post-treatment control data collection followed three complete exchanges of the bath with VH. Results Data are reported as percent change from control amplitude; 3.75 μm AP4 enhanced EJP amplitude 30%, with a subsequent depression of 35% in 3.75 μm AP4 and 25 μm D3. The final washout returned to within 0.27% of precontrol. Qualitatively similar results were found in three additional preparations. Discussion 25 μm D3 can suppress 3.75 μm of AP4-induced EJP potentiation. This further suggests that D3 may be a useful agent in suppressing glutamate excitotoxicity induced by NMDA receptor stimulation. Supported by the Arnold C., Barbara M. and Georgianna Fossa Spinal Cord Injury Research Fund, the IU School of Medicine Brain and Spinal Cord Injury Research Program, and the Hillblom Foundation (L.D.).

Actions of MDMA at glutamatergic neuromuscular junctions.

3,4-Methylenedioxymethamphetamine (MDMA, "Ecstasy") compels mammalian serotonergic neurons to release serotonin (5-HT). In this study, MDMA altered synaptic transmission presynaptically by enhancing quantal release in two model glutamatergic synapses-the neuromuscular junction (NMJ) of the crayfish opener muscle, which is enhanced by exogenous 5-HT application, and the NMJ of a larval body wall muscle in Drosophila melanogaster, which is insensitive to exogenous 5-HT application. At the crayfish NMJ, MDMA mimicked the actions of 5-HT but only at a substantially higher concentration. At the Drosophila NMJ, MDMA altered synaptic transmission but not through a 5-HT receptor. Using simple invertebrate preparations, we have demonstrated an additional non-serotonergic mechanism of MDMA activity that has not yet been addressed in vertebrate systems and that may play an important role in understanding the mechanism of action for a commonly abused drug.

Phosphorylation and Local Presynaptic Protein Synthesis in Calcium- and Calcineurin-Dependent Induction of Crayfish Long-Term Facilitation

Long-term facilitation at the crayfish opener muscle is elicited by prolonged high frequency stimulation, and arises from an increase in functional active zones, resulting in increased transmitter release. LTF induction depends critically upon presynaptic calcium accumulation and calcineurin (PP2B) activity. The protein synthesis dependence of this synaptic strengthening was investigated. LTF occurred without transcription, but the translation inhibitors cycloheximide and anisomycin, or local presynaptic injection of mRNA cap analog m 7GpppG, impaired LTF expression. Both MAP kinase and phosphatidylinositol 3-OH kinase (PI3K) activation are implicated in this rapamycin-sensitive synaptic potentiation. This study defines an important role for protein synthesis in the expression of activity-dependent plasticity, and provides mechanistic insight for the induction of this process at presynaptic sites.

Invertebrate connectin spans as much as 3.5 microm in the giant sarcomeres of crayfish claw muscle.

In crayfish claw closer muscle, the giant sarcomeres are 8.3 microm long at rest, four times longer than vertebrate striated muscle sarcomeres, and they are extensible up to 13 microm upon stretch. Invertebrate connectin (I-connectin) is an elastic protein which holds the A band at the center of the sarcomere. The entire sequence of crayfish I-connectin was predicted from cDNA sequences of 53 424 bp (17 352 residues; 1960 kDa). Crayfish I-connectin contains two novel 68- and 71-residue repeats, and also two PEVK domains and one kettin region. Kettin is a small isoform of I-connectin. Immunoblot tests using antibody to the 68-residue repeats revealed the presence of I-connectin also in long sarcomeres of insect leg muscle and barnacle ventral muscle. Immunofluorescence microscopy demonstrated that the two repeats, the long spacer and the two PEVK domains contribute to sarcomere extension. These regions rich in charged amino acids, occupying 63% of the crayfish I-connectin molecule, may allow a span of a 3.5 microm distance as a new class of composite spring.

Intracellular pH of crab and crayfish muscle fibre types during GABA application and inhibitory nerve stimulation.

The inhibitory motoneurons of crustaceans form synapses both with the sarcolemma of muscle fibres and with the very distal branchings of the excitatory motoneurons. The transmitter of these synapses is GABA (gamma-aminobutyric acid) which is known to open Cl- channels. Studies on the dactyl opener muscle of crayfish suggest that application of GABA not only leads to an increase in the Cl- permeability but also to a considerable HCO3- conductance that causes an intracellular acidification. To investigate possible physiological implications, we measured the intracellular pH of various muscle fibre types of crayfish and crab using pH-sensitive microelectrodes. Independent of the presence or absence of inhibitory innervation, bath application of 10(-5) mol l(-1) GABA led to acidification in all fibre types (pH change: 0.14 +/- 0.08, n = 11). In no preparation was a change in intracellular pH observed upon stimulation of specific or common inhibitory motoneurons with 1040 pulses s(-1) for 2-5 min. The results suggest that HCO3- conductance cannot be activated through synaptic GABA receptors. However, all crustacean muscle fibre types seem to possess extrasynaptic GABA-sensitive channels that exhibit a considerable HCO3- conductance. The physiological importance of these channels remains to be elucidated.

Evaluation of the Number of Agonist Molecules Needed to Activate a Ligand-Gated Channel from the Current Rising Phase

We propose a new method for calculating the number of agonist binding sites ( n) in ligand-gated receptor channels from the initial phase of the current. This method is based on the fact that the relation between the current ( I) and its first-time derivative ( I′) at the beginning of the current reflects the number of transitions that lead to channel opening. We show that, for constant agonist concentration, the above relationship at t → 0 provides the number of steps leading to channel opening. When the agonist concentration is not constant but rather increases linearly with time, the corresponding value can be obtained using a slightly modified procedure. The analytical results were compared with computer simulations and a good match between the two was obtained. The theoretical procedure was then validated experimentally using the nicotinic receptor, because, for this receptor, the number of binding sites is well established. Indeed, the expected number of two binding sites was obtained. The method was then tested for the quisqualate-type glutamate receptor channel from the opener muscle of crayfish. The number of this receptor's binding sites is not fully resolved. Our results suggest that, for this glutamate receptor as well, two binding sites must be occupied to open the channel.

Role of alpha-SNAP in promoting efficient neurotransmission at the crayfish neuromuscular junction.

In this manuscript, we address the role of the soluble N-ethylmaleimide sensitive factor attachment protein (alpha-SNAP) in synaptic transmission at the neuromuscular junction of the crayfish opener muscle. Immunochemical methods confirm the presence of alpha-SNAP in these preparations and show that it is concentrated in the synaptic areas. Microinjection and electrophysiological studies show that alpha-SNAP causes an increase in neurotransmitter release yet does not significantly affect the kinetics. More specific quantal analysis, using focal, macropatch, synaptic current recordings, shows that alpha-SNAP increases transmitter release by increasing the probability of exocytosis but not the number of potential release sites. These data demonstrate that the role of alpha-SNAP is to increase the efficiency of neurotransmission by increasing the probability that a stimulus will result in neurotransmitter release. What this suggests is that alpha-SNAP is critical for the formation and maintenance of a "ready release" pool of synaptic vesicles.

Fast BK-type channel mediates the Ca(2+)-activated K(+) current in crayfish muscle.

The role of the Ca(2+)-activated K(+) current (I(K(Ca))) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca(2+), K(+), and Ca(2+)-dependent K(+) currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I(K(Ca)). These voltage- and Ca(2+)-activated channels had a mean single-channel conductance of approximately 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca(2+) concentration. Intracellular loading with the Ca(2+) chelator bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (< or =560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I(K(Ca)). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I(K(Ca)), which probably reflects temporal Ca(2+) variations in the whole muscle fiber. We conclude that the channels mediating I(K(Ca)) in crayfish muscle are voltage- and Ca(2+)-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca(2+) sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.

Neuromodulators Enhance Transmitter Release by Two Separate Mechanisms at the Inhibitor of Crayfish Opener Muscle

A presynaptic voltage control method has been used to investigate the modulatory effects of serotonin (5-HT) and okadaic acid (OA) on the inhibitory junction of the crayfish opener muscle. Instead of using action potentials, we used 20 msec pulses depolarized to 0 mV to activate transmitter release. This approach allowed us to monitor two separate physiological parameters related to the release process. The first parameter, transmitter release kinetics, is characterized as the delay when inhibitory postsynaptic conductance reaches its half-maximum (IPSG50). The second parameter, the total area of IPSG (IPSGarea), estimates total transmitter output. We have reported previously that the F2 component of synaptic facilitation is associated with a decrease in IPSG50 but without a change in IPSGarea. These results raised the possibility that IPSG50 and IPSGarea could be mediated by separate mechanisms that were modulated independently. To explore this possibility, we investigated the effects of 5-HT (100-200 nM) and OA (2.5 microM) on the two parameters. 5-HT and OA enhanced IPSG neither by changing the sensitivity of postsynaptic receptors, as tested by iontophoretically ejected GABA, nor by elevating resting and action potential-activated presynaptic free calcium, as monitored by fura-2 imaging. 5-HT and OA decreased IPSG50 by 3.0 +/- 1.4 and 3.6 +/- 1.1 msec, respectively, and increased IPSGarea by 50 +/- 21 and 37 +/- 6%, respectively. The ability of F2 facilitation to accelerate release kinetics was reduced in the presence of the modulators, suggesting that the mechanism underlying the accelerated release kinetics was shared by the two modes of synaptic enhancement. This report demonstrates that the acceleration in release kinetics and the increase in total release are two separate mechanisms for enhancing transmitter output and that these two mechanisms can be activated without changes in presynaptic calcium dynamics.

Effects of replacing extracellular chloride with formate on the inhibitory neuromuscular junction of the crayfish opener muscle

The effect of partially replacing extracellular chloride by formate on the inhibitory junction of the crayfish opener muscle is investigated. Inhibitory postsynaptic potential (IPSP) amplitude, recorded in muscle fibers and presynaptic axons, increases significantly in the formate saline whereas input resistance and resting membrane potential of muscle fibers are not affected. The increase in IPSP amplitude is mainly due to an increase in IPSP driving force while the GABA mediated conductance change underlying IPSP is not altered. The waveform of presynaptic action potential is slightly altered by formate substitution where an after-depolarizing potential is decreased. This change does not seem to affect the probability of transmitter release because the magnitude of synaptic facilitation is unchanged. In conclusion, formate substitution significantly increases IPSP amplitudes by increasing its driving force without affecting presynaptic release mechanisms.

Voltage-gated and Ca2+-activated conductances mediating and controlling graded electrical activity in crayfish muscle.

Crayfish opener muscle fibers provide a unique preparation to quantitatively evaluate the relationships between the voltage-gated Ca2+ (ICa) and Ca2+-activated K+ (IK(Ca)) currents underlying the graded action potentials (GAPs) that typify these fibers. ICa, IK(Ca), and the voltage-gated K+ current (IK) were studied using two-electrode voltage-clamp applying voltage commands that simulated the GAPs evoked in current-clamp conditions by 60-ms current pulses. This methodology, unlike traditional voltage-clamp step commands, provides a description of the dynamic aspects of the interaction between different conductances participating in the generation of the natural GAP. The initial depolarizing phase of the GAP was due to activation of the ICa on depolarization above approximately -40 mV. The resulting Ca2+ inflow induced the activation of the fast IK(Ca) (<3 ms), which rapidly repolarized the fiber (<6 ms). Because of its relatively slow activation, the contribution of IK to the GAP repolarization was delayed. During the final steady GAP depolarization ICa and IK(Ca) were simultaneously activated with similar magnitudes, whereas IK aided in the control of the delayed sustained response. The larger GAPs evoked by higher intensity stimulations were due to the increase in ICa. The resulting larger Ca2+ inflow increased IK(Ca), which acted as a negative feedback that precisely controlled the fiber's depolarization. Hence IK(Ca) regulated the Ca2+-inflow needed for the contraction and controlled the depolarization that this Ca2+ inflow would otherwise elicit.

GABA A receptors affect directly the release boutons in the neuromuscular junction of the crayfish opener muscle

GABA A receptors affect directly the release boutons in the neuromuscular junction of the crayfish opener muscle

Change of transmitter release kinetics during facilitation revealed by prolonged test pulses at the inhibitor of the crayfish opener muscle.

A presynaptic voltage-control method was used to study synaptic facilitation at the inhibitory neuromuscular synapse of the crayfish opener muscle. The expression of the F2 component of facilitation, monitored 150 ms after a conditioning stimulus, was examined by systematically changing the duration of the presynaptic test pulse. (Test pulses in all experiments were depolarized to 0 mV.) Control and facilitated inhibitory postsynaptic potentials (IPSPs) exhibited identical time courses when test pulse duration was brief (approximately 2 ms). When the duration of the test pulse was increased beyond 2 ms, the transmitter release time course shifted to an earlier point in time during facilitation. Meanwhile, the increase in total transmitter release, measured from inhibitory postsynaptic conductance (IPSG) area (total release), became less pronounced with increasing duration of the test pulse. With a 20-ms test pulse, facilitation did not cause any detectable change in total release but the half-maximal point of the facilitated IPSG shifted by 3 ms (release shift). This change in release kinetics was not associated with a decrease in minimal synaptic delay. Furthermore, the relationship between total release and presynaptic pulse duration suggested that the transmitter release activated by a 20-ms pulse can be defined as a distinct component of continuous transmitter release (early component). The facilitation process accelerated the release kinetics of the early component but did not modify its total transmitter content. To test the hypothesis that the release shift is indeed mediated by the same mechanism that increases IPSP amplitude during facilitation, we investigated the correlation between the release shift and IPSP amplitude change. The two parameters were significantly correlated when the magnitude of facilitation was changed 1) during the decay of facilitation and 2) by varying the strength of the conditioning stimulus. The experimental approach reported here provides two new physiological parameters, release shift and total release, for the analysis of synaptic facilitation.

Sustained GABA-induced regulation of the L-type Ca2+ conductance in crustacean muscle fibers.

The sustained effects of gamma-aminobutyric acid (GABA) on voltage-gated conductances, and excitatory and inhibitory postsynaptic currents (EPSC and IPSC, respectively) in crayfish opener muscle fibers were analyzed using the two-electrode voltage-clamp technique. GABA (1.0 mM) was applied for 1-2 min and measurements were performed 30 min after restoring control Ringer solution. The L-type Ca2+ current (ICa) was reduced by > 33%. The ICa conductance (gCa) was reduced and the activation and inactivation were slowed down by GABA. The ICa regulation outlasted GABA superfusion (150 min). A small decrease (< 19%) of the Ca2+-activated K+ current (IKCa), due to the ICa reduction, was also recorded. The leak (IL), the delayed-rectifier (IK) and the hyperpolarization-activated (IAB) currents were not affected. Picrotoxin (0.5 mM) and bicuculline (0.2 mM) blocked the ICa reduction. Neither the GABAB antagonist saclofen (1.0 mM) nor the agonist baclofen (1.0 mM) had any effect. Therefore, the ICa regulation was probably mediated through GABAA receptors. EPSCs, but not IPSCs, were reduced (30%) for prolonged periods (> 100 min.) after GABA application. We describe a new, potentially functional, role for GABA receptors in the mediation of a sustained reduction of presynaptic and postsynaptic excitability in crustacean muscle.

Activation and detection of facilitation as studied by presynaptic voltage control at the inhibitor of the crayfish opener muscle.

Facilitation at the crayfish neuromuscular inhibitor synapse was investigated with the use of a presynaptic voltage control method in which 5-ms presynaptic pulses were used to activate and monitor facilitation. A single 5-ms pulse was able to activate facilitation with a decay time constant similar to that of the F2 component of facilitation activated by action potentials. The quality of the control of presynaptic potential during F2 facilitation was evaluated by measuring the amplitude of presynaptic pulses and by analyzing the shape of the depolarization-release coupling plot during facilitation. Both approaches suggested that neither the amplitude of presynaptic depolarizations nor the space clamp of the presynaptic axon was changed during F2 facilitation. The activation of facilitation was examined by changing the amplitude of conditioning pulses systematically and using a test pulse of a constant amplitude to monitor facilitation. We found that a significant amount of facilitation could be activated by conditioning pulses that were subthreshold to the activation of transmitter release. Facilitation plateaued before the inhibitory postsynaptic potentials (IPSPs) activated by conditioning pulses reached their maximum. A double logarithm plot of facilitation magnitude against the conditioning IPSP amplitude yielded a slope of 0.34, which implies that the calcium ion cooperativity of activating facilitation is about one third of the secretion process. These findings enabled us to activate near maximal facilitation, by a burst of subthreshold conditioning pulses, without any conditioning transmitter release, and, therefore, to avoid complications associated with previous transmitter release. The detection of facilitation was examined by changing test pulse amplitude systematically to evaluate the ability of the test pulse to detect a constant level of facilitation. The magnitude of normalized facilitation decreased with increasing test pulse amplitude. The magnitude of absolute facilitation (the amplitude of the facilitated minus the control IPSP) increased with increasing test pulse amplitude. A double logarithm plot between facilitated and control IPSPs gave rise to a slope of 0.77, which suggests that the calcium cooperativity of transmitter release was decreased during facilitation.

Study of the inhibitor of the crayfish neuromuscular junction by presynaptic voltage control.

The inhibitor of the crayfish opener muscle was investigated by a presynaptic voltage control method. Two microelectrodes were inserted into the inhibitor and the amplitude and duration of presynaptic depolarization were controlled by a voltage-clamp amplifier. The inhibitory postsynaptic potential (IPSP) was measured from a muscle fiber located near the presynaptic voltage electrode. Nonlinear summation of IPSP amplitudes was corrected after chloride equilibrium potential was measured. With the use of 5-ms presynaptic pulses, the depolarization-release coupling (D-R) curve constructed from IPSP peak amplitudes (IPSPcor) had a threshold of about -35 mV and reached its maximal level at -5 to -10 mV. Depolarization beyond the maximum led to a suppression of neurotransmitter release. When transmitter release during a presynaptic pulse was completely suppressed, IPSPs activated by tail current could be identified with an average synaptic delay of 2.5 ms. Transmitter secretion triggered by a calcium current activated during the 5-ms pulses (IPSPon) was also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5-ms pulses. D-R coupling plots measured from IPSPon exhibited a more pronounced suppression than that obtained from IPSPcor. The effect of presynaptic pulse duration on the level of transmitter release was analyzed. Transmitter release increased with increasing duration and was nearly saturated by 20-ms pulses depolarized to 0 mV. The following conditions were identified as necessary to obtain a consistent D-R curve with a clear suppression: 1) small animals, 3.8 cm head to tail, 2) 15 degrees C, 3) 40 mM tetraethylammonium and 1 mM 4-aminopyridine, 4) an extracellular calcium concentration of < or = 10 mM. In addition, a consistent correlation was found among the branching pattern of the inhibitor, the placement of the presynaptic electrode, and the characteristics of the D-R curves. An ideal presynaptic electrode configuration involved placing the voltage electrode in a secondary branch, approximately 100 microns from the main branch point, and placing the current electrode at the branch point. Postsynaptically, optimal recordings were obtained from muscle fibers innervated by a single branch of the inhibitor that originated from a point near the presynaptic voltage electrode. A cable-release model was constructed to evaluate the relationship between the shape of the D-R coupling curves and the space constants of the presynaptic terminals. A comparison between the model and the D-R coupling curves suggested that the space constant of an inhibitor branch on a muscle fiber is > or = 8 times longer than its actual length. Therefore the upper limit estimate of the space constant of a typical preparation is approximately 3 mm. Results reported here outline morphological and physiological conditions needed to achieve optimal control of the presynaptic branch of the crayfish inhibitor. The cable-release model quantitatively defines the extent of presynaptic voltage control.

Patch clamp study and kinetic modeling on the most abundant chloride channel on distal fibers of crayfish opener muscle

Whereas the inhibitory innervation of the deep extensor abdominal muscle in crayfish is mediated by a weakly acting common inhibitor, the opener muscle exhibits a stronger inhibition. In the present study the most abundant γ-aminobutyric acid-activated chloride channel on distal fibers of crayfish opener muscle was characterized by measuring the current responses after applying pulses of γ-aminobutyric acid to outside-out patches. The results were compared to those obtained earlier with the chloride channel on the deep extensor abdominal muscle of the same species. The double logarithmic plot of the dose-response relationship had a slope of n H = 2.2 in contrast to n H = 5.3 for the channel on the deep extensor abdominal muscle. The rise time of the current response declined to 1 ms at a γ-aminobutyric acid concentration of 50 mmol · l−1. With lower concentrations the rise time increased to a maximal value of 280 ms. No peak of the rise time at low γ-aminobutyric acid concentrations, as observed for the channel on the deep extensor abdominal muscle, was obvious. The open and closed times were similar to those of the channel of the deep extensor abdominal muscle. Different reaction schemes were discussed to describe the kinetics of the chloride channel of the opener muscle.

Presynaptic calcium currents at voltage-clamped excitor and inhibitor nerve terminals of crayfish.

1. A two-electrode voltage clamp was used to record calcium currents from the excitatory and inhibitory nerve terminals that innervate the crayfish (Procambarus spp.) opener muscle. Other voltage-dependent currents were blocked with tetrodotoxin, 3,4-diaminopyridine, 4-aminopyridine and tetraethylammonium. 2. The presynaptic calcium current at both excitatory and inhibitory synapses was blocked by cadmium and omega-agatoxin IVA but was not affected by omega-conotoxin GVIA, omega-conotoxin MVIIC or nifedipine, suggesting that the calcium currents flow through P-type calcium channels. 3. Current-voltage (I-V) relations at both excitatory and inhibitory synapses are similar, with current activation near -40 mV, peak current near -10 mV and current reversal at membrane potentials greater than +25 mV. I-V relations were scaled along the current axis by partial calcium current blockade with cobalt, suggesting that series resistance and space-clamp errors were small. 4. A subset of terminals on one muscle fibre was locally superfused with a physiological saline containing barium; the rest of the preparation was superfused with a physiological saline containing calcium channel antagonists. Under such conditions the characteristics of the I-V relation were very similar to the I-V relations recorded when the entire preparation was bathed in physiological levels of calcium, suggesting that the space clamp was adequate. 5. Calcium channel activation, as determined from tail current analyses, was similar when the entire preparation was bathed in physiological levels of calcium or if terminals on one muscle fibre were locally superfused with barium. 6. During a 30 ms depolarization, calcium currents inactivated to a greater extent in inhibitory than in excitatory terminals. The inactivation was of small magnitude (< 20%) and was eliminated by intracellular injection of the calcium chelator BAPTA, suggesting that the inactivation was calcium dependent. 7. These data show that biophysical and pharmacological properties of calcium currents at crayfish neuromuscular junctions resemble those found at stellate synapses in squid.