The understanding of neurotransmitter release at vertebrate synapses has been hampered by the paucity of preparations in which presynaptic ionic currents and postsynaptic responses can be monitored directly. We used cultured embryonic Xenopus neuromuscular junctions and simultaneous pre-and postsynaptic patch-clamp current-recording procedures to identify the major presynaptic conductances underlying the initiation of neurotransmitter release.Step depolarizations and action potential waveforms elicited Na and K currents along with Ca and Ca-activated K (K Ca ) currents. The onset of K Ca current preceded the peak of the action potential. The predominantly -CgTX GVIA-sensitive Ca current occurred primarily during the falling phase, but there was also significant Ca 2ϩ entry during the rising phase of the action potential. The postsynaptic current began a mean of 0.7 msec after the time of maximum rate of rise of the Ca current. -CgTX also blocked K Ca currents and transmitter release during an action potential, suggesting that Ca and K Ca channels are colocalized at presynaptic active zones. In double-ramp voltage-clamp experiments, K Ca channel activation is enhanced during the second ramp. The 1 msec time constant of decay of enhancement with increasing interpulse interval may reflect the time course of either the deactivation of K Ca channels or the diffusion/removal of Ca 2ϩ from sites of neurotransmitter release after an action potential. Key word: neuromuscular junction; nerve terminal; calcium channel; charybdotoxin; conotoxin; synaptic delayNeurotransmitter release from nerve terminals is triggered by the entry of Ca 2ϩ through voltage-gated Ca channels (Katz, 1969;Augustine et al., 1987). Our understanding of the relationship between the presynaptic ionic currents and release is based largely on studies of the squid giant synapse (Katz and Miledi, 1967;Llinás et al., 1981;Charlton et al., 1982; Augustine et al., 1985a,b). Several critical questions remain, however, that one would like to address with equivalent biophysical rigor at a vertebrate synapse in which the presynaptic ionic currents and postsynaptic currents can be measured simultaneously and release can be resolved at the single quantum level. In this report we take advantage of a neuromuscular synapse preparation in which this can be done.Among the important pending questions are the timing and delay between Ca 2ϩ entry during an action potential and release, the roles of different Ca and Ca-activated K (K Ca ) channels in the release process, and the quantitative relationship between Ca 2ϩ influx and release. In squid, it has been shown that Ca 2ϩ enters principally during the repolarization phase of the action potential (Llinás et al., 1982). Physiological studies of various excitable secretory cells have shown that different types of Ca channels play a dominant role in triggering release in different terminals, and often multiple Ca channel types are present in any given terminal (Kerr and Yoshikami, 1984;Pfrieger et al., 1992, Luebke et al...

The stretch of a frog muscle within the physiological range can more than double the spontaneous and evoked release of neurotransmitter from its motor nerve terminals. Here, stretch enhancement of release was suppressed by peptides containing the sequence arginine-glycine-aspartic acid (RGD), which blocks integrin binding. Integrin antibodies also inhibited the enhancement obtained by stretching. Stretch enhancement depended on intraterminal calcium derived both from external calcium and from internal stores. Muscle stretch thus might enhance the release of neurotransmitters either by elevating internal calcium concentrations or by increasing the sensitivity of transmitter release to calcium in the nerve terminal.

Neurotransmitter release during action potentials is thought to require transient, localized [Ca2+]i as high as hundreds of micromolar near presynaptic release sites. Most experimental attempts to characterize the magnitude and time course of these Ca2+ domains involve optical methods that sample large volumes, require washout of endogenous buffers and often affect Ca2+ kinetics and transmitter release. Endogenous calcium-activated potassium (KCa) channels colocalize with presynaptic Ca2+ channels in Xenopus nerve-muscle cultures. We used these channels to quantify the rapid, dynamic changes in [Ca2+]i at active zones during synaptic activity. Confirming Ca2+-domain predictions, these KCa channels revealed [Ca2+]i over 100 microM during synaptic activity and much faster buildup and decay of Ca2+ domains than shown using other techniques.

SUMMAARY1. A short-latency interaction between motoneurones has been studied with intracellular and root potential recordings from the isolated spinal cord of the frog. Antidromic stimulation of one ventral root causes brief depolarization (VR-EPSP) of the motoneurones of adjacent, non-excited motoneurones. The summed activity of many such VR-EPSPs can be seen as a brief depolarization (VR-VRP) passing out an adjacent ventral root.2. Both intracellular and root-recorded signs of this interaction are graded in amplitude.3. It was found that this interaction decreased with increasing temperature. This is in contrast to the behaviour of the ventral root potential resulting from dorsal root stimulation (DR-VRP) or the dorsal root potentials resulting from either dorsal root (DR-DRP) or ventral root (VR-DRP) stimulation, all of which increased in amplitude from below 10 to about 17' C.4. Pharmacological evidence suggests that the interaction between motoneurones is not chemically mediated. The VR-VRP was not affected by a large variety of transmitter blocking agents, including curare, dihydro-,/-erythroidine, atropine, succinylcholine, hexamethonium and DOPA, while the VR-DRP, which probably originates with the release of ACh from an axon collateral, was consistently blocked.5. Mg2+ suppressed the VR-VRP more slowly than the other potentials, and this suppression was increased by adding Ca2+, rather than reversed, as in the case of the other root potentials, which are presumably mediated by chemical transmission.6. The interaction between motoneurones is strongly facilitated by orthodromic depolarization of the motoneurones being antidromically * Present address.INTERACTION BETWEEN FROG MOTONEURONES 613 stimulated. Extracellular recordings within the cord support the conclusion that this facilitation is a result of the enhancement of antidromic invasion, perhaps especially of the dendrites, by slight depolarization.7. One VR-VRP (or VR-EPSP) first suppresses response to another (for about 10 msec), then facilitates response to the second, with maximum effect around 20-40 msec. This is the case whether both stimuli go to the same or to different ventral roots, although occlusion is less and facilitation greater in the latter case. Occlusion of the VR-EPSP also results from full excitation of the cell in which recording is being done.8. The mechanism of this interaction remains uncertain, but it would seem likely that overlapping dendrites of adjacent motoneurones interact with each other electrically through close apposition or specialized contacts. Occlusion would result from the refractoriness of strongly depolarized dendrites, facilitation from the enhancement of invasion of antidromically stimulated motoneurones by the weaker (or residual) depolarization occurring after earlier activity of motoneurones or their dendrites.

Neurotransmitter release from frog motor nerve terminals is strongly modulated by change in muscle length. Over the physiological range, there is an approximately 10% increase in spontaneous and evoked release per 1% muscle stretch. Because many muscle fibers do not receive suprathreshold synaptic inputs at rest length, this stretch-induced enhancement of release constitutes a strong peripheral amplifier of the spinal stretch reflex. The stretch modulation of release is inhibited by peptides that block integrin binding of natural ligands. The modulation varies linearly with length, with a delay of no more than approximately 1-2 msec and is maintained constant at the new length. Moreover, the stretch modulation persists in a zero Ca2+ Ringer and, hence, is not dependent on Ca2+ influx through stretch activated channels. Eliminating transmembrane Ca2+ gradients and buffering intraterminal Ca2+ to approximately normal resting levels does not eliminate the modulation, suggesting that it is not the result of release of Ca2+ from internal stores. Finally, changes in temperature have no detectable effect on the kinetics of stretch-induced changes in endplate potential (EPP) amplitude or miniature EPP (mEPP) frequency. We conclude, therefore, that stretch does not act via second messenger pathways or a chemical modification of molecules involved in the release pathway. Instead, there is direct mechanical modulation of release. We postulate that tension on integrins in the presynaptic membrane is transduced mechanically into changes in the position or conformation of one or more molecules involved in neurotransmitter release, altering sensitivity to Ca2+ or the equilibrium for a critical reaction leading to vesicle fusion.

When hunting for fish Noctilio leporinus uses several strategies. In high search flight it flies within 20-50 cm of the water surface and emits groups of two to four echolocation signals, always containing at least one pure constant frequency (CF) pulse and one mixed CF-FM pulse consisting of a CF component which is followed by a frequency-modulated (FM) component. The pure CF signals are the longest, with an average duration of 13.3 ms and a maximum of 17 ms. The CF component of the CF-FM signals averages 8.9 ms, the FM sweeps 3.9 ms. The CF components have frequencies of 52.8-56.2 kHz and the FM components have an average bandwidth of 25.9 kHz. A bat in high search flight reacts to jumping fish with "pointed dips" at the spot where a fish has broken the surface. As it descends to the water surface the bat shows the typical approach pattern of all bats with decreasing pulse duration and pulse interval. A jumping fish reveals itself by a typical pattern of temporary echo glints, reflected back to the bat from its body and from the water disturbance. In low search flight N. leporinus drops to a height of only 4~ 10 cm, with body parallel to the water, legs extended straight back and turned slightly downward, and feet cocked somewhat above the line of the legs and poised within 2-4 cm of the water surface. In this situation N. leporinus emits long series of short CF-FM pulses with an average duration of 5.6 ms (CF 3.1 and FM 2.6) and an average pulse interval of 20 ms, indicating that it is looking for targets within a short range. N. leporinus also makes pointed dips during low search flight by rapidly snapping the feet into the water at the spot where it has localized a jumping fish or disturbance. In the random rake mode, N. leporinus drops to the water surface, lowers its feet and drags its claws through the water in relatively straight lines for up to 10 m. The echolocation behavior is similar to that of high search flight. This indicates that in this hunting mode N. leporinus is not pursuing specific targets, and that raking is a random or statistical search for surface fishes. When raking, the bat uses two strategies. In directed random rake it rakes through patches of water where fish jumping activity is high. Our interpretation is that the bat detects this activity by echolocation but prefers not to concentrate on a single jumping fish. In the absence of jumping fish, after flying for several minutes without any dips, N. leporinus starts to make very long rakes in areas where it has hunted successfully before (memorydirected random rake). Hunting bats caught a fish approximately once in every 50~00 passes through the hunting area.

Neuromuscular connections have long served as models of synaptic structure and function. They also provide illuminating insights into the dynamic cell-cell interactions governing synaptogenesis, neuromuscular differentiation, and the maintenance of effective function. This paper reviews recent advances in our understanding of the regulatory and inductive interactions involved in motor axon pathfinding, target recognition, bidirectional control of gene expression during synapse formation, motoneuron cell death, terminal rearrangement, and the ongoing remodeling of synaptic number, structure, and function to adjust to growth and changes in use.

1. Nerve terminals in two different muscles of the frog, the sartorius and cutaneous pectoris (c.p.), have been found to differ sharply in safety factor. This difference is shown to be attributable to corresponding disparities in the amount of transmitter released, without evident correlated morphological differences. 2. In Ringer containing 0.3 mM‐Ca2+ and 1 mM‐Mg2+, quantal content of c.p. junctions exceeded that of sartorius junctions by 3‐4 times. 3. When quantal content was corrected for nerve terminal size, c.p. terminals still released 2‐4 times more transmitter per unit terminal length. 4. Light and electron microscopic examination of junctional morphology in the two muscles revealed no significant difference in the spacing of presynaptic active zones, the width of synaptic contact, or the density of presynaptic vesicles and mitochondria. It seems likely, therefore, that the greater release at c.p. junctions is due to a ‘physiological’ difference between the two populations of terminals. 5. No evidence could be found that action potential invasion of the terminal was less complete in the sartorius than in the c.p. 6. The dependence of evoked and spontaneous release on Ca2+ concentration was of similar slope for terminals in the two muscles, but of different absolute value, consistent with the observed difference in release.