The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments "Type I" endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements ("boutons"); whereas muscles 12 and 13 have in addition "Type II" terminals, which are more widely distributed and have smaller "boutons". Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the "Type II" endings seen in the light microscope. Type I endings are thought to generate the 'fast' and 'slow' junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

SUMMARYAND CONCLUSIONS1. In a model synaptic system, the excitatory neuromuscular junction of the freshwater crayfish, the nerve terminals possess synapses that vary in structural complexity, with numbers of active zones ranging from zero to five. Active zones on individual synapses show a wide range of separation distances. We tested the hypothesis that two active zones of a single synapse in close proximity can enhance the localized increase in free calcium ion concentration, thus enhancing the probability of neurotransmission at that synapse. We evaluated the increase in calcium ion concentration as a function of distance between adjacent active zones.2. To test this hypothesis, a reaction-diffusion model for Ca2+ entering the presynaptic terminals was used. This test was used because 1) present measurement techniques are inadequate to resolve quantitatively the highly localized, transient calcium microdomains at synaptic active zones; and 2) there is presently no suitable preparation for physiological recording from isolated synapses with varying distances between active zones. Included in the model were intracellular buffer and a typical distribution of voltage-activated Ca2+ channels for an active zone, estimated from freeze-fracture micrographs.3. The model indicated that localized Ca2+ clouds from discrete active zones can overlap to create spatial enhancement of Ca2+ concentration. The degree of interaction between two active zones depends on the distance between them. When two typical active zones are separated by 5200 nm, the maximum intracellular Ca2+ concentration ( [ Ca2'li) is greater at 1) the midpoint between them, and 2) the center of each one, than at the corresponding positions for a single isolated active zone. Enhanced [ Ca2+]i at the edge of the active zone where "docked' ' synaptic vesicles occur would be expected to have an effect on transmitter release.4. When the model includes no intracellular buffer, the increase in [Ca"]i is a linear function of calcium channel current, but is a nonlinear function of the number of conducting calcium channels in an active zone. With immobile buffer included, the increase in [ Ca2+li is nonlinear with respect to both channel current and number of conducting channels.5. Inclusion of immobile buffer in the model provides "released" residual calcium that slowly accumulates during a train of current pulses. Released residual calcium accumulates more rapidly at paired active zones separated by 5200 nm than at single isolated active zones.6. We propose that the probability of release is enhanced at synapses with closely associated active zones. Synapses of this type ( "complex' ' synapses) could be selectively recruited when the neuron is active at low frequencies. At higher frequencies of neuronal activity, more distant active zones may interact and acquire a greater probability of releasing quanta. This would provide the nerve terminal with one component of a mechanism for frequency facilitation, because the number of quanta released by the terminal as a...