Nested transcripts of gap junction gene have distinct expression patterns.

The shaking B locus (shakB, or Passover) codes for structural molecules of gap junctions in Drosophila. This report describes the complex set of transcripts from the shakB locus. A nested set of five transcripts is described. The transcripts share 3' exons, but each has its own 5' exon. The transcripts are arrayed as a series in the genomic DNA stretching over 60 kb. The 5' end of each successive transcript lies further proximal on the chromosome. Each new transcript shares all the 3' exons with the one preceding it, but adds one or two more 5' exons. The different transcripts are expressed in a wide variety of locations in the nervous system and in non-neural tissues. Some tissues express more than one transcript, and the expression pattern of each is developmentally regulated. Within the adult central nervous system (CNS), these transcripts have an expression pattern that is restricted to the giant fiber system (GFS). The GFS is a small set of neurons which mediates the visually induced escape jump. shakB is required for function of the GFS electrical synapses. The transcript previously defined as active in the giant fiber is not, in fact, expressed in that cell. Instead, we find that another transcript, shakB(N3), and perhaps shakB(N4) as well, is expressed in the GFS; this transcript is not expressed elsewhere in the adult CNS. Two other transcripts, shakB(N1) and shakB(N2), are expressed in the optic lamina but not elsewhere in the CNS. This expression pattern explains the neurophysiological and behavioral defects in escape exhibited in mutants of shakB.

Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster.

The ability to tolerate a low-O2 environment varies widely among species in the animal kingdom. Some animals, such as Drosophila melanogaster, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O2, we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O2 deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.

Neurons of the Drosophila giant fiber system: I. Dorsal longitudinal motor neurons.

The giant fiber system (GFS) mediates the startle response of Drosophila. This response includes an activation of the dorsal longitudinal wing-depressor muscles (DLMs). However, the morphology of the motor neurons innervating these muscles has not been well studied. Even the location of the somata of these motor neurons has been a source of controversy. This paper identifies the somata and provides a morphological description of these motoneurons. The DLM is comprised of six muscle fibers, named a through f (dorsal to ventral). Each muscle fiber is singly innervated. Each of the four ventral muscle fibers is innervated by a separate motor neuron (DLMn c-f), but the two dorsal fibers share an axon (DLMn a/b). Motor neurons were back filled by introducing horseradish peroxidase (HRP) into individual muscle fibers. The cell body of DLMn a/b is extraordinarily large (32 microm) and lies dorsal and contralateral. In this hemiganglion, it does not have a fixed position; it can be found anywhere from the midline to the extreme lateral edge of the ganglion. The position is not genetically controlled: We find no strain differences, and, within a single individual, the right and left cells may take different positions. The neuritic arborization fills a shallow dorsal cap of the ganglion, with branches arrayed like a feather. The cell bodies of the four motor neurons c-f lie in an ipsilateral and ventral cluster. Each soma occupies a fixed corner of this quadrilaterally shaped cluster. The neurites ramify in the same dorsal region as DLMn a/b.

Behavioral and Electrophysiologic Responses of Drosophila melanogaster to Prolonged Periods of Anoxia.

Sensitivity to anoxia varies tremendously among phyla and species. Most mammals are exquisitely sensitive to low concentrations of inspired oxygen, while some fish, turtles and crustacea are very resistant. To determine the basis of anoxia tolerance, it would be useful to utilize a model system which can yield mechanistic answers. We studied the fruit fly, Drosophila melanogaster, to determine its anoxia resistance since this organism has been previously studied using a variety of approaches and has proven to be very useful in a number of areas of biology. Flies were exposed to anoxia for periods of 5-240 min, and, after 1-2 min in anoxia, Drosophila lost coordination, fell down, and became motionless. However, they tolerated a complete nitrogen atmosphere for up to 4 h following which they recovered. In addition, a nonlinear relation existed between time spent in anoxia and time to recovery. Extracellular recordings from flight muscles in response to giant fiber stimulation revealed complete recovery of muscle-evoked response, a response that was totally absent during anoxia. Mean O(2) consumption per gram of tissue was substantially reduced in low O(2) concentrations (20% of control). We conclude from these studies that: (1) Drosophila melanogaster is very resistant to anoxia and can be useful in the study of mechanisms of anoxia tolerance; and (2) the profound decline in metabolic rate during periods of low environmental O(2) levels contributes to the survival of Drosophila. Copyright 1997 Elsevier Science Ltd. All rights reserved

Passover eliminates gap junctional communication between neurons of the giant fiber system in Drosophila. off.

The Passover-related gene family plays significant roles in cellular connectivity. Mutations in three family members from Drosophila and from Caenorhabditis elegans alter a few specific electrical synapses. The passage of cobalt between Drosophila neurons was used to assay the presence of gap junctional connections. The giant fiber in the wild type has specific gap junctional connections in the brain and in the thorax. In flies mutant for Passover, cobalt cannot pass into or out of the giant fiber in either the anterograde or the retrograde directions. A large number of other gap junctional connections remain unaffected. This demonstrates that the Passover gene is necessary for gap-junctional communication between the neurons of the Drosophila giant fiber system.

Reevaluation of electrophoresis in the Drosophila egg chamber.

To evaluate the hypothesis of electrophoretic transport of cytoplasmic components, the transfollicle potentials of Drosophila oocytes and nurse cells were measured using improved techniques. We found input resistances 20 to 1000 times higher than those in previous reports. Measurements were made in a large variety of conditions: in external potassium concentrations from 1 to 100 mM, over the concomitant membrane potential range -84 to -23 mV, from developmental stages 5 to 10, and with or without using hemolymph, anesthetics, or collagenase. In all of these circumstances, no voltage gradient was detectable with intracellular microelectrodes from nurse cells to oocyte or between nurse cells. No voltage gradient was detected with external suction electrodes. Our results do not support the electrophoretic theory.

Studies of nerve-muscle interactions in Xenopus cell culture: analysis of early synaptic currents.

We have studied the spontaneous and nerve-evoked synaptic currents during the initial period of nerve-muscle contact in Xenopus cell cultures. The precise timing of the contact was achieved by physically manipulating embryonic muscle cells into contact with co-cultured spinal neurons. Previous studies have shown that physical contact of the muscle membrane induces pulsatile release of acetylcholine (ACh) from the growth cone of these neurons, resulting in spontaneous synaptic currents (SSCs) in the muscle cell within seconds following the contact. In the present work, we first showed that these SSCs at the manipulated nerve-muscle contacts are similar to those observed at naturally occurring synapses. We then examined the possible cellular mechanisms responsible for the marked variation in SSC amplitude and showed that it most likely results from differences in either the amount of ACh contained in each release event or the extent of close membrane apposition near the release sites. During the first 20 min following the nerve-muscle contact, there was an increase in the frequency and mean amplitude of the SSCs. During a similar period, the evoked synaptic currents (ESCs), which were induced by suprathreshold electrical stimulation of the neuronal soma, also showed an increase in the mean amplitude and a reduction in the delay of onset following the stimulus. These postcontact changes in the efficacy of synaptic transmission may be related to an increase in the total area of close membrane apposition between the nerve and muscle cells. This was suggested by the finding that neurite-muscle adhesion increases over a similar postcontact period. The transition from low- to high-efficacy transmission during the early phase of contact may reflect the process of selective adhesion between the cells, and thus signify the formation of specific synapse. Analysis of the fluctuation in the ESC amplitude at the early nerve-muscle contact suggests that evoked release of ACh occurs as multiples of a quantal unit. However, this unit is apparently related to only a small subpopulation of SSCs of relatively high amplitudes.(ABSTRACT TRUNCATED AT 400 WORDS)

Studies of nerve-muscle interactions in Xenopus cell culture: fine structure of early functional contacts.

We have studied the fine structure of nerve-muscle contacts during the first few hours of synaptogenesis in embryonic Xenopus cell cultures. The precise timing of contact was achieved by manipulating isolated spherical myocytes (myoballs) into contact with growth cones or neurites of co-cultured spinal neurons. The contacts were shown to be functional by whole-cell voltage-clamp recording of nerve-evoked synaptic currents in the muscle cell. The ultrastructure of these functional contacts was examined by thin-section electron microscopy. In total, 20 nerve-muscle pairs were studied with contact periods ranging from 20 min to 12 hr, during which time a substantial increase in the amplitude of synaptic currents occurred. The structure of noncontacting cells and of nerve-muscle contacts formed between the cells by natural encounters in 1-d-old cultures were also examined in order to identify the features and the time course of morphological differentiation of early functional contacts. Prominent features of the contact area during the first few hours included: close apposition of the nerve and muscle membranes, greater frequency of coated pits and vesicles, and thickening of postsynaptic muscle membrane. Occasionally, clusters of clear vesicles occurred near presynaptic membrane, but no further sign of active zone differentiation was observed. In comparison, definitive active zone structure, well-formed extracellular basal lamina, and widened cleft were seen in natural contacts less than 24 hr old. This study of the identified functional contacts may help us to understand the structural basis for early nerve-muscle interaction and the functional significance of various synaptic specializations.

Evoked release of acetylcholine from the growing embryonic neuron.

An excised patch of embryonic muscle membrane was used as a probe for measuring the release of acetylcholine (AcCho) from growing spinal neurons in Xenopus cell culture. The neuron was stimulated extracellularly at the soma, and the evoked AcCho release was monitored at the growth cone, along the neurite, and near the soma. For a majority of the neurons studied, a brief suprathreshold stimulation of the soma triggered a pulse of AcCho release from the growth cone. This release showed many of the characteristics reminiscent of the transmitter release at the nerve terminal of a mature neuromuscular synapse: it occurs within a few ms following the stimulation, depends on extracellular Ca2+ concentration, and exhibits depression and potentiation during and after high-frequency stimulation, respectively. Similar evoked release was also observed only at selected points along the neurite, and prolonged suprathreshold stimulus was required to induce release from the soma. These results indicate that some of the growing spinal neurons have acquired a substantial number of AcCho molecules as well as an efficient mechanism for excitation-secretion coupling at the growth cone, ready for establishing functional contact with the target muscle cell. This notion was further supported by the finding that the evoked AcCho release is capable of inducing suprathreshold excitation of the muscle cell within the first minute following neurite-muscle contact.

Non-quantal release of acetylcholine at a developing neuromuscular synapse in culture.

Local, pulsed application of d-tubocurarine at neuromuscular synapses in embryonic Xenopus nerve-muscle culture resulted in a transient hyperpolarization of muscle membrane potential. Miniature endplate potentials (MEPPs) were abolished during the hyperpolarization and recovered after the return of resting membrane potential. The magnitude of hyperpolarization was independent of the frequency of MEPPs before curarization, and it had an average peak value of 4.3 mV in medium containing physiological levels of Ca2+. Prolonged application of curare or alpha-bungarotoxin led to sustained hyperpolarizations up to 8 mV in magnitude. Denervation produced by mechanically removing the neurite from the muscle cell also produced similar hyperpolarization, and curarization after denervation was without significant hyperpolarizing effect. Increasing the extracellular Ca2+ concentration to about 8 mM abolished the curare-induced hyperpolarization response, in sharp contrast to its effect in elevating the frequency of MEPPs. Taken together, our results indicate that innervated embryonic muscle cells were maintained at a depolarized state relative to that of uninnervated muscle cells by a steady, spontaneous release of acetylcholine (ACh) from the innervating neurite. The cellular mechanism underlying this mode of ACh release appears to be different from that of the quantal ACh release responsible for MEPPs.

Three types of transmitter release from embryonic neurons.

Prior to the contact with their target muscle cells in culture, growth cones of many isolated Xenopus embryonic neurons release acetylcholine (ACh) spontaneously. Using patch clamp techniques, this release can be detected by an outside-out patch of muscle membrane placed near the growth cone. Intracellular recording from innervated muscle cells showed spontaneous miniature endplate potentials (MEPPs) of varying amplitudes. Amplitude histograms showed a skewed distribution with multiple peaks, suggesting the existence of subunits in either the quantal packages of ACh released by the nerve terminal or in the postsynaptic muscle response. In addition to the quantal ACh release reflected by MEPPs, nerve terminal also release a large amount of ACh in a non-quantal fashion. This non-quantal ACh release is revealed by the hyperpolarization of the muscle membrane following extracellular application of curare or alpha-bungarotoxin, as well as by denervation of the muscle cell.

Spontaneous potentials of embryonic epithelium in urodeles (Cynops orientalis).

The spontaneous potentials (SPs) of nerveless embryonic epithelium of Cynops orientalis were observed by conventional intracellular micro-electrode technique. The amplitude and frequency of SPs of epithelial cells and the course of initiation and decline were recorded. The SPs can occur repeatedly at the stages when the embryonic epithelium is able to conduct excitation. The SP is very similar to the evoked propagatable potential, but has shorter duration. The amplitudes and frequencies of SPs differ in the different embryos and in different epithelial cells in the same embryo. The excitable epithelial cells of the embryo may lose their conductivity when the SPs come to a stable phase both in amplitude and frequency. And the conductivity can recover again after the subsiding of SPs. The SPs can be eliminated by tetrodotoxin (TTX), but they are not affected by the treatment of cobalt chloride.