Patricia K. Rivlin

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors). In neurons, the t‐SNARE SNAP‐25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP‐25 temperature‐sensitive paralytic mutant in Drosophila, SNAP‐25ts. The mutation causes a Gly50 to Glu change in SNAP‐25s first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro‐generated 70 kDa SNARE complexes containing SNAP‐25ts are thermally stable but the mutant SNARE multimers (of ∼120 kDa) rapidly dissociate at 37°C. The SNAP‐25ts mutant has two effects on neurotransmitter release depending upon temperature. At 22°C, evoked release of neurotransmitter in SNAP‐25ts larvae is greatly increased, and at 37°C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22°C the mutation causes the SNARE complex to be more fusion competent but, at 37°C the same mutation leads to SNARE multimer instability and fusion incompetence.

Morphology and molecular organization of the adult neuromuscular junction of Drosophila

While the larval neuromuscular junction (NMJ) of Drosophila has emerged as a model system to study synaptic function and development, little attention has been given to the study of the adult NMJ. Here we report an immunocytochemical and morphological characterization of an adult NMJ preparation of the prothorax. All muscles examined were innervated by small, uniform type II terminals (0.5–1.5 μm), a subset of which contained octopamine. Terminals classified as type I varied in their morphology across different muscles, ranging from strings or clusters of boutons (0.8–5.5 μm) to an elongate terminal (80–100 μm long) with few branches and contiguous swellings (3–15 μm) along its length. Analysis of the molecular composition of the NMJs during the first 5 days after eclosion revealed four major findings: 1) type I boutons increase in size during early adulthood; 2) Fasciclin II‐immunoreactivity is not detectable at type I terminals, while DLG‐immunoreactivity is observed at the synapse; 3) a Shaker‐GFP fusion protein that localizes to all type I boutons in the larva is differentially localized at adult prothoracic NMJs; and 4) while all type I terminals contain glutamate, the glutamate receptor subunits, DGluRIIA and DGluRIIB, are expressed and clustered in only a subset of muscles. These findings suggest that maturation of the adult NMJ occurs during early adulthood and that muscle‐specific properties may play a role in organizing synaptic components in the adult. Furthermore, these results demonstrate that there are major differences in the molecular organization of the adult and larval NMJs. J. Comp. Neurol. 468:596–613, 2004.

Tissue remodeling: a mating-induced differentiation program for the Drosophila oviduct

BackgroundIn both vertebrates and invertebrates, the oviduct is an epithelial tube surrounded by visceral muscles that serves as a conduit for gamete transport between the ovary and uterus. While Drosophila is a model system for tubular organ development, few studies have addressed the development of the flys oviduct. Recent studies in Drosophila have identified mating-responsive genes and proteins whose levels in the oviduct are altered by mating. Since many of these molecules (e.g. Muscle LIM protein 84B, Coracle, Neuroglian) have known roles in the differentiation of muscle and epithelia of other organs, mating may trigger similar differentiation events in the oviduct. This led us to hypothesize that mating mediates the last stages of oviduct differentiation in which organ-specific specializations arise.ResultsUsing electron- and confocal-microscopy we identified tissue-wide post-mating changes in the oviduct including differentiation of cellular junctions, remodeling of extracellular matrix, increased myofibril formation, and increased innervation. Analysis of once- and twice-mated females reveals that some mating-responsive proteins respond only to the first mating, while others respond to both matings.ConclusionWe uncovered ultrastructural changes in the mated oviduct that are consistent with the roles that mating-responsive proteins play in muscle and epithelial differentiation elsewhere. This suggests that mating triggers the late differentiation of the oviduct. Furthermore, we suggest that mating-responsive proteins that respond only to the first mating are involved in the final maturation of the oviduct while proteins that remain responsive to later matings are also involved in maintenance and ongoing function of the oviduct. Taken together, our results establish the oviduct as an attractive system to address mechanisms that regulate the late stages of differentiation and maintenance of a tubular organ.

Classical conditioning through auditory stimuli in Drosophila: methods and models

SUMMARY The role of sound in Drosophila melanogaster courtship, along with its perception via the antennae, is well established, as is the ability of this fly to learn in classical conditioning protocols. Here, we demonstrate that a neutral acoustic stimulus paired with a sucrose reward can be used to condition the proboscis-extension reflex, part of normal feeding behavior. This appetitive conditioning produces results comparable to those obtained with chemical stimuli in aversive conditioning protocols. We applied a logistic model with general estimating equations to predict the dynamics of learning, which successfully predicts the outcome of training and provides a quantitative estimate of the rate of learning. Use of acoustic stimuli with appetitive conditioning provides both an alternative to models most commonly used in studies of learning and memory in Drosophila and a means of testing hearing in both sexes, independently of courtship responsiveness.

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration.

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (DeltaV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.

Genetic feminization of the thoracic nervous system disrupts courtship song in male Drosophila melanogaster.

Abstract: Despite the growing research investigating the sex-specific organization of courtship behavior in Drosophila melanogaster, much remains to be understood about the sex-specific organization of the motor circuit that drives this behavior. To investigate the sex-specification of a tightly patterned component of courtship behavior, courtship song, the authors used the GAL4/UAS targeted gene expression system to feminize the ventral ganglia in male Drosophila and analyzed the acoustic properties of courtship song. More specifically, the authors used the thoracic-specifying teashirt promoter (tshGAL4) to express feminizing transgenes specifically in the ventral ganglia. When tshGAL4 drove expression of transformer (tra), males were unable to produce prolonged wing extensions. Transgenic expression of an RNAi construct directed against male-specific fruitless (fruM) transcripts resulted in normal wing extension, but highly defective courtship song, with 58% of males failing to generate detectable courtship song. Of those that did sing, widths of individual pulses were significantly broader than controls, suggesting thoracic fruM function serves to mediate proprioceptive-dependent wing vibration damping during pulse song. However, the most critical signal in the song, the interpulse interval, remained intact. The inability to phenocopy this effect by reducing fruM expression in motor neurons and proprioceptive neurons suggests thoracic interneurons require fruM for proper pulse song execution and patterning of pulse structure, but not for pulse timing. This provides evidence that genes establishing sex-specific activation of complex behaviors may also be used in establishing pattern-generating motor networks underlying these sex-specific behaviors.

The role of Ultrabithorax in the patterning of adult thoracic muscles in Drosophila melanogaster.

Abstract. Mutations in the homeotic gene, Ultrabithorax (Ubx), result in the transformation of the third thoracic (T3) segment into the second thoracic (T2) segment. Although it has been well established that these mutations have striking effects on adult epidermal structures in T3, the effect of these mutations on the adult musculature has been controversial. In this study, a series of Ubx regulatory mutations, anterobithorax, bithorax, postbithorax, and bithoraxoid, as well as combinations of these alleles were used to reevaluate the role of Ubx in the patterning of the T3 musculature. Homeotic indirect and direct flight muscles (IFMs and DFMs) were identified in the transformed T3 segment of all alleles and allelic combinations with the exception of postbithorax. We critically evaluated the pattern and amount of these muscles and found that while the amount and/or quality of homeotic IFMs increased, the amount of homeotic DFMs did not vary significantly as the severity of the ectodermal transformation increased. Because Ubx is not expressed in the adult mesoderm of T3, these results suggest that inductive cues play a major role in the patterning of adult thoracic muscles. We provide a model that illustrates the central role of inductive cues in generating the final adult muscle pattern in the thorax.