No abstract

In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased (ϳ10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

A protein-trap screen using the Drosophila neuromuscular junction (NMJ) as a model synapse was performed to identify genes that control synaptic structure or plasticity. We found that Shaggy (Sgg), the Drosophila homolog of the mammalian glycogen synthase kinases 3 ␣ and ␤, two serine-threonine kinases, was concentrated at this synapse. Using various combinations of mutant alleles of shaggy, we found that Shaggy negatively controlled the NMJ growth. Moreover, tissue-specific expression of a dominant-negative Sgg indicated that this kinase is required in the motoneuron, but not in the muscle, to control NMJ growth. Finally, we show that Sgg controlled the microtubule cytoskeleton dynamics in the motoneuron and that Futsch, a microtubule-associated protein, was required for Shaggy function on synaptic growth.

Identification of the neurotransmitters in clock neurons is critical for understanding the circuitry of the neuronal network that controls the daily behavioral rhythms in Drosophila. Except for the neuropeptide pigment-dispersing factor, no neurotransmitters have been clearly identified in the Drosophila clock neurons. Here we show that glutamate and its metabotropic receptor, DmGluRA, are components of the clock circuitry and modulate the rhythmic behavior pattern of Drosophila. The dorsal clock neurons, DN1s in the larval brain and some DN1s and DN3s in the adult brain, were immunolabeled with antibodies against Drosophila vesicular glutamate transporter (DvGluT), suggesting that they are glutamatergic. Because the DN1s may communicate with the primary pacemaker neurons, s-LN(v)s, we tested glutamate responses of dissociated larval s-LN(v)s by means of calcium imaging. Application of glutamate dose dependently decreased intracellular calcium in the s-LN(v)s. Pharmacology of the response suggests the presence of DmGluRA on the s-LN(v)s. Antibodies against DmGluRA labeled dissociated s-LN(v)s and the LN(v) dendrites in the intact larval and adult brain. The role of metabotropic glutamate signaling was tested in behavior assays in transgenic larvae and flies with altered DmGluRA expression in the LN(v)s and other clock neurons. Larval photophobic behavior was enhanced in DmGluRA mutants. For adults, we could induce altered activity patterns in the dark phase under LD conditions and increase the period during constant darkness by knockdown of DmGluRA expression in LN(v)s. Our results suggest that a glutamate signal from some of the DNs modulates the rhythmic behavior pattern via DmGluRA on the LN(v)s in Drosophila.