Puckered and JNK signaling in pioneer neurons coordinates the motor activity of the Drosophila embryo.

Central nervous system organogenesis is a complex process that obeys precise architectural rules. The impact that nervous system architecture may have on its functionality remains, however, relatively unexplored. To clarify this problem, we analyze the development of the Drosophila embryonic Ventral Nerve Cord (VNC). VNC morphogenesis requires the tight control of Jun kinase (JNK) signaling in a subset of pioneer neurons, exerted in part via a negative feedback loop mediated by the dual specificity phosphatase Puckered. Here we show that the JNK pathway autonomously regulates neuronal electrophysiological properties without affecting synaptic vesicle transport. Manipulating JNK signaling activity in pioneer neurons during early embryogenesis directly influences their function as organizers of VNC architecture and, moreover, uncovers a role in the coordination of the embryonic motor circuitry that is required for hatching. Together, our data reveal critical links, mediated by the control of the JNK signaling cascade by Puckered, between the structural organization of the VNC and its functional optimization.

A Poly-Glutamine Region in the Drosophila VAChT Dictates Fill-Level of Cholinergic Synaptic Vesicles.

While the primary role of vesicular transporters is to load neurotransmitters into synaptic vesicles (SVs), accumulating evidence suggests that these proteins also contribute to additional aspects of synaptic function, including vesicle release. In this study, we extend the role of the VAChT to include regulating the transmitter content of SVs. We report that manipulation of a C-terminal poly-glutamine (polyQ) region in the Drosophila VAChT is sufficient to influence transmitter content, and release frequency, of cholinergic vesicles from the terminals of premotor interneurons. Specifically, we find that reduction of the polyQ region, by one glutamine residue (13Q to 12Q), results in a significant increase in both amplitude and frequency of spontaneous cholinergic miniature EPSCs (mEPSCs) recorded in the aCC and RP2 motoneurons. Moreover, this truncation also results in evoked synaptic currents that show increased duration: consistent with increased ACh release. By contrast, extension of the polyQ region by one glutamine (13Q to 14Q) is sufficient to reduce mEPSC amplitude and frequency and, moreover, prevents evoked SV release. Finally, a complete deletion of the polyQ region (13Q to 0Q) has no obvious effects to mEPSCs, but again evoked synaptic currents show increased duration. The mechanisms that ensure SVs are filled to physiologically-appropriate levels remain unknown. Our study identifies the polyQ region of the insect VAChT to be required for correct vesicle transmitter loading and, thus, provides opportunity to increase understanding of this critical aspect of neurotransmission.

Split-QF System for Fine-Tuned Transgene Expression in Drosophila.

The Q-system is a binary expression system that works well across species. Here, we report the development and demonstrate the applications of a split-QF system that drives strong expression in Drosophila, is repressible by QS, and is inducible by a small nontoxic molecule (quinic acid). The split-QF system is fully compatible with existing split-GAL4 and split-LexA lines, thus greatly expanding the range of possible advanced intersectional experiments and anatomical, physiological, and behavioral assays in Drosophila, and in other organisms.

The VAChTY49N mutation provides insecticide-resistance but perturbs evoked cholinergic neurotransmission in Drosophila.

Global agriculture and the control of insect disease vectors have developed with a heavy reliance on insecticides. The increasing incidence of resistance, for virtually all insecticides, threatens both food supply and effective control of insect borne disease. CASPP ((5-chloro-1'-[(E)-3-(4-chlorophenyl)allyl]spiro[indoline-3,4'-piperidine]-1-yl}-(2-chloro-4-pyridyl)methanone)) compounds are a potential new class of neuroactive insecticide specifically targeting the Vesicular Acetylcholine Transporter (VAChT). Resistance to CASPP, under laboratory conditions, has been reported following either up-regulation of wildtype VAChT expression or the presence of a specific point mutation (VAChTY49N). However, the underlying mechanism of CASPP-resistance, together with the consequence to insect viability of achieving resistance, is unknown. In this study, we use electrophysiological characterisation of cholinergic release at Drosophila larval interneuron→motoneuron synapses to investigate the physiological implications of these two identified modes of CASPP resistance. We show that both VAChT up-regulation or the expression of VAChTY49N increases miniature (mini) release frequency. Mini frequency appears deterministic of CASPP activity. However, maintenance of SV release is not indicative of resistance in all cases. This is evidenced through expression of syntaxin or complexin mutants (sytx3-61/cpxSH1) that show similarly high mini release frequency but are not resistant to CASPP. The VAChTY49N mutation additionally disrupts action potential-evoked cholinergic release and fictive locomotor patterning through depletion of releasable synaptic vesicles. This observation suggests a functional trade-off for this point mutation, which is not seen when wildtype VAChT is up-regulated.

Central cholinergic synaptic vesicle loading obeys the set-point model in Drosophila.

Experimental evidence shows that neurotransmitter release, from presynaptic terminals, can be regulated by altering transmitter load per synaptic vesicle (SV) and/or through change in the probability of vesicle release. The vesicular acetylcholine transporter (VAChT) loads acetylcholine into SVs at cholinergic synapses. We investigated how the VAChT affects SV content and release frequency at central synapses in Drosophila melanogaster by using an insecticidal compound, 5Cl-CASPP, to block VAChT and by transgenic overexpression of VAChT in cholinergic interneurons. Decreasing VAChT activity produces a decrease in spontaneous SV release with no change to quantal size and no decrease in the number of vesicles at the active zone. This suggests that many vesicles are lacking in neurotransmitter. Overexpression of VAChT leads to increased frequency of SV release, but again with no change in quantal size or vesicle number. This indicates that loading of central cholinergic SVs obeys the "set-point" model, rather than the "steady-state" model that better describes loading at the vertebrate neuromuscular junction. However, we show that expression of a VAChT polymorphism lacking one glutamine residue in a COOH-terminal polyQ domain leads to increased spontaneous SV release and increased quantal size. This effect spotlights the poly-glutamine domain as potentially being important for sensing the level of neurotransmitter in cholinergic SVs.