Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth.

Drosophila Frequenin (Frq) and its mammalian and worm homologue, NCS-1, are Ca(2+)-binding proteins involved in neurotransmission. Using site-specific recombination in Drosophila, we created two deletions that removed the entire frq1 gene and part of the frq2 gene, resulting in no detectable Frq protein. Frq-null mutants were viable, but had defects in larval locomotion, deficient synaptic transmission, impaired Ca(2+) entry and enhanced nerve-terminal growth. The impaired Ca(2+) entry was sufficient to account for reduced neurotransmitter release. We hypothesized that Frq either modulates Ca(2+) channels, or that it regulates the PI4Kbeta pathway as described in other organisms. To determine whether Frq interacts with PI4Kbeta with consequent effects on Ca(2+) channels, we first characterized a PI4Kbeta-null mutant and found that PI4Kbeta was dispensable for synaptic transmission and nerve-terminal growth. Frq gain-of-function phenotypes remained present in a PI4Kbeta-null background. We conclude that the effects of Frq are not due to an interaction with PI4Kbeta. Using flies that were trans-heterozygous for a null frq allele and a null cacophony (encoding the alpha(1)-subunit of voltage-gated Ca(2+) channels) allele, we show a synergistic effect between these proteins in neurotransmitter release. Gain-of-function Frq phenotypes were rescued by a hypomorphic cacophony mutation. Overall, Frq modulates Ca(2+) entry through a functional interaction with the alpha(1) voltage-gated Ca(2+)-channel subunit; this interaction regulates neurotransmission and nerve-terminal growth.

Modular neuropile organization in the Drosophila larval brain facilitates identification and mapping of central neurons.

Elucidating how neuronal networks process information requires identification of critical individual neurons and their connectivity patterns. For this purpose, we used the third-instar Drosophila larval brain and applied reverse-genetic tools, immunolabeling procedures, and 3D digital reconstruction software. Consistent topological definition of neuropile compartments in the larval brain can be obtained through simple fluorescence-immunolabeling methods. The modular neuropiles can be used as a fiducial framework for mapping the projection patterns of individual neurons labeled with green fluorescent protein (GFP). GFP-labeled neurons often exhibit dendrite-like arbors as well as clustered varicose terminals on neurite branches that innervate identifiable neuropile compartments. We identified candidate cholinergic interneurons in genetic mosaic brains that overlap with the larval optic nerve terminus. By using the neuropile framework, we demonstrate that the candidate visual interneurons are not a subset of the previously identified circadian pacemaker neurons that also contact the larval optic nerve terminus; they may represent parallel pathways in the processing of visual inputs. Thus, in the Drosophila larval brain, modular neuropiles can be used as a framework for systematically identifying, mapping, and classifying interneurons; understanding their roles in behavior can then be pursued further.

Microfluidic devices for imaging neurological response of Drosophila melanogaster larva to auditory stimulus.

Two microfluidic devices (pneumatic chip and FlexiChip) have been developed for immobilization and live-intact fluorescence functional imaging of Drosophila larva's Central Nervous System (CNS) in response to controlled acoustic stimulation. The pneumatic chip is suited for automated loading/unloading and potentially allows high throughput operation for studies with a large number of larvae while the FlexiChip provides a simple and quick manual option for animal loading and is suited for smaller studies. Both chips were capable of significantly reducing the endogenous CNS movement while still allowing the study of sound-stimulated CNS activities of Drosophila 3rd instar larvae using genetically encoded calcium indicator GCaMP5. Temporal effects of sound frequency (50-5000 Hz) and intensity (95-115 dB) on CNS activities were investigated and a peak neuronal response of 200 Hz was identified. Our lab-on-chip devices can not only aid further studies of Drosophila larva's auditory responses but can be also adopted for functional imaging of CNS activities in response to other sensory cues. Auditory stimuli and the corresponding response of the CNS can potentially be used as a tool to study the effect of chemicals on the neurophysiology of this model organism.