Immunofluorescence and image analysis pipeline for Drosophila motor neurons

The neuromuscular junction (NMJ) of larval Drosophila is widely used as a genetic model for basic neuroscience research. The presynaptic side of the NMJ is formed by axon terminals of motor neurons, the soma of which reside in the ventral ganglion of the central nervous system (CNS). Here we describe a streamlined protocol for dissection and immunostaining of the Drosophila CNS and NMJ that allows processing of multiple genotypes within a single staining tube. We also present a computer script called Automated Image Analysis with Background Subtraction which facilitates identification of motor nuclei, quantification of pixel intensity, and background subtraction. Together, these techniques provide a pipeline for neuroscientists to compare levels of different biomolecules in motor nuclei. We conclude that these methods should be adaptable to a variety of different cell and tissue types for the improvement of efficiency, reproducibility, and throughput during data quantification.

Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca2+ signalling in glutamatergic interneurons

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. Here, we report a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. We find that nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, we show that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. We further show that IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.

Electrophysiological and Morphological Characterization of Identified Motor Neurons in the Drosophila Third Instar Larva Central Nervous System

We have used dye fills and electrophysiological recordings to identify and characterize a cluster of motor neurons in the third instar larval ventral ganglion. This cluster of neurons is similar in position to the well-studied embryonic RP neurons. Dye fills of larval dorsomedial neurons demonstrate that individual neurons within the cluster can be reproducibly identified by observing their muscle targets and bouton morphology. The terminal targets of these five neurons are body wall muscles 6/7, 1, 14, and 30 and the intersegmental nerve (ISN) terminal muscles (1, 2, 3, 4, 9, 10, 19, 20). All cells except the ISN neuron, which has a type Is ending, display type Ib boutons. Two of these neurons appear to be identical to the embryonic RP3 and aCC cells, which define the most proximal and distal innervations within a hemisegment. The targets of the other neurons in the larval dorsomedial cluster do not correspond to embryonic targets of the neurons in the RP cluster, suggesting rewiring of this circuit during early larval stages. Electrophysiological studies of the five neurons in current clamp revealed that type Is neurons have a longer delay in the appearance of the first spike compared with type Ib neurons. Genetic, biophysical, and pharmacological studies in current and voltage clamp show this delay is controlled by the kinetics and voltage sensitivity of inactivation of a current whose properties suggest that it may be the Shal IA current. The combination of genetic identification and whole cell recording allows us to directly explore the cellular substrates of neural and locomotor behavior in an intact system.

Histochemical mapping of NADPH diaphorase in the nervous system of the parasitic nematode Ascaris suum

SUMMARYNADPH diaphorase has recently been discovered to be responsible for neuronal nitric oxide (NO) synthase activity in mammals. It thus serves as a histochemical marker for the localization of NO synthase in the nervous system. The histochemical technique was used to map out potential NO-producing neurones in the nervous system of the parasitic nematode, Ascaris suum. Positive staining for NADPH diaphorase was present in various parts of the central nervous system, in particular within selective cell bodies and fibres in the ventral ganglion, the retrovesicular ganglion, ventral and dorsal cords and sublateral lines. Intense staining was also present in the motorneurone commissures, indicating a potential role for NO as a neurotransmitter at the neuromuscular junction. NADPH disphorase-positive neurones were not confined to the central nervous system. Selective staining was also present in the enteric nervous system, in particular the pharynx and in the peripheral nervous system innervating the sensory organs.

Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system

Mutations in the complex gene locus optomotor-blind (omb) can lead to defects in the development of both the optic lobes and external features of the adult fly. We describe here the expression of omb in the developing and adult nervous system using in situ hybridization. During embryogenesis, omb expression is first observed in the optic lobe anlagen. It later expands to a larger part of the developing larval brain and to the gnathal lobes. Cells in the ventral and peripheral nervous systems begin to express omb after completion of germ band extension. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres. During the larval and pupal stages, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. At these stages, only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk. In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes. In the mutation In(1)ombH31, 40 kb of regulatory DNA, downstream from the transcription unit, are removed from the omb gene. In(1)ombH31 is characterized by the lack of a set of giant interneurons from the lobula plate of the adult optic lobes. We find that, already during embryogenesis, there is a drastic difference between wild type and In(1)ombH31 in the level of the omb transcript in the optic lobe primordia. The adult mutant phenotype may thus be caused by omb misexpression during embryonic development.