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Identifying the cellular origins and mapping the dendritic and axonal arbors of neurons have been century old quests to understand the heterogeneity among these brain cells. Current Brainbow based transgenic animals take the advantage of multispectral labeling to differentiate neighboring cells or lineages, however, their applications are limited by the color capacity. To improve the analysis throughput, we designed Bitbow, a digital format of Brainbow which exponentially expands the color palette to provide tens of thousands of spectrally resolved unique labels. We generated transgenic Bitbow Drosophila lines, established statistical tools, and streamlined sample preparation, image processing, and data analysis pipelines to conveniently mapping neural lineages, studying neuronal morphology and revealing neural network patterns with unprecedented speed, scale, and resolution.

During the embryonic development of Drosophila melanogaster many transcriptional activators are involved in the formation of the embryonic brain. In our study we show that the transcription factor Homeobrain (Hbn), a member of the 57B homeobox gene cluster, is an additional factor involved in the formation of the embryonic Drosophila brain. Using a Hbn antibody and specific cell type markers a detailed expression analysis during embryonic brain development was conducted. We show that Hbn is expressed in several regions in the protocerebrum, including fibre tract founder cells closely associated with the supraesophageal brain commissure and also in the mushroom bodies. During the formation of the supraesophageal commissure, Hbn and FasII-positive founder cells build an interhemispheric bridge priming the commissure and thereby linking both brain hemispheres. The Hbn expression is restricted to neural but not glial cells in the embryonic brain. In a mutagenesis screen we generated two mutant hbn alleles that both show embryonic lethality. The phenotype of the hbn mutant alleles is characterized by a reduction of the protocerebrum, a loss of the supraesophageal commissure and mushroom body progenitors and also by a dislocation of the optic lobes. Extensive apoptosis correlates with the impaired formation of the embryonic protocerebrum and the supraesophageal commissure. Our results show that Hbn is another important factor for embryonic brain development in Drosophila melanogaster.

International audience; Mitochondrial defects associated with respiratory chain complex I deficiency lead to heterogeneous fatal syndromes. While the role of NDUFS8, an essential subunit of the core assembly of the complex I, is established in mitochondrial diseases, the mechanisms underlying neuropathology are poorly understood. We developed a Drosophila model of NDUFS8 deficiency by knocking down the expression of its fly homologue in neurons or in glial cells. Downregulating ND23 in neurons resulted in shortened lifespan, and decreased locomotion. Although total brain ATP levels were decreased, histological analysis did not reveal any signs of neurodegeneration except for photoreceptors of the retina. Interestingly, ND23 deficiency-associated phenotypes were rescued by overexpressing the glucose transporter hGluT3 demonstrating that boosting glucose metabolism in neurons was sufficient to bypass altered mitochondrial functions and to confer neuroprotection. We then analyzed the consequences of ND23 knockdown in glial cells. In contrast to neuronal knockdown, loss of ND23 in glia did not lead to significant behavioral defects nor to reduced lifespan, but induced brain degeneration, as visualized by numerous vacuoles found all over the nervous tissue. This phenotype was accompanied by the massive accumulation of lipid droplets at the cortex-neuropile boundaries, suggesting an alteration of lipid metabolism in glia. These results demonstrate that complex I deficiency triggers metabolic alterations both in neurons and glial cells which may contribute to the neuropathology.

To understand the brain, molecular details need to be overlaid onto neural wiring diagrams so that synaptic mode, neuromodulation and critical signaling operations can be considered. Single-cell transcriptomics provide a unique opportunity to collect this information. Here we present an initial analysis of thousands of individual cells from Drosophila midbrain, that were acquired using Drop-Seq. A number of approaches permitted the assignment of transcriptional profiles to several major brain regions and cell-types. Expression of biosynthetic enzymes and reuptake mechanisms allows all the neurons to be typed according to the neurotransmitter or neuromodulator that they produce and presumably release. Some neuropeptides are preferentially co-expressed in neurons using a particular fast-acting transmitter, or monoamine. Neuromodulatory and neurotransmitter receptor subunit expression illustrates the potential of these molecules in generating complexity in neural circuit function. This cell atlas dataset provides an important resource to link molecular operations to brain regions and complex neural processes.

Contains fulltext : 156933.pdf (Publisher’s version ) (Open Access) Drosophila represents a key model organism for dissecting neuronal circuits that underlie innate and adaptive behavior. However, this task is limited by a lack of tools to monitor physiological parameters of spatially distributed, central synapses in identified neurons. We generated transgenic fly strains that express functional fluorescent reporters targeted to either pre- or postsynaptic compartments. Presynaptic Ca(2+) dynamics are monitored using synaptophysin-coupled GCaMP3, synaptic transmission is monitored using red fluorescent synaptophysin-pHTomato, and postsynaptic Ca(2+) dynamics are visualized using GCaMP3 fused with the postsynaptic matrix protein, dHomer. Using two-photon in vivo imaging of olfactory projection neurons, odor-evoked activity across populations of synapses is visualized in the antennal lobe and the mushroom body calyx. Prolonged odor exposure causes odor-specific and differential experience-dependent changes in pre- and postsynaptic activity at both levels of olfactory processing. The approach advances the physiological analysis of synaptic connections across defined groups of neurons in intact Drosophila.

Octopamine is an important neuromodulator in the insect nervous system, influencing memory formation, sensory perception and motor control. In this study, we compare the distribution of octopamine-like immunoreactive neurons in two parasitic wasp species of the Nasonia genus, N. vitripennis and N. giraulti. These two species were previously described as differing in their learning and memory formation, which raised the question as to whether morphological differences in octopaminergic neurons underpinned these variations. Immunohistochemistry in combination with confocal laser scanning microscopy was used to reveal and compare the somata and major projections of the octopaminergic neurons in these wasps. The brains of both species showed similar staining patterns, with six different neuron clusters being identified in the brain and five different clusters in the subesophageal ganglion. Of those clusters found in the subesophageal ganglion, three contained unpaired neurons, whereas the other three consisted in paired neurons. The overall pattern of octopaminergic neurons in both species was similar, with no differences in the numbers or projections of the ventral unpaired median (VUM) neurons, which are known to be involved in memory formation in insects. In one other cluster in the brain, located in-between the optic lobe and the antennal lobe, we detected more neurons in N. vitripennis compared with N. giraulti. Combining our results with findings made previously in other Hymenopteran species, we discuss possible functions and some of the ultimate factors influencing the evolution of the octopaminergic system in the insect brain.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown. The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.