Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.

Neurotransmitter receptor recruitment at postsynaptic specializations is key in synaptogenesis, since this step confers functionality to the nascent synapse. The Drosophila neuromuscular junction (NMJ) is a glutamatergic synapse, similar in composition and function to mammalian central synapses. Various mechanisms regulating the extent of postsynaptic ionotropic glutamate receptor (iGluR) clustering have been described, but none are known to be essential for the initial localization and clustering of iGluRs at postsynaptic densities (PSDs). We identified and characterized the Drosophila neto (neuropilin and tolloid-like) as an essential gene required for clustering of iGluRs at the NMJ. Neto colocalizes with the iGluRs at the PSDs in puncta juxtaposing the active zones. neto loss-of-function phenotypes parallel the loss-of-function defects described for iGluRs. The defects in neto mutants are effectively rescued by muscle-specific expression of neto transgenes. Neto clustering at the Drosophila NMJ coincides with and is dependent on iGluRs. Our studies reveal that Drosophila Neto is a novel, essential component of the iGluR complexes and is required for iGluR clustering, organization of PSDs, and synapse functionality.

Motor neuron diseases (MNDs) are progressive neurodegenerative disorders characterized by selective death of motor neurons leading to spasticity, muscle wasting and paralysis. Human VAMP-associated protein B (hVAPB) is the causative gene of a clinically diverse group of MNDs including amyotrophic lateral sclerosis (ALS), atypical ALS and late-onset spinal muscular atrophy. The pathogenic mutation is inherited in a dominant manner. Drosophila VAMP-associated protein of 33 kDa A (DVAP-33A) is the structural homologue of hVAPB and regulates synaptic remodeling by affecting the size and number of boutons at neuromuscular junctions. Associated with these structural alterations are compensatory changes in the physiology and ultrastructure of synapses, which maintain evoked responses within normal boundaries. DVAP-33A and hVAPB are functionally interchangeable and transgenic expression of mutant DVAP-33A in neurons recapitulates major hallmarks of the human diseases including locomotion defects, neuronal death and aggregate formation. Aggregate accumulation is accompanied by a depletion of the endogenous protein from its normal localization. These findings pinpoint to a possible role of hVAPB in synaptic homeostasis and emphasize the relevance of our fly model in elucidating the patho-physiology underlying motor neuron degeneration in humans.

General anesthesia remains a mysterious phenomenon, even though a number of compelling target proteins and processes have been proposed [1]. General anesthetics such as isoflurane abolish behavioral responsiveness in all animals, and in the mammalian brain, these diverse compounds probably achieve this in part by targeting endogenous sleep mechanisms [2, 3]. However, most animals sleep [4], and they are therefore likely to have conserved sleep processes. A decade of neurogenetic studies of arousal in Drosophila melanogaster have identified a number of different neurons and brain structures that modulate sleep duration in the fly brain [5-9], but it has remained unclear until recently whether any neurons might form part of a dedicated circuit that actively controls sleep and wake states in the fly brain, as has been proposed for the mammalian brain [10]. We studied general anesthesia in Drosophila by measuring stimulus-induced locomotion under isoflurane gas exposure. Using a syntaxin1A gain-of-function construct, we found that increasing synaptic activity in different Drosophila neurons could produce hypersensitivity or resistance to isoflurane. We uncover a common pathway in the fly brain controlling both sleep duration and isoflurane sensitivity, centered on monoaminergic modulation of sleep-promoting neurons of the fan-shaped body.

Transgenic manipulation of subsets of brain cells is increasingly used for studying behaviors and their underlying neural circuits. In Drosophila, the GAL4-upstream activating sequence (UAS) binary system is powerful for gene manipulation, but GAL4 expression is often too broad for fine mapping of neural circuits. Here, we describe the development of unique molecular genetic tools to restrict GAL4 expression patterns. Building on the GAL4-UAS system, our method adds two components: a collection of enhancertrap recombinase, Flippase (ET-FLP), transgenic lines that provide inheritable, reproducible, and tissue-specific FLP and an FRT-dependent GAL80 "flip-in" construct that converts FLP expression into tissue-specific repression of GAL4 by GAL80. By including a UASencoded fluorescent protein, circuit morphology can be simultaneously marked while the circuit function is assessed using another UAS transgene. In a proof-of-principle analysis, we applied this ET-FLP-induced intersectional GAL80/GAL4 repression (FINGR) method to map the neural circuitry underlying fly wing inflation. The FINGR system is versatile and powerful in combination with the vast collection of GAL4 lines for neural circuit mapping as well as for clonal analysis based on the infusion of the yeast-derived FRT/FLP system of mitotic recombination into Drosophila. The strategies and tactics underlying our FINGR system are also applicable to other genetically amenable organisms in which transgenes including the GAL4, UAS, GAL80, and FLP factors can be applied.U nderstanding the neural substrates underlying behavior remains a major goal in neuroscience. One effective approach is altering behavioral outputs in animals through genetic manipulation of neurons or neural circuits. This approach is exemplified by numerous studies carried out in mice, zebrafish, Caenorhabditis elegans, and Drosophila (1). In Drosophila, extensive studies of learning, memory, circadian rhythm, courtship, wing inflation, and climbing have provided valuable insights into the neural substrates of these behaviors (2-8). These fly activities are easily observed, are amenable to genetic perturbations, and can be quantitatively analyzed to correlate manipulations of circuits with changes in behavior.Manipulating neural structures underlying these behaviors has principally depended on imaginative designs and applications of molecular-genetic entities. The yeast-derived GAL4-upstream activating sequence (UAS) binary transgene expression system laid the foundation for many of these manipulations (reviewed in ref. 9). In this system, GAL4 can be expressed in a tissue-specific manner via either enhancer-traps or gene-specific promoters. Wherever GAL4 is made active, a protein-coding sequence under the control of UAS is also expressed (10, 11) (Fig. 1A). Thousands of tissue-specific GAL4 enhancer-trap lines have been created.Despite the enormous diversity of available tissue-specific lines, GAL4 expression patterns are rarely restrictive enough to map key elements of neural circuitry. Hen...