To characterize Ca(2+)-mediated synaptic vesicle fusion, we analyzed Drosophila synaptotagmin I mutants deficient in specific interactions mediated by its two Ca(2+) binding C2 domains. In the absence of synaptotagmin I, synchronous release is abolished and a kinetically distinct delayed asynchronous release pathway is uncovered. Synapses containing only the C2A domain of synaptotagmin partially recover synchronous fusion, but have an abolished Ca(2+) cooperativity. Mutants that disrupt Ca(2+) sensing by the C2B domain have synchronous release with normal Ca(2+) cooperativity, but with reduced release probability. Our data suggest the Ca(2+) cooperativity of neurotransmitter release is likely mediated through synaptotagmin-SNARE interactions, while phospholipid binding and oligomerization trigger rapid fusion with increased release probability. These results indicate that synaptotagmin is the major Ca(2+) sensor for evoked release and functions to trigger synchronous fusion in response to Ca(2+), while suppressing asynchronous release.

No abstract

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein that results in intracellular aggregate formation and neurodegeneration. Pathways leading from polyglutamine tract expansion to disease pathogenesis remain obscure. To elucidate how polyglutamine expansion causes neuronal dysfunction, we generated Drosophila transgenic strains expressing human huntingtin cDNAs encoding pathogenic (Htt-Q128) or nonpathogenic proteins (Htt-Q0). Whereas expression of Htt-Q0 has no discernible effect on behavior, lifespan, or neuronal morphology, pan-neuronal expression of Htt-Q128 leads to progressive loss of motor coordination, decreased lifespan, and time-dependent formation of huntingtin aggregates specifically in the cytoplasm and neurites. Huntingtin aggregates sequester other expanded polyglutamine proteins in the cytoplasm and lead to disruption of axonal transport and accumulation of aggregates at synapses. In contrast, Drosophila expressing an expanded polyglutamine tract alone, or an expanded polyglutamine tract in the context of the spinocerebellar ataxia type 3 protein, display only nuclear aggregates and do not disrupt axonal trafficking. Our findings indicate that nonnuclear events induced by cytoplasmic huntingtin aggregation play a central role in the progressive neurodegeneration observed in Huntington's disease.

SUMMARY Retrograde signals from postsynaptic targets are critical during development and plasticity of synaptic connections. These signals serve to adjust the activity of presynaptic cells according to postsynaptic cell outputs and to maintain synaptic function within a dynamic range. Despite their importance, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are unknown. Here we show that a retrograde signal mediated by Synaptotagmin 4 (Syt4) is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Thus, by transferring an essential component of retrograde signaling through exosomes, presynaptic cells enable retrograde signaling.

Summary Many feeding behaviors represent stereotyped, organized sequences of motor patterns that have been the subject of neuroethological studies1,2 such as electrophysiological characterization of neurons governing prey capture in toads1,3. Technical limitations, however, have prevented detailed study of the functional role of these neurons as in other studies on vertebrate organisms. Complexities involved in studies of whole animal behavior can be resolved in Drosophila, where remote activation of brain cells by genetic means4 allows one to interrogate the nervous system in freely moving animals to identify neurons that govern a specific behavior, and then to repeatedly target and manipulate these neurons to characterize their function. Here we show finding of neurons that generate the feeding motor program in Drosophila. We performed an unbiased screen using remote neuronal activation and identified a critical pair of brain cells that induces the entire feeding sequence when activated. These Fdg (feeding)-neurons are also essential for normal feeding as their suppression or ablation eliminates the sugar-induced feeding behavior. Activation of a single Fdg-neuron induced asymmetric feeding behavior and ablation of a single Fdg-neuron distorted the sugar-induced feeding behavior to be asymmetric, indicating the direct role of these neurons in shaping motor program execution. Simultaneously recording neuronal activity with calcium imaging during feeding behavior5 further revealed that the Fdg-neurons respond to food presentation, but only in starved flies. Our results demonstrate that Fdg-neurons operate firmly within the sensori-motor watershed, downstream of sensory and metabolic cues and at the top of the feeding motor hierarchy to execute the decision to feed.