The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

The blood-brain barrier (BBB) ensures brain function in vertebrates and insects by maintaining ionic integrity of the neuronal bathing fluid. Without this barrier, paralysis and death ensue. The structural analogs of the BBB are occlusive (pleated-sheet) septate and tight junctions between perineurial cells, glia and perineurial cells, and possibly between glia. Immature Diptera have such septate junctions (without tight junctions) while both junctional types are found in the imago. Genetic and molecular biology of these junctions are discussed, namely tight (occludin) and pleated-sheet septate (neurexin IV). A temporal succession of blood barriers form in immature Diptera. The first barrier forms in the peripheral nervous system where pleated-sheet septate junctions bond cells of the nascent (embryonic) chordotonal organs in early neurogenesis. At the end of embryonic life, the central nervous system is fully vested with a blood-brain barrier. A blood-eye barrier arises in early pupal life. Future prospects in blood-barrier research are discussed.

Six morphologically distinct glial cell layers are described in the housefly lamina ganglionaris, a region previously thought to be composed of only three. 1. The external glial layer abuts the basement membrane of the retina. The cells of this layer have a highly involuted surface membrane and an abundance of ribosomes and rough endoplasmic reticulum (ER) throughout their cytoplasm. They envelop the traversing photoreceptor and mechanoreceptor axons as well as the large tracheoblast cells of the fenestrated layer. They are referred to as the fenestrated layer glia. 2. The second glial layer is composed of large, horizontally elongated cells with large elongate nuclei. They contain large membrane-bounded vacuoles and extensive arrays of parallel-running microtubules and smooth ER. These glia invest the photoreceptor axons through much of the multiple chiasmatic (pseudocartridge) region and are thus designated as the pseudocartridge glia. 3-4. Satellite glia comprise the third and fourth glial layers. Thin cytoplasmic processes of these multipolar glia intervene between the tightly packed monopolar neuron somata and the photoreceptor axons of the nuclear layer. The satellite glia are distinguished into two sub-groups: distal and proximal. The distal satellite glia are exclusively responsible for the large glial invaginations of the type I monopolar cell bodies. Multilaminated processes of the proximal layer of satellite glia surround the photoreceptor axons and the neurite neck of the monopolar neurons prior to their entry into the plexiform layer. The proximal satellite glia also contain prominent lipid deposits. 5. Epithelial glia are columnar cells that occupy the plexiform layer. They envelop the optic cartridges of the neuropil and are the substrate for two characteristic glial-neuronal invaginations; i.e. the capitate projection and the 'gnarl'. The cytoplasm of the epithelial glia is electron dense and contains numerous stacked arrays of infolded membrane. 6. Marginal glia form the proximal boundary of the optic neuropil. They invest the axons entering or leaving through the base of the lamina ganglionaris. Marginal glia contain large numbers of parallel microtubules and numerous polyribosomes. Fine structural evidence is presented relevant to the role of these six glial layers in the maintenance of ionic and metabolic homeostasis across the retina-lamina barrier.