Vertebrate synapsins constitute a family of synaptic proteins that participate in the regulation of neurotransmitter release. Information on the presence of synapsin homologs in invertebrates has been inconclusive. We have now cloned a Drosophila gene coding for at least two inferred proteins that both contain a region with 50% amino acid identity to the highly conserved vesicle- and actin-binding "C" domain of vertebrate synapsins. Within the C domain coding sequence, the positions of two introns have been conserved exactly from fly to human. The positions of three additional introns within this domain are similar. The Drosophila synapsin gene (Syn) is widely expressed in the nervous system of the fly. The gene products are detected in all or nearly all conventional synaptic terminals. A single amber (UAG) stop codon terminates the open reading frame (ORF1) of the most abundant transcript of the Syn gene 140 amino acid codons downstream of the homology domain. Unexpectedly, the stop codon is followed by another 443 in-frame amino acid codons (ORF2). Using different antibodies directed against ORF1 or ORF2, we demonstrate that in the adult fly small and large synapsin isoforms are generated. The small isoforms are only recognized by antibodies against ORF1; the large isoforms bind both kinds of antibodies. We suggest that the large synapsin isoform in Drosophila may be generated by UAG read-through. Implications of such an unconventional mechanism for the generation of protein diversity from a single gene are discussed.

Vertebrate synapsins are abundant synaptic vesicle phosphoproteins that have been proposed to fine-regulate neurotransmitter release by phosphorylation-dependent control of synaptic vesicle motility. However, the consequences of a total lack of all synapsin isoforms due to a knock-out of all three mouse synapsin genes have not yet been investigated. In Drosophila a single synapsin gene encodes several isoforms and is expressed in most synaptic terminals. Thus the targeted deletion of the synapsin gene of Drosophila eliminates the possibility of functional knock-out complementation by other isoforms. Unexpectedly, synapsin null mutant flies show no obvious defects in brain morphology, and no striking qualitative changes in behaviour are observed. Ultrastructural analysis of an identified 'model' synapse of the larval nerve muscle preparation revealed no difference between wild-type and mutant, and spontaneous or evoked excitatory junction potentials at this synapse were normal up to a stimulus frequency of 5 Hz. However, when several behavioural responses were analysed quantitatively, specific differences between mutant and wild-type flies are noted. Adult locomotor activity, optomotor responses at high pattern velocities, wing beat frequency, and visual pattern preference are modified. Synapsin mutant flies show faster habituation of an olfactory jump response, enhanced ethanol tolerance, and significant defects in learning and memory as measured using three different paradigms. Larval behavioural defects are described in a separate paper. We conclude that Drosophila synapsins play a significant role in nervous system function, which is subtle at the cellular level but manifests itself in complex behaviour.

The "cysteine string protein" (CSP) genes of higher eukaryotes code for a novel family of proteins characterized by a "J" domain and an unusual cysteine-rich region. Previous studies had localized the proteins in neuropil and synaptic terminals of larval and adult Drosophila and linked the temperature-sensitive paralysis of the mutants described here to conditional failure of synaptic transmission. We now use the null mutants as negative controls in order to reliably detect even low concentrations of CSPs by immunohistochemistry, employing three monoclonal antibodies. In wild-type flies high levels of cysteine string proteins are found not only in apparently all synaptic terminals of the embryonic, larval, and adult nervous systems, but also in the "tall cells" of the cardia, in the follicle cells of the ovary, in specific structures of the female spermatheca, and in the male testis and ejaculatory bulb. In addition, low levels of CSPs appear to be present in all tissues examined, including neuronal perikarya, axons, muscles, Malpighian tubules, and salivary glands. Western blots of isolated tissues demonstrate that of the four isoforms expressed in heads only the largest is found in non-neural organs. The wide expression of CSPs suggests that at least some of the various phenotypes of the null mutants observed at permissive temperatures, such as delayed development, short adult lifespan, modified electroretinogram, and optomotor behavior, may be caused by the lack of CSPs outside synaptic terminals.

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The synapse-associated protein of 47 kDa (SAP47) is a member of a phylogenetically conserved gene family of hitherto unknown function. In Drosophila, SAP47 is encoded by a single gene (Sap47) and is expressed throughout all synaptic regions of the wild-type larval brain; specifically, electron microscopy reveals anti-SAP47 immunogold labeling within 30 nm of presynaptic vesicles. To analyze SAP47 function, we used the viable and fertile deletion mutant Sap47 156 , which suffers from a 1.7 kb deletion in the regulatory region and the first exon. SAP47 cannot be detected by either immunoblotting or immunohistochemistry in Sap47 156 mutants. These mutants exhibit normal sensory detection of odorants and tastants as well as normal motor performance and basic neurotransmission at the neuromuscular junction. However, short-term plasticity at this synapse is distorted. Interestingly, Sap47 156 mutant larvae also show a 50% reduction in odorant-tastant associative learning ability; a similar associative impairment is observed in a second deletion allele (Sap47 201 ) and upon reduction of SAP47 levels using RNA interference. In turn, transgenically restoring SAP47 in Sap47 156 mutant larvae rescues the defect in associative function. This report thus is the first to suggest a function for SAP47. It specifically argues that SAP47 is required for proper behavioral and synaptic plasticity in flies-and prompts the question whether its homologs are required for proper behavioral and synaptic plasticity in other species as well.