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.

Hydroxyurea (HU) treatment of on targets in the calyx. Hence, the ablated interneuearly first instar larvae in Drosophila was previously rons were derived from the LNb, whereas the HUshown to ablate a single dividing lateral neuroblast (LNb) in the brain. Early larval HU application to resistant elements originated from neuroblasts which P[GAL4] strains that label specific neuron types enbegin to divide later in larval life. Developmental abled us to identify the origins of the two major classes GAL4 expression patterns suggested that differentiof interneurons in the olfactory system. HU treatment ated RI are present at the larval stage already and resulted in the loss of antennal lobe local interneurons may be retained through metamorphosis. ᭧ 1997 John and of a subset of relay interneurons (RI), elements Wiley & Sons, Inc. J Neurobiol 32: 443-456, 1997 usually projecting to the calyx and the lateral protoc-Keywords: olfactory interneurons; cell lineage; pererebrum (LPR). Other RI were resistant to HU and sisting larval neurons; P[GAL4] enhancer trap lines; still projected to the LPR. However, they formed no hydroxyurea ablation; Drosophila melanogaster collaterals in the calyx region (which was also ab-

Antibodies can be used to identify tissue- and stage-specifically expressed genes. A monoclonal antibody MAB ab49 from a hybridoma library screened for immunohistochemical staining in the adult nervous system of Drosophila melanogaster was found to selectively bind to all neuropil regions and to synaptic boutons of motor neurons. In Western blots of homogenized brains the antibody recognizes two proteins of 32 and 34 kD. Using this antibody we have isolated seven cDNA clones that derive from two polyadenylated mRNA splice variants of a gene located at 79E1-2 on polytene chromosomes. The two mRNAs code for two inferred proteins of 249 and 223 amino acids, respectively, which are identical except for their C-terminals and a central deletion of 21 amino acids in the second protein. Both contain a contiguous string of 11 cysteine residues. In situ hybridization to frozen head sections detects expression of this gene in retina and neuronal perikarya. The 32 and 34 kD brain proteins that presumably are localized predominantly in synaptic terminals of photoreceptors and most if not all neurons may correspond to two variant cysteine-string proteins as they are of similar molecular weight and share an antigenic binding site for MAB ab49.

We have studied the function of the major central olfactory pathway in fruit flies. Key elements of this pathway, the projection neurons (PNs), connect the antennal lobes with the lateral protocerebrum both directly and indirectly, the latter via the mushroom bodies (MBs). Transgenic expression of tetanus toxin in the majority of PNs and few MB neurons leads to defects in odor detection and male courtship. Considering behavioral data from flies lacking MBs, our results argue that the direct PN-to-lateral protocerebrum pathway is necessary and sufficient to process these experienceindependent behaviors. Moreover, the involvement of an olfactory pathway in male courtship suggests a role of volatile attractive female pheromones in Drosophila. Providing structure-function relationships is a central issue in behavioral neuroscience (1, 2). The development of the enhancer trap technique in Drosophila (3, 4) made it possible to manipulate subsets of identified neurons in a noninvasive manner. In particular, the P[GAL4]͞UAS system allows ectopic expression of any gene of interest (4). When expressing tetanus toxin light chain (TeTxLC) (5), synaptic transmission is locally blocked, and the behavioral consequences can be observed (6-9).We investigate the function of projection neurons (PNs), the major elements of the central olfactory pathway of Drosophila melanogaster ( Fig. 6) (10). Olfactory receptor neurons project to the antennal lobes (ALs), the first-order olfactory brain area. The information is then transmitted by PNs via the antennocerebral tract to two target areas, the mushroom bodies (MBs), involved in experience-dependent olfactory processing (2, 11), and the lateral protocerebrum (LPR) (12). Because subsets of MB extrinsic neurons project to the LPR (13, 14), the PNs provide the LPR with olfactory information both directly and, indirectly, via the MBs.Flies with an intact direct pathway, lacking the indirect one, are impaired in experience-dependent but not -independent olfactory behavior (15). We have generated a P[GAL4] line, GH146, which enables us to shut down about two-thirds of all PNs (Figs. 1 and 6) (16) through ectopic expression of TeTxLC. Both the direct and the indirect input into the LPR are thus blocked, which allows us to investigate possible effects on experience-independent olfactory processing. We show that odor detection and male courtship are severely impaired. We also test gustatory processing to address the question of effect specificity. Furthermore, neurons showing reporter gene expression in the visual system are identified as necessary for movement detection. Materials and MethodsDrosophila Strains. P[GAL4] line GH146 was described in ref. 16. UAS-reporter strains were UAS-lacZ (4), UAS-tau (17), UAStra (18), and the UAS-TeTxLC line TNT-E (called TNT for simplicity) (5). These lines and the wild-type strain Canton-S (CS) were used as controls in behavioral tests.Immunocytochemistry. ␤-Galactosidase and antibody staining of whole mount preparations and sections was done according to...

GAL4-driven targeted expression of tetanus toxin light chain (UAS-TeTxLC) in a subset of chemosensory neurons of the larval antennomaxillary complex (AMC) and pharynx causes abnormal chemosensory behavior in Drosophila melanogaster. Consistent with strongest staining in the dorsal organ (DO), the presumed olfactory organ of the AMC, tetanus toxin-expressing larvae subjected to an olfactory preference assay show anosmic behavior to most volatile substances tested. Furthermore, we observed reduced responses to sodium chloride, fructose, and sucrose in gustatory plate assays. Surprisingly, the entire subset of labeled sensory neurons from the terminal (maxillary) organ (TO) of the AMC was found to project via the antennal nerve to the larval antennal lobe region. The maxillary nerve remained completely unstained. Hence, a subset of neurons from the TO builds an anatomical entity with projections from the DO. Our results suggest that the AMC contains both olfactory and gustatory sensilla, and that the DO is the main olfactory organ in larvae.