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A panel of 148 monoclonal antibodies directed against Drosophila neural antigens has been prepared by using mice immunized with homogenates ofDrosophila tissue. Antibodies were screened immunohistochemically on cryostat sections of fly heads. A large diversity of staining patterns was observed. Some antigens were broadly distributed among tissues; others were highly specific to nerve fibers, neuropil, muscle, the tracheal system, cell nuclei, photoreceptors, or other structures. The antigens for many of the antibodies have been identified on immunoblots. Monoclonal antibodies that identify specific molecules within the nervous system should prove useful in the study of the molecular genetics of neural development.The development and function of the nervous system involves the participation of various genetically encoded macromolecules whose nature and identities are largely unknown. An incisive approach toward identifying them is offered by the hybridoma technique (1), which can produce monoclonal antibodies (MAbs) against specific molecules singled out from a complex mixture of immunogens. For example, by immunization with leech segmental ganglia, Zipser and McKay (2) found MAbs that recognized antigens expressed in different subsets of neurons. Trisler et al. (3) used immunization with chick retinal segments and identified a molecule that occurs in a gradient across the developing retina.As a step toward elucidating, at the molecular level, the relationship between the genome and the nervous system, we have produced a battery of MAbs showing specificity for the Drosophila nervous system and related tissues. Drosophila offers the advantage that both classical genetic and recombinant DNA techniques can be used to identify the gene that encodes the antigen. Mutations in the gene that alter or delete the antigen can provide insight into its role in development, physiology, and behavior.MATERIALS AND METHODS Generation of MAbs. Hybridomas were obtained from five fusions. Immunogens were homogenates of heads, brains, or retinas dissected from Drosophila melanogaster (C-S strain) adult flies previously frozen at -90°C and dehydrated at -200C in acetone. Each BALB/c mouse received, over a period of 4-20 months, five injections (four intraperitoneal and one intravenous). Each injection contained material dissected from 20-50 flies. Standard procedures using NS-1 myeloma cells (4) were followed to generate hybridomas, which were cloned by limiting dilution. The MAbs described here are IgGs, except 3F12 which is IgM and 3H6 and 4E9 for which the class is undetermined.Immunohistochemistry. Cryostat sections (2-10 ,um) of fly heads were prepared and stored on coverslips at -20'C over silica gel. To eliminate eye pigments, which can cause fluorescent background, cinnabar brown mutant flies were used. Staining was done at room temperature. The sections were fixed with 2% formalin in 75 mM Na phosphate buffer (pH 7.0) for 30 min and then rinsed for 5 min in 10 mM Tris HCl, pH 7.5/ 130 mM NaCl/5 mM KCI/5 mM NaN3/1 mM...

The Shaker locus of Drosophila contains a very large transcription unit. It is expressed predominantly in the nervous system by multiple, differential as well as alternative, splicing mechanisms into different, but functionally related proteins. The structure of the Shaker transcription unit and the properties of the encoded Shaker protein family provide a molecular basis for A channel diversity in excitable cells.

In an attempt to correlate behavioral and neuronal changes, we examined the structural and functional effects of odor exposure in Drosophila. Young adult flies were exposed to a high concentration of the selected odor, usually benzaldehyde or isoamyl acetate, for 4 d and subsequently tested for their olfactory response to a variety of odorants and concentrations. The behavioral response showed specific adaptation to the exposed odor. By contrast, olfactory transduction, as measured in electroantennograms, remained normal. In vivo volume measurements were performed on olfactory glomeruli, the anatomical and functional units involved in odor processing. Preexposed flies exhibited volume reduction of certain glomeruli, in an odor-selective manner. Of a sample of eight glomeruli measured, dorsal medial (DM) 2 and ventral (V) were affected by benzaldehyde exposure, whereas DM6 was affected by isoamyl acetate. Estimation of the number of synapses indicates that volume reduction involves synapse loss that can reach 30% in the V glomerulus of flies adapted to benzaldehyde. Additional features of odorant-induced adaptation, including concentration dependence and perdurance, also show correlation, because both effects are elicited by high odor concentrations and are long-lasting (Ͼ1 week). Finally, the dunce mutant fails to develop behavioral adaptation as well as morphological changes in the olfactory glomeruli after exposure. These neural changes thus appear to require the cAMP signaling pathway. Key words: adaptation; olfactory glomeruli; memory; synapse number; dunce; DrosophilaA striking property of nervous systems is their ability to adapt structural and functional features to the input they receive during lifetime. Behavioral changes, including learning and memory, correlate with modulation of neuronal activity that can eventually lead to changes in gene expression and synapse number (Bailey and Kandel, 1993;Martin and Kandel, 1996;Milner et al., 1998;Corriveau, 1999;Yuste and Sur, 1999). Experience-related changes are usually detected in response to complex environments, however, making it difficult to establish a direct correlation between synapse modification and storage of specific information (for review, see Moser, 1999). The brain of the insect imago undergoes experience-dependent modifications (Bulloch and Ridgway, 1989). Integration centers such as the mushroom bodies (Heisenberg, 1998) and the central complex (Davis, 1996;Strausfeld, 1999) have been well studied in this respect (Brandon and Coss, 1982;Withers et al., 1993;Durst et al., 1994;Gronenberg et al., 1996;Barth and Heisenberg, 1997;Fahrbach et al., 1998;Barth, 1999). Additional brain structures also undergo behaviordependent changes, in particular the antennal lobes (AL), the insect olfactory centers (Masson and Mustaparta, 1990). In the honeybee, shifting to new behavioral tasks in the hive is accompanied by specific AL structural changes, albeit the triggering stimulus remains unknown (Winnington et al., 1996;Sigg et al., 1997).The relevance of...

The possibility of changing the number of synapses may be an important asset in the treatment of neurological diseases. In this context, the synaptogenic role of the phosphoinositide-3-kinase (PI3K) signaling cascade has been previously demonstrated in Drosophila. This study shows that treatment with a PI3K-activating transduction peptide is able to promote synaptogenesis and spinogenesis in primary cultures of rat hippocampal neurons, as well as in CA1 hippocampal neurons in vivo. In culture, the peptide increases synapse density independently of cell density, culture age, dendritic complexity, or synapse type. The induced synapses also increase neurotransmitter release from cultured neurons. The synaptogenic signaling pathway includes PI3K-Akt. Furthermore, the treatment is effective on adult neurons, where it induces spinogenesis and enhances the cognitive behavior of treated animals in a fear-conditioning assay. These findings demonstrate that functional synaptogenesis can be induced in mature mammalian brains through PI3K activation.

Synapses are specialized communication points between neurons, and their number is a major determinant of cognitive abilities. These dynamic structures undergo developmental-and activity-dependent changes. During brain aging and certain diseases, synapses are gradually lost, causing mental decline. It is, thus, critical to identify the molecular mechanisms controlling synapse number. We show here that the levels of phosphoinositide 3 kinase (PI3K) regulate synapse number in both Drosophila larval motor neurons and adult brain projection neurons. The supernumerary synapses induced by PI3K overexpression are functional and elicit changes in behavior. Remarkably, PI3K activation induces synaptogenesis in aged adult neurons as well. We demonstrate that persistent PI3K activity is necessary for synapse maintenance. We also report that PI3K controls the expression and localization of synaptic markers in human neuroblastoma cells, suggesting that PI3K synaptogenic activity is conserved in humans. Thus, we propose that PI3K stimulation can be applied to prevent or delay synapse loss in normal aging and in neurological disorders.

Genes of the RBR family are characterized by the RBR signature (two RING finger domains separated by an IBR/DRIL domain). The RBR family is widespread in eukaryotes, with numerous members in animals (mammals, Drosophila, Caenorhabditis) and plants (Arabidopsis). But yeasts, such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, contain only two RBR genes. We determined the phylogenetic relationships and the most likely orthologs in different species of several family members for which functional data are available. These include: (1) parkin, whose mutations are involved in forms of familial Parkinson's disease; (2) the ariadne genes, recently characterized in Drosophila and mammals; (3) XYbp and Dorfin, two mammalian genes whose products interact with the centrosome; (4) XAP3, RBCK1, and UIP28, mammalian genes encoding Protein Kinase-C-binding proteins; and (5) ARA54, an androgen receptor coactivator. Because several of these genes are involved in ubiquitination, we used phylogenetic and structural analyses to explore the hypothesis that all RBR proteins might play a role in ubiquitination. We show that the involvement of RBR proteins in ubiquitination predates the animals-plants-fungi divergence. On the basis of the evidence provided by cases of gene fusion, we suggest that Ariadne proteins interact with cullin domain-containing proteins to form complexes with ubiquitin-ligase activity.

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