We examined tyrosine hydroxylase (TH-GAL4) expression and anti-TH immunoreactivity in the Drosophila protocerebrum and characterized single cell clones of the TH-GAL4 neurons. Eight clusters of putative dopaminergic neurons were characterized. Neurons in three of the clusters project to the mushroom body neuropil: PAM neurons project to the medial portion of the horizontal lobes; PPL1 neurons project to the vertical lobes, the junction area, the heel and distal peduncle; and PPL2ab neurons project to the calyx. Five types of PPL1 neurons were discovered that innervate different zones of the mushroom body lobes. Functional imaging experiments showed that the dopaminergic processes in four of the zones differ in response properties to odor, electric shock, or following the pairing of odor and electric shock. These results indicate that distinct dopaminergic neurons define separate zones of the mushroom body lobes and are probably involved in different functions. Differences in functional response properties of these neurons suggest that they are involved in different behavioral processes.

The olfactory nervous system of insects and mammals exhibits many similarities, which suggests that the mechanisms for olfactory learning may be shared. Molecular genetic investigations of Drosophila learning have uncovered numerous genes whose gene products are essential for olfactory memory formation. Recent studies of the products of these genes have continued to expand the range of molecular processes known to underlie memory formation. Recent research has also broadened the neuroanatomical areas thought to mediate olfactory learning to include the antennal lobes in addition to a previously accepted and central role for the mushroom bodies. The roles for neurons extrinsic to the mushroom body neurons are becoming better defined. Finally, the genes identified to participate in Drosophila olfactory learning have conserved roles in mammalian organisms, highlighting the value of Drosophila for gene discovery.

Four putative adenylyl cyclase genes from Drosophila melanogaster were identified by virtue of their extensive sequence homology with mammalian cyclases. One corresponds to the learning and memory gene rutabaga and is most similar to the mammalian brain Ca2+/calmodulin (CaM)-responsive cyclase. In a mammalian expression system, rutabaga cyclase activity was stimulated approximately 5-fold by the presence of Ca2+/CaM. A point mutation, identified at this locus in rut1 mutant flies, resulted in loss of detectable adenylyl cyclase activity. New P element insertion-induced rutabaga mutations mapped to within 200 nucleotides of the 5' end of the rutabaga cDNA. These data confirm the identity of the rutabaga locus as the structural gene for the Ca2+/CaM-responsive adenylyl cyclase and show that the inactivation of this cyclase leads to a learning and memory defect.

We have developed a method for turning on and off the expression of transgenes within Drosophila in both time and space. Two different enhancer detector elements carrying an RU486-inducible form of the yeast transcription factor GAL4 were constructed and used to generate enhancer detector lines. These lines were screened for RU486-inducible reporter gene expression in the adult head. We identified lines that exhibit inducible expression in many cell and tissue types, verifying that the elements respond to nearby enhancers. No expression was detected in the absence of the ligand. The P{Switch1} element responded to genomic enhancers less efficiently than P{Switch2} but produced more specific patterns of expression. Two P{Switch} lines were used to ablate fat body tissue in adult females through the induced expression of diphtheria toxin. These females were sterile, which correlates with fat body loss, and they died prematurely.

The mushroom bodies of the Drosophila brain are important for olfactory learning and memory. To investigate the requirement for mushroom body signaling during the different phases of memory processing, we transiently inactivated neurotransmission through this region of the brain by expressing a temperature-sensitive allele of the shibire dynamin guanosine triphosphatase, which is required for synaptic transmission. Inactivation of mushroom body signaling through α/β neurons during different phases of memory processing revealed a requirement for mushroom body signaling during memory retrieval, but not during acquisition or consolidation.