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The modulatory neurotransmitters that trigger biochemical cascades underlying olfactory learning in Drosophila mushroom bodies have remained unknown. To identify molecules that may perform this role, putative biogenic amine receptors were cloned using the polymerase chain reaction (PCR) and single-strand conformation polymorphism analysis. One new receptor, DAMB, was identified as a dopamine D1 receptor by sequence analysis and pharmacological characterization. In situ hybridization and immunohistochemical analyses revealed highly enriched expression of DAMB in mushroom bodies, in a pattern coincident with the rutabaga-encoded adenylyl cyclase. The spatial coexpression of DAMB and the cyclase, along with DAMB's capacity to mediate dopamine-induced increases in cAMP make this receptor an attractive candidate for initiating biochemical cascades underlying learning.

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Volado is a new memory mutant of Drosophila. The locus encodes two isoforms of a new alpha-integrin, a molecule that dynamically mediates cell adhesion and signal transduction. The Volado gene is expressed preferentially in mushroom body cells, which are neurons known to mediate olfactory learning in insects. Volado proteins are concentrated in the mushroom body neuropil, brain areas that contain mushroom body processes in synaptic contact with other neurons. Volado mutants display impaired olfactory memories within 3 min of training, indicating that the integrin is required for short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. Thus the Volado integrin is essential for the physiological processes underlying memory. We propose a model in which integrins act as dynamic regulators of synapse structure or the signalling events underlying short-term memory formation.

Although the Drosophila melanogaster (fly) model is a popular platform for investigating diet-related phenomena, it can be challenging to measure the volume of agar-based food media flies consume. We addressed this challenge by developing a dye-based method called Consumption-Excretion (Con-Ex). In Con-Ex studies, flies consume solid food labeled with dye, and the volume of food consumed is reflected by the sum of the dye inside of and excreted by flies. Flies consumed-excreted measurable amounts of FD&C Blue No. 1 (Blue 1) and other dyes in Con-Ex studies, but only Blue 1 was readily detectable at concentrations that had no discernable effect on consumption-excretion. In studies with Blue 1, consumption-excretion (i) increased linearly with feeding duration out to 24 h at two different laboratory sites, (ii) was sensitive to starvation, mating status and strain, and (iii) changed in response to alteration of media composition as expected. Additionally, the volume of liquid Blue 1 consumed from capillary tubes was indistinguishable from the volume of Blue 1 excreted by flies, indicating that excreted Blue 1 reflects consumed Blue 1. Our results demonstrate that Con-Ex with Blue 1 as a food tracer is a useful method for assessing ingestion of agar-based food media in adult flies.

Age-related locomotor impairment in humans is important clinically because it is associated with several co-morbidities and increased risk of death. One of the hallmarks of age-related locomotor impairment in humans is a decrease in walking speed with age. Genetically tractable model organisms such as Drosophila are essential for delineating mechanisms underlying age-related locomotor impairment and age-related decreases in locomotor speed. Negative geotaxis, the ability of flies to move vertically when startled, is a common measure of locomotor behavior that declines with age in Drosophila. Toward further developing Drosophila as a model for age-related locomotor impairment, we investigated whether negative geotaxis reflects climbing or a combination of climbing and other behaviors such as flying and jumping. Additionally, we investigated whether locomotor speed in negative geotaxis assays declines with age in flies as found for walking speed in humans. We find that the vast majority of flies climb during negative geotaxis assays and that removal of hind legs, but not wings, impairs the behavior. We also find that climbing speed decreases with age in four wild type genetic backgrounds, in flies housed at different temperatures, and in control and long-lived flies harboring a mutation in OR83b. The decreases in climbing speed correlate with the age-related impairments in the distance climbed. These studies establish negative geotaxis in Drosophila as a climbing behavior that declines with age due to a decrease in climbing speed. Agerelated decreases in locomotor speed are common attributes of locomotor senescence in flies and humans.

Volado, the gene encoding the Drosophila ␣PS3-integrin, is required for normal short-term memory formation (Grotewiel et al., 1998), supporting a role for integrins in synaptic modulation mechanisms. We show that the Volado protein (VOL) is localized to central and peripheral larval Drosophila synapses. VOL is strongly concentrated in a subpopulation of synaptic boutons in the CNS neuropil and to a variable subset of synaptic boutons at neuromuscular junctions (NMJs). Mutant morphological and functional synaptic phenotypes were analyzed at the NMJ. Key words: integrins; synaptic plasticity; synaptic signaling; adhesion; learning and memory; neuromuscular junction; DrosophilaA recent screen in Drosophila for new learning and memory mutants (Grotewiel et al., 1998) added a particularly intriguing new gene, Volado, to the growing list of genes involved in various phases of the learning-to-memory process (Boynton and Tully, 1992;Davis et al., 1995;Davis, 1996;Skoulakis and Davis, 1996;Dubnau and Tully, 1998). Volado, also known as ␣PS3 (Stark et al., 1997), encodes two ␣-integrin proteins differing only in their first 63 amino acids (Stark et al., 1997;Grotewiel et al., 1998). The Volado (VOL) proteins have enriched expression in the adult mushroom bodies (Grotewiel et al., 1998), synapse-dense brain structures that serve as insect olfactory memory centers and have long been implicated in cAMP signaling-dependent forms of behavioral learning and memory (Davis et al., 1995;Davis, 1996;Dubnau and Tully, 1998). Viable Volado mutants have a dominant effect on adult olfactory memory, reducing short-term memory (STM) assessed 3-15 min after training by ϳ50%. The mutant STM defect is reversibly rescued by conditional VOL expression just 3 hr before training (Grotewiel et al., 1998), suggesting a dynamic role for integrins in behavior modulation. These results indicate an exciting new potential role for integrins in mediating persistent changes in synaptic efficacy thought to accompany memory formation.Integrins function as ␣␤ receptor heterodimers capable of interacting with a variety of extracellular matrix (ECM) and cytoskeletal proteins, mediating cell-cell and cell-ECM adhesion interactions and bidirectional signaling across cell membranes (Hynes, 1992;Diamond and Springer, 1994;Clarke and Brugge, 1995;Jones, 1996). Cellular studies using peptide inhibitors of integrin-ECM ligand interactions have recently implicated synaptic integrins in rapid (minutes) and reversible consolidation of long-term potentiation (LTP) in the mammalian hippocampus (Bahr et al., 1997;Staubli et al., 1998), providing evidence that integrins function in long-term physiological plasticity related to learning. Dynamic changes in integrin-dependent adhesive interactions or intracellular signaling activity could mediate alterations in synapse morphology or number, or modulate transmission strength at existing synapses. Synaptic integrins are thus strategically positioned to have roles in both morphological and functional synaptic plasticity mecha...

Haploinsufficiency of RAI1 results in Smith-Magenis syndrome (SMS), a disorder characterized by intellectual disability, multiple congenital anomalies, obesity, neurobehavioral abnormalities, and a disrupted circadian sleep-wake pattern. An inverted melatonin rhythm (i.e., melatonin peaks during the day instead of at night) and associated sleep-phase disturbances in individuals with SMS, as well as a short-period circadian rhythm in mice with a chromosomal deletion of Rai1, support SMS as a circadian-rhythm-dysfunction disorder. However, the molecular cause of the circadian defect in SMS has not been described. The circadian oscillator temporally orchestrates metabolism, physiology, and behavior largely through transcriptional modulation. Data support RAI1 as a transcriptional regulator, but the genes it might regulate are largely unknown. Investigation into the role that RAI1 plays in the regulation of gene transcription and circadian maintenance revealed that RAI1 regulates the transcription of circadian locomotor output cycles kaput (CLOCK), a key component of the mammalian circadian oscillator that transcriptionally regulates many critical circadian genes. Data further show that haploinsufficiency of RAI1 and Rai1 in SMS fibroblasts and the mouse hypothalamus, respectively, results in the transcriptional dysregulation of the circadian clock and causes altered expression and regulation of multiple circadian genes, including PER2, PER3, CRY1, BMAL1, and others. These data suggest that heterozygous mutation of RAI1 and Rai1 leads to a disrupted circadian rhythm and thus results in an abnormal sleep-wake cycle, which can contribute to an abnormal feeding pattern and dependent cognitive performance. Finally, we conclude that RAI1 is a positive transcriptional regulator of CLOCK, pinpointing a novel and important role for this gene in the circadian oscillator.