ABSTRACT Background Like most animals, insects rely on their highly sensitive olfactory systems for survival. Olfaction plays a primary role in finding food and mates as well as in the avoidance of noxious chemicals and predators. Insect olfactory neurons typically express an odor-specific odorant receptor (OR) along with Orco, the olfactory co-receptor. Orco binds ORs and permits their trafficking to the ciliated dendrites of antennal olfactory sensory neurons (OSNs), where together, they form heteromeric, ligand-gated, non-selective cation channels. Orco is highly conserved across insect orders, and one particularly well-conserved region of Orco is predicted to be a Calmodulin (CaM) binding site (CBS). In this study, we explore the relationship between Orco and CaM _in vivo_ in the olfactory neurons of _Drosophila melanogaster_. Results OSN-specific knock-down of CaM at the onset of OSN development dramatically reduces olfactory responsiveness and Orco trafficking to OSN dendrites without affecting OSN morphology. We next generated a series of Orco CBS mutants and used them to rescue the _Orco1_ null mutant. While wild type Orco rescues the _Orco1_ defect in trafficking ORs to OSN dendrites, all of the Orco CBS mutants remain stuck in the OSN cell bodies, precluding even the smallest odor-evoked response. Finally, we found CaM's modulation of OR trafficking is activity-dependent. Knock-down of CaM in all Orco-positive OSNs after OR expression is well-established has relatively little effect on olfactory responsiveness alone. When combined with an extended exposure to a given odor, however, this late-onset CaM knock-down dramatically reduces both olfactory sensitivity and dendritic Orco trafficking only in OSNs that respond to that specific odor. Conclusions In this study, we show Calmodulin regulates OR trafficking and olfactory responsiveness _in vivo_ in _Drosophila_ olfactory neurons via a highly conserved binding site on the olfactory co-receptor Orco. As CaM's modulation of Orco seems to be activity-dependent, we propose a model in which the CaM/Orco interaction allows insect OSNs to maintain appropriate dendritic levels of OR regardless of environmental odor concentration.
Mammalian T-type Ca2+ channels are encoded by three separate genes (Cav3.1, 3.2, 3.3). In mammals, T-type channels are reported to be sleep stabilizers that are important in the generation of the delta rhythms of deep sleep, but controversy remains. Progress in identifying the precise physiological functions of the T-type channels has been hindered by many factors, including possible compensation between the products of these three genes and a lack of specific pharmacological inhibitors. Invertebrates have only one T-type channel gene and its physiological functions are less well-studied. We cloned Ca-α1T, the only Cav3 channel gene in the _Drosophila melanogaster_ genome, expressed it in _Xenopus_ oocytes or HEK-293 cells, and verified that it is capable of passing typical T-type currents. Voltage-clamp analysis revealed that the biophysical properties of Ca-α1T show mixed similarity, sometimes falling closer to Cav3.1, sometimes to Cav3.2, and sometimes to Cav3.3. We found that Ca-α1T is broadly expressed across the adult fly brain in a pattern vaguely reminiscent of mammalian T-type channels. In addition, flies lacking Ca-α1T show an abnormal increase in sleep duration that is most pronounced during subjective day under continuous dark conditions despite normal oscillations of the circadian clock. Thus, our study suggests invertebrate T-type Ca2+ channels promote wakefulness rather than stabilizing sleep.
The _Drosophila_ olfactory system is highly stereotyped in form and function; olfactory sensory neurons (OSNs) expressing a specific odorant receptor (OR) always appear in the same antennal location and the axons of OSNs expressing the same OR converge on the same antennal lobe glomeruli. Although some transcription factors have been implicated in a combinatorial code specifying OR expression and OSN identity, it is clear other players remain unidentified. To mitigate some of the challenges of genome-wide screening, we propose a two-tiered approach comprising a primary “pooling” screen for miRNAs whose tissue-specific over-expression causes a phenotype of interest followed by a focused secondary screen using gene-specific RNAi. Since miRNAs down-regulate their target mRNAs, miRNA over-expression phenotypes should be attributable to target loss-of-function. Since miRNA-target pairing is sequence-dependent, predicted targets of miRNAs identified in the primary screen are candidates for the secondary screen. Since miRNAs are short, however, miRNA misexpression will likely uncover non-biological miRNA-target relationships. Rather than focusing on miRNA function itself where these non-biological relationships could be misleading, we propose using miRNAs as tools to focus a more traditional RNAi-based screen. Here we describe a proof-of-concept miRNA-based screen that uncovers a role for Atf3 in the expression of the odorant receptor Or47b.