, 2003 and Nässel and Elekes, 1992). To identify the specific TH-Gal4 neurons that trigger proboscis extension, we employed a genetic mosaic analysis to restrict dTRPA1 expression to small subsets of TH-Gal4 neurons ( Gordon and Scott, 2009). Briefly, the repressor Gal80 flanked by FRT recombination sites was expressed ubiquitously to inhibit Gal4-dependent expression. Induction of Flp recombinase under the control of a heat-shock promoter led to the stochastic excision of Gal80 and the expression of UAS-dTRPA1 in different TH-Gal4 subpopulations ( Gordon and Scott, 2009). The inclusion of UAS-mCD8-GFP allowed for visualization of cells expressing dTRPA1. Mosaic animals were tested for

proboscis extension to heat and classified as extenders and nonextenders. The neurons labeled in Selleckchem Bosutinib the brains and thoracic ganglia of extenders and nonextenders were Verteporfin compared to test whether specific TH-Gal4 neurons were associated with the extension phenotype. Eleven different cell populations were frequently labeled by this method ( Figure 4). Most cell

populations showed a similar frequency distribution in both behavioral categories; however, one cell was present in 93% of extenders (51/55) and rarely present in nonextenders (1%; 1/99). In addition, three extenders showed Gal4 expression in just two cells in the entire nervous system; each contained the cell found in 93% of extenders and a second cell that was different in each fly. These results argue that a single TH-Gal4 cell is sufficient to drive proboscis extension. Other cells

may modestly influence proboscis extension but would not be uncovered by mosaic analyses. Instead, the mosaic analysis is biased toward identifying single neurons sufficient to activate proboscis extension. The TH-Gal4 neuron that generates extension shows broad arborizations in the ventral anterior SOG, the primary taste relay ( Figure 5 and Figure S2). This brain region receives gustatory axons from the proboscis, mouthparts, legs, and motor neuron dendrites that drive proboscis extension ( Figure S2). Previous studies characterizing the anatomy of TH-Gal4 neurons have classified this neuron as a ventral unpaired medial neuron based on cell-body position ( Nässel and Elekes, 1992). We name this Liothyronine Sodium neuron TH-VUM. As expected, TH-VUM expresses tyrosine hydroxylase by immunohistochemistry ( Figure 5C), demonstrating that it is indeed a dopaminergic neuron. To determine whether processes are dendrites or axons, a marker for presynaptic terminals, Synaptobrevin-GFP (Syn-GFP) ( Estes et al., 2000), was expressed in single-cell TH-Gal4 clones. Syn-GFP labeled all arbors of TH-VUM, suggesting that the neuron releases transmitter throughout the SOG ( Figure 5D). Based on its localization in the primary taste region and its extensive arborizations, the TH-VUM neuron is well situated to modulate taste behaviors.