T evoked synaptic activity in these neurons is independent of intracellular 
Ca2+ signaling and SOCE.reduced GFP positive cells in the T2 segment (Fig. 7B) and this trend was also observed in the 5-HT positive cells (Fig. 7C). Because of the observed variation amongst T2 neurons, individual cells were counted in this region and compared across 10 control and 10 TRH/TNT animals. In TNTvif controls, TNT fliers and non-fliers, T2a9 and b9 neurons were nearly always present with the exception of one individual in TNT non-fliers (sample 2; Fig. 7D ) where all four T2 cells were absent. Variation existed in T2c9 and d `neurons. Based on cell numbers observed with antiGFP and anti-5-HT staining, the T2d’ cells were absent in 6/10 individuals of TNT non-fliers (Fig. 7 F), and the T2c9 cells were absent in 4/10 such individuals. Moreover, in the TNT populations, fewer anti-GFP cells were marked by anti-5-HTInhibition of synaptic function affects number of serotonergic neurons in the second thoracic segmentTo understand how inhibition of synaptic function in TRH neurons during pupal development affects flight, we visualized TRH positive neurons in TNT expressing flier and non-flier populations, and compared these with animals expressing inactive TNT (UASTNTvif). For this purpose a recombinant strain was generated expressing a membrane bound GFP (UASmCD8GFP) with TRHGAL4. Initially, third instar larval brains from animals expressing Tetanus toxin (UASTNTH) and control animals expressing inactive tetanus toxin (UASTNTvif) were visualized. These showed no significant difference in serotonergic cell populations as judged by anti-GFP and anti-5-hydroxytryptamine (5-HT, serotonin) immunostaining (Fig. 4A, B). The number of cells observed in each defined neural segment, were similar to earlier reports (Fig. 4C, D) [25,26]. Next, numbers of serotonergic neurons were quantified in the central brain of adults expressing TNT or TNTvif (Fig. 5A ). The numbers of previously identified 5-HT positive neurons (Fig. 5D), were no different in TNT expressing fliers and non-fliers as well as TNTvif Nafarelin controls (Fig. 5E). However, 6 GFP-positive medial cells, 1 cell in the Lp1 cluster, 2 cells in the LP2 cluster, 1 cell in SE1 and 1 cell in 1081537 the SE3 clusters were observed in the brain which did not stain with anti-5-HT (Fig. 5A ). These neurons were of a larger size as compared with other neurons. Similar non-5-HT positive medial cells have been observed in another TRHGAL4 strain [27], implying that these neurons are TRH positive but don’t synthesize 5-HT at detectable levels. Overall, there was no significant difference in the number of cells between controls and the brains of either fliers or non-fliers expressing TNT in TRH neurons (Fig. 5E). Next, serotonergic neurons in the thoracic segments were quantified, since in principle they were most likely to PD168393 modulate the flight central pattern generator (CPG) [28]. Variation in the number of dopaminergic and serotonergic cells has been observed in thoracic segments amongst animals of the same genotype [29]. In the first 16574785 thoracic segment (T1), 4 cells (denoted as a, b, c and d) were observed in nearly all the samples, including non-fliers of the TRH/TNT genotype (Fig. 6A ). The T2 region also had 4 cells, a9, b9, c9 and d9. In controls and TRH/TNT fliers, 1/10 flies had a fifth cell in the T2 region marked by anti-GFP, although this extra cell did not counter stain with anti-5-HT (denoted as T2e9) (Fig. 6B). Thus, on an aver.T evoked synaptic activity in these neurons is independent of intracellular Ca2+ signaling and SOCE.reduced GFP positive cells in the T2 segment (Fig. 7B) and this trend was also observed in the 5-HT positive cells (Fig. 7C). Because of the observed variation amongst T2 neurons, individual cells were counted in this region and compared across 10 control and 10 TRH/TNT animals. In TNTvif controls, TNT fliers and non-fliers, T2a9 and b9 neurons were nearly always present with the exception of one individual in TNT non-fliers (sample 2; Fig. 7D ) where all four T2 cells were absent. Variation existed in T2c9 and d `neurons. Based on cell numbers observed with antiGFP and anti-5-HT staining, the T2d’ cells were absent in 6/10 individuals of TNT non-fliers (Fig. 7 F), and the T2c9 cells were absent in 4/10 such individuals. Moreover, in the TNT populations, fewer anti-GFP cells were marked by anti-5-HTInhibition of synaptic function affects number of serotonergic neurons in the second thoracic segmentTo 
understand how inhibition of synaptic function in TRH neurons during pupal development affects flight, we visualized TRH positive neurons in TNT expressing flier and non-flier populations, and compared these with animals expressing inactive TNT (UASTNTvif). For this purpose a recombinant strain was generated expressing a membrane bound GFP (UASmCD8GFP) with TRHGAL4. Initially, third instar larval brains from animals expressing Tetanus toxin (UASTNTH) and control animals expressing inactive tetanus toxin (UASTNTvif) were visualized. These showed no significant difference in serotonergic cell populations as judged by anti-GFP and anti-5-hydroxytryptamine (5-HT, serotonin) immunostaining (Fig. 4A, B). The number of cells observed in each defined neural segment, were similar to earlier reports (Fig. 4C, D) [25,26]. Next, numbers of serotonergic neurons were quantified in the central brain of adults expressing TNT or TNTvif (Fig. 5A ). The numbers of previously identified 5-HT positive neurons (Fig. 5D), were no different in TNT expressing fliers and non-fliers as well as TNTvif controls (Fig. 5E). However, 6 GFP-positive medial cells, 1 cell in the Lp1 cluster, 2 cells in the LP2 cluster, 1 cell in SE1 and 1 cell in 1081537 the SE3 clusters were observed in the brain which did not stain with anti-5-HT (Fig. 5A ). These neurons were of a larger size as compared with other neurons. Similar non-5-HT positive medial cells have been observed in another TRHGAL4 strain [27], implying that these neurons are TRH positive but don’t synthesize 5-HT at detectable levels. Overall, there was no significant difference in the number of cells between controls and the brains of either fliers or non-fliers expressing TNT in TRH neurons (Fig. 5E). Next, serotonergic neurons in the thoracic segments were quantified, since in principle they were most likely to modulate the flight central pattern generator (CPG) [28]. Variation in the number of dopaminergic and serotonergic cells has been observed in thoracic segments amongst animals of the same genotype [29]. In the first 16574785 thoracic segment (T1), 4 cells (denoted as a, b, c and d) were observed in nearly all the samples, including non-fliers of the TRH/TNT genotype (Fig. 6A ). The T2 region also had 4 cells, a9, b9, c9 and d9. In controls and TRH/TNT fliers, 1/10 flies had a fifth cell in the T2 region marked by anti-GFP, although this extra cell did not counter stain with anti-5-HT (denoted as T2e9) (Fig. 6B). Thus, on an aver.
