Fly mutants of the inositol 1,4,5 trisphosphate receptor gene, itpr, are unable to evoke air-puff stimulated flight, even though physiological responses on stimulation of the giant-fiber pathway remain unaltered. Previous work has demonstrated that expression of an itpr+ cDNA in aminergic neurons rescued loss of flight in itpr mutants close to wildtype levels and blocking of synaptic activity in aminergic neurons by tetanus toxin expression reduced flight to 45%. Moreover, an adult requirement for the serotonergic component of aminergic neurons was indicated, since a flight deficit of 33% was observed in wild-type adult flies fed for 5 days with a serotonin synthesis inhibitor, parachlorophenylalanine. Thus a role for synaptic activity in aminergic neurons was indicated, with a possible requirement for serotonin both during AMN107 clinical trial development and in adult flight. More recently, it was shown that intracellular Ca2+ signaling through IP3R and storeoperated Ca2+ entry in neurons are important for air-puff induced flight, suggesting that aminergic neuron function in Drosophila flight might require IP3R mediated Ca2+ signals. In this study, we have studied the effect of blocking synaptic function and reduced intracellular Ca2+ signaling specifically in serotonergic neurons, on air-puff stimulated flight. It is known that the insect flight circuit is formed during pupal development. Therefore, the effect of blocking synaptic activity in serotonergic neurons during pupal development and in adults was assessed. We show that blocking synaptic activity in serotonergic neurons either during flight circuit development or in adults reduces air-puff induced flight significantly. Our data suggest that synaptic activity affects the number of flight modulating serotonergic neurons in the second thoracic segment, but modulation of flight by these neurons does not require the IP3R or SOCE. In locusts, serotonin acts on the fast extensor and flexor tibiae motor neurons and this results in potentiation of synaptic transmission between these neurons, thereby modulating their neuronal properties and synaptic strengths. The partial flight deficit observed in Drosophila by synaptic inhibition of serotonergic neurons could be due to loss or reduction in similar modulatory effects of serotonin on as yet unidentified neurons of the flight circuit. Studies in locusts have also shown that a flight central pattern generator residing in the thorax, drives the motoneurons and maintains the phase relationship among the motor units of each muscle. Biogenic amines, such as octopamine and tyramine have been shown to modulate the flight CPG in locusts, Manduca and other moths. Though precise components of flight CPG are unknown, it is thought to be activated by a muscarinic cholinergic mechanism in locusts. Because octopaminergic modulation of Drosophila flight CPG has already been shown, it is likely that the flight CPG is modulated by multiple neuromodulators including serotonin. Our data suggest that the function of these neuromodulators can be compensated by each other. Temporal blocking of synaptic function in TRH neurons by expressing a temperature sensitive dynamin transgene, UASShits demonstrated a greater requirement for synaptic activity in serotonergic neurons during pupal development, followed by a reduced requirement in adults. In Drosophila, components of indirect flight motoneurons undergo dendritic and axonal remodeling during early pupal stages.