The goal of this research is to define and characterize the neural networks governing two sequentially-linked behavioral programs in the fly. The first is an adaptive, environmentally-sensitive program that mediates the search for safe surroundings, and the second is a hormonally-driven program that serves to expand the recently developed wings of the newly emerged adult. The first program is used to find a safe perch from which the immobile fly can expand its wings, and the second program initiates expansion. Because wing expansion must be undertaken within several hours of emergence, the need for safety must be balanced by the imperative to expand and each individual fly must decide when (and under what environmental circumstances) to expand. Wing expansion thus provides a behavioral paradigm for studying decision-making, the most fundamental aspect of behavioral integration, and for understanding how hormonal and environmental factors act, individually and in concert, to recruit motor patterns to assemble behavioral sequences. Such understanding should, in turn, shed light on the deficits in behavioral organization that lie at the root of many mental disorders, including obsessive-compulsive disorder, schizophrenia, and bipolar disorder. As indicated above, the behavioral program that governs wing expansion is a primary focus of our work and characterization of the neural network that controls this process is a priority. Our previous work identified a """"""""command system"""""""" of approximately 50 peptidergic neurons that is distributed throughout the central nervous system and is both necessary for wing expansion and sufficient to induce that process when stimulated after emergence (Peabody et al., 2009). During the past year, our efforts have focused on three principal questions regarding the function of the command system: 1) What is the minimal cell group within the command system that is sufficient to activate the wing expansion program? 2) At what developmental stage does the command system become competent to drive wing expansion? and 3) What neuronal effectors lie downstream of the command system? We have made considerable progress on each of these questions. A manuscript describing our investigation of the first question is currently in review, and a manuscript related to the second question is in the final stages of preparation. To identify the minimal cell group within the command system, we have followed up on earlier suppression studies, which identified 16 neurons that express the hormone bursicon as essential components of the wing expansion circuitry. We have now shown that stimulation of these neurons by the genetically targeted activator UAS-TRPM8 elicits the entire wing expansion motor program in newly emerged adult flies. Remarkably, we also find that stimulation of a single pair of the bursicon-expressing neurons located in the subesophageal ganglion (i.e. the BSEG) can drive the wing expansion program, indicating that these two neurons act as command neurons for wing expansion. This finding was made possible by combining our previously developed UAS-TRPM8 technique for neuronal activation (Peabody et al., 2009) with our Split Gal4 system for refined transgene targeting (Luan et al., 2006). By using the Split Gal4 system in conjunction with suppressors of excitability we were also able to perform targeted neuronal silencing of the BSEG to show that they were necessary for the performance of the motor patterns that normally underlie wing expansion. In fact, silencing the BSEG abrogated the ability of flies to expand their wings under all conditions when environmental inhibition was present. Surprisingly, however, in the absence of environmental inhibition, flies lacking BSEG function were able to expand, but they used an alternate set of motor patterns to do so. We speculate that the """"""""compensatory"""""""" wing expansion program used by BSEG-suppressed flies when they are unperturbed may represent the vestige of an ancestral wing expansion program, similar to the one used by insects other than Drosophila that lack the ability to delay expansion in response to environmental conditions. To investigate the question of when the command system becomes competent to drive the wing expansion program, we have attempted to induce wing expansion at developmentally ectopic times. We find that while stimulation of either the BSEG or the full complement of bursicon-expressing neurons is sufficient to induce wing expansion in newly emerged flies, none of these manipulations induces wing expansion in flies that have not yet emerged. Not only do such flies not expand their wings, they do not show evidence of bursicon release. These results suggest that prior to emergence, the bursicon-expressing neurons are electrically unexcitable. This lack of excitability may reflect intrinsic membrane properties of these neurons, or may reflect active suppression by inhibitory synaptic bombardment. Regardless of the means of suppression, our experiments make clear that in response to emergence, or some process closely associated with it, bursicon release and its behavioral sequelae are suppressed. Furthermore, they indicate that the mechanisms of suppression differ from those that mediate environmental inhibition after emergence, demonstrating an added layer of complexity in the regulation of wing expansion behavior. While the specific mechanisms of both pre- and post-emergence inhibition remain to be determined, our results promise to provide insight into how nervous systems temporally assemble motor programs to form behavioral sequences. The final focus of our characterization of the command system for wing expansion involves identifying its downstream cellular effectors. Here, we have focused on identifying neurons that express the bursicon receptor, which is encoded by the rickets gene. In order to both identify and characterize the functions of these neurons, we have used a novel strategy that exploits the """"""""self-cleaving"""""""" T2A peptide to co-express the Gal4 transcription factor in cells that express rickets. The resulting """"""""rickets-Gal4"""""""" driver line has allowed us to identify rickets-expressing neurons (by driving the expression of the reporter, UAS-EGFP) and also to confirm their identity (by driving a UAS-rickets rescue transgene to rescue the behavioral deficits of rickets null mutants). In on-going work, we are trying to identify the functional roles of various subsets of rickets-expressing neurons in the wing expansion circuit. In this work, we are exploiting a tool for refined transgene targeting, similar to our earlier Split Gal4 technique, called the Split LexA system. This system, which we developed in collaboration with the laboratory of Dr. Chi-hon Lee (Ting et al., 2011), allows the expression patterns of Gal4 driver lines, such as rickets-Gal4, to be parsed into subpatterns. In this way, subsets of rickets-expressing neurons can be independently manipulated to understand their functions. In summary, we have made substantial progress in functionally characterizing essential components of the circuit that governs post-emergence behavior in Drosophila using a palette of tools we have developed. As we extend and refine our analysis of this circuit, our work should provide insight into the principles used by all nervous systems to generate and organize behavior. In generating a functional map of the wing expansion circuit, we will also position ourselves to initiate an analysis of its developmental determinants at the level of individual cells and cell-fate decisions by leveraging the powerful molecular genetic tools available for studying development in Drosophila.

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