Manipulating feeding circuits in freely moving animals is challenging, in part because the timing of sensory inputs is affected by the animal’s behavior. To address this challenge in Drosophila, we developed the Sip-Triggered Optogenetic Behavior Enclosure (‘STROBE’). The STROBE is a closed-looped system for real-time optogenetic activation of feeding flies, designed to evoke neural excitation coincident with food contact. We previously demonstrated the STROBE’s utility in probing the valence of fly sensory neurons (Jaeger et al., 2018). Here we provide a thorough characterization of the STROBE system, demonstrate that STROBE-driven behavior is modified by hunger and the presence of taste ligands, and find that mushroom body dopaminergic input neurons and their respective post-synaptic partners drive opposing feeding behaviors following activation. Together, these results establish the STROBE as a new tool for dissecting fly feeding circuits and suggest a role for mushroom body circuits in processing naïve taste responses.

SUMMARYTastes are typically thought to evoke innate appetitive or aversive behaviours, prompting food acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses can be modified through experience-dependent changes in mushroom body circuits. In this study, we develop a novel taste learning paradigm using closed-loop optogenetics. We find that appetitive and aversive taste memories can be formed by pairing gustatory stimuli with optogenetic activation of sensory or dopaminergic neurons associated with reward or punishment. As with olfactory memories, distinct dopaminergic subpopulations drive the parallel formation of short- and long-term appetitive memories. Long-term memories are protein synthesis-dependent and have energetic requirements that are satisfied by a variety of caloric food sources or by direct stimulation of MB-MP1 dopaminergic neurons. Our paradigm affords new opportunities to probe plasticity mechanisms within the taste system and understand the extent to which taste responses are experience dependent.

Connexins (Cxs) and pannexins (Panxs) are highly regulated large-pore channel-forming proteins that participate in cellular communication via small molecular exchange with the extracellular microenvironment, or in the case of connexins, directly between cells. Given the putative functional overlap between single membrane-spanning connexin hemichannels and Panx channels, and cardiovascular system prevalence, we generated the first Cx40−/−Panx1−/− mouse with the anticipation that this genetic modification would lead to a severe cardiovascular phenotype. Mice null for both Cx40 and Panx1 produced litter sizes and adult growth progression similar to wild-type (WT), Cx40−/− and Panx1−/− mice. Akin to Cx40−/− mice, Cx40−/−Panx1−/− mice exhibited cardiac hypertrophy and elevated systolic, diastolic, and mean arterial blood pressure compared with WT and Panx1−/− mice; however assessment of left ventricular ejection fraction and fractional shortening revealed no evidence of cardiac dysfunction between groups. Furthermore, Cx40−/−, Panx1−/−, and Cx40−/−Panx1−/− mice demonstrated impaired endothelial-mediated vasodilation of aortic segments to increasing concentrations of methacholine (MCh) compared with WT, highlighting roles for both Cx40 and Panx1 in vascular endothelial cell (EC) function. Surprisingly, elevated kidney renin mRNA expression, plasma renin activity, and extraglomerular renin-producing cell populations found in Cx40−/− mice was further exaggerated in double knockout mice. Thus, while gestation and gross development were conserved in Cx40−/−Panx1−/− mice, they exhibit cardiac hypertrophy, hypertension, and impaired endothelial-mediated vasodilation that phenocopies Cx40−/− mice. Nevertheless, the augmented renin homeostasis observed in the double knockout mice suggests that both Cx40 and Panx1 may play an integrative role.

Manipulating feeding circuits in freely moving animals is challenging, in part because the timing of sensory inputs is affected by the animal's behavior. To address this challenge in Drosophila, we developed the Sip-Triggered Optogenetic Behavior Enclosure ("STROBE"). The STROBE is a closed-looped system for real-time optogenetic activation of feeding flies, designed to evoke neural excitation coincident with food contact. We demonstrate that optogenetic stimulation of sweet sensory neurons in the STROBE drives attraction to tasteless food, while activation of bitter sensory neurons promotes avoidance. Moreover, feeding behavior in the STROBE is modified by the fly's internal state, as well as the presence of chemical taste ligands. We also find that mushroom body dopaminergic neurons and their respective post-synaptic partners drive opposing feeding behaviors following activation. Together, these results establish the STROBE as a new tool for dissecting fly feeding circuits and suggest a role for mushroom body circuits in processing naïve taste responses.

Feeding is an essential part of animal life that is greatly impacted by the sense of taste. Although the characterization of taste-detection at the periphery has been extensive, higher-order taste and feeding circuits are still being elucidated. Here, we use an automated closed-loop optogenetic activation screen to detect novel taste and feeding neurons in Drosophila melanogaster. Out of 122 Janelia FlyLight Project GAL4 lines preselected based on expression pattern, we identify six lines that acutely promote feeding and 35 lines that inhibit it. As proof of principle, we follow up on R70C07-GAL4, which labels neurons that strongly inhibit feeding. Using split-GAL4 lines to isolate subsets of the R70C07-GAL4 population, we find both appetitive and aversive neurons. Furthermore, we show that R70C07-GAL4 labels putative second-order taste interneurons that contact both sweet and bitter sensory neurons. These results serve as a resource for further functional dissection of fly feeding circuits.