Cardiovascular Characterization of Cx40/Panx1 Single and Double Knockout Mice

Connexins (Cxs) and pannexins (Panxs) are protein families that form large-pore channels which exist at the plasma membrane for both intracellular and extracellular signaling. Given their potential for overlapping cellular signaling functions we proposed that mice lacking both a connexin and a pannexin would have a severe phenotype. To investigate this possibility we crossed Panx1 null mice with Cx40 knockout mice and characterized the first global connexin/pannexin double knockout mouse. Intriguingly, the combined ablation of both Cx40 and Panx1 caused decreased prenatal and newborn survival, but did not affect the fertility or lifespan of surviving mice. Cx40-/- and Cx40-/-Panx1-/- mice had cardiac hypertrophy, and furthermore, combined channel ablation led to increasing severe hypertension and decreased endothelium dependent vasodilation in Cx40-/-Panx1-/- mice. Overall, these studies suggest that even though Panx1 and Cx40 act via differential mechanisms, they have a co-regulatory role in certain physiological processes such as vascular response

Ablation of both Cx40 and Panx1 results in similar cardiovascular phenotypes exhibited in Cx40 knockout mice

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

Using light to investigate taste reward circuits in Drosophila

The refinement of foraging and feeding behavior through experience is a vital process in an animal’s life, leading to increased overall fitness and survival. Yet, little is known about how initial taste perceptions are transformed in higher order neural circuits to produce lasting changes in behavior. Using Drosophila melanogaster, we investigate the neurobiology of taste memory formation. We find that flies form appetitive and aversive short- and long-term taste memories, which are processed in the mushroom body (MB), an associative learning neuropil. Moreover, appetitive short- and long-term memory formation is regulated by distinct subpopulations of protocerebral anterior medial neurons (PAMs), and long-term memory formations requires a caloric unconditioned stimulus (the US), which we hypothesize activates of MB-MP1 neurons. Transmission of the US signal from the primary taste center in the fly brain to the extrinsic PAM neurons of MB is regulated in part by sTPNs and lTPNs, two newly discovered taste projection neurons. sTPNs respond to a variety of sweet tastants, and when silenced flies fail to form short-term memories in a simple light memory task. Contrastingly, lTPNs respond to sucrose only upon ingestion, and when silenced fail to form long-term light memories. Interestingly, sTPNs neuronal activation dynamics mirrors that of PAM DANs arborizing on the horizontal tip of the MB lobes, and lTPN signaling shows similarities to PAM-α1 neurons. Finally, we investigated the modulatory role that discrete DAN/MBON cell types play in the innate acceptance or rejection of a meal source. Upon activation, we found that most DAN/MBON pairs show a similar activation patterns to those previously shown to initiate the approach or avoidance of an odor. This implies that the valence of these discrete MB associated neurons is fixed and instructs similar output behaviors among different sensory systems.Science, Faculty ofZoology, Department ofGraduat

Optogenetic induction of appetitive and aversive taste memories in Drosophila

Tastes typically evoke innate behavioral responses that can be broadly categorized as acceptance or rejection. However, research in Drosophila melanogaster indicates that taste responses also exhibit plasticity 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 neurons or dopaminergic neurons encoding 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 depend on experience