Chemogenetic Modulation of Thermoregulation in Arctic Ground Squirrels Using AAV9-DREADDs

The non-model organism, Arctic Ground Squirrel (AGS), demonstrates unique thermoregulatory abilities, including survival at subzero temperatures during hibernation. To obtain more information on the thermoregulatory system of this unique species for future translational therapeutic use, we evaluated the feasibility of modulating thermoregulation via Adeno-Associated Virus (AAV) carrying plasmid for Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) technology. The target for AAV injection was the raphe pallidus (RPa), a critical brain region containing sympathetic premotor neurons for the control of brown adipose tissue (BAT) thermogenesis. We hypothesized 1) positive viral expression in RPa and 2) that activation of DREADD receptors would modulate sympathetic outflow to BAT. Four AGS (2M, 2F) underwent stereotaxic injection, in RPa, of AAV9 vectors containing the plasmid encoding either the inhibitory (pAAV-hSyn-hM4D(Gi)-mCherry) or excitatory (pAAV-hSyn-hM3D(Gq)-mCherry) DREADD construct. iButtons were implanted intraperitoneally to monitor body temperature. Following 4 weeks of recovery to allow DREADD expression in targeted neurons, all non-hibernating AGS were placed in metabolic chambers where oxygen consumption and core body temperature were measured. To evaluate the effect of RPa inhibition on thermogenesis, AGS were exposed to 2°C overnight to maintain active thermogenesis and elevated metabolic rates, while to evaluate the effect of RPa activation, AGS were kept at room temperature to maintain inhibited thermogenesis. Each AGS received clozapine-N-oxide (1 mg/kg i.p.) and saline as a control after a 96-hour washout period. Following a period of four hours, animals were euthanized and perfused with 4% formaldehyde for brain extraction and following immunostaining. Confocal microscopy confirmed a scattered mCherry expression in the RPa region of all AGS, demonstrating the feasibility of the use of DREADD technology and good tropism for AAV9 in AGS. However, when compared with vehicle injection, no significant differences in body temperature or oxygen consumption were observed following clozapine-N-oxide injection. We speculate that the lack of physiological response to CNO treatment may be due to the low number of RPa neurons infected, insufficient expression of the DREADD construct, or lack of internalization of the M3 and M4 in the neuron’s membrane. We are currently investigating AAV serotype tropism in AGS and optimizing methods to enhance viral infection and expression. TRIM P20GM130443 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.