AAV-mediated gene transfer to colon-innervating primary afferent neurons

Investigation of neural circuits underlying visceral pain is hampered by the difficulty in achieving selective manipulations of individual circuit components. In this study, we adapted a dual AAV approach, used for projection-specific transgene expression in the CNS, to explore the potential for targeted delivery of transgenes to primary afferent neurons innervating visceral organs. Focusing on the extrinsic sensory innervation of the mouse colon, we first characterized the extent of dual transduction following intrathecal delivery of one AAV9 vector and intracolonic delivery of a second AAV9 vector. We found that if the two AAV9 vectors were delivered one week apart, dorsal root ganglion (DRG) neuron transduction by the second vector was greatly diminished. Following delivery of the two viruses on the same day, we observed colocalization of the transgenes in DRG neurons, indicating dual transduction. Next, we delivered intrathecally an AAV9 vector encoding the inhibitory chemogenetic actuator hM4D(Gi) in a Cre-recombinase dependent manner, and on the same day injected an AAV9 vector carrying Cre-recombinase in the colon. DRG expression of hM4D(Gi) was demonstrated at the mRNA and protein level. However, we were unable to demonstrate selective inhibition of visceral nociception following hM4D(Gi) activation. Taken together, these results establish a foundation for development of strategies for targeted transduction of primary afferent neurons for neuromodulation of peripheral neural circuits

Neurodevelopmental Effects of Octopamine on Isolation-Induced Social Behaviors in Pheidole dentata

Isolation increases the performance of social behaviors when ants are reunited with nestmates. Octopamine (OA) mediates isolation induced social behaviors in ants. To study the behavioral mechanism of OA on social behavior, we conducted two sets of experiments. First, we acutely treated adult Pheidole dentata workers with OA following 5-day isolation, and recorded their social interactions. In the second experiment, the same behavioral process was employed but ants were treated during the pupal stage and tested as adults to evaluate the developmental effects of OA. Based on prior research we expected decreased social behaviors following OA treatment in adults, and increased social behaviors in adults developmentally treated with OA. Contrary to our expectations, we found an increase in the duration of social behaviors in OA treated adults, and no effect of OA on the duration of social behaviors compared to controls in the developmental group. However, vehicle treatment during development significantly reduced social behaviors as adults, suggesting a protective role of OA that rescued behaviors back to control levels. Therefore acute OA treatment differentially affects social behaviors in normal and developmentally treated adult ants

Targeting the somatosensory system with AAV9 and AAV2retro viral vectors.

Adeno-associated viral (AAV) vectors allow for site-specific and time-dependent genetic manipulation of neurons. However, for successful implementation of AAV vectors, major consideration must be given to the selection of viral serotype and route of delivery for efficient gene transfer into the cell type being investigated. Here we compare the transduction pattern of neurons in the somatosensory system following injection of AAV9 or AAV2retro in the parabrachial complex of the midbrain, the spinal cord dorsal horn, the intrathecal space, and the colon. Transduction was evaluated based on Cre-dependent expression of tdTomato in transgenic reporter mice, following delivery of AAV9 or AAV2retro carrying identical constructs that drive the expression of Cre/GFP. The pattern of distribution of tdTomato expression indicated notable differences in the access of the two AAV serotypes to primary afferent neurons via peripheral delivery in the colon and to spinal projections neurons via intracranial delivery within the parabrachial complex. Additionally, our results highlight the superior sensitivity of detection of neuronal transduction based on reporter expression relative to expression of viral products