Gore Reshma, Esmail Tina, Pflepsen Kelsey, Marron Fernandez de Velasco Ezequiel, Kitto Kelley F, Riedl Maureen S, Karlen Andrea, McIvor R Scott, Honda Christopher N, Fairbanks Carolyn A, Vulchanova Lucy
Department of Neuroscience, University of Minnesota, Minneapolis, MN, United States.
Department of Pharmaceutics, University of Minnesota, Minneapolis, MN, United States.
Front Pain Res (Lausanne). 2023 Aug 4;4:1225246. doi: 10.3389/fpain.2023.1225246. eCollection 2023.
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.
对内脏痛相关神经回路的研究因难以对单个回路组件进行选择性操作而受阻。在本研究中，我们采用了一种双重腺相关病毒（AAV）方法（用于中枢神经系统中特定投射的转基因表达），以探索将转基因靶向递送至支配内脏器官的初级传入神经元的可能性。以小鼠结肠的外在感觉神经支配为重点，我们首先对鞘内注射一种AAV9载体和结肠内注射第二种AAV9载体后的双重转导程度进行了表征。我们发现，如果两种AAV9载体相隔一周递送，第二种载体对背根神经节（DRG）神经元的转导会大大减少。在同一天递送两种病毒后，我们观察到转基因在DRG神经元中共定位，表明存在双重转导。接下来，我们以Cre重组酶依赖的方式鞘内递送编码抑制性化学遗传激活剂hM4D(Gi)的AAV9载体，并在同一天在结肠中注射携带Cre重组酶的AAV9载体。在mRNA和蛋白质水平均证实了hM4D(Gi)在DRG中的表达。然而，我们未能证明hM4D(Gi)激活后对内脏伤害感受的选择性抑制。综上所述，这些结果为开发针对初级传入神经元进行靶向转导以调节外周神经回路的策略奠定了基础。
