Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion, expected concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools, however, provide the potential for cell-type specific neural stimulation using genetic targeting and/or spatially shaped excitation light. Here, we demonstrate light-activated stimulation of the endocrine pancreas by targeting parasympathetic (cholinergic) axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to (1) ultrasound image-guided optical stimulation of axon terminals in the pancreas or (2) optical stimulation of axons of the cervical vagus nerve. Measurements were made in basal-glucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and cervical vagal stimulation, while a rapid reduction in glycemic levels were observed under pancreatic stimulation. Additionally, ultrasound-based measurements of blood flow in the pancreas were increased under pancreatic stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest its therapeutic potential for the control of insulin secretion and glucose homeostasis.

Vagus nerve stimulation has shown many benefits for disease therapies but current approaches involve imprecise electrical stimulation that gives rise to off-target effects, while the functionally relevant pathways remain poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate targeted neural communication with light-sensitive actuators (opsins) and can be targeted to organs of interest based on the location of viral delivery. Here, we tested whether retrograde adeno-associated virus (rAAV2-retro) injected in the heart can be used to selectively express opsins in vagus nerve fibers controlling cardiac function. Furthermore, we investigated whether perturbations in cardiac function could be achieved with photostimulation at the cervical vagus nerve. Viral injection in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Photostimulation using both one-photon stimulation and two-photon holography with a GRIN-lens incorporated nerve cuff, was tested on the pilot-cohort of injected mice. Changes in heart rate, surface electrocardiogram, and respiratory responses were observed in response to both one- and two-photon photostimulation. The results demonstrate feasibility of retrograde labeling for organ targeted optical neuromodulation.

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AbstractTargeting specifics subsets of peripheral pathways of the autonomic nervous system will enable new avenues to study organ control and develop new disease therapies. Vagus nerve stimulation (VNS) has shown many therapeutic benefits but current approaches involve imprecise electrical stimulation that gives rise to adverse effects, and the functionally relevant pathways are poorly understood. One method to overcome these limitations is the use of optogenetic techniques, which facilitate highly specific neural communication with light-sensitive actuators (opsins). Opsins can be targeted to cell populations of interest based on the location of viral delivery and genetic control of expression. Here, we tested whether holographic photostimulation of subsets of axons of the cervical vagus nerve that innervate the heart can be used to modulate cardiac function. Viral injection of retrograde adeno-associated virus (rAAV2-retro) in the heart resulted in robust, primarily afferent, opsin reporter expression in the vagus nerve, nodose ganglion, and brainstem. Selective holographic photostimulation of axons resulted in changes in heart rate, surface cardiac electrogram, and respiratory responses that were different from responses elicited by whole nerve photostimulation.

Previous studies have demonstrated stimulation of endocrine pancreas function by vagal nerve electrical stimulation. While this increases insulin secretion; concomitant reductions in circulating glucose do not occur. A complicating factor is the non-specific nature of electrical nerve stimulation. Optogenetic tools enable high specificity in neural stimulation using cell-type specific targeting of opsins and/or spatially shaped excitation light. Here, we demonstrate lightactivated stimulation of the endocrine pancreas by targeting vagal parasympathetic axons. In a mouse model expressing ChannelRhodopsin2 (ChR2) in cholinergic cells, serum insulin and glucose were measured in response to both ultrasound image-guided optical stimulation of axon terminals in the pancreas and optical stimulation of axons of the cervical vagus nerve, together with ultrasound-based measures of pancreas blood flow. Measurements were made in basalglucose and glucose-stimulated conditions. Significant increases in plasma insulin occurred relative to controls under both pancreas and vagal stimulation, accompanying rapid reductions in glycemic levels. Additionally, a significant increase in pancreatic blood flow was measured following optical stimulation. Together, these results demonstrate the utility of in-vivo optogenetics for studying the neural regulation of endocrine pancreas function and suggest therapeutic potential for the control of insulin secretion and glucose homeostasis.