Understanding the function of specific neuronal populations within the peripheral nervous system, particularly the vagus nerve, is crucial for advancing neuroscience. The vagus nerve plays a vital role in autonomic functions and behaviors, but its complex architecture and the challenges of manipulating specific components in freely behaving animals have hindered progress. Optogenetics offers a powerful tool for precise neural manipulation, but its application to the peripheral nervous system has been limited by the constraints of fiber-optic delivery. Existing methods often lack the necessary stability, flexibility, and organ specificity for chronic studies in awake animals. This study addresses these limitations by developing a wireless, implantable device capable of targeted optogenetic stimulation within specific organs, enabling investigation of peripheral neural circuits in freely moving animals. The study specifically focuses on the vagus nerve, where the ability to selectively manipulate different sensory fiber types within specific organs (like the stomach) is essential to understand their distinct roles in complex physiological processes like appetite regulation.

Previous research has explored optogenetic manipulation of vagal afferents, but these studies were often limited by the use of fiber optics under anesthesia, hindering the investigation of long-term behavioral and physiological effects. Wireless technologies have advanced, enabling miniaturized devices for brain applications, but organ-restricted illumination in the periphery remained a challenge. Existing wireless devices lacked the durability for chronic studies and often suffered from light back-scatter and non-specific activation of surrounding tissues. This paper builds upon previous advancements in wireless optogenetics, addressing the limitations of existing technologies to provide a more robust and versatile platform for peripheral neural stimulation.

The researchers developed a fully implantable, wireless optogenetic device consisting of a miniaturized circuit for RF energy harvesting, a flexible tether delivering current to a micro-LED (μLED), and a unique tether fabrication method to enhance durability. The μLED is positioned within the target organ, minimizing light scatter and ensuring precise stimulation. To improve the device's longevity and ability to withstand the harsh conditions within organs, a novel pre-curved, sandwiched tether design was implemented. This design significantly reduced mechanical strain compared to traditional methods. The researchers used a three-dimensional (3D) modeling to analyze the mechanics of the tether and optimize its design for enhanced durability. Rigorous mechanical testing, including cyclical load tests, confirmed the improved durability of the device. Waterproof testing demonstrated continued functionality for over two months, even in extreme temperature conditions. Thermal assessment and specific absorption rate (SAR) calculations confirmed that the device's operation remained within safe limits. For high-throughput studies, a multiplexer approach was developed to power eight individual devices using a single RF transmitter. An advanced dual-coil antenna system improved wireless coverage, eliminating the need for high RF power and reducing heat generation. A reed switch mechanism allowed for multimodal operation, enabling remote control of different stimulation parameters. The device's efficacy was demonstrated by targeting Calca+ vagal afferents in the stomach of CalcrCre transgenic mice using AAV9-DIO-ChR2-tdTomato viral vectors. The researchers performed behavioral experiments, including food intake measurements, place preference tests, and open-field assays to assess the impact of optogenetic stimulation on appetite and behavior. Statistical analysis was used to compare experimental groups, utilizing techniques such as two-tailed t-tests and two-way repeated-measures ANOVA.

The newly developed wireless device demonstrated significant improvements in durability, lasting over a month in vivo, compared to a post-curved design which failed within three days. The multiplexing and dual-coil antenna system enabled high-throughput, precise optogenetic manipulation in multiple animals simultaneously. The device allowed for organ-specific, light delivery, minimizing light scatter and ensuring targeted stimulation. The application of this technology to the study of gastric vagal afferents revealed a novel role for Calca+ vagal sensory fibers in suppressing appetite. Optogenetic activation of these fibers reduced food intake and induced an aversion to sucrose, suggesting an appetite-suppressing mechanism based on negative valence. These findings challenge previous assumptions about the role of specific vagal afferent populations in appetite regulation.

The findings of this study significantly advance the field of peripheral optogenetics by providing a robust, versatile, and scalable platform for the manipulation of peripheral neural circuits in freely behaving animals. The development of the durable, multimodal wireless device overcomes previous limitations associated with fiber optics and allows for precise and chronic studies not possible before. The discovery of the role of Calca+ vagal sensory fibers in suppressing appetite through a negative valence mechanism provides new insights into the complex interplay between the gut and brain in regulating feeding behavior. This platform also offers significant advantages for high-throughput studies, reducing the cost and complexity of optogenetic experiments.

This study presents a significant advancement in the field of peripheral optogenetics by developing a durable, multimodal, and high-throughput wireless device for precise neural manipulation. The successful application of this technology to the study of gastric vagal afferents demonstrates its potential for investigating the role of other peripheral neural circuits in various physiological processes. Future research could explore the application of this device to other organs and neural pathways, potentially leading to the development of novel therapeutic targets for various disorders.

While the study demonstrates the efficacy of the device, future work could explore the effects of chronic, long-term activation of Calca+ vagal afferents. Additionally, the study focused on a specific subset of vagal afferents; further investigation could examine other genetically distinct populations. The sample sizes in some behavioral experiments were relatively small, and replication with larger cohorts would further strengthen the conclusions.