Intermittent KHz-frequency electrical stimulation selectively engages small unmyelinated vagal afferents

Afferent and efferent vagal fibers mediate bidirectional communication between the brain and visceral organs. Small, unmyelinated C-afferents constitute the majority of vagal fibers, play critical roles in numerous interoceptive circuits and autonomic reflexes in health and disease and may contribute to the efficacy and safety of vagus nerve stimulation (VNS). Selective engagement of C-afferents with electrical stimuli has not been feasible, due to the default fiber recruitment order: larger fibers first, smaller fibers last. Here, we determine and optimize an electrical stimulus that selectively engages vagal C-afferents. Intermittent KHz-frequency electrical stimulation (KES) activates motor and, preferentially, sensory vagal neurons in the brainstem. During KES, asynchronous activity of C-afferents increases, while that of larger fibers remains largely unchanged. In parallel, KES effectively blocks excitability of larger fibers while moderately suppressing excitability of C-afferents. By compiling selectivity indices in individual animals, we find that optimal KES parameters for C-afferents are >5KHz frequency and 7-10 times engagement threshold (×T) intensity in rats, 15-25×T in mice. These effects can be explained in computational models by how sodium channel responses to KES are shaped by axonal size and myelin. Our results indicate that selective engagement of vagal C-afferents is attainable by intermittent KES. Introduction Homeostasis in organisms is maintained through orchestrated operation of immune, endocrine and neural processes. The autonomic nervous system (ANS) plays central role in homeostatic control, through an interconnected network of fast-acting reflexes that regulate the function of internal organs in real time. Autonomic reflexes comprise sensing and effector mechanisms in body organs, integrator systems in the brain, and peripheral nerves, which convey information between them. The vagus nerve is the main neural conduit of body-brain communication, mediating bidirectional transmission of sensory (afferent) and motor (efferent) information. The vast majority of nerve fibers in the vagus are small, unmyelinated, slowly conducting, C-type afferents(1). Vagal C-afferents mediate numerous and diverse functions, including sensing of nutrients(2) and regulation of appetite and glucose metabolism(3), effects of gut microbiome on brain function and cognition(4), neural regulation of breathing(5), modulation of immune responses to lung infections(6), and shaping of emotional responses by bodily feedback(7). Vagal C-afferents also constitute the afferent arm of cardiovascular reflexes(8), neuroimmune circuits in the gut(9), and the inflammatory reflex itself(10) , a neuroimmune circuit that maintains immunological homeostasis throughout the body(11). Engagement of C-afferents by vagus nerve stimulation (VNS) may have therapeutic implications for neurostimulation-based therapies of arthritis(12), inflammatory bowel disease(13, 14), heart failure(15) and obesity(16). Controlled, selective engagement of distinct nerve fiber types, separately from other fiber populations in the same nerve, is required for the study of their physiological and translational roles(17). Selective activation of C-afferents in the vagus is possible using optogenetic nerve stimulation(18) but that is currently only practical with mice in acute experiments, with limited value in preclinical models of chronic disease and unclear clinical applicability. On the other hand, electrical stimulation of the vagus can be delivered acutely or chronically, in various animal models, including mice(19), and in humans. However, there is no known electrical stimulus that selectively activates vagal C-fibers. That is mainly because of the natural recruitment order of nerve fibers: larger fibers (Aand B-type) are activated well before smaller fibers can be engaged(20), and at high stimulus intensities, required to activate C-fibers, larger fibers are also maximally activated(21, 22). The lack of a C-fiber-selective electrical stimulus hinders the study of the many interoceptive functions and autonomic reflexes in which Cafferents are involved, the translational testing of VNS in animal models of disease and, ultimately, the therapeutic potential of VNS. Kilohertz-frequency electrical stimulation (KES) blocks nerve conduction (23-26) and is used in the vagus nerve for treating obesity(27) and in somatic sensory nerves for treating pain(28, 29), with an assumed mechanism of action that involves blocking of C-afferents. Using electrical and optogenetic stimulation, imaging, physiological and computational methods, we show here that KES of the vagus nerve can instead preferentially activate C-afferents while simultaneously blocking larger fibers, in a reliable and reversible manner. We also describe a method to select optimal KES frequency and intensity for C-afferent fiber activation in real time, in individual subjects.