Vagus nerve stimulation (VNS) paired with forelimb training drives robust, specific reorganization of movement representations in the motor cortex. This effect is hypothesized to be mediated by VNS-dependent engagement of neuromodulatory networks. VNS influences activity in the locus coeruleus (LC) and dorsal raphe nucleus (DRN), but the involvement of these neuromodulatory networks in VNS-directed plasticity is unknown. We tested the hypothesis that cortical norepinephrine and serotonin are required for VNS-dependent enhancement of motor cortex plasticity. Rats were trained on a lever pressing task emphasizing proximal forelimb use. Once proficient, all rats received a surgically implanted vagus nerve cuff and cortical injections of either immunotoxins to deplete serotonin or norepinephrine, or vehicle control. Following surgical recovery, rats received half second bursts of 0.8 mA or sham VNS after successful trials. After five days of pairing intracortical microstimulation (ICMS) was performed in the motor cortex contralateral to the trained limb. VNS paired with training more than doubled cortical representations of proximal forelimb movements. Depletion of either cortical norepinephrine or serotonin prevented this effect. The requirement of multiple neuromodulators is consistent with earlier studies showing that these neuromodulators regulate synaptic plasticity in a complimentary fashion.

Peripheral nerve stimulation has emerged as a platform therapy to treat a wide range of disorders. Continued development and translation of these strategies requires that researchers have access to reliable, customizable electrodes for nerve stimulation. Here, we detail procedures to build three different configurations of cuff electrodes with varying numbers and orientations of contacts for nerve stimulation in rats. These designs are built with simple, widely available materials, using platinum-iridium electrodes assembled into polyurethane tubing. Moreover, the designs can easily be customized to increase versatility and individualize for specific stimulation applications. This protocol provides a resource to facilitate the construction and customization of stimulation cuffs to support preclinical nerve stimulation research.

Vagus nerve stimulation (VNS) has shown promise as an adjuvant treatment for posttraumatic stress disorder (PTSD), as it enhances fear extinction and reduces anxiety symptoms in multiple rat models of this condition. Yet, identification of the optimal stimulation paradigm is needed to facilitate clinical translation of this potential therapy. Using an extinction-resistant rat model of PTSD, we tested whether varying VNS intensity and duration could maximize extinction learning while minimizing the total amount of stimulation. We confirmed that sham rats failed to extinguish after a week of extinction training. Delivery of the standard LONG VNS trains (30 s) at 0.4 mA enhanced extinction and reduced anxiety but did not prevent fear return. Increasing the intensity of LONG VNS trains to 0.8 mA prevented fear return and attenuated anxiety symptoms. Interestingly, delivering 1, 4 or 16 SHORT VNS bursts (0.5 s) at 0.8 mA during each cue presentation in extinction training also enhanced extinction. LONG VNS trains or multiple SHORT VNS bursts at 0.8 mA attenuated fear renewal and reinstatement, promoted extinction generalization and reduced generalized anxiety. Delivering 16 SHORT VNS bursts also facilitated extinction in fewer trials. This study provides the first evidence that brief bursts of VNS can enhance extinction training, reduce relapse and support symptom remission using much less VNS than previous protocols. These findings suggest that VNS parameters can be adjusted in order to minimize total charge delivery and maximize therapeutic effectiveness.

Pairing vagus nerve stimulation (VNS) with rehabilitation has emerged as a potential strategy to improve recovery after neurological injury, an effect ascribed to VNS-dependent enhancement of synaptic plasticity. Previous studies demonstrate that pairing VNS with forelimb training increases forelimb movement representations in motor cortex. However, it is not known whether VNS-dependent enhancement of plasticity is restricted to forelimb training or whether VNS paired with other movements could induce plasticity of other motor representations. We tested the hypothesis that VNS paired with orofacial movements associated with chewing during an unskilled task would drive a specific increase in jaw representation in motor cortex compared to equivalent behavioral experience without VNS. Rats performed a behavioral task in which VNS at a specified intensity between 0 and 1.2 mA was paired with chewing 200 times per day for five days. Intracortical microstimulation (ICMS) was then used to document movement representations in motor cortex. VNS paired with chewing at 0.8 mA significantly increased motor cortex jaw representation compared to equivalent behavioral training without stimulation (Bonferroni-corrected unpaired t-test, p < 0.01). Higher and lower intensities failed to alter cortical plasticity. No changes in other movement representations or total motor cortex area were observed between groups. These results demonstrate that 0.8 mA VNS paired with training drives robust plasticity specific to the paired movement, is not restricted to forelimb representations, and occurs with training on an unskilled task. This suggests that moderate intensity VNS may be a useful adjuvant to enhance plasticity and support benefits of rehabilitative therapies targeting functions beyond upper limb movement.

A variety of skilled reaching tasks have been developed to evaluate forelimb function in rodent models. The single pellet skilled reaching task and pasta matrix task have provided valuable insight into recovery of forelimb function in models of neurological injury and disease. Recently, several automated measures have been developed to reduce the cost and time burden of forelimb assessment in rodents. Here, we provide a within-subject comparison of three common forelimb assessments to allow direct evaluation of sensitivity and efficiency across tasks. Rats were trained to perform the single pellet skilled reaching task, the pasta matrix task, and the isometric pull task. Once proficient on all three tasks, rats received an ischemic lesion of motor cortex and striatum to impair use of the trained limb. On the second week post-lesion, all three tasks measured a significant deficit in forelimb function. Performance was well-correlated across tasks. By the sixth week post-lesion, only the isometric pull task measured a significant deficit in forelimb function, suggesting that this task is more sensitive to chronic impairments. The number of training days required to reach asymptotic performance was longer for the isometric pull task, but the total experimenter time required to collect and analyze data was substantially lower. These findings suggest that the isometric pull task represents an efficient, sensitive measure of forelimb function to facilitate preclinical evaluation in models of neurological injury and disease.

Peripheral nerve injury is a common cause of lifelong disability in the United States. Although the etiology varies, most traumatic nerve injuries occur in the upper limb and include damage to the radial nerve. In conjunction with the well-described effects of peripheral damage, nerve injuries are accompanied by changes in the central nervous system. A comprehensive understanding of the functional consequences of nerve injury is necessary to develop new therapeutic interventions.

Vagus nerve stimulation (VNS) paired with motor rehabilitation enhances recovery of function after neurological injury in rats and humans. This effect is ascribed to VNS-dependent facilitation of plasticity in motor networks. Previous studies document an inverted-U relationship between VNS intensity and cortical plasticity, such that moderate intensities increase plasticity, while low or high intensity VNS does not. We tested the interaction of moderate and high intensity VNS trains to probe the mechanisms that may underlie VNS-dependent plasticity. Rats performed a behavioral task where VNS was paired with jaw movement during chewing. For five days, subjects received 100 pairings of moderate intensity VNS (Standard VNS), 100 pairings alternating between moderate and high intensity VNS (Interleaved VNS), or 50 pairings of moderate intensity VNS (Short VNS) approximately every 8 s. After the final behavioral session, intracortical microstimulation (ICMS) was used to evaluate movement representations in motor cortex. 100 pairings of moderate intensity VNS enhanced motor cortex plasticity. Replacing half of moderate intensity stimulation with high intensity VNS blocked this enhancement of plasticity. Removing high intensity stimulation, leaving only 50 pairings of moderate intensity VNS, reinstated plasticity. These results demonstrate that there is a period for at least 8 s after high intensity stimulation in which moderate intensity VNS is not able to engage mechanisms required for synaptic reorganization. More importantly, this study demonstrates that changes in stimulation parameters are a critical determinant of the magnitude of plasticity and likely the efficacy of VNS-enhanced recovery.

Nerve injury affecting the upper limb is a leading cause of lifelong disability. Damage to the nerves in the arm often causes weakness and somatosensory dysfunction ranging from numbness to pain. Previous studies show that combining brief bursts of electrical vagus nerve stimulation (VNS) with motor or tactile rehabilitation can restore forelimb function after median and ulnar nerve injury, which causes hyposensitivity of the ventral forelimb. Here, we sought to determine whether this approach would be similarly effective in a model of radial nerve injury that produces allodynia in the ventral forelimb. To test this, rats underwent complete transection of the radial nerve proximal to the elbow followed by tubular repair. In the first experiment, beginning ten weeks after injury, rats received six weeks of tactile rehabilitation, consisting of mechanical stimulation of either the dorsal or ventral region of the forepaw in the injured limb, with or without concurrent VNS. In a second experiment, a separate cohort of rats underwent six weeks of forelimb motor rehabilitative training with or without paired VNS. Contrary to findings in previous models of hyposensitivity, VNS therapy fails to improve recovery of either somatosensory or motor function in the forelimb after radial nerve injury. These findings describe initial evidence that pain may limit the efficacy of VNS therapy and thus highlight a characteristic that should be considered in future studies that seek to develop this intervention.

Advanced age is associated with a higher incidence of stroke and worse functional outcomes. Vagus nerve stimulation (VNS) paired with rehabilitative training has emerged as a potential method to improve recovery after brain injury but to date has only been evaluated in young rats. Here, we evaluated whether VNS paired with rehabilitative training would improve recovery of forelimb function after ischemic lesion of the motor cortex in rats 18 months of age. Rats were trained to perform the isometric pull task, an automated, quantitative measure of volitional forelimb strength. Once proficient, rats received an ischemic lesion of the motor cortex and underwent rehabilitative training paired with VNS for 6 weeks. VNS paired with rehabilitative training significantly enhances recovery of forelimb function after lesion. Rehabilitative training without VNS results in a 34% ± 19% recovery, whereas VNS paired with rehabilitative training yields a 98% ± 8% recovery of prelesion of forelimb function. VNS does not significantly reduce lesion size. These findings demonstrate that VNS paired with rehabilitative training enhances motor recovery in aged subjects in a model of stroke and may suggest that VNS therapy may effectively translate to elderly stroke patients.

Vagus nerve stimulation (VNS) has rapidly gained interest as a treatment for a variety of disorders. A number of methods have been employed to stimulate the vagus nerve, but the most common relies on a cuff electrode implanted around the cervical branch of the nerve. Recently, two non-invasive methods have increased in popularity: transcutaneous cervical VNS (tcVNS) and transcutaneous auricular VNS (taVNS). Despite promising clinical results, there has been little direct comparison of these methods to stimulation delivered via an implanted device. In this study, we directly compared both non-invasive strategies to stimulation with an implanted cuff electrode on activation of the Hering-Breuer (HB) reflex, a non-invasive biomarker of A-fiber activation in the vagus. Stimulation was delivered across a wide range of parameters using tcVNS, taVNS, and an implanted cuff electrode in female rats. Activation of the HB reflex, changes in heart rate, and neck muscle twitch force were recorded. Consistent with low thresholds reported in previous studies, we found that the threshold to activate the HB reflex using an implanted cuff electrode was 0.406 ± 0.066 mA. tcVNS was capable of activating the HB reflex, but the threshold was 34.18 ± 1.86 mA, over 15 fold higher than the stimulation intensity that caused twitching of the neck muscles (2.09 ± 0.16 mA). No activation of the HB reflex was observed with taVNS at any parameters. These results describe activation of the HB reflex with each strategy and provide initial evidence regarding differences in the activation of the vagus nerve with invasive and non-invasive methods.

Rett syndrome is an X-linked neurodevelopmental disorder caused by a mutation in the gene MECP2. Individuals with Rett syndrome display developmental regression at an early age, and develop a range of motor, auditory, cognitive, and social impairments. Several studies have successfully modeled some aspects of dysfunction and Rett syndrome-like phenotypes in transgenic mouse and rat models bearing mutations in the MECP2 gene. Here, we sought to extend these findings and characterize skilled learning, a more complex behavior known to be altered in Rett syndrome.

Loss of sensory function is a common consequence of neurological injury. Recent clinical and preclinical evidence indicates vagus nerve stimulation (VNS) paired with tactile rehabilitation, consisting of delivery of a variety of mechanical stimuli to the hyposensitive skin surface, yields substantial and long-lasting recovery of somatosensory function after median and ulnar nerve transection and repair. Here, we tested the hypothesis that a specific component of the tactile rehabilitation paired with VNS is necessary for recovery of somatosensory function. In a second experiment in a separate cohort, we investigated whether VNS paired with tactile rehabilitation could improve skilled forelimb motor function. Elements of the study design, including planned sample size, assessments, and statistical comparisons, were preregistered prior to beginning data collection (https://osf.io/3tm8u/). Animals received a peripheral nerve injury (PNI) causing chronic sensory loss. Eight weeks after injury, animals were given a VNS implant followed by six weeks of tactile rehabilitation sessions consisting of repeated application of one of two distinct mechanical stimuli, a filament or a paintbrush, to the previously denervated forepaw. VNS paired with either filament indentation or brushing of the paw significantly improved recovery of forelimb withdrawal thresholds after PNI compared to tactile rehabilitation without VNS. The effect size was twice as large when VNS was paired with brushing compared to VNS paired with point indentation. An independent replication in a second cohort confirmed that VNS paired with brush restored forelimb withdrawal thresholds to normal. These rats displayed significant improvements in performance on a skilled forelimb task compared to rats that did not receive VNS. These findings support the utility of pairing VNS with tactile rehabilitation to improve recovery of somatosensory and motor function after neurological injury. Additionally, this study demonstrates that the sensory characteristics of the rehabilitation paired with VNS determine the degree of recovery.

Recovery from serious neurological injury requires substantial rewiring of neural circuits. Precisely-timed electrical stimulation could be used to restore corrective feedback mechanisms and promote adaptive plasticity after neurological insult, such as spinal cord injury (SCI) or stroke. This study provides the first evidence that closed-loop vagus nerve stimulation (CLV) based on the synaptic eligibility trace leads to dramatic recovery from the most common forms of SCI. The addition of CLV to rehabilitation promoted substantially more recovery of forelimb function compared to rehabilitation alone following chronic unilateral or bilateral cervical SCI in a rat model. Triggering stimulation on the most successful movements is critical to maximize recovery. CLV enhances recovery by strengthening synaptic connectivity from remaining motor networks to the grasping muscles in the forelimb. The benefits of CLV persist long after the end of stimulation because connectivity in critical neural circuits has been restored.

Cervical spinal cord injury (cSCI) often causes chronic upper extremity disability. Reliable measurement of arm function is critical for development of therapies to improve recovery after cSCI. In this study, we report a suite of automated rehabilitative tools to allow simple, quantitative assessment of hand and wrist motor function.

Chronic impairment of the arm and hand is a common consequence of stroke. Animal and human studies indicate that brief bursts of vagus nerve stimulation (VNS) in conjunction with rehabilitative training improve recovery of motor function after stroke. In this study, we tested whether VNS could promote generalization, long-lasting recovery, and structural plasticity in motor networks.

Enhanced metabotropic glutamate receptor subunit 5 (mGluR5) function is causally associated with the pathophysiology of fragile X syndrome, a leading inherited cause of intellectual disability and autism. Here we provide evidence that altered mGluR5-Homer scaffolds contribute to mGluR5 dysfunction and phenotypes in the fragile X syndrome mouse model, Fmr1 knockout (Fmr1(-/y)). In Fmr1(-/y) mice, mGluR5 was less associated with long Homer isoforms but more associated with the short Homer1a. Genetic deletion of Homer1a restored mGluR5-long Homer scaffolds and corrected several phenotypes in Fmr1(-/y) mice, including altered mGluR5 signaling, neocortical circuit dysfunction and behavior. Acute, peptide-mediated disruption of mGluR5-Homer scaffolds in wild-type mice mimicked many Fmr1(-/y) phenotypes. In contrast, Homer1a deletion did not rescue altered mGluR-dependent long-term synaptic depression or translational control of target mRNAs of fragile X mental retardation protein, the gene product of Fmr1. Our findings reveal new functions for mGluR5-Homer interactions in the brain and delineate distinct mechanisms of mGluR5 dysfunction in a mouse model of cognitive dysfunction and autism.

Nerve damage can cause chronic, debilitating problems including loss of motor control and paresthesia, and generates maladaptive neuroplasticity as central networks attempt to compensate for the loss of peripheral connectivity. However, it remains unclear if this is a critical feature responsible for the expression of symptoms. Here, we use brief bursts of closed-loop vagus nerve stimulation (CL-VNS) delivered during rehabilitation to reverse the aberrant central plasticity resulting from forelimb nerve transection. CL-VNS therapy drives extensive synaptic reorganization in central networks paralleled by improved sensorimotor recovery without any observable changes in the nerve or muscle. Depleting cortical acetylcholine blocks the plasticity-enhancing effects of CL-VNS and consequently eliminates recovery, indicating a critical role for brain circuits in recovery. These findings demonstrate that manipulations to enhance central plasticity can improve sensorimotor recovery and define CL-VNS as a readily translatable therapy to restore function after nerve damage.

The majority of available systems for vagus nerve stimulation use helical stimulation electrodes, which cover the majority of the circumference of the nerve and produce largely uniform current density within the nerve. Flat stimulation electrodes that contact only one side of the nerve may provide advantages, including ease of fabrication. However, it is possible that the flat configuration will yield inefficient fiber recruitment due to a less uniform current distribution within the nerve. Here we tested the hypothesis that flat electrodes will require higher current amplitude to activate all large-diameter fibers throughout the whole cross-section of a nerve than circumferential designs. Computational modeling and in vivo experiments were performed to evaluate fiber recruitment in different nerves and different species using a variety of electrode designs. Initial results demonstrated similar fiber recruitment in the rat vagus and sciatic nerves with a standard circumferential cuff electrode and a cuff electrode modified to approximate a flat configuration. Follow up experiments comparing true flat electrodes to circumferential electrodes on the rabbit sciatic nerve confirmed that fiber recruitment was equivalent between the two designs. These findings demonstrate that flat electrodes represent a viable design for nerve stimulation that may provide advantages over the current circumferential designs for applications in which the goal is uniform activation of all fascicles within the nerve.

Chronic sensory loss is a common and undertreated consequence of many forms of neurological injury. Emerging evidence indicates that vagus nerve stimulation (VNS) delivered during tactile rehabilitation promotes recovery of somatosensation.

Studies have indicated that vagus nerve stimulation (VNS) enhances extinction learning in rodent models. Here, we investigated if pairing VNS with the conditioned stimulus is required for the enhancing effects of VNS. Adult Sprague-Dawley rats were exposed to intense stress followed by fear conditioning training to produce resistant fear. Rats were then implanted with a cuff electrode around the left vagus. After recovery, rats underwent extinction training paired with VNS (0.5 s, 0.8 mA, 100 µs, and 30 Hz) or with Sham VNS (0 mA). VNS rats were randomized into the following subgroups: During VNS (delivered during presentations of the conditioned stimulus, CS), Between VNS (delivered between CS presentations), Continuous VNS (delivered during the entire extinction session), and Dispersed VNS (delivered at longer inter-stimulation intervals across the extinction session). Sham VNS rats failed to extinguish the conditioned fear response over 5 days of repeated exposure to the CS. Rats that received Between or Dispersed VNS showed modest improvement in conditioned fear at the retention test. During and Continuous VNS groups displayed the greatest reduction in conditioned fear. These findings indicate that delivering VNS paired precisely with CS presentations or continuously throughout extinction promotes the maximum enhancement in extinction learning.