The vagus nerve (VN) plays an important role in regulating physiological conditions in the gastrointestinal (GI) tract by communicating via the parasympathetic pathway to the enteric nervous system (ENS). However, the lack of knowledge in the neurophysiology of the VN and GI tract limits the development of advanced treatments for autonomic dysfunctions related to the VN. To better understand the complicated underlying mechanisms of the VN-GI tract neurophysiology, it is necessary to use an advanced device enabled by microfabrication technologies. Among several candidates including intraneural probe array and extraneural cuff electrodes, microchannel electrode array devices can be used to interface with smaller numbers of nerve fibers by securing them in the separate channel structures. Previous microchannel electrode array devices to interface teased nerve structures are relatively bulky with thickness around 200 µm. The thick design can potentially harm the delicate tissue structures, including the nerve itself. In this paper, we present a flexible thin film based microchannel electrode array device (thickness: 11.5 µm) that can interface with one of the subdiaphragmatic nerve branches of the VN in a rat. We demonstrated recording evoked compound action potentials (ECAP) from a transected nerve ending that has multiple nerve fibers. Moreover, our analysis confirmed that the signals are from C-fibers that are critical in regulating autonomic neurophysiology in the GI tract.

Neuromodulation by vagus nerve stimulation (VNS) provides therapeutic benefits in multiple medical conditions, including epilepsy and clinical depression, but underlying mechanisms of action are not well understood. Cervical vagus nerve biopsies were procured from transplant organ donors for high resolution light microscopy (LM) and transmission electron microscopy (TEM) to map the human fascicular and sub-fascicular organization. Cervical vagal segments show laterality with right sided dominance in fascicle numbers and cross-sectional areas as well as sexual dimorphism with female dominance in fascicle numbers. The novel and unprecedented detection of numerous small fascicles by high resolution LM and TEM expand the known fascicle size range and morphological diversity of the human vagus nerve. Ground truth TEM quantification of all myelinated and unmyelinated axons within individual nerve fascicles show marked sub-fascicular heterogeneity of nerve fiber numbers, size, and myelination. A heuristic action potential interpreter (HAPI) tool predicts VNS-evoked compound nerve action potentials (CNAPs) generated by myelinated and unmyelinated nerve fibers and validates functional dissimilarity between fascicles. Our findings of laterality, sexual dimorphism, and an expanded range of fascicle size heterogeneity provide mechanistic insights into the varied therapeutic responses and off-target effects to VNS and may guide new refinement strategies for neuromodulation. Ultrastructural mapping of the human vagus nerve and compound nerve action potential predictions to vagus nerve stimulation suggest that vagal projectome features, including laterality, sexual dimorphism, and heterogeneity, shape neuromodulation.

Vagal preganglionic neurons innervate myenteric ganglia. These autonomic efferents are distributed so densely within the ganglia that it has been impractical to track individual vagal axons through the myenteric plexus with tracer labeling. To evaluate whether vagal efferent axons evidence selectivity, particularly for nitrergic or non-nitrergic myenteric neurons within the plexus, we limited the numbers and volumes of brainstem dextran biotin tracer injections per animal. Reduced labeling and the use of immunohistochemistry generated cases in which some individual axons could be distinguished and traced in three dimensions (Neurolucida) within and among successive (up to 46) myenteric ganglia. In the myenteric plexus of all stomach regions, the majority (∼86%) of vagal efferents were organized into two distinct subtypes. One subtype (∼24% of dextran-labeled efferents, designated “primarily nitrergic”) selectively contacted and linked—both within and between ganglia—nitric oxide synthase positive (nNOS+) neurons into presumptive motor modules. A second subtype (∼62% of efferents, designated “primarily non-nitrergic”) appeared to selectively contact and link—both within and between ganglia—non-nitrergic enteric neurons into a second type of effector ensemble. A third candidate type (∼14% of labeled preganglionics), appeared to lack “nitrergic selectivity” and to contact both nNOS+ and nNOS− enteric neurons. In addition to the quantitative assessment of the efferent axons in stomach, qualitative observations of the proximal duodenum indicated similar selective vagal efferent projections, in proportions comparable with those evaluated in the stomach. Limited injections of tracer, three-dimensional (3-D) tracing of individual axons, and histochemistry of myenteric neurons might distinguish additional efferent phenotypes.

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin-biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: (1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. (2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. (3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric neurons and the circular muscle, exhibiting polymorphic terminal structures. (4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

A thorough understanding of the neuroanatomy of peripheral nerves is required for a better insight into their function and the development of neuromodulation tools and strategies. In biophysical modeling, it is commonly assumed that the complex spatial arrangement of myelinated and unmyelinated axons in peripheral nerves is random, however, in reality the axonal organization is inhomogeneous and anisotropic. Present quantitative neuroanatomy methods analyze peripheral nerves in terms of the number of axons and the morphometric characteristics of the axons, such as area and diameter. In this study, we employed spatial statistics and point process models to describe the spatial arrangement of axons and Sinkhorn distances to compute the similarities between these arrangements (in terms of first- and second-order statistics) in various vagus and pelvic nerve cross-sections. We utilized high-resolution transmission electron microscopy (TEM) images that have been segmented using a custom-built high-throughput deep learning system based on a highly modified U-Net architecture. Our findings show a novel and innovative approach to quantifying similarities between spatial point patterns using metrics derived from the solution to the optimal transport problem. We also present a generalizable pipeline for quantitative analysis of peripheral nerve architecture. Our data demonstrate differences between male- and female-originating samples and similarities between the pelvic and abdominal vagus nerves.

Nociceptive afferent axons innervate the stomach and send signals to the brain and spinal cord. Peripheral nociceptive afferents can be detected with a variety of markers [e.g., substance P (SP) and calcitonin gene-related peptide (CGRP)]. We recently examined the topographical organization and morphology of SP-immunoreactive (SP-IR) axons in the whole mouse stomach muscular layer. However, the distribution and morphological structure of CGRP-IR axons remain unclear. We used immunohistochemistry labeling and applied a combination of imaging techniques, including confocal and Zeiss Imager M2 microscopy, Neurolucida 360 tracing, and integration of axon tracing data into a 3D stomach scaffold to characterize CGRP-IR axons and terminals in the whole mouse stomach muscular layers. We found that: 1) CGRP-IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP-IR axons densely innervated the blood vessels. 3) CGRP-IR axons ran in parallel with the longitudinal and circular muscles. Some axons ran at angles through the muscular layers. 4) They also formed varicose terminal contacts with individual myenteric ganglion neurons. 5) CGRP-IR occurred in DiI-labeled gastric-projecting neurons in the dorsal root and vagal nodose ganglia, indicating CGRP-IR axons were visceral afferent axons. 6) CGRP-IR axons did not colocalize with tyrosine hydroxylase (TH) or vesicular acetylcholine transporter (VAChT) axons in the stomach, indicating CGRP-IR axons were not visceral efferent axons. 7) CGRP-IR axons were traced and integrated into a 3D stomach scaffold. For the first time, we provided a topographical distribution map of CGRP-IR axon innervation of the whole stomach muscular layers at the cellular/axonal/varicosity scale.

Vagus nerve stimulation (VNS) has the potential to treat various peripheral dysfunctions, but the traditional cuff electrodes for VNS are susceptible to off-target effects. Microelectrodes may enable highly selective VNS that can mitigate off-target effects, but they suffer from the increased impedance. Recent studies on microelectrodes with non-Euclidean geometries have reported higher energy efficiency in neural stimulation applications. These previous studies use electrodes with mm/cm-scale dimensions, mostly targeted for myelinated fibers. This study evaluates fractal microelectrodes for VNS in a rodent model (N = 3). A thin-film device with fractal and circle microelectrodes is fabricated to compare their neural stimulation performance on the same radial coordinate of the nerve. The results show that fractal microelectrodes can activate C-fibers with up to 52% less energy (p = 0.012) compared to circle microelectrodes. To the best of the knowledge, this work is the first to demonstrate a geometric advantage of fractal microelectrodes for VNS in vivo.

This protocol describes the methods used to generate samples of human vagus tissue suitable for TEM imaging.

More than 50 million Americans suffer from chronic pain. However, the anatomical and physiological mechanisms of peripheral nociceptive processes have not been well elucidated which has seriously impeded the progress of designing novel bioengineering manipulations/treatments for chronic pain. In this study, we performed a comprehensive topographical mapping of pain-related neural circuitry in the flat-mount of whole mouse stomach (male, n=8, 3-5 months). We used Substance P (SP) as a marker for nociceptive axons and applied a combination of state-of-the-art techniques, including flat-mount tissue processing and immunohistochemistry of the whole stomach, confocal microscopy, Zeiss Imager microscopy to determine the distribution and morphology of SP-IR axons and terminals in the whole stomach. We found that 1) SP-IR axons formed extensive terminal networks in both the ventral and dorsal stomachs. 2) SP-IR axons were much denser in the antrum and corpus regions than in the fundus and cardia. 3) SP-IR varicose axons ran in parallel with the circular and longitudinal muscle layers. 4) SP-IR axons innervated blood vessels. 5) SP-IR varicose axons formed terminals wrapping around individual myenteric neurons. 6) There were no confirmed SP-IR myenteric neurons. 7) SP-IR afferent innervation of the stomach showed a similar pattern of SP-IR axons and terminals between the ventral and dorsal stomachs. In control mice (n=8), we injected tracer DiI into the ventral and dorsal stomach muscular layers or Fluorogold injection (i.p) and found that the main extrinsic source of SP-IR afferent axons in the stomach was from the T7-T11 DRG and to a lesser extent the VNG, but not from the celiac ganglia and dorsal motor nucleus of vagus. Our data provide for the first time a comprehensive topographical map for SP-IR axons and terminals in the whole stomach with single cell/axon/synapse resolution. This work will contribute to a 3D digital representation of a brain-stomach nociceptive atlas in a stomach scaffold, thus providing a novel anatomical foundation for functional mapping of nociceptive afferent axons and their pathological remodeling in the stomach. The first two authors contributed equally to this work.

Background and Hypothesis: Vagus nerve stimulation (VNS) is used for treatment of epilepsy in over 100,000 patients worldwide and is a potential treatment for many inflammatory disorders. VNS-evoked compound nerve action potentials (CNAPs) may have applications as a biomarker of VNS treatment efficacy, but transcutaneous measurement of this activity is obscured by muscle artifacts. A more precise understanding of muscle activation patterns could improve recording and analysis protocols for isolating the vagus nerve (VN) CNAP. We hypothesize that analyzing multi-electrode array (MEA) surface recordings overlying the VN will allow us to characterize this muscle artifact. Project Methods: Five patients undergoing VNS therapy for epilepsy were enrolled in a clinical study at Indiana University School of Medicine (IRB #2006075899). A custom-made MEA with a grid of 32 soft foam electrodes was placed on the skin overlying the VN on each side of the neck. Surface potentials were recorded for approximately 20 minutes at the patient’s established device settings. Results: VNS-evoked potentials were visualized in all five patients. Two probable muscle artifacts were identified, defined as non-propagating features in the mean response to n > 600 pulses of VNS. The first had an onset latency of ~1-3 ms, and the second had a latency of ~7-10 ms. Both artifacts appeared primarily in the electrodes overlying the laryngeal muscles. Conclusion and Potential Impact: The short latency of the first muscle artifact suggests stimulus signal leakage activating the superior laryngeal nerve. The second artifact with longer latency is likely caused by the recurrent laryngeal nerve. Previous analyses may have mischaracterized the first muscle artifact as nerve activity. The use of MEA-based recordings clarifies our understanding of the VN’s response to VNS, which may lead to better treatment efficacy and the eventual development of personalized VNS therapies for epilepsy and a range of inflammatory disorders.

Nociceptive afferents innervate the stomach and send nociceptive signals centrally to the brain and locally to the enteric nervous system. Nociceptive afferents can be detected with a variety of different markers (e.g., CGRP, SP, TRPV1). However, the distribution and morphological structure of nociceptive axons and terminals have not yet been well determined in the flat‐mounts of the whole stomach. In this study, we used calcitonin gene‐related peptide (CGRP) as a marker to label nociceptive afferent axons and terminals in the in the flat mounts of the whole ventral and dorsal stomachs of mouse (n = 6/each side, 3‐5 months). We applied a combination of state‐of‐the‐art techniques, including confocal microscopy, Zeiss Imager microscopy, flat‐mount tissue processing of the whole organ and immunohistochemistry, Neurolucida 360 tracing and integration of the tracing data into a 3D stomach scaffold to determine the distribution and morphology of CGRP‐IR axons and terminals in the whole stomach. We found that 1) CGRP‐IR axons formed extensive terminal networks in both ventral and dorsal stomachs. 2) CGRP‐IR axons dramatically innervated the blood vessels. 3) In longitudinal and circular muscles, CGRP‐IR varicose axons ran in parallel with the direction of the muscles. 4) CGRP‐IR axons formed a complex network between the longitudinal and circular muscle layers. 5) In the myenteric ganglia, CGRP‐IR axons formed varicose terminal contacts with individual myenteric neurons. 6) We did not observe any significant CGRP‐IR myenteric neurons. 7) CGRP‐IR axon innervation of the blood vessels were traced and digitized and integrated into a 3D scaffold. This is the first time that we provided a topographical map of CGRP‐IR innervation of the whole stomach at single cell/axons resolution. The work provides an anatomical foundation for functional studies of CGRP‐IR axons in various regions of the stomach and their remodeling in diseases. The first two authors contributed equally to this work.

Nociceptive afferents innervate the stomach and send signals centrally to the brain and locally to stomach tissues. Nociceptive afferents can be detected with a variety of different markers. In particular, substance P (SP) is a neuropeptide and is one of the most commonly used markers for nociceptive nerves in the somatic and visceral organs. However, the topographical distribution and morphological structure of SP‐immunoreactive (SP‐IR) axons and terminals in the whole stomach have not yet been fully determined. In this study, we labeled SP‐IR axons and terminals in flat mounts of the ventral and dorsal halves of the stomach of mice. Flat‐mount stomachs, including the longitudinal and circular muscular layers and the myenteric ganglionic plexus, were processed with SP primary antibody followed by fluorescent secondary antibody and then scanned using confocal microscopy. We found that (1) SP‐IR axons and terminals formed an extensive network of fibers in the muscular layers and within the ganglia of the myenteric plexus of the whole stomach. (2) Many axons that ran in parallel with the long axes of the longitudinal and circular muscles were also immunoreactive for the vesicular acetylcholine transporter (VAChT). (3) SP‐IR axons formed very dense terminal varicosities encircling individual neurons in the myenteric plexus; many of these were VAChT immunoreactive. (4) The regional density of SP‐IR axons and terminals in the muscle and myenteric plexus varied in the following order from high to low: antrum‐pylorus, corpus, fundus, and cardia. (5) In only the longitudinal and circular muscles, the regional density of SP‐IR axon innervation from high to low were: antrum‐pylorus, corpus, cardia, and fundus. (6) The innervation patterns of SP‐IR axons and terminals in the ventral and dorsal stomach were comparable. Collectively, our data provide for the first time a map of the distribution and morphology of SP‐IR axons and terminals in the whole stomach with single‐cell/axon/synapse resolution. This work will establish an anatomical foundation for functional mapping of the SP‐IR axon innervation of the stomach and its pathological remodeling in gastrointestinal diseases.

Post‐Acute Sequelae of SARS‐CoV‐2 infection (PASC) is now recognized as a constellation of symptoms such as postural hypotension, anxiety, and “brain fog” in addition to autonomic nervous system dysfunction such as tachycardia and labile hypertension. Better quantification of these conditions, specifically, autonomic nervous system dysfunction, is desired for future diagnostics, treatment modalities, and gaining a further understanding of PASC. This research has led to application of a novel autonomic activity tracking algorithm and GUI suite from previous work, see Figure 1, to study the physiological autonomic activity in six patients who had recovered from acute SARS‐CoV‐2 infection a mean of six months prior but were still experiencing symptoms. By measuring skin sympathetic nervous activity (SKNA), heart rate variability, and the cutaneous electrogastrogram (EGG), all before and after a water meal challenge, the data was fed into a custom analysis pipeline, shown in Figure 1. Each of the PASC patients were compared to a mean response of 34 healthy controls, each undergoing a 20‐minute baseline recording and another 20‐minute recording after ingestion of an 8 oz water test meal. All six patients showed significantly abnormal heart rate variability on frequency domain analysis in predominantly the low frequency (LF) and very low frequency (VLF) bands, and less so in the high frequency (HF) band, suggesting sympathetic nerve dysfunction. Three patients showed a significant decrease in SKNA while two showed a significant increase. All patients showed an abnormal cutaneous EGG. As shown in Figure 2, the temporal responses of aSKNA, VLF, LF, HF, and EGG for the PASC patients revealed that on average they were statistically different (p<0.05) from the healthy controls’ responses respectively during 98.4%, 78.4%, 86.76%, 47.9%, and 86.1% of the 40‐minute time period in testing, 20 minutes of baseline and 20 minutes after ingestion. In conclusion, we are looking at the sympathetic, parasympathetic, and enteric nervous systems synced temporally for applications with classification and further stratification of PASC based on the temporal dynamics of their autonomic nervous system mediated coordination from digestion. This shows that SARS‐CoV02 infection appears to have a significant effect on sympathetic and parasympathetic autonomic nervous system function and may be responsible for the disturbances noted in PASC. This work provides the framework and example of use for further applications in autonomic disorder physiological response exploration and furthermore can be expanded to other areas of neuromodulation.

Axonal characterizations of connectomes in healthy and disease phenotypes are surprisingly incomplete and biased because unmyelinated axons, the most prevalent type of fibers in the nervous system, have largely been ignored as their quantitative assessment quickly becomes unmanageable as the number of axons increases. Herein, we introduce the first prototype of a high-throughput processing pipeline for automated segmentation of unmyelinated fibers. Our team has used transmission electron microscopy images of vagus and pelvic nerves in rats. All unmyelinated axons in these images are individually annotated and used as labeled data to train and validate a deep instance segmentation network. We investigate the effect of different training strategies on the overall segmentation accuracy of the network. We extensively validate the segmentation algorithm as a stand-alone segmentation tool as well as in an expert-in-the-loop hybrid segmentation setting with preliminary, albeit remarkably encouraging results. Our algorithm achieves an instance-level F-1 score of between 0.7 and 0.9 on various test images in the stand-alone mode and reduces expert annotation labor by 80% in the hybrid setting. We hope that this new high-throughput segmentation pipeline will enable quick and accurate characterization of unmyelinated fibers at scale and become instrumental in significantly advancing our understanding of connectomes in both the peripheral and the central nervous systems.

This protocol described the steps to acquire and analyze 4D gastric magnetic resonance imaging data under fast and fed states in healthy human subjects and patients with gastroparesis.

BACKGROUND:Time-sequenced magnetic resonance imaging (MRI) of the stomach is an emerging technique for non-invasive assessment of gastric emptying and motility. However, an automated and systematic image processing pipeline for analyzing dynamic 3D (ie, 4D) gastric MRI data has not been established. This study uses an MRI protocol for imaging the stomach with high spatiotemporal resolution and provides a pipeline for assessing gastric emptying and motility. METHODS:Diet contrast-enhanced MRI images were acquired from seventeen healthy humans after they consumed a naturalistic contrast meal. An automated image processing pipeline was developed to correct for respiratory motion, to segment and compartmentalize the lumen-enhanced stomach, to quantify total gastric and compartmental emptying, and to compute and visualize gastric motility on the luminal surface of the stomach. KEY RESULTS:The gastric segmentation reached an accuracy of 91.10 ± 0.43% with the Type-I error and Type-II error being 0.11 ± 0.01% and 0.22 ± 0.01%, respectively. Gastric volume decreased 34.64 ± 2.8% over 1 h where the emptying followed a linear-exponential pattern. The gastric motility showed peristaltic patterns with a median = 4 wave fronts (range 3-6) and a mean frequency of 3.09 ± 0.07 cycles per minute. Further, the contractile amplitude was stronger in the antrum than in the corpus (antrum vs. corpus: 5.18 ± 0.24 vs. 3.30 ± 0.16 mm; p < 0.001). CONCLUSIONS & INFERENCES:Our analysis pipeline can process dynamic 3D MRI images and produce personalized profiles of gastric motility and emptying. It will facilitate the application of MRI for monitoring gastric dynamics in research and clinical settings.

Objective. Gastric electrical stimulation (GES) is a bioelectric intervention for gastroparesis, obesity, and other functional gastrointestinal disorders. In a potential mechanism of action, GES activates the nerve endings of vagal afferent neurons and induces the vago-vagal reflex through the nucleus tractus solitarius (NTS) in the brainstem. However, it is unclear where and how to stimulate in order to optimize the vagal afferent responses. Approach. To address this question with electrophysiology in rats, we applied mild electrical currents to two serosal targets on the distal forestomach with dense distributions of vagal intramuscular arrays (IMAs) that innervated the circular and longitudinal smooth muscle layers. During stimulation, we recorded single and multi-unit responses from gastric neurons in NTS and evaluated how the recorded responses depended on the stimulus orientation and amplitude. Main results. We found that NTS responses were highly selective to the stimulus orientation for a range of stimulus amplitudes. The strongest responses were observed when the applied current flowed in the same direction as the IMAs in parallel with the underlying smooth muscle fibers. Our results suggest that gastric neurons in NTS may encode the orientation-specific activity of gastric smooth muscles relayed by vagal afferent neurons. Significance. This finding suggests that the orientation of GES is critical to effective engagement of vagal afferents and should be considered in light of the structural phenotypes of vagal terminals in the stomach.

The vagus nerve provides motor, sensory, and autonomic innervation of multiple organs, and electrical vagus nerve stimulation (VNS) provides an adjunctive treatment option for e.g. medication-refractory epilepsy and treatment-resistant depression. The mechanisms of action for VNS are not known, and high-resolution anatomical mapping of the human vagus nerve is needed to better understand its functional organization. Electron microscopy (EM) is required for the detection of both myelinated and unmyelinated axons, but access to well-preserved human vagus nerves for ultrastructural studies is sparse. Intact human vagus nerve samples were procured intra-operatively from deceased organ donors, and tissues were immediately immersion fixed and processed for EM. Ultrastructural studies of cervical and sub-diaphragmatic vagus nerve segments showed excellent preservation of the lamellated wall of myelin sheaths, and the axolemma of myelinated and unmyelinated fibers were intact. Microtubules, neurofilaments, and mitochondria were readily identified in the axoplasm, and the ultrastructural integrity of Schwann cell nuclei, Remak bundles, and basal lamina was also well preserved. Digital segmentation of myelinated and unmyelinated axons allowed for determination of fiber size and myelination. We propose a novel source of human vagus nerve tissues for detailed ultrastructural studies and mapping to support efforts to refine neuromodulation strategies, including VNS.