Elsevier

Brain Research

Volume 1060, Issues 1–2, 26 October 2005, Pages 153-161
Brain Research

Research Report
Thoracic cross-over pathways of the rat vagal trunks

https://doi.org/10.1016/j.brainres.2005.08.037Get rights and content

Abstract

It is very difficult to study the independent contributions of the afferent and efferent pathways of the subdiaphragmatic vagus to physiology and behavior. Total subdiaphragmatic vagotomy can confound the interpretation of experimental results because it destroys both afferent and efferent vagal fibers. One approach to address this problem involves producing a total ablation of afferent (or efferent) vagal fibers while retaining half of the efferent (or afferent) vagal fibers by making a unilateral rhizotomy plus contralateral subdiaphragmatic vagotomy. However, the completeness of this afferent (or efferent) lesion is based on the assumption that there are no cross-over pathways within the thoracic cavity between the vagal trunks of the rat. To directly test for the presence of vagal cross-over pathways in the rat, we recorded the compound action potentials from the ventral and dorsal trunks of the subdiaphragmatic vagus following electrical stimulation of the left or right cervical vagi. C-fiber cross-over pathways comprised an average of 9% of the total nerve responses (range was 0 to 29%, n = 20). Direct application of the anesthetic bupivacaine to the vagus completely blocked the recorded signals. The vagal cross-over pathways were also demonstrated using capsaicin as a stimulus. These results indicate the presence of thoracic cross-over pathways between vagal trunks in the rat and demonstrate that for most animals it is not possible to produce a “complete” ablation of afferent (or efferent) components of the subdiaphragmatic vagus using unilateral rhizotomy combined with contralateral subdiaphragmatic vagotomy.

Introduction

The vagus is a mixed nerve containing afferent and efferent components that connect the brain to almost all of the internal organs (for review of the vagal afferent system see [2]). Considerable focus has been directed toward understanding the sensory functions of the subdiaphragmatic vagus because it is likely to play important roles in providing information to the brain for nutrition, immune signaling, and detection of toxins. Total subdiaphragmatic vagotomy is commonly used to investigate the role of the subdiaphragmatic vagus in physiology and behavior in the rat. However, results from studies using this lesion method are difficult to interpret because both the vagal afferent and efferent nerve fibers are sectioned. A primary side effect of total subdiaphragmatic vagotomy is gastrointestinal stasis and dysfunction that results from cutting vagal efferent fibers [23]. Total subdiaphragmatic vagotomized animals typically have bloated stomachs and must be fed a liquid diet to return body weight and food intake from reduced to normal levels [17].

Three techniques have been developed to selectively lesion the afferent fibers of the rat vagus. First, kainic acid injections into the nodose ganglia result in destruction of sensory cell bodies and appear not to damage efferent fibers of passage [20]; but, perhaps because of the relative difficulty of surgically isolating and injecting the nodose ganglia, this technique has not been widely used. Second, the neurotoxin capsaicin injected systemically or applied directly to the vagus results in destruction of vagal afferent fibers (e.g., [5], [31]). Unfortunately, many vagal afferent fibers are capsaicin-resistant and are not destroyed by this treatment [3]. A third technique uses a unilateral rhizotomy (cutting the afferent vagal rootlets as they exit the cranium before entering the nodose ganglion) combined with a contralateral subdiaphragmatic vagotomy [19], [24], [26], [28], [34], [35], [36], [37], [39]. The result of this method is a bilateral ablation of vagal afferent fibers and a unilateral lesion of vagal efferent fibers. This method can also be used to investigate the efferent system by selectively cutting the efferent vagal rootlets [39]. Unilateral rhizotomy plus contralateral subdiaphragmatic vagotomy is an improvement over the total subdiaphragmatic vagotomy because it does not produce noticeable gastrointestinal motor impairments and animals have normal levels of food intake and body weight shortly after surgery [26], [37], [39]. This methodology for selective ablation of the afferent (or efferent) vagal fibers relies on the assumption that there are no cross-over pathways between vagal trunks within the thoracic cavity of the rat. However, vagal cross-over pathways are well established for a another species, i.e., the thoracic communicating branch in the ferret [8], [9].

Although it is frequently stated that unilateral afferent rootlet rhizotomy combined with contralateral subdiaphragmatic vagotomy is a “complete” or “total” deafferentation of the subdiaphragmatic vagal system, there is some evidence from electrophysiology and tract-tracing studies for vagal cross-over pathways in the rat. Sauter and colleagues reported that electrical stimulation of either the left or right cervical vagus evoked compound action potentials from both the dorsal and ventral subdiaphragmatic vagal trunks [33]. Of the six rats tested, five were reported to show a minor left cross-over pathway and four a minor right cross-over pathway [33] (see Fig. 1 for a schematic of the minor and major pathways of the vagal system). Sauter et al. indicate that the minor vagal pathways accounted for approximately 20% of the total responses in those animals that showed minor pathway responses [33].

Anatomical tracing also suggests that there might be thoracic cross-over pathways between the rat vagal trunks. In contrast to the heavy staining occurring in the ipsilateral nodose ganglion, there were a few cells observed in the contralateral nodose ganglion when HRP (horseradish peroxidase) was applied to a specific subdiaphragmatic vagal trunk [27]. However, evidence suggests that HRP is a less sensitive method for tract tracing and could potentially underestimate the magnitude of a neural pathway (e.g., [11], [38]). More recently, a viral tract-tracing study from the stomach to the brainstem suggests that not all of the vagal afferent fibers are destroyed when rats receive a unilateral afferent rhizotomy combined with contralateral subdiaphragmatic vagotomy [32].

The current experiments were conducted to make a more complete assessment of the vagal cross-over pathways in the rat using electrophysiological techniques. Our experiments were designed to address five issues: (1) use a large group of animals to show descriptive statistics for any cross-over pathways, (2) eliminate sources of signal artifacts, potentially produced by animal movements and neural feedback, by paralyzing animals with pancuronium bromide and cutting the central and distal connections of the vagi, (3) greatly reduce the stimulator produced artifact, probably caused by capacitance discharge through electrode cables, by using biphasic current pulses (see [25]) and offline data processing to remove a slow artifact signal, using a 0.05 Hz high pass filter, (4) use a local anesthetic, bupivacaine, to determine if the electrically evoked compound action potential is neurally mediated, and (5) demonstrate cross-over pathways using a different stimulus, namely direct application of capsaicin to the vagus. This last issue is particularly important since electrical stimulation can produce electromyographic artifacts that can confound interpretations of compound action potential recordings from the vagus nerve (see [33]), but this should not be a problem when using a chemical stimulus, such as capsaicin. Capsaicin was used as a stimulus because it is known to activate a large portion of afferent fiber population of the vagus.

Section snippets

Subjects

Experiments were conducted on male Sprague–Dawley rats (350–500 g; Charles River, Kingston, NY, USA). Surgery and anesthesia is based on established methodology [13], [14]. Animals were euthanized at the conclusion of an experiment by jugular vein infusion of sodium pentobarbital (60 mg). All experiments conformed to established standards of the NIH and the Monell Center IACUC.

Surgery and electrophysiological recordings

Rats were initially anesthetized with sodium pentobarbital (i.p.; 50 mg/kg) followed by a continuous infusion of sodium

Experiment 1

Fig. 2 shows the raw data from a representative recording. A stimulus of 1 mA was used for all analyses because this produced the most reliable responses. Electrical stimulation of the cervical vagi evoked responses from the ventral and dorsal trunks that were dominated by two distinct phases of activity. A statistical analysis of the signals from 1 to 150 ms revealed that electrical stimulation produced statistically significant differences in responses from the major pathways compared to the

Discussion

The current electrophysiology experiments provide strong evidence for vagal cross-over pathways in the rat. It is likely that these cross-over pathways are within the thoracic cavity like they are in the ferret [8], [9]. Electrical stimulation of the left or right cervical vagi revealed C-fiber cross-over pathways that account for an average of 9% of the total response, but there was a great amount of variability (range = 0 to 29%) (see Fig. 6). A neural origin for these responses is indicated

Acknowledgments

This work was supported by grants from the National Institutes of Health (DK065971 and DK36339). The authors are grateful for the initial instruction in recording compound action potentials from the vagi of the rat and for the excellent comments on the manuscript by Dr. Ralph Norgren, Pennsylvania State College of Medicine. The authors also acknowledge the excellent technical assistance of Mr. Marc Ciucci.

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