Tetrodotoxin-resistant voltage-dependent sodium channels in identified muscle afferent neurons

Baker Laboratory of Pharmacology, Dept. of Pharmacology, Kirksville College of Osteopathic Medicine, AT Still Univ. of Health Sciences, Kirksville, MO 63501. .
Journal of Neurophysiology (Impact Factor: 2.89). 08/2012; 108(8):2230-41. DOI: 10.1152/jn.00219.2012
Source: PubMed


Muscle afferents are critical regulators of motor function (Group I and II) and cardiovascular responses to exercise (Group III and IV). However, little is known regarding the expressed voltage-dependent ion channels. We identified muscle afferent neurons in dorsal root ganglia (DRGs), using retrograde labeling to examine voltage-dependent sodium (Na(V)) channels. In patch-clamp recordings, we found that the dominant Na(V) current in the majority of identified neurons was insensitive to tetrodotoxin (TTX-R), with Na(V) current in only a few (14%) neurons showing substantial (>50%) TTX sensitivity (TTX-S). The TTX-R current was sensitive to a Na(V)1.8 channel blocker, A803467. Immunocytochemistry demonstrated labeling of muscle afferent neurons by a Na(V)1.8 antibody, which further supported expression of these channels. A portion of the TTX-R Na(V) current appeared to be noninactivating during our 25-ms voltage steps, which suggested activity of Na(V)1.9 channels. The majority of the noninactivating current was insensitive to A803467 but sensitive to extracellular sodium. Immunocytochemistry showed labeling of muscle afferent neurons by a Na(V)1.9 channel antibody, which supports expression of these channels. Further examination of the muscle afferent neurons showed that functional TTX-S channels were expressed, but were largely inactivated at physiological membrane potentials. Immunocytochemistry showed expression of the TTX-S channels Na(V)1.6 and Na(V)1.7 but not Na(V)1.1. Na(V)1.8 and Na(V)1.9 appear to be the dominant functional sodium channels in small- to medium-diameter muscle afferent neurons. The expression of these channels is consistent with the identification of these neurons as Group III and IV, which mediate the exercise pressor reflex.

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    • "The NaV1.8 channel has also been found in the soma of small diameter sensory C and Aδ neurons [24] which have been shown to be involved in nociception in models of neuropathic and inflammatory pain [25]. One study reported that the NaV1.8 channel is present in 86% of the muscle sensory afferent fibers, which makes it a useful marker of putative muscle nociceptors [26], and recently it has been found in trigeminal ganglion neurons innervating the rat masseter muscle [27]. Taken together, this evidence suggests that NaV1.8 channel mainly found in the peripheral sensory neurons and their expression on nerve fibers provides a method to identify putatively nociceptive afferent fibers [24,28]. "
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    ABSTRACT: Background: Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. Methods: Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. Results: A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly higher in patients (42.6%) compared to healthy controls (12.0%) (P < .001). Conclusions: This study showed that the 5-HT3A-receptor is highly expressed in human masseter and tibialis muscles and that there are more nerve fibers that express 5-HT3A-receptors in the masseter of women with myofascial TMD compared to healthy women. These findings indicate that 5-HT3-receptors might be up-regulated in myofascial TMD and could serve as potential biomarkers of chronic muscle pain.
    The Journal of Headache and Pain 09/2014; 15(1):63. DOI:10.1186/1129-2377-15-63 · 2.80 Impact Factor
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    • "Images were captured using a Nikon Eclipse 80i epifluorescence microscope, and neurons were measured using ImageJ ( Cell size was calculated , and positive fluorescent labeling was determined as described previously (Ramachandra et al. 2012). For immunohistochemistry, rats were killed as described above, and both gastrocnemius muscles were dissected out along with the tendons. "
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    ABSTRACT: The Exercise Pressor Reflex (EPR) is generated by group III and IV muscle afferents during exercise to increase cardiovascular function. Muscle contraction is triggered by acetylcholine (ACh), which is metabolized into choline that could serve as a signal of exercise-induced activity. We demonstrate that ACh can induce current in muscle afferents neurons isolated from male Sprague-Dawley rats. The nicotinic ACh receptors (nAChR) appear to be expressed by some group III-IV neurons since capsaicin (TRPV1) and/or ATP (P2X) induced current in 56% of ACh-responsive neurons. α7 and α4ß2 nAChRs have been shown to be expressed in sensory neurons. An α7 nAChR antibody stained 83% of muscle afferent neurons. Functional expression was demonstrated by using the specific α7 nAChR blockers α-conotoxin IMI (IMI) and methyllycaconitine (MLA). MLA inhibited ACh responses in 100% of muscle afferent neurons, while IMI inhibited ACh responses in 54% of neurons. Dihydro-ß-erythroidine, an α4ß2 nAChR blocker, inhibited ACh responses in 50% of muscle afferent neurons, but recovery from block was not observed. Choline, an α7 nAChR agonist, elicited a response in 60% of ACh responsive neurons. Finally, we demonstrated the expression of α7 nAChR by peripherin labeled (group IV) afferent fibers within gastrocnemius muscles. Some of these α7 nAChR positive fibers were also positive for P2X3 receptors. Thus, choline could serve as an activator of the EPR by opening α7 nAChR expressed by group IV (and possible group III) afferents. nAChRs could become pharmacological targets for suppressing the excessive EPR activation in patients with peripheral vascular disease.
    Journal of Neurophysiology 06/2014; 112(6). DOI:10.1152/jn.00035.2014 · 2.89 Impact Factor
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    ABSTRACT: Sensory neurons in the dorsal root ganglia (DRG) express a subset of voltage dependent sodium channels (NaV) including NaV 1.1, 1.6, 1.7, 1.8 and 1.9. Previous work supported preferential localization of NaV 1.8 channels to small-medium diameter, nociceptive afferent neurons. However, we recently published evidence that NaV 1.8 was the dominant NaV channel expressed in the somas of small, medium and large diameter muscle afferent neurons, which is consistent with other reports. Here, we extend those results to show that NaV 1.8 expression is not correlated with afferent neuron diameter. Using immunocytochemistry, we found NaV 1.8 expression in ~50% of sensory afferent neurons with diameters ranging from 20 to 70 µm. In addition, electrophysiological analysis shows that the kinetic and inactivation properties of NaV 1.8 current are invariant with neuron size. These data add further support to the idea that NaV 1.8 contributes to the electrical excitability of both nociceptive and non-nociceptive sensory neurons.
    Channels (Austin, Tex.) 10/2012; 7(1). DOI:10.4161/chan.22445 · 2.32 Impact Factor
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