Spinal muscarinic receptors are activated during low or high frequency TENS-induced antihyperalgesia in rats

Abstract

Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin–carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 μg), non-selective muscarinic antagonist atropine (30 μg) or one of the muscarinic subtype antagonists: pirenzepine (M₁, 10 μg), methoctramine (M₂, 10 μg), 4-DAMP (M₃, 10 μg), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M₁ and M₃ muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.

Introduction

Transcutaneous electrical nerve stimulation (TENS) is a therapeutic modality, clinically used to relieve acute and chronic pain (Johnson et al., 1992). TENS is easy to use for the patient and is devoid of major side effects. Although the use of TENS is very common, its analgesic mechanism is not fully understood. Two types of TENS are used clinically, low frequency TENS (frequency of stimulation<10 Hz) and high frequency TENS (frequency>50 Hz). Different theories have been proposed for the mechanism of action of TENS, the popular one being the gate control theory proposed by Melzack and Wall (1965). According to gate control theory, the nociceptive information from small diameter afferents is overridden by the stimulation of large diameter fibers and the pain stimulus is prevented from reaching supraspinal centers. However, neuropharmacological studies point towards neurotransmitter-related spinal and supraspinal mechanisms in TENS-induced analgesia. Endogenous opioids released in the central nervous system are implicated in the analgesic mechanism of TENS by various investigators (see Sluka and Walsh, 2003).

The antihyperalgesic effect of TENS predominantly involves central (spinal and supraspinal) mechanisms (Sluka et al., 1999, Kalra et al., 2001) rather than peripheral mechanisms (Janko and Trontelj, 1980). Spinally, opioid and serotonin receptors mediate TENS antihyperalgesia (Sluka et al., 1999, Radhakrishnan et al., 2003). Further, sensitization of dorsal horn neurons induced by carrageenan inflammation is reversed by high and low frequency TENS (Ma and Sluka, 2001). Descending inhibitory systems from the periaqueductal grey (PAG) and rostral ventromedial medulla (RVM) mediate analgesia through opioid, adrenergic and serotonin receptors spinally (Fields and Basbaum, 1999). In the spinal cord, inhibition also involves cholinergic receptors and they are clearly involved in spinal antinociception (Zhuo and Gebhart, 1991, Fang and Proudfit, 1996; reviewed by Eisenach, 1999, Pinardi et al., 2003). Among the spinal receptors, cholinergic receptors are unique in that they mediate antinociception through interactions with most of the inhibitory receptors, i.e. 5-HT, adrenergic or opioid receptors, in the spinal cord (Chiang and Zhuo, 1989, Gordh et al., 1989, Li et al., 1994, Obata et al., 2002, Chen and Pan, 2001, Honda et al., 2002). Both cholinergic nicotinic (Arimatsu et al., 1981, Ninkovic and Hunt, 1983; see review by Coggeshall and Carlton, 1997) and cholinergic muscarinic (Kayaalp and Neff, 1980; see review by Coggeshall and Carlton, 1997, Eisenach, 1999) receptors are localized to the dorsal horn, in laminae I–IV. Although nicotinic receptors are located in the spinal cord, their role in spinal antinociception is controversial. Thus, spinal cholinergic receptors are important in the inhibition of nociception through activation of opioid and 5-HT receptors.

Since there is compelling evidence for spinal and/or supraspinal opioid and 5-HT receptor mediation in TENS analgesia, we investigated the involvement of spinal cholinergic nicotinic and muscarinic receptors in the antihyperalgesic mechanisms of low and high frequency TENS, using selective antagonists. Selectivity and functional differences of the spinal cholinergic receptor subtypes were studied using selective antagonists against the spinal antinociceptive effects of muscarinic agonists.