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 microg), non-selective muscarinic antagonist atropine (30 microg) or one of the muscarinic subtype antagonists: pirenzepine (M1, 10 microg), methoctramine (M2, 10 microg), 4-DAMP (M3, 10 microg), 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.

Fig. 1

Bar graphs showing effects of saline or cholinergic antagonists viz. mecamylamine (50 μg), atropine (30 μg), pirenzepine (10 μg), methoctramine (10 μg) or 4-DAMP (10 μg), on the antihyperalgesia produced by low frequency TENS. Intrathecal treatment with atropine, pirenzepine and 4-DAMP significantly (*, p < 0.01, Tukey’s post hoc test) attenuated the antihyperalgesic effects of low frequency TENS compared to intrathecal treatment with saline. Values are mean ± S.E.M. Contra = contralateral, Ipsi = Ipsilateral.

Fig. 2

Bar graphs showing effects of saline or cholinergic antagonists viz. mecamylamine (50 μg), atropine (30 μg), pirenzepine (10 μg), methoctramine (10 μg) or 4-DAMP (10 μg), on the antihyperalgesia produced by high frequency TENS. Atropine, pirenzepine and 4-DAMP significantly (*, p < 0.01, Tukey’s post hoc test) attenuated the antihyperalgesic effects of high frequency TENS, compared to saline group. Data are mean ± S.E.M. Contra = contralateral, Ipsi = Ipsilateral.

Fig. 3

Bar graphs showing the effects of intrathecal administration of saline or cholinergic antagonists viz. mecamylamine (50 μg), atropine (30 μg), pirenzepine (10 μg), methoctramine (10 μg) or 4-DAMP (10 μg) on kaolin/carrageenan-induced hyperalgesia, without TENS application. There was no effect on the hyperalgesia by any antagonist or saline. Values are mean ± S.E.M. Contra = contralateral, Ipsi = ipsilateral.

Fig. 4

Effects of muscarinic antagonists viz. atropine (30 μg), pirenzepine (10 μg), methoctramine (10 μg) or 4-DAMP (10 μg) on the antihyperalgesia caused by spinal carbachol (500 ng) (left panel) and effects of methoctramine on the antinociception caused by arecaidine (20 μg) (right panel). Methoctramine did not affect the antihyperalgesia caused by carbachol but reversed the antihyperalgesia caused by arecaidine. *Significantly different from saline effects (p≤0.01, Tukey’s) post hoc test, ^#Significantly different from saline control (p = 0.01, Independent samples t-test). Data shown are mean ± S.E.M.