Elsevier

Neuroscience

Volume 140, Issue 1, 2006, Pages 259-268
Neuroscience

Pain mechanism
Inhibitory M2 muscarinic receptors are upregulated in both axotomized and intact small diameter dorsal root ganglion cells after peripheral nerve injury

https://doi.org/10.1016/j.neuroscience.2006.02.013Get rights and content

Abstract

Acetylcholine reduces nociceptive input in part by activating inhibitory M2 muscarinic receptors on primary sensory neurons, and acetylcholinesterase inhibitors and muscarinic agonists produce analgesia in humans and animals. M2 muscarinic receptors are upregulated in animals with diabetic neuropathy, but their level of expression and function after peripheral nerve injury has not been previously examined. This study tested, using intracellular Ca2+ response to membrane depolarization, the effect of the M2 muscarinic receptor agonist bethanechol on individual dorsal root ganglion cells from normal and L5-6 spinal nerve-ligated rats, followed by M2 muscarinic receptor immunostaining. We also examined functional transient receptor potential for vanilloids-1 activity by determining intracellular Ca2+ response evoked by capsaicin in M2 muscarinic receptor immunoreactive cells. In normal dorsal root ganglion cells, bethanechol inhibited the Ca2+ response in a concentration-related fashion, and this inhibition was blocked by the M2 muscarinic receptor antagonist gallamine. Cells expressing M2 muscarinic receptors by immunostaining were significantly inhibited by bethanechol, whereas those lacking positive staining were not. The proportion of studied dorsal root ganglion neurons with positive M2 muscarinic receptor staining increased significantly in the injured ipsilateral L5-6 and the uninjured ipsilateral L4 ganglia, but not in the contralateral dorsal root ganglion neurons compared with normals. In contrast, the proportion of neurons responding to capsaicin significantly decreased in the injured ipsilateral L5-6 dorsal root ganglion cells. These results suggest that inhibitory M2 muscarinic receptors are upregulated in small- and medium-sized axotomized dorsal root ganglion neurons and their uninjured neighbors following nerve injury, and may represent an appropriate target for analgesia in this setting.

Section snippets

Animals

Male Sprague–Dawley rats (Harlan Industries, Indianapolis, IN, USA), weighing 200–250 g, were used in this study. Animal surgery was confirmed with the Wake Forest University Guidelines on the ethical use of animals, and studies were performed under Animal Care and Use Committee approval. All experiments conformed with guidelines regarding the study of animals published by the International Association for the Study of Pain, including minimizing the number of animals studied and their

General observations and response to potassium stimulation

The L5 and L6 spinal nerve ligation strongly decreased the withdrawal threshold of the hindpaw ipsilateral to ligation from 40±4.1 g to 1.9±0.2 g (P<0.001, n=27). The withdrawal threshold in the contralateral hindpaw was also significantly decreased (P<0.001) in accordance with previous reports (Kim and Chung, 1992). Withdrawal threshold in the contralateral hindpaw (14±1.9 g) was nonetheless higher than our criterion for hypersensitivity (<4 g), and significantly higher than that of the paw

Discussion

This study utilized a nonspecific membrane depolarizing stimulus (KCl) to survey the populations of small- and medium-sized DRG neurons which were inhibited by bethanechol in normal animals and DRG neurons which were axotomized as well as their uninjured neighbors after spinal nerve ligation. Two key findings were observed: that the pharmacology and immunohistochemistry was consistent with inhibitory M2 mAChRs on these neurons in normal animals, and that the proportion of small to medium size

Conclusion

In summary, the current study, utilizing a functional and immunologic definition, supports the presence of inhibitory M2 mAChRs on sensory neurons and for the first time to our knowledge, demonstrates upregulation of these inhibitory receptors in uninjured DRG neurons whose axons intermingle with injured afferents following peripheral nerve injury. Increase in M2 AChR immunoreactivity and function but decreased TRPV1 agonist response following nerve injury argues that these proteins are

Acknowledgments

This work was supported in part by NIH grants GM35523 and NS42386.

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