["alpha2-Adrenergic agonists reduce mechanical and thermal hypersensitivity in animals with nerve injury and effectively treat neuropathic pain in humans. Previous studies indicate a reliance of alpha2-adrenergic agonists in this setting on spinal cholinergic activation and stimulation of muscarinic receptors. The subtype(s) of muscarinic receptors in the spinal cord that produces antinociception in normal animals is controversial, and those involved in reducing hypersensitivity and interacting with alpha2-adrenergic systems after nerve injury are unstudied. To examine this, the left L5 and L6 spinal nerves were tightly ligated in rats, resulting in reduction in withdrawal threshold to punctate mechanical stimuli. Intrathecal clonidine, 15 micro g, returned the withdrawal threshold to normal. Using highly specific m1 and m4 antagonists, we observed no reduction in the effect of clonidine by the m1 antagonist, but inhibition of clonidine's effect by the m4 antagonist. Western analysis revealed no difference in quantitative expression of m1 and m4 receptor protein in the dorsal spinal cord of spinal nerve-injured animals compared with sham-operated controls, suggesting this interaction with m4 receptors does not reflect an increase in receptor expression."] Does intrathecal clonidine reduce hypersensitivity after nerve injury by a mechanism involving spinal m4 muscarinic receptors?