Involvement of M 3 muscarinic receptors of the spinal cord in formalin-induced nociception in mice

Abstract

Subcutaneous injection of formalin into a paw of mice caused two distinct phases of licking and biting, first phase (1–5 min) and the second phase (7–30 min) after the injection. The muscarinic antagonist atropine (0.1–10 ng, i.t.) and the M 3 receptor antagonist 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP) (0.1–20 ng, i.t.) inhibited the second phase of this response, whereas higher doses of atropine (20–100 ng, i.t.) did not cause inhibition. The M 1 muscarinic receptor antagonist pirenzepine (10–100 ng, i.t.) did not inhibit either the first or the second phase response, but a high dose of pirenzepine (1000 ng, i.t.) tended to inhibit the second phase response. On the other hand, the M 2 muscarinic receptor antagonist 11-{(2-[(diethylamino)methyl]-1-piperidinyl}acetyl)-5,11-dihydro-6 H-pyrido(2,3- b)(1,4)benzodiazepine-6-one (AF-DX116; 10–1000 ng, i.t.) had no effect on either the first or the second phase of response. The opioid receptor antagonist naloxone did not affect the 4-DAMP-induced anti-nociceptive response. The i.t. injection of the acetylcholinesterase inhibitor neostigmine (25 ng) significantly inhibited only the second phase. The acetylcholine (ACh) depletor hemicholinium-3 (HC-3) (1 μg, i.t.) completely abolished the 4-DAMP-induced anti-nociceptive response. The ACh content of the spinal cord was significantly increased 14 min after formalin injection. This significant increase in the ACh content was inhibited by pretreatment with 4-DAMP (10 ng, i.t.). These results suggest that endogenous ACh in the spinal cord acts as a transmitter anti-nociception, and that ACh release regulated by presynaptic M 3 muscarinic receptors in the spinal cord is involved in the second phase of nociception induced by formalin.