Muscarinic excitatory and inhibitory mechanisms involved in afferent fibre‐evoked depolarization of motoneurones in the neonatal rat spinal cord
Authors: Kurihara Takashi , Suzuki Hidenori , Yanagisawa Mitsuhiko , Yoshioka Koichi 
Published: July 2012

The involvement of acetylcholine and muscarinic receptors in spinal synaptic responses evoked by electrical and noxious sensory stimuli was investigated in the neonatal rat spinal cord in vitro.
Potentials were recorded extracellularly from a ventral root (L3–L5) of the isolated spinal cord, spinal cord‐cutaneous nerve, and spinal cord‐skin preparations of 1‐ to 4‐day‐old rats. Spinal reflexes were elicited by electrical stimulation of the ipsilateral dorsal root or the cutaneous saphenous nerve, or by noxious skin stimulation.
Single shock stimulation of supramaximum intensity of a dorsal root induced a mono‐synaptic reflex in the corresponding ventral root. Bath‐application of the muscarinic agonists, muscarine (0.3–30 μm) and (+)‐cis‐dioxolane (0.1–100 μm), produced an inhibition of the mono‐synaptic reflex and a depolarization of motoneurones. Other muscarinic agonists, arecoline (10 nm–10 μm) and oxotremorine (10 nm–1 μm), inhibited the mono‐synaptic reflex with little or no depolarization of motoneurones. Repetitive stimulation of the saphenous nerve at C‐fibre strength induced a slow depolarizing response lasting about 30 s of the L3 ventral root. This slow ventral root potential (VRP) was also inhibited by arecoline (10 nm–10 μm) and oxotremorine (10 nm–1 μm).
In the spinal cord‐saphenous nerve‐skin preparation, a slow VRP was evoked by application of capsaicin (0.5 μm), bradykinin (3 μm), or noxious heat (47°C) to skin. This slow VRP was depressed by the muscarinic agonists, arecoline (3 μm) and oxotremorine (1 μm).
Of the (+)‐cis‐dioxolane‐induced inhibition of mono‐synaptic reflex and motoneurone depolarization, the M2 antagonists, AF‐DX 116 (0.1–1 μm) and methoctramine (100–300 nm), preferentially blocked the former response, whereas the M3 antagonists, 4‐DAMP (3–10 nm) and p‐F‐HHSiD (0.3–3 μm), preferentially blocked the latter response. AF‐DX 116 (0.1–1 μm) and methoctramine (100–300 nm) also effectively antagonized the arecoline‐ and oxotremorine‐induced inhibition of the slow VRP. The pA2 values of AF‐DX 116 and methoctramine against the arecoline‐induced inhibition of the mono‐synaptic reflex were both 6.79, and that of 4‐DAMP against the (+)‐cis‐dioxolane‐induced motoneurone depolarization was 8.16.
In the spinal cord‐cutaneous nerve preparation, the saphenous nerve‐evoked slow VRP was augmented by the anticholinesterase, edrophonium (5 μm). AF‐DX 116 (1 μm) and methoctramine (100 nm) also potentiated the slow VRP, whereas 4‐DAMP (10 nm) depressed the response. 4‐DAMP (5–10 nm) depressed the capsaicin‐induced slow VRP in the spinal cord‐skin preparation.
Oxotremorine (0.3 μm) and arecoline (1 μm) markedly depressed the depolarization of motoneurones evoked by application of capsaicin (3 μm) to the spinal cord, whereas they depressed only slightly the depolarization induced by substance P (10 nm).
The present study suggests that both excitatory (via M3‐type receptors) and inhibitory (via M2‐type receptors) muscarinic mechanisms are involved in afferent fibre‐evoked nociceptive transmissions in the neonatal rat spinal cord.