Role of presynaptic muscarinic and GABA(B) receptors in spinal glutamate release and cholinergic analgesia in rats

Abstract

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors can produce effective pain relief. However, the analgesic mechanisms and the site of actions of cholinergic agents in the spinal cord are not fully understood. In this study, we investigated the mechanisms underlying cholinergic presynaptic regulation of glutamate release onto spinal dorsal horn neurons. The role of spinal GABA(B) receptors in the antinociceptive action of muscarine was also determined. Whole-cell voltage-clamp recordings were performed on visualized dorsal horn neurons in the lamina II in the spinal cord slice preparation of rats. The miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded in the presence of tetrodotoxin. The evoked EPSCs (eEPSCs) were obtained by electrical stimulation of the dorsal root entry zone or the attached dorsal root. Nociception in rats was measured using a radiant heat stimulus and the effect of intrathecal administration of drugs tested. Acetylcholine (10-100 microM) reduced the amplitude of monosynaptic eEPSCs in a concentration-dependent manner. Acetylcholine also significantly decreased the frequency of non-NMDA receptor-mediated mEPSCs, which was antagonized by atropine but not mecamylamine. The frequency of GABA(A) receptor-mediated mIPSCs was significantly increased by acetylcholine and this excitatory effect was abolished by atropine. Existence of presynaptic M(2) muscarinic receptors in the spinal dorsal horn was further demonstrated by immunocytochemistry staining and dorsal rhizotomy. CGP55845, a GABA(B) receptor antagonist, significantly attenuated the inhibitory effect of acetylcholine on the frequency of mEPSCs and the amplitude of monosynaptic eEPSCs in lamina II neurons. Furthermore, the antinociceptive action produced by intrathecal muscarine was significantly reduced by CGP55845 pretreatment in rats. Therefore, data from this integrated study provide new information that acetylcholine inhibits the glutamatergic synaptic input to lamina II neurons through presynaptic muscarinic receptors. Inhibition of glutamate release onto lamina II neurons by presynaptic muscarinic and GABA(B) heteroreceptors in the spinal cord probably contributes to the antinociceptive action of cholinergic agents.

Figure 1. Identification of mono- and polysynaptic EPSCs in lamina II neurons evoked by electrical stimulation of the dorsal root entry zone in spinal cord slices

A, representative recordings showing monosynaptic eEPSCs in a lamina II neuron elicited at 0.2 and 20 Hz (0.3 ms, 0.4 mA). Note the constant latency and lack of conduction failure when stimulated at 0.2 and 20 Hz. The peak amplitude was reduced after the first eEPSC (indicated by an arrow) during stimulation at 20 Hz. B, polysynaptic eEPSCs in a different lamina II neuron in response to stimulation at 0.2 and 20 Hz (0.3 ms, 0.3 mA). Note the presence of polysynaptic components at 0.2 and 20 Hz. When stimulated at 20 Hz, the latency of eEPSCs was increased and conduction failure was revealed (indicated by an arrow). In each panel, 10 consecutive eEPSCs traces were superimposed.

Figure 2. A concentration-dependent effect of acetylcholine on the amplitude of eEPSCs in lamina II neurons

A, original recordings of eEPSCs of a lamina II neuron during control conditions and with application of different concentrations of acetylcholine. These traces are averages of 8 consecutive responses. The stimulus artifacts are indicated by the arrows. B, summary data showing the effect of acetylcholine on the peak amplitude of eEPSCs in 14 lamina II neurons. *P < 0.05 compared to the control (Friedman ANOVA test with Dunn's post hoc test).

Figure 3. Effect of mecamylamine and atropine on acetylcholine-induced inhibition of eEPSCs in lamina II neurons

A, application of a muscarinic receptor antagonist, atropine (10 μM, ATR) blocked the inhibitory effect of acetylcholine (100 μM, ACh) on the amplitude of eEPSCs in 1 lamina II neuron. B, a nicotinic receptor antagonist, mecamylamine (10 μM, MCM) did not affect acetylcholine-induced inhibition of eEPSCs in 1 neuron. C, summary data showing the effect of acetylcholine and atropine or mecamylamine on the peak amplitude of eEPSCs in 7 lamina II neurons. *P < 0.05 compared to the control (Friedman ANOVA test with Dunn's post hoc test).

Figure 4. The inhibitory effect of acetylcholine on mEPSCs in lamina II neurons

A, raw tracings during control conditions, application of acetylcholine (100 μM) and application of CNQX (20 μM). Note that CNQX completely eliminated mEPSCs. B and C, cumulative plot analysis of mEPSCs of the same neuron showing the distribution of the inter-event interval (B) and amplitude (C) during control conditions and acetylcholine application. Acetylcholine increased the inter-event interval of mEPSCs (P < 0.05, Kolmogorov-Smirnov test) without changing the distribution of the amplitude. D, superimposed averages of 200 consecutive mEPSCs obtained during control conditions and acetylcholine application. The decay time constant of mEPSCs during control and acetylcholine perfusion was identical, being 1.91 ms. E and F, summary data showing the effect of acetylcholine on the frequency (E) and the amplitude (F) of mEPSCs of 14 lamina II neurons. Data presented as means ± s.e.m.*P < 0.05 compared to the control (Wilcoxon signed rank test).

Figure 5. Effect of atropine and mecamylamine on the inhibitory effect of 100 μM acetylcholine on mEPSCs of lamina II neurons

A, summary data showing the effect of acetylcholine and atropine (10 μM) on the frequency and amplitude of mEPSCs of 7 lamina II neurons. B, summary data showing the effect of acetylcholine and mecamylamine (10 μM) on the frequency and amplitude of mEPSCs of 9 lamina II neurons. Data presented as means ± s.e.m.*P < 0.05 compared to the control (Wilcoxon signed rank test).

Figure 6. Light microscopic view (×200) of M₂ immunoreactivity in the spinal cord dorsal horn

A, a control dorsal spinal cord section (no primary antibody control). B, M₂ immunoreactivity was present in the spinal dorsal horn with a high density in the superficial laminae in a sham-operated rat. C, unilateral dorsal rhizotomy (left side) caused a dramatic reduction in M₂ receptor immunoreactivity in the superficial laminae of the lumbar spinal cord compared to the intact side.

Figure 7. Effect of acetylcholine on GABA-mediated mIPSCs of lamina II neurons

A, raw tracings during control conditions and application of acetylcholine (100 μM, ACh) in a lamina II neuron. B and D, summary data showing the effect of acetylcholine on the frequency (B) and the amplitude (D) of mIPSCs of 6 lamina II neurons. C and E, atropine (10 μM, ATR) blocked the effect of acetylcholine on mIPSCs in 6 lamina II neurons. The mIPSCs were recorded in the presence of TTX (1 μM), CNQX (20 μM) and strychnine (2 μM). Data presented as means ± s.e.m.*P < 0.05 compared to the control (Wilcoxon signed rank test).

Figure 8. Effect of CGP55845 on the inhibitory effect of acetylcholine on eEPSCs in lamina II neurons

A, original tracings showing attenuation of 100 μM acetylcholine-induced inhibition of eEPSCs by 1 μM CGP55845 in 1 neuron. B, summary data showing the effect of acetylcholine (100 μM) alone and CGP55845 (0.2–5 μM, CGP) plus acetylcholine on the peak amplitude of eEPSCs in 8 lamina II neurons. *P < 0.05 compared to the control; **P < 0.05 compared to acetylcholine alone (Friedman ANOVA test with Dunn's post hoc test).

Figure 9. Effect of CGP55845 on acetylcholine-induced inhibitory effect on mEPSCs in lamina II neurons

A, original recordings showing mEPSCs during control conditions, application of acetylcholine (100 μM, ACh) and application of CGP55845 (1 μM, CGP) plus acetylcholine in 1 lamina II neuron. B and C, cumulative probability plot showing the distribution of the inter-event interval (B) and amplitude (C) of this neuron during control conditions, application of acetylcholine and application of CGP55845 plus acetylcholine. D and E, summary data showing the modulation by CGP55845 of the effect of acetylcholine on the frequency (D) and amplitude (E) of mEPSCs in 10 lamina II neurons. *P < 0.05 compared to the control; **P < 0.05 compared to acetylcholine alone (Friedman ANOVA test with Dunn's post hoc test).

Figure 10. Effect of intrathecal CGP55845 (0.1, 1 and 10 μg, n = 7–8) or saline (n = 7) on the antinociceptive action of intrathecal injection of muscarine (8 μg) in rats

The nociceptive threshold was determined by the withdrawal response of the hindpaw to a noxious heat stimulus. Data presented as means ± s.e.m.*P < 0.05 compared to the baseline control (time 0); **P < 0.05 compared to the saline-treated group.

Figure 11. Simplified scheme showing the direct and indirect inhibitory effects of acetylcholine on glutamate release onto lamina II neurons

Lamina II neurons receive both glutamatergic excitatory and GABAergic inhibitory inputs. Acetylcholine can inhibit glutamate release through presynaptic muscarinic receptors located on the glutamatergic terminals. Also, acetylcholine can activate muscarinic receptors on the GABAergic terminals to evoke synaptic GABA release. Increased GABA can spill over to the neighbouring glutamatergic terminals, which inhibits glutamate release through GABA_B receptors located on the glutamatergic nerve terminals. Glu: glutamate.