Experimental Study Of The Regulation Of Muscarinic Receptors On Glycinergic Inputs To Spinal Lamina â…¡ Neurons

Experiment 1 Effect of oxo-M on glycinergic sIPSCs and mlPSCsObjective To investigate the effect of oxo-M on glycinergic sIPSCs and mlPSCs in lamina II neurons in spinal cord. Method Recordings of postsynaptic currents were performed using the whole-cell voltage-clamp method. At the holding potential of 0 mV, the spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in the presence of 10μM bicuculline (GABA_a receptor antagonist), and 20 μM CNQX (a non-NMDA antagonist). To record the miniature inhibitory postsynaptic currents (mlPSCs), lμM. tetrodotoxin (TTX) was added in the perfusion solution. The non-selective mAChR agonist oxo-M was applied through bath perfusion. The effect of oxo-M on sIPSCs and mlPSCs was examined at the concentration of 1, 3, 5, and 10 uM. Result In a total of 22 lamina II neurons tested, oxo-M at the concentration of 3 μM significantly increased the frequency of sIPSCs without changing amplitude in 16 of 22 neurons (72.73%). Interestingly, when the concentrations of oxo-M were increased to 5 and 10 uM, the potentiating effect of oxo-M on the frequency of sIPSCs was attenuated compared with that of 3 uM. oxo-M in the above 16 neurons. The sIPSCs were completely abolished by 2 uM strychnine. Atropine, the specific mAChR antagonist , completely blocked the effect of oxo-M on the frequency of sIPSCs. In 9 additional lamina II neurons, 1 — 10 uM oxo-M failed to alter significantly the frequency and amplitude of glycinergic mlPSCs. Conclusion Activation of mAChRs on the somatodendritic site of glycinergicinterneurons increased the synaptic glycine release to spinal dorsal horn neurons not in dose-dependently manner. While Oxo-M at the higher concentration decreased the glycine release in lamian II neurons.Experiment 2 Effect of oxo-M on glycinergic sIPSCs and mlPSCs inPTX-treated ratsObjective To investigate the effect of oxo-M on glycinergic sIPSCs and mlPSCs in lamina II neurons in PTX-treated rat. Method Recordings of postsynaptic currents were the same as Part I. 10 Male SD rats weighting 160-180g were randomly classified into two groups (n=5). After the spinal cord was not injuryed by catheter, we pre-treated rats with intrathecal 1 \ig or 2 ^g PTX to inactivate Gi/o protein 5 to 7 days before the final electrophysiological experiments. Result In 15 lamina II neurons obtained from rats pre-treated with intrathecal 2 jug PTX, 3-10 uM oxo-M significantly increased the frequency of glycinergic sIPSCs in a dose-dependent manner without altering the amplitude. In another 8 lamina II neurons recorded from PTX (2 ng) pre-treated rats, 3 uM oxo-M initially induced a significant increase in the frequency of sIPSCs. However, in the presence of 1 uM TTX, 3 uM oxo-M had no significant effect on the frequency and amplitude of mlPSCs in above 8 cells tested. A similar effect was observed in 10 lamina II neurons from rats pre-treated with intrathecal 1 u.g PTX. The dose-response effect of oxo-M on glycinergic IPSCs was not significantly different between rats pretreated with 1 and 2 jag PTX (p> 0.05). Conclusion inactivation of Gj/0 proteins with PTX increases the effectiveness of the mAChR agonist at higher concentrations. These data indicatedthat inhibitory effect of oxo-M at higher concentration is relative to the Gi/oproteins.Experiment 3 Influence of GABAb receptors on the effect of oxo-M onglycinergic sIPSCsObjective To investigate influence of GABAb receptors on the effect of oxo-M on glycinergic sIPSCs in lamina II neurons, and further determine if GABAb receptors are involved in the attenuated effect of oxo-M at higher concentrations on glycinergic sIPSCs. Method Recordings of postsynaptic currents were the same as Part I. We used 1 uM CGP-55845 at the holding potential of 0 mV, a specific GABAb receptor antagonist, to block the GABAb receptor in lamina II neurons. We recored the effect of 1-10 uM oxo-M on glycinergic sIPSCs in spinal dorsal horn neurons. Results In the presence of 1 uM. CGP-55845, oxo-M significantly increased the frequency of sIPSCs in a dose-dependent manner in 13 neurons. Notably, the effect of oxo-M on the frequency of sIPSCs reached the maximum at 3 uM, and this effect was sustained at 5 and 10 uM. Conclusion These data show that the effect of oxo-M on synaptic glycine release is influenced by GABAb receptors, likely through increased GABA release due to concurrent stimulation of mAChRs on adjacent GABAergic interneurons in the lamina II, which decreased the glycine release through the inhibitory effect of presynaptic GABAb receptors located on glycinergic neurons.Experiment 4 Role of M₂ and M4 mAChR subtypes in Oxo-M-inducedincrease in glycinergic sIPSCsObjective To further determine the role of M₂ and M4 mAChR subtypes in oxo-M-induced increase in glycinergic sIPSCs in glycinergic neurons of lamina II. Method Recordings of postsynaptic currents were the same as Part I.. This part was classified two parts. The lamina II neurons were randomly classified into two groups in first part: (1) Perfusing 10 μM AFDX-116 - an M₂-preferring mAChR antagonist, to further delineate the role of the M₂ mAChR subtype in the effect of oxo-M on glycinergic sIPSCs. (2) Using 1 μM himbacine, an M2- and M₄-preferring mAChR antagonist, to assess the role of M4 subtypes in the effect of oxo-M on synaptic glycine release to lamina II neurons. We recored the effect of 1-10 μM on glycinergic sIPSCs in spianl dorsal horn neurons at holding potential of 0 mV. The seconed part is finished in the presence of 1 uM CGP-55845 to block GABA_B receptors. We first blocked GABA_B receptors (to remove the indirect effect of oxo-M on GABAergic neurons) and then tested the effect of 5 uM oxo-M on glycinergic IPSCs before and after blocking M₂ (10 μM AFDX-116) mAChRs or M₂/M₄(1 μM himbacine). Two groups were randomly classified from the lamina II neurons: group (1) perfusing 10 uM AFDX-116;group (2) perfusing 1 μ M himbacine. Result In the first part: 15 neurons were recored in the AFDX-116 group, 3-10 μM. oxo-M concentration-dependently increased the frequency of sIPSCs in 15 cells;while 17 neurons were recored in the Himbacine group, effect of 3-10 μM oxo-M on glycinergic sIPSCs was similar to that of 3-10 μ.M oxo-M in the AFDX-116 group, there were no significant difference between the two groups (p>0.05). In the seconed part: l|iM . CGP-55845 was administered consistently. 16 lamina II neurons were recorded in perfusing AFDX-116 group, initial application of 5 \iM oxo-M significantlyincreased the frequency of sIPSCs in the presence of 1 uM CGP-55845. AFDX-116 (10 uM) alone did not have significant effect on sIPSCs, but significantly reduced the potentiating effect of 5 uM oxo-M on the frequency of sIPSCs in the above 16 cells. Another 15 cells were tested in perfusing 1 uM himbacine group, 1 uM himbacine also significantly decreased the stimulating effect of 5 uM oxo-M on the frequency of glycinergic sIPSCs in the presence of 1 uM CGP-55845. Bath application of AFDX-116 alone had no significant effect on sIPSCs in all the neurons tested, there were no significant difference between the two groups (p>0.05). Conclusion M2 and M4 mAChR subtypes are involved in the decreasing effect of oxo-M at higher concentration on synaptic glycine release to lamina II neurons. Furthermore, after removing the presynaptic inhibitory effect of GABAb receptors, stimulation of M2 mAChR subtypes on glycinergic intemeurons increases synaptic glycine release to spinal lamina II neurons,while activation of M4. mAChR subtypes on it had no significantly effect on glycine release.Experiment 5 Role of M3 mAChR subtype in oxo-M-induced increase in glycinergic sIPSCsObjective To determine the role of M3 mAChR subtype in oxo-M-induced increase in glycinergic sIPSCs in lamina II neurons. Method Recordings of postsynaptic currents were same to Part I. The spinal dorsal horn neurons were randomly classified into two groups, (1) lamina II neurons in normal rats (2) lamina II neurons in PTX-treated rats. The PTX-treated method was the same as experiment II. 4-DAMP, an M3-preferring mAChR antagonist, was perfused toblock the M3 subtype, we determined if the effect of oxo-M on potentiation of glycinergic sIPSCs is mediated by the M3 subtype in the spinal cord. At the holding potential of 0 mV, all spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in the presence of 10 uM bicuculline and 20 uM CNQX, 3 u M oxo-M plus 25 nM 4-DAMP were employed after the increasing effect of 3 fiM oxo-M on glycinergic sIPSCs was washed out. Result 9 spianl dorsal horn neurons in normal rats and 8 neurons in PTX-treated rats were recored, we found that 3uM oxo-M significantly increased the frequency of sIPSCs, bath application of 25 nM 4-DAMP completely blocked the potentiating effect of 3 uM oxo-M on the frequency of sIPSCs in all cells tested. There were no defference between the noraml and PTX-treated rats. Conclusion M3 mAChR subtype plays a predominant role in potentiation of glycinergic inhibitory tone in the spinal cord by the mAChR agonist, and is key receptor in lamian II interneurons.