Activation of the Mammalian Target of Rapamycin in the Rostral Ventromedial Medulla Contributes to the Maintenance of Nerve Injury-Induced Neuropathic Pain in Rat

Figure 5

The neuronal excitability of 5-HTergic neurons in the RVM was significantly increased in SNI rats, which could be effectively impaired by rapamycin via a postsynaptic mechanism. (a) In vitro brain slice infusion revealed that rapamycin (250 μM) rapidly inhibited the activation of mTOR in SNI rats. Consistently, in the absence of rapamycin, SNI produced a significant increase in the levels of phosphorylated mTOR and S6K ( compared to the sham + vehicle group, ). After treatment with rapamycin (250 μM) on day 7, the upregulation of p-mTOR and p-S6K levels was remarkably decreased ( compared to the SNI + vehicle group, ) in brain slices from SNI rats. (b) One representative whole-cell patched neuron in the RVM was injected with biocytin (labeled with Alexa 594, red). This cell also showed 5-HT immunoreactivity (Alexa 488, green). The same neuron is also pictured during whole-cell patching (RMg: raphe magnus nucleus; RPa: raphe pallidus nucleus). Scale bars = 100 μm (above) and 25 μm (below). (c)–(e) Superimposed samples and cumulative fraction results showing that rapamycin inhibited the amplitude rather than the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in the RVM 5-HTergic neurons. (c), (e) Bath application of rapamycin (250 μM) had no effect on the frequency and amplitude of the sEPSCs in rats with sham surgery. (d), (e) Rapamycin (250 μM) inhibited the frequency but not the amplitude of sEPSCs in rats with SNI. (f) Summarized results for the effects of rapamycin on sEPSCs in rats with SNI or sham surgery ( versus sham , ; + rapamycin versus sham + rapamycin, , ). (g) Sample traces and average results showing that the number of action potentials (APs) in a train induced by the injection of step currents (400 ms, 0–60 pA) was not affected by rapamycin in the sham group (, , two-way repeated measures ANOVA). (h) Sample traces and average results showing that the number of APs in a train induced by the injection of step currents (400 ms, 0–60 pA) was significantly reduced by rapamycin in the SNI group (, , two-way repeated measures ANOVA). The Holm-Sidak post hoc test indicated that rapamycin decreased the spike number when currents of 20 (, ), 30 (, ), 40 (, ), 50 (, ), and 60 pA (, ) were applied. It is worth noting that the resting membrane potential (RMP) was slightly hyperpolarized and that the amplitude of spikes was also slightly decreased in the SNI group.