During development, axon branching is influenced by sensory-evoked and spontaneous neural activity. We studied the molecular mechanism that underlies activity-dependent branch formation at horizontally elongating axons (horizontal axons) in the upper cortical layers, focusing on Rho family small GTPases. Axonal labeling with enhanced yellow fluorescent protein showed that horizontal axons formed several branches in organotypic slice cultures. This branch formation was considerably increased by introducing constitutively active RhoA and was slightly inhibited by dominant-negative RhoA. Activators and inhibitors of endogenous RhoA signaling also promoted and inhibited branching, respectively. Daily imaging of horizontal axon growth further demonstrated that constitutively active RhoA increased the dynamic addition and loss of branches. Moreover, the amount of active RhoA relative to the total amount of RhoA was examined by a pull-down assay in cortical slices treated with sodium channel or glutamate receptor blockers to reduce neural activity. Activity blockade significantly decreased active RhoA compared with normal culture conditions, in which spontaneous firing is prominent. These findings suggest that RhoA signaling acts as a positive regulator for activity-dependent axon branching in cortical neurons.

Activity-dependent increases in the responsiveness of spinal neurons to their normal afferent input, termed central sensitization, have been suggested to play a key role in abnormal pain sensation. We investigated the role of distinct voltage-dependent calcium channel (VDCC) subtypes in the long-term potentiation (LTP) of C-fiber-evoked field potentials (FPs) recorded in the spinal dorsal horn of rats, that is, a synaptic model to describe central sensitization. When spinally applied, we observed that omega-conotoxin GVIA (ω-CgTx), an N-type VDCC antagonist, produced a dose-dependent and prolonged inhibition of basal C-fiber-evoked FPs in naïve animals. ω-CgTx did not perturb the induction of LTP by high-frequency stimulation (HFS) of the sciatic nerve; however, potentiation was maintained at a lower level. Following the establishment of spinal LTP in naïve animals, the inhibitory effect of ω-CgTx on C-fiber-evoked FPs was significantly increased. Furthermore, in animals with chronic pain produced via peripheral nerve injury, where spinal LTP was barely induced by HFS, basal C-fiber-evoked FPs were strongly inhibited by ω-CgTx. As a result, ω-CgTx exerted a similar inhibitory profile on C-fiber-evoked FPs following the establishment of spinal LTP and chronic pain. In contrast, spinally administered omega-agatoxin IVA (ω-Aga-IVA), a P/Q-type VDCC antagonist, showed little effect on C-fiber-evoked FPs either before or after the establishment of LTP, but strongly suppressed LTP induction. These results demonstrate the requirement of N- and P/Q-type VDCCs in the maintenance and induction of LTP in the spinal dorsal horn, respectively, and their distinct contribution to nociceptive synaptic transmission and its plasticity. In vivo electrophysiological studies demonstrate the distinct and predominant functions of voltage-dependent calcium channel subtypes for spinal long-term potentiation and chronic pain.

Keywords5-HT and noradrenaline reuptake inhibitor; spinal cord; long-term potentiation; neuropathic pain; C-fibre-evoked field potential; in vivo electrophysiology ------------------------------- BACKGROUND AND PURPOSEThe analgesic action of 5-HT and noradrenaline reuptake inhibitors (SNRIs) on nociceptive synaptic transmission in the spinal cord is poorly understood. We investigated the effects of milnacipran, an SNRI, on C-fibre-evoked field potentials (FPs) in spinal long-term potentiation (LTP), a proposed synaptic mechanism of hypersensitivity, and on the FPs in a neuropathic pain model. EXPERIMENTAL APPROACHC-fibre-evoked FPs by electrical stimulation of the sciatic nerve fibres were recorded in the spinal dorsal horn of anaesthetized adult rats, and LTP was induced by high-frequency stimulation of the sciatic nerve fibres. A rat model of neuropathic pain was produced by L5 spinal nerve ligation and transection. KEY RESULTSMilnacipran produced prolonged inhibition of C-fibre-evoked FPs when applied spinally after the establishment of LTP of C-fibre-evoked FPs in naïve animals. In the neuropathic pain model, spinal administration of milnacipran clearly reduced the basal C-fibre-evoked FPs. These inhibitory effects of milnacipran were blocked by spinal administration of methysergide, a 5-HT1/2 receptor antagonist, and yohimbine or idazoxan, a2-adrenoceptor antagonists. However, spinal administration of milnacipran in naïve animals did not affect the basal C-fibre-evoked FPs and the induction of spinal LTP. CONCLUSION AND IMPLICATIONSMilnacipran inhibited C-fibre-mediated nociceptive synaptic transmission in the spinal dorsal horn after the establishment of spinal LTP and in the neuropathic pain model, by activating both spinal 5-hydroxytryptaminergic and noradrenergic systems. The condition-dependent inhibition of the C-fibre-mediated transmission by milnacipran could provide novel evidence regarding the analgesic mechanisms of SNRIs in chronic pain. AbbreviationsCNS, central nervous system; FP, field potential; HFS, high-frequency stimulation; LTP, long-term potentiation; SNL, spinal nerve ligation; SNRI, 5-HT and noradrenaline reuptake inhibitor; SSRI; selective serotonin reuptake inhibitor BJP British Journal of Pharmacology