Inhibition of α5 subunit-containing GABAA receptors facilitated spinal nociceptive transmission and plasticity.

BACKGROUND: γ-Aminobutyric acid (GABA) type A receptors (GABAA Rs) locate at both synaptic and extrasynaptic membrane, which generate phasic and tonic inhibition, respectively. In spinal cord dorsal horn, the phasic inhibition produced by transient activation of synaptic GABAA Rs plays an important role in the gating control over nociceptive conveyance. Although extrasynaptic GABAA Rs that contain α5 subunits (α5-GABAA Rs) are also detectable in spinal dorsal horn, much less is known about the function of these receptors. METHODS: The C fibre-evoked field potentials were recorded in superficial dorsal horn of spinal cord, and the effects of α5-GABAA R inverse agonist L-655708 on basal synaptic transmission and long-term potentiation (LTP) of C-fibre responses were examined. The possible changes of glutamate receptor function and pain sensitivity after α5-GABAA R inhibition were investigated by western blot and behavioural tests. RESULTS: Inhibition of α5-GABAA Rs by L-655708 boosted the basal synaptic transmission and facilitated the induction of N-methyl-d-aspartate subtype glutamate receptors (NMDARs)-dependent LTP. L-655708 was found to enhance the phosphorylation and synaptic accumulation of NMDARs and α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid receptors (AMPARs). Intrathecal L-655708 injection also decreased the pain thresholds of intact mice in a dose-dependent manner. CONCLUSIONS: α5-GABAA Rs were critical for the tonic inhibition of glutamatergic neurotransmission and plasticity in spinal dorsal horn. SIGNIFICANCE: Tonic inhibition generated by α5-GABAA Rs is important for information processing. However, whether and how α5-GABAA Rs regulate the conveyance of nociceptive signals in spinal cord is largely unknown. Here, we revealed a negative control by α5-GABAA Rs over nociceptive transmission and plasticity.

Noradrenergic α2 receptor attenuated inflammatory pain through STEP61/ERK signalling.

BACKGROUND: Activation of noradrenergic α2 receptor in spinal dorsal horn effectively alleviates the pathological pain. However, the precise mechanisms underlying noradrenergic pain suppression are not fully understood. Convincing evidence has indicated that extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a key role in spinal sensitization. The present study investigated the potential influence of noradrenergic α2 receptor agonist clonidine on ERK1/2 activity. METHOD: Clonidine was intrathecally given after intraplantar injection of complete Freund's adjuvant (CFA) in mice. The possible changes of ERK1/2 signalling were detected by Western blot, immunohistochemistry, co-immunoprecipitation and behavioural tests. RESULTS: CFA significantly enhanced ERK1/2 activity in spinal dorsal horn, which was, however, greatly attenuated by clonidine application. Pretreatment with pertussis toxin abolished the inhibitory effect of clonidine on ERK1/2, suggesting the involvement of Giα subunit (Gi protein). Noradrenergic α2 receptor/Gi protein might repress ERK1/2 through cAMP-dependent protein kinase (PKA) pathway, because direct ERK1/2 activation by PKA agonist forskolin was also suppressed by clonidine. We found that 61 kD isoform of striatal-enriched protein phosphatase (STEP61) was a key intermediary for α2 receptor/Gi protein/PKA signalling to manipulate ERK1/2 activity. By reducing PKA-mediated phosphorylation of STEP61 at Ser221, clonidine significantly resumed the inhibition conferred by STEP61 on ERK1/2. Direct expression of STEP61 mutant devoid of Ser221 phosphorylation mimicked clonidine by inhibiting ERK1/2 and pain sensitization in CFA-injected mice. CONCLUSION: The analgesic action produced by noradrenergic α2 receptor agonist clonidine involved the reversal of ERK1/2 hyperactivity in spinal dorsal horn of inflamed mice.

Fyn kinase-regulated NMDA receptor- and AMPA receptor-dependent pain sensitization in spinal dorsal horn of mice.

BACKGROUND: Inhibition of Src-family protein tyrosine kinases (SFKs) in spinal dorsal horn has been established as an effective strategy for the alleviation of chronic pathological pain. As one of the important SFKs members, Fyn kinase is critical for synaptic plasticity and many pathophysiological processes. However, whether Fyn is involved in spinal sensitization is far from being elucidated. METHOD: We manipulated Fyn activity by expressing a constitutively active Fyn mutant [Fyn(Y528F) ] or a catalytically null mutant [Fyn(K296M) ] in the spinal dorsal horn of mice, and performed behavioural and biochemical experiments to investigate the role of Fyn in regulating the nociceptive responses and the synaptic expression of ionotropic glutamate receptors. RESULTS: Spinal expression of Fyn(Y528F) alone in intact mice was sufficient to elicit persistent mechanical allodynia and thermal hyperalgesia, which lasted for at least 12 days. Fyn(Y528F) simultaneously enhanced the concentrations of N-methyl-D-aspartic acid (NMDA)-subtype and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype glutamate receptors at synaptosomal membrane fraction. Pharmacological inhibition of NMDA receptors or AMPA receptors greatly alleviated Fyn(Y528F)-induced pain hypersensitivity. To evaluate the contribution of Fyn to inflammatory pain, we expressed Fyn(K296M) before intradermal injection of complete Freund's adjuvant (CFA), finding that Fyn(K296M) had no effect on the induction of inflammatory pain within 3 h post-CFA injection, which, however, repressed the synaptic accumulation of NMDA receptors and AMPA receptors to attenuate the maintenance of chronic pain states. CONCLUSION: Fyn played a key role in the sustained sensitization of nociceptive behaviours by up-regulating the functions of ionotropic glutamate receptors in spinal dorsal horn.