Molecular Mechanisms For Spinal GABAergic Disinhibition To Evoke Allodynia In Mice

Peripheral tissue or nerve injury simultaneously impairs GABAergic inhibition and enhances NMDA receptor-mediated nociceptive synaptic transmission in spinal dorsal horn, which have been proposed as two critical components responsible for chronic pathological pain. However, the intrinsic relationship between GABAergic disinhibition and glutamatergic hyperactivity is still largely unknown. The present study hypothesized that the reduced inhibition might serve as a key initiator for NMDA receptor hyperfunction involved in central sensitization.To test this hypothesis, the present study intrathecally injected GABAA receptor antagonist bicuculline in naive mice to mimic the GABAergic disinhibition and investigated whether bicuculline evoked mechanical allodynia by regulating NMDA receptor phosphorylation, synaptic concentration and its total protein expression. Meanwhile, the present study also examined whether intrathecal application of GABAA receptor agonist diazepam to resume GABAergic inhibition could alleviated inflammatory pain by repressing tissue injury-induced NMDA receptor hyperfunction.Our data found that:(1) Intrathecal administration of bicuculline dose-dependently elicited mechanical allodynia in naive mice in response to Von Frey filament stimuli. (2) The mechanical allodynia induced by bicuculline in naive mice could be attenuated by intrathecal NMDA receptor antagonist D-APV in a dose-dependent manner, suggesting that GABAergic disinhibition indeed caused NMDA receptor hyperfunction to alter the nociceptive reflex sensitization. (3) The mechanical allodynia induced by bicuculline in naive mice didn't require the changes in the total expression level of NMDA receptor NR1, NR2A and NR2B subunit in spinal dorsal horn. (4) However, bicuculline was able to enhance the localization of NR1 and NR2B subunit at PSD-enriched fraction, with that of NR2A unaltered, suggesting that the specific recruitment of NR2B-containing NMDA receptor (NR2BR) at synapses to facilitate NR2BR-mediated nociceptive synaptic transmission might operate as a key step for the reduced inhibition to trigger NMDA receptor hyperfunction. (5) Interestingly, inhibition of cAMP-dependent protein kinase (PKA) completely abolished bicuculline-induced synaptic expression of NR2BR, while the total protein levels of NR2BR were insensitive to PKA inhibitor. These data implicated that PKA might act downstream of GABAergic disinhibition to boost NR2BR synaptic concentration. (6) The molecular mechanisms underlying PKA action might involve the phosphorylation of NR1 subunit at Serine 897 (S897) to promote the forward trafficking of NR2BR toward synapses. (7) In inflamed mice, intrathecal application of GABAA receptor agonist diazepam to resume GABAergic inhibition dose-dependently alleviated mechanical allodynia, supporting the importance of the disinhibition in inflammatory pain. (8) Diazepam potently inhibited NR1 phosphorylation at S897 and depressed the synaptic accumulation of NR2BR, which might contribute to the analgesia produced by diazepam.Taken together, these results suggested that the impaired GABAergic inhibition following tissue injury exerted a critical role in inducing NMDA receptor hyperfunction and mechanical allodynia. Our data thus provided strong evidence for the intimate interaction of GABAergic system with glutamatergic system in the integration and transduction of sensory signalings in spinal dorsal horn.