Tonic GABA and Glycine Inhibition Control Pain Hypersensitivity via Limiting α2δ-1– and mGluR5–Dependent NMDA Receptor Activity at Primary Afferent→Excitatory Neuron Synapses

Impaired synaptic inhibition by GABA and glycine contributes to excitatory-inhibitory imbalance in the spinal cord associated with chronic neuropathic pain; however, the underlying mechanisms remain unclear. Here, we investigated how GABAergic and glycinergic inputs regulate synaptic <i>N</i>-methyl-d-aspartate receptor (NMDAR) activity in excitatory and inhibitory neurons of the spinal dorsal horn in male and female mice. Vesicular glutamate transporter 2 (VGluT2)-expressing excitatory neurons and vesicular γ-aminobutyric acid transporter (VGAT)-expressing inhibitory neurons exhibited comparable mixed GABAergic and glycinergic inhibitory postsynaptic currents. Blockade of GABA_A receptors with gabazine or glycine receptors with strychnine potentiated NMDAR-mediated miniature excitatory postsynaptic current (mEPSC) frequency, the amplitude of EPSCs monosynaptically evoked from the dorsal root, and puff NMDA currents in VGluT2, but not VGAT, neurons. These effects were abolished by silencing neuronal activity with tetrodotoxin or in <i>Cacna2d1</i> knock-out (KO) mice. In mice with conditional <i>Grin1</i> KO in primary sensory neurons (<i>Grin1</i>-cKO), gabazine and strychnine did not affect mEPSC frequency but still enhanced puff NMDA currents in dorsal horn neurons. Furthermore, intrathecal gabazine- or strychnine-induced nociceptive hypersensitivity was diminished by <i>Grin1</i>-cKO, <i>Cacna2d1</i> KO, or α2δ-1 C-terminus peptide. Additionally, blocking metabotropic glutamate receptor 5 (mGluR5) prevents gabazine- and strychnine-induced increases in NMDAR-mediated mEPSC frequency, evoked EPSCs, and puff NMDA currents in VGluT2 neurons as well as nociceptive hypersensitivity. Our findings reveal that GABAergic and glycinergic inhibition tonically suppresses both presynaptic and postsynaptic NMDAR activity at primary afferent→excitatory neuron synapses. α2δ-1 and mGluR5 are essential for disinhibition-induced nociceptive hypersensitivity and synaptic NMDAR hyperactivity in the spinal cord.