抄録
Somatosensory information from the periphery through primary afferents is properly processed and integrated by neuronal circuits in the spinal dorsal horn (SDH). Excitatory and inhibitory SDH interneurons are crucial for this processes. However, the effects of the acute and selective inactivation of inhibitory SDH interneurons on pain processing are not fully understood. In the present study, we employed designer receptors exclusively activated by designer drugs (DREADD) technology to examine the effects of silencing SDH inhibitory interneurons on somatosensory information processing at both the synaptic and behavioral levels.
Male heterozygous Vgat-Cre mice were used. For the selective silencing of SDH inhibitory interneurons, we unilaterally microinjected an AAV including genes encoding a modified human muscarinic Gi protein-coupled receptor (hM4Di) and mCherry (AAV2/9-EF1α-FLEX-hM4Di-P2A-mCherry) into the SDH of Vgat-Cre mice at the fourth lumbar segment. Using whole-cell patch-clamp recording, we examined the effect of silencing inhibitory interneurons on synaptic responses by clozapine-N-oxide (CNO) application in spinal cord slices. In the behavioral study, we measured spontaneous nocifensive behaviors (licking, biting and flinching) after CNO intraperitoneal injection and examined the effect of analgesics on these nocifencive behaviors.
Four weeks after viral injection, mCherry fluorescence was detected exclusively in the ipsilateral SDH and immunolabeled with paired box 2 (PAX2), a marker for inhibitory interneurons. Bath application of CNO rapidly hyperpolarized the resting membrane potentials of Vgat-Cre positive SDH neurons, and CNO treatment rapidly induced spontaneous nocifensive behaviors in these mice. In mCherry negative SDH neurons, A fiber-mediated polysynaptic EPSCs was facilitated by CNO, which was eliminated by co-application of N-methyl-D-aspartate (NMDA) receptor antagonist. NMDA receptor antagonism also reduced the CNO-induced nocifensive behaviors. Moreover, these nocifensive behaviors were suppressed by pregabalin but resistant to morphine.
We demonstrated that the rapid and selective silencing of inhibitory SDH interneurons using DREADD technology induced morphine-resistant spontaneous nocifensive behaviors. These results suggest that this approach may provide a useful model for understanding the mechanisms of opioid-resistant pain signalling and for developing novel analgesics.
