Abstract
Inhibitory interneurons in the spinal dorsal horn (SDH) are crucial for processing somatosensory information originating in the periphery. However, the effects of the acute and selective inactivation of GABAergic SDH interneurons on pain processing are not fully understood. In this study, we used designer receptors exclusively activated by designer drugs (DREADD) technology and vesicular GABA transporter-Cre (Vgat-Cre) mice to selectively express a modified human muscarinic Gi protein-coupled receptor (hM4Di) in Vgat-Cre+ GABAergic SDH interneurons in the fourth lumbar segment. We found that clozapine-N-oxide (CNO) treatment rapidly hyperpolarized these neurons and induced spontaneous nocifensive behaviours in these mice. In Vgat-Creneg lamina II neurons, CNO produced facilitation of A fibre-mediated polysynaptic excitatory responses, an effect that required N-methyl-D-aspartate (NMDA) receptor activation. The CNO-induced nocifensive behaviours were also reduced by NMDA receptor antagonism. Moreover, these nocifensive behaviours were suppressed by pregabalin but resistant to morphine. Our findings indicate that Vgat-Cre+ SDH interneurons play an important role in morphine-resistant nocifensive behaviours and suggest that this approach may provide a useful model for understanding the mechanisms of opioid-resistant pain signalling and for developing novel analgesics.
Highlights
Somatosensory information from the periphery is conveyed to the spinal dorsal horn (SDH) via primary afferents[1]
The exact role of the SDH inhibitory interneurons in somatosensory processing is not fully understood because GABA and glycine in the spinal cord are released from SDH interneurons and from neurons descending from the brainstem, such as from the rostral ventromedial medulla (RVM)[3, 4]
Double immunostaining showed that 97.6 ± 1.1% of mCherry+ (Vgat-Cre+) cells were immunolabelled with paired box 2 (PAX2) (Fig. 1c), a marker for inhibitory interneurons13. mCherry fluorescence in the SDH was observed in 25.1 ± 3.3% of total PAX2+ cells tested
Summary
Somatosensory information from the periphery is conveyed to the spinal dorsal horn (SDH) via primary afferents[1]. For the selective manipulation of SDH inhibitory interneurons, we used the flip-excision (FLEX) system[9, 10] in which an adeno-associated virus (AAV) encoding a modified human muscarinic Gi protein-coupled receptor (hM4Di) was microinjected using our established minimally invasive injection method into the SDH11 of Vgat-Cre mice[12] containing genetically targeted inhibitory interneurons. Using this system, we examined the effects of silencing SDH inhibitory interneurons on somatosensory information processing at both the synaptic and behavioural levels
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