Cholinergic Neurotransmission in the Posterior Insular Cortex Is Altered in Preclinical Models of Neuropathic Pain: Key Role of Muscarinic M2 Receptors in Donepezil-Induced Antinociception.

Neuropathic pain is one of the most debilitating pain conditions, yet no therapeutic strategy has been really effective for its treatment. Hence, a better understanding of its pathophysiological mechanisms is necessary to identify new pharmacological targets. Here, we report important metabolic variations in brain areas involved in pain processing in a rat model of oxaliplatin-induced neuropathy using HRMAS (1)H-NMR spectroscopy. An increased concentration of choline has been evidenced in the posterior insular cortex (pIC) of neuropathic animal, which was significantly correlated with animals' pain thresholds. The screening of 34 genes mRNA involved in the pIC cholinergic system showed an increased expression of the high-affinity choline transporter and especially the muscarinic M2 receptors, which was confirmed by Western blot analysis in oxaliplatin-treated rats and the spared nerve injury model (SNI). Furthermore, pharmacological activation of M2 receptors in the pIC using oxotremorine completely reversed oxaliplatin-induced mechanical allodynia. Consistently, systemic treatment with donepezil, a centrally active acetylcholinesterase inhibitor, prevented and reversed oxaliplatin-induced cold and mechanical allodynia as well as social interaction impairment. Intracerebral microdialysis revealed a lower level of acetylcholine in the pIC of oxaliplatin-treated rats, which was significantly increased by donepezil. Finally, the analgesic effect of donepezil was markedly reduced by a microinjection of the M2 antagonist, methoctramine, within the pIC, in both oxaliplatin-treated rats and spared nerve injury rats. These findings highlight the crucial role of cortical cholinergic neurotransmission as a critical mechanism of neuropathic pain, and suggest that targeting insular M2 receptors using central cholinomimetics could be used for neuropathic pain treatment. SIGNIFICANCE STATEMENT: Our study describes a decrease in cholinergic neurotransmission in the posterior insular cortex in neuropathic pain condition and the involvement of M2 receptors. Targeting these cortical muscarinic M2 receptors using central cholinomimetics could be an effective therapy for neuropathic pain treatment.

Paracetamol is a centrally acting analgesic using mechanisms located in the periaqueductal grey.

BACKGROUND AND PURPOSE: We previously demonstrated that paracetamol has to be metabolised in the brain by fatty acid amide hydrolase enzyme into AM404 (N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide) to activate CB1 receptors and TRPV1 channels, which mediate its analgesic effect. However, the brain mechanisms supporting paracetamol-induced analgesia remain unknown. EXPERIMENTAL APPROACH: The effects of paracetamol on brain function in Sprague-Dawley rats were determined by functional MRI. Levels of neurotransmitters in the periaqueductal grey (PAG) were measured using in vivo 1 H-NMR and microdialysis. Analgesic effects of paracetamol were assessed by behavioural tests and challenged with different inhibitors, administered systemically or microinjected in the PAG. KEY RESULTS: Paracetamol decreased the connectivity of major brain structures involved in pain processing (insula, somatosensory cortex, amygdala, hypothalamus, and the PAG). This effect was particularly prominent in the PAG, where paracetamol, after conversion to AM404, (a) modulated neuronal activity and functional connectivity, (b) promoted GABA and glutamate release, and (c) activated a TRPV1 channel-mGlu5 receptor-PLC-DAGL-CB1 receptor signalling cascade to exert its analgesic effects. CONCLUSIONS AND IMPLICATIONS: The elucidation of the mechanism of action of paracetamol as an analgesic paves the way for pharmacological innovations to improve the pharmacopoeia of analgesic agents.

Supra-spinal FAAH is required for the analgesic action of paracetamol in an inflammatory context.

Paracetamol (acetaminophen) is the most commonly used analgesic in the world. Recently, a new view of its action has emerged: that paracetamol would be a pro-drug that should be metabolized by the FAAH enzyme into AM404, its active metabolite. However, this hypothesis has been demonstrated only in naive animals, a far cry from the clinical pathologic context of paracetamol use. Moreover, FAAH is a ubiquitous enzyme expressed both in the central nervous system and in the periphery. Thus, we explored: (i) the involvement of FAAH in the analgesic action of paracetamol in a mouse model of inflammatory pain; and (ii) the contributions of central versus peripheral FAAH in this action. The analgesic effect of paracetamol was evaluated in thermal hyperalgesia, mechanical allodynia and hyperalgesia induced by an intra-plantar injection of carrageenan (3%) in FAAH knock-out mice or their littermates. Moreover, the contribution of the central and peripheral enzymes was explored by comparing the effect of a global FAAH inhibitor (URB597) to that of a peripherally restricted FAAH inhibitor (URB937) on paracetamol action. Here, we show that in a model of inflammatory pain submitted to different stimuli, the analgesic action of paracetamol was abolished when FAAH was genetically or pharmacologically inhibited. Whereas a global FAAH inhibitor, URB597 (0.3 mg/kg), reduced the anti-hyperalgesic action of paracetamol, a brain-impermeant FAAH inhibitor, URB937 (0.3 mg/kg), had no influence. However, administered intracerebroventricularly, URB937 (5 µg/mouse) reduced the action of paracetamol. These results demonstrate that the supra-spinally-located FAAH enzyme is necessary for the analgesic action of paracetamol.