Primers used in the present study were custom-synthesized

damide degradation in rat brain homogenates is between two and three orders of magnitude lower than the potency of this compound in assays of TRPV1 activity. The selectivity towards TRPV1 is even larger for arvanil and olvanil, which both are weak inhibitors of anandamide uptake and degradation. This is in line with the global brain levels of endocannabinoids being unaffected by systemic administration of 4-aminophenol or paracetamol. It is therefore intriguing that the cannabinoid CB1 receptor antagonist AM251 could prevent the antinociceptive effect of 4-aminophenol in the rat formalin and paw pressure tests. We have previously reported similar findings with respect to paracetamol. Unsaturated long chain N-acyl-hydroxyphenylamides and -vanillylamides, including AM404, arvanil and olvanil, are poor cannabinoid receptor agonists. It is, Analgesic TRPV1 Active Drug Metabolites in Brain thus, unlikely that sufficient amounts of AM404 and arvanil plus olvanil are generated from 4-aminophenol and HMBA in the brain, respectively, to sustain a direct activation of the cannabinoid CB1 receptor. A recent study, addressing the intriguing antinociceptive effect of the TRPV1 activator capsaicin in the periaqueductal gray, an important midbrain region for regulation of descending pain inhibitory pathways projecting to the dorsal horn, has provided evidence of the recruitment of cannabinoid CB1 receptors downstream of TRPV1 present on excitatory glutamatergic neurons in the ventrolateral PAG. Microinjection of capsaicin into the rat ventrolateral PAG reduced the nocifensive behaviour in the hot plate test, an effect that was inhibited by co-injection of capsaicin with the cannabinoid CB1 receptor antagonist AM251 as well as with the selective TRPV1 blocker SB 366791. Patch-clamp recordings in brain slices of the ventrolateral PAG demonstrated that capsaicin produced not only a glutamate-dependent neuronal excitation, but also an endocannabinoid-mediated retrograde inhibition of GABAergic neurons, an effect (-)-Blebbistatin reversed by AM251. Such a dual effect of TRPV1 activators in the ventrolateral PAG may explain the seemingly contradictory finding 10355733 that pharmacological or genetic inactivation of the cannabinoid CB1 receptor is able to inhibit the antinociceptive effects 4-aminophenol and paracetamol. Bulbospinal serotonergic pathways originate in several brainstem nuclei, including the nucleus raphe magnus. These pathways receive input from PAG and contribute to the descending regulation of nociceptive signalling via serotonin release and activation of its cognate receptors on dorsal horn neurons. Our findings that intervention with spinal serotoninergic mechanisms prevents the antinociceptive effects of 4-aminophenol and paracetamol provide additional evidence that supraspinal TRPV1 may regulate nociceptive neurotransmission via activation of descending bulbospinal pathways. TRPV1 activation also facilitates glutamate neurotransmission in the dorsolateral PAG and 18194435 in other brain areas of potential importance for the regulation of descending pain inhibition, including the locus coeruleus and paraventricular nucleus. Thus, the identity and localization of the TRPV1-expressing Analgesic TRPV1 Active Drug Metabolites in Brain neurons activated by the lipids metabolites of paracetamol, 4aminophenol and HMBA, and the relationship between these neurons and the descending bulbospinal serotonergic pathways remain to be elucidated. Several factors may influence the biotran