Peripheral cannabinoid receptors exert a powerful inhibitory control over pain initiation, but the endocannabinoid signal that normally engages this intrinsic analgesic mechanism is unknown. To address this question, we developed a peripherally restricted inhibitor of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of the endocannabinoid anandamide. The compound, called URB937, suppresses FAAH activity and increases anandamide levels outside the central nervous system (CNS). Despite its inability to access brain and spinal cord, URB937 attenuates behavioral responses indicative of persistent pain in rodent models of peripheral nerve injury and inflammation, and prevents noxious stimulus-evoked neuronal activation in spinal cord regions implicated in nociceptive processing. CB1 cannabinoid receptor blockade prevents these effects. The results suggest that anandamide-mediated signaling at peripheral CB1 receptors controls the access of pain-related inputs to the CNS. Brain-impenetrant FAAH inhibitors, which strengthen this gating mechanism, might offer a new approach to pain therapy.

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here we show that foot shock stress produces antinociception in rats by activating type-5 metabotropic glutamate receptors (mGlu5) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu5 in the dlPAG with (S)-3,5-dihydroxyphenylglycine (DHPG) triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB1 cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 and (−)-tetrahydrolipstatin (THL), which inhibit activity of DGL-α and DGL-β isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-α co-localized with mGlu5 at postsynaptic sites of the dlPAG, whereas CB1 was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally-mediated silencing of DGL-α, but not DGL-β, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu5–DGL-α cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

Monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) degrade the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (AEA), respectively. Pharmacological inhibition of these enzymes in the periphery may elucidate the role of endocannabinoids in controlling nociceptive transmission. We compared effects of the MGL inhibitor JZL184, the FAAH inhibitor URB597, and the endocannabinoid uptake inhibitor VDM11, administered locally in the paw, on behavioral hypersensitivities produced by capsaicin, the pungent ingredient in hot chili peppers. Intradermal capsaicin (10 μg i.pl.) produced nocifensive behavior, thermal hyperalgesia, and mechanical allodynia in rats. JZL184 (100 μg i.pl.) suppressed capsaicin-induced nocifensive behavior and thermal hyperalgesia without altering capsaicin-evoked mechanical allodynia. Effects of JZL184 were blocked by either the CB1 antagonist AM251 (80 μg i.pl.) or the CB2 antagonist AM630 (25 μg i.pl.). URB597 (75 μg i.pl.) suppressed capsaicin-induced mechanical allodynia without altering capsaicin-evoked thermal hyperalgesia or nocifensive behavior. Effects of URB597 were blocked by AM251 (80 μg i.pl.), but not by AM630 (25 μg i.pl.). VDM11 (100 μg i.pl.) suppressed capsaicin-evoked hypersensitivity for all three dependent measures (nocifensive behavior, thermal hyperalgesia, and mechanical allodynia), suggesting an additive effect following putative elevation of both AEA and 2-AG. The VDM11-induced suppression of capsaicin-evoked nocifensive behavior and thermal hyperalgesia was blocked by either AM251 (80 μg i.pl.) or AM630 (25 μg i.pl.), as observed with JZL184. The VDM11-induced suppression of capsaicin-evoked mechanical allodynia was blocked by AM251 (25 μg i.pl.) only, as observed with URB597. Thus, peripheral inhibition of enzymes hydrolyzing 2-AG and AEA suppresses capsaicin-evoked behavioral sensitization with distinct patterns of pharmacological specificity and in a non-overlapping and modality-specific manner. Modulation of endocannabinoids in the periphery suppressed capsaicin-evoked nocifensive behavior and thermal hyperalgesia through either CB1 or CB2 receptor mechanisms but suppressed capsaicin-evoked mechanical allodynia through CB1 mechanisms only. Inhibition of endocannabinoid transport was more effective in suppressing capsaicin-induced sensitization compared to inhibition of either FAAH or MGL alone. These studies are the first to unveil the effects of pharmacologically increasing peripheral endocannabinoid levels on capsaicin-induced behavioral hypersensitivities. Our data suggest that 2-AG, the putative product of MGL inhibition, and AEA, the putative product of FAAH inhibition, differentially suppress capsaicin-induced nociception through peripheral cannabinoid mechanisms.

Broiler breeder hens are typically provided a restricted amount of feed once a day. This feed is rapidly consumed; therefore, the hens fast for an extended period of time before their next feeding. In the current research, the effects on reproductive performance of implementing a twice-a-day vs. a once-a-day feeding program after photostimulation were investigated. Pullets and cockerels were reared on a skip-a-day feeding program. Pullets were weighed at 20 wk of age and then distributed into 30 laying pens such that each pen had a similar BW distribution. Each individual laying pen consisted of 35 hens and 4 roosters. At 21 wk of age, the birds were photostimulated for reproduction and 15 of the laying pens were placed on a once-a-day feeding schedule, whereas the other 15 pens were placed on a twice-a-day feeding schedule. The total amount of feed provided per day to all the laying pens was the same. Birds fed once a day received all their feed at 0630 h, whereas birds fed twice a day received 60% of their total feed allotment at 0630 h and the other 40% at 1500 h. Even though both treatment groups began egg production at the end of wk 23, birds fed twice a day laid more (P < or = 0.05) eggs through 42 wk of age than those fed once a day. Additionally, the average egg weight for the entire production period, which lasted until the birds were 60 wk of age, was greater for hens fed twice a day. Overall BW uniformity for the entire experiment was significantly better for hens fed twice a day vs. once a day. However, cumulative mortality was significantly higher for hens fed twice a day than for those fed once a day. The results indicate that feeding broiler breeder hens twice a day after photostimulation may enhance reproductive performance during the early lay period.

Cannabinoids suppress nocifensive behaviors in rodents. We presently investigated peripheral endocannabinoid modulation of itch- and pain-related behaviors elicited from facial vs. spinally-innervated skin of rats. Intradermal (id) injection of the pruritogen serotonin (5-HT) elicited significantly more hindlimb scratch bouts, and longer cumulative time scratching, when injected in the rostral back compared to the cheek. Pretreatment of skin with inhibitors of degrading enzymes for the endocannabinoids anandamide (URB597) or 2-arachidonoylglycerol (JZL184) significantly reduced scratching elicited by 5-HT in the rostral back. These effects were prevented by co-treatment with antagonists of the CB1 (AM251) or CB2 receptor (AM630), implicating both receptor subtypes in endocannabinoid suppression of scratching in spinally-innervated skin. Conversely, pretreatment with either enzyme inhibitor, or with AM630 alone, increased the number of scratch bouts elicited by id 5-HT injection in the cheek. Moreover, pretreatment with JZL184 also significantly increased pain-related forelimb wipes directed to the cheek following id injection of the algogen, allyl isothiocyanate (AITC; mustard oil). Thus, peripheral endocannabinoids have opposite effects on itch-related scratching behaviors in trigeminally- vs. spinally-innervated skin. These results suggest that increasing peripheral endocannabinoid levels represents a promising therapeutic approach to treat itch arising from the lower body, but caution that such treatment may not relieve, and may even exacerbate, itch and pain arising from trigeminally-innervated skin of the face or scalp.