Spared nerve injury decreases motivation in long-access homecage-based operant tasks in mice.

ABSTRACT: Neuropathic pain causes both sensory and emotional maladaptation. Preclinical animal studies of neuropathic pain-induced negative affect could result in novel insights into the mechanisms of chronic pain. Modeling pain-induced negative affect, however, is variable across research groups and conditions. The same injury may or may not produce robust negative affective behavioral responses across different species, strains, and laboratories. Here, we sought to identify negative affective consequences of the spared nerve injury model on C57BL/6J male and female mice. We found no significant effect of spared nerve injury across a variety of approach-avoidance conflict, hedonic choice, and coping strategy assays. We hypothesized these inconsistencies may stem in part from the short test duration of these assays. To test this hypothesis, we used the homecage-based Feeding Experimentation Device version 3 to conduct 12-hour, overnight progressive ratio testing to determine whether mice with chronic spared nerve injury had decreased motivation to earn palatable food rewards. Our data demonstrate that despite equivalent task learning, spared nerve injury mice are less motivated to work for a sugar pellet than sham controls. Furthermore, when we normalized behavioral responses across all the behavioral assays we tested, we found that a combined normalized behavioral score is predictive of injury state and significantly correlates with mechanical thresholds. Together, these results suggest that homecage-based operant behaviors provide a useful platform for modeling nerve injury-induced negative affect and that valuable pain-related information can arise from agglomerative data analyses across behavioral assays-even when individual inferential statistics do not demonstrate significant mean differences.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Perinatal oxycodone exposure causes long-term sex-dependent changes in weight trajectory and sensory processing in adult mice.

RATIONALE: In utero opioid exposure is associated with lower weight and a neonatal opioid withdrawal syndrome (NOWS) at birth, along with longer-term adverse neurodevelopmental outcomes and mood disorders. While NOWS is sometimes treated with continued opioids, clinical studies have not addressed if long-term neurobehavioral outcomes are worsened with continued postnatal exposure to opioids. In addition, pre-clinical studies comparing in utero only opioid exposure to continued post-natal opioid administration for withdrawal mitigation are lacking. OBJECTIVES: Here, we sought to understand the impact of continued postnatal opioid exposure on long term behavioral consequences. METHODS: We implemented a rodent perinatal opioid exposure model of oxycodone (Oxy) exposure that included Oxy exposure until birth (short Oxy) and continued postnatal opioid exposure (long Oxy) spanning gestation through birth and lactation. RESULTS: Short Oxy exposure was associated with a sex-specific increase in weight gain trajectory in adult male mice. Long Oxy exposure caused an increased weight gain trajectory in adult males and alterations in nociceptive processing in females. Importantly, there was no evidence of long-term social behavioral deficits, anxiety, hyperactivity, or memory deficits following short or long Oxy exposure. CONCLUSIONS: Our findings suggest that offspring with prolonged opioid exposure experienced some long-term sequelae compared to pups with opioid cessation at birth. These results highlight the potential long-term consequences of opioid administration as a mitigation strategy for clinical NOWS symptomology and suggest alternatives should be explored.

Characterisation of peroxisome proliferator-activated receptor signalling in the midbrain periaqueductal grey of rats genetically prone to heightened stress, negative affect and hyperalgesia.

The stress-hyperresponsive Wistar-Kyoto (WKY) rat strain exhibits a hyperalgesic phenotype and is a useful genetic model for studying stress-pain interactions. Peroxisome proliferator-activated receptor (PPAR) signalling in the midbrain periaqueductal grey (PAG) modulates pain. This study characterised PPAR signalling in the PAG of WKY rats exposed to the formalin test of inflammatory pain, versus Sprague-Dawley (SD) controls. Formalin injection reduced levels of the endogenous PPAR ligands N-palmitoylethanolamide (PEA) and N-oleoylethanolamide (OEA) in the lateral(l) PAG of SD rats, but not WKY rats which exhibited higher levels of these analytes compared with formalin-injected SD counterparts. Levels of mRNA coding for fatty acid amide hydrolase (FAAH; catabolises PEA and OEA) were lower in the lPAG of WKY versus SD rats. PPARγ mRNA and protein levels in the lPAG were higher in saline-treated WKY rats, with PPARγ protein levels reduced by formalin treatment in WKY rats only. In the dorsolateral(dl) or ventrolateral(vl) PAG, there were no effects of formalin injection on PEA or OEA levels but there were some differences in levels of these analytes between saline-treated WKY and SD rats and some formalin-evoked alterations in levels of PPARα, PPARγ or FAAH mRNA in WKY and/or SD rats. Pharmacological blockade of PPARγ in the lPAG enhanced formalin-evoked nociceptive behaviour in WKY, but not SD, rats. These data indicate differences in the PPAR signalling system in the PAG of WKY versus SD rats and suggest that enhanced PEA/OEA-mediated tone at PPARγ in the lPAG may represent an adaptive mechanism to lower hyperalgesia in WKY rats.

N-palmitoylethanolamide in the anterior cingulate cortex attenuates inflammatory pain behaviour indirectly via a CB1 receptor-mediated mechanism.

The neural substrates and mechanisms mediating the antinociceptive effects of the endogenous bioactive lipid, N-palmitoylethanolamide (PEA), require further investigation. We investigated the effects of exogenous PEA administration into the anterior cingulate cortex (ACC), an important brain region linked with cognitive and affective modulation of pain, on formalin-evoked nociceptive behaviour in rats. Potential involvement of peroxisome proliferator-activated receptor isoforms (PPAR) α and γ or endocannabinoid-mediated entourage effects at cannabinoid1 (CB1) receptors or transient receptor potential subfamily V member 1 (TRPV1) in mediating the effects of PEA was also investigated. Intra-ACC administration of PEA significantly attenuated the first and early second phases of formalin-evoked nociceptive behaviour. This effect was attenuated by the CB1 receptor antagonist AM251, but not by the PPARα antagonist GW6471, the PPARγ antagonist GW9662, or the TRPV1 antagonist 5'-iodo resiniferatoxin. All antagonists, administered alone, significantly reduced formalin-evoked nociceptive behaviour, suggesting facilitatory/permissive roles for these receptors in the ACC in inflammatory pain. Post-mortem tissue analysis revealed a strong trend for increased levels of the endocannabinoid anandamide in the ACC of rats that received intra-ACC PEA. Expression of c-Fos, a marker of neuronal activity, was significantly reduced in the basolateral nucleus of the amygdala, but not in the central nucleus of the amygdala, the rostral ventromedial medulla or the dorsal horn of the spinal cord. In conclusion, these data indicate that PEA in the ACC can reduce inflammatory pain-related behaviour, possibly via AEA-induced activation of CB1 receptors and associated modulation of neuronal activity in the basolateral amygdala.

Genotype-dependent responsivity to inflammatory pain: A role for TRPV1 in the periaqueductal grey.

Negative affective state has a significant impact on pain, and genetic background is an important moderating influence on this interaction. The Wistar-Kyoto (WKY) inbred rat strain exhibits a stress-hyperresponsive, anxiety/depressive-like phenotype and also displays a hyperalgesic response to noxious stimuli. Transient receptor potential subfamily V member 1 (TRPV1) within the midbrain periaqueductal grey (PAG) plays a key role in regulating both aversive and nociceptive behaviour. In the present study, we investigated the role of TRPV1 in the sub-columns of the PAG in formalin-evoked nociceptive behaviour in WKY versus Sprague-Dawley (SD) rats. TRPV1 mRNA expression was significantly lower in the dorsolateral (DL) PAG and higher in the lateral (L) PAG of WKY rats, compared with SD counterparts. There were no significant differences in TRPV1 mRNA expression in the ventrolateral (VL) PAG between the two strains. TRPV1 mRNA expression significantly decreased in the DLPAG and increased in the VLPAG of SD, but not WKY rats upon intra-plantar formalin administration. Intra-DLPAG administration of either the TRPV1 agonist capsaicin, or the TRPV1 antagonist 5'-Iodoresiniferatoxin (5'-IRTX), significantly increased formalin-evoked nociceptive behaviour in SD rats, but not in WKY rats. The effects of capsaicin were likely due to TRPV1 desensitisation, given their similarity to the effects of 5'-IRTX. Intra-VLPAG administration of capsaicin or 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, and similar effects were seen with 5'-IRTX in WKY rats. Intra-LPAG administration of 5'-IRTX reduced nociceptive behaviour in a moderate and transient manner in SD rats, but not in WKY rats. These results indicate that modulation of inflammatory pain by TRPV1 in the PAG occurs in a sub-column-specific manner. The data also provide evidence for differences in the expression of TRPV1, and differences in the effects of pharmacological modulation of TRPV1 in specific PAG sub-columns, between WKY and SD rats, suggesting that TRPV1 expression and/or functionality in the PAG plays a role in hyper-responsivity to noxious stimuli in a genetic background prone to negative affect.

Supraspinal Transient Receptor Potential Subfamily V Member 1 (TRPV1) in Pain and Psychiatric Disorders.

The transient receptor potential subfamily V member 1 (TRPV1) belongs to the diverse transient receptor potential (TRP) family of cation channels. It was first characterized in primary afferent fibres as a receptor for capsaicin. Peripheral TRPV1 has a very well-described role in nociception. However, TRPV1 is now recognized to have a broader distribution and function, with supraspinal/brain TRPV1 known to modulate pain processing. Recently, studies employing histological, genetic and pharmacological approaches have provided evidence that supraspinal TRPV1 also modulates brain neurobiology and behaviours related to anxiety, depression and schizophrenia. Key brain regions involved in TRPV1-mediated modulation of pain and affect include the periaqueductal grey, hippocampus and medial prefrontal cortex. Thus, TRPV1 in the brain is emerging as an important molecular substrate which is dually implicated in both pain and psychiatric disorders, and represents a novel therapeutic target for these conditions and their comorbidity.

Impaired endocannabinoid signalling in the rostral ventromedial medulla underpins genotype-dependent hyper-responsivity to noxious stimuli.

Pain is both a sensory and an emotional experience, and is subject to modulation by a number of factors including genetic background modulating stress/affect. The Wistar-Kyoto (WKY) rat exhibits a stress-hyper-responsive and depressive-like phenotype and increased sensitivity to noxious stimuli, compared with other rat strains. Here, we show that this genotype-dependent hyperalgesia is associated with impaired pain-related mobilisation of endocannabinoids and transcription of their synthesising enzymes in the rostral ventromedial medulla (RVM). Pharmacological blockade of the Cannabinoid1 (CB1) receptor potentiates the hyperalgesia in WKY rats, whereas inhibition of the endocannabinoid catabolising enzyme, fatty acid amide hydrolase, attenuates the hyperalgesia. The latter effect is mediated by CB1 receptors in the RVM. Together, these behavioural, neurochemical, and molecular data indicate that impaired endocannabinoid signalling in the RVM underpins hyper-responsivity to noxious stimuli in a genetic background prone to heightened stress/affect.