ABSTRACT:Noxious cold sensation is commonly associated with peripheral neuropathies; however, there has been limited progress in understanding the mechanism. Here, we identify a novel role for peripherally expressed kappa opioid receptors [KORs] in noxious cold hypersensitivity in a mouse model of chemotherapy. In this model, we show that oxaliplatin-induced cold hypersensitivity is attenuated using norbinaltorphimine (NorBNI, KOR antagonist (10 mg/kg, i.p.) in both male and female mice. To further examine the role of KORs in cold hypersensitivity, we show that activation of KOR with U50,488 (KOR agonist, 5 mg/kg i.p.) significantly increases the number of jumps on a cold plate at 3°C as compared to controls, which is attenuated by KOR antagonist NorBNI. We have determined that this effect is mediated through peripheral KORs using both pharmacology and a peripherally restricted KOR agonist, ff(nle)r-NH2. Using a conditional knockout mouse model of KOR in the dorsal root ganglia (using PirtCre;Oprk fl/fl ), we see reduced U50488-induced noxious cold hypersensitivity as compared to controls in male and female mice, confirming the role of KORs in the dorsal root ganglia in mediating cold hypersensitivity. To confirm the role of peripheral KORs in oxaliplatin-induced cold allodynia, we use the PirtCre;Oprk fl/fl in our oxaliplatin-induced cold allodynia model. Here, we show that in the absence of KORs in dorsal root ganglia, oxaliplatin-induced cold hypersensitivity is attenuated in male and female mice. Overall, our data suggest that peripheral KORs modulate cold hypersensitivity in a mouse model of chemotherapy.

The concept of ligand bias is based on the premise that different agonists can elicit distinct responses by selectively activating the same receptor. These responses often determine whether an agonist has therapeutic or undesirable effects. Therefore, it would be highly advantageous to have agonists that specifically trigger the therapeutic response. The last two decades have seen a growing trend towards the consideration of ligand bias in the development of ligands to target the κ-opioid receptor (κOR). Most of these ligands selectively favor G-protein signaling over β-arrestin signaling to potentially provide effective pain and itch relief without adverse side effects associated with κOR activation. Importantly, the specific role of β-arrestin 2 in mediating κOR agonist-induced side effects remains unknown, and similarly the therapeutic and side-effect profiles of G-protein-biased κOR agonists have not been established. Furthermore, some drugs previously labeled as G-protein-biased may not exhibit true bias but may instead be either low-intrinsic-efficacy or partial agonists. In this review, we discuss the established methods to test ligand bias, their limitations in measuring bias factors for κOR agonists, as well as recommend the consideration of other systematic factors to correlate the degree of bias signaling and pharmacological effects. This article is part of the Special Issue on "Ligand Bias".

Multimodal neural interfaces open new opportunities in brain research by enabling more sophisticated and systematic neural circuit dissection. Integrating complementary features across distinct functional domains, these multifunctional neural probes have greatly advanced the interrogation of complex neural circuitry. However, introducing multiple functionalities into a compact form factor for freely behaving animals presents substantial design hurdles that complicate the device or require more than one device. Moreover, fixed functionality poses challenges in meeting the dynamic needs of chronic neuroscience inquiry, such as replacing consumable parts like batteries or drugs. To address these limitations, the modular implantable neural device (MIND) is introduced with a one-touch magnetic assembly mechanism. Leveraging the seamless exchange of neural interface modules such as optical stimulation, drug delivery, and electrical stimulation, MIND ensures functional adaptability, reusability, and scalability. The versatile design of MIND will facilitate brain research by enabling simplified access to multiple functional modalities as needed.

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.

Noxious cold sensation is commonly associated with peripheral neuropathies, however, there has been limited progress in understanding the mechanism of cold pain. Here we identify a role for kappa opioid receptors (KOR) in driving noxious cold hypersensitivity. First, we show that systemic activation of KOR by the agonist U50,488 (U50), increases the latency to jump and the number of jumps on a cold plate at 3°C, and that the KOR antagonist NorBNI attenuates U50-induced noxious cold hypersensitivity. However, the central administration of NorBNI does not block U50-induced noxious cold hypersensitivity, suggesting that peripheral KOR may modulate this effect. To directly test this, we use the peripherally-restricted KOR agonist, ff(nle)r-NH2 and also show selective activation of peripheral KOR causes noxious cold hypersensitivity. To begin to understand how peripheral KOR drive noxious cold hypersensitivity we investigated whether KOR interact with transient receptor potential ankyrin 1(TRPA1) channels, known to facilitate the perception of noxious cold, in dorsal root ganglion (DRG). Using fluorescent in situ hybridization, we show that KOR mRNA colocalizes with the transcripts for the cold-activated TRPA1 channels in DRG. We also show a potentiation in intracellular calcium release in DRG neurons during the simultaneous application of the TRPA1 agonist, mustard oil (MO), and a KOR agonist, U50, when compared to MO alone. Together our data suggest that peripheral KOR may induce noxious cold hypersensitivity through modulation of TRPA1 channels.### Competing Interest StatementThe authors have declared no competing interest.

Mu-opioid receptor (mu OR) 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 overdose(1). Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site(2) found in mu OR3 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 mu OR highlight the key interactions between the guanidine of the ligands and the key Asp(2.50) residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at G(i) subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest G(z) efficacy among the panel of mu OR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed mu OR-dependent antinociception with attenuated adverse effects, supporting the mu OR 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 G(i), G(o) and G(z) subtypes and arrestins, thus modulating their in vivo pharmacology.

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.

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, 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. Further, 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.

Opioid agonists have been used in the field of pain management for centuries, but are considered a major public health concern, especially in the US. One of the biggest challenges consists in understanding the ability to target specifically the different subtypes of opioid receptors and to trigger a specific functional response correlating with an in vivo analgesic response devoid of side‐effects.These opioid receptors are part of the GPCRs family, which is known to have common features across different subfamilies. One important feature of these receptors is the presence of allosteric binding pockets, which is progressively gaining momentum as a novel target for current therapeutic strategies. We hypothesized that targeting an allosteric site could potentially allow for receptor functional selectivity, helping to reduce the overall side effects profile. One site, in particular, known as the Na+ binding pocket, is known to play a key role in GPCR function and is thought to be the center of the functional mechanism, with several bias switches characterized in the pocket residues (Katritch et al., Trends Biochem Sci. 2014).Here we report structure‐based rational drug design of bitopic ligands targeting the Na+ pocket of mu (MOR) and kappa opioid receptors (KOR). Our designed bitopics show high affinity, subtype selectivity, G‐protein bias and analgesic actions in mice. Mutations in Na+ binding pocket diminished functional potency validating allosteric site targeting. Together, results suggest the possibility of achieving subtype selectivity and identification of an additional subpocket in both MOR and KOR to reduce βarrestin‐2 recruitment by targeting an allosteric pocket in the opioid receptors. The highly conserved constitution of the Na+ binding pocket opens the opportunity for rational discovery of new GPCR modulators with desired functional, and potentially therapeutic profile for opioids as well as other class A GPCRs.Support or Funding InformationWe gratefully acknowledge the financial support from Faculty Research Incentive Fund, NIDA, NIAAA and US department of defense. We also would like to thank the Philippe Foundation for their financial support.

: The Transient Receptor Potential Subfamily V Member 1 (TRPV1) belongs to the diverse group of the transient receptor potential (TRP) family of cation channels. It was first characterized in primary afferent fibers 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 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 co-morbidity.

: 21 Negative affective state has a significant impact on pain, and genetic background is an 22 important moderating influence on this interaction. The Wistar–Kyoto (WKY) inbred rat strain 23 exhibits a stress-hyperresponsive, anxiety/depressive-like phenotype and also displays a 24 hyperalgesic response to noxious stimuli. Transient receptor potential subfamily V member 1 25 (TRPV1) within the midbrain periaqueductal grey (PAG) plays a key role in regulating both 26 aversive and nociceptive behaviour. In the present study, we investigated the role of TRPV1 in 27 the sub-columns of the PAG in formalin-evoked nociceptive behaviour in WKY versus 28 Sprague-Dawley (SD) rats. TRPV1 mRNA expression was significantly lower in the 29 dorsolateral (DL) PAG and higher in the lateral (L) PAG of WKY rats, compared with SD 30 counterparts. There were no significant differences in TRPV1 mRNA expression in the 31 ventrolateral (VL) PAG between the two strains. TRPV1 mRNA expression significantly 32 decreased in the DLPAG and increased in the VLPAG of SD, but not WKY rats upon intra- 33 plantar formalin administration. Intra-DLPAG administration of either the TRPV1 agonist 34 capsaicin, or the TRPV1 antagonist 5’-Iodoresiniferatoxin (5’-IRTX), significantly increased 35 formalin-evoked nociceptive behaviour in SD rats, but not in WKY rats. The effects of 36 capsaicin were likely due to TRPV1 desensitisation, given their similarity to the effects of 5’- 37 IRTX. Intra-VLPAG administration of capsaicin or 5’-IRTX reduced nociceptive behaviour in 38 a moderate and transient manner in SD rats, and similar effects were seen with 5’-IRTX in 39 WKY rats. Intra-LPAG administration of 5’-IRTX reduced nociceptive behaviour in a 40 moderate and transient manner in SD rats, but not in WKY rats. These results indicate that 41 modulation of inflammatory pain by TRPV1 negative affect.

Publication Information Madasu, M. K., Roche, M., & Finn, D. P. (2015). Supraspinal Transient Receptor Potential Subfamily V Member 1 (TRPV1) in Pain and Psychiatric Disorders. In Finn, David P. & Leonard, Brian E. (Eds.), Pain in Psychiatric Disorders: Modern Trends Pharmacopsychiatry (Vol. 30, pp. 80-93). Basel: Karger. doi: 10.1159/000435934 Publisher Karger Link to publisher's version https://doi.org/10.1159/000435934

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.

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.