Genetic mouse models in opioid research: current status and future directions.

Synthetic and semi-synthetic opioids are prescribed for the management of severe pain conditions, but their long-term use is often leading to physical dependence and addiction disorders. Understanding the complex neurobiology of the opioid system in preclinical models will be essential for the development of safe and efficacious analgesics. With rising numbers of synthetic opioid users and overdose cases, a better understanding of the neuroanatomical and cellular pathways associated with physical dependence and addiction is expected to guide treatment approaches for opioid use disorders. In this commentary, we highlight the importance of advanced genetic mouse models for studying the regional effects of opioid receptors, and we discuss the need of genetic mouse models for the investigation of the regional, circuit and cell compartment-specific role of intracellular mediators of opioid actions.

Desipramine induces anti-inflammatory dorsal root ganglion transcriptional signatures in the murine spared nerve injury model.

Monoamine-targeting antidepressants serve as frontline medications for chronic pain and associated comorbidities. While persistent anti-allodynic properties of antidepressants generally require weeks of treatment, several groups have demonstrated acute analgesic effects within hours of administration, suggesting a role in non-mesocorticolimbic pain processing regions such as the peripheral nervous system. To further explore this possibility, after four weeks of spared nerve injury or sham surgeries, we systemically administered desipramine or saline for an additional three weeks and performed whole transcriptome RNA sequencing on L3-6 dorsal root ganglia. Along with alterations in molecular pathways associated with neuronal activity, we observed a robust immunomodulatory transcriptional signature in the desipramine treated group. Cell subtype deconvolution predicted that these changes were associated with A- and C-fibers. Of note, differentially expressed genes from the dorsal root ganglia of DMI-treated, injured mice were largely unique compared to those from the nucleus accumbens of the same animals. These observations suggest that, under peripheral nerve injury conditions, desipramine induces specific gene expression changes across various regions of the nociceptive circuitry.

tRNA epitranscriptomic alterations associated with opioid-induced reward-seeking and long-term opioid withdrawal in male mice.

DNA cytosine methylation has been documented as a potential epigenetic mechanism of transcriptional regulation underlying opioid use disorder. However, methylation of RNA cytosine residues, which would drive another level of biological influence as an epitranscriptomic mechanism of gene and protein regulation has not been studied in the context of addiction. Here, we probed whether chronic morphine exposure could alter tRNA cytosine methylation (m5C) and resulting expression levels in the medial prefrontal cortex (mPFC), a brain region crucial for reward processing and executive function that exhibits opioid-induced molecular restructuring. We identified dynamic changes in glycine tRNA (tRNAGlyGCC) cytosine methylation, corresponding to altered expression levels of this tRNA at multiple timepoints following 15 days of daily morphine. Additionally, a robust increase in methylation, coupled with decreased expression, was present after 30 days of withdrawal, suggesting that repeated opioid administration produces changes to the tRNA regulome long after discontinuation. Furthermore, forebrain-wide knockout of neuronal Nsun2, a tRNA methyltransferase, was associated with disruption of opioid conditioned place preference, and this effect was recapitulated by regional mPFC Nsun2 knockout. Taken together, these studies provide a foundational link between the regulation of tRNA cytosine methylation and opioid reward and highlight the tRNA machinery as a potential therapeutic target in addiction.

RGS4 Actions in Mouse Prefrontal Cortex Modulate Behavioral and Transcriptomic Responses to Chronic Stress and Ketamine.

The signal transduction protein, regulator of G protein signaling 4 (RGS4), plays a prominent role in physiologic and pharmacological responses by controlling multiple intracellular pathways. Our earlier work identified the dynamic but distinct roles of RGS4 in the efficacy of monoamine-targeting versus fast-acting antidepressants. Using a modified chronic variable stress (CVS) paradigm in mice, we demonstrate that stress-induced behavioral abnormalities are associated with the downregulation of RGS4 in the medial prefrontal cortex (mPFC). Knockout of RGS4 (RGS4KO) increases susceptibility to CVS, as mutant mice develop behavioral abnormalities as early as 2 weeks after CVS resting-state functional magnetic resonance imaging I (rs-fMRI) experiments indicate that stress susceptibility in RGS4KO mice is associated with changes in connectivity between the mediodorsal thalamus (MD-THL) and the mPFC. Notably, RGS4KO also paradoxically enhances the antidepressant efficacy of ketamine in the CVS paradigm. RNA-sequencing analysis of naive and CVS samples obtained from mPFC reveals that RGS4KO triggers unique gene expression signatures and affects several intracellular pathways associated with human major depressive disorder. Our analysis suggests that ketamine treatment in the RGS4KO group triggers changes in pathways implicated in synaptic activity and responses to stress, including pathways associated with axonal guidance and myelination. Overall, we show that reducing RGS4 activity triggers unique gene expression adaptations that contribute to chronic stress disorders and that RGS4 is a negative modulator of ketamine actions. SIGNIFICANCE STATEMENT: Chronic stress promotes robust maladaptation in the brain, but the exact intracellular pathways contributing to stress vulnerability and mood disorders have not been thoroughly investigated. In this study, the authors used murine models of chronic stress and multiple methodologies to demonstrate the critical role of the signal transduction modulator regulator of G protein signaling 4 in the medial prefrontal cortex in vulnerability to chronic stress and the efficacy of the fast-acting antidepressant ketamine.

Annulus Fibrosus Injury Induces Acute Neuroinflammation and Chronic Glial Response in Dorsal Root Ganglion and Spinal Cord-An In Vivo Rat Discogenic Pain Model.

Chronic painful intervertebral disc (IVD) degeneration (i.e., discogenic pain) is a major source of global disability needing improved knowledge on multiple-tissue interactions and how they progress in order improve treatment strategies. This study used an in vivo rat annulus fibrosus (AF) injury-driven discogenic pain model to investigate the acute and chronic changes in IVD degeneration and spinal inflammation, as well as sensitization, inflammation, and remodeling in dorsal root ganglion (DRG) and spinal cord (SC) dorsal horn. AF injury induced moderate IVD degeneration with acute and broad spinal inflammation that progressed to DRG to SC changes within days and weeks, respectively. Specifically, AF injury elevated macrophages in the spine (CD68) and DRGs (Iba1) that peaked at 3 days post-injury, and increased microglia (Iba1) in SC that peaked at 2 weeks post-injury. AF injury also triggered glial responses with elevated GFAP in DRGs and SC at least 8 weeks post-injury. Spinal CD68 and SC neuropeptide Substance P both remained elevated at 8 weeks, suggesting that slow and incomplete IVD healing provides a chronic source of inflammation with continued SC sensitization. We conclude that AF injury-driven IVD degeneration induces acute spinal, DRG, and SC inflammatory crosstalk with sustained glial responses in both DRGs and SC, leading to chronic SC sensitization and neural plasticity. The known association of these markers with neuropathic pain suggests that therapeutic strategies for discogenic pain need to target both spinal and nervous systems, with early strategies managing acute inflammatory processes, and late strategies targeting chronic IVD inflammation, SC sensitization, and remodeling.

Tianeptine promotes lasting antiallodynic effects in a mouse model of neuropathic pain.

Tricyclic antidepressants (TCAs), such as desipramine (DMI), are effective at managing neuropathic pain symptoms but often take several weeks to become effective and also lead to considerable side effects. Tianeptine (TIAN) is an atypical antidepressant that activates the mu-opioid receptor but does not produce analgesic tolerance or withdrawal in mice, nor euphoria in humans, at clinically-relevant doses. Here, we evaluate the efficacy of TIAN at persistently alleviating mechanical allodynia in the spared nerve injury (SNI) model of neuropathic pain, even well after drug clearance. After finding an accelerated onset of antiallodynic action compared to DMI, we used genetically modified mice to gain insight into RGS protein-associated pathways that modulate the efficacy of TIAN relative to DMI in models of neuropathic pain. Because we observed similar behavioral responses to both TIAN and DMI treatment in RGS4, RGSz1, and RGS9 knockout mice, we performed RNA sequencing on the NAc of TIAN- and DMI-treated mice after prolonged SNI to further clarify potential mechanisms underlying TIANs faster therapeutic actions. Our bioinformatic analysis revealed distinct transcriptomic signatures between the two drugs, with TIAN more directly reversing SNI-induced differentially expressed genes, and further predicted several upstream regulators that may be implicated in onset of action. This new understanding of the molecular pathways underlying TIAN action may enable the development of novel and more efficacious pharmacological approaches for the management of neuropathic pain.

Oxycodone withdrawal induces HDAC1/HDAC2-dependent transcriptional maladaptations in the reward pathway in a mouse model of peripheral nerve injury.

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. We developed a mouse model of oxycodone exposure and subsequent withdrawal in the presence or absence of chronic neuropathic pain. Oxycodone withdrawal alone triggered robust gene expression adaptations in the nucleus accumbens, medial prefrontal cortex and ventral tegmental area, with numerous genes and pathways selectively affected by oxycodone withdrawal in mice with peripheral nerve injury. Pathway analysis predicted that histone deacetylase (HDAC) 1 is a top upstream regulator in opioid withdrawal in nucleus accumbens and medial prefrontal cortex. The novel HDAC1/HDAC2 inhibitor, Regenacy Brain Class I HDAC Inhibitor (RBC1HI), attenuated behavioral manifestations of oxycodone withdrawal, especially in mice with neuropathic pain. These findings suggest that inhibition of HDAC1/HDAC2 may provide an avenue for patients with chronic pain who are dependent on opioids to transition to non-opioid analgesics.

SARS-CoV-2 airway infection results in the development of somatosensory abnormalities in a hamster model.

Although largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and chronic phenotypes. To gain insight on the molecular basis of these sensory abnormalities, we used the golden hamster model to characterize and compare the effects of infection with SARS-CoV-2 and influenza A virus (IAV) on the sensory nervous system. We detected SARS-CoV-2 transcripts but no infectious material in the cervical and thoracic spinal cord and dorsal root ganglia (DRGs) within the first 24 hours of intranasal virus infection. SARS-CoV-2-infected hamsters exhibited mechanical hypersensitivity that was milder but prolonged compared with that observed in IAV-infected hamsters. RNA sequencing analysis of thoracic DRGs 1 to 4 days after infection suggested perturbations in predominantly neuronal signaling in SARS-CoV-2-infected animals as opposed to type I interferon signaling in IAV-infected animals. Later, 31 days after infection, a neuropathic transcriptome emerged in thoracic DRGs from SARS-CoV-2-infected animals, which coincided with SARS-CoV-2-specific mechanical hypersensitivity. These data revealed potential targets for pain management, including the RNA binding protein ILF3, which was validated in murine pain models. This work elucidates transcriptomic signatures in the DRGs triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities.

Spinal Cord Sensitization and Spinal Inflammation from an In Vivo Rat Endplate Injury Associated with Painful Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration with Modic-like changes is strongly associated with pain. Lack of effective disease-modifying treatments for IVDs with endplate (EP) defects means there is a need for an animal model to improve understanding of how EP-driven IVD degeneration can lead to spinal cord sensitization. This rat in vivo study determined whether EP injury results in spinal dorsal horn sensitization (substance P, SubP), microglia (Iba1) and astrocytes (GFAP), and evaluated their relationship with pain-related behaviors, IVD degeneration, and spinal macrophages (CD68). Fifteen male Sprague Dawley rats were assigned into sham or EP injury groups. At chronic time points, 8 weeks after injury, lumbar spines and spinal cords were isolated for immunohistochemical analyses of SubP, Iba1, GFAP, and CD68. EP injury most significantly increased SubP, demonstrating spinal cord sensitization. Spinal cord SubP-, Iba1- and GFAP-immunoreactivity were positively correlated with pain-related behaviors, indicating spinal cord sensitization and neuroinflammation play roles in pain responses. EP injury increased CD68 macrophages in the EP and vertebrae, and spinal cord SubP-, Iba1- and GFAP-ir were positively correlated with IVD degeneration and CD68-ir EP and vertebrae. We conclude that EP injuries result in broad spinal inflammation with crosstalk between spinal cord, vertebrae and IVD, suggesting that therapies must address neural pathologies, IVD degeneration, and chronic spinal inflammation.

A Regional and Projection-Specific Role of RGSz1 in the Ventrolateral Periaqueductal Grey in the Modulation of Morphine Reward.

Opioid analgesics exert their therapeutic and adverse effects by activating µ opioid receptors (MOPR); however, functional responses to MOPR activation are modulated by distinct signal transduction complexes within the brain. The ventrolateral periaqueductal gray (vlPAG) plays a critical role in modulation of nociception and analgesia, but the exact intracellular pathways associated with opioid responses in this region are not fully understood. We previously showed that knockout of the signal transduction modulator Regulator of G protein Signaling z1 (RGSz1) enhanced analgesic responses to opioids, whereas it decreased the rewarding efficacy of morphine. Here, we applied viral mediated gene transfer methodology and delivered adeno-associated virus (AAV) expressing Cre recombinase to the vlPAG of RGSz1fl\fl mice to demonstrate that downregulation of RGSz1 in this region decreases sensitivity to morphine in the place preference paradigm, under pain-free as well as neuropathic pain states. We also used retrograde viral vectors along with flippase-dependent Cre vectors to conditionally downregulate RGSz1 in vlPAG projections to the ventral tegmental area (VTA) and show that downregulation of RGSz1 prevents the development of place conditioning to low morphine doses. Consistent with the role for RGSz1 as a negative modulator of MOPR activity, RGSz1KO enhances opioid-induced cAMP inhibition in periaqueductal gray (PAG) membranes. Furthermore, using a new generation of bioluminescence resonance energy transfer (BRET) sensors, we demonstrate that RGSz1 modulates Gαz but not other Gαi family subunits and selectively impedes MOPR-mediated Gαz signaling events invoked by morphine and other opioids. Our work highlights a regional and circuit-specific role of the G protein-signaling modulator RGSz1 in morphine reward, providing insights on midbrain intracellular pathways that control addiction-related behaviors. SIGNIFICANCE STATEMENT: This study used advanced genetic mouse models to highlight the role of the signal transduction modulator named RGSz1 in responses to clinically used opioid analgesics. We show that RGSz1 controls the rewarding efficacy of opioids by actions in ventrolateral periaqueductal gray projections to the ventral tegmental area, a key component of the midbrain dopamine pathway. These studies highlight novel mechanisms by which pain-modulating structures control the rewarding efficacy of opioids.

Comparative Transcriptional Analyses in the Nucleus Accumbens Identifies RGS2 as a Key Mediator of Depression-Related Behavior.

BACKGROUND: Major depressive disorder is one of the most commonly diagnosed mental illnesses worldwide, with a higher prevalence in women than in men. Although currently available pharmacological therapeutics help many individuals, they are not effective for most. Animal models have been important for the discovery of molecular alterations in stress and depression, but difficulties in adapting animal models of depression for females has impeded progress in developing novel therapeutic treatments that may be more efficacious for women. METHODS: Using the California mouse social defeat model, we took a multidisciplinary approach to identify stress-sensitive molecular targets that have translational relevance for women. We determined the impact of stress on transcriptional profiles in male and female California mouse nucleus accumbens (NAc) and compared these results with data from postmortem samples of the NAc from men and women diagnosed with major depressive disorder. RESULTS: Our cross-species computational analyses identified Rgs2 (regulator of G protein signaling 2) as a transcript downregulated by social defeat stress in female California mice and in women with major depressive disorder. RGS2 plays a key role in signal regulation of neuropeptide and neurotransmitter receptors. Viral vector-mediated overexpression of Rgs2 in the NAc restored social approach and sucrose preference in stressed female California mice. CONCLUSIONS: These studies show that Rgs2 acting in the NAc has functional properties that translate to changes in anxiety- and depression-related behavior. Future studies should investigate whether targeting Rgs2 represents a novel target for treatment-resistant depression in women.

SARS-CoV-2 Airway Infection Results in Time-dependent Sensory Abnormalities in a Hamster Model.

Despite being largely confined to the airways, SARS-CoV-2 infection has been associated with sensory abnormalities that manifest in both acute and long-lasting phenotypes. To gain insight on the molecular basis of these sensory abnormalities, we used the golden hamster infection model to characterize the effects of SARS-CoV-2 versus Influenza A virus (IAV) infection on the sensory nervous system. Efforts to detect the presence of virus in the cervical/thoracic spinal cord and dorsal root ganglia (DRGs) demonstrated detectable levels of SARS-CoV-2 by quantitative PCR and RNAscope uniquely within the first 24 hours of infection. SARS-CoV-2-infected hamsters demonstrated mechanical hypersensitivity during acute infection; intriguingly, this hypersensitivity was milder, but prolonged when compared to IAV-infected hamsters. RNA sequencing (RNA-seq) of thoracic DRGs from acute infection revealed predominantly neuron-biased signaling perturbations in SARS-CoV-2-infected animals as opposed to type I interferon signaling in tissue derived from IAV-infected animals. RNA-seq of 31dpi thoracic DRGs from SARS-CoV-2-infected animals highlighted a uniquely neuropathic transcriptomic landscape, which was consistent with substantial SARS-CoV-2-specific mechanical hypersensitivity at 28dpi. Ontology analysis of 1, 4, and 30dpi RNA-seq revealed novel targets for pain management, such as ILF3. Meta-analysis of all SARS-CoV-2 RNA-seq timepoints against preclinical pain model datasets highlighted both conserved and unique pro-nociceptive gene expression changes following infection. Overall, this work elucidates novel transcriptomic signatures triggered by SARS-CoV-2 that may underlie both short- and long-term sensory abnormalities while also highlighting several therapeutic targets for alleviation of infection-induced hypersensitivity. One Sentence Summary: SARS-CoV-2 infection results in an interferon-associated transcriptional response in sensory tissues underlying time-dependent hypersensitivity.

SARS-CoV-2 infection in hamsters and humans results in lasting and unique systemic perturbations after recovery.

The host response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in prolonged pathologies collectively referred to as post-acute sequalae of COVID-19 (PASC) or long COVID. To better understand the mechanism underlying long COVID biology, we compared the short- and long-term systemic responses in the golden hamster after either SARS-CoV-2 or influenza A virus (IAV) infection. Results demonstrated that SARS-CoV-2 exceeded IAV in its capacity to cause permanent injury to the lung and kidney and uniquely affected the olfactory bulb (OB) and olfactory epithelium (OE). Despite a lack of detectable infectious virus, the OB and OE demonstrated myeloid and T cell activation, proinflammatory cytokine production, and an interferon response that correlated with behavioral changes extending a month after viral clearance. These sustained transcriptional changes could also be corroborated from tissue isolated from individuals who recovered from COVID-19. These data highlight a molecular mechanism for persistent COVID-19 symptomology and provide a small animal model to explore future therapeutics.

A promising chemical series of positive allosteric modulators of the µ-opioid receptor that enhance the antinociceptive efficacy of opioids but not their adverse effects.

Positive allosteric modulators (PAMs) of the µ-opioid receptor (MOR) have been proposed to exhibit therapeutic potential by maximizing the analgesic properties of clinically used opioid drugs while limiting their adverse effects or risk of overdose as a result of using lower drug doses. We herein report in vitro and in vivo characterization of two small molecules from a chemical series of MOR PAMs that exhibit: (i) MOR PAM activity and receptor subtype selectivity in vitro, (ii) a differential potentiation of the antinociceptive effect of oxycodone, morphine, and methadone in mouse models of pain that roughly correlates with in vitro activity, and (iii) a lack of potentiation of adverse effects associated with opioid administration, such as somatic withdrawal, respiratory depression, and analgesic tolerance. This series of MOR PAMs holds promise for the development of adjuncts to opioid therapy to mitigate against overdose and opioid use disorders.

Regulators of G Protein Signaling in Analgesia and Addiction.

Regulator of G protein signaling (RGS) proteins are multifunctional proteins expressed in peripheral and neuronal cells, playing critical roles in development, physiologic processes, and pharmacological responses. RGS proteins primarily act as GTPase accelerators for activated Gα subunits of G-protein coupled receptors, but they may also modulate signal transduction by several other mechanisms. Over the last two decades, preclinical work identified members of the RGS family with unique and critical roles in intracellular responses to drugs of abuse. New information has emerged on the mechanisms by which RGS proteins modulate the efficacy of opioid analgesics in a brain region- and agonist-selective fashion. There has also been progress in the understanding of the protein complexes and signal transduction pathways regulated by RGS proteins in addiction and analgesia circuits. In this review, we summarize findings on the mechanisms by which RGS proteins modulate functional responses to opioids in models of analgesia and addiction. We also discuss reports on the regulation and function of RGS proteins in models of psychostimulant addiction. Using information from preclinical studies performed over the last 20 years, we highlight the diverse mechanisms by which RGS protein complexes control plasticity in response to opioid and psychostimulant drug exposure; we further discuss how the understanding of these pathways may lead to new opportunities for therapeutic interventions in G protein pathways. SIGNIFICANCE STATEMENT: Regulator of G protein signaling (RGS) proteins are signal transduction modulators, expressed widely in various tissues, including brain regions mediating addiction and analgesia. Evidence from preclinical work suggests that members of the RGS family act by unique mechanisms in specific brain regions to control drug-induced plasticity. This review highlights interesting findings on the regulation and function of RGS proteins in models of analgesia and addiction.

HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice.

BACKGROUND: HDAC6 is a class IIB histone deacetylase expressed at many levels of the nociceptive pathway. This study tested the ability of novel and selective HDAC6 inhibitors to alleviate sensory hypersensitivity behaviors in mouse models of peripheral nerve injury and peripheral inflammation. METHODS: We utilized the murine spared nerve injury (SNI) model for peripheral nerve injury and the Complete Freund's Adjuvant (CFA) model of peripheral inflammation. We applied the Von Frey assay to monitor mechanical allodynia. RESULTS: Using the SNI model, we demonstrate that daily administration of the brain-penetrant HDAC6 inhibitor, ACY-738, abolishes mechanical allodynia in male and in female mice. Importantly, there is no tolerance to the antiallodynic actions of these compounds as they produce a consistent increase in Von Frey thresholds for several weeks. We observed a similar antiallodynic effect when utilizing the HDAC6 inhibitor, ACY-257, which shows limited brain expression when administered systemically. We also demonstrate that ACY-738 and ACY-257 attenuate mechanical allodynia in the CFA model of peripheral inflammation. CONCLUSIONS: Overall, our findings suggest that inhibition of HDAC6 provides a promising therapeutic avenue for the alleviation of mechanical allodynia associated with peripheral nerve injury and peripheral inflammation.

Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2.

Tissue repair after spinal cord injury requires the mobilization of immune and glial cells to form a protective barrier that seals the wound and facilitates debris clearing, inflammatory containment and matrix compaction. This process involves corralling, wherein phagocytic immune cells become confined to the necrotic core, which is surrounded by an astrocytic border. Here we elucidate a temporally distinct gene signature in injury-activated microglia and macrophages (IAMs) that engages axon guidance pathways. Plexin-B2 is upregulated in IAMs and is required for motor sensory recovery after spinal cord injury. Plexin-B2 deletion in myeloid cells impairs corralling, leading to diffuse tissue damage, inflammatory spillover and hampered axon regeneration. Corralling begins early and requires Plexin-B2 in both microglia and macrophages. Mechanistically, Plexin-B2 promotes microglia motility, steers IAMs away from colliding cells and facilitates matrix compaction. Our data therefore establish Plexin-B2 as an important link that integrates biochemical cues and physical interactions of IAMs with the injury microenvironment during wound healing.