Pain States In Mice Research Articles

126 Regulator of G Protein Signaling 4 (RGS4) Gene Expression is Upregulated in the Superficial Spinal Dorsal Horn and in Dorsal Root Ganglion Neurons in a Murine Model of Neuropathic Pain

INTRODUCTION: Regulator of G protein signaling 4 (RGS4), a potent negative regulator of G-protein coupled receptor signaling, is a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and is upregulated in response to peripheral inflammation and nerve injury. METHODS: To understand the dynamics of RGS4 gene expression after nerve injury, we perform the spared-nerve injury (SNI) model of neuropathic pain in C57BL/6. At 21 days (DRG) or 2.5 months (spinal cord) post-surgery (SNI vs sham), animals were sacrificed and specimens were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RESULTS: RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (p = 0.0384). In sham-treated mice, there were no differences in cell-type-specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p = 0.4170). Cell-type-specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; p = 0.0015; one-way ANOVA) following SNI. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p = 0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p = 0.0012). In addition, neuronal-specific RGS4 expression was significantly increased in the DRG in SNI vs Sham-injured mice (p = 0.0050). CONCLUSION: Our data suggest that upregulation of RGS4 expression in the superficial dorsal horn and DRG may play a crucial role in maintenance of chronic pain following nerve injury.

Chronic pain‐mediated Regulator of G protein signaling 4 (RGS4) gene expression in superficial dorsal horn of spinal cord

Regulator of G protein signaling 4 (RGS4) is a potent negative regulator of G-protein coupled receptor signaling duration. Recent studies have identified RGS4 as a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and several studies have demonstrated upregulation of RGS4 mRNA in response to both peripheral inflammation and nerve injury. Here, we tested the hypothesis that peripheral nerve injury alters RGS4 neuronal expression broadly, and with specific expression-patterning among excitatory and inhibitory interneuron populations within the superficial dorsal horn of the mouse spinal cord. Chronic neuropathic pain was induced in C57BL/6 male mice via spared-nerve injury (SNI). At 2.5 months post-surgery (SNI vs sham), animals were sacrificed and lumbar spinal cords were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (P =0.0384). RGS4 mRNA remained unchanged (SNI vs sham) among expression-positive dorsal horn neurons (p=0.6327). In sham-treated mice, there were no differences in cell-type specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p=0.4170). Cell-type specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; P =0.0015; one-way ANOVA) following spared-nerve injury. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p=0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p=0.0012). These data demonstrate that nerve injury induces upregulation of RGS4 in the superficial dorsal horn, and suggest that RGS4-positive inhibitory interneurons may be implicated in influencing persistent pain signaling within the dorsal horn of the spinal cord.

The Sigma-2 receptor / transmembrane protein 97 (σ2R/TMEM97) modulator JVW-1034 reduces heavy alcohol drinking and associated pain states in male mice

Alcohol Use Disorder (AUD) is a chronic relapsing disorder characterized by compulsive alcohol intake, loss of control over alcohol intake, and a negative emotional state when access to alcohol is prevented. AUD is also closely tied to pain, as repeated alcohol drinking leads to increased pain sensitivity during withdrawal. The sigma-2 receptor, recently identified as transmembrane protein 97 (σ2R/TMEM97), is an integral membrane protein involved in cholesterol homeostasis and lipid metabolism. Selective σ2R/Tmem97 modulators have been recently shown to relieve mechanical hypersensitivity in animal models of neuropathic pain as well as to attenuate alcohol withdrawal signs in C. elegans and to reduce alcohol drinking in rats, suggesting a potential key role for this protein in alcohol-related behaviors. In this study, we tested the effects of a potent and selective σ2R/TMEM97 ligand, JVW-1034, on heavy alcohol drinking and alcohol-induced heightened pain states in mice using an intermittent access model. Administration of JVW-1034 decreased both ethanol intake and preference for ethanol, without affecting water intake, total fluid intake, or food intake. Notably, this effect was specific for alcohol, as JVW-1034 had no effect on sucrose intake. Furthermore, JVW-1034 reduced both thermal hyperalgesia and mechanical hypersensitivity in ethanol withdrawn mice. Our data provide important evidence that modulation of σ2R/TMEM97 with small molecules can mediate heavy alcohol drinking as well as chronic alcohol-induced heightened pain sensitivity, thereby identifying a promising novel pharmacological target for AUD and associated pain states.

RGS4 Maintains Chronic Pain Symptoms in Rodent Models

Regulator of G-protein signaling 4 (RGS4) is a potent modulator of G-protein-coupled receptor signal transduction that is expressed throughout the pain matrix. Here, we use genetic mouse models to demonstrate a role of RGS4 in the maintenance of chronic pain states in male and female mice. Using paradigms of peripheral inflammation and nerve injury, we show that the prevention of RGS4 action leads to recovery from mechanical and cold allodynia and increases the motivation for wheel running. Similarly, RGS4KO eliminates the duration of nocifensive behavior in the second phase of the formalin assay. Using the Complete Freud's Adjuvant (CFA) model of hindpaw inflammation we also demonstrate that downregulation of RGS4 in the adult ventral posterolateral thalamic nuclei promotes recovery from mechanical and cold allodynia. RNA sequencing analysis of thalamus (THL) from RGS4WT and RGS4KO mice points to many signal transduction modulators and transcription factors that are uniquely regulated in CFA-treated RGS4WT cohorts. Ingenuity pathway analysis suggests that several components of glutamatergic signaling are differentially affected by CFA treatment between RGS4WT and RGS4KO groups. Notably, Western blot analysis shows increased expression of metabotropic glutamate receptor 2 in THL synaptosomes of RGS4KO mice at time points at which they recover from mechanical allodynia. Overall, our study provides information on a novel intracellular pathway that contributes to the maintenance of chronic pain states and points to RGS4 as a potential therapeutic target.SIGNIFICANCE STATEMENT There is an imminent need for safe and efficient chronic pain medications. Regulator of G-protein signaling 4 (RGS4) is a multifunctional signal transduction protein, widely expressed in the pain matrix. Here, we demonstrate that RGS4 plays a prominent role in the maintenance of chronic pain symptoms in male and female mice. Using genetically modified mice, we show a dynamic role of RGS4 in recovery from symptoms of sensory hypersensitivity deriving from hindpaw inflammation or hindlimb nerve injury. We also demonstrate an important role of RGS4 actions in gene expression patterns induced by chronic pain states in the mouse thalamus. Our findings provide novel insight into mechanisms associated with the maintenance of chronic pain states and demonstrate that interventions in RGS4 activity promote recovery from sensory hypersensitivity symptoms.

(245) Electrophysiological and Morphological Characterization of Genetically Distinct Nociceptive Neurons in the Central Amygdala

The central amygdala (CeA) has key functions in modulating pain-related behaviors and exhibits experience-dependent plasticity in the context of chronic pain. Recent work from our lab has further revealed that distinct CeA cell types contribute differently to behavioral outcomes in pathological pain states in mice. Cells expressing Protein Kinase C delta (CeA-PKCδ), for example, promote hypersensitivity and display increased excitability following injury. By contrast, somatostatin-expressing CeA (CeA-Som) neurons contribute to an endogenous analgesic tone at baseline and display suppressed excitability during nerve-injury. The cellular mechanisms driving this opposing modulation of pain-related behaviors are still poorly understood. We hypothesized that the convergence of genetic identity, intrinsic membrane properties and morphological characteristics of distinct subpopulations of neurons contributes to the functional heterogeneity in the CeA. Here, we begin to test this hypothesis by characterizing the electrophysiological and morphological properties of CeA-PKCδ and CeA-Som neurons. Preliminary results from whole-cell patch-clamp electrophysiology experiments in acute amygdala slices show that the electrophysiological properties of CeA-PKCδ and CeA-Som neurons are distinct. Firing properties in CeA-PKCδ neurons, for example, are highly heterogeneous, with spontaneously active, accommodating, and non-accommodating cells found among this population. In contrast, the overwhelming majority of CeA-Som neurons are either spontaneously active or non-accommodating. Notably, non-accommodating CeA-Som neurons are more excitable than non-accommodating CeA-PKCδ neurons, displaying increased firing responses to depolarizing current injection, reduced latencies to first spike and after hyperpolarizations and changes in resting membrane potentials. Ongoing experiments are evaluating the morphological properties of these genetically distinct cells, with the end goal of correlating pain-related behaviors with morphological properties, genetic identity and firing properties. Altogether, our results demonstrate that the output gain of CeA-Som cells is significantly greater than that of CeA-PKCδ neurons, suggesting that cell-type-specific differences in neuronal excitability might contribute to functional heterogeneity in the CeA.

(260) Examining Sex Differences in Analgesic Tolerance to a Peripherally-Restricted Opioid combination

The development of analgesic tolerance to opioid pharmacotherapies presents a persistent issue in the treatment of chronic pain, resulting in dose escalation and increased risk for dependence and respiratory depression. Recent studies have demonstrated the involvement of peripheral opioid receptors in the development of opioid tolerance to classical opioids like morphine and oxycodone. There have also been reports of differences in opioid analgesia, tolerance and addiction between males and females. Therefore, we studied whether peripherally-restricted opioid combinations would differentially induce tolerance in a rodent model of neuropathic pain. For these studies, we used loperamide (Lo), a peripherally-restricted mu-opioid agonist, in combination with oxymorphindole or N-benzyl-oxymorphindole (OMI, BOMI), which are partial and full agonists at the delta-opioid receptor, respectively. The Lo-OMI and Lo-BOMI combinations have been shown to synergistically reverse neuropathic pain states in mice and rats. After the induction of a neuropathic pain phenotype with spared nerve injury, male or female mice and rats were given once daily injections of Lo-OMI, Lo-BOMI or morphine for 10 days, and daily mechanical sensitivity was monitored by an electronic von Frey apparatus. Initial experiments indicate that male mice do not develop tolerance to either peripherally-restricted combination, but that female mice rapidly become tolerant to the analgesic effects of Lo-BOMI. Based on these results, we sought to determine whether the same difference persists in rats. We conclude that in female mice—but not males—the delta-opioid receptor component of the mu-delta synergistic combination is involved in the development of analgesic tolerance.

Prospective administration of anti–nerve growth factor treatment effectively suppresses functional connectivity alterations after cancer-induced bone pain in mice

Cancer-induced bone pain is abundant among advanced-stage cancer patients and arises from a primary tumor in the bone or skeletal metastasis of common cancer types such as breast, lung, or prostate cancer. Recently, antibodies targeting nerve growth factor (NGF) have been shown to effectively relieve neuropathic and inflammatory pain states in mice and in humans. Although efficacy has been shown in mice on a behavioral level, effectiveness in preventing pain-induced functional rearrangements in the central nervous system has not been shown. Therefore, we assessed longitudinal whole-brain functional connectivity using resting-state functional magnetic resonance imaging in a mouse model of cancer-induced bone pain. We found functional connectivity between major hubs of ascending and descending pain pathways such as the periaqueductal gray, amygdala, thalamus, and cortical somatosensory regions to be affected by a developing cancer pain state. These changes could be successfully prevented through prospective administration of a monoclonal anti-NGF antibody (mAb911). This indicates efficacy of anti-NGF treatment to prevent pain-induced adaptations in brain functional networks after persistent nociceptive input from cancer-induced bone pain. In addition, it highlights the suitability of resting-state functional magnetic resonance imaging readouts as an indicator of treatment response on the basis of longitudinal functional network changes.