A parabrachial hub for the prioritization of survival behavior.

Long-term sustained pain in the absence of acute physical injury is a prominent feature of chronic pain conditions. While neurons responding to noxious stimuli have been identified, understanding the signals that persist without ongoing painful stimuli remains a challenge. Using an ethological approach based on the prioritization of adaptive survival behaviors, we determined that neuropeptide Y (NPY) signaling from multiple sources converges on parabrachial neurons expressing the NPY Y1 receptor to reduce sustained pain responses. Neural activity recordings and computational modeling demonstrate that activity in Y1R parabrachial neurons is elevated following injury, predicts functional coping behavior, and is inhibited by competing survival needs. Taken together, our findings suggest that parabrachial Y1 receptor-expressing neurons are a critical hub for endogenous analgesic pathways that suppress sustained pain states.

Alleviation of neuropathic pain with neuropeptide Y requires spinal Npy1r interneurons that coexpress Grp.

Neuropeptide Y targets the Y1 receptor (Y1) in the spinal dorsal horn (DH) to produce endogenous and exogenous analgesia. DH interneurons that express Y1 (Y1-INs; encoded by Npy1r) are necessary and sufficient for neuropathic hypersensitivity after peripheral nerve injury. However, as Y1-INs are heterogenous in composition in terms of morphology, neurophysiological characteristics, and gene expression, we hypothesized that a more precisely defined subpopulation mediates neuropathic hypersensitivity. Using fluorescence in situ hybridization, we found that Y1-INs segregate into 3 largely nonoverlapping subpopulations defined by the coexpression of Npy1r with gastrin-releasing peptide (Grp/Npy1r), neuropeptide FF (Npff/Npy1r), and cholecystokinin (Cck/Npy1r) in the superficial DH of mice, nonhuman primates, and humans. Next, we analyzed the functional significance of Grp/Npy1r, Npff/Npy1r, and Cck/Npy1r INs to neuropathic pain using a mouse model of peripheral nerve injury. We found that chemogenetic inhibition of Npff/Npy1r-INs did not change the behavioral signs of neuropathic pain. Further, inhibition of Y1-INs with an intrathecal Y1 agonist, [Leu31, Pro34]-NPY, reduced neuropathic hypersensitivity in mice with conditional deletion of Npy1r from CCK-INs and NPFF-INs but not from GRP-INs. We conclude that Grp/Npy1r-INs are conserved in higher order mammalian species and represent a promising and precise pharmacotherapeutic target for the treatment of neuropathic pain.

Thoracic Dorsal Root Ganglion Application of Resiniferatoxin Reduces Myocardial Ischemia-Induced Ventricular Arrhythmias.

BACKGROUND: A myocardial ischemia/reperfusion (IR) injury activates the transient receptor potential vanilloid 1 (TRPV1) dorsal root ganglion (DRG) neurons. The activation of TRPV1 DRG neurons triggers the spinal dorsal horn and the sympathetic preganglionic neurons in the spinal intermediolateral column, which results in sympathoexcitation. In this study, we hypothesize that the selective epidural administration of resiniferatoxin (RTX) to DRGs may provide cardioprotection against ventricular arrhythmias by inhibiting afferent neurotransmission during IR injury. METHODS: Yorkshire pigs (n = 21) were assigned to either the sham, IR, or IR + RTX group. A laminectomy and sternotomy were performed on the anesthetized animals to expose the left T2-T4 spinal dorsal root and the heart for IR intervention, respectively. RTX (50 µg) was administered to the DRGs in the IR + RTX group. The activation recovery interval (ARI) was measured as a surrogate for the action potential duration (APD). Arrhythmia risk was investigated by assessing the dispersion of repolarization (DOR), a marker of arrhythmogenicity, and measuring the arrhythmia score and the number of non-sustained ventricular tachycardias (VTs). TRPV1 and calcitonin gene-related peptide (CGRP) expressions in DRGs and CGRP expression in the spinal cord were assessed using immunohistochemistry. RESULTS: The RTX mitigated IR-induced ARI shortening (-105 ms ± 13 ms in IR vs. -65 ms ± 11 ms in IR + RTX, p = 0.028) and DOR augmentation (7093 ms2 ± 701 ms2 in IR vs. 3788 ms2 ± 1161 ms2 in IR + RTX, p = 0.020). The arrhythmia score and VT episodes during an IR were decreased by RTX (arrhythmia score: 8.01 ± 1.44 in IR vs. 3.70 ± 0.81 in IR + RTX, p = 0.037. number of VT episodes: 12.00 ± 3.29 in IR vs. 0.57 ± 0.3 in IR + RTX, p = 0.002). The CGRP expression in the DRGs and spinal cord was decreased by RTX (DRGs: 6.8% ± 1.3% in IR vs. 0.6% ± 0.2% in IR + RTX, p < 0.001. Spinal cord: 12.0% ± 2.6% in IR vs. 4.5% ± 0.8% in IR + RTX, p = 0.047). CONCLUSIONS: The administration of RTX locally to thoracic DRGs reduces ventricular arrhythmia in a porcine model of IR, likely by inhibiting spinal afferent hyperactivity in the cardio-spinal sympathetic pathways.

Endogenous µ-opioid-Neuropeptide Y Y1 receptor synergy silences chronic postoperative pain in mice.

Tissue injury creates a delicate balance between latent pain sensitization (LS) and compensatory endogenous analgesia. Inhibitory G-protein-coupled receptor (GPCR) interactions that oppose LS, including µ-opioid receptor (MOR) or neuropeptide Y Y1 receptor (Y1R) activity, persist in the spinal cord dorsal horn (DH) for months, even after the resolution of normal pain thresholds. Here, we demonstrate that following recovery from surgical incision, a potent endogenous analgesic synergy between MOR and Y1R activity persists within DH interneurons to reduce the intensity and duration of latent postoperative hypersensitivity and ongoing pain. Failure of such endogenous GPCR signaling to maintain LS in remission may underlie the transition from acute to chronic pain states.

Contribution of µ Opioid Receptor-expressing Dorsal Horn Interneurons to Neuropathic Pain-like Behavior in Mice.

BACKGROUND: Intersectional genetics have yielded tremendous advances in our understanding of molecularly identified subpopulations and circuits within the dorsal horn in neuropathic pain. The authors tested the hypothesis that spinal µ opioid receptor-expressing neurons (Oprm1-expressing neurons) contribute to behavioral hypersensitivity and neuronal sensitization in the spared nerve injury model in mice. METHODS: The authors coupled the use of Oprm1Cre transgenic reporter mice with whole cell patch clamp electrophysiology in lumbar spinal cord slices to evaluate the neuronal activity of Oprm1-expressing neurons in the spared nerve injury model of neuropathic pain. The authors used a chemogenetic approach to activate or inhibit Oprm1-expressing neurons, followed by the assessment of behavioral signs of neuropathic pain. RESULTS: The authors reveal that spared nerve injury yielded a robust neuroplasticity of Oprm1-expressing neurons. Spared nerve injury reduced Oprm1 gene expression in the dorsal horn as well as the responsiveness of Oprm1-expressing neurons to the selective µ agonist (D-Ala2, N-MePhe4, Gly-ol)-enkephalin (DAMGO). Spared nerve injury sensitized Oprm1-expressing neurons, as reflected by an increase in their intrinsic excitability (rheobase, sham 38.62 ± 25.87 pA [n = 29]; spared nerve injury, 18.33 ± 10.29 pA [n = 29], P = 0.0026) and spontaneous synaptic activity (spontaneous excitatory postsynaptic current frequency in delayed firing neurons: sham, 0.81 ± 0.67 Hz [n = 14]; spared nerve injury, 1.74 ± 1.68 Hz [n = 10], P = 0.0466), and light brush-induced coexpression of the immediate early gene product, Fos in laminae I to II (%Fos/tdTomato+: sham, 0.42 ± 0.57% [n = 3]; spared nerve injury, 28.26 ± 1.92% [n = 3], P = 0.0001). Chemogenetic activation of Oprm1-expressing neurons produced mechanical hypersensitivity in uninjured mice (saline, 2.91 ± 1.08 g [n = 6]; clozapine N-oxide, 0.65 ± 0.34 g [n = 6], P = 0.0006), while chemogenetic inhibition reduced behavioral signs of mechanical hypersensitivity (saline, 0.38 ± 0.37 g [n = 6]; clozapine N-oxide, 1.05 ± 0.42 g [n = 6], P = 0.0052) and cold hypersensitivity (saline, 6.89 ± 0.88 s [n = 5] vs. clozapine N-oxide, 2.31 ± 0.52 s [n = 5], P = 0.0017). CONCLUSIONS: The authors conclude that nerve injury sensitizes pronociceptive µ opioid receptor-expressing neurons in mouse dorsal horn. Nonopioid strategies to inhibit these interneurons might yield new treatments for neuropathic pain.

Mechanical and Heat Hyperalgesia upon Withdrawal From Chronic Intermittent Ethanol Vapor Depends on Sex, Exposure Duration, and Blood Alcohol Concentration in Mice.

Approximately half of patients with alcohol use disorder report pain and this can be severe during withdrawal. Many questions remain regarding the importance of biological sex, alcohol exposure paradigm, and stimulus modality to the severity of alcohol withdrawal-induced hyperalgesia. To examine the impact of sex and blood alcohol concentration on the time course of the development of mechanical and heat hyperalgesia, we characterized a mouse model of chronic alcohol withdrawal-induced pain in the presence or absence the alcohol dehydrogenase inhibitor, pyrazole. Male and female C57BL/6J mice underwent chronic intermittent ethanol vapor ± pyrazole exposure for 4 weeks, 4 d/wk to induce ethanol dependence. Hind paw sensitivity to the plantar application of mechanical (von Frey filaments) and radiant heat stimuli were measured during weekly observations at 1, 3, 5, 7, 24, and 48 hours after cessation of ethanol exposure. In the presence of pyrazole, males developed mechanical hyperalgesia after the first week of chronic intermittent ethanol vapor exposure, peaking at 48 hours after cessation of ethanol. By contrast, females did not develop mechanical hyperalgesia until the fourth week; this also required pyrazole and did not peak until 48 hours. Heat hyperalgesia was consistently observed only in females exposed to ethanol and pyrazole; this developed after the first weekly session and peaked at 1 hour. We conclude that Chronic alcohol withdrawal-induced pain develops in a sex-, time-, and blood alcohol concentration-dependent manner in C57BL/6J mice. PERSPECTIVE: Alcohol withdrawal-induced pain is a debilitating condition in individuals with AUD. Our study found mice experience alcohol withdrawal-induced pain in a sex and time course specific manor. These findings will aid in elucidating mechanisms of chronic pain and AUD and will help individuals remain abstinent from alcohol.

A Parabrachial-to-Amygdala Circuit That Determines Hemispheric Lateralization of Somatosensory Processing.

BACKGROUND: The central amygdala (CeA) is a bilateral hub of pain and emotional processing with well-established functional lateralization. We reported that optogenetic manipulation of neural activity in the left and right CeA has opposing effects on bladder pain. METHODS: To determine the influence of calcitonin gene-related peptide (CGRP) signaling from the parabrachial nucleus on this diametrically opposed lateralization, we administered CGRP and evaluated the activity of CeA neurons in acute brain slices as well as the behavioral signs of bladder pain in the mouse. RESULTS: We found that CGRP increased firing in both the right and left CeA neurons. Furthermore, we found that CGRP administration in the right CeA increased behavioral signs of bladder pain and decreased bladder pain-like behavior when administered in the left CeA. CONCLUSIONS: These studies reveal a parabrachial-to-amygdala circuit driven by opposing actions of CGRP that determines hemispheric lateralization of visceral pain.

Spinal neuropeptide Y Y1 receptor-expressing neurons are a pharmacotherapeutic target for the alleviation of neuropathic pain.

Peripheral nerve injury sensitizes a complex network of spinal cord dorsal horn (DH) neurons to produce allodynia and neuropathic pain. The identification of a druggable target within this network has remained elusive, but a promising candidate is the neuropeptide Y (NPY) Y1 receptor-expressing interneuron (Y1-IN) population. We report that spared nerve injury (SNI) enhanced the excitability of Y1-INs and elicited allodynia (mechanical and cold hypersensitivity) and affective pain. Similarly, chemogenetic or optogenetic activation of Y1-INs in uninjured mice elicited behavioral signs of spontaneous, allodynic, and affective pain. SNI-induced allodynia was reduced by chemogenetic inhibition of Y1-INs, or intrathecal administration of a Y1-selective agonist. Conditional deletion of Npy1r in DH neurons, but not peripheral afferent neurons prevented the anti-hyperalgesic effects of the intrathecal Y1 agonist. We conclude that spinal Y1-INs are necessary and sufficient for the behavioral symptoms of neuropathic pain and represent a promising target for future pharmacotherapeutic development of Y1 agonists.

Postsurgical Latent Pain Sensitization Is Driven by Descending Serotonergic Facilitation and Masked by µ-Opioid Receptor Constitutive Activity in the Rostral Ventromedial Medulla.

Following tissue injury, latent sensitization (LS) of nociceptive signaling can persist indefinitely, kept in remission by compensatory µ-opioid receptor constitutive activity (MORCA) in the dorsal horn of the spinal cord. To demonstrate LS, we conducted plantar incision in mice and then waited 3-4 weeks for hypersensitivity to resolve. At this time (remission), systemic administration of the opioid receptor antagonist/inverse agonist naltrexone reinstated mechanical and heat hypersensitivity. We first tested the hypothesis that LS extends to serotonergic neurons in the rostral ventral medulla (RVM) that convey pronociceptive input to the spinal cord. We report that in male and female mice, hypersensitivity was accompanied by increased Fos expression in serotonergic neurons of the RVM, abolished on chemogenetic inhibition of RVM 5-HT neurons, and blocked by intrathecal injection of the 5-HT3R antagonist ondansetron; the 5-HT2AR antagonist MDL-11 939 had no effect. Second, to test for MORCA, we microinjected the MOR inverse agonist d-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) and/or neutral opioid receptor antagonist 6ß-naltrexol. Intra-RVM CTAP produced mechanical hypersensitivity at both hindpaws; 6ß-naltrexol had no effect by itself, but blocked CTAP-induced hypersensitivity. This indicates that MORCA, rather than an opioid ligand-dependent mechanism, maintains LS in remission. We conclude that incision establishes LS in descending RVM 5-HT neurons that drives pronociceptive 5-HT3R signaling in the dorsal horn, and this LS is tonically opposed by MORCA in the RVM. The 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.SIGNIFICANCE STATEMENT Surgery leads to latent pain sensitization and a compensatory state of endogenous pain control that is maintained long after tissue healing. Here, we show that either chemogenetic inhibition of serotonergic neuron activity in the RVM or pharmacological inhibition of 5-HT3 receptor signaling at the spinal cord blocks behavioral signs of postsurgical latent sensitization. We conclude that MORCA in the RVM opposes descending serotonergic facilitation of LS and that the 5-HT3 receptor is a promising therapeutic target for the development of drugs to prevent the transition from acute to chronic postsurgical pain.

Endogenous µ-opioid receptor activity in the lateral and capsular subdivisions of the right central nucleus of the amygdala prevents chronic postoperative pain.

Tissue injury induces a long-lasting latent sensitization (LS) of spinal nociceptive signaling that is kept in remission by an opposing µ-opioid receptor (MOR) constitutive activity. To test the hypothesis that supraspinal sites become engaged, we induced hindpaw inflammation, waited 3 weeks for mechanical hypersensitivity to resolve, and then injected the opioid receptor inhibitors naltrexone, CTOP or ß-funaltrexamine subcutaneously, and/or into the cerebral ventricles. Intracerebroventricular injection of each inhibitor reinstated hypersensitivity and produced somatic signs of withdrawal, indicative of LS and endogenous opioid dependence, respectively. In naïve or sham controls, systemic naloxone (3 mg/kg) produced conditioned place aversion, and systemic naltrexone (3 mg/kg) increased Fos expression in the central nucleus of the amygdala (CeA). In LS animals tested 3 weeks after plantar incision, systemic naltrexone reinstated mechanical hypersensitivity and produced an even greater increase in Fos than in sham controls, particularly in the capsular subdivision of the right CeA. One third of Fos+ profiles co-expressed protein kinase C delta (PKCδ), and 35% of PKCδ neurons co-expressed tdTomato+ in Oprm1Cre ::tdTomato transgenic mice. CeA microinjection of naltrexone (1 µg) reinstated mechanical hypersensitivity only in male mice and did not produce signs of somatic withdrawal. Intra-CeA injection of the MOR-selective inhibitor CTAP (300 ng) reinstated hypersensitivity in both male and female mice. We conclude that MORs in the capsular subdivision of the right CeA prevent the transition from acute to chronic postoperative pain.

Sphingosine-1-phosphate receptor 1 agonist SEW2871 alters membrane properties of late-firing somatostatin expressing neurons in the central lateral amygdala.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR1) has been shown to be the primary mediator of S1P signaling, we utilized S1PR1 agonist SEW2871 and S1PR1 antagonist NIBR to determine a potential role of S1PR1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR1. Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR1 activation, which may have implications for how S1P alters supraspinal circuits.

Sex Differences in Protein Kinase A Signaling of the Latent Postoperative Pain Sensitization That Is Masked by Kappa Opioid Receptors in the Spinal Cord.

Latent sensitization (LS) of pain engages pronociceptive signaling pathways in the dorsal horn that include NMDA receptor (NMDAR)→adenylyl cyclase-1 (AC1)→protein kinase A (PKA), and exchange proteins directly activated by cyclic AMP (Epacs). To determine whether these pathways operate similarly between males and females or are under the inhibitory control of spinal κ opioid receptors (KOR), we allowed hyperalgesia to resolve after plantar incision and then blocked KOR with intrathecal administration of LY2456302, which reinstated hyperalgesia and facilitated touch-evoked immunoreactivity of phosphorylated extracellular signal-regulated kinase (pERK) in neurons (NeuN) but not astrocytes (GFAPs) nor microglia (Iba1). LY2456302 reinstated hyperalgesia even when administered 13 months later, indicating that chronic postoperative pain vulnerability persists for over a year in a latent state of remission. In both sexes, intrathecal MK-801 (an NMDAR competitive antagonist) prevented LY2456302-evoked reinstatement of hyperalgesia as did AC1 gene deletion or the AC1 inhibitor NB001. NB001 also prevented stimulus-evoked pERK. In both sexes, the Epac inhibitor ESI-09 prevented reinstatement, whereas the Epac activator 8-CPT reinstated hyperalgesia. By contrast, the PKA inhibitor H89 prevented reinstatement only in male mice, whereas the PKA activator 6-bnz-cAMP itself evoked reinstatement at all doses tested (3-30 nmol, i.t.). In neither sex did incision change gene expression of KOR, GluN1, PKA, or Epac1 in dorsal horn. We conclude that sustained KOR signaling inhibits spinal PKA-dependent mechanisms that drive postoperative LS in a sex-dependent manner. Our findings support the development of AC1, PKA, and Epac inhibitors toward a new pharmacotherapy for chronic postoperative pain.SIGNIFICANCE STATEMENT Because of neural mechanisms that are not well understood, men and women respond differently to treatments for chronic pain. We report that surgical incision recruits a pronociceptive latent pain sensitization that persisted for over a year and was kept in check by the sustained analgesic activity of κ opioid receptors. NMDAR→AC1→cAMP→Epac signaling pathways in the dorsal horn of the spinal cord maintain latent sensitization in both males and females; however, only males recruit a PKA-dependent mechanism. This work presents a novel male-specific mechanism for the promotion of chronic postoperative pain.

Fast A-type currents shape a rapidly adapting form of delayed short latency firing of excitatory superficial dorsal horn neurons that express the neuropeptide Y Y1 receptor.

KEY POINTS: Neuropeptide Y Y1 receptor-expressing neurons in the dorsal horn of the spinal cord contribute to chronic pain. For the first time, we characterized the firing patterns of Y1-expressing neurons in Y1eGFP reporter mice. Under hyperpolarized conditions, most Y1eGFP neurons exhibited fast A-type potassium currents and delayed, short-latency firing (DSLF). Y1eGFP DSLF neurons were almost always rapidly adapting and often exhibited rebound spiking, characteristics of spinal pain neurons under the control of T-type calcium channels. These results will inspire future studies to determine whether tissue or nerve injury downregulates the channels that underlie A-currents, thus unmasking membrane hyperexcitability in Y1-expressing dorsal horn neurons, leading to persistent pain. ABSTRACT: Neuroanatomical and behavioural evidence indicates that neuropeptide Y Y1 receptor-expressing interneurons (Y1-INs) in the superficial dorsal horn (SDH) are predominantly excitatory and contribute to chronic pain. Using an adult ex vivo spinal cord slice preparation from Y1eGFP reporter mice, we characterized firing patterns in response to steady state depolarizing current injection of GFP-positive cells in lamina II, the great majority of which expressed Y1 mRNA (88%). Randomly sampled (RS) and Y1eGFP neurons exhibited five firing patterns: tonic, initial burst, phasic, delayed short-latency <180 ms (DSLF) and delayed long-latency >180 ms (DLLF). When studied at resting membrane potential, most RS neurons exhibited delayed firing, while most Y1eGFP neurons exhibited phasic firing. A preconditioning membrane hyperpolarization produced only subtle changes in the firing patterns of RS neurons, but dramatically shifted Y1eGFP neurons to DSLF (46%) and DLLF (24%). In contrast to RS DSLF neurons, which rarely exhibited spike frequency adaptation, Y1eGFP DSLF neurons were almost always rapidly adapting, a characteristic of nociceptive-responsive SDH neurons. Rebound spiking was more prevalent in Y1eGFP neurons (6% RS vs. 32% Y1eGFP), indicating enrichment of T-type calcium currents. Y1eGFP DSLF neurons exhibited fast A-type potassium currents that are known to delay or limit action potential firing and exhibited smaller current density as compared to RS DSLF neurons. Our results will inspire future studies to determine whether tissue or nerve injury downregulates channels that contribute to A-currents, thus potentially unmasking T-type calcium channel activity and membrane hyperexcitability in Y1-INs, leading to persistent pain.

Prostaglandin 15d-PGJ2 targets PPARγ and opioid receptors to prevent muscle hyperalgesia in rats.

Pharmacological agents directed to either opioid receptors or peroxisome proliferator-activated receptor gamma (PPARγ) at peripheral tissues reduce behavioral signs of persistent pain. Both receptors are expressed in muscle tissue, but the contribution of PPARγ activation to muscle pain and its modulation by opioid receptors remains unknown. To address this question, we first tested whether the endogenous PPARγ ligand 15d-PGJ2 would decrease mechanical hyperalgesia induced by carrageenan administration into the gastrocnemius muscle of rats. Next, we used receptor antagonists to determine whether the antihyperalgesic effect of 15-deoxyΔ-12,14-prostaglandin J2 (15d-PGJ2) was PPARγ- or opioid receptor-dependent. Three hours after carrageenan, muscle hyperalgesia was quantified with the Randall-Selitto test. 15d-PGJ2 prevented carrageenan-induced muscle hyperalgesia in a dose-dependent manner. The antihyperalgesic effect of 15d-PGJ2 was dose-dependently inhibited by either the PPARγ antagonist, 2-chloro-5-nitro-N-phenylbenzamide, or by the opioid receptor antagonist, naloxone. We conclude that 15d-PGJ2 targets PPARγ and opioid receptors to prevent muscle hyperalgesia. We suggest that local PPARγ receptors are important pharmacological targets for inflammatory muscle pain.

Targeting spinal neuropeptide Y1 receptor-expressing interneurons to alleviate chronic pain and itch.

An accelerating basic science literature is providing key insights into the mechanisms by which spinal neuropeptide Y (NPY) inhibits chronic pain. A key target of pain inhibition is the Gi-coupled neuropeptide Y1 receptor (Y1). Y1 is located in key sites of pain transmission, including the peptidergic subpopulation of primary afferent neurons and a dense subpopulation of small, excitatory, glutamatergic/somatostatinergic interneurons (Y1-INs) that are densely expressed in the dorsal horn, particularly in superficial lamina I-II. Selective ablation of spinal Y1-INs with an NPY-conjugated saporin neurotoxin attenuates the development of peripheral nerve injury-induced mechanical and cold hypersensitivity. Conversely, conditional knockdown of NPY expression or intrathecal administration of Y1 antagonists reinstates hypersensitivity in models of chronic latent pain sensitization. These and other results indicate that spinal NPY release and the consequent inhibition of pain facilitatory Y1-INs represent an important mechanism of endogenous analgesia. This mechanism can be mimicked with exogenous pharmacological approaches (e.g. intrathecal administration of Y1 agonists) to inhibit mechanical and thermal hypersensitivity and spinal neuron activity in rodent models of neuropathic, inflammatory, and postoperative pain. Pharmacological activation of Y1 also inhibits mechanical- and histamine-induced itch. These immunohistochemical, pharmacological, and cell type-directed lesioning data, in combination with recent transcriptomic findings, point to Y1-INs as a promising therapeutic target for the development of spinally directed NPY-Y1 agonists to treat both chronic pain and itch.

Effect of Sex on Motor Function, Lesion Size, and Neuropathic Pain after Contusion Spinal Cord Injury in Mice.

Spinal cord injury (SCI) causes neurodegeneration, impairs locomotor function, and impacts the quality of life particularly in those individuals in whom neuropathic pain develops. Whether the time course of neurodegeneration, locomotor impairment, or neuropathic pain varies with sex, however, remains understudied. Therefore, the objective of this study in male and female C57BL/6 mice was to evaluate the following outcomes for six weeks after a 75-kdyn thoracic contusion SCI: locomotor function using the Basso Mouse Scale (BMS); spinal cord tissue sparing and rostral-caudal lesion length; and mechanical allodynia and heat hyperalgesia using hindpaw application of Von Frey filaments or radiant heat stimuli, respectively. Although motor function was largely similar between sexes, all of the males, but only half of the females, recovered plantar stepping. Rostral-caudal lesion length was shorter in females than in males. Mechanical allodynia and heat hyperalgesia after SCI developed in all animals, regardless of sex; there were no differences in pain outcomes between sexes. We conclude that contusion SCI yields subtle sex differences in mice depending on the outcome measure but no significant differences in behavioral signs of neuropathic pain.

Neuropeptide Y tonically inhibits an NMDAR➔AC1➔TRPA1/TRPV1 mechanism of the affective dimension of chronic neuropathic pain.

Transection of the sural and common peroneal branches of the sciatic nerve produces cutaneous hypersensitivity at the tibial innervation territory of the mouse hindpaw that resolves within a few weeks. We report that interruption of endogenous neuropeptide Y (NPY) signaling during remission, with either conditional NPY knockdown in NPYtet/tet mice or intrathecal administration of the Y1 receptor antagonist BIBO3304, reinstated hypersensitivity. These data indicate that nerve injury establishes a long-lasting latent sensitization of spinal nociceptive neurons that is masked by spinal NPY-Y1 neurotransmission. To determine whether this mechanism extends beyond the sensory component of nociception, we used conditioned place aversion and preference assays to evaluate the affective component of pain. We found that BIBO3304 produced place aversion in mice when administered during remission. Furthermore, the analgesic drug gabapentin produced place preference after NPY knockdown in NPYtet/tet but not control mice. We then used pharmacological agents and deletion mutant mice to investigate the cellular mechanisms of neuropathic latent sensitization. BIBO3304-induced reinstatement of mechanical hypersensitivity and conditioned place aversion could be prevented with intrathecal administration of an N-methyl-d-aspartate receptor antagonist (MK-801) and was absent in adenylyl cyclase type 1 (AC1) deletion mutant mice. BIBO3304-induced reinstatement could also be prevented with intrathecal administration an AC1 inhibitor (NB001) or a TRPV1 channel blocker (AMG9801), but not vehicle. Intrathecal administration of a TRPA1 channel blocker (HC030031) prevented the reinstatement of neuropathic hypersensitivity produced either by BIBO3304, or by NPY knockdown in NPYtet/tet but not control mice. Our results confirm new mediators of latent sensitization: TRPA1 and TRPV1. We conclude that NPY acts at spinal Y1 to tonically inhibit a molecular NMDAR➔AC1 intracellular signaling pathway in the dorsal horn that is induced by peripheral nerve injury and drives both the sensory and affective components of chronic neuropathic pain.

Role of 1-Deoxysphingolipids in docetaxel neurotoxicity.

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients' symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-DeoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein-coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySL. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Sex differences in kappa opioid receptor inhibition of latent postoperative pain sensitization in dorsal horn.

Tissue injury produces a delicate balance between latent pain sensitization (LS) and compensatory endogenous opioid receptor analgesia that continues for months, even after re-establishment of normal pain thresholds. To evaluate the contribution of mu (MOR), delta (DOR), and/or kappa (KOR) opioid receptors to the silencing of chronic postoperative pain, we performed plantar incision at the hindpaw, waited 21 days for the resolution of hyperalgesia, and then intrathecally injected subtype-selective ligands. We found that the MOR-selective inhibitor CTOP (1-1000 ng) dose-dependently reinstated mechanical hyperalgesia. Two DOR-selective inhibitors naltrindole (1-10 µg) and TIPP[Ψ] (1-20 µg) reinstated mechanical hyperalgesia, but only at the highest dose that also produced itching, licking, and tail biting. Both the prototypical KOR-selective inhibitors nor-BNI (0.1-10 µg) and the newer KOR inhibitor with more canonical pharmocodynamic effects, LY2456302 (0.1-10 µg), reinstated mechanical hyperalgesia. Furthermore, LY2456302 (10 µg) increased the expression of phosphorylated signal-regulated kinase (pERK), a marker of central sensitization, in dorsal horn neurons but not glia. Sex studies revealed that LY2456302 (0.3 µg) reinstated hyperalgesia and pERK expression to a greater degree in female as compared to male mice. Our results suggest that spinal MOR and KOR, but not DOR, maintain LS within a state of remission to reduce the intensity and duration of postoperative pain, and that endogenous KOR but not MOR analgesia is greater in female mice.

Neuropeptide Y release in the rat spinal cord measured with Y1 receptor internalization is increased after nerve injury.

Neuropeptide Y (NPY) modulates nociception in the spinal cord, but little is known about its mechanisms of release. We measured NPY release in situ using the internalization of its Y1 receptor in dorsal horn neurons. Y1 receptor immunoreactivity was normally localized to the cell surface, but addition of NPY to spinal cord slices increased the number of neurons with Y1 internalization in a biphasic fashion (EC50s of 1 nM and 1 µM). Depolarization with KCl, capsaicin, or the protein kinase A activator 6-benzoyl-cAMP also induced Y1 receptor internalization, presumably by releasing NPY. NMDA receptor activation in the presence of BVT948, an inhibitor of protein tyrosine phosphatases, also released NPY. Electrical stimulation of the dorsal horn frequency-dependently induced NPY release; and this was decreased by the Y1 antagonist BIBO3304, the Nav channel blocker lidocaine, or the Cav2 channel blocker ω-conotoxin MVIIC. Dorsal root immersion in capsaicin, but not its electrical stimulation, also induced NPY release. This was blocked by CNQX, suggesting that part of the NPY released by capsaicin was from dorsal horn neurons receiving synapses from primary afferents and not from the afferent themselves. Mechanical stimulation in vivo, with rub or clamp of the hindpaw, elicited robust Y1 receptor internalization in rats with spared nerve injury but not sham surgery. In summary, NPY is released from dorsal horn interneurons or primary afferent terminals by electrical stimulation and by activation of TRPV1, PKA or NMDA receptors in. Furthermore, NPY release evoked by noxious and tactile stimuli increases after peripheral nerve injury.