Opposing Effects on Descending Control of Nociception by µ and κ Opioid Receptors in the Anterior Cingulate Cortex.

BACKGROUND: The efficiency of descending pain modulation, commonly assessed with the conditioned pain modulation procedure, is diminished in patients with chronic pain. The authors hypothesized that the efficiency of pain modulation is controlled by cortical opioid circuits. METHODS: This study evaluated the effects of µ opioid receptor activation in the anterior cingulate cortex on descending control of nociception, a preclinical correlate of conditioned pain modulation, in male Sprague-Dawley rats with spinal nerve ligation-induced chronic pain or in sham-operated controls. Additionally, the study explored the consequences of respective activation or inhibition of κ opioid receptor in the anterior cingulate cortex of naive rats or animals with neuropathic pain. Descending control of nociception was measured as the hind paw withdrawal response to noxious pressure (test stimulus) in the absence or presence of capsaicin injection in the forepaw (conditioning stimulus). RESULTS: Descending control of nociception was diminished in the ipsilateral, but not contralateral, hind paw of rats with spinal nerve ligation. Bilateral administration of morphine in the anterior cingulate cortex had no effect in shams but restored diminished descending control of nociception without altering hypersensitivity in rats with neuropathic pain. Bilateral anterior cingulate cortex microinjection of κ opioid receptor antagonists, including nor-binaltorphimine and navacaprant, also re-established descending control of nociception in rats with neuropathic pain without altering hypersensitivity and with no effect in shams. Conversely, bilateral injection of a κ opioid receptor agonist, U69,593, in the anterior cingulate cortex of naive rats inhibited descending control of nociception without altering withdrawal thresholds. CONCLUSIONS: Anterior cingulate cortex κ opioid receptor activation therefore diminishes descending control of nociception both in naive animals and as an adaptive response to chronic pain, likely by enhancing net descending facilitation. Descending control of nociception can be restored by activation of µ opioid receptors in the anterior cingulate cortex, but also by κ opioid receptor antagonists, providing a nonaddictive alternative to opioid analgesics. Navacaprant is now in advanced clinical trials.

Associations Between Vitamin D Deficiency/Insufficiency and Depression Expose Health Disparities in Older Rural West Texans: A Project FRONTIER Study.

OBJECTIVE: To determine associations between Vitamin D (VD) levels and clinical depression through the Geriatric Depression Scale (GDS) and its questions and subdomains, stratified by demographics and Hispanic/Latino ethnicity (HLE). DESIGN, SETTING, AND PARTICIPANTS: A cohort of 299 Project FRONTIER participants aged 62.6 ± 11.7 years old, 70.9% female, and 40.5% HLE were used. Standard correlation and regression analyses were employed. MEASUREMENTS: The main outcome measures were VD (serum 25(OH)-VD) level, GDS-30 (30-item questionnaire), GDS-30 subfactors and questions, and HLE status. VD categories were defined as VD deficiency (VDD; ≤20 ng/mL), VD insufficiency (VDI; 21-29 ng/mL), VD sufficiency (30-38 ng/mL) and high VD sufficiency (>38 ng/mL). RESULTS: The majority (61.5%) of samples fell into VDD/VDI categories. A significant negative association was found between VD level and GDS-30 total score. VD level was negatively correlated with Dysphoria and Meaninglessness GDS-30 subfactors. Although GDS subfactors were similar between HLE and non-HLE groups, VD levels were significantly lower in HLE samples. Finally, HLE/non-HLE groups were differentially stratified across VD categories. Only 4% of HLEs fell into the high VD sufficient category, suggesting low VD supplementation. CONCLUSION: A significant negative association between VD level and depressive symptoms was revealed in our aging Project FRONTIER participants. HLE individuals were overrepresented in VDD/VDI samples, and VDD/VDI was associated primarily with the Dysphoria GDS subdomain. Regression analysis predicted high VD sufficiency (95.5 ng/mL) to be associated with no depressive symptoms (GDS=0). Our results underscore troubling disparities in VD-related depressive symptoms between HLE and non-HLE populations.

Chronic pain recruits hypothalamic dynorphin/kappa opioid receptor signalling to promote wakefulness and vigilance.

Increased vigilance in settings of potential threats or in states of vulnerability related to pain is important for survival. Pain disrupts sleep and conversely, sleep disruption enhances pain, but the underlying mechanisms remain unknown. Chronic pain engages brain stress circuits and increases secretion of dynorphin, an endogenous ligand of the kappa opioid receptor (KOR). We therefore hypothesized that hypothalamic dynorphin/KOR signalling may be a previously unknown mechanism that is recruited in pathological conditions requiring increased vigilance. We investigated the role of KOR in wakefulness, non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep in freely moving naïve mice and in mice with neuropathic pain induced by partial sciatic nerve ligation using EEG/EMG recordings. Systemic continuous administration of U69,593, a KOR agonist, over 5 days through an osmotic minipump decreased the amount of NREM and REM sleep and increased sleep fragmentation in naïve mice throughout the light-dark sleep cycle. We used KORcre mice to selectively express a Gi-coupled designer receptor activated by designer drugs (Gi-DREADD) in KORcre neurons of the hypothalamic paraventricular nucleus, a key node of the hypothalamic-pituitary-adrenal stress response. Sustained activation of Gi-DREADD with clozapine-N-oxide delivered in drinking water over 4 days, disrupted sleep in these mice in a similar way as systemic U69,593. Mice with chronic neuropathic pain also showed disrupted NREM and total sleep that was normalized by systemic administration of two structurally different KOR antagonists, norbinaltorphimine and NMRA-140, currently in phase II clinical development, or by CRISPR/Cas9 editing of paraventricular nucleus KOR, consistent with endogenous KOR activation disrupting sleep in chronic pain. Unexpectedly, REM sleep was diminished by either systemic KOR antagonist or by CRISPR/Cas9 editing of paraventricular nucleus KOR in sham-operated mice. Our findings reveal previously unknown physiological and pathophysiological roles of dynorphin/KOR in eliciting arousal. Physiologically, dynorphin/KOR signalling affects transitions between sleep stages that promote REM sleep. Furthermore, while KOR antagonists do not promote somnolence in the absence of pain, they normalized disrupted sleep in chronic pain, revealing a pathophysiological role of KOR signalling that is selectively recruited to promote vigilance, increasing chances of survival. Notably, while this mechanism is likely beneficial in the short-term, disruption of the homeostatic need for sleep over longer periods may become maladaptive resulting in sustained pain chronicity. A novel approach for treatment of chronic pain may thus result from normalization of chronic pain-related sleep disruption by KOR antagonism.

Pain and aging: A unique challenge in neuroinflammation and behavior.

Chronic pain is one of the most common, costly, and potentially debilitating health issues facing older adults, with attributable costs exceeding $600 billion annually. The prevalence of pain in humans increases with advancing age. Yet, the contributions of sex differences, age-related chronic inflammation, and changes in neuroplasticity to the overall experience of pain are less clear, given that opposing processes in aging interact. This review article examines and summarizes pre-clinical research and clinical data on chronic pain among older adults to identify knowledge gaps and provide the base for future research and clinical practice. We provide evidence to suggest that neurodegenerative conditions engender a loss of neural plasticity involved in pain response, whereas low-grade inflammation in aging increases CNS sensitization but decreases PNS sensitivity. Insights from preclinical studies are needed to answer mechanistic questions. However, the selection of appropriate aging models presents a challenge that has resulted in conflicting data regarding pain processing and behavioral outcomes that are difficult to translate to humans.

Hmgb1 Silencing in the Amygdala Inhibits Pain-Related Behaviors in a Rat Model of Neuropathic Pain.

Chronic pain presents a therapeutic challenge due to the highly complex interplay of sensory, emotional-affective and cognitive factors. The mechanisms of the transition from acute to chronic pain are not well understood. We hypothesized that neuroimmune mechanisms in the amygdala, a brain region involved in the emotional-affective component of pain and pain modulation, play an important role through high motility group box 1 (Hmgb1), a pro-inflammatory molecule that has been linked to neuroimmune signaling in spinal nociception. Transcriptomic analysis revealed an upregulation of Hmgb1 mRNA in the right but not left central nucleus of the amygdala (CeA) at the chronic stage of a spinal nerve ligation (SNL) rat model of neuropathic pain. Hmgb1 silencing with a stereotaxic injection of siRNA for Hmgb1 into the right CeA of adult male and female rats 1 week after (post-treatment), but not 2 weeks before (pre-treatment) SNL induction decreased mechanical hypersensitivity and emotional-affective responses, but not anxiety-like behaviors, measured 4 weeks after SNL. Immunohistochemical data suggest that neurons are a major source of Hmgb1 in the CeA. Therefore, Hmgb1 in the amygdala may contribute to the transition from acute to chronic neuropathic pain, and the inhibition of Hmgb1 at a subacute time point can mitigate neuropathic pain.

Contribution of Corticotropin-Releasing Factor Receptor 1 (CRF1) to Serotonin Receptor 5-HT2CR Function in Amygdala Neurons in a Neuropathic Pain Model.

The amygdala plays a key role in emotional-affective aspects of pain and in pain modulation. The central nucleus (CeA) serves major amygdala output functions related to emotional-affective behaviors and pain modulation. Our previous studies implicated the corticotropin-releasing factor (CRF) system in amygdala plasticity and pain behaviors in an arthritis model. We also showed that serotonin (5-HT) receptor subtype 5-HT2CR in the basolateral amygdala (BLA) contributes to increased CeA output and neuropathic pain-like behaviors. Here, we tested the novel hypothesis that 5-HT2CR in the BLA drives CRF1 receptor activation to increase CeA neuronal activity in neuropathic pain. Extracellular single-unit recordings of CeA neurons in anesthetized adult male rats detected increased activity in neuropathic rats (spinal nerve ligation model) compared to sham controls. Increased CeA activity was blocked by local knockdown or pharmacological blockade of 5-HT2CR in the BLA, using stereotaxic administration of 5-HT2CR short hairpin RNA (shRNA) viral vector or a 5-HT2CR antagonist (SB242084), respectively. Stereotaxic administration of a CRF1 receptor antagonist (NBI27914) into the BLA also decreased CeA activity in neuropathic rats and blocked the facilitatory effects of a 5-HT2CR agonist (WAY161503) administered stereotaxically into the BLA. Conversely, local (BLA) knockdown of 5-HT2CR eliminated the inhibitory effect of NBI27914 and the facilitatory effect of WAY161503 in neuropathic rats. The data suggest that 5-HT2CR activation in the BLA contributes to neuropathic pain-related amygdala (CeA) activity by engaging CRF1 receptor signaling.

Metabotropic Glutamate Receptor 5 and 8 Modulate the Ameliorative Effect of Ultramicronized Palmitoylethanolamide on Cognitive Decline Associated with Neuropathic Pain.

This study investigated whether metabotropic glutamate receptor (mGluR) 5 and 8 are involved in the effect of ultramicronizedpalmitoylethanolamide (um-PEA) on the cognitive behavior and long term potentiation (LTP) at entorhinal cortex (LEC)-dentate gyrus (DG) pathway in mice rendered neuropathic by the spare nerve injury (SNI). SNI reduced discriminative memory and LTP. Um-PEA treatment started after the development of neuropathic pain had no effects in sham mice, whereas it restored cognitive behavior and LTP in SNI mice. 2-Methyl-6-(phenylethynyl) pyridine (MPEP), a selective mGluR5 antagonist, improved cognition in SNI mice and produced a chemical long term depression of the field excitatory postsynaptic potentials (fEPSPs) in sham and SNI mice. After theta burst stimulation (TBS) MPEP restored LTP in SNI mice. In combination with PEA, MPEP antagonized the PEA effect on discriminative memory and decreased LTP in SNI mice. The (RS)-4-(1-amino-1-carboxyethyl)phthalic acid (MDCPG), a selective mGluR8 antagonist, did not affect discriminative memory, but it induced a chemical LTP and prevented the enhancement of fEPSPs after TBS in SNI mice which were treated or not treated with PEA. The effect of PEA on LTP and cognitive behavior was modulated by mGluR5 and mGluR8. In particular in the SNI conditions, the mGluR5 blockade facilitated memory and LTP, but prevented the beneficial effects of PEA on discriminative memory while the mGluR8 blockade, which was ineffective in itself, prevented the favorable action of the PEA on LTP. Thus, although their opposite roles (excitatory/inhibitory of the two receptor subtypes on the glutamatergic system), they appeared to be required for the neuroprotective effect of PEA in conditions of neuropathic pain.

5-HT2C Receptor Knockdown in the Amygdala Inhibits Neuropathic-Pain-Related Plasticity and Behaviors.

Neuroplasticity in the amygdala drives pain-related behaviors. The central nucleus (CeA) serves major amygdala output functions and can generate emotional-affective behaviors and modulate nocifensive responses. The CeA receives excitatory and inhibitory inputs from the basolateral nucleus (BLA) and serotonin receptor subtype 5-HT2CR in the BLA, but not CeA, has been implicated anxiogenic behaviors and anxiety disorders. Here, we tested the hypothesis that 5-HT2CR in the BLA plays a critical role in CeA plasticity and neuropathic pain behaviors in the rat spinal nerve ligation (SNL) model. Local 5-HT2CR knockdown in the BLA with stereotaxic injection of 5-HT2CR shRNA AAV vector decreased vocalizations and anxiety- and depression-like behaviors and increased sensory thresholds of SNL rats, but had no effect in sham controls. Extracellular single-unit recordings of CeA neurons in anesthetized rats showed that 5-HT2CR knockdown blocked the increase in neuronal activity (increased responsiveness, irregular spike firing, and increased burst activity) in SNL rats. At the synaptic level, 5-HT2CR knockdown blocked the increase in excitatory transmission from BLA to CeA recorded in brain slices from SNL rats using whole-cell patch-clamp conditions. Inhibitory transmission was decreased by 5-HT2CR knockdown in control and SNL conditions to a similar degree. The findings can be explained by immunohistochemical data showing increased expression of 5-HT2CR in non-GABAergic BLA cells in SNL rats. The results suggest that increased 5-HT2CR in the BLA contributes to neuropathic-pain-related amygdala plasticity by driving synaptic excitation of CeA neurons. As a rescue strategy, 5-HT2CR knockdown in the BLA inhibits neuropathic-pain-related behaviors.SIGNIFICANCE STATEMENT Neuroplasticity in the amygdala has emerged as an important pain mechanism. This study identifies a novel target and rescue strategy to control abnormally enhanced amygdala activity in an animal model of neuropathic pain. Specifically, an integrative approach of gene transfer, systems and brain slice electrophysiology, behavior, and immunohistochemistry was used to advance the novel concept that serotonin receptor subtype 5-HT2C contributes critically to the imbalance between excitatory and inhibitory drive of amygdala output neurons. Local viral vector-mediated 5-HT2CR knockdown in the amygdala normalizes the imbalance, decreases neuronal activity, and inhibits neuropathic-pain-related behaviors. The study provides valuable insight into serotonin receptor (dys)function in a limbic brain area.

Fear extinction learning ability predicts neuropathic pain behaviors and amygdala activity in male rats.

Background The amygdala plays a key role in fear learning and extinction and has emerged as an important node of emotional-affective aspects of pain and pain modulation. Impaired fear extinction learning, which involves prefrontal cortical control of amygdala processing, has been linked to neuropsychiatric disorders. Here, we tested the hypothesis that fear extinction learning ability can predict the magnitude of neuropathic pain. Results We correlated fear extinction learning in naive adult male rats with sensory and affective behavioral outcome measures (mechanical thresholds, vocalizations, and anxiety- and depression-like behaviors) before and after the induction of the spinal nerve ligation model of neuropathic pain compared to sham controls. Auditory fear conditioning, extinction learning, and extinction retention tests were conducted after baseline testing. All rats showed increased freezing responses after fear conditioning. During extinction training, the majority (75%) of rats showed a decline in freezing level to 50% in 5 min (fear extinction+), whereas 25% of the rats maintained a high freezing level (>50%, fear extinction-). Fear extinction- rats showed decreased open-arm preference in the elevated plus maze, reflecting anxiety-like behavior, but there were no significant differences in sensory thresholds, vocalizations, or depression-like behavior (forced swim test) between fear extinction+ and fear extinction- types. In the neuropathic pain model (four weeks after spinal nerve ligation), fear extinction- rats showed a greater increase in vocalizations and anxiety-like behavior than fear extinction+ rats. Fear extinction- rats, but not fear extinction+ rats, also developed depression-like behavior. Extracellular single unit recordings of amygdala (central nucleus) neurons in behaviorally tested rats (anesthetized with isoflurane) found greater increases in background activity, bursting, and evoked activity in fear extinction- rats than fear extinction+ rats in the spinal nerve ligation model compared to sham controls. Conclusion The data may suggest that fear extinction learning ability predicts the magnitude of neuropathic pain-related affective rather than sensory behaviors, which correlates with differences in amygdala activity changes.

Rescue of Impaired mGluR5-Driven Endocannabinoid Signaling Restores Prefrontal Cortical Output to Inhibit Pain in Arthritic Rats.

The medial prefrontal cortex (mPFC) serves executive functions that are impaired in neuropsychiatric disorders and pain. Underlying mechanisms remain to be determined. Here we advance the novel concept that metabotropic glutamate receptor 5 (mGluR5) fails to engage endocannabinoid (2-AG) signaling to overcome abnormal synaptic inhibition in pain, but restoring endocannabinoid signaling allows mGluR5 to increase mPFC output hence inhibit pain behaviors and mitigate cognitive deficits. Whole-cell patch-clamp recordings were made from layer V pyramidal cells in the infralimbic mPFC in rat brain slices. Electrical and optogenetic stimulations were used to analyze amygdala-driven mPFC activity. A selective mGluR5 activator (VU0360172) increased pyramidal output through an endocannabinoid-dependent mechanism because intracellular inhibition of the major 2-AG synthesizing enzyme diacylglycerol lipase or blockade of CB1 receptors abolished the facilitatory effect of VU0360172. In an arthritis pain model mGluR5 activation failed to overcome abnormal synaptic inhibition and increase pyramidal output. mGluR5 function was rescued by restoring 2-AG-CB1 signaling with a CB1 agonist (ACEA) or inhibitors of postsynaptic 2-AG hydrolyzing enzyme ABHD6 (intracellular WWL70) and monoacylglycerol lipase MGL (JZL184) or by blocking GABAergic inhibition with intracellular picrotoxin. CB1-mediated depolarization-induced suppression of synaptic inhibition (DSI) was also impaired in the pain model but could be restored by coapplication of VU0360172 and ACEA. Stereotaxic coadministration of VU0360172 and ACEA into the infralimbic, but not anterior cingulate, cortex mitigated decision-making deficits and pain behaviors of arthritic animals. The results suggest that rescue of impaired endocannabinoid-dependent mGluR5 function in the mPFC can restore mPFC output and cognitive functions and inhibit pain. Significance statement: Dysfunctions in prefrontal cortical interactions with subcortical brain regions, such as the amygdala, are emerging as important players in neuropsychiatric disorders and pain. This study identifies a novel mechanism and rescue strategy for impaired medial prefrontal cortical function in an animal model of arthritis pain. Specifically, an integrative approach of optogenetics, pharmacology, electrophysiology, and behavior is used to advance the novel concept that a breakdown of metabotropic glutamate receptor subtype mGluR5 and endocannabinoid signaling in infralimbic pyramidal cells fails to control abnormal amygdala-driven synaptic inhibition in the arthritis pain model. Restoring endocannabinoid signaling allows mGluR5 activation to increase infralimbic output hence inhibit pain behaviors and mitigate pain-related cognitive deficits.

Plasma Membrane Na⁺-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy.

SLC13A5 is a Na⁺-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na⁺:citrate3- stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 µM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150-200 µM. Li⁺ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-d-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.

Differential contributions of vasopressin V1A and oxytocin receptors in the amygdala to pain-related behaviors in rats.

Neuroplastic changes in the amygdala account for emotional-affective aspects of pain and involve neuropeptides such as calcitonin gene-related peptide and corticotropin-releasing factor. Another neuropeptide system, central arginine vasopressin, has been implicated in neuropsychiatric disorders, but its role in pain-related emotional expression and neuroplasticity remains to be determined. Here, we tested the hypothesis that arginine vasopressin in the amygdala contributes to pain-related emotional-affective responses, using stereotaxic applications of arginine vasopressin and antagonists for G-protein coupled vasopressin V1A and oxytocin receptors in adult male Sprague-Dawley rats. In normal animals, arginine vasopressin increased audible and ultrasonic vocalizations and anxiety-like behavior (decreased open-arm preference in the elevated plus maze). The facilitatory effects were blocked by a selective V1A antagonist (SR 49059, Relcovaptan) but not by an oxytocin receptor antagonist (L-371,257). L-371,257 had some facilitatory effects on vocalizations. Arginine vasopressin had no effect in arthritic rats (kaolin/carrageenan knee joint pain model). SR 49059 inhibited vocalizations and anxiety-like behavior (elevated plus maze) in arthritic, but not normal, rats and conveyed anxiolytic properties to arginine vasopressin. Arginine vasopressin, SR 49059, and L-371,257 had no significant effects on spinal reflexes. We interpret the data to suggest that arginine vasopressin through V1A in the amygdala contributes to emotional-affective aspects of pain (arthritis model), whereas oxytocin receptors may mediate some inhibitory effects of the vasopressin system.

Small-conductance calcium-activated potassium (SK) channels in the amygdala mediate pain-inhibiting effects of clinically available riluzole in a rat model of arthritis pain.

BACKGROUND: Arthritis pain is an important healthcare issue with significant emotional and affective consequences. Here we focus on potentially beneficial effects of activating small-conductance calcium-activated potassium (SK) channels in the amygdala, a brain center of emotions that plays an important role in central pain modulation and processing. SK channels have been reported to regulate neuronal activity in the central amygdala (CeA, output nucleus). We tested the effects of riluzole, a clinically available drug for the treatment of amyotrophic lateral sclerosis, for the following reasons. Actions of riluzole include activation of SK channels. Evidence in the literature suggests that riluzole may have antinociceptive effects through an action in the brain but not the spinal cord. Mechanism and site of action of riluzole remain to be determined. Here we tested the hypothesis that riluzole inhibits pain behaviors by acting on SK channels in the CeA in an arthritis pain model. RESULTS: Systemic (intraperitoneal) application of riluzole (8 mg/kg) inhibited audible (nocifensive response) and ultrasonic (averse affective response) vocalizations of adult rats with arthritis (5 h postinduction of a kaolin-carrageenan monoarthritis in the knee) but did not affect spinal withdrawal thresholds, which is consistent with a supraspinal action. Stereotaxic administration of riluzole into the CeA by microdialysis (1 mM, concentration in the microdialysis fiber, 15 min) also inhibited vocalizations, confirming the CeA as a site of action of riluzole. Stereotaxic administration of a selective SK channel blocker (apamin, 1 µM, concentration in the microdialysis fiber, 15 min) into the CeA had no effect by itself but inhibited the effect of systemic riluzole on vocalizations. Off-site administration of apamin into the basolateral amygdala (BLA) as a placement control or stereotaxic application of a selective blocker of large-conductance calcium-activated potassium (BK) channels (charybdotoxin, 1 µM, concentration in the microdialysis fiber, 15 min) into the CeA did not affect the inhibitory effects of systemically applied riluzole. CONCLUSIONS: The results suggest that riluzole can inhibit supraspinally organized pain behaviors in an arthritis model by activating SK, but not BK, channels in the amygdala (CeA but not BLA).

Amygdala pain mechanisms.

A limbic brain area, the amygdala plays a key role in emotional responses and affective states and disorders such as learned fear, anxiety, and depression. The amygdala has also emerged as an important brain center for the emotional-affective dimension of pain and for pain modulation. Hyperactivity in the laterocapsular division of the central nucleus of the amygdala (CeLC, also termed the "nociceptive amygdala") accounts for pain-related emotional responses and anxiety-like behavior. Abnormally enhanced output from the CeLC is the consequence of an imbalance between excitatory and inhibitory mechanisms. Impaired inhibitory control mediated by a cluster of GABAergic interneurons in the intercalated cell masses (ITC) allows the development of glutamate- and neuropeptide-driven synaptic plasticity of excitatory inputs from the brainstem (parabrachial area) and from the lateral-basolateral amygdala network (LA-BLA, site of integration of polymodal sensory information). BLA hyperactivity also generates abnormally enhanced feedforward inhibition of principal cells in the medial prefrontal cortex (mPFC), a limbic cortical area that is strongly interconnected with the amygdala. Pain-related mPFC deactivation results in cognitive deficits and failure to engage cortically driven ITC-mediated inhibitory control of amygdala processing. Impaired cortical control allows the uncontrolled persistence of amygdala pain mechanisms.

Nasal application of neuropeptide S inhibits arthritis pain-related behaviors through an action in the amygdala.

Recently discovered neuropeptide S (NPS) has anxiolytic and pain-inhibiting effects in rodents. We showed previously that NPS increases synaptic inhibition of amygdala output to inhibit pain behaviors. The amygdala plays a key role in emotional-affective aspects of pain. Of clinical significance is that NPS can be applied nasally to exert anxiolytic effects in rodents. This study tested the novel hypothesis that nasal application of NPS can inhibit pain-related behaviors in an arthritis model through NPS receptors (NPSR) in the amygdala. Behaviors and electrophysiological activity of amygdala neurons were measured in adult male Sprague Dawley rats. Nasal application of NPS, but not saline, inhibited audible and ultrasonic vocalizations and had anxiolytic-like effects in the elevated plus-maze test in arthritic rats (kaolin/carrageenan knee joint arthritis model) but had no effect in normal rats. Stereotaxic application of a selective non-peptide NPSR antagonist (SHA68) into the amygdala by microdialysis reversed the inhibitory effects of NPS. NPS had no effect on hindlimb withdrawal thresholds. We showed previously that intra-amygdala application of an NPSR antagonist alone had no effect. Nasal application of NPS or stereotaxic application of NPS into the amygdala by microdialysis inhibited background and evoked activity of amygdala neurons in arthritic, but not normal, anesthetized rats. The inhibitory effect was blocked by a selective NPSR antagonist ([D-Cys(tBu)5]NPS). In conclusion, nasal application of NPS can inhibit emotional-affective, but not sensory, pain-related behaviors through an action in the amygdala. The beneficial effects of non-invasive NPS application may suggest translational potential.

CB1 augments mGluR5 function in medial prefrontal cortical neurons to inhibit amygdala hyperactivity in an arthritis pain model.

The medial prefrontal cortex (mPFC) serves executive control functions and forms direct connections with subcortical areas such as the amygdala. Our previous work showed abnormal inhibition of mPFC pyramidal cells and hyperactivity of amygdala output neurons in an arthritis pain model. To restore mPFC activity and hence control pain-related amygdala hyperactivity this study focused on CB1 and mGluR5 receptors, which are important modulators of cortical functions. Extracellular single-unit recordings of infralimbic mPFC pyramidal cells and of amygdala output neurons in the laterocapsular division of the central nucleus (CeLC) were made in anesthetised adult male rats. mPFC neurons were classified as 'excited' or 'inhibited' based on their response to brief innocuous and noxious test stimuli. After arthritis pain induction, background activity and evoked responses of excited neurons and background activity and inhibition of inhibited neurons decreased. Stereotaxic application of an mGluR5-positive allosteric modulator (N-cyclobutyl-6-((3-fluorophenyl)ethynyl) nicotinamide hydrochloride, VU0360172) into the mPFC increased background and evoked activity of excited, but not inhibited, mPFC neurons under normal conditions but not in arthritis. A selective CB1 receptor agonist (arachidonyl-2-chloroethylamide) alone had no effect but restored the facilitatory effects of VU0360172 in the pain model. Coactivation of CB1 and mGluR5 in the mPFC inhibited the pain-related activity increase of CeLC neurons but had no effect under normal conditions. The data suggest that excited mPFC neurons are inversely linked to amygdala output (CeLC) and that CB1 can increase mGluR5 function in this subset of mPFC neurons to engage cortical control of abnormally enhanced amygdala output in pain.

Alcohol withdrawal and pain: Peripheral mechanisms join central circuits.

Negative affective aspects of alcohol withdrawal and pain involve converging brain circuits. In this issue of Neuron, Son et al.1 identify a peripheral mechanism of an alcohol-withdrawal-induced headache-like condition, which is centered on mast-cell-specific receptor MrgprB2 activated by corticotropin-releasing factor (CRF) in dura mater to drive nociception.

Impaired amygdala astrocytic signaling worsens neuropathic pain-associated neuronal functions and behaviors.

Introduction: Pain is a clinically relevant health care issue with limited therapeutic options, creating the need for new and improved analgesic strategies. The amygdala is a limbic brain region critically involved in the regulation of emotional-affective components of pain and in pain modulation. The central nucleus of amygdala (CeA) serves major output functions and receives nociceptive information via the external lateral parabrachial nucleus (PB). While amygdala neuroplasticity has been linked causally to pain behaviors, non-neuronal pain mechanisms in this region remain to be explored. As an essential part of the neuroimmune system, astrocytes that represent about 40-50% of glia cells within the central nervous system, are required for physiological neuronal functions, but their role in the amygdala remains to be determined for pain conditions. In this study, we measured time-specific astrocyte activation in the CeA in a neuropathic pain model (spinal nerve ligation, SNL) and assessed the effects of astrocyte inhibition on amygdala neuroplasticity and pain-like behaviors in the pain condition. Methods and Results: Glial fibrillary acidic protein (GFAP, astrocytic marker) immunoreactivity and mRNA expression were increased at the chronic (4 weeks post-SNL), but not acute (1 week post-SNL), stage of neuropathic pain. In order to determine the contribution of astrocytes to amygdala pain-mechanisms, we used fluorocitric acid (FCA), a selective inhibitor of astrocyte metabolism. Whole-cell patch-clamp recordings were performed from neurons in the laterocapsular division of the CeA (CeLC) obtained from chronic neuropathic rats. Pre-incubation of brain slices with FCA (100 µM, 1 h), increased excitability through altered hyperpolarization-activated current (Ih) functions, without significantly affecting synaptic responses at the PB-CeLC synapse. Intra-CeA injection of FCA (100 µM) had facilitatory effects on mechanical withdrawal thresholds (von Frey and paw pressure tests) and emotional-affective behaviors (evoked vocalizations), but not on facial grimace score and anxiety-like behaviors (open field test), in chronic neuropathic rats. Selective inhibition of astrocytes by FCA was confirmed with immunohistochemical analyses showing decreased astrocytic GFAP, but not NeuN, signal in the CeA. Discussion: Overall, these results suggest a complex modulation of amygdala pain functions by astrocytes and provide evidence for beneficial functions of astrocytes in CeA in chronic neuropathic pain.

Investigating the Relationship Between Marital Status and Ethnicity on Neurocognitive Functioning in a Rural Older Population: A Project FRONTIER Study.

OBJECTIVES: Research indicates being married is related to better physical and psychological health. Little is known regarding the relationship between marital status and neurocognitive functioning and whether it differs based on ethnicity (Hispanic vs non-Hispanic). This is the first study to examine this relationship in a sample of aging adults in rural Texas. METHODS: Data from 1,864 participants (Mage = 59.68, standard deviation [SD]age = 12.21), who were mostly Hispanic (n = 1,053), women (n = 1,295), and married (n = 1,125) from Project Facing Rural Obstacles to Healthcare Now Through Intervention, Education, & Research were analyzed. Neuropsychological testing comprised Repeatable Battery for the Assessment of Neuropsychological Status, Trails Making Test, and Clock Drawing. Participants were dichotomized, married, and unmarried. RESULTS: There was a significant interaction between Hispanic identity and marital status on overall neurocognitive functioning (F(1, 1,480) = 4.79, p < .05, ηp2 = 0.003). For non-Hispanic individuals, married individuals had higher overall neurocognitive functioning compared to unmarried individuals, whereas neurocognitive functioning for Hispanic individuals did not significantly differ between married and unmarried individuals. There were significant main effects as married individuals (M = 84.95, SD = 15.56) had greater overall neurocognitive functioning than unmarried individuals (M = 83.47, SD = 15.86; F(1, 1,480) = 14.67, p < .001, ηp2 = 0.01), Hispanic individuals (M = 78.02, SD = 14.25) had lower overall neurocognitive functioning than non-Hispanic individuals (M = 91.43, SD = 15.07; F(1, 1,480) = 284.99, p < .001, ηp2 = 0.16). DISCUSSION: Hispanics living in rural areas experience additional stressors that could lead to worse neurocognitive functioning, which is supported by the Lifespan Biopsychosocial Model of Cumulative Vulnerability and Minority Health, which postulates that race/ethnicity/socioeconomic-status-related stressors exacerbate the impact of other life stressors. Reduction of stress on rural Hispanics should be a priority as it could positively affect their neurocognitive functioning.

Chemogenetic Manipulation of Amygdala Kappa Opioid Receptor Neurons Modulates Amygdala Neuronal Activity and Neuropathic Pain Behaviors.

Neuroplasticity in the central nucleus of the amygdala (CeA) plays a key role in the modulation of pain and its aversive component. The dynorphin/kappa opioid receptor (KOR) system in the amygdala is critical for averse-affective behaviors in pain conditions, but its mechanisms are not well understood. Here, we used chemogenetic manipulations of amygdala KOR-expressing neurons to analyze the behavioral consequences in a chronic neuropathic pain model. For the chemogenetic inhibition or activation of KOR neurons in the CeA, a Cre-inducible viral vector encoding Gi-DREADD (hM4Di) or Gq-DREADD (hM3Dq) was injected stereotaxically into the right CeA of transgenic KOR-Cre mice. The chemogenetic inhibition of KOR neurons expressing hM4Di with a selective DREADD actuator (deschloroclozapine, DCZ) in sham control mice significantly decreased inhibitory transmission, resulting in a shift of inhibition/excitation balance to promote excitation and induced pain behaviors. The chemogenetic activation of KOR neurons expressing hM3Dq with DCZ in neuropathic mice significantly increased inhibitory transmission, decreased excitability, and decreased neuropathic pain behaviors. These data suggest that amygdala KOR neurons modulate pain behaviors by exerting an inhibitory tone on downstream CeA neurons. Therefore, activation of these interneurons or blockade of inhibitory KOR signaling in these neurons could restore control of amygdala output and mitigate pain.