How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a functional assessment.

BACKGROUND: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic disorders. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs remain key challenges to study human nociception in vitro. Here, we report a detailed functional characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Anatomic's commercially available RealDRG™ were further characterized for both functional and expression phenotyping of key nociceptor markers. METHODS: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Manual patch clamp was used to functionally characterize both control and patient-derived neurons. High throughput techniques were further used to demonstrate that RealDRGs™ derived from the Anatomic protocol are amenable to high throughput technologies for disease modelling. RESULTS: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. Chambers protocol results in predominantly tonic firing when compared to Anatomic protocol. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. RealDRG™ sensory neurons show heterogeneity of nociceptive markers indicating that the cells may be useful as a humanized model system for translational studies. CONCLUSIONS: We validated the efficiency of two differentiation protocols and their potential application for functional assessment and thus understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Highly specific σ2R/TMEM97 ligand FEM-1689 alleviates neuropathic pain and inhibits the integrated stress response.

The sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal antinociceptive effect is approximately 24 h following dosing. We sought to understand this unique antineuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout mice for Tmem97, we find that a σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce antinociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

How to differentiate induced pluripotent stem cells into sensory neurons for disease modelling: a comparison of two protocols.

Background: Human induced pluripotent stem cell (iPSC)-derived peripheral sensory neurons present a valuable tool to model human diseases and are a source for applications in drug discovery and regenerative medicine. Clinically, peripheral sensory neuropathies can result in maladies ranging from a complete loss of pain to severe painful neuropathic symptoms. Sensory neurons are located in the dorsal root ganglion and are comprised of functionally diverse neuronal types. Low efficiency, reproducibility concerns, variations arising due to genetic factors and time needed to generate functionally mature neuronal populations from iPSCs for disease modelling remain key challenges to study human nociception in vitro. Here, we report a detailed characterization of iPSC-derived sensory neurons with an accelerated differentiation protocol ("Anatomic" protocol) compared to the most commonly used small molecule approach ("Chambers" protocol). Methods: Multiple iPSC clones derived from different reprogramming methods, genetics, age, and somatic cell sources were used to generate sensory neurons. Expression profiling of sensory neurons was performed with Immunocytochemistry and in situ hybridization techniques. Manual patch clamp and high throughput cellular screening systems (Fluorescence imaging plate reader, automated patch clamp and multi-well microelectrode arrays recordings) were applied to functionally characterize the generated sensory neurons. Results: The Anatomic protocol rendered a purer culture without the use of mitomycin C to suppress non-neuronal outgrowth, while Chambers differentiations yielded a mix of cell types. High throughput systems confirmed functional expression of Na+ and K+ ion channels. Multi-well microelectrode recordings display spontaneously active neurons with sensitivity to increased temperature indicating expression of heat sensitive ion channels. Patient-derived nociceptors displayed higher frequency firing compared to control subject with both, Chambers and Anatomic differentiation approaches, underlining their potential use for clinical phenotyping as a disease-in-a-dish model. Conclusions: We validated the efficiency of two differentiation protocols and their potential application for understanding the disease mechanisms from patients suffering from pain disorders. We propose that both differentiation methods can be further exploited for understanding mechanisms and development of novel treatments in pain disorders.

Developing a 3-D computational model of neurons in the central amygdala to understand pharmacological targets for pain.

Neuropathic and nociplastic pain are major causes of pain and involve brain areas such as the central nucleus of the amygdala (CeA). Within the CeA, neurons expressing protein kinase c-delta (PKCδ) or somatostatin (SST) have opposing roles in pain-like modulation. In this manuscript, we describe our progress towards developing a 3-D computational model of PKCδ and SST neurons in the CeA and the use of this model to explore the pharmacological targeting of these two neural populations in modulating nociception. Our 3-D model expands upon our existing 2-D computational framework by including a realistic 3-D spatial representation of the CeA and its subnuclei and a network of directed links that preserves morphological properties of PKCδ and SST neurons. The model consists of 13,000 neurons with cell-type specific properties and behaviors estimated from laboratory data. During each model time step, neuron firing rates are updated based on an external stimulus, inhibitory signals are transmitted between neurons via the network, and a measure of nociceptive output from the CeA is calculated as the difference in firing rates of pro-nociceptive PKCδ neurons and anti-nociceptive SST neurons. Model simulations were conducted to explore differences in output for three different spatial distributions of PKCδ and SST neurons. Our results show that the localization of these neuron populations within CeA subnuclei is a key parameter in identifying spatial and cell-type pharmacological targets for pain.

Highly specific σ2R/TMEM97 ligand alleviates neuropathic pain and inhibits the integrated stress response.

The Sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands alleviate mechanical hypersensitivity in mouse neuropathic pain models with a time course wherein maximal anti-nociceptive effect is approximately 24 hours following dosing. We sought to understand this unique anti-neuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons? Using male and female conventional knockout (KO) mice for Tmem97, we find that a new σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce anti-nociception in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion (DRG) neurons, we demonstrate that FEM-1689 inhibits the integrated stress response (ISR) and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.

Barbamide Displays Affinity for Membrane-Bound Receptors and Impacts Store-Operated Calcium Entry in Mouse Sensory Neurons.

Marine cyanobacteria are a rich source of bio-active metabolites that have been utilized as leads for drug discovery and pharmacological tools for basic science research. Here, we describe the re-isolation of a well-known metabolite, barbamide, from Curaçao on three different occasions and the characterization of barbamide's biological interactions with targets of the mammalian nervous system. Barbamide was originally discovered as a molluscicidal agent from a filamentous marine cyanobacterium. In our hands, we found little evidence of toxicity against mammalian cell cultures. However, barbamide showed several affinities when screened for binding affinity for a panel of 45 receptors and transporters known to be involved in nociception and sensory neuron activity. We found high levels of binding affinity for the dopamine transporter, the kappa opioid receptor, and the sigma receptors (sigma-1 and sigma-2 also known as transmembrane protein 97; TMEM97). We tested barbamide in vitro in isolated sensory neurons from female mice to explore its functional impact on calcium flux in these cells. Barbamide by itself had no observable impact on calcium flux. However, barbamide enhanced the effect of the TRPV1 agonist capsaicin and enhanced store-operated calcium entry (SOCE) responses after depletion of intracellular calcium. Overall, these results demonstrate the biological potential of barbamide at sensory neurons with implications for future drug development projects surrounding this molecule.

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.

Design, synthesis, and biological evaluation of C6-difluoromethylenated epoxymorphinan Mu opioid receptor antagonists.

The recent widespread abuse of high potency synthetic opioids, such as fentanyl, presents a serious threat to individuals affected by substance use disorder. Synthetic opioids generally exhibit prolonged in vivo circulatory half-lives that can outlast the reversal effects of conventional naloxone-based overdose antidotes leading to a life-threatening relapse of opioid toxicity known as renarcotization. In this manuscript, we present our efforts to combat the threat of renarcotization by attempting to extend the half-life of traditional MOR antagonists through the design of novel, fluorinated 4,5-epoxymorphinans possessing increased lipophilicity. Analogues were prepared via a concise synthetic strategy highlighted by decarboxylative Wittig olefination of the C6 ketone to install a bioisosteric 1,1-difluoromethylene unit. C6-difluoromethylenated compounds successfully maintained in vitro potency against an EC90 challenge of fentanyl and were predicted to have enhanced circulatory half-life compared to the current standard of care, naloxone. Subsequent in vivo studies demonstrated the effective blockade of fentanyl-induced anti-nociception in mice.

Slow-sustained delivery of naloxone reduces typical naloxone-induced precipitated opioid withdrawal effects in male morphine-dependent mice.

Thousands of individuals die each year from opioid-related overdoses. While naloxone (Narcan®) is currently the most widely employed treatment to reverse opioid toxicity, high or repeated doses of this antidote often lead to precipitated opioid withdrawal (POW). We hypothesized that a slow linear release of naloxone from a nanoparticle would induce fewer POW symptoms compared to high-dose free naloxone. First, we measured the acute impact of covalent naloxone nanoparticles (Nal-cNPs) on morphine-induced antinociception in the hotplate test. We found that Nal-cNP treatment blocked the antinociceptive effect of morphine within 15 min of administration. Next, we tested the impact of Nal-cNPs on POW symptoms in male morphine-dependent mice. To induce morphine dependence, mice were treated with 5 mg/kg morphine (or saline) twice-daily for six consecutive days. On day 7 mice received 5 mg/kg morphine (or saline) injections 2 hr prior to receiving treatment of either unmodified free naloxone, a high or low dose of Nal-cNP, empty nanoparticle (cNP-empty), or saline. Behavior was analyzed for 0-6 hr followed by 24 and 48 hr time points after treatment. As expected, free naloxone induced a significant increase in POW behavior in morphine-dependent mice compared to saline-treated mice upon free naloxone administration. In comparison, reduced POW behavior was observed with both doses of Nal-cNP. Side effects of Nal-cNP on locomotion and fecal boli production were measured and no significant side-effects were observed. Overall, our data show that sustained release of naloxone from a covalent nanoparticle does not induce severe POW symptoms in morphine-dependent mice.

Agent-based modeling of the central amygdala and pain using cell-type specific physiological parameters.

The amygdala is a brain area involved in emotional regulation and pain. Over the course of the last 20 years, multiple researchers have studied sensory and motor connections within the amygdala in trying to understand the ultimate role of this structure in pain perception and descending control of pain. A number of investigators have been using cell-type specific manipulations to probe the underlying circuitry of the amygdala. As data have accumulated in this research space, we recognized a critical need for a single framework to integrate these data and evaluate emergent system-level responses. In this manuscript, we present an agent-based computational model of two distinct inhibitory neuron populations in the amygdala, those that express protein kinase C delta (PKCδ) and those that express somatostatin (SOM). We utilized a network of neural links to simulate connectivity and the transmission of inhibitory signals between neurons. Type-specific parameters describing the response of these neurons to noxious stimuli were estimated from published physiological and immunological data as well as our own wet-lab experiments. The model outputs an abstract measure of pain, which is calculated in terms of the cumulative pro-nociceptive and anti-nociceptive activity across neurons in both hemispheres of the amygdala. Results demonstrate the ability of the model to produce changes in pain that are consistent with published studies and highlight the importance of several model parameters. In particular, we found that the relative proportion of PKCδ and SOM neurons within each hemisphere is a key parameter in predicting pain and we explored model predictions for three possible values of this parameter. We compared model predictions of pain to data from our earlier behavioral studies and found areas of similarity as well as distinctions between the data sets. These differences, in particular, suggest a number of wet-lab experiments that could be done in the future.

Visceral pressure stimulator for exploring hollow organ pain: a pilot study.

BACKGROUND: The regulation and control of pressure stimuli is useful for many studies of pain and nociception especially those in the visceral pain field. In many in vivo experiments, distinct air and liquid stimuli at varying pressures are delivered to hollow organs such as the bladder, vagina, and colon. These stimuli are coupled with behavioral, molecular, or physiological read-outs of the response to the stimulus. Care must be taken to deliver precise timed stimuli during experimentation. For example, stimuli signals can be used online to precisely time-lock the stimulus with a physiological output. Such precision requires the development of specialized hardware to control the stimulus (e.g., air) while providing a precise read-out of pressure and stimulus signal markers. METHODS: In this study, we designed a timed pressure regulator [termed visceral pressure stimulator (VPS)] to control air flow, measure pressure (in mmHg), and send stimuli markers to online software. The device was built using a simple circuit and primarily off-the-shelf parts. A separate custom inline analog-to-digital pressure converter was used to validate the real pressure output of the VPS. RESULTS: Using commercial physiological software (Spike2, CED), we were able to measure mouse bladder pressure continuously during delivery of unique air stimulus trials in a mouse while simultaneously recording an electromyogram (EMG) of the overlying abdominal muscles. CONCLUSIONS: This device will be useful for those who need to (1) deliver distinct pressure stimuli while (2) measuring the pressure in real-time and (3) monitoring stimulus on-off using physiological software.

Integrated Meditation and Exercise Therapy: A Randomized Controlled Pilot of a Combined Nonpharmacological Intervention Focused on Reducing Disability and Pain in Patients with Chronic Low Back Pain.

OBJECTIVE: This pilot trial examined the effects of a combined intervention of mindfulness meditation followed by aerobic walking exercise compared with a control condition in chronic low back pain patients. We hypothesized that meditation before exercise would reduce disability, pain, and anxiety by increasing mindfulness prior to physical activity compared with an audiobook control group. PARTICIPANTS: Thirty-eight adults completed either meditation and exercise treatment (MedExT) (n=18) or an audiobook control condition (n=20). SETTING: Duquesne University Exercise Physiology Laboratory. DESIGN: A pilot, assessor-blinded, randomized controlled trial. METHODS: Over a 4-week period, participants in the MedExT group performed 12-17 minutes of guided meditation followed by 30 minutes of moderate-intensity walking exercise 5 days per week. Measures of disability, pain, mindfulness, and anxiety were taken at baseline and postintervention. Pain perception measurements were taken daily. RESULTS: Compared with the control group, we observed larger improvements in disability in the MedExT intervention, although the changes were modest and not statistically significant (mean between-group difference, -1.24; 95% confidence interval [CI], -3.1 to 0.6). For secondary outcome measures, MedExT increased mindfulness (within-group) from pre-intervention to postintervention (P=0.0141). Additionally, mean ratings of low back pain intensity and unpleasantness significantly improved with time for the MedExT group compared with that of the control group, respectively (intensity P=0.0008; unpleasantness P=0.0022). CONCLUSION: . Overall, 4 weeks of MedExT produced suggestive between-group trends for disability, significant between-group differences for measures of pain, and significant within-group increases in mindfulness.

Left and right hemispheric lateralization of the amygdala in pain.

Hemispheric asymmetries within the brain have been identified across taxa and have been extensively studied since the early 19th century. Here, we discuss lateralization of a brain structure, the amygdala, and how this lateralization is reshaping how we understand the role of the amygdala in pain processing. The amygdala is an almond-shaped, bilateral brain structure located within the limbic system. Historically, the amygdala was known to have a role in the processing of emotions and attaching emotional valence to memories and other experiences. The amygdala has been extensively studied in fear conditioning and affect but recently has been shown to have an important role in processing noxious information and impacting pain. The amygdala is composed of multiple nuclei; of special interest is the central nucleus of the amygdala (CeA). The CeA receives direct nociceptive inputs from the parabrachial nucleus (PBN) through the spino-parabrachio-amygdaloid pathway as well as more highly processed cortical and thalamic input via the lateral and basolateral amygdala. Although the amygdala is a bilateral brain region, most data investigating the amygdala's role in pain have been generated from the right CeA, which has an overwhelmingly pro-nociceptive function across pain models. The left CeA has often been characterized to have no effect on pain modulation, a dampened pro-nociceptive function, or most recently an anti-nociceptive function. This review explores the current literature on CeA lateralization and the hemispheres' respective roles in the processing and modulation of different forms of pain.

Framework for advancing rigorous research.

There is a pressing need to increase the rigor of research in the life and biomedical sciences. To address this issue, we propose that communities of 'rigor champions' be established to campaign for reforms of the research culture that has led to shortcomings in rigor. These communities of rigor champions would also assist in the development and adoption of a comprehensive educational platform that would teach the principles of rigorous science to researchers at all career stages.

The dosing of aerobic exercise therapy on experimentally-induced pain in healthy female participants.

Knowledge of efficacious dosing respective to exercise type and pain condition is extremely limited in the literature. This study aimed to determine the impact of dose of moderate intensity treadmill walking on experimentally-induced pain in healthy human participants. Forty females were divided into 4 groups: control (no exercise), low dose exercise (3×/wk), moderate dose exercise (5×/wk) or high dose exercise (10×/wk). Over a 7-day period, subjects performed treadmill walking during assigned exercise days. Both qualitative and quantitative measures of pain were measured at baseline, during the trial, and 24 hrs post-final intervention session via sensitivity thresholds to painful thermal and painful pressure stimulation. Significant effects of treatment were found post-intervention for constant pressure pain intensity (p = 0.0016) and pain unpleasantness ratings (p = 0.0014). Post-hoc tests revealed significant differences between control and moderate and control and high dose groups for constant pressure pain intensity (p = 0.0015), (p = 0.0094), respectively and constant pressure pain unpleasantness (p = 0.0040), (p = 0.0040), respectively. Moderate and high dose groups had the greatest reductions in ratings of pain, suggesting that our lowest dose of exercise was not sufficient to reduce pain and that the moderate dose of exercise may be a sufficient starting dose for exercise-based adjuvant pain therapy.

Correction: Lipoprotein Receptor LRP1 Regulates Leptin Signaling and Energy Homeostasis in the Adult Central Nervous System.

[This corrects the article DOI: 10.1371/journal.pbio.1000575.].

Exercise-induced hypoalgesia: A meta-analysis of exercise dosing for the treatment of chronic pain.

OBJECTIVE: Increasing evidence purports exercise as a first-line therapeutic for the treatment of nearly all forms of chronic pain. However, knowledge of efficacious dosing respective to treatment modality and pain condition is virtually absent in the literature. The purpose of this analysis was to calculate the extent to which exercise treatment shows dose-dependent effects similar to what is seen with pharmacological treatments. METHODS: A recently published comprehensive review of exercise and physical activity for chronic pain in adults was identified in May 2017. This report reviewed different physical activity and exercise interventions and their effectiveness in reducing pain severity and found overall modest effects of exercise in the treatment of pain. We analyzed this existing data set, focusing specifically on the dose of exercise intervention in these studies. We re-analyzed data from 75 studies looking at benefits of time of exercising per week, frequency of exercise per week, duration of intervention (in weeks), and estimated intensity of exercise. RESULTS: Analysis revealed a significant positive correlation with exercise duration and analgesic effect on neck pain. Multiple linear regression modeling of these data predicted that increasing the frequency of exercise sessions per week is most likely to have a positive effect on chronic pain patients. DISCUSSION: Modest effects were observed with one significant correlation between duration and pain effect for neck pain. Overall, these results provide insufficient evidence to conclude the presence of a strong dose effect of exercise in pain, but our modeling data provide tes predictions that can be used to design future studies to explicitly test the question of dose in specific patient populations.

Cyanobacterial extract with serotonin receptor subtype 7 (5-HT7 R) affinity modulates depression and anxiety-like behavior in mice.

Marine cyanobacteria represent a unique source in the field of drug discovery due to the secondary metabolites they produce and the structural similarity these compounds have to endogenous mammalian receptor ligands. A series of cyanobacteria were subjected to extraction, fractionation by column chromatography and screened for affinity against CNS targets with a focus on serotonin receptors (5-HTRs). Out of 276 fractions screened, 21% had activity at 5-HTRs and/or the 5-HT transporter (SERT). One sample, a cyanobacterium identified by 16S rRNA sequencing as Leptolyngbya from Las Perlas archipelago in Panama, contained a fraction with noted affinity for the 5-HT7 receptor (5-HT7 R). This fraction (DUQ0002I) was screened via intracerebroventricular (ICV) injections in mice using depression and anxiety assays including the forced swim, tail suspension, elevated zero maze, and light-dark preference tests. DUQ0002I decreased depression and anxiety-like behaviors in males and did not have effects in 5-HT7 R knockout or female mice. Administration of DUQ0002I to the CA1 of the hippocampus induced antidepression-like, but not anxiolytic-like behaviors. Testing of further purified materials showed no behavioral effects, leading us to hypothesize that the behavioral effects are likely caused by a synergistic effect between multiple compounds in the fraction. Finally, DUQ0002I was used in a model of neuropathic pain with comorbid depression (spared nerve injury-SNI). DUQ0002I had a similar antidepressant effect in animals with SNI, suggesting a role for the 5-HT7 R in the development of comorbid pain and depression. These results demonstrate the potential that cyanobacterial metabolites have in the field of neuropharmacognosy.

Central amygdala activation of extracellular signal-regulated kinase 1 and age-dependent changes in inflammatory pain sensitivity in mice.

Aging populations are more sensitive to noxious stimuli as a result of altered somatosensory systems. In these experiments, we examined pain-like behaviors in young, middle-aged, and old mice during peripheral inflammation to determine if the same sensitivity exists in preclinical animal models. Immediately following injury, middle-aged and old mice exhibited more spontaneous pain-like behaviors than young mice, matching pain prevalence in clinical populations. Middle-aged and old mice also developed persistent mechanical hypersensitivity in the injured paw. Furthermore, old mice developed mechanical hypersensitivity in the noninjured paw suggesting age-dependent changes in central nociceptive systems. To address this end, pain-related protein expression was examined in the central nucleus of the amygdala, a limbic brain region that modulates somatic pain. Following injury, increased phosphorylation of extracellular signal-regulated kinase 1, a protein with known nociceptive functions, was observed in the right central nucleus of the amygdala of old mice and not middle-aged or young animals. These findings suggest that age-dependent changes in supraspinal nociceptive systems may account for increased pain-like behaviors in aging populations.