Mark H. Pitcher

PKMζ is essential for spinal plasticity underlying the maintenance of persistent pain

BackgroundChronic pain occurs when normally protective acute pain becomes pathologically persistent. We examined here whether an isoform of protein kinase C (PKC), PKMζ, that underlies long-term memory storage in various brain regions, also sustains nociceptive plasticity in spinal cord dorsal horn (SCDH) mediating persistent pain.ResultsCutaneous injury or spinal stimulation produced persistent increases of PKMζ, but not other atypical PKCs in SCDH. Inhibiting spinal PKMζ, but not full-length PKCs, reversed plasticity-dependent persistent painful responses to hind paw formalin and secondary mechanical hypersensitivity and SCDH neuron sensitization after hind paw capsaicin, without affecting peripheral sensitization-dependent primary heat hypersensitivity after hind paw capsaicin. Inhibiting spinal PKMζ, but not full-length PKCs, also reversed mechanical hypersensitivity in the rat hind paw induced by spinal stimulation with intrathecal dihydroxyphenylglycine. Spinal PKMζ inhibition also alleviated allodynia 3 weeks after ischemic injury in rats with chronic post-ischemia pain (CPIP), at a point when allodynia depends on spinal changes. In contrast, spinal PKMζ inhibition did not affect allodynia in rats with chronic contriction injury (CCI) of the sciatic nerve, or CPIP rats early after ischemic injury, when allodynia depends on ongoing peripheral inputs.ConclusionsThese results suggest spinal PKMζ is essential for the maintenance of persistent pain by sustaining spinal nociceptive plasticity.

Spinal NKCC1 blockade inhibits TRPV1-dependent referred allodynia

BackgroundThe Na+, K+, 2Cl- type I cotransporter (NKCC1) and TRPV1 receptors, at the level of the dorsal horn, have been implicated in mediating allodynia in response to an inflammatory insult. The NKCC1 cotransporter regulates intracellular [Cl-] and thus the magnitude and polarity of GABAA receptor responses in neurons. TRPV1 receptors transduce diverse chemical and natural stimuli in nociceptors and are critical for inflammatory hyperalgesia.ResultsHere we have tested the role of spinal NKCC1 cotransporters and TRPV1 receptors in referred allodynia in a model of visceral hyperalgesia in mice. Intrathecal (IT) injection of the NKCC1 inhibitor bumetanide (BUM, 1 nmol) inhibited referred, abdominal allodynia evoked by an intracolonic capsaicin injection. BUM was effective when injected IT either before or up to 4 hrs after the establishment of referred allodynia. The TRPV1 antagonist AMG 9810 (1 nmol) also inhibited referred allodynia in this model suggesting the involvement of an endogenous TRPV1 agonist in the dorsal horn in referred allodynia. In support of this suggestion, the endovanilloid TRPV1 agonist, narachidonoyl- dopamine (NADA, 1 or 10 nmol, IT) evoked stroking allodynia in the hindpaw that was blocked by co-treatment with AMG 9810 (1 nmol). The TRPV1-dependent stroking allodynia caused by NADA appeared to be functionally linked to NKCC1 because BUM (1 nmol) also inhibited NADA-evoked stroking allodynia.ConclusionOur findings indicate that spinal NKCC1 and TRPV1 are critical for referred allodynia mediated by a painful visceral stimulus. Moreover, they suggest that endogenous TRPV1 agonists, released in the CNS in painful conditions, might stimulate TRPV1 receptors on primary afferents that, in turn, play a role in increasing NKCC1 activity leading to allodynia.

Effects of inflammation on the ultrastructural localization of spinal cord dorsal horn group I metabotropic glutamate receptors

Inflammatory pain is thought to induce functional plasticity of spinal dorsal horn neurons and may produce changes in glutamate receptor expression. Plasticity of group I metabotropic glutamate receptors (mGluR1 and mGluR5) is important in various neuronal systems, and these receptors are also known to modulate nociceptive neurotransmission in the spinal dorsal horn. The present study aimed at determining whether persistent inflammatory pain produces alterations in intracellular and plasma membrane‐associated mGluR1α and mGluR5 in spinal cord dorsal horn. Persistent inflammation was induced in male Long Evans rats by a unilateral intraplantar injection of 100 μL of complete Freunds adjuvant (CFA). Three days after the CFA injection thermal withdrawal latencies were obtained prior to processing of transverse spinal cord sections for preembedding immunogold labeling after incubation in primary antibody for mGluR1α or mGluR5. Using electron microscopy, we quantified immunogold‐labeled mGluR1α and mGluR5 profiles, located in lamina V and I–II, respectively, of both CFA‐treated rats and untreated control rats. Compared to untreated rats, CFA‐treated rats had a significant increase in the number of plasma membrane‐associated mGluR5 immunogold‐labeled particles in lamina I–II neurons of the spinal cord. Although no changes to mGluR1α expression were found in CFA‐treated rats, plasma membrane‐associated mGluR1α was significantly closer to the synapse. Therefore, in CFA‐treated rats there was a specific increase in the ratio of plasma membrane‐associated versus intracellular immunogold‐labeled particles for mGluR5, and lateral movement of mGluR1α toward the synapse, indicating that peripheral inflammation‐induced trafficking of group I mGluRs in spinal dorsal horn neurons may be an important factor in the development of plastic changes associated with inflammation‐induced chronic pain. J. Comp. Neurol. 505:412–423, 2007.

Role of the NKCC1 co-transporter in sensitization of spinal nociceptive neurons.

&NA; The Na+, K+, 2Cl− co‐transporter type 1 (NKCC1) plays a pivotal role in hyperalgesia associated with inflammatory stimuli. NKCC1 contributes to maintain high [Cl−]i in dorsal root ganglia (DRG) neurons which cause primary afferent depolarization (PAD) when GABAA receptors are activated. Enhanced GABA‐induced depolarization, through increased NKCC1 activity, has been hypothesized to produce orthodromic spike activity of sufficient intensity to account for touch‐induced pain. In the present study, we investigate this hypothesis using in vivo electrophysiology on rat dorsal horn neurons; the effects of spinal blockade of NKCC1 on intraplantar capsaicin‐induced sensitization of dorsal horn neurons were examined. Single wide dynamic range (WDR) and nociceptive specific (NS) neuron activity in the dorsal horn was recorded using glass microelectrodes in anesthetized rats. Dorsal horn neurons with a receptive field on the plantar surface of the hindpaw were studied. Neuronal responses to mechanical stimuli (brush, von Frey filaments) were recorded ten minutes before intraplantar injection of 0.3 ml 0.1% capsaicin (CAP), 40 min after CAP and 15 min after local application of the NKCC1 blocker bumetanide (BTD; 500 &mgr;M) on the spinal cord. After CAP, low and high threshold stimulation of the cutaneous receptive field produced a significant enhancement in spike frequency over pre‐CAP values in both WDR and NS neurons. Spinal BTD application reduced the spike frequency to baseline levels as well as attenuated the CAP‐induced increases in background activity. Our data support the hypothesis that NKCC1 plays an important role in the sensitization of dorsal horn neurons following a peripheral inflammatory insult.

Effect of environment on the long-term consequences of chronic pain.

Abstract Much evidence from pain patients and animal models shows that chronic pain does not exist in a vacuum but has varied comorbidities and far-reaching consequences. Patients with long-term pain often develop anxiety and depression and can manifest changes in cognitive functioning, particularly with working memory. Longitudinal studies in rodent models also show the development of anxiety-like behavior and cognitive changes weeks to months after an injury causing long-term pain. Brain imaging studies in pain patients and rodent models find that chronic pain is associated with anatomical and functional alterations in the brain. Nevertheless, studies in humans reveal that lifestyle choices, such as the practice of meditation or yoga, can reduce pain perception and have the opposite effect on the brain as does chronic pain. In rodent models, studies show that physical activity and a socially enriched environment reduce pain behavior and normalize brain function. Together, these studies suggest that the burden of chronic pain can be reduced by nonpharmacological interventions.

Translational control of nociception via 4E-binding protein 1

Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception. DOI: http://dx.doi.org/10.7554/eLife.12002.001

Restraint training for awake functional brain scanning of rodents can cause long-lasting changes in pain and stress responses.

Abstract With the increased interest in longitudinal brain imaging of awake rodents, it is important to understand both the short-term and long-term effects of restraint on sensory and emotional processing in the brain. To understand the effects of repeated restraint on pain behaviors and stress responses, we modeled a restraint protocol similar to those used to habituate rodents for magnetic resonance imaging scanning, and studied sensory sensitivity and stress hormone responses over 5 days. To uncover lasting effects of training, we also looked at responses to the formalin pain test 2 weeks later. We found that while restraint causes acute increases in the stress hormone corticosterone, it can also cause lasting reductions in nociceptive behavior in the formalin test, coupled with heightened corticosterone levels and increased activation of the “nociceptive” central nucleus of the amygdala, as seen by Fos protein expression. These results suggest that short-term repeated restraint, similar to that used to habituate rats for awake functional brain scanning, could potentially cause long-lasting changes in physiological and brain responses to pain stimuli that are stress-related, and therefore could potentially confound the functional activation patterns seen in awake rodents in response to pain stimuli.

Modest Amounts of Voluntary Exercise Reduce Pain- and Stress-Related Outcomes in a Rat Model of Persistent Hind Limb Inflammation

Aerobic exercise improves outcomes in a variety of chronic health conditions, yet the support for exercise-induced effects on chronic pain in humans is mixed. Although many rodent studies have examined the effects of exercise on persistent hypersensitivity, the most used forced exercise paradigms that are known to be highly stressful. Because stress can also produce analgesic effects, we studied how voluntary exercise, known to reduce stress in healthy subjects, alters hypersensitivity, stress, and swelling in a rat model of persistent hind paw inflammation. Our data indicate that voluntary exercise rapidly and effectively reduces hypersensitivity as well as stress-related outcomes without altering swelling. Moreover, the level of exercise is unrelated to the analgesic and stress-reducing effects, suggesting that even modest amounts of exercise may impart significant benefit in persistent inflammatory pain states. PERSPECTIVE Modest levels of voluntary exercise reduce pain- and stress-related outcomes in a rat model of persistent inflammatory pain, independently of the amount of exercise. As such, consistent, self-regulated activity levels may be more relevant to health improvement in persistent pain states than standardized exercise goals.

eIF2α phosphorylation controls thermal nociception

Significance Distinct cellular stresses converge on the translation initiation factor, eukaryotic initiation factor 2α (eIF2α) to modulate the rate of protein synthesis. Increased phosphorylation of eIF2α has been described in peripheral neurons from neuropathic and diabetic rats. However, the role of eIF2α phosphorylation in pain has not been reported. Here we show that phosphorylation of eIF2α controls thermal, but not mechanical, sensation via modulation of the activity of a major heat transducer, transient receptor potential vanilloid 1. We also find that chronic inflammation-induced eIF2α phopshorylation contributes to inflammation-induced thermal hypersensitivity. These results demonstrate that eIF2α phosphorylation plays a major role in controlling noxious heat sensitivity. A response to environmental stress is critical to alleviate cellular injury and maintain cellular homeostasis. Eukaryotic initiation factor 2 (eIF2) is a key integrator of cellular stress responses and an important regulator of mRNA translation. Diverse stress signals lead to the phosphorylation of the α subunit of eIF2 (Ser51), resulting in inhibition of global protein synthesis while promoting expression of proteins that mediate cell adaptation to stress. Here we report that eIF2α is instrumental in the control of noxious heat sensation. Mice with decreased eIF2α phosphorylation (eIF2α+/S51A) exhibit reduced responses to noxious heat. Pharmacological attenuation of eIF2α phosphorylation decreases thermal, but not mechanical, pain sensitivity, whereas increasing eIF2α phosphorylation has the opposite effect on thermal nociception. The impact of eIF2α phosphorylation (p-eIF2α) on thermal thresholds is dependent on the transient receptor potential vanilloid 1. Moreover, we show that induction of eIF2α phosphorylation in primary sensory neurons in a chronic inflammation pain model contributes to thermal hypersensitivity. Our results demonstrate that the cellular stress response pathway, mediated via p-eIF2α, represents a mechanism that could be used to alleviate pathological heat sensation.

Stimulation of Cutaneous Low Threshold Mechanoreceptors in Mice After Intracolonic Capsaicin Increases Spinal c-Fos Labeling in an NKCC1-Dependent Fashion

UNLABELLED Stimulation of peripheral nociceptors results in increased c-Fos labeling in spinal cord regions associated with nociceptive processing. Accordingly, intracolonic capsaicin, which generates robust secondary (referred) allodynia on the abdomen of mice, also causes an increased spinal c-Fos labeling. In naïve rodents, low intensity innocuous stimulation does not affect c-Fos labeling in spinal nociceptive regions. However, after persistent noxious input, low intensity stimulation of the inflamed region further enhances c-Fos labeling, suggesting that low threshold mechanosensitive fibers gain access to the nociceptive channel after persistent inflammation. We have previously proposed that afferent activity in low threshold sensory fibers activates nociceptive sensory fibers through Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) -mediated enhanced primary afferent depolarization. Here, we show that intracolonic capsaicin enhances spinal c-Fos labeling and secondary allodynia in an NKCC1-dependent manner. Furthermore, we demonstrate that gently brushing the abdomen, the region of secondary allodynia, further increased spinal c-Fos levels, an effect that can be prevented by spinal NKCC1 blockade. These findings provide evidence that increased NKCC1 activity contributes to secondary allodynia and that innocuous touch can access the nociceptive channel in part through enhanced NKCC1 activity. PERSPECTIVE While touch normally soothes acute pain, we demonstrate that following peripheral inflammation, touch evokes pain (allodynia) through the switching of a normally inhibitory spinal pathway into an excitatory pathway. Activation of low threshold mechanoreceptors activates spinal nociceptive neurons following inflammation-induced enhancement of NKCC1 expression, as measured by spinal c-Fos labeling.