Activity Of Dorsal Horn Neurons Research Articles

Amitriptyline and duloxetine attenuate activities of superficial dorsal horn neurons in a rat reserpine-induced fibromyalgia model

Fibromyalgia (FM) is an intractable disease with a chief complaint of chronic widespread pain. Amitriptyline (AMI) and duloxetine (DLX), which are antidepressant drugs, have been reported to ameliorate pain in patients with FM and pain-related behaviors in several rodent models of FM. However, the mechanisms of action of AMI and DLX are not yet fully understood. Here, we examined the effects of these drugs on the responsiveness of superficial dorsal horn (SDH) neurons in the spinal cord, using a rat FM model developed by injecting a biogenic amine depleter (reserpine). Extracellular recordings of SDH neurons in vivo demonstrated that bath application of AMI and DLX at concentrations of 0.1–1.0 mM on the dorsal surface of the spinal cord markedly suppressed spontaneous discharge and von Frey filament-evoked mechanical firing in SDH neurons. The suppression induced by the drugs was noted in a concentration-dependent manner and the suppressive effects resolved after washing the spinal cord surface. These results show that SDH neurons are the site of action for AMI and DLX in a rat reserpine-induced FM model. Spinal mechanisms may underlie the therapeutic effects of these drugs in patients with FM.

Tiam1-mediated maladaptive plasticity underlying morphine tolerance and hyperalgesia.

Opioid pain medications, such as morphine, remain the mainstay for treating severe and chronic pain. Prolonged morphine use, however, triggers analgesic tolerance and hyperalgesia (OIH), which can last for a long period after morphine withdrawal. How morphine induces these detrimental side effects remains unclear. Here, we show that morphine tolerance and OIH are mediated by Tiam1-coordinated synaptic structural and functional plasticity in the spinal nociceptive network. Tiam1 is a Rac1 GTPase guanine nucleotide exchange factor that promotes excitatory synaptogenesis by modulating actin cytoskeletal dynamics. We found that prolonged morphine treatment activated Tiam1 in the spinal dorsal horn and Tiam1 ablation from spinal neurons eliminated morphine antinociceptive tolerance and OIH. At the same time, the pharmacological blockade of Tiam1-Rac1 signalling prevented the development and reserved the established tolerance and OIH. Prolonged morphine treatment increased dendritic spine density and synaptic NMDA receptor activity in spinal dorsal horn neurons, both of which required Tiam1. Furthermore, co-administration of the Tiam1 signalling inhibitor NSC23766 was sufficient to abrogate morphine tolerance in chronic pain management. These findings identify Tiam1-mediated maladaptive plasticity in the spinal nociceptive network as an underlying cause for the development and maintenance of morphine tolerance and OIH and provide a promising therapeutic target to reduce tolerance and prolong morphine use in chronic pain management.

Emerging Molecular and Synaptic Targets for the Management of Chronic Pain Caused by Systemic Lupus Erythematosus.

Patients with systemic lupus erythematosus (SLE) frequently experience chronic pain due to the limited effectiveness and safety profiles of current analgesics. Understanding the molecular and synaptic mechanisms underlying abnormal neuronal activation along the pain signaling pathway is essential for developing new analgesics to address SLE-induced chronic pain. Recent studies, including those conducted by our team and others using the SLE animal model (MRL/lpr lupus-prone mice), have unveiled heightened excitability in nociceptive primary sensory neurons within the dorsal root ganglia and increased glutamatergic synaptic activity in spinal dorsal horn neurons, contributing to the development of chronic pain in mice with SLE. Nociceptive primary sensory neurons in lupus animals exhibit elevated resting membrane potentials, and reduced thresholds and rheobases of action potentials. These changes coincide with the elevated production of TNFα and IL-1β, as well as increased ERK activity in the dorsal root ganglion, coupled with decreased AMPK activity in the same region. Dysregulated AMPK activity is linked to heightened excitability in nociceptive sensory neurons in lupus animals. Additionally, the increased glutamatergic synaptic activity in the spinal dorsal horn in lupus mice with chronic pain is characterized by enhanced presynaptic glutamate release and postsynaptic AMPA receptor activation, alongside the reduced activity of glial glutamate transporters. These alterations are caused by the elevated activities of IL-1β, IL-18, CSF-1, and thrombin, and reduced AMPK activities in the dorsal horn. Furthermore, the pharmacological activation of spinal GPR109A receptors in microglia in lupus mice suppresses chronic pain by inhibiting p38 MAPK activity and the production of both IL-1β and IL-18, as well as reducing glutamatergic synaptic activity in the spinal dorsal horn. These findings collectively unveil crucial signaling molecular and synaptic targets for modulating abnormal neuronal activation in both the periphery and spinal dorsal horn, offering insights into the development of analgesics for managing SLE-induced chronic pain.

Optogenetic inhibition of spinal inhibitory neurons facilitates mechanical responses of spinal wide dynamic range neurons and causes mechanical hypersensitivity

Inhibitory interneurons in the spinal dorsal horn (DH) play a major role in regulating innocuous and noxious information. Reduction in inhibitory synaptic transmission is thought to contribute to the development of touch-evoked pain (allodynia), a common symptom of neuropathic pain. However, it is not fully understood how inhibitory neurons in the DH regulate sensory responses in surrounding neurons and modulate sensory transmission. In this study, we established a novel experimental method to analyze temporal activity of DH neurons during the optogenetically induced disinhibition state by combining extracellular recording and optogenetics. We investigated how specific and temporally restricted dysfunction of DH inhibitory neurons affected spinal neuronal activities evoked by cutaneous mechanical stimulation. In behavioral experiments, the specific and temporally restricted spinal optogenetic suppression of DH inhibitory neurons induced mechanical hypersensitivity. Furthermore, this manipulation enhanced the mechanical responses of wide dynamic range (WDR) neurons, which are important for pain transmission, in response to brush and von Frey stimulation but not in response to nociceptive pinch stimulation. In addition, we examined whether a neuropathic pain medication, mirogabalin, suppressed these optogenetically induced abnormal pain responses. We found that mirogabalin treatment attenuated the abnormal firing responses of WDR neurons and mechanical hypersensitivity. These results suggest that temporally restricted and specific reduction of spinal inhibitory neuronal activity facilitates the mechanical responses of WDR neurons, resulting in neuropathic-like mechanical allodynia which can be suppressed by mirogabalin. Our optogenetic methods could be useful for developing novel therapeutics for neuropathic pain.

TNFα Enhances Calcium Influx by Interacting with AMPA Receptors in the Spinal Dorsal Horn Neurons.

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine and has been implicated in pain regulation. Neuronal activity in the spinal dorsal horn contributes to nociceptive transmission. However, it is not fully understood how TNFα affects the activity of spinal dorsal horn neurons. In the present study, we used calcium imaging to characterize the mechanism by which TNFα regulates calcium influx in the cultured spinal dorsal horn neurons. We observed thatTNFα incubation caused an increase in fluorescent intensity of Fura-2, a specific intracellular calcium indicator, in the cultured spinal dorsal horn neurons, and such effect was significantly inhibited by co-incubation with R7050, a selective TNFα receptor antagonist, which suggests thatTNFα can enhance calcium influx and increase neuronal activity via activating TNFα receptors in the spinal dorsal horn. Using double immunofluorescence staining, we showed that TNFα receptors were co-expressed with a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor subunit GluA1 in the spinal dorsal horn neurons. We further observed that treatment with 1-naphthyl acetyl spermine (NASPM), a specific calcium-permeable AMPA receptor blocker, completely blocked the effect of TNFα incubation on calcium influx in the cultured neurons. Moreover, lipopolysaccharide (LPS)-induced calcium influx was inhibited by co-incubation with R7050. Together, our results suggest thatTNFα in the spinal dorsal horn can increase calcium-indicated neuronal activity through the interaction between its receptor and calcium-permeable AMPA receptors.

Surround Inhibition Mediates Pain Relief by Low Amplitude Spinal Cord Stimulation: Modeling and Measurement.

Low-frequency (<200 Hz), subperception spinal cord stimulation (SCS) is a novel modality demonstrating therapeutic efficacy for treating chronic neuropathic pain. When stimulation parameters were carefully titrated, patients experienced rapid onset (seconds–minutes) pain relief without paresthesia, but the mechanisms of action are unknown. Using an integrated computational model and in vivo measurements in urethane-anesthetized rats, we quantified how stimulation parameters (placement, pulse width, frequency, and amplitude) influenced dorsal column (DC) axon activation and neural responses in the dorsal horn (DH). Both modeled and recorded DC axons responded with irregular spiking patterns in response to low-amplitude SCS. Maximum inhibition of DH neurons occurred at ∼80% of the predicted sensory threshold in both modeled and recorded neurons, and responses were strongly dependent on spatially targeting of stimulation, i.e., the complement of DC axons activated, and on stimulation parameters. Intrathecal administration of bicuculline shifted neural responses to low-amplitude stimulation in both the model and experiment, suggesting that analgesia is dependent on segmental GABAergic mechanisms. Our results support the hypothesis that low-frequency subperception SCS generates rapid analgesia by activating a small number of DC axons which inhibit DH neuron activity via surround inhibition.

Evaluating optimized temporal patterns of spinal cord stimulation (SCS)

BackgroundTemporal patterns of stimulation represent a novel dimension for improving the efficacy of spinal cord stimulation to treat chronic neuropathic pain. ObjectiveWe hypothesized that nonregular temporal patterns of stimulation designed using a computational model would be superior to conventional stimulation at constant frequencies or completely random patterns of stimulation. MethodsUsing a computational model of the dorsal horn network and an optimization algorithm based on biological evolution, we designed an optimized pattern of spinal cord stimulation with comparable efficacy and increased efficiency relative to constant frequency (CF) stimulation. We evaluated the effect of different temporal patterns on individual neurons recorded in the dorsal horn of urethane-anesthetized rats. ResultsThe optimized pattern and 50 Hz CF stimulation produced greater inhibition of spontaneously firing neurons recorded in vivo than random 50 Hz stimulation or a pattern designed intentionally with poor fitness. Spinal Cord Stimulation (SCS) led to significant changes in the firing patterns of recorded units, and stimulation patterns that generated significant inhibition also tended to reduce entropy and regularize the firing patterns of units, suggesting that patterns of dorsal horn neuron activity may be important for pain perception in addition to the firing rate. ConclusionsThese results demonstrate that the computational model can be used as a tool for optimizing stimulation parameters and suggest that optimized temporal patterns may increase the efficacy of spinal cord stimulation.

Spinal cord stimulation mitigates the myocardial ischemia induced sympathoexcitation by suppressing the spinal neural synchrony and intermediolateral nucleus hyperactivity

BackgroundMyocardial ischemia disrupts the cardiospinal neural network which leads to cardiac sudden death. Spinal cord stimulation (SCS) suppresses the sympathoexcitation caused by myocardial ischemia. However, how SCS modulates the spinal neural network is not known.HypothesisWe hypothesize that SCS decreases myocardial ischemia‐induced sympathoexcitation by suppressing the spinal neural synchrony and intermediolateral nucleus (IML) activity.Materials and MethodsEight Yorkshire pigs (50‐55 kg) with chronic left circumflex coronary artery myocardial infarction (MI) were anesthetized and underwent laminectomy and a sternotomy, 5 weeks post MI. Extracellular in‐vivo spinal neural recordings were done by using a 64 channel microelectrode array inserted at the T2 segment of the spinal cord. SCS was performed at 1 kHz, 0.03 ms, 90% motor threshold for 30 mins. Acute (3 min) left anterior descending coronary artery occlusion (LAD CAO) was performed pre‐ and 1 min post‐SCS to evaluate the impact of SCS on spinal neural network processing during myocardial ischemia. Dorsal horn (DH) and IML neural interactions, including normalized neuronal synchrony, activation recovery interval (ARI), and dispersion of repolarization (DOR) (markers of cardiac arrhythmogenesis) were evaluated during myocardial ischemia pre‐ vs. post‐ SCS.ResultsFiring rate of 1283 recorded spinal neurons increased during 3 mins LAD CAO and 1 min reperfusion pre‐SCS (baseline(BL): 0.516 ± 0.038 Hz; LAD CAO: 0.559 ± 0.038 Hz ,P&lt;0.001 vs. BL; reperfusion: 0.524 ± 0.037 Hz ,P&lt;0.001 vs. BL). SCS at 1kHz, mitigated the spinal neural network response to the LAD CAO (BL: 0.509 ± 0.039 Hz; LAD CAO: 0.486 ± 0.036 Hz; reperfusion: 0.489 ± 0.038 Hz, P&lt;0.001 vs. BL). This suppression of neural firing by SCS was observed in both IML (pre‐SCS: BL: 0.208 ± 0.033 Hz; LAD CAO: 0.308 ± 0.042Hz ,P&lt;0.001 vs. BL; reperfusion: 0.374 ± 0.048 Hz ,P&lt;0.001 vs. BL; post‐SCS: BL: 0.069 ± 0.010 Hz; LAD CAO: 0.080 ± 0.011Hz; reperfusion: 0.109 ± 0.018 Hz) and DH neurons (pre‐SCS: BL: 0.033 ± 0.003 Hz; LAD CAO: 0.039 ± 0.003 Hz ,P&lt;0.001 vs. BL; reperfusion: 0.041 ± 0.003 Hz ,P&lt;0.001 vs. BL; post‐SCS: BL: 0.036 ± 0.003 Hz; LAD CAO: 0.035 ± 0.003 Hz; reperfusion: 0.029 ± 0.002 Hz, P&lt;0.001 vs. BL). ARI shortening due to LAD CAO was mitigated by SCS (pre‐SCS: ‐43.200 ± 5.206 ms; post‐SCS: ‐29.810 ± 6.495 ms, P&lt; 0.05 vs. pre‐SCS). SCS decreased the LAD CAO induced augmented DOR (pre‐SCS: 2461.00 ± 350.40 ms2; post‐SCS: 1996.00 ± 302.00 ms2, P&lt;0.05 vs. pre‐SCS)ConclusionMyocardial ischemia induces sympathoexcitation by increasing the IML activity which is caused by activation of DH neurons and increased spinal neural synchrony. SCS mitigates the adverse effect of myocardial ischemia by suppressing the spinal neural synchrony and IML hyperactivity.

Pain Modulation from the Locus Coeruleus in a Model of Hydrocephalus: Searching for Oxidative Stress-Induced Noradrenergic Neuroprotection.

Pain transmission at the spinal cord is modulated by noradrenaline (NA)-mediated actions that arise from supraspinal areas. We studied the locus coeruleus (LC) to evaluate the expression of the cathecolamine-synthetizing enzyme tyrosine hydroxylase (TH) and search for local oxidative stress and possible consequences in descending pain modulation in a model of hydrocephalus, a disease characterized by enlargement of the cerebral ventricular system usually due to the obstruction of cerebrospinal fluid flow. Four weeks after kaolin injection into the cisterna magna, immunodetection of the catecholamine-synthetizing enzymes TH and dopamine-β-hydroxylase (DBH) was performed in the LC and spinal cord. Colocalization of the oxidative stress marker 8-OHdG (8-hydroxyguanosine; 8-OHdG), with TH in the LC was performed. Formalin was injected in the hindpaw both for behavioral nociceptive evaluation and the immunodetection of Fos expression in the spinal cord. Hydrocephalic rats presented with a higher expression of TH at the LC, of TH and DBH at the spinal dorsal horn along with decreased nociceptive behavioral responses in the second (inflammatory) phase of the formalin test, and formalin-evoked Fos expression at the spinal dorsal horn. The expression of 8-OHdG was increased in the LC neurons, with higher co-localization in TH-immunoreactive neurons. Collectively, the results indicate increased noradrenergic expression at the LC during hydrocephalus. The strong oxidative stress damage at the LC neurons may lead to local neuroprotective-mediated increases in NA levels. The increased expression of catecholamine-synthetizing enzymes along with the decreased nociception-induced neuronal activation of dorsal horn neurons and behavioral pain signs may indicate that hydrocephalus is associated with alterations in descending pain modulation.

Hypoxia-induced carbonic anhydrase mediated dorsal horn neuron activation and induction of neuropathic pain.

Neuropathic pain, such as that seen in diabetes mellitus, results in part from central sensitisation in the dorsal horn. However, the mechanisms responsible for such sensitisation remain unclear. There is evidence that disturbances in the integrity of the spinal vascular network can be causative factors in the development of neuropathic pain. Here we show that reduced blood flow and vascularity of the dorsal horn leads to the onset of neuropathic pain. Using rodent models (type 1 diabetes and an inducible endothelial-specific vascular endothelial growth factor receptor 2 knockout mouse) that result in degeneration of the endothelium in the dorsal horn, we show that spinal cord vasculopathy results in nociceptive behavioural hypersensitivity. This also results in increased hypoxia in dorsal horn neurons, depicted by increased expression of hypoxia markers such as hypoxia inducible factor 1α, glucose transporter 3, and carbonic anhydrase 7. Furthermore, inducing hypoxia through intrathecal delivery of dimethyloxalylglycine leads to the activation of dorsal horn neurons as well as mechanical and thermal hypersensitivity. This shows that hypoxic signalling induced by reduced vascularity results in increased hypersensitivity and pain. Inhibition of carbonic anhydrase activity, through intraperitoneal injection of acetazolamide, inhibited hypoxia-induced pain behaviours. This investigation demonstrates that induction of a hypoxic microenvironment in the dorsal horn, as occurs in diabetes, is an integral process by which neurons are activated to initiate neuropathic pain states. This leads to the conjecture that reversing hypoxia by improving spinal cord microvascular blood flow could reverse or prevent neuropathic pain.

A novel cell tool for α2δ-1-NMDAR target-based analgesic drug discovery

The α2δ-1 protein coded by Cacna2d1 is dramatically up-regulated in dorsal root ganglion (DRG) neurons and spinal dorsal horn following sensory nerve injury in various animal models of neuropathic pain. Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. The α2δ-1-NMDAR interaction promotes surface trafficking and synaptic targeting of NMDARs in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury, as well as in other pathological conditions such as in the paraventricular nucleus (PVN) with neurogenic hypertension and in the brain with ischemic stroke. The lentiviral transfection method was used to construct a human embryonic kidney HEK293T cell line that could stably express α2δ-1-NMDAR complex. A stably transfected cell line was observed by florescence microscope, and identified by RT-qPCR and Western blotting. The results showed that the HEK293T cell line was successfully transfected and the genes could be stably expressed. Subsequently, the transfected cell line was successfully developed into a target drug screening system using patch clamp techniques. It provides a promising cell model for further research on the interaction mechanism of α2δ-1-NMDAR complex and drug screening for chronic pain and related diseases with low side effects.

Discrete field potentials produced by coherent activation of spinal dorsal horn neurons.

In addition to the action potentials generated by the ongoing activation of single dorsal horn neurons in the anesthetized cat, we often recorded small negative field potentials with a fast-rising phase and a slow decay (dIFPs). These potentials could be separated in different classes, each with a specific and rather constant shape and amplitude. They were largest in spinal laminae III-V and gradually faded at deeper locations, without showing the polarity reversal displayed at these depths by the focal potentials produced by stimulation of muscle and cutaneous afferents. We propose that the dIFPs are postsynaptic field potentials generated by strongly coupled sets of dorsal horn neurons displaying a spatial orientation that generates closed field potentials in response to stimulation of high-threshold cutaneous and muscle afferents. These neuronal sets could form part of the spinal inhibitory circuitry that mediates presynaptic inhibition and Ib non-reciprocal postsynaptic inhibition and could be involved in the sensory-motor transformations activated by stimulation of high-threshold cutaneous afferents.

Toward rebalancing blood pressure instability after spinal cord injury with spinal cord electrical stimulation: A mini review and critique of the evolving literature.

High-level spinal cord injury commonly leads to blood pressure instability. This manifests clinically as orthostatic hypotension (OH), where blood pressure can drop to the point of loss of consciousness, and autonomic dysreflexia (AD), where systolic blood pressure can climb to over 300 mmHg in response to an unperceived noxious stimulus. These blood pressure fluctuations can occur multiple times a day, contributing to increased vessel shear stress and heightened risk of cardiovascular disease. The pathophysiology of both of these conditions is rooted in impairments in regulation of spinal cord sympathetic preganglionic neurons, which control blood pressure by mediating vascular resistance and catecholamine release. Recently, spinal cord electrical stimulation has provided evidence that it may modulate these blood pressure imbalances. Early proposed mechanisms suggest activation of spinal cord dorsal horn neurons that ultimately act upon the sympathetic preganglionic neuronal pathways. For OH, spinal cord stimulation likely induces local activation of these neurons to generate baseline sympathetic tone and accompanying vasoconstriction. The mechanisms for spinal stimulation regulating AD are less clear, though some suggest it activates inhibitory circuits to dampen the overactive sympathetic response. While questions remain, spinal cord electrical stimulation is an intriguing new modality that may restore blood pressure regulation following spinal cord injury.

Neuronal Sensitization and Synaptic Facilitation in the Superficial Dorsal Horn of a Rat Reserpine-induced Pain Model

Chronic widespread pain is one of the important issues to be solved in medical practice. Impaired spinal descending pain inhibitory system due to decreased monoamine neurotransmitters is assumed to cause nociceptive hypersensitivities in chronic painful conditions like that described in patients with fibromyalgia (FM). However, response behaviors and synaptic transmission of the spinal dorsal horn neurons in response to reserpine remain to be clarified. Here we examined the activities of superficial dorsal horn (SDH) neurons, as well as excitatory and inhibitory postsynaptic inputs to SDH neurons, using a putative rat model of FM that was established by injecting reserpine. Extracellular recordings in vivo revealed that SDH neurons were sensitized to mechanical stimulation applied to the neurons’ receptive fields, and the mechanically sensitized neurons were spontaneously more active. The sensitizing effect was evident 1 day and 3 days after the reserpine treatment, but subsided 5 days after the treatment or later. Using patch-clamp recordings in vivo, spontaneous excitatory postsynaptic currents (sEPSCs) to SDH neurons were found to increase in the pain model, while spontaneous inhibitory postsynaptic currents (sIPSCs) to SDH neurons decreased. These results demonstrate that the SDH neurons were strongly sensitized in response to the reserpine treatment, and that increased excitatory and decreased inhibitory postsynaptic inputs could be responsible for the spinal nociceptive hypersensitivity in the putative FM model.

Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity.

N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.

Differential Activation of Colonic Afferents and Dorsal Horn Neurons Underlie Stress-Induced and Comorbid Visceral Hypersensitivity in Female Rats

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions. PerspectiveChronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity.

Activation of MrgprA3 and MrgprC11 on Bladder-Innervating Afferents Induces Peripheral and Central Hypersensitivity to Bladder Distension.

Understanding the sensory mechanisms innervating the bladder is paramount to developing efficacious treatments for chronic bladder hypersensitivity conditions. The contribution of Mas-gene-related G protein-coupled receptors (Mrgpr) to bladder signaling is currently unknown. Using male and female mice, we show with single-cell RT-PCR that subpopulations of DRG neurons innervating the mouse bladder express MrgprA3 (14%) and MrgprC11 (38%), either individually or in combination, with high levels of coexpression with Trpv1 (81%-89%). Calcium imaging studies demonstrated MrgprA3 and MrgprC11 agonists (chloroquine, BAM8-22, and neuropeptide FF) activated subpopulations of bladder-innervating DRG neurons, showing functional evidence of coexpression between MrgprA3, MrgprC11, and TRPV1. In ex vivo bladder-nerve preparations, chloroquine, BAM8-22, and neuropeptide FF all evoked mechanical hypersensitivity in subpopulations (20%-41%) of bladder afferents. These effects were absent in recordings from Mrgpr-clusterΔ-/- mice. In vitro whole-cell patch-clamp recordings showed that application of an MrgprA3/C11 agonist mixture induced neuronal hyperexcitability in 44% of bladder-innervating DRG neurons. Finally, in vivo instillation of an MrgprA3/C11 agonist mixture into the bladder of WT mice induced a significant activation of dorsal horn neurons within the lumbosacral spinal cord, as quantified by pERK immunoreactivity. This MrgprA3/C11 agonist-induced activation was particularly apparent within the superficial dorsal horn and the sacral parasympathetic nuclei of WT, but not Mrgpr-clusterΔ-/- mice. This study demonstrates, for the first time, functional expression of MrgprA3 and MrgprC11 in bladder afferents. Activation of these receptors triggers hypersensitivity to distension, a critically valuable factor for therapeutic target development.SIGNIFICANCE STATEMENT Determining how bladder afferents become sensitized is the first step in finding effective treatments for common urological disorders such as overactive bladder and interstitial cystitis/bladder pain syndrome. Here we show that two of the key receptors, MrgprA3 and MrgprC11, that mediate itch from the skin are also expressed on afferents innervating the bladder. Activation of these receptors results in sensitization of bladder afferents, resulting in sensory signals being sent into the spinal cord that prematurely indicate bladder fullness. Targeting bladder afferents expressing MrgprA3 or MrgprC11 and preventing their sensitization may provide a novel approach for treating overactive bladder and interstitial cystitis/bladder pain syndrome.

Chronic BDNF simultaneously inhibits and unmasks superficial dorsal horn neuronal activity

Brain-derived neurotrophic factor (BDNF) is critically involved in the pathophysiology of chronic pain. However, the mechanisms of BDNF action on specific neuronal populations in the spinal superficial dorsal horn (SDH) requires further study. We used chronic BDNF treatment (200 ng/ml, 5–6 days) of defined-medium, serum-free spinal organotypic cultures to study intracellular calcium ([Ca2+]i) fluctuations. A detailed quantitative analysis of these fluctuations using the Frequency-independent biological signal identification (FIBSI) program revealed that BDNF simultaneously depressed activity in some SDH neurons while it unmasked a particular subpopulation of ‘silent’ neurons causing them to become spontaneously active. Blockade of gap junctions disinhibited a subpopulation of SDH neurons and reduced BDNF-induced synchrony in BDNF-treated cultures. BDNF reduced neuronal excitability assessed by measuring spontaneous excitatory postsynaptic currents. This was similar to the depressive effect of BDNF on the [Ca2+]i fluctuations. This study reveals novel regulatory mechanisms of SDH neuronal excitability in response to BDNF.

The superoxide scavenger tempol attenuates DNA oxidative injury and spontaneous pain-like behavior in chronic post-cast pain model rats

The mechanism of severe pain occurring because of physical disuse, such as complex regional pain syndrome Type I, has not been elucidated so far. Therefore, to investigate this mechanism, we have developed a model called a chronic post-cast pain (CPCP) model. Oxidative stress-related factors generated in a fixed limb may be triggers for nociceptive signals due to physical disuse. On the basis of the results of our previous studies, we speculated that oxidative stress-related factors in immobilized hind limbs may also be triggers of nociceptive signals due to physical disuse. In this study, we aimed to clarify whether an oxidative stress-related factor is involved in the induction of nociceptive signals. The time course of oxidative damage in the soleus (slow-twitch fiber) and gastrocnemius (fast-twitch fiber) muscles was evaluated by immunostaining of 8-hydroxy-2′-deoxyguanosine (a marker of oxidative damage in DNA). We also investigated the effects of tempol, a scavenger of superoxide, on oxidative damage in DNA, spontaneous pain-related behaviors (licking and/or biting and flinching), and the activation of spinal dorsal horn neurons (c-Fos). Systemic administration of tempol before cast removal attenuated oxidative damage to DNA in immobilized skeletal muscles, suppressed spontaneous pain-related behavior, and suppressed the activation of spinal dorsal horn neurons. We suggest that superoxide generated in immobilized skeletal muscles after cast removal is one of the peripheral factors that trigger nociceptive signals.

Dorsal horn disinhibition and movement-induced behaviour in a rat model of inflammatory arthritis

Alterations beyond joint inflammation such as changes in dorsal horn (DH) excitability contribute to pain in inflammatory arthritis (IA). More complete understanding of specific underlying mechanisms will be important to define novel targets for the treatment of IA pain. Pre-clinical models are useful, but relevant pain assays are vital for successful clinical translation. For this purpose, a method is presented to assess movement-induced pain-related behaviour changes that was subsequently used to investigate DH disinhibition in IA. IA was induced by intra-articular injection of complete Freund's adjuvant (CFA) in male rats, and weight distribution was assessed before and after walking on a treadmill. To confirm increased activity in nociception-related pathways, fos expression was assessed in the superficial DH, including in nociceptive neurons, identified by neurokinin 1 (NK1) immunoreactivity, and interneurons. Inhibitory terminal density onto NK1+ neurons was assessed and lastly, a cohort of animals was treated for 3 days with gabapentin. At 4 weeks post-CFA, walking reduced weight distribution to the affected joint and increased DH fos expression, including in NK1+ neurons. Neuronal activity in inhibitory cells and inhibitory terminal density on NK1+ neurons were decreased in CFA-treated animals compared with controls. Treatment with gabapentin led to recovered behaviour and DH neuronal activity pattern in CFA-treated animals. We describe an assay to assess movement-induced pain-related behaviour changes in a rodent IA model. Furthermore, our results suggest that disinhibition may contribute to pain related to movement in IA.