Excitability Of Dorsal Horn Neurons Research Articles

Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord.

K+-Cl- cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In Cacna2d1 KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.Significance statementImpaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1–NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.

Brief Opioid Exposure Paradoxically Augments Primary Afferent Input to Spinal Excitatory Neurons via α2δ-1-Dependent Presynaptic NMDA Receptors.

Treatment with opioids not only inhibits nociceptive transmission but also elicits a rebound and persistent increase in primary afferent input to the spinal cord. Opioid-elicited long-term potentiation (LTP) from TRPV1-expressing primary afferents plays a major role in opioid-induced hyperalgesia and analgesic tolerance. Here, we determined whether opioid-elicited LTP involves vesicular glutamate transporter-2 (VGluT2) or vesicular GABA transporter (VGAT) neurons in the spinal dorsal horn of male and female mice and identified underlying signaling mechanisms. Spinal cord slice recordings revealed that µ-opioid receptor (MOR) stimulation with DAMGO initially inhibited dorsal root-evoked EPSCs in 87% VGluT2 neurons and subsequently induced LTP in 49% of these neurons. Repeated morphine treatment increased the prevalence of VGluT2 neurons displaying LTP with a short onset latency. In contrast, DAMGO inhibited EPSCs in 46% VGAT neurons but did not elicit LTP in any VGAT neurons even in morphine-treated mice. Spinal superficial laminae were densely innervated by MOR-containing nerve terminals and were occupied by mostly VGluT2 neurons and few VGAT neurons. Furthermore, conditional Grin1 knockout in dorsal root ganglion neurons diminished DAMGO-elicited LTP in lamina II neurons and attenuated hyperalgesia and analgesic tolerance induced by repeated treatment with morphine. In addition, DAMGO-elicited LTP in VGluT2 neurons was abolished by protein kinase C inhibition, gabapentin, Cacna2d1 knockout, or disrupting the α2δ-1-NMDA receptor interaction with an α2δ-1 C terminus peptide. Thus, brief MOR stimulation distinctively potentiates nociceptive primary afferent input to excitatory dorsal horn neurons via α2δ-1-coupled presynaptic NMDA receptors, thereby causing hyperalgesia and reducing analgesic actions of opioids.SIGNIFICANCE STATEMENT Opioid drugs are potent analgesics for treating severe pain and are commonly used during general anesthesia. However, opioid use often induces pain hypersensitivity, rapid loss of analgesic efficacy, and dose escalation, which can cause dependence, addiction, and even overdose fatality. This study demonstrates for the first time that brief opioid exposure preferentially augments primary sensory input to genetically identified glutamatergic excitatory, but not GABAergic/glycinergic inhibitory, neurons in nociceptive dorsal horn circuits. This opioid-elicited synaptic plasticity is cell type specific and mediated by protein kinase C-dependent and α2δ-1-dependent activation of NMDA receptors at primary sensory nerve terminals. These findings elucidate how intraoperative use of opioids for preemptive analgesia paradoxically aggravates postoperative pain and increases opioid consumption and suggest new strategies to improve opioid analgesic efficacy.

Inhibition of Spinal 5-HT3 Receptor and Spinal Dorsal Horn Neuronal Excitability Alleviates Hyperalgesia in a Rat Model of Parkinson's Disease.

Pain in Parkinson's disease (PD) is increasingly recognized as a major factor associated with poor life quality of PD patients. However, classic therapeutic drugs supplying dopamine have limited therapeutic effects on PD-related pain. This suggests that there is a mechanism outside the dopamine system that causes pain in PD. Our previous study demonstrated that 6-OHDA induced PD model manifested hyperalgesia to thermal and mechanical stimuli and decreased serotonin (5-hydroxytryptamine; 5-HT) in the spinal dorsal horn (SDH). Several 5-HT receptor subtypes have been confirmed to be associated with nociception in the spinal cord, such as 5-HT1A receptor, 5-HT1B receptor, 5-HT2 receptor, 5-HT3 receptor, and 5-HT7 receptor. Most research has shown that 5-HT1A receptor and 5-HT3 receptor play a key role in pain transmission in the spinal cord. We hypothesized that hyperalgesia of 6-OHDA rats may be related to increased excitability of SDH neurons, and functional change of 5-HT3 receptor may reverse the hyperalgesia of 6-OHDA lesioned rats and decrease cell excitability of SDH neurons. To test this hypothesis, we used whole-cell patch-clamp and pharmacological methods to evaluate the effect of 5-HT3 receptor and 5-HT1A receptor on the hyperalgesia of 6-OHDA rats. The results suggested that increased excitability in SDH neurons could be reversed by 5-HT3 receptor antagonist ondansetron (20μmol/L) and palosetron (10μmol/L), but not 5-HT3 receptor agonist m-CPBG (30μmol/L) and SR 57,727 (10μmol/L), 5-HT1A receptor agonist 8-OH DPAT (10μmol/L) and eptapirone (10μmol/L) and 5-HT1A receptor antagonist WAY-100635 (10μmol/L) and p-MPPI (10μmol/L). Intrathecal injection of ondansetron (0.1mg/kg) but not m-CPBG (0.1mg/kg), 8-OH DPAT (0.1mg/kg), and WAY-100635 (0.1mg/kg) significantly attenuated the mechanical hyperalgesia and thermal hyperalgesia in 6-OHDA lesioned rats. In conclusion, the present study suggests that inhibition of spinal 5-HT3 receptor and SDH neuronal excitability alleviates hyperalgesia in PD rats. Our study provides a novel mechanism or therapeutic strategy for pain in patients with PD.

Short-Term Effects of Corticosterone on the Excitability of the Mouse Superficial Dorsal Horn

Mounting preclinical and clinical evidence demonstrate that the behavioral and cellular consequences of stress vary across different stages of development. Cortisol, or corticosterone (CORT) in rodents, is a major stress hormone and which can rapidly modulate neuronal excitability in multiple brain regions. However, its short-term effects on the function of nociceptive circuits in the developing spinal superficial dorsal horn (SDH) are poorly understood. Here we examined the expression of glucocorticoid receptors (GR) in the adolescent SDH and explored the effects of CORT on intrinsic membrane excitability and synaptic transmission in GABAergic and presumed glutamatergic SDH neurons using Glutamic acid decarboxylase 67-Enhanced Green Fluorescent Protein (Gad67-EGFP) mice. In situ hybridization revealed that Gad67-EGFP-negative SDH neurons exhibited a significantly higher number of GR mRNA puncta compared to adjacent Gad67-EGFP-positive SDH neurons. In vitro patch clamp recordings from spinal cord slices suggested that the acute application of CORT (100 nM) reduced glutamate or GABA release onto a subset of SDH neurons regardless of cell type. However, incubation of spinal cord slices with CORT for 90 minutes failed to influence the intrinsic membrane properties (including rheobase, resting potential and repetitive firing frequency) or miniature postsynaptic currents (mPSCs) in either neuronal population compared to the vehicle control. Collectively, while these results suggest that GR activation fails to exert intermediate effects on the excitability of the adolescent SDH network, the abundance of GR expression in the region raises the possibility that CORT can evoke slow changes in the excitability of dorsal horn neurons via genomic mechanisms. Grant support from NS072202.

Theta-Burst Stimulation of Primary Afferents Drives Long-Term Potentiation in the Spinal Cord and Persistent Pain via α2δ-1-Bound NMDA Receptors.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury.SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain.

Neuron-specific spinal cord translatomes reveal a neuropeptide code for mouse dorsal horn excitatory neurons

The spinal dorsal horn harbors a sophisticated and heterogeneous network of excitatory and inhibitory neurons that process peripheral signals encoding different sensory modalities. Although it has long been recognized that this network is crucial both for the separation and the integration of sensory signals of different modalities, a systematic unbiased approach to the use of specific neuromodulatory systems is still missing. Here, we have used the translating ribosome affinity purification (TRAP) technique to map the translatomes of excitatory glutamatergic (vGluT2+) and inhibitory GABA and/or glycinergic (vGAT+ or Gad67+) neurons of the mouse spinal cord. Our analyses demonstrate that inhibitory and excitatory neurons are not only set apart, as expected, by the expression of genes related to the production, release or re-uptake of their principal neurotransmitters and by genes encoding for transcription factors, but also by a differential engagement of neuromodulator, especially neuropeptide, signaling pathways. Subsequent multiplex in situ hybridization revealed eleven neuropeptide genes that are strongly enriched in excitatory dorsal horn neurons and display largely non-overlapping expression patterns closely adhering to the laminar and presumably also functional organization of the spinal cord grey matter.

Evaluation of the GABAA Receptor Expression and the Effects of Muscimol on the Activity of Wide Dynamic Range Neurons Following Chronic Constriction Injury of the Sciatic Nerve in Rats

Introduction: The modality of γ-aminobutyric acid receptors (GABAA) in control of dorsal horn neuronal excitability and inhibition of sensory information is ambiguous. The aim of the present study was to investigate the expression of GABAA receptor and the effects of its agonist muscimol on wide dynamic range (WDR) neuronal activity in the chronic constriction injury (CCI) model of neuropathic pain. Methods: Adult male Wistar rats weighing 200 to 250 g were used for the induction of CCI neuropathy. 14 days after surgery, muscimol (0.5, 1, and 2 mg/kg i.p.) was injected. Then, the behavioral tests were performed. Thereafter, the animals were sacrificed, and the lumbar segments of the spinal cords were collected for Western blot analysis of the GABAA receptor α1 subunit expression. The electrophysiological properties of WDR neurons were studied by single unit recordings in separate groups on the 14th day after CCI. Results: The outcomes indicated the development of thermal hyperalgesia and mechanical allodynia after neuropathy; nonetheless, the expression of GABAA receptor α1 subunit did not change significantly. Moreover, the evoked responses of the WDR neurons to electrical, mechanical, and thermal stimuli were significantly increased. 14 days after CCI, muscimol administration decreased thermal hyperalgesia, mechanical allodynia, and hyper-responsiveness of the WDR neurons in CCI rats. Conclusion: It confirms that the modulation of the spinal GABAA receptors after nerve injury can offer further insights to design new therapeutic agents in order to reduce the neuropathic pain symptoms.

Evaluation of the GABAA Receptor Expression and the Effects of Muscimol on the Activity of Wide Dynamic Range Neurons Following Chronic Constriction Injury of Sciatic Nerve in Rats.

Introduction:The modality of γ-aminobutyric acid type a receptors (GABAA) controls dorsal horn neuronal excitability and inhibits sensory information. This study aimed to investigate the expression of the GABAA receptor and the effects of its agonist muscimol on Wide Dynamic Range (WDR) neuronal activity in the Chronic Constriction Injury (CCI) model of neuropathic pain.Methods:Adult male Wistar rats weighing 200 to 250 g were used to induce CCI neuropathy. Fourteen days after surgery, muscimol (0.5, 1, and 2 mg/kg IP) was injected. Then, the behavioral tests were performed. After that, the animals were killed, and the lumbar segments of the spinal cords were collected for Western blot analysis of the GABAA receptor α1 subunit expression. The electrophysiological properties of WDR neurons were studied by single-unit recordings in separate groups 14 days after CCI.Results:The outcomes indicated the development of thermal hyperalgesia and mechanical allodynia after neuropathy; nonetheless, the expression of the GABAA receptor α1 subunit did not change significantly. Moreover, the evoked responses of the WDR neurons to electrical, mechanical, and thermal stimuli increased considerably. Fourteen days after CCI, muscimol administration decreased thermal hyperalgesia, mechanical allodynia, and hyper-responsiveness of the WDR neurons in CCI rats.Conclusion:The modulation of the spinal GABAA receptors after nerve injury can offer further insights to design new therapeutic agents to reduce neuropathic pain symptoms.

Sensory neuron-derived NaV1.7 contributes to dorsal horn neuron excitability.

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno-electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system-specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis.

Histamine H4 receptor stimulation in the locus coeruleus attenuates neuropathic pain by promoting the coeruleospinal noradrenergic inhibitory pathway

The locus coeruleus (LC) adrenergic nuclei constitute a pain-control inhibitory system nucleus implicated in descending modulation of pain through the action on spinal α2-adrenoceptors. Histaminergic innervation from the tuberomammillary nucleus of the LC increases firing of noradrenergic neurons and might contribute to pain control. Here we evaluated the contribution of LC histaminergic innervation in descending modulation of neuropathic hypersensitivity, by investigating the role of the histamine H4 receptor subtype in a mouse model of neuropathic pain. Intra LC administration of the H4 agonist VUF 8430 attenuated mechanical and thermal allodynia of mice that underwent spared nerve injury (SNI). Similarly, histamine in the LC showed mechanical and thermal anti-hypersensitivity. Pretreatment of LC with JNJ 10191584 (H4 antagonist) prevented the beneficial effect of VUF 8430 and histamine on nociceptive behaviour. Comparable results were obtained after intrathecal administration of drugs. The intrathecal administration of the α2-adrenoceptor agonist clonidine ameliorated mechanical and thermal allodynia in SNI mice. The clonidine-induced anti-hypersensitivity effect was prevented by intra LC pretreatment with JNJ 10191584. In addition, clonidine failed to suppress neuropathic pain in H4 deficient mice. LC H4 receptors showed a ubiquitous distribution within LC, a neuronal localization and H4 immunostaining was detected on noradrenergic neurons expressing phosphorylated cAMP response element-binding protein (CREB), a marker of neuronal activation. Under pain pathological conditions H4 stimulation might promote the activation of the coeruleospinal noradrenergic neurons that exert an inhibitory control over spinal dorsal horn neuronal excitability. Thus, histamine H4 receptor stimulation may represent a perspective for neuropathic pain management.

CircAnks1a in the spinal cord regulates hypersensitivity in a rodent model of neuropathic pain

Circular RNAs are non-coding RNAs, and are enriched in the CNS. Dorsal horn neurons of the spinal cord contribute to pain-like hypersensitivity after nerve injury in rodents. Here we show that spinal nerve ligation is associated with an increase in expression of circAnks1a in dorsal horn neurons, in both the cytoplasm and the nucleus. Downregulation of circAnks1a by siRNA attenuates pain-like behaviour induced by nerve injury. In the cytoplasm, we show that circAnks1a promotes the interaction between transcription factor YBX1 and transportin-1, thus facilitating the nucleus translocation of YBX1. In the nucleus, circAnks1a binds directly to the Vegfb promoter, increases YBX1 recruitment to the Vegfb promoter, thereby facilitating transcription. Furthermore, cytoplasmic circAnks1a acts as a miRNA sponge in miR-324-3p-mediated posttranscriptional regulation of VEGFB expression. The upregulation of VEGFB contributes to increased excitability of dorsal horn neurons and pain behaviour induced by nerve injury. We propose that circAnks1a and VEGFB are regulators of neuropathic pain.

Receptor dependence of BDNF actions in superficial dorsal horn: relation to central sensitization and actions of macrophage colony stimulating factor 1

Peripheral nerve injury elicits an enduring increase in the excitability of the spinal dorsal horn. This change, which contributes to the development of neuropathic pain, is a consequence of release and prolonged exposure of dorsal horn neurons to various neurotrophins and cytokines. We have shown in rats that nerve injury increases excitatory synaptic drive to excitatory neurons but decreases drive to inhibitory neurons. Both effects, which contribute to an increase in dorsal horn excitability, appear to be mediated by microglia-derived BDNF. We have used multiphoton Ca2+ imaging and whole cell recording of spontaneous excitatory postsynaptic currents in defined-medium organotypic cultures of GAD67-GFP+ mice spinal cord to determine the receptor dependence of these opposing actions of BDNF. In mice, as in rats, BDNF enhances excitatory transmission onto excitatory neurons. This is mediated via presynaptic TrkB and p75 neurotrophin receptors and exclusively by postsynaptic TrkB. By contrast with findings from rats, in mice BDNF does not decrease excitation of inhibitory neurons. The cytokine macrophage colony-stimulating factor 1 (CSF-1) has also been implicated in the onset of neuropathic pain. Nerve injury provokes its de novo synthesis in primary afferents, its release in spinal cord, and activation of microglia. We now show that CSF-1 increases excitatory drive to excitatory neurons via a BDNF-dependent mechanism and decreases excitatory drive to inhibitory neurons via BDNF-independent processes. Our findings complete missing steps in the cascade of events whereby peripheral nerve injury instigates increased dorsal horn excitability in the context of central sensitization and the onset of neuropathic pain. NEW & NOTEWORTHY Nerve injury provokes synthesis of macrophage colony-stimulating factor 1 (CSF-1) in primary afferents and its release in the dorsal horn. We show that CSF-1 increases excitatory drive to excitatory dorsal horn neurons via BDNF activation of postsynaptic TrkB and presynaptic TrkB and p75 neurotrophin receptors. CSF-1 decreases excitatory drive to inhibitory neurons via a BDNF-independent processes. This completes missing steps in understanding how peripheral injury instigates central sensitization and the onset of neuropathic pain.

Inflammatory-induced spinal dorsal horn neurons hyperexcitability is mediated by P2X4 receptors.

Introduction:Purinergic ionotropic P2X receptors (P2RX) are involved in normal and pathological pain transmission. Among them, P2X4 are expressed in dorsal root ganglion and in the spinal cord. Their activation during nerve injury or chronic peripheral inflammation modifies pain sensitivity that leads to the phenomenon of allodynia and hyperalgesia.Objectives:We study here, in vivo, the role of P2X4 on the excitability of dorsal horn neurons (DHNs) in naive or pathological context.Methods:We recorded DHNs in vivo in anesthetized wild-type or P2RX4−/− mice. We measured nociceptive integration and short-term sensitization by DHNs both in naive and inflamed mice.Results:Our results indicate that P2X4 alter neuronal excitability only in the pathological context of peripheral inflammation. Consequently, excitability of DHNs from inflamed P2RX4−/− mice remains similar to naive animals.Conclusion:These results confirm the prominent role of P2X4 in inflammatory pain context and demonstrate that P2X4 are also involved in the hyperexcitability of DHNs.

Contribution of Plasma Membrane Calcium ATPases to neuronal maladaptive responses: Focus on spinal nociceptive mechanisms and neurodegeneration.

Plasma membrane calcium ATPases (PMCAs) are ion pumps that expel Ca2+ from cells and maintain Ca2+ homeostasis. Four isoforms and multiple splice variants play important and non-overlapping roles in cellular function and integrity and have been implicated in diseases including disorders of the central nervous system (CNS). In particular, one of these isoforms, PMCA2, is critical for spinal cord (SC) neuronal function. PMCA2 expression is decreased in SC neurons at onset of symptoms in animal models of multiple sclerosis. Decreased PMCA2 expression affects the function and viability of SC neurons, with motor neurons being the most vulnerable population. Recent studies have also shown that PMCA2 could be an important contributor to pain processing in the dorsal horn (DH) of the SC. Pain sensitivity was altered in female, but not male, PMCA2+/- mice compared to PMCA2+/+ littermates in a modality-dependent manner. Changes in pain responsiveness in the female PMCA2+/- mice were paralleled by female-specific alterations in the expression of effectors, which have been implicated in the excitability of DH neurons, in mechanisms governing nociception and in the transmission of pain signals. Other PMCA isoforms and in particular, PMCA4, also contribute to the excitability of neurons in the dorsal root ganglia (DRG), which contain the first-order sensory neurons that convey nociceptive information from the periphery to the DH. These findings suggest that specific PMCA isoforms play specialized functions in neurons that mediate pain processing. Further investigations are necessary to unravel the precise contribution of PMCAs to mechanisms governing pathological pain in models of injury and disease.

Modulation of Pain and Itch by Spinal Glia

Chronic pain and itch are a pathological operation of the somatosensory system at the levels of primary sensory neurons, spinal cord and brain. Pain and itch are clearly distinct sensations, and recent studies have revealed the separate neuronal pathways that are involved in each sensation. However, the mechanisms by which these sensations turn into a pathological chronic state are poorly understood. A proposed mechanism underlying chronic pain and itch involves abnormal excitability in dorsal horn neurons in the spinal cord. Furthermore, an increasing body of evidence from models of chronic pain and itch has indicated that synaptic hyperexcitability in the spinal dorsal horn might not be a consequence simply of changes in neurons, but rather of multiple alterations in glial cells. Thus, understanding the key roles of glial cells may provide us with exciting insights into the mechanisms of chronicity of pain and itch, and lead to new targets for treating chronic pain and itch.

The inhibition of spinal synaptic plasticity mediated by activation of AMP-activated protein kinase signaling alleviates the acute pain induced by oxaliplatin

Our recent findings demonstrated that oxaliplatin entering CNS may directly induce spinal central sensitization, and contribute to the rapid development of CNS-related side effects including acute pain during chemotherapy. However, the mechanism is largely unclear. In the current study, we found that the amplitude of C-fiber-evoked field potentials was significantly increased and the expression of phosphorylated mammalian AMP-activated protein kinase α (AMPKα) was markedly decreased following high frequency stimulation (HFS) or single intraperitoneal injection of oxaliplatin (4mg/kg). Spinal local application of AMPK agonist metformin (25μg) prevented the long term potentiation (LTP) induction and the activation of mTOR/p70S6K signal pathway, and significantly attenuated the acute thermal hyperalgesia and mechanical allodynia following single oxaliplatin treatment. Importantly, we found that incubation of low concentration oxaliplatin at dose of 6.6nM (the detected concentration in CSF following a single intraperitoneal injection of oxaliplatin) also significantly inhibited the AMPKα activation and increased the amplitude of sEPSCs, the number of action potential, and the expression of p-mTOR and p-p70S6K in spinal cord slices. Metformin (25μg) or rapamycin (2μg) inhibited the increased excitability of dorsal horn neurons and the decrease of p-AMPKα expression induced by low concentration oxaliplatin incubation. Furthermore, spinal application of AMPK inhibitor compound C (5μg) induced the spinal LTP, thermal hyperalgesia and mechanical allodynia, and rapamycin attenuated the spinal LTP, the thermal hyperalgesia and mechanical allodynia following oxaliplatin treatment (i.p.). Local application of metformin significantly decreased the mTOR and p70S6K activation induced by tetanus stimulation or oxaliplatin (i.p.). These results suggested that the decreased AMPKα activity via negatively regulating mTOR/p70S6K signal pathway enhanced the synaptic plasticity and contributed to acute pain induced by low concentration of oxaliplatin entering CNS.

Clinically relevant concentration of pregabalin has no acute inhibitory effect on excitation of dorsal horn neurons under normal or neuropathic pain conditions: An intracellular calcium-imaging study in spinal cord slices from adult rats

Pregabalin is thought to exert its therapeutic effect in neuropathic pain via binding to α2δ−1 subunits of voltage-gated calcium (Ca2+) channels. However, the exact analgesic mechanism after its binding to α2δ−1 subunits remains largely unknown. Whether a clinical concentration of pregabalin (≈10μM) can cause acute inhibition of dorsal horn neurons in the spinal cord is controversial. To address this issue, we undertook intracellular Ca2+-imaging studies using spinal cord slices with an intact attached L5 dorsal root, and examined if pregabalin acutely inhibits the primary afferent stimulation-evoked excitation of dorsal horn neurons in normal rats and in rats with streptozotocin-induced painful diabetic neuropathy. Under normal conditions, stimulation of a dorsal root evoked Ca2+ signals predominantly in the superficial dorsal horn. Clinically relevant (10μM) and a very high concentration of pregabalin (100μM) did not affect the intensity or spread of dorsal root stimulation-evoked Ca2+ signals, whereas an extremely high dose of pregabalin (300μM) slightly but significantly attenuated Ca2+ signals in normal rats and in diabetic neuropathic (DN) rats. There was no difference between normal rats and DN rats with regard to the extent of signal attenuation at all concentrations tested. These results suggest that the activity of dorsal horn neurons in the spinal cord is not inhibited acutely by clinical doses of pregabalin under normal or DN conditions. It is very unlikely that an acute inhibitory action in the dorsal horn is the main analgesic mechanism of pregabalin in neuropathic pain states.

Spinal Cord Stimulation What Do We Know?

sia. Mechanism of Action: SCS works in multiple domains Neurochemical effects: There is no relation between SCS and opioid release, there is an increase dorsal horn GABA expression seen in animal model after SCS stimulation, levels of Substance P and serotonin are shown to be elevated by SCS. Studies have proven that excitatory amino acid release is suppressed by SCS via action on GABA-B receptors and intra-thecal adenosine as well baclofen may potentiate the effect of SCS. The net effect of these neurochemical effects reduces dorsal horn neuronal excitability [3]. Neurophysiological effects: SCS application modulates nociceptive response via an action on abnormal pain-mediating Abeta fibre function. The response of wide dynamic range of neurons (WDR) to chemical, thermal and mechanical stimuli in animal neuropathic models has shown suppression with application of SCS. Various models have shown specific role played by remote nervous system like anterior pretectal nucleus which modulates the descending pain inhibitory pathways due to SCS stimulation [3]. Cardiac/Vascular effects: In angina pectoris, SCS has been demonstrated to reduce noxious input to pain pathway, inhibit the afferent sympathetic nervous system and stabilize intra-cardiac neuronal activity. In ischemic pain models SCS is associated with peripheral vasodilation, mediated via the release of calcitonin gene-related peptide (CGRP).

Microglia in the spinal cord and neuropathic pain.

In contrast to physiological pain, pathological pain is not dependent on the presence of tissue-damaging stimuli. One type of pathological pain - neuropathic pain - is often a consequence of nerve injury or of diseases such as diabetes. Neuropathic pain can be agonizing, can persist over long periods and is often resistant to known painkillers. A growing body of evidence shows that many pathological processes within the central nervous system are mediated by complex interactions between neurons and glial cells. In the case of painful peripheral neuropathy, spinal microglia react and undergo a series of changes that directly influence the establishment of neuropathic pain states. After nerve damage, purinergic P2X4 receptors (non-selective cation channels activated by extracellular adenosine triphosphate) are upregulated in spinal microglia in a manner that depends on the transcription factors interferon regulatory factor8 and 5, both of which are expressed in microglia after peripheral nerve injury. P2X4 receptor expression on the cell surface of microglia is also regulated at the post-translational level by signaling from CC chemokine receptor chemotactic cytokine receptor2. Furthermore, spinal microglia in response to extracellular stimuli results in signal transduction through intracellular signaling cascades, such as mitogen-activated protein kinases, p38 and extracellular signal-regulated protein kinase. Importantly, inhibiting the function or expression of these microglial molecules suppresses the aberrant excitability of dorsal horn neurons and neuropathic pain. These findings show that spinal microglia are a central player in mechanisms for neuropathic pain, and might be a potential target for treating the chronic pain state.

Hoxb8 intersection defines a role for Lmx1b in excitatory dorsal horn neuron development, spinofugal connectivity, and nociception.

Spinal cord neurons respond to peripheral noxious stimuli and relay this information to higher brain centers, but the molecules controlling the assembly of such pathways are poorly known. In this study, we use the intersection of Lmx1b and Hoxb8::Cre expression in the spinal cord to genetically define nociceptive circuits. Specifically, we show that Lmx1b, previously shown to be expressed in glutamatergic dorsal horn neurons and critical for dorsal horn development, is expressed in nociceptive dorsal horn neurons and that its deletion results in the specific loss of excitatory dorsal horn neurons by apoptosis, without any effect on inhibitory neuron numbers. To assess the behavioral consequences of Lmx1b deletion in the spinal cord, we used the brain-sparing driver Hoxb8::Cre. We show that such a deletion of Lmxb1 leads to a robust reduction in sensitivity to mechanical and thermal noxious stimulation. Furthermore, such conditional mutant mice show a loss of a subpopulation of glutamatergic dorsal horn neurons, abnormal sensory afferent innervations, and reduced spinofugal innervation of the parabrachial nucleus and the periaqueductal gray, important nociceptive structures. Together, our results demonstrate an important role for the intersection of Lmx1b and Hoxb8::cre expression in the development of nociceptive dorsal horn circuits critical for mechanical and thermal pain processing.