Responses Of Dorsal Horn Neurons Research Articles

A mouse model of peripheral postischemic dysesthesia: involvement of reperfusion-induced oxidative stress and TRPA1 channel.

Peripheral postischemic dysesthesia was examined behaviorally in mice and we investigated the underlying molecular mechanism with a focus on oxidative stress. Hind-paw ischemia was induced by tight compression of the ankle with a rubber band, and reperfusion was achieved by cutting the rubber tourniquet. We found that reperfusion after ischemia markedly provoked licking of the reperfused hind paw, which was significantly inhibited by systemic administration of the antioxidant N-acetyl-l-cysteine and the transient receptor potential (TRP) A1 channel blocker HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide]. Postischemic licking was also significantly inhibited by an intraplantar injection of another antioxidant, phenyl-N-tert-butylnitrone. The TRPV1 channel blocker BCTC [N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide] did not inhibit postischemic licking. An intraplantar injection of hydrogen peroxide elicited hind-paw licking, which was inhibited by N-acetyl-l-cysteine, phenyl-N-tert-butylnitrone, and HC-030031. Postischemic licking was not affected by chemical depletion of sensory C-fibers, but it was inhibited by morphine, which has been shown to inhibit the C- and Aδ-fiber-evoked responses of dorsal horn neurons. Interestingly, postischemic licking was not inhibited by gabapentin and pregabalin, which have been shown to inhibit the C-fiber- but not Aδ-fiber-evoked response. The present results suggest that ischemia-reperfusion induces oxidative stress, which activates TRPA1 channels to provoke postischemic licking. It has been suggested that this behavior is mediated by myelinated (probably Aδ-type) afferent fibers. Oxidative stress and TRPA1 channels may be potential targets to treat peripheral ischemia-associated dysesthesia.

A preclinical physiological assay to test modulation of knee joint pain in the spinal cord: effects of oxycodone and naproxen.

Sensory processing in the spinal cord during disease states can reveal mechanisms for novel treatments, yet very little is known about pain processing at this level in the most commonly used animal models of articular pain. Here we report a test of the prediction that two clinically effective compounds, naproxen (an NSAID) and oxycodone (an opiate), are efficacious in reducing the response of spinal dorsal horn neurons to noxious knee joint rotation in the monosodium iodoacetate (MIA) sensitized rat. The overall objective for these experiments was to develop a high quality in vivo electrophysiology assay to confidently test novel compounds for efficacy against pain. Given the recent calls for improved preclinical experimental quality we also developed and implemented an Assay Capability Tool to determine the quality of our assay and ensure the quality of our results. Spinal dorsal horn neurons receiving input from the hind limb knee joint were recorded in anesthetized rats 14 days after they were sensitized with 1 mg of MIA. Intravenous administered oxycodone and naproxen were each tested separately for their effects on phasic, tonic, ongoing and afterdischarge action potential counts in response to innocuous and noxious knee joint rotation. Oxycodone reduced tonic spike counts more than the other measures, doing so by up to 85%. Tonic counts were therefore designated the primary endpoint when testing naproxen which reduced counts by up to 81%. Both reductions occurred at doses consistent with clinically effective doses for osteoarthritis. These results demonstrate that clinically effective doses of standard treatments for osteoarthritis reduce pain processing measured at the level of the spinal cord for two different mechanisms. The Assay Capability Tool helped to guide experimental design leading to a high quality and robust preclinical assay to use in discovering novel treatments for pain.

Synergistic effect of 5-hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic and visceral pain.

Synergistic activity has been observed between serotonergic 5-hydroxytryptamine 3 (5-HT3) and tachykinergic neurokinin 1 (NK1) receptor-mediated responses. This study investigated the efficacy of a 5-HT3 antagonist, palonosetron, and a NK1 antagonist, netupitant, alone or in combination in rodent models of somatic and visceral colonic hypersensitivity. In a rat model of experimental neuropathic pain, somatic hypersensitivity was quantified by the number of ipsilateral paw withdrawals to a von Frey filament (6g). Electrophysiologic responses were recorded in the dorsal horn neurons after mechanical or thermal stimuli. Acute colonic hypersensitivity was induced experimentally in rats by infusing dilute acetic acid (0.6%) directly into the colon. Colonic sensitivity was assessed by a visceromotor behavioral response quantified as the number of abdominal contractions in response to graded isobaric pressures (0-60 mm Hg) of colorectal distension. Palonosetron or netupitant was administered alone or in combination via oral gavage. When dosed alone, both significantly reduced somatic sensitivity, decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation, and caused significant (P < 0.05) inhibition of colonic hypersensitivity in a dose-dependent manner. The combined administration of palonosetron and netupitant at doses that were ineffective alone significantly reduced both somatic and visceral sensitivity and decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation. In summary, the combination of palonosetron with a NK1 receptor antagonist showed synergistic analgesic activity in rodent models of somatic and visceral hypersensitivity, and may prove to be a useful therapeutic approach to treat pain associated with irritable bowel syndrome.

Behavioral model of itch, alloknesis, pain and allodynia in the lower hindlimb and correlative responses of lumbar dorsal horn neurons in the mouse

We have further developed a behavioral model of itch and pain in the lower hindlimb (calf) originally reported by LaMotte et al. (2011) that allows comparisons with responses of lumbar dorsal horn neurons to pruritic and noxious stimuli. Intradermal (id) microinjection of the pruritogens histamine, SLIGRL-NH2 (agonist of PAR-2 and MrgprC11) and chloroquine (agonist of MrgprA3) into the calf of the lower limb elicited significant biting and a small amount of licking directed to the injection site, over a 30-min time course. Following id injection of histamine, low-threshold mechanical stimuli reliably elicited discrete episodes of biting (alloknesis) over a longer time course; significantly less alloknesis was observed following id injection of SLIGRL-NH2. Capsaicin injections elicited licking but little biting. Following id injection of capsaicin, low-threshold mechanical stimuli elicited discrete hindlimb flinches (allodynia) over a prolonged (>2h) time course. In single-unit recordings from superficial lumbar dorsal horn neurons, low-threshold mechanically evoked responses were significantly enhanced, accompanied by receptive field expansion, following id injection of histamine in histamine-responsive neurons. This was not observed in histamine-insensitive neurons, or following id injection of saline or SLIGRL-NH2, regardless of whether the latter activated the neuron or not. These results suggest that itch-responsive neurons are selectively sensitized by histamine but not SLIGRL-NH2 to account for alloknesis. The presently described “calf” model appears to distinguish between itch- and pain-related behavioral responses, and provides a basis to investigate lumbar spinal neural mechanisms underlying itch, alloknesis, pain and allodynia.

Altered nociceptive, endocrine, and dorsal horn neuron responses in rats following a neonatal immune challenge

The neonatal period is characterized by significant plasticity where the immune, endocrine, and nociceptive systems undergo fine-tuning and maturation. Painful experiences during this period can result in long-term alterations in the neurocircuitry underlying nociception, including increased sensitivity to mechanical or thermal stimuli. Less is known about the impact of neonatal exposure to mild inflammatory stimuli, such as lipopolysaccharide (LPS), on subsequent inflammatory pain responses. Here we examine the impact of neonatal LPS exposure on inflammatory pain sensitivity and HPA axis activity during the first three postnatal weeks. Wistar rats were injected with LPS (0.05mg/kg IP, Salmonella enteritidis) or saline on postnatal days (PNDs) 3 and 5 and later subjected to the formalin test at PNDs 7, 13, and 22. One hour after formalin injection, blood was collected to assess corticosterone responses. Transverse spinal cord slices were also prepared for whole-cell patch clamp recording from lumbar superficial dorsal horn neurons (SDH). Brains were obtained at PND 22 and the hypothalamus was isolated to measure glucocorticoid (GR) and mineralocorticoid receptor (MR) transcript expression using qRT-PCR. Behavioural analyses indicate that at PND 7, no significant differences were observed between saline- or LPS-challenged rats. At PND 13, LPS-challenged rats exhibited enhanced licking (p<.01), and at PND 22, increased flinching in response to formalin injection (p<.05). LPS-challenged rats also displayed increased plasma corticosterone at PND 7 and PND 22 (p<.001) but not at PND 13 following formalin administration. Furthermore, at PND 22 neonatal LPS exposure induced decreased levels of GR mRNA and increased levels of MR mRNA in the hypothalamus. The intrinsic properties of SDH neurons were similar at PND 7 and PND 13. However, at PND 22, ipsilateral SDH neurons in LPS-challenged rats had a lower input resistance compared to their saline-challenged counterparts (p<.05). These data suggest neonatal LPS exposure produces developmentally regulated changes in formalin-induced behavioural responses, corticosterone levels, and dorsal horn neuron properties following noxious stimulation later in life. These findings highlight the importance of immune activation during the neonatal period in shaping pain sensitivity later in life. This programming involves both spinal cord neurons and the HPA axis.

Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain

ObjectivesBlockade of transient receptor potential vanilloid 1 (TRPV1) with systemic antagonists attenuates osteoarthritis (OA) pain behaviour in rat models, but on-target-mediated hyperthermia has halted clinical trials. The present study investigated...

Roles of glutamate, substance P, and gastrin-releasing peptide as spinal neurotransmitters of histaminergic and nonhistaminergic itch

We investigated roles for substance P (SP), gastrin-releasing peptide (GRP), and glutamate in the spinal neurotransmission of histamine-dependent and -independent itch. In anesthetized mice, responses of single superficial dorsal horn neurons to intradermal (i.d.) injection of chloroquine were partially reduced by spinal application of the α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate acid (AMPA)/kainate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Co-application of CNQX plus a neurokinin-1 (NK-1) antagonist produced stronger inhibition, while co-application of CNQX, NK-1, and GRP receptor (GRPR) antagonists completely inhibited firing. Nociceptive-specific and wide dynamic range-type neurons exhibited differential suppression by CNQX plus either the GRPR or NK-1 antagonist, respectively. Neuronal responses elicited by i.d. histamine were abolished by CNQX alone. In behavioral studies, individual intrathecal administration of a GRPR, NK-1, or AMPA antagonist each significantly attenuated chloroquine-evoked scratching behavior. Co-administration of the NK-1 and AMPA antagonists was more effective, and administration of all 3 antagonists abolished scratching. Intrathecal CNQX alone prevented histamine-evoked scratching behavior. We additionally employed a double-label strategy to investigate molecular markers of pruritogen-sensitive dorsal root ganglion (DRG) cells. DRG cells responsive to histamine and/or chloroquine, identified by calcium imaging, were then processed for co-expression of SP, GRP, or vesicular glutamate transporter type 2 (VGLUT2) immunofluorescence. Subpopulations of chloroquine- and/or histamine-sensitive DRG cells were immunopositive for SP and/or GRP, with >80% immunopositive for VGLUT2. These results indicate that SP, GRP, and glutamate each partially contribute to histamine-independent itch. Histamine-evoked itch is mediated primarily by glutamate, with GRP playing a lesser role. Co-application of NK-1, GRP, and AMPA receptor antagonists may prove beneficial in treating chronic itch.

Scratching inhibits serotonin-evoked responses of rat dorsal horn neurons in a site- and state-dependent manner

Scratching inhibits pruritogen-evoked responses of neurons in the superficial dorsal horn, implicating a spinal site for scratch inhibition of itch. We investigated if scratching differentially affects neurons depending on whether they are activated by itchy vs. painful stimuli, and if the degree of inhibition depends on the relative location of scratching. We recorded from rat lumbar dorsal horn neurons responsive to intradermal (id) microinjection of serotonin (5-hydroxytryptamine, 5-HT). During the response to 5-HT, scratch stimuli (3mm, 300mN, 2Hz, 20s) were delivered at the injection site within the mechanosensitive receptive field (on-site), or 4–30mm away, outside of the receptive field (off-site). During off-site scratching, 5-HT-evoked firing was significantly attenuated followed by recovery. On-site scratching excited neurons, followed by a significant post-scratch decrease in 5-HT-evoked firing. Most neurons additionally responded to mustard oil (allyl isothiocyanate). Off-site scratching had no effect, while on-site scratching excited the neurons. These results indicate that scratching exerts a state-dependent inhibitory effect on responses of spinal neurons to pruritic but not algesic stimuli. Moreover, on-site scratching first excited neurons followed by inhibition, while off-site scratching immediately evoked the inhibition of pruritogen-evoked activity. This accounts for the suppression of itch by scratching at a distance from the site of the itchy stimulus.

Neural coding of nociceptive stimuli—from rat spinal neurones to human perception

Translational studies are key to furthering our understanding of nociceptive signalling and bridging the gaps between molecules and pathways to the patients. This requires use of appropriate preclinical models that accurately depict outcome measures used in humans. Whereas behavioural animal studies classically involve reports related to nociceptive thresholds of, for example, withdrawal, electrophysiological recordings of spinal neurones that receive convergent input from primary afferents permits investigation of suprathreshold events and exploration of the full-range coding of different stimuli. We explored the central processing of nociceptive inputs in a novel parallel investigation between rats and humans. Using radiant laser pulses, we first compared the electrophysiological responses of deep wide dynamic range and superficial nociceptive-specific neurones in the rat dorsal horn with human psychophysics and cortical responses. Secondly, we explored the effects of spatial summation using laser pulses of identical energy and different size. We observed 3 main findings. Firstly, both rodent and human data confirmed that neodymium–yttrium aluminium perovskite laser stimulation is a nociceptive-selective stimulus that never activates Aβ afferents. Secondly, graded laser stimulation elicited similarly graded electrophysiological and behavioural responses in both species. Thirdly, there was a significant degree of spatial summation of laser nociceptive input. The remarkable similarity in rodent and human coding indicates that responses of rat dorsal horn neurones can translate to human nociceptive processing. These findings suggest that recordings of spinal neuronal activity elicited by laser stimuli could be a valuable predictive measure of human pain perception.

Pharmacodynamic Effects of a d-Amino Acid Oxidase Inhibitor Indicate a Spinal Site of Action in Rat Models of Neuropathic Pain

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund's adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

The Antinociceptive and Antihyperalgesic Effects of Topical Propofol on Dorsal Horn Neurons in the Rat

Propofol (2,6-diisopropylphenol) is an IV anesthetic used for general anesthesia. Recent evidence suggests that propofol-anesthetized patients experience less postoperative pain, and that propofol has analgesic properties when applied topically. We presently investigated the antinociceptive effects of topical propofol using behavioral and single-unit electrophysiological methods in rats. In behavioral experiments with rats, we assessed the effect of topical hindpaw application of propofol (1%-25%) on heat and mechanically evoked paw withdrawals. In electrophysiological experiments, we recorded from lumbar dorsal horn wide dynamic range (WDR)-type neurons in pentobarbital-anesthetized rats. We assessed the effect of topical application of propofol to the ipsilateral hindpaw on neuronal responses elicited by noxious heat, cold, and mechanical stimuli. We additionally tested whether propofol blocks heat sensitization of paw withdrawals and WDR neuronal responses induced by topical application of allyl isothiocyanate (AITC; mustard oil). Topical application of propofol (1%-25%) significantly increased the mean latency of the thermally evoked hindpaw withdrawal reflex on the treated (but not opposite) side in a concentration-dependent manner, with no effect on mechanically evoked hindpaw withdrawal thresholds. Propofol also prevented shortening of paw withdrawal latency induced by AITC. In electrophysiological experiments, topical application of 10% and 25% propofol, but not 1% propofol or vehicle (10% intralipid), to the ipsilateral hindpaw significantly attenuated the magnitude of responses of WDR neurons to noxious heating of glabrous hindpaw skin with no significant change in thermal thresholds. Maximal suppression of noxious heat-evoked responses was achieved 15 minutes after application followed by recovery to the pre-propofol baseline by 30 minutes. Responses to skin cooling or graded mechanical stimuli were not significantly affected by any concentration of propofol. Topical application of AITC enhanced the noxious heat-evoked response of dorsal horn neurons. This enhancement of heat-evoked responses was attenuated when 10% propofol was applied topically after application of AITC. The results indicate that topical propofol inhibits responses of WDR neurons to noxious heat consistent with analgesia, and reduced AITC sensitization of WDR neurons consistent with an antihyperalgesic effect. These results are consistent with clinical studies demonstrating reduced postoperative pain in surgical patients anesthetized with propofol. The mechanism of analgesic action of topical propofol is not clear, but may involve desensitization of TRPV1 or TRPA1 receptors expressed in peripheral nociceptive nerve endings, engagement of endocannabinoids, or activation of peripheral γ-aminobutyric acid A receptors.

Roles of Phosphotase 2A in Nociceptive Signal Processing

Multiple protein kinases affect the responses of dorsal horn neurons through phosphorylation of synaptic receptors and proteins involved in intracellular signal transduction pathways, and the consequences of this modulation may be spinal central sensitization. In contrast, the phosphatases catalyze an opposing reaction of de-phosphorylation, which may also modulate the functions of crucial proteins in signaling nociception. This is an important mechanism in the regulation of intracellular signal transduction pathways in nociceptive neurons. Accumulated evidence has shown that phosphatase 2A (PP2A), a serine/threonine specific phosphatase, is implicated in synaptic plasticity of the central nervous system and central sensitization of nociception. Therefore, targeting protein phosphotase 2A may provide an effective and novel strategy for the treatment of clinical pain. This review will characterize the structure and functional regulation of neuronal PP2A and bring together recent advances on the modulation of PP2A in targeted downstream substrates and relevant multiple nociceptive signaling molecules.

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Electrical Stimulation of Large Myelinated Afferents Inhibits Responses of Rat Spinal Dorsal Horn Neurons to Noxious and Innocuous Cutaneous Stimulation

Sex differences in spinal processing of transient and inflammatory colorectal stimuli in the rat

Sex differences in the spinal processing of somatic and visceral stimuli contribute to greater female sensitivity in many pain disorders. The present study examined spinal mechanisms that contribute to sex differences in visceral sensitivity. The visceromotor response to colorectal distention (CRD) was more robust in normal female rats and after intracolonic mustard oil compared with that in male rats. No sex difference was observed in the CRD-evoked response of lumbosacral (LS) and thoracolumbar (TL) colonic afferents in normal and mustard oil–treated rats, but there was a sex difference in spontaneous activity that was exacerbated by intracolonic mustard oil. The response of visceroceptive dorsal horn neurons to CRD was greater in normal female rats in the LS and TL spinal segments. The effect of intracolonic mustard oil on the CRD-evoked response of different phenotypes of visceroceptive dorsal horn neurons was dependent on sex and segment. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response in normal rats with greater effect in male rats. Correspondingly, there was greater cell membrane expression of the GluN1 subunit in dorsal horn extracts in female rats. After intracolonic mustard oil, there was no longer a sex difference in the effect of APV nor GluN1 expression in LS segments, but greater female expression in TL segments. These data document a sex difference in spinal processing of nociceptive visceral stimuli from the normal and inflamed colon. Differences in dorsal horn neuronal activity and NMDA receptor expression contribute to the sex differences in the visceral sensitivity observed in awake rats.

Estrogen Receptor β Activation Is Antinociceptive in a Model of Visceral Pain in the Rat

The mechanism underlying estrogen modulation of visceral pain remains unclear. Our previous studies indicate that activation of estrogen receptor α (ERα) enhances visceral pain. The purpose of the present study was to investigate the role of estrogen receptor β (ERβ) activation in spinal processing of visceral stimuli. The effects of selective ERβ agonists on the visceromotor response (VMR) and dorsal horn neuronal responses to colorectal distention (CRD) were tested in ovariectomized and intact female rats. The magnitude of the VMR to CRD was significantly attenuated by ERβ agonists diarylpropionitrile (DPN) and WAY-200070 4 hours after subcutaneous injection. Pretreatment with the estrogen receptor antagonist ICI 182,780 obscured the DPN-evoked attenuation. There was no effect of DPN on the VMR at earlier time points. Subcutaneous and spinal administration of DPN attenuated the response of visceroceptive dorsal horn neurons with a comparable time course. DPN attenuated the VMR in intact rats regardless of estrous cycle stage. The time course of effect of ERβ activation on the visceromotor response and neuronal activity is consistent with transcriptional or translational modulation of neuronal activity. PerspectiveActivation of ERβ is antinociceptive in the colorectal distention model of visceral pain, which may provide a therapeutic target to manage irritable bowel syndrome in the clinic.

Site‐dependent and state‐dependent inhibition of pruritogen‐responsive spinal neurons by scratching

The relief of itch by scratching is thought to involve inhibition of pruritogen-responsive neurons in the spinal cord. We recorded the responses of superficial dorsal horn neurons in mice to intradermal injection of the pruritogens chloroquine and histamine. Scratching within an area 5-17 mm distant from the injection site, outside of the units' mechanoreceptive fields (off-site), significantly inhibited chloroquine-evoked and histamine-evoked responses without affecting capsaicin-evoked firing. This is consistent with observations that scratching at a distance from a site of itch is antipruritic. In contrast, scratching directly at the injection site (within the receptive field; on-site) had no effect on chloroquine-evoked neuronal firing, but enhanced the same neurons' responses to intradermal injection of the algogen capsaicin. Moreover, neuronal responses to histamine were enhanced during on-site scratching, and this was followed by suppression of firing below baseline levels after termination of scratching. Scratching thus inhibits pruritogen-responsive neurons in a manner that depends on the input modality (i.e. pain vs. histamine-dependent or histamine-independent itch) and skin location.

Sprouting of colonic afferent central terminals and increased spinal mitogen‐activated protein kinase expression in a mouse model of chronic visceral hypersensitivity

Visceral pain following infection or inflammation is a major clinical problem. Although we have knowledge of how peripheral endings of colonic afferents change in disease, their central projections have been overlooked. With neuroanatomical tracing and colorectal distension (CRD), we sought to identify colonic afferent central terminals (CACTs), the dorsal horn (DH) neurons activated by colonic stimuli in the thoracolumbar (T10-L1) DH, and determine how they are altered by postinflammatory chronic colonic mechanical hypersensitivity. Retrograde tracing from the colon identified CACTs in the DH, whereas immunohistochemistry for phosphorylated MAP kinase ERK 1/2 (pERK) identified DH neurons activated by CRD (80 mmHg). In healthy mice, CACTs were located primarily in DH laminae I (LI) and V (LV) and projected down middle and lateral DH collateral pathways. CRD evoked pERK immunoreactivity in DH neurons, the majority of which were located in LI and LV, the same regions as CACTs. In postinflammatory mice, CACTs were significantly increased in T12-L1 compared with healthy mice. Although CACTs remained abundant in LI, they were more widespread and were now present in deeper laminae. After CRD, significantly more DH neurons were pERK-IR postinflammation (T12-L1), with abundant expression in LI and deeper laminae. In both healthy and postinflammatory mice, many pERK neurons were in close apposition to CACTs, suggesting that colonic afferents can stimulate specific DH neurons in response to noxious CRD. Overall, we demonstrate that CACT density and the number of responsive DH neurons in the spinal cord increase postinflammation, which may facilitate aberrant central representation of colonic nociceptive signaling following chronic peripheral hypersensitivity.

Effective Connectivity Among Brain Regions Associated With Slow Temporal Summation of C-Fiber-Evoked Pain in Fibromyalgia Patients and Healthy Controls

Temporal summation of “second pain” (TSSP) or “windup” results from the summation of C-fiber-evoked responses of dorsal-horn neurons. This phenomenon is dependent on stimulus frequency (≥.33 Hz) and relevant to central sensitization and chronic pain. Our previous neuroimaging studies characterized brain regions associated with TSSP in normal control (NC) and fibromyalgia (FM) groups. During an fMRI scan, subjects received sensitivity-adjusted repetitive heat pulses at .33 on the right foot. FM subjects required significantly lower stimulus intensities than NC to achieve similar TSSP and no significant group differences in the pain-related brain activity were detected. In our current study, we asked whether the effective connectivity among a set of TSSP-related brain regions identified in our previous work differs amongst FM and NC groups. Structural equation modeling was used to characterize the effective connectivity amongst a priori selected brain areas, including the thalamus, S1, S2, posterior insula, and the anterior midcingulate cortex (aMCC) within the left and right hemispheres. This analysis confirmed our a priori models of effective connectivity among these regions mainly confirmed those hypothesized, yet some unpredicted connections were additionally identified (thalamus to aMCC and aMCC to S1). While the models of effective connectivity were not identical in the FM and NC groups, they were very similar. Additionally, the TSSP related effective connectivity of right and left hemisphere regions was very similar. These results provide evidence for significant overlap of the fundamental brain mechanisms that process sensory and affective information related to TSSP in NC and FM groups. PerspectiveModels of effective connectivity involving pain-related processes were estimated with fMRI data from chronic pain and healthy populations. Models were estimated in both hemispheres, and although similar, fibromyalgia was associated with unique models of pain-related processes. Group differences involved the left hemisphere and S1, S2, and posterior insula.

Spontaneous firing and evoked responses of spinal nociceptive neurons are attenuated by blockade of P2X3 and P2X2/3 receptors in inflamed rats

P2X3 and P2X2/3 receptors are selectively expressed on primary afferent nociceptors and have been implicated in modulating nociception in different models of pathological pain, including inflammatory pain. In an effort to delineate further the role of P2X3 receptors (homomeric and heteromeric) in the modulation of nociceptive transmission after a chronic inflammation injury, A-317491, a potent and selective P2X3-P2X2/3 antagonist, was administered to CFA-inflamed rats in order to examine its effects on responses of spinal dorsal horn neurons to mechanical and thermal stimulation. Systemic injection of A-317491 (30 μmol/kg, i.v.) reduced the responses of wide-dynamic-range (WDR) and nociceptive specific (NS) neurons to both high-intensity mechanical (pinch) and heat (49°C) stimulation. A-317491 also decreased low-intensity (10 g von Frey hair) mechanically evoked activity of WDR neurons but did not alter WDR neuronal responses to cold stimulation (5°C). Spontaneous firing of WDR neurons in CFA-inflamed rats was also significantly attenuated by A-317491 injection. By using immunohistochemistry, P2X3 receptors were demonstrated to be enhanced in lamina II of the spinal dorsal horn after inflammation. In summary, blockade of P2X3 and P2X2/3 receptors dampens mechanical- and heat-related signaling, as well as nonevoked activity of key classes of spinal nociceptive neurons in inflamed animals. These data suggest that P2X3 and/or P2X2/3 receptors have a broad contribution to somatosensory/nociceptive transmission in rats with a chronic inflammatory injury and are consistent with previous behavioral data demonstrating antiallodynic and antihyperalgesic effects of receptor antagonists.

Functional significance of M-type potassium channels in nociceptive cutaneous sensory endings.

M-channels carry slowly activating potassium currents that regulate excitability in a variety of central and peripheral neurons. Functional M-channels and their Kv7 channel correlates are expressed throughout the somatosensory nervous system where they may play an important role in controlling sensory nerve activity. Here we show that Kv7.2 immunoreactivity is expressed in the peripheral terminals of nociceptive primary afferents. Electrophysiological recordings from single afferents in vitro showed that block of M-channels by 3 μM XE991 sensitized Aδ- but not C-fibers to noxious heat stimulation and induced spontaneous, ongoing activity at 32°C in many Aδ-fibers. These observations were extended in vivo: intraplantar injection of XE991 selectively enhanced the response of deep dorsal horn (DH) neurons to peripheral mid-range mechanical and higher range thermal stimuli, consistent with a selective effect on Aδ-fiber peripheral terminals. These results demonstrate an important physiological role of M-channels in controlling nociceptive Aδ-fiber responses and provide a rationale for the nocifensive behaviors that arise following intraplantar injection of the M-channel blocker XE991.