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

  • Mark H. Pitcher
    Address reprint requests to Mark H. Pitcher, Anesthesia Research Unit, McGill University, 3655 Promenade Sir William Osler (room 1207), Montréal, Québec, Canada H3G 1Y6.
    Anesthesia Research Unit, Faculty of Medicine, McGill University, Montréal, Québec, Canada

    Alan Edwards Center for Research on Pain, McGill University, Montréal, Québec, Canada
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  • Francisco R. Nieto
    Department of Pharmacology, Faculty of Medicine and Institute of Neuroscience, Biomedical Research Center, University of Granada, Granada, Spain
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  • Fernando Cervero
    Anesthesia Research Unit, Faculty of Medicine, McGill University, Montréal, Québec, Canada

    Alan Edwards Center for Research on Pain, McGill University, Montréal, Québec, Canada
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Published:November 29, 2012DOI:


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


      While touch normally soothes acute pain, we demonstrate that following peripheral inflammation, touch evokes pain (allodynia) through the switching of a normally inhibitory spinal pathway into an excitatory pathway. Activation of low threshold mechanoreceptors activates spinal nociceptive neurons following inflammation-induced enhancement of NKCC1 expression, as measured by spinal c-Fos labeling.

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        • Alvarez-Leefmans F.J.
        • Gamino S.M.
        • Giraldez F.
        • Nogueron I.
        Intracellular chloride regulation in amphibian dorsal root ganglion neurones studied with ion-selective microelectrodes.
        J Physiol. 1988; 406: 225-246
      1. Alvarez-Leefmans F.J. Alvarez-Leefmans F.J. Russell J.M. Chloride Channels and Carriers in Nerve, Muscle, and Glial Cells. Plenum Publishing Corp, New York, NY1990: 109-158
        • Alvarez-Leefmans F.J.
        • Nani A.
        • Marquez S.
        Presynaptic inhibition and neural control.
        in: Rudomin P. Romo R. Mendell L.M. Chloride Transport, Osmotic Balance, and Presynaptic Inhibition. Oxford University Press, New York, NY1998: 50-79
        • Bester H.
        • Beggs S.
        • Woolf C.J.
        Changes in tactile stimuli-induced behavior and c-Fos expression in the superficial dorsal horn and in parabrachial nuclei after sciatic nerve crush.
        J Comp Neurol. 2000; 428: 45-61
        • Brater D.C.
        Clinical pharmacology of loop diuretics in health and disease.
        Eur Heart J. 1992; 13: 10-14
        • Catheline G.
        • Le Guen S.
        • Honoré P.
        • Besson J.M.
        Are there long-term changes in the basal or evoked c-Fos expression in the dorsal horn of the spinal cord of the mononeuropathic rat?.
        Pain. 1999; 80: 347-357
        • Catheline G.
        • Le Guen S.
        • Besson J.M.
        Intravenous morphine does not modify dorsal horn touch-evoked allodynia in the mononeuropathic rat: a c-Fosstudy.
        Pain. 2001; 92: 389-398
        • Cervero F.
        • Laird J.M.
        Mechanisms of touch-evoked pain (allodynia): a new model.
        Pain. 1996; 68: 13-23
        • Cervero F.
        • Laird J.M.
        • García-Nicas E.
        Secondary hyperalgesia and presynaptic inhibition: an update.
        Eur J Pain. 2003; 7: 345-351
        • Chabwine J.N.
        • Talavera K.
        • Verbert L.
        • Eggermont J.
        • Vanderwinden J.M.
        • De Smedt H.
        • Van Den Bosch L.
        • Robberecht W.
        • Callewaert G.
        Differential contribution of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 to chloride handling in rat embryonic dorsal root ganglion neurons and motor neurons.
        FASEB J. 2009; 23: 1168-1176
        • Coggeshall R.E.
        c-Fos, nociception and the dorsal horn.
        Prog Neurobiol. 2005; 77: 299-352
        • Coull J.A.
        • Boudreau D.
        • Bachand K.
        • Prescott S.A.
        • Nault F.
        • Sík A.
        • De Koninck P.
        • De Koninck Y.
        Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain.
        Nature. 2003; 424: 938-942
        • Eijkelkamp N.
        • Kavelaars A.
        • Elsenbruch S.
        • Schedlowski M.
        • Holtmann G.
        • Heijnen C.J.
        Increased visceral sensitivity to capsaicin after DSS-induced colitis in mice: spinal cord c-Fos expression and behavior.
        Am J Physiol Gastrointest Liver Physiol. 2007; 293: G749-G757
        • Eijkelkamp N.
        • Heijnen C.J.
        • Elsenbruch S.
        • Holtmann G.
        • Schedlowski M.
        • Kavelaars A.
        G protein-coupled receptor kinase 6 controls post-inflammatory visceral hyperalgesia.
        Brain Behav Immun. 2009; 23: 18-26
        • Funk K.
        • Woitecki A.
        • Franjic-Wurtz C.
        • Gensch T.
        • Mohrlen F.
        • Frings S.
        Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons.
        Mol Pain. 2008; 4: 32
        • Galan A.
        • Laird J.M.A.
        • Cervero F.
        In vivo recruitment by painful stimuli of AMPA receptor subunits to the plasma membrane of spinal cord neurons.
        Pain. 2004; 112: 315-323
        • Galan A.
        • Cervero F.
        Painful stimuli induce in vivo phosphorylation and membrane mobilization of mouse spinal cord NKCC1 co-transporter.
        Neuroscience. 2005; 133: 245-252
        • Gilbert D.
        • Franjic-Wurtz K.
        • Funk C.
        • Gensch T.
        • Frings S.
        • Mohrlen F.
        Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system.
        Int J Devl Neuroscience. 2007; 25: 479-489
        • Goransson I.
        • Cederlund R.
        A study of the effect of desensitization on hyperaesthesia in the hand and upper extremity after injury or surgery.
        Hand Therapy. 2011; 16: 12-18
        • Harris J.A.
        Using c-Fos as a neural marker of pain.
        Brain Res Bull. 1998; 45: 1-8
        • Hayashi E.
        • Kobayashi T.
        • Shiroshita Y.
        • Kuratani K.
        • Kinoshita M.
        • Hara H.
        An automated evaluation system for analyzing antinociceptive effects on intracolonic capsaicin-induced visceral pain-related licking behavior in mice.
        J Pharmaco Toxico Meth. 2011; 64: 119-123
        • Honore P.
        • Rogers S.D.
        • Schwei M.J.
        • Salak-Johnson J.L.
        • Luger N.M.
        • Sabino M.C.
        • Clohisy D.R.
        • Mantyh P.W.
        Murine models of inflammatory, neuropathic and cancer pain each generates a unique set of neurochemical changes in the spinal cord and sensory neurons.
        Neuroscience. 2000; 98: 585-598
        • Isenring P.
        • Jacoby S.C.
        • Forbush III, B.
        The role of transmembrane domain 2 in cation transport by the Na–K–Cl cotransporter.
        Proc Natl Acad Sci U S A. 1998; 95: 7179-7184
        • Jankowska E.
        • McCrea D.
        • Rudomin P.,
        • Sykova E.
        bservations on neuronal pathways subserving primary afferent depolarization.
        J Neurophysiol. 1981; 46: 506-516
        • Kosai K.
        • Tateyama S.
        • Ikeda T.
        • Uno T.
        • Nishimori T.
        • Takasaki M.
        MK-801 reduces non-noxious stimulus-evoked c-Fos-like immunoreactivity in the spinal cord of rats with chronic constriction nerve injury.
        Brain Res. 2001; 910: 12-18
        • Laird J.M.
        • García-Nicas E.
        • Delpire E.J.
        • Cervero F.
        Presynaptic inhibition and spinal pain processing in mice: a possible role of the NKCC1 cation-chloride co-transporter in hyperalgesia.
        Neurosci Lett. 2004; 361: 200-203
        • Laird J.M.
        • Martinez-Caro L.
        • Garcia-Nicas E.
        • Cervero F.
        A new model of visceral pain and referred hyperalgesia in the mouse.
        Pain. 2001; 92: 335-342
        • Laird J.M.A.
        • Olivar C.
        • Roza C.
        • De Felipe C.
        • Hunt S.P.
        • Cervero F.
        Deficits in visceral pain and hyperalgesia of mice with a disruption of the tachykinin NK1 receptor gene.
        Neuroscience. 2000; 98: 345-352
        • Laird J.M.A.
        • Souslova V.
        • Wood J.N.
        • Cervero F.
        Deficits in visceral pain and referred hyperalgesia in Nav1.8 (SNS/ PN3)-null mice.
        J Neurosci. 2002; 22: 8352-8356
        • Li Y.Q.
        • Li H.
        • Wei J.
        • Qu L.
        • Wu L.A.
        Expression changes of K+-Cl- co- transporter 2 and Na+-K+-Cl- co-transporter1 in mouse trigeminal subnucleus caudalis following pulpal inflammation.
        Brain Res Bull. 2010; 81: 561-564
        • Ma Q.P.
        • Woolf C.J.
        Basal and touch-evoked c-Fos-like immunoreactivity during experimental inflammation in the rat.
        Pain. 1996; 67: 307-316
        • Mansikka H.
        • Erbs E.
        • Borrelli E.
        • Pertovaara A.
        Influence of the dopamine D2 receptor knockout on pain-related behavior in the mouse.
        Brain Res. 2008; 1052: 82-87
        • Mitrovic M.
        • Shahbazian A.
        • Bock E.
        • Pabst M.A.
        • Holzer P.
        Chemo-nociceptive signalling from the colon is enhanced by mild colitis and blocked by inhibition of transient receptor potential ankyrin 1 channels.
        Brit J Pharmaco. 2010; 160: 1430-1442
        • Molander C.
        • Hongpaisan J.
        • Grant G.
        Changing pattern of c-FOS expression in spinal cord neurons after electrical stimulation of the chronically injured sciatic nerve in the rat.
        Neuroscience. 1992; 50: 223-236
        • Molander C.
        • Hongpaisan J.
        • Grant G.
        Distribution of c-Fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve.
        Brain Res. 1994; 644: 74-82
        • Morales-Aza B.M.
        • Chillingworth N.L.
        • Payne J.A.
        • Donaldson L.F.
        Inflammation alters cation chloride cotransporter expression in sensory neurons.
        Neurobiol Dis. 2004; 17: 62-69
        • Paxinos G.
        • Watson C.
        The rat brain in stereotaxic coordinates.
        Elsevier Academic Press, London, UK2005
        • Payne J.A.
        Functional characterization of the neuronal-specific K-Cl cotransporter: implications for [K+]o regulation.
        Am J Physiol. 1997; 273: C1516-C1525
        • Pieraut S.
        • Laurent-Matha V.
        • Sar C.
        • Hubert T.
        • Méchaly I.
        • Hilaire C.
        • Mersel M.
        • Delpire E.
        • Valmier J.
        • Scamps F.
        NKCC1 phosphorylation stimulates neurite growth of injured adult sensory neurons.
        J Neurosci. 2007; 27: 6751-6759
        • Pitcher M.H.
        • Cervero F.
        Role of the NKCC1 co-transporter in sensitization of spinal nociceptive neurons.
        Pain. 2010; 151: 756-762
        • Pitcher M.H.
        • Price T.J.
        • Entrena J.M.
        • Cervero F.
        Spinal NKCC1 blockade inhibits TRPV1-dependent referred allodynia.
        Mol Pain. 2007; 3: 17
        • Price T.J.
        • Hargreaves K.M.
        • Cervero F.
        Protein expression and mRNA cellular distribution of the NKCC1 cotransporter in the dorsal root and trigeminal ganglia of the rat.
        Brain Res. 2006; 1112: 146-158
        • Rocha-González H.I.
        • Mao S.
        • Alvarez-Leefmans F.J.
        Na+, K+,2Cl- cotransport and intracellular chloride regulation in rat primary sensory neurons: thermodynamic and kinetic aspects.
        J Neurophysiol. 2008; 100: 169-184
        • Shortland P.
        • Molander C.
        The time-course of abeta-evoked c-Fosexpression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury.
        Brain Res. 1998; 810: 288-293
        • Shortland P.
        • Molander C.
        Alterations in the distribution of stimulus-evoked c-Fosin the spinal cord after neonatal peripheral nerve injury in the rat.
        Brain Res Dev Brain Res. 2000; 119: 243-250
        • Sung K.W.
        • Kirby M.
        • McDonald M.P.
        • Lovinger D.M.
        • Delpire E.
        Abnormal GABAA receptor-mediated currents in dorsal root ganglion neurons isolated from Na-K-2Cl cotransporter null mice.
        J Neurosci. 2000; 20: 7531-7538
        • Tokunaga A.
        • Kondo E.
        • Fukuoka T.
        • Miki K.
        • Dai Y.
        • Tsujino H.
        • Noguchi K.
        Excitability of spinal cord and gracile nucleus neurons in rats with chronically injured sciatic nerve examined by c-Fosexpression.
        Brain Res. 1999; 847: 321-331
        • Treede R.D.
        • Meyer R.A.
        • Raja S.N.
        • Campbell J.N.
        Peripheral and central mechanisms of cutaneous hyperalgesia.
        Prog Neurobiol. 1992; 38: 397-421
        • Tsai Y.C.
        • So E.C.
        • Chen H.H.
        • Wang L.K.
        • Chien C.H.
        Effect of intrathecal octreotide on thermal hyperalgesia and evoked spinal c-Fosexpression in rats with sciatic constriction injury.
        Pain. 2002; 99: 407-413
        • Valencia-de Ita S.
        • Lawand N.B.
        • Lin Q.
        • Castañeda-Hernandez G.
        • Willis W.D.
        Role of the Na+ K+ 2Cl- cotransporter in the development of capsaicin-induced neurogenic inflammation.
        J Neurophysiol. 2006; 95: 3553-6351
        • Wei F.
        • Dubner R.
        • Ren K.
        Dorsolateral funiculus-lesions unmask inhibitory or disfacilitatory mechanisms which modulate the effects of innocuous mechanical stimulation on spinal c-Fosexpression after inflammation.
        Brain Res. 1999; 820: 112-116
        • Willis W.D.
        Dorsal root potentials and dorsal root reflexes: a double-edged sword.
        Exp Brain Res. 1999; 124: 395-421