Elsevier

Neuroscience

Volume 256, 3 January 2014, Pages 392-402
Neuroscience

Neonatal tissue injury reduces the intrinsic excitability of adult mouse superficial dorsal horn neurons

https://doi.org/10.1016/j.neuroscience.2013.10.057Get rights and content

Highlights

  • Neonatal injury alters the passive membrane properties of adult dorsal horn neurons.

  • Early incision reduces intrinsic membrane excitability in the mature dorsal horn.

  • Kir channels regulate membrane excitability in adult mouse dorsal horn cells.

  • Neonatal incision up-regulates Kir currents selectively in adult GABAergic neurons.

Abstract

Tissue damage during the neonatal period evokes long-lasting changes in nociceptive processing within the adult spinal cord which contribute to persistent alterations in pain sensitivity. However, it remains unclear if the observed modifications in neuronal activity within the mature superficial dorsal horn (SDH) following early injury reflect shifts in the intrinsic membrane properties of these cells. Therefore, the present study was undertaken to identify the effects of neonatal surgical injury on the intrinsic excitability of both GABAergic and presumed glutamatergic neurons within lamina II of the adult SDH using in vitro patch clamp recordings from spinal cord slices prepared from glutamic acid decarboxylase-green fluorescent protein (Gad-GFP) mice. The results demonstrate that hindpaw surgical incision at postnatal day (P) 3 altered the passive membrane properties of both Gad-GFP and adjacent, non-GFP neurons in the mature SDH, as evidenced by decreased membrane resistance and more negative resting potentials in comparison to naïve littermate controls. This was accompanied by a reduction in the prevalence of spontaneous activity within the GABAergic population. Both Gad-GFP and non-GFP neurons displayed a significant elevation in rheobase and decreased instantaneous firing frequency after incision, suggesting that early tissue damage lowers the intrinsic membrane excitability of adult SDH neurons. Isolation of inward-rectifying K+ (Kir) currents revealed that neonatal incision significantly increased Kir conductance near physiological membrane potentials in GABAergic, but not glutamatergic, lamina II neurons. Overall, these findings suggest that neonatal tissue injury causes a long-term dampening of intrinsic firing across the general population of lamina II interneurons, but the underlying ionic mechanisms may be cell-type specific.

Introduction

The processing of noxious stimuli within the CNS begins in the superficial dorsal horn (SDH) of the spinal cord, where a complex network of excitatory and inhibitory interneurons integrates sensory inputs and strongly regulates the output of the spinal pain circuit by modulating the excitability of a small population of neurons which send ascending projections to the brain (Todd, 2010). Mounting evidence suggests that the level of activity within mature dorsal horn neurons is significantly influenced by sensory experience during the early postnatal period. For example, in vivo electrophysiological studies using extracellular recordings have demonstrated that skin wounding in the newborn rat leads to enlarged receptive fields in dorsal horn neurons at 6 weeks post-injury (Torsney and Fitzgerald, 2003). Elevated rates of spontaneous activity and exaggerated firing in response to mechanical stimulation have also been reported in the adult dorsal horn in vivo after peripheral inflammation during the neonatal period (Peng et al., 2003). This documented hyperexcitability following early tissue damage could be explained by long-term alterations in the balance of synaptic excitation vs. inhibition onto adult SDH neurons and/or modifications in their intrinsic membrane properties which in turn modulate their excitability in a cell-autonomous manner. While in vivo extracellular recordings are invaluable in measuring the responses of dorsal horn cells to natural sensory stimuli, this technique cannot distinguish between these potential underlying mechanisms.

Recent studies have focused on identifying changes in synaptic connectivity occurring within the mature SDH network following transient injuries sustained during the neonatal period. Deficits in both phasic and tonic glycinergic transmission have been observed in the adult SDH following neonatal surgical injury (Li et al., 2013a), while stronger descending inhibition to the mature dorsal horn has been reported after peripheral inflammation during early life (Zhang et al., 2010), which may be mediated by a potentiation in opioidergic tone in the CNS (Laprairie and Murphy, 2009). However, it remains unclear whether neonatal tissue damage evokes persistent alterations in the intrinsic firing properties of developing SDH neurons. It is known that the intrinsic membrane properties of SDH neurons are developmentally regulated in a cell-type specific manner (Walsh et al., 2009, Li and Baccei, 2011, Li and Baccei, 2012), and significant changes in the transcription of genes encoding voltage-dependent and voltage-independent ion channels occur during the first postnatal weeks (Blankenship et al., 2013). Given the clear importance of neuronal activity in the modulation of gene expression (Lyons and West, 2011), perturbations in sensory input resulting from injuries during this sensitive developmental period may have long-term consequences for the electrophysiological phenotype of mature SDH neurons.

Therefore, the present study was undertaken to elucidate the persistent effects of neonatal surgical injury on the intrinsic membrane excitability of both inhibitory and presumed excitatory interneurons within lamina II of the adult mouse spinal cord.

Section snippets

Ethical approval

All experiments adhered to animal welfare guidelines established by the University of Cincinnati Institutional Animal Care and Use Committee which approved this study.

Hindpaw surgical incision

At postnatal day (P)3, female glutamic acid decarboxylase-green fluorescent protein (Gad-GFP) mice (FVB-Tg(GadGFP)4570Swn; Jackson Labs, Bar Harbor, ME, USA), which express enhanced GFP (eGFP) under the control of the GAD67 promoter (Oliva et al., 2000), were anesthetized with isoflurane (2–3%) and a small incision made through

Neonatal tissue injury modifies the intrinsic membrane properties of adult SDH neurons

To identify long-term changes in the intrinsic excitability of mature SDH neurons after neonatal tissue damage, unilateral hindpaw surgical incision (Brennan et al., 1996) was administered at postnatal day (P)3 in Gad-GFP mice, which selectively express eGFP in GABAergic neurons (Oliva et al., 2000). Naïve littermate-matched controls (handled in an identical manner including exposure to anesthesia) were used for all experiments. At P49–63, in vitro whole-cell patch clamp recordings were

Discussion

This study demonstrates, for the first time, that the intrinsic membrane properties of adult SDH neurons are shaped by sensory experience during early postnatal development. Surgical injury during the neonatal period evoked a reduction in neuronal excitability across multiple subpopulations within lamina II of the mature spinal cord. These results also confirm that Kir channels are potent regulators of membrane excitability in adult SDH neurons and further suggest that injury-evoked changes in

Future directions

It will ultimately be important to elucidate the somatotopy of these changes in intrinsic excitability within the mature SDH following early tissue damage. In other words, are these alterations in membrane properties restricted to the regions of the SDH which receive direct projections from primary afferents innervating the injury site, or do similar shifts in excitability occur throughout the rostrocaudal axis of the spinal cord? Notably, the long-term hypoalgesia seen in rodents following

Conclusions

The present findings demonstrate that the intrinsic firing properties of neurons within adult spinal pain circuits are highly sensitive to noxious sensory experience during the neonatal period. As a result, this study adds to the growing body of work illustrating that early trauma can have lifelong consequences for nociceptive processing in the CNS.

Acknowledgments

This work was supported by the U.S. National Institutes of Health (NS072202 to M.L.B.). The authors would also like to thank Elizabeth Kritzer for technical assistance.

References (52)

  • E. Polgar et al.

    Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain

    Pain

    (2003)
  • K. Ren et al.

    Characterization of basal and re-inflammation-associated long-term alteration in pain responsivity following short-lasting neonatal local inflammatory insult

    Pain

    (2004)
  • R.S. Saliba et al.

    Blocking L-type voltage-gated Ca2+ channels with dihydropyridines reduces gamma-aminobutyric acid type A receptor expression and synaptic inhibition

    J Biol Chem

    (2009)
  • A.J. Watt et al.

    Activity coregulates quantal AMPA and NMDA currents at neocortical synapses

    Neuron

    (2000)
  • T. Yasaka et al.

    Populations of inhibitory and excitatory interneurons in lamina II of the adult rat spinal dorsal horn revealed by a combined electrophysiological and anatomical approach

    Pain

    (2010)
  • Y.H. Zhang et al.

    Effects of neonatal inflammation on descending modulation from the rostroventromedial medulla

    Brain Res Bull

    (2010)
  • L. Bremner et al.

    Functional GABA(A)-receptor-mediated inhibition in the neonatal dorsal horn

    J Neurophysiol

    (2006)
  • T.J. Brennan et al.

    Characterization of a rat model of incisional pain

    Pain

    (1996)
  • J. Burrone et al.

    Multiple forms of synaptic plasticity triggered by selective suppression of activity in individual neurons

    Nature

    (2002)
  • W.A. Coetzee et al.

    Molecular diversity of K+ channels

    Ann N Y Acad Sci

    (1999)
  • D. Derjean et al.

    Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states

    Nat Neurosci

    (2003)
  • A.S. Dhamoon et al.

    Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current

    Circ Res

    (2004)
  • S.A. Goldstein et al.

    Potassium leak channels and the KCNK family of two-P-domain subunits

    Nat Rev Neurosci

    (2001)
  • B.A. Graham et al.

    Recording temperature affects the excitability of mouse superficial dorsal horn neurons, in vitro

    J Neurophysiol

    (2008)
  • T.J. Grudt et al.

    Correlations between neuronal morphology and electrophysiological features in the rodent superficial dorsal horn

    J Physiol

    (2002)
  • G.J. Hathway et al.

    The changing balance of brainstem-spinal cord modulation of pain processing over the first weeks of rat postnatal life

    J Physiol

    (2009)
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