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