Neonatal tissue damage facilitates nociceptive synaptic input to the developing superficial dorsal horn via NGF-dependent mechanisms

Abstract

Tissue injury during a critical period of early life can facilitate spontaneous glutamatergic transmission within developing pain circuits in the superficial dorsal horn (SDH) of the spinal cord. However, the extent to which neonatal tissue damage strengthens nociceptive synaptic input to specific subpopulations of SDH neurons, as well as the mechanisms underlying this distinct form of synaptic plasticity, remains unclear. Here we use in vitro whole-cell patch clamp recordings from rodent spinal cord slices to demonstrate that neonatal surgical injury selectively potentiates high-threshold primary afferent input to immature lamina II neurons. In addition, the increase in the frequency of miniature excitatory postsynaptic currents after hindpaw incision was prevented by neonatal capsaicin treatment, suggesting that early tissue injury enhances glutamate release from nociceptive synapses. This occurs in a widespread manner within the developing SDH, as incision elevated miniature excitatory postsynaptic current frequency in both GABAergic and presumed glutamatergic lamina II neurons of Gad-GFP transgenic mice. The administration of exogenous nerve growth factor into the rat hindpaw mimicked the effects of early tissue damage on excitatory synaptic function, while blocking trkA receptors in vivo abolished the changes in both spontaneous and primary afferent-evoked glutamatergic transmission following incision. These findings illustrate that neonatal tissue damage can alter the gain of developing pain pathways by activating nerve growth factor-dependent signaling cascades, which modify synaptic efficacy at the first site of nociceptive processing within the central nervous system.

Fig. 1

Neonatal hindpaw incision enhances spontaneous excitatory signaling within the female rat SDH. (A) Examples of mEPSCs recorded at a holding potential (V_h) of −70 mV (top) and mIPSCs isolated at a V_h of 0 mV (bottom) from the same lamina II neuron. (B) Surgical injury at postnatal day (P) 3 significantly increased the frequency (left; **p = 0.004; Mann-Whitney test) but not amplitude (right) of mEPSCs across the general population of lamina II cells at P5–6. (C, D) Separation of the data according to the sex of the pup revealed a selective increase in mEPSC frequency within the female SDH (C; left; **p = 0.009; Mann-Whitney test) using this model of tissue damage.

Fig. 2

Minimal stimulation of low-threshold vs. high-threshold sensory inputs to developing SDH neurons. (A) Plot of monosynaptic EPSC amplitude vs. intensity of primary afferent stimulation illustrating an example of an EPSC mediated by low-threshold sensory inputs to the immature rat SDH (see inset). Note that increasing the level of dorsal root stimulation to 5X threshold failed to recruit any additional synaptic inputs to this lamina II neuron. Scale bar: 50 pA, 5 ms. (B) A similar step-like increase in EPSC amplitude is seen in a different lamina II cell (inset), but at a ~50-fold higher threshold than seen in A. Scale bar: 25 pA, 5 ms. (C) Histogram showing the distribution of monosynaptic EPSC thresholds across the sampled population of lamina II neurons at P3–4.

Fig. 3

Early tissue damage selectively increases the amplitude of high-threshold (HT) primary afferent inputs to lamina II neurons. (A – C) Surgical incision of the rat hindpaw at P2 failed to significantly alter the amplitude (A) and coefficient of variation (B) of EPSCs mediated by low-threshold sensory afferents, as well as the response to paired stimulation (C). (D) P2 incision significantly increased the amplitude of monosynaptic EPSCs evoked by high-threshold stimulation of the attached dorsal root in the same spinal cord slices (**p = 0.005; Mann-Whitney test). (E) Neonatal injury also decreased the CV of HT-mediated EPSCs in the same lamina II neurons (*p = 0.015; Mann-Whitney test). (F) The paired-pulse ratio of HT-mediated EPSCs was unaffected by the hindpaw incision.

Fig. 4

TRPV1-expressing primary afferents are involved in the facilitation of spontaneous glutamatergic signaling within the rat SDH following neonatal incision. (A) While mEPSC frequency in lamina II neurons is significantly elevated following P3 hindpaw incision in pups receiving vehicle injections from birth (left; *p = 0.012; Mann-Whitney test), no such increase is seen in pups pre-treated with systemic capsaicin at P0–1 (right). (B) Incision at P3 failed to change mEPSC amplitude in both the vehicle (left) and capsaicin-treated (right) pups.

Fig. 5

Early surgical injury strengthens glutamatergic drive onto both inhibitory and presumed excitatory interneurons within lamina II of the developing spinal cord. (A, B) Examples of sagittal spinal cord sections at P4–5 illustrating the distribution of Gad-GFP neurons throughout the L4–L5 dorsal horn. Scale bars: A, 250 μm; B, 50 μm. (C, D) Hindpaw incision at P3 increased mEPSC frequency (left; *p < 0.05; Mann-Whitney test) in both GABAergic (C) and presumed glutamatergic (D) lamina II neurons without altering mEPSC amplitude (right).

Fig. 6

Exogenous NGF delivered to the hindpaw is sufficient to evoke an age-dependent facilitation of excitatory signaling in the immature SDH and the effects of surgical injury on glutamatergic function are not observed after NGF pretreatment. (A) Injection of NGF into the hindpaw at P3–4 significantly increased mEPSC frequency in rat lamina II neurons at P5–6 compared to vehicle-treated littermates (left; **p = 0.001; Mann-Whitney test) without altering mEPSC amplitude (right). (B) NGF administration from P17–18 failed to modulate spontaneous glutamatergic transmission compared to vehicle controls at P19–20. (C, D) mEPSC frequency is significantly elevated by P3 hindpaw incision in pups injected with vehicle from P2–4 (C, left; *p = 0.042; Mann-Whitney test), but not in pups treated with NGF during the same period (D, left).

Fig. 7

Disruption of trkA signaling in vivo abolishes the effects of neonatal surgical injury on spontaneous glutamatergic transmission in the developing SDH. (A) Hindpaw incision significantly increased the mEPSC frequency (left), but not amplitude (right), in pups receiving injections of a vehicle solution at P2–3 (**p = 0.005; Mann-Whitney test). (B) This increase failed to occur when the pups were treated with the selective trkA inhibitor VMD902 during the same time period (left; p = 0.678).

Fig. 8

trkA inhibition prevents the enhancement of high-threshold (HT) primary afferent input to the SDH following early tissue damage. (A – C) There were no significant effects of P2 hindpaw incision on EPSC amplitude (A), coefficient of variation (B) or paired-pulse ratio (C) in pups which were treated with VMD902 prior to the injury.