Repeated touch and needle-prick stimulation in the neonatal period increases the baseline mechanical sensitivity and postinjury hypersensitivity of adult spinal sensory neurons

Abstract

Noxious stimulation at critical stages of development has long-term consequences on somatosensory processing in later life, but it is not known whether this developmental plasticity is restricted to nociceptive pathways. Here, we investigate the effect of repeated neonatal noxious or innocuous hind paw stimulation on adult spinal dorsal horn cutaneous mechanical sensitivity. Neonatal Sprague-Dawley rats of both sexes received 4 unilateral left hind paw needle pricks (NPs, n = 13) or 4 tactile (cotton swab touch) stimuli, per day (TC, n = 11) for the first 7 days of life. Control pups were left undisturbed (n = 17). When adult (6-8 weeks), lumbar wide-dynamic-range neuron activity in laminae III-V was recorded using in vivo extracellular single-unit electrophysiology. Spike activity evoked by cutaneous dynamic tactile (brush), pinch and punctate (von Frey hair) stimulation, and plantar receptive field areas were recorded, at baseline and 2 and 5 days after left plantar hind paw incision. Baseline brush receptive fields, von Frey hair, and pinch sensitivity were significantly enhanced in adult NP and TC animals compared with undisturbed controls, although effects were greatest in NP rats. After incision, injury sensitivity of adult wide-dynamic-range neurons to both noxious and dynamic tactile hypersensitivity was significantly greater in NP animals compared with TC and undisturbed controls. We conclude that both repeated touch and needle-prick stimulation in the neonatal period can alter adult spinal sensory neuron sensitivity to both innocuous and noxious mechanical stimulation. Thus, spinal sensory circuits underlying touch and pain processing are shaped by a range of early-life somatosensory experiences.

Figure 1.

Mechanical sensitivity during the first week of life after repeated neonatal procedures. (A) The time course of the experiment. Rat pups (littermates) either received 4 tactile stimuli (cotton swab to the left hind paw, N = 9 males and 2 females) or noxious NPs (N = 10 males and 3 females). Mechanical withdrawal thresholds in the ipsilateral and contralateral paws were tested daily using calibrated von Frey filaments, at baseline (before any repeated neonatal procedures), and at 1, 3, and 5 hours after. (B and C) Changes in mechanical withdrawal thresholds after repeated neonatal procedures in the ipsilateral (B) and contralateral (C) hind paw. Mechanical withdrawal threshold increased after the third postnatal day in tactile controls. Baseline mechanical sensitivity was comparable between the 2 neonatal procedure groups. However, after NPs, ipsilateral mechanical withdrawal thresholds were significantly lower compared with tactile controls, indicating the development of mechanical allodynia from postnatal day 3 to 7. In addition, neonatal NPs led to significantly lower mechanical thresholds in the contralateral hind paw on postnatal day 6 and 7. BL, baseline measurement; NP, needle prick; P(0-7), postnatal day 0 to 7; _1, _3, and _5: measurement 1, 3, and 5 hours after NP/tactile stimulus; TC, tactile control stimulus. Data presented as mean ± SEM, *P < 0.05, **P < 0.01, ****P < 0.0001, between treatment comparisons.

Figure 2.

Baseline sensitivity to cutaneous brush and noxious pinch stimulation in adult dorsal horn neurons after neonatal repeated procedures. (A) Time course of the experiment. Pups received repeated NP or TC neonatal stimuli as shown in Figure 1. A different group of pups were left undisturbed (UC). At 6 to 8 weeks of age, single-unit extracellular recordings were performed in the ipsilateral (B–E) and contralateral (F) spinal dorsal horn. Cutaneous brush– and pinch-evoked spike activity and receptive field areas were recorded in wide-dynamic-range neurons. (B) Brush-evoked spike activity was significantly greater in NP animals compared with UC. (C) Brush receptive field areas were significantly larger in NP and TC animals compared with UC, and significantly larger in NP compared with TC. (D) Pinch-evoked firing activity was significantly greater in NP compared with UC. (E) Repeated neonatal procedures significantly increased pinch receptive fields in adulthood, and this effect was greatest in NP animals. (F) Brush receptive fields were largest in the contralateral dorsal horn of NP animals. NP, needle prick; px², pixel²; TC, tactile control stimulus. Data presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001, ****P < 0.0001, between treatment comparisons.

Figure 3.

Postinjury sensitivity to cutaneous brush and noxious pinch stimulation in adult dorsal horn neurons after neonatal repeated procedures. (A) Time course of the experiment. Pups received neonatal stimulation or were left undisturbed. At 6 to 8 weeks of age, animals received an incision to the left hind paw on the plantar surface. Single-unit extracellular recordings were performed in the ipsilateral (B–E) or contralateral (F and G) spinal dorsal horn 2 and 5 days later. Cutaneous brush– and pinch-evoked spike activity and receptive field areas of wide-dynamic-range neurons were recorded. (B) No changes were observed in brush responses between groups, but brush-evoked firing was significant higher in naive animals, at both 2 and 5 days after incision compared with baseline (before incision, shown as transparent coloured bars). (C) After incision, brush receptive sizes were significantly larger in all animals compared with baseline. Between-group differences were also observed. At 5 days after incision, brush receptive fields were larger in TC and NP animals compared with naive. (D) Pinch-evoked firing activity after incision increased in NP animals compared with UC at 5 days after incision. (E) Pinch receptive field postincision was also significantly larger in all animals after incision compared with baseline. At 2 days, pinch receptive fields were significantly larger in TC and NP animals compared with naive. At 5 days, pinch receptive fields were larger in NP animals compared with naive. (F) Significant increases in postincision brush responses were also observed for NP animals in the contralateral dorsal horn after 2 days. (G) Contralateral pinch-evoked firing activity after incision was significantly enhanced in NP animals at day 2 postincision. NP, needle prick; px², pixel²; TC, tactile control stimulus; UC, undisturbed. Data presented as mean ± SEM, transparent coloured bars represent the baseline responses of each group. ^P < 0.05, ^^P < 0.01, ^^^P < 0.001, ^^^^P < 0.0001, within treatment comparison, compared with baseline. *P < 0.05, ***P < 0.001, ****P < 0.0001, between treatment comparisons. #P < 0.05, between postincision day comparison.

Figure 4.

Baseline and postinjury sensitivity to cutaneous punctate mechanical stimulation in adult dorsal horn neurons after neonatal repeated procedures. (A) Time course of the experiment. Responses in WDR neurons were recorded after graded von Frey hair (vFh) stimulation at baseline and postinjury in the ipsilateral (left panel) and contralateral (right panel) dorsal horn. Data were plotted as stimulus response curves (B) Baseline vFh sensitivity ipsilateral to injury was increased in NP animals and TC animals compared with UC. (C) Needle-prick animals displayed significantly increased vFh-evoked firing at 26 g in contralateral WDRs. (D) Two days after incision injury, ipsilateral stimulus response curves indicated slight hypersensitivity in NP compared with TC and UC at the highest vFh strength. (E) Two days after incision, NP animals were hypersensitive compared with TC and UC littermates in contralateral WDRs. (F) Five days after incision, NP animals were significantly more sensitive in ipsilateral WDRs than TC and UC. Changes were also observed in the contralateral dorsal horn. (G) Five days after incision, vFh-evoked contralateral neuronal firing was highest in the NP group. NP, needle prick; TC, tactile control stimulus; UC, undisturbed; vFh/VF, von Frey filament; WDR, wide-dynamic range. In all figures: data presented as mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, between treatment comparisons. Asterisks are colour coded; dark blue asterisks represent between neonatal NP and undisturbed control comparisons; purple asterisks represent between neonatal tactile control and undisturbed control comparisons.