Development of nociceptive synaptic inputs to the neonatal rat dorsal horn: glutamate release by capsaicin and menthol

Abstract

To study the postnatal development of nociceptive synaptic inputs in the superficial dorsal horn of the neonatal rat spinal cord, we examined the effect of capsaicin and menthol on glutamatergic mEPSCs in postnatal day (P) 0-1, P5-6 and P9-11 slices of spinal cord. Capsaicin (100 nM to 2 microM) increased the mEPSC frequency in a concentration-dependent manner at all ages tested, with a significant enhancement of the effect between P5 and P10. This effect was sensitive to vanilloid receptor (VR) antagonists. The elevation in mEPSC frequency occurred at concentrations of capsaicin (100 nM) that did not alter the distribution of mEPSC amplitudes and was abolished by a dorsal rhizotomy, demonstrating that capsaicin acts via presynaptic VR1 receptors localized on primary afferents. Menthol significantly increased the mEPSC frequency with a similar developmental pattern to capsaicin without consistently affecting mEPSC amplitude. The increase in mEPSC frequency following capsaicin did not depend on transmembrane calcium influx since it persisted in zero [Ca2+]o. The facilitation of spontaneous glutamate release by capsaicin was sufficient to evoke action potentials in neonatal dorsal horn neurons but was accompanied by a block of EPSCs evoked by electrical stimulation of the dorsal root. These results indicate that VR1-expressing nociceptive primary afferents form functional synaptic connections in the superficial dorsal horn from birth and that activation of the VR1 receptor increases spontaneous glutamate release via an undetermined mechanism. In addition, the data suggest that immature primary afferents express functional menthol receptors that are capable of modulating transmitter release. These results have important functional implications for infant pain processing.

Figure 1. Capsaicin increases the frequency of mEPSCs in neonatal dorsal horn neurons

A, mean frequency of spontaneous EPSCs (recorded in aCSF) and miniature EPSCs (recorded in 500 nm TTX) increase with age over the first 10 postnatal days. † Significant difference between the frequency of sEPSCs and mEPSCs at a given age (P < 0.0001, t test). B, top panel, example of recording from V_h=−70 mV demonstrating the slow inward current and large increase in mEPSC frequency that result from the bath application of 2 μm capsaicin in a P10 neuron in the presence of 10 μm bicuculline and 0.5 μm strychnine. Subsequent perfusion with 10 μm NBQX abolished the mEPSCs. B, bottom panel, different time scale illustrating the kinetics of mEPSCs before and after 2 μm capsaicin in another P10 cell (scale bars: 20 pA, 50 ms). C, cumulative probability function showing an example of the altered distribution of mEPSC inter-event intervals after capsaicin treatment. D, lower concentrations of capsaicin (100 nm), which significantly increase mEPSC frequency, did not change the mean mEPSC amplitude in P9–P10 neurons (n = 8). E, dose dependence of capsaicin effect on mEPSC frequency. * Significant differences in mEPSC frequency compared to baseline conditions (*P < 0.05; ***P < 0.001; one-way ANOVA). F, the average frequency of mEPSCs following 2 μm capsaicin was significantly greater in the P9–P10 group compared to other age groups (**P < 0.01; ANOVA). G, the peak amplitude of the slow inward current evoked by 2 μm capsaicin did not significantly vary according to postnatal age.

Figure 2. VR1 receptors on primary afferents mediate capsaicin-evoked increase in mEPSC frequency

A, the effect of 2 μm capsaicin on mEPSC frequency was abolished by prior application of the non-competitive VR1 antagonist Ruthenium Red (RR; 10 μm; n = 4) but persisted in the presence of the competitive antagonist capsazepine (CPZ; 10 μm; n = 5; **P < 0.001). For purposes of comparison, the modulation of mEPSC frequency by 2 μm capsaicin in the absence of antagonists is illustrated to the left (n = 7; P < 0.001). B, the facilitation of mEPSC frequency observed with 100 nm capsaicin (*P < 0.05) was completely blocked by CPZ (n = 5). C, capsaicin (2 μm) had no significant effect on mEPSC frequency in P8–P11 dorsal horn neurons located ipsilateral to a previous dorsal rhizotomy (n = 7), while a dramatic elevation was witnessed on the contralateral side (n = 5; **P < 0.001).

Figure 3. Menthol increases mEPSC frequency in P10–P11 neurons of the superficial dorsal horn

A, cumulative probability function describing the shift in mEPSC inter-event intervals seen after application of 500 μm (−)menthol in a P10 cell. The intervals were significantly reduced following menthol treatment (P < 0.05; Kolmogorov-Smirnov two-sample test) in all P10–P11 neurons examined (n = 6). B, menthol (500 μm) did not significantly affect mEPSC amplitudes in a population of P10–P11 neurons (n = 6; P > 0.05; paired t test). C, facilitation of mEPSC frequency by menthol is dependent on postnatal age (*P < 0.01; Student's unpaired t test).

Figure 4. Effect of capsaicin on mEPSC frequency does not require external Ca²⁺

A, plot of mEPSC frequency vs. time for a representative P10 neuron perfused in zero Ca²⁺ and 3 mm Mg²⁺ solution, illustrating a significant rise in mEPSC frequency after application of 2 μm capsaicin (bar). B, at P10, prior application of dantrolene sodium (30 μm; n = 3), caffeine (20–30 mm; n = 8) or FCCP (2 μm; n = 5) failed to prevent the significant increase in mEPSC frequency by capsaicin (2 μm) under zero-Ca²⁺ conditions (**P < 0.001; ANOVA). The capsaicin effect also persists despite pre-exposure of the slice to the membrane permeable Ca²⁺ chelator BAPTA AM (50 μm) for 10–15 min (n = 6; **P < 0.001).

Figure 5. Capsaicin inhibits electrically evoked EPSCs in the superficial laminae of the neonatal dorsal horn

A, examples of evoked EPSCs (eEPSCs) recorded in a lamina I-II neuron at P10 in response to electrical stimulation of the attached dorsal root (threshold: 69 μA, 50 μs; latency: 10.5 ms). In this cell, a complete block of both the early and late components of the eEPSC accompanies the increased sEPSC frequency seen after capsaicin (2 μm). B, representative EPSCs evoked in a deep (lamina III-IV) dorsal horn neuron (threshold: 70 μA, 50 μs; latency: 3.4 ms) illustrating the lack of effect of capsaicin on eESPC amplitude.

Figure 6. Capsaicin elicits action potentials in superficial dorsal horn neurons during the first postnatal week

In current clamp, cells were maintained at V_m=−60 to −65 mV with DC injection and 2 μm capsaicin was subsequently bath applied at the indicated times to P1 (A) or P5 (B) neurons.