Substance P-driven feed-forward inhibitory activity in the mammalian spinal cord

Terumasa Nakatsuka, Meng Chen, Daisuke Takeda, Christopher King, Jennifer Ling, Hong Xing, Toyofumi Ataka, Charles Vierck, Robert Yezierski, Jianguo G Gu, Terumasa Nakatsuka, Meng Chen, Daisuke Takeda, Christopher King, Jennifer Ling, Hong Xing, Toyofumi Ataka, Charles Vierck, Robert Yezierski, Jianguo G Gu

Abstract

In mammals, somatosensory input activates feedback and feed-forward inhibitory circuits within the spinal cord dorsal horn to modulate sensory processing and thereby affecting sensory perception by the brain. Conventionally, feedback and feed-forward inhibitory activity evoked by somatosensory input to the dorsal horn is believed to be driven by glutamate, the principle excitatory neurotransmitter in primary afferent fibers. Substance P (SP), the prototypic neuropeptide released from primary afferent fibers to the dorsal horn, is regarded as a pain substance in the mammalian somatosensory system due to its action on nociceptive projection neurons. Here we report that endogenous SP drives a novel form of feed-forward inhibitory activity in the dorsal horn. The SP-driven feed-forward inhibitory activity is long-lasting and has a temporal phase distinct from glutamate-driven feed-forward inhibitory activity. Compromising SP-driven feed-forward inhibitory activity results in behavioral sensitization. Our findings reveal a fundamental role of SP in recruiting inhibitory activity for sensory processing, which may have important therapeutic implications in treating pathological pain conditions using SP receptors as targets.

Figures

Figure 1
Figure 1
Feed-forward inhibitory activity in the absence of glutamatergic driving force. a, Rat spinal cord slice with attached dorsal root. A portion of the root is sucked into a stimulation electrode. Recordings were made in lamina V. b, Five consecutive traces show EPSCs evoked by electrical stimulation (top). The EPSCs were abolished in the presence of 20 μM CNQX plus 50 μM APV (bottom). Vh = -60 mV. c, In the same cell, stimulation evoked IPSCs (top), which were abolished in the presence of 20 μM CNQX and 50 μM APV (bottom). Vh = -10 mV. d, In the same cell, trains of stimulation (20 Hz for 1 min) increased IPSCs in the presence of 20 μM CNQX plus 50 μM APV. Top trace was IPSCs recorded before and after electrical stimulation. The bottom 3 traces are at an expanded scale. Vh = -10 mV. e&f, Time course of IPSC frequency (e) and amplitude (f). Horizontal bars indicate stimulation. Overall, at peak responses, IPSC frequency increased to 376 ± 47% of control (n = 5, P < 0.05); IPSC amplitude increased to 228 ± 74% of control (n = 5, P < 0.05). Similar results were also obtained in the presence of 3 mM kynurenic acid (see Figure 2c). g–j, Capsaicin-induced increases in inhibitory activity in the absence of glutamatergic driving force. g, The top trace is a continuous recording of IPSCs from a rat lamina V neuron before and following the application of 2 μM capsaicin in the presence of 3 mM kynurenic acid. The bottom 2 traces are at an expanded scale. h, The time course of IPSC frequency in (g). bin width: 10s. i&j, Capsaicin-induced increases in IPSC frequency (i) and amplitude (j) recorded from 6 rat lamina V neurons in the presence of 3 mM kynurenic acid.
Figure 2
Figure 2
Feed-forward inhibitory activity driven by SP through NK1 receptor activation. a, Effects of exogenously applied neuropeptides on IPSCs recorded in rat lamina V neurons. Neuropeptides tested include galanin (0.3 μM, n = 4), somatostatin (2 μM, n = 4), NPY (1 μM, n = 4), CGRP (0.5 μM, n = 5), and substance P (1 μM, n = 6). b, Antagonism of capsaicin-induced increases in inhibitory activity in rat lamina V neurons. Capsaicin was applied in the presence of neurokinin receptor antagonists APTL (10 μM, n = 5), L-733, 060 (2 μM, n = 4), L-732,138 (100 μM, n = 5), SB222200 (2 μM, n = 8), and the Gi/o protein blockers pertussis toxin (2 μg/ml, n = 4) and NEM (100 μM, n = 5). c, Antagonism of electrical stimulation-induced increases in inhibitory activity by the SP antagonist APTL (10 μM, n = 7). Recordings were from rat lamina V neurons. d, Effects of capsaicin and SP on IPSCs in NK1R+/+ mice (n = 8 for capsaicin, n = 10 for SP) and NK1R-/- mice (n = 21 for cap, n = 11 for SP). Experiments were performed in the presence of 3 mM kynurenic acid (a–d) or 20 μM CNQX plus 50 μM APV (some experiments in a&b). e, Images show a cultured GIN mice EGFP neuron (arrow indicated) before (left) and after loading the Ca2+ indicator Fluor-3 (middle), and following application of 100 nM SP (Right). The experiment was performed in the presence of 500 nM TTX and 3 mM kynurenic acid. Similar results were obtained from 22 EGFP neurons. f, The florescence image shows a spinal cord slice obtained from a GIN mouse. An EGFP neuron in lamina V is indicated by a small box and enlarged in a bigger box. g, Non-adaptive action potential firing induced by depolarizing current (50 pA) in the EGFP neuron. h, Application of 1 μM SP produced a prolonged depolarization and action potential firings (top) in the same cell. The dotted line (bottom) shows, at expanded scale, the membrane depolarization (action potentials are omitted for clarity). Kynurenic acid (3 mM) was present throughout the experiments (n = 7).
Figure 3
Figure 3
SP-driven inhibitory activity under conditions when glutamatergic driving force is intact. All experiments were performed in bath solution without glutamate receptor antagonists. a, A continuous recording of IPSCs from a lamina V neuron of a NK1R+/+ mouse. Four traces (bottom) show, at an expanded scale, the IPSCs before, during, 1 sec after, and 9 min after trains of stimulation. The trace during stimulation is at a more expanded scale to show pulse-by-pulse eIPSCs. b, Same as a except the experiment was performed on a NK1R-/- mouse. The pulse-by-pulse eIPSCs (second trace of lower panel) were similar to those of NK1R+/+ mice, but ISPCs returned to the basal level immediately after termination of the train stimulation (third trace of lower panel). c, Time course of IPSC frequency (top) and amplitude (bottom). IPSCs during stimulation are not included. d, Peak IPSC frequency and amplitude after trains of stimulation in NK1R+/+ (n = 6) and NK1R-/- mice (n = 8). In a–d, stimulation was applied at intensity of 500 μA and a frequency of 20 Hz. e, Capsaicin-induced changes of IPSCs in lamina V neurons of NK1R+/+ mice (n = 6) and NK1R-/- mice (n = 16).
Figure 4
Figure 4
Assessment of the role of SP-driven inhibitory activity in behavioral responses to noxious stimuli. a&b, Micrographs show NK1 receptor immunoreactivity in the lamina I region of a normal rat (a) and 14 days following intrathecal application of SP-SAP (b). c, Capsaicin-induced increases of IPSCs recorded from lamina V neurons of SP-SAP treated rats (n = 5). d, The first set of bars show baseline of reflexive lick/guard response to heat stimuli at 44.5°C in normal (open bar, n = 8) and SP-SAP rats (solid bar, n = 8). The second set of bars show sensitization of behavioral responses by capsaicin in normal rats (n = 8) and attenuation of the behavioral response in SP-SAP rats (n = 8). The third set of bars show that the NK1 receptor antagonist CP97 attenuated behavioral responses in normal rats (n = 8) but sensitize behavioral response in SP-SAP rats (n = 8) (see Additional file: 1).

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