Noxious cutaneous thermal stimuli induce a graded release of endogenous substance P in the spinal cord: imaging peptide action in vivo

Abstract

Dorsal root ganglia (DRG) neurons synthesize and transport substance P (SP) to the spinal cord where it is released in response to intense noxious somatosensory stimuli. We have shown previously that SP release in vivo causes a rapid and reversible internalization of SP receptors (SPRs) in dorsal horn neurons, which may provide a pharmacologically specific image of neurons activated by SP. Here, we report that noxious heat (43 degrees, 48 degrees, and 55 degrees C) and cold (10 degrees, 0 degrees, -10 degrees, and -20 degrees C) stimuli, but not innocuous warm (38 degrees C) and cold (20 degrees C) stimuli, applied to the hindpaw of anesthetized rats induce SPR internalization in spinal cord neurons that is graded with respect to the intensity of the thermal stimulus. Thus, with increasing stimulus intensities, both the total number of SPR+ lamina I neurons showing SPR internalization and the number of internalized SPR+ endosomes within each SPR immunoreactive neuron showed a significant increase. These data suggest that thermal stimuli induce a graded release of SP from primary afferent terminals and that agonist dependent receptor endocytosis provides evidence of a spatially and pharmacologically unique "neurochemical signature" after specific somatosensory stimuli.

Fig. 1.

Confocal images of SPR immunoreactivity in the rat lumbar spinal cord. A, Confocal image of the rat spinal cord (L4 segment) 8 min after the plantar surface of the right hindpaw was stimulated for 30 sec with a 55°C thermode. Theboxes show the areas of the dorsal horn of the spinal cord that were sampled on the contralateral and stimulated sides. This medial aspect of the spinal cord is also the area that receives nociceptive inputs from the plantar surface of the hindpaw. InA the SPR immunoreactivity appears white, whereas in B and C panels the SPR immunoreactivity appears gray (low levels) andwhite (high levels). B₁,B₂, Confocal images, projected from 11 optical sections acquired at 0.7 μm intervals, of SPR immunoreactivity in the contralateral and ipsilateral sides, respectively, of the lumbar spinal cord after a −20°C stimulus to the right hindpaw. In the contralateral, unstimulated dorsal horn (B₁), the SPR immunoreactivity is present on the plasma membrane, whereas on the stimulated side (B₂), the SPR immunoreactivity is associated mainly with SPR+ endosomes.C1–C6, Confocal photomicrographs, projected from 22 optical sections acquired at 0.7 μm intervals, of SPR+ lamina I cell bodies 8 min after a single thermal stimulus was delivered to the hindpaw. In the contralateral control (C3) and in the ipsilateral spinal cord after the 32°C stimulus (C4), the SPR immunoreactivity is associated with the cell surface. After noxious thermal stimuli, the cell bodies experience a loss of immunoreactivity from the cell surface and an increase in the number of SPR+ endosomes in the neuronal cell body (C1, C2, C5,C6), suggesting that there is a graded release of SP from the primary afferents that is correlated with the intensity of the noxious thermal stimulation. Scale bars: A, 0.4 mm;B1–B2, 35 μm; C1–C6, 20 μm.

Fig. 2.

A, Mean proportion of SPR immunoreactive lamina I cell bodies exhibiting more than five SPR immunoreactive endosomes per SPR immunoreactive lamina I cell body as a function of stimulus intensity. B, Mean (±SEM) number of SPR+ endosomes per cell body in SPR immunoreactive lamina I neurons. In both cases the thermal stimulus group was compared with the 32°C contralateral control group, and the neurons sampled were present in the medial aspect of the rat L4 spinal segment. *p< 0.05.