CO-LOCALIZATION OF SUBSTANCE P RECEPTOR (NK1) AND PREPROTACHYKININ (PPT) MRNA IN RAT DORSAL HORN NEURONS

Abstract

S358 INTRODUCTION: Substance P (SP) and related tachykinins modulate pain processing in the dorsal horn of the spinal cord via release of SP from primary afferents terminating in superficial laminae. SP also is synthesized by intrinsic dorsal horn neurons located in superficial laminae (primarily lamina I) and in deeper laminae (primarily lamina V), and many of these are spinothalamic neurons [1]. The effects of SP on nociception are likely mediated by the NK1 tachykinin receptor, since NK1 antagonists block behavioral nociceptive sensitization resulting from tissue injury [2]. Dorsal horn neurons containing NK1 receptor are located in superficial laminae (lamina I) and in deeper laminae (laminae III-V), and many of these are spinothalamic neurons [3]. Release of SP from terminals in the dorsal horn is modulated by opioid peptides which produce their actions, in part, through mu opioid receptors (MOR1), which are expressed both by primary afferents and by intrinsic dorsal horn neurons. In order to determine if SP is synthesized by dorsal horn neurons expressing NK1 or MOR1 receptors, we performed double-label in situ hybridization (ISH) on sections of lumbar spinal cord. METHODS: Fresh frozen spinal cord tissue from male Sprague Dawley rats was cryostat sectioned at 12 um and processed for ISH. RNA probes antisense for preprotachykinin (PPT) were transcribed using digoxigenin-UTP and probes antisense for NK1 or MOR1 mRNA were transcribed using33 P-UTP, and the PPT probe was combined with either the NK1 or the MOR1 probe and spinal cord sections were hybridized with this mixture overnight, washed, processed for immunohistochemical detection of digoxigenin labeled cells (Boehringer Mannheim), and then processed for autoradiographic detection of (33) P-UTP labeled cells. Spinal cord sections were examined under brightfield and darkfield illumination for the presence of blue-black reaction product associated with digoxigenin labeling (PPT cells) or the presence of silver grains associated with33 P labeling (NK1 or MOR1 cells), and for colocalization of digoxigenin and33 P labeling in the same cell. RESULTS: In the dorsal horn PPT positive cells were primarily located in lamina I and in deeper lamina V, which partially overlapped the distribution of NK1 positive cells, which were densely distributed in lamina I and more loosely distributed in laminae III-V. MOR1 positive cells were primarily located in lamina II and in laminae III-V. In lamina I approximately 70% of NK1 positive cells were also positive for PPT, but in deeper laminae only 20% of NK1 cells expressed PPT. No MOR1 positive cells contained PPT mRNA, either in superficial or deep laminae. DISCUSSION: These results describe co-localization of mRNA coding for the SP receptor NK1 and for the neuropeptide SP in dorsal horn neurons. In contrast, dorsal horn neurons containing mRNA coding for the mu opioid receptor do not co-localize with SP mRNA. Since spinothalamic neurons are located in both superficial (I-II) and deep (III-V) laminae of the dorsal horn and approximately 75% of laminae I-II spinothalamic neurons and 35% of laminae III-V spinothalamic neurons contain NK1 receptors [3], these data suggest that most lamina I cells and a significant percentage of lamina V cells co-localizing NK1 and SP project directly to thalamic pain nuclei. Together these data suggest a disynaptic SPergic pathway transmitting nociceptive information from the periphery to the thalamus. Although it is well established that mu opioid receptors modulate SP transmission in primary sensory terminals at the first synapse in laminae I-II by presynaptic mechanisms [4], the absence of co-localization of SP mRNA with MOR1 mRNA in dorsal horn neurons suggests that opioid modulation of SP transmission in second order sensory neurons projecting to the thalamus is mediated postsynaptically by MOR1 expressing dorsal horn interneurons.