Visualization and quantification of gene expression involved in pain transmission and regulation with molecular probes

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For the purpose to increase the sensitivity of an in situ hybridization technique with nonradioactive probes, we examined the several methods for nonradioactive labeling of RNA probes. Simiar sensitivities were obtained between digoxigenin- and fluorescein-labeled antisense RNA probes to detect the target mRNA,but the background signals were lower in the results with digoxigenin-labeled probe than with fluorescein-labeled one. For an analysis of the results of double in situ hybridization histochemistry, we developed an apparatus to simultaneously obtain both bright- and dark-field images, which were transferred to a personal computer and analyzed using a software for image analysis, NIH image. Coexistence of the mu-opioid receptor mRNA with the mRNA for preprotachykinin A (PPTA), a precursor protein of substance P,in the rat dorsal root ganglia was examined by this system, and we could quantitatively analyze the expression of mu-opioid receptor mRNA,which was detected by a ^<35>S-labeled probe, specifically in the cells expressing PPTA mRNA,which was visualized by a digoxigenin-labeled probe. Furthermore, we developed a method to analyze the of film-autoradiogram, and showed that expressions of mu-and kappa-, but not delta-, opioid receptor mRNAs were enhanced in the superfacial layrs of spinal dorsal horn of the arthritic rats.