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Immediate early genes are rapidly and transiently induced by many stimuli and produce their transcription factors. Of the immediate early genes, the proto-oncogene c-fos and its product Fos play a role in cell proliferation, differentiation and general signal transmission as the 'third messenger' regulating the transcription of other genes. Even minute stimuli such as the attachment of electrodes, needle injection and saline administration increase the level of c-fos mRNA expression in animal brains. It is, therefore, necessary to investigate the effects of using anesthetics and solvents for drug administration on c-fos expression in order to accurately assess net c-fos induction after stimulation. Furthermore, experimental procedures might themselves affect c-fos mRNA expression in the brain after in vivo stimulation. In this review, we discuss technical pitfalls in assessing in vivo c-fos expression. Rough handling with repeated saline administration enhanced cortical c-fos mRNA expression in the rat brain after a single saline injection by increasing baseline c-fos mRNA levels. In contrast, gentle handling with repeated saline administration diminished c-fos mRNA expression after a single injection by decreasing baseline c-fos mRNA levels. These two types of handling with the repeated injection led to diametrically opposite results on c-fos mRNA expression after a single stimulation. Our results suggest that c-fos mRNA induction after a single stimulation might be affected by the types or intensities of animal handling and that effects of animal handling must be considered when estimating c-fos mRNA induction.

Pitfalls in Assessment of c-fos mRNA Expression in the Brain: Effects of Animal Handling