Figure 6.

Tetracycline-controlled overexpression of constitutively active Dexras1 in the SCN abrogates MAPK activation in response to nocturnal light. A, Experimental schematic to drive SCN-specific expression of the constitutively active Dexras1 (A178V) mutant using the tetracycline-inducible system. A single transgenic construct containing two genes, constitutively active Dexras1 harboring the A178V point mutation and eGFP, in a polycistronic unit under the control of the tetracycline-responsive (tetO) promoter was generated. The Dexras1 (A178V)-IRES-eGFP transgenic mice were bred with the CaMKIIα-tTA mice. In the absence of the tetracycline analog doxycycline (Dox), double transgenic mice coexpress Dexras1(A178V) and eGFP in a tissue-specific manner. The Dexras1(A178V)-IRES-eGFP founder line #3901 shown here expresses specifically in the SCN. GFP immunoreactivity was found throughout the SCN, marking cells that express the Dexras1(A178V) transgene. B, Expression of eGFP in double transgenic mice generated from the breeding of the CaMKIIα-tTA strain to Dexras1(A178V)-IRES-eGFP founder lines #3901 (left) and #3601 (right). Founder line #3901 exhibited SCN-specific expression, whereas transgene expression was more broadly expressed in founder line #3601 and observed in the SCN, cortex, hippocampus, and striatum. C, Light-induced MAPK activation in double transgenic CaMKIIα-tTA::Dexras1(A178V)-IRES-eGFP mice. Double transgenic mice received a brief light pulse (15 min; 100 lux) at CT 20, and brain sections were processed 30 min later for expression of p-ERK (red) and GFP (green) using immunofluorescent labeling and confocal microscopy was used to visualize the signals. Cells that express the transgenes do not exhibit p-ERK immunoreactivity after light treatment (left panel). The absence of colocalization of GFP and p-ERK expression is more evident under higher magnification (right panel).