A time-dependent role for the transcription factor CREB in neuronal allocation to an engram underlying a fear memory revealed using a novel in vivo optogenetic tool to modulate CREB function

摘要

a Schematic of opto-DN-CREB construct. The photoreceptor PYP (photoactive yellow protein) is bound to a dominant-negative inhibitor of CREB (A-CREB). In the lights-off condition, A-CREB (fused with PYP) binds to endogenous CREB, thereby blocking CREB function. Upon blue light (BL) stimulation, PYP undergoes a conformational change, sequestering A-CREB, thereby liberating endogenous CREB and increasing CREB function. b (Top) Schematic of HSV-opto-DN-CREB construct. (Bottom) HSV-GFP control construct. c Exogenous addition of the PYP chromophore (CHR) is not required for opto-DN-CREB to increase CREB function in HEK cells. As expected, in HEK cells transfected with opto-DN-CREB, addition of the adenylyl cyclase activator forskolin (FSK) increased expression of the CREB target gene Nurr1 compared to control cells (Cntrl, transfected with opto-DN-CREB, no FSK). Nurr1 expression was further increased with BL, whether exogenous chromophore (CHR) was (Left) added (+CHR) [Cntrl (n = 4), FSK (n = 7), FSK + BL (n = 6)] (Right) or not (−CHR) [Cntrl (n = 4), FSK (n = 6), FSK + BL (n = 6)]. d Normalization of FSK + BL groups from c with, and without, exogenous CHR show similar increases in Nurr1 expression, indicating exogenous chromophore is not required for opto-DN-CREB function. e (Left) Experimental design for experiment showing opto-DN-CREB enhances CREB function. (Right) In cultured hippocampal neurons expressing opto-DN-CREB, 1 h of BL increased Creb expression [HSV-GFP (n = 3), HSV-opto-DN-CREB (n = 4)]. All error bars represent SEM; n.s. represents p > 0.05, * represents p < 0.05, ** represents p < 0.001.