In Vivo Imaging of the Coupling between Neuronal and CREB Activity in the Mouse Brain

Abstract

Sensory experiences cause long-term modifications of neuronal circuits by modulating activity-dependent transcription programs that are vital for regulation of long-term synaptic plasticity and memory. However, it has not been possible to precisely determine the interaction between neuronal activity patterns and transcription factor activity. Here we present a technique using two-photon fluorescence lifetime imaging (2pFLIM) with new FRET biosensors to chronically image in vivo signaling of CREB, an activity-dependent transcription factor important for synaptic plasticity, at single-cell resolution. Simultaneous imaging of the red-shifted CREB sensor and GCaMP permitted exploration of how experience shapes the interplay between CREB and neuronal activity in the neocortex of awake mice. Dark rearing increased the sensitivity of CREB activity to Ca 2+ elevations and prolonged the duration of CREB activation to more than 24 h in the visual cortex. This technique will allow researchers to unravel the transcriptional dynamics underlying experience-dependent plasticity in the brain.

A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3

“…2b). We designated this protein mCyRFP3, since the name mCyRFP3 was already assigned to a different mCyRFP1 variant 12 . mCyRFP3 exhibited absorbance and excitation centered near 500 nm and peak emission of 586 nm, slightly red-shifted from mCyRFP1 (Fig.…”

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

“…In cell culture models, these signalling pathways have been dissected extensively, through the use of genetically-encoded fluorescent and bioluminescent biosensors. Some of these biosensors have recently been used in the in vivo context, to link specific signalling patterns with behavioral outcomes 2 – 6 .…”

“…Many intracellular signalling biosensors utilize changes in Förster resonance energy transfer (FRET) between two fluorescent proteins 7 to report levels or activity of second messengers, protein kinases, GTPases, post-translational modifications and protein–protein interactions 8 – 11 . These tools have been widely used to dissect signaling pathways in cultured cells and have recently begun to be applied in vivo , aided by recent technological developments in intravital imaging 10 , 12 , microendoscopy 3 , 2-photon microscopy 13 – 15 and fluorescence lifetime measurement 16 . Here we report a ratiometric fiber-photometry approach for real-time recording of genetically-encoded FRET biosensors in freely moving rodents.…”

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors

“…In cell culture, these signalling pathways have been dissected extensively, through the use of genetically-encoded fluorescent and bioluminescent biosensors. Some of these biosensors have recently been applied in vivo , to link specific signalling patterns with behavioral outcomes 2–6 .…”

“…Many intracellular signalling biosensors have been developed which utilize changes in Förster resonance energy transfer (FRET) between two fluorescent proteins 11 , in order to report levels or activity of second messengers, protein kinases, GTPases, post-translational modifications and protein-protein interactions 12–15 . These tools have been widely used to dissect signaling pathways in cultured cells and have recently begun to be applied in vivo , aided by recent technological developments such as intravital imaging 14,16 , microendoscopy 3 , 2-photon microscopy 2,5,6,17 and fluorescence lifetime measurement 4 . Here we report a ratiometric fiber-photometry approach for real-time recording of genetically-encoded FRET biosensors in freely moving rodents.…”