Every day, the neuronal connections within our brains change depending on what we learn and experience in our daily lives. Specialized proteins, called activity-dependent transcription factors, activate genes within the cells of the brain to help it translate this rapid incoming signaling into lasting neuronal changes. Until now, it has not been possible to directly monitor transcription factors activity. Scientists at the Max Planck Florida Institute for Neuroscience (MPFI) have designed and developed novel biosensors that will allow the simultaneous, in vivo study of both sensory-evoked neuronal activity and transcription factor dynamics. The biosensors couple the specialized techniques of two-photon calcium imaging with two-photon fluorescence lifetime imaging (2pFLIM), to enable scientists to investigate how transcription factors function in a living brain. “Transcription factor activity in the brain isn’t a static, but rather a very dynamic process that can occur on the order of hours to days after a sensory experience,” Tal Laviv, research fellow in the Yasuda Lab at MPFI, said. “We wanted to develop a new way to study how this process is actually occurring in a living brain and chose to study CREB [cAMP response-element binding protein] due to its strong involvement in plasticity, learning, and memory.” MPFI scientists demonstrated a technique using 2pFLIM with fluorescence resonance energy transfer (FRET) biosensors to chronically image in vivo signaling of the cAMP response-element binding protein, or CREB at single-cell resolution. They started by creating 2pFLIM biosensors designed to report the direct activity of CREB. They packaged the sensors and expressed them in a population of neurons within the somatosensory cortex of mice. Using the 2pFLIM CREB sensor, the team monitored CREB activity in the same population of neurons while mice experienced an enriched environment. The enriched environment caused a significant increase in overall CREB activity. When mice were removed from the enriched environment for an extended period of time, CREB activity returned to normal levels, indicating sensory experience as a driver for the sustained activity. To better understand how sensory experiences and neuronal activation shape CREB activity in the living brain, the team expressed the CREB sensor and a sensor of neuronal activity (a calcium sensor) in the visual cortex of mice. Visual cortical neurons were imaged in dark-reared mice during presentation of visual stimuli. Calcium and CREB dynamics in single cells were simultaneously imaged for prolonged periods of time. The results revealed a dynamic regulation of CREB activity in the visual cortex: Dark-reared mice displayed dramatically increased levels of visually evoked CREB compared to mice raised in normal light/dark conditions. In addition, CREB activity levels were maintained for a period of at least one day in dark-reared mice. This elevated CREB activity was not due to elevated calcium levels within individual neurons, indicating that sensory experience can finely tune the sensitivity of activity-dependent transcription in the living brain. The use of 2pFLIM biosensors could be broadly applied to many different types of transcription factors in the future, the researchers believe, and could provide an opportunity to unravel the transcriptional dynamics underlying experience-dependent plasticity in the brain. The research was published in Neuron (www.doi.org/10.1016/j.neuron.2019.11.028). Researchers at MPFI have developed a new approach for studying the experience-driven activity of transcription factors in vivo. Courtesy of Max Planck Florida Institute for Neuroscience.