In vivo two-photon imaging reveals stochastic and synchronized extracellular signal-regulated kinase dynamics in neurons of awake mice

The neocortex is essential for higher-order brain functions such as cognition, perception, language, and motor control. Although extracellular signal-regulated kinase (ERK) activity in neurons has been well studied for its role in memory formation, its activity patterns in the awake mice brain remain unclear. This study aimed to investigate the spatiotemporal dynamics of ERK activity in neocortical neurons of awake mice using in vivo imaging. We used Förster resonance energy transfer (FRET)-based biosensor and two-photon microscopy to examine ERK activity in vivo through a cranial window. Visual cortical neurons exhibited NMDA receptor-dependent ERK activation in response to visual stimuli. Beyond stimulus-induced activation, we discovered spontaneous and stochastic ERK activation patterns categorized as single, multi-pulsatile, and sustained activations, which were consistent across various cortical regions, including the visual and somatosensory cortices. Furthermore, synchronized ERK activation among neurons was observed, suggesting a coordinated ERK activation mechanism within neuronal networks that may contribute to sensory information processing. These findings reveal a highly dynamic ERK activation in neocortical cells and provide new insights regarding the functional role of ERK in the awake brain. • In vivo two-photon FRET imaging revealed ERK dynamics in the awake mouse brain. • Visual stimulation evoked NMDA receptor-dependent ERK activation in cortical neurons. • Spontaneous and stochastic ERK activations occurred in the brain without stimuli. • Distinct dynamic patterns of ERK activation were observed in the mouse brain. • Subsets of neurons show synchronized ERK activation.