Two-photon calcium imaging of medial prefrontal cortex and hippocampus without cortical invasion

In vivo two-photon calcium imaging currently allows us to observe the activity of multiple neurons up to [~]900 m below the cortical surface without cortical invasion. However, many other important brain areas are located deeper than this. Here, we used a 1100 nm laser, which underfilled the back aperture of the objective, and red genetically encoded calcium indicators to establish two-photon calcium imaging of the intact mouse brain and detect neural activity up to 1200 m from the cortical surface. This imaging was obtained from the medial prefrontal cortex (the prelimbic area) and the hippocampal CA1 region. We found that the neural activity related to reward prediction is higher in the prelimbic area than in layer 2/3 of the secondary motor area, while it is negligible in the hippocampal CA1 region. Reducing the invasiveness of imaging is an important strategy to reveal the brain processes active in cognition and memory.