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Two-photon calcium imaging has previously only been useful for imaging ongoing neuronal activity in the superficial cortical layers in vivo. Here the authors describe technology that enables imaging of sensory-evoked neuronal activity in layer 5 of adult mouse somatosensory cortex. Multiphoton imaging (MPI) is widely used for recording activity simultaneously from many neurons in superficial cortical layers in vivo. We combined regenerative amplification multiphoton microscopy (RAMM) with genetically encoded calcium indicators to extend MPI of neuronal population activity into layer 5 (L5) of adult mouse somatosensory cortex. We found that this approach could be used to record and quantify spontaneous and sensory-evoked activity in populations of L5 neuronal somata located as much as 800 μm below the pia. In addition, we found that RAMM could be used to simultaneously image activity from large (∼80) populations of apical dendrites and follow these dendrites down to their somata of origin.
Mittmann et al. (Sun,) studied this question.