, 2010), tunable lenses (Grewe et al., 2011), or multibeam systems (Amir et al., 2007 and Cheng et al., 2011). To rapidly obtain two-photon imaging data from a larger range of depths, we have previously shown that insertion of a sharp, 1 mm glass microprism into the neocortex of an anesthetized mouse can be used for acute, single-session two-photon imaging of anatomical structures across all six cortical layers, including the soma and dendrites of cortical layer 5 pyramidal cells, in a single field-of-view (Chia and Levene, 2009a,

Chia and Levene, 2009b and Chia and Levene, 2010). Here, we describe an improved approach that has enabled chronic anatomical and functional imaging of hundreds of individual neurons and neuronal processes CX-5461 in vitro simultaneously across all cortical layers. The structure and function of neurons at distances >150 μm from the prism face were not qualitatively different after prism insertion and remained stable for months after prism insertion. We also demonstrate that microprisms can be used for simultaneous, high-speed calcium imaging CT99021 datasheet from neurons in layers 2 to 6 during locomotion and for imaging visual responses in long-range axon terminals in deep cortical layers. This approach complements traditional in vivo electrophysiological methods by enabling high-yield,

simultaneous chronic monitoring of subcellular structure and neural activity in superficial and deep-layer cortical neurons in behaving mice. Two parallel approaches have dominated the study of neocortical circuits.

One approach involves recordings in living coronal brain slices (typically ∼400 μm thick). Farnesyltransferase Although many long-range axonal inputs to the cortical columns within each slice are severed, this reductionist approach has provided a wealth of insights regarding the layer-specific physiological properties of neurons and the interlaminar flow of neural impulses, using multiple intracellular and extracellular recordings (e.g., Adesnik and Scanziani, 2010, Sanchez-Vives and McCormick, 2000 and Thomson, 2010), two-photon calcium imaging (MacLean et al., 2006), and voltage-sensitive dye imaging (Petersen and Sakmann, 2001). A second common approach involves neuronal recordings from the intact brain, where it is possible to correlate neural activity with sensory perception and with behavior. Two-photon imaging has provided a means for monitoring neural activity and structural plasticity across days and weeks in awake animals (e.g., Dombeck et al., 2007, Andermann et al., 2010, Mank et al., 2008 and Trachtenberg et al., 2002). However, many aspects of cortical processing remain out of reach because of the challenges in imaging deep-layer neurons and in simultaneous in vivo imaging across all layers.