These results provided the impetus for determining whether two-photon imaging of calcium activity in neurons via a microprism could be achieved in the visual cortex of awake behaving mice. We obtained stable, chronic recordings of calcium activity across cortical layers as follows: first, a headpost was affixed to the skull and a 5 mm craniotomy was performed over mouse

visual cortex. A standard cranial VX-809 datasheet window was then installed (8 mm round coverslip glued above two 5 mm round coverslips to slightly compress the brain, reducing brain motion and regrowth; see Figures S1A–S1D; Andermann et al., 2011). Cortical expression of the genetically-encoded calcium indicator, GCaMP3 (Tian et al., 2009), was achieved using adeno-associated virus (AAV) injection in layers 2/3, 5, and 6. Following recovery from surgery, mice were trained to tolerate several hours of head restraint (see Experimental Procedures and Andermann et al., 2011). Subsequently, the original cranial window (Figures S1A and S1B) was removed under anesthesia BGB324 cell line and replaced by a microprism assembly (Figures S1C and S1D; Experimental Procedures) consisting of a microprism glued to three layers of coverglass.

Gluing the microprism in a specific, predetermined location relative to the cranial window allowed (1) targeted insertion in posterior V1 near the site of unless GCaMP3 expression, (2) minimization of damage to large surface vasculature, and (3) orientation toward posterior and lateral cortex (Figure 1B) to minimize damage to thalamocortical axons from the lateral geniculate nucleus (which traverse cortex from lateral to medial, below layer 6, before ascending into their target cortical column; Antonini et al., 1999). Before imaging GCaMP3 activity through a microprism, we evaluated how the implant of a prism influences the sensory response properties of nearby neurons. We first measured the visual response properties of neurons

through a standard cranial window and then assessed the response properties of the same neurons following insertion of a microprism at a distance of 350 μm away (Figure 2A, white dashed squares, and 2B). We used an identical approach across sessions—two-photon volume imaging of visual responses of GCaMP3-expressing layer 2/3 neurons through the cranial window in an awake, head-fixed mouse that was free to run on a linear trackball (Experimental Procedures; Glickfeld et al., 2013). The insertion of the microprism resulted in the accumulation of some blood at the brain surface and prism surface, which cleared up over the course of several days (Figures 2A and S2H–S2M). Major surface vessels >150 μm from the prism face remained intact, and no obvious changes in blood flow through these vessels were observed during or after prism insertion.