We demonstrated chronic two-photon imaging of neurons for months

after prism implantation. Although images can be obtained on the day of surgery (Chia and Levene, 2009b), they were sometimes less clear and required somewhat higher laser power. In these cases, imaging clarity improves over subsequent days. This initial clouding could be due in part to gliosis (Barretto et al., 2011). However, the time course of immediate clouding and subsequent improvement in clarity is more consistent with clearing of blood at the prism and cortical surfaces (Figures S2H–S2M; see also Chia and Levene, 2009b). The chronic implantation of a 1 mm prism involves severing of some horizontal cortical connections. Thus, as with live imaging studies using coronal brain Selleckchem BAY 73-4506 sections, it is important to carefully assess whether the local cortical circuitry near

the prism imaging face is sufficiently preserved to provide meaningful anatomical and functional data. We therefore undertook a multitiered approach, using functional and anatomical imaging, microelectrode recordings, and histological staining, to show that the basic cell health and receptive field properties of neurons at >100–150 μm lateral to the prism face are qualitatively similar to what is observed in experiments not involving chronically ZD1839 in vivo implanted prisms. Multiunit electrophysiological recordings from barrel cortex demonstrated that cortical neurons close to the prism face were endogenously active and responsive to whisker stimulation at 10 min, 3 days, and 120 days postimplant. Even immediately following

prism insertion, ketamine-induced oscillations in spiking persisted, with normal tactile response latencies and intensities. We did note a temporary increase in sustained spiking activity immediately following prism insertion (suggesting damage to a subset of neurons), but we did not observe spreading depression or acute silencing of cortical activity. Epifluorescence and two-photon imaging demonstrated blood perfusion in intact Thiamine-diphosphate kinase radial vessels at distances of >50 μm from the prism face (Figures 2A, 2B, and S2H–S2M; Chia and Levene, 2009b). Functional and anatomical imaging of the same neurons across weeks (Figures 2, 3, and S1) and across sessions both prior to and following prism insertion, suggests that most neurons at >150 μm from the prism face did not undergo damage-induced cell degeneration or death. Consistent with previous studies (Niell and Stryker, 2008, Niell and Stryker, 2010 and Olsen et al., 2012), we observed sharp orientation tuning in neurons in all cortical layers of mouse V1 (Figure 3B), at distances of ∼200 μm from the prism face. Critically, orientation and direction tuning in the same neurons was largely stable across days, both prior to and following prism implant.