, 2009b and Royer et al., 2010), to silicon probes for multi-site recording in awake, behaving animals (Royer et al., 2010). An issue with all of these extracellular methods is that there is no guarantee that recorded spikes are arising from photosensitive cells, rather than from indirectly recruited cells. Normally this is not a concern, and optrode recordings still provide extremely
useful feedback on the activity in the local circuit during control Galunisertib order that could never be obtained with electrical stimulation. However, care must be taken not to overinterpret (for example) latencies to spiking, which can be highly variable in vivo due to differences in illumination intensity, as predictive of whether a unit is directly or indirectly driven by light. Latencies as long as 10–12 ms or greater are certainly possible for directly driven cells, while conversely latencies as short as 3–4 ms should be possible even for indirectly driven (nonphotosensitive) cells. The concept of all-optical interrogation of neural circuits (Deisseroth et al., 2006 and Scanziani and Häusser, 2009) is appealing since spatial distribution and cell-type information can be more readily extracted from imaging data
than from electrophysiology. Dye-based imaging has been conducted in combination with optogenetic control in a number of studies, using Ca2+ dyes such as fura-2 (Zhang et al., 2007) and Fluo-5F (Zhang and Oertner, 2007), and voltage-sensitive check details dyes such as RH-155 (Airan et al., 2007, Airan et al., 2009 and Zhang et al., 2010). The development of new and improved genetically encoded sensors for neural activity (Lundby et al., 2008, Dreosti et al., 2009, Dreosti and Lagnado, 2011, Lundby et al., 2010 and Tian et al., 2009) opens up a new class of possibilities for capitalizing on cell-type-specific readout information that would complement the cell-type-specific play-in of information provided by optogenetics. Although channelrhodopsin action spectra through overlap to some extent with the excitation spectra of these fluorophores, one can minimize photoactivation during imaging by minimizing irradiance used to excite the fluorophores, and by using scanning microscopy (confocal or two-photon
based). When using scanning laser microscopy, the rapid ChR kinetics that initially posed challenges for two-photon activation (Rickgauer and Tank, 2009) are actually favorable since Ca2+ imaging can be performed by two-photon excitation with minimal photoactivation of ChRs. Indeed, Zhang and Oertner used two-photon imaging of the Ca2+ dye Fluo-5F to record dendritic calcium transients evoked with either ChR2 photostimulation or direct current injections in individual neurons in the slice culture preparation (Zhang and Oertner, 2007), while Guo et al. used GCaMP2 in C.elegans neurons, using a low wide-field light power density for imaging GCaMP (488 nm; 0.01 mW/mm2; Guo et al., 2009) to avoid unwanted photostimulation by the fluorescence excitation light.
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