Optogenetics enables the controlled excitation of defined neuron populations in animals that have been genetically sensitized to light. The ReaChR channelrhodopsin variant used in such studies incorporates portions of VChR1 and VChR2 (channelrhodopsins from Volvox carteri), ChIEF (the transmembrane domains of Chlamydomonas reinhardtii channelrhodopsins 1 and 2 modified with an Ile to Val mutation), and a point mutation (L171I) to enhance activation by red light (~590 nm). Axons expressing the protein become selectively inactivated during continuous excitation.

Although ReaChR viral vector models for neurological circuit mapping exist, they demonstrate some variability in expression around the injection site. To achieve more uniform expression, ReaChR tagged with fluorescent protein mCitrine was integrated into the mouse genome. Conditional expression from the Gt(ROSA)26Sor promoter is enabled upon excision of both loxP- and Frt-flanked stop cassettes. If bred to ROSA26-Cre (e.g. Stock# ) or ROSA26-Flp mice (e.g. Stock No. 003946) to ubiquitously remove one of the two conditional stop cassettes, expression of ReaChR in offspring can be made dependent on the introduction of a single recombinase directed at the remaining stop cassette.

Progeny of animals crossed with a ROSA26-Flp strain that are injected with AAV-Syn-iCre (viral vector Cre) in the primary somatosensory cortex (S1) demonstrate fluorescence at the injection site as well as in S1-originating exons extending to the primary motor cortex (M1).

In vitro recording of coronal sections from Flp-excised animals infected with AAV-Syn-iCre (virus expressing iCre from a synapsin promoter) reveal uniform current responses from L2/3 pyramidal neurons stimulated with 590 nm light.

Applications for cortical circuit mapping with dual photostimulation (2CRACM) have been demonstrated.