Abstracts

Rationale: The concentration of intracellular calcium, which is involved in many cellular processes, can be tracked to monitor CNS activity. Alterations in the epileptic brain may manifest through altered calcium activity. Genetically encoded calcium indicators (GECIs) allow for chronic imaging and expression in genetically defined cell types. GCaMP5G, a GFP-based GECI, is fast, has low baseline fluorescence, exhibits high signal-to-noise ratio and is, therefore, ideal for recording CNS activity. In order to express GCaMP5G in specific CNS cell types, we generated a powerful and flexible Cre-dependent mouse reporter line with GCaMP5G-IRES-tdTomato in the RNA Polymerase II (Polr2a) locus. This reporter line should prove valuable in the study of mouse models of epilepsy.Methods: We constructed the Polr2a-ITR-CAG-LSL-GCaMP5G-IRES-tdTomato-WPRE-ITR (PC::G5-tdT) targeting vector with the strong ubiquitous CAG promoter, a loxP-flanked inverted neomycin resistance gene and a transcription stop (LSL) cassette, a GCaMP5G gene, a FRT-flanked internal ribosomal entry site (IRES) driving tdTomato expression and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The cassette was flanked with piggyBac inverted terminal repeats (ITR) from the cabbage looper moth Trichoplusia ni. In vivo and slice imaging was performed using two-photon microscopy. Intact awake animal experiments were implemented with an epifluorescence microscope. Results: The PC::G5-tdT reporter mouse line exhibits several notable features including: An improved GCaMP to reliably detect CNS cell activity in vivo; Polr2a locus (chromosome 11) targeting allowing breeding without any linkage issues with Rosa26 (chromosome 6) targeted mice; tdTomato for detection of abeyant GCaMP5-positive cells; FRT-flanked IRES-tdTomato which can be removed with FLP and potential increase in expression level following exposure to piggyBac transposase. PC::G5-tdT crosses to HoxB8-Cre, GFAP-CreER and Emx1-Cre lines displayed no obvious behavioral or morphological abnormalities. We tested the mouse in a range of preparations. KCl-evoked astrocyte calcium transients (up to 350% F/F0) were observed in acute hippocampal slices obtained from a GFAP-CreER cross. In vivo imaging, using an epifluorescence microscope and 4x objective, in an awake P2 Emx1-Cre mouse, with both skin and bone intact, revealed spontaneous calcium transients (70%) throughout the cortex. Spontaneous transients were recorded from astrocytes (150%) in the prefrontal cortex and odor-evoked calcium transients (250%) in olfactory bulb glomeruli of an anaesthetized Emx1-Cre cross were recorded in vivo. Presentation of different odors (methyl valerate vs. ethyl butyrate) reliably elicited different patterns of glomeruli activation. Conclusions: Cre-lox technology makes it straightforward to image calcium transients in a wide variety of cell types. The PC::G5-tdT reporter mouse is an extremely valuable tool for the study of CNS activity in the healthy and epileptic brain.