Local and CNS-wide astrocyte intracellular calcium signalling attenuation in vivo with CalExflox mice: experimental and computational exploration

In this study, we generated knock-in CalExflox mice (Calcium Extrusion) for Cre-dependent expression of mCherry-hPMCA2w/b to attenuate astrocyte calcium signaling in genetically defined cells in vivo. We crossed CalExflox mice to astrocyte specific Aldh1l1-Cre/ERT2 mice in order to achieve inducible global CNS-wide calicum signaling attenuation. Within six days of induction in the bigenic mice, we observed profoundly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality, which based on imaging, behavioural, and RNA-seq analyses was likely to be partly due to significant cerebellar defects. CalExflox mice were crossed to astrocyte specific Aldh1l1-Cre/ERT2 mice to generate bigenic mice carrying both alleles (CalExflox; Aldh1l1-Cre/ERT2). CalExflox mice were used as controls. Both groups of mice received a single dose tamoxifen (75 mg/kg) and the cerebellar and the striata were dissected out 5 days later. The tissues were homogenized in ice-cold lysis buffer and the cerebellar RNA was extracted using a mirVana miRNA isolation kit. RNA samples with RNA integrity number (RIN) greater than 7 were used for multiplexed library preparation with Nugen Ovation RNA-Seq System V2. All samples were multiplexed into a single pool in order to avoid batch effects, and sequencing was performed on Illumina NextSeq 4000 for 2 × 75 yielding at least 45 million reads per sample.