Fig. 4

Activity dependent alternative splicing of Rcan1. a Rcan1 is encoded by 6 exons (yellow boxes, not to scale). The first three exons are mutually exclusive and their alternative usage results in transcript 1, 2 and 3 (green). The location of two alternative probes used for in situ hybridization (ISH) are shown in black. b Visualization of the microarray data. The plot shows the log2 ratio calculated from the mean normalized intensities of transformed expression values over the course of probesets per exon. Green lines represent indicated expression kinetics. Blue boxes represent the probesets per exon present on the microarrays. c Validation of Rcan1 variant expression in hippocampus of control mice or 1, 4 or 8 h after seizure onset by RNA sequencing. Shown are coverage plots (blue) from paired-end reads. The reference gene track is depicted below (black). d Sashimi plot of RNA sequencing data. Numbers indicate the counts of RNA sequencing reads that span the respective exon junctions. e Validation of splicing events by RT-PCR. Numbers indicate exons. Exon-spanning primer pairs (arrows) were used to assess expression of exons in total RNA of hippocampi of control mice (−) and of mice sacrificed 4 h after onset of seizures (+). Note that the observed transcript size in the left panel indicates exclusion of exon 3 and that the forward primer in the right panel corresponds to the 5′-UTR of exon 3. f Autoradiograms of ISH of parallel sagittal sections of E16 embryonic mice with the variant specific Rcan1 probes on parallel sections. g Autoradiograms of ISH of parallel coronal brain sections of mice sacrificed at indicated time points after seizure onset. ISH of one specific antisense probe was conducted in parallel on one glass slide. CA1 field CA1 of the hippocampus, cc cerebral cortex, dg dentate gyrus, he heart, l lung, md midbrain