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New transgenic mouse lines for selectively targeting astrocytes and for studying calcium signals in astrocyte processes in situ and in vivo

ABSTRACT: The Khakh laboratory developed and characterized a new Aldh1l1-Cre/ERT2 transgenic mouse line to selectively target astrocytes in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and analyses of the cortical astrocyte transcriptome from adults.

ORGANISM(S): Mus musculus

PROVIDER: GSE84540 | GEO | 2016/11/07

SECONDARY ACCESSION(S): PRJNA329559

REPOSITORIES: GEO

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Neural circuit-specialized astrocytes: genomic, proteomic, morphological and functional evidence

Project description:The Khakh laboratory used Aldh1l1-Cre/ERT2 transgenic mouse line crossed with RiboTag mice to sequence actively translated mRNAs and compare striatal and hippocampal astrocyte transcriptomes from adults.

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Astrocyte alterations that accompany neuroinflammation induced anhedonia

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New Transgenic Mouse Lines for Selectively Targeting Astrocytes and Studying Calcium Signals in Astrocyte Processes In Situ and In Vivo.

Project description:Astrocytes exist throughout the nervous system and are proposed to affect neural circuits and behavior. However, studying astrocytes has proven difficult because of the lack of tools permitting astrocyte-selective genetic manipulations. Here, we report the generation of Aldh1l1-Cre/ERT2 transgenic mice to selectively target astrocytes in vivo. We characterized Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections, and crosses to RiboTag, Ai95, and new Cre-dependent membrane-tethered Lck-GCaMP6f knockin mice that we also generated. Two to three weeks after tamoxifen induction, Aldh1l1-Cre/ERT2 selectively targeted essentially all adult (P80) brain astrocytes with no detectable neuronal contamination, resulting in expression of cytosolic and Lck-GCaMP6f, and permitting subcellular astrocyte calcium imaging during startle responses in vivo. Crosses with RiboTag mice allowed sequencing of actively translated mRNAs and determination of the adult cortical astrocyte transcriptome. Thus, we provide well-characterized, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios.

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

Project description: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.

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Transcriptional analysis of murine cortical astrocytes with adeno-associated viral transduction with and without 4x6T miR cassette, vs transgenically labeled astrocytes

Project description:We generated adeno-associated Cre viral vectors with improved astrocyte-specific expression by including a cassette of miRNA targeting sequences (4x6T) to decrease off-target expression. We delivered these viruses in Ribotag mice using the PHP.eB serotype, either intracortically or systemically. We compared these samples with mice that received intracortical AAV2/5::GfaABC1D-Cre and transgenic Aldh1l1-CreERT2/Ribotag mice. We sequenced whole-tissue RNA as well as ribosomally-loaded astrocyte-enriched mRNA to assess cell-type specificity of viruses as well as virus-related transcriptomic changes.

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Slc1a3-2A-CreERT2 knock-in mouse to study astrocyte gene expression and calcium signaling

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Riding the spermatogenic wave: Profiling gene expression within neonatal germ and Sertoli cells during a synchronized initial wave of spermatogenesis

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Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis.

Project description:Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.

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