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

Abstract

Summary 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 characterised Aldh1l1-Cre/ERT2 mice using imaging, immunohistochemistry, AAV-FLEX-GFP microinjections and crosses to RiboTag, Ai95 and new Cre-dependent membrane tethered Lck-GCaMP6f knock-in 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 characterised, easy-to-use resources with which to selectively study astrocytes in situ and in vivo in multiple experimental scenarios.

Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis

“…Next, we mined gene expression databases 12-14 to identify astrocytic cell adhesion molecules (CAMs) known to interact with neuronal and synaptic proteins. Intriguingly, astrocytes express three members of the NL family, NL1, NL2, and NL3, at levels comparable to, or higher than, neurons (Extended Data Fig.…”

“…d , Average FPKM values of cell-type specific transcripts from specimens listed in c (all data from 14 ). e , Average FPKM values of NL mRNAs that are engaged with translating ribosomes from P80 mouse astrocytes (red) and total input (black) using the Aldh1L1-creER T2 /Ribo-tag mice (all data from 12 ). f , Average FPKM values of cell-type specific transcripts from specimens listed in e (all data from 12 ).…”

“…e , Average FPKM values of NL mRNAs that are engaged with translating ribosomes from P80 mouse astrocytes (red) and total input (black) using the Aldh1L1-creER T2 /Ribo-tag mice (all data from 12 ). f , Average FPKM values of cell-type specific transcripts from specimens listed in e (all data from 12 ). g , Single optical section confocal images (top) and 3D surface renderings (bottom) of fluorescent in-situ hybridization (FISH) experiments of V1 L1 astrocytes (green) from P21 Aldh1L1-EGFP mice.…”

Genetic targeting of astrocytes in the gliovascular unit of the adult brain

“…Currently the most common method to selectively target astrocytes is through transgenic mouse lines. For example, researchers have expressed Cre in astrocytes using astrocyte-specific promoters (Glial fibrillary acid protein (Gfap), Glutamate-aspartate transporter (Glast), among others) 3,4 . These astrocyte transgenic mice lines have given us insight into how astrocytes contribute to a variety of behaviors and neurovascular properties 5,6 .…”

“…2+ indicators (GECIs) have enabled several groups to examine the dynamics of fine-process Ca 2+ signaling during spontaneous and evoked activity both in vivo and in situ 4,36 . Recent further optimization of GECIs has allowed for increased monitoring of Ca 2+ signals in fine processes 37 , increased temporal specificity and sensitivity 38 , and simultaneous imaging of astrocyte Ca 2+ signaling changes across multiple cellular compartments 39 .…”