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. 2016 Dec 21;92(6):1181-1195.
doi: 10.1016/j.neuron.2016.11.030. Epub 2016 Dec 8.

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

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Free PMC article

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

Rahul Srinivasan et al. Neuron. .
Free PMC article

Abstract

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.

Keywords: Aldh1l1; Cre/ERT2; GCaMP; Lck-GCaMP; astrocyte; calcium; hippocampus; striatum; visual cortex.

Figures

Figure 1
Figure 1. Creation and characterization of Aldh1l1-Cre/ERT2 BAC transgenic mice
A. Schematic showing the BAC targeting construct used to create the Aldh1l1-Cre/ERT2 transgenic mice (key primer locations are also shown). B. Representative montage of the hippocampus from the Aldh1l1-Cre/ERT2 transgenic mouse injected with AAV FLEX-GFP virus with no tamoxifen injection. The white arrows show leaky expression of GFP in neurons. C. Representative montage of the hippocampus from the mGfap-Cre transgenic mouse injected with AAV FLEX-GFP virus stained for GFP (green) and S100β (red). There was expression of GFP in astrocytes; the white arrows show expression of GFP in neurons within the CA1, CA3 and dentate gyrus. D. Representative montage of the hippocampus from a Slc1a3-Cre/ERT2 transgenic mouse injected with AAV FLEX-GFP virus, followed by 75 mg/kg i.p. tamoxifen for 5 consecutive days. E. Representative montage of the hippocampus from an Aldh1l1-Cre/ERT2 transgenic mouse injected with AAV FLEX-GFP virus, followed by 75 mg/kg i.p. tamoxifen for 5 days. There was abundant, widespread expression in astrocytes; the white arrow shows GFP expression in one neuron. F. Bar graphs showing the number of astrocytes and neurons in hippocampal montages from each transgenic line following AAV FLEX-GFP virus injection. White bars show the total number of S100β positive astrocytes, the number of neurons observed in controls without tamoxifen injection and the total number of NeuN positive neurons in hippocampal montages. G–J. Representative high magnification images of CA1 astrocytes in the Aldh1l1-Cre/ERT2 mouse showing co-staining for S100β (G), GFAP (H), Glt1 (I), but not NeuN (J). In B–G, the sections were stained for GFP (green) and S100β (red).
Figure 2
Figure 2. Creation of Lck-GCaMP6fflox knock-in mice and characterization of Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice
A. Lck-GCaMP6f localizes to the plasma membrane and detects near membrane calcium signals. B. Schematic of the targeting construct used to create Lck-GCaMP6fflox knock-in mice. C. For initial characterization, Lck-GCaMP6fflox knock-in mice and Ai95 mice were injected with AAV2/5-GfaABC1D Cre AAVs or crossed with Cre/ERT2 transgenic mice. D. Representative montage of Lck-GCaMP6fflox x Slc1a3-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained for Lck-GCaMP6f. The yellow arrow indicates expression of Lck-GCaMP6f and cyto-GCaMP6f in granule cells. The pink arrow shows expression of Lck-GCaMP6f in the mossy fiber pathway. E. Representative montage of Ai95 x Slc1a3-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained for cyto-GCaMP6f. F. Representative hippocampal montage of Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained for Lck-GCaMP6f. The white arrows point to astrocytes that lack Lck-GCaMP6f expression. G. Representative hippocampal montage of Ai95 x Aldh1l1-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained with a GFP antibody shows GCaMP6f expression in nearly all astrocytes of the hippocampus. H. Representative image of the hippocampal CA1 region from a Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained for Lck-GCaMP6f (green) and S100β (red); the pyramidal cell layer (Pyr) and Stratum radiatum (S.r) are indicated. The white arrow shows an astrocyte with S100β that lacks Lck-GCaMP6f expression. The panels to the right separately show Lck-GCaMP6f staining using a GFP antibody and S100β staining. I. Representative image of the hippocampal CA1 region from a Ai95 x Aldh1l1-Cre/ERT2 double transgenic mouse injected with tamoxifen and stained for cyto-GCaMP6f (green) and S100β (red). The white arrow shows an astrocyte with S100β lacking cyto-GCaMP6f expression. The panels to the right separately show cyto-GCaMP6f staining using a GFP antibody and S100β staining. J–K and L–M. As in H–I, but for V1 of the visual cortex and striatum, respectively.
Figure 3
Figure 3. Comparison of Slc1a3-Cre/ERT2 and Aldh1l1-Cre/ERT2 transgenic mice
A. Bar graph showing the percentage of S100β+ astrocytes that express Lck-GCaMP6f in the Lck-GCaMP6fflox x Slc1a3-Cre/ERT2 and the Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice in the hippocampal CA1, CA3 and DG areas, in the V1 visual cortex and dorsolateral striatum. B. As in A, but for Ai95 x Slc1a3-Cre/ERT2 and Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice. C. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the hippocampal CA1 region of Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice. D. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the hippocampal CA1 region of Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice. E. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the hippocampal CA1 region of Lck-GCaMP6fflox x Slc1a3-Cre/ERT2 double transgenic mice. F. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the hippocampal CA1 region of Ai95 x Slc1a3-Cre/ERT2 double transgenic mice. G. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the V1 visual cortex of Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice. H. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the V1 visual cortex of Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice. I. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the V1 visual cortex of Lck-GCaMP6fflox x Slc1a3-Cre/ERT2 double transgenic mice. J. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the V1 visual cortex of Ai95 x Slc1a3-Cre/ERT2 double transgenic mice. K. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the striatum of Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice. L. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the striatum of Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice. M. Representative staining for Lck-GCaMP6f (green) and S100β (red) in the striatum of Lck-GCaMP6fflox x Slc1a3-Cre/ERT2 double transgenic mice. N. Representative staining for cyto-GCaMP6f (green) and S100β (red) in the striatum of Ai95 x Slc1a3-Cre/ERT2 double transgenic mice.
Figure 4
Figure 4. Visual cortex astrocytes from Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 and Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice display spontaneous and PE-evoked calcium signals
A. Schematic of the approach/workflow for imaging of astrocyte calcium signals in brain slices. B. Representative images of baseline calcium signals in V1 visual cortex astrocytes, before and during 10 μM phenylephrine (PE) in Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mice. C. As in B, but for Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice expressing cyto-GCaMP6f. D. Representative traces of calcium signals in the soma and processes of the astrocyte from the Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 double transgenic mouse shown in B. E. Representative traces of Ca2+ signals in the soma and processes of the astrocyte from the Ai95 x Aldh1l1-Cre/ERT2 double transgenic mouse shown in C. F–G. Scatter plots of PE-evoked calcium signal amplitude and decay times from the somata (F) and processes (G) of visual cortex astrocytes of the Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 and Ai95 x Aldh1l1-Cre/ERT2 double transgenic mice, respectively. H. Scatter plots of spontaneous calcium signal properties in astrocyte processes from Lck-GCaMP6fflox x Aldh1l1-Cre/ERT2 (Lck-G6f) and Ai95 x Aldh1l1-Cre/ERT2 (cyto-G6f) double transgenic mice. I. Representative traces and average data for the effect of Ca2+ free buffers on Ca2+ signals measured with Lck- and cyto-GCaMP6f in astrocyte somata and processes. The data are from 5 mice in panels A–H and for 4 mice for panel I. Sometimes the bars representing the s.e.m. are smaller than the symbol used for the mean.
Figure 5
Figure 5. Comparison of in vivo astrocyte calcium signals measured with Lck-GCaMP6f and cyto-GCaMP6f driven by Aldh1l1-Cre/ERT2 mice
A. Schematic drawings showing the workflow for in vivo 2PLSM in head-fixed, awake mice. In brief, after the mice were injected with tamoxifen for 7 days, a lightweight metal head bar was glued to their skull and a 3 mm cranial window was made above the visual cortex. Mice were then head-fixed onto a spherical treadmill where they were free to rest or run. A 40 x objective lens as part of a 2-photon laser scanning imaging microscope was positioned above the cranial window. An air pump outside the microscope enclosure was used to generate an unexpected air puff, which evoked startle response. B & C. Top: Representative pseudo-colored images showing the fluorescence increase of membrane-tethered Lck-GCaMP6f (B) and cyto-GCaMP6f (C) in astrocytes of mouse visual cortex before and during startle. Bottom: representative ΔF/F traces from 8 randomly selected ROIs (10 μm2 each) in Lck-GCaMP6f x Aldh1l1-Cre/ERT2 mice (orange) and cyto-GCaMP6f x Aldh1l1-Cre/ERT2 (blue). The gray vertical line indicates the air puff. The trend for the baseline is shown in black. D. The average F/F trace of 118 ROIs from four Lck-GCaMP6f x Aldh1l1-Cre/ERT2 mice (s.e.m. shown with gray lines for every 5th time point). E. The average F/F trace of 96 ROIs from four cyto-GCaMP6f x Aldh1l1-Cre/ERT2 mice (s.e.m. shown with gray lines for every 5th time point). F. Comparison of calcium signals detected by Lck-GCaMP6f (113 events, n = 4 mice) and cyto-GCaMP6f (94 events, n = 4 mice) during startle. G. Comparison of calcium signals detected by Lck-GCaMP6f and cyto-GCaMP6f during voluntary locomotion (without air puff). H. Comparisons of calcium signals detected by Lck-GCaMP6f and cyto-GCaMP6f during the resting phase (without air puff and locomotion).
Figure 6
Figure 6. Aldh1l1-Cre/ERT2 mice permit high-density imaging of calcium signals in vivo and in brain slices
A. Representative images and average data of 10 μM PE-evoked astrocyte calcium signals in visual cortex brain slices when cyto-GCaMP6f was driven by Slc1a3-Cre/ERT2 or by Aldh1l1-Cre/ERT2. B. As in A, but for in vivo startle-evoked response in the visual cortex. In both cases, many more astrocytes were detected when cyto-GCaMP6f was driven by the Aldh1l1-Cre/ERT2 mouse.
Figure 7
Figure 7. Aldh1l1-Cre/ERT2 x RiboTag mice and the determination of the cortical astrocyte transcriptome at P80
A. Representative photomicrographs of IHC data showing strong colocalisation between S100β and Rpl22HA in the visual cortex. B. Representative photomicrographs of IHC data showing no colocalisation between NeuN and Rpl22HA. C. Schematic of the workflow. D. The representative Western blot shows that Rpl22HA was preserved in the IP sample, whereas β-actin was depleted in relation to input. In contrast, there was no Rpl22HA in the supernatant. In the IP lane, the 25 and 50 kD bands are the light and heavy chains of the anti-HA antibody that was used in the IP. E. The RNAseq FPKM values of well-established markers of astrocytes, neurons, oligodendrocytes, and microglia in the IP samples are plotted as mean ± s.e.m. from four biological replicates (n = 4 mice). F. Graphs comparing expression of 4727 transcripts enriched in either P80 IP (2-fold enriched over input FDR < 0.05) or P7 astrocytes (2-fold enriched over average of all other cell types) ranked based on FPKM percentile. Genes that were not sequenced in both datasets were excluded from this list. Left: Scatter plot representing the rank of each gene in the P80 (x-axis) vs. the P7 (y-axis) dataset. Clustering along the diagonal indicates similar rank in both datasets. Right: Rank-rank hypergeometric overlap (RRHO) heatmap. Each pixel represents the significance of overlap between the two datasets (-log10(pvalue), hypergeometric test, bin size = 50). Red cells represent highly significant overlap. Color scale (right) represents a range between -log10(pvalue) = 0 (p = 1) and 350 (p = 10−350) G. A heatmap showing relative expression (row z-score) of the 32 genes whose percentile FPKM differ by at least 0.1 between highly expressed P80 and P7 cortical astrocytes determined by RRHO algorithm. These genes and their FPKM values are reported in Supp Table 2. All genes for the analysis in panel F and all the raw data are provided as part of Supplementary Excel file 1. The data used for P7 were from Zhang et al., (2014)

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