Deletion of neuroligins from astrocytes does not detectably alter synapse numbers or astrocyte cytoarchitecture by maturity
- Department of Molecular and Cellular Physiology, Stanford University School of Medicine, United States
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, United States
- Department of Neurosciences, University of California San Diego, United States
- Department of Neurobiology, University of California San Diego, United States
- Department of Pathology, Stanford University School of Medicine, United States
- Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, United States
- Howard Hughes Medical Institute, Stanford University School of Medicine, United States
Figures
Figure 1
Neuroligin genes (Nlgn1–3) are abundantly expressed by neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) in the brain as determined by analyses of publicly available RNAseq datasets.
Analysis of Nlgn1, Nlgn2, and Nlgn3 mRNA levels in neurons (green), astrocytes (blue), oligodendrocyte lineage cells (orange), microglia (red), and other cell types in the brain (yellow) using the single-cell RNAseq dataset published from the McCarroll lab (Saunders et al., 2018, https://www.dropviz.org) (A), Chan Zuckerberg Initiative (The Tabula Muris Consortium et al., 2018) (B), Wu lab (Zhang et al., 2014) (C), and Linnarson lab (Zeisel et al., 2018, https://www.mousebrain.org) (D). Note that although relative expression levels vary greatly between datasets, all datasets support the conclusion that Nlgn1, Nlgn2, and Nlgn3 are broadly expressed in astrocytes and OPCs. (E) Analysis of Nlgn1, Nlgn2, and Nlgn3 mRNA levels in astrocytes in three different brain regions (cortex, hippocampus, and striatum) using the bulk RNAseq datasets published by the Khakh lab (Chai et al., 2017; Srinivasan et al., 2016, https://www.astrocyternaseq.org) that examined RiboTag-purified mRNAs. (F) Analysis of Nlgn1, Nlgn2, and Nlgn3 mRNA levels in astrocytes as a function of age in mice using the bulk RNAseq datasets published by the Barres lab (Clarke et al., 2018; https://www.brainrnaseq.org). Astrocyte mRNA was purified from three different brain regions (cortex, hippocampus, and striatum) by RiboTag pulldowns in Aldh1l1-eGFP-L10a mice. Note that Nlgn4 is not measured in the RNAseq experiments shown, probably because its expression levels are low and because its mRNA is very GC rich.
Figure 2
Nlgn1–3 are efficiently and selectively deleted in astrocytes by crossing triple Nlgn1–3 conditional KO mice with Aldh1l1-CreERT2 driver mice and inducing Cre-activity with tamoxifen early during postnatal development.
(A) Breeding strategy. Triple conditional KO mice carrying floxed Nlgn1, Nlgn2, and Nlgn3 alleles or mice with a Cre-sensitive tdTomato (tdT) reporter allele (Ai14) were crossed with pan-astrocyte, tamoxifen-inducible Aldh1l1-CreERT2 BAC transgenic mice. Nlgn1–3 cKO mice were crossed for multiple generations until homozygosity was reached (females: Nlgn1f/f 2f/f 3f/f, males: Nlgn1f/f 2f/f 3f/y). (B, C) Two different tamoxifen-induced Cre-activation schedules were used to delete Nlgn1–3 in astrocytes. Aldh1l1-CreERT2 mice and controls (littermate Nlgn1–3 cKO mice lacking the Aldh1l1-CreERT2 allele) were injected with tamoxifen at P10 and P11 (B) (Trotter et al., 2021) or at P1 (C). Mice were sacrificed at least 4 (B) or 5 weeks (C) post Cre induction to ensure complete deletion of neuroligins and decay of any astrocyte-specific neuroligin proteins. (D, E) To confirm the specificity and efficiency of the deletion of target genes in astrocytes using the Aldh1l1-CreERT2 BAC transgenic mouse line via tamoxifen injection at P1, Cre-recombination was visualized in the hippocampus (D) and primary visual cortex (E) via expression of tdT in reporter mice (magenta). Sections were additionally labeled for NeuN to mark neurons (green) and S100β to mark astrocytes (blue). (F) The Aldh1l1-CreERT2-induced deletion of floxed genes produced by P1 tamoxifen injections is effective and selective for astrocytes as quantified using expression of tdTomato in reporter mice. tdTomato expression was quantified in the CA1 region of the hippocampus (the S. oriens, S. pyramidale, S. radiatum, and S. lacunosum-moleculare), the dentate gyrus (molecular layer [ML], granule cell layer [GCL], and hilus), and layer IV of the primary visual cortex.
Figure 3
Quantification of the Nlgn1–3 conditional KO efficiency in astrocytes by qRT-PCR using RiboTag-mediated isolation of astrocytic mRNAs.
(A) Diagram of the experimental strategy. AAVs (AAV2/5) encoding the RiboTag (Rpl22-HA) and Lck-mVenus (a membrane-targeted fluorescent reporter) driven by the astrocyte-specific GfaABC1D promoter and additionally including a 4x6T cassette of mIR-124 targeting sequences (Gleichman et al., 2023) were injected at P35-40 into the CA1 region of mice with a P1 induction of the astrocyte-specific Nlgn1–3 KO or of control mice. Mice were analyzed 4 weeks later. (B) Representative coronal brain section (counterstained with DAPI) showing successful viral targeting of CA1 and adjacent regions indicated by the presence of Lck-mVenus (green) and Rpl22-HA (purple) astrocytes. (C–E) quantitative RT-PCR validation of the successful pull-down of highly enriched astrocyte ribosome-bound mRNA from micro-dissected cortical tissue (i.e., Rpl22-HA). Enrichment of astrocyte marker, Aqp4, and de-enrichment of other cell marker genes is expressed as levels in pull-down (i.e., IP) relative to total mRNA (i.e., input). (D) Confirmation in astro-Nlgn123 cKO mice of reduced levels of Nlgn1, Nlgn2, and Nlgn3 using assays that recognized floxed exons here expressed as levels in IP relative to input. (E) Same as D, except expressed as a % change relative to wild-type based on the 2−ΔΔCt method. (F–H) Same as C–E, but for the hippocampal CA1 region. Data in summary graphs are means ± SEM with each data point representing individual animals. Unpaired two-tailed t-tests were used to test statistical significance of data in D and G, whereas a one-sample t-test was used to test statistical significance of data in E and H (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Figure 4
Conditional deletion of Nlgn1–3 in astrocytes throughout the brain at early postnatal timepoints (P10/P11 or P1) has only modest effects on overall neuroligin protein levels and does not significantly alter the synaptic proteome.
Representative immunoblots and quantifications of Nlgn1, Nlgn2, and Nlgn3 protein levels from hippocampal (A) and cortical lysates (B) of astrocyte Nlgn1–3 cKO and littermate control mice injected with tamoxifen at P10 and P11 and sacrificed at P38. Proteins were quantified on immunoblots using fluorescent secondary antibodies, with protein levels normalized to β-actin and then to control levels (n = 5, all male). (C, D) Representative immunoblots and (D) quantification for various synaptic protein levels from hippocampal and cortical lysates of astrocyte Nlgn1–3 cKO and littermate control mice injected with tamoxifen at P10 and P11 and sacrificed at P38. Protein is quantified using fluorescent secondary antibodies, with protein levels normalized to β-actin and then to control levels (n = 5, all male). (E–H) Same as (A–D) except mice were injected with tamoxifen at P1 and sacrificed at P35 (n = 4, 2 male and 2 female). Numerical data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (**p < 0.01).
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Figure 4—source data 1
PDF file containing original western blots for Figure 4A, C, E, G indicating the relevant bands and experimental groups.
- https://cdn.elifesciences.org/articles/87589/elife-87589-fig4-data1-v1.zip
-
Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4A, C, E, G.
- https://cdn.elifesciences.org/articles/87589/elife-87589-fig4-data2-v1.zip
Figure 5 with 1 supplement
Conditional deletion of Nlgn1–3 in astrocytes starting at P1 does not alter excitatory or inhibitory synapse numbers in the hippocampus as assessed by immunohistochemistry with antibodies to synaptic markers.
(A) Representative images of CA1 and dentate gyrus hippocampal sections from astrocyte Nlgn1–3 cKO and littermate control mice, injected with tamoxifen at P1 and sacrificed at P35, stained for dendritic marker MAP2 (magenta), excitatory presynaptic marker vGluT1 (red), excitatory postsynaptic marker Homer1 (green), and DAPI (blue), and taken at ×20 (top) and ×60 (bottom) magnifications. (B) Quantification of the total vGluT1 immunofluorescence signal for low- (×20; top) and high-magnification imaging (×60; bottom) across the layers of the hippocampus (S. oriens, S. pyramidale, S. radiatum, S. lacunosum-moleculare, dentate gyrus molecular layer), first internally normalized to MAP2 and then to the average vGluT1 immunofluorescence levels in the S. oriens of control mice. (C) Quantification of the total Homer1 immunofluorescence signal for low- (×20; top) and high-magnification imaging (×60; bottom) in the CA1 S. pyramidale (left) and S. radiatum (right), first internally normalized to MAP2 and then to the average Homer1 immunofluorescence level in control mice. (D) Representative images of CA1 and dentate gyrus hippocampal sections from astrocyte Nlgn1–3 cKO and littermate control mice stained for dendritic marker MAP2 (magenta), excitatory presynaptic marker vGluT2 (red), excitatory postsynaptic marker Homer1 (green), and DAPI (blue), and taken at ×20 (top) and ×60 (bottom) magnifications. (E) Quantification of total vGluT2 immunofluorescence signal for low- (×20; top) and high-magnification imaging (×60; bottom) across the layers of the hippocampus, first internally normalized to MAP2 and then to average vGluT2 immunofluorescence level in S. oriens of control mice. (F) Quantification of total Homer1 immunofluorescence signal for high-magnification imaging (×60) in the CA1 S. lacunosum-moleculare (left) and dentate gyrus molecular layer (right), first internally normalized to MAP2 and then to the average Homer1 immunofluorescence level in control mice. (G) Representative images of CA1 and dentate gyrus hippocampal sections from astrocyte Nlgn1–3 cKO and littermate control mice stained for dendritic marker MAP2 (magenta), inhibitory postsynaptic marker Gephyrin (red), inhibitory presynaptic marker GAD67 (green), and DAPI (blue), and taken at ×20 (top) and ×60 (bottom) magnifications. (H) Quantification of total Gephyrin (top) and GAD67 (bottom) immunofluorescence signal for low-magnification imaging (×20) across the layers of the hippocampus, first internally normalized to MAP2 and then to average Gephyrin (top) or GAD67 (bottom) immunofluorescence level in S. oriens of control mice. (I) Quantification of puncta density for Gephyrin (left) and for Gephyrin having GAD67 (right) for high-magnification imaging (×60) in the CA1 S. radiatum. (J) Same as (I), except that the density of GAD67-positive puncta and of puncta positive for GAD67 having Gephyrin were quantified. Data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (n = 4, 2 male and 2 female).
Figure 5—figure supplement 1
Conditional deletion of astrocytic Nlgn1–3 at P1 does not alter the number or size of inhibitory synapses in the CA1-region Stratum Radiatum.
(A) Quantification of total Gephyrin (left) or GAD67 (right) immunofluorescence in CA1 Str. Radiatum from astrocyte Nlgn1–3 cKO and littermate control mice. Images of hippocampal sections were taken at ×60 magnification. Total immunofluorescent signal was first internally normalized to MAP2 and then to average Gephyrin (left) or GAD67 (right) immunofluorescence level in control mice. (B) Quantification of puncta size for Gephyrin (left) and GAD67 (right) CA1 Str. Radiatum from astrocyte Nlgn1–3 cKO and littermate control mice. Representative images are shown in Figure 5G. Data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (n = 4, 2 male and 2 female).
Figure 6 with 1 supplement
Conditional deletion of Nlgn1–3 in astrocytes starting at P1 has no major effect on basal excitatory or inhibitory neurotransmission monitored in hippocampal CA1 pyramidal neurons.
(A) Representative traces for miniature EPSCs (mEPSCs) from CA1 pyramidal neurons in acute slices from astrocyte Nlgn1–3 cKO and littermate controls injected with tamoxifen at P1 and recorded at P44–P50. (B) Cumulative distribution and summary graph of the mEPSC amplitude and (C) frequency. (D) Summary graph of mEPSC rise (left) and decay (right) times (n = 14–15 cells/3 mice per genotype). (E–H) Same as (A–D) except for miniature IPSCs (mIPSCs) (n = 16 cells/3 mice per genotype). Data in summary graphs are means ± SEM with each data point representing individual cells. Unpaired two-tailed t-tests were used to test statistical significance of data in bar graphs, and Kolmogorov–Smirnov tests were used for cumulative curves (****p < 0.0001).
Figure 6—figure supplement 1
Conditional deletion of astrocytic Nlgn1–3 at P1 does not alter CA1 pyramidal neuron membrane properties.
Summary graphs of membrane capacitance (left) and membrane resistance (right) from CA1 pyramidal neurons in acute slices from astrocyte Nlgn1–3 cKO and littermate controls injected with tamoxifen at P1 and recorded at P44–P50. Data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (n = 14–15 cells/3 mice per genotype).
Figure 7
Conditional deletion of Nlgn1–3 in astrocytes starting at P1 does not alter excitatory or inhibitory synapse numbers in layer IV of the primary visual cortex as assessed by immunohistochemistry with antibodies to synaptic markers.
(A) Representative images of primary visual cortex (V1) layer IV (L4) astrocyte Nlgn1–3 cKO and littermate control mice, injected with tamoxifen at P1 and sacrificed at P35, stained for the dendritic marker MAP2 (magenta), excitatory presynaptic marker vGluT1 (red), excitatory postsynaptic marker Homer1 (green), and DAPI (blue) and taken at ×60 magnification. (B) Quantification of total vGluT1 immunofluorescence signal in V1L4 first internally normalized to MAP2 and then to the average vGluT1 immunofluorescence level in control mice. (C) Representative images of V1L4 astrocyte Nlgn1–3 cKO and littermate control mice stained for dendritic marker MAP2 (magenta), excitatory presynaptic marker vGluT2 (red), excitatory postsynaptic marker Homer1 (green), and DAPI (blue) taken at ×60 magnification. (D) Quantification of total vGluT2 (top) and Homer1 (bottom) immunofluorescence signal in V1L4 first internally normalized to MAP2 and then to the average vGluT2 (top) and Homer1 (bottom) immunofluorescence levels in control mice. (E) Representative images of V1L4 astrocyte Nlgn1–3 cKO and littermate control mice stained for dendritic marker MAP2 (magenta), inhibitory postsynaptic marker Gephyrin (red), inhibitory presynaptic marker GAD67 (green), and DAPI (blue) and taken at ×60 magnification. (F) Quantification of total Gephyrin (left) or GAD67 (right) immunofluorescence signal in V1L4 first internally normalized to MAP2 and then to average Gephyrin (left) or GAD67 (right) immunofluorescence level in control mice. (G) Quantification of Gephyrin (left), GAD67 (middle left), Gephyrin having GAD67 (middle right), and GAD67 having Gephyrin (right) puncta density in V1L4. Data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (*p < 0.05) (n = 4, 2 male and 2 female).
Figure 8
Conditional deletion of Nlgn1–3 in astrocytes starting at P1 does not detectably alter the proteome or the cytoarchitecture of astrocytes in the hippocampus or layer IV of the primary visual cortex.
(A) Representative immunoblots and (B) quantification of various glial protein levels from hippocampal and cortical lysates of astrocyte Nlgn1–3 cKO and littermate control mice injected with tamoxifen at P1 and sacrificed at P35. Proteins were quantified using fluorescent secondary antibodies, with protein levels normalized to β-actin and then to control levels (n = 4, 2 male and 2 female). Note that some blots in A exhibit similar shapes but are not copy-paste errors but truly independent blots. (C) Representative images of CA1 and dentate gyrus hippocampal sections from astrocyte Nlgn1–3 cKO and littermate control mice, injected with tamoxifen at P1 and sacrificed at P35, stained for astrocytic marker GFAP (white) and DAPI (blue), and taken at ×20 magnification. (D) Quantification of the total GFAP immunofluorescence signal across the layers of the hippocampus (S. oriens, S. pyramidale, S. radiatum, S. lacunosum-moleculare, dentate gyrus molecular layer), normalized to the average GFAP immunofluorescence level in the S. oriens of control mice (n = 4, 2 male and 2 female). (E) To measure astrocyte volume, astrocyte Nlgn1–3 cKO and littermate control mice were injected with tamoxifen at P1, subjected to stereotactic injections into the hippocampus of AAVs expressing membrane-tethered mVenus in astrocytes at P14, and sacrificed at P35. Representative images of mVenus-expressing astrocytes in the CA1 S. radiatum are shown with a corresponding 3D volume reconstruction performed in Imaris. (F) Summary graph of CA1 S. radiatum astrocyte volumes shown averaged per animal with means ± SEM on the bar graph, as well as data points for individual astrocyte volumes. Statistical significance determined by unpaired two-tailed t-test of data averaged per animal (n = 3, 1 male and 2 female). (G, H) Same as E and F, except for primary visual cortex layer IV astrocytes. Numerical data are means ± SEM. Dots in bar graphs represent independent biological replicates; in F and H, larger dots are independent biological replicates and smaller dots are pseudoreplicates since these are commonly reported in papers to boost statistical significance.
-
Figure 8—source data 1
PDF file containing original western blots for Figure 8A, indicating the relevant bands and experimental groups.
- https://cdn.elifesciences.org/articles/87589/elife-87589-fig8-data1-v1.zip
-
Figure 8—source data 2
Original files for western blot analysis displayed in Figure 8A.
- https://cdn.elifesciences.org/articles/87589/elife-87589-fig8-data2-v1.zip
Figure 9 with 1 supplement
Deletion of Nlgn1–4 in mouse glia co-cultured with wild-type human neurons does not significantly alter spontaneous synaptic events (mEPSCs) mediated by the co-cultured neurons.
(A) Schematic of the experimental strategy. (B–D) Measurements of the mouse Nlgn1, Nlgn2, and Nlgn3 mRNA levels in the mouse glia culture as a function of the expression of ΔCre (Ctrl., negative control) or Cre (cKO) reveal complete deletion of the expression of all neuroligins (n = 3 independent cultures). (E–G) mEPSC measurements in human neurons co-cultured with mouse glia expressing (Ctrl.) or lacking neuroligins (cKO) fail to uncover any changes as a function of the neuroligin expression in the glia, (E, sample traces; F, cumulative probability plot of the mEPSC amplitudes [inset, summary graph of the mean mEPSC amplitude]; G, cumulative probability plot of the mEPSC interevent intervals [inset, summary graph of the mean mEPSC frequency]). Numerical data are means ± SEM. Dots in bar graphs in F and G represent cells (n = 3 independent cultures).
Figure 9—figure supplement 1
Further data characterizing the effect of a deletion of glial Nlgn1–3 on co-cultured human neurons.
(A) Representative images of primary mouse glia cultures from Nlgn1–4 quadruple cKO mice infected with lentiviruses encoding FLP-EGFP (Ctrl.) or Cre-EGFP fusion proteins and stained for GFP, DAPI, and GFAP as glial marker. Note that the levels of EGFP expression differ among glia cells but that based on qRT-PCR measurements the lower levels of Cre-EGFP in some glial cells are sufficient for complete recombination of Nlgn1, Nlgn2, and Nlgn3 genes (Nlgn4 is constitutively deleted). (B) Summary graph of the ratio of EGFP-expressing to DAPI-stained cells demonstrates that nearly all cells in the culture are infected by the lentiviruses. (C) Summary graphs of the membrane capacitance (left) and input resistance (right) monitored in human neurons that are co-cultured with mouse glia expressing or lacking all neuroligins (Nlgn1–4). Data are means ± SEM with statistical significance determined by unpaired two-tailed t-test (n = 14–15 cells/3 mice per genotype).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Aldh1l1-CreERT2 BAC transgenic mice | The Jackson Laboratory | RRID:IMSR_JAX:029655 | |
| Strain, strain background (M. musculus) | Nlgn123 cKO mice | Südhof lab | ||
| Cell line (Homo sapiens) | HEK293T cells | ATCC | RRID:CVCL_0063 | |
| Cell line (H. sapiens) | Human H1 embryonic stem cells (H1 hESCs) | WiCell Research Resources | WA01; male; RRID:CVCL_9771 | |
| Antibody | Anti-Nlgn1 (mouse monoclonal) | Synaptic Systems | 129111, RRID:AB_887747 | 1:2000 |
| Antibody | Anti-Nlgn2 (rabbit polyclonal) | Synaptic Systems | 129203, RRID:AB_993014 | 1:1000 |
| Antibody | Anti-Nlgn3 (mouse monoclonal) | Synaptic Systems | 129311, RRID:AB_2151947 | 1:2000 |
| Antibody | Anti-β Actin (mouse monoclonal) | Sigma-Aldrich | A1978, RRID:AB_476692 | 1:5000 |
| Antibody | Anti-VGluT2 (guinea pig, polyclonal) | Millipore Sigma | AB2251, RRID:AB_1587626 | 1:1000 |
| Antibody | Anti-VGluT1 (rabbit polyclonal) | TCS | YZ6089, RRID:AB_2861224 | 1:1000 |
| Antibody | Anti-Synaptotagmin 1 (rabbit polyclonal) | TCS | V216 | 1:1000 |
| Antibody | Anti-Synaptobrevin 2 (rabbit polyclonal) | TCS | P939 | 1:1000 |
| Antibody | Anti-SNAP25 (rabbit polyclonal) | TCS | P913, RRID:AB_2861227 | 1:1000 |
| Antibody | Anti-PSD95 (mouse monoclonal) | Synaptic Systems | 124011, RRID:AB_10804286 | 1:1000 |
| Antibody | Anti-Mint1 (rabbit polyclonal) | TCS | P730 | 1:1000 |
| Antibody | Anti-GRIP (mouse monoclonal) | BD Transduction Laboratories | 611319, RRID:AB_398845 | 1:1000 |
| Antibody | Anti-GluR2 (mouse monoclonal) | Neuromab | 75-002, RRID:AB_2232661 | 1:1000 |
| Antibody | Anti-GluR1 (mouse monoclonal) | Neuromab | 75-327, RRID:AB_2315840 | 1:1000 |
| Antibody | Anti-Gephyrin (mouse monoclonal) | Neuromab | 75-444, RRID:AB_2636852 | 1:1000 |
| Antibody | Anti-CASK (mouse monoclonal) | Neuromab | 75-000, RRID:AB_2068730 | 1:1000 |
| Antibody | Anti-Calbindin (mouse monoclonal) | Sigma | C9848, RRID:AB_476894 | 1:2000 |
| Antibody | Anti-Mouse SPARC-like1 (goat polyclonal) | R&D Systems | BAF2836, RRID:AB_2195096 | 0.5 µg/ml |
| Antibody | Anti-SPARC (mouse monoclonal) | DSHB | mAB 236, RRID:AB_2617208 | 1 µg/ml |
| Antibody | Anti-Myelin Basic Protein (chicken polyclonal) | EnCOR Biotechnology | CPCA-MBP, RRID:AB_2572352 | 1:5000 |
| Antibody | Anti-Kir4.1 (rabbit polyclonal) | Millipore Sigma | AB5818, RRID:AB_92053 | 1:1000 |
| Antibody | Anti-EEAT1 (rabbit polyclonal) | Abcam | ab416, RRID:AB_304334 | 1:1000 |
| Antibody | Anti-MAP2 (chicken polyclonal) | EnCOR Biotechnology | CPCA-MAP2, RRID:AB_2138173 | 1:500 |
| Antibody | Anti-Gephyrin (guinea pig monoclonal) | Synaptic Systems | 147318, RRID:AB_2661777 | 1:200 |
| Antibody | Anti-GAD67 (mouse monoclonal) | Millipore | MAB5406, RRID:AB_2278725 | 1:1000 |
| Antibody | Anti-VGluT1 (guinea pig polyclonal) | Millipore | AB5905, RRID:AB_2301751 | 1:1000 |
| Antibody | Anti-MAP2 (mouse monoclonal) | Sigma-Aldrich | M1406, RRID:AB_477171 | 1:1000 |
| Antibody | Anti-Homer1 (rabbit polyclonal) | TCS | YZ6081 | 1:1000 |
| Antibody | Anti-NeuN (mouse monoclonal) | Millipore Sigma | MAB377, RRID:AB_2298772 | 1:1000 |
| Antibody | Anti-S100 (rabbit polyclonal) | Abcam | ab868, RRID:AB_306716 | 1:1000 |
| Antibody | Anti-GFP (rabbit polyclonal) | Invitrogen | A-11122, RRID:AB_221569 | 1:1000 |
| Antibody | Anti-HA.11 Epitope Tag-Alexa647 (mouse monoclonal) | BioLegend | 682404, RRID:AB_2566616 | 1:250 |
| Antibody | Alexa fluor 647, goat anti-rabbit IgG | Invitrogen | A-21245, RRID:AB_141775 | 1:1000 |
| Antibody | Alexa fluor 546, goat anti-rabbit IgG | Invitrogen | A-11035, RRID:AB_143051 | 1:1000 |
| Antibody | Alexa fluor 633, goat anti-mouse IgG | Invitrogen | A-21052, RRID:AB_2535719 | 1:1000 |
| Antibody | Alexa fluor 488, goat anti-guinea pig IgG | Invitrogen | A11073, RRID:AB_2534117 | 1:1000 |
| Antibody | Alexa fluor 647, goat anti-chicken IgG | Invitrogen | A21449, RRID:AB_1500594 | 1:1000 |
| Antibody | Alexa fluor 546, goat anti-guinea pig IgG | Invitrogen | A-11074, RRID:AB_2534118 | 1:1000 |
| Antibody | Alexa fluor 488, goat anti-mouse IgG | Invitrogen | A-11001, RRID:AB_2534069 | 1:1000 |
| Antibody | IRDye 800CW donkey anti-chicken IgG | Licor | 926-32218, RRID:AB_1850023 | 1:10,000 |
| Antibody | IRDye 800CW donkey anti-goat IgG | Licor | 926-32214, RRID:AB_621846 | 1:10,000 |
| Antibody | IRDye 680RD donkey anti-guinea pig IgG | Licor | 926-68077, RRID:AB_10956079 | 1:10,000 |
| Antibody | IRDye 800CW donkey anti-rabbit IgG | Licor | 926-32213, RRID:AB_621848 | 1:10,000 |
| Antibody | IRDye 680LT donkey anti-mouse IgG | Licor | 926-68022, RRID:AB_10715072 | 1:10,000 |
| Antibody | IRDye 800CW donkey anti-mouse IgG | Licor | 926-32212, RRID:AB_621847 | 1:10,000 |
| Chemical compound, drug | Picrotoxin | Tocris | 1128 | |
| Chemical compound, drug | CNQX | Tocris | 1045 | |
| Chemical compound, drug | D-AP5 | Tocris | 0106 | |
| Chemical compound, drug | Tetrodotoxin | Cayman Chemical | 14964 | |
| Chemical compound, drug | Tamoxifen | Sigma | T5648-1G | |
| Chemical compound, drug | Corn Oil | Sigma | C8267 | |
| Sequence-based reagent | Actb PrimeTime qPCR Assay | IDT | Mm.PT.39a.22214843g | |
| Sequence-based reagent | Aqp4 PrimeTime qPCR Assay | IDT | Mm.PT.58.9080805 | |
| Sequence-based reagent | Sox9 PrimeTime qPCR Assay | IDT | Mm.PT.58.42739087 | |
| Sequence-based reagent | Pdgfra PrimeTime qPCR Assay | IDT | Mm.PT.56a.5639577 | |
| Sequence-based reagent | MBP PrimeTime qPCR Assay | IDT | Mm.PT.58.28532164 | |
| Sequence-based reagent | P2ry12 PrimeTime qPCR Assay | IDT | Mm.PT.58.43542033 | |
| Sequence-based reagent | Rbfox3 PrimeTime qPCR Assay | IDT | Mm.PT.58.11398454 | |
| Sequence-based reagent | Nlgn1 PrimeTime qPCR Assay | IDT | Mm.PT.58.5704919 | |
| Sequence-based reagent | Nlgn2 PrimeTime qPCR Assay | IDT | Mm.pt.58.41142345 | |
| Sequence-based reagent | Nlgn3 PrimeTime qPCR Assay | IDT | Mm.PT.58.31138258 | |
| Software, algorithm | Clampfit | Molecular Devices | N/A | |
| Software, algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | |
| Software, algorithm | Prism | Graphpad Software Inc | RRID:SCR_002798 | |
| Software, algorithm | Image Studio Lite | Licor | RRID:SCR_014211 | |
| Software, algorithm | Imaris | Oxford Instruments | RRID:SCR_007370 | |
| Software, algorithm | NIS-Elements Basic Research | Nikon | RRID:SCR_002776 |
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Deletion of neuroligins from astrocytes does not detectably alter synapse numbers or astrocyte cytoarchitecture by maturity
eLife 12:RP87589.
https://doi.org/10.7554/eLife.87589.4
