Inhibitory input directs astrocyte morphogenesis through glial GABA B R

Abstract

Communication between neurons and glia plays an important role in establishing and maintaining higher order brain function. Astrocytes are endowed with complex morphologies which places their peripheral processes in close proximity to neuronal synapses and directly contributes to their regulation of brain circuits. Recent studies have shown that excitatory neuronal activity promotes oligodendrocyte differentiation; whether inhibitory neurotransmission regulates astrocyte morphogenesis during development is unknown. Here we show that inhibitory neuron activity is necessary and sufficient for astrocyte morphogenesis. We found that input from inhibitory neurons functions through astrocytic GABA _B R and that its deletion in astrocytes results in a loss of morphological complexity across a host of brain regions and disruption of circuit function. Expression of GABA _B R in developing astrocytes is regulated in a region-specific manner by SOX9 or NFIA and deletion of these transcription factors results in region-specific defects in astrocyte morphogenesis, which is conferred by interactions with transcription factors exhibiting region-restricted patterns of expression. Together our studies identify input from inhibitory neurons and astrocytic GABA _B R as universal regulators of morphogenesis, while further revealing a combinatorial code of region-specific transcriptional dependencies for astrocyte development that is intertwined with activity-dependent processes.

Figure 1.. Inhibitory neuron activity regulates astrocyte morphogenesis

a. Schematic of DREADD-based activation of inhibitory neurons in post-natal Aldh1l1-GFP mice. b. Slice electrophysiological recordings of DREADD-expressing (hM3Dq or hM4Di) inhibitory neurons at P21, with and without CNO activation. Traces are representative of neuronal firing. c-e. Imaging of Aldh1l1-GFP astrocytes after hM3Dq activation of inhibitory neurons; quantification using Scholl analysis, branch number, and total processes at P21; n = 3 pairs of animals (47, 51 cells; d, generalized linear mixed-effects (GLME) model with Sidak’s multiple comparisons test, **P = 0.001; e, linear mixed-effect (LME) model, ***P = 0.00012, 0.00013). f. RNAscope imaging and quantitative analysis for Gabbr1 expression in Alhd1l1-GFP expressing astrocytes at P21; n = 3 pairs of animals (49, 59 cells; LME model, ***P = 0.00090). Dashed circle denotes astrocyte with Gabbr1. g-i. Imaging of Aldh1l1-GFP astrocytes after hM4Di inhibition of inhibitory neurons; quantification using Scholl analysis, branch number, and total processes at P21; n = 3 and 4 animals (44, 71 cells; h, GLME model with Sidak’s multiple comparisons test, **P = 0.0013; i-j, GLME model, *P = 0.0327, **P = 0.0014). j. RNAscope imaging and quantitative analysis for Gabbr1 expression in Alhd1l1-GFP expressing astrocytes at P21; n = 3 and 4 animals (49, 64 cells; LME model, P = 0.1014). Dashed circle denotes astrocyte with Gabbr1. Scale bars, 20 μm (c, g, j) and 10 μm (f). Data represent mean ± s.d. (d, h), median, minimum value, maximum value and interquartile range (IQR) (e-f bottom, i-j).

Figure 2.. Gabbr1 is required for astrocyte morphogenesis

a. Experimental timeline and mouse lines rendering astrocyte-specific knockout of Gabbr1. b. RNA-Scope imaging of Gabbr1 within Aldh1l1-GFP astrocytes from control and Gabbr1-cKO mouse lines; quantification derived from n = 3 pairs of animals (control: OB 18, CX 18, HC 16; cKO: OB 17, CX 19, HC 17 cells; LME model, ***P = 0.00020, ****P = 3.07e-06, **P = 0.0030). Dashed circle denotes astrocyte with Gabbr1. c. Imaging of Aldh1l1-GFP astrocytes from the cortex, CA1 of the hippocampus, and olfactory bulb at P28; quantification via Scholl analysis derived from n = 3 pairs of animals (control: OB 43, CX 27, HC 32, cKO: OB 31, CX 33, HC 30 cells; GLME model with Sidak’s multiple comparisons test, **P = 0.0011, *** P = 0.0006, **P = 0.0037). d. Imaging of GCaMP6s activity in control and Gabbr1-cKO astrocytes from the cortex at P28; quantification is derived from n = 3 pairs of animals (24,33 cells; GLME model, P = 0.6361, 0.2239). e. Imaging of GCaMP6s activity in the presence of TTX and baclofen; quantification derived from n = 40 cells from 3 pairs of animals (two-tailed Wilcoxon matched-pairs signed rank test, *P = 0.022, P = 0.89, ***P = 0.0006, P = 0.32). f. DREADD-based activation of inhibitory neurons in Gabbr1-cKO mice. g-i. Imaging of Aldh1l1-GFP astrocytes from Gabbr1-cKO (and control) after hM3Dq activation of inhibitory neurons; quantification using Scholl analysis, branch number, and total processes at P21; n = 3 and 5 animals (50, 80 cells; h, GLME model with Sidak’s multiple comparisons test, **P = 0.0011, i; GLME model, *P = 0.034, **P = 0.0026). Scale bars, 10 μm (b, d), 30 μm (c), and 20 μm (g). Data represent mean ± s.d. (c, h), median, minimum value, maximum value and IQR (b, d, i).

Figure 3.. Loss of astrocytic Gabbr1 disrupts cortical circuit function

a. CellChat interaction diagram illustrating astrocyte interactions with neurons in the cortex from P28 Gabbr1-cKO mice; width of colored arrow indicates scale of interaction. See Extended Data Figure 4. b-c. KEGG pathway analysis of neurons from Gabbr1-cKO scRNA-Seq (b, analyzed by Enrichr) and dot plot of differentially expressed genes from KEGG (c, analyzed by Seurat FindMarkers). d. Schematic of viral labeling of inhibitory neurons and experimental timeline. e-h. Representative traces of spontaneous EPSCs and IPSCs from excitatory and inhibitory neurons from cortex of Gabbr1-cKO and controls. Associated cumulative and bar plots demonstrate quantification of sEPSC and sIPSC from 3 pairs of animals (e, n = 13, 15 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.8207, **P = 0.003; f, n = 15, 12 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.1995, 0.5888; g, n = 13, 15 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.7856, 0.0504; h, n = 11, 9 cells; Kolmogorov-Smirnov test, **** P < 0.0001; two-tailed Mann-Whitney test, P = 0.2299, 0.3796). i. Experimental timeline for behavioral analysis. j. 3-chamber social interaction and pre-pulse inhibition studies on Gabbr1-cKO and control mice from 10 animals in control group and 11 animals in cKO group (left, GLME model with Sidak’s multiple comparisons test, *P = 0.015; right, two-tailed Mann-Whitney test, *P = 0.043). Data represent mean ± s.e.m. (e-h), s.d. (j).

Figure 4.. Gabbr1 regulates astrocyte morphology through Ednrb1

a. Volcano plots from RNA-Seq analysis of control and Gabb1-cKO astrocytes from cortex, hippocampus, and OB. b. Table of the number of differentially expressed genes (DEGs) from each region. c. Gene Ontology (GO) analysis of DEGs performed with Enrichr. d. Immunostaining for EDNRB in P28 astrocytes from Gabbr1-cKO and control astrocytes. e. Quantification of EDNRB expression in Gabbr1-cKO and control from n = 6 pairs of animals (two-tailed Mann-Whitney test, *P = 0.041, 0.015, P = 0.1320). f. Schematic and timeline of selective deletion of Ednrb in cortical astrocytes. g-i. Imaging of virally labeled astrocytes from the P28 cortex of mice where Ednrb has been knocked out using guideRNAs in the ROSA-LSL-Cas9-EGFP mouse line; quantification via Scholl analysis was derived from n = 3 pairs of animals (53, 49 cells; h, GLME model with Sidak’s multiple comparisons test, **P = 0.001; i, GLME model, **P = 0.001, ***P = 0.0002). Scale bars, 20 μm (d), 30 μm (g). Data represent mean ± s.d. (h), median, minimum value, maximum value and IQR (e, i).

Figure 5.. Region-specific regulation of Gabbr1 by SOX9 and NFIA

a. Homer transcription factor motif analysis on differentially expressed genes (DEGs) from P1 and P14 timepoints from astrocytes isolated from the cortex, hippocampus, and olfactory bulb. b. Schematic depicting mouse lines and experimental timelines. c-h. RNAscope imaging of Gabbr1 expression in Aldh1l1-GFP astrocytes from Nfia-cKO, Sox9-cKO and associated controls at P28; quantitative analysis of Gabbr1 expression is derived from n = 3 pairs of animals (d, 50, 50, 29, and 33 cells; GLME model, *P = 0.022; LME model ***P = 0.0002; e, 29, 19, 19, and 25 cells; LME model, P = 0.17, 0.27; f, 26, 25, 26, and 29 cells; GLME model, P = 0.91, 0.95; g, 30, 29, 29, 30 cells; LME model, **P = 0.0094, *P = 0.014). Dashed circle denotes astrocyte with Gabbr1. h. Chromatin immunoprecipitation of NFIA from P28 cortex or SOX9 from P28 olfactory bulb (OB), followed by PCR detection of association with motif in proximal promoter region of Gabbr1. i-j. Imaging of GCaMP6s activity from the cortex of Nfia-cKO mice or the OB from Sox9-cKO mice (and controls) in the presence of TTX and baclofen; quantification is derived from n = 19–26 cells from 3 pairs of animals (i, 23, 26 cells, two-tailed Wilcoxon matched-pairs signed rank test, ***P = 0.0001, P = 0.53, *P = 0.048, P = 0.37; j, 19 20 cells, two-tailed Wilcoxon matched-pairs signed rank test, P = 0.65, 0.45, *P = 0.012, P = 0.81). Scale bars, 10 μm (c) and 20 μm (i-j). Data represent median, minimum value, maximum value and IQR (d-g).

Figure 6.. Regulation of astrocyte morphogenesis by region-specific mechanisms

a. Timeline and mouse lines rendering astrocyte-specific knockout of Sox9 and Nfia. b. Imaging of Aldh1l1-GFP astrocytes at P28 from the Sox9-cKO and Nfia-cKO; quantification via Scholl analysis was derived from n = 3 pairs of animals (Nfia control: OB 56, CX 52, Nfia cKO: OB 60, CX 48, Sox9 control: OB 29, CX 35, Sox9 cKO: OB 39, CX 33 cells; GLME model with Sidak’s multiple comparisons test, *P = 0.015, P = 0.41, 0.60, **P = 0.0097). c. CX-specific DEGs increased between P7–P14. d. Immunostaining for LHX2 in P28 astrocytes quantified from n = 3 pairs of animals (LME model, ****P = 1.31e-12). e. Immunoprecipitation of LHX2 and immunoblot of LHX2 and NFIA from the P28 cortex. f. OB-specific DEGs increased between P7–P14. g. Immunostaining for NPAS3 in P28 astrocytes quantified from n = 3 pairs of animals (GLME model, **P = 0.0013). h. Immunoprecipitation of NPAS3 and immunoblot of NPAS3 and SOX9 from P28 cortex. i. Schematic of Lhx2 deletion in cortical astrocytes. j-l. Imaging of virally labeled astrocytes lacking Lhx2 from P28 cortex; quantification via Scholl analysis was derived from n = 3 pairs of animals (41,41 cells; k, GLME model with Sidak’s multiple comparisons test, ****P = 1.29e-24; l, GLME model, ***P = 0.00099, ****P = 3.98e-05). m. RNAscope for Gabbr1 expression in Cas9-EGFP cortical astrocytes lacking Lhx2 and controls at P28; quantitative analysis demonstrating reduction of Gabbr1 expression is derived from n = 3 pairs of animals (51,55 cells; GLME model, *P = 0.015; LME model, **P = 0.0003). Scale bars, 30 μm (b, j), 20 μm (d, g, m). Data represent mean ± s.d. (b, k), median, minimum value, maximum value and IQR (d, g, l, m).