Region and layer-specific expression of GABAA receptor isoforms and KCC2 in developing cortex

Abstract

Introduction: γ-Aminobutyric acid (GABA) type A receptors (GABA_ARs) are ligand-gated Cl-channels that mediate the bulk of inhibitory neurotransmission in the mature CNS and are targets of many drugs. During cortical development, GABA_AR-mediated signals are significantly modulated by changing subunit composition and expression of Cl-transporters as part of developmental processes and early network activity. To date, this developmental evolution has remained understudied, particularly at the level of cortical layer-specific changes. In this study, we characterized the expression of nine major GABA_AR subunits and K-Cl transporter 2 (KCC2) in mouse somatosensory cortex from embryonic development to postweaning maturity.

Methods: We evaluated expression of α1-5, β2-3, γ2, and δ GABA_AR subunits using immunohistochemistry and Western blot techniques, and expression of KCC2 using immunohistochemistry in cortices from E13.5 to P25 mice.

Results: We found that embryonic cortex expresses mainly α3, α5, β3, and γ2, while expression of α1, α2, α4, β2, δ, and KCC2 begins at later points in development; however, many patterns of nuanced expression can be found in specific lamina, cortical regions, and cells and structures.

Discussion: While the general pattern of expression of each subunit and KCC2 is similar to previous studies, we found a number of unique temporal, regional, and laminar patterns that were previously unknown. These findings provide much needed knowledge of the intricate developmental evolution in GABA_AR composition and KCC2 expression to accommodate developmental signals that transition to mature neurotransmission.

Keywords: GABA-A receptors; GABAA subtypes; Western blot (WB); cortical development; developmental expression pattern; immunohistochemistry (IHC); potassium chloride co-transporter 2 (KCC2).

Figure 1

Sample P26 images showing region-specific immunolabeling patterns of GABA_AR subunits in the CNS. Regions with the highest expression were used for setting acquisition settings and the maximum grading threshold for quantifying subunit expression within cortex.

Figure 2

Expression of GABA_AR α1-5 subunits in developing cortex. Coronal sections are overlaid from embryonic day E13.5/E15.5 on the bottom left to postnatal day 26 on the top right separately for each α subunit. All sections are oriented from ventral bottom to dorsal top, with lateral cortex on the right. Separate spatial scaling has been used for sections E13.5-P1 (black scale bar) and P5-P26 (red scale bar), separately for each subunit. Signal is represented using a subunit-specific heat map lookup table to highlight differences in regional expression. Heatmap intensity scaling is shown by the bar in top left.

Figure 3

GABA_AR α1 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of α1 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal α1 signal that could be detected in brain at P26. (C) Quantification of GABA_AR α1 protein in cortical samples by Western blot (WB), with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E15.5, 8/P1, 3/P5, 4/P10, 4/P15, 4/P18, and 4/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; ND, not determined; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 4

GABA_AR α2 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of α2 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal α2 signal that could be detected in brain at P26. (C) Quantification of GABA_AR α2 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001, ^**P < 0.01 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5; 3/E15.5, 3/E17.5, 6/P1, 4/P3, 5/P5, 5/P7, 4/P10, 3/P15, 4/P18, and 5/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 5

Fibers of radial glia co-localize with GABA_AR α2, but not GABA_AR α3. (A) Both GABA_AR α2 and α3 immunolabeling showed a pattern of ascending cortical fibers, which are prominent in the CP/developing layers 2/3 for α3 (right) and are found throughout the cortical column for α2 (left) in embryonic and early postnatal period. These fibers resemble radial glia marked by RC2 (middle). (B) The radial glia marker RC2 co-localized with α2-immunopositive fibers (left, arrows), but not with α3-immunopositive fibers (right, arrows). Note that white arrows denote fibers immunopositive for α2 and α3, while green empty arrows indicate radial glia fibers. (C) Subcortical expression of α3-positive fibers has a different pattern than radial glia. The yellow dotted line in (A, C) indicates the basal margin of the subplate, while red dotted line in (C) indicates the margin between intermediate zone/white matter and the striatum. All images are oriented basal bottom to apical top.

Figure 6

GABA_AR α3 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of α3 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal α3 signal that could be detected in brain at P26. (C) Quantification of GABA_AR α3 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. *P < 0.05 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5; 3/E15.5, 3/E17.5, 6/P1, 4/P3, 6/P5, 6/P7, 4/P10, 3/P15, 4/P18, 6/P25. L1-L6, layer 1-layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 7

GABA_AR α3 expression in fibers of the internal capsule during embryonic development. (A) E17.5 coronal section showing prominent α3 immunolabeling of internal capsule fibers in the white matter/intermediate zone located below the subplate and running medioventrally through the striatum. These regions of interest are enlarged in the inset. (B) α3-immunolabeled fibers are absent in the α3-knockout mouse (α3 KO, right), but consistently present in sections from wildtype mice (left). The yellow dotted line in (A, B) indicates the basal margin of the subplate. All images are oriented ventral bottom to dorsal top, with lateral cortex on the right.

Figure 8

GABA_AR α4 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of α4 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal α4 signal that could be detected in brain at P26. (C) Quantification of GABA_AR α4 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5; 3/E15.5, 3/E17.5, 6/P1, 3/P3, 4/P5, 3/P7, 6/P10, 3/P15, 5/P18, and 4/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 9

GABA_AR α5 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of α5 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal α5 signal that could be detected in brain at P26. (C) Quantification of GABA_AR α5 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5; 3/E15.5, 3/E17.5, 3/P1, 3/P3, 4/P5, 3/P7, 5/P10, 3/P15, 7/P18, and 6/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; ND, not determined; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 10

Expression of GABA_AR β2, β3, δ, and γ2 subunits in developing cortex. Coronal sections are overlaid from embryonic day E13.5/E15.5 on the bottom left to postnatal day 26 on the top right separately for each subunit. All sections are oriented from ventral bottom to dorsal top, with lateral cortex on the right. Separate scaling has been used for sections E13.5-P1 (black scale bar) and P5-P26 (red scale bar), separately for each subunit. Signal is represented using a heat map lookup table to highlight differences in regional expression. Heatmap intensity scaling is shown by the bar in top left.

Figure 11

GABA_AR δ expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of δ immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal δ signal that could be detected in brain at P26. (C) Quantification of GABA_AR δ protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 4/E13.5; 3/E15.5, 4/P1, 4/P5, 3/P10, 4/P15, 5/P18, and 3/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; ND, not determined; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 12

GABA_AR γ2 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of γ2 immunoreactivity in barrel field cortex across development. Of note, the sections shown here were stained at the same time, but L1 was damaged in the P12 sections. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal γ2 signal that could be detected in brain at P26. (C) Quantification of GABA_AR γ2 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001, ^***P < 0.001, ^**P < 0.01 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5, 3/E15.5, 3/E17.5, 6/P1, 3/P3, 3/P5, 3/P7, 3/P10, 4/P15, 4/P18, and 4/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; ND, not determined; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 13

GABA_AR β2 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of β2 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal β2 signal that could be detected in brain at P26. (C) Quantification of GABA_AR β2 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001, ^***P < 0.001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 3/E13.5; 3/E15.5, 3/E17.5, 6/P1, 3/P3, 3/P5, 3/P7, 4/P10, 3/P15, 4/P18, and 3/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 14

GABA_AR β3 expression in barrel field cortex from embryonic age to maturity. (A) Exemplar images of β3 immunoreactivity in barrel field cortex across development. Scale bar = 250 μm. (B) Grading of immunofluorescence intensity across cortical lamina, where + + ++ is the maximal β3 signal that could be detected in brain at P26. (C) Quantification of GABA_AR β3 protein in cortical samples by Western blot, with a timeline of all points on the left and binned stages tested for significance on the right. ^****P < 0.0001 by one-way ANOVA with Šídák's multiple comparisons; n in mice/age: 4/E13.5, 4/E15.5, 4/E17.5, 4/P1, 4/P5, 4/P10, 4/P15, 4/P18, and 4/P25. L1–L6, layer 1–layer 6; MZ, marginal zone; SP, subplate; CP, cortical plate; also refer to “Lamina in Grading Table and Figures” section in Methods.

Figure 15

KCC2 expression in developing cortex. Coronal sections are overlaid from embryonic day E13.5 on the bottom left to postnatal day 19 on the top right, and concensus grading of immunofluorescence intensity is on bottom right. All sections are oriented from ventral bottom to dorsal top, with lateral cortex on the right. Separate scaling has been used for sections E13.5-P1 (black scale bar) and P4-P19 (red scale bar). Signal is represented using a heat map lookup table to highlight differences in regional expression. Heatmap intensity scaling is shown by the bar in top left. For grading of immunofluorescence intensity, + + ++ is the maximal KCC2 signal that could be detected in brain at P19+. L1–L6, layer 1–layer 6; SP, subplate; also refer to “Lamina in Grading Table and Figures” section in Methods.