Gao J et al. (2022), Epigenetic regulation of GABAergic differentiat...

XB-ART-59380

Front Cell Neurosci 2022 Jan 01;16:988732. doi: 10.3389/fncel.2022.988732.

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Epigenetic regulation of GABAergic differentiation in the developing brain.

Gao J , Luo Y , Lu Y , Wu X , Chen P , Zhang X , Han L , Qiu M , Shen W .

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In the vertebrate brain, GABAergic cell development and neurotransmission are important for the establishment of neural circuits. Various intrinsic and extrinsic factors have been identified to affect GABAergic neurogenesis. However, little is known about the epigenetic control of GABAergic differentiation in the developing brain. Here, we report that the number of GABAergic neurons dynamically changes during the early tectal development in the Xenopus brain. The percentage of GABAergic neurons is relatively unchanged during the early stages from stage 40 to 46 but significantly decreased from stage 46 to 48 tadpoles. Interestingly, the histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 (about 3.5 days). Chronic application of valproate acid (VPA), a broad-spectrum histone deacetylase (HDAC) inhibitor, at stage 46 for 48 h increases the acetylation of H3K9 and the number of GABAergic cells in the optic tectum. VPA treatment also reduces apoptotic cells. Electrophysiological recordings show that a VPA induces an increase in the frequency of mIPSCs and no changes in the amplitude. Behavioral studies reveal that VPA decreases swimming activity and visually guided avoidance behavior. These findings extend our understanding of histone modification in the GABAergic differentiation and neurotransmission during early brain development.

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Species referenced: Xenopus laevis
Genes referenced: camk2a ctrl elavl4 gad1.1 tecta.2
GO keywords: brain development [+]
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	Figure 1. GABA+ cells are developmentally downregulated from stages 46 to 48 in the optic tectum. (A) The optic tectum was immunostained with anti-GABA and anti-CaMKIIα antibodies at stage 48. Arrowheads indicate the GABA+ and CaMKIIα– cells. Arrows indicate the GABA– and CaMKIIα+ cells. Scale bar = 50 μm. (B) The optic tectum was immunostained with an anti-GABA antibody at stages (St) 40 (B1–3), 46 (B4–6), and 48 (B7–9), respectively. The white line indicates the zoomed-in images that GABA is expressed in the cytoplasm (up-left corner, B1–3). The dotted line indicated the ventricular cell layer (B3,B6,B9). Scale bar = 50 μm. (C) The data shows the total number of GABA+ cells in the optic tectum. (D) The summary graph shows the total number of DAPI+ cells. (E) The summary graph shows the ratio of GABA+ cells to DAPI+ cells. (GABA: St40, 468 ± 27.0, N = 5, St46, 1103.8 ± 62.9, N = 5, St48, 723 ± 28.7, N = 6; DAPI: St40, 2227.4 ± 48.9, N = 5, St46, 4859 ± 125.5, N = 5, St48, 5515 ± 91.9, N = 7; p < 0.01, *p < 0.001). N represents the number of tadpoles.
	Figure 2. Histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 optic tectum. (A) Western blot analysis of H3K9 protein expression using an anti-H3K9ac antibody at St42, St44, St46, and St48. (B) The data shows that the relative intensity of H3K9 at St48 was significantly decreased compared to St42 and St46 tadpoles (N = 3, p < 0.05, p < 0.01, **p < 0.001). N represents the number of independent experiments.
	Figure 3. The number of GABA+ cells was increased by VPA treatment. (A) Representative images showing that the control (A1–3) and VPA-treated (A4–6) brains were immunostained with an anti-GABA antibody at stage 48. Arrows indicate the GABA+ cells. Scale bar = 50 μm. (B) The data shows that the GABA+ cells were significantly increased by VPA treatment. GABA: Ctrl, 690.2 ± 13.4, N = 5; VPA, 909.7 ± 28.3, N = 6). N represents the number of tadpoles. (C,D) Western blot of H3K9ac and summary of data showing that the relative intensity of H3K9 to GAPDH was significantly increased by VPA treatment. N = 12. ***p < 0.001. N represents the number of independent experiments.
	Figure 4. Valproate acid-induced GABA+ cells are HuD+ differentiated neurons. (A) Control (A1–4) and VPA-reared (A5–8) optic tecta were immunostained with anti-GABA and anti-HuD antibodies at stage 48. Red/white lines indicate zoomed-in images showing that GABA and HuD are overlapped in the cell body layer. Arrows indicate that GABA+ and HuD+ cells are distributed in the neuropil. Scale bar = 50 μm. (B) The data shows that the number of GABA+ cells was significantly increased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 688.2 ± 13.3, N = 5; VPA, 858.8 ± 22.3, N = 5; **p < 0.001). (C) The data shows that the number of HuD+ cells was significantly increased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 4379.8 ± 134.8, N = 5; VPA, 4791.8 ± 89.7, N = 5; p < 0.05). (D) Representative images of apoptosis in control and VPA-reared optic tecta. Arrows indicate the apoptotic cells. Scale bar = 50 μm. (E) The data shows that the number of apoptotic cells was significantly decreased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 62.2 ± 3.5, N = 6; VPA, 30.6 ± 5.5, N = 5; ***p < 0.001). (F) Control and VPA-reared optic tecta were performed TUNEL analysis and immunostained with an anti-GABA antibody at stage 48. Arrows indicate that the apoptotic cells are not stained with GABA. (Ctrl, N = 6; VPA, N = 7). Scale bar = 50 μm. N represents the number of tadpoles.
	Figure 5. Valproate acid increases GABAergic synapses and the frequency of mIPSCs. (A,B) Western blot analysis and data summary show that the relative intensities of GAD67 (A) and VGAT (B) to GAPDH were significantly increased in VPA-reared tadpoles compared to Ctrl. GAD67, N = 6; VGAT, N = 6. N represents the number of independent experiments. p < 0.05, p < 0.01. (C) Representative traces were showing the recordings of mIPSCs from Ctrl and VPA-treated animals. Scale bar = 10 pA, 0.1 s. (D–F) The data shows that the frequency of mIPSCs was significantly increased in VPA-reared tadpoles compared to control tadpoles (D). p < 0.01. The cumulative probability plot shows that the inter-event-interval of mIPSCs was significantly decreased in VPA-treated tadpoles compared to ctrl (E). Kolmogorov–Smirnov test, *p < 0.01. The data shows that the amplitude of mIPSCs was not altered in VPA-reared tadpoles compared to control tadpoles (F). Ctrl, N = 24, n = 5; VPA, N = 24, n = 4. N represents the number of neurons and n represents the number of tadpoles.
	Figure 6. Valproate acid-reared tadpoles show impaired free-swimming and visual avoidance behaviors. (A) Representative images showing the swimming traces of a tadpole within 60 s in Ctrl (left) and VPA-reared (right) tadpoles. Scale bar = 2 mm. (B) Summary of data showing the total swimming distance was decreased in VPA-raised tadpoles compared to ctrl tadpoles. (Ctrl, 120.3 ± 16.3 mm, N = 47; VPA, 51.1 ± 6.3 mm, N = 41; *p < 0.001). (C) The data summary shows that the total swimming distance decreased in VPA-injected tadpoles compared to ddH2O-injected tadpoles. (ddH2O, 187.4 ± 16.9 mm, N = 20; VPA, 78.5 ± 6.5 mm, N = 45; p < 0.001). (D) A sequence of time-lapse images shows that a tadpole makes an avoiding turning in response to a moving spot (4 mm diameter) within 0.6 s. White arrows indicate the moving direction of the spot. Red arrows indicate the turning direction of the tadpole. The panel on the upper right shows the swim trajectories of the animal, with the start point marked as 0 s and the end point marked as 0.6 s. Spot luminance: 90 cd/m2. Scale bar = 2 mm. (E) Summary of data showing that the avoidance index (AI,%) was significantly decreased in VPA-treated tadpoles compared to Ctrl tadpoles. (Ctrl, 30.8 ± 2.2, N = 24; VPA, 23.8 ± 2.5, N = 24; p < 0.05). (F) The data shows that the avoidance index was significantly decreased in VPA-injected tadpoles compared to ddH2O-injected tadpoles. (ddH2O, 28.5 ± 2.8, N = 20; VPA, 21.1 ± 2.0, N = 36; *p < 0.05). N represents the number of tadpoles.

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	Figure 1. GABA+ cells are developmentally downregulated from stages 46 to 48 in the optic tectum. (A) The optic tectum was immunostained with anti-GABA and anti-CaMKIIα antibodies at stage 48. Arrowheads indicate the GABA+ and CaMKIIα– cells. Arrows indicate the GABA– and CaMKIIα+ cells. Scale bar = 50 μm. (B) The optic tectum was immunostained with an anti-GABA antibody at stages (St) 40 (B1–3), 46 (B4–6), and 48 (B7–9), respectively. The white line indicates the zoomed-in images that GABA is expressed in the cytoplasm (up-left corner, B1–3). The dotted line indicated the ventricular cell layer (B3,B6,B9). Scale bar = 50 μm. (C) The data shows the total number of GABA+ cells in the optic tectum. (D) The summary graph shows the total number of DAPI+ cells. (E) The summary graph shows the ratio of GABA+ cells to DAPI+ cells. (GABA: St40, 468 ± 27.0, N = 5, St46, 1103.8 ± 62.9, N = 5, St48, 723 ± 28.7, N = 6; DAPI: St40, 2227.4 ± 48.9, N = 5, St46, 4859 ± 125.5, N = 5, St48, 5515 ± 91.9, N = 7; p < 0.01, *p < 0.001). N represents the number of tadpoles.
	Figure 2. Histone acetylation of H3K9 is developmentally decreased from stage 42 to 48 optic tectum. (A) Western blot analysis of H3K9 protein expression using an anti-H3K9ac antibody at St42, St44, St46, and St48. (B) The data shows that the relative intensity of H3K9 at St48 was significantly decreased compared to St42 and St46 tadpoles (N = 3, p < 0.05, p < 0.01, **p < 0.001). N represents the number of independent experiments.
	Figure 3. The number of GABA+ cells was increased by VPA treatment. (A) Representative images showing that the control (A1–3) and VPA-treated (A4–6) brains were immunostained with an anti-GABA antibody at stage 48. Arrows indicate the GABA+ cells. Scale bar = 50 μm. (B) The data shows that the GABA+ cells were significantly increased by VPA treatment. GABA: Ctrl, 690.2 ± 13.4, N = 5; VPA, 909.7 ± 28.3, N = 6). N represents the number of tadpoles. (C,D) Western blot of H3K9ac and summary of data showing that the relative intensity of H3K9 to GAPDH was significantly increased by VPA treatment. N = 12. ***p < 0.001. N represents the number of independent experiments.
	Figure 4. Valproate acid-induced GABA+ cells are HuD+ differentiated neurons. (A) Control (A1–4) and VPA-reared (A5–8) optic tecta were immunostained with anti-GABA and anti-HuD antibodies at stage 48. Red/white lines indicate zoomed-in images showing that GABA and HuD are overlapped in the cell body layer. Arrows indicate that GABA+ and HuD+ cells are distributed in the neuropil. Scale bar = 50 μm. (B) The data shows that the number of GABA+ cells was significantly increased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 688.2 ± 13.3, N = 5; VPA, 858.8 ± 22.3, N = 5; **p < 0.001). (C) The data shows that the number of HuD+ cells was significantly increased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 4379.8 ± 134.8, N = 5; VPA, 4791.8 ± 89.7, N = 5; p < 0.05). (D) Representative images of apoptosis in control and VPA-reared optic tecta. Arrows indicate the apoptotic cells. Scale bar = 50 μm. (E) The data shows that the number of apoptotic cells was significantly decreased in VPA-raised tadpoles compared to Ctrl. (Ctrl, 62.2 ± 3.5, N = 6; VPA, 30.6 ± 5.5, N = 5; ***p < 0.001). (F) Control and VPA-reared optic tecta were performed TUNEL analysis and immunostained with an anti-GABA antibody at stage 48. Arrows indicate that the apoptotic cells are not stained with GABA. (Ctrl, N = 6; VPA, N = 7). Scale bar = 50 μm. N represents the number of tadpoles.
	Figure 5. Valproate acid increases GABAergic synapses and the frequency of mIPSCs. (A,B) Western blot analysis and data summary show that the relative intensities of GAD67 (A) and VGAT (B) to GAPDH were significantly increased in VPA-reared tadpoles compared to Ctrl. GAD67, N = 6; VGAT, N = 6. N represents the number of independent experiments. p < 0.05, p < 0.01. (C) Representative traces were showing the recordings of mIPSCs from Ctrl and VPA-treated animals. Scale bar = 10 pA, 0.1 s. (D–F) The data shows that the frequency of mIPSCs was significantly increased in VPA-reared tadpoles compared to control tadpoles (D). p < 0.01. The cumulative probability plot shows that the inter-event-interval of mIPSCs was significantly decreased in VPA-treated tadpoles compared to ctrl (E). Kolmogorov–Smirnov test, *p < 0.01. The data shows that the amplitude of mIPSCs was not altered in VPA-reared tadpoles compared to control tadpoles (F). Ctrl, N = 24, n = 5; VPA, N = 24, n = 4. N represents the number of neurons and n represents the number of tadpoles.
	Figure 6. Valproate acid-reared tadpoles show impaired free-swimming and visual avoidance behaviors. (A) Representative images showing the swimming traces of a tadpole within 60 s in Ctrl (left) and VPA-reared (right) tadpoles. Scale bar = 2 mm. (B) Summary of data showing the total swimming distance was decreased in VPA-raised tadpoles compared to ctrl tadpoles. (Ctrl, 120.3 ± 16.3 mm, N = 47; VPA, 51.1 ± 6.3 mm, N = 41; *p < 0.001). (C) The data summary shows that the total swimming distance decreased in VPA-injected tadpoles compared to ddH2O-injected tadpoles. (ddH2O, 187.4 ± 16.9 mm, N = 20; VPA, 78.5 ± 6.5 mm, N = 45; p < 0.001). (D) A sequence of time-lapse images shows that a tadpole makes an avoiding turning in response to a moving spot (4 mm diameter) within 0.6 s. White arrows indicate the moving direction of the spot. Red arrows indicate the turning direction of the tadpole. The panel on the upper right shows the swim trajectories of the animal, with the start point marked as 0 s and the end point marked as 0.6 s. Spot luminance: 90 cd/m2. Scale bar = 2 mm. (E) Summary of data showing that the avoidance index (AI,%) was significantly decreased in VPA-treated tadpoles compared to Ctrl tadpoles. (Ctrl, 30.8 ± 2.2, N = 24; VPA, 23.8 ± 2.5, N = 24; p < 0.05). (F) The data shows that the avoidance index was significantly decreased in VPA-injected tadpoles compared to ddH2O-injected tadpoles. (ddH2O, 28.5 ± 2.8, N = 20; VPA, 21.1 ± 2.0, N = 36; *p < 0.05). N represents the number of tadpoles.