Tfap2b acts in GABAergic neurons to control sleep in mice

Abstract

Sleep is a universal state of behavioral quiescence in both vertebrates and invertebrates that is controlled by conserved genes. We previously found that AP2 transcription factors control sleep in C. elegans, Drosophila, and mice. Heterozygous deletion of Tfap2b, one of the mammalian AP2 paralogs, reduces sleep in mice. The cell types and mechanisms through which Tfap2b controls sleep in mammals are, however, not known. In mice, Tfap2b acts during early embryonic stages. In this study, we used RNA-seq to measure the gene expression changes in brains of Tfap2b^-/- embryos. Our results indicated that genes related to brain development and patterning were differentially regulated. As many sleep-promoting neurons are known to be GABAergic, we measured the expression of GAD1, GAD2 and Vgat genes in different brain areas of adult Tfap2b^+/- mice using qPCR. These experiments suggested that GABAergic genes are downregulated in the cortex, brainstem and cerebellum areas, but upregulated in the striatum. To investigate whether Tfap2b controls sleep through GABAergic neurons, we specifically deleted Tfap2b in GABAergic neurons. We recorded the EEG and EMG before and after a 6-h period of sleep deprivation and extracted the time spent in NREM and in REM sleep as well as delta and theta power to assess NREM and REM sleep, respectively. During baseline conditions, Vgat-tfap2b^-/- mice exhibited both shortened NREM and REM sleep time and reduced delta and theta power. Consistently, weaker delta and theta power were observed during rebound sleep in the Vgat-tfap2b^-/- mice after sleep deprivation. Taken together, the results indicate that Tfap2b in GABAergic neurons is required for normal sleep.

Figure 1

Genes differentially expressed in Tfap2b^−/− developing mouse brains. We compared gene expression between homozygous mutant embryos (E14.5) and their wild-type littermates. Volcano plots showing the significantly up-regulated (red) and down-regulated genes (blue) in female SP (A) and DMH (B). Up regulated genes are presented in orange for male (C) and DMH (D). We compared gene expression between homozygous mutant embryos (E14.5) and their wild-type littermates. Significantly changed genes as defined by an FDR (False Discovery Rate) < 0.2 and a logFC > 0.5 were highlighted and the most robustly regulated genes were labeled (FDR < 0.01; logFC > 2). Female E14.5 embryos tfap2b^+/+, n = 3; female E14.5 embryos tfap2b^−/−, n = 2 (based on PCA analysis shown in Fig. S1, the sample named “mut3” was excluded from the DE genes analysis); male E14.5 embryos tfap2b^+/+, n = 3; male E14.5 embryos tfap2b, n = 3; SP, secondary prosencephalon; DMH, diencephalon, midbrain and hindbrain.

Figure 2

Functionally grouped gene ontology and pathway annotation networks for the genes that were differentially expressed in the Tfap2b^−/− female DMH. We classified downregulated genes by ClueGO into networks of embryonic brain development (A) and neurotransmission (B). We classified upregulated genes into major groups that are called “negative regulation of glial cell differentiation” and “forebrain generation of neurons” (C). We categorized all genes by biological function with GO terms as nodes where the most significant terms were highlighted. Poorly grouped terms (< 10 nodes) are not shown. Table 1 shows all of the genes found under each GO term, which are represented by different colors as specified in Table 1.

Figure 3

Expression of GAD67, GAD65 and Vgat genes was altered in the brains of adult Tfap2b^+/− mice. Data were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test and were shown as the mean ± SEM, *p < 0.05, **p < 0.01. Tfap2b^+/+ (n = 3) and Tfap2b^+/− (n = 3). CTX, cortex; HP, hippocampus; HY, hypothalamus; STR, striatum; BS, brainstem; CB, cerebellum. Asterisks (*) with short lines identify significance for pairwise comparisons for specific GABA-related genes in CTX and STR. In the BS and CB no significance could be found individual GABA-related genes. In the BS and CB, the asterisks with long lines identify significance in total variations of all three GABA-related genes combined.

Figure 4

Sleep time is shortened in female GABA neuron-specific Tfap2b homozygous knockdowns. (A) Time spent in sleep in female Tfap2b^fl/fl, Vgat-cre, Vgat-tfap2b^−/− mice over the Zeitgeber time (ZT) course. (B) Sleep quantity during 24 h, light and dark phase. ZT course NREMS quantification (C) and total NREMS amount during 24 h, light and dark phase (D). REMS quantification over the ZT course (E) and during 24 h, light and dark phases (F). All data were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test and were shown as the mean ± SEM. Significant pairwise comparisons were marked with *P < 0.05, **P < 0.01. Female Tfap2b^fl/fl (n = 5), Vgat-cre (n = 5), Vgat-tfap2b^−/− (n = 5).

Figure 5

Reduced delta and theta power during NREM and REM sleep, respectively, in female Vgat-tfap2b^−/− mice. NREMS power spectra in 24 h scale (A) and delta power (0.5 – 4.0 Hz) changes over ZT course (B). REMS power spectra in 24 h scale (C) and theta power (5 – 10 Hz) changes over ZT course (D). Wake power spectra and 0.2—10 Hz power changes over ZT course. Data in (A, C, E) were analyzed using Friedman test followed by Dunn’s multiple comparisons test. Data in (B, D, F) were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test and were shown as the mean ± SEM. Highlighted is the 0.5–10 Hz frequency band (A, C, E), which was used for statistical analysis. Significant pairwise comparisons of Tfap2b^fl/fl vs. Vgat-tfap2b^−/− were marked with ***P < 0.001, ****P < 0.0001. Female Tfap2b^fl/fl (n = 5), Vgat-cre (n = 5), Vgat-tfap2b^−/− (n = 5).

Figure 6

Delta and theta power during rebound sleep are weaker in female Vgat-tfap2b^−/− mice. Linear regression plots of accumulative delta (A) and theta power (B) following 6 h of SD. Equality of slopes or intercepts was tested by linear regression analysis. Significant differences were detected between groups and marked with *P < 0.05, ***P < 0.001. Female Tfap2b^fl/fl (n = 5), Vgat-cre (n = 5), Vgat-tfap2b^−/− (n = 5).

Figure 7

Sleep time is shortened in male GABAergic specific Tfap2b homozygous knockouts in baseline recording. (A) Sleep quantity changes in male Tfap2b^fl/fl, Vgat-cre, Vgat-tfap2b^−/− mice over the Zeitgeber time (ZT) course. (B) Sleep quantity during 24 h, light and dark phase. (C) ZT course NREMS quantification and (D) total NREMS amount during 24 h, light and dark phase. REMS quantification over the ZT course (E) and during 24 h, light and dark phases (F). All data were analyzed by two-way ANOVA followed by Sidak’s multiple comparisons test and were shown as the mean ± SEM. Significant pairwise comparisons were marked with *P < 0.05. Male Tfap2b^fl/fl (n = 5), Vgat-cre (n = 4), Vgat-tfap2b^−/− (n = 6).