Maternal care and DNA methylation of a glutamic acid decarboxylase 1 promoter in rat hippocampus

Abstract

Parenting and the early environment influence the risk for various psychopathologies. Studies in the rat suggest that variations in maternal care stably influence DNA methylation, gene expression, and neural function in the offspring. Maternal care affects neural development, including the GABAergic system, the function of which is linked to the pathophysiology of diseases including schizophrenia and depression. Postmortem studies of human schizophrenic brains have revealed decreased forebrain expression of glutamic acid decarboxylase 1 (GAD1) accompanied by increased methylation of a GAD1 promoter. We examined whether maternal care affects GAD1 promoter methylation in the hippocampus of adult male offspring of high and low pup licking/grooming (high-LG and low-LG) mothers. Compared with the offspring of low-LG mothers, those reared by high-LG dams showed enhanced hippocampal GAD1 mRNA expression, decreased cytosine methylation, and increased histone 3-lysine 9 acetylation (H3K9ac) of the GAD1 promoter. DNA methyltransferase 1 expression was significantly higher in the offspring of low- compared with high-LG mothers. Pup LG increases hippocampal serotonin (5-HT) and nerve growth factor-inducible factor A (NGFI-A) expression. Chromatin immunoprecipitation assays revealed enhanced NGFI-A association with and H3K9ac of the GAD1 promoter in the hippocampus of high-LG pups after a nursing bout. Treatment of hippocampal neuronal cultures with either 5-HT or an NGFI-A expression plasmid significantly increased GAD1 mRNA levels. The effect of 5-HT was blocked by a short interfering RNA targeting NGFI-A. These results suggest that maternal care influences the development of the GABA system by altering GAD1 promoter methylation levels through the maternally induced activation of NGFI-A and its association with the GAD1 promoter.

Figure 1.

Influence of maternal care on hippocampal GAD1 expression. Mean ± SEM levels of GAD1 mRNA quantified using qRT-PCR analysis of the amplified GAD1 mRNA (relative to the β2M standard) in the postnatal day 4 and adult offspring of high- or low-LG mothers (n = 4–8/group; *p < 0.05).

Figure 2.

Maternal regulation of NGFI-A binding to the GAD1 promoter. Mean ± SEM qPCR data of the amplified GAD1 promoter sequence from NGFI-A immunoprecipitated hippocampal extract as a ratio of that from the input fraction. Hippocampal samples were obtained from the male, postnatal day 4 pups of high- or low-LG mothers immediately after a nursing bout (ON) or 25 min after the termination of a nursing bout (without any subsequent mother–pup interaction; OFF; n = 4–6/group; *p < 0.05 from all other groups).

Figure 3.

Regulation of GAD1 expression in primary hippocampal neuronal cell cultures. a, Mean ± SEM levels of GAD₆₇ expression (relative optical density) quantified using Western blotting with samples from hippocampal cultures treated with serotonin (5-HT) (1 μm) or medium alone (*p < 0.01). b, Representative Western blots showing bands corresponding to GAD₆₇ and α-tubulin (used to control for loading variation). c, Physical map of the CMV/V5-His-NGFI-A expression vector. d, Mean ± SEM levels of GAD1 mRNA quantified using qRT-PCR analysis of the amplified GAD1 mRNA (relative to the β2M standard) in primary hippocampal neuronal cultures treated with a lentiviral vector containing an NGFI-A expression plasmid (OE), the empty viral vector (EV), and a reference, no-treatment group (NT) (n = 8/group; *p < 0.05). e, Mean ± SEM levels of GAD1 mRNA amplified from primary hippocampal neuronal cultures, quantified using qRT-PCR (with β2M as standard), treated with medium alone plus empty viral vector (EV–CTL), 5-HT in medium plus empty viral vector (EV–5-HT), medium alone plus the viral vector containing an NGFI-A siRNA (SI–CTL), and 5-HT in medium plus the viral vector containing an NGFI-A siRNA (SI–5-HT; n = 4–6/group) in sample (*p < 0.01 compared with all other groups).

Figure 4.

Maternal effect on GAD1 promoter methylation. a, A sequence map of the GAD1 promoter showing the NGFI-A consensus sequence. b, Mean ± SEM percentage of cloned GAD1 promoter sequences revealing one or more methylated CpG sites as determined using NaBis mapping (10–20 clones per sample, with n = 5/group; *p < 0.01). c, Mean ± SEM percentage of cloned GAD1 promoter sequences revealing one or more methylated CpG sites as determined using NaBis mapping for each of the individual CpG dinucleotides within the GAD1 promoter region. Statistical analysis reveals an overall group effect.

Figure 5.

Maternal regulation of the epigenetic state of the GAD1 promoter. a, Mean ± SEM levels of DNMT1 mRNA quantified using qRT-PCR analysis of the amplified DNMT1 mRNA (relative to the β2M standard) in hippocampal samples derived from adult offspring of high- or low-LG mothers (n = 5/group; *p < 0.05). b, Mean ± SEM qPCR data of the amplified GAD1 promoter sequence immunoprecipitated from hippocampal samples with an antibody to H3-K9ac or NGFI-A and normalized to the input values (*p < 0.05; n = 4 samples/group). c, Representative images of GAD1 and β2M qPCR products on agarose gel confirming the amplification of a single specific product from chromatin immunoprecipitation assays described above.