Variations in postnatal maternal care and the epigenetic regulation of metabotropic glutamate receptor 1 expression and hippocampal function in the rat

Abstract

Variations in maternal care in the rat affect hippocampal morphology and function as well as performance on hippocampal-dependent tests of learning and memory in the offspring. Preliminary genome-wide analyses of gene transcription and DNA methylation of the molecular basis for such maternal effects suggested differences in the epigenetic state and transcriptional activity of the Grm1 gene in the rat as a function of maternal care. Grm1 encodes the type I metabotropic glutamate receptor (mGluR1), and we found increased mGluR1 mRNA and protein in hippocampus from the adult offspring of mothers showing an increased frequency of pup licking/grooming (i.e., high-LG mothers) that was associated with a decrease in the methylation of Grm1. ChIP assays showed increased levels of histone 3 lysine 9 acetylation and histone 3 lysine 4 trimethylation of Grm1 in hippocampus from the adult offspring of high-LG compared with low-LG mothers. These histone posttranslational modifications were highly correlated, and both associate inversely with DNA methylation and positively with transcription. Studies of mGluR1 function showed increased hippocampal mGluR1-induced long-term depression in the adult offspring of high-LG compared with low-LG mothers, as well as increased paired-pulse depression (PPD). PPD is an inhibitory feedback mechanism that prevents excessive glutamate release during high-frequency stimulation. The maternal effects on both long-term depression and PPD were eliminated by treatment with an mGluR1-selective antagonist. These findings suggest that variations in maternal care can influence hippocampal function and cognitive performance through the epigenetic regulation of genes implicated in glutamatergic synaptic signaling.

Fig. 1.

Group I mGluR expression in the hippocampus. (A) qRT-PCR analysis of mGluR1 and mGluR5 mRNA levels in hippocampus from adult offspring of high-LG and low-LG mothers (n = 5 per group) expressed as the mean + SEM signal normalized using β₂ microglobulin (B2M). *P < 0.05. (B and C) Protein levels of mGluR1 and mGluR5 were measured by Western blot analysis in hippocampal lysates prepared from high-LG and low-LG offspring (n = 10–12 per group) and normalized to actin. Representative blots show the expression of mGluR1 and mGluR5. (D) Mean + SEM levels of mGluR1 and mGluR5 immunoreactivity expressed as OD unit in hippocampal samples from the adult offspring or high-LG and low-LG mothers. *P < 0.05.

Fig. 2.

DNA methylation analysis of the Grm1 gene. (A) Site map depicts the region and sequence (CpG dinucleotides in boldface) of the Grm1 gene analyzed for DNA methylation using sodium bisulfite sequencing. The bar covers the region in part of the coding and untranslated regions of exon 2 (MA, methylation assay). (B) Mean + SEM percentage of methylated clones in hippocampal samples from adult offspring of high-LG and low-LG mothers (n = 5 per group). *P < 0.01. (C) Mean + SEM levels of methylation across the 14 individual CpG sites of the analyzed region of the Grm1 gene as a function of maternal care (n = 5 per group). (D) Representative bead-on-string models depict methylated (○) or unmethylated (●) CpG sites across individual clones of one sample from the hippocampus of high-LG or low-LG mothers. The models depict the increased frequency of methylation typical in samples from the low-LG offspring, with a sporadic distribution. (E) Mean + SEM level of mGluR1 mRNA determined using qRT-PCR and normalized to β₂ microglobulin (B2M) in cultured primary hippocampal neurons treated with 0, 1, or 5 μM 5-aza-cytosine (n = 4–5 per group). *P < 0.05.

Fig. 3.

ChIP analysis of histone posttranslational modifications. (A) Schema depicts the regions (02A, 1,393 from TSS) and (−01A, −254 from TSS) of the Grm1 gene analyzed for various histone modifications. Chr, chromosome. (B) Mean + SEM levels of H3K9ac IP of regions 02A and −01A of the Grm1 gene in hippocampal samples from the adult offspring of high-LG and low-LG mothers (n = 6–8 per group). *P < 0.05. (C) Mean + SEM levels of H3K4me3 IP of regions 02A and −01A of the Grm1 gene in hippocampal samples from the adult offspring of high-LG and low-LG mothers (n = 6–8 per group). *P < 0.05. (D) Mean + SEM levels of H3K27me3 IP of regions 02A and −01A of the Grm1 gene in hippocampal samples from the adult offspring of high-LG and low-LG mothers (n = 6–8 per group). (E and F) Scatterplots of the relation between H3K9ac and H3K4me3 across all samples in regions 02A and −01A of the Grm1 gene in hippocampal samples from the adult offspring of high-LG and low-LG mothers (n = 14 total).

Fig. 4.

mGluR1-dependent LTD in high-LG and low-LG offspring. (A) Scatterplots of normalized AMPAR- field excitatory postsynaptic potential (fEPSP) slope vs. time before and 1 h after LTD induction in low-LG and high-LG offspring. (Upper) Traces show representative examples of AMPAR-fEPSP before (Left) and 1 h after (Right) DHPG application (50 μM, 10 min). [Adjacent scale bars: 0.4 mV (vertical) and 40 ms (horizontal).] Dashed lines indicate baseline fEPSP amplitude (traces) or fEPSP slope (scatterplot). Note that mGluR-dependent LTD was induced in hippocampal slices from high-LG offspring only. (B) Scatterplots of AMPAR-fEPSP slope vs. time before and after LTD induction in high-LG offspring in the presence of vehicle or an mGluR1 antagonist, JNJ 16259685 (JNJ; 1 μM). Note that DHPG-induced LTD was abolished by mGluR1 blockade. (C) Mean + SEM changes in fEPSP slope 1 h after DHPG application. Compared with low-LG offspring, high-LG offspring displayed an attenuated fEPSP slope after DHPG. (D) DHPG-induced LTD in high-LG offspring was blocked by JNJ 16259685. *P < 0.05.

Fig. 5.

mGluR1-dependent PPD in low-LG offspring. (A) Mean + SEM paired-pulse ratio reflects the effect of an mGluR1 antagonist, JNJ 16259685 (JNJ; 1 μM), on the paired-pulse ratio of the AMPAR-field excitatory postsynaptic potential (fEPSP) slope in high-LG and low-LG offspring. PPD (interpulse interval is 20 ms) of AMPAR-fEPSP was inducible in hippocampal slices from high-LG offspring only. This short-term plastic change was abolished by JNJ 16259685 (JNJ). *P < 0.05. (B) Representative traces of paired pulse-induced AMPAR-fEPSP recorded from low-LG and high-LG offspring in the presence of vehicle (artificial cerebrospinal fluid; Upper) and JNJ 16259685 (Lower).