Prenatal SAMe Treatment Changes via Epigenetic Mechanism/s USVs in Young Mice and Hippocampal Monoamines Turnover at Adulthood in a Mouse Model of Social Hierarchy and Depression

Abstract

The role of hippocampal monoamines and their related genes in the etiology and pathogenesis of depression-like behavior, particularly in impaired sociability traits and the meaning of changes in USVs emitted by pups, remains unknown. We assessed the effects of prenatal administration of S-adenosyl-methionine (SAMe) in Sub mice that exhibit depressive-like behavior on serotonergic, dopaminergic and noradrenergic metabolism and the activity of related genes in the hippocampus (HPC) in adulthood in comparison to saline-treated control Sub mice. During postnatal days 4 and 8, we recorded and analyzed the stress-induced USVs emitted by the pups and tried to understand how the changes in the USVs' calls may be related to the changes in the monoamines and the activity of related genes. The recordings of the USVs showed that SAMe induced a reduction in the emitted flat and one-frequency step-up call numbers in PND4 pups, whereas step-down type calls were significantly increased by SAMe in PND8 pups. The reduction in the number of calls induced by SAMe following separation from the mothers implies a reduction in anxiety, which is an additional sign of decreased depressive-like behavior. Prenatal SAMe increased the concentrations of serotonin in the HPC in both male and female mice without any change in the levels of 5HIAA. It also decreased the level of the dopamine metabolite DOPAC in females. There were no changes in the levels of norepinephrine and metabolites. Several changes in the expression of genes associated with monoamine metabolism were also induced by prenatal SAMe. The molecular and biochemical data obtained from the HPC studies are generally in accordance with our previously obtained data from the prefrontal cortex of similarly treated Sub mice on postnatal day 90. The changes in both monoamines and their gene expression observed 2-3 months after SAMe treatment are associated with the previously recorded behavioral improvement and seem to demonstrate that SAMe is effective via an epigenetic mechanism.

Keywords: S-adenosyl-methionine; USVs; brain monoamine metabolism; depression.

Figure 1

The mean of call numbers differs between the groups. (A)—bar charts representing the average number of calls emitted by pups on postnatal days 4 and 8, respectively. Bars reflect mean values, error bars represent SEM. Two-Way ANOVA, **—p-value of multiple comparisons < 0.01. (B)—representative oscillogram of a three-second sample of the USV recording. USV oscillogram of SAMe-treated offspring (blue trace) overlaid with that of Saline-treated offspring (orange trace), reflecting similarity in the emission intensity between groups reflecting a higher number of the calls during a certain time. (C)—representative sonograms obtained from sample recording of a postnatal day 4 SAMe- (upper panel) or Saline (lower panel)-treated offspring, reflecting differences in emission frequency in the treatment groups, with the SAMe-treated pups emitting vocalizations much more rarely than the Saline-treated ones.

Figure 2

Call type distribution between experimental groups. The charts reflect the percentage distribution of the different call types within the treatment groups and ages, sorted in a decreasing order. (A)—Saline-treated offspring’s call distribution at postnatal day 4. (B)—SAMe-treated pups’ call distribution at PND4. (C)—Saline-treated pups’ calls at PND8. (D)—distribution of the calls within the SAMe group at PND8.

Figure 3

Prenatal treatment with SAMe leads to the alternation of the specific syllables types. SAMe given prenatally, changed the number of flat, frequency step down and step up calls emitted by pups at PND4: (A)—The number of emitted flat calls were significantly lowered by SAMe, compared to the control group (n = 21) at PND4, but not on day 8; (B)—SAMe increased the number of emitted one frequency step down calls at PND8. (C)—The number of frequency step up call emissions was reduced by SAMe at PND4 compared to control. Bar charts represent mean of call numbers, error bars represent SEM; Two-Way ANOVA, *—p-value of multiple comparisons < 0.01, **—p-value < 0.001, ****—p-value < 0.0001, n = 32 pups for SAMe and 21 for Saline group, respectively.

Figure 4

SAMe effect on Serotonin turnover in the Hippocampus. SAMe treatment significantly elevated the levels of serotonin in the HPC of both male and female mice (A). The levels of HIAA (B) and of the 5-HIAA/5-HT ratio (C) remained unchanged in both sexes. The data are presented as mean ± SEM. Levels of 5-HT and 5-HIAA are presented in ng/mg, n = 6 for each group. * p < 0.05 compared to control mice of the same gender.

Figure 5

SAMe effect on dopamine metabolism (A). The levels of dopamine in the hippocampus of Sub mice were unchanged by SAMe treatment (B). The levels of DA catabolic product, DOPAC, were higher in the hippocampus of female Sub mice; however, the differences in the ratio of DOPAC/DA did not reach statistical significance (C). The levels of HVA (D), HVA/DOPAC index (E), and overall turnover of DA were unchanged (F). The data are presented as mean ± SEM. The levels of DA, DOPAC and HVA are presented in ng/mg, n = 6 for each group. * p < 0.05 compared to control mice of the same gender.

Figure 6

SAMe effect on norepinephrine turnover. NE (A), MHPG levels (B) and MHPG to NE ratio (C) in the PFC and hippocampus of Sub mice treated with SAMe did not deviate from control mice. The data are presented as mean ± SEM. Levels of NE and MHPG are presented in ng/mg, n = 6 for each group.

Figure 7

Tph2, Aldh and Mao-b genes expression in the hippocampus. No effect of SAMe treatment was observed for the expression of the Tph2 (A), Aldh (B) and Mao-b (C) evaluated genes. The data are presented as means ± SEM. n = 6 for each group.

Figure 8

SAMe effect on Htr2a, Sert and Drd2 genes expression in the hippocampus. (A) The expression levels of Htr2a were significantly decreased in both male and female hippocampi by SAMe; (B) SAMe treatment significantly increased the Sert gene expression only in male hippocampi, demonstrating the sex differences in response to treatment; (C) Drd2 expression levels were changed in a sex-depended manner by SAMe, as a dramatic elevation in gene expression was measured in female hippocampi compared to controls. The data are presented as means ± SEM., * p < 0.05, n = 6 for each group.

Figure 9

Chart flow demonstrating the flow of the experimental design.