Alteration of selective neurotransmitters in fetal brains of prenatally alcohol-treated C57BL/6 mice: quantitative analysis using liquid chromatography/tandem mass spectrometry

Abstract

We previously demonstrated that prenatal alcohol exposure results in brain defects at different embryonic stages. This study is aimed at characterizing the influence of prenatal alcohol exposure on the levels of several neurotransmitters at early embryonic stage 13 (E13). Pregnant C57BL/6 mice were exposed to either a 25% ethanol derived calorie diet (ALC) or pair-fed (PF) liquid diet from E7 to E13. At E13, fetal brains were collected from dams of the ALC and PF groups. Liquid chromatography/tandem mass spectrometry (LC-MS) was then used to evaluate neurotransmitter levels. This approach involved the use of an LC column in conjunction with multiple-reaction monitoring mass spectrometry. Quantitative analyses of catecholamines, idolamine, and amino acid neurotransmitters revealed significant reductions in the levels of dopamine (p=0.004), norepinephrine (p=0.0009), epinephrine (p=0.0002), serotonin (p=0.004), and GABA (p=0.002) in the ALC group compared to the PF group. However, there was no significant change in the levels of glutamate in E13 fetal brains. These findings demonstrate that prenatal alcohol exposure reduces the concentrations of some catecholamines, idolamine, and amino acid neurotransmitters in E13 fetal brains. This study suggests that alterations of selective neurotransmitters may be the cause of abnormalities in brain function and behavior found in fetal alcohol spectrum disorders.

Figure 1

Dot-plots illustrating the changes in the levels of dopamine (a), epinephrine (b), norepinephrine (c), GABA (d), glutamate (e) and serotonin (f) as a result of prenatal alcohol exposure. Asterisks depict the statistically significance differences.

Figure 2

LC/MSMS chromatograms of catecholamines: dopamine (a), epinephrine (b) and norepinephrine (c) depicting significant reduction in the levels of these neurotransmitters in E13 fetal brains of alcohol-treated prenatally mice. In all cases, high signal (blue) is associated with PF control group and low signal (red) is associated with ALC group.

Figure 3

LC/MSMS chromatograms of amino acids: GABA (a) and glutamate (b) depicting significant reduction in the levels of GABA in E13 fetal brain of alcohol-treated prenatally mice. High signal (blue) is associated with PF control group and low signal (red) is associated with ALC group. However, no significance difference in the level of glutamate was found between ALC and PF groups.

Figure 4

LC/MSMS chromatograms show the levels of serotonin in E13 fetal brain as a result of prenatal alcohol exposure. Significant down-regulation of the level of serotonin was found in the ALC group compared to PF group. Upper trace is that of PF group (blue) and lower trace is that of ALC group (red).