Exploring epigenetic modification of the stress-related FKBP5 gene in mice exposed to alcohol during early postnatal development.

Early developmental exposure to alcohol has been implicated in adverse effects on the brain, often associated with the onset of neurodevelopmental disorders. Moreover, maternal alcohol consumption during pregnancy has been linked to the manifestation of mental health disorders, such as depression and anxiety, in subsequent generations. These mood disturbances may be attributed to alterations in protein expressions related to depression and anxiety within the hippocampus. While the precise mechanisms remain elusive, it is likely that pre- and postnatal exposure to alcohol induces changes in hippocampus, potentially through epigenetic modifications. The FKBP5 gene, known to modulate the stress response, is particularly relevant in this context. We postulate that alcohol-induced methylation of the FKBP5 gene disrupts HPA axis function, thereby prompting individuals to anxiety-like and depressive-like behaviors. To investigate this hypothesis, female C57BL/6 pups were subjected to early alcohol exposure via intubation with ethanol mixed in artificial milk from Postnatal Day 3 to Day 20. The intubation control pups were subjected to the same procedures without ethanol or milk, and a non-intubated control group included. Anxiety-like and depressive-like behaviors were assessed using the open field test, plus maze test, forced swim test, and tail suspension test when the pups reached 3 months of age. For epigenetic analysis of the FKBP5 gene, genomic DNA was isolated from hippocampal tissues and subjected to bisulfite conversion to distinguish methylated and unmethylated cytosines. Then, methylation-specific PCR was performed to assess methylation levels. Pups exposed to early postnatal alcohol exhibited increased levels of depression-like behavior and susceptibility to anxiety-like behavior during adolescence, as verified by behavioral assessments. Methylation profiling revealed higher rates of methylation within the stress-associated gene FKBP5 in both the early postnatal alcohol-exposed cohort (13.82%) and the intubation control group (3.93%), in contrast to the control cohort devoid of stress or alcohol exposure. These findings suggest a potential epigenetic mechanism underlying the observed behavioral alterations, implicating FKBP5 methylation as a candidate mediator of the increased vulnerability to mood disorders following early postnatal alcohol exposure.

Developmental exposure to the Fox River PCB mixture modulates behavior in juvenile mice.

Developmental exposures to PCBs are implicated in the etiology of neurodevelopmental disorders (NDDs). This observation is concerning given the continued presence of PCBs in the human environment and the increasing incidence of NDDs. Previous studies reported that developmental exposure to legacy commercial PCB mixtures (Aroclors) or single PCB congeners found in Aroclors caused NDD-relevant behavioral phenotypes in animal models. However, the PCB congener profile in contemporary human samples is dissimilar to that of the legacy Aroclors, raising the question of whether human-relevant PCB mixtures similarly interfere with normal brain development. To address this question, we assessed the developmental neurotoxicity of the Fox River Mixture (FRM), which was designed to mimic the congener profile identified in fish from the PCB-contaminated Fox River that constitute a primary protein source in the diet of surrounding communities. Adult female C57BL/6 J mouse dams (8-10 weeks old) were exposed to vehicle (peanut oil) or FRM at 0.1, 1.0, or 6.0 mg/kg/d in their diet throughout gestation and lactation, and neurodevelopmental outcomes were assessed in their pups. Ultrasonic vocalizations (USVs) and measures of general development were quantified at postnatal day (P) 7, while performance in the spontaneous alternation task and the 3-chambered social approach/social novelty task was assessed on P35. Triiodothyronine (T3) and thyroxine (T4) were quantified in serum collected from the dams when pups were weaned and from pups on P28 and P35. Developmental exposure to FRM did not alter pup weight or body temperature on P7, but USVs were significantly decreased in litters exposed to FRM at 0.1 or 6.0 mg/kg/d in the maternal diet. FRM also impaired male and female pups' performance in the social novelty task. Compared to sex-matched vehicles, significantly decreased social novelty was observed in male and female pups in the 0.1 and 6.0 mg/kg/d dose groups. FRM did not alter performance in the spontaneous alternation or social approach tasks. FRM increased serum T3 levels but decreased serum T4 levels in P28 male pups in the 1.0 and 6.0 mg/kg/d dose groups. In P35 female pups and dams, serum T3 levels decreased in the 6.0 mg/kg/d dose group while T4 levels were not altered. Collectively, these findings suggest that FRM interferes with the development of social communication and social novelty, but not memory, supporting the hypothesis that contemporary PCB exposures pose a risk to the developing brain. FRM had sex, age, and dose-dependent effects on serum thyroid hormone levels that overlapped but did not perfectly align with the FRM effects on behavioral outcomes. These observations suggest that changes in thyroid hormone levels are not likely the major factor underlying the behavioral deficits observed in FRM-exposed animals.

The impact of continuous and intermittent ketogenic diets on cognitive behavior, motor function, and blood lipids in TgF344-AD rats.

Studies suggest that ketogenic diets (KD) may improve memory in mouse models of aging and Alzheimer's disease (AD). This study determined whether a continuous or intermittent KD (IKD) enhanced cognitive behavior in the TgF344-AD rat model of AD. At 6 months-old, TgF344-AD and wild-type (WT) littermates were placed on a control (CD), KD, or IKD (morning CD and afternoon KD) provided as two meals per day for 2 or 6 months. Cognitive and motor behavior and circulating ß-hydroxybutyrate (BHB), AD biomarkers and blood lipids were assessed. Animals on a KD diet had elevated circulating BHB, with IKD levels intermediate to CD and KD. TgF344-AD rats displayed impaired spatial learning memory in the Barnes maze at 8 and 12 months of age and impaired motor coordination at 12 months of age. Neither KD nor IKD improved performance compared to CD. At 12 months of age, TgF344-AD animals had elevated blood lipids. IKD reduced lipids to WT levels with KD further reducing cholesterol below WT levels. This study shows that at 8 or 12 months of age, KD or IKD intervention did not improve measures of cognitive or motor behavior in TgF344-AD rats; however, both IKD and KD positively impacted circulating lipids.

The investigation of the effects of postnatal alcohol exposure on molecular content and antioxidant capacity of mice liver tissue.

One of the most common causes of fetal alcohol spectrum disease (FASD) characterized with neurodevelopmental disorder and growth retardation, is the postnatal alcohol consumption. Since studies in literature are mainly focused on alcohol-induced effects on brain tissues, the molecular effects of postnatal alcohol consumption on fetal liver are not clarified yet. The aim of this study is to determine the postnatal alcohol consumption-induced structural and compositional changes on liver tissue and the antioxidant capacity of liver. Newborn mice were divided into 3 groups as control group without any treatment, alcohol group treated with 3.0 g/kg of ethanol in 0.02 ml/g of artificially enriched milk between Postnatal Days (PD) 3-20 and intragastric intubation control group which was intragastrically intubated in the same method as the alcohol group but without ethanol/milk. These postnatal days in mice refers prenatal period (third trimester) of gestation in human. The biomolecular changes were determined by ATR-FTIR spectral analysis of the samples, besides the biochemical measurement of total protein content and antioxidant capacity of liver tissue. The result of the current study shows that while there was a slight increase in total lipid content, significant decrease in unsaturated lipid and total protein contents and total antioxidant capacity of liver were observed in alcohol-treated group. Thus, it is concluded that postnatal alcohol treatment causes significant changes in tissue proteins and lipids by inducing lipid peroxidation and changes in protein conformations of the liver tissue. In addition to that alcohol consumption also reduce the antioxidant capacity of liver tissue.

The structural effects of Vitamin A deficiency on biological macromolecules due to ethanol consumption and withdrawal: An FTIR study with chemometrics.

The structural effects of vitamin A-deficiency were examined on the molecular profiles of biomolecules of male rat hippocampus during prolonged ethanol intake/withdrawal using FT-IR spectroscopy coupled with chemometrics. Liquid ethanol diet with/without vitamin A was maintained to adult rats for 3-months. The rats were decapitated at different ethanol withdrawal times and FT-IR spectra were obtained. Ethanol consumption/withdrawal produced significant changes in proteins' conformations, while having insignificant structural effects on lipids. In vitamin A deficiency, ethanol produced structural changes in lipids by lipid ordering especially in the early-ethanol withdrawal. Furthermore, an increase in lipid and protein content, saturated/unsaturated lipid ratio, a decrease in nucleic acids content and decrease in membrane fluidity were observed. These changes were less severe in the presence of Vitamin A. This study is clinically important for individuals with vitamin A deficiency because they have to be more cautious when consuming alcohol to protect themselves from cognitive dysfunctions.

Effects of prenatal binge-like ethanol exposure and maternal stress on postnatal morphological development of hippocampal neurons in rats.

BACKGROUND: Alcohol is one of the most commonly used drugs of abuse negatively affecting human health and it is known as a potent teratogen responsible for fetal alcohol syndrome (FAS), which is characterized by cognitive deficits especially pronounced in juveniles but ameliorating in adults. Searching for the potential morphological correlates of these effects, in this study, we compared the course of developmental changes in the morphology of principal hippocampal neurons in fetal-alcohol (A group), intubated control (IC group), and intact control male rats (C group) over a protracted period of the first two postnatal months. METHODS: Ethanol was administered to the pregnant Wistar dams intragastrically, throughout gestation days (GD) 7-20, at a total dose of 6g/kg/day resulting in the mean blood alcohol concentration (BAC) of 246.6±40.9mg/dl. Ten morphometric parameters of Golgi-stained hippocampal neurons (pyramidal and granule) from CA1, CA3, and DG areas were examined at critical postnatal days (PD): at birth (PD1), at the end of the brain growth spurt period (PD10), in juveniles (PD30), and in young adults (PD60). RESULTS: During postnatal development, the temporal pattern of morphometric changes was shown to be region-dependent with most significant alterations observed between PD1-30 in the CA region and between PD10-30 in the DG region. It was also parameter-dependent with the soma size (except for CA3 pyramids), number of primary dendrites, dendrite diameter, dendritic tortuosity and the branch angle demonstrating little changes, while the total dendritic field area, dendritic length, number of dendritic bifurcations, and spine density being highly increased in all hippocampal regions during the first postnatal month. Moderate ethanol intoxication and the maternal intubation stress during gestation, showed similar, transient effects on the neuron development manifested as a smaller soma size in granule cells, reduced dendritic parameters and lower spine density in pyramidal neurons at PD1. Full recovery from these effects took place within the first 10 postnatal days. CONCLUSIONS: This study showed regional and temporal differences in the development of different morphometric features of principal hippocampal neurons in intact subjects over a protracted 2-months postnatal period. It also demonstrated an overlap in the effects of a moderate fetal ethanol intoxication and a mild maternal stress produced by the intragastric intubation, a commonly used method of ethanol administration to the pregnant dams. Fast recovery from the adverse effects on the soma size, dendritic arborization and spines density observed at birth indicates towards the fetal ethanol/stress induced developmental retardation.

Examination of age-dependent effects of fetal ethanol exposure on behavior, hippocampal cell counts, and doublecortin immunoreactivity in rats.

Ethanol is known as a potent teratogen having adverse effects on brain and behavior. However, some of the behavioral deficits caused by fetal alcohol exposure and well expressed in juveniles ameliorate with maturation may suggest some kind of functional recovery occurring during postnatal development. The aim of this study was to reexamine age-dependent behavioral impairments in fetal-alcohol rats and to investigate the changes in neurogenesis and gross morphology of the hippocampus during a protracted postnatal period searching for developmental deficits and/or delays that would correlate with behavioral impairments in juveniles and for potential compensatory processes responsible for their amelioration in adults. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7-21 gestation days at daily dose of 6 g/kg. Isocaloric intubation and intact control groups were included. Locomotor activity, anxiety, and spatial learning tasks were applied to juvenile and young-adult rats from all groups. Unbiased stereological estimates of hippocampal volumes, the total number of pyramidal and granular cells, and double cortin expressing neurons were carried out for postnatal days (PDs) PD1, PD10, PD30, and PD60. Alcohol insult during second trimester equivalent caused significant deficits in the spatial learning in juvenile rats; however, its effect on hippocampal morphology was limited to a marginally lower number of granular cells in dentate gyrus (DG) on PD30. Thus, initial behavioral deficits and the following functional recovery in fetal-alcohol subjects may be due to more subtle plastic changes within the hippocampal formation but also in other structures of the extended hippocampal circuit. Further investigation is required.

Effects of early postnatal alcohol exposure on the developing retinogeniculate projections in C57BL/6 mice.

Previous studies on the adverse effects of perinatal exposure to ethanol (EtOH) on the developing visual system mainly focused on retinal and optic nerve morphology. The aim of the present study was to investigate whether earlier reported retinal and optic nerve changes are accompanied by anomalies in eye-specific fiber segregation in the dorsal lateral geniculate nucleus (dLGN). C57BL/6 mice pups were exposed to ethanol by intragastric intubation at either 3 or 4 g/kg from postnatal days (PD) 3-10, the third trimester equivalent to human gestation. Control (C) and intubation control (IC) groups not exposed to ethanol were included. On PD9, retinogeniculate projections were labeled by intraocular microinjections of cholera toxin-ß (CTB) either conjugated to Alexa 488 (green) or 594 (red) administrated to the left and right eye, respectively. Pups were sacrificed 24 h after the last CTB injection. The results showed that ethanol exposure decreased the total number of dLGN neurons and significantly reduced the total dLGN projection as well as the contralateral and ipsilateral projection areas.

Effects of early postnatal exposure to ethanol on retinal ganglion cell morphology and numbers of neurons in the dorsolateral geniculate in mice.

BACKGROUND: The adverse effects of fetal and early postnatal ethanol intoxication on peripheral organs and the central nervous system are well documented. Ocular defects have also been reported in about 90% of children with fetal alcohol syndrome, including microphthalmia, loss of neurons in the retinal ganglion cell (RGC) layer, optic nerve hypoplasia, and dysmyelination. However, little is known about perinatal ethanol effects on retinal cell morphology. Examination of the potential toxic effects of alcohol on the neuron architecture is important because the changes in dendritic geometry and synapse distribution directly affect the organization and functions of neural circuits. Thus, in the present study, estimations of the numbers of neurons in the ganglion cell layer and dorsolateral geniculate nucleus (dLGN), and a detailed analysis of RGC morphology were carried out in transgenic mice exposed to ethanol during the early postnatal period. METHODS: The study was carried out in male and female transgenic mice expressing yellow fluorescent protein (YFP) controlled by a Thy-1 (thymus cell antigen 1) regulator on a C57 background. Ethanol (3 g/kg/d) was administered to mouse pups by intragastric intubation throughout postnatal days (PDs) 3 to 20. Intubation control (IC) and untreated control (C) groups were included. Blood alcohol concentration was measured in separate groups of pups on PDs 3, 10, and 20 at 4 different time points, 1, 1.5, 2, and 3 hours after the second intubation. Numbers of neurons in the ganglion cell layer and in the dLGN were quantified on PD20 using unbiased stereological procedures. RGC morphology was imaged by confocal microscopy and analyzed using Neurolucida software. RESULTS: Binge-like ethanol exposure in mice during the early postnatal period from PDs 3 to 20 altered RGC morphology and resulted in a significant decrease in the numbers of neurons in the ganglion cell layer and in the dLGN. In the alcohol exposure group, out of 13 morphological parameters examined in RGCs, soma area was significantly reduced and dendritic tortuosity significantly increased. After neonatal exposure to ethanol, a decrease in total dendritic field area and an increase in the mean branch angle were also observed. Interestingly, RGC dendrite elongation and a decrease in the spine density were observed in the IC group, as compared to both ethanol-exposed and pure control subjects. There were no significant effects of alcohol exposure on total retinal area. CONCLUSIONS: Early postnatal ethanol exposure affects development of the visual system, reducing the numbers of neurons in the ganglion cell layer and in the dLGN, and altering RGCs' morphology.