Prenatal Ethanol Exposure and Postnatal Environmental Intervention Alter Dopaminergic Neuron and Microglia Morphology in the Ventral Tegmental Area During Adulthood

Abstract

Background: Prenatal ethanol exposure (PE) impairs midbrain dopaminergic (DA) neuron function, which might contribute to various cognitive and behavioral deficits, including attention deficits and increased addiction risk, often observed in individuals with fetal alcohol spectrum disorders. Currently, the underlying mechanisms for PE-induced deficits are unclear. PE could lead to neuroinflammation by activating microglia, which play an important role in synaptic function. In the present study, we investigated PE effects on microglial activation and DA neuron density and morphology in the ventral tegmental area (VTA). Since postnatal environmental enrichment can reduce neuroinflammation and ameliorate several PE-induced behavioral deficits, we examined if a postnatal environmental intervention strategy using neonatal handling and postweaning complex housing could reverse PE effects on VTA DA neurons and microglia.

Methods: Pregnant rats received 0 or 6 g/kg/d ethanol by 2 intragastric intubations on gestation days 8 to 20. After birth, rats were reared in the standard laboratory or enriched condition. Male adult rats (8 to 12 weeks old) were used for immunocytochemistry.

Results: The results showed that PE decreased VTA DA neuron body size in standardly housed rats. Moreover, there was a significant decrease in numbers of VTA microglial branches and junctions in PE rats, suggesting morphological activation of microglia and possible neuroinflammation. The PE effects on microglia were normalized by postnatal environmental intervention, which also decreased the numbers of microglial branches and junctions in control animals, possibly via reduced stress.

Conclusions: Our findings show an association between PE-induced morphological activation of microglia and impaired DA neuron morphology in the VTA. Importantly, postnatal environmental intervention rescues possible PE-induced microglial activation. These data support that environmental intervention can be effective in ameliorating cognitive and behavioral deficits associated with VTA DA neuron dysfunctions, such as attention deficits and increased addiction risk.

Keywords: Dopamine; Environmental Enrichment; Fetal Alcohol Spectrum Disorders; Microglia; Ventral Tegmental Area.

Figure 1.. Quantifying ventral tegmental area (VTA) cells after prenatal ethanol exposure (PE).

Representative sections were obtained from 8–12-weeks-old male rats after PE and stained for tyrosine hydroxylase (TH; dopamine/DA neuron marker) and ionized calcium binding adaptor molecule 1 (IBA-1; microglia marker) in the anterior VTA (A) and middle VTA (B). The region of interest (ROI) for counting DA neurons and microglia is shown in (C) from the anterior VTA (area of the ROI: 78002.665 μm²). All the images in this figure are derived from a 2D projection of 6 Z-stack sections (0.54 μm between sections). The projection images were used to quantify the density of DA neurons and microglia with Fiji. This method was also used to measure DA cell body size in the VTA.

Figure 2.. Prenatal ethanol exposure (PE) and postnatal environmental intervention effects on dopamine (DA) neuron density in the ventral tegmental area (VTA).

The results show that PE and postnatal environmental intervention did not exert any effects on DA neuron density in the anterior or middle VTA. Neuron density is measured by number of neurons within the region of interest. Data are presented as Mean ± SEM.

Figure 3.. Prenatal ethanol exposure (PE) and postnatal environmental intervention effects on ventral tegmental area (VTA) dopamine (DA) neuron body size.

The results showed that PE led to a significant decrease in DA cell body size in standardly housed animals in both the anterior and middle VTA. This effect was not reversed by postnatal environmental intervention.. Data are presented as Mean ± SEM. *: p < 0.05, control vs. PE in the same rearing condition; ##: p < 0.01, standard vs. enriched controls

Figure 4.. Prenatal ethanol exposure (PE) and postnatal environmental intervention effects on ventral tegmental area (VTA) microglia density and body size.

(A) Microglia density. There were no differences in microglia density between control and PE rats in either the anterior or middle VTA. Postnatal environmental intervention did not exert any effects on microglia density in either the anterior or the middle VTA. Microglia density is measured by number of microglia within the region of interest. (B) Microglia body size. There were no differences in microglia body size between control and PE rats in either the anterior or the middle VTA. Postnatal environmental intervention did not have any effects on microglia body size. Data are presented as Mean ± SEM.

Figure 5.. Prenatal ethanol exposure (PE) and postnatal environmental intervention effects on ventral tegmental area (VTA) microglia branching patterns.

(A) Representative 2-D projection images from 3-D Z-stack images of VTA microglia. The quantification of microglia branching patterns was achieved using the 3-D skeleton module in Fiji. (B), (C), & (D) Microglial branch number, junction number, and average branch length, respectively. In standardly housed animals, PE decreased the number of branches and number of junctions while the branch length was not altered in both the anterior and middle VTA. Postnatal environmental intervention reversed the PE-induced reductions in numbers of microglial branches and junctions observed in standardly housed rats. In addition, in control animals, small but significant decreases in numbers of branches and junctions in the anterior and middle VTA were observed in rats with postnatal environmental intervention, relative to their standardly housed counterparts. Data are presented as Mean ± SEM. *: p < 0.05; ***: p < 0.001, control vs. PE rats in the same rearing condition. #: p < 0.05; ##: p < 0.01; ###: p < 0.001, standardly housed vs. rats undergoing environmental intervention with the same prenatal treatment.