Functional and Structural Correlates of Impaired Enrichment-Mediated Adult Hippocampal Neurogenesis in a Mouse Model of Prenatal Alcohol Exposure

Abstract

Background: Fetal alcohol spectrum disorders (FASDs) are associated with a wide range of cognitive deficiencies.

Objective: We previously found that gestational exposure to moderate levels of alcohol in mice throughout the 1st-2nd human trimester-equivalents for brain development results in profound impairment of the hippocampal neurogenic response to enriched environment (EE) in adulthood, without altering baseline neurogenesis rate under standard housing (SH). However, the functional and structural consequences of impaired EE-mediated neurogenesis in the context of prenatal alcohol exposure (PAE) have not been determined.

Results: Here, we demonstrate that PAE-EE mice display impaired performance on a neurogenesis-dependent pattern discrimination task, broadened behavioral activation of the dentate gyrus, as assessed by expression of the immediate early gene, c-Fos, and impaired dendritic branching of adult-generated dentate granule cells (aDGCs).

Conclusions: These studies further underscore the impact of moderate gestational alcohol exposure on adult hippocampal plasticity and support adult hippocampal neurogenesis as a potential therapeutic target to remediate certain neurological outcomes in FASD.

Keywords: Pattern discrimination learning; dentate gyrus; fetal alcohol spectrum disorders; immediate-early genes.

Fig. 1

PAE impairs EE-mediated improvement A-B context fear discrimination learning. A. Experimental timeline. SAC and PAE Nestin-CreERT2:tdTomato mice received tamoxifen dosing to induce reporter expression in aDGCs and were subjected to SH or EE housing conditions for 10 weeks prior to behavioral testing. B. Experimental design for the A-B context fear discrimination task. Mice received two 2-second 0.5 mA foot shocks separated by a 90 second interval in Context A only. The first shock occurred 90 seconds following placement into the chamber and mice were removed from the chamber 30 seconds following the 2^nd foot shock. In the non-shock context B, mice received no foot shock or other aversive event. Each mouse experienced one daily session in Context A and one daily session in Context B, separated by 3 hours. The order of context exposure was alternated each day for 7 days. Daily discrimination scores were calculated as: (freezing time context A - freezing time context B) / (freezing time context A + freezing time context B) during the first 90 seconds of each testing session. C. Discrimination learning over time. Data are plotted as the mean daily discrimination scores per group±SEM. Group n’s were as follows: SAC-SH (n = 8 mice) and SAC-EE (n = 8 mice) sampled across 5 separate litters; PAE-SH (n = 15 mice) and PAE-EE (n = 15 mice) sampled across 7-8 separate litters. Three way ANOVA statistics: testing day [F(6,250) = 325, p < 0.0001], alcohol treatment [F(1,250) = 175, p < 0.0001], housing [F(1,250) = 12.49, p = 0.0005)], alcohol treatment x housing [F(1,250) = 98.73, p < 0.0001], alcohol treatment x housing x day [F(6,250) = 18.38, p < 0.0001]. *p < 0.01 SAC-EE vs. all other groups (Tukey’s multiple comparison). D. Mean discrimination scores at testing day 7. *p < 0.01 SAC-EE vs. all other groups (Tukey’s post-hoc comparison). E. Freeze time in Context A (shock) and Context B (non-shock) across all experimental groups on testing day 7. Data expressed as mean±SEM.

Fig. 2

Impaired EE-mediated discrimination learning in PAE mice is directly correlated with impaired neurogenesis. A. Representative confocal microscopy images of coronal sections through the dorsal dentate gyrus demonstrating tdTomato⁺ aDGCs (red), NeuN⁺ mature postmitotic neurons (green), DAPI⁺ nuclear counterstain (blue). B. Number of tdTomato+/NeuN+aDGCs across groups (means±SEM). Two-way ANOVA statistics: alcohol treatment [F (1,11) = 5.83, p = 0.03)], housing [F(1,11) = 6.74, p = 0.02], alcohol treatment x housing interaction [F(1,11) = 8.07, p = 0.01]. *p = 0.02 SAC-SH vs. SAC-EE; p < 0.01 SAC-EE vs. PAE-EE (Tukey’s post-hoc analysis). N = 4 mice/group. C. Behavioral performance as a function of neurogenesis. Colored circles correspond to data from individual mice from SAC-SH (blue), SAC-EE (red), PAE-SH (black) and PAE-EE (green); i.e., color convention as in B. Pearson correlation, R² = 0.9422.

Fig. 3

PAE-EE mice display broadened novelty-induced c-Fos expression within the dentate gyrus compared to SAC-EE mice. A. Experimental timeline. SAC and PAE C57Bl6/J mice were exposed to EE housing conditions for 8 weeks (SAC-EE and PAE-EE). Mice were then exposed to a novel environment for 30 minutes and sacrificed 80 minutes following return to home cage. SAC-EE and PAE-EE mice not exposed to novelty remained in home cage until sacrifice and served as controls. B. c-Fos immunofluorescence. Representative confocal images of c-Fos immunoreactivity (pink) within dorsal dentate gyrus in SAC-EE and PAE-EE mice following exposure to novel environment. Histological sections were counterstained with DAPI nuclear dye (blue). C. Quantification of c-Fos⁺ nuclei within the suprapyramidal blade of the dorsal dentate gyrus across groups (mean±SEM). Two-way ANOVA statistics: alcohol treatment x novelty interaction [[F (1,21) = 10.03, p = 0.005]. Tukey’s post-hoc analysis revealed a significant impact of novelty on c-Fos+expression only in PAE-EE mice (*p = 0.01). Group n’s are as follows: SAC-EE home cage (n = 5 mice across 5 litters) and SAC-EE novelty (n = 7 mice across 7 litters); PAE-EE home cage (n = 5 mice across 5 litters) and PAE-EE novelty (n = 8 mice across 8 litters).

Fig. 4

PAE impairs EE-mediated dendritic branching in aDGCs. A. Sholl analysis of dendritic branching complexity in tdTomato⁺ aDGCs across experimental groups. Three way ANOVA statistics: alcohol treatment [F(1,216) = 12.27, p < 0.0006], housing [F(1,162) = 9.40, p < 0.0025], alcohol treatment x housing interaction [(F(1,162) = 24.70), p < 0.0001]. n’s were as follows: SAC-SH (n = 4 mice; 14 neurons sampled) and SAC-EE (n = 6 mice; 21 neurons sampled) across 6 separate litters; PAE-SH (n = 4 mice; 16 neurons sampled) and PAE-EE (n = 4 mice; 14 neurons sampled) across 2 separate litters. 2-5 tdTomato⁺ cells were sampled/mouse and averaged such that n = mouse constitutes the unit of statistical determination. B. Representative Neurolucida^tm traces from compressed Z-stack confocal images for each group. C. Quantification of cell soma size of tdTomato+aDGCs across groups. Mean±SEM *p < 0.02 Tukey’s post-hoc. D. Quantification of total dendritic length in tdTomato⁺ aDGCs across groups. Mean±SEM.

Fig. 5

Comparison of spine density and morphology in tdTomato⁺ aDGCs from SAC-EE vs. PAE-EE mice. A. Total dendritic spine densities in SAC-EE vs. PAE-EE mice. SAC-EE (n = 6 mice from 5 litters), PAE-EE (n = 4 mice from 2 litters). Mean #spines/μm±SEM. B. Densities of stubby, long thin, filopodial and mushroom protrusions in SAC-EE vs. PAE-EE mice. Classification are based on criteria as outlined in Methods. Mean #spines/μm±SEM. *p < 0.01 unpaired t-test with Welch’s correction. C. Mean percent distribution of spine classes in tdTomato+aDGCs from SAC-EE and PAE-EE mice. D. Representative 3D reconstruction of dendritic segment with filopodial, long thin, stubby and mushroom spines. Image reconstructed with Imaris^tm Filament Tracer software.