Donepezil Reverses Dendritic Spine Morphology Adaptations and Fmr1 Epigenetic Modifications in Hippocampus of Adult Rats After Adolescent Alcohol Exposure

Abstract

Background: Adolescent intermittent ethanol (AIE) exposure produces persistent impairments in cholinergic and epigenetic signaling and alters markers of synapses in the hippocampal formation, effects that are thought to drive hippocampal dysfunction in adult rodents. Donepezil (Aricept), a cholinesterase inhibitor, is used clinically to ameliorate memory-related cognitive deficits. Given that donepezil also prevents morphological impairment in preclinical models of neuropsychiatric disorders, we investigated the ability of donepezil to reverse morphological and epigenetic adaptations in the hippocampus of adult rats exposed to AIE. Because of the known relationship between dendritic spine density and morphology with the fragile X mental retardation 1 (Fmr1) gene, we also assessed Fmr1 expression and its epigenetic regulation in hippocampus after AIE and donepezil pretreatment.

Methods: Adolescent rats were administered intermittent ethanol for 16 days starting on postnatal day 30. Rats were treated with donepezil (2.5 mg/kg) once a day for 4 days starting 20 days after the completion of AIE exposure. Brains were dissected out after the fourth donepezil dose, and spine analysis was completed in dentate gyrus granule neurons. A separate cohort of rats, treated identically, was used for molecular studies.

Results: AIE exposure significantly reduced dendritic spine density and altered morphological characteristics of subclasses of dendritic spines. AIE exposure also increased mRNA levels and H3-K27 acetylation occupancy of the Fmr1 gene in hippocampus. Treatment of AIE-exposed adult rats with donepezil reversed both the dendritic spine adaptations and epigenetic modifications and expression of Fmr1.

Conclusions: These findings indicate that AIE produces long-lasting decreases in dendritic spine density and changes in Fmr1 gene expression in the hippocampal formation, suggesting morphological and epigenetic mechanisms underlying previously reported behavioral deficits after AIE. The reversal of these effects by subchronic, post-AIE donepezil treatment indicates that these AIE effects can be reversed by up-regulating cholinergic function.

Keywords: Fmr1; Adolescent alcohol; Dendritic Spines; Donepezil; Epigenetics; Hippocampus; Histone Acetylation.

Conflict of interest statement

CONFLICT OF INTEREST: The authors have no conflicts of interest.

Copyright © 2018 by the Research Society on Alcoholism.

Figures

Fig. 1

Effects of adolescent intermittent ethanol (AIE) exposure and donepezil (DZ) post-treatment on dendrites and total dendritic spine density in adult dentate gyrus granule neurons. (A) Representative 3D image of a DiI labeled granule neuron in the dorsal dentate gyrus. Yellow arrow denotes axon stemming from the soma and yellow box represents a typical section of dendrite used for analysis. (B) Representative labeling of a section of dendrite showing the Imaris filament recreation of the dendritic shaft (shown in yellow) and spines (shown in blue). Quantitation of (C) dendrite diameter (main effect of AIE: F (1,20) = 35.77, ***p < 0.0001) and (D) total dendritic spine density (interaction: F (1,19) = 5.56, p = 0.0293; ***p < 0.001 vs Water/Water; **p < 0.01 vs AIE/Water) in granule neurons from adult rats exposed to AIE and DZ. For all dendrite and dendritic spine analysis, there were n = 5–6 rats/group, n = 3 – 6 dendritic sections/rat, and n = 111 total dendritic sections.

Fig. 2

Adaptations in dendritic spine subclasses in adult rats exposed to adolescent intermittent ethanol (AIE) and donepezil (DZ) post-treatment. (A) Schematic illustrating parameters used to classify spines based on their morphological characteristics (HD = head diameter; L = length; ND = neck diameter; NL = neck length). (B) Changes in density of dendritic spine subclasses in AIE and DZ treated rats (Long spines: *p = 0.0131, Water vs AIE; Stubby spines: **p = 0.0056, Water vs DZ; Mushroom spines: **p < 0.001, Water/vs AIE).

Fig. 3

Morphological adaptations in spine subclass (A) volume (Filopodia: *p = 0.0196, Water/Water vs AIE/Water; Long spines: ***p < 0.0001, Water/Water vs AIE/Water, *p = 0.0313, AIE/Water vs AIE/DZ; Stubby spines: ***p < 0.0001, Water/Water vs AIE/Water, *p = 0.046, AIE/Water vs AIE/DZ), (B) diameter (Filopodia: *p = 0.032, Water/Water vs AIE/Water; Long spines: ***p < 0.001, Water/Water vs AIE/Water; **p < 0.01, AIE/Water vs AIE/DZ; Stubby spines: **p < 0.01, Water/Water vs AIE/Water, *p = 0.0176, AIE/Water vs AIE/DZ), and (C) length in adult rats exposed to AIE and DZ (Filopodia: *p = 0.0279, Water vs DZ).

Fig. 4

Donepezil reverses AIE-induced morphological adaptations in dendritic spine terminal point (A) diameter (Filopodia: *p = 0.0215, Water/Water vs AIE/Water; Long spines: ***p < 0.0001, Water/Water vs AIE/Water; **p = 0.0029, AIE/Water vs AIE/DZ; Stubby spines: ***p < 0.0001, Water/Water vs AIE/Water, *p = 0.0336, AIE/Water vs AIE/DZ, *p = 0.0456, Water/Water vs AIE/DZ) and (B) volume (Long spines: ***p < 0.0001, Water/Water vs AIE/Water, **p = 0.0088, AIE/Water vs AIE/DZ; Stubby spines: ***p < 0.0001, Water/Water vs AIE/Water, *p = 0.0362, AIE/Water vs AIE/DZ).

Fig. 5

Morphological characteristics of dendritic spine necks after AIE exposure and donepezil post-treatment. Changes in spine neck (A) volume (Filopodia: *p = 0.0221, Water/Water vs AIE/Water; Long spines: **p = 0.0017, Water vs AIE; Stubby spines: *p = 0.073, Water vs AIE), (B) diameter (Filopodia: *p = 0.0161, Water/Water vs AIE/Water; Long spines: ***p = 0.0001, Water/Water vs AIE/Water; **p = 0.0036, AIE/Water vs AIE/DZ; Stubby spines: *p = 0.031, Water vs AIE), and (C) length (Filopodia: *p = 0.0455, Water vs DZ; Long spines: **p = 0.0025, Water/Water vs AIE/Water; **p = 0.0027, AIE/Water vs AIE/DZ; Stubby spines: *p = 0.0203, Water/Water vs AIE/Water; Mushroom: *p = 0.047, Water vs DZ) in dentate gyrus granule neurons.

Fig. 6

The spine head diameter frequency distribution was significantly changed by AIE exposure and donepezil post-treatment (0.2 μm: ***p < 0.0001, Water/Water vs AIE/Water, **p = 0.0078, AIE/Water vs AIE/DZ; 0.36–0.5 μm: ***p < 0.0001, Water/Water vs AIE/Water, **p = 0.0093, AIE/Water vs AIE/DZ; 0.51–0.65 μm: ***p = 0.0008, Water vs AIE).

Fig. 7

Effects of AIE and donepezil treatment on Fmr1 gene expression and its regulation by histone modifications (H3K9/14 and H3K27 acetylation) in the hippocampus in adulthood. (A) Fmr1 gene expression data is represented as fold changes in mRNA levels. (*p = 0.012, Water/Water vs AIE/Water; **p = 0.0047, AIE/Water vs AIE/DZ). (B,C) ChIP data is represented as fold changes in the occupancy of H3K9/14 and H3K27 acetylation of both promoter and gene body of Fmr1 (CREB binding site: *p = 0.027, Water/Water vs AIE/Water; **p = 0.0004, AIE/Water vs AIE/DZ). All values mean ± SEM and derived from n = 5–8/group.