Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice

Abstract

Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical role of mTOR in both autophagy and synaptic plasticity in the developing brain, the present study aims to evaluate whether binge ethanol treatment in adolescence would induce dysfunctions in synaptic plasticity and cognitive functions and if mTOR inhibition with rapamycin is capable of restoring both effects. Using C57BL/6 adolescent female and male mice (PND30) treated with ethanol (3 g/kg) on two consecutive days at 48-hour intervals over 2 weeks, we show that binge ethanol treatment alters the density and morphology of dendritic spines, effects that are associated with learning and memory impairments and changes in the levels of both transcription factor CREB phosphorylation and miRNAs. Rapamycin administration (3 mg/kg) prior to ethanol administration restores ethanol-induced changes in both plasticity and behavior dysfunctions in adolescent mice. These results support the critical role of mTOR/autophagy dysfunctions in the dendritic spines alterations and cognitive alterations induced by binge alcohol in adolescence.

Keywords: adolescence; autophagy; binge ethanol treatment; cognitive function; dendritic spines; mTOR; synaptic pruning.

Figure 1

Rapamycin restores the ethanol‐induced impairment of mTOR phosphorylation in the hippocampus of adolescent female and male mice. The immunoblot analysis and the quantification of mTOR phosphorylation (p‐mTOR) in the hippocampus of PND44 female and male animals intermittently treated with saline, rapamycin, ethanol or rapamycin plus ethanol. Figure S2 includes the full‐length Coomassie blue staining as the loading control. A representative immunoblot is shown. Values represent mean ± SEM, n = 6 mice/group. **P < 0.01, compared to their respective saline‐treated group; ^### P < 0.001 compared to their respective ethanol‐treated group.

Figure 2

Illustration of the brain area and spine classification, used to study Dil staining. (A) In the top panel, a schematic of brain slices is shown at distances of 2.80, 2.46 and 2.06 mm from the bregma. The green line denotes the granule cells layer (GCL) of the dentate gyrus of the hippocampus, where images were taken. (B) A representative image is provided from a granule cell in the dentate gyrus of the hippocampus. (C) Illustration represents a common morphological classification of dendritic spines. The spines with a head/neck diameter ratio above 1.1 µm are considered thin or mushroom. The spines that do not meet the neck ratio value and have a length to spine to head diameter above 2.5 µm are classified as thin, otherwise as stubby. The spines that meet the neck ratio value and have a head diameter that equals or exceeds 0.35 µm are labeled as mushroom, otherwise as stubby. (D) The NeuronStudio process used previously to analyze the spine morphology.

Figure 3

Rapamycin restores ethanol‐induced alterations in the spine morphology of the dentate gyrus of granule cells in the hippocampus of adolescent female and male mice treated with ethanol. Representative images of the Dil stain on the medial granular dendrites of each group are shown. Arrowheads indicate thin spines in female mice and arrows indicate stubby spines in male mice. Graph bars denote the quantification of spine density in each spine type and in the total amount of spines (spines/µm). Data represent mean ± SEM, n = 6 mice/group. *P < 0.05, ****P < 0.0001 compared to their respective saline‐treated group; ^# P < 0.05, ^## P < 0.01, ^#### P < 0.0001 compared to their respective ethanol‐treated group. Scale bar = 1 µm.

Figure 4

Rapamycin restores ethanol‐induced cognitive dysfunction in adolescent female and male mice. (A) Bars represent the mean (± SEM, n = 12 mice/group) of the time to reach the goal in the difficult and easy mazes in the Hebb–Williams mazes. **P < 0.01 compared to their respective saline‐treated group. ^## P < 0.01 compared to their respective ethanol‐treated group. (B) Bars represent the mean (± SEM, n = 12 mice/group) of the discrimination index during the novel object recognition task. ***P < 0.001 compared to their respective saline‐treated group. ^### P < 0.01 compared to their respective ethanol‐treated group. (C) Bars represent the time taken to enter the dark compartment of the passive avoidance test during the training and test sessions (24 h and 72 h after training). Data are presented as mean (± SEM), n = 12 mice/group. **P < 0.01 compared to ethanol‐treated groups and their control counterparts. ^## P < 0.01 compared to rapamycin‐treated groups and their control counterparts.

Figure 5

Rapamycin restores the ethanol‐induced impairment of transcription factor CREB in female and male mice. The immunoblot analysis and quantification of p‐CREB, p‐ERK and p‐Akt in the hippocampus lysates of the PND44 female and male animals intermittently treated with saline, rapamycin, ethanol or rapamycin plus ethanol. Figure S2 includes the full‐length Coomassie blue staining as the loading control. A representative immunoblot of each protein is shown. Values represent mean ± SEM, n = 6 mice/group. *P < 0.05 compared to their respective saline‐treated group. ^# P < 0.05 compared to their respective ethanol‐treated group.

Figure 6

Rapamycin is able to restore the differential levels of miRNAs induced by binge‐like ethanol treatment in adolescent female and male mice. Bars represent mean ± SEM, n = 6. *P < 0.05, **P < 0.01, ***P < 0.0001 compared to their respective saline‐treated group. ^# P < 0.05 compared to their respective ethanol‐treated group. ⁺⁺ P < 0.01, ⁺⁺⁺⁺ P < 0.0001 sex or interaction differences between females and males.