Chronic intermittent ethanol during adolescence and adulthood alters dendritic spines in the primary motor and visual cortex in rats

Abstract

Prolonged adolescent binge drinking can disrupt sleep quality and increase the likelihood of alcohol-induced sleep disruptions in young adulthood in rodents and in humans. Striking changes in spine density and morphology have been seen in many cortical and subcortical regions after adolescent alcohol exposure in rats. However, there is little known about the impact of alcohol exposure on dendritic spines in the same motor and sensory cortices that EEG sleep is typically recorded from in rats. The aim of this study is to investigate whether an established model of chronic intermittent ethanol vapor in rats that has been demonstrated to disrupt sleep during adolescence or adulthood, also significantly alters cortical dendritic spine density and morphology. To this end, adolescent and adult Wistar rats were exposed to 5 weeks of ethanol vapor or control air exposure. After a 13-day withdrawal, primary motor cortex (M1) and primary/secondary visual cortex (V1/V2) layer V dendrites were analyzed for differences in spine density and morphology. Spines were classified into four categories (stubby, long, filopodia, and mushroom) based on the spine length and the width of the spine head and neck. The main results indicate an age-specific effect of adolescent intermittent ethanol exposure decreasing spine density in the M1 cortex compared to age-matched controls. Reductions in the density of M1 long-shaped spine subclassifications were seen in adolescent ethanol-exposed rats, but not adult-exposed rats, compared to their air-controls. Irrespective of age, there was an overall reduction produced by ethanol exposure on the density of filopodia and the length of long-shaped spines in V1/V2 cortex as compared to their air-exposed controls. Together, these data add to growing evidence that some cortical circuits are vulnerable to the effects of alcohol during adolescence and begin to elucidate potential mechanisms that may influence brain plasticity following early alcohol use.

Keywords: Adolescence; Alcohol; Dendrites; Motor cortex; Sleep; Spines; Visual cortex.

Figure 1.

(A) Representative fluorescent labeling of a dendritic section in the visual cortex acquired using a super-resolution confocal microscope. (B) Imaris software was used to generate a 3D reconstruction of the dendritic shaft and spines from the fluorescent signal using the Filament function. An example of a dendritic section from the white box in Panel A shows the Imaris Filament and the four distinct morphological subtypes of dendritic protrusions: mushroom spines, long spines, stubby spines, and filopodia.

Figure 2.

Effects of ethanol vapor exposure in adolescent (AIE) and adult (CIE) rats on dendrite diameter and total dendritic spine density in primary motor (M1) cortical neurons. (A) Quantitation of total dendrite spine density and (B) dendrite diameter in M1 cortex from AIE and CIE rats or age-matched control. Ethanol exposure decreased M1 spine density in AIE rats compared to air-control rats, an effect not seen in CIE rats. Irrespective of exposure, M1 dendritic diameter was significantly increased in the adult rat group compared to adolescent group. **p > 0.05 compared to age-matched control, # p > 0.05 main effect of age.

Figure 3.

Adaptations in dendritic morphology in M1 cortical neurons after almost two weeks of withdrawal from ethanol vapor in adolescent (AIE) and adult (CIE) rats or age-matched air-control exposure. Panel (A) depicts total dendrite spine density for mushroom, long, stubby and filopodia in primary motor cortex of AIE and CIE rats or air-control. The density of long spines in M1 was significantly reduced in AIE rats compared to age-matched controls. Quantification of M1 spine (B) diameter (C) length (D) neck length and (E) neck diameter resulted in a significant increase in the long spine subtype of CIE and AIE rats. Irrespective of age, ethanol exposure significantly increased stubby spine neck diameter compared to air-control. (F) M1 spine volume was not impacted by age or exposure group. ***p > 0.001 compared to age-matched control, # p > 0.05 main effect of age.

Figure 4.

Effects of ethanol vapor exposure in adolescent (AIE) and adult (CIE) rats on dendrites and total dendritic spine density in adult V1/V2 cortical neurons. Quantitation of total (A) dendrite spine density and (B) dendrite diameter in AIE and CIE rats or age-matched control. Decreases in V1/V2 spine density were seen in CIE rats compared to air-controls, an effect not seen in AIE rats. **p > 0.05 compared to age-matched control.

Figure 5.

Adaptations in dendritic morphology in V1/V2 cortical neurons after withdrawal from ethanol vapor in adolescent (AIE) and adults (CIE) or air-control exposure. Figure depicts quantification of mushroom, long, stubby, and filopodia spine subtypes. Quantification of (A) spine density in V1/V2 resulted in a significant decrease in filopodia density in ethanol vapor rats compared to air-control. There was also an age-dependent effect in subby spines with a reduction in density in adult compared to adolescent exposed rats. (B) V1/V2 diameter of the spines and (E) diameter of the spine neck subtypes did not result in any differences. However, differences were seen in (C) spine length and (D) spine neck length subclassifications. Length of spine and spine neck for the long subclass were longer in adult rats compared to adolescent. There was a significant decrease in long spine length for ethanol animals compared to controls. Additionally, there is a main effect of age with greater long spine length in adult compared to adolescent rats. (E) Age of group did impact stubby spine volume with deceases with age. **p > 0.05 compared to age-matched control, # p > 0.05 main effect of age.