Effect of chronic sleep restriction on ethanol preference and cortical structural plasticity

Abstract

Sleep loss is associated with a potential risk of using drugs such as cocaine, methamphetamines, and alcohol. Recently, our group showed that chronic sleep restriction (CSR) for 7 days/4 h induces a significant increase in ethanol intake and delta FosB immunoreactivity in the rat's prefrontal cortex. However, whether CSR promotes changes in structural plasticity that explain ethanol consumption is unknown. Therefore, the present study aimed to determine if CSR induces changes in the dendritic length, branching of the dendritic tree, and spine morphology of the pyramidal neurons from the prelimbic cortex and whether these structural changes are associated with ethanol consumption. For this purpose, adult male Wistar rats were divided into four experimental groups: control, CSR for 7 days/4 h daily, CSR + ethanol exposure, and ethanol exposure. The two-bottle free-choice paradigm was used to measure ethanol intake, and the gentle handling method was used for CSR. At the end of the experiment, the rats were euthanized, and their brains were dissected and processed by Golgi-Cox staining. Sholl analysis was used to characterize structural plasticity. Results show that CSR induced an increase in the ethanol index preference. In addition, ethanol intake and ethanol + CSR increased the total dendritic length, dendritic tree branching, and mushroom spines in prelimbic cortex neurons. In conclusion, changes in structural plasticity associated with CSR and continuous access to ethanol may translate into neuroadaptive changes that favor drug preference and subsequently reinforce addictive behavior.

Keywords: Dendritic spines; Ethanol intake; Neuronal plasticity; Sleep.

Graphical abstract

Fig. 1

Diagram of the experimental design. Control group (Ctrl) rats without manipulation. Chronic sleep restriction group (CSR) rats with sleep restriction for 4 hours / 7 days using gentle handling method. Ethanol consumption group (EtOH) rats were exposed to ethanol using a two-bottle free-choice drinking paradigm. EtOH + CSR group rats were exposed to the ethanol and sleep-restricted in the sixth week. After meticulous manipulations, all rats were euthanized, and their brains were processed for Golgi-Cox staining. The structural plasticity was determined using the Sholl analysis.

Fig. 2

Basal values of the ethanol intake and effect of chronic sleep restriction (CSR). Panel A shows the mean basal values of ethanol intake during the fourth- and fifth weeks vs. the sixth week in the EtOH group. Panel B shows the mean basal values of ethanol intake during the fourth- and fifth weeks vs. the sixth week in the EtOH + CSR group. Panel C shows the ethanol intake in the sixth week, observing that CSR induces a slight increase in ethanol consumption. Panel D shows the ethanol intake mean induced by CSR. Panel E shows the increase in the ethanol preference percentage due to the influence of the day variable in the EtOH + CSR group. Panel F highlights the increase in the percentage preference for ethanol-induced by CSR. Panel G shows that water intake is reduced in the EtOH + CSR vs. EtOH group during the sixth week of the experimental protocol. The values show means ± S.E.M. ∗ p < 0.05, ∗∗p <0.009, ∗∗∗ p < 0.0001. Each experimental group n = 5.

Fig. 3

Ethanol (EtOH) intake and chronic sleep restriction (CSR) increase total dendritic length in the prelimbic cortex. Panel A represents the average value of total dendritic length in the four experimental groups. Panel B shows micrographs of representative cortical neurons (red arrows) in each experimental group. The values show means ± S.E.M. ∗p <0.0001, indicating a thorough data analysis and robust results.

Fig. 4

Chronic sleep restriction (CSR) and ethanol (EtOH) affect branching order in neurons from the prelimbic cortex. Panel A shows the branching order in each experimental group, observing that EtOH + CSR significantly increases the dendritic branching of the neurons. The values indicate means ± S.E.M. Statistical differences between groups are indicated by different letters above the bars. Bars that share the same letter are not significantly different, whereas bars with different letters indicate statistically significant differences between groups. The corresponding p-values are reported in Section 3.2.2 of the manuscript. Panel B shows drawings of reconstructed pyramidal neurons from four experimental groups. Colors represent different branching orders.

Fig. 5

The total number and type of spines in neurons of the prelimbic cortex. Panel A underscores the role of ethanol (EtOH) intake and chronic sleep restriction (CSR) in increasing the total number of spines. No differences were observed between CSR compared to the control (Ctrl). The values show means ± S.E.M.∗p < 0.05; ∗∗p < 0.01. Panel B highlights that EtOH and EtOH + CSR increase the percentage of mushroom-type (M) dendritic spines. Interestingly, the percentage of the thin (T) and stubby (S) spines is reduced in EtOH and EtOH + CSR groups. Statistical differences between groups are indicated by different letters above the bars, p < 0.05. M=mushroom, T=thin, S=stubby, U=unclassified, and B=bifurcated.

Fig. 6

Serum corticosterone (CORT) levels in the experimental groups. Neither ethanol (EtOH) nor chronic sleep restriction (CSR) alone induced significant changes in serum CORT concentrations compared to the control (Ctrl) group. However, the combined EtOH + CSR treatment resulted in an increased in CORT levels. The values indicate means ± S.E.M. ∗p < 0.01.