Abstinence and Fear Experienced during This Period Produce Distinct Cortical and Hippocampal Adaptations in Alcohol-Dependent Rats

Abstract

Previous studies demonstrate that ethanol dependence induced by repeating cycles of chronic intermittent ethanol vapor exposure (CIE) followed by protracted abstinence produces significant gray matter damage via myelin dysfunction in the rodent medial prefrontal cortex (mPFC) and alterations in neuronal excitability in the mPFC and the dentate gyrus (DG) of the hippocampus. Specifically, abstinence-induced neuroadaptations have been associated with persistent elevated relapse to drinking. The current study evaluated the effects of forced abstinence for 1 day (d), 7 d, 21 d, and 42 d following seven weeks of CIE on synaptic plasticity proteins in the mPFC and DG. Immunoblotting revealed reduced expression of CaMKII in the mPFC and enhanced expression of GABA_A and CaMKII in the DG at the 21 d time point, and the expression of the ratio of GluN2A/2B subunits did not change at any of the time points studied. Furthermore, cognitive performance via Pavlovian trace fear conditioning (TFC) was evaluated in 3 d abstinent rats, as this time point is associated with negative affect. In addition, the expression of the ratio of GluN2A/2B subunits and a 3D structural analysis of neurons in the mPFC and DG were evaluated in 3 d abstinent rats. Behavioral analysis revealed faster acquisition of fear responses and reduced retrieval of fear memories in CIE rats compared to controls. TFC produced hyperplasticity of pyramidal neurons in the mPFC under control conditions and this effect was not evident or blunted in abstinent rats. Neurons in the DG were unaltered. TFC enhanced the GluN2A/2B ratio in the mPFC and reduced the ratio in the DG and was not altered by abstinence. These findings indicate that forced abstinence from CIE produces distinct and divergent alterations in plasticity proteins in the mPFC and DG. Fear learning-induced changes in structural plasticity and proteins contributing to it were more profound in the mPFC during forced abstinence.

Keywords: GluN; Golgi–Cox; alcohol dependence; dentate gyrus; prefrontal cortex; trace fear conditioning.

Figure 1

Abstinence-induced alterations in plasticity-related proteins in the mPFC and DG of the hippocampus. (a) Schematic of CIE weeks and time points during abstinence when brain tissue was harvested. (b,c) Coronal section of the rat brain indicating areas of tissue collected for immunoblotting analysis ((b), mPFC +3.7 from bregma; (c), DG −4.3 from bregma). (d) Representative immunoblots from all experimental groups in mPFC-enriched tissue. Coomassie was used as a loading control. (e,f) Quantitative data (mean ± SE) indicated as dot plots of GABA_A, pCaMKII, tCaMKII, GluN2A/2B in the mPFC (e) and DG (f) indicated as percent change from the control condition. The control condition is indicated as a dashed line at 100%; * p < 0.05 vs. control condition. PND, postnatal day; AB, abstinence.

Figure 2

Abstinence alters the retrieval of trace fear memories. (a) Experimental groups with a timeline of CIE and TFC or sham conditioning. (b,c) Schematic of the TFC procedure, indicating details of the training during acquisition (b) and retrieval (c). Three days after the cessation of CIE, age-matched controls (CIE naïve) and CIE rats were trained on TFC (e,f), and 24 h after training, all animals were tested for CS retrieval (g). Freezing was monitored during the 3 min baseline session and during CS and trace intervals (d). (d) Quantitative data indicating freezing behavior during baseline. (e,f) Quantitative data indicating percent change in freezing from baseline during tone (e) and trace (f) periods. (g) Quantitative data indicating freezing behavior during retrieval. * p < 0.05 vs. control; * p < 0.05 vs. CIE. Data shown are represented as mean ± SEM. Control, n = 7; CIE, n = 5; control + TFC, n = 9; CIE + TFC, n = 12. CIE, chronic intermittent ethanol exposure; TFC, trace fear conditioning; CS, conditioned stimulus; US, unconditioned stimulus; FS, foot shock; ITI, intertrial interval; AB, abstinence; d, days; h, hours; m, minutes; s, seconds.

Figure 3

Abstinence and TFC produce distinct alterations in the structural plasticity of pyramidal neurons. (a) Example tracing of a pyramidal neuron with 3D sholl ring. Arrowhead points to apical dendrites and thin arrow points to basal dendrites. (b,c) Quantitative analysis of basal (b) and apical (c) dendrites plotted as the number of intersections on the x-axis and the distance from the soma on the y-axis. (d) Example tracing of a granule cell neuron in the DG with 3D sholl ring. Arrowhead points to apical dendrites. (e) Quantitative analysis of apical dendrites plotted as the number of intersections on the x-axis and the distance from the soma on the y-axis. PN, pyramidal neurons; GCN, granule cell neurons; TFC, trace fear conditioning. Signs after the vertical line in (b,c)—p < 0.05 (#) interaction, ($) main effect of distance, and (*) main effect of groups by ANOVA. Data shown are represented as mean ± SEM. Control, n = 4; CIE, n = 3; control + TFC, n = 4; CIE + TFC, n = 4.

Figure 4

GluN2A/2B ratio is differentially altered in the mPFC and DG by TFC. (a) Representative immunoblots of GluN2A and GluN2B from all experimental groups in mPFC-enriched tissue. (b,c) Density of GluN2A/2B expression in mPFC (b) and DG (c). Main effect of TFC by ANOVA followed by post hoc tests is indicated with lines. Data shown are represented as mean ± SEM. Control, n = 7; CIE, n = 5; control + TFC, n = 9; CIE + TFC, n = 12.