Adolescent alcohol exposure reduces behavioral flexibility, promotes disinhibition, and increases resistance to extinction of ethanol self-administration in adulthood

Abstract

The prefrontal cortex (PFC) is a brain region that is critically involved in cognitive function and inhibitory control of behavior, and adolescence represents an important period of continued PFC development that parallels the maturation of these functions. Evidence suggests that this period of continued development of the PFC may render it especially vulnerable to environmental insults that impact PFC function in adulthood. Experimentation with alcohol typically begins during adolescence when binge-like consumption of large quantities is common. In the present study, we investigated the effects of repeated cycles of adolescent intermittent ethanol (AIE) exposure (postnatal days 28-42) by vapor inhalation on different aspects of executive functioning in the adult rat. In an operant set-shifting task, AIE-exposed rats exhibited deficits in their ability to shift their response strategy when the rules of the task changed, indicating reduced behavioral flexibility. There were no differences in progressive ratio response for the reinforcer suggesting that AIE did not alter reinforcer motivation. Examination of performance on the elevated plus maze under conditions designed to minimize stress revealed that AIE exposure enhanced the number of entries into the open arms, which may reflect either reduced anxiety and/or disinhibition of exploratory-like behavior. In rats that trained to self-administer ethanol in an operant paradigm, AIE increased resistance to extinction of ethanol-seeking behavior. This resistance to extinction was reversed by positive allosteric modulation of mGluR5 during extinction training, an effect that is thought to reflect promotion of extinction learning mechanisms within the medial PFC. Consistent with this, CDPPB was also observed to reverse the deficits in behavioral flexibility. Finally, diffusion tensor imaging with multivariate analysis of 32 brain areas revealed that while there were no differences in the total brain volume, the volume of a subgroup of regions (hippocampus, thalamus, dorsal striatum, neocortex, and hypothalamus) were significantly different in AIE-exposed adults compared with litter-matched Control rats. Taken together, these findings demonstrate that binge-like exposure to alcohol during early to middle adolescence results in deficits in PFC-mediated behavioral control in adulthood.

Figures

Figure 1

Experimental paradigm and ethanol vapor exposure model. Depicted is a schematic representation of the time line of the vapor exposure and behavioral procedures in relation to the postnatal day of the rat.

Figure 2

Characterization of the level of intoxication and BEC of the adolescent intermittent ethanol (AIE) vapor exposure procedure. (a) Comparison of the level of intoxication in rats at the end of each cycle of ethanol exposure by vapor inhalation. Immediately after removal from the vapor chambers, rats were assessed for their level of behavioral intoxication using an intoxication rating of 1–5. The target level of intoxication was a rating of 2 (slight motor impairment) to 3 (moderate motor impairment). Shown are the average±SEM of intoxication rating at each cycle of exposure. *Indicates significant decrease (p<0.05) from day 1 of cycles 1–3; # indicates significant increase (p<0.05), and ## indicates significant decrease (p<0.001) from day 1 of cycle 1–3. (b) BEC values were obtained from tail vein blood drawn immediately following chamber exposure on days 3, 7, 11, and 15. Shown on the graph are the mean±BEC for each of the four cycles. (c) Comparison of BEC levels from tail and trunk blood obtained from the same rat revealed they are highly consistent between the two compartments. (d) The level of intoxication based upon the intoxication rating scale strongly correlated with the BEC. Shown is the BEC (mean±SEM) combined from AIE cycles one and four taken at the end of day 2 of each cycle. Statistical analysis revealed a highly significant correlation of the BEC reading with the level of intoxication (p=0.001).

Figure 3

Adolescent alcohol exposure results in alterations in volume of a subset of brain regions in the adult rat assessed by diffusion tensor imaging (DTI). Volumetric measurements of 32 different regions were subjected to partial least squares discriminative analysis (PLS-DA) from which the selectivity ratio (SR) and variable importance (VIP) values were obtained. As shown in the figure, plotting the SR versus VIP resulted in a group of five brain regions that are clearly separate (boxed area). Nominal tests of VIP over its bootstrap error revealed significance (p<0.01) for all five of these brain regions. The brain areas that were included in the analysis were as follows: amygdala, anterior commissure, anterior piriform, aqueduct, brainstem, central gray, cerebellum, corpus callosum, entorhinal cortex, external capsule, fimbria, fornix, fourth ventricle, genu, hippocampus, hypothalamus, inferior colliculus, internal capsule, lateral ventral, medial prefrontal cortex, neocortex, olfactory bulb, orbital cortex, rest of forebrain, rest of midbrain, splenium, striatum, substantia nigra, superior colliculus, thalamus, third ventricle, ventral tegmental area.

Figure 4

AIE exposure results in deficits in performance on an operant set-shifting task in adulthood. (a) There were no differences between the groups in the number of trials required to reach criteria on the first rule. However, when tested for their ability to switch to the new rule (location rule), AIE-exposed rats required more trials to reach criterion. (b) The increase in the number of trials required to shift to the new rule was associated with a significant increase in the number of total errors. (c) Control and AIE-exposed rats showed equal reinforcer motivation when tested on a progressive ratio responding procedure. (*p<0.05 vs Control; Control, n=13; AIE, n=14).

Figure 5

Administration of CDPPB immediately prior to testing in the operant set-shifting task attenuates AIE exposure-induced deficits in behavioral flexibility in adulthood. (a) There were no differences between the groups in the number of trials required to reach criteria on the first (visual) rule (all rats learned to perform this task within 30 trials). When tested for their ability to switch to the new rule (location), AIE+vehicle-treated rats required more trials to reach criterion (*p<0.05 compared with all other groups). Treatment with CDPPB prior to the set-shift significantly reduced the number of trials required for criterion in AIE-exposed rats to a level similar to Control rats (#p<0.05). (b) Although AIE+saline rats displayed more perseverative errors compared with all other groups, these increases were not significantly different and there were no differences among the groups in total regressive errors during the set-shift procedure (AIE and Controls, n=10).

Figure 6

Adult AIE-exposed rats exhibit reduced anxiety and/or disinhibitory-like behavior on the elevated plus maze. (a) AIE exposure resulted in a significant increase in percent time in the open arms compared with Controls (*p<0.05 vs Control). (b) There was also a significant increase in the percent entries into the open arms in AIE-exposed rats (*p<0.05 vs Control). (c) There were no differences in the total number of arm entries between AIE and Control rats (p>0.05; Control, n=20; AIE, n=22).

Figure 7

Exposure to AIE results in increased ethanol self-administration in adulthood. (a) Adult rats exposed to AIE showed both an increase in active lever responding for ethanol and ethanol reinforcements (*p<0.05 vs Control). (b) In spite of the greater number of ethanol reinforcements delivered, the BEC measured at the end of the operant session were not different. (c) While not statistically significant, there was a clear trend (p=0.08) toward front loading of ethanol during the first 3 min of the self-administration session in AIE-exposed rats compared with Control rats.

Figure 8

Exposure to AIE results in increased resistance to the extinction of ethanol-seeking behavior in adulthood. (a) Rats exposed to AIE displayed an increased resistance to extinction as evidenced by increased responding on the previously active lever on multiple days of extinction (*p<0.05 vs all other groups). Treatment with CDPPB prior to each extinction session attenuated the increased resistance to extinction observed in AIE-exposed rats and facilitated extinction in Control rats (#p<0.05 vs Control). (b) AIE exposure increased the number of trials required to reach extinction criteria (*p<0.05 vs Control). Treatment with CDPPB prior to extinction training attenuated the increased resistance to extinction in AIE rats (**p<0.05 vs AIE) and reduced the number of sessions required for extinction in Control rats (#p<0.05 vs Control).