Adolescent binge ethanol exposure alters specific forebrain cholinergic cell populations and leads to selective functional deficits in the prefrontal cortex

Abstract

Adolescence has been identified as a vulnerable developmental time period during which exposure to drugs can have long-lasting, detrimental effects. Although adolescent binge-like ethanol (EtOH) exposure leads to a significant reduction in forebrain cholinergic neurons, EtOH's functional effect on acetylcholine (ACh) release during behavior has yet to be examined. Using an adolescent intermittent ethanol exposure model (AIE), rats were exposed to binge-like levels of EtOH from postnatal days (PD) 25 to 55. Three weeks following the final EtOH exposure, cholinergic functioning was assessed during a spontaneous alternation protocol. During maze testing, ACh levels increased in both the hippocampus and prefrontal cortex. However, selectively in the prefrontal cortex, AIE rats displayed reduced levels of behaviorally relevant ACh efflux. We found no treatment differences in spatial exploration, spatial learning, spatial reversal, or novel object recognition. In contrast, AIE rats were impaired during the first attentional set shift on an operant set-shifting task, indicative of an EtOH-mediated deficit in cognitive flexibility. A unique pattern of cholinergic cell loss was observed in the basal forebrain following AIE: Within the medial septum/diagonal band there was a selective loss (30%) of choline acetyltransferase (ChAT)-positive neurons that were nestin negative (ChAT+/nestin-); whereas in the Nucleus basalis of Meynert (NbM) there was a selective reduction (50%) in ChAT+/nestin+. These results indicate that early adolescent binge EtOH exposure leads to a long-lasting frontocortical functional cholinergic deficit, driven by a loss of ChAT+/nestin+ neurons in the NbM, which was associated with impaired cognitive flexibility during adulthood.

Fig. 1. Adolescent Intermittent Ethanol (AIE) Exposure protocol

Schematic outlining the age range, AIE exposure protocol, and the timeline for cannula implantation surgery, behavioral testing and tissue collection (IHC: immunohistochemistry). Blood ethanol concentrations (BECs) following the first (1) and eighth (8) intraoral gastric gavage are illustrated in the chart insert. BECs did not significantly differ between the first and eighth gavage during AIE treatment.

Fig. 2. AIE leads to blunted acetylcholine (ACh) efflux in the prefrontal cortex (PFC) during spontaneous alternation, but does not impair alternation behavior

(A) Average percent alternation scores by AIE treatment. There was no effect of AIE on alternation behavior. (B) Arm entries during spontaneous alternation. There was no significant difference in activity between AIE and control groups. (C) Hippocampal ACh efflux before (baseline), during (maze) and after spontaneous alternation behavior (post maze). There was a main effect of phase, wherein ACh efflux significantly increased during maze behavior compared to baseline and post maze levels (p<0.05). AIE did not affect ACh efflux in the hippocampus. (D) Prefrontal cortical ACh efflux measured during baseline, maze behavior and post maze. There was a main effect of phase, with ACh levels increasing during maze behavior (p<0.05). There was also a significant interaction between AIE treatment and phase: AIE blunted the rise in ACh efflux during maze behavior in the PFC (T; p<0.05).

Fig. 3. Cannulae implant location

Schematic demonstrating the typical location of cannula implantation (left panels: cresyl violet stain) for the prefrontal cortex (A) and hippocampus (B). The black dot on the right panels illustrate the target location for the end of the cannula (the probes extended 2 mm for the frontal cortex and 3 mm in the hippocampus). Right panel diagrams are taken from (Paxinos and Watson, 2013).

Fig. 4. Cell Counting Regions of Interest

Schematic demonstrating representative cell counting contours for the medial septum/diagonal band (A), which began at 1.6 mm relative to Bregma and extended through −0.4 mm relative to Bregma. The light grey contours outline the medial septum, the dark grey outline denote the vertical limb of the diagonal band and the grey countours outline the horizontal limb of the diagonal band. Panel (B) illustrates the contours made for cell counts in the Nucleus basalis of Mynert, which extended from −0.96 mm to −2.64 mm relative to Bregna. All diagrams are taken from (Paxinos and Watson, 2013).

Fig. 5. AIE does not impair novel object recognition or Barnes maze behavior

(A) Discrimination index (novel-familiar/novel+familiar) for the 24-hour and 7- day retention test. AIE did not significantly impair recognition memory at either delay. At 24-hours, both experimental groups spent more time exploring the novel object compared to the familiar object (*; p<0.05), an effect not seen at the 7-day retention test (with the discrimination index, a score of zero denotes no object preference). (B) Latency to reach the goal box during Barnes maze testing. Both experimental groups learned the spatial location of the goal box across 11 training trials. All rats took significantly longer to reach the new location of the goal box following the first reversal (*; p<0.05), an effect not seen following the second reversal. AIE did not significantly alter latency results during training or reversals.

Fig. 6. AIE leads to an impairment in attentional set shifting

(A) Average trials required to reach criterion (10 consecutive presses on the rewarded lever) during operant discriminations. AIE led to a significant increase in the number of trials required to reach criterion on the first set shift (response to cued discrimination; T; p<0.05). AIE did not affect rule learning, additional set shifts or a response reversal. All rats required significantly more trials to reach criterion after the response reversal (R; p<0.05). (B) Average errors (responding to the non- rewarded lever) committed during operant set shifting. AIE led to a significant increase in errors following the first set shift (response to cued discrimination; T; p<0.05). AIE did not affect rule learning, additional set shifts or a response reversal. All rats committed significantly more errors following the response reversal (R; p<0.05). (C) Average lever presses during operant set shifting. There were no significant group differences in lever pressing activity.

Fig. 7. AIE leads to decreased choline acetyltransferase positive and nestin negative (ChAT+/ nestin−) cell populations and smaller ChAT+/ nestin+ soma areas in the medial septum/diagonal band (MS/DB)

(A) Estimated total number of ChAT+/ nestin− and ChAT+/ nestin+ cells in the MS/DB. AIE lead to a significant reduction in the estimated ChAT+/ nestin− cell population and did not affect the ChAT+/ nestin+ cell population in the MS/DB (T; p<0.05). (B) Number of cells (per mm²) in the MS/DB. AIE leads to a significant reduction in the total number of immunoreactive cells in the MS/DB (T; p<0.05), and a significant reduction in the number of ChAT+/ nestin− cells (T; p<0.05). AIE did not affect the number of ChAT+/ nestin+ cells. (C) Average soma area of ChAT+/ nestin− and ChAT+/ nestin+ cells in the MS/DB. AIE leads to a significant reduction in the soma area of ChAT+/ nestin+ cells in the MS/DB (T; p<0.05). ChAT+/ nestin+ soma areas were significantly larger compared to ChAT+/ nestin− soma areas in control rats (*; p<0.05). (D) Image depicting ChAT and nestin immunohistological stain at 40x magnification in the MS/DB. Representative ChAT+/ nestin− (^) cells are stained red and ChAT+/ nestin+ (*) cells are stained purple.

Fig. 8. AIE leads to decreased ChAT+/ nestin+ cell populations in the Nucleus basalis of Meynert (NbM)

(A) Estimated total number of ChAT+/ nestin− and ChAT+/ nestin+ cells in the NbM. AIE significantly decreased the ChAT+/ nestin+ cell population in the NbM (T; p<0.05). There was a trend for AIE to decrease the estimated ChAT+/ nestin− cell population (t; p=0.08). (B) Number of cells (per mm²) in the NbM. AIE leads to a significant reduction in the total number of cholinergic cells in the NbM (T; p<0.05), and a significant reduction in the number of ChAT+/ nestin+ cells (T; p<0.05). There was a trend in the reduction of ChAT+/ nestin− cells per mm² as a result of AIE (t; p=.06). (C) Average soma area of ChAT+/ nestin− and ChAT+/ nestin+ cells in the NbM. AIE did not +significantly affect soma area in either cell population.