Executive functioning-specific behavioral impairments in a rat model of human third trimester binge drinking implicate prefrontal-thalamo-hippocampal circuitry in Fetal Alcohol Spectrum Disorders

Abstract

Individuals diagnosed with Fetal Alcohol Spectrum Disorders (FASD) often display behavioral impairments in executive functioning (EF). Specifically, the domains of working memory, inhibition, and set shifting are frequently impacted by prenatal alcohol exposure. Coordination between prefrontal cortex and hippocampus appear to be essential for these domains of executive functioning. The current study uses a rodent model of human third-trimester binge drinking to identify the extent of persistent executive functioning deficits following developmental alcohol by using a behavioral battery of hippocampus- and prefrontal cortex-dependent behavioral assays in adulthood. Alcohol added to milk formula was administered to Long Evans rat pups on postnatal days 4-9 (5.25 g/kg/day of ethanol; intragastric intubation), a period when rodent brain development undergoes comparable processes to human third-trimester neurodevelopment. Procedural control animals underwent sham intubation, without administration of any liquids (i.e., alcohol, milk solution). In adulthood, male rats were run on a battery of behavioral assays: novel object recognition, object-in-place associative memory, spontaneous alternation, and behavioral flexibility tasks. Alcohol-exposed rats demonstrated behavioral impairment in object-in-place preference and performed worse when the rule was switched on a plus maze task. All rats showed similar levels of novel object recognition, spontaneous alternation, discrimination learning, and reversal learning, suggesting alcohol-induced behavioral alterations are selective to executive functioning domains of spatial working memory and set-shifting in this widely-utilized rodent model. These specific behavioral alterations support the hypothesis that behavioral impairments in EF following prenatal alcohol exposure are caused by distributed damage to the prefrontal-thalamo-hippocampal circuit consisting of the medial prefrontal cortex, thalamic nucleus reuniens, and CA1 of hippocampus.

Keywords: Development; Fetal alcohol spectrum disorders; Set-shifting; Spatial working memory.

Fig 1:. Experimental timeline for all manipulations.

While litters initially contained both male and female rat pups, all behavioral assays were performed on male Long Evans rats. The number in parentheses below each behavioral task on the timeline indicates the number of days over which the task occurred.

Fig 2:. Schematic Diagram illustrating Behavioral Flexibility task progression.

Start arms are labeled with an “S”. Goal arms are labeled with a “G”. The rewarded goal arm is indicated with a shaded arrow, with the curved path illustrating the path from start point (black circle) to the reward.

Fig 3:. Rats with higher BACs on PD4 show lower levels of novel object preference.

SI and AE groups did not differ in the total amount of time exploring objects in either the sample, or first minute of the probe phase (3A). Both SI and AE groups did display preference for a novel object, and discrimination index (DI) between postnatal treatments did not significantly differ (3B). The x-axis in 3B indicates a “chance” DI of 0 (similar amounts of time exploring both the novel and familiar object). Data are presented as bars representing mean ± SEM, with sample size in parentheses either within the panel 3A legend, or on the x-axis in panel 3B. Grey bars represent the SI procedural control group, while white bars represent the AE experimental group. # p ≤ 0.050 relative to chance (DI of 0.0)

Fig 4:. PD 4–9 alcohol exposure eliminates moved object preference.

SI and AE groups did not differ in the total amount of time exploring objects in either the sample, or first minute of the probe phase (4A). There was no significant preference for the objects that would be moved during the sample phase, prior to moving (4B). The SI group did display preference for moved objects, while the AE group did not, although the SI and AE groups did not significantly differ in their preference for moved objects (4C). The dotted lines in 4B and 4C indicate a “chance” DI of 0 (similar amounts of time exploring both the novel and familiar object). Blood alcohol content (BAC) at 90 minutes following alcohol administration on PD4 showed a strong negative correlation with discrimination index during the OIP task. Data in 4A, 4B, and 4C are presented as bars representing mean ± SEM, with sample size in parentheses within the panel 4A legend, or on the x-axis in panels 4B and 4C. Grey bars represent the SI procedural control group, while white bars represent the AE experimental group. Individual data points are plotted in 4D, while the dashed line indicates the line-of-best-fit for the linear correlation. #p ≤ 0.050 relative to chance (DI of 0.0), n.s. = p>0.050 relative to chance (DI of 0.0)

Fig 5:. PD 4–9 alcohol exposure did not alter alternation or activity patterns beyond control levels in a plus-maze spontaneous alternation task in males.

Alternation score did not significantly differ between AE and SI groups (5A). Total number of arm entries did not significantly differ between AE and SI groups (5B). Bars represent mean ± SEM, with sample sizes in parentheses next to group name on the x-axis. The dotted line in 5A represents chance alternation (9.4%). Grey bars represent the SI procedural control group, while white bars represent the AE experimental group.

Fig 6:. AE males manifest deficits in behavioral flexibility through impaired rule switching.

AE group required approximately twice the number of trials that the SI group required to learn a rule switch (RS) (6A). This was due to an increase in the number of successful trials during the RS (6B), but no difference in the number of errors (6C). There were no significant differences on any of these measures for the initial discrimination (Disc), first reversal (Rev1), second reversal (Rev2), or post-rule switch reversal (Rev3). Bars represent mean ± SEM, with sample sizes in parentheses within the panel 6A legend. Grey bars represent the SI procedural control group, while white bars represent the AE experimental group. *p ≤ 0.050