Neonatal alcohol exposure reduces number of parvalbumin-positive interneurons in the medial prefrontal cortex and impairs passive avoidance acquisition in mice deficits not rescued from exercise

Abstract

Developmental alcohol exposure causes a host of cognitive and neuroanatomical abnormalities, one of which is impaired executive functioning resulting from medial prefrontal cortex (mPFC) damage. This study determined whether third-trimester equivalent alcohol exposure reduced the number of mPFC GABAergic parvalbumin-positive (PV+) interneurons, hypothesized to play an important role in local inhibition of the mPFC. The impact on passive avoidance learning and the therapeutic role of aerobic exercise in adulthood was also explored. Male C57BL/6J mice received either saline or 5g/kg ethanol (two doses, two hours apart) on PD 5, 7, and 9. On PD 35, animals received a running wheel or remained sedentary for 48days before behavioral testing and perfusion on PD 83. The number of PV+ interneurons was stereologically measured in three separate mPFC subregions: infralimbic, prelimbic and anterior cingulate cortices (ACC). Neonatal alcohol exposure decreased number of PV+ interneurons and volume of the ACC, but the other regions of the mPFC were spared. Alcohol impaired acquisition, but not retrieval of passive avoidance, and had no effect on motor performance on the rotarod. Exercise had no impact on PV+ cell number, mPFC volume, or acquisition of passive avoidance, but enhanced retrieval in both control and alcohol-exposed groups, and enhanced rotarod performance in the control mice. Results support the hypothesis that part of the behavioral deficits associated with developmental alcohol exposure are due to reduced PV+ interneurons in the ACC, but unfortunately exercise does not appear to be able to reverse any of these deficits.

Keywords: anterior cingulate cortex; executive function; fear; fetal alcohol spectrum disorders; inhibition; running.

Figure 1. mPFC subregion identification and staining

A) Methylene blue stained coronal mPFC section (2.5× magnification) with example tracings of the different subregions (top: anterior cingulate (ACC); middle: prelimbic (PL); bottom: infralimbic (IL)). B) mPFC PV/DAB labeled GABAergic interneurons (20× magnification).

Figure 2. Persistent impact of PD 5, 7, and 9 alcohol exposure evident in brain weight as adults

A. When measured in adulthood, the brains of mice exposed to a PD 5, 7, and 9 alcohol exposure are significantly smaller than those of their saline-exposed mice littermates. B. The negative impact of PD 5, 7, and 9 alcohol exposure on brain weight is still evident even when animals are matched for body size. All values represent mean ± SEM. *p < 0.05.

Figure 3. Wheel Running Levels

A. Data demonstrate the average running distance (km) per 24 hours of alcohol- and saline-exposed mice. Animals exposed to a PD5, 7, and 9 alcohol exposure ran significantly less than their saline-exposed littermates. B. Daily running distance (km) of alcohol-exposed and saline-exposed mice throughout the entirety of the intervention. All values represent mean ± SEM. *p < 0.05.

Figure 4. Neonatal alcohol exposure has no impact on Rotarod performance, while previous exposure to exercise enhances performance

PD 5, 7, and 9 alcohol exposure fails to impact Rotarod performance in adulthood on both Days of Rotarod testing. Running wheel access improved Rotarod performance in both saline and alcohol-exposed mice on Day 1. On Day 2, Alcohol Runners failed to show behavioral improvements on the task, while Saline Runners showed a significant improvement across days. All values represent mean ± SEM. *p < 0.05.

Figure 5. PD 5, 7, and 9 alcohol exposure impairs passive avoidance performance

A. Latency to reach criterion on Day 1. When tested in adulthood, mice exposed to a PD 5, 7, and 9 alcohol exposure required a significantly longer time to reach criterion compared to their saline-exposed littermates, regardless of Intervention. B. Latency to cross over into the dark chamber on Day 2. Mice given access to a running wheel demonstrate significantly longer latencies to cross over than their sedentary littermates, regardless of Postnatal Treatment condition. All values represent mean ± SEM. *p < 0.05, ^p < 0.001.

Figure 6. PD 5, 7, and 9 administration has long-term impact on not the overall volume, but rather distinct mPFC subregions

A. Neither PD 5, 7, and 9 alcohol exposure nor exercise impacts mPFC volume. B. PD 5, 7, and 9 alcohol exposure significantly reduces the volume of the ACC of the mPFC. Exercise has no impact on ACC volume. C. Neither PD 5, 7, and 9 alcohol exposure nor exercise impacts the volume of the PL/IL. All values represent mean ± SEM. *p < 0.05.

Figure 7. PD 5, 7, and 9 administration has long-term impact on the number of mPFC PV+ interneurons

A. PD 5, 7, and 9 alcohol exposure significantly reduces the number of GABAergic PV+ interneurons in the mPFC. Exercise has no impact on number of PV+ interneurons. B. PD 5, 7, and 9 alcohol exposure significantly reduces the number of PV+ interneurons in the ACC. No effect of exercise. C. Neither PD 5, 7, and 9 alcohol exposure nor exercise impacts the number of PV+ GABAergic interneurons in the PL/IL. All values represent mean ± SEM. *p < 0.05, ^p < 0.001.