Binge Ethanol Effects on Prefrontal Cortex Neurons, Spatial Working Memory and Task-Induced Neuronal Activation in Male and Female Rats

Abstract

Excessive alcohol intake is associated with a multitude of health risks, especially for women. Recent studies in animal models indicate that the female brain is more negatively affected by alcohol, compared to the male brain. Among other regions, excessive alcohol consumption damages the frontal cortex, an area important for many functions and decision making of daily life. The objective of the present study was to determine whether the medial prefrontal cortex (mPFC) in female rats is selectively vulnerable to alcohol-induced damage. In humans, loss of prefrontal grey matter resulting from heavy alcohol consumption has been documented, however this volume loss is not necessarily due to a decrease in the number of neurons. We therefore quantified both number and nuclear volume of mPFC neurons following binge alcohol, as well as performance and neuronal activation during a prefrontal-dependent behavioral task. Adult male and female Long-Evans rats were assigned to binge or control groups and exposed to ethanol using a well-established 4-day model of alcohol-induced neurodegeneration. Both males and females had significantly smaller average neuronal nuclei volumes than their respective control groups immediately following alcohol binge, but neither sex showed a decrease in neuron number. Binged rats of both sexes initially showed spatial working memory deficits. Although they eventually achieved control performance, binged rats of both sexes showed increased c-Fos labeling in the mPFC during rewarded alternation, suggesting decreased neural efficiency. Overall, our results substantiate prior evidence indicating that the frontal cortex is vulnerable to alcohol, but also indicate that sex-specific vulnerability to alcohol may be brain region-dependent.

1.0 Introduction

Excessive alcohol intake contributes to at least 30 different diseases affecting nearly all systems of the body¹. Globally, among people ages 15–49, alcohol use is the leading risk factor for premature death and disability². In addition to the well-known short-term health risks, excessive alcohol use can also lead to the development of serious problems such as heart disease, stroke, cancer, cognitive impairment, depression, anxiety, unemployment, social problems, alcohol dependence, and brain damage³. For women especially, there is an increased risk of negative consequences from alcohol consumption, including alcohol-related incident fatalities, heart disease, stroke, suicide, liver cirrhosis, and death⁴. Although women tend to consume less alcohol and have a shorter duration of intake than men, long term heavy consumption is more likely to harm a woman’s health and, potentially, damage the brain⁴. Both human and animal studies suggest that females may be uniquely vulnerable to the harmful neurological effects of alcohol^5–12, although not all studies show this^13,14.

Using a rodent model of alcohol-induced neurodegeneration, we have recently shown sex differences in hippocampal damage and cognitive impairment¹⁵. Like the hippocampus, the medial prefrontal cortex (mPFC) is alcohol-vulnerable, and loss of prefrontal grey matter has been well-documented in human alcoholics^16–18. Some volume recovery occurs with abstinence^19,20, and this combined with evidence showing that volume loss is not equivalent to neuronal loss^21,22, suggests that the primary issue is with cell shrinkage, not loss. In the present study, we used archived tissue from our study of sex differences in alcohol-induced hippocampal damage and quantified the number and volume of neuron nuclei in the mPFC. The karyoplasmic ratio, or the ratio between cell body and nucleus volume, is maintained at a constant value within the cell ^23–25. Therefore, we hypothesized that binged female rats, but not male, would show a decrease in nuclear volume in the mPFC when compared to same-sex controls.

We have also recently shown that sex differences in hippocampal damage after binge alcohol are associated with spatial learning impairment in female rats, but not males¹⁵. This finding prompted us to investigate whether binge alcohol would result in sex differences in a prefrontal cortex-mediated cognitive task. The frontal cortex is critical for a wide variety of tasks, including motor function, emotional processing, problem solving, spontaneity, memory, language, spatial orientation, judgment, impulse control, working memory, and social behavior^26–28. To test for sex differences in mPFC functioning following alcohol exposure, we tested rewarded alternation (RA) performance with a short inter-trial interval to target working memory²⁹, in a second cohort of male and female rats. We predicted that binged females, but not males, would show impairment compared to same-sex controls.

Cognitive efficiency is another consideration when evaluating alcohol-related damage. Even in the absence of observed cognitive or behavioral deficits, brain damage may still have occurred, as is evident in other forms of brain injury like asymptomatic traumatic brain injury or stroke^30–32. Increased frontal activation has been observed in human frequent binge drinkers during tests of working memory³³, cognitive control³⁴, and spatial attention³⁵, indicating the recruitment of more neuronal resources to achieve behavioral performance comparable to controls. The neural efficiency hypothesis is that more intelligent individuals require less neural activation to achieve the same performance outcome^36,37. Following this line of logic, increased neuronal activation during a cognitive task would be indicative of decreased efficiency. In the present study, we analyzed activation of mPFC neurons in animals that underwent the RA task. It was predicted that binged females, but not males, would show increased c-Fos expression compared to control animals during the rewarded alternation test, indicating decreased cognitive efficiency resulting from binge alcohol.

2.0 Material and Methods

2.1 Animals

All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Houston (#14-013). To assess mPFC neuron number and volume directly after binge alcohol, archived tissue from 27 male and female rats (8 female binge, 6 female control, 7 male binge, 6 male control) from a prior study¹⁵ was used (Cohort 1). To probe for sex differences in binge alcohol effects on spatial working memory, mPFC neuronal number and volume and task-induced activation, 39 male and female rats (9 female binge, 10 female control, 10 male binge, 10 male control) were used (Cohort 2). Except where noted, procedures were identical between the cohorts.

Upon arrival (from Harlan Sprague Dawley, Indianapolis, IN, USA) at 8 weeks of age, rats spent one week acclimating to vivarium conditions, which included same-sex housing (2–3 per cage) in clear Plexiglas cages, ad libitum rat chow and water and a reversed light/dark cycle (lights off at 9:00/on at 21:00). During the acclimation period, rats were handled to make them accustomed to the experimenters. Next, rats were randomly assigned to the following groups: male control, male binge, female control and female binge. Rats then underwent 4 days of binge alcohol (or control diet) and were either sacrificed the day the binge ended (Cohort 1), or following 4 days of abstinence and 5 days of rewarded alternation testing (Cohort 2).

2.2 Binge Ethanol Administration

Food was removed from both control and experimental groups prior to the first dose of ethanol or control solution and replaced after the final dose, although water was always available. Animals received either the ethanol diet (25% ethanol w/v in nutritionally complete diet; vanilla Ensure Plus^™) or isocaloric control diet (dextrose in vanilla Ensure Plus^™) given every 8 hours over 4 days via intragastric gavage, according to a paradigm modified from Majchrowicz³⁸ as previously described^8,39,40.

The initial loading dose of ethanol diet was 5 g/kg. Thereafter, animals were dosed according to their behavioral intoxication, based on the Majchrowicz scale (0, normal rat; 1, hypoactive; 2, ataxia; 3, ataxia with dragging abdomen and/or delayed righting reflex; 4, loss of righting reflex; 5, loss of eye blink reflex). Each of these scores has an accompanying dose of ethanol between 0 and 5 g/kg, such that a higher score (higher observed behavioral intoxication) corresponds to a lower dose. Behavioral intoxication was assessed immediately before each dose of alcohol was administered. This method maintains consistent intoxication relevant to an alcohol use disorder (AUD) while avoiding mortality^39,41. Control animals were given the average volume of fluid that their same-sex alcohol group received for each dose. To determine blood ethanol concentration (BEC), blood was drawn from the lateral saphenous vein 90 minutes following the seventh dose of ethanol. Samples were immediately centrifuged, then extracted serum was stored at −80°C until analysis. Samples were analyzed using an AM1 Analyzer (Analox, MA, USA), based on external standards.

Some evidence suggests that female rats may have increased ethanol sensitivity during certain phases of the estrous cycle^42,43. Thus, in order to control for this variability in animals undergoing cognitive testing, all female rats in Cohort 2 received their first dose of control or alcohol diet when they were in the diestrus stage of their cycle. Vaginal smears of both female binge and control groups were taken between 8:00 and 9:00 daily, beginning 5–7 days prior to the first alcohol or control dose. Each sample was placed on a glass slide, stained with cresyl violet, and then coverslipped. Stage of estrous was determined by examining samples under a light microscope at 10x magnification.

2.3 Withdrawal

Because Cohort 2 was sacrificed 9 days post-binge, spontaneous withdrawal symptoms were monitored every 30 minutes for hours 10–26 after the last dose, which encompasses the peak withdrawal period³⁸. Every 30 minutes, the most severe observed withdrawal symptom was scored using the scale created by Penland and colleagues⁴⁴. Red lights were used during the dark phase of the light cycle so as not to disrupt the animals’ circadian rhythms.

2.4 Spatial Working Memory Task

The rewarded alternation (RA) task relies on rodents’ natural inclination to alternate areas of exploration in a new environment and rewards them for correctly alternating choice arms. Working memory, a prefrontal cortex dependent function, is engaged by using a short inter-trial interval (ITI), as rats must recall which arm they most recently visited to obtain access to the food pellet reward^29,45. Prior to the 4-day binge, all rats were habituated in home cage groups to the T-maze, which is an apparatus made of clear Plexiglas, with three guillotine doors secured in metal brackets which can be raised and lowered by the experimenter. During habituation, all doors were raised and unlimited food reward pellets (chocolate 45mg Dustless Precision Pellets®, Bio-Serv, NJ, USA) were present in both food dishes located at the end of each choice arm. Also during this time, food pellets were placed into each home cage so rats could become accustomed to searching out pellets in a familiar environment. Throughout behavior testing, rats were food-restricted so that they were adequately motivated to seek out the food reward. Because all rats (controls and binged) lose body weight (9–20%) during the 4-day binge, afterwards, each animal was either food restricted or fed ad libitum to reach 85–90% of their pre-binge body weight by the start of the spatial working memory task. During behavior testing, each rat was weighed daily and given food rations accordingly to maintain the range of 85–90% of their original free-feeding weight.

Day 1 of behavioral testing began on the fourth day of alcohol abstinence and was conducted at the beginning of the animals’ active cycle. Each rat began testing by being placed in the starting area, and allowed to settle behind the starting guillotine door. Rats received one forced trial followed by ten choice trials in succession each day, for five days. For the forced trial, one of the two reward arms (randomly chosen) was opened, forcing the rat to visit that arm. For the choice trials, the guillotine door was lowered behind the animal once both hindlimbs crossed into a chosen arm, thus confining it to that arm. Once the rat had spent either 10 seconds in the reward arm or eaten all the reward pellets, it was immediately picked up and placed back at the start. Trials 2–11 were conducted with a 15 second ITI, with the rats able to freely choose which arm they visited. Rats found a three-pellet reward only in the bowl in the arm opposite that of which they visited on the previous trial, however, pellet dust was present in both reward dishes so rats could not use olfactory cues to determine which dish contained the reward. Thus, the animal had to employ the use of working memory to remember which arm they most recently visited and choose the other arm to receive a reward ^29,45. The maze was spot cleaned between trials and wiped down with a 70% ethanol solution between animals.

2.5 Immunohistochemistry (IHC)

One hour after completing the last RA trial, rats were overdosed with anesthetic and intracardially perfused with saline, then 4% paraformaldehyde until the neck was stiff. Brains were extracted, post-fixed overnight and then stored at 4°C in 30% sucrose. Serial coronal sections (50 μm) were collected on a freezing sliding microtome (Leica, Bannockburn, IL) and stored in 96-well microtiter plates at −20°C in cryoprotectant.

To visualize neuronal cell bodies, a 1-in-6 series of tissue sections from the mPFC (~6 sections per animal, spaced 300 μm apart) were immunohistochemically processed for the neuronal marker, NeuN. Sections were rinsed 3 times (10 minutes each) in 0.1 M Tris buffered saline (TBS) at room temperature (RT), followed by a 30-minute treatment with 0.6% hydrogen peroxide. After this, sections were once again rinsed in TBS three times for 10 minutes each. Next, they were blocked for 60 minutes in 3% normal donkey serum (Sigma-Aldrich, St. Louis, MO), then incubated in the primary antibody (guinea pig anti-NeuN, EMD Millipore, Billerica, MA; 1:1,000) at 4°C. After 72 hours, the sections were rinsed twice (15 minutes each) in TBS, and then incubated in secondary antibody (donkey anti-guinea pig biotinylated, Jackson ImmunoResearch, West Grove, PA, USA, 1:250) for 24 hours at RT. Sections were then underwent another 3 10-minute TBS washes before being treated for 60 minutes in avidin-biotin complex (ABC, Vector Labs, Burlingame, CA). After 3 more 10- minute rinses in TBS, the sections were reacted in diaminobenzidine (DAB). After a final four 10-minute TBS rinses, the sections were mounted on gelatin-coated slides, counterstained with methyl green, cover-slipped and coded so that the investigator performing cell counts remained blind to experimental condition.

For visualization of c-Fos, the IHC protocol of Cholvin and colleagues⁴⁶ was used. Sections were first floated three times in 0.05 M Phosphate Buffer (PBS) for ten minutes each. After the PBS rinses, sections were floated in sodium citrate for 10 minutes at 90°C. Following another 3 PBS rinses, sections were placed in a blocking solution of 5% normal donkey serum for 1 hour. Following blocking, sections were incubated in the primary antibody (mouse anti-c-Fos, Santa Cruz Biotechnology, CA, USA, 1:100) at RT for 18 hours. Next, sections were washed 3 times for 10 minutes each in PBS and then incubated in the secondary antibody (biotinylated donkey anti-rabbit, Jackson ImmunoResearch, PA; 1:250) for 1 hour. Following three more PBS washes, the sections were incubated in ABC for 45 minutes, given two more rinses in PBS, then a single ten-minute rinse in TBS. Next, sections were reacted in DAB and then rinsed another three times in PBS before being mounted. Slides were cleared in xylene for 5 minutes, then cover slipped and coded.

2.6 Stereology

The mPFC area for cellular analysis included the infralimbic, prelimbic, and anterior cingulate cortices between Bregma 3.9 mm and 2.2 mm⁴⁷ (see Figure 1A). The total population of NeuN+ cells in the mPFC was estimated using the optical fractionator, and neuronal volume estimated using the nucleator probe, both of which were applied via StereoInvestigator (MBF Bioscience, Williston, VT). The mPFC was traced at 4X and NeuN+ cells counted within 2-dimensional counting frames (40 × 40 μm) at 100x, using a grid size of 400 × 400 μm. Total population (N) of NeuN+ cells was estimated using the following formula:

$$\mathrm{N}=\mathrm{\sum }\mathrm{Q}-\times \frac{1}{\mathrm{h}}\times \frac{1}{\text{asf}}\times \frac{1}{\text{ssf}}$$

where Q- is the number of cells counted, h is the mounted section thickness, asf is the area sampling fraction and ssf is the section sampling fraction. The volume of each counted NeuN+ cell nucleus was determined by the nucleator probe, which works within the optical fractionator workflow in order to measure the area and volume of each nucleus counted within the sampling frames. The probe creates rays at a random orientation that emanate from the center point of the cell, which is arbitrarily designated by the investigator, and the points at which the rays intersect with the boundary of the cell is marked (Figure 1). Nuclear volume is calculated using the formula for the volume of a sphere, where the radius of the sphere was determined by using the average length of measured rays for each cell. To estimate the total population of c fos+ cells, similar optical fractionator procedures were followed, except that the counting frame size was 80 × 80 μm.

Figure 1.

Area of interest for cell quantification in the mPFC, which included the infralimbic, prelimbic, and anterior cingulate cortices (A). Screenshot of nucleator probe working within the optical fractionator workflow in StereoInvestigator software (B). Points at which the rays intersect the boundary of the nuclei are manually marked and the average radius is used to estimate the volume of each nucleus.

2.7 Statistical Analysis

Male and female ethanol doses and BECs were compared using an independent group t-test. Behavioral intoxication and spontaneous withdrawal scores are ordinal data and were therefore compared between males and females using Mann-Whitney testing. Pearson correlations were performed to examine the relationship between behavioral intoxication and mean withdrawal score, with correlation significance determined by using the critical value table for Pearson’s Correlation Coefficient. A three-way mixed factorial design with Time as the repeated measure was used to evaluate T-maze percent correct alternations for the variables Time, Sex and Diet as well as the interactions of these three variables. Neuronal data was analyzed using two-way ANOVA with the variables Sex, Diet and the Sex x Diet interaction. All values are presented as mean ± standard error of the mean. Planned Bonferroni-corrected post hoc comparisons were used when appropriate. Results from statistical tests were deemed significant if the p-value was less than 0.05 and all analyses were run using SAS 9.4.

3.0 Results

3.1 Neuronal number and volume in the mPFC immediately post-binge

Figure 2 depicts results from analysis of tissue harvested immediately following 4-day binge alcohol. The tissue analyzed was archived mPFC sections from a prior study of the hippocampus following binge alcohol¹⁵. As previously reported, males and females did not differ in behavioral intoxication, dose administered, BEC or withdrawal score¹⁵. There were no significant main effects of Diet [F(1,23)=2.73, p=.11] or Sex [F(1,23)=.71, p=.41] and no significant interaction [F(1,23)=.01, p=.92] on the estimated total population of NeuN+ cells present in the mPFC. There was, however, a significant main effect of Diet on neuronal volume [F(1,23)=.44, p=.0004], with no significant main effect of Sex [F(1,23)=.44, p=.52] or Diet x Sex interaction [F(1,23)=.56, p=.46]. The mean nuclear volume for both male and female binged rats was 19.9% smaller than those of their respective control groups.

Figure 2.

There was no significant effect of binge alcohol on mPFC neuronal number (A), however both female and male binged rats had a significant reduction in nucleus volume (B) in the mPFC in comparison to respective control groups immediately following a four-day binge. ****p<.0001 for Diet

3.2 Behavioral intoxication and withdrawal in rats used for behavioral analysis

Contrary to what is commonly observed in the human population⁴⁸, female rats acted less behaviorally intoxicated than male rats, and thus received higher doses of alcohol [t(17)=4.81, p=.0002] (Table 1). However, despite males receiving a lower alcohol dose, male and female rats did not have significantly different blood ethanol concentrations (mg/dl) [t(17)=.18, p=.67]. Males and females also did not differ on withdrawal severity [t(17)=.57, p=.58]. There was no significant correlation between mean behavioral intoxication and mean withdrawal score [r²=.14, p=.58].

Table 1.

Alcohol Administration Measures

Sex	Average Behavioral Intoxication	Alcohol Dose (g/kg)	Average Withdrawal Score	BEC (mg/dl)
Male	2.18 ± 0.06	2.79 ± 0.06	1.0 ± 0.01	404.6 ± 13.8
Female	1.68 ± 0.08*	3.31 ± 0.08*	1.1 ± 0.01	397.8 ± 8.0

^*p < 0.05 significant effect of Sex

3.3 Rewarded alternation behavior following alcohol abstinence

For percentage of correct alternations on the RA working memory task, there was a significant main effect of Diet [F(1,191)=31.24, p<.0001], and Day x Diet interaction [F(4,1946)=2.73, p=.03]. There was a significant effect of Diet on the first [F(1,191)=34.17, p<.0001], second [F(1,191)=17.28, p=.0002] and fourth [F(1,191)=5, p=.02], days of testing. (Figure 3A). Additionally, there were no significant effects of Diet or Sex for the percent of trials in which reward pellets were eaten [F(1, 191)=.05, p= 0.9760].

Figure 3.

Both female and male binged rats had fewer correct alternations than control groups, indicating impairment in spatial working memory (A). On trials 1–5 of the first testing day, both binged and control animals began with similar percentages of correct alternations. However, while control animals chose the rewarded arm correctly more often by trials 6–10 (B), binged animals did not show improvement in correct arm alternations until the third day of testing.

3.4 Neuronal number, volume and activation in the mPFC following alcohol abstinence

Following 9 days of alcohol abstinence, there were no significant effects of either Diet or Sex on mPFC neuronal population [F(1,35)=.12, p=.94] or nuclear volume [F(1,35)=.12, p=.95] (Figure 4). There was, however, a significant main effect of Diet [F(1,35)=15.25, p=.0004], on number of c fos+ cells in the mPFC (Figure 5), but no significant effect of Sex [F(1,35)=.02, p=.88] or Diet x Sex interaction [F(1,35)=.29, p=.59].

Figure 4.

After nine days of alcohol abstinence, there were no detectable differences in either neuronal number (A), or nucleus volume (B) in the mPFC based on stereological assessment.

Figure 5.

Image of c-Fos staining in the mPFC at Bregma 2.70 mm (A). Although there was no significant difference in percent correct alternations on the final day of testing, binged animals had more c-Fos+ cells in the mPFC (B), suggesting reduced neuronal efficiency. ****p<.0001 for Diet.

4.0 Discussion

Decades of data substantiate that women are more vulnerable than men to the health-related consequences of alcohol, including increased rates of organ damage⁴⁹ and mortality ^50,51. Prior research also indicates that the female brain may be selectively vulnerable to the harmful effects of alcohol^11,12,15,52. Using a model of alcohol-induced neurodegeneration, we have recently found damage in the female hippocampus (but not the male), accompanied by spatial navigation impairment¹⁵. In the present study, we used this same model to probe for sex differences in vulnerability of the prefrontal cortex. We found males and females to be equally affected in terms of nuclear volume and number of prefrontal cortex neurons early after binge alcohol. Moreover, we found binge-induced spatial working memory impairments in both males and females. Finally, although binged animals of both sexes ultimately achieved control performance, it was associated with more neuronal activation, suggesting decreased efficiency.

The nuclei of neurons in the mPFC of binged animals of both sexes had reduced volume. Since cells retain a relatively constant ratio between nuclear volume and cell body volume^23–25, this nuclear volume loss is likely indicative of grey matter atrophy, which has been previously observed in other brain areas using this model⁵³. Although nuclear shrinking is usually indicative of cell death^54,55, we did not find a reduction in the number of remaining neurons. Moreover, the reduction in nuclear volume was temporary, since after nine days of abstinence there were no detectable differences between binged and control animals. This is consistent with the natural recovery reported to occur with abstinence in humans^19,20, which in the frontal cortex likely stems primarily from normalization of cell size, versus the hippocampus, in which recovery could be supported by the generation of new neurons. Moreover, it is possible that experience contributed to nuclear volume recovery, as it was coincident with achievement of control performance on the spatial working memory task.

Consistent with the lack of sex differences in alcohol-induced mPFC damage, we found no sex differences in performance on the rewarded alternation task. A short inter-trial interval required rats to employ working memory^29,45, a hallmark function of the frontal lobe. Control animals performed very well on this task, reaching maximum performance by the second block of trials on day 1 (Figure 3B). As with any spatial working memory task, the entire fronto-hippocampal circuit is involved, including the hippocampus, which plays a key role in spatial working memory, and damage to it can negatively affect performance on learning, memory and spatial navigation tasks^56–59. All spatial working memory tasks likely rely on the use of the hippocampus and mPFC working in conjunction, however, and consistent with this, alcohol-induced damage to the prefrontal cortex has previously been associated with impairments in this spatial working memory task²⁹. Indeed, the task appeared difficult for the binged animals. They showed no improvement across trials on day one, and during the first two days of testing they had overall 20% fewer correct alternations than control rats. Ultimately however, the binged groups did achieve control performance. The mPFC contributes to cognition, motivation and risk-taking behavior^26–28, and impairments in any of these could contribute to the decreased performance we observed on the spatial working memory task. Although we cannot completely rule out the possibility that binge alcohol-induced mPFC damage altered risk-taking behavior or motivation, animals were familiarized with the T-maze apparatus prior to the 4-day binge, so it was not a novel environment for them. Moreover, no changes in food-related motivation were noted, as binged and control animals were equally likely to consume the food reward pellets during behavior testing.

Compensatory metabolic changes have been observed in human binge drinkers during cognitive tasks. For example, despite behavioral performance comparable to controls, binge drinkers showed increased brain activity in response to a working memory task³³. This suggests that a history of binge alcohol requires the brain to use more energy to perform at baseline levels, indicative of less efficient cognitive processing. Therefore, despite the similar performance between control animals and binged on the last day of rewarded alternation testing, we reasoned that binged animals might show more neuronal activation, which we assessed by quantifying c-Fos+ cells. Binged rats had more c-Fos+ cells in the mPFC in response to rewarded alternation testing. This suggests that even in the absence of detectable cognitive deficits or grey matter loss, persistent changes in brain efficiency can still occur as a result of binge alcohol. It is important to note that alcohol withdrawal has also been shown to induce neural activation^60,61, which may have contributed to the observed c-Fos activation in the mPFC of binged animals during behavioral testing. However, this is unlikely, as these studies showed that Fos activity was increased early (7–24 hours) after withdrawal, and in the current study we assessed c-Fos activation 7 days after the end of withdrawal.

A possible reason that we did not find sex differences in the present study is that female vulnerability to alcohol may be brain-region specific. To illustrate, a sex difference in alcohol-induced hippocampal damage has been reported⁵, but a recent study of pontocerebellar volume in male and female alcoholics showed none¹⁴. Moreover, although the frontal cortex is an area commonly found to be damaged by excessive alcohol consumption^59,62–64, it is possible that sex-specific vulnerability is more prominent in the white matter of this area of the cortex. It would be advantageous for future studies to examine both gray and white matter following alcohol exposure in order to develop a more comprehensive understanding of how the female and male brains differ in reaction to alcohol.

5.0 Conclusions

The results of this study indicate that binge ethanol caused a temporary volume decrease in the nuclei of neurons in the mPFC of both male and female rats, which disappeared with abstinence. Additionally, binged animals of both sexes showed transient deficits in spatial working memory. Although they ultimately achieved control performance levels, binged animals of both sexes showed increased task-induced neuronal activation, suggesting decreased efficiency. Thus, our results show frontal cortex susceptibility to alcohol damage in both sexes, but indicate that sex differences in alcohol vulnerability may be brain region-specific.

Highlights.

• Binge ethanol induces temporary volume loss in prefrontal cortex neuronal nuclei

• Binge ethanol impairs spatial working memory and decreases task-induced neuronal efficiency

• The effects of binge ethanol on prefrontal neurons and rewarded alternation are not sexdependent