Formation of emotional memories are an important mechanism for how animals, including humans, learn to avoid threatening and aversive environmental stimuli. While fear learning and memory is typically a healthy process, it can also be maladaptive and pathological, and is an important etiological factor in many psychological disorders. In animal studies, Pavlovian auditory fear conditioning is a leading model for investigating associative fear learning and memory. In this paradigm, a neutral stimulus (auditory tone) is temporally paired with an aversive stimulus (e.g. a foot shock) such that repeated tone-shock pairings lead to a learned association. Subsequent presentation of the conditioned stimulus (CS) alone elicits a defensive response (e.g. freezing) even without presentation of the unconditioned stimulus (US). However, when the CS is repeatedly presented without being paired with the US, the conditioned response can be extinguished. Extinction is not simply the unlearning or forgetting of the original associative fear memory, but instead involves the formation a new inhibitory memory (Tovote et al., 2015; Bouton et al., 2021). Importantly, while this inhibitory extinction memory may initially dominate, the original fear memory remains and can subsequently be reactivated in response to environmental context and cues.

Adolescence is characterized as a period of increased impulsivity, risk-taking and sensation seeking, and as such is also a time when many individuals begin experimenting with alcohol (Spear, 2018; Crews et al., 2019). Of particular concern is that alcohol consumption in this age group frequently occurs in repeated episodes of “extreme” binge drinking that result in high levels of intoxication. For example, a recent national survey of adolescent youths revealed that 4.5% of 8th graders and 16.8% of 12th graders reported having engaged in “extreme” binge drinking, which was defined as having consumed 10 or more drinks in a single session (Patrick and Terry-McElrath, 2019). Adolescence is also a period of continued brain remodeling and rapid behavioral development that includes maturation of top-down executive control over behavior (Casey et al., 2008; Drzewiecki and Juraska, 2020).

While the developing adolescent brain is highly plastic, this feature may also enhance its vulnerability to adverse environmental insults such as those associated with repeated episodes of extreme binge intoxication that can produce long-lasting changes in brain structure, function, and behavior. In support of this, recent studies in rodent models of adolescent alcohol binge intoxication have reported a number of deficits in behavioral control and decision-making in adulthood that are consistent with a general reduction of behavioral flexibility and adaptive decision-making (Spear, 2018). Changes that have been reported include deficits in set-shifting (Gass et al., 2014a; Fernandez and Savage, 2018), reversal learning (Coleman et al., 2014), habitual behaviors (Barker et al., 2017; Towner and Spear, 2021), increases in risky decision-making (Clark et al., 2012), resistance to extinction of ethanol self-administration (Gass et al., 2014b), and increases in anxiety-like behaviors (Pandey et al., 2015). Interestingly, it has become clear in recent years that many of the fundamental principles of learning and memory also play a major role in the development and maintenance of deficits associated with addictions and trauma and anxiety-related disorders (Tipps et al., 2014). Previous findings from our lab have shown that pharmacological manipulation of the metabotropic glutamate receptor 5 (mGlu5) can attenuate behavioral deficits associated with chronic alcohol and stress (Gass et al., 2014a; Smiley et al., 2020; Smiley et al., 2021a). However, the impact of adolescent alcohol exposure on fear learning in adulthood has not been widely investigated, and even less is known regarding sex-specific differences as most previous studies have been conducted in males. It is of particular interest that many of the key brain regions and neurocircuits implicated in different aspects of monitoring of threats (Bouton et al., 2021) also undergo important developmental changes during adolescence, and thus may be particularly vulnerable to repeated episodes of excessive ethanol intoxication during adolescence. Therefore, the present study utilized a well-characterized model of binge-like alcohol exposure and a Pavlovian fear conditioning paradigm to determine the impact of adolescent alcohol exposure on fear learning and memory in adult male and female rats. An additional aim of this study was to examine the ability of mGlu5 modulation to prevent or attenuate deficits in fear-related behaviors following chronic alcohol exposure during the adolescent period.

The goal of the present study was to determine the effects of repeated episodes of alcohol intoxication during adolescence on fear conditioning, extinction, and recovery in adulthood. The experimental approach employed a model of intermittent alcohol exposure by vapor inhalation that is designed to simulate repeat episodes of binge-like alcohol exposure. The procedure involved subjecting adolescent rats to 5 intermittent cycles of ethanol vapor between PD 28–44, a period that encompasses the early to middle stage of adolescence. Behavioral intoxication and blood ethanol concentration (BEC) were assessed at the end of each of the 5 exposure cycles. The average intoxication scores using the 1-5 point rating scale was 2.66 ± 0.16 for male rats and 2.4 ± 0.09 for female rats, which represents a moderate level of intoxication on the rating scale. The corresponding BEC values were 375 ± 9.6 mg/dl for the male rats and 271 ± 12.7 mg/dl for the female rats. Unpaired t-test comparisons indicated the BEC levels in the males was significantly higher than in the females (p < 0.0001).

To determine whether AIE impacted conditioned fear behaviors assessed during adulthood, the Air control and AIE exposed adult rats were subjected to 3 consecutive days of tone-shock pairing. For male rats (Figure 2A), a two-way ANOVA revealed a significant main effect of Conditioning Day (F (2, 78) = 54.73, p < 0.0001) and treatment (AIE vs Air) (F (1, 78) = 18.28, p < 0.0001). Sidak’s post-hoc analysis revealed a significant difference of Air control rats at Conditioning Day 1 compared to all groups on Conditioning Day 2 and 3 (all p values < 0.0001). Post-hoc analysis also revealed a significant increase of freezing in the AIE rats compared to the Air control rats at Conditioning Day 1 (p = 0.0295). For female rats (Figure 2B), ANOVA revealed a significant main effect of Conditioning Day (F (2, 102) = 20.23, p < 0.0001). Sidak’s post-hoc test indicated that freezing by both Air and AIE rats at Condition Day 1 was significantly lower than freezing at Conditioning Days 2 and 3 (all p values < 0.01). The ANOVA also indicated that AIE had no effect on freezing in the female rats (F (1, 102) = 0.05625, p = 0.8130). To determine if there was a significant sex difference in the acquisition of conditioned fear, we next performed an ANOVA comparing freezing at Conditioning Day 3 for the male versus female rats. As shown in Figure 2C, this revealed that male rats froze significantly more than their female counterparts (F (1, 48) = 18.41, p < 0.0001).

Following completion of tone-shock fear conditioning, rats were subjected to 5 daily sessions of fear extinction in a novel environment that involved 10 presentations of the CS (tone) in the absence of the US (shock). As shown in Figures 3A–E, ANOVA of freezing during fear extinction by male rats revealed a day by session-dependent reduction in freezing behavior as evidenced by a main effect of the number of CS (CS#) presentations {Day 1: F (9, 260) = 5.504, p < 0.0001; Day 2: F (9, 260) = 31.94, p < 0.0001; Day 3: F (9, 260) = 34.38, p < 0.0001; Day 4: F (9, 260) = 29.70, p < 0.0001; Day 5: F (9, 260) = 39.23, p < 0.0001}. There was also a significant main effect of AIE at all extinction days except Day 4 {Day 1: F (1, 78) = 18.28, p < 0.0001; Day 2: F (1, 260) = 17.68, p < 0.0001; Day 3: F (1, 260) = 5.494, p = 0.019; Day 5: F (1, 260) = 71.24, p < 0.0001}. Post-hoc analysis also revealed both increases and decreases in freezing in response to the CS that depended on the day of extinction (all p values < 0.05). However, while comparison of the average freezing across the 10 CS for each extinction day (Figure 3F) indicated there was a significant main effect of Extinction Day (F (4, 130) = 98.80, p < 0.0001), there was no main effect of treatment (Air vs AIE) (F (1, 130) = 1.838, p = 0.1775). For fear extinction in female rats (Figures 4A–E), there was a significant effect of CS# on Extinction Day 1 (F (9, 340) = 3.652, p = 0.0002) and Day 2 (F (9, 340) = 4.657, p < 0.0001), but no effect of AIE on any of the extinction days. Comparison of the average freezing across the 10 CS of each extinction day (Figure 4F) confirmed there was a significant effect of Extinction Day (F (4, 170) = 6.605, p < 0.0001), but no effect of treatment (F (1, 170) = 0.4692, p = 0.4943).

Following completion of extinction training, animals were sequentially tested for fear recall, context renewal, and spontaneous recovery 21 days after completion of extinction training. For recall (Figure 5A) ANOVA revealed that female rats exhibited significantly lower levels of freezing compared to male rats (F (1, 60) = 31.88, p < 0.0001). Post-hoc analysis indicated that AIE exposed male rats froze significantly more than the corresponding male Air controls (p = 0.0288). With context renewal (Figure 5B), female rats again exhibited significantly lower levels of freezing compared to the male rats (F (1, 60) = 125.4, p < 0.0001). Post-hoc analysis also indicated that male AIE rats froze significantly less than male Air control rats (p < 0.0001). For spontaneous recovery (Figure 5C), ANOVA also indicated that female rats froze significantly less than male rats (F (1, 60) = 3296, p < 0.0001). Post-hoc analysis further revealed that AIE significantly increased freezing in the male rats (p < 0.0001) but not female rats.

Taken together, the above results suggest that AIE exposed male, but not female, adult rats exhibit significant alterations in freezing behavior associated with conditioned fear learning. However, the results also revealed that female rats as a group exhibited significantly lower levels of freezing compared to male rats. We therefore examined whether the observed lack of effect of AIE on fear and extinction behaviors in female rats might have been associated with lower levels of fear acquisition exhibited by some of the female rats compared to male rats. To test this, we separated the female rats into two groups based upon their level of freezing during fear conditioning. A cutoff of 73.3% freezing was used for classification as either a high conditioning (HC) or low conditioning (LC) rat. This classification criteria represented one standard deviation below the average percent freezing observed on conditioning on days 2 and 3 of the male rats. None of the male rats fell below this cutoff, and thus all male rats were considered to be high conditioners. The BEC levels for the HC group were 287 ± 19.7 mg/dl and 253 ± 15.85 mg/dl for the LC group. Unpaired t-test analysis revealed these were not significantly different (p = 0.0789). As shown in Figure 6A, comparison of freezing at Conditioning Day 3 for the HC and LC female rats revealed a significant group difference (F (1, 32) = 46.17, p < 0.0001). Post-hoc comparisons confirmed the level of freezing by both the Air and AIE HC rats was significantly higher than freezing by the Air and AIE LC rats (all p values < 0.001). When examining the time-course of fear conditioning across days, the HC group of female rats acquired fear conditioning at a similar rate to the male rats (compare Figure 6B with Figure 2A). ANOVA revealed a significant main effect of Conditioning Day (F (2, 39) = 177.3, p < 0.0001). Post-hoc analysis further indicated the level of freezing for both Air and AIE rats on Conditioning Day 1 was significantly different from freezing on Conditioning Day 2 and 3 (all p values < 0.0001). In contrast, while ANOVA of the LC female rats (Figure 6C) revealed a significant main effect of Conditioning Day (F (2, 57) = 8.825, p = 0.0005), post-hoc analysis indicated there were no significant differences across any of the conditioning days. ANOVA also indicated that AIE had no effect on freezing in either the HC (F (1, 39) = 1.116, p = 0.2973) or LC (F (1, 57) = 1.881, p = 0.1756) rats.

We next compared freezing in the HC and LC rats across the 5 days of extinction training. With the HC group of female rats (Figures 7A–E), ANOVA revealed a significant effect of CS# at Extinction Day 1 (F (9, 130) = 3.418, p = 0.0008) and Day 2 (F (9, 130) = 7.664, p < 0.0001), but no significant main effect of CS# at Extinction Days 3–5. There was also a significant increase in freezing in the AIE rats compared to the Air control rats at Extinction Day 1, 4 and 5 (Day 1: F (1, 130) = 8.739, p = 0.0037; Day 4: F (1, 130) = 18.92, p < 0.0001; Day 5: F (1, 130) = 9.542, p = 0.0025). When comparing the average freezing across each extinction day (Figure 7F), ANOVA revealed there was a significant effect of Extinction Day (F (4, 65) = 4.991, p = 0.0014). However, while there was a trend towards an increase in freezing in the AIE HC rats compared to the Air HC rats, this was not a statistically significant effect (p = 0.069). Analysis of freezing across the 5 days of extinction in the LC female rats (Figures 8A–E) revealed a significant effect of CS# at Extinction Day 1 (F (9, 190) = 3.604, p = 0.0004) and Day 2 (F (9, 190) = 3.220, p = 0.0012), but no significant main effect of CS# at Extinction Days 3–5. ANOVA also revealed there was a significant decrease in freezing in the AIE LC rats compared to the Air control LC rats at Extinction Day 2, 3 and 4 (Day 2: F (1, 190) = 4.883, p = 0.0283; Day 3: F (1, 190) = 9.553, p = 0.0023; Day 4: F (1, 190) = 9.271, p = 0.0027). When comparing the average freezing across the time-course of extinction training (Figure 8F), there was a significant effect of Extinction Day (F (4, 95) = 6.086, p = 0.0002), but no effect of treatment (Air vs AIE) (F (1, 95) = 3.148, p = 0.0792).

We next examined the level of freezing by HC and LC rats in the tests of fear recovery that followed extinction training. As shown in Figures 9A–C, this analysis indicated there was no significant main effect of group (HC vs LC) or treatment (Air vs AIE) with Recall (Group: F (1, 32) = 0.9783, p = 0.3300; Treatment: F (1, 32) = 0.5235, p = 0.4746 ), Context Renewal (Group: F (1, 32) = 0.2455, p = 0.6237; Treatment: F (1, 32) = 1.198, p = 0.2818), or Spontaneous Recovery (Group: F (1, 32) = 2.565, p = 0.1191; Treatment: F (1, 32) = 1.579, p = 0.2180). Taken together, the above analysis suggests that the significantly lower level of fear conditioning exhibited by a subgroup of female rats did not appear to underlie the absence of an AIE effect on measures of fear recovery in females.

We have previously shown that administration of the mGlu5 PAM CDPPB can reverse AIE-induced deficits in behavioral flexibility. Therefore, we performed a final set of experiments to determine if administration of CDPPB prior to each extinction session could prevent AIE-induced alterations of fear conditioning and recovery that was observed in male AIE exposed rats. These studies were conducted only in male rats since AIE did not alter fear behaviors in the female rats. As shown in Figure 10A, a three-way ANOVA revealed a significant main effect of Conditioning Day (F (2, 60) = 73.93, p < 0.0001), AIE (F (1, 60) = 31.93, p < 0.0001), and CDPPB (F (2, 60) = 6.146, p = 0.0037). Post-hoc comparisons further indicated that on Conditioning Day 1, the AIE and AIE+CDPPB rats froze significantly more than all other groups of rats (all p values < 0.05). When comparing the average freezing on each day of extinction training (Figure 10B), a three-way ANOVA confirmed there was a main effect of Extinction Day (F (4, 180) = 151.9, p < 0.0001), AIE (F (1, 180) = 9.857, p = 0.0020), and CDPPB (F (1, 180) = 81.55, p < 0.0001). Post-hoc analysis indicated that CDPPB significantly reduced freezing behavior compare to the Air control and/or AIE rats at Extinction Days 2–4 (all p values < 0.05).

Following completion of extinction training, the rats were subjected to our standard method of testing for fear recovery. For recall (Figure 10C), a one-way ANOVA indicated a significant group difference (F (3, 20) = 11.69, p = 0.0001). Post-hoc analysis further revealed that both groups of rats that received CDPPB froze significantly less compared to AIE exposed rats (all p values < 0.001). While there was a trend towards an effect of AIE compared to the Air control rats (p = 0.0505), this just missed the minimum criteria to be considered a statistically significant effect. Analysis of contextual renewal (Figure 10D) also showed a significant group difference (F (3, 20) = 5.310, p = 0.0074). Post-hoc analysis indicated that AIE exposure resulted in a significant reduction in freezing compared to all other groups, and that this reduction was absent in the CDPPB treated rats (all p values < 0.05). For spontaneous recovery (Figure 10E), ANOVA again revealed a significant group difference (F (3, 20) = 25.29, p < 0.0001). Post-hoc analysis indicated that AIE exposed rats froze significantly more compared to all other groups, and that the AIE-induced increase was absent in the CDPPB group (all p values < 0.0002). Post-hoc analysis also revealed that freezing by the AIR+CDPPB rats was significantly lower compared to the Air control rats (p = 0.0487).

The present study utilized Pavlovian conditioning to examine the impact of repeated episodes of binge-like alcohol exposure during adolescence on fear learning and memory in male and female adult Long-Evans rats. The fear procedure involved a sequential ABBAB contextual design separated into three phases: an initial conditioned fear acquisition phase performed in context A, a cued-fear extinction phase performed in context B, and a fear recovery phase in which testing was performed in either the conditioning context (renewal) or extinction context (recall and spontaneous recovery). Our results revealed both sex differences and sex-specific effects of AIE on fear conditioning and fear recovery.

The conditioning procedure used consisted of a total of the 9 tone-shock pairings separated into 3 presentations per day for 3 consecutive days. This multi-day conditioning approach has an advantage over single-session conditioning as it introduces a more protracted temporal component to the acquisition of conditioned fear, which can serve as a form of context. As expected, we observed that the percent time male Air control rats spent freezing in response to tone-shock presentations increased over conditioning days, with the largest increase occurring between conditioning day 1 and 2. Male AIE exposed rats exhibited increases in freezing during tone-shock presentations compared to the male Air control rats. While this increase was observed across the 3 days of conditioning (e.g., an overall main effect of AIE), the AIE-induced increase in freezing during the tone-shock presentation was particularly prominent on conditioning day 1, which then stabilized on conditioning days 2–3 at substantially elevated levels (>80%) in both Air and AIE exposed groups. Previous studies have shown that AIE exposure is associated with increased anxiety-like behaviors in adulthood (Kyzar et al., 2019). This increase was also shown to be dependent upon AIE-induced epigenetic reprograming in the amygdala. Since the amygdala is a key brain region of the fear neurocircuitry, it is reasonable to suggest that the AIE-induced increase in fear conditioning is consistent with changes in how the amygdala processes information of potential environmental threats.

Similar to the multi-day conditioning procedure, extinction training was conducted over a 5-day period that also introduced a protracted temporal component to extinction learning. We observed that male Air control rats displayed both within-session and between-session reductions in the percentage of time spent freezing in response to CS presentation. This observation is consistent with previous studies that also utilized a protracted extinction protocol (Elias et al., 2010; Smiley et al., 2020; Smiley et al., 2021a). While some within session differences in freezing between the Air control and AIE exposed rats were noted, there was no significant difference in extinction of conditioned freezing when examined across days of extinction training.

While the neurocircuitry of fear and extinction is complex, several brain areas are thought to play particularly important roles in this neuronal network. The basolateral amygdala (BLA) functions as a central hub region where activity-dependent plasticity critically shapes fear learning and memory (Ciocchi et al., 2010; Amano et al., 2011). Inputs from the ventral hippocampus (vHP) to the BLA provides contextual information of the threat that is critical for the expression of conditioned fear, while subregions of the mPFC differentially regulate fear conditioning and extinction. In particular, excitatory inputs from the prelimbic (PrL) sub-region to the BLA promote the expression of conditioned fear whereas inputs from the infralimbic (IfL) sub-region promote fear extinction (Tovote et al., 2015; Day and Stevenson, 2020). Previous reports that AIE exposure leads to deficits in behavioral flexibility (Spear, 2018), including resistance to the extinction of ethanol self-administration (Gass et al., 2014b), lead us to hypothesize that AIE exposure might also be associated with resistance to extinction of conditioned fear. Adding further support to this suggestion, the enhanced freezing we observed in AIE exposed rats during fear conditioning might also be expected to lead to differences in fear extinction, especially during the initial time-course of extinction training. However, our observations that male AIE exposed rats extinguished conditioned freezing at a similar rate and extent compared to male Air rats did not support this idea.

An interesting observation in the present study was that male AIE exposed rats exhibited significant alterations in freezing on all three tests of fear recovery in comparison to the male Air control rats. The AIE-induced alterations in fear recovery included increased freezing during fear recall and spontaneous recovery, and reductions in freezing during fear renewal. Therefore, in contrast to a lack of effect of AIE on fear extinction, AIE exposed male rats exhibited significant alterations during assessment of post-extinction fear memory. The reduction in recall (observed as an increase in freezing) appeared to represent a protracted change since an increase in freezing was also observed during spontaneous recovery assessed 21 days after extinction training. A previous study conducted in male Sprague-Dawley rats also reported that intermittent ethanol exposure during early adolescence disrupted fear conditioning in adulthood (Broadwater and Spear, 2013). While that study did not observed an effect of AIE on the acquisition of condition fear, they did report reductions of contextual renewal similar to the present study. A number of previous studies, mostly conducted in male rats, have shown that repeated episodes of binge-like adolescent alcohol exposure leads to deficits in behavior control in adulthood (Spear, 2018). Therefore, given the overlap of the neurocircuitry of fear (e.g., mPFC, HP, BLA) with the neurocircuitry of cognitive and emotional behaviors, it is not surprising that AIE-induced alterations in anxiety and cognition also extend to long-term alterations in threat responding. Adolescence is a period during which threat monitoring/response and the neurocircuits that mediate this behavior are highly plastic and undergoing rapid changes (Gerhard et al., 2021), and emotional experiences during this time can profoundly shape long-term functioning of the neurocircuitry that mediates affective behavior. Thus, our observation may have important translational implications as difficulties in assessing and appropriately responding to potential threats is a key feature of many psychiatric disorders that may be impacted by a history of adolescent alcohol abuse.

Another interesting observation in the present study was the striking differences between male and female rats in fear conditioning and recovery. In humans, the vulnerability to develop fear and anxiety-related disorders is twice as high in females compared to males (Day and Stevenson, 2020). However, results from studies investigating sex differences in rodent are less clear. While studies of Pavlovian fear learning suggest that female rodents typically exhibit reductions in contextual fear conditioning (Maren et al., 1994; Pryce et al., 1999; Wiltgen et al., 2001; Kosten et al., 2005; Gresack et al., 2009; Ribeiro et al., 2010), the results of fear extinction have been mixed (Velasco et al., 2019). Regardless, a number of studies have reported reduced contextual fear conditioning in females compared to males as evidenced by reductions in freezing during later retrieval tests of contextual fear memory. In the present study, female rats exhibited significant reductions in conditioned freezing compared to males, more rapid extinction of freezing, lower levels of freezing during recall and contextual renewal, and a near complete absence of freezing during spontaneous recovery. The latter observation of the reduction in spontaneous recovery in female rats appears to be in contrast to a previous report that females exhibit increases in spontaneous recovery (Fenton et al., 2014). However, considerable methodological and procedural differences between the two studies caution against a direct comparison of the results. For the most part, our observations are in general agreement with the studies showing female rats exhibit reduced acquisition of conditioned freezing and contextual fear retention compared to males. They are also in agreement with a recent study showing that AIE exposed male mice exhibited enhanced contextual fear acquisition compared to the male Air controls (Kasten et al., 2020). This effect was sex-specific as AIE did not alter fear acquisition in female mice. To ascertain whether the lower level of fear conditioning observed in the female rats may have contributed to an apparent reduction in fear extinction and responding, we classified female rats as either high conditioning subjects or low conditioning subjects. This analysis revealed that both the high and low conditioning groups of female rats displayed similarly low levels of freezing during test of recall, renewal and spontaneous recovery.

It is worth noting that caution should be exercised when interpreting results of sex differences based solely on freezing behavior. The amount of time an animal spends freezing during the CS is typically interpreted to reflect the strength of the tone-shock association, with low freezing reflecting a weak associative memory. However, female rats have been reported to be more active than male rats and may adopt more active avoidance strategies compared to less active male rats (Beatty and Beatty, 1970; Beatty and Fessler, 1976; Hyde and Jerussi, 1983; Steenbergen et al., 1990; Fernandes et al., 1999; File, 2001). Consistent with this, a novel form of active fear avoidance—termed “darting”—has been observed in females rats during fear conditioning (Gruene et al., 2015; Colom-Lapetina et al., 2019). Animals that display this form of coping behavior are also less likely to freeze in response to the tone, leading to the suggestion that darting behavior in females may represent an active coping response (Gruene et al., 2015; Jones and Monfils, 2016). Furthermore, these animals also exhibited more rapid reductions in both darting and freezing behaviors during extinction, and thus darting may be a form of active coping that promotes long-term reductions in fear. Studies have also shown that female rats tend to be more risk aversive than male rats and display greater active avoidance (Orsini et al., 2016; Orsini et al., 2021), which may contribute to greater vulnerability to anxiety-related disorders in women compared to men (Li and Graham, 2017).

Another caveat to interpretation of our data relates to potential differences in sensitivity between males and females to foot shock. As with most studies that compare fear conditioning in males and females, we used the same shock intensity (0.75 mA) during conditioning in both sexes. While we cannot rule out that differences in sensitivity to the shock contributed to our observations of sex difference in fear conditioning and recovery, we believe it is unlikely to have played a significant role. In fact, female rats have been reported to be more sensitive, not less sensitive, to shock than males (Beatty and Fessler, 1976; Kosten et al., 2005; Dalla and Shors, 2009).

In contrast to observations in adult male rats, adult AIE exposed female rats did not exhibit differences in freezing compared to the Air control females during of fear conditioning, extinction, or fear recovery. In our AIE procedure, we quantify the level of behavioral intoxication immediately upon removal of the animals from the chambers, and then use that information to guide subsequent dosing adjustments in the amount of alcohol vapor delivered to the chambers during the next exposure cycle. This approach resulted in only slightly different levels of intoxication in the female rats compared to the male rats (2.4 ± 0.09 for female rats versus 2.66 ± 0.16 for male rats on the intoxication rating scale). However, the corresponding blood ethanol levels in the females were significantly lower than the levels in males (271 ± 12.7 mg/dl for the female rats versus 383 ± 9.6 mg/dl for the male rats). While the lower levels of blood ethanol in the female rats could in theory have differentially impacted fear conditioning in adulthood, it should be noted that these blood ethanol levels are relatively high and both model extreme binge intoxication in humans.

As noted above, AIE exposure results in impairments in behavioral flexibility and cognitive control in the adults. We have also previously demonstrated that administration of CDPPB, a PAM of mGlu5 receptors with pro-cognitive actions, can reverse these deficits (Gass et al., 2014a). In the present study, we extended these observations to threat responding by demonstrating that PAM of mGlu5 receptors completely prevented the alterations in fear recovery in the male AIE exposed rats. Importantly, while CDPPB was administered only during fear extinction training, its effect on fear recovery was long-lasting as it normalized the subsequent AIE-induced alterations in fear recall, renewal, and spontaneous recovery. This observation may have important clinical implications for pharmacological treatments aimed at preventing the re-emergence of maladaptive fear and intrusive memories that are problematic in many psychiatric disorders. Due to the overlapping neurocircuitry associated with AUD and trauma-related disorders such as PTSD, current behavioral therapies utilize basic learning and memory principles (e.g., extinction-based exposure therapy). While they have been used as a treatment for both PTSD (Kessler et al., 1995) and drug addiction (Conklin and Tiffany, 2002), they have shown limited efficacy. The results from these studies suggest that a combination of behavioral and pharmacological treatment may be a more effective therapy for individuals who suffer from these debilitating disorders.