Peri-adolescent ethanol exposure differentially affects mechanical and thermal sensitivity in rats.

Male and female Long Evans rats bred at Binghamton University were used in this study. Rats were weaned at PD 21 and pair-housed with an age-matched and same-sex cagemate from another litter and placed on a 12-hour light-dark cycle with lights on at 7:00 am and off at 7:00 pm. Male and female rats were split into two exposure groups – AIR and aCIE (details below). To minimize litter effects, no more than 1–2 pups per litter were assigned to each experimental condition. Rats were given access to standard rat chow (Purina Lab Diet Irradiated 5LOD) and water ad libitum . All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Binghamton University, an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited institution. Male and female Long Evans rats were exposed to air (AIR; male, n = 16; female, n = 16) or ethanol vapor (aCIE; male, n = 14; female, n = 16) throughout adolescence and emerging adulthood (PD 28 −68, Fig. 1A ) for a total of ten cycles. Each cycle consisted of two days ON (12-hour AIR or aCIE exposure at the onset of the dark cycle each day) followed by two days OFF (exposure to room air, withdrawal period). Because food pellets absorb ethanol vapor, after each AIR or aCIE exposure, food pellets were replaced. After the second day of exposure within a cycle, cages were changed. Ethanol concentration in vapor chambers was adjusted systematically to maintain blood ethanol concentrations approximately at 200 mg/dL. Two ethanol exposed males were excluded from all experiments due to intoxication-induced hypothermia and dehydration during exposure paradigm. To verify ethanol exposure, blood was collected from the tail vein immediately following the second exposure of the first, fourth, seventh, and tenth cycles. Blood samples were centrifuged at 4°C for 20 minutes at 14,800 RPMs to separate serum from plasma. Once separated, serum was used to quantify blood ethanol concentration (BEC) using ANALOX (Model Number AM1; Analox Instruments Ltd. London, UK). A two-way mixed effects ANOVA comparing BECs across the four timepoints between the two sexes ( Fig. 1B ) revealed a main effect of cycle (F (3, 109) = 20.19, p <0.0001 ) and a significant cycle X sex interaction (F (3, 109) = 8.175, p = <0.0001). Šídák’s multiple comparisons test revealed significantly higher BECs at cycle 1 in aCIE-exposed female compared to male rats ( p = 0.0301 ) and significantly higher BECs at cycle 7 in aCIE-exposed male compared to female rats ( p = 0.0003). We conducted three behavioral experiments to characterize this aCIE exposure model for mechanical sensitivity and thermal hyperalgesia, as well as aversion learning and retention ( Fig. 1C ). The protocol used produced 4 approximately equal groups, AIR (n = 16) and aCIE (n = 14) male rats and AIR (n = 16) and aCIE (n = 16) females. Experiments 1 and 2 were counterbalanced and conducted each week. Mechanical and thermal sensitivity testing began one week following the last alcohol exposure and continued weekly until the fourth week of abstinence. For both mechanical and thermal sensitivity, the right hind paw was tested. Because this is not an injury model, we did not have a priori rationale to measure bilaterality. Rats were tested on all three experiments at the onset of their light cycle each day. To assess mechanical sensitivity, we used the same procedure as our previous study 14 . Briefly, rats were placed in Plexiglas enclosures (18” (Length) X 9.5” (Width) X 12” (Height)) with a metal grid floor and habituated for 30 mins per day for two days at the time when testing would occur. Testing was conducted on the third day after a 10-minute habituation period. During the test, the plantar surface of the right hind paw was probed using Von Frey filaments (Touch Test Sensory Probes; 58011; Stoelting IL), calibrated to exert target forces ranging from 0.008 g to 300 g. Filaments were applied in an ascending manner until a behavioral response of paw withdrawal or licking behavior was observed. Once a filament elicited a response, a lower filament was then tested to identify the paw withdrawal threshold. If the lower filament did not elicit a response, the higher filament was tested again. This up-down process was repeated until a paw withdrawal response was elicited for the same filament for three consecutive trials. The value of this filament was then recorded as the paw withdrawal threshold. Mechanical sensitivity was measured during acute and protracted abstinence – once per week from weeks one through four following the last air or ethanol vapor exposure. This behavioral assessment was counterbalanced with an assessment of thermal hyperalgesia (described below). For scientific rigor, Von Frey assessment was conducted by an experimenter blinded to experimental conditions. To assess thermal hyperalgesia, we used the Hargreaves plantar test (UGO Basile Hargreaves Apparatus, Gemonio VA, Italy). Rats were placed in a Plexiglas enclosure (10.5” (Length) X 12” (Width) X 12” (Height)) with a glass bottom (31.5” (Length) X 12” (Width)) and habituated for 30 minutes per day for two days prior to testing at the time testing would occur. Testing was conducted on the third day after a 10-minute habituation period. During the test, an IR beam (52%; power: 201mW/cm 2 , UGO Basile Heat-Flux I.R. Radiometer Cat. 37300, Gemonio VA, Italy; approximate temperature: 30°C) was used to apply heat through the glass on the plantar surface of right hind paw. This IR setting was selected based on prior literature 33 , 34 . Latency to withdraw the paw was recorded following application of the heat stimulus; the trial was ended after 20 seconds in the absence of a response to avoid tissue damage to the plantar surface. An average withdrawal latency of three trials was recorded. Hargreaves was conducted once per week from weeks one through four following the last air or ethanol vapor exposure. The experimenter was blinded to exposure conditions to increase rigor. Because hypersensitivity to mechanical or thermal stimulation could be aversive, we reasoned that the mechanical and thermal stimulation mediated nociceptive response could be generalized to conditioned fear responses. Therefore, in the same group of rats, during the sixth week of abstinence, we conducted a three-day context-dependent fear conditioning protocol using chambers (32 × 25 × 25 cm) made of clear polycarbonate (top, front walls), white acrylic (back wall), and stainless steel (sides, shock grids, drop pan) material. The grid floors consisted of 19 parallel 4.8-mm diameter rods situated 1 cm apart. Fear conditioning proceeded as previously described 35 . On Day 1, after a 5-minute chamber acclimation period (pre-shock) rats were administered with three shocks (1 mA for 1 sec each) delivered at 6, 7.5, and 9 minutes, followed by a 4-minute consolidation period (post-shock) to form an association between the unconditioned stimulus (shock) and the context (conditioned stimulus). Twenty-four hours later, rats were returned to chambers for 15 minutes in the absence of foot shocks to test retention (TOR) of the context. Finally, 24 hours later rats were again returned to the chamber for 15 minutes to assess extinction (EXT). During these testing days freezing behavior was recorded using VideoFreeze ™ Video Fear Conditioning Software (Med Associates, SOF-843). The data were quantified as percentage of the time spent freezing for the following time bins: Pre-Shock (Minutes 1–5), Shock (Minutes 6–9), post-shock (Minutes 10–13), for each condition. TOR and EXT are representative of minutes 1–15 during each testing day. We then calculated total time spent freezing in 1-minute bins to compare percent of the total time spent freezing on each testing day. All data in this study were analyzed using Graphpad Prism 10.5 (GraphPad Software, La Jolla, CA, USA). All data are reported as mean and standard error of the mean (SEM). Because we observed a significant difference in BECs in male and female rats across the exposure paradigm, data from male and female groups were analyzed separately. All Von Frey withdrawal threshold and Hargreaves withdrawal latency data were assessed using a repeated measures (RM) two-way analysis of variance (ANOVA), with exposure or sex and abstinence time as the independent variables and paw withdrawal threshold force (Von Frey) or paw withdrawal latency (Hargreaves) as the dependent variable. We then used Pearson’s R correlations on all Von Frey and Hargreaves data to assess association between pain modalities across the 4 weeks. For fear conditioning data, to assess freezing time differences during the shock day we used RM two-way ANOVA, with exposure or sex, and event (pre-shock, shock, post-shock) as independent variables and percent time spent freezing as the dependent variable. Furthermore, we assessed differences in time spent engaged in freezing behaviors across the three-day procedure, with day, and exposure or sex as independent variables and percent of time spent freezing as the dependent variable. We used an unpaired Student’s t -test to compare freezing behavior during TOR between AIR-exposed male and female rats or between AIR- and aCIE-exposed male or female rats. All RM two-way ANOVAs were followed by Sidak’s post-hoc pairwise comparisons. The statistical significance value was set to p <0.05 and a statistical trend was defined as p <0.08.

To assess the effect of aCIE exposure on mechanical sensitivity we used Von Frey filaments and measured the force at which the male and female rats exposed to AIR or aCIE exhibited paw withdrawal ( Fig. 2A ), referred to as the ‘Withdrawal Threshold Force’. A RM two-way ANOVA comparing AIR-exposed male and female rats ( Fig. 2B ) revealed main effects of sex (F (1,30) =25.53; p <0.0001) and week of abstinence (F (3,90) =5.226; p =0.0023), whereby AIR-exposed female rats exhibited a significantly lower paw withdrawal threshold compared to AIR-exposed male rats. However, there was no significant interaction between sex and week of abstinence (F (3, 90) = 1.292; p =0.2822). Given the paw withdrawal threshold differences in AIR-exposed male and female rats, and the sex differences observed in the BECs, we analyzed aCIE exposure-associated changes in mechanical sensitivity separately in male and female rats. A RM two-way ANOVA comparing paw withdrawal threshold exhibited by AIR- and aCIE-exposed male rats revealed an interaction between exposure and week of abstinence (F (3, 84) = 3.048; p =0.0331) and a main effect of exposure (F (1,28) =10.75; p =0.0028), and a main effect of week of abstinence (F (3,84) =4.198; p =0.0081) suggesting that the aCIE exposure produced mechanical hypersensitivity in an abstinence time-dependent manner ( Fig. 2C ). Indeed, Šídák’s multiple comparisons test revealed that the aCIE-exposure-associated mechanical hypersensitivity exacerbated during protracted withdrawal – abstinence weeks 1 ( p =0.0450), 3 ( p =0.0058) and 4 ( p =0.0399) in male rats. A RM two-way ANOVA comparing AIR- and aCIE-exposed female rats ( Fig. 2D ) revealed a main effect of exposure (F (1, 30) =7.232; p =0.0116) such that aCIE-exposed female rats exhibited heightened mechanical sensitivity during withdrawal. However, neither an effect of abstinence week (F (3, 90) =1.852; p =0.1435) nor an interaction between exposure and week of exposure (F (3, 90) =1.144; p =0.3357) were observed. Overall, aCIE exposure augmented mechanical sensitivity during forced abstinence in both sexes. To assess thermal hyperalgesia following aCIE exposure, we used the Hargreaves plantar test during weeks 1–4 of forced abstinence ( FIG. 2A ). A RM two-way ANOVA comparing AIR-exposed male and female rats ( FIG. 2E ) revealed a main effect of sex (F (1,30) =6.385; p =0.0170) whereby AIR-exposed female rats exhibited a significantly lower paw withdrawal latency compared to AIR-exposed male rats. However, there was no significant main effect of week of abstinence (F (3,90) =0.8065; p =0.4935) or an interaction between sex and week of abstinence (F (3, 90) =0.8007; p =0.4967). We analyzed aCIE exposure-associated changes in thermal hyperalgesia separately in male and female rats given the sex differences in thermal sensitivity and BECs. A RM two-way ANOVA comparing paw withdrawal latency exhibited by AIR- and aCIE-exposed male rats ( Fig. 2F ) revealed a statistically significant interaction between exposure and week of abstinence (F (3, 84) = 4.440; p =0.0060) and a main effect of week of abstinence (F (3, 84) = 3.450; p =0.0202), but no significant effects of exposure (F (1,28) =3.341; p =0.0782). Indeed, Šídák’s multiple comparisons test revealed that aCIE exposure augmented thermal sensitivity in males during week 3 ( p =0.0458) and week 4 ( p =0.0283) of forced abstinence. A RM two-way ANOVA comparing AIR- and aCIE-exposed female rats ( Fig. 2G ) revealed no significant effect of exposure (F (1, 30) = 0.7131; p =0.4051), but no effect of week of abstinence (F (3, 90) = 0.4854; p =0.6933) or an interaction between exposure and week of abstinence (F (3, 90) =1.052; p =0.3736). Overall, aCIE exposure selectively enhanced thermal hyperalgesia in males during protracted abstinence with no effect of exposure in females. We next examined a possible correlation between mechanical sensitivity and thermal nociception during early abstinence (Week 1) and protracted abstinence (Week 4) using Pearson’s R correlations. During Week 1 of abstinence (early abstinence) in male rats we observed a statistically significant negative correlation ( r (28) = −0.4844; p= 0.0067) such that a higher paw withdrawal threshold during mechanical sensitivity assessment was associated with a lower paw withdrawal latency in thermal hyperalgesia ( Fig. 3A ). Interestingly, this correlation reversed during protracted abstinence (Week 4); we observed a positive correlation ( r (28) =0.2001; p =0.2890; Fig. 3B ) though this was not significant. In contrast, in female rats ( Fig. 3C ) we observed no significant correlations ( r (30) =0.1435; p =0.4335) during early abstinence (Week 1) nor during protracted abstinence (Week 4) ( r (30) = −0.009567; p =0.9586; Week 4; Fig. 3D ). Overall, these data suggest that the association between pain modalities varies depending on sex and ethanol withdrawal timepoint whereby during Week 1 of abstinence, male rats display a transient negative association during Week 1 of abstinence. During the Hargreaves test, we observed heightened avoidance behaviors and general hyperactivity in the female rats. Thus, in the same group of rats examined on Von Frey and Hargreaves, we used a 3-day context-dependent fear conditioning paradigm during Week 6 of forced abstinence ( Fig. 4A ) and assessed possible differences in fear learning. A RM two-way ANOVA comparing percentage of time spent freezing during shock day between AIR-exposed male and female rats ( Fig. 4B ) revealed a main effect of the conditioning period (F (2,44) =512.2; p < 0.0001) such that no freezing was observed during acclimation (pre-shock period), but freezing behavior increased significantly during the application of foot shocks (shock period) and remained high during the post-shock period in both male and female rats. This freezing behavior effect was not influenced by sex (F (1, 22) = 0.04517; p =0.8337), there was no interaction between sex and conditioning period (F (2, 44) = 0.4412; p =0.6461). Similar to the AIR-exposed male and female groups comparison, a RM two-way ANOVA comparing unconditioned freezing between AIR- and aCIE exposed male rats ( Fig. 4C ) revealed a significant main effect of conditioning period (F (2,40) =483.6; p <0.0001) such that freezing behavior increased after the shock period in both AIR- and aCIE-exposed male rats. Exposure did not affect freezing (F (1,20) =427.8; p =0.1293), and there was no interaction between exposure and conditioning period (F (2,40) =2.503; p =0.1293). A similar RM two-way ANOVA comparing unconditioned freezing between AIR- and aCIE-exposed female rats ( Fig. 4D ) showed no freezing during acclimation (pre-shock) but a significant increase during (shock) and after the shock (post-shock; main effect of conditioning period; F (2,44) =239.9; p <0.0001). As with the other comparisons, there were no effects of exposure (F (1,22) =1.357; p =0.2565) or interaction between exposure and conditioning period (F (2,44) =1.491; p =0.2363). Overall, this suggests that aCIE exposure did not alter the acquisition of fear conditioning. Qualitatively, the data suggested some minor (albeit insignificant) differences in freezing during the shock period. Therefore, in order to fully assess the unconditioned freezing, we quantified freezing in 1-minute time bins and analyzed the 13-minute time course on Day 1 – conditioning day ( Supplementary Fig. 1A - C ). While we observed a main effect of time, neither sex nor exposure affected freezing in response to shock. To assess differences in memory retention, we measured the conditioned freezing response 24 hours after the shock day (TOR). An unpaired t -test comparison between AIR-exposed male and female rats showed that female rats spent significantly less time freezing than male rats ( t (22) =2.840; p =0.0095; Fig. 4E ). When comparing the effect of aCIE exposure in male rats, there was no significant difference in the conditioned freezing response ( t (20) =0.3127; p =0.7577; Fig. 4F ). Similarly, a comparison of the effect of aCIE exposure in female rats, also revealed no difference in conditioned freezing ( t (22) =0.8509; p =0.4040; Fig. 4G ). We then assessed percentage time spent freezing across the three trial days which are represented as average percent time spent freezing for the following windows, during the shock time period (6–9 minutes) and the full 15-minute trial for TOR and extinction. A RM two-way ANOVA comparing percentage of time spent freezing during shock on shock day, TOR, extinction (EXT) day between AIR-exposed male and female rats ( Fig. 4H ) revealed a significant interaction between sex and day (F (2, 44) =6.035; p =0.0048), as well as main effects of day (F (2, 44) =7.305; p =0.0018) and sex (F (1, 22) =4.783; p =0.0397) suggesting that freezing behavior was greater in male rats compared to female rats. Indeed, Šídák’s multiple comparisons test revealed that during the TOR, AIR-exposed male rats spent significantly more time freezing compared to female rats ( p =0.0030 ). A similar comparison with RM two-way ANOVA between AIR- and aCIE-exposed male rats ( Fig. 4I ) revealed a main effect of day (F (2, 40) =12.22; p < 0.0001), but no effect of exposure (F (1, 20) =0.6051; p =0.4457) or interaction between exposure and day (F (2, 40) =2.694; p =0.0798). Similarly, A RM two-way ANOVA comparing freezing behavior between AIR- and aCIE-exposed female rats ( Fig. 4J ) revealed a significant main effect of day (F (2, 44) =23.38; p < 0.0001), but no effect of exposure (F (1, 22) =0.8119; p =0.3773) and no interaction between day and exposure (F (2, 44) =0.2344; p =0.7920). The full-time course of percent time freezing for TOR and EXT are shown in Supplementary Figure 2A - F . Together these data suggest that while sex influenced conditioned freezing during TOR, neither unconditioned nor conditioned freezing behavior was affected by aCIE exposure.

Overall, aCIE exposure promotes heightened pain sensitivity during protracted withdrawal. These findings suggest a significant pain-related health risk for individuals with a history of alcohol misuse during adolescence. Chronic pain has high comorbidity with depression and anxiety 76 – 78 , as well as an increased risk of substance use disorders 79 – 81 . Furthermore, individuals with AUD are at a particularly elevated risk of poor pain management and health outcomes 5 , 7 , 12 , 13 , making the interaction between these disorders an important area of research to improve pain management.

In this study, we aimed to behaviorally characterize the aCIE exposure model with a particular focus on mechanical and thermal sensitivities. The aCIE exposure resulted in BECs that varied in a cycle- and sex-dependent manner, and produced mechanical hypersensitivity during early and protracted withdrawal in both sexes, but thermal hyperalgesia selectively in male rats during protracted abstinence. Further, freezing behaviors during shock, time of retention, and extinction were not affected by aCIE exposure in either sex. Together, these results suggest that aCIE exposure alters nociception, but responsivity is not generalized to an aversive stimulus (shock) applied to the footpad. These novel findings show that nociceptive effects of aCIE exposure are sex and pain modality specific. BECs observed in female and male rats in the current study were within range of those typically achieved for vapor exposure (175–250mg/dL) 16 , 36 – 38 . However, a time course of BEC comparison between male and female rats revealed higher BECs in female rats during Cycle 1, an effect that inverted at Cycle 7. This suggests that ethanol sensitivity may shift across developmental periods differently in male and female rats. Following an 8 day 2 hour-access drinking paradigm beginning at PD 28, females tended to have lower BECs than males, despite higher levels of ethanol consumption 39 , indicating that females require higher levels of exposure to ethanol to produce BECs comparable to their male counterparts. Interestingly, 18 year old women in a clinical study using intravenous-self administration of alcohol exhibited lower blood alcohol levels compared to age-matched men, despite comparable differences in subjective ratings of intoxication symptoms 40 . Together these studies suggest ethanol dose-dependently affects BECs by sex across development, but this has not been systematically examined. Congruent with previous rodent and human clinical reports 14 , 41 – 43 , AIR-exposed female rats in the current study exhibited greater mechanical and thermal hyperalgesia compared to male rats, suggesting baseline sex-differences in pain sensitivity across multiple modalities. In both chronic pain and AUD treatment-seeking population, women display heightened pain sensitivity and prevalence for pain disorders compared to men 13 , 43 – 45 , sex differences potentially driven by neuroendocrine interactions. Both clinical and preclinical studies suggest an estrogen-dependent augmentation in proinflammatory biomarkers and pain responsivity, and testosterone-associated protection against pain. For example, gonadectomized male rats exhibit formalin-induced hyperalgesia, an effect further potentiated with estradiol administration 46 . Moreover, estrogen application to cultured trigeminal neurons from ovariectomized rats elevates calcitonin gene-related peptide, a proinflammatory neuropeptide associated pain 47 . Further, high levels of prolactin are implicated in chronic migraines and endometriosis, conditions with a higher prevalence in women 48 , 49 . In preclinical research, plantar incision elevates serum prolactin levels in male and female mice, with exacerbation in the females 50 , and the prolactin system activation promotes mechanical allodynia, with disruption reducing post-operative hyperalgesia selectively in female mice 50 , 51 . Together, these findings suggest that neuroendocrine function may be a critical regulator of female-specific pain sensitivity. Consistent with our previous report 14 and others 15 – 17 , 52 , here we showed that aCIE exposure augmented mechanical sensitivity during protracted abstinence in male and female rodents. Interestingly however, in one study male rats exhibited heightened mechanical sensitivity during acute and protracted abstinence, but female rats showed this effect only transiently during protracted abstinence 17 . This difference is likely due to strain differences (Wistar rats 17 vs. Long Evans, current study), and differences in behavioral assessment (testing during acute withdrawal in between exposures through adolescence and four weeks of abstinence 17 vs. testing only in adulthood after cessation of the exposure protocol, current study). It is possible that repeated testing during acute withdrawal throughout the exposure could have differentially primed pain behaviors or unsensitized the animals in a sex-dependent manner. In general, adolescent alcohol exposure likely dysregulates the nociceptive system and promotes central sensitization, which is responsiveness of nociceptive neurons to subthreshold stimuli 53 , 54 , in this case, von Frey filaments. For example, following chronic low dose ethanol exposure during adulthood, female mice display augmented mechanical allodynia to an inflammatory challenge, accompanied by dorsal root ganglion inflammation and sensitization to pain 55 . Moreover, both sexes of mice exposed to four day drinking in the dark protocol, and Marchigian Sardinian alcohol-preferring rats exhibit persisting mechanical allodynia 56 , 57 . Clinically, AUD is associated with pain-related central sensitization 53 . This hyperresponsivity of pain processing commonly co-occurs with nerve damage, resulting in painful chronic neuropathy and heightened pain perception 58 . Neuropathy affects both large and small nerve fibers in patients with AUD 59 , 60 , highlighting the disruption of both non-noxious tactile sensation and noxious pain sensation, respectively 61 , 62 . This central sensitization may in fact result from maladaptive neuroplasticity in the overlapping AUD and chronic pain circuitry 11 , 63 . Unlike reports of mechanical sensitivity, the reports assessing the impact of adolescent alcohol exposure on thermal hyperalgesia are inconsistent. Here we observed aCIE-associated thermal hyperalgesia selectively in aCIE-exposed male rats during protracted abstinence when compared to their AIR-exposed counterparts. While one previous study reports no effect of adolescent alcohol exposure on thermal hyperalgesia in male or female rats 16 , others report thermal hyperalgesia in male rodents 15 , 17 , 18 . Interestingly, chronic ethanol vapor exposure in adults promotes thermal hyperalgesia during acute abstinence selectively in male rats 64 . These findings along with the data in the current study may suggest that adolescent alcohol exposure induced thermal hyperalgesia may be male specific. Ultimately, there is a scarcity in literature dissecting these sex differences and more research in this area is needed. The associations between mechanical and thermal modalities are sex and abstinence timepoint specific. In male rats, during Week 1 of testing, mechanical and thermal sensitivities are negatively correlated, but notably this association is no longer present by Week 4 of assessment. This may be explained by a few factors. First, the data suggest that AIR-exposed male rats habituate to the thermal stimulus, while the aCIE-exposed male rats develop hypersensitivity dissociating the correlation observed at initial testing (Week 1). Additionally, both preclinical 65 – 68 and clinical 69 , 70 studies have highlighted that pain is heterogenous in symptom presentation and in molecular signatures across modalities. It is therefore possible that adolescent alcohol exposure differentially alters these diverging pain-regulating neural mechanisms in both a sex- and abstinence time-specific manner, accounting for the lack of consistent association between the two pain modalities. Anecdotally, we observed thermal sensor avoidance-associated freezing behavior and heightened locomotion during the Hargreaves assay. Freezing and hyperlocomotion or darting behaviors are characteristics of fear responses in male and female rats, respectively 71 . We therefore hypothesized that aCIE-associated changes in fear learning may generalize to an aversive stimulus (shock). However, we did not observe aCIE specific effects on unconditioned (during shock) or conditioned (retention) freezing behavior, or extinction of fear memory in either sex. This was surprising as previous studies report aCIE exposure associated disruptions in unconditioned and conditioned fear responses, as well as fear memory 28 , 32 . These effects are dependent on age-of-ethanol exposure during adolescence, sex, and length of the abstinence period, with attenuation in fear memory during early and protracted abstinence, and disruptions in fear learning during early abstinence. Ethanol exposure during mid-late adolescence does not impair contextual fear conditioning, but promotes resistance to fear memory extinction 28 . In the current study, aCIE exposure occurred throughout adolescence (early-late) and contextual fear learning, retention, and extinction was measured six-weeks into abstinence. Thus, it is possible that our exposure covering the entirety of adolescence masked any early-adolescent ethanol exposure associated changes. Other potential explanations for this discrepancy include the mode of ethanol administration (inhalation), strain of rats used, or the duration of abstinence from the final ethanol exposure. One limitation of the present work is that fear conditioning was tested six weeks into abstinence, two-weeks post termination of mechanical and thermal testing. Finally, it should be noted that the shock intensity used in tests of contextual fear conditioning was 1.0 mA. While this shock intensity is within the range of what is commonly used, assessing fear conditioning across a range of shock intensities might yield a more sensitive discrimination of aversive conditioning in aCIE-exposed rats in future studies. Clinical evidence suggests that chronic pain promotes avoidance behaviors, particularly when faced with fear of experiencing pain, whereby individuals have a goal of avoiding future pain 72 , 73 . Recent preclinical studies have used the platform-mediated avoidance task to assess goal-directed threat avoidance 74 , 75 . Interestingly, adolescent alcohol exposure promotes goal-directed avoidance behaviors on initial test day (learning) in male rats, but during extinction (memory) in female rats compared to their respective air-exposed counterparts 75 . This assay may be more translatable to the escape-like behaviors we observed during the Hargreaves test and should be incorporated in future studies to assess goal-directed threat avoidance.

Adolescent alcohol use is a public health concern in the United States with reports stating that 7 million youth (ages 12–17) consumed alcohol within the past month and about 7.6% of youth 12–20 report engaging in binge drinking in 2024 1 , 2 . Alcohol misuse potentiates pain in both adult 3 – 8 and adolescent populations 9 , 10 . Altogether this elevation in pain perception can in turn potentiate negative affect and further perpetuate cycles of alcohol misuse elevating the risk of alcohol use disorder (AUD) 11 . Alcohol misuse affects multiple modalities of pain in the clinical population. For example, treatment seeking individuals experience thermal hyperalgesia during alcohol withdrawal 12 and binge-level alcohol consumption reduces mechanical pain threshold 8 , with a higher prevalence of pain among women (62.57%) compared to men (47.35%) with AUD 13 . AUD complicates pain management, especially in individuals with a history of adolescent alcohol use. Our goal in the current study was to characterize a murine model of adolescent ethanol exposure that modifies clinical pain outcomes. Preclinical studies examining the impact of chronic ethanol exposure during adolescence on pain sensitivity in adulthood across different pain modalities reported heightened mechanical sensitivity during withdrawal in male rodents 14 – 17 . However, data in female rodents is mixed with some studies reporting heightened sensitivity 14 , 16 and others showing no effect 17 . Similarly, assessments of thermal hyperalgesia showed either a reduction in thermal hyperalgesia, no change, or potentiation depending on abstinence time and sex 15 – 18 . For example, adolescent ethanol exposure promoted thermal hyperalgesia during acute 17 and protracted 15 , 17 abstinence in male rodents, with only a transient emergence during protracted abstinence in female rats 17 . Interestingly however, another study found no impact of adolescent ethanol exposure on thermal hyperalgesia in male or female rats during early or protracted abstinence 16 . Given these mixed findings, we assessed mechanical sensitivity and thermal hyperalgesia with an inhalation model of adolescent chronic intermittent ethanol (aCIE) exposure in male and female rats. In addition to changes in pain sensitivity, alcohol exposure during adolescence disrupts typical development of neural circuits involved in affect-related processing, often resulting in negative affect behaviors in humans 7 , 11 , 19 and rodents 20 – 22 . In murine models, though adolescent ethanol exposure-associated disruption in anxiety-like behaviors 23 – 27 and social interaction 28 – 31 were observed in both sexes, changes in fear conditioning behaviors depend on sex, age of ethanol exposure, and duration of abstinence. For example, ethanol exposure between PD 28–44/48 (early-mid adolescence) disrupted acquisition of contextual fear conditioning after 16 days 32 , but not 22 days 28 of abstinence in male rats. With respect to retention, PD 28–44/48 exposure attenuated retention after 16 and 22 days of abstinence in male rats 28 , 32 . Neither of these effects were found in female rats 32 . Surprisingly, ethanol exposure from PD 35–55 (mid-late adolescence) did not affect acquisition or retention, but prevented extinction of fear memory after 22 days of abstinence 28 . Overall, these studies suggest that adolescent alcohol exposure influences aversion-based learning, albeit in an age-of-exposure and sex-specific manner. Given these findings, we reasoned that repeated testing of mechanical sensitivity and thermal nociception, could be generalized to conditioned fear responses. Thus, we assessed the impact of aCIE exposure on context-dependent fear conditioning, retention, and extinction, using shock-based fear conditioning procedures, which involve application of an aversive stimulus to the paws activating overlapping somatosensory and nociceptive inputs. In summary, here we further characterized the aCIE rat model that we developed previously 14 . Our overarching hypothesis was that aCIE exposure would augment mechanical sensitivity and thermal nociception. Furthermore, we expected to observe an aCIE-associated persistence of fear memory.