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Published in final edited form as: Psychopharmacology (Berl). 2024 Sep 30;241(12):2513–2523. doi: 10.1007/s00213-024-06693-8

BNST CRF RECEPTOR TYPE 1 MODULATES MECHANICAL HYPERSENSITIVITY INDUCED BY ADOLESCENT ALCOHOL EXPOSURE IN ADULT FEMALE MICE

Natalia B Bertagna a,b, Eleanor B Holmgren a,c, Sheila A Engi a,d, Linh Ha a, Fabio C Cruz b, Lucas Albrechet-Souza a,d, Tiffany A Wills a,d,e
PMCID: PMC12884584  NIHMSID: NIHMS2137142  PMID: 39348004

Abstract

Rationale

Alcohol exposure during adolescence has been linked to long-lasting behavioral consequences, contributing to the development of alcohol use disorder. Negative affect and chronic pain during alcohol withdrawal are critical factors influencing problematic alcohol use and relapse. Our previous research demonstrated that adolescent intermittent ethanol (AIE) vapor exposure elicits sex-specific negative affect-like behavior in adult mice following stress exposure. Additionally, AIE induces persistent mechanical hypersensitivity, which is accompanied by increased activation of corticotropin-releasing factor receptor type 1 (CRFR1) neurons in the dorsolateral bed nucleus of the stria terminalis (dlBNST).

Objectives

This study extends previous work by investigating plasma corticosterone levels and CRFR1 protein expression in the dlBNST following restraint stress exposure in adult mice with an AIE history. We also aim to explore the role of dlBNST CRFR1 signaling in mediating negative affect-like behavior and mechanical hypersensitivity.

Results

Female mice exhibited elevated plasma corticosterone levels compared to males following restraint stress. Moreover, females with AIE history showed higher expression of CRFR1 protein in the dlBNST compared to air controls. Antagonism of CRFR1 in the dlBNST blocked AIE-induced mechanical hypersensitivity in adult females but did not affect stress-induced negative affect-like behavior. In alcohol-naïve females, intra-dlBNST administration of a CRFR1 agonist induced mechanical hypersensitivity.

Conclusions

These findings provide new insights into the neurobiological mechanisms underlying stress-induced negative affect and pain-related behavior, both influenced by a history of adolescent alcohol exposure. The results suggest that CRFR1 antagonists warrant further investigation for their potential in addressing alcohol-related chronic pain.

Keywords: alcohol, adolescent, pain, bed nucleus of the stria terminalis, negative affect, mechanical hypersensitivity, corticotropin-releasing factor, corticotropin-releasing factor receptor type 1

Introduction

Adolescence is a critical period of brain development, during which individuals may be particularly vulnerable to the long-lasting consequences of repeated alcohol exposure (Lees et al. 2020). Early initiation of alcohol use significantly increases the risk of developing alcohol use disorder (AUD) and other adverse outcomes (Grant and Dawson 1997). AUD is characterized by cycles of excessive alcohol consumption followed by periods of abstinence, over time leading to persistent neuroadaptations that drive continued alcohol use (Koob and Le Moal 2005; Koob and Volkow 2016).

One proposed mechanism underlying AUD is negative reinforcement, where alcohol consumption temporarily alleviates the negative emotional states experienced during abstinence (Koob 2020; Koob and Le Moal 2001). Additionally, heightened pain sensitivity may play a role in AUD, as individuals with a history of chronic alcohol use often report more severe pain and a greater tendency to use alcohol for pain management compared to social drinkers (Brennan et al. 2005; Egli et al. 2012). The term hyperkatifeia has been introduced to describe the increased negative emotional and motivational symptoms, including pain pathology, observed during withdrawal from drugs of abuse (Koob 2021). While negative affect and psychiatric disorders are associated with problematic alcohol consumption in both genders, these factors are particularly relevant for women (Peltier et al. 2019). Indeed, women were shown to be more likely than men to consume alcohol during unpleasant emotional circumstances (Abulseoud et al. 2013).

Our laboratory has shown that adult female mice with a history of adolescent intermittent ethanol (AIE) vapor exhibit increased latency to consume a palatable reinforcer in the novelty-induced hypophagia (NIH) test following restraint stress. This response was not observed in female air controls or male mice (Kasten et al. 2020). Interestingly, chemogenetic stimulation of the dorsolateral bed nucleus of the stria terminalis (dlBNST) using designer receptors exclusively activated by designer drugs (DREADDs) produces a similar behavioral phenotype, mimicking the effects of restraint stress (Albrechet-Souza et al. 2024). Additionally, AIE exposure leads to persistent mechanical hypersensitivity, accompanied by increased activation of corticotropin-releasing factor receptor type 1 (CRFR1) neurons in the dlBNST (Bertagna et al. 2024). These findings suggest that adolescent alcohol exposure may induce a persistent hyperkatifeia-like state, potentially driving future alcohol consumption.

This study builds on our previous work by examining the role of dlBNST CRFR1 signaling in mediating negative affect-like behavior and mechanical hypersensitivity in adult mice with a history of adolescent alcohol exposure. In Experiment 1, we measured plasma corticosterone levels and CRFR1 protein expression in the dlBNST following restraint stress in adult male and female mice with a history of AIE or air exposure. Experiment 2 focused on the role of dlBNST CRFR1 signaling in negative affect-like behavior and mechanical hypersensitivity in females with and without an AIE history. These findings provide novel insights into the neurobiological mechanisms altered by adolescent alcohol exposure that contribute to the development of negative affect and pain-related behaviors, key factors driving AUD.

Materials and methods

Subjects

A total of 112 three-week-old and 12 eight-week old C57BL/6J mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Male and female mice were housed separately, in groups of four or five, with food and water available ad libitum, in a humidity- and temperature-controlled (22°C) vivarium on a 12-hours light-dark cycle (lights on at 7:00 AM). All procedures were approved by the Institutional Animal Care and Use Committee of the Louisiana State University Health Sciences Center and were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory animals (NIH Publications No. 8023, revised 1978).

Study design

In Experiment 1, adult male and female mice with a history of AIE or air controls were subjected to 1 hour of restraint stress. Trunk blood samples were collected immediately after the stressor for the assessment of plasma corticosterone levels. In a separate cohort of mice, euthanasia was performed 1 hour after restraint stress to examine the expression of CRFR1 protein in the dlBNST using western blot analysis (Fig. 1A).

Fig. 1.

Fig. 1

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General timelines. A Experiment 1: adolescent male and female mice underwent two 4-day cycles of 16-hour ethanol vapor exposure per day, separated by 3 days of no exposure, from postnatal day (PND) 27 to 38. Mice were then left undisturbed until adulthood (PND 70+) and subjected to 1 hour of restraint stress. Trunk blood was collected immediately after the stressor for plasma corticosterone analysis. In a separate cohort of mice, euthanasia was performed 1 hour after the stress for the measurement of CRFR1 protein expression in the dorsolateral BNST. B Experiment 2: adolescent female mice underwent ethanol vapor exposure and were left undisturbed until adulthood. Baseline mechanical sensitivity was measured using the von Frey (VF) test before the implantation of bilateral guide-cannulas. Mice received intra-dorsolateral BNST microinjections of the CRFR1 antagonist NBI or vehicle prior to exposure to restraint stress and were tested for negative affect-like behavior using the novelty-induced hypophagia (NIH) test. Next, the animals received a microinjection of NBI or vehicle and were tested for mechanical sensitivity. A separate cohort of alcohol-naïve adult female mice received intra-dorsolateral BNST microinjections of the CRFR1 agonist Stressin-1 (STR) or vehicle and were tested for mechanical sensitivity. AIE, adolescent intermittent ethanol

In Experiment 2, adult female mice with a history of AIE or air controls received intra-dlBNST microinjections of the CRFR1 antagonist NBI or vehicle prior to exposure to restraint stress and were tested for negative affect-like behavior using the NIH test. Next, these animals received a microinjection of NBI or vehicle and were tested for mechanical sensitivity using the von Frey test. A separate cohort of alcohol-naïve adult female mice received intra-dlBNST microinjections of the CRFR1 agonist Stressin-1 or vehicle and were tested for mechanical sensitivity (Fig. 1B).

Ethanol vapor exposure

Mice were exposed to AIE following the protocol described in previous studies (Albrechet et al. 2024; Bertagna et al. 2024; Kasten et al. 2020; 2021). In brief, the exposure to ethanol vapor occurred from 4:00 PM to 8:00 AM the following day, allowing for consistent blood ethanol concentrations ranging from 200 to 250 mg/dL. The AIE protocol consisted of two cycles, each lasting four days, with 16 hours of in-chamber sessions followed by 8 hours of out-of-chamber sessions. These cycles were separated by three days of no exposure, starting from postnatal day (PND) 27 and ending on PND 38. Prior to each ethanol vapor session, mice received an injection of the alcohol dehydrogenase inhibitor pyrazole (air control groups, 1 mmol/kg) or pyrazole + ethanol (AIE groups, 1 mmol/kg + 0.8 g/kg). In mouse models of alcohol vapor exposure, pyrazole is required to achieve blood ethanol concentrations at levels necessary to achieve dependence (Brandner et al., 2023). Male and female mice were exposed separately in dedicated vapor chambers. Control animals underwent an identical regimen, with the exception that they were exposed to water vapor instead of ethanol.

Acute restraint stress

Adult mice (PND 70+) were restrained in well-ventilated 50-mL conical tubes for 1 hour during the light phase of the light/dark cycle, between 9:00 AM and 12:00 PM. After the restraint period, mice were either immediately euthanized for plasma corticosterone analysis or left undisturbed for 1 hour before undergoing euthanasia for western blot analysis or the NIH test.

Plasma corticosterone response

Immediately after exposure to restraint stress, mice were euthanized by decapitation under isoflurane anesthesia, and trunk blood samples were collected in EDTA-covered tubes. The samples were centrifuged at 4000 rpm for 14 minutes at 4°C, and plasma was extracted and stored at −80°C. Total corticosterone levels were quantified in duplicate using the DetectX ELISA kit (Arbor Assays; Ann Arbor, MI, USA) in accordance with the manufacturer’s instructions (Albrechet-Souza et al. 2020).

Western blot analysis

One hour after exposure to restraint stress, mice were euthanized by decapitation under isoflurane anesthesia. Brains were promptly dissected, snap-frozen in −30°C isopentane, and stored at −80°C until microdissection. Tissue samples from the dlBNST were extracted from frozen coronal brain sections (500 μm thick) using a 15-gauge punch tool on a cryostat, in accordance with Paxinos and Franklin (2001). Tissue punches were stored at −80°C until homogenization. Proteins were separated using SDS-PAGE (10%) and transferred to nitrocellulose membranes, which were then blocked in 5% milk in TBST and incubated with CRFR1 antibody (1:1000; 720290; Thermo Fisher Scientific; Waltham, MA, USA). Infrared-conjugated secondary antibodies (LiCor Biosciences; Lincoln, NE, USA) were employed for detection using the Odyssey system (LiCor Biosciences). Densitometry analysis was performed using Image J (National Institutes of Health, Bethesda, MD, USA) on images that were linearly adjusted for brightness and contrast. The signals were normalized to α-tubulin (1:1000; PA529444; Thermo Fisher Scientific).

Stereotaxic surgery and microinjection procedure

Adult female mice were anesthetized using isoflurane and positioned on an Angle Two Small Animal Stereotaxic Instrument (Leica Microsystems; Wetzlar, Germany). They received a subcutaneous injection of meloxicam-SR (4 mg/kg) for pain relief. A dual-cannula system (P1 Technologies; Roanoke, VA, USA) was then implanted bilaterally to target the dlBNST. The stereotaxic coordinates used were as follows: 0.14 mm anterior to bregma, ±0.8 mm lateral to the midline, and 4.14 mm ventral to the skull (Albrechet-Souza et al. 2024; Paxinos and Franklin 2001). The guide cannulas were fitted with dummy cannulas and dust caps, matching their length, while the dual injectors protruded 0.1 mm past the guide cannulas.

The CRFR1 antagonist NBI 27914 (1 nmol, Tocris Bioscience; Ellisville, MO, USA) and the CRFR1 agonist Stressin-1 (0.2 μg, Tocris Bioscience) were freshly dissolved in physiological saline solution. Seven days following the surgical procedure, mice received bilateral infusions of vehicle or drug (0.2 μl/side, infused at 0.1 μl/min), and the injectors were kept in place for 1 minute after the end of the infusion to facilitate diffusion and prevent capillary action. Mice were administered NBI 5 minutes before exposure to restraint stress or the von Frey test. Stressin-1 was administered 15 minutes before the von Frey test.

Novelty-induced hypophagia test

On the first day of the NIH test, adult female mice were given 2-hour access to 50-ml bottles containing Ensure vanilla-flavored shake (Abbott Laboratories; Abbott Park, IL, USA) in their home cages in a dimly lit room (50 lux). The following day, we conducted home cage testing to determine the latency for Ensure consumption. Mice that did not drink during the 15-minute home cage testing were excluded from further NIH test analysis. On the day of the novel cage test, mice received either a vehicle or NBI injection into the dlBNST and, after 5 minutes, they were exposed to restraint stress and then left undisturbed for 1 hour. Mice were then placed individually into clean cages without shavings, with bottles containing Ensure positioned. The novel cage test took place under bright lighting (420 lux), and it lasted for 30 minutes. We calculated the change in latency by subtracting the latency to drink during the home cage test from the latency to drink during the novel cage test, measured in seconds (Albrechet-Souza et al. 2024; Kasten et al. 2020; 2021).

von Frey test

We employed a modified up-down method to measure mechanical sensitivity following the protocol by Chaplan et al. (1994). Mice were individually placed in cup-like containers (dimensions: length 8.2 cm, width 8.2 cm, height 9.5 cm) on a wire-mesh rack. After an acclimation period of at least 1 hour, a set of von Frey monofilaments with logarithmic intervals ranging from 0.4 to 6 grams (0.4, 0.6, 1, 1.4, 2, 4, and 6 grams; Aesthesio®, DanMic Global; San Jose, CA, USA) was used to determine the threshold that elicits a hind paw withdrawal response. The mechanical withdrawal threshold is defined as the minimum von Frey monofilament that elicits a withdrawal reflex. Each stimulus was applied three times to the center of the plantar surface of both left and right paws at 5-minute intervals. The average of these six measurements was used to calculate the percentage of withdrawal threshold ([average/6g]*100). All testing was conducted during the light phase of the light/dark cycle. For females with an AIE history or air controls, mechanical withdrawal thresholds were measured twice before the stereotaxic surgery, and the average of these measurements was taken as the baseline. Testing occurred 5 minutes after the intra-dlBNST infusion of either vehicle or NBI. For the alcohol-naïve females, mechanical withdrawal thresholds were measured 15 minutes after the intra-dlBNST infusion of either vehicle or Stressin-1.

Histological confirmation

After completing the von Frey test, mice were euthanized by decapitation under isoflurane anesthesia. Brains were dissected, snap-frozen in −30°C isopentane, and stored at −80°C until sectioning. Coronal sections of 30 μm containing the dlBNST were obtained using a cryostat, mounted on Super Frost Plus slides (Thermo Fisher Scientific), and stained with Cresyl Violet. To confirm the accurate placement of cannulas, we conducted verification using light microscopy. Mice with injector tracks that did not terminate within the dlBNST were excluded from the analysis (n = 8).

Statistical analysis

Data analysis was performed using GraphPad Prism 10.0.2 (GraphPad; La Jolla, CA, USA), and is expressed as the mean ± standard error of the mean (SEM). Plasma corticosterone levels and CRFR1 expression were analyzed using a two-way analysis of variance (ANOVA), with sex and alcohol exposure as between-subjects factors. Post hoc unpaired Student’s t-tests were used to compare CRFR1 expression between AIE and air-exposed mice in each sex. Latency to consume the appetitive reinforcer on the NIH home cage test and change in latency on the NIH novel cage test were analyzed using two-way ANOVA, with alcohol exposure and dlBNST injection as between-subjects factors. von Frey-NBI data were analyzed using two-way repeated measures ANOVA, with alcohol exposure as a between-subjects factor and dlBNST injection as a within-subject factor, followed by Bonferroni’s multiple comparisons test. von Frey-Stressin-1 data were analyzed using a paired Student’s t-test.

Results

Experiment 1: Females exhibit higher plasma corticosterone levels than males after exposure to restraint stress

The general timeline for Experiment 1 is depicted in Figure 1A. Our laboratory has shown that a history of AIE combined with stress exposure elicits sex-specific negative affect-like behaviors in adult mice (Kasten et al. 2020). However, it was unclear whether the corticosterone response to stress is altered in adult mice with an AIE history. A two-way ANOVA revealed a significant main effect of sex (F1, 35 = 8.29, p = 0.0067) on plasma corticosterone levels following restraint stress (Fig. 2A). However, there was no significant effect of alcohol exposure (F1, 35 = 1.01, p = 0.3219, n.s) or interaction between sex and alcohol exposure (F1, 35 = 1.48, p = 0.2319, n.s). These findings indicate that females exhibit higher levels of plasma corticosterone than males following restraint stress, regardless of alcohol history.

Fig 2.

Fig 2

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Plasma corticosterone levels and CRFR1 protein expression in the dorsolateral BNST of adult mice subjected to restraint stress with a history of adolescent intermittent ethanol (AIE) vapor exposure or air controls. A Plasma corticosterone levels measured immediately after restraint stress in male and female mice. B Representative western blot images depicting two isoforms of CRFR1 and α-tubulin in female (F) and male (M) mice. C Quantification of the optical densities of the 48 kDa CRFR1 band normalized to the optical densities of α-tubulin in male and female mice. D Quantification of the optical densities of the 50 kDa CRFR1 band normalized to the optical densities of α-tubulin in male and female mice. Data are presented as mean ± SEM. N = 7-10 mice/group. *Denotes p < 0.05; **Denotes p < 0.01

Experiment 1: Exposure to restraint stress upregulates CRFR1 protein expression in the dlBNST in females with AIE history

Western blot analysis using a CRFR1 antibody revealed two bands at approximately 48 kDa and 50 kDa (Fig. 2B). According to the manufacturer, CRFR1 predicted band size is 48 kDa. A two-way ANOVA revealed a marginally significant main effect of alcohol exposure (F1, 26 = 4.10, p = 0.0531) on the CRFR1 48 kDa isoform expression in the dlBNST. There was no significant effect of sex (F1, 26 = 1.66, p = 0.2084, n.s) or interaction between sex and alcohol exposure (F1, 26 = 2.95, p = 0.0977, n.s). To further explore potential differences between air controls and alcohol-exposed mice, post hoc t-tests were conducted within each sex. Females with a history of AIE exhibited significantly higher CRFR1 48 kDa isoform expression compared to air control females (Fig. 2C; t = 2.62, p = 0.0223). No significant differences were observed in males (Figure 2C; t = 0.22, p = 0.8280, n.s). Regarding the expression of the CRFR1 50 kDa isoform in the dlBNST (Fig. 2D), a two-way ANOVA revealed no significant main effect of alcohol exposure (F1, 27 = 0.24, p = 0.687, n.s), sex (F1, 27 = 2.51, p = 0.1249, n.s) or interaction between sex and alcohol exposure (F1, 27 = 0.21, p = 0.6527, n.s). These results demonstrate that females with AIE history display higher expression of CRFR1 protein in the dlBNST in comparison to air controls following restraint stress.

Experiment 2: The antagonism of CRFR1 in the dlBNST fails to reverse stress-induced negative affect-like behavior in females with AIE history

The general timeline for Experiment 2 is illustrated in Figure 1B. Only female mice were utilized in this experiment, given that the increase in CRFR1 expression after restraint stress was not observed in male mice. Additionally, the increase in latency to consume a palatable reinforcer in the NIH test following restraint stress or dlBNST activation has been previously demonstrated to occur specifically in females with AIE history (Albrechet-Souza et al. 2024, Kasten et al., 2020, 2021).

A detailed NIH test timeline is presented in Fig. 3A. A diagram showing the correct placements of intra-dlBNST cannulas in female mice is displayed in Fig. 3B. A two-way ANOVA revealed no significant effects of alcohol exposure or treatment groups before dlBNST injection on the latency to consume the appetitive reinforcer during the home cage test (Fig. 3C; alcohol exposure effect: F1, 29 = 1.44, p = 0.2400, n.s; treatment effect: F1, 29 = 0.71, p = 0.4076, n.s). On the novel cage test, a two-way ANOVA revealed that AIE history significantly increased the latency to consume the palatable reinforcer (Fig. 3D; alcohol exposure effect: F1, 29 = 4.71, p = 0.0383). However, there were no significant effects of dlBNST injection (F1, 29 = 0.20, p = 0.6572, n.s) or interaction between alcohol exposure and dlBNST injection (F1, 29 = 1.64, p = 0.2105, n.s). These findings confirm that females with AIE history exhibit heightened latency to consume an appetitive reinforcer in a novel and mildly anxiogenic environment following restraint stress. However, the blockade of CRFR1 in the dlBNST does not reverse this response.

Fig 3.

Fig 3

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The antagonism of CRFR1 in the dorsolateral BNST does not alter negative affect-like behavior in female mice with adolescent intermittent ethanol (AIE) history. A Detailed timeline of the novelty-induced hypophagia (NIH) test: adult female mice with AIE history or air controls underwent a habituation phase with access to an appetitive reinforcer for 2 hours. The following day, home cage test was conducted to measure the latency to consume. On the novel cage test day, mice received an injection of the CRFR1 antagonist NBI or vehicle into the dorsolateral BNST and were subjected to restraint stress for 1 hour. Following the stress exposure, mice were placed in a novel cage under bright light, and the latency to consume the reinforcer was measured for 30 minutes. B Correct placements of intra-dorsolateral BNST cannulas in female mice. The number of points in the figure is less than the total number of animals because of overlapping injection sites. C Latency to consume the appetitive reinforcer during the home cage test in female mice with AIE history and air controls. D Change in latency to consume the appetitive reinforcer (calculated as the difference between the latency to consume during the novel cage test and the latency to consume during the home cage test) in female mice with AIE history and air controls injected with intra-dorsolateral BNST vehicle or NBI before exposure to restraint stress. Data are presented as mean ± SEM. N = 8-9 mice/group. *Denotes p < 0.05

Experiment 2: The antagonism of CRFR1 in the dlBNST blocks mechanical hypersensitivity in females with AIE history

Fig. 4A illustrates the von Frey withdrawal thresholds in adult female mice with a history of AIE or air controls during baseline and after the microinjection of vehicle or NBI into the dlBNST, in a design that counterbalanced vehicle and drug treatments. A two-way repeated measures ANOVA revealed significant main effects of alcohol exposure (F1, 35 = 12.86, p = 0.0010), BNST injection (F1.922, 67.27 = 14.61, p < 0.0001), and interaction between alcohol exposure and BNST injection (F1.922, 67.27 = 14.61, p < 0.0001) on the paw withdrawal threshold in female mice. Bonferroni’s multiple comparisons test revealed that females with AIE history exhibited significantly lower paw withdrawal thresholds compared to air control females at the baseline and following vehicle infusion (p < 0.0001 and p = 0.0007, respectively). However, the significant difference between air controls and AIE females was no longer present after NBI injection (p = 0.4402, n.s). These findings confirm that AIE exposure induces long-lasting mechanical hypersensitivity in female mice. Importantly, this effect can be blocked by injecting the CRFR1 antagonist NBI into the dlBNST.

Fig 4.

Fig 4

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Mechanical hypersensitivity in female mice. A The CRFR1 antagonist NBI injected into the dorsolateral BNST blocks adolescent intermittent ethanol (AIE)-induced mechanical hypersensitivity in females. The graph illustrates the von Frey withdrawal thresholds in adult female mice with AIE history and air controls during baseline (before the stereotaxic surgery) and after the injection of vehicle (Veh) or NBI into the dorsolateral BNST (N = 18-19 mice/group). *Denotes p < 0.05, air control group compared to the AIE group at the corresponding treatment; #Denotes p < 0.05, AIE-NBI compared to AIE-baseline and AIE-vehicle. Data are presented as mean ± SEM. B The CRFR1 agonist Stressin-1 (STR) injected into the dorsolateral BNST induces mechanical hypersensitivity in alcohol-naïve female mice. The graph illustrates the von Frey thresholds in adult female mice after the injection of Veh or STR into the dorsolateral BNST (N = 10 mice). ****Denotes p < 0.0001

Experiment 2: The CRFR1 agonist in the dlBNST induces mechanical hypersensitivity in alcohol-naïve females

Fig. 4B illustrates the von Frey withdrawal thresholds in alcohol-naïve adult female mice following the microinjection of vehicle or Stressin-1 into the dlBNST. The experimental design counterbalanced vehicle and drug treatments. A paired Student’s t-test revealed that the CRFR1 agonist significantly decreased the paw withdrawal threshold compared to vehicle treatment (t = 12.87, p < 0.0001). These findings demonstrate that CRFR1 activation in the dlBNST is sufficient to promote mechanical hypersensitivity in female mice.

Discussion

Attempts at abstinence often lead to negative affect and hyperalgesia, increasing the risk of relapse (Egli et al. 2012; Koob 2020). Our findings demonstrated that female mice exhibited elevated plasma corticosterone levels following restraint stress compared to males. Additionally, females with a history of AIE and adult restraint stress showed increased expression of CRFR1 protein in the dlBNST compared to air controls, a response not observed in their male counterparts. Adolescent ethanol exposure induced long-lasting mechanical hypersensitivity in adult females, which was effectively blocked by a CRFR1 antagonist administered into the dlBNST. Furthermore, administration of a CRFR1 agonist into the dlBNST induced mechanical hypersensitivity in alcohol-naïve females. Stress-induced negative affect-like behavior, as measured by the NIH test, was not affected by the CRFR1 antagonism in the dlBNST.

In Experiment 1, we observed that plasma corticosterone levels were higher in adult females compared to males immediately after exposure to restraint stress, and a history of adolescent alcohol did not alter the corticosterone response to the stressor. These findings are consistent with previous research indicating that female rodents exhibit a heightened activation of the hypothalamic-pituitary-adrenal axis in response to different stressors (Albrechet-Souza et al. 2020; Kudielka and Kirschbaum 2005; Nguyen et al. 2020; Oyola and Handa 2017; Rivier 1999). However, a recent study reported that the corticosterone response to a mild stressor during acute withdrawal is blunted in female mice with a history of two-bottle choice alcohol consumption (Neira et al. 2023). It is important to note that in our investigation, corticosterone levels were assessed approximately 40 days after the last exposure to ethanol. This temporal distinction between the studies suggests that the neuroendocrine alterations induced by alcohol may primarily manifest during the early withdrawal period, potentially reflecting the dynamic nature of the neuroendocrine response and the progression of alcohol-related adaptations.

Women are more likely than men to have anxiety and mood conditions, as well as comorbid alcohol/substance abuse disorders (Back et al. 2011; Conway et al. 2006; Kessler et al. 2005). Moreover, negative emotions, drinking to regulate negative affect, and stress are among the factors associated with increasing rates of AUD in women (Fox and Sinha 2009). Following exposure to restraint stress, we found a higher expression of CRFR1 protein in the dlBNST in adult female mice with a history of AIE compared to air controls. Notably, this AIE effect was not observed in males. The increased CRFR1 expression following stress exposure in females with an AIE history could render them more susceptible to the effects of CRF release, shifting them more easily into a dysregulated state of stress reactivity (Bangasser 2013). In humans, there is evidence that elevated CRF and CRFR1 levels contribute to anxiety and affective disorders in clinical populations (Risbrough and Stein 2006). Interestingly, stress reactivity and anxiety are blunted in transgenic mice that do not express CRFR1 (Bale and Vale 2004; Müller et al. 2003). It is worth noting that although the CRFR1 antibody utilized in this study underwent systematic validation by Zegers-Delgado et al. (2022), it targets amino acids 425-445 of the human CRFR1 protein. This epitope exhibits a high level of sequence similarity with amino acids 392-411 of the human CRFR2 protein (UniProt IDs: P34998-CRFR1_HUMAN; Q13324-CRFR2_HUMAN). Therefore, we cannot entirely rule out the possibility of cross-reactivity between them.

The current study confirms previous findings from our laboratory, indicating that female mice exposed to AIE displayed an increased latency to consume an appetitive reinforcer in a novel and mildly anxiogenic environment during the NIH test following exposure to restraint stress (Kasten et al. 2020; 2021). However, the blockade of CRFR1 in the dlBNST did not alter this response. A prior investigation examining the role of CRFR1 in contextual fear conditioning and unconditioned fear to predator odor revealed that intra-dlBNST administration of the CRFR1 antagonist antalarmin disrupted the retention of contextual fear but had no effect on freezing to the predator odor in rats, indicating a diverging role for dlBNST CRFR1 in conditioned vs. unconditioned fear responses (Asok et al. 2016). In a comprehensive translational study involving anxious alcohol-dependent women, researchers reported a lack of significant effects of the CRFR1 antagonist verucerfont on physiological and psychological stress responses, as well as subjective craving induced by alcohol-associated cues and stressors (Schwandt et al. 2016). These findings are consistent with another study from the same group using the CRFR1 antagonist pexacerfont (Kwako et al. 2015).

A pivotal finding in this study is that antagonism of CRFR1 in the dlBNST effectively blocked the mechanical hypersensitivity induced by AIE in adult females. However, it should be emphasized that mice treated with NBI during the first von Frey test still exhibited mechanical hypersensitivity during the second test. This indicates that while the CRFR1 antagonist acutely blocked this response, it did not fully reverse it. Additionally, the administration of the selective CRFR1 agonist Stressin-1 into the dlBNST was sufficient to promote mechanical hypersensitivity in alcohol-naïve female mice. These results not only support but also extend prior findings from our laboratory indicating that mechanical hypersensitivity in mice with a history of AIE is associated with increased activation of CRFR1 neurons in the dlBNST (Bertagna et al. 2024). Consistent with our data, administration of a CRFR1 antagonist into the anterolateral BNST reduced the number of abdominal contractions in response to colorectal distension in a model of visceral pain in rats (Tran et al. 2014). Furthermore, a study using a viral approach to selectively reduce CRF expression in the dorsal BNST demonstrated decreased thermal nociceptive sensitivity in mice (Yu et al. 2021).

Previous research has demonstrated that manipulating CRF signaling in the BNST can influence several aspects of pain-related behavior (Ide et al. 2013; Kaneko et al. 2016; Tran et al. 2014). However, the source of this CRF remains unclear, as the BNST contains locally produced CRF and receives CRF inputs from the central nucleus of the amygdala (CeA) (Dong et al. 2001; Sakanaka et al. 1986). Our future work will explore the involvement of CeA-BNST projections in mechanical and thermal sensitivity during alcohol withdrawal under baseline conditions, as well as in response to noxious stimuli such as inflammatory pain. Furthermore, it is crucial to comprehend how the BNST modulates the activity of nociresponsive neurons in downstream targets, such as the ventrolateral periaqueductal gray.

In conclusion, our findings reveal that female mice exhibited elevated plasma corticosterone levels compared to males following restraint stress. Additionally, female mice with a history of AIE and adult stress displayed increased expression of CRFR1 protein in the dlBNST compared to air controls, a response not observed in male mice. These increased CRFR1 expression may contribute to the heightened susceptibility of females with an alcohol history to stress-related conditions, including co-occurring AUD and anxiety disorders. Although the administration of the CRFR1 antagonist NBI directly into the dlBNST did not attenuate experimental measures of negative emotionality in adult females exposed to adolescent alcohol, it acutely blocked pain-related behavior. Moreover, the CRFR1 agonist Stressin-1 administered into the dlBNST was sufficient to induce mechanical hypersensitivity in alcohol-naïve females. These findings enhance our understanding of the neurobiological mechanisms underlying AUD, reveling a potential dissociation in the mechanisms involving in stress-induced negative affect and pain-related behavior, both influenced by adolescent alcohol use and key factors driving AUD.

Funding

This work was supported by the National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (AA028011, TAW). LSUHSC Research Enhancement Program (TAW) and São Paulo Research Foundation (FAPESP-Brazil 2021/13317-9, NBB; 2018/15505-4, FCC).

Financial support:

National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (AA028011, TAW); LSUHSC Research Enhancement Program (TAW); São Paulo Research Foundation (FAPESP-Brazil 2021/13317-9, NBB; 2018/15505-4, FCC).

Footnotes

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Data availability

The data that support the findings of this study are available upon request from the corresponding author.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available upon request from the corresponding author.

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