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. Author manuscript; available in PMC: 2023 Sep 14.
Published in final edited form as: Neuropharmacology. 2021 Mar 2;188:108512. doi: 10.1016/j.neuropharm.2021.108512

Moderate adolescent chronic intermittent ethanol exposure sex-dependently disrupts synaptic transmission and kappa opioid receptor function in the basolateral amygdala of adult rats

Kathryn R Przybysz a,b, Meredith E Gamble a,b, Marvin R Diaz a,b
PMCID: PMC10500544  NIHMSID: NIHMS1928405  PMID: 33667523

Abstract

Adolescent alcohol exposure is associated with many consequences in adulthood, including altered affective and reward-related behaviors. However, the long-term neurological disruptions underlying these behaviors are not fully understood. Shifts in the excitatory/inhibitory balance in the basolateral amygdala (BLA) relate to the expression of these behaviors and changes to BLA physiology are seen during withdrawal immediately following adolescent ethanol exposure, but no studies have examined whether these changes persist long-term. The kappa opioid receptor (KOR) neuromodulatory system mediates negative affective behaviors, and alterations of this system are implicated in behavioral changes following adult and adolescent chronic ethanol exposure. In the BLA, the KOR system undergoes functional changes across development, but whether BLA KOR function is disrupted by adolescent ethanol exposure is unknown. In this study, male and female Sprague-Dawley rats were exposed to a vapor model of moderate adolescent chronic intermittent ethanol (aCIE) and assessed for long-term effects on GABAergic and glutamatergic neurotransmission within the adult BLA and KOR modulation of these systems. aCIE exposure increased presynaptic glutamate transmission in females but had no effect in males or on GABA transmission in either sex. Additionally, aCIE exposure disrupted male KOR modulation of GABA release, with no effects in females or on glutamate transmission. These data suggest that aCIE produces sex-dependent and long-term changes to BLA physiology and KOR function. This is the first study to examine these persistent adaptations following adolescent alcohol exposure and opens a broad avenue for future investigation into other adolescent ethanol-induced disruptions of these systems.

Keywords: Adolescent alcohol, Adult, Basolateral amygdala, Kappa opioid receptor, Sex differences, Electrophysiology

Introduction

Throughout adolescence individuals undergo extensive maturation, marked by major neurodevelopment and behavioral changes, as they transition into adulthood. During this period, adolescents show increased risk-taking and reward-seeking behaviors, and especially the initiation of drug use (Casey, Getz, & Galvan, 2008; Masten, Faden, Zucker, & Spear, 2009; Spear, 2000). Alcohol, in particular, is consumed at alarming rates during adolescence, with 7.4 million individuals under the age of twenty-one reporting drinking in the past month (SAMHSA, 2019). Young adolescence appears to be a crucial time for the initiation of problematic alcohol use; many individuals who begin drinking during early adolescence tend to rapidly escalate their use, with more than ten percent accelerating to meet alcohol use disorder (AUD) criteria within the first year of initiation (Faden, 2006; Forman-Hoffman, Edlund, Glasheen, & Ridenour, 2017; Masten et al., 2009). This early introduction to alcohol use has been associated with numerous long-lasting alterations to brain function, brain structure, and behavior (Crews, Vetreno, Broadwater, & Robinson, 2016; Spear, 2018; Squeglia, Jacobus, & Tapert, 2014), including increased rates of anxiety, depression, and AUD later on (Duncan, Alpert, Duncan, & Hops, 1997; Grant & Dawson, 1997; Hawkins et al., 1997; Pitkanen, Lyyra, & Pulkkinen, 2005; Sung, Erkanli, Angold, & Costello, 2004; Wolford-Clevenger & Cropsey, 2019). Our understanding of the effects of adolescent alcohol comes largely from studies that have used exposures yielding BECs of 150–250 mg/dL, far exceeding the definition of binge-level exposure [80 mg/dL, (NIAAA, 2004)]. However, adolescents do not routinely drink to these levels (SAMHSA, 2019).

According to a 2018 report, of the 18.8% of adolescents between the ages of twelve and twenty that reported drinking in the past month, approximately 5.1% did not indicate binge or heavy use (Center for Behavioral Health Statistics and Quality. 2018 National Survey on Drug Use and Health Public Use File Codebook; Substance Abuse and Mental Health Services Administration: Rockville, MD, USA, 2019). Additionally, moderate levels of alcohol drinking do appear to put individuals at higher risk for several significant health consequences (Poli et al., 2013). Thus, there is a critical need to further examine the neurobiological impacts of moderate levels of intoxication, as these are understudied and still relevant to this adolescent population.

Animal models have been used to examine the long-term effects of adolescent alcohol use and have found varying results regarding negative affect and reward-related behaviors, regardless of the level of ethanol exposure. While some research has shown increased adult voluntary drinking and anxiety-like behavior following exposure to heavy amounts of ethanol (BECs >180 mg/dL per exposure) throughout most of the adolescent period (Alaux-Cantin et al., 2013; Kyzar, Zhang, & Pandey, 2019; Pandey, Sakharkar, Tang, & Zhang, 2015; Sakharkar et al., 2019), others have identified decreases or no changes in these behaviors (Gass et al., 2014; Nentwig, Starr, Chandler, & Glover, 2019; Torcaso, Asimes, Meagher, & Pak, 2017). Low to moderate levels of intoxication throughout the majority of adolescence have also been shown to increase voluntary drinking (Amodeo, Kneiber, Wills, & Ehlers, 2017; Broadwater, Varlinskaya, & Spear, 2013) and social anxiety-like responses (Varlinskaya, Kim, & Spear, 2017a) in adulthood. Conversely, we recently found that a relatively moderate exposure to ethanol every other day from postnatal days (P) 30–40 (5 total exposures) was not sufficient to alter social or non-social anxiety-like behaviors or ethanol intake in adulthood; however, exposure to forced swim stress in adulthood increased ethanol intake only in adolescent ethanol-exposed males (Gamble & Diaz, 2020). Interestingly, restraint stress in adulthood enhanced social anxiety-like responses only in controls, an effect that was blunted in adolescent ethanol-exposed subjects that already exhibited enhanced social anxiety-like responses (Varlinskaya et al., 2017a). Thus, low-to-moderate exposure to ethanol during adolescence may alter sensitivity to future stress exposure by altering stress systems within affect and reward-related brain structures.

The kappa opioid receptor (KOR) system, in particular, is heavily implicated in stress and addiction due to its role in aversive, dysphoric, and anxiety-related behaviors. Numerous studies have demonstrated that acute exposure to stress or alcohol activate the KOR system, while chronic exposures lead to various neuroadaptations in the KOR system [see extensive reviews by (Bruchas, Land, & Chavkin, 2010; Chavkin & Ehrich, 2014; Chavkin & Koob, 2016; Crowley & Kash, 2015; Karkhanis & Al-Hasani, 2020)]. Interestingly, there is accumulating evidence indicating that early-life exposure to alcohol through adolescence may produce different neuroadaptations in the KOR system when compared to the canonical effects observed in adult male subjects [reviewed in (Diaz, Przybysz, & Rouzer, 2017)]. For example, it was recently shown that adolescent exposure to relatively high levels of ethanol either during early or late adolescence produced sex-specific changes in KOR-mediated inhibition of dopamine release within the nucleus accumbens of adult subjects that were dependent on the timing of the exposure (Spodnick et al., 2020). Consistent with differential effects of early-life versus adult exposures on KOR function, the observed effects following late adolescent ethanol exposure in males was opposite to that found following adult ethanol exposure in males (Karkhanis, Huggins, Rose, & Jones, 2016). However, it is unclear whether exposure to moderate levels of ethanol during adolescence may also lead to long-term alterations in KOR function.

The basolateral nucleus of the amygdala (BLA) plays a significant role in affect and reward processing (Janak & Tye, 2015; Tye et al., 2011) and is a major target of ethanol, particularly following exposures during adolescence. Increased glutamate transmission (Christian, Alexander, Diaz, Robinson, & McCool, 2012; Läck, Diaz, Chappell, DuBois, & McCool, 2007; McGinnis, Parrish, Chappell, Alexander, & McCool, 2020; McGinnis, Parrish, & McCool, 2020; Morales, McGinnis, Robinson, Chappell, & McCool, 2018) and decreased GABA transmission (Diaz, Christian, Anderson, & McCool, 2011) were observed 24 hours into withdrawal from 10 days of exposure to relatively high levels of ethanol during adolescence (based on weight). Furthermore, lesions of the BLA following adolescent ethanol exposure reduced ethanol intake in adulthood (Moaddab, Mangone, Ray, & McDannald, 2017), suggesting long-term effects of adolescent ethanol exposure on BLA function. Importantly, we have found that the KOR system within the BLA modulates GABA transmission in ethanol-naïve adolescent males, but not adult males, or the glutamate system at either age (Przybysz, Werner, & Diaz, 2017). We also recently showed that adolescent exposure to forced swim stress leads to short-term alterations in KOR modulation of BLA GABA transmission in males that was associated with social anxiety-like alterations (Varlinskaya, Johnson, Przybysz, Deak, & Diaz, 2020). These previous findings suggest that adolescent ethanol exposure may lead to persistent alterations in BLA function and disrupt the neurodevelopmental trajectory of KOR function within the BLA. To test this, we investigated basal GABA and glutamate transmission and the effects of U69593, a KOR agonist, on synaptic transmission of adult male and female rats that were exposed to our previously characterized (Gamble & Diaz, 2020) moderate adolescent chronic intermittent ethanol (aCIE) paradigm.

Methods

Animals

This study included male and female Sprague-Dawley rats that were bred and reared in our animal colony at Binghamton University, with breeding pairs originating from Envigo (Indianapolis, IN). Animals were housed in a temperature-controlled (22°C) vivarium, given ad libitum access to standard lab chow (LabDiet 5L0D. PicoLab Laboratory Rodent Diet, ScottPharma Solutions, Marlborough, MA) and water, and maintained on a 12:12 h light: dark cycle (lights on at 0700 h). Litters were culled to 12 pups (six males and six females) whenever possible on postnatal day (P) 2 and reared with their mothers until weaning on P21. Following weaning, animals were group-housed in groups of 2–3 animals of each sex per cage. All animal procedures were approved by the Binghamton University Institutional Animal Care and Use Committee.

Moderate adolescent Chronic Intermittent Ethanol (aCIE) Exposure

We utilized a moderate adolescent chronic intermittent ethanol (aCIE) exposure as we previously characterized (Gamble & Diaz, 2020). Briefly, starting on P30, animals in their home cages were transferred to vapor inhalation chambers for ten consecutive days. Animals were exposed to either room air (control group) or vaporized ethanol (aCIE group) for 12 h overnight (2000–0800 h) every other night during this ten-day period, resulting in five cycles of 12 h ethanol on, 36 h ethanol off (Figure 1). All animal husbandry (food, water, and bedding changes) occurred during the “off” periods, and no handling or movement of the animals occurred during the 12-hour exposure periods. Vapor levels were recorded just prior to the end of each exposure day to ensure consistent exposure throughout the 10-day exposure period and across cohorts. Rat weight was also monitored throughout the exposure period, with rats being weighed before being placed into the vapor chambers, twice at regular intervals during the 10-day exposure period, and following the end of the last exposure. Rat chow was replaced after each exposure to ethanol to avoid consumption of ethanol saturated chow during the periods of abstinence. Following the final cycle, animals were transferred back to the animal colony until adulthood (~P70).

Figure 1.

Figure 1.

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Diagram of aCIE exposure paradigm

Timeline of aCIE exposure paradigm. Rats were placed into ethanol vapor exposure chambers on P30, and were exposed to vaporized ethanol or room air for twelve hours (8:00 PM to 8:00 AM) every other night for ten days (five exposures). On P40, rats were returned to the animal colony room, and electrophysiological recordings began on P70.

Whole-cell patch-clamp electrophysiology

All electrophysiological procedures were done as previously described ((Przybysz et al., 2017; Varlinskaya et al., 2020). Briefly, adult rats (P70–100) were sedated with ketamine (250 mg/kg) and quickly decapitated. Brains were rapidly removed and immersed in cold oxygenated (95% O2, 5% CO2) sucrose artificial cerebrospinal fluid (ACSF) cutting solution containing (in mM): sucrose (220), KCl (2), NaH2PO4 (1.25), NaHCO3 (26), glucose (10), MgSO4 (12), CaCl2 (0.2), and ketamine (0.43). 300 μm brain slices containing BLA were made using a Vibratome (Leica Microsystems, Bannockburn, IL, USA). Slices were incubated in oxygenated normal ACSF containing (in mM): NaCl (126), KCl (2), NaH2PO4 (1.25), NaHCO3 (26), glucose (10), CaCl2 (2), MgSO4 (1), and ascorbic acid (0.4), and allowed to recover for at least 40 min at 34°C before recording. Slices remained at 34°C and all experiments were performed 1–4 h after slice preparation.

Following incubation, slices were transferred to a recording chamber in which oxygenated ACSF was warmed to 32°C and superfused over the slice at 3 mL/min. All recordings were made from pyramidal neurons within the BLA that were visualized using infrared-differential interference contrast microscopy (Olympus America, Center Valley, PA). Pyramidal neurons were identified based on morphology and capacitance (> 150 pF) as previously described (Przybysz et al., 2017; Varlinskaya et al., 2020); with a threshold for acceptable access resistance as less than 30 MΩ. Spontaneous inhibitory postsynaptic current (sIPSC) recordings were collected with patch pipettes filled with KCl internal solution containing (in mM): KCl (135), HEPES (10), MgCl2 (2), EGTA (0.5), Mg-ATP (5), Na-GTP (1), and QX314-Cl (1), pH of 7.25, and osmolarity of 280–290 mOsm. Spontaneous excitatory postsynaptic current (sEPSC) recordings were made with a K-gluconate internal solution, containing (in mM): K-Gluconate (120), KCl (15), EGTA (0.1), HEPES (10), MgCl2 (4), MgATP (4), Na3GTP (0.3), phosphocreatine (7), and QX-314 Br (1.5), pH of 7.3, and osmolarity of 295–305 mOsm. Data were acquired with a MultiClamp 700B (Molecular Devices, Sunnyvale, CA) at 10 kHz, filtered at 1 kHz, and stored for later analysis using pClamp software (Molecular Devices). For recordings of GABAA receptor-mediated sIPSCs, we pharmacologically blocked AMPA and NMDA glutamate receptors using 1 mM kynurenic acid and 50 μM DL-APV, respectively. For sEPSC recordings, we blocked GABAA receptors with 10 mM gabazine.

Upon rupturing the cell membrane, neurons were allowed to equilibrate for at least 5 min before a baseline was recorded. We have previously shown that the KOR agonist U69593 produces a stable response ~1 min into drug application (Przybysz et al., 2017; Varlinskaya et al., 2020), so following 3 min of baseline recording, we recorded at least 4 min of continuous U69593 (1 μM) application. Given potential variability in timing of U69593 reaching the slice and instability of the baseline in the first 1 minute of recording, we discarded the first minute of recording and only used the subsequent minute to quantify the baseline. To account for slight variability in drug wash-on times and to allow one minute for U69593 to take effect, we also discarded the first 1 minute of the drug period, and used the second minute of U69593 application to quantify the effect of KOR activation (The approximate drug application is highlighted in the time-course figures, with the period used for quantification of average baseline and drug effects indicated in brackets. Access resistance was monitored throughout each recording, and only recordings in which the access resistance changed <20% were kept for analysis.

Drugs and Chemicals

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Kynurenic acid, DL-APV, and QX314-Cl were purchased from Tocris/R&D Systems (Bristol, UK).

Statistics

Electrophysiological data were analyzed using MiniAnalysis (Synaptosoft Inc.) and were analyzed statistically with Prism 8 (GraphPad, San Diego, CA). Given evidence that BLA physiology is substantially different between males and females (Blume et al., 2017), data from males and females were analyzed separately. Power analysis (G*Power 3.1.9.2) based on preliminary data and an alpha of 0.05 indicated a sample size of at least 8 units per group. Sample sizes indicate number of individual cells, but no more than 2 cells were recorded from a single animal in any given experiment (total number of rats used = 26 males and 26 females). Data were first analyzed using the Pearson omnibus and Kolmogorov-Smirnov (K-S) normality tests. If data followed a normal distribution, parametric tests were used; otherwise, nonparametric tests were used. Grubbs outlier tests were run on all basal and U69593 data sets. Using this analysis we identified two outliers in the air-exposed female group for the sEPSC recordings, including one outlier for basal sEPSC frequency and one outlier for basal sEPSC amplitude. These two cells were removed from this and all further analyses. To identify how each individual cell responded to U69593 application, we calculated inter-event intervals across the baseline and KOR agonist recordings, then compared the cumulative distributions of the U69593 period compared to baseline using the Kolmogorov-Smirnov Test to compare cumulative distributions for each individual cell. Cells for which the test was nonsignificant were sorted into the “No Change” group, indicating the agonist had no effect on sIPSC or sEPSC frequency. If the K-S for a cell was significantly different, we assessed whether that cell’s average sIPSC or sEPSC frequency with the KOR agonist was greater or less than its respective basal sIPSC/sEPSC frequency, and used this information to sort each cell into the “potentiated” or “inhibited” groups, respectively. All data are presented as mean ± SEM, with p ≤ 0.05 considered statistically significant.

Results

Membrane properties of BLA pyramidal cells across aCIE exposure in males and females

To determine whether our aCIE exposure paradigm shifted the membrane properties of BLA pyramidal cells, we compared the membrane capacitance and membrane resistance values from air- and EtOH-exposed cell recordings, separating comparisons in males and females and using different internal solutions. In males, aCIE did not alter the membrane capacitance (t = 0.29, df = 16, n = 9, p = 0.77) or membrane resistance (t = 1.05, df = 16, n = 9, p = 0.31) using the KCl internal solution. However, when using the K-Gluconate internal solution, ethanol-exposed males showed a significant reduction in membrane capacitance (t = 2.55, df = 19, n = 10–11, p = 0.02) with no change in membrane resistance (t = 0.22, df = 19, n = 10–11, p = 0.83). In females, no difference was observed in membrane capacitance using KCl internal solution (t = 0.89, df = 19, n = 10–11, p = 0.38) or K-Gluconate internal solution (t = 0.11, df = 16, n = 9, p = 0.91). However, membrane resistance was significantly reduced in EtOH-exposed females using KCl internal solution (t = 3.47, df = 19, n = 10–11, p = 0.003), but no difference was observed between air- and EtOH-exposed females using K-Gluconate internal solution (t = 0.11, df = 16, n = 10–9, p = 0.91). These findings are summarized in Table 1.

Table 1.

Cell properties of BLA pyramidal cells across sex and exposure using either KCl internal solution (sIPSCs) or K-Gluconate internal solution (sEPSCs).

Membrane
Properties		 KCL
Membrane
Capacitance(pF)		 KCl
Membrane
Resistance(MΩ)		 K-Gluc
Membrane
Capacitance(pF)		 K-Gluc
Membrane
Resistance(MΩ)
Air Male 229.1 (13.6) 87.2(12.0) 293.0 (22.4) 123.5(21.9)
EtOH Male 223.4(13.6) 116.1 (24.7) 226.4(11.9)* 128.9(9.2)
Air Female 194(17.1) 115.5(12.0) 278.8(11.6) 110.1 (12.6)
EtOH Female 211.6(8.5) 68.1 (5.5)* 303.9(19.6) 112.3(15.3)
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*

indicates significant main effect of exposure (p < 0.05).

Basal GABA transmission in adult BLA

We performed electrophysiological experiments in the BLA of adult rats that had undergone the aCIE exposure paradigm. Assessment of basal GABA transmission in the BLA of males showed that aCIE exposure did not alter sIPSC frequency (Fig. 2A&C: t = 1.78, df = 16, n = 9, p = 0.09) or amplitude (Fig. 2B&C: t = 1.62, df = 9.4, n = 9, p = 0.14). In females, there was also no effect of aCIE on sIPSC frequency (Fig. 2D&F: t = 0.22, df = 19, n = 10–11, p = 0.83) or amplitude (Fig. 2E&F: t = 0.81, df = 19, n = 10–11, p = 0.43).

Figure 2.

Figure 2.

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Exposure to aCIE does not alter adult BLA GABA transmission.

Comparison of BLA sIPSC frequency (A, D) and amplitude (B, E) between air and aCIE-exposed adult males (top) and females (bottom), with exemplar traces demonstrating no effect of aCIE exposure in either sex (C, F). Bars depict mean ± SEM.

aCIE shifts GABA responsiveness to U69593

We next assessed the effect of U69593 (1 μM) on GABA transmission within the BLA. This U69593 concentration was chosen based on previous work from our lab (Przybysz, et al., 2017, Varlinskaya et al., 2020). When averaging across all cells, in neurons from air-exposed males there was no effect of 1 μM U69593 on sIPSC frequency (Fig. 3A: 12.34 ± 12.86% change from baseline, n = 9), consistent with our previously reported findings from naïve adult males using 1 μM U69593 (Przybysz et al., 2017). However, in ethanol-exposed males, 1 μM U69593 significantly decreased sIPSC frequency (Fig 3A: −17.68 ± 6.48% change from baseline, n = 9, p = 0.03 compared to 0). There was no effect of 1 μM U69593 on sIPSC amplitude in air- or ethanol-exposed males (Fig. 3B: air: 15.32 ± 10.78% change from baseline, n = 9, p = 0.19 compared to 0; EtOH: 0.95 ± 8.74% change from baseline, n = 9, p = 0.92 compared to 0). Graphs showing individual within-cell responses to U69593 are displayed in Supplementary Figure 1A–D. Despite these aCIE-induced differences in U69593 modulation of sIPSC frequency, we observed high variability in controls, consistent with our previous observations (Przybysz et al., 2017). Therefore, to determine whether aCIE exposure shifted the responsiveness of individual cells to U69593, we ran Kolmogorov-Smirnov (K-S) tests on each recording as we have done previously (Varlinskaya et al., 2020). This analysis revealed that the aCIE exposure shifted the proportion of cells that responded to U69593. In air-exposed males, 3/9 cells were non-responsive, while sIPSC frequency was inhibited in 2/9 cells and was potentiated in 4/9 cells. However, in EtOH-exposed males, 4/9 cells were unresponsive, but sIPSC frequency was inhibited in all other cells (5/9); no cells were potentiated by U69593. Component graphs illustrating this shift are shown in Fig. 3C, and time courses for each of the groups from air- and EtOH-exposed animals are displayed in Fig. 3D and 3E, respectively. Time-courses for sIPSC amplitudes are shown in Fig. 3F.

Figure 3.

Figure 3.

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Exposure to aCIE alters KOR modulation of GABA transmission and individual cell responsiveness to KOR activation in adult male BLA.

Change from baseline (%) of sIPSC frequency (A) and amplitude (B) for all cells following application of KOR agonist U69593 (1 μM) in air- and aCIE-exposed males. * = p < 0.05, significant change from baseline. (C) Component chart indicating an aCIE-induced shift in the percentage of BLA pyramidal cells that were inhibited (orange), not changed (blue), or potentiated (green) by KOR activation. (D, E) Frequency time-courses of U69593 application for air- (D) or aCIE- (E) exposed males separated by directional change. (F) Amplitude time-course of U69593 application for air- and aCIE-exposed males averaged across all cells in each exposure group. Light gray shading in D, E, and F indicates the period of U69593 application. Brackets in D and E correspond to the 60 s period used to calculate average baseline sIPSC frequency and frequency % change from baseline, shown in A, and brackets in F correspond to the 60 s period used to calculate average baseline sIPSC amplitude and amplitude % change from baseline, shown in B.

In neurons from females, 1 μM U69593 had no effect on sIPSC frequency (Fig. 4A: air: −8.31 ± 6.52% change from baseline, n = 11, p = 0.23 compared to 0; EtOH: −5.55 ± 6.51% change from baseline, n = 10, p = 0.42 compared to 0) or amplitude (Fig 4B: air: -8.14 ± 9.26% change from baseline, n = 11, p = 0.40 compared to 0; EtOH: −0.79 ± 11.61% change from baseline, n = 10, p = 0.95 compared to 0). Graphs showing individual within-cell responses to U69593 are displayed in Supplementary Figure 1E–H. When analyzing for shifts in individual cell responsivity using the K-S test, we found subtle evidence for an aCIE-induced shift. In air-exposed females, an equal number of cells were inhibited by U69593 as did not respond to U69593 (5/11), while one cell was potentiated by U69593. In EtOH-exposed females, this proportion was shifted so that the majority of cells were nonresponsive (6/10), three cells were inhibited, and one cell was potentiated. Component graphs and time courses illustrating this shift can be found in Fig. 4C, 4D, and 4E.

Figure 4.

Figure 4.

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Exposure to aCIE does not alter KOR modulation of GABA transmission but shifts individual cell responsiveness to KOR activation in adult female BLA.

Change from baseline (%) of sIPSC frequency (A) and amplitude (B) for all cells following application of KOR agonist U69593 (1 μM) in air- and aCIE-exposed females. (C) Component chart indicating an aCIE-induced shift in the percentage of BLA pyramidal cells that were inhibited (orange), not changed (blue), or potentiated (green) by KOR activation. (D, E) Frequency time-courses of U69593 application for air- (D) or aCIE- (E) exposed females separated by directional change. (F) Amplitude time-course of U69593 application for air- and aCIE-exposed females averaged across all cells in each exposure group. Light gray shading in D, E, and F indicates the period of U69593 application. Brackets in D and E correspond to the 60 s period used to calculate average baseline sIPSC frequency and frequency % change from baseline, shown in A, and brackets in F correspond to the 60 s period used to calculate average baseline sIPSC amplitude and amplitude % change from baseline, shown in B.

Basal glutamate transmission in adult BLA

Assessment of aCIE exposure effects on glutamate transmission in the BLA showed that in males neither sEPSC frequency (Fig. 5A: t = 0.34, df = 19, n = 10–11, p = 0.74) nor amplitude (Fig. 5B: t = 1.32, df = 19, n = 10–11, p = 0.20) was affected by aCIE. However, in females, aCIE exposure significantly increased sEPSC frequency (Fig. 5D: t = 2.42, df = 9.04, n = 9, p = 0.04) and sEPSC amplitude (Fig. 5E: t = 2.67, df = 16, n = 9, p = 0.02).

Figure 5.

Figure 5.

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Exposure to aCIE increases adult female BLA glutamate transmission.

Comparison of BLA sEPSC frequency (A, D) and amplitude (B, E) between air- and aCIE-exposed adult males (top) and females (bottom), * = p < 0.05 between air and aCIE groups. (C, F) Exemplar traces demonstrating no aCIE-induced difference in males (C) but a significant increase in sEPSC frequency and sEPSC amplitude in aCIE females (F).

U69593-mediated glutamate transmission

When assessing the effect of 1 μM U69593 on BLA glutamate transmission, we found no effect on sEPSC frequency in air- or ethanol-exposed males (Fig. 6A: air: −3.41 ± 8.65 % change from baseline, n = 11, p = 0.70 compared to 0; EtOH: 0.72 ± 11.9 % change from baseline, n = 10, p = 0.95 compared to 0). 1 μM U69593 also did not affect sEPSC amplitude in air- or ethanol-exposed males (Fig. 6B: air: −0.30 ± 4.10 % change from baseline, n = 11, p = 0.94 compared to 0; EtOH: −3.79 ± 6.90% change from baseline, n = 10, p = 0.60 compared to 0). Graphs showing individual within-cell responses to U69593 are displayed in Supplementary Figure 2A–D. When using the K-S test to assess shift in responsivity to U69593, we found a slight change caused by aCIE exposure. In air-exposed males, all cells but two were nonresponsive (9/11), with the two responsive cells being inhibited. In EtOH-exposed males, all but two cells were also nonresponsive (8/10), with one cell being inhibited and one cell being potentiated.

Figure 6.

Figure 6.

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KOR activation does not alter KOR modulation of glutamate transmission but causes a slight shift in individual cell responsiveness to KOR activation in adult male BLA.

Change from baseline (%) of sEPSC frequency (A) and amplitude (B) for all cells following application of KOR agonist U69593 (1 μM) in air- and aCIE-exposed males. C) Component chart indicating a slight aCIE-induced shift in the percentage of BLA pyramidal cells that were inhibited (orange), not changed (blue), or potentiated (green) by KOR activation. (D, E) Frequency time-courses of U69593 application for air- (D) or aCIE- (E) exposed males separated by directional change. (F) Amplitude time-course of U69593 application for air- and aCIE-exposed males averaged across all cells in each exposure group. Light gray shading in D, E, and F indicates the period of U69593 application. Brackets in D and E correspond to the 60 s period used to calculate average baseline sEPSC frequency and frequency % change from baseline, shown in A, and brackets in F correspond to the 60 s period used to calculate average baseline sEPSC amplitude and amplitude % change from baseline, shown in B.

In both air- and ethanol-exposed females, 1 μM U69593 had no effect on sEPSC frequency (Fig. 7A: air: −11.07 ± 11.31% change from baseline, n = 9, p = 0.36 compared to 0; EtOH: −12.14 ± 10.39% change from baseline, n = 9, p = 0.28 compared to 0) or sEPSC amplitude (Fig. 7B: air: −3.62 ± 4.25% change from baseline, n = 9, p = 0.42 compared to 0; EtOH: −0.77 ± 8.2% change from baseline, n = 9, p = 0.93 compared to 0). Graphs showing individual within-cell responses to U69593 are displayed in Supplementary Figure 2E–H. When analyzing individual cell responsiveness to U69593 using the K-S test, we found little evidence of an aCIE-induced shift. The majority of cells from air-exposed females were nonresponsive (5/9), with 3/9 cells being inhibited and 1/9 cells being potentiated. In ethanol-exposed females, 4/9 cells were nonresponsive, while 3/9 cells were inhibited by U69593 and 2/9 cells were potentiated by U69593.

Figure 7.

Figure 7.

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Exposure to aCIE does not alter KOR modulation of glutamate transmission but causes a slight shift in individual cell responsiveness to KOR activation in adult female BLA.

Change from baseline (%) of sEPSC frequency (A) and amplitude (B) for all cells following application of KOR agonist U69593 (1 μM) in air- and aCIE-exposed females. (C) Component chart indicating an aCIE-induced shift in the percentage of BLA pyramidal cells that were inhibited (orange), not changed (blue), or potentiated (green) by KOR activation. (D, E) Frequency time-courses of U69593 application for air- (D) or aCIE- (E) exposed females separated by directional change. (F) Amplitude time-course of U69593 application for air- and aCIE-exposed females averaged across all cells in each exposure group. Light gray shading in D, E, and F indicates the period of U69593 application. Brackets in D and E correspond to the 60 s period used to calculate average baseline sEPSC frequency and frequency % change from baseline, shown in A, and brackets in F correspond to the 60 s period used to calculate average baseline sEPSC amplitude and amplitude % change from baseline, shown in B.

Correlation between basal GABA/glutamate transmission and magnitude of 1 μM U69593 response

To examine whether the cellular response to U69593 differed as a function of basal frequency or amplitude, we ran Pearson correlations between sIPSC frequency/amplitude or sEPSC frequency/amplitude and their corresponding % change with 1 μM U69593. When looking at GABA transmission, we found a significant negative correlation with basal sIPSC frequency in air-exposed males (r = −0.67, p = 0.05) and with sIPSC amplitude in air-exposed females (r = −0.60, p = 0.05). For glutamate transmission, we found significant negative correlations with sEPSC frequency in air females (r = −0.69, p = 0.04), and significant negative correlations with sEPSC amplitude in air-exposed males (r = −0.69, p = 0.02) and EtOH-exposed males (r = −0.66, p = 0.04), as well as air-exposed females (r = −0.60, p = 0.01). A summary of all correlation coefficients and associated p-values are displayed in Table 2. Together with our other cell-responsiveness data, these indicate that aCIE exposure may disrupt the way individual cells respond to U69583 in both males and females.

Table 2.

Correlations between baseline sIPSCs or sEPSCs and the magnitude of the response to U69593 (% change). Bold correlation coefficients indicate significant (p < 0.05) correlations.

Baseline X U69593
Response		 Air
Male
Frequency		 Air
Male
Amplitude		 EtOH
Male
Frequency		 EtOH
Male
Amplitude		 Air
Female
Frequency		 Air
Female
Amplitude		 EtOH
Female
Frequency		 EtOH
Female
Amplitude
slPSC r value −0.67 −0.65 0.01 −0.27 −0.06 −0.60 0.26 −0.44
p value 0.05 0.06 0.98 0.49 0.85 0.05 0.46 0.20
sEPSC r value −0.31 −0.69 −0.01 −0.66 −0.69 −0.77 0.14 −0.31
p value 0.36 0.02 0.97 0.04 0.04 0.01 0.72 0.42
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Discussion

Numerous studies have demonstrated that exposure to alcohol during adolescence has long-term consequences, including increased risk for developing an anxiety disorder or alcohol use disorder in adulthood (Crews et al., 2016). Despite the well-established role of the BLA in disorders of this type, few studies have investigated the long-term effects of adolescent alcohol exposure on BLA physiology, particularly using moderate levels of ethanol. Therefore, one goal of the present study was to understand how exposure to alcohol during adolescence may affect adult inhibitory (GABAergic) and excitatory (glutamatergic) neurotransmission in the BLA. Our findings indicate that adult males who had gone through aCIE exposure exhibited no changes to basal glutamate transmission or basal GABA transmission compared to air-exposed controls. Females exposed to aCIE also showed no change in GABA transmission but did exhibit a significant increase in sEPSC frequency and amplitude compared to air-exposed controls, together suggesting a persistent increase in glutamate transmission in females following adolescent alcohol exposure.

The present work is the first to examine long-term changes to BLA physiology following alcohol exposure during adolescence. However, studies examining the effects of adolescent vapor exposure followed by acute withdrawal have provided evidence that adolescent alcohol exposure disrupts excitatory and inhibitory transmission in the male BLA. For example, one study that exposed adolescent (based on weight) male Sprague Dawley rats to intermittent vapor ethanol for 10 days found enhanced presynaptic glutamate release and enhanced postsynaptic NMDA receptor function 24 hours into withdrawal (Läck et al., 2007). This group has also identified different alterations on specific glutamatergic inputs into the BLA, finding that projections from the prefrontal cortex (Christian et al., 2012) and agranular insular cortex (McGinnis, Parrish, & McCool, 2020) have enhanced postsynaptic AMPA receptor function. They also found that glutamatergic projections from the dorsal and ventral medial prefrontal cortex (mPFC) to the BLA are differentially impacted, showing that presynaptic glutamate release from the dorsal mPFC is increased while it is decreased from the ventral mPFC 24 hours into withdrawal (McGinnis, Parrish, Chappell, et al., 2020). While our sEPSC findings in males do not reflect those of these studies, a major difference is the period of abstinence from ethanol, as our rats underwent prolonged (30+ days) abstinence following exposure. Two studies that exposed adolescent rats to eight cycles of 16 hours on and 8 hours off from vaporized ethanol found no apparent effects on group 1 mGluR-mediated long term depression in the BNST 30 days after exposure (Carzoli et al., 2019; Kasten et al., 2020), despite observing alterations to this and increased sEPSC frequency 24 hours after exposure (Carzoli et al., 2019). Based on these studies in the BNST and our own findings, it seems likely that the disrupted mechanisms identified during acute withdrawal undergo additional compensatory adaptations during protracted abstinence. In the present study, we did not examine any potential changes to intrinsic excitability of BLA neurons, nor did we explore possible input-specific or population-specific effects as was done in those studies described above. It is possible that alterations may depend on the neuronal population or specific synapses being sampled, as these previous studies directly examined (Christian, Alexander, Diaz, & McCool, 2013; Christian et al., 2012; McGinnis, Parrish, Chappell, et al., 2020; McGinnis, Parrish, & McCool, 2020; Morales et al., 2018). Additionally, the studies outlined above used a greater total number of exposures or had exposures that induced greater BECs in their animals compared to our aCIE exposure, which also likely plays an important role in the effects observed (more discussion on this below). Investigation of these variables in future studies would provide a more comprehensive understanding of how our aCIE exposure, as well as a more protracted abstinence period following adolescent alcohol exposure, influences glutamate transmission in the male BLA.

Interestingly, our sEPSC findings in our female animals are somewhat consistent with these previous works; we also found increased sEPSC frequency and amplitude in aCIE females, suggesting a general elevation of glutamate transmission within the BLA. A study by Morales et al. found that females also exhibited pre- and postsynaptic changes to the BLA glutamate system following a 10-day exposure and acute withdrawal (Morales et al., 2018). Interestingly, females were more resistant to adolescent alcohol exposure-induced alterations to the glutamate system, in that females required a full 10-day exposure before exhibiting the changes described above, whereas males required only a 7-day exposure to begin exhibiting alterations (Morales et al., 2018). In contrast to these findings in the BLA including the current study, in the BNST of females sEPSC frequency was enhanced and NMDA receptor-mediated long term depression was attenuated during acute withdrawal, with this effect being absent 30 days later (Carzoli et al., 2019; Kasten et al., 2020). Given that few studies have examined physiological alterations following adolescent alcohol exposure in females, these studies, along with our findings, point toward important sex-dependent adaptations in neurotransmission in anxiety-related brain areas. It is likely that these sex-dependent changes may underlie, in part, some of the well-described sex differences in behavior associated with adolescent alcohol exposure.

In contrast to the glutamate system, we did not find aCIE-induced alterations to GABA transmission in either males or females. Although long-term alterations to inhibitory transmission in the BLA following adolescent alcohol exposure have not been examined, studies assessing GABA transmission following acute withdrawal from chronic intermittent ethanol exposure in presumptive adolescent males showed that presynaptic GABA release was suppressed 24 hours into abstinence, and this was specific to feed-forward inhibitory synapses that mediate top-down regulation of BLA excitability (Diaz et al., 2011). In this same study, local feedback inhibitory synapses did not exhibit this decrease in GABA release, indicating that alterations to inhibitory transmission are synapse dependent. Postsynaptic alterations to the GABA system at both synapse types were also found during acute withdrawal (24 hours into forced abstinence), although this appeared to differentially affect specific GABAA receptor subunits at different synapses (Diaz et al., 2011). Importantly, these synapses are localized on different compartments on pyramidal cells within the BLA, with feedforward inhibitory synapses located along the external capsule and synapse onto distal dendrites, while local feedback inhibitory synapses are spread throughout the BLA and synapse onto proximal dendrites (Marowsky, Yanagawa, Obata, & Vogt, 2005; Silberman, Ariwodola, & Weiner, 2009; Woodruff & Sah, 2007). Because of the location of these different synapses onto pyramidal cells, sIPSCs are most likely derived from GABA release from local interneurons (Silberman et al., 2009). In the present study, our sIPSC recordings were therefore likely sampled from local feedback inhibitory synapses, although GABAergic inputs from other brain structures cannot be completely ruled out. Nevertheless, our finding that aCIE did not alter sIPSC frequency in males is potentially consistent with that from Diaz and colleagues. Additionally, since we did not observe changes in sIPSC amplitude, it appears that aCIE does not produce long-term postsynaptic changes to GABA transmission within the BLA; however, we did not examine any receptor- or subunit-specific mechanisms so we cannot rule out potential postsynaptic adaptations. As with our assessment of the glutamate system, future exploration of these and other potential mechanisms would provide a more thorough understanding of how our aCIE paradigm and protracted abstinence may influence this system. The present study is also the only study to examine adolescent alcohol exposure-induced alterations to GABA transmission in the BLA of females. While our GABA findings from males and females were similar, basal differences in BLA physiology have previously been found between males and females (Blume et al., 2017), so further exploration of this system in females is necessary.

Given that exposure to ethanol at any point in the lifespan leads to neuroadaptations to the KOR system, a well-established stress system, and that our model of moderate aCIE produced interactive effects with adult stress exposure (Gamble & Diaz, 2020), we assessed whether aCIE exposure would alter BLA KOR function. In control males, we found that KOR activation did not change GABA or glutamate transmission, a finding consistent with previous work from our lab (Przybysz et al., 2017). Interestingly, in aCIE males, KOR activation significantly reduced sIPSC frequency, which we found to be a result of an increase in the number of individual cells that were inhibited by the agonist. In contrast, we did not observe any effects of aCIE on KOR modulation of glutamate transmission in males, although it is worth mentioning that there were mixed effects when analyzing the effect of U69593 on sEPSCs in individual neurons. These data indicate that aCIE exposure disrupts BLA KOR function in males by producing an overall suppressing effect on GABA transmission. Interestingly, although we found slight differences in the number of cells that responded to U69593 in control and aCIE females, on average, this did not produce significant changes in GABA or glutamate transmission. While this is the first study to examine the impact of adolescent ethanol exposure on KOR modulation of synaptic function in the BLA, several studies have investigated the effects of chronic ethanol exposure on the KOR system in adulthood in various brain regions including the extended amygdala. One study using repeated binge-level administration of ethanol found that KOR mRNA was increased relative to controls in tissue containing both BLA and central amygdala (D’Addario et al., 2013). Increased dynorphin-A immunoreactivity and dynorphin-A stimulated G-protein receptor coupling were also found in the central amygdala following induction of ethanol dependence and acute withdrawal (Kissler et al., 2014). Consistent with findings in the amygdala, KOR mRNA expression was also increased in the BNST during acute withdrawal from chronic ethanol exposure (Erikson, Wei, & Walker, 2018). Finally, withdrawal from chronic ethanol exposure increased KOR regulation of dopamine transmission in the nucleus accumbens which may underlie reduced dopamine release in ethanol-dependent rats (Karkhanis et al., 2016; Rose et al., 2016). However, it was recently reported that adolescent ethanol exposure during early adolescence (P25–45) leads to enhanced KOR-mediated suppression of dopamine release in adult females, but not males, whereas exposure during late adolescence (P45–65) resulted in reduced KOR-mediated suppression of dopamine release in adult males, but not females (Spodnick et al., 2020). Thus, the current study provides additional evidence that ethanol exposure, regardless of the age, produces sex-specific adaptations to the KOR system.

Notably, the present study differs from the work described above in that our electrophysiological testing occurred after a protracted abstinence period, rather than during acute withdrawal. Studies examining changes to expression of the KOR system have found that dynorphin-B peptide levels were upregulated 21 days following adolescent alcohol exposure (Granholm, Segerstrom, & Nylander, 2018; Lindholm, Ploj, Franck, & Nylander, 2000), but current studies examining long-term changes to KOR function following chronic alcohol exposure are limited. One study examining the role of the KOR system in the alcohol deprivation effect following 18 days of abstinence found that the selective KOR antagonist JDTic reduced the reinstatement of alcohol intake (Uhari-Väänänen et al., 2019), while another found that the KOR system was still actively involved in mediating abstinence-induced stress responsiveness six weeks after chronic alcohol exposure (Gillett, Harshberger, & Valdez, 2013). Another study specifically examining the role of central amygdala KORs in alcohol intake during acute and protracted withdrawal found that infusion of a KOR antagonist into the central amygdala reduced alcohol consumption during acute withdrawal, and that this effect lasted throughout protracted abstinence (Kissler & Walker, 2016). These studies together indicate that the KOR system is both altered by chronic alcohol exposure in adulthood and remains in this altered state for up to six weeks following termination of exposure. Our results, together with those in the nucleus accumbens (Spodnick et al., 2020), compliment this work showing that KOR function is disrupted at least 30 days following termination of adolescent ethanol exposure. We also expand upon this work by revealing these effects in a brain region that has not previously been studied in this context. The BLA is heavily involved in behaviors associated with the long-term effects of both adult and adolescent alcohol exposure including anxiety, stress responsiveness, and reward processing, so this work opens a broad avenue for future investigation into the behavioral implications of KOR dysfunction following alcohol exposure at multiple developmental ages.

Work investigating the role of the KOR system in females is much less abundant than work using males, but studies including both sexes have consistently demonstrated that females may be less sensitive to KOR manipulation [for review, see (Chartoff & Mavrikaki, 2015)]. Notably, this has been shown in behaviors related to affective states such as anxiety, social interaction, depression, and reward sensitivity (Przybysz, Varlinskaya, & Diaz, 2020; Russell et al., 2014; Varlinskaya, Spear, & Diaz, 2018), all of which engage the BLA in some capacity. It is therefore interesting that we did not observe differences in how KOR activation modulates BLA neurotransmission between control males and females. On the other hand, in aCIE-exposed animals, we found that KOR activation altered neurotransmission in males but had no effect in females. Conversely, KOR suppression of dopamine release was enhanced in the nucleus accumbens following ethanol exposure during early adolescence (Spodnick et al., 2020). Interestingly, females are resistant to acute KOR-mediated responses to manipulations like stress, as we found that females with a history of restraint stress were less sensitive to the socially-inhibiting effects of KOR activation (Varlinskaya et al., 2018). We also recently showed that social behavior in either adolescent or adult females was not affected by forced swim stress, a manipulation that has been shown to engage the KOR system (Varlinskaya et al., 2020). While the present study adds to our understanding of how long-term abstinence from adolescent alcohol exposure may disrupt KOR function in females, it is clear that additional work is needed to fully understand these effects across the brain and their influence on sex-specific behavioral profiles.

Another important distinction between the present study and previous work examining the effects of adolescent alcohol exposure is that we used a moderate-level exposure paradigm. Adolescents who consume alcohol often consume it in a binge-like pattern (Spear, 2014), but according to the National Drug Use and Health report from 2018, a similar percentage of adolescents consume alcohol in amounts that do not reach binge levels (Substance Abuse and Mental Health Services Administration, 2018). Despite this, the majority of preclinical work dedicated to understanding the neural consequences of adolescent alcohol exposure has used binge-level exposure, with very few studies explicitly examining the effects of low-moderate exposure. In our behavioral assessment of the moderate aCIE paradigm used in this study, we did not observe any significant effects of moderate aCIE on social or non-social anxiety-like behaviors or on ethanol intake in either sex (Gamble & Diaz, 2020). Similarly, another study that ran a battery of behavioral assays following 4-week voluntary intermittent alcohol consumption, an exposure that likely yielded moderate-level BECs, found no differences in novel object recognition or anxiety-like behavior two weeks following the final exposure (Sanchez-Marin et al., 2020). However, exposure to ethanol during adolescence can lead to heightened sensitivity and reactivity to stress later in life (Allen et al., 2016; Logrip et al., 2013; Varlinskaya, Kim, & Spear, 2017b; Vore, Doremus-Fitzwater, Gano, & Deak, 2017), an effect that we found following exposure to forced swim stress in adulthood, which increased ethanol intake only in aCIE-exposed males, while having no effect in females (Gamble & Diaz, 2020). The finding that moderate aCIE only altered KOR modulation of synaptic transmission in the BLA of adult males is in line with our behavioral findings, as we predict that the KOR system is engaged by stress exposure. More specifically, our findings indicate that KOR activation in aCIE males would reduce inhibitory tone in the BLA, leading to over-excitation of this structure and potentially driving increased ethanol intake. Future studies should directly test these potential mechanisms and determine why females may be resilient to moderate exposure to ethanol in adolescence.

Taken together, the current study found that moderate aCIE exposure followed by protracted abstinence enhanced BLA glutamate transmission in females and produced functional alterations to the BLA KOR system in males. This study is the first to demonstrate that adolescent alcohol exposure produces long-term changes to BLA physiology and provides further evidence of the vulnerability of the KOR system to insults during developmentally sensitive periods of the lifespan [for review, see (Diaz et al., 2017)]. Overall, advancing our understanding of how adolescent alcohol exposure produces alterations to the excitatory/inhibitory balance in the brain and the systems that modulate it, many of which may confer vulnerability to affective dysfunction and alcohol use disorder in adulthood, will be critical to developing better therapeutic tools to better treat these disorders across the population.

Supplementary Material

Supplementary Figure 1

Supplemental Figure 1. Before-after graphs showing individual within-cell effects of KOR activation on sIPSC frequency and amplitude in males (top) and females (bottom). U69593 caused a significant reduction of sIPSC amplitude in aCIE-exposed males (D) and females (H). Paired t-test, * = p < 0.05.

Supplementary Figure 2

Supplemental Figure 2. Before-after graphs showing individual within-cell effects of KOR activation on sEPSC frequency and amplitude in males (top) and females (bottom). U69593 caused a significant reduction of sEPSC amplitude in both air- (F) and aCIE-exposed (H) females. Paired t-test, * = p < 0.05.

Funding:

This work was supported by NIAAA grant AA024890 and Binghamton University Presidential Diversity Research Grant.

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Associated Data

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

Supplementary Materials

Supplementary Figure 1

Supplemental Figure 1. Before-after graphs showing individual within-cell effects of KOR activation on sIPSC frequency and amplitude in males (top) and females (bottom). U69593 caused a significant reduction of sIPSC amplitude in aCIE-exposed males (D) and females (H). Paired t-test, * = p < 0.05.

NIHMS1928405-supplement-Supplementary_Figure_1.docx (201.6KB, docx)
Supplementary Figure 2

Supplemental Figure 2. Before-after graphs showing individual within-cell effects of KOR activation on sEPSC frequency and amplitude in males (top) and females (bottom). U69593 caused a significant reduction of sEPSC amplitude in both air- (F) and aCIE-exposed (H) females. Paired t-test, * = p < 0.05.

NIHMS1928405-supplement-Supplementary_Figure_2.docx (210.1KB, docx)

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