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. Author manuscript; available in PMC: 2026 Feb 12.
Published in final edited form as: Pharmacol Biochem Behav. 2024 Aug 7;243:173840. doi: 10.1016/j.pbb.2024.173840

Kappa-opioid receptor antagonism in the nucleus accumbens shell distinguishes escalated alcohol consumption and negative affective-like behavior from physiological withdrawal in alcohol-dependence

Gaetan Lepreux 1,*, Angela M Henricks 2,*, Gengze Wei 1,2, Bok Soon Go 3, Chloe M Erikson 2, Rachel M Abella 2, Amy Pham 1, Brendan M Walker 1,2,4,5
PMCID: PMC12893421  NIHMSID: NIHMS2131884  PMID: 39096973

Abstract

Alcohol use disorder (AUD) is a chronic relapsing disease that is deleterious at individual, familial, and societal levels. Although AUD is one of the highest preventable causes of death in the USA, therapies for the treatment of AUD are not sufficient given the heterogeneity of the disorder and the limited number of approved medications. To provide better pharmacological strategies, it is important to understand the neurological underpinnings of AUD. Evidence implicates the endogenous dynorphin (DYN) / κ-opioid receptor (KOR) system recruitment in dysphoric and negative emotional states in AUD to promote maladaptive behavioral regulation. The nucleus accumbens shell (AcbSh), mediating motivational and emotional processes that is a component of the mesolimbic dopamine system and the extended amygdala, is an important site related to alcohol’s reinforcing actions (both positive and negative) and neuroadaptations in the AcbSh DYN / KOR system have been documented to induce maladaptive symptoms in AUD. We have previously shown that in other nodes of the extended amygdala, site-specific KOR antagonism can distinguish different symptoms of alcohol dependence and withdrawal. In the current study, we examined the role of the KOR signaling in the AcbSh of male Wistar rats in operant alcohol self-administration, measures of negative affective-like behavior, and physiological symptoms during acute alcohol withdrawal in alcohol-dependence. To induce alcohol dependence, rats were exposed to chronic intermittent ethanol vapor for 14h/day for three months, during which stable escalation of alcohol self-administration was achieved and pharmacological AcbSh KOR antagonism ensued. The results showed that AcbSh KOR antagonism significantly reduced escalated alcohol intake and negative affective-like states but did not alter somatic symptoms of withdrawal. Understanding the relative contribution of these different drivers is important to understand and inform therapeutic efficacy approaches in alcohol dependence and further emphasis the importance of the KOR/DYN system as a target for AUD therapeutics.

1. INTRODUCTION

Alcohol use disorder (AUD), defined by the inability to control or cease alcohol use despite personal and societal harm, was diagnosable in 11.2% of the United States population age 18 and older in 2021 (SAMSHA, 2023) and adversely impacts society in the United States with over $200 billion USD in annual costs (Sacks et al., 2015). Moreover, not only have AUD diagnoses been increasing in contemporary times, but deaths attributable to AUD have as well with a 29% increase from 2016–2021 (Esser et al., 2024) which is staggering given that AUD has been estimated as the third-most preventable cause of death in the United States (Mokdad et al., 2004). Current pharmacotherapeutic strategies for AUD in the United States have remained unchanged since acamprosate was approved in 2004 by the United States Food and Drug Administration (FDA), and along with disulfiram and naltrexone as the three FDA-approved medications, are not efficacious in all those utilizing pharmacotherapy given the heterogeneity of AUD development and expression (Heilig and Egli, 2006; Walker et al., 2012; Akbar et al., 2018). AUD is associated with increased risk of injuries, diseases and disorders of affect induced by AUD, such as anxiety and depression (Driessen et al., 2001; Boden and Fergusson, 2011; Sánchez-Peña et al., 2012) that are relatively unaddressed by currently-approved strategies, but can drive self-medication with alcohol. Once alcohol-dependent, negative affective alterations contribute to the shift from using alcohol for positive reinforcing properties to self-medicating according to negative reinforcement principles (Khantzian, 1990; Koob and Le Moal, 1997; Markou et al., 1998; Walker, 2012) that involve the removal of aversive stimuli, such as anxiety and depression associated with acute and protracted withdrawal (e.g., Williams et al., 2012) being reinforcing.

The endogenous opioid peptide system is involved in the regulation of motivation and emotion and has been linked to the consumption of abused drugs, including alcohol (Bodnar, 2022). Several opioid receptors were historically identified, each with a preferred endogenous ligand and include the mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR) that preferentially bind β-endorphin, enkephalin and dynorphin (DYN), respectively (Chavkin et al., 1982; Fowler and Fraser, 1994) with nociceptin opioid receptor (NOP) and its ligand, nociceptin/orphanin FQ (N/OFQ) later included as a fourth opioid receptor / peptide system (Mollereau et al., 1994; Meunier et al., 1995; for review, see Toll et al., 2016). Endogenous opioid systems in the brain have long been a target for the treatment of AUD with naltrexone (Volpicelli et al., 1992), a general opioid receptor antagonist with primarily MOR-related efficacy in AUD (Walker and Koob, 2008; Akbar et al., 2018). Preclinical studies have compared opioid ligands for the ability to reduce operant alcohol self-administration and comparisons between naltrexone and nalmefene, an opioid receptor ligand with both MOR antagonist and KOR partial agonist effects (that could be considered a functional antagonist under conditions of high endogenous ligand presence), highlighted the potential importance of the KOR and DYN in AUD therapeutics (Walker and Koob, 2008) in non-dependent and alcohol-dependent rats. Further testing substantiated a role for the KOR by testing the KOR antagonist nor-binaltorphimine (nor-BNI) that selectively reduced escalated alcohol self-administration in alcohol-dependent rats compared to non-dependent rats and supported the concept of DYN / KOR system recruitment in alcohol dependence that could serve as a therapeutic target for the treatment of AUD. Indeed, KOR activation is involved in aversion and dysphoria (McLaughlin et al., 2003; Land et al., 2008; Berger et al., 2013; Al-Hasani et al., 2015) and has been shown to promote depressive-like behavior (Todtenkopf et al., 2004; Carlezon et al., 2006; Nestler and Carlezon, 2006; Bruchas et al., 2010; Carlezon and Miczek, 2010; Knoll and Carlezon, 2010), while KOR antagonists have anti-depressant properties (Pliakas et al., 2001; Mague et al., 2003b; Carr et al., 2010; Bruchas et al., 2011) and dysregulation of the KOR/DYN system is involved in motivational, negative emotional, and executive function psychopathologies associated with AUD (for reviews, see Sirohi et al., 2012; Walker et al., 2012; Karkhanis and Al-Hasani, 2020).

The extended amygdala (Alheid and Heimer, 1988) is a functionally-interconnected network that is comprised of the nucleus accumbens shell (AcbSh), central nucleus of the amygdala (CeA), and bed nucleus of the stria terminalis (BNST), and has been shown to be an important network for the regulation of motivation and emotion and is greatly impacted in AUD and other addictive disorders to drive maladaptive behavioral regulation (Koob, 2009; Berridge and Kringelbach, 2015; Koob and Volkow, 2016; Centanni et al., 2019). As a result, a tremendous amount of research from our lab and others has focused on the extended amygdala to understand the role DYN / KOR system dysregulation plays in alcohol dependence and AUD in general. Alcohol can increase DYN release in nuclei of the extended amygdala (Lindholm et al., 2000b; Marinelli et al., 2006; Lam et al., 2008) and induce neuroadaptations in the DYN/KOR system under conditions of chronicity that involve increased DYN peptide and KOR function / sensitivity (Kissler et al., 2014; Rose et al., 2016; Siciliano et al., 2016). These effects can putatively contribute to hypodopaminergia that could lead to a depressive state (Nestler and Carlezon, 2006) that drives self-medication with alcohol through negative reinforcement mechanisms (Walker, 2012).

In order to further understand extended amygdala DYN / KOR dysregulation in the context of specific AUD-related phenotypes, we systematically began to evaluate a host of behaviors implicated as drivers of self-medication (e.g., negative affective-like behavior vs physiological withdrawal; Williams et al., 2012) following site-specific KOR antagonism in the extended amygdala in non-dependent and alcohol-dependent rats (Nealey et al., 2011; Kissler et al., 2014; Kissler and Walker, 2016; Erikson et al., 2018). To date, KOR antagonism in the CeA and BNST have revealed an interesting dissociation between the effects of KOR antagonism on physiological withdrawal symptoms and escalated alcohol self-administration was observed in the CeA (Kissler and Walker, 2016), but a trend towards a reduction in somatic withdrawal signs was observed following dorsolateral BNST KOR antagonism (Erikson et al., 2018). The AcbSh is the final node of our extended amygdala investigation for these phenotypic drivers of escalated alcohol self-administration and the focus of this study. Specifically, the purpose of the current study was to assess the role of AcbSh KORs in alcohol withdrawal-induced escalated drinking, negative affective states, and physiological withdrawal. We evaluated whether site-specific intra-AcbSh nor-BNI, according to a between-group dose-response curve, attenuates acute alcohol withdrawal-induced escalated drinking in an operant alcohol self-administration paradigm, 22-kHz USVs as an index of negative affective-like behavior followed by a discrete evaluation of specific contributors to negative affective-like behavior via measurement of depressive- or anxiety-like behavior, and signs of physiological (somatic) withdrawal in rats made dependent on alcohol following exposure to chronic intermittent alcohol vapor.

2. METHODS

2.1. Animals

A total of fifty-eight male Wistar rats approximately 70 days old (bred from Charles River Laboratory (Hollister, CA) breeding pairs) were used for this experiment according to the timeline found in Fig. 1. Animals were handled for 5 days before the beginning of the study and housed in groups of two or three rats per cage for the length of the experiments other than a 5-day recovery period following guide cannula implantation (see below). The environment was controlled for temperature (21±2 1C) and humidity with a 12-h reverse light cycle and ad libitum food and water. Animal care adhered to the National Research Council’s Guide for the Care and Use of Laboratory Animals (2011) and Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (2003), with all procedures approved by the Washington State University and University of South Florida Institutional Animal Care and Use Committees.

Fig 1. Experimental organization and timeline.

Fig 1.

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2.2. Acquisition of Operant Alcohol Self-administration

Animals were trained to self-administer 10% alcohol using a sweetener-fade technique during 30-minute operant sessions using methods previously published (Valenstein et al., 1967; Walker and Koob, 2007; Ji et al., 2008; Nealey et al., 2011; Williams et al., 2012). Briefly, operant training took place in standard operant conditioning chambers (Med Associates, St Albans, VT) with custom drinking wells (Behavioral Pharma, La Jolla, CA) using a fixed ration 1 (FR-1) schedule of reinforcement. Acquisition of the operant response occurred using a sweetened fluid as the reinforcer (0.125% saccharin and 3% glucose), which does not require food or water deprivation. Then, 10% alcohol (w/v) was added to the sweetened fluid and over 3 weeks, sweetener was slowly removed until only 10% alcohol (w/v) was left in the solution. After the responding for alcohol stabilized (<10% deviation in lever-presses over three sessions), animals underwent AcbSh cannulation.

2.3. Surgical Procedures

All animals were anesthetized with isoflurane gas (maintained at ~2%) and bilateral guide cannulae were implanted into the AcbSh using stereotaxic coordinates (from bregma: AP +1.8, DV −5.8, and ML ±1.0), as published previously (Walker and Ettenberg, 2007; Nealey et al., 2011). AcbSh guide cannulae were secured in place using acrylic dental cement and machine screws used to anchor the acrylic. The open ends of the guide cannulae were sealed with bilateral obturators (PlasticsOne, Roanoke, VA) to reduce risk of infection and maintain guide cannulae patency. Obturators and cannula guides were protected with stainless-steel cap nuts. Postoperative analgesics (flunixin; MWI Veterinary Supply, Meridian, ID) were administered daily for a total of 5 days following surgery. Animals were allowed to recover for at least 1 week before chronic intermittent alcohol vapor exposure began. Cannula patency was verified throughout the experiment by ensuring that the proper volume of solution had been infused into each animal’s internal cannula during pharmacological testing, and also by histology at the conclusion of the experiment by injecting cresyl violet (see histology section) and extracting the brain to confirm dye had penetrated the AcbSh.

2.4. Intermittent Alcohol Vapor Exposure

Chronic intermittent alcohol vapor exposure (14 h on/10 h off; 7 days/week) was carried out in custom designed vapor chambers (La Jolla Alcohol Research, La Jolla, CA). 95% alcohol is vaporized and introduced into the airflow of the sealed environmental chambers in which animals are housed. The experimenter can adjust the rate of 95% alcohol vaporized such that the blood ethanol concentration (BEC) of the animals is kept within a desired range (175–225mg%) which was determined bi-weekly by collecting blood from the tails (~50 μl). After centrifugation, plasma samples were assayed for alcohol content using the Analox GL5 (Analox Instruments, Lunenburg, MA).

2.5. Drugs

Nor-binaltorphimine (nor-BNI) dihydrochloride was purchased from Tocris Bioscience, Ellisville, MO, and was soluble at the concentrations tested in artificial cerebral spinal fluid (aCSF) as described previously (Alvarez-Jaimes et al., 2009; Nealey et al., 2011; Kissler et al., 2014; Kissler and Walker, 2016; Erikson et al., 2018; Wei et al., 2022).

2.6. Post-vapor Intra-AcbSh Pharmacology and Behavior

For all behavioral tests conducted throughout this study, the animals were tested at a time point corresponding to 6–10 h in acute withdrawal. Following daily vapor exposure using this paradigm, this time-point has been shown to coincide with complete elimination of BECs (Wei et al., 2022) and is ideal for behavioral testing due to the elimination of the confounding presence of alcohol. There were two experiments conducted: 1) Operant alcohol self-administration with concomitant physiological withdrawal and 22-kHz ultrasonic vocalization (USV) measurement. 2) Physiological withdrawal measurement with concomitant elevated plus maze (EPM) and forced swim test (FST) evaluations.

2.6.1. Post-vapor operant alcohol self-administration

Following 4 weeks of chronic intermittent alcohol vapor exposure, animals were allowed to self-administer (FR-1) ethanol for 30 min twice a week during acute withdrawal (6–8 hrs after the vapor turned off). Once stable responding was achieved, animals were parsed into the three different treatment groups representing nor-BNI dosing (0, 2, and 6 μg). There were three phases of testing: 1) sham infusions 2) aCSF infusions and 3) a pharmacological challenge with nor-BNI. Sham infusions were used to habituate the animals to the infusion process and consisted of inserting a bilateral internal cannulae (28GA, Plastics One) for 2 min, with no infusions at that time. After the 2 min period, the animal was transferred to the operant testing room and given a 5 min waiting period before the self-administration session began. After stable sham responding was achieved (<10% deviation for 2 self-administration sessions), the same procedure was followed for aCSF infusions (0.5 μl/side over 2 min, plus an additional two minute). The aCSF infusions occurred until stable responding was established (<10% deviation for 2 self-administration sessions). Sham and aCSF infusions demonstrated that no changes in ethanol responding occurred due to the infusion procedure or nor-BNI vehicle and that any changes in self-administration were due to the pharmacological challenge. After each self-administration session, the animals were returned to the alcohol vapor-chambers for their intermittent exposure regimen such that future testing was conducted under identical conditions of alcohol dependence and withdrawal. Pharmacological infusions of nor-BNI occurred as described above for the aCSF infusions, however, since nor-BNI has an extended duration of action (Picker et al., 1996; Walker et al., 2011), animals were given a one-time infusion of nor-BNI (0, 2, or 6 μg; n=5–7/grp) immediately prior to self-administration sessions with the addition of a 5-min waiting period. We have previously shown that when administered peripherally, centrally, and site-specifically in the extended amygdala, nor-BNI does not affect alcohol drinking in non-dependent animals using this limited-access operant alcohol self-administration paradigm (Walker and Koob, 2008; Nealey et al., 2011; Walker et al., 2011; Kissler et al., 2014; Erikson et al., 2018) and thus, an air-exposed group was not used in this experiment to avoid unnecessary use of animals.

2.6.2. Post-vapor 22-kHz Ultrasonic Vocalizations

Forty-eight hours after testing in the self-administration paradigm (following intra-AcbSh nor-BNI or aCSF infusions), production of 22-kHz USVs were assessed during acute withdrawal using methods published previously (Williams et al., 2012; Berger et al., 2013; Kissler et al., 2014; Erikson et al., 2018). USVs were measured in a quiet room with dim lighting (15 lux) by administering an air-puff (60 psi) to the nape of the animal’s neck. Air-puffs have been validated as a non-painful method of inducing 22-kHz USVs in rodents (Knapp and Pohorecky, 1995). Vocalizations were recorded by a microphone affixed 15cm above the animal’s head. Each test consisted of two trials, and each trial consisted of 15 air-puffs every 15 seconds, or until the animal vocalized. Once an animal vocalized, the experimenter waited until the animal stopped vocalizing for 1min before beginning a new trial. If an animal vocalized for 10 min, the trial was stopped and a new trial began. Vocalizations were recorded with a P48 Electret Ultrasound Microphone (Avisoft Bioacoustics, Germany), E-MU Systems Audio/MIDI Interface (Scotts Valley, CA), and Avisoft Bioacoustics software (created by AEST, Italy). The number and duration of 22-kHz USVs from the second trial were objectively counted with an Avisoft Bioacoustics software automation protocol (Berlin, Germany) and used for all data analyses.

2.6.3. Post-vapor physiological withdrawal

In order to determine whether changes in post-dependent self-administration and affective behavior were co-incident with alterations in physiological withdrawal, separate groups of alcohol dependent rats (n=5/grp) were tested in a parallel treatment regimen to those in the operant self-administration / USV paradigms and received AcbSh (0 or 6μg) infusions of nor-BNI or aCSF during acute withdrawal. Physiological withdrawal signs, indicative of an alcohol-dependent state, were assessed using the methods described previously (Schulteis et al., 1995; Williams et al., 2012; Kissler and Walker, 2016; Erikson et al., 2018; Wei et al., 2022). Four behaviors were scored on a scale of 0–2 (2 indicating severe or persistent presence of the symptom): 1) hyperirritability upon touch, 2) presence of the ventromedial distal flexion response (measured by gently grasping the rat by the scruff of the neck and checking the retraction of the limbs towards the body), 3) tail stiffness/rigidity, and 4) abnormal posture or gait. Animals were observed for 3 min and given scores for all symptoms were combined to obtain a single composite score for physiological withdrawal symptoms ranging from 0 to 8. In addition, physiological withdrawal was also assessed in the animals tested in the forced swim test and elevated plus-maze following chronic intermittent alcohol vapor exposure (see below) to ensure a dependence-like state was realized by the alcohol vapor exposure regimen when tested during acute withdrawal.

2.6.4. Post-vapor elevated plus maze (EPM)

Following a vapor exposure duration consistent with those animals in the operant self-administration / USV experiments (i.e., three months of vapor exposure) and consistent with the timing of the USV assessment (i.e., forty-eight hours following nor-BNI or aCSF infusions), animals were tested in the EPM paradigm during acute withdrawal (0, 2, or 6 μg; n=8–9/grp) 5 min after physiological withdrawal testing as described above. The EPM consists of a raised Plexiglas platform (50 cm high) with two open arms and two closed arms of equal length (47×10 cm each) and a 10×10-cm center platform. The floors of the EPM as well as the walls of the closed arms were opaque (40 cm high). Each animal was placed in the center of the platform facing the same direction and allowed to explore the maze for 5 min. Illumination in all arms was approximately 15 lux. Each animal was recorded by video and AnyMaze video tracking software (Stoelting Co, Wood Dale, IL) was used to score the amount of time spent in the open and closed arms, as well as open and closed arm entries and distance traveled (m). The maze was cleaned with Quatricide® and dried between each animal.

2.6.5. Post-vapor forced swim test (FST)

Forty-eight hours after testing in the EPM, the same animals tested for physiological withdrawal and EPM performance were tested in the FST paradigm. The FST test was conducted subsequent to the EPM because forced swim stress could influence EPM performance and reduce experimental control. The FST apparatus was a custom-built clear Plexiglas® cylinder (diameter= 34 cm, height= 79.5 cm). The cylinder was filled to 53 cm with water temperature at 24±2 °C. The illumination at the surface of the water was approximately 25 lux. Animals were placed in the cylinder and a 15 min video was recorded and analyzed with AnyMaze video tracking software (Stoelting Co, Wood Dale, IL). Immobility was defined as a lack of active swimming with animals floating to maintain their head above water with only minor paw movement. Swimming was defined as active movement of the animals with all four paws and climbing was defined by active attempts to climb the walls of the apparatus to escape.

2.7. Histology

Following the completion of the pharmacological challenges and behavioral testing, all animals were infused with 0.5 μl/side of 0.6% cresyl violet and euthanized. The brains were then removed, immediately snap-frozen in 2-methylbutane (isopentane) and stored at −80°C until histology was verified. On the day of histological examination, the brains were frozen to −20°C and mounted on specimen discs to be sliced using a cryostat (Leica 1850, Bannockburn, IL). 40 mm sections were mounted on slides and the injection sites were evaluated for appropriate intra-AcbSh placement (See Fig 2).

Figure 2: Nucleus accumbens histology.

Figure 2:

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Filled black circles denote internal cannula placement in animals from the alcohol self-administration / USV experiment with filled black triangles indicative of surgical misses used as negative controls with complementary dark gray indicative of physiological withdrawal data. Light gray circles denote internal cannula placement in the second physiological withdrawal/FST/EPM experiment.

2.8. Statistical Analysis

A priori power analyses for sample size (using α=0.05 and β = 0.2) were conducted using previous effect sizes generated through between group site-specific nor-BNI infusions (Kissler and Walker, 2016) which showed that our group sizes were appropriate and for prudence, we included additional animals to account for attrition. In the self-administration/USV component of the experiments, three animals were lost due to head stage complications and two animals administered nor-BNI (one from the 2-μg and one from the 6-μg doses) were removed from the study for incorrect cannula placement and used as negative controls, with data included in Fig 3. Furthermore, during a laboratory move, three brains from the self-administration / USV experiment were lost due to a logistical error, but their data was included in the final analysis because the data was consistent with their group assignment - namely, one of the three that was in the aCSF condition showed typical vapor-induced escalation while the other two that were administered nor-BNI displayed dose-related suppression of escalated alcohol self-administration. Taken together with the negative control data showing no effect of KOR antagonism outside the target region and the confirmed experimental data, the data from these animals was included in the data analysis. Lastly, in the experiment evaluating physiological withdrawal in conjunction with FST and EPM measurement, one animal in the aCSF control condition lost a cannula guide and was euthanized prior to site-specific pharmacological evaluation.

Fig. 3: The effect of site-specific KOR antagonism in the nucleus accumbens shell on operant alcohol self-administration and physiological withdrawal.

Fig. 3:

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Left Panel: Following alcohol dependence induction (denoted by the dashed line), escalation of operant alcohol self-administration was observed under intra-AcbSh sham and aCSF-treated conditions (***p < 0.001). Administration of nor-BNI resulted in significant reduction in alcohol self-administration with post-hoc tests indicating the 6-μg dose significantly reduced escalated alcohol self-administration (** p=0.013) when compared to the 0-μg condition. Moreover, when considering percent change from aCSF treatment for dependence-induced escalation (see left panel inset), escalated alcohol self-administration was dose-dependently reduced by intra-AcbSh nor-BNI with both doses inducing significant reductions in the percent change following nor-BNI infusion into the AcbSh (‡ = p= 0.029 and ‡‡‡= p = 0.0008). Right Panel: Intra-AcbSh nor-BNI (6 μg) did not alter physiological withdrawal scores. aCSF = artificial cerebrospinal fluid, NC = negative control, nor-BNI = nor-binaltorphimine.

In order to assess the effect of intra-AcbSh nor-BNI on vapor-induced behavioral change with and without KOR antagonism, a univariate analysis of variance (ANOVA) assessed operant alcohol self-administration (g/kg) and percent change in alcohol consumption (calculated as the percent change between nor-BNI test day and each animal’s previous aCSF self-administration session) following nor-BNI administration (0, 2, and 6 μg). Moreover, a multivariate ANOVA was used to assess the number and duration of air-puff induced 22-kHz USVs and somatic withdrawal signs following AcbSh nor-BNI challenge. If there was a main effect of dose, post-hoc Tukey Honest Significant Difference (HSD) tests were used to compare experimental treatments to the vehicle condition. Multivariate ANOVAs were also used to compare nor-BNI doses (0, 2, and 6 μg) as the between-group factor in the secondary FST, EPM and somatic withdrawal tests.

3. RESULTS

3.1. Experiment 1: Self-administration, 22-kHz ultrasonic vocalizations and Physiological Withdrawal

Chronic intermittent alcohol vapor exposure induced a significant escalation of alcohol self-administration as evidenced by both the sham- and aCSF-treated grouped performance (F (1,17) = 67.960, p<0.001 and F (1, 17) = 29.228, p<0.001, respectively; see Fig. 3). Importantly, there was a main effect of nor-BNI to reduce operant alcohol self-administration (g/kg; F (2, 13) = 5.685, p = 0.017). Post-hoc Tukey HSD comparisons showed that that the 6-μg dose of nor-BNI significantly decreased self-administration (Fig. 3; p= 0.013) with dependence-induced physiological signs showing zero impact from intra-AcbSh nor-BNI (F (1, 8) = 0.065, p=0.806).

When evaluating the percent change from aCSF responding following the three nor-BNI doses (0, 2, and 6 μg), the ANOVA identified a significant main effect of nor-BNI dose (Fig 3; F, 2, 13) = 12.078, p = 0.0011 with post-hoc Tukey HSD tests clearly confirming a dose-dependent reduction with the 2 μg (p = 0.029) and 6 μg (p= 0. 0008) doses differing significantly from the aCSF (0.0 μg) control condition. The individual animal data under aCSF and nor-BNI conditions that was used to generate the percent change data has been included in Supplemental Fig. S1.

When 22-kHz USVs were assessed in the self-administering, alcohol-dependent animals during acute withdrawal, a multivariate ANOVA showed significant main effects of nor-BNI dose for both the total number (Fig. 4A; F (2, 12) = 18.710, p = 0.0002) and duration (F (2, 12) = 15.760, p = 0.0004; Mean = 107.42 s, 10.1015 s, and 15.6205 s for the nor-BNI 0.0, 2.0 and 6.0 μg doses, respectively; data not shown) of USVs emitted. When compared to the control aCSF infusions, post-hoc Tukey HSD tests identified both doses of nor-BNI significantly attenuating the number (Fig. 4A; 2-μg, p = 0. 0005; 6-μg, p = 0.0005) and duration (0-μg: mean = 107.43, S.E.M. = 14.93; 2-μg: mean = 10.102, S.E.M. = 10.1, p = 0.0008; 6-μg, mean = 15.62, S.E.M. =15.62, p = 0.0013; data not shown) of 22-kHz USVs.

Fig. 4: The effect of site-specific KOR antagonism in the nucleus accumbens shell on 22-kHz USVs, physiological withdrawal, and performance in the EPM and FST.

Fig. 4:

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Panel A: Following alcohol dependence induction and operant alcohol self-administration testing, nor-BNI significantly attenuated 22-kHz USVs (***p < 0.001). Conversely, nor-BNI did not have an impact on dependence-induced physiological withdrawal scores (Panel B) or performance in the EPM (Panel C) or FST (Panel D). nor-BNI = nor-binaltorphimine, USV = ultrasonic vocalization.

3.2. Experiment 2: EPM, FST and Physiological Withdrawal.

Having found that intra-AcbSh nor-BNI infusions could significantly reduce 22-kHz USVs in (Fig. 4A), in addition to measurement of physiological withdrawal, we attempted to identify the relative anxiety- and depressive-like behavioral contributions to the compound negative affective-like behavior that 22-kHz USV measurement provides. The multivariate ANOVA that was conducted identified that neither physiological withdrawal symptoms (Fig 4B; F (2, 23) = 0.129, p = 0.879), any of the EPM parameters (Fig. 3C; F (2, 23) = 0.216 – 0.610, p = 0.552 – 0.807), including distance travelled (0-μg: mean = 6.9, S.E.M. = 0.79; 2-μg: mean = 7.05, S.E.M. = 0.7; 6-μg, mean = 5.18, S.E.M. =1.096; data not shown), nor FST performance (Fig. 4D; F (2, 23) = 0.073 – 0.844, p = 0.443 – 0.930) showed effects of nor-BNI.

DISCUSSION

Consistent with our and other’s previous findings showing that chronic intermittent alcohol vapor exposure induces escalated alcohol self-administration during acute withdrawal under fixed- and progressive-ratio schedules of reinforcement (e.g., O’Dell et al., 2004; Walker and Koob, 2007; Walker and Koob, 2008; Williams et al., 2012), alcohol-dependent rats in this study showed a significant increase in post-dependence alcohol self-administration during acute withdrawal under sham- and aCSF-treated conditions when compared to their baseline responding (Fig 3). Importantly, intra-AcbSh infusion of nor-BNI dose-dependently reduced escalated alcohol self-administration when evaluating percent change in the animals, an effect consistent with our previous investigations in the extended amygdala showing that site-specific KOR antagonism rescues dependence-induced escalation of operant alcohol self-administration (Nealey et al., 2011; Kissler et al., 2014; Kissler and Walker, 2016; Erikson et al., 2018; Wei et al., 2022). However, the primary purpose of these experiments was to evaluate the co-incidence of reduced escalation of alcohol self-administration in conjunction with reductions in other symptoms of AUD, including physiological withdrawal and negative affective-like behavior.

Counter to the effects of intra-AcbSh nor-BNI on operant alcohol self-administration, physiological withdrawal in the two separate experiments was unaffected by KOR antagonism at any dose. While this effect of KOR antagonism is consistent with results we previously established in the CeA (Kissler and Walker, 2016), we showed that within the dorsolateral BNST, in addition to significant reductions in operant alcohol self-administration and reduced 22-kHz USVs, KOR antagonism produced a trend towards reducing physiological withdrawal symptoms (Erikson et al., 2018). Consistent with our BNST-restricted effects, recent evidence has also shown that physiological withdrawal associated with alcohol dependence can be reduced via systemic KOR antagonism (Flores-Ramirez et al., 2024), an effect also shown previously following peripheral KOR antagonism when measuring nicotine withdrawal (Tejeda et al., 2012). It could be that KOR antagonism in the BNST is involved in the reduced physiological withdrawal reported following systemic KOR antagonism, although other sites related to KOR-mediated modulation of BNST-related circuitry, including innervation by the ventral noradrenergic bundle stemming from the locus coeruleus (LC; Aston-Jones et al., 1999), could also play a role given that KOR modulation of noradrenergic activity in the LC (Al-Hasani et al., 2013) has modified behavior related to addictive disorders. Moreover, the KOR has been shown to pre- and post-synaptically modulate multiple neurotransmitter systems that include DA, GABA, glutamate and serotonin (Werling et al., 1988; Spanagel et al., 1992; Thompson et al., 2000; Hjelmstad and Fields, 2003; Margolis et al., 2003; Margolis et al., 2006; Land et al., 2008; Grilli et al., 2009; Kallupi et al., 2013; Kang-Park et al., 2013; Tejeda et al., 2013; Gilpin et al., 2014; Tejeda et al., 2015; Karkhanis et al., 2016; Siciliano et al., 2016), with many implicated in dependence and withdrawal such that only future experiments will be able to delineate the precise roles these systems contribute to AUD maladaptive behavioral regulation and future therapeutic endeavors. Nonetheless, the present data are our final investigation in the physiological vs psychological withdrawal as drivers of escalated alcohol self-administration in the extended amygdala. Studies have confirmed that after most acute physiological symptoms of alcohol dependence have subsided, the psychological discomfort of anxiety and depression experienced during withdrawal plays a more important role in relapse and continued excessive alcohol use (for review, see Koob, 2003) which is consistent with the concept of a chronic relapsing disorder that extends well beyond physiological withdrawal symptoms. Indeed, we previously showed that under extended conditions of vapor exposure, we were able to observe protracted abstinence-induced alterations in escalated alcohol self-administration that remained sensitive to nor-BNI administration as far as 30-days (the maximum length tested) into withdrawal (Kissler and Walker, 2016).

22-kHz USVs are considered a measure of negative affective states in rats (Knutson et al., 2002; Burgdorf et al., 2020) and we previously showed that an intracerebroventricular infusion of nor-BNI significantly attenuated alcohol withdrawal-induced increases in 22-kHz USVs. Here, we show that KOR antagonism attenuates the number and duration of 22-kHz USVs during acute alcohol withdrawal when nor-BNI was specifically infused into the AcbSh (Fig. 4A). These data are consistent with the effects of nor-BNI in the dorsolateral BNST on USV production (Erikson et al., 2018). It is our view that 22-kHZ USVs can be considered an compound affective stimulus with conditions of alarm, anxiety, behavioral despair, etc. represented (Brudzynski, 2007; Burgdorf et al., 2008; Brudzynski, 2009, 2015, 2019; Silkstone and Brudzynski, 2019; Burgdorf et al., 2020) that have been implicated in withdrawal from alcohol and benzodiazepines (Knapp et al., 1993; Vivian et al., 1994), but a precise delineation of the negative affective-like contributors is difficult (with that being said, the concept of ‘negative affect’ routinely has an overlapping etiology based in anxiety and depression). As this experiment has demonstrated that nor-BNI infused into the AcbSh decreases alcohol withdrawal-induced negative affective-like behavior, but not physiological withdrawal, we posit that KOR-mediated signaling in the AcbSh, CeA, and BNST drives escalated alcohol drinking through primarily negative affective-like mechanisms to induce escalated alcohol self-administration. Furthermore, once an alcohol-dependent state is eliminated through abstinence or reduction in daily alcohol consumption or heavy drinking days, physiological withdrawal would clearly not be an important factor and negative affective states would drive maladaptive behavioral regulation in alcohol dependence (Walker, 2012; Walker et al., 2012).

Interestingly, when attempting to conduct a refined analysis of the respective negative affective-like contributors to the increased 22-kHZ ultrasonic vocalizations observed in the present study, intra-AcbSh nor-BNI failed to ameliorate anxiety- and depressive-like symptoms in the EPM and FST. However, this is not the first time we have observed increased sensitivity of 22-kHz USVs compared to traditional, but less than desired when considering ethologically viability, affective-like measurement following site-specific KOR antagonism (Erikson et al., 2018).

One pharmacological caveat that is important to note is that in the present experiment, intra-AcbSh nor-BNI was infused five minutes prior to an alcohol self-administration session. Although in mice, nor-BNI has affinity for the MOR at acute time points (up to 2 hours following nor-BNI administration; Endoh et al., 1992; Broadbear et al., 1994), nor-BNI does not attenuate alcohol drinking in non-dependent rats (Walker and Koob, 2008; Nealey et al., 2011; Walker et al., 2011; Kissler et al., 2014; Erikson et al., 2018) and its affinity for the MOR has not been replicated in rats (Picker et al., 1996). Thus, we concluded that the effects of nor-BNI on alcohol self-administration seen in this study are the result of selective KOR antagonism. An additional caveat related to site-specific nor-BNI infusion in the AcbSh is whether there could be locomotor effects of nor-BNI that are masquerading as motivational effects. However, when evaluating distance travelled in the EPM, there were no effects of nor-BNI observed. Lastly, the sole use of males in these experiments is a limitation of the current study and these results should be verified using female Wistar rats.

Along with the current data, previous studies have demonstrated that the KOR/DYN system in the Acb is a vital regulator of alcohol dependence and withdrawal (Berridge and Kringelbach, 2015; Corre et al., 2018). For example, rats exposed to alcohol for four weeks show increased prodynorphin (Pdyn; gene for DYN) mRNA in the Acb following 24 and 48 hours of withdrawal (Przewlocka et al., 1997) and 13 days of alcohol exposure leads to increased DYN B in the Acb both 30 minutes and 21 days into withdrawal (Lindholm et al., 2000a) following ethanol an extended alcohol administration protocol. Importantly, modulation of negative affect by AcbSh KOR-mediated signaling may be due to the ability of the KOR to modulate other neurotransmitter systems. Alcohol self-administration is associated with an increase in dopamine in the Acb, but this effect is blocked by a systemic injection of nor-BNI (Doyon et al., 2006). This implies that the KOR/DYN system modulates alcohol-induced dopamine release in this region, and it has been proposed that dysregulation of the mesolimbic dopamine system facilitates depression (Nestler and Carlezon, 2006). Further, it has been shown that dopamine transmission in the Acb is altered after chronic mild stress in rats (Di Chiara et al., 1999) and rats selectively bred for depressive-like phenotypes exhibit altered dopamine levels in the Acb following a FST test (Yadid et al., 2001), further indicating that altered mesolimbic dopamine transmission may contribute to symptoms of depression. Moreover, KOR-antagonists have anti-depressant properties (Newton et al., 2002; Mague et al., 2003a), and collectively, it has been suggested that KORs regulate mesolimbic dopamine transmission (Shippenberg and Rea, 1997; Nestler and Carlezon, 2006) to induce depressive-like states. Indeed, we recently showed that in alcohol dependent rats, Oprk1 mRNA expression was significantly elevated in the ventral tegmental area (VTA; primary dopaminergic source projecting the ACbSh) of alcohol-dependent compared to non-dependent rats and when leveraging Cre-Lox technology in conjunction with transgenic TH::Cre rats, overexpression of Oprk1 mRNA in VTA dopamine neurons recapitulated both depressive-like behavior and escalated alcohol self-administration associated with alcohol dependence in non-dependent ‘social’ drinking rats.

One extremely interesting development in KOR-mediated neurobiology showed that dynorphinergic neurons in the ventral part of the AcbSh regulated aversion while preference and reward seeking was mediated by DYN in the dorsal AcbSh region (Al-Hasani et al., 2015). As we target the ventral portions of the AcbSh with our stereotaxic coordinates, it is consistent that we routinely see amelioration of dependence-induced maladaptive behavioral regulation through our AcbSh manipulations. Additional recent investigations into the functional morphology of KOR-mediated effects on AUD-related behaviors along the rostral / caudal AcbSh axis showed that KOR agonism in the AcbSh influenced two-bottle choice alcohol consumption according to AcbSh subregion, sex, and ethanol intake levels with multiple possible interpretations of the data (Pirino et al., 2024).

The present study is important for understanding the neurobiology of KOR-mediated reductions in symptoms of alcohol dependence and should help inform therapeutic development efforts that focus on the modulation of specific brain circuitry. Additional evidence for a role of the DYN / KOR system in AUD stems from alcohol drinking being associated with increased DYN release in the Acb (Mitchell et al., 2012), and a variation of OPRK1, the gene that codes for the KOR, is associated with alcohol dependence in humans (Edenberg et al., 2008). As mentioned, overexpression of Oprk1 in non-dependent rats recapitulates symptoms of alcohol dependence (Lepreux et al., 2023). Additionally, while AUD and depression are highly comorbid (Boden and Fergusson, 2011), it has also been suggested that alcohol dependence may cause depression in humans as a subset of alcoholics report substance-induced depression (Schuckit et al., 1997). Therefore, the data presented here is relevant to the treatment of alcohol withdrawal-induced negative emotional states such as depression and continues to highlight the importance of the KOR/DYN system as an important target for AUD therapeutics.

Supplementary Material

Supplemental Figure

Acknowledgements

The authors would like to thank the USF Department of Psychiatry and Behavioral Neurosciences and the WSU Psychology Department as well as members in the Laboratory of Alcoholism and Addiction Neuroscience for their assistance and the vivarium staff for their continued support.

Funding

Support for this research was provided in part by R01AA020394 and R01AA031171 from the National Institute on Alcohol Abuse and Alcoholism and grants from the WSU Alcohol and Drug Abuse Research Program according to the State of Washington Initiative Measure No. 171 to BMW. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism, the National Institutes of Health, or the States of Washington or Florida.

Footnotes

Declaration of Interest

The authors declare no conflict of interest.

References

  1. Akbar M, Egli M, Cho YE, Song BJ, Noronha A (2018) Medications for alcohol use disorders: An overview. Pharmacol Ther 185:64–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Al-Hasani R, McCall JG, Foshage AM, Bruchas MR (2013) Locus coeruleus kappa-opioid receptors modulate reinstatement of cocaine place preference through a noradrenergic mechanism. Neuropsychopharmacology 38:2484–2497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Al-Hasani R, McCall JG, Shin G, Gomez AM, Schmitz GP, Bernardi JM, Pyo CO, Park SI, Marcinkiewcz CM, Crowley NA, Krashes MJ, Lowell BB, Kash TL, Rogers JA, Bruchas MR (2015) Distinct Subpopulations of Nucleus Accumbens Dynorphin Neurons Drive Aversion and Reward. Neuron 87:1063–1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Alheid GF, Heimer L (1988) New perspectives in basal forebrain organization of special relevance for neuropsychiatric disorders: the striatopallidal, amygdaloid, and corticopetal components of substantia innominata. Neuroscience 27:1–39. [DOI] [PubMed] [Google Scholar]
  5. Alvarez-Jaimes L, Stouffer DG, Parsons LH (2009) Chronic ethanol treatment potentiates ethanol-induced increases in interstitial nucleus accumbens endocannabinoid levels in rats. J Neurochem 111:37–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Aston-Jones G, Delfs JM, Druhan J, Zhu Y (1999) The bed nucleus of the stria terminalis. A target site for noradrenergic actions in opiate withdrawal. Ann N Y Acad Sci 877:486–498. [DOI] [PubMed] [Google Scholar]
  7. Berger AL, Williams AM, McGinnis MM, Walker BM (2013) Affective cue-induced escalation of alcohol self-administration and increased 22-kHz ultrasonic vocalizations during alcohol withdrawal: role of kappa-opioid receptors. Neuropsychopharmacology 38:647–654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berridge Kent C, Kringelbach Morten L (2015) Pleasure Systems in the Brain. Neuron 86:646–664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Boden JM, Fergusson DM (2011) Alcohol and depression. Addiction 106:906–914. [DOI] [PubMed] [Google Scholar]
  10. Bodnar RJ (2022) Endogenous opiates and behavior: 2020. Peptides 151:170752. [DOI] [PubMed] [Google Scholar]
  11. Broadbear JH, Negus SS, Butelman ER, de Costa BR, Woods JH (1994) Differential effects of systemically administered nor-binaltorphimine (nor-BNI) on kappa-opioid agonists in the mouse writhing assay. Psychopharmacology (Berl) 115:311–319. [DOI] [PubMed] [Google Scholar]
  12. Bruchas MR, Land BB, Chavkin C (2010) The dynorphin/kappa opioid system as a modulator of stress-induced and pro-addictive behaviors. Brain Res 1314:44–55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Bruchas MR, Schindler AG, Shankar H, Messinger DI, Miyatake M, Land BB, Lemos JC, Hagan CE, Neumaier JF, Quintana A, Palmiter RD, Chavkin C (2011) Selective p38alpha MAPK deletion in serotonergic neurons produces stress resilience in models of depression and addiction. Neuron 71:498–511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Brudzynski SM (2007) Ultrasonic calls of rats as indicator variables of negative or positive states: acetylcholine-dopamine interaction and acoustic coding. Behav Brain Res 182:261–273. [DOI] [PubMed] [Google Scholar]
  15. Brudzynski SM (2009) Communication of adult rats by ultrasonic vocalization: biological, sociobiological, and neuroscience approaches. ILAR J 50:43–50. [DOI] [PubMed] [Google Scholar]
  16. Brudzynski SM (2015) Pharmacology of Ultrasonic Vocalizations in adult Rats: Significance, Call Classfication and neural substrate. Current Neuropharmacology 13:180–192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Brudzynski SM (2019) Emission of 22 kHz vocalizations in rats as an evolutionary equivalent of human crying: Relationship to depression. Behav Brain Res 363:1–12. [DOI] [PubMed] [Google Scholar]
  18. Burgdorf J, Kroes RA, Moskal JR, Pfaus JG, Brudzynski SM, Panksepp J (2008) Ultrasonic vocalizations of rats (Rattus norvegicus) during mating, play, and aggression: Behavioral concomitants, relationship to reward, and self-administration of playback. J Comp Psychol 122:357–367. [DOI] [PubMed] [Google Scholar]
  19. Burgdorf JS, Brudzynski SM, Moskal JR (2020) Using rat ultrasonic vocalization to study the neurobiology of emotion: from basic science to the development of novel therapeutics for affective disorders. Curr Opin Neurobiol 60:192–200. [DOI] [PubMed] [Google Scholar]
  20. Carlezon WA, Miczek KA (2010) Ascent of the kappa-opioid receptor in psychopharmacology. Psychopharmacology (Berl) 210:107–108. [DOI] [PubMed] [Google Scholar]
  21. Carlezon WA, Beguin C, DiNieri JA, Baumann MH, Richards MR, Todtenkopf MS, Rothman RB, Ma Z, Lee DY, Cohen BM (2006) Depressive-like effects of the kappa-opioid receptor agonist salvinorin A on behavior and neurochemistry in rats. J Pharmacol Exp Ther 316:440–447. [DOI] [PubMed] [Google Scholar]
  22. Carr GV, Bangasser DA, Bethea T, Young M, Valentino RJ, Lucki I (2010) Antidepressant-like effects of kappa-opioid receptor antagonists in wistar kyoto rats. Neuropsychopharmacology 35:752–763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Centanni SW, Bedse G, Patel S, Winder DG (2019) Driving the Downward Spiral: Alcohol-Induced Dysregulation of Extended Amygdala Circuits and Negative Affect. Alcohol Clin Exp Res 43:2000–2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Chavkin C, James IF, Goldstein A (1982) Dynorphin is a specific endogenous ligand of the kappa opioid receptor. Science 215:413–415. [DOI] [PubMed] [Google Scholar]
  25. Corre J, van Zessen R, Loureiro M, Patriarchi T, Tian L, Pascoli V, Luscher C (2018) Dopamine neurons projecting to medial shell of the nucleus accumbens drive heroin reinforcement. Elife 7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Council NR (2011) Guide for the care and use of laboratory animals, 8th ed Edition. Washington, D.C: National Academies Press. [Google Scholar]
  27. Di Chiara G, Loddo P, Tanda G (1999) Reciprocal changes in prefrontal and limbic dopamine responsiveness to aversive and rewarding stimuli after chronic mild stress: implications for the psychobiology of depression. Biol Psychiatry 46:1624–1633. [DOI] [PubMed] [Google Scholar]
  28. Doyon WM, Howard EC, Shippenberg TS, Gonzales RA (2006) Kappa-opioid receptor modulation of accumbal dopamine concentration during operant ethanol self-administration. Neuropharmacology 51:487–496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Driessen M, Meier S, Hill A, Wetterling T, Lange W, Junghanns K (2001) The course of anxiety, depression and drinking behaviours after completed detoxification in alcoholics with and without comorbid anxiety and depressive disorders. Alcohol and Alcoholism 36:249–255. [DOI] [PubMed] [Google Scholar]
  30. Edenberg HJ, Wang J, Tian H, Pochareddy S, Xuei X, Wetherill L, Goate A, Hinrichs T, Kuperman S, Nurnberger JI Jr., Schuckit M, Tischfield JA, Foroud T (2008) A regulatory variation in OPRK1, the gene encoding the kappa-opioid receptor, is associated with alcohol dependence. Hum Mol Genet 17:1783–1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Endoh T, Matsuura H, Tanaka C, Nagase H (1992) Nor-binaltorphimine: a potent and selective kappa-opioid receptor antagonist with long-lasting activity in vivo. Arch Int Pharmacodyn Ther 316:30–42. [PubMed] [Google Scholar]
  32. Erikson CM, Wei G, Walker BM (2018) Maladaptive behavioral regulation in alcohol dependence: Role of kappa-opioid receptors in the bed nucleus of the stria terminalis. Neuropharmacology 140:162–173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Esser MB, Sherk A, Liu Y, Naimi TS (2024) Deaths from Excessive Alcohol Use - United States, 2016–2021. MMWR Morb Mortal Wkly Rep 73:154–161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Flores-Ramirez FJ, Illenberger JM, Pascasio G, Terenius L, Martin-Fardon R (2024) LY2444296, a kappa-opioid receptor antagonist, selectively reduces alcohol drinking in male and female Wistar rats with a history of alcohol dependence. Sci Rep 14:5804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Fowler CJ, Fraser GL (1994) Mu-, Delta-, Kappa-Opioid Receptors and Their Subtypes - a Critical-Review with Emphasis on Radioligand Binding Experiments. Neurochemistry International 24:401–426. [DOI] [PubMed] [Google Scholar]
  36. Gilpin NW, Roberto M, Koob GF, Schweitzer P (2014) Kappa opioid receptor activation decreases inhibitory transmission and antagonizes alcohol effects in rat central amygdala. Neuropharmacology 77:294–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Grilli M, Neri E, Zappettini S, Massa F, Bisio A, Romussi G, Marchi M, Pittaluga A (2009) Salvinorin A exerts opposite presynaptic controls on neurotransmitter exocytosis from mouse brain nerve terminals. Neuropharmacology 57:523–530. [DOI] [PubMed] [Google Scholar]
  38. Guidelines NRCUCo (2003) Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research. Washington, D.C.: National Academy Press. [PubMed] [Google Scholar]
  39. Heilig M, Egli M (2006) Pharmacological treatment of alcohol dependence: target symptoms and target mechanisms. Pharmacol Ther 111:855–876. [DOI] [PubMed] [Google Scholar]
  40. Hjelmstad GO, Fields HL (2003) Kappa opioid receptor activation in the nucleus accumbens inhibits glutamate and GABA release through different mechanisms. J Neurophysiol 89:2389–2395. [DOI] [PubMed] [Google Scholar]
  41. Ji D, Gilpin NW, Richardson HN, Rivier CL, Koob GF (2008) Effects of naltrexone, duloxetine, and a corticotropin-releasing factor type 1 receptor antagonist on binge-like alcohol drinking in rats. Behav Pharmacol 19:1–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kallupi M, Wee S, Edwards S, Whitfield TW Jr., Oleata CS, Luu G, Schmeichel BE, Koob GF, Roberto M (2013) Kappa opioid receptor-mediated dysregulation of gamma-aminobutyric acidergic transmission in the central amygdala in cocaine addiction. Biol Psychiatry 74:520–528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Kang-Park M, Kieffer BL, Roberts AJ, Siggins GR, Moore SD (2013) kappa-Opioid receptors in the central amygdala regulate ethanol actions at presynaptic GABAergic sites. J Pharmacol Exp Ther 346:130–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Karkhanis AN, Al-Hasani R (2020) Dynorphin and its role in alcohol use disorder. Brain Res 1735:146742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Karkhanis AN, Huggins KN, Rose JH, Jones SR (2016) Switch from excitatory to inhibitory actions of ethanol on dopamine levels after chronic exposure: Role of kappa opioid receptors. Neuropharmacology 110:190–197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Khantzian EJ (1990) The Self Regulation and Self Medication Factor in alcoholism and the addictions. [PubMed] [Google Scholar]
  47. Kissler JL, Walker BM (2016) Dissociating Motivational From Physiological Withdrawal in Alcohol Dependence: Role of Central Amygdala kappa-Opioid Receptors. Neuropsychopharmacology 41:560–567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Kissler JL, Sirohi S, Reis DJ, Jansen HT, Quock RM, Smith DG, Walker BM (2014) The one-two punch of alcoholism: role of central amygdala dynorphins/kappa-opioid receptors. Biol Psychiatry 75:774–782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Knapp DJ, Pohorecky LA (1995) An air-puff stimulus method for elicitation of ultrasonic vocalizations in rats. J Neurosci Methods 62:1–5. [DOI] [PubMed] [Google Scholar]
  50. Knapp DJ, Saiers JA, Pohorecky LA (1993) Observations of novel behaviors as indices of ethanol withdrawal-induced anxiety. Alcohol Alcohol Suppl 2:489–493. [PubMed] [Google Scholar]
  51. Knoll AT, Carlezon WA (2010) Dynorphin, stress, and depression. Brain Res 1314:56–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Knutson B, Burgdorf J, Panksepp J (2002) Ultrasonic vocalizations as indices of affective states in rats. Psychological Bulletin 128:961–977. [DOI] [PubMed] [Google Scholar]
  53. Koob GF (2003) Alcoholism: allostasis and beyond. Alcohol Clin Exp Res 27:232–243. [DOI] [PubMed] [Google Scholar]
  54. Koob GF (2009) Brain stress systems in the amygdala and addiction. Brain Res 1293:61–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Koob GF, Le Moal M (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278:52–58. [DOI] [PubMed] [Google Scholar]
  56. Koob GF, Volkow ND (2016) Neurobiology of addiction: a neurocircuitry analysis. Lancet Psychiatry 3:760–773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Lam MP, Marinelli PW, Bai L, Gianoulakis C (2008) Effects of acute ethanol on opioid peptide release in the central amygdala: an in vivo microdialysis study. Psychopharmacology (Berl) 201:261–271. [DOI] [PubMed] [Google Scholar]
  58. Land BB, Bruchas MR, Lemos JC, Xu M, Melief EJ, Chavkin C (2008) The dysphoric component of stress is encoded by activation of the dynorphin kappa-opioid system. J Neurosci 28:407–414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Lepreux G, Shinn GE, Wei G, Suko A, Concepcion G, Sirohi S, Soon Go B, Bruchas MR, Walker BM (2023) Recapitulating phenotypes of alcohol dependence via overexpression of Oprk1 in the ventral tegmental area of non-dependent TH::Cre rats. Neuropharmacology 228:109457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Lindholm S, Ploj K, Franck J, Nylander I (2000a) Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain. Alcohol 22:165–171. [DOI] [PubMed] [Google Scholar]
  61. Lindholm S, Ploj K, Franck J, Nylander I (2000b) Repeated ethanol administration induces short- and long-term changes in enkephalin and dynorphin tissue concentrations in rat brain. Alcohol 22:165–171. [DOI] [PubMed] [Google Scholar]
  62. Mague SD, Pliakas AM, Todtenkopf MS, Tomasiewicz HC, Zhang Y, Stevens WC Jr., Jones RM, Portoghese PS, Carlezon WA Jr. (2003a) Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J Pharmacol Exp Ther 305:323–330. [DOI] [PubMed] [Google Scholar]
  63. Mague SD, Pliakas AM, Todtenkopf MS, Tomasiewicz HC, Zhang Y, Stevens WC Jr., Jones RM, Portoghese PS, Carlezon WA Jr. (2003b) Antidepressant-like effects of kappa-opioid receptor antagonists in the forced swim test in rats. J Pharmacol Exp Ther 305:323–330. [DOI] [PubMed] [Google Scholar]
  64. Margolis EB, Hjelmstad GO, Bonci A, Fields HL (2003) Kappa-opioid agonists directly inhibit midbrain dopaminergic neurons. J Neurosci 23:9981–9986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Margolis EB, Lock H, Chefer VI, Shippenberg TS, Hjelmstad GO, Fields HL (2006) Kappa opioids selectively control dopaminergic neurons projecting to the prefrontal cortex. Proc Natl Acad Sci U S A 103:2938–2942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Marinelli PW, Lam M, Bai L, Quirion R, Gianoulakis C (2006) A microdialysis profile of dynorphin A(1–8) release in the rat nucleus accumbens following alcohol administration. Alcohol Clin Exp Res 30:982–990. [DOI] [PubMed] [Google Scholar]
  67. Markou A, Kosten TR, Koob GF (1998) Neurobiological similarities in depression and drug dependence: a self-medication hypothesis. Neuropsychopharmacology 18:135–174. [DOI] [PubMed] [Google Scholar]
  68. McLaughlin JP, Marton-Popovici M, Chavkin C (2003) Kappa opioid receptor antagonism and prodynorphin gene disruption block stress-induced behavioral responses. J Neurosci 23:5674–5683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Meunier JC, Mollereau C, Toll L, Suaudeau C, Moisand C, Alvinerie P, Butour JL, Guillemot JC, Ferrara P, Monsarrat B, et al. (1995) Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377:532–535. [DOI] [PubMed] [Google Scholar]
  70. Mitchell JM, O’Neil JP, Janabi M, Marks SM, Jagust WJ, Fields HL (2012) Alcohol consumption induces endogenous opioid release in the human orbitofrontal cortex and nucleus accumbens. Sci Transl Med 4:116ra116. [DOI] [PubMed] [Google Scholar]
  71. Mokdad AH, Marks JS, Stroup DF, Gerberding JL (2004) Actual causes of death in the United States, 2000. JAMA 291:1238–1245. [DOI] [PubMed] [Google Scholar]
  72. Mollereau C, Parmentier M, Mailleux P, Butour JL, Moisand C, Chalon P, Caput D, Vassart G, Meunier JC (1994) ORL1, a novel member of the opioid receptor family. Cloning, functional expression and localization. FEBS Lett 341:33–38. [DOI] [PubMed] [Google Scholar]
  73. Nealey KA, Smith AW, Davis SM, Smith DG, Walker BM (2011) kappa-opioid receptors are implicated in the increased potency of intra-accumbens nalmefene in ethanol-dependent rats. Neuropharmacology 61:35–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Nestler EJ, Carlezon WA (2006) The mesolimbic dopamine reward circuit in depression. Biol Psychiatry 59:1151–1159. [DOI] [PubMed] [Google Scholar]
  75. Newton SS, Thome J, Wallace TL, Shirayama Y, Schlesinger L, Sakai N, Chen J, Neve R, Nestler EJ, Duman RS (2002) Inhibition of cAMP response element-binding protein or dynorphin in the nucleus accumbens produces an antidepressant-like effect. J Neurosci 22:10883–10890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. O’Dell LE, Roberts AJ, Smith RT, Koob GF (2004) Enhanced alcohol self-administration after intermittent versus continuous alcohol vapor exposure. Alcohol Clin Exp Res 28:1676–1682. [DOI] [PubMed] [Google Scholar]
  77. Picker MJ, Mathewson C, Allen RM (1996) Opioids and rate of positively reinforced behavior: III. Antagonism by the long-lasting kappa antagonist norbinaltorphimine. Behav Pharmacol 7:495–504. [PubMed] [Google Scholar]
  78. Pirino BE, Hawks A, Carpenter BA, Candelas PG, Gargiulo AT, Curtis GR, Karkhanis AN, Barson JR (2024) Kappa-opioid receptor stimulation in the nucleus accumbens shell and ethanol drinking: Differential effects by rostro-caudal location and level of drinking. Neuropsychopharmacology. [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Pliakas AM, Carlson RR, Neve RL, Konradi C, Nestler EJ, Carlezon WA Jr. (2001) Altered responsiveness to cocaine and increased immobility in the forced swim test associated with elevated cAMP response element-binding protein expression in nucleus accumbens. J Neurosci 21:7397–7403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Przewlocka B, Turchan J, Lason W, Przewlocki R (1997) Ethanol withdrawal enhances the prodynorphin system activity in the rat nucleus accumbens. Neurosci Lett 238:13–16. [DOI] [PubMed] [Google Scholar]
  81. Rose JH, Karkhanis AN, Chen R, Gioia D, Lopez MF, Becker HC, McCool BA, Jones SR (2016) Supersensitive Kappa Opioid Receptors Promotes Ethanol Withdrawal-Related Behaviors and Reduce Dopamine Signaling in the Nucleus Accumbens. Int J Neuropsychopharmacol 19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  82. Sacks JJ, Gonzales KR, Bouchery EE, Tomedi LE, Brewer RD (2015) 2010 National and State Costs of Excessive Alcohol Consumption. Am J Prev Med 49:e73–e79. [DOI] [PubMed] [Google Scholar]
  83. SAMSHA (2023) Results from the 2022 National Survey on Drug Use and Health. Office of Applied Studies, DHHS Publication, Rockville, MD. [Google Scholar]
  84. Sánchez-Peña JF, Alvarez-Cotoli P, Rodríguez-Solano JJ (2012) Psychiatric disorders associated with alcoholism: 2 year follow-up of treatment. Actas Esp Psiquiatr 40:129–135. [PubMed] [Google Scholar]
  85. Schuckit MA, Tipp JE, Bergman M, Reich W, Hesselbrock VM, Smith TL (1997) Comparison of induced and independent major depressive disorders in 2,945 alcoholics. Am J Psychiatry 154:948–957. [DOI] [PubMed] [Google Scholar]
  86. Schulteis G, Markou A, Cole M, Koob GF (1995) Decreased brain reward produced by ethanol withdrawal. Proc Natl Acad Sci U S A 92:5880–5884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Shippenberg TS, Rea W (1997) Sensitization to the behavioral effects of cocaine: modulation by dynorphin and kappa-opioid receptor agonists. Pharmacol Biochem Behav 57:449–455. [DOI] [PubMed] [Google Scholar]
  88. Siciliano CA, Calipari ES, Yorgason JT, Lovinger DM, Mateo Y, Jimenez VA, Helms CM, Grant KA, Jones SR (2016) Increased presynaptic regulation of dopamine neurotransmission in the nucleus accumbens core following chronic ethanol self-administration in female macaques. Psychopharmacology (Berl) 233:1435–1443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Silkstone M, Brudzynski SM (2019) The antagonistic relationship between aversive and appetitive emotional states in rats as studied by pharmacologically-induced ultrasonic vocalization from the nucleus accumbens and lateral septum. Pharmacol Biochem Behav 181:77–85. [DOI] [PubMed] [Google Scholar]
  90. Sirohi S, Bakalkin G, Walker BM (2012) Alcohol-induced plasticity in the dynorphin/kappa-opioid receptor system. Front Mol Neurosci 5:95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  91. Spanagel R, Herz A, Shippenberg TS (1992) Opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. Proc Natl Acad Sci U S A 89:2046–2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Tejeda HA, Natividad LA, Orfila JE, Torres OV, O’Dell LE (2012) Dysregulation of kappa-opioid receptor systems by chronic nicotine modulate the nicotine withdrawal syndrome in an age-dependent manner. Psychopharmacology (Berl) 224:289–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Tejeda HA, Hanks AN, Scott L, Mejias-Aponte C, Hughes ZA, O’Donnell P (2015) Prefrontal Cortical Kappa Opioid Receptors Attenuate Responses to Amygdala Inputs. Neuropsychopharmacology 40:2856–2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Tejeda HA, Counotte DS, Oh E, Ramamoorthy S, Schultz-Kuszak KN, Backman CM, Chefer V, O’Donnell P, Shippenberg TS (2013) Prefrontal cortical kappa-opioid receptor modulation of local neurotransmission and conditioned place aversion. Neuropsychopharmacology 38:1770–1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Thompson AC, Zapata A, Justice JB Jr., Vaughan RA, Sharpe LG, Shippenberg TS (2000) Kappa-opioid receptor activation modifies dopamine uptake in the nucleus accumbens and opposes the effects of cocaine. J Neurosci 20:9333–9340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Todtenkopf MS, Marcus JF, Portoghese PS, Carlezon WA Jr. (2004) Effects of kappa-opioid receptor ligands on intracranial self-stimulation in rats. Psychopharmacology (Berl) 172:463–470. [DOI] [PubMed] [Google Scholar]
  97. Toll L, Bruchas MR, Calo G, Cox BM, Zaveri NT (2016) Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems. Pharmacol Rev 68:419–457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  98. Valenstein ES, Cox VC, Kakolewski JW (1967) Polydipsia elicited by the synergistic action of a saccharin and glucose solution. Science 157:552–554. [DOI] [PubMed] [Google Scholar]
  99. Vivian JA, Farrell WJ, Sapperstein SB, Miczek KA (1994) Diazepam withdrawal: effects of diazepam and gepirone on acoustic startle-induced 22 kHz ultrasonic vocalizations. Psychopharmacology (Berl) 114:101–108. [DOI] [PubMed] [Google Scholar]
  100. Volpicelli JR, Alterman AI, Hayashida M, O’Brien CP (1992) Naltrexone in the treatment of alcohol dependence. Arch Gen Psychiatry 49:876–880. [DOI] [PubMed] [Google Scholar]
  101. kWalker BM (2012) Conceptualizing withdrawal-induced escalation of alcohol self-administration as a learned, plasticity-dependent process. Alcohol 46:339–348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Walker BM, Ettenberg A (2007) Intracerebroventricular ethanol-induced conditioned place preferences are prevented by fluphenazine infusions into the nucleus accumbens of rats. Behav Neurosci 121:401–410. [DOI] [PubMed] [Google Scholar]
  103. Walker BM, Koob GF (2007) The gamma-aminobutyric acid-B receptor agonist baclofen attenuates responding for ethanol in ethanol-dependent rats. Alcohol Clin Exp Res 31:11–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  104. Walker BM, Koob GF (2008) Pharmacological evidence for a motivational role of kappa-opioid systems in ethanol dependence. Neuropsychopharmacology 33:643–652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Walker BM, Zorrilla EP, Koob GF (2011) Systemic kappa-opioid receptor antagonism by nor-binaltorphimine reduces dependence-induced excessive alcohol self-administration in rats. Addict Biol 16:116–119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  106. Walker BM, Valdez GR, McLaughlin JP, Bakalkin G (2012) Targeting dynorphin/kappa opioid receptor systems to treat alcohol abuse and dependence. Alcohol 46:359–370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Wei G, Sirohi S, Walker BM (2022) Dysregulated kappa-opioid receptors in the medial prefrontal cortex contribute to working memory deficits in alcohol dependence. Addict Biol 27:e13138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  108. Werling LL, Frattali A, Portoghese PS, Takemori AE, Cox BM (1988) Kappa receptor regulation of dopamine release from striatum and cortex of rats and guinea pigs. J Pharmacol Exp Ther 246:282–286. [PubMed] [Google Scholar]
  109. Williams AM, Reis DJ, Powell AS, Neira LJ, Nealey KA, Ziegler CE, Kloss ND, Bilimoria JL, Smith CE, Walker BM (2012) The effect of intermittent alcohol vapor or pulsatile heroin on somatic and negative affective indices during spontaneous withdrawal in Wistar rats. Psychopharmacology (Berl) 223:75–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  110. Yadid G, Overstreet DH, Zangen A (2001) Limbic dopaminergic adaptation to a stressful stimulus in a rat model of depression. Brain Res 896:43–47. [DOI] [PubMed] [Google Scholar]

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