Blockade of orexin receptors in the infralimbic cortex prevents stress‐induced reinstatement of alcohol‐seeking behaviour in alcohol‐dependent rats

A major problem managing alcohol use disorder is the high vulnerability to relapse, even after long periods of abstinence. Chronic alcohol use dysregulates stress responsivity, rendering this system hyporesponsive and making individuals vulnerable to relapse. Orexin (hypocretin) plays a role in diverse physiological processes, including stress. Orexin neurons in the hypothalamus, project to the infralimbic cortex. This study asked does infralimbic cortex orexin transmission play a significant role in stress‐induced reinstatement of alcohol‐seeking behaviour in alcohol‐dependent rats.


| INTRODUCTION
Alcohol use disorder (AUD) is a major health concern, given its contribution to disability and preventable deaths globally (Witkiewitz et al., 2019). In the United States alone, $30% of all adults will meet the criteria for alcohol use disorder during their lifetime (Grant et al., 2015). Although there have been major advances in the treatment and management of other chronic health conditions, such as diabetes and heart disease, effective treatments for alcohol use disorder remain elusive (Pierce et al., 2012). A central problem in treating alcohol use disorder is the prevalence of relapse to alcohol use, even after protracted periods of forced or self-imposed abstinence. Stress is a major factor that contributes to the chronic relapsing and compulsive nature of drug addiction, given its long-lasting ability to elicit craving in individuals who are recovering from substance use disorder and the ability to reinstate drug seeking in rodents (Stephens & Wand, 2012;Stewart, 2008). Indeed, patients with a diagnosis of alcohol use disorder often relapse during episodes of heightened stress (Breese et al., 2005;Sinha, 2007). Nevertheless, the exact mechanisms that link stress to greater vulnerability to alcohol relapse are not thoroughly understood.
Altogether, this literature implicates the orexin system in alcohol intake and alcohol-seeking behaviour.
Orexin is exclusively produced in the hypothalamus, including the lateral hypothalamus, dorsomedial hypothalamus and perifornical area (Baldo et al., 2003;DiLeone et al., 2003;Winsky-Sommerer et al., 2004). Orexin neurons project throughout the brain, densely innervating diverse brain regions that are involved in arousal, motivation and responsivity to stress-related stimuli (Baldo et al., 2003;Grafe & Bhatnagar, 2018;Peyron et al., 1998). Among these regions, the medial prefrontal cortex emerged as playing an important role in compulsive drug-seeking behaviour (Kalivas, 2008;Koob, 2008). The rodent medial prefrontal cortex is an anatomically heterogeneous structure with functional dichotomy between the dorsal division that consists of the prelimbic cortex and ventral What is already known • Stress is a major contributor to the chronic relapsing and compulsive nature of alcohol addiction.
• The orexin system is involved in alcohol use and plays a role in stress regulation.

What does this study add
• Blocking orexin receptors in the infralimbic cortex prevents stress-induced relapse in alcohol-dependent subjects.
• Alcohol dependence may result in the dysregulation of orexin transmission in the infralimbic cortex.

What is the clinical significance
• Dual orexin receptor antagonists are a strong candidate for the treatment of alcohol use disorder. division that consists of the infralimbic cortex. The prelimbic cortex is believed to play a significant role in the execution of executive behaviours. The infralimbic cortex has been described as playing a more meaningful role in response inhibition . Indeed, activation of the prelimbic cortex has been shown to initiate and promote drug-seeking behaviour, and activation of the infralimbic cortex has been shown to inhibit it Van den Oever et al., 2010). Additionally, chronic intermittent alcohol exposure has been shown to produce a greater effect on neuronal excitability and neurotransmission in the infralimbic cortex compared with the prelimbic cortex, highlighting the possibility that the infralimbic cortex has a more pronounced influence than the prelimbic cortex on alcohol-seeking behaviour (Pleil et al., 2015). Notably, the infralimbic cortex receives orexin inputs from the hypothalamus (Date et al., 1999). Hcrtr1 and Hcrtr2 messenger ribonucleic acid (mRNA) expression has been detected in the infralimbic cortex (Marcus et al., 2001). Furthermore, projections from the infralimbic cortex to the hypothalamus have also been described (Floyd et al., 2001;Vertes, 2004). These projections primarily originate from infralimbic cortex layer 5 pyramidal neurons that are directly gated by γ-aminobutyric acid (GABA) transmission, which controls regional output and can lead to the generation of local oscillatory activity (Buzsaki & Wang, 2012;Gabbott et al., 2005;Guyon et al., 2021). These findings raise the hypothesis that the dysregulation of orexin inputs to the infralimbic cortex that is induced by alcohol dependence might participate in the overall dysregulation of infralimbic cortex function (Pfarr et al., 2018) following chronic alcohol exposure. This dysregulation, in turn, could play a central role in compulsive alcohol seeking that is exacerbated by episodes of heightened stress.
To verify the implication of orexin transmission in the infralimbic cortex during the stress-induced reinstatement of alcohol-seeking behaviour, the present study tested (1), the ability of intra-infralimbic cortex administration of the OX 1/2 receptor antagonist TCS 1102 to reverse the stress-induced reinstatement of alcohol-seeking behaviour in male and female rats with a history of alcohol dependence, (2), evaluated whether chronic alcohol exposure alters Hcrtr1 and Hcrtr2 mRNA expression in the infralimbic cortex and Hcrt mRNA expression in the hypothalamus and (3), tested the hypothesis that alcohol dependence alters orexin influence over infralimbic cortex output by regulating inhibitory GABAergic transmission onto layer 5 pyramidal neurons.

| Animals
A total of 112 male and female (n = 66 each) Wistar rats (Charles River Laboratories, Hollister, CA, USA), weighing 150-170 g upon arrival, were housed two per cage in a temperature-and humiditycontrolled vivarium on a reverse 12/12 h light/dark cycle (lights OFF at 8:00 AM; lights ON at 8:00 PM) with ad libitum access to food and water. All animal procedures were conducted in strict adherence to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Research Council, 2003) and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute. Animal studies are reported in compliance with the ARRIVE guidelines (Percie du Sert et al., 2020) and with the recommendations made by the British Journal of Pharmacology (Lilley et al., 2020). Before the experiments began, the animals were given 1 week to acclimate to the housing and handling conditions.

| Alcohol self-administration training
Alcohol self-administration training was conducted as previously described (Matzeu & Martin-Fardon, 2020). Importantly, no fading (saccharin or sucrose) was required to induce voluntary alcohol intake.
After the 1 week housing acclimation period and for the remainder of the alcohol self-administration training procedure (Figure 1a), the rats were given access to alcohol in standard operant conditioning chambers (29 Â 24 Â 19.5 cm; Med Associates, St. Albans, VT, USA) during daily 30 min self-administration sessions on a fixed-ratio 1 (FR1) schedule of reinforcement. In these sessions, responses on the right lever resulted in the delivery of 0.1 ml of 10% (w/v) alcohol (prepared in tap water from 95% w/v alcohol) and the brief (0.5 s) illumination of a cue light above the lever. Responses on the left inactive lever resulted in no programmed consequences. Responses on both levers were recorded. All rats underwent self-administration training in parallel and were then randomly assigned to the stress-induced reinstatement experiment, quantitative polymerase chain reaction (qPCR) assay or whole-cell voltage-clamp electrophysiology studies after confirming that the different groups did not differ in their selfadministration performance (i.e. number of active lever responses) at the end of the training period.

| Infralimbic cortex cannulation
To confirm that infralimbic cortex cannulation had no unexpected effects on alcohol self-administration, 2 weeks after selfadministration training began, the animals were implanted with bilateral guide cannulas (22-gauge, 15 mm, Plastics One, Roanoke, VA, USA) using stereotaxic equipment. While under isoflurane anesthesia (5% for induction, 1-3% for maintenance), the cannulas were aimed at the infralimbic cortex (anterior/posterior, +3.2 mm from bregma; medial/lateral, ±0.75 mm; dorsal/ventral, À2.6 mm from dura; Paxinos & Watson, 1998) and positioned 2 mm above the target injection point. In order to minimize post-surgical discomfort, animals were administered the antibiotic Cefazolin (300 mg/kg, SC) and the nonsteroidal antiinflamatory analgesic Flunixin (2.5 mg/kg, SC) at the end of the surgery. During recovery from anesthesia, body temperature was maintained at 37 o C by means of a heating pad. Operated rats were monitored until they are fully recovered from anesthesia.
After 7 days of recovery from surgery, all rats resumed selfadministration training for 1 week ( Figure 1a) to verify that the alcohol self-administration baseline was not modified by the bilateral implantation of the guide cannulas.

| Chronic intermittent alcohol vapour exposure
After self-administration training (21 days) was completed, half of the rats were made alcohol dependent via chronic intermittent alcohol vapour exposure, whereas the other half were exposed to air only (nondependent group). During dependence induction (6 weeks;  et al. (1996), including measures of ventromedial limb retraction, vocalization (i.e. irritability in response to touch), tail stiffness, abnormal gait and body tremors. Each of these behaviours was assigned a score of 0-2, based on severity: 0 = no signs; 1 = moderate; and 2 = severe. To confirm alcohol dependence and assess withdrawal severity, the sum of the five scores (0-10) was used as a quantitative measure. This approach was used because this model of alcohol dependence is well known to lead to motivational and somatic signs of withdrawal in rats (Vendruscolo & Roberts, 2014). At Weeks 4-6 of alcohol vapour exposure (Figure 1c), the animals underwent a 30 min fixed-ratio 1 alcohol self-administration session at an acute abstinence point (i.e. 8 h after the alcohol vapour was turned OFF and when blood and brain alcohol levels are negligible) three times per week (Monday, Wednesday and Friday). Air-exposed animals were subjected to the same approach. To further corroborate alcohol dependence, blood alcohol levels were measured after the last selfadministration session on weeks 4-6 of chronic intermittent alcohol vapour exposure.
F I G U R E 1 Timeline of the experimental procedures. (a) After 2 weeks of self-administration training, rats received bilateral infralimbic cortex (IL) cannulations, were given 1 week to recover and underwent another week of self-administration training. Baseline somatic withdrawal signs (WDS) and blood alcohol levels (BALs) were recorded upon the completion of training. (b) Between Weeks 1 and 3 of chronic intermittent alcohol vapour exposure, the rats were scored for WDS during acute abstinence (8 h after the vapour was turned OFF on Wednesday) and BALs were recorded 30 min before the alcohol vapours were turned OFF (on Thursdays). (c) Between Weeks 4 and 6, the rats underwent self-administration sessions three times per week (Monday, Wednesday and Friday) during acute abstinence (8 h after alcohol vapour was turned OFF). (d) Between Weeks 7 and 8, the rats underwent daily extinction sessions during acute abstinence (8 h after alcohol vapour was turned OFF). (e) Representation of injection sites. o, rats with correct injection sites. Â, rats with missed injection sites. W, week 2.6 | Extinction training On weeks 7 and 8 of chronic intermittent alcohol vapour exposure ( Figure 1d), the rats (24 females and 24 males) underwent daily 30 min extinction sessions. These extinction sessions were identical to the alcohol self-administration sessions, but alcohol was withheld. For habituation to the footshock stress procedure, the rats were placed in the operant chambers 15 min before each session. At the end of this 15 min period, both levers were extended into the operant chambers and the extinction session began. The rats underwent extinction training for a total of 10 sessions. Before the last extinction session, the rats received a sham intracranial injection to allow for habituation to the microinjection procedure. This consisted of inserting an injector for 2 min (Plastics One, Roanoke, VA, USA) into the guide cannula that extended into the infralimbic cortex. After the sham injections, the rats were returned to their home cages for 2 min and then placed in the operant chamber for 15 min. At the end of this 15 min period, both levers were extended into the operant chambers and the rats were tested under extinction conditions.

| Stress-induced reinstatement
Twenty-four hours after the sham injection, the rats received an intra- injectors that extended 2.0 mm beyond the guide cannula. The injectors were delicately inserted into the guide cannula and intracerebral injections were made at a flow rate of 0.5 μlÁmin À1 over 1 min. The rats were held gently during the injection procedure to prevent the injectors from moving to not cause any stress-like response to the injections themselves. Although the pump was shut off, the injectors were left in the guide cannula for an additional 1 min to allow diffusion away from the injector tip. Once the injectors were removed, the animals were returned to their home cages for 2 min and then placed in the operant chambers to undergo footshock stress (15 min; variable intermittent electric footshock, 0.5 mA; duration, 0.5 s; mean shock interval, 40 s; range, 10-70 s; Flores-Ramirez et al., 2022;Martin-Fardon et al., 2000;Matzeu & Martin-Fardon, 2020;Sidhpura et al., 2010;Zhao et al., 2006). Two minutes after the termination of footshock, the levers were extended into the chamber and responses were recorded for 30 min. Studies of stress-induced reinstatement typically are conducted using the extinction-reinstatement model with footshock stress having been the predominant model for over more than two decades (Flores-Ramirez et al., 2022;Le et al., 1998Le et al., , 1999Le et al., , 2000Martin-Fardon et al., 2000;Matzeu & Martin-Fardon, 2020;Zhao et al., 2006); it has been shown to be a reliable animal model of relapse and has been instrumental in the identification of brain regions that are recruited by stress and that may play a pivotal role in stress-induced drug seeking (Erb & Stewart, 1999;Wang et al., 2005Wang et al., , 2006Wang et al., , 2007Zhao et al., 2006). In the present study, each animal was tested only once with vehicle or TCS 1102 according to a between-subjects design. To verify the injection sites, the rats were deeply anaesthetized via carbon dioxide (CO 2 ) inhalation, rapidly decapitated, and their brains were harvested, snap frozen in methylbutane and cut into 30 μM coronal sections using a cryostat (Leica CM3050S, Leica Biosystems Nussloch GmbH, Heidelberg, Germany).
Using an adult rat brain atlas as a reference (Paxinos & Watson, 1998), the injector tips were plotted from clear representative sections to determine their exact location. Off-target cannulations were excluded from the study (Figure 1e).

| qPCR procedure
Rats (n = 16; 8 females and 8 males) that were used for the gene expression analysis were prepared in parallel, in which they underwent the same behavioural procedure that is described above, including alcohol-dependence induction, but they did not undergo infralimbic cortex cannulation, did not receive infralimbic cortex injections and did not undergo reinstatement testing. Twenty-four hours after the last extinction session, at 8 h of abstinence, corresponding to the time when the behavioural group of rats was tested for stress-induced reinstatement, rats that were designated for qPCR analysis were deeply anaesthetized via CO 2 inhalation and rapidly decapitated, and their brains were rapidly harvested, snap frozen in methylbutane and stored at À80 C. An additional group of age-matched rats (n = 8; 4 females and 4 males) that were experimentally naive to all experimental conditions (i.e. alcohol selfadministration and dependence induction) but were handled similarly to the behavioural groups was also deeply anaesthetized and rapidly decapitated, and their brains were rapidly harvested, snap frozen in methylbutane and stored at À80 C. Brains from all groups were dissected into serial coronal sections, and the infralimbic cortex and the whole hypothalamus were collected using rapid core tissue punches (World Precision Instruments, Sarasota, FL, USA). RNA isolation was performed using RNA Concentrator-5 kits (Zymo
Infralimbic layer 5 pyramidal glutamatergic neurons were visualized with infrared differential interference contrast (IR-DIC) optics, a 60Â water immersion objective (Olympus BX51WI, Tokyo, Japan) and charge-coupled device camera (EXi Aqua, QImaging, Surrey, BC, Canada). Layer 5 pyramidal neurons were identified by size and shape as previously described (Varodayan, Sidhu, et al., 2018). Whole-cell voltage-clamp recordings from 86 neurons were performed in gapfree acquisition mode with a sampling rate per signal of 20 kHz and low-pass filtered at 10 kHz using a MultiClamp 700B amplifier and Digidata 1440A and pClamp 10.2 software (all from Molecular Devices, Sunnyvale, CA, USA).
Each experiment was conducted using tissue from a minimum of six rats, with 1-2 cells per animal for a single treatment group. sIPSC frequencies, amplitudes, rise times and decay times were analysed using Mini Analysis software (Synaptosoft, Fort Lee, NJ, USA) and visually confirmed, with events <5 pA excluded. The average characteristics from a minimum of 60 events during a 3 min interval were calculated. In these experiments, increases in sIPSC frequencies are associated with higher release probabilities and changes in amplitudes and kinetics are linked to alterations of postsynaptic receptor function (Otis et al., 1994;Roberto et al., 2003).

| Statistical analysis
Self-administration during chronic intermittent alcohol vapour exposure (i.e., baseline vs. Weeks 4-6) was analysed using two-way

| Materials
Orexin-A was obtained from American Peptide, Sunnyvale, CA, USA.

| RESULTS
Among the rats that were designated for the stress-induced reinstatement study, 16 were excluded (four never acquired self-administration, six had cannula misplacements and six had health complications), thus reducing the total number of animals to 96 (n = 48 for stressinduced reinstatement experiment; n = 24 for qPCR assay; and n = 24 for the electrophysiology studies).

| Alcohol self-administration and escalation
Over 21 sessions of self-administration training (30 minÁday À1 ), the rats (n = 88) acquired alcohol self-administration ( Figure 2a). The Bonferroni post hoc test confirmed that active lever presses were significantly higher than inactive lever presses starting at Session 2 (P < 0.05; Figure 2a).
During Weeks 4-6 of chronic intermittent alcohol vapour exposure, alcohol-dependent animals exhibited a significant escalation of alcohol intake, a measure that was obtained by averaging the intake data that were recorded Monday, Wednesday and Friday of that week (Figure 2b). During Weeks 4-6 of alcohol dependence, animals exhibited significantly higher somatic withdrawal signs at an acute abstinence point (8 h after vapours were off; Figure 2c). Furthermore, alcohol-dependent animals exhibited significantly higher blood alcohol levels after the self-administration sessions of Weeks 4-6 ( Figure 2d).

| Extinction and stress-induced reinstatement
The behavioural pattern of extinction between nondependent and dependent rats was similar (three-way mixed factors ANOVA; session:-F 9, 828 = 16.84, P < 0.05; alcohol dependence: F 1, 92 = 16.62, P < 0.05; lever: F 1, 92 = 71.06; session Â alcohol-dependence interaction: P > 0.05; alcohol-dependence Â lever interaction: P > 0.05; session Â alcohol-dependence Â lever interaction: P > 0.05). Therefore, the data from the nondependent and dependent rats were combined for the analysis of the 10 day extinction training (Figure 3a) during which the number of active lever responses gradually and significantly decreased until the total number of responses was ≤10 (Figure 2b).
The number of responses at the active lever remained higher compared to the inactive lever responses from the first to last extinction sessions (Bonferroni post hoc test, P < 0.05). Following extinction, the sham injections (designed to habituate the rats to the central F I G U R E 2 Time course of alcohol self-administration over 21 sessions of training and escalation of drinking during Weeks 4-6 of chronic intermittent alcohol vapour exposure. (a) Rats exhibited stable self-administration over the last seven training sessions. (b) At Weeks 4-6 of chronic intermittent alcohol vapour exposure, dependent rats exhibited the escalation of alcohol drinking. (c) During acute abstinence, an increase in somatic withdrawal signs (WDS) was observed in dependent rats on Weeks 4-6 of chronic intermittent alcohol vapour exposure. (d) After the self-administration sessions at Weeks 4-6 of chronic intermittent alcohol vapour exposure, alcohol-dependent rats had higher blood alcohol levels (BALs) than nondependent rats. The data are expressed as mean + SEM. * P < 0.05, versus respective baseline. # P < 0.05, versus inactive lever. Blue dots denote data from male rats and red dots represent data from females. B, baseline; W, week injections) did not reinstate or suppress any behaviour (Figure 3b). In contrast, exposure to stress precipitated the reinstatement of alcoholseeking behaviour in nondependent and dependent rats that received the VEH injection, which was prevented by the intra-infralimbic cortex injection of TCS 1102 in animals with a history of alcohol dependence ( Figure 3b). No differences in inactive lever responses were observed regardless of history of alcohol-dependence and treatment condition (Figure 3b).

| qPCR
Analyses of mRNA expression in the infralimbic cortex demonstrated that alcohol dependence significantly increased Hcrtr1 and Hcrtr2 mRNA expression (Figure 4a,b). Similarly, an increase in Hcrt mRNA expression was observed in the hypothalamus in alcohol-dependent rats following extinction (Figure 4c).

| Electrophysiology
The impact of OX receptor signalling on medial prefrontal cortex activity was examined next, specifically focusing on inhibitory control of infralimbic cortex layer 5 pyramidal neurons because they are the main output cells of the region. The baseline characteristics of GABAergic transmission were not significantly different in nondependent and dependent rats (Table 1). Orexin-A application (1 μM for 15 min; Schmeichel et al., 2017) had no effect on the sIPSC frequency F I G U R E 3 Extinction training and effect of TCS 1102 on the stress-induced reinstatement of alcohol-seeking behaviour in rats with a history of alcohol dependence. (a) Extinction of alcohol-seeking behaviour over 10 sessions. (b) Intermittent footshock stress precipitated alcohol-seeking behaviour in rats that received vehicle (VEH) in both dependent and nondependent groups. The administration of TCS 1102 in the infralimbic cortex (IL) prevented the stress-induced reinstatement of alcohol seeking in dependent rats but not in nondependent rats. No differences in inactive lever responses were observed. The data are expressed as mean + SEM. * P < 0.05, versus VEH. * P < 0.05, versus inactive lever presses. + P < 0.05, versus respective EXT. Blue dots denote data from male rats and red dots represent data from females. E, extinction; S, sham; T, TCS 1102; V, vehicle F I G U R E 4 Effects of chronic intermittent alcohol vapour exposure on the messenger ribonucleic acid (mRNA) expression of Hcrt in the hypothalamus (HYP) and Hcrtr1 and Hcrtr2 in the infralimbic cortex (IL). Chronic intermittent alcohol vapour exposure-induced dependence increased Hcrt mRNA expression in (a) the HYP and (b) Hcrtr1 and (c) Hcrtr2 mRNA expression in the IL. The data are expressed as mean + SEM. * P < 0.05, versus naive. + P < 0.05, versus nondependent animals. Blue dots denote data from male rats and red dots represent data from females. D, dependent; N, naive; ND, nondependent in nondependent rats but significantly increased it (suggesting increased GABA release) in the alcohol-dependent group (Figure 5a,b). This led to a significant difference in orexin-A response across the two rat groups (nondependent: mdn = 104. 2, 94.3-137.4; dependent: mdn = 143.1, 123.3-190.4; U = 51, P < 0.01, Mann-Whitney test). There were no group differences in sIPSC amplitude or kinetics, although orexin-A increased the rise time in dependent rats and decay time in nondependent rats (Figure 5c,d).
Application of the OX 1/2 antagonist TCS 1102 (10 μM for 15 min) had the converse effect and significantly decreased the sIPSC frequency (suggesting decreased GABA release) in the nondependent group but had no effect in dependent rats (Figure 5a

| DISCUSSION
The goal of the present study was to assess the implication of OX receptor signalling in the infralimbic cortex during the stress-induced reinstatement of alcohol-seeking behaviour in animals with a history of alcohol dependence. Furthermore, to explore whether alcohol dependence causes molecular changes OX receptors, Hcrtr1 and Hcrtr2 mRNA expressions were measured in the infralimbic cortex and Hcrt mRNA expression was measured in the hypothalamus at an acute abstinence time point (8 h after the alcohol vapour was OFF).
Consistent with several previous studies (Matzeu et al., 2018;Matzeu & Martin-Fardon, 2020;O'Dell et al., 2004;Vendruscolo & Roberts, 2014), alcohol-dependent rats exhibited an increase (i.e. escalation) in alcohol self-administration during alcohol dependence that was induced by chronic intermittent alcohol vapour exposure. Consistent with previous reports (Le et al., 1999;Martin-Fardon et al., 2000;Matzeu & Martin-Fardon, 2020), intermittent footshock stress reinstated extinguished alcohol-seeking behaviour. Interestingly, intra-infralimbic cortex administration of the OX 1/2 antagonist TCS 1102 significantly blocked the stress-induced reinstatement of alcohol-seeking behaviour in alcohol-dependent rats but not in nondependent rats as observed previously (Flores-Ramirez et al., 2022). Preclinical studies demonstrated that rats with a history of alcohol dependence exhibit an increase in alcohol self-administration (escalation) and somatic and motivational signs of withdrawal that are characteristic of dependence, observable at 6-8 h of abstinence from alcohol (Matzeu et al., 2018;Matzeu & Martin-Fardon, 2020;O'Dell et al., 2004;Roberts et al., 1996;Vendruscolo & Roberts, 2014). Likewise, the dependent rats in the present study exhibited the escalation of alcohol self-administration, an increase in somatic withdrawal signs and high blood alcohol levels after self-administration sessions during

Notably, increases in
Week 6 of alcohol vapour exposure (Figure 2b-d). Overall, the present results, combined with previous reports, add to the evidence that intermittent exposure to alcohol vapour elicits behavioural and neurobiological signs of dependence. Changes in neurocircuitry systems, specifically those that are involved in response inhibition, reward and stress, may contribute to hyperkatifeia and thus negative reinforcement, in which relief from negative symptoms that are associated with alcohol withdrawal motivates an individual to seek and take alcohol, particularly during times of stress (Koob, 2014).
Intermittent footshock induced alcohol-seeking behaviour in nondependent rats regardless of treatment, as well as alcohol-dependent rats treated with VEH. Furthermore, intra-infralimbic cortex administration of TCS 1102 prevented stress-induced reinstatement in dependent rats only. A possible behavioural confound following TCS 1102 injection in the infralimbic cortex could be a diffusion into the adjacent prelimbic cortex, making it possible for TCS 1102 to exert nonspecific/additional behavioural effects through the prelimbic cortex. However, the accuracy of the injections (depicted in Figure 1b) together with our earlier study that used a similar approach (Flores-Ramirez et al., 2022) to demonstrate that administration of a corticotropin-releasing factor receptor 1 antagonist in the infralimbic cortex (but not in the prelimbic cortex) prevented the stress-induced reinstatement of alcohol-seeking behaviour strongly dispute this T A B L E 1 Basal GABA A receptor-spontaneous inhibitory postsynaptic currents (sIPSC) characteristics Note: Baseline sIPSC frequencies, amplitudes, rise times and decay times in infralimbic cortex layer 5 pyramidal neurons of nondependent and dependent rats. There were no significant differences across groups. All data are presented as mean ± SEM.
F I G U R E 5 Legend on next page. possibility and provide evidence that TCS 1102 ability to prevent OX 1 and OX 2 receptors play significant roles in drug self-administration and the reinstatement of drug-seeking behaviour (Plaza-Zabala et al., 2012Uslaner et al., 2014), and involvement of the orexin system may be associated with dependence (Moorman & Aston-Jones, 2009;Moorman et al., 2017). Evidence that the orexin system is involved in alcohol self-administration and seeking is particularly compelling (Lawrence et al., 2006;Matzeu & Martin-Fardon, 2020), given that systemic administration of the OX 1 antagonist SB-334867 was reported to decrease voluntary home-cage alcohol consumption (Anderson et al., 2014;Moorman & Aston-Jones, 2009), reduce operant self-administration (Jupp et al., 2011;Lawrence et al., 2006;Moorman et al., 2017;Richards et al., 2008) and blunt the olfactory cue-induced reinstatement of alcohol-seeking behaviour in alcoholpreferring rats (Lawrence et al., 2006). Importantly, OX 1 blockade decreased reinstatement that was induced by alcohol-related stimuli (Brown et al., 2016;Martin-Fardon & Weiss, 2014) and stress (Richards et al., 2008) in nondependent rats. Similarly, the pharmacological blockade of OX 2 with a peripheral injection of LSN2424100, a selective OX 2 antagonist, decreased home-cage alcohol consumption in alcohol-preferring (P) rats and alcohol-preferring C57BL/6J mice (Anderson et al., 2014). Systemic JNJ-10397049 administration reduced operant alcohol self-administration in nondependent rats (Shoblock et al., 2011). Furthermore, an intra-ventral tegmental area (VTA) infusion of the OX 1/2 antagonist almorexant attenuated alcohol self-administration (Srinivasan et al., 2012) and intra-pPVT administration of the OX 1/2 antagonist TCS 1102, which was tested herein, also prevented the stress-induced reinstatement of alcohol-seeking behaviour in alcohol-dependent rats only (Matzeu & Martin-Fardon, 2020).
Overall, the present results strongly indicate that orexin transmission throughout the brain, particularly in regions that are affected by drug dependence (e.g. the ventral tegmental area, pPVT and infralimbic cortex), is implicated in the stress-induced reinstatement of alcohol seeking in animals with a history of alcohol dependence.
The present study showed that the blockade of OX 1 and OX 2 receptors in the infralimbic cortex with the OX 1/2 antagonist TCS 1102 prevented the stress-induced reinstatement of alcohol-seeking behaviour only in alcohol-dependent rats. Interestingly, the ability of ability to prevent stress-induced reinstatement in alcohol-dependent rats. Notably, a recent study from our laboratory showed that intra-pPVT TCS 1102 administration also prevented the stress-induced reinstatement of alcohol-seeking behaviour selectively in alcoholdependent rats (Matzeu & Martin-Fardon, 2020). The present study extends these findings to the infralimbic cortex. Similar effects of TCS 1102 are unsurprising when considering the interconnectedness of the hypothalamus, pPVT and infralimbic cortex, but further studies are required to determine the unique functional consequences of orexin elevations within each region.
Hcrt mRNA expression in the hypothalamus and Hcrtr1/2 mRNA expression in the infralimbic cortex increased in animals with a history of alcohol dependence at the time of testing (i.e., 24 h after the last extinction session) during acute abstinence. Interestingly, the nondependent group of rats did not exhibit any changes in Hcrt mRNA or Hcrtr1/2 mRNA expression when compared to the naive control. This suggests that self-administration of alcohol per se did not induce any changes in gene expressions on its own. Moreover, together with an earlier study from this group that used a nondrug alternative reinforcer (i.e. sweetened condensed milk) the data further suggest that the operant procedure itself did not alter gene expression, given that F I G U R E 5 Effects of chronic intermittent alcohol vapour exposure on orexin signalling at γ-aminobutyric acid (GABA) synapses in the infralimbic cortex (IL). (a) Representative GABA A receptor-mediated spontaneous inhibitory postsynaptic current (sIPSC) traces from IL layer 5 pyramidal neurons before and during 1 μM orexin-A (Orx-A), 10 μM TCS 1102 and Orx-A in TCS 1102 application in nondependent and alcohol-dependent rats. (b) Orx-A had no effect on the sIPSC frequency in nondependent rats but increased it in alcohol-dependent rats ( * P < 0.05 vs. baseline; # P < 0.05 nondependent vs. dependent). (c) Orx-A did not alter sIPSC amplitudes. (d) Orx-A slightly increased the rise time in dependent rats and the decay time in nondependent rats ( * P < 0.05 vs. baseline). (e) TCS 1102 decreased the sIPSC frequency in nondependent rats but had no effect in alcohol-dependent rats ( * P < 0.05 vs. baseline; # P < 0.05 nondependent vs. dependent). (f) TCS 1102 did not alter sIPSC amplitudes. (g) TCS 1102 slightly increased the decay time in both nondependent and dependent rats ( * P < 0.05 vs. baseline). (h-j) Orx-A did not alter sIPSCs after pretreatment with TCS 1102. n = 13-18 cells from 10-12 rats per experimental group. Blue squares denote cells from male rats and red squares represent cells from females. D, dependent; ND, nondependent in nondependent rats self-administering sweetened condensed milk Hcrt mRNA and Hcrtr1/2 mRNA expression were not different to what was measured in experimentally naive rats (Matzeu & Martin-Fardon, 2020). The results obtained in the alcohol-dependent rats are consistent with previous work that reported increases in Hcrt mRNA expression in the lateral hypothalamus in genetically selected alcoholpreferring rats (Lawrence et al., 2006) and an increase in Hcrtr2 expression in the anterior PVT (Barson et al., 2015). Additionally, in a previous study from our laboratory, significant increases in Hcrt mRNA expression were observed in the hypothalamus and increases in Hcrtr1/2 mRNA expression were observed in the pPVT at the same point of testing. The present results extend these earlier findings (Matzeu & Martin-Fardon, 2020)  One limitation of the present study is that the qPCR analyses were conducted in animals that did not undergo stress-induced reinstatement and did not receive either VEH or TCS 1102 in the infralimbic cortex. Assessing changes in mRNA in animals that are concomitantly exposed to dependence, stress and pharmacological treatment would certainly be an interesting line of investigation. The rationalization for our approach was to obtain a clearer picture of molecular changes that are induced specifically by alcohol dependence itself and set the stage for TCS 1102 ability to prevent reinstatement. Furthermore, only assessing changes in the expression of mRNA and not actual orexin neuropeptides/proteins or receptor density is also a limitation. Increases in gene expression do not necessarily result in higher protein levels and may not be representative of increases in the activation of these molecules Mehra et al., 2003). Thus, future studies of actual levels of orexin and OX 1 and OX 2 receptor density in groups of rats that are made dependent, undergo stress-induced reinstatement (e.g. Flores-Ramirez et al., 2022) and are administered with TCS1102 are warranted.
Overall, the present findings support the hypothesis that orexin transmission in the infralimbic cortex is involved in the stress-induced reinstatement of alcohol-seeking behaviour in subjects with a history of alcohol dependence. Changes in mRNA that were observed in both the hypothalamus and infralimbic cortex, as well as the decreased basal effect of OX receptor signalling over infralimbic cortex GABAergic synapses, as well as its increased stimulated effect, support the notion that the orexin system undergoes long-lasting maladaptations that result from repeated cycles of alcohol exposure, underscoring the dynamic nature of neurocircuitry systems that render subjects vulnerable to drinking, craving and relapse. Because stress is one of the most well-known risk factors for relapse in patients who suffer from alcohol use disorder, the present study highlights the importance of considering pharmacological targets that interact with stress processing when developing therapies for alcohol use disorder. However, knowing that environmental stimuli that are repeatedly paired with drug consumption acquire incentive-motivational value, evoking the expectation of drug availability and memories of past drug experience, what remains to be determined is the mediation by OX receptor signalling in the infralimbic cortex of the incentive-motivational value of alcoholrelated stimuli to induce alcohol-seeking behaviour. Additionally, if, as suggested by the present findings, orexin transmission in the infralimbic cortex is compromised by alcohol dependence, another aspect that could be tested is whether an injection of TCS 1102 in the infralimbic cortex would improve the physical signs of alcohol withdrawal and consequently prevent the escalation of alcohol drinking. These are aspects that will need to be addressed in the future. Ultimately, targeting both OX 1/2 receptors using OX 1/2 antagonists could have strong potential to reduce hyperkatifeia that is elicited by alcohol dependence and withdrawal and attenuate pathological drug seeking that is induced by stress in individuals who suffer from alcohol use disorder.

ACKNOWLEDGEMENTS
This is Publication Number 30169 from The Scripps Research Institute. The authors thank Michael Arends for editorial assistance.

CONFLICTS OF INTEREST
The authors declare no competing interests.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.