Behavioral Domain-Specific Effects of Positive Modulation of α5 and α6 GABA(A) Receptors in a Rat Double-Hit Stress Model.

Behavioral Domain‐Specific Effects of Positive Modulation of α5 and α6 GABAA Receptors in a Rat Double‐Hit Stress Model - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice Pharmacol Res Perspect . 2026 Apr 17;14(3):e70245. doi: 10.1002/prp2.70245 Search in PMC Search in PubMed View in NLM Catalog Add to search Behavioral Domain‐Specific Effects of Positive Modulation of α5 and α6 GABA A Receptors in a Rat Double‐Hit Stress Model Đorđe Đorović Đorđe Đorović 1 Institute of Anatomy “Niko Miljanić”, School of Medicine, University of Belgrade, Belgrade, Serbia Find articles by Đorđe Đorović 1 , Jovana Aranđelović Jovana Aranđelović 2 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Find articles by Jovana Aranđelović 2 , Jana Ivanović Jana Ivanović 2 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Find articles by Jana Ivanović 2 , Kristina Jezdić Kristina Jezdić 2 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Find articles by Kristina Jezdić 2 , Bojan Batinić Bojan Batinić 3 Department of Physiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Find articles by Bojan Batinić 3 , Dishary Sharmin Dishary Sharmin 4 Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin, Milwaukee, Wisconsin, USA Find articles by Dishary Sharmin 4 , Prithu Mondal Prithu Mondal 4 Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin, Milwaukee, Wisconsin, USA Find articles by Prithu Mondal 4 , James M Cook James M Cook 4 Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin, Milwaukee, Wisconsin, USA Find articles by James M Cook 4 , Miroslav M Savić Miroslav M Savić 2 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Find articles by Miroslav M Savić 2, ✉ Author information Article notes Copyright and License information 1 Institute of Anatomy “Niko Miljanić”, School of Medicine, University of Belgrade, Belgrade, Serbia 2 Department of Pharmacology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia 3 Department of Physiology, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia 4 Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin, Milwaukee, Wisconsin, USA * Correspondence: Miroslav M. Savić ( [email protected] ) ✉ Corresponding author. Revised 2026 Mar 15; Received 2025 Aug 17; Accepted 2026 Mar 25; Collection date 2026 Jun. © 2026 The Author(s). Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. PMC Copyright notice PMCID: PMC13088882  PMID: 41995413 ABSTRACT Impulsivity is an understudied area of post‐traumatic stress disorder, a debilitating disorder specifically associated with stress. We examined reward‐related impulsive behavior, anxiety‐like behavior, locomotor activity and social behavior in the absence and presence of protracted pharmacological positive modulation of α5‐ and α6‐GABA A receptors (GL‐II‐73 and DK‐I‐56‐1, respectively) in male Sprague–Dawley rats exposed to a combination of maternal deprivation (MD) and single prolonged stress (SPS). While locomotor and anxiety‐like behavior were not affected in the SPS and MD+SPS groups, the double‐hit group treated with DK‐I‐56‐1 exhibited a higher locomotor distance compared with MD+SPS and a higher percentage of open‐arm time in the elevated plus maze compared with the control group. In the variable delay‐to‐signal task of impulsivity, the total number of successful trials and premature responses (PR) in the first stage of the test day were reduced in all groups exposed to stress compared with the controls. Based on PR rates in the first and second set of trials, motor impulsivity was apparently suppressed in all stressed groups, while delay intolerance was suppressed only in the MD+SPS+GL‐II‐73 group, respectively. In the three‐chamber test, social interaction was completely normal, while social recognition was preserved in the MD+SPS+GL‐II‐73 group. In the resident‐intruder test, social play was reduced only in the SPS group. The evaluation of impulsive behavior in the used complex task was hindered by the lack of motivation of stressed rats, which in the case of omission percentage was ameliorated by positive modulation of α5 and α6 GABA A receptors. Keywords: DK‐I‐56‐1, GL‐II‐73, impulsivity, motivation, rat model of double‐hit stress, variable delay‐to‐signal paradigm, α5 GABA A receptors, α6 GABA A receptors A double‐hit stress model in Sprague–Dawley rats induces alterations in motivation, motor impulsivity, and social recognition that are partially ameliorated by α5‐ and α6‐GABA A receptor modulation, suggesting domain‐specific effects of this modulation on the stress‐induced phenotype. 1. Introduction Post‐traumatic stress disorder (PTSD) is a debilitating psychiatric condition characterized by intrusive re‐experiencing, hyperarousal, avoidance, and negative alterations in mood and cognition that can develop after a traumatic event [ 1 ]. Growing evidence suggests that impulsivity [ 1 ] and social cognition [ 2 ] remain insufficiently explored in PTSD, limiting progress in understanding the disorder and improving pharmacological treatments. Social cognition is a multifactorial construct influenced by mood and motivation and may contribute to alterations in social play behavior [ 3 ]. On the other hand, impulsivity is a complex psychological trait and state described by a tendency toward rapid, unplanned reactions to stimuli without due consideration of the negative outcomes [ 4 ]. It has been posited to be composed of attentional impulsivity, motor impulsivity, and non‐planning impulsivity [ 5 ] or, according to the five‐factor model, lack of premeditation, lack of perseverance, sensation seeking, and positive and negative urgency [ 6 ]. Efforts to define the neurocognitive correlates of impulsivity have led to the distinction between motor impulsivity (behavioral impulsivity or impulsive action) and cognitive impulsivity (impulsive choice), encompassed in the delay discounting concept and representing a preference for a smaller/immediate reward over a larger/delayed reward [ 7 , 8 , 9 ]. Neurochemical deficits related to GABA were commonly identified in PTSD [ 10 ], and dysfunction of GABA‐ergic signaling is thought to play a central role in processes affecting emotional regulation and impulsivity [ 11 , 12 ]. Among GABA A receptors, those that can be localized both synaptically and extrasynaptically are of particular interest, as they contribute to both phasic and tonic inhibition of neurons, such as receptors composed of the subunits α5βγ2 and α6βγ2 [ 13 ]. The α5 subunit‐containing GABA A receptors (α5GABA A Rs) are predominantly localized in the hippocampus and prefrontal cortex, suggesting a specific role in cognitive processes [ 14 ]. These receptors are potential therapeutic targets for a range of neuropsychiatric conditions, including depression [ 15 ], schizophrenia [ 16 ], cognitive impairments associated with aging [ 17 ] and neurodegenerative diseases [ 18 ]. GABA A receptors containing the α6 subunit (α6GABA A Rs) are predominantly expressed in the granule cells of the cerebellum, playing a role in both motor and cognitive processes [ 19 ]. The α6 subunit expressed in other brain regions at lower levels may be involved in various neurophysiological processes [ 20 , 21 , 22 ]. Impulsive behavior and the effects of α5‐ or α6‐GABA A R modulators have not been studied in a PTSD animal model. The aim of this work was to investigate impulsive behavior, anxiety‐like behavior, emotional reactivity, and social memory in the absence and presence of pharmacological positive modulation of α5 and α6‐GABA A Rs in a double‐hit stress model in rats [ 23 ]. The model combined postnatal 24‐h maternal deprivation with severe stress in early adulthood in the form of single prolonged stress, which are in themselves widely used animal models of early life stress or PTSD [ 23 ]. 2. Materials and Methods 2.1. Animals and Housing Male and nulliparous female Sprague–Dawley rats, bred from a colony of Charles River (Italy), were housed and maintained in the vivarium of the Faculty of Pharmacy, University of Belgrade, Serbia. All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, approved by the Ethics Committee for Animal Experiments at the University of Belgrade—Faculty of Pharmacy and the Ministry of Agriculture, Forestry and Water Management—Veterinary Directorate (approval number: 323‐07‐10 046/2020‐05, dated 23.09.2020). The animals were housed in pairs of males and females in standard Plexiglas cages (26 × 42 × 15 cm) with sawdust bedding at controlled temperature (23°C ± 1°C), humidity and a 12‐h light–dark cycle (lights on 07:00–19:00) and had ad libitum access to food and water. After 2 weeks, the males were removed and the females were checked twice daily for delivery. The day of birth was designated as postnatal day 0 (P0). 2.2. Maternal Deprivation (MD) and Single Prolonged Stress (SPS) The overall design of the study is shown in Figure 1 . Maternal deprivation (MD) was performed on the P9: The dams were removed at 10:00 am, the pups were weighed and returned to their home cage at room temperature for 24 h. On the P10, the pups were weighed again and dams were returned. In the control litters, the dams were taken out for weighing for 3 min at both P9 and P10. Litters were left undisturbed until weaning on P21, apart from routine cage maintenance when rats were sexed. Only male rats were used in the study and they were housed in groups (4 per cage) in new cages with sawdust bedding. FIGURE 1. Open in a new tab Experimental design. Rats were exposed to two different stressors at separate time points: Maternal deprivation (MD) during the early postnatal period (postnatal day 9; P9) and single prolonged stress (SPS) in early adulthood (P60), or were not subjected to any stress procedure (control animals; CTRL). Seven days before SPS, all animals underwent training for the variable delay‐to‐stimulus test, which was used for impulsivity testing (IT). After SPS was applied to a subset of CTRL animals and all MD animals, a 14‐day consolidation phase followed. The treatment of animals started 3 days before behavioral testing. CTRL and SPS animals received vehicle (VEH), while MD+SPS animals were treated with either vehicle, α5, or α6 GABA A receptor positive allosteric modulators (GL‐II‐73 and DK‐I‐56‐1, respectively). Five groups were formed: CTRL‐VEH ( n = 29), SPS‐VEH ( n = 19), MD+SPS‐VEH ( n = 18), MD+SPS‐GL‐II‐73 ( n = 18), and MD+SPS‐DK‐I‐56‐1 ( n = 17). Seventeen days after SPS, behavioral testing began, consisting of IT, EPM, SLA, RIT, and 3CT. 3CT, three chamber test; EPM, elevated plus maze; P, postnatal day; RIT, resident intruder test; SLA, spontaneous locomotor activity. At P60, the single prolonged stress (SPS) procedure was applied to all animals exposed to MD and to 19 animals unexposed to MD. The remaining animals unexposed to MD were left undisturbed in their cages. Rats were restrained for 2 h in custom‐made clear plastic tubes and then placed in a transparent acrylic cylinder (24 cm diameter, 50 cm height) two‐thirds filled with water at 24°C to perform a 20‐min forced swim. After a 15‐min recovery period, the rats were exposed to diethyl ether until loss of consciousness and then returned to their home cages for 14 days before treatment began. 2.3. Drug Treatment MD+SPS animals were treated with either GL‐II‐73 or DK‐I‐56‐1 or vehicle (VEH) (MD+SPS‐VEH, MD+SPS‐GL‐II‐73, MD+SPS‐DK‐I‐56‐1), while the other two groups were treated with vehicle (CTRL‐VEH and SPS‐VEH). GL‐II‐73 and DK‐I‐56‐1 are positive allosteric modulators (PAMs) selective for α5‐ and α6‐GABA A receptors, respectively, synthesized by the group of Dr. James M. Cook (University of Wisconsin‐Milwaukee, USA). The animals received intraperitoneal injections of GL‐II‐73, DK‐I‐56‐1 or vehicle (SOL: 85% distilled water, 14% propylene glycol, 1% Tween 80) at 2 mg/mL at a dose of 10 mg/kg/day for 7 days, according to previously published studies [ 21 , 24 ]. Treatment started 14 days after SPS and 3 days before the behavioral tests. The mean weight at the start of treatment was 291.4 g (range 255.12–327.69 g). The following 5 experimental groups were formed from three consecutive subcohorts: CTRL‐VEH ( n = 29), SPS‐VEH ( n = 19), MD+SPS‐VEH ( n = 18), MD+SPS‐GL‐II‐73 ( n = 18), MD+SPS‐DK‐I‐56‐1 ( n = 17) (Figure S1 ). 2.4. Elevated Plus Maze (EPM) The EPM consisted of two open arms (50 × 10 cm, 0.3 cm high ledges) and two closed arms (50 × 10 × 40 cm) connected by a central area (10 × 10 cm) that was 50 cm above the floor. The maze was illuminated with a red neon ceiling light (10 lx in the closed arms). Each rat was placed in the center of the maze facing a closed arm and allowed to explore the maze for 5 min. The standard spatiotemporal variables were recorded and analyzed using the ANY‐maze Video Tracking System software (Stoelting Co., Wood Dale, IL, USA) in accordance with previous studies [ 25 , 26 ]. Anxiety indices included the percentage of open‐arm entries and time spent in the open arms. The parameter related to motor activity was the number of closed arm entries. An entry into an open arm or closed arm was scored when 90% of the animal crossed the virtual line separating the neighboring zones, whereas an exit occurred when more than 90% of the animal left the respective zone. 2.5. Spontaneous Locomotor Activity (SLA) Locomotor activity was measured in four blurred Plexiglas chambers (40 × 25 × 35 cm) under indirect white light (60 lx). Each trial lasted 90 min, without prior habituation. Parameters that were quantified are total distance traveled and number of rotations, which were automatically assessed via ANY‐maze Software, Stoelting Co. 2.6. Variable Delay to Signal (VDS) Task The variable delay‐to‐signal (VDS) paradigm is used to assess different dimensions of impulsivity. In this study, a modified version of the protocol by Leite‐Almeida et al. [ 27 ] was implemented using ABET software on four touchscreen‐based testing chambers (Campden Instruments Ltd., Lafayette, IN, USA). Each chamber had a trapezoidal prism shape (24 × 13 × 33.5 × 30 cm: longer base × shorter base × lateral sides × height). The front wall, extending from the longer base, housed a touchscreen featuring a 3 × 3 cm interactive area in the lower center. Opposite this, the rear wall contained a pellet tray that delivered 60 mg sugar pellets—manufactured in‐house—upon correct responses. Animal movement within the chamber was monitored via infrared (IR) beams located on the sides. Rats were maintained on a restricted diet (65 g of pelleted food per cage/day) starting 1 week prior to and throughout the VDS protocol. The automated procedure consisted of two main phases: training and testing. The training phase, lasting 7 days, comprised three steps focused on procedural learning. Animals were trained to respond to a white light cue appearing in the designated touchscreen area (against a black background) to receive a sugar pellet reward. On the first day, animals were habituated to the chambers for 15 min, during which the white light cue appeared intermittently, and sugar pellets were dispensed freely to facilitate association between stimulus and reward. On the second day, a “must touch” protocol was used for 20 min, requiring animals to actively touch the light cue to receive a reward. Activation of the food tray IR sensor by pellet retrieval triggered the 6‐s intertrial interval (ITI). For the following 5 days, animals completed 80 trials within 30 min each day. ITI between the reward and the touchscreen being turned on was 6 s. Touching the stimulus light led to a reward, whereas premature screen interactions during the ITI were recorded as premature responses (PRs), following Leite‐Almeida et al. [ 27 ]. Such responses were punished by switching off the overhead light for 5 s, with repeated infractions prolonging this dark period. In all other cases, the chamber light remained on. Failure to respond to the light cue within 60 s was scored as an omission, and the punishment was administered. On the eighth day, the test phase was conducted, consisting of up to 100 trials or lasting a maximum of 1 h, whichever came first. The test session was divided into three phases with variable ITIs: (1) an initial 20‐trial phase identical to training (ITI = 6 s; ITI6si), except that premature responses were not punished; (2) a delay tolerance assessment phase consisting of 60 randomly distributed trials with a prolonged ITI of either 9 s or 15 s (30 trials with ITI9s and 30 trials with ITI15s); and (3) a final phase of 20 trials identical to the initial phase (ITI6sf). Four parameters were quantified: total number of trials, number of premature responses (PR), PR rate, and percentage of omissions. PR rate was calculated according to the formula [ 27 ]: PRrate = PR _ stage / time _ of _ ITI _ per _ stage × N _ trials _ stage × 60 where PR_stage was the number of premature responses in the stage ITI6si, ITI9s, ITI15s, or ITI6sf; time_of_ITI_per_stage was the duration of the respective stage: 6, 9, 15, or 6 s; and N_trials_stage was the number of completed trials within the stage: 20, 30, 30, or 20, respectively. 2.7. Three‐Chamber Test (3CT) The 3CT apparatus consisted of three non‐transparent Plexiglas chambers: a central compartment and two identical side chambers [ 18 ]. The protocol included habituation (5 min, only in the center), the social interaction test (SIT, 10 min, one side chamber with an unfamiliar conspecific under a wire cage, the other chamber empty) and the social recognition test (SRT, 10 min, new unfamiliar conspecific presented). The time in the interaction zones (area around each cage) spent with a conspecific rather than in an empty chamber (social exploration) or with an unfamiliar conspecific (social recognition) was recorded and analyzed with the ANY‐maze Software, Stoelting Co to assess social preference and memory. 2.8. Resident Intruder Test (RIT) The RIT was used to assess stress‐related social behavior [ 28 ]. Three days before the test, the rats were housed individually. On the test day, an unfamiliar, age‐ and weight‐matched intruder was introduced into the resident's home cage, with which social interaction took place for 10 min in dim red light during the dark phase. During the test, the activity of the rats in the cage was recorded and later quantified using the program AnyMaze Software, Stoelting Co. The parameters analyzed were social exploration (allogrooming) and social play (wrestling/crawling/boxing/chasing/pining down). 2.9. Statistical Analysis With the exception of the VDS protocol, behavioral data were recorded and analyzed using ANY‐maze software (Stoelting Co., Wood Dale, IL, USA). Data from three subcohorts were pooled. Two separate statistical analyses were performed to evaluate the effects of stress exposure and pharmacological treatment. First, CTRL‐VEH, SPS‐VEH, and MD+SPS‐VEH groups were compared to evaluate the effects of stress exposure. Second, CTRL‐VEH, MD+SPS‐VEH, MD+SPS‐GL‐II‐73, and MD+SPS‐DK‐I‐56‐1 groups were analyzed to assess the effects of the pharmacological interventions on the stress‐induced phenotype. Because the study design does not include all combinations of stress exposure and treatment conditions, a fully factorial analysis was not applicable. Statistical analyses included one‐way ANOVA with Tukey's post hoc test (IT, SLA, EPM), two‐way repeated‐measures ANOVA with Sidak's post hoc test (IT, 3CT), and Kruskal–Wallis test with Dunn's post hoc test (RIT, due to variance of medians in social play). Post hoc tests were performed to compare all groups within each analysis; however, only significant group differences are reported. As the CTRL‐VEH and MD+SPS‐VEH groups were included in both analyses, results from the stress‐effect and treatment‐effect comparisons were displayed within the same graphs. Distinct color‐coded significance markers were used to indicate the outcomes of the two predefined statistical comparisons. Significant main effects (between‐subject factor: model or model+treatment and/or within‐subjects factor: ITI or chamber) are shown in the figure legends. Group differences are indicated by asterisks (* adjusted p < 0.05, ** adjusted p < 0.01, *** adjusted p < 0.001 for the Tukey or Dunn test, and * p < 0.05, ** p < 0.01, *** p < 0.001 for the Sidak post hoc comparisons), and statistical trends (0.05 < adjusted p < 0.10) are also noted. The adjusted p values are reported in Results section together with q values where applicable. All analyses were performed using GraphPad Prism (version 10.1.0; GraphPad Software, La Jolla, CA, USA). 3. Results 3.1. Locomotor Activity and Anxiety‐Like Behavior In the SLA, total distance traveled and rotations did not differ among the vehicle‐treated CTRL, SPS, and MD+SPS groups (Figure 2a ). MD+SPS animals receiving DK‐I‐56‐1 traveled a longer distance (adjusted p = 0.0361, q = 3.901; Figure 2a ) and showed a trend toward more rotations (adjusted p = 0.075, q = 3.479; Figure S2 ) compared with MD+SPS‐VEH. FIGURE 2. Open in a new tab Effects of stress and treatment on spontaneous locomotor activity and anxiety‐like behavior in the elevated plus maze (EPM). Two types of comparisons were made. First, CTRL‐VEH, SPS‐VEH, and MD+SPS‐VEH (vehicle‐treated) groups were compared to evaluate the effects of stress exposure. Second, CTRL‐VEH, MD+SPS‐VEH, MD+SPS‐GL‐II‐73, and MD+SPS‐DK‐I‐56‐1 groups were analyzed to assess the effects of pharmacological interventions. Distance traveled during 90 min of testing in the spontaneous locomotor activity (SLA) test is shown (a). Three parameters evaluated in the elevated plus maze test (EPM) are displayed: Number of closed arm entries (b), percentage of open arm entries (c), and percentage of open arm time (d). Data were analyzed using one‐way ANOVA followed by Tukey's post hoc test. Comparisons between MD+SPS‐VEH, SPS‐VEH, and CTRL‐VEH are shown in red, while those between MD+SPS‐GL‐II‐73, MD+SPS‐DK‐I‐56‐1, MD+SPS‐VEH, and CTRL‐VEH are shown in green, if significant. Corresponding F and p values are shown in the upper right corner of each graph if significant. Asterisks in the graphs represent significant differences between groups (* for 0.01 < adjusted p < 0.05). Differences at the trend level are indicated by the exact adjusted p value. Technical outliers due to impaired tracking by the ANY‐maze software were excluded from the statistical analysis. No significant group differences were found for EPM entries into the closed arm (Figure 2b ). The MD+SPS‐VEH group showed a trend toward more entries into the open arm compared with CTRL‐VEH, but this effect was not observed when the treatment groups (MD+SPS‐GL‐II‐73 and MD+SPS‐DK‐I‐56‐1) were included in the analysis (adjusted p = 0.092, q = 3.019; Figure 2c ). No difference in time spent in the open arm was observed between vehicle groups (Figure 2d ), but MD+SPS animals treated with DK‐I‐56‐1 tended to spend more time in the open arm than MD+SPS‐VEH (adjusted p = 0.0532, q = 3.691; Figure 2d ). 3.2. Impulsivity MD+SPS‐VEH (adjusted p = 0.0003, q = 5.943) and SPS‐VEH animals (adjusted p = 0.0264, q = 3.764) completed fewer trials than CTRL‐VEH (Figure 3a ). The same was observed for MD+SPS animals treated with GL‐II‐73 or DK‐I‐56‐1 (vehicle‐treated: adjusted p = 0.0001, q = 6.367; GL‐II‐73: adjusted p = 0.001, q = 5.555; DK‐I‐56‐1: adjusted p = 0.0024, q = 5.209; Figure 3a ). FIGURE 3. Open in a new tab Effects of stress and treatment on reward‐related impulsivity in the variable delay‐to‐signal test (VDS). Two types of comparisons were made. First, CTRL‐VEH, SPS‐VEH, and MD+SPS‐VEH (vehicle‐treated) groups were compared to evaluate the effects of stress exposure. Second, CTRL‐VEH, MD+SPS‐VEH, MD+SPS‐GL‐II‐73 and MD+SPS‐DK‐I‐56‐1 groups were analyzed to assess the effects of pharmacological interventions. Four parameters measured in this task are presented: Total number of trials (a), number of premature responses (PR) normalized to the total number of trials (b), PR rate at ITI6si, ITI9s, ITI15s for animals that completed more than half of the trials (c), and percentage of omissions (d). Data were analyzed by one‐way ANOVA followed by Tukey post hoc test (a, b, d) and two‐way ANOVA with repeated measures followed by Sidak post hoc test (c). Comparisons between MD+SPS‐VEH, SPS‐VEH, and CTRL‐VEH are shown in red, while those between MD+SPS‐GL‐II‐73, MD+SPS‐DK‐I‐56‐1, MD+SPS‐VEH, and CTRL‐VEH are shown in green, if significant. The corresponding F and p values are indicated in the upper right corner of each graph, if significant, for the between‐subject factor model (m), the combined factor model+treatment (m+t), and/or within‐subject factor intertrial interval (ITI). Asterisks in the graphs represent significant differences compared with CTRL‐VEH (* for 0.01 < adjusted p < 0.05, ** for 0.001 < adjusted p < 0.01, *** for adjusted p < 0.001). Differences at the trend level are indicated by the exact adjusted p value. A statistical outlier occurred in the CTRL‐VEH group according to Grubb's test. Both stressed vehicle‐treated groups made fewer premature responses (PRs) normalized to total trials compared with CTRL‐VEH (SPS‐VEH: adjusted p = 0.0304, q = 3.685; MD+SPS‐VEH: adjusted p = 0.0061, q = 4.522; Figure 3b ). A similar reduction was observed in MD+SPS animals treated with GL‐II‐73 or DK‐I‐56‐1 (vehicle‐treated: adjusted p = 0.0044, q = 4.929; GL‐II‐73: adjusted p = 0.0022, q = 5.233; DK‐I‐56‐1: adjusted p = 0.0103, q = 4.538; Figure 3b ). For animals completing > 50 trials, the PR rate during ITI6s was lower in the MD+SPS‐VEH group (adjusted p = 0.0051, q = 5; Figure 3c ) and showed a trend toward reduction in SPS‐VEH (adjusted p = 0.0708, q = 3.254) compared with CTRL‐VEH. In animals completing > 20 trials, SPS‐VEH also showed a lower PR rate than CTRL‐VEH (adjusted p = 0.0464, q = 3.448; Figure S3a ). The PR rate during ITI6s was reduced in all MD+SPS groups (vehicle and drug‐treated) compared with CTRL‐VEH (> 50 trials: vehicle‐treated—adjusted p = 0.0093, q = 5; GL‐II‐73: adjusted p = 0.0161, q = 4.624; DK‐I‐56‐1: adjusted p = 0.0093, q = 5; Figure 3c ; > 20 trials: vehicle‐treated—adjusted p = 0.0096, q = 4.582; GL‐II‐73: adjusted p = 0.0089, q = 4.622; DK‐I‐56‐1: adjusted p = 0.0167, q = 4.309; Figure S3a ). MD+SPS‐ GL‐II‐73 animals also showed a lower PR rate at ITI15s compared with CTRL‐VEH (adjusted p = 0.0056, q = 5.136; Figure 3c ). For ITI9s and ITI15s, reduced PR rates were observed only in MD+SPS‐ GL‐II‐73 animals compared with CTRL‐VEH (adjusted p = 0.0261, q = 4.059; Figure S3b ). Animals completing > 78 trials within the SPS‐VEH group showed a trend toward a lower ITI15s PR rate compared with CTRL‐VEH (adjusted p = 0.0648; Figure S3c ). MD+SPS‐VEH animals had more omissions than CTRL‐VEH (adjusted p = 0.0354, q = 3.906; Figure 3d ) and correspondingly fewer correct trials (adjusted p = 0.0446; Figure S3d ). Test duration was longer in all experimental groups compared with CTRL‐VEH (SPS‐VEH: adjusted p = 0.0067, q = 4.473; MD+SPS‐VEH: adjusted p = 0.0027, q = 5.143; GL‐II‐73: adjusted p = 0.0009, q = 5.592; DK‐I‐56‐1: adjusted p = 0.0032, q = 5.069; Figure S3e ). 3.3. Social Behavior The main effect of chamber was significant in both phases of the 3CT, with animals spending more time in the chamber containing a conspecific than in the chamber with an empty cage (Figure 4a,b ). In the SIT phase of the 3CT, all groups spent more time with a conspecific than in the empty chamber (CTRL‐VEH: adjusted p < 0.0001; SPS‐VEH: adjusted p < 0.0001; MD+SPS‐VEH: adjusted p < 0.0001; GL‐II‐73: adjusted p = 0.0227; DK‐I‐56‐1: adjusted p = 0.0007; Figure 4a ). FIGURE 4. Open in a new tab Effects of stress and treatment on social behavior in the three‐chamber test (3CT) and resident intruder test (RIT). Two types of comparisons were made. First, CTRL‐VEH, SPS‐VEH, and MD+SPS‐VEH (vehicle‐treated) groups were compared to evaluate the effects of stress exposure. Second, CTRL‐VEH, MD+SPS‐VEH, MD+SPS‐GL‐II‐73, and MD+SPS‐DK‐I‐56‐1 groups were analyzed to assess the effects of pharmacological interventions. Two parameters were evaluated in the 3CT: Social interaction in the first phase (a) and social recognition in the second phase (d). Social interaction represents the time spent in a chamber containing a conspecific (animal) in a cage versus an empty cage (empty), while social recognition represented time spent in the chamber with a novel (new) versus familiar (old) animal. Two parameters evaluated in the RIT are presented: Time spent in social exploration (c) and time spent in social play (d). Data were analyzed by two‐way ANOVA with repeated measures followed by Sidak post hoc test (a, b) and Kruskal‐Wallis test followed by Dunn post hoc test (c, d). Comparisons between MD+SPS‐VEH, SPS‐VEH, and CTRL‐VEH are shown in red, while those between MD+SPS‐GL‐II‐73, MD+SPS‐DK‐I‐56‐1, MD+SPS‐VEH, and CTRL‐VEH are shown in green, if significant. The corresponding F and p values are indicated in the upper right corner of each graph, if significant, for the between‐subject factor model, the combined factor model+treatment, and/or the within‐subject factor chamber. Asterisks in the graphs represent significant differences between groups (* for 0.01 < adjusted p < 0.05, ** for 0.001 < adjusted p < 0.01, *** for adjusted p < 0.001). Differences at the trend level are indicated by the exact adjusted p value. Technical outliers related to impaired tracking by the ANY‐maze software were excluded from the statistical analysis. In the SRT phase, SPS‐VEH animals showed a trend toward a preference for the novel conspecific compared with the familiar one ( p = 0.0739; Figure S4a ), whereas MD+SPS‐VEH animals showed no preference. MD+SPS animals treated with GL‐II‐73 spent more time in the chamber containing the novel conspecific than in the chamber with the familiar conspecific (adjusted p = 0.0238; Figure 4b ; adjusted p = 0.0160; Figure S4a ), similar to CTRL‐VEH animals during the first 5 min of the SRT phase (adjusted p = 0.0461; Figure 4b ). In the RIT, vehicle‐treated stressed animals did not differ from CTRL‐VEH animals in social exploration (Figure 4c ). Time spent in social play differed between vehicle‐treated stressed groups (Kruskal–Wallis p = 0.0022; Figure 4d ): it was higher in MD+SPS‐VEH than in SPS‐VEH (adjusted p = 0.0076) and lower in SPS‐VEH than in CTRL‐VEH (adjusted p = 0.0056; Figure 4d ). GABAergic treatments (GL‐II‐73 or DK‐I‐56‐1) in MD+SPS animals had no effect on social exploration or play compared with vehicle‐treated MD+SPS animals (Figure 4c,d ). No differences were found in total social interaction in the RIT across groups, although DK‐I‐56‐1‐treated MD+SPS animals showed a trend toward reduced interaction compared with CTRL‐VEH (adjusted p = 0.0714; Figure S4b ). 4. Discussion Early life adversities are thought to be associated with increased susceptibility to developing PTSD when paired with additional stress in adulthood [ 29 ], leading to a range of behavioral deficits including hyperarousal, impaired cognition, and sociability. Thus, we developed a double‐hit stress model to investigate the effects of early life stress in the form of maternal deprivation on single prolonged stress in early adulthood in rats [ 23 ]. In the current study we assessed changes in emotionality and mood (spontaneous locomotor activity and elevated plus maze tests), reward‐related impulsivity (variable delay‐to‐signal test) and social behavior (three‐chamber and resident intruder tests) in male Sprague–Dawley rats exposed to these stress paradigms, revealing distinct effects across behavioral domains. Animals exposed to SPS or MD+SPS had similar changes in reward‐related impulsivity, while the effects on social play did not match (SPS caused a significant decrease in social play behavior compared with both MD+SPS group and unstressed controls). We also examined the effects of GABA‐modulating treatments on these behavioral domains in the MD+SPS model. Decreased blood plasma GABA levels after trauma have been shown to significantly predict the development of PTSD [ 30 ], while postmortem prefrontal cortex tissue from individuals with PTSD shows a significant decrease in transcripts encoding GABA transporters and GABA‐related neuropeptides [ 31 ]. Both findings support the rationale for assessing GABA‐enhancing treatments in PTSD models. Likely due to compensatory adaptations, postmortem prefrontal cortex tissue shows an increase in subunits and anchoring proteins that mediate the major synaptic effects of GABA [ 31 ]. Therefore, we chose to assess the influence of modulating receptors with extrasynaptic locations. GL‐II‐73 and DK‐I‐56‐1, which are behaviorally and electrophysiologically well‐characterized PAMs for α5‐ and α6‐GABAARs, respectively [ 32 , 33 ], produced discrete effects in different behavioral domains in the present MD+SPS model. 4.1. Locomotor Activity and Anxiety‐Like Behavior Spontaneous locomotor activity and anxiety‐like behavior were unaffected in the SPS, MD+SPS and GL‐II‐73 treated rats in our experiment. This contrasts with our previous work on the MD+SPS model in Wistar rats where MD+SPS animals displayed decreased locomotor activity in the SLA [ 23 ], suggesting strain‐specific effects of stress. In other stress‐related protocols, such finding has already been reported [ 34 ]. α6‐GABA A R PAM showed a role in modulating this behavior in MD+SPS rats. Interestingly, DK‐I‐56‐1 treatment in MD+SPS animals increased locomotor activity in the SLA test compared with vehicle treated MD+SPS animals, suggesting an increased exploratory drive [ 33 ] and showed a tendency to increase percentage of open‐arm time in the EPM compared with the CTRL group. It has recently been demonstrated that the cerebellum can influence the initiation of movements and locomotor activity through direct connections with dopaminergic neurons of the substantia nigra pars compacta [ 35 ], and therefore the hyperlocomotor response induced by DK‐I‐56‐1 could potentially involve this pathway. α6‐GABA A Rs in the striatum have been implicated in the regulation of anxiety‐like behavior through interactions with dopaminergic D3 receptors [ 36 ]. Notably, both cerebellar projections to substantia nigra pars compacta and ventral tegmental area and D3 receptors in the striatum have also been demonstrated to influence reward‐related signals [ 35 , 37 , 38 ], although the contribution of α6‐GABA A R modulation to these processes remains to be clarified. 4.2. Impulsivity A remarkable result of this study was that the animals in all groups exposed to stress completed a significantly lower number of trials during the test. It also showed that the percentage of omissions was significantly higher in the MD+SPS group. This means that the animals in this group not only had higher latencies to feed and response latencies; they also failed to touch the illuminated touchscreen more often during the entire 60‐s period. Because measurement of the exact values of latencies to feed and response latencies was not programed, it is not possible to separate the motivational and cognitive components (cf. [ 39 ]) that may have contributed to this profound stress‐induced incapacitation, nor to attribute the discrete recovering influences of GL‐II‐73 and DK‐I‐56‐1 to a specific behavioral domain. Nonetheless, these results demonstrate a robust deteriorative effect of severe stress that has a profound impact on impulsivity‐related behaviors. The effect of stress on motivation is well documented in the literature [ 40 ]. It has been suggested it is primarily related to effort, not anhedonia or suppressed appetite [ 41 , 42 , 43 ] and it has been termed stress‐induced anergia [ 40 ]. Namely, this phenomenon has been linked to incentive salience, which is defined as the attribution of a motivational component (“wanting” as opposed to “liking”) to a rewarding stimulus [ 44 ]. PTSD patients show motivational deficits and an altered reward system, although the exact nature of this deficit is not yet clear [ 45 , 46 ]. Reduced activity in the reward pathway in PTSD has been associated with motivational deficits [ 47 ], and PTSD patients display decreased willingness to invest effort in hedonic activities [ 48 ]. Rats are sensitive to cognitive effort [ 49 ]. The sustained attention required during the VDS task constitutes a mental strain [ 50 ], which may explain the lack of perseverance of stressed animals in our study. Interestingly, this facet of impulsivity has been highlighted as a trait unrelated to motor or cognitive impulsivity [ 51 ]. Decreased incentive salience of reward‐related cues with increased appetitive motivation was detected in rats exposed to SPS [ 52 ]. The effects of SPS on motivation have been related to changes in the mesolimbic dopaminergic system [ 52 , 53 ], and interestingly, the ability to modulate dopamine activity and/or hyperdopaminergic states has been demonstrated for both GL‐II‐73 [ 54 ] and DK‐I‐56‐1 [ 55 ]. All stressed groups showed decreased premature response rates during the 6 s ITI. It has already been demonstrated that the PR rate during the first stage of the VDS test correlates with motor impulsivity in the 5‐choice serial reaction time task (5‐CSRTT) [ 27 ]. Lovic et al. [ 56 ] investigated the relationship between incentive salience and impulsivity and concluded that rats that attribute higher incentive salience to reward predicting cues display more premature responses in tests of impulsive action. The suppressive effect of stress on incentive salience could therefore explain our finding of a reduced premature response rate during the first stage of the VDS test in stressed animals. During the second stage of the VDS protocol, with prolonged and variable ITIs, all stressed groups increased their PR rate to the level of CTRL, with the exception of GL‐II‐73‐treated rats, which displayed a significantly reduced PR rate compared with CTRL during the 15 s but not 9 s ITI, suggesting that this treatment improved their ability either to inhibit prepotent responses or to wait for the response signal [ 57 ]. α5‐GABA A R inverse agonists in non‐stressed rats have been found to impair attention and decrease premature responses in the 5‐CSRTT [ 58 ]. Due to the poor performance of the stressed rats in our experiment, we were unable to obtain results from the final (6 s ITI) stage of the VDS test, which previous studies have shown to be strongly correlated with behavioral impulsivity in the delay discounting task [ 27 ]. 4.3. Social Behavior Both direct and indirect social interaction was normal in the stressed groups in 3CT and RIT, respectively. The 3CT revealed impaired social recognition in all stressed groups, an effect that was ameliorated by GL‐II‐73. This is consistent with a previous study in healthy rats, in which the active metabolite of GL‐II‐73, MP‐III‐22, improved social memory [ 59 ]. Furthermore, the α2/3‐ and α5‐selective positive allosteric modulator L‐838417 alleviated impaired social behavior in a mouse model of autism [ 60 ], suggesting that modulation of GABA A receptors containing these subunits may influence social behavior in this disorder. In PTSD, abnormalities in social cognition are strongly implicated and are considered a major contributor to the difficulties patients experience in maintaining social relationships [ 2 ]. In patients, these deficits have been associated with alterations in the dorsomedial subsystem of the default mode network, which plays a key role in social cognitive processing [ 61 ]. Notably, α5‐GABA A Rs are highly expressed in hippocampal and cortical circuits involved in memory and social behavior [ 14 ]. Therefore, the beneficial effects of positive modulation of α5‐GABA A Rs in the MD+SPS model suggest that this receptor subtype may represent a therapeutic target for social dysfunction in stress‐related disorders as well. In the current study, social play was reduced in the SPS animals compared with both MD+SPS and CTRL groups, whereas MD+SPS animals displayed normal levels of this behavior. The deteriorative effect of SPS on rat social play aligns with a previous study [ 62 ]. Interestingly, stress exposure during early adolescence has been reported to promote resilience in certain behavioral domains in Sprague–Dawley rats subjected to SPS in adulthood [ 63 ]. In Wistar rats [ 23 ], a 2 × 2 design with MD and SPS as factors revealed a main effect of SPS in the social interaction test, although SPS and MD+SPS groups showed similarly reduced social interaction. The different behavioral pattern observed in Sprague–Dawley rats in the present study may reflect a slightly different testing protocol or strain‐specific effects, where conceivably MD in this strain could have acted to enhance resilience to impairments in social play after later stress (cf. [ 64 ]). 4.4. Limitations The present study should be interpreted in light of several design considerations. A maternal deprivation (MD)–only group was not included, which limits the ability to isolate the independent contribution of early‐life stress and to distinguish additive from interaction effects between MD and SPS. In addition, treatment‐only control groups were not examined, precluding assessment of potential baseline effects of the pharmacologic interventions in non‐stressed animals. Furthermore, because the study did not employ a fully factorial design including all combinations of early‐life stress, adult stress, and treatment, interaction effects between these factors could not be formally tested. Finally, the study was conducted exclusively in male rats, and therefore the findings may not necessarily generalize to females. 5. Conclusion In the VDS paradigm assessing reward‐related impulsivity, animals exposed to SPS or the combined stressors (MD+SPS) showed reduced motivation and motor impulsivity compared with control animals. Notably, stressed animals treated with positive allosteric modulators of α5‐ or α6‐containing GABA A Rs did not show the stress‐associated increase in omissions observed in vehicle‐treated MD+SPS animals. These findings suggest that modulation of these predominantly extrasynaptic receptors may exert a subtle protective influence against the profound effects of severe stress in this double‐hit stress model. Author Contributions Đorđe Đorović: writing – original draft, methodology, investigation, conceptualization. Jovana Aranđelović: writing – original draft, visualization, validation, methodology, investigation, conceptualization, formal analysis, data curation. Jana Ivanović: methodology, investigation. Kristina Jezdić: methodology, investigation. Bojan Batinić: software, methodology. Dishary Sharmin: synthesis. Prithu Mondal: synthesis. James M. Cook: writing – review and editing, supervision, resources. Miroslav M. Savić: writing – review and editing, supervision, project administration, methodology, investigation, funding acquisition, conceptualization. Funding This work was supported by Science Fund of the Republic of Serbia (Grant 7749108), National Institutes of Health (Grants R01 NS076517 and R01 MH096463), and National Science Foundation (Grant CHE‐1625735). Ethics Statement The research was approved by the Ethics Committee for Animal Experiments of the University of Belgrade—Faculty of Pharmacy, Serbia and the Ministry of Agriculture, Forestry and Water Management—Veterinary Directorate, within the project Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma‐2 receptors: In vitro/in vivo delineation by nano‐ and hiPSC‐based platforms (NanoCellEmoCog), funded by the Science Fund of the Republic of Serbia, Grant No. 7749108, approval No. 323‐07‐10 046/2020‐05, 23/09/2020. Conflicts of Interest J.M.C. and M.M.S. are co‐inventors or listed on patent applications that cover GABAergic ligands, including GL‐II‐73 and DK‐I‐56‐1, and their use in brain disorders. The authors have no other conflicts of interest or competing financial interests to declare that are relevant to the content of this article. Supporting information Figure S1: Experimental design. Figure S2: Locomotor activity (SLA). Figure S3: Reward‐related impulsivity (VDS). Figure S4: Social behavior (3CT and RIT). PRP2-14-e70245-s001.docx (315.5KB, docx) Acknowledgments This research was funded by the Science Fund of the Republic of Serbia, Grant No. 7749108, Neuroimmune aspects of mood, anxiety and cognitive effects of leads/drug candidates acting at GABAA and/or sigma‐2 receptors: In vitro/in vivo delineation by nano‐ and hiPSC‐based platform—NanoCellEmoCog. 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Supplementary Materials Figure S1: Experimental design. Figure S2: Locomotor activity (SLA). Figure S3: Reward‐related impulsivity (VDS). Figure S4: Social behavior (3CT and RIT). PRP2-14-e70245-s001.docx (315.5KB, docx) Data Availability Statement Data will be made available on request to the corresponding author. 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