Diazepam selectively biases approach during threat–reward conflict while sparing reactive avoidance

Diazepam selectively biases approach during threat–reward conflict while sparing reactive avoidance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Diazepam selectively biases approach during threat–reward conflict while sparing reactive avoidance Óscar Enciso-Pablo, Félix Sierra-Ramírez, Francisco Sotres-Bayón This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8928345/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Rationale: Benzodiazepines are widely used to treat anxiety; however, their influence on defensive behavior when threats compete with rewards remains unclear. Objectives Here, we tested the effects of diazepam on reactive avoidance (no conflict), threat–reward conflict resolution, and behavioral flexibility. Methods Using an integrated platform-mediated avoidance paradigm in female and male rats, we assessed the effects of a low dose systemically administered diazepam (1 mg/kg). Results Diazepam did not alter the reactive avoidance memory expression when threat cues were presented without a concurrent reward cue. In contrast, during the cued threat–reward conflict, diazepam reduced platform avoidance and increased reward engagement, shifting the avoidance/approach balance toward the approach. Diazepam also impaired behavioral flexibility during the reversal from conflict to reactive avoidance, maintaining an approach-dominant strategy when avoidance should be reinstated. Sex differences were observed selectively during the flexibility assessment, reflecting a quantitatively stronger diazepam-induced shift toward reward engagement in males compared with females. Control assays showed no significant effects of diazepam on threat or reward memory retrieval, sucrose intake, open-field locomotion, or anxiety-like behavior. Conclusions These findings indicate that diazepam selectively reshapes approach–avoidance arbitration under motivational competition and alters subsequent flexibility while sparing reactive avoidance. Diazepam Approach–avoidance conflict Behavioral flexibility Decision-making Active avoidance Sex difference Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Benzodiazepines such as diazepam remain a cornerstone in the pharmacological treatment of anxiety and related disorders, acting primarily through enhancement of GABAergic inhibition (Shader and Greenblatt 1993 ; Benson et al. 1998 ; Rudolph and Knoflach 2011 ; Möhler 2012 ; Griebel and Holmes 2013 ). Although their clinical efficacy is well established, the behavioral mechanisms through which benzodiazepines influence motivated behavior remain unclear. Notably, benzodiazepines have also been characterized by ‘anticonflict’ effects in punished-responding assays, increasing approach behavior even when it is associated with threat or punishment (Vogel et al. 1971 ; File 1985 ). In addition to modulating threat-related defensive responses, these drugs can influence decisions that involve weighing potential threats against rewards (Hartley and Phelps 2012 ; Calhoon and Tye 2015 ). However, it remains unclear whether benzodiazepines primarily dampen avoidance responses across conditions or bias behavior, specifically when avoidance and approach motives compete. The platform-mediated avoidance (PMA) paradigm provides a framework for dissociating learned avoidance from conflict-driven decision-making under approach-avoidance conditions. In its traditional configuration, PMA measures active avoidance elicited by a conditioned threat cue in the absence of explicit competing reward cues (Bravo-Rivera et al. 2014 , 2015 ; Diehl et al. 2018 , 2019 , 2020 ; Velazquez-Hernandez and Sotres-Bayon 2021 ; Halcomb et al. 2024 ; López-Moraga et al. 2024 , 2025 , 2026 ; Ruble et al. 2024a , b ; Kramer et al. 2025 ; Enriquez-Traba et al. 2025 ; St Laurent et al. 2025 ; Zeidler et al. 2025 ). More recent extensions incorporate the simultaneous presentation of threat and reward cues, generating a motivational conflict that requires the integration of defensive and appetitive drives (Bravo-Rivera and Sotres-Bayon, 2020 ; Bravo-Rivera et al., 2021 ). Importantly, recent work has emphasized that approach–avoidance conflict is not well captured by averaged behavioral measures but instead reflects the emergence of distinct behavioral strategies, providing a framework in which pharmacological manipulations may bias the balance between avoidance- and reward-directed responses (Bravo-Rivera et al., 2021 ; Illescas-Huerta et al., 2021 ). This conflict version of the PMA enables the quantification of behavioral allocation toward avoidance or approach and provides an opportunity to assess behavioral flexibility when contingencies change, a process that is often disrupted in anxiety- and stress-related conditions (Hartley and Phelps 2012 ). To capture these shifts, avoidance and approach can be quantified in parallel and summarized using an avoidance/approach balance index derived from the time allocated to the safety platform versus the reward zone. Here, we used an integrated PMA design to examine how diazepam modulates reactive avoidance (no conflict), threat–reward conflict resolution, and behavioral flexibility in adult male and female rats. Diazepam (1 mg/kg) was selected based on prior evidence showing minimal effects on locomotion, motor coordination, and basal anxiety-like behavior while allowing the detection of behavioral modulation under approach–avoidance conflict conditions (Illescas-Huerta et al., 2021 ). We hypothesized that diazepam would spare learned avoidance when behavior is guided by a single threat cue, bias behavior toward reward during threat–reward conflict, and impair behavioral flexibility during the reversal from conflict to reactive avoidance. Control assays were included to rule out non-specific effects on locomotion, feeding, or memory retrieval, which could otherwise account for shifts in approach–avoidance allocation. Using this integrated design, we show that diazepam spares reactive avoidance expression under no-conflict conditions but shifts behavioral allocation toward reward during threat–reward conflict and alters subsequent behavioral flexibility. METHODS Subjects. A total of 72 adult Wistar rats (3 months of age), equally distributed by sex, were obtained from the breeding colony of the Institute of Cellular Physiology and used in this study to investigate potential sex-dependent effects of diazepam´s modulation of motivated behaviors. At the beginning of the experiments, the females weighed 240–290 g (n = 36) and the males weighed 280–320 g (n = 36). The animals were individually housed under a 12 h light/dark cycle with ad libitum access to water. To motivate reward-seeking behavior, rats were food restricted (12–16 g/day of standard laboratory chow) to maintain body weight at approximately 85% of baseline; food allowance was increased by 5 g/week to allow for normal growth. All experimental procedures were approved by the Bioethics Committee of the Institute of Cellular Physiology, National Autonomous University of Mexico, and were conducted in accordance with institutional and national guidelines. Platform-mediated avoidance task A schematic representation of the platform-mediated avoidance task and experimental timeline is shown in Fig. 1 . The PMA task was used to dissociate reactive avoidance from decision-making in a threat–reward conflict. In the traditional PMA task, threat cues are presented in the absence of concurrent reward cues, resulting in minimal competition between avoidance and approach behaviors. This condition is referred to as reactive avoidance. In contrast, threat–reward conflict is generated by the simultaneous presentation of threat and reward cues, producing a genuine motivational conflict (Bravo-Rivera et al., 2021 ). An integrated design was used in which the same animals underwent reactive avoidance training and testing, followed by threat–reward conflict training and testing. A separate group was used for the flexibility (reversal) memory test. Reactive avoidance training and memory test Phase 1: Reward learning. Rats were trained to press a lever to obtain sucrose pellets (Bio-Serv) under a variable-interval schedule (VI-30). The animals received five daily training sessions and reached a minimum response rate of 12 presses/minute during the final session. Phase 2: Cued threat learning. Avoidance training was conducted as previously described (Bravo-Rivera et al. 2014 ) using standard operant conditioning chambers (Coulbourn Instruments) housed in sound-attenuating enclosures (Med Associates). The chamber floor consisted of stainless-steel bars capable of delivering foot shocks. Rats were conditioned to a tone (30 s, 4 kHz, 75 dB) that co-terminated with a foot shock (2 s, 0.4 mA). Animals received nine tone–shock pairings per session at variable intervals averaging 180 s for 10 consecutive days. Sucrose pellets were available throughout the training period via lever pressing on a VI-30 schedule. A square safety platform (14.0 × 14.0 cm) was positioned in the corner opposite the lever, allowing the rats to avoid foot shocks. Each session included an initial and final 5 min period without tone presentations to maintain lever responding and minimize contextual fear. Reactive avoidance memory test : Twenty-four hours after completion of training, rats were presented with two tone cues in the absence of footshock (intertone interval: 180 s), with sucrose pellets available under a VI-30 reinforcement schedule. This setup required animals to revert from the conflict strategy to reactive avoidance. The test was conducted 30 min after diazepam or vehicle administration was performed. Threat–reward conflict training and memory test Phase 1: Cued reward learning. On the day following the reactive avoidance memory test, the rats were placed in the same chambers with sucrose pellets available only during 30 s periods, signaled by illuminating the cue light above the lever. Each lever press was reinforced with a single sucrose pellet. Sessions consisted of 20 trials with inter-trial intervals of approximately 180 s and were conducted once daily for three days. Phase 2: Conflict learning. Conflict training commenced 24 h after completing the reward training. During each trial, threat (tone) and reward (light) cues were presented for 30 s each. The animals received nine tone–light co-presentation trials per session, with inter-trial intervals averaging 180 s. Training continued for 15 consecutive days to allow for the stabilization of the behavioral strategy. Each session included initial and final 5 min periods without cue presentations. Threat–reward conflict memory test. Twenty-four hours after the final training session, the rats were presented with two simultaneous tone–light cue presentations in the absence of foot shock (inter-trial interval: 180 s). The test was conducted 30 min after diazepam or vehicle administration was performed. Flexibility (reversal) memory test : An independent group of rats was trained using the same reactive avoidance and threat–reward conflict protocol but did not receive diazepam or vehicle during the prior testing. Twenty-four hours after completion of the threat–reward conflict memory test, the animals were returned to the conditioning chambers and subjected to a reactive avoidance test consisting of two-tone presentations (30 s each) in the absence of foot shock (intertone interval: 180 s), with sucrose pellets available under a VI-30 schedule. This test assessed behavioral flexibility (reversal learning), defined as the ability to reinstate avoidance behavior following a rule change from threat–reward conflict to reactive avoidance. The test was conducted 30 min after diazepam or vehicle administration. Control behavioral tests Threat conditioning memory retrieval test. Auditory threat conditioning was performed in standard operant chambers in a separate cohort of animals. On day 1, the rats received five tone presentations (30 s, 4 kHz, 75 dB), each co-terminating with a foot shock (2 s, 0.4 mA), with intertone intervals averaging 120 s. On day 2, memory retrieval was assessed using two tone presentations in the absence of a foot shock. The freezing behavior was then quantified. Testing was conducted 30 min after diazepam or vehicle administration. Open-field test. Locomotor activity and anxiety-like behavior were assessed in a modified open-field arena (90 × 90 × 60 cm) located in a dark testing room with separate cohorts of rats. The arena consisted of a safe peripheral zone (60 × 60 cm) and a central threat zone (30 × 30 cm) illuminated with high-intensity light (1500 lx). The rats were placed at the center, and their behavior was recorded for 5 min. The total distance traveled and mean velocity were used as measures of locomotion, and the time spent in the center was used as an index of anxiety-like behaviors. Testing was performed 30 min after the drug administration. Instrumental conditioning memory reward test : Rats were placed in the conditioning chambers and presented with two light cues (30 inter-trial interval: 180 s). These rats were used for the flexibly experiment. Lever presses during cue presentation were quantified. Testing was conducted 30 min after diazepam or vehicle administration was conducted. Food intake test. To assess basal motivation for reward consumption, rats were placed in their home cage with access to 3 g of sucrose pellets for 1 min, starting from their first approach to the food dish. These rats were used for the flexibly experiment. Pellet consumption (g) was quantified. Testing was performed 30 min after drug administration. Systemic drug administration Diazepam (DZP; 1 mg/kg, s.c. Valium ® (Roche, Mexico)) was dissolved in saline (5 mg/ml) and administered via subcutaneous injection 30 min before behavioral testing. The control animals received an equivalent volume of saline (vehicle). The selected dose was based on a prior study showing that 1 mg/kg produces reliable anxiolytic effects without sedative or locomotor impairment in rats tested in approach–avoidance conflict paradigms, whereas a higher dose (2 mg/kg) can induce motor suppression and reduce task engagement (Illescas-Huerta et al., 2021 ). During the pre-test period, the animals remained in their home cages without access to food or water. Data collection and analysis All behavioral responses were recorded using digital video cameras (Logitech) and automatically analyzed using a commercial software (ANY-maze; Stoelting). Behavioral measures were extracted during cue presentation and during predefined time windows before and after cue onset, as specified for each behavioral test. All behavioral measures reflected the average of the two cue presentations, whereas the number of platform entries represented the cumulative total across both presentations. Avoidance behavior was quantified as the percentage of time spent on the safety platform, number of platform entries, and latency to enter the platform for the first time after the footshock. Time-resolved avoidance dynamics were assessed by computing platform occupancy in 3-s bins before, during, and after cue presentation. Approach behavior was quantified as the time spent in the reward zone (lever and food dish area) and the number of lever presses during cue presentation. To capture the overall behavioral strategy during conflict and flexibility tests, we computed an avoidance/approach balance index by combining the percentage of time spent on the safety platform (avoidance) and the percentage of time spent in the reward zone (approach). In this two-dimensional representation, greater displacement along the y-axis reflected increased avoidance, whereas greater displacement along the x-axis reflected increased approach behavior. To quantitatively summarize the relative dominance of these competing responses, a normalized balance index was calculated as (Avoidance - Approach) / (Avoidance + Approach), such that positive values indicated avoidance-dominant allocation, negative values indicated approach-dominant allocation, and values near zero reflected balanced engagement of both behaviors. This index was treated as a dependent variable and analyzed using t-tests. For the control behavioral tests, lever presses during cue presentation were used to assess instrumental reward-seeking behavior. In the open-field test, the total distance traveled and mean velocity were used as indices of locomotor activity, whereas the time spent in the center of the arena was used as an index of anxiety-like behaviors. In the threat conditioning test, freezing was defined as the absence of all movements except respiration and was expressed as a percentage of the tone duration spent freezing. For the reward intake test, sucrose consumption (g) was quantified for a 1-min period. Statistical analyses were performed using commercial software (Prism; GraphPad). Group comparisons of mean behavioral measures were analyzed using unpaired two-tailed Student’s t-tests or two-way ANOVAs, as appropriate. Time-resolved behavioral dynamics were analyzed using a two-way repeated-measures ANOVA with treatment and time as factors. Sex-dependent effects were analyzed using two-way ANOVA, with sex and treatment as factors. Post-hoc comparisons were conducted using the Bonferroni multiple comparison test when appropriate. Statistical significance was sep at P < 0.05. All data are presented as the mean ± SEM. RESULTS An overview of the platform-mediated avoidance (PMA) task and the integrated experimental design used in this study are shown in Fig. 1 . Diazepam does not alter reactive avoidance (no-conflict) memory expression We first examined whether diazepam affected the expression of reactive avoidance under conditions involving minimal competition between threats and rewards (Fig. 2 ). During the reactive avoidance memory test, diazepam did not alter the time spent on the safety platform during tone presentation compared to vehicle-treated animals (Fig. 2 A, left ; VEH, n = 16; DZP, n = 16; t(30) = 0.39, p = 0.69). Similarly, the number of platform entries during tone presentation did not differ between the groups (Fig. 2 A, right ; t(30) = 1.10, p = 0.27). Analysis of avoidance dynamics revealed comparable temporal profiles of platform occupancy before, during, and after tone presentation in vehicle- and diazepam-treated rats (Fig. 2 B, left panel). Two-way repeated-measures ANOVA revealed no main effect of treatment in the pre-tone (F(1, 30) = 1.12, p = 0.30), tone (F(1, 30) = 0.16, p = 0.57), or post-tone periods (F(1, 30) = 0.16, p = 0.57). Likewise, the treatment × time interaction was not significant in any phase (pre-tone: F(9, 270) = 1.49, p = 0.15; tone: F(9, 270) = 0.85, p = 0.69; post-tone: F(9, 270) = 0.85, p = 0.69), indicating that diazepam did not affect the temporal pattern of reactive avoidance response. Consistent with this, the latency to the first platform entry during the tone was not affected by diazepam administration (Fig. 2 B, right ; t(30) = 0.43, p = 0.66). Diazepam did not alter the approach-related measures during reactive avoidance. The time spent in the reward zone and the number of lever presses during tone presentation were comparable between vehicle- and diazepam-treated animals (Fig. 2 C; t(30) = 0.66, p = 0.51 reward zone ; t(30) = 0.67, p = 0.50 lever presses ). Representative movement tracking and occupancy heat maps further illustrated similar spatial allocation patterns across the groups (Fig. 2 D). Accordingly, the integrated avoidance/approach balance index did not differ between the treatments under reactive avoidance conditions (Fig. 2 E, t(30) = 0.30, p = 0.76). Together, these results indicate that diazepam does not disrupt the expression of reactive avoidance when threat cues are presented without concurrent cue rewards. Diazepam biases behavior toward reward during threat–reward conflict Next, we assessed the effects of diazepam during the threat–reward conflict, in which threat and reward cues were presented simultaneously (Fig. 3 ). Under these conditions, diazepam significantly reduced avoidance behavior, as reflected by the decreased time spent on the safety platform during cue co-presentation compared to vehicle-treated animals (Fig. 3 A, left ; t(30) = 6.92, p < 0.0001). The number of platform entries was also reduced following diazepam administration (Fig. 3 A, right ; t(30) = 3.35, p = 0.0022). Time-resolved analysis revealed that diazepam markedly attenuated the sustained platform occupancy during the conflict period (Fig. 3 B, left ). In the conflict condition, diazepam significantly altered avoidance across the pre-tone, tone, and post-tone periods. Two-way repeated-measures ANOVA revealed a significant main effect of treatment during the pre-tone (F(1, 30) = 6.70, p = 0.015), tone (F(1, 30) = 48.03, p < 0.0001), and post-tone periods (F(1, 30) = 8.14, p = 0.0078). The treatment × time interaction was not significant during the pre-tone phase (F(9, 270) = 0.87, p = 0.55), but was robustly significant during the tone (F(9, 270) = 8.51, p < 0.0001) and post-tone periods (F(9, 270) = 3.51, p = 0.0004), indicating that diazepam dynamically shifted the temporal pattern of avoidance responding during the threat–reward conflict. In addition, diazepam increased the latency to the first platform entry during conflict trials (Fig. 3 B, right ; t(30) = 5.41, p < 0.0001), consistent with reduced avoidance engagement. In contrast to its effects on avoidance, diazepam enhanced approach-related behavior during the threat–reward conflict. Diazepam-treated animals spent more time in the reward zone and exhibited increased lever pressing during cue co-presentation compared to vehicle-treated controls (Fig. 3 C; t(30) = 6.64, p < 0.0001, reward zone ; t(30) = 3.01, p = 0.0052, lever presses ).Representative tracking data and heat maps illustrated a shift away from the safety platform and toward the reward area after diazepam treatment (Fig. 3 D). Consistent with these effects, the avoidance/approach balance index revealed a robust shift toward approach-dominant behavior in diazepam-treated rats compared to controls (Fig. 3 E; t(30) = 7.19, p < 0.0001 ). These findings demonstrate that diazepam selectively biases behavior toward reward engagement when threats and rewards compete. Diazepam impairs behavioral flexibility during reversal from conflict to reactive avoidance To determine whether diazepam affected the ability to adapt behavior following a rule change, we assessed performance in a flexibility (reversal) memory test in which animals were required to reinstate avoidance behavior after threat–reward conflict training (Fig. 4 ). During this test, diazepam-treated rats spent significantly less time on the safety platform than vehicle-treated animals (Fig. 4 A, left ; VEH, n = 12; DZP, n = 12; t(22) = 3.40, p = 0.0025), whereas the number of platform entries did not differ between the groups (Fig. 4 A, right ; t(22) = 0.51, p = 0.60). Time-resolved analysis revealed attenuated avoidance expression across the tone and post-tone periods in diazepam-treated animals compared to that in controls (Fig. 4 B). During the flexibility assessment, diazepam produced phase-specific effects on avoidance response. Two-way repeated-measures ANOVA showed no significant main effect of treatment during the pre-tone period (F(1, 22) = 3.79, p = 0.064), and the treatment × time interaction was also not significant (F(9, 198) = 1.88, p = 0.057). In contrast, during the tone period, diazepam significantly affected avoidance (F(1, 22) = 11.85, p = 0.0023), and the treatment × time interaction was significant (F(9, 198) = 2.76, p = 0.0047). In the post-tone period, the main effect of treatment was not significant (F(1, 22) = 2.04, p = 0.17), but the treatment × time interaction remained robust (F(9, 198) = 4.03, p < 0.0001), indicating persistent temporal modulation of avoidance dynamics during the flexibility testing. However, the latency to the first platform entry during the tone was not affected by diazepam administration (Fig. 2 B, right ; t(22) = 1.18, p = 0.072). In parallel, diazepam increased approach-related behavior during the flexibility test, as reflected by greater reward zone occupancy and lever pressing compared with vehicle-treated rats (Fig. 4 C; t(22) = 3.70, p = 0.0012, reward zone; t(22) = 3.52, p = 0.001,9 lever presses) ). Representative movement tracking and heat maps illustrated impaired reinstatement of avoidance after diazepam administration (Fig. 4 D). The avoidance/approach balance index confirmed a persistent shift toward approach-dominant behavior in diazepam-treated animals during the flexibility test ( Fig. 4 E; t(22) = 3.68, p = 0.0013), consistent with reduced behavioral flexibility. Pre-test balance measures were comparable across groups prior to each memory test ( Supplementary Figure S1 ), indicating that the treatment effects reflected altered behavioral allocation during testing rather than pre-existing baseline group differences. Sex-dependent effects of diazepam across behavioral conditions Next, we examined whether females and males differed in their avoidance/approach balance within each behavioral condition ( Fig. 5 ). Balance index values were comparable between females and males during the reactive avoidance memory test under both vehicle and diazepam treatment ( Fig. 5 A; t(14) = 1.23, p = 0.23, vehicle; t(14) = 0.23, p = 0.81, diazepam) and during the threat–reward conflict within each treatment condition (Fig. 5 B; t(14) = 0.80, p = 0.43, vehicle; t(14) = 0.47, p = 0.64, diazepam). In contrast, during the flexibility (reversal) memory test, the balance index differed between females and males under diazepam treatment but not under vehicle treatment (Fig. 5 C; t(10) = 2.09, p = 0.07, vehicle; t(10) = 3.37, p = 0.007 diazepam). This finding indicates sex-dependent modulation of behavioral allocation, specifically during the reversal from conflict to reactive avoidance. Decomposition of balance index components suggested that, under diazepam, males exhibited a stronger reduction in avoidance output together with a larger increase in reward engagement compared with females, consistent with a quantitatively stronger shift toward approach during flexibility rather than a qualitatively different behavioral pattern across sexes ( Supplementary Figure S2 ). Control behavioral tests To rule out non-specific explanations for the diazepam-induced reallocation of avoidance and approach behaviors, we assessed additional behavioral parameters. Diazepam did not significantly alter conditioned freezing during threat memory retrieval, lever pressing during reward memory retrieval, sucrose intake, open-field locomotion, or anxiety-like behavior ( Fig. 6 A–D; all p-values > 0.05 ). These findings indicate that diazepam-induced shifts in the PMA task were not specific changes attributed to locomotion, baseline motivation, or threat-related defensive response. DISCUSSION In the present study, we examined how diazepam modulates reactive avoidance (no conflict), threat–reward conflict resolution, and behavioral flexibility using an integrated PMA paradigm. Our results show that a low dose of diazepam (1 mg/kg) does not disrupt the expression of learned reactive avoidance when behavior is guided by threat cues. In contrast, when threat and reward cues were presented concurrently, diazepam biased behavior toward reward engagement, reduced avoidance, and shifted the avoidance/approach balance toward the approach. This selective behavioral effect extended to a flexibility test in which diazepam disrupted adaptive updating following conflict training, producing a persistent approach-dominant allocation instead of reinstating avoidance. Notably, sex-dependent differences emerged specifically during flexibility, indicating that the reversal from conflict to reactive avoidance may be differentially sensitive to diazepam in males and females. A central finding was the dissociation between preserved reactive avoidance and altered behavior under threat–reward conflict. Traditional descriptions of benzodiazepine action often emphasize anxiolysis as a reduction in threat-related defensive responding (Blanchard et al. 1990 ; Zhao et al. 2018 ; Groenink et al. 2023 ). However, diazepam did not suppress defensive responses across conditions. Instead, its effects emerged specifically when animals were required to arbitrate between competing motivational needs. Under conflict, rats must evaluate the relative costs and benefits of avoidance versus reward pursuit, and diazepam shifts this evaluation toward approach and reward-seeking. This pattern is consistent with frameworks in which approach–avoidance conflict reflects a decision process involving the selection among competing strategies rather than a graded modulation of avoidance strength (Eliot, 2008; Rangel et al. 2008 ; Choi and Kim 2010 ; Mitchell et al. 2011 ; Friedman et al. 2015 ; Hamel et al. 2017 ; Piantadosi et al. 2017 ; Bercovici et al. 2018 ; Walters et al. 2019 ; Illescas-Huerta et al. 2021 ; Calvin et al. 2025 ), and supports the view that conflict engages additional cognitive and motivational control operations beyond those required for reactive avoidance (Botvinick et al. 2001 ; Aupperle and Paulus 2010 ; Hartley and Phelps 2012 ; Aupperle et al. 2015 ; Calhoon and Tye 2015 ; Jackson et al. 2016 ). Importantly, the behavioral reallocation observed under diazepam cannot readily explain the non-specific changes in locomotion, basal motivation, or global suppression of defensive responses. In the present dataset, diazepam did not significantly alter conditioned freezing during threat memory retrieval, lever pressing during reward memory retrieval, sucrose intake, open-field locomotion or anxiety-like behavior. These results complement those of previous studies showing that this dose does not induce sedation or motor impairment in standard assays (Savić et al., 2009 ; Illescas-Huerta et al., 2021 ; Pádua-Reis et al., 2021 ). Together, these results support the interpretation that diazepam primarily modulates how threat-related information constrains action selection during motivational competition rather than disrupting threat memory retrieval per se or broadly disinhibiting behavior. In addition to conflict resolution, diazepam alters the behavioral flexibility. During the reversal-like flexibility memory test, diazepam reduced platform occupancy and maintained a shift toward an approach-dominant allocation. While benzodiazepines are typically studied for acute anxiolytic actions, this result suggests that enhancing GABAergic inhibition can also bias animals toward the persistence of reward-directed allocation following prior conflict experience, reducing adaptive updating when task contingencies require reinstatement of avoidance. This observation aligns with the broader idea that stress- and anxiety-related states can bias behavioral selection and updating (Aupperle and Paulus 2010 ; Smith et al. 2012 ; Park and Moghaddam 2017 ). However, in the present task, the drug effect manifested as reduced adaptation across motivational contexts, rather than a generalized performance deficit. In the present study, we also observed sex-dependent differences in the effects of diazepam on behavioral flexibility. Whereas females and males showed comparable balance indices during reactive avoidance and threat–reward conflict, the balance index diverged by sex during the flexibility memory test. The decomposition of the balance index into avoidance and approach components suggests that this difference reflects a quantitatively stronger diazepam-induced shift toward reward engagement in males rather than a qualitatively distinct behavioral pattern across sexes. These findings add to the evidence that sex can modulate the pharmacological effects on motivated behavior and decision processes (Orsini et al., 2016 ; Islas-Preciado et al., 2020 ; Ecevitoglu et al., 2024 ; Faraji et al. 2025 ) and highlight the value of explicitly considering sex as a biological variable when characterizing the effects of drugs on flexibility under threat. In summary, these findings refine our understanding of how benzodiazepines influence defensive behaviors in complex motivational settings. Rather than broadly reducing threat-related defensive responding or avoidance, diazepam selectively biases behavioral allocation during threat–reward conflict and alters subsequent flexibility while maintaining reactive avoidance. This dissociation suggests that benzodiazepines may alleviate anxiety-related conflict by shifting the resolution of competing motivational drives rather than by suppressing defensive responses per se. More generally, reducing the constraining influence of threat on action selection may favor reward engagement during conflict but may also promote the persistence of approach-dominant strategies when conditions require flexible reinstatement of avoidance. Future studies using dose-response approaches will be crucial to fully characterize the therapeutic window and potential side effects across a range of doses, as our current study utilized a single dose (1mg/kg). While this dose was carefully selected to avoid confounding locomotor or sedative effects, it restricts the generalizability of these specific findings to broader clinical applications and varying degrees of anxiety, as the observed behavioral specificity might be dose-dependent. Additionally, circuit-specific manipulations (e.g., targeting prefrontal–striatal and amygdala networks) will be important for identifying the neural mechanisms that enable diazepam to reshape approach–avoidance arbitration and flexibility in the face of threat. Declarations Author Contributions: OE-P and FS-B designed the study; OE-P and FS-R performed the experiments; OE-P and FS-B analyzed the data; OE-P and FS-B wrote the manuscript. Conflict of interest: The authors declare no conflicts of interest. Funding sources: Secretaría de Ciencia, Humanidades, Tecnología e Innovación (SECIHTI; grant CF-2023-I-225), Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México (UNAM; grants: IN214223 and IN226026) to FS-B. OE-P is a doctoral student at the Programa de Doctorado en Ciencias Bioquímicas at UNAM and is supported by the SECIHTI fellowship (1047792). Acknowledgements: We thank Christian Bravo-Rivera and the SotresLab for helpful discussions and comments on a previous version of the manuscript. Data availability The data supporting the findings of this study are available from the corresponding author upon reasonable request. Animal ethics approval All experimental procedures were approved by the Bioethics Committee of the Institute of Cellular Physiology, National Autonomous University of Mexico (UNAM), and were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals. Consent to participate Not applicable. Consent for publication Not applicable. References Aupperle RL, Melrose AJ, Francisco A et al (2015) Neural substrates of approach-avoidance conflict decision-making. Hum Brain Mapp 36:449–462. https://doi.org/10.1002/hbm.22639 Aupperle RL, Paulus MP (2010) Neural systems underlying approach and avoidance in anxiety disorders. Dialogues Clin Neurosci 12:517–531. https://doi.org/10.31887/DCNS.2010.12.4 Benson JA, Löw K, Keist R et al (1998) Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits. FEBS Lett 431:400–404. https://doi.org/10.1016/s0014-5793(98)00803-5 Bercovici DA, Princz-Lebel O, Tse MT et al (2018) Optogenetic Dissection of Temporal Dynamics of Amygdala-Striatal Interplay during Risk/Reward Decision Making. https://doi.org/10.1523/ENEURO.0422-18.2018 . eNeuro 5:ENEURO.0422-18.2018 Blanchard DC, Blanchard RJ, Tom P, Rodgers RJ (1990) Diazepam changes risk assessment in an anxiety/defense test battery. Psychopharmacology 101:511–518. https://doi.org/10.1007/BF02244230 Botvinick MM, Braver TS, Barch DM et al (2001) Conflict monitoring and cognitive control. Psychol Rev 108:624–652. https://doi.org/10.1037/0033-295x.108.3.624 Bravo-Rivera C, Roman-Ortiz C, Brignoni-Perez E et al (2014) Neural Structures Mediating Expression and Extinction of Platform-Mediated Avoidance. J Neurosci 34:9736–9742. https://doi.org/10.1523/JNEUROSCI.0191-14.2014 Bravo-Rivera C, Roman-Ortiz C, Montesinos-Cartagena M, Quirk GJ (2015) Persistent active avoidance correlates with activity in prelimbic cortex and ventral striatum. Front Behav Neurosci 9:184. https://doi.org/10.3389/fnbeh.2015.00184 Bravo-Rivera C, Sotres-Bayon F (2020) From Isolated Emotional Memories to Their Competition During Conflict. https://doi.org/10.3389/fnbeh.2020.00036 . Front Behav Neurosci 14: Bravo-Rivera H, Rubio Arzola P, Caban-Murillo A et al (2021) Characterizing Different Strategies for Resolving Approach-Avoidance Conflict. Front Neurosci 15:608922. https://doi.org/10.3389/fnins.2021.608922 Calhoon GG, Tye KM (2015) Resolving the neural circuits of anxiety. Nat Neurosci 18:1394–1404. https://doi.org/10.1038/nn.4101 Calvin OL, Erickson MT, Walters CJ, Redish AD (2025) Dorsal hippocampus represents locations to avoid as well as locations to approach during approach-avoidance conflict. PLoS Biol 23:e3002954. https://doi.org/10.1371/journal.pbio.3002954 Choi J-S, Kim JJ (2010) Amygdala regulates risk of predation in rats foraging in a dynamic fear environment. Proceedings of the National Academy of Sciences 107:21773–21777. https://doi.org/10.1073/pnas.1010079108 Diehl MM, Bravo-Rivera C, Quirk GJ (2019) The study of active avoidance: A platform for discussion. Neurosci Biobehavioral Reviews 107:229–237. https://doi.org/10.1016/j.neubiorev.2019.09.010 Diehl MM, Bravo-Rivera C, Rodriguez-Romaguera J et al (2018) Active avoidance requires inhibitory signaling in the rodent prelimbic prefrontal cortex. Elife 7:e34657. https://doi.org/10.7554/eLife.34657 Diehl MM, Iravedra-Garcia JM, Morán-Sierra J et al (2020) Divergent projections of the prelimbic cortex bidirectionally regulate active avoidance. Elife 9:e59281. https://doi.org/10.7554/eLife.59281 Ecevitoglu A, Beard KR, Srynath S et al (2024) Pharmacological characterization of sex differences in the effects of dopaminergic drugs on effort-based decision making in rats. Psychopharmacology 241:2033–2044. https://doi.org/10.1007/s00213-024-06615-8 Elliot AJ (2008) Handbook of approach and avoidance motivation. Psychology, New York Enriquez-Traba J, Arenivar M, Yarur-Castillo HE et al (2025) Dissociable control of motivation and reinforcement by distinct ventral striatal dopamine receptors. Nat Neurosci 28:105–121. https://doi.org/10.1038/s41593-024-01819-9 Faraji M, Bizon JL, Setlow B (2025) A novel cost-benefit decision-making task involving cued punishment: Effects of sex and psychostimulant administration. Behav Brain Res 495:115781. https://doi.org/10.1016/j.bbr.2025.115781 File SE (1985) Tolerance to the behavioral actions of benzodiazepines. NeurosciBiobehav Rev 9:113–121. https://doi:10.1016/0149-7634(85)90037-5 Friedman A, Homma D, Gibb LG et al (2015) A Corticostriatal Path Targeting Striosomes Controls Decision-Making under Conflict. Cell 161:1320–1333. https://doi.org/10.1016/j.cell.2015.04.049 Griebel G, Holmes A (2013) 50 years of hurdles and hope in anxiolytic drug discovery. Nat Rev Drug Discov 12:667–687. https://doi.org/10.1038/nrd4075 Groenink L, Verdouw PM, Zhao Y et al (2023) Pharmacological modulation of conditioned fear in the fear-potentiated startle test: a systematic review and meta-analysis of animal studies. Psychopharmacology 240:2361–2401. https://doi.org/10.1007/s00213-022-06307-1 Halcomb CJ, Philipp TR, Dhillon PS et al (2024) Sex divergent behavioral responses in platform-mediated avoidance and glucocorticoid receptor blockade. Psychoneuroendocrinology 159:106417. https://doi.org/10.1016/j.psyneuen.2023.106417 Hamel L, Thangarasa T, Samadi O, Ito R (2017) Caudal Nucleus Accumbens Core Is Critical in the Regulation of Cue-Elicited Approach-Avoidance Decisions. https://doi.org/10.1523/ENEURO.0330-16.2017 . eNeuro 4:ENEURO.0330-16.2017 Hartley CA, Phelps EA (2012) Anxiety and Decision-Making. Biol Psychiatry 72:113–118. https://doi.org/10.1016/j.biopsych.2011.12.027 Illescas-Huerta E, Ramirez-Lugo L, Sierra RO et al (2021) Conflict Test Battery for Studying the Act of Facing Threats in Pursuit of Rewards. Front Neurosci 15:645769. https://doi.org/10.3389/fnins.2021.645769 Islas-Preciado D, Wainwright SR, Sniegocki J et al (2020) Risk-based decision making in rats: Modulation by sex and amphetamine. Horm Behav 125:104815. https://doi.org/10.1016/j.yhbeh.2020.104815 Jackson F, Nelson BD, Hajcak G (2016) The uncertainty of errors: Intolerance of uncertainty is associated with error-related brain activity. Biol Psychol 113:52–58. https://doi.org/10.1016/j.biopsycho.2015.11.007 Kramer C, Ruble S, Fort TD et al (2025) Modifying the platform-mediated avoidance task: A new protocol to study active avoidance within a social context in rats. PLoS ONE 20:e0321776. https://doi.org/10.1371/journal.pone.0321776 Li CJ, Pineda D, Reimer AE et al (2025) Sex Based Differences in Active Avoidance and Approach Strategy in the Platform Mediated Avoidance Task. https://doi.org/10.1101/2025.10.01.679640 . bioRxiv 2025.10.01.679640 López-Moraga A, De Ceuninck M, Van der Heyden Y et al (2026) Differential effects of chronic restraint stress on two active avoidance tasks in rats. Prog Neuropsychopharmacol Biol Psychiatry 144:111586. https://doi.org/10.1016/j.pnpbp.2025.111586 López-Moraga A, Luyten L, Beckers T (2024) A history of avoidance does not impact extinction learning in male rats. NPJ Sci Learn 9:11. https://doi.org/10.1038/s41539-024-00223-z López-Moraga A, Luyten L, Beckers T (2025) Generalization and extinction of platform-mediated avoidance in male and female rats. Sci Rep 15:9730. https://doi.org/10.1038/s41598-025-94265-x Mitchell MR, Vokes CM, Blankenship AL et al (2011) Effects of acute administration of nicotine, amphetamine, diazepam, morphine, and ethanol on risky decision-making in rats. Psychopharmacology 218:703–712. https://doi.org/10.1007/s00213-011-2363-8 Möhler H (2012) The GABA system in anxiety and depression and its therapeutic potential. Neuropharmacology 62:42–53. https://doi.org/10.1016/j.neuropharm.2011.08.040 Orsini CA, Willis ML, Gilbert RJ et al (2016) Sex differences in a rat model of risky decision-making. Behav Neurosci 130:50–61. https://doi.org/10.1037/bne0000111 Pádua-Reis M, Nôga DA, Tort ABL, Blunder M (2021) Diazepam causes sedative rather than anxiolytic effects in C57BL/6J mice. Sci Rep 11:9335. https://doi.org/10.1038/s41598-021-88599-5 Park J, Moghaddam B (2017) Impact of anxiety on prefrontal cortex encoding of cognitive flexibility. Neuroscience 345:193–202. https://doi.org/10.1016/j.neuroscience.2016.06.013 Piantadosi PT, Yeates DCM, Wilkins M, Floresco SB (2017) Contributions of basolateral amygdala and nucleus accumbens subregions to mediating motivational conflict during punished reward-seeking. Neurobiol Learn Mem 140:92–105. https://doi.org/10.1016/j.nlm.2017.02.017 Rangel A, Camerer C, Montague PR (2008) Neuroeconomics: The neurobiology of value-based decision-making. Nat Rev Neurosci 9:545–556. https://doi.org/10.1038/nrn2357 Ruble S, Kramer C, West L et al (2024a) Active avoidance recruits the anterior cingulate cortex regardless of social context in male and female rats. https://doi.org/10.21203/rs.3.rs-3750422/v2 . Res Sq rs.3.rs-3750422 Ruble S, Payne K, Kramer C et al (2024b) Social context modulates active avoidance: Contributions of the anterior cingulate cortex in male and female rats. Neurobiol Stress 34:100702. https://doi.org/10.1016/j.ynstr.2024.100702 Rudolph U, Knoflach F (2011) Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 10:685–697. https://doi.org/10.1038/nrd3502 Savić MM, Milinković MM, Rallapalli S et al (2009) The differential role of alpha1-and alpha5-containing GABA(A) receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats. Int J Neuropsychopharmacol 12:1179–1193. https://doi.org/10.1017/S1461145709000108 Shader RI, Greenblatt DJ (1993) Use of benzodiazepines in anxiety disorders. N Engl J Med 328:1398–1405. https://doi.org/10.1056/NEJM199305133281907 Smith KS, Engin E, Meloni EG, Rudolph U (2012) Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice. Neuropharmacology 63:250–258. https://doi.org/10.1016/j.neuropharm.2012.03.001 St Laurent R, Kusche KM, Rein B et al (2025) Intercalated Amygdala Dysfunction Drives Avoidance Extinction Deficits in the Sapap3 Mouse Model of Obsessive-Compulsive Disorder. Biol Psychiatry 97:707–720. https://doi.org/10.1016/j.biopsych.2024.10.021 Velazquez-Hernandez G, Sotres-Bayon F (2021) Lateral Habenula Mediates Defensive Responses Only When Threat and Safety Memories Are in Conflict. https://doi.org/10.1523/ENEURO.0482-20.2021 . eNeuro 8:ENEURO.0482-20.2021 Vogel JR, Beer B, Clody DE (1971) A simple and reliable conflict procedure testing anti-anxiety agents. Psychopharmacologia 21:1–7. https://doi:10.1007/BF00403989 Walters CJ, Jubran J, Sheehan A et al (2019) Avoid-approach conflict behaviors differentially affected by anxiolytics: implications for a computational model of risky decision-making. Psychopharmacology 236:2513–2525. https://doi.org/10.1007/s00213-019-05197-0 Zeidler Z, Gomez MF, Gupta TA et al (2025) Dopaminergic projections to the prefrontal cortex are critical for rapid threat avoidance learning. bioRxiv 2024.05.02.592069. https://doi.org/10.1101/2024.05.02.592069 Zhao Y, Bijlsma EY, Verdouw PM et al (2018) The contribution of contextual fear in the anxiolytic effect of chlordiazepoxide in the fear-potentiated startle test. Behav Brain Res 353:57–61. https://doi.org/10.1016/j.bbr.2018.06.03 Additional Declarations No competing interests reported. Supplementary Files SupplementaryMaterial.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8928345","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596519538,"identity":"e992db16-0e85-4049-893a-5c031cb8ae88","order_by":0,"name":"Óscar Enciso-Pablo","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Óscar","middleName":"","lastName":"Enciso-Pablo","suffix":""},{"id":596519539,"identity":"fa14b100-34d4-4c07-96c1-22180ee39f40","order_by":1,"name":"Félix Sierra-Ramírez","email":"","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":false,"prefix":"","firstName":"Félix","middleName":"","lastName":"Sierra-Ramírez","suffix":""},{"id":596519540,"identity":"a6a26240-4057-4302-a565-08d19064dabc","order_by":2,"name":"Francisco Sotres-Bayón","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxUlEQVRIiWNgGAWjYPACGx72BuJVM4OINB6eAyRqOcxAvBb59v5jHz78OS/Dw36ATbqgZhuD7owE/FoMzhxmnjmz7TYPD08Cm/SMY7cZzM4QsM9AIpmZmbfhNo+9BAObNA8bUMvxBgIOmwHU8ufPOR4esJZ/QC2HCXnmBlALA9sBiBbeNiJsAfrFmLG3LRnol8Rma96+2zwE/SLf3viY4ccfO3se9sMHb/N8uy1ndiOBkMvggBHsIB6i1Y+CUTAKRsEowA0AIAc6R1w5yVYAAAAASUVORK5CYII=","orcid":"","institution":"National Autonomous University of Mexico","correspondingAuthor":true,"prefix":"","firstName":"Francisco","middleName":"","lastName":"Sotres-Bayón","suffix":""}],"badges":[],"createdAt":"2026-02-20 18:23:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8928345/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8928345/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103391822,"identity":"5536e4c2-d032-4683-b49a-a25da7b04bf1","added_by":"auto","created_at":"2026-02-25 07:57:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45928126,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated PMA design and behavioral readouts. \u003c/strong\u003e(A) Schematic of the platform-mediated avoidance (PMA) chamber depicting the safety platform, shock grid, threat cue (tone), and reward cue/light linked to lever press and sucrose delivery. (B) Traditional PMA configuration assessing reactive avoidance under no-conflict conditions and an extended configuration incorporating simultaneous threat and reward cues to generate a motivational conflict. (C) Integrated experimental timeline used in this study, including reactive avoidance, conflict, and flexibility (reversal) memory testing. (D) Behavioral measures used to quantify avoidance (platform occupancy/entries), approach (reward zone occupancy and lever presses), and avoidance/approach balance index derived from time allocation to the safety platform versus the reward zone.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/26acc8e5252ad31733fc2ffa.png"},{"id":103391780,"identity":"6ca373a2-7678-4918-b944-3f1c39c82f84","added_by":"auto","created_at":"2026-02-25 07:57:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33166440,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiazepam does not alter the expression of reactive avoidance (no conflict) memory.\u003c/strong\u003e Reactive avoidance memory test (VEH, n = 16; DZP, n = 16). (A) Time spent on the safety platform (%) and number of platform entries during tone presentation. (B) Time course of platform occupancy across pre-tone, tone, and post-tone bins (left) and latency to the first platform entry (right). (C) Approach measures: time in the reward zone (%) and lever presses during tone presentation. (D) Representative movement-tracking trajectories and occupancy heat maps. (E) Avoidance/approach relationship during reactive avoidance shown in a two-dimensional plot (reward-zone time vs. platform time), with a summary panel showing the normalized avoidance/approach balance index used for group comparisons. Data are presented as mean ± SEM, with individual data points shown.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/6dc5a9676b0e679db033ab79.png"},{"id":103391776,"identity":"1f76365d-2e7f-4941-9aad-4fae4cbac96e","added_by":"auto","created_at":"2026-02-25 07:57:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32421347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiazepam biases behavior toward rewards during threat–reward conflict.\u003c/strong\u003e Conflict memory test (VEH, n = 16; DZP, n = 16). (A) Avoidance measures: time on the safety platform (%) and number of platform entries during cue co-presentation. (B) Time course of platform occupancy across pre-, cue, and post-cue bins (left) and latency to the first platform entry (right panel). (C) Approach measures: time in the reward zone (%) and lever presses during cue co-presentation. (D) Representative movement-tracking trajectories and occupancy heat maps. (E) Avoidance/approach relationship during conflict shown in a two-dimensional plot (reward-zone time vs. platform time), with a summary panel showing the normalized avoidance/approach balance index used for the group. Data are presented as mean ± SEM, with individual data points shown for each group. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/0b9b4a4995288ac8d2cbaf31.png"},{"id":103391866,"identity":"6861274c-01c6-4b18-83be-8f1ab2473255","added_by":"auto","created_at":"2026-02-25 07:57:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":32784599,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiazepam impairs behavioral flexibility during conflict to reactive avoidance reversal. \u003c/strong\u003eFlexibility (reversal) memory test (VEH, n = 12; DZP, n = 12). (A) Avoidance measures: time spent on the safety platform (%) and number of platform entries during tone presentation. (B) Time course of platform occupancy across pre-tone, tone, and post-tone bins. (C) Approach measures: time in the reward zone (%) and lever presses during tone presentation. (D) Representative movement-tracking trajectories and occupancy heat maps. (E) Avoidance/approach relationship during flexibility shown in a two-dimensional plot (reward zone time vs. platform time), with a summary panel showing the normalized avoidance/approach balance index used for group comparisons. Data are presented as mean ± SEM, with individual data points shown. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/ace91cbe1d36ad2947c64997.png"},{"id":103391789,"identity":"ef657400-cc82-4737-a4a3-80942fd21d4b","added_by":"auto","created_at":"2026-02-25 07:57:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":33582623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex-dependent effects of diazepam on the balance index across behavioral conditions. \u003c/strong\u003eThe avoidance/approach balance index is shown separately for females and males under vehicle (females, n = 8; males, n = 8; top row) and diazepam (females, n = 8; males, n = 8; bottom row) during (A) reactive avoidance (no conflict), (B) threat–reward conflict, and (C) flexibility (reversal) memory tests. For flexibility, the group sizes were as follows: vehicle (females, n = 6; males, n = 6) and diazepam (females, n = 6; males, n = 6). Each plot includes an embedded panel displaying the normalized avoidance/approach balance index used for group comparison. Data are presented as mean ± SEM, with individual data points shown. **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/e6195c6224569a4248e6fa26.png"},{"id":103391843,"identity":"aa932551-2318-4f06-bfd0-253be689f7e3","added_by":"auto","created_at":"2026-02-25 07:57:33","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":23007834,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eControl behavioral tests. \u003c/strong\u003eDiazepam effects on control measures, including (A) threat memory retrieval (freezing; vehicle, n = 8; diazepam, n = 8), (B) reward memory retrieval (lever presses; vehicle, n = 12; diazepam, n = 12), (C) feeding behavior (sucrose pellets consumed; vehicle, n = 12; diazepam, n = 12), and (D) open-field measures (distance traveled, speed, and time in the center; vehicle, n = 8; diazepam, n = 8). Data are presented as mean ± SEM with individual data points.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/7064d147cb62d79a517a6504.png"},{"id":103391860,"identity":"fa771ae2-0b99-408e-8749-ff83e3204dec","added_by":"auto","created_at":"2026-02-25 07:57:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":446541,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8928345/v1/accef761db7cffd8ba66432f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Diazepam selectively biases approach during threat–reward conflict while sparing reactive avoidance","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBenzodiazepines such as diazepam remain a cornerstone in the pharmacological treatment of anxiety and related disorders, acting primarily through enhancement of GABAergic inhibition (Shader and Greenblatt \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Benson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Rudolph and Knoflach \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; M\u0026ouml;hler \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Griebel and Holmes \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although their clinical efficacy is well established, the behavioral mechanisms through which benzodiazepines influence motivated behavior remain unclear. Notably, benzodiazepines have also been characterized by \u0026lsquo;anticonflict\u0026rsquo; effects in punished-responding assays, increasing approach behavior even when it is associated with threat or punishment (Vogel et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; File \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1985\u003c/span\u003e). In addition to modulating threat-related defensive responses, these drugs can influence decisions that involve weighing potential threats against rewards (Hartley and Phelps \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Calhoon and Tye \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, it remains unclear whether benzodiazepines primarily dampen avoidance responses across conditions or bias behavior, specifically when avoidance and approach motives compete.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe platform-mediated avoidance (PMA) paradigm provides a framework for dissociating learned avoidance from conflict-driven decision-making under approach-avoidance conditions. In its traditional configuration, PMA measures active avoidance elicited by a conditioned threat cue in the absence of explicit competing reward cues (Bravo-Rivera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Diehl et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Velazquez-Hernandez and Sotres-Bayon \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Halcomb et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; L\u0026oacute;pez-Moraga et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2025\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2026\u003c/span\u003e; Ruble et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003eb\u003c/span\u003e; Kramer et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Enriquez-Traba et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; St Laurent et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zeidler et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). More recent extensions incorporate the simultaneous presentation of threat and reward cues, generating a motivational conflict that requires the integration of defensive and appetitive drives (Bravo-Rivera and Sotres-Bayon, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Bravo-Rivera et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eImportantly, recent work has emphasized that approach\u0026ndash;avoidance conflict is not well captured by averaged behavioral measures but instead reflects the emergence of distinct behavioral strategies, providing a framework in which pharmacological manipulations may bias the balance between avoidance- and reward-directed responses (Bravo-Rivera et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Illescas-Huerta et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This conflict version of the PMA enables the quantification of behavioral allocation toward avoidance or approach and provides an opportunity to assess behavioral flexibility when contingencies change, a process that is often disrupted in anxiety- and stress-related conditions (Hartley and Phelps \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). To capture these shifts, avoidance and approach can be quantified in parallel and summarized using an avoidance/approach balance index derived from the time allocated to the safety platform versus the reward zone.\u003c/p\u003e \u003cp\u003eHere, we used an integrated PMA design to examine how diazepam modulates reactive avoidance (no conflict), threat\u0026ndash;reward conflict resolution, and behavioral flexibility in adult male and female rats. Diazepam (1 mg/kg) was selected based on prior evidence showing minimal effects on locomotion, motor coordination, and basal anxiety-like behavior while allowing the detection of behavioral modulation under approach\u0026ndash;avoidance conflict conditions (Illescas-Huerta et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). We hypothesized that diazepam would spare learned avoidance when behavior is guided by a single threat cue, bias behavior toward reward during threat\u0026ndash;reward conflict, and impair behavioral flexibility during the reversal from conflict to reactive avoidance. Control assays were included to rule out non-specific effects on locomotion, feeding, or memory retrieval, which could otherwise account for shifts in approach\u0026ndash;avoidance allocation. Using this integrated design, we show that diazepam spares reactive avoidance expression under no-conflict conditions but shifts behavioral allocation toward reward during threat\u0026ndash;reward conflict and alters subsequent behavioral flexibility.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e \u003cb\u003eSubjects.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA total of 72 adult Wistar rats (3 months of age), equally distributed by sex, were obtained from the breeding colony of the Institute of Cellular Physiology and used in this study to investigate potential sex-dependent effects of diazepam\u0026acute;s modulation of motivated behaviors. At the beginning of the experiments, the females weighed 240\u0026ndash;290 g (n\u0026thinsp;=\u0026thinsp;36) and the males weighed 280\u0026ndash;320 g (n\u0026thinsp;=\u0026thinsp;36). The animals were individually housed under a 12 h light/dark cycle with \u003cem\u003ead libitum\u003c/em\u003e access to water. To motivate reward-seeking behavior, rats were food restricted (12\u0026ndash;16 g/day of standard laboratory chow) to maintain body weight at approximately 85% of baseline; food allowance was increased by 5 g/week to allow for normal growth. All experimental procedures were approved by the Bioethics Committee of the Institute of Cellular Physiology, National Autonomous University of Mexico, and were conducted in accordance with institutional and national guidelines.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlatform-mediated avoidance task\u003c/h2\u003e \u003cp\u003eA schematic representation of the platform-mediated avoidance task and experimental timeline is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The PMA task was used to dissociate reactive avoidance from decision-making in a threat\u0026ndash;reward conflict. In the traditional PMA task, threat cues are presented in the absence of concurrent reward cues, resulting in minimal competition between avoidance and approach behaviors. This condition is referred to as reactive avoidance. In contrast, threat\u0026ndash;reward conflict is generated by the simultaneous presentation of threat and reward cues, producing a genuine motivational conflict (Bravo-Rivera et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). An integrated design was used in which the same animals underwent reactive avoidance training and testing, followed by threat\u0026ndash;reward conflict training and testing. A separate group was used for the flexibility (reversal) memory test.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReactive avoidance training and memory test\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhase 1: Reward learning.\u003c/span\u003e Rats were trained to press a lever to obtain sucrose pellets (Bio-Serv) under a variable-interval schedule (VI-30). The animals received five daily training sessions and reached a minimum response rate of 12 presses/minute during the final session.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhase 2: Cued threat learning.\u003c/span\u003e Avoidance training was conducted as previously described (Bravo-Rivera et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) using standard operant conditioning chambers (Coulbourn Instruments) housed in sound-attenuating enclosures (Med Associates). The chamber floor consisted of stainless-steel bars capable of delivering foot shocks. Rats were conditioned to a tone (30 s, 4 kHz, 75 dB) that co-terminated with a foot shock (2 s, 0.4 mA). Animals received nine tone\u0026ndash;shock pairings per session at variable intervals averaging 180 s for 10 consecutive days. Sucrose pellets were available throughout the training period via lever pressing on a VI-30 schedule. A square safety platform (14.0 \u0026times; 14.0 cm) was positioned in the corner opposite the lever, allowing the rats to avoid foot shocks. Each session included an initial and final 5 min period without tone presentations to maintain lever responding and minimize contextual fear.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eReactive avoidance memory test\u003c/span\u003e: Twenty-four hours after completion of training, rats were presented with two tone cues in the absence of footshock (intertone interval: 180 s), with sucrose pellets available under a VI-30 reinforcement schedule. This setup required animals to revert from the conflict strategy to reactive avoidance. The test was conducted 30 min after diazepam or vehicle administration was performed.\u003c/p\u003e\n\u003ch3\u003eThreat–reward conflict training and memory test\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhase 1: Cued reward learning.\u003c/span\u003e On the day following the reactive avoidance memory test, the rats were placed in the same chambers with sucrose pellets available only during 30 s periods, signaled by illuminating the cue light above the lever. Each lever press was reinforced with a single sucrose pellet. Sessions consisted of 20 trials with inter-trial intervals of approximately 180 s and were conducted once daily for three days.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePhase 2: Conflict learning.\u003c/span\u003e Conflict training commenced 24 h after completing the reward training. During each trial, threat (tone) and reward (light) cues were presented for 30 s each. The animals received nine tone\u0026ndash;light co-presentation trials per session, with inter-trial intervals averaging 180 s. Training continued for 15 consecutive days to allow for the stabilization of the behavioral strategy. Each session included initial and final 5 min periods without cue presentations.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThreat\u0026ndash;reward conflict memory test.\u003c/span\u003e Twenty-four hours after the final training session, the rats were presented with two simultaneous tone\u0026ndash;light cue presentations in the absence of foot shock (inter-trial interval: 180 s). The test was conducted 30 min after diazepam or vehicle administration was performed.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFlexibility (reversal) memory test\u003c/span\u003e: An independent group of rats was trained using the same reactive avoidance and threat\u0026ndash;reward conflict protocol but did not receive diazepam or vehicle during the prior testing. Twenty-four hours after completion of the threat\u0026ndash;reward conflict memory test, the animals were returned to the conditioning chambers and subjected to a reactive avoidance test consisting of two-tone presentations (30 s each) in the absence of foot shock (intertone interval: 180 s), with sucrose pellets available under a VI-30 schedule. This test assessed behavioral flexibility (reversal learning), defined as the ability to reinstate avoidance behavior following a rule change from threat\u0026ndash;reward conflict to reactive avoidance. The test was conducted 30 min after diazepam or vehicle administration.\u003c/p\u003e\n\u003ch3\u003eControl behavioral tests\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eThreat conditioning memory retrieval test.\u003c/span\u003e Auditory threat conditioning was performed in standard operant chambers in a separate cohort of animals. On day 1, the rats received five tone presentations (30 s, 4 kHz, 75 dB), each co-terminating with a foot shock (2 s, 0.4 mA), with intertone intervals averaging 120 s. On day 2, memory retrieval was assessed using two tone presentations in the absence of a foot shock. The freezing behavior was then quantified. Testing was conducted 30 min after diazepam or vehicle administration.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eOpen-field test.\u003c/span\u003e Locomotor activity and anxiety-like behavior were assessed in a modified open-field arena (90 \u0026times; 90 \u0026times; 60 cm) located in a dark testing room with separate cohorts of rats. The arena consisted of a safe peripheral zone (60 \u0026times; 60 cm) and a central threat zone (30 \u0026times; 30 cm) illuminated with high-intensity light (1500 lx). The rats were placed at the center, and their behavior was recorded for 5 min. The total distance traveled and mean velocity were used as measures of locomotion, and the time spent in the center was used as an index of anxiety-like behaviors. Testing was performed 30 min after the drug administration.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eInstrumental conditioning memory reward test\u003c/span\u003e: Rats were placed in the conditioning chambers and presented with two light cues (30 inter-trial interval: 180 s). These rats were used for the flexibly experiment. Lever presses during cue presentation were quantified. Testing was conducted 30 min after diazepam or vehicle administration was conducted.\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eFood intake test.\u003c/span\u003e To assess basal motivation for reward consumption, rats were placed in their home cage with access to 3 g of sucrose pellets for 1 min, starting from their first approach to the food dish. These rats were used for the flexibly experiment. Pellet consumption (g) was quantified. Testing was performed 30 min after drug administration.\u003c/p\u003e\n\u003ch3\u003eSystemic drug administration\u003c/h3\u003e\n\u003cp\u003eDiazepam (DZP; 1 mg/kg, s.c. Valium \u0026reg; (Roche, Mexico)) was dissolved in saline (5 mg/ml) and administered via subcutaneous injection 30 min before behavioral testing. The control animals received an equivalent volume of saline (vehicle). The selected dose was based on a prior study showing that 1 mg/kg produces reliable anxiolytic effects without sedative or locomotor impairment in rats tested in approach\u0026ndash;avoidance conflict paradigms, whereas a higher dose (2 mg/kg) can induce motor suppression and reduce task engagement (Illescas-Huerta et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). During the pre-test period, the animals remained in their home cages without access to food or water.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData collection and analysis\u003c/h2\u003e \u003cp\u003eAll behavioral responses were recorded using digital video cameras (Logitech) and automatically analyzed using a commercial software (ANY-maze; Stoelting). Behavioral measures were extracted during cue presentation and during predefined time windows before and after cue onset, as specified for each behavioral test.\u003c/p\u003e \u003cp\u003eAll behavioral measures reflected the average of the two cue presentations, whereas the number of platform entries represented the cumulative total across both presentations. Avoidance behavior was quantified as the percentage of time spent on the safety platform, number of platform entries, and latency to enter the platform for the first time after the footshock. Time-resolved avoidance dynamics were assessed by computing platform occupancy in 3-s bins before, during, and after cue presentation. Approach behavior was quantified as the time spent in the reward zone (lever and food dish area) and the number of lever presses during cue presentation. To capture the overall behavioral strategy during conflict and flexibility tests, we computed an avoidance/approach balance index by combining the percentage of time spent on the safety platform (avoidance) and the percentage of time spent in the reward zone (approach). In this two-dimensional representation, greater displacement along the y-axis reflected increased avoidance, whereas greater displacement along the x-axis reflected increased approach behavior. To quantitatively summarize the relative dominance of these competing responses, a normalized balance index was calculated as (Avoidance - Approach) / (Avoidance\u0026thinsp;+\u0026thinsp;Approach), such that positive values indicated avoidance-dominant allocation, negative values indicated approach-dominant allocation, and values near zero reflected balanced engagement of both behaviors. This index was treated as a dependent variable and analyzed using t-tests.\u003c/p\u003e \u003cp\u003eFor the control behavioral tests, lever presses during cue presentation were used to assess instrumental reward-seeking behavior. In the open-field test, the total distance traveled and mean velocity were used as indices of locomotor activity, whereas the time spent in the center of the arena was used as an index of anxiety-like behaviors. In the threat conditioning test, freezing was defined as the absence of all movements except respiration and was expressed as a percentage of the tone duration spent freezing. For the reward intake test, sucrose consumption (g) was quantified for a 1-min period. Statistical analyses were performed using commercial software (Prism; GraphPad). Group comparisons of mean behavioral measures were analyzed using unpaired two-tailed Student\u0026rsquo;s t-tests or two-way ANOVAs, as appropriate. Time-resolved behavioral dynamics were analyzed using a two-way repeated-measures ANOVA with treatment and time as factors. Sex-dependent effects were analyzed using two-way ANOVA, with sex and treatment as factors. Post-hoc comparisons were conducted using the Bonferroni multiple comparison test when appropriate. Statistical significance was sep at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAn overview of the platform-mediated avoidance (PMA) task and the integrated experimental design used in this study are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eDiazepam does not alter reactive avoidance (no-conflict) memory expression\u003c/h3\u003e\n\u003cp\u003eWe first examined whether diazepam affected the expression of reactive avoidance under conditions involving minimal competition between threats and rewards (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). During the reactive avoidance memory test, diazepam did not alter the time spent on the safety platform during tone presentation compared to vehicle-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cb\u003eleft\u003c/b\u003e; VEH, n\u0026thinsp;=\u0026thinsp;16; DZP, n\u0026thinsp;=\u0026thinsp;16; t(30)\u0026thinsp;=\u0026thinsp;0.39, p\u0026thinsp;=\u0026thinsp;0.69). Similarly, the number of platform entries during tone presentation did not differ between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cb\u003eright\u003c/b\u003e; t(30)\u0026thinsp;=\u0026thinsp;1.10, p\u0026thinsp;=\u0026thinsp;0.27).\u003c/p\u003e \u003cp\u003eAnalysis of avoidance dynamics revealed comparable temporal profiles of platform occupancy before, during, and after tone presentation in vehicle- and diazepam-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eleft\u003c/b\u003e panel). Two-way repeated-measures ANOVA revealed no main effect of treatment in the pre-tone (F(1, 30)\u0026thinsp;=\u0026thinsp;1.12, p\u0026thinsp;=\u0026thinsp;0.30), tone (F(1, 30)\u0026thinsp;=\u0026thinsp;0.16, p\u0026thinsp;=\u0026thinsp;0.57), or post-tone periods (F(1, 30)\u0026thinsp;=\u0026thinsp;0.16, p\u0026thinsp;=\u0026thinsp;0.57). Likewise, the treatment \u0026times; time interaction was not significant in any phase (pre-tone: F(9, 270)\u0026thinsp;=\u0026thinsp;1.49, p\u0026thinsp;=\u0026thinsp;0.15; tone: F(9, 270)\u0026thinsp;=\u0026thinsp;0.85, p\u0026thinsp;=\u0026thinsp;0.69; post-tone: F(9, 270)\u0026thinsp;=\u0026thinsp;0.85, p\u0026thinsp;=\u0026thinsp;0.69), indicating that diazepam did not affect the temporal pattern of reactive avoidance response. Consistent with this, the latency to the first platform entry during the tone was not affected by diazepam administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eright\u003c/b\u003e; t(30)\u0026thinsp;=\u0026thinsp;0.43, p\u0026thinsp;=\u0026thinsp;0.66).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDiazepam did not alter the approach-related measures during reactive avoidance. The time spent in the reward zone and the number of lever presses during tone presentation were comparable between vehicle- and diazepam-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC; t(30)\u0026thinsp;=\u0026thinsp;0.66, p\u0026thinsp;=\u0026thinsp;0.51 \u003cem\u003ereward zone\u003c/em\u003e; t(30)\u0026thinsp;=\u0026thinsp;0.67, p\u0026thinsp;=\u0026thinsp;0.50 \u003cem\u003elever presses\u003c/em\u003e). Representative movement tracking and occupancy heat maps further illustrated similar spatial allocation patterns across the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Accordingly, the integrated avoidance/approach balance index did not differ between the treatments under reactive avoidance conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, t(30)\u0026thinsp;=\u0026thinsp;0.30, p\u0026thinsp;=\u0026thinsp;0.76). Together, these results indicate that diazepam does not disrupt the expression of reactive avoidance when threat cues are presented without concurrent cue rewards.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDiazepam biases behavior toward reward during threat\u0026ndash;reward conflict\u003c/h2\u003e \u003cp\u003eNext, we assessed the effects of diazepam during the threat\u0026ndash;reward conflict, in which threat and reward cues were presented simultaneously (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Under these conditions, diazepam significantly reduced avoidance behavior, as reflected by the decreased time spent on the safety platform during cue co-presentation compared to vehicle-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eleft\u003c/b\u003e; t(30)\u0026thinsp;=\u0026thinsp;6.92, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The number of platform entries was also reduced following diazepam administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cb\u003eright\u003c/b\u003e; t(30)\u0026thinsp;=\u0026thinsp;3.35, p\u0026thinsp;=\u0026thinsp;0.0022).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTime-resolved analysis revealed that diazepam markedly attenuated the sustained platform occupancy during the conflict period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cb\u003eleft\u003c/b\u003e). In the conflict condition, diazepam significantly altered avoidance across the pre-tone, tone, and post-tone periods. Two-way repeated-measures ANOVA revealed a significant main effect of treatment during the pre-tone (F(1, 30)\u0026thinsp;=\u0026thinsp;6.70, p\u0026thinsp;=\u0026thinsp;0.015), tone (F(1, 30)\u0026thinsp;=\u0026thinsp;48.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and post-tone periods (F(1, 30)\u0026thinsp;=\u0026thinsp;8.14, p\u0026thinsp;=\u0026thinsp;0.0078). The treatment \u0026times; time interaction was not significant during the pre-tone phase (F(9, 270)\u0026thinsp;=\u0026thinsp;0.87, p\u0026thinsp;=\u0026thinsp;0.55), but was robustly significant during the tone (F(9, 270)\u0026thinsp;=\u0026thinsp;8.51, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and post-tone periods (F(9, 270)\u0026thinsp;=\u0026thinsp;3.51, p\u0026thinsp;=\u0026thinsp;0.0004), indicating that diazepam dynamically shifted the temporal pattern of avoidance responding during the threat\u0026ndash;reward conflict. In addition, diazepam increased the latency to the first platform entry during conflict trials (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cb\u003eright\u003c/b\u003e; t(30)\u0026thinsp;=\u0026thinsp;5.41, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), consistent with reduced avoidance engagement.\u003c/p\u003e \u003cp\u003eIn contrast to its effects on avoidance, diazepam enhanced approach-related behavior during the threat\u0026ndash;reward conflict. Diazepam-treated animals spent more time in the reward zone and exhibited increased lever pressing during cue co-presentation compared to vehicle-treated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC; t(30)\u0026thinsp;=\u0026thinsp;6.64, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, \u003cem\u003ereward zone\u003c/em\u003e; t(30)\u0026thinsp;=\u0026thinsp;3.01, p\u0026thinsp;=\u0026thinsp;0.0052, \u003cem\u003elever presses\u003c/em\u003e).Representative tracking data and heat maps illustrated a shift away from the safety platform and toward the reward area after diazepam treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Consistent with these effects, the avoidance/approach balance index revealed a robust shift toward approach-dominant behavior in diazepam-treated rats compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; t(30)\u0026thinsp;=\u0026thinsp;7.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 ). These findings demonstrate that diazepam selectively biases behavior toward reward engagement when threats and rewards compete.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDiazepam impairs behavioral flexibility during reversal from conflict to reactive avoidance\u003c/h2\u003e \u003cp\u003eTo determine whether diazepam affected the ability to adapt behavior following a rule change, we assessed performance in a flexibility (reversal) memory test in which animals were required to reinstate avoidance behavior after threat\u0026ndash;reward conflict training (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). During this test, diazepam-treated rats spent significantly less time on the safety platform than vehicle-treated animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eleft\u003c/b\u003e; VEH, n\u0026thinsp;=\u0026thinsp;12; DZP, n\u0026thinsp;=\u0026thinsp;12; t(22)\u0026thinsp;=\u0026thinsp;3.40, p\u0026thinsp;=\u0026thinsp;0.0025), whereas the number of platform entries did not differ between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cb\u003eright\u003c/b\u003e; t(22)\u0026thinsp;=\u0026thinsp;0.51, p\u0026thinsp;=\u0026thinsp;0.60).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTime-resolved analysis revealed attenuated avoidance expression across the tone and post-tone periods in diazepam-treated animals compared to that in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). During the flexibility assessment, diazepam produced phase-specific effects on avoidance response. Two-way repeated-measures ANOVA showed no significant main effect of treatment during the pre-tone period (F(1, 22)\u0026thinsp;=\u0026thinsp;3.79, p\u0026thinsp;=\u0026thinsp;0.064), and the treatment \u0026times; time interaction was also not significant (F(9, 198)\u0026thinsp;=\u0026thinsp;1.88, p\u0026thinsp;=\u0026thinsp;0.057). In contrast, during the tone period, diazepam significantly affected avoidance (F(1, 22)\u0026thinsp;=\u0026thinsp;11.85, p\u0026thinsp;=\u0026thinsp;0.0023), and the treatment \u0026times; time interaction was significant (F(9, 198)\u0026thinsp;=\u0026thinsp;2.76, p\u0026thinsp;=\u0026thinsp;0.0047). In the post-tone period, the main effect of treatment was not significant (F(1, 22)\u0026thinsp;=\u0026thinsp;2.04, p\u0026thinsp;=\u0026thinsp;0.17), but the treatment \u0026times; time interaction remained robust (F(9, 198)\u0026thinsp;=\u0026thinsp;4.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), indicating persistent temporal modulation of avoidance dynamics during the flexibility testing. However, the latency to the first platform entry during the tone was not affected by diazepam administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eright\u003c/b\u003e; t(22)\u0026thinsp;=\u0026thinsp;1.18, p\u0026thinsp;=\u0026thinsp;0.072).\u003c/p\u003e \u003cp\u003eIn parallel, diazepam increased approach-related behavior during the flexibility test, as reflected by greater reward zone occupancy and lever pressing compared with vehicle-treated rats (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; t(22)\u0026thinsp;=\u0026thinsp;3.70, p\u0026thinsp;=\u0026thinsp;0.0012, reward zone; t(22)\u0026thinsp;=\u0026thinsp;3.52, p\u0026thinsp;=\u0026thinsp;0.001,9 lever presses) ). Representative movement tracking and heat maps illustrated impaired reinstatement of avoidance after diazepam administration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The avoidance/approach balance index confirmed a persistent shift toward approach-dominant behavior in diazepam-treated animals during the flexibility test \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE; t(22)\u0026thinsp;=\u0026thinsp;3.68, p\u0026thinsp;=\u0026thinsp;0.0013), consistent with reduced behavioral flexibility. Pre-test balance measures were comparable across groups prior to each memory test (\u003cb\u003eSupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e), indicating that the treatment effects reflected altered behavioral allocation during testing rather than pre-existing baseline group differences.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSex-dependent effects of diazepam across behavioral conditions\u003c/h2\u003e \u003cp\u003eNext, we examined whether females and males differed in their avoidance/approach balance within each behavioral condition \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u003cb\u003e).\u003c/b\u003e Balance index values were comparable between females and males during the reactive avoidance memory test under both vehicle and diazepam treatment \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA; t(14)\u0026thinsp;=\u0026thinsp;1.23, p\u0026thinsp;=\u0026thinsp;0.23, vehicle; t(14)\u0026thinsp;=\u0026thinsp;0.23, p\u0026thinsp;=\u0026thinsp;0.81, diazepam) and during the threat\u0026ndash;reward conflict within each treatment condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; t(14)\u0026thinsp;=\u0026thinsp;0.80, p\u0026thinsp;=\u0026thinsp;0.43, vehicle; t(14)\u0026thinsp;=\u0026thinsp;0.47, p\u0026thinsp;=\u0026thinsp;0.64, diazepam). In contrast, during the flexibility (reversal) memory test, the balance index differed between females and males under diazepam treatment but not under vehicle treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC; t(10)\u0026thinsp;=\u0026thinsp;2.09, p\u0026thinsp;=\u0026thinsp;0.07, vehicle; t(10)\u0026thinsp;=\u0026thinsp;3.37, p\u0026thinsp;=\u0026thinsp;0.007 diazepam). This finding indicates sex-dependent modulation of behavioral allocation, specifically during the reversal from conflict to reactive avoidance.\u003c/p\u003e \u003cp\u003eDecomposition of balance index components suggested that, under diazepam, males exhibited a stronger reduction in avoidance output together with a larger increase in reward engagement compared with females, consistent with a quantitatively stronger shift toward approach during flexibility rather than a qualitatively different behavioral pattern across sexes (\u003cb\u003eSupplementary Figure S2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eControl behavioral tests\u003c/h2\u003e \u003cp\u003eTo rule out non-specific explanations for the diazepam-induced reallocation of avoidance and approach behaviors, we assessed additional behavioral parameters. Diazepam did not significantly alter conditioned freezing during threat memory retrieval, lever pressing during reward memory retrieval, sucrose intake, open-field locomotion, or anxiety-like behavior \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D; all p-values\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003cb\u003e).\u003c/b\u003e These findings indicate that diazepam-induced shifts in the PMA task were not specific changes attributed to locomotion, baseline motivation, or threat-related defensive response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn the present study, we examined how diazepam modulates reactive avoidance (no conflict), threat\u0026ndash;reward conflict resolution, and behavioral flexibility using an integrated PMA paradigm. Our results show that a low dose of diazepam (1 mg/kg) does not disrupt the expression of learned reactive avoidance when behavior is guided by threat cues. In contrast, when threat and reward cues were presented concurrently, diazepam biased behavior toward reward engagement, reduced avoidance, and shifted the avoidance/approach balance toward the approach. This selective behavioral effect extended to a flexibility test in which diazepam disrupted adaptive updating following conflict training, producing a persistent approach-dominant allocation instead of reinstating avoidance. Notably, sex-dependent differences emerged specifically during flexibility, indicating that the reversal from conflict to reactive avoidance may be differentially sensitive to diazepam in males and females.\u003c/p\u003e \u003cp\u003eA central finding was the dissociation between preserved reactive avoidance and altered behavior under threat\u0026ndash;reward conflict. Traditional descriptions of benzodiazepine action often emphasize anxiolysis as a reduction in threat-related defensive responding (Blanchard et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Groenink et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, diazepam did not suppress defensive responses across conditions. Instead, its effects emerged specifically when animals were required to arbitrate between competing motivational needs. Under conflict, rats must evaluate the relative costs and benefits of avoidance versus reward pursuit, and diazepam shifts this evaluation toward approach and reward-seeking. This pattern is consistent with frameworks in which approach\u0026ndash;avoidance conflict reflects a decision process involving the selection among competing strategies rather than a graded modulation of avoidance strength (Eliot, 2008; Rangel et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Choi and Kim \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Mitchell et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Friedman et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Hamel et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Piantadosi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bercovici et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Walters et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Illescas-Huerta et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Calvin et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and supports the view that conflict engages additional cognitive and motivational control operations beyond those required for reactive avoidance (Botvinick et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Aupperle and Paulus \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Hartley and Phelps \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Aupperle et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Calhoon and Tye \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Jackson et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eImportantly, the behavioral reallocation observed under diazepam cannot readily explain the non-specific changes in locomotion, basal motivation, or global suppression of defensive responses. In the present dataset, diazepam did not significantly alter conditioned freezing during threat memory retrieval, lever pressing during reward memory retrieval, sucrose intake, open-field locomotion or anxiety-like behavior. These results complement those of previous studies showing that this dose does not induce sedation or motor impairment in standard assays (Savić et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Illescas-Huerta et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; P\u0026aacute;dua-Reis et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Together, these results support the interpretation that diazepam primarily modulates how threat-related information constrains action selection during motivational competition rather than disrupting threat memory retrieval per se or broadly disinhibiting behavior.\u003c/p\u003e \u003cp\u003eIn addition to conflict resolution, diazepam alters the behavioral flexibility. During the reversal-like flexibility memory test, diazepam reduced platform occupancy and maintained a shift toward an approach-dominant allocation. While benzodiazepines are typically studied for acute anxiolytic actions, this result suggests that enhancing GABAergic inhibition can also bias animals toward the persistence of reward-directed allocation following prior conflict experience, reducing adaptive updating when task contingencies require reinstatement of avoidance. This observation aligns with the broader idea that stress- and anxiety-related states can bias behavioral selection and updating (Aupperle and Paulus \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Smith et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Park and Moghaddam \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, in the present task, the drug effect manifested as reduced adaptation across motivational contexts, rather than a generalized performance deficit.\u003c/p\u003e \u003cp\u003eIn the present study, we also observed sex-dependent differences in the effects of diazepam on behavioral flexibility. Whereas females and males showed comparable balance indices during reactive avoidance and threat\u0026ndash;reward conflict, the balance index diverged by sex during the flexibility memory test. The decomposition of the balance index into avoidance and approach components suggests that this difference reflects a quantitatively stronger diazepam-induced shift toward reward engagement in males rather than a qualitatively distinct behavioral pattern across sexes. These findings add to the evidence that sex can modulate the pharmacological effects on motivated behavior and decision processes (Orsini et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Islas-Preciado et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ecevitoglu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Faraji et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and highlight the value of explicitly considering sex as a biological variable when characterizing the effects of drugs on flexibility under threat.\u003c/p\u003e \u003cp\u003eIn summary, these findings refine our understanding of how benzodiazepines influence defensive behaviors in complex motivational settings. Rather than broadly reducing threat-related defensive responding or avoidance, diazepam selectively biases behavioral allocation during threat\u0026ndash;reward conflict and alters subsequent flexibility while maintaining reactive avoidance. This dissociation suggests that benzodiazepines may alleviate anxiety-related conflict by shifting the resolution of competing motivational drives rather than by suppressing defensive responses per se. More generally, reducing the constraining influence of threat on action selection may favor reward engagement during conflict but may also promote the persistence of approach-dominant strategies when conditions require flexible reinstatement of avoidance. Future studies using dose-response approaches will be crucial to fully characterize the therapeutic window and potential side effects across a range of doses, as our current study utilized a single dose (1mg/kg). While this dose was carefully selected to avoid confounding locomotor or sedative effects, it restricts the generalizability of these specific findings to broader clinical applications and varying degrees of anxiety, as the observed behavioral specificity might be dose-dependent. Additionally, circuit-specific manipulations (e.g., targeting prefrontal\u0026ndash;striatal and amygdala networks) will be important for identifying the neural mechanisms that enable diazepam to reshape approach\u0026ndash;avoidance arbitration and flexibility in the face of threat.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cu\u003eAuthor Contributions:\u003c/u\u003e OE-P and FS-B designed the study; OE-P and FS-R performed the experiments; OE-P and FS-B analyzed the data; OE-P and FS-B wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cu\u003eConflict\u0026nbsp;of\u0026nbsp;interest:\u003c/u\u003e The authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eFunding sources: Secretar\u0026iacute;a de Ciencia, Humanidades, Tecnolog\u0026iacute;a e Innovaci\u0026oacute;n (SECIHTI; grant CF-2023-I-225), Direcci\u0026oacute;n General de Asuntos del Personal Acad\u0026eacute;mico de la Universidad Nacional Aut\u0026oacute;noma de M\u0026eacute;xico (UNAM; grants: IN214223\u0026nbsp;and\u0026nbsp;IN226026) to\u0026nbsp;FS-B. OE-P\u0026nbsp;is a\u0026nbsp;doctoral\u0026nbsp;student at the\u0026nbsp;Programa\u0026nbsp;de Doctorado en Ciencias Bioqu\u0026iacute;micas at UNAM and is supported by the SECIHTI fellowship (1047792).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cu\u003eAcknowledgements:\u003c/u\u003e We thank Christian Bravo-Rivera and the SotresLab for helpful discussions and comments on a previous version of the manuscript.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal ethics approval\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;All experimental procedures were approved by the Bioethics Committee of the Institute of Cellular Physiology, National Autonomous University of Mexico (UNAM), and were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;Not applicable.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAupperle RL, Melrose AJ, Francisco A et al (2015) Neural substrates of approach-avoidance conflict decision-making. Hum Brain Mapp 36:449\u0026ndash;462. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/hbm.22639\u003c/span\u003e\u003cspan address=\"10.1002/hbm.22639\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAupperle RL, Paulus MP (2010) Neural systems underlying approach and avoidance in anxiety disorders. Dialogues Clin Neurosci 12:517\u0026ndash;531. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.31887/DCNS.2010.12.4\u003c/span\u003e\u003cspan address=\"10.31887/DCNS.2010.12.4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBenson JA, L\u0026ouml;w K, Keist R et al (1998) Pharmacology of recombinant gamma-aminobutyric acidA receptors rendered diazepam-insensitive by point-mutated alpha-subunits. FEBS Lett 431:400\u0026ndash;404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0014-5793(98)00803-5\u003c/span\u003e\u003cspan address=\"10.1016/s0014-5793(98)00803-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBercovici DA, Princz-Lebel O, Tse MT et al (2018) Optogenetic Dissection of Temporal Dynamics of Amygdala-Striatal Interplay during Risk/Reward Decision Making. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/ENEURO.0422-18.2018\u003c/span\u003e\u003cspan address=\"10.1523/ENEURO.0422-18.2018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. eNeuro 5:ENEURO.0422-18.2018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanchard DC, Blanchard RJ, Tom P, Rodgers RJ (1990) Diazepam changes risk assessment in an anxiety/defense test battery. Psychopharmacology 101:511\u0026ndash;518. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02244230\u003c/span\u003e\u003cspan address=\"10.1007/BF02244230\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBotvinick MM, Braver TS, Barch DM et al (2001) Conflict monitoring and cognitive control. Psychol Rev 108:624\u0026ndash;652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1037/0033-295x.108.3.624\u003c/span\u003e\u003cspan address=\"10.1037/0033-295x.108.3.624\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBravo-Rivera C, Roman-Ortiz C, Brignoni-Perez E et al (2014) Neural Structures Mediating Expression and Extinction of Platform-Mediated Avoidance. J Neurosci 34:9736\u0026ndash;9742. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/JNEUROSCI.0191-14.2014\u003c/span\u003e\u003cspan address=\"10.1523/JNEUROSCI.0191-14.2014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBravo-Rivera C, Roman-Ortiz C, Montesinos-Cartagena M, Quirk GJ (2015) Persistent active avoidance correlates with activity in prelimbic cortex and ventral striatum. Front Behav Neurosci 9:184. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnbeh.2015.00184\u003c/span\u003e\u003cspan address=\"10.3389/fnbeh.2015.00184\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBravo-Rivera C, Sotres-Bayon F (2020) From Isolated Emotional Memories to Their Competition During Conflict. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnbeh.2020.00036\u003c/span\u003e\u003cspan address=\"10.3389/fnbeh.2020.00036\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Front Behav Neurosci 14:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBravo-Rivera H, Rubio Arzola P, Caban-Murillo A et al (2021) Characterizing Different Strategies for Resolving Approach-Avoidance Conflict. Front Neurosci 15:608922. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnins.2021.608922\u003c/span\u003e\u003cspan address=\"10.3389/fnins.2021.608922\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalhoon GG, Tye KM (2015) Resolving the neural circuits of anxiety. Nat Neurosci 18:1394\u0026ndash;1404. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nn.4101\u003c/span\u003e\u003cspan address=\"10.1038/nn.4101\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalvin OL, Erickson MT, Walters CJ, Redish AD (2025) Dorsal hippocampus represents locations to avoid as well as locations to approach during approach-avoidance conflict. PLoS Biol 23:e3002954. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pbio.3002954\u003c/span\u003e\u003cspan address=\"10.1371/journal.pbio.3002954\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi J-S, Kim JJ (2010) Amygdala regulates risk of predation in rats foraging in a dynamic fear environment. Proceedings of the National Academy of Sciences 107:21773\u0026ndash;21777. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1010079108\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1010079108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiehl MM, Bravo-Rivera C, Quirk GJ (2019) The study of active avoidance: A platform for discussion. Neurosci Biobehavioral Reviews 107:229\u0026ndash;237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neubiorev.2019.09.010\u003c/span\u003e\u003cspan address=\"10.1016/j.neubiorev.2019.09.010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiehl MM, Bravo-Rivera C, Rodriguez-Romaguera J et al (2018) Active avoidance requires inhibitory signaling in the rodent prelimbic prefrontal cortex. Elife 7:e34657. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.34657\u003c/span\u003e\u003cspan address=\"10.7554/eLife.34657\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiehl MM, Iravedra-Garcia JM, Mor\u0026aacute;n-Sierra J et al (2020) Divergent projections of the prelimbic cortex bidirectionally regulate active avoidance. Elife 9:e59281. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.7554/eLife.59281\u003c/span\u003e\u003cspan address=\"10.7554/eLife.59281\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEcevitoglu A, Beard KR, Srynath S et al (2024) Pharmacological characterization of sex differences in the effects of dopaminergic drugs on effort-based decision making in rats. Psychopharmacology 241:2033\u0026ndash;2044. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00213-024-06615-8\u003c/span\u003e\u003cspan address=\"10.1007/s00213-024-06615-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElliot AJ (2008) Handbook of approach and avoidance motivation. Psychology, New York\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEnriquez-Traba J, Arenivar M, Yarur-Castillo HE et al (2025) Dissociable control of motivation and reinforcement by distinct ventral striatal dopamine receptors. Nat Neurosci 28:105\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41593-024-01819-9\u003c/span\u003e\u003cspan address=\"10.1038/s41593-024-01819-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFaraji M, Bizon JL, Setlow B (2025) A novel cost-benefit decision-making task involving cued punishment: Effects of sex and psychostimulant administration. Behav Brain Res 495:115781. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbr.2025.115781\u003c/span\u003e\u003cspan address=\"10.1016/j.bbr.2025.115781\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFile SE (1985) Tolerance to the behavioral actions of benzodiazepines. NeurosciBiobehav Rev 9:113\u0026ndash;121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1016/0149-7634(85)90037-5\u003c/span\u003e\u003cspan address=\"https://doi:10.1016/0149-7634(85)90037-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFriedman A, Homma D, Gibb LG et al (2015) A Corticostriatal Path Targeting Striosomes Controls Decision-Making under Conflict. Cell 161:1320\u0026ndash;1333. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cell.2015.04.049\u003c/span\u003e\u003cspan address=\"10.1016/j.cell.2015.04.049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGriebel G, Holmes A (2013) 50 years of hurdles and hope in anxiolytic drug discovery. Nat Rev Drug Discov 12:667\u0026ndash;687. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrd4075\u003c/span\u003e\u003cspan address=\"10.1038/nrd4075\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroenink L, Verdouw PM, Zhao Y et al (2023) Pharmacological modulation of conditioned fear in the fear-potentiated startle test: a systematic review and meta-analysis of animal studies. Psychopharmacology 240:2361\u0026ndash;2401. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00213-022-06307-1\u003c/span\u003e\u003cspan address=\"10.1007/s00213-022-06307-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalcomb CJ, Philipp TR, Dhillon PS et al (2024) Sex divergent behavioral responses in platform-mediated avoidance and glucocorticoid receptor blockade. Psychoneuroendocrinology 159:106417. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.psyneuen.2023.106417\u003c/span\u003e\u003cspan address=\"10.1016/j.psyneuen.2023.106417\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHamel L, Thangarasa T, Samadi O, Ito R (2017) Caudal Nucleus Accumbens Core Is Critical in the Regulation of Cue-Elicited Approach-Avoidance Decisions. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/ENEURO.0330-16.2017\u003c/span\u003e\u003cspan address=\"10.1523/ENEURO.0330-16.2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. eNeuro 4:ENEURO.0330-16.2017\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHartley CA, Phelps EA (2012) Anxiety and Decision-Making. Biol Psychiatry 72:113\u0026ndash;118. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopsych.2011.12.027\u003c/span\u003e\u003cspan address=\"10.1016/j.biopsych.2011.12.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIllescas-Huerta E, Ramirez-Lugo L, Sierra RO et al (2021) Conflict Test Battery for Studying the Act of Facing Threats in Pursuit of Rewards. Front Neurosci 15:645769. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fnins.2021.645769\u003c/span\u003e\u003cspan address=\"10.3389/fnins.2021.645769\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIslas-Preciado D, Wainwright SR, Sniegocki J et al (2020) Risk-based decision making in rats: Modulation by sex and amphetamine. Horm Behav 125:104815. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.yhbeh.2020.104815\u003c/span\u003e\u003cspan address=\"10.1016/j.yhbeh.2020.104815\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJackson F, Nelson BD, Hajcak G (2016) The uncertainty of errors: Intolerance of uncertainty is associated with error-related brain activity. Biol Psychol 113:52\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopsycho.2015.11.007\u003c/span\u003e\u003cspan address=\"10.1016/j.biopsycho.2015.11.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKramer C, Ruble S, Fort TD et al (2025) Modifying the platform-mediated avoidance task: A new protocol to study active avoidance within a social context in rats. PLoS ONE 20:e0321776. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0321776\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0321776\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi CJ, Pineda D, Reimer AE et al (2025) Sex Based Differences in Active Avoidance and Approach Strategy in the Platform Mediated Avoidance Task. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2025.10.01.679640\u003c/span\u003e\u003cspan address=\"10.1101/2025.10.01.679640\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. bioRxiv 2025.10.01.679640\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Moraga A, De Ceuninck M, Van der Heyden Y et al (2026) Differential effects of chronic restraint stress on two active avoidance tasks in rats. Prog Neuropsychopharmacol Biol Psychiatry 144:111586. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pnpbp.2025.111586\u003c/span\u003e\u003cspan address=\"10.1016/j.pnpbp.2025.111586\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Moraga A, Luyten L, Beckers T (2024) A history of avoidance does not impact extinction learning in male rats. NPJ Sci Learn 9:11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41539-024-00223-z\u003c/span\u003e\u003cspan address=\"10.1038/s41539-024-00223-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026oacute;pez-Moraga A, Luyten L, Beckers T (2025) Generalization and extinction of platform-mediated avoidance in male and female rats. Sci Rep 15:9730. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-025-94265-x\u003c/span\u003e\u003cspan address=\"10.1038/s41598-025-94265-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell MR, Vokes CM, Blankenship AL et al (2011) Effects of acute administration of nicotine, amphetamine, diazepam, morphine, and ethanol on risky decision-making in rats. Psychopharmacology 218:703\u0026ndash;712. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00213-011-2363-8\u003c/span\u003e\u003cspan address=\"10.1007/s00213-011-2363-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026ouml;hler H (2012) The GABA system in anxiety and depression and its therapeutic potential. Neuropharmacology 62:42\u0026ndash;53. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neuropharm.2011.08.040\u003c/span\u003e\u003cspan address=\"10.1016/j.neuropharm.2011.08.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrsini CA, Willis ML, Gilbert RJ et al (2016) Sex differences in a rat model of risky decision-making. Behav Neurosci 130:50\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1037/bne0000111\u003c/span\u003e\u003cspan address=\"10.1037/bne0000111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eP\u0026aacute;dua-Reis M, N\u0026ocirc;ga DA, Tort ABL, Blunder M (2021) Diazepam causes sedative rather than anxiolytic effects in C57BL/6J mice. Sci Rep 11:9335. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-88599-5\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-88599-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark J, Moghaddam B (2017) Impact of anxiety on prefrontal cortex encoding of cognitive flexibility. Neuroscience 345:193\u0026ndash;202. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neuroscience.2016.06.013\u003c/span\u003e\u003cspan address=\"10.1016/j.neuroscience.2016.06.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePiantadosi PT, Yeates DCM, Wilkins M, Floresco SB (2017) Contributions of basolateral amygdala and nucleus accumbens subregions to mediating motivational conflict during punished reward-seeking. Neurobiol Learn Mem 140:92\u0026ndash;105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.nlm.2017.02.017\u003c/span\u003e\u003cspan address=\"10.1016/j.nlm.2017.02.017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangel A, Camerer C, Montague PR (2008) Neuroeconomics: The neurobiology of value-based decision-making. Nat Rev Neurosci 9:545\u0026ndash;556. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrn2357\u003c/span\u003e\u003cspan address=\"10.1038/nrn2357\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuble S, Kramer C, West L et al (2024a) Active avoidance recruits the anterior cingulate cortex regardless of social context in male and female rats. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21203/rs.3.rs-3750422/v2\u003c/span\u003e\u003cspan address=\"10.21203/rs.3.rs-3750422/v2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Res Sq rs.3.rs-3750422\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuble S, Payne K, Kramer C et al (2024b) Social context modulates active avoidance: Contributions of the anterior cingulate cortex in male and female rats. Neurobiol Stress 34:100702. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ynstr.2024.100702\u003c/span\u003e\u003cspan address=\"10.1016/j.ynstr.2024.100702\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRudolph U, Knoflach F (2011) Beyond classical benzodiazepines: Novel therapeutic potential of GABAA receptor subtypes. Nat Rev Drug Discov 10:685\u0026ndash;697. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrd3502\u003c/span\u003e\u003cspan address=\"10.1038/nrd3502\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavić MM, Milinković MM, Rallapalli S et al (2009) The differential role of alpha1-and alpha5-containing GABA(A) receptors in mediating diazepam effects on spontaneous locomotor activity and water-maze learning and memory in rats. Int J Neuropsychopharmacol 12:1179\u0026ndash;1193. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S1461145709000108\u003c/span\u003e\u003cspan address=\"10.1017/S1461145709000108\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShader RI, Greenblatt DJ (1993) Use of benzodiazepines in anxiety disorders. N Engl J Med 328:1398\u0026ndash;1405. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJM199305133281907\u003c/span\u003e\u003cspan address=\"10.1056/NEJM199305133281907\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith KS, Engin E, Meloni EG, Rudolph U (2012) Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice. Neuropharmacology 63:250\u0026ndash;258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neuropharm.2012.03.001\u003c/span\u003e\u003cspan address=\"10.1016/j.neuropharm.2012.03.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSt Laurent R, Kusche KM, Rein B et al (2025) Intercalated Amygdala Dysfunction Drives Avoidance Extinction Deficits in the Sapap3 Mouse Model of Obsessive-Compulsive Disorder. Biol Psychiatry 97:707\u0026ndash;720. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biopsych.2024.10.021\u003c/span\u003e\u003cspan address=\"10.1016/j.biopsych.2024.10.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVelazquez-Hernandez G, Sotres-Bayon F (2021) Lateral Habenula Mediates Defensive Responses Only When Threat and Safety Memories Are in Conflict. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1523/ENEURO.0482-20.2021\u003c/span\u003e\u003cspan address=\"10.1523/ENEURO.0482-20.2021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. eNeuro 8:ENEURO.0482-20.2021\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVogel JR, Beer B, Clody DE (1971) A simple and reliable conflict procedure testing anti-anxiety agents. Psychopharmacologia 21:1\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi:10.1007/BF00403989\u003c/span\u003e\u003cspan address=\"https://doi:10.1007/BF00403989\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWalters CJ, Jubran J, Sheehan A et al (2019) Avoid-approach conflict behaviors differentially affected by anxiolytics: implications for a computational model of risky decision-making. Psychopharmacology 236:2513\u0026ndash;2525. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00213-019-05197-0\u003c/span\u003e\u003cspan address=\"10.1007/s00213-019-05197-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeidler Z, Gomez MF, Gupta TA et al (2025) Dopaminergic projections to the prefrontal cortex are critical for rapid threat avoidance learning. bioRxiv 2024.05.02.592069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1101/2024.05.02.592069\u003c/span\u003e\u003cspan address=\"10.1101/2024.05.02.592069\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Y, Bijlsma EY, Verdouw PM et al (2018) The contribution of contextual fear in the anxiolytic effect of chlordiazepoxide in the fear-potentiated startle test. Behav Brain Res 353:57\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbr.2018.06.03\u003c/span\u003e\u003cspan address=\"10.1016/j.bbr.2018.06.03\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Diazepam, Approach–avoidance conflict, Behavioral flexibility, Decision-making, Active avoidance, Sex difference","lastPublishedDoi":"10.21203/rs.3.rs-8928345/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8928345/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eRationale:\u003c/h2\u003e \u003cp\u003eBenzodiazepines are widely used to treat anxiety; however, their influence on defensive behavior when threats compete with rewards remains unclear.\u003c/p\u003e\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eHere, we tested the effects of diazepam on reactive avoidance (no conflict), threat\u0026ndash;reward conflict resolution, and behavioral flexibility.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eUsing an integrated platform-mediated avoidance paradigm in female and male rats, we assessed the effects of a low dose systemically administered diazepam (1 mg/kg).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDiazepam did not alter the reactive avoidance memory expression when threat cues were presented without a concurrent reward cue. In contrast, during the cued threat\u0026ndash;reward conflict, diazepam reduced platform avoidance and increased reward engagement, shifting the avoidance/approach balance toward the approach. Diazepam also impaired behavioral flexibility during the reversal from conflict to reactive avoidance, maintaining an approach-dominant strategy when avoidance should be reinstated. Sex differences were observed selectively during the flexibility assessment, reflecting a quantitatively stronger diazepam-induced shift toward reward engagement in males compared with females. Control assays showed no significant effects of diazepam on threat or reward memory retrieval, sucrose intake, open-field locomotion, or anxiety-like behavior.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings indicate that diazepam selectively reshapes approach\u0026ndash;avoidance arbitration under motivational competition and alters subsequent flexibility while sparing reactive avoidance.\u003c/p\u003e","manuscriptTitle":"Diazepam selectively biases approach during threat–reward conflict while sparing reactive avoidance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-25 07:55:09","doi":"10.21203/rs.3.rs-8928345/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae4e1458-a9ea-4a05-a7e1-1b3ec9263277","owner":[],"postedDate":"February 25th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-13T21:23:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-25 07:55:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8928345","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8928345","identity":"rs-8928345","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}