Behavioral characterization of alprazolam withdrawal in female Wistar rats: social interaction and exploratory activity

Introduction

Benzodiazepine withdrawal is characterized by a broad range of psychological and somatic symptoms, posing a considerable challenge in clinical practice. In animal models, withdrawal is associated with heightened anxiety-like behavior and altered exploratory activity, while its effects on social behavior remain less understood. In our experimental model, abrupt alprazolam discontinuation produces a pronounced anxiety-like behavioral phenotype approximately 36 h after the last administration. This study extended the behavioral characterization of the model by examining social interaction and social-approach dynamics during alprazolam withdrawal.

Methods

Adult female Wistar rats received intraperitoneal alprazolam twice daily for eight days according to an escalating-dose regimen (3, 5, 7 and 9 mg/kg; each dose for two consecutive days, n = 23) or vehicle (n = 17). Approximately 36 h after abrupt discontinuation, animals underwent behavioral testing. Each animal first completed a 10-min open-field test, which simultaneously served as a habituation phase to the 100 × 100 cm arena. Two identical perforated objects were then positioned diagonally, and animals explored them for 5 min to minimize novelty-related object exploration. An unfamiliar, age- and sex-matched stranger rat was subsequently placed inside one object, after which the experimental animal was returned for a 10-min social interaction test. Behavior was analyzed in real time using ANY-maze software v.7.40. Social parameters were analyzed separately for the first and second 5-min periods following stranger introduction. Whole-test locomotor parameters were compared using two-tailed unpaired t-tests whereas within-test time-dependent effects were evaluated using mixed-effects models followed by Šídák-adjusted multiple comparisons.

Results

Across the entire 15-min object-familiarization and social-interaction session, alprazolam-withdrawn rats showed reduced locomotor activity, with shorter total distance travelled [t(38) = 3.309, p = 0.0021] and lower average speed [t(38) = 3.294, p = 0.0021]. Distance travelled within the social interaction zone showed effects of treatment and time (both p < 0.0001) and a treatment × time interaction (p = 0.0034). Withdrawal animals travelled less within this zone during object familiarization before stranger introduction (p = 0.0015) and during the first 5 min after stranger introduction (p = 0.0007), but not during the second 5 min (p = 0.8974). Social-interaction zone entries also showed a treatment × time interaction (p = 0.0018), with fewer entries in withdrawal animals during the first 5 min after stranger introduction (p = 0.0008), but not during the second 5 min (p = 0.9755). Time spent in the social-interaction zone showed no significant treatment × time interaction (p = 0.1152), although a numerical reduction was observed in withdrawal animals during the first 5 min after stranger introduction.

Conclusions

Alprazolam withdrawal is associated with a dynamic behavioral phenotype characterized by reduced exploratory activity and attenuated initial social approach. Reduced social-interaction zone entries suggest altered initiation of social approach; however, reduced exploration preceding stranger introduction indicates that this effect cannot yet be fully dissociated from broader exploratory suppression. Further analysis of additional social, spatial, and exploratory parameters will more comprehensively characterize social behavior during alprazolam withdrawal and help distinguish general exploratory alterations from social-specific behavioral changes.

Acknowledgment:Supported by NITRA, grant: 451-03-33/2026-03/ 200017.

References

No references.