Safety Signals Enable Single-Session Active Avoidance paradigm and Expose Threat Generalization in Tuberous Sclerosis Complex

Animals must flexibly discriminate between threat and non-threat to deploy adaptive defensive strategies. We introduce a single-session differential signaled active avoidance (DSAA) paradigm that temporally dissociates acquisition, consolidation, and retrieval of instrumental avoidance memory. Interleaving a behaviorally noncontingent neutral cue (CS-) with a threat-predictive, behaviorally contingent cue (CS+) enhanced long-term memory without altering acquisition, demonstrating that differential contingency structure selectively reinforces memory consolidation rather than influencing learning performance. Naïve mice of either sex inferred contingencies within a single session, and discrimination achieved during training predicted retrieval precision. However, discrimination operated within defined boundary conditions: Extended training or elevated threat intensity destabilized cue specificity and promoted persistent avoidance generalization. Under high-threat conditions, freezing and shuttling co-emerged as complementary defensive responses, indicating a shift from precise cue-based encoding to a generalized defensive state. Remote retrieval recruited oxytocin receptor-expressing cells in the medial prefrontal cortex and activated mTORC1-dependent translational signaling, implicating protein synthesis in maintenance of discriminative avoidance memory. In a Tuberous Sclerosis Complex mouse model with Tsc2 haploinsufficiency in oxytocin-responsive cells, males displayed intact acquisition but generalized avoidance at both recent and remote time points, a deficit not rescued by additional training. These findings identify oxytocin-modulated translational control as a molecular gate stabilizing threat-safety discrimination and show that disruption of this axis - by excessive threat or reduced Tsc2 gene dosage - biases memory toward pathological generalization, providing a mechanistic framework for safety-learning deficits in neurodevelopmental and anxiety-related disorders. Significance Statement Adaptive behavior requires distinguishing between cues that signal threat and safety. Here we introduce a single-episode Differential Signaled Active Avoidance (DSAA) paradigm that separates acquisition, consolidation, and retrieval of avoidance memory. Interleaving a neutral safety cue with a threat cue strengthens long-term memory consolidation without affecting learning during training. However, excessive threat or overtraining destabilizes cue specificity and promotes avoidance generalization. Remote retrieval engages oxytocin receptor-expressing neurons in the medial prefrontal cortex and activates mTORC1-dependent translational signaling. In a Tsc2 haploinsufficiency model, disruption of this pathway produces persistent avoidance generalization despite intact learning. These findings identify oxytocin-modulated translational control as a mechanism stabilizing threat-safety discrimination and provide a framework for understanding maladaptive fear generalization in neurodevelopmental and anxiety disorders.