Safety Signals Enable Single-Episode 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-episode 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 animals inferred contingencies within a single episode, and discrimination achieved during training predicted retrieval precision. However, discrimination operated within defined boundary conditions: overtraining 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 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.