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Astrocytic role in second-order threat conditioning in the amygdala–piriform cortex circuit

Poster Session C - Sunday, March 8, 2026, 5:00 – 7:00 pm PDT, Fairview/Kitsilano Ballroom

Negar Nazari¹ (), Tayebeh Sepahvand¹, Tian Qin¹, Qi Yuan¹; ¹Faculty of Medicine, Memorial University of Newfoundland

Fear- and anxiety-related disorders involve maladaptive associative learning, where neutral cues acquire emotional salience through indirect associations. Second-order conditioning (SOC) models this phenomenon by pairing a previously conditioned cue (CS1) predicting an aversive event with a novel neutral cue (CS2). Our prior work identified reciprocal connectivity between the basolateral amygdala (BLA) and piriform cortex (PC) as a key substrate for SOC. Here, we investigated whether astrocytic signaling within the BLA contributes to SOC formation, hypothesizing that disrupting astrocyte activity would impair SOC and attenuate BLA–PC circuit engagement. Adult rats received viral vectors targeting astrocytes and projection-specific neurons in the BLA. For astrocyte inhibition, AAV2/5-GfaABC1D-hM4Di-mCherry was infused into the BLA; controls received AAV2/5-GfaABC1D-mCherry. To label BLA–PC projection neurons, a retrograde AAV-retro-CaMKIIα-Cre was injected into the PC, and a Cre-dependent virus into the BLA. After three weeks, animals underwent a three-day olfactory-auditory SOC paradigm (tone-shock pairing, odor-tone pairing, CS2 recall). Clozapine-N-oxide (CNO) was administered 30 minutes before training or recall to inhibit astrocytic activity. Freezing to CS2 was the behavioral index of SOC memory. Brains were collected for c-Fos immunolabeling to assess neuronal activation. Silencing astrocytes in the BLA significantly reduced freezing to CS2 during both SOC acquisition and recall compared to controls. Analysis of c-Fos expression in BLA neurons and BLA–PC projection cells is ongoing. Preliminary findings indicate that astrocytic activity in the BLA is essential for encoding and retrieval of second-order threat memories. These results highlight a previously unrecognized glial contribution to higher-order associative learning within the amygdala-piriform cortex network.

Topic Area: LONG-TERM MEMORY: Other