The basolateral amygdala (BLA) is one of the key brain areas involved in aversive learning, especially fear memory formation. Studies of aversive learning in BLA have largely focused on neuronal function, while the role of BLA astrocytes in aversive learning remains largely unknown. It has long been known that astrocytes express multiple G-protein-coupled receptors (GPCRs), which allow them to sense neurotransmitters and neuromodulators in the extracellular space from local or long-range projections. However, as to whether and how astrocytic GPCRs regulate fear memory remains unclear.

To address this issue, we manipulated the BLA astrocytes by expressing Gq-coupled receptor hM3Dq and discovered that astrocytic Gq-modulation during fear conditioning promoted auditory cue fear memory but did not affect the less-stress memory or induce anxiety-like behavior. Chemogenetic activation of BLA astrocytes during memory retrieval had no effect on fear memory expression. In addition, astrocytic Gq-activation increased c-Fos expression in BLA and medial prefrontal cortex (mPFC) in fear conditioning, but not in the homecage. Using the retrograde virus tracing, we found that mPFC-projecting BLA neurons activity showed significant enhancement after astrocytic Gq activation during fear conditioning. Electrophysiology recordings showed that activating astrocyte Gq in BLA promoted spike field coherence and phase locking percentage not only within BLA, but also between BLA and mPFC. Finally, direct chemogenetic activation of BLA-to-mPFC projection during learning enhanced cued fear memory. Taken together, our data suggest that astrocytes in BLA may contribute to aversive learning by modulating the amygdala-mPFC communication.

Given that BLA astrocytes play important roles in fear learning and memory, the specific role of BLA astrocytes in fear memory acquisition, memory consolidation, and memory retrieval remains unclear. Using in vivo free moving fiber photometry calcium recording, we observed that BLA astrocytic calcium signaling was robustly increased during fear learning. We manipulated the BLA astrocytes by expressing melanopsin, a Gq GPCR photopigment and discovered that optogenetic activation of astrocytic Gq during fear conditioning promoted auditory cue fear memory but not context fear memory. Also, optogenetic activation of astrocytic Gq during memory consolidation or memory recall had no effect on memory performance. In addition, optogenetic activation of astrocytic Gq did not affect locomotor ability or anxiety like behavior. Electrophysiology recordings showed that optogenetic activating astrocytic Gq in BLA promoted spike field coherence and phase locking percentage not only within BLA, but also between BLA and mPFC. Reducing calcium signaling with an astrocyte-specific calcium extruder significantly impaired auditory-cue fear memory without effect on locomotor ability or anxiety like behavior. Finally, specific reducing BLA astrocytic Gq calcium signaling by iβARK suppressed the fear memory enhancement induced by optogenetic Gq activation of BLA astrocytes, suggesting that the calcium induced by astrocytic Gq activation is crucial for fear memory formation. Taken together, these data demonstrates that astrocytic Gq GPCR and calcium signaling in BLA contribute to fear memory formation but not memory consolidation or memory retrieval.

The mPFC and β-adrenoceptors (βARs, Gs GPCR) have been implicated in modulating anxiety-like behavior. However, the specific contributions of the β2-AR subtype in mPFC in anxiety are still unclear. To address this issue, we used optogenetic and microRNA-based (miRNA) silencing to dissect the role of β2-AR in anxiety-like behavior. On the one hand, we use a chimeric rhodopsin/β2-AR (Opto-β2-AR) with in vivo optogenetic techniques to selectively activate β2-adrenergic signaling in excitatory neurons of the mPFC. We found that opto-activation of β2-AR is sufficient to induce anxiety-like behavior and reduce the social interaction. On the other hand, we utilize the miRNA silencing technique to specifically knock down the β2-AR in mPFC excitatory neurons. We found that the β2-AR knock down induces anxiolytic-like behavior and promotes the social interaction compared to the control group. These data suggest that β2-AR signaling in the mPFC has a critical role in anxiety-like states. These findings suggest that inhibiting of β2-AR signaling in the mPFC may be an effective treatment of emotional disorders.