Adaptive behavior relies on modifying innate responses through learning, especially in response to environmental threats. Learned fear arises when a neutral stimulus (CS) becomes associated with an aversive stimulus (US). Fear extinction, the reduction of conditioned fear responses when the US is absent, involves learning that the CS no longer predicts the US. Extinction doesn't erase the original fear memory but creates a new memory representation. However, deficits in extinction can lead to excessive fear, mirroring PTSD in humans. Fear extinction involves complex interactions between brain regions, including the amygdala, hippocampus, nucleus accumbens, medial prefrontal cortex (mPFC), and ventral tegmental area (VTA). Dopamine plays a critical role in fear extinction, signaling the omission of the expected aversive US, but the underlying neural circuits and molecular mechanisms remain largely unknown. This study focuses on the gastrin-releasing peptide (GRP), enriched in BLA excitatory neurons and VTA dopaminergic neurons, to investigate its potential role in regulating dopamine function in the BLA during fear extinction. GRP and dopamine have opposing effects on conditioned fear inhibition, suggesting a potential interaction in fear processing. This research uses a stress-enhanced fear learning (SEFL) paradigm and *Grp* knockout mice to explore the role of GRP in fear extinction.

Extensive research has established the amygdala's central role in fear processing. Studies have shown the importance of the amygdala, hippocampus, NAc, mPFC, and VTA in fear extinction. Deficits in fear extinction are a hallmark of PTSD. While rodent models offer valuable tools to investigate impaired fear extinction, the underlying molecular mechanisms and specific cell populations involved remain poorly understood. A growing body of evidence emphasizes dopamine's significance in fear extinction, with studies demonstrating its role in signaling the omission of expected aversive US across various species. Dopamine neurons, projecting from the VTA and substantia nigra (SNc) to the amygdala, are implicated in fear circuit modulation. However, the precise mechanisms by which dopaminergic inputs to the amygdala process fear extinction, the specific cell types and molecular pathways involved, are not well characterized. The role of the GRP system in fear extinction and PTSD is not well-established, although it is known to modulate the inhibitory processes in the amygdala.

The study employed *Grp* knockout (*Grp⁻⁻*) mice, generated by deleting most of exon 1 and replacing it with GFP. The researchers mapped GRPergic neural circuitry using retrograde tracing with rAAV2-retro-CaMKII-tdTomato. Fear conditioning and extinction were assessed using standard protocols, including cued and contextual fear tests, and short-term and long-term memory assessments. The SEFL paradigm, combining stress exposure (2 hours of restraint) and fear extinction, was used to model PTSD. In vivo fiber photometry with dLight1.2 was employed to record dopamine signals in the BLA during SEFL. Ex vivo electrophysiology combined with optogenetics (AAV-hSyn-ChR2-EGFP injection into the VTA) assessed VTA-BLA connectivity. RNA-seq and qPCR were used to analyze gene expression in the BLA, focusing on dopamine-related genes after SEFL memory recall. Anxiety and pain sensitivity were assessed using the elevated plus maze, open field, light-dark box, and pain sensitivity tests. In addition, bombesin-saporin injections were used to selectively ablate GRPR-expressing interneurons in the BLA of wildtype mice to investigate the amygdala-specific effects of GRP signaling.

1. *Grp⁻⁻* mice exhibited significantly enhanced long-term fear memory in both cued and contextual fear conditioning paradigms, but short-term memory was unaffected. 2. Ablation of GRPR cells in the BLA of wildtype mice using bombesin-saporin also resulted in enhanced cued fear memory. 3. *Grp⁻⁻* mice showed increased susceptibility to SEFL, exhibiting higher freezing during the recall test compared to wildtype mice. This effect was specific to the SEFL paradigm, and was not observed in animals undergoing standard fear learning. 4. In vivo fiber photometry demonstrated enhanced dopamine release in the BLA of *Grp⁻⁻* mice during fear conditioning and early extinction phases. 5. Optogenetic stimulation revealed occluded presynaptic VTA-BLA connectivity in *Grp⁻⁻* mice, suggesting dysregulated dopaminergic input from the VTA in the absence of GRP. 6. RNA-seq and qPCR analyses revealed a concerted downregulation of dopamine-related genes (*Th*, *Nurr1*, *Drd1*, *Drd2*) in the BLA of *Grp⁻⁻* mice following SEFL memory recall, suggesting a compensatory mechanism. 7. Co-immunostaining showed co-expression of GRP and tyrosine hydroxylase in the VTA, indicating that a subset of VTA dopaminergic neurons also express GRP.

The findings demonstrate that GRP plays a critical and complex role in regulating dopamine signaling during fear processing. The opposite directions of modulation (enhanced dopamine release early, downregulation of dopamine-related genes late) suggest a dynamic interplay between GRP and dopamine across different phases of fear learning and extinction. The enhanced dopamine release in *Grp⁻⁻* mice during fear learning and extinction, combined with the subsequent downregulation of dopamine-related genes during recall, may reflect a compensatory mechanism to cope with excessive dopamine signaling. The occluded VTA-BLA connectivity observed in *Grp⁻⁻* mice indicates the importance of GRP for the integrity of dopaminergic input to the BLA. These findings provide new insights into the molecular mechanisms underlying fear extinction deficits and may have implications for understanding the pathophysiology of PTSD. The study strongly suggests that GRP acts as a critical node linking stress and fear processing, and the GRP-dopamine interaction is crucial for both acquisition and extinction of fear memories.

This study identifies GRP as a crucial regulator of dopaminergic control of fear extinction, impacting both early (increased dopamine release) and late (downregulation of dopamine-related genes) stages of fear processing. The *Grp⁻⁻* mouse model exhibits PTSD-like symptoms, particularly enhanced susceptibility to SEFL. The findings highlight GRP's potential as a functional biomarker of learned fear processing and suggest therapeutic strategies targeting GRP and dopamine signaling pathways for PTSD treatment. Future research should investigate the temporal dynamics of GRP-dopamine interaction and explore the potential of GRP-based therapies in combination with exposure-based therapies.

The use of a global *Grp* knockout approach might limit the precise identification of GRP's action sites and the extent to which compensatory mechanisms influence the observed effects. While the bombesin-saporin experiments partially address amygdala-specific effects, it still doesn't rule out possible developmental contributions. Future research using conditional knockouts and cell-type specific manipulations will further clarify the precise role of GRP in different brain regions and cell types. The study primarily focused on the GRP-dopamine interaction, and further research is needed to explore interactions with other neurotransmitter systems and signaling pathways.