Dopamine release and dopamine-related gene expression in the amygdala are modulated by the gastrin-releasing peptide in opposite directions during stress-enhanced fear learning and extinction

Adaptive behavior necessitates learning to navigate environmental threats, which can be innate or learned. Learned fear involves associating a neutral stimulus (CS) with an aversive unconditioned stimulus (US), leading to a conditioned fear response. Fear extinction, the reduction of this response after the threat ceases, involves creating a new memory representing the lack of current threat. However, deficits in extinction can lead to excessive fear, mirroring PTSD in humans. Fear extinction involves interactions between brain regions like the amygdala, hippocampus, nucleus accumbens, medial prefrontal cortex, and ventral tegmental area (VTA). While the dopaminergic system is linked to reward learning, growing evidence suggests its importance in fear extinction, signaling the omission of expected aversive US. Dopamine neurons influence fear circuits through projections to the amygdala. Understanding how these inputs process fear extinction is crucial. This study focused on the gastrin-releasing peptide (GRP) gene, enriched in BLA excitatory neurons and VTA dopaminergic neurons. GRP and dopamine have opposing effects on conditioned fear inhibition. The study aimed to investigate the role of BLA and VTA GRPergic neurons in fear extinction using a Stress-Enhanced Fear Learning (SEFL) paradigm and *Grp* knockout mice.

The literature extensively documents the neural circuitry involved in fear conditioning and extinction, emphasizing the amygdala's central role. Studies highlight the interaction of the amygdala with other brain regions like the hippocampus, prefrontal cortex, and VTA in regulating fear responses. The involvement of dopamine in fear extinction is increasingly recognized, with research demonstrating its role in signaling the absence of expected aversive outcomes. The opposing effects of GRP and dopamine on fear inhibition have been described, setting the stage for investigating their interaction. Rodent models of PTSD, characterized by impaired fear extinction, provide valuable tools for studying this phenomenon. Prior research has investigated specific brain circuits but lacked detailed knowledge of the molecular mechanisms and cell types involved in fear extinction and PTSD. The GRP's known presence in specific neuronal populations of the amygdala and VTA, along with its opposing effects relative to dopamine, made it a prime candidate for investigation.

The study used *Grp* knockout (*Grp*) mice, where exon 1 of the *Grp* gene was replaced with GFP, enabling the tracking of GRP expression. Immunohistochemistry mapped GFP expression, revealing its enrichment in the amygdala, hippocampus, and mPFC. Retrograde tracing with rAAV2 mapped GRPergic neuron circuitry. Fear conditioning protocols assessed short-term memory (STM) and long-term memory (LTM) in *Grp* mice, and double knockouts (*Grp*/GRPR KO) were analyzed to understand GRP/GRPR interaction. Selective GRPR ablation in adult wildtype mice was performed using bombesin-saporin injections. The Stress-Enhanced Fear Learning (SEFL) paradigm, combining restraint stress and fear extinction, assessed the role of GRP in stress-fear interaction. In vivo fiber photometry using dLight recorded dopamine signals in the BLA during SEFL. Ex vivo electrophysiology examined VTA-BLA connectivity using optogenetics. RNA-seq and qPCR assessed dopamine-related gene expression in the BLA. Statistical analyses involved two-way ANOVAs, t-tests, and post-hoc tests.

The *Grp* mice exhibited enhanced LTM in both cued and contextual fear conditioning, but STM was unaffected. *Grp*/GRPR double KO mice showed even stronger LTM enhancement, indicating potential secondary ligands/receptors. Selective GRPR ablation also increased freezing, confirming the amygdala's role in GRP-mediated fear memory. *Grp* mice showed increased c-Fos and Arc expression in the BLA after fear conditioning. Anxiety and pain sensitivity were normal in *Grp* mice. In the SEFL paradigm, *Grp* mice displayed increased susceptibility to stress-enhanced fear learning, with higher freezing during recall. qPCR analysis revealed downregulation of dopamine-related genes (*Th*, *Nurr1*, *Drd1*) in the BLA of *Grp* mice after SEFL recall. In vivo photometry showed enhanced dopamine responses to shock and learned fear cues in *Grp* mice during training and early extinction. Optogenetics and ex vivo electrophysiology indicated occluded presynaptic VTA-BLA connectivity in *Grp* mice. RNA-seq confirmed the qPCR results and identified additional downregulated genes, including *Drd2* and *Ppm1f*, associated with stress and PTSD. The study highlights that neither stress alone nor *Grp* deletion had significant effects; rather, a synergistic effect was observed.

This study identifies GRP as a crucial regulator of dopaminergic control over fear learning and extinction. Disrupting this link enhances dopamine release in the amygdala during fear learning and early extinction, yet decreases dopamine-related gene transcription after extinction memory recall. The opposing effects of GRP on dopamine underscore its role in fear processing. The findings support the role of dopamine in salience signaling, suggesting GRP's involvement in initial fear memory encoding and long-term memory recall. The study maps the GRP-positive auditory pathway to the amygdala, revealing that only the indirect thalamo-cortico-amygdala pathway, not the direct thalamo-amygdala pathway, contains GRP. The *Grp* mouse model suggests a potential genetic link to PTSD, showing PTSD-like symptoms without altered anxiety or pain sensitivity. The study implies that enhancing long-term dopamine function, potentially by increasing transcription of dopamine-related genes, could improve extinction of undesired fear, with implications for PTSD treatment.

This research establishes GRP as a key regulator of dopaminergic control of fear extinction. GRP knockout mice exhibit enhanced fear memory and susceptibility to stress-enhanced fear learning, due to dysregulated dopamine signaling and VTA-BLA connectivity. Downregulation of dopamine-related genes following extinction memory recall is observed in these mice. The study identifies GRP as a potential biomarker for learned fear processing and proposes the *Grp* mouse as a model for PTSD, suggesting novel therapeutic strategies targeting GRP and dopamine.

The use of classical gene knockout approach in *Grp* mice might have developmental effects impacting interpretation. Future studies with conditional knockouts and more precise temporal control of GRP manipulation would further refine understanding of its role. While the study focuses on the GRP-dopamine link, other neurotransmitters might be involved; future investigations into other neurotransmitter systems in these mice are needed. The mixed-group housing of knockout and wildtype mice may have affected behavior, so future experiments might consider single-housing.