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

The study addresses how dopamine signaling contributes to fear extinction and PTSD-like phenotypes, focusing on molecular and circuit mechanisms within amygdala-centered networks. Fear extinction involves new learning that suppresses conditioned responses, and impairments are linked to PTSD. Dopamine has been implicated in signaling aversive US omission and modulating extinction across species, yet the dopaminergic inputs to the amygdala and their molecular regulators are not well defined. The gastrin-releasing peptide (Grp) gene is enriched in excitatory neurons in the BLA and in VTA dopaminergic populations, with GRP and dopamine exerting opposing effects on BLA inhibition. The authors hypothesize that GRP regulates dopamine function in the BLA during fear learning and extinction, and that loss of GRP would dysregulate dopaminergic signaling, enhancing stress-enhanced fear learning and impairing extinction.

Existing literature shows that extinction depends on amygdala, hippocampus, mPFC, NAc, and VTA circuitry, with deficits contributing to PTSD. Beyond reward, dopamine signals the omission of expected aversive outcomes and modulates extinction learning in flies, rodents, and humans. VTA/SNc projections innervate the amygdala, gating plasticity via suppression of feedforward inhibition and influencing intercalated cells. However, the specific molecular pathways within these dopaminergic circuits are unclear. GRP is expressed in BLA excitatory neurons and colocalizes with dopamine markers in VTA; GRP enhances inhibition via GRPR on GABAergic interneurons, whereas dopamine reduces BLA inhibition. Prior work indicates GRPR loss enhances LTP and impairs extinction. The authors build upon these findings to test whether GRP acts as a molecular regulator of amygdala dopamine function during stress-related fear learning and extinction.

- Animals: Generation of Grp knockout (Grp−/−) mice by deleting most of exon 1 and inserting GFP; verification of normal development. Crosses with GRPR KO to create double KO. Adult selective ablation of GRPR-expressing interneurons in BLA using bombesin-saporin. - Circuit mapping: rAAV2-retro-CaMKII-tdTomato retrograde tracing in Grp−/− to map GRPergic projections among auditory thalamus/cortex, amygdala nuclei, vHP, and mPFC; GFP reporter used to mark GRP+ cells. - Behavior: Standard cued and contextual fear conditioning (1 CS-US pairing for some experiments; 2 pairings for SEFL), tests of short-term memory (1 h) and long-term memory (24 h), anxiety-like behavior (EPM, OF, LD), shock reactivity (movement, vocalization, jump). SEFL paradigm: 2 h restraint stress, 7-day interval, cued fear conditioning, extinction in novel context 4 days later (two sessions), recall at 30 days post-shock. Post-shock freezing used to stratify stressed mice into stress-susceptible and stress-resilient. - Neural activity markers: qRT-PCR of IEGs (c-Fos, Arc) in BLA 30 min after fear conditioning. - Dopamine monitoring: In vivo fiber photometry with fluorescent dopamine sensor dLight1.2 targeted to BA/BLA; signals recorded during training and extinction across SEFL protocol. - Optogenetics and electrophysiology: AAV-hSyn-ChR2-EGFP injected into VTA to stimulate VTA-BLA axons; ex vivo whole-cell recordings from BLA principal neurons measured sEPSPs/sIPSPs at baseline and with light stimulation; resting membrane potential and spike activity assessed; TH immunostaining used to confirm dopaminergic projections. - Gene expression: qRT-PCR on BLA tissue 30 min post-Recall for dopamine-related genes (Th, Nurr1, Drd1, Drd2) normalized to Gapdh and β-actin; Western blot for TH protein; RNA-seq on separate cohorts at the same recall time point; DESeq2 differential expression, TPM comparisons, GO enrichment analyses. - Statistics: Two-way and three-way repeated measures ANOVAs, post-hoc tests (Tukey, Bonferroni, Holm-Sidak), t-tests, correlation analyses (R2).

- Memory phenotypes: Grp−/− mice showed enhanced long-term fear memory with intact short-term memory. Contextual LTM increased (p=0.031). Cued LTM two-way ANOVA showed genotype effect (F(1,80)=6.64, p=0.011) with near-threshold CS comparison (p=0.0508). STM unaffected. GRP/GRPR double KO further enhanced contextual and cued LTM (context p=0.018; cued p=0.015). - Adult BLA GRPR interneuron ablation increased cued LTM freezing (Treatment×Stimulus F(1,58)=6.45, p=0.0138; Tukey p=0.0062), with ~50% reduction of GRPR+ cells, confirming adult circuit role. - SEFL behavior: During extinction, genotype main effect (F1,64=18.137, p<0.001). Recall showed Genotype×Stress interaction (F1,64=4.269, p=0.042), with KO-SEFL freezing higher than KO-FL (p=0.0196), WT-SEFL (p=0.0003), and WT-FL (p=0.001). In WT-SEFL, stress-susceptible vs resilient differed during extinction and recall (extinction susceptibility F1,18=11.380, p=0.003; recall p=0.008), but not in KO-SEFL (extinction susceptibility F1,18=1.711, p=0.206; recall p=0.698). KO mice, regardless of susceptibility category, froze more than WT. - Amygdala activity: After fear conditioning, BLA IEGs c-Fos and Arc mRNA increased in Grp−/− vs WT; no differences in naive mice. Anxiety assays and shock sensitivity showed no genotype differences. - Dopamine gene expression post-Recall (qPCR): In BLA, Grp−/− SEFL (KO-SEFL) showed decreased Th (Genotype×Stress interaction, #p<0.05), decreased Nurr1 with stress in KO, and decreased Drd1 with GRP knockout; Drd1 also decreased in KO-FL. No differences in naive VTA/BLA for Th, Nurr1, Drd1, Drd2. - DA anatomy: GRP (GFP) co-expressed with TH in VTA subpopulations; TH+ fibers present in BLA; VTA dopaminergic projections target BA, not LA/BMA GRP+ cells. - DA photometry (dLight1.2): During training, both genotypes showed increases to tone and shock (event F(2,49)=19.47, p<0.0001). KO showed larger shock responses than WT (Group×Event F(2,48)=4.60, p<0.05; KO vs WT shock p=0.0007; another analysis p=0.0187). During extinction day 1, KO had larger tone responses (Group F(1,16)=6.054, p=0.026; Extinction 1 tone p=0.0439; not different on day 2, p=0.7228). Tone responses during extinction correlated with prior shock-evoked DA during training (R2=0.70, p<0.001) but not with prior tone responses (R2=0.096, p=0.33). - VTA-BLA connectivity (optogenetics/ephys): In WT, light stimulation increased sEPSP frequency (Tukey WT OFF vs WT ON p=0.023). In KO, baseline sEPSP frequency matched WT under light and did not increase with light; WT OFF vs KO OFF p=0.0016; WT OFF vs KO ON p=0.0004. sEPSP amplitude unchanged across conditions. sIPSP frequency elevated at KO baseline vs WT baseline (p=0.0034) and remained elevated with light; amplitude unchanged. KO neurons had ~4 mV more depolarized resting membrane potential and increased spike activity. Findings indicate occluded presynaptic VTA-BLA excitatory drive and heightened inhibition baseline in KO. - RNA-seq post-Recall: KO-SEFL vs KO-FL: 452 genes altered (q<0.05); KO-SEFL vs WT-SEFL: 336 genes altered. Th TPM lower in KO-SEFL; Nurr1 lower in SEFL vs FL (significant in KO); Drd1 lower in KO-SEFL vs WT-SEFL; additional decreases: Drd2 and Grik2 in KO-SEFL vs WT-SEFL. Ppm1f decreased in KO-SEFL vs WT-SEFL (stress/PTSD-linked gene). GO: downregulation of synaptic transmission modules (including presynaptic modulation; negative regulation of glutamatergic transmission) and upregulation of microtubule/cilium assembly categories.

Findings demonstrate that GRP is a molecular regulator of dopaminergic control over fear learning and extinction within the VTA–BLA circuit. Loss of GRP enhances dopamine release to aversive shock during conditioning and to shock-paired cues during early extinction, while long-term recall after SEFL produces downregulation of dopamine-related transcripts (Th, Nurr1, Drd1/Drd2) in the BLA. This suggests a biphasic regulation: acute hyperdopaminergic signaling during learning/early extinction and compensatory transcriptional downregulation during remote memory recall. Optogenetics and ex vivo physiology reveal presynaptic VTA-BLA connectivity is functionally occluded in Grp−/− mice, indicating dysregulated dopaminergic input contributes to BLA hyperexcitability and altered inhibitory tone. Behavioral outcomes align with these circuit and molecular changes: enhanced long-term fear memory, increased susceptibility in SEFL, and impaired extinction recall in KO-SEFL. The GRP-dopamine linkage positions GRP as a potential functional biomarker of learned fear processing and stress susceptibility. The RNA-seq data reinforce a specific impact on synaptic transmission genes and microtubule-associated pathways, consistent with observed synaptic physiology alterations. Overall, the work supports dopamine’s role in signaling salient aversive events and regulating extinction, with GRP gating this process to prevent long-term maladaptive fear responses.

This study identifies GRP as a key modulator of dopamine signaling in fear circuits, showing opposite-direction effects: increased dopamine release during conditioning and early extinction, and decreased dopamine-pathway gene expression during remote extinction recall in Grp−/− mice. The results integrate behavioral (enhanced LTM, SEFL susceptibility and impaired recall), circuit (dysregulated VTA-BLA presynaptic drive), and molecular (downregulated Th, Nurr1, Drd1/Drd2; synaptic transmission gene changes) evidence to position GRP as the first gene regulating dopaminergic control of fear extinction. GRP may serve as a biomarker for learned fear processing and a genetic model for PTSD-like phenotypes. Future research should employ temporally and cell-type-specific manipulations to localize GRP/GRPR contributions, map precise timing of transcriptional changes, test causality of dopamine transcriptional modulation on extinction, and evaluate therapeutic strategies that enhance GRP signaling and/or dopamine-related gene expression to improve extinction-based interventions.

- Global knockout: Grp was deleted systemically from early development, raising concerns about developmental and off-target effects. Adult BLA GRPR interneuron ablation partially addressed this but does not fully resolve timing/cell-type specificity. - Incomplete mapping of affiliations aside, experimental: Other neurotransmitter systems (e.g., glutamate) were not comprehensively interrogated, though electrophysiology suggests downstream glutamatergic changes. - Housing effects: Mixed housing of WT and KO might have influenced behavioral phenotypes. - Stress effects in WT group-level SEFL extinction deficits were not replicated here, potentially due to cohort/housing variables. - Mechanistic gap: The compensatory link between acute DA hyperrelease and later transcriptional downregulation remains inferential; more causal experiments are needed (e.g., manipulating DA synthesis/receptors during SEFL). - Fiber photometry implants may alter mobility, potentially affecting extinction freezing measurements.