Dorsal Raphe to Basolateral Amygdala Corticotropin-Releasing Factor Circuit Regulates Cocaine-Memory Reconsolidation

Abstract

Environmental stimuli elicit drug craving and relapse in cocaine users by triggering the retrieval of strong cocainerelated contextual memories. Retrieval can also destabilize drug memories, requiring reconsolidation, a protein synthesis-dependent storage process, to maintain memory strength. Corticotropin-releasing factor (CRF) signaling in the basolateral amygdala (BLA) is necessary for cocainememory reconsolidation. We have hypothesized that a critical source of CRF in the BLA is the dorsal raphe nucleus (DR) based on its neurochemistry, anatomical connectivity, and requisite involvement in cocaine-memory reconsolidation. To test this hypothesis, male and female Sprague-Dawley rats received adeno-associated viruses to express Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) selectively in CRF neurons of the DR and injection cannulae directed at the BLA. The rats were trained to self-administer cocaine in a distinct environmental context then received extinction training in a different context. They were then briefly reexposed to the cocaine-predictive context to destabilize (reactivate) cocaine memories. Intra-BLA infusions of the DREADD agonist deschloroclozapine (DCZ; 0.1 mM, 0.5 μL/hemisphere) after memory reactivation attenuated cocaine-memory strength, relative to vehicle infusion. This was indicated by a selective, DCZ-induced and memory reactivation-dependent decrease in drug-seeking behavior in the cocaine-predictive context in DREADD-expressing males and females at test compared to respective controls. Notably, BLA-projecting DR CRF neurons that exhibited increased c-Fos expression during memory reconsolidation co-expressed glutamatergic and serotonergic neuronal markers. Together, these findings suggest that the DR_CRF → BLA circuit is engaged to maintain cocaine-memory strength after memory destabilization, and this phenomenon may be mediated by DR CRF, glutamate, and/or serotonin release in the BLA.

Figure 1.. Validation study results.

(A) Corticotropin-releasing factor (CRF) antibody specificity as indicated by colocalization between CRF mRNA, visualized using fluorescence in situ hybridization (CRF ISH), and CRF protein, labeled using immunohistochemistry (CRF IHC), in 97.1 ± 0.9 % of the DR cell bodies examined. (B) Schematic of the cell- and circuit-specific chemogenetic approach. Rats received an infusion of a cocktail of viruses (200 nL) into the midline dorsal raphe (DR) to express the hM4Di fused with mCherry (Gi) or mCherry alone (Control; Ctrl) Cre-dependently, under the CRF promoter. Guide cannulae were implanted into the basolateral amygdala (BLA) to permit bilateral infusions of deschloroclozapine (DCZ) in the terminal region of the target circuit. (C) Representative hM4Di-mCherry expression in the DR and colocalization of hM4Di-mCherry expression with CRF-immunoreactivity seen in 96.9 ± 0.6 % of DR mCherryexpressing neurons examined. (D) Representative mCherry expression in the DR and colocalization of mCherry expression with CRFimmunoreactivity. (E) Representative mCherry-expressing DR CRF axon terminals in the BLA and adjacent central amygdaloid nucleus (CeA) and injection cannula tract in the BLA. (F) Experimental timeline for DREADD efficacy and anatomical selectivity assessment. A subset of rats received a memory-reactivation session ~6 days after their last test session in Experiment 1 or 2. Memory reactivation was followed immediately by intra-BLA VEH or DCZ treatment [(Gi) VEH: n = 6 males, 7 females; (Ctrl) DCZ: n = 2 males, 7 females; (Gi) DCZ: n = 5 males, 4 females)]. Brain tissue was collected 2 hours later to assess Zif268 expression in the BLA and adjacent CeA. (G) Representative images of Zif268-immunoreactive nuclei and mCherry expression in the BLA. (H) Density of Zif268 expression (mean number of nuclei/mm² ± SEM) in the BLA (upper panel) and CeA (lower panel). Symbols: ANOVA *treatment group main effect or t-test; ^⚥sex main effect (underlined). All ps < 0.05. Abbreviations: Aq, cerebral aqueduct.

Figure 2.. Chemogenetic DRCRF → BLA circuit inhibition prior to memory reconsolidation weakens cocaine-memory strength.

(A) Timeline of Experiment 1. The Gi-coupled DREADD hM4Di fused with mCherry (Gi) or mCherry alone (control; Ctrl) was expressed in dorsal raphe (DR) corticotropin-releasing factor (CRF) neurons and guide cannulae were directed at the basolateral amygdala (BLA). Rats then received cocaine self-administration training in a salient environmental context and extinction training in a different context. Next, they received 15-minute re-exposure to the cocaine-predictive context (i.e., memory reactivation) followed by bilateral intra-BLA microinjections of vehicle (VEH) [(Gi) VEH; n = 10 males, 11 females)] or the DREADD agonist deschloroclozapine (DCZ) [(Ctrl) DCZ: n = 8 males, 8 females; (Gi) DCZ: n = 10 males, 8 females)]. Non-reinforced lever responding in the extinction and cocaine-predictive contexts was tested 24 and 72 hours later, respectively. (B) Injection cannula placements in the BLA. The numbers between the schematics indicate AP distance from Bregma in millimeters. (C) Cocaine infusions and active- and inactive-lever responses (mean/2 h ± SEM) during cocaine self-administration training (last 10 sessions) and extinction training. (D) Active-lever responses during the memory-reactivation session (mean/15 minutes ± SEM) immediately prior to intra-BLA treatment. (E) Active-lever responses (mean/2 hours ± SEM) upon first re-exposure to the extinction and cocaine-predictive contexts after intra-BLA treatment. (F) Active-lever response latency (mean ± SEM) in the cocaine-predictive context at test. (G) Time course of active-lever responses (mean/20 min ± SEM) in the cocaine-predictive context at test. Symbols: ANOVA ^‡time main (underlined) or simple main effect (see details for pairwise comparisons in Results); ^⚥sex simple main effect; ^#context simple main effect; *treatment group simple main effect. All ps ≤ 0.05.

Figure 3.. Chemogenetic inhibition of the DRCRF → BLA circuit after memory reconsolidation does not alter cocaine-memory strength.

(A) Timeline of Experiment 2. The Gi-coupled DREADD hM4Di fused with mCherry (Gi) was expressed in dorsal raphe (DR) corticotropin-releasing factor (CRF) neurons and guide cannulae were directed at the basolateral amygdala (BLA). Rats then received cocaine self-administration training in a distinct context and extinction training in a different context. Next, they received 15-minute reexposure to the cocaine-predictive context (i.e., memory reactivation). Six hours later, the rats received bilateral intra-BLA injections of vehicle (VEH) [(Gi) VEH; n = 6 males, 5 females)] or the DREADD agonist deschloroclozapine (DCZ) [(Gi) DCZ; n = 5 males, 5 females)]. Non-reinforced lever responding in the extinction and cocaine-predictive contexts was tested 24 and 72 hours later, respectively. (B) Injection cannula placements in the BLA. The numbers between the schematics indicate AP distance from Bregma in millimeters. (C) Cocaine infusions and active- and inactive-lever responses (mean/2 h ± SEM) during cocaine self-administration training (last 10 sessions) and extinction training. (D) Active-lever responses during the memory-reactivation session (mean/15 minutes ± SEM) 6 hours prior to intra-BLA treatment. (E) Active-lever responses (mean/2 hours ± SEM) upon first re-exposure to the extinction and cocainepredictive contexts after delayed intra-BLA treatment. (F) Active-lever response latency (mean ± SEM) in the cocaine-predictive context at test. (G) Time course of active-lever responses (mean/20 min ± SEM) in the cocaine-predictive context at test. Symbols: ^‡time main (underlined) or simple main effect (see details for pairwise comparisons in Results); ^#context main effect; All ps < 0.05.

Figure 4.. BLA-projecting DR CRF neurons activated during cocaine-memory reconsolidation co-express vGlut3 and TPH2.

(A) Timeline of Experiment 3. GFP was expressed in basolateral amygdala (BLA) -projecting corticotropin-releasing factor (CRF) cell bodies in the dorsal raphe (DR) using a combinatorial viral approach. Rats then received cocaine self-administration training in a distinct context and extinction in a different context. Next, they received 15-minute re-exposure to the cocaine-predictive context to reactive cocaine memories (MR; n = 4 males, 4 females) or remained in their home cages (No-MR; n = 4 males, 4 females). Brain tissue was collected 2 hours later to capture c-Fos expression approximately 30 minutes into memory reconsolidation. (B) Cocaine infusions and active- and inactive-lever responses (mean/2 h ± SEM) during cocaine self-administration training (last 10 sessions), extinction training sessions, and memory reactivation. (C) GFP-expressing (BLA-projecting CRF) cell body density in the DR (mean cell bodies/mm² ± SEM) in the No-MR and MR groups. (D) Representative image of GFP expression in BLA-projecting DR neurons and in their axon terminals within the BLA of the same subject. (E) Colocalization between GFP-expression and CRF-immunoreactivity seen in 98 ± 0.2% of DR neurons examined. (F, upper panel) Density of c-Fos⁺ GFP-expressing (GFP⁺) neuronal populations in the DR, following No-MR and MR (mean cell bodies/mm² ± SEM). Data are shown collapsed across sex where sex differences were not observed. (F, Inset) Proportion of c-Fos⁺ GFP-expressing neurons following No-MR and MR. (F, lower panel) Representative images of c-Fos⁺ GFP-expressing cell types observed in the DR. (G, upper panel) Density of c-Fos⁺ GFP-non-expressing (GFP⁻) cell types following No-MR and MR (mean cell bodies/mm² ± SEM). Data are shown collapsed across sex where sex differences were not observed. (G, Inset) Total c-Fos expression (mean GFP⁺ plus GFP⁻ cell bodies/mm² ± SEM) following No-MR and MR. Black segment represents cFos⁺ GFP⁺ neurons collapsed across cell type. (G, lower panel) Representative images of c-Fos⁺ GFP-non-expressing cell types observed in the DR. Symbols: ^‡time main effect; *treatment group simple main effect (underlined) or t-test, No-MR vs MR; ^⚥sex main effect; All p < 0.05. Abbreviations: Aq, cerebral aqueduct; dDR, dorsal subregion of dorsal raphe; lDR, lateral subregion of dorsal raphe; vDR, ventral subregion of dorsal raphe; PAG, periaqueductal gray.

Figure 5.. Colocalization of c-Fos with neuronal phenotype markers in the DR.

Stitched confocal microscopy images of c-Fosimmunoreactivity and cell type markers in the dorsal raphe (DR) of representative rats in the memory reactivation (MR) and no-memory reactivation (No-MR) groups. White arrows indicate colocalization of c-Fos with a cell type marker: (A) GFP (BLA-projecting CRF neurons), (B) vGlut3 (glutamate cell marker), (C) TPH2 (serotonin cell marker), and (D) TH⁺ (dopamine cell marker in the DR). (E) Merged image of c-Fos and all cell type markers. Abbreviations: Aq, cerebral aqueduct; dDR, dorsal subregion of dorsal raphe; lDR, lateral subregion of dorsal raphe; vDR, ventral subregion of dorsal raphe; PAG, periaqueductal gray.