Research ArticleNeuroscience

β-Arrestin–biased β-adrenergic signaling promotes extinction learning of cocaine reward memory

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Sci. Signal.  09 Jan 2018:
Vol. 11, Issue 512, eaam5402
DOI: 10.1126/scisignal.aam5402
  • Fig. 1 The β-adrenergic receptor antagonist propranolol, but not carvedilol, impaired extinction learning of cocaine-CPP and extinction learning–induced ERK activation.

    (A) Experimental scheme. After cocaine–cocaine-conditioned place preference (CPP) training, mice were subjected to a daily extinction session for 7 days, and propranolol, carvedilol, or vehicle was infused in the infralimbic prefrontal cortex (IL-PFC) within 10 min after each extinction trial. For Western blotting, the IL-PFC was collected 15 min after the fourth extinction trial. (B) Effect of propranolol on extinction learning of cocaine-CPP (propranolol, 14 mice; vehicle, 19 mice). **P < 0.01 compared to vehicle; #P < 0.05 as indicated. (C) Effect of carvedilol on extinction learning of cocaine-CPP (carvedilol, 27 mice; vehicle, 30 mice). *P < 0.05 compared to vehicle. (D) Effect of propranolol on extinction-induced extracellular signal–regulated kinase (ERK) activation in the IL-PFC. p, phosphorylated. ***P < 0.005 compared to control/vehicle; ###P < 0.005 compared to extinction/vehicle. (E) Effect of carvedilol on extinction-induced ERK activation in the IL-PFC. For (D) and (E), the number of mice per group is indicated in the bars. ***P < 0.005 compared to control/vehicle; ###P < 0.005 compared to vehicle. Error bars denote ±SEM. See table S2 for additional statistical test details and results.

  • Fig. 2 β-Arrestin2 local knockout in the IL-PFC blocks extinction learning of cocaine-CPP.

    (A) Experimental scheme. AAV-EF1α:eGFP-T2A-Cre or AAV-EF1α:eGFP was infused into the IL-PFC of Arrb2flox/flox mice after cocaine-CPP training. After a 2-week recovery, the mice were subjected to daily extinction sessions. (B) Infection of neurons in the IL-PFC by adeno-associated virus (AAV) and immunostaining for neuronal nuclei (NeuN) 2 weeks after bilateral AAV injection. Scale bars, 100 μm. (C) Arrb2 messenger RNA (mRNA) expression 2 weeks after injection of AAV-EF1α:eGFP (eGFP) or AAV-EF1α:eGFP-T2A-Cre (Cre) into the IL-PFC of Arrb2flox/flox mice. Number of mice per group is indicated in the bars. **P < 0.01 compared to enhanced green fluorescent protein (eGFP). (D) Effect of β-arrestin2 deletion in the IL-PFC on extinction learning of cocaine-CPP (eGFP/Arrb2flox/flox, 16 mice; Cre/Arrb2flox/flox, 18 mice). *P < 0.05, ***P < 0.005 compared to eGFP/Arrb2flox/flox; #P < 0.05 as indicated. (E) Correlation of the percentage of eGFP+ neurons in the IL-PFC of Arrb2flox/flox mice with the extent of cocaine-CPP extinction (eGFP/Arrb2flox/flox, 8 mice; Cre/Arrb2flox/flox, 10 mice). (F) Experimental scheme. After cocaine-CPP training, AAVs were infused in the IL-PFC of Arrb2flox/flox mice. Two weeks later, these mice were subjected to retrieval-extinction sessions, and the memory retention test was performed 1 day later. (G) Effect of β-arrestin2 ablation in the IL-PFC on the memory retention test after retrieval-extinction session of cocaine-CPP (eGFP/Arrb2flox/flox, 17 mice; Cre/Arrb2flox/flox, 16 mice). ***P < 0.005 compared to Cre/Arrb2flox/flox in pretest; ###P < 0.005 as indicated; &&&P < 0.005 compared to eGFP/Arrb2flox/flox in retrieval. (H) Correlation of the percentage of eGFP+ neurons in the IL-PFC of Arrb2flox/flox mice with the extent of the extinction of cocaine-CPP. Error bars denote ±SEM (eGFP/Arrb2flox/flox, nine mice; Cre/Arrb2flox/flox, eight mice). See table S3 for additional statistical test details and results.

  • Fig. 3 Overexpression of β-arrestin2 in the IL-PFC promotes extinction learning of cocaine-CPP.

    (A) Experimental scheme. After cocaine-CPP training, AAVs were infused into the IL-PFC. After a 2-week recovery, daily extinction sessions were carried out. (B) Construction of recombinant AAV encoding hemagglutinin (HA)–tagged β-arrestin2 under the control of a human synapsin promoter inserted between inverted terminal repeats (ITR) (AAV-hSyn:HA-Arrb2-T2A-eGFP). (C) Effect of injection of AAV-hSyn:HA-Arrb2-T2A-eGFP or AAV-hSyn:HA-galactosidase (Gal)-T2A-eGFP on Arrb2 mRNA abundance. Number of mice per group is indicated in the bars. *P < 0.05 compared to Gal. (D) Left: The expression of eGFP in IL-PFC. Right: Co-immunostaining of HA and NeuN in the IL-PFC. Scale bars, 100 μm. (E) Effect of β-arrestin2 overexpression on extinction learning of cocaine-CPP (Arrb2-eGFP, 31 mice; Gal-eGFP, 16 mice). *P < 0.05, ***P < 0.005, compared to AAV-Gal-eGFP; #P < 0.05 as indicated. (F) Correlation of the percentage of eGFP+ neurons in the IL-PFC with the extent of the extinction of cocaine-CPP (Arrb2-eGFP, 10 mice; Gal-eGFP, 11 mice). Error bars denote ±SEM. See table S4 for additional statistical test details and results.

  • Fig. 4 Extinction learning of cocaine-CPP depends on β-arrestin2–biased β-adrenergic signaling.

    (A) Experimental scheme. AAV-hSyn:HA-Arrb2-T2A-eGFP or AAV-EF1α:eGFP-T2A-Cre was infused bilaterally in the IL-PFC of C57 mice or Arrb2flox/flox mice after cocaine-CPP training. Propranolol or carvedilol was infused in the IL-PFC within 10 min after each extinction trial. (B) Effect of propranolol on the enhanced extinction caused by β-arrestin2 overexpression in the IL-PFC (vehicle; 17 mice; propranolol, 15 mice). *P < 0.05, ***P < 0.005 compared to vehicle; #P < 0.05 as indicated. (C) Effect of carvedilol on the impaired CPP extinction caused by β-arrestin2 knockout in the IL-PFC (Cre/Arrb2flox/flox, 16 mice; eGFP/Arrb2flox/flox, 15 mice). *P < 0.05, **P < 0.01 compared to eGFP/Arrb2flox/flox; #P < 0.05 as indicated. Error bars denote ±SEM. See table S5 for additional statistical test details and results.

  • Fig. 5 β-arrestin2 deletion in excitatory neurons in the IL-PFC blocks extinction learning of cocaine reward memory.

    (A) Experimental scheme. AAV-CaMKIIα:GFP-Cre was infused into the IL-PFC of Arrb2flox/flox mice and their wild-type (WT) littermates after cocaine-CPP training. In another cohort of mice, CAV2-Cre was infused in the nucleus accumbens (NAc) shell to infect IL-PFC neurons projecting to the NAc shell in a retrograde fashion, and AAV-hSyn:Flex-tdTomato was infused in the IL-PFC of Arrb2flox/flox mice and their WT littermates. After a 2-week recovery, the mice were subjected to daily extinction sessions. (B) Immunostaining for NeuN 2 weeks after bilateral AAV injection. Scale bars, 100 μm. (C) Effect of β-arrestin2 deletion in excitatory neurons in the IL-PFC on extinction learning of cocaine-CPP (WT, 16 mice; Arrb2flox/flox, 15 mice). *P < 0.05, **P < 0.01 compared to WT; #P < 0.05 as indicated. (D) AAV injection sites (left) and expression of tdTomato (right) in the IL-PFC. Scale bar, 100 μm. CPu, caudate putamen; AcbSh, NAc shell. (E) Effect of β-arrestin2 deletion in IL-PFC neurons projecting to the NAc shell after the acquisition of cocaine-CPP on extinction learning (WT, 18 mice; Arrb2flox/flox, 17 mice). **P < 0.01, ***P < 0.005 compared to WT; #P < 0.05 as indicated. (F) Experimental scheme. LV-DIO-Arrb2-shRNA (short hairpin–mediated RNA) or LV-DIO–scramble shRNA was infused into the IL-PFC of CaMKIIα:Cre mice before cocaine self-administration training. (G) Effect of LV-DIO-Arrb2-shRNA and LV-DIO–scramble shRNA injection on cocaine self-administration (0.5 mg/kg per infusion) training on fixed-ratio 1 (FR-1) and FR-3 schedule. Active, portal producing a cocaine injection; inactive, portal yielding no cocaine injection (scramble shRNA, 19 mice; Arrb2-shRNA, 19 mice). (H) Effect of β-arrestin2 knockdown in excitatory neurons in the IL-PFC on extinction learning of cocaine self-administration in nose poke extinction session (scramble shRNA, 19 mice; Arrb2-shRNA, 19 mice). *P < 0.05, ***P < 0.005 compared to scramble shRNA; #P < 0.05 as indicated. (I) Effect of β-arrestin2 knockdown in excitatory neurons in the IL-PFC on cue-induced reinstatement after cue extinction session. Number of mice per group is indicated in the bars. *P < 0.05 compared to scramble shRNA/cue extinction; ##P < 0.01 compared to scramble shRNA/no cue extinction. Error bars denote ±SEM. See table S6 for additional statistical test details and results.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/512/eaam5402/DC1

    Fig. S1. The IL-PFC was activated during extinction learning of cocaine-induced CPP.

    Fig. S2. Generation of β-arrestin2 conditional knockout mice.

    Fig. S3. Conditional knockout of β-arrestin2 in IL-PFC inhibitory neurons did not impair extinction learning of cocaine-CPP.

    Table S1. Summary of statistics for fig. S1.

    Table S2. Summary of statistics for Fig. 1.

    Table S3. Summary of statistics for Fig. 2.

    Table S4. Summary of statistics for Fig. 3.

    Table S5. Summary of statistics for Fig. 4.

    Table S6. Summary of statistics for Fig. 5.

    Table S7. Summary of statistics for fig. S3.

  • Supplementary Materials for:

    β-Arrestin–biased β-adrenergic signaling promotes extinction learning of cocaine reward memory

    Bing Huang, Youxing Li, Deqin Cheng, Guanhong He, Xing Liu,* Lan Ma*

    *Corresponding author. Email: lanma{at}fudan.edu.cn (L.M.); xingliu{at}fudan.edu.cn

    This PDF file includes:

    • Fig. S1. The IL-PFC was activated during extinction learning of cocaine-induced CPP.
    • Fig. S2. Generation of β-arrestin2 conditional knockout mice.
    • Fig. S3. Conditional knockout of β-arrestin2 in IL-PFC inhibitory neurons did not impair extinction learning of cocaine-CPP.
    • Table S1. Summary of statistics for fig. S1.
    • Table S2. Summary of statistics for Fig. 1.
    • Table S3. Summary of statistics for Fig. 2.
    • Table S4. Summary of statistics for Fig. 3.
    • Table S5. Summary of statistics for Fig. 4.
    • Table S6. Summary of statistics for Fig. 5.
    • Table S7. Summary of statistics for fig. S3.

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    Citation: B. Huang, Y. Li, D. Cheng, G. He, X. Liu, L. Ma, β-Arrestin–biased β-adrenergic signaling promotes extinction learning of cocaine reward memory. Sci. Signal. 11, eaam5402 (2018).

    © 2018 American Association for the Advancement of Science