Delayed noradrenergic activation in the dorsal hippocampus promotes the long-term persistence of extinguished fear - PubMed (original) (raw)

. 2014 Jul;39(8):1933-45.

doi: 10.1038/npp.2014.42. Epub 2014 Feb 19.

Jian-Feng Liu 2, Yan-Xue Xue 3, Chang Yang 4, Wei Yan 2, Hui-Min Wang 2, Yi-Xiao Luo 2, Hai-Shui Shi 5, Ji-Shi Wang 4, Yan-Ping Bao 3, Shi-Qiu Meng 2, Zeng-Bo Ding 3, Xue-Yi Wang 6, Lin Lu 7

Affiliations

Delayed noradrenergic activation in the dorsal hippocampus promotes the long-term persistence of extinguished fear

Ning Chai et al. Neuropsychopharmacology. 2014 Jul.

Abstract

Fear extinction has been extensively studied, but little is known about the molecular processes that underlie the persistence of extinction long-term memory (LTM). We found that microinfusion of norepinephrine (NE) into the CA1 area of the dorsal hippocampus during the early phase (0 h) after extinction enhanced extinction LTM at 2 and 14 days after extinction. Intra-CA1 infusion of NE during the late phase (12 h) after extinction selectively promoted extinction LTM at 14 days after extinction that was blocked by the β-receptor antagonist propranolol, protein kinase A (PKA) inhibitor Rp-cAMPS, and protein synthesis inhibitors anisomycin and emetine. The phosphorylation levels of PKA, cyclic adenosine monophosphate response element-binding protein (CREB), GluR1, and the membrane GluR1 level were increased by NE during the late phase after extinction that was also blocked by propranolol and Rp-cAMPS. These results suggest that the enhancement of extinction LTM persistence induced by NE requires the activation of the β-receptor/PKA/CREB signaling pathway and membrane GluR1 trafficking. Moreover, extinction increased the phosphorylation levels of Erk1/2, CREB, and GluR1, and the membrane GluR1 level during the late phase, and anisomycin/emetine alone disrupted the persistence of extinction LTM, indicating that the persistence of extinction LTM requires late-phase protein synthesis in the CA1. Propranolol and Rp-cAMPS did not completely disrupt the persistence of extinction LTM, suggesting that another β-receptor/PKA-independent mechanism underlies the persistence of extinction LTM. Altogether, our results showed that enhancing hippocampal noradrenergic activity during the late phase after extinction selectively promotes the persistence of extinction LTM.

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Figures

Figure 1

Figure 1

Norepinephrine in the CA1 during the early or late phase after extinction enhanced extinction memory. (a) Timeline for this experiment. Norepinephrine was microinfused into the CA1 subarea of the dorsal hippocampus at 0, 6, 12, and 24 h after extinction. Extinction memory was tested 2 and 14 days later. After the second extinction LTM test, a footshock (0.8 mA, 1 s) was given, and fear reinstatement was tested 1 day later. (b) Representative cannula placement in the CA1 of rat. (c) Norepinephrine at doses of 0.3 and 1.0 μg/μl microinfused into the CA1 at 0 h after extinction enhanced the extinction LTM test 14 days after extinction (both p<0.05), and NE at a dose of 1.0 μg/μl inhibited the reinstatement test (p<0.05). (d) Norepinephrine microinfused into the CA1 at 6 h after extinction did not affect the extinction LTM test or reinstatement test. (e) Norepinephrine at 1.0 μg/μl microinfused into the CA1 at 12 h after extinction enhanced the extinction LTM test 14 days later and NE at a dose of 0.3 and 1.0 μg/μl reduced the subsequent reinstatement test (all p<0.05). (f) Norepinephrine microinfused into the CA1 at 24 h after extinction had no effect on the subsequent extinction LTM test or reinstatement test. The data are expressed as mean±SEM (_n_=8–10 per group). *P<0.05, compared with 0 μg NE group. CFC, contextual fear conditioning; LTM, long-term memory; NE, norepinephrine.

Figure 2

Figure 2

Norepinephrine in the CA1 during the late phase after extinction selectively enhanced the persistence of extinction LTM. (a) Timeline for this experiment. Norepinephrine (1.0 μg/μl) was microinfused into the CA1 at 0 or 12 h after weak extinction training (only 20 min). Extinction LTM was tested 2 or 14 days later. After the extinction LTM test 2, a footshock (0.8 mA, 1 s) was given, and fear reinstatement was tested 1 day later. A separate group without any drug treatment was used as a control. (b) At 2 days after weak extinction, compared with No NE group, the 0 h NE (p<0.05) but not 12 h NE (_p_>0.05) group of rats showed reduced freezing time, and both the 0 h NE group and 12 h NE group exhibited lower fear in extinction LTM test 2 and reinstatement test (all p<0.05). These results suggest that NE microinfused into the CA1 at 0 h after extinction enhanced the formation of extinction LTM, whereas NE treatment 12 h after extinction selectively promoted the persistence of extinction LTM. The data are expressed as mean±SEM (_n_=8–10 per group). *P<0.05, compared with control group.

Figure 3

Figure 3

Norepinephrine in the CA1 during the late phase after extinction had no effect on anxiety-like behavior or locomotion. (a) At 14 days after NE (1.0 μg/μl) microinfusion into the CA1, the rats underwent elevated plus maze test and locomotor activity test. Norepinephrine did not affect the numbers of entries into or time spent on the open arms in the elevated plus maze (all _p_>0.05). (b) Norepinephrine did not affect the total distance traveled in the locomotor activity test (_p_>0.05). The data are expressed as mean±SEM (_n_=9–10 per group).

Figure 4

Figure 4

Propranolol blocked the NE-induced enhancement of extinction LTM persistence. (a) Timeline for this experiment. Norepinephrine (1.0 μg/μl) was microinfused into the CA1 at 12 h after extinction training, and the _β_-receptor blocker propranolol was microinfused into the CA1 30 min before. Extinction LTM was tested 2 or 14 days later. After the extinction LTM test 2, a footshock (0.8 mA, 1 s) was given, and fear reinstatement was tested 1 day later. (b) Norepinephrine microinfused into the CA1 at 12 h after extinction enhanced the extinction LTM test 14 days after extinction and the reinstatement test (p<0.05) that were blocked by propranolol (_p_<0.05). Propranolol microinfused alone did not affect the extinction LTM test or reinstatement test (both _p_>0.05). The data are expressed as mean±SEM (_n_=10–11 per group). *P<0.05 compared with Saline+Saline group.

Figure 5

Figure 5

Rp-cAMPS blocked the NE-induced enhancement of extinction LTM persistence. (a) Timeline for this experiment. Norepinephrine (1.0 μg/μl) was microinfused into the CA1 at 12 h after extinction training, and the PKA inhibitor Rp-cAMPS was microinfused into the CA1 60 min before. Extinction LTM was tested 2 or 14 days later. After the extinction LTM test 2, a footshock (0.8 mA, 1 s) was given, and fear reinstatement was tested 1 day later. (b) Norepinephrine microinfused into the CA1 at 12 h after extinction enhanced the extinction LTM test 14 days after extinction and the reinstatement test (p<0.05) that were blocked by Rp-cAMPS (_p_<0.05). Rp-cAMPS treatment alone did not affect the extinction LTM test or reinstatement test (both _p_>0.05). The data are expressed as mean±SEM (_n_=8 per group). *P<0.05 compared with Saline+Saline group.

Figure 6

Figure 6

The persistence of extinction LTM requires late-phase protein synthesis in the CA1. (a) Timeline for this experiment. Norepinephrine (1.0 μg/μl) was microinfused into the CA1 at 12 h after extinction training, and the protein synthesis inhibitor anisomycin was microinfused into the CA1 at 1 h before. Extinction LTM was tested 2 or 14 days later. After the extinction LTM test 2, a footshock (0.8 mA, 1 s) was given, and fear reinstatement was tested 1 day later. (b) Anisomycin blocked the effect of NE on extinction LTM test 2 and the reinstatement test (p<0.05), suggesting that the effect of NE on extinction LTM required new protein synthesis (p<0.05). Moreover, anisomycin alone significantly promoted fear recovery in extinction LTM test 2, suggesting a protein synthesis-dependent process during the late phase after extinction (p<0.05). (c) Emetine disrupted extinction LTM in test 2 and prevented the extinction-enhancing effect of NE (p<0.05). The data are expressed as mean±SEM (_n_=8–10 per group). *P<0.05 compared with Saline+Saline group or DMSO+Saline group.

Figure 7

Figure 7

NE activated the _β_-receptor/PKA signaling pathway in the CA1 during the late phase after extinction. (a) Representative western blots. (b) Extinction training significantly increased synaptosomal membrane GluR1 expression in the CA1 during the late phase of extinction (p<0.05). NE augmented the alteration of GluR1 that was blocked by propranolol and Rp-cAMPS (both _p_<0.05). (c) The level of pGluR1 (Ser 845) in the CA1 during the late phase of extinction was increased by NE that was prevented by propranolol and Rp-cAMPS (all _p_<0.05). (d) NE had no effect on total GluR1 level (_p_>0.05). (e) NE increased pPKA level in the CA1 during the late phase of extinction that was blocked by propranolol and Rp-cAMPS (all p<0.05). (f) NE had no effect on total PKA level (_p_>0.05). (g) pErk1/2 was only increased by extinction (p<0.05) and unaffected by NE, propranolol, and Rp-cAMPS (all _p_>0.05). (h) Extinction did not affect total Erk1/2 level (_p_>0.05). (i) NE promoted the increase in pCREB level in the CA1 induced by extinction training (p<0.05). Propranolol and Rp-cAMPS blocked the enhanced expression of pCREB (both _p_<0.05) but had no effect on the increase in pCREB level induced by extinction training (both _p_>0.05). (j) NE had no effect on total CREB level (_p_>0.05). The data are expressed as mean±SEM (_n_=8 per group). *P<0.05 compared with Extinction+Saline+Saline group; #p<0.05 compared with No Extinction+Saline+Saline group. pGluR1, phosphorylated GluR1; tGluR1, total GluR1; pPKA, phosphorylated PKA; tPKA, total PKA; pErk1/2, phosphorylated Erk1/2; tErk1/2, total PKA; pCREB, phosphorylated CREB; tCREB, total CREB.

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