Journal: eLife
Article Title: The entorhinal cortex modulates trace fear memory formation and neuroplasticity in the mouse lateral amygdala via cholecystokinin
doi: 10.7554/elife.69333
Figure Lengend Snippet: Figure 7. In vivo knockdown of Cck expression blocks auditory evoked potential (AEP)-long-term potentiation (LTP) induction in the LA. (a) Schematic diagram of the experiment. CCK-Cre mice were injected in the EC with an AAV expressing short hairpin RNA (shRNA) (anti-Cck or anti-Scramble) and ChR2. In vivo recording was conducted in the LA (left). After Cre-mediated recombination, EC-CCK neurons were transfected with shRNA targeting Cck (anti-Cck) or nonsense sequence (anti-Scramble) as well as the excitatory opsin ChR2 variant E123T/T159C (right). AAV, adeno-associated virus; EC, entorhinal cortex; LA, lateral amygdala; REC, recording; ITR, inverted terminal repeat; mU6, mouse U6 promoter; CAG, CMV enhancer, chicken β-actin promoter; WPRE, woodchuck hepatitis virus (WHP) posttranscriptional regulatory element. (b) Post hoc verification of the electrode tracks and optic fiber. (c) Post hoc immunofluorescent staining targeting ChR2 in the EC (left) as well as in the CCK-positive projections distributed in the LA (right). Magnified images are shown in the bottom insets. (d) Quantification of the expression of Cck mRNA in CCK-Cre mice injected with anti-Cck or anti-Scramble shRNA in the EC. Samples with extremely low RNA concentration (<26.7 ng/μL) were discarded. **p < 0.01, NS, not significant, two-way RM ANOVA with post hoc Bonferroni pairwise comparison. EC in anti-scramble group, N = 5; AC in anti-scramble group, N = 2; EC in anti-Cck group, N = 5; AC in anti-Cck group, N = 6. (e) Time course plot of the normalized AEP slope before and after pairing in mice expressing anti-Cck (n = 19) or anti-Scramble (n = 14) shRNA. (f) Representative traces of the averaged AEP before (–10 to 0 min, dotted line) and after (10–20 min, solid line) induction in the two groups. Anti-Scramble is indicated in blue, and anti-Cck is indicated in red. (g) The average normalized slopes 10 min before pairing (–10 to 0 min, before) and 10 min after pairing (10–20 min, after) in the two groups. ***p < 0.001, two-way RM ANOVA with Bonferroni post hoc pairwise test; RM ANOVA, repeated-measures analysis of variance; NS, not significant; fEPSP, field excitatory postsynaptic potential. (h) The average raw slopes 10 min before pairing (–10 to 0 min, before) and 10 min after pairing (10–20 min, after) in the two groups. **p < 0.01, two-way RM ANOVA with Bonferroni post hoc pairwise test.
Article Snippet: 69333 24 of 33 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Recombinant DNA reagent AAV8- Cre- ON- ChR2antiScramble This paper N/A AAV virus expressing Cre- dependent ChR2 and Cre- dependent shRNA targeting nonsense Scramble Recombinant DNA reagent retroAAV- hSyn- FLEXjGcamp7s Addgene RRID:Addgene_104491 N/A Recombinant DNA reagent AAV- hSyn- CCK2.0 Vigene Bioscience, Jing et al., 2019 Construct is from Prof. Yulong Li’s Lab at Peking University Recombinant DNA reagent pAAV- CAG- Flex- tdTomato Addgene RRID:Addgene_28306 N/A Recombinant DNA reagent PUC57- mU6 with TATALox BGI, Ventura et al., 2004 N/A Recombinant DNA reagent PUC57- CAG- DIOChR2(E123T/T159C)- Flag Addgene RRID:Addgene_35509; Addgene_101766 N/A Recombinant DNA reagent pUC57- CAG- DIO- mCherryEYFP (inverted) Addgene RRID:Addgene_34582; Addgene_98750 N/A Recombinant DNA reagent AAV8- hSyn- FLEX- Jaws- GFP UNC, Chuong et al., 2014 N/A Sequence- based reagent Cck BGI shRNA Target GACT CCCA GACC TAAT GTTGC Sequence- based reagent Scramble BGI shRNA Target GTTG GCTC CTAG CAGA TCCTA Sequence- based reagent Primers for genotyping Cck/- mice BGI PCR primers 5'- ATGCAGGCAAATTTTGGTGT- 3'; 5'- GAGCGGACACCCTTACCTTT- 3'; 5'-GACTTCTGTGTGCGGGACTT- 3 Sequence- based reagent Gapdh (Forward) BGI qPCR primer 5'- AGGT CGGT GTGA ACGG ATTTG- 3' Sequence- based reagent Gapdh (Reverse) BGI qPCR primer 5'- TGTA GACC ATGT AGTT GAGGTCA- 3' Sequence- based reagent Cck (Forward) BGI qPCR primer 5'- ATCT GTCC AGAG TGTG CAATGC- 3' Sequence- based reagent Cck (Reverse) BGI qPCR primer 5'- TGAG GGGC AGAA GGAA ATCTCT- 3' Chemical compound, drug Urethane Sigma- Aldrich Cat# U2500 N/A Chemical compound, drug Pentobarbital Alfasan International B.V. 20% Dorminal Chemical compound, drug CCK- 4 Abcam, Cambridge, UK Cat# ab141328 N/A Chemical compound, drug Dil Stain Thermo Fisher Scientific Cat# D282 N/A Chemical compound, drug Clozapine Sigma- Aldrich Cat# C6305 N/A Peptide, recombinant proteins Alexa Fluor 647- conjugated Cholera Toxin Subunit B Thermo Fisher Scientific Cat# C34778 N/A Genetic reagent (Mus musculus) Mouse: C57BL/6 The Laboratory Animal Services Centre, Chinese University of Hong Kong N/A N/A Continued Continued on next page Feng et al. eLife 2021;10:e69333.
Techniques: In Vivo, Knockdown, Expressing, Injection, shRNA, Transfection, Sequencing, Variant Assay, Virus, Staining, Concentration Assay, Comparison
Addgene inc is a verified supplier 
