Mice at ages of 6 to 7 weeks were anesthetized using 5% chloral hydrate with single intraperitoneal injection (0.01 ml/g body weight) and mounted in a stereotactic frame with nonrupture ear bars (RWD Life Science). After making an incision to the midline of the scalp, small bilateral craniotomies were performed using a microdrill with 0.5-mm burrs. Glass pipettes, with tip diameters of 10 to 20 μm, were made by P-97 Micropipette Puller (Sutter Glass pipettes) for AAV microinjection. The microinjection pipettes were first filled with silicone oil and then connected to a microinjector pump (KDS 310, KD Scientific) with full air exclusion. AAV-containing solutions were loaded into the tips of pipettes and injected at the following coordinates (posterior to bregma, AP; lateral to the midline, ML; below the bregma, DV; in millimeters): BLA: AP −1.60, ML ±3.30, DV −4.80; mPFC: AP +1.70, ML ±0.40, DV −2.60; dHPC: AP −2.00, ML ±1.50, DV −1.25; vHPC: AP −3.16, ML ±3.20, DV −4.75; dHPC: AP −2.00, ML ±1.50, DV −1.25; mPFC: AP +1.70, ML ±0.40, DV −2.60. Virus-containing solutions were injected bilaterally into the BLA (0.3 μl per side), mPFC (0.3 μl per side), vHPC (1 μl per side), or dHPC (0.3 μl per side) at a rate of 0.1 μl/min. After injection, the pipettes were left in place for an additional 10 min to allow the injectant to diffuse adequately. To inactivate Asic1a gene and overexpress BDNF simultaneously in vHPC, a mixture of AAV-Syn-BDNF-mCherry (1.0 × 1011 VG/ml) and AAV-Syn-Cre-GFP (>1.0 × 1012 VG/ml) (ratio, 1:3) were bilaterally injected into vHPC (1 μl per side). To inactivate Asic1a gene and allow optical stimulation of excitatory neurons simultaneously in vHPC, a mixture of AAV-CaMKIIα-ChR2-mCherry (>1.0 × 1012 VG/ml) and AAV-Syn-Cre-GFP (>1.0 × 1012 VG/ml) was bilaterally injected into vHPC (1 μl per side). For controls, AAV-Syn-BDNF-mCherry and AAV-Syn-Cre-GFP were replaced with AAV-Syn-mCherry and AAV-Syn-GFP, respectively. Mice were allowed to recover for at least 4 weeks before behavioral and other tests. The injection sites of virus solutions were examined after experiments by the expression of the fluorescent protein, GFP, YFP, or mCherry.

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