Paav camkiiα hm4di mcherry

5XFAD mice were anaesthetized with isoflurane in the stereotaxic frame for the entire surgery, and their body temperature was maintained with a heating pad. In order to inhibit neuronal activity in the mammillary body (MB), 200 nl of adendo-associated virus carrying inhibitory DREADD, hM4Di and mCherry (pAAV-CamKIIα-hM4Di-mCherry, Addgene, catalog #50477-AAV8) were injected into the MB (From bregma: A|P: −2.0 mm, M|L: 0.0 mm, D|V: −5.6 mm). Injections were performed at a rate of 50 nl per min. The needle was allowed to sit in the target location for 3 min prior to the start of viral infusion and for 5 min after injection was completed. 5XFAD mice injected with AAV-CamKIIα-EGFP (AAV-CamKIIa-GFP, University of North Carolina Vector Core) in the MB were used as control group. We allowed for 3 weeks of postoperative recovery and time for expression of viral vectors. Mice were then given i.p. injections of clozapine-n-oxide (CNO, Tocris, catalog #4936/50) at 3 mg/kg body weight, daily for 5 weeks. CNO was dissolved in saline with a working solution at 0.5 mg/ml. To control the CNO effect, both control and mice with expression of inhibitory DREADD were injected with CNO in the experiment.

DNA plasmids encoding pAAV-CaMKIIα-hM3Dq-internal ribosomal entry site (IRES)-mCitrine (Addgene, plasmid #50466), pAAV-CaMKIIα-hM4Di-mCherry (Addgene, plasmid #50477), pAAV-CaMKIIα-hChR2(H134R)-EYFP (Addgene, plasmid #26969), pAAV-CaMKIIα-EGFP (Addgene, plasmid #50469), and pAAV-Ubi-eGFP (Addgene, plasmid #62518) were obtained from Addgene. pAAV-CaMKIIα-Cre-GFP was constructed from Cre-GFP empty vector (Addgene, plasmid #20781) into pAAV-CaMKIIα-hChR2-EYFP (Addgene, plasmid #26969). Plasmid DNA was amplified, purified, and collected using a standard plasmid maxiprep kit (Qiagen). The purified plasmids were mixed into CaCl₂ solution with the DNA plasmid coding AAV-DJ and co-transfected into HEK293T cells using calcium phosphate precipitation methods. Transfected cells were harvested at 72 h after transfection and the virus was purified using the AAV purification mega kit (Cell Biolabs, Inc.). Viral titers were 5 × 10¹² particles/ml and stored in aliquots at −80 °C until use.