Materials and reagents

1. Mice [all animals were housed in standard laboratory conditions with ad libitum access to food and water, as well as a 12/12 h light/dark cycle (light: 7:00 AM–7:00 PM), a temperature range of 22–26 °C, and a humidity level of 55%–60%. The mice used in this study were cared for in accordance with the authorized protocols of Southeast University, Nanjing, China)].

   1. FAD^4T mice [C57BL/6 genetic background, 10–12 weeks old. FAD^4T mice co-expressed human APP with the Swedish (KM670/671NL) and India (V717F) variants, together with human mutant PS1 (M146L, L286V), driven by the mouse Thy1 promoter (Gempharmatech Co., Ltd, catalog number: T053302)]

   2. Mice used for novel stimulation (N1–N7) and social test (S1–S2) (C57BL/6J genetic background, 10–12 weeks old, 22–25 g) (Gempharmatech Co., Ltd, catalog number: N000013, Jiangsu, China)

2. Nitrile laboratory gloves (Guangming, China)

3. Paper towels (Breeze, China)

4. 75% Ethanol (Alladin, catalog number: A171299)

Equipment

1. Mouse breeding room with constant temperature system, ventilation system, and automatic light timing system

2. Home cage (380 mm × 180 mm × 170 mm, with a height under the partition bar inside the cage of 13 cm, which meets the requirements of the GB14925 national standard. It can accommodate 5–8 experimental mice weighing 20–30 g) (SHINVA, China)

3. Small electronic scale (127 mm × 106 mm × 19 mm) (Kubei, China)

4. Metal pen holder (golden, diameter 8 cm, height 10.3 cm, weight 140 g) (Wuling, China)

5. Timer (82 mm × 62 mm × 24 mm) (Anytime, model: XL-009B)

6. Spray bottle (500 mL) (LDPE, Sangon Biotech, catalog number: F505008)

7. Objects for novel object recognition (NOR) test (two bright-red metal cubes, 83 mm × 83 mm × 83 mm, were used as similar objects; one light-purple metal cylinder, 85 mm in diameter and 84 mm in height, was used as the novel object) (Aiziling, China)

8. Mouse behavioral testing room with constant temperature system, ventilation system, and automatic light timing system

9. Behavioral testing apparatus (Xinruan, China)

   1. NOR apparatus

      A square Plexiglas box (50 cm × 50 cm × 50 cm) with four 50 cm-high sidewalls and an open roof. In addition, two metal boxes with heavy objects inside were used as similar objects, which maintained consistency in size, color, and shape. Another metal box with a heavy object, similar in size but different in color and shape, was used as the novel object

   2. 3-chamber apparatus

      A rectangular box made of Plexiglas (50 cm × 25 cm) with a detachable base, creating three distinct compartments divided by two partitions. Each partition contained a small door (10 cm × 5 cm) to facilitate mouse’s movement between the different chambers. In addition, there were two metal pen holders that were identical in size, shape, and color, used for restraining stranger mice.

10. Computer (Lenovo, model number: Tianyi 510pro-14IMB)

11. Camera (MOKOSE, model number: HDC10)

Software and datasets

1. Video tracking and analyzing software (Noldus, EthoVision XT 13 software)

2. Statistical analysis software (Prism 8.0.1, GraphPad, https://www.graphpad.com/)

Procedure