Behavioral analysis

WT mice injected with PBS, Thy1:SNCA/Snca^–/– mice injected with PBS, and Thy1:SNCA/Snca^–/– mice injected with ampLB were subjected to behavioral tests from 6 to 8MPI. Before every test, the mice were habituated to the experimental environment for more than 30 min. Samples that encountered technical problems were removed from the analyses.

Mice were placed at the center of the field inside an open field apparatus (36 × 36 cm) and allowed to move freely for 15 min. The distance traveled and time spent in the center area (18 × 18 cm) were recorded using video tracking software EthoVision XT 15 (Noldus).

Mice were placed at the end of one arm of the Y-maze apparatus (San Diego Instruments) and allowed to move freely for 5 min. The distance traveled and series of arm entries were recorded using video tracking software EthoVision XT 15 (Noldus). An alternation was defined as entries into all three arms on consecutive occasions. The number of maximum alternations was therefore the total number of arm entries minus two, and the percentage of alternations was calculated.

The Barnes maze test was conducted on a white circular surface with 20 holes equally spaced along the perimeter (Supplementary Fig. ^6e). For acquisition trials, a shelter was placed under one of the holes, i.e., the “target hole”. Mice were placed in the center and allowed to move freely for up to 3 min. The latency to reach the target, the number of holes visited other than the target, and the distance traveled were recorded using the video tracking software EthoVision XT 15 (Noldus). Mice were subjected to acquisition trials twice a day for 7 days, and then to probe tests 24 h and 10 days after the last acquisition trial. In the probe tests without the shelter, mice were placed in the center and allowed to move freely for 3 min. The time spent around each hole, the number of visiting each hole, and the distance traveled were recorded. “Target zone” was defined as the target hole plus the 2 holes on either side of the target hole.

Conditioning and test sessions were performed in a standard operant chamber (Med Associates) equipped with a tone generator and house light. Mice were handled for habituation in front of the apparatus for 2 min per day for 3 days. On day 0, conditioning was performed (Supplementary Fig. ^6f). Mice were placed in a test chamber inside a sound-attenuated cabinet and allowed to explore freely for 150 s. A white noise, which is a conditioned stimulus, was presented for 30 s, followed by a foot shock (2 s, 1 mA) serving as an unconditioned stimulus. On days 1 and 10, contextual fear memory and auditory-cued fear memory tests were performed. For the contextual fear memory test, mice were placed in the same chamber in the same context as the conditioning and immobile time and distance traveled were recorded for 5 min. For the auditory-cued fear memory test, mice were placed in a different chamber in a different context from the conditioning. Mice were allowed to move freely for 2.5 min, and then the white noise was presented for 2.5 min. Immobile time and distance traveled were recorded automatically.