GoKawiilBats
Fifteen adult male Egyptian fruit bats (Rousettus aegyptiacus) were included in this study for neural recording experiments (weight, 160–200 g). Information on individual bats is summarized in Extended Data Table 1. All experimental procedures for the neural recordings were approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science. An additional 12 bats were included in a behavioural-control experiment in a smaller setup, without neural recordings (see below).
Behavioural setups for neural recordings
Four different behavioural setups were used in this study, shown in Extended Data Fig. 1a,b. All of the setups used tunnels with identical cross-section shape, with a width of 2.3 m and maximal height of 2.35 m (ref. 12), with uniform illumination (5 lux). Details on individual bats in each setup are found in Extended Data Table 1.
(1) Linear flight sessions in long tunnel: ten of the recorded bats flew back and forth in fixed-size linear tunnels of lengths 200 m or 130 m, shuttling between two landing balls positioned at the two ends of the tunnel (Extended Data Table 1, bats 1, 3 and 7–14; Extended Data Fig. 1a). Note that these lengths are rounded: The 200-m tunnel had an effective length of 194 m, and the 130-m tunnel had a length that varied between 129–134 m across sessions. (2) Flight sessions in short linear segment: four bats were recorded in an additional separate session in which they flew in a short segment of the tunnel: either 6 m (bats 1, 14 and 15) or 15 m (bat 4). These segments were blocked with opaque curtains or solid blocks at both ends. (3) Landmark perturbation: two bats (bats 2 and 4) performed two recording sessions, separated by a short sleep session of 5–10 min. A single prominent landmark was moved 7.5 m between session 1 and session 2 (the positions of the perturbed landmark are shown in Extended Data Fig. 1b (orange triangles); photograph of the perturbed landmark is shown in Extended Data Fig. 1d). The perturbed landmark was moved daily between the exact same two positions for these two bats. (4) Switching between multicompartment session and linear-flight session in long tunnel: four bats (bats 2, 4, 5 and 6) performed two recording sessions, separated by a short sleep session. In the first recording session (session 1) the bats flew in the large multicompartment tunnel (Extended Data Fig. 1b, top); 193 m length) between 3 landing balls positioned at the 3 ends of the tunnel. Rewards were given equally after landing at each landing ball, and there were therefore no correct or incorrect choices in this paradigm. In the second recording session (session 2), the junction leading to the two short compartments was blocked with a black opaque curtain, and a landing ball was positioned just before the curtain, leaving a 180 m straight tunnel for the bats to fly back and forth between the two landing balls (Extended Data Fig. 1b, bottom). Note that the 180-m tunnel had an effective length of 174 m.
Each behavioural session started and ended with sleep sessions (each sleep session lasting 5–10 min). For the sleep sessions the bat was placed inside a small covered cage, which was positioned in a quiet location inside the tunnel.
In all of the experiments, human experimenters were sitting at the ends of the tunnel (beyond the landing balls). The humans were outside of the area in which the bats were flying.
Training of bats and recording sessions
All 15 bats were initially pretrained for a few days in a flight-room or in a short segment of the tunnel (6 m or 10 m), with the aim of getting used to handling by humans and learning to perform direct flights between two landing balls. Then, 12 bats were further trained for an average of 3 weeks in the 200 m tunnel (8 bats) or in the 180 m multicompartment environment (4 bats) to fly continuous long flights. After this training, the neural recordings began. Two additional bats were recorded in the 130 m tunnel: Neural recordings in these bats were conducted from the first day of exposure to the long tunnel; we note that in our previous study12, we did not find any change in neural coding in CA1 along days in these two bats. Three of these 14 bats were also neuronally recorded in shorter tunnels (6 m or 15 m; see Extended Data Table 1). One additional bat (bat 15) was neuronally recorded only in the short 6 m tunnel.
After training, all of the bats were implanted with a microdrive for electrophysiological recordings in the dorsal hippocampus (see below).
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