Fig. 3. Controlling context-dependent fear conditioning and social interaction behavior.

a Schematic illustration of virus injection and fear conditioning experiments. A mixture of viruses expressing ST-KA2, M13-TEV-C, and TetO-NpHR-EYFP was injected into the dorsal hippocampus. Fiber optics were implanted bilaterally above the viral injection site. Short pulses of blue light (5 s × 3 times) were delivered for labeling active neurons, and yellow light was shined during the probe test. Reporter gene expression was confirmed by taking confocal images. b The percentage of freezing was compared before and after conditioning, and with or without 589 nm light during the retrieval period. *p < 0.05. Graphs expressed as mean ± SEM. The sample size presents the number of independent mice. c Representative image of NpHR-EYFP expression. Scale bars, 50 μm. d Freezing score was analyzed by two independent people in a blind manner. The freezing percentage was scored every 10 s and crossed-checked with correlation analysis. e The extent of virus injection and NpHR-EYFP expression is plotted across several coronal sections of the brain. Images illustrate a series of coronal schematics showing the extent of AAV expression (red) and activity-dependent labeling (green). The extents were traced based on fluorescent images taken at low magnification (2.5×) for each animal (n = 5 independent mice). Darkness represents coincidence from different animals. f Schematic illustration of the experimental procedure. Viruses expressing ST-KA2, M13-TEV-C, and TetO-ChrimsonR-EGFP were injected into the dorsal hippocampus CA1 area bilaterally. Short pulses of blue light (5 s × 3 times) were delivered with a 1-min interval for labeling, and 589 nm yellow light was shined for testing behavioral causality. g The percentage of freezing was compared before and after conditioning. During the reactivation session, freezing behavior in a novel context (context B) was compared before and after the delivery of 589 nm light. Reactivation of ST-Cal-Light-labeled neurons was sufficient to trigger freezing in context B. h Virus injection and fiber optic implantation scheme. Viruses were injected into layer 5/6 of the mPFC. i Cartoon for social interaction experiments. Whenever the mouse entered a social zone, a blue laser connected to the fiber optics was switched on. j Sample images of active neuron labeling in the mPFC by ST-Cal-Light. Scale bars, 100 μm. k Graphical demonstration of positive correlation between the number of blue light illumination/social interactions and G/R ratio. Each data point corresponds to one mouse. l When active neurons were labeled with EGFP reporter, 589 nm light did not decrease social interaction. Data are presented as mean values ± SEM. Statistical significance is judged by a two-tailed paired t-test. m NpHR reporter gene was expressed during the labeling process, social interaction behavior was significantly inhibited during the probe test. Source data and statistics are provided in the Source Data file.