Application Cases

Experiment Name: The Role of CPLX1 Gene and PV+ Inhibitory Neurons in Ultrasonic Perception

Research Direction: Ultrasonic perception is essential for the daily lives of some mammalian species. For example, echolocating microbats and toothed whales use ultrasound for navigation and prey localization, while mice and rats use ultrasound for social communication. However, it remains unclear how the brain perceives ultrasound. With over 1,400 species and the widest range of auditory sensitivity frequencies (from 8 kHz to above 200 kHz), bats are ideal for studying mammalian ultrasonic perception. Based on morphology, bats can be divided into microbats and megabats. Microbats use ultrasonic echolocation for navigation and foraging in space, while megabats mainly rely on their well-developed vision and perception of human-audible sounds in daily life. Therefore, the diversity of species and auditory frequency ranges makes bats a unique model for studying the molecular mechanisms of ultrasonic perception.

Ultrasonic perception is crucial for many mammals. For example, small bats with echolocation abilities and toothed whales use ultrasound for navigation and prey localization, while mice and rats use ultrasound for social activities. The auditory cortex, as the site of transformation from auditory scenes to perceptual representations, is vital for generating higher-level cognitive information. However, research on ultrasonic perception in the auditory cortex is relatively limited. Therefore, this study aims to identify cell populations and marker genes related to ultrasonic perception by constructing high-quality reference genomes of bats and single-cell maps of the auditory cortex, revealing the molecular mechanisms of ultrasonic perception.

Experiment Objective: To explore the role of the CPLX1 gene and PV+ inhibitory neurons in ultrasonic perception, uncover the molecular mechanisms of ultrasonic perception in mammals, provide clues for improving age-related hearing loss, and serve as an example for using single-cell omics to study the unique traits of specialized animals.

Testing Equipment: ATA-3090B power amplifier, signal generator, speaker, oscilloscope

Experiment Process: This experiment used the XR-XC404 regulation system on adult male mice three weeks after stereotactic injection of AAV. Each mouse was placed in an operational chamber located in a soundproof compartment, allowing it to freely explore for 2 minutes. Three tones (70-90 dB SPL, 16 or 63 kHz, lasting 30 seconds) were played, followed by a 0.4 mA, 2-second electric shock with a 1-minute interval. Pure tones were generated in the waveform generator, powered by the ATA-3090B power amplifier, and played through an ultrasonic speaker placed 15 cm above the floor. After 24 hours, the mice underwent two consecutive tests: context change test and tone test. Specifically, the context change meant transferring the mice to another environment to limit the influence of the environment. The mice were transferred to the changed box for 4 minutes to measure freezing behavior, followed by playing pure tones (70-90 dB SPL, 16 or 63 kHz) for 1 minute to test freezing during the sound. To modify the modified box, the metal grid floor was replaced with a plastic board, red stripes were painted on the white plastic walls, and 3% acetic acid was used instead of 20% ethanol as the background odor.

Figure 1-1: Schematic diagram of the experiment on the role of CPLX1 gene and PV+ inhibitory neurons in ultrasonic perception

Experiment Results: Cross-species comparisons revealed significant differences in Parvalbumin (PV)+ inhibitory neurons between microbats and megabats. Fiber photometry calcium imaging experiments showed that PV+ inhibitory neurons exhibited distinct calcium signal responses to 63 kHz ultrasonic signals. In mouse behavioral experiments, silencing PV+ inhibitory neurons led to an inability of mice to perceive ultrasonic signals, indicating that PV+ inhibitory neurons in the auditory cortex are associated with ultrasonic perception. In PV+ inhibitory neurons of the auditory cortex, the expression level of the CPLX1 gene (encoding complexin-1 protein) in microbats was significantly higher than that in megabats. Perturbation experiments on mouse Cplx1 found that this severely affected their ultrasonic perception ability. The study also found that CPLX1 functions throughout the entire auditory circuit in microbats but not in megabats.

Figure 1-2: Heatmap of representative individual mouse GCaMP signals exposed to 16 kHz to 63 kHz. The heatmap is sorted by ΔF/F (%). The color scale represents ΔF/F (%).

Product Recommendation: ATA-3090C Power Amplifier

Figure: ATA-3090C Power Amplifier Specifications