ReadMe The uploaded files are the main code for 'Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway' project. The functions for measuring the entropy are stored in 0.1Functions_for_entropy_from_github.R, which was downloaded from https://github.com/sestanlab/Cross-species-PFC-snRNA-seq/tree/main/EvolutionPatterns and I modified a little bit for this project. The self-defined functions for Seurat downstream analysis, drawing venn plot etc. are stored in 0.2self_defined_functions.R . The doubletFinder code is in 1.doublet_finder_code.R. The clustering and annotation of four bats AUD cells is in 2.bat_aud_clustering_anno.R . The clustering and annotation of four bats AUD excitatory neurons is in 3.Exc_subclustering.R . The clustering and annotation of four bats AUD inhibitory neurons is in 4.Inh_subclustering.R. The clustering and annotation for somatosensory cortex cells is in 5.Somatosensory cortex data analysis.R. The differential analysis between Rs/Ms and Rl/Cs PS neuron is in 6.Difference between PS between myotis_Rsin and Rle_CS in AC.R. The clustering and annotation for DCN, VCN, SO, IC, MGB is in 7.DCN,VCN,SO,IC,MGB cells clustering and annotation.R. The spatial transcriptome data analysis is in 8.ST data analysis.R. Article abstract Unlike megabats, which rely on a well-developed sense of vision, microbats utilize ultrasonic echolocation to navigate and locate prey. To study ultrasound perception, we compared the auditory cortices of microbats and megabats by constructing reference genomes and single-nucleus atlases for four species. We found that parvalbumin (PV)+ neurons exhibited evident cross-species differences and could respond to ultrasound signals, and their silencing severely affected ultrasound perception in mouse auditory cortex. Moreover, megabat PV+ neurons expressed low levels of complexins (CPLX1-4), which can facilitate neurotransmitter release, while microbat PV+ neurons highly expressed CPLX1 to improve neurotransmission efficiency. Further perturbation of Cplx1 in PV+ neurons impaired ultrasound perception in mouse auditory cortex. In addition, CPLX1 functioned in other parts of the auditory pathway in microbats but not megabats and exhibited convergent evolution between echolocating microbats and whales. Altogether, we conclude that CPLX1 expression throughout the entire auditory pathway can enhance mammalian ultrasound neurotransmission. .