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Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway

Nature Genetics volume 56, pages 1503–1515 (2024)Cite this article

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Abstract

Unlike megabats, which rely on well-developed vision, microbats use ultrasonic echolocation to navigate and locate prey. To study ultrasound perception, here 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, whereas their silencing severely affected ultrasound perception in the mouse auditory cortex. Moreover, megabat PV+ neurons expressed low levels of complexins (CPLX1–CPLX4), which can facilitate neurotransmitter release, while microbat PV+ neurons highly expressed CPLX1, which improves neurotransmission efficiency. Further perturbation of Cplx1 in PV+ neurons impaired ultrasound perception in the 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.

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Fig. 1: Reference-quality genomes and ACx atlases of microbats and megabats.
Fig. 2: Distinct differences between microbats and megabats in ACx PV+ inhibitory neurons.
Fig. 3: Important role of PV+ inhibitory neurons in ultrasound perception.
Fig. 4: CPLX1 is differentially expressed between microbats and megabats.
Fig. 5: Cplx1 perturbation in the mouse ACx impairs ultrasound perception.
Fig. 6: Differential complexin expression between microbats and megabats.
Fig. 7: CPLX1 functions in other parts of the auditory pathway.
Fig. 8: CPLX1 exhibits convergent evolution between echolocating microbats and whales.

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Data availability

Raw and processed data are deposited in the Genome Sequence Archive under BioProject accession no. PRJCA008842. Source data are provided with this paper.

Code availability

The R code needed to reproduce the main results of this study is available via Zenodo at https://doi.org/10.5281/zenodo.11044010 (ref. 63).

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Acknowledgements

We thank M. Guo, Y. Zhang and P. Luo for helping with field sampling. This work was supported by the Research Foundation for Advanced Talents from Guangzhou National Laboratory (no. YW-JCYJ0604 to J.D.) and Bioland Laboratory (no. 1102101216 to J.D.), a China Postdoctoral Science Foundation fellowship (no. 2021M692241 to M.L.) and the National Key R&D Program of China (no. 2021ZD0202403 to C.S.). L.Z. was supported by the Guangdong Provincial Science and Technology Program (nos. 2021B1212110003 and 2021B1212050021).

Author information

Author notes

  1. These authors contributed equally: Meiling Liu, Changliang Wang, Lifang Huo, Jie Cao, Xiuguang Mao, Ziqing He, Chuanxia Hu, Haijian Sun.

Authors and Affiliations

  1. GMU-GIBH Joint School of Life Sciences, The Guangdong-Hong Kong-Macau Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou National Laboratory, Guangzhou Medical University, Guangzhou, China

    Meiling Liu, Changliang Wang, Lifang Huo, Jie Cao, Ziqing He, Chuanxia Hu, Haijian Sun, Wenjun Deng, Weiya He, Yifu Chen, Meifeng Gu, Jiayu Liao, Ning Guo, Congping Shang & Ji Dong

  2. Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou, China

    Meiling Liu, Lifang Huo, Jie Cao, Chuanxia Hu, Congping Shang & Ji Dong

  3. School of Ecological and Environmental Sciences, East China Normal University, Shanghai, China

    Xiuguang Mao

  4. Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong Public Laboratory of Wild Animal Conservation and Utilization, Institute of Zoology, Guangdong Academy of Sciences, Guangzhou, China

    Xiangyang He & Libiao Zhang

  5. State Key Laboratory of Cognitive Neuroscience and Learning, IDG/McGovern Institute for Brain Research, Beijing Normal University, Beijing, China

    Qian Wu & Xiaoqun Wang

  6. CAS Key Laboratory of Regenerative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China

    Jiekai Chen

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Contributions

J.D., C.S., X.W. and L.Z. conceived and designed the study. M.L., L.H., J. Cao, C.H., W.D., W.H., Y.C., M.G., J.L., N.G., X.H., Q.W. and J. Chen. performed the experiments. C.W., Z.H., H.S. and X.M. conducted the bioinformatics analyses. J.D. and M.L. wrote the paper with help from all the authors. All the authors reviewed the paper.

Corresponding authors

Correspondence to Libiao Zhang, Xiaoqun Wang, Congping Shang or Ji Dong.

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Nature Genetics thanks Erich Jarvis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Construction of bat reference genomes and ACx atlases.

(a) List of bat species included in the study with details relating to echolocation call type and acoustic energy. (b) Spectrograms of four bat species, among which, R. leschenaultia is referred from a previous study (see Methods for details). (c) Strategies for genome assembly with bat muscle tissue. (d) Hi-C maps for the genomes of two bat species prior to (left) and post manual curation (right). The dotted circle shows the position needed to be corrected. (e) QV base call accuracy values of the final assemblies. (f) Overview of genome annotation with 13 bat tissues. (g) Gene number and transcript number detected in each library. (h) UMAP visualization of ACx cells in each bat species. Cell types are indicated by colors. Exc, excitatory neuron; Inh, inhibitory neuron; Olig, oligodendrocyte; OPC, oligodendrocyte precursor cell; Astro, astrocyte; Endo, endothelial cell; Micro, microglia; Epend, ependymal cell. (i) Heatmap of top 10 marker genes (y axis) for each cell type (x axis) in 4 bat species. The color key from purple to yellow indicates low to high expression levels, respectively. (j) Cell type proportion in each sample. (k) Statistical analysis of the proportion of different cell types in left and right ACx. n = 7 for left ACx and n = 8 for right ACx. Data are mean values ± SD.

Source data

Extended Data Fig. 2 The differences in neuronal populations between microbats and megabats.

(a) UMAP visualization of excitatory neuron subclusters in each bat species. Cell types are indicated by colors. (b) Dot plot showing expression pattern of representative marker genes in each excitatory neuron subcluster. Dot size and color represent the percentage of marker gene expression (Perc. Expr.) and average expression level (Aver. Expr.), respectively. (c) Heatmap of top 10 marker genes (y axis) for excitatory subclusters (x axis) in 4 bat species. (d) Heatmap of top 10 marker genes (y axis) for inhibitory subclusters (x axis) in 4 bat species. (e) UMAP visualization of inhibitory neuron subclusters split by species. (f) PCA visualization of PS+ inhibitory neurons in different bat species. (g) UMAP visualization of inhibitory neuron subclusters in four bat species by using different integration resolutions.

Extended Data Fig. 3 Construction of bat SCx atlas.

(a) Gene number and transcript number detected in each library. (b) UMAP visualization of all SCx cells in two bats (left) and in each species (right). Cell types are indicated by colors. Exc, excitatory neuron; Inh, inhibitory neuron; Olig, oligodendrocyte; OPC, oligodendrocyte precursor cell; Astro, astrocyte; Endo, endothelial cell; Micro, microglia; Fibro, Fibroblast. (c) Dot plot showing the expression patterns of representative marker genes for each cell type of bat SCx. Dot size and color represent the percentage of marker gene expression (Perc. Expr.) and average expression level (Aver. Expr.), respectively. (d) Heatmap of top 10 marker genes (y axis) for SCx each cell type (x axis) in each bat species. The color key from purple to yellow indicates low to high expression levels, respectively. (e) UMAP visualization of inhibitory neuron subclusters in two bats. (f) Dot plot showing the expression patterns of representative marker genes for each subcluster of inhibitory neurons in bat SCx. Dot size and color represent the percentage of marker gene expression (Perc. Expr.) and average expression level (Aver. Expr.), respectively.

Extended Data Fig. 4 Validation of PV+ inhibitory neurons on ultrasound perception in mouse ACx.

(a) Dot plot showing the expression patterns of PVALB, VIPR2, PDE3A and SYT2 in bat inhibitory neurons. Dot size and color represent the percentage of marker gene expression (Perc. Expr.) and average expression level (Aver. Expr.), respectively. (b) UMAP visualization (left) of all cell types of mouse ACx based on a previous study and violin plots of Syt2 and Pde3a expression patterns in all cell types (right). Gluta: glutamatergic neuron; Astro, astrocyte; Endo, endothelial cell; Micro-PVM: microglia-perivascular macrophage; Olig, oligodendrocyte; SMC-Peri: smooth muscle cell-pericyte; VLMC: vascular/leptomeningeal cell. (c) Gene ontology (GO) categories enriched in PS+ (top) or PP+ (bottom) neuron-specific DEGs. One-sided hypergeometric test ‘p-value’ was used and then adjusted for multiple comparisons. (d) Predicted cross-species cluster similarities of PS+ and PP+ inhibitory neurons between bat and mouse. (e) Heatmap of DEGs in PS+ and PP+ inhibitory neurons in bat and mouse. (f) Heatmaps of GCaMP signals in representative individual mice when exposed to 16 kHz or 63 kHz cue. Heatmaps are sorted by the ΔF/F (%). Color scale indicates ΔF/F (%).

Extended Data Fig. 5 DEGs of PS+ inhibitory neurons between microbats and megabats.

(a) Venn diagram showing downregulated gene numbers in PS+ inhibitory neurons of microbats compared with those of megabats. (b) Heatmap of 369 downregulated genes in PS+ inhibitory neurons of microbats. (c) KEGG enrichment analysis using 369 downregulated genes in PS+ inhibitory neurons of microbats. One-sided hypergeometric test ‘p-value’ was used and then adjusted for multiple comparisons. (d) Synaptic vesicle cycle, the most enriched KEGG term of all 320 upregulated genes. (e) Spatial distribution of the expression patterns of synaptic vesicle cycle related genes in the ACx of M. ricketti and C. sphinx.

Extended Data Fig. 6 Validating the role of Cplx1 in ultrasound perception of mouse ACx.

(a) Fluorescence-labeled siRNA transfection in P19 cell line. Representative images from 3 independent experiments. (b) Relative normalized expression of Cplx1 in 3 different Cplx1 siRNA transfected P19 cell line detected by RT-qPCR. n = 3 biologically independent samples. (c) Example micrographs showing the range of AAV infection labeled by eGFP. Green regions (middle) represent the ACx. Representative images from 4 independent experiments. (d) RT-qPCR validation of KD efficiency of Cplx1 in mouse ACx. Two (Cplx1-KD) or three (NC) biological replicates, each with three technical repeats, were conducted in each sample. Data are mean values ± SD. Two-sided t-test P values are indicated. (e) Representative example of locomotion of NC and Cplx1 KD mice tested with 16 kHz or 63 kHz cue in context B box. Dots indicate the ___location of the mouse every 0.04 s. (f) Relative normalized expression of Cplx1 and smCplx1 in rescued mice by bulk RNA-seq analysis. n = 4 biologically independent samples. (g) UMAP visualization of mouse ACx inhibitory neurons based on a previous study and the expression patterns of Pvalb and Cplx1. (h) Representative flow cytometry results of sorting mCherry+ cells in PV-ires-Cre mouse ACx. (i) RT-qPCR validation of specific KD efficiency of Cplx1 in PV-ires-Cre mouse ACx. Two biological replicates, each with three technical repeats, were conducted in each group. Data are mean values ± SD. Two-sided t-test P values are indicated. (j) Fluorescence-labeled sgRNA transfection in P19 cell line. Representative images from 3 independent experiments. (k) T7EI assays for assessing the CRISPR/Cas9 mediated indels formation of Cplx1 in P19 cell line. Representative images from 2 independent experiments.

Source data

Extended Data Fig. 7 Expression ratio and perturbation of CPLX1 and CPLX2.

(a) Expression ratio of CPLX1 and CPLX2 in excitatory, inhibitory (without PS+) and PS+ neurons of bat ACx. Neurons with TPM expression level >= 1 were counted. Exc, excitatory neuron; Inh, inhibitory neuron. (b) Example traces of IPSC amplitude triggered by different current pulse frequencies.

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Extended Data Fig. 8 Important role of Cplx1 in ultrasonic perception in the cochlea.

(a) UMAP visualization of cochlear cells in each bat species and mouse. Cell types are indicated by colors. HC, Hair cell. BC, Basal cell. IC, Intermedaite cell. MC, Marginal cell. Fibro, Fibroblast. Imm, Immune cell. Endo, Endothelial cell. Sch, Schwann cell. SGN-I, Type I spiral ganglion cell. SGN- II, Type II spiral ganglion cell. SC, Supporting cell. OSC/DC/IPC/IBC, Outer sulcus cell/Deiters’ cell/Inner pillar cel/Inner border cell. DC/OSC, Deiters’ cell/Outer sulcus cell. HeC, Hensen’s cell. (b) Dot plot showing expression patterns of representative marker genes for each cochlear cell type. Dot size and color represent the percentage of marker gene expression (Perc. Expr.) and average expression level (Aver. Expr.), respectively. (c) UMAP visualization of the expression patterns of two marker genes for SGN-I (NEFL) and SGN- II (TMEM132E), respectively. (d) Immunofluorescence staining of CPLX1 in cochleae of Cplx1 KD and NC mice. Scale bar: 100 μm. Cells infected by AAV would turn into green (eGFP), and cells with successful Cplx1 KD would exhibit green but less red (CPLX1 antibody), indicating the success of perturbation experiments. (e) RT-qPCR validation of KD efficiency of Cplx1 in mouse cochlea. Three (Cplx1 KD) or two (NC) biological replicates, each with four technical repeats, were conducted in each sample. Data are mean values ± SD. Two-sided t-test P values are indicated. (f) Representative example of mABR signals in a NC (left) and a Cplx1 KD (right) mouse.

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Extended Data Fig. 9 Identification of cell types in the bat auditory pathway.

(a) Violin plots showing expression pattern of representative marker genes for each cell type in five key auditory pathway components of R. sinicus. DCN, dorsal cochlear nucleus. VCN, ventral cochlear nucleus. SO, superior olive. IC, inferior colliculus. MGB, medial geniculate body. (b) Violin plots showing expression pattern of representative marker genes for each cell type in five key auditory pathway components of C. sphinx.

Extended Data Fig. 10 CPLX1 aa sequence analysis among mammals.

(a) Full-length aa sequence alignment of CPLX1 in mammals. The 90th aa was boxed in red. (b) Predicted CPLX1 protein structure of C. sphinx by AlphaFold. Different colors correspond to different function domains: NT, N-terminus; AH, α-helix; CH, central helix; CT, C-terminus. (c) Example traces of IPSC amplitude triggered by different current pulse frequencies.

Supplementary information

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Supplementary Video 1

Ultrasound cue-associated freezing behavior test in Cplx1 KD mice at 16 kHz.

Supplementary Video 2

Ultrasound cue-associated freezing behavior test in Cplx1 KD mice at 63 kHz.

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Liu, M., Wang, C., Huo, L. et al. Complexin-1 enhances ultrasound neurotransmission in the mammalian auditory pathway. Nat Genet 56, 1503–1515 (2024). https://doi.org/10.1038/s41588-024-01781-z

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