Abstract
Vertebrates rely on rod photoreceptors for vision in low-light conditions. The specialized downstream circuit for rod signalling, called the primary rod pathway, is well characterized in mammals, but circuitry for rod signalling in non-mammals is largely unknown. Here we demonstrate that the mammalian primary rod pathway is conserved in zebrafish, which diverged from extant mammals ~400 million years ago. Using single-cell RNA sequencing, we identified two bipolar cell types in zebrafish that are related to mammalian rod bipolar cell (RBCs), the only bipolar type that directly carries rod signals from the outer to the inner retina in the primary rod pathway. By combining electrophysiology, histology and ultrastructural reconstruction of the zebrafish RBCs, we found that, similar to mammalian RBCs, both zebrafish RBC types connect with all rods in their dendritic territory and provide output largely onto amacrine cells. The wiring pattern of the amacrine cells postsynaptic to one RBC type is strikingly similar to that of mammalian RBCs and their amacrine partners, suggesting that the cell types and circuit design of the primary rod pathway emerged before the divergence of teleost fish and mammals. The second RBC type, which forms separate pathways, was either lost in mammals or emerged in fish.
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Data availability
No new scRNA-seq data were generated in this paper.
Code availability
Computational scripts detailing scRNA-seq analysis reported in this paper are available on GitHub at https://github.com/shekharlab/ZebrafishBC. We have also provided R markdown (Rmd) files that show step-by-step reproduction of the key results on GitHub at https://github.com/shekharlab/ZebrafishBC.
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Acknowledgements
We thank the Vision Core at the University of Washington for processing zebrafish retina samples and acquiring serial images for SBFSEM, and R. N. Swanstrom for helping with cell tracing of the EM volume. Funding was provided by the MCSA fellowship (‘ColourFish’ 748716) from the EU Horizon 2020 and Research to Prevent Blindness Career Development Award to T.Y.; the NIH EY14358 to R.O.W.; U01MH105960 and R01 EY022073 to J.R.S.; EY01730 (Vision Core grant, PI M. Neitz) Developmental Biology Training Grant HD07183 to F.D.D.; the Wellcome Trust 220277/Z20/Z, European Research Council ERC-StG 677687, UKRI BBSRC, BB/R014817/1 and BB/W013509/1, Leverhulme Trust PLP-2017-005, RPG-2021-026 to T.B.; NIH grant R00EY028625, Hellmann Foundation Fellowship, and the McKnight Foundation Fellowship to K.S.; the DFG through TRR-274, TP C04 (project ID 408885537) to L.G.; travel grant from the Graduate School in Systemic Neuroscience to Y.K., and from the Max Planck Society to Y.K. and H.B.
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A.M.H., P.M., J.H., Y.K., K.S., J.R.S., H.B., T.B., R.O.W. and T.Y. designed the study; Y.K. performed scRNA-seq under the supervision of H.B. and J.R.S. with guidance from K.S.; J.H. processed and analysed the scRNA-seq data under the supervision of K.S.; the data were further interpreted by J.H., K.S., H.B. and J.R.S.; T.Y., S.C.S. and L.G. generated new plasmids; T.Y. and F.D.D. generated novel lines; T.Y. performed experiments, collected and analysed the data for light microscopy with help from O.L.; P.M. and F.R. performed whole-cell patch recordings and interpreted the data; F.D.D. prepared the sample for SBFSEM; A.M.H., O.L. and T.Y. traced the EM images; T.Y. analysed and interpreted the EM data; all results including transcriptional, physiological and anatomical analysis were further interpreted by K.S., J.R.S., H.B., T.B., R.O.W. and T.Y.; T.Y. wrote the manuscript with input from all authors.
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Extended data
Extended Data Fig. 1 Expression patterns of the identified marker genes in BC clusters.
The size of each circle depicts the percentage of cells in the cluster in which the marker was detected (≥1 UMI), and its grey scale depicts the scaled average expression level of cells within the cluster.
Extended Data Fig. 2 Dendritic tiling of RBC1 and RBC2 BCs across the retina.
a,b, Confocal images of retinal flat mount at outer plexiform layer level from Tg(vsx1:memCerulean)q19 (vsx1:memCer) (a) and Tg(vsx2:memCerulean)wst01 (vsx2:memCer) (b). Note that the vsx1:memCer line occasionally labels OFF BCs. These BCs were distinguished by tracing the cells to the axon terminals in the confocal image volumes.
Extended Data Fig. 3 RBC1 and RBC2 are ON BCs.
Example traces of voltage responses of RBC1 and RBC2 after a cone activating light flash (arrow heads). Inhibitory neurotransmitter receptors were blocked (inh lock) by a bath application of gabazine, strychnine, and TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid).
Extended Data Fig. 4 Identification of RBC1 and RBC2 postsynaptic neurons in SBFSEM volume.
a, A partial image of an example SEM image of an adult zebrafish retina. OPL (outer plexiform layer), INL (inner nuclear layer), IPL (inner plexiform layer), GCL (ganglion cell layer). b, Magnified image of the region within the black box in a at the bottom layer of the IPL. Characteristic large bipolar cell axons are painted in light yellow and green. c, Ribbon synapse distributions in a RBC1 and a RBC2. The locations of ribbon synapses are marked in red. Arrow heads indicate the locations of example ribbon synapses (arrows) shown in the insets. d, Reconstruction of all RBC1s and RBC2s in the EM volume. Postsynaptic neurons of a centrally located RBC1 (open arrow head) and RBC2 (closed arrow head) were reconstructed in Figs. 5, 6, and S5–9. e,f, Traces of neuronal processes and the location of somas of cells that are post-synaptic to RBC1 and RBC2 cells. Individual cells were color coded. IPL: inner plexiform layer.
Extended Data Fig. 5 Gallery of mono-stratifying AC making reciprocal synapses with RBC1.
En face and side views of individual cells. The numbers of input (open bar) and output (closed bar) synapses with each RBC1 (blue) and RBC2 (red) terminal are indicated as the height of the bars.
Extended Data Fig. 6 Gallery of mono-stratifying AC without reciprocal synapses with RBC1.
En face and side views of individual cells. The numbers of input (open bar) and output (closed bar) synapses with each RBC1 (blue) and RBC2 (red) terminal are indicated as the height of the bars.
Extended Data Fig. 7 Gallery of bi-stratifying AC and RGC contacted to RBC1.
En face and side views of individual cells. The numbers of input (open bar) and output (closed bar) synapses with each RBC1 (blue) and RBC2 (red) terminal are indicated as the height of the bars.
Extended Data Fig. 8 Gallery of mono-stratifying AC connected to RBC2.
En face and side views of individual cells. The numbers of input (open bar) and output (closed bar) synapses with each RBC1 (blue) and RBC2 (red) terminal are indicated as the height of the bars.
Extended Data Fig. 9 Gallery of bi-stratifying AC and RGC connected to RBC2.
En face and side views of individual cells. The numbers of input (open bar) and output (closed bar) synapses with each RBC1 (blue) and RBC2 (red) terminal are indicated as the height of the bars.
Extended Data Fig. 10 Cx35 is highly expressed in two layers of the IPL and co-localizes with ON stratifying BC axon terminals.
a, Confocal images of retinal slices from Tg(vsx1:memCerulean)q19 (vsx1:memCer). Immunolabeling for Connexin35 (cx35) and PKCα are in cyan and magenta, respectively. IPL: inner plexiform layer. b, Distribution patterns of Cx35 immunostaining across the IPL. Cx35 labeling is enriched in the layers where A2-like AC dendrites stratify (grey shades). The blue thick line is mean values and shades are S.E.M. n=3 animals. c, Distribution of cx35 puncta in the PKCα positive ON BC axons. Cx35 labeling outside the axons was digitally masked (removed) in the image on the right.
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Hellevik, A.M., Mardoum, P., Hahn, J. et al. Ancient origin of the rod bipolar cell pathway in the vertebrate retina. Nat Ecol Evol (2024). https://doi.org/10.1038/s41559-024-02404-w
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DOI: https://doi.org/10.1038/s41559-024-02404-w