Isoe, Yasuko 1 ; Vohra, Sumit2 ; Tavhelidse-Suck, Tinatini3 ; Knoblich, Simon4 ; Seleit, Ali4; Boulanger-Weill, Jonathan1 ; Chambule, Sydney1 ; Aspiras, Ariel1 ; Santoriello, Cristina1 ; Randlett, Owen5 ; Wittbrodt, Joachim3 ; Lichtman, Jeff 1 ; Fishman, Mark 1 ; Hege, Hans-Christian2 ; Baum, Daniel2 ; Engert, Florian 1

Research facility: Harvard University

Published Jul 29, 2026 on Dryad. https://doi.org/10.5061/dryad.zw3r228hw

Abstract

Comparative brain analyses across different species can uncover general principles of neural circuits and behaviour. To advance such efforts, we developed a unified brain atlas platform that allowed us to quantitatively compare neural structures across two teleost larvae, medaka (Oryzias latipes) and zebrafish (Danio rerio). A novel feature of the platform is that it enables the effortless integration of user-generated imaging volumes, which facilitates quantitative comparison of anatomical, transcriptional, and functional data across species and laboratories. Using the integrated toolsets, we confirmed that we were able to identify anatomical differences in brain regions, as well as differentially expressed genes in both species. We further demonstrated the existence of functionally distinct regions in the telencephalon of both species that showed differential responses to social stimuli. The web-based and interactive nature of this atlas platform will encourage exchange of data, information, and knowledge across the teleost community. Its readily scalable nature enables easy contributions by other groups and allows for expansion into a continuously growing and globally accessible database.

Fish maintenance

All medaka fish and zebrafish wild-type and transgenic lines used in this research were raised in methylene blue water for a few days and then in filtered fish facility water on a 14 h light, 10 h dark cycle at 28°C. We kept the density of embryos around 30 fish per Petri dish. Under 28°C incubation, medaka hatch at 6-7 days post-fertilisation (dpf), while zebrafish hatch at 2 dpf (Signore et al. 2009). We used 0 or 1 days post-hatch larvae for medaka and 5-7 dpf larvae for zebrafish in the experiments.

in situ hybridization

In situ hybridisation experiments were performed according to previously published procedures (Vauti et al. 2020; Shainer et al., n.d.). In brief, after fixing fish larvae in 4% PFA/PBS overnight, the larvae were dehydrated with 100% methanol. Then we washed with ethanol and performed permeabilization with ethanol/xylol solution. Then we rehydrated with a sequence of ethanol/H2O with 0.1% Tween-20. Then we permeabilized larvae with 80% acetone/H2O at -20C for 30 min and washed larvae with PBST (0.1% Tween-20, PBS). We pre-hybridized the larvae with a prewarmed hybridization buffer for 4 hours and incubated them with the HCR probe for 60 hours at 37 C. Then, the larvae were washed with a 5× SSC buffer and incubated with an amplification buffer for 30min. After that, larvae were mixed with a hairpin that was heated up at 95 C for 90 seconds and incubated overnight at RT. After washing with 5 x SCCT, we cleared the larvae with a series of glycerol and stored them at -20C. We always stained with snap25, which was used as a whole-neuron marker for registration. We ordered HCR-RNA bundles from Molecular Instruments, Inc.

c-fos experiments and analysis

For the social stimuli assay, two fish larvae were placed in a Petri dish at room temperature with light on. For the feeding assay, the fish larvae were placed in a dish, and paramecia were added. For the MS222 assay, 60 µg/ml of MS222 (Sigma-Aldrich) was dissolved in filtered fish water. Samples were collected 40 min after treatment and immediately fixed with 4% PFA/PBS overnight. For the vibration assay, the dish containing fish larvae was placed on to the shaker (SCILOGEX, SCI-0180-S) at a speed of 60 rpm for 1 hour. Then HCR experiments were then performed with c-fos and snap25 probes as described above. Snap25 was used to normalise and quantify the c-fos signal as it is widely expressed in the nervous system.

To analyse the neural activity across samples in each treatment, we first created a snap25 channel for the reference brain. After registering the image stacks to the reference brain by aligning to this snap25 channel, we calculated the average value of c-fos and snap25 staining across all MECE and MISC masks. For each mask, the c-fos value divided by snap25 value was calculated and normalised with the average of all the snap25 values across the whole brain and plotted in the Manhattan plot. The image stacks were generated by averaging each pixel across samples and then displaying the maximum projected image. The lookup table was chosen so that the highest and lowest values matched the maximum and minimum of the lookup table. For the c-fos analysis in the telencephalon, the same analysis was performed using the MISC masks created for the telencephalon.

Confocal imaging

Samples whose subpopulations are labeled were imaged with confocal microscopy (Zeiss LSM780). We used a 10x objective, and the spatial resolution of the image stacks was 0.59um x 0.59um x 2 um or 0.59um x 0.59um x 1.5 um.

Brain registration

After the imaging step, all imaging volumes were registered to the reference brain using the Advanced Normalisation Tools (ANTs) (Ahrens et al. 2012). We performed the registration with rigid [-m GC, -c 300x300x300x0, 1e-8,10, -f 12x8x4x2, -s 4x3x2x1], affine [-m GC, -c 300x300x300x0, 1e-8,10, -f 12x8x4x2, -s 4x3x2x1] and SyN (diffeomorphic symmetric) [-m CC, -c 300x300x300x0, 1e-8,10, -f 12x8x4x2x1, -s 4x3x2x1x0] transformations, with the specified parameters. The registrations were carried out on a high-performance cluster with 300 GB of memory and 150 CPU cores. The tutorial with a detailed description of the parameters and step-by-step instructions for the registration with the Python script will be available on the FishExplorer website (https://zebrafishatlas.zib.de/tutorial/image-registration.html).

For the immunostained samples, we always stained with Hoechst dye and used the Hoechst reference brain stacks for registration. To register HCR-stained samples with the reference brain, we imaged a few stacks of snap25- and Hoechst-stained brains and made a snap25 channel as the reference brain. HCR-stained samples always had snap25 staining to register other staining channels to the reference brain. After registration, we created the average stacks that were used to determine the region masks.

A cross-species atlas platform for comparative neuroscience in teleost fish

Data files

Abstract

README: A cross-species atlas platform for comparative neuroscience in teleost fish

Description of the data and file structure

Files

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