DOI: 10.5061/dryad.02v6wwqg3
title: Evolution of a central dopamine circuit underlies adaptation of light-evoked sensorimotor response in the blind cavefish, <em>Astyanax mexicanus</em>

description: Adaptive behaviors emerge in novel environments through functional changes
 in neural circuits. While relationships between circuit function and
 behavior have been well studied, how evolution shapes those circuits and
 leads to behavioral adaptation is poorly understood. The Mexican cavefish,
 Astyanax mexicanus, provides a unique genetically amendable model system,
 equipped with above-ground eyed surface fish and multiple evolutionarily
 divergent populations of blind cavefish that have evolved in complete
 darkness. These differences in environment and vision provide an
 opportunity to examine how a neural circuit is functionally influenced by
 the presence of light. Here, we examine differences in the detection and
 behavioral response induced by non-visual light reception. Both
 populations exhibit photokinetic behavior, with surface fish becoming
 hyperactive following sudden darkness and cavefish becoming hyperactive
 following sudden illumination. To define these photokinetic neural
 circuits, we integrated whole-brain functional imaging with our Astyanax
 brain atlas for surface and cavefish responding to light changes. We
 identified the caudal posterior tuberculum as the central modulator for
 both light and dark-stimulated photokinesis. To uncover how spatiotemporal
 neuronal activity differed between surface fish and cavefish, we used
 stable pan-neuronal GCaMPAstyanaxtransgenics to show that a subpopulation
 of darkness-sensitive neurons in surface fish is now light sensitive in
 cavefish. Further functional analysis revealed that this integrative
 switch is dependent on dopamine signaling, suggesting a key role for
 dopamine and a highly conserved dopamine circuit in modulating the
 evolution of a circuit driving an essential behavior. Together, these data
 shed light on how neural circuits evolved to adapt to novel settings and
 reveal the power of Astyanax as a model to elucidate mechanistic insights
 underlying sensory adaptation. Abstract Figure
description: # Evolution of a central dopamine circuit underlies adaptation of
 light-evoked sensorimotor response in the blind cavefish, *Astyanax
 mexicanus* Data for this study includes behavioral data (tracking, time
 binned analysis, and summary statistics .txt), imaging data for anatomy
 (raw and downsampled .nii.gz; brain segmentations, iWarp.nii.gz; affine
 brain masks **.mat), live imaging (**.tiff, .avi), and custom Fiji,
 MATLAB, and bash scripts used for all analyses. All imaging data was
 registered to the Astyanax Atlas using Advanced Normalization Tools
 (ANTs), behavior was analyzed using matlab, anatomy was analyzed using
 cobraZ
 ([https://www.nichd.nih.gov/research/atNICHD/Investigators/burgess/software](https://www.nichd.nih.gov/research/atNICHD/Investigators/burgess/software)), fixed brain mapping was analyzed using MAP-mapping ([https://github.com/owenrandlett/Z-Brain](https://github.com/owenrandlett/Z-Brain)), live imaging was analyzed using Mesmerize ([https://github.com/kushalkolar/MESmerize](https://github.com/kushalkolar/MESmerize)), and all statistics were generated and applied using the program Prism (GraphPad, Inc.) #### Description of the Data and file structure Repository: Kozol2024KinesisData.tar.gz contains all the files described in this document. BEHAVIOR: 6 6-day-old surface fish, cavefish, and surface to cave hybrid video analysis. Raw tracking (**.txt) was then analyzed using the 5-minute binned.m script. ANATOMY: 6-day-old surface, cave, and surface to cave hybrid Astyanax mexicanus fish were euthanized with 250mg/L MS222 dissolved in Astyanax system water, then fixed with formaldehyde, stained with antibodies and RNA probes, and imaged on a confocal microscope. These files were then run through a pipeline of brain normalization tools, which produced multiple files needed for morphometric analysis programs CobraZ and morpho. Neuroanatomical volumetric comparisons. nifti (.nii.gz) files are tETERRK-stainedrains sections of surface, pachon, and surface to pachon hybrids used for anatomical analyses. Nifti files were registered to the astyanax F2 standard brain (SPF2_021921_010.nii.gz) using a custom script (ANTs1.sh) calling Advanced Normalization Tools (ANTs; [http://stnava.github.io/ANTs/](http://stnava.github.io/ANTs/)) binaries. The ANTs1.sh script segments each ttERK-stained brain using inverse registration and prints a registered NIFTI file, MATLAB affine brain mask (GenericAffine.mat), and segmentation file (iWarp.nii.gz) that were then run using the CobraZ software ([https://www.nichd.nih.gov/research/atNICHD/Investigators/burgess/software](https://www.nichd.nih.gov/research/atNICHD/Investigators/burgess/software)). Final outputs from cobraZ were analyzed for the different scales of brain segmentation using the following Python CobraZ_transform_Rob.py (26 regions). CobraZ outputs include Holm's corrected t-tests comparing the two analyzed groups. pEPERK_MAPPING6-day-old surface fish and cavefish were treated the same as larvae for ANATOMY, but exposed to a stimulus before euthanasia. tERK and ppERK-stained larvae were then imaged using a confocal microscope, and raw image stacks were then downsampled, sorted according to treatment, and run using the makethemapmap.mat script. Significant differences in files were then analyzed for statistically significant activity in different brain regions across populations. LLIVE_IMAGING 6-day-old GCaMP6s positive surface fish and cavefish were immobilized 2% low agarose agar via bungarotoxin. Larvae were then imaged and exposed to 330-second cycle light changes. Stacks were then loaded as a dataframe, normalized, corrected for movement, and analyzed for hierarchical clustering in the program Mesmerizeapps. of neurons were then exported and used for analysis and figures. ABLATIONS: 6 6-day-old CaMP6s positive surface fish and cavefish were immobilized 2% low agarose agar via bungarotoxin. Bilateral ovoid ROIs were then placed in hypothalamic (test) and non-hypothalamic (sham) regions. Cells were then ablated using a 2-photon microscope, with pre- and post-ablation images captured (**.tiff). Fish were then allowed to recover for 24 hours and tested for behavior. Following behavior, larvae were euthanized as previously described and stained and analyzed for tTh1-positive neurons and ablation location. All output is named for specific populations or genetic lines of Astyanax mexicanus. Wildtype populations include surface rio choy (surface), Pachon cavefish (Pachon), hybrid populations; surface x Pachon F1 hybrid (SPF1) and surface x Pachon F2 hybrid (SPF2). These abbreviations appear in source data, statistical tables, figures, and manuscript text. For more detailed materials and methods, see bioRxiv Kozol et al. 2024 doi: [https://doi.org/10.1101/2024.07.25.605141](https://doi.org/10.1101/2024.07.25.605141) Directory trees and descriptions for the repository of data Kozol2024KinesisData.tar.gz: Kozol2024KinesisData/behavior/ Behavior files are filed by population. Each subdirectory contains the video files (avi), raw and binned tracking data (txt). ##Raw folders contain ethovision project file names, followed by trial name and well number, --Project: Track-astyanax sensory screen; Trial name: -Trial 1; Well number:-4-Subject 1. E.g.,       " Track-astyanax sensory screen-Trial 1-4-Subject 1.txt" Binned data contain the same naming convention as raw data, with a "Displ_daynight" extension that characterizes binning into light phase "day" and dark phase "night". E.g. ", Track-astyanax sensory screen-Trial 1-4-Subject 1_Displ_daynight.txt" Kozol2024KinesisData/anatomy_correlations/iWarp/ Contains segment files for all (**.iWarp.nii.gz) for each brain analyzed in this study. These files are the whole-brain segments for each larlarvad that need to be added to the iWarp folder in cobraZ to run volumetric comparisons. Data files are named for population "e.g., SPF2 P2 (surface to cave f2 hybrids; breeding pair 2)", data "e.g., 011522", condition of stimulus "e.g., off" for lights-off, well number "e.g., 006," and antibody channel (-01 for total-ERK and -02 for phospho-ERK). E.g. SMF2_012522_Off_006-01.nii.gz Kozol2024KinesisData/anatomy_correlations/affine/ Contains affine brain masks for all (**.mat) for each brain analyzed in this study. The affine brain mask provides the whole-brain volume that is needed to calculate the normalization of each brain region in cobraZ. These need to be added to the affine folder in cobraZ to run volumetric comparisons. Data files have the same naming convention as iWarp files and correspond to the same larva's brain. Kozol2024KinesisData/anatomy_correlations/nifti/ Contains tERK-stained brain z-stacks (**.nii.gz) for each brain analyzed in this study. Each tERK brain was used in the ANTs normalization and inverse registration to produce the affine and iWarp files needed for cobraZ analysis. Data files have the same naming convention as iWarp files and correspond to the same larva's brain. Kozol2024KinesisData/gcamp/ Live imaging time series are filed by population. Each population folder contains the live imaging files. TIF files are named by population, transgenic, date, larva number a, nd then exposed to light cycle or no light cycle (sham). Population: surface; line: gcamp; date: 032023; larva: 001; condition: \_sham. E.g. surface_gcamp_032023_001_sham.nd2. Fixed folders provide post-live imaging fixed and stained larva. These files have the same naming convention as the live imaged larva. Kozol2024KinesisData/mapmapping/ Brain maps are filed by population. Each folder contains prenormalized and normalized versions of files, including output from the makethemapmap script. Significant differences. Tiff files were then segmented to determine regional variation in brain activity. Each file is an output from the makethemapmap.m script that is defined by the light condition "on" or "off". "ZScores.tif" provides a full brain stack with colored pixels that correspond to significantly different normalized pERK staining. Green for lights-on and magenta for lights-off. "medianStack.tif" provides a median stack that was calculated by taking all of the total-ERK and phospho-ERK stacks for one condition (e.g., off), normalizing by dividing phospho-ERK/total-ERK, then calculating a median of all normalized stacks for that condition. "SignificantDeltaMedians.tif" provides the delta Median value at each pixel found to be above the FDR threshold. "StdStack.tif" provides the output of randomization and calculations for setting a false discovery rate. Kozol2024KinesisData/ablation/ Ablations are filed as pachon (cavefish). Each folder contains ablation imaging for each individual that was ablated. Each folder is an individual larv, and each series consists of the first file, pre-ablation, and the second file, post-ablation. E.g. Kozol2024KinesisData/ablation/pachon/2/ablation_2.nd2 = pre-ablation, Kozol2024KinesisData/ablation/pachon/2/ablation_2001.nd2 = post-ablation. Kozol2024KinesisData/statistics/ Contains .xlsx file with all statistical tables and .docx file that defines the tables region, test, and associated figure in this study. FFileis named Cavefish_photokinesis_supplementary_tables.xlsx and consists of every statistical table described in the manuscript. Sharing/access Information The repository is available for the public domain via the Creative Commons Zero (CC0) license. Any use or dissemination of data in this repository should cite bioRxiv Kozol et al. 2024 doi: [https://doi.org/10.1101/2024.07.25.605141](https://doi.org/10.1101/2024.07.25.605141) as the source. Additional information or questions can be addressed to Dr. Robert Kozol at [rkozol@fau.edu](mailto:rkozol@fau.edu)