DOI: 10.5061/dryad.bk3j9kdpg
title: The organization of serotonergic fibers in the Pacific angelshark brain: Neuroanatomical and supercomputing analyses

description: Serotonergic axons (fibers) are a universal feature of all vertebrate
 brains. They form meshworks, typically quantified with regional density
 measurements, and appear to support neuroplasticity. The self-organization
 of this system remains poorly understood, partly because of the strong
 stochasticity of individual fiber trajectories. In an extension to our
 previous analyses of the mouse brain, serotonergic fibers were
 investigated in the brain of the Pacific angelshark (Squatina
 californica), a representative of a unique (ray-like) lineage of the
 squalomorph sharks. First, the fundamental cytoarchitecture of the
 angelshark brain was examined, including the expression of ionized
 calcium-binding adapter molecule 1 (Iba1, AIF-1) and the mesencephalic
 trigeminal nucleus. Second, serotonergic fibers were visualized with
 immunohistochemistry, which showed that fibers in the forebrain have the
 tendency to move toward the dorsal pallium and also accumulate at higher
 densities at pial borders. Third, a population of serotonergic fibers was
 modeled inside a digital model of the angelshark brain by using a
 supercomputing simulation. The simulated fibers were defined as sample
 paths of reflected fractional Brownian motion (FBM), a continuous-time
 stochastic process. The regional densities generated by these simulated
 fibers reproduced key features of the biological serotonergic fiber
 densities in the telencephalon, a brain division with a considerable
 physical uniformity and no major “obstacles” (dense axon tracts). These
 results demonstrate that the paths of serotonergic fibers may be
 inherently stochastic, and that a large population of such paths can give
 rise to a consistent, non-uniform, and biologically-realistic fiber
 density distribution. Local densities may be induced by the constraints of
 the three-dimensional geometry of the brain, with no axon guidance cues.
 However, they can be further refined by anisotropies that constrain fiber
 movement (e.g., major axon tracts, active self-avoidance, chemical
 gradients). In the angelshark forebrain, such constraints may be reduced
 to an attractive effect of the dorsal pallium, suggesting that
 anatomically complex distributions of fiber densities can emerge from the
 interplay of a small set of stochastic and deterministic processes.
description: # The Organization of Serotonergic Fibers in the Pacific Angelshark Brain:
 Neuroanatomical and Supercomputing Analyses Authors: Skirmantas Janusonis,
 Ralf Metzler, Thomas Vojta --- #### FILE:
 PacificAngelshark_CoronalSectionSet_Nissl.zip This set of images is a
 series of coronal sections through a Pacific angelshark (Squatina
 californica) adult female brain. The specimen was collected in Santa
 Barbara County (California, USA) in September 2017. The brain was embedded
 in gelatin (in two blocks) and sectioned at 40 micrometers thickness on a
 freezing microtome. The sections have been stained with a Nissl dye
 (thionine). The sections are in the sequential rostro-caudal order,
 separated by 160 micrometers. The first section
 ("PacificAngelshark_Sp24_Thionine_1s1") contains a scale bar
 that applies to all sections with no magnification in the name. These
 images have been imaged in bright field with a 1X objective. Some images
 have two partially overlapping parts ("p1" and "p2").
 Some details are shown at higher magnifications. These images have been
 captured in bright field and contain a scale bar (their names also include
 the magnification; e.g., "_5X"). Each physical slide had 6
 mounted sections. The file name
 "PacificAngelshark_Sp24_Thionine_AsB" refers to the shark
 specimen number ("Sp24"), the slide number (A), and the section
 number (B) on this slide. Some sections in the middle of the set are
 incomplete (e.g., "14s1") because they were at the beginning of
 the second block. The imaging system: Zeiss AxioImager Z1, with the
 following objectives: 1X (Plan-Neofluar, NA = 0.025), 5X (EC
 Plan-Neofluar, NA = 0.16), 10X (Plan-Apochromat, NA = 0.45), and 20X
 (Plan-Apochromat, NA = 0.80). All sections have been processed and imaged
 by Dr. Skirmantas Janusonis (University of California, Santa Barbara). ---
 #### FILE: PacificAngelshark_CoronalSectionSet_UnstainedWet.zip This set
 of images is a series of coronal sections through a Pacific angelshark
 (Squatina californica) adult female brain. The specimen was collected in
 Santa Barbara County (California, USA) in September 2017. The brain was
 embedded in gelatin (in two blocks) and sectioned at 40 micrometer
 thickness on a freezing microtome. The sections have been imaged on glass
 slides, unstained and uncoverslipped, after a quick dip in water. The
 sections are in the sequential rostro-caudal order, separated by 160
 micrometers. The first section
 ("PacificAngelshark_Sp24_Thionine_1s1") contains a scale bar
 that applies to all sections with no magnification in the name. These
 images have been imaged in bright field with a 1X objective. Some images
 have two partially overlapping parts ("p1" and "p2").
 Each physical slide had 6 mounted sections. The file name
 "PacificAngelshark_Sp24_Thionine_AsB" refers to the shark
 specimen number ("Sp24"), the slide number (A), and the section
 number (B) on this slide. Some sections in the middle of the set are
 incomplete (e.g., "14s1") because they were at the beginning of
 the second block. The imaging system: Zeiss AxioImager Z1, with a 1X
 objective (Plan-Neofluar, NA = 0.025). All sections have been processed
 and imaged by Dr. Skirmantas Janusonis (University of California, Santa
 Barbara). --- ### FILE: PacificAngelshark_SimulatedFiberDensities.zip This
 dataset contains the simulated densities of 4800 serotonergic fibers
 modeled as fractional Brownian motion paths with Hurst index H = 0.8. Each
 path consists of 2^25 (approximately 33.6 million) steps. In the
 simulations, distances were measured in "pixel units" whose size
 corresponds to the size of the pixels in the images used to create the
 3D-brain model. One pixel unit corresponds to 12.5 micrometers in the
 physical angelshark brain. The fiber densities were recorded using a cubic
 grid with cells of linear size of 25.0 micrometers (i.e., 2 pixel-units).
 The densities are stored in the files 011.dat through 999.dat. Each file
 contains the densities at one rostrocaudal level (one coronal section).
 The filename indicates the rostrocaudal position in pixel units. Each data
 file is organized in columns. The first three columns (igx, igy, igz)
 contain the labels (counters) of the grid cells in the three dimensions.
 The next three columns (x, y, x) contain the position of the center of
 each cell in pixel units. The seventh column (distrib) contains the fiber
 density, and the eighth column (log(distrib)) contains the natural log of
 distrib (or -100 if the density is zero). The densities are normalized as
 probability densities (i.e., the integral of the density over the entire
 shape is normalized to unity). --- #### FILE:
 Reader_SimulatedFiberDensities.nb This script (written in Wolfram
 Mathematica 14) reads simulated fiber densities (in the *.dat format) and
 outputs them as TIFF images. --- #### FILE:
 ContourGenerator_OneSection.zip This set contains the scripts (written in
 Wolfram Mathematica 14) used to generate brain contours for the
 supercomputing simulations. It is a two-step process, with a validation
 step (Steps 1-3). One brain section (#22) is shown as an example
 (PacificAngelshark_22.tif, with manually outlined contours in the other
 TIFF files). The set includes the executed Mathematica notebooks saved as
 PDF files and Excel (xlsx) files generated in the process (these files can
 be regenerated by running the scrips (nb), but they can also be opened
 without Mathematica). --- #### FILE:
 PacificAngelshark_CoronalSections_Serotonin-IHC.zip High-resolution
 (tiled) images of serotonergic fibers at eight coronal brain levels of an
 adult female Pacific angelshark (Squatina californica). The sections have
 been immunostained for serotonin (5-HT; ImmunoStar #20079) with the Cy3
 fluorophore and imaged in color with a 5X objective (EC Plan-Neofluar, NA
 = 0.16) on a Zeiss AxioImager Z1 system. For details, see Janusonis et al.
 (2025)
 ([https://doi.org/10.3389/fnins.2025.1602116](https://doi.org/10.3389/fnins.2025.1602116)). The sections have been processed and imaged by Dr. Skirmantas Janusonis (University of California, Santa Barbara). ## Change Log **21 Aug 2025:** two files were added: ContourGenerator_OneSection.zip and PacificAngelshark_CoronalSections_Serotonin-IHC.zip