The self-organization of the serotonergic matrix, a massive axon meshwork in all vertebrate brains, is driven by the structural and dynamical properties of its constitutive elements. Each of these elements, a single serotonergic axon (fiber), has a unique trajectory and can be supported by a soma that executes one of the many available transcriptional programs. This “individuality” of serotonergic neurons necessitates the development of specialized methods for single-fiber analyses, both at the experimental and theoretical levels. We developed an integrated platform that facilitates experimental isolation of single serotonergic fibers in brain tissue, including regions with high fiber densities, and demonstrated the potential of their quantitative analyses based on stochastic modeling. Single fibers were visualized using two transgenic mouse models, one of which is the first implementation of the Brainbow toolbox in this system. The trajectories of serotonergic fibers were automatically traced in the three spatial dimensions with a novel algorithm, and their properties were captured with a single parameter associated with the directional von Mises-Fisher probability distribution. The system represents an end-to-end workflow that can be imported into various studies, including those investigating serotonergic dysfunction in brain disorders. It also supports new research directions inspired by single-fiber analyses in the serotonergic matrix, including supercomputing simulations and modeling in physics.

The z-stack named "zStack_EGFP.zip" is a zipped 3D-image of the inferior colliculus in the brain of an adult mouse that expresses EGFP (enhanced green fluorescent protein) in serotonergic fibers (after Tph2-dependent Cre recombination). The image was acquired on a Leica SP8 resonant scanning confocal system with a 60xoil (NA 1.40) objective. Each image is 3144 x 3144 pixels, with the XY-scaling of 59 nm/pixel and the Z-step (between consecutive images) of 299 nm. The images have been converted to grayscale and autocontrasted. The set includes a maximum-intensity projection (an RGB image). Three Wolfram Mathematica notebooks (executable code) are included ("SingleFiberAnalysis_Step1_EGFP-Trace1.nb", "SingleFiberAnalysis_Step2.nb", "SingleFiberAnalysis_Step3.nb") that trace and analyze one fiber in the image. The notebooks can be used in other datasets.
 
The z-stack named "zStack_Brainbow.zip" is a zipped 3D-image of the basolateral amygdala in the brain of an adult mouse that expresses Brainbow fluorophores in serotonergic fibers (after Tph2-dependent Cre recombination). The image was acquired on a Leica SP8 resonant scanning confocal system with a 60xoil (NA 1.40) objective. Each image is 2552 x 2552 pixels, with the XY-scaling of 72 nm/pixel and the Z-step (between consecutive images) 299 nm. The original (not autocontrasted) images in the three fluorophore channels (ch0, ch1, ch2) are named "Brainbow_M18_AF488_AF594_AF647_AmygdalaBLARegion" and the merged channels (converted to grayscale and autocontrasted) are named "BLA". The set includes a maximum-intensity projection (an RGB image). Three Wolfram Mathematica notebooks (executable code) are included ("SingleFiberAnalysis_Step1_Brainbow-Trace(1-3).nb") that trace three fibers in the image. The notebooks can be used in other datasets.

The z-stack named "zStack_Brainbow-Subset.zip" is an XY-subset of "zStack_Brainbow." Specifically, the original z-stack has been subsetted in the XY-plane but retains all optical sections (in the Z-dimension). The pixel scaling is the same as in the original image. The set includes the merged channels (converted to grayscale and autocontrasted), which are named "Subset_BLA", and a maximum-intensity projection (an RGB image). Twenty Wolfram Mathematica notebooks (executable code) are included ("SingleFiberAnalysis_Step1_BrainbowSubset-Trace(1-20).nb") that trace twenty fibers in the image. The notebooks can be used in other datasets. The traced fibers can be analyzed as a set with "SingleFiberAnalysis_Step2_BrainbowSubset.nb" and "SingleFiberAnalysis_Step3_BrainbowSubset.nb".  
 
The Wolfram Mathematica notebooks "Simulator_SingleFiber.nb" and "Simulator_BrainbowFibers.nb" generate a single monochrome fiber and a set of Brainbow fibers, respectively.