An experimental platform for stochastic analyses of single serotonergic fibers in the mouse brain

Abstract

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.

Qualitative Observations of Transgenically Individualized Serotonergic Fibers in the Mouse Telencephalon

“…26 Theoretical tools have been recently developed to model this and similar dynamics. 25,26 The high-tortuosity mode may be a property intrinsic to the fiber, but it may also be induced by its environment. For example, it may be associated with pericellular baskets that serotonergic neurons fibers can produce.…”

“…Four animals (all males) were used in the study. The study did not include any of the animals used in the previous study 26 (all AAV injections were performed in 2023).…”

“…They were immediately immersion-fixed in 4% paraformaldehyde overnight at 4ºC, cryoprotected in 30% sucrose for two days at 4ºC, and sectioned coronally into PBS at 40 µm thickness on a freezing microtome. Sections through the rostral raphe were examined unstained with epifluorescence microscopy, and two mice (100 and 111 days post-surgery) with excellent fluorophore expression in the entire raphe region were selected for triple-label immunohistochemistry (with some modifications of the original protocol 26 ). At the time of the brain dissection, the mice were 17 and 17.5 months of age, respectively.…”

“…23,24 Some aspects of this self-organization can be captured by models that treat the trajectories of serotonergic fibers as paths of stochastic processes. 15,25,26 Ultimately, a comprehensive mechanistic explanation should bridge the dynamics of single serotonergic fibers – as they extend, branch, and react to chemical and mechanical signals – and their resultant densities in the adult brain. These densities may represent a dynamic equilibrium rather than a static distribution: serotonergic fibers can robustly regenerate in the adult mammalian brain, with new paths, 27,28 which suggests that some individual fiber segments may continue to grow in the normal (injury-free) brain, with the accompanying degeneration of other segments.…”

“…We have recently developed a transgenic approach in which individual serotonergic fibers express random intensity combinations of three fluorophores with different spectral characteristics. 26 This approach is an implementation of the Brainbow 3.2 toolbox, 32 which is uniquely well-suited for this purpose. In particular, it allows for the identification of fiber segments that belong to the same or different neurons – even if these segments intersect, intertwine, or temporarily leave the imaging volume.…”