High-resolution spatiotemporal analysis of single serotonergic axons in an in vitro system

Abstract

Vertebrate brains have a dual structure, composed of (i) axons that can be well-captured with graph-theoretical methods and (ii) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.

Qualitative Observations of Transgenically Individualized Serotonergic Fibers in the Mouse Telencephalon

“…Limited information can be extracted from fixed tissue, but these high-resolution images suggest that fiber branches can develop by lateral "budding" (in which one branch is developmentally older than the other one) and by bifurcation (where both branches are of the same age). Both mechanisms have been proposed in previous studies, [36][37][38] but this question can be definitely answered only with modern live imaging methods (e.g., 3D-holotomography 29 ). In layer 1, fibers of distinctly different identities traveled together (separated by only several micrometers), with no detectable interference (Fig.…”

“…A key challenge is this effort is that serotonergic fibers are thin, with a diameter that is near the limit of optical resolution, and that typically many fibers intertwine in the same three-dimensional volume. In vitro studies of isolated serotonergic neurons can provide some essential information, 29 but cell-culture environments are radically different from those in the natural brain parenchyma. It should also be noted that analyses of the transcriptomes of individual serotonergic neurons have revealed an enormous diversity of transcriptional programs, 1,30,31 further highlighting limitations of population-level studies.…”

Predicting the Distribution of Serotonergic Axons: A Supercomputing Simulation of Reflected Fractional Brownian Motion in a 3D-Mouse Brain Model

“…In primary cell cultures, adjacent axon segments of serotonergic neurons can strongly vary in their caliber, varicosity size, and other features (Hingorani et al, 2022). This variability is likely partially stochastic, as a consequence of the strongly stochastic properties of neural tissue at the microscopic level (Jang et al, 2010; Nicholson and Hrabetova, 2017; Hrabetova et al, 2018; Hingorani et al, 2022). Furthermore, different neuron clusters may show preference for different targets; however, individual serotonergic axons within the same cluster may differ in their collateralization and target specificity (Gagnon and Parent, 2014; Ren et al, 2019).…”

“…However, without accurate experimental information about the relative dynamics of both processes such simulations are unlikely to produce robust results. State-of-the-art imaging techniques have provided new insights into the growth dynamics of single serotonergic axons (Jin et al, 2016; Hingorani et al, 2022), but live-imaging of serotonergic fibers in the intact developing brain requires further technological advances.…”

“…Several classes of serotonergic axons have been defined based on their morphology (Kosofsky and Molliver, 1987), but recent studies have demonstrated that axonal morphology may undergo significant changes in a regionally- and developmentally-dependent manner (Gagnon and Parent, 2014; Maddaloni et al, 2017; Nazzi et al, 2019; Okaty et al, 2019; Andersson et al, 2020). In primary cell cultures, adjacent axon segments of serotonergic neurons can strongly vary in their caliber, varicosity size, and other features (Hingorani et al, 2022). This variability is likely partially stochastic, as a consequence of the strongly stochastic properties of neural tissue at the microscopic level (Jang et al, 2010; Nicholson and Hrabetova, 2017; Hrabetova et al, 2018; Hingorani et al, 2022).…”