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Serotonergic axons (fibers) have a ubiquitous distribution in vertebrate brains, where they form meshworks with well-defined, regionally-specific densities. In humans, perturbations of these densities have been associated with abnormal neural processes, including neuropsychiatric conditions. The self-organization of serotonergic meshworks depends on the cumulative behavior of many serotonergic axons, each one of which has a virtually unpredictable trajectory. In order to bridge the high stochasticity at the microscopic level and the regional stability at the mesoscopic level, we are developing tunable hydrogel systems that can support causal modeling of these processes. These same systems can support future restorative efforts in neural tissue because serotonergic axons are nearly unique in their ability to robustly regenerate in the adult brain. In the study, we extended our research in 2D-primary brainstem cultures (Hingorani et al., 2022) to 3D-hydrogels. Tunable hydrogel scaffolds can closely mimic the mechanical and biochemical properties of actual neural tissue in all three dimensions and are therefore qualitatively different from 2D-environments. However, the integration of these scaffolds with highly sensitive neurons poses unique challenges. As the first step in building a hydrogel-based platform for the bioengineering of serotonergic axons, we studied primary brainstem neurons in several commercially available hydrogel platforms. The viability and dynamics of serotonergic somata and neurites were analyzed at different days in vitro with immunocytochemistry and high-resolution confocal microscopy. In addition, live imaging of neuron growth cones was performed, and the observed dynamics was compared to our extensive database of holotomographic (refractive index-based) recordings in 2D-cultures. The progress and key problems will be discussed. This research was funded by NSF CRCNS (#1822517 and #2112862), NIMH (#MH117488), and the California NanoSystems Institute. 

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.