Background

Results

Conclusions

All neural processes in the human brain take place in a dense matrix of thin fibers (axons) that release serotonin, an ancient neurotransmitter that supports the plasticity of neural tissue 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. Previous work in our laboratory has demonstrated that 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. However, visualizing this dynamic behavior in vivo is currently extremely difficult. Recent technological advances (e.g., digital holotomography, three-dimensional (3D)-cell culture systems, microphysiological systems) have changed the way complex neuronal systems can be studied and offer promising alternatives to in vivo approaches. This research examined single serotonergic axons in culture systems (co-cultures and mono-cultures), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. Several 3D-hydrogel systems were additionally tested. This research shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of the 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 computational approaches.

Recent evidence in immunology has indicated that a peripheral player may be contributing to central serotonergic signaling. Researchers have shown that transmigration of blood platelets across the blood brain barrier (BBB) occurs during times of inflammation in the CNS, but none have suggested whether or not this process impacts central serotonin signaling. This paper addresses current research determining the transmigration of platelets into the CNS under natural conditions and addresses future work to solidify any contribution by platelets to CNS 5-HT signaling. Using immunohistochemistry and confocal imaging we mapped the serotonergic varicosities of the mouse brain and used them in comparison to a swellshark (lacking a peripheral serotonin system) to determine any significant differences. It was observed that the ‘loose-varicosities’ seen in the mouse did not appear to be present in the sharks, indicating that these may actually be transmigrated platelets. In order to determine whether or not platelets that cross the BBB contribute significantly to 5-HT signaling we must first determine an accurate and efficient sensor to extracellular changes of 5-HT. In the presence of Fluoxetine (a serotonin reuptake inhibitor), RTq-PCR was used on a variety of receptors to determine sensitivity. Findings were inconclusive for all but one receptor (ITGB3), showing a significant increase in mRNA transcripts. Our results require further verification of platelets from the microvasculature in the CNS using more definitive labeling techniques, elucidation of additional markers as efficient serotonin sensors, and assessing changes in receptor expression as a function of pharmacological platelet depletion.

The current understanding of monoaminergic neurotransmission relies upon the ability to image and analyze the components of these systems. Yet, the efforts to understand these systems are inhibited from a combination of the unique properties of axons that release monoamines and limitations in imaging technologies. Axons from monoaminergic cells can be thin (~ 1 μm in diameter) and cannot be viewed directly, requiring both fluorescent labeling and high-resolution imaging. Adding further complications are that the axons travel the entirety of the brain, appear to intertwine freely with each other, and are often difficult to track individually. Here, we successfully designed an experimental process for monoaminergic axon research that endows the ability for axon differentiation with the use of Brainbow AAV and Cre technologies. These approaches were validated with a new algorithm that can reliably trace individual axons in various brain regions. To complement these methods and provide information on axon trajectories in the natural 3D-space, we also designed a process for high-resolution light-sheet microscopy using tissue clearing technologies for imaging of axons without sectioning tissue combined with machine learning algorithms for automated tracing. These methods allow for unprecedented access to individual axon trajectories and support their modeling as paths of spatial stochastic processes. This project focused on the serotonin system, however, these methods can be extended and applied to research using other monoamines. This is the first research on serotonin or monoamines in general with the capability of individual axon discrimination and can help answer many previously elusive questions within this field of research.