Recent experimental and theoretical work by our group has shown that the self-organization of the brain serotonergic matrix is strongly driven by the spatiotemporal dynamics of single serotonergic axons (fibers). The trajectories of these axons are often stochastic in character and can be described by step-wise random walks or time-continuous processes (e.g., fractional Brownian motion). The success of these modeling efforts depends on experimental data that can validate the proposed mathematical frameworks and constrain their parameters. In particular, further progress requires reliable experimental tracking of individual serotonergic axons in time and space. Visualizing this dynamic behavior in vivo is currently extremely difficult because of the high axon densities and other resolution limitations. In this study, we used in vitro systems of mouse primary brainstem neurons to examine serotonergic axons with unprecedented spatiotemporal precision. The high-resolution methods included confocal microscopy, STED super-resolution microscopy, and live imaging with holotomography. We demonstrate that the extension of developing serotonergic axons strongly relies on discrete attachments points on other, non-serotonergic neurons. These membrane anchors are remarkably stable but can be stretched into nano-scale tethers that accommodate the axon’s transitions from neuron to neuron, as it advances through neural tissue. We also show that serotonergic axons can be flat (ribbon-like) and produce screw-like rotations along their trajectory, perhaps to accommodate mechanical constraints. We conclude that the stochastic dynamics of serotonergic axons may be conditioned by the stochastic geometry of neural tissue and, consequently, may reflect it. Our current research includes hydrogels to better understand these processes in controlled artificial environments. Since serotonergic axons are nearly unique in their ability to regenerate in the adult mammalian brain and they support neural plasticity, this research not only advances fundamental neuroscience but can also inform efforts to restore injured neural tissue.
This research was funded by NSF CRCNS (#1822517 and #2112862), NIMH (#MH117488), and the California NanoSystems Institute.
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Imaging neuronal structure dynamics using 2‐photon super‐resolution patterned excitation reconstruction microscopy
Visualizing fine neuronal structures deep inside strongly light‐scattering brain tissue remains a challenge in neuroscience. Recent nanoscopy techniques have reached the necessary resolution but often suffer from limited imaging depth, long imaging time or high light fluence requirements. Here, we present two‐photon super‐resolution patterned excitation reconstruction (2P‐SuPER) microscopy for 3‐dimensional imaging of dendritic spine dynamics at a maximum demonstrated imaging depth of 130 μm in living brain tissue with approximately 100 nm spatial resolution. We confirmed 2P‐SuPER resolution using fluorescence nanoparticle and quantum dot phantoms and imaged spiny neurons in acute brain slices. We induced hippocampal plasticity and showed that 2P‐SuPER can resolve increases in dendritic spine head sizes on CA1 pyramidal neurons following theta‐burst stimulation of Schaffer collateral axons. 2P‐SuPER further revealed nanoscopic increases in dendritic spine neck widths, a feature of synaptic plasticity that has not been thoroughly investigated due to the combined limit of resolution and penetration depth in existing imaging technologies.
- NSF-PAR ID:
- 10046490
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Journal of Biophotonics
- Volume:
- 11
- Issue:
- 3
- ISSN:
- 1864-063X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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