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ABSTRACT The considerably slow pace of human brain development correlates with an evolutionary increase in brain size, cell numbers, and expansion of neuronal structures, with axon tracts undergoing an even greater evolutionary increase than other neuronal domains. However, whether tempo is responsible for these differences in magnitude, and how, remains to be determined. Here, we used brain organoids to investigate this and observed that human axon tracts spend more time growing and extend farther compared to those of mice, independent of their tissue environment. Single cell RNA sequencing analysis pointed to a subset of calcium-permeable ion channels expressed throughout neuron development, including during axon tract outgrowth. Calcium imaging during early neuron development consistently revealed a reduced calcium influx in human neurons compared to mouse neurons. Stimulating calcium influx and increasing cAMP levels resulted in premature halting of axon tract outgrowth and shorter axon tracts, mimicking the mouse phenotype, while abrogating calcium influx led to an even longer phase of axon tract outgrowth and longer axon tracts in humans. Thus, evolutionary differences in calcium regulation set the tempo of neuronal development, by extending the time window to foster the more elaborated human neuron morphology.
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Multi-curvature micropatterns unveil distinct calcium and mitochondrial dynamics in neuronal networks
Tangential curvatures are a key geometric feature of tissue folds in the human cerebral cortex. In the brain, these smoother and firmer bends are called gyri and sulci and form distinctive curved tissue patterns imposing a mechanical stimulus on neuronal networks. This stimulus is hypothesized to be essential for proper brain cell function but lacks in most standard neuronal cell assays. A variety of soft lithographic micropatterning techniques can be used to integrate round geometries in cell assays. Most microfabricated patterns, however, focus only on a small set of defined curvatures. In contrast, curvatures in the brain span a wide physical range, leaving it unknown which precise role distinct curvatures may play on neuronal cell signaling. Here we report a hydrogel-based multi-curvature design consisting of over twenty bands of distinct parallel curvature ranges to precisely engineer neuronal networks' growth and signaling under patterns of arcs. Monitoring calcium and mitochondrial dynamics in primary rodent neurons grown over two weeks in the multi-curvature patterns, we found that static calcium signaling was locally attenuated under higher curvatures ( k > 0.01 μm −1 ). In contrast, to randomize growth, transient calcium signaling showed higher synchronicity when neurons formed networks in confined multi-curvature patterns. Additionally, we found that mitochondria showed lower motility under high curvatures ( k > 0.01 μm −1 ) than under lower curvatures ( k < 0.01 μm −1 ). Our results demonstrate how sensitive neuronal cell function may be linked and controlled through specific curved geometric features. Furthermore, the hydrogel-based multi-curvature design possesses high compatibility with various surfaces, allowing a flexible integration of geometric features into next-generation neuro devices, cell assays, tissue engineering, and implants.
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- Award ID(s):
- 1846271
- PAR ID:
- 10228375
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 21
- Issue:
- 6
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 1164 to 1174
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D0LC01205J
