Substrate Topography Determines Neuronal Polarization and Growth In Vitro

Abstract

The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. In vivo, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. In vitro, many studies have shown the possibility to influence neuronal morphology and growth by biophysical, i.e. topographic, signaling. In this work we have taken this approach one step further and investigated the impact of substrate topography in the very early differentiation stages of developing neurons, i.e. when the cell is still at the round stage and when the first neurite is forming. For this purpose we fabricated micron sized pillar structures with highly reproducible feature sizes, and analyzed neurons on the interface of flat and topographic surfaces. We found that topographic signaling was able to attract the polarization markers of mouse embryonic neurons -N-cadherin, Golgi-centrosome complex and the first bud were oriented towards topographic stimuli. Consecutively, the axon was also preferentially extending along the pillars. These events seemed to occur regardless of pillar dimensions in the range we examined. However, we found differences in neurite length that depended on pillar dimensions. This study is one of the first to describe in detail the very early response of hippocampal neurons to topographic stimuli.

Brain cells and neuronal networks: Encounters with controlled microenvironments

“…Moreover, when confronted with a differential surface topography, neurons specify an axon preferentially on nanopillars. Such effects were reported by Micholt et al [70] on micron sized silicon pillars (Fig. 4b and c), with in addition the demonstration that the first sprout formed from the soma just after plating faces the nanopillared surface.…”

“…By triggering the formation of actin patches between which neurites might build mechanical forces (Fig. 4c), this adhesive structure might foster a larger neurite growth [70]. Interestingly, this hypothesis brings together (i) the several lines of evidence suggesting that the fast elongating axon (relatively to the other neuronal processes) is the most tensed neurite [74,21] and (ii) the recent discovery of the existence of periodic actin rings along the axon of hippocampal neurons [75].…”

“…Another hypothesis relies on a possible channeling effect provided by topographies. Neurite directional choices were either clearly [70], or indirectly [69] evidenced on pillared surfaces, which might reduce the time required for growth cone decision-making and consequently might trigger a faster elongation rate. In support of this channeling effect hypothesis, a mechanism for accelerated growth based on the integration of signals emanating from non-aligned and aligned filopodia on a grooved surface was proposed [76].…”

Nanoscale Surface Topography Reshapes Neuronal Growth in Culture

“…This result was explained in terms of possible boosts of growth at pillar contacts 6 . In our study, it is interesting to note that, although directional choices are made by neurites in their positioning on the top of nano-pillars, the accelerated elongation that occurs in this somehow point-like, zero-dimensional adhesive configuration is observed in the absence any channeling effect such as the one provided by the one-dimensional topography of grooves or the presence of micro-pillars.…”

Neuronal Growth on a-Si and Au Nanopillars

“…1,2 Among several different topographies, micrometer sized pillars have been used for different purposes such as to promote neuronal axonal growth and orientation [3][4][5] and cellular differentiation. [5][6][7][8] Smaller diameter pillars, nanopillars, are applied for different purposes, for example, examination of neuronal development 9 and pinning neurons. [10][11][12] Nanopillars have also been used to access cellular membranes for electroporation, 13,14 which led them to the successful measurement of neuronal intracellular action potentials.…”