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.

Guidance of neuronal extensions is a complex process essential for linking neurons into complex functional networks underlying the workings of the neural system. Decades of research have suggested the ability of neuronal growth cones to integrate multiple types of cues during the extension process, but also have raised numerous still unanswered questions about synergy or antagonism between the superimposed chemical and mechanical signaling inputs. In this study, using a novel microfabricated analysis platform, we investigate the response of primary mouse embryonic hippocampal neurons to superimposed topographic and soluble chemical cues. We find that an optimal spatial frequency of topographic cues exists, maximizing the precision of the neurite extension. This optimal frequency can help the extending neurites navigate a topographically complex environment, providing pronounced directional selectivity. We also demonstrate that this cue can synergistically enhance attractive and suppress repulsive guidance by the bi-functional soluble cue Netrin-1, and eliminate the repulsive guidance by a chemorepellent Semaphorin3A (Sema3A). These results suggest that topographic cues can provide optimal periodic input into the guidance signaling processes involved in growth cone chemoattraction and can synergistically interact with chemical gradients of soluble guidance cues, shedding light on complex events accompanying the development of the functional nervous system.

These results indicate that silicon micro-pillar substrates create a permissive environment for the growth of primary auditory neurons promoting neurite sprouting and are a promising technology for future high-density three-dimensional CMOS-based auditory neuro-electronic interfaces.