Possible model for interaction of young neurons with topography for polarization and axon formation.

<p>(<b>A</b>) When a neuron is located on the interface between a pillar array and a flat area (step 1) the N-cadherin patch is accumulated at the topographical contact, also F-actin is located at pillar contact via an unknown mechanism (step 2). After first sprout formation (step 3), the Golgi and centrosome are also recruited to the interface (step 4), from which at a later time point the axon starts to form (step 5). (<b>B</b>) Pillar contact gives rise to growth signaling, if this is repeated frequently enough during outgrowth of the neurite this leads to an overall growth encouragement. On the other hand if the pillars are spaced further apart, this neurite will not be able to exploit its full growth potential.</p

Substrate Topography Determines Neuronal Polarization and Growth <i>In Vitro</i>

<div><p>The establishment of neuronal connectivity depends on the correct initial polarization of the young neurons. <i>In vivo</i>, developing neurons sense a multitude of inputs and a great number of molecules are described that affect their outgrowth. <i>In vitro,</i> 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.</p></div

Effect of pillar contact on N-cadherin distribution.

<p>(<b>A</b>) Example of a neuron with the soma covering at least one pillar at 1 hour in culture, immunostained for N-cadherin (GC-4). Its N-cadherin crescent is oriented towards the pillar contacts. For 0.6–1 n = 112, 1.2–2 n = 115, 2.4–7 n = 67. (<b>B</b>) Example of a neuron with the soma touching the pillars 1 hour in culture. The N-cadherin crescent is in the process of being recruited towards the pillar contact.*, significantly different from random (dashed line, 50% for random positioning) using X²-test (p<0.05), ^# indicates p<0.1.For 0.6–1 n = 68, 1.2–2 n = 58, 2.4–7 n = 109. Green: CMFDA Cell tracker, Blue: Hoechst. Scale bars are 5 µm.</p

Substrate Lay-out.

<p>(<b>A</b>) The substrate consisted of individual areas decorated with pillars of different dimensions. The pillar width ranged from 1–5.6 µm (1, 1.2, 1.4, 1.6, 1.8, 2, 2.4, 2.8, 4, 5.6 µm) in the vertical direction, while the spacing ranged from 0.6–15 µm (0.6, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.4, 3.2, 4, 5, 7, 10, 15 µm) and the height was kept constant at 3 µm. (<b>B</b>) Scanning electron microscopy images of different width and spacing of pillars. Left is W = 1 µm, S = 1 µm, middle is W = 1.6 µm, S = 1.6 µm, right is W = 5.6 µm, S = 10 µm. Scale bars are 10 µm.</p

Parameters for the construction of Fig. 7B.

<p>Per spacing the values for the measured mean distance of the patches to the pillar center are given, together with higher and lower border of the simulated confidence intervals. The last column shows values for the density of patches on the neurites.</p

Axon position for neurons sensing topography.

<p>(<b>A</b>) Examples of axon position for neuronal soma touching the pillars (left) and covering at leat one pillar (right) (green: tau-1 axon specific staining, red: MAP-2 dendrite specific staining, blue: Hoechst). (<b>B</b>) Axon position analysis for both touching and covering conditions. (<b>C</b>) Golgi position analysis for both touching and covering conditions (dashed line, 25% for random positioning).*, significantly different from random. For ‘soma on interface’, 0.6–1 n = 16, 1.2–2 n = 32, 2.4–7 n = 23. For ‘soma touching’, 0.6–1 n = 16, 1.2–2 n = 41, 2.4–7 n = 28.</p

Growth cone morphology on different substrates.

<p>(<b>A</b>) Examples of time lapses of growth cones on pillar beds with different parameters. The growth cone morphology is stable over longer time (here: 800 s), confirmed by the outlined overlay images. The locations of the pillars are outlined. Scale bar is 5 µm. (<b>B</b>) Average growth cone area on different pillar spacings (Student t-test, p<0.05).</p

Analysis of first bud and Golgi-centrosome complex position.

<p>(<b>A</b>) Time lapse of neurons expressing the F-actin probe GFP-UtrCH, located on the edge of a pillar bed. Scale bar is 10 µm. (<b>B</b>) Analysis of the first sprout positioned towards (ON) or away (OFF) from the pillars. Dashed line indicates the 50% level. Examples of cells with first sprout ON are shown at the right (<b>C</b>) Analysis of the Golgi positioned towards (ON) or away (OFF) from the pillars. Examples of cells with Golgi ON are shown at the right. Scale bars are 10 µm. *, significantly different from random using X² test (p<0.05). Blue, Nuclei (Hoechst), Green, Golgi apparatus (GPP130), red, microtubules (tuj-1), grey, substrate. For (B), 0.6–1 n = 31, 1.2–2 n = 37, 2.4–7 n = 18. For (C), 0.6–1 n = 53, 1.2–2 n = 53, 2.4–7 n = 43.</p

Signaling events at pillar contacts.

<p><b>(</b>A) Example of a hippocampal neuron grown on a pillar substrate (W = 1.6 µm, S = 1.2 µm) stained for phosphotyrosine (PY, green) and tuj-1 (red). Scale bar is 5 µm. (<b>B</b>) Comparison of the mean patch distance from the pillar center (red marks) to the confidence interval for the simulated patches (gray shaded area). For a constant W = 1.6 µm, the PY patches are outside of the confidence interval for S = 1.2 µm and S = 2 µm. This is indicated by a *. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066170#pone-0066170-t001" target="_blank">Table 1</a> for detailed parameter values. Individual cases are tabulated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066170#pone.0066170.s004" target="_blank">Table S1</a>. The y-axis values were drawn as shown in a representative example in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066170#pone.0066170.s003" target="_blank">Fig. S3B</a>. (<b>C</b>) Co-localization of actin filaments and phosphorylated tyrosine positions. Cyan β_III-tubulin, green actin, red phosphotyrosine. (<b>D</b>) Paxillin-GFP shows the same expression pattern than phosphotyrosine. Cyan β_III-tubulin, green Pax-GFP, red PY. Scale bars are 5 µm.</p

Analysis of neurite length on different pillar parameters.

<p>(<b>A</b>) Axon and average neurite length at different time points after plating. The black line shows the growth evolution on a flat substrate, red shows W = 4 µm, S = 2 µm, blue shows W = 1.8 µm, S = 1.6 µm. The process lengths for W = 1.8 µm, S = 1.6 µm, and W = 4 µm, S = 2 µm are significantly (p<0.05) higher than on flat after 4, 8, 12 and 20 h. (<b>B</b>) Examples of neuronal morphology on flat and on the previously summed pillar parameters. Scale bars are 10 µm. (<b>C</b>) Surface plot of axon length in function of width and spacing after 20 h. Maximal axon length at 20 h is achieved by pillars of W = 1–2 µm and S = 1–2 µm. (<b>D</b>) Time lapse imaging (GFP-UtrCH) of a minor neurite extending its processes. See Movie S1 for full time lapse. W = 2.8 µm, S = 1.6 µm. Scale bar is 10 µm.</p