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. 2022 Oct 20;12(1):17513.
doi: 10.1038/s41598-022-21742-y.

Comparison of fractal and grid electrodes for studying the effects of spatial confinement on dissociated retinal neuronal and glial behavior

Saba Moslehi  1   2 , Conor Rowland  1   2 , Julian H Smith  1   2 , Willem Griffiths  3 , William J Watterson  1   2 , Cristopher M Niell  3   4 , Benjamín J Alemán  1   2   5   6 , Maria-Thereza Perez  7   8 , Richard P Taylor  9   10   11
Affiliations

Comparison of fractal and grid electrodes for studying the effects of spatial confinement on dissociated retinal neuronal and glial behavior

Saba Moslehi et al. Sci Rep. .

Abstract

Understanding the impact of the geometry and material composition of electrodes on the survival and behavior of retinal cells is of importance for both fundamental cell studies and neuromodulation applications. We investigate how dissociated retinal cells from C57BL/6J mice interact with electrodes made of vertically-aligned carbon nanotubes grown on silicon dioxide substrates. We compare electrodes with different degrees of spatial confinement, specifically fractal and grid electrodes featuring connected and disconnected gaps between the electrodes, respectively. For both electrodes, we find that neuron processes predominantly accumulate on the electrode rather than the gap surfaces and that this behavior is strongest for the grid electrodes. However, the 'closed' character of the grid electrode gaps inhibits glia from covering the gap surfaces. This lack of glial coverage for the grids is expected to have long-term detrimental effects on neuronal survival and electrical activity. In contrast, the interconnected gaps within the fractal electrodes promote glial coverage. We describe the differing cell responses to the two electrodes and hypothesize that there is an optimal geometry that maximizes the positive response of both neurons and glia when interacting with electrodes.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The lithography mask designs and scanning electron microscopy (SEM) images of the grid and fractal electrodes. (a,b) Binary masks of the grid and fractal electrodes respectively. (c,d) the zoom-in of the red boxes in (a) and (b) showing some of the geometric parameters of the two electrodes (see “Materials and methods” section and Supplementary Information for definitions). (e,f) SEM images of VACNTs in a region equivalent to the red boxes in (a) and (b) for the grid and fractal electrodes, respectively. The mask sizes in (a) and (b) are to scale (see Table 1 for relative sizes). Scale bars in (e) and (f) are 50 and 200 µm, respectively.
Figure 2
Figure 2
Representative examples of fluorescence images of retinal cells interacting with the grid and fractal electrodes at 17 DIV (green = GFAP labelled glia; red = β-tubulin III labelled neurons; blue = DAPI labeled nuclei). Glia on the VACNT surfaces of the (a) grid and (b) fractal electrodes. Glia accumulating on the SiO2 surfaces of the (c) grid and (d) fractal electrodes. The structure of glia on the VACNT surfaces of the (e) grid and (f) fractal electrodes as well as the SiO2 surfaces of the (g) grid and (h) fractal electrodes. Neuron processes following the VACNT electrodes of the (i) grid and (j) fractal electrodes. (k) Neuron clusters inside the grid chambers sending processes towards the VACNT sidewalls. (l) Large neuron clusters on the SiO2 surface connecting to the neurons on the VACNT surface of a fractal electrode. Neuron processes following the VACNT electrodes of the (m) grid and (n) fractal electrodes. (o) Neuron cluster attached to the VACNT sidewall of a grid chamber sending processes onto both the SiO2 and VACNT surfaces. (p) Neuron clusters and connecting processes on the SiO2 and VACNT surfaces of a fractal electrode. The images in (c) and (k) show the same FOV, as do (d) and (l). Electrode edges are highlighted with white lines except for panels (i), (j), (m), and (n) which concentrate on the behavior of processes along the edges because the lines would have obscured these processes. Scale bars are: 10 µm in (e) and (f); 20 µm in (g), (h), (m), (n), and (o); 40 µm in (p); 50 µm in (a), (b), (c), (i), (j), and (k); and 100 µm in (d) and (l).
Figure 3
Figure 3
Comparison of glial and neuronal behavior on the SiO2 and VACNT surfaces for the grid and fractal electrodes at 17 DIV. Statistical analysis showing boxplots of GSi (left) compared with GCNT (right) for the (a) grids and (c) fractals, as well as NSi (left) compared with NCNT (right) for the (b) grids and (d) fractals. The y axes of (a) and (c) display the range of GSi and GCNT values and the y axes of (b) and (d) display the range of NSi and NCNT values. Stars in panels (b–d) indicate the degrees of significance: *** and **denote p ≤ 0.001 and p ≤ 0.01, respectively. The red plus in panel (d) is an outlier. Note that GSi and GCNT are unitless and NSi and NCNT have units of µm−1 (see “Materials and methods” section).
Figure 4
Figure 4
Study of the relationship of GSi with GCNT and NCNT with NSi for the grid and fractal electrodes. (a) Scatterplot of GSi versus GCNT for the grids (red) and fractals (blue). (b) Scatterplot of NCNT versus NSi for the grids (red) and fractals (blue). The solid black lines represent the GSi = GCNT and NCNT = NSi conditions in (a) and (b), respectively. The solid red and blue lines are fits through zero for the grids and fractals, respectively.
Figure 5
Figure 5
Comparison between the grid and fractal electrodes at 17 DIV in terms of the glial and neuronal behavior on the SiO2 and VACNT surfaces. Statistical analysis showing boxplots of (a) GSi, (b) GCNT, (c) NSi, and (d) NCNT between the grid and fractal electrodes. Stars in all panels indicate the degrees of significance: ** and *denote p ≤ 0.01 and p ≤ 0.05, respectively. The red plus in panel (c) is an outlier.
Figure 6
Figure 6
Study of the relationship of GCNT with NCNT for the grid and fractal electrodes. Scatterplot of GCNT versus NCNT for the grids (red) and fractals (blue).
Figure 7
Figure 7
Study of the relationship of NCNT with GSi for the grid and fractal electrodes. Scatterplot of NCNT versus GSi for the grids (red) and fractals (blue). The solid red and blue lines are fits through zero for the grids and fractals, respectively.
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