Optimization of a 96-Well Electroporation Assay for Postnatal Rat CNS Neurons Suitable for Cost-Effective Medium-Throughput Screening of Genes that Promote Neurite Outgrowth

Abstract

Following an injury, central nervous system (CNS) neurons show a very limited regenerative response which results in their failure to successfully form functional connections with their original target. This is due in part to the reduced intrinsic growth state of CNS neurons, which is characterized by their failure to express key regeneration-associated genes (RAGs) and by the presence of growth inhibitory molecules in CNS environment that form a molecular and physical barrier to regeneration. Here we have optimized a 96-well electroporation and neurite outgrowth assay for postnatal rat cerebellar granule neurons (CGNs) cultured upon an inhibitory cellular substrate expressing myelin-associated glycoprotein or a mixture of growth inhibitory chondroitin sulfate proteoglycans. Optimal electroporation parameters resulted in 28% transfection efficiency and 51% viability for postnatal rat CGNs. The neurite outgrowth of transduced neurons was quantitatively measured using a semi-automated image capture and analysis system. The neurite outgrowth was significantly reduced by the inhibitory substrates which we demonstrated could be partially reversed using a Rho Kinase inhibitor. We are now using this assay to screen large sets of RAGs for their ability to increase neurite outgrowth on a variety of growth inhibitory and permissive substrates.

Keywords: 96-well; CNS neuron; RAG; electroporation; inhibitory substrate; neurite outgrowth; neuronal regeneration; transfection.

Figure 1

A typical field of view showing electroporated CGNs cultured on a monolayer of growth-permissive CHO-R2 cells. (A) A typical field of view taken using the IN Cell Analyzer 1000 semi-automated cell imager with a 10× Nikon ApoPlan objective. (B) DAPI stained nuclei of CHO-R2 cells and CGNs. (C) CGNs stained for beta-III-tubulin. (D) eGFP positive, transfected CGNs expressing GFP. (E) The merged image; transfected neurons appear yellow. Scale bars: 100 μm.

Figure 2

Electroporation optimization of postnatal rat CGNs. (A) Voltage optimization. The mean number of CGNs was significantly decreased at all voltages compared to the non-electroporated control. The mean number of transfected CGNs significantly increased at voltages higher than 200 V compared to the non-electroporated control. (B) Pulse length optimization. The mean number of CGNs was significantly decreased at pulse lengths higher than 0.4 ms. The mean number of transfected CGNs significantly increased using pulse lengths between 0.4 and 1 ms. (C) Pulse number optimization. The mean number of CGNs was significantly decreased at all pulse numbers. The mean number of transfected CGNs was significantly increased using 1 pulse. Values represent mean and SD; analysis was performed using one way ANOVA with Dunnett’s post hoc tests comparing with the non-electroporated control (*P < 0.05, ***P < 0.001), n = 8.

Figure 3

Chinese hamster ovary-MAG cells inhibit CGN neurite outgrowth, which can be partially reversed by the ROCK inhibitor Y-27632. (A) Quantification of the mean neurite length per transfected neuron of CGNs cultured on CHO-MAG or CHO-R2 cells. The mean neurite length per transfected CGN was significantly longer when cultured on CHO-R2 cells compared to CHO-MAG cells. The CHO-MAG inhibition could be partially reversed with 20 μM Y-27632. The mean neurite length per transfected CGN was significantly longer when cultured on CHO-MAG cells in the presence of Y-27632 compared to CGNs cultured on CHO-MAG cells without Y-37632. Values represent mean and SD; analysis was performed using one way ANOVA with Dunnett’s post hoc tests comparing to CHO-MAG cells (***P < 0.001), n = 8. (B) Non-inhibitory CHO-R2 cell substrate. (C) CGN neurite outgrowth is inhibited by the CHO-MAG cell substrate. (D) The ROCK inhibitor Y-27632 reverses the MAG inhibition and promotes neurite outgrowth. Scale bar: 100 μm.

Figure 4

CSPG inhibits postnatal CGN neurite outgrowth, which can be partially reversed by the ROCK inhibitor Y-27632. (A) Quantification of the mean neurite length per transfected neuron of CGNs cultured on Laminin and CSPG substrates. CGNs cultured on laminin had significantly longer neurites than CGNs cultured on a CSPG substrate. The CSPG inhibition could be partially reversed using 20 μM Y-27632. The mean neurite length per transfected CGN was significantly longer when cultured on the CSPG substrate in the presence of Y-27632 compared to CGNs cultured on the CSPG substrate without Y-37632. Values represent mean and SD; analysis was performed using one way ANOVA with Dunnett’s post hoc tests comparing to CSPG substrate (***P < 0.001, **P < 0.01), n = 8. (B) Non-inhibitory laminin substrate. (C) Neurite outgrowth is inhibited by the CSPG substrate. (D) The ROCK inhibitor Y-27632 can promote CGN neurite outgrowth and overcome the CSPG inhibition. Scale bar: 100 μm.

Figure 5

Eighty four percent co-expression of mCherry and GFP was obtained using a 4:1 DNA ratio. (A) Quantification of the percentage of cells co-expressing GFP and mCherry. The percentage of cells co-expressing both fluorescent proteins increased with the ratio of mCherry to GFP, peaking at a ratio of 4:1 where 84% of the GFP expressing cells also expressed mCherry. Values represent mean and SD, n = 8. (B) CGNs co-transfected with mCherry and GFP using a ratio of 4:1. CGNs co-expressing GFP (green) and mCherry (red) appear yellow. Scale bar: 100 μm.

Figure 6

Regeneration-associated genes are successfully over-expressed following electroporation, and over-expression of KLF-7 but not ATF-3 was able to enhance neurite outgrowth on an inhibitory MAG substrate. (A) Quantification of the relative level of ATF-3 mRNA in electroporated CGNs. Significantly higher levels (2700-fold) of ATF-3 were detected in CGNs transfected with the ATF-3 plasmid compared to CGNs transfected with the mCherry plasmid. Values represent mean and SD; analysis was performed using an independent samples Student’s t-test (**P < 0.01), n = 4. (B) Quantification of the mean neurite length per transfected neuron of CGNs cultured on CHO-MAG cells. The CHO-MAG inhibition could be partially reversed by over-expressing KLF-7 or addition of 20 μM Y-27632 but not by over-expressing ATF-3. Values represent mean and SD; analysis was performed using one way ANOVA with Dunnett’s post hoc tests comparing to CHO-MAG cells (***P < 0.001), n = 8.