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Characterization of compound mechanisms in native neuronal DRG cultures applying a high capacity approach

Susanne Lardell, MS – External Collaborations Manager, Cellectricon AB

Paul Karila

Christina Nodin, PhD – External Collaborations Manager, Cellectricon AB


Background and Aims : In vitro cultures of dorsal root ganglion (DRG) neurons is a relevant model system for identifying compounds that putatively modify chronic pain conditions and to obtain a mechanistic understanding of drug action. Existing models often have low capacity, in terms of throughput, and are limited to a single readout.
The aim of this study was to investigate the possibility of using Cellectricon’s Discovery Platform (Sidders et al. 2018) and a multitude of stimuli and read-outs to answer more specific research questions, taking advantage of the platform’s combination of high capacity testing of compounds in a multi well-format with high resolution readouts. In addition, we demonstrate how different stimuli may be combined with multiple readouts to create in vitro assays with maximal utility depending on the scientific question under investigation.

Methods : DRGs from adult rats were dissected and cultured. The possibility of using ligand-induced stimuli was investigated using the TRPA1 agonist allyl isothiocyanate (AITC) as well as the TRPV1 agonist capsaicin. The ligands were added acutely in concentration-response format to the DRG cultures during simultaneous imaging on a high-throughput electrophysiology platform. The response was evaluated using a calcium sensitive probe as well as capsaicin-induced CGRP release. In addition, small molecule antagonists were evaluated for their ability to inhibit the response induced by the agonists to demonstrate the specificity of the response. Cultures were preincubated with the antagonists in concentration response-format followed by addition of an EC50-EC80 concentration of the respective agonist.
The above acute ligand application approach is suitable for e.g. ligand-gated ion channels but less ideal for e.g. investigation of compound effects on voltage-gated channels or more long-term neurotrophic actions of compounds. For those applications, electric field stimulation (EFS) was applied to demonstrate changes in excitability caused by sodium channel blockers and neurotrophic factors. To investigate if compound effects on excitability could be quantified also using morphological readouts, DRG cultures were incubated with paclitaxel, a drug known to induce morphological effects in vitro. The effect was evaluated using a calcium sensitive probe during EFS as well as using a morphological, high content imaging readout to assess changes on e.g. neuronal count and neurite density.

Results : A clear concentration-dependent effect was seen for both AITC and capsaicin. The response induced by capsaicin was successfully measured using two different readouts generating EC50 values within the same range. The agonist-induced responses could be reversed with preincubation of small molecule antagonists.
Paclitaxel decreased the EFS response after 24 h incubation, and in addition, the number of β-III tubulin positive cell bodies as well as the total neurite length was decreased in a concentration-dependent manner.

Conclusions : Using our Discovery Platform, we are able to characterize compound effects applying tailored stimulation paradigms in combination with a wide range of read-outs depending on the specific research question. With this approach, we were also able to couple changes in excitability to morphological changes. We hereby show that the platform, originally developed for high throughput compound screening using electric field stimulation, is also highly suitable for mechanistic studies where subtle modulatory effects need to be determined.

References : Sidders B, Karlsson A, Kitching L, Torella R, Karila P, Phelan A, J Mol Biol. 2018 Sep 14;430(18 Pt A):3005-3015. doi: 10.1016/j.jmb.2018.07.016. Epub 2018 Jul 18.

Conflicts of Interest : No conflict of interest

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