Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models
-
1
Natural and Medical Sciences Institute, Germany
-
2
Natural and Medical Sciences Institute, Germany
-
3
NMI Technologie Transfer GmbH, Germany
-
4
Hochschule Albstadt-Sigmaringen, Life Sciences Faculty, Germany
Motivation
Chronic Neuropathic Pain (NP) is frequently associated with peripheral nerve injury or disease. It affects 6-8% of the population, and current treatments are inadequate, since they have limited efficacy.
Interest in understanding the mechanisms that underpin neuropathic pain has been growing in the last years, but the exact mechanisms haven’t been clarified yet. Experimental models of neuropathic pain showed that non-neuronal cells, in particular Schwann cells and satellite glia cells (SGCs), play a very active role in the development of sensory abnormalities. What is less clear is exactly how these non-neuronal cells induce the hyper-excitable state in pain signaling neurons associated with neuropathic pain condition.
Materials and methods
Mice between p13 and p20 were euthanized with CO2, and the vertebral column was removed. Each column was cut along its main axis, and for each half dorsal root ganglia (DRG) were removed. DRG were dissociated enzymatically, and dead cells and debris were removed by gradient centrifugation. Pure sensory neurons were obtained using the Neuron Isolation Kit (Miltenyi Biotec). Co-cultures of neurons-glia and cultures of pure sensory neurons were plated on micro electrode arrays (MEA) and glass coverslips coated with polyethyleneimine (PEI) 0.075 g/ml, and laminin 20 µg/ml, supplied with modified Neurobasal-A medium and kept at 37°C with 5% CO2. After 2-4 days in vitro, the electrical activity of each MEA was recorded using MEA2100 system (Multichannel System), and calcium imaging was performed.
Results
By using a high-density MEA system, we were able to record the electrical activity of both pure sensory neurons and co-cultures of neurons-glia derived from murine dorsal root ganglia (DRG). To analyse the differential excitability of co-cultures of neurons-glia and pure neurons, capsaicin was applied to every cell culture. Co-cultures showed an increase in electrical excitability in response to capsaicin already after 1 day in vitro. Cultures of pure sensory neurons showed electrical activity in response to capsaicin after 1 day in vitro, although lower if compared to that of the co-cultures. Surprisingly, this activity was completely lost after 3 days in vitro. To further investigate this effect, calcium imaging experiments were performed on these cultures. Application of capsaicin could evoke in both cases an increase in intracellular calcium concentration and no significant difference was noticed between purified and non-purified cultures. This demonstrates that purified sensory neurons do actually express the TRPV1 receptor although they are somehow unable to generate action potentials in response to capsaicin application.
Discussion and conclusion
It has been demonstrated that sensory neurons possess electrical activity in response to different excitatory stimuli already after 1 day in vitro; however, in the case of purified neurons, this activity disappears after 2 days in vitro. This cannot be explained by a lack in the capsaicin receptor TRPV1, whose presence has been proved by calcium-imaging experiment. The ability of purified sensory neurons to maintain their electrical activity shortly after being plated suggests the presence of a “memory effect”, that allows them to behave as in vivo, effect that disappears as the culture timeline increases, as noticed from the MEA recording data. All these data combined lead to the hypothesis that non-neuronal cells, such as Schwann cells and satellite glial cells, play an active role in the activity of sensory neurons, and their absence lead to a loss of functionality in these neurons. Future studies, which include perforated patch-clamp experiments, will be required to understand the loss of electrical activity in purified sensory neurons, identify proteins that are dysregulated in purified sensory neurons, and their relationship with non-neuronal cells.
Figure legends:
Figure1: Co-culture of sensory neurons and glia on MEA.
Figure 2: Culture of purified sensory neurons on MEA.
Acknowledgements
This project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No 116072. This Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA.
Keywords:
neuropathic pain,
dorsal root ganglia,
sensory neurons,
Neuroglia,
MEA
Conference:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018.
Presentation Type:
Poster Presentation
Topic:
Microphysiological systems
Citation:
Izzi
F,
Loser
D and
Cesare
P
(2019). Micro electrode arrays to investigate neuron-glia crosstalk in neuropathic pain in-vitro models.
Conference Abstract:
MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays.
doi: 10.3389/conf.fncel.2018.38.00029
Copyright:
The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers.
They are made available through the Frontiers publishing platform as a service to conference organizers and presenters.
The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated.
Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed.
For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions.
Received:
27 Mar 2018;
Published Online:
17 Jan 2019.
*
Correspondence:
Mrs. Francesca Izzi, Natural and Medical Sciences Institute, Reutlingen, 72770, Germany, Francesca.Izzi@nmi.de