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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Neuroscience

External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures

Published: May 7th, 2017

DOI:

10.3791/54357

1Laboratory of Genetics, The Salk Institute for Biological Studies, 2Department of Physics and SEAS, Harvard University, 3Department of Physics of Complex Systems, Weizmann Institute of Science

Neuronal cultures are a good model for studying emerging brain stimulation techniques via their effect on single neurons or a population of neurons. Presented here are different methods for stimulation of patterned neuronal cultures by an electric field produced directly by bath electrodes or induced by a time-varying magnetic field.

A neuron will fire an action potential when its membrane potential exceeds a certain threshold. In typical activity of the brain, this occurs as a result of chemical inputs to its synapses. However, neurons can also be excited by an imposed electric field. In particular, recent clinical applications activate neurons by creating an electric field externally. It is therefore of interest to investigate how the neuron responds to the external field and what causes the action potential. Fortunately, precise and controlled application of an external electric field is possible for embryonic neuronal cells that are excised, dissociated and grown in cultures. This allows the investigation of these questions in a highly reproducible system.

In this paper some of the techniques used for controlled application of external electric field on neuronal cultures are reviewed. The networks can be either one dimensional, i.e. patterned in linear forms or allowed to grow on the whole plane of the substrate, and thus two dimensional. Furthermore, the excitation can be created by the direct application of electric field via electrodes immersed in the fluid (bath electrodes) or by inducing the electric field using the remote creation of magnetic pulses.

The interaction between neurons and external electric fields has fundamental implications as well as practical ones. While it is known since the times of Volta that an externally applied electric field can excite tissue, the mechanisms responsible for the production of a resultant action potential in neurons are only recently starting to be unraveled 1,2,3,4. This includes finding answers to questions regarding the mechanism that causes depolarization of membrane potential, the role of membrane properties and of ion channels, and even the re....

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Ethics Statement: Procedures involving animal handling were done in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Weizmann Institute of Science, and the appropriate Israeli law. The Weizmann Institute is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). The Weizmann Institutional Animal Care and Use Committee approved this study, conducted with hippocampal neurons.

1. Preparation of .......

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The protocol presented allows for easy patterning of neuronal cultures. When it is combined with several methods we developed for stimulation, it enables to make measurements of some intrinsic neuron properties such as Chronaxie and Rheobase5, to compare properties of healthy and diseased neurons27, to find optimal ways to stimulate cultures as a function of their structure and many more novel approaches. Some examples are presented in the n.......

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1D patterning is an important tool that can be used for a variety of applications. For example, we have used 1D patterning for creating logic gates from neuronal cultures 29 and more recently to measure the Chronaxie and Rheobase of rat hippocampal neurons 5, and the slowing down of signal propagation velocity of firing activity in Down syndrome hippocampal neurons compared to the wild type (WT) hippocampal neurons 27. The suggested protocol for 1D p.......

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The authors thank Ofer Feinerman, Fred Wolf, Menahem Segal, Andreas Neef and Eitan Reuveny for very helpful discussions. The authors thank Ilan Breskin and Jordi Soriano for developing early versions of the technology. The authors thank Tsvi Tlusty and Jean-Pierre Eckmann for help with the theoretical concepts. This research was supported by the Minerva Foundation, the Ministry of Science and Technology, Israel, and by Israel Science Foundation grant 1320/09 and the Bi-National Science Foundation grant 2008331.

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Name Company Catalog Number Comments
APV Sigma-Aldrich A8054 Disconnect the network. Mentioned in Section 2.4.2
B27 supp Gibco 17504-044 Plating medium. Mentioned in Section 1.1.1
bicuculline Sigma-Aldrich 14343 Disconnect the network . Mentioned in Section 2.4.2
Borax (sodium tetraborate decahydrate) Sigma-Aldrich S9640 Borate buffer. Mentioned in Section 1.1.2
Boric acid Frutarom LTD 5550710 Borate buffer. Mentioned in Section 1.1.2
CaCl2 , 1M Fluka  21098 Extracellular recording solution . Mentioned in Section 1.5.2
CNQX Sigma-Aldrich C239 Disconnect the network . Mentioned in Section 2.4.2
COMSOL COMSOL Inc Multiphysics 3.5 Numerical simulation. Mentioned in Section 3.5.2
D-(+)-Glucose, 1M Sigma-Aldrich 65146 Plating medium, Extracellular recording solution . Mentioned in Section 1.1.1    1.5.2
D-PBS Sigma-Aldrich D8537 Cell Cultures. Mentioned in Section 1.2.4    1.2.6
FCS(FBS) Gibco 12657-029 Plating medium. Mentioned in Section 1.1.1
Fibronectin Sigma-Aldrich F1141 Bio Coating. Mentioned in Section 1.2.6
Fluo4, AM Life technologies F14201 Imaging of spontaneous or evoked activity . Mentioned in Section 1.5.1    1.5.3    1.5.5
FUDR Sigma-Aldrich F0503 Changing medium. Mentioned in Section 1.4.1
Gentamycin Sigma-Aldrich G1272 Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1
GlutaMAX 100X Gibco 35050-038 Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1
Hepes, 1M Sigma-Aldrich H0887 Extracellular recording solution . Mentioned in Section 1.5.2
HI HS  BI 04-124-1A Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1    1.4.1    1.4.2
KCl,  3M Merck 1049361000 Extracellular recording solution. Mentioned in Section 1.5.2
Laminin  Sigma-Aldrich L2020 Bio Coating. Mentioned in Section 1.2.6
MEM x 1 Gibco 21090-022 Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1    1.4.2
MgCl2 , 1M Sigma-Aldrich M1028 Extracellular recording solution. Mentioned in Section 1.5.2
NaCl, 4M Bio-Lab 19030591 Extracellular recording solution . Mentioned in Section 1.5.2
Octadecanethiol Sigma-Aldrich 01858 Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3
Pluracare F108 NF Prill BASF Corparation  50475278 Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4    1.2.6
Poly-L-lysine 0.01% solution  Sigma-Aldrich  P47075 Promote cell division. Mentioned in Section 1.1.4
Sucrose, 1M Sigma-Aldrich S1888 Extracellular recording solution . Mentioned in Section 1.5.2
Thiol  Sigma-Aldrich 1858 Bio-Rejection Coating. Mentioned in Section 1.2.3
URIDINE Sigma-Aldrich U3750 Changing medium. Mentioned in Section 1.4.1
Sputtering machine AJA International, Inc ATC Orion-5Series  coating glass with thin layers of metal. Mentioned in Section 1.2.2
Pen plotter  Hewlett Packard  HP 7475A Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5
Electrodes wires  A-M Systems, Carlsborg WA 767000 Electric stimulation of neuronal cultures. Mentioned in Section 2.1    2.2    2.3   2.4.5
Signal generator BKPrecision 4079 Shaping of the electric signal. Mentioned in Section 2.3
Amplifier Homemade Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3
Power supply Matrix  MPS-3005 LK-3  Power supply to the sputtering machine. Mentioned in Section 1.2.2.3
Transcranial magnetic stimulation Magstim, Spring Gardens, UK Rapid 2 Magnetic stimulation of neuronal culture. Mentioned in Section 3.1   3.3   3.4
Epoxy Cognis Versamid 140 Casting of homemade coils. Mentioned in Section 3.4
Epoxy Shell EPON 815  Casting of homemade coils. Mentioned in Section 3.4
Platinum wires 0.005'' thick; A-M Systems,   Carlsborg WA  767000 Electric stimulation of neuronal cultures. Mentioned in Section 2.1
Circular magnetic coil Homemade Magnetic stimulation of neuronal culture. Mentioned in Section 3.3
WaveXpress SW B&K Precision  Waveform editing software. Mentioned in Section 2.1.32
Xion Ultra 897 Andor Sensitive EMCCD camera. Mentioned in Section 2.4.4

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