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Method Article
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 region in the neuron that responds to the electric field 2,5. Therapeutic neurostimulation 6,7,8,9,10 methodologies are particularly dependent on this information, which can be crucial for targeting the afflicted areas and for understanding the outcome of the therapy. Such understanding can also help in developing treatment protocols and new approaches for stimulation of different areas in the brain.
Measuring the interaction within the in vivo brain adds an important component to this understanding, but is hampered by the imprecision and low controllability of measurements within the skull. In contrast, measurements in cultures can easily be performed in high volume with high precision, excellent signal to noise performance and a high degree of reproducibility and of control. Using cultures a large variety of neuronal properties of collective network behavior can be elucidated 11,12,13,14,15,16. Similarly, this well controlled system is expected be highly efficient in elucidating the mechanism by which other stimulation methods work, for example how channel opening during optical stimulation in optogenetically active neurons 17,18,19 is responsible for creating action potential.
Here the focus is on describing the development and understanding of tools that can efficiently excite the neuron via an external electric field. In this paper we describe the preparation of two-dimensional and one-dimensional patterned hippocampal cultures, stimulation using different configurations and orientation of a directly applied electric field by bath electrodes, and finally stimulation of two-dimensional and patterned one-dimensional cultures by a time-varying magnetic field, which induces an electric field 5,20,21.
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 Two-dimensional (2D) and One-dimensional (1D) Hippocampal Cultures
2. Electric Stimulation of Cultures
NOTE: The basic setup for electric stimulation is shown in Figure 1. A cover slip on which the neuronal culture has been grown for about 14 days is placed in a Petri dish under a fluorescence microscope. Electrical activity of the neurons is imaged using calcium sensitive dyes while a voltage is applied via two pairs of bath electrodes that are positioned outside the culture. The electrodes are driven by a signal generator whose output is amplified by a dual channel amplifier. Voltage control for stimulation is preferred over the more standard current control25,26 because the electric field vectors are determined directly, thus enabling straightforward vector addition and combination. This does require a careful check of the uniformity of the electric field, which can be performed over the whole sample for the case of voltage control. When using voltage control care should be taken to avoid any ground loops and the homogeneity of the electric field should be verified (see 2.2 below).
3. Magnetic Stimulation of Cultures
Note: The basic setup for magnetic stimulation is shown in Figure 2. On top right is shown an inverted fluorescence microscope that is used to image calcium sensitive dyes in the neurons. The magnetic coil (blue circles) is positioned about 5 mm concentrically above a neuronal ring culture, (blue outline). A pickup coil (red circle) on the circumference of the Petri dish monitors the voltage induced by the magnetic pulse. On the top left is shown the measured dynamics of the magnetic stimulator (MS) coil with a capacitor voltage load of 5,000 kV, as integrated from the pickup coil. The induced electric field (calculated for a ring radius of 14 mm) is depicted in green while the magnetic field is depicted in blue. On the bottom are shown images of the neuronal culture. At bottom left is a bright field image of a patterned 24-mm coverslip. The white areas are the neurons. The photographed pattern consists of concentric ring cultures with different radii. At the bottom right is a zoom onto a short segment of the rings, showing individual neurons. For a scale, the rings' width is about 200 µm.
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...
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...
The authors declare that they have no competing financial interests.
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.
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 , 1 M | 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, 1 M | Sigma-Aldrich | 65146 | Plating medium, Extracellular recording solution. Mentioned in Sections 1.1.1 and 1.5.2 |
D-PBS | Sigma-Aldrich | D8537 | Cell Cultures. Mentioned in Sections 1.2.4 and 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 |
Fluo4AM | Life technologies | F14201 | Imaging of spontaneous or evoked activity. Mentioned in Sections 1.5.1, 1.5.3, and 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, 1 M | 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 Sections 1.1.1, 1.4.1, and 1.4.2 |
KCl, 3 M | 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 , 1 M | Sigma-Aldrich | M1028 | Extracellular recording solution. Mentioned in Section 1.5.2 |
NaCl, 4 M | 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 Sections 1.2.4 and 1.2.6 |
Poly-L-lysine 0.01% solution | Sigma-Aldrich | P47075 | Promote cell division. Mentioned in Section 1.1.4 |
Sucrose, 1 M | 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 Sections 2.1, 2.2, 2.3, and 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 Sections 3.1, 3.3, and 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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