Name: external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures
Uploaded: 2017-05-07T14:00:00
Description: Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

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.

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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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 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.

<table>
	<tbody>
		<tr>
			<td>APV</td>
			<td>Sigma-Aldrich</td>
			<td>A8054</td>
			<td>Disconnect the network. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>B27 supp</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>bicuculline</td>
			<td>Sigma-Aldrich</td>
			<td>14343</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>Borax (sodium tetraborate decahydrate)</td>
			<td>Sigma-Aldrich</td>
			<td>S9640</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Frutarom LTD</td>
			<td>5550710</td>
			<td>Borate buffer. Mentioned in Section 1.1.2</td>
		</tr>
		<tr>
			<td>CaCl2 , 1M</td>
			<td>Fluka&#160;</td>
			<td>21098</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Sigma-Aldrich</td>
			<td>C239</td>
			<td>Disconnect the network&#160;. Mentioned in Section 2.4.2</td>
		</tr>
		<tr>
			<td>COMSOL</td>
			<td>COMSOL Inc</td>
			<td>Multiphysics 3.5</td>
			<td>Numerical simulation. Mentioned in Section 3.5.2</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution&#160;. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.5.2</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Cell Cultures. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>FCS(FBS)</td>
			<td>Gibco</td>
			<td>12657-029</td>
			<td>Plating medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>Fluo4, AM</td>
			<td>Life technologies</td>
			<td>F14201</td>
			<td>Imaging of spontaneous or evoked activity&#160;. Mentioned in Section 1.5.1&#160;&#160;&#160; 1.5.3&#160;&#160;&#160; 1.5.5</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>GlutaMAX 100X</td>
			<td>Gibco</td>
			<td>35050-038</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>HI HS&#160;</td>
			<td>BI</td>
			<td>04-124-1A</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.1.1&#160;&#160;&#160; 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>KCl,&#160; 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Laminin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Bio Coating. Mentioned in Section 1.2.6</td>
		</tr>
		<tr>
			<td>MEM x 1</td>
			<td>Gibco</td>
			<td>21090-022</td>
			<td>Plating medium, Changing medium, Final medium. Mentioned in Section 1.4.1&#160;&#160;&#160; 1.4.2</td>
		</tr>
		<tr>
			<td>MgCl2 , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Octadecanethiol</td>
			<td>Sigma-Aldrich</td>
			<td>01858</td>
			<td>Cleaning Cr-Au coated coverslips (1D cultures). Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>Pluracare F108 NF Prill</td>
			<td>BASF Corparation&#160;</td>
			<td>50475278</td>
			<td>Bio-Rejection Coating, Bio Coating. Mentioned in Section 1.2.4&#160;&#160;&#160; 1.2.6</td>
		</tr>
		<tr>
			<td>Poly-L-lysine 0.01% solution&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;P47075</td>
			<td>Promote cell division. Mentioned in Section 1.1.4</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution&#160;. Mentioned in Section 1.5.2</td>
		</tr>
		<tr>
			<td>Thiol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>1858</td>
			<td>Bio-Rejection Coating. Mentioned in Section 1.2.3</td>
		</tr>
		<tr>
			<td>URIDINE</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Changing medium. Mentioned in Section 1.4.1</td>
		</tr>
		<tr>
			<td>Sputtering machine</td>
			<td>AJA International, Inc</td>
			<td>ATC Orion-5Series&#160;</td>
			<td>coating glass with thin layers of metal. Mentioned in Section 1.2.2</td>
		</tr>
		<tr>
			<td>Pen plotter&#160;</td>
			<td>Hewlett Packard&#160;</td>
			<td>HP 7475A</td>
			<td>Etching of pattern to the coated coverslip. Mentioned in Section 1.2.5</td>
		</tr>
		<tr>
			<td>Electrodes wires&#160;</td>
			<td>A-M Systems, Carlsborg WA</td>
			<td>767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1&#160;&#160;&#160; 2.2&#160;&#160;&#160; 2.3&#160;&#160; 2.4.5</td>
		</tr>
		<tr>
			<td>Signal generator</td>
			<td>BKPrecision</td>
			<td>4079</td>
			<td>Shaping of the electric signal. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Homemade</td>
			<td></td>
			<td>Voltage amplification of the signal from the signal generator to the electrodes. Mentioned in Section 2.3</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix&#160;</td>
			<td>MPS-3005 LK-3&#160;</td>
			<td>Power supply to the sputtering machine. Mentioned in Section 1.2.2.3</td>
		</tr>
		<tr>
			<td>Transcranial magnetic stimulation</td>
			<td>Magstim, Spring Gardens, UK</td>
			<td>Rapid 2</td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.1&#160;&#160; 3.3&#160;&#160; 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Cognis</td>
			<td>Versamid 140</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Shell</td>
			<td>EPON 815&#160;</td>
			<td>Casting of homemade coils. Mentioned in Section 3.4</td>
		</tr>
		<tr>
			<td>Platinum wires 0.005&#39;&#39; thick; A-M Systems,&#160;</td>
			<td>&#160;Carlsborg WA</td>
			<td>&#160;767000</td>
			<td>Electric stimulation of neuronal cultures. Mentioned in Section 2.1</td>
		</tr>
		<tr>
			<td>Circular magnetic coil</td>
			<td>Homemade</td>
			<td></td>
			<td>Magnetic stimulation of neuronal culture. Mentioned in Section 3.3</td>
		</tr>
		<tr>
			<td>WaveXpress SW</td>
			<td>B&#38;K Precision&#160;</td>
			<td></td>
			<td>Waveform editing software. Mentioned in Section 2.1.32</td>
		</tr>
		<tr>
			<td>Xion Ultra 897</td>
			<td>Andor</td>
			<td></td>
			<td>Sensitive EMCCD camera. Mentioned in Section 2.4.4</td>
		</tr>
	</tbody>
</table>

external excitation of neurons using electric and magnetic fields in one- and two-dimensional cultures

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 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...

Watch this Scientific Journal Video about External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures at JoVE.com

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

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 ...

The overall goal of this protocol for the electric and magnetic stimulation of one-dimensional and two-dimensional cultures is to use neuronal cultures efficiently as a reduced model for brain stimulation from which we can extract insight and important parameters. Using our electric stimulation protocol with one dimensional cultures allows us to measure specific parameters related to neural excitation, for example, chronaxie and rheobase in different compartments of the neuron. The magnetic stimulation technique allows us to compare the effect of magnetic versus electric fields on neuronal cultures.This model also allows us to better diagnose and treat brain disorders. We propose an effective and easy method for culturing pattern neuronal networks in any design pattern even one-dimensional networks. We can then orient electric and time-varying magnetic fields with respect to these networks to separate and observe contributions from different compartments of the neuron.The main advantage of this technique is that it does not require human subjects or animal models enabling extensive and exhaustive search of the right parameters for neuronal stimulation. Demonstrating the procedure will be Yuri Burnishev, a technician from my laboratory and Ami Eisen, a PhD student in the laboratory. To begin, prepare patterned neuronal cultures by seeding neural cells on glass coverslips partially pre-coated with a biorejection layer.To do this, clean 25 millimeter coverslips. And using a Sputtering Machine, coat the coverslip with a biorejecting layer. Adjust the paramaters for the deposition process to create a layer consisting of a thin chrome film of six to 10 angstroms, that ensures coating stability and a 30 angstrom thick gold layer providing the coating with thiol reactivity.Once the process is completed, remove the coverslip from the Sputtering Machine and examine the obtained coating. Next, assemble a modified pen plotter system by replacing an ink tip of a plotter pen with a sharp metal etching needle. Place a rubber sheet glued to a stiff plate into the plotter so that a sturdy and high-friction surface is ensured during etching.Then, adjust the sheet's position by pressing the buttons provided on the plotter. Once the rubber sheet is in place, pipette a small drop of water onto its surface and place the pre-coated coverslip over the drop. Next, using the plotter, etch through the metal layers by repetitive scratching generating 200 micrometer-thick lines to create the desired patterns.Prior to an electric stimulation, place the seeded coverslip into an imaging chamber. To stimulate the cells with an electric field of a constant direction, use two parallel electrodes separated by 11 millimeters and located at opposite sides of the culture plated on the 13 millimeter coverslips. Then, assemble the culture imaging chamber and matching electrode holder such that the wire electrodes are immersed in the extracellular media and positioned one millimeter above the culture.Then, plug the single electrode pair to the operational amplifier that amplifies the signal from the generator. Apply a single pulse of bipolar square-wave with a 50%duty cycle and amplitude of 22 volts. Use a bipolar wave with no DC component to avoid electrolysis that may occur at the electrodes.Apply the pulse lasting from 10 microseconds to four milliseconds to ensure effective stimulation but prevent cell injury. To stimulate the cells with electric fields of a changing direction, use two perpendicular pairs of electrodes. Make sure that the two pairs of electrodes as well as their power and monitoring terminals are completely electrically isolated from each other and are floating from earth ground.To change the electric field orientation, vary the amplitude of the voltage applied to the two electrode pairs. Then, determine the intensity and the direction of the resulting field by vector addition. To generate a fixed amplitude rotating field, apply a sine wave to one electrode pair while feeding the other pair with a cosine wave.To begin magnetic stimulation, position a neuronal culture, grown in a circular-shaped pattern, under the microscope. Stimulate the culture using a standard circular magnetic stimulation coil and a supporting stimulator which are commercially available. To stimulate the cultures with non-standard magnetic field intensities, connect a custom-made magnetic stimulation coil to the custom-built stimulator.Then, with an aid of a laser pointer, align the center of the culture to the center of the coil to ensure successful stimulation. Adjust the coil positions so that it remains approximately five millimeters above the center of the culture. Once the stimulation coil is properly positioned, load the stimulator to a potential of up to five kilovolts.Then, using a high current, high voltage switch, discharge the high voltaging current through the stimulation coil to generate a magnetic pulse that stimulates the activity of the cultured neurons. Finally, mount a pickup coil at the same position as the neuronal culture and measure the intensity of the generated magnetic field. This schematic shows that by applying different amplitudes of voltage pulses to the two pairs of electrodes, the electric fields can be oriented to every angle without the need to manually turn the culture.Here are the example recordings of calcium transients with electric stimulation at different-applied voltages. When applying a constant duration of electric field, the number of neurons that fire and responds to the electric field shows a cumulative Gaussian distribution as a function of the amplitude of the voltage. And this is an example of a strength-duration curve obtained while employing this protocol.For the ring cultures responding to the magnetic field, the success rate of stimulation grows with the culture radius. This graph depicts the relation between the ring size and the magnetic field strength. Most successful excitations of the cultures lie in the overlap between the space of the experimentally accessible phase and the high probability region.Once mastered, the preparation of one-dimensional cultures can be performed within three to four hours. When performing electric and magnetic stimulation, it's important to remember to allow full recovery of the culture from the stimulation step and to make sure that all equipment is floating with respect to the electric ground. By following this procedure, other questions can be addressed such as culturing neurons from different areas of the brain, measuring statistics, for example, of cortical neurons.Another possibility is to measure parameters of any disease model neuron. After its development, this technique can pave the way for the optimization of the strength and durations needed for effective and safe brain stimulation. After watching this video, you should have a good understanding of how to create any desired pattern of neuronal cultures and to be able to stimulate them electrically or magnetically for the study of interactions of neurons with electromagnetic fields.

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SCIENTISTS IN ACTION

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

The Use of Magnetic Resonance Spectroscopy as a Tool for the Measurement of Bi-hemispheric Transcranial Electric Stimulation Effects on Primary Motor Cortex Metabolism

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of neurological and psychiatric disorders such as stroke and depression. Yet, the mechanisms underlying its ability to modulate brain excitability to improve clinical symptoms remains poorly understood 33. To help improve this understanding, proton magnetic resonance spectroscopy (1H-MRS) can be used as it allows the in vivo quantification of brain metabolites such as &#947;-aminobutyric acid (GABA) and glutamate in a region-specific manner 41. In fact, a recent study demonstrated that 1H-MRS is indeed a powerful means to better understand the effects of tDCS on neurotransmitter concentration 34. This article aims to describe the complete protocol for combining tDCS (NeuroConn MR compatible stimulator) with 1H-MRS at 3 T using a MEGA-PRESS sequence. We will describe the impact of a protocol that has shown great promise for the treatment of motor dysfunctions after stroke, which consists of bilateral stimulation of primary motor cortices 27,30,31. Methodological factors to consider and possible modifications to the protocol are also discussed.

This article aims to describe a basic protocol for combining transcranial direct current stimulation (tDCS) with proton magnetic resonance spectroscopy (1H-MRS) measurements to investigate the effects of bilateral stimulation on primary motor cortex metabolism.

Transcranial direct current stimulation (tDCS) is a neuromodulation technique that has been increasingly used over the past decade in the treatment of ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...