Name: Vídeo: Estimulação de culturas neuronais padronizadas por um campo magnético - Conceito
Uploaded: 2024-10-31T13:46:39.000+00:00
Duration: 1 min 19 s
Description: 40 visualizações. Fonte: Stern, S., et al., Excitação externa de neurônios usando campos elétricos e magnéticos em culturas unidimensionais e bidimensionais. J. Vis. Exp. (2017) Este vídeo demonstra um método para estimular culturas neuronais cultivadas em um anel circular padronizado em uma lamínula, usando um campo magnético variável no tempo.

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1. Preparation of coverslips for 1D cultures.
<ol>
	<li>Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia solution, and 25 mL 30% hydrogen peroxide. Place the solution with the glass coverslips on a heating plate at ~ 50&#176;C for 30 min, and then dry the glass coverslips with nitrogen.</li>
	<li>First, coverslips are coated with a thin chrome film (99.999%) of 6 &#197; thickness, followed by a 30 &#197; layer of gold (99.999%), using either vapor or sputter deposition.
	<ol>
		<li>To achieve a sputtering rate of 0.05 &#8211; 1 &#197; /s, use a sputtering machine with 2 e-beams and a vacuum system of 260 l/s with target sizes 2&#34; and 4&#34;. Use a rotation stage that can go from 0 to 100 rounds per minute (RPM). Use a direct current (DC) sputter power of 0 to 750 Watts.</li>
		<li>To get plasma at 10 mTorr pressure in the chamber, rotate the device at 30 rpm and use argon at 99.999%.</li>
		<li>Operate the power supply of the sputtering guns at 40 W DC sputter power for the chrome, leading to ~ 0.12 &#197;/s coverage rate, and at 10 W DC sputter power for the gold, leading to ~ 0.28 &#197;/s.</li>
	</ol>
	</li>
	<li>Using ultrasound for 30 min, dissolve 0.1 g 1-octadecanethiol in 100 mL ABS AR ethanol. Place Cr-Au coated coverslips in this solution for 2 h, then wash with ethanol ABS AR and dry with nitrogen.</li>
	<li>Prepare a solution of 100 mL Dulbecco&#39;s phosphate buffered saline (D-PBS) and 3.5 g of a tri-block co-polymer (see Table of Materials/Reagents) by stirring for 1 - 2 h at 600 - 700 rpm. Place coverslips in the solution for 1 h. Dry coverslips with nitrogen.</li>
	<li>Mechanically etch the desired pattern by scratching the bio-rejection layer. Do this using a pen plotter, where the pen is replaced by an etching needle. Scratch the pattern through the metal layers to reach the underlying glass. Control this process by a computer to achieve a replicated desired pattern. Patterns formed by this process are demonstrated in Figure 1 and Figure 2.</li>
	<li>Prepare a bio-compatible layer of 100 mL D-PBS, 3.5 g tri-block co-polymer, 35.7 &#181;L/mL fibronectin, and 29 &#181;l/mL laminin.
	<ol>
		<li>Sterilize coverslips in ultra-violet light for at least 10 min. Incubate coverslips in the prepared bio-compatible solution overnight. 
		&#8203;NOTE: The bio-compatible layer will form only where the bio-rejection layer has been etched off in the previous step.</li>
		<li>The next day, wash the coverslips two times with P-DBS. Incubate them in PM overnight. The coverslips are now ready for cell plating.</li>
	</ol>
	</li>
</ol>
2. Maintenance of the neuronal cultures.
<ol>
	<li>Prepare changing medium (CM) composed of (per mL): 0.9 mL MEM+3G, 0.1 mL HI HS, 10 &#181;L 5-fluoro-2&#8242;-deoxyuridine (FUDR) with uridine 100x.</li>
	<li>Prepare final medium (FM) composed of (per mL): 0.9 mL MEM+3G and 0.1 mL HI HS.</li>
	<li>Replace PM with 1-1.5 mL CM after 4 days in vitro (DIV). At 6 DIV, replace 50% of the CM with fresh CM. At DIV 8, change the medium to 1.5 mL FM, followed by a 50% change of FM every 2 days. After about one week spontaneous synchronous activity emerges.</li>
</ol>
3. Imaging of spontaneous or evoked activity in neuronal cultures with fluorescent dyes.
<ol>
	<li>Prepare a solution of 50 &#181;g calcium-sensitive fluorescent dye (see Table of Materials/Reagents) in 50 &#181;L DMSO (dimethyl sulfoxide).</li>
	<li>Prepare extracellular recording solution (EM) containing (in mM) 10 HEPES, 4 KCl, 2 CaCl2, 1 MgCl2, 139 NaCl, 10 D-glucose, 45 sucrose (pH 7.4).</li>
	<li>Incubate neuronal culture in 2 mL EM with 8 &#181;L of the calcium-sensitive fluorescent dye solution for 1 h. Protect from light and gently rotate to ensure homogenous spread of the dye to the cells.</li>
	<li>Replace solution with fresh EM prior to imaging. The fluorescent imaging is demonstrated in Figure 3.</li>
	<li>Image in a fluorescence microscope with optical filters for calcium fluorescence imaging (excitation peak at 488 nm, emission peak at 520 nm), using a camera and software capable of quantifying the intensity of any region of interest (ROI) within the field of view of the microscope.</li>
</ol>
4. Magnetic Stimulation of Cultures
Note: The basic setup for magnetic stimulation is shown in Figure 1. On the 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&#39; width is about 200 &#181;m.
<ol>
	<li>Grow the neurons in a circular ring pattern (etched as described in 1.5) for 1D culture stimulation. Use a calcium-sensitive fluorescent dye for calcium imaging (as described in section 3) with a 1D culture patterned on thin (170 &#181;m), long (10 mm) lines.
	<ol>
		<li>Use a circular magnetic coil and position a Petri dish approximately 5 mm below and concentric with the coil. Use a custom coil of approximately 30-mm (inner diameter, 46-mm outer diameter) coil with an inductance of L = 90 mH driven by a homemade or commercial MS loaded with a maximal voltage of 5 kV.</li>
		<li>To magnetically stimulate neuronal cultures, a high-voltage and current are discharged through a conducting coil using a high-current-high-voltage switch. The magnetic stimulator (MS) can be built using large capacitors, on the order of 100 mF, to obtain a high voltage of 1 - 5 kV. Alternatively, use a commercially available MS (see Table of Materials/Reagents). 
		NOTE: Use a 0.254 mm thick and 6.35 mm wide polyester-coated rectangular copper wire to fabricate a homemade coil. Turn wires on custom-made frames, insulated with glass fibers, and cast in epoxy (see Table of Materials/Reagents). Alternatively use commercially available coils (see Table of Materials/Reagents).</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CaCl2 , 1M</td>
			<td>Fluka</td>
			<td>21098</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>65146</td>
			<td>Plating medium, Extracellular recording solution</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Cell Cultures</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Sigma-Aldrich</td>
			<td>F1141</td>
			<td>Bio Coating</td>
		</tr>
		<tr>
			<td>Fluo4, AM</td>
			<td>Life technologies</td>
			<td>F14201</td>
			<td>Imaging of spontaneous or evoked activity</td>
		</tr>
		<tr>
			<td>FUDR</td>
			<td>Sigma-Aldrich</td>
			<td>F0503</td>
			<td>Changing medium</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>Plating medium, Changing medium, Final medium.</td>
		</tr>
		<tr>
			<td>Hepes, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>HI HS</td>
			<td>BI</td>
			<td>04-124-1A</td>
			<td>Plating medium, Changing medium, Final medium</td>
		</tr>
		<tr>
			<td>KCl, 3M</td>
			<td>Merck</td>
			<td>1049361000</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>L2020</td>
			<td>Bio Coating.</td>
		</tr>
		<tr>
			<td>MEM x 1</td>
			<td>Gibco</td>
			<td>21090-022</td>
			<td>Plating medium, Changing medium, Final medium</td>
		</tr>
		<tr>
			<td>Magnesium chloride , 1M</td>
			<td>Sigma-Aldrich</td>
			<td>M1028</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>NaCl, 4M</td>
			<td>Bio-Lab</td>
			<td>19030591</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>Octadecanethiol</td>
			<td>Sigma-Aldrich</td>
			<td>1858</td>
			<td>Cleaning Cr-Au coated coverslips (1D cultures)</td>
		</tr>
		<tr>
			<td>Sucrose, 1M</td>
			<td>Sigma-Aldrich</td>
			<td>S1888</td>
			<td>Extracellular recording solution</td>
		</tr>
		<tr>
			<td>Uridine</td>
			<td>Sigma-Aldrich</td>
			<td>U3750</td>
			<td>Changing medium</td>
		</tr>
		<tr>
			<td>Sputtering machine</td>
			<td>AJA International, Inc</td>
			<td>ATC Orion-5Series</td>
			<td>Coating glass with thin layers of metal</td>
		</tr>
		<tr>
			<td>Pen plotter</td>
			<td>Hewlett Packard</td>
			<td>HP 7475A</td>
			<td>Etching of pattern to the coated coverslip</td>
		</tr>
		<tr>
			<td>Power supply</td>
			<td>Matrix</td>
			<td>MPS-3005 LK-3</td>
			<td>Power supply to the sputtering machine</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Cognis</td>
			<td>Versamid 140</td>
			<td>Casting of homemade coils</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Shell</td>
			<td>EPON 815</td>
			<td>Casting of homemade coils</td>
		</tr>
	</tbody>
</table>

stimulation of patterned neuronal cultures by a magnetic field

Source: Stern, S., et al., <a href="https://app.jove.com/v/54357">External Excitation of Neurons Using Electric and Magnetic Fields in One- and Two-dimensional Cultures.</a> J. Vis. Exp. (2017)
This video demonstrates a method for stimulating neuronal cultures grown in a patterned circular ring on a coverslip, using a time-varying magnetic field.

<img alt="Figure 1" src="/files/ftp_upload/22716/22716_Figure1.jpg" /> 
Figure 1: Schematic of the Setup Used for Magnetic Stimulation of Neuronal Cultures. A. At the top is shown the magnetic coil (blue circles), which is located 5 mm concentrically above the neuronal ring culture, placed in a Petri dish (blue outline). A pickup coil (red circle) positioned on the circumference of the Petri dish measures the voltage induced by the magnetic pulse. At the bottom, the measured dynamics of the magnetic stimulator coil are shown (using an MS 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 B. An inverted microscope images fluorescent dyes sensitive to calcium transients of neurons reacting to magnetic pulses. C. Neurons grown on a pattern of concentric rings, used for effective stimulation by the ring magnetic stimulator. D. Bright field microscope image of neurons grown on one line of the pattern.
<img alt="Figure 2" src="/files/ftp_upload/22716/22716_Figure2.jpg" /> 
Figure 2: Examples of Patterns of 1D Neuronal Cultures Used for Orienting the Electric Field with the Direction of Axonal Growth. A. circular pattern is used for the circular magnetic coil, when the induced electric field has a circular orientation. B. Line patterns are used when the induced or direct electric field has a single orientation.
<img alt="Figure 3" src="/files/ftp_upload/22716/22716_Figure3.jpg" /> 
Figure 3: Example Traces of Calcium Transients Imaged During Synchronous Network Bursts. A. An image of neurons that were dyed previous to the experiment with a calcium dye. B. Traces of intensity vs. time of the ROIs in A with the color of the trace representing the color of the border of the ROI in A. A large increase in intensity synchronized within the three ROIs represents a network burst.

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

1. Preparation of coverslips for 1D cultures.

	Clean glass coverslips by immersion in a base piranha solution consisting of 75 mL DDW, 25 mL 25% ammonia ...

Take a patterned coverslip with a neuronal ring culture within a culture plate containing an extracellular recording solution.
These cells are preincubated with a calcium-sensitive fluorescent dye to measure intracellular calcium levels.
Place the plate on a fluorescence microscope stage.
Position the circular magnetic coil connected to a magnetic stimulator concentrically at a suitable distance above the culture to ensure optimal stimulation.
Using the magnetic stimulator, apply high voltage and current through the coil to generate a time-varying magnetic field.
This magnetic field induces an electric field in the neurons, generating an action potential.
Upon reaching the nerve terminal, the action potential activates the voltage-gated calcium channels, allowing calcium influx into the cell and neurotransmitter release at the synapse.
This release facilitates signal transmission to the neighboring neurons through synaptic connections.
Due to the calcium ion influx, these ions bind to the intracellular calcium-sensitive dye, increasing fluorescence indicative of neuronal activity.

stimulation-of-patterned-neuronal-cultures-by-a-magnetic-field

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Stimulation and Modulation Techniques

40 visualizações. Fonte: Stern, S., et al., Excitação externa de neurônios usando campos elétricos e magnéticos em culturas unidimensionais e bidimensionais. J. Vis. Exp. (2017) Este vídeo demonstra um método para estimular culturas neuronais cultivadas em um anel circular padronizado em uma lamínula, usando um campo magnético variável no tempo. 

Vídeo: Estimulação de culturas neuronais padronizadas por um campo magnético - Conceito

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, which is unlike the protocols used for human subjects. We report a successful application of rTMS to the unilateral hemisphere of rat brain. The rTMS was delivered with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol to one side of the brain by using a small 25-mm figure-8 coil. We placed the center of the coil 1 cm lateral to the vertex on the biauricular line and angulated the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex. We also used an in-house water cooling system to enable repetitive magnetic stimulation for more than 20 min, even at a 20-Hz stimulation frequency. Increases in the transcriptions of immediate early genes (Arc, Junb, and Egr2) were greater after rTMS than after sham stimulation. After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in stimulated cortex than in contralateral side. The model presented herein will elucidate the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

We applied repetitive transcranial magnetic stimulation (rTMS) to the unilateral hemisphere of rat brain, by placing a 25-mm figure-8 coil 1 cm lateral to the vertex on the biauricular line and angulating the coil by 45&#176;. An in-house water cooling system was used for rTMS for more than 20 min.

Estimulação Magnética Transcraniana repetitiva para o Hemisfério unilateral do cérebro de rato

Previous rodent models of repetitive transcranial magnetic stimulation (rTMS) adopted whole-brain stimulation instead of unilateral hemispheric rTMS, ...

JoVE Journal

Comportamento

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

Protocolo de estimulação magnética transcraniana on-line para medir Cortical fisiologia associada com inibição de resposta

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

Neurociência

Combined Transcranial Magnetic Stimulation and Electroencephalography of the Dorsolateral Prefrontal Cortex

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic field pulses. The initiation of cortical activation or its modulation depends on the background activation of the neurons of the cortical region activated, the characteristics of the coil, its position and its orientation with respect to the head. TMS combined with simultaneous electrocephalography (EEG) and neuronavigation (nTMS-EEG) allows for the assessment of cortico-cortical excitability and connectivity in almost all cortical areas in a reproducible manner. This advance makes nTMS-EEG a powerful tool that can accurately assess brain dynamics and neurophysiology in test-retest paradigms that are required for clinical trials. Limitations of this method include artifacts that cover the initial brain reactivity to stimulation. Thus, the process of removing artifacts may also extract valuable information. Moreover, the optimal parameters for dorsolateral prefrontal (DLPFC) stimulation are not fully known and current protocols utilize variations from the motor cortex (M1) stimulation paradigms. However, evolving nTMS-EEG designs hope to address these issues. The protocol presented here introduces some standard practices for assessing neurophysiological functioning from stimulation to the DLPFC that can be applied in patients with treatment resistant psychiatric disorders that receive treatment such as transcranial direct current stimulation (tDCS), repetitive transcranial magnetic stimulation (rTMS), magnetic seizure therapy (MST) or electroconvulsive therapy (ECT).

The protocol presented here is for TMS-EEG studies utilizing intracortical excitability test-retest design paradigms. The intent of the protocol is to produce reliable and reproducible cortical excitability measures for assessing neurophysiological functioning related to therapeutic interventions in the treatment of neuropsychiatric diseases such as major depression.

Estimulação magnética transcraniana combinada e Eletroencefalografia do córtex pré-frontal Dorsolateral

Transcranial magnetic stimulation (TMS) is a non-invasive method that produces neural excitation in the cortex by means of brief, time-varying magnetic ...

Stimulation of Patterned Neuronal Cultures by a Magnetic Field

Transcrição

Stimulation of Patterned Neuronal Cultures by a Magnetic Field