Name: repetitive transcranial magnetic stimulation to the unilateral hemisphere of rat brain
Uploaded: 2016-10-22T13:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain at JoVE.com

This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2008-313-E00458). The authors thank Jin-Joo Lee for the technical assistance.

All of the procedures using animals were reviewed and approved by the Institutional Animal Care and Use Committee of Seoul National University Hospital.
1. Experimental Setup
<ol>
	<li>Animal preparation
	<ol>
		<li>Allow male Sprague-Dawley rats 1 week to adapt to their new environment before starting the experiment. 
		NOTE: Although 8-week old rats were used in the present study, a developing or adult brain can be chosen according to the research hypotheses.</li>
	</ol>
	</li>
	<li>Inhalation anesthesia for induction
	<ol>
		<li>Induce and maintain anesthesia with 5% and 2% of isoflurane dissolved in 40%/60% and 25%/75% oxygen/nitrogen via a chamber and nose cone, respectively. Adjust anesthesia depth to the level of abolishing the pedal withdrawal reflex to toe pinch to confirm proper anesthetization. 
		NOTE: Using awoken animals can be a better choice in translational terms, but there are difficulty to restrain during rTMS and they are prone to excessive stress.</li>
		<li>Monitor body temperature with a rectal probe and maintain it at 37 &#176;C by using a homeothermic blanket. Monitor anesthetic depth using pedal withdrawal reflex, temperature, respiratory rate and heart rate.</li>
	</ol>
	</li>
	<li>Switch-over to intravenous anesthesia for maintenance
	<ol>
		<li>Prepare the tail with an alcohol swab. Catheterize a lateral tail vein with a 24-gauge venous catheter for transition to IV anesthesia (Figure 1A). Load propofol intravenously (1 mg/[kg&#183;min] over 10 min, using 10 mg/ml emulsion) to the animals. Discontinue isoflurane 5 min after starting propofol loading.</li>
		<li>Maintain propofol sedation at an infusion rate of 500 - 700 &#181;g/(kg&#183;min) throughout the experiment, as in a previous study16. Supplement oxygen at 0.8 L/min via a nose cone. 
		NOTE: Anesthesia with propofol is to reduce potential suppression of cortical excitability by the inhalation agent17-19. However, anesthesia is not mandatory in rTMS experiments, and awoken animals can also be used. Anesthesia methods should be decided in consideration of the research hypotheses.</li>
		<li>Use veterinary ointment on eyes to prevent dryness while under anesthesia.</li>
		<li>Apply magnetic stimulation (see Section2) 10 min after complete transition to i.v. anesthesia.</li>
	</ol>
	</li>
	<li>Recovery conditions
	<ol>
		<li>Monitor vital signs during recovery phase. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. If an animal has undergone surgery, do not return it to the company of other animals until fully recovered. 
		NOTE: If surgery for a disease model is performed, post-surgical pain management is necessary. However, pain management is not needed for this rTMS experiment.</li>
	</ol>
	</li>
</ol>
2. Repetitive Transcranial Magnetic Stimulation
<ol>
	<li>Stimulator and coil
	<ol>
		<li>Apply stimulation by using a repetitive stimulator that delivers biphasic stimuli via a 25 mm figure-8 coil. Locate the center of the coil 0.5 cm lateral to the vertex on the biauricular line, and angulate the coil 45&#176; to the ground. 
		NOTE: The maximal magnetic field strength of the coil is 4.0 T. The magnetic coil is mounted firmly on a built-in holder.</li>
	</ol>
	</li>
	<li>Motor threshold
	<ol>
		<li>Determine the motor threshold (MT) at the hot spot, with the center of the coil positioned 0.5 cm lateral to the vertex on the biauricular line and with the surface flat onto the calvaria. This is the same methodology used in a previous study20. 
		NOTE: Define MT as the minimum stimulus intensity evoking 5 or more palpable contractions on the contralateral forepaw by 10 consecutive stimuli. Verify whether the stimulation is primarily causing contralateral muscle contraction to ensure unilateral stimulation.</li>
	</ol>
	</li>
	<li>Application of rTMS
	<ol>
		<li>Apply rTMS 10 min after stabilization of deep anesthesia. Place the center of the coil at the target rTMS site, selected from the cerebral cortices depending on research questions. Then, tilt the coil to ensure direct contact between the coil center and the surface of skull at the stimulation point. 
		NOTE: For example, angulate the coil 45&#176; to the ground to minimize a potential direct effect of rTMS on the contralateral cortex (Figure 1B and 1C).</li>
		<li>Subject the animals to a session of 20-min rTMS of the unilateral hemisphere. Using the software console, deliver rTMS with a low-frequency (1 Hz), high-frequency (20 Hz), or sham stimulation protocol, and set the stimulation intensity at 100 - 110% of the MT.</li>
		<li>Perform 1 Hz stimulation without rest. Using the software console input &#34;1,200&#34; shots for &#34;20&#34; min). For 20 Hz stimulation, conduct 2 sec of stimulation followed by 28 sec of rest. Using the software console input &#34;1,600&#34; shots for &#34;20&#34; min.</li>
		<li>For sham stimulation, tilt the coil perpendicular (90&#176; rotation) to the calvaria and place the coil edge 2 cm apart from head surface (Figure 1D). Fix the coil holder firmly to the main apparatus; there is no need to hold the coil by hand during the experiment. 
		NOTE: To compensate for acoustic and other non-specific effects, distinct sham protocols should be used for distinct stimulation protocols. For example, 1-Hz sham stimulation can be used for 1-Hz rTMS experiments.</li>
	</ol>
	</li>
	<li>Cooling the coil
	<ol>
		<li>Use a water cooling system to enable repetitive magnetic stimulation for more than 20 min at 1- and 20-Hz stimulation frequencies (Figure 2). Circulate icy water surrounding the whole length of the coil during the experiment to prevent overheating, although the temperature of the coil or stimulator is not monitored. 
		NOTE: Commercially available cooled rat coils can also be used.</li>
		<li>If possible, monitor the coil temperature by viewing the heating gauge of the rTMS machine.&#160; NOTE: There were no adverse consequences related to the rTMS stimulation. There is, however, a potential burn risk if metal ear identification tags are used near the stimulating coil.</li>
	</ol>
	</li>
</ol>
3. Micro Positron Emission Tomography
<ol>
	<li>Animal preparation
	<ol>
		<li>Conduct inhalation anesthesia for induction and i.v. anesthesia for maintenance (see step 1.2.1 and 1.3.1). Apply 1-Hz rTMS to an animal for 10 min at a stimulation intensity of 100-110% of the MT.</li>
		<li>Five minutes after finishing the rTMS stimulation, inject 1 mCi of 2-[F-18]fluoro-deoxyglucose (18FDG) dissolved in 0.5 ml of normal saline intravenously by using a tail vein catheter. Allow 30 min for 18FDG uptake. NOTE: Place the rat under anesthesia during the entire micro-PET experiment.</li>
	</ol>
	</li>
	<li>Image analysis
	<ol>
		<li>Use a PET scanner for brain imaging to reaffirm the unilaterality of the stimulation. Reconstruct images with a 3-D iterative algorithm. To assess the changes in metabolism induced by rTMS, identify regions of interest (ROIs) in the images of the transverse brain sections21.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>After performing micro-PET imaging, euthanize the rats in a chamber prefilled with carbon dioxide while the rats are in deep anesthesia.</li>
	</ol>
	</li>
</ol>
4. mRNA Microarray
<ol>
	<li>Euthanasia
	<ol>
		<li>Induce and maintain anesthesia with 5% and 2% of isoflurane dissolved in 40%/60% and 25%/75% oxygen/nitrogen via a chamber and nose cone, respectively. Anesthetize deeply to the level of abolishing the pedal withdrawal reflex to toe pinch before being decapitated.</li>
		<li>Decapitate the rats for euthanasia 5 min after 1 session of 1-Hz rTMS.</li>
	</ol>
	</li>
	<li>Tissue harvest
	<ol>
		<li>Lay out materials and surgical instruments in the order of use, including folded paper towels, a bone rongeur, microscissors, larger surgical scissors, a microforcep, a No. 10 or 11 scalpel blade, a lidded 10-cm glass petri dish filled with ice, and 1.5-ml tubes. Prepare a plastic bag for disposal of carcass.</li>
		<li>Make a midline skin incision in the skull anterioposteriorly. Bluntly dissect the soft tissue and surrounding muscles with surgical scissors, and remove the skull bone piece using a bone rongeur. Quickly dissect the fresh brain carefully from the skull. Then, lay it on ice by using the microforceps and microscissors. Rinse the brain tissue in ice-cold normal saline.</li>
		<li>Transfer the brain to dry ice immediately, and subsequently store it at -80 &#176;C in a tube until further processing.</li>
		<li>Thaw the brain tissue before harvest.</li>
		<li>Place the brain dorsal side up, and harvest the brain tissue from the stimulated cerebral cortex (around the hot spot in the primary motor cortex) on ice by using the microforceps and microscissors. Put the harvested tissue in the 1.5-ml tube.</li>
	</ol>
	</li>
	<li>RNA preparation
	<ol>
		<li>Extract total RNA from tissue homogenates using lysis reagent22. Process with DNase digestion and clean-up procedures. Quantify the RNA samples and aliquots and store them at -80 &#176;C until use.</li>
		<li>For quality control, evaluate RNA purity and integrity by denaturing gel electrophoresis, at a OD ratio of 260:280, and analyze them on a commercially available analyzer.</li>
	</ol>
	</li>
	<li>Labeling and purification
	<ol>
		<li>Amplify and purify total RNA by using a commercially available RNA amplification kit to yield biotinylated cRNA. Briefly, reverse-transcribe 550 ng of total RNA to cDNA by using a T7 oligo(dT) primer. Synthesize and in vitro transcribe the second-strand cDNA and then label it with biotin-NTP.</li>
		<li>After purification, quantify cDNA by using a spectrophotometer.</li>
	</ol>
	</li>
	<li>Hybridization and data export23
	<ol>
		<li>Use the expression beadchip for mRNA expression analysis. Hybridize the labeled 750-ng cDNA samples to each rat-12 expression bead array for 16 - 18 hr at 58 &#176;C. Carry out detection of array signal by using streptavidin-Cy3.</li>
		<li>Scan arrays with a confocal scanner. Perform array data export processing and analysis by using a commercially available software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The primary purpose of this study was to introduce an animal model of unilateral rTMS. Although unilateral stimulation is one of the most fundamental characteristics of human rTMS research, many studies have not adopted it in small animals. However, Rotenberg et al.15 recorded contralateral MEPs with stimulation of 100% MT using a figure-8 coil with an outside lobe diameter of 20 mm, whereas stimulation with 112.5% and 133.3% MT produced ipsilateral as well as contralateral MEPs. This might be because the large induced electric field can affect the contralateral hemisphere. Thus, our study is an extension of this previous work15,24, by moving the coil more lateral and tilting it to accentuate unilateral stimulation. The primary aim of this study was achieved because we confirmed that micro-PET revealed a local increase in the glucose metabolism in the stimulated cerebral cortex after rTMS (Figure 3).
Location and angulation of the coil are critical steps in this experiment. Unilateral stimulation is possible by placing the center of the rTMS coil 1 cm lateral to the vertex on the biauricular line and angulating the coil 45&#176; to the ground. The stimulation site can be different from the primary motor cortex (M1), depending on the condition that investigators want to target with rTMS. For instance, to ameliorate depression, the dorsolateral prefrontal cortex (DLPFC) is stimulated with rTMS, but the motor threshold, which is also measured in M1, determines the stimulation intensity even for DLPFC rTMS. Likewise, the hotspot&#160;&#8212; 0.5 cm lateral to the vertex on the biauricular line&#160;&#8212; was used to determine the motor threshold in the present study. The more lateral cortex &#8212; 1 cm lateral to the vertex&#160;&#8212; was intentionally selected to ensure the unilaterality of stimulation and investigate rTMS-induced molecular changes.
As for the magnetic field strength within the tissue, in a previous finite element modeling study on the induced electrical field in the mouse brain, the induced electrical field by the 70 mm figure-8 coil at 75% MSO reached approximately 150 V/m on the brain surface and in the cortex. The electric field strength dropped dramatically as distance increased, showing the maximum depth with greater than 100 V/m strength was just 1.9 mm for the 70 mm figure-8 coil25. In another rat study, at 10 mm depth the induced electrical field strength decreased to 25% of that on the brain surface26. Interestingly, the half power region (HPR) was as broad as ~7 x 7 mm (0.51 cm2) even when a 25 mm figure-8 coil was used25. Although concrete numbers were not provided for the 70 mm figure-8 coil, Salvador and Miranda commented that the HPR for the 70 mm coil was larger than that of the 25 mm coil. Since we wanted to prevent the HPR from covering the contralateral hemisphere, we selected a spot 1 cm lateral to the midline. Tilting was inevitable to ensure direct contact between the coil center and the surface of the skull at the stimulation point.
Anesthesia can potentially depress neuronal excitability, glucose metabolism, and gene expression. Haghighi et al. revealed that isoflurane at concentration of 0.5% significantly depressed electrical transcranial MEPs recorded from rats17. On the other hand, MEPs were preserved during propofol infusion as high as 40 mg/[kg&#183;hr], with amplitudes remaining large in rats18. In a human study, no compound muscle action potentials (CMAP) were detected during isoflurane anesthesia. However, 333 Hz, four-pulse magnetic stimulation evoked CMAP in the hypothenar muscle in 75% of patients, and in the anterior tibial muscle in 65% of patients, during propofol anesthesia19. Using awoken animals can be a better choice in the physiological aspects, but they are not easy to restrain during rTMS and are prone to stressful conditions.
As troubleshooting, a simple cooler that used a water-circulating pump enabled us to extend the stimulation duration for more than 20 min even at a 20-Hz stimulation frequency. This is important because it enables as many stimulations as in rTMS protocols for human subjects. Cooling the figure-8 coil with only a handheld ice-cold water bag was not sufficient to ensure stimulation of more than 20 min. Long rTMS duration in small animals will provide the opportunity for in-depth investigation of the molecular mechanisms of rTMS. Commercially available cooled rat coils will be reasonable alternatives.
There were several limitations in this experiment. First, only a biphasic pulse was available, which was a limitation of the rTMS machine we used. Future studies investigating the effect of various pulses and waveforms will be needed. Second, we adopted a pragmatic approach to determine the motor threshold by palpation. Although this method may be inferior to EMG techniques in terms of accuracy, it is readily reproducible and applicable to many research hypotheses. For example, if the primary purpose of a researcher were to investigate differences between the primary motor cortex and adjacent subcortices in rTMS-induced gene or protein expression, more precise determination of motor threshold would be necessary. If a researcher, however, wanted to analyze rTMS-induced gene expression profiles in the dorsolateral prefrontal cortical tissue, the present pragmatic approach may suffice, because the distance and angle between the target tissue and the coil can vary slightly during the movement of the coil from the M1 to the DLPFC area. Third, although we successfully applied rTMS on the unilateral hemisphere of the rat brain, still the stimulation is not as focal as rTMS in human research. The induced strong electric field of ~ 0.5 cm2 on less than 10 cm2 of the rat brain surface seems relatively more diffuse than that in the human hemispheric surface of ~ 2,500 cm2 27. We believe, however, that the model presented herein can be used for elucidating the molecular mechanisms of rTMS by allowing analysis of the inter-hemispheric difference in its effect.

Repetitive transcranial magnetic stimulation (rTMS), a tool for non-invasive brain stimulation and neuromodulation, has been applied in the treatment of various conditions such as central pain1,2, depression3, migraine4, and even stroke5-7. Rapidly changing electrical current through coils on the head induces an electrical field on the cerebral cortex and a resultant neuronal activation. The excitability of the cerebral cortex can be modulated by rTMS, which can last for more than 30 min after the stimulation is terminated.
Suggested mechanisms of the rTMS after-effect include long-term potentiation/depression-like effect8, transient shift in ionic balance9, and metabolic changes10. In addition, Di Lazzaro et al. suggest that intermittent theta-burst stimulation affects the excitatory synaptic inputs to pyramidal tract neurons, both in the stimulated and the contralateral hemisphere11.
Significant limitations, however, have hindered researchers from translating on-bench evidence to clinical situations. First, in previous animal studies, rTMS was used for whole-brain stimulation12. Whole-brain stimulation is quite different from the protocols used in human studies9. The other problem is related with the stimulation duration. This is at least partly attributable to the fact that an effective cooling system was unavailable for small coils in the past.
In recent years, seminal articles have been published suggesting ways for overcoming these difficulties in the rTMS experiment on the small animal brain. By these animal models, it was revealed that the rat brain also shows similar cortical excitability changes as in human in response to low-frequency rTMS13. More importantly, cellular and molecular mechanisms of rTMS are increasingly being investigated using animal models of rTMS. A case in point is that a distinct type of inhibitory interneuron is known to be most sensitive to intermittent theta burst stimulation14. Rodent models of rTMS, thus, offer new opportunities for exploring much-sought questions on the molecular underpinnings of rTMS-induced changes. If small animal models of rTMS can be used in more laboratories, it may greatly accelerate and strengthen research in this area.
We now describe how to apply rTMS to the unilateral hemisphere of rat brain, an extension of the previous work15. Stimulation-induced changes were evaluated by using micro-positron emission tomography (PET) and mRNA microarrays to study rTMS-induced changes in the stimulated cerebral cortex.

<table><tbody><tr><td>Homeothermic blanket with a rectal probe</td><td>Harvard apparatus</td><td>507222F</td><td></td></tr><tr><td>Isoflurane (Forane sol.)</td><td>Choongwae</td><td></td><td></td></tr><tr><td>Propofol (Provive Inj. 1% 20 ml)</td><td>Claris Lifesciences</td><td></td><td></td></tr><tr><td>Repetitive magnetic stimulator (Magstim Rapid&lt;sup&gt;2&lt;/sup&gt;)</td><td>Magstim Company Ltd</td><td></td><td></td></tr><tr><td>25 mm figure-of-8 coil</td><td>Magstim Company Ltd</td><td>1165-00</td><td></td></tr><tr><td>PET-CT</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>QIAzol Lysis Reagent</td><td>Qiagen</td><td>(US Patent No. 5,346,994)</td><td></td></tr><tr><td>RNeasy Lipid Tissue Mini Kit</td><td>Qiagen</td><td>74804</td><td></td></tr><tr><td>RNeasy Mini Spin Columns</td><td>Qiagen</td><td>(Mat No. 1011708)</td><td></td></tr><tr><td>Agilent 2100 Bioanalyzer</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>Ambion Illumina RNA amplification kit</td><td>Ambion</td><td></td><td></td></tr><tr><td>Nanodrop Spectrophotometer</td><td>NanoDrop</td><td>ND-1000</td><td></td></tr><tr><td>Illumina RatRef-12 Expression BeadChip</td><td>Illumina, Inc.</td><td></td><td></td></tr><tr><td>Amersham fluorolink streptavidin-Cy3</td><td>GE Healthcare Bio-Sciences</td><td></td><td></td></tr></tbody></table>

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.

Fifteen 8-week old male Sprague-Dawley rats were used for a separate inter-rater reliability analysis of MT determination. Using palpation of muscle twitching, the MTs were obtainable in all rats and measured as 33.00 &#177; 4.21% maximal stimulator output (% MSO) and 33.93 &#177; 0.88% MSO, respectively, by two independent researchers. Bland-Altman bias was -0.93, and the 95% limits of agreement were -9.13 to 7.26%.
In the micro-PET experiment on six 8-week old rats (n= 4 in the 1-Hz rTMS, and n= 2 in the sham rTMS group), the uptake of 18F-FDG in the ROIs was calculated as the averaged nCi/cc after calibration of both ipsilateral and contralateral cerebral cortices in the same images. The radioactivity in the contralateral area was used as a reference to normalize data obtained in the ipsilateral area, and the differential uptake ratio (DUR) was calculated. The mean DURs obtained from three consecutive transverse images were averaged to obtain the DURs for the rats. This is the same methodology used in a previous study21. 18FDG-PET images showed a focal increase in glucose metabolism in the stimulated left cortical area in the 1-Hz group, supporting the unilaterality of the rTMS (Figure 3).
In the mRNA microarray study, the quality of hybridization and overall chip performance were monitored by visual inspection of both the internal quality control checks and raw scanned data. Array data were filtered according to a detection p value of &#60; 0.05 (similar to the signal-to-noise ratio) in at least 50% samples (a higher signal value was required to obtain a detection p value of &#60; 0.05). The selected gene signal value was transformed by logarithm and normalized by using a quantile method. The statistical significance of the expression data was determined by using the Mann-Whitney U test and fold change, in which the null hypothesis was that no difference exists between the 1-Hz rTMS (n = 4) and sham groups (n = 4). The false discovery rate was controlled by adjusting the p value by using the Benjamini-Hochberg algorithm. After normalization and filtering, mRNAs showing significant differential expressions (|fold change| 1.2, p &#60; 0.05) were selected. As a result, the expression levels of the immediate early genes were significantly higher in the rTMS group than in the sham group, with the expressions of the Arc, Junb, and Egr2 genes upregulated (Figure 4A).
In addition, we measured bdnf mRNA expressions in the stimulated and contralateral cortex after 5 consecutive days of 20-min rTMS (n = 5 each in the 1-Hz and 20-Hz groups). After 1-Hz stimulation, bdnf mRNA expression was significantly higher in the stimulated cortex than in the contralateral one (Figure 4B). This revealed differential rTMS-induced changes in the stimulated and contralateral cerebral cortices.
<img alt="Figure 1" src="/files/ftp_upload/54217/54217fig1.jpg" /> 
Figure 1. Experimental Settings. (A) An intravenous catheter is inserted at a lateral tail vein (arrow), and a nose cone is used for anesthesia with isoflurane as well as for oxygen supplement after a switch-over to intravenous propofol. (B) Dorsal anterolateral view during rTMS. (C) Dorsal posterior view. The surface of a figure-of-8 coil is angulated 45&#176; to the ground to minimize the potential direct stimulation of the contralateral cortex. (D) A schematic illustration of sham rTMS. The coil is placed 2 cm away from and tilted perpendicular (90&#176; rotation) to the calvaria. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54217/54217fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54217/54217fig2.jpg" /> 
Figure 2. The Cooling System uses a Water-circulating Pump with Motor. Ice packing on the copper wires of the coil is not needed, as the cooling system enwrapping the cable of the coil is sufficient to cool the heat produced at the copper wires. The surface of the coil is not in direct contact with the iced water. The cooling system is active during stimulation sessions.
<img alt="Figure 3" src="/files/ftp_upload/54217/54217fig3.jpg" /> 
Figure 3. Positron Emission Tomography (PET) Image. (A) The coronal sections of micro-PET images of a rat obtained using 2-[F-18]fluoro-deoxyglucose, showing increased local glucose metabolism in the stimulated cortex after 1-Hz rTMS for 10 min at 100% of the MT (arrows). (B) The ratio of FDG uptake in stimulated/contralateral cortex in the 1-Hz (n= 4) and sham rTMS group (n= 2). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54217/54217fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54217/54217fig4.jpg" /> 
Figure 4. The mRNA Microarray of the Immediate Early Genes and bdnf. (A) Arc, Junb, and Egr2 were differentially expressed, which were identified on the microarray obtained 5 min after 1 session of 1-Hz rTMS, ordered by fold change. The expression levels of the genes were significantly higher in the rTMS group (n = 4) than in the sham group (n = 4) (p &#60; 0.05 with Mann-Whitney U test), with the expressions of the Arc, Junb, and Egr2 genes upregulated. (B) After 5 consecutive days of 20-min 1-Hz rTMS, bdnf mRNA expression was significantly higher in the stimulated cortex than in the contralateral side (*p &#60; 0.05, Wilcoxon signed-rank test). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54217/54217fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain at JoVE.com

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

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.

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

repetitive-transcranial-magnetic-stimulation-to-unilateral-hemisphere

Nanyang Technological University

Research

JoVE Journal

Behavior

12.4K Views.  Chungnam National University Hospital, Daejeon. 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°. An in-house water cooling system was used for rTMS for more than 20 min. 

Video: Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Medicine

Neuronavigation-guided Repetitive Transcranial Magnetic Stimulation for Aphasia

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential therapeutic effects. Brain excitability is non-invasively modulated by rTMS, and rTMS to the language areas has proved its potential effects on treatment of aphasia. In our protocol, we aim to artificially induce virtual aphasia in healthy subjects by inhibiting Brodmann area 44 and 45 using neuronavigational TMS (nTMS), and F3 of the International 10-20 EEG system for conventional TMS (cTMS). To measure the degree of aphasia, changes in reaction time to a picture naming task pre- and post-stimulation are measured and compare the delay in reaction time between nTMS and cTMS. Accuracy of the two TMS stimulation methods is compared by averaging the Talairach coordinates of the target and the actual stimulation. Consistency of stimulation is demonstrated by the error range from the target. The purpose of this study is to demonstrate use of nTMS and to describe the benefits and limitations of the nTMS compared to those of cTMS.

This study is designed to test the hypothesis that neuronavigational system-guided transcranial magnetic stimulation has higher accuracy for targeting the intended target as demonstrated by eliciting a greater degree of virtual aphasia in healthy subjects, measured by delay in reaction time to picture naming.

Repetitive transcranial magnetic stimulation (rTMS) is widely used for several neurological conditions, as it has gained acknowledgement for its potential ...

Simultaneous Transcranial Alternating Current Stimulation and Functional Magnetic Resonance Imaging

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs frequency-specific stimulation of the human brain through current applied to the scalp with surface electrodes. Most current knowledge of the technique is based on behavioral studies; thus, combining the method with brain imaging holds potential to better understand the mechanisms of tACS. Because of electrical and susceptibility artifacts, combining tACS with brain imaging can be challenging, however, one brain imaging technique that is well suited to be applied simultaneously with tACS is functional magnetic resonance imaging (fMRI). In our lab, we have successfully combined tACS with simultaneous fMRI measurements to show that tACS effects are state, current, and frequency dependent, and that modulation of brain activity is not limited to the area directly below the electrodes. This article describes a safe and reliable setup for applying tACS simultaneously with visual task fMRI studies, which can lend to understanding oscillatory brain function as well as the effects of tACS on the brain.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations, though its effects are not completely understood. This article describes a safe and reliable setup for applying tACS simultaneously with functional magnetic resonance imaging, which can increase understanding oscillatory brain function and effects of tACS.

Transcranial alternating current stimulation (tACS) is a promising tool for noninvasive investigation of brain oscillations. TACS employs ...

Utilizing Repetitive Transcranial Magnetic Stimulation to Improve Language Function in Stroke Patients with Chronic Non-fluent Aphasia

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this experiment, we demonstrate the administration of low-frequency repetitive TMS (rTMS) to an optimal stimulation site in the right hemisphere in patients with chronic non-fluent aphasia. A battery of standardized language measures is administered in order to assess baseline performance. Patients are subsequently randomized to either receive real rTMS or initial sham stimulation. Patients in the real stimulation undergo a site-finding phase, comprised of a series of six rTMS sessions administered over five days; stimulation is delivered to a different site in the right frontal lobe during each of these sessions. Each site-finding session consists of 600 pulses of 1 Hz rTMS, preceded and followed by a picture-naming task. By comparing the degree of transient change in naming ability elicited by stimulation of candidate sites, we are able to locate the area of optimal response for each individual patient. We then administer rTMS to this site during the treatment phase. During treatment, patients undergo a total of ten days of stimulation over the span of two weeks; each session is comprised of 20 min of 1 Hz rTMS delivered at 90% resting motor threshold. Stimulation is paired with an fMRI-naming task on the first and last days of treatment. After the treatment phase is complete, the language battery obtained at baseline is repeated two and six months following stimulation in order to identify rTMS-induced changes in performance. The fMRI-naming task is also repeated two and six months following treatment. Patients who are randomized to the sham arm of the study undergo sham site-finding, sham treatment, fMRI-naming studies, and repeat language testing two months after completing sham treatment. Sham patients then cross over into the real stimulation arm, completing real site-finding, real treatment, fMRI, and two- and six-month post-stimulation language testing.

We explore the use of repetitive transcranial magnetic stimulation (rTMS) to improve language abilities in patients with chronic stroke and non-fluent aphasia. After identifying a site in the right frontal gyrus for each patient that responds optimally to stimulation, we target this site during ten days of rTMS treatment.

Transcranial magnetic stimulation (TMS) has been shown to significantly improve language function in patients with non-fluent aphasia1. In this ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

Neuroscience

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 ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Functional Magnetic Resonance Spectroscopy at 7 T in the Rat Barrel Cortex During Whisker Activation

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to technological developments over the last decade and the increase in magnetic field strength, it is now possible to obtain good resolution spectra in vivo in the rat brain. Neuroenergetics (i.e., the study of brain metabolism) and, especially, metabolic interactions between the different cell types have attracted more and more interest in recent years. Among these metabolic interactions, the existence of a lactate shuttle between neurons and astrocytes is still debated. It is, thus, of great interest to perform functional proton magnetic resonance spectroscopy (1H-MRS) in a rat model of brain activation and monitor lactate. However, the methyl lactate peak overlaps lipid resonance peaks and is difficult to quantify. The protocol described below allows metabolic and lactate fluctuations to be monitored in an activated brain area. Cerebral activation is obtained by whisker stimulation and 1H-MRS is performed in the corresponding activated barrel cortex, whose area is detected using blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI). All steps are fully described: the choice of anesthetics, coils, and sequences, achieving efficient whisker stimulation directly in the magnet, and data processing.

After checking by blood-oxygen-level-dependent functional magnetic resonance imaging (BOLD fMRI) that the corresponding somatosensory barrel field cortex area (called S1BF) is correctly activated, the main goal of this study is to quantify lactate content fluctuations in the activated rat brains by localized proton magnetic resonance spectroscopy (1H-MRS) at 7 T.

Nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to measure cerebral metabolite contents in vivo and noninvasively. Thanks to ...

A Pilot Study on the Repetitive Transcranial Magnetic Stimulation of A&#946; and Tau Levels in Rhesus Monkey Cerebrospinal Fluid

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in the brain. As a non-invasive technique, repetitive transcranial magnetic stimulation (rTMS) has been applied for the treatment of neurodegenerative disorders. This study explored the effects of rTMS on A&#946; and tau levels in rhesus monkey cerebrospinal fluid (CSF). This is a single-blind, self-controlled study. Three different frequencies (low frequency, 1 Hz; high frequencies, 20 Hz and 40 Hz) of rTMS were used to stimulate the bilateral-dorsolateral prefrontal cortex (DLPFC) of the rhesus monkey. A catheterization method was used to collect CSF. All samples were subjected to liquid chip detection to analyze CSF biomarkers (A&#946;42, A&#946;42/A&#946;40, tTau, pTau). CSF biomarker levels changed with time after stimulation by rTMS. After stimulation, the A&#946;42 level in CSF showed an upward trend at all frequencies (1 Hz, 20 Hz, and 40 Hz), with more significant differences for the high-frequencies (p &lt; 0.05) than for the low frequency. 
After high-frequency rTMS, the total Tau (tTau) level of CSF immediately increased at the post-rTMS timepoint (p &lt; 0.05) and gradually decreased by 24 h. Moreover, the results showed that the level of phosphorylated Tau (pTau) increased immediately after 40 Hz rTMS (p &lt; 0.05). The ratio of A&#946;42/A&#946;40 showed an upward trend at 1 Hz and 20 Hz (p &lt; 0.05). There was no significant difference in the tau levels with low-frequency (1 Hz) stimulation. Thus, high-frequencies (20 Hz and 40 Hz) of rTMS may have positive effects on A&#946; and tau levels in rhesus monkey CSF, while low-frequency (1 Hz) rTMS can only affect A&#946; levels.

Here, we describe the procedure for a pilot study to explore the effect of repetitive transcranial magnetic stimulation with different frequencies (1 Hz/20 Hz/40 Hz) on A&#946; and tau metabolism in rhesus monkey cerebrospinal fluid.

Previous studies have demonstrated that a non-invasive light-flickering regime and auditory tone stimulation could affect A&#946; and tau metabolism in ...

Stimulating Mouse Brain with a Low-Cost EEG and Millimeter-Sized Coil

Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, utilizing a millimeter-sized coil. Using conventional screw electrodes combined with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain. In addition, we explain how a millimeter-sized coil is produced using low-cost equipment usually found in laboratories. Practical procedures for fabricating the flexible multielectrode array substrate and the surgical implantation technique for screw electrodes are also presented, which are necessary to produce low-noise EEG signals. Although the methodology is useful for recording from the brain of any small animal, the present report focuses on electrode implementation in an anesthetized mouse skull. Furthermore, this method can be easily extended to an awake small animal that is connected with tethered cables via a common adapter and fixed with a TMS device to the head during recording.The present version of the EEG-TMS system, which can include a maximum of 32 EEG channels (a device with 16 channels is presented as an example with fewer channels) and one TMS channel device, is described. Additionally, typical results obtained by the application of the EEG-TMS system to anesthetized mice are briefly reported.

Author Spotlight: Low-Cost Electroencephalographic Recording System Combined with a Millimeter-Sized Coil to Transcranially Stimulate the Mouse Brain In Vivo  

A low-cost electroencephalographic recording system combined with a millimeter-sized coil is proposed to drive transcranial magnetic stimulation of the mouse brain in vivo. Using conventional screw electrodes with a custom-made, flexible, multielectrode array substrate, multi-site recording can be carried out from the mouse brain in response to transcranial magnetic stimulation.

A low-cost electroencephalographic (EEG) recording system is proposed here to drive transcranial magnetic stimulation (TMS) of the mouse brain in vivo, ...

Repetitive Transcranial Magnetic Stimulation to the Unilateral Hemisphere of Rat Brain

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