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 ...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+and+safety+of+transcranial+magnetic+stimulation+in+the+acute+treatment+of+major+depression:+a+multisite+randomized+controlled+trial.">Efficacy and safety of transcranial magnetic stimulation in the acute treatment of major depression: a multisite randomized controlled trial.</a> Biol Psychiatry. 62 (11), 1208-1216 (2007).</li><li>Brighina, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facilitatory+effects+of+1+Hz+rTMS+in+motor+cortex+of+patients+affected+by+migraine+with+aura.">Facilitatory effects of 1 Hz rTMS in motor cortex of patients affected by migraine with aura.</a> Exp Brain Res. 161 (1), 34-38 (2005).</li><li>Lefaucheur, J. 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R., Pascual-Leone, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+impact+on+14C-2-deoxyglucose+brain+metabolism+following+patterns+of+high+and+low+frequency+repetitive+transcranial+magnetic+stimulation+in+the+posterior+parietal+cortex.">Opposite impact on 14C-2-deoxyglucose brain metabolism following patterns of high and low frequency repetitive transcranial magnetic stimulation in the posterior parietal cortex.</a> Exp Brain Res. 176 (4), 603-615 (2007).</li><li>Di Lazzaro, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+basis+of+the+effects+of+intermittent+theta+burst+stimulation+of+the+human+motor+cortex.">The physiological basis of the effects of intermittent theta burst stimulation of the human motor cortex.</a> J Physiol. 586 (16), 3871-3879 (2008).</li><li>Post, A., Keck, M. 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R., Seo, K. S., Oh, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+use+of+granulocyte-colony+stimulating+factor+with+repetitive+transcranial+magnetic+stimulation+did+not+enhance+recovery+of+function+in+the+early+subacute+stroke+in+rats.">Concurrent use of granulocyte-colony stimulating factor with repetitive transcranial magnetic stimulation did not enhance recovery of function in the early subacute stroke in rats.</a> Neurol Sci. 36 (5), 771-777 (2015).</li><li>Haghighi, S. S., Green, K. D., Oro, J. J., Drake, R. K., Kracke, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depressive+effect+of+isoflurane+anesthesia+on+motor+evoked+potentials.">Depressive effect of isoflurane anesthesia on motor evoked potentials.</a> Neurosurgery. 26, 993-997 (1990).</li><li>Fishback, A. S., Shields, C. B., Linden, R. D., Zhang, Y. P., Burke, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+propofol+on+rat+transcranial+magnetic+motor+evoked+potentials.">The effects of propofol on rat transcranial magnetic motor evoked potentials.</a> Neurosurgery. 37 (5), 969-974 (1995).</li><li>Rohde, V., Krombach, G. A., Baumert, J. H., Kreitschmann-Andermahr, I., Weinzierl, M., Gilsbach, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+motor+evoked+potentials+following+repetitive+magnetic+motor+cortex+stimulation+during+isoflurane+or+propofol+anaesthesia.">Measurement of motor evoked potentials following repetitive magnetic motor cortex stimulation during isoflurane or propofol anaesthesia.</a> Br J Anaesth. 91 (4), 487-492 (2003).</li><li>Lee, S. A., Oh, B. 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J., Pascual-Leone, A., Jensen, F. E., Rotenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+measure+of+cortical+inhibition+by+mechanomyography+and+paired-pulse+transcranial+magnetic+stimulation+in+unanesthetized+rats.">A new measure of cortical inhibition by mechanomyography and paired-pulse transcranial magnetic stimulation in unanesthetized rats.</a> J Neurophysiol. 107 (3), 966-972 (2012).</li><li>Salvador, R., Miranda, P. 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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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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% 20ml)</td>
			<td>Claris Lifesciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Repetitive magnetic stimulator (Magstim Rapid2)</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 micr...

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, ...

The overall goal of this experiment is to conduct a unilateral repetitive transcranial magnetic stimulation, or rTMS, in a small animal model. This method can help answer key questions regarding molecular mechanisms of rTMS, by enabling unilateral hemispheric stimulation to the rat brain. The main advantage of this technique is that it provides an opportunity to explore the interhemispheric differences of molecular events, in response to unilateral rTMS.Individuals new to this method may struggle because the rTMS coil can be so large that they stimulate the whole brain, and because the timing of the motor threshold is time consuming and laborious. An experienced technician from our laboratory, Jin-Joo Lee, will be demonstrating the procedure. Place a rectal probe in an anesthetized adult male rat to monitor the body temperature, using a homeothermic blanket to maintain the temperature at 37 degrees Celsius.Apply ointment to the animal's eyes. Then, 10 minutes after the complete transition to IV anesthesia, mark a point 0.5 centimeters lateral to the vertex on the biauricular line, and fix the coil holder firmly to the apparatus. Then, locate the center of a 25 millimeter figure of eight coil at the point, and angulate the coil 45 degrees to the ground.Holding the coil surface flat against the calvaria, gradually increase the stimulation intensity to determine the minimum stimulus intensity required to evoke five or more palpable contralateral forepaw contractions, by ten consecutive biphasic stimuli, through the coil. Verify that the stimulation is primarily causing the contralateral muscle contraction, without any ipsilateral contraction, to ensure a unilateral stimulation. Use a water-cooling system to cool the coil for 10 minutes.Maintaining the coil temperature with the cooling system, use the software console to set the stimulation intensity at 100 to 110%of the motor threshold. Then, to apply the rTMS at a one hertz stimulation, first set the input to 1200 shots, for 20 minutes. Then, move the center of the coil to the target site on one cerebral cortex, and tilt the coil, to ensure the direct contact between the coil center and the surface of the skull at the point of stimulation.For a 20 hertz stimulation, set the input to 1600 shots over 20 minutes, and conduct two seconds of stimulation, followed by 28 seconds of rest. For a sham stimulation, tilt the coil at a 90 degree rotation to the calvaria, and place the edge of the coil two centimeters from the head surface. At the end of the stimulation, allow the animal to recover with full monitoring.In this representative experiment, six eight-week-old rats were imaged by micro-positron emission tomography to evaluate the uptake of a radio isotope in the cerebral cortices regions of interest. The radioactivity in the contralateral area was used as a reference to normalize the data obtained in the ipsilateral area. The mean differential uptake ratios obtained from three consecutive transverse images, were then averaged to calculate the differential uptake ratio for the rats, demonstrating a focal increase in the glucose metabolism in the stimulated left cortical area, in the one hertz group.Supporting the unilaterality of the rTMS. In this mRNA microarray study, the expression levels of several immediate early genes were observed to be significantly higher in the rTMS animals than in the sham group. In addition, after a one hertz stimulation, brain-derived neurotrophic factor mRNA expression was also significantly higher in the stimulated cortex, revealing differential rTMS-induced changes in the stimulated versus the contralateral cerberal cortices.Once administered, the rTMS technique can be completed in one hour, if it's performed properly. While attempting this procedure, it's important to take care to properly position the rTMS coil, and to prepare the contra and ipsilateral forepaw contractions to confirm the unilateral stimulation of the hemispheres. This technique will be useful in the field of translational neurorehabilitation research, for exploring the molecular mechanisms of rTMS, through the analysis of interhemispheric differences, and the various pathologic conditions.After watching this video, you should have a good understanding of how to conduct unilateral rTMS, in a small animal model.

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12.3K visualizações.  Chungnam National University Hospital, Daejeon. The overall goal of this experiment is to conduct a unilateral repetitive transcranial magnetic stimulation, or rTMS, in a small animal model. This method can help answer key questions regarding molecular mechanisms of rTMS, by enabling unilateral hemispheric stimulation to the rat brain. The main advantage of this technique is that it provides an opportunity to explore the interhemispheric differences of molecular events, in response to unilateral rTMS.Individuals new to this method m...