This work was in part supported by the Intramural Research Fund from the Japanese Ministry of Health, Labour and Welfare; Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI 15H01820, 15H04966, 18H04088, 20K21778, 21H04866, 21K11330, 20K19367); MEXT-Supported Program for the Strategic Research Foundation at Private Universities, 2015-2019 from the Japanese Ministry of Education, Culture, Sports, Science and Technology (S1511017); the Naito Science &#38; Engineering Foundation. This research also received funding from the Alliance for Regenerative Rehabilitation Research &#38; Training (AR3T), which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Neurological Disorders and Stroke (NINDS), and National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health under Award Number P2CHD086843.

All animal experiments were approved by the Institutional Animal Care and Use Committee of National Rehabilitation Center for Persons with Disabilities. 8-9 weeks old male Sprague-Dawley rats were used to measure accelerations at the head during treadmill running and PHM. 9-10 weeks old male C57BL/6 mice were used for behavior tests and histological analyses of the PFC. The animals were obtained from commercial sources (see Table of Materials).
1. Measurement of magnitud...

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The authors declare that there is no competing interest associated with the work described in this paper.

Using the developed PHM application system, we have shown that 5-HT signaling in their PFC neurons is mechanically regulated. Because of the complexity of exercise effects, it has been difficult to precisely dissect the consequence of exercise in the context of health promotion. The focus is on mechanical aspects to preclude the involvement or contribution of metabolic events that may occur with or subsequently to exercise activities, such as energy consumption. The method described here is expected to be more broadly us...

Exercise is beneficial to treat or prevent several physical disorders, including lifestyle diseases such as diabetes mellitus and essential hypertension1. Related to this, evidence has also been accumulated regarding the positive effects of exercise on brain functions2. However, molecular mechanisms underlying the benefits of exercise for the brain remain primarily unelucidated. Most physical activities and workouts generate mechanical accelerations at the head, at least to some extent. Whereas various physiological phenomena are mechanically regulated, the importance of mechanical loading has, in most cases, been documented in musculoskeletal system3,4,5. Although the brain is also subjected to mechanical forces during physical activities, particularly so-called impacting exercises, mechanical regulation of physiological brain function has rarely been studied. Because the generation of mechanical accelerations at the head is relatively common to physical workouts, it has been speculated that mechanical regulation might be implicated in the benefits of exercise to brain functions.
5-HT2A receptor signaling is essential in regulating emotions and behaviors among various biochemical signals that function in the nervous system. It is involved in multiple psychiatric diseases6,7,8, on which exercise has been proven to be therapeutically effective. 5-HT2A&#160;receptor is a subtype of 5-HT2 receptor that belongs to the serotonin family and is also a member of the G-protein-coupled receptor (GPCR) family, the signaling of which is modulated by its internalization, either ligand-dependent or -independent9. Head twitching is a characteristic behavior of rodents, the quantity (frequency) of which explicitly represents the intensity of 5-HT2A receptor signaling in their prefrontal cortex (PFC) neurons10,11. Taking advantage of the strict specificity of this hallucinogenic response to administrated 5-HT (head-twitch response, hereafter referred to as HTR; see Supplementary Movie 1), the hypothesis mentioned above on mechanical implications in exercise effects on brain functions was tested. Thus, we analyzed and compared the HTR of mice subjected to either forced exercise (treadmill running) or exercise-mimicking mechanical intervention (PHM).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-hydroxytryptophan (5-HTP)</td>
			<td>Sigma-Aldrich</td>
			<td>H9772</td>
			<td>Serotonin (5-HT) precursor</td>
		</tr>
		<tr>
			<td>Brushless motor driver</td>
			<td>Oriental motor</td>
			<td>BMUD30-A2</td>
			<td>Speed changer build-in motor driver</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Oriental yeast company</td>
			<td>C57BL/6J</td>
			<td>Mice used in this study</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM33050S</td>
			<td>Microtome to cut frozen samples</td>
		</tr>
		<tr>
			<td>DC Motor</td>
			<td>Oriental motor</td>
			<td>BLM230-GFV2</td>
			<td>Motor</td>
		</tr>
		<tr>
			<td>Donkey anti-goat Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A-11057</td>
			<td>Secondary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>A-31571</td>
			<td>Secondary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-21206</td>
			<td>Secondary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma-Aldrich</td>
			<td>S30-100ML</td>
			<td>Blocker of non-specific binding of antibodies in immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Keyence</td>
			<td>BZ-9000</td>
			<td>Fluorescence microscope</td>
		</tr>
		<tr>
			<td>Goat polyclonal anti-5-HT2A receptor</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-15073</td>
			<td>Primary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Pfizer</td>
			<td>v002139</td>
			<td>Inhalation anesthetic</td>
		</tr>
		<tr>
			<td>KimWipe</td>
			<td>NIPPON PAPER CRECIA</td>
			<td>S-200</td>
			<td>Paper cloth for cleaning surfaces, parts, instruments in labratory</td>
		</tr>
		<tr>
			<td>Liquid Blocker</td>
			<td>Daido Sangyo</td>
			<td>PAP-S</td>
			<td>Marker used to make the slide surface water-repellent</td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-NeuN (clone A60)</td>
			<td>EMD Millipore (Merck)</td>
			<td>MAB377</td>
			<td>Primary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>NinjaScan-Light</td>
			<td>Switchscience</td>
			<td>SSCI-023641</td>
			<td>Accelerometer to measure accelerations</td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Sakura Finetek</td>
			<td>45833</td>
			<td>Embedding agent for preparing frozen tissue sections</td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant</td>
			<td>Invitrogen</td>
			<td>P36934</td>
			<td>Mounting medium to prevent flourscence fading</td>
		</tr>
		<tr>
			<td>Rabbit polyclonal anti-c-Fos</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-52</td>
			<td>Primary antibody used for immunohistochemical staining</td>
		</tr>
		<tr>
			<td>Slide box</td>
			<td>AS ONE</td>
			<td>03-448-1</td>
			<td>Opaque box to store slides</td>
		</tr>
		<tr>
			<td>Spike2</td>
			<td>Cambridge electronic design limited (CED)</td>
			<td>N/A</td>
			<td>Application software used to analyze acceleration</td>
		</tr>
		<tr>
			<td>Sprague-Dawley rats</td>
			<td>Japan SLC</td>
			<td>Slc:SD</td>
			<td>Rats used in this study</td>
		</tr>
		<tr>
			<td>Treadmill machine</td>
			<td>Muromachi</td>
			<td>MK-680</td>
			<td>System used in experiments of forced running of rats and mice</td>
		</tr>
	</tbody>
</table>

application of passive head motion to generate defined accelerations at the heads of rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The peak magnitude of the vertical accelerations at the rats&#39; heads during their treadmill running at a moderate velocity (20 m/min) was approximately 1.0 &#215; g (Figure 1C). The PHM system (Figure 1D) was set up to generate vertical acceleration peaks of 1.0 &#215; g at the rodents&#39; heads.
PHM application (2 Hz, 30 min/day for 7 days) to mice significantly attenuated their HTR as compared to the control mi...

Watch this Scientific Journal Video about Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents at JoVE.com

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Our protocol helps answer the questions of how physical exercise supports organism homeostasis. The systems allowed us to dissect direct mechanical effects on the brain from complex consequences of physical activities such as walking and jogging. To begin, fix the accelerometer on top of the head of a rat using surgical tape.After complete recovery from anesthesia, place the rat in the treadmill machine. Adjust the treadmill to a moderate velocity at 20 meters per minute. Use the application software to measure the magnitude of vertical accelerations along X, Y, and Z axis when the rat is running the treadmill.Match the values obtained from the software to the magnitude in frequency of vertical acceleration in the PHM system. Preset the amplitude of oscillation of the platform and the rotation speed of the propeller shaped cam. Then place the mouse in a prone position with the head located on the oscillatable and the rest of the body on the static platforms.Turn on the motor to oscillate the platform vertically and apply PHM to the mouse. In the transparent plastic case, set up the video camera at a frame rate of 24 FPS to record the entire space. Next, intraperitoneally administer five hydroxytryptophan to a mouse.Place the mouse in the transparent cage and start recording for 30 minutes. Review the recorded video at 0.5x speed and count the head twitching manually. Retrieve the cryosections of the mouse brain from the slide box.Leave the slides on clean wipes at room temperature until the samples dehydrate entirely. Use a liquid blocker pen to draw a circle around the cryosectioned tissue. Then add TBS-T solution.On the bottom of a tray, place dampened wipes to create a moist environment for the slides. After a permeablization with TBS-T, add 4%donkey serum for blocking and incubate at room temperature for one hour. Rinse the slides once by immersion in TBS-T for five minutes.Then apply 100 microliters of appropriately diluted primary antibody and dappy mix on each slide. Cover the tray and incubate at room temperature overnight. After incubation, rinse with TBS-T thrice for five minutes each.Apply 100 microliters of appropriately diluted species matched fluorescent secondary antibody and incubate at room temperature for one hour. Afterrinse with TBS-T thrice for five minutes each, mount the slides with mounting medium and cover the slides with cover slips. After mounting, view the sample under a fluorescence microscope.The PHM system was adjusted to produce vertical acceleration peaks at approximately 1.0 times G which was equivalent to those of the heads of rats during treadmill running. PHM application to mice significantly attenuated their head twitch response as compared to the control mice representing a suppressive effect of PHM on 5-HT2A receptor signaling in the prefrontal cortex neurons. Treadmill running in PHM significantly enhanced 5-HT2A receptor internalization and mouse prefrontal cortex neurons.Quantification of internalized and membrane associated 5-HT2A receptor positive areas relative to the new and positive area confirmed this observation. Immunosstaining and quantification of c-Fos expression in 5-HT2A receptor positive neurons showed that treadmill running in PHM downregulated 5-HTP induced c-Fos expression. This is the downstream cellular event of 5-HT2A receptor activation and mouse PFC neurons.Mechanical intervention can be applied to the body parts other than the head to investigate the mechanical aspects of exercise effects.

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1.6K 조회수.  National Rehabilitation Center for Persons with Disabilities. 우리의 프로토콜은 신체 운동이 유기체 항상성을 지원하는 방법에 대한 질문에 답하는 데 도움이됩니다. 이 시스템을 통해 걷기 및 조깅과 같은 신체 활동의 복잡한 결과로부터 뇌에 대한 직접적인 기계적 영향을 해부 할 수있었습니다. 시작하려면 수술 용 테이프를 사용하여 쥐의 머리 위에 가속도계를 고정하십시오.마취에서 완전히 회복 된 후, 쥐를 러닝 머신 기계?...