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

Summary

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.

Abstract

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-HT_2A) 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.

Introduction

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 system^3,4,5. 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-HT_2A 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 diseases^6,7,8, on which exercise has been proven to be therapeutically effective. 5-HT_2A 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-HT_2A receptor signaling in their prefrontal cortex (PFC) neurons^10,11. 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).

Protocol

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 magnitudes of accelerations along x-, y-, and z-axes during treadmill running

1. Anesthetize the rat with inhalation of 1.5% isoflurane.
   NOTE: The rats were used after at least 1 week of acclimation to the laboratory environment. Ensure that the rat is not responsive to a hindlimb toe pinch.
2. Fix the accelerometer (see Table of Materials) on top of the rat's head using surgical tape.
3. After complete recovery from anesthesia, place the rat in the treadmill machine (see Table of Materials), and adjust the treadmilling to a moderate velocity (20 m/min)¹² (Figure 1A).
   NOTE: It took at least 20 min to confirm the full recovery of the rat from anesthesia after the termination of isoflurane inhalation and start the treadmill experiment. Ensure that the rat is responsive to a hindlimb toe pinch, being able to walk or run without apparent staggering.
4. Measure the magnitude of vertical accelerations during rat treadmill running using the application software following the manufacturer's instructions (see Table of Materials).
   NOTE: Extract 10 serial waves and individually calculate the average accelerations along the 3-dimensional axes (x-, y-, and z-axes, Figure 1B). Peak magnitudes were quantified by defining stepping-synchronized waves (~2 Hz frequency) as treadmill running-induced accelerations (Figure 1C). Rats were used for this study as their larger body size was suitable for reliably measuring the vertical acceleration at the head, which was not possible in mice. However, mice were used for further studies because of the ease and reliability concerning the quantitative analysis of head-twitch response.

2. Adjustment of the PHM system and application of PHM to mice

1. Pre-set the amplitude of the platform's oscillation and the propeller-shaped cam's rotation speed in the PHM system (Figure 1D) so the magnitude and frequency of vertical acceleration match the values obtained in step 1.4.
   NOTE: The PHM system comprises a metal framework and wooden platform. The motor speed can be changed and controlled by adjusting the dial connected to the build-in driver (see Table of Materials). The dial scale of 600 corresponds to 2 Hz, Figure 1E. The propeller-shaped cam has four blades with 5 mm step heights (Figure 1F).
2. Anesthetize the mouse via inhalation of 1.2% isoflurane.
   NOTE: Mice were used after at least 1 week of acclimation to the laboratory environments. Ensure that the mouse is not responsive to a hindlimb toe pinch.
3. Place the mouse in a prone position with the head and the rest of the body located on the oscillatable and static platforms, respectively.
   NOTE: Keep the mouse anesthetized (1.2% isoflurane).
4. Turn the motor on to oscillate the platform vertically, and apply PHM to the mouse.
   ​NOTE: The motor speed was adjusted to oscillate the platform at 2 Hz (see step 2.1). Anesthetize and place the control mouse on the PHM platform likewise, but leave the motor off.

3. Running of the mouse on the treadmill

1. Place the mouse on the treadmill machine and adjust the treadmilling to a moderate velocity (10 m/min)¹³.

4. Quantification of mouse head-twitch response (HTR)

1. Set up the video camera (frame rate: 24 fps) to record the entire space in the transparent plastic case.
   NOTE: The plastic cage was used to keep the mouse in the field of video recording.
2. Intraperitoneally administer 5-hydroxytryptophan (5-HTP) (100 mg/kg) (see Table of Materials), the precursor to 5-HT, to a mouse.
3. Place the mouse in the transparent cage and start recording for 30 min.
4. Review the recorded video (1/2x or 1/3x speed), counting the head twitching manually.
   NOTE: The analysts were not blinded to the experimental procedure. Characteristic "tic-like" rapid motion of mouse (see Supplementary Movie 1) was counted as head twitching, which rarely occurs under normal breeding environment.

5. Immunohistochemical analysis of mouse PFC

1. Once HTR tests are completed, anesthetize the mouse by administrating the mixture of midazolam (4.0 mg/kg), butorphanol (4.0 mg/kg), and medetomidine (0.3 mg/kg), perfuse with 4% paraformaldehyde (PFA) in PBS, and then excise the brain following previously published reports^14,15.
2. Post-fix the brains in 4% PFA in PBS for an additional 24 h at 4 °C, and store in 30% sucrose/PBS until they sink. Freeze the fixed-in optimal cutting temperature compound (OCT compound, see Table of Materials).
3. Retrieve the cryo-sections of the mouse brain from the slide box (see Table of Materials). Leave the slides on clean wipes at room temperature until the samples dehydrate entirely.
   NOTE: Twenty-micrometer-thick sagittal sections (Lateral +0.5–1.5 mm) were prepared from frozen samples embedded in OCT compound using a cryostat (see Table of Materials).
4. Use a liquid blocker pen (see Table of Materials) to draw a circle around the cryo-sectioned tissue on the slide to confine the spread area of the solution (0.1% Tween-20 in Tris-buffered saline (TBS-T).
5. Place dampened wipes on the bottom of a tray holding the slides to create a moist environment.
6. After permeabilization with TBS-T, block with 4% donkey serum (see Table of Materials) at room temperature for 1 h.
7. Rinse the slides once by 5 min immersion in TBS-T.
8. Apply 100 µL of appropriately diluted primary antibody and DAPI (see Table of Materials) mix on each slide, cover the tray to avoid drying the sample, and incubate overnight at room temperature.
9. Rinse with TBS-T three times (5 min incubation each).
10. Apply 100 µL of appropriately diluted species-matched fluorescent secondary antibody (conjugated with Alexa Fluor 488, 568, or 645) (see Table of Materials) on each slide and incubate for 1 h at room temperature.
11. Rinse with TBS-T three times (5 min incubation each).
12. Mount the slides with mounting medium (see Table of Materials). Cover the slides with coverslips.
13. View the sample under a fluorescence microscope.

Results

The peak magnitude of the vertical accelerations at the rats' heads during their treadmill running at a moderate velocity (20 m/min) was approximately 1.0 × g (Figure 1C). The PHM system (Figure 1D) was set up to generate vertical acceleration peaks of 1.0 × g at the rodents' heads.

PHM application (2 Hz, 30 min/day for 7 days) to mice significantly attenuated their HTR as compared to the control mi...

Discussion

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...

Materials

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