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 magnitudes of accelerations along x-, y-, and z-axes during treadmill running
<ol>
	<li>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.</li>
	<li>Fix the accelerometer (see Table of Materials) on top of the rat&#39;s head using surgical tape.</li>
	<li>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.</li>
	<li>Measure the magnitude of vertical accelerations during rat treadmill running using the application software following the manufacturer&#39;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.</li>
</ol>
2. Adjustment of the PHM system and application of PHM to mice
<ol>
	<li>Pre-set the amplitude of the platform&#39;s oscillation and the propeller-shaped cam&#39;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).</li>
	<li>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.</li>
	<li>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).</li>
	<li>Turn the motor on to oscillate the platform vertically, and apply PHM to the mouse. 
	&#8203;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.</li>
</ol>
3. Running of the mouse on the treadmill
<ol>
	<li>Place the mouse on the treadmill machine and adjust the treadmilling to a moderate velocity (10 m/min)13.</li>
</ol>
4. Quantification of mouse head-twitch response (HTR)
<ol>
	<li>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.</li>
	<li>Intraperitoneally administer 5-hydroxytryptophan (5-HTP) (100 mg/kg) (see Table of Materials), the precursor to 5-HT, to a mouse.</li>
	<li>Place the mouse in the transparent cage and start recording for 30 min.</li>
	<li>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 &#34;tic-like&#34; rapid motion of mouse (see Supplementary Movie 1) was counted as head twitching, which rarely occurs under normal breeding environment.</li>
</ol>
5. Immunohistochemical analysis of mouse PFC
<ol>
	<li>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 reports14,15.</li>
	<li>Post-fix the brains in 4% PFA in PBS for an additional 24 h at 4 &#176;C, and store in 30% sucrose/PBS until they sink. Freeze the fixed-in optimal cutting temperature compound (OCT compound, see Table of Materials).</li>
	<li>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&#8211;1.5 mm) were prepared from frozen samples embedded in OCT compound using a cryostat (see Table of Materials).</li>
	<li>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).</li>
	<li>Place dampened wipes on the bottom of a tray holding the slides to create a moist environment.</li>
	<li>After permeabilization with TBS-T, block with 4% donkey serum (see Table of Materials) at room temperature for 1 h.</li>
	<li>Rinse the slides once by 5 min immersion in TBS-T.</li>
	<li>Apply 100 &#181;L 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.</li>
	<li>Rinse with TBS-T three times (5 min incubation each).</li>
	<li>Apply 100 &#181;L 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.</li>
	<li>Rinse with TBS-T three times (5 min incubation each).</li>
	<li>Mount the slides with mounting medium (see Table of Materials). Cover the slides with coverslips.</li>
	<li>View the sample under a fluorescence microscope.</li>
</ol>

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E., 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=Treadmill+exercise+prevents+aging-induced+failure+of+memory+through+an+increase+in+neurogenesis+and+suppression+of+apoptosis+in+rat+hippocampus.">Treadmill exercise prevents aging-induced failure of memory through an increase in neurogenesis and suppression of apoptosis in rat hippocampus.</a> Experimental Gerontology. 45 (5), 357-365 (2010).</li><li>Li, H., 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=Regular+treadmill+running+improves+spatial+learning+and+memory+performance+in+young+mice+through+increased+hippocampal+neurogenesis+and+decreased+stress.">Regular treadmill running improves spatial learning and memory performance in young mice through increased hippocampal neurogenesis and decreased stress.</a> Brain Research. 1531, 1-8 (2013).</li><li>Gonz&#225;lez-Maeso, J., 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=Hallucinogens+recruit+specific+cortical+5-HT2A+receptor-mediated+signaling+pathways+to+affect+behavior.">Hallucinogens recruit specific cortical 5-HT2A receptor-mediated signaling pathways to affect behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Ryu, Y., 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=Mechanical+regulation+underlies+effects+of+exercise+on+serotonin-induced+signaling+in+the+prefrontal+cortex+neurons.">Mechanical regulation underlies effects of exercise on serotonin-induced signaling in the prefrontal cortex neurons.</a> iScience. 23 (2), 100874 (2020).</li><li>Shefer, G., Rauner, G., Stuelsatz, P., Benayahu, D., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate-intensity+treadmill+running+promotes+expansion+of+the+satellite+cell+pool+in+young+and+old+mice.">Moderate-intensity treadmill running promotes expansion of the satellite cell pool in young and old mice.</a> FEBS Journal. 280 (17), 4063-4073 (2013).</li><li>Wang, J., 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=Moderate+exercise+has+beneficial+effects+on+mouse+ischemic+stroke+by+enhancing+the+functions+of+circulating+endothelial+progenitor+cell-derived+exosomes.">Moderate exercise has beneficial effects on mouse ischemic stroke by enhancing the functions of circulating endothelial progenitor cell-derived exosomes.</a> Experimental Neurology. 330, 113325 (2020).</li><li>Pac&#225;k, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressor-specific+activation+of+the+hypothalamic-pituitary-adrenocortical+axis.">Stressor-specific activation of the hypothalamic-pituitary-adrenocortical axis.</a> Physiological Research. 49, 11-17 (2000).</li><li>Okamoto, M., Soya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+exercise+model+for+enhancement+of+hippocampal+neurogenesis:+A+possible+candidate+for+promotion+of+neurogenesis.">Mild exercise model for enhancement of hippocampal neurogenesis: A possible candidate for promotion of neurogenesis.</a> The Journal of Physical Fitness and Sports Medicine. 1 (4), 585-594 (2012).</li></ol>

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...

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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 ...

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1.6K Views.  National Rehabilitation Center for Persons with Disabilities. 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

Intracavernosal Pressure Recording to Evaluate Erectile Function in Rodents

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. Rodents are employed by many studies to research the physiology/pathology of erectile function. Erectile function in rodents can be evaluated by measuring the intracavernosal pressure (ICP). In practice, ICP can be monitored following electrical stimulation of the cavernous nerves (CNs). The arterial pressure of the carotid artery (the mean arterial pressure) is used as the reference for ICP. Using ICP recording protocols, many key parameters of erectile function can be measured from the ICP response curve. The ICP measurement provides more information than the apomorphine-induced penile erection test, and is cheaper than telemetric monitoring of the corpus spongiosum penis, making this method the most popular one to evaluate erectile function. However, compared to the easily-performed APO-induced erectile function test, successful ICP recordings require attention to detail, practice, and adherence to the operation method. In this work, an introduction to ICP recording in rats is provided to complement the procedure efficiently.

Intracavernosal pressure recording (ICP) is an important method to evaluate the erectile function of experimental animals. Here, a detailed protocol is demonstrated for the recording procedure of ICP by catheterizing the crura penis and then electrically stimulating the cavernous nerves in rats.

Erectile dysfunction (ED) is defined as the inability to attain or keep an erection of the penis, and this has become a prevalent male sexual disorder. ...

Diagonal Method to Measure Synergy Among Any Number of Drugs

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many doses, allow sensitive measurement of drug interactions. However, these assays are costly and do not scale well for measuring interaction among many drugs. Several recent studies have reported drug interaction measurements using a diagonal sampling of the traditional checkerboard assay. This alternative methodology greatly decreases the cost of drug interaction experiments and allows interaction measurement for combinations with many drugs. Here, we describe a protocol to measure the three pairwise interactions and one three-way interaction among three antibiotics in duplicate, in five days, using only three 96-well microplates and standard laboratory equipment. We present representative results showing that the three-antibiotic combination of Levofloxacin + Nalidixic Acid + Penicillin G is synergistic. Our protocol scales up to measure interactions among many drugs and in other biological contexts, allowing for efficient screens for multi-drug synergies against pathogens and tumors.

In this protocol, we describe how to make Loewe additivity-based drug interaction measurements for pairwise and three-way drug combinations.

A synergistic drug combination has a higher efficacy compared to the effects of individual drugs. Checkerboard assays, where drugs are combined in many ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...