We wish to thank the animal care personnel at the University at Buffalo Animal Laboratory Animal Facility. This research was supported by a Veteran Affairs Rehabilitation Research and Development Grant RX001066 and the Indian Trail Foundation.

All studies and experimental protocols were approved by and in compliance with guidelines of the University at Buffalo and VA Western New York Animal Care and Use Committees.
1. Experiment Setup and General Advice
NOTE: A total of twenty-four female mice on a C57BL/6J background were used in this protocol starting at 23&#160;months of age. The mice carried&#160;a conditional SIRTloxp-exon4-loxp mutation29, however, this was not induced in this experiment.
<ol>
	<li>Ensure that mice receive permanent identifiers such as ear tags, RFID chips, or tail tattoos. 
	NOTE: It is recommended to also use temporary markings (e.g., permanent marker to mark tails) for quick identification during assessment periods.</li>
	<li>Clean the treadmill with 0.25-0.5% bleach (v/v) or 70% (v/v) ethanol at the end of the day or to remove feces or urine between experimental trials. Dry solutions completely before initiating a new trial. 
	NOTE: Ethanol may increase wear to the treadmill belt. It is recommended that the treadmill be cleaned between each run if working with group-housed male mice in order to minimize in cage fighting. It is recommended to perform treadmill assessments at the same time of day at each time point in longitudinal studies30,31,32, and if other assessments are being performed, the order should remain the same. The investigator running the experiment should also be blind to the group designations of the mice.</li>
</ol>
2. Acclimatizing Mice to Treadmill Apparatus
NOTE: Initiate acclimatization of mice 1&#160;month prior to baseline experiments.
<ol>
	<li>Set up the initial training program using the treadmill software (v3.4.7) in Manual mode (Figure 1 and Table 1).
	<ol>
		<li>Open the treadmill software. Then, click on the file and open the experiment.</li>
		<li>Input the values indicated in Table 1 on the Manual tab (Figure 1) under Acclimation.</li>
		<li>Input the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20. 
		NOTE: Shock intensity for this initial run is set at level 1 (0.46&#160;mA).</li>
	</ol>
	</li>
	<li>Set the inclination of the treadmill to 0&#176; (flat).</li>
	<li>Hold mice by the tail when placing in the treadmill and place mice directly on the belt to avoid starting mice on the shock grid.</li>
	<li>Use a brush or tongue depressor to keep mice away from the shock grid when training or assessment begins. Nudge mice to begin running to avoid unintended shocks.</li>
	<li>After each mouse is given the initial training program, repeat the program as above with the shock intensity increased to level 2 (0.59&#160;mA) with at least 15&#160;min between trainings.</li>
	<li>One to two days after the initial training program administer the uphill treadmill acclimatization.
	<ol>
		<li>Open the treadmill software. Then click on the file and then open the experiment.</li>
		<li>Input the values on the Basic tab as indicated in Table 2 under Acclimation.</li>
		<li>Under Shock Detection, input the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20.</li>
		<li>Click on the Profile Mode tab (Figure 2) and for Step 1, input a start speed of 0&#160;m/min and an end speed of 5&#160;m/min with a period of 5&#160;s.
		<ol>
			<li>Add a warm up step 2 with a start speed of 5&#160;m/min and end speed of 5&#160;m/min for 30&#160;seconds. Add a transition step that starts at 5&#160;m/min and an end speed of 6&#160;m/min for 5&#160;s.</li>
			<li>Add a test speed step that starts at 6&#160;m/min and ends at 6&#160;m/min for 20&#160;s. Add a transition step that starts at 6&#160;m/min and ends at 5&#160;m/min.</li>
			<li>Add a recovery interval that starts at 5&#160;m/min and ends at 5&#160;m/min for 20&#160;s (Figure 2).</li>
		</ol>
		</li>
		<li>Repeat step 2.6.4 to add test speed steps as indicated in Table 2 under acclimation.</li>
	</ol>
	</li>
	<li>Two weeks prior to baseline assessments, continue acclimatization by providing two treadmill flat aerobic capacity assessments as described in section 3, given on consecutive days.</li>
	<li>One to two days after the second endurance training, provide two treadmill uphill anaerobic capacity assessments as described in section 4, given on the same day with a minimum of 30&#160;min between trainings.</li>
</ol>
3. Flat Continuous Treadmill Assessment
<ol>
	<li>Create a treadmill program as described in step 2.1, using values indicated in Table 1 under Assessment. Under Shock Detection, set the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 10, and the number of shocks as 20.</li>
	<li>Monitor the mice throughout the trial and remove mice from the instrument that reach the endpoint criteria. 
	NOTE: This step is to avoid mice inadvertently touching the shock grid of neighboring mice and affecting data. Parameters include the total time on belt, the distance travelled, and the shocks to visits ratio. It is recommended to perform two assessments at each time point, separated by at least one day between assessments.</li>
</ol>
4. Uphill Interval Treadmill Assessment
<ol>
	<li>Create a treadmill program as described in step 2.6, using values indicated in Table 2 under Assessment. Under Shock Detection, set the session number as 1, the number of active channels as 1 to 6 depending on the number of mice, the number of visits to the grid as 5, and the number of shocks as 10.</li>
	<li>Set the interval field in the treadmill program to 0.5. 
	NOTE: This allows for data points to be collected every second instead of every minute, which aids in identifying the speed at endpoint for each mouse. The program will automatically stop after the 50&#160;m/min stage due to software limitations. Parameters include the time on belt, the distance travelled, and the test speed of the last successfully completed stage before endpoint. The latter is used for determining baseline intensity for the HIIT regimen described in step 5. It is recommended to perform two assessments per time point, separated by at least 30&#160;min.</li>
</ol>
5. Short session High Intensity Interval Training
<ol>
	<li>Set the treadmill uphill (25&#176;) and remove the plastic cover.</li>
	<li>Determine the intensity for each mouse. Use the speed of the last successfully completed stage (Step 4.5) and find the intensity group and corresponding Base, Sprint, and Dash speeds using Table 3.</li>
	<li>Open the treadmill software and click on file to create a new program.
	<ol>
		<li>Click on the Profile Mode tab and for Step 1, input a start speed of 0&#160;m/min and an end speed as indicated under Base speed as indicated in Table 3 with a period of 5&#160;s.</li>
		<li>Add a warm up step 2 with a start speed at Base speed and end speed at Base speed for 180&#160;s.</li>
		<li>Input 3 intervals each with 1) a transition step that goes from Base to Sprint for 5&#160;s, 2) an step at Sprint speed for 60s, 3) a transition step that goes from &#8220;sprint&#8221; to Base over 5&#160;s, and 4) a step for a recovery interval at Base speed for 60&#160;s.</li>
		<li>Add a transition step that goes from Base to Sprint speed over 5&#160;s and a final step that goes from Sprint to Dash speed over 60&#160;s. 
		NOTE: Programs can be saved, modified, and reloaded.</li>
	</ol>
	</li>
	<li>Perform exercise three days a week with at least one day of rest between sessions (i.e., Monday, Wednesday, Friday).</li>
	<li>Rewrite exercise programs to increase Base, Sprint, and Dash speeds 1&#160;m/min every two weeks. 
	NOTE: No electric shocks are given during the exercise.</li>
	<li>Motivate mice to run using a makeup brush (recommended) or tongue depressor to lightly motivate mice that fall near or on the shock grid. If a mouse does not respond retry in 5&#160;s, then 10&#160;s, and then retry every 30&#160;s or during a recovery interval until the end of the session. 
	NOTE: consider removing mice from the study that do not run on the treadmill at any intensity or are unable to complete the first three intervals at the intensity defined for the lowest intensity group (Table 3)</li>
	<li>Move mice that cannot be motivated to complete the first three intervals in two consecutive exercise sessions to lower intensity groups (Table 3).</li>
	<li>Move mice that do not require motivation in two consecutive exercise sessions to higher intensity groups (Table 3).</li>
	<li>Take sedentary control mice in cages place beside the treadmill as it runs. Alternatively, sedentary controls can be placed in the lanes of a non-moving treadmill for 10&#160;min.</li>
</ol>

The authors have nothing to disclose.

Benefits from short sessions are a key aspect of high intensity interval training that captures scientific and public interest. However, animal studies rarely investigate HIIT regimens that are 10 min or less. Here, we describe a protocol for a 10 min short session HIIT uphill treadmill exercise regimen that enhanced treadmill performance in aged female mice and which we previously have shown to increase physical performance in aged male mice17. A strength of our protocol is that in addition to the protocol being only 10 min in length, the design is modular such that the number of intervals can be added or removed to make protocols that are even one min in length. Additionally, the length of the intervals can be modified providing multiple strategies to titrate exercise and evaluate the impacts.
Another strength of this protocol is that we include a system to tailor the exercise regimen to the physical capabilities of individual mice, which to our knowledge only one other study33 aside from our report in aged male mice17 has used. Although individually tailoring an exercise intervention introduces variability into the experimental design, an important advantage of this method is that it better models the administration of prescribed exercise in human clinical settings. Furthermore, animals do not train at intensities that are too easy or difficult, which could be a significant factor in experiments where the animal population exhibits diversity in exercise capacity (i.e. during aging). One other advantage of this methodology is that it does not require the use of metabolic treadmills to determine VO2max as the exercise intensity is tailored to the performance of the animal, a concept that is further examined by Picoli et al.34.
This protocol includes two assessments for treadmill performance, in the form of a slowly accelerating flat treadmill test and an inclined treadmill incrementing interval test, respectively. There are multiple protocols that have been published for determining continuous treadmill performance in mice using treadmills16,34,35, including a JoVE article36. Multiple parameters differ across these studies and across the literature, including: inclination (usually 0&#176; or 5&#176; for endurance testing), shock intensity (ranging from 0.25-1.12 mA), and rules for defining exhaustion. It was also noticed that 5 consecutive shocks were commonly used to define exhaustion across multiple studies. Although this strategy may induce greater exhaustion than the rules we have used in our study and this protocol, this system also assumes all mice have similar pain thresholds, which may not be true depending on experimental conditions. Our definition of exhaustion of 10 visits or 20 total shocks provides a framework to assess if there are different pain thresholds. Ultimately, there are advantages and disadvantages to both strategies and the decision for defining the endpoint should be best aligned with the goals of the study. Additionally, some treadmill assessment protocols for mice have been designed without the use of shocks as a stimulus37. Although there are advantages to this approach37, some drawbacks to consider include the use of a determination of exhaustion that is subjective and the potential difficulty of assessing multiple mice simultaneously.
Additionally, others have used work and power as parameters to describe treadmill performance35,38,39. The speed of the belt and rate of acceleration are also diversely represented in the literature. As most protocols feature increasing speed, the result will yield a blend of aerobic and anaerobic capacity in the mouse, with slower protocols providing more focus on the aerobic component. We therefore designed our uphill treadmill interval test to provide greater focus on the anaerobic component17. To achieve this, our protocol features short test intervals with active recovery between tests, allowing for mice to achieve higher speeds and therefore greater anaerobic utilization relative to our flat continuous treadmill endurance assessment. For our protocol we used an active recovery period of 20 s between intervals, the half time for reoxygenation of muscle tissue in human tissue40. However, we note a limitation of this method is that the specific impacts on anaerobic metabolism have yet to be elucidated, and including an examination of anaerobic parameters such as glycolytic metabolism, phosphocreatine utilization, and lactate kinetics would strengthen this method. In addition, new studies that investigate the impacts of changing stage and active rest durations, inclination, and different speed increments would also strengthen this method.
The methodology described in this protocol, including the two treadmill assessments, are designed for older animals in particular, and as such includes greater time for acclimation of mice to the protocols. Proper acclimation is a critical step to experimental design to ensure endpoints are due to exhaustion and not insufficient learning by the mouse. Improper acclimation is likely apparent in longitudinal studies - yet, importantly, may not be noticed in crosssectional studies. Although, shorter acclimation protocols might be possible for younger cohorts, in our experience older mice require greater acclimation and a published protocol for exercise training in aged rats initiates acclimation 1 month prior with 10 total days given for treadmill acclimatization41. However, we agree with Castro et al. that providing excessive acclimation may impact the behavior of and impacts on the mice35 and his recommendation of 3 to 10 days total35, for which our acclimatization protocol is in line with these recommendations. Furthermore, older animals also exhibit greater diversity in exercise performance and for this reason we include two trials per assessment time point. Younger animals display greater homogeneity in performance and therefore a single trial may be sufficient. Furthermore, the diversity of aged animals may make direct cross-sectional comparisons of an intervention difficult to interpret, and more success may be achieved by comparing the change from baseline as was done in the analysis of the aged female mice. We also indicate as a caveat that we have not tested our short session HIIT protocol in younger mice, where it is possible a ceiling effect in performance may mask the benefits of our short session HIIT protocol. The protocol was also used only in the C57BL/6J mouse strain, and therefore the impact of this exercise in other mouse strains remains to be elucidated. Additionally, the C57BL6/J mice used in this experiment carried&#160;a SIRT1loxp-exon4-loxp mutation29, that was not induced. These mice were aged in house from birth; however, insufficient numbers were available at 24 months of age to power 4 groups. We therefore focused the experiment on just HIIT and sedentary groups. We note that both the present cohort of aged female mice and our previously published cohort of aged male mice17 both improved in physical performance using this short session HIIT protocol.

Regular exercise is effective at preventing or delaying many age-associated diseases such as sarcopenia and frailty1,2,3,4. However, less than 15% of those 65 and older meet recommendations of 150 min a week of moderate intensity exercise plus strength 
training5,6. As the lack of time and lengthy sessions are common barriers to exercise, high intensity interval training (HIIT) is emerging as an alternative to traditional regimens. HIIT features multiple short bursts of intense activity that are interspersed with brief periods of active recovery, and there has been recent interest in identifying the shortest regimens that still yield beneficial outcomes. Such studies include 3 day a week regimens featuring total session times of 4 min7, 2-3 min8, 1.5 min9, a single min10, and even 40 s11.
Likewise, there has been substantial interest in HIIT animal models. A majority of studies used mice12,13,14,15,16,17,18,19,20,21 or rats22,23,24,25 and were performed using a treadmill, although a few others used swimming protocols26,27,28. A majority of these studies use VO2max to set the initial intensity of the exercise13,14,19,21,24. Additionally, although an often described benefit of HIIT is having shorter regimens, almost all of these identified studies feature regimens that last 30&#160;min or longer11,12,13,14,15,18,19,21, with the exception of one with a slightly longer than 10&#160;min regimen20, and another with 19 min across three different intensities16. To our knowledge, there are no reported animal studies that examine a 10&#160;min or less HIIT regimen, or tailor the regimen to individual animals, with the exception of our published study17 that serves as the basis for this protocol.
Here, we describe a protocol for HIIT in aged mice designed to model individualized, short session (&#8804;10&#160;min) variants used recently in human studies7,8,9,10,11. The method includes a 10 min regimen on an inclined (25&#176;) treadmill with a 3 min warm up, and four 1 min intervals at high intensity, interspersed with three 1 min active recovery segments. Advantages of the protocol include greater clinical relevance as it features strategies for tailoring intensity to individual animals, setting intensities that are not based on VO2max, thereby avoiding the need for metabolic treadmills, and modular design whereby the number of intervals and timings are easily adjustable. Additionally, within this protocol we provide instructions for two strategies for treadmill assessment, which include flat continuous and uphill interval, to examine endurance. Using these methods, we extend our previous findings that short session HIIT increased functional capacity in aged male mice17, and now demonstrate HIIT increases treadmill performance in aged female mice.

<table border="0" cellpadding="0" cellspacing="0" style="width:1076px;" width="1077">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Exer-3/6 Open Treadmill w/ Shock, Detection, auto-calibration and PC Interface/Software</td>
			<td style="width:119px;">Columbus Instruments</td>
			<td style="width:119px;">1055-SDRM</td>
			<td style="width:623px;">The Columbus Instruments 3/6 treadmill allow up to 6 mice or 3 rats simultaneously. 
			The device comes with controllers to allow manual control of treadmill belt speed and shock intensity, or connections to a computer and software to run and control these elements.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach</td>
			<td style="width:119px;">Varies</td>
			<td style="width:119px;">Varies</td>
			<td>0.25-0.5% Bleach solution (V/V) is used to clean the treadmill belt between sessions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:119px;">Varies</td>
			<td style="width:119px;">Varies</td>
			<td>70% ethanol solution (V/V) can alternatively be used to clean treadmill belt between runs and sesions.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Make-up Brush (large)</td>
			<td style="width:119px;">Varies</td>
			<td style="width:119px;">Varies</td>
			<td>A make-up brush provides a soft surface and ample length to motivate mice to continue exercise.</td>
		</tr>
	</tbody>
</table>

short session high intensity interval training and treadmill assessment in aged mice

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, with regimens less than or equal to 10 min is of particular interest as several human studies feature routines as short as a few minutes a couple times a week. However, there is a paucity of animal studies that model the impacts of short session HIIT. Here, we describe a methodology for an individually tailored and progressive short session HIIT regimen of 10 min given 3 days a week for aged mice using an inclined treadmill. Our methodology also includes protocols for treadmill assessment. Mice are initially acclimatized to the treadmill and then given baseline flat and uphill treadmill assessments. Exercise sessions begin with a 3 min warm-up, then three intervals of 1 min at a fast pace, followed by 1 min at an active recovery pace. Following these intervals, the mice are given a final segment that starts at the fast pace and accelerates for 1 min. The HIIT protocol is individually tailored as the speed and intensity for each mouse are determined based upon initial anaerobic assessment scores. Additionally, we detail the conditions for increasing or decreasing the intensity for individual mice depending on performance. Finally, intensity is increased for all mice every two weeks. We previously reported in this protocol enhanced physical performance in aged male mice and here show it also increases treadmill performance in aged female mice. Advantages of our protocol include low administration time (about 15 min per 6 mice, 3 days a week), strategy for individualizing for mice to better model prescribed exercise, and a modular design that allows for the addition or removal of the number and length of intervals to titrate exercise benefits.

A total of twenty-five female mice were bred and aged in house. The C57BL/6J background mice carried&#160;a SIRT1loxp-exon4-loxp mutation29; however, this&#160;conditional knockout was not induced and therefore all mice exhibited full length Sirtuin1 (data not shown). At 24 months of age, mice were assessed for treadmill endurance and uphill sprint capacity prior to and after the administration of two months of HIIT exercise (n = 14), or remaining cage sedentary (n = 11). Our data show all 14 mice in the HIIT group increased treadmill time on belt compared to 7 of 11 of the SED mice (Figure 4A, paired student&#39;s T-Test p &#60; 0.0001 for HIIT and p &#60; 0.14 for SED). In total HIIT mice exhibited greater improvement in time on belt based on the better of two trials (HIIT: 18.2 &#177; 10.5 min to 31.8 &#177; 13.7 min, delta 13.6 &#177; 7.5 min versus SED: 19.9 &#177; 10.0 min to 25.3 &#177; 7.3 min, delta 5.3 &#177; 11.4 min, unpaired student&#39;s T-test p &#60; 0.0391).
Additionally, we detected greater increase in uphill treadmill capacity as 12 of 14 HIIT mice increased maximal speed while we observed decline in 8 of 11 SED mice (Figure 4B, paired student&#39;s T-Test p &#60; 0.0022 for HIIT and p &#60; 0.85 for SED). HIIT mice also demonstrated greater improvement in maximal speed compared to SED mice based on the better of two trials (HIIT: 14.6 &#177; 4.3 m/min to 17.6 &#177; 5.5 m/min, delta 3.0 &#177; 3.0 m/min versus SED: 16.9 &#177; 3.8 m/min to 16.5 &#177; 5.0 m/min, delta 0.5 &#177; 5.0 m/min, unpaired student&#39;s T-test p = 0.0441). Shock tolerance was similar between the two groups of mice (Shock to grid visit ratio baseline - SED: 1.2 &#177; 0.1 versus HIIT: 1.4 &#177; 0.4, p=0.24; endpoint - SED: 1.2 &#177; 0.4 versus HIIT: 1.3 &#177; 0.4, p=0.46).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59138/59138fig1.jpg" /> 
Figure 1: Treadmill software manual mode parameters for treadmill endurance assessment. The treadmill software manual mode allows adjustment for protocols involving continuous belt acceleration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59138/59138fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59138/59138fig2.jpg" /> 
Figure 2: Treadmill software profile mode program parameters for uphill sprint assessment. The treadmill software profile mode, used in conjunction with the options on the manual mode tab (Figure 1), allows the ability to create protocols with custom speed intervals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59138/59138fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59138/59138fig3.jpg" /> 
Figure 3: High Intensity Interval Training regimen schematic. Base, Sprint, and Dash speeds are indicated in Table 3. Speeds increase by 1 m/min every two weeks. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59138/59138fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59138/59138fig4.jpg" /> 
Figure 4. Impacts of HIIT on treadmill performance in aged mice. Twenty-four month old female mice were given HIIT (n=14) or remained sedentary (SED, n=11) for 2 months. Treadmill endurance (A) was assessed before and after the exercise intervention as a best of two trials given on consecutive days on a flat treadmill. Uphill sprint capacity (B) was assessed as the best of two trials administered with no less than 30 min of rest on an inclined (25&#176;) treadmill. Lines indicate individual mice. B is baseline and E is endpoint, *** indicate a p-value &#60; 0.001 and **** indicates p &#60; 0.0001 as determined by a paired student&#39;s T-test comparing within group change between baseline and endpoint. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59138/59138fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Program parameters for treadmill endurance acclimatization and assessment.



<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Program parameter</td>
			<td>Acclimation</td>
			<td>Assessment</td>
		</tr>
		<tr>
			<td>Interval</td>
			<td>1</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Duration</td>
			<td>5</td>
			<td>90</td>
		</tr>
		<tr>
			<td>Enable Profile Mode</td>
			<td>Unchecked</td>
			<td>Unchecked</td>
		</tr>
		<tr>
			<td>Accel Step</td>
			<td>1.0 m/min</td>
			<td>1 m/min</td>
		</tr>
		<tr>
			<td>Accel Int</td>
			<td>60 seconds</td>
			<td>120 seconds</td>
		</tr>
		<tr>
			<td>Max Speed</td>
			<td>10 m/min</td>
			<td>35 m/min</td>
		</tr>
		<tr>
			<td>S_speed</td>
			<td>5 m/min</td>
			<td>5 m/min</td>
		</tr>
		<tr>
			<td>Shock Rep Rate</td>
			<td>1 hz</td>
			<td>1 hz</td>
		</tr>
		<tr>
			<td>Shock Intensity</td>
			<td>1 then 2</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
Table 2: Program parameters for uphill sprint acclimatization and assessment.



<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Program parameter</td>
			<td>Acclimation</td>
			<td>Assessment</td>
		</tr>
		<tr>
			<td>Interval</td>
			<td>0.5</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Duration</td>
			<td>10</td>
			<td>90</td>
		</tr>
		<tr>
			<td>Enable Profile Mode</td>
			<td>Checked</td>
			<td>Checked</td>
		</tr>
		<tr>
			<td>Accel Step</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Accel Int</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Max Speed</td>
			<td>15 m/min</td>
			<td>35 m/min</td>
		</tr>
		<tr>
			<td>S_speed</td>
			<td>5 m/min</td>
			<td>5 m/min</td>
		</tr>
		<tr>
			<td>Shock Rep Rate</td>
			<td>1 hz</td>
			<td>1 hz</td>
		</tr>
		<tr>
			<td>Shock Intensity</td>
			<td>2</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="3">PROFILE MODE (Refer to Figures 1 and 2 to set up program parameters)</td>
		</tr>
		<tr>
			<td>Test intervals</td>
			<td>8, 10, 10, 10 m/min</td>
			<td>6, 8, 10, 11, 12 &#8230; 51, 52 m/min</td>
		</tr>
	</tbody>
</table>
Table 3: Intensity designations for HIIT exercise mice.



<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Intensity Group</td>
			<td rowspan="2">Baseline Uphill Sprint Score</td>
			<td colspan="3">Initial Exercise Speeds</td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Sprint</td>
			<td>Dash</td>
		</tr>
		<tr>
			<td>0</td>
			<td>&#60; 8 m/min</td>
			<td>5 m/min</td>
			<td>7 m/min</td>
			<td>10 m/min</td>
		</tr>
		<tr>
			<td>1</td>
			<td>8-16 m/min</td>
			<td>5 m/min</td>
			<td>10 m/min</td>
			<td>15 m/min</td>
		</tr>
		<tr>
			<td>2</td>
			<td>17-25 m/min</td>
			<td>8 m/min</td>
			<td>13 m/min</td>
			<td>18 m/min</td>
		</tr>
		<tr>
			<td>3</td>
			<td>&#62;25 m/min</td>
			<td>11 m/min</td>
			<td>16 m/min</td>
			<td>21 m/min</td>
		</tr>
	</tbody>
</table>

Watch this Scientific Journal Video about Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice at JoVE.com

Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

Short session (&#8804;10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

High intensity interval training (HIIT) is emerging as a therapeutic approach to prevent, delay, or ameliorate frailty. In particular short session HIIT, ...

short-session-high-intensity-interval-training-treadmill-assessment

Nanyang Technological University

Research

JoVE Journal

Behavior

9.7K Views.  University at Buffalo and Research Service, Veterans Affairs Western New York Healthcare System. Short session (≤10 min) high intensity interval training (HIIT) is emerging as an alternative to longer exercise modalities, yet the shorter variants are rarely modeled in animal studies. Here, we describe a 10 min, 3 day a week, uphill treadmill HIIT protocol that enhances physical performance in male and female aged mice.

Video: Short Session High Intensity Interval Training and Treadmill Assessment in Aged Mice

Assessment of Cocaine-induced Behavioral Sensitization and Conditioned Place Preference in Mice

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to drugs of abuse make lasting alterations in the brain function of vulnerable individuals, and these persistent alterations likely serve to maintain the maladaptive drug seeking and taking behaviors characteristic of addiction/dependence2. In rodents, reward experience and contextual associations are frequently measured using the conditioned place preference assay, or CPP, wherein preference for a previously drug-paired context is measured. Behavioral sensitization, on the other hand, is an increase in a drug-induced behavior that develops progressively over repeated exposures. Since sensitized behaviors can often be measured after several months of drug abstinence, depending on the dose and length of initial exposure, they are considered observable correlates of lasting drug-induced plasticity. Researchers have found these assays useful in determining the neurobiological substrates mediating aspects of addiction as well as assessing the potential of different interventions in disrupting these behaviors. This manuscript describes basic, effective protocols for mouse CPP and locomotor behavioral sensitization to cocaine.

This protocol is intended to enable researchers to conduct experiments designed to test these aspects of addiction using the conditioned place preference and locomotor behavioral sensitization assays.

It is thought that rewarding experiences with drugs create strong contextual associations and encourage repeated intake. In turn, repeated exposures to ...

Pavlovian Conditioned Approach Training in Rats

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the attribution of incentive salience, or the transformation of neutral stimuli into attractive, &#34;wanted&#39; stimuli capable of motivating behavior. Pavlovian conditioned approach (PCA) develops after the response-independent presentation of a conditioned stimulus (CS; e.g., a lever) that predicts the delivery of an unconditioned stimulus (US; e.g., a food pellet) and can be used to measure incentive salience. During training, three patterns of conditioned responses (CRs) can develop: sign-tracking behavior (CS-directed CR), goal-tracking behavior (US-directed CR), and an intermediate response (both CRs). Sign-trackers attribute incentive salience to reward-related cues and are more vulnerable to cue-induced reinstatement of drug-seeking as well as other addiction-related behaviors, making PCA a potentially valuable procedure for studying addiction vulnerability. Here, we describe materials and methods used to elicit PCA behavior from rats as well as analyze and interpret PCA behavior in individual experiments.

Here, we present a protocol to elicit Pavlovian conditioned approach behavior in rats. This procedure can be used to measure individual differences in the tendency to approach and attribute incentive salience to reward-related cues and investigate addiction vulnerability.

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of current interventions to adequately treat fatigue in all patients leaves a need for new treatment options. Despite the therapeutic need and importance of preclinical research in helping identify promising novel treatments, few preclinical assays of fatigue are available. Moreover, the most common preclinical assay used to assess fatigue-like behavior, voluntary wheel running, is not suitable for use with some strains of mice, may not be sensitive to drugs that reduce fatigue, and has relatively low throughput. The current protocol describes a novel, non-voluntary preclinical assay of fatigue-like behavior, the treadmill fatigue test, and provides evidence of its efficacy in detecting fatigue-like behavior in mice treated with a chemotherapy drug known to cause fatigue in humans and fatigue-like behavior in animals. This assay may be a beneficial alternative to wheel running, as fatigue-like behavior and potential interventions can be assessed in a greater number of mice over a shorter time frame, thus permitting faster discovery of new therapeutic options.

Fatigue is a common, undertreated and frequently poorly-understood symptom in many diseases and disorders. New preclinical assays of fatigue may help to improve current understanding and future treatment of fatigue. To that end, the current protocol provides a novel means of measuring fatigue-like behavior in the mouse.

Fatigue is a prominent symptom in many diseases and disorders and reduces quality of life for many people. The lack of clear pathogenesis and failure of ...

Behavioral Assessment of Hearing in 2 to 4 Year-old Children: A Two-interval, Observer-based Procedure Using Conditioned Play-based Responses

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human auditory development for these age groups. This paper describes an observer-based procedure for measuring hearing sensitivity with a two-interval, two-alternative forced-choice paradigm. Young children are trained to perform a play-based, motor response (e.g., putting a block in a bucket) whenever they hear a target signal. An experimenter observes the child&#39;s behavior and makes a judgment about whether the signal was presented during the first or second observation interval; the experimenter is blinded to the true signal interval, so this judgment is based solely on the child&#39;s behavior. These procedures were used to test 2 to 4 year-olds (n = 33) with no known hearing problems. The signal was a 1,000 Hz warble tone presented in quiet, and the signal level was adjusted to estimate a threshold corresponding to 71%-correct detection. A valid threshold was obtained for 82% of children. These results indicate that the two-interval procedure is both feasible and reliable for use with toddlers and preschoolers. The two-interval, observer-based procedure described in this paper is a powerful tool for evaluating hearing in young children because it guards against response bias on the part of the experimenter.

This paper describes a procedure for measuring hearing sensitivity in 2 to 4 year-old children. Children are trained to perform play-based responses when they hear a target signal. Thresholds are then estimated in a two-interval, two-alternative forced-choice paradigm, based on observations of the child&#39;s behaviors.

Collecting reliable behavioral data from toddlers and preschoolers is challenging. As a result, there are significant gaps in our understanding of human ...

Introducing Clicker Training as a Cognitive Enrichment for Laboratory Mice

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous research determined a profound impact of gentle handling protocols on the well-being of laboratory mice. By introducing clicker training to the keeping of mice, not only do we promote the amicable treatment of mice, but we also enable them to experience cognitive enrichment. Clicker training is a form of positive reinforcement training using a conditioned secondary reinforcer, the "click" sound of a clicker, which serves as a time bridge between the strengthened behavior and an upcoming reward. The effective implementation of the clicker training protocol with a cohort of 12 BALB/c inbred mice of each sex proved to be uncomplicated. The mice learned rather quickly when challenged with tasks of the clicker training protocol, and almost all trained mice overcame the challenges they were given (100% of female mice and 83% of male mice). This study has identified that clicker training for mice strongly correlates with reduced fear in the mice during human-mice interactions, as shown by reduced anxiety-related behaviors (e.g., defecation, vocalization, and urination) and fewer depression-like behaviors (e.g., floating). By developing a reliable protocol that can be easily integrated into the daily routine of the keeping of laboratory mice, the lifetime experience of welfare in the mice can be improved substantially.

The development of new refinement strategies for laboratory mice is a challenging task that contributes towards fulfilling the 3R principle. This protocol introduces clicker training as a cognitive enrichment program for laboratory mice.

Establishing new refinement strategies in laboratory animal science is a central goal in fulfilling the requirements of Directive 2010/63/EU. Previous ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Classical Short-Delay Eyeblink Conditioning in One-Year-Old Children

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a puff of air to the cornea). Eyeblink conditioning is often used experimentally to detect abnormalities in cerebellar-dependent learning and memory that underlies this type of associative learning. While experiments in adults and older children are relatively simple to administer using commercial equipment, eyeblink conditioning in infants is more challenging due to their poor compliance, which makes correct positioning of the equipment difficult. To achieve conditioning in one-year-old infants, a custom-made or an adapted commercial system can be used to deliver the air puff to the infant's cornea. The main challenge lies in successfully detecting and classifying the behavioral responses. We report that automated blink detection methods are unreliable in this population, and that conditioning experiments should be analyzed using frame-by-frame analysis of supplementary video camera recordings. This method can be applied to study developmental changes in eyeblink conditioning and to examine whether this paradigm can detect children with neurological disorders.

This protocol describes an eyeblink conditioning paradigm appropriate for experiments with one-year-old infants. Commercial or custom-made equipment can be used to deliver the stimuli, and data collection and analysis should be performed on the video recordings.

Classical eyeblink conditioning (EBC) refers to the learned association between a conditioned stimulus (an auditory tone) and an unconditioned stimulus (a ...

A Vibrotactile Feedback Device for Seated Balance Assessment and Training

Postural perturbations, motion tracking, and sensory feedback are modern techniques used to challenge, assess, and train upright sitting, respectively. The goal of the developed protocol is to construct and operate a sitting platform that can be passively destabilized while an inertial measurement unit quantifies its motion and vibrating elements deliver tactile feedback to the user. Interchangeable seat attachments alter the stability level of the device to safely challenge sitting balance. A built-in microcontroller allows fine-tuning of the feedback parameters to augment sensory function. Posturographic measures, typical of balance assessment protocols, summarize the motion signals acquired during timed balance trials. No dynamic sitting protocol to date provides variable challenge, quantification, and sensory feedback free of laboratory constraints. Our results demonstrate that non-disabled users of the device exhibit significant changes in posturographic measures when balance difficulty is altered or vibrational feedback provided. The portable, versatile device has potential applications in rehabilitation (following skeletal, muscular, or neurological injury), training (for sports or spatial awareness), entertainment (via virtual or augmented reality), and research (of sitting-related disorders).

A sitting platform has been developed and assembled that passively destabilizes sitting posture in humans. During the user&#39;s stabilizing task, an inertial measurement unit records the device&#39;s motion, and vibrating elements deliver performance-based feedback to the seat. The portable, versatile device may be used in rehabilitation, assessment, and training paradigms.

Postural perturbations, motion tracking, and sensory feedback are modern techniques used to challenge, assess, and train upright sitting, respectively. ...

Supramaximal Intensity Hypoxic Exercise and Vascular Function Assessment in Mice

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by international guidelines for patients suffering from cardiovascular diseases, more specifically, lower extremity artery diseases, where the patients' walking capacity is considerably altered, affecting their quality of life. 
Traditionally, both low continuous exercise and interval training have been used. Recently, supramaximal training has also been shown to improve athletes' performances via vascular adaptations, amongst other mechanisms. The combination of this type of training with hypoxia could bring an additional and/or synergic effect, which could be of interest for certain pathologies. Here, we describe how to perform supramaximal intensity training sessions in hypoxia on healthy mice at 150% of their maximal speed, using a motorized treadmill and a hypoxic box. We also show how to dissect the mouse in order to retrieve organs of interest, particularly the pulmonary artery, the abdominal aorta, and the iliac artery. Finally, we show how to perform ex vivo vascular function assessment on the retrieved vessels, using isometric tension studies.

High-intensity training in hypoxia is a protocol that has been proven to induce vascular adaptations potentially beneficial in some patients and to improve athletes&#39; repeated sprint ability. Here, we test the feasibility of training mice using that protocol and identify those vascular adaptations using ex vivo vascular function assessment.

Exercise training is an important strategy for maintaining health and preventing many chronic diseases. It is the first line of treatment recommended by ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...