The authors would like to thank Angela Le, Sarah Ruby, and Marisa Mickey for their work maintaining the animal colonies. This work was funded by 1R15 HD076379 (L.R.D.), 3R15 HD076379 (L.R.D. to support CNR), and the McDevitt Undergraduate Research Fellowship in Natural Sciences (BEE).

All procedures were approved by the Le Moyne College Institutional Animal Care and Use Committee. All use of animals was in agreement with the policies described in the Guide for the Care and Use of Laboratory Animals8.
NOTE: (Critical) Prior to experimentation, obtain all necessary approvals and training required for animal use. It is important the experimenters are familiarized with the mouse behaviors and activity levels, including signs of sleep, distress, and/or movement artifact vs. normal sniffing and breathing.
1. Whole-body Barometric Plethysmography Chamber
<ol>
	<li>Read the appropriate user manuals for the barometric plethysmography chamber, including connectors, O-rings, etc., and create a standard protocol file to define analyzers (e.g., metabolic) and parameters specific to the software.</li>
	<li>Make sure all hoses and tubes are connected to the chamber. Connect a gas flow tube (flow-in) and a vacuum tube (flow-out) directly to the barometric plethysmography chamber. 
	NOTE: The inflow must be attached to the opening marked bias flow.</li>
	<li>Attach CO2, O2, and N2 gas tanks to the gas mixer. Make sure all gas tanks are in the open position prior to experimentation.</li>
</ol>
2. Calibration of the Barometric Plethysmograph Chamber
<ol>
	<li>Calibrate a high and a low flow of gas by selecting the 7700-Amplifier Setup under the Hardware tab of the barometric plethysmography software.</li>
	<li>Set a vacuum (flow out of the chamber) appropriate for the experimental design&#160;and gas analyzers (~0.1 L/min). 
	NOTE: The outflow rate must remain the same throughout the calibrations and experiment for accurate metabolic recordings.</li>
	<li>Set a low flow of air by removing the flow tube from the chamber and turning off the vacuum.</li>
	<li>Record the zero flow by entering a 0 into the Low Unit cell for the corresponding chamber. Double-click the Low Cal cell, change the time to 3 s, and hit Measure.</li>
	<li>Reattach the flow tube and allow gas (20.93% O2, balanced N2) to flow through the barometric plethysmography chamber from the gas mixer.</li>
	<li>Convert the inflow from liters/minutes into milliliters/second. Click the High Unit cell for the corresponding chamber and enter the value in milliliters/second. Double-click High Cal, change the time to 3 s, and click Measure.</li>
	<li>Leave the 7700-Amplifier Setup tab open to calibrate the metabolic analyzers to the barometric plethysmography software.</li>
</ol>
3. Metabolic Analyzer Calibration
<ol>
	<li>In the gas mixer program, set the gas mixer to release a flow of gas containing 20.93% O2 and 79.07% N2.</li>
	<li>On the metabolic analyzers, set the O2 calibration level to 20.93% and the CO2 to read 0%. Turn the dial back to Sample once the appropriate values are entered.</li>
	<li>Set the high O2 percentage. Click on the ABCD-4 tab of the barometric plethysmography software and then enter 20.93 under High Unit of the C2 line. Under High Cal, change the time to 3 s and hit Measure.</li>
	<li>Set the low CO2 percentage. Enter 0 under Low Cal of the C3 line, and then change the time to 3 s and click Measure under Low Cal.</li>
	<li>In the gas mixer program, change the O2 value to 10% and the CO2 value to 5%. Wait several minutes for the gas flow to adjust to these values. On the metabolic analyzers, turn the adjustment knobs to calibrate CO2 equal to 5%. Be sure to turn the dial back to Sample once the values are calibrated.</li>
	<li>Set the high CO2 percentage. Ensure the analyzer readings are stable before inserting appropriate values into the O2 and CO2 on the barometric plethysmography software. Click High Unit under C3 and enter 5. Change High Cal to 3 s and hit Measure.</li>
	<li>Set the low O2 percentage. Click Low Unit under the C2 option and enter 10. Click Low Cal, input 3 s, and click Measure.</li>
	<li>Change the gas values on the gas mixer back to 20.93% O2 and 79.07% N2. Wait several minutes for the chamber to adjust to these values. Repeat the steps 3.1&#8210;3.7 if the metabolic analyzers do not automatically read 20.93% O2 and 0% CO2, to ensure proper calibration. Routinely confirm proper calibration with certified gas tanks.</li>
	<li>Recheck the flow meters connected to the barometric plethysmography chamber. Adjust the air flow into and out of the chamber to rates appropriate for the experiment (typically, 0.1&#8211;0.3 L/min).</li>
	<li>Once all settings have been applied to the barometric plethysmography software, click OK to begin recording.</li>
</ol>
4. Unrestrained Barometric Plethysmography
<ol>
	<li>Record the mouse&#8217;s weight and initial body temperature. Wait 10 min before placing the mouse in the chamber, to collect O2 and CO2 data from an empty chamber. Work in a quiet area familiar to the mice so noise and smells do not interfere with the data collection. Avoid any possible disruptions, including the opening and closing of doors or personnel moving in/out of the data collection room. 
	NOTE: This specific protocol employed 22 month-old male C57BL/6J mouse.</li>
	<li>During the first hour, document the behaviors of the mouse and take detailed notes, including specific values of the flow in/out of the chamber.</li>
	<li>After 60 min of chamber habituation, watch for segments of quiet breathing for the following 60 min. List any segments lasting at least 15 s in length without sniffing and grooming. Take body temperature measures every 10 min when using an implantable device.</li>
	<li>At the end of the experiment, remove the mouse from the chamber and place it back in its cage. All equipment should be cleaned and wiped down thoroughly. If droplets of water remain, use pressurized air to remove them.</li>
</ol>
5. Analysis of Pattern of Breathing and Metabolism
<ol>
	<li>Open the barometric plethysmography review file and consult the recorded notes for the animal of interest.</li>
	<li>Open the Metabolic panel in the software and take the average of the first 10 min of O2 and CO2, when the chamber was empty. Record these values as the FiO2 and FiCO2.</li>
	<li>View the Flow panel of the barometric plethysmography software. Right-click Analyze Attributes and set appropriate parameters. Under the Meta 1 tab, enter the FiO2 and FiCO2 from step 5.2, as well as the flow into the chamber under Meta 2, to calculate VO2 and VCO2.</li>
	<li>For pattern of breathing analysis, confirm the times for the 15 seconds of quiet breathing using notes about animal behavior as well as the flow panel tracing.&#160;Enter the times for the 15 s intervals of quiet breathing under Open Data Parser Dialogue from the Data Parser tab. Click Parser View Mode to only show the specific 15 s segments of interest.</li>
	<li>Click Save Parsed Derived Data. Open the data file in a spreadsheet to obtain the binned data.</li>
</ol>
6. Analysis of Apneas and Augmented Breaths
<ol>
	<li>In the open review file, exit Parser View Mode. Go into the Graph Setup option under Setup &#62; P3 Setup and select Page View under Type. Select 5 for the number of panes. Enter -2 into the box labeled Low and 2 into the box labeled High for flow measures in milliliters/second. Apply the changes.</li>
	<li>Scroll to the 30 min mark on the flow tracings panel.</li>
	<li>Count apneas and augmented breaths for the 30&#8211;60 min after the mouse was placed in the chamber. Quantify periods of suspended breathing lasting longer than or equal to 0.5 s, indicative of an apnea. Augmented breaths are indicated by a sharp rise in the breathing trace above 1.25 mL/s followed by a sharp decrease below -0.75 mL/s.</li>
</ol>

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R., 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=Neural+deficits+contribute+to+respiratory+insufficiency+in+Pompe+disease.">Neural deficits contribute to respiratory insufficiency in Pompe disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (23), 9419-9424 (2009).</li><li>Ogier, M., 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=Brain-derived+neurotrophic+factor+expression+and+respiratory+function+improve+after+ampakine+treatment+in+a+mouse+model+of+Rett+syndrome.">Brain-derived neurotrophic factor expression and respiratory function improve after ampakine treatment in a mouse model of Rett syndrome.</a> Journal of Neuroscience. 27 (40), 10912-10917 (2007).</li><li>Ohshima, 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=Hypoxic+ventilatory+response+during+light+and+dark+periods+and+the+involvement+of+histamine+H1+receptor+in+mice.">Hypoxic ventilatory response during light and dark periods and the involvement of histamine H1 receptor in mice.</a> American Journal of Physiology-Regulatory Integrative and Comparative Physiology. 293 (3), 1350-1356 (2007).</li><li>van Schaik, S. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+Blood+Sampling+in+Male+CD-1+and+C57BL/6J+Mice+with+1%+Isoflurane+is+Similar+to+Awake+Mice.">Arterial Blood Sampling in Male CD-1 and C57BL/6J Mice with 1% Isoflurane is Similar to Awake Mice.</a> Journal of Applied Physiology. , (2018).</li><li>Receno, C. N., Eassa, B. E., Reilly, D. P., Cunningham, C., DeRuisseau, L. 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The authors have nothing to disclose.

The protocol provides information regarding a quiet breathing baseline in mice, as well as collecting data about central apneas and augmented breaths. The representative results show that a 10 min quiet baseline has a similar pattern of breathing when compared to an average of four 15 s bouts for a cohort of old mice. Importantly, the 15 s bouts are not statistically different, nor do these groups have differences in variation from one another using Levene&#8217;s test. These data demonstrate that even one short bout is sufficient for monitoring quiet breathing. However, it is entirely possible that analyzing individual variation within a mouse at 15 s vs. 10 min may result in different findings, as the 10 min bout could encompass minimal sniffing and grooming activities. However, using Levene&#8217;s test for a comparison of individual mouse baseline segments provides a different analysis than the one described in this protocol. Overall, the design of this methodology uses 15 s breathing segments that can be acquired during minutes 60&#8211;120 in the chamber, versus having to wait for each mouse to achieve longer durations of quiet baseline.
The shorter duration required for baseline allows for more anxious/agitated strains of mice to be tested for quiet breathing. The use of a longer breathing segment (i.e., 10 or 2 min) lengthens the protocol duration, to a point where a trial may need to be abandoned if the mice do not display a quiet breathing trace within 3 h. Since many experimental designs also incorporate respiratory challenges (i.e., hypoxia), the extended time allotted for other gases highlights the need for baseline collection time to be standardized. The use of a single 15 s bout of quiet breathing helps to relieve the concern of working with mice (and strains of mice) that may be particularly excitable in the chamber. While working with barometric plethysmography, we found that ~10% of mice per study had to be excluded because of their inability to perform as little as 2 min of continuous quiet breathing within the chamber. The implementation of previous familiarization trials was unsuccessful in getting mice to calm down faster when placed in the chamber on the day of experimentation. However, because different strains, sexes, and ages of mice may all react differently to the chamber environment10,11, it is possible that habituation techniques may be helpful12,13 for some cohorts. Our familiarization trials consisted of placing the mice in the UBP chamber in the testing room for 1&#8211;2 h for several days prior to experimentation. While we observed no changes in animal behavior following this procedure, a previous study has shown that 24 h of habituation was needed to eliminate novelty effects resulting in spontaneous physical activity in mice12. Additionally, Kabir et al. found that placing plastic cylinders similar in size to the barometric plethysmography chamber in the home cage was advantageous in getting rats to familiarize themselves with the setups prior to experimentation13.
This protocol also uncovers possible respiratory dysfunction in mice via the quantification of central apneas, indicative of neural control issues. Thirty minutes of observation prior to the baseline pattern-of-breathing collection showed that all 16 aged mice displayed a high number of apneic episodes and augmented breaths (represented in Figure 1C). The numerous apneas in this aged mouse cohort highlight the ability of this protocol to quantify another important breathing measure without adding additional time to experimentation. It should be noted that age and disease progression (if applicable) can affect the presence and number of apneic episodes.
In order to characterize quiet breathing, it is important to continuously observe the barometric plethysmography chamber and mouse throughout the duration of the protocol. For the quantification of quiet breathing, mice should be awake but not partake in any active behaviors such as sniffing, grooming, or exploring (represented in Figure 1A). Since patterns of breathing during sleep can differ from those in an awake animal14,15, the collection of calm breathing during the awake state is critical. It is possible that longer segments of quiet breathing could include periods of sleep, which may not be desired depending on the experimental design. In this case, shorter segments of quiet breathing would be ideal to document, as the likelihood of data collection during sleep is reduced when active segments flank the short (15 s) quiet breathing segments. We have observed that longer segments of quiet breathing can be challenging to acquire in the mouse model, as mouse behavior in the chamber seems to be very different compared to that of rats. It is important both to critically observe mouse respiratory flow for appropriate breathing segments and to document animal behavior. In cases of reduced ventilation or unstable breathing, this method can still be utilized. In these instances, it is essential the experimenter is blinded to the cohort when selecting the 15 s segments. The software program should distinguish the breaths, with an acceptance rate of 100% during the 15 s period. We advise taking note of the breathing tracings in addition to ensuring that the animals meet the behavioral criteria for baseline since it is possible that stationary mice may still be anxious. A previous study reported that although rats exhibited calm behavior, they still showed altered breathing patterns (i.e., increased frequency) in response to controlled stimuli within the testing room13.
Measures of frequency, VT, VE, inspiratory and expiratory time, and VE/VCO2 are all quantified using analyzers and the UBP software and are frequently reported in the literature. Particularly, VT and VE calculations use the Drorbaugh and Fenn equation16, which requires known values for body temperature, ambient chamber temperature, humidity, and barometric pressure. It is recommended to collect these measures throughout the experiment for the most accurate VT and VE values. Other variables that are calculated by the system should be used with caution. UBP is not a direct measure of pulmonary mechanics; thus, variables related to airway resistance (e.g., enhanced pause [Penh]) should be interpreted with this caveat in mind5. Additional components of the UBP setup that can impact variables calculated by the software include flow rates and the general calibration of the system. Confirm seals and gaskets are working properly (no leaks) and ensure proper connection of all equipment to the barometric plethysmography chamber (Figure 5). Flow rates in and out of the chamber should be kept consistent. Required flow rates can differ between UBP setups, so it is important to check these values prior to experimentation. The flow rate into the chamber should be enough to provide fresh air or gas challenges in a timely manner. The flow rate should also be sufficient for allowing the metabolic analyzers to measure O2 and CO2 without having CO2 build up within the chamber environment, which poses the risk of a changing pattern of breathing. Similarly, gas mixer/analyzer calibrations need to be regularly implemented to ensure that the metabolic parameters are accurately measured.
Other considerations for conscious UBP include reducing distractions within the experimental room while animals are being tested. Loud noises, different smells, and the presence of nonessential personnel in the room can all add to anxious behaviors exhibited by mice. Using smaller rooms as testing areas may help, but if this is not possible, cardboard walls (with a small viewing window) can be set up surrounding the chamber to lessen distractions for the mice. Electrical activity within the room should be kept at a minimum to prevent additional noise within the barometric plethysmography tracings. Therefore, it is important to take note of the flow tracings during the 10 min period when the software is collecting data from an empty chamber. These tracings should remain flat, and any interruptions or slight waves are signs of noise and should be addressed. Pressure changes from opening and closing doors or from HVAC functioning can also add to erroneous fluctuations, and ensuring that these actions occur minimally (and noting them when they do occur) is critical. Humidity can also affect the calculated tidal volume and minute ventilation, making it very important to confirm that the chamber and connecting tubes are dried before use. If necessary, the use of Drierite beads in sequence with the flow-in tubes can help remove all condensation in the air prior to chamber entrance. This step would be instituted in cases when the humidity has routinely been higher than levels listed in The Guide for the Care and Use of Animals8 (30%&#8211;70%, ideally within 10% of setpoint). Humidity can also build up in the chamber due to the presence of the animal. Although some humidity is normal, it may continue to build if the animal is excessively active or placed in the chamber for longer durations. If humidity levels reach maximum levels (99.99%), the chamber may need to be opened and wiped down during experimentation to maintain comparable breathing measures. The software accounts for changes in barometric pressure, ambient temperature, animal temperature, and humidity. Best practice is to maintain the values within a reasonable range so that &#8220;apples to apples&#8221; are being compared across ages, strains, and sexes. Moreover, the circadian cycle of mice and the time range of testing, as well as specific lighting conditions of the room, are important details to consider13,17. For instance, we typically test mice in lighting similar to their housing room (either light or dark cycle) and within a 3 h range18. Experimenters should also remain blinded to the animal groups during data collection and analyses to prevent differences in the selection of a baseline. When possible, the same experimenter should collect all data and/or analyze all tracings in a given experiment. Steps to keep experimenters blinded to the animal groups, as well as randomization and testing during similar times of the day, are crucial to the rigor of investigation. Ultimately, there are extraneous factors that may alter the flow tracings, and these concerns should be considered when performing UBP.
The UBP method is a technique used to characterize the pattern of breathing in mice. Baseline measures can be collected within 2 h when using a 15 s breathing segment. Here we report a method which can be performed with aged mice, who are often more agitated in the chamber than younger mice, suggesting that other anxious or active mouse strains could also be tested with this protocol. The data collected from UBP is noninvasive and allows for testing over multiple time points, which is useful for studies about aging, drug therapy, and disease progression.

UBP is a common technique for the assessment of breathing patterns1,2,3,4. In this method, mice are placed in a closed chamber where pressure differences between the main chamber (where the animal is housed) and a reference chamber are filtered through a pneumotachograph to obtain values. The resulting UBP setup is noninvasive and unrestrained and allows for respiratory measures to be assessed without the requirement of anesthesia or surgery. Additionally, this technique is suitable for studies requiring multiple measurements in the same mouse over time. Variables such as breathing frequency, tidal volume, and minute ventilation can be quantified with this method, during a single trial or over several trials. Whole-body UBP also provides measures of peak flows and respiratory cycle duration. Together, these parameters quantify the pattern of breathing. The recorded breathing traces also make it possible to review the data and count the number of central apneas displayed within a given time period. This count can be used alongside an analysis of tidal volume and inspiratory times to gauge other alterations in the pattern of breathing.
While several noninvasive plethysmography techniques exist for the direct assessment of pulmonary physiological parameters, whole-body UBP allows for a way to screen for respiratory function with minimal undue stress to the mouse. Head-out plethysmography, which utilizes tidal midexpiratory flow measures and is also noninvasive, relies on restraint, like many other types of plethysmography (e.g., double-chamber plethysmography). While these methods have been used in rodent models to measure airway responsiveness5, the use of neck collars or small restraint tubes may take mice (vs. other species) longer to acclimate to and return their breathing to resting levels.
Obtaining an optimal air-breathing segment is an important consideration for baseline comparisons. The increased use of commercially available plethysmography systems makes collecting pattern-of-breathing data possible in many laboratories. Importantly, pattern of breathing is variable throughout the collection period, particularly for mice. With that said, it is necessary to standardize baseline analysis as a means of ensuring that the training level of experimenters does not confound results. There are numerous ways to collect an air-breathing segment, serving as one area of variation between experimental designs. One example includes averaging the final 10&#8211;30 min of data following a previously defined set of time within the chamber1, while another method involves waiting until the mouse is visibly calm for 5&#8211;10 min6. The latter can take 2&#8211;3 h to achieve and in some cases, a trial may need to be abandoned if the mouse is not calm for long enough. This concern is an especially important consideration for strains of mice where observed behaviors are more anxious and excitable7. These mice may take longer to adjust to the chamber environment and only remain calm for short bursts of time. Limiting the time devoted to baseline collection standardizes the chamber time for each mouse.
It is crucial that experimenters obtain a suitable baseline that encompasses resting behavior values in the mouse but also occurs in a timely manner. Hence, the goal of this report is to provide a description of methods used to obtain short quiet baseline values for breathing parameters in mice. Moreover, we report that apneas and augmented breaths can be quantified during the first hour in the chamber.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Carbon Dioxide Analyzer</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">CD-3A&#160;</td>
			<td style="width:727px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Carbon Dioxide Sensor</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">&#160;P-61B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Computer</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td>must be compliant with Ponemah requirements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Drierite beads</td>
			<td style="width:135px;">PermaPure LLC</td>
			<td style="width:119px;">DM-AR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Flow Control</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">R-1</td>
			<td>vacuum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Flowmeter</td>
			<td style="width:135px;">TSI</td>
			<td align="right" style="width:119px;">4100</td>
			<td>need one per chamber and one for vacuum</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Gas Mixer</td>
			<td style="width:135px;">MCQ Instruments</td>
			<td style="width:119px;">GB-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Gas Tanks</td>
			<td style="width:135px;">Haun</td>
			<td style="width:119px;"></td>
			<td>100% oxygen, 100% carbon dioxide, 100% nitrogen - food grade, or pre-mixed tanks for nomal room air and gas challenges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Oxygen Analyzer</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">S-3A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Oxygen Sensor</td>
			<td style="width:135px;">AEI Technologies</td>
			<td style="width:119px;">&#160;N-22M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Polyurethane Tubing</td>
			<td style="width:135px;">SMC</td>
			<td style="width:119px;">TUS 0604 Y-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Ponemah Software</td>
			<td style="width:135px;">DSI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Small Rodent Chamber</td>
			<td style="width:135px;">Buxco/DSI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Thermometer (LifeChip System)</td>
			<td style="width:135px;">Destron-Fearing</td>
			<td style="width:119px;"></td>
			<td>any type of thermometer to take accurate body temperatures is appropriate, but the use of implantable chips allows for minimal disturbance to animal for taking several body temperature measurements while the animal is still in the UBP chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Transducers</td>
			<td style="width:135px;">Validyne</td>
			<td style="width:119px;">DP45</td>
			<td>need one per chamber&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:351px;">Whole Body Plethysmography System&#160;</td>
			<td style="width:135px;">Data Science International (DSI)</td>
			<td style="width:119px;"></td>
			<td>Includes ACQ-7700, pressure/temperature probes, etc.&#160;</td>
		</tr>
	</tbody>
</table>

method to obtain pattern of breathing in senescent mice through unrestrained barometric plethysmography

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and minute ventilation are routinely reported. Moreover, information can be collected regarding the neural output of breathing, including the existence of central apneas and augmented breaths. An important consideration for UBP is obtaining a breathing segment with a minimal impact of anxious or active behaviors, to elucidate the response to breathing challenges. Here, we present a protocol that allows for short, quiet baselines to be obtained in aged mice, comparable to waiting for longer bouts of quiet breathing. The use of shorter time segments is valuable, as some strains of mice may be increasingly excitable or anxious, and longer periods of quiet breathing may not be achieved within a reasonable timeframe. We placed 22 month-old mice in a UBP chamber and compared four 15 s quiet breathing segments between minutes 60&#8211;120 to a longer 10 min quiet breathing period that took&#160;2&#8211;3 h to acquire. We also obtained counts of central apneas and augmented breaths prior to the quiet breathing segments, following a 30 min familiarization period. We show that 10 min of quiet breathing is comparable to using a much shorter 15 s duration. Additionally, the time leading up to these 15 s quiet breathing segments can be used to gather data regarding apneas of central origin. This protocol allows investigators to collect pattern-of-breathing data in a set amount of time and makes quiet baseline measures feasible for mice that may exhibit increased amounts of excitable behavior. The UBP methodology itself provides a useful and noninvasive way to collect pattern-of-breathing data and allows for mice to be tested over several time points.

The results of UBP as an evaluation of pattern of breathing in 16 aged (22-month-old) mice performed under normal air gas (20.93% O2 with balanced N2) are reported. The analysis first included a comparison of a longer 10 min quiet breathing segment (which took over 2 h to obtain) versus the average of four short 15 s segments (quantified within minutes 60&#8211;120). A representative flow tracing of quiet breathing, where breathing is consistent with no active breathing behaviors, is provided in Figure 1A. When similar tracings are collected from animals, 100% of the breaths should be accepted by the software. However, Figure 1B represents breathing from a more active segment, where the mice are exploring the chamber, sniffing, and/or grooming. Tracings similar to that shown in Figure 1B are less likely to be accepted by the software and are not ideal for the type of breathing collection used and explained by this methodology. The parameters selected for the assessment of possible pattern-of-breathing differences between the two time points were breathing frequency (Figure 2A), tidal volume (VT, Figure 2B), minute ventilation (VE, Figure 2C), tidal volume/inspiratory time ratio (VT/Ti, Figure 2D), and minute ventilation/expelled carbon dioxide ratio (VE/VCO2/g, Figure 2E), which were all calculated using the barometric plethysmography software and Drorbaugh and Fenn equation. Values reported for these measures are within the range of what we have previously reported for the mouse model6,9. No significant differences were elucidated between groups; post hoc corrections for multiple comparisons of breathing frequency and VT data were accounted for with Bonferroni (p &#60; 0.025 was considered significant). These results show that the use of a simplified protocol using 15 s baselines provides similar results to that of a longer baseline protocol.
Further analysis was performed with each of the four 15 s baseline segments for frequency, VT, VE, VT/Ti, and VE/VCO2/g (Figure 3). No significant differences (p &#62; 0.05) between any of the time points were found. There were also no differences in the variability between any of the four time segments for any pattern-of-breathing measure. Additionally, we tested the variability of the segment of the 15 s group vs. the 10 min group and found no significant differences using Levene&#8217;s test when comparing the averaged group data.
The number of apneas and augmented breaths observed for each animal during minutes 30&#8211;60 of the UBP protocol are presented in Figure 4. These results show that aged animals showcase a high number of apneas and the presence of augmented breaths within a 30 min span (tracing shown in Figure 1C). The data are indicative of changes during the aging process, as these findings were observed in 22 month-old mice. To confirm interrater reliability for apnea and augmented breath analyses, Pearson correlation was calculated for two different investigators. A high degree of agreement between raters was found, as indicated by a value of r = .99 for apneas and r = .86 for augmented breaths. In future studies, an increased number of apneas compared to a control group would be telling of a breathing dysfunction stemming from a neural component.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59393/59393fig1v2.jpg" /> 
Figure 1: Representative flow tracings. (A) Flow tracing from a quiet baseline, where the mouse does not showcase any active behaviors such as sniffing or grooming. (B) Flow tracing from an active breathing period not included in our analyses, where mice are moving about the chamber and many breaths are not routinely accepted. (C) Flow tracing displaying an augmented breath followed by a period of apnea. A 5 s window is shown for all traces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59393/59393fig1v2large.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/59393/59393fig2v3.jpg" /> 
Figure 2: Breathing parameters are similar for calm breathing segments of 10 min and 15 s in 22-month-old mice. Barometric plethysmography was used to collect breathing data in aged mice (n = 16, 22 months old). Breathing data were calculated for mice during two different time points, namely the average of four 15 s calm intervals within the 60&#8211;120 min mark of the mouse being in the chamber and for 10 min of consistent calm breathing. (A) Breathing frequency (breaths/minute). (B) Tidal volume (VT; milliliters/breath). (C) Minute ventilation (VE; milliliters/minute). (D) Ratio of tidal volume to inspiration time (VT/Ti; milliliters/second). (E) Ratio of minute ventilation to carbon dioxide expelled, normalized to weight (VE/VCO2/g). There are no statistically significant differences between the groups after post hoc corrections (p &#62; 0.025). Values of &#62;3 SD above the mean were considered outliers and removed from the data set. Data are presented as mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59393/59393fig2v3large.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/59393/59393fig3.jpg" /> 
Figure 3: Comparison of four 15 s intervals. Breathing data were calculated in calm breathing mice (n = 16, 22 months old) for four separate 15 s intervals within the 60&#8211;120 min of chamber placement. (A) Breathing frequency (breaths/minute). (B) Tidal volume (VT; milliliters/breath). (C) Minute ventilation (VE; milliliters/minute). (D) Ratio of tidal volume to inspiration time (VT/Ti; milliliters/second). (E) Ratio of minute ventilation to carbon dioxide expelled, normalized to weight (VE/VCO2/g). There are no statistically significant differences between the time segments (p &#62; 0.05). Outliers are defined as &#62;3 SD above the mean and removed. Data are presented as mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59393/59393fig3large.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/59393/59393fig4.jpg" /> 
Figure 4: Apnea and augmented breath counts in mice. Apneas (&#8805;0.5 s without breathing) and augmented breaths (ABs; a sharp increase in inhalation over 1.25 mL/s followed by a sharp exhalation below -0.75&#160;mL/s) were counted in aged mice (n = 16, 22 months old) between 30&#8211;60 min. The counts were analyzed over 30 min and the total for that time period are reported. Data are presented as mean &#177; SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59393/59393fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59393/59393fig5a.jpg" /> 
Figure 5: Schematic of the unrestrained barometric plethysmography (UBP) setup. The overall UBP setup should be similar to that described in the figure. Flow measurements must be measured for the gases entering and leaving the chamber, and the gas composition must be known for data interpretation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59393/59393fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography at JoVE.com

Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Unrestrained barometric plethysmography (UBP) is a method for quantifying the pattern of breathing in mice, where breathing frequency, tidal volume, and ...

method-to-obtain-pattern-breathing-senescent-mice-through

Nanyang Technological University

Research

JoVE Journal

Behavior

6.5K Views.  Le Moyne College. Unrestrained barometric plethysmography is used to quantify the pattern of breathing in awake mice. We show that 15 s segments under a standardized protocol display similar values to an extended duration of quiet breathing. This methodology also allows for the quantification of apnea and augmented breaths during the first hour in the chamber.

Video: Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography

The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

The Rodent Psychomotor Vigilance Test (rPVT): A Method for Assessing Neurobehavioral Performance in Rats and Mice

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article describes a rodent version of the PVT&#8212;termed the &#34;rPVT&#34;&#8212;that measures similar aspects of attention (i.e., performance accuracy, motor speed, premature responding, and lapses in attention). Data are presented that demonstrate both the short- and long-term usefulness of the rPVT when employed with laboratory rats. Rats easily learn the rPVT, and learning to perform the basic procedure takes less than two weeks of training. Once acquired, rat performances in the rPVT show a high degree of similarity to these same performance measures in the human PVT, including similarities in, lapses in attention, reaction times, vigilance decrements across session time (i.e., the human &#34;time-on-task&#34; effects), and the response-stimulus interval (RSI) effect described for humans. Thus the rPVT can be an extremely valuable tool for assessing the effects of a wide range of variables on sustained attention quite similar to human PVT performances, and thus can be useful for developing novel treatments for neurobehavioral dysfunctions.

The Rodent Psychomotor Vigilance Test rPVT: A Method for Assessing Neurobehavioral Performance in Rats and Mice

A rat version of the human Psychomotor Vigilance Test (PVT) is described that measures aspects of attention similar to those measured with the human PVT, including aspects of human vigilance such as performance accuracy, motor speed, premature responding, and lapses in attention.

The human Psychomotor Vigilance Test (PVT) is a widely used procedure for measuring changes in fatigue and sustained attention. The present article ...

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

A Laboratory Method to Measure Contagious Yawning in Rats

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. Communication is also the way sexes interact during courtship and how rivals settle disputes without fighting. However, there are some behavioral patterns for which it is difficult to test the existence of a communicatory function, because several types of sensory modalities are likely involved. For example, contagious yawning is a communicatory act in mammals that potentially occurs through sight, hearing, smell, or a combination of these senses depending on whether the animals are familiar to one another. Therefore, to test hypotheses about the possible communicatory role of such behaviors, a suitable method is necessary to identify the participating sensory modalities.
The method proposed here aims to obtain yawn contagion curves for familiar and unfamiliar rats and evaluate the relative participation of visual and olfactory sensory modalities. The method uses inexpensive materials, and with some minor changes, it can also be used with other rodent species such as mice. Overall, the method involves the substitution of clear dividers (with or without holes) with opaque dividers (with or without holes) that either allow or prevent communication between rats placed in adjacent cages with holes in adjoining sides. Accordingly, four conditions can be tested: olfactory communication, visual communication, both visual and olfactory communication, and neither visual nor olfactory communication. As social interaction occurs between the rats, these test conditions simulate what may occur in a natural environment. In this respect, the method proposed here is more effective than traditional methods that rely on video presentations whose biological validity can raise concerns. Nonetheless, it does not discriminate between the potential role of hearing and roles of smell and vision in yawn contagion.

The method described here aims to obtain yawn contagion curves in pairs of familiar or unfamiliar male rats. Cages with holes separated by either clear or opaque partitions with (or without) holes are used to detect whether visual, olfactory, or both types of sensory cues can stimulate yawn contagion.

Communication is an essential aspect of animal social life. Animals may influence one another and come together in schools, flocks, and herds. ...

Investigation into Deep Breathing through Measurement of Ventilatory Parameters and Observation of Breathing Patterns

In this protocol, two deep breathing patterns were shown to 15 participants to determine an easy yet effective method of breathing exercise for future application in a clinical setting. The women in their twenties were seated comfortably in a chair with back support. They were fitted with an airtight mask connected to a&#160;gas analyzer. Three electrodes were placed on the chest connected to a wireless transmitter for relaying to the electrocardiograph. They executed a 5 min rest phase, followed by 5 min of deep breathing with a natural breathing pattern, terminating with a 5 min rest phase. This was followed by a 10 min intermission before commencing the second instruction phase of substituting the natural breathing pattern with the diaphragmatic breathing pattern. Simultaneously, the following took place: a) continuous collection, measurement and analysis of the expired gas to assess the ventilatory parameters on a breath-by-breath basis; b) measurement of the heart rate by an electrocardiograph; and c) videotaping of the participant&#8217;s thoracoabdominal movement from a lateral aspect. From the video capture, the investigators carried out visual observation of the fast-forward motion-images followed by classification of the breathing patterns, confirming that the participants had carried out the method of deep breathing as instructed. The amount of oxygen uptake revealed that, during deep breathing, the work of breathing decreased. The results from the expired minute ventilation, respiration rate and tidal volume confirmed increased ventilatory efficiency for deep breathing with the natural breathing pattern compared to that with the diaphragmatic breathing pattern. This protocol suggests a suitable method of instruction for assessing deep breathing exercises on the basis of oxygen consumption, ventilatory parameters, and chest wall excursion.

Here, we present a protocol to assess two deep breathing patterns of natural and diaphragmatic breathing for their effectiveness and ease of execution. Fifteen participants were selected, utilizing an electrocardiograph and expired gas analyzer for measurement of the ventilatory parameters, together with visual assessment by video capture of thoracoabdominal movement.

In this protocol, two deep breathing patterns were shown to 15 participants to determine an easy yet effective method of breathing exercise for future ...

Training Dogs for Awake, Unrestrained Functional Magnetic Resonance Imaging

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for acquisition of functional and structural data. This method of training dogs for awake, unrestrained fMRI employs a generalization procedure of stationing in several dissimilar locations to facilitate transfer of the stationing behavior to the real MRI scan environment; it does so without the need for extensive training time in the MRI scan environment, which can be expensive. Further, this method splits the training of a stationing (i.e., chin rest) behavior from desensitization to the MRI environment (i.e., 100+ decibel scan audio), the latter accomplished during dedicated Auditory Exposure conditioning sessions. The complete training and testing protocol required 14 hours and resulted in immediate transfer to novel locations. We also present examples of canine fMRI data that have been acquired from visual face processing and olfactory discrimination paradigms.

Magnetic resonance imaging (MRI) on unrestrained awake dogs is a new method with several advantages over imaging with physical or chemical restraint. This protocol introduces a cost-effective training method that minimizes training in the MRI environment, which can be expensive, and maximizes the subject pool available for canine functional MRI.

We present a canine functional Magnetic Resonance Imaging (fMRI) training protocol that can be done in a cost-effective manner, with high-energy dogs, for ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...