Name: method to obtain pattern of breathing in senescent mice through unrestrained barometric plethysmography
Uploaded: 2020-04-28T13:00:00
Description: Watch this Scientific Journal Video about Method to Obtain Pattern of Breathing in Senescent Mice through Unrestrained Barometric Plethysmography at JoVE.com

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, di...

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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 ...

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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			<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>
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			<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>
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			<td height="21" style="height:21px;width:351px;">Computer</td>
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			<td>must be compliant with Ponemah requirements</td>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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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 <st...

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 ...

This protocol can be used to measure patterns of breathing in mice. Importantly, it can minimize the impact of active behaviors in genetically modified strains that can confound results. The main advantages of this technique are that it is noninvasive and that it does not require the use of anesthetic or stress-inducing restraints.Begin by making sure that all of the hoses and tubes are connected to the barometric plethysmograph chamber and by connecting a gas inflow tube and a vacuum outflow tube directly to the barometric plethysmography chamber. To calibrate the low flow into the chamber, set the flow into and out of the chamber to zero and remove the flow tube into the chamber. Turn off the vacuum and enter a zero into the low unit cell to record the zero flow in the 7700 amplifier setup of the barometric plethysmograph software.Then reattach the inflow tube and turn on vacuum flow to calibrate the high flow and to allow 20.93%oxygen balanced nitrogen gas to flow through the barometric plethysmography chamber from the gas mixer. Next, convert the inflow value measured from the flow meter from liters per minute into milliliters per second and click the high unit cell to enter the value in milliliters per second. Double-click high cal, change the time to three seconds and click Measure.Then leave the 7700 amplifier setup tab open to calibrate the metabolic analyzers to the barometric plethysmography software. To calibrate the metabolic analyzer, in the gas mixer program, set the gas mixer to 20.93%oxygen and 79.07%nitrogen. And on the metabolic analyzer, set the oxygen calibration level to 20.93%and the carbon dioxide to 0%Turn the dial back to sample once the appropriate values have been entered on the gas analyzers to set the high oxygen and low carbon dioxide percentage.Click on the ABCD 4 tab of the barometric plethysmography software and enter 20.93 under high unit of the C2 line for oxygen. Under high cal, change the time to three seconds and click Measure. Enter zero under low cal of the C3 line for carbon dioxide.Then change the time to three seconds and click Measure under low cal. In the gas mixer program, change the oxygen value to 10%and the carbon dioxide value to 5%and wait several minutes for the gas flow to adjust to these values. On the metabolic analyzers, turn the adjustment knobs to calibrate the carbon dioxide to 5%taking care to turn the dial back to sample once the values have been calibrated.After checking that the analyzer readings are stable, click high unit under C3 and enter five for carbon dioxide. Then change high cal to three seconds and click Measure. Click low unit under the C2 option and enter 10 for oxygen and click low cal, input three seconds and click Measure.Change the gas mixer back to 20.93%oxygen and 79.07%nitrogen and wait several minutes for the chamber to adjust to these values. Routinely run additional calibrations with certified gas tanks to confirm the flow mixer and oxygen-carbon dioxide analyzers are performing properly. When the metabolic analyzers have been calibrated, recheck the flow meters connected to the barometric plethysmograph chamber and adjust the air flow into and out of the chamber to rates appropriate for the experiment.When all of the settings have been applied to the barometric plethysmography software, click Close in the Acquisition tab. Hit Start Acquisition, name the file and click OK to begin recording. For unrestrained barometric plethysmography, record the weight and initial body temperature of the mouse before returning the mouse to its home cage.Wait 10 minutes to collect the background oxygen and carbon dioxide data from the empty chamber before placing the mouse into the chamber. During the first hour, document the behavior of the animal taking detailed notes that include the specific values of the in and outflow of the chamber. At the end of the chamber habituation, watch for segments of quiet breathing for the next 60 minutes taking body temperature measurements every 10 minutes when using an implantable device.Take care to become familiar with general mouse behaviors so that instances of calm breathing with no sniffing, grooming or exploring can be appropriately identified. At the end of the experiment, return the mouse to its cage and thoroughly clean and wipe down the equipment. For a metabolism analysis, open the metabolic panel in the software and obtain the average of the first 10 minutes of the oxygen and carbon dioxide levels from when the chamber was empty.View the flow panel of the barometric plethysmography software and right-click Analyze Attribute to set appropriate parameters. For a 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. Then enter the times under open parser dialogue from the data parser tab and click Parser View Mode to show only the specific 15-second segments of interest.To analyze apneas and augmented breath, in the open review file, exit the parser view mode and click Setup P3 Setup and Graph Setup to select Page View as the type and five as the number of panes. Enter minus two into the box labeled low and two into the box labeled high for the flow measures in milliliters per second and apply the changes. Then scroll to the 30-minute mark on the flow tracings panel and manually count the apneas and augmented breaths for the 30 to 60-minute period after the mouse was placed in the chamber.For this analysis, periods of suspended breathing lasting longer than or equal to 0.5 seconds are indicative of an apnea. Augmented breaths are indicated by a sharp rise in the breathing trace above 1.25 milliliters per second followed by a sharp decrease below minus 0.75 milliliters per second. This representative flow tracing of quiet breathing in a 22-month-old mouse is typical of a pattern in which the breathing is consistent with no active breathing behaviors.This breathing pattern is from a more active segment during which the mice were exploring the chamber sniffing and/or grooming and is not ideal for the type of breathing collection used for this methodology. The parameter selected for the assessment of possible pattern of breathing differences between the two time points were breathing frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio. Further analysis was performed with each of the four 15-second baseline segments for frequency, tidal volume, minute ventilation, tidal volume inspiratory time ratio, and minute ventilation expelled carbon dioxide ratio.No significant differences between any of the time points and no differences in the variability between any of the four time segments for any pattern of breathing measure were found. Quantification of the number of apneas and augmented breaths observed for each animal during minutes 30 to 60 of the unrestrained barometric plethysmography protocol revealed that aged animals showcased a high number of apneas and the presence of augmented breaths within a 30-minute span. Researchers can also employ gas challenges such as hypoxia or hypercapnia to document any changes in the breathing pattern resulting from an intervention or therapy.This technique provides a means for characterizing patterns of breathing in small rodents and genetically modified animal strains that may exhibit distinct behavioral differences.

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Behavior

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 ...

Psychophysical Tracking Method to Measure Taste Preferences in Children and Adults

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to adulthood. The method, which is identical for children, adolescents, and adults, is of short duration (&#60; 15 min), does not rely on sustained attention or place demands on memory (which would yield spurious age differences), and minimizes the impact of language development, making this method amenable to the cognitive limitations of pediatric populations. In this whole-mouth tasting method, subjects are asked to taste (without swallowing) pairs of solutions of different sucrose concentrations and to point to the solution they prefer. Each subsequent pair contains the participant&#39;s preceding preferred concentration and an adjacent stimulus concentration. The procedure continues until the subject chooses either a given concentration of sucrose when paired with both a higher and a lower concentration, or the highest or lowest concentration two consecutive times. Subjects are prevented from reaching response criteria on the basis of first or second position bias by the two-series design of the method, which counterbalances the order of solution presentation within each pair between the series (the weaker concentration is presented first in Series 1, second in Series 2). The geometric mean of the two sucrose concentrations chosen in Series 1 and 2 is an estimate of the participant&#39;s most preferred level of sucrose. Sucrose preference as determined with this laboratory-based measure has been shown to be associated with preference for sugars in foods and beverages and with taste receptor genotype, family history of alcoholism, and race/ethnicity, as well as depressive symptomatology among pediatric populations. The method has real-world relevance and has been applied to determine most preferred level of other tastes (e.g., salt), making it a valuable psychophysical tool.

Sweet taste has powerful hedonic appeal among people of all ages, particularly children. Described herein is a reliable and valid method that can be used to determine the level of sweetness most preferred, making it a valuable psychophysical tool for scientists.

The Monell two-series, forced-choice, paired-comparison tracking method provides a reliable measure of sweet taste preferences from childhood to ...

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 ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

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 ...