The authors thank Dr. Shimpachiro Ogiwara, former Professor at the University of Kanazawa, and Mrs. Sandra M. Ogiwara, CSP (UK), BScPT (C), for English editing of the manuscript.

This protocol was in accordance with the ethical principles of the Declaration of Helsinki. The procedure was explained to all participants before commencement of the study.
1. Participant screening
<ol>
	<li>Recruit 15 healthy women in their twenties through convenience sampling. Check medical history verbally. Exclude participants with cardiopulmonary disease.</li>
	<li>Explain the procedure to the participant.</li>
	<li>Ask the participant to refrain from eating and drinking 2 h prior to the commencement of the measurement and to bring a tight-fitting black shirt.</li>
</ol>
2. Procedure
<ol>
	<li>Preparation for the procedure
	<ol>
		<li>Calibrate the gas analyzer that has integrated components of a pneumotachograph and oxygen/carbon dioxide concentration meters 15 &#8211; 30 min before the measurement. Follow manufacturer&#39;s protocols.</li>
		<li>Attach a video camera to a tripod at a distance of 1.5 m from the chair the participant will sit on. Prepare to record a lateral view of the sitting participant in a range from the top of the&#160;cranium to the seat of the chair.</li>
		<li>Instruct the waiting participant to put on the tight-fitting black shirt in a cubicle, and when ready, stand by the side of the chair in the laboratory.</li>
		<li>Place three electrodes (positive, negative&#160;and ground) on the skin of the chest in standing, each with a wire to connect to a transmitter that relays to the electrocardiograph.</li>
		<li>Seat the participant comfortably for 5 min in a chair with a back support at an angle of 70&#176; and, if necessary, insert a small cushion in the neck and/or&#160;lumbar region.</li>
		<li>Explain to the participant deep breathing with a natural breathing (NB) pattern of slow and deep breaths, breathing in through the nose and blowing out through the mouth with no consideration or knowledge given on specific movement of the chest.</li>
		<li>Ask the participant to take a natural deep breath without any guidance. Prepare to begin measurement if the investigator is satisfied with breathing performance. Observe the participant&#39;s thoracoabdominal movement during inspiration and expiration.</li>
	</ol>
	</li>
	<li>Measurement of deep breathing with the NB pattern
	<ol>
		<li>Fit the participant with a sampling mask over the mouth and nose for measurement of the expired gas. Perform a seal test: close the hole for the sampling tube of the mask with a finger and ask the participant to breathe out gently and confirm whether air leaks from the mask. Connect a sampling tube to the mask for measurement of the ventilatory parameters.</li>
		<li>Ask the participant to refrain from talking during the procedure.</li>
		<li>Instruct the participant to rest for 5 min, and simultaneously begin recording the expired gas and heart rate, together with video capture. After the 5 min rest phase, instruct the participant to begin deep breathing for 5 min with NB pattern. On termination, instruct the participant to rest for 5 min.</li>
		<li>Continue recording and measuring throughout the three phases.</li>
		<li>Perform only one three-phase trial for each participant.</li>
	</ol>
	</li>
	<li>Rest phase
	<ol>
		<li>Inform the participant that the experimenter will take off the mask and allow her a 10 min intermission phase.</li>
		<li>Instruct the participant that she may sit and talk in the laboratory, but not drink. Begin timing the intermission phase with a stopwatch at the moment of taking off the mask.</li>
	</ol>
	</li>
	<li>Measurement of deep breathing with the diaphragmatic breathing (DB) pattern
	<ol>
		<li>Seat the participant as in step 2.1.5.</li>
		<li>Explain to the participant deep breathing with a diaphragmatic breathing (DB) pattern. Ask the participant to lace her fingers, place them on her abdomen and take a deep breath in through the nose, expanding the abdomen under the hands and then blowing out through the mouth and gently retracting the abdomen.</li>
		<li>Instruct the participant to practice this deep breathing with the DB pattern until the investigator is satisfied.&#160;Observe that thoracoabdominal expansion takes place during inspiration followed by its contraction on expiration.</li>
		<li>Fit the participant with a sampling mask over the mouth and nose for measurement of the expired gas. Perform a seal test: close the hole for the sampling tube of the mask with a finger and ask the participant to breathe out gently and confirm whether air leaks from the mask. Connect a sampling tube to the mask for measurement of the ventilatory parameters.</li>
		<li>Ask the participant to refrain from talking during the measurement.</li>
		<li>Instruct the participant to rest for 5 min, and simultaneously begin recording the expired gas and heart rate, together with video capture. After the 5 min rest phase, instruct the participant to begin deep breathing for 5 min with the DB pattern. On termination, instruct the participant to rest for 5 min. Continue recording and measuring throughout the three phases.</li>
		<li>Take the mask off the participant following the 5 min rest phase.</li>
		<li>Ask the participant immediately which of the two deep breathing techniques was more comfortable. Record the participant&#8217;s response on a spreadsheet.</li>
		<li>Remove the electrodes, leads and transmitter from the participant and let her leave.</li>
		<li>Perform only one three-phase trial for each participant.</li>
	</ol>
	</li>
</ol>
3. Measurement of the Ventilatory Parameters
<ol>
	<li>Sample the breath-by-breath expired gas using a&#160;gas analyzer (see Table of Materials and Figure 2).
	<ol>
		<li>Measure the following ventilatory parameters: oxygen uptake (VO2), carbon dioxide output (VCO2), expired minute ventilation (VE), respiratory rate (f), tidal volume (VT), expiratory time (Te), and inspiratory time (Ti).</li>
		<li>Measure the heart rate using a medical telemetry sensor for the electrocardiograph (see Table of Materials and Figure 2), which is connected to the gas analyzer. 
		NOTE: The&#160;gas analyzer is operated using computer software provided by the manufacturer (Table of Materials).</li>
	</ol>
	</li>
	<li>Collect the data for each 5 min phase of rest and deep breathing for NB and DB patterns. Save the data on the ventilatory parameters in CSV format using computer software (see Table of Materials and Figure 3,4).
	<ol>
		<li>Collect mean data for each 5 min phase of this protocol. Average data for the optionally set phase is obtained on a breath-by-breath basis.</li>
	</ol>
	</li>
	<li>Enter the data for each participant into the spreadsheet program (see Table of Materials and Figure 5) and determine the mean and standard deviation (SD) for the initial rest and deep breathing phases for NB and DB.</li>
</ol>
4. Assessment of the Breathing Pattern
<ol>
	<li>Record thoracoabdominal movement from a lateral view of the participant using a video camera (Table of Materials).</li>
	<li>Make sure that the background color is in sharp contrast to the participant&#8217;s silhouette.</li>
	<li>Record the video image at 1/30 s per frame, which is the standard speed for the video camera used.</li>
	<li>Upload the motion-images into a personal computer using video editing software 1 (Table of Materials).</li>
	<li>Observe 5 min video images of the deep breathing phases at double speed under visual assessment and classify the breathing patterns as upper costal, diaphragmatic or thoracoabdominal. Use video editing software 2 (Table of Materials). 
	NOTE: Video images are analyzed by a cardiopulmonary physical therapist (MY).</li>
</ol>
5. Participants&#8217; preferred pattern for deep breathing
<ol>
	<li>Prepare a spreadsheet for the participant&#39;s response.</li>
	<li>Ask the participant which of the two deep breathing techniques is more comfortable following the measurement of the DB pattern.</li>
	<li>Fill in the spreadsheet with the participant&#39;s response.</li>
	<li>Be ready to listen to the participant if she wants to talk about the procedure. Do not include the participant&#8217;s comments in the analysis.</li>
</ol>
6. Statistical Analysis
NOTE: Perform statistical analysis using commercial computer software (Table of Materials), and then provide all the button clicks.
<ol>
	<li>Ventilatory parameters
	<ol>
		<li>Do not analyze the 5 min rest phase following the two deep breathing phases in this protocol.</li>
		<li>Determine the mean and SD for the initial rest phases and deep breathing phases for each parameter.</li>
		<li>Employ two-way repeated-measures analysis of variance (2-way ANOVA) to evaluate the ventilatory parameters and heart rate for the initial rest phases and two deep breathing phases. 
		NOTE: Factor &#34;instruction&#34;&#160;includes two levels NB and DB, and factor &#34;phase&#34;&#160;has two levels rest phase and deep breathing phase.</li>
		<li>Using the Bonferroni method, evaluate between each factor for parameter measurement, yielding significant interaction following 2-way ANOVA.</li>
	</ol>
	</li>
	<li>Classification of breathing patterns exhibited by the participants including their preferred pattern for deep breathing.
	<ol>
		<li>Categorize the number of participants according to their breathing pattern in upper costal, diaphragmatic or thoracoabdominal breathing.</li>
		<li>Compile from the spreadsheet the number of participants according to their preferred pattern of deep breathing.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Yokogawa, 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=Comparison+of+two+instructions+for+deep+breathing+exercise:+non-specific+and+diaphragmatic+breathing.">Comparison of two instructions for deep breathing exercise: non-specific and diaphragmatic breathing.</a> Journal of Physical Therapy Science. 30, 614-618 (2018).</li><li>Lewis, L. K., Williams, M. T., Olds, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+effect+on+outcomes+related+to+the+mechanism+of+intervention+and+physiological+outcomes+but+insufficient+evidence+of+clinical+benefits+for+breathing+control:+a+systematic+review.">Short-term effect on outcomes related to the mechanism of intervention and physiological outcomes but insufficient evidence of clinical benefits for breathing control: a systematic review.</a> Australian Journal of Physiotherapy. 53, 219-227 (2007).</li><li>Cahalin, L. P., Braga, M., Matsuo, Y., Hernandez, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+diaphragmatic+breathing+in+persons+with+chronic+obstructive+pulmonary+disease:+A+review+of+the+literature.">Efficacy of diaphragmatic breathing in persons with chronic obstructive pulmonary disease: A review of the literature.</a> Journal of Cardiopulmonary Rehabilitation. 22, 7-21 (2002).</li><li>Sackner, M. A., Gonzalez, H. F., Jenouri, G., Rodriguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+abdominal+and+thoracic+breathing+on+breathing+pattern+components+in+normal+subjects+and+in+patients+with+chronic+obstructive+pulmonary+disease.">Effects of abdominal and thoracic breathing on breathing pattern components in normal subjects and in patients with chronic obstructive pulmonary disease.</a> The American Review of Respiratory Disease. 130, 584-587 (1984).</li><li>Gosselink, R. A., Wagenaar, R. C., Rijswijk, H., Sargeant, A. J., Decramer, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diaphragmatic+breathing+reduces+efficiency+of+breathing+in+patients+with+chronic+obstructive+pulmonary+disease.">Diaphragmatic breathing reduces efficiency of breathing in patients with chronic obstructive pulmonary disease.</a> American Journal of Respiratory and Critical Care Medicine. 151, 1138-1142 (1995).</li><li>Adachi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+Exercise+Test.">Cardiopulmonary Exercise Test.</a> International Heart Journal. 58, 654-665 (2017).</li><li>Guazzi, M., Bandera, F., Ozemek, C., Systrom, D., Arena, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiopulmonary+Exercise+Testing:+What+Is+Its+Value.">Cardiopulmonary Exercise Testing: What Is Its Value.</a> Journal of the American College of Cardiology. 70, 1618-1636 (2017).</li><li>Jones, A. Y., Dean, E., Chow, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+oxygen+cost+of+breathing+exercises+and+spontaneous+breathing+in+patients+with+stable+chronic+obstructive+pulmonary+disease.">Comparison of the oxygen cost of breathing exercises and spontaneous breathing in patients with stable chronic obstructive pulmonary disease.</a> Physical Therapy. 83, 424-431 (2003).</li><li>Ito, M., Kakizaki, F., Tsuzura, Y., Yamada, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+effect+of+respiratory+muscle+stretch+gymnastics+and+diaphragmatic+breathing+on+respiratory+pattern.">Immediate effect of respiratory muscle stretch gymnastics and diaphragmatic breathing on respiratory pattern.</a> Internal Medicine. 38, 126-132 (1999).</li><li>Jansson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+significance+of+the+respiratory+exchange+ratio+after+different+diets+during+exercise+in+man.">On the significance of the respiratory exchange ratio after different diets during exercise in man.</a> Acta Physiologica Scandinavica. 114, 103-110 (1982).</li><li>Yokogawa, 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=Effects+of+food+intake+on+physiological+responses+to+cardiopulmonary+exercise+testing.">Effects of food intake on physiological responses to cardiopulmonary exercise testing.</a> Journal of Physical Therapy Science. 19, 145-150 (2007).</li><li>Romei, 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=Effects+of+gender+and+posture+on+thoraco-abdominal+kinematics+during+quiet+breathing+in+healthy+adults.">Effects of gender and posture on thoraco-abdominal kinematics during quiet breathing in healthy adults.</a> Respiratory Physiology &amp; Neurobiology. 172, 184-191 (2010).</li><li>Binazzi, B., 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=Breathing+pattern+and+kinematics+in+normal+subjects+during+speech,+singing+and+loud+whispering.">Breathing pattern and kinematics in normal subjects during speech, singing and loud whispering.</a> Acta Physiologica. 186, 233-246 (2006).</li><li>Faul, F., Erdfelder, E., Lang, A. -. G., Buchner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G*Power+3:+A+flexible+statistical+power+analysis+program+for+the+social,+behavioral,+and+biomedical+sciences.">G*Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences.</a> Behavior Research Method. 39, 175-191 (2007).</li><li>Reychler, G., 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=Incentive+spirometry+and+positive+expiratory+pressure+improve+ventilation+and+recruitment+in+postoperative+recovery:+A+randomized+crossover+study.">Incentive spirometry and positive expiratory pressure improve ventilation and recruitment in postoperative recovery: A randomized crossover study.</a> Physiotherapy Theory and Practice. 35, 199-205 (2019).</li></ol>

The authors declare that they have no competing financial interests.

By the use of this protocol, effective instruction for deep breathing can be examined through oxygen consumption, ventilatory parameters, and chest wall excursion. The participants had a mean age of 21.6 years, mean body mass of 51.9 kg, mean height of 159.3 cm and a body mass index of 20.5 kg/m2. No incentives were offered to partake in this protocol. There are three critical steps within the protocol. First, concerning the control of food intake, the&#160;ratio of carbon dioxide output to oxygen uptake provi...

The cardiopulmonary physical therapist normally treats the patient according to the individual&#39;s needs and requirements. However, in general, the patient is left to carry out preoperative deep breathing exercise by him/herself. Therefore, it is imperative to find a simple and effective instruction method for the patient to carry out deep breathing exercises1.
Diaphragmatic breathing is such a breathing exercise and one method of breathing control2,3. The therapeutic outcome of this method includes a reduction in work of breathing and improvement in efficiency of breathing2,3, and this brings about an increase in tidal volume, resulting in a reduction in respiratory rate. However, some researchers have pointed out that diaphragmatic breathing exercise may cause asynchronous and paradoxical motion of the rib cage due to abdominal excursions in some patients4,5. In such cases, the use of a patient&#39;s natural breathing pattern may be efficacious. Regarding the question of deep breathing being effective as a means of a reduction in the mechanical work of breathing and improvement of ventilatory efficiency, it may be useful to quantify ventilatory parameters by the use of a&#160;gas analyzer.
It is well known that cardiopulmonary exercise testing is carried out using a gas analyzer6,7. Some investigators8,9 have reported measurement for diaphragmatic breathing with a gas analyzer in&#160;patients with chronic obstructive pulmonary disease. Jones et al.8 compared diaphragmatic breathing, pursed-lip breathing, and a combination of both, with that of spontaneous breathing. During these three methods of breathing, oxygen consumption (VO2) and respiratory rate (f) were measured, which showed that a&#160; higher resting VO2 may be explained by the increased mechanical work of breathing8. Ito et al.9 examined the immediate effect of diaphragmatic breathing or respiratory muscle stretch on VO2, f and tidal volume (VT). We may expect from the results of the aforementioned studies that similar evidence could be obtained by application of similar breathing exercises to confirm an effective deep breathing method of instruction.
This protocol describes the method for measurement of ventilatory parameters and chest wall excursion in deep breathing with two breathing patterns, together with their results and analysis. Continuous and quantitative sampling of ventilatory parameters can measure breathing precisely compared to alternative techniques. VO2 obtained in this protocol can be regarded as an indicator of work of breathing8. Further, f, VT, and minute ventilation are related to ventilatory efficiency.&#160;Information on breathing pattern can also be obtained from these ventilator parameters plus inspiratory and expiratory time. This protocol also involves assessment of chest wall excursion through video capture, which corresponds to observation by a physical therapist of the patient&#39;s chest wall excursion during breathing exercise. The overall goal of this study was to find a viable and efficient method of deep breathing exercise based on analysis of oxygen consumption, ventilatory parameters, and chest wall excursion.

<table>
	<tbody>
		<tr>
			<td>Expired gas analyzer</td>
			<td>Minato Medical Science, Osaka, Japan</td>
			<td>AE-300S</td>
			<td></td>
		</tr>
		<tr>
			<td>Expired gas analyzing software</td>
			<td>Minato Medical Science, Osaka, Japan</td>
			<td>AT for Windows</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical telemetry sensor for electrocardiograph</td>
			<td>Nihon Kohden, Tokyo, Japan</td>
			<td>BSM-2401</td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet program</td>
			<td>Microsoft, https://www.microsoft.com/ja-jp</td>
			<td>Excel</td>
			<td></td>
		</tr>
		<tr>
			<td>SPSS Statistical Software</td>
			<td>IBM, https://www.ibm.com/jp-ja/analytics/spss-statistics-software</td>
			<td>Version 23.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony, Tokyo, Japan</td>
			<td>DCR-SR 100</td>
			<td></td>
		</tr>
		<tr>
			<td>Video editing software 1</td>
			<td>Sony, Tokyo, Japan</td>
			<td>PlayMemories Home</td>
			<td></td>
		</tr>
		<tr>
			<td>Video editing software 2</td>
			<td>Adobe, https://www.adobe.com/jp/</td>
			<td>Premiere Elements 11</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The Ventilatory Parameters and Heart Rate 
Based on the data (Figure 5), the NB and DB patterns were analyzed statistically (Figure 6 and Table 1). The f, VT and Te were found to have a significant interaction (p&#60;0.05, respectively). A significant decrease in the f was found for both the NB and DB patterns during deep breathing compared to the initial rest phases (p&#60;0.05, respectively), and, during deep b...

Watch this Scientific Journal Video about Investigation into Deep Breathing through Measurement of Ventilatory Parameters and Observation of Breathing Patterns at JoVE.com

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

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 ...

investigation-into-deep-breathing-through-measurement-ventilatory

Research

Education

JoVE Journal

JoVE Core

Anatomy and Physiology

Nursing

Behavior

Unit 1

The Respiratory System

Vital Signs: Respiration

SCIENTISTS IN ACTION

11.5K Views.  Kanazawa University. 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.

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

Corticospinal Excitability Modulation During Action Observation

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone else's action becomes anticipatory simulation of a complementary act. TMS was delivered to the left primary motor cortex corresponding to the hand to induce the highest level of MEP activity from the abductor digiti minimi (ADM; the muscle serving little finger abduction) as well as the first dorsal interosseus (FDI; the muscle serving index finger flexion/extension) muscles. A neuronavigation system was used to maintain the position of the TMS coil, and electromyographic (EMG) activity was recorded from the right ADM and FDI muscles. Producing original data with regard to motor resonance, the combined TMS/MEP technique has taken research on the perception-action coupling mechanism a step further. Specifically, it has answered the questions of how and when observing another person's actions produces motor facilitation in an onlooker's corresponding muscles and in what way corticospinal excitability is modulated in social contexts.

Single-pulse transcranial magnetic stimulation over the primary motor cortex, neuronavigation, and registration of electromyographic activity of hand muscles were used in this study to explore corticospinal excitability while participants were observing action sequences.

This study used the transcranial magnetic stimulation/motor evoked potential (TMS/MEP) technique to pinpoint when the automatic tendency to mirror someone ...

Measurement of Neurophysiological Signals of Ignoring and Attending Processes in Attention Control

Attention control is the ability to selectively attend to some sensory signals while ignoring others. This ability is thought to involve two processes: enhancement of sensory signals that are to be attended and the attenuation of sensory signals that are to be ignored. The overall strength of attentional modulation is often measured by comparing the amplitude of a sensory neural response to an external input when attended versus when ignored. This method is robust for detecting attentional modulation, but precludes the ability to assess the separate dynamics of attending and ignoring processes. Here, we describe methodology to measure independently the neurophysiological signals of attending and ignoring using the intermodal attention task (IMAT). This task, when combined with electroencephalography, isolates neurophysiological sensory responses in auditory and visual modalities, when either attending or ignoring, with respect to a passive control. As a result, independent dynamics of attending and of a ignoring can be assessed in either modality. Our results using this task indicate that the timing and cortical sources of attending and ignoring effects differ, as do their contributions to the attention modulation effect, pointing to unique neural trajectories and demonstrating sample utility of measuring them separately.

Attention control comprises enhancement of target signals and attenuation of distractor signals. We describe an approach to measure separately but concurrently, the neurophysiology of attending and ignoring in sustained intermodal attention, utilizing a passive control condition during which neither process is continuously engaged.

Attention control is the ability to selectively attend to some sensory signals while ignoring others. This ability is thought to involve two processes: ...

A General Method for Evaluating Deep Brain Stimulation Effects on Intravenous Methamphetamine Self-Administration

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people. There is a need for novel, effective and practical treatment strategies that are validated in animal models. Neuromodulation, including deep brain stimulation (DBS) therapy, refers to the use of electricity to influence pathological neuronal activity and has shown promise for psychiatric disorders, including drug dependence. DBS in clinical practice involves the continuous delivery of stimulation into brain structures using an implantable pacemaker-like system that is programmed externally by a physician to alleviate symptoms. This treatment will be limited in methamphetamine users due to challenging psychosocial situations. Electrical treatments that can be delivered intermittently, non-invasively and remotely from the drug-use setting will be more realistic. This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence. Methamphetamine dependence is rapidly developed in rodents using an operant paradigm of intravenous (IV) self-administration that incorporates a period of extended access to drug and demonstrates both escalation of use and high motivation to obtain drug.

This article describes the delivery of intracranial electrical stimulation that is temporally and spatially separate from the drug-use environment for the treatment of IV methamphetamine dependence.

Substance use disorders, particularly to methamphetamine, are devastating, relapsing diseases that disproportionally affect young people.  There is a need ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Visualizing Motion Patterns in Acupuncture Manipulation

Acupuncture manipulation varies widely among practitioners in clinical settings, and it is difficult to teach novice students how to perform acupuncture manipulation techniques skillfully. The Acupuncture Manipulation Education System (AMES) is an open source software system designed to enhance acupuncture manipulation skills using visual feedback. Using a phantom acupoint and motion sensor, our method for acupuncture manipulation training provides visual feedback regarding the actual movement of the student's acupuncture manipulation in addition to the optimal or intended movement, regardless of whether the manipulation skill is lifting, thrusting, or rotating. Our results show that students could enhance their manipulation skills by training using this method. This video shows the process of manufacturing phantom acupoints and discusses several issues that may require the attention of individuals interested in creating phantom acupoints or operating this system.

Here, we present a protocol for using the Acupuncture Manipulation Education System (AMES) in the training of acupuncture manipulation skills using phantom acupoints.

Acupuncture manipulation varies widely among practitioners in clinical settings, and it is difficult to teach novice students how to perform acupuncture ...

Novel Object Recognition Test for the Investigation of Learning and Memory in Mice

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is fairly simple and can be completed over 3 days: habituation day, training day, and testing day. During training, the mouse is allowed to explore 2 identical objects. On test day, one of the training objects is replaced with a novel object. Because mice have an innate preference for novelty, if the mouse recognizes the familiar object, it will spend most of its time at the novel object. Due to this innate preference, there is no need for positive or negative reinforcement or long training schedules. Additionally, the ORT can also be modified for numerous applications. The retention interval can be shortened to examine short-term memory, or lengthened to probe long-term memory. Pharmacological intervention can be used at various times prior to training, after training, or prior to recall to investigate different phases of learning (i.e., acquisition, early or late consolidation, or recall). Overall, the ORT is a relatively low-stress, efficient test for memory in mice, and is appropriate for the detection of neuropsychological changes following pharmacological, biological, or genetic manipulations.

The object recognition test (ORT) is a simple and efficient assay for evaluating learning and memory in mice. The methodology is described below.

The object recognition test (ORT) is a commonly used behavioral assay for the investigation of various aspects of learning and memory in mice. The ORT is ...

Using Clicker Training and Social Observation to Teach Rats to Voluntarily Change Cages

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on well-being and can affect the comparability and reproducibility of research results, the amount of stress experienced by laboratory animals should be minimized and avoided when possible. Further, the direct contact between the rat and animal caretaker during the cage change bears hygiene risks and therefore possibly negatively impacts the well-being of the rats and the quality of the research. 
Our protocol aims to improve the routinely performed cage changing procedure. For this reason, we present a feasible protocol that enables rats to learn via clicker training and observation to voluntarily change to a clean cage. This training helps to reduce stress caused by the physical disturbance and handling associated with the cage changes and concurrently enables a reduction in direct contact between animal and animal caretaker after the training phase is completed.
The implementation of clicker training to rats is fast and easy. Rats are generally interested in the training and efficiently learn the desired behavior, which entails changing cages through a pipe. Even without training, the rats learn to perform the desired behavior by observation, as 80% of the observational learning group successfully changed cages when tested. The training further helps to establish a relationship of trust between trainer and animal. As hygiene and well-being are both very important in animal experiments, this protocol might also help to improve high-quality research.

This protocol introduces a method of cage change for rats via clicker training. Rats learn the desired behavior not only by direct training but also by observational learning. The implementation of this fast and easy protocol might help to improve well-being and hygiene in rodent facilities.

Cage cleaning is a routinely performed husbandry procedure and is known to induce stress in laboratory rats. As stress can have a negative impact on ...

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.

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 ...

Application of 3D Printing in the Construction of Burr Hole Ring for Deep Brain Stimulation Implants

3D printing has been widely applied in the medical field since the 1980s, especially in surgery, such as preoperative simulation, anatomical learning and surgical training. This raises the possibility of using 3D printing to construct a neurosurgical implant. Our previous works took the construction of the burr hole ring as an example, described the process of using softwares like computer aided design (CAD), Pro/Engineer (Pro/E) and 3D printer to construct physical products. That is, a total of three steps are required, the drawing of 2D-image, the construction of 3D-image of burr hole ring, and using a 3D printer to print the physical model of burr hole ring. This protocol shows that the burr hole ring made of carbon fiber can be rapidly and accurately molded by 3D printing. It indicated that both CAD and Pro/E softwares can be used to construct the burr hole ring via integrating with the clinical imaging data and further applied 3D printing to make the individual consumables.

Here, we present a protocol to demonstrate 3D printing in the construction of deep brain stimulation implants.

3D printing has been widely applied in the medical field since the 1980s, especially in surgery, such as preoperative simulation, anatomical learning and ...

Measurement of Spatial Stability in Precision Grip

The purpose of the protocol is to indirectly evaluate the direction of the finger force during manipulation of a handheld object based on the biomechanical relationships in which deviated force direction causes center of pressure (COP) replacement. To evaluate this, a thin, flexible, and high spatial resolution pressure sensor sheet is used. The system allows measurement of the COP trajectory in addition to the force amplitude and its temporal regulation. A series of experiments found that increased trajectory length reflected a sensorimotor deficit in stroke patients, and that decreased COP trajectory reflects a compensatory strategy to avoid an object slipping from the hand grip in the elderly. Moreover, the COP trajectory could also be decreased by dual task interference. This article describes the experimental procedure and discusses how finger COP contributes to an understanding of the physiology and pathophysiology of grasping.

The goal of this protocol is to measure the center of pressure (COP) replacement using a high spatial resolution sensor sheet to reflect the spatial stability in a precision grip. The use of this protocol could contribute to greater understanding of the physiology and pathophysiology of grasping.