This work was partially supported by a grant from JSPS KAKENHI Grant Number JP17K00209. The author thanks Takayuki Hoshino and Kazuhiro Shigeta for technical assistance.

All methods described here have been approved by the Ethics Committee of Tokyo Denki University. For every participant, informed consent was obtained after the participant received a detailed explanation of the protocol.
1. Setup of the Left-Right Reversed Audition System
<ol>
	<li>Setup of the Reversed Audition System without a Participant 
	<ol>
		<li>Prepare a linear pulse-code-modulation (LPCM) recorder, binaural microphones, and binaural in-ear earphones.</li>
		<li>Connect the left and right lines of the microphones crossly to the LPCM recorder so that left-right reversed analogue sound signals are digitalized. Moreover, connect the left and right lines of the earphones straight through to the recorder so that the reversed digitalized signals are immediately played. 
		NOTE: In the case of employing the binaural earphone-microphones as binaural earphones, do not use the earphone parts in order to reduce the spillover of the sounds that go through the microphone parts.</li>
		<li>Put the bodies of the microphones and the earphones together for each ear with slight isolation by sound proofing materials, and cover the microphones with dedicated windscreens for suppressing the wind noise.</li>
		<li>Insert rechargeable batteries and a large-capacity high-speed memory card into the LPCM recorder and turn it on. Set the recording conditions properly in such a manner that the sound signals are recorded on the memory card as an LPCM format at a sampling rate of 96 kHz with a 24-bit depth.</li>
		<li>Place the body of the system into a pocket-sized bag.</li>
	</ol>
	</li>
	<li>Setup of the Reversed Audition System with a Participant 
	<ol>
		<li>Instruct a participant to insert the earphones of the reversed audition system tightly into the ear canals.</li>
		<li>Disconnect the lines for the left and right microphones and connect the dominant-ear side of the microphone straight through to the recorder.</li>
		<li>Instruct the participant to take off and put on the dominant-ear side of the system repetitively while adjusting the sound volume of the recorder to make the subjective loudness of direct (normal) and indirect (reversed) sounds equal (as close as possible).</li>
		<li>Check the loudness for the non-dominant ear as well, and connect all the lines of the system back again.</li>
		<li>Place the system into the participant&#8217;s pocket, fix the cords on the participant&#8217;s clothes appropriately to prevent them from becoming entangled, and pick up unwanted noises.</li>
	</ol>
	</li>
</ol>
2. Validation of the Left-Right Reversed Audition System
NOTE: Perform the following steps to validate the left-right reversed audition system, irrespective of experiments studying adaptation to left-right reversal.
<ol>
	<li>Validation of the Sound Source Localization of the Reversed Audition System
	<ol>
		<li>Locate a digital angle protractor whose initial direction is defined as 0&#176; at the center of an anechoic room, and assume a virtual circle centered at this point with a radius of 2 m. Along the virtual circle, mark 72 possible sound sources at every 5&#176; from -180&#176; to 175&#176; in a clockwise manner, and set up plane-wave speakers at these points directed towards the center of the circle.</li>
		<li>Set up a video camera near the center of the room to record the display of the digital protractor. 
		NOTE: Since the display of the protractor moves with the protractor&#8217;s body, the field of view of the video should be large enough to cover all the possible areas. Moreover, the video camera should be carefully placed in order to not disturb the participant&#8217;s sitting position and the sound presentation.</li>
		<li>Prepare for two sessions of sound source localization: in the first session, the participant does not put on the reversed audition system. In the second session, the participant puts on the equipment, calibrates it, and checks the system (as explained in step 1.2) as quickly as possible.</li>
		<li>Guide the participants to sit comfortably and blindfolded at the center of the circle facing a 0&#176;&#160;sound source and wait for the experiment to start.</li>
		<li>Conduct two sessions of sound source localization. In both sessions, have the participant use the protractor to indicate the perceived sound direction as precisely as possible without moving the head.</li>
		<li>For each session, start video-recording the angle display of the protractor, and present 1000-Hz sounds at 65-dB sound pressure level (SPL) from any of the sound sources: the sound at one location is randomly switched to the sound at another location every 10 s in such a way that each location is used once. 
		NOTE: Here we use MATLAB with the Psychophysics Toolbox16,17,18. Although this toolbox is commonly used to present sounds, any reliable stimulation software can also be used.</li>
		<li>After each session, stop the video-recording and instruct the participants to take a break for sufficient amount of time.</li>
		<li>Read the trial-by-trial perceptual angles displayed on the protractor from the recorded video, and evaluate the spatial performance of the reversed audition system by comparing the perceptual angles in the normal and the reversed conditions against the physical angles defined by the direction of sound sources.</li>
	</ol>
	</li>
	<li>Validation of the Delay of the Reversed Audition System
	<ol>
		<li>Put the reversed audition system on a desk in a calm room with no participants.</li>
		<li>Disconnect a line to the left microphone, and place a plane-wave speaker and the left earphone as close as possible to the right microphone.</li>
		<li>Start recording direct (normal) sounds from the speaker and indirect (reversed) sounds from the left earphone simultaneously through the right microphone.</li>
		<li>Present 1-ms click sounds from the speaker with a moderate inter-stimulus interval at 65-dB SPL.</li>
		<li>After a sufficient number of trials, stop presenting and recording the sounds.</li>
		<li>In order to confirm the symmetrical configuration of the system, repeat the same steps above using the right earphone and the left microphone.</li>
		<li>Read the recorded sound data using software (e.g., MATLAB) and evaluate the difference between the onset timings of the direct (normal) sounds and indirect (reversed) sounds, which corresponds to a potential delay caused by the time spent passing through the electrical path in the system.</li>
	</ol>
	</li>
</ol>
3. Studying the Adaptation to Left-Right Reversed Audition
<ol>
	<li>Procedure of the Exposure to Reversed Audition
	<ol>
		<li>Remind the participants repeatedly of their right to quit the exposure at any time. 
		NOTE: Stop the exposure as soon as possible if the participant reports sickness or if an observer notices any sign that the participant wants to quit the exposure for any reason.</li>
		<li>Prepare a sufficient number of spare rechargeable batteries and large-capacity high-speed memory cards to allow the participant to replace them at anytime.</li>
		<li>Instruct the participant to wear, calibrate, and check the reversed audition system by themselves during the exposure period, as explained in step 1.2. Perform the same procedure each time the participant wears the system after each interruption.</li>
		<li>Instruct the participant to perform daily-life activities while wearing the system continuously for approximately a month, except while sleeping, bathing, neuroimaging, and other emergency times. In these cases, ask participants to remove the system and immediately insert earplugs into their ears to prevent recovery of adaptation. 
		NOTE: Although it is ideal for the participant to wear the system all day and night, it is strongly recommended that the system not be worn while sleeping and bathing in order to prevent unexpected loud noises and electrical shocks, respectively.</li>
		<li>Replace the batteries and memory cards routinely before battery exhaustion and memory overcapacity, respectively. Remove the system and replace it with earplugs quickly in a silent place without producing any sound.</li>
		<li>When a participant needs to move around outside, drive the participant in a car, accompany the participant on the move, or ask them to use safe means of transportation for acts performed alone. 
		NOTE: Great care should be taken by the researcher in order to not endanger the participant&#8217;s safety during the exposure period, especially when the participant goes outside. Prohibit the participant from performing any dangerous behaviors.</li>
		<li>In order to facilitate adaptation, instruct the participant to experience situations involving high auditory input, such as walking in a shopping mall or a campus, having a conversation with more than two persons, and playing 3D video games, for as long as possible.</li>
		<li>Instruct the participant to keep a diary or provide a subjective report to an observer as frequently as possible about perceptual and behavioral changes, experienced events, and anything that the participant notices.</li>
		<li>After the target exposure period, instruct the participant to take off the reversed audition system. 
		NOTE: It is also important to follow up about the perceptual and behavioral changes in order to examine the recovery process from the adaption to left-right reversed audition.</li>
	</ol>
	</li>
	<li>Neuroimaging During the Exposure to Reversed Audition
	<ol>
		<li>Instruct the participant to train on a task that will be used during the neuroimaging experiments as sufficiently as possible.
		<ol>
			<li>For example, train the participant to perform a selective reaction time task in two conditions, compatible and incompatible15. The compatible condition consists of responding immediately to the right-ear sound with the right index finger and to the left-ear sound with the left index finger. The incompatible condition consists of responding immediately to the right-ear sound with the left index finger and to the left-ear sound with the right index finger.</li>
			<li>Use 1000-Hz sounds at 65-dB SPL for 0.1 s with an inter-stimulus interval of 2.5 &#8211; 3.5 s, which appears pseudorandomly on either ear side.</li>
		</ol>
		</li>
		<li>Before the exposure to reversed audition, conduct a neuroimaging experiment under the trained task.
		<ol>
			<li>For example, record either MEG or EEG responses, as well as the left and right finger responses under the selective reaction time task15. The task consists of two compatible and two incompatible blocks that are alternatively arranged with an inter-block interval of at least 30 s, and with sounds appearing 80 times for each block through the inserted earphones with plastic ear tubes. 
			NOTE: Although a 122-channel MEG system was used in Aoyama and Kuriki15, a multi-channel EEG system is also suitable for this protocol.</li>
			<li>For the MEG/EEG recording, set the sampling rate at 1 kHz and the analog recording passband at 0.03 &#8211; 200 Hz.</li>
		</ol>
		</li>
		<li>During approximately a 1-month exposure to reversed audition, conduct neuroimaging experiments under the trained task every week without the reversed audition system in exactly the same way as in the pre-exposure experiment (step 3.2.2). 
		NOTE: The system is removed immediately before and put on immediately after each experiment.</li>
		<li>One week after the exposure, conduct a neuroimaging experiment under the trained task in exactly the same way as the pre-exposure experiment (step 3.2.2).</li>
		<li>Analyze the collected data before, during, and after the exposure to left-right reversed audition.
		<ol>
			<li>For example, after rejecting the epochs contaminated with eye-related artifacts, removing the offset in the pre-stimulus interval, and setting the low-pass filtering at 40 Hz, average the MEG/EEG data from 100 ms before to 500 ms after the sound onset for the stimulus-response compatible and incompatible conditions15.</li>
			<li>Using an MNE software package19,20, estimate the sources of the brain activity with dynamic statistical parametric maps (dSPMs) overlaid on cortical surface images and quantify the intensities of brain activity with minimum-norm estimates (MNEs) for each time point of the averaged data.</li>
			<li>Calculate the auditory-motor functional connectivity from single-trial zero-mean MEG/EEG data from 90 to 500 ms after the sound onset for each condition 
			NOTE: Here we use&#160;MATLAB with the Multivariate Granger Causality Toolbox21.</li>
			<li>For the behavioral data, calculate the mean reaction times for the stimulus-response compatible and incompatible conditions.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The author has nothing to disclose.

The proposed protocol aimed to establish a methodology for studying adaptation to left-right reversed audition as an effective tool for uncovering the adaptability of humans to a novel auditory environment. As evidenced by the representative results, the constructed apparatus achieved left-right reversed audition with high spatiotemporal accuracy. Although the previous apparatuses for reversed audition11,12,13,14 were mostly reliable in the front auditory field, this protocol provides high performance in a 360&#186;&#160;sound source localization coupled with hearing characteristics. Moreover, a potential delay of 2 ms lost through the electrical path in the system, which has never been evaluated in other electronic apparatuses13,14, is considered to be negligible due to the human temporal auditory acuity22. Unlike the traditional apparatus of curved trumpets11,12 with a strange appearance and uncomfortable fit, the reversed audition system used in the present protocol looks like a mobile music player and enables a participant to focus on daily life without arousing curiosity or drawing attention of other individuals. At this point, it is even superior to the apparatuses for reversed vision using prisms1,2,3,4,5,6,7. Indeed, as evidenced by the representative results, around 1 month of wearing the apparatus achieved adaptation to left-right reversed audition at the perceptual, behavioral, and neural levels. As in previous protocols11,12,13,14, it was quite challenging to perform experiments with many participants, due to the long research period and difficulties in participant recruitment. However, individual results provided reliable, rich and valuable information about auditory adaptation (for details, see Aoyama and Kuriki15). Therefore, the present protocol is much better suited for facilitating the adaptation to reversed audition than any other previous protocols that have failed to noticeably advance knowledge about the adaptation11,12,13,14.
As a basic premise, the highest priority in the proposed protocol should be the participant&#39;s safety, health, and will during the exposure to the reversed audition. In order to preserve these, an observer must take great care and communicate with the participant as much as possible, especially during and immediately after the exposure period. If any of the conditions are unsatisfactory, an observer must stop the exposure immediately. Apart from that, one of the most critical steps of the protocol is to instruct the participant to experience situations involving high auditory input for as long as possible. Unlike visual cases where the retinal input has fine spatial resolution23,24, exposure to reversed audition is less effective due to low auditory spatial resolution25,26. In addition, non-environmental auditory events rarely occur in daily life, unless a person is subjected to high auditory inputs. Moreover, it is not enough for sounds to be directional and lateralized, but the sounds should also be accompanied by other sensory information or movement to facilitate the adaptation. Without this step, lower, or even no adaptive effect, is expected. Another critical step is to instruct the participant to train on a task as sufficiently as possible before the first neuroimaging experiment so that task performance converges at a certain level. This is necessary for a precise evaluation of the adaptive effect on behavioral and neural responses, because it is quite difficult to dissociate between the adaptive and the task learning effects over time. Preliminary reduction of the task learning effect thus promotes further analysis of the adaptation.
The present protocol can be flexibly modified, depending on the availability of experimental equipment and the purpose of study. For example, to validate the sound source localization of the reversed audition system, it is acceptable to employ another established method for sound source localization, instead of the digital angle protractor, and a sufficiently calm soundproof room, instead of an anechoic room. To study the adaptation to left-right reversed audition, the exposure period can be either shortened or prolonged and the frequency of neuroimaging can be either lower or higher, according to the situation. For further study, it is recommended to perform neuroimaging more frequently after the exposure period to investigate the recovery process after the adaptation. If neuroimaging is unavailable, it is possible to replace neuroimaging experiments by behavioral experiments. In this protocol, there is a possibility that a participant will request temporary suspension of the exposure due to inevitable reasons. Unless the participant agrees to insert earplugs into the ears during the suspended period, the exposure should be terminated due to unknown recovery effects on readaptation; a new experiment should be started with another participant. Another possible issue is that a balance of subjective loudness between left and right sounds becomes uncertain due to physical contact with the system or for other reasons. In that case, it is recommended for the participant to confirm, with the eyes closed, if the sounds emanating from the front are only localized at the front before readjusting the volume.
Even though the present apparatus showed high performance in 360&#176; sound source localization, the results indicated a slight perceptual bias toward the counterclockwise rotation, especially for sounds coming from the right-front and the left-back directions. Assuming that the earphones are properly inserted into the participant&#39;s ear canals, two possibilities are considered for the asymmetrical distortion of the localization: individual acoustic characteristics and spillover of unreversed sounds. Acoustic characteristics are typically modeled as head-related transfer functions (HRTFs)27, and common HRTFs are used for any participant in the current version of the apparatus without specific optimization. Thus, there is room to improve the apparatus by implementing individual HRTFs for each ear and participant. In contrast, slight spillover of unreversed sounds is relatively uncontrollable. Although separation of microphone and earphone parts of the system reduces the spillover and usual sounds are unlikely to generate perceptible bone conduction28, it is technically difficult to prevent the spillover completely in a wearable way. Moreover, during the exposure, it is almost impossible to control bone-conducted self-produced voices; thus, there is nothing to do but to assume a symmetric distribution for them. Therefore, it is considered that the implementation of individual HRTFs is the priority to improve the apparatus and achieve more effective adaptation.
To our knowledge, this is the first successful protocol established for studying the long-term adaptation to precise left-right reversed audition with neuroimaging. In addition, this protocol has a great potential for extensive applicability in both auditory and multisensory research. For example, the system incorporating a microcomputer could be set up to induce different alterations in auditory space, such as an overall rightward shift or a compression of auditory space toward the center. Since spatial information is concordantly processed across sensory modalities, altered auditory space could be a strong tool to reveal mechanisms of multisensory spatial recalibration in a way similar to Zwiers et al.29, who reported the effects of wearing prism lenses with spatially compressed vision on sound source localization. Nowadays, it is becoming increasingly popular to use currently available techniques in a multimodal manner, such as the simultaneous use of EEG and fMRI30, and a delayed combined use of transcranial brain stimulation and EEG/MEG31. While the simultaneous use of two neuroimaging techniques compensates for their weaknesses reciprocally, the delayed combined use of neurostimulation and neuroimaging techniques reveals brain functions related to after-effects caused by the neurostimulation using the neuroimaging. Notably, an experimental scheme of the present protocol can be regarded as an expanded version of the latter case. Similar to the neurostimulation techniques, continuous wearing of a wearable apparatus with unusual sensory space causes after-effects of adaptation. These effects can be then measured by a neuroimaging technique. Therefore, the delayed combined use of a wearable apparatus and a neuroimaging technique reveals brain functions related to adaptation (as briefly pointed out in Aoyama and Kuriki15). From a general point of view, this scheme can provide new insights into neuroimaging studies with a variety of adaptive effects. In conclusion, the present protocol, under this scheme, provides a promising methodology for studying left-right reversed audition as a tool to uncover the adaptability of humans to a novel environment in the auditory domain.

Adaptability to a novel environment is one of the fundamental functions for humans to live robustly in any situation. One effective tool for uncovering the mechanism of environmental adaptability in humans is an unusual sensory space that is artificially produced by apparatuses. In the majority of the previous studies dealing with this topic, special spectacles with prisms have been used to achieve left-right reversed vision1,2,3,4,5 or up-down reversed vision6,7. Furthermore, exposure to such vision from a few days to more than a month has revealed perceptual and behavioral adaptation1,2,3,4,5,6,7 (e.g., capability to ride a bicycle2,5,7). Moreover, periodic measurements of the brain activity using neuroimaging techniques, such as electroencephalography (EEG)1, magnetoencephalography (MEG)3, and functional magnetic resonance imaging (fMRI)2,4,5,7, have detected changes in the neural activity underlying the adaptation (e.g., bilateral visual activation for unilateral visual stimulation4,5). Although the participant&#39;s appearance becomes strange to some extent and great care is needed for the observer to maintain the participant&#39;s safety, reversed vision with prisms provides precise three-dimensional (3D) visual information without any delay in a wearable manner. Therefore, the methodology for uncovering the mechanism of environmental adaptability is relatively established in the visual domain.
In 1879, Thompson proposed a concept of pseudophone, &#34;an instrument for investigating the laws of binaural audition by means of the illusions it produces in the acoustic perception of space&#34;8. However, in contrast to the visual cases1,2,3,4,5,6,7, few attempts have been made to study the adaptation to unusual auditory spaces, and no noticeable knowledge has been obtained to date. Despite a long history of developing virtual auditory displays9,10, wearable apparatuses for controlling 3D audition have rarely been developed. Hence, only a few reports examined the adaptation to left-right reversed audition. One traditional apparatus consists of a pair of curved trumpets that are crossed and inserted into a participant&#39;s ear canals in a contrariwise manner11,12. In 1928, Young first reported the use of these crossed trumpets and wore them continuously for 3 days at most or a total of 85 h to test adaptation to left-right reversed audition. Willey et al.12 retested the adaptation in three participants wearing the trumpets for 3, 7, and 8 days, respectively. The curved trumpets easily provided left-right reversed audition, but had an issue with the reliability of spatial accuracy, wearability, and strange appearance. A more advanced apparatus for the reversed audition is an electronic system in which left and right lines of head/earphones and microphones are reversely connected13,14. Ohtsubo et al.13 achieved auditory reversal using the first ever binaural headphone-microphones that were connected to a fixed amplifier and evaluated its performance. More recently, Hofman et al.14 cross-linked complete-in-canal hearing aids and tested adaptation in two participants that wore the aids for 49 h in 3 days and 3 weeks, respectively. Although these studies have reported high performance of sound source localization in the front auditory field, the sound source localization in the backfield and a potential delay of electrical devices have never been evaluated. Especially in Hofman et al.&#39;s study, the spatial performance of the hearing aids was guaranteed for the front 60&#176; in the head-fixed condition and for the front 150&#176; in the head-free condition, suggesting unknown omniazimuth performance. Moreover, the exposure period may be too short to detect phenomena related to the adaptation as compared with the longer cases of reversed vision2,4,5. None of these studies have measured brain activity using neuroimaging techniques. Therefore, the uncertainty in spatiotemporal accuracy, the short exposure periods, and the non-utilization of neuroimaging could be reasons for the small number of reports and the limited amount of knowledge on adaptation to left-right reversed audition.
Thanks to the recent advances in wearable acoustic technology, Aoyama and Kuriki15 succeeded in constructing a left-right reversed 3D audition using only wearable devices that recently became available and achieved the omniazimuth system with high spatiotemporal accuracy. Moreover, approximately a 1-month exposure to reversed audition using the apparatus exhibited some representative results for MEG measurements. Based on this report, we describe, in this article, a detailed protocol to set-up, validate and use the system, and to test the adaptation to left-right reversed audition with the help of neuroimaging that is performed periodically without the system. This approach is effective for uncovering the adaptability of humans to a novel environment in the auditory domain.

<table>
	<tbody>
		<tr>
			<td>Linear pulse-code-modulation recorder</td>
			<td>Sony</td>
			<td>PCM-M10</td>
			<td></td>
		</tr>
		<tr>
			<td>Binaural microphones</td>
			<td>Roland</td>
			<td>CS-10EM</td>
			<td></td>
		</tr>
		<tr>
			<td>Binaural in-ear earphones</td>
			<td>Etymotic Research</td>
			<td>ER-4B</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital angle protractor</td>
			<td>Wenzhou Sanhe Measuring Instrument</td>
			<td>5422-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Plane-wave speaker</td>
			<td>Alphagreen</td>
			<td>SS-2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Video camera</td>
			<td>Sony</td>
			<td>HDR-CX560</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2012a, R2015a</td>
			<td>R2012a for stimulation and R2015a for analysis</td>
		</tr>
		<tr>
			<td>Psychophysics Toolbox</td>
			<td>Free</td>
			<td>Version 3</td>
			<td>http://psychtoolbox.org</td>
		</tr>
		<tr>
			<td>Insert earphones</td>
			<td>Etymotic Research</td>
			<td>ER-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetoencephalography system</td>
			<td>Neuromag</td>
			<td>Neuromag-122 TM</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroencephalography system</td>
			<td>Brain Products</td>
			<td>acti64CHamp</td>
			<td></td>
		</tr>
		<tr>
			<td>MNE</td>
			<td>Free</td>
			<td>MNE Software Version 2.7, 
			MNE 0.13</td>
			<td>https://martinos.org/mne/stable/index.html</td>
		</tr>
		<tr>
			<td>The Multivariate Granger Causality Toolbox</td>
			<td>Free</td>
			<td>mvgc_v1.0</td>
			<td>http://www.sussex.ac.uk/sackler/mvgc/</td>
		</tr>
	</tbody>
</table>

a method to study adaptation to left-right reversed audition

An unusual sensory space is one of the effective tools to uncover the mechanism of adaptability of humans to a novel environment. Although most of the previous studies have used special spectacles with prisms to achieve unusual spaces in the visual domain, a methodology for studying the adaptation to unusual auditory spaces has yet to be fully established. This study proposes a new protocol to set-up, validate, and use a left-right reversed stereophonic system using only wearable devices, and to study the adaptation to left-right reversed audition with the help of neuroimaging. Although individual acoustic characteristics are not yet implemented, and slight spillover of unreversed sounds is relatively uncontrollable, the constructed apparatus shows high performance in a 360&#176; sound source localization coupled with hearing characteristics with little delay. Moreover, it looks like a mobile music player and enables a participant to focus on daily life without arousing curiosity or drawing attention of other individuals. Since the effects of adaptation were successfully detected at the perceptual, behavioral, and neural levels, it is concluded that this protocol provides a promising methodology for studying adaptation to left-right reversed audition, and is an effective tool for uncovering the adaptability of humans to a novel environments in the auditory domain.

The representative results shown here are based on Aoyama and Kuriki15. The present protocol achieved left-right reversed audition with high spatiotemporal accuracy. Figure 1 shows the sound source localization in directions over 360&#176; before and immediately after putting on the left-right reversed audition system (Figure 1A), in six participants, as indicated by the cosine similarity. As shown in Figure 1B, the perceptual angles in the normal condition were quite well correlated with the physical angles (positive correlation, adjusted R2 = 0.99). The perceptual angles in the reversed condition were also well correlated with the physical angles (negative correlation, adjusted R2 = 0.96; see also Figure 4 in Aoyama and Kuriki15), although there existed a slight perceptual bias toward the counterclockwise rotation, especially for sounds coming from the right-front and the left-back directions. Notably, the perceptual angles in the reversed condition were more correlated with the oppositely arranged perceptual angles in the normal condition (adjusted R2 = 0.98) than the physical angles, as shown in Figure 1C. Furthermore, a potential delay of the system was estimated to be a constant 2 ms. The present protocol also achieved a natural wearing appearance, like listening to music with a mobile music player, thereby avoiding any stress of being noticed by other individuals.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56808/56808fig1.jpg" /> 
Figure 1: Sound source localization in 360&#176; directions, before and immediately after putting on the left-right reversed audition system, in six participants. (A) The constructed left-right reversed audition system. (B) Cosine similarity between perceptual angles and sign-regulated physical angles in the normal (blue) and reversed (red) conditions plotted against (unregulated) physical angles, respectively. While the physical angles are directly used for the cosine similarity in the normal condition, the signs of physical angles are inverted in the reversed condition. (C) Cosine similarity between perceptual angles in the reversed condition and oppositely arranged perceptual angles in the normal condition plotted against physical angles (purple). This figure has been modified from Aoyama and Kuriki15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56808/56808fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The present protocol revealed perceptual changes to the reversed audition from a relatively early stage during the approximately 1-month exposure. Although a feeling of strangeness was reported just after the exposure, it began to decrease within a week of the exposure and continued to drop further over time. Mirror-image sounds were gradually perceived as normal, which also occurred with visual information and movements. One week after the end of the exposure period, all changes returned to the pre-exposure level. The present protocol detected not only perceptual but also behavioral and neural changes underlying the adaptation. Figure 2 shows changes in behavioral and neural responses during the selective reaction time task over the exposure time in a representative participant. As shown in Figure 2A, the mean reaction times for response-incompatible sounds were overall longer than those for response-compatible sounds from the pre-exposure period to the third week, but became slightly shorter in the fourth week. This relative inversion followed the transient elongation of the mean reaction times irrespective of compatibility in the second week. After the exposure, all mean reaction times returned to the initial level. The MNE intensities of the left and right N1m components exhibited similar trends to the mean reaction times, as shown in Figure 2B, although the compatible-incompatible relationship was inversed. The N1m components are distinct auditory evoked fields observed at about 90 ms after sound onset, and their source was confirmed to be located in the bilateral superior temporal planes using dSPMs. Overall, the intensities in the stimulus-response compatible conditions were higher than those in the incompatible conditions from the pre-exposure period to the third week, but were slightly lower in the fourth week. This relative inversion followed the transient enhancement of the intensities irrespective of compatibility and laterality in the second week. After the exposure, they returned to the initial levels.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56808/56808fig2v2.jpg" /> 
Figure 2: Behavioral and neural responses during the selective reaction time task in a representative participant. (A) Mean reaction times for stimulus-response compatible and incompatible conditions. (B) Left and right auditory N1m intensities for stimulus-response compatible and incompatible conditions, as evaluated by minimum-norm estimates. Yellow zones indicate a period exposed to left-right reversed audition. This figure has been modified from Aoyama and Kuriki15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56808/56808fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Furthermore, the present protocol revealed changes in the functional connectivity across the left and right auditory and motor areas during the selective reaction time task in two participants, as shown in Figure 3. The functional connectivity was tested with the Granger causality test at a threshold of p &#60; 0.05. Initially, these auditory-motor areas communicated with each other irrespective of stimulus and response. However, after exposure to the reversed audition, the auditory-motor connectivity became unstable. Notably, in the second week, the auditory-motor connectivity was disrupted drastically, especially in the right motor-to-auditory feedback and left-to-right motor communication. Immediately after that, the connectivity recovered at the level of the first week, and returned to the initial level after the exposure.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56808/56808fig3.jpg" /> 
Figure 3: Auditory-motor functional connectivity as tested by Granger causality tests during the selective reaction time task in two participants. Red, yellow, and no arrow(s) indicate the number of participants who showed significance at a threshold of p &#60; 0.05 (N = 2, 1, and 0, respectively). LM and RM denote left and right motor areas, respectively, and LA and RA denote left and right auditory areas, respectively. This figure has been modified from Aoyama and Kuriki15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56808/56808fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Method to Study Adaptation to Left-Right Reversed Audition at JoVE.com

A Method to Study Adaptation to Left-Right Reversed Audition

The present study proposes a protocol to investigate the adaptation to left-right reversed audition achieved only by wearable devices, using neuroimaging, which can be an effective tool for uncovering the adaptability of humans to a novel environment in the auditory domain.

An unusual sensory space is one of the effective tools to uncover the mechanism of adaptability of humans to a novel environment. Although most of the ...

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6.4K Views.  Keio University. The present study proposes a protocol to investigate the adaptation to left-right reversed audition achieved only by wearable devices, using neuroimaging, which can be an effective tool for uncovering the adaptability of humans to a novel environment in the auditory domain.

Video: A Method to Study Adaptation to Left-Right Reversed Audition

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 ...

Rodent Brain Microinjection to Study Molecular Substrates of Motivated Behavior

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as appropriate models, partly because the response to behaviorally relevant stimuli and the circuitry parsing stimulus-action outcomes is astonishingly similar between humans and rodents. In studying molecular substrates of complex behaviors, the microinjection of reagents that modify, augment, or silence specific systems is an invaluable technique. However, it is crucial that the microinjection site is precisely targeted in order to aid interpretation of the results. We present a method for the manufacture of surgical implements and microinjection needles that enables accurate microinjection and unlimited customizability with minimal cost. Importantly, this technique can be successfully completed in awake rodents if conducted in conjunction with other JoVE articles that covered requisite surgical procedures. Additionally, there are many behavioral paradigms that are well suited for measuring motivation. The progressive ratio is a commonly used method that quantifies the efficacy of a reinforcer to maintain responding despite an (often exponentially) increasing work requirement. This assay is sensitive to reinforcer magnitude and pharmacological manipulations, which allows reinforcing efficacy and/ or motivation to be determined. We also present a straightforward approach to program operant software to accommodate a progressive ratio reinforcement schedule.

Rodents are an appropriate model to investigate the molecular substrates of behavior and complex psychiatric disorders. Brain microinjection in awake rodents can be used to elucidate disease substrates. An efficient and customizable brain microinjection method as well as the execution of an operant paradigm that quantifies motivation is presented.

Brain microinjection can aid elucidation of the molecular substrates of complex behaviors, such as motivation. For this purpose rodents can serve as ...

Visualizing Visual Adaptation

Many techniques have been developed to visualize how an image would appear to an individual with a different visual sensitivity: e.g., because of optical or age differences, or a color deficiency or disease. This protocol describes a technique for incorporating sensory adaptation into the simulations. The protocol is illustrated with the example of color vision, but is generally applicable to any form of visual adaptation. The protocol uses a simple model of human color vision based on standard and plausible assumptions about the retinal and cortical mechanisms encoding color and how these adjust their sensitivity to both the average color and range of color in the prevailing stimulus. The gains of the mechanisms are adapted so that their mean response under one context is equated for a different context. The simulations help reveal the theoretical limits of adaptation and generate &#34;adapted images&#34; that are optimally matched to a specific environment or observer. They also provide a common metric for exploring the effects of adaptation within different observers or different environments. Characterizing visual perception and performance with these images provides a novel tool for studying the functions and consequences of long-term adaptation in vision or other sensory systems.

This article describes a novel method for simulating and studying adaptation in the visual system.

Many techniques have been developed to visualize how an image would appear to an individual with a different visual sensitivity: e.g., because of optical ...

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 ...

Using a Split-belt Treadmill to Evaluate Generalization of Human Locomotor Adaptation

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. However, studying human locomotor learning can be challenging. During infancy and childhood, the neuromuscular system is quite immature, and it is unlikely that locomotor learning during early stages of development is governed by the same mechanisms as in adulthood. By the time humans reach maturity, they are so proficient at walking that it is difficult to come up with a sufficiently novel task to study de novo locomotor learning. The split-belt treadmill, which has two belts that can drive each leg at a different speed, enables the study of both short- (i.e., immediate) and long-term (i.e., over minutes-days; a form of motor learning) gait modifications in response to a novel change in the walking environment. Individuals can easily be screened for previous exposure to the split-belt treadmill, thus ensuring that all experimental participants have no (or equivalent) prior experience. This paper describes a typical split-belt treadmill adaptation protocol that incorporates testing methods to quantify locomotor learning and generalization of this learning to other walking contexts. A discussion of important considerations for designing split-belt treadmill experiments follows, including factors like treadmill belt speeds, rest breaks, and distractors. Additionally, potential but understudied confounding variables (e.g., arm movements, prior experience) are considered in the discussion.

We describe a protocol for investigating human locomotor adaptation using the split-belt treadmill, which has two belts that can drive each leg at a different speed. We specifically focus on a paradigm designed to test the generalization of adapted locomotor patterns to different walking contexts (e.g., gait speeds, walking environments).

Understanding the mechanisms underlying locomotor learning helps researchers and clinicians optimize gait retraining as part of motor rehabilitation. ...

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors remains poorly understood. In Drosophila melanogaster, environmental cues, such as food availability, affect mating activity offering a tractable system to investigate the mechanisms modulating sexual behavior. In D. melanogaster, environmental cues are often sensed via the chemosensory gustatory and olfactory systems. Here, we present a method to test the effect of environmental chemical cues on mating behavior. The assay consists of a small mating arena containing food medium and a mating couple. The mating frequency for each couple is continuously monitored for 24 h. Here we present the applicability of this assay to test environmental compounds from an external source through a pressurized air system as well as manipulation of the environmental components directly in the mating arena. The use of a pressurized air system is especially useful to test the effect of very volatile compounds, while manipulating components directly in the mating arena can be of value to ascertain a compound's presence. This assay can be adapted to answer questions about the influence of genetic and environmental cues on mating behavior and fecundity as well as other male and female reproductive behaviors.

A Method to Test the Effect of Environmental Cues on Mating Behavior in Drosophila melanogaster

We demonstrate an assay to analyze the environmental and genetic cues that influence mating behavior in the fruit fly Drosophila melanogaster.

An individual's sexual drive is influenced by genotype, experience and environmental conditions. How these factors interact to modulate sexual behaviors ...

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

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. ...

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

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

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

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