This work was supported by the Hakubi Project of Kyoto University, Grant-in-Aid for Challenging Research (Pioneering) (No.22K18644), Grant-in-Aid for Scientific Research (C) (No. 22K12745 ), Grant-in-Aid for Scientific Research (B) (No. 21H02851), and Grant-in-Aid for Scientific Research (A) (No. 19H01039). We would like to thank Editage (www.editage.jp) for English language editing.

All experiments were approved by the Animal Experimentation Committee of EHUB (No.2022-003, 2023-104) and conducted in accordance with the Guide for Care and Use of Laboratory Primates published by EHUB. Nine common marmosets (Callithrix jacchus, six males and three females, 2-12 years old, weighing 330-490 g) were used for the experiment. Four of the nine marmosets were housed in pairs or families, while five were housed individually. Marmosets in the colony typically remain in family housing until two years of age, after which they are transitioned to either pair or individual housing based on the success of pair formation.
1. Animals
<ol>
	<li>House the marmosets in single cages equipped with nest boxes, wooden perches, and other enrichment devices.</li>
	<li>Maintain the rooms under a 12 h light-dark cycle, with temperature and humidity maintained at 28 &#177; 2 &#176;C and 40 &#177; 20%, respectively.</li>
	<li>Feed the animals 14 g of New World monkey pellets twice a day, supplemented with food such as gum arabic and mealworms. Provide water ad libitum.</li>
	<li>Perform all the experiments in a sound-attenuated box in an experimental room.</li>
</ol>
2. Equipment (Figure 1B and Table of Materials)
<ol>
	<li>Use 4 mm silver electrodes. The AgCl coating on the electrode surface prevents polarization and ensures stable recording.</li>
	<li>Use an amplifier to record the EEG signals. Bandpass filter (0.016-250 Hz) and sample the data at 1,000 Hz.</li>
	<li>Connect a 64-channel Electrode Input Box to the amplifier, placing it in front of the subject.</li>
	<li>Place the speaker 30 cm from the marmoset head and control the sound level at 65-75 dB, as measured in the ear position. Deliver the auditory stimuli via this speaker.</li>
	<li>Place the camera in front of the subject to monitor their condition during the EEG recording.</li>
	<li>The primate chair is constructed of acrylic plates and synthetic resin posts. For the experiments, have the researcher hold the animals as they sit on the footplate; at this point, insert a neckpiece and secure it to the neck panel, and insert and secure the waist piece to the waist panel. Ensure that the entire body of each marmoset is loosely secured.</li>
</ol>
3. Anesthesia
<ol>
	<li>Anesthetize the animals with an intramuscular injection of alfaxalone (6-8 mg/kg) and atropine (0.05 mg/kg). This protocol allows anesthesia to be maintained for approximately 20 min. Administer additional doses of alfaxalone to prolong the duration of anesthesia if the procedure is longer. Also administer antiemetic agents (Maropitant 1 mg/kg, subcutaneous injection) beforehand to counter the nausea, which is a side effect of alfaxalone. 
	NOTE: Marmosets must be under injection or inhalation anesthesia during the procedures and should be allowed to recover immediately after hair shaving.</li>
	<li>Monitor the vitals with a pulse oximeter and administer oxygen-air mixture (O2 0.5 L/min, air 0.5 L/min) if necessary.</li>
	<li>Maintain the room temperature above 27 &#176;C, and wrap a warming cloth around the body of the marmoset to prevent hypothermia.</li>
</ol>
4. Hair removal
<ol>
	<li>Shave the entire head (including behind the auricularia) with an electric shaver.</li>
	<li>Apply hair removal cream for sensitive skin. Wipe off the cream with a wet gauze after 5 min.</li>
</ol>
5. Mask preparation
<ol>
	<li>Process a thermoplastic mask in advance to fit the size of the monkey chair. Specifically, drill the part of the back that protrudes from the vertical length of the chair with four screw holes such that it can be fixed to the chair&#39;s neck plate. 
	NOTE: Thermoplastic masks are safe and can be used to immobilize the head during radiation therapy in patients. We used a small mask designed for children and cut it to fit the size of the marmosets. This mask softens when placed in warm water at 75 &#176;C and hardens as the temperature reduces when it is removed from the water.</li>
	<li>For the experiments, place the animals in a primate chair. Support the anesthetized animal using neck and waist plates and secure the animal and these plates to the chair.</li>
	<li>Warm the mask in hot water and then mold it to fit the head of the marmoset. After removing the mask from the warm water, wait for the temperature to drop (approximately in the 50s &#176;C range) to prevent burns, and then place it on the subject&#39;s head to mold the mask.</li>
	<li>After cooling and hardening, detach the mask from the animal and cut out the top of the head and ear parts of the mask to expose the area for the electrode setting. 
	NOTE: Once this mask was made, it could be used on other individuals.</li>
</ol>
6. Chair and mask adaptation (30 min/day for 3 days)
<ol>
	<li>Habituate the animal to the chair under awake conditions by placing it in a chair and rewarding it for approximately 30 min. Repeat this procedure for 3 days.</li>
	<li>Habituate the animal to head fixation using the mask for 2 days. 
	NOTE: This adaptation process was customized to the individual&#39;s condition. After the adaptation, the animals may resist temporarily during capture and head-mask setting, but once they are seated in the chair and their head is fixed, they become calm.</li>
	<li>During chair adaptation, evaluate the following behavioral parameters: i) emission of anxious or alarming noises, ii) rejection of the offered reward, and iii) violent movements. If any of these behaviors are observed, terminate the session for that day, allowing the marmoset to gradually adapt to the experimental environment.</li>
</ol>
7. EEG recording (2 h/day)
<ol>
	<li>Subject preparation
	<ol>
		<li>Attach a transfer cage to a small window of the home cage and move the marmoset from the home cage to the transfer cage, usually by itself. Cover the carrying cage with a cloth and transfer it to the experimental room.</li>
		<li>Head fixation
		<ol>
			<li>Capture the marmoset using protective gloves and place it in the special chair.</li>
			<li>Place the prepared mask on the marmoset&#39;s head. Pass the screw body attached to the chair through the hole in the mask and fix it using thumb screws.</li>
			<li>Secure the head and mask lightly with a band just below the nasion. 
			NOTE: The subject&#39;s head did not need to be completely fixed as long as the electrodes were not removed by movement. Therefore, we used the same mask across all subjects.</li>
		</ol>
		</li>
		<li>Definition of the location of electrodes
		<ol>
			<li>Use the nasion, inion, and earlobes as anatomical landmarks to determine the location of the electrodes according to the International 10-20 method32,33,34,35,36,37. Measure the distance between the nasion and inion in the mid-line with a tape measure. Define the location of Cz in the middle of the distance. Position the other electrodes (Fz, Pz, Oz, F3, or F4) with 20% of the inion-neion length as the electrode spacing. Mark the electrode positions on the scalp using an oil-based dermatograph.</li>
		</ol>
		</li>
		<li>Skin preparation
		<ol>
			<li>Rub the marked area with a thin cotton swab dipped in rubbing alcohol to remove dirt and sebum from the scalp.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Electrode settings 
	NOTE: Perform steps 7.2.1-7.2.3 at all electrode positions to place electrodes at Fz, Cz, C3, C4, Pz, A1, A2, and F3 (or F4).
	<ol>
		<li>Using a tube cutter, cut a piece of silicon tubing with an inner diameter of 4 mm and an outer diameter of 7 mm into a length of approximately 20 mm.</li>
		<li>Apply adhesive to the edges of the cut tube and adhere it to the scalp.</li>
		<li>Fill the inside of the tube with EEG gel using a syringe and a non-pointed syringe needle.</li>
		<li>Connect the reference electrode to Pz and the ground electrodes to F3 or F4 (Figure 1C).</li>
		<li>Insert electrodes into the tube and connect the electrode cable to the input box.</li>
		<li>Launch the application for EEG recordings to measure electrode impedance, and adjust the parameters to ensure all electrodes are below 5 k&#937;.</li>
		<li>Bundle the electrode cables to reduce noise.</li>
	</ol>
	</li>
	<li>Give rewards during the head immobilization procedure and between task sessions by manually administering 1-3 mL of the liquid reward (gum or nutrition) using a syringe.</li>
	<li>Specify the file to be saved on the EEG recording software and press the Start Recording button. Run the script for stimulus presentation immediately after the EEG recording is started. Press the stop button on the EEG recording when the stimulus presentation script execution is complete to end all recordings. When the recording is completed, remove the electrodes and head mask.</li>
	<li>Capture the animals and return them to their carrying cages. Leave the tubes on the scalp; all the tubes fell off naturally in approximately 1 day. 
	NOTE: In the early periods, we used acetone to dissolve the adhesive glue and remove the tube after the recording sessions; however, there were cases of skin injury; therefore, we subsequently returned the animals to their home cages without removing the tube. In the dozens of experiments, we have conducted thus far, marmosets have not shown any interest in the removed tubes, and no accidental ingestion has ever occurred.</li>
	<li>Stimulus
	<ol>
		<li>Record natural simple and compound calls in the captive or experimental room from marmosets that are not used in subsequent EEG recordings. Extract three species-specific simple and compound calls (Phee, Tsik-Ek, and Tsik-String5 calls) from the recorded files.</li>
		<li>In addition to the call stimuli, create white noise using a function in the programming software and use that as a stimulus.</li>
		<li>To follow this protocol, use three auditory files (16 bit, 48 kHz) of marmoset calls in the experiment. Control the task using a custom script. 
		NOTE: Each block contains 50 calls for each stimulus, for a total of 200 calls. Each recording block will last approximately 10 min. Each participant must perform two blocks. The inter-call interval is 3 s. Of the four audio stimulus files (see Supplemental File 1), the Phee call stimulus was approximately 2 s long, whereas the other three were approximately 1 s long. This was because a single Phee call is still a long-lasting call (Figure 1D).</li>
		<li>In each sound stimulus file, the right channel contains the marmoset call or noise data, and the left channel contains the trigger signal for the onset of the stimulus. Send this trigger signal to the EEG recording system via a synchronization device and record it as event time.</li>
	</ol>
	</li>
</ol>
8. Data analysis
NOTE: The original code written in the Programming software and toolbox was used to postprocess the EEG data, as outlined below (Supplemental File 2)37.
<ol>
	<li>Preprocessing
	<ol>
		<li>Re-reference to the linked ear reference.</li>
		<li>High-pass filter at 2 Hz.</li>
		<li>Epoch from 100 ms before to 1000 ms after stimulus onset.</li>
		<li>Baseline-correct to the mean of the 100-ms prestimulus period. Reject artifacts using a criterion of &#177;150 &#181;V.</li>
	</ol>
	</li>
	<li>Plotting event-related potential (ERP)
	<ol>
		<li>Average all trial data for each subject.</li>
		<li>Obtain group-averaged waveforms by averaging all subjects.</li>
		<li>To compare the averaged ERPs between call types and noise stimuli, apply a one-way analysis of variance (ANOVA) with stimuli as the between-subjects factor in Cz response.</li>
		<li>Apply post hoc multiple comparison analysis with Tukey&#39;s method.</li>
	</ol>
	</li>
	<li>Plotting event-related spectral perturbation (ERSP)
	<ol>
		<li>Calculate the ERSP to visualize the mean event-related change in spectral power over time at a broad frequency range using Equation (1). Fk (f,t) is the spectral estimate of trial k at frequency f and time t: 
		<img alt="Equation 1" src="/files/ftp_upload/66869/66869eq01.jpg" style="margin: auto;" />&#160; &#160; (1)</li>
		<li>Apply time-frequency decomposition to the activities using sinusoidal wavelet transforms, with three cycles of length at the lowest frequency (10 Hz), increasing linearly with frequency up to 32 cycles at the highest frequency (120 Hz).</li>
		<li>Define the initial and transient responses in a 150 ms period after stimulus onset at 2-30 Hz, and the sustained responses in an 800 ms period from 151 to 950 ms after stimulus onset at 40-100 H.</li>
		<li>To test the differences in initial and sustained responses in the Fz and Cz among subject ages and call types, perform a two-way ANOVA using call type as the within-subject factor and ages as the between-subject factor.</li>
	</ol>
	</li>
</ol>

Noninvasive EEG for Comparative Studies of Vocal Perception in Marmosets

The authors have no conflicts of interest to disclose.

Points to note about anesthesia 
Both ketamine and xylazine administration have been attempted, and while these are analgesic and therefore suitable for long painful tasks, marmosets tend to experience decreases in blood oxygen levels without oxygen inhalation44. In short, alfaxalon is probably best suited for painless tasks such as shaving or mask making. In addition, for shaving-, which takes only 10-15 min, inhalation anesthesia would be the most suitable. Isoflurane was n...

Primates use species-specific vocalizations to convey biologically important information, such as the caller&#39;s emotional state or intention to maintain social bonds, the presence of predators, or other dangerous situations. Investigation of the neural mechanisms underlying the perception of vocalization in vocal-rich nonhuman primates may provide us with critical clues to better understand the evolutionary origins of human language.
Common marmosets are small primates native to South America. In recent years, marmosets have been increasingly used as model animals, alongside macaque monkeys, because of their high reproductivity, ease of use owing to their small size, and the development of useful transgenic techniques1,2,3. In addition to their utility as disease models, rich vocal communication within groups is another unique characteristic of this species4,5,6,7. Marmosets routinely exchange vocal signals to communicate with invisible conspecifics in the forest. By examining the brain activity involved in vocal perception and production in marmosets, we can determine how they process the auditory information of their own or conspecific calls in the brain and identify which neural circuits are involved. Previous studies have demonstrated neural activity in the primary auditory cortex8,9,10,11,12 and frontal cortex13,14 involved in vocal production in marmosets. Furthermore, these excited and suppressed neuronal responses were modulated by auditory-vocal interactions in the primary auditory cortex8,10. These studies provided detailed neural activity data at the single-neuron level using invasive recording methods. Numerous studies have further examined the neural activity involved in marmoset vocal production; however, vocal perception remains poorly understood15,16.
Several noninvasive brain imaging studies have elucidated the neural mechanisms of vocal processing in marmosets17,18,19; their high spatial resolution is an advantage, however, keeping animals in the awake state during scanning requires advanced techniques. However, more recently, Jafari et al. identified frontotemporal regions involved in vocal perception in awake marmosets using functional magnetic resonance imaging (fMRI)19. Almost all experiments to elucidate the brain functions involved in vocal perception and production in humans have been conducted using noninvasive methods, such as scalp electroencephalography (EEG), magnetoencephalography (MEG)20,21, and fMRI22,23,24. Numerous studies in humans have investigated brain activity related to vocal perception using EEG. Most of these studies have focused on emotional information25,26,27 and the saliency of emotional words28, with the results revealing changes in event-related potentials during vocal perception29. Electrocorticography (ECoG) and single-neuron recordings using intracranially implanted electrodes in humans have only been conducted in a limited number of experiments in patients undergoing neurosurgical treatment30,31.
An evolutionary perspective comparing humans with monkeys is important when understanding the unique neural mechanisms underlying vocal perception and production that have developed in humans. To directly compare the neural mechanisms involved in speech perception and vocalization in vocal-rich nonhuman primates, such as the marmoset, with humans, it is important to compare data between the two species using the same method. Functional MRI allows whole-brain imaging and has a high spatial resolution. It has the advantage of recording activity perpendicular to the skull or in deep regions that are difficult to record with EEG or MEG. However, the MRI machine is expensive to install and maintain, and there are many restrictions on the stimuli that can be presented due to the nature of the device. In comparison, EEG, event-related potentials (ERPs), and MEG have a high temporal resolution, making them useful for analyzing time-series vocal processing. In particular, EEG has the advantages of high mobility and the ability to be used in a variety of experimental settings, relatively low cost, and the requirement for just a single operator.
Since a large amount of EEG data has already been obtained in humans, EEG measurement methods using non-invasive paradigms are needed for non-human primates. Our research group developed a unique noninvasive EEG recording method using tubes32 for macaques and marmosets. Here, we report several novel findings regarding auditory processing in nonhuman primates33,34,35,36,37. To characterize brain activity in response to species-specific vocalizations in marmosets, we constructed an experimental system to noninvasively record brain activity using electrodes placed on the scalp. In this study, we describe the EEG measurement method for marmosets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alfaxalone</td>
			<td>Meiji Animal Health</td>
			<td>Alfaxan</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Brain Products</td>
			<td>BrainAmp</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Fuso Pharmaceutical Industries</td>
			<td>Atropine Sulfate Injection</td>
			<td></td>
		</tr>
		<tr>
			<td>Audio editor</td>
			<td>Adobe</td>
			<td>Adobe Audition</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processing software</td>
			<td>MathWorks</td>
			<td>MATLAB</td>
			<td>version R2023a</td>
		</tr>
		<tr>
			<td>Data processing toolbox</td>
			<td>University of California-SanDiego</td>
			<td>EEGLAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Data processing toolbox</td>
			<td>University of California-Davis</td>
			<td>ERPLAB</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Panasonic</td>
			<td>ER803PPA</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode</td>
			<td>Unique Medical</td>
			<td>UL-3010</td>
			<td>AgCl coated (custom)</td>
		</tr>
		<tr>
			<td>Electrode gel</td>
			<td>Neurospec AG</td>
			<td>V16 SuperVisc</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode input box</td>
			<td>Brain Products</td>
			<td>EIB64-DUO</td>
			<td>64ch</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td>3M</td>
			<td>Scotch 7005S</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removering cream</td>
			<td>Kracie</td>
			<td>epilat for sensitive skin</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Bussan Animal Health</td>
			<td>ds isoflurane</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid gum</td>
			<td>San-ei Yakuhin Boeki</td>
			<td>Arabic Call SS</td>
			<td>Gum arabic+water</td>
		</tr>
		<tr>
			<td>Liquid nutrition</td>
			<td>Nestl&#233; Health Science Company</td>
			<td>Isocal 1.0 Junior</td>
			<td>Polymeric formula</td>
		</tr>
		<tr>
			<td>Maropitant</td>
			<td>Zoetis&#160;</td>
			<td>Cerenia injectable solution</td>
			<td></td>
		</tr>
		<tr>
			<td>Monitor Camera</td>
			<td>Intel</td>
			<td>RealSense LiDAR Camera L515</td>
			<td></td>
		</tr>
		<tr>
			<td>Monkey pellets</td>
			<td>Oriental Yeast</td>
			<td>SPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Primate chair</td>
			<td>Natsume Seisakusho</td>
			<td>Order made</td>
			<td></td>
		</tr>
		<tr>
			<td>Pulse oximeters</td>
			<td>Covident</td>
			<td>Nellcor</td>
			<td>PM10N</td>
		</tr>
		<tr>
			<td>Skin prepping pasta&#160;</td>
			<td>Mammendorfer Institut f&#252;r Physik und Medizin</td>
			<td>NeuPrep</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicon tube</td>
			<td>AsONE</td>
			<td>&#934;4 x 7mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Speaker</td>
			<td>Fostex</td>
			<td>PM0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Synchronization device</td>
			<td>Brain Vision</td>
			<td>StimTrak</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermoplastic mask</td>
			<td>CIVCO</td>
			<td>MTAPU Type Uniframe Thermoplastic Mask 2.4mm</td>
			<td></td>
		</tr>
	</tbody>
</table>

electroencephalography measurements in awake marmosets listening to conspecific vocalizations 

Vocal communication plays a crucial role in the social interactions of primates, particularly in survival and social organization. Humans have developed a unique and advanced vocal communication strategy in the form of language. To study the evolution of human language, it is necessary to investigate the neural mechanisms underlying vocal processing in humans, as well as to understand how brain mechanisms have evolved by comparing them with those in nonhuman primates. Herein, we developed a method to noninvasively measure the electroencephalography (EEG) of awake nonhuman primates. This recording method allows for long-term studies without harming the animals, and, importantly, allows us to directly compare nonhuman primate EEG data with human data, providing insights into the evolution of human language. In the current study, we used the scalp EEG recording method to investigate brain activity in response to species-specific vocalizations in marmosets. This study provides novel insights by using scalp EEG to capture widespread neural representations in marmosets during vocal perception, filling gaps in existing knowledge.

Author Spotlight: Investigating Vocal Information Representation in Small Primates and Its Alteration by Psychiatric Disorders Using Noninvasive EEG

Electroencephalography Measurements in Awake Marmosets Listening to Conspecific Vocalizations 

We would like to know how vocal information is represented in the brain of small primates which are rich in vocal communications. We would like to illustrate how the representation will be altered by psychiatric disorders such as schizophrenia in the future. We compared non-invasive EEGs across four primate species, and found that the latency of auditory cortex to respond to sounds is longer in humans than in other primates.This would have a significant impact on the perception of complex sounds, such as calls and speech sounds. Few experimental methods allow direct comparison of brain activity obtained from human and animals. We hope to quantify the process of human language evolution by direct comparison, or directly comparing data among various animal species using the non-invasive measurement method.We hope to address this challenge with our protocol.

First, we plotted the average event-related potentials (ERPs) for each auditory stimulus in the marmosets (Figure 2). The auditory evoked potential (AEP) was prominent in the Noise condition, reflecting the clear onset of the stimuli (see Figure 1D). To compare the averaged ERPs between call types and noise stimuli, we applied a one-way analysis of variance (ANOVA) with stimuli as the between-subjects factor in Cz response. We found a significant main e...

To study the evolution of language, comparing brain mechanisms in humans with those in nonhuman primates is important. We developed a method to noninvasively measure the electroencephalography (EEG) of awake animals. It allows us to directly compare EEG data between humans and animals for the long term without harming them.

Vocal communication plays a crucial role in the social interactions of primates, particularly in survival and social organization. Humans have developed a ...

electroencephalography-measurements-awake-marmosets-listening-to

Research

JoVE Journal

Neuroscience

Development and Implementation of Noninvasive EEG Recording Methods for Auditory Processing Studies in Marmosets

After anesthetizing the marmoset, monitor the vitals with a pulse oximeter. If necessary, administer an oxygen air mixture. Maintain the room temperature above 27 degrees Celsius, and wrap a warming cloth around the body of the marmoset to prevent hypothermia.Using an electric shaver, shave the head. Apply hair removal cream for sensitive skin. After five minutes, wipe off the cream with wet gauze.Drill four screw holes into the protruding back portion of the mask to align with the vertical length of the chair. Next place the anesthetized marmoset on the primate chair. Secure the animal on the chair using neck and waist plates.Place the thermoplastic mask plate in hot water to soften it to fit the head of the marmoset. Remove the mask plate from the water and allow it to cool before placing it on the subject's head. After hardening, remove the mask from the marmoset head.Cut out the top of the head and ear parts of the mask to expose the area for the electrode setting. Habituate the marmoset to the chair under awake conditions and reward it for approximately 30 minutes. Connect a 64 channel electrode input box to the amplifier.Arrange the speaker near the amplifier and control the sound level at 65 to 75 decibels to deliver auditory stimuli. Place the camera in front of the subject to monitor their condition during the EEG recording. Then arrange the primate chair in front of the camera.Attach a transfer cage to a small window of the home cage and allow the marmoset to move to the transfer cage. Cover the carrying cage with a cloth and transfer it to the experimental room. Hold the mart using protective gloves and place it on the primate chair.Put the previously prepared mask on the mart's head. Pass the screws attached to the chair through the hole in the mask and fix it using thumb screws. Use the nasion, inion, and ear lobes as anatomical landmarks to determine the location of the electrodes according to the international 10:20 method.Measure the distance between the nasion and inion in the midline with a tape. Define the location of Cz in the middle of the distance. Position the other electrodes with 20%of the inion/nasion length as the electrode spacing.Mark the electrode positions on the scalp using an oil-based dermatograph. Rub the marked area with a thin cotton swab dipped in alcohol to remove dirt and sebum from the scalp. Using a tube cutter, cut a piece of silicon tubing into a length of approximately seven to nine millimeters.Apply adhesive to the edges of the cut tube and adhere it to the scalp. Using a syringe with a non-pointed needle, fill the inside of the tube with EEG gel. Connect the reference electrode to PZ and the ground electrodes to F4 or F3.Insert electrodes into the tube and connect the electrode cable to the input box.Launch the EEG recording application. Adjust the parameters to ensure all electrodes are below five kilo-Ohms and measure the electrode impedance. During head immobilization and between task sessions, administer one to three milliliters of liquid reward using a syringe.Specify the file to be saved on the EEG recording software and press the start recording button. Immediately run the script for stimulus presentation. When the stimulus presentation script execution is completed, press the stop button to end all recordings.Then remove the electrodes and head mask from the marmoset. Capture the animal and place it back in the carrying cage. Record simple and compound calls from marmoset that are not used in subsequent EEG recordings.Extract three species-specific simple and compound calls from the recorded files. Next, create white noise using a function in the programming software and use that as a stimulus. To follow this, use three auditory files of marmoset calls in the experiment.Control the task using a custom script. In each sound stimulus file, the right channel contains the marmoset call or noise data, and the left channel contains the trigger signal for the onset of the stimulus. Send the trigger signal to the EEG system via a synchronization device and record it as event time.The auditory evoked potential was prominent in the noise condition reflecting the clear onset of the stimuli. The time frequency analysis showed that event related spectral power increased at a lower frequency immediately after stimulus onset. The CZ region showed a decrease in gamma range power lasting for one second post stimulus onset in younger subjects.The initial transient responses in the FZ region revealed significant main effects of stimulus, type, and age with no significant interaction. Sustained responses in CZ showed a significant effect of stimulus type, but no effect of age or interaction.

Preparation of an Awake Mouse for Recording Neural Responses and Injecting Tracers

It is well known that anesthesia alters neural response properties in various regions of the brain.13. In the auditory system, fundamental response properties of brainstem neurons including threshold, frequency specificity, and inhibitory sidebands are altered in significant ways under anesthesia1-2. These observations prompted physiologists to seek ways to record from single neurons without the contaminating effects of anesthesia. One result was a decerebrate preparation, where the brainstem was completely transected at the level of the midbrain4. The drawbacks of this preparation are a formidable surgery, the elimination of descending projections from the forebrain, and an inability to use sensory stimulation to examine structures above the midbrain. A different strategy has been to implant electrode arrays chronically to record from single neurons and multiunit clusters while the animal is awake and/or behaving5,6. These techniques however are not compatible with injecting tracer dyes after first electrophysiologically characterizing a brain structure. To avoid altering neural response properties with anesthetics while recording electrophysiological response properties from single neurons, we have adapted a head restraint technique long used in bats7-9 to mouse10-12. Using this method, we are able to conduct electrophysiological recordings over several days in the unanesthetized mouse. At the end of the recording sessions, we can then inject a dye to reconstruct electrode positions and recording sites or inject a tracer so that pathways to and from the recording loci can be determined. This method allows for well isolated single neuron recordings over multiple days without the use anesthetics.

Electrophysiological characterization of neuronal responses is important for understanding brain function and for guiding the placement of dyes for pathway tracing. However, many studies are performed in anesthetized animals. To understand brain function without anesthetics, we developed a method to record neuronal response properties and inject dyes in awake mouse.

It is well known that anesthesia alters neural response properties in various regions of the brain.13.  In the auditory system, fundamental response ...

Simultaneous Monitoring of Wireless Electrophysiology and Memory Behavioral Test as a Tool to Study Hippocampal Neurogenesis

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on animals and humans. Adult neurogenesis mechanism is also linked to memory and learning improvement. Traditionally, researchers used to assess learning and memory parameters in rodent models by behavioral tasks. Therefore, the simultaneous monitoring of behavioral changes and EEG is particularly interesting in correlating data between brain activity and task-related behaviors. However, most of the equipment required to perform both studies are either complex, expensive, or uses a wired setup network that hinders the natural animals&#8217; movement. In this study, EEG was recorded with a wireless electrophysiology device along with the execution of a novel object recognition task (NORT). The animal&#8217;s behavior was monitored simultaneously by a video tracking system. Both recordings were analyzed offline by their timestamps which were synched to link EEG signals with the animal&#8217;s actions. Subjects consist of adult Wistar rats after medium-term environmental enrichment treatment. Six skull screw electrodes were fixed in pairs on both hemispheres over frontal, central, and parietal regions and were referenced to an electrode located posterior of the nasal bone. NORT protocol consists of exposing the animal to two identical objects for 10 min. After 2 h and 24 h, one of the objects was replaced with a novel one. Exploration time for each object was monitored by a behavioral tracking software (BTS) and EEG data recording. The analysis of the EEG synced with behavioral data consists of estimations of alpha and beta relative band power and comparisons between novel object recognition versus familiar object exploration, between three experimental stages. In this manuscript, we have discussed electrodes manufacturing process, epidural electrodes implantation surgery, environmental enrichment protocol, NORT protocol, BTS setup, EEG &#8211; BTS coupling for simultaneous monitoring in real-time, and EEG data analysis based on automatic events detection.

The protocol presented here provides information on the simultaneous electroencephalography (EEG) and behavioral assessment in real-time. We have discussed all steps involved in this protocol as an attractive solution for researchers in many fields of neuroscience, particularly in learning and memory areas.

Brainwaves amplitude obtained from electroencephalography (EEG) has been well-recognized as a basis for cognitive capacity, memory, and learning on ...

Behavior

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Chronic Implantation of Whole-cortical Electrocorticographic Array in the Common Marmoset

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent development of thin, flexible ECoG electrodes has enabled conduction of stable recordings of large-scale cortical activity. We have developed a whole-cortical ECoG array for the common marmoset. The array continuously covers almost the entire lateral surface of cortical hemisphere, from the occipital pole to the temporal and frontal poles, and it captures whole-cortical neural activity in one shot. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain. Marmosets have two advantages regarding ECoG recordings, one being the homologous organization of anatomical structures in humans and macaques, including frontal, parietal, and temporal complexes. The other advantage is that the marmoset brain is lissencephalic and contains a large number of complexes, which are more difficult to access in macaques with ECoG, that are exposed to the brain surface.These features allow direct access to most cortical areas beneath the surface of the brain. This system provides an opportunity to investigate global cortical information processing with high resolutions at a sub-millisecond order in time and millimeter order in space.

We have developed a whole-cortical electrocorticographic array for the common marmoset that continuously covers almost the entire lateral surface of cortex, from the occipital pole to the temporal and frontal poles. This protocol describes a chronic implantation procedure of the array in the epidural space of the marmoset brain.

Electrocorticography (ECoG) allows the monitoring of electrical field potentials from the cerebral cortex with high spatiotemporal resolution. Recent ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...

Multi-system Monitoring for Identification of Seizures, Arrhythmias and Apnea in Conscious Restrained Rabbits

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease and arrhythmias in people with epilepsy (i.e., epileptic heart.) Additionally, cardiac and autonomic disturbances have been reported surrounding seizures. 1:1,000 epilepsy patients/year die of sudden unexpected death in epilepsy (SUDEP). The mechanisms for SUDEP remain incompletely understood. Electroencephalograms (EEG) and electrocardiograms (ECG) are two techniques routinely used in the clinical setting to detect and study the substrates/triggers for seizures and arrhythmias. While many studies and descriptions of this methodology are in rodents, their cardiac electrical activity differs significantly from humans. This article provides a description of a non-invasive method for recording simultaneous video-EEG-ECG-oximetry-capnography in conscious rabbits. As cardiac electrical function is similar in rabbits and humans, rabbits provide an excellent model of translational diagnostic and therapeutic studies. In addition to outlining the methodology for data acquisition, we discuss the analytical approaches for examining neuro-cardiac electrical function and pathology in rabbits. This includes arrhythmia detection, spectral analysis of EEG and a seizure scale developed for restrained rabbits.

Using simultaneous video-EEG-ECG-oximetry-capnography, we developed a methodology to evaluate the susceptibility of rabbit models to develop provoked arrhythmias and seizures. This novel recording system establishes a platform to test the efficacy and safety of therapeutics and can capture the complex cascade of multi-system events that culminate in sudden death.

Patients with ion channelopathies are at a high risk of developing seizures and fatal cardiac arrhythmias. There is a higher prevalence of heart disease ...

Medicine

520 Views.  Kyoto University.  To study the evolution of language, comparing brain mechanisms in humans with those in nonhuman primates is important. We developed a method to noninvasively measure the electroencephalography (EEG) of awake animals. It allows us to directly compare EEG data between humans and animals for the long term without harming them.

Noninvasive EEG for Comparative Studies of Vocal Perception in Marmosets

Summary