Electroencephalography Measurements in Awake Marmosets Listening to Conspecific Vocalizations

Abstract

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.

Introduction

Primates use species-specific vocalizations to convey biologically important information, such as the caller'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 techniques^1,2,3. In addition to their utility as disease models, rich vocal communication within groups is another unique characteristic of this species^4,5,6,7. 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 cortex^8,9,10,11,12 and frontal cortex^13,14 involved in vocal production in marmosets. Furthermore, these excited and suppressed neuronal responses were modulated by auditory-vocal interactions in the primary auditory cortex^8,10. 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 understood^15,16.

Several noninvasive brain imaging studies have elucidated the neural mechanisms of vocal processing in marmosets^17,18,19; 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 fMRI^22,23,24. Numerous studies in humans have investigated brain activity related to vocal perception using EEG. Most of these studies have focused on emotional information^25,26,27 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 treatment^30,31.

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 primates^{33,34,35,36,37}. 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.

Protocol

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.

1. Animals

1. House the marmosets in single cages equipped with nest boxes, wooden perches, and other enrichment devices.
2. Maintain the rooms under a 12 h light-dark cycle, with temperature and humidity maintained at 28 ± 2 °C and 40 ± 20%, respectively.
3. 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.
4. Perform all the experiments in a sound-attenuated box in an experimental room.

2. Equipment (Figure 1B and Table of Materials)

1. Use 4 mm silver electrodes. The AgCl coating on the electrode surface prevents polarization and ensures stable recording.
2. Use an amplifier to record the EEG signals. Bandpass filter (0.016-250 Hz) and sample the data at 1,000 Hz.
3. Connect a 64-channel Electrode Input Box to the amplifier, placing it in front of the subject.
4. 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.
5. Place the camera in front of the subject to monitor their condition during the EEG recording.
6. 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.

3. Anesthesia

1. 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.
2. Monitor the vitals with a pulse oximeter and administer oxygen-air mixture (O₂ 0.5 L/min, air 0.5 L/min) if necessary.
3. Maintain the room temperature above 27 °C, and wrap a warming cloth around the body of the marmoset to prevent hypothermia.

4. Hair removal

1. Shave the entire head (including behind the auricularia) with an electric shaver.
2. Apply hair removal cream for sensitive skin. Wipe off the cream with a wet gauze after 5 min.

5. Mask preparation

1. 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'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 °C and hardens as the temperature reduces when it is removed from the water.
2. 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.
3. 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 °C range) to prevent burns, and then place it on the subject's head to mold the mask.
4. 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.

6. Chair and mask adaptation (30 min/day for 3 days)

1. 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.
2. Habituate the animal to head fixation using the mask for 2 days.
   NOTE: This adaptation process was customized to the individual'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.
3. 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.

7. EEG recording (2 h/day)

1. Subject preparation
   1. 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.
   2. Head fixation
      1. Capture the marmoset using protective gloves and place it in the special chair.
      2. Place the prepared mask on the marmoset's head. Pass the screw body attached to the chair through the hole in the mask and fix it using thumb screws.
      3. Secure the head and mask lightly with a band just below the nasion.
         NOTE: The subject'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.
   3. Definition of the location of electrodes
      1. Use the nasion, inion, and earlobes as anatomical landmarks to determine the location of the electrodes according to the International 10-20 method^{32,33,34,35,36,37}. 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.
   4. Skin preparation
      1. Rub the marked area with a thin cotton swab dipped in rubbing alcohol to remove dirt and sebum from the scalp.
2. 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).
   1. 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.
   2. Apply adhesive to the edges of the cut tube and adhere it to the scalp.
   3. Fill the inside of the tube with EEG gel using a syringe and a non-pointed syringe needle.
   4. Connect the reference electrode to Pz and the ground electrodes to F3 or F4 (Figure 1C).
   5. Insert electrodes into the tube and connect the electrode cable to the input box.
   6. Launch the application for EEG recordings to measure electrode impedance, and adjust the parameters to ensure all electrodes are below 5 kΩ.
   7. Bundle the electrode cables to reduce noise.
3. 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.
4. 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.
5. 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.
6. Stimulus
   1. 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.
   2. In addition to the call stimuli, create white noise using a function in the programming software and use that as a stimulus.
   3. 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).
   4. 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.

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.

1. Preprocessing
   1. Re-reference to the linked ear reference.
   2. High-pass filter at 2 Hz.
   3. Epoch from 100 ms before to 1000 ms after stimulus onset.
   4. Baseline-correct to the mean of the 100-ms prestimulus period. Reject artifacts using a criterion of ±150 µV.
2. Plotting event-related potential (ERP)
   1. Average all trial data for each subject.
   2. Obtain group-averaged waveforms by averaging all subjects.
   3. 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.
   4. Apply post hoc multiple comparison analysis with Tukey's method.
3. Plotting event-related spectral perturbation (ERSP)
   1. Calculate the ERSP to visualize the mean event-related change in spectral power over time at a broad frequency range using Equation (1). F_k (f,t) is the spectral estimate of trial k at frequency f and time t:
          (1)
   2. 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).
   3. 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.
   4. 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.

Representative Results

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 effect of stimuli on Cz activity at 13-18 ms, 28-36 ms, and 45-88 ms after stimulus onset, respectively (p < 0.05). Post hoc multiple comparison analysis with Tukey's method showed that the difference was between noise and other calls (p < 0.05), but there was no difference between marmoset call types. The result suggests that differences in brain activity by call type could not be observed from the event-related potentials alone.

Next, we conducted a time-frequency analysis for each subject. Figure 3 shows an example of the time-frequency maps for the Tsik-string call obtained by subject R (elder, Figure 3A) and subject Y (younger, Figure 3B). We found that event-related spectral power increased at a lower frequency of approximately 20-50 Hz immediately after stimulus onset. These responses were prominently observed in the Cz. In contrast, the gamma range power (over 30 Hz) decreased after stimulus onset compared to the baseline period. In addition, this decline lasted for 1 s. No decrease in event-related power was observed in elder individuals over 8 years. In these examples, the elder individuals had a stronger initial response to the call in the vertex region (Cz), while the younger individuals showed a sustained decrease in γ-band activity during the call presentation. The results suggest that there are differences in initial and sustained responses depending on the subject's age.

Finally, we investigated the relationship between subject age and event-related spectral perturbation (ERSP) power in the initial transient response (Figure 4A) and sustained response (Figure 4B). A two-way ANOVA with Stimulus type as a within-subject factor and age as a between-subject factor was conducted to determine the contribution of the type of auditory stimulus and the subject's age to EEG activity. The initial, transient responses in the Fz showed significant main effects of Stimulus type (F (3,24) = 9.020, p < 0.001) and Age (F (8,24) = 3.934, p = 0.004). However, there was no significant interaction between the Stimulus type and Age (p = 0.144). In the transient responses in Cz, there was a significant main effect of Stimulus type (F (3,24) = 8.533, p < 0.001), but no effect of Age (F (8,24) = 2.215, p = 0.073), and no interaction (p = 0.228). For sustained responses, there were significant main effects of both Stimulus type and Age (F (3,24) = 9.020, p < 0.001; F (8,24) = 3.934, p = 0.004, respectively) on Fz. No significant interaction was observed (P = 0.144). The sustained responses in Cz showed a significant main effect of Stimulus type (F (3,24) = 8.533, p < 0.001) but no main effect of Age (F(8,24) = 2.215, p = 0.073) or interaction (p = 0.228). These results suggest that in the middle-frontal area (Fz), initial transient responses to call and noise stimuli varied greatly with increasing age, and sustained responses were suppressed in younger age groups. These may reflect the functional maturation of the frontal region.

Previous neurophysiological studies have reported neuronal responses in the primary auditory cortex during vocalization in marmosets^9,38. In addition, more than half of these neurons exhibit an inhibitory response that persists during vocalization^9,38. Furthermore, previous electrophysiological studies in nonhuman primates and humans have shown that high gamma band activity in the local field potential (LFP) and ECoG correlates well with firing rates in neurons^39,40,41,42. Scalp EEG is a spatiotemporally smoothed version of the LFP, integrated over an area of 1 cm² or more⁴³. Although the high-gamma component of the EEG has a lower correlation with firing rates than LFP and ECoG, it is thought to code the output signal as an integrated range of several centimeters. Therefore, the sustained decrease in gamma band power observed at Cz in our experiments may reflect the activity of neuronal clusters showing suppressed activity during call emission, which is found in the auditory cortices. In contrast, previous electrophysiological studies have reported that more neurons in the frontal cortex, mainly in the premotor cortex, exhibit excitatory responses during call vocalization¹³. Interestingly, sustained inhibitory activity was observed even when the Fz was placed in the frontal area in our experiments, although distinct neural mechanisms were observed between vocal perception and production. A recent fMRI study has further identified several subregions in the frontal cortex, including the anterior cingulate cortex as well as the premotor cortex, as 'vocal patches' that respond to species-specific calls in marmosets¹⁹. Our results reflect the overall brain activity in these regions.

In the current experiment, we used only midline positions for the exploration electrodes (Fz, Cz, Pz, and Oz); therefore, we cannot mention any differences between the right and left EEG activity of auditory processing. In the future, we need to investigate the laterality of the neural activity underlying vocal processing.

Prior studies have reported that low gamma activity in the LFP and ECoG is generated by synaptic inputs to pyramidal cells. Thus, a high gamma activity reflects a signal component closer to the output, whereas a low gamma activity reflects those closer to the input³⁹. In the present study, we observed transient activity in the beta and low-gamma bands immediately following exposure to a call. These responses may reflect sensory input signals to the cortex. The advantage of our method is that it can capture the brain activity from different neuronal populations as dynamic changes with high temporal resolution. To our knowledge, this is the first study to reveal how scalp EEG signals change during species-specific vocal perception in marmosets. The present results provide new insights into the integration of neural representations through recordings of a wide range of brain regions.

Figure 1: Experimental setup. (A) An exemplar image of a subject during recording. The marmoset is seated in a chair and the head is fixed to the chair by a mask. The mouth is maintained open to facilitate breathing and drinking reward fluids, and the electrodes were attached to the top of the head. (B) Equipment: Sound stimuli are presented through a speaker. An amplifier, electrode input box, and a monitoring camera were also installed. (C) Location of electrodes: Electrodes were placed on Fz, Cz, Pz, Oz, A1, and A2 according to the International 10-20 System. We defined the location of electrodes using the inion, nasion, and bilateral preauricular points as anatomical landmarks. The C3 or C4 electrode was used as the ground electrode. (D) Sonograms (left panel) and spectrograms (left panel) for all audio stimuli. The Phee call is a single long call lasting less than 2 s. The Tsik-Ek call is a combination call of a Tsik followed by an Ek. Two sets of the compound call presented approximately 1 s. The Tsik-string call is a repetitive call of Tsik⁵, and four Tsik were presented approximately for 1 s in the stimulus. As a non-call stimulus, we used a white noise signal generated by a custom script that lasted about 1 s. The sound onset latencies were visually inspected on a digital audio editor. The red arrows indicate each latency, Phee call 49 ms, Tsik-Ek call 35 ms, Tsik-string call 16 ms, and white noise 0 ms. Please click here to view a larger version of this figure.

Figure 2: Grand-averaged event-related potentials to the Phee, Tsik-Ek, and Tsik-string calls and noise. (n = 9) The activity was aligned to the onset of each call or white noise. The black horizontal arrow indicates the period for the stimulus presentation. The Phee call lasted for approximately 2 s, the rest lasted for 1 s. The red horizontal lines at Cz indicate the periods with significant differences between the auditory stimuli. All of these were between the Noise and the other calls, and there were no differences between the calls. Please click here to view a larger version of this figure.

Figure 3: Example of time-frequency maps for calls. (A) The ERSP map for the Tsik-string call in subject R (145 months old); (B) The EPSP map for the same Tsik-string call in subject Y (23 months old). The left and right panels show the data recorded from the Fz and Cz electrodes, respectively. The red vertical lines indicate the timing of the onset of the auditory file, not the call onset. Abbreviation: EPSP = event-related spectral perturbation. Please click here to view a larger version of this figure.

Figure 4: Relationship between mean event-related power and age of subjects. (A) Relationship between mean event-related power at α and β-band and age of subjects. The mean ERSP power was calculated at 8-29 Hz from a 1-150 ms period from each stimulus onset compared to those in the baseline period (-200 to 0 ms before stimulus onset). The left and right panels show the data recorded from the Fz and Cz electrodes, respectively. (B) Relationship between mean event-related power at γ-band and age of subjects. The mean ERSP power was calculated at 30-100 Hz from a 151-950 ms period from each stimulus onset compared to those in the baseline period (-200 to 0 ms before stimulus onset). Thus, negative ERSP values indicate a decrease in power compared to that before the stimulus presentation. Abbreviation: ERSP = event-related spectral perturbation. Please click here to view a larger version of this figure.

Supplemental File 1: A zip file containing four audio files used in the experiments. The PH.wav file includes a stimulus with one long Phee call; TE.wav contains two Tsik-Ek calls with an interval between them; MB.wav contains a Tsik-string call stimulus with four consecutive Tsik (also called mobbing call); NO.wav contains a program-generated white Gaussian noise. Please click here to download this File.

Supplemental File 2: A zip file containing postprocessing code. Please click here to download this File.

Discussion

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 not used during intubation due to its short duration and low concentration (approximately 1%).

Advantages of EEG measurement
Scalp EEG is a method of recording brain activity through the skin, skull, and subcutaneous tissues, which has a lower spatial resolution than recording methods targeted at single neurons or intracranial electrodes with higher spatial resolution. Despite these disadvantages, there are several advantages to measuring scalp EEG in nonhuman primates. First, the method is noninvasive and does not injure the animals. For example, one may want to measure the brain activity of very valuable animal models of a disease created using a very difficult genetic engineering procedure. In such cases, these techniques can be safely used to measure brain activity without requiring surgical intervention. Indeed, this method enabled us to acquire neural data from the same subject over a long period without injury. In addition, the number of subjects can be increased compared with invasive neurophysiological methods because it is easier and less time-consuming. Second, EEG data obtained from nonhuman primates using this technique can be directly compared with previously obtained human EEG data using the same behavioral paradigm as humans. This advantage allowed us to examine cognitive neural activity from an evolutionary perspective. Third, our recording method allows recordings from awake primates. Anesthetic agents significantly attenuate cortical activity, although the effects of attenuation, degree, and pattern of inhibition vary depending on the agents. This method can record brain activity while awake, without any influence of drugs. Furthermore, by administering drugs that act on the central nervous system and observing changes in brain activity, this technique can be applied in experiments involving neural mechanisms.

Functional brain imaging is another noninvasive method for exploring the brain mechanisms associated with vocal processing in humans and nonhuman primates. Recent technological advances have enabled the application of fMRI in animals in an awake state. Functional brain imaging has the advantage of allowing whole-brain exploration with high spatial resolution. However, the equipment and its running costs are expensive to install and maintain. Conversely, EEG has the advantage of high temporal resolution and reveals more dynamic frequency-specific brain activity. In addition, the equipment has a relatively low cost and high portability and can be performed by a single experimenter. Furthermore, various stimuli and equipment could be easily introduced. Taking advantage of these methods and integrating findings from different techniques will provide a more detailed understanding of the neural mechanisms involved in vocal perception in marmosets.

Comparison with human or macaque data
In EEG, the signals generated in the brain must pass through the dura mater, subcutaneous tissue, and skull before being recorded on the scalp. These tissues act as low-pass filters. In particular, high-frequency components such as spikes are significantly attenuated compared to the alpha and beta band components. Therefore, unlike electrocorticography (ECoG), some components of brain activity cannot be detected on the scalp. This is a limitation of scalp EEG. Marmosets have thinner skulls, subcutaneous tissues, and dura mater than macaque monkeys and humans, while the size of the head is also smaller. Therefore, care must be taken when comparing results obtained in humans and macaques. For example, signals that originate in the deep brain, such as the brainstem, can be recorded on the scalp as relatively large signals because of the lower attenuation rate and relatively close distance between the signal source and electrodes.

In addition, marmosets have very few, if any, shallow cerebral sulci. This makes them quite distinct from humans, who have many sulci, and in whom the cortex is internalized as the gyrus. The number and depth of the cerebral sulci differ among humans, macaques, and marmosets. Electrical signals are generated perpendicular to the cortex and measured using electrodes placed over the cerebral gyri, where the apical dendrites of pyramidal neurons, which are the major sources of EEG signals, are aligned perpendicular to the electrodes. In the sulci, signals are attenuated and projected onto distant electrodes because they are generated horizontally with respect to the brain surface. When comparing results in humans with those in macaques and marmosets, it is necessary to consider the location of the EEG recordings and the anatomy of the area.

Materials

Name	Company	Catalog Number	Comments
Alfaxalone	Meiji Animal Health	Alfaxan
Amplifier	Brain Products	BrainAmp
Atropine	Fuso Pharmaceutical Industries	Atropine Sulfate Injection
Audio editor	Adobe	Adobe Audition
Data processing software	MathWorks	MATLAB	version R2023a
Data processing toolbox	University of California-SanDiego	EEGLAB
Data processing toolbox	University of California-Davis	ERPLAB
Electric shaver	Panasonic	ER803PPA
Electrode	Unique Medical	UL-3010	AgCl coated (custom)
Electrode gel	Neurospec AG	V16 SuperVisc
Electrode input box	Brain Products	EIB64-DUO	64ch
Glue	3M	Scotch 7005S
Hair removering cream	Kracie	epilat for sensitive skin
Isoflurane	Bussan Animal Health	ds isoflurane
Liquid gum	San-ei Yakuhin Boeki	Arabic Call SS	Gum arabic+water
Liquid nutrition	Nestlé Health Science Company	Isocal 1.0 Junior	Polymeric formula
Maropitant	Zoetis	Cerenia injectable solution
Monitor Camera	Intel	RealSense LiDAR Camera L515
Monkey pellets	Oriental Yeast	SPS
Primate chair	Natsume Seisakusho	Order made
Pulse oximeters	Covident	Nellcor	PM10N
Skin prepping pasta	Mammendorfer Institut für Physik und Medizin	NeuPrep
Slicon tube	AsONE	Φ4 x 7mm
Speaker	Fostex	PM0.3
Synchronization device	Brain Vision	StimTrak
Thermoplastic mask	CIVCO	MTAPU Type Uniframe Thermoplastic Mask 2.4mm