development and implementation of noninvasive eeg recording methods for auditory processing studies in marmosets

Noninvasive EEG for Comparative Studies of Vocal Perception in Marmosets

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.

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.

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.

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JoVE Journal

Neuroscience

46 ビュー. マーモセットに麻酔をかけた後、パルスオキシメータでバイタルをモニターします。必要に応じて、酸素混合気を投与します。室温を27°C以上に保ち、マーモセットの体に温めた布を巻き付けて低体温症を防ぎます。電気シェーバーを使用して、頭を剃ります。敏感肌用の脱毛クリームを塗ります。5分後、濡れたガーゼでクリームを拭き取...