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.

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...

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

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.

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 ...

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.

electroencephalography-measurements-awake-marmosets-listening-to

Research

JoVE Journal

Neuroscience

490 Views.  Kyoto University. 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 comp...

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

Assessment of Ultrasonic Vocalizations During Drug Self-administration in Rats

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and relapse. Various types of behavioral activity are commonly utilized as measures of drug motivation in animals. However, a crucial component of drug abuse relapse in abstinent cocaine users is "drug craving", which is difficult to model in animals, as it often occurs in the absence of overt behaviors. Yet, it is possible that a class of ultrasonic vocalizations (USVs) in rats may be a useful marker for affective responses to drug administration, drug anticipation and even drug craving. Rats vocalize in ultrasonic frequencies that serve as a communicatory function and express subjective emotional states. Several studies have shown that different call frequency ranges are associated with negative and positive emotional states. For instance, high frequency calls ("50-kHz") are associated with positive affect, whereas low frequency calls ("22-kHz") represent a negative emotional state. This article describes a procedure to assess rat USVs associated with daily cocaine self-administration. For this procedure, we utilized standard single-lever operant chambers housed within sound-attenuating boxes for cocaine self-administration sessions and utilized ultrasonic microphones, multi-channel recording hardware and specialized software programs to detect and analyze USVs. USVs measurements reflect emotionality of rats before, during and after drug availability and can be correlated with commonly assessed drug self-administration behavioral data such lever responses, inter-response intervals and locomotor activity. Since USVs can be assessed during intervals prior to drug availability (e.g., anticipatory USVs) and during drug extinction trials, changes in affect associated with drug anticipation and drug abstinence can also be determined. In addition, determining USV changes over the course of short- and long-term drug exposure can provide a more detailed interpretation of drug exposure effects on affective functioning.

Drug self-administration and ultrasonic vocalizations (USV) are used as behavioral assessments in animal research, but rarely in combination. The purpose of this article is to describe the advantages of recording USVs during drug self-administration procedures to assess affective responses to drug experience.

Drug self-administration procedures are commonly used to study behavioral and neurochemical changes associated with human drug abuse, addiction and ...

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

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 ...

Concurrent Recording of Co-localized Electroencephalography and Local Field Potential in Rodent

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the neurogenesis of EEG is still very limited. Local field potentials (LFPs) recorded via a multi-laminar microelectrode can provide a more detailed account of simultaneous neural activity across different cortical layers in the neocortex, but the technique is invasive. Combining EEG and LFP measurements in a pre-clinical model can greatly enhance understanding of the neural mechanisms involved in the generation of EEG signals, and facilitate the derivation of a more realistic and biologically accurate mathematical model of EEG. A simple procedure for acquiring concurrent and co-localized EEG and multi-laminar LFP signals in the anesthetized rodent is presented here. We also investigated whether EEG signals were significantly affected by a burr hole drilled in the skull for the insertion of a microelectrode. Our results suggest that the burr hole has a negligible impact on EEG recordings.

This protocol describes a simple method for concurrent recording of co-localized electroencephalography (EEG) and multi-laminar local field potential in an anesthetized rat. A burr hole drilled in the skull for the insertion of a microelectrode is shown to produce negligible distortion of the EEG signal.

Although electroencephalography (EEG) is widely used as a non-invasive technique for recording neural activities of the brain, our understanding of the ...

Reliable Acquisition of Electroencephalography Data during Simultaneous Electroencephalography and Functional MRI

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG (good temporal resolution) and fMRI (good spatial resolution) to measure neuronal activity during an electrographic event, through hemodynamic responses known as blood-oxygen-level-dependent (BOLD) changes. It is a non-invasive research tool that is utilized in neuroscience research and is highly beneficial to the clinical community, especially for the management of neurological diseases, provided that proper equipment and protocols are administered during data acquisition. Although recording EEG-fMRI is apparently straightforward, the correct preparation, especially in placing and securing the electrodes, is not only important for safety but is also critical in ensuring the reliability and analyzability of the EEG data obtained. This is also the most experience-demanding part of the preparation. To address these issues, a straightforward protocol that ensures data quality was developed. This article provides a step-by-step guide for acquiring reliable EEG data during EEG-fMRI using this protocol that utilizes readily available medical products. The presented protocol can be adapted to different applications of EEG-fMRI in research and clinical settings, and may be beneficial to both inexperienced and expert operators.

This article provides a straightforward protocol for acquiring good quality electroencephalography (EEG) data during simultaneous EEG and functional magnetic resonance imaging by utilizing readily available medical products.

Simultaneous electroencephalography (EEG) and functional magnetic resonance imaging (fMRI), EEG-fMRI, combines the complementary properties of scalp EEG ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their activity in vivo in behaving mice. This paper describes a method for the implantation of a chronic cranial window to allow for the longitudinal imaging of brain cells in awake, head-restrained mice. In combination with genetic strategies and viral injections, it is possible to label specific cells and regions of interest with structural or physiological markers. This protocol demonstrates how to combine viral injections to label neurons in the vicinity of GCaMP6-expressing astrocytes in the cortex for simultaneous imaging of both cells through a cranial window. Multiphoton imaging of the same cells can be performed for days, weeks, or months in awake, behaving animals. This approach provides researchers with a method for viewing cellular dynamics in real time and can be applied to answer a number of questions in neuroscience.

Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

Presented here is a protocol for the implantation of a chronic cranial window for the longitudinal imaging of brain cells in awake, head-restrained mice.

To fully understand the cellular physiology of neurons and glia in behaving animals, it is necessary to visualize their morphology and record their ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Head Implants for the Neuroimaging of Awake, Head-Fixed Rats

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular functions of the brain and can adversely influence neurophysiological results. The use of awake animals for research studies is advantageous but poses the major challenge of keeping the animals calm and stationary to minimize motion artifacts throughout data acquisition. Awake imaging in smaller-sized rodents (e.g., mice) is very common but remains scant in rats as rats are bigger, stronger, and have a greater tendency to oppose movement restraints and head fixation over the long durations required for imaging. A new model of neuroimaging of awake, head-fixed rats using customized hand-sewn slings, 3D-printed head implants, head caps, and a headframe is described. The results acquired following a single trial of single-whisker stimulation suggest an increase in the intensity of the evoked functional response. The acquisition of the evoked functional response from awake, head-fixed rats is faster than that from anesthetized rats, reliable, reproducible, and can be used for repeated longitudinal studies.

A detailed new procedure for functional imaging of awake, head-fixed rats is described.

Anesthetics, commonly used in preclinical and fundamental scientific research, have a depressive influence on the metabolic, neuronal, and vascular ...