Awake Marmosets Research Articles

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.

Distributed representations of prediction error signals across the cortical hierarchy are synergistic

A relevant question concerning inter-areal communication in the cortex is whether these interactions are synergistic. Synergy refers to the complementary effect of multiple brain signals conveying more information than the sum of each isolated signal. Redundancy, on the other hand, refers to the common information shared between brain signals. Here, we dissociated cortical interactions encoding complementary information (synergy) from those sharing common information (redundancy) during prediction error (PE) processing. We analyzed auditory and frontal electrocorticography (ECoG) signals in five common awake marmosets performing two distinct auditory oddball tasks and investigated to what extent event-related potentials (ERP) and broadband (BB) dynamics encoded synergistic and redundant information about PE processing. The information conveyed by ERPs and BB signals was synergistic even at lower stages of the hierarchy in the auditory cortex and between auditory and frontal regions. Using a brain-constrained neural network, we simulated the synergy and redundancy observed in the experimental results and demonstrated that the emergence of synergy between auditory and frontal regions requires the presence of strong, long-distance, feedback, and feedforward connections. These results indicate that distributed representations of PE signals across the cortical hierarchy can be highly synergistic.

Mapping of facial and vocal processing in common marmosets with ultra-high field fMRI

Primate communication relies on multimodal cues, such as vision and audition, to facilitate the exchange of intentions, enable social interactions, avoid predators, and foster group cohesion during daily activities. Understanding the integration of facial and vocal signals is pivotal to comprehend social interaction. In this study, we acquire whole-brain ultra-high field (9.4 T) fMRI data from awake marmosets (Callithrix jacchus) to explore brain responses to unimodal and combined facial and vocal stimuli. Our findings reveal that the multisensory condition not only intensifies activations in the occipito-temporal face patches and auditory voice patches but also engages a more extensive network that includes additional parietal, prefrontal and cingulate areas, compared to the summed responses of the unimodal conditions. By uncovering the neural network underlying multisensory audiovisual integration in marmosets, this study highlights the efficiency and adaptability of the marmoset brain in processing facial and vocal social signals, providing significant insights into primate social communication.

Tonotopic organization of auditory cortex in awake marmosets revealed by multi-modal wide-field optical imaging

Tonotopic organization of the auditory cortex has been extensively studied in many mammalian species using various methodologies and physiological preparations. Tonotopy mapping in primates, however, is more limited due to constraints such as cortical folding, use of anesthetized subjects, and mapping methodology. Here we applied a combination of through-skull and through-window intrinsic optical signal imaging, wide-field calcium imaging, and neural probe recording techniques in awake marmosets (Callithrix jacchus), a New World monkey with most of its auditory cortex located on a flat brain surface. Coarse tonotopic gradients, including a recently described rostral-temporal (RT) to parabelt gradient, were revealed by the through-skull imaging of intrinsic optical signals and were subsequently validated by single-unit recording. Furthermore, these tonotopic gradients were observed with more detail through chronically implanted cranial windows with additional verifications on the experimental design. Moreover, the tonotopy mapped by the intrinsic-signal imaging methods was verified by wide-field calcium imaging in an AAV-GCaMP labeled subject. After these validations and with further effort to expand the field of view more rostrally in both windowed and through-skull subjects, an additional putative tonotopic gradient was observed more rostrally to the area RT, which has not been previously described by the standard model of tonotopic organization of the primate auditory cortex. Together, these results provide the most comprehensive data of tonotopy mapping in an awake primate species with unprecedented coverage and details in the rostral proportion and support a caudal-rostrally arranged mesoscale organization of at least three repeats of functional gradients in the primate auditory cortex, similar to the ventral stream of primate visual cortex.

Representation of conspecific vocalizations in amygdala of awake marmosets.

Human speech and animal vocalizations are important for social communication and animal survival. Neurons in the auditory pathway are responsive to a range of sounds, from elementary sound features to complex acoustic sounds. For social communication, responses to distinct patterns of vocalization are usually highly specific to an individual conspecific call, in some species. This includes the specificity of sound patterns and embedded biological information. We conducted single-unit recordings in the amygdala of awake marmosets and presented calls used in marmoset communication, calls of other species and calls from specific marmoset individuals. We found that some neurons (47/262) in the amygdala distinguished 'Phee' calls from vocalizations of other animals and other types of marmoset vocalizations. Interestingly, a subset of Phee-responsive neurons (22/47) also exhibited selectivity to one out of the three Phees from two different 'caller' marmosets. Our findings suggest that, while it has traditionally been considered the key structure in the limbic system, the amygdala also represents a critical stage of socially relevant auditory perceptual processing.

A vocalization-processing network in marmosets

Vocalizations play an important role in the daily life of primates and likely form the basis of human language. Functional imaging studies have demonstrated that listening to voices activates a fronto-temporal voice perception network in human participants. Here, we acquired whole-brain ultrahigh-field (9.4 T) fMRI in awake marmosets (Callithrix jacchus) and demonstrate that these small, highly vocal New World primates possess a similar fronto-temporal network, including subcortical regions, that is activated by the presentation of conspecific vocalizations. The findings suggest that the human voice perception network has evolved from an ancestral vocalization-processing network that predates the separation of New and Old World primates.

High Spectral and Temporal Acuity in Primary Auditory Cortex of Awake Cats

Most accounts of single- and multi-unit responses in auditory cortex under anesthetized conditions have emphasized V-shaped frequency tuning curves and low-pass sensitivity to rates of repeated sounds. In contrast, single-unit recordings in awake marmosets also show I-shaped and O-shaped response areas having restricted tuning to frequency and (for O units) sound level. That preparation also demonstrates synchrony to moderate click rates and representation of higher click rates by spike rates of non-synchronized tonic responses, neither of which are commonly seen in anesthetized conditions. The spectral and temporal representation observed in the marmoset might reflect special adaptations of that species, might be due to single- rather than multi-unit recording, or might indicate characteristics of awake-versus-anesthetized recording conditions. We studied spectral and temporal representation in the primary auditory cortex of alert cats. We observed V-, I-, and O-shaped response areas like those demonstrated in awake marmosets. Neurons could synchronize to click trains at rates about an octave higher than is usually seen with anesthesia. Representations of click rates by rates of non-synchronized tonic responses exhibited dynamic ranges that covered the entire range of tested click rates. The observation of these spectral and temporal representations in cats demonstrates that they are not unique to primates and, indeed, might be widespread among mammalian species. Moreover, we observed no significant difference in stimulus representation between single- and multi-unit recordings. It appears that the principal factor that has hindered observations of high spectral and temporal acuity in the auditory cortex has been the use of general anesthesia.

An integrated resource for functional and structural connectivity of the marmoset brain

Comprehensive integration of structural and functional connectivity data is required to model brain functions accurately. While resources for studying the structural connectivity of non-human primate brains already exist, their integration with functional connectivity data has remained unavailable. Here we present a comprehensive resource that integrates the most extensive awake marmoset resting-state fMRI data available to date (39 marmoset monkeys, 710 runs, 12117 mins) with previously published cellular-level neuronal tracing data (52 marmoset monkeys, 143 injections) and multi-resolution diffusion MRI datasets. The combination of these data allowed us to (1) map the fine-detailed functional brain networks and cortical parcellations, (2) develop a deep-learning-based parcellation generator that preserves the topographical organization of functional connectivity and reflects individual variabilities, and (3) investigate the structural basis underlying functional connectivity by computational modeling. This resource will enable modeling structure-function relationships and facilitate future comparative and translational studies of primate brains.

A radiofrequency coil to facilitate task-based fMRI of awake marmosets

BackgroundThe small common marmoset (Callithrix jacchus) is an ideal nonhuman primate for awake fMRI in ultra-high field small animal MRI scanners. However, it can often be challenging in task-based fMRI experiments to provide a robust stimulus within the MRI environment while using hardware (an RF coil and restraint system) that is compatible with awake imaging. New methodHere we present an RF coil and restraint system that permits unimpeded access to an awake marmoset’s head subsequent to immobilization, thereby permitting the setup of peripheral devices and stimuli proximal to the head. ResultsAs an example application, an fMRI experiment probing whole-brain activation in response to marmoset vocalizations was conducted—this paradigm showed significant bilateral activation in the inferior colliculus, medial lateral geniculate nucleus, and auditory cortex. Comparison with existing method(s)The coil performance was evaluated and compared to a previously published restraint system with integrated RF coil. The image and temporal SNR were improved by up to 58 % and 27 %, respectively, in the peripheral cortex and by 30 % and 3 % in the centre of the brain. The restraint-system topology limited head motion to less than 100 µm of translation and 0.30° of rotation when measured over a 15-minute acquisition. ConclusionsThe proposed hardware solution provides a versatile approach to awake-marmoset imaging and, as demonstrated, can facilitate task-based fMRI.

Selective corticofugal modulation on sound processing in auditory thalamus of awake marmosets.

Cortical feedback has long been considered crucial for the modulation of sensory perception and recognition. However, previous studies have shown varying modulatory effects of the primary auditory cortex (A1) on the auditory response of subcortical neurons, which complicate interpretations regarding the function of A1 in sound perception and recognition. This has been further complicated by studies conducted under different brain states. In the current study, we used cryo-inactivation in A1 to examine the role of corticothalamic feedback on medial geniculate body (MGB) neurons in awake marmosets. The primary effects of A1 inactivation were a frequency-specific decrease in the auditory response of most MGB neurons coupled with an increased spontaneous firing rate, which together resulted in a decrease in the signal-to-noise ratio. In addition, we report for the first time that A1 robustly modulated the long-lasting sustained response of MGB neurons, which changed the frequency tuning after A1 inactivation, e.g. some neurons are sharper with corticofugal feedback and some get broader. Taken together, our results demonstrate that corticothalamic modulation in awake marmosets serves to enhance sensory processing in a manner similar to center-surround models proposed in visual and somatosensory systems, a finding which supports common principles of corticothalamic processing across sensory systems.

Distinct neuronal types contribute to hybrid temporal encoding strategies in primate auditory cortex.

Studies of the encoding of sensory stimuli by the brain often consider recorded neurons as a pool of identical units. Here, we report divergence in stimulus-encoding properties between subpopulations of cortical neurons that are classified based on spike timing and waveform features. Neurons in auditory cortex of the awake marmoset (Callithrix jacchus) encode temporal information with either stimulus-synchronized or nonsynchronized responses. When we classified single-unit recordings using either a criteria-based or an unsupervised classification method into regular-spiking, fast-spiking, and bursting units, a subset of intrinsically bursting neurons formed the most highly synchronized group, with strong phase-locking to sinusoidal amplitude modulation (SAM) that extended well above 20 Hz. In contrast with other unit types, these bursting neurons fired primarily on the rising phase of SAM or the onset of unmodulated stimuli, and preferred rapid stimulus onset rates. Such differentiating behavior has been previously reported in bursting neuron models and may reflect specializations for detection of acoustic edges. These units responded to natural stimuli (vocalizations) with brief and precise spiking at particular time points that could be decoded with high temporal stringency. Regular-spiking units better reflected the shape of slow modulations and responded more selectively to vocalizations with overall firing rate increases. Population decoding using time-binned neural activity found that decoding behavior differed substantially between regular-spiking and bursting units. A relatively small pool of bursting units was sufficient to identify the stimulus with high accuracy in a manner that relied on the temporal pattern of responses. These unit type differences may contribute to parallel and complementary neural codes.

Mesoscopic landscape of cortical functions revealed by through-skull wide-field optical imaging in marmoset monkeys

The primate cerebral cortex is organized into specialized areas representing different modalities and functions along a continuous surface. The functional maps across the cortex, however, are often investigated a single modality at a time (e.g., audition or vision). To advance our understanding of the complex landscape of primate cortical functions, here we develop a polarization-gated wide-field optical imaging method for measuring cortical functions through the un-thinned intact skull in awake marmoset monkeys (Callithrix jacchus), a primate species featuring a smooth cortex. Using this method, adjacent auditory, visual, and somatosensory cortices are noninvasively parcellated in individual subjects with detailed tonotopy, retinotopy, and somatotopy. An additional pure-tone-responsive tonotopic gradient is discovered in auditory cortex and a face-patch sensitive to motion in the lower-center visual field is localized near an auditory region representing frequencies of conspecific vocalizations. This through-skull landscape-mapping approach provides new opportunities for understanding how the primate cortex is organized and coordinated to enable real-world behaviors.

A silent two-photon imaging system for studying in vivo auditory neuronal functions

Two-photon laser-scanning microscopy has become an essential tool for imaging neuronal functions in vivo and has been applied to different parts of the neural system, including the auditory system. However, many components of a two-photon microscope, such as galvanometer-based laser scanners, generate mechanical vibrations and thus acoustic artifacts, making it difficult to interpret auditory responses from recorded neurons. Here, we report the development of a silent two-photon imaging system and its applications in the common marmoset (Callithrix Jacchus), a non-human primate species sharing a similar hearing range with humans. By utilizing an orthogonal pair of acousto-optical deflectors (AODs), full-frame raster scanning at video rate was achieved without introducing mechanical vibrations. Imaging depth can be optically controlled by adjusting the chirping speed on the AODs without any mechanical motion along the Z-axis. Furthermore, all other sound-generating components of the system were acoustically isolated, leaving the noise floor of the working system below the marmoset’s hearing threshold. Imaging with the system in awake marmosets revealed many auditory cortex neurons that exhibited maximal responses at low sound levels, which were not possible to study using traditional two-photon imaging systems. This is the first demonstration of a silent two-photon imaging system that is capable of imaging auditory neuronal functions in vivo without acoustic artifacts. This capacity opens new opportunities for a better understanding of auditory functions in the brain and helps isolate animal behavior from microscope-generated acoustic interference.

An evolutionary gap in primate default mode network organization

SUMMARYThe human default mode network (DMN) is engaged at rest and in cognitive states such as self-directed thoughts. Interconnected homologous cortical areas in primates constitute a network considered as the equivalent. Here, based on a cross-species comparison of the DMN between humans and non-hominoid primates (macaques, marmosets, and mouse lemurs), we report major dissimilarities in connectivity profiles. Most importantly, the medial prefrontal cortex (mPFC) of non-hominoid primates is poorly engaged with the posterior cingulate cortex (PCC), though strong correlated activity between the human PCC and the mPFC is a key feature of the human DMN. Instead, a fronto-temporal resting-state network involving the mPFC was detected consistently across non-hominoid primate species. These common functional features shared between non-hominoid primates but not with humans suggest a substantial gap in the organization of the primate’s DMN and its associated cognitive functions.

An open access resource for functional brain connectivity from fully awake marmosets

The common marmoset (Callithrix jacchus) is quickly gaining traction as a premier neuroscientific model. However, considerable progress is still needed in understanding the functional and structural organization of the marmoset brain to rival that documented in longstanding preclinical model species, like mice, rats, and Old World primates. To accelerate such progress, we present the Marmoset Functional Brain Connectivity Resource (marmosetbrainconnectome.org), currently consisting of over 70 h of resting-state fMRI (RS-fMRI) data acquired at 500 µm isotropic resolution from 31 fully awake marmosets in a common stereotactic space. Three-dimensional functional connectivity (FC) maps for every cortical and subcortical gray matter voxel are stored online. Users can instantaneously view, manipulate, and download any whole-brain functional connectivity (FC) topology (at the subject- or group-level) along with the raw datasets and preprocessing code. Importantly, researchers can use this resource to test hypotheses about FC directly – with no additional analyses required – yielding whole-brain correlations for any gray matter voxel on demand. We demonstrate the resource's utility for presurgical planning and comparison with tracer-based neuronal connectivity as proof of concept. Complementing existing structural connectivity resources for the marmoset brain, the Marmoset Functional Brain Connectivity Resource affords users the distinct advantage of exploring the connectivity of any voxel in the marmoset brain, not limited to injection sites nor constrained by regional atlases. With the entire raw database (RS-fMRI and structural images) and preprocessing code openly available for download and use, we expect this resource to be broadly valuable to test novel hypotheses about the functional organization of the marmoset brain.

Corticofugal modulation of temporal and rate representations in the inferior colliculus of the awake marmoset.

Temporal processing is crucial for auditory perception and cognition, especially for communication sounds. Previous studies have shown that the auditory cortex and the thalamus use temporal and rate representations to encode slowly and rapidly changing time-varying sounds. However, how the primate inferior colliculus (IC) encodes time-varying sounds at the millisecond scale remains unclear. In this study, we investigated the temporal processing by IC neurons in awake marmosets to Gaussian click trains with varying interclick intervals (2-100ms). Strikingly, we found that 28% of IC neurons exhibited rate representation with nonsynchronized responses, which is in sharp contrast to the current view that the IC only uses a temporal representation to encode time-varying signals. Moreover, IC neurons with rate representation exhibited response properties distinct from those with temporal representation. We further demonstrated that reversible inactivation of the primary auditory cortex modulated 17% of the stimulus-synchronized responses and 21% of the nonsynchronized responses of IC neurons, revealing that cortico-colliculus projections play a role, but not a crucial one, in temporal processing in the IC. This study has significantly advanced our understanding of temporal processing in the IC of awake animals and provides new insights into temporal processing from the midbrain to the cortex.

Effects of Cortical Cooling on Sound Processing in Auditory Cortex and Thalamus of Awake Marmosets.

The auditory thalamus is the central nexus of bottom-up connections from the inferior colliculus and top-down connections from auditory cortical areas. While considerable efforts have been made to investigate feedforward processing of sounds in the auditory thalamus (medial geniculate body, MGB) of non-human primates, little is known about the role of corticofugal feedback in the MGB of awake non-human primates. Therefore, we developed a small, repositionable cooling probe to manipulate corticofugal feedback and studied neural responses in both auditory cortex and thalamus to sounds under conditions of normal and reduced cortical temperature. Cooling-induced increases in the width of extracellularly recorded spikes in auditory cortex were observed over the distance of several hundred micrometers away from the cooling probe. Cortical neurons displayed reduction in both spontaneous and stimulus driven firing rates with decreased cortical temperatures. In thalamus, cortical cooling led to increased spontaneous firing and either increased or decreased stimulus driven activity. Furthermore, response tuning to modulation frequencies of temporally modulated sounds and spatial tuning to sound source location could be altered (increased or decreased) by cortical cooling. Specifically, best modulation frequencies of individual MGB neurons could shift either toward higher or lower frequencies based on the vector strength or the firing rate. The tuning of MGB neurons for spatial location could both sharpen or widen. Elevation preference could shift toward higher or lower elevations and azimuth tuning could move toward ipsilateral or contralateral locations. Such bidirectional changes were observed in many parameters which suggests that the auditory thalamus acts as a filter that could be adjusted according to behaviorally driven signals from auditory cortex. Future work will have to delineate the circuit elements responsible for the observed effects.

Distinct neuron populations for simple and compound calls in the primary auditory cortex of awake marmosets.

Marmosets are highly social non-human primates that live in families. They exhibit rich vocalization, but the neural basis underlying this complex vocal communication is largely unknown. Here we report the existence of specific neuron populations in marmoset A1 that respond selectively to distinct simple or compound calls made by conspecific marmosets. These neurons were spatially dispersed within A1 but distinct from those responsive to pure tones. Call-selective responses were markedly diminished when individual domains of the call were deleted or the domain sequence was altered, indicating the importance of the global rather than local spectral-temporal properties of the sound. Compound call-selective responses also disappeared when the sequence of the two simple-call components was reversed or their interval was extended beyond 1 s. Light anesthesia largely abolished call-selective responses. Our findings demonstrate extensive inhibitory and facilitatory interactions among call-evoked responses, and provide the basis for further study of circuit mechanisms underlying vocal communication in awake non-human primates.

Neural network of social interaction observation in marmosets.

A crucial component of social cognition is to observe and understand the social interactions of other individuals. A promising nonhuman primate model for investigating the neural basis of social interaction observation is the common marmoset (Callithrix jacchus), a small New World primate that shares a rich social repertoire with humans. Here, we used functional magnetic resonance imaging acquired at 9.4 T to map the brain areas activated by social interaction observation in awake marmosets. We discovered a network of subcortical and cortical areas, predominately in the anterior lateral frontal and medial frontal cortex, that was specifically activated by social interaction observation. This network resembled that recently identified in Old World macaque monkeys. Our findings suggest that this network is largely conserved between New and Old World primates and support the use of marmosets for studying the neural basis of social cognition.

Whole brain mapping of somatosensory responses in awake marmosets investigated with ultra-high-field fMRI.

The common marmoset (Callithrix jacchus) is a small-bodied New World primate that is becoming an important model to study brain functions. Despite several studies exploring the somatosensory system of marmosets, all results have come from anesthetized animals using invasive techniques and postmortem analyses. Here, we demonstrate the feasibility for getting high-quality and reproducible somatosensory mapping in awake marmosets with functional magnetic resonance imaging (fMRI). We acquired fMRI sequences in four animals, while they received tactile stimulation (via air-puffs), delivered to the face, arm, or leg. We found a topographic body representation with the leg representation in the most medial part, the face representation in the most lateral part, and the arm representation between leg and face representation within areas 3a, 3b, and 1/2. A similar sequence from leg to face from caudal to rostral sites was identified in areas S2 and PV. By generating functional connectivity maps of seeds defined in the primary and second somatosensory regions, we identified two clusters of tactile representation within the posterior and midcingulate cortex. However, unlike humans and macaques, no clear somatotopic maps were observed. At the subcortical level, we found a somatotopic body representation in the thalamus and, for the first time in marmosets, in the putamen. These maps have similar organizations, as those previously found in Old World macaque monkeys and humans, suggesting that these subcortical somatotopic organizations were already established before Old and New World primates diverged. Our results show the first whole brain mapping of somatosensory responses acquired in a noninvasive way in awake marmosets.NEW & NOTEWORTHY We used somatosensory stimulation combined with functional MRI (fMRI) in awake marmosets to reveal the topographic body representation in areas S1, S2, thalamus, and putamen. We showed the existence of a body representation organization within the thalamus and the cingulate cortex by computing functional connectivity maps from seeds defined in S1/S2, using resting-state fMRI data. This noninvasive approach will be essential for chronic studies by guiding invasive recording and manipulation techniques.