Face-Selective Patches in Marmosets Are Involved in Dynamic and Static Facial Expression Processing.

The correct identification of facial expressions is critical for understanding the intention of others during social communication in the daily life of all primates. Here we used ultra-high-field fMRI at 9.4 T to investigate the neural network activated by facial expressions in awake New World common marmosets from both male and female sex, and to determine the effect of facial motions on this network. We further explored how the face-patch network is involved in the processing of facial expressions. Our results show that dynamic and static facial expressions activate face patches in temporal and frontal areas (O, PV, PD, MD, AD, and PL) as well as in the amygdala, with stronger responses for negative faces, also associated with an increase of the respiration rates of the monkey. Processing of dynamic facial expressions involves an extended network recruiting additional regions not known to be part of the face-processing network, suggesting that face motions may facilitate the recognition of facial expressions. We report for the first time in New World marmosets that the perception and identification of changeable facial expressions, vital for social communication, recruit face-selective brain patches also involved in face detection processing and are associated with an increase of arousal.SIGNIFICANCE STATEMENT Recent research in humans and nonhuman primates has highlighted the importance to correctly recognize and process facial expressions to understand others' emotions in social interactions. The current study focuses on the fMRI responses of emotional facial expressions in the common marmoset (Callithrix jacchus), a New World primate species sharing several similarities of social behavior with humans. Our results reveal that temporal and frontal face patches are involved in both basic face detection and facial expression processing. The specific recruitment of these patches for negative faces associated with an increase of the arousal level show that marmosets process facial expressions of their congener, vital for social communication.

Improving preclinical to clinical translation of cognitive function for aging-related disorders: the utility of comprehensive touchscreen testing batteries in common marmosets.

Concerns about poor animal to human translation have come increasingly to the fore, in particular with regards to cognitive improvements in rodent models, which have failed to translate to meaningful clinical benefit in humans. This problem has been widely acknowledged, most recently in the field of Alzheimer's disease, although this issue pervades the spectrum of central nervous system (CNS) disorders, including neurodevelopmental, neuropsychiatric, and neurodegenerative diseases. Consequently, recent efforts have focused on improving preclinical to clinical translation by incorporating more clinically analogous outcome measures of cognition, such as touchscreen-based assays, which can be employed across species, and have great potential to minimize the translational gap. For aging-related research, it also is important to incorporate model systems that facilitate the study of the long prodromal phase in which cognitive decline begins to emerge and which is a major limitation of short-lived species, such as laboratory rodents. We posit that to improve translation of cognitive function and dysfunction, nonhuman primate models, which have conserved anatomical and functional organization of the primate brain, are necessary to move the field of translational research forward and to bridge the translational gaps. The present studies describe the establishment of a comprehensive battery of touchscreen-based tasks that capture a spectrum of domains sensitive to detecting aging-related cognitive decline, which will provide the greatest benefit through longitudinal evaluation throughout the prolonged lifespan of the marmoset.

A model of marmoset monkey vocal turn-taking.

Vocal turn-taking has been described in a diversity of species. Yet, a model that is able to capture the various processes underlying this social behaviour across species has not been developed. To this end, here we recorded a large and diverse dataset of marmoset monkey vocal behaviour in social contexts comprising one, two and three callers and developed a model to determine the keystone factors that affect the dynamics of these natural communicative interactions. Notably, marmoset turn-taking did not abide by coupled-oscillator dynamics, but rather call timing was overwhelmingly stochastic in these exchanges. Our features-based model revealed four key factors that encapsulate the majority of patterns evident in the behaviour, ranging from internal processes, such as particular states of the individual driving increased calling, to social context-driven suppression of calling. These findings indicate that marmoset vocal turn-taking is affected by a broader suite of mechanisms than previously considered and that our model provides a predictive framework with which to further explicate this natural behaviour at both the behavioural and neurobiological levels, and for direct comparisons with the analogous behaviour in other species.

Early molecular events of autosomal-dominant Alzheimer's disease in marmosets with PSEN1 mutations.

INTRODUCTION: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan. METHODS: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures. RESULTS: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood. DISCUSSION: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression. HIGHLIGHTS: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.

Rabies virus variants from bats closely related to variants found in marmosets (Callithrix jacchus), a neglected source of human rabies infection in Brazil.

Rabies is a fatal viral zoonosis caused by rabies virus (RABV). RABV infects the central nervous system and triggers acute encephalomyelitis in both humans and animals. Endemic in the Brazilian Northeast region, RABV emergence in distinct wildlife species has been identified as a source of human rabies infection and as such, constitutes a public health concern. Here, we performed post-mortem RABV analyses of 144 encephalic tissues from bats sampled from January to July 2022, belonging to 15 different species. We identified phylogenetically distinct RABV from Phyllostomidae and Molossidae bats circulating in Northeastern Brazil. Phylogenetic clustering revealed the close evolutionary relationship between RABV viruses circulating in bats and variants hosted in white-tufted marmosets, commonly captured to be kept as pets and linked to human rabies cases and deaths in Brazil. Our findings underline the urgent need to implement a phylogenetic-scale epidemiological surveillance platform to track multiple RABV variants which may pose a threat to both humans and animals.

A tale of two hierarchies: Hormonal and behavioral factors underlying sex differences in social dominance in cooperative breeding callitrichids.

Callitrichid primates are recognized for high levels of sociality in small groups, their great behavioral flexibility, and single-female dominant hierarchies. Previous work has highlighted that dominant, breeding callitrichids engage in behavioral and hormonal reproductive suppression of related and unrelated subordinates by both producing more offspring, having higher levels of ovulatory hormones, and accessing more sociosexual opportunities. This suppression constitutes a nexus of changes in pituitary responsiveness, ovarian cyclicity, sexual behavior, affiliation, and aggression. In this review, I will highlight important features that characterize callitrichid social hierarchies across broad social contexts. Dominant females sometimes exert reproductive suppression on subordinate nonbreeding females, but this suppression varies across callitrichids based on social stability and changes in group composition, particularly related to the number, experience, and age of nonbreeding subordinates. Meanwhile, dominant males may induce suppression of reproduction in subordinate males, but these effects occur by different behavioral and endocrine mechanisms and to a much lesser extent than their female counterparts; While dominant female callitrichids usually show higher levels of aggression relative to their male counterparts, callitrichids show a general absence of intersexual dominance, likely as an effort of maintaining a cohesive breeding pair within a stable social group and social cooperation. Future efforts are needed to identify precise neuroendocrine mechanisms underlying the presence of sex differences in callitrichid behavior separate from peripheral reproductive function. This is especially important with regard to parental experience, social relationships, development and aging, with larger implications toward understanding sex differences in overall health and wellbeing.

A comparison between common marmosets (Callithrix jacchus) and human infants sheds light on traits proposed to be at the root of human octave equivalence.

Two notes separated by a doubling in frequency sound similar to humans. This "octave equivalence" is critical to perception and production of music and speech and occurs early in human development. Because it also occurs cross-culturally, a biological basis of octave equivalence has been hypothesized. Members of our team previousy suggested four human traits are at the root of this phenomenon: (1) vocal learning, (2) clear octave information in vocal harmonics, (3) differing vocal ranges, and (4) vocalizing together. Using cross-species studies, we can test how relevant these respective traits are, while controlling for enculturation effects and addressing questions of phylogeny. Common marmosets possess forms of three of the four traits, lacking differing vocal ranges. We tested 11 common marmosets by adapting an established head-turning paradigm, creating a parallel test to an important infant study. Unlike human infants, marmosets responded similarly to tones shifted by an octave or other intervals. Because previous studies with the same head-turning paradigm produced differential results to discernable acoustic stimuli in common marmosets, our results suggest that marmosets do not perceive octave equivalence. Our work suggests differing vocal ranges between adults and children and men and women and the way they are used in singing together may be critical to the development of octave equivalence. RESEARCH HIGHLIGHTS: A direct comparison of octave equivalence tests with common marmosets and human infants Marmosets show no octave equivalence Results emphasize the importance of differing vocal ranges between adults and infants.

Arm movements induced by noninvasive optogenetic stimulation of the motor cortex in the common marmoset.

Optogenetics is now a fundamental tool for investigating the relationship between neuronal activity and behavior. However, its application to the investigation of motor control systems in nonhuman primates is rather limited, because optogenetic stimulation of cortical neurons in nonhuman primates has failed to induce or modulate any hand/arm movements. Here, we used a tetracycline-inducible gene expression system carrying CaMKII promoter and the gene encoding a Channelrhodopsin-2 variant with fast kinetics in the common marmoset, a small New World monkey. In an awake state, forelimb movements could be induced when Channelrhodopsin-2-expressing neurons in the motor cortex were illuminated by blue laser light with a spot diameter of 1 mm or 2 mm through a cranial window without cortical invasion. Forelimb muscles responded 10 ms to 50 ms after photostimulation onset. Long-duration (500 ms) photostimulation induced discrete forelimb movements that could be markerlessly tracked with charge-coupled device cameras and a deep learning algorithm. Long-duration photostimulation mapping revealed that the primary motor cortex is divided into multiple domains that can induce hand and elbow movements in different directions. During performance of a forelimb movement task, movement trajectories were modulated by weak photostimulation, which did not induce visible forelimb movements at rest, around the onset of task-relevant movement. The modulation was biased toward the movement direction induced by the strong photostimulation. Combined with calcium imaging, all-optical interrogation of motor circuits should be possible in behaving marmosets.

Efficiency of whole-exome sequencing in old world and new world primates using human capture reagents.

BACKGROUND: Whole-exome sequencing (WES) can expedite research on genetic variation in non-human primate (NHP) models of human diseases. However, NHP-specific reagents for exome capture are not available. This study reports the use of human-specific capture reagents in WES for olive baboons, marmosets, and vervet monkeys. METHODS: Exome capture was carried out using the SureSelect Human All Exon V6 panel from Agilent Technologies, followed by high-throughput sequencing. Capture of protein-coding genes and detection of single nucleotide variants were evaluated. RESULTS: Exome capture and sequencing results showed that more than 97% of old world and 93% of new world monkey protein coding genes were detected. Single nucleotide variants were detected across the genomes and missense variants were found in genes associated with human diseases. CONCLUSIONS: A cost-effective approach based on commercial, human-specific reagents can be used to perform WES for the discovery of genetic variants in these NHP species.

Contextual complexity of chemical signals in callitrichids.

In nearly four decades our research and that of others on chemical signaling in callitrichid primates suggest a high degree of contextual complexity in both the use of signals and the response to these signals. We describe our research including observational field studies, behavioral bioassays ("playbacks"), functional imaging, and conditioning studies. Scent marking in both captivity and the wild is used for more than just territorial marking. Social contextual effects are seen in responses by subordinate females responding with ovulatory inhibition only to chemical signals from familiar dominant reproductive females. Males detect ovulation through changes in scent marks. Males responded behaviorally and hormonally to chemical signals of novel ovulating females as a function of their reproductive status (fathers, males paired with a female but not fathers, and single males). Multiple brain areas are activated in males by female chemical signals of ovulation including areas relating to memory, evaluation, and motivation. Furthermore, males can be conditioned to respond sexually to a nonsexual odor demonstrating that learning plays an important role in response to chemical signals. Male androgen and estrone levels changed significantly in response to infant chemical signals as a function of whether the males were fathers or not, whether the odors were from their own versus other infants, as well as the infant's stage of development. Chemical signals in callitrichids are providing a rich source of understanding the context and function of the chemical sensory system and its stimulation of neural, behavioral, and hormonal actions in the recipients.

Pygmy Marmoset Exudate Feeding Stimulates Exudate Production.

Exudates are an important renewable resource for many primates. Exudate renewability is based on observations of primates repeatedly depleting exudate sites and measures of exudate trees' daily replenishment rates, but the role of the consumer in the renewal process is unclear. Trees' exudate production may be independent of the consumer, remaining unchanged regardless of depletion frequency, but since trees produce exudates as a physiological response to fungal infection, they may produce more exudates with more frequent depletion. To test these competing hypotheses, we employed a within-subjects experimental design in which we exposed pygmy marmosets' exudate holes to two treatment conditions: collecting exudates after 5 h and collecting exudates every hour for five consecutive hours. To compare production outputs between treatments, we used generalised linear mixed modelling in which log-transformed production data were a function of treatment with exudate holes nested within trees as a random effect. The model indicated that the cumulative production of hourly exudate removal was significantly greater than the amount accumulated after 5 h. Furthermore, the random effect of holes nested within trees had the greatest impact on variation in differences between treatments, but another unknown source also contributed to the observed variation. These results support the hypothesis that consumers partly drive exudate production, and although it is unknown what other factors, such as fungal load and healing trajectory, may influence variation between treatments, we conclude that pygmy marmosets can stimulate exudate production by consuming exudates.

A platform for semiautomated voluntary training of common marmosets for behavioral neuroscience.

Generally behavioral neuroscience studies of the common marmoset employ adaptations of well-established training methods used with macaque monkeys. However, in many cases these approaches do not readily generalize to marmosets indicating a need for alternatives. Here we present the development of one such alternate: a platform for semiautomated, voluntary in-home cage behavioral training that allows for the study of naturalistic behaviors. We describe the design and production of a modular behavioral training apparatus using CAD software and digital fabrication. We demonstrate that this apparatus permits voluntary behavioral training and data collection throughout the marmoset's waking hours with little experimenter intervention. Furthermore, we demonstrate the use of this apparatus to reconstruct the kinematics of the marmoset's upper limb movement during natural foraging behavior.NEW & NOTEWORTHY The study of marmosets in neuroscience has grown rapidly and presents unique challenges. We address those challenges with an innovative platform for semiautomated, voluntary training that allows marmosets to train throughout their waking hours with minimal experimenter intervention. We describe the use of this platform to capture upper limb kinematics during foraging and to expand the opportunities for behavioral training beyond the limits of traditional training sessions. This flexible platform can easily incorporate other tasks.

Hypervirulent Klebsiella pneumoniae as Unexpected Cause of Fatal Outbreak in Captive Marmosets, Brazil.

After the sudden death of captive marmosets in São Paulo, Brazil, we conducted a histologic and microbiologic study. We found hyperacute septicemia caused by hypermucoviscous sequence type 86 K2 Klebsiella pneumoniae. We implemented prophylactic antimicrobial therapy, selected dedicated staff for marmoset interactions, and sanitized the animals' fruit to successfully control this outbreak.

Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.

Not all fine-branch locomotion is equal: Grasping morphology determines locomotor performance on narrow supports.

Fine-branch models have long played a central role in primate evolutionary research. Nevertheless, recent studies of positional behavior in nonprimate arboreal mammals have challenged the idea that synapomorphic primate features, such as grasping extremities, uniquely facilitated access to the fine-branch zone. We test the alternative hypothesis that grasping extremities specifically improve locomotor performance in a fine-branch environment by examining how support diameter influences locomotor mechanics in one sciurid rodent (Sciurus carolinensis) and two platyrrhine primates (Callithrix jacchus and Saimiri boliviensis). These species were chosen to broadly model different stages in the evolution of primate grasping morphology. The results showed that transitioning from broad to narrower supports required the greatest kinematic adjustment in squirrels and the least adjustment in squirrel monkeys, with marmosets displaying an intermediate level of adjustment. Moreover, on any given support, squirrels' locomotor mechanics differed from marmosets' in a manner consistent with a greater need for stability, despite superficial ecomorphological similarities between sciurid rodents and callitrichine primates. Morphological analyses of autopodial size and proportions suggest that variation in locomotor performance more closely tracked variation in overall hand and foot size rather than digit length per se. Indeed, a broad comparative analysis revealed that for their body mass, primates have longer hands than similarly sized arboreal rodents and marsupials (although only the primate-rodent comparison was significant after incorporating phylogenetic relatedness). Inclusion of fossil stem primates (plesiadapiforms) and euprimates (adapiforms) in these analyses suggests that this primate-wide grade shift in relative autopodial size must have occurred early in the evolutionary history of the group. Overall, our findings show that basal primate morphological adaptations may have specifically facilitated improved locomotor performance in a fine-branch niche, rather than merely permitting access to the environment. As such, future adaptive hypotheses of primate origins should incorporate the import of primate-like morphology on locomotor performance as well.

Marmoset prosociality is intentional.

Marmoset monkeys show high levels of proactive prosociality, a trait shared with humans, presumably because both species rely on allomaternal care. However, it is not clear whether the proximate regulation of this convergent trait is also similar, in particular with regard to intentionality, which is a defining characteristic of prosocial behavior in the human literature. The aim of this paper was to investigate whether marmoset monkeys' prosociality fulfils the criteria of intentionality developed in primate communication research. The results show that marmoset prosocial behavior (i) has some degree of flexibility, since individuals can use multiple means to reach their goal and adjust them to specific conditions, (ii) depends on the presence of an audience, i.e. potential recipients (social use), and (iii) is goal-directed, because (a) it continues exactly until the putative goal is reached, and (b) individuals check back and look at/for their partner when their prosocial actions do not achieve the putative goal (i.e. if their actions don't lead to the expected outcome, this elicits distinct reactions in the actor). These results suggest that marmoset prosociality is under some degree of voluntary, intentional control. They are in line with other findings that marmosets perceive each other as intentional agents, and only learn socially from actions that are perceived as intentional. The most parsimonious conclusion is, therefore, that prosocial behavior is fundamentally under voluntary control in marmosets, just as it is in humans, even though our more sophisticated cognitive abilities allow for a far more complex integration of prosociality into a broader variety of contexts and of behavioral goals.

c-Jun expression after cerebral hyperstimulation differs between rats and marmosets.

Immediate early genes (IEGs) are a fundamental element in the way we respond and adapt to a variety of stimuli. We have recently reported that IEG response, as measured by c-Fos expression, is different between rodents and primates. Here, we further extend this analysis by assessing the expression of c-Jun, one of the main complements of c-Fos, under the same stimulation protocol. For this, we investigated the immunohistochemical expression of c-Jun (and compared with that previously shown for c-Fos) after stimulation with pentylenetetrazol in the cingulate gyrus, motor cortex, piriform cortex, inferior temporal cortex, and visual cortex of rats and marmosets (Callithrix jacchus), both male and female. Overall the immunohistochemical expression of c-Jun was more intense but remained elevated for a shorter duration in marmosets as compared to rats. These results are in contrast to what we had previously shown for c-Fos. Furthermore, in terms of the temporal profile, c-Fos and c-Jun expression occurred in a complementary manner in rats-the peak of c-Fos expression coincided with low levels of c-jun expression-and in a superimposed manner in marmosets-the peak of c-Fos expression coincided with the peak of c-Jun expression. Since Fos proteins may form dimers with Jun proteins and together control late gene expressions in the cell nucleus, this different expression profile between primates and rodents may bear meaningful impact for how the nervous system reacts and adapts to stimulation.

Comparative genomics of Bifidobacterium species isolated from marmosets and humans.

The genus Bifidobacterium is purported to have beneficial consequences for human health and is a major component of many gastrointestinal probiotics. Although species of Bifidobacterium are generally at low relative frequency in the adult human gastrointestinal tract, they can constitute high proportions of the gastrointestinal communities of adult marmosets. To identify genes that might be important for the maintenance of Bifidobacterium in adult marmosets, ten strains of Bifidobacterium were isolated from the feces of seven adult marmosets, and their genomes were sequenced. There were six B. reuteri strains, two B. callitrichos strains, one B. myosotis sp. nov. and one B. tissieri sp. nov. among our isolates. Phylogenetic analysis showed that three of the four species we isolated were most closely related to B. bifidum, B. breve and B. longum, which are species found in high abundance in human infants. There were 1357 genes that were shared by at least one strain of B. reuteri, B. callitrichos, B. breve, and B. longum, and 987 genes that were found in all strains of the four species. There were 106 genes found in B. reuteri and B. callitrichos but not in human bifidobacteria, and several of these genes were involved in nutrient uptake. These pathways for nutrient uptake appeared to be specific to Bifidobacterium from New World monkeys. Additionally, the distribution of Bifidobacterium in fecal samples from captive adult marmosets constituted as much as 80% of the gut microbiome, although this was variable between individuals and colonies. We suggest that nutrient transporters may be important for the maintenance of Bifidobacterium during adulthood in marmosets.

Generation of genetically engineered non-human primate models of brain function and neurological disorders.

Research with non-human primates (NHP) has been essential and effective in increasing our ability to find cures for a large number of diseases that cause human suffering and death. Extending the availability and use of genetic engineering techniques to NHP will allow the creation and study of NHP models of human disease, as well as broaden our understanding of neural circuits in the primate brain. With the recent development of efficient genetic engineering techniques that can be used for NHP, there's increased hope that NHP will significantly accelerate our understanding of the etiology of human neurological and neuropsychiatric disorders. In this article, we review the present state of genetic engineering tools used in NHP, from the early efforts to induce exogeneous gene expression in macaques and marmosets, to the latest results in producing germline transmission of different transgenes and the establishment of knockout lines of specific genes. We conclude with future perspectives on the further development and employment of these tools to generate genetically engineered NHP.

Investigating the spatiotemporal characteristics of the deoxyhemoglobin-related and deoxyhemoglobin-unrelated functional hemodynamic response across cortical layers in awake marmosets.

Blood oxygenation level dependent (BOLD) functional magnetic resonance imaging (fMRI) has become a major tool to map neural activity. However, the spatiotemporal characteristics of the BOLD functional hemodynamic response across the cortical layers remain poorly understood. While human fMRI studies suffer from low spatiotemporal resolution, the use of anesthesia in animal models introduces confounding factors. Additionally, inflow contributions to the fMRI signal become non-negligible when short repetition times (TRs) are used. In the present work, we mapped the BOLD fMRI response to somatosensory stimulation in awake marmosets. To address the above technical concerns, we used a dual-echo gradient-recalled echo planar imaging (GR-EPI) sequence to separate the deoxyhemoglobin-related response (absolute T2* differences) from the deoxyhemoglobin-unrelated response (relative S0 changes). We employed a spatial saturation pulse to saturate incoming arterial spins and reduce inflow effects. Functional GR-EPI images were obtained from a single coronal slice with two different echo times (13.5 and 40.5ms) and TR=0.2s. BOLD, T2*, and S0 images were calculated and their functional responses were detected in both hemispheres of primary somatosensory cortex, from which five laminar regions (L1+2, L3, L4, L5, and L6) were derived. The spatiotemporal distribution of the BOLD response across the cortical layers was heterogeneous, with the middle layers having the highest BOLD amplitudes and shortest onset times. ΔT2* also showed a similar trend. However, functional S0 changes were detected only in L1+2, with a fast onset time. Because inflow effects were minimized, the source of S0 functional changes in L1+2 could be attributed to a reduction of cerebrospinal fluid volume fraction due to the functional increase in cerebral blood volume and to unmodeled T2* changes in the extra- and intra-venous compartments. Caution should be exercised when interpreting laminar BOLD fMRI changes in superficial layers as surrogates of underlying neural activity.