DNA metabarcoding reveals the seasonal variation of dietary composition of Taihangshan macaque (Macaca mulatta tcheliensis), Jiyuan, north China.

Dietary analysis in wildlife is fundamental for understanding their flexible response to seasonal changes and developing effective conservation management measures. Taihangshan macaque (Macaca mulatta tcheliensis) is the northernmost population of rhesus macaque, currently only distributed in the southern Mt. Taihangshan area. This area belongs to a semi-arid region resulting in limited plant food availability for Taihangshan macaques, with seasonal variation. Herein, we used a chloroplast trnL DNA metabarcoding approach to identify the plant diet diversity and composition from 100 fecal samples of Taihangshan macaque in four seasons (spring, summer, autumn, and winter) from 2020 to 2021. The results revealed that (1) a total of 48 distinct families, 88 genera, and 52 species within the 105 food items that were consumed by Taihangshan macaques throughout the year; (2) the diversity of food items exhibited significant differences across the four seasons; (3) Rosaceae, Rhamnaceae, Fagaceae, and Poaceae are the preferential food items for Taihangshan macaques and have different relative abundances, fluctuating with seasonal variation. DNA metabarcoding can expand our understanding of the repertoire of food items consumed by Taihangshan macaques by detecting some consumed food items in this population that were not yet discovered using traditional methods. Therefore, the integrative results from traditional methods and DNA metabarcoding can provide a fundamental understanding of dietary composition to guide the conservation management of Taihangshan macaques.

Macaque Brainnetome Atlas: A multifaceted brain map with parcellation, connection, and histology.

The rhesus macaque (Macaca mulatta) is a crucial experimental animal that shares many genetic, brain organizational, and behavioral characteristics with humans. A macaque brain atlas is fundamental to biomedical and evolutionary research. However, even though connectivity is vital for understanding brain functions, a connectivity-based whole-brain atlas of the macaque has not previously been made. In this study, we created a new whole-brain map, the Macaque Brainnetome Atlas (MacBNA), based on the anatomical connectivity profiles provided by high angular and spatial resolution ex vivo diffusion MRI data. The new atlas consists of 248 cortical and 56 subcortical regions as well as their structural and functional connections. The parcellation and the diffusion-based tractography were evaluated with invasive neuronal-tracing and Nissl-stained images. As a demonstrative application, the structural connectivity divergence between macaque and human brains was mapped using the Brainnetome atlases of those two species to uncover the genetic underpinnings of the evolutionary changes in brain structure. The resulting resource includes: (1) the thoroughly delineated Macaque Brainnetome Atlas (MacBNA), (2) regional connectivity profiles, (3) the postmortem high-resolution macaque diffusion and T2-weighted MRI dataset (Brainnetome-8), and (4) multi-contrast MRI, neuronal-tracing, and histological images collected from a single macaque. MacBNA can serve as a common reference frame for mapping multifaceted features across modalities and spatial scales and for integrative investigation and characterization of brain organization and function. Therefore, it will enrich the collaborative resource platform for nonhuman primates and facilitate translational and comparative neuroscience research.

Effects of Seasonality and Pregnancy on Hair Loss and Regrowth in Rhesus Macaques.

Several studies have examined the etiology of alopecia, or hair loss, in rhesus macaques. While outcomes differ across studies, some commonalities have emerged. Females, particularly pregnant females, show more alopecia than males, and alopecia follows a seasonal pattern. Much research has explored causes of hair loss; however, alopecia can result from lack of hair growth in addition to hair loss. To better understand how sex, reproductive state, and season affect alopecia, we followed 241 rhesus macaques (Macaca mulatta) in outdoor breeding groups over one year, recording both alopecia severity and presence of hair regrowth. We found that both alopecia and hair regrowth followed a seasonal pattern; alopecia was highest in spring and lowest in late summer, while regrowth started in spring and peaked in late summer. Reproductive state also correlated with both alopecia and hair growth. Females in their third trimester had the highest average level of alopecia and the lowest amount of hair regrowth. Regrowth resumed postpartum, regardless of whether females were rearing an infant. Results indicate that the seasonal pattern of alopecia is due in part to the seasonal limitations on hair regrowth, and that breeding, which also occurs seasonally in rhesus macaques, may further suppress hair regrowth.

Pterion variation in the skulls of rhesus macaques from Cayo Santiago: Inheritance, development, and pathology.

The pterion is the sutural juncture of the frontal, parietal, sphenoidal, temporal, and zygomatic bones on the lateral aspect of the cranium. As a craniometric landmark, the pterion has a taxonomic valence, in addition to a common neurosurgical entry point in medicine. Variation in the articulation patterns at the pterion have been documented between primate species yet have a high degree of uniformity within species, suggesting a genetic control for this complex region of the skull. In this study, pterion pattern variation was investigated in 1627 Rhesus macaque crania of the Cayo Santiago colony. The colony's associated skeletal collections accompany known age, sex, and maternal lineages. Pterion pattern prevalence rates were tested against matrilines, as well as cranial shape, and cranial sutural fusion ages (including individuals with prematurely fused sutures). Five patterns were identified, the most prominent being the prevailing Old World Monkey frontotemporal (FT) articulation (83.4%). The relative frequency of those not exhibiting the FT pattern was found to vary considerably between matrilineal families (p = 0.037), ranging from 5.3% to 34.2%. Mothers with the non-FT pterion pattern were three times as likely to bear non-FT offspring. Cranial shape additionally varied with pterion type. Males exhibiting zygomaticotemporal (ZT) and sphenoparietal (SP) articulations possessed a relatively longer and narrower cranium than those with the default FT type (p = < 0.001). Cranial sutural fusion ages were not found to differ between pterion types, though all individuals with craniosynostosis (6; 0.38%) exhibited the FT type. The study provided strong evidence for a genetic source for pterion pattern as well as outlining a relatively novel relationship with cranial shape and sutural fusion ages. A unifying explanation may lie in those genes involved in both sutural and craniofacial development, or in the variation of brain growth processes channeling sutural articulation at the pterion. Both may be heritable and responsible for producing observed matrilineal differences in the pterion.

Analysis of cell populations in the normal rhesus macaque (Macaca mulatta) lower intestinal tract and diagnostic thresholds for chronic enterocolitis.

Rhesus macaques (Macaca mulatta) are used extensively in biomedical research, often with a focus on the gastrointestinal tract, and yet a full characterization of their normal resident intestinal cell populations has not been published. In addition, chronic enterocolitis (CE), also known as idiopathic chronic diarrhea, affects up to 25% of colony-housed rhesus macaques, often requiring euthanasia for welfare concerns and severely limiting their value as a breeding animal or research subject. We aimed to characterize subjective and objective variables in sections of the ileum, cecum, colon, and rectum in 16 healthy rhesus macaques and compare these results with a cohort of 37 animals euthanized for CE to produce relevant diagnostic thresholds and to improve case definitions for future studies. We found neutrophils to be an infrequent but expected component of the large intestinal leukocyte population. Animals with CE had significantly increased total leukocyte populations between crypts in the cecum, colon, and rectum; variable increases in specific cell populations across all levels of the distal intestinal tract; and significantly increased intraepithelial CD3+ T cells in the colon and rectum. Concentrations of enteroendocrine cells, enterochromaffin cells, and intestinal mast cells were not significantly different between healthy and affected individuals. This study characterizes individual leukocyte populations in the rhesus macaque lower intestinal tract, is the first to evaluate rhesus macaque intestinal mast cells, and provides key diagnostic thresholds for evaluating animals with potential CE.

Identification of microRNAs Derived from Transposable Elements in the Macaca mulatta (Rhesus Monkey) Genome.

Transposable elements (TEs) are mobile DNA entities that can move within the host genome. Over long periods of evolutionary time, TEs are typically silenced via the accumulation of mutations in the genome, ultimately resulting in their immobilization. However, they still play an important role in the host genome by acting as regulatory elements. They influence host transcription in various ways, one of which as the origin of the generation of microRNAs (miRNAs), which are so-called miRNAs derived from TEs (MDTEs). miRNAs are small non-coding RNAs that are involved in many biological processes by regulating gene expression at the post-transcriptional level. Here, we identified MDTEs in the Macaca mulatta (rhesus monkey) genome, which is phylogenetically close species to humans, based on the genome coordinates of miRNAs and TEs. The expression of 5 out of 17 MDTEs that were exclusively registered in M. mulatta from the miRBase database (v22) was examined via quantitative polymerase chain reaction (qPCR). Moreover, Gene Ontology analysis was performed to examine the functional implications of the putative target genes of the five MDTEs.

Nascent transcription upon interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines.

OBJECTIVES: The interferon-triggered innate immune response has been observed to be under strong diversifying selection to counteract the many pathogens hosts have to defend against. In particular, rewiring of gene transcription regulation allows organisms to rapidly acquire new phenotypes by removing and adding genes into the innate immune gene network. Dissecting the molecular processes by which this rewiring takes place, either by changing the DNA regulatory elements or by changing the activity of the regulators across species, is key to better understand this evolutionary process. DATA DESCRIPTION: To better comprehend the evolutionary dynamics that have occurred in the initial transcriptional response to interferon in primates, we present Precision Run-On (PRO-seq) datasets made after 1 h of interferon-α2 stimulation on human and rhesus macaque lymphoblastoid cell lines. Further, we tested the difference between using either species' cognate interferon versus using the other orthologous interferon to account for any potential impacts in the interaction of the orthologous interferons with their cellular membrane receptors. This data provides insights into the regulatory mechanisms that drive species-specific responses to environmental perturbations, such as the one driven by the interactions of pathogens and their hosts.

Evidence of potential Campylobacter jejuni zooanthroponosis in captive macaque populations.

Non-human primates share recent common ancestry with humans and exhibit comparable disease symptoms. Here, we explored the transmission potential of enteric bacterial pathogens in monkeys exhibiting symptoms of recurrent diarrhoea in a biomedical research facility in China. The common zoonotic bacterium Campylobacter jejuni was isolated from macaques (Macaca mulatta and Macaca fascicularis) and compared to isolates from humans and agricultural animals in Asia. Among the monkeys sampled, 5 % (44/973) tested positive for C. jejuni, 11 % (5/44) of which displayed diarrhoeal symptoms. Genomic analysis of monkey isolates, and 1254 genomes from various sources in Asia, were used to identify the most likely source of human infection. Monkey and human isolates shared high average nucleotide identity, common MLST clonal complexes and clustered together on a phylogeny. Furthermore, the profiles of putative antimicrobial resistance genes were similar between monkeys and humans. Taken together these findings suggest that housed macaques became infected with C. jejuni either directly from humans or via a common contamination source.

Histomorphological analysis of the superficial musculoaponeurotic system in Macaca mulatta species.

INTRODUCTION: The superficial musculoaponeurotic system (SMAS) is a well described facial functional unit in humans. SMAS connects mimic musculature to the skin having many implication in facial mimic expression. One of the various morphological and physiological analogies in human and Macaca mulatta species is the facial mimic. The present study analyzed Macaca mulatta species SMAS morphology and its facial topographical differences and compared this with human SMAS tissue morphology. MATERIAL AND METHODS: Macaca mulatta full-graft tissue blocks of skin, subcutaneous tissue and mimic muscles from five topographical different facial regions (Regio Temporalis, Regio Buccalis, Regio Infraorbitalis, Regio Angulus Oris and Regio Mandibularis) were collected postmortem from eight individuals (n = 8) at the German Primate Center, Leibniz Institute for Primate Research in Göttingen (DPZ) and studied histologically. Haematoxylin-eosin and azan stained histological serial sections of full-graft tissue blocks were analyzed and SMAS topographical differences evaluated. RESULTS: SMAS typical tissue morphology was recognized in all Macaca mulatta histological serial sections (n = 780). Regio Infraorbitalis Macaca mulatta SMAS (MmSMAS) morphology was similar to human infraorbital SMAS morphology (type I SMAS). Suborbicularis oculi fat pad was recognized in Macaca mulatta samples. Human type I similar SMAS morphology was demonstrated over Macaca mulatta Regio Temporalis and Regio Buccalis. Regio Angulus Oris and the cranial area of the Regio Mandibularis presented human type II similar SMAS morphology. Type IV MmSMAS was closely related to the parotid gland tissue presence. The cervical area of the Regio Mandibularis presented human type V similar SMAS morphology. CONCLUSIONS: SMAS is a complex fibro-musculo-adipose tissue network and probably an important pivot in Macaca mulatta facial system supporting mimic expression. This study provided insights into MmSMAS typology and similarity with human SMAS tissue morphology.

Skeletal age during hurricane impacts fluctuating asymmetry in Cayo Santiago rhesus macaques.

As natural disasters become more frequent due to climate change, understanding the biological impact of these ecological catastrophes on wild populations becomes increasingly pertinent. Fluctuating asymmetry (FA), or random deviations from bilateral symmetry, is reflective of developmental instability and has long been positively associated with increases in environmental stress. This study investigates craniofacial FA in a population of free-ranging rhesus macaques (Macaca mulatta) that has experienced multiple Category 3 hurricanes since the colony's inception on Cayo Santiago, including 275 individuals from ages 9 months to 31 years (F = 154; M = 121). Using geometric morphometrics to quantify FA and a linear mixed-effect model for analysis, we found that sex, age, and decade of birth did not influence the amount of FA in the individuals included in the study, but the developmental stage at which individuals experienced these catastrophic events greatly impacted the amount of FA exhibited (p = .001). Individuals that experienced these hurricanes during fetal life exhibited greater FA than any other post-natal developmental period. These results indicate that natural disasters can be associated with developmental disruption that results in long-term effects if occurring during the prenatal period, possibly due to increases in maternal stress-related hormones.

A medida que los desastres naturales se vuelven más frecuentes debido al cambio climático, entender el impacto biológico de estas catástrofes ecológicas en poblaciones silvestres va en aumento pertinente. La asimetría fluctuante (AF), o desviaciones aleatorias de simetría bilateral, es reflejo de inestabilidad durante el desarrollo y se ha asociado positivamente con incrementos en estrés ambiental durante mucho tiempo. Este estudio investiga AF craneofacial en una población de macacos rhesus (Macaca mulatta) en libertad que ha experimentado múltiples huracanes categoría 3 desde el inicio de la colonia en Cayo Santiago, e incluye 275 individuos de 9 meses a 31 años de edad (F = 154; M = 121). Usando morfometría geométrica para cuantificar AF y un modelo lineal de efectos mixtos para análisis, encontramos que el sexo, la edad y la década de nacimiento no influyeron en la cantidad de AF en los individuos incluidos en el estudio, pero la etapa de desarrollo en la que los individuos experimentaron estos eventos catastróficos impactó altamente la cantidad de AF exhibida (p = .001). Los individuos que experimentaron estos huracanes durante el período fetal exhibieron mayor AF que cualquier otro período de desarrollo posnatal. Estos resultados indican que los desastres naturales pueden asociarse con trastornos del desarrollo que tienen efectos a largo plazo si ocurren durante el período prenatal, posiblemente debido al aumento de hormonas maternas relacionadas con el estrés.

Complete genome sequence of Shigella flexneri strain P099 from the gum of an adult rhesus monkey, Macaca mulatta, with gingivitis.

Shigella flexneri was associated with gingivitis, a periodontal disease, in the rhesus monkey. We report the circularized 4.8-Mbp complete genome of Shigella flexneri strain P099 isolated from the gum of an adult rhesus monkey, Macaca mulatta, with clinical symptoms of gingivitis.

Association between seroprevalence of measles virus in monkeys and degree of human-monkey contact in Bangladesh.

Measles infections can cause significant morbidity and mortality in human and monkey populations. The endemicity of measles in human populations and viral circulation within populations of free-living monkeys may have important repercussions for potential zoonotic transmission events and for the long-term health of monkey populations. Yet, there has not yet been a rigorous investigation of the dynamics of measles transmission where human and monkey populations coexist. In this study, to determine the difference in seroprevalence of the measles virus across different contexts of human-monkey contact, we analyzed serum samples collected from 56 apparently healthy Macaca mulatta monkeys who occupied diverse contexts, with different degrees of human-monkey contact, in Bangladesh. This is the first report of measles virus seroprevalence in monkeys in Bangladesh. We found a clear association between measles virus seropositivity in monkeys and the context in which they interact with humans. Seroprevalence was the lowest in wild areas (0.0%) and increased in shrines (4.8%), urban areas (5.9%), and was highest among monkeys who are used as performance animals (50.0%). This work suggests that a One Health approach informed by local interspecies transmission dynamics is necessary to develop strategies that both improve measles vaccination coverage, achieve long-term surveillance in monkey populations, and prevent measles spillback to monkeys. This approach aims to inform conservation efforts and protect the long-term health of human and monkey populations.

Diet and feeding behavior of a group of high-altitude rhesus macaques: high adaptation to food shortages and seasonal fluctuations.

Diet and feeding behavior data are crucial to a deep understanding of the behavioral response and adaptation of primates to a high-altitude environment. From August 2019 to June 2021, we collected data on the feeding behavior of a high-altitude rhesus macaque Macaca mulatta group from Yajiang County, Western Sichuan Plateau, which has an altitude of over 3,500 m. The results showed that feeding (33.0 ± 1.8%) and moving (28.3 ± 2.6%) were the dominant behavior of rhesus macaques. Macaques ate 193 food items, comprising 11 food categories from 90 species. Our study found that plant roots (30.9 ± 30.1%) and young leaves (28.0 ± 33.1%) were the main foods eaten by macaques. The preferred foods of rhesus macaques were young leaves, fruits, and seeds, and the consumption of these items was positively correlated with its food availability. When the availability of preferred foods was low, macaques took plant roots, barks, and fallen leaves as fallback foods. In particular, roots were a dominant food item in winter, and this way of feeding became a key survival strategy. Our results suggest that, facing the relative scarcity and strong seasonal fluctuations of food resources in high-altitude habitat, macaques adopt active foraging strategies, relying on a variety of food species and adjusting flexibly their food choices based on food availability, which may help to maximize the energy efficiency of high-altitude macaques.

Behavioral effects of triazolam and pregnanolone combinations: reinforcing and sedative-motor effects in female rhesus monkeys.

Introduction: Benzodiazepines (BZs) are prescribed as anxiolytics, but their use is limited by side effects including abuse liability and daytime drowsiness. Neuroactive steroids are compounds that, like BZs, modulate the effects of GABA at the GABAA receptor. In a previous study, combinations of the BZ triazolam and neuroactive steroid pregnanolone produced supra-additive (i.e., greater than expected effects based on the drugs alone) anxiolytic effects but infra-additive (i.e., lower than expected effects based on the drugs alone) reinforcing effects in male rhesus monkeys, suggestive of an improved therapeutic window. Methods: Female rhesus monkeys (n=4) self-administered triazolam, pregnanolone, and triazolam-pregnanolone combinations intravenously under a progressive-ratio schedule. In order to assess characteristic sedative-motor effects of BZ-neuroactive steroid combinations, female rhesus monkeys (n=4) were administered triazolam, pregnanolone, and triazolam-pregnanolone combinations. Trained observers, blinded to condition, scored the occurrence of species-typical and drug-induced behaviors. Results: In contrast to our previous study with males, triazolam-pregnanolone combinations had primarily supra-additive reinforcing effects in three monkeys but infra-additive reinforcing effects in one monkey. Scores for deep sedation (i.e., defined as atypical loose-limbed posture, eyes closed, does not respond to external stimuli) and observable ataxia (any slip, trip, fall, or loss of balance) were significantly increased by both triazolam and pregnanolone. When combined, triazolam-pregnanolone combinations had supra-additive effects for inducing deep sedation, whereas observable ataxia was attenuated, likely due to the occurrence of robust sedative effects. Discussion: These results suggest that significant sex differences exist in self-administration of BZ-neuroactive steroid combinations, with females likely to show enhanced sensitivity to reinforcing effects compared with males. Moreover, supra-additive sedative effects occurred for females, demonstrating a higher likelihood of this adverse effect when these drug classes are combined.

Congenital adactyly and oligodactyly with metatarsal hypoplasia in two rhesus monkeys.

Congenital digital defects, particularly adactyly and oligodactyly, have been reported frequently in humans, however, their occurrence in rhesus monkeys is rare, mainly in the hind limbs. We present here for the first time, two female rhesus monkeys with unilateral congenital toe defects with metatarsal involvement. One showing adactyly and the other oligodactyly. In this report, we present the clinical/radiological details of these cases as well as discuss the risk factors possibly involved, such as contact with pesticides and genetic factors.

Assembly of novel microbial genomes from gut metagenomes of rhesus macaque (Macaca mulatta).

Rhesus macaque (RM, Macaca mulatta), as an important model animal, commonly suffers from chronic diarrheal disease, challenging the breeding of RMs. Gut microbiomes play key roles in maintaining intestinal health of RMs. However, it is still unclear about more features of gut microbiome as responsible for intestinal health of RMs. In this study, we performed de novo assembly of metagenome-assembled genomes (MAGs) based on fecal metagenomes from chronic diarrheal RMs and asymptomatic individuals. In total of 731 non-redundant MAGs with at least 80% completeness were reconstructed in this study. More than 97% MAGs were novel genomes compared with more than 250,000 reference genomes. MAGs of Campylobacter and Helicobacteraceae from RM guts mainly carried flagella-associated virulence genes and chemotaxis-associated virulence genes, which might mediate motility and adhesion of bacteria. Comparing to MAGs of Campylobacter from humans, distributions and functions of these MAGs of Campylobacter from RMs exhibited significant differences. Most members of Bacteroidota, Spirochaetota, Helicobacteraceae, Lactobacillaceae and Anaerovibrio significantly decreased in guts of chronic diarrhea RMs. More than 92% MAGs in this study were not contained in 2,985 MAGs previously reported from other 22 non-human primates (NHPs), expanding the microbial diversity in guts of NHPs. The distributions and functions of gut microbiome were prominently influenced by host phylogeny of NHPs. Our results could help to more clearly understand about the diversity and function of RMs gut microbiome.

Monkeys do not show sex differences in toy preferences through their individual choices.

BACKGROUND: As interest in evaluating sex differences in nonhuman animals grows, the finding that male and female monkeys have toy preferences that differ, and that parallel those documented in human children, has garnered significant attention and is leveraged as an argument in favor of a biological contribution for human sex differences. To date, however, only two studies have investigated sex differences in monkeys' toy preferences, both documenting that males prefer toys considered to be "masculine" (such as vehicles) and females prefer toys considered to be "feminine" (such as dolls). Monkeys in these studies were tested in their social groups, making it hard to determine if the sex differences reported reflect actual individual preferences or result from social dynamics present at the time of testing. METHOD: Here, we assessed the preferences of 14 rhesus macaques (N = 7 males; N = 7 females) who were singly tested in a choice test with a variety of toys characterized as masculine (hard non-zoomorphic wheeled toys), feminine (zoomorphic soft toys), neutral (hard non-zoomorphic toys) and ambiguous (zoomorphic or plush vehicles) based on criteria from previous studies. RESULTS: Males and females showed similar preferences for neutral and "masculine" toys and preferred them (i.e., were more likely to interact with them) to "feminine" and sex-ambiguous toys. When they interacted with the toys, both males and females interacted more with neutral than with "masculine" toys. Females, but not males, interacted more with neutral and "masculine" toys than with "feminine" toys. The highest frequency of interaction for any single toy for the male monkeys was with the doll-standing is stark contrast to previous findings. CONCLUSIONS: Our results contrast greatly with the previous study in rhesus monkeys, as well as findings in human children, suggesting that the previously documented sex differences are likely context dependent, and question the existence of a strong biological basis to sex differences in toy preferences.

Composition of Bacteriophages for Therapeutic and Prophylactic Use in Monkeys.

Keeping rhesus monkeys as laboratory animals requires timely prevention and treatment of infections, including diseases of bacterial etiology. Based on our own studies of the microflora of healthy and sick monkeys, as well as analysis of published reports, we identified clinically significant representatives of pathogenic and opportunistic bacteria: E. coli, Staphylococcus aureus, Klebsiella spp., Proteus spp. The isolates of these bacterial species and genera circulating in monkeys kept in the enclosure were isolated, four virulent bacteriophage strains with a wide spectrum of lytic activity against these isolates were selected and newly isolated. The composition based on virulent bacteriophage strains was tested on monkeys with assessment of its safety and its dynamics of detection of phage-specific DNA.

Functional topography of pulvinar-visual cortex networks in macaques revealed by INS-fMRI.

The pulvinar in the macaque monkey contains three divisions: the medial pulvinar (PM), the lateral pulvinar (PL), and the inferior pulvinar (PI). Anatomical studies have shown that connections of PM are preferentially distributed to higher association areas, those of PL are biased toward the ventral visual pathway, and those of PI are biased with the dorsal visual pathway. To study functional connections of the pulvinar at mesoscale, we used a novel method called INS-fMRI (infrared neural stimulation and functional magnetic resonance imaging). This method permits studies and comparisons of multiple pulvinar networks within single animals. As previously revealed, stimulations of different sites in PL and PI produced topographically organized focal activations in visual areas V1, V2, and V3. In contrast, PM stimulation elicited little or diffuse response. The relative activations of areas V1, V2, V3A, V3d, V3v, V4, MT, and MST revealed that connections of PL are biased to ventral pathway areas, and those of PI are biased to dorsal areas. Different statistical values of activated blood-oxygen-level-dependent responses produced the same center of activation, indicating stability of connectivity; it also suggests possible dynamics of broad to focal responses from single stimulation sites. These results demonstrate that infrared neural stimulation-induced connectivity is largely consistent with previous anatomical connectivity studies, thereby demonstrating validity of our novel method. In addition, it suggests additional interpretations of functional connectivity to complement anatomical studies.