Distribution pattern of the celiac, cranial mesenteric, and caudal mesenteric arteries to the gastrointestinal tract in the common marmoset (Callithrix jacchus).

Gastrointestinal diseases pose problems to captive common marmosets. Therefore, knowledge of the anatomy of the arterial supply to the gastrointestinal tract is an important prerequisite for implementing appropriate veterinary care. The common marmoset's intestinal tract has a well-developed cecum specialized for the fermentative digestion of tree gums. This specialized gastrointestinal tract may have a unique pattern of arterial distribution. This study aimed to elucidate the species-specific gastrointestinal tract arterial anatomy of the common marmoset. We traced the celiac, cranial mesenteric, and caudal mesenteric arteries in six male and nine female common marmosets using the latex injection method. We found that the celiac artery gave rise to the left gastric, common hepatic, splenic, and caudal pancreaticoduodenal arteries. In addition to these arteries, the celiac artery gave origin to the middle colic or jejunal arteries in seven or one cases, respectively. The branches of the cranial mesenteric artery consisted of 3-6 arteries, including the middle colic, caudal pancreaticoduodenal, jejunal, right colic, ileocolic, and ileal arteries, as well as a common trunk of the ventral cecal and ileal branches, and the dorsal cecal and colic branches. In four cases, the cranial mesenteric artery gave rise to the jejunal, ileocolic, and ileal arteries. In one of the 13 cases, the celiac and cranial mesenteric arteries formed a common trunk. The caudal mesenteric artery branched into the left colic, sigmoid, and cranial rectal arteries in all the cases. These findings provide an anatomical basis for gastrointestinal veterinary care of common marmosets.

Anxious about rejection, avoidant of neglect: Infant marmosets tune their attachment based on individual caregiver's parenting style.

Children's secure attachment with their primary caregivers is crucial for physical, cognitive, and emotional maturation. Yet, the causal links between specific parenting behaviors and infant attachment patterns are not fully understood. Here we report infant attachment in New World monkeys common marmosets, characterized by shared infant care among parents and older siblings and complex vocal communications. By integrating natural variations in parenting styles and subsecond-scale microanalyses of dyadic vocal and physical interactions, we demonstrate that marmoset infants signal their needs through context-dependent call use and selective approaches toward familiar caregivers. The infant attachment behaviors are tuned to each caregiver's parenting style; infants use negative calls when carried by rejecting caregivers and selectively avoid neglectful and rejecting caregivers. Family-deprived infants fail to develop such adaptive uses of attachment behaviors. With these similarities with humans, marmosets offer a promising model for investigating the biological mechanisms of attachment security.

A calcitonin receptor-expressing subregion of the medial preoptic area is involved in alloparental tolerance in common marmosets.

Like humans, common marmoset monkeys utilize family cooperation for infant care, but the neural mechanisms underlying primate parental behaviors remain largely unknown. We investigated infant care behaviors of captive marmosets in family settings and caregiver-infant dyadic situations. Marmoset caregivers exhibited individual variations in parenting styles, comprised of sensitivity and tolerance toward infants, consistently across infants, social contexts and multiple births. Seeking the neural basis of these parenting styles, we demonstrated that the calcitonin receptor-expressing neurons in the marmoset medial preoptic area (MPOA) were transcriptionally activated during infant care, as in laboratory mice. Further, site-specific neurotoxic lesions of this MPOA subregion, termed the cMPOA, significantly reduced alloparental tolerance and total infant carrying, while sparing general health and other social or nonsocial behaviors. These results suggest that the molecularly-defined neural site cMPOA is responsible for mammalian parenting, thus provide an invaluable model to study the neural basis of parenting styles in primates.

Hippocampal Connectivity of the Presubiculum in the Common Marmoset (Callithrix jacchus).

The marmoset (a New World monkey) has recently received much attention as an experimental animal model; however, little is known about the connectivity of limbic regions, including cortical and hippocampal memory circuits, in the marmoset. Here, we investigated the neuronal connectivity of the marmoset, especially focusing on the connectivity between the hippocampal formation and the presubiculum, using retrograde and anterograde tracers (cholera toxin-B subunit and biotin dextran amine). We demonstrated the presence of a direct projection from the CA1 pyramidal cell layer to the deep layers of the presubiculum in the marmoset, which was previously identified in the rabbit brain, but not in the rat. We also found that the cells of origin of the subiculo-presubicular projections were localized in the middle part along the superficial-to-deep axis of the pyramidal cell layer of the distal subiculum in the marmoset, which was similar to that in both rats and rabbits. Our results suggest that, compared to the rat and rabbit brains, connections between the hippocampal formation and presubiculum are highly organized and characteristic in the marmoset brain.

A Farnesyltransferase Inhibitor Restores Cognitive Deficits in Tsc2+/- Mice through Inhibition of Rheb1.

Tuberous sclerosis complex (TSC) is caused by mutations in Tsc1 or Tsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability, and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in cultured Tsc2+/- neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in cultured Tsc2+/- neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1 and MAP (mitogen-activated protein (MAP)) kinase signaling, and restored dendritic spine morphology in the hippocampi of male Tsc2+/- mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in male Tsc2+/- mice in vivo Heterozygous Rheb1 knockout in male Tsc2+/- mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments in Tsc2+/- mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders.SIGNIFICANCE STATEMENT Tuberous sclerosis complex (TSC) is an autosomal-dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID has been reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found that Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used the farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1 and recovered proper synapse formation and plasticity-related Arc (activity-regulated cytoskeleton-associated protein) expression in TSC neurons. Furthermore, in vivo lonafarnib treatment restored contextual memory and Arc induction in TSC mice. Together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.

Chemogenetic inactivation reveals the inhibitory control function of the prefronto-striatal pathway in the macaque brain.

The lateral prefrontal cortex (LPFC) has a strong monosynaptic connection with the caudate nucleus (CdN) of the striatum. Previous human MRI studies have suggested that this LPFC-CdN pathway plays an important role in inhibitory control and working memory. We aimed to validate the function of this pathway at a causal level by pathway-selective manipulation of neural activity in non-human primates. To this end, we trained macaque monkeys on a delayed oculomotor response task with reward asymmetry and expressed an inhibitory type of chemogenetic receptors selectively to LPFC neurons that project to the CdN. Ligand administration reduced the inhibitory control of impulsive behavior, as well as the task-related neuronal responses observed in the local field potentials from the LPFC and CdN. These results show that we successfully suppressed pathway-selective neural activity in the macaque brain, and the resulting behavioral changes suggest that the LPFC-CdN pathway is involved in inhibitory control.

Normal tension glaucoma-like degeneration of the visual system in aged marmosets.

The common marmoset (Callithrix jacchus) is a non-human primate that provides valuable models for neuroscience and aging research due to its anatomical similarities to humans and relatively short lifespan. This study was carried out to examine whether aged marmosets develop glaucoma, as seen in humans. We found that 11% of the aged marmosets presented with glaucoma-like characteristics; this incident rate is very similar to that in humans. Magnetic resonance imaging showed a significant volume loss in the visual cortex, and histological analyses confirmed the degeneration of the lateral geniculate nuclei and visual cortex in the affected marmosets. These marmosets did not have elevated intraocular pressure, but showed an increased oxidative stress level, low cerebrospinal fluid (CSF) pressure, and low brain-derived neurotrophic factor (BDNF) and TrkB expression in the retina, optic nerve head and CSF. Our findings suggest that marmosets have potential to provide useful information for the research of eye and the visual system.

A Single Pheromone Receptor Gene Conserved across 400 My of Vertebrate Evolution.

Pheromones are crucial for eliciting social and sexual behaviors in diverse animal species. The vomeronasal receptor type-1 (V1R) genes, encoding members of a pheromone receptor family, are highly variable in number and repertoire among mammals due to extensive gene gain and loss. Here, we report a novel pheromone receptor gene belonging to the V1R family, named ancient V1R (ancV1R), which is shared among most Osteichthyes (bony vertebrates) from the basal lineage of ray-finned fishes to mammals. Phylogenetic and syntenic analyses of ancV1R using 115 vertebrate genomes revealed that it represents an orthologous gene conserved for >400 My of vertebrate evolution. Interestingly, the loss of ancV1R in some tetrapods is coincident with the degeneration of the vomeronasal organ in higher primates, cetaceans, and some reptiles including birds and crocodilians. In addition, ancV1R is expressed in most mature vomeronasal sensory neurons in contrast with canonical V1Rs, which are sparsely expressed in a manner that is consistent with the "one neuron-one receptor" rule. Our results imply that a previously undescribed V1R gene inherited from an ancient Silurian ancestor may have played an important functional role in the evolution of vertebrate vomeronasal organ.

Evolution of vomeronasal receptor 1 (V1R) genes in the common marmoset (Callithrix jacchus).

Pheromones are crucial for eliciting innate responses and inducing social and sexual behaviors in mammals. The vomeronasal receptor 1 genes, V1Rs, encode members of a pheromone receptor family that are mainly expressed in the vomeronasal organ (VNO). The V1R family shows extraordinary variety in gene number among vertebrates owing to successive gene gains and losses during evolution. Such diversity is believed to reflect a degree of dependence on the VNO. We investigated V1R evolution in primate lineages closely related to humans because these VNOs show a trend toward degeneration. We performed extensive phylogenetic analyses for V1Rs from a broad range of primate species. Although the decline of intact genes was evident in anthropoids (hominoids, Old World monkeys and New World monkeys), we found that a certain number of intact genes persist in New World monkeys. In one New World monkey species, the common marmoset (Callithrix jacchus), we examined seven putatively functional V1Rs using in situ hybridization and reverse transcription-PCR. Based on their mRNA expression patterns in the VNO and other organs, two types of V1Rs emerged: the canonical class with VNO-specific expression, and a second group having more ubiquitous expression in various organs as well as VNO. Furthermore, phylogenetic analysis revealed that the class with the more widespread expression had been retained longer in evolution than the VNO-specific type. We propose that the acquisition of a novel non-VNO-related function(s) may have led to the survival of a small but persistent number of V1Rs in anthropoid primates.

The olfactory bulb and the number of its glomeruli in the common marmoset (Callithrix jacchus).

The olfactory system has been well studied in mammals such as mice and rats. However, few studies have focused on characterizing this system in diurnal primates that rely on their sense of smell to a lesser extent due to their ecological environment. In the present study, we determined the histological organization of the olfactory bulb in the common marmoset (Callithrix jacchus). We then constructed 3-dimensional models of the glomeruli of the olfactory bulb, and estimated the number of glomeruli. Olfactory glomeruli are the functional units of olfactory processing, and have been investigated in detail using mice. There are approximately 1800 glomeruli in a mouse hemibulb, and olfactory sensory neurons expressing one selected olfactory receptor converge onto one or two glomeruli. Because mice have about 1000 olfactory receptor genes, it is proposed that the number of glomeruli in mammals is nearly double that of olfactory receptor genes. The common marmoset carries only about 400 intact olfactory receptor genes. The present study revealed that the number of glomeruli in a marmoset hemibulb was approximately 1500-1800. This result suggests that the number of glomeruli is not positively correlated with the number of intact olfactory receptor genes in mammals.

Three-dimensional reconstruction of electron micrographs reveals intrabulbar circuit differences between accessory and main olfactory bulbs.

Three-dimensional (3D) reconstruction of synaptic arrangement on a particular dendrite provides essential information regarding neuronal properties and neural microcircuits. Unconventional synapses are particularly good candidates for such steric attribution. In main and accessory olfactory bulbs (MOBs and AOBs), there are dendrodendritic reciprocal synapses (RSs) between excitatory projection neurons and inhibitory interneurons. Although the fine structure and configuration of these synapses have been investigated in MOB, their characteristics in AOB were unknown. In this study, we performed 3D AOB reconstruction using serial section transmission electron microscopy. We found numerous RSs on primary dendrites from glomeruli to mitral/tufted (MT) cell somas. These synapses formed between dendritic shafts of MT cells and large dendritic spines, or so-called gemmules, of granule (Gr) cells. This indicates that chemical signals received by a glomerulus are regulated in the primary dendrite of an MT cell before reaching its soma. In MOB, RSs are located on secondary dendrites and act as lateral and self-inhibiting following mitral cell depolarization. Our results indicate that AOB intrabulbar microcircuitry is quite different from that in the MOB.

Social interaction influences blood cortisol values and brain aromatase genes in the protandrous false clown anemonefish, Amphiprion ocellaris.

Anemonefish, Amphiprion spp., are socially controlled, protandrous sex changers with a monogamous mating system. Under certain conditions, sexually immature anemonefish with ambisexual gonads differentiate directly into males or females. Formation and maintenance of social rank in a group are considered key requirements for the induction of sex change or differentiation. Generally, each animal living in a social group experiences a different level of social stress in accordance with its social rank, and we hypothesize that the stress situation of individual anemonefish influences its sex determination. Groups of three sexually immature anemonefish were placed into each of five experimental tanks and kept for 10 days to allow for social rank formation and behavioral observation. The fish were then euthanized, and blood and brain samples were collected from each fish. The social rank of each individual was distinguishable from day 1 of the experiment. Aggressive behaviors were most frequent and blood Cortisol values were higher in dominant individuals. The transcription of mRNA for stress-related genes, i.e., those encoding for glucocorticoid and arginine vasotocin receptors, was higher in the brains of dominant individuals than in other social ranks. Furthermore, we detected higher transcription levels of gonad and brain aromatase genes, which encode the enzyme that converts androgens into estrogens, in the brains of dominant individuals. These results suggest that social rank reflects the blood Cortisol value, which in turn leads to sex differentiation by manipulating transcription of genes, including aromatase genes, in the brain.

Experimental techniques for neuroscience research using common marmosets.

The common marmoset (Callithrix jacchus) is a species of New World monkeys. Because of its ease of maintenance and breeding in laboratories, use of the marmoset is growing rapidly in biomedical research. In neuroscience, the marmosets are attracting more attention, since they have the developed cerebral cortex which plays a key role in higher brain functions. In this review on neuroscience research using the marmoset, experimental techniques developed in our laboratory are summarized. We introduce artificial rearing of neonates, stereotaxic surgery, neuroanatomy including virtual microscopy based on the Internet technology, behavioral study using a large number of marmosets, and primary neuron culture study.

Sexually dimorphic effects of estrogen on spines in cultures of accessory olfactory bulb.

A sex difference has been reported in the responsiveness of the vomeronasal (VN) system to pheromones. In the present study, to clarify a direct and acute influence of 17ß-estradiol (E2) on the accessory olfactory bulb (AOB) neurons, we investigated the effect of E2 on dendritic spines in cultured AOB cells derived from male and female neonatal rats. After 17-18 days in vitro (DIV), cultured AOB cells were transfected with GFP expression vectors. At 21-23 DIV, cells were treated with E2, and time-lapse images of transfected AOB neurons identified as granule cells were taken under a confocal laser scanning microscope for 3h. The dendritic spine head area of granule cells was quantitatively evaluated, and spine heads were classified into larger (≥ 1 µm²) and smaller (<1 µm²) ones before E2-treatment (0 h). In cultured cells derived from both sexes, the larger spines were not significantly changed at 1, 2 and 3 h after E2-treatment. In contrast, E2-treatment significantly enlarged the head area of the smaller spines of granule cells derived from the female, whereas E2 did not cause any significant effects on those from the male. Our results provide evidence for the sexually-dimorphic effect of E2 on spine development in AOB granule cells.

Vomeronasal neurons promote synaptic formation on dendritic spines but not dendritic shafts in primary culture of accessory olfactory bulb neurons.

To investigate the morphological changes of accessory olfactory bulb (AOB) neurons arising from pheromonal signals, a coculture system of AOB neurons and vomeronasal (VN) neurons had been established. Our previous study indicates that under coculture condition, the density of dendritic spines of an AOB neuron is less and the individual spine-head volume is larger than those under monoculture condition. In this study, to determine whether these differences in the dendrites of AOB neurons reflect the differences in synapse formation and synaptic properties, we observed these cultured cells by electron microscopy. Various synapses were observed under each culture condition. Synapses were classified on the basis of their postsynaptic structure and the size of postsynaptic density (PSD) was measured. Under the coculture condition with VN neurons, synapses on dendritic spines, which formed between AOB neurons, were observed frequently. In contrast, many synapses were formed on dendritic shafts under monoculture condition. The PSD of asymmetrical synapses on the spines under coculture condition was larger than that under monoculture condition. Moreover, some dendrodendritic reciprocal synapses were found only in coculture. We confirmed synapse formation between VN axons and AOB dendrites by immunohistochemical electron microscopy; thus, the characteristics of synapses between AOB neurons are considered to be modified by the synaptic contacts with VN axons.

Cyclic phosphatidic acid is produced by autotaxin in blood.

Cyclic phosphatidic acid (cPA), an analog of lysophosphatidic acid (LPA), was previously identified in human serum. Although cPA possesses distinct physiological activities not elicited by LPA, its biochemical origins have scarcely been studied. In the present study, we assayed cPA formation from lysophosphatidylcholine in fetal bovine serum and found significant activity of transphosphatidylation that generated cPA. The cPA-producing enzyme was purified from fetal bovine serum using five chromatographic steps yielding a 100-kDa protein with cPA biosynthetic activity. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of its tryptic peptides revealed that the enzyme shared identical fragments with human autotaxin, a serum lysophospholipase D that produces LPA. Western blot analysis demonstrated that the 100-kDa protein was specifically recognized by an anti-human autotaxin antibody. Moreover, recombinant rat autotaxin was found to generate cPA in addition to LPA. No significant cPA- or LPA-producing activity was detected in autotaxin-depleted serum from bovine or human prepared by immunoprecipitation with an anti-autotaxin monoclonal antibody. These results indicate that the generation of cPA and LPA in serum is mainly attributed to autotaxin.

Modification of synapse formation of accessory olfactory bulb neurons by coculture with vomeronasal neurons.

Previously, a coculture system of accessory olfactory bulb (AOB) neurons and vomeronasal (VN) neurons was established for studying the functional roles of AOB neurons in pheromonal signal processing. In this study, the effect of VN neurons on the development of AOB neurons was examined in a coculture system. Spine density was quantitatively measured for various culture periods of 21, 28, 36, and 42 days in vitro. The densities of dendritic spines were lower in the coculture than in single culture for all periods in vitro. Synapse formation on spines was analyzed immunocytochemically using an anti-synaptophysin antibody. The ratio of the density of synaptophysin-immunopositive spine/total spine density was larger in the coculture than in the single culture. The volume of spine head was larger in the coculture than in single culture. These changes were not observed in the coculture in which there was no physical contact between AOB neurons and VN neurons. These observations suggest that synapse formation on the spines of AOB neurons is modified by physical contact with VN neurons.

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons.

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.