An eye tracking system for monitoring face scanning patterns reveals the enhancing effect of oxytocin on eye contact in common marmosets.

Eye tracking systems are used to investigate eyes position and gaze patterns presumed as eye contact in humans. Eye contact is a useful biomarker of social communication and known to be deficient in patients with autism spectrum disorders (ASDs). Interestingly, the same eye tracking systems have been used to directly compare face scanning patterns in some non-human primates to those in human. Thus, eye tracking is expected to be a useful translational technique for investigating not only social attention and visual interest, but also the effects of psychiatric drugs, such as oxytocin, a neuropeptide that regulates social behavior. In this study, we report on a newly established method for eye tracking in common marmosets as unique New World primates that, like humans, use eye contact as a mean of communication. Our investigation was aimed at characterizing these primates face scanning patterns and evaluating the effects of oxytocin on their eye contact behavior. We found that normal common marmosets spend more time viewing the eyes region in common marmoset's picture than the mouth region or a scrambled picture. In oxytocin experiment, the change in eyes/face ratio was significantly greater in the oxytocin group than in the vehicle group. Moreover, oxytocin-induced increase in the change in eyes/face ratio was completely blocked by the oxytocin receptor antagonist L-368,899. These results indicate that eye tracking in common marmosets may be useful for evaluating drug candidates targeting psychiatric conditions, especially ASDs.

Effects of the 5-HT1A receptor agonists buspirone and 8-OH-DPAT on pupil size in common marmosets.

As pupil size is affected by psychotropic drugs in all mammals, it has been used as a well-established clinical indicator for the preclinical and clinical development of novel drugs. It has been reported that activation of the serotonin (5-HT)1A receptor differently affects pupil response in rodents (mydriasis) and humans (miosis). Thus, it is important to establish a quantitative system for measuring pupil size using other species, such as nonhuman primates. Common marmosets have recently attracted a great deal of attention as suitable experimental animals in the psychoneurological field because of handling ease compared with other nonhuman primates and the requirement for small amounts of test drugs. In this study, we constructed a system for measuring changes in pupil size using an infrared eye-tracking camera and evaluated the effects on pupil size of the 5-HT1A receptor agonists buspirone, 8-OH-DPAT and buspirone active metabolite 1-(2-pyrimidinyl) piperazine. Our results show that both buspirone and 8-OH-DPAT significantly decrease pupil size in a dose-dependent manner. The 5-HT1A receptor antagonist WAY 100635 completely blocked both buspirone and 8-OH-DPAT-induced miosis, whereas 1-(2-pyrimidinyl) piperazine had no effect on pupil size. These results suggest that measurement of pupil size may be a useful biomarker for predicting the pharmacodynamics of new 5-HT1A receptor agonists.

The atypical antipsychotic blonanserin reverses (+)-PD-128907- and ketamine-induced deficit in executive function in common marmosets.

Antagonism of the dopamine D3 receptor is considered a promising strategy for the treatment of cognitive impairment associated with schizophrenia. We have previously reported that the atypical antipsychotic blonanserin, a dopamine D2/D3 and serotonin 5-HT2A receptor antagonist, highly occupies dopamine D3 receptors at its antipsychotic dose range in rats. In the present study, we evaluated the effects of blonanserin on executive function in common marmosets using the object retrieval with detour (ORD) task. The dopamine D3 receptor-preferring agonist (+)-PD-128907 at 1mg/kg decreased success rate in the difficult trial, but not in the easy trial. Since the difference between the two trials is only cognitive demand, our findings indicate that excess activation of dopamine D3 receptors impairs executive function in common marmosets. Blonanserin at 0.1mg/kg reversed the decrease in success rate induced by (+)-PD-128907 in the difficult trial. This finding indicates that blonanserin has beneficial effect on executive function deficit induced by activation of the dopamine D3 receptor in common marmosets. Next, and based on the glutamatergic hypothesis of schizophrenia, the common marmosets were treated with the N-methyl-d-aspartate (NMDA) receptor antagonist ketamine. Ketamine at sub-anesthetic doses decreased success rate in the difficult trial, but not in the easy trial. Blonanserin at 0.1mg/kg reversed the decrease in success rate induced by ketamine in the difficult trial. The findings of this study suggest that blonanserin might have beneficial effect on executive dysfunction in patients with schizophrenia.

The dopamine D1 receptor agonist SKF-82958 effectively increases eye blinking count in common marmosets.

Eye blinking is a spontaneous behavior observed in all mammals, and has been used as a well-established clinical indicator for dopamine production in neuropsychiatric disorders, including Parkinson's disease and Tourette syndrome [1,2]. Pharmacological studies in humans and non-human primates have shown that dopamine agonists/antagonists increase/decrease eye blinking rate. Common marmosets (Callithrix jacchus) have recently attracted a great deal of attention as suitable experimental animals in the psychoneurological field due to their more developed prefrontal cortex than rodents, easy handling compare to other non-human primates, and requirement for small amounts of test drugs. In this study, we evaluated the effects of dopamine D1-4 receptors agonists on eye blinking in common marmosets. Our results show that the dopamine D1 receptor agonist SKF-82958 and the non-selective dopamine receptor agonist apomorphine significantly increased common marmosets eye blinking count, whereas the dopamine D2 agonist (+)-PHNO and the dopamine D3 receptor agonist (+)-PD-128907 produced somnolence in common marmosets resulting in a decrease in eye blinking count. The dopamine D4 receptor agonists PD-168077 and A-41297 had no effect on common marmosets' eye blinking count. Finally, the dopamine D1 receptor antagonist SCH 39166 completely blocked apomorphine-induced increase in eye blinking count. These results indicate that eye blinking in common marmosets may be a useful tool for in vivo screening of novel dopamine D1 receptor agonists as antipsychotics.

Behavioral and neurophysiological effects of Ro 10-5824, a dopamine D4 receptor partial agonist, in common marmosets.

RATIONALE: Growing evidence suggests that dopamine D4 receptors (D4Rs) are involved in controlling executive functions. We have previously demonstrated that Ro 10-5824, a D4R partial agonist, improves the performance of common marmosets in the object retrieval detour (ORD) task. However, the neural mechanisms underlying this improvement are unknown. OBJECTIVES: We investigated the behavioral and neurophysiological effects of Ro 10-5824 in common marmosets. METHODS: The effects of Ro 10-5824 on cognitive function were evaluated using the ORD task. The neurophysiological effects of Ro 10-5824 were investigated by quantitative electroencephalography, especially on baseline gamma band activity in the frontal cortex. The effects of Ro 10-5824 on spontaneous locomotion were also assessed. RESULTS: Systemic administration of Ro 10-5824 at 3 mg/kg significantly increased the success rate in the ORD task. At doses of 1 and 3 mg/kg, Ro 10-5824 increased baseline gamma band activity in the frontal cortex. Ro 10-5824 had no effect on spontaneous locomotion. CONCLUSIONS: Activation of D4R by Ro 10-5824 improves the success rate in the ORD task and increases baseline gamma band activity in the frontal cortex without affecting locomotion in common marmosets. These findings highlight the role of D4R in gamma oscillations of non-human primates. As gamma oscillations are thought to be involved in attention and behavioral inhibition, our results suggest D4R agonists may improve these cognitive functions by modulating baseline gamma band activity in the frontal cortex.

Phosphorylation of myosin II regulatory light chain by ZIP kinase is responsible for cleavage furrow ingression during cell division in mammalian cultured cells.

Zipper-interacting protein kinase (ZIPK) is known to regulate several functions such as apoptosis, smooth muscle contraction, and cell migration. While exogenously expressed GFP-ZIPK localizes to the cleavage furrow, role of ZIPK in cytokinesis is obscure. Here, we show that ZIPK is a major MRLC kinase during mitosis. Moreover, ZIPK siRNA-mediated knockdown causes delay of cytokinesis. The delay in cytokinesis of ZIPK-knockdown cells was rescued by the exogenous diphosphorylation-mimicking MRLC mutant. Taken together, these findings suggest that ZIPK plays a role in the progression and completion of cytokinesis through MRLC phosphorylation.

Effects of lurasidone on ketamine-induced joint visual attention dysfunction as a possible disease model of autism spectrum disorders in common marmosets.

Infants with autism have difficulties performing joint visual attention (JVA), defined as following another person's pointing gesture and gaze. Some non-human primates (NHPs) can also perform JVA. Most preclinical research on autism spectrum disorders (ASD) has used rodents as animal models of this social interaction disorder. However, models using rodents fail to capture the complexity of social interactions that are disrupted in ASD. Therefore, JVA impairment in NHPs might be a more useful model of ASD. The aim of this study was to develop an appropriate and convenient ASD model with common marmosets. We first tested whether marmosets were capable of performing JVA. Subsequently, we administered ketamine, an N-methyl-d-aspartate (NMDA) receptor antagonist, to induce JVA impairment and investigated the effects of lurasidone, a newer antipsychotic agent, on the JVA impairments. An apparatus was constructed using 4 white boxes, which were attached to the corners of a frame. All boxes had a hinged door, and marmosets could easily obtain a reward by pushing the door. An experimenter pointed and gazed at the boxes to inform the marmosets which box contained the reward. Their behavior was scored according to the number of incorrect choices. The JVA score was significantly higher in the cued vs. uncued tasks. Ketamine significantly decreased the JVA score, but lurasidone significantly reversed this effect. These findings suggest that this experimental system could be a useful animal model of neuropsychiatric disorders characterized by NMDA-receptor signaling, including ASD, and that lurasidone might be effective for some aspects of ASD.

Generation of colonic IgA-secreting cells in the caecal patch.

Gut-associated lymphoid tissues are responsible for the generation of IgA-secreting cells. However, the function of the caecal patch, a lymphoid tissue in the appendix, remains unknown. Here we analyse the role of the caecal patch using germ-free mice colonized with intestinal bacteria after appendectomy. Appendectomized mice show delayed accumulation of IgA(+) cells in the large intestine, but not the small intestine, after colonization. Decreased colonic IgA(+) cells correlate with altered faecal microbiota composition. Experiments using photoconvertible Kaede-expressing mice or adoptive transfer show that the caecal patch IgA(+) cells migrate to the large and small intestines, whereas Peyer's patch cells are preferentially recruited to the small intestine. IgA(+) cells in the caecal patch express higher levels of CCR10. Dendritic cells in the caecal patch, but not Peyer's patches, induce CCR10 on cocultured B cells. Thus, the caecal patch is a major site for generation of IgA-secreting cells that migrate to the large intestine.

CognitionMaster: an object-based image analysis framework.

BACKGROUND: Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired. RESULTS: In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose "analysis pipelines" that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept. CONCLUSIONS: We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.

Systemic circulation and bone recruitment of osteoclast precursors tracked by using fluorescent imaging techniques.

Osteoclasts are bone-resorbing polykaryons differentiated from monocyte/macrophage-lineage hematopoietic precursors. It remains unclear whether osteoclasts originate from circulating blood monocytes or from bone tissue-resident precursors. To address this question, we combined two different experimental procedures: 1) shared blood circulation "parabiosis" with fluorescently labeled osteoclast precursors, and 2) photoconversion-based cell tracking with a Kikume Green-Red protein (KikGR). In parabiosis, CX(3)CR1-EGFP knock-in mice in which osteoclast precursors were labeled with EGFP were surgically connected with wild-type mice to establish a shared circulation. Mature EGFP(+) osteoclasts were found in the bones of the wild-type mice, indicating the mobilization of EGFP(+) osteoclast precursors into bones from systemic circulation. Receptor activator for NF-κB ligand stimulation increased the number of EGFP(+) osteoclasts in wild-type mice, suggesting that this mobilization depends on the bone resorption state. Additionally, KikGR(+) monocytes (including osteoclast precursors) in the spleen were exposed to violet light, and 2 d later we detected photoconverted "red" KikGR(+) osteoclasts along the bone surfaces. These results indicate that circulating monocytes from the spleen entered the bone spaces and differentiated into mature osteoclasts during a certain period. The current study used fluorescence-based methods clearly to demonstrate that osteoclasts can be generated from circulating monocytes once they home to bone tissues.

[Bone and calcium update; bone research update. Trafficking of osteoclast precursors visualized by intravital imaging technology].

Osteoclasts are 'bone-resorbing' giant polykaryons that differentiate from mononuclear macrophage/monocyte-lineage hematopoietic precursors. However, it has remained unclear how osteoclast precursors migrate into the bone surface and what controls their migratory behaviors in vivo . To investigate these questions, we utilized an advanced imaging system for visualizing live bone tissues with intravital multiphoton microscopy that we have recently established. By means of the system we have recently succeeded in visualization of osteoclast precursors in live bones and revealed that sphingosine-1-phosphate (S1P) , a lipid mediator, dynamically regulates migration and localization of osteoclasts and their precursors in vivo . Here we show the latest data and the detailed methodology of intravital imaging of bone tissues, and also discuss its further application.

Monoclonal antibody specific for Dhx9/NDHII/RHA.

Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression.