Efficient embryo transfer in the common marmoset monkey (Callithrix jacchus) with a reduced transfer volume: a non-surgical approach with cryopreserved late-stage embryos.

Among primates, the common marmoset is suitable for primate embryology research. Its small body size, however, has delayed the technical development of efficient embryo transfer. Furthermore, three factors have been determined to adversely affect the performance of marmoset embryo transfer: nonsurgical approaches, the use of cryopreserved embryos, and the use of late-stage embryos. Here we performed embryo transfer under conditions that included the above three factors and using either a small (1 µl or less) or a large volume (2-3 µl) of medium. The pregnancy and birth rates were 50% (5/10) and 27% (3/11), respectively, when using the large volume, and 80% (8/10) and 75% (9/12), respectively, when using the small volume. The latter scores exceed those of previous reports using comparable conditions. Thus, it appears that these three previously considered factors could be overcome, and we propose that reducing the transfer volume to 1 µl or less is essential for successful marmoset embryo transfer.

Adult-like action potential properties and abundant GABAergic synaptic responses in amygdala neurons from newborn marmosets.

The amygdala plays an important role in the processing of emotional events. This information processing is altered by development, but little is known about the development of electrophysiological properties of neurons in the amygdala. We studied the postnatal development of electrophysiological properties of neurons in the basolateral amygdala (BLA) of the common marmoset (Callithrix jacchus). Whole-cell patch-clamp recordings were obtained from BLA pyramidal neurons in brain slices prepared from developing and adult marmosets, and electrophysiological properties known to change during development in rats were analysed. Two passive electrical properties of the neuronal membrane - the input resistance (R(in)) and the membrane time constant () - significantly decreased with postnatal development. In contrast, the action potential only showed a slight decrease in duration during the first month of life, whereas the amplitude did not change after birth. Passive electrical properties and action potentials in neurons of 4-week-old marmosets were similar to those in neurons of 4-year-old marmosets. The development of the action potential duration was not correlated with the development of R(in) or , whereas the development of R(in) and was correlated with each other. Abundant spontaneous and noradrenaline-induced GABAergic currents were present immediately after birth and did not change during postnatal development. These results suggest that newborn infant marmoset BLA pyramidal neurons possess relatively mature action potentials and receive vigorous GABAergic synaptic inputs, and that they acquire adult-like electrophysiological properties by the fourth week of life.

Pump-Free Microfluidic Hemofiltration Device.

Hemofiltration removes water and small molecules from the blood via nanoporous filtering membranes. This paper discusses a pump-free hemofiltration device driven by the pressure difference between the artery and the vein. In the design of the filtering device, oncotic pressure needs to be taken into consideration. Transmembrane pressure (TMP) determines the amount and direction of hemofiltration, which is calculated by subtracting the oncotic pressure from the blood pressure. Blood pressure decreases as the channels progress from the inlet to the outlet, while oncotic pressure increases slightly since no protein is removed from the blood to the filtrate in hemofiltration. When TMP is negative, the filtrate returns to the blood, i.e., backfiltration takes place. A small region of the device with negative TMP would thus result in a small amount of or even zero filtrates. First, we investigated this phenomenon using in vitro experiments. We then designed a hemofiltration system taking backfiltration into consideration. We divided the device into two parts. In the first part, the device has channels for the blood and filtrate with a nanoporous membrane. In the second part, the device does not have channels for filtration. This design ensures TMP is always positive in the first part and prevents backfiltration. The concept was verified using in vitro experiments and ex vivo experiments in beagle dogs. Given the simplicity of the device without pumps or electrical components, the proposed pump-free hemofiltration device may prove useful for either implantable or wearable hemofiltration.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms.

Age-associated neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and the polyglutamine (polyQ) diseases, are becoming prevalent as a consequence of elongation of the human lifespan. Although various rodent models have been developed to study and overcome these diseases, they have limitations in their translational research utility owing to differences from humans in brain structure and function and in drug metabolism. Here, we generated a transgenic marmoset model of the polyQ diseases, showing progressive neurological symptoms including motor impairment. Seven transgenic marmosets were produced by lentiviral introduction of the human ataxin 3 gene with 120 CAG repeats encoding an expanded polyQ stretch. Although all offspring showed no neurological symptoms at birth, three marmosets with higher transgene expression developed neurological symptoms of varying degrees at 3-4 months after birth, followed by gradual decreases in body weight gain, spontaneous activity, and grip strength, indicating time-dependent disease progression. Pathological examinations revealed neurodegeneration and intranuclear polyQ protein inclusions accompanied by gliosis, which recapitulate the neuropathological features of polyQ disease patients. Consistent with neuronal loss in the cerebellum, brain MRI analyses in one living symptomatic marmoset detected enlargement of the fourth ventricle, which suggests cerebellar atrophy. Notably, successful germline transgene transmission was confirmed in the second-generation offspring derived from the symptomatic transgenic marmoset gamete. Because the accumulation of abnormal proteins is a shared pathomechanism among various neurodegenerative diseases, we suggest that this new marmoset model will contribute toward elucidating the pathomechanisms of and developing clinically applicable therapies for neurodegenerative diseases.

Relationship between corneal aberrations and contrast sensitivity after hyperopic laser in situ keratomileusis.

PURPOSE: To evaluate the relationship between corneal aberrations and contrast sensitivity (CS) after hyperopic laser in situ keratomileusis (H-LASIK). METHODS: In 13 patients (13 eyes) who underwent H-LASIK, we measured CS and corneal topography preoperatively and at 1 year postoperatively. Photopic and scotopic CS values were measured at 3, 6, and 12 cycles/degree (cpd) using an MCT-8000 contrast tester. Corneal aberrations were determined from the data on corneal topography using CTView. The corneal high-order aberrations were defined as the sum of the third- and fourth-order aberrations in the 4-mm zone and the sum of the third- to sixth-order aberrations in the 6-mm-zone. RESULTS: Under scotopic conditions at 12 cpd, the changes in CS significantly correlated with changes in the corneal aberrations. Scotopic CS was significantly deteriorated by glare, but photopic CS was not significantly changed. H-LASIK induced a significant increase in corneal aberrations that positively correlated with the amount of correction, regardless of the improvement in logMAR corrected visual acuity. LogMAR corrected visual acuity did not significantly correlate with corneal aberrations. Furthermore, decentration significantly correlated with the changes in the 6-mm zone corneal aberrations. CONCLUSIONS: In eyes after H-LASIK, the changes in scotopic CS significantly correlated with those in the corneal aberrations, which might have resulted from decentration or ablation profiles in H-LASIK and a relatively small optical zone. Further studies will be needed to validate this relationship.

More effective induction of anesthesia using midazolam-butorphanol-ketamine-sevoflurane compared with ketamine-sevoflurane in the common marmoset monkey (Callithrix jacchus).

The common marmoset has been increasingly used for research in the biomedical field; however, there is little information available regarding effective methods of anesthesia in this species. This study retrospectively analyzed 2 regimens of anesthesia induction: intramuscular injection of ketamine followed by inhalation of 5% sevoflurane, and intramuscular injection of midazolam, butorphanol and ketamine followed by inhalation of 5% sevoflurane. Anesthetic depth did not reach the surgical anesthesia stage in 7 out of 99 animals receiving the former regimen, whereas there were only 2 such animals out of 273 receiving the latter regimen. The latter regimen, when followed by maintenance anesthesia with 3% sevoflurane inhalation, was successfully used in various nociceptive procedures. These results indicate that the injection of a combination of midazolam, butorphanol and ketamine followed by inhalation of a high concentration of sevoflurane is effective for anesthesia induction in marmosets.

Cryopreservation of ovaries from neonatal marmoset monkeys.

The ovary of neonatal nonhuman primates contains the highest number of immature oocytes, but its cryopreservation has not yet been sufficiently investigated in all life stages. In the current study, we investigated cryodamage after vitrification/warming of neonatal ovaries from a nonhuman primate, the common marmoset (Callithrix jacchus). A Cryotop was used for cryopreservation of whole ovaries. The morphology of the vitrified/warmed ovaries was found to be equivalent to that of fresh ovaries. No significant difference in the number of oocytes retaining normal morphology per unit area in histological sections was found between the two groups. In an analysis of dispersed cells from the ovaries, however, the cell viability of the vitrified/warmed group tended to be decreased. The results of a comet assay showed no significant differences in DNA damage. These results show that cryopreservation of neonatal marmoset ovaries using vitrification may be useful as a storage system for whole ovaries.

Identification of a virulence determinant that is conserved in the Jawetz and Heyl biotypes of [Pasteurella] pneumotropica.

[Pasteurella] pneumotropica is a ubiquitous bacterium frequently isolated from laboratory rodents. Although this bacterium causes various diseases in immunosuppressed animals, little is known about major virulence factors and their roles in pathogenicity. To identify virulence factors, we sequenced the genome of [P.] pneumotropica biotype Heyl strain ATCC 12555, and compared the resulting non-contiguous draft genome sequence with the genome of biotype Jawetz strain ATCC 35149. Among a large number of genes encoding virulence-associated factors in both strains, four genes encoding for YadA-like proteins, which are known virulence factors that function in host cell adherence and invasion in many pathogens. In this study, we assessed YadA distribution and biological activity as an example of one of virulence-associated factor shared, with biotype Jawetz and Heyl. More than half of mouse isolates were found to have at least one of these genes; whereas, the majority of rat isolates did not. Autoagglutination activity, and ability to bind to mouse collagen type IV and mouse fibroblast cells, was significantly higher in YadA-positive than YadA-negative strains. To conclude, we identified a large number of candidate genes predicted to influence [P.] pneumotropica pathogenesis.

Posterior corneal surface changes after hyperopic laser in situ keratomileusis.

PURPOSE: To evaluate posterior corneal surface topographic changes after hyperopic laser in situ keratomileusis (H-LASIK) using Orbscan I (Orbtek, Inc.). SETTING: Department of Ophthalmology, Nara Medical University, Nara, Japan. METHODS: In 25 eyes of 15 patients who had H-LASIK, the posterior corneal surface was measured with slit-scanning corneal topography (Orbscan I) preoperatively and 1 year postoperatively. The center as a fit zone and calculated posterior corneal surface changes were taken at 4 points: nasal, temporal, superior, and inferior sides in the 5.0 mm diameter. The posterior corneal topographic changes were analyzed using an analysis of variance. The postoperative:preoperative magnification ratio of the posterior corneal surface was calculated in a theoretical eye model. RESULTS: When a "+" reading was defined as the forward displacement and "-" was defined as the backward displacement, the mean posterior corneal topographic changes were -2.8 microm +/- 27.9 (SD) at the nasal side, -4.5 +/- 27.8 microm at the temporal side, -3.9 +/- 20.1 microm at the superior side, and -2.3 +/- 20.1 microm at the inferior side. The posterior corneal surface between any 2 examined points showed no significant difference after H-LASIK. In addition, the hypothetical change in the posterior cornea was -8.3 microm after +3.0 diopter H-LASIK, which was approximately closer to the study results. In each side, the amount of the attempted correction was significantly correlated with the posterior corneal topographic change. CONCLUSIONS: Clinical measurement of the posterior corneal displacement after H-LASIK with Orbscan revealed a backward shift. This change corresponded to the hypothetical artifactual changes with Orbscan; that is, changes in the magnification ratio.

Association of food location with biological cues in the macaque monkey.

Many animal species including humans are endowed with the ability to use biological cues and can extract information by observing other individuals. This study explored whether the macaque monkey could use biological cue to find a hidden target. When the experimenter hid food in one hand and crossed and uncrossed hands quickly, the monkey had no difficulty in finding the food and correctly reached for the baited hand. However, when the food was hidden in one of two cups and the cups were shuffled, the monkey could correctly select the baited cup only at an equal level of luck. These results indicate that the macaque monkey could associate the location of food with a biological cue better than a non-biological cue and keep it in memory when the target was unseen.

Tool-use learning induces BDNF expression in a selective portion of monkey anterior parietal cortex.

Learning but not execution of tool-use induced expression of brain-derived neurotrophic factor (BDNF). The expression was highest in the anterior bank of the intraparietal sulcus, especially in the region posteriorly adjacent to the somatosensory shoulder and forearm region in area 3b, suggesting that BDNF plays a role in altering the body image of the hand to include the repeatedly used tool as its extension.

Tool-use learning selectively induces expression of brain-derived neurotrophic factor, its receptor trkB, and neurotrophin 3 in the intraparietal multisensorycortex of monkeys.

When humans repeatedly use a tool, our body image alters until the tool finally becomes a part or an extension of the body. This alteration of body image perhaps results from re-integration of somatosensory and visual signals. We trained Japanese monkeys to use a rake-shaped tool to retrieve a distant food pellet, then used a novel tissue-sampling method to suction brain tissue from the anterior bank of their intraparietal sulcus, where somatosensory and visual signals converge. Examination of the messenger RNA expression levels of neurotrophins and their receptors using real-time quantitative polymerase chain reaction revealed learning-selective induction in the expression of brain-derived neurotrophic factor, its receptor trkB, and NT-3 during, but not after, the learning. These results suggest that these factors are involved in the reorganization of the somatosensory and visual signals in the anterior bank of the intraparietal sulcus when monkeys are learning the use of the tool.

Evaluation of the corneal endothelium after hyperopic laser in situ keratomileusis.

PURPOSE: To evaluate corneal endothelial changes after hyperopic laser in situ keratomileusis (LASIK) considering overestimation and underestimation of the cell count measurement. SETTING: Department of Ophthalmology, Nara Medical University, Nara, Japan. METHODS: The data were from the clinical trial of the Nidek EC-5000 excimer laser for hyperopic LASIK. The mean correction was 3.59 diopters (D) +/- 1.54 (SD) (range 2.0 to 6.0 D). Using noncontact specular microscopy, the corneal endothelial changes in 25 eyes of 15 patients who had hyperopic LASIK were measured. Follow-up ranged from 6 months (n = 25) to 1 year (n = 21). The overestimation and underestimation of the corneal endothelial cell count that would occur after +5.0 D hyperopic LASIK was hypothetically calculated. RESULTS: The measured endothelial cell count per 1.0 mm(2) did not significantly decrease up to 1 year after hyperopic LASIK (preoperatively, 2508 +/- 395; at 1 year, 2814 +/- 349). The hypothetical calculation revealed that a +5.0 D hyperopic correction corresponded to a 0.1% underestimation of the corneal endothelial cell count. CONCLUSIONS: Underestimation of the corneal endothelial cell count after hyperopic LASIK was negligible. Hyperopic LASIK with the Nidek EC-5000 excimer laser did not significantly decrease corneal endothelial cells up to 1 year after surgery.

Natural imitation induced by joint attention in Japanese monkeys.

To examine whether joint attention enables Japanese monkeys to imitate human actions, we presented an oral action and manual actions directed towards targets to three monkeys who had joint attention through communicative eye-gaze and pointing gestures and to one monkey who had incomplete joint attention and who had acquired imperative pointing but not the use of eye-gaze gestures. Two of the monkeys who were already capable of joint attention were also able to imitate naturally, while the monkey who did not previously show joint attention was not able to imitate until acquiring joint attention capacity. We suggest that joint attention induces natural imitation during interaction between different species--in this case, between monkeys and humans--while individuals not showing joint attention but only attention to a target or movement are only able to follow motion. The monkey may be endowed by nature with motion-following capacity. We speculate that motion-following capacity is developed and controlled through joint attention, and is connected with natural imitation.

Effect of the size of zona pellucida opening on hatching in the common marmoset monkey (Callithrix jacchus) embryo.

The use of the common marmoset monkey in biomedical research has increased recently, and further attention has been devoted to this model after the successful production of transgenic marmosets. To extend genetic engineering approaches to widespread biomedical research fields, efficient prolonged in vitro culturing of embryo development is necessary. We aimed to evaluate the effects of the size of the zona pellucida opening on promoting the hatching process in the marmoset embryo. Piezo-microdrilling of a 6-µm opening in eight embryos resulted in four partially hatched embryos and one hatched embryo after 5 days of culture. Piezo-microdrilling a 20-µm opening in 11 embryos resulted in nine partial hatchings and no hatched embryos. Piezo-scraping an 80-µm opening in six embryos resulted in no partially hatched embryos and five hatched embryos. These results suggest that an 80-µm opening, rather than 6-µm or 20-µm openings, is suitable to complete the hatching process in the marmoset embryo.

Ultrasound-guided non-surgical embryo collection in the common marmoset.

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.

Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAV1 and rAAV9.

Profiles of recombinant adeno-associated virus (rAAV)-mediated transduction show interspecies differences for each AAV serotype. Robust long-term transgene expression is generally observed in rodents, whereas insufficient transduction is seen in animals with more advanced immune systems. Non-human primates, including the common marmoset, could provide appropriate models for neuromuscular diseases because of their higher brain functions and physiological resemblance to humans. Strategies to induce pathologies in the neuromuscular tissues of non-human primates by rAAV-mediated transduction are promising; however, transgene expression patterns with rAAV transduction have not been elucidated in marmosets. In this study, transduction of adult marmoset skeletal muscle with rAAV9 led to robust and persistent enhanced green fluorescent protein (EGFP) expression that was independent of the muscle fiber type, although lymphocyte infiltration was recognized. Systemic rAAV injection into pregnant marmosets led to transplacental fetal transduction. Surprisingly, the intraperitoneal injection of rAAV1 and rAAV9 into the neonatal marmoset resulted in systemic transduction and persistent transgene expression without lymphocyte infiltration. Skeletal and cardiac muscle were effectively transduced with rAAV1 and rAAV9, respectively. Interestingly, rAAV9 transduction led to intense EGFP signaling in the axons of the corpus callosum. These transduction protocols with rAAV will be useful for investigating gene functions in the neuromuscular tissues and developing gene therapy strategies.Molecular Therapy-Nucleic Acids (2013) 2, e95; doi:10.1038/mtna.2013.21; published online 28 May 2013.

A cross-species socio-emotional behaviour development revealed by a multivariate analysis.

Recent progress in affective neuroscience and social neurobiology has been propelled by neuro-imaging technology and epigenetic approach in neurobiology of animal behaviour. However, quantitative measurements of socio-emotional development remains lacking, though sensory-motor development has been extensively studied in terms of digitised imaging analysis. Here, we developed a method for socio-emotional behaviour measurement that is based on the video recordings under well-defined social context using animal models with variously social sensory interaction during development. The behaviour features digitized from the video recordings were visualised in a multivariate statistic space using principal component analysis. The clustering of the behaviour parameters suggested the existence of species- and stage-specific as well as cross-species behaviour modules. These modules were used to characterise the behaviour of children with or without autism spectrum disorders (ASDs). We found that socio-emotional behaviour is highly dependent on social context and the cross-species behaviour modules may predict neurobiological basis of ASDs.

Reading marmoset behavior 'semantics' under particular social context by multi-parameters correlation analysis.

Social interactions are a fundamental aspect of human and animal behavior. Although neuroimaging and other non-invasive methods have progressed recently, the neurobiology of social behavior requires the use of animal models. Here, we introduced a multi-behavior parameter integration method and applied it to female-male interaction of adult common marmosets (Callithrix jacchus). Based on the correlated parameters and meeting context, we found that the behavioral endpoints clustered in four distinct categories, which could be interpreted as active, freeze, alert, and affinity emotional states. The relevance of this interpretation was supported as the female behavior category change positively correlated with serum cortisol and progesterone levels after social interaction. Thus, our multi-behavior parameter integration method may be useful to evaluate social emotionality in animal models, as well as to quantify social behavior in human psychiatric disorders.

Efficient gene transfer into neurons in monkey brain by adeno-associated virus 8.

Although the adeno-associated virus (AAV) vector is a promising tool for gene transfer into neurons, especially for therapeutic purposes, neurotropism in primate brains is not fully elucidated for specific AAV serotypes. Here, we injected AAV serotype 8 (AAV8) vector carrying the enhanced green fluorescent protein (EGFP) gene under a ubiquitous promoter into the cerebral cortex, striatum and substantia nigra of common marmosets. Robust neuronal EGFP expression was observed at all injected sites. Cell typing with immunohistochemistry confirmed efficient AAV8-mediated gene transfer into the pyramidal neurons in the cortex, calbindin-positive medium spiny neurons in the striatum and dopaminergic neurons in the substantia nigra. The results indicate a preferential tropism of AAV8 for subsets of neurons, but not for glia, in monkey brains.