Hidetoshi Ishibashi

Local and retrograde gene transfer into primate neuronal pathways via adeno-associated virus serotype 8 and 9.

Viral vector-mediated gene transfer has become increasingly valuable for primate brain research, in particular for application of genetic methods (e.g. optogenetics) to study neuronal circuit functions. Neuronal cell tropisms and infection patterns are viable options for obtaining viral vector-mediated transgene delivery that is selective for particular neuronal pathways. For example, several types of viral vectors can infect axon terminals (retrograde infections), which enables targeted transgene delivery to neurons that directly project to a particular viral injection region. Although recent studies in rodents have demonstrated that adeno-associated virus serotype 8 (AAV8) and 9 (AAV9) efficiently transduce neurons, the tropisms and infection patterns remain poorly understood in primate brains. Here, we constructed recombinant AAV8 or AAV9, which expressed an enhanced green fluorescent protein (EGFP) gene driven by a ubiquitous promoter (AAV8-EGFP and AAV9-EGFP, respectively), and stereotaxically injected it into several brain regions in marmosets and macaque monkeys. Immunohistochemical analyses revealed almost exclusive colocalization of EGFP fluorescence via AAV9-mediated gene transfer with a neuron-specific marker, indicating endogenous neuronal tropism of AAV9, which was consistent with our previous results utilizing AAV8. Injections of either AAV8-EGFP or AAV9-EGFP into the marmoset striatum resulted in EGFP expression in local striatal neurons as a result of local infection, as well as expression in dopaminergic neurons of the substantia nigra via retrograde transport along nigrostriatal axonal projections. Retrograde infections were also observed in the frontal cortex and thalamus, which are known to have direct projections to the striatum. These local and retrograde gene transfers were further demonstrated in the geniculocortical pathway of the marmoset visual system. These findings indicate promising capabilities of AAV8 and AAV9 to deliver molecular tools into a range of primate neural systems in pathway-specific manners through their neuronal tropisms and infection patterns.

Transgenic Monkey Model of the Polyglutamine Diseases Recapitulating Progressive Neurological Symptoms

Abstract 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.

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.

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.

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.

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

ABSTRACT 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.

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.

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, but little is known about the postnatal development of electrophysiological properties of amygdala neurons. •  We performed slice patch‐clamp recordings from basolateral amygdala (BLA) pyramidal neurons in the developing and adult common marmoset (Callithrix jacchus). •  The marmoset is a non‐human primate that has been increasingly used as an animal model of human disease, but no previous studies have used brain slice patch‐clamp techniques in marmosets. •  We found that BLA pyramidal neurons from newborn marmosets possess relatively mature action potentials and receive vigorous GABAergic synaptic inputs, and that marmoset BLA neurons acquire adult‐like electrophysiological properties within the first 4 weeks of life. •  Our study provides novel information on several features of postnatal development of BLA pyramidal neurons in marmosets.

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.

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.