Arata Oh-Nishi

Organization of the marmoset cerebellum in three‐dimensional space: Lobulation, aldolase C compartmentalization and axonal projection

The cerebellar cortex is organized by transverse foliation and longitudinal compartmentalization. Although the latter, which is recognized through the molecular expression in subsets of Purkinje cells (PCs), is closely related to topographic axonal projection and represents functional divisions, the details have not been fully clarified in mammals other than rodents. Therefore, we examined folial and compartmental organization of the marmoset cerebellum, which resembles the macaque cerebellum, and compared it with that of the rodent cerebellum by aldolase C immunostaining, three‐dimensional reconstruction of the PC layer, and labeling of olivocerebellar and corticonuclear projections. Longitudinal stripes of different aldolase C expression intensities separated the entire cerebellar cortex into multiple compartments. Lobule VIIAb–d was equivalent to rodent lobule VIc in that it contained a transverse gap in the cortical layers and served as the rostrocaudal boundary for compartments and axonal branching. Olivocortical and corticonuclear projection patterns in major compartments indicated that the compartmental organization in the marmoset cerebellum was generally equivalent to that in the rodent cerebellum, although two compartments were missing in the pars intermedia and several compartments that have not been seen in rodents were recognized in the flocculus, nodulus, and the most lateral hemisphere. Reconstruction showed that the paraflocculus and flocculus were formed by a single longitudinal sheet, the axis of which was parallel to the aldolase C compartments, PC dendrites, and olivocerebellar climbing fiber distribution. The results indicate that molecular compartmentalization in the marmoset cerebellum reflected both the common fundamental organization of the mammalian cerebellum and species‐dependent differentiation. J. Comp. Neurol. 518:1764–1791, 2010.

Maternal immune activation by polyriboinosinic-polyribocytidilic acid injection produces synaptic dysfunction but not neuronal loss in the hippocampus of juvenile rat offspring.

It has been suggested that maternal immune activation increases the risk of psychiatric disorders such as schizophrenia in offspring. There are many reports about hippocampal structural pathology in schizophrenia. Antipsychotic drug administration in adolescence prevented postpubertal hippocampal structural pathology in the maternal immune activation animal model. These findings suggest the possibility that maternal immune activation induces hippocampal dysfunction in juvenile offspring. To test this hypothesis, we investigated hippocampal function in juvenile offspring of maternal immune activation model rat. A synthetic double-stranded RNA polyriboinosinic-polyribocytidilic acid (Poly I:C; 4 mg/kg/day, I.P.) was injected to pregnant rats on gestation days 15 and 17, in order to cause immune activation by stimulating Toll-like receptor 3. Hippocampal synaptic function and morphology in their juvenile offspring (postnatal days 28-31) were compared to those in vehicle-injected control offspring. Field responses were recorded in the hippocampal CA1 region by stimulating commissural/Schaffer collaterals. Pre-synaptic fiber volley amplitudes (mV) and field excitatory post-synaptic potential slopes (mV/ms) were significantly lower in treated offspring. In addition, short-term synaptic plasticity, namely, the paired-pulse facilitation ratio, was significantly higher and long-term synaptic plasticity (long-term potentiation) was significantly impaired in treated offspring. Furthermore, major pre-synaptic protein (synaptophysin) expressions were decreased, but not major post-synaptic proteins (GluR1, GluR2/3, and NR1), in hippocampal CA1 of treated offspring, whereas neuronal loss was not detected in the hippocampal CA1-CA3 regions. These results indicate that maternal immune activation leads to synaptic dysfunction without neuronal loss in the hippocampus of juvenile offspring, and this may be one of the early stages of schizophrenia pathologies.

Detailed Expression Pattern of Aldolase C (Aldoc) in the Cerebellum, Retina and Other Areas of the CNS Studied in Aldoc-Venus Knock-In Mice

Aldolase C (Aldoc, also known as “zebrin II”), a brain type isozyme of a glycolysis enzyme, is expressed heterogeneously in subpopulations of cerebellar Purkinje cells (PCs) that are arranged longitudinally in a complex striped pattern in the cerebellar cortex, a pattern which is closely related to the topography of input and output axonal projections. Here, we generated knock-in Aldoc-Venus mice in which Aldoc expression is visualized by expression of a fluorescent protein, Venus. Since there was no obvious phenotypes in general brain morphology and in the striped pattern of the cerebellum in mutants, we made detailed observation of Aldoc expression pattern in the nervous system by using Venus expression in Aldoc-Venus heterozygotes. High levels of Venus expression were observed in cerebellar PCs, cartwheel cells in the dorsal cochlear nucleus, sensory epithelium of the inner ear and in all major types of retinal cells, while moderate levels of Venus expression were observed in astrocytes and satellite cells in the dorsal root ganglion. The striped arrangement of PCs that express Venus to different degrees was carefully traced with serial section alignment analysis and mapped on the unfolded scheme of the entire cerebellar cortex to re-identify all individual Aldoc stripes. A longitudinally striped boundary of Aldoc expression was first identified in the mouse flocculus, and was correlated with the climbing fiber projection pattern and expression of another compartmental marker molecule, heat shock protein 25 (HSP25). As in the rat, the cerebellar nuclei were divided into the rostrodorsal negative and the caudoventral positive portions by distinct projections of Aldoc-positive and negative PC axons in the mouse. Identification of the cerebellar Aldoc stripes in this study, as indicated in sample coronal and horizontal sections as well as in sample surface photos of whole-mount preparations, can be referred to in future experiments.

PET Analysis of Dopaminergic Neurodegeneration in Relation to Immobility in the MPTP-Treated Common Marmoset, a Model for Parkinson's Disease

Background Positron Emission Tomography (PET) measurement was applied to the brain of the common marmoset, a small primate species, treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The marmoset shows prominent Parkinson’s disease (PD) signs due to dopaminergic neural degeneration. Recently, the transgenic marmoset (TG) carrying human PD genes is developing. For phenotypic evaluations of TG, non-invasive PET measurement is considered to be substantially significant. As a reference control for TG, the brain of the MPTP-marmoset as an established and valid model was scanned by PET. Behavioral analysis was also performed by recording locomotion of the MPTP-marmoset, as an objective measure of PD signs. Methodology/Principal Findings Marmosets received several MPTP regimens (single MPTP regimen: 2 mg/kg, s.c., per day for 3 consecutive days) were used for PET measurement and behavioral observation. To measure immobility as a central PD sign, locomotion of marmosets in their individual living cages were recorded daily by infrared sensors. Daily locomotion counts decreased drastically after MPTP regimens and remained diminished for several months or more. PET scan of the brain, using [11C]PE2I as a ligand of the dopamine (DA) transporter, was performed once several months after the last MPTP regimen. The mean binding potential (BPND) in the striatum (putamen and caudate) of the MPTP-marmoset group was significantly lower than that of the MPTP-free control group (n = 5 for each group). In the MPTP-marmosets, the decrease of BPND in the striatum closely correlated with the decrease in locomotion counts (r = 0.98 in putamen and 0.91 in caudate). Conclusion/Significance The present characterization of neural degeneration using non-invasive PET imaging and of behavioral manifestation in the MPTP marmoset mimics typical PD characteristics and can be useful in evaluating the phenotype of TG marmosets being developed.

Compartmentalization of the chick cerebellar cortex based on the link between the striped expression pattern of aldolase C and the topographic olivocerebellar projection.

The avian cerebellum is organized into multiple longitudinal stripes defined by expression profiles of aldolase C (zebrin II) in Purkinje cells. The relationship between the aldolase C striped pattern and the olivocerebellar projection pattern is crucial in understanding cerebellar functional compartmentalization. We identified all aldolase C stripes across all lobules with the serial section alignment analysis method and then looked at this relationship by anterograde and retrograde labeling of olivocerebellar axons in the chick cerebellum. Aldolase C stripes were generally consistent and continuous from lobule I through VII and to the medial part of lobules VIII–IXb. The dorsal and ventral lamellas (DL, VL) of the inferior olive projected to the stripes in these areas with a simple mediolateral topographic relation. A few aldolase C stripes appeared at the lateral edge of lobules VI–VIII. Several more stripes were added in the lateral parts of lobules IXa–IXb and IXc–X. The medial column (MC) of the inferior olive projected to the stripes in lobules VIII–X, including the added lateral stripes, with a complex topographic relation. Sharp boundaries between aldolase C‐positive and ‐negative stripes often accompanied a gap in the Purkinje cell layer and bordered topographically distinct groups of axons. Although the compartmental organization of the chick cerebellum is comparable to that of the mammalian cerebellum, several significant differences in the organization suggest partly separate evolutionary lineages of the mammalian and avian cerebella. We propose that rostral lobules may be evolved by rostral extension of medial stripes from caudal lobules in the avian cerebellum. J. Comp. Neurol. 523:1886–1912, 2015

PET imaging-guided chemogenetic silencing reveals a critical role of primate rostromedial caudate in reward evaluation.

The rostromedial caudate (rmCD) of primates is thought to contribute to reward value processing, but a causal relationship has not been established. Here we use an inhibitory DREADD (Designer Receptor Exclusively Activated by Designer Drug) to repeatedly and non-invasively inactivate rmCD of macaque monkeys. We inject an adeno-associated viral vector expressing the inhibitory DREADD, hM4Di, into the rmCD bilaterally. To visualize DREADD expression in vivo, we develop a non-invasive imaging method using positron emission tomography (PET). PET imaging provides information critical for successful chemogenetic silencing during experiments, in this case the location and level of hM4Di expression, and the relationship between agonist dose and hM4Di receptor occupancy. Here we demonstrate that inactivating bilateral rmCD through activation of hM4Di produces a significant and reproducible loss of sensitivity to reward value in monkeys. Thus, the rmCD is involved in making normal judgments about the value of reward.

A high-yield automated radiosynthesis of the alpha-7 nicotinic receptor radioligand [18F]NS10743

[18F]NS10743, a promising and highly competitive α7 nAChR radioligand has been synthesized so far by microwave irradiation using a manual single-mode device followed by a palladium-catalyzed reduction of remaining nitro-precursor for HPLC separation purposes. For further preclinical and clinical use, regulated production of [18F]NS10743 by fully automated radiosynthesis is a crucial requirement. Therefore, we chose a commercial synthesis module and developed the automated radiosynthesis of [18F]NS10743. Besides evaluation of several radiosynthesis procedures, we performed an extensive HPLC study for quantitative separation of [18F]NS10743 from the corresponding nitro precursor. After implementation of the optimized procedure on a TRACERlabTM FX F-N synthesis module, [18F]NS10743 was obtained in high radiochemical purity (≥99%) with an overall radiochemical yield of 32.2±7% (n=3). The specific activities at the end of the synthesis were 571±17GBq/µmol (n=3).

Use of human methylation arrays for epigenome research in the common marmoset (Callithrix jacchus)

We examined the usefulness of commercially available DNA methylation arrays designed for the human genome (Illumina HumanMethylation450 and MethylationEPIC) for high-throughput epigenome analysis of the common marmoset, a nonhuman primate suitable for research on neuropsychiatric disorders. From among the probes on the methylation arrays, we selected those available for the common marmoset. DNA methylation data were obtained from genomic DNA extracted from the frontal cortex and blood samples of adult common marmosets as well as the frontal cortex of neonatal marmosets. About 10% of the probes on the arrays were estimated to be useful for DNA methylation assay in the common marmoset. Strong correlations existed between human and marmoset DNA methylation data. Illumina methylation arrays are useful for epigenome research using the common marmoset.

PM371. Measurement of genomic copy number and DNA methylation levels of LINE-1 in the brains of poly(I:C) mouse model

We previously reported the increased copy number of retrotransposon LINE-1 in neurons of schizophrenia as well as animal models of psychiatric disorders including poly(I:C) model. Exposure of poly(I:C), a chemical analogue of double-stranded RNA, in the prenatal stage of animal induces the behavioral impairment in its later developmental stage. Therefore, this animal model provides the opportunity to elucidate the molecular mechanism of increased LINE-1 retrotransposition during prenatal stage. Here we examined LINE-1 copy number and DNA methylation levels of brain tissues in poly(I:C) mouse. We injected poly(I:C) with various doses to pregnant mice, and found that LINE-1 copy number of pups was increased in a dose-dependent manner. Especially, pregnant mice with consecutive poly(I:C) injection showed most significant increase of LINE-1 copy number. Interestingly, DNA methylation levels of active LINE-1 subfamilies in the adult stage showed subfamily-dependent methylation changes, suggesting the distinctive functional roles among the active subfamilies. PM372 Functional change of serotonin 2C receptors in N-Methyl-D-aspartate (NMDA) receptor hypofunctional condition Takahiro Tsutsumi, Ken Inada, Jun Ishigooka, Masahiko Kawano, Takaaki Kawano, Hiroyuki Muraoka, Hidehiro Oshibuchi, Makiko

A possible serologic biomarker for maternal immune activation-associated neurodevelopmental disorders found in the rat models

Epidemiological studies have shown that maternal infection during early pregnancy increases the risk of neurodevelopmental disorders (i.e., schizophrenia or autism) in offspring. Recently, diagnostic/stratification biomarkers for the maternal immune activation background in patients with neurodevelopmental disorders have been energetically searched for in the patient blood. Here, we report a novel serologic marker candidate for the disorders found in the maternal immune activation (MIA) rat model. Serum proteome analysis of the MIA rat showed that the immunoglobulin (Ig) light chain is reproducibly augmented. The Ig light chain in sera takes two forms - free form or bound to the Ig heavy chain. Only the former is an inflammatory disease marker, but pro-inflammatory cytokine levels in the sera of the MIA rats were below detectable limits of the ELISA protocol we used. We thereby carried out serum assays of Ig light chains and pro-inflammatory cytokines of commercially available schizophrenia patient sera for research. Although the number of samples was limited, we found augmentation of free Ig light chains but not pro-inflammatory cytokines in sporadic schizophrenia patient sera. Our findings suggest that Ig light chain assay of the schizophrenia/autism patient sera would be worthy to be validated in larger scale.