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IntroductionThe cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive.Methods and resultsWe show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer.DiscussionHence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Animal models have been used to model human diseases, and among them, small fishes have been highlighted for their usefulness in various ways, such as the low cost of maintenance, ease of genetic modification, small size for easy handling, and strength in imaging studies due to their relative transparency. Recently, the use of turquoise killifish, Nothobranchius furzeri, which is known to exhibit various aging phenotypes in a short period, has attracted attention in research on aging and age-related diseases. However, when using animal models, it is important to keep their genetic background and interspecies differences in mind for translating them into human diseases. In this article, we obtained the gene symbols of protein-coding genes of turquoise killifish, medaka, zebrafish, and humans from NCBI datasets and extracted common shared genes among four species to explore the potential of interspecies translational research and to apply small fish models for human age-related disorders. Common shared protein-coding genes were analyzed with the Reactome Pathway Database to determine the coverage of these genes in each pathway in humans. We applied common shared genes to the Orphanet database to establish a list of human diseases that contain common shared genes among the four species. As examples, the senescence-related pathways and some pathways of human age-related diseases, such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, nonalcoholic fatty liver disease, progeria, hepatocellular carcinoma, and renal cell carcinoma, were extracted from the curated pathway and disease list to discuss the further utility of fish models for human age-related disorders.

Neurodegenerative disorders are characterized by the progressive loss of neuronal structure or function, resulting in memory loss and movement disorders. Although the detailed pathogenic mechanism has not been elucidated, it is thought to be related to the loss of mitochondrial function in the process of aging. Animal models that mimic the pathology of a disease are essential for understanding human diseases. In recent years, small fish have become ideal vertebrate models for human disease due to their high genetic and histological homology to humans, ease of in vivo imaging, and ease of genetic manipulation. In this review, we first outline the impact of mitochondrial dysfunction on the progression of neurodegenerative diseases. Then, we highlight the advantages of small fish as model organisms, and present examples of previous studies regarding mitochondria-related neuronal disorders. Lastly, we discuss the applicability of the turquoise killifish, a unique model for aging research, as a model for neurodegenerative diseases. Small fish models are expected to advance our understanding of the mitochondrial function in vivo, the pathogenesis of neurodegenerative diseases, and be important tools for developing therapies to treat diseases.

The African turquoise killifish (N. furzeri) is a useful model organism for studying aging, age-related diseases, and embryonic diapause. CRISPR/Cas9-mediated gene knockout and Tol2 transposon-mediated transgenesis in N. furzeri have been reported previously. However, these methods take time to generate knockout and transgenic fish. In addition, knock-in technology that inserts large DNA fragments as fluorescent reporter constructs into the target gene in N. furzeri has not yet been established. Here, we show that triple-target CRISPR-mediated single gene disruption efficiently produces whole-body biallelic knockout and enables the examination of gene function in the F0 generation. In addition, we developed a method for creating the knock-in reporter N. furzeri without crossing by optimizing the CRISPR/Cas9 system. These methods drastically reduce the experimental duration and we think that these will accelerate aging and developmental studies using N. furzeri.

Parkinson's disease is a neurodegenerative disorder that causes motor and nonmotor symptoms due to the loss of dopaminergic nerves and is characterized by the presence of Lewy bodies, which are mainly composed of α-synuclein. Glucosylceramidase beta (GBA), which is a causative gene of autosomal recessive Gaucher disease, is also known to be a risk gene for Parkinson's disease. In this study, we tried to detect synergistic effects of α-synuclein accumulation and gba depletion on dopaminergic neurodegeneration in zebrafish.We generated a transgenic line of zebrafish overexpressing the A53T α-synuclein and gba mutant fish, and analyzed pathologies of α-synuclein aggregation and neurodegeneration.Zebrafish overexpressing the A53T α-synuclein did not exhibit α-synuclein aggregate formation. After the loss of gba function in this mutant α-synuclein transgenic line, we observed the marked presence of α-synuclein aggregates. Loss of gba function in zebrafish resulted in dopaminergic and noradrenergic neurodegeneration but this level of neurodegeneration was not exacerbated by overexpression of mutant α-synuclein.These results indicate that loss of gba function was sufficient to generate a neurodegenerative phenotype in zebrafish regardless of the expression of α-synuclein.

ATP13A2 is the autosomal recessive causative gene for juvenile-onset Parkinson’s disease (PARK9, Parkinson’s disease 9), also known as Kufor-Rakeb syndrome. The disease is characterized by levodopa-responsive Parkinsonism, supranuclear gaze palsy, spasticity, and dementia. Previously, we have reported that Atp13a2 deficient medaka fish showed dopaminergic neurodegeneration and lysosomal dysfunction, indicating that lysosome-autophagy impairment might be one of the key pathogeneses of Parkinson’s disease. Here, we established Atp13a2 deficient zebrafish using CRISPR/Cas9 gene editing. We found that the number of TH + neurons in the posterior tuberculum and the locus coeruleus significantly reduced (dopaminergic neurons, 64 % at 4 months and 37 % at 12 months, p < 0.001 and p < 0.05, respectively; norepinephrine neurons, 52 % at 4 months and 40 % at 12 months, p < 0.001 and p < 0.05, respectively) in Atp13a2 deficient zebrafish, proving the degeneration of dopaminergic neurons. In addition, we found the reduction (60 %, p < 0.05) of cathepsin D protein expression in Atp13a2 deficient zebrafish using immunoblot. Transmission electron microscopy analysis using middle diencephalon samples from Atp13a2 deficient zebrafish showed lysosome-like bodies with vesicle accumulation and fingerprint-like structures, suggesting lysosomal dysfunction. Furthermore, a significant reduction (p < 0.001) in protein expression annotated with vesicle fusion with Golgi apparatus in Atp13a2 deficient zebrafish by liquid-chromatography tandem mass spectrometry suggested intracellular trafficking impairment. Therefore, we concluded that Atp13a2 deficient zebrafish exhibited degeneration of dopaminergic neurons, lysosomal dysfunction and the possibility of intracellular trafficking impairment, which would be the key pathogenic mechanism underlying Parkinson’s disease.

The presence of ectopic DNA in the cytoplasm induces inflammation and cell death. It has been widely reported that leakage of nuclear DNA into the cytoplasm can mainly be sensed by cyclic GMP-AMP synthase (cGAS). We recently reported that mitochondria-derived cytoplasmic double-stranded DNA (dsDNA) that has escaped lysosomal degradation induces significant cytotoxicity in cultured cells and in vivo. Cytoplasmic mitochondrial DNA is assumed to be involved in various diseases and disorders, and more and more papers have been published confirming this. On the other hand, the current method for evaluating mitochondrial DNA in the cytoplasm may not be quantitative. Here, we introduce in detail a method to evaluate ectopic mitochondrial DNA in cells. This method is useful in basic research as well as in the study of aging, Parkinson’s disease, Alzheimer’s disease, heart failure, autoimmune diseases, cancer, and other conditions.

In larviculture of marine finfish, Tisochrysis lutea cultured cells have a potential to be a sustainable enrichment diet for rotifers due to its de novo synthesis of docosahexaenoic acid (DHA). To improve its effectiveness in rotifer enrichment for later larviculture, we determined which growth phase of T. lutea can enhance DHA in polar lipids (PLs) in Brachionus plicatilis sp. complex rotifers without reducing their vigor. Tisochrysis lutea was harvested at logarithmic growth phase, deceleration growth phase, and stationary phase. The ambient concentrations of phosphorus (P) and nitrogen (N), and fatty acid composition of microalgae were measured. P was limited between logarithmic and deceleration growth phases, followed by N starvation in stationary phase. Total lipid DHA was lowest in logarithmic growth phase, whereas DHA levels increased in deceleration growth phase and maintained at high levels even in stationary phase. PL-DHA also increased during deceleration growth phase, although its abundance dropped during stationary phase. In a rotifer enrichment trial, T. lutea harvested in each phase were fed to B. plicatilis sp. complex rotifers for 12 h. The highest PL-DHA value for cells in deceleration growth phase reflected the concentrations of the rotifers. No significant differences were found in rotifer growth rate and egg ratio. Rotifers fed cells in deceleration growth phase exhibited higher swimming speeds than those fed the other cells, potentially linking to increased larval capture success. Therefore, we recommend the deceleration growth phase as an optimal timing for T. lutea harvest for the effective enrichment of B. plicatilis sp. complex rotifers.

In recent years, small fishes such as zebrafish and medaka have been widely recognized as model animals. They have high homology in genetics and tissue structure with humans and unique features that mammalian model animals do not have, such as transparency of embryos and larvae, a small body size and ease of experiments, including genetic manipulation. Zebrafish and medaka have been used extensively in the field of neurology, especially to unveil the mechanisms of neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease, and recently, these fishes have also been utilized to understand neurodevelopmental disorders such as autism spectrum disorder. The turquoise killifish has emerged as a new and unique model animal, especially for ageing research due to its unique life cycle, and this fish also seems to be useful for age-related neurological diseases. These small fishes are excellent animal models for the analysis of human neurological disorders and are expected to play increasing roles in this field. Here, we introduce various applications of these model fishes to improve our understanding of human neurological disorders.

Mitochondrial dysfunction and lysosomal dysfunction have been implicated in Parkinson’s disease (PD), but the links between these dysfunctions in PD pathogenesis are still largely unknown. Here we report that cytosolic dsDNA of mitochondrial origin escaping from lysosomal degradation was shown to induce cytotoxicity in cultured cells and PD phenotypes in vivo. The depletion of PINK1, GBA and/or ATP13A2 causes increases in cytosolic dsDNA of mitochondrial origin and induces type I interferon (IFN) responses and cell death in cultured cell lines. These phenotypes are rescued by the overexpression of DNase II, a lysosomal DNase that degrades discarded mitochondrial DNA, or the depletion of IFI16, which acts as a sensor for cytosolic dsDNA of mitochondrial origin. Reducing the abundance of cytosolic dsDNA by overexpressing human DNase II ameliorates movement disorders and dopaminergic cell loss in gba mutant PD model zebrafish. Furthermore, IFI16 and cytosolic dsDNA puncta of mitochondrial origin accumulate in the brain of patients with PD. These results support a common causative role for the cytosolic leakage of mitochondrial DNA in PD pathogenesis., Mitochondrial and lysosomal dysfunction are central to Parkinson’s disease (PD) pathogenesis. Here, the authors show mitochondrial dsDNA in the cytosol in cellular and Zebrafish models of PD induces cytotoxicity and neurodegeneration; knock-down of IFI16, a cytosolic dsDNA sensor, rescues cytotoxicity, as does overexpression of lysosomal DNAse II.

Euryhaline rotifer belonging to the genera Brachionus are often used as live feed for first feeding marine finfish larviculture. Two common rotifer enrichments, a single usage of docosahexaenoic acid (DHA)-enriched Chlorella vulgaris and a combination of DHA-enriched C. vulgaris and salmon roe emulsion oil were used to assess: (i) nutritional status of rotifers and, (ii) performance when rotifers were fed to Pagrus major and Paralichthys olivaceus larvae from 2 to 15 days after hatching (DAH). Both rotifer enrichment treatments were found to be nutritionally effective with high unsaturated fatty acid (HUFA) content, especially in the combined treatment with significantly higher eicosapentaenoic acid (EPA). Despite deficient levels of EPA, DHA-enriched C. vulgaris was found to be a suitable enrichment for rotifer and also had positive response in survival, growth and trypsin activity in both P. major and P. olivaceus. DHA-enriched C. vulgaris only treatment had higher rotifer population growth (13% higher), egg bearing capacity (7% higher), almost double the soluble protein content and generally higher free amino acid content. Oxidation of lipids in the combined diet due to its high-saturated fatty acid content could be a possible cause to the inferior performance of the combined treatment. Both P. major (20.1 ± 10.1%) and P. olivaceus (12.3 ± 3.87%) suffered low survival and lower gut content in the combined treatment especially during periods of significant growth in both species (7–11 DAH). Despite high HUFA content in the combined treatment, this study revealed that consideration of free amino acids and soluble protein content as parameters of nutritional indicators for first feeding P. major and P. olivaceus should not be discounted.

Summary: Parkinson’s disease (PD) is a neurodegenerative disease characterized by α-synuclein-positive inclusion bodies and loss of neurons, including dopaminergic neurons. Difficulty in replicating PD phenotypes using animal models partly limits the understanding of PD and the therapy required. Although PD is strongly associated with aging, most experimental animals may not exhibit age-related symptoms. Herein, we demonstrate that Nothobranchius furzeri, a rapidly aging teleost with a short life span, exhibits age-dependent degeneration of dopaminergic and noradrenergic neurons and progression of α-synuclein pathologies. These pathological phenotypes are similar to those observed in human patients with PD. Amelioration of the cell loss by genetic depletion of α-synuclein suggests that α-synuclein is not a bystander but a causative protein of neurodegeneration. N. furzeri can reveal mechanisms underlying PD, especially of the idiopathic form that affects a majority of patients with PD, including α-synuclein-dependent neurodegeneration, age-dependent phenotypes, and progression of α-synuclein pathology. : Matsui et al. find that annual killifish, Nothobranchius furzeri, reveals age-dependent degeneration of dopamine neurons and accumulation of α-synuclein. Genetic depletion of α-synuclein rescues these phenotypes. Keywords: Nothobranchius furzeri, aging, Parkinson’s disease, α-synuclein

Efficient production of beneficial metabolites by microalgae using light-emitting diodes (LED) is attracting more interest. In batch cultures, metabolic changes that occur with nutrient deficiency over time must be monitored to estimate the metabolite content. Our previous study showed that nutrient deficiency in Nannochloropsis oculata can be briefly evaluated by measuring the absorption ratio at 490 and 680 nm (Abs490/Abs680). However, little is known about the relation with this ratio and metabolic change, particularly in LED light-conditioned cultures. In this study, we investigate if Abs490/Abs680 can be an indicator of the biochemical status of N. oculata cultured under various LED color regimes. Three LED treatments were used: Blue (B), Blue + Red (BR), and Red (R). In all LED treatments, the Abs490/Abs680 decreased for several days and then increased when phosphate was limited or non-existent. The trends in the C16:0 and C20:5ɷ3 proportions and chlorophyll a content commonly changed at the turning points of the Abs490/Abs680. Furthermore, significant correlations were found among the metabolic state and absorption ratio, except in treatment R. We suggest that spectrophotometry can be used for monitoring metabolic changes in N. oculata cultures under various LED irradiance, unless there is excessive 630 nm-red light exposure.

The euryhaline copepod Pseudodiaptomus inopinus play important roles in coastal waters as vectors of docosahexanoic (DHA) and eicosapentaenoic (EPA) acids for larval fish. While DHA and EPA in polar lipids (PLs) are more effective for fish larval development than non-polar lipid forms (NLs), there is little knowledge how much these lipids are accumulated in copepods from microalgae and are effective for early development of fish larvae. We report PLs fatty acid profiles of P. inopinus fed DHA-poor microalgae and evaluate its significance as a food source for larvae development of Pagrus major, compared with DHA-enriched rotifers. Copepods and rotifers were fed a mixed diet of three algal species (Phaeodactylum tricornutum, Tisochrysis lutea, and Pavlova lutheri), in addition of DHA-supplemented Super Fresh Chlorella (SFC) for rotifers. Compared with SFC, the algal mixture had higher EPA but lower DHA. Copepods had higher DHA and EPA in total lipids than rotifers fed each diet. Copepod PLs were specifically enriched with DHA and their contents were higher than both rotifers. On the other hand, PLs EPA contents were comparable between preys, indicating that copepods selectively fortified the PLs. Fish culture experiment showed that larvae fed copepods had higher growth than those fed SFC-enriched rotifers. Principal component analysis for each organism fatty acid composition emphasized trophic modification of DHA by copepods toward larval fish. This study highlighted that P. inopinus contribute to enhanced growth of coastal larval fish by efficiently transferring DHA via copepod fatty acid metabolism.

Chronic intestinal pseudo-obstruction (CIPO) is a severe and refractory intestinal motility disorder. However, due to its rarity and difficult histological investigation, its pathophysiology has not been characterized. Therefore, in this study, we aimed to determine the role of esophageal high-resolution manometry (HRM) in CIPO and the histological and clinical characteristics of the disease. Patients with CIPO were analyzed for clinical characteristics; histological findings; and clinical courses after therapeutic intervention. In addition, HRM was performed to determine the esophageal involvement. Eleven patients were diagnosed with CIPO, and five required the long period of parenteral nutrition showing impaired esophageal motility including achalasia and absent contractility diagnosed with HRM. The four of these five cases showed acute onset of the CIPO following the triggering events of pregnancy, appendicitis, and surgery. In contrast, other six patients with normal or Jackhammer esophagus on HRM had moderate severity of CIPO with gradual onset. The histological analyses revealed that the loss of the intestinal neural ganglion cells and layers by inflammation, destruction, and atrophy are related to the severity of the clinical course of the disease and esophageal HRM findings of achalasia and absent contractility. HRM may be useful to diagnose the severity of the clinical course and to determine the therapeutic options for CIPO.

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p

Parkinson's disease is a neurodegenerative disorder that involves movement discloses, degeneration of dopaminergic neurons, and presence of cytoplasmic inclusion bodies. Various animal models have been developed and small fish including zebrafish and medaka fish have recently been employed as a new model for Parkinson disease. In this review, we summarize fish models of Parkinson's disease mainly using our own findings and explain two major hypotheses of PD: lysosome dysfunction theory and mitochondrial dysfunction theory. Finally, we discuss the potential for future application of small fish model.

Small teleost fish including zebrafish and medaka have been used as animal models for research because of their small body size, vast amounts of eggs produced, their rapid development, low husbandry costs, and transparency during embryogenesis. Although the body size and appearance seem different, fish and mammals including human still possess anatomical and functional similarities in their brains. This review summarizes the similarities of brain structures and functions between teleost fish and mammalian brains, focusing on the dopamine system, functional regionalization of the cerebellum, and presence of the nucleus ruber.

The effective use of distillery by-products of the shochu sweet potato has long been considered as a rich source of protein, fiber, amino acids, fatty acids, probiotics with a great potential as a functional feed for animals. This study applied fermented by-product of Shochu distillery (FBPSD) and salmon roe emulsion oil for rotifer enrichment to evaluate its effects on the: (i) nutritional composition of rotifers and (ii) feeding responses of Paralichthys olivaceus larvae when fed with enriched rotifers. The salmon roe oil enrichment was set as a control diet and FBPSD was supplemented at different doses (1, 5 and 10 % based on the salmon roe oil amount). The results showed that the rotifers supplemented with FBPSD at 5 and 10 % significantly enhanced protein, carbohydrate, and HUFA content. Similarly, the activity and expression profiles of targeted enzymes including trypsin, chymotrypsin, lipase and amylase effectively responded at early and middle-metamorphosis of the larvae to 5 and 10 % enrichment regiment. These two treatments were also found to be better supported in energy metabolism evidenced by Cytochrome-C-Oxidase (cox) and ATPase-pk (atp) gene expression in the larvae. Moreover, the expression levels of immune-related genes (IL-10, IL-1β, TNF-α, P2Y6) and antioxidant-related genes (SOD, CAT) were significantly improved at 5 and 10 % of FBPSD. Cessation of feeding and delay in growth during the metamorphic process was also molecularly demonstrated. Larvae suffered low survival (24.5–25.3 %) in control and 1 % supplementation of FBPSD, but significantly improved at 5 and 10 % (36.4–38.7%). However, the growth performance of larval fish was not remarkable after 40 days of culture. The results of this study revealed that FBPSD could be considered as a potential supplementary enrichment for live feed in fish larviculture, especially at 5 and 10 % rotifer enrichment dosage.

Kawakawa tuna (Euthynnus affinis) is a novel target species for aquaculture in Japan and currently there is very limited knowledge on its early ontogenetic development in captivity. The ontogeny of E. affinis larvae from hatching to 20 days after hatching (DAH) was illustrated in the present study through an integrated approach of histology, biochemistry and molecular approaches. Larvae were fed enriched L-type rotifer Brachionus plicatilis species complex from 2 to 10 DAH, followed by enriched Artemia nauplii. Target digestive enzymes; trypsin, pepsin, lipase and amylase were assessed in order to evaluate nutritional condition. The larvae began exogenous feeding from 2 DAH, coinciding with the opening of the oral capacity and anus. The yolk sac was rapidly consumed and completely resorbed at 3 DAH, while the oil globule disappeared by 5 DAH. Enzymatic activity and expression of the target enzymes were detectable at hatching, except pepsin. With the development of the gastric glands, pepsin activity was then detected at 10 DAH, which was followed by rapid increase in pepsin activity at 15 DAH. The larvae showed rapid growth, however, high mortality also occurred at 6–10 DAH which has been highlighted as a critical stage in the larviculture of E. affinis. From 15 to 20 DAH, the histological structures of the digestive tract were completely segmented with the digestive tract resembling that of its juvenile stages. The activity of trypsin, pepsin, and lipase tended to increase sharply, while amylase dramatically dropped, possibly indicating a change in dietary preference. Findings from the present study provides a preliminary insight into the early development of the digestive system and associated digestive enzymes of E. affinis larvae and establishes an initial step towards the rearing and feeding protocol optimization of this novel target species.

BACKGROUND Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

The microalga Nannochloropsis oculata is used as a diet for rotifers and can enrich the highly unsaturated fatty acid (HUFA) proportion of rotifer polar lipids (PL-HUFA). For efficient enrichment of PL-HUFAs in rotifers, we evaluated nutrient–sufficient or –deficient cell populations to clarify when the alga should be harvested and fed to rotifers. The nutrient conditions for N. oculata were spectrophotometrically monitored during cultivation and cells with nutrient sufficiency (NS) and phosphorous deficiency (PD) were harvested. The PD cells exhibited reduced chlorophyll a content, photosynthetic activity, and growth rate and accumulated total lipids as non-polar lipids (NLs). A decrease in the proportion of HUFA to total lipids between two nutrient states corresponded to a similar phenomenon observed in NLs. On the other hand, PLs, including glycolipids and phospholipids, showed different trends. Glycolipids did not change fatty acid composition between two states. Phospholipids, however, showed significantly higher contents of arachidonic acid (ArA) and eicosapentaenoic acid (EPA) only in the early periods of PD culture. It was concluded that phosphorous deficiency stimulated cells to increase the fatty acid desaturation rate of non-plastidial membranes. Mitochondria in the PD cells decreased in number but increased in size and mass. Such adaptation of mitochondria can be associated with high desaturation of non-plastidial membranes. When the early P–limited phase culture was used for the enrichment of rotifers, PL-ArA and EPA of the rotifers were enhanced by twice as much as those fed on NS cells. This study provides evidence that the beginning of phosphorous deficiency is the optimum time to harvest N. oculata cells for enhancing rotifer PL-HUFAs.

This study aimed to determine whether enteric neurons are involved in multiple system atrophy (MSA). Four-μm-thick slices of small intestine were prepared from 10%-formalin-fixed and paraffin-embedded materials obtained from autopsied cases. Enteric neurons were stained using an anti-peripherin antibody. Immunostaining of phosphorylated α-synuclein was also performed. Areas of the cytoplasm and nucleus that showed nucleoli were measured using computer software. Both areas of myenteric neurons were significantly smaller in MSA cases (n = 3) than in control subjects (n = 3) (P 0.0001); however, no deposits of phosphorylated α-synuclein were observed. These findings suggest that myenteric neurons in MSA are affected independent of α-synuclein accumulation.

A modified method of cerebrospinal fluid injection was developed for the efficient and reliable administration of substances to the zebrafish central nervous system. The accuracy of this modified method was evaluated using Alexa Fluor dye injection. A high survival ratio was achieved due to the simplicity of the procedure and ice-tricaine combined anaesthesia. To validate this new method, we injected ammonium chloride, which successfully blocked lysosome function resulting in elevated LC3-II and the accumulation of ubiquitinated proteins. Injection of human α-synuclein fibrils initiated a prion-like propagation of α-synuclein pathology in zebrafish. This method can be used to investigate the effects of various substances and the propagation of α-synuclein in the central nervous system.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output. Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon's start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output. Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon's start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Teleost fish have recently been employed as a model for human neurodegenerative diseases. We used toxin exposure and genetic engineering to develop models of Parkinson's disease (PD) in the teleost fish, medaka. Among the toxins examined, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors, and tunicamycin all induced important features of PD in medaka. Specifically, these agents induced dopaminergic cell loss and reduced spontaneous movement, and the latter three toxins produced inclusion bodies that were ubiquitously distributed in the medaka brain. Despite the extensive distribution of these inclusion bodies, the middle diencephalic dopaminergic neurons were particularly susceptible to the effect of the toxins, suggesting that this cluster of dopaminergic neurons is analogous to the human substantia nigra. We have also created a variety of different genetic models using the Targeting Induced Local Lesions in Genomes (TILLING) method, and found that neither PTEN-induced putative kinase 1 (PINK1) mutants nor Parkin mutants disclosed significant dopaminergic cell loss. Surprisingly however, PINK1 and Parkin double mutants exhibited selective dopamine cell loss, as well as aggregation and deficit of mitochondrial activity. Another mutant, the ATP13A2 mutant, also expressed a PD phenotype, exhibiting marked cathepsin D reduction and fingerprint-like structures that are generally found in lysosome storage diseases. Taken together, these data indicate that medaka fish can serve as a new animal model for PD. In this review, we summarize our data and discuss the potential for future application of this animal model.

Small teleost fish including zebrafish and medaka have been used as animal models in basic science research due to the relative ease of handling and transparency during embryogenesis. Current advances in genetic engineering and progress in disease genetics allowed utilization of these fish to study neurological diseases and psychiatric disorders. This review summarizes the advantages and disadvantages of using fish for neuropsychiatric research using primarily our own studies as examples. We discuss how fish belong to a class of vertebrates, are feasible for imaging, and include diverse species with multiple research possibilities yet to be discovered.

Finding a physical approach for increasing the superconducting transition temperature (Tc) is a challenge in the field of material science. Shear strain effects on the superconductivity of rhenium were investigated using magnetic measurements, X-ray diffraction, transmission electron microscopy, and first-principles calculations. A large shear strain reduces the grain size and simultaneously expands the unit cells, resulting in an increase in Tc. Here we show that this shear strain approach is a new method for enhancing Tc and differs from that using hydrostatic strain. The enhancement of Tc is explained by an increase in net electron–electron coupling rather than a change in the density of states near the Fermi level. The shear strain effect in rhenium could be a successful example of manipulating Bardeen–Cooper–Schrieffer-type Cooper pairing, in which the unit cell volumes are indeed a key parameter.

We measured the electrical conductivity σ of aluminum specimen consisting of submicron-grains by observing the AC magnetic susceptibility resulting from the eddy current. By using a commercial platform for magnetic measurement, contactless measurement of the relative electrical conductivity σ n of a nonmagnetic metal is possible over a wide temperature (T) range. By referring to σ at room temperature, obtained by the four-terminal method, σ n(T) was transformed into σ(T). This approach is useful for cylinder specimens, in which the estimation of the radius and/or volume is difficult. An experiment in which aluminum underwent accumulative roll bonding, which is a severe plastic deformation process, validated this method of evaluating σ as a function of the fraction of high-angle grain boundaries.

The teleost fish has been widely used in creating neurodegenerative models. Here we describe the teleost medaka fish Parkinson's disease (PD) models we developed using toxin treatment and genetic engineering. 1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors and tunicamycin treatment in our model fish replicated some salient features of PD: selective dopamine cell loss and reduced spontaneous movement with the last three toxins producing inclusion bodies ubiquitously in the brain. Despite the ubiquitous distribution of the inclusion bodies, the middle diencephalic dopaminergic neurons were particularly vulnerable to these toxins, supporting the idea that this dopamine cluster is similar to the human substantia nigra. PTEN-induced putative kinase 1 (PINK1) homozygous mutants also showed reduced spontaneous swimming movements. These data indicate that medaka fish can serve as a new model animal of PD. In this review we summarize our previous data and discuss future prospects.

Perturbations in protein folding and degradation are key pathological mechanisms in neurodegenerative diseases, including Parkinson's disease (PD). Recent evidence suggests that mishandling of proteins may play an important role in the pathogenesis of PD. We have utilized medaka fish to monitor the effects of injecting neurotoxins into the CSF space. In this study, ammonium chloride, tunicamycin, and lactacystin were tested for their ability to disturb lysosomal proteolysis, N-glycosylation in the endoplasmic reticulum, and proteasomal degradation, respectively. All of the substances tested induced selective loss of dopaminergic neurons, movement disorders and inclusion bodies. Among them, the features of the inclusion bodies that developed after ammonium chloride injection mimicked those of PD: co-localization of ubiquitin and phosphorylated α-synuclein, as well as the presence of LC3 protein in the inclusion bodies. Our study demonstrated that medaka fish are useful for examining the effects of environmental toxins and lysosome inhibition, and lysosome inhibitors may be factors in the development of PD.

Recent findings suggest that a defect in the ubiquitin-proteasome system plays an important role in the pathogenesis of Parkinson's disease (PD). A previous report (McNaught et al. 2004) demonstrated that rats systemically injected with proteasome inhibitors exhibited PD-like clinical symptoms and pathology. However, because these findings have not been consistently replicated, this model is not commonly used to study PD. We used medaka fish to test the effect of systemic administration of proteasome inhibitors because of the high level of accessibility of the cerebrospinal fluid in fish. We injected lactacystin or epoxomicin into the CSF of medaka. With proteasome inhibition in the medaka brain, selective dopaminergic and noradrenergic cell loss was observed. Furthermore, treated fish exhibited reduced spontaneous movement. Treatment with proteasome inhibitors also induced the formation of inclusion bodies resembling Lewy bodies, which are characteristic of PD. Treatment with 6-OHDA also induced dopaminergic cell loss but did not produce inclusion bodies. These findings in medaka are consistent with previous results reporting that non-selective proteasome inhibition replicates the cardinal features of PD: locomotor dysfunction, selective dopaminergic cell loss, and inclusion body formation.

Parkinson's disease is a neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. The PTEN-induced kinase 1 gene (PINK1) is responsible for recessive inherited familial Parkinson's disease (PARK6). Neither the function of PINK1 nor its role in the prevention of Parkinson's disease is fully understood. Gene disruption of PINK1 causes remarkably different phenotypes in animal models such as Drosophila melanogaster, zebrafish, and mouse, none of which recapitulate Parkinson's-disease-like symptoms. We established PINK1-gene-disrupted medaka fish. These mutant fish grew normally at first, then developed significant decrease in the frequency of spontaneous swimming movements in the late-adult stage. Although the mutants did not show any dopaminergic cell loss, the amount of 3,4-dihydroxyphenylacetic acid, a major metabolite of dopamine, decreased. Thus, PINK1 contributes to the maintenance of dopamine metabolism, even before the selective death of dopaminergic neurons. Our animal model is therefore a valuable tool to detect pathogenesis in Parkinson's patients in the early stages.

Objective : Auditory P300 has been reported to be abnormal in demented patients with Parkinson's disease. However, it is still controversial which factors in Parkinson's disease influence P300 parameters. Methods : Forty patients with Parkinson's disease were included. Patients were divided into two groups: patients with dementia (PDD) and without dementia (PDND). An ‘odd-ball’ paradigm was used for auditory event-related potentials. Results : P300 latency was markedly delayed in PDD patients. Age and DRS1 (attention) were the most important factors influencing P300 latency. Conclusions : Although there have been reports of P300 in the past, its abnormalities reflect the deficit of attention in Parkinson's disease.

Objective We compared brain perfusion image using 3D-SSP analysis of 123I-IMP SPECT between Parkinson's disease patients with auditory verbal hallucination and those without auditory hallucination. Methods Eighty-three cases with Parkinson's disease were studied. In 6 of these patients, auditory hallucination was noted. Among them, four cases had verbal hallucination and two other cases had elementary hallucination. Auditory hallucination was not found in the other 77 cases. Results Right thalamic perfusion was significantly increased in the verbal hallucination group compared to the group that lacked auditory hallucination. Conclusion In Parkinson's disease, the right thalamic hyperactive state may be related to verbal hallucination.

The pathophysiology of depression and anxiety in Parkinson’s disease remains obscure. We aimed to compare the fractional anisotropy (FA) values of Parkinson’s disease (PD) patients with and without depression to investigate the nature of depression in PD. Twenty-eight patients were divided into two groups: those with depression and those without. Diagnosis of depression was made using the DSM-IV criteria. Patients in the two groups were matched for Hoehn Yahr stage. There were significant reductions in FA values in the bilateral frontal ROIs possibly representing anterior cingulate bundles. The anterior cingulate bundles play an important role in depression in PD, and some aspects of depression in PD have pathological processes in common with de novo depression.

Background Few imaging studies investigated frontal dysfunctions in Parkinson's disease. Objectives We investigated relationships between Wisconsin Card Sorting Test (WCST) and brain perfusion in patients with non-demented Parkinson's disease. Methods Patients were divided into two groups according to WCST score: (1) CA (number of category achieved)≤2 or (2) CA≥3. We performed three-dimensional stereotactic surface projection and volume of interest analysis using 123I-IMP scintigraphy. Results Hypoperfusions of the bilateral posterior cingulate, rostrodorsal prefrontal, and left frontopolar cortices were shown in CA≤2 group, with the left cingulate and right rostrodorsal prefrontal cortices showing prominent hypoperfusion. CA and PE (perseverative errors) scores significantly correlated with perfusion of the left posterior cingulate cortex. PE score also correlated with perfusion of the right rostrodorsal prefrontal cortex. Conclusion Data indicated participations of various regions in task achievement. Careful interpretation of WCST scores is required.

Background Although excessive daytime sleepiness (EDS) in Parkinson's disease (PD) has received a great deal of attention, the underlying pathological mechanism of its development and the relative contributions of brain function to this process are poorly understood. Objective To compare the fractional anisotropy (FA) values of PD patients with EDS and those without EDS, and to investigate the mechanism of EDS in this disease. Methods Eleven patients with PD with EDS, 26 patients with PD without EDS and 10 controls participated in this study. Patients with an ESS (Epworth Sleepiness Scale) score ≥10 were defined as having EDS. The FA values of 5 regions of interest (ROI) including the fornix were compared between the three groups. Results There were significant reductions in FA values of the fornix fiber in patients with EDS. ESS scores had significant correlation with FA values of the fornix. Conclusions Our study is the first to find the fornix fiber degeneration in PD patients with EDS. These results indicate that fornix dysfunction may have some correlations with EDS in PD.

Pathophysiology of hallucinations in Parkinson’s disease is poorly understood. This study investigated relationships between visual hallucinations and visual acuity. Twenty-six consecutive patients with Parkinson’s disease participated in this study. Patients were divided into two groups: patients with visual hallucinations (VH group) and those without visual hallucinations (no-VH group). Unaided and corrected eyesight was evaluated in all patients, and if frequent use of prescription glasses or contact lenses was involved, eyesight using these lenses was also measured as the patient’s own best eyesight. If a patient did not use prescription glasses or contact lenses, the patient’s own best eyesight was defined as the unaided eyesight. Multivariate regression analysis demonstrated that agonist use and best eyesight were different after the backward elimination method. Visual hallucinations were closely related not to uncorrected eyesight or unaided eyesight but to the patient’s best eyesight. It is suggested that impaired visual acuity is a risk factor for visual hallucinations.

The red nucleus is located in the rostral midbrain of the vertebrate brain and controls motor coordination during locomotion. It receives input from the cerebellum and sends its output to the spinal cord. The presence of the red nucleus is well established in tetrapods, and its existence has also been suggested in teleosts but its presence and position has still been under discussion. By using wheat germ agglutinin (WGA) as a genetically encoded anterograde tracer, we recently identified contralateral projections from the cerebellum to a putative red nucleus in the zebrafish midbrain tegmentum. In this report we further revealed red nucleus derived from this contralateral afferent from the cerebellum using WGA and contralateral projections to the hindbrain-spinal cord junction site using DiI-mediated retrograde tracing. Thus the structure that we have identified by anterograde and retrograde tracing fulfills the anatomical demands for the red nucleus: the location in the midbrain tegmentum, contralateral afferent from the cerebellum (cerebello- ruber projection) and contralateral efferent to the spinal cord (rubro-spinal projection).

Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function.We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response.The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

Objective – To compare brain perfusion image using three-dimensional stereotactic surface projection (3D-SSP) analysis of N-isopropyl-p-123I iodoamphetamine (123I-IMP) single photon emission computed tomography (SPECT) between patients with Parkinson's disease with orthostatic hypotension and those without orthostatic hypotension. Materials and methods – Fifteen patients with Parkinson's disease and orthostatic hypotension and 13 patients with Parkinson's disease without orthostatic hypotension were studied. We compared brain perfusion image between the two groups by 3D-SSP. Results – Bilateral anterior cingulate gyrus perfusion of the patients with orthostatic hypotension was significantly decreased compared to that of the patients without orthostatic hypotension. Conclusions – The disorder of anterior cingulate gyrus may participate in the autonomic failure in Parkinson's disease.

Background: Decreased cardiac metaiodobenzylguanidine (MIBG) uptake was reported in Parkinson’s disease and this contributes to the differential diagnosis between Parkinson’s disease and other forms of parkinsonism such as multiple system atrophy. However, decreased MIBG uptake of the thyroid has not been demonstrated. Objective: To compare MIBG uptake of the thyroid among Parkinson’s disease, multiple system atrophy and controls. Methods: Twenty-six patients with Parkinson’s disease, 11 patients with multiple system atrophy and 14 controls were examined in this study. Planar images were taken 15 minutes (early images) and 3 hours (late images) after intravenous injection of 111 MBq 123 I-MIBG. Results: MIBG uptake of the thyroid on early images decreased significantly in Parkinson’s disease compared to controls (p < 0.0001) and multiple system atrophy (p = 0.018). MIBG uptake of the thyroid on early images decreased significantly also in multiple system atrophy compared to controls (p = 0.027). On late images, thyroid uptake differed significantly only between Parkinson’s disease and controls (p = 0.010). Conclusions: Our study is the first to demonstrate decreased MIBG uptake of the thyroid in Parkinson’s disease. Sympathetic nervous denervation of Parkinson’s disease occurred not only in the heart but also in the thyroid.