Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Alternative exon skipping biases substrate preference of the deubiquitylase USP15 for mysterin/RNF213, the moyamoya disease susceptibility factor.

The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-ß signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping.

Neuromuscular regulation in zebrafish by a large AAA+ ATPase/ubiquitin ligase, mysterin/RNF213.

Mysterin (also known as RNF213) is a huge intracellular protein with two AAA+ ATPase modules and a RING finger ubiquitin ligase domain. Mysterin was originally isolated as a significant risk factor for the cryptogenic cerebrovascular disorder moyamoya disease, and was found to be involved in physiological angiogenesis in zebrafish. However, the function and the physiological significance of mysterin in other than blood vessels remain largely unknown, although mysterin is ubiquitously expressed in animal tissues. In this study, we performed antisense-mediated suppression of a mysterin orthologue in zebrafish larvae and revealed that mysterin-deficient larvae showed significant reduction in fast myofibrils and immature projection of primary motoneurons, leading to severe motor deficits. Fast muscle-specific restoration of mysterin expression cancelled these phenotypes, and interestingly both AAA+ ATPase and ubiquitin ligase activities of mysterin were indispensable for proper fast muscle formation, demonstrating an essential role of mysterin and its enzymatic activities in the neuromuscular regulation in zebrafish.

Moyamoya disease-associated protein mysterin/RNF213 is a novel AAA+ ATPase, which dynamically changes its oligomeric state.

Moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. We recently identified mysterin/RNF213 as its first susceptibility gene, which encodes a 591-kDa protein containing enzymatically active P-loop ATPase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. Here we demonstrate that mysterin further contains two tandem AAA+ ATPase modules and forms huge ring-shaped oligomeric complex. AAA+ ATPases are known to generally mediate various biophysical and mechanical processes with the characteristic ring-shaped structure. Fluorescence correlation spectroscopy and biochemical evaluation suggested that mysterin dynamically changes its oligomeric forms through ATP/ADP binding and hydrolysis cycles. Thus, the moyamoya disease-associated gene product is a unique protein that functions as ubiquitin ligase and AAA+ ATPase, which possibly contributes to vascular development through mechanical processes in the cell.

Effects of preparation methods for multi-wall carbon nanotube (MWCNT) suspensions on MWCNT induced rat pulmonary toxicity.

Since there is a possibility of inhaling the fibers of multi-wall carbon nanotube (MWCNT) without any agglomeration, it is important that the pulmonary toxicity is evaluated by intratracheal instillation without agglomeration. MWCNT suspended in an artificial lung surfactant (ALS) with or without grinding in an agate mortar was instilled once intratracheally to rats to determine whether differences of the effects to pulmonary toxicity by different amounts of agglomerated MWCNT particle. The MWCNT suspension preparation method with grinding was effective at reducing agglomerates and in increasing uniform dispersion of the fibers. The ground MWCNT induced higher LDH levels and neutrophil ratios in the bronchoalveolar lavage fluid (BALF). There were no remarkable responses in rats in the non-ground MWCNT group, with the exception of inflammatory responses in the early phase. Some histopathological findings varied between rats given the ground MWCNT and non-ground MWCNT. A major difference was an MWCNT-laden macrophage infiltration site in the lung, which were in the alveolus in the ground MWCNT group, and in the interstitium in non-ground MWCNT group. Accordingly, the preparation method with grinding is considered to be effective at reducing agglomerates and ensuring uniform dispersion of the fibers. These findings lead us to conclude that the amount of agglomerates in the suspension is an important factor affecting the pulmonary toxicity of MWCNT.

Malignant Mesothelioma in the Thoracic Cavity of a Crj:CD(SD) Rat Characterized by Round Hyalinous Stroma.

Spontaneous malignant mesothelioma was found in a 104-week-old male Crj:CD(SD) rat. The tumor was scattered on the surface of the lung, heart, mediastinal pleura and thoracic wall and metastasized to the alveolar septa. Histopathologically, small flattened or cuboidal tumor cells proliferated with stroma, formed almost normal papillary structures and reacted positively to colloidal iron stain and immunohistochemical staining for mesothelin. Round hyalinous stromata were pronounced, which is a characteristic feature, and the possible reason for this is as follows; at first, a small amount of collagen fibers was formed in the center of the clusters of several tumor cells, and then the cell clusters expanded like balloons with an increase in the collagen fibers.

Mixed Thymoma in a Young Cynomolgus Monkey (Macaca fascicularis).

A mass with a diameter of 1.5 cm was found in the thymus of a 4-year and 3-month-old male cynomolgus monkey. Microscopically, the mass consisted of two different patterns of proliferation, dense or fascicular proliferation of elongated spindle cells in a sporadic storiform pattern and dense proliferation of thymic cortex-like lymphoid cells in which the multifocal pale nests resembling the thymic medulla were distributed. In these pale nests, large dendriform cells sometimes forming Hassall's corpuscles were present. The proliferating spindle cells were positive for cytokeratin. The lymphoid cells in the mass were positive for CD3. We concluded that the mass consisted of the neoplastic thymic epithelium with thymocytes proliferation containing medullary differentiation. The mass was diagnosed as a mixed thymoma according to the WHO classification of thymomas in humans. Mixed thymoma is characterized as a mixture of two types of proliferative lesions, spindle-shaped epithelial proliferation and a lymphocyte predominant lesion with or without medullary differentiation. To the best of our knowledge, this is the first report concerning thymoma in monkeys.

Deposition process of eosinophilic substance in mouse nasal septum.

An eosinophilic substance is usually observed in the mouse nasal septum, and its volume increases with age. In contrast to descriptions in textbooks defining the eosinophilic substance as amyloid, our previous report revealed that the observed eosinophilic substance is not amyloid, but consisted of collagen and an amorphous material. Furthermore, it was suggested that the amorphous material was produced by the clear hematoxylin and eosin (HE)-stained nasal gland epithelial cells. In this study, we investigated the deposition process of the amorphous material produced by nasal gland epithelial cells in the interstitium morphologically. In most cases, the amorphous materials in the clear HE-stained nasal gland epithelial cells accumulated at the basal portion. Collagen fibers surrounding the nasal glands partially disappeared, whereas the amorphous material in contact with the rough endoplasmic reticulum of the nasal gland epithelial cells continued to the amorphous material in the interstitium. These findings suggested that the amorphous material produced by the clear HE-stained nasal gland epithelial cells migrated to the interstitium through the partial opening of the basement membrane.

Experimental induction of amyloidosis by bovine amyloid fibrils in Sore Hock rabbits.

We report the experimental amyloidosis associated with administration of bovine amyloid fibrils in rabbits afflicted by Sore Hock (SH), which is ulcerative pododermatitis. Two groups of SH-afflicted rabbits were subjected to five inflammatory stimulations at intervals of 4 days by intraepithelial injection of a mixture consisting of Freund's complete adjuvant and lipopolysaccharide. One group of rabbits was administered amyloid in conjunction with the last inflammatory stimulation and the other group was not. For additional control, two groups were designed. A third group consisted of rabbits without SH, which were subjected to five stimulations and were administered amyloid. A fourth group consisted of SH-afflicted rabbits, subjected to 0-4 stimulations and administered amyloid. Amyloid depositions were observed in SH-afflicted rabbits, which had been stimulated five times and given amyloid (18/18). In the 4th group, only one rabbit, which had been subjected to four stimulations, showed amyloid depositions. No amyloid depositions were observed in the other rabbits. These results suggest that bovine AA amyloid fibrils have an amyloid-enhancing factor-like effect on SH-afflicted rabbits.

Immunohistochemical distribution of amyloid deposits in 25 cows diagnosed with systemic AA amyloidosis.

The distribution of amyloid deposits was histopathologically and immunohistochemically examined in 25 cows aged 5 to 10 years that had been diagnosed with systemic AA amyloidosis. This examination revealed that amyloid deposits were also present in the hypophysis, ovary, uterus, mammary gland and skeletal muscle, in addition to the liver, kidney, spleen, pancreas, thyroid gland, adrenal gland, gastrointestinal mucosa, heart, lung and lymph nodes. The examined cows tended to have chronic inflammations, including chronic mastitis (six cases) or chronic pneumonia (four cases), which is thought of as a causative agent of AA amyloidosis. In contrast, five cases did not exhibit any chronic inflammation.