A rapid and bidirectional reporter of neural activity reveals neural correlates of social behaviors in Drosophila.

Neural activity is modulated over different timescales encompassing subseconds to hours, reflecting changes in external environment, internal state and behavior. Using Drosophila as a model, we developed a rapid and bidirectional reporter that provides a cellular readout of recent neural activity. This reporter uses nuclear versus cytoplasmic distribution of CREB-regulated transcriptional co-activator (CRTC). Subcellular distribution of GFP-tagged CRTC (CRTC::GFP) bidirectionally changes on the order of minutes and reflects both increases and decreases in neural activity. We established an automated machine-learning-based routine for efficient quantification of reporter signal. Using this reporter, we demonstrate mating-evoked activation and inactivation of modulatory neurons. We further investigated the functional role of the master courtship regulator gene fruitless (fru) and show that fru is necessary to ensure activation of male arousal neurons by female cues. Together, our results establish CRTC::GFP as a bidirectional reporter of recent neural activity suitable for examining neural correlates in behavioral contexts.

A neural theory for counting memories.

Keeping track of the number of times different stimuli have been experienced is a critical computation for behavior. Here, we propose a theoretical two-layer neural circuit that stores counts of stimulus occurrence frequencies. This circuit implements a data structure, called a count sketch, that is commonly used in computer science to maintain item frequencies in streaming data. Our first model implements a count sketch using Hebbian synapses and outputs stimulus-specific frequencies. Our second model uses anti-Hebbian plasticity and only tracks frequencies within four count categories ("1-2-3-many"), which trades-off the number of categories that need to be distinguished with the potential ethological value of those categories. We show how both models can robustly track stimulus occurrence frequencies, thus expanding the traditional novelty-familiarity memory axis from binary to discrete with more than two possible values. Finally, we show that an implementation of the "1-2-3-many" count sketch exists in the insect mushroom body.

A Case of Methimazole-Induced Acute Pancreatitis With an HLA Allele Causing Antithyroid Drug-Induced Agranulocytosis.

Among the side effects of methimazole (MMI) for the treatment of Graves' disease, MMI-induced acute pancreatitis (MIP) is a rare adverse reaction, with only 7 cases being reported to date. However, 2 large-scale population-based studies recently revealed that the risk of MIP was significantly higher, ranging from 0.02% to 0.56%. Although MIP is common in middle-aged and elderly Asian women, its pathogenesis remains largely unknown. We herein present a case of a 72-year-old Japanese woman with Graves' disease who developed MIP 12 days after the initiation of MMI. The MMI was discontinued, the patient was switched to propylthiouracil (PTU) therapy, and pancreatitis gradually resolved. Serological human leukocyte antigen (HLA) typing identified HLA-DRB1*08:03:02. This HLA allele was previously detected in a patient with MIP and is one of the major risk factors for agranulocytosis induced by antithyroid drugs, including PTU as well as MMI. In cases of MIP, PTU is being considered as an alternative to MMI; however, its safety needs further investigation and patients require close monitoring after the switch to PTU. Further studies are warranted, particularly on the relationship between MIP and the presence of HLA alleles causing antithyroid drug-induced agranulocytosis.

Leiomyosarcoma of the splenic vein with a history of bilateral retinoblastoma: A case report and review of the literature.

Diagnosing leiomyosarcomas of the splenic vein is challenging, and a treatment strategy has not yet been established for this condition. We herein report the case of a 45-year-old female patient with a history of retinoblastoma who underwent pancreatosplenectomy for a primary leiomyosarcoma originating from the splenic vein and transcatheter arterial chemoembolization for metastatic hepatic lesions observed 5 months postoperatively. An initial medical check-up using abdominal ultrasound revealed a 40-mm mass behind the pancreatic tail. Imaging tests revealed a well-circumscribed mass compressing the pancreas posteriorly, without invasion into the pancreatic duct. The splenic vein was torn, with the epiploic veins developed as collateral blood vessels, which was an atypical finding for carcinoma. The patient was followed up regularly. At 5 years after the first visit, an abdominal ultrasound showed that the mass had increased in size to 50 mm, and had developed into a tumor embolus within the splenic vein, causing an obstruction of the vessel. Pancreatosplenectomy was performed due to suspicion of malignancy. The final diagnosis was leiomyosarcoma arising from the splenic vein. The patient was discharged on postoperative day 15 and was followed up regularly thereafter. Imaging studies performed 5 months postoperatively revealed four hepatic lesions. The hepatic masses were histologically diagnosed as metastatic leiomyosarcomas, and transcatheter arterial chemoembolization was performed using epirubicin. Since then, regular follow-ups have been conducted without observed recurrence. Leiomyosarcoma of the splenic vein is exceedingly rare, and the number of reported cases is not sufficient to establish clinical guidelines. Therefore, it is crucial to collect more reports on the occurrence and treatment of this disease.

Solid pseudopapillary neoplasm of the pancreas in a young male with main pancreatic duct dilatation: A case report.

BACKGROUND: Solid pseudopapillary neoplasms (SPNs) are rare tumors of the pancreas. Typically, they occur in young females, often have characteristic imaging features, such as cystic components and calcification, and have few effects on the pancreatic duct. CASE SUMMARY: A 31-year-old man was admitted to our hospital with the chief complaint of epigastric pain. There was only mild tenderness in his upper abdomen, and blood tests showed only a slight increase in alkaline phosphatase. Contrast-enhanced computed tomography showed a 40-mm-diameter, hypovascular mass in the head of the pancreas, and the main pancreatic duct upstream of the mass was severely dilated. Magnetic resonance imaging showed low intensity on T1-weighted images, with high intensity on T2-weighted image in some parts. Pancreatic ductal adenocarcinoma was the primary differential diagnosis. Portal vein infiltration could not be ruled out, so this case was a candidate for neoadjuvant chemotherapy. Subsequently, endoscopic ultrasound-guided fine needle aspiration was performed, and pathological evaluation and immunostaining suggested a diagnosis of SPN. Thus, pancreatoduodenectomy was performed. One year after the operation, the patient is alive with no recurrence. CONCLUSION: Main pancreatic duct dilatation is usually a finding of suspected pancreatic cancer. However, pancreatic duct dilatation can occur in SPN depending on the location and growth speed. Therefore, SPN should be considered in the differential diagnosis of tumors with pancreatic duct dilatation, and pathological evaluation by endoscopic ultrasound-guided fine needle aspiration should be actively performed.

Pancreatic ductal adenocarcinoma with mismatch repair deficiency resected after long-term observation.

Pancreatic ductal adenocarcinoma (PDAC) with mismatch repair (MMR) deficiency is a rare subtype, clinicopathological features of which have not been fully understood. A 70-year-old woman was admitted for the investigation of a 20-mm pancreatic tumor in the pancreatic head, detected during the cause scrutiny of exacerbation of diabetes mellitus and panhypopituitarism. The tumor decreased in size after administration of hydrocortisone for panhypopituitarism. Autoimmune pancreatitis, complicated with hypophysitis, was suspected, and prednisolone treatment was administered. The tumor did not show enlargement for 3 years during which a dose of prednisolone was maintained. However, 1.5 years after the cessation of prednisolone administration, the tumor size increased again. On endoscopic ultrasonography, the tumor was found to be a 25.2-mm mass lesion with almost uniformly low echogenicity and blood flow signal, and anisonucleosis on cytodiagnosis was revealed. Pancreatoduodenectomy was performed, and on histological analysis, moderately differentiated tubular adenocarcinoma with massive lymphocytic infiltration was observed. Immunohistochemistry revealed a concomitant loss of MSH2 and MSH6 in the tumor cells, which implicated mutant MSH2 gene. She has remained well with no recurrence for 2.9 years since her surgery. We herein report a case of PDAC with MMR deficiency, resected after long-term observation.

Representations of Novelty and Familiarity in a Mushroom Body Compartment.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.

The neuronal architecture of the mushroom body provides a logic for associative learning.

We identified the neurons comprising the Drosophila mushroom body (MB), an associative center in invertebrate brains, and provide a comprehensive map describing their potential connections. Each of the 21 MB output neuron (MBON) types elaborates segregated dendritic arbors along the parallel axons of ∼2000 Kenyon cells, forming 15 compartments that collectively tile the MB lobes. MBON axons project to five discrete neuropils outside of the MB and three MBON types form a feedforward network in the lobes. Each of the 20 dopaminergic neuron (DAN) types projects axons to one, or at most two, of the MBON compartments. Convergence of DAN axons on compartmentalized Kenyon cell-MBON synapses creates a highly ordered unit that can support learning to impose valence on sensory representations. The elucidation of the complement of neurons of the MB provides a comprehensive anatomical substrate from which one can infer a functional logic of associative olfactory learning and memory.

An isothermal absorptiometric assay for viable microbes using the redox color indicator 2,6-dichlorophenolindophenol.

A simple and rapid isothermal absorptiometric assay for detection of viable microbes using the redox color indicator 2,6-dichlorophenolindophenol (DCPIP) was studied. The absorbance of DCPIP decreased at 600 nm because of a redox reaction occurring between DCPIP and the surface membrane of viable microbes and was inversely proportional to the viable cell density. The redox reaction was found not only with bacteria, but also with yeast and a mixture of bacteria and yeast. In this assay, the influence of light scattering and absorption caused by microbial cells and coexisting substances in the sample was excluded by a time difference method. The assay required only 10 min for one incubation mixture, and highly repeatable results from three consecutive measurements were obtained by isothermal incubation for specific times at 30 °C using a thermostable three-cuvette-stir system. Thus, the cell density of microbial cell suspensions or growth medium was successfully determined, and a practical lower detection limit for food inspection was obtained at 104-106 cfu/ml. Single-cell effects on DCPIP reduction were evaluated and compared between species. Consequently, this assay is expected to be a useful tool for the rapid measurement of viable microbes as a preliminary assay for the Hazard Analysis Critical Control Point program.

Robust discrimination between self and non-self neurites requires thousands of Dscam1 isoforms.

Down Syndrome cell adhesion molecule (Dscam) genes encode neuronal cell recognition proteins of the immunoglobulin superfamily. In Drosophila, Dscam1 generates 19,008 different ectodomains by alternative splicing of three exon clusters, each encoding half or a complete variable immunoglobulin domain. Identical isoforms bind to each other, but rarely to isoforms differing at any one of the variable immunoglobulin domains. Binding between isoforms on opposing membranes promotes repulsion. Isoform diversity provides the molecular basis for neurite self-avoidance. Self-avoidance refers to the tendency of branches from the same neuron (self-branches) to selectively avoid one another. To ensure that repulsion is restricted to self-branches, different neurons express different sets of isoforms in a biased stochastic fashion. Genetic studies demonstrated that Dscam1 diversity has a profound role in wiring the fly brain. Here we show how many isoforms are required to provide an identification system that prevents non-self branches from inappropriately recognizing each other. Using homologous recombination, we generated mutant animals encoding 12, 24, 576 and 1,152 potential isoforms. Mutant animals with deletions encoding 4,752 and 14,256 isoforms were also analysed. Branching phenotypes were assessed in three classes of neurons. Branching patterns improved as the potential number of isoforms increased, and this was independent of the identity of the isoforms. Although branching defects in animals with 1,152 potential isoforms remained substantial, animals with 4,752 isoforms were indistinguishable from wild-type controls. Mathematical modelling studies were consistent with the experimental results that thousands of isoforms are necessary to ensure acquisition of unique Dscam1 identities in many neurons. We conclude that thousands of isoforms are essential to provide neurons with a robust discrimination mechanism to distinguish between self and non-self during self-avoidance.

Dscam-mediated cell recognition regulates neural circuit formation.

The Dscam family of immunoglobulin cell surface proteins mediates recognition events between neurons that play an essential role in the establishment of neural circuits. The Drosophila Dscam1 locus encodes tens of thousands of cell surface proteins via alternative splicing. These isoforms exhibit exquisite isoform-specific binding in vitro that mediates homophilic repulsion in vivo. These properties provide the molecular basis for self-avoidance, an essential developmental mechanism that allows axonal and dendritic processes to uniformly cover their synaptic fields. In a mechanistically similar fashion, homophilic repulsion mediated by Drosophila Dscam2 prevents processes from the same class of cells from occupying overlapping synaptic fields through a process called tiling. Genetic studies in the mouse visual system support the view that vertebrate DSCAM also promotes both self-avoidance and tiling. By contrast, DSCAM and DSCAM-L promote layer-specific targeting in the chick visual system, presumably through promoting homophilic adhesion. The fly and mouse studies underscore the importance of homophilic repulsion in regulating neural circuit assembly, whereas the chick studies suggest that DSCAM proteins may mediate a variety of different recognition events during wiring in a context-dependent fashion.

Absorption-based highly sensitive and reproducible biochemical oxygen demand measurement method for seawater using salt-tolerant yeast Saccharomyces cerevisiae ARIF KD-003.

Salt-tolerant yeast Saccharomyces cerevisiae ARIF KD-003 was applied to highly sensitive and reproducible absorbance-based biochemical oxygen demand (BOD(AB-ScII)) measurement for seawater. In the previous work, we have studied the BOD(AB-ScI) method using normal Baker's yeast S. cerevisiae, and the excellent feature of the Baker's yeast as uniformly sustainable in solution could successfully be utilized. However, the BOD(AB-ScI) responses were disappeared by the existence of chloride ion as well as seawater. In the present method, uniformity in solution was also observed with S. cerevisiae ARIF KD-003, and salt-tolerance of the yeast was observed even in saturate concentration of sodium chloride. Next, characterizations of the influences of pH and incubation temperature were investigated. After optimum conditions were obtained, two calibration curves were made between 0.33 and 22 mg O2 L(-1) BOD using standard solution of glucose glutamic acid (GGA) or mixture of GGA and artificial seawater. Then, excellent reproducibility as the averages of relative standard deviation (R.S.D.(av)) in two calibration curves (nine points each) was successfully obtained at 1.10% at pure water or 1.03% at artificial seawater standard, respectively. In addition, the 3 sigma lower detection limit was calculated to be 0.07 mg O2 L(-1) BOD, and 0.11 mg O2 L(-1) BOD was experimentally detected by increase of the sample volume at 1.5-folds. The storage stability of the S. cerevisiae ARIF KD-003 was obtained at least 4 weeks.

Dscam diversity is essential for neuronal wiring and self-recognition.

Neurons are thought to use diverse families of cell-surface molecules for cell recognition during circuit assembly. In Drosophila, alternative splicing of the Down syndrome cell adhesion molecule (Dscam) gene potentially generates 38,016 closely related transmembrane proteins of the immunoglobulin superfamily, each comprising one of 19,008 alternative ectodomains linked to one of two alternative transmembrane segments. These ectodomains show isoform-specific homophilic binding, leading to speculation that Dscam proteins mediate cell recognition. Genetic studies have established that Dscam is required for neural circuit assembly, but the extent to which isoform diversity contributes to this process is not known. Here we provide conclusive evidence that Dscam diversity is essential for circuit assembly. Using homologous recombination, we reduced the entire repertoire of Dscam ectodomains to just a single isoform. Neural circuits in these mutants are severely disorganized. Furthermore, we show that it is crucial for neighbouring neurons to express distinct isoforms, but that the specific identity of the isoforms expressed in an individual neuron is unimportant. We conclude that Dscam diversity provides each neuron with a unique identity by which it can distinguish its own processes from those of other neurons, and that this self-recognition is essential for wiring the Drosophila brain.

Dendrite self-avoidance is controlled by Dscam.

Dendrites distinguish between sister branches and those of other cells. Self-recognition can often lead to repulsion, a process termed "self-avoidance." Here we demonstrate that dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus. By alternative splicing, Dscam encodes a vast number of cell-surface proteins of the immunoglobulin superfamily. We demonstrate that interactions between identical Dscam isoforms on the cell surface underlie self-recognition, while the cytoplasmic tail converts this recognition to dendrite repulsion. Sister dendrites expressing the same isoforms engage in homophilic repulsion. By contrast, Dscam diversity ensures that inappropriate repulsive interactions between dendrites sharing the same receptive field do not occur. The selectivity of Dscam-mediated cell interactions is likely to be widely important in the developing fly nervous system, where processes of cells must distinguish between self and nonself during the construction of neural circuits.

Got diversity? Wiring the fly brain with Dscam.

The Drosophila gene Dscam, encoding Down syndrome cell-adhesion molecule, is required for the development of neural circuits. Alternative splicing of Dscam mRNA potentially generates 38016 isoforms of a cell-surface recognition protein of the immunoglobulin superfamily. These isoforms include 19008 different ectodomains joined to one of two alternative transmembrane segments. Each ectodomain comprises a unique combination of three variable immunoglobulin domains. Biochemical studies support a model in which each isoform preferentially binds to the same isoform on opposing cell surfaces. This homophilic binding requires matching at all three variable immunoglobulin domains. These findings raise the intriguing possibility that specificity of binding by the Dscam isoforms mediates cell-surface recognition events required for wiring the fly brain.

DNA methylation controls the timing of astrogliogenesis through regulation of JAK-STAT signaling.

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. We report here that conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. Our study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation.

Identification of target genes for the Xenopus Hes-related protein XHR1, a prepattern factor specifying the midbrain-hindbrain boundary.

The midbrain-hindbrain boundary (MHB) acts as a local organizer in the development of the CNS in vertebrates. Previously, we identified an MHB-specific bHLH-WRPW transcriptional repressor gene, Xenopus Hes-related 1 (XHR1), which is initially expressed in the presumptive MHB (pre-MHB) region at the early gastrula stage. To better understand the gene cascades involved in MHB formation, we investigated the genes downstream from XHR1 by differential screening using a Xenopus cDNA macroarray and a dexamethasone (DEX)-inducible, dominant-negative transcriptional activator construct of XHR1 (XHR1-VP16-GR). Among the newly identified candidate target genes of XHR1 were Enhancer of split-related genes (ESR1, ESR3/7, and ESR9) and Xenopus laevis cleavage 2 (XLCL2). XHR1-VP16-GR induced the expression of the ESR genes and XLCL2 as well as Xdelta1, Xngnr1, and XHR1 itself in the presence of DEX even after pretreatment with the protein synthesis inhibitor, cycloheximide. This suggests that these genes are direct targets of XHR1. XHR1-knockdown experiments with antisense morpholino oligos and ectopic expression of wild-type XHR1 revealed that XHR1 is necessary and sufficient to repress ESR genes in the pre-MHB region. Misexpression of the ESR genes in the pre-MHB region repressed the MHB marker gene, Pax2, suggesting that the repression of the ESR genes by XHR1 is at least partly required for the early development of the pre-MHB. Our data also show that XHR1 is not activated by Notch signaling, differing from ESR genes. Taken together, we propose a model in which XHR1 defines the pre-MHB region as a prepattern gene by repressing those possible direct target genes.

Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies.

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.