Systems neuroscience in Drosophila: Conceptual and technical advantages.

The fruit fly Drosophila melanogaster is ideally suited for investigating the neural circuit basis of behavior. Due to the simplicity and genetic tractability of the fly brain, neurons and circuits are identifiable across animals. Additionally, a large set of transgenic lines has been developed with the aim of specifically labeling small subsets of neurons and manipulating them in sophisticated ways. Electrophysiology and imaging can be applied in behaving individuals to examine the computations performed by each neuron, and even the entire population of relevant neurons in a particular region, because of the small size of the brain. Moreover, a rich repertoire of behaviors that can be studied is expanding to include those requiring cognitive abilities. Thus, the fly brain is an attractive system in which to explore both computations and mechanisms underlying behavior at levels spanning from genes through neurons to circuits. This review summarizes the advantages Drosophila offers in achieving this objective. A recent neurophysiology study on olfactory behavior is also introduced to demonstrate the effectiveness of these advantages.

Innervation and activity dependent dynamics of postsynaptic oxidative metabolism.

Despite extensive investigations into the mechanisms of aerobic respiration in mitochondria, the spontaneous metabolic activity of individual cells within a whole animal has not been observed in real time. Consequently, little is known about whether and how the level of mitochondrial energy metabolism is regulated in a cell during development of intact systems. Here we studied the dynamics of postsynaptic oxidative metabolism by monitoring the redox state of mitochondrial flavoproteins, an established indicator of energy metabolism, at the developing Drosophila neuromuscular junction. We detected transient and spatially synchronized flavoprotein autofluorescence signals in postsynaptic muscle cells. These signals were dependent on the energy substrates and coupled to changes in mitochondrial membrane potential and Ca2+ concentration. Notably, the rate of autofluorescence signals increased during synapse formation through contact with the motoneuronal axon. This rate was also influenced by the magnitude of synaptic inputs. Thus, presynaptic cells tightly regulate postsynaptic energy metabolism presumably to maintain an energetic balance during neuromuscular synaptogenesis. Our results suggest that flavoprotein autofluorescence imaging should allow us to begin assessing the progress of synapse formation from a metabolic perspective.

Synaptic components necessary for retrograde signaling triggered by calcium/calmodulin-dependent protein kinase II during synaptogenesis.

The development and function of presynaptic terminals are tightly controlled by retrograde factors presented from postsynaptic cells. However, it remains elusive whether major constituents of synapses themselves are necessary for retrograde modulation during synaptogenesis. Here we show that the homophilic cell adhesion molecule Fasciclin II (FasII) as well as the scaffolding protein Discs large (DLG) is indispensable for retrograde signaling initiated by calcium/calmodulin-dependent protein kinase II (CaMKII) at developing Drosophila neuromuscular junctions. Postsynaptic activation of CaMKII increased the area of nerve terminals, the number of active zones, and the frequency of miniature excitatory synaptic currents in wild-type animals. However, all of these retrograde effects were abolished in the fasII or dlg mutant background. On the other hand, the retrograde effects remained in null mutants of the glutamate receptor subunit GluRIIA. Furthermore, we show that CaMKII-induced modulation was independent of the bone morphogenetic protein signaling that is important for retrograde control at mature larvae. These results highlight a novel function of FasII as well as DLG, and more broadly, illustrate that prime synaptic components are necessary for transferring target-derived signals to presynaptic cells at a certain developing synapse.

Developmental stage-dependent modulation of synapses by postsynaptic expression of activated calcium/calmodulin-dependent protein kinase II.

In Drosophila neuromuscular junctions, there is a unique system which consists of two neighboring muscles (M6 and M7) innervated by the same neurons and a gene of interest can be expressed in only M6 or in both muscles by GAL4-upstream activating sequence expression system. By using this system, we previously demonstrated that expression of activated calcium/calmodulin-dependent protein kinase II (CaMKII) in the muscle cell promotes coordinated maturation of pre- and postsynaptic sites of larvae just after hatching (JAH larvae) in a synapse-specific manner. Here we show that the promotive effects are no longer seen in the older larvae, 8-10 h after hatching (8 h AH larvae). Morphological studies indicate that CaMKII activation in fact reduces postsynaptic sites at 8 h AH. This is opposite to the effect observed in JAH larvae. These results suggest that the mode of CaMKII function switches during development, and that regulation of postsynaptic CaMKII activity is necessary for normal synaptic development. Finally, we report that in 8 h AH but not in JAH larvae, synapses on M7, in which CaMKII activity is not manipulated, are affected by the expression of activated CaMKII in M6. This suggests the interesting possibility that at certain developmental stages only, modification of synapses on one target cell can influence the synapses on another target cell innervated by the same neurons.

Postsynaptic activation of calcium/calmodulin-dependent protein kinase II promotes coordinated pre- and postsynaptic maturation of Drosophila neuromuscular junctions.

The interaction between a neuron and its target cell(s) is essential for the development of synapses. To elucidate the role of target cells in synaptogenesis, the activity of postsynaptic calcium/calmodulin-dependent protein kinase II (CaMKII) was manipulated in a mosaic manner and its specific effect was examined at the developing Drosophila neuromuscular junction. We found that postsynaptic expression of constitutively active CaMKII augmented the amplitude of excitatory synaptic currents (ESCs) and the frequency of miniature ESCs. It also promoted morphological maturation of presynaptic as well as postsynaptic specializations, which presumably underlie the enhancement of synaptic activities. Expression of an inhibitory peptide of CaMKII in the postsynaptic cell partially affected the synaptic maturation. These results suggest two significant functions of postsynaptic CaMKII in synaptogenesis-retrograde modulation of presynaptic properties and coordinated regulation of pre- and postsynaptic maturation.

Determination of residual double bonds in resin-dentin interface by Raman spectroscopy.

OBJECTIVES: The quality of the hybrid layer is believed to be more important than the thickness of this layer. The purpose of this study was to investigate a method to analyze the percentage of adhesive resin residual double bonds in the dentin-resin interface using laser Raman spectroscopy. METHODS: Bovine dentin was treated with dentin adhesives and resin composite was bonded according to the manufacturers' instructions. The specimens were sectioned parallel to dentinal tubules and the surfaces were then polished to 1 microm diamond pastes. Raman spectra were recorded along a line perpendicular to the dentin-resin interface in steps of 0.2 microm. The measurement of residual C=C bond was made on a relative basis by comparing the C=C unpolymerized methacrylate stretching vibration (1638 cm(-1)) against the C=O stretching mode of the ester group (1719 cm(-1)). The percentage of residual double bonds including pendant and monomeric double bonds was calculated by comparing the obtained ratio with that of uncured adhesive resin. RESULTS: The amount of residual double bonds in the hybrid layer varied from 10 to 25% compared to the uncured adhesives, a relatively higher percentage was detected for Fluoro Bond (12.3-23.6%) and Single Bond (9.5-21.8%), and lower for Mac Bond II (10.6-18.0%) and Mega Bond (10.7-16.3%). No relationship was seen between the percentage of remaining double bonds and the location within the resin-dentin interface. SIGNIFICANCE: Laser Raman microscopy used was a useful tool for measuring the residual double bonds in the dentin-resin interface.

Age-related changes in skin wrinkles assessed by a novel three-dimensional morphometric analysis.

BACKGROUND: A system has been developed whereby the morphology of the skin surface can be evaluated directly in three dimensions. This system employs a non-invasive device that utilizes white light of halogen origin, and which allows the computation of wrinkle depth and width, and other parameters of skin surface morphology. Using innovative engineering, an optical system has been devised so that light is transmitted via a slit and can be used to measure not only replicas of the skin but also the skin surface directly. The measurement area is 6.4 x 6.4 mm, and the theoretical resolution with a x 50 magnification lens is within 12.5 micro m. OBJECTIVES: To use this system to study age-related changes in the morphology of wrinkles at the eye corner areas of women of varying ages. METHODS: One hundred and one healthy women (age range 20-80 years) residing in the Tokyo area were the subjects used in this study. RESULTS: Wrinkles demonstrated a rapid increase in depth in women aged 40 years or older, and plateaued at the age of 60 years. Surface morphology parameters yielded results similar to those of age-related changes in wrinkles. CONCLUSIONS: This new analytical system provides a rapid and convenient non-invasive method to evaluate skin surface morphology in three dimensions, especially with respect to wrinkle formation. The results obtained using this system provide a deeper insight into the mechanistic relationship between wrinkles and skin elasticity.

Ex vivo whole-embryo culture of caspase-8-deficient embryos normalize their aberrant phenotypes in the developing neural tube and heart.

Caspase-8 plays the role of initiator in the caspase cascade and is a key molecule in death receptor-induced apoptotic pathways. To investigate the physiological roles of caspase-8 in vivo, we have generated caspase-8-deficient mice by gene targeting. The first signs of abnormality in homozygous mutant embryos were observed in extraembryonic tissue, the yolk sac. By embryonic day (E) 10.5, the yolk sac vasculature had begun to form inappropriately, and subsequently the mutant embryos displayed a variety of defects in the developing heart and neural tube. As a result, all mutant embryos died at E11.5. Importantly, homozygous mutant neural and heart defects were rescued by ex vivo whole-embryo culture during E10.5-E11.5, suggesting that these defects are most likely secondary to a lack of physiological caspase-8 activity. Taken together, these results suggest that caspase-8 is indispensable for embryonic development.

[Successful combined therapy with ATG, cyclosporin and G-CSF for both liver dysfunction and bone marrow failure in hepatitis-associated aplastic anemia].

A 28-year-old man developed cryptogenic hepatitis in January 1999, and treatment with glycyrrhizic acid improved his liver function. From June, however, pancytopenia began to develop gradually. The patient received G-CSF against leukocytopenia (WBC 1,100/microliter, neutrophils 590/microliter) and was transferred to our hospital in August 1999. A diagnosis of hepatitis-associated aplastic anemia was made on the basis of liver dysfunction (AST 156 IU/l, ALT 386 IU/l), hypoplastic bone marrow, and pancytopenia (WBC 4,400/microliter, neutrophils 3,340/microliter under G-CSF administration, Hb 9.8 g/dl, platelets 2.4 x 10(4)/microliter, reticulocytes 4.7 x 10(4)/microliter). Immediately after starting combined therapy with ATG, cyclosporin, and G-CSF, his liver function began to improve and was normalized on day 7. Pancytopenia began to ameliorate on day 9, and blood parameters on day 60 were WBC 4,200/microliter (without G-CSF administration), Hb 12.0 g/dl, platelets 9.0 x 10(4)/microliter, and reticulocytes 4.1 x 10(4)/microliter. Although the prognosis of hepatitis-associated aplastic anemia is generally poor, immunosuppressive therapy was markedly effective for both pancytopenia and hepatic dysfunction in the present case.

[Successful treatment of idiopathic pure red cell aplasia with antithymocyte globulin].

An 18-year-old woman was admitted to our hospital because of severe anemia on October 16, 1999. Laboratory data included hemoglobin 3.5 g/dl, reticulocytes 2,200/microliter, WBC 3,500/microliter, and Plt 38.5 x 10(4)/microliter. Bone marrow aspiration showed a normocellular marrow with severe erythroid hypoplasia, suggesting a diagnosis of pure red cell aplasia. Methylprednisolone pulse therapy was started on October 20, but there was no response. Administration of cyclosporine A (CyA; 400-450 mg) was begun on November 1, but again there was no response. Antithymocyte globulin (ATG; 800 mg/day for 5 days, 15 mg/kg) was started from December 1 in addition to prednisolone (60 mg/day) and CyA (450 mg/day). On day 7 of ATG therapy, the reticulocyte count began to increase, and reached a peak of 32.6 x 10(4)/microliter on day 20. The patient's hemoglobin level started to increase on day 13, and reached 8.5 g/dl on day 27. A complete response has been maintained up to the time of writing, and the hemoglobin level was 11.9 g/dl on December 14, 2000. This is the first detailed Japanese case report of successful treatment of pure red cell aplasia using ATG.

Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase.

By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.

Ecotropic viral integration site-1 is activated during, and is sufficient for, neuroectodermal P19 cell differentiation.

Expression of the ecotropic viral integration site-1 (Evi1) proto-oncogene during murine embryonal development is observed by in situ hybridization in primary head folds and neural crest-derived cells associated with the peripheral nervous system and embryonic mesoderm. To elucidate whether expression of Evi1 is involved in early neuroectodermal or mesodermal differentiation, we used murine embryonal carcinoma P19 cells as a model for the study of early embryonic differentiation. After retinoic acid (RA) treatment with aggregation, expression of Evi1 was detected during neural differentiation in P19 cells. However, Evi1 was not expressed in P19 cells during mesodermal differentiation after DMSO treatment with aggregation. Enforced expression of Evi1 in P19 cells induced neuron-specific microtubule-associated protein-2 microtubule-associated protein-2 and TrkA expression in the absence of RA under monolayer culture. After incubation with RA with aggregation, the Evi1 clones expressed microtubule-associated protein-2 continuously but did not express glial fibrillary acidic protein as an astrocyte marker protein until 12 days of culture. Thus, the overexpression of Evi1 leads to neural differentiation of P19 cells and blocks further differentiation into astrocytes by RA treatment, suggesting that Evi1 might be an important transcription factor for regulation of early neuroectodermal differentiation.

Characterization of the antiseptic-resistance gene qacE delta 1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1.

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.

Incomplete Sjögren-Larsson syndrome in two Japanese siblings.

Sjögren-Larsson syndrome (SLS) is a rare, autosomal recessive disorder characterized by ichthyosis, spastic diplegia and mental retardation. Biochemical studies have pinpointed the pathogenesis resulting in the deficiency of the fatty aldehyde dehydrogenase (FALDH) component of the fatty alcohol NAD+ oxidoreductase complex. Histochemical analysis revealed a reduction in alcohol dehydrogenase (AD) activity in the skin. SLS patients have been categorized biochemically into two groups: complete and incomplete reduction according to the degree of FALDH deficiency. Our patients demonstrated incomplete clinical features, including a 1/3 reduction in FALDH activity, and decreased AD activity in the ichthyotic lesion. The phenotypical differences between our cases and classic SLS are probably due to the partial FALDH deficiency.

Distribution of the antiseptic-resistance gene qacE delta 1 in gram-positive bacteria.

The distribution of the antiseptic-resistance genes qacE and qacE delta 1, originally isolated from Gram-negative bacteria, was studied in a large number of Gram-positive bacteria by a method that included the polymerase chain reaction. A total of 151 strains of Staphylococcus and Enterococcus, isolated from clinical sources and obtained from the Japanese Collection of Microorganisms, was used in this analysis. We found the qacE delta 1 gene in 36 of 103 strains of Staphylococcus and in nine of 48 strains of Enterococcus. All of the strains in which we detected the qacE delta 1 gene were clinical isolates. The qacE gene was not detected in any of the strains examined in this study. The nucleotide sequences of the qacE delta 1 genes from the strains of Staphylococcus and Enterococcus were identical to that of the gene located on integron InC in Pseudomonas aeruginosa. These results indicate that the antiseptic-resistance gene qacE delta 1 is present in Gram-positive, as well as Gram-negative, bacteria.

Isolation of an Escherichia coli mutant which permits the high-level expression of a streptomycin-resistance gene derived from Pseudomonas aeruginosa.

Pretreatment with streptomycin at a low concentration influenced the susceptibility to streptomycin of a strain of Escherichia coli carrying a streptomycin-resistance plasmid, pSA1700, derived from Pseudomonas aeruginosa. This phenomenon was due to a mutation that occurred at about 10(-8)-10(-10) of frequency in a regulatory gene involved in gene expression on the chromosome of E. coli. A product encoded by the regulatory gene on the chromosome of E. coli might normally repress gene expression by binding to part of the promoter region of the streptomycin-resistance gene derived from P. aeruginosa.

Distribution of the antiseptic-resistance genes qacE and qacE delta 1 in gram-negative bacteria.

The distribution of the antiseptic-resistance genes qacE and qacE delta 1 was studied in a large number of Gram-negative bacteria by a method that included the polymerase chain reaction (PCR). A total of 117 strains of Gram-negative bacteria, isolated from clinical or environmental sources, was used in this analysis. We demonstrated the presence of these genes in 48 of 78 strains of Pseudomonas, in 20 of 26 strains of Vibrio, and in four of 13 strains of other species. These results indicate that the antiseptic-resistance genes are present in a broad range of species of Gram-negative bacteria.