杉本 大樹

BACKGROUND: Cortical spreading depression is thought to be the underlying mechanism of migraine aura. In 2006, three relatives having the point mutation E700K in ATP1A2 exon 15 were diagnosed with familial hemiplegic migraine 2 characterized by complicated forms of aura. Here, we generated a transgenic mouse model having the human E700K mutation in the Atp1a2 orthologous gene. OBJECTIVE: To investigate the characteristics of cortical spreading depression in a mouse model with E700K mutation in the Atp1a2. METHODS: Cortical spreading depression was induced by applying stepwise increases of KCl concentration or electrical stimulation intensity to C57BL/6J-Tg(Atp1a2*E700K)9151Kwk mice (Tg, both sexes) and corresponding wild-type animals. Under urethane anesthesia, the responsiveness and threshold to cortical spreading depression were examined and the distribution of c-Fos expression, a neuronal activity marker, was immunohistochemically determined. RESULTS: Overall, Tg mice showed significantly faster propagation velocity (p < 0.01) and longer full-width-at-half-maximum (p < 0.01) than wild-type animals, representing a slower recovery from direct current potential deflection. The cortical spreading depression threshold tended to be lower in Tg, especially in females. c-Fos-positive cells were significantly enhanced in the ipsilateral somatosensory cortex, piriform cortex, amygdala and striatum (each p < 0.05 vs. contralateral side). Numbers of c-Fos positive cells were significantly higher in the ipsilateral amygdala of Tg, as compared with wild-type animals (p < 0.01). CONCLUSION: The effect of cortical spreading depression may be greater in E700K transgenic mice than that in wild-type animals, while the threshold for cortical spreading depression shows little change. Higher c-Fos expression in the amygdala may indicate alterations of the limbic system in Tg, suggesting an enhanced linkage between cortical spreading depression and amygdala connectivity in familial hemiplegic migraine 2 patients.

The ATP1A2 coding α2 subunit of Na,K-ATPase, which is predominantly located in astrocytes, is a causative gene of familial hemiplegic migraine type 2 (FHM2). FHM2 model mice (Atp1a2tmCKwk/+ ) are susceptible to cortical spreading depression (CSD), which is profoundly related to migraine aura and headache. However, astrocytic properties during CSD have not been examined in FHM2 model mice. Using Atp1a2tmCKwk/+ crossed with transgenic mice expressing G-CaMP7 in cortical neurons and astrocytes (Atp1a2+/- ), we analyzed the changes in Ca2+ concentrations during CSD. The propagation speed of Ca2+ waves and the percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- were higher than those in wild-type mice. Increased percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- may contribute to FHM2 pathophysiology.

Atp1a3 is the Na-pump alpha3 subunit gene expressed mainly in neurons of the brain. Atp1a3-deficient heterozygous mice (Atp1a3+/−) show altered neurotransmission and deficits of motor function after stress loading. To understand the function of Atp1a3 in a social hierarchy, we evaluated social behaviors (social interaction, aggression, social approach and social dominance) of Atp1a3+/− and compared the rank and hierarchy structure between Atp1a3+/− and wild-type mice within a housing cage using the round-robin tube test and barbering observations. Formation of a hierarchy decreases social conflict and promote social stability within the group. The hierarchical rank is a reflection of social dominance within a cage, which is heritable and can be regulated by specific genes in mice. Here we report: (1) The degree of social interaction but not aggression was lower in Atp1a3+/− than wild-type mice, and Atp1a3+/− approached Atp1a3+/− mice more frequently than wild type. (2) The frequency of barbering was lower in the Atp1a3+/− group than in the wild-type group, while no difference was observed in the mixed-genotype housing condition. (3) Hierarchy formation was not different between Atp1a3+/− and wild type. (4) Atp1a3+/− showed a lower rank in the mixed-genotype housing condition than that in the wild type, indicating that Atp1a3 regulates social dominance. In sum, Atp1a3+/− showed unique social behavior characteristics of lower social interaction and preference to approach the same genotype mice and a lower ranking in the hierarchy.

Despite high estimates of the heritability of aggressiveness, the genetic basis for individual differences in aggression remains unclear. Previously, we showed that the wild-derived mouse strain MSM/Ms (MSM) exhibits highly aggressive behaviors, and identified chromosome 15 (Chr 15) as the location of one of the genetic factors behind this escalated aggression by using a panel of consomic strains of MSM in a C57BL/6J (B6) background. To understand the genetic effect of Chr 15 derived from MSM in detail, this study examined the aggressive behavior of a Chr 15 consomic strain towards different types of opponent. Our results showed that both resident and intruder animals had to have the same MSM Chr 15 genotype in order for attack bites to increase and attack latency to be reduced, whereas there was an intruder effect of MSM Chr 15 on tail rattle behavior. To narrow down the region that contains the genetic loci involved in the aggression-eliciting effects on Chr 15, we established a panel of subconsomic strains of MSM Chr 15. Analysis of these strains suggested the existence of multiple genes that enhance and suppress aggressive behavior on Chr 15, and these loci interact in a complex way. Regression analysis successfully identified four genetic loci on Chr 15 that influence attack latency, and one genetic locus that partially elicits aggressive behaviors was narrowed down to a 4.1-Mbp region (from 68.40 Mb to 72.50 Mb) on Chr 15.

Dystonia is a neurological disorder with involuntary and simultaneous contractions of agonist and antagonist muscles. Rapid-onset dystonia parkinsonism (RDP), one of the heredity forms of dystonia, is caused by mutations of Na,K-ATPase alpha 3 subunit gene (ATP1A3). The abrupt onset of bulbar and limb symptoms of RDP are often triggered by physical and/or emotional stress. We reported previously that Atp1a3-deficient heterozygous mice showed higher locomotor activity and developed enhanced dystonia symptoms after kainate injection into the cerebellum, but not spontaneous movement disorder like RDP patients. Here we show that Atp1a3-deficient heterozygous mice exhibited shorter stride length at 4 weeks of age without stress and at later stages under chronic restraint stress loading. Shorter hanging time in the hanging box test was also observed after stress loading. Shorter stride length and hanging time may be relevant to certain phenotypes, such as gait abnormality, observed in RDP patients. Atp1a3 was widely expressed in the brain, including basal ganglia and cerebellum, and spinal cord of young mice, and the expression pattern was compatible with movement abnormalities under lack of one of alleles. Our results demonstrated the usefulness of Atp1a3-deficient heterozygous mice as an animal model of RDP and its potential use to explore the pathophysiology of movement abnormality in this disorder. (C) 2014 Elsevier B.V. All rights reserved.

Background: Owing to their complex nature, social interaction tests normally require the observation of video data by a human researcher, and thus are difficult to use in large-scale studies. We previously established a statistical method, a hidden Markov model (HMM), which enables the differentiation of two social states ("interaction" and "indifference"), and three social states ("sniffing", "following", and "indifference"), automatically in silico. New method: Here, we developed freeware called DuoMouse for the rapid evaluation of social interaction behavior. This software incorporates five steps: (1) settings, (2) video recording, (3) tracking from the video data, (4) HMM analysis, and (5) visualization of the results. Results: Using DuoMouse, we mapped a genetic locus related to social interaction. We previously reported that a consomic strain, B6-Chr6C(MSM), with its chromosome 6 substituted for one from MSM/Ms, showed more social interaction than C57BL/6 (B6). We made four subconsomic strains, C3, C5, C6, and C7, each of which has a shorter segment of chromosome 6 derived from B6-Chr6C, and conducted social interaction tests on these strains. DuoMouse indicated that C6, but not C3, C5, and C7, showed higher interaction, sniffing, and following than B6, specifically in males. Comparison with existing method: The data obtained by human observation showed high concordance to those from DuoMouse. The results indicated that the MSM-derived chromosomal region present in C6 but not in C3, C5, and C7-associated with increased social behavior. Conclusions: This method to analyze social interaction will aid primary screening for difference in social behavior in mice. (C) 2014 Elsevier B.V. All rights reserved.

Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and β subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

Male mice emit ultrasonic vocalizations (USVs) towards females during male-female interaction. It has been reported that USVs of adult male mice have the capability of attracting females. Although the waveform pattern of USVs is affected by genetic background, differences among strains with respect to USV and the effects of these differences on courtship behavior have not been analyzed fully. We analyzed USV patterns, as well as actual social behavior during USV recording, in 13 inbred mouse strains, which included laboratory and wild-derived strains. Significant effects of strain were observed for the frequency of USV emission, duration, and frequency of the waveform category. Principal component (PC) analysis showed that PC1 was related to frequency and duration, and PC2-4 were related to each waveform. In the comparison of USV patterns and behaviors among strains, wild-derived KJR mice displayed the highest scores for PC2-4, and female mice paired with KJR males did not emit rejection-related click sounds. It is assumed that the waveforms emitted by KJR males have a positive effect in male-female interaction. Therefore, we extracted waveforms in PC2-4 from the USV recordings of KJR mice to produce a sound file, "HIGH2-4". As a negative control, another sound file ("LOW2-4") was created by extracting waveforms in PC2-4 from strains with low scores for these components. In the playback experiments using these sound files, female mice were attracted to the speaker that played HIGH2-4 but not the speaker that played LOW2-4. These results highlight the role of strain differences in the waveforms of male USVs during male-female interaction. The results indicated that female mice use male USVs as information when selecting a suitable mate.

Sorogena stoianovitchae is the only ciliate that undergoes fruiting body development. Previously, we demonstrated that the developmental process is divided into five distinct stages: aggregation, compact aggregation, secretion of a mucous matrix, stalk elongation, and completion of the fruiting body. When S. stoianovitchae is mildly starved, several hundreds of cells aggregate beneath the water surface, and the aggregate develops into an aerial fruiting body. Essential requirements for fruiting body development are high-cell density, a light-dark cycle, and a dark period of more than 8 consecutive hours. In addition, the initial aggregation begins during the night, and light stimulation at sunrise triggers subsequent development. To elucidate the genes involved in fruiting body development, we carried out a reverse transcription-polymerase chain reaction (RT-PCR) in cells before and after such development. Thirty-six sequences with stage-specific expression patterns were cloned and partially sequenced. BLASTX search revealed that sequences with high identity for extracellular proteins (mucin, proteophosphoglycan) or membrane proteins (surface protein, TM9SF) are likely candidates for aggregation material, mucous matrix, and stalk material. Other sequences showed similarities to proteins, such as the casein kinase related to exocytosis in Paramecium, suggesting that they are involved in exocytosis signaling pathways for fruiting body development.

The fruiting body-forming ciliate Sorogena stoianovitchae is a protist that is multicellular in one stage of its life cycle. When nutrient levels are depleted, a number of Sorogena cells aggregate beneath the water surface to form an aerial fruiting body. Based on morphologies and the inhibition of protein synthesis, fruiting body development is divided into five distinct stages: (1) aggregation before sunrise, (2) compact aggregation after sunrise, (3) secretion of mucous matrix, (4) stalk-elongation, and (5) completion of the fruiting-body. In the aggregation stage, the cells were trapped in a matrix material that stained orange with 4',6-diamino-2-phenylindole (DAPI), but differed from the mucous matrix in the later stage. A short interruption of the dark period, at 6-8 h after the onset of dark, inhibited fruiting body development. Irrespective of the length of the dark period (10-16 h), the cells remained in the aggregation stage until the beginning of the light period. Therefore, an uninterrupted dark period of more than 8 h is critical for the initial aggregation of cells, but subsequent development is triggered by light.