Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria

Using the mitochondrial potential (ΔΨm) marker JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) and high-resolution imaging, we functionally analyzed mitochondria in cultured rat hippocampal astrocytes. Ratiometric detection of JC-1 fluorescence identified mitochondria with high and low ΔΨm. Mitochondrial density was highest in the perinuclear region, whereas ΔΨm tended to be higher in peripheral mitochondria. Spontaneous ΔΨm fluctuations, representing episodes of increased energization, appeared in individual mitochondria or synchronized in mitochondrial clusters. They continued upon withdrawal of extracellular Ca2+, but were antagonized by dantrolene or 2-aminoethoxydiphenylborate (2-APB). Fluo-3 imaging revealed local cytosolic Ca2+ transients with similar kinetics that also were depressed by dantrolene and 2-APB. Massive cellular Ca2+ load or metabolic impairment abolished ΔΨm fluctuations, occasionally evoking heterogeneous mitochondrial depolarizations. The detected diversity and ΔΨm heterogeneity of mitochondria confirms that even in less structurally polarized cells, such as astrocytes, specialized mitochondrial subpopulations coexist. We conclude that ΔΨm fluctuations are an indication of mitochondrial viability and are triggered by local Ca2+ release from the endoplasmic reticulum. This spatially confined organelle crosstalk contributes to the functional heterogeneity of mitochondria and may serve to adapt the metabolism of glial cells to the activity and metabolic demand of complex neuronal networks. The established ratiometric JC-1 imaging—especially combined with two-photon microscopy—enables quantitative functional analyses of individual mitochondria as well as the comparison of mitochondrial heterogeneity in different preparations and/or treatment conditions

The 580-520 Ma Gondwana suture of Madagascar and its continuation into Antarctica and Africa

U-Pb age data from southwest Madagascar provide a compelling case that the pre-Gondwana Indian plate was stitched with the arc terranes of the Arabian Nubian Shield along a suture that closed between 580. Ma and 520. Ma. The key observations supportive of this interpretation are: (1) metamorphism dated to 630-600. Ma is manifested only on the west side of the suture in rocks that have affinities with the oceanic and island arc terranes of the Arabian Nubian Shield, or which represent continental rocks welded to these terranes prior to the amalgamation of Gondwana, and (2) orogenesis at 580-520. Ma is manifest in rocks on both sides of the suture, an observation taken to mark the timing of collision and to reflect spatial continuity across the suture. In southwest Madagascar the distribution of metamorphic ages places the suture along the Beraketa high-strain zone, the tectonic boundary between the Androyen and Anosyen domains. Similar age relationships allow for the extrapolation of this tectonic boundary into both East Antarctica and Africa

The tectonic domains of southern and western Madagascar

Southern and western Madagascar is comprised of five tectonic provinces that, from northeast to southwest, are defined by the: (i) Ikalamavony, (ii) Anosyen, (iii) Androyen, (iv) Graphite and (v) Vohibory Domains. The Ikalamavony, Graphite and Vohibory Domains all have intermediate and felsic igneous protoliths of tonalite-trondhjemite-granodiorite-granite composition, with positive εNd, and low Sr and Pb isotopic ratios. All three domains are interpreted to be the products of intra-oceanic island arc magmatism. The protoliths of the Ikalamavony and Graphite Domains formed repectively between c. 1080–980 Ma and 1000–920 Ma, whereas those of the Vohibory Domain are younger and date to between c. 670–630 Ma. Different post-formation geologic histories tie the Vohibory-Graphite and Ikalamavony Domains to opposite sides of the pre-Gondwana Mozambique Ocean. By contrast, the Androyen and Anosyen Domains record long crustal histories. Intermediate to felsic igneous protoliths in the Androyen Domain are of Palaeoproterozoic age (c. 2200–1800 Ma), of tonalite-trondhjemite-granodiorite-granite composition, and show negative εNd, moderate to high 87Sr/86Sr and variable Pb isotopic compositions. The felsic igneous protoliths of the Anosyen Domain are of granitic composition and, when compared to felsic gneisses of the Androyen Domain, show consistently lower Sr/Y and markedly higher Sr and Pb isotope ratios. Like the Vohibory and Graphite Domains, the Androyen Domain can be linked to the western side of the Mozambique Ocean, while the Anosyen Domain shares magmatic and detrital zircon commonalities with the Ikalamavony Domain. It is consequently linked to the opposing eastern side of this ocean. The first common event observed in all domains dates to c. 580–520 Ma and marks the closure of the Mozambique Ocean. The trace of this suture lies along the boundary between the Androyen and Anosyen Domains and is defined by the Beraketa high-strain zone.The majority of the research was funded by the International Development Agency (Crédit No.: 3754 MAG) and undertaken under the auspices of the Projet de Gouvernance des Ressources Minérales (PRGM – Madagascar). Sr–Nd–Pb isotopic analysis was undertaken with support of an internal University of Melbourne Grant (ECR602698) awarded to SDB

SEROSURVEY OF SELECTED ARBOVIRAL PATHOGENS IN FREE-RANGING, TWO-TOED SLOTHS ( CHOLOEPUS HOFFMANNI

We screened for antibodies to 16 arboviruses in four populations of free-ranging sloths in Costa Rica. Blood samples were taken from 16 Hoffman’s two-toed sloths (HTSs; CHOLOEPUS HOFFMANNI) and 26 brown-throated sloths (BTSs; BRADYPUS VARIEGATUS) over a 3-yr period. We used serologic assays to detect antibodies against 10 arboviruses previously described in sloths (St. Louis encephalitis [SLEV], Changuinola, Venezuelan equine encephalitis, Ilheus [ILHV], Oropouche, Mayaro, Utinga, Murutucu, Punta Toro, and vesicular stomatitis [VSV] viruses) and six arboviruses not described in sloths (Rio Grande, West Nile [WNV], eastern equine encephalitis, Piry, Munguba, and La Crosse viruses). Overall, 80% of sloths had detectable antibodies to SLEV, 67% had antibodies to ILHV, 32% to Punta Toro virus, 30% to Changuinola virus, 15% to WNV, 14% to VSV, 11% to Venezuelan equine encephalitis virus, and 10% to Rio Grande virus. No samples had detectable antibodies to the remaining eight viruses. We found a significant increase in prevalence of antibody to VSV in HTSs between 2005 and 2007, and for WNV antibody between 2005 and 2006. We found no significant differences in the prevalences of antibodies to the sampled viruses between the two locations. Antibody prevalences were significantly higher in HTSs than in BTSs for SLEV in 2005. Antibody-positive results for ILHV were likely due to cross-reaction with SLEV. The novel finding of antibodies to Rio Grande virus in sloths could be due to cross-reaction with another phlebovirus. These findings might have implications for land management and domestic animal health. Due to the nature of the study, we could not determine whether sloths could represent amplification hosts for these viruses, or whether they were only exposed and could be used as sentinel species. Further studies are needed to fully characterize arboviral exposure in sloths

SEROSURVEY OF SELECTED ARBOVIRAL PATHOGENS IN FREE-RANGING, TWO-TOED SLOTHS (CHOLOEPUS HOFFMANNI) AND THREE-TOED SLOTHS (BRADYPUS VARIEGATUS) IN COSTA RICA, 200507

We screened for antibodies to 16 arboviruses in four populations of free-ranging sloths in Costa Rica. Blood samples were taken from 16 Hoffman's two-toed sloths (HTSs; Choloepus hoffmanni ) and 26 brown-throated sloths (BTSs; Bradypus variegatus ) over a 3-yr period. We used serologic assays to detect antibodies against 10 arboviruses previously described in sloths (St. Louis encephalitis [SLEV], Changuinola, Venezuelan equine encephalitis, Ilheus [ILHV], Oropouche, Mayaro, Utinga, Murutucu, Punta Toro, and vesicular stomatitis [VSV] viruses) and six arboviruses not described in sloths (Rio Grande, West Nile [WNV], eastern equine encephalitis, Piry, Munguba, and La Crosse viruses). Overall, 80% of sloths had detectable antibodies to SLEV, 67% had antibodies to ILHV, 32% to Punta Toro virus, 30% to Changuinola virus, 15% to WNV, 14% to VSV, 11% to Venezuelan equine encephalitis virus, and 10% to Rio Grande virus. No samples had detectable antibodies to the remaining eight viruses. We found a significant increase in prevalence of antibody to VSV in HTSs between 2005 and 2007, and for WNV antibody between 2005 and 2006. We found no significant differences in the prevalences of antibodies to the sampled viruses between the two locations. Antibody prevalences were significantly higher in HTSs than in BTSs for SLEV in 2005. Antibody-positive results for ILHV were likely due to cross-reaction with SLEV. The novel finding of antibodies to Rio Grande virus in sloths could be due to cross-reaction with another phlebovirus. These findings might have implications for land management and domestic animal health. Due to the nature of the study, we could not determine whether sloths could represent amplification hosts for these viruses, or whether they were only exposed and could be used as sentinel species. Further studies are needed to fully characterize arboviral exposure in sloths