The original version of this Article contained two errors in the Results section, in which the di... more The original version of this Article contained two errors in the Results section, in which the discussion of prior work published in reference 48 inaccurately referred to skin temperature, rather than tail temperature. The sentence 'It has recently been reported that optogenetic stimulation of axon terminals of PBel-CGRP+ (calcitonin gene-related peptide) neurons in PSTh induces a reduction in skin temperature 48 ' was inaccurate. The correct version replaces 'skin temperature' by 'tail temperature'. The sentence 'These results are consistent with a recent report that opto-stimulation of the PBel-PSTh or PBel-CeA pathway can cause hypothermia 48 ' was incorrect, as the study in reference 48 studied tail temperature and not skin temperature and is thus not directly related to the findings on hypothermia reported in the original Article. This sentence has been removed. This has been corrected in both the PDF and HTML versions of the Article.
Sleep disturbances have been recognized as a core symptom of post-traumatic stress disorders (PTS... more Sleep disturbances have been recognized as a core symptom of post-traumatic stress disorders (PTSD). However, the neural basis of PTSD-related sleep disturbances remains unclear. It has been challenging to establish the causality link between a specific brain region and traumatic stress-induced sleep abnormalities. Here, we found that single prolonged stress (SPS) could induce acute changes in sleep/wake duration as well as short-and long-term electroencephalogram (EEG) alterations in the isogenic mouse model. Moreover, the medial prefrontal cortex (mPFC) showed persistent high number of c-fos expressing neurons, of which more than 95% are excitatory neurons, during and immediately after SPS. Chemogenetic inhibition of the prelimbic region of mPFC during SPS could specifically reverse the SPS-induced acute suppression of delta power (1-4 Hz EEG) of non-rapid-eye-movement sleep (NREMS) as well as most of long-term EEG abnormalities. These findings suggest a causality link between hyper-activation of mPFC neurons and traumatic stress-induced specific sleep-wake EEG disturbances.
Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted t... more Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for under...
Intravital microscopy is a powerful tool for discovery and analysis in cell biology, neurobiology... more Intravital microscopy is a powerful tool for discovery and analysis in cell biology, neurobiology, immunology and oncology and has increasingly become a method used in most biomedical research. Recently, advances in imaging system, fluorescence labeling and tissue immobilization techniques have made it possible to in vivo image subcellular structure dynamics and cellular processes in virtually all abdominal organs of live mice. In this chapter, we describe the considerations that comprise subcellular intravital imaging in mouse intraperitoneal organs. After discussing the topics such as choosing the suitable imaging modalities and optics, labeling strategies, tissue stabilization, anesthesia and animal handling, examples of imaging various organs with subcellular resolution using a tissue stabilization device made by the authors are presented. This chapter will be helpful to scientists who are interested in adding intravital microscopy to their technology toolbox. We have focused our discussions on in vivo imaging in abdominal organs, however, most of the points we make are relevant to imaging of any organs.
Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has re... more Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in realtime as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.
Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerat... more Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site's time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O L) suggested that the most probable site lies within the ND3 gene. By adopting this O L position we calculated times of exposure for 0 FD (nondegenerate), 2 FD (twofold degenerate), and 4 FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4 FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2 FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0 FD sites with exposure time, which is explicable as the result of biased usage of 4 FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes.
In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry matern... more In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two-and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.
Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial g... more Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from ...
The original version of this Article contained two errors in the Results section, in which the di... more The original version of this Article contained two errors in the Results section, in which the discussion of prior work published in reference 48 inaccurately referred to skin temperature, rather than tail temperature. The sentence 'It has recently been reported that optogenetic stimulation of axon terminals of PBel-CGRP+ (calcitonin gene-related peptide) neurons in PSTh induces a reduction in skin temperature 48 ' was inaccurate. The correct version replaces 'skin temperature' by 'tail temperature'. The sentence 'These results are consistent with a recent report that opto-stimulation of the PBel-PSTh or PBel-CeA pathway can cause hypothermia 48 ' was incorrect, as the study in reference 48 studied tail temperature and not skin temperature and is thus not directly related to the findings on hypothermia reported in the original Article. This sentence has been removed. This has been corrected in both the PDF and HTML versions of the Article.
Sleep disturbances have been recognized as a core symptom of post-traumatic stress disorders (PTS... more Sleep disturbances have been recognized as a core symptom of post-traumatic stress disorders (PTSD). However, the neural basis of PTSD-related sleep disturbances remains unclear. It has been challenging to establish the causality link between a specific brain region and traumatic stress-induced sleep abnormalities. Here, we found that single prolonged stress (SPS) could induce acute changes in sleep/wake duration as well as short-and long-term electroencephalogram (EEG) alterations in the isogenic mouse model. Moreover, the medial prefrontal cortex (mPFC) showed persistent high number of c-fos expressing neurons, of which more than 95% are excitatory neurons, during and immediately after SPS. Chemogenetic inhibition of the prelimbic region of mPFC during SPS could specifically reverse the SPS-induced acute suppression of delta power (1-4 Hz EEG) of non-rapid-eye-movement sleep (NREMS) as well as most of long-term EEG abnormalities. These findings suggest a causality link between hyper-activation of mPFC neurons and traumatic stress-induced specific sleep-wake EEG disturbances.
Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted t... more Observations of rapid shifts in mitochondrial DNA (mtDNA) variants between generations prompted the creation of the bottleneck theory. A prevalent hypothesis is that a massive reduction in mtDNA content during early oogenesis leads to the bottleneck. To test this, we estimated the mtDNA copy number in single germline cells and in single somatic cells of early embryos in mice. Primordial germ cells (PGCs) show consistent, moderate mtDNA copy numbers across developmental stages, whereas primary oocytes demonstrate substantial mtDNA expansion during early oocyte maturation. Some somatic cells possess a very low mtDNA copy number. We also demonstrated that PGCs have more than 100 mitochondria per cell. We conclude that the mitochondrial bottleneck is not due to a drastic decline in mtDNA copy number in early oogenesis but rather to a small effective number of segregation units for mtDNA in mouse germ cells. These results provide new information for mtDNA segregation models and for under...
Intravital microscopy is a powerful tool for discovery and analysis in cell biology, neurobiology... more Intravital microscopy is a powerful tool for discovery and analysis in cell biology, neurobiology, immunology and oncology and has increasingly become a method used in most biomedical research. Recently, advances in imaging system, fluorescence labeling and tissue immobilization techniques have made it possible to in vivo image subcellular structure dynamics and cellular processes in virtually all abdominal organs of live mice. In this chapter, we describe the considerations that comprise subcellular intravital imaging in mouse intraperitoneal organs. After discussing the topics such as choosing the suitable imaging modalities and optics, labeling strategies, tissue stabilization, anesthesia and animal handling, examples of imaging various organs with subcellular resolution using a tissue stabilization device made by the authors are presented. This chapter will be helpful to scientists who are interested in adding intravital microscopy to their technology toolbox. We have focused our discussions on in vivo imaging in abdominal organs, however, most of the points we make are relevant to imaging of any organs.
Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has re... more Intravital imaging of brain and bone marrow cells in the skull with subcellular resolution has revolutionized neurobiology, immunology and hematology. However, the application of this powerful technology in studies of abdominal organs has long been impeded by organ motion caused by breathing and heartbeat. Here we describe for the first time a simple device designated 'microstage' that effectively reduces organ motions without causing tissue lesions. Combining this microstage device with an upright intravital laser scanning microscope equipped with a unique stick-type objective lens, the system enables subcellular-level imaging of abdominal organs in live mice. We demonstrate that this technique allows for the quantitative analysis of subcellular structures and gene expressions in cells, the tracking of intracellular processes in realtime as well as three-dimensional image construction in the pancreas and liver of the live mouse. As the aforementioned analyses based on subcellular imaging could be extended to other intraperitoneal organs, the technique should offer great potential for investigation of physiological and disease-specific events of abdominal organs. The microstage approach adds an exciting new technique to the in vivo imaging toolbox.
Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerat... more Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site's time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O L) suggested that the most probable site lies within the ND3 gene. By adopting this O L position we calculated times of exposure for 0 FD (nondegenerate), 2 FD (twofold degenerate), and 4 FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4 FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2 FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0 FD sites with exposure time, which is explicable as the result of biased usage of 4 FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes.
In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry matern... more In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two-and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.
Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial g... more Species of the mussel genus Mytilus possess maternally and paternally transmitted mitochondrial genomes. In the interbreeding taxa Mytilus edulis and M. galloprovincialis, several genomes of both types have been fully sequenced. The genome consists of the coding part (which, in addition to protein and RNA genes, contains several small noncoding sequences) and the main control region (CR), which in turn consists of three distinct parts: the first variable (VD1), the conserved (CD), and the second variable (VD2) domain. The maternal and paternal genomes are very similar in gene content and organization, even though they differ by >20% in primary sequence. They differ even more at VD1 and VD2, yet they are remarkably similar at CD. The complete sequence of a genome from the closely related species M. trossulus was previously reported and found to consist of a maternal-like coding part and a paternal-like and a maternal-like CR. From this and from the fact that it was extracted from ...
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