Projection Neuron Circuits Resolved Using Correlative Array Tomography

Assessment of three-dimensional morphological structure and synaptic connectivity is essential for a comprehensive understanding of neural processes controlling behavior. Different microscopy approaches have been proposed based on light microcopy (LM), electron microscopy (EM), or a combination of both. Correlative array tomography (CAT) is a technique in which arrays of ultrathin serial sections are repeatedly stained with fluorescent antibodies against synaptic molecules and neurotransmitters and imaged with LM and EM (Micheva and Smith, 2007). The utility of this correlative approach is limited by the ability to preserve fluorescence and antigenicity on the one hand, and EM tissue ultrastructure on the other. We demonstrate tissue staining and fixation protocols and a workflow that yield an excellent compromise between these multimodal imaging constraints. We adapt CAT for the study of projection neurons between different vocal brain regions in the songbird. We inject fluorescent tracers of different colors into afferent and efferent areas of HVC in zebra finches. Fluorescence of some tracers is lost during tissue preparation but recovered using anti-dye antibodies. Synapses are identified in EM imagery based on their morphology and ultrastructure and classified into projection neuron type based on fluorescence signal. Our adaptation of array tomography, involving the use of fluorescent tracers and heavy-metal rich staining and embedding protocols for high membrane contrast in EM will be useful for research aimed at statistically describing connectivity between different projection neuron types and for elucidating how sensory signals are routed in the brain and transformed into a meaningful motor output

Correlative Microscopy of Densely Labeled Projection Neurons Using Neural Tracers

Three-dimensional morphological information about neural microcircuits is of high interest in neuroscience, but acquiring this information remains challenging. A promising new correlative technique for brain imaging is array tomography (Micheva and Smith, 2007), in which series of ultrathin brain sections are treated with fluorescent antibodies against neurotransmitters and synaptic proteins. Treated sections are repeatedly imaged in the fluorescence light microscope (FLM) and then in the electron microscope (EM). We explore a similar correlative imaging technique in which we differentially label distinct populations of projection neurons, the key routers of electrical signals in the brain. In songbirds, projection neurons can easily be labeled using neural tracers, because the vocal control areas are segregated into separate nuclei. We inject tracers into areas afferent and efferent to the main premotor area for vocal production, HVC, to retrogradely and anterogradely label different classes of projection neurons. We optimize tissue preparation protocols to achieve high fluorescence contrast in the FLM and good ultrastructure in the EM (using osmium tetroxide). Although tracer fluorescence is lost during EM preparation, we localize the tracer molecules after fixation and embedding by using fluorescent antibodies against them. We detect signals mainly in somata and dendrites, allowing us to classify synapses within a single ultrathin section as belonging to a particular type of projection neuron. The use of our method will be to provide statistical information about connectivity among different neuron classes, and to elucidate how signals in the brain are processed and routed among different areas

Gemtelligence: Accelerating Gemstone classification with Deep Learning

The value of luxury goods, particularly investment-grade gemstones, is greatly influenced by their origin and authenticity, sometimes resulting in differences worth millions of dollars. Traditionally, human experts have determined the origin and detected treatments on gemstones through visual inspections and a range of analytical methods. However, the interpretation of the data can be subjective and time-consuming, resulting in inconsistencies. In this study, we propose Gemtelligence, a novel approach based on deep learning that enables accurate and consistent origin determination and treatment detection. Gemtelligence comprises convolutional and attention-based neural networks that process heterogeneous data types collected by multiple instruments. Notably, the algorithm demonstrated comparable predictive performance to expensive laser-ablation inductively-coupled-plasma mass-spectrometry (ICP-MS) analysis and visual examination by human experts, despite using input data from relatively inexpensive analytical methods. Our innovative methodology represents a major breakthrough in the field of gemstone analysis by significantly improving the automation and robustness of the entire analytical process pipeline

Dicer and Hsp104 Function in a Negative Feedback Loop to Confer Robustness to Environmental Stress

SummaryEpigenetic mechanisms can be influenced by environmental cues and thus evoke phenotypic variation. This plasticity can be advantageous for adaptation but also detrimental if not tightly controlled. Although having attracted considerable interest, it remains largely unknown if and how environmental cues such as temperature trigger epigenetic alterations. Using fission yeast, we demonstrate that environmentally induced discontinuous phenotypic variation is buffered by a negative feedback loop that involves the RNase Dicer and the protein disaggregase Hsp104. In the absence of Hsp104, Dicer accumulates in cytoplasmic inclusions and heterochromatin becomes unstable at elevated temperatures, an epigenetic state inherited for many cell divisions after the heat stress. Loss of Dicer leads to toxic aggregation of an exogenous prionogenic protein. Our results highlight the importance of feedback regulation in building epigenetic memory and uncover Hsp104 and Dicer as homeostatic controllers that buffer environmentally induced stochastic epigenetic variation and toxic aggregation of prionogenic proteins

B and T cell acute lymphoblastic leukemia evade chemotherapy at distinct sites in the bone marrow

Persistence of residual disease after induction chemotherapy is a strong predictor of relapse in acute lymphoblastic leukemia (ALL). The bone marrow microenvironment may support treatment escape. Using 3D fluorescence imaging of 10 primary ALL xenografts we identify sites of predilection in the bone marrow for resistance to induction with dexamethasone, vincristine and doxorubicin. We detect B-cell precursor ALL cells predominantly in the perisinusoidal space at early engraftment and after chemotherapy. The spatial distribution of T-ALL cells was more widespread with contacts to endosteum, nestin+ pericytes and sinusoids. Dispersion of T-ALL cells in the bone marrow increased under chemotherapeutic pressure. A subset of slowly dividing ALL cells was transiently detected upon short-term chemotherapy, but not at residual disease after chemotherapy, challenging the notion that ALL cells escape treatment by direct induction of a dormant state in the niche. These lineage-dependent differences point to niche interactions that may be more specifically exploitable to improve treatment

EDAM-bioimaging : The ontology of bioimage informatics operations, topics, data, and formats

International audienceThe ontology of bioimage informatics operations, topics, data, and formats What? EDAM-bioimaging is an extension of the EDAM ontology, dedicated to bioimage analysis, bioimage informatics, and bioimaging. Why? EDAM-bioimaging enables interoperable descriptions of software, publications, data, and workflows, fostering reliable and transparent science. How? EDAM-bioimaging is developed in a community spirit, in a welcoming collaboration between numerous bioimaging experts and ontology developers. How can I contribute? We need your expertise! You can help by reviewing parts of EDAM-bioimaging, posting comments with suggestions, requirements, or needs for clarification, or participating in a Taggathon or another hackathon. Please see https://github.com/edamontology/edam-bioimaging#contributing. EDAM-bioimaging is developed in an interdisciplinary open collaboration supported by the hosting institutions, participating individuals, and NEUBIAS COST Action (CA15124) and ELIXIR-EXCELERATE (676559) funded by the Horizon 2020 Framework Programme of the European Union. https://github.com/edamontology/edam-bioimaging @edamontology /edamontology/edam-bioimagin

The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Aldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na+ reabsorption via up-regulation of the epithelial Na+ channel (ENaC) and the Na+-K+- ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in ~20% of renal tubule cells (MR/X mice), in which MR-positive (MRwt) and -negative (MRko) cells can be studied sideby- side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MRwt and MRko cells and dietary Na+ restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MRko cells in the CS did not show any detectable alpha- ENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na+ restriction. Furthermore, Na+-K+-ATPase expression was unaffected in MRko cells of the DCT, while it was lost in MRko cells of the CS. In conclusion, MR is crucial for ENaC and Na+-K+-ATPase regulation in the CS, but is dispensable for NCC and Na+-K+-ATPase regulation in the DCT

Drosophila β-Tubulin 97EF is upregulated at low temperature and stabilizes microtubules

Cells in ectotherms function normally within an often wide temperature range. As temperature dependence is not uniform across all the distinct biological processes, acclimation presumably requires complex regulation. The molecular mechanisms that cope with the disruptive effects of temperature variation are still poorly understood. Interestingly, one of five different β-tubulin paralogs, βTub97EF, was among the genes upregulated at low temperature in cultured Drosophila cells. As microtubules are known to be cold sensitive, we analyzed whether βTub97EF protects microtubules at low temperatures. During development at the optimal temperature (25°C), βTub97EF was expressed in a tissue-specific pattern primarily in the gut. There, as well as in hemocytes, expression was increased at low temperature (14°C). Although βTub97EF mutants were viable and fertile at 25°C, their sensitivity within the well-tolerated range was slightly enhanced during embryogenesis specifically at low temperatures. Changing β-tubulin isoform ratios in hemocytes demonstrated that β-Tubulin 97EF has a pronounced microtubule stabilizing effect. Moreover, βTub97EF is required for normal microtubule stability in the gut. These results suggest that βTub97EF upregulation at low temperature contributes to acclimation by stabilizing microtubules