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

Use of Genuine Sources of Ergot Resistance in Species of the \u3ci\u3edilatata\u3c/i\u3e Group of \u3ci\u3ePaspalum\u3c/i\u3e

Paspalum dilatatum Poir. (dallisgrass) is an excellent C4 forage for summer production, highly productive with a wide distribution within warm-temperate regions. P. dilatatum is native to South America with special relevance for milk and meat production since its forage quality is superior to that of other C4 forage grasses and it shows tolerance to frost and water stress. In situations of temperate and warm temperate climate, the presence of these species plays a key role in the ecological stability of the system due to the complementarity of the growth cycles between winter and summer grasses. Claviceps paspali (ergot) is a fungus that mainly parasitizes Paspalum spp., generating a structure called sclerotia, in which indole-diterpenoid alkaloids are isolated. Despite the excellent forage characteristics of Paspalum species, there is a need for the generation of varieties able to overcome two major limitations that were identified early on in this species, such as ergot susceptibility and seed production. With this objective, selected genotypes were crossed between apomictic and sexual species of different ploidy using P. malacophyllum as a source of immunity. Immunity to ergot has been evaluated in the field for two years in different representative regions of Uruguay and the accessions that did not get sick were selected as pollen donors. Crosses made with P. flavescens showed a germination percentage ranging between 1.7 and 7.09, while in P. dilatatum var. Chirú the range was reduced between 0.99 and 1.25 according to the employed parents. The hybrid nature of the progeny is being verified by microsatellites and functional markers associated with immunity and DNA content estimated by flow cytometry. This work aims to generate the basis to transfer immunity from P. malacophyllum in selected genotypes of Paspalum species predominant in Pampa biome and to improve seed production

Posidonia oceanica restoration, a relevant strategy after boat anchoring degradation?

Trabajo presentado al 7th Mediterranean Symposium on Marine Vegetation, celebrado en Génova (Italia), los días 19 y 20 de septiembre de 2022.The anchoring of large pleasure boats constitutes one of the main threats in shallow marine habitats and particularly for seagrass beds. In the Mediterranean, this activity has seen constant development during the last decades, causing major physical disturbances in Posidonia oceanica meadows and associated ecosystem services, notably in terms of climate change mitigation (i.e. carbon fixation and sequestration capacities). In this context, the aims of the present study are to estimate the impact of these anchoring activities on P. oceanica meadows in a particularly highlyfrequented area (Sant’Amanza gulf, SE Corsica Island) and to set up a strategy to restore this major carbon sink. Since the last decade, time-series of marine habitat maps revealed an important regression of P. oceanica meadows, with a loss of 72.9 ha, corresponding to 11% of the meadow surface and 9% decline in the total carbon fixation and sequestration performed each year. Moreover, in the most impacted part of the bay (Balistra bay), a loss of 16.6 ha (28%) has been recorded between 2011 and 2022. Following recent enforcement of anchoring regulation, prohibiting the anchoring of large units (greater than 24 m), and the lockdown linked to the COVID-19 pandemic, anchorages in the seagrass reduced by 92%, between 2018 and 2022. Natural recovery was observed at the edge of the meadow (plagiotropic rhizomes) but this growth is slow and the areas to recover are large. As a result, a transplant experiment, from cuttings harvested from the adjacent meadows, was initiated in the spring 2021. Four restoration techniques are being tested in the some pilot sites and the development of these transplants will be monitored and compared to the natural recovery.This research has been co-financed by INTERREG program (GIREPAM), Collectivity of Corsica Corse (PADDUC-CHANGE), SETEC Foundation, Environment Office of Corsica and the French Office of Biodiversity (RenforC program)

Studying synapses in human brain with array tomography and electron microscopy

Postmortem studies of synapses in human brain are problematic due to the axial resolution limit of light microscopy and the difficulty preserving and analyzing ultrastructure with electron microscopy. Array tomography overcomes these problems by embedding autopsy tissue in resin and cutting ribbons of ultrathin serial sections. Ribbons are imaged with immunofluorescence, allowing high-throughput imaging of tens of thousands of synapses to assess synapse density and protein composition. The protocol takes approximately 3 days per case, excluding image analysis, which is done at the end of the study. Parallel processing for transmission electron microscopy (TEM) using a protocol modified to preserve structure in human samples allows complimentary ultrastructural studies. Incorporation of array tomography and TEM into brain banking is a potent way of phenotyping synapses in well-characterized clinical cohorts to develop clinico-pathological correlations at the synapse level. This will be important for research in neurodegenerative disease, developmental diseases, and psychiatric illness

Pentoxifylline Does Not Decrease Short-term Mortality but Does Reduce Complications in Patients With Advanced Cirrhosis

Background & AimsPentoxifylline, an inhibitor of tumor necrosis factor-α, is given to patients with liver diseases, but its effects in patients with advanced cirrhosis are unknown. We performed a randomized, placebo-controlled, double-blind trial of its effects in patients with cirrhosis. Methods A total of 335 patients with cirrhosis (Child–Pugh class C) were assigned to groups given either pentoxifylline (400 mg, orally, 3 times daily; n = 164) or placebo (n = 171) for 6 months. The primary end point was mortality at 2 months. Secondary end points were mortality at 6 months and development of liver-related complications. Results By 2 months, 28 patients in the pentoxifylline group (16.5%) and 31 in the placebo group (18.2%) had died (P = .84). At 6 months, 50 patients in the pentoxifylline group (30.0%) and 54 in the placebo group (31.5%) had died (P = .75). The proportions of patients without complications (eg, bacterial infection, renal insufficiency, hepatic encephalopathy, or gastrointestinal hemorrhage) were higher in the pentoxifylline group than in the placebo group at 2 months (78.6% vs 63.4%; P = .006) and 6 months (66.8% vs 49.7%; P = .002). The probability of survival without complications was higher in the pentoxifylline group than in the placebo group at 2 and 6 months (P = .04). In multivariate analysis, the factors associated with death were age, the Model for End-Stage Liver Disease score, and presence of early-stage carcinoma. Treatment with pentoxifylline was the only factor associated with liver-related complications. Conclusions Although pentoxifylline does not decrease short-term mortality in patients with advanced cirrhosis, it does reduce the risk of complications

Deregulation, Privatisation and Marketisation of Nordic Comprehensive Education : Social Changes Reflected in Schooling

Peer reviewe

Identification, characterisation and expression analysis of natural killer receptor genes in Chlamydia pecorum infected koalas (Phascolarctos cinereus)

BACKGROUND: Koalas (Phascolarctos cinereus), an iconic Australian marsupial, are being heavily impacted by the spread of Chlamydia pecorum, an obligate intracellular bacterial pathogen. Koalas vary in their response to this pathogen, with some showing no symptoms, while others suffer severe symptoms leading to infertility, blindness or death. Little is known about the pathology of this disease and the immune response against it in this host. Studies have demonstrated that natural killer (NK) cells, key components of the innate immune system, are involved in the immune response to chlamydial infections in humans. These cells can directly lyse cells infected by intracellular pathogens and their ability to recognise these infected cells is mediated through NK receptors on their surface. These are encoded in two regions of the genome, the leukocyte receptor complex (LRC) and the natural killer complex (NKC). These two families evolve rapidly and different repertoires of genes, which have evolved by gene duplication, are seen in different species. METHODS: In this study we aimed to characterise genes belonging to the NK receptor clusters in the koala by searching available koala transcriptomes using a combination of search methods. We developed a qPCR assay to quantify relative expression of four genes, two encoded within the NK receptor cluster (CLEC1B, CLEC4E) and two known to play a role in NK response to Chalmydia in humans (NCR3, PRF1). RESULTS: We found that the NK receptor repertoire of the koala closely resembles that of the Tasmanian devil, with minimal genes in the NKC, but with lineage specific expansions in the LRC. Additional genes important for NK cell activity, NCR3 and PRF1, were also identified and characterised. In a preliminary study to investigate whether these genes are involved in the koala immune response to infection by its chlamydial pathogen, C. pecorum, we investigated the expression of four genes in koalas with active chlamydia infection, those with past infection and those without infection using qPCR. This analysis revealed that one of these four, CLEC4E, may be upregulated in response to chlamydia infection. CONCLUSION: We have characterised genes of the NKC and LRC in koalas and have discovered evidence that one of these genes may be upregulated in koalas with chlamydia, suggesting that these receptors may play a role in the immune response of koalas to chlamydia infection