Reconstructing neural circuits using multiresolution correlated light and electron microscopy

Correlated light and electron microscopy (CLEM) can be used to combine functional and molecular characterizations of neurons with detailed anatomical maps of their synaptic organization. Here we describe a multiresolution approach to CLEM (mrCLEM) that efficiently targets electron microscopy (EM) imaging to optically characterized cells while maintaining optimal tissue preparation for high-throughput EM reconstruction. This approach hinges on the ease with which arrays of sections collected on a solid substrate can be repeatedly imaged at different scales using scanning electron microscopy. We match this multiresolution EM imaging with multiresolution confocal mapping of the aldehyde-fixed tissue. Features visible in lower resolution EM correspond well to features visible in densely labeled optical maps of fixed tissue. Iterative feature matching, starting with gross anatomical correspondences and ending with subcellular structure, can then be used to target high-resolution EM image acquisition and annotation to cells of interest. To demonstrate this technique and range of images used to link live optical imaging to EM reconstructions, we provide a walkthrough of a mouse retinal light to EM experiment as well as some examples from mouse brain slices

Gravity Waves from Quantum Stress Tensor Fluctuations in Inflation

We consider the effects of the quantum stress tensor fluctuations of a conformal field in generating gravity waves in inflationary models. We find a non-scale invariant, non-Gaussian contribution which depends upon the total expansion factor between an initial time and the end of inflation. This spectrum of gravity wave perturbations is an illustration of a negative power spectrum, which is possible in quantum field theory. We discuss possible choices for the initial conditions. If the initial time is taken to be sufficiently early, the fluctuating gravity waves are potentially observable both in the CMB radiation and in gravity wave detectors, and could offer a probe of transplanckian physics. The fact that they have not yet been observed might be used to constrain the duration and energy scale of inflation.Comment: 17 -pages, no figure

Distinct patterns of temporal and directional connectivity among intrinsic networks in the human brain

To determine the spatiotemporal relationships among intrinsic networks of the human brain, we recruited seven neurosurgical patients (four males and three females) who were implanted with intracranial depth electrodes. We first identified canonical resting-state networks at the individual subject level using an iterative matching procedure on each subject's resting-state fMRI data. We then introduced single electrical pulses to fMRI pre-identified nodes of the default network (DN), frontoparietal network (FPN), and salience network (SN) while recording evoked responses in other recording sites within the same networks. We found bidirectional signal flow across the three networks, albeit with distinct patterns of evoked responses within different time windows. We used a data-driven clustering approach to show that stimulation of the FPN and SN evoked a rapid (&lt;70 ms) response that was predominantly higher within the SN sites, whereas stimulation of the DN led to sustained responses in later time windows (85–200 ms). Stimulations in the medial temporal lobe components of the DN evoked relatively late effects (&gt;130 ms) in other nodes of the DN, as well as FPN and SN. Our results provide temporal information about the patterns of signal flow between intrinsic networks that provide insights into the spatiotemporal dynamics that are likely to constrain the architecture of the brain networks supporting human cognition and behavior.SIGNIFICANCE STATEMENTDespite great progress in the functional neuroimaging of the human brain, we still do not know the precise set of rules that define the patterns of temporal organization between large-scale networks of the brain. In this study, we stimulated and then recorded electrical evoked potentials within and between three large-scale networks of the brain, the default network (DN), frontoparietal network (FPN), and salience network (SN), in seven subjects undergoing invasive neurosurgery. Using a data-driven clustering approach, we observed distinct temporal and directional patterns between the three networks, with FPN and SN activity predominant in early windows and DN stimulation affecting the network in later windows. These results provide important temporal information about the interactions between brain networks supporting human cognition and behavior.</jats:p

Activation of β-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptors δ (PPARδ) in Neonatal Rat Cardiomyocytes

Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs) in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ) in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI) phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells

Recombinant lipidated dengue-4 envelope protein domain III elicits protective immunity

AbstractThe combination of recombinant protein antigens with an immunostimulator has the potential to greatly increase the immunogenicity of recombinant protein antigens. In the present study, we selected the dengue-4 envelope protein domain III as a dengue vaccine candidate and expressed the protein in lipidated form using an Escherichia coli-based system. The recombinant lipidated dengue-4 envelope protein domain III folded into the proper conformation and competed with the dengue-4 virus for cellular binding sites. Mice immunized with lipidated dengue-4 envelope protein domain III without exogenous adjuvant had higher frequencies of dengue-4 envelope protein domain III-specific B cells secreting antibodies than mice immunized with the nonlipidated form. Importantly, lipidated dengue-4 envelope protein domain III-immunized mice demonstrated a durable neutralizing antibody response and had reduced viremia levels after challenge. The study demonstrates that lipidated dengue-4 envelope protein domain III is immunogenic and may be a potential dengue vaccine candidate. Furthermore, the lipidation strategy can be applied to other serotypes of dengue virus

Lipopolysaccharide-stimulated Leukocytes Contribute to Platelet Aggregative Dysfunction, Which is Attenuated by Catalase in Rats

Endotoxemia causes several hematological dysfunctions, including platelet degranulation or disseminated intravascular coagulation, which lead to thrombotic and hemorrhagic events. Here, we tested the hypothesis that bacterial lipopolysaccharide (LPS)-stimulated leukocytes contribute to platelet aggregative dysfunction, and this function is attenuated by antioxidants. Plateletrich plasma (PRP) was prepared from whole blood of normal and endotoxemic rats. The ability of platelet aggregation was measured by an aggregometer. LPS (50–100 μg/mL) was incubated with PRP, whole blood and PRP with polymorphonuclear leukocytes (PMNs) for 30 minutes, 60 minutes and 90 minutes, and platelet aggregation was detected. LPS-induced platelet aggregative dysfunction was undetectable in intact PRP which was isolated from normal whole blood, whereas it was detected in PRP isolated from endotoxemic rats and LPS-treated whole blood. Moreover, the effect of LPS-induced platelet aggregative dysfunction on intact PRP was observed when the PMNs were added. LPS-induced platelet aggregative dysfunction was significantly attenuated by catalase alone and in combination with NG-nitro-L-arginine methyl ester, but not by NG-nitro-L-arginine methyl ester alone. These results indicate that LPS-stimulated PMNs modulate platelet aggregation during LPS treatment and the effects are reversed by antioxidants. PMNs serve as an approach to understand LPS-induced platelet aggregative dysfunction during endotoxemia. During this process, the generation of reactive oxygen species, hydrogen peroxide especially, from LPS-stimulated PMNs could be an important potential factor in LPS-induced platelet aggregative dysfunction. Catalase contributes to the prevention of platelet dysfunction during LPS-induced sepsis