Locus coeruleus to basolateral amygdala noradrenergic projections promote anxiety-like behavior

Increased tonic activity of locus coeruleus noradrenergic (LC-NE) neurons induces anxiety-like and aversive behavior. While some information is known about the afferent circuitry that endogenously drives this neural activity and behavior, the downstream receptors and anatomical projections that mediate these acute risk aversive behavioral states via the LC-NE system remain unresolved. Here we use a combination of retrograde tracing, fast-scan cyclic voltammetry, electrophysiology, and in vivo optogenetics with localized pharmacology to identify neural substrates downstream of increased tonic LC-NE activity in mice. We demonstrate that photostimulation of LC-NE fibers in the BLA evokes norepinephrine release in the basolateral amygdala (BLA), alters BLA neuronal activity, conditions aversion, and increases anxiety-like behavior. Additionally, we report that β-adrenergic receptors mediate the anxiety-like phenotype of increased NE release in the BLA. These studies begin to illustrate how the complex efferent system of the LC-NE system selectively mediates behavior through distinct receptor and projection-selective mechanisms

Methods for the determination of the structures and dynamics of proteins by solid-state NMR spectroscopy

Protein molecules perform a vast array of functions in living organisms and the characterisation of their structures and dynamics is a key step towards a full understanding of many biological processes. Magic angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy has emerged as a uniquely powerful technique for the extraction of such information at atomic resolution, with mounting successes founded on continual developments in methodology and technology. In this thesis, a number of new approaches for probing the structures and dynamics of proteins are presented, towards the aim of overcoming current challenges regarding sensitivity, spectral resolution and a shortage of quantitative experimental observables. A streamlined method for simultaneously obtaining long-distance homonuclear (13C-13C) and heteronuclear (15N-13C) contacts is introduced that relies on the third spinassisted recoupling (TSAR) mechanism. The experiment, dubbed "time-shared TSAR" (TSTSAR), effectively doubles the information content of spectra and reduces the required experimental time to that needed for just one of the equivalent PAR or PAINCP experiments. An approach for the quantitative study of large proteins and complexes is presented, relying on a combination of proton detection at "ultrafast" (≥55 kHz) MAS frequencies, sample deuteration and optional paramagnetic doping. This is successfully employed for the characterisation of a >300 kDa precipitated complex of the protein GB1 with full length human immunoglobulin (IgG), with only a few nanomoles of sample. Recent advances in MAS technology have enabled spinning frequencies of 100 kHz and above to be obtained. Using the dipeptide β-Asp-Ala, it is found that under such conditions, protons lines are narrowed to an extent similar to that achievable using contemporary homonuclear decoupling methods, leading to a time-efficient method for obtaining resolved spectra of small, natural-abundance molecules. Similar experiments with a GB1-IgG complex sample confirm the technology’s applicability to non-model biological systems, despite the tiny rotor volume of 0.7 μL (≤3 nanomoles of complex). 15N R1ρ relaxation rates are measured for the same complex and compared with identical measurements in crystalline GB1, allowing for a direct comparison between the slow (ns-ms) dynamics of the protein in different molecular environments. Motions on this time scale are found to be more prevalent in the complex, possibly evidence of an overall collective molecular motion. An approach for the measurement of aliphatic 13C relaxation rates in fully protonated samples is presented, based on a combination of ultrafast MAS rates and alternately labelled samples. Sample spinning at ≥80 kHz enables resolved 13Cα-1H correlations, forming a base for 13Cα relaxation experiments that are subsequently performed on crystalline [1,3-13C,15N]GB1 and analysed using a simple model-free (SMF) treatment. It is noted that without further data, this analysis is likely inadequate for an accurate description of the dynamics of the protein. The measurement of 13C’ R1ρ relaxation rates at ultrafast MAS rates is introduced as a probe of backbone protein dynamics in fully protonated samples. 13C and 15N R1 and R1ρ relaxation rates are measured in crystalline [U-13C,15N]GB1 and analysed using the SMF formalism. An examination of simulated spectral densities rationalises the apparent inconsistencies that arise from this and reveals that motions in GB1 occur on at least two time scales. A combined 15N/13C extended model-free (EMF) analysis is conducted for peptide plane motions in GB1, whereupon it is found that the addition of 13C data helps to remove fitting artefacts present in a 15N-only analysis

Intermolecular interactions and protein dynamics by solid-state NMR spectroscopy

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1–antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific 15N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond–nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex

Unraveling the complexity of protein backbone dynamics with combined 13C and 15N solid-state NMR relaxation measurements

Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce 13C spin–lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of 13C′ R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz 1H Larmor frequencies and compare them to 13C′ R1, 15N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when 15N data is used alone. We also discuss how internal motions characterized by different time scales contribute to 15N and 13C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s Disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intra- and inter-molecular contacts, backbone and side chain torsion angles, relaxation measurements, and calculations of chemical shifts. These reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are co-assembled from differently structured monomers, which we describe as a type of ‘intrinsic’ polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. Weshow that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain, and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms

Strengthening the development of the short-rotation plantations bioenergy sector: Policy insights from six European countries

This paper, based on a participatory methodological framework involving expert stakeholders and researchers from six European countries (Germany, Ireland, Poland, Spain, Sweden and UK), analyses the priority issues for the development of short-rotation plantations (SRP), and proposes a series of policy strategies to strengthen this development. The results indicate that there is a lack of awareness of the multifaceted benefits of SRP at the level of farmers, policy makers and public authorities. More research is required to put a value on the multifunctionality of SRP and justify its public support. Small-scale projects using established technologies are also required with energy crops introduced in a phased manner. The simultaneous dissemination of this knowledge upwards to policy makers and downwards to producers and farmers is critical in the success of SRP. Also, greater financial support on both the supply and demand side is highlighted as being necessary: on the supply side linking multifunctional benefits of SRP and targeted payments, along with increased long-term contractual arrangements between farmers and energy plant operators; demand side incentives should overcome any difference in price between fossil fuels and energy crops. Groups to lobby for the uptake and support of SRP and bioenergy are also of necessary

Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules

International audienceMagic-angle spinning solid-state NMR spectroscopy is an important technique to study mo- lecular structure, dynamics and interactions, and is rapidly gaining importance in biomolecu- lar sciences. Here we provide an overview of experimental approaches to study molecular dy- namics by MAS solid-state NMR, with an emphasis on the underlying theoretical concepts and differences of MAS solid-state NMR compared to solution-state NMR. The theoretical foundations of nuclear spin relaxation are revisited, focusing on the particularities of spin re- laxation in solid samples under magic-angle spinning. We discuss the range of validity of Redfield theory, as well as the inherent multi-exponential behavior of relaxation in solids. Ex- perimental challenges for measuring relaxation parameters in MAS solid-state NMR and a few recently proposed relaxation approaches are discussed, which provide information about time scales and amplitudes of motions ranging from picoseconds to milliseconds. We also discuss the theoretical basis and experimental measurements of anisotropic interactions (chemical-shift anisotropies, dipolar and quadrupolar couplings), which give direct infor- mation about the amplitude of motions. The potential of combining relaxation data with such measurements of dynamically-averaged anisotropic interactions is discussed. Although the focus of this review is on the theoretical foundations of dynamics studies rather than their ap- plication, we close by discussing a small number of recent dynamics studies, where the dy- namic properties of proteins in crystals are compared to those in solution

Restoring the Balance: Implementing a National Strategy to Legally Integrate Traditional and Modern Medicine in Senegal

Mentor: Wilmetta Toliver-Diallo From the Washington University Undergraduate Research Digest: WUURD, Volume 6, Issue 2, Spring 2011. Published by the Office of Undergraduate Research, Joy Zalis Kiefer Director of Undergraduate Research and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor