A photocaged orexin-B for spatiotemporally precise control of orexin signaling

Orexin neuropeptides carry out important neuromodulatory functions in the brain, yet tools to precisely control the activation of endogenous orexin signaling are lacking. Here, we developed a photocaged orexin-B (photo-OXB) through a C-terminal photocaging strategy. We show that photo-OXB is unable to activate its cognate receptors in the dark but releases functionally active native orexin-B upon uncaging by illumination with UV-visible (UV-vis) light (370–405 nm). We established an all-optical assay combining photo-OXB with a genetically encoded orexin biosensor and used it to characterize the efficiency and spatial profile of photo-OXB uncaging. Finally, we demonstrated that photo-OXB enables optical control over orexin signaling with fine temporal precision both in vitro and ex vivo. Thus, our photocaging strategy and photo-OXB advance the chemical biological toolkit by introducing a method for the optical control of peptide signaling and physiological function

UV (IUE) spectra of the central stars of high latitude planetary nebulae Hb7 and Sp3

We present an analysis of the UV (IUE) spectra of the central stars of Hb7 and Sp3. Comparison with the IUE spectrum of the standard star HD 93205 leads to a spectral classification of O3V for these stars, with an effective temperature of 50,000 K. From the P-Cygni profiles of CIV (1550 A), we derive stellar wind velocities and mass loss rates of -1317 km/s +/- 300 km/s and 2.9X10^{-8} solar mass yr^{-1} and -1603 km/s +/- 400 km/s and 7X10^{-9} solar mass yr^{-1} for Hb7 and Sp3 respectively. From all the available data, we reconstruct the spectral energy distribution of Hb7 and Sp3.Comment: 4 pages, 3 figures, latex, accepted for publication in Astronomy & Astrophysic

On the Luminosities and Temperatures of Extended X-ray Emission from Planetary Nebulae

We examine mechanisms that may explain the luminosities and relatively low temperatures of extended X-ray emission in planetary nebulae. By building a simple flow structure for the wind from the central star during the proto, and early, planetary nebulae phase, we estimate the temperature of the X-ray emitting gas and its total X-ray luminosity. We conclude that in order to account for the X-ray temperature and luminosity, both the evolution of the wind from the central star and the adiabatic cooling of the post-shocked wind's material must be considered. The X-ray emitting gas results mainly from shocked wind segments that were expelled during the early planetary nebulae phase, when the wind speed was moderate. Alternatively, the X-ray emitting gas may result from a collimated fast wind blown by a companion to the central star. Heat conduction and mixing between hot and cool regions are likely to occur in some cases and may determine the detailed X-ray morphology of a nebula, but are not required to explain the basic properties of the X-ray emitting gas.Comment: ApJ, submitted; 16 page

Physical Structure of Planetary Nebulae. I. The Owl Nebula

The Owl Nebula is a triple-shell planetary nebula with the outermost shell being a faint bow-shaped halo. We have obtained deep narrow-band images and high-dispersion echelle spectra in the H-alpha, [O III], and [N II] emission lines to determine the physical structure of each shell in the nebula. These spatio-kinematic data allow us to rule out hydrodynamic models that can reproduce only the nebular morphology. Our analysis shows that the inner shell of the main nebula is slightly elongated with a bipolar cavity along its major axis, the outer nebula is a filled envelope co-expanding with the inner shell at 40 km/s, and the halo has been braked by the interstellar medium as the Owl Nebula moves through it. To explain the morphology and kinematics of the Owl Nebula, we suggest the following scenario for its formation and evolution. The early mass loss at the TP-AGB phase forms the halo, and the superwind at the end of the AGB phase forms the main nebula. The subsequent fast stellar wind compressed the superwind to form the inner shell and excavated an elongated cavity at the center, but has ceased in the past. At the current old age, the inner shell is backfilling the central cavity.Comment: 10 pages, 6 figures, 1 table, to appear in the Astronomical Journa

Optical tools for visualizing and controlling human GLP-1 receptor activation with high spatiotemporal resolution

The glucagon-like peptide-1 receptor (GLP1R) is a broadly expressed target of peptide hormones with essential roles in energy and glucose homeostasis, as well as of the blockbuster weight-loss drugs semaglutide and liraglutide. Despite its large clinical relevance, tools to investigate the precise activation dynamics of this receptor with high spatiotemporal resolution are limited. Here, we introduce a novel genetically encoded sensor based on the engineering of a circularly permuted green fluorescent protein into the human GLP1R, named GLPLight1. We demonstrate that fluorescence signal from GLPLight1 accurately reports the expected receptor conformational activation in response to pharmacological ligands with high sensitivity (max ΔF/F$_{0}$=528%) and temporal resolution (τ$_{ON}$ = 4.7 s). We further demonstrated that GLPLight1 shows comparable responses to glucagon-like peptide-1 (GLP-1) derivatives as observed for the native receptor. Using GLPLight1, we established an all-optical assay to characterize a novel photocaged GLP-1 derivative (photo-GLP1) and to demonstrate optical control of GLP1R activation. Thus, the new all-optical toolkit introduced here enhances our ability to study GLP1R activation with high spatiotemporal resolution

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires measuring spatiotemporally precise neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators

The Detection of Wind Variability in Magellanic Cloud O Stars

We present Far Ultraviolet Explorer (FUSE) spectra for three Magellanic Cloud O stars (Sk 80, Sk -67 05 and Sk -67 111) with repeated observations. The data demonstrate the capabilities of FUSE to perform time-resolved spectroscopy on extragalactic stars. The wavelength coverage of FUSE provides access to resonance lines due to less abundant species, such as sulfur, which are unsaturated in O supergiants. This allows us to examine wind variability at all velocities in resonance lines for stars with higher mass loss rates than can be studied at longer (lambda > 1150 A) wavelengths. The FUSE wavelength range also includes resonance lines from ions which bracket the expected dominant ionization stage of the wind. Our observations span 1-4 months with several densely sampled intervals of 10 hours or more. These observations reveal wind variability in all of the program stars and distinctive differences in the ionization structure and time scales of the variability. Sk -67 111 demonstrates significant wind variability on a time scale less than 10 hours and the coolest O star (Sk -67 05) exhibits the largest variations in O VI.Comment: 3 pages of text and 3 JPG figures. To be included in the FUSE ApJ Letters special issu

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca2+ influx via NMDA receptors. Here, we show that Ca2+/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca 2+-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca2+-induced dissociation of PSD-95 from the postsynaptic membrane. Synopsis Ca2+ influx promotes Ca2+/calmodulin binding to the N-terminus of PSD-95, which blocks PSD-95 palmitoylation leading to reduced retention of PSD-95 at synapses. This effect will likely decrease postsynaptic glutamate receptor content and thereby synaptic strength. Ca2+/calmodulin forms a collapsed structure around the N-terminal helix of PSD-95 that sequesters the palmitoylation sites (Cys3 and Cys5) and a key tyrosine (Tyr12). Binding of Ca2+/calmodulin to the N-terminus of PSD-95 decreases its palmitoylation to release PSD-95 from postsynaptic sites Binding of Ca 2+/calmodulin also displaces the serine/threonine kinase CDKL5 from PSD-95, which otherwise helps augment synaptic strength A point mutation of PSD-95 that prevents Ca2+/calmodulin binding turns the Ca 2+-induced reduction in PSD-95 at synapses into an increase, uncovering the existence of a second mechanism that augments postsynaptic PSD-95 enrichment upon Ca2+ influx. Ca2+ influx promotes Ca 2+/calmodulin binding to the N-terminus of PSD-95, which blocks PSD-95 palmitoylation leading to reduced retention of PSD-95 at synapses