Reversible low-light induced photoswitching of crowned spiropyran-DO3A complexed with gadolinium(III) ions.

Photoswitchable spiropyran has been conjugated to the crowned ring system DO3A, which improves its solubility in dipolar and polar media and stabilizes the merocyanine isomer. Adding the lanthanide ion gadolinium(III) to the macrocyclic ring system leads to a photoresponsive magnetic resonance imaging contrast agent that displays an increased spin-lattice relaxation time (T₁) upon visible light stimulation. In this work, the photoresponse of this photochromic molecule to weak light illumination using blue and green light emitting diodes was investigated, simulating the emission spectra from bioluminescent enzymes. Photon emission rate of the light emitting diodes was changed, from 1.75 × 10¹⁶ photons·s⁻¹ to 2.37 × 10¹² photons·s⁻¹. We observed a consistent visible light-induced isomerization of the merocyanine to the spiropyran form with photon fluxes as low as 2.37 × 10¹² photons·s⁻¹ resulting in a relaxivity change of the compound. This demonstrates the potential for use of the described imaging probes in low light level applications such as sensing bioluminescence enzyme activity. The isomerization behavior of gadolinium(III)-ion complexed and non-complexed spiropyran-DO3A was analyzed in water and ethanol solution in response to low light illumination and compared to the emitted photon emission rate from over-expressed Gaussia princeps luciferase

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli

Development of a Three-Dimensional In Vitro Model for Longitudinal Observation of Cell Behavior: Monitoring by Magnetic Resonance Imaging and Optical Imaging

Purpose: The aim of this study is the development of a three-dimensional multicellular spheroid cell culture model for the longitudinal comparative and large-scale screening of cancer cell proliferation with noninvasive molecular imaging techniques under controlled and quantifiable conditions. Procedures: The human glioblastoma cell line Gli36ΔEGFR was genetically modified to constitutively express the fluorescence protein mCherry, and additionally labeled with iron oxide nanoparticles for high-field MRI detection. The proliferation of aggregates was longitudinally monitored with fluorescence imaging and correlated with aggregate size by light microscopy, while MRI measurements served localization in 3D space. Irradiation with γ-rays was used to detect proliferational response. Results: Cell proliferation in the stationary three-dimensonal model can be observed over days with high accuracy. A linear relationship of fluorescence intensity with cell aggregate size was found, allowing absolute quantitation of cells in a wide range of cell amounts. Glioblastoma cells showed pronounced suppression of proliferation for several days following high-dose γ-irradiation. Conclusions: Through the combination of two-dimensional optical imaging and 3D MRI, the position of individual cell aggregates and their corresponding light emission can be detected. This allows an exact quantification of cell proliferation, with a focus on very small cell amounts (below 100 cells) using high resolution noninvasive techniques as a well-controlled basis for further cell transplantation studies

Sleep is required to consolidate odor memory and remodel olfactory synapses

Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here, we show that, although the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear if, in any system, sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures

Reversible Low-Light Induced Photoswitching of Crowned Spiropyran-DO3A Complexed with Gadolinium(III) Ions

Photoswitchable spiropyran has been conjugated to the crowned ring system DO3A, which improves its solubility in dipolar and polar media and stabilizes the merocyanine isomer. Adding the lanthanide ion gadolinium(III) to the macrocyclic ring system leads to a photoresponsive magnetic resonance imaging contrast agent that displays an increased spin-lattice relaxation time (T1) upon visible light stimulation. In this work, the photoresponse of this photochromic molecule to weak light illumination using blue and green light emitting diodes was investigated, simulating the emission spectra from bioluminescent enzymes. Photon emission rate of the light emitting diodes was changed, from 1.75 × 1016 photons·s−1 to 2.37 × 1012 photons·s−1. We observed a consistent visible light-induced isomerization of the merocyanine to the spiropyran form with photon fluxes as low as 2.37 × 1012 photons·s−1 resulting in a relaxivity change of the compound. This demonstrates the potential for use of the described imaging probes in low light level applications such as sensing bioluminescence enzyme activity. The isomerization behavior of gadolinium(III)-ion complexed and non-complexed spiropyran-DO3A was analyzed in water and ethanol solution in response to low light illumination and compared to the emitted photon emission rate from over-expressed Gaussia princeps luciferase

Reversible Low-Light Induced Photoswitching of Crowned Spiropyran-DO3A Complexed with Gadolinium(III) Ions

molecule

Contribution of the cyclic nucleotide gated channel subunit, CNG-3, to olfactory plasticity in Caenorhabditis elegans.

In Caenorhabditis elegans, the AWC neurons are thought to deploy a cGMP signaling cascade in the detection of and response to AWC sensed odors. Prolonged exposure to an AWC sensed odor in the absence of food leads to reversible decreases in the animal's attraction to that odor. This adaptation exhibits two stages referred to as short-term and long-term adaptation. Previously, the protein kinase G (PKG), EGL-4/PKG-1, was shown necessary for both stages of adaptation and phosphorylation of its target, the beta-type cyclic nucleotide gated (CNG) channel subunit, TAX-2, was implicated in the short term stage. Here we uncover a novel role for the CNG channel subunit, CNG-3, in short term adaptation. We demonstrate that CNG-3 is required in the AWC for adaptation to short (thirty minute) exposures of odor, and contains a candidate PKG phosphorylation site required to tune odor sensitivity. We also provide in vivo data suggesting that CNG-3 forms a complex with both TAX-2 and TAX-4 CNG channel subunits in AWC. Finally, we examine the physiology of different CNG channel subunit combinations

Aversive Behavior in the Nematode <i>C</i>. <i>elegans</i> Is Modulated by cGMP and a Neuronal Gap Junction Network

<div><p>All animals rely on their ability to sense and respond to their environment to survive. However, the suitability of a behavioral response is context-dependent, and must reflect both an animal’s life history and its present internal state. Based on the integration of these variables, an animal’s needs can be prioritized to optimize survival strategies. Nociceptive sensory systems detect harmful stimuli and allow for the initiation of protective behavioral responses. The polymodal ASH sensory neurons are the primary nociceptors in <i>C</i>. <i>elegans</i>. We show here that the guanylyl cyclase ODR-1 functions non-cell-autonomously to downregulate ASH-mediated aversive behaviors and that ectopic cGMP generation in ASH is sufficient to dampen ASH sensitivity. We define a gap junction neural network that regulates nociception and propose that decentralized regulation of ASH signaling can allow for rapid correlation between an animal’s internal state and its behavioral output, lending modulatory flexibility to this hard-wired nociceptive neural circuit.</p></div

Model for ODR-1 modulation of ASH-mediated nociceptive signaling.

<p>(A) Our working model is that the transmembrane guanylyl cyclase ODR-1 functions in the AWB, AWC and ASI sensory neurons to decrease <i>C</i>. <i>elegans</i> behavioral sensitivity to the bitter tastant quinine (and possibly the volatile odorant octanol). Removal of food leads to cGMP accumulation, at least in the AWCs, likely by direct/indirect activation of ODR-1 in these neurons. Based on the re-annotated wiring diagram (WormWiring.org), we propose that cGMP then flows via gap junction connections from the site of its production in the ODR-1-expressing AWB/AWC/ASI sensory neurons, through ADF, AFD and AIA, to the ASH nociceptors. Once in ASH, cGMP activates the cGMP-dependent protein kinase EGL-4, which likely directly phosphorylates the regulator of G protein signaling proteins RGS-2 and RGS-3, stimulating their activity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref058" target="_blank">58</a>]. RGS-2 and RGS-3 downregulate Gα proteins that signal downstream of G protein-coupled receptors (GPCRs) that are activated by ligands such as quinine and octanol. When animals are well-fed, cGMP levels are low and there is only minimal inhibition of G protein-coupled signaling in ASH. Upon food remove, cGMP influx into ASH activates EGL-4, resulting in diminished nociceptive behavioral sensitivity to a subset of ASH-detected aversive stimuli. Decentralized modulation of ASH sensitivity may allow an animal to integrate multiple environmental cues with its internal state to maximize the appropriateness of its response to its surroundings. (B) The same diagram depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.g005" target="_blank">Fig 5A</a> is shown here, but with only those gap junction connections originally reported in White et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref002" target="_blank">2</a>] included. (C) The left column shows a list of the neurons reported by White et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref002" target="_blank">2</a>] to make gap junctions with ASH, while the right column lists the neurons currently annotated at WormWiring.org to make gap junctions with ASH. The neurons shown in bold are those that are common to both lists.</p

ODR-1 functions in adult sensory neurons.

<p>(A) ODR-1 expression in the AWB, AWC and ASI sensory neurons is sufficient to rescue the behavioral hypersensitivity of <i>odr-1(lof)</i> animals. The <i>str-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref066" target="_blank">66</a>], <i>ceh-36p3</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref065" target="_blank">65</a>], <i>gpa-4</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref064" target="_blank">64</a>], <i>trx-1</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref063" target="_blank">63</a>] and <i>srbc-66</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref062" target="_blank">62</a>] promoters were used to drive expression of wild-type <i>odr-1</i> (genomic sequence) in <i>odr-1(lof)</i> animals. These promoters drive expression in the following cells: <i>str-1</i> (AWB), <i>ceh-36p3</i> (AWC), <i>gpa-4</i> (ASI), <i>trx-1</i> (ASJ and intestinal cells), <i>srbc-66</i> (ASK). While more <i>odr-1(lof)</i> animal respond to 1 mM quinine than do wild-type animals, co-expressing <i>odr-1</i> in the AWB, AWC and ASI sensory neurons returned response to wild-type levels (p > 0.1). (B) ODR-1 functions in adult animals to regulate behavioral sensitivity. Adult <i>odr-1(lof)</i> animals expressing <i>odr-1</i> (genomic sequence) under the control of a heat shock inducible promoter (hsp) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006153#pgen.1006153.ref068" target="_blank">68</a>] were tested without heat shock (white bars) or 4 hours after heat shock treatment (grey bars). While <i>odr-1(lof)</i> animals have a hypersensitive response to dilute (1 mM) quinine, heat shock induced expression of <i>odr-1</i> in adult <i>odr-1(lof)</i> animals abolished this hypersensitivity and returned quinine response to the degree seen in wild-type animals (p > 0.1 when comparing <i>odr-1(lof)</i> animals with heat shock treatment to wild-type animals either with or without heat shock). (C) The extracellular domain of ODR-1 is not required for regulation of quinine sensitivity. In <i>odr-1(lof)</i> animals, the <i>odr-1</i> promoter was used to drive expression of either wild-type ODR-1 (genomic sequence), ODR-1 lacking its extracellular domain (ΔECD) or ODR-1 with a point mutation (E874A) that abolishes GTP binding in the catalytic domain. ODR-1(ΔECD) rescued the hypersensitivity of <i>odr-1(lof)</i> animals as well as wild-type ODR-1 (p > 0.1). Expression of ODR-1(E874A) had no effect on response sensitivity (p > 0.5 when compared to <i>odr-1(lof)</i> animals). The percentage of animals responding is shown. The combined data of ≥ 3 independent lines and n ≥ 120 transgenic animals is shown in each panel. Allele used: <i>odr-1(n1936)</i> loss-of-function. WT = the N2 wild-type strain. lof = loss-of-function. n.s. = not significant.</p