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.

Bright split red fluorescent proteins for the visualization of endogenous proteins and synapses.

Self-associating split fluorescent proteins (FPs) are split FPs whose two fragments spontaneously associate to form a functional FP. They have been widely used for labeling proteins, scaffolding protein assembly and detecting cell-cell contacts. Recently developments have expanded the palette of self-associating split FPs beyond the original split GFP1-10/11. However, these new ones have suffered from suboptimal fluorescence signal after complementation. Here, by investigating the complementation process, we have demonstrated two approaches to improve split FPs: assistance through SpyTag/SpyCatcher interaction and directed evolution. The latter has yielded two split sfCherry3 variants with substantially enhanced overall brightness, facilitating the tagging of endogenous proteins by gene editing. Based on sfCherry3, we have further developed a new red-colored trans-synaptic marker called Neuroligin-1 sfCherry3 Linker Across Synaptic Partners (NLG-1 CLASP) for multiplexed visualization of neuronal synapses in living C. elegans, demonstrating its broad applications.

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.

The receptor protein tyrosine phosphatase CLR-1 is required for synaptic partner recognition.

During neural circuit formation, most axons are guided to complex environments, coming into contact with multiple potential synaptic partners. However, it is critical that they recognize specific neurons with which to form synapses. Here, we utilize the split GFP-based marker Neuroligin-1 GFP Reconstitution Across Synaptic Partners (NLG-1 GRASP) to visualize specific synapses in live animals, and a circuit-specific behavioral assay to probe circuit function. We demonstrate that the receptor protein tyrosine phosphatase (RPTP) clr-1 is necessary for synaptic partner recognition (SPR) between the PHB sensory neurons and the AVA interneurons in C. elegans. Mutations in clr-1/RPTP result in reduced NLG-1 GRASP fluorescence and impaired behavioral output of the PHB circuit. Temperature-shift experiments demonstrate that clr-1/RPTP acts early in development, consistent with a role in SPR. Expression and cell-specific rescue experiments indicate that clr-1/RPTP functions in postsynaptic AVA neurons, and overexpression of clr-1/RPTP in AVA neurons is sufficient to direct additional PHB-AVA synaptogenesis. Genetic analysis reveals that clr-1/RPTP acts in the same pathway as the unc-6/Netrin ligand and the unc-40/DCC receptor, which act in AVA and PHB neurons, respectively. This study defines a new mechanism by which SPR is governed, and demonstrates that these three conserved families of molecules, with roles in neurological disorders and cancer, can act together to regulate communication between cells.

C. elegans avoids toxin-producing Streptomyces using a seven transmembrane domain chemosensory receptor.

Predators and prey co-evolve, each maximizing their own fitness, but the effects of predator-prey interactions on cellular and molecular machinery are poorly understood. Here, we study this process using the predator Caenorhabditis elegans and the bacterial prey Streptomyces, which have evolved a powerful defense: the production of nematicides. We demonstrate that upon exposure to Streptomyces at their head or tail, nematodes display an escape response that is mediated by bacterially produced cues. Avoidance requires a predicted G-protein-coupled receptor, SRB-6, which is expressed in five types of amphid and phasmid chemosensory neurons. We establish that species of Streptomyces secrete dodecanoic acid, which is sensed by SRB-6. This behavioral adaptation represents an important strategy for the nematode, which utilizes specialized sensory organs and a chemoreceptor that is tuned to recognize the bacteria. These findings provide a window into the molecules and organs used in the coevolutionary arms race between predator and potential prey.

Soil-applied imidacloprid translocates to ornamental flowers and reduces survival of adult Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens lady beetles, and larval Danaus plexippus and Vanessa cardui butterflies.

Integrated Pest Management (IPM) is a decision making process used to manage pests that relies on many tactics, including cultural and biological control, which are practices that conserve beneficial insects and mites, and when needed, the use of conventional insecticides. However, systemic, soil-applied neonicotinoid insecticides are translocated to pollen and nectar of flowers, often for months, and may reduce survival of flower-feeding beneficial insects. Imidacloprid seed-treated crops (0.05 mg AI (active ingredient) /canola seed and 1.2 mg AI/corn seed) translocate less than 10 ppb to pollen and nectar. However, higher rates of soil-applied imidacloprid are used in nurseries and urban landscapes, such as 300 mg AI/10 L (3 gallon) pot and 69 g AI applied to the soil under a 61 (24 in) cm diam. tree. Translocation of imidacloprid from soil (300 mg AI) to flowers of Asclepias curassavica resulted in 6,030 ppb in 1X and 10,400 ppb in 2X treatments, which are similar to imidacloprid residues found in another plant species we studied. A second imidacloprid soil application 7 months later resulted in 21,000 ppb in 1X and 45,000 ppb in 2X treatments. Consequently, greenhouse/nursery use of imidacloprid applied to flowering plants can result in 793 to 1,368 times higher concentration compared to an imidacloprid seed treatment (7.6 ppb pollen in seed- treated canola), where most research has focused. These higher imidacloprid levels caused significant mortality in both 1X and 2X treatments in 3 lady beetle species, Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens, but not a fourth species, Coccinella septempunctata. Adult survival were not reduced for monarch, Danaus plexippus and painted lady, Vanessa cardui, butterflies, but larval survival was significantly reduced. The use of the neonicotinoid imidacloprid at greenhouse/nursery rates reduced survival of beneficial insects feeding on pollen and nectar and is incompatible with the principles of IPM.

Non-invasive assessment of oxidative capacity in young Indian men and women: a 31P magnetic resonance spectroscopy study.

It is generally assumed that men display greater strength and muscle capacity than women. However, previous biochemical and histological studies have shown that men have greater capacity for anaerobic metabolism and women have higher or similar oxidative metabolism. Therefore, in the present study, we estimated oxidative capacity of gastrocnemius muscle and compared in Indian men and women using non-invasive in vivo 31P magnetic resonance spectroscopy (MRS). Healthy subjects (8 young males and 9 females, age-matched) performed plantar flexion exercise within a magnet and MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr), ADP, and pH of the calf muscles were carried out using a 1.5 T whole-body MRI system. PCr values during recovery were fitted to an exponential curve, and oxidative capacity was calculated using rate constant (k(PCr)), as an index of oxidative phosphorylation. When men and women were compared for different metabolic ratios, ADP, pH, k(PCr) and oxidative capacity, all parameters turned out to be statistically insignificant. The results showed no gender effect on skeletal muscle oxidative metabolism. The study demonstrated the usefulness of such non-invasive method to indirectly measure the oxidative capacity of the muscle based on PCr recovery.