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Caenorhabditis elegans Olfaction
Summary and Keywords
To survive, animals must properly sense their surrounding environment. The types of sensation that allow for detecting these changes can be categorized as tactile, thermal, aural, or olfactory. Olfaction is one of the most primitive senses, involving the detection of environmental chemical cues. Organisms must sense and discriminate between abiotic and biogenic cues, necessitating a system that can react and respond to changes quickly. The nematode, Caenorhabditis elegans, offers a unique set of tools for studying the biology of olfactory sensation.
The olfactory system in C. elegans is comprised of 14 pairs of amphid neurons in the head and two pairs of phasmid neurons in the tail. The male nervous system contains an additional 89 neurons, many of which are exposed to the environment and contribute to olfaction. The cues sensed by these olfactory neurons initiate a multitude of responses, ranging from developmental changes to behavioral responses. Environmental cues might initiate entry into or exit from a long-lived alternative larval developmental stage (dauer), or pheromonal stimuli may attract sexually mature mates, or repel conspecifics in crowded environments. C. elegans are also capable of sensing abiotic stimuli, exhibiting attraction and repulsion to diverse classes of chemicals. Unlike canonical mammalian olfactory neurons, C. elegans chemosensory neurons express more than one receptor per cell. This enables detection of hundreds of chemical structures and concentrations by a chemosensory nervous system with few cells. However, each neuron detects certain classes of olfactory cues, and, combined with their synaptic pathways, elicit similar responses (i.e., aversive behaviors). The functional architecture of this chemosensory system is capable of supporting the development and behavior of nematodes in a manner efficient enough to allow for the genus to have a cosmopolitan distribution.
Keywords: olfaction, chemosensation, connectomics, GPCRs, neural circuits
In its simplest form, olfaction requires a receptor to detect a chemical and transduce a signal within the cell detecting the cue. The evolution of eukaryotic nervous systems has led to the production of specialized cells, the olfactory neurons, which sense, process, and communicate chemical changes sensed in the environment and transmit information to the rest of the organism.
Studies of olfaction in humans and other mammals have found that the olfactory neurons are extremely specialized, and express only one odorant receptor per cell, following a "one neuron, one receptor" rule (Bargmann, 2006b; Bear, Lassance, Hoekstra, & Datta, 2016; Chess, Simon, Cedar, & Axel, 1994; Serizawa et al., 2003). However, these receptors are not specific themselves, and may detect multiple odorants. While much can be learned studying these systems, researchers also investigate evolutionarily distinct olfactory systems, using the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans. Both species offer useful benefits, such as nervous systems that are easily manipulated. The nervous system of Drosophila contains approximately 150,000 neurons (Jenett et al., 2012), compared to approximately 86 billion neurons found in human brains (Azevedo et al., 2009). The C. elegans nervous system, meanwhile, has only 302 neurons (White, Southgate, Thomson, & Brenner, 1986) whose lineal origins and connectivity are known, and therefore is an extremely compact and experimentally tractable nervous system. In C. elegans, approximately 36 neurons contribute to olfaction, and the "one neuron, one receptor" rule is not followed; multiple olfactory receptors are present in individual neurons. Despite this, the molecular mechanisms of olfaction remain conserved, with G protein-coupled receptors, receptor guanylate cyclases, and intracellular signaling cascades sensing and initiating responses to many chemical cues in the environment (Bargmann, 2006a). Studying the C. elegans olfactory network will allow for generation of an in-depth understanding of a complex neural coding strategy: How does this small number of olfactory neurons sense and integrate a myriad of olfactory signals to generate the robust behaviors observed?
Caenorhabditis elegans
The nematode Caenorhabditis elegans is eutelic—every animal has the same number of somatic cells. As such, every cell lineage has been mapped, from fertilized zygote to fully developed adult (Sulston & Horvitz, 1977). As such, the structure of the entire nervous system, comprised of 302 neurons, has been determined using electron micrographs (Ward, Thomson, White, & Brenner, 1975; Ware, Clark, Crossland, & Russell, 1975; White, Southgate, et al., 1986). In recent years, researchers have started characterizing the network's functional connections(Azulay, Itskovits, & Zaslaver, 2016; Bargmann, 2012; Bargmann & Marder, 2013; Hong & Park, 2016; Rengarajan & Hallem, 2016; Sohn, Choi, Ahn, Lee, & Jeong, 2011; Towlson, Vertes, Ahnert, Schafer, & Bullmore, 2013).
A small proportion of C. elegans are male (0.01%–0.02%) (Hodgkin, 1983), which are morphologically distinct from hermaphrodites. Males do not carry eggs and exhibit a fan-shaped tail (Barr & Garcia, 2006). Besides these gross morphological differences, the male nervous system is comprised of 385 neurons, 89 of which are sex-specific (Sammut et al., 2015; Sulston, Albertson, & Thomson, 1980). Only a small number of these sex-specific neurons are thought to function in olfaction. How the addition of these sex-specific neurons changes the connectome of the nervous system has not yet been fully explained (Fagan & Portman, 2014; Garcia & Portman, 2016; M. P. Hart & Hobert, 2015; Portman, 2017; Serrano-Saiz et al., 2017).
C. elegans has become a leading model for understanding olfactory nervous systems, as it has a fully mapped hermaphroditic nervous system, is transparent, and is amenable to genetic manipulations. The nematode allows for the study of mechanisms underlying olfaction using genetically encoded calcium indicators (GECIs) to measure real-time neural dynamics in live animals (Chokshi, Bazopoulou, & Chronis, 2010; Hilliard, Apicella, Kerr, Suzuki, Bazzicalupo, & Schafer, 2005; Schrodel, Prevedel, Aumayr, Zimmer, & Vaziri, 2013; Tatro, 2014; Touhara & Vosshall, 2009). Combining the robust behavioral responses of nematodes with the ease of generating genetic mutants, understanding olfactory mechanisms has dramatically increased through the use of this roundworm.
In their natural milieu—usually rotting fruits and vegetables (Félix & Braendle, 2010; Schulenburg & Felix, 2017; Teotonio, Estes, Phillips, & Baer, 2017)—C. elegans individuals are exposed to a large variety of chemical cues, which signal information about their environment. These cues range from gases and volatiles, to water-soluble compounds, and from abiotic to biogenic cues.
Olfactory Neuronal Anatomy
Although olfaction occurs at both ends of the nematode (Hilliard, Bargmann, & Bazzicalupo, 2002), most research has focused on the neurons within major anterior sensilla, the amphids. The amphids are a pair of channels that each contain sensory dendrites of 12 sensory neurons (Ward et al., 1975; Ware et al., 1975; White et al., 1986) (Figure 1A). Eleven of the amphid neuron-types are chemosensory. In addition to amphid olfactory neurons, the head contains other olfactory neurons: BAG and URX, which are involved in sensing carbon dioxide and oxygen, respectively (Figure 1B), with the aid of the AQR and PQR neurons (Gray et al., 2004).
The IL2 neurons, located anterior to the amphid olfactory neurons, are not present as a bilateral pair, but instead are present as a set of six neurons, with dorsal, ventral, and lateral pairs (Figure 1B). The lateral neurons display a connectivity different from that of the remaining four neurons, further complicating the elucidation of their role (Wang, Schwartz, & Barr, 2010). There are neurons in the tail (phasmids) that sense odorants, though the number of chemosensory neurons in the tail is drastically lower than that seen in the anterior, with only two pairs of phasmid neurons being present that contain cilia exposed to the external environment (Figure 1C).
The Male Nervous System
Of the 89 sex-specific neurons present in the male nervous system, only four located in the amphid region participate in olfaction: the cephalic male (CEM) neurons (White, Nicholas, Gritton, Truong, Davidson, & Jorgensen, 2007). These radially symmetric neurons exhibit dorsal/ventral as well as left/right symmetry, with cilia exposed to the external environment alongside the hermaphroditic amphid olfactory neurons (Figure 1A). The clearly distinguishable male-tail is heavily involved in the mating process and includes the majority of the male-specific neurons. Thirty-six ray neurons innervate this structure (Liu & Sternberg, 1995; White et al., 2007).
The Nature of Amphid Olfactory Neurons
Winged Cilia Olfactory Neurons
Although not exposed to the external environment, winged ciliated neurons are involved in the sensation of volatile cues, which diffuse across the cuticle (Wes & Bargmann, 2001; White et al., 1986). C. elegans is able to distinguish between seven classes of these odors. Most of these volatiles were found to be products of bacterial metabolism, suggesting a biologically relevant role for AWA and AWC—food chemotaxis (Bargmann, Hartwieg, & Horvitz, 1993). Most neurons in C. elegans are present as anatomically symmetrical, bilateral pairs. However, AWC has been shown to exhibit stochastic asymmetry in its gene expression and sensing abilities (Cochella et al., 2014; Pierce-Shimomura, Morse, & Lockery, 1999; Troemel, Kimmel, & Bargmann, 1997; Wes & Bargmann, 2001; Yu, Avery, Baude, & Garbers, 1997).
One of the repulsion-driving olfactory neurons, AWB, also senses volatiles, such as 1-octanol (Troemel, Kimmel, & Bargmann, 1997). The biological relevance of 1-octanol in natural environments can be debated, as it has only been found in the extracts from some enteric Gram negative bacteria (Elgaali, Hamilton-Kemp, Newman, Collins, Yu, & Archbold, 2002). However, given that the volatile metabolites emitted by bacteria are complex mixtures (Hamilton-Kemp, Newman, Collins, Elgaali, Yu, & Archbold, 2005), and the natural food sources of nematodes are incompletely known, 1-octanol serves as a reliable stimulus for AWB in experimental conditions.
The Single- and Double-Rod Ciliated Amphid Neurons
Water-soluble attractants and repellents are sensed by the single- and double-rod ciliated amphid neurons (Table 1). These neurons contain rod-shaped cilia which extend through the amphid sheath and cuticle to sense odorants in the external environment (Bargmann, 2006a; Ward et al., 1975). Like AWC, ASE is asymmetric, but it is more consistent in its asymmetry than AWC, with the right neuron (ASER) always sensing Cl- and K+ ions, and ASEL sensing Na+ (Pierce-Shimomura et al., 1999).
ASH is unique among the amphid sensilla in that it is polymodal and acts as the key nociceptor in C. elegans (Chatzigeorgiou, Bang, Hwang, & Schafer, 2013; de Bono & Maricq, 2005; Hukema, Rademakers, Dekkers, Burghoorn, & Jansen, 2006; Komuniecki, Harris, Hapiak, Wragg, & Bamber, 2012; Walker, Vázquez-Manrique, Gower, Gregory, Schafer, & Baylis, 2009). While most neurons sense an odorant and elicit a general response downstream, ASH's response is more complex, as the downstream signaling exhibits specificity to the stimulus sensed. For example, while OCR-2 and OSM-9 (see Downstream Ion Channels Involved in Olfaction section) are required for all responses within the ASH Neurons, the g-proteins utilized for different GPCRs varies (i.e., either GPA-3 or ODR-3 can function to generate a neuronal response) (Hilliard, Bergamasco, Arbucci, Plasterk, & Bazzicalupo, 2004; Walker et al., 2009).
Largely involved in developmental processes, ASI assists in controlling entry into dauer. ASI is the sole source of DAF-7 and TGF-β in C. elegans, which act to prevent dauer entry (Meisel, Panda, Mahanti, Schroeder, & Kim, 2014; Ren, Lim, Johnsen, Albert, Pilgrim, & Riddle, 1996; Schackwitz, Inoue, & Thomas, 1996). This signaling pathway is regulated by the availability of food, population density, carbon dioxide levels, the presence of dauer pheromone, and temperature, while more recent work has expanded this list to include mRNA decay pathways (Androwski, 2017; Borbolis, Flessa, Roumelioti, Diallinas, Stravopodis, & Syntichaki, 2017). ASI is also required for proper dauer exit and resumption of normal development after stress conditions are mitigated (Ren et al., 1996), as well as being required for withdrawal from noxious stimuli(Mills et al., 2016). The olfactory regulation of dauer control arises through ASI sensation of the majority of daumone constituents: ascr#2, ascr#3, and ascr#5 (Kim et al., 2009; McGrath, Xu, Ailion, Garrison, Butcher, & Bargmann, 2011; Park et al., 2012).
The double-ciliated neuron, ADL, has been shown to sense ascr#3 as well. However, instead of initiating dauer entry or avoidance behaviors, ADL regulates body fat content (Hussey et al., 2017).
The ASK neuron functions in driving both avoidance and attractive behaviors. Removal of ASH through laser ablation experiments results in ASK gaining the ability to sense many aversive stimuli (Hilliard et al., 2002; Hukema et al., 2006; Sambongi et al., 1999). ASK plays a major role in sensing attractive biogenic cues. Icas#1, #3, and #9, indolated derivatives of ascr#1, #3, and #9, respectively, were shown to attract C. elegans of both sexes (Srinivasan et al., 2012). However, this attraction is extremely concentration-dependent across the sexes, with males no longer attracted to low concentrations. ASK has also been shown to sense ascr#3, which is repulsive to hermaphrodites at concentrations that attract males. This likely arises from the combined output of ASK in males with the chemoattraction that is driven by the male-specific CEM neurons (Narayan et al., 2016; Pungaliya et al., 2009; Srinivasan et al., 2008; White & Jorgensen, 2012).
ADF is unique in that it is the only serotonergic sensory neuron in C. elegans hermaphrodites (Sze, Victor, Loer, Shi, & Ruvkun, 2000). Little is known about what ADF may be sensing in the surrounding environment, although it has been shown to contribute to dauer control, as animals lacking ADF display aberrant dauer repression (Schackwitz et al., 1996).
Little has been studied concerning the role of the ASG and ASJ neurons. ASJ is known to contribute to dauer entry and recovery, but what exactly it senses in the environment to initiate these developmental changes remains unknown. ASJ has also been shown to be involved in the sensation of pathogenic bacteria (Meisel et al., 2014). It is likely that, like ASI, these neurons sense a combination of pheromone cues, population density cues, and information about food availability. This, however, remains to be determined.
Male-Specific Ciliated Amphid Olfactory Neurons
Male C. elegans contain four extra ciliated neurons that function as olfactory neurons—the CEM neurons. Ascarosides #3 and #8, both of which contribute to the induction of dauer, also elicit repulsion of hermaphrodites, yet attract males, are sensed by these neurons (Jang et al., 2012; Macosko et al., 2009; Narayan et al., 2016; Pungaliya et al., 2009; Srinivasan et al., 2008).
Oxygen and Carbon Dioxide Sensing Neurons
Water soluble and volatile compounds are not the only stimuli sensed by olfactory neurons. Oxygen and carbon dioxide levels are sensed by a small subset of sensory neurons not included in the classical set of amphid olfactory neurons (Carrillo & Hallem, 2015).
The ciliated BAG neurons sense CO2 levels (Bretscher, Busch, & de Bono, 2008; Bretscher et al., 2011; Busch et al., 2012; Zimmer et al., 2009), and drive avoidance behaviors in situations in which there are elevated levels of the gas (Hallem & Sternberg, 2008). With help from ASE, and the canonical thermosensory neuron AFD, BAG is the main sensor of CO2 in the nematode (Bretscher et al., 2011).
The URX neurons, which are not ciliated, also develop in the amphid region and contain dendrites that are exposed to the pseudocoelomic fluid (Styer, Singh, Macosko, Steele, Bargmann, & Aballay, 2008). In combination with the AQR and PQR neurons, URX helps C. elegans aerotax towards ideal O2 levels (Chang, Chronis, Karow, Marletta, & Bargmann, 2006; Gray et al., 2004).
Other Amphid Olfactory Neurons
The IL2 neurons have cilia exposed to the external environment, but what they sense and what they communicate is unknown. In dauer larvae, IL2 cilia regulate a dispersal behavior, though it remains unknown how the cilia are activated to induce this output (Lee et al., 2012; Schroeder et al., 2013).
The Nature of Phasmid Olfactory Neurons
Although the roles of PHA and PHB have been determined (Barrios, Ghosh, Fang, Emmons, & Barr, 2012; Hilliard et al., 2002), what these neurons are sensing remains largely unknown. Many of the sex-specific neurons present in males are located in the tail, increasing the overall number of neurons exposed to the environment, increasing phasmid olfactory capability (White et al., 2007). Three pairs of ray neurons also contribute to an increase in the number of serotonergic olfactory neurons (Jafari, Xie, Kullyev, Liang, & Sze, 2011; White et al., 2007). It has not been yet determined exactly what these olfactory neurons sense.
The distal tips of the copulatory spicules contain the exposed cilia of the SPD and SPV neurons. Given the spicules' role during mating, it is proposed that these neurons sense vulval pheromones to manage release of sperm into the correct location (LeBoeuf, Correa, Jee, & García, 2014; Liu & Sternberg, 1995; Schindelman, Whittaker, Thum, Gharib, & Sternberg, 2006). To date, there has been no identification of these proposed vulval pheromones.
Assays Used in the Study of C. elegans Olfaction
Behavioral Assays
The most ethologically relevant output of sensation to study is behavior. With obtainable results on short timescales, the effect of a stimulus on C. elegans behavior can be studied both in fine detail and in a high-throughput manner (Ghosh, Nitabach, Zhang, & Harris, 2017; Maruyama, 2016). The simplest of observable behaviors are attraction and repulsion. In fact, this chemotaxis was studied in C. elegans even before Brenner's initial push for the use of the nematode as a standard model system (Ward, 1973).
The eutelic and transparent nature of C. elegans allows for reliable identification and laser ablation of specific cells. In these ablations, a laser is focused and pulsed at the nucleus of a neuron of interest, effectively killing the cell (Fang-Yen, Gabel, Samuel, Bargmann, & Avery, 2012). Nematodes undergo this laser surgery as larvae, and are then allowed to develop in the absence of the ablated cell, after which, the animal can be assayed, and changes in outputs observed.
In 1973, Ward et al. showed that C. elegans are able to respond to attractants by moving up a concentration gradient, accumulating in the area of the cue, and then habituating to the cue (Figure 2A) (Ward, 1973). In the now-canonical population chemotaxis assay developed by the Bargmann lab, the number of C. elegans at selected time points within specified attractant areas are counted to generate a chemotaxis index (Figure 2A) (Bargmann et al., 1993). Changes in this index amongst mutants or laser-ablated animals have helped identify neurons and neuromodulators that play roles in attractive olfaction.
C. elegans utilize two independent mechanisms to chemotax towards ideal concentrations of odorants: pirouettes (klinokinesis) and weathervaning (klinotaxis). Characterization of changes in the uses of these methods of chemotaxis in mutant animals has helped to identify the underpinnings of how C. elegans reach an ideal environment (Chalasani et al., 2007; Lockery, 2011; Luo, Gabel, Ha, Zhang, & Samuel, 2008). The pirouette mechanism is comprised of "bouts of sharp turns," usually including a reversal and/or ω turn (Iino & Yoshida, 2009). These sharp turns are initiated upon a sensation of a decrease in the concentration of an attractant, such as salt (Pierce-Shimomura et al., 1999), although they also utilize this method in response to volatile odorants (Chalasani et al., 2007).
In contrast to pirouettes, C. elegans also employ a more gradual curving towards an attractant, termed weathervaning (Iino & Yoshida, 2009). This behavior was first proposed in 1973, but there was no further evidence supporting this behavior until over three decades later (Iino & Yoshida, 2009; Ward, 1973). To generate this curve, the amphid sensory neurons were found to sense changes in odorant concentrations at each apex of the head swing during the sinusoidal movement of the worm (Izquierdo & Lockery, 2010; Kato, Xu, Cho, Abbott, & Bargmann, 2014; Larsch, Flavell, Liu, Gordus, Albrecht, & Bargmann, 2015). Iino and Yoshida proposed that slightly larger head swings are then generated in the direction of an attractant, generating the curving motion (Iino & Yoshida, 2009).
Avoidance assays have also been established to aid in the understanding of how C. elegans senses chemical cues in its environment. A drop assay was developed by Hilliard et al., which exposes a forward moving animal to a drop of a soluble chemical cue (Figure 2C) (Hilliard et al., 2002; Hilliard, Bergamasco, Arbucci, Plasterk, & Bazzicalupo, 2004). An avoidance index is then calculated by determining the fraction of worms that initiate reversals upon exposure to the cue. There is also a population-level aversion assay in which worms are placed on one side of a line of aversive chemical. A volatile attractant is then placed on the opposite side of the aversive chemical line (Figure 2D). An aversion index is calculated by dividing the number of worms that cross the boundary in one hour by the total number of worms on the plate (Hart, 2006).
C. elegans senses oxygen and carbon dioxide levels through olfactory neurons. Aerotaxis studies involve specialized devices, in which a gas gradient can be generated by pumping opposing gases into a chamber housing an agar arena (Figure 2E) (Gray et al., 2004). A score is given based upon where animals are found within the gradient, and mutants can easily be determined by changes in this distribution.
Assays allowing for the study of more complex behaviors (such as aggregation, foraging, and dispersal) have also been developed, as advancements in worm tracking and technological capabilities have been made (Greene et al., 2016b; Milward, Busch, Murphy, de Bono, & Olofsson, 2011; Sambongi et al., 1999). With greater software and coding capabilities, worm tracking is now common, and even a marketable process. Many labs have developed their own tracking software (Albrecht & Bargmann, 2011; Buckingham, Partridge, & Sattelle, 2014; Chalasani et al., 2007; Cronin, Feng, & Schafer, 2006; Faumont et al., 2011; Feng, Cronin, Wittig, Sternberg, & Schafer, 2004; Kawano, Po, Gao, Leung, Ryu, & Zhen, 2011; Leifer, Fang-Yen, Gershow, Alkema, & Samuel, 2011; Stirman, Crane, Husson, Gottschalk, & Lu, 2012; Swierczek, Giles, Rankin, & Kerr, 2011; Tsechpenakis, Bianchi, Metaxas, & Driscoll, 2008; Tsibidis & Tavernarakis, 2007), and companies exist yhat offer user friendly software for purchase, such as WormLab—developed by MBF Bioscience. There are options available for the tracking of single worms or populations of worms, either on agar plates, in suspension, and more. Analyses with these trackers include parameters such as speed, turning frequencies, and bending angles, with multi-worm trackers even being able to distinguish between two worms that come into contact with each other. In-depth descriptions of available worm-trackers, with side-by-side comparisons are available (Husson, Costa, Schmitt, & Gottschalk, 2012).
Developmental Assays
As a result of processing information about their immediate environment and their developmental state, nematodes emit small molecules to communicate information to conspecifics. To do so, they utilize a class of biogenic, small-molecule pheromones, termed ascarosides. To date, ascarosides have been found to mediate entry into dauer, an alternative and environmentally persistent developmental state (the collective mix of ascarosides that signal dauer formation are termed "daumone" (Golden & Riddle, 1982)), foraging behavior, avoidance behavior, and mate recognition (Butcher, 2017a, 2017b; Chute & Srinivasan, 2014; Ludewig & Schroeder, 2013).
In dauer-formation assays (Figure 3A), worms are allowed to develop on plates containing chemicals that may induce dauer development. Since dauer worms survive harsh conditions, the worms surviving after treatment can be scored and compared to the pre-treatment population to calculate the fraction of induced dauer larvae (Jeong et al., 2005). Alternatively, dauers can also be identified "based on size, shape, and lack of pharyngeal pumping" (Butcher, Fujita, Schroeder, & Clardy, 2007).
In dauer recovery assays (Figure 3B), worms are cultured in liquid media and allowed to form dauers. Non-dauer worms are removed from the culture, and the dauers are then placed on an agar plate containing chemicals that may induce exit from dauer. After two days, dauer and non-dauer worms are visually scored (Butcher et al., 2007).
Imaging of Neural Activity in Olfactory Neurons
With the development of genetically encoded calcium indicators (GECIs), visualization of neuronal activity in vivo has become possible (Nakai, Ohkura, & Imoto, 2001). These proteins allow for real-time readout of intra-cellular calcium levels. Original GECIs utilized Green Fluorescent Protein (GFP) as their fluorescent protein in the fusion (generating the GCaMP family of GECIs), although recent developments have incorporated red-shifted chromophores, such as RFP (RCaMPs) (Akerboom et al., 2012, 2009; Sun et al., 2013).
By expressing GECIs in olfactory neurons, and exposing C. elegans to an olfactory stimulus, the depolarization and hyperpolarization of sensory neurons can not only be visualized, but measured (Hilliard et al., 2005). Microfluidic devices allow for the containment of nematodes in a manner that allows for capture of the fluorescent dynamics of GECIs upon exposure to olfactory cues (Reilly, Lawler, Albrecht, & Srinivasan, 2017). Animals can be assayed while "trapped" in devices, or allowed to roam in "arenas" (Chronis, Zimmer, & Bargmann, 2007; Lagoy & Albrecht, 2015; Larsch, Ventimiglia, Bargmann, & Albrecht, 2013).
Mechanisms of Olfaction
Molecular and Cellular Mechanisms
G Protein-Coupled Receptors
The C. elegans genome encodes approximately 1,200 G protein-coupled receptors (GPCRs), over half of which are proposed to be chemosensory in nature. Only a handful of receptors have been directly linked to sensation of specific cues (Bastiani & Mendel, 2006). This has likely arisen due to the redundancy of olfactory neurons in the nematode. With only approximately three dozen neurons participating in olfaction, there is a remarkable amount of GPCR co-expression within olfactory neurons. As such, the redundant nature of these receptors has caused genetic screens to be ineffective in linking receptors to biological roles. In the two decades since the finding that ODR-10 senses diacetyl (Sengupta, Chou, & Bargmann, 1996), only a handful of GCPRs have been linked to specific targets: SRBC-64 and -66, DAF-37 and -38, SRG-36 and -37 sense dauer inducing ascarosides (Kim et al., 2009; McGrath et al., 2011; Park et al., 2012), SRX-43 and SRX-44 sense icas#9 to control foraging behaviors (Greene et al., 2016b; Greene, Dobosiewicz, Butcher, McGrath, & Bargmann, 2016a), while SRI-14 senses high concentrations of diacetyl alongside the low-concentration sensing ODR-10 (Sengupta et al., 1996; Taniguchi, Uozumi, Kiriyama, Kamizaki, & Hirotsu, 2014).
GPCRs can bind ligands present in the worm's environment, and, in conjunction with associated G proteins, propagate signaling cascades. The remaining GPCRs, which are not expected to be chemosensory in nature, likely play other roles in neurotransmission (Bargmann, 1998; Bargmann, 2006a).
The original method of determining GPCR expression patterns was through the generation of transcriptional fusions, wherein the promoter of the GPCR gene of interest drives expression of GFP. These first tests showed that GPCRs in C. elegans are expressed in either single or multiple neuronal classes (Taniguchi et al., 2014; Troemel, Chou, Dwyer, Colbert, & Bargmann, 1995). As expected, it was confirmed that GCPRs, which function as chemoreceptors in these olfactory neurons, are localized to the cilia of these neurons (Dwyer, Troemel, Sengupta, & Bargmann, 1998; Troemel et al., 1995).
This has continued to hold true with the advent of ascaroside receptor discoveries, using both fusions and immunostaining (Greene et al., 2016b; Greene, Dobosiewicz, et al., 2016a; Kim et al., 2009; McGrath et al., 2011; Park et al., 2012). GPCRs have been shown to function as heterodimers, with two unique receptors involved in the sensation of each cue. In this sensation method, some receptors function as primary binders of specific targets, while the other member may function as a partner for multiple primary receptors in the sensation of stimuli (Park et al., 2012). Despite the relatedness of ascarosides in terms of core structure, the receptors identified to date are widespread across the GPCR families present in C. elegans; genes have been discovered to be responsible for ascaroside sensation in the srbc, srg, srw, and srx families (Greene et al., 2016b; Greene, Dobosiewicz, et al., 2016a; Kim et al., 2009; McGrath et al., 2011; Park et al., 2012).
Stimuli sensed by GPCRs need not be as structurally complex as ascaroside pheromones, however. In fact, the ketone diacetyl was found to be sensed by two different GPCRs, dependent on the concentration of the stimuli: ODR-10 for low concentrations and SRI-14 for high concentrations. Diacetyl elicits an attractive response when sensed by the AWA neurons and bound to ODR-10 (Sengupta et al., 1996). However, high concentrations are sensed by SRI-14, expressed in the nociceptive ASH neuron, and produce aversive behaviors (Taniguchi et al., 2014).
G protein-coupled receptors would not function properly without the related G proteins and downstream intracellular signaling cascades. Within the C. elegans genome, there are twenty Gα, two Gβ, and two Gγ subunits (Jansen, Thijssen, Werner, van derHorst, Hazendonk, & Plasterk, 1999). Due to the presence of hundreds of GPCRs in the olfactory neurons, it is understandable that multiple subunits may be expressed in individual neurons. Because of this, the roles of individual Gα subunits vary in importance (Bastiani & Mendel, 2006).
Downstream Ion Channels Involved in Olfaction
Downstream of GPCRs and their related G-protein complexes(Cuppen, van der Linden, Jansen, & Plasterk, 2003), C. elegans use two canonical intracellular signaling cascades to elicit depolarizations (Bargmann, 2006a): cyclic nucleotide-gated channels, and transient receptor potential (TRP) channels, which are utilized in two separate subsets of olfactory neurons (Table 1) (Coburn & Bargmann, 1996; Colbert & Bargmann, 1997; Komatsu, Mori, Rhee, Akaike, & Ohshima, 1996; Tobin et al., 2002).
Olfactory neurons utilizing cyclic nucleotide-gated channels to initiate depolarizations express TAX-2 and TAX-4 as a heterodimer in the cilia of the neuron (Table 1). This channel is downstream of receptor-type guanylate cyclases, which induce influxes of cGMP, and lead to a flood of sodium or calcium into the neuron (Ortiz et al., 2006). These channels are also functional in the oxygen and carbon dioxide sensing neurons, where they are activated by soluble guanylate cyclases (Bretscher et al., 2011; Carrillo, Guillermin, Rengarajan, Okubo, & Hallem, 2013; Cheung, Arellano-Carbajal, Rybicki, & de Bono, 2004; Gray et al., 2004; Hallem et al., 2011; Hallem & Sternberg, 2008; Yu et al., 1997).
While receptor-type guanylate cyclases (rGCs) are crucial toward generating cGMP flux downstream of GCPRs in TAX-2/TAX-4 neurons (Ortiz et al., 2006), there are a few instances wherein they act as chemoreceptors themselves. For example, GCY-9 functions to directly bind CO2 in the BAG neurons, while GCY-14 serves as a pH indicator in the left ASE neuron (Murayama & Maruyama, 2013; Murayama, Takayama, Fujiwara, & Maruyama, 2013; Smith, Martinez-Velazquez, & Ringstad, 2013). The large, nematode specific expansion of rGCs (Fitzpatrick, O'Halloran, & Burnell, 2006) suggests that there are more playing as yet unknown but direct roles in chemosensation.
Conversely, olfactory neurons can utilize heterodimeric TRP channels to provoke depolarizations in the sensory cilia. OSM-9/OCR-2 form a heterodimeric channel in these TRP-utilizing cilia (Table 1). In Table 1, there is no overlap between cells expressing both tax-2 and tax-4, and cells expressing both osm-9 and ocr-2, but there is some overlap between tax-2, tax-4 and osm-9 alone. In this signaling cascade, G proteins regulate lipid metabolism which, in turn, activate the TRP channel, leading to the desired depolarization (Kahn-Kirby et al., 2004).
Table 1. Olfactory Neurons and Cilium Structures
Olfactory Cues Sensed
Intracellular Components
osm-9, ocr-2
tax-2, tax-4, osm-9
Single Rod
tax-2, tax-4, osm-9
Single Rod
Dauer Control
tax-2, tax-4, osm-9
Single Rod
Aversive Stimuli
osm-9, ocr-2
Single Rod
Dauer Control/Foraging
Daumone, Icas#9
tax-2, tax-4, osm-9
Single Rod
Dauer Control
tax-2, tax-4, osm-9
Single Rod
Icas, ascr#3
tax-2, tax-4, osm-9
Double Rods
Dauer Control
osm-9, ocr-2
Double Rods
Water solubes, ascr#3, icas#9
osm-9, ocr-2
tax-2, tax-4
Single Rod
osm-9, ocr-2
Male Attraction
ascr#3, ascr#8
Vulval Pheromones
Circuit-Level Analysis
Integration of Olfactory Inputs
Unlike other known olfactory systems studied in neuroscience, such as that of the mouse, C. elegans olfaction relies on only a small subset of neurons, requiring co-expression of receptors within individual neurons. This is in juxtaposition to the one-neuron-one-receptor system observed in mammals (Bargmann, 2006b; Chess et al., 1994; Serizawa et al., 2003). The majority of this limited number of olfactory neurons converges onto a small subset of interneurons, largely responsible for integration of sensation. Without this integration, proper olfactory responses would be impossible. This layer of neuronal networks is critical when investigating olfactory mechanisms.
With the exception of ASJ and URX, all of the amphid olfactory neurons synapse onto at least one member of the "first-layer interneurons": AIA, AIB, AIY, and AIZ (Ward et al., 1975; Ware et al., 1975; White, Southgate, Thomson, et al., 1986). It is these neurons that are speculated to integrate the information from the upstream neurons, and initiate proper behavioral or developmental responses. These "first-layer interneurons" are responsible for summing and integrating sensory input, before passing the information downstream onto either motor neurons or the command interneurons which control forward and reverse locomotory circuitries (Figure 4) (Chalfie et al., 1985). The complex and recurrent connectivity among these four neurons allows for fine-tuning and integration of sensory input prior to initiating downstream signaling.
Interneurons such as RMG have are thought to serve as hubs for integration of olfactory input. RMG receives inputs from six amphid olfactory neurons, and modulates aggregation and other social behaviors (Figure 5A) (Macosko et al., 2009). Likewise, neurons such as AWA and ASH modulate the RIM interneuron to initiate backward and inhibit forward movements (Figure 5B) (Ghosh et al., 2016). While they are not directly responsible for sensing olfactory stimuli in the environment, it is critical to include these synaptic partners in studies of olfaction, as the regulatory roles of these neurons contribute immensely to the complexity of olfactory circuits.
A Functional Foraging Circuit
Icas#9, a weak inducer of dauer, promotes foraging behavior. Work by Greene, Dobosiewicz, et al. (2016a) unveiled a circuit that senses icas#9 through three olfactory neurons: ASI, ASJ, and ADL (Figure 5C) (Greene et al., 2016a, 2016b). ASI promotes roaming when sensing icas#9 in opposition to ADL (Figure 5C). ASJ senses icas#9 through a different receptor than that present in ASI, yet drives the same behavior (Greene, Dobosiewicz, et al., 2016a). ADL, utilizing the same receptor found in ASJ, represses the foraging behavior promoted by ASI and ASJ.
Responding to Volatile Odors
The AWC-AIA-AIY circuit is a complex circuit that tunes responses to volatile odors. AWC functions in an "odor-off" fashion; when odors are removed, AWC inhibition of the downstream interneuron AIY is relieved (Figure 5D) (Chalasani et al., 2007). Upon removal of the odor, glutamatergic inhibition of AIY resumes. In turn, the response of AWC to these odors is modulated by the AIA interneuron (Figure 5D) (Chalasani et al., 2010).
Nociception and Avoidance
ASI and ASH both sense copper to drive avoidance behaviors. The "reciprocal inhibition" observed in these neurons tunes this avoidance behavior. ASI activates ADF, which, in turn, functions to inhibit ASH (Figure 5E). ASH, meanwhile, activates the RIC interneuron, which then works to inhibit ASI signaling (Guo et al., 2015).
ASH has also been shown to sense 1-octanol and to activate the AIB interneuron and the AVA motor neuron, which drives backwards locomotion (Figure 4). AWC also contributes towards the rate of spontaneous reversals by activating the AIB interneuron (Figure 5D). However, AWC is inhibited by both 1-octanol and food, preventing it from activating the AIB interneuron. The removal of food alters internal 5-HT levels (Harris et al., 2011, 2009), which in turn also inhibits the AIB interneuron. This cumulative inhibition of the AIB interneuron results in inhibition of reversals and, upon completion of a reversal, re-initiation of forward movement (Summers, Layne, Ortega, Harris, Bamber, & Komuniecki, 2015).
Male Ascaroside Sensation
Many functional connectomes remain to be understood. Male-specific CEM neurons sense ascarosides #3 and #8 to attract males to receptive hermaphrodites (Srinivasan et al., 2008). However, while the class of neurons is required for proper response to these cues, not all four CEM neurons elicit the same changes in calcium levels (Narayan et al., 2016). A subset of CEM neurons depolarize, others hyperpolarize, and others seem to not respond at all. These subsets vary from animal to animal, suggesting that the cumulative output of the CEM neurons is more important than individual CEM neuron response. Given that the four neurons respond differently, it is likely that there is cross-talk occurring between the four neurons, possibly similar to the ASI-ASH "reciprocal inhibition."
Future Directions
Given the complexity of the olfactory system in C. elegans, genetic and molecular approaches need to be complemented with functional circuit studies. Identifying receptors responsible for the sensation of olfactory stimuli coupled with elucidating the functional connections of the nematode, will improve the understanding of the olfactory connectome.
Functional Connectomics
The initial wave of C. elegans neuronal circuit dissections focused heavily on understanding the physical connectome. Many studies have since shown that neurons communicate heavily outside of synaptic connections suggesting a much bigger picture of how neural circuits function (Bargmann, 2012; Bentley et al., 2016; Rajendran et al., 2014). Canonically, only neuropeptide signals have been known to function in a long-range, extra-synaptic fashion (Leinwand & Chalasani, 2014; Mills et al., 2012). However, recent studies have shown that the monoaminergic nervous system communicates in context-dependent and extra-synaptic manners (Hardaway et al., 2015; Komuniecki, Hapiak, Harris, & Bamber, 2014). Also, extracellular vesicles are playing increasingly important roles in extra-synaptic cell-to-cell signaling and circuit regulation (Rajendran et al., 2014; Wang et al., 2015).
Increasingly, the physiological state or age of C. elegans is being found to influence the functional aspects of the connectome (Chao, Komatsu, Fukuto, Dionne, & Hart, 2004; Ghosh et al., 2016; Leinwand, Yang, Bazopoulou, Chronis, Srinivasan, & Chalasani, 2015). To completely elucidate the functional connectome, factors such as age, sex, physiological state, and life history, must be taken into account and assayed independently. A likely outcome of these studies is that many overlapping functional connectomes will be generated providing deeper insights into the role of neuroregulators.
Receptor Identification
To fully understand olfaction, the mechanistic underpinnings of the process must be revealed. To do this, the receptors that physically sense and bind these cues need to be identified. Due to the inherent complexity that arises from co-expression (Hostettler, Grundy, Kaser-Pebernard, Wicky, Schafer, & Glauser, 2017; Troemel et al., 1995) and heterodimerization (Park et al., 2012) of GPCRs, fully identifying receptor complexes will require an immense amount of investigation. With multiple receptors being required for sensing different concentrations of the same cue, the mechanisms of olfaction have become even more complex than initially believed (Taniguchi et al., 2014).
There are many approaches currently available for identifying receptors, ranging from RNAi knockdowns to quantitative trait locus mapping for identification of chromosomal regions responsible for proper olfactory response (Greene et al., 2016b; Greene, Dobosiewicz, et al., 2016a; Taniguchi et al., 2014). The development of biochemical probes increases the scope by which identifying olfactory receptors can be achieved. These probes are modified cues, containing functional groups that can be photo-activated to covalently bind to the receptor, allowing for direct confirmation of binding. These probes can also be constructed to allow for FRET analysis following covalent binding, removing the need for purification to identify the receptor (Park et al., 2012). Recently, genomic and transcriptomic investigations have been used to match receptor expression profiles with ligand targets (Greene et al., 2016b; Greene, Dobosiewicz, et al., 2016a; Greer et al., 2016).
While much has been discovered about molecular and circuit mechanisms of olfaction, much remains to be understood. C. elegans' experimental amenability and defined connectome offers an ideal tool for addressing these gaps by providing a "systems-level" analysis of olfaction. Increases in neuronal imaging capabilities allows for unrestrained live imaging with minimal alterations being made to the natural state of the animal. This allows for circuit-level read-outs to be deciphered. Recent studies have focused on understanding global control of olfaction (Kato et al., 2015; Nguyen et al., 2016; Venkatachalam et al., 2016). Future work will likely focus on uncovering the regulation of olfactory circuits that integrate olfactory stimuli along with targeted identification of individual GPCRs necessary for olfactory sensation.
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text:
Day's Worst: Greenhouse gas emissions hit record high
Word that greenhouse gas emissions hit a record high in 2011 is bad news for the planet.
It's also another indication that more urgency must be injected into discussions of how to reduce the concentration of this principal global warming pollutant.
Concentrations of the gas, which traps heat near the earth's surface, are up 40 percent since the beginning of the Industrial Age, according to the World Meteorological Organization.
As it so happens, the United Nations begins a new round of climate change talks next week in the Qatari capital of Doha.
World leaders must come to agreement on a way to stabilize greenhouse gas emissions so the world's climate does not become dangerously unstable.

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Dedicated do-it-yerselfer with 15 years experience. I know enough to avoid screwing it up most of the time. I also know when to ask advice. I can sweat a pipe or de-sweat a pipe. Install a hot water heater, toilet, sink, dishwasher, stove, alternater, starter, CPU power supply, replace a motherboard, write a macro, sell a house a car a boat a camper, install a toilet, work with black pipe, pvc, install a sprinkler head, frame a wall, sheet rock a wall, install a header, run wiring, install a new or old work high hat.
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So, I can do a little of everything. If your reading this you are probably wondering who I am that's about it.


text:
RAID: It's alive
RAID is still relevant today, possibly even more than in the past. However, it has become a transparent, standard feature that isn't often discussed. This tip outlines the state of RAID today.
In 2006, we saw the celebration of the 50th anniversary of the magnetic hard disk drive (HDD). And, in 2007, we have the 20th anniversary of the publication of the University of California Berkley RAID white paper A Case for Redundant Arrays of Inexpensive Disks (RAID)." Given the focus on virtualization (server, storage, networks and I/O), continuous data protection (CDP), grids, deduplication, iSCSI, VTLs, NAS and object-based storage, it's no wonder RAID has become lost in the crowd. Or, has it?
RAID has fallen out of the limelight as a feature that vendors spend a lot of time talking about, which speaks to its level of maturity. In fact, some have even declared RAID as a dead or "zombie" technology that is no longer relevant even though it continues to grow in adoption on enterprise, midrange, small and midsized businesses (SMB) and small office/home office (SOHO) storage systems, adapter cards and via RAID on Chip (ROC), and RAID on motherboard (ROMB) deployments (Figure-1). In other words, with continued enhancements, RAID has reached the plateau of productivity having left the hype cycle evidenced by the industry association RAID Advisory Board (RAB) falling by the side of the road several years ago.
Figure-1: - Various types of storage and RAID controllers (Chips, Adapters, Systems)
RAID should be thought of as a tool to enhance and delivery various level of storage services, including performance and availability. On one hand, disk drives continue to become more reliable with larger capacities. However, more data is being stored, which raises exposure risks when a disk drive fails. In addition to the availability aspect of RAID and sibling data protection technologies, including local and remote mirroring or replication to other storage systems, RAID also provides performance enhancements to help address the ever growing storage performance I/O gap between available capacity and performance.
Two of the most common knocks on RAID-6 and multidrive parity is the performance impact during writes or rebuilds along with the cost of an extra disk drive for parity compared to RAID-5. RAID-6 does in fact use twice the number of drives for parity compared to RAID-5. However, it still uses fewer drives than a mirror set or hybrid mirror and parity scheme like RAID-51.
RAID-6 (dual parity) and other multidrive parity schemes, including Network Appliance Inc.'s (NetApp) RAID-DP, that are targeted at reducing data availability exposure during rebuild of large capacity Fibre Channel, SAS and SATA disk drives, have come under pressure lately, similar to how RAID-5 was criticized 10 years ago. The issue with RAID-6 and any multidrive parity scheme is that of the performance overhead when doing parity calculations, writing data or rebuilding from a failed disk drive.
Keep the parity and "protection overhead" story in perspective to the level of service, cost and protection needed for your applications. Regarding the performance impact, different RAID controllers (ROCs, adapters and external controllers) handle RAID-6, other RAID levels and application workloads differently. Some controllers have offload and performance acceleration for RAID-5 and RAID-6 rebuild or write operations, some are optimized for OLTP, while others are optimized for large sequential stream applications.
Check out the entire RAID Handbook.
About the author: Greg Schulz is founder and senior analyst with the IT infrastructure analyst and consulting firm StorageIO Group. Greg is also the author and illustrator of Resilient Storage Networks (Elsevier) and has contributed material to Storage magazine and other TechTarget venues.


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Winter holiday in Merano in South Tyroll
On the slopes in just 15 minutes from the hotel
Tobogganing, snow shoe hiking and skiing in South Tyrol
Skiing and snowboarding in the new and modern Merano 2000 ski region is a real highlight. At up to 2,300 metres altitude, winter sports enthusiasts have the time of the lives from December to March in this winter paradise.
A shuttle bus will take you from the hotel to the ski region's cable car base station in just 15 minutes - several times a day. Short distances and the best snow conditions await you during your winter holiday in Merano.
5* hotel in the Merano 2000 ski region
Merano 2000 ski region - enjoyment for the whole family
Merano 2000 - that's over 40 kilometers of slopes at an altitude of up to 2,300 meters directly on our doorstep. This family-friendly ski region is just 15 minutes from our Hotel Ansitz Plantitscherhof. Once you have arrived on the high plateau above Merano, you will be rewarded with perfectly-prepped slopes and an extraordinarily beautiful 360° panorama view. The wide variety of slopes range from difficult to easy.
Whether you are an experienced skier or have just begun skiing, the Merano 200 ski region provides you with guaranteed fun in the snow. Snowboarders will appreciate the wide slopes and delight in the thrills of the Snow Park. Snowboard beginners can try out their first moves and jumps at their own pace.
You will also love the Merano 2000 ski region if you are into snowshoeing, ski tours or cross-country skiing. The long days of skiing at this sunny altitude and the wide selection of the highest quality cuisine at the variety of catering stop-offs are unique. Particularly convenient for families: the ski depot. You can keep all your equipment and things here for the next day.
Spend your next winter holiday with us at our hotel in the Merano 2000 ski region. Find the offers below.
Christmas market in Merano
roll through the Christmas market in Merano and try out all the South Tyrolean specialities such as goulash, dumplings and barley soup - of course all washed down with a delicious mulled wine. Festively decorated art and crafts stands, lovingly restored carousels of yesteryear and traditional music bands invite you to stroll and stay around.
A visit to the Merano Christmas market will stay in the your memory for a long time.
Summer Holiday
Summer Holiday


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Voting's not good enough
As an anti-war demonstrator, I agree with Michael Burns' criticism of demonstration street-blocking tactics [Letter, "A better way to speak: Vote," April 10, 2003] (http://www.readthehook.com/93296/letter-better-way-speak-vote).
There are many of us who don't hide behind the First Amendment but observe the Ninth Amendment as well ("My right to demonstrate in the street ends where your right to get to the hospital begins"). The aim of demonstrations, after all, should be to remind complacent people that war is ugly and appalling in its present application, not to recreate it.
But Burns' call to leave the street altogether and just vote (once a year at most) is not a satisfactory option for many people today. Personally, I didn't vote in the last election because my ability to trust in any individual candidate for a lengthy term has been seriously and forever damaged by scandals, broken promises, and unilateral ill-considered actions– like one guy starting a war.
Our representative democracy was a fine compromise back in the days before planes and automobiles and the Internet. But today we have incredible interactive technologies that move information faster than the speed of sound at a Massachusetts Bay Colony town meeting.
We need to be moving to a system where we don't have middlemen. I'm not talking about abolishing Congress or having 280 million folks cast votes in nightly referenda. Rather, Congressmen should eventually become ceremonial figures like presidential electors, required by law to poll their constituency daily by all available means to find out exactly where the citizens stand on an issue, then be bound to vote their district's preference in session.
Until I find a candidate who wants to improve the accuracy and efficiency of democracy, I will be using every one of my powers not delegated to the United States (Tenth Amendment), including freedom of speech. Voting for fallible media-anointed figures every couple of years is not real enticing.
Kristopher Rikken


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Alyssa Milano, who grew up before America's eyes, is still getting used to the leaps and bounds her 22-month old son Milo Thomas is making.
"He's been here [in New York] with me for the last seven weeks since I'm hosting Project Runway All Stars and the difference is amazing," Milano, 40, told PEOPLE at the MLB.com and Nivea Men's All Star Bash on Sunday.
"I brought a baby and I'm going home with a little boy. It's so crazy! He's gotten so big," she adds. "I take it day by day and enjoy all of my time with him. I'm blessed to have the kind of job that he can be with me every day. But it's still freaking exhausting!"
Though the actress – who is a longtime Dodgers season ticket holder and designer of a women's MLB apparel line – clearly is an advocate for the nation's pastime, she's not putting pressure on her son to find his way to a baseball diamond.
"He seems to be gravitating the most towards basketball," she shares, adding proudly, "He's really good. He gets it in the basket and everything! Whatever he decides to do, I'm totally all for it."
As for her steamy work on ABC's Mistresses, Milano's character Savi Davis will find herself in even more hot water as the season progresses. After having an affair with her coworker, she finds out that she's pregnant and finally confesses her indiscretion (but not the baby news!) to her husband. It's only going to go downhill from there.
"She has a pretty big cliffhanger at the end of the season," says Milano. "It gets progressively worse for her. It's from all sides – her mom comes into town. Everything that could possibly go wrong, goes wrong."
– Mary Margaret


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The Reno Red Baby Bedding is a gender neutral set with contemporary styling. A soft gray polka dot print is coupled with a simple but sophisticated red and white scale print to make a soothing set with rich color. The crisp white bumper will surround your little one with a serene space for sleeping while the rest of the set lends itself to global trend design, contemporary design, and eclectic nurseries.
The skirt has a 16" drop and the matching changing pad, decorative pillow, and extra crib sheets are sold as options.
All set pieces are made in the USA.
Would you like us to help you find a similar item? Talk with our Design Consultants toll-free at1-800-927-5164.
*Shipping transit time varies based on destination. Free shipping offers are limited to the 48 contiguous states of the USA.
International Customers (including Alaska and Hawaii) please contact a Customer Service Representative if you have any questions about placing an order with PoshTots.
Promotion codes cannot be used for sale item purchases.
*Free shipping offers are limited to the 48 contiguous states of the USA.


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Seattle, WashingtonSeattle is a young city with a rich history. Settlers first landed at Alki Point in 1851 and named the area after Sealth, the Suquamish Indian chief who befriended them. Rebuilt after the Great Fire of 1889, "The Emerald City" has a legacy of vision and strength. Seattle has hosted two World's Fairs (1909 and 1962) and is the birthplace of two modern marvels, Boeing and Microsoft.
Known for its rainy climate, Seattle actually averages less annual rainfall than many East Coast cities. The mild weather, spectacular natural surroundings and rich cultural diversity attract visitors from around the world.
Seattle tours are available to passengers with afternoon flights or an overnight stay in Seattle after their cruise.
Top things to do and see in Seattle, Washington: View Excursions
1Pike Place Market
Home of the world-famous flying fish, Pike Place Market is the oldest open-air farmer's market on the West Coast and boasts hundreds of artisans, local farmers, fishmongers and curio shops in a vibrant atmosphere.
2Space Needle
The Space Needle, a futuristic fixture of the Seattle skyline, was built for the 1962 World's Fair. Spectacular panoramic views of the Emerald City's natural beauty abound from its 520-foot high observation deck.
3Pioneer Square
Seattle's oldest neighborhood is a 17-square-block National Historic District located in the southwest corner of downtown. Rebuilt after the Great Fire of 1889, beautifully restored architectural masterpieces can be found around every corner.
4Wine Tasting
Woodinville, less than 30 minutes from downtown Seattle, boasts over 100 wineries. Choose from the friendly tasting rooms and many tours, including the famed Columbia Winery and Chateau Ste Michelle.
5Boeing's Future of Flight Aviation Center
This facility offers the only opportunity to tour a commercial jet assembly plant in North America and learn about the dynamics of flight, new aviation innovations and go behind the scenes at Boeing.
6Hiram Chittenden Locks
Built in 1911 this complex of locks provides a link for sailboats, motorboats, tugs and barges between Puget Sound and the Ship Canal, which connects eastward to Lake Union and Lake Washington.
7International District
One of the city's oldest neighborhoods, this multiethnic enclave is rich with history and culture. Highlights include delectable restaurants, unique specialty shops and diverse heritage sites.
8Lake Union
An urban gem and popular recreational spot close to downtown Seattle. Seaplanes, competitive rowboats and pleasure craft ply the waters and a series of parks and floating houseboats dot its shores.


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He was rushed to nearby Elmhurst Hospital that night, where he would later die.
"I fight with anger all the time," said Greene's mother, 52-year-old Debra Greene. "Someone had to have seen something."
It was roughly 5 a.m. on April 20, 2012, when Greene was returning home to LeFrak City from a night out at a Manhattan night club with friends. After they dropped him off on the corner, he found himself running from a hailstorm of bullets.
Wiping tears away Debra Geene said of the older of her two sons, "he had plans, he was a smart boy."
Police still have no suspects or leads in the investigation. Last week, local elected officials called on local residents to come forward with any information that could help solve the case.
"It is up to the community to do everything it can to help the NYPD help catch the killer," said Councilman Daniel Dromm. "By increasing the reward amount, we are sending a strong message that we are not giving up on our search."
Dromm has already pledged $1,000 of his own money, while State Senator Jose Peralta has secured $500 from the Bogota Service Corporation and an anonymous donor is pledging another $5000.
The total reward for information leading to the arrest and conviction of Greene's killers is $24,000.
Elected officials also called on the state legislature to require microstamping, which creates a fingerprint-type etching into a bullet casing in order to trace a gun to the owner.
"We call upon the state legislature, in particular the senate Republicans, to pass microstamping," said Peralta, who said he believes this crime would have been solved already had microstamping been in practice in New York.
Peralt said that while the gun, the individual and even the car were never found or identified, the bullet casings were found on the ground at the scene of the crime. For a minimal cost, according to Peralta, the NYPD could have traced its way back to the murder weapon's owner.
"I want justice," Ms. Greene concluded. "Closure and justice."


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42nd Annual Head Start Conference and Expo
Mar. 29 - Apr. 2, 2015
About this Conference NHSA's Annual Head Start Conference and Expo is the largest national event devoted to the Head Start and Early Head Start community. This year more than 5,000 executive directors, directors, administrators, managers, teachers, policy council members, and parents from every state will gather in Washington, D.C. March 29 - April 2, 2015.
Resources for Children
sidebar page
Research For fifty years, research and evaluation have documented Head Start's impact on school readiness, academic achievement, health, family stability, and long-term success. As a nonprofit membership and advocacy organization, NHSA sees its role in research as:
Research by Topic
Research by Topic Below are links to compilations of research on some of the most important topics for Head Start practitioners. While these aim to gather useful resources and research, they are by no means exhaustive. For additional research sources and strategies for navigating them, visit Accessing and Reading Research.
blog article
Bridging the Word Gap Challenge
What is the Word Gap and Why do we Need to Address it?
2017 Annual National Head Start Conference and Expo
April 7 - 10, 2017


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Genre: Drama
Plot: The title of this series is Dispatches from Elsewhere which was first released in the year 2020 and takes part in the drama genres. The episodes last around minutes and you can enjoy them here for free. FMovies will add new episode once it is available online. You can choose which episode to watch below.
Next Episode Date: Apr 14th