text:
Feeding is a fundamental activity of all animals that can be regulated by internal energy status or external sensory signals. We have characterized a zinc finger transcription factor, klumpfuss (klu), which is required for food intake in Drosophila larvae. Microarray analysis indicates that expression of the neuropeptide gene hugin (hug) in the brain is altered in klu mutants and that hug itself is regulated by food signals. Neuroanatomical analysis demonstrates that hug-expressing neurons project axons to the pharyngeal muscles, to the central neuroendocrine organ, and to the higher brain centers, whereas hug dendrites are innervated by external gustatory receptor-expressing neurons, as well as by internal pharyngeal chemosensory organs. The use of tetanus toxin to block synaptic transmission of hug neurons results in alteration of food intake initiation, which is dependent on previous nutrient condition. Our results provide evidence that hug neurons function within a neural circuit that modulates taste-mediated feeding behavior.
Citation: Melcher C, Pankratz MJ (2005) Candidate Gustatory Interneurons Modulating Feeding Behavior in the Drosophila Brain. PLoS Biol 3(9): e305. doi:10.1371/journal.pbio.0030305
Academic Editor: Michael Bate, University of Cambridge, United Kingdom
Received: September 24, 2004; Accepted: June 30, 2005; Published: August 30, 2005
Copyright: © 2005 Melcher et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: CC/CA, corpora cardiaca/corpora allata; CNS, central nervous system; GFP, green fluorescent protein; GR, gustatory receptor; hug , hugin ; klu , klumpfuss ; OR, olfactory receptor; POD, peroxidase; ppl , pumpless ; SOG, subesophageal ganglion; TH, tyrosine hydroxylase; VNC, ventral nerve cord; YFP, yellow fluorescent protein
All animals must be able to evaluate their nutrient requirement, as well as the nutrient supply offered by the environment, and translate the resulting information into appropriate behavioral responses. These can range from deciding to stop or continue feeding, or to look for alternate food sources. The nutrient signals can derive internally, reflecting the body's energy state and metabolic need, or through external sensory inputs, such as olfactory and gustatory signals. The sensory modalities further provide the basis for many types of higher brain functions, such as learning and memory. Feeding behavior, in turn, decisively influences almost all aspects of animal growth and reproduction. The role of the central nervous system (CNS) in integrating an animal's feeding behavior with sensory signals on the availability and quality of nutrients is, although undisputed, insufficiently understood [1].
Drosophila provides a genetically accessible system to study the molecular mechanisms that coordinate feeding behavior with sensory signals. This organism has an array of feeding characteristics that can be exploited for behavioral analysis, and insects in general have been used extensively as models for a wide range of behavioral and physiological studies [2,3]. In this context, the identification of genes encoding chemosensory receptors in Drosophila has provided a major impetus in understanding sensory signal transduction [4–8]. These genes have been broadly divided as encoding olfactory or gustatory receptors (ORs and GRs, respectively). Olfactory sensory neurons expressing specific ORs in the external mouth region project axons to distinct glomeruli of the antennal lobe [8–12]. Projection neurons then connect the antennal lobe to the mushroom body, where central processing of olfactory information occurs [13–15]. Gustatory sensory neurons are located not only in the external mouth region, but also internally in the pharynx; both types project to the subesophageal ganglion (SOG), a region implicated in feeding and taste response [8,16–18]. As compared with the antennal lobe, much less is known about the organization of the SOG—for example, whether it is also organized in glomerular structure. The neurons that connect the SOG to higher brain centers, in a manner analogous to the olfactory projection neurons, have also not been identified.
In both olfactory and gustatory cases, the knowledge is even sparser concerning the identity of interneurons that act between the sensory neurons and motor or neuroendocrine outputs and how they might influence feeding behavior. Studies in different insects have shown that various parts of the CNS are interconnected with the neuroendocrine organs and the enteric (stomatogastric) nervous system, which innervates the feeding apparatus [19,20]. The mouth parts have also been shown to be innervated by nerves from the SOG [21]. Nevertheless, an integrated map of the neurons comprising these circuits and their function in mediating a behavioral response has been lacking.
We have previously identified a gene, pumpless (ppl), that is required for food intake behavior in the Drosophila larvae [22]. It encodes a subunit of the glycine cleavage system and is expressed exclusively in the fat body. Although not feeding, ppl mutant larvae do not show characteristics of starving larvae, as assayed both by molecular markers and behavioral characteristics; furthermore, feeding high levels of amino acids can phenocopy several aspects of the ppl feeding phenotype. These observations led to a model in which amino acid-dependent signals from the fat body to the brain can signal cessation of feeding. In this study, we characterize another mutant, klumpfuss (klu), with a phenotype very similar to ppl mutants. Through microarray analysis, we identified the neuropeptide gene hugin (hug) as being deregulated in klu mutants. hug is expressed in a small assembly of about 20 neurons in the SOG. Connectivity mapping and behavioral studies suggest that hug-expressing neurons function in a neural circuitry in the brain that modulates chemosensory signal-dependent feeding behavior.
Molecular Characterization of Larval Mutant Defective in Feeding Behavior
In a screen for Drosophila mutant larvae defective in feeding, we identified the P-element line P(9036). These animals fail to pump food from the pharynx into the esophagus (Figure 1A), which is not due to a morphological block in the esophagus. The failure to feed is also not due to a general illness of the animal or global locomotory defects, because they can move around with the same vigor as wild-type or heterozygote siblings. P(9036) larvae also display wandering-like behavior, in which they move away from the food (Figure 1B and 1C). During this wandering-like phase, P(9036) larvae move about with food lodged in their pharynx, further supporting the view that the feeding defect is not due to a general body movement defect. Wandering behavior is observed in wild-type larvae when they stop feeding and move away from food shortly before pupariation [23]. These feeding behavior defects have also been observed for ppl mutants [22]. ppl encodes an amino acid catabolizing enzyme that is expressed exclusively in the fat body, an organ analogous to the vertebrate liver. Thus, P(9036) and ppl mutants, as immature first instar larvae, display feeding behaviors characteristic of sated, full-grown, third instar larvae. We characterized the gene corresponding to P(9036) and found it to be klu, a zinc finger protein-encoding gene that is expressed specifically in the developing nervous system [24,25]. P(9036) fails to complement the lethality of all klu alleles tested, and trans-heterozygotes also show the characteristic feeding defect (Figure 1D).
(A) P(9036)-mutant larvae show feeding and growth defects as compared to wild-type (wt) controls. In wild-type, red food fills the gut, whereas it accumulates in the pharynx of P(9036) mutants. Physically, the food can pass the mutant pharynx (see arrow).
(B) P(9036) mutants show a wandering behavior, in which they leave the food source (depicted in a schematic snapshot drawing; dots represent larvae outside the food source, which is shown in red).
(C) Quantification of the P(9036) wandering phenotype shows that from 50 individuals, 23 ± 4 mutants can be found outside the food source, in contrast to only 2 ± 1 for the wild-type control at a given time point (n = 8 time points).
(D) The known klu alleles kluXR19, kluR51C, and P(1741) fail to complement P(9036), and trans-heterozygous larvae display the feeding phenotype.
(E) Microarray analysis of P(9036) revealed several neuropeptide genes as being deregulated in P(9036) mutants compared to wild-type controls (×-fold changes in P(9036) are listed; genes with more than 2-fold change are boxed).
(F–H) Semi-quantitative in situ hybridizations to first instar larval brains verify the upregulation of the neuropeptide gene hug in P(9036) klu mutants in comparison to wild-type controls, and show an upregulation of hug in ppl feeding mutants as well. The expression of hug is in the SOG. Anterior points to the upper left corner; ventral views are shown.
(I–K) Semi-quantitative in situ hybridizations to late second instar larval brains demonstrate a downregulation of hug in wild-type under starvation (PBS) and sugar conditions (20% sucrose), in comparison to normal feeding condition (yeast). Anterior points to the upper left corner; ventral views are shown.
The Neuropeptide Gene hug Expression is Altered in klu Mutants and in Amino Acid-Deficient Conditions
To study the central control process that could underlie the feeding defect of klu mutants, we performed microarray analysis of klu mutants with a focus on neuropeptide genes. We reasoned that their expression patterns in the brain would be specific enough for analysis at single-cell resolution. Furthermore, neuropeptides have been shown to influence food intake in different organisms, including mammals [1]. RNA from klu mutant larvae and wild-type larvae were isolated and hybridized to three Affymetrix chips each and compared. Figure 1E lists Drosophila neuropeptide genes and their expression profile in klu mutants, relative to wild-type first instar larvae. We then performed in situ hybridizations on wild-type larval brains with the six highest upregulated genes. We decided to focus our efforts on hug because it had the most specific expression pattern in the larval brain. While all others showed staining in different parts of the brain or in the ventral nerve cord (VNC) (unpublished data), hug showed staining in only a cluster of about 20 cells in the SOG of the larval brain, with no staining anywhere else (Figures 1F and 2). Its expression in embryos is also highly restricted in the brain [26]. hug encodes a prepropeptide capable of generating at least two neuropeptides, Drm-PK2 and hug-γ. The former encodes a myostimulatory peptide while the latter shows homology to ecdysis-triggering hormone-1, ETH-1 [26]. Both can activate a G-protein–coupled receptor belonging to the vertebrate neuromedin U group [27]. A hug homolog is also found in Anopheles gambiae [28].
(A) A schematic drawing of the Drosophila larval CNS, showing the relative positions of the two brain hemispheres (BH) and the VNC, with the neuroendocrine ring gland (RG) located dorsoanterior to the CNS. The esophagus goes through the brain, and we have termed the hole through which the esophagus passes as the foramen (F). Positions of the SOG, the larval antennal lobes (AL), the mushroom bodies (MB), and the median neurosecretory cells (mNSC) are depicted.
(B) hug expression (shown in green) is restricted to the SOG. adipokinetic hormone (akh, shown in red) serves as a ring gland marker; the CNS is false-colored in blue.
(C) Detection of marker gene expression under hug promoter-Gal4 (hugS3) construct (shown in green) labels hug cell bodies and neuronal projections relative to the neuropil (22C10, shown in red) and nuclei of post-mitotic neurons (elav, shown in blue).
(D–F) The endogenous hug expression pattern (shown in red in [D]) is reproduced by the hugS3 expression pattern (shown in [E]). Note double-positive cell bodies in (F). akh serves as ring gland marker; CNS, ring gland, and foramen are outlined.
(G and H) Immunofluorescent (G) as well as direct (H) detection of GFP expressed under the hug promoter-Ga14 construct reveals putative hug dendrites dorsoanterior of the hug cell bodies, where spherical structures are innervated. The hug cell bodies in the depicted blow-up of SOG region are out of focus in (H).
(I) hug axons innervating the protocerebrum cross the midline, showing ipsilateral and contralateral innervation of mushroom body region.
(J–L) Expression of eGFP under hugS3 shows hug axons leaving the SOG laterally (see arrows). These can be followed to the cephalopharyngeal complex of the larvae (K and L) onto the pharyngeal muscles (axons shown in green overlayed with transmission light picture; note the mouth hook apparatus in black). Dorsal (K) and lateral views (L) are shown, anterior points to upper left corner.
All fluorescence images in this and subsequent figures are Z-stack projections, and all scales bars are 50 μm, unless otherwise noted.
To confirm the microarray data, we performed semi-quantitative in situ hybridization in wild-type and klu mutant larval brains. hug is upregulated in klu mutants (Figure 1F and 1G). We then investigated whether hug expression is also regulated in ppl larvae, which display a similar feeding defect as klu. There is also an upregulation of hug in ppl mutants (Figure 1H). We next investigated whether hug expression is regulated by different nutrient signals. We therefore placed wild-type larvae in starvation and sugar conditions (that is, both being amino acid-deficient diets) and monitored hug expression. hug was downregulated in both conditions (Figure 1I, 1J, and 1K), indicating a response to nutrient signals distinct from simple lack of energy. As hug is upregulated in klu and in ppl mutants, both of which do not feed and wander about, a higher hug level correlates with decrease of food intake and food-seeking behavior. For hug downregulation under starvation and sugar conditions, a lower hug level correlates with increased food-seeking behavior since Drosophila larvae become hyperactive and disperse when food is removed.
hug-Expressing Neurons Project to the Ring Gland, the Pharyngeal Muscles, and the Protocerebrum
As mentioned above, hug is expressed specifically in a small group of neurons in the SOG (Figure 2A and 2B). To gain insight into the physiological processes that hug-expressing neurons (referred to as hug neurons) could be involved in, we wanted to determine their connectivity pattern. Therefore, we constructed a hug promoter-Gal4 line (hugS3) in order to express different versions of green fluorescent protein (GFP) marker genes for neuroanatomical studies. This approach revealed hug neuron projection to the ring gland (Figure 2C). The ring gland, as the master neuroendocrine organ of Drosophila larvae, controls metabolism and growth. For example, median neurosecretory cells of the pars intercerebralis that express Drosophila insulin like peptides (dilps), also project to the ring gland, whereas adipokinetic hormone (akh), thought to be a glucagon homolog, is produced by the ring gland [29–31].
In addition to the ring gland, we also observed hug neuron projection to the protocerebrum, near the median neurosecretory cells and the mushroom bodies (Figure 2D–2F), which comprise the center for olfactory learning and memory [13,32]. The axons projecting to the protocerebrum also cross at the midline just above the foramen (Figure 2I). We also noticed an intriguing glomerular-like structure of what are most likely hug dendrites in the SOG, just dorsoanterior to the hug cell bodies (Figure 2G and 2H). Singh [17] has described glomerular organization in the SOG of adult Drosophila that relays gustatory information. Such a glomerular organization has not been previously recognized for the larval SOG, but it would be analogous to the glomerular organization in the antennal lobes that relay olfactory information.
Strikingly, there is also projection of hug neurons to the pharyngeal muscles (Figure 2J, 2K, and 2L), which pump food into the mouth atrium. These arise from axons that leave the brain (Figure 2J) and project anteriorly along each side of the dorsal pharyngeal muscles, and terminate near the anterior end of the pharynx (Figures 2K, 2L, and 4). There has been no previous case of identified neurons in the larval SOG that project to motor outputs. At this point, we do not know whether the pharyngeal muscles are actually innervated by these axons. Taken together, these results demonstrate that hug neurons in the larvae project to key organs regulating feeding and growth—namely, the pharynx and the ring gland—as well as to higher brain centers.
hug Dendrites Innervate GR-Expressing Sensory Organs and Chemosensory Organs of the Pharynx
The projection of hug neurons to the mushroom body region, together with the fact that hug is expressed specifically in the SOG, which relays gustatory information, suggested that hug neurons could be involved in mediating chemosensory signals. Therefore, we investigated whether hug neurons receive direct input from the chemosensory organs (Figure 3A–3D). It has been demonstrated that sensory organs in the larval head that express ORs or GRs send their axons either to the antennal lobe or the SOG [8] (Figure 3A and 3B). Recently, an enhancer trap line MJ94 was used to label putative chemosensory organs of the internal pharynx [18]. As internal pharyngeal sensory organs are good candidates for transducing gustatory signals, we wondered if these sensory organs terminate at hug dendrites. As shown in Figure 3E and 3F, they indeed terminate in the contact region of hug dendrites.
(A) Schematic drawing of the head region of a Drosophila larva with external as well as internal chemosensory neurons in antennomaxillary complex and internal mouth region innervating the larval CNS (depicted in green). hug neurons in the SOG (shown in red) project to the ring gland (RG), pharyngeal muscle (PM) region, and the protocerebrum (PC). Relative positions of external chemosensory sensillae (dorsal organ [do] and terminal organ [to]), internal chemosensory sensillae (ventral pharyngeal sense organ [vps], dorsal pharyngeal sense organ [dps], dorsal pharyngeal organ [dpo], and posterior pharyngeal sense organ [pps]), and major projections to the CNS are shown.
(B) GR21D1-positive sensory neurons (shown by X-Gal staining) in the dorsal organ project axons to the CNS (see arrow).
(C and D) Optical section through median CNS (composed of ten confocal 1-μm sections) shows hug arborizations in the SOG and mushroom body region (shown in green) relative to general neuropil (red) and cortical (DNA marker Draq5, blue) landmarks. Note the labeling of corpora allata cells in the ring gland (arrow; see Materials and Methods and Figure S2). Boxed area is shown at higher magnification in (D), revealing spherically organized neuropil regions lateral to foramen (partially outlined).
(E) Expression of nSyb-GFP under MJ94 enhancer trap construct labels axon terminals of internal gustatory sensory neurons (shown in green), innervating SOG and VNC.
(F) Close-up of SOG region shows co-localization of spherically organized axon terminals of MJ94 positive gustatory neurons (green) and hug neuronal arborizations (red).
(G) Axon terminals of GR66C1-positive chemosensory neurons (shown in green) can be detected in the vicinity of hug cell bodies (shown in red). dilp3 staining (blue) serves as morphological landmark.
(H) Optical section (composed of ten confocal 1-μm sections) containing the GR66C1 axon terminals (shown in green) also include the spherical hug arborizations. hug cell bodies are out of focus in (H); a close-up of the SOG is depicted.
(I and J) Axon terminals of GR21D1 positive chemosensory neurons (shown in green) also project to the vicinity of hug cell bodies (shown in red in [I]), but the optical section comprising these terminals does not contain the hug arborizations (note the absence of spherical hug dendrites in [J] as compared to [H]).
(K–M) Comparison of axon tracts used by olfactory projection neurons, which can be labeled by enhancer trap line GH146 (shown by GFP real color in [K] and in green in [L], marked by asterisks), with those used by hug neurons (shown by YFP real color in [K] and in red in [L], marked by arrows), indicate that hug neurons are distinct from olfactory projection neurons (OPN). These differences are summarized in (M). LAN, larval antennal nerve; LMN, larval maxillary nerve; LPN, larval pharyngeal nerve.
To further test this, we checked to see if chemosensory neurons that express specific GRs also project to hug dendrites. For example, it has been shown that GR66C1-positive neurons project to the SOG, whereas GR21D1-positive neurons project to the antennal lobe [8]. To see if these sensory projections terminate at or near hug neurons, we first performed staining of GR66C1- and GR21D1-positive axon terminals with hug in situ hybridization. GR66C1 receptor neurons indeed project to the vicinity of hug-expressing cells (Figure 3G). To see if these axons may potentially make synaptic contacts with hug dendrites, we used a hug promoter–yellow fluorescent protein (YFP) line (in which YFP was placed directly under the hug promoter), in combination with GR promoters driving nSyb-GFP [8,33], thus allowing simultaneous visualization of GR axon terminals and hug dendrites. As shown in Figure 3H, GR66C1-positive neurons project to the glomerular-like SOG region contacted by hug dendrites. GR21D1-positive neurons also project near the hug cells (Figure 3I), but by contrast to GR66C1-positive neurons, do not contact hug dendrites (Figure 3J). Rather, they terminate dorsoanterior to the hug dendrites, where the antennal lobes are located [8]. Taken together, these results suggested that hug neurons may act as second-order interneurons that relay gustatory information.
To further distinguish the relationship between hug neurons and the olfactory or gustatory systems, we determined whether hug neurons share the same axon tracts to the mushroom bodies as the second-order neurons that relay olfactory sensory input. The dendrites of these olfactory projection neurons underlie the glomerular structure of the antennal lobes and vertically transduce olfactory information for processing to the mushroom bodies. These projections can be visualized by the enhancer trap line GH146 [14]. As shown in Figure 3K and 3L, the axon projections of hug neurons are distinct from olfactory projection neurons: hug neurons project to a more dorsomedial region in the protocerebrum than olfactory projection neurons, and they use different axon tracts. These results essentially rule out hug neurons being olfactory projection neurons. Projection neurons transducing gustatory signals to higher brain centers have not yet been identified. In this context, hug neurons could act as gustatory projection neurons that connect gustatory sensory neurons via SOG with the protocerebrum (Figure 3M).
Subpopulation of hug Neurons Project to Distinct Targets
We have also noticed a difference in the projection specificity among the hug neurons. A series of enhancer trap lines have been isolated that label cells projecting their axons to the ring gland [34], one of which (Okt30) is co-expressed with hug (Figure 4A and 4B). When we use our hug promoter-YFP line (to distinguish it from GFP reporter constructs) in combination with the Okt30 ring gland enhancer trap line, we find that a distinct set of hug neurons project to only the ring gland and not to the protocerebrum, the pharynx, or the ventral cord (Figure 4C).
(A and B) Enhancer trap line Okt30 (shown in green) labels SOG neurons projecting to ring gland. Okt30 expression pattern colocalizes with hug expression (shown in red in [B]). There are four double positive cells in (B). dilp3 staining (blue) serves as morphological landmark.
(C) Direct detection and false colorization of GFP expressed in Okt30 positive cells (green) and YFP expressed under hug promoter (red) reveals only the axons to ring gland as double positive.
(D) TH promoter construct labels dopaminergic CNS neurons (shown in green). Four TH-positive SOG neurons also express hug (shown in red). capa staining (blue) serves as morphological landmark.
(E) Combination of projection patterns of TH positive cells (green) with hug cells (red) reveals the axons innervating the pharyngeal muscles as the only double positive ones.
(F) Schematic summary of (A–E) showing distinct subpopulations of hug neurons projecting to the ring gland only (green), pharynx only (blue), and the remaining targets (red).
(G–I) Projection pattern of hug is unaffected in klu mutants. Direct detection and false coloring of hug promoter-YFP (shown in green) in the klu background reveals the pharyngeal muscles (PM), the ring gland (RG), and the VNC as being targeted in feeding mutants. Trachea are false-colored (red) in composite figure (I).
Another subpopulation of hug neurons is revealed by using the TH-Gal4 line. This drives reporter gene expression under the promoter of tyrosine hydroxylase (TH), a key enzyme in dopamine synthesis [35]. As shown in Figure 4D, specific hug neurons express TH-Gal4 reporter gene, indicating that a subset of hug neurons might be dopaminergic. When TH-Gal4-driven lacZ is used in combination with hug promoter-YFP, we observe that TH-positive hug neurons project to only the pharynx and not to the protocerebrum, the ring gland, or the VNC (Figure 4E). These results indicate that at least three distinct subpopulations of hug neurons exist: those projecting to only the ring gland, those projecting to only the pharyngeal muscles, and those projecting to the protocerebrum and/or the VNC (Figure 4F). The distinct target specificity suggests differences in the function of the hug subpopulations. In the honeybee Apis, the subesophageal-calycal tract neurons are located in the SOG, send axons to the protocerebrum, and receive input from the sensory neurons of the proboscis; these neurons are thought to transduce gustatory information [36]. Some of the hug neurons could act similarly to these honeybee neurons. Based on the connectivity map of the hug neurons, we also wondered if the global targeting of these neurons was altered in klu mutant larvae. Therefore, we crossed the hug promoter-YFP construct into klu mutant background (Figures 4G and 4H). Although we cannot rule out subtle local differences, the basic connectivity pattern is retained in the mutants (Figure 4I).
hug Neuron Connectivity Pattern Is Similar in Larvae and Adults
To see if hug neurons might also have a function in the adults, we determined the connectivity pattern in adult animals. There are some noticeable morphological differences in the feeding apparatus and neuroendocrine organs between adults and larvae (Figure 5A). One is the presence of the crop in the adult but not in the larva. The crop is a food storage organ, and its absence in the larvae most likely reflects a difference in the feeding habits; whereas adults are intermittent feeders, the larvae feed continuously. Another is the relocation of the neuroendocrine organs. The corpora cardiaca/corpora allata (CC/CA) complex, which comprises part of the ring gland in the larvae, is located right above the proventriculus in the adults, at the junction between the gut and the crop. This is in contrast to the larvae, where it is located on top of the brain hemispheres. Monitoring hugS3 expression in adults, we observe axon projections to the protocerebrum, the CC/CA complex, and the ventral cord (Figure 5B–5H). A subpopulation of hug neurons may also be dopaminergic, as in the larvae (Figure 5I). To further characterize the projections to the protocerebrum, we used the OK107 enhancer trap Gal4 line [37] together with hug promoter-YFP. These stainings indicate that hug axons traverse along the median neurosecretory cells in the pars intercerebralis, and terminate near the mushroom bodies (Figure 5J and 5K). A similar pattern is observed in the larvae (Figure 5L and 5M). The precise targets of hug neurons projecting to the protocerebrum remain to be determined. Taken together, despite the morphological differences, the connectivity pattern of hug neurons is remarkably similar between larvae and adults.
(A) Relative positions of adult CNS composed of brain hemispheres (BH) and VNC, as well as esophagus passing the brain through the foramen (F), proventriculus (P), crop (C), and gut (G) are depicted schematically. The neuroendocrine CC/CA complex is located on top of the proventriculus (arrow).
(B) Localization of hug neurons and projections in adult CNS relative to nuclear marker. Note projections to the protocerebrum (top of the head), VNC, and CC/CA (arrow).
(C) hug neuronal projections relative to general neuropil marker (22c10, shown in red).
(D–F) Close-ups of different optical sections (composed of confocal 1- to 2.5-μm sections) show spherical hug arborizations in median SOG region (green in [D]), arborizations in lateral SOG region (green in [E], arrow) and in protocerebrum (green in [F]).
(G) Transmission light image of CC/CA complex (arrow) on top of proventriculus (P).
(H) Immunofluorescent close-up of the area boxed in (G). Nuclei of elav positive CC cells (blue) and of CA (green, see arrow), hug axon terminals (green, see arrowhead).
(I) TH positive SOG neurons (green) co-express hug (red); capa staining serves as morphological landmark.
(J and K) Different optical sections (composed of confocal 1- to 2.5-mm sections) show hug (green) projections above the mushroom bodies and adjacent to the median neurosecretory cells marked by OK107 (mushroom body and median neurosecretory cell marker, red); elav marker, blue. (K) Close-up of the region in (J), showing the medial neurosecretory cells. Scale bars equal 20 μ.
(L and M) Different optical sections (composed of confocal 1-to 2.5-mm sections) showing OK107 (green) and hug (red) in larva. (M) Close-up of the median neurosecretory cell region. Scale bars equal 20 μ.
Blocking Synaptic Transmission of hug Neurons Alters Food Intake Behavior
Based on the connectivity map of hug neurons and the alteration in hug expression under different nutrient and feeding conditions, we initiated a series of experiments to explore the role of hug in regulating feeding. As hug mutants have not yet been identified, we tested the effects of overexpressing hug in the larvae. We first used hugS3 to drive hug expression but did not observe any phenotype (unpublished data). This is most likely because using an endogenous promoter does not result in high enough overexpression of hug in cells that already express physiological levels of hug. We therefore used a strong ubiquitous promoter (tubulin-gal4). There was a strong reduction in growth (Figure 6A), with no larvae surviving to pupal stage; we also observed defects in food intake, although not to the same strong degree as with klu mutants (Figure 6A). This is consistent with the view outlined earlier that high hug levels correlate with decreased food intake.
(A) Overexpression of hug neuropeptide gene under ubiquitous promoter leads to reduced feeding and growth (compare size of UAS-hug larvae and controls of same age) as well as to larval lethality. Note the individual phenocopying the klu feeding phenotype (arrow).
(B) Partial rescue of klu feeding defect by blocking hug neuronal activity. n= 5; error bars represent standard deviation; see Materials and Methods for details.
(C) Schematic of adult internal morphology with proventriculus, crop, and gut. Crop is depicted in full and empty state.
(D) Feeding behavior of adult flies monitored by the presence of red food in the midgut and crop (marked by arrows). Starting from the same feeding status (empty crop at time point 0 min), experimental flies expressing TeTxLC under hugS3 construct initiate uptake of red food immediately (notice large amount of red food in crop and gut after 5 min) when confronted with red yeast paste after feeding on standard fly food overnight (overnight normal). In contrast, control flies (hugS3 flies and TeTxLC flies crossed with wild-type) initiate food uptake after 15–35 min (notice traces of red food in midgut) when confronted with red yeast paste. The same feeding status is detected in experimental as well as in control flies after long feeding period on red yeast (overnight normal, 180 min). Overnight starvation equalizes feeding behavior when flies are confronted with red yeast paste for 15 min (notice same amount of red food in all cases at overnight starvation 15 min). Two representative samples of each time point and genotype are displayed. Two independent experiments were carried out, and for each experimental set ten individuals were taken out randomly and dissected. From 20 total flies, 16 ± 2 showed the phenotype displayed here o/n, overnight.
In order to gain further information on the function of hug neurons, we then blocked synaptic transmission in these cells using tetanus toxin light chain (TeTxLC) [38]. We first carried out the experiments in the larvae but did not see any difference (unpublished data). However, we reasoned that any potential increase in feeding response may not be readily detectable in the larvae because they feed continuously, already at a maximal rate. Therefore, we tested whether blocking synaptic transmission of hug neurons could suppress the feeding defect of klu mutants. We indeed observed a significant rescue of klu mutant feeding phenotype (Figure 6B).
We then carried out behavioral analysis on adults, since they are discontinuous feeders and thus may display an increased feeding behavior. Furthermore, one can visualize the quantity of food eaten by the size of the crop (Figure 6C). Experimental and control flies were placed in food vials containing standard fly food for several days. They were then transferred to yeast paste containing red dye. A striking result was observed. After 5 min, the experimental flies had a completely filled crop (Figure 6D, left column), whereas the control lines (Figure 6D, middle and right columns) had very little food in the crop. Even after 30 min, the control flies had very little food in their crops and only traces of red food were detectable in the midgut. By 180 min, both experimental and control flies showed the same degree of feeding. These results suggested that hug neurons are involved in regulating the initiation phase of feeding: control flies wait for a certain period before initiating feeding on the new food source, whereas decreasing hug neuronal signaling results in flies initiating their feeding immediately. When flies were transferred from yeast to colored yeast, or normal food to colored normal food, no difference was seen between experimental and control flies (unpublished data), indicating that hug neurons are not simply affecting the rate of feeding per se; we also did not observe a difference when transferring from yeast to normal food, indicating that the hug neuron-dependent behavioral effect is also not due to a simple fact of changing food sources. When flies were transferred from normal food into yeast containing 1M quinine (quinine is an aversive tastant), experimental flies again filled their crops earlier than controls (Figure S1). However, when flies were kept on yeast containing 1M quinine, and then transferred to yeast without the quinine, both experimental and control flies filled their crops with the new yeast within 5 min (Figure S1); analogously, when flies were starved before placing them on red yeast, both control and experimental flies filled their crops at about the same rate (Figure 6D, bottom row). These results suggest that the quality of previous food condition plays a role in defining hug neuronal function. Taken together, our studies support the view that hug neurons act within a neural circuitry in the brain that modulates feeding behavior based on chemosensory and nutrient signals.
Central Relay of Gustatory Information
The identification of candidate chemosensory receptors in mammals and invertebrates has provided major insights into the molecular mechanisms underlying sensory information processing. In the Drosophila olfactory system, projections of OR-expressing sensory organs terminate at specific glomerular structures in the antennal lobe. The olfactory projection neurons then act in a second relay to convey the information to the mushroom bodies in the higher brain region. The gustatory organs, expressing specific GRs, project to a different brain region, the SOG, which has been implicated in gustatory signal transduction and feeding response in different insects. Our results indicate that the neurons that express the hug neuropeptide gene are likely candidates for acting as interneurons that transduce gustatory information. These comprise an assembly of about 20 neurons in the SOG. The close proximity of their dendrites with the axon terminals of gustatory sensory organs of the external head, and chemosensory organs of the internal pharynx, suggests a synaptic contact, but this requires functional verification. Whether the SOG is also organized into glomerular structure, like the antennal lobe, is not known. Such an organization has been suggested in adult Drosophila [17], although data on larvae have been lacking. Our results on the dendritic pattern of hug neurons also suggest a glomerular structure of the larval SOG, but this remains an open issue.
The hug neurons, in turn, send axons to at least three distinct targets: the ring gland, the pharyngeal muscles, and the protocerebrum. The projections to the ring gland and the pharyngeal muscles suggest that hug neurons coordinate sensory information with growth, metabolism, and food intake; the axon tracts to the protocerebrum suggest a role of hug neurons in transducing sensory signals for processing in the higher brain centers. These axon tracts are distinct from those of the olfactory projection neurons, projecting to a more dorsomedial region of the mushroom body, and adjacent to the median neurosecretory cells of the pars intercerebralis. Thus, hug neurons are ideally connected to undertake the role of integrating gustatory sensory signals with higher brain functions and feeding behavior.
Chemosensory Adaptation, Nutrient Status, and Food Intake Response
The chemosensory systems of all animals play critical roles in modulating feeding behavioral response. Feeding behavior can have diverse aspects, including locating a food source, evaluating food for nutritional appropriateness, choosing between different food sources, and deciding to initiate or terminate feeding. Blocking synaptic transmission by tetanus toxin in the hug neurons alters a specific aspect of the feeding behavioral response. When transferred to a certain new food source, the control flies wait for a period before initiating feeding, whereas experimental flies start feeding almost immediately. In both cases, the size of the crop after a longer feeding period does not change, meaning that no difference is seen in the termination phase of feeding. It is interesting to note that GR66C1 (also named GR66a) neurons, which project to hug dendrites, have recently been shown to mediate aversive taste response [39,40]. This is consistent with the behavior of flies in which hug signaling is decreased, since they lose their "aversive" response, as manifested in the elimination of a wait period before feeding. This behavior is dependent on internal nutrient status, as well as food quality, since if animals are starved or given food with an aversive tastant beforehand (such as yeast with quinine), control flies also start feeding immediately on the new yeast source.
Insects have evolved a wide variety of feeding behaviors based on food identity, quality, and availability. Some of these are innate, whereas others are acquired through experience. For example, food preference in the tobacco hornworm is dependent on what they initially encounter after hatching. They are capable of growing on a wide variety of sources, but once they have fed on a particular food type, they will maintain this food preference [41,42]. In this context, a possible scenario is that Drosophila associate feeding with a particular food source with which they become familiar. When they encounter a different food source, they must first re-evaluate it, perhaps for nutrient content, or adapt to it, before initiating feeding. Therefore, hug neurons appear to regulate the decision to initiate feeding based on previous food experience.
Central Integration of Feeding Behavior and Growth
In animals with a developed endocrine system, there is an intricate interdependence among feeding, growth, and neuroendocrine activity. Drosophila larvae are characterized by continuous feeding and a huge increase in organismal growth; in the adult, although no growth at organismal level takes place, a large cellular growth is required in the female for egg production. Both are highly dependent on feeding and the quality of food, such as protein content, and are under neuroendocrine control [2]. klu and ppl represent two genes that are required for food intake and growth in Drosophila. Mutations in both genes result in reduced food intake and growth. In addition, as young larvae, mutants display a wandering-like behavior, which is reminiscent of full-grown wild-type larvae, which stop feeding and move away from the food source just prior to pupariation, a process dependent on the neuroendocrine system [23]. Mutations in either of the genes lead to an upregulation of hug neuropeptide gene expression in the brain, whereas hug expression is downregulated in the absence of food signals.
What could be the function of the hug neuropeptides? hug encodes at least two distinct neuropeptides [26]. One (hug-γ) has homology to an ecdysone triggering hormone, while the second (Drm-PK-2) is a pyrokinin with myostimulatory activity. hug-γ could be involved in controlling growth and metabolism. This view is supported by projection of hug neurons to the ring gland, the master neuroendocrine organ. In addition, overexpression of hug has been shown to cause molting defects [26]. Drm-PK-2, on the other hand, may play a role in modifying the mechanical aspect of food intake, which is supported by the projection of hug neurons to the pharyngeal muscles. One interesting possibility is that the different neuropeptides are translated or trafficked to different targets in subset of hug neurons. In this case, a common gene expression pattern can be utilized to send out different signals to the different targets, such as to the higher brain center, feeding apparatus, and neuroendocrine organ. This would be a mechanism for coordinating different growth-dependent processes with a common input signal, for example, from a particular food signal. In this context, one way to explain the upregulation of the hug gene in klu and ppl mutants would be that the level of hug gene differentially correlates with the degree of food-seeking response. High levels, as in the mutants that do not feed, would reflect lower feeding and food-seeking response, whereas low levels, as in the absence of food sensory input, would reflect increased food-seeking response. This would also be consistent with hug overexpression studies and with the correlation seen between decreasing hug neuronal activity and increased feeding (Figure 7). Further functional studies, including imaging analysis [43,44], should increase our understanding of how the hug neural circuit coordinates sensory perception, feeding behavior, and growth.
The hug neurons, which express the neuropeptide gene hug and which interconnect gustatory sensillae via the SOG to the pharyngeal muscles, the protocerebrum, and the neuroendocrine organ, modulate chemosensory dependent feeding behavior. Increased hug signaling correlates with decreased feeding, whereas decreased hug signaling correlates with increased feeding (see Discussion section for details).
Materials and Methods
Feeding behavior assay
The larval feeding behavior assay was done as described previously [22]. Flies were allowed to lay eggs on apple juice agar plates containing colored yeast paste (150 mg Carmen Red, Sigma-Aldrich [St. Louis, Missouri, United States] per 100 g yeast paste). Given numbers of larvae from overnight egg collections were allowed to develop for 24 h at 25 °C and subsequently (2-h intervals) monitored for feeding and wandering phenotypes under a dissection microscope. For starvation experiments, wild-type larvae of late second instar were placed in petri dishes, containing filter paper that was soaked with either PBS (for complete starvation) or PBS containing 20% sucrose. For normal feeding conditions, fresh yeast paste was given. All feeding experiments were done at room temperature for 6 h. Overexpression studies were done using UAS-hug and tub-Gal4/TM3 fly lines [26], and heterozygote siblings were used as controls.
For larval rescue experiments, lines used were P(9036)/TM3 (parental line 1), UAS-TeTxLC; P(9036)/TM3 (parental line 2), and hugS3, P(9036)/TM3 (parental line 3). The genotypes assayed were +/UAS-TeTxLC; and P(9036)/P(9036) for control 1 (C1) + hugS3, P(9036)/P(9036) for control 2 (C2) and +/UAS-TeTxLC/+; hugS3, P(9036) for experimental (see Figure 6B). Feeding phenotypes were counted from 100 eggs per collection. Five independent collections per genotype were carried out.
For the adult feeding assay, experimental flies (hug promoter construct driving UAS-TeTxLC expression) and control flies (hugS3-Gal4 flies and UAS-TeTxLC flies crossed with wild-type) derived from 0- to 4-h egg collections were allowed to develop on indicated food for several days. After overnight feeding (for example, on standard fly food or PBS only), flies were allowed to feed on apple juice agar plates containing red-colored food being assayed. At given time points, randomly chosen individuals per genotype were removed and dissected under a dissection microscope. Preparations were fixed and mounted in Mowiol (see below) for microscopic analysis.
Molecular and microarray analyses
The hug construct was made by cloning a 1.5-kb PCR fragment containing the hug regulatory region (amplified from genomic DNA using 5′– CTTCAGGGCCTTGGCTG and 5′– GGGACAACTGATGCCACG as primers) into a pCaSpeR-AUG-Gal4 vector [10]. The direct hug promoter YFP construct was made by replacing AUG-Gal4 of the hug-pCaSpeR construct, with a YFP fragment derived from YFP-pCS2+ vector (Clontech, Palo Alto, California, United States). Transgenic flies were obtained following standard injection protocols.
Microarray experiments were, in principle, done as described previously [45] using Affymetrix (Santa Clara, California, United States) GeneChips representing some 13,500 genes. Egg collections (0–4 h) of the P(9036)/TM3-GFP line were allowed to develop for an additional 22 h at 25 °C. Homozygous P(9036) larvae were hand-picked under a fluorescence microscope, and total RNA was isolated using the NucleoSpin RNA II Kit (Macherey-Nagel, Düren, Germany). GeneChip hybridization and data analysis was done as described [45].
Histochemistry and fluorescence microscopy
Histochemical in situ hybridizations were done following standard protocols, with the slight modification of replacing the proteinase K digest with an overnight incubation of the dissected and fixed larval brains in methanol at −20 °C prior to hybridization. Samples were mounted either in Canada balsam or in Mowiol (12 ml glycerol, 4.8 g Mowiol 40–88, 12 ml H2O, and 24 ml 200 mM Tris [pH 8.5]), and images were taken using a Zeiss (Oberkochen, Germany) LSM 510 META in transmission mode. Fluorescence in situ hybridizations were done using the Tyramide Signal Amplification Kit (PerkinElmer, Wellesley, California, United States) and following the manufacturer's instructions. Overnight incubation with digoxygenin- and/or fluorescein-labeled riboprobes was followed by post-hybridization for additional 2 h. Detection of the first riboprobe with peroxidase (POD)-coupled antibody was performed by overnight incubation at 4 °C, followed by the first staining reaction using fluorescein-tyramide at 1:150 dilution and allowing the reaction to run for 10 min at room temperature. After inactivation of POD by incubation with 10 mM HCl in Drosophila Ringer's solution for 10 min, the second riboprobe was detected by overnight incubation with POD-coupled antibody at 4 °C. The second staining reaction was performed by applying Cy3-tyramide at 1:150 dilution for 10 min at room temperature. In cases of dual marker protein detection, primary antibodies (α–ßGal, Cappel, or α–GFP [Abcam, Cambridge, United Kingdom], used at 1:1,000) were applied together with first POD-antibody and secondary fluorescent antibody (Cy5-coupled α-rabbit, diluted at 1:200 [Jackson Immunoresearch, West Grove, Pennsylvania, United States]) was applied together with second POD-antibody. Samples were mounted in Mowiol and evaluated using a Zeiss LSM 510 META in confocal multitracking mode, generating optical 1- to 1.5-μm sections (using a Zeiss 40×/1.2W C-Apochromat lens) or 2.5-μm sections (using a Zeiss 25×/0.8Imm Plan-Neofluar lens). For direct detection and unmixing of GFP/YFP fluorescence, larval brains of appropriate genotype were dissected in chilled Drosophila Ringer's solution on ice, and mounted without fixation in PBS, using coverslips as spacers and nail polish as sealant. Immediate analysis was performed by emission fingerprinting, using a Zeiss LSM 510 META in confocal lambda mode. Other antibodies used for immunofluorescence were 22C10 diluted 1:100 (Developmental Studies Hybridoma Bank, Iowa City, Iowa, United States) and α–elav, diluted 1:300 (Developmental Studies Hybridoma Bank), as well as Alexa488-coupled α-rat and α-mouse antibodies, each diluted 1:200 (Molecular Probes, Eugene, Oregon, United States) and Cy3-coupled α-rat and α-rabbit antibodies diluted 1:200 (Jackson Immunoresearch, West Grove, Pennsylvania, United States). Nuclear counterstaining was performed using Draq5 (Biostatus Ltd., Leicestershire, United Kingdom), diluted 1:1,000 together with secondary antibodies. The GFP antibody co-labeling the corpora allata nuclei (Torrey Pines Biolabs, Houston, Texas, United States) was used at 1:1,000 dilution. The 3D reconstruction of optical sections and figure post-processing were done using Volocity 2.6 (Improvision, Lexington, Massachusetts, United States) and Photoshop 7.0 (Adobe Systems, San Jose, California, United States) on a Mac G4 computer (Apple Computer, Sunnyvale, California, United States).
Supporting Information
Figure S1. Qualitative Graphical Representation of Feeding Analysis
Flies were scored (none, traces, or full) based on amount of red food color in the gut and crop. Overnight treatment was for 12 h. Arrows represent transfer to fresh food, either of the same type or different. For each feeding regimen, two independent experiments were carried out, and for each experimental set and time point, ten individuals were taken out randomly and dissected. The top two graphs are graphical representations of the results depicted in Figure 6D. For experiments with quinine yeast as test food, 12 ± 2 individuals showed displayed phenotypes.
Figure S2. Fortuitous Staining of Corpora Allatum
Fortuitous staining of corpora allatum nuclei (shown in green, arrows) by α-GFP antibody from Torrey Pines Biolabs in wild-type larvae (A) and adults (B and C) relative to the neuropile (22C10, shown in red) and the cortex (DNA marker Draq5, shown in blue). We do not know the reason for this, as α-GFP antibodies from other sources do not show this cross-reactivity.
We thank G. Wahlström, R. F. Stocker, K. Scott, D. Schmucker, L. B. Vosshall, B. Gerber, S. Noselli, T. Klein, C. O'Kane, and T. Siegmund for fly lines and vectors, and T. Kastilan for help with the transgenics. This work was supported by Forschungszentrum Karlsruhe and by grants from DFG (Deutsche Forschungsgemeinschaft) to MJP.
Author Contributions
CM and MJP conceived, designed, and performed the experiments, analyzed the data, contributed reagents/materials/analysis tools, and wrote the paper.
- 1. Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG (2000) Central nervous system control of food intake. Nature 404: 661–671.
- 2.
Pflugfelder O (1958) Entwicklungsphysiologie der Insekten. Leipzig (Germany): Akademische Verlagsgesellschaft Geest & Portig. 490 p.
- 3.
Dethier VG (1976) The hungry fly. Cambridge: (Harvard University Press). 489 p.
- 4. Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim J, et al. (1999) A novel family of divergent seven-transmembrane proteins: Candidate odorant receptors in Drosophila. Neuron 22: 327–338.
- 5. Gao Q, Chess A (1999) Identification of candidate Drosophila olfactory receptors from genomic DNA sequence. Genomics 60: 31–39.
- 6. Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R (1999) A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725–736.
- 7. Clyne PJ, Warr CG, Carlson JR, et al. (2000) Candidate taste receptors in Drosophila. Science 287: 1830–1834.
- 8. Scott K, Brady R, Cravchik A, Morozov P, Rzhetsky A (2001) A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila. Cell 104: 661–673.
- 9. Gao Q, Yuan B, Chess A (2000) Convergent projections of Drosophila olfactory neurons to specific glomeruli in the antennal lobe. Nat Neurosci 3: 780–785.
- 10. Vosshall LB, Wong AM, Axel R (2000) An olfactory sensory map in the fly brain. Cell 102: 147–159.
- 11. Marin EC, Jefferis GS, Komiyama T, Zhu H, Luo L (2002) Representation of the glomerular olfactory map in the Drosophila brain. Cell 109: 243–255.
- 12. Wong AM, Wang JW, Axel R (2002) Spatial representation of the glomerular map in the Drosophila protocerebrum. Cell 109: 229–241.
- 13. Heisenberg M, Borst A, Wagner S, Byers D (1985) Drosophila mushroom body mutants are deficient in olfactory learning. J Neurogenet 2: 1–30.
- 14. Stocker RF, Heimbeck G, Gendre N, de Belle JS (1997) Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons. J Neurobiol 32: 443–456.
- 15. Stocker RF (1994) The organization of the chemosensory system in Drosophila melanogaster: A review. Cell Tissue Res 275: 3–26.
- 16. Stocker RF, Schorderet M (1981) Cobalt filling of sensory projections from internal and external mouthparts in Drosophila. Cell Tissue Res 216: 513–523.
- 17. Singh NR (1997) Neurobiology of the gustatory systems in Drosophila and some terrestrial insects. Microsc Res Tech 275: 3–26.
- 18. Gendre N, Luer K, Friche S, Grillenzoni N, Ramaekers A, et al. (2004) Integration of complex larval chemosensory organs into the adult nervous system of Drosophila. Development 131: 83–92.
- 19.
Penzlin H (1985) Stomatogastric nervous system. In: Kerkut GA, Gilbert LI, editors. Comprehensive insect physiology. Oxford: Pergamon Press. pp. 371–406.
- 20. Hartenstein V, Tepass U, Gruszynski-Defeo E (1994) Embryonic development of the stomatogastric nervous system in Drosophila. J Comp Neurol 350: 367–381.
- 21. Aubele E, Klemm N (1977) Origin, destination and mapping of tritocerebral neurons of locust. Cell Tissue Res 178: 199–219.
- 22. Zinke I, Kirchner C, Chao LC, Tetzlaff MT, Pankratz MJ (1999) Suppression of food intake and growth by amino acids in Drosophila: The role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126: 5275–5284.
- 23.
Riddiford L (1993) Hormones and Drosophila development. In: Bate M, Arias AM, editors. The development of Drosophila. Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press. pp. 899–939.
- 24. Klein T, Campos-Ortega JA (1997) Klumpfuss, a Drosophila gene encoding a member of the EGR family of transcription factors, is involved in bristle and leg development. Development 124: 3123–3134.
- 25. Yang X, Bahri S, Klein T, Chia W (1997) Klumpfuss, a putative Drosophila zinc finger transcription factor, acts to differentiate between the identities of two secondary precursor cells within one neuroblast lineage. Genes Dev 11: 1396–1408.
- 26. Meng X, Wahlstrom G, Immonen T, Kolmer M, Tirronen M, et al. (2002) The Drosophila hugin gene codes for myostimulatory and ecdysis-modifying neuropeptides. Mech Dev 117: 5–13.
- 27. Park Y, Kim YJ, Adams ME (2002) Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor co-evolution. Proc Natl Acad Sci U S A 99: 11423–11428.
- 28. Riehle MA, Garczynski SF, Crim JW, Hill CA, Brown MR (2002) Neuropeptides and peptide hormones in Anopheles gambiae. Science 298: 172–175.
- 29. Brogiolo W, Stocker H, Ikeya T, Rintelen F, Fernandez R (2001) An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Curr Biol 11: 213–221.
- 30. Ikeya T, Galic M, Belawat P, Nairz K, Hafen E (2002) Nutrient-dependent expression of insulin-like peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr Biol 12: 1293–1300.
- 31. Rulifson EJ, Kim SK, Nusse R (2002) Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes. Science 296: 1118–1120.
- 32. Zars T, Fischer M, Schulz R, Heisenberg M (2000) Localization of a short-term memory in Drosophila. Science 288: 672–675.
- 33. Estes PS, Ho GL, Narayanan R, Ramaswami M (2000) Synaptic localization and restricted diffusion of a Drosophila neuronal synaptobrevin–green fluorescent protein chimera in vivo. J Neurogenet 13: 233–255.
- 34. Siegmund T, Korge G (2001) Innervation of the ring gland of Drosophilamelanogaster. J Comp Neurol 431: 481–491.
- 35. Friggi-Grelin F, Coulom H, Meller M, Gomez D, Hirsh J, et al. (2003) Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J Neurobiol 54: 618–627.
- 36. Schroter U, Menzel R (2003) A new ascending sensory tract to the calyces of the honeybee mushroom body, the subesophageal-calycal tract. J Comp Neurol 465: 168–178.
- 37. Connolly JB, Roberts IJ, Armstrong JD, Kaiser K, Forte M, et al. (1996) Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies. Science 274: 2104–2107.
- 38. Sweeney ST, Broadie K, Keane J, Niemann H, O'Kane CJ (1995) Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects. Neuron 14: 341–351.
- 39. Thorne N, Chromey C, Bray S, Amrein H (2004) Taste perception and coding in Drosophila. Curr Biol 14: 1065–1079.
- 40. Wang Z, Singhvi A, Kong P, Scott K (2004) Taste representations in the Drosophila brain. Cell 117: 981–991.
- 41. del Campo ML, Miles CI, Schroeder FC, Mueller C, Booker R, et al. (2001) Host recognition by the tobacco hornworm is mediated by a host plant compound. Nature 411: 186–189.
- 42. del Campo ML, Miles CI (2003) Chemosensory tuning to a host recognition cue in the facultative specialist larvae of the moth Manduca sexta. J Exp Biol 206: 3979–3990.
- 43. Fiala A, Spall T, Diegelmann S, Eisermann B, Sachse S, et al. (2002) Genetically expressed chameleon in Drosophilamelanogaster is used to visualize olfactory information in projection neurons. Curr Biol 12: 1877–1884.
- 44. Wang JW, Wong AM, Flores J, Vosshall LB, Axel R (2003) Two-photon calcium imaging reveals an odor-evoked map of activity in the fly brain. Cell 112: 271–282.
- 45. Zinke I, Schutz CS, Katzenberger JD, Bauer M, Pankratz MJ (2002) Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. Embo J 21: 6162–6173.


text:
Hypocrites have overrun country
Published: Friday, April 29, 2005 at 4:30 a.m.
Last Modified: Friday, April 29, 2005 at 12:00 a.m.
To The Editor: From the oceans to the prairies, this country is a nation of irresponsible hypocrites. I love America the country, but sometimes I wonder about America, the People.
We complain about gas prices as we drive our gas-sucking SUVs and trucks. We complain about someone's spoiled, lazy and irresponsible children (yours are too) who demand everything now, when we should look in a mirror to see who teaches them to be this way!
Our children cannot pass eighth-grade math and science when they graduate from high school, then we complain when technical jobs are going overseas. Pseudo-Christianity in this country has become a "fad"; all the while we murder innocent people in other countries in the name of "anti-terrorism" and then wonder why those countries hate us.
How did you feel on 9/11? Yet, we support the attack of Iraq, killing their innocents by the thousands! We complain about those politicians we helped get elected into office!
As a wise woman told me years ago when I would complain about something that was caused by my actions (or nonactions), "Boy, you made your bed, now lie in it." What a smart woman she is.
Don Howell
Reader comments posted to this article may be published in our print edition. All rights reserved. This copyrighted material may not be re-published without permission. Links are encouraged.
Comments are currently unavailable on this article

subset: commoncrawl


text:
Latest Video Reviews
Battle Supremacy: Evolution iPad Review
Dead Effect 2 Review
Afterpulse iPad Review
Brothers: A Tale of Two Sons iPad Review
FIFA 16 Ultimate Team� Review
Lara Croft GO Review
FINAL FANTASY VII iPad Review
Loot & Legends iPad Review
To-Fu Fury iPad Review
Warhammer 40,000: Deathwatch - Tyranid Invasion iPad Review
This War of Mine iPad Review
Nono Islands iPad Review
Latest Articles
Cave Bowling Review
After a while it's almost comforting to play a game by Donut Games, if only because you know almost exactly what you're getting in to before you even load the game. After loading up Cave Bowling I'm greeted by the same menus, the same options and the same 3-star system common to just about every one of their games, but once you get in to the game it's clear to see that Donut Games are learning some new tricks. Cave Bowling is all about smashing a ball across the screen after setting a…Watch The Video Review
Everybody Hates NASA: Pro Review
Sometimes you have to wonder if you get what you deserve when you make a wish and it's eventually answered. I often wish for developers to try their hands at something whacky and out of the ordinary, if only to give me something interesting to review. Everybody Hates NASA by Inert Soap could certainly be defined as 'whacky' and 'out of the ordinary' and while it's not a perfect game, the way in which it seems to be able to mash seemingly disparate elements together is impressive. At i…Watch The Video Review
Creatures & Castles Review
Do you fancy yourself to have a steady hand and god-like skills of timing? If so you might just be one of the few people Creatures & Castles by hiive intends to target as you plunge in to the labyrinthine depths of this puzzle title.The premise is a simple one that's easy to get behind as you're presented with room after room of maze-like designs that need to be explored to collect a treasure before exiting the level. The catch is that you can only move on a path that you have draw…Watch The Video Review
Sonic Spinball Review
Sonic the Hedgehog had an odd period in his career where he was placed in all sorts of strange predicaments and situations that have (in retrospect) been considered fairly questionable. Though strangely enough, the idea of Sonic in a pinball game wasn't much of a stretch and in 1993 gamers were given Sonic the Hedgehog Spinball (or Sonic Spinball as it's now known on the App Store) by SEGA. While regular pinball titles were often content with simple physics engines and skill-shots to…Watch The Video Review
Zombie Trailer Park Review
Zombie Trailer Park by Ninja Kiwi is (for those who haven't come across it before) another free Flash title that has made the jump to the App Store. Interestingly, the game has been converted with almost no changes (barring the interface) and to compensate for this the price tag is a surprisingly reasonable '$0'. The game itself is a fairly typical castle defense setup, with cash being earned over time and units being purchased to run blindly in to their opposition until you've pushed…Watch The Video Review
Farts VS Zombies Review
I'll be perfectly honest here and say that I had no idea what to expect when I jumped in to Farts VS Zombies by Mutant Games. Was it meant to be ironic? Was it simply a cash-in on keywords? In short I asked myself, 'How self-aware is this game?' and the answer was fairly clear. It knows exactly what it's doing and I'm not sure that manages to make the experience any better. You'll have to take control of Colonel John Farter (resident Chuck Norris look-alike) as he creates multicolored…Watch The Video Review
Trouble in Tin Town Review
With so much variety available on the App Store, it's odd that more turn-based strategies haven't cropped up. Developer Jovian Minds has clearly seen an opportunity in the lack of strategic titles and has paired their game with a retinue of witty wind-up toys that are just itching to jump in to a fight. Though perhaps calling it 'strategy' is stretching things a little bit as the game often boils down to punching ranged units with your melee units and shooting melee units with your ra…Watch The Video Review
Pocket HalfPipe Review
It may not be the next 'Angry Birds', but Pocket HalfPipe by NaffRobot is the kind of casual title that balances on a fine edge of skill and simplicity, resulting in a game that's immediately addictive even without the need for overly-complicated gameplay or multiple levels. There are three competitive modes along with a fourth free-skating mode to play in and the goal is to simply beat the score that has been set, unlocking a new item of clothing, skateboard or even a special move to…Watch The Video Review
DeathFall Review
If you haven't heard the term 'ear worm' before, let me explain: Have you ever had a moment when you realize you have a small loop of a song stuck in your head and it's almost impossible to get your brain to skip over it? That's an 'ear worm'. DeathFall by Jurgis Games is the gaming equivalent of an 'ear worm' and while there could be something grand beyond the walls of this game, you feel like you're stuck in some never ending loop that never attempts to break free. The journey you t…Watch The Video Review
The Blocks Cometh Review
UPDATE: It has come to our attention that this game may have been produced without the consent of Halfbot (developers of the original free Flash game of the same name). Until this is resolved, please support the original developers by holding off on your purchase of this title and feel free to check out the original at: http://www.halfbot.com/?page_id=279 Finding that next great addictive Arcade title isn't always an easy task, but searching for games that boil down other games to the…Watch The Video Review
Block Rogue Review
Block Rogue by Buzz Monkey Software isn't your typical Sokoban-style puzzler, but not because you play the game in some unique way. You'll find yourself hitting switches, pushing blocks and rolling boulders much like any similar puzzler, but for your little character there's a clear motivation for attempting to complete all 325 levels. That's right, 325 levels - and we're not talking easy levels either; you'll have to avoid the usual set of problems that will force you to slap the und…Watch The Video Review
Top 10 iOS Games of 2010 - AppSpy Edition
Hi everyone! With the holiday period having come to a close we've finally made some time to let our readers know what we consider to be the best releases of 2010. As with almost any 'top list' it should be taken with a grain of salt, but to clarify things further this is more of a personal expression of what rocked my world and what I believe is important in terms of the growth of the iPhone as a serious handheld gaming device. SPOILER NOTE: I've placed a text transcript of the audio below the video. If you'd prefer not to spoil things, don't scroll down too far ;) 2010 was, for iOS device users, a crazy year packed with highlights that ranged from incredible highs to disturbing…
Space Hedgehogs vs. Space Cacti Review
Attempting to improve on the elegant simplicity of Galcon Labs' real-time Risk style gameplay is no easy task, but titles like Civilizations Wars and Modern Conflict have certainly held their ground. Space Hedgehogs vs. Space Cacti by Sad Cat Software adds a 4X (eXplore, eXpand, eXploit, eXterminate) like features to the mix, and while it's not Master of Orion, you'll still need to outwit and outplay your opponents while balancing your resources for research. Forces can be deployed to…Watch The Video Review
Mad Skills Motocross Review
If you never had a chance to play the classic motocross racer 'Excitebike' back in the day then you may have missed out on a genuinely (and I'm sorry to say it) exciting racer that challenged players with its semi-realistic style that rewarded players that carefully managed their bikes instead of simply smashing the accelerator. Mad Skills Motocross by Turborilla feels like a serious revival of this classic racer, pitting players against AI opponents while using minimalistic controls t…Watch The Video Review
Garage Inc. Review
Sure you may have powdered a few noses, treated a few people to spas and served them tasty food in the past, but have you pulled up your sleeves to repair some vintage vehicles? Garage Inc. by Breakthrough Entertainment is a management style title with a classic twist and you'll have to work heard to break out of your debt with the local mobsters. After taking a loan from his cousin, Angelo Marito opens up a garage in Chicago and shortly thereafter is told about where that money came…Watch The Video Review


text:
I'm in a wicked music rut and could use some suggestions. The last CD I bought was the Black Keys which I love but it's only like 30 minutes long so I've listened to it about 5,000 times and it's getting a little stale. I've been reverting to all my old cds which is fine, but I miss listening to new music. Thanks!
Way OT - What's everyone listening to for music these days?
i have sirius satelite in my car so i don't really listen to CD's. My hubby bought and suprised me with my SUV when i got home from the hospital after having our baby, I got a Adele CD and its been in the car ever since. so if i'm sick of the radio i listn to Adele. or a lullabye baby Pearl Jam CD for the baby. but i actully think he preferrs the adele CD. he seems to love music.
Kellen 5.18.11
Baby on Board 11.15.12
i love the black keys!
I Love my baby boy!! 5/6/2011
Expecting my princess!! 8/18/2012
Gold boobies ( . Y . )
I have Sirius as well, so it's Howard Stern all the time, but right now, it's Beastie Boys- RIP MCA
Mommy to 3 babies- 1 with fur, 1 without & 1 with wings
I know, terrible #cancersucks
I'm listening to a station on Sirius that's doing a tribute to him
Mommy to 3 babies- 1 with fur, 1 without & 1 with wings
mumford & sons, and lots of classics lately, sinatra, simon and garfunkle, chicago, journey, beatles, phil collins, but then i also mix in afi, matisyahu, the neptunes. yeah i'm pretty random
DUBSTEP!!! Loooooove me some good techno beats like that!!! Check out like Bass Cannon by FluxPavillion, Skrillex, or anything by Vaski. There is sooooo much good stuff outt here. I also love me some LMFAO, Nicki Minaj, Eminem, Lil Wayne, etc.
Some people just need a high five.
In the face.
With a chair.
Have Sirius, too, love it! I just bought Adele which is great and I also always have the best of Heart in my car and several 80's compelations and my ipod is full of stuff - everyting from Yanni to Journey to Elvis.
Thanks everyone. I have Sirius too and I love it, but sometimes I get tired because they play all the same new songs. I know there are other new bands out there that I'd probably love, but just can't figure out how to find them...
Learn more
Look how far your child has come.
Review your child's growth milestones.
Read about 5 years 1 month : Week 2.
Your growing child
Your 5-year-old: Telling lies
Your 5-year-old: Boundless energy
Your 5-year-old: Speaking skills
Your 5-year-old: Healthy weight
Your 5-year-old: Loveys
Learn more
What's next for your growing child?
Get a glimpse of what's to come.
Read about 5 years 3 months.


text:
I just started doing the Strong-lifts program this week, and while researching the correct movement for each exercise I got a little confused with the barbell row. in some videos I saw it being done with an underhand grip, and in others with an overhand grip. Is there a difference between the grips? If there is, then which one is recommended to do in order to get the best results from the Strong-lifts program?
Physical Fitness Stack Exchange is a question and answer site for physical fitness professionals, athletes, trainers, and those providing health-related needs. Join them; it only takes a minute:
Sign up
Sign up
Here's how it works:
- Anybody can ask a question
- Anybody can answer
- The best answers are voted up and rise to the top
I do barbell rows overhand, like exrx recommends and like Arnold does and like this StrongLifts-doing guy does. I think underhand would turn them into curls when they're challenging.
StrongLifts specifically recommends against the underhand grip.
I am glad that you are venturing into the world of barbell rows. They are a great row in my opinion. To address your question, I have to go with the overhand grip.
To support my answer, I recommend taking a look at the video presented in this article The Bent Over Barbell Row.
You'll notice that during the bent over barbell row you lean over the bar and lift up, so using an overhand grip gives you a sturdier hold and a better angle to lift upwards.
I hope this helps you- Neil Cook


text:
Fresh flakes bring crowds to mountain
Conditions spotty, but betterUPDATED 8:52 PM EST Jan 19, 2014
Fresh snow is a welcome sight for ski mountains, ski shops and skieers across Burlington.
More Video
CommentsThe views expressed are not those of this site, this station or its affiliated companies. By posting your comments you agree to accept our terms of use.blog comments powered by Disqus


text:
Amnesty rush eases off at Asian embassies
Abu Dhabi: As the amnesty declared by the UAE for illegal workers ends on Sunday, the number of Asian workers who have sought emergency certificates [out-passes] from their embassies has dropped in comparison to the previous amnesty in 2007.
Asians constitute the majority of the workforce in the UAE.
The Pakistani embassy in Abu Dhabi issued 5,867 emergency certificates to amnesty seekers — the highest number among Asian nations, followed by the Bangladesh and Indian missions — 4,500 and 2,600 respectively.
Amnesty seekers who are not in a position to produce their passports have to get the emergency certificates from their embassies or consulates to prove their nationality.
After the applicants receive out-passes from their respective embassy or consulate, the UAE immigration authorities issue an exit permit that allows them to leave the country, provided they had entered the UAE on a valid visa.
Those amnesty seekers who have a passport can directly approach the UAE immigration authorities to get the exit permit. Between December 4 and January 4, about 20,000 illegal residents from 106 nations approached the immigration authorities and more than 9,400 of them already left the country, the UAE Ministry of Interior announced on January 5. The updated figures were not available from the ministry.
The Pakistani embassy issued 5,867 emergency certificates as of Monday, January 28, said Jamil Ahmad Khan, the Pakistani ambassador. About 30,000 Pakistanis in the UAE had availed themselves of the amnesty in 2007 but, this year, the number will be less, Khan said.
The Bangladeshi embassy issued about 4,500 out-passes and the consulate in Dubai about 12,000 as of Sunday, said Mohammad Nazmul Quanine, Bangladesh's ambassador to the UAE. The total number of applicants may not cross 25,000 by the end of the deadline whereas 40,500 Bangladeshis availed of the amnesty in 2007 through the embassy and the consulate, the envoy said. A few thousands more might have directly approached the immigration authorities during the previous amnesty, he added.
The number of amnesty seekers from India, who constitute the largest expatriate community in the UAE, has gone down drastically compared to 2007, according to M.K Lokesh, India's ambassador to the UAE.
The Indian embassy in Abu Dhabi and the consulate in Dubai had issued 2,600 out-passes as of Sunday, Lokesh said. About 40,000 Indians took advantage of the 2007 amnesty, but the number this year will reach nowhere near that figure, he added.
The Philippine embassy in Abu Dhabi has issued about 1,571 out-passes as of January 27, said Grace Relucio Princessa, Philippine ambassador to the UAE.
The Sri Lankan embassy, despite repeated attempts, did not comment on the number of people who had approached it for out-passes.
The Indonesian and Nepalese embassies said they had received roughly the same number of out-pass applicants as during the previous amnesty. The Indonesian embassy issued about 850 out-passes as of February 27, said Salman Al Farisi, Indonesia's ambassador.
"Last time [2007], we issued about 1,000 out-passes and the number may reach the same this time," he said.
The Nepalese embassy issued 107 out-passes as of Sunday, said Deepak Adhikari, deputy head of the mission. He said the figure was not much different during the previous amnesty.


text:
iHerb カスタマーレビュー
のセーブ: ¥335 (23 % オフ)
TERRIFIC cough syrup!
Reviewer2031172により Sep 10, 2010 に掲載
My husband and I have been using this cough syrup for a couple of years now. We discovered it here on iherb. We both have asthma and it works to soothe and calm coughs for whatever reason. In July of this year, my husband had a quadruple bypass. Anybody who knows what this is knows that you have a breathing tube down your throat and his was in a little longer than usual. When they pulled it out, it hurt his throat more than the average person. He's had a frog in there for awhile. It didn't dawn on me to try this syrup out on him because it's not like it's a cold or asthma issue. But out of desperation we tried it and it calms down the frog and weird cough he has from the surgery so he gets some relief. I give this cough syrup AAAAAAAAAAAAAAAa+++++++++++++ !! Better than anything OTC.
mrkq10により Oct 30, 2007 に掲載
it helps to relief cough especialy at night.
Daytime cough syrup by B&T
Reviewer1363563により Jul 24, 2008 に掲載
This has been the most effective cough syrup for my fmaily! Honey is the key ingredient.
Boericke & Tafel Daytime cough syrup
Reviewer1153068により Aug 19, 2008 に掲載
Wonderful product! I am a singer and this product helps sooth the throat so well and of course it helps with a cough/ cold symptoms. Please continue to offer both the night and daytime cough syrups.They are fantsatic!!!
Reviewer1231783により Dec 22, 2007 に掲載
Works very well! And, it has no harmful side effects.
Great Stuff!
Reviewer3351687により Jul 13, 2008 に掲載
I was skeptical when my daughter sent me my first bottle, but found out it is great when your cough is just starting. Reduces symptoms right away.
Great Product!
Reviewer1705774により Jun 09, 2011 に掲載
Started with nighttime, add this after it worked so well.
soothing on the throat
bbchrisにより Jan 08, 2010 に掲載
I was delighted to find a gentle cough syrup to keep on hand in medicine cabinet. Thank you iherb!
Katarinatanにより Nov 11, 2012 に掲載
I got this for my mom's persistent coughs. She seemed to be better now.
Tastes good
Reviewer3350679により Nov 08, 2008 に掲載


text:
Filter by:
Content type:
Stage 1&2
Stage 2&3
Stage 3&4
Stage 4&5
Challenge level:
This article explains how to make your own magic square to mark a special occasion with the special date of your choice on the top line.
Replace each letter with a digit to make this addition correct.
This addition sum uses all ten digits 0, 1, 2...9 exactly once.
Find the sum and show that the one you give is the only
What is the largest number you can make using the three digits 2, 3
and 4 in any way you like, using any operations you like? You can
only use each digit once.
Crosses can be drawn on number grids of various sizes. What do you notice when you add opposite ends?
Find a great variety of ways of asking questions which make 8.
Arrange the numbers 1 to 16 into a 4 by 4 array. Choose a number.
Cross out the numbers on the same row and column. Repeat this
process. Add up you four numbers. Why do they always add up to 34?
A combination mechanism for a safe comprises thirty-two tumblers
numbered from one to thirty-two in such a way that the numbers in
each wheel total 132... Could you open the safe?
In this 100 square, look at the green square which contains the numbers 2, 3, 12 and 13. What is the sum of the numbers that are diagonally opposite each other? What do you notice?
The sum of the first 'n' natural numbers is a 3 digit number in which all the digits are the same. How many numbers have been summed?
Use your logical reasoning to work out how many cows and how many
sheep there are in each field.
What is the sum of all the digits in all the integers from one to
one million?
Three dice are placed in a row. Find a way to turn each one so that the three numbers on top of the dice total the same as the three numbers on the front of the dice. Can you find all the ways to do. . . .
How can we help students make sense of addition and subtraction of negative numbers?
Do you notice anything about the solutions when you add and/or
subtract consecutive negative numbers?
There are exactly 3 ways to add 4 odd numbers to get 10. Find all
the ways of adding 8 odd numbers to get 20. To be sure of getting
all the solutions you will need to be systematic. What about. . . .
In the following sum the letters A, B, C, D, E and F stand for six
distinct digits. Find all the ways of replacing the letters with
digits so that the arithmetic is correct.
What are the missing numbers in the pyramids?
Mrs Morgan, the class's teacher, pinned numbers onto the backs of
three children. Use the information to find out what the three
numbers were.
Find the numbers in this sum
If you wrote all the possible four digit numbers made by using each
of the digits 2, 4, 5, 7 once, what would they add up to?
I was looking at the number plate of a car parked outside. Using my special code S208VBJ adds to 65. Can you crack my code and use it to find out what both of these number plates add up to?
Who said that adding couldn't be fun?
Is it possible to rearrange the numbers 1,2......12 around a clock
face in such a way that every two numbers in adjacent positions
differ by any of 3, 4 or 5 hours?
Investigate $1^n + 19^n + 20^n + 51^n + 57^n + 80^n + 82^n $ and $2^n + 12^n + 31^n + 40^n + 69^n + 71^n + 85^n$ for different values of n.
Can you substitute numbers for the letters in these sums?
Can you explain how this card trick works?
This article suggests some ways of making sense of calculations involving positive and negative numbers.
If each of these three shapes has a value, can you find the totals
of the combinations? Perhaps you can use the shapes to make the
given totals?
Delight your friends with this cunning trick! Can you explain how
it works?
Choose any three by three square of dates on a calendar page.
Circle any number on the top row, put a line through the other
numbers that are in the same row and column as your circled number.
Repeat. . . .
Use the numbers in the box below to make the base of a top-heavy
pyramid whose top number is 200.
You have four jugs of 9, 7, 4 and 2 litres capacity. The 9 litre
jug is full of wine, the others are empty. Can you divide the wine
into three equal quantities?
Can you arrange the digits 1,2,3,4,5,6,7,8,9 into three 3-digit
numbers such that their total is close to 1500?
Annie cut this numbered cake into 3 pieces with 3 cuts so that the
numbers on each piece added to the same total. Where were the cuts
and what fraction of the whole cake was each piece?
For this challenge, you'll need to play Got It! Can you explain the strategy for winning this game with any target?
Four bags contain a large number of 1s, 3s, 5s and 7s. Pick any ten numbers from the bags above so that their total is 37.
What is happening at each box in these machines?
Find out what a Deca Tree is and then work out how many leaves
there will be after the woodcutter has cut off a trunk, a branch, a
twig and a leaf.
Add the sum of the squares of four numbers between 10 and 20 to the
sum of the squares of three numbers less than 6 to make the square
of another, larger, number.
Using 3 rods of integer lengths, none longer than 10 units and not
using any rod more than once, you can measure all the lengths in
whole units from 1 to 10 units. How many ways can you do this?
Arrange three 1s, three 2s and three 3s in this square so that
every row, column and diagonal adds to the same total.
A lady has a steel rod and a wooden pole and she knows the length
of each. How can she measure out an 8 unit piece of pole?
Can you make square numbers by adding two prime numbers together?
The clockmaker's wife cut up his birthday cake to look like a clock
face. Can you work out who received each piece?
An environment which simulates working with Cuisenaire rods.
Tell your friends that you have a strange calculator that turns
numbers backwards. What secret number do you have to enter to make
141 414 turn around?
Place the digits 1 to 9 into the circles so that each side of the
triangle adds to the same total.
Cassandra, David and Lachlan are brothers and sisters. They range
in age between 1 year and 14 years. Can you figure out their exact
ages from the clues?
Well now, what would happen if we lost all the nines in our number
system? Have a go at writing the numbers out in this way and have a
look at the multiplications table.


text:
Online Class: Crystal Therapy
no certificate
with CEU Certificate*
18Exams &
average time
Course Description
In this new age where we seek alternative ways to treat and cure our ailments, our understanding and awareness of crystals and their healing powers have flourished. In fact, you may have noticed that something magical happens when we use crystals as part of our healing process. This Crystal Therapy course will take you on a journey of self-discovery and self-healing as we explore crystal intelligence and how crystals synchronize with the seven chakras for healing and clearing of energy fields.
Crystals and gemstones have been used for thousands of years as powerful healing aids. We may not always be conscious of it, but Crystal Therapy is part of our everyday life. We use the power of crystals in the form of rock salt and season our food using table salt crystals. Many people wear precious, semi-precious stones and different minerals not only as jewelry but for their healing benefits as well.
We humans respond to colors, light refraction and rainbows. Even animals are attracted by light play. Is it any wonder that crystal healing is a powerful, yet gentle therapy that works on all levels of the body, mind and spirit? Crystals have the potential to help all of us in a specialized way. You'll discover how they can draw in and radiate healing energy specific to each symptom of illness.
In addition, this course explains how to store and care for your crystals by cleaning, clearing and charging them to amplify their healing abilities. We'll also focus on some of the ways crystals and other stones are used in healing methods, enhancing meditation, and promoting spiritual development.
- Completely Online
- Self-Paced
- Instructor Feedback
- IACET CEUs
- 6 Months to Complete
- 24/7 Availability
- Start Anytime
- PC & Mac Compatible
- Android & iOS Friendly
Request More Information
Course Lessons
Lesson 1: History of Crystal TherapyCrystals and gemstones have been used by man for thousands of years as powerful healing aids.
Lesson 2: Crystals as Healing HelpersEnergy is channeled through the body by way of paths and, eventually, culminates in various areas of the body called energy centers (chakras).
Lesson 3: Choosing Your CrystalsLet's examine what is the most important criterion when selecting a crystal - a vibrational match to your purpose.
Lesson 4: Processing Crystals for TherapyWhen you purchase any new crystal, or if a crystal is gifted to you, it should be cleansed and cleared before use.
Lesson 5: Crystal Therapy TechniquesA crystal altar is a place where you keep your crystals and stones for meditation, healing therapy, or to restore your energy.
Lesson 6: The Light and Dark Side of CrystalsIt is believed every stone and crystal, large or small, has Deva consciousness - or an individual spiritual intelligence.
Lesson 7: Crystal HarmonyCrystals are used to assist men and women of every age for all stages of healing.
Lesson 8: Crystal LinksMost of us have a tough time deciding wall colors, let alone learning aura colors.
Lesson 9: Color Therapy, Balancing, and the MetaphysicalTo restore body balance, healers recommend using crystals along the body for a few minutes each day. Crystal Therapy is crystal vibrations that affect the flow of energy around it, and may help resolve concerns in the body.
Lesson 10: Crystal Journeys and Grid SystemsCrystal journey sessions provide a self-healing journey back to other times and places to better understand your current life.
Lesson 11: Evocative Crystal TherapyYou may or may not be interested in one or more of the healing methods explained in this lesson; however, it's often useful to be aware of the options. If one method doesn't work for you, try something else.
Lesson 12: Essential Crystals and Crystal HealersBring a list of basic and essential crystals you'll need for your kit. Stay as grounded as possible and open to invitations from the stones themselves.
Additional Course Information
- Document Your Lifelong Learning Achievements
- Earn an Official Certificate Documenting Course Hours and CEUs
- Verify Your Certificate with a Unique Serial Number Online
- View and Share Your Certificate Online or Download/Print as PDF
- Display Your Certificate on Your Resume and Promote Your Achievements Using Social Media
Choose Your Subscription Plan
No Certificate / No CEUs
This course only
|Includes certificate
|Includes CEUs
|Instructor support
|Time to complete
|6 months
|No. of courses
|1 course
Certificate & CEUs
This course only
|Includes certificate
|Includes CEUs
|Instructor support
|Time to complete
|6 months
|No. of courses
|1 course
Certificates & CEUs
Includes all 500+ courses
|Includes certificate
|Includes CEUs
|Instructor support
|Time to complete
|12 Months
|No. of courses
Certificates & CEUs
Includes all 500+ courses
|Includes certificate
|Includes CEUs
|Instructor support
|Time to complete
|No. of courses
Student Testimonials
- "I enjoyed the class, I picked up a few things, so learning is always a positive thing." -- Suzanne P.
- "Well as I am someone very well aware of crystal healing I was glad seeing these therapy mentioned here with so much well organized lectures and very useful things that we can learn both self and while sharing." -- Emrit C.
- "The instruction of this course creates positive attitude among the students and students are more likely to be involved in this course." -- Sankar V.


text:
Are you getting THE DISPLACED DISPATCH?
CLICK HERE TO REGISTER for THE DISPLACED DISPATCH. It's delivered to your inbox every other Sunday (summer schedule) and features:
* new works by expats or other international creatives;
* debate-worthy topics in the expat realm;
* a few surprising discoveries expats have made; &
* the latest Displaced Nation posts.
SIGN UP NOW. (NEXT ISSUE: July 31st)
We have interviewed many displaced creatives: memoirists, novelists, entrepreneurs & artists of various kinds. Check out the collection for possible soulmates!
About The Displaced Nation
The founders of The Displaced Nation share a passion for what we call the "displaced life" of global residency and travel—particularly when it leads to creative pursuits, be it writing, art, food, business or even humo(u)r.
Displaced Nation Columnists
Popular Posts
Follow us on Twitter
Our latest tweets
- A Canadian-Colombian couple who met thru @DateMissTravel (@JessonaJourney) buff.ly/2a1sMiT #travel #truelove https://t.co/aHx06oDbOJ..... 1 hour ago
- Who's who in the #Yangon #expat zoo? 5 kinds of foreigners now seen in #Myanmar, @TheMyanmarTimes: buff.ly/2a40p0u..... 12 hours ago
- TKX @helenahalme & @rovingjay for RTs! The travail of #travel abroad with a group of middle schoolers (1/2) thedisplacednation.com/2016/07/22/wor…..... 16 hours ago
We talk about:
Blog Stats
- 308,454 hits


text:
Your products, customized just the way you want them!
Looking for
Team Uniforms,
Embroidered Clothing?
Groups & Teams apparel on our sister site
Transform your creativity into beautiful products, sell them on CafePress and make money. It's easy and fun.
letter O, black, white, black and white, pink, hot pink, zebra, stripe, animal print, animal pattern, zebra stripe, zebra stripes, letter, monogram, monogrammed, personal, personalized,custom,initial,


text:
Easter Sunday Worship At First United Methodist Church
- When Sunday, April 20, 2014 at 8 a.m. , 9:30 a.m. and 11 a.m.
- Where First United Methodist Church, 2111 Camino del Rio South, San Diego Map | Website
- Age limit All ages
- Categories Family, Holidays, Vocal Music
- Cost Free
Above: "Lift High the Cross" Lent, Holy Week and Easter 2014 at First United Methodist Church of San Diego.
Celebrate Easter at First United Methodist Church of San Diego on Sunday, April 20, at the 8, 9:30 or 11 AM sanctuary worship featuring choirs, brass quartet, timpani and organ; or modern worship at The Water's Edge service in The Cove at 9:30 or F5 Church in Linder Hall at 11.
Sunday School for children age 3 through 5th at 9:30 or 11 with special egg hunt in each classroom. Nursery care available for all services.
Call 619-297-4366 or visit www.fumcsd.org for more information.
Location: First United Methodist Church 2111 Camino Del Rio S. San Diego, CA 92108 Google Map


text:
I'm in need of some feedback regarding our current approach with template designs please.
They are required to be advanced for features like multiple database table projects where a project uses data from more than one source. The table names need to be in the tokens so that CSV 2 POST know which tables data replaces the tokens, a precaution for when two or more tables share the same column name but actually hold different table.
This sort of thinking is for our most advanced users, but we need another approach and I would like the idea to sit for a while before I get started.
The Idea
I'm considering allowing users to create their own column replacement tokens. It would be an optional approach, so that the current approach does not vanish and confuse current users. The current approach is also quicker anyway.
Users would type in tokens until they have a token for each place the need data to be inserted. They would then pair database table columns with each of their tokens.
The Advantage
The current system does not allow a template design to be used with multiple projects in certain situations, something I was trying to avoid but it is not really possible. Explaining these situations and teaching users what situation they are in with their project and the limitations that apply and why; is not an easy job. It is a level of support that leans towards consultation, it just gets so advanced.
The approach of creating our own tokens would allow a template to be used for many projects. It would require more meta data being stored with the templates i.e. a separate set of relationships (columns to tokens) for each project. Actually a lot easier than it might sound and I'm looking forward to implementing it.
How does this sound to you?
If you have a need for sharing designs between projects please let me know.
Why Use Template Designs In Multiple Projects?
If you need to ask, you probably don't have a need for the ability. However it never hurts to know what CSV 2 POST is capable, there is a lot of hidden ability.
Some users import data to multiple tables, not a multiple table project, just multiple database tables holding different types of product data. They want the data to be used in different ways, different templates and different category setup but still on the same blog.