However, considering that an increase in the efficiency of serotonin uptake is likely to result in a decrease in the level of extracellular serotonin that can activate SER-7 in MC and that restoring serotonin in NSM in the tph-1 null mutant or killing NSM in wild type did not affect the feeding response on familiar food, it is more likely that the greater increase is due to increased serotonin release from ADF.
In conclusion, only familiar bacteria activate ADFs, which increases serotonin release from the neurons and subsequently activates the feeding response. Worms may recognize familiar bacteria by taste, smell or texture. To get insight into the mechanism, we tested if worms recognize familiar bacteria by their taste or smell. For this, we examined feeding rates of worms on bacteria mixed with LB broth or with medium conditioned by one of the bacteria Figure 14A. The conditioned media do not contain any bacterial particles, thus, if the media alter the feeding responses to familiar food or novel food, it suggests that gustatory or olfactory cues in the media were sensed by worms and affected discrimination of familiar food from novel food.
We found that the media from bacteria that are familiar to the tested worms did not alter the feeding responses to the novel bacteria Figure 14B,C. Each condition is coded by three symbols. The first and the second letter above the bar represent training and test food in order. The third symbol, below the bar, represents the conditioned media that was mixed with the test food. B — C Conditioned media from novel bacteria override the stimulatory effect of familiar bacteria on feeding.
The average values of the feeding rates presented in B are Exposure to a particular food plays a significant role in shaping the pattern of food intake by altering subsequent consumption of the food. Here, using the simple animal model C. We first showed that regulation of feeding by familiarity discrimination is conserved in C. We speculate that the C. The pathogenic bacteria kill worms in 4 hr Zhang et al. Our hypothesis can be tested by testing whether worms increase the feeding response to the pathogenic bacteria after experience.
By combining genetic analysis with imaging and immunohistochemistry, we found that recognition of familiar bacteria activates a pair of chemosensory neurons ADF, which transmits an endocrine serotonin signal that activates SER-7 in MC pharyngeal motor neurons, whose activation increases the feeding rate via cholinergic transmission from MC to the pharyngeal muscles Figure Given that NSM is a prominent reservoir of serotonin in the pharynx, and that NSM is implicated in regulating the enhanced slowing response on food Sawin et al.
One plausible explanation is that NSM releases little or no serotonin, which is insufficient to activate MC neurons in the pharynx in response to familiar food. This explanation does not contradict previously reported NSM function in the enhanced slowing response Sawin et al.
An endocrine serotonin signal from ADF, not a local serotonin signal from NSM, may have been employed for the feeding activation on familiar food to systemically control multiple behaviors and physiological adaptations.
To test this possibility, it would be informative to study whether familiarity of food affects behaviors Horvitz et al. Further studies will be helpful to understand how recognition of familiar food contributes to survival in C. How then are ADF and the downstream serotonin feeding signal controlled to increase feeding on particular bacteria after experience? Given that familiar food substantially increased ADF activity compared to the baseline Figure 12A,B , at least two neural pathways should act antagonistically in controlling the activity of ADF or its upstream neurons.
The simplest model would be that ADF or its upstream neurons are positively regulated by perception of food and negatively regulated by perception of olfactory and gustatory cues of novel bacteria Figure Our study predicts that novel bacteria dictate activation of ADF and the subsequent serotonin-dependent feeding response in the natural habitat where more than one bacterial type are likely to grow mixed together.
Preliminary data show that C. The following observations suggest that ADF releases serotonin and increases the feeding response when worms encounter familiar bacteria: 1 Serotonin from ADF increases the feeding response Figure 8A ; 2 ADF is activated selectively by familiar bacteria within 1 min Figure 12A,B ; 3 ADF releases more serotonin in response to familiar bacteria than novel bacteria Figure 11D. Interestingly, serotonin transmission from ADF was also shown to be critical for the learned aversion to pathogenic bacteria Zhang et al.
For the aversive learning, it is not yet clear when the serotonin signal from ADF acts. It will be interesting to understand how serotonin signaling from ADF and the physiological context are integrated to produce seemingly opposite experience-dependent behaviors. Further studies to understand regulation of the two seemingly opposite behaviors at the neural circuit level will also help us understand how the C. Many questions remain to be answered to fully understand the mechanism underlying recognition of familiar bacteria in C.
How do worms sense different bacteria? What changes in the nervous system underlie the process of becoming familiar to particular bacteria during experience? Further quests to explore these unanswered questions may deepen our understanding of sensory information processing and familiarity discrimination. Except when stated otherwise, C.
Except in Figure 10 , all worms used were hermaphrodites. In the main text only the gene name is shown. Fred Ausubel and Dr. Gary Ruvkun, respectively. Removal of bacteria was checked by the absence of traces of bacteria on the track. We starved the animals for the following reasons: First, the 7—8 hr of starvation synchronized the nutritional status of worms. Second, the 7- to 8-hr gap between the test and the last exposure to the training bacteria allows us to test if C.
Then, individual animals were transferred to test food for measuring feeding rates after 7—8 hr of starvation on unseeded NGM plates at room temperature RT. The feeding rate of each animal pumps per min was calculated by averaging the three measures from each animal pumps per 30 s and subsequently by multiplying by 2.
For each experiment, preparation of worms and reagents and feeding assays were performed in the same way in a designated place, mostly using the same batch of reagents. In contrast, conditions for feeding assays for different experiments varied in several ways, for instance, temperature and amount of test bacteria. As a result, we got consistent range of pumping rates within experiments, but not among different experiments.
The ser-7b promoter 2. The flp promoter and the flp-2 promoter were kind gifts from Dr. Chris Li Kim and Li, For DA, the extrachromosomal array was integrated into the chromosome by gamma irradiation 6 krad. The integration line was outcrossed five times against DA The ceh-2 promoter and the srh promoter were kind gifts from Dr.
Cori Bargmann. The ceh-2 promoter spans 1. To minimize the variation caused by sodium azide, we retrieved all the mock-operated and the putative ADF-ablated animals from the agarose pad after the same incubation time 12 min. Mock-operated groups were treated in the same way except that the laser was not fired. The retrieved animals were cultured on HB until adulthood.
One-day-old adults were placed in a T-shaped microfluidic chamber Figure 16 , with the tip of their nose exposed to constantly flowing LB broth. ADF neurons were visualized through a Zeiss plan-apochromat 63X, 1. The camera was controlled by Micromanager Edelstein et al. Briefly, fluorescence intensity I in the and nm wavelength images was measured in a circular region of interest ROI centered on the neuron. Background fluorescence I' was measured in a second ROI surrounding the first one. The main branch of the channel was connected to an inlet and outlet, allowing LB or bacterial solutions to flow through.
Switching from LB to the bacterial solutions was achieved via an upstream valve. A smaller branch orthogonal to the first one contained the worm.
This tapered channel allowed immobilization of the animal while exposing only the tip of the nose to the flowing LB or bacterial solutions. Samples of DA and DA were prepared as for the feeding assay. Just after the 7-hr starvation we divided each group into three equal subgroups and fixed one.
The remaining two groups were separately refed on either DA or HB and fixed after 1 hr. This assay could not be done immediately after training because most of the serotonin-uptaking cells were serotonin positive in DA as in wild type worms. The background was too high to detect any increase in serotonin release. In DA, the control strain that is defective in serotonin uptake, serotonin signal was found only in ADF.
To calculate the increase in the average number of serotonin-positive serotonin-uptaking cells during 1 hr of refeeding for each group Figure 11D , we first blindly counted the number of serotonin positive AIMs and RIH from each animal and calculated the average number for each group.
To examine serotonin effects on feeding rate in absence of bacteria, feeding rates were quantified from 3- to5-hr-old L1 larvae that had never been exposed to bacteria. The feeding assay was performed with L1 larvae because it was easier to examine feeding responses of developmentally synchronized worms that are free from bacteria in large numbers.
The strategy is particularly useful for the developmentally retarded mutants that carry eat-2 ad , which makes comparisons of the feeding rates in adults rather difficult. We confirmed that the effects of single null mutations of the serotonin receptors in response to serotonin are consistent in L1 and adults data not shown and thus, it is likely that our observations made in L1 larvae are still valid in adults. After collecting embryos by egg preparation, we incubated them on unseeded NGM plates for 2 hr.
Newly hatched L1 larvae 0- to 2-hr-old were then transferred to unseeded NGM plates and incubated for 3 hr. Each experiment continued for 1 hr. Feeding rates shown in Figures 4G and 8E were calculated by averaging two measurements per animal pumps per 55 s. Except the data that are analysed by one-way ANOVA or by Student t test, data were statistically analysed by both the unpaired t -test and the Mann—Whitney U test two-tailed.
The two tests produced the same conclusions for all data analyses. For data presentation, the more conservative p value was selected. The ser-7 effect on the feeding rate shown in Figure 4H for each food condition was calculated by subtracting the averaged feeding rate of the ser-7 mutant from the rate of wild-type worms that were tested under each food condition. GraphPad Prism version 5. The effects of inhibitory serotonin signal on feeding rates in HH and DD groups shown in Figure 7F were calculated and compared as for the ser-7 effect using the feeding rates of the ser-7 single mutant and the ser-4; mod-1; ser-7 triple mutant.
To compare the increase in the numbers of serotonin positive cells during 1 hr of refeeding on familiar food with the increase on novel food, we tested the data shown in Figure 11D using Fisher's method Fisher, for combining the results of several independent tests bearing upon the same overall hypothesis.
We first compared the difference in the increase in the number of serotonin positive cells between HH and DH groups, and between DD and HD groups in each experiment using Student's t test two-tailed. For clarity, some results are presented in more than one panel. The feeding rates of wild type and the ser-7 null mutant in presence of serotonin are shown in Figures 4G and 6.
The feeding rates of the ser-7 null mutant on familiar food and novel food are presented in Figures 4E,F and 7A—D. The feeding rates of the ser-4; mod-1; ser-7 triple mutant on familiar food and novel food are presented in Figures 4E,F and 7C,D. In Figure 4H , comparison of the differences in the feeding rates between wild-type N2 and the ser-7 null mutant animals on familiar food with the differences on novel food using Student t -test. STEP 1. Calculation of the parameters means and standard error of the means of the differences in the feeding rates between N2 and ser-7 under HH, DH, DD and HD for the comparisons using Student t -test.
Mean X Y is the mean of the feeding rate of animals of genotype X under Y condition; Var X Y is the variance of the feeding rate of animals of genotype X under Y condition; n X Y is number of animals of genotype X that were tested under Y condition. STEP 2.
Comparison of the differences in the feeding rates between wild-type N2 and the ser-7 null mutant animals on familiar food with the differences on novel food using Student t -test. These statistical analyses concluded that the difference in the feeding rates between wild-type N2 and ser-7 is greater on familiar food than the difference on novel food, suggesting that serotonin signaling via SER-7 is more active on familiar food than novel food. In Figure 7F , comparison of the differences in the feeding rates between the ser-4; mod-1; ser-7 OT and the ser-7 null mutant animals on familiar food using Student t -test was done in the same way.
Mean X is mean of number of serotonin-positive serotonin-uptaking cells in group X; Var X is variance of number of serotonin-positive serotonin-uptaking cells in group X; n X is sample number of group X. These statistical analyses conclude that the increases in the average number of serotonin positive serotonin-uptaking neurons of HH and DD groups are greater than the increases of DH and HD groups, respectively. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown.
Reviewers have the opportunity to discuss the decision before the letter is sent see review process. Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.
Your article has been evaluated by a Senior editor and 3 reviewers, one of whom is a member of our Board of Reviewing Editors. Peggy Mason served as the Reviewing editor. The Reviewing editor and two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments based on the reviewers' reports.
Your work is thorough, interesting, and novel. Attention to the following points would further strengthen the manuscript:. Depending on how the experiments were done, this could be of serious concern or simply puzzling. It should be stated explicitly whether experiments and controls for every comparison were done on the same day, using the same reagents.
The variability in baseline pumping rate appears as an issue in much of the data and is of particular concern with regard to drawing negative conclusions e.
Do ADF::gfp animals display similar rates of bleaching? The average values of the individual yellow and cyan channels should also be presented. A description of the design of the microfluidic chip or a reference should be given. How much time passed from picking to imaging? One issue that still concerns us is the day-to-day variation issue. Of course we understand that variation happens. Future studies investigating the precise amounts of particular amino acid species in the dietary E. A uracil auxotroph derived from E.
HT DE3. Derived from E. Derived from OP50 [41]. For bacterial cultures used to seed worm plates and for nutrient composition analysis, E.
NGM plates 6 cm were seeded with 0. Early embryos were isolated from gravid adults by alkaline hypochlorite [9] and plated on nematode growth media NGM.
For each biological replicate, approximately 20, young adult nematodes were harvested, washed, and aliquots were removed for protein determination. The extract was washed with 0. Neutral lipids were separated by thin layer chromatography on Silica gel plates as described in [42].
Triacylglycerol and phospholipid fractions were scraped for fatty acid methyl ester derivatization and analyzed by gas chromatography [43]. Live Nile Red staining of C. Fixed Nile Red staining of C. Approximately — nematodes are suspended in 1 ml of water. Worms are allowed to settle, washed once with M9 buffer, and allowed to settle again. Nile Red images were acquired using identical settings and exposure times to allow direct comparisons.
L4 worms were transferred to fresh plates at the beginning of the experiment, day 0 [15]. No FUdR or antibiotics were included in the plates. Worms were transferred to fresh plates daily until they stopped laying eggs, after which they were transferred every 4—5 days. Worms were scored daily for viability, and worms that crawled off the plate or burst at the vulva were excluded from the analysis.
The mean and maximum lifespans were determined by the average of three to five independent trials, each using 40— animals. For all assays, at least three independent bacterial growth experiments were performed, and each assay was repeated in triplicate for each growth.
Carbohydrate: Total sugars were determined with the anthrone method. Reducing and non-reducing sugars react with anthrone reagent under acidic conditions to yield a blue-green color [44]. Bacterial lawns were washed off of NGM plates with water.
Aliquots of bacteria and glucose standards were added to 3 ml of anthrone solution 0. Absorbance of cooled samples was measured at nm. Fatty acids were extracted with hexane and analyzed by gas chromatography as described in [43].
Total progeny were determined as described in [47]. We thank Greg Hermann for suggesting Nile Red staining of fixed nematodes. We thank Young-jai You and Leon Avery for helpful conversations and communication of unpublished data.
We are grateful to Heidi Tissenbaum for the gift of tub-1 nr and tub-1 nr strains. Other C. Conceived and designed the experiments: JLW. Wrote the paper: JLW. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Background The nematode Caenorhabditis elegans has emerged as an important model for studies of the regulation of fat storage.
Conclusions These studies demonstrate that nutritional cues perceived in the intestine regulate fat storage levels independently of neuroendocrine cues. Introduction Many components regulating human metabolism are conserved in the nematode C.
Results Fat storage levels in C. Download: PPT. Figure 1. Dietary E. Figure 2. Comparison of live Nile Red staining, fixed Nile Red staining, and triacylglycerol stores in wild type and mutants. Lifespan is not markedly affected by bacterial diets Many long-lived mutants of C. Table 1. Lifespan analysis of wild-type C. Carbohydrate levels in four dietary bacterial strains inversely correlate with fat stores in C.
Figure 3. Characterization of cell number, dry weight, and macronutrient composition of E. Fatty Acid composition differences in C. Figure 4. Fatty acid composition differences in E. Figure 5. Relationship between branched chain fatty acid C17iso levels in triacylglycerol stores and total fat stores. Peptide transport in the intestine is necessary for fat storage differences The only strain examined in this study that showed no difference in levels of fat storage when growing on OP50 and HB was a mutant pept-1 , previously called opt-2 and pep-2 , which carries a deletion in a gene encoding an intestinal peptide transporter [30].
Table 2. Fatty acid composition of total lipids of wild type and pept Reduced fertility in pept-1 growing on HB Finally, we asked whether the differences in fat stores in worms grown on OP50 or HB affected reproductive success by counting the number of live progeny produced from individuals of various genotypes raised on either HB or OP50 E.
Discussion Obesity is a disorder in energy homeostasis that develops when energy intake exceeds energy expenditure. Materials and Methods C. Lipid analysis of C. Nile Red staining of C. Lifespan analysis of C. Macronutrient analysis of E. Fertility analysis of C. References 1. Jones KT, Ashrafi K Caenorhabditis elegans as an emerging model for studying the basic biology of obesity. Dis Model Mech 2: — View Article Google Scholar 2. Biochim Biophys Acta — View Article Google Scholar 3.
Watts JL Fat synthesis and adiposity regulation in Caenorhabditis elegans. Trends Endocrinol Metab. View Article Google Scholar 4. Mol Cell Endocrinol 51— View Article Google Scholar 5. Trends Endocrinol Metab — View Article Google Scholar 6. Science — View Article Google Scholar 7. WormBook 1— View Article Google Scholar 8.
Cell Metab 8: — The left end of the first three fragments is bp downstream of the ATG of exon 1a. The right ends are at base pairs , , and , respectively. The last PCR fragment begins 1. The PCR products were injected along with rol-6 as an injection marker. At least two transgenic lines were established for each fragment. Rescuing fragments restored pumping rates to wild-type levels in all transgenic animals analyzed. The smallest rescuing fragment begins at position and ends at Eat encodes a predicted amino-acid protein with an intracellular amino terminus, a transmembrane domain, and a short extracellular carboxy-terminal end.
The transmembrane domain is indicated by a bar above the sequence. Asterisks above the sequence indicate nucleotides affected in eat mutants. We sequenced cDNAs isolated from transcripts that use the alternative first exon 1b. When a transcript is made using this exon, a stop codon is generated in the second exon, resulting in an open reading frame that would produce a amino-acid protein. Translation initiation from exon 1b is in a different frame from exon 1a, resulting in the stop codon in the second exon.
To determine if this small open reading frame was eat , we fused a cDNA composed of exons 1b, 2, and 3 to 1 kb of genomic sequence upstream of exon 1b Figure 4.
We injected this fusion product into eat ad and found that it rescued the slow-pumping defect. All transgenic animals from two independent lines analyzed were rescued. This shows that eat is encoded by this open reading frame and that the genomic sequence just upstream of exon 1b is the eat promoter. Sequence analysis of the predicted protein from this transcript suggests that it contains a amino-acid intracellular domain at the amino-terminal end, a transmembrane domain, and a amino-acid, carboxy-terminal, extra cellular domain and has no similarities to previously described proteins Figure 5 ; Sonnhammer et al.
A Northern blot was performed to determine the size of the eat transcript. A sequence corresponding to exon 1b was used to probe RNA isolated from wild-type worms. We have three mutant alleles of eat Two, ad and nu , are recessive, and one, adsd , is semidominant Raizen et al. We sequenced the coding region of YE8A. Mutations in all three affect exon 1b Figure 4. On the basis of the nature of the mutations, we conclude that ad and nu are both null alleles.
EAT is expressed in pharyngeal muscle: To determine where eat is expressed, we fused GFP in frame to exon 1b and injected the fusion product into wild-type worms. We examined the transgenic worms and observed GFP expression in pharyngeal muscle and pharyngeal neuron M5 Figure 7.
There is also very faint GFP expression in five to six unidentified neurons in the extrapharyngeal nervous system not shown. The expression pattern of EATGFP supports the conclusion that the pharyngeal muscle is the main site of eat function. EAT-2 is correctly localized in eat mutants: One possible role for eat is that it is required for folding or trafficking of the nicotinic receptor.
This result indicates that eat is not required for folding or trafficking of the EAT-2 channel. A A bright-field image of the pharynx. B Fluorescent image of the pharynx. Fluorescence posterior to the pharynx is auto-fluorescence from the intestine.
Pharynxes from eat mutants, however, are resistant to this concentration of nicotine. A possible explanation for this result is that eat18 is required for the function of other nicotinic receptors in the pharynx in addition to eat The location of the receptors can be seen by using fluorescence microscopy.
In wild-type worms, there is extensive labeling of the pharynx Figure 8. Top A differential interference contrast image of a worm pharynx.
Arrow points to the location of the MC synapse. Bottom A fluorescence image showing the localization of the fusion protein at the MC synapse arrow. MC mechanism: Previous work in our lab led to the proposal that the MC neurotransmitter is acetylcholine Avery and Horvitz ; Raizen et al.
This is supported by the finding that eat-2 encodes a nicotinic acetylcholine receptor subunit. The observation that EAT-2 functions in the pharyngeal muscle demonstrates that MC stimulates the muscle directly, using fast synaptic transmission to control pharyngeal pumping rate. This leads to the opening of a voltage-activated calcium channel and subsequent muscle contraction Lee et al. The rate of MC firing controls the rate of pharyngeal pumping. The role of eat in nicotinic neurotransmission: Mutations in eat cause the same defect in pumping as mutations in eat-2 : worms are incapable of rapid pharyngeal pumping and EPSPs from the excitatory motor neuron MC are not present Raizen et al.
Several lines of evidence suggest that eat could be a component of the pharyngeal nicotinic receptor. First, an eat ::GFP fusion is expressed in pharyngeal muscle, suggesting that this may be the site of eat activity.
Also, we showed previously that pharynxes dissected from eat mutants were resistant to bath-applied nicotine, indicating that nicotinic receptors in the pharyngeal muscle are defective Raizen et al. We have observed allele-specific genetic interactions between eat-2 and eat , indicating that they could be members of the same protein complex.
Worms that are trans -heterozygous for eat adsd and two alleles of eat-2, ad and ad , have a wild-type pumping rate. In this case, the eat-2 mutants are able to suppress the intermediate slow-pumping phenotype of eat ad Another eat-2 allele, eat-2 ad , when trans -heterozygous with eat adsd , enhances the intermediate slow-pumping phenotype.
Taken together, these data indicate that EAT could be a component of pharyngeal nicotinic receptor. A possible reason for the lack of acetylcholine binding to eat mutant pharynxes is that EAT is required for the formation of the acetylcholine-binding site. This would also be consistent with the inability of MC to cause excitatory postsynaptic potentials in eat mutants. Arrows point to staining in the pharynx and arrowheads point to staining outside the pharynx. There are alternative possibilities for the role of eat in the function of the nicotinic receptor.
One alternative is that eat could be required for inserting the nicotinic receptor into the postsynaptic membrane. In this model, the nicotinic receptor would be targeted correctly to the location of the synapse, but it would remain in a subsynaptic pool of receptors. The role of eat could be to move the receptor from the subsynaptic pool and insert it into the postsynaptic membrane.
The resolution of the light microscope could not distinguish between receptors in the subsynaptic pool and those that had been inserted into the membrane. This model would also explain the inability of MC to activate the receptor: acetylchololine released by MC would not have access to receptors in the subsynaptic pool. Ultrastructural analysis of the MC synapse would be required to determine the exact location of EAT-2 in wild-type and eat mutant worms.
It is interesting that eDf7 complements eat ad but does not complement eat adsd although the deficiency breakpoint does not overlap the adsd lesion. We think several things are responsible for this. Although eat is encoded by a small ORF, it is part of a large transcript. We think that this reduces the amount of EAT protein that is made because the shortened transcript from the deficiency chromosome is likely to be less stable than the full-length wild-type transcript.
The decreased eat expression from the deficiency chromosome is still enough to supply wild-type levels of eat activity in the presence of the recessive allele ad The semidominant allele, adsd , behaves like a dominant negative mutation Raizen et al. If eat expression from the eDf7 chromosome is reduced, there might not be enough activity to overcome the dominant negative effect of adsd , resulting in the slow-pumping phenotype. Other genes required for ion channel function in C. Although EAT is also a small transmembrane protein, in contrast to RIC-3, it appears to be required for formation of the acetylcholine-binding site.
Additionally, although ric-3 is involved in nicotinic neurotransmission in several cell types, eat appears to be specifically required for pharyngeal nicotinic acetylcholine receptors. Chelur et al. Biochemical and electrophysiological experiments will be needed to further define the role that eat plays in regulating pharyngeal nicotinic receptors. We thank Renee McKay and members of the Avery lab for comments on the manuscript.
We thank Tim Niacaris and Wayne Davis for technical help. Josh Kaplan provided eat nu Yuji Kohara provided the eat-2 cDNA. B : — Google Scholar. Avery L , The genetics of feeding in Caenorhabditis elegans. Genetics : —
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