BACKGROUND
the nematode caenorhabditis elegans is useful for studying the effects of a gene on behavior through facilitated behavioral analyses and well-developed genetic analyses.
in this organism, various cell-specific promoters can be utilized for cell-specific expression to determine the function of a gene in a specific cell.
recently, temporal control of cell-specific expression using heat shock factor- <dig>  mutants has been reported  <cit> .
in addition, the effects of gene knockdown can be assessed using various loss-of-function mutants, and researchers recently developed a new method of generating loss-of-function mutations in targeted genes in c. elegans  <cit> .
rnai is one of the most powerful tools for gene knockdown.
rnai-mediated cell-specific knockdown in c. elegans is a currently available technique  <cit>  based on driving the expression of double-stranded rna in target cells via cell-specific promoters.
this method identifies the cells in which the target gene functions.
moreover, gene functions can be analyzed by this system because of cell-specificity, even if mutants of the gene show fatal phenotypes.
in contrast to cell-specific promoters, heat shock promoters drive the expression of genes at arbitrary timing via heat shock  <cit> .
a previous report has shown that rna hairpins, which are driven by the heat shock promoter, induce knockdown of the target gene  <cit> , suggesting the possibility of time-specific knockdown.
however, heat shock promoters drive global expression, meaning that the knockdown is performed in the majority of cells, thereby removing vital cellular functions and thus inducing lethality when essential genes are knocked down by this method.
therefore, for detailed analysis of multifunctional genes, such as the components of the ras-mapk pathway described below, simultaneous time-specific and cell-specific knockdown is necessary.
to our knowledge, however, such a method has not yet been developed in c. elegans.
intracellular signal transduction pathways play an essential role in regulating diverse cellular functions.
most of these pathways have widespread functions in various tissues and at different stages.
for example, the ras-mapk signaling pathway controls cell proliferation and differentiation at the embryonic stage  <cit>  and regulates synaptic formation required for neural plasticity at the adult stage  <cit> .
to distinguish and clearly understand the multiple functions of these signal transduction pathways, conditional knockdown systems, such as time- and cell-specific knockdown, are required.
the ras-mapk pathway in c. elegans plays a role in various processes, such as vulval induction, olfaction and germ-line apoptosis  <cit> .
however, whether or not the signaling pathway is involved in the control of locomotion behavior is unknown.
animals use various strategies according to the circumstance in which they are placed to gain food efficiently.
under enriched environmental conditions in which animals are near food resources, efficient exploration is achieved by their use of perception, which incorporates olfaction, vision and touch.
however, animals roam randomly to gain information when food resources are far from them.
c. elegans has a simple nervous system.
this worm is composed of only  <dig> neurons  <cit>  and uses diverse strategies to obtain food.
a recent study has shown that switching between dwelling and roaming activities according to the surroundings is controlled by a neural circuit and two neuromodulators, serotonin and neuropeptide pigment dispersing factor , which exhibit opposing effects  <cit> .
in the neural circuit, serotonergic signaling initiates and extends the dwelling states, while pdf signaling affects roaming states by acting on each target neuron.
while roaming, c. elegans uses two strategies to approach food resources efficiently in enriched environmental conditions.
the first approach is klinotaxis , in which the animals gradually curve according to the gradient of chemoattractants  <cit> , and the second is klinokinesis , in which they turn and change the direction of locomotion when a decrease in the concentration of chemoattractants is sensed  <cit> .
the strategies for switching between dwelling and roaming activities, and those of approaching food under enriched environmental conditions, are well studied and understood.
however, there is a scarcity of information regarding the strategy used by c. elegans when roaming under poor environmental conditions.
constitutive and exploratory head movements  might be one possible strategy.
il <dig> and olq mechanosensory neurons are known to be involved in transducing signals during both aversive head-withdrawal and the control of foraging  <cit> .
further, of the motor neurons connecting to il <dig> and olq neurons, rmd motor neurons possess the main synaptic projection  <cit> , and thus play important roles in regulating head movements  <cit> .
the neural circuit composed of il <dig>  olq and rmd neurons is essential for the regulation of spontaneous foraging and head-withdrawal movements.
however, the role of foraging behavior on roaming and locomotion under poor environmental conditions, how head movements are regulated in foraging, and which signal transduction pathways are involved in the regulation of foraging are all unknown.
in this study, we analyzed the effect of the ras-mapk signaling pathway on the regulation of foraging behavior via an rnai method with simultaneous time-specific and cell-specific knockdown of ras.
we found that mutants of the ras-mapk signaling pathway exhibit abnormal locomotion behavior  under poor environmental conditions in which they are far from food resources.
cell-specific knockdown of the ras gene revealed that the ras protein was functional in il <dig>  olq and rmd neurons, and thereby controlled foraging behavior.
further, results indicate that ras regulated the localization of glr- <dig> glutamate receptors in rmd neurons.
moreover, we demonstrated that the function of ras at the adult stage was important for the regulation of glr- <dig> localization in rmd neurons by using time- and cell-specific rnai .
this method enabled cell-specific knockdown of gene functions at arbitrary timing.
RESULTS
mutations in the ras-mapk pathway lead to circular locomotion instead of normal exploratory foraging
under poor environmental conditions in which c. elegans is far from food resources, it crawled around the field to search for food resources.
a previous study quantitatively characterized this behavior and reported that a locomotory behavior under these conditions has a long-range directionality and cannot be explained by a simple random isotropic model of locomotion  <cit> .
wild-type animals moved in an apparently unbiased manner when placed on a plate without any chemicals or food .
however, ras mutants showed abnormal locomotion behavior on the blank plates.
as the loss-of-function  mutants of ras, let- <dig>  <cit>  continued to move in a loopy pattern , we named this phenotype ;circular locomotion’ .
we defined an animal that exhibited cl as one showing loopy tracks in more than 30% of the whole track left during the one hour test period .
a large proportion of let- <dig> mutants were categorized with cl but exhibited milder phenotypes  compared to neuron-specific let- <dig> rnai strains described below, probably due to n <dig> being a weak allele.
ras and the downstream mapk pathway are essential for numerous physiological cellular functions, and are highly conserved in diverse organisms  <cit> .
although the ras-mapk pathway in c. elegans regulates a number of processes  <cit> , its involvement of the pathway controlling locomotion behavior is unknown.figure  <dig>
ras mutants exhibit abnormal locomotion behavior under poor circumstances.
tracks representative of wild type and the let- <dig> mutant in blank plates.
the wild-type crawls around the field randomly, whereas the let- <dig> mutant continues to move in a loopy pattern, termed circular locomotion .
average curving rate of wild type and let- <dig> mutants for a fixed time   or a fixed distance   of exploratory behavior .
positive or negative values indicate curves toward ventral or dorsal sides, respectively.
asterisks indicate significant differences compared with wild-type animals .
histograms of curving rate in wild type and let- <dig> mutants in the fixed distance .
quantitative analysis of directional locomotion.
the median of log <dig> at the distance of size δ in wild-type and let- <dig> mutants is shown in log-log scale  when analyzed in the fixed distance .
error bars represent the first and the third quartile.
proportion of animals exhibiting circular locomotion  in mutants of the ras-mapk pathway
genotype
cl
number
let-23
sem-5
let-60
let-60
lin-45
mek-2
mpk-1
to analyze cl quantitatively, we measured the curving rate, which is defined as the change in direction of locomotion per unit length of an animal’s advancement  <cit> , of the locomotion under poor conditions.
wild-type animals exhibited mostly random but slightly biased curves toward the ventral side .
in contrast, let- <dig> mutants exhibited significantly larger and directional curves toward the dorsal side  .
given that each animal moved at a different velocity , we calculated the average curving rate in the first 30 mm of each track.
in this analysis, let- <dig> mutants also exhibited significantly larger curves  than wild type .
these results suggest that cl may reflect large, directional curves.
in addition, to quantitatively measure the extent to which animals made directional tracks, we performed the analysis based on fractal analysis as described previously  <cit> .
briefly, the trajectory of each animal was divided into segments with length of size δ , and the sum of linear distances of adjacent dividing points was computed as lδ .
we then calculated lδ/l <dig> , in which l <dig>  indicates the length of the track.
again, the analysis was performed on the first 30 mm of each track, thus l0 = 30 mm.
when animals tend to move in narrower areas, such as with cl, lδ/l <dig> is expected to decrease.
lδ/l <dig> values for animals with let- <dig> mutations tended to be lower than those of wild type animals , indicating that the former tended to move in narrower areas than the latter.
taken together, cl is characterized as large, directional curves and in migration relatively narrow areas.
in c. elegans, components of the ras-mapk pathway: ras, mapkkk , mapkk , and mapk  are encoded by the let- <dig>  lin- <dig>  mek- <dig> and mpk- <dig> genes, respectively  <cit> .
each lf or null  mutant of the pathway, let- <dig>  lin- <dig>  mek- <dig> and mpk- <dig>  frequently exhibited cl .
further, mutants of the upstream components of the signaling pathway, let- <dig> and sem- <dig> also exhibit cl .
the genes let- <dig> and sem- <dig> encode an egf-receptor-family transmembrane tyrosine kinase and an adaptor protein orthologous to human grb <dig>  respectively  <cit> .
however, let- <dig> mutants  did not exhibit cl, suggesting that inactivation, and not hyperactivation, of the pathway caused cl.
these results indicate that the ras-mapk pathway modulated locomotion behavior under poor environmental conditions in c. elegans.
we previously reported that the ras-mapk pathway controls both klinotaxis, a migratory behavior towards odorants, and olfactory plasticity  <cit> .
awc olfactory neuron- and aiy interneuron-specific expression of the let- <dig> gene rescues the defects of klinotaxis and plasticity, respectively, of let- <dig> mutants  <cit> .
however, our results revealed that the expression of let- <dig> in awc and aiy did not reduce the number of the let- <dig> mutants exhibiting cl .
let- <dig> mutants have no abnormality in chemotaxis for sodium chloride  <cit> .
we observed that mutants rarely exhibited cl when they moved towards the chemoattractants.
a previous study reported that mutants exhibited moderate to severe defects in chemotaxis in response to 1 μl of 10− <dig>  10− <dig> and 10− <dig> dilutions of isoamyl alcohol on 9 cm plates   <cit> .
when mutants were placed 3 cm from 1 μl of a 10− <dig> or 10− <dig> dilution of isoamyl alcohol, the mutants exhibited cl frequently, as they were on plates without stimuli.
in contrast, the proportion of those exhibiting cl significantly decreased  when mutants were exposed to 10− <dig> isoamyl alcohol .
these results suggest that cl occurs independently of the defects of klinotaxis and olfactory plasticity.
the ras-mapk pathway functions in neural circuits underlying foraging behavior to control direction of locomotion
to elucidate the mechanism underlying impairment of the ras-mapk pathway-mediated cl, we analyzed the locomotion behavior of let- <dig> mutants in more detail.
let- <dig> mutants exhibited abnormal foraging behavior in which their head movements were slightly asymmetrical and over-bent.
this observation led us to assess whether abnormal foraging behavior induced cl.
previous reports have shown that the rate of spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: il <dig>  olq, and rmd  <cit> .
il <dig> and olq are mechanosensory neurons, and rmd are motor neurons that receive main connections from il <dig> and olq neurons, and innervate the head muscles  <cit> .
therefore, we examined whether blocking the activity of these neurons causes cl.
unc- <dig> was used to inhibit the activity of these neurons.
this gene encodes a constitutively active form of the potassium channel unc- <dig> and can reduce the neuronal activity by hyperpolarization  <cit> .
we found that il <dig>  olq or rmd neuron-specific expression of unc- <dig> in wild-type animals significantly  caused cl , indicating that inhibition of these neurons induced locomotion behavior in a loopy pattern in c. elegans, even if one class of neuron was impaired.
a previous report indicated that the osm- <dig> promoter we utilized for the expression in olq also drives the expression in awa and adl sensory neurons  <cit> .
however, awa- or adl-ablated animals did not exhibit cl , indicating the importance of olq in the regulation of locomotion.
these results suggest that the neural circuit  involved in foraging behavior played an essential role in determining the direction of locomotion.figure  <dig>
let- <dig> ras in il <dig>  olq and rmd neurons controls the direction of locomotion.
the proportion of animals exhibiting cl in wild type, let- <dig> mutants and transgenic animals in which il <dig>  olq or rmd neurons were inactivated by unc- <dig> .
rescue experiments in which the wild-type let- <dig> gene was expressed in each or all the neurons regulating foraging in let- <dig> mutants .
il1-, olq- or rmd-specific rnai of let- <dig> causes frequent cl .
definition of ‘head angle’ as the angle between two lines where one passes the worm centroid and the terminal bulb of pharynx and the other the terminal bulb of pharynx and nose tip.
the ventral or the dorsal bends of the head are defined as positive or negative values, respectively.
head angles of ventral and dorsal bends in wt and rmd::let-60rnai animals.
n ≥ <dig> bends.
error bars represent sem.
asterisks indicate significant differences compared with wild-type animals  or let- <dig> mutants  .
n.s., not significant.
cl, circular locomotion; sem, standard error of the mean; wt, wild type.
these results led us to investigate whether the ras-mapk pathway plays a role in the neural circuit involved in foraging behavior to control locomotion.
we, therefore, conducted rescue experiments where the wild-type let- <dig> gene ) was expressed in neurons regulating foraging behavior in let- <dig> mutants.
we found that expression of let- <dig> in all three neuronal classes rescued the cl phenotype in let- <dig> mutants, which was not found when the expression of let- <dig> occurred in a single class of neurons .
these results suggested that let- <dig> ras functions in all classes of neurons, which regulate foraging behavior.
further, this suggestion was consistent with the finding that impairment of let- <dig> in a single class of neurons caused cl , as described below.
we next performed cell-specific knockdown experiments using rnai  <cit> .
cell-specific knockdown of the let- <dig> gene in each class of neurons  caused cl .
in particular, knockdown of let- <dig> in il <dig> or rmd neurons strongly  induced cl, suggesting that let- <dig> ras largely contributed to the function of il <dig> and rmd neurons.
taken together, these results indicate that functions of let- <dig> ras in all il <dig>  olq and rmd neurons were required for foraging behavior to control the direction of locomotion.
compared to animals with inhibition of neurons, neuron-specific let- <dig> rnai strains exhibited milder phenotypes , suggesting that pathways other than ras signaling might also be involved in the regulation of locomotion, although the possibility that rnai is at least somewhat effective cannot be excluded.
we directly observed and quantified foraging behavior in wild type and animals with rmd-specific rnai knockdown of let- <dig>  we monitored the head angle of worms during locomotion on plates without any chemicals or food.
this angle was defined as that between two lines in which one passes the worm centroid and the terminal bulb of pharynx and the other the terminal bulb of pharynx and nose tip .
ventral bends were counted as positive and dorsal bends as negative values, respectively .
the mean head angle of dorsal bends in animals with rmd-specific let- <dig> rnai was significantly  larger than in wild type animals, while that of ventral bends was not .
this result seems to be consistent with the finding that let- <dig> mutants tended to exhibit dorsal-biased large turns in exploratory behavior  and indicates that ras in rmd neurons is important for foraging behavior, suggesting a correlation between foraging and exploratory behavior.
the ras-mapk pathway regulates the localization of glutamate receptors in rmd neurons
we next explored the role of the ras-mapk pathway in il <dig>  olq and rmd neurons.
the ras-mapk pathway is essential for neurogenesis in fruit flies and mice  <cit> .
to clarify if this pathway was required for neurogenesis of these neurons, we observed the morphology of the neurons in let- <dig> mutants.
these mutants did not exhibit any apparent morphological defects in il <dig>  olq or rmd neurons , suggesting that the ras-mapk pathway was not involved in the morphogenesis of these neurons.
the ras-mapk pathway also has important roles in synapse formation and the control of synaptic protein localization  <cit> .
to examine whether this pathway regulates formation of pre-synapses in il <dig> and olq neurons, we observed the localization of synaptobrevin, snb- <dig>  <cit> , in the axons of these neurons.
snb- <dig> is a synaptic vesicle protein essential for vesicle docking or fusion, which is required for synaptic transmission  <cit> .
we observed that snb- <dig> localization was not affected by il1- or olq-specific knockdown of let- <dig> .
therefore, the ras-mapk pathway might not be involved in presynaptic formation of these neurons.
previous studies have shown that ras signaling drives the synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid  receptors in post-synapses  <cit> .
in c. elegans, the glr- <dig> gene  is expressed in neurons, including rmd  <cit> .
our results revealed that glr- <dig> mutants also displayed cl .
the expression of glr- <dig> by its own promoter restored the defect in locomotion behavior of glr- <dig> mutants .
moreover, rmd-specific expression of glr-1::gfp significantly  rescued the cl phenotype of glr- <dig> mutants .
these results suggest that glr- <dig> in rmd neurons played an important role in controlling the direction of locomotion and also indicated that glr-1::gfp was functional.
we next investigated whether the ras-mapk pathway was associated with glr- <dig> for the regulation of locomotion behavior.
to assess the effect of this pathway on glr- <dig>  we investigated the localization of glr-1::gfp in neurites of rmd neurons in wild-type and animals with rmd-specific knockdown of let- <dig> via fluorescence.
glr-1::gfp was expressed by mgl- <dig> promoter, which drives the expression in nsm, aia and rmd neurons  <cit> .
distinguishing the neurites of rmd and aia neurons in the nerve ring is difficult.
thus, we targeted the neurites of rmdd neurons because these neurons are distinct from those of aia .
in wild-type animals, punctate localization of clustered glr- <dig> was observed in neurites of rmd neurons .
however, in rmd neurons of rmd-specific let- <dig> knockdown animals, glr-1::gfp clusters were very faint  or not detected  .
we confirmed that the expression of gfp by the mgl- <dig> promoter was not affected by the knockdown of the let- <dig> gene , suggesting that let- <dig> ras did not act on the mgl- <dig> promoter.
these results indicated that ras was required for normal localization of glr- <dig> in post-synapses of rmd neurons.figure  <dig>
let- <dig> ras mediates the localization of glr- <dig> glutamate receptors in rmd neurons.
the proportion of wild type, glr- <dig> mutants and glr- <dig> mutants with expression of glr- <dig> gene by its own promoter exhibiting cl .
the proportion of wild type, glr- <dig> mutants and glr- <dig> mutants with expression of glr-1::gfp by rig-5a promoter exhibiting cl .
error bars represent sem.
asterisks indicate significant differences .
mgl- <dig> promoter drives expression in nsm, rmdv, rmdd and aia neurons.
neurites of rmdd neurons  were investigated by analyzing the clusters of glr-1::gfp in rmd neurons specifically.
glr-1::gfp clusters expressed by the mgl- <dig> promoter in wild type  and wild type with rmd-specific rnai of let- <dig> .
enlarged images show neurites of rmdd neurons .
average intensity of glr-1::gfp clusters in neurites of rmdd neurons is significantly reduced by rmd-specific knockdown of let- <dig> .
error bars represent sem.
an asterisk indicates significant differences .
representative images of mgl-1p::gfp in wild type  and wild type with rmd-specific rnai of let- <dig> .
scale bars = 10 μm.
cl, circular locomotion; sem, standard error of the mean.
time- and cell-specific rnai is useful for analyses of the temporal profile of gene functions in the specific cell
we then analyzed the manner in which the ras-mapk pathway modulated the localization of glr- <dig>  the ras-mapk pathway is involved in morphogenesis, synaptic formation, and neural plasticity  <cit> .
therefore, our results led us to test two hypotheses:  the ras-mapk pathway plays a role in the development of synapses or synaptic connections of rmd neurons at the embryonic stage, and  the signaling pathway is involved in glr- <dig> localization machinery at the adult stage.
to examine these hypotheses, we analyzed the stage at which let-60ras mediated the control of glr- <dig> localization in rmd neurons.
to dissect the function of a gene which has different roles according to the stages, such as ras, a method to regulate the function of the gene spatially and temporally is needed.
rnai is one of the powerful tools for gene knockdown, and a recent study has introduced the cell-specific knockdown technique using rnai  <cit> .
the method of cell-specific knockdown is based on the expression of double-stranded rna by the cell-specific promoter   <cit> .
however, this method is incapable of performing time-specific knockdown and dissecting the stage at which the target protein functions in a specific cell.
to perform simultaneous time-specific and cell-specific knockdown, we modified the previous method  <cit> .
the heat shock promoter hsp16- <dig>  <cit> , which drives the expression in almost all tissues, is able to activate gene expression at arbitrary timing by heat shock.
by introducing the heat shock promoter to the cell-specific rnai technique, we established a new method for time-specific and cell-specific knockdown of genes, in which expression of one rna strand  was driven by a cell-specific promoter, and the expression of the other rna strand was induced by a heat shock promoter .
under normal conditions , single stranded rna was expressed in the target cells by the cell-specific promoter, whereas under the heat shock-inducing condition , double-stranded rna was expressed in only target cells by the heat shock promoter and the cell-specific promoter.
overall, this new technique was expected to elicit simultaneous time-specific and cell-specific knockdown by heat shock.
therefore, this method was termed time-specific and cell-specific rnai .
to confirm the effect of the t.c.rnai method, we examined whether expression of gfp can be decreased in the time and cell-specific manner by gfp t.c.rnai.
to clearly observe the effect of rnai, we photobleached pre-existing gfp, which otherwise has a relatively long half life  <cit> , before t.c.rnai and observed the recovery of gfp fluorescence after t.c.rnai.
this way we can assess the decrease of mrna abundance caused by rnai.
to quantitatively compare the gfp intensity before and after t.c.rnai, we measured the gfp intensity in the same individuals before photobleaching , just after photobleaching and after a heat shock for 30 minutes at 33°c and a recovery for one hour at 20°c.
then we calculated the rate of change in the fluorescence intensity of each animal.
based on this rate, the recovery ratio of gfp intensity compared to controls  was measured .
we first analyzed the effect of gfp t.c.rnai in awc sensory neurons.
gfp was expressed by the gcy- <dig> promoter which drives the expression in awc, awb and i <dig>  <cit>  and monitored the gfp intensity in cell bodies of awc.
in adult animals expressing both gcy-10::gfp and hsp::gfp, the recovery ratio of gfp intensity in awc significantly decreased after the heat shock compared to that after the mock treatment .
the expression of double-stranded rna by the reciprocally exchanged promoter also induced gfp knockdown after the heat shock .
the gfp intensity in awc was normally recovered after the heat shock in animals without expression of the rnai constructs, suggesting the decrease of the recovery ratio was not due to an influence of heat shock on the gcy- <dig> promoter itself .
we also confirmed that expression of only a single rna strand driven by a heat shock promoter or a cell-specific promoter could not decrease the recovery ratio .figure  <dig>
confirmation of the effect of the novel method, time- and cell-specific rnai .
the average recovery ratio of gfp intensity of awc neurons in animals without rnai constructs, animals with only gcy-10p::gfp, gcy-10p::gfp, hsp16-2::gfp, hsp16-2::gfp, animals with both gcy-10p::gfp and hsp16-2::gfp or both hsp16-2::gfp and gcy-10p::gfp after heat-shocked or control condition.
.
the average recovery ratio of gfp intensity of awb and awc neurons in animals with srd-17p::gfp and hsp16-2::gfp after heat-shocked or control condition .
representative images of gcy-10p::gfp in animals with srd-17p::gfp and hsp16-2::gfp before  and after  photobleaching and after heat-shocked condition .
arrow heads indicate awb neurons, and arrows indicate awc neurons.
scale bars = 10 μm.
the average recovery ratio of gfp intensity of anterior or lateral protrusions of vulva in animals without rnai constructs or animals with myo-3p::gfp and hsp16-2::gfp after heat-shocked or control condition .
chemotaxis to isoamyl alcohol  in wild-type animals, odr- <dig> mutants and wild-type animals with odr-3 t.c.rnai .
awc:: and hsp:: mean gcy-10p::odr- <dig> and hsp16-2::odr- <dig>  error bars represent sem and asterisks indicate significant differences .
sem, standard error of the mean.
moreover, to verify the cell-specificity of the gene knockdown by t.c.rnai, we monitored gfp intensity in both awc and awb neurons at the same time.
we used gcy- <dig> promoter for the expression of gfp in both awc and awb neurons, and srd- <dig> promoter for the expression of a single-stranded rna specifically in awc neurons.
in animals that expressed srd-17p::gfpand hsp::gfp, recovery of gfp intensity in awc was reduced but not in awb after the heat shock , indicating the cell-specific effect of t.c.rnai.
taken together, t.c.rnai could induce time- and cell-specific knockdown of gfp in awc neurons effectively.
next, to investigate whether t.c.rnai could cause gene knockdown in tissues other than neurons, we performed gfp knockdown in vulval muscles.
we found a decrease of the recovery ratio of gfp intensity in vulval muscles after heat shock treatment, but not after control treatment in animals with myo-3::gfp, myo-3::gfp and hsp::gfp .
we confirmed that gfp intensity in animals without expression of rnai constructs was recovered normally after the heat shock .
these results suggested that t.c.rnai could cause gene knockdown in muscles as well as neurons in a cell-specific manner.
in addition, to assess whether t.c.rnai can be applied to endogenous genes, we performed knockdown of odr- <dig> by t.c.rnai.
the odr- <dig> gene encodes a g protein α which mainly functions in awc chemosensory neurons and is essential for olfactory responses to odorants, including isoamyl alcohol, which is sensed by awc  <cit> .
we found that animals in which odr- <dig> was knocked down in awc neurons by t.c.rnai at the adult stage showed a defect in the response to isoamyl alcohol .
however, the adult transgenic animals that had undergone heat shock treatment at the l <dig> larval stage exhibited a normal response .
these results indicated that odr- <dig> worked in awc neurons at the adult stage, which is consistent with the well-known function of odr- <dig> in the olfactory signaling pathway.
these results suggested that t.c.rnai could induce time- and cell-specific knockdown of endogenous genes as well as transgenes, and the possibility that our new method could be applied widely.
t.c.rnai revealed the stage at which ras functions in rmd neurons to control locomotion behavior
using our new method, t.c.rnai, we investigated the effects of let- <dig> knockdown on the localization of glr- <dig> in rmd neurons at the embryonic and adult stage.
our results indicated that glr- <dig> localization was not affected by let- <dig> knockdown at the embryonic stage .
however, knockdown of let- <dig> at the adult stage caused significant abnormality in the localization of glr- <dig> , which was the same phenotype induced by cell-specific rnai .
we confirmed that the rig-5a cell-specific promoter also drove rmd-specific expression at the embryonic stage .
these results indicated that in rmd neurons at the adult stage, the ras-mapk pathway controlled glr- <dig> localization.figure  <dig>
t.c.rnai provides detailed profiles of let-60ras in rmd neurons.
representative images of glr-1::gfp in neurites of rmdd of transgenic animals with let- <dig> time- and rmd-specific rnai  .
average intensity of glr-1::gfp fluorescence in rmdd neurons of transgenic animals with let-60 t.c.rnai after heat shock at the embryonic or adult stage .
the proportion of wild type, let- <dig> mutants, and animals with let-60 t.c.rnai after heat shock treatment exhibiting cl .
in  and , rmd:: and hsp:: mean rig-5ap::let- <dig> and hsp16-2::let- <dig>  respectively.
error bars represent sem and asterisks indicate significant differences .
sem, standard error of the mean.
we next analyzed the stage at which the ras-mapk pathway in rmd neurons regulated locomotion behavior.
the knockdown of let- <dig> in rmd neurons at the l <dig> larva stage resulted in no defects in locomotion behavior, whereas the expression of let- <dig> double-stranded rna at the adult stage significantly  increased the rate of cl , indicating that ras regulated locomotion at the adult stage in rmd.
the same result was obtained even when the sense and the anti-sense strand were expressed by the reciprocally changed promoter .
these results were consistent with the earlier finding that ras in rmd neurons at the adult stage was necessary for the normal localization of glr- <dig> .
however, knockdown of let- <dig> at the embryonic stage also significantly induced cl , suggesting a role for ras other than the regulation of glr- <dig> localization at the embryonic stage to control locomotion behavior.
neither wild-type animals exposed to heat shock nor transgenic animals without heat shock showed cl .
further, we confirmed that expression of only a single rna strand by a heat shock promoter or a cell-specific promoter cannot cause knockdown of the target gene .
discussion
in the present study, we developed a novel method, t.c.rnai that achieves simultaneous time- and cell-specific knockdown.
we confirmed that t.c.rnai causes time- and cell-specific knockdown of gfp.
we further show that t.c.rnai caused abnormal locomotion behavior and mislocalization of glr- <dig> by adult stage- and rmd neurons-specific knockdown of let-60ras, and aberrant responses to an odorant by adult stage- and awc neurons-specific knockdown of odr- <dig> via heat shock.
temporal and spatial restriction of the effects of knockdown is necessary for the analysis of various and crucial genes, such as ras.
let-60ras determines the fate of the excretory duct cell, which is required for osmoregulation  <cit> .
moreover, null mutants are lethal in the l <dig> larval stage  <cit> .
nevertheless, in the present study, the effects of let- <dig> knockdown at both the embryonic and l <dig> larval stage was observed by t.c.rnai without lethality.
overall, t.c.rnai might provide a basis for understanding temporal and special profiles of essential genes.
this approach was used in the present study for ras, which is involved in various physiological activities, such as development, signal transduction and neural plasticity.
we analyzed the exploratory behavior of c. elegans.
under enriched conditions, c. elegans exhibited klinokinesis and klinotaxis .
our results indicated that in contrast to these responses, foraging behavior was used by c. elegans to search for food resources over wide-ranging areas and determined the direction of locomotion under poor conditions.
we found that cl occurred in let- <dig> mutants under poor conditions, whereas the approach to sodium chloride of the mutants was normal  <cit> .
therefore, c. elegans may exhibit foraging behavior to search for food cues specifically under poor environmental conditions.
moreover, once animals detect food stimuli, the possible contribution of klinokinesis and klinotaxis may play a greater role than foraging to determine both locomotion direction and the movement towards food resources.
our results showed that the ras-mapk pathway was functional in il <dig>  olq and rmd neurons.
in il <dig> and olq neurons, let- <dig> knockdown did not cause abnormal localization of snb- <dig>  suggesting roles other than the regulation of synaptic vesicle localization for the ras-mapk pathway.
in pre-synapses, this pathway controls the localization of transporter vesicles, in addition to other roles  <cit> .
a previous study showed that mapk modulates the release probability at glutamatergic synapses in rats  <cit> .
in c. elegans, eat- <dig> encodes the ortholog of bnpi vesicular glutamate transporter that appears to have a role in the synthesis of glutamate or concentrate glutamate into synaptic vesicles, and eat- <dig> is expressed in il <dig> and olq neurons  <cit> .
we previously reported that pkc- <dig> which controls synaptic transmission functions downstream of ras signaling in awc neurons in which glutamate is released as a neural transmitter and it is mediated by eat- <dig>  <cit> .
in the present study, we found that cl occurred in mutants of eat- <dig> .
therefore, the ras-mapk pathway may be associated with the regulation of eat- <dig> directly via phosphorylation, or indirectly via other molecules, including transcription factors in il <dig> and olq neurons to control foraging behavior.
in rmd neurons, our findings show that let-60ras plays an essential role in the localization of glr- <dig>  although lin- <dig> or lin- <dig> encodes a transcription factor that functions downstream of mapk in vulval induction  <cit> , the mapk signaling pathway unlikely activates these transcription factors to control the expression of glr- <dig>  because our current findings from t.c.rnai experiments indicated that glr- <dig> mislocalization was induced within 30 minutes by heat shock at the adult stage.
that mapk might directly phosphorylate and regulate glr- <dig> or the proteins required for its localization is possible.
a recent report shows that auxiliary subunits are important for regulating ampa receptor trafficking to the synaptic membrane  <cit> .
further, the cytoskeletal protein, α-adducin- <dig> , is expressed in rmd neurons  <cit> .
our present results from the t.c.rnai analyses show that ras in rmd neurons at the embryonic stage is required for the regulation of foraging, but not for control of glr- <dig> localization.
these findings show that let-60ras was neither involved in morphogenesis of rmd neurons nor formation of post-synapses, both of which are necessary for the normal localization of glr- <dig> in rmd.
therefore, we speculate that let- <dig> ras might regulate the formation of, or play a role at, the neuromuscular junction in rmd motor neurons during the embryonic stage.
a previous study has shown that the ras-mapk pathway is triggered by trans-synaptic signaling, and is responsible for the formation of the neuromuscular junction and its functions  <cit> .
in c. elegans, the acetylcholine transporter and choline acetyltransferase play a role at the neuromuscular junction and are encoded by unc- <dig>  <cit>  and cha- <dig>  <cit> , respectively.
further, these two genes are localized in the synaptic region  <cit> .
therefore, exploring the functional role of unc- <dig> and cha- <dig> in rmd neurons may shed more light on the function of the ras-mapk pathway at the embryonic stage in rmd neurons.
CONCLUSIONS
our results demonstrate that the ras-mapk pathway functions in the neural circuit underlying the foraging behavior essential for the control of the direction of locomotion under poor environmental conditions in c. elegans.
the pathway regulates localization of glr- <dig> glutamate receptors in rmd neurons.
we established a time- and cell-specific rnai method which demonstrated the temporal profile of the signaling pathway in specific neurons.
the control of glr- <dig> localization in rmd requires ras at the adult stage.
this method can be applied to other genes.

 the nematode caenorhabditis elegans is useful for studying the effects of a gene on behavior through facilitated behavioral analyses and well-developed genetic analyses. in this organism, various cell-specific promoters can be utilized for cell-specific expression to determine the function of a gene in a specific cell. recently, temporal control of cell-specific expression using heat shock factor- mutants has been reported. in addition, the effects of gene knockdown can be assessed using various loss-of-function mutants, and researchers recently developed a new method of generating loss-of-function mutations in targeted genes in c. elegans. rnai is one of the most powerful tools for gene knockdown. rnai-mediated cell-specific knockdown in c. elegans is a currently available technique based on driving the expression of double-stranded rna in target cells via cell-specific promoters. this method identifies the cells in which the target gene functions. moreover, gene functions can be analyzed by this system because of cell-specificity, even if mutants of the gene show fatal phenotypes. in contrast to cell-specific promoters, heat shock promoters drive the expression of genes at arbitrary timing via heat shock. a previous report has shown that rna hairpins, which are driven by the heat shock promoter, induce knockdown of the target gene, suggesting the possibility of time-specific knockdown. however, heat shock promoters drive global expression, meaning that the knockdown is performed in the majority of cells, thereby removing vital cellular functions and thus inducing lethality when essential genes are knocked down by this method. therefore, for detailed analysis of multifunctional genes, such as the components of the ras-mapk pathway described below, simultaneous time-specific and cell-specific knockdown is necessary. to our knowledge, however, such a method has not yet been developed in c. elegans. intracellular signal transduction pathways play an essential role in regulating diverse cellular functions. most of these pathways have widespread functions in various tissues and at different stages. for example, the ras-mapk signaling pathway controls cell proliferation and differentiation at the embryonic stage and regulates synaptic formation required for neural plasticity at the adult stage. to distinguish and clearly understand the multiple functions of these signal transduction pathways, conditional knockdown systems, such as time- and cell-specific knockdown, are required. the ras-mapk pathway in c. elegans plays a role in various processes, such as vulval induction, olfaction and germ-line apoptosis. however, whether or not the signaling pathway is involved in the control of locomotion behavior is unknown. animals use various strategies according to the circumstance in which they are placed to gain food efficiently. under enriched environmental conditions in which animals are near food resources, efficient exploration is achieved by their use of perception, which incorporates olfaction, vision and touch. however, animals roam randomly to gain information when food resources are far from them. c. elegans has a simple nervous system. this worm is composed of only neurons and uses diverse strategies to obtain food. a recent study has shown that switching between dwelling and roaming activities according to the surroundings is controlled by a neural circuit and two neuromodulators, serotonin and neuropeptide pigment dispersing factor, which exhibit opposing effects. in the neural circuit, serotonergic signaling initiates and extends the dwelling states, while pdf signaling affects roaming states by acting on each target neuron. while roaming, c. elegans uses two strategies to approach food resources efficiently in enriched environmental conditions. the first approach is klinotaxis, in which the animals gradually curve according to the gradient of chemoattractants, and the second is klinokinesis, in which they turn and change the direction of locomotion when a decrease in the concentration of chemoattractants is sensed. the strategies for switching between dwelling and roaming activities, and those of approaching food under enriched environmental conditions, are well studied and understood. however, there is a scarcity of information regarding the strategy used by c. elegans when roaming under poor environmental conditions. constitutive and exploratory head movements might be one possible strategy. il and olq mechanosensory neurons are known to be involved in transducing signals during both aversive head-withdrawal and the control of foraging. further, of the motor neurons connecting to il and olq neurons, rmd motor neurons possess the main synaptic projection, and thus play important roles in regulating head movements. the neural circuit composed of il olq and rmd neurons is essential for the regulation of spontaneous foraging and head-withdrawal movements. however, the role of foraging behavior on roaming and locomotion under poor environmental conditions, how head movements are regulated in foraging, and which signal transduction pathways are involved in the regulation of foraging are all unknown. in this study, we analyzed the effect of the ras-mapk signaling pathway on the regulation of foraging behavior via an rnai method with simultaneous time-specific and cell-specific knockdown of ras. we found that mutants of the ras-mapk signaling pathway exhibit abnormal locomotion behavior under poor environmental conditions in which they are far from food resources. cell-specific knockdown of the ras gene revealed that the ras protein was functional in il olq and rmd neurons, and thereby controlled foraging behavior. further, results indicate that ras regulated the localization of glr- glutamate receptors in rmd neurons. moreover, we demonstrated that the function of ras at the adult stage was important for the regulation of glr- localization in rmd neurons by using time- and cell-specific rnai. this method enabled cell-specific knockdown of gene functions at arbitrary timing.  mutations in the ras-mapk pathway lead to circular locomotion instead of normal exploratory foraging under poor environmental conditions in which c. elegans is far from food resources, it crawled around the field to search for food resources. a previous study quantitatively characterized this behavior and reported that a locomotory behavior under these conditions has a long-range directionality and cannot be explained by a simple random isotropic model of locomotion. wild-type animals moved in an apparently unbiased manner when placed on a plate without any chemicals or food. however, ras mutants showed abnormal locomotion behavior on the blank plates. as the loss-of-function mutants of ras, let- continued to move in a loopy pattern, we named this phenotype;circular locomotion’. we defined an animal that exhibited cl as one showing loopy tracks in more than 30% of the whole track left during the one hour test period. a large proportion of let- mutants were categorized with cl but exhibited milder phenotypes compared to neuron-specific let- rnai strains described below, probably due to n being a weak allele. ras and the downstream mapk pathway are essential for numerous physiological cellular functions, and are highly conserved in diverse organisms. although the ras-mapk pathway in c. elegans regulates a number of processes, its involvement of the pathway controlling locomotion behavior is unknown.figure ras mutants exhibit abnormal locomotion behavior under poor circumstances. tracks representative of wild type and the let- mutant in blank plates. the wild-type crawls around the field randomly, whereas the let- mutant continues to move in a loopy pattern, termed circular locomotion. average curving rate of wild type and let- mutants for a fixed time or a fixed distance of exploratory behavior. positive or negative values indicate curves toward ventral or dorsal sides, respectively. asterisks indicate significant differences compared with wild-type animals. histograms of curving rate in wild type and let- mutants in the fixed distance. quantitative analysis of directional locomotion. the median of log at the distance of size δ in wild-type and let- mutants is shown in log-log scale when analyzed in the fixed distance. error bars represent the first and the third quartile. proportion of animals exhibiting circular locomotion in mutants of the ras-mapk pathway genotype cl number let-23 sem-5 let-60 let-60 lin-45 mek-2 mpk-1 to analyze cl quantitatively, we measured the curving rate, which is defined as the change in direction of locomotion per unit length of an animal’s advancement, of the locomotion under poor conditions. wild-type animals exhibited mostly random but slightly biased curves toward the ventral side. in contrast, let- mutants exhibited significantly larger and directional curves toward the dorsal side. given that each animal moved at a different velocity, we calculated the average curving rate in the first 30 mm of each track. in this analysis, let- mutants also exhibited significantly larger curves than wild type. these results suggest that cl may reflect large, directional curves. in addition, to quantitatively measure the extent to which animals made directional tracks, we performed the analysis based on fractal analysis as described previously. briefly, the trajectory of each animal was divided into segments with length of size δ, and the sum of linear distances of adjacent dividing points was computed as lδ. we then calculated lδ/l, in which l indicates the length of the track. again, the analysis was performed on the first 30 mm of each track, thus l0 = 30 mm. when animals tend to move in narrower areas, such as with cl, lδ/l is expected to decrease. lδ/l values for animals with let- mutations tended to be lower than those of wild type animals, indicating that the former tended to move in narrower areas than the latter. taken together, cl is characterized as large, directional curves and in migration relatively narrow areas. in c. elegans, components of the ras-mapk pathway: ras, mapkkk, mapkk, and mapk are encoded by the let- lin- mek- and mpk- genes, respectively. each lf or null mutant of the pathway, let- lin- mek- and mpk- frequently exhibited cl. further, mutants of the upstream components of the signaling pathway, let- and sem- also exhibit cl. the genes let- and sem- encode an egf-receptor-family transmembrane tyrosine kinase and an adaptor protein orthologous to human grb respectively. however, let- mutants did not exhibit cl, suggesting that inactivation, and not hyperactivation, of the pathway caused cl. these results indicate that the ras-mapk pathway modulated locomotion behavior under poor environmental conditions in c. elegans. we previously reported that the ras-mapk pathway controls both klinotaxis, a migratory behavior towards odorants, and olfactory plasticity. awc olfactory neuron- and aiy interneuron-specific expression of the let- gene rescues the defects of klinotaxis and plasticity, respectively, of let- mutants. however, our results revealed that the expression of let- in awc and aiy did not reduce the number of the let- mutants exhibiting cl. let- mutants have no abnormality in chemotaxis for sodium chloride. we observed that mutants rarely exhibited cl when they moved towards the chemoattractants. a previous study reported that mutants exhibited moderate to severe defects in chemotaxis in response to 1 μl of 10− 10− and 10− dilutions of isoamyl alcohol on 9 cm plates. when mutants were placed 3 cm from 1 μl of a 10− or 10− dilution of isoamyl alcohol, the mutants exhibited cl frequently, as they were on plates without stimuli. in contrast, the proportion of those exhibiting cl significantly decreased when mutants were exposed to 10− isoamyl alcohol. these results suggest that cl occurs independently of the defects of klinotaxis and olfactory plasticity. the ras-mapk pathway functions in neural circuits underlying foraging behavior to control direction of locomotion to elucidate the mechanism underlying impairment of the ras-mapk pathway-mediated cl, we analyzed the locomotion behavior of let- mutants in more detail. let- mutants exhibited abnormal foraging behavior in which their head movements were slightly asymmetrical and over-bent. this observation led us to assess whether abnormal foraging behavior induced cl. previous reports have shown that the rate of spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: il olq, and rmd. il and olq are mechanosensory neurons, and rmd are motor neurons that receive main connections from il and olq neurons, and innervate the head muscles. therefore, we examined whether blocking the activity of these neurons causes cl. unc- was used to inhibit the activity of these neurons. this gene encodes a constitutively active form of the potassium channel unc- and can reduce the neuronal activity by hyperpolarization. we found that il olq or rmd neuron-specific expression of unc- in wild-type animals significantly caused cl, indicating that inhibition of these neurons induced locomotion behavior in a loopy pattern in c. elegans, even if one class of neuron was impaired. a previous report indicated that the osm- promoter we utilized for the expression in olq also drives the expression in awa and adl sensory neurons. however, awa- or adl-ablated animals did not exhibit cl, indicating the importance of olq in the regulation of locomotion. these results suggest that the neural circuit involved in foraging behavior played an essential role in determining the direction of locomotion.figure let- ras in il olq and rmd neurons controls the direction of locomotion. the proportion of animals exhibiting cl in wild type, let- mutants and transgenic animals in which il olq or rmd neurons were inactivated by unc-. rescue experiments in which the wild-type let- gene was expressed in each or all the neurons regulating foraging in let- mutants. il1-, olq- or rmd-specific rnai of let- causes frequent cl. definition of ‘head angle’ as the angle between two lines where one passes the worm centroid and the terminal bulb of pharynx and the other the terminal bulb of pharynx and nose tip. the ventral or the dorsal bends of the head are defined as positive or negative values, respectively. head angles of ventral and dorsal bends in wt and rmd::let-60rnai animals. n ≥ bends. error bars represent sem. asterisks indicate significant differences compared with wild-type animals or let- mutants. n.s., not significant. cl, circular locomotion; sem, standard error of the mean; wt, wild type. these results led us to investigate whether the ras-mapk pathway plays a role in the neural circuit involved in foraging behavior to control locomotion. we, therefore, conducted rescue experiments where the wild-type let- gene ) was expressed in neurons regulating foraging behavior in let- mutants. we found that expression of let- in all three neuronal classes rescued the cl phenotype in let- mutants, which was not found when the expression of let- occurred in a single class of neurons. these results suggested that let- ras functions in all classes of neurons, which regulate foraging behavior. further, this suggestion was consistent with the finding that impairment of let- in a single class of neurons caused cl, as described below. we next performed cell-specific knockdown experiments using rnai. cell-specific knockdown of the let- gene in each class of neurons caused cl. in particular, knockdown of let- in il or rmd neurons strongly induced cl, suggesting that let- ras largely contributed to the function of il and rmd neurons. taken together, these results indicate that functions of let- ras in all il olq and rmd neurons were required for foraging behavior to control the direction of locomotion. compared to animals with inhibition of neurons, neuron-specific let- rnai strains exhibited milder phenotypes, suggesting that pathways other than ras signaling might also be involved in the regulation of locomotion, although the possibility that rnai is at least somewhat effective cannot be excluded. we directly observed and quantified foraging behavior in wild type and animals with rmd-specific rnai knockdown of let- we monitored the head angle of worms during locomotion on plates without any chemicals or food. this angle was defined as that between two lines in which one passes the worm centroid and the terminal bulb of pharynx and the other the terminal bulb of pharynx and nose tip. ventral bends were counted as positive and dorsal bends as negative values, respectively. the mean head angle of dorsal bends in animals with rmd-specific let- rnai was significantly larger than in wild type animals, while that of ventral bends was not. this result seems to be consistent with the finding that let- mutants tended to exhibit dorsal-biased large turns in exploratory behavior and indicates that ras in rmd neurons is important for foraging behavior, suggesting a correlation between foraging and exploratory behavior. the ras-mapk pathway regulates the localization of glutamate receptors in rmd neurons we next explored the role of the ras-mapk pathway in il olq and rmd neurons. the ras-mapk pathway is essential for neurogenesis in fruit flies and mice. to clarify if this pathway was required for neurogenesis of these neurons, we observed the morphology of the neurons in let- mutants. these mutants did not exhibit any apparent morphological defects in il olq or rmd neurons, suggesting that the ras-mapk pathway was not involved in the morphogenesis of these neurons. the ras-mapk pathway also has important roles in synapse formation and the control of synaptic protein localization. to examine whether this pathway regulates formation of pre-synapses in il and olq neurons, we observed the localization of synaptobrevin, snb-, in the axons of these neurons. snb- is a synaptic vesicle protein essential for vesicle docking or fusion, which is required for synaptic transmission. we observed that snb- localization was not affected by il1- or olq-specific knockdown of let-. therefore, the ras-mapk pathway might not be involved in presynaptic formation of these neurons. previous studies have shown that ras signaling drives the synaptic delivery of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in post-synapses. in c. elegans, the glr- gene is expressed in neurons, including rmd. our results revealed that glr- mutants also displayed cl. the expression of glr- by its own promoter restored the defect in locomotion behavior of glr- mutants. moreover, rmd-specific expression of glr-1::gfp significantly rescued the cl phenotype of glr- mutants. these results suggest that glr- in rmd neurons played an important role in controlling the direction of locomotion and also indicated that glr-1::gfp was functional. we next investigated whether the ras-mapk pathway was associated with glr- for the regulation of locomotion behavior. to assess the effect of this pathway on glr- we investigated the localization of glr-1::gfp in neurites of rmd neurons in wild-type and animals with rmd-specific knockdown of let- via fluorescence. glr-1::gfp was expressed by mgl- promoter, which drives the expression in nsm, aia and rmd neurons. distinguishing the neurites of rmd and aia neurons in the nerve ring is difficult. thus, we targeted the neurites of rmdd neurons because these neurons are distinct from those of aia. in wild-type animals, punctate localization of clustered glr- was observed in neurites of rmd neurons. however, in rmd neurons of rmd-specific let- knockdown animals, glr-1::gfp clusters were very faint or not detected. we confirmed that the expression of gfp by the mgl- promoter was not affected by the knockdown of the let- gene, suggesting that let- ras did not act on the mgl- promoter. these results indicated that ras was required for normal localization of glr- in post-synapses of rmd neurons.figure let- ras mediates the localization of glr- glutamate receptors in rmd neurons. the proportion of wild type, glr- mutants and glr- mutants with expression of glr- gene by its own promoter exhibiting cl. the proportion of wild type, glr- mutants and glr- mutants with expression of glr-1::gfp by rig-5a promoter exhibiting cl. error bars represent sem. asterisks indicate significant differences. mgl- promoter drives expression in nsm, rmdv, rmdd and aia neurons. neurites of rmdd neurons were investigated by analyzing the clusters of glr-1::gfp in rmd neurons specifically. glr-1::gfp clusters expressed by the mgl- promoter in wild type and wild type with rmd-specific rnai of let-. enlarged images show neurites of rmdd neurons. average intensity of glr-1::gfp clusters in neurites of rmdd neurons is significantly reduced by rmd-specific knockdown of let-. error bars represent sem. an asterisk indicates significant differences. representative images of mgl-1p::gfp in wild type and wild type with rmd-specific rnai of let-. scale bars = 10 μm. cl, circular locomotion; sem, standard error of the mean. time- and cell-specific rnai is useful for analyses of the temporal profile of gene functions in the specific cell we then analyzed the manner in which the ras-mapk pathway modulated the localization of glr- the ras-mapk pathway is involved in morphogenesis, synaptic formation, and neural plasticity. therefore, our results led us to test two hypotheses: the ras-mapk pathway plays a role in the development of synapses or synaptic connections of rmd neurons at the embryonic stage, and the signaling pathway is involved in glr- localization machinery at the adult stage. to examine these hypotheses, we analyzed the stage at which let-60ras mediated the control of glr- localization in rmd neurons. to dissect the function of a gene which has different roles according to the stages, such as ras, a method to regulate the function of the gene spatially and temporally is needed. rnai is one of the powerful tools for gene knockdown, and a recent study has introduced the cell-specific knockdown technique using rnai. the method of cell-specific knockdown is based on the expression of double-stranded rna by the cell-specific promoter. however, this method is incapable of performing time-specific knockdown and dissecting the stage at which the target protein functions in a specific cell. to perform simultaneous time-specific and cell-specific knockdown, we modified the previous method. the heat shock promoter hsp16-, which drives the expression in almost all tissues, is able to activate gene expression at arbitrary timing by heat shock. by introducing the heat shock promoter to the cell-specific rnai technique, we established a new method for time-specific and cell-specific knockdown of genes, in which expression of one rna strand was driven by a cell-specific promoter, and the expression of the other rna strand was induced by a heat shock promoter. under normal conditions, single stranded rna was expressed in the target cells by the cell-specific promoter, whereas under the heat shock-inducing condition, double-stranded rna was expressed in only target cells by the heat shock promoter and the cell-specific promoter. overall, this new technique was expected to elicit simultaneous time-specific and cell-specific knockdown by heat shock. therefore, this method was termed time-specific and cell-specific rnai. to confirm the effect of the t.c.rnai method, we examined whether expression of gfp can be decreased in the time and cell-specific manner by gfp t.c.rnai. to clearly observe the effect of rnai, we photobleached pre-existing gfp, which otherwise has a relatively long half life, before t.c.rnai and observed the recovery of gfp fluorescence after t.c.rnai. this way we can assess the decrease of mrna abundance caused by rnai. to quantitatively compare the gfp intensity before and after t.c.rnai, we measured the gfp intensity in the same individuals before photobleaching, just after photobleaching and after a heat shock for 30 minutes at 33°c and a recovery for one hour at 20°c. then we calculated the rate of change in the fluorescence intensity of each animal. based on this rate, the recovery ratio of gfp intensity compared to controls was measured. we first analyzed the effect of gfp t.c.rnai in awc sensory neurons. gfp was expressed by the gcy- promoter which drives the expression in awc, awb and i and monitored the gfp intensity in cell bodies of awc. in adult animals expressing both gcy-10::gfp and hsp::gfp, the recovery ratio of gfp intensity in awc significantly decreased after the heat shock compared to that after the mock treatment. the expression of double-stranded rna by the reciprocally exchanged promoter also induced gfp knockdown after the heat shock. the gfp intensity in awc was normally recovered after the heat shock in animals without expression of the rnai constructs, suggesting the decrease of the recovery ratio was not due to an influence of heat shock on the gcy- promoter itself. we also confirmed that expression of only a single rna strand driven by a heat shock promoter or a cell-specific promoter could not decrease the recovery ratio.figure confirmation of the effect of the novel method, time- and cell-specific rnai. the average recovery ratio of gfp intensity of awc neurons in animals without rnai constructs, animals with only gcy-10p::gfp, gcy-10p::gfp, hsp16-2::gfp, hsp16-2::gfp, animals with both gcy-10p::gfp and hsp16-2::gfp or both hsp16-2::gfp and gcy-10p::gfp after heat-shocked or control condition.. the average recovery ratio of gfp intensity of awb and awc neurons in animals with srd-17p::gfp and hsp16-2::gfp after heat-shocked or control condition. representative images of gcy-10p::gfp in animals with srd-17p::gfp and hsp16-2::gfp before and after photobleaching and after heat-shocked condition. arrow heads indicate awb neurons, and arrows indicate awc neurons. scale bars = 10 μm. the average recovery ratio of gfp intensity of anterior or lateral protrusions of vulva in animals without rnai constructs or animals with myo-3p::gfp and hsp16-2::gfp after heat-shocked or control condition. chemotaxis to isoamyl alcohol in wild-type animals, odr- mutants and wild-type animals with odr-3 t.c.rnai. awc:: and hsp:: mean gcy-10p::odr- and hsp16-2::odr- error bars represent sem and asterisks indicate significant differences. sem, standard error of the mean. moreover, to verify the cell-specificity of the gene knockdown by t.c.rnai, we monitored gfp intensity in both awc and awb neurons at the same time. we used gcy- promoter for the expression of gfp in both awc and awb neurons, and srd- promoter for the expression of a single-stranded rna specifically in awc neurons. in animals that expressed srd-17p::gfpand hsp::gfp, recovery of gfp intensity in awc was reduced but not in awb after the heat shock, indicating the cell-specific effect of t.c.rnai. taken together, t.c.rnai could induce time- and cell-specific knockdown of gfp in awc neurons effectively. next, to investigate whether t.c.rnai could cause gene knockdown in tissues other than neurons, we performed gfp knockdown in vulval muscles. we found a decrease of the recovery ratio of gfp intensity in vulval muscles after heat shock treatment, but not after control treatment in animals with myo-3::gfp, myo-3::gfp and hsp::gfp. we confirmed that gfp intensity in animals without expression of rnai constructs was recovered normally after the heat shock. these results suggested that t.c.rnai could cause gene knockdown in muscles as well as neurons in a cell-specific manner. in addition, to assess whether t.c.rnai can be applied to endogenous genes, we performed knockdown of odr- by t.c.rnai. the odr- gene encodes a g protein α which mainly functions in awc chemosensory neurons and is essential for olfactory responses to odorants, including isoamyl alcohol, which is sensed by awc. we found that animals in which odr- was knocked down in awc neurons by t.c.rnai at the adult stage showed a defect in the response to isoamyl alcohol. however, the adult transgenic animals that had undergone heat shock treatment at the l larval stage exhibited a normal response. these results indicated that odr- worked in awc neurons at the adult stage, which is consistent with the well-known function of odr- in the olfactory signaling pathway. these results suggested that t.c.rnai could induce time- and cell-specific knockdown of endogenous genes as well as transgenes, and the possibility that our new method could be applied widely. t.c.rnai revealed the stage at which ras functions in rmd neurons to control locomotion behavior using our new method, t.c.rnai, we investigated the effects of let- knockdown on the localization of glr- in rmd neurons at the embryonic and adult stage. our results indicated that glr- localization was not affected by let- knockdown at the embryonic stage. however, knockdown of let- at the adult stage caused significant abnormality in the localization of glr-, which was the same phenotype induced by cell-specific rnai. we confirmed that the rig-5a cell-specific promoter also drove rmd-specific expression at the embryonic stage. these results indicated that in rmd neurons at the adult stage, the ras-mapk pathway controlled glr- localization.figure t.c.rnai provides detailed profiles of let-60ras in rmd neurons. representative images of glr-1::gfp in neurites of rmdd of transgenic animals with let- time- and rmd-specific rnai. average intensity of glr-1::gfp fluorescence in rmdd neurons of transgenic animals with let-60 t.c.rnai after heat shock at the embryonic or adult stage. the proportion of wild type, let- mutants, and animals with let-60 t.c.rnai after heat shock treatment exhibiting cl. in and, rmd:: and hsp:: mean rig-5ap::let- and hsp16-2::let- respectively. error bars represent sem and asterisks indicate significant differences. sem, standard error of the mean. we next analyzed the stage at which the ras-mapk pathway in rmd neurons regulated locomotion behavior. the knockdown of let- in rmd neurons at the l larva stage resulted in no defects in locomotion behavior, whereas the expression of let- double-stranded rna at the adult stage significantly increased the rate of cl, indicating that ras regulated locomotion at the adult stage in rmd. the same result was obtained even when the sense and the anti-sense strand were expressed by the reciprocally changed promoter. these results were consistent with the earlier finding that ras in rmd neurons at the adult stage was necessary for the normal localization of glr-. however, knockdown of let- at the embryonic stage also significantly induced cl, suggesting a role for ras other than the regulation of glr- localization at the embryonic stage to control locomotion behavior. neither wild-type animals exposed to heat shock nor transgenic animals without heat shock showed cl. further, we confirmed that expression of only a single rna strand by a heat shock promoter or a cell-specific promoter cannot cause knockdown of the target gene. discussion in the present study, we developed a novel method, t.c.rnai that achieves simultaneous time- and cell-specific knockdown. we confirmed that t.c.rnai causes time- and cell-specific knockdown of gfp. we further show that t.c.rnai caused abnormal locomotion behavior and mislocalization of glr- by adult stage- and rmd neurons-specific knockdown of let-60ras, and aberrant responses to an odorant by adult stage- and awc neurons-specific knockdown of odr- via heat shock. temporal and spatial restriction of the effects of knockdown is necessary for the analysis of various and crucial genes, such as ras. let-60ras determines the fate of the excretory duct cell, which is required for osmoregulation. moreover, null mutants are lethal in the l larval stage. nevertheless, in the present study, the effects of let- knockdown at both the embryonic and l larval stage was observed by t.c.rnai without lethality. overall, t.c.rnai might provide a basis for understanding temporal and special profiles of essential genes. this approach was used in the present study for ras, which is involved in various physiological activities, such as development, signal transduction and neural plasticity. we analyzed the exploratory behavior of c. elegans. under enriched conditions, c. elegans exhibited klinokinesis and klinotaxis. our results indicated that in contrast to these responses, foraging behavior was used by c. elegans to search for food resources over wide-ranging areas and determined the direction of locomotion under poor conditions. we found that cl occurred in let- mutants under poor conditions, whereas the approach to sodium chloride of the mutants was normal. therefore, c. elegans may exhibit foraging behavior to search for food cues specifically under poor environmental conditions. moreover, once animals detect food stimuli, the possible contribution of klinokinesis and klinotaxis may play a greater role than foraging to determine both locomotion direction and the movement towards food resources. our results showed that the ras-mapk pathway was functional in il olq and rmd neurons. in il and olq neurons, let- knockdown did not cause abnormal localization of snb- suggesting roles other than the regulation of synaptic vesicle localization for the ras-mapk pathway. in pre-synapses, this pathway controls the localization of transporter vesicles, in addition to other roles. a previous study showed that mapk modulates the release probability at glutamatergic synapses in rats. in c. elegans, eat- encodes the ortholog of bnpi vesicular glutamate transporter that appears to have a role in the synthesis of glutamate or concentrate glutamate into synaptic vesicles, and eat- is expressed in il and olq neurons. we previously reported that pkc- which controls synaptic transmission functions downstream of ras signaling in awc neurons in which glutamate is released as a neural transmitter and it is mediated by eat-. in the present study, we found that cl occurred in mutants of eat-. therefore, the ras-mapk pathway may be associated with the regulation of eat- directly via phosphorylation, or indirectly via other molecules, including transcription factors in il and olq neurons to control foraging behavior. in rmd neurons, our findings show that let-60ras plays an essential role in the localization of glr- although lin- or lin- encodes a transcription factor that functions downstream of mapk in vulval induction, the mapk signaling pathway unlikely activates these transcription factors to control the expression of glr- because our current findings from t.c.rnai experiments indicated that glr- mislocalization was induced within 30 minutes by heat shock at the adult stage. that mapk might directly phosphorylate and regulate glr- or the proteins required for its localization is possible. a recent report shows that auxiliary subunits are important for regulating ampa receptor trafficking to the synaptic membrane. further, the cytoskeletal protein, α-adducin-, is expressed in rmd neurons. our present results from the t.c.rnai analyses show that ras in rmd neurons at the embryonic stage is required for the regulation of foraging, but not for control of glr- localization. these findings show that let-60ras was neither involved in morphogenesis of rmd neurons nor formation of post-synapses, both of which are necessary for the normal localization of glr- in rmd. therefore, we speculate that let- ras might regulate the formation of, or play a role at, the neuromuscular junction in rmd motor neurons during the embryonic stage. a previous study has shown that the ras-mapk pathway is triggered by trans-synaptic signaling, and is responsible for the formation of the neuromuscular junction and its functions. in c. elegans, the acetylcholine transporter and choline acetyltransferase play a role at the neuromuscular junction and are encoded by unc- and cha-, respectively. further, these two genes are localized in the synaptic region. therefore, exploring the functional role of unc- and cha- in rmd neurons may shed more light on the function of the ras-mapk pathway at the embryonic stage in rmd neurons.  our results demonstrate that the ras-mapk pathway functions in the neural circuit underlying the foraging behavior essential for the control of the direction of locomotion under poor environmental conditions in c. elegans. the pathway regulates localization of glr- glutamate receptors in rmd neurons. we established a time- and cell-specific rnai method which demonstrated the temporal profile of the signaling pathway in specific neurons. the control of glr- localization in rmd requires ras at the adult stage. this method can be applied to other genes. 
in particular, rnai is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible rnai or cell-specific rnai. to investigate the temporal- and cell-specific profiles of the functions of ras, we developed a new rnai method that enables simultaneous time- and cell-specific knockdown. we have established a new rnai method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the ras-mapk pathway in the control of exploratory behavior under poor environmental conditions. in this method, one rna strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded rna in the target cell when heat shock is induced. here, we investigated the ras gene for its role in exploratory behavior in c. elegans. c. elegansexploratory behaviorglutamate receptorrnai methodthe ras-mapk pathwayissue-copyright-statement© the author 2015 the nematode worm caenorhabditis elegans, in which loss-of-function mutants and rna interference models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: il olq, and rmd, and we found that ras functions in this neural circuit to modulate the direction of locomotion. we further observed that ras plays an essential role in the regulation of glr- glutamate receptor localization in rmd neurons. we found that, under poor environmental conditions, mutations in the ras-mapk signaling pathway lead to circular locomotion instead of normal exploratory foraging. this technique revealed that control of glr- localization in rmd neurons requires ras at the adult stage.
 the nematode worm caenorhabditis elegans, in which loss-of-function mutants and rna interference models are available, is a model organism useful for analyzing effects of genes on various life phenomena, including behavior. in particular, rnai is a powerful tool that enables time- or cell-specific knockdown via heat shock-inducible rnai or cell-specific rnai. however, conventional rnai is insufficient for investigating pleiotropic genes with various sites of action and life stage-dependent functions.  here, we investigated the ras gene for its role in exploratory behavior in c. elegans. we found that, under poor environmental conditions, mutations in the ras-mapk signaling pathway lead to circular locomotion instead of normal exploratory foraging. spontaneous foraging is regulated by a neural circuit composed of three classes of neurons: il olq, and rmd, and we found that ras functions in this neural circuit to modulate the direction of locomotion. we further observed that ras plays an essential role in the regulation of glr- glutamate receptor localization in rmd neurons. to investigate the temporal- and cell-specific profiles of the functions of ras, we developed a new rnai method that enables simultaneous time- and cell-specific knockdown. in this method, one rna strand is expressed by a cell-specific promoter and the other by a heat shock promoter, resulting in only expression of double-stranded rna in the target cell when heat shock is induced. this technique revealed that control of glr- localization in rmd neurons requires ras at the adult stage. further, we demonstrated the application of this method to other genes.  we have established a new rnai method that performs simultaneous time- and cell-specific knockdown and have applied this to reveal temporal profiles of the ras-mapk pathway in the control of exploratory behavior under poor environmental conditions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords c. elegansexploratory behaviorglutamate receptorrnai methodthe ras-mapk pathwayissue-copyright-statement© the author 2015 