Drosophila neuropeptide F (NPF), a homolog of vertebrate neuropeptide Y, functions in feeding and coordination of behavioral changes in larvae and in modulation of alcohol sensitivity in adults, suggesting diverse roles for this peptide. To gain more insight into adult-specific NPF neuronal functions, how npf expression is regulated in the adult brain was studied. npf expression is regulated in both sex-nonspecific and male-specific manners. The data show that male-specific npf (ms-npf) expression is controlled by the transformer (tra)-dependent sex-determination pathway. Furthermore, fruitless, one of the major genes functioning downstream of tra, is apparently an upstream regulator of ms-npf transcription. Males lacking ms-npf expression (through traF-mediated feminization) or npf-ablated male flies display significantly reduced male courtship activity, suggesting that one function of ms-npf neurons is to modulate fruitless-regulated sexual behavior. Interestingly, one of the ms-npf neuronal groups belongs to the previously defined clock-controlling dorsolateral neurons. Such ms-npf expression in the dorsolateral neurons is absent in arrhythmic ClockJrk and cycle02 mutants, suggesting that npf is under dual regulation by circadian and sex-determining factors. Based on these data, it is proposed that NPF also plays a role in clock-controlled sexual dimorphism in adult Drosophila (Lee, 2006).
Sexual dimorphism of brain structure and function generates differential neural circuitries, ultimately leading to the production of gender-specific behaviors. In Drosophila, fru is an essential neural sex determinant responsible for male courtship behavior. Because fru-encoded FRUM protein is a BTB-Zn-finger transcription factor, FRUM likely regulates expression of an array of genes to establish neural substrates controlling male behavior. However, such downstream targets of the FRUM are poorly known, hampering understanding of the molecular mechanisms underlying fru-controlled establishment of male-specific neural circuitry. Intriguingly, these studies identified npf as an at least indirect target of the FRUM, suggesting that NPF is a neurochemical factor mediating FRUM functions (Lee, 2006).
Recent studies on the expression of sex-specific fru transcripts and of reporter expression driven by fru-gal4 suggest that fru acts in establishing sexually dimorphic anatomical differences and in rendering male-specific functions to neurons that are commonly present in both. Thus, one important question is whether ms-npf is due to the lack of corresponding neurons in the female brains or to cell-specific transcriptional activation of npf by FRUM in males. The data support the argument that the latter is the case, at least for L1-s neurons, because a comparable number of such neurons independently marked by anti-TIM was observed in both males and females, and because FRUM is persistently present in well differentiated ms-npf neurons. Therefore, it is suggested that one way of masculizing neurons directed by fru is to establish sex-specific production of neurosignaling molecules, which are likely to deliver male-specific neuronal functions. In line with this suggestion, it is notable that male-specific serotonin production in a group of eight neurons in the abdominal ganglion is also controlled by fru, and such serotonergic neurons innervate male reproductive organs to control appropriate male mating activities. These data indicate that ms-NPF is another neurosignaling molecule for fru-controlled male courtship. Although the neuronal targets of ms-npf are unknown, prolonged courtship-initiation latencies and general attenuation of courtship activities caused by the absence of ms-npf suggest that ms-NPF is involved in the central processing of courtship-activating stimuli or in an 'output pathway' that mediates courtship actions (Lee, 2006).
Sexually dimorphic NPY expression has been described in the rat hypothalamus. Large populations of hypothalamic NPY mRNA-producing cells are localized within the arcuate nucleus. Interestingly, the caudal region of the arcuate nucleus contains significantly more NPY cells in males than in females. Further studies suggested that the male gonadal hormone testosterone is a positive regulator of male-biased NPY expression. Similar sexually dimorphic NPY/NPF expression in the brains of distantly related species suggests that these neuropeptides play conserved roles associated with male-specific CNS functions that underlie sexual behavior (Lee, 2006).
Dual regulation of npf by sex and clock factors within a subset of male LNd neurons suggests that NPF is associated with clock-controlled sexually dimorphic behavioral performance. In light/dark cycles, the circadian timing system directs bimodal daily peaks of locomotion, occurring at lights-on (morning) and at lights-off (evening) transitions in WT flies. Interestingly, a distinct sexual difference was observed in the peak of the morning locomotion, which occurs ~1 h earlier in males than in females. However, this male-specific phase of the morning activity was unaffected by npf-ablation, suggesting that npf is not associated with this aspect of sexual dimorphism (Lee, 2006).
The brain-behavioral system is also capable of anticipating photic transitions, as demonstrated by the gradual increase in activity levels before lights-on or lights-off. Among six groups of clock neurons defined in the Drosophila adult brain, clock-relevant functions are relatively well studied for the s-LNvs and the LNds. In the former cell type, clock-controlled pigment-dispersing factor production is essential for circadian locomotor activity rhythms as well as lights-on anticipation, whereas the LNds are required for anticipation of lights-off. The data implicate NPF as a neuromodulatory substance within a subset of the LNds, involved in this 'late-day' component of the locomotor cycle in males (Lee, 2006).
Although the biological meaning of LNd-regulated lights-off anticipation remains unknown, it is notable that the flies’ increasing locomotion at dusk is temporally coincident with especially vigorous mating activities of fruit flies. This finding, then, raises the possibility that anticipatory activity at dusk is causally connected with maximum mating propensity. In this respect, that there are sex- and clock-controlled npf expressions in the LNds provides the first glimpse of this neuropeptide as a putative output factor, which would participate in certain aspects of the clock-controlled reproductive behavior. These actions are intimately connected to evolutionary fitness, which may be one reason for the circadian system of Drosophila to have evolved and been refined (Lee, 2006).
It was postulated that an abundance of Npf in the CNS might promote feeding, whereas npf downregulation could facilitate switching to the nonfeeding state. To test this hypothesis, transgenic flies deficient in Npf signaling were generated by using the Drosophila GAL4-UAS binary expression system. The npf-gal4 constructs containing 4.5 or 1 kb npf upstream regulatory sequences, when crossed to a UAS-GFP (green fluorescent protein) reporter line, each directed a GFP expression pattern that reflected endogenous npf expression in the CNS of young third instar larvae (74 hr after egg laying; 74 hr AEL). Two independent transgenic fly lines containing the 1 kb npf promoter (npf-gal4-1 and npf-gal4-2) were further used to direct the ablation of Npf neurons by crossing them with flies harboring a UAS-DTI transgene that encodes an attenuated diphtheria toxin. The larval progeny (74 hr AEL) from the control cross (y w × UAS-DTI) showed the normal Npf immunostaining pattern consisting of four protocerebral neurons, whereas the progeny (74 hr AEL) from crosses between npf-gal4 and UAS-DTI completely lost the Npf immunoreactivity in the CNS. Npf expression was examined in larvae containing UAS-DTI driven by either a Drosophila CCAP or npfr1 promoter (CCAP-gal4 × UAS-DTI and npfr1-gal4-1 × UAS-DTI). Comparable levels of Npf immunoactivity were observed in these larvae. Moreover, the immunostaining of Drosophila insulin-like peptides in the CNS of npf-gal4, npfr1-gal4, CCAP-gal4, and y w × UAS-DTI larvae were very similar. Thus, there appears to be little or no leaky expression of DTI by the UAS-DTI construct alone (Wu, 2003).
The feeding response of transgenic larvae ablated of the Npf neurons was tested. A solid agar medium containing 10% glucose was used as the food source. This feeding level was chosen for two reasons. (1) 10% glucose solution can be utilized by larvae as the food source; (2) the Npf neuronal network is known to respond to chemosensory stimulation by 10% glucose (Shen, 2001). Well-fed young third instars normally showed little feeding response to the medium, unless they had been held on water-agar for an extended period. Larvae were routinely withheld from food for 2 hr. Thirty synchronized young third instar larvae (74 hr AEL) were used in each of the three separate trials. The larvae were transferred onto a glucose-agar disc (35 mm in diameter) placed on a large food-free agar plate. The feeding assay was designed to allow larvae to forage freely. Approximately 70% or more of control larvae (y w × UAS-DTI, y w, and UAS-DTI) remained on the disc after a test period of 20 min. In contrast, only 5%-12% of the larvae from experimental crosses, npf-gal4-1 × UAS-DTI and npf-gal4-2 × UAS-DTI, stayed under the same condition. Thus, loss of Npf neurons causes premature insensitivity in the feeding response. The larvae ablated of Npf neurons may be less motivated to respond to food that is not readily accessible (e.g., glucose imbedded in agar). This possibility was tested by feeding the larvae with liquid foods (yeast paste or 10% glucose-agarose paste). The assay conditions were the same as above except that a 0.5 ml aliquot of the liquid food was added to the center of the agar disc surface. Upon transfer to the periphery of the yeast or glucose paste, all larvae from both the control and experimental crosses stayed within the food and fed immediately. At the end of 20 min, 85%-95% of the larvae still remained inside the food. To rule out the possibility that larvae lacking the Npf system are deficient in sensing glucose, the ability of experimental (npf-gal4-1 and npfr1-gal4-1 × UAS-DTI) and control (y w × UAS-DTI) larvae to discriminate glucose-agar paste from water-agar paste was tested. The two-choice preference test showed that 100% of both control and experimental larvae preferred to feed in 4% glucose-agar over water-agar paste, and 80% of the larvae fed in 1% glucose-agar. However, the larvae showed difficulty in discriminating 0.25% glucose-agar from water-agar. Taken together, these results indicate that the Npf neural system is not an essential part of the basal feeding machinery but is crucial for normal food response under deprived circumstances (motivational feeding) (Wu, 2003).
In mammalian models, feeding motivation can be determined by measuring animals' willingness to press a lever to obtain food. An assay was designed to quantitatively assess the motivation of Npf and NPFR1 neuron-deficient larvae to extract embedded nutrients (glucose) from an agar block. Synchronized young third instars (72 hr AEL) were rinsed with water, withheld from food for 3 hr, and subsequently transferred into a plate containing glucose-agar blocks. Before the assay, many small cuts were made in the vertical surface of the agar blocks to accommodate one larva in each crack. Within 10 min, about 50% of the larvae voluntarily crawled into a crack, which invariably triggered larval feeding. The frequency of agar scraping by larval mouth hook was determined by counting the number of mouth hook contractions over a 30 s period. The data showed that larvae deficient in Npf signaling (npf-gal4 or npfr1-gal4 × UAS-DTI) have much lower frequency of mouth hook contractions than the y w × UAS-DTI controls. However, all three groups of larvae showed similarly high frequency of mouth hook contraction in liquid food (10% glucose-agar paste), indicating the larvae have no deficits in motions related to food ingestion. In combination, these results strongly suggest that larvae lacking Npf signaling are less motivated to extract glucose from the agar than normal larvae. The growth rate of Npf neuron-deficient and control larvae was compared under well-fed conditions, and no obvious abnormality in growth and development was observed (Wu, 2003).
A Drosophila NPY receptor homolog encoded by the npfr1 gene has been pharmacologically characterized to be a Npf receptor (NPFR1) (Garczynski, 2002). It was reasoned that if Npf neuron-deficient larval phenotypes are due to the specific loss of Npf action, similar phenotypes might also be expected from larvae lacking NPFR1 cells. Two independent transgenic lines containing an npfr1-gal4 construct were generated, in which a 6.6 kb npfr1 upstream sequence is fused with gal4. To localize the cells that express NPFR1, the CNS tissues of young transgenic third instars (74 hr AEL) were incubated simultaneously with mouse anti-NPFR1 and rabbit anti-Npf antiserum. The double immunostaining revealed that in the control larvae (y w × UAS-DTI), NPFR1 cells are located in the dorsomedial surface of the subesophageal and abdominal ganglia, while the Npf neurons extend their axons into the brain hemispheres as well as along the midline of the ventral nerve cord (VNC). Thus, the NPFR1 cells appear to be aligned appropriately for the reception of locally released Npf in the segmented ventral ganglia. In contrast, the larvae from the experimental crosses (npfr1-gal4-1 and -2 × UAS-DTI) showed no NPFR1 immunoreactivity. The Npf immunostaining pattern in NPFR1 neuron-deficient larvae appeared to be indistinguishable from that seen in normal y w × UAS-DTI larvae, suggesting that the Npf neural circuit remained intact. Like Npf neuron-deficient and control larvae, NPFR1 neuron-deficient larvae showed no obvious abnormality in growth and development. Tests to the feeding revealed that these larvae display the altered feeding behaviors similar to those of NPF-deficient larvae. It is therefore concluded that the regulatory activity of Npf in food response is mediated by the NPFR1 neurons, which, in turn, may modulate the activities of head and abdominal muscles involved in larval feeding and foraging (Wu, 2003).
The Npf neuron-deficient larvae showed a strong tendency to wander away from the glucose-agar disc. This behavior was examined by analyzing the effects of the loss of Npf signaling on larval locomotion in the presence and absence of food. Semisolid agarose paste was used, since it is a medium that is soft enough to reveal the track of larva movement but hard enough to prevent potential burrowing by larvae. Before the assay, 2-hr-old third instars were rinsed with water until their bodies were free of visible food particles, and food was withheld for 2 hr. Each larva was first allowed to crawl on the surface of semisolid agarose medium for 6 min. The same larva was then transferred to a 10% glucose-agarose medium and allowed to crawl for another 6 min. The track lengths of each larva left on the surface of both media were measured. About 90% of y w or control larvae (y w × UAS-DTI) crawled more actively on the food-free agarose than on the glucose agarose. In contrast, more than 90% of the Npf signaling-deficient larvae crawled more on the glucose agarose than on the food-free agarose. The average of the path lengths of the larvae from the different groups was also calculated. The average crawling distances of the four Npf or NPFR1 neuron-deficient larvae were shorter than the control larvae on food-free agarose; when Npf signaling-deficient larvae were placed on glucose agarose, however, they crawled similar or greater distances than the control larvae. These results revealed that Npf signaling-deficient larvae have no general muscle defects for movement; they are less active in foraging than normal larvae on agarose but are hyperactive in locomotion on glucose-agarose medium. The experiments also demonstrate that one of the roles of Npf in facilitating larval foraging on a poor food source is to suppress the locomotion. The effect of glucose on larval locomotion can be quantified more accurately by eliminating variations among individuals. This can be achieved by measuring the ratio of path length on glucose agarose over that on agarose of each larva (defined as food response index, FRI). Remarkably, the FRI values for experimental larvae range from 1.7 to 2.2, which are at least 2-fold higher than normal y w larvae, while the control larvae showed a FRI value similar to the wild-type, indicating that the Npf system indeed exerts a strong inhibitory effect on larval locomotion in the presence of food. It is suggested that glucose elicits a broad central excitation as well as Npf-mediated neural inhibition that normally counterbalances it, and thus loss of Npf signaling causes hypermobility upon exposure to the glucose-agarose medium (Wu, 2003).
npf expression in the CNS has been shown to be promptly downregulated at the end of the feeding phase when older third instar larvae are moving away from food. It was of interest know whether Npf signaling plays a role in modifying larval response to food. To investigate this possibility, wild-type (Canton S) young (CS 72 hr AEL) and older third instars (CS 98 hr AEL), respectively, were placed on a glucose-agar plate (45 mm in diameter) coated with a thin layer of yeast paste. The larval distribution on the plate was recorded after a period of 45 min. In a central zone that accounts for 65% of the total area, about 70% of the young third instars were found to browse evenly across the medium surface. In contrast, only about 10% of the older larvae remained in the central zone; among those animals (90%) that migrated to the periphery (bordering), 82% of them participated in clumping that involved multiple animals. Apparently, the older third instar larvae with naturally downregulated npf expression in the brain display bordering and clumping phenotypes similar to C. elegans social strains containing a less-active form of NPY receptor-like protein (de Bono, 1998). Further tests were performed with the young third instar progeny from a control and four experimental crosses (y w, npf-gal4-1, -2 and npfr1-gal4-1, -2 × UAS-DTI). The control larvae forage uniformly on the assay plate, showing no preference to aggregate. In contrast, the Npf or NPFR1 neuron-deficient larvae display bordering and clumping phenotypes similar to those of older Canton S larvae. Thus, the loss of Npf signaling is sufficient to cause the premature onset of bordering and clumping behavior (Wu, 2003).
If the natural downregulation of Npf signaling is sufficient to trigger bordering and clumping in older wild-type larvae, could these phenotypes be suppressed by overexpressing Npf? This question was addressed by crossing the UAS-npf fly with a 386Y-gal4 enhancer-trap line that drives a broad expression pattern including numerous peptidergic neurons. The third instar progeny (100 hr AEL) from the cross indeed browsed evenly across the assay plate, while control larvae (y w × UAS-npf, 100 hr AEL) showed the phenotypes of bordering and clumping. Moreover, the Npf-overexpressing larvae remain solitary throughout the third instar life. The suppression of bordering and clumping by Npf overexpression cannot be due to locomotory deficits, since the path length of 386Y-gal4 × UAS-npf larvae crawling on the water-agarose paste was similar to that of normal larvae. Therefore, it is concluded that the Npf signaling system is programmed to regulate the onset of a possible social behavior during larval development (Wu, 2003).
The Npf signaling system appears to play a central role in regulating the developmental switch from feeding to food aversion in third instar larvae. To provide further support to the notion that larvae deficient in Npf signaling prematurely display behaviors associated with older nonfeeding larvae, the food response and bordering and clumping behaviors were analyzed quantitatively in NPF or NPFR1 neuron-deficient and control larvae at three different ages (72, 84, and 96 hr AEL). The Npf signaling-deficient larvae (npf-gal4 and npfr1-gal4 × UAS-DTI) from the three different age groups displayed insensitivity to solid food (10% glucose embedded in agar) as well as strong bordering and clumping phenotypes. In contrast, younger control larvae (Canton S and y w × UAS-DTI) of 72 hr and 84 hr AEL showed sensitive response to the solid food and displayed neither bordering nor clumping. However, as expected, the control larvae did display food aversion as well as bordering and clumping at 96 hr AEL. Therefore, these results indicate that the Npf signaling system is essential for proper temporal regulation of the onset of behaviors associated with older larvae (Wu, 2003).
The bordering and clumping phenotypes of C. elegans have been suggested to reflect social foraging behaviors (de Bono, 1998). However, it is evident that the bordering and clumping behaviors of nonfeeding Drosophila larvae cannot be associated with foraging or feeding. Then what is the biological significance of the developmental activation of social behavior in Drosophila larvae? Third instars exiting the feeding phase often aggregate while burrowing into apple juice-agar. It was speculated that the social behavior might be required for larval burrowing activity. A larval burrowing assay was developed to test the hypothesis. Wild-type nonfeeding third instar larvae (CS 98 hr AEL) were placed on an apple juice-agar plate and monitored over a 60 min period. The larvae dispersed initially, began to swarm toward the periphery, and subsequently burrowed in clumps under lifted agar pieces within 20 min. The burrowing activity of Npf-overexpressing solitary larvae (386Y-gal4 × UAS-npf; 100 hr AEL) was tested under the same conditions. In all five trials, the solitary larvae displayed no social interaction and completely failed to burrow into the agar, even after 1 hr; the control larvae (y w × UAS-npf; 100 hr AEL), however, always displayed social burrowing and penetrated into the agar in less than 30 min. Approximately 70%-90% of the social larvae congregated in burrowing sites in each case, whereas no obvious burrowing was observed for the solitary 386Y-gal4 × UAS-npf larvae. The lack of burrowing by the solitary larvae is unlikely due to a deficit in agar cutting, since the same larvae, under crowded conditions, were able to cut apple juice-agar into numerous small pieces (Wu, 2003).
To better understand the larval social burrowing behavior, attempts were made to determine how social larvae initiate the burrowing process. Thirty larvae from each group were transferred onto the apple juice-agar plate, and monitored for 10 min. While the solitary 386Y-gal4 × UAS-npf larvae crawled around in a random manner, control larvae (386Y-gal4 × y w or H1-lacZ) aggregated at one or more sites. Social larvae did not show any preference to the area that was just occupied by 15 to 20 congregating larvae for about 10 min until agar digging just began. These results indicate that social larvae were able to aggregate by attracting each other well before a specific digging site(s) had been marked. It was observed that, within minutes after aggregation, a small number of larvae began to adopt a unique drilling motion; these larvae swung their bodies back and forth in a vertical, upside-down position while digging into the agar. More larvae gradually joined in digging, resulting in the lift of an agar piece. The 386Y-gal4 × UAS-npf larvae that overexpress Npf never displayed such drilling motion. Thus, the food-dependent larval aggregation appears to be critical to turn on the drilling motion which is, in turn, required for efficient penetration through the agar. The minimal time required for the onset of social burrowing (Tmin) by larvae was determined at the different population densities. The onset of burrowing is defined as the time when one or more larvae begin to engage in the drilling motion. For example, about 15 and 48 min are needed for detecting social burrowing at densities of 60 and 5 larvae per plate, respectively. These results strongly suggest that the social interaction can effectively induce larvae to initiate digging behavior. The social burrowing behavior may allow larvae to penetrate obstacles efficiently, thereby facilitating their migration from feeding sites to desirable locations to form puparia (Wu, 2003).
Whether solitary larvae overexpressing Npf and control social larvae can interact with each other was tested. Five solitary larvae (386Y-gal4 × UAS-npf) and five social larvae (386Y-gal4 × y w) were added to each plate, and the Tmin of the larvae was determined. It was found that mixing five solitary and five social larvae together significantly reduced the Tmin in comparison to that of five social larvae alone, although Tmin was still much longer than that of ten social larvae together. Therefore, this result suggests that Npf-overexpressing larvae may be normal in transmitting signals for social interaction but deficient in receiving the communication from peers. Consistent with this notion, it was also observed that solitary larvae failed to display bordering and clumping in the presence of social larvae. Interestingly, the study in C. elegans showed that solitary worms also failed to group with social worms (de Bono, 1998), suggesting that a similar sensory mechanism might be involved in both cases (Wu, 2003).
The loss of Npf signaling in early third instar larvae leads to motivational feeding deficits and food-conditioned hypermobility, suggesting that the Npf system enhances food response. In earlier experiments, it was found that Npf-overexpressing and control larvae (386Y-gal4 × UAS-npf or H1-lacZ) developed similarly during the feeding phase and have similar body sizes at 98 hr AEL. It was of interest to know how Npf overexpresssion might affect food response in older larvae that normally exhibit food aversion. Synchronized 386Y-gal4 × UAS-npf and two control larvae (w11i8 and 386Y-gal4 × H1-lacZ) were fed with blue yeast paste and monitored for their food intake, starting from 92 hr AEL. As expected, control larvae showed a normal feeding profile. The third instar larvae ceased their feeding activity when they became 1 day old (or about 96 hr AEL), and formed puparia close to the end of the second day. However, the 386Y-gal4 × UAS-npf larvae overexpressing Npf did not stop feeding until 12-24 hr later than the controls. In addition, these larvae were also late to reach puparium by about the same amount of time. Importantly, in the absence of food, the well-fed early third instars (74 hr AEL) of both 386Y-gal4 × UAS-npf larvae and 386Y-gal4 × H1-lacZ controls developed into puparia at the same rate. Taken together, these results indicate that Npf overexpression increases larval feeding time, and larvae lacking or overexpressing Npf display reciprocal phenotypes. The fact that Npf prolongs feeding in the presence but not the absence of food suggests that Npf overexpression does not destruct the mechanisms for metamorphoses per se; rather it appears to delay the onset of larval metamorphosis by maintaining the positive energy flow (Wu, 2003).
Feeding larvae lacking Npf or NPFR1 neurons displayed precocious social behavior, suggesting that two complex behaviors, foraging and social burrowing, appear to be independently regulated by the Npf system. To further address this issue, a developmental analysis of the impact of Npf overexpression was performed on the behaviors of older third instar larvae that ceased feeding. Thirty nonfeeding larvae overexpressing Npf (120 hr AEL) were placed on an apple juice-agar plate. These larvae dispersed randomly across the agar surface, displaying neither food aversion nor social burrowing, indicating that suppression of social behavior can be regulated independently, regardless of whether they are feeding or not. How the loss of food aversion and social burrowing might impact the selection of pupation sites was evaluated. It was found that the vast majority of the 386Y-gal4 × UAS-npf larvae formed puparia on the moist food-containing agar surface, whereas most control larvae picked the dry plastic surface for pupariation. Drosophila pupae, which remain immobile for 4-5 days at the ambient temperature, are highly susceptible to the killing by mold and bacterial overgrowth. Since a moist food-containing environment is much more conducive to mold and bacterial growth than a dry and food-free environment, the Npf-overexpressing flies are likely to be disadvantaged in their fitness to survive. Thus, the developmental downregulation of Npf signaling at the end of the larval feeding phase probably has a critical biological function: it initiates both aversive response to food and cooperative burrowing activity, thereby greatly increasing the odds of larval survival into adulthood (Wu, 2003).
Alcohol is likely to affect neurons nonselectively, and the understanding of its action in the CNS requires elucidation of underlying neuronal circuits and associated cellular processes. A Drosophila signaling system has been identified, comprising neurons expressing neuropeptide F (NPF, a homolog of mammalian neuropeptide Y) and its receptor, NPFR1, that acutely mediates sensitivity to ethanol sedation. Flies deficient in NPF/NPFR1 signaling showed decreased alcohol sensitivity, whereas those overexpressing NPF exhibited the opposite phenotype. Furthermore, controlled functional disruption of NPF or NPFR1 neurons in adults rapidly confers resistance to ethanol sedation. Finally, the NPF/NPFR1 system selectively mediates sedation by ethanol vapor but not diethyl ether, indicating that the observed NPF/NPFR1 activity reflects a specialized response to alcohol sedation rather than a general response to intoxication by sedative agents. Together, these results provide the molecular and neural basis for the strikingly similar alcohol-responsive behaviors between flies and mammals (Wen, 2005).
The NPF distribution in the whole adult brain was analyzed by using immunofluorescence staining. To obtain high specificity, the anti-NPF antiserum was preabsorbed against an amidated oligopeptide (C8) corresponding to the C-terminal structure of NPF. The specificity of the preabsorbed antiserum was also verified genetically, and by in situ RNA hybridization. The immunofluorescence staining revealed that NPF is prominently expressed in two pairs of neurons in the posterior side of the central brain, and NPF-positive projections display a largely symmetrical pattern that is stereotypic in both male and female brains. Extensive arborizations were observed at several regions in the posterior side of the central brain, whereas no NPF immunoactivity was detected in the optical lobes. One of the sites that exhibits intense NPF immunostaining is the fan-shaped body of the central complex, implicated in coordinating motor activities. The NPF neuronal projections also innervate contralaterally the subesophageal ganglion, which might be important for regulating feeding and walking. NPF-containing arbors were also found in two lateral areas of the lower part of the central brain that appear to harbor the giant commissural interneurons of the giant fiber pathway. These data suggest that NPF neurons might coordinately modulate diverse sensory and motor neurons important for feeding, flight, and locomotion (Wen, 2005).
It was asked whether overexpression of NPF could cause increased alcohol sensitivity. An enhancer-trap gal4 line (386Y-gal4) was chosen because it has been shown to drive reporter expression in brain cells, including peptidergic neurons. Transgenic flies containing UAS-npf driven by 386Y-gal4 showed strong ectopic expression of NPF in the adult brain and were tested for alcohol resistance. In this case, a 31% ethanol solution was used, because it conferred a weak sedative effect on control flies in earlier experiments. Because the influence of the genetic background on fly ethanol response was more pronounced at the reduced ethanol concentration, more control crosses were added to exclude other trivial reasons that might cause increased sensitivity to ethanol. Three different types of control flies were included, 386Y-gal4 X y w or H1-lacZ, and Canton S X UAS-npf, each of which contains either 386Y-gal4 or UAS-npf alone, and also provides an assessment of the effects due to variations in genetic background. Flies overexpressing NPF were shown to be highly sensitive to alcohol, even at the reduced concentration, whereas all of the control flies showed high resistance to ethanol vapor under the same condition. The ethanol sensitivity was examined of npf-gal4 X UAS-npf flies that overexpress NPF in the NPF neurons. In this experiment, all transgenic flies are in the y w background. Again, the experimental flies (npf-gal4 X UAS-npf) were much more sensitive to ethanol than were the control flies. Taken together, these results provide direct evidence that NPF is a critical component for acute modulation of ethanol response (Wen, 2005).
The NPF neuronal circuit has also been shown to respond to chemosensory stimuli by sugar, and promote feeding response. The present work demonstrates that it has a role in acute response to ethanol. Therefore, the NPF system appears to be capable of regulating fly responses to diverse external cues. It is proposed that the NPF neuronal circuit should be a useful platform for unraveling the physiological roles of genes in behavioral regulation in the context of functionally characterized neurons (Wen, 2005).
Parallel activities have been reported between mammalian NPY and Drosophila NPF in feeding regulation. This study demonstrates another functional parallel between the two signaling molecules in ethanol response (Thiele, 1998; Thiele, 2002). To many animals, including fruit flies, rodents, and humans, ethanol is a natural component in a variety of their food sources. At lower concentrations, ethanol is likely to serve as a volatile food cue. The Drosophila NPY-like signaling system has been shown to enhances the motivation of food acquisition. Therefore, it is inviting to speculate that the NPY-like system might be evolved to function as an alcohol sensor to facilitate food searching. It would be interesting to test whether NPY/NPF signaling is activated by ethanol at a low concentration (Wen, 2005).
In C. elegans, an NPY-like receptor, NPR-1, has been recently reported to suppress the development of acute tolerance of ethanol intoxication (Davies, 2003). However, the ligand of NPR-1 is FMRFamide-like, rather than NPY-like, and its role in acute alcohol sensitivity has not been reported. Therefore, future work will be needed to clarify the functional relationship among mammalian NPY, Drosophila NPF, and nematode NPR-1, and to determine whether the NPY and NPY-like systems regulate different aspects of behavioral response to alcohol (Wen, 2005).
Several NPY receptor subtypes have been identified that mediate NPY action through coupling to heterotrimeric G proteins that down-regulate the intracellular level of cAMP. Consistent with this work, pharmacological analysis has shown that binding of NPF to the NPFR1 receptor inhibits forskolin-stimulated adenylate cyclase activity in cultured cells. It is proposed that Y1- and NPFR1-mediated alcohol sensitivity in mammals and fruit flies may require down-regulation of second-messenger cAMP and subsequent reduction of protein kinase A activity (Wen, 2005).
Immunofluorescence staining reveals that NPF-positive axon arbors are widely present in the central brain. Interestingly, a 6.6-kb npfr1 promoter sequence drives GFP expression in a small number of neurons that appear to be adjacent to, or overlap with, the NPF arbors in the subesophageal and dorsolateral regions. The subesophageal ganglia have been implicated in coordinated locomotion, whereas the neurobiological role of neurons in the dorsolateral regions is not clear. It would also be interesting to determine whether ethanol-responsive NPFR1 neurons play a role in mediating the fly wake-sleep cycle (Wen, 2005).
It remains unclear how the NPF neuronal circuit responds to ethanol but not to ether. One possible explanation is that the cell membrane components of NPF neurons may selectively interact with volatile agents. For example, the binding of ethanol, but not ether, to NPF neurons may elicit an intracellular response(s) that subsequently leads to increased release of NPF and possibly other neurotransmitter(s) as well. In support of this notion, ethanol has been shown to selectively interact with the Drosophila potassium channel protein, Shaw2. It will be interesting to determine whether Shaw2 is expressed in NPF neurons, and if so, whether it might mediate the selective response to ethanol vapor. Furthermore, a number of cell membrane proteins have been biochemically identified as potential molecular targets of volatile anesthetics. The NPF neuronal circuit offers a useful platform to test the physiological relevance of these targets in alcohol response (Wen, 2005).
Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2005a).
The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2005a).
When fed ad libitum, normal larvae (w1118) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2005a).
dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6KDN, encoding a dominant negative, and UAS-dS6KACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6KDN or UAS-dS6KACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6KDN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6KACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2005a).
DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6KACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2005a).
Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInRACT and UAS-dInRDN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3KDN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInRDN, UAS-dPTEN, or UAS-dPI3KDN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInRACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2005a).
A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6KDN and UAS-dS6KACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6KDN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInRDN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6KDN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6KACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2005a).
The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2005a).
npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2005a).
A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shits1) driven by npf-gal4 or npfr1-gal4. The shits1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shits1) and paired controls (y w x UAS-shits1 and npf-gal4 and npfr1-gal4 x w1118) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2005a).
This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2005a).
The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2005a).
Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2005a).
Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2005a).
Omnivores, including humans, have an inborn tendency to avoid noxious or unfamiliar foods. Such defensive foraging behaviors are modifiable, however, in response to physiological needs. This study describes method for assessing risk-sensitive food acquisition in Drosophila. Food-deprived fly larvae become more likely to feed on noxious foods (adulterated with quinine) as the duration of deprivation increases. The neuropeptide F receptor NPFR1, a mammalian neuropeptide Y (NPY) receptor homolog, centrally regulates the response to noxious food in D. melanogaster. Overexpression of NPFR1 is sufficient to cause nondeprived larvae to more readily take in noxious food, whereas loss of NPFR1 signaling leads to the opposite phenotype. Moreover, NPFR1 neuronal activity may be directly regulated by the insulin-like signaling pathway. Upregulation of insulin-like receptor signaling in NPFR1 cells suppresses the feeding response to noxious food. These results suggest that the coordinated activities of the conserved NPY- and insulin-like receptor signaling systems are essential for the dynamic regulation of noxious food intake according to the animal's energy state (Wu, 2005b).
Drosophila larvae can be used in genetic and neurobiological analyses of various food-related behaviors. Synchronized early third-instar larvae (74 h after egg laying; 74 h AEL) feed voraciously for rapid growth. Nondeprived wild-type larvae display a strong feeding response to a liquid diet containing sugar, yeast paste and green food dyes. Addition of the noxious substance quinine to the diet, however, elicits a strong aversive response. For example, nondeprived larvae largely reject the 0.5% quinine food that they initially encounter and continue to search for new food sources. In contrast, a much larger proportion of larvae deprived for 40 or 120 min (about 30% and 80%, respectively) continued to feed in the 0.5% quinine food. Thus, the larval response to noxious food appears to be a useful model for assessing risk tolerance associated with food/reward seeking (Wu, 2005b).
A neuronal circuit consisting of four to six NPF neurons and a small number of NPFR1 neurons in the larval CNS shows neural plasticity in response to chemosensory stimuli, and it promotes deprivation-motivated feeding behaviors. To test whether NPF and NPFR1 neurons play a role in the larval response to noxious food, a controlled disruption of NPF/NPFR1 neuronal activities was performed in feeding larvae. Two drivers, npf-gal4 and npfr1-gal4, were used to direct expression of a temperature-sensitive allele of shibire (shits1), which encodes a semidominant-negative form of dynamin capable of blocking neurotransmitter release at a restrictive temperature (>29°C). At 23°C, nondeprived control larvae (y w;UAS-shits1, w;npf-gal4 and w;npfr1-gal4) show a gradual reduction in feeding response as the quinine concentration increases from 0 to 0.05% to 0.2% and finally to 0.5%. Their feeding responses to quinine foods remains the same even at the restrictive temperature of 30°C. Nondeprived experimental larvae (npf-gal4;UAS-shits1 and npfr1-gal4;UAS-shits1) behave much like controls, except for a small but significant reduction in feeding response by the npf-gal4;UAS-shits1 larvae at 30°C, suggesting that the NPF pathway may be weakly active in nondeprived larvae. In control larvae that were food deprived for 120 min at both 23°C and 30°C, at least 70% showed enhanced feeding in the 0.5% quinine diet, whereas none of their nondeprived counterparts did at either temperature. Such deprivation-induced intake of 0.5% quinine food is drastically reduced in both npf-gal4;UAS-shits1 and npfr1-gal4;UAS-shits1 larvae after a 15-min incubation at 30°C, but it remains normal at the permissive temperature of 23°C. These findings suggest that the NPF/NPFR1 neuronal circuit is centrally involved in suppressing aversion to noxious foods in deprived animals. The data also provide functional evidence that NPF release may be coupled to a dynamin-mediated process. Consistent with this notion, dynamin has been implicated in the formation of transport vesicles from the trans-Golgi network. It has also been reported that pharmacological inhibition of norepinephrine uptake by neurons that release both NPY and norepinephrine markedly reduces NPY release (Wu, 2005b).
To determine whether NPF and NPFR1 directly regulate aversive responses to noxious food, npfr1 was selectively knocked down by expressing its double-stranded RNA (dsRNA) in NPFR1 cells, as well as in the nervous system. A UAS-npfr1dsRNA line was used that has been shown to cause reduced npfr1 expression and NPFR1 signaling deficiency. Nondeprived experimental larvae (npfr1-gal4;UAS-npfr1dsRNA, elav-gal4;UAS-npfr1dsRNA or appl-gal4;UAS-npfr1dsRNA) and controls (for example, w;npfr1-gal4 and y w;UAS-npfr1dsRNA) shows similar responses to quinine foods. However, after 2 h of food deprivation, the experimental, but not the control, larvae showed attenuated feeding responses that were virtually identical to those seen in npf-gal4;UAS-shits1 and npfr1-gal4;UAS-shits1 larvae at 30°C. For example, about 80% of the control larvae ate the 0.5% quinine food, whereas less than 40% of the experimental larvae did. Therefore, these findings further indicate that neural NPFR1 is critical for regulating aversive responses to noxious food (Wu, 2005b).
The potential effect of overexpressing an npfr1cDNA in NPFR1 neurons on feeding of quinine-containing foods was also tested. Nondeprived control larvae (e.g., y w;UAS-npfr1) rejected the 0.5% quinine food. In contrast, at least 50% of the nondeprived experimental larvae (npfr1-gal4;UAS-npfr1) displayed active feeding in the same food. Furthermore, experimental and control larvae deprived for 120 min also showed significant feeding of food containing 0.2% or 0.5% quinine. When the quinine concentration was increased to 0.8%, significantly more NPFR1-overexpressing larvae (>50%) remained feeding relative to controls . Thus, larvae overexpressing NPFR1 show opposite feeding behavior phenotypes compared with larvae deficient in NPF/NPFR1 signaling (Wu, 2005b).
It was postulated that NPFR1 neurons may centrally mediate deprivation-induced intake of noxious food. To test this idea, the role of Drosophila p70 ribosomal S6 kinase (dS6K), a cell-autonomous effector of nutrient-sensing pathways, was evaluated in NPFR1 neurons. The npfr1-gal4 driver was used to direct two transgenes, UAS-dS6KDN and UAS-dS6KACT, which encode a dominant-negative and constitutively active form of dS6K, respectively. In the absence of food deprivation, npfr1-gal4;UAS-dS6KDN larvae still ate the 0.5% quinine-adulterated food, similar to npfr1-gal4;UAS-npfr1 larvae. Moreover, npfr1-gal4;UAS-dS6KDN larvae ate the 0.8% quinine-containing food under deprivation. In contrast, food-deprived larvae overexpressing dS6K (npfr1-gal4;UAS-dS6KACT) showed a decrease in feeding response to the 0.5% quinine food. Taken together, these results suggest that dS6K is essential for the transduction of hunger stimuli in NPFR1 neurons (Wu, 2005b).
It has been shown that dS6K is a downstream effector of the Drosophila insulin-like receptor (dInR) pathway. Therefore, whether dInR signaling regulates the NPFR1 pathway was examined. A dominant-negative and a constitutively active form of dInR (dInRDN and dInRACT, respectively) were expressed under the control of npfr1-gal4. Without food deprivation, more than 40% of the npfr1-gal4;UAS-InRDN larvae showed a feeding response to the 0.5% quinine food. In contrast, less than 10% of control larvae showed a feeding response to the same food. Moreover, like controls, the npfr1-gal4;UAS-InRACT larvae showed no feeding response. Phosphatidylinositol 3-kinase (dPI3K) and phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN) are key components of the dInR pathway. Disruption of dInR signaling by overexpressing dPTEN or a dominant-negative form of dPI3K also led to increased feeding response to noxious food by nondeprived larvae. Under food-deprived conditions, dInR/dPI3K signaling-deficient larvae showed significantly stronger feeding response to 0.8% quinine than did control larvae. Conversely, deprived larvae expressing dInRACT or Dp110 (encoding a catalytic subunit of dPI3K) in NPFR1 neurons showed a reduced feeding response to 0.5% quinine food relative to paired controls. These results suggest that dInR signaling is critical for transducing hunger stimuli in NPFR1 neurons (Wu, 2005b).
Among the seven insulin-like genes, dilp2 is most abundantly expressed in two clusters of neurosecretory cells in the larval CNS (Rulifson, 2002). dilp2 was overexpressed in the nervous system using neuron-specific elav-gal4. Nondeprived elav-gal4;UAS-dilp2 larvae showed a normal response to foods containing various concentrations of quinine. The same larvae deprived for 120 min, however, showed significantly attenuated feeding at 0.5% quinine concentration in comparison with controls. Taken together, these results suggest that DILP2 negatively regulates the signaling activity of NPFR1 neurons (Wu, 2005b).
The overexpression of NPFR1 or dS6K in NPFR1 cells has a dominant but opposite effect on larval response to noxious food. To delineate the functional relationship between the dS6K and NPFR1 signaling pathways, UAS-dS6KDN and UAS-npfr1dsRNA were simultaneously expressed using the npfr1-gal4 driver. The experimental and control larvae were individually assayed for their feeding response to quinine diets under deprived and nondeprived conditions. As expected, about 50% of the npfr1-gal4;UAS-dS6KDN larvae showed a feeding response to the 0.5% quinine food. In contrast, more than 80% of the controls as well as larvae coexpressing dS6KDN and npfr1dsRNA rejected the same food. Under deprived conditions, the npfr1-gal4;UAS-dS6KDN larvae also showed a stronger feeding response to 0.8% quinine food than did larvae coexpressing npfr1dsRNA and UAS-dS6KDN (Wu, 2005b).
In parallel, UAS-dS6KACT and UAS-npfr1 were also coexpressed using the npfr1-gal4 driver. Under nondeprived conditions, more than 70% of the npfr1-ga4;UAS-npfr1 larvae as well as larvae that coexpress dS6KACT and npfr1 cDNA showed significant feeding activity in the 0.5% quinine food; in contrast, virtually all of the control larvae as well as npfr1-gal4;UAS-dS6KACT larvae rejected the same food. Therefore, nondeprived larvae overexpressing npfr1 cDNA in NPFR1 cells alone or together with dS6KACT exhibit similar responses to noxious food. The same groups of larvae were also tested under deprived conditions. About 50% of the npfr1-ga4;UAS-npfr1 as well as larvae coexpressing npfr1 cDNA and dS6KACT showed feeding response to 0.8% quinine food, whereas virtually all of the npfr1-gal4;UAS-dS6KACT larvae rejected the same food. Taken together, these results indicate that NPFR1 activity exerts a dominant effect over that of dS6K in NPFR1 cells (Wu, 2005b).
This study demonstrates a previously uncharacterized role of the insulin signaling pathway in regulating aversive response to noxious food. A model is proposed to illustrate how DILP and NPF neural signaling pathways coordinately regulate feeding in the presence of noxious food. NPFR1 neurons respond to two extracellular cues, DILPs and NPF. DILP/dInR signaling is likely responsible for transducing hunger stimuli in NPFR1 neurons through dS6K, which in turn negatively regulates the NPFR1 signaling pathway. Furthermore, the observation that NPFR1 overexpression is sufficient to override the inhibitory effect of constitutively active dS6K suggests that dS6K may suppress directly or indirectly the activity of NPFR1 (Wu, 2005b).
It remains to be determined how NPFR1 neurons regulate the tolerance of noxious food. One possibility is that the NPF/NPFR1 system might be part of the neural circuit regulating risky behaviors. Consistent with this idea, NPY, the mammalian homolog of NPF, is involved in suppressing anxiety and fear (Thorsell, 2000). For example, NPY knockout mice show less activity when placed in the center of an open field and an increased startle response to auditory stimuli (Thorsell, 2000; Bannon, 2000). Future experiments will be needed to test whether the NPY and insulin receptor pathways coordinately regulate risk-sensitive foraging behavior in mammals. In contrast, the NPF/NPFR1 system, which is upregulated in deprived animals, may strengthen the reinforcing effect (reward value) of food, thereby reducing the avoidance of noxious stimuli (Levine, 2003; Levine, 2004). For example, in the absence of food deprivation, NPFR1 overexpression is sufficient to trigger motivated feeding on less-accessible solid medium, which is ordinarily rejected by normal larvae (Wu, 2005b). Given its prominent role in behavioral adaptation to adverse feeding conditions, the insulin/NPY-like signaling network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective life styles (Wu, 2005b).
Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner [IC(50) = 65 nM]. DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly [IC(50) = 51 nM]. Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF (Garczynski, 2002).
Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, the activation of GPCRs in living cells can be visualized. This approach was used to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. The identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (neuropeptide F receptor, abbreviated as NPFR1 or CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702), were identified. Also CG6515 and CG7887 were de-orphaned by showing that these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. CG14484, encoding a receptor related to vertebrate bombesin receptors, respond specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, it was shown that dromyosuppressin also stimulates GTPgammaS binding and inhibits cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors (Johnson, 2003).
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