Drosophila Neuropeptide F (Npf), a human NPY homolog, coordinates larval behavioral changes during development. The brain expression of npf is high in larvae attracted to food, whereas its downregulation coincides with the onset of behaviors of older larvae, including food aversion, hypermobility, and cooperative burrowing. Loss of Npf signaling in young transgenic larvae leads to the premature display of behavioral phenotypes associated with older larvae. Conversely, Npf overexpression in older larvae prolongs feeding, and suppresses hypermobility and cooperative burrowing behaviors. The neuropeptide F receptor (NPFR1) has also been characterized, and has been found to be expressed in a pair of dorsolateral neurons in the central brain and a small number of neurons in the subesophageal ganglion (Garczynski, 2002; Wen, 2005). The Npf system provides a new paradigm for studying the central control of cooperative behavior (Wu, 2003).

Neuropeptide Y (NPY), a conserved 36 amino acid neuromodulator, is enriched in the hypothalamus that is responsible for the central regulation of feeding in vertebrates (Beck, 2001; Williams, 2001). Pharmacological studies have implicated hypothalamic NPY as a prominent stimulator for appetitive behavior. Chronic administration of NPY in the paraventricular nucleus induces uncontrolled food intake in rats, which subsequently develop severe obesity (Stanley, 1986). In a leptin-deficient (ob/ob) mouse model, loss of NPY attenuates the obesity syndrome (Erickson, 1996a). However, the physiological role of NPY in feeding regulation has been difficult to establish. In most cases, NPY knockout mice displayed no obvious reduction in food intake under either well-fed or fasting conditions and had normal body weight (e.g., Erickson, 1996b and Qian, 2002). Recently, the NPY-deficient mice in a C57BL/6 background were shown to exhibit mild obesity (Segal-Lieberman, 2003). Transgenic rodents overexpressing NPY also showed no significant difference in food intake and body weight from the control counterparts (Thiele, 1998; Inui, 2000; Thorsell 2000), except in one case where NPY-overexpressing mice exhibited mild obesity after 50% sucrose feeding (Kaga, 2001). Moreover, the phenotypes of transgenic mice lacking NPY receptor subtypes, Y1, Y2, or Y5, have not provided clear support for a role of the NPY system in promoting food intake and body weight, as predicted by the pharmacological data (Naveilhan, 1999; Marsh, 1998 and Pedrazzini, 1998). Thus, much work is still needed to determine the physiological significance of NPY in feeding behavior (Wu, 2003).

It has been postulated that NPY might play a motivational role in foraging behavior (de Bono, 2002; Tecott, 1998). Genetic and pharmacological studies have provided consistent evidence for a role of NPY in suppressing anxiety, fear, and responsiveness to aversive/stress stimuli (Thorsell, 2002; Wahlestedt, 1993; Palmiter, 1998; Bannon, 2000; El Bahh, 2001; Li 2002). For example, NPY knockout mice exhibited less center activity in an open-field test and increased startling response to an acoustic stimulus (Bannon, 2000), whereas mice overexpressing NPY showed increased tolerance to stress and lack of fear suppression of behavior (Thorsell, 2000). Interestingly, mice injected with NPY are more willing to work for food and display increased tolerance to the aversive taste of quinine-adulterated milk (Jewett, 1995; Flood, 1991). These properties of NPY appear to bode well for its speculated role in promoting food searching and acquisition, especially under adverse conditions (Wu, 2003).

Drosophila might be a simpler genetic model for studying the physiological role of the NPY system. The Drosophila genome contains a single coding sequence for the NPY homolog, Npy. The Npy neuronal network has been characterized in the CNS of Drosophila larvae (Shen, 2001; Brown 1999). The Npf neural system comprises four to six Npf neurons located in the brain and subesophageal ganglia. In response to chemosensory stimulation by sugar, the Npf neuronal circuit undergoes long-term, dose-dependent modifications through npf activation and an increase in the number of Npf-positive varicosities. These properties of the Npf neurons support its potential role in the regulation of food-related behaviors. Although an NPY homolog has not been identified in C. elegans, a genetic study has implicated a conserved NPY-like signaling system in regulating food-conditioned foraging behavior in the worm (de Bono, 1998). Natural isolates of the nematode display either solitary or social foraging. The solitary foragers browse slowly across a bacterial lawn, while the social foragers move rapidly toward the edge of a bacterial lawn and aggregate into clumps. Remarkably, a single nucleotide substitution in a putative NPY receptor-like gene, npr-1, is sufficient to account for the two distinct foraging patterns (Wu, 2003).

This study reports that the npf gene is highly expressed in larvae attracted to food but is turned off in older larvae that exhibit food aversion, increased mobility, food-dependent clumping, and cooperative burrowing. Transgenic larvae deficient in Npy signaling precociously exhibit the phenotypes of food aversion and social behaviors normally displayed by older nonfeeding larvae. Conversely, Npy overexpression in the larval CNS prolongs the feeding activity and suppresses the social behavior in older larvae. Evidence is provided that one of the physiological roles of the Npy-like system is to sustain larval foraging activity under adverse feeding conditions. Moreover, there is a striking parallel between the food response and social behavior of larvae deficient in Npf signaling and C. elegans lacking an NPY receptor-like gene (de Bono, 1998). These results indicate that the conserved Npf signaling system is developmentally programmed to modify foraging and social behavior in Drosophila larvae (Wu, 2003).

Targeted ablation of Npf and NPFR1 neurons using an attenuated diphtheria toxin (DTI) has proven to be effective for the dissection and functional characterization of the Npf neuropeptide signaling cascade. This study compared the behavioral phenotypes between larvae deficient in NPF or NPFR1 neurons and larvae that selectively express tetanus toxin light chains in Npf neurons or double-stranded npfr1 RNA in NPFR1 neurons. Blocking of neurotransmission by Npf neurons or disrupting npfr1 function through RNA interference each altered foraging and social behaviors in patterns similar to those of Npf or NPFR1 neuron-deficient larvae (Wu, 2003).

Npf signaling is developmentally regulated to switch on/off two opposing complex behaviors related to food: foraging and food aversion. The npf expression in the brain is strong in feeding larvae, and the loss of Npf signaling leads to the phenotypes of premature insensitivity in feeding response, food-conditioned hypermobility, and precocious social interaction. These behavioral phenotypes display a striking resemblance to those of C. elegans strains lacking an NPY receptor-like gene (de Bono, 1998). Conversely, in older nonfeeding larvae, the brain expression of npf is developmentally downregulated, and ectopic expression of a npf cDNA can delay larval entry into the nonfeeding phase and suppress the food aversion and cooperative burrowing behaviors normally displayed by these older larvae. These results demonstrate that the conserved NPY signaling system modulates foraging and social behavior in flies and likely in worms as well. Interestingly, Drosophila rover larvae, which have shown elevated activity of a for-encoded cGMP-dependent protein kinase, also exhibit similar behavioral responses to food (Osborne, 1997). Like Npf signaling-deficient larvae, the rovers show no reduction in locomotion on a food surface versus a food-free surface. It is possible that the for product might be a component of the Npf signaling pathway. In this regard, it would be interesting to know if for is expressed in NPFR1 neurons. It was recently reported that the increase of for expression is associated with honeybee transition from hive work to foraging activity (Ben-Shahar, 2002). It is suggested that the conserved NPY system may regulate foraging and social behavior in many different animals (Wu, 2003).

The burrowing behavior of nonfeeding Drosophila larvae is genetically programmed and is often unique to different species. This study shows that individual larvae of D. melanogaster work cooperatively to dig through apple juice-agar in search of food-free sites suitable for pupation. Cooperative social behaviors have been observed across diverse species. Such cooperation provides the members of an animal group unique superiorities in foraging, feeding, self-defense, and aggression that are otherwise impossible to achieve by one or a few animals. The Npf system provides an excellent model for studying the central control of cooperative social behavior within an animal group (Wu, 2003).

It is revealing to compare how Drosophila and C. elegans have exploited the use of the conserved NPY signaling system to their respective advantages. In Drosophila larvae, Npf signaling is dynamically regulated during development, thereby providing a temporal mechanism to restrict the onset of social behavior in nonfeeding larvae. The downregulation of C. elegans NPY signaling was achieved through a more permanent mechanism by genetically mutating a putative NPY receptor-like gene. Furthermore, in Drosophila larvae, the increased social interaction can lead to more effective burrowing and therefore a better chance of survival to the adult stage. However, the social variants of C. elegans may have adopted the social behavior as a part of an evolutionarily advantageous feeding strategy. It is suggested that the NPY signaling system may regulate innate social behaviors associated with different biological functions on either a short- or long-term basis in diverse organisms. How does the Npf system regulate larval social behavior? It is possible that the loss of Npf signaling might lead to the synthesis of a chemotactant(s) that is secreted by larvae while burrowing or crawling on food. Alternatively, the Npf signaling could suppress larval response to the chemical cue(s) that triggers larval social interaction. Solitary larvae overexpressing Npf can facilitate cooperative digging by social larvae; in contrast, solitary larvae are unable to display bordering and clumping behaviors even in the presence of social larvae. These observations appear to support a role for Npf in blocking larval response to social signals (Wu, 2003).

The food-conditioned grouping by C. elegans has been shown to involve nociceptive neurons that detect an aversive signal(s) from bacteria (food to the worm) as well as other antagonistic sensory neurons (de Bono, 2002; Coates, 2002). These results suggest that similar neuronal circuits could also operate in Drosophila. The social behavior of Drosophila larvae is food dependent. On water-agar surface, social larvae do not display the bordering and clumping phenotypes. Moreover, the social behavior is normally turned on in older third instar larvae exiting the feeding phase and beginning to seek a food-free surface. Thus, like in C. elegans, an aversive stimulus from food appears to be needed to initiate the social behavior. Evidence is provided that the activity of the Npf system is necessary and sufficient to suppress the onset of food aversion and social interaction by Drosophila larvae. In mammals, NPY exerts neuronal inhibitory activity and reduces sensitivity to nociceptive stimulation (Erickson 1996b; El Bahh, 2001; Li, 2002). It is suggestd that Npf may also exert inhibitory effects on the sensory circuits that transduce aversive/stress stimuli and chemical cues for triggering social interaction. Further work will be needed to determine whether the regulation of social behavior in Drosophila larvae involves a complex network of sensory neuronal pathways similar to that in C. elegans (Wu, 2003).

This study has provided evidence for the physiological role of Npf in regulating food-related behaviors. The larvae lacking or overexpressing Npf display two reciprocal feeding behavioral phenotypes. Drosophila larvae deficient in Npf signaling displayed normal baseline feeding, similar to NPY knockout mice. The intake rate of liquid food (e.g., yeast paste) was also similar between Npf neuron-deficient and control larvae. These observations indicate that the NPY system is not an essential component of the basal feeding machinery in both vertebrates and invertebrates. However, fasting larvae ablated of Npf neurons exhibit deficits in foraging; these larvae are much less motivated in extracting food from solid agar than their control counterparts. Although the role of mammalian NPY in motivational feeding is still controversial, two recent reports have suggested that NPY knockout mice in a C57BL/6 background also had reduced feeding after prolonged fasting (Segal-Lieberman, 2003; Bannon, 2000). The mouse NPY receptor Y1 is widely distributed in the brain and has been implicated in fast-induced hyperphagia (Pedrazzini, 1998). In Drosophila, the activity of NPFR1 neurons is essential for motivational feeding under deprived conditions. These observations suggest that NPFR1 may have a role parallel to the mammalian Y1 receptor. Some important neurological deficits of mice lacking NPY activity include increased anxiety and seizure susceptibility (Erickson, 1996b; Wahlestedt, 1993; El Bahh, 2001). The glucose-induced hyperactivity of Npf neuron-deficient larvae may also be caused by the loss of Npf-mediated neuronal inhibition. Consistent with this notion, Npf overexpression suppresses the food aversion behavior normally associated with nonfeeding larvae, in which the npf expression is downregulated. In summary, there is now substantial evidence for the functional conservation of the NPY signaling system in Drosophila and mammals, further validating the use of Drosophila as a model for studying molecular and neural mechanisms underlying behavior control (Wu, 2003).

The larvae deficient in Npf signaling offered a unique opportunity to examine how sugar impacts the nervous system. It is somewhat puzzling that, although Npf neuron-deficient larvae are hyperactive on solid glucose-agarose medium, they do feed normally on glucose-containing liquid food. However, these apparently conflicting observations can be best explained by the fact that Npf neuron-deficient larvae still have a Npf-independent mechanism that can effectively suppress larval locomotion while engaging in food uptake. Previous reports by others indicate that sugar can induce a central excitatory state in flies that enhances their feeding activity. In Npf neuron-deficient larvae, however, the excitatory effect of glucose is excessive and detrimental to feeding activity. Apparently, the Npf-mediated neural inhibition is also food dependent, since Npf neuron-deficient larvae behaved normally on water-agar. The Npf action may be essential to refine and limit food-elicited excitatory effects to intended action sites (e.g., muscles required for food intake). Such excitation-inhibition interplay is perhaps a general mechanism underlying the neural control of feeding responses in metazoans (Wu, 2003).

GENE STRUCTURE

cDNA clone length - 493

Bases in 5' UTR - 74

Exons - 3

Bases in 3' UTR - 113

PROTEIN STRUCTURE

Amino Acids - 102

Structural Domains

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila melanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF is expressed in the brain and midgut of fly larvae and adults (Brown, 1999).

date revised: 30 May 2006 neuropeptide F: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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