Gene name - neuropeptide F
Cytological map position - 89D5
Function - peptide hormone
Symbol - npf
FlyBase ID: FBgn0027109
Genetic map position - 3R
Classification - neuropeptide Y family
Cellular location - secreted
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).
Behavioral expression of food-associated memory in fruit flies is constrained by satiety and promoted by hunger, suggesting an influence of motivational state. This study identified a neural mechanism that integrates the internal state of hunger and appetitive memory. Stimulation of neurons that express neuropeptide F (dNPF), an ortholog of mammalian NPY, mimics food deprivation and promotes memory performance in satiated flies. Robust appetitive memory performance requires the dNPF receptor in six dopaminergic neurons that innervate a distinct region of the mushroom bodies. Blocking these dopaminergic neurons releases memory performance in satiated flies, whereas stimulation suppresses memory performance in hungry flies. Therefore, dNPF and dopamine provide a motivational switch in the mushroom body that controls the output of appetitive memory (Krashes, 2009).
Drosophila can be efficiently trained to associate odorants with sucrose reward. Importantly, fruit flies have to be hungry to effectively express appetitive memory performance (Krashes, 2008). This apparent state dependence implies that signals for hunger and satiety may interact with memory circuitry to regulate the behavioral expression of learned food-seeking behavior. The mushroom body (MB) in the fly brain is a critical site for appetitive memory. Synaptic output from the MB α′β′ neurons is required to consolidate appetitive memory whereas output from the αβ subset is specifically required for memory retrieval (Krashes, 2007; Krashes, 2008). This anatomy provides a foundation for understanding neural circuit integration between systems representing a motivational state and those for memory (Krashes, 2009).
Neuropeptide Y (NPY) is a highly conserved 36 amino acid neuromodulator that stimulates food-seeking behavior in mammals. NPY messenger RNA (mRNA) levels are elevated in neurons in the arcuate nucleus of the hypothalamus of food-deprived mice. Most impressively, ablation of NPY-expressing neurons from adult mice leads to starvation. NPY exerts its effects through a family of NPY receptors and appears to have inhibitory function. NPY therefore must repress the action of inhibitory pathways in order to promote feeding behavior. Drosophila neuropeptide F (dNPF) is an ortholog of NPY, which has a C-terminal amidated phenylalanine instead of the amidated tyrosine in vertebrates. Evidence suggests that dNPF plays a similar role in appetitive behavior in flies. dNPF overexpression prolongs feeding in larvae and delays the developmental transition from foraging to pupariation. Furthermore, overexpression of a dNPF receptor gene, npfr1, causes well-fed larvae to eat bitter-tasting food that wild-type larvae will only consume if they are hungry (Krashes, 2009 and references therein).
This study exploited dNPF to identify a neural circuit that participates in motivational control of appetitive memory behavior in adult fruit flies. Stimulation of dNPF neurons promotes appetitive memory performance in fed flies, mimicking the hungry state. npfr1 is required in dopaminergic (DA) neurons that innervate the MB for satiety to suppress appetitive memory performance. Directly blocking the DA neurons during memory testing reveals performance in fed flies, whereas stimulating them suppresses performance in hungry flies. These data suggest that six DA neurons are a key module of dNPF-regulated circuitry, through which the internal motivational states of hunger and satiety are represented in the MB (Krashes, 2009).
It is critical to an animal's survival that behaviors are expressed at the appropriate time. Motivational systems provide some of this behavioral control. Apart from the observation that motivational states are often regulated by hormones or neuromodulatory factors, little is known about how motivational states modulate specific neural circuitry. Hungry fruit flies form appetitive long-term memory, after a 2 min pairing of odorant and sucrose, and memory performance is only robust if the flies remain hungry (Krashes, 2008). Therefore, this paradigm includes key features of models for motivational systems: the conditioned odor provides the incentive cue predictive of food, there is a learned representation of the goal object (odorant/sucrose), and the expression of learned behavior depends on the internal physiological state (hunger and not satiety). This study identified a neural circuit mechanism that integrates hunger/satiety and appetitive memory (Krashes, 2009).
The signals that ordinarily control dNPF-releasing neurons is unknown. In mammals, NPY-expressing neurons are a critical part of a complex hypothalamic network that regulates food intake and metabolism. In times of adequate nutrition, NPY-expressing neurons are inhibited by high levels of leptin and insulin that are transported into the brain after release from adipose tissue and the pancreas (Figlewicz, 2009). In hungry mice, leptin and insulin levels fall, leading to loss of inhibition of NPY neurons. Flies do not have leptin, but they have several insulin-like peptides, that may regulate dNPF neurons. Some NPY-expressing neurons are directly inhibited by glucose (Levin, 2006). Fly neurons could sense glucose with the Bride of Sevenless receptor (Kohyama-Koganeya, 2008). In blowflies, satiety involves mechanical tension of the gut and abdomen. Lastly, it will be interesting to test the role of other extracellular signals implicated in fruit fly feeding behavior, including the Hugin and Take-out neuropeptides (Krashes, 2009).
NPY inhibits synaptic function in mammals, and the data from this study suggest that dNPF promotes appetitive memory performance by suppressing inhibitory MB-MP neurons [named according to the regions of the MB that they innervate: medial lobe and pedunculus (MP)]. A model is proposed in which MB-MP neurons gate MB output. Appetitive memory performance is low in fed flies because the MB αβ and γ neurons are inhibited by tonic dopamine release from MB-MP neurons. Hence, when the fly encounters the conditioned odorant during memory testing, the MB neurons encoding that olfactory memory respond, but the signal is not propagated beyond the MB because of the inhibitory influence of MB-MP neurons. However, when the flies are food deprived, dNPF levels rise, and dNPF disinhibits MB-MP neurons, and other circuits, through the action of NPFR1. dNPF disinhibition of the MB-MP neurons opens the gate on the MB. Therefore, when hungry flies encounter the conditioned odorant during memory testing, the relevant MB neurons are activated and the signal propagates to downstream neurons, leading to expression of the conditioned behavior (Krashes, 2009).
Satiety and hunger are not absolute states. Sometimes above-chance performance scores are observed in fed flies, and shorter periods of feeding after training suggest that inhibition of performance is graded. This could be accounted for by a competitive push-pull inhibitory mechanism between dNPF and MB-MP neurons (Krashes, 2009).
By gating the MB through the MB-MP neurons, hunger and satiety are likely affecting the relative salience of learned odor cues in the fly brain. However, MB-MP neurons are unlikely to change the sensory representation of odor in the MB because flies trained with stimulated MB-MP neurons perform normally when tested for memory without stimulation. Therefore, odors are likely perceived the same irrespective of MB-MP neuron activity. Furthermore, the MB-MP neurons did not affect naive responses to the specific odorants used. It will be interesting to test whether MB-MP neurons change responses to other odorants and/or modulate arousal, visual stimulus salience, and attention-like phenomena (Krashes, 2009).
There are eight different morphological classes of DA neurons that innervate the MB (Mao, 2009), and the current data imply functional subdivision. Previous studies concluded that DA neurons convey aversive reinforcement (Krashes, 2009).
This study specifically manipulated the MB-MP DA neurons. MB-MP neurons are not required for acquisition of aversive olfactory memory, consistent with a distinct function in controlling the expression of appetitive memory. Since several studies have implicated the MB α lobe in memory, other DA neurons in protocerebral posterior lateral 1 (PPL1) that innervate the α lobe (like those labeled in MBGAL80;krasavietz) may provide reinforcement. The MB-MP neurons may also be functionally divisible and independently regulated to gate MB function. The idea that a specific DA circuit restricts stimulus-evoked behavior is reminiscent of literature tying dopamine to impulse control in mammals. Previous studies of DA neurons in Drosophila have simultaneously manipulated all, or large numbers of DA neurons. The current data suggest that the DA neurons should be considered as individuals or small groups (Krashes, 2009).
Flies have to be hungry to efficiently acquire appetitive memory, but whether this reflects a state-dependent neural mechanism or results from the failure to ingest enough sugar is unclear. Stimulation of MB-MP neurons in hungry flies did not impair appetitive memory formation, and therefore MB-MP neurons are unlikely to constrain learning in fed flies. Other dNPF-regulated neurons may provide this control since NPY has been implicated in learning (Krashes, 2009).
The dNPF-expressing neurons innervate broad regions of the brain and may simultaneously modulate distinct neural circuits to promote food seeking. MB-MP neurons represent a circuit through which the salience of learned food-relevant odorant cues is regulated by relative nutritional state. Given the apparent role of the MB as a locomotor regulator, MB-MP neurons may also generally promote exploratory behavior. There are likely to be independent circuits for other elements of food-seeking behavior including those that potentiate gustatory pathway sensitivity and promote ingestion (Krashes, 2009).
NPY stimulates feeding but inhibits sexual behavior in rats. Modulators exerting differential effects could provide a neural mechanism to establish a hierarchy of motivated states and coordinate behavioral control. dNPF may potentiate activity in food seeking-related circuits while suppressing circuits required for other potentially competing behaviors, e.g., sexual pursuit (Krashes, 2009).
This study has provided the first multilevel neural circuit perspective for a learned motivated behavior in fruit flies. The work demonstrates a clear state-dependence for the expression of appetitive memory. Odorants that evoke conditioned appetitive behavior in hungry flies are ineffective at evoking appetitive behavior in satiated flies. Therefore, the fly brain is not simply a collection of input-output reflex units and includes neural circuits through which the internal physiological state of the animal establishes the appropriate context for behavioral expression (Krashes, 2009).
It has been proposed that a satiated fly receives maximum inhibitory feedback so that sensory input is behaviorally ineffective. As deprivation increases inhibition wanes and sensory input becomes increasingly effective in initiating feeding. The current data provide experimental evidence that this prediction is also likely to be accurate for expression of appetitive memory in the fruit fly where the mechanism involves neuromodulation in the central brain. The DA MB-MP neurons inhibit the expression of appetitive memory performance in satiated flies, whereas dNPF disinhibits the MB-MP neurons in food-deprived flies. The likelihood that appetitive behavior is triggered by the conditioned odorant is therefore determined by the competition between inhibitory systems in the brain. The concept that continuously active inhibitory forces in the insect brain control behavioral expression has also be proposed many years ago. This study provides evidence that these neurons exist and that their hierarchical arrangement is a key determinant of behavioral control (Krashes, 2009).
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
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