InteractiveFly: GeneBrief

neuropeptide F: Biological Overview | Regulation | Developmental Biology | Evolutionary Homologs | References


Gene name - neuropeptide F

Synonyms -

Cytological map position - 89D5

Function - peptide hormone

Keywords - foraging behavior, locomotory behavior, neuropeptide hormone, larval feeding behavior

Symbol - NPF

FlyBase ID: FBgn0027109

Genetic map position - 3R

Classification - neuropeptide Y family

Cellular location - secreted



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Rohwedder, A., Selcho, M., Chassot, B. and Thum, A. S. (2015). Neuropeptide F neurons modulate sugar reward during associative olfactory learning of Drosophila larvae. J Comp Neurol [Epub ahead of print]. PubMed ID: 26234537
Summary:
All organisms continuously have to adapt their behavior according to changes in the environment in order to survive. Experience-driven changes in behavior are usually mediated and maintained by modifications in signaling within defined brain circuits. Given the simplicity of the larval brain of Drosophila and its experimental accessibility on the genetic and behavioral level, this study has analyzed if neuropeptide F (dNPF) neurons are involved in classical olfactory conditioning. dNPF is an ortholog of the mammalian neuropeptide Y, a highly conserved neuromodulator that stimulates food-seeking behavior. This study provides a comprehensive anatomical analysis of the dNPF neurons on the single cell level. Artificial activation of dNPF neurons inhibits appetitive olfactory learning by modulating the sugar reward signal during acquisition. No effect is detectable for the retrieval of an established appetitive olfactory memory. The modulatory effect is based on the joint action of three distinct cell types that, if tested on single cell level, inhibit and invert the conditioned behavior. Taken together, this work describes anatomically and functionally a new part of the sugar reinforcement signaling pathway for classical olfactory conditioning in Drosophila larvae.

Slade, J. D. and Staveley, B. E. (2016). Manipulation of components that control feeding behavior in Drosophila melanogaster increases sensitivity to amino acid starvation. Genet Mol Res 15. PubMed ID: 26909968
Summary:
Feeding is a complex behavior that is regulated by several internal mechanisms. Neuropeptides are able to survey quantities of stored energy and inform the organism if nutrient intake is required. Neuropeptide F (NPF), a homolog of the mammalian neuropeptide Y, acts to induce feeding within the homeostatic regulation of this behavior. Drosophila and other insects bear a shorter form of NPF known as short NPF (sNPF) that can influence feeding. A neural hormone regulator, the dopamine transporter (DAT), works to clear dopamine from the synapses. This action may manipulate the post-feeding reward circuit in that lowered dopamine levels depress feeding, and excess dopamine levels encourage feeding. This study overexpressed and impaired the activities of NPF, sNPF, and DAT in Drosophila and examined their ability to survive during conditions of amino acid starvation. Too much or too little NPF or sNPF, which are key players in homeostatic feeding regulation, leads to increased sensitivity to amino acid starvation and diminished survivorship when compared to controls. When DAT, a member of the post-feeding reward system, is either overexpressed or reduced via mutation, Drosophila has increased sensitivity to amino acid starvation. Taken together, these results indicate that subtle variation in the expression of key components of these systems impacts survivorship during adverse nutrient conditions.

Erion, R., King, A.N., Wu, G., Hogenesch, J.B. and Sehgal, A. (2016). Neural clocks and Neuropeptide F/Y regulate circadian gene expression in a peripheral metabolic tissue. Elife [Epub ahead of print]. PubMed ID: 27077948
Summary:
Metabolic homeostasis requires coordination between circadian clocks in different tissues. Also, systemic signals appear to be required for some transcriptional rhythms in the mammalian liver and the Drosophila fat body. This study shows that free-running oscillations of the fat body clock require clock function in the PDF-positive cells of the fly brain. Interestingly, rhythmic expression of the cytochrome P450 transcripts, sex-specific enzyme 1 (sxe1) and Cyp6a21, which cycle in the fat body independently of the local clock, depends upon clocks in neurons expressing neuropeptide F (NPF). NPF signaling itself is required to drive cycling of sxe1 and Cyp6a21 in the fat body, and its mammalian ortholog, Npy, functions similarly to regulate cycling of cytochrome P450 genes in the mouse liver. These data highlight the importance of neuronal clocks for peripheral rhythms, particularly in a specific detoxification pathway, and identify a novel and conserved role for NPF/Npy in circadian rhythms.

Chung, B. Y., Ro, J., Hutter, S. A., Miller, K. M., Guduguntla, L. S., Kondo, S. and Pletcher, S. D. (2017). Drosophila Neuropeptide F signaling independently regulates feeding and sleep-wake behavior. Cell Rep 19(12): 2441-2450. PubMed ID: 28636933
Summary:
Proper regulation of sleep-wake behavior and feeding is essential for organismal health and survival. While previous studies have isolated discrete neural loci and substrates important for either sleep or feeding, how the brain is organized to coordinate both processes with respect to one another remains poorly understood. This study provides evidence that the Drosophila Neuropeptide F (NPF) network forms a critical component of both adult sleep and feeding regulation. Activation of NPF signaling in the brain promotes wakefulness and adult feeding, likely through its cognate receptor NPFR. Flies carrying a loss-of-function NPF allele do not suppress sleep following prolonged starvation conditions, suggesting that NPF acts as a hunger signal to keep the animal awake. NPF-expressing cells, specifically those expressing the circadian photoreceptor cryptochrome, are largely responsible for changes to sleep behavior caused by NPF neuron activation, but not feeding, demonstrating that different NPF neurons separately drive wakefulness and hunger.
Shao, L., Saver, M., Chung, P., Ren, Q., Lee, T., Kent, C. F. and Heberlein, U. (2017). Dissection of the Drosophila neuropeptide F circuit using a high-throughput two-choice assay. Proc Natl Acad Sci U S A 114(38): E8091-e8099. PubMed ID: 28874527
Summary:
In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted the development of a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. Optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments, and the preference for NPF neuron activation is dependent on NPF signaling. Finally, a small subset of NPF-expressing neurons located in the dorsomedial posterior brain was identified that are sufficient to elicit preference. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.
BIOLOGICAL OVERVIEW

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).

A neural circuit mechanism integrating motivational state with memory expression in Drosophila

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).


REGULATION

Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search

Internal physiological states influence behavioral decisions. This study investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food search behavior in Drosophila. It was found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food search behavior (Root, 2011).

This study reports that a state of starvation modulates olfactory sensitivity at the first synapse in a form of presynaptic facilitation. Starvation increases sNPFR1 transcription in ORNs, which is both necessary and sufficient for presynaptic facilitation. It has been well established that fluctuation of insulin is a key metabolic cue to maintain energy homeostasis. This study implicates that a low insulin signal via the PI3K pathway increases sNPFR1 expression. Interestingly, a subset of glomeruli exhibit starvation-dependent presynaptic facilitation that depends on intraglomerular sNPF signaling, while selective knockdown of sNPF or sNPFR1 in only the DM1 glomerulus affects food search behavior. This finding corroborates previous work revealing that the DM1 glomerulus is hardwired for innate odor attraction (Semmelhack, 2009). Thus, an internal state of starvation, with insulin as a global satiety signal acting on sensory neurons through a local sNPF signal, shifts the odor map. Starvation modulation of the odor map increases the saliency of glomerular activity to match the changing physiological needs of an organism (Root, 2011).

The Or42b sensory neurons may be considered as a neural substrate for appetitive choices because they integrate internal and external cues to influence an important innate behavior. In this integration a highly conserved neuropeptide plays an important role in the peripheral olfactory system. A similar presynaptic facilitation mechanism may exist in vertebrates as well. In an aquatic salamander, NPY has been shown to enhance electrical responses of cells in the olfactory epithelium to a food related odorant in starved animals. In addition, NPY immunoreactivity has been observed in the olfactory epithelium of mouse and zebrafish. In the nematode C. elegans, elevated activity levels of an NPY-like receptor cause a change in foraging pattern (Macosko, 2009). This study demonstrates that a fluctuating metabolic cue controls sNPFR1 levels in Or42b neurons, which in turn modulates appetitive behavior. However, it remains to be determined whether other ORNs mediate attraction behavior and whether they are subject to sNPF mediated modulation. Given the ubiquitous use of insulin as a metabolic cue, modulation by NPY/sNPF receptors in the early olfactory system could be a conserved mechanism between different animal species (Root, 2011).

The internal state of an organism influences its behavior. There is abundant evidence indicating that the global metabolic cue, insulin, works together with local neuropeptides in specific neural circuits to generate state-dependent behavioral responses. In Drosophila, the tolerance of a noxious food source is suppressed by insulin signaling and enhanced by NPF signaling such that these two peptides exert their opposing effects on the same neurons that mediate the behavior. In the mammalian hypothalamus, expression of the orexigenic NPY is suppressed in the satiety state via insulin signaling. Results from this study indicate that olfactory response in the periphery is reduced in the satiety state, in which insulin suppresses NPFR1 expression to alter neuronal excitability. Insulin's upstream control over sNPFR1 expression, however, appears to be specific to select neuronal types. Previous work in Drosophila has shown that sNPFR1 signaling exerts upstream control of insulin production in the Dilp2 neuroendocrine cells (Lee, 2008). In C. elegans, the release of an insulin-like peptide in an interneuron is downstream of a neuropeptide involved in promoting behavioral adaptation to food odors (Chalasani, 2010). Thus, different neuronal subtypes may adopt the same neuropeptides for unique and divergent molecular responses. Peptidergic modulation provides a rich repertoire of functional states for the same neural circuit to meet the demand of different internal states (Root, 2011).

Central mechanisms to control appetitive behavior, similar to the well-documented modulation of the hypothalamus by NPY, also appear to be important in Drosophila. A recent study demonstrates that appetitive memory requires the NPF receptor in the dopaminergic neurons that innervate specific mushroom body lobes (Krashes, 2009). This poses the question: what functions are subserved by starvation modulation of multiple neural substrates? It is interesting to note that sensitization of Or42b ORNs is sufficient to enhance food search behavior in fed flies. Perhaps central modulation by starvation is not necessary for food search behavior. Modulation in the periphery may serve to gate an animals’ sensitivity to specific food odorants, while central modulation may serve to enhance an animal’s ability to remember the relevant cues in finding a particular food source (Root, 2011).

Sex- and clock-controlled expression of the neuropeptide F gene in Drosophila

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).

The dNPF system regulates food response

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).

Mapping and ablation of Npf-Receptor cells

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).

Npf suppresses hyperexcitation

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 controls onset of social behavior

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 time course of larval behavioral changes

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).

Solitary larvae are deficient in burrowing

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).

Cooperative interaction between solitary and social larvae

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).

Npf overexpression prolongs the feeding phase

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).

Drosophila neuropeptide F and its receptor, NPFR1, define a signaling pathway that acutely modulates alcohol sensitivity

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).

Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila: Potential role of Neuropeptide F pathway

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).

Regulation of aversion to noxious food by Drosophila neuropeptide Y- and insulin-like systems

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).

A PDF/NPF neuropeptide signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged mating

A primary function of males for many species involves mating with females for reproduction. Drosophila melanogaster males respond to the presence of other males by prolonging mating duration to increase the chance of passing on their genes. To understand the basis of such complex behaviors, this study examined the genetic network and neural circuits that regulate rival-induced Longer-Mating-Duration (LMD). This study identified a small subset of clock neurons in the male brain that regulate LMD via neuropeptide signaling. LMD requires the function of pigment-dispersing factor (PDF) in four s-LNv neurons and its receptor PDFR in two LNd neurons per hemisphere, as well as the function of neuropeptide F (NPF) in two neurons within the sexually dimorphic LNd region and its receptor NPFR1 in four s-LNv neurons per hemisphere. Moreover, rival exposure modifies the neuronal activities of a subset of clock neurons involved in neuropeptide signaling for LMD (Kim, 2013).

This study provides evidence for the crucial involvement of two neuropeptides, PDF and NPF, in the modulation of reproductive behavior by the male's prior experience with other males. By identifying neurons required for this neuropeptide modulation, this study delineates the central neuronal circuitry and finds that the crucial neurons expressing a neuropeptide are not in synaptic contact with the crucial neurons expressing its receptor, providing further evidence for the long-range influence of neuropeptides. Remarkably, sharing housing with male rivals alters the activity of a subset of clock neurons, including those neurons expressing PDF and NPF that are crucial for this behavioral modulation. It was also found that these altered neuronal activities of PDF- and NPF-expressing neurons in group-reared males are dependent on the signaling by NPF and PDF, respectively (Kim, 2013).

LMD requires PDF expression in four s-LNv neurons, and it also requires the expression of the NPF receptor, NPFR1, in those four s-LNv neurons. These four s-LNv neurons thus appear to act in the LMD generation as a relay station to receive NPF neuropeptide signaling and to transmit PDF neuropeptide signaling to neurons expressing the PDF receptor PDFR (Kim, 2013).

Unlike PDF-expressing neurons with well-known functions for circadian rhythm behavior, much less is known about neurons expressing PDFR. To search for the PDFR-expressing cells involved in LMD, a small number of CRY-positive, but PDF-negative, neurons required to generate LMD were identified. Various pdfR-GAL4 lines were used to identify LNd neurons and PI neurons as candidate PDFR-expressing neurons. After the involvement of PI neurons was ruled out, it was demonstrated that expressing PDFR in LNd neurons of pdfR mutants was sufficient to rescue the LMD deficits. Among this small group of CRY-positive, but PDF-negative, LNd neurons, two cells that express PDFR, but not NPF, and another distinct group of two sexually dimorphic cells that express NPF, but not PDFR, in each hemisphere are required for LMD. Moreover, the neuronal activities of these male-specific LNd neurons that express NPF were increased by the exposure to rivals, whereas the neuronal activity of PDF-expressing s-LNv neurons appeared to be decreased by rival exposure. These four s-LNv neurons also express NPFR1, which is coupled to Gi to mediate inhibition of adenylyl cylcase. Given that the rival exposure-induced alteration of s-LNv neuronal activity requires NPFR1 function, one plausible scenario is that rival exposure increases the activity of NPF-expressing LNd neurons, which release NPF to activate NFPR1 on s-LNv neurons so as to reduce the activity of these PDF-expressing s-LNv neurons (Kim, 2013).

PDF appears to be released in a paracrine fashion to activate the G-protein-coupled receptor PDFR. PDFR is not found in the four s-LNv neurons that express PDF. One LNd neuron is known to be PDFR-positive, though its PDFR signaling has not been characterized. The two PDFR-expressing LNd neurons per hemisphere were found to be crucial for LMD, and they do not form direct synaptic contact with the s-LNv dorsal projections, consistent with the previous report that presynaptic terminals of PDF-expressing neurons have no direct contact with LNd neurons. Expression of the secreted form of PDF via an s-LNv-specific GAL4 driver in pdf01 mutant could rescue the disrupted LMD; however, expression of a membrane-tethered form of PDF could not. In contrast, expression of a membrane-tethered form of PDF via pdfR(D)-GAL4(2), with restricted expression in LNd and PI neurons, could rescue the disrupted LMD phenotype of pdf01 mutants. These results indicate that PDF secreted from s-LNv neurons can activate PDFR in LNd neurons to generate LMD. The dendrites of PDFR-positive LNd neurons labeled by pdfR(D)-GAL4(2) are located near the dorsal projections of PDF-expressing neurons. The dendrites of LNd neurons labeled by 50y-GAL4, which could impair LMD when it drives the expression of pdfR-siRNA to reduce PDFR activity in LNd neurons, also are located near these PDF-expressing neuronal projections. It has been reported that neuropeptide signaling does not require synaptic contacts. The released peptide may diffuse over tens of micrometers to reach its receptors, and the action of a peptide is limited by dilution as well as degradation/ inactivation by membrane-bound peptidases. Thus, PDF released from s-LNv neuronal projections may signal nearby PDFR-positive LNd neurons via diffusion rather than direct synaptic contact. In summary, this study has identified two PDFR-positive LNd neurons per hemisphere that are responsible for generating LMD via PDF/PDFR signaling. It is suggested that PDF released from s-LNv is responsible for PDFR signaling in these LNd neurons (Kim, 2013).

This study reveals that PDF and NPF signaling is crucial for the mating duration that is controlled by the male's experience with rivals. Moreover, rival exposure greatly reduced the activity of the s-LNv neurons normalized by that of l-LNv neurons both expressing PDF but increased the activity of LNd neurons normalized by that of D2 neurons both expressing NPF. Interestingly, this increase in neuronal activity of NPF-positive LNd neurons in group-rearing conditions is not observed in pdfR mutant animals. Given that PDFR and NPF are expressed by two distinct populations of LNd neurons, the requirement of PDFR function for the rival-induced modulation of NPF-expressing neuronal activity in the LNd region raises the intriguing question of whether neuronal signaling (perhaps involving another as-yet-unidentified neuropeptide) is involved in LMD (Kim, 2013).

A recent study has identified four abdominal ganglion (AG) interneurons (INs) that contain the neuropeptide corazonin (Crz) and modulate copulation duration. These neurons might play a role as a final set of effectors for the convergent effects of acute and chronic rival competition on the copulation duration. Elucidating the neural circuitry between these AG neurons and clock neurons would be helpful in furthering understanding of how male flies regulate mating duration in response to rivals (Kim, 2013).

Recent studies have shown that sexually dimorphic responses to pheromones in the nematode Caenorhabditis elegans may arise from differences in the balance of neural circuits during development or in the adult via neuromodulation. The current study adds to this emerging body of literature, illustrating the importance of sexually dimorphic neuromodulation via neuropeptide signaling in social behavior (Kim, 2013).

Drosophila Life Span and Physiology Are Modulated by Sexual Perception and Reward

Sensory perception modulates aging and physiology across taxa. This study found that perception of female sexual pheromones through a specific gustatory receptor expressed in a subset of foreleg neurons in male fruit flies rapidly and reversibly decreases fat stores, reduces resistance to starvation, and limits life span together with neurons that express the reward-mediating neuropeptide F. High-throughput RNA-seq experiments revealed a set of molecular processes that were impacted by the activity of the longevity circuit, thereby identifying new candidate cell non-autonomous aging mechanisms. Mating reversed the effects of pheromone perception, suggesting a model where life span is modulated through integration of sensory and reward circuits and where healthy aging may be compromised when the expectations defined by sensory perception are discordant with ensuing experience (Gendron, 2013).

Sensory perception can modulate aging and physiology in multiple species. In Drosophila, exposure to food-based odorants partially reverses the anti-aging effect of dietary restriction, whereas broad reduction in olfactory function promotes longevity and alters fat metabolism. Even the well-known relation between body temperature and life span may have a sensory component (Gendron, 2013).

To identify sensory cues and neuronal circuitry that underlie the effects of sensory perception on aging, this study focused on the perception of potential mates. Social interactions are prevalent throughout nature, and the influence of social context on health and longevity is well-known in several species, including humans. Such influences include behavioral interactions with mates and broader physiological 'costs of reproduction,' which often form the basis for evolutionary models of aging (Gendron, 2013).

In Drosophila, the presence of potential mates is perceived largely through non-volatile cuticular hydrocarbons, which are produced by cells called oenocytes and are secreted to the cuticular surface where they function as pheromones. To test whether differential pheromone exposure influenced life span or physiology, 'experimental' flies of the same genotype were housed with 'donor' animals of the same sex that either expressed normal pheromone profiles or were genetically engineered to express pheromone profiles characteristic of the opposite sex. Donor males with feminized pheromone profiles were generated by targeting expression of the sex determination gene, tra, to the oenocytes (via OK72-GAL4 or Prom-E800-Gal4), whereas masculinization of female flies was accomplished by expressing tra-RNAi in a similar way. This design allowed manipulation of the experimental animals' perceived sexual environment without introducing complications associated with mating itself (Gendron, 2013).

In Drosophila, sensory manipulations can affect life span, fat storage (as determined by baseline measures of triacylglyceride-TAG), and certain aspects of stress resistance. This study has found that flies exposed to pheromones of the opposite sex showed differences in these phenotypes. Experimental male flies exposed to male donor pheromone had higher amounts of TAG, were substantially more resistant to starvation, and exhibited a significantly longer life span than genetically identical male siblings exposed to female donor pheromone. Females exhibited similar phenotypes in response to male donor pheromone, but the magnitude of the effects was smaller. Subsequent experiments were therefore focused on males (Gendron, 2013).

The characteristics of pheromone exposure were indicative of a mechanism involving sensory perception. Effects were similar in several genetic backgrounds, including a strain recently collected in the wild, and were largely unaffected by cohort composition. Pheromone-induced phenotypes were detected after as little as two days exposure to donor animals, persisted with longer manipulations, and were progressively reversed when female donor pheromone was removed. Pheromone effects appeared not to be mediated by aberrant or aggressive interactions with donor flies because no significant differences were observed in such behaviors and because continuous, vigorous agitation of the vials throughout the exposure period, which effectively disrupted observed behaviors, had no effect on the impact of donor pheromone. Furthermore, exposure of experimental males to the purified female pheromone 7-11-heptacosadiene (7-11 HD) produced physiological changes in the absence of donor animals (Gendron, 2013).

To explore the sensory modality through which donor pheromone exerts its effects, this study tested whether a broadly-expressed olfactory co-receptor, Or83b, whose loss of function renders flies largely unable to smell, was required for pheromone effects. Or83b mutant flies exhibited similar changes in starvation resistance in response to donor pheromone as did control animals, indicating that olfaction was not required. To test whether taste perception was involved, flies were tested that were mutant for the gene Pox neuro (Poxn), a null mutation that putatively transforms all chemosensory neurons into mechanosensory neurons. Drosophila taste neurons are present in the mouthparts and distributed on different body parts including the wings, legs, and genitals, which allow sensation by contact. When the Poxn null mutation is coupled with a partially rescuing transgene, Poxn ΔM22-B5-ΔXB, flies are generally healthy, but gustatory perception is eliminated in the labelum, the legs, and the wing margins. Poxn ΔM22-B5XB flies showed no pheromone-induced changes in starvation resistance, TAG amounts, or life span. However, Poxn mutant flies that carried a transgene that restores taste function to the legs and wing margins (but not labelum; PoxnΔM22-B5-Full1 responses were similar to those of control flies. Thus, the effects of pheromone exposure appear to be mediated by taste perception through gustatory neurons outside of the mouthparts (Gendron, 2013).

To identify specific gustatory receptors and neurons that might mediate the pheromone effects, candidate pheromone receptors were tested. Of the mutants that were examined, only flies that carried a loss of function mutation in the gene pickpocket 23 (ppk23) were resistant to the effects of pheromone exposure. Further analysis verified that ppk23 was required for the effects of pheromone exposure on starvation resistance, TAG amounts, and life span. Silencing ppk23-expressing neurons only during exposure to donor males by expressing a temperature-sensitive dominant negative allele of the dynamin gene shibire (via ppk23-GAL4; UAS-shits) also eliminated the differential response to pheromones. In male Drosophila, the transcription factor fruitless (fru) is expressed with ppk23 in pheromone-sensing neurons located in the animals' forelegs, and silencing fru-expressing neurons during exposure (via fru-GAL4;UAS-shits) abrogated pheromone effects. Consistent with a requirement for these neurons, it was found that surgical amputation of the forelegs, but not injury alone, was sufficient to reproducibly eliminate the effects of pheromone exposure. Moreover, acute, targeted activation of ppk23-expressing neurons using a temperature-sensitive TRPA1 channel (ppk23-GAL4;UAS-TRPA1) was sufficient to mimic the effects of female pheromone without exposure. Together, these data indicate that pheromone-sensing neurons in the foreleg of the male fly that express the gustatory receptor, ppk23, and the transcription factor, fruitless, influence stress resistance, physiology, and life span in response to perception of female pheromones (Gendron, 2013).

To examine brain circuits that may function in transducing pheromone perception, UAS-shits was selectively expressed to block synaptic transmission in various neuro-anatomical regions with the goal of disrupting the physiological effects of donor pheromone exposure. The effects were abrogated when UAS-shits was driven in neurons characterized by expression of neuropeptide F (NPF, as represented by npf-GAL4). Further analysis verified that pheromone-induced changes in starvation resistance and TAG abundance were lost following silencing of npf-expressing neurons. Consistent with a possible role in transducing pheromone information, npf expression was significantly increased by 30% in experimental males after exposure to feminized donor males, and activation of npf-expressing neurons was sufficient to decrease life span in the absence of pheromone exposure (Gendron, 2013).

NPF may function as a mediator of sexual reward in Drosophila, and its mammalian counterpart, neuropeptide Y (NPY), has been associated with sexual motivation and psychological reward. Tests were performed to see whether the effects of pheromone perception might be rescued by allowing males to successfully mate with females. Neither a small number of conjugal visits with virgin females nor housing with wild-type females in a 1:1 ratio was sufficient to ameliorate the effects of pheromone exposure. In this context, decreased longevity may be a consequence of pheromone perception and not of mating itself. Male Drosophila are willing and able to copulate up to five times in rapid succession before requiring a refractory period. It was found that supplementing donor cohorts with an excess of mating females (in a 5:1 ratio) was sufficient to significantly reduce the effects on mortality and TAG caused by female donor pheromone early in life. The benefits of mating on age-specific mortality decreased with age, suggesting that aging may reduce mating efficiency or may diminish effective mating reward (Gendron, 2013).

To identify how sexual perception and reward may alter physiological responses in peripheral tissues, changes in gene expression were examined using whole-genome RNA-seq technology. 195 genes were found with significantly different expression (using an experiment-wise error rate of 0.05) in control male flies that were exposed to feminized or control donor males for 48 hours. Nearly all (188/195 = 96%) of the changes appeared to be due to pheromone perception because they were not observed in identical experiments using ppk23 mutant flies. Males exposed to female pheromones decreased transcription of genes encoding odorant-binding proteins and increased transcription of several genes with lipase activity. A significant enrichment was observed in secreted molecules, which includes genes encoding proteins mediating immune- and stress-responses. Many of these genes and pathways were highlighted in a recent meta-analysis of gene expression changes in response to stress and aging (Gendron, 2013).

Activities of insulin and target of rapamycin (TOR) signaling, which modulate aging across taxa, increase sexual attractiveness in flies. The current demonstration that perception of sexual characteristics is sufficient to modulate life span and physiology suggests aging pathways in one individual may modulate health and life span in another. These types of indirect genetic effects have the potential to be influential agents of natural selection, suggesting that expectation/reward imbalance may have broad effects on health and physiology in humans and may present a potent evolutionary force in nature (Gendron, 2013).

Central peptidergic modulation of peripheral olfactory responses

Drosophila neuropeptide F (NPF) modulates the responses of a specific population of antennal olfactory sensory neurons (OSNs) to food-derived odors. Knock-down of NPF in NPF neurons specifically reduces the responses of the ab3A neurons to ethyl butyrate, a volatile ester found in apples and other fruits. Knock-down of the NPF receptor (NPFR) in the ab3A neuron reduces their responses and disrupts the ability of the flies to locate food. A sexual dimorphism was identified in ab3A responsiveness: ab3A neurons in females immediately post-eclosion are less responsive to ethyl butyrate than those of both age-matched males and older females. Not only does this change correlate with brain NPF levels, but also NPFR mutants show no such sexual dimorphism. Finally, by way of mechanism, mutation of NPFR seems to cause intracellular clustering of OR22a, the odorant receptor expressed in the ab3A neurons. This modulation of the peripheral odorant responsiveness of the ab3A neurons by NPF is distinct from the modulation of presynaptic gain in the ab3A neurons previously observed with the similarly named but distinct neuropeptide sNPF. Rather than affecting the strength of the output at the level of the first synapse in the antennal lobe, NPF-NPFR signaling may affect the process of odorant detection itself by causing intracellular OR clustering (Lee, 2017).

This study has identified a role in Drosophila for neuropeptide F (NPF) and its receptor NPFR in modulating the peripheral responses of the ab3A class of olfactory sensory neurons (OSNs). These neurons detect a range of fruity-smelling esters associated with the fruits that provide Drosophila food and a place to lay their eggs. Loss of NPF in NPF neurons reduces odor-evoked spiking of ab3A neurons in response to apple odors (e.g., ethyl butyrate and methyl butyrate) without affecting their spontaneous activity. It does not, however, affect the responses of ab1A/B or ab2A neurons to their preferred ligands or several other neuron classes to ethyl butyrate (i.e., ab2B, ab8A/B, and pb1A). The study has shown that ab3A neurons must express NPFR themselves to exhibit this increase in olfactory responsiveness; ab3A-specific expression of NPFR rescues the reduced ab3A responses of NPFR c01896 mutant flies. This modulation of olfactory responses by NPF and NPFR also affects olfactory-guided behaviors, as ab3A neuron-specific knock-down of NPFR reduces attraction of flies to apple juice baits (Lee, 2017).

Root (2011) found that hunger enhances the responses of DM1, DM2, and DM4-the antennal lobe glomeruli that receive input from ab1A, ab3A, and ab2A neurons, respectively. They found that these neurons produce sNPF, which when released, alters the calcium responses of their own presynaptic terminals. This acts as a sort of gain control, enhancing the activation of the corresponding second-order olfactory projection neurons. In the ab1A neurons, Root unambiguously attributed this presynaptic gain control to the sNPF receptor sNPFR1 and showed that insulin signaling and starvation both alter sNPFR1 expression. This means that the ab1A neurons, which respond to food-related esters like the ab3A neurons, induce larger responses in their associated projection neurons in times of starvation, enhancing foraging behavior. The NPF-mediated modulation of ab3A neurons discovered in this study seems to differ from this sNPF-mediated gain control. Rather than magnifying presynaptic calcium responses as sNPFR activation seems to do, NPFR activation increases the number of odor-evoked action potentials in the ab3A neurons. It is unclear how these two pathways relate and what other implications these distinctions may have, but they suggest that the modulation of ab3A neurons by NPF and sNPF act through different molecular mechanisms. It is distinct from NPF's modulation of sugar taste detection, which was discovered by Inagaki (2014). That study found that hunger and NPF increase sugar responses indirectly by modulating the influence of upstream dopaminergic neurons on the GR5a-positive GRNs in the labellum (Lee, 2017).

Lin (2016) recently reported evidence that juvenile hormone (JH), which is secreted into the circulating hemolymph by the corpora allata, acts on its receptor Methoprene-tolerant (Met) in the pheromone-sensitive antennal trichoid at4 neurons to sensitize them. While hormones are secreted into the circulation to act on distant target tissues, neuropeptides are typically secreted from peptide-producing neurons onto neighboring cells. No one had previously reported NPFergic innervation of the antennae or antennal lobes, it was initially suspected that NPF may be similarly secreted into the circulation. But by combining two copies of NPF-GAL4, it was possible to visualize NPF-GAL4-positive innervation of the antennal lobes. Since this study also found that knock-down of NPF in the ab3A neurons themselves reduces their responses to EB, NPF seems to be acting locally (Lee, 2017).

To address the mechanism by which NPF-NPFR signaling modulates ab3A neurons, NPFR mutant antennae were stained with an antiserum that recognizes OR22a, the odorant receptor expressed by the ab3A neurons. No difference was detected in OR22a staining in the outer ab3A dendrites where odorant binding takes place. Still, compared to control antennae, NPFR mutant antennae show dramatically more OR22a-positive puncta near the ciliary dilations that separate the inner and outer ab3A dendritic segments. It is unclear what these OR22a-positive puncta are, but it is expected that they represent either OR22a molecules whose trafficking to the ciliated outer dendrites is being blocked or those whose internalization from the periphery is being enhanced. These puncta strongly resemble the OR22a-positive puncta that appear in the absence of the olfactory co-receptor Orco required for OR trafficking to the ciliated outer dendrites and co-localize with markers of the endoplasmic reticulum. This could support the former hypothesis-a specific reduction in dendritic OR22a trafficking in the absence of NPFR-but the OR22a staining in the outer dendrites of NPFR c01896 antennae seems to be unaffected (Lee, 2017).

Although the molecular dynamics of OR dendritic surface localization, odorant binding, internalization, deactivation, and recycling remain somewhat unclear, it is speculated that NPFR may modulate OR recycling. If NPFR acts to stabilize active odorant/OR complexes at the cell surface, effectively delaying receptor inactivation, each odorant stimulus would elicit more action potentials. Reduced signaling through NPFR could accelerate the internalization of the active odorant/OR complexes, effectively reducing the length of time they spend on the outer dendritic membrane. If this speculation proves true, the wild-type levels of OR22a in the NPFR c01896 mutant antennae suggest that there may be a compensatory increase in the trafficking of new OR complexes to the outer segment. In other words, if this speculation is true, there should be a tight coupling between OR externalization and internalization. Since NPFR typically inhibits adenylyl cyclase and reduces neuronal activity, it is unclear how such a modulation of OR recycling would occur. Future studies should address the precise mechanisms that guide the movement of ORs in and out of the outer dendrites and how the various peptidergic signaling pathways may modulate those movements (Lee, 2017).

This study also found that female flies show lower ab3A responses immediately post-eclosion than male flies, but this difference disappears as the flies age. A clear correlation was found between ab3A responses and NPF staining in young female versus male brains, and this sexual dimorphism was shown to be absent in NPFR c01896 mutants. Unfortunately, it was not possible to directly compare males and females in the trap assay, especially when they were young. Young males and females have dramatically different levels of body fat and appetites. Because of this, females require much longer periods of starvation to motivate them to move through the trap than males. This is why only males were used for this behavioral assay and why it was only possible to focus on the role of NPFR in the OSNs rather than on sex-specific differences in behavior. The function of this sexual dimorphism is unclear, but it may enhance dispersal of young females to new and more palatable food sources. Once Drosophila larvae reach their final larval instar, they stop foraging and move out of their food to find a dry location to pupate. A piece of fruit suitable for laying eggs before a single round of the 10- to 14-day Drosophila life cycle may be less suitable for a second. Thus, the reduction in olfactory responses to fruit odors observe in young female flies may help encourage them to find new food sources for egg laying. It will be interesting to test this hypothesis in future studies (Lee, 2017).

The majority of the impact insects have on human society stems from their feeding behaviors (e.g., destroying crops or transmitting disease through infectious bites). Since insect feeding behaviors are guided by olfaction, this study focused in how insect olfactory systems change in response to internal and external cues. This showed that in genetic model insect Drosophila melanogaster NPF acts on its receptor NPFR to sensitize a specific population of antennal olfactory neurons that detect an important food-related odorant. This peripheral olfactory modulation by NPF and NPFR is sexually dimorphic in young adult flies and it affects olfactory-guided attraction to food odors. Since homologues of NPF and NPFR exist across insect species, it will be interesting to see whether these homologues also modulate olfactory food detection in these species. If so, this modulation may represent another potential target for future pest control strategies (Lee, 2017).

Protein Interactions

Neuropeptide F receptor

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).


DEVELOPMENTAL BIOLOGY

The conserved neuropeptide Y (NPY) signaling pathway has been strongly implicated in the stimulation of food uptake in vertebrates as well as in the regulation of food conditioned foraging behaviors of Caenorhabditis elegans. Using in situ RNA hybridization and immunocytochemistry, the study reports the neuronal network of Drosophila neuropeptide F (dNPF), a human NPY homologue, in the larval central nervous system and its food-dependent modifications. Indications are provided that gustatory stimulation by sugar, but not its ingestion or metabolism, is sufficient to trigger long-term, dose-dependent alterations of the dNPF neuronal circuit through both dnpf activation and increased synaptic transmission. These results strongly suggest that the dNPF neuronal circuit is an integral part of the sensory system that mediates food signaling, providing the neural basis for understanding how invertebrate NPY regulates food response (Shen, 2001).

The Npf neural system is comprised of 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 (Shen, 2001; Brown 1999).

The possible role of Npf in regulating feeding activity was investigated in the third instar larva. Young third instar larvae feed voraciously, but their feeding activity subsides as they mature and become increasingly mobile. Under controlled growth conditions, the larval transition from feeding to nonfeeding is largely completed by the first 24 hr, when larvae are moving away from yeast paste on apple juice-agar. To determine the relationship between the feeding activity and npf neural expression, the npf RNA level in the CNS tissues of feeding and nonfeeding larvae was examined by whole-mount in situ RNA hybridization using a digoxigenin-labeled antisense npf RNA probe. Two-hour-old third instars were either harvested immediately or withheld from food for an additional 24 hr before tissue dissection and fixation. In both cases, strong fluorescence staining was detected in the four neurons in the brain lobes, indicating that the npf RNA level remains high in larvae that are attracted to food, regardless of their age and feeding state. Quantification of fluorescence staining in the four neurons showed that the npf RNA levels were comparable in both types of tissues. The npf expression was examined in synchronized 24-hr-old third instars that were fed continuously. Before tissue collection, the natural cessation of food intake was confirmed by the absence of dyed yeast paste in the gut. The fluorescence staining in the brain was undetectable in most of the nonfeeding larvae, while the rest showed greatly diminished staining, indicating that the downregulation of npf expression in the brain coincides with the cessation of larval feeding

More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2

Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the Drosophila and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing beta-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. A specific polyclonal antiserum was also made to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, expression in the midgut was found to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced (Veenstra, 2014).


EVOLUTIONARY HOMOLOGS

A second set of neuropeptide F peptides in Drosophila and their receptor: Short neuropeptide F precursor and Neuropeptide F-like Receptor 76F

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y2 receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs (Mertens, 2002).

Since the Drosophila genome encodes at least 4 receptors belonging to the NPY subgroup of receptors, the Drosophila EST database was searched for the presence of EST clones, encoding one of the NPY-type receptors. PCR amplification of the EST clone (GH23382) with oligonucleotide primers specific for the predicted ORF of CG7395 (Neuropeptide F-like Receptor 76F) produced a single product of approximately 1800 bp. Sequence determination of the TA-cloned PCR product revealed a DNA insert of 1803 bp, corresponding to the sequence and size of the predicted receptor in the cDNA database of BDGP. The deduced protein encoded by the ORF of Drm-sNPF-R is 600 amino acids long. Analysis by the TMHMM program revealed that this protein is predicted to have seven transmembrane domains along with the intracellular and extracellular loops, consistent with the known G-protein coupled receptors. The N-terminal extracellular region exhibits no O-glycosylation, 2 N-glycosylation sites, along with 7 Ser/Thr phosphorylation sites (Mertens, 2002).

A phylogenetic tree based on Clustal W alignment of Drm-sNPF-R and various known NPY receptors indicates that Drm-sNPF-R is most closely related to the vertebrate neuropeptide Y2 receptors, i.e., of the domestic guinea pig (33% identity and 49% homology), humans (33% identity and 49% homology), the domestic pig (33% identity and 48% homology), and the rat (33% identity and 48% homology). An NPY-like orphan GPCR of C. elegans (C53C7.1) displays 33% identity and 47% homology. Sequence conservation among Drm-sNPF-R, the human Y2 receptor, and the C. elegans orphan receptor is depicted by similarities shown in their alignment by the AlignX program (Mertens, 2002).

Short NPFs and 'head' peptides display substantial sequence similarities and appear to belong to the same family. All NPFs have a typical R(K)–X1–R–X2amide motif, where the first amino acid of this motif is always a basic amino acid residue such as Arg or Lys. X1 can be L, T or P and X2 is always an aromatic amino acid residue such as Phe or Trp. It is proposed to (re)name all peptides with the R(K)–X1–R–X2amide C-terminal motif, as short NPFs; these peptides do not only occur in the head or central nervous system, but instead reach 10 times higher amounts in the abdomen and the midgut. In addition, their precursor in the Drosophila genome is annotated as the short NPF precursor. The present identification of a specific short NPF receptor in Drosophila is in favor of the presence of functional short NPFs. Several reports indicate that short NPFs have a hormonal function in insects, associated with reproduction and digestion. The expression of the short NPF receptor not only in the nervous system, but also in peripheral targets (ovaries, gut) is in agreement with a hormonal function of short NPFs. Hemolymph from sugar-fed mosquito females contains 414 fmol/μl immunoreactive short NPF. Short NPFs are also abundantly present in endocrine cells of the midgut, suggesting that they might have a function in digestion. The demonstration of the presence of the short NPF receptor transcript in the midgut favors this hypothesis (Mertens, 2002).

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (Neuropeptide F-like Receptor 76F, NPFR76F, or GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62%-66% similarity and 32%-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila (Feng, 2003).

Since the NPFR76F receptor was maximally activated by a short insect NPF-like sequence from Leptinotarsa, genome mining was used to search for Drosophila peptides which might be functionally equivalent to (or better than) the Leptinotarsa I peptide at activating NPFR76F. Initially, one precursor sequence, a gene (npf) encoding Drosophila NPF was identified at chromosome location 89D3. This gene was found by blasting an incomplete version of the Drosophila genome (October 1999) with the amino acid sequence of Aplysia NPY. This revealed a precursor molecule encoding a 36 amino acid peptide with sequence similarity to NPF. The completed Drosophila genome sequence has now been searched and no other potential NPF precursors were identified. Since this peptide (NPF-A1) contained a potential dibasic amino acid cleavage site within its sequence, both the shorter 28 amino acid form (NPF-A2) and the full-length peptide (NPF-A1) were sequenced for testing (Feng, 2003).

A second open reading frame in the Drosophila genome encodes a precursor peptide for two short NPF-like peptides (Drm-sNPF-1, AQRSPSLRLRFamide and Drm-sNPF-2, WFGDVNQKPIRSPSLRLRFamide). The precursor for these peptides is encoded by the short NPF precursor (sNPF) gene (CG13968) and maps to position 38A7 on the left arm of Drosophila chromosome 2. The WFGDVNQKPIRSPSLRLRFamide peptide (Drm-sNPF-2) contains a potential single basic amino acid-processing site. This peptide (Drm-sNPF-2), its shorter form (peptide Drm-sNPF-2 s, PIRSPSLRLRFamide) and the Drm-sNPF-1 peptide (all encoded by the sNPF gene) were synthesized. The same precursor was predicted to include the sequences for two other short peptides, PQRLRWamide and PMRLRWamide, which have been designated Drm-sNPF-3 and Drm-sNPF-4, respectively. These peptides were synthesized and tested (Feng, 2003).

When tested at 1 µm, the shorter NPF-like peptides derived from the sNPF gene (peptides Drm-sNPF-1 and Drm-sNPF-2) were more effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents in Xenopus oocytes expressing NPFR76F and Galpha16. Shortening of peptide Drm-sNPF-2 to the Drm-sNPF-2s form may slightly increase its effectiveness. The longer Drosophila peptides derived from the precursor gene npf at 89D3 were much less effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents. In addition, the PQRLRWamide (Drm-sNPF-3) and PMRLRWamide (Drm-sNPF4) peptides were much less effective than the original Leptinotarsa I sequence at inducing inward currents (Feng, 2003).

Dose-response curves for the shorter endogenous Drosophila NPF-like peptides encoded by the sNPF gene at 38A7 reveal that AQRSPSLRLRFamide (peptide Drm-sNPF-1) is the most potent peptide tested (pEC50 = -8.84). It showed a threshold for the generation of inward currents between 100 pm and 1 nm and a maximal effect at 100 nm. The second putative endogenous Drosophila NPF-like peptide encoded by the sNPF gene, PIRSPSLRFamide (peptide Drm-sNPF-2 s) (pEC50 = -7.62), and the original Leptinotarsa I NPF-like sequence, ARGPQLRLRFamide (pEC50 = -7.83), were an order of magnitude less potent than AQRSPSLRLRFamide. These results justify the classification of NPFR76F as a NPF-like receptor and suggest that the short peptide AQRSPSLRLRFamide (sNPF-1) may be the endogenous agonist for this receptor. The other two short peptides encoded by the sNPF gene, PQRLRWamide (pEC50 = -6.1) and PMRLRWamide (pEC50 = -7.30), were 2.7 and 1.5 orders of magnitude, respectively, less potent than the AQRSPSLRLRFamide sequence, leading to questioning of their designation as true short NPF-like peptides (Feng, 2003).

Expression of NPFR76F transcripts was assessed by Northern blot analysis of poly(A)+ RNA prepared from adult body parts. A single transcript of 6.5 kb was detected in both heads and appendages (legs and antennae), suggesting that NPFR76F is expressed in both the central and peripheral nervous systems. In addition to finding transcript in heads and appendages, trace amounts were also seen in bodies. When compared with the amount of RNA loaded from each body part (as indicated by the ubiquitous rp49 loading control), the relative abundance of transcript in bodies is very low. This distribution of the NPFR76F transcript is consistent with a role for this NPF-like receptor in the Drosophila nervous system (Feng, 2003).

To further refine the NPFR76F receptor transcript expression, in situ hybridization with a digoxigenin-labelled antisense RNA probe to whole mounts of the mature embryos was used. The central nervous system in embryos is composed of two dorsal brain hemispheres and a fused ventral ganglion. The NPFR76F receptor is expressed both in the dorsal brain and in the ventral ganglion. In the brain the receptor is strongly expressed in the specific cells in the dorsal posterior region. It is estimated that there are 22-24 cells in each brain lobe expressing NPFR76F, including the strongly expressing cells. In the ventral ganglion, pairs of cells along the ventral midline, as well as cells found in a bilaterally symmetric pattern in a more lateral position from the midline, also express receptor mRNA. In each full segment of the ventral ganglion, the receptor is strongly expressed in eight to 12 cells, including a pair of cells at the midline in each segment (Feng, 2003).

In the peripheral nervous system the receptor is expressed in a subset of sensilla and in the anterior sensory complex. It is estimated that there are 10-14 cells in the anterior sensory complex, including the antennomaxillary complex, the labral sensory complex and the labial sensory complex, that express NPFR76F. Finally, in the posterior sensilla, there are eight cells that express NPFR76F. The expression pattern of the NPFR76F receptor in many specific cells in the dorsal brain, the ventral ganglion, lateral sensilla, the anterior sensory complex and the posterior sensilla suggests that this receptor is involved in a widespread modulation of neuronal activity (Feng, 2003).

A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides are more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system (Reale, 2004).

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. This study reports the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster (Lee, 2004).

Various evidence suggests that sNPF and dNPF peptides have different functions. The sNPF peptide is found only in nervous systems, whereas the neuropeptide F (dNPF) is found as the Drosophila brain-gut peptide. The expression patterns of sNPF and dNPF differ in the larval brain. For example, the sNPF expression is found in the anterior dorsal neurons of the brain, whereas the dNPF expression is detected in the four neurons of larval brain. In the feeding behavior analysis, overexpression of sNPF in wandering larvae did not extend the feeding period, contrary to the extension of the feeding period in the wandering larval stage by overexpression of dNPF. At the receptor level, each peptide works in different receptors; for example, the NPFR76F receptor is for sNPF peptides, and the DmNPFR1 receptor is for the dNPF. These differences indicate that the dNPF and sNPF peptides function in different neurons and may regulate different aspects of feeding behaviors in Drosophila (Lee, 2004).

Like other neuropeptides, the sNPF peptide may be involved in regulating various physiological processes other than regulating food intake because the sNPF peptide and transcript were expressed during all developmental stages, and the sNPF is localized in the mushroom body calyx and medulla of the adult brain. The mushroom body is involved in learning and memory. These unknown multi-functions of the sNPF peptide in various biological processes are the subjects of future studies (Lee, 2004).

Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids are clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs (Garczynski, 2006).

The sNPFs of D. melanogaster differ in their interactions with the sNPF receptor Drm-NPFR76F, as analyzed directly by radioreceptor assay. A wide variety of D. melanogaster sNPFs were assayed for their ability to inhibit the binding of 125I-[D-Y1]-Drm-sNPF1 to membranes prepared from cells stably transfected with Drm-NPFR76F. Two distinctive classes of activity of sNPFs were readily apparent. The sNPF peptides containing nine or more amino acids typically exhibited high affinity, as judged by an IC50 < 1 nM, whereas peptides containing eight for fewer amino acids exhibited IC50 values of >5 nM. Those exhibiting lower affinity included sNPF3 and sNPF4, peptides with the C-terminal RLRWa sequence. The minimum length of the highly active group was represented by sNPF211–19. The sequence of this sNPF211–19 (RSPSLRLRFa) differs from sNPF14–11 (SPSLRLRFa) only by a single arginine residue, which appears to confer a substantial increase in binding affinity. To test this hypothesis, an alanine substituted analog, Drm-sNPF211–19R11A, was assayed and found to exhibit a substantial drop in activity, with an IC50 of only 12.5 nM, indicating the crucial role of this arginine residue (Garczynski, 2006).

For further tests of these apparent structure-function relations, putative sNPFs identified in the genomes of A. gambiae and A. aegypti also were examined in the Drm-NPFR76F radioreceptor assay. Each of these mosquito sNPF peptides conformed to the distinctive pattern of length-associated activity established previously for those of D. melanogaster. In contrast, the A. aegypti head peptides which partly resemble sNPFs were either weakly active, Aea-HP-I, or inactive, Aea-HP-III, despite being of sufficient length and having the requisite arginine. Accordingly, additional structural features in the C-terminus common to sNPF peptides appear important for high affinity binding (Garczynski, 2006).

Neuropeptide F in other insects

The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50 approximately 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF>Aea-NPF>Drm-NPF>Drm-NPF8-36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito (Garczynski, 2005).

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal (Stanek, 2002).

Neuropeptide Y receptor in C. elegans

Natural isolates of C. elegans exhibit either solitary or social feeding behavior. Solitary foragers move slowly on a bacterial lawn and disperse across it, while social foragers move rapidly on bacteria and aggregate together. A loss-of-function mutation in the npr-1 gene, which encodes a predicted G protein-coupled receptor similar to neuropeptide Y receptors, causes a solitary strain to take on social behavior. Two isoforms of NPR-1 that differ at a single residue occur in the wild. One isoform, NPR-1 215F, is found exclusively in social strains, while the other isoform, NPR-1 215V, is found exclusively in solitary strains. An NPR-1 215V transgene can induce solitary feeding behavior in a wild social strain. Thus, isoforms of a putative neuropeptide receptor generate natural variation in C. elegans feeding behavior (de Bono, 1998).

Wild isolates of Caenorhabditis elegans can feed either alone or in groups. This natural variation in behaviour is associated with a single residue difference in NPR-1, a predicted G-protein-coupled neuropeptide receptor related to Neuropeptide Y receptors. The NPR-1 isoform associated with solitary feeding acts in neurons exposed to the body fluid to inhibit social feeding. Furthermore, suppressing the activity of these neurons, called AQR, PQR and URX, using an activated K(+) channel, inhibits social feeding. NPR-1 activity in AQR, PQR and URX neurons seems to suppress social feeding by antagonizing signalling through a cyclic GMP-gated ion channel encoded by tax-2 and tax-4. Mutations in tax-2 or tax-4 disrupt social feeding, and tax-4 is required in several neurons for social feeding, including one or more of AQR, PQR and URX. The AQR, PQR and URX neurons are unusual in C. elegans because they are directly exposed to the pseudocoelomic body fluid. The data suggest a model in which these neurons integrate antagonistic signals to control the choice between social and solitary feeding behaviour (Coates, 2002).

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of C. elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations (Davies, 2003).

Mutation of mammalian neuropeptide Y

Neuropeptide Y (NPY), a 36-amino-acid transmitter distributed throughout the nervous system, is thought to function as a central stimulator of feeding behaviour. NPY has also been implicated in the modulation of mood, cerebrocortical excitability, hypothalamic-pituitary signalling, cardiovascular physiology and sympathetic function. However, the biological significance of NPY has been difficult to establish owing to a lack of pharmacological antagonists. Mice deficient for NPY have normal food intake and body weight, and become hyperphagic following food deprivation. Mutant mice decrease their food intake and lose weight, initially to a greater extent than controls, when treated with recombinant leptin. Occasional, mild seizures occur in NPY-deficient mice and mutants are more susceptible to seizures induced by a GABA (gamma-aminobutyric acid) antagonist. These results indicate that NPY is not essential for certain feeding responses or leptin actions but is an important modulator of excitability in the central nervous system (Erickson, 1996a).

The obesity syndrome of ob/ob mice results from lack of leptin, a hormone released by fat cells that acts in the brain to suppress feeding and stimulate metabolism. Neuropeptide Y (NPY) is a neuromodulator implicated in the control of energy balance and is overproduced in the hypothalamus of ob/ob mice. To determine the role of NPY in the response to leptin deficiency, ob/ob mice deficient for NPY were generated. In the absence of NPY, ob/ob mice are less obese because of reduced food intake and increased energy expenditure, and are less severely affected by diabetes, sterility, and somatotropic defects. These results suggest that NPY is a central effector of leptin deficiency (Erickson, 1996b).

An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception (Bannon, 2000).

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. AgRP-deficient (Agrp-/-) mice were generated to examine the physiological role of AgRP. Agrp-/- mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp-/- mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp-/- mice. Since AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, AgRP and NPY double-knockout (Agrp-/-;Npy-/-) mice were generated to determine whether NPY or AgRP plays a compensatory role in Agrp-/- or NPY-deficient (Npy-/-) mice, respectively. Similar to mice deficient in either AgRP or NPY, Agrp-/-;Npy-/- mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. These results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY (Qian, 2003).

Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy-/- mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. On a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy-/- mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity (Segal-Lieberman, 2003).

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy-/-) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, food intake was assessed during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy-/- mice was reduced by 33% during this period. In contrast, intake did not differ between genotypes when measured over a 24-h period. Furthermore, reduced dark cycle 4h food intake in Npy-/- mice was not evident after a 24-h fast, despite a pronounced delay in the initiation of feeding. To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source. By comparison, HP intake was markedly reduced in Npy-/- mice during the first week, although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction (Sindelar, 2005).

Biological effects of NPY in mammals

Exogenous neuropeptide Y (NPY) reduces experimental anxiety in a wide range of animal models. The generation of an NPY-transgenic rat has provided a unique model to examine the role of endogenous NPY in control of stress and anxiety-related behaviors using paradigms previously used by pharmacological studies. Locomotor activity and baseline behavior on the elevated plus maze were normal in transgenic subjects. Two robust phenotypic traits were observed. (1) Transgenic subjects showed a markedly attenuated sensitivity to behavioral consequences of stress, in that they were insensitive to the normal anxiogenic-like effect of restraint stress on the elevated plus maze and displayed absent fear suppression of behavior in a punished drinking test. (2) A selective impairment of spatial memory acquisition was found in the Morris water maze. Control experiments suggest these traits to be independent. These phenotypic traits were accompanied by an overexpression of prepro-NPY mRNA and NPY peptide and decreased NPY-Y1 binding within the hippocampus, a brain structure implicated both in memory processing and stress responses. Data obtained using this unique model support and extend a previously postulated anti-stress action of NPY and provide novel evidence for a role of NPY in learning and memory (Thorsell, 2000).

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. NPY-overexpressing mice have been developed by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but the obese phenotype was not observed in the heterozygote. In the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibit significantly increased body weight gain with transiently increased food intake after 50% sucrose-loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of the observation period (Kaga, 2001).

Despite numerous experiments showing that administration of neuropeptide Y (NPY) to rodents stimulates feeding and obesity, whereas acute interference with NPY signaling disrupts feeding and promotes weight loss, NPY-null mice have essentially normal body weight regulation. These conflicting observations suggest that chronic lack of NPY during development may lead to compensatory changes that normalize regulation of food intake and energy expenditure in the absence of NPY. To test this idea, gene targeting was used to introduce a doxycycline (Dox)-regulated cassette into the Npy locus, such that NPY would be expressed until the mice were given Dox, which blocks transcription. Compared with wild-type mice, adult mice bearing this construct expressed approximately 4-fold more Npy mRNA, which fell to approximately 20% of control values within 3 days after treatment with Dox. NPY protein also fell approximately 20-fold, but the half-life of approximately 5 days was surprisingly long. The biological effectiveness of these manipulations was demonstrated by showing that overexpression of NPY protected against kainate-induced seizures. Mice chronically overexpressing NPY had normal body weight, and administration of Dox to these mice did not suppress feeding. Furthermore, the refeeding response of these mice after a fast was normal. It is concluded that, if there is compensation for changes in NPY levels, then it occurs within the time it takes for Dox treatment to deplete NPY levels. These observations suggest that pharmacological inhibition of NPY signaling is unlikely to have long-lasting effects on body weight (Ste Marie, 2005).

Central neuropeptide Y (NPY) injection has been reported to cause hyperphagia and in some cases also hypometabolism or hypothermia. Chronic central administration induced a moderate rise of short duration in body weight, without consistent metabolic/thermal changes. In the present studies the acute and subsequent subacute ingestive and metabolic/thermal changes were studied following intracerebroventricular (i.c.v.) injections of NPY in cold-adapted and non-adapted rats, or the corresponding chronic changes following i.c.v. NPY infusion. Besides confirming basic earlier data, this study demonstrated novel findings: a temporal relationship for the orexigenic and metabolic/thermal effects, and differences of coordination in acute/subacute/chronic phases or states. The acute phase (30-60 min after injection) was anabolic: coordinated hyperphagia and hypometabolism/hypothermia. NPY evoked a hypothermia by suppressing any (hyper)metabolism in excess of basal metabolic rate, without enhancing heat loss. Thus, acute hypothermia was observed in sub-thermoneutral but not thermoneutral environments. The subsequent subacute catabolic phase exhibited opposite effects: slight increase in metabolic rate, rise in body temperature, reaching a plateau within 3-4 h after injection -- this was maintained for at least 24 h; meanwhile the food intake decreased and the normal daily weight gain stopped. This rebound is only indirectly related to NPY. Chronic (7-day long) i.c.v. NPY infusion induced an anabolic phase for 2-3 days, followed by a catabolic phase and fever, despite continued infusion. In cold-adaptation environment the primary metabolic effect of the infusion induced a moderate hypothermia with lower daytime nadirs and nocturnal peaks of the circadian temperature rhythm, while at near-thermoneutral environments in non-adapted rats the infusion attenuated only the nocturnal temperature rise by suppressing night-time hypermetabolism. Further finding is that in cold-adapted animals, the early feeding effect of NPY-infusion was enhanced, whereas the early hypothermic effect in cold was limited by interference with competing thermoregulatory mechanisms (Szekely, 2005)

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. The effect was examined of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorates diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound does not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, since the compound does not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity (Ishihara, 2006).

Mutation of NPY receptor

Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter that is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. This study describes mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake (Pedrazzini, 1998).

Transcriptional regulation of NPY

Neuropeptide Y (NPY) and Agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. It is hypothesized that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, it has been demonstrated that 17beta-estradiol differentially regulates estrogen receptor (ER)alpha and ERbeta levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ERalpha and ERbeta or ERalpha alone, respectively, whereas the induction of NPY and AgRP in N-38 neurons is strictly ERbeta-dependent, as assessed by ER-specific agonists and siRNA knockdown of ERalpha or ERbeta. Through transient transfection analysis in N-38 neurons, the estrogen-mediated repression of NPY was mapped to within -1078 of the 5' regulatory region of the NPY gene. These results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERbeta to ERalpha. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction (Titolo, 2006).

Ablation of NPY neurons

Hypothalamic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP) are thought to be critical regulators of feeding behavior and body weight. To determine whether NPY/AgRP neurons are essential in mice, the human diphtheria toxin receptor was targeted to the Agrp locus, which allows temporally controlled ablation of NPY/AgRP neurons to occur after an injection of diphtheria toxin. Neonatal ablation of NPY/AgRP neurons has minimal effects on feeding, whereas their ablation in adults causes rapid starvation. These results suggest that network-based compensatory mechanisms can develop after the ablation of NPY/AgRP neurons in neonates but do not readily occur when these neurons become essential in adults (Luquiet, 2005).

Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. This study demonstrates that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis (Gropp, 2005).

Agouti-related protein (AgRP) and neuropeptide Y (NPY) are colocalized in arcuate nucleus (arcuate) neurons implicated in the regulation of energy balance. Both AgRP and NPY stimulate food intake when administered into the third ventricle and are up-regulated in states of negative energy balance. However, mice with targeted deletion of either NPY or AgRP or both do not have major alterations in energy homeostasis. Using bacterial artificial chromosome (BAC) transgenesis expression of a neurotoxic CAG expanded form of ataxin-3 has been targeted to AgRP-expressing neurons in the arcuate. This resulted in a 47% loss of AgRP neurons by 16 weeks of age, a significantly reduced body weight, and reduced food intake. Transgenic mice had significantly reduced total body fat, plasma insulin, and increased brown adipose tissue UCP1 expression. Transgenic mice failed to respond to peripherally administered ghrelin but retained sensitivity to PYY 3-36. These data suggest that postembryonic partial loss of AgRP/NPY neurons leads to a lean, hypophagic phenotype (Bewick, 2006).

Ethanol consumption and resistance are inversely related to neuropeptide Y levels

Genetic linkage analysis of rats that were selectively bred for alcohol preference identified a chromosomal region that includes the neuropeptide Y (NPY) gene. Alcohol-preferring rats have lower levels of NPY in several brain regions compared with alcohol-non-preferring rats. Alcohol consumption by mice that completely lack NPY as a result of targeted gene disruption was studied. NPY-deficient mice show increased consumption, compared with wild-type mice, of solutions containing 6%, 10% and 20% (v/v) ethanol. NPY-deficient mice are also less sensitive to the sedative/hypnotic effects of ethanol, as shown by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations do not differ significantly from those of controls. In contrast, transgenic mice that overexpress a marked NPY gene in neurons that usually express it have a lower preference for ethanol and are more sensitive to the sedative/hypnotic effects of this drug than controls. These data are direct evidence that alcohol consumption and resistance are inversely related to NPY levels in the brain (Thiele, 1998).

Voluntary ethanol consumption and resistance to ethanol-induced sedation are inversely related to neuropeptide Y (NPY) levels in NPY-knock-out (NPY-/-) and NPY-overexpressing mice. Knock-out mice completely lacking the NPY Y1 receptor (Y1-/-) were studied to further characterize the role of the NPY system in ethanol consumption and neurobiological responses to this drug. Male Y1-/- mice show increased consumption of solutions containing 3, 6, and 10% (v/v) ethanol when compared with wild-type control mice. Female Y1-/- mice show increased consumption of a 10% ethanol solution. In contrast, Y1-/- mice show normal consumption of solutions containing either sucrose or quinine. Relative to Y1(+/+) mice, male Y1-/- mice were found to be less sensitive to the sedative effects of 3.5 and 4.0 gm/kg ethanol as measured by more rapid recovery from ethanol-induced sleep, although plasma ethanol levels did not differ significantly between the genotypes. Finally, male Y1-/- mice showed normal ethanol-induced ataxia on the rotarod test after administration of a 2.5 gm/kg dose. These data suggest that the NPY Y1 receptor regulates voluntary ethanol consumption and some of the intoxicating effects caused by administration of ethanol (Thiele, 2002).



EVOLUTIONARY HOMOLOGS

A second set of neuropeptide F peptides in Drosophila and their receptor: Short neuropeptide F precursor and Neuropeptide F-like Receptor 76F

A seven transmembrane G-protein coupled receptor has been cloned from Drosophila melanogaster. This receptor shows structural similarities to vertebrate Neuropeptide Y2 receptors and is activated by endogenous Drosophila peptides, recently designated as short neuropeptide Fs (sNPFs). sNPFs have so far been found in neuroendocrine tissues of four other insect species and of the horseshoe crab. In locusts, they accelerate ovarian maturation, and in mosquitoes, they inhibit host-seeking behavior. Expression analysis by RT-PCR shows that the sNPF receptor (Drm-sNPF-R) is present in several tissues (brain, gut, Malpighian tubules and fat body) from Drosophila larvae as well as in ovaries of adult females. All 4 Drosophila sNPFs clearly elicited a calcium response in receptor expressing mammalian Chinese hamster ovary cells. The response is dose-dependent and appeared to be very specific. The short NPF receptor was not activated by any of the other tested arthropod peptides, not even by FMRFamide-related peptides (also ending in RFamide), indicating that the Arg residue at position 4 from the amidated C-terminus appears to be crucial for the response elicited by the sNPFs (Mertens, 2002).

Since the Drosophila genome encodes at least 4 receptors belonging to the NPY subgroup of receptors, the Drosophila EST database was searched for the presence of EST clones, encoding one of the NPY-type receptors. PCR amplification of the EST clone (GH23382) with oligonucleotide primers specific for the predicted ORF of CG7395 (Neuropeptide F-like Receptor 76F) produced a single product of approximately 1800 bp. Sequence determination of the TA-cloned PCR product revealed a DNA insert of 1803 bp, corresponding to the sequence and size of the predicted receptor in the cDNA database of BDGP. The deduced protein encoded by the ORF of Drm-sNPF-R is 600 amino acids long. Analysis by the TMHMM program revealed that this protein is predicted to have seven transmembrane domains along with the intracellular and extracellular loops, consistent with the known G-protein coupled receptors. The N-terminal extracellular region exhibits no O-glycosylation, 2 N-glycosylation sites, along with 7 Ser/Thr phosphorylation sites (Mertens, 2002).

A phylogenetic tree based on Clustal W alignment of Drm-sNPF-R and various known NPY receptors indicates that Drm-sNPF-R is most closely related to the vertebrate neuropeptide Y2 receptors, i.e., of the domestic guinea pig (33% identity and 49% homology), humans (33% identity and 49% homology), the domestic pig (33% identity and 48% homology), and the rat (33% identity and 48% homology). An NPY-like orphan GPCR of C. elegans (C53C7.1) displays 33% identity and 47% homology. Sequence conservation among Drm-sNPF-R, the human Y2 receptor, and the C. elegans orphan receptor is depicted by similarities shown in their alignment by the AlignX program (Mertens, 2002).

Short NPFs and 'head' peptides display substantial sequence similarities and appear to belong to the same family. All NPFs have a typical R(K)–X1–R–X2amide motif, where the first amino acid of this motif is always a basic amino acid residue such as Arg or Lys. X1 can be L, T or P and X2 is always an aromatic amino acid residue such as Phe or Trp. It is proposed to (re)name all peptides with the R(K)–X1–R–X2amide C-terminal motif, as short NPFs; these peptides do not only occur in the head or central nervous system, but instead reach 10 times higher amounts in the abdomen and the midgut. In addition, their precursor in the Drosophila genome is annotated as the short NPF precursor. The present identification of a specific short NPF receptor in Drosophila is in favor of the presence of functional short NPFs. Several reports indicate that short NPFs have a hormonal function in insects, associated with reproduction and digestion. The expression of the short NPF receptor not only in the nervous system, but also in peripheral targets (ovaries, gut) is in agreement with a hormonal function of short NPFs. Hemolymph from sugar-fed mosquito females contains 414 fmol/μl immunoreactive short NPF. Short NPFs are also abundantly present in endocrine cells of the midgut, suggesting that they might have a function in digestion. The demonstration of the presence of the short NPF receptor transcript in the midgut favors this hypothesis (Mertens, 2002).

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (Neuropeptide F-like Receptor 76F, NPFR76F, or GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62%-66% similarity and 32%-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila (Feng, 2003).

Since the NPFR76F receptor was maximally activated by a short insect NPF-like sequence from Leptinotarsa, genome mining was used to search for Drosophila peptides which might be functionally equivalent to (or better than) the Leptinotarsa I peptide at activating NPFR76F. Initially, one precursor sequence, a gene (npf) encoding Drosophila NPF was identified at chromosome location 89D3. This gene was found by blasting an incomplete version of the Drosophila genome (October 1999) with the amino acid sequence of Aplysia NPY. This revealed a precursor molecule encoding a 36 amino acid peptide with sequence similarity to NPF. The completed Drosophila genome sequence has now been searched and no other potential NPF precursors were identified. Since this peptide (NPF-A1) contained a potential dibasic amino acid cleavage site within its sequence, both the shorter 28 amino acid form (NPF-A2) and the full-length peptide (NPF-A1) were sequenced for testing (Feng, 2003).

A second open reading frame in the Drosophila genome encodes a precursor peptide for two short NPF-like peptides (Drm-sNPF-1, AQRSPSLRLRFamide and Drm-sNPF-2, WFGDVNQKPIRSPSLRLRFamide). The precursor for these peptides is encoded by the short NPF precursor (sNPF) gene (CG13968) and maps to position 38A7 on the left arm of Drosophila chromosome 2. The WFGDVNQKPIRSPSLRLRFamide peptide (Drm-sNPF-2) contains a potential single basic amino acid-processing site. This peptide (Drm-sNPF-2), its shorter form (peptide Drm-sNPF-2 s, PIRSPSLRLRFamide) and the Drm-sNPF-1 peptide (all encoded by the sNPF gene) were synthesized. The same precursor was predicted to include the sequences for two other short peptides, PQRLRWamide and PMRLRWamide, which have been designated Drm-sNPF-3 and Drm-sNPF-4, respectively. These peptides were synthesized and tested (Feng, 2003).

When tested at 1 µm, the shorter NPF-like peptides derived from the sNPF gene (peptides Drm-sNPF-1 and Drm-sNPF-2) were more effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents in Xenopus oocytes expressing NPFR76F and Galpha16. Shortening of peptide Drm-sNPF-2 to the Drm-sNPF-2s form may slightly increase its effectiveness. The longer Drosophila peptides derived from the precursor gene npf at 89D3 were much less effective than the original Leptinotarsa I NPF-like sequence at inducing inward currents. In addition, the PQRLRWamide (Drm-sNPF-3) and PMRLRWamide (Drm-sNPF4) peptides were much less effective than the original Leptinotarsa I sequence at inducing inward currents (Feng, 2003).

Dose-response curves for the shorter endogenous Drosophila NPF-like peptides encoded by the sNPF gene at 38A7 reveal that AQRSPSLRLRFamide (peptide Drm-sNPF-1) is the most potent peptide tested (pEC50 = -8.84). It showed a threshold for the generation of inward currents between 100 pm and 1 nm and a maximal effect at 100 nm. The second putative endogenous Drosophila NPF-like peptide encoded by the sNPF gene, PIRSPSLRFamide (peptide Drm-sNPF-2 s) (pEC50 = -7.62), and the original Leptinotarsa I NPF-like sequence, ARGPQLRLRFamide (pEC50 = -7.83), were an order of magnitude less potent than AQRSPSLRLRFamide. These results justify the classification of NPFR76F as a NPF-like receptor and suggest that the short peptide AQRSPSLRLRFamide (sNPF-1) may be the endogenous agonist for this receptor. The other two short peptides encoded by the sNPF gene, PQRLRWamide (pEC50 = -6.1) and PMRLRWamide (pEC50 = -7.30), were 2.7 and 1.5 orders of magnitude, respectively, less potent than the AQRSPSLRLRFamide sequence, leading to questioning of their designation as true short NPF-like peptides (Feng, 2003).

Expression of NPFR76F transcripts was assessed by Northern blot analysis of poly(A)+ RNA prepared from adult body parts. A single transcript of 6.5 kb was detected in both heads and appendages (legs and antennae), suggesting that NPFR76F is expressed in both the central and peripheral nervous systems. In addition to finding transcript in heads and appendages, trace amounts were also seen in bodies. When compared with the amount of RNA loaded from each body part (as indicated by the ubiquitous rp49 loading control), the relative abundance of transcript in bodies is very low. This distribution of the NPFR76F transcript is consistent with a role for this NPF-like receptor in the Drosophila nervous system (Feng, 2003).

To further refine the NPFR76F receptor transcript expression, in situ hybridization with a digoxigenin-labelled antisense RNA probe to whole mounts of the mature embryos was used. The central nervous system in embryos is composed of two dorsal brain hemispheres and a fused ventral ganglion. The NPFR76F receptor is expressed both in the dorsal brain and in the ventral ganglion. In the brain the receptor is strongly expressed in the specific cells in the dorsal posterior region. It is estimated that there are 22-24 cells in each brain lobe expressing NPFR76F, including the strongly expressing cells. In the ventral ganglion, pairs of cells along the ventral midline, as well as cells found in a bilaterally symmetric pattern in a more lateral position from the midline, also express receptor mRNA. In each full segment of the ventral ganglion, the receptor is strongly expressed in eight to 12 cells, including a pair of cells at the midline in each segment (Feng, 2003).

In the peripheral nervous system the receptor is expressed in a subset of sensilla and in the anterior sensory complex. It is estimated that there are 10-14 cells in the anterior sensory complex, including the antennomaxillary complex, the labral sensory complex and the labial sensory complex, that express NPFR76F. Finally, in the posterior sensilla, there are eight cells that express NPFR76F. The expression pattern of the NPFR76F receptor in many specific cells in the dorsal brain, the ventral ganglion, lateral sensilla, the anterior sensory complex and the posterior sensilla suggests that this receptor is involved in a widespread modulation of neuronal activity (Feng, 2003).

A Drosophila melanogaster G-protein-coupled receptor (NPFR76F) that is activated by neuropeptide F-like peptides has been expressed in Xenopus oocytes to determine its ability to regulate heterologously expressed G-protein-coupled inwardly rectifying potassium channels. The activated receptor produced inwardly rectifying potassium currents by a pertussis toxin-sensitive G-protein-mediated pathway and the effects were reduced in the presence of proteins, such as the betaARK 1 carboxy-tail fragment and alpha-transducin, which bind G-protein betagamma-subunits. Short Drosophila NPF-like peptides are more potent than long NPF-like peptides at coupling the receptor to the activation of inwardly rectifying potassium channels. The putative endogenous short Drosophila NPF-like peptides showed agonist-specific coupling depending on whether their actions were assessed as the activation of the inwardly rectifying potassium channels or as the activation of endogenous inward chloride channels through a co-expressed promiscuous G-protein, Galpha16. As inwardly rectifying potassium channels are known to be encoded in the Drosophila genome and the NPFR76F receptor is widely expressed in the Drosophila nervous system, the receptor could function to control neuronal excitability or slow wave potential generation in the Drosophila nervous system (Reale, 2004).

Neuropeptides regulate a wide range of animal behavior including food consumption, circadian rhythms, and anxiety. Recently, Drosophila neuropeptide F, which is the homolog of the vertebrate neuropeptide Y, was cloned, and the function of Drosophila neuropeptide F in feeding behaviors was well characterized. However, the function of the structurally related short neuropeptide F (sNPF) was unknown. This study reports the cloning, RNA, and peptide localizations, and functional characterizations of the Drosophila sNPF gene. The sNPF gene encodes the preprotein containing putative RLRF amide peptides and was expressed in the nervous system of late stage embryos and larvae. The embryonic and larval localization of the sNPF peptide in the nervous systems revealed the larval central nervous system neural circuit from the neurons in the brain to thoracic axons and to connective axons in the ventral ganglion. In the adult brain, the sNPF peptide was localized in the medulla and the mushroom body. However, the sNPF peptide was not detected in the gut. The sNPF mRNA and the peptide were expressed during all developmental stages from embryo to adult. From the feeding assay, the gain-of-function sNPF mutants expressed in nervous systems promoted food intake, whereas the loss-of-function mutants suppressed food intake. Also, sNPF overexpression in nervous systems produced bigger and heavier flies. These findings indicate that the sNPF is expressed in the nervous systems to control food intake and regulate body size in Drosophila melanogaster (Lee, 2004).

Various evidence suggests that sNPF and dNPF peptides have different functions. The sNPF peptide is found only in nervous systems, whereas the neuropeptide F (dNPF) is found as the Drosophila brain-gut peptide. The expression patterns of sNPF and dNPF differ in the larval brain. For example, the sNPF expression is found in the anterior dorsal neurons of the brain, whereas the dNPF expression is detected in the four neurons of larval brain. In the feeding behavior analysis, overexpression of sNPF in wandering larvae did not extend the feeding period, contrary to the extension of the feeding period in the wandering larval stage by overexpression of dNPF. At the receptor level, each peptide works in different receptors; for example, the NPFR76F receptor is for sNPF peptides, and the DmNPFR1 receptor is for the dNPF. These differences indicate that the dNPF and sNPF peptides function in different neurons and may regulate different aspects of feeding behaviors in Drosophila (Lee, 2004).

Like other neuropeptides, the sNPF peptide may be involved in regulating various physiological processes other than regulating food intake because the sNPF peptide and transcript were expressed during all developmental stages, and the sNPF is localized in the mushroom body calyx and medulla of the adult brain. The mushroom body is involved in learning and memory. These unknown multi-functions of the sNPF peptide in various biological processes are the subjects of future studies (Lee, 2004).

Among insects, short neuropeptide Fs (sNPF) have been implicated in regulation of reproduction and feeding behavior. For Drosophila melanogaster, the nucleotide sequence for the sNPF precursor protein encodes four distinctive candidate sNPFs. In the present study, all four peptides were identified by mass spectrometry in body extracts of D. melanogaster; some also were identified in hemolymph, suggesting potential neuroendocrine roles. Actions of sNPFs in D. melanogaster are mediated by the G protein-coupled receptor Drm-NPFR76F. Mammalian CHO-K1 cells were stably transfected with the Drm-NPFR76F receptor for membrane-based radioreceptor studies. Binding assays revealed that longer sNPF peptides comprised of nine or more amino acids are clearly more potent than shorter ones of eight or fewer amino acids. These findings extend understanding of the relationship between structure and function of sNPFs (Garczynski, 2006).

The sNPFs of D. melanogaster differ in their interactions with the sNPF receptor Drm-NPFR76F, as analyzed directly by radioreceptor assay. A wide variety of D. melanogaster sNPFs were assayed for their ability to inhibit the binding of 125I-[D-Y1]-Drm-sNPF1 to membranes prepared from cells stably transfected with Drm-NPFR76F. Two distinctive classes of activity of sNPFs were readily apparent. The sNPF peptides containing nine or more amino acids typically exhibited high affinity, as judged by an IC50 < 1 nM, whereas peptides containing eight for fewer amino acids exhibited IC50 values of >5 nM. Those exhibiting lower affinity included sNPF3 and sNPF4, peptides with the C-terminal RLRWa sequence. The minimum length of the highly active group was represented by sNPF211–19. The sequence of this sNPF211–19 (RSPSLRLRFa) differs from sNPF14–11 (SPSLRLRFa) only by a single arginine residue, which appears to confer a substantial increase in binding affinity. To test this hypothesis, an alanine substituted analog, Drm-sNPF211–19R11A, was assayed and found to exhibit a substantial drop in activity, with an IC50 of only 12.5 nM, indicating the crucial role of this arginine residue (Garczynski, 2006).

For further tests of these apparent structure-function relations, putative sNPFs identified in the genomes of A. gambiae and A. aegypti also were examined in the Drm-NPFR76F radioreceptor assay. Each of these mosquito sNPF peptides conformed to the distinctive pattern of length-associated activity established previously for those of D. melanogaster. In contrast, the A. aegypti head peptides which partly resemble sNPFs were either weakly active, Aea-HP-I, or inactive, Aea-HP-III, despite being of sufficient length and having the requisite arginine. Accordingly, additional structural features in the C-terminus common to sNPF peptides appear important for high affinity binding (Garczynski, 2006).

Neuropeptide F in other insects

The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50 approximately 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF>Aea-NPF>Drm-NPF>Drm-NPF8-36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito (Garczynski, 2005).

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal (Stanek, 2002).

Neuropeptide Y receptor in C. elegans

Natural isolates of C. elegans exhibit either solitary or social feeding behavior. Solitary foragers move slowly on a bacterial lawn and disperse across it, while social foragers move rapidly on bacteria and aggregate together. A loss-of-function mutation in the npr-1 gene, which encodes a predicted G protein-coupled receptor similar to neuropeptide Y receptors, causes a solitary strain to take on social behavior. Two isoforms of NPR-1 that differ at a single residue occur in the wild. One isoform, NPR-1 215F, is found exclusively in social strains, while the other isoform, NPR-1 215V, is found exclusively in solitary strains. An NPR-1 215V transgene can induce solitary feeding behavior in a wild social strain. Thus, isoforms of a putative neuropeptide receptor generate natural variation in C. elegans feeding behavior (de Bono, 1998).

Wild isolates of Caenorhabditis elegans can feed either alone or in groups. This natural variation in behaviour is associated with a single residue difference in NPR-1, a predicted G-protein-coupled neuropeptide receptor related to Neuropeptide Y receptors. The NPR-1 isoform associated with solitary feeding acts in neurons exposed to the body fluid to inhibit social feeding. Furthermore, suppressing the activity of these neurons, called AQR, PQR and URX, using an activated K(+) channel, inhibits social feeding. NPR-1 activity in AQR, PQR and URX neurons seems to suppress social feeding by antagonizing signalling through a cyclic GMP-gated ion channel encoded by tax-2 and tax-4. Mutations in tax-2 or tax-4 disrupt social feeding, and tax-4 is required in several neurons for social feeding, including one or more of AQR, PQR and URX. The AQR, PQR and URX neurons are unusual in C. elegans because they are directly exposed to the pseudocoelomic body fluid. The data suggest a model in which these neurons integrate antagonistic signals to control the choice between social and solitary feeding behaviour (Coates, 2002).

Variation in the acute response to ethanol between individuals has a significant impact on determining susceptibility to alcoholism. The degree to which genetics contributes to this variation is of great interest. Allelic variation that alters the functional level of NPR-1, a neuropeptide Y (NPY) receptor-like protein, can account for natural variation in the acute response to ethanol in wild strains of C. elegans. NPR-1 negatively regulates the development of acute tolerance to ethanol, a neuroadaptive process that compensates for effects of ethanol. Furthermore, dynamic changes in the NPR-1 pathway provide a mechanism for ethanol tolerance in C. elegans. This suggests an explanation for the conserved function of NPY-related pathways in ethanol responses across diverse species. Moreover, these data indicate that genetic variation in the level of NPR-1 function determines much of the phenotypic variation in adaptive behavioral responses to ethanol that are observed in natural populations (Davies, 2003).

Mutation of mammalian neuropeptide Y

Neuropeptide Y (NPY), a 36-amino-acid transmitter distributed throughout the nervous system, is thought to function as a central stimulator of feeding behaviour. NPY has also been implicated in the modulation of mood, cerebrocortical excitability, hypothalamic-pituitary signalling, cardiovascular physiology and sympathetic function. However, the biological significance of NPY has been difficult to establish owing to a lack of pharmacological antagonists. Mice deficient for NPY have normal food intake and body weight, and become hyperphagic following food deprivation. Mutant mice decrease their food intake and lose weight, initially to a greater extent than controls, when treated with recombinant leptin. Occasional, mild seizures occur in NPY-deficient mice and mutants are more susceptible to seizures induced by a GABA (gamma-aminobutyric acid) antagonist. These results indicate that NPY is not essential for certain feeding responses or leptin actions but is an important modulator of excitability in the central nervous system (Erickson, 1996a).

The obesity syndrome of ob/ob mice results from lack of leptin, a hormone released by fat cells that acts in the brain to suppress feeding and stimulate metabolism. Neuropeptide Y (NPY) is a neuromodulator implicated in the control of energy balance and is overproduced in the hypothalamus of ob/ob mice. To determine the role of NPY in the response to leptin deficiency, ob/ob mice deficient for NPY were generated. In the absence of NPY, ob/ob mice are less obese because of reduced food intake and increased energy expenditure, and are less severely affected by diabetes, sterility, and somatotropic defects. These results suggest that NPY is a central effector of leptin deficiency (Erickson, 1996b).

An extensive behavioral characterization was conducted with mice lacking the gene for neuropeptide Y (NPY) including response to 24 and 48 h fast and challenge with small molecule antagonists of NPY receptors implicated in mediating the feeding effects of NPY (i.e., Y1 and Y5). In addition, wildtype (WT) and NPY knockout (KO) mice were tested in locomotor monitors, elevated plus maze, inhibitory avoidance, acoustic startle, prepulse inhibition, and hot plate assays. One of the major findings was that the NPY KO mice have a reduced food intake relative to WT controls in response to fasting. Also, based on data from the behavioral models, the NPY KO mice may have an anxiogenic-like phenotype, and appear to be hypoalgesic in the hot plate paradigm. The data from these studies provide further evidence of involvement of NPY in energy balance, anxiety, and possibly nociception (Bannon, 2000).

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. AgRP-deficient (Agrp-/-) mice were generated to examine the physiological role of AgRP. Agrp-/- mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp-/- mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp-/- mice. Since AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, AgRP and NPY double-knockout (Agrp-/-;Npy-/-) mice were generated to determine whether NPY or AgRP plays a compensatory role in Agrp-/- or NPY-deficient (Npy-/-) mice, respectively. Similar to mice deficient in either AgRP or NPY, Agrp-/-;Npy-/- mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. These results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY (Qian, 2003).

Neuropeptide Y (NPY) is an orexigenic (appetite-stimulating) peptide that plays an important role in regulating energy balance. When administered directly into the central nervous system, animals exhibit an immediate increase in feeding behavior, and repetitive injections or chronic infusions lead to obesity. Surprisingly, initial studies of Npy-/- mice on a mixed genetic background did not reveal deficits in energy balance, with the exception of an attenuation in obesity seen in ob/ob mice in which the NPY gene was also deleted. On a C57BL/6 background, NPY ablation is associated with an increase in body weight and adiposity and a significant defect in refeeding after a fast. This impaired refeeding response in Npy-/- mice resulted in a deficit in weight gain in these animals after 24 h of refeeding. These data indicate that genetic background must be taken into account when the biological role of NPY is evaluated. When examined on a C57BL/6 background, NPY is important for the normal refeeding response after starvation, and its absence promotes mild obesity (Segal-Lieberman, 2003).

Neuropeptide Y (NPY) is a potent orexigenic peptide that is implicated in the feeding response to a variety of stimuli. The current studies employed mice lacking NPY (Npy-/-) and their wild-type (Npy+/+) littermates to investigate the role of this peptide in the feeding response to circadian and palatability cues. To investigate the response to a circadian stimulus, food intake was assessed during the 4-h period following dark onset, a time of day characterized by maximal rates of food consumption. Compared to Npy+/+ controls, intake of Npy-/- mice was reduced by 33% during this period. In contrast, intake did not differ between genotypes when measured over a 24-h period. Furthermore, reduced dark cycle 4h food intake in Npy-/- mice was not evident after a 24-h fast, despite a pronounced delay in the initiation of feeding. To investigate the role of NPY in the feeding response to palatability cues, mice were presented with a highly palatable diet (HP) for 1h each day (in addition to having ad libitum access to chow) for 18 days. Npy+/+ mice rapidly increased daily HP intake such that by the end of the first week, they derived a substantial fraction of daily energy from this source. By comparison, HP intake was markedly reduced in Npy-/- mice during the first week, although it eventually increased (by Day 9) to values comparable to those of Npy+/+ controls. These experiments suggest that NPY contributes to the mechanism whereby food intake increases in response to circadian and palatability cues and that mechanisms driving food intake in response to these stimuli differ from those activated by energy restriction (Sindelar, 2005).

Biological effects of NPY in mammals

Exogenous neuropeptide Y (NPY) reduces experimental anxiety in a wide range of animal models. The generation of an NPY-transgenic rat has provided a unique model to examine the role of endogenous NPY in control of stress and anxiety-related behaviors using paradigms previously used by pharmacological studies. Locomotor activity and baseline behavior on the elevated plus maze were normal in transgenic subjects. Two robust phenotypic traits were observed. (1) Transgenic subjects showed a markedly attenuated sensitivity to behavioral consequences of stress, in that they were insensitive to the normal anxiogenic-like effect of restraint stress on the elevated plus maze and displayed absent fear suppression of behavior in a punished drinking test. (2) A selective impairment of spatial memory acquisition was found in the Morris water maze. Control experiments suggest these traits to be independent. These phenotypic traits were accompanied by an overexpression of prepro-NPY mRNA and NPY peptide and decreased NPY-Y1 binding within the hippocampus, a brain structure implicated both in memory processing and stress responses. Data obtained using this unique model support and extend a previously postulated anti-stress action of NPY and provide novel evidence for a role of NPY in learning and memory (Thorsell, 2000).

Neuropeptide Y (NPY), one of the most abundant peptide transmitters in the mammalian brain, is assumed to play an important role in feeding and body weight regulation. However, there is little genetic evidence that overexpression or knockout of the NPY gene leads to altered body weight regulation. NPY-overexpressing mice have been developed by using the Thy-1 promoter, which restricts NPY expression strictly within neurons in the central nervous system, but the obese phenotype was not observed in the heterozygote. In the homozygous mice, overexpression of NPY leads to an obese phenotype, but only after appropriate dietary exposure. NPY-overexpressing mice exhibit significantly increased body weight gain with transiently increased food intake after 50% sucrose-loaded diet, and later they developed hyperglycemia and hyperinsulinemia without altered glucose excursion during 1 year of the observation period (Kaga, 2001).

Despite numerous experiments showing that administration of neuropeptide Y (NPY) to rodents stimulates feeding and obesity, whereas acute interference with NPY signaling disrupts feeding and promotes weight loss, NPY-null mice have essentially normal body weight regulation. These conflicting observations suggest that chronic lack of NPY during development may lead to compensatory changes that normalize regulation of food intake and energy expenditure in the absence of NPY. To test this idea, gene targeting was used to introduce a doxycycline (Dox)-regulated cassette into the Npy locus, such that NPY would be expressed until the mice were given Dox, which blocks transcription. Compared with wild-type mice, adult mice bearing this construct expressed approximately 4-fold more Npy mRNA, which fell to approximately 20% of control values within 3 days after treatment with Dox. NPY protein also fell approximately 20-fold, but the half-life of approximately 5 days was surprisingly long. The biological effectiveness of these manipulations was demonstrated by showing that overexpression of NPY protected against kainate-induced seizures. Mice chronically overexpressing NPY had normal body weight, and administration of Dox to these mice did not suppress feeding. Furthermore, the refeeding response of these mice after a fast was normal. It is concluded that, if there is compensation for changes in NPY levels, then it occurs within the time it takes for Dox treatment to deplete NPY levels. These observations suggest that pharmacological inhibition of NPY signaling is unlikely to have long-lasting effects on body weight (Ste Marie, 2005).

Central neuropeptide Y (NPY) injection has been reported to cause hyperphagia and in some cases also hypometabolism or hypothermia. Chronic central administration induced a moderate rise of short duration in body weight, without consistent metabolic/thermal changes. In the present studies the acute and subsequent subacute ingestive and metabolic/thermal changes were studied following intracerebroventricular (i.c.v.) injections of NPY in cold-adapted and non-adapted rats, or the corresponding chronic changes following i.c.v. NPY infusion. Besides confirming basic earlier data, this study demonstrated novel findings: a temporal relationship for the orexigenic and metabolic/thermal effects, and differences of coordination in acute/subacute/chronic phases or states. The acute phase (30-60 min after injection) was anabolic: coordinated hyperphagia and hypometabolism/hypothermia. NPY evoked a hypothermia by suppressing any (hyper)metabolism in excess of basal metabolic rate, without enhancing heat loss. Thus, acute hypothermia was observed in sub-thermoneutral but not thermoneutral environments. The subsequent subacute catabolic phase exhibited opposite effects: slight increase in metabolic rate, rise in body temperature, reaching a plateau within 3-4 h after injection -- this was maintained for at least 24 h; meanwhile the food intake decreased and the normal daily weight gain stopped. This rebound is only indirectly related to NPY. Chronic (7-day long) i.c.v. NPY infusion induced an anabolic phase for 2-3 days, followed by a catabolic phase and fever, despite continued infusion. In cold-adaptation environment the primary metabolic effect of the infusion induced a moderate hypothermia with lower daytime nadirs and nocturnal peaks of the circadian temperature rhythm, while at near-thermoneutral environments in non-adapted rats the infusion attenuated only the nocturnal temperature rise by suppressing night-time hypermetabolism. Further finding is that in cold-adapted animals, the early feeding effect of NPY-infusion was enhanced, whereas the early hypothermic effect in cold was limited by interference with competing thermoregulatory mechanisms (Szekely, 2005)

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. The effect was examined of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorates diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound does not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, since the compound does not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity (Ishihara, 2006).

Mutation of NPY receptor

Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter that is widely distributed throughout the central and peripheral nervous system. NPY involvement has been suggested in various physiological responses including cardiovascular homeostasis and the hypothalamic control of food intake. At least six subtypes of NPY receptors have been described. Because of the lack of selective antagonists, the specific role of each receptor subtype has been difficult to establish. This study describes mice deficient for the expression of the Y1 receptor subtype. Homozygous mutant mice demonstrate a complete absence of blood pressure response to NPY, whereas they retain normal response to other vasoconstrictors. Daily food intake, as well as NPY-stimulated feeding, are only slightly diminished, whereas fast-induced refeeding is markedly reduced. Adult mice lacking the NPY Y1 receptor are characterized by increased body fat with no change in protein content. The higher energetic efficiency of mutant mice might result, in part, from the lower metabolic rate measured during the active period, associated with reduced locomotor activity. These results demonstrate the importance of NPY Y1 receptors in NPY-mediated cardiovascular response and in the regulation of body weight through central control of energy expenditure. In addition, these data are also indicative of a role for the Y1 receptor in the control of food intake (Pedrazzini, 1998).

Transcriptional regulation of NPY

Neuropeptide Y (NPY) and Agouti-related peptide (AgRP) stimulate feeding, whereas NPY also facilitates the estrogen-mediated preovulatory GnRH surge. In addition to regulating reproductive function, estrogen also acts as an anorexigenic hormone, although it is not yet known which hypothalamic neurons are involved in this process. It is hypothesized that estrogen may directly control hypothalamic NPY and/or AgRP synthesis to influence energy homeostasis. Using two clonal, murine hypothalamic neuronal cell models, N-38 and N-42, it has been demonstrated that 17beta-estradiol differentially regulates estrogen receptor (ER)alpha and ERbeta levels, as well as NPY and AgRP gene expression in a manner that is temporally coordinated with the changes in ER abundance. The estrogen-mediated repression of NPY and AgRP mRNA levels in N-38 and N-42 neurons require either ERalpha and ERbeta or ERalpha alone, respectively, whereas the induction of NPY and AgRP in N-38 neurons is strictly ERbeta-dependent, as assessed by ER-specific agonists and siRNA knockdown of ERalpha or ERbeta. Through transient transfection analysis in N-38 neurons, the estrogen-mediated repression of NPY was mapped to within -1078 of the 5' regulatory region of the NPY gene. These results provide the first evidence that NPY and AgRP gene expression is directly regulated by estrogen in specific hypothalamic neurons, and that this regulation is dependent upon the ratio of ERbeta to ERalpha. The biphasic control of neuronal NPY/AgRP transcription may be a mechanism by which estrogen has distinct effects on both energy homeostasis and reproduction (Titolo, 2006).

Ablation of NPY neurons

Hypothalamic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP) are thought to be critical regulators of feeding behavior and body weight. To determine whether NPY/AgRP neurons are essential in mice, the human diphtheria toxin receptor was targeted to the Agrp locus, which allows temporally controlled ablation of NPY/AgRP neurons to occur after an injection of diphtheria toxin. Neonatal ablation of NPY/AgRP neurons has minimal effects on feeding, whereas their ablation in adults causes rapid starvation. These results suggest that network-based compensatory mechanisms can develop after the ablation of NPY/AgRP neurons in neonates but do not readily occur when these neurons become essential in adults (Luquiet, 2005).

Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. This study demonstrates that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis (Gropp, 2005).

Agouti-related protein (AgRP) and neuropeptide Y (NPY) are colocalized in arcuate nucleus (arcuate) neurons implicated in the regulation of energy balance. Both AgRP and NPY stimulate food intake when administered into the third ventricle and are up-regulated in states of negative energy balance. However, mice with targeted deletion of either NPY or AgRP or both do not have major alterations in energy homeostasis. Using bacterial artificial chromosome (BAC) transgenesis expression of a neurotoxic CAG expanded form of ataxin-3 has been targeted to AgRP-expressing neurons in the arcuate. This resulted in a 47% loss of AgRP neurons by 16 weeks of age, a significantly reduced body weight, and reduced food intake. Transgenic mice had significantly reduced total body fat, plasma insulin, and increased brown adipose tissue UCP1 expression. Transgenic mice failed to respond to peripherally administered ghrelin but retained sensitivity to PYY 3-36. These data suggest that postembryonic partial loss of AgRP/NPY neurons leads to a lean, hypophagic phenotype (Bewick, 2006).

Ethanol consumption and resistance are inversely related to neuropeptide Y levels

Genetic linkage analysis of rats that were selectively bred for alcohol preference identified a chromosomal region that includes the neuropeptide Y (NPY) gene. Alcohol-preferring rats have lower levels of NPY in several brain regions compared with alcohol-non-preferring rats. Alcohol consumption by mice that completely lack NPY as a result of targeted gene disruption was studied. NPY-deficient mice show increased consumption, compared with wild-type mice, of solutions containing 6%, 10% and 20% (v/v) ethanol. NPY-deficient mice are also less sensitive to the sedative/hypnotic effects of ethanol, as shown by more rapid recovery from ethanol-induced sleep, even though plasma ethanol concentrations do not differ significantly from those of controls. In contrast, transgenic mice that overexpress a marked NPY gene in neurons that usually express it have a lower preference for ethanol and are more sensitive to the sedative/hypnotic effects of this drug than controls. These data are direct evidence that alcohol consumption and resistance are inversely related to NPY levels in the brain (Thiele, 1998).

Voluntary ethanol consumption and resistance to ethanol-induced sedation are inversely related to neuropeptide Y (NPY) levels in NPY-knock-out (NPY-/-) and NPY-overexpressing mice. Knock-out mice completely lacking the NPY Y1 receptor (Y1-/-) were studied to further characterize the role of the NPY system in ethanol consumption and neurobiological responses to this drug. Male Y1-/- mice show increased consumption of solutions containing 3, 6, and 10% (v/v) ethanol when compared with wild-type control mice. Female Y1-/- mice show increased consumption of a 10% ethanol solution. In contrast, Y1-/- mice show normal consumption of solutions containing either sucrose or quinine. Relative to Y1(+/+) mice, male Y1-/- mice were found to be less sensitive to the sedative effects of 3.5 and 4.0 gm/kg ethanol as measured by more rapid recovery from ethanol-induced sleep, although plasma ethanol levels did not differ significantly between the genotypes. Finally, male Y1-/- mice showed normal ethanol-induced ataxia on the rotarod test after administration of a 2.5 gm/kg dose. These data suggest that the NPY Y1 receptor regulates voluntary ethanol consumption and some of the intoxicating effects caused by administration of ethanol (Thiele, 2002).


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date revised: 15 April 2014

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