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1.  Behavioral phenotyping of Neuropeptide S receptor knockout mice 
Behavioural brain research  2009;205(1):1-9.
Central administration of Neuropeptide S (NPS) in rodents induces arousal and prolonged wakefulness as well as anxiolytic-like effects. NPS has also been implicated in modulation of cognitive functions and energy homeostasis. Here we present a comprehensive phenotypical analysis of mice carrying a targeted mutation in the NPS receptor (NPSR) gene. NPSR knockout mice were found to exhibit reduced exploratory activity when challenged with a novel environment, which might indicate attenuated arousal. We also observed attenuated late peak wheel running activity in NPSR knockout mice, representing reduced activity during the subjective evening. These mice also displayed increased anxiety-like behaviors when compared to their wildtype littermates, although analysis of anxiety behaviors was limited by genetic background influences. Unexpectedly, NPSR knockout mice showed enhanced motor performance skills. No phenotypical differences were detected in the forced-swim test, startle habituation and pre-pulse inhibition paradigms. Together, these data indicate that the endogenous NPS system might be involved in setting or maintaining behavioral arousal thresholds and that the NPS system might have other yet undiscovered physiological functions.
doi:10.1016/j.bbr.2009.07.024
PMCID: PMC2753742  PMID: 19646487
arousal; circadian; anxiety; motor performance; neuropeptide
2.  Neuropeptide S attenuates neuropathological, neurochemical and behavioral changes induced by the NMDA receptor antagonist MK-801 
Neuropharmacology  2009;58(1):166-172.
Neuropeptide S (NPS) and its cognate receptor were reported to mediate anxiolytic-like and arousal effects. NPS receptors are predominantly expressed in the brain, especially in limbic structures, including amygdala, olfactory nucleus, subiculum and retrosplenial cortex. In contrast, the NPS precursor is expressed in only a few brainstem nuclei where it is co-expressed with various excitatory transmitters, including glutamate. The current study investigates interactions of the NPS system with glutamatergic neurotransmission. It has been suggested that dysfunctions in glutamatergic neurotransmission via N-methyl-D-aspartate (NMDA) receptors might be involved in the pathophysiology of schizophrenia since NMDA receptor antagonists, such as MK-801, have been shown to induce psychotic-like behavior in humans and animal models. Also, MK-801 is known to produce histological changes such as cytoplasmic vacuoles in retrosplenial cortex neurons where NPS receptors are highly expressed. In this study we show that NPS is able to alleviate neuropathological, neurochemical and behavioral changes produced by NMDA receptor antagonists. NPS treatment attenuated MK-801-induced vacuolization in the rat retrosplenial cortex in a dose dependent manner that can be blocked by an NPS receptor-selective antagonist. NPS also suppressed MK-801-induced increases of extracellular acetylcholine levels in the retrosplenial cortex. In the prepulse inhibition (PPI) assay, animals pretreated with NPS recovered significantly from MK-801-induced disruption of PPI. Our study suggests that NPS may have protective effects against the neurotoxic and behavioral changes produced by NMDA receptor antagonists and that NPS receptor agonists may elicit antipsychotic effects.
doi:10.1016/j.neuropharm.2009.06.027
PMCID: PMC2783386  PMID: 19576911
glutamate; NMDA; microdialysis; neuropeptide; schizophrenia; prepulse inhibition; retrosplenial cortex
3.  Importance of extracellular loop one of the Neuropeptide S receptor for biogenesis and function 
Peptides  2009;31(1):130.
Neuropeptide S (NPS) is the endogenous ligand of a formerly orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain. NPS promotes arousal and induces anxiolytic-like effects after central administration in rodents. Previously, we have reported that the N107I polymorphism in the human NPS receptor results in a gain-of-function characterized by an increase in agonist potency without changing agonist binding affinity. We have extended our findings by investigating pharmacological and biochemical consequences of mutations in the vicinity of position 107. Alanine substitutions were made for D105 and N101, and stable clones were analyzed for agonist-induced changes of intracellular Ca2+. Receptor protein expression was monitored by Western blot and flow cytometry. The mutation D105A produced receptors that have a ∼200-fold higher EC50 despite elevated total receptor protein and surface expression compared to cell lines expressing the parental receptor NPSR-N107. The mutation N101A resulted in slightly reduced agonist potency without affecting the ability of the protein to form functional receptors. Stable NPSR-A101 clones show little expression of the fully glycosylated form. However, NPSR-A101 receptors are expressed on the cell surface and are functional, suggesting that full glycosylation is not required for receptor function. Our studies suggest that N-linked glycosylation is not important for receptor biogenesis or function, and that residue D105 might be critical for receptor binding.
doi:10.1016/j.peptides.2009.10.015
PMCID: PMC2814945  PMID: 19874863
Neuropeptide S; GPCR; extracellular loop one; biogenesis; mutagenesis; pharmacological chaperone
4.  Synthesis and pharmacological in vitro and in vivo profile of SHA 68 (3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide), a selective antagonist of the Neuropeptide S receptor 
Neuropeptide S (NPS) has been shown to modulate arousal, sleep-wakefulness, anxiety-like behavior and feeding after central administration of the peptide agonist to mice or rats. We report here the chemical synthesis and pharmacological characterization of SHA 66 (3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid benzylamide) and SHA 68 (3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide), two closely related bicyclic piperazines with antagonistic properties at the NPS receptor (NPSR). The compounds block NPS-induced Ca2+-mobilization and SHA 68 shows displaceable binding to NPSR in the nanomolar range. The antagonistic activity of SHA 68 appears to be specific as it does not affect signaling at fourteen unrelated G protein-coupled receptors. Analysis of pharmacokinetic parameters of SHA 68 demonstrates that the compound reaches pharmacologically relevant levels in plasma and brain following intraperitoneal (i.p.) administration. Furthermore, peripheral administration of SHA 68 in mice (50 mg/kg, i.p.) is able to antagonize NPS-induced horizontal and vertical activity as well as stereotypic behavior. Therefore, SHA 68 could be a useful tool to characterize physiological functions and pharmacological parameters of the NPS system in vitro and in vivo.
doi:10.1124/jpet.107.135103
PMCID: PMC2583099  PMID: 18337476
5.  Phylogenetic appearance of Neuropeptide S precursor proteins in tetrapods 
Peptides  2007;28(4):830-837.
Sleep and emotional behavior are two hallmarks of vertebrate animal behavior, implying that specialized neuronal circuits and dedicated neurochemical messengers may have been developed during evolution to regulate such complex behaviors. Neuropeptide S (NPS) is a newly identified peptide transmitter that activates a typical G protein-coupled receptor. Central administration of NPS produces profound arousal, enhances wakefulness and suppresses all stages of sleep. In addition, NPS can alleviate behavioral responses to stress by producing anxiolytic-like effects. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates with the exception of fish. A high level of sequence conservation, especially of aminoterminal structures was detected, indicating stringent requirements for agonist-induced receptor activation. Duplication of the NPS precursor gene was only found in one out of two marsupial species with sufficient genome coverage (Monodelphis domestica; opossum), indicating that the duplicated opossum NPS sequence might have arisen as an isolated event. Pharmacological analysis of both Monodelphis NPS peptides revealed that only the closely related NPS peptide retained agonistic activity at NPS receptors. The duplicated precursor might be either a pseudogene or could have evolved different receptor selectivity. Together, these data show that NPS is a relatively recent gene in vertebrate evolution whose appearance might coincide with its specialized physiological functions in terrestrial vertebrates.
doi:10.1016/j.peptides.2007.01.008
PMCID: PMC1868679  PMID: 17293003
neuropeptide; evolution; tetrapods; arousal; sleep
6.  Synthesis and separation of the enantiomers of the Neuropeptide S receptor antagonist (9R/S)-3-Oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68) 
Journal of medicinal chemistry  2011;54(8):2738-2744.
This study reports the synthesis, chromatographic separation and pharmacological evaluation of the two enantiors of the neuropeptide S receptor (NPSR) antagonist (9R/S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (SHA 68). The (9R)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10) and (9S)-3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide (compound 10a) were synthesized and their purity assessed by chiral chromatography. The absolute configuration of the enantiomer 10 has been assigned from the crystal structure of the corresponding (S)-phenyl ethyl amine derivative 8. Calcium mobilization studies performed on cells expressing the recombinant NPSR demonstrated that compound 10 is the active enantiomer while the contribution of 10a to the NPSR antagonist properties of the racemic mixture is negligible.
doi:10.1021/jm200138r
PMCID: PMC3095364  PMID: 21466221
7.  Neuropeptide S Enhances Memory During the Consolidation Phase and Interacts with Noradrenergic Systems in the Brain 
Neuropsychopharmacology  2010;36(4):744-752.
Neuropeptide S (NPS) has been shown to promote arousal and anxiolytic-like effects, as well as facilitation of fear extinction. In rodents, NPS receptors (NPSR) are prominently expressed in brain structures involved in learning and memory. Here, we investigate whether exogenous or endogenous NPS signaling can modulate acquisition, consolidation, or recall of emotional, spatial, and contextual memory traces, using two common behavioral paradigms, inhibitory avoidance (IA) and novel object recognition. In the IA paradigm, immediate and delayed post-training central NPS administration dose dependently enhanced memory retention in mice, indicating that NPS may act during the consolidation phase to enhance long-term memory. In contrast, pre-training or pre-test NPS injections were ineffective, suggesting that NPS had no effect on IA memory acquisition or recall. Peripheral administration of a synthetic NPSR antagonist attenuated NPS-induced IA memory enhancement, showing pharmacological specificity. NPS also enhanced hippocampal-dependent non-aversive memory in the novel object recognition task. In contrast, NPSR knockout mice displayed deficits in IA memory, novel object recognition, and novel place or context recognition, suggesting that activity of the endogenous NPS system is required for memory formation. Blockade of adrenergic signaling by propranolol attenuated NPS-induced memory enhancement in the IA task, indicating involvement of central noradrenergic systems. These results provide evidence for a facilitatory role of NPS in long-term memory, independent of memory content, possibly by acting as a salience signal or as an arousal-promoting factor.
doi:10.1038/npp.2010.207
PMCID: PMC3037424  PMID: 21150909
neuropeptide; memory; consolidation; inhibitory avoidance; object recognition; mice; learning & memory; neuropeptides; behavioral science; catecholamines; neuropeptide; NPSR antagonist; memory; inhibitory avoidance; novel object recognition
8.  Neuropeptide S: A transmitter system in the brain regulating fear and anxiety 
Neuropharmacology  2009;58(1):29-34.
Summary
The recently discovered Neuropeptide S (NPS) and its cognate receptor represent a highly interesting system of neuromodulation with unique physiological effects. On one hand, NPS increases wakefulness and arousal. On the other, NPS produces anxiolytic-like effects by acutely reducing fear responses as well as modulating long-term aspects of fear memory, such as attenuation of contextual fear or enhancement of fear extinction. The main sources of NPS in the brain are a few clusters of NPS-producing neurons in the brainstem. NPS binds to a G-protein-coupled receptor that is highly conserved among vertebrates and stimulates mobilization of intracellular Ca2+ as well as activation of protein kinases. In synaptic circuits within the amygdala, which are important for processing of acute fear as well as formation and expression of fear memories, NPS causes increased release of the excitatory transmitter glutamate, especially in synaptic contacts to a subset of GABAergic interneurons. Polymorphisms in the human NPS receptor gene have been associated with altered sleep behavior and panic disorder. In conclusion, the NPS system displays a unique physiological profile with respect to the specificity and time course of its actions. These functions could provide interesting opportunities for both basic research and clinical applications.
doi:10.1016/j.neuropharm.2009.06.001
PMCID: PMC2784192  PMID: 19523478
Neuropeptide S; G-Protein-coupled receptor; Amygdala; Fear Behavior; Anxiety Disorder
9.  Further studies at neuropeptide S position 5: discovery of novel neuropeptide S receptor antagonists 
Journal of medicinal chemistry  2009;52(13):4068-4071.
Neuropeptide S (NPS) regulates various biological functions by activating the NPS receptor (NPSR). Previous studies demonstrated that the substitution of Gly5 with D-amino acids generates NPSR antagonists. Eleven [D-Xaa5]NPS derivatives were synthesized and pharmacologically tested measuring [Ca2+]i in HEK293mNPSR cells. The present results confirmed that the [D-Xaa5] substitution promotes antagonist activity with potency inversely related to the side chain size and allowed to identify the novel potent NPSR peptide antagonist [tBu-D-Gly5]NPS.
doi:10.1021/jm900604g
PMCID: PMC2732566  PMID: 19473027
10.  In vitro and in vivo pharmacological characterization of the neuropeptide S receptor antagonist [D-Cys(tBu)5]NPS 
Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as NPSR. In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)5]NPS was identified. [D-Cys(tBu)5]NPS up to 100 μM did not stimulate calcium mobilization in HEK293 cells stably expressing the mouse NPSR (HEK293mNPSR), however, the peptide inhibited in a concentration dependent manner the stimulatory effects elicited by 10 and 100 nM NPS (pK 6.62). In Schild analysis experiments [D-Cys(tBu)5]NPS (0.1 - 100 μM) produced a concentration dependent and parallel rightward shift of the concentration response curve to NPS showing a pA2 value of 6.44. 10 μM [D-Cys(tBu)5]NPS did not affect signalling at seven NPSR unrelated G-protein coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmole intracerebroventricularly reduced the percent of animals losing the RR in response to diazepam 15 mg/kg and their sleeping time. [D-Cys(tBu)5]NPS (1-10 nmoles) was inactive per se but dose dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal promoting action of 1 nmole NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)5]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist, but with moderate potency. Moreover, using this tool together with receptor knockout mice studies we demonstrated that the arousal-promoting action of NPS is due to the selective activation of the NPSR protein.
doi:10.1124/jpet.108.143867
PMCID: PMC2630366  PMID: 18971372
11.  Synthesis and biological activity of human neuropeptide S analogues modified in position 5 
Journal of medicinal chemistry  2009;52(2):524-529.
Neuropeptide S (NPSa), the endogenous ligand of a previously orphan receptor now named NPSR, regulates various biological functions in the brain, including arousal, locomotion, anxiety, and food intake. Here we report on a focused structure-activity study of Gly5 which has been replaced with L and D amino acids. Fifteen NPS related peptides were synthesized and pharmacologically tested for intracellular calcium mobilization using HEK293 cells stably expressing the mouse NPSR. The results of this study demonstrated that peptide potency is inversely related to the side chain size while peptide efficacy strongly depends on the relative L and D configuration with the L aminoacids favoring agonist while D aminoacids displaying antagonist pharmacological activity. [D-Val5]NPS behaved as NPSR pure antagonist in HEK293mNPSR cells showing the highest potency (pKB 7.56) among this series of peptides. The antagonist action of [D-Val5]NPS was confirmed in vivo in mice where the peptide at a dose of 10 nmoles completely blocked the stimulatory effect of 0.1 nmole NPS on locomotor activity.
doi:10.1021/jm8012294
PMCID: PMC2653091  PMID: 19113861
12.  Neuropetide S-mediated control of fear expression and extinction: role of intercalated GABAergic neurons in the amygdala 
Neuron  2008;59(2):298-310.
Summary
A deficient extinction of memory is particularly important in the regime of fear, where it limits the beneficial outcomes of treatments of anxiety disorders. Fear extinction is thought to involve inhibitory influences of the prefrontal cortex on the amygdala, although the detailed synaptic mechanisms remain unknown. Here we report that neuropeptide S (NPS), a recently discovered transmitter of ascending brainstem neurons, evokes anxiolytic effects and facilitates extinction of conditioned fear responses when administered into the amygdala in mice. An NPS receptor antagonist exerts functionally opposing responses, indicating that endogenous NPS is involved in anxiety behavior and extinction. Cellularly, NPS increases glutamatergic transmission to intercalated GABAergic neurons in the amygdala via presynaptic NPS receptors on connected principal neurons. These results identify mechanisms of NPS in the brain, a key role of intercalated neurons in the amygdala for fear extinction, and a potential pharmacological avenue for treating anxiety disorders.
doi:10.1016/j.neuron.2008.07.002
PMCID: PMC2610688  PMID: 18667157
13.  Effect of Orphanin FQ/Nociceptin (OFQ/N) and Isoflurane on the Prolactin Secretory Response in OFQ/N Knockout Mice 
Peptides  2007;28(8):1611-1614.
The prolactin secretory response to subcutaneous injection of orphanin FQ/nociceptin (OFQ/N) was measured in wild-type and OFQ/N knockout mice. These injections were given with and without isoflurane anesthesia, to determine if isoflurane would affect the prolactin secretory response. OFQ/N injection significantly increased prolactin levels in males and females, regardless of genotype, with a more robust response in females. Isoflurane pretreatment did not affect prolactin levels in controls or in animals injected with OFQ/N. This is the first report that exogenously administered OFQ/N stimulates prolactin secretion in mice and that brief isoflurane exposure does not significantly affect this response.
doi:10.1016/j.peptides.2007.07.008
PMCID: PMC2128045  PMID: 17698246
opiates; opioid; anesthesia; gender; stress

Results 1-13 (13)