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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.

We have recently shown that Neuropeptide S (NPS) can promote arousal and induce anxiolytic-like effects after central administration in rodents. Another study reported a number of natural polymorphisms in the human NPS receptor gene. Some of these polymorphisms were associated with increased risk of asthma and possibly other forms of atopic diseases, but the physiological consequences of the mutations remain unclear. One of the polymorphisms produces an Asn-Ile exchange in the first extracellular loop of the receptor protein, and a C-terminal splice variant of the NPS receptor was found overexpressed in human asthmatic airway tissue. We sought to study the pharmacology of the human receptor variants in comparison with the murine receptor protein. Here, we report that the N107I polymorphism in the human NPS receptor results in a gain-offunction characterized by an increase in agonist potency without changing binding affinity in NPSR Ile 107 . In contrast, the C-terminal splice variant of the human NPS receptor shows a pharmacological profile similar to NPSR Asn 107 . The mouse NPS receptor, which also carries an Ile residue at position 107, displays an intermediate pharmacological profile. Structureactivity relationship studies show that the amino terminus of NPS is critical for receptor activation. The altered pharmacology of the Ile 107 isoform of the human NPS receptor implies a mechanism of enhanced NPS signaling that might have physiological significance for brain function as well as peripheral tissues that express NPS receptors.Neuropeptide S (NPS) is the endogenous ligand of an orphan G protein-coupled receptor (GPCR). The NPS receptor (NPSR) belongs to the subfamily of peptide GPCRs and is widely expressed in the brain, with highest levels found in hypothalamus, amygdala, endopiriform nucleus, cortex, subiculum, and nuclei of the thalamic midline. In contrast, the NPS precursor mRNA is found in only a few brain structures (Xu et al., 2004). Highest levels of NPS precursor expression were detected in a novel nucleus located in between the noradrenergic locus coeruleus and Barrington's nucleus in the pontine area of the rat brain stem. Other brain regions of high NPS precursor expression include the lateral parabrachial nucleus, sensory principle 5 nucleus, and a few scattered neurons in the amygdala and dorsomedial hypothalamic nucleus. In addition, we found high expression of NPS and NPSR mRNA in endocrine tissues, including thyroid, mammary, and salivary glands, but did not observe significant levels in rat lung tissue.Central administration of NPS promotes behavioral arousal and suppresses all stages of sleep in rodents. Furthermore, NPS was found to produce anxiolytic-like effects in a battery of four different tests that measure behavioral responses of rodents to novelty or stress. NPS was shown to induce transient increases of intracellular Ca 2ϩ , indicating that it might have excitatory effects at the cellular level (Xu et al., 2004).Recently, a number of polymorphisms in the human NPS rece...

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 Ca 2ϩ mobilization, and SHA 68 shows displaceable binding to NPSR in the nanomolar range. The antagonistic activity of SHA 68 seems to be specific because it does not affect signaling at 14 unrelated G protein-coupled receptors. Analysis of pharmacokinetic parameters of SHA 68 demonstrates that the compound reaches pharmacologically relevant levels in plasma and brain after i.p. administration. Furthermore, peripheral administration of SHA 68 in mice (50 mg/kg i.p.) is able to antagonize NPSinduced 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.Neuropeptide S (NPS) and its receptor, NPSR, are a recently identified transmitter system that modulates a number of brain functions . NPS is a small peptide of 20 amino acids that occurs in all tetrapod vertebrates but is absent from fish (Reinscheid, 2007). Activation of NPSR produces transient increases in intracellular Ca 2ϩ and cAMP and thus increases cellular excitability (Reinscheid et al., 2005). Expression of NPS precursors and receptors is found in specific brain areas that have been associated with arousal, emotional processing, energy, and hormonal homeostasis, as well as learning and memory (Xu et al., 2004(Xu et al., , 2007. In the rat, NPS precursor transcripts are expressed in only a few brainstem structures; in particular, in a previously uncharacterized nucleus situated between the noradrenergic locus coeruleus and Barrington's nucleus. Besides the pericoerulear region, NPS mRNA is only found in the lateral parabrachial nucleus and the principle sensory 5 nucleus of the rat brainstem. A few scattered cells expressing NPS precursor transcripts are also detected in the amygdala and hypothalamus. In the brainstem, the majority of NPSexpressing neurons coexpress other excitatory transmitters, such as glutamate, acetylcholine, or corticotropin-releasing factor (Xu et al., 2007). NPSR mRNA is found at high levels in hypothalamus, thalamus, amygdala, various cortical regions, and the parahippocampal formation. Central administration of NPS was shown to produce profound arousal that is independent of novelty (Xu et al., 2004).

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