Arousal and anxiety are behavioral responses that involve complex neurocircuitries and multiple neurochemical components. Here, we report that a neuropeptide, neuropeptide S (NPS), potently modulates wakefulness and could also regulate anxiety. NPS acts by activating its cognate receptor (NPSR) and inducing mobilization of intracellular Ca2+. The NPSR mRNA is widely distributed in the brain, including the amygdala and the midline thalamic nuclei. Central administration of NPS increases locomotor activity in mice and decreases paradoxical (REM) sleep and slow wave sleep in rats. NPS was further shown to produce anxiolytic-like effects in mice exposed to four different stressful paradigms. Interestingly, NPS is expressed in a previously undefined cluster of cells located between the locus coeruleus (LC) and Barrington's nucleus. These results indicate that NPS could be a new modulator of arousal and anxiety. They also show that the LC region encompasses distinct nuclei expressing different arousal-promoting neurotransmitters.

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.

Clozapine N-oxide (CNO) is a ligand for a powerful chemogenetic system that can selectively inhibit or activate neurons; the so-called Designer Receptors Exclusively Activated by Designer Drugs (DREADD) system. This system consists of synthetic G-protein-coupled receptors, which are not believed to be activated by any endogenous ligand, but are activated by the otherwise inert CNO. However, it has previously been shown that the administration of CNO in humans and rats leads to detectable levels of the bioactive compounds clozapine and N-desmethylclozapine (N-Des). As a follow-up, experiments were conducted to investigate the effects of CNO in male Long–Evans rats. It was found that 1 mg/kg CNO reduced the acoustic startle reflex but had no effect on prepulse inhibition (PPI; a measure of sensorimotor gating). CNO (2 and 5 mg/kg) had no effect on the disruption to PPI induced by the NMDA antagonist phencyclidine or the muscarinic antagonist scopolamine. In locomotor studies, CNO alone (at 1, 2, and 5 mg/kg) had no effect on spontaneous locomotion, but 5 mg/kg CNO pretreatment significantly attenuated d-amphetamine-induced hyperlocomotion. In line with the behavioral results, fast-scan cyclic voltammetry found that 5 mg/kg CNO significantly attenuated the d-amphetamine-induced increase in evoked dopamine. However, the effects seen after CNO administration cannot be definitively ascribed to CNO because biologically relevant levels of clozapine and N-Des were found in plasma after CNO injection. Our results show that CNO has multiple dose-dependent effects in vivo and is converted to clozapine and N-Des emphasizing the need for a CNO-only DREADD-free control group when designing DREADD-based experiments.

Recovering alcoholics with insomnia can achieve better sleep by applying cognitive-behavioural strategies.

Neuropeptide S (NPS) is the endogenous ligand for GPR154, now referred to as neuropeptide S receptor (NPSR). Physiologically, NPS has been characterized as a modulator of arousal and has been shown to produce anxiolytic-like effects in rodents. Neuroanatomical analysis in the rat revealed that the NPS precursor mRNA is strongly expressed in the brainstem in only three distinct regions: the locus coeruleus area, the principal sensory trigeminal nucleus, and the lateral parabrachial nucleus. NPSR mRNA expression in the rat is widely distributed, with the strongest expression in the olfactory nuclei, amygdala, subiculum, and some cortical structures, as well as various thalamic and hypothalamic regions. Here we report a comprehensive map of NPS precursor and receptor mRNA expression in the mouse brain. NPS precursor mRNA is only expressed in two regions in the mouse brainstem: the Kölliker-Fuse nucleus and the pericoerulear area. Strong NPSR mRNA expression was found in the dorsal endopiriform nucleus, the intra-midline thalamic and hypothalamic regions, the basolateral amgydala, the subiculum, and various cortical regions. In order to elucidate projections from NPS-producing nuclei in the brainstem to NPSR-expressing structures throughout the brain, we performed immunohistochemical analysis in the mouse brain by using two polyclonal anti-NPS antisera. The distribution of NPS-immunopositive fibers overlaps well with NPSR mRNA expression in thalamic and hypothalamic regions. Mismatches between NPSR expression and NPS-immunoreactive fiber staining were observed in hippocampal, olfactory, and cortical regions. These data demonstrate that the distribution pattern of the central NPS system is only partially conserved between mice and rats.

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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, demonstrating 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.

Dopamine receptors are G protein-coupled receptors that are critically involved in locomotion, reward, and cognitive processes. The D2 class of dopamine receptors (DRD2, -3, and -4) is the target for antipsychotic medication. DRD4 has been implicated in cognition, and genetic studies have found an association between a highly polymorphic repeat sequence in the human DRD4 coding region and attention deficit hyperactivity disorder. Using DRD4 as a model, we show that antipsychotics can function as potent pharmacological chaperones up-regulating receptor expression and can also rescue a non-functional DRD4 folding mutant. This chaperone-mediated up-regulation involves reduced degradation by the 26 S proteasome; likely via the stabilization of newly synthesized receptor in the endoplasmic reticulum. Dopamine itself can function as a chaperone when shuttled into the cell by means of the dopamine transporter. Furthermore, different repeat variants of DRD4 display differential sensitivity to this chaperone effect. These data suggest that folding efficiency may be rate-limiting for dopamine receptor biogenesis and that this efficiency differs between receptor variants. Consequently, the clinical profile of dopaminergic ligands, including antipsychotics, may include their ability to serve as pharmacological chaperones.