Anxiolytic-like effect of neuropeptide S in the rat defensive burying

Abstract

Neuropeptide S (NPS) has been recently identified as the endogenous ligand of a previously orphan G-protein-coupled receptor now named NPSR. Both NPS and its receptor are expressed in the brain, where they modulate different functions. In particular, it has been demonstrated that intracerebroventricular (i.c.v.) injection of NPS in rodents increases wakefulness and promotes anxiolytic-like effects. In the present study we used the defensive burying (DB) test in rats to further investigate the action of human NPS (0.1–10 nmol, i.c.v.) on anxiety-related behaviors. Diazepam (1.5 mg/kg, i.p.) and caffeine (20 mg/kg, i.p.) were used in parallel experiments as standard anxiolytic and anxiogenic drugs, respectively. None of the tested drugs produced statistical differences in the latency to contact the probe, burying behavior latency, number of shocks received or immobility/freezing duration. Caffeine increased cumulative burying behavior and the buried bedding height in a statistically significant manner thus promoting anxiogenic like effects. Opposite results were obtained with diazepam that significantly reduced these behavioral parameters. The anxiolytic-like action of diazepam was mimicked by NPS that reduced cumulative burying behavior in a dose dependent manner. Collectively, robust anxiolytic-like effects were recorded in response to NPS in the DB test. These results are of particular interest since the outcome of this assay is marginally influenced by drug effects on locomotor activity. In conclusion, we provide further evidence that NPS evokes genuine anxiolytic-like effects in the rat; therefore NPSR selective agonists are worthy of development as innovative drugs for the treatment of anxiety disorders.

Introduction

Neuropeptide S (NPS) is a newly identified peptide transmitter that is involved in the regulation of anxiety and wakefulness [33]. Reverse pharmacology studies led to the identification of NPS as the endogenous ligand of the NPS receptor (NPSR), a former orphan G-protein-coupled receptor [33]. NPS has strong sequence conservation across mammalian species [22] and is named after a highly conserved N-terminal serine at position 1. As an agonist of NPSR, NPS produces an increase in free intracellular calcium and cyclic adenosine mono-phosphate accumulation [13], [33] indicating a coupling with both Gq and Gs proteins [21].

NPSR is widely expressed throughout the nervous system: highest levels of the receptor are found in cortex, thalamus, hypothalamus and amygdala and low levels in the brainstem while the NPS precursor mRNA is expressed strongly in a few brain areas including the locus coeruleus (LC) area, the principle sensory trigeminal nucleus and the lateral parabrachial nucleus of the brainstem [33], [32].

This pattern of expression may be consistent with a role for NPS in arousal, regulation of food intake and anxiety [3], [27], [33]. NPS appears to induce behavioral activation and induce wakefulness, but at the same time, produces an anxiolytic-like effect, a paradox shared also by nicotine [16]. This suggests that arousal and stress are not always closely linked indicating that diverse sets of responses may be involved and different neurotransmitter systems implicated in these conditions [12]. NPS was repeatedly shown to produce anxiolytic-like effects in mice exposed to several different anxiety-predictive paradigms and increase locomotor activity [33], [23], [18].

Indeed, centrally administered NPS could produce an anxiolytic-like profile that was independent of the motor-activating effects of the peptide [33]. Since hyperlocomotion may be associated to emotional consequences, particularly in stressful conditions, the present study was designed to investigate the effect of NPS with the aim of minimizing its possible confounding effect on locomotion. For this purpose, we chose the rat defensive burying (DB) test. This assay measures fear-like responses of composite nature, generated by exposure to stressful environmental conditions and is established for its sensitivity to conventional anxiolytic tranquilizers and anxiogenic compounds [11].

The DB test had been previously validated [30], under the present experimental conditions, with prototypical anxiolytic (i.e. diazepam) and anxiogenic (i.e. caffeine) compounds on the basis of dose-response curves in anxiety tests [15], [14] and controlled for effects on sensorimotor function (results not shown). In the current experiments, the timing scheduled in this behavioral assay in the rat was assessed using the highest dose of NPS (10 nmol/rat) in preliminary time-effect experiments on the basis of personal and other groups’ [27] observations. The effects elicited by NPS were compared to those evoked, under the same experimental conditions, by the aforementioned reference stimulant and anxiolytic drugs, caffeine (20 mg/kg, i.p.) and diazepam (1.5 mg/kg, i.p.).

Our hypothesis was to demonstrate a clear cut anxiolytic effect of NPS using the rat defensive burying test, an assay that is marginally affected by locomotor changes.