The key novelty of this is study is the finding that nasal application of recently discovered neuropeptide S (NPS) can inhibit pain behaviors in an arthritis model. This effect involves NPS receptors in the amygdala, specifically the intercalated cell (ITC) area that serves as a gate keeper of excitatory drives to amygdala output neurons in the CeA. Moreover, new electrophysiology results show that NPS administered nasally or directly into the ITC area inhibits nociceptive processing in CeA neurons that are known to play an important role in the generation and modulation of pain behaviors [3, 4]. While our previous work identified ITC-driven inhibition of CeA neurons as the synaptic mechanism of action of NPS in the amygdala , the effect of NPS on nociceptive processing in amygdala neurons in the intact animal was not known. The behavioral and electrophysiological results are significant because the beneficial effects obtained with a non-invasive application method may suggest potential usefulness in the clinical setting, although NPS functions in humans remain to be determined. However, human NPS and NPS receptors exist and the primary sequence of NPS is highly conserved among vertebrates especially at the N-terminus with the amino acid serine (S) hence the name neuropeptide S [24, 25, 31].
NPS is primarily localized to discrete brainstem areas, including the lateral parabrachial nucleus and the peri-locus coeruleus . These brainstem areas interact closely with the amygdala, for example through corticotropin releasing factor (CRF) expressing neurons in the lateral parabrachial nucleus and in the amygdala (CeA) [41–44]. CRF can activate NPS neurons in the locus coeruleus . The amygdala CRF system plays an important role in pain modulation and generation of pain behaviors [6, 46, 47]. The amygdala is also one of the brain areas where NPSR is found in highest abundance . NPS has been shown to control amygdala output by increasing synaptic feedforward inhibition mediated by a cluster of inhibitory ITC interneurons as a mechanism to exert anxiolytic effects . Recent work from our group suggests that a similar mechanism may also account for pain-inhibiting effects of NPS . Thus, NPS could be part of a homeostatic circuit of emotions involving limbic and brainstem areas .
In support of this notion, behavioral studies showed anxiolytic effects of NPS in the brain. Intracerebroventricular administration of NPS had anxiolytic effects in various assays and facilitated the extinction of conditioned fear responses [23, 30, 31, 36, 49]. Administration of an NPS receptor antagonist (SHA68) into the amygdala reversed the effects of NPS, suggesting a major role of the amygdala in NPS function. Intracerebroventricular administration of NPS also had antinociceptive effects in the tail-flick, hot-pate and formalin tests [34, 35]. We showed previously that direct application of NPS into the ITC of the amygdala inhibited emotional-affective responses and anxiety-like behavior in an arthritis pain model but had no effect on sensory aspects (mechanical withdrawal thresholds) , suggesting the involvement of NPS in the amygdala in discrete aspects of pain modulation. The results of the present study provide further support of this concept.
Importantly, recent evidence suggests that NPS can be administered nasally to exert anxiolytic effects [36, 37]. Topical application of NPS on the rhinarium (glabrous area around the nostrils) of rats increased novel object recognition, a common method for assessing cognitive memory enhancing effects, and had anxiolytic effects in the elevated plus-maze . A study in mice reported that application of NPS to each nostril had anxiolytic effects on the elevated plus-maze and dark-light tests . Locomotor activity was not affected in these studies. To the best of our knowledge, the present study is the first to examine the effects of nasal NPS on pain behaviors and amygdala activity. We also show that inhibitory effects of NPS in the pain model are mediated through NPS receptors in the amygdala, because stereotaxic application of a selective NPSR antagonist reversed the effects of NPS. Importantly, NPS had no effect under normal conditions, which is consistent with our previous study using stereotaxic application of NPS into the amygdala . We interpret our data as evidence for a compensatory change in NPSR function and/or expression, which would be consistent with homeostatic functions of the NPS amygdala system. Although high levels of NPSR mRNA are found in the ITC of rats , NPSR protein expression appears to be absent in this area under normal conditions . NPSR protein expression in the arthritis pain model remains to be determined.
Some methodological aspects need to be considered. We selected the 45 min time point after nasal NPS for the following reasons: Previous studies detected behavioral effects 30 min after nasal NPS [36, 37]. Internalization of the NPS receptor-ligand complex in the brain, including the amygdala, was detected 30 min after nasal application of fluorophore-conjugated NPS . In our preliminary pilot experiments, behavioral effects of nasal NPS were detected 30 min after application but became more pronounced after 45 min and were still present at 60 min. It should be noted that a previous study showed that NPS (total of 20 μl) was completely absorbed within 2 min after topical nasal application.
The dose of 14 nmol NPS was selected based on data in the literature. In rats, nasal application of NPS (4 nmol) was effective on object discrimination (memory-enhancing effect) whereas anxiolytic effects were detected with NPS (40 nmol) but not NPS (4 nmol) . A study in mice tested intranasal NPS (7, 14 and 28 nmol) and found that NPS (14 nmol) was the optimum dose for anxiolytic effects . We considered, but did not perform, a dose-response analysis because it would have significantly increased the number of animals without changing the key finding of this study, which is the beneficial effect of nasal NPS through an action on NPSR in the amygdala. Still, we acknowledge this as a potential shortcoming of our study.
Drug concentrations for stereotaxic application by microdialysis were derived from data in the literature and our own work . In brain slices from arthritic rats, NPS (1 μM) inhibited synaptic plasticity in CeA neurons significantly through a direct action on ITC cells. In mice brain slices, NPS (10 μM) had near maximum effects on synaptic activation of ITC cells but the mechanism of action was presynaptic to ITC cells . The rather indirect synaptic effect and species differences may explain differences in effective concentrations. Our previous studies comparing drug effects in brain slices and microdialysis drug application in intact animals determined that microdialysis required a 100-fold higher concentration than that needed in the tissue because of the concentration gradient across the dialysis membrane and diffusion in the tissue [29, 40, 46, 47, 50]. Drug concentrations for microdialysis application were adjusted accordingly in the present study. Importantly, NPS effects were blocked by competitive NPSR antagonists, confirming the appropriateness of the selected concentrations. We showed previously that [D-Cys(tBu)5]NPS, a well-established selective NPSR antagonist , blocked synaptic effects of NPS in the amygdala slice preparation (10 μM) and behavioral effects of NPS when applied by microdialysis (1 mM) into the ITC area , and so we used these concentrations in the present study. SHA68, a selective non-peptide NPSR antagonist [52, 53], has become commercially available only recently. In vitro assays of calcium mobilization showed that SHA68 (10 nM to 1 μM) antagonized the effects of NPS with IC50 values of about 50 nM but was inactive per se [49, 52]. In radioligand binding experiments a Ki value of about 50 nM was also found as well . The concentration used in the microdialysis probe in our studies (50 μM) was based on the assumption that a submaximal concentration would be needed to fully antagonize the effects of NPS. This concentration is relatively low compared to those used in other studies where the injection of SHA68 (10 μM) into the amygdala antagonized behavioral effects of NPS (10 μM) and SHA68 (100 μM) antagonized the synaptic effects of NPS in mice . Differences in the mode of action of NPS in the amygdala of rats (directly on ITC) and mice (indirectly, presynaptic to ITC) may explain the lower concentration needed in our study.
Finally, we did not perform placement control injections in this study. Drug injections targeted the ITC area in behavioral and electrophysiological experiments. Our previous studies on synaptic effects of NPS in brain slices showed a direct action on ITC cells but not on CeA or BLA neurons (synaptic inhibition of CeA neurons was driven by NPSR activation on ITC cells) . Accordingly, administration of NPS into the CeA as a control in our previous behavioral studies had no effect  and therefore we did not repeat these experiments here. Although NPSR antagonist injection into the ITC area blocked the effect of nasal NPS completely, we cannot rule out the contribution of NPSR in other brain areas. For example NPSR activation by nasal NPS has been found in the ventral hippocampus .