We assessed the functional role of NPSR1-deficiency in locomotion, stress reactivity, and learning and memory, and tested the role of NPSR1 in NPS-stimulated locomotion, anxiety, and HPA axis response through NPSR1 deletion. The principal finding in NPSR1-deficient mice was that males exhibited increased depression-like behavior in the forced swim test and reduced acoustic startle reactivity, both indicative of sensorimotor reactivity down-regulation to aversive stimuli. Why this effect was restricted only to males and was not seen in females is unknown, but it is of interest that an NPSR1 SNP (Asn/Ile107
) was associated with panic disorder in male Japanese patients (Okamura et al., 2007
). After NPS ICV injection, NPSR1-deficient mice did not respond to NPS-induced stimulation during tests of locomotion, anxiety, or corticosterone release compared with WT NPS-treated mice, identifying a non-redundant role of NPSR1 as the receptor for NPS. It should be noted that NPSR1-deficient mice were tested in a battery of tests to characterize their basic phenotype. Possible carry-over effects of one test on another may be a factor in influencing the observed effects, however, we arranged the tests in order from least to most apparent stress to minimize such influences and it is evident by the findings that the effects were highly specific and hence unlikely to be attributable to experience-related transference.
The FST and TST were used to evaluate depression-like behavior. The development of immobility disengages the animal from active coping when confronted with an inescapable stressor (Lucki et al. 2001
) and these tests have proven predictive for antidepressant efficacy. NPSR1 deletion in and of itself did not affect locomotor activity or swimming in the MWM or TST immobility. These data suggest that the depressive phenotype in the FST in male NPSR1-deficient mice is not attributable to reduced locomotor activity or a generalized suppression effect. The FST and TST findings are not necessarily contradictory. Cryan et al. (2005)
have reviewed the literature and found that some drugs and gene deletions show divergent outcomes on these two tests. While the reasons for such divergence on tests thought to assess similar functions remains unknown, divergence itself does not imply the presence of a false positive finding in the FST.
Elevated zero maze, light-dark exploration, open-field locomotor activity, and marble burying are the most frequently employed behavioral methods to determine anxiety states in response to novelty (Van Meer and Raber, 2005
). Our data show that NPSR1-deficient mice did not have a changed response when evaluated in these tests indicating that absence of the receptor does not alter anxiety under basal conditions. Clinically, antidepressants are often effective in treating anxiety and it is well established that anxiety and mood disorders exhibit significant comorbidity. Yet the link between these functions remains poorly understood. The lack of anxiety differences in NPSR1-deficient mice may represent a mechanism with different effects on anxiety-like versus depression-like behaviors. In reviewing the literature, based on neurotransmitter changes and dysregulation of HPA axis in humans and animals, Boyer (2000)
suggested that anxiety and depressive disorders showed differences in regulating the release of several peptides or hormones of the HPA axis except for increases corticotrophin-releasing factor (CRF) in the cerebrospinal fluid which was common across conditions. Another possibility for explaining this may be the compensatory effect of lifelong NPSR1 gene deletion. Thus, for future studies an antagonist specific for NPSR1 could be used to confirm the role of NPSR1 in anxiety and depression.
NPSR1 mRNA is expressed in the input and output pathways of the hippocampus, which is involved in regulation of learning and memory. Recently Han et al. (2009)
reported that ICV administration of NPS facilitated spatial memory in the MWM without altering latency to the target or swimming speed. We also examined spatial learning and memory in NPSR1-defficient and WT mice using the MWM. Our data showed mild changes in NPSR1-deficient mice, suggesting that endogenous NPS may not modulate spatial learning and memory under basal conditions. Garau et al. (2009)
examined fear conditioning in NPSR-deficient mice and showed differences in NPSR-deficient mice compared to WT mice. Factors resulting in these different results may be related to differences in endogenous NPS release under different testing conditions or different mechanisms between the two types of learning and memory processes.
When NPS was administered ICV, clear anxiolytic effects were induced that are consistent with recent data (Xu et al., 2004
; Leonard et al., 2008
). What is unique in the present experiment is the demonstration that the anxiolytic, locomotor facilitation, and corticosterone response to NPS are dependent on NPSR1 since NPSR1-deficient mice were unchanged from aCSF-treated WT controls on these measures in contrast to the activating effects of NPS in WT mice. These data therefore provide independent evidence that NPSR1 is the primary and perhaps sole receptor mediating NPS effects on anxiety, locomotor activation, and a significant contributor to stress-induced corticosterone release. These findings are also consistent with other lines of evidence. For example, by reverse pharmacology, NPS has previously been suggested as the endogenous ligand for NPSR1 but whether other receptors existed was unclear. Several groups reported that compounds that bind to NPSR1 inhibit the stimulatory effect of NPS on locomotor activity (Okamura et al., 2008
and Guerrini et al., 2009
) and the arousal promoting effect during the righting reflex recovery test (Camarda at al., 2009
) but the specificity of these effects was not entirely clear since these antagonists could be binding to other yet unidentified receptors. By using NPSR1 gene targeting, the current experiment provides more specific evidence that NPSR1 is the receptor for NPS-induced locomotor activation and anxiolytic effects at least to the extent that is reflected by the tests used herein.
In the elevated zero maze, NPS-treated WT mice had increased time in open areas and head dips and shorter latencies to enter the first open quadrant, all consistent with a reduced anxiety phenotype. In addition, NPS-treated WT mice showed increased rearing (vertical activity) and central time activity, also consistent with reduced anxiety. Each of these NPS-induced effects was absent in NPSR1-deficient NPS-treated mice. We also examined the effect of NPS on light-dark exploration. At the beginning of the test we put the mice in the lighted area. NPS-treated mice showed increased horizontal activity in both light and dark areas and increased transitions between the light and dark sides. This pattern suggests the predominance of the locomotor activating effect of NPS rather than a specific anxiolytic effect. However, Leonard et al. (2008)
have shown that the locomotor activating and anxiolytic effects of NPS can be distinguished by comparing the effects of NPS to the effects of the indirect dopaminergic agonist (+)-amphetamine and to the benzodiazepine type GABAergic anxiolytics. For example, they showed that (+)-amphetamine, while increasing locomotor activity does not increase open time in the elevated zero maze or increase punished crossings in the four-plate test, as does NPS. Therefore, locomotor activation per se is not a confounder when the two effects co-occur but originate from distinct processes. In addition, Leonard et al. (2008)
showed that NPS inhibits rectal probe stress-induced hyperthermia, suggesting that the effects of NPS are selective. The weakness of the tests we used here to identify activity of NPSR1 in anxiety is that it is not completely excluded that the reduced anxiety phenotype in the elevated zero maze and open-field test observed in NPS-treated WT mice is partially due to the activation of general locomotion. Future experiments should test NPSR1-deficient NPS-treated mice using tests such as the four-plate test to further evaluate the role of NPSR1 in stress reactivity.
We also examined the effect of NPS on anxiety in the marble burying test (not presented) but saw no NPS-induced anxiolytic effect and hence had no basis for testing NPSR1-deficient mice with this procedure. This is in contrast to Xu et al. (2004)
who reported that ICV NPS reduced marble burying. The reason for this discrepancy is not known but may be the result of methodological differences. Xu et al. (2004)
tested the mice for 30 min whereas here, the mice were tested for 20 min. It is also unclear whether Xu et al. (2004)
performed other tests before marble burying which could contribute to outcome differences. More importantly than this one difference is the fact that overall our anxiety test data are consistent with those of Xu et al. (2004)
and the more recent data of Leonard et al. (2008)
, suggesting that the role of NPS in anxiety and locomotor stimulation are robust.
The HPA axis is activated in response to stressors, and when the stress effect is sufficient results in an increase in plasma corticosterone levels in rodents. Corticosterone regulates a variety of adaptations at the level of neuroendocrine, autonomic, immunological and behavioral responses. A physical stressor (forced swim) and some drugs (methamphetamine) can activate the HPA axis (Müller et al., 2000
and Prickaerts et al., 2006
). After forced swim or methamphetamine challenge and open-field exposure, NPSR1-deficient mice and WT littermates had similar corticosterone levels, suggesting that NPSR1 deletion did not disrupt HPA axis response mechanisms. Smith et al. (2006)
reported that NPS stimulates the HPA axis and we showed this effect also. These data are consistent with the finding of Leonard et al. (2008)
that ICV NPS attenuates stress-induced hyperthermia and demonstrates the importance of NPS in stress adaption as well as in anxiety.
In conclusion, the present findings provide the first direct evidence that NPSR1 is the dominant central receptor mediating NPS-induced locomotor activation, anxiolysis, and interaction with corticosterone release and supports the view expressed by Leonard et al. (2008)
that the NPS-NPSR1 pathway represents a novel pharmacological target for therapeutic agents for the treatment of anxiety-related disorders.