University of Cincinnati
Date: 8/20/2010
I, Hongyan Zhu , hereby submit this original work as part of the requirements for the degree of Doctor of Philosophy in Developmental Biology.
It is entitled: Deep breath and relax: a study of NPS/NPSR1
Student's name: Hongyan Zhu
This work and its defense approved by:
Committee chair: Marc Rothenberg, MD, PhD
Committee member: Simon Hogan, PhD
Committee member: Timothy Lecras, PhD
Committee member: Michael Williams, PhD
Committee member: Charles Vorhees, PhD
Committee member: David Hildeman, PhD
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Last Printed:2/21/2011 Document Of Defense Form Deep Breath and Relax: a study of NPS/NPSR1
A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati
In partial fulfillment of the requirements for the degree of Doctor of Philosophy In the Graduate Program in Molecular and Developmental Biology of the College of Medicine.
2010 By Hongyan Zhu MD, Hubei College of Traditional Chinese Medicine, 1992 MS, Beijing University of Traditional Chinese Medicine, 1999
Committee Chair: Marc E. Rothenberg, M.D., Ph.D. Committee: David Hildeman Ph.D. Simon Hogan Ph.D. Tim LeCras Ph.D. Charles Vorhees Ph.D. Michael Williams Ph.D.
Abstract
Asthma is a complex disorder involving the interaction between host genetic susceptibility and environmental factors. Psychological stress contributes to the exacerbation of asthma. NPSR1 has been linked with asthma, including airway hyperresponsiveness (AHR) and panic disorders, by a genomic association study. In this thesis, we aimed to characterize the role of NPSR1 in pulmonary and stress responses. We examined the Npsr1-deficient mice in two models of asthma and found that Npsr1 deletion had no impact on airway inflammation or airway hyperresponsiveness (AHR) in ovalbumin- or Aspergillus fumigatus- induced experimental asthma. Further, Npsr1 had a very low level of expression, based on RT-PCR analysis, in the basal or allergen-challenged lung, supporting that NPSR1’s direct role in the lung may be limited. Behavioral studies revealed that Npsr1-deficient mice had an impaired stress response and failed to show increases in locomotion, anxiolysis, or corticosterone release induced by intracerebroventricular (ICV) administration of neuropeptide S (NPS), the putative endogenous ligand of NPSR1, suggesting that NPS/NPSR1 signaling is involved in stress response and has anxiolytic effect. Nps and Npsr1 are expressed in brain regions that regulate respiratory rhythm, and clinical evidence show association of respiration changes with psychological stress and emotional diseases. Thus, we investigated whether NPS/NPSR1 regulated respiratory function through a central-mediated pathway. Analysis of breathing patterns by whole-body plethysmography revealed that ICV NPS, as compared with the artificial cerebrospinal fluid control, increased respiratory frequency and
T decreased tidal volume in an NPSR1-dependent manner but did not affect enhanced pause (Penh), a parameter that correlates with lung obstruction.
Following serial methacholine inhalation, ICV NPS increased respiratory frequency in WT mice, but not Npsr1-deficient mice, and had no effect on tidal volume. ICV NPS significantly reduced AHR to methacholine as measured by whole-body plethysmography, whereas ICV NPS did not alter airway mechanics in response to methacholine as measured by invasive plethysmography.
Collectively, these data support the hypothesis that NPS and NPSR1 regulate respiratory functions likely via a central-mediated pathway suggesting that a central nervous system pathway may potentially mediate the underlying association of NPSR1 with asthma and AHR.
U
V Acknowledgements
I would first like to express gratitude to my mentor and advisor, Dr. Marc Rothenberg, for his extensive guidance and encouragement. Most importantly, he trained me to be an independent and creative scientist. He was always challenging me to expand my thinking. As a role model, he has been teaching me the skills of career development in Biomedical Science. Dr. Rothenberg is famous for the saying - NME (no more excuses) and his working style - getting work done perfectly and in the shortest amount of time. All of these teachings will be invaluable for the rest of my life. Thanks again for Dr. Rothenberg’s investment of time and money for my training.
I am greatly indebted to Dr. Charles Vorhees and Dr. Michael Williams because without their unwavering support, this dissertation would not have been possible. They brought me into the world of behavioral studies. Beyond providing the facilities and training me in the techniques, they encouraged me and helped me overcome difficulties during rough times. They dedicated lots of time and effort to improve my writing of the behavioral study manuscript.
In addition to Dr. Vorhees and Dr. Williams, I would like to thank Drs. David Hildeman, Simon Hogan, and Tim LeCras, members of my dissertation defense committees, for their expertise and guidance that helped in shaping me as a scientist. Their passion for research also strongly influenced me. They also gave me suggestions for personal or career development during my graduate training.
I would also like to thank Dr. Fred Finkelman for providing the equipment and facility for the respiration study and giving critique on the respiration manuscript. Also I would like to thank Dr. Nives Zimmermann for her suggestions on the experimental design.
Another person I want to specifically mention is Dr. Patricia Fulkerson. She mentored me during my rotation in the Rothenberg lab. Her passion for science, ability of management and productiveness were so impressive. Her great accomplishments, completion of MD/PD while taking good care of 3 kids have been encouraging me.
Many thanks go to the past and present members in the Rothenberg lab. They are smart guys and crazy in the pursuit of science as well as providing an enjoyable research environment. I was very lucky to have chosen this lab. In particular, extend my appreciation for all those who helped me conduct these studies: Dr. Carine Blanchard, Dr. Eric Brandt, Dr. Yoshiyuki Yamada, Ms. Melissa Mingler and Ms. Melissa Mcbride. I would also like to thank Dr. Julie Caldwell and Ms. Shawna Hottinger for help in paper and thesis reviewing.
W I am forever grateful to the support of MDB program. Without their recruiting me into the program and their successful running this program, it would have been impossible for me to have performed this work.
This dissertation is dedicated to my family. The unconditional love from my parents, sisters, husband and son provided the solid foundation to support me in completing this program.
X Table of Contents
Abstract 2 Acknowledgements 5 Table of Contents 7
Chapter 1. Introduction 9
Asthma and asthma-associated genes 9 NPS and NPSR1 10 Stress, asthma, and respiration 18 In vivo assessment of respiratory function 33 Anxiety and depression mouse model 34 References 40
Chapter 2. Abnormal response to stress and impaired NPS-induced hyperlocomotion, anxiolytic effect and corticosterone increase in mice lacking NPSR1 58
Abstract 59 Introduction 60 Materials and Methods 62 Results 75 Discussion 86 Acknowledgements 93 Figure legends 94 Figures 99 References 105
Chapter 3. Neuropeptide S and its receptor NPSR1: a correlation of pulmonary response with stress response 112
Abstract 113 Introduction 115 Methods 117 Results 122 Discussion 126 Acknowledgements 130 Table 131 Figure legends 132 Figures 135 References 139
Y Chapter 4. Discussion and future directions 145
Role of NPSR1 in NPS-induced anxiolytic effect 145 Interpretation of sex-biased behavioral phenotypes in WT and Npsr1-deficient Mice 146 Endogenous NPS expression in the brain and interaction with other neurotransmitters under stressful conditions in the WT and Npsr1-deficient mice 148 NPS and fight-or-flight response 149 Role of NPS and NPSR1 in respiration and asthma 150 Limitations of Npsr1-deficient mouse model and asthma mouse model 154 Development of pharmaceutical drugs for NPS/NPSR1 system 155 Reference 156
Z Chapter 1
Introduction
1. Asthma and asthma-associated genes
Allergic bronchial asthma is a common chronic inflammatory disease of the airway, characterized by recurring wheezing, cough, short of breath, airflow obstruction, and bronchospasm. The underlying pathogenesis includes allergen- induced Th2 immune response, elevated IgE levels; airway inflammation with an influx of lymphocytes, monocytes, and eosinophils; goblet cell hyperplasia; airway structural remodeling with increased collagen and airway smooth muscle, and a thicker sub-epithelial region due to abnormal injury–repair response
(Djukanovi , 2000; Kay, 2001). Although intensive research is being conducted, the incidence of asthma is still rising in western society.
Asthma is a complex disorder involving the interaction between host genetic susceptibility and environmental factors. Genome-wide screening and positional cloning, a reverse genetic study method, have identified 4 genes (ADAM33,
DPP10, GPRA, and PHF11) as asthma susceptibility genes (Weiss et al., 2009).
The functions of these genes in asthma pathogenesis are still unknown. The full name for GPRA is G protein coupled receptor for asthma susceptibility. Initially, single nucleotide polymorphisms (SNPs) and haplotypes of the GPRA gene were associated with asthma or airway hyperresponsiveness (AHR) in Finnish and
Canadian populations (Laitinen et al., 2004). Recently, this genetic association has been confirmed in Italian, Chinese, German, European-American, and
[ Hispanic populations (Kormann et al., 2005; Melén et al., 2005; Feng et al., 2006;
Hersh et al., 2007; Malerba et al., 2007; Zhu et al., 2007). NPSR1 is the official name for GPRA because neuropeptide S (NPS) has been identified as the putative ligand for GPRA by a reverse pharmacological strategy (Xu et al., 2004).
2. NPS and NPSR1
2.1. Introduction
Human NPS is a 20- amino-acid peptide with a highly conserved sequence across mammalian Figure 1. A) Alignment of amino acids of human, rat, and mouse NPS peptide. Amino acids different from human NPS are highlighted in red. B) species (Xu et al., Primary structure of the human NPS precursor. Hydrophobic signal peptide is shown in green box. Mature NPS peptide (in red box) is 2004; Reinscheid, released by endoprotease cleavage at KR (in blue box)
2007). The name was given because of the N-terminal serine residue conserved in all species analyzed so far. As with other neuropeptides, NPS is cleaved from a larger precursor protein containing a hydrophobic signal peptide and proteolytic processing sites (Reinscheid et al., 2005) (Figure 1).
The human NPS receptor, NPSR1, belongs to the G protein-coupled receptor family (Figure 2B). NPS-NPSR1 binding causes increased intracellular calcium mobilization and cAMP accumulation and also stimulates phosphorylation of mitogen-activated protein kinase (Gupte et al., 2004; Xu et al., 2004; Reinscheid et al., 2005).
SR Figure 2. Genomic organization of mouse Npsr1 and alignment of murine and human NPSR1. (A) Schematic representation of the genomic organization of murine Npsr1 is shown according to the GenBank ( NC_000075) sequence. Exons are depicted as boxes and introns as lines. Coding regions of exons are shown as shaded boxes. The length of exons and introns are marked as well. (B) Alignment of murine and human NPSR1. The amino acids identical between 3 proteins are shown in shaded boxes. The amino acid sequence, which is deleted in the Npsr1-deficient mice described in chapter 2, is marked with green line. The 7 transmembrane domains are marked with blue lines. The site of Asn/Ile107 SNP in human NPSR1 is shown in red box. Mouse NPSR1 conserves Ile107.
The human NPSR1 gene encodes 8 variants by alternative splicing. Among them, only NPSR1-A and NPSR1-B, full-length splice variants are functional receptors, supported by the evidence that they are located on the cell membrane
(Vendelin et al., 2005) (Figure 2B). NPSR1-A and NPSR1-B differ only in the cytoplasmic domain (Figure 2B). An Asn/Ile107 SNP in the first extracellular loop of NPSR1 (Figure 2B) has been associated with increased asthma susceptibility
(Laitinen et al., 2004). The Asn/Ile107 SNP increases both the agonist efficacy as
SS well as intracellular trafficking of the receptor to the cell membrane without affecting binding affinity (Reinscheid et al., 2005; Bernier et al., 2006).
Classical structure-reactivity relationship studies show that NPS-(1-6) is the shortest sequence required to activate NPSR1 in vitro with similar potency and efficacy as full-length peptide. The residues Phe2, Arg3, and Asn4 of NPS are crucial for biological activity, whereas the sequence Thr8-Gly9-Met10 is important for receptor binding. The sequence Val6-Gly7 acts as a hinge region between the two above-mentioned domains. The stimulatory effect of ICV NPS on mouse locomotor activity was not fully mimicked by NPS-(1-10), suggesting that the C-terminal region of the peptide is important for in vivo activity.
Furthermore, part of a nascent helix within the peptide, spanning residues 5 through 13, acts as a regulatory region that inhibits receptor activation. Notably, this inhibition is absent in the asthma-linked Asn/Ile107 variant of NPSR, suggesting that residue 107 interacts with the aforementioned regulatory region of NPS (Roth et al., 2006; Bernier et al., 2006).
2.2. Expression and functional study in the lung
Immunohistochemistry of bronchial biopsies suggests that NPSR1-A is predominantly expressed by smooth muscle cells in the bronchus of both healthy and asthmatic individuals, whereas human NPSR1-B is expressed by epithelial cells of healthy individuals but by epithelial and smooth muscle cells of asthma patients. However, these results have not been consistently confirmed (Laitinen et al., 2004).
ST
In rodents, only one NPSR1 exists, and its amino acid sequence shows higher similarity to NPSR1-A and conserves Ile107. Allen et al. (2006) used an Npsr1 gene-targeted mouse, in which exon 4 containing Ile107 was deleted, to assess the role of NPSR1 in asthma development. They found no evidence that NPSR1 contributed to asthma development in an ovalbumin (OVA)-induced experimental asthma model or LPS-mediated airway inflammation model. However, they also did not detect pulmonary expression of NPSR1 in healthy individuals or asthmatic patients or of Npsr1 in OVA-sensitized, saline-challenged or OVA- challenged mice by real-time PCR. These results are in contrast to the initial findings by Laitinen et al., who used an unconventional OVA sensitization model
(OVA in combination with Stachybotrys chartarum) and found pulmonary upregulation of Npsr1 in mice with allergic lung disease (Laitinen et al., 2004).
Although implicated in human and murine asthma, the lack of evidence for a functional role of NPSR1 in a known immunological or asthmatic pathway raises questions about its contribution to asthma and the mechanism by which it may operate.
Through microarray analysis of downstream gene targets regulated by NPSR1 upon NPS stimulation in NPSR1-transfected HEK-293H cell line, the gene categories 'cell proliferation', 'morphogenesis', and 'immune response' were among the most altered. Among the up-regulated genes, matrix metallopeptidase 10 (MMP10), INHBA (activin A), interleukin 8 (IL8), and EPH
SU receptor A2 (EPHA2) exhibited a significant NPS dose-response relationship as confirmed by quantitative reverse-transcriptase–PCR. Immunohistochemical analyses revealed that the NPSR1-A regulated genes MMP10 and TIMP metallopeptidase inhibitor 3 (TIMP3) were co-localized with NPSR1-A in the bronchial epithelium. Furthermore, MMP10 was also co-localized with NPSR1-A in macrophages and eosinophils of sputum samples from asthmatics and controls (Vendelin et al., 2006). Orsmark-Pietras et al. (2008) identified Tenascin
C (TNC), a risk gene for asthma, as a target gene of NPS-NPSR1 signaling.
Polymorphisms within TNC were genetically associated to childhood asthma- and allergy-related phenotypes, with the strongest association to rhinoconjunctivitis, and there appears to be significant epistasis between NPSR1 and TNC altering the risk of disease.
2.3. Expression and functional study in the brain
In the rat, both quantitative real-time PCR and in situ hybridization studies reveal that Nps precursor mRNA and Npsr1 mRNA are widely expressed throughout the central nervous system (CNS). In situ hybridization studies reveal that the highest level of Nps precursor mRNA is found in the brain stem, mainly in the principle sensory 5 nucleus, the lateral parabrachial nucleus, and a previously undescribed area adjacent to the locus ceruleus. Low levels of Nps precursor mRNA expression are also found in the dorsomedial hypothalamic nucleus and the amygdala. In the brainstem, Nps precursor mRNA is co-expressed with excitatory or stimulatory transmitters, such as glutamate, acetylcholine, and
SV corticotrophin-releasing factor (CRF); all of these neurotransmitters have an important role in emotional regulation. The highest levels of Npsr1 mRNA were found in the hypothalamus, thalamus, amygdala, various cortical regions, and the parahippocampal formation, suggesting that NPSR1 is involved in the modulation of anxiety, arousal, energy balance, hormonal homeostasis, and learning and memory (Xu et al., 2004; Xu et al., 2007), as well as respiration.
A susceptibility locus for panic disorder was mapped to the human NPSR1 locus
(chromosome 7p15) (Knowles et al., 1998; Crowe et al., 2001; Logue et al.,
2003) and an NPSR1 SNP (Asn/Ile107) was associated with panic disorder in male Japanese patients (Okamura et al., 2007). Consistent with this genetic association study, intracerebroventricular (ICV) administration of human NPS showed anxiolytic-like effects in mouse models measuring responses to stressful or unfamiliar environments (open-field, light-dark, elevated plus maze, marble burying) (Xu et al. 2004; Leonard et al., 2008). These anxiolytic-like effects of
NPS were recently confirmed in the mouse four-plate and stress-induced hyperthermia tests (Leonard et al., 2008). Recent mechanistic studies demonstrated that the amygdala might represent the crucial brain area for NPS anxiolytic-like effects and facilitating extinction of conditioned fear (Jüngling et al.,
2008; Meis et al., 2008) and that CRF1 mediates the cocaine-seeking and locomotor stimulant effects of NPS, but not its effects on anxiety-like behavior
(Pañeda et al., 2009). Moreover, ICV NPS increased spontaneous locomotor activity and produced profound arousal that was independent of novelty. ICV
SW NPS was also able to induce wakefulness by suppressing all stages of sleep as demonstrated by electroencephalographic recordings in rats (Xu et al., 2004).
Several groups developed NPSR1 antagonists to characterize the physiological function of the NPS/NPSR1 system (Okamura et al., 2008; Camarda et al., 2009;
Guerrini et al., 2009). Their results showed that an NPS antagonist [D-Cys(tBu)5] dose-dependently antagonized the arousal-promoting action of NPS in mice
(Camarda et al., 2009) and that peripheral administration of antagonist SHA 68 in mice was able to block NPS-induced horizontal and vertical activity, as well as stereotypic behavior (Okamura et al., 2008). However, because it is unclear whether these antagonists specifically blocked NPSR1 and whether NPS has other receptors in the brain, the role of NPSR1 in NPS-induced functions in the brain is still unclear. At present, only one group used Npsr1-mutant mice (the same mice as Allen et al. used) to understand the function of NPSR1 in the brain, showing that the arousal-promoting action of 1 nmol NPS could be detected in wild-type (WT) but not in mutant mice (Camarda et al., 2009).
2.4. Expression in other tissues or cells
Pulkkinen et al. (2006) identified monocyte/macrophages and eosinophils as
NPSR1-positive cells in human blood and sputum cells. In peripheral blood mononuclear cells (PBMCs), monocyte activation with LPS, but not T cell activation with anti-CD3/CD28 antibodies, resulted in increased NPS and NPSR1 expression. NPS was capable of inducing phagocytosis of unopsonized bacteria.
SX This suggested that the NPS-NPSR1 pathway might be involved in regulating immune system.
NPSR1 gene polymorphism has been associated with inflammatory bowel disease (IBD) susceptibility (D'Amato et al., 2007) and colonic transit in functional gastrointestinal disorders (FGID) (Camilleri et al., 2010). NPSR1-A and NPS were found to be expressed in the crypt epithelial enteroendocrine cells of the small intestine. In NPSR1-A overexpressing cells, NPS stimulation upregulated the expression of the CGA, TAC1, NTS and GAL genes, which encode peptide hormone products of the gastric mucosal and intestinal enteroendocrine cells.
Stimulation with the pro-inflammatory cytokines TNF and IFN increased
NPSR1 expression in the THP1 monocytic cells. These results suggested that
NPS-NPSR1 signalling might have a dual role along the gut–brain axis and participate in the regulation of the peptide hormone production in enteroendocrine cells of the small intestine (Sundman et al., 2010). Camilleri et al. (2010) also reported that NPS-NPSR1 signaling upregulated neuropeptide expression, such as cholecystokinin, vasoactive intestinal peptide, peptide YY, and somatostatin in an NPSR1-A overexpressing cell line.
In addition, high expression of Nps and Npsr1 mRNA were found in endocrine tissues, including thyroid, mammary, and salivary glands. Central administration of NPS could modulate food intake and stress hormone (Beck et al., 2005; Niimi,
2006; Smith et al., 2006; Cannella et al., 2009)
SY
In summary, Npsr1 is highly expressed in the brain regions involved in stress response and regulation of respiratory function and endocrine tissues as well, whereas low levels of Npsr1 are expressed in the lung. Allen’s study showed that the direct role of NPSR1 in the development of experimental asthma was limited (Allen et al., 2006). Central NPS can modulate anxiety-related behaviors and regulate HPA axis. This evidence suggests that NPSR1 may be involved in the pathogenesis of asthma via CNS-mediated pathway.
3. Stress, asthma, and respiration
3.1. Link of stress with asthma
Stress can be defined as the psychophysiological reaction of the body to a variety of emotional or physical stimuli that are perceived to be threatening and unmanageable (Cohen et al., 1995). Normal reactions to stress generate an adaptive individual response aiming to reduce the negative impact of the stressor, such as fight-or-flight. If the host-coping abilities are judged as inadequate to confront the threatening situation, the stress is termed distress
(Lazarus et al., 1984). This results in negative emotional responses, which disturb the regulation of the hypothalamic-pituitary-adrenocortical (HPA) axis and the sympathetic and adrenomedullary (SAM) system, thereby inducing neuronal and humoral changes (Maier et al., 1998). Once activated, the stress response affects the regulation of the behavioral and biological functions, including
SZ cardiovascular activity, immune function, and further negative health consequences (Lazarus et al., 1984).
Depending on the duration of stress exposure, the stress is classified into acute and chronic stress. Acute stress (lasting several hours to several days), also called the fight-or-flight response or positive stress, is time-limited in duration and typically has a clear onset and offset, like an immediate threat of an exam.
Chronic stress lasts several months to lifetime and elicits prolonged responses
(Chen et al., 2007). In humans, these stressors include negative life events, such as reduced social status, death of a friend or family member, and loss of a job, or long-term negative emotions, such as anxiety and depression. In humans, chronic stress has been shown to play an important role in both onset of certain disorders and the exacerbation of symptoms, primarily in inflammatory diseases, such as cardiac ischemia (Krantz et al., 2000), IBD (Mawdsley et al.,
2005), atopic dermatitis, and allergic asthma (Wright, 2005; Montoro et al., 2009), or in autoimmune diseases, such as rheumatoid arthritis, and others (Potter et al., 1997; Martinelli, 2000; Mizokami et al., 2004).
From the historical perspective, asthma was originally believed to be psychogenic. Accumulating evidence have substantiated that stress contributes to exacerbations of asthma. Clinicians have observed that between 20% and
35% of subjects with asthma experience exacerbations of symptoms during periods of stress (Isenberg et al., 1992). Some asthmatic patients experience
S[ increased bronchoconstriction during acutely distressful situations (Lehrer, 1998).
Stress associated with final examinations can act as a cofactor to increase eosinophilic airway inflammation to antigen challenge in college students Figure 3. Psychological stress response and its effect on respiratory with mild asthma (Liu function. Normal reactions to acute stress generate an adaptive response aiming to reduce the negative impact of the stressor, such as fight-or-flight (e.g. increased minute ventilation, locomotion, exploration, corticosterone et al., 2002). High production, and heart rate). Chronic stress induces negative emotional responses, further altering the inflammatory process and respiratory levels of psychosocial function and, therefore, may potentially affect the development of asthma. Some neuropeptides have been implicated in the stress response and stress are correlated pathophysiology of anxiety and depression. with the high onset and morbidity of asthma (Sandberg et al., 2000). Psychologic distress in children has been associated with asthma and reduced clinical response to immunotherapy (Ippoliti et al., 2006), with more frequent and longer admissions to the hospital (Kaptein, 1982), and with greater functional disability
(Gutstand et al., 1989). Differences in stress coping behavior lead to different impact on asthma symptoms. Passive stressful tasks, like watching bloody surgeries or stress-inducing videos, cause increased airway resistance in asthmatic patients in comparison to active stressful tasks like arithmetic (Lehrer et al., 1996). Similarly, relieving stress by writing about stressful experiences improves a number of physiological parameters of asthma for long periods
TR (Smyth et al., 1999). Psychological stress has the capacity to alter the inflammation process or respiratory function and, therefore, may potentially affect the development of asthma (Figure 3). In addition, chronic allergic asthma may have a psychological effect by disrupting daily life, which could further deteriorate asthma.
3.2. Stress and lung inflammation
Possible links of stress with asthma include stress-induced modulation of immune and inflammatory processes and psycho-neuro-immunological imbalances as a possible cause of asthma exacerbations.
Psychological stress affects lung inflammation through the HPA axis and the autonomic nervous system. The latter is composed of sympathetic and parasympathetic system and originated in the CNS with norepinephrine and acetylcholine as neurotransmitters, respectively. Psychological stress induces the activation of the paraventricular nucleus of the hypothalamus, which secrete corticotropin-releasing hormone (CRH). CRH travels to the anterior pituitary gland, which secrets adrenocorticotropin hormone (ACTH). ACTH travels to the adrenal glands through the peripheral circulation, inducing the secretion of corticoids by the adrenal cortex and of catecholamines (epinephrine, norepinephrine, dopamine) by the adrenal medulla. Catecholamines and corticoids modulate inflammation distinctively depending on the duration of stress. On the other hand, psychological stress activates the locus coeruleus in
TS the brain stem, inducing secretion of norepinephrine, activating the sympathetic nervous system, and releasing norepinephrine in the sympathetic nerve endings.
In the lung, sympathetic activation facilitates pulmonary bronchodilation and regulates various features of the humoral response involved in asthma, including
Th2 cytokine responses, mast cell activation, and eosinophil activation.
Parasympathetic fibers innervate the airways and release acetylcholine after activation, facilitating bronchoconstriction and mucus secretion. In patients with asthma, stress is associated with hyperresponsiveness of both the parasympathetic and alpha-sympathetic pathways. Psychological stress and lung inflammation bidirectionally communicate through these hormones, neurotransmitters, neuropeptides, and cytokines (Chen et al., 2007; Montoro et al., 2009).
Acute psychological stress activates the HPA axis, resulting in secretion of corticoids and catecholamines. These stress hormones reduce inflammation and plasma exudation. Acute stress augments the immune response and induces an adrenal hormone-mediated redistribution of immune cells from the blood to the bone marrow, lymph nodes, and skin (Umland et al., 2002). Acute stress also facilitates antigen specific cell-mediated immunity, alters populations of T-cell subsets, and modulates mononuclear cell trafficking (Dhabhar et al., 1996). As time passes, exposure to chronic stress leads to adaptation of the HPA axis response, including glucocorticoid receptor down-regulation, loss of negative feedback on secretion of CRF and ACTH hormone by the hypothalamic and
TT pituitary gland, respectively (Makino et al., 2002), and disabling of sensitivity to endogenous corticosteroids; asthmatic patients demonstrate the same consequences as stress-related glucocorticoid resistance (Wittert et al., 1996;
Makino et al., 2002). Chronic stress seems to inhibit the migration of immune cells from the blood, correlating with the attenuation of responsiveness to corticosteroids (Dhabhar, 2000). In ways other than glucocorticoid resistance, exposure to high doses of corticoids can disrupt Th1/Th2 balance and amplify the
Th2 cytokine response (Marshall et al., 2000), which favor the onset and exacerbation of asthma following exposure to a trigger. The classic effect of chronic stress on the immune response has been reported as suppression of cellular immunity, including natural killer cell cytotoxicity, lymphocyte proliferation to mitogens, and in vitro production of lFN- , leading to increased susceptibility to infection, which further exacerbates asthma (Marshall et al., 2004). In the patient with asthma, long-term exposure to high levels of epinephrine and norepinephrine due to chronic stress leads to down-regulation of 2-adrenergic receptors in pulmonary and lymphoid tissues (Miller et al., 2006). This contributes to exacerbation of asthma symptoms by amplifying Th2 cytokines production, mast cell degranulation, and eosinophil activation, as well as diminishing the beta-agonist’s bronchodilatory effects in treatment.
Studies in mice indicate that abnormal regulation of CRH and endogenous glucocorticoids has been implicated in the pathogenesis of asthma. Silverman
(2004) reported that CRH deficiency disrupted endogenous glucocorticoid
TU production and enhanced allergen-induced airway inflammation and lung mechanical dysfunction in mice. There are distinct effects of acute and chronic psychological stress on airway inflammation in murine models of allergic asthma.
Acute stress improved the airway inflammation in a glucocorticoid-dependent manner, whereas chronic psychological stress induced the exacerbation of allergic airway inflammation, which was independent of the glucocorticoid response (Forsythe et al., 2004). Okuyama (2007) reported a similar phenomenon and emphasized that acute stress modification of the allergic airway responses varied depending upon the mouse genetic background.
Psychological stress affects asthma development distinctly depending on the type of stress and the genetic background of the host (Wright, 2005). Genetic differences may explain the fact that only a subgroup of patients with asthma experience increased severity under stressful conditions. This finding is supported by strain differences in mice in behavioral responses to stress and in effects of stress on asthma exacerbation (Beuzen et al., 1995; Crawley et al.,
1997; Griebel et al., 2000; Tang et al., 2002; Okuyama et al., 2007). Okuyama et al. (2007) reported that acute stress inhibited inflammatory cells in the bronchoalveolar lavage fluid (BALF) in BALB/c mice, but not in C57BL/6 mice whereas chronic stress significantly increased pulmonary inflammation in both mouse strains. In addition, in utero and postnatal environmental factors, independent of genetic susceptibility, affect expression of asthma. Maternal stress and exposure to stress during early childhood could result in elevated
TV corticoid levels and dysfunctional behaviors, affect Th1/Th2 differentiation, and increase the susceptibility to asthma in susceptible children that are evident later in life (Essex et al., 2002; von Hertzen, 2002; Wright et al., 2004; Meaney et al.,
2005).
3.3. Stress and respiration
Some neuropetides (substance P), neurotransmitters (acetylcholine, norepinephrine, histamine), and cytokines secreted through inflammation processes are involved in regulation of respiratory function. In the following, we will focus on the structures of CNS responsible for respiration and review the literature regarding the association of psychological stress and emotional diseases with respiration.
In the CNS, there are two neurogenic systems, voluntary and involuntary, responsible for establishing the basic respiratory rhythm. Medulla pons, limbic systems, hypothalamus, and other subcortical structures are involved in involuntary respiration. Voluntary respiration is initiated by the cerebral cortex.
Breathing is initiated in the brainstem by a network of neurons that project neuromuscular output to muscles in the chest wall and upper airway. The coordinated action of these muscles lead to mechanical forces that both move the chest wall to inflate (or deflate) the lungs and ensure that the size of the upper airway permits airflow in and out of the lungs.
TW Limbic systems are responsible for the respiratory regulation in emotion.
Grossman (1983) summarized that respiration and emotional states have been linked in the following ways: 1) stressors have an effect on respiratory function;
2) specific patterns of respiratory variables are associated with dispositional personality traits; and 3) breathing pattern may be a good physiological index of subjective feeling of anxiety. During episodes of stress, respiratory alterations are manifested as increase in respiratory rate, minute ventilation volume and irregularity of breathing, alteration in tidal volume of respiration, and decrease in blood and alveolar CO2 levels as compared to baseline conditions (Garssen,
1980; Suess et al., 1980). Abdominal-diaphragmatic breathing is enhanced by relaxation and suppressed under emotional stress (Faulkner, 1941). Increased ventilation characterized by shortness of breath, palpitations, dizziness, faintness, or tingling in extremities is a component of the fight-or-flight response, an adaptive response to danger. However, if this increased ventilation is not followed by sufficient physical activity, the level of arterial PCO2 falls.
Hyperventilation itself produces the change of mood and symptoms observed in anxiety disorders (Lum, 1987). Enlow et al. (2009) showed that there were associations of maternal lifetime trauma exposure and traumatic stress during the perinatal period with disrupted infant cardiorespiratory regulation and behavioral distress in response to psychological challenge. Infants of mothers with low exposure to trauma and perinatal traumatic stress showed expected increases in behavioral distress and cardiorespiratory activation from baseline to stressor and decreases in these parameters from stressor to recovery. Infants of
TX mothers exposed to multiple traumas and with elevated perinatal traumatic stress showed similar patterns of activation from baseline to stressor but failed to show decreases during recovery.
Respiration is thought to be psychophysiological, a mediator between mind and body for a variety of disorders. Interactions between emotional states, respiratory behaviors, and physiological changes in chemical blood composition and autonomic nervous system regulation have a role in psychological diseases.
Jason (1994) showed that there was an association between reported respiratory symptoms and psychological status; however, there was no evidence that patients with bronchial asthma had more anxiety and depression than those without asthma as assessed by a structural interview, spirometry, methacholine challenge, peak flow diary, skin prick test, measurement of eosinophil activity in peripheral blood, and determination of the psychological status by the questionnaire of hospital anxiety and depression scale. Experimental exposure to emotional stimuli has also been shown to increase respiratory resistance in asthma (Ritz et al., 2000). Thus, anxiety and depression are likely not causes of asthma, but instead are modulators of asthma severity and/or occurance.
Panic disorders are a set of severe and chronic anxiety disorders. Besides recurrent spontaneous anxiety attacks, panic disorders are dominated by respiratory symptoms such as hyperventilation, a feeling of being smothered, and shortness of breath. Hypersensitivity of brainstem nuclei regulating respiratory
TY activity has been proposed in patients with panic disorder (Gorman et al.,1988), and respiratory dysregulation has been identified as a biological marker for panic disorder (Hegel et al., 1997; Holt et al., 1998). Breathing training treatment can reduce symptoms of anxiety and hyperventilation compared with controls (Bessel et al., 1997). Thus, a variety of clinical psychological therapies, including cognitive-behavioral treatment, contain breathing training (Barlow et al., 1989).
Smoller (1996) showed that the symptoms of panic attacks and pulmonary disease overlap. Patients with pulmonary disease, particularly those with obstructive lung disease, have a high rate of panic symptoms. Successful treatment of panic can help these patients improve functional status and quality of life.
3.4. Neuropeptides, asthma, and emotional disorder
Growing evidence has emerged indicating neuronal dysregulation in allergic asthma. A large number of Figure 4. Role of pulmonary neuropeptides in the pathogenesis of asthma. Sensory nerves innervate lung and lymphoid organs. Allergen neuropeptides have exposure leads to enhanced synthesis of neuropeptides from the pulmonary sensory nerve ending and cause airway smooth muscle been identified in contraction, mucus secretion, increased airway microvascular permeability and exudation of plasma into the airway lumen. Neuropeptides modulate airways and have immune cell functions by binding to receptors on the immune cells, resulting in cellular activation and neurogenetic inflammation. Activation of immune cells and release of neuropeptides sensitize sensory nerve fibers an important role in and increase neuropeptide production.
TZ the pathogenesis of asthma (Figure 4). These neuropeptides mediate local interactions between the immune system, lung structural cells, and the autonomic nervous system within the lung, contributing not only to airway smooth muscle contraction, but also to the modulation of allergic airway inflammation by direct interaction with immune cells (Barnes, 2001).
Recent studies indicate that allergen exposure leads to sensory neuroplasticity in the airways (Barnes, 1995). Sensory neuroplasticity is characterized by an enhanced synthesis of neuropeptides in nodose primary afferent neurons.
Neuro-immune interactions are facilitated by the innervations of lung and lymphoid organs by sensory nerves. Pulmonary sensory nerves, which can be found in and around the bronchi, bronchioles, and occasionally in alveoli
(Lundberg et al., 1984), form a dense network surrounding the airway smooth muscle, submucosal gland, epithelium, and blood vessel (Lamb et al., 2002).
Sensory nerve fibers often form close contacts with mast, macrophage/monocyte cell lineage and lymphoid cells (Nohr et al., 1991). Neuropeptides are released from the sensory nerve ending upon contact with an irritant and cause airway smooth muscle contraction, mucus secretion, increased airway microvascular permeability and exudation of plasma into the airway lumen, and modulation of immune cell functions, leading to neurogenic inflammation. Neuropeptides bind to the receptors on the immune cells, resulting in chemotaxis; activation and degranulation of eosinophils; release of histamine, leukotrienes C4, and prostaglandin D2 by mast cells; increased lymphocytes proliferation, chemotaxis
T[ and adhesion; and inhibition of Th1 cytokine synthesis (De Swert et al., 2006). In the airways, activation of immune cells and release of neuropeptides not only increase airway inflammation but also sensitize sensory nerve fibers and increase neuropeptide production. Stimulation of sensory nerves in the airways results in CNS reflex responses consistent with asthma symptoms (e.g., bronchoconstriction, cough, mucous secretion) (Wright et al., 2005). Jacoby et al.
(1993) report that eosinophil granule proteins such as major basic protein (MBP) and eosinophil peroxidase (EPO) are selective allosteric antagonists at
muscarinic M2 receptor. Loss of function of these M2 receptors leads to an increase in vagally mediated airway hyperreactivity as seen in some patients with asthma and in the animal models of hyperreactivity.
Among neuropeptides, vasoactive intestinal peptide (VIP) and peptide histidine methionine are potent bronchodilators. In asthma, their effect might contribute to exaggerated bronchial responsiveness if these peptides are broken down more rapidly by peptidase from inflammatory cells (Barnes et al., 1987). Sensory neuropeptides, such as substance P, neurokinin A, and calcitonin gene-related peptide (CGRP) might contribute to the pathology of asthma. Substance P is the primary tachykinin that acts on the neurokinin-1 (NK-1) receptor. In experimental studies, substance P has been linked to neurogenic inflammation and regulation of stress hormonal pathways and has been implicated in asthma (Bienenstock et al., 2003). Substance P has a role in bronchial hyperresponsiveness, airway inflammation, mast cell activation, cytokine secretion, and mucous secretion
UR (Rosenkranz, 2007; Elenkov, 2008). Substance P is increased in the lungs of asthmatic patients (Rosenkranz, 2007). Stress leads to the release of substance
P (Joachim et al., 2006). NK-1 receptor is present in the human lung and submucosal glands and on rodent mast cells, lymphocytes, and macrophages. A
NK-1 receptor antagonist improves stress-induced exacerbation of asthma in a murine model of asthma (Joachim et al., 2004). Substance P deletion by chronic treatment with capsaicin is associated with reduced bronchoconstrictor response to allergen in sensitized animals (Saria et al., 1983). This evidence suggests that neuropeptides may be associated with the symptoms of asthma and stress induced exacerbation of asthma.
Some neuropeptides, for example, substance P, have been implicated in the pathophysiology in a subset of anxiety and depression sufferers (Figure 3).
Depressed patients in multiple studies have shown increased serum and cerebrospinal fluid (CSF) levels of substance P. Substance P is located in the brain regions involved in processing stress and evokes behavioral indications of anxiety, fear, and aversion. Substance P activity is also aberrant in some individuals with anxiety and depression. Blocking substance P receptor or lowering substance P levels has effectively been used in the treatment of anxiety and depression (Rosenkranz, 2007). Asthma has a high co-incidence with anxiety and depression. Stress and negative emotion have also been shown to trigger and exacerbate symptoms in these diseases. Substance P mediates the communications between the lung, immune system, endocrine system, and the
US brain and evokes symptoms of asthma and emotional diseases. Chronic allergic asthma and these emotional disorders may share a common pathophysiological pathway.
NPSR1 has been associated with asthma and panic disorder. Although there was no direct evidence showing that NPSR1 was involved in the pathogenesis of experimental asthma, the expression pattern of Npsr1 in the brain and endocrine tissues, and NPS’s function in regulation of anxiety and HPA axis suggest that
NPSR1 may be involved in the pathogenesis of asthma through a central- mediated pathway. In this thesis (Figure 5), we first elucidated the functional role of NPSR1 in stress process at baseline and then determined if NPSR1 mediates
NPS-induced anxiolytic effects or corticosterone increases using a novel Npsr1- targeted mouse in which exon 2 was deleted (Chapter 2). Nps and Npsr1 were mainly expressed in the brain regions involved in regulating respiration, suggesting that NPS and NPSR1 have a role in this process.
In chapter 3, we investigated the action of ICV- administered NPS on basal and methacholine-challenged respiration in Npsr1- deficient and wildtype mice. In addition, Npsr1-deficient mice were used to determine if NPSR1 was directly involved in allergen-induced (OVA or
UT Aspergillus fumigatus) experimental asthma, including AHR.
4. In vivo assessment of respiratory function
Currently, there are noninvasive and invasive methods to assess respiratory function. The whole-body plethysmography, a noninvasive method, involves placing an unrestrained animal in a chamber and utilizing a barometric analysis technique to quantify pulmonary physiological values. From pressure changes in the chamber, the plethysmograph enables calculations of ventilatory parameters, including minute ventilation, tidal volume, frequency, and patterns of breathing under basal conditions as well as analysis of changes during exposure to varying concentrations of gas and other stimuli. Without bronchoconstriction, it monitors the basic flow-derived parameters. With methacholine challenge, enhanced pause (Penh) represents an index of AHR. The advantages of whole-body plethysmography include repeated recordings in the same animal without the need of anesthesia or restraint and noninvasive measurement of breathing pattern during routine behaviors (e.g., rest, exercise, grooming), during treatments or conditioning periods, and in various states of consciousness (e.g., asleep, awake, anesthetized) (Uchida et al., 1996; Cieslewicz et al., 1999). The major drawbacks of this method include the lack of direct assessment of pulmonary mechanics and its susceptibility to artifacts, such as movement, temperature, and sniffing. It is important to avoid confounding environmental effects by studying the animals in a relatively quiet room that is not used for
UU autopsy, surgery, or dissections (DeLorme et al., 2002; Sly et al., 2004; Baekey et al., 2009).
Invasive methods are most appropriate when there is a need to precisely localize airway mechanics for the study of a particular disease or disorder. They typically involve the insertion of measuring devices (a tracheostomy) and require surgeries and anesthesia. The flexiVent system, an invasive method, permits sensitive and specific analysis of pulmonary mechanics and enables identification of anatomical relationships in the lung/chest wall and upper airway.
However, the invasive method is affected by the anesthesia required for the surgical procedures and repeated assessments in the same animal are not possible because these procedures are terminal, necessitating large numbers of animals (DeLorme et al., 2002; Sly et al., 2004).
In the chapter 3 of this thesis, we aimed to identify whether ICV NPS had directly mechanical effect on airway or centrally changed respiratory pattern using aforementioned two methods.
5. Anxiety and depression mouse model
The role of NPSR1 in stress response and whether NPSR1 is required for NPS induced anxiolytic effect are still unknown. To answer these questions, we screened anxiety and depression related behaviors in NPSR1-deficent mice in the chapter 2 of this thesis.
UV 5.1. Models for assessing anxiety-like behavior
Classical mouse models of measuring anxiety-like behavior include unconditioned response tests that do not involve pain or discomfort and conditioned responses to stressful or painful events (e.g. exposure to an electric footshock). Unconditioned response tests include exploration-based approach – avoidance conflict tests (open-field, elevated zero maze, and light/dark exploration tests) and social and other tests such as acoustic startle response, mouse defense test battery, and stress-induced change in physiological parameters (Belzung et al., 1995). Exploration-based approach – avoidance conflict tests are widely used because they can quickly screen drug treatments or mouse genotypes related to anxiety modulation without mouse training or complex schedules. All of these procedures are based on the exposure of subjects to unfamiliar places (Cryan and Holmes, 2005).
5.1.1. Open-field test
Locomotor activity is the foundation of every mouse behavioral paradigm. To prevent false interpretation of the behavior, it is important to first perform spontaneous locomotion before starting any further behavior test screening. The open-field test measures locomotion and exploration, evaluating total amount of movement and type of spontaneous activity over 60-minute periods. The open- field can be also used to measure anxiety-like behaviors. The novel environment provided by the open-field may prompt mice to produce naturally conflicting motivations related to fear and exploration. Upon initial exposure to the open-
UW field, mice express anxiety-like behaviors such as lower levels of locomotion and more time in the margin area of the open-field. As time in the open-field increases, mice exhibit greater exploratory behavior, reflected in higher levels of locomotion, more time in the central area of the open-field, and increased rearing
(Tang et al., 2004). Increasing background noise or illumination level results in decreased activity and increases the stressful properties of the open-field.
5.1.2. Elevated zero maze
The facility of the elevated zero maze is a running circle composed of 2 open areas and 2 enclosed areas. Based on the conflict between the tendency of mice to explore a novel environment and the aversive properties of an open, elevated visual cliff (Crawley et al., 1997; Cryan and Holmes, 2005), the non- drug-treated mice are expected to avoid open areas and prefer to remain in the enclosed areas for most of the testing period. Anxiolytic drugs increase the number of entries into the open areas, time spent in the open areas and head dips.
5.1.3. Light-dark exploration test
The facility for the light-dark exploration test is the same open chamber as used for the open-field test divided into 2 parts, 1 lit area and 1 dark area, by inserting a black box. There is an opening through which mice can run between the lit and dark area. The test is based on the conflict between the tendency of mice to explore a novel environment and the aversive properties of a brightly lit open
UX field. Anxiolytic drugs increase exploratory behaviors, such as the number of transitions and/or time spent in the dark (Crawley et al., 1997).
The above exploration-based tests (open-field, elevated zero maze, light-dark exploration) do not dissociate decreased anxiety-like behavior from increased novelty seeking or impulsivity-related approach behavior. In addition, these tests rely on intact mouse sensory and motor function.
5.1.4. Marble burying test
The marble burying test measures the defensive behaviors in response to external threatening stimuli, such as marbles. The mice spontaneously bury the external marbles placed in their cages. The anxiolytic drugs tend to decrease defensive behaviors (Njung’e et al., 1991).
5.1.5. Acoustic startle response
The acoustic startle response can be elicited by sudden, loud acoustic stimuli and is characterized by a coordinated contraction of the muscles of the eyelid, neck, and extremities. Sensorimotor gating is assessed by prepulse inhibition: a weak, non-startling stimulus (prepulse) diminishes the response to a startle stimulus, which is presented 30-500 ms later. Prepulse inhibition deficits can be seen in the patients with neuropsychiatric disease like schizophrenia,
Huntington’s disease, attention deficit disorder, and post-traumatic stress disorder (Plappert et al., 2001).
UY
5.2. Models for assessing antidepressant activity
Experimental models for assessing depression-like behaviors include despair paradigms (e.g., tail suspension, forced swim tests), olfactory bulbectomy, maternal/social deprivation, and “anhedonic” chronic mild stress (Kalueff et al.,
2007). Tail suspension and forced swim tests are the most widely used models for measuring antidepressant-like activities in mice. These models are based on the fact that mice exposed to the short-term, inescapable stress of being suspended by its tail or being in an inescapable cylinder filled with water will develop immobility posture, a sign of behavioral despair and giving up hope of escaping. The immobility responses are analogous to depressed patients lacking sustained expenditure of effort, reflecting pronounced psychomotor impairments (Cryan and Holmes, 2005). This behavior can be countered by pharmacological or genetic manipulation related to depression and antidepressant action. Although these two tests have similar intentions, the tail suspension test is not the dry-land version of the forced swim test. Compared with the forced swim test, the tail suspension test avoids hypothermic exposure and the need of mice to swim. The latter can be especially important if the gene- modified mice have recognized or unrecognized compromised motor activity.
The responses to antidepressant drugs in both tests are not consistent. Some drugs seem to affect activity in the forced swim test, but not in the tail suspension test (Cryan, Mombereau and Vassout; 2005).
UZ The Morris water maze is known as a hippocampus memory paradigm. Because cognitive mechanism plays an important role in stress pathogenesis (Van Der
Kolk et al., 1996; Ohl, 2005), the Morris water maze has been used to assess depression like-behavior (Schulz et al., 2004). Visual activity and swimming ability are important for spatial learning tasks.
Although the mechanism of anxiety and depression has not been fully understood, anxiety and depression show overlap and co-occurrence (Kalueff et al., 2007) in their symptoms and response to the same treatment, suggesting they share common neurobiological dysfunction.
5.3. Mouse strain differences in the behavioral tests
Emerging findings report that differences in anxiety or depression-related behaviors vary among commonly used mouse strains and are attributed to the genetic background (Beuzen et al., 1995; Crawley et al., 1997; Griebel et al.,
2000; Tang et al., 2002). BALB/c mice have greater home cage activity than
C57BL/6 mice. However, in the novel environment tests such as the open-field and elevated zero maze tests, BALB/c mice appeared to be more anxious than
C57BL/6 mice, showing less horizontal and vertical movement, less time spent in the center at initial exposure to the open-field, greater numbers of stretched- attend postures, fewer head dips and transitions, and less time spent in the open in the elevated zero maze test. For the light-dark exploration test, C57BL/6 mice show the highest numbers of baseline light-dark transitions and strongest
U[ responses to anxiolytics whereas BALB/c mice have the lowest baseline transitions and weakest responses to anxiolytics among 4 inbred and 3 outbred strains of mice. In the Morris water maze, C57BL/6 mice show good performance whereas BALB/c mice perform poorly. Among 11 inbred strains of mice, the C57BL/6 and BALB/c mice strains show medium levels of acoustic startle response. For prepulse inhibition, C57BL/6 mice have the lowest level, and BALB/c mice have medium levels. Mouse strain distributions of behavioral activities provide the information to optimally choose a mouse strain to test the phenotype of gene-mutated mice or interpret the results properly.
Mouse emotional behaviors in the tests are influenced by different kinds of variables such as test procedures, experimental surroundings, age, gender, and housing conditions. The tests are also influenced by previous test history
(McIlwain et al., 2001). Thus, results should be interpreted with caution and in the context of the mouse strain, and the potential effect of the previous test should be considered when arranging the order of a series of behavior tests.
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WY Chapter 2
Abnormal response to stress and impaired NPS-induced hyperlocomotion, anxiolytic effect and corticosterone increase in mice lacking NPSR1
Hongyan Zhua, b, Melissa K. Minglera, Melissa L. McBridea, Andrew J. Murphyc, David M. Valenzuelac, George D. Yancopoulosc, Michael T. Williamsd, Charles V. Vorheesd, *, and Marc E. Rothenberga, *
aDivision of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA. bGraduate Program of Molecular and Developmental Biology, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA. cRegeneron Pharmaceuticals, Inc, Tarrytown, New York 10591, USA. dDivision of Neurology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA.
*Correspondence should be addressed to Marc E. Rothenberg, Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA; Tel.: (+1) 513 636-7210; Fax: (+1) 513 636-3310; E-mail: [email protected] or Charles V. Vorhees, Division of Neurology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA; Tel.: (+1) 513 636 8622; Fax: (+1) 513 636 3912; E-mail: [email protected].
Previously published in Psychoneuroendocrinology 2010; 35(8): 1119-1132
WZ Abstract
NPSR1 is a G protein coupled receptor expressed in multiple brain regions involved in modulation of stress. Central administration of NPS, the putative endogenous ligand of NPSR1, can induce hyperlocomotion, anxiolytic effects and activation of the HPA axis. The role of NPSR1 in the brain remains unsettled. Here we used NPSR1 gene-targeted mice to define the functional role of NPSR1 under basal conditions on locomotion, anxiety- and/or depression-like behavior, corticosterone levels, acoustic startle with prepulse inhibition, learning and memory, and under NPS-induced locomotor activation, anxiolysis, and corticosterone release. Male, but not female, NPSR1-deficient mice exhibited enhanced depression-like behavior in a forced swim test, reduced acoustic startle response, and minor changes in the Morris water maze. Neither male nor female NPSR1-deficient mice showed alterations of baseline locomotion, anxiety- like behavior, or corticosterone release after exposure to a forced swim test or methamphetamine challenge in an open-field. After intracerebroventricular (ICV) administration of NPS, NPSR1-deficient mice failed to show normal NPS-induced increases in locomotion, anxiolysis, or corticosterone release compared with WT
NPS-treated mice. These findings demonstrate that NPSR1 is essential in mediating NPS effects on behavior.
Keywords: neuropeptide S; neuropeptide S receptor; NPSR1; stress; anxiety; depression; locomotor activity; corticosterone
W[ 1. Introduction
NPSR1 (also known as GPR154, GPRA) is a G protein coupled receptor (GPCR) expressed in multiple brain regions suggested to be involved in the modulation of anxiety, arousal, energy balance, and hormonal homeostasis, as well as learning and memory (Xu et al., 2007). Human NPSR1 mRNA encodes 8 variants by alternative splicing, only 2 of which, hNPSR1-A and hNPSR1-B, are functional receptors supported by the evidence that they are located on the cell membrane
(Vendelin et al., 2005). In rodents, one single NPSR1 exists and its amino acid sequence shows higher similarity to hNPSR1-A. A susceptibility locus for panic disorder was mapped to the human NPSR1 locus (chromosome 7p15) (Knowles et al., 1998; Crowe et al., 2001 and Logue et al., 2003) and an NPSR1 SNP
(Asn/Ile107) was associated with panic disorder in male Japanese patients
(Okamura et al., 2007).
By a reverse pharmacological strategy, neuropeptide S (NPS) was identified as a putative ligand for NPSR1 since binding causes increased intracellular calcium mobilization and cAMP accumulation (Xu et al., 2004). An Asn/Ile107 SNP in
NPSR1 increases both the agonist efficacy as well as intracellular trafficking of the receptor to the cell membrane without affecting binding affinity (Reinscheid et al., 2005 and Bernier et al., 2006). Real-time PCR shows NPS to be mainly expressed in the brain and endocrine tissues (Xu et al., 2004). NPS precursor mRNA is co-expressed with excitatory or stimulatory transmitters, such as glutamate, acetylcholine, and corticotrophin-releasing factor (CRF) in the
XR brainstem (Xu et al., 2007); all these neurotransmitters have an important role in emotional regulation. Central administration of NPS modulates locomotor activity, arousal, anxiety, the hypothalamic-pituitary-adrenal (HPA) axis, and food intake (Xu et al., 2004; Beck et al., 2005; Niimi, 2006; Smith et al., 2006; Leonard et al., 2008; Rizzi et al., 2008; Vitale et al., 2008). Together, these data suggest that NPSR1 may be involved in these activities induced by NPS. Although several groups developed NPSR1 antagonists to characterize the physiological function of the NPS/NPSR1 system (Okamura et al., 2008; Camarda et al., 2009 and Guerrini et al., 2009), the role of NPSR1 in the brain is still unclear.
The current investigation was undertaken to elucidate the functional role of
NPSR1 in locomotor activity, anxiety and/or depression, startle reactivity, corticosterone release, spatial learning ability, and response to methamphetamine challenge at baseline (without extraneous NPS stimulation).
Animals were also examined to determine if NPSR1 mediates NPS-induced locomotor activation, anxiolytic effects, or corticosterone increases using a novel
NPSR1-targeted mouse in which exon 2 was deleted.
XS 2. Materials and Methods
2.1. Mice
NPSR1-deficient mice were established by gene targeting using VelocigeneTM technology (Regeneron Pharmaceuticals, Inc. Tarrytown, NY) (Valenzuela et al.,
2003). The main strategy was based on replacing the 3’ 128 bp of exon 2 and the first 1510 bp of intron 2 with a translational-fusion -Gal reporter gene and a neomycin resistance gene. Mice with the genetic background of 129Sv/J ×
C57BL/6 cross were backcrossed 7 generations with C57BL/6 mice. Genotypes were determined by PCR of tail DNA using primers specific for the wild type (WT) allele (sense, 5'-CATCCAGGAAACGT GAGCAC-3'; antisense, 5'-
TCTAGCATCGGCACAGTCTG-3') and the gene deleted allele (sense, 5'-
CATCCAGGAAACGTGAGCAC-3'; antisense, 5'-GTCTGTCCTAGCTTCCTCACT
G-3'). Behavioral testing under baseline conditions were carried out on 8- to 10- week-old NPSR1-deficient mice and WT littermates derived from crossing
NPSR1 heterozygous mice. The NPSR1-deficient mice used for behavioral studies after NPS ICV injection were generated using NPSR1 homozygous female and male matings, and age and sex matched C57BL/6 WT mice bred in- house were used as control. All the mice were maintained and housed 4 per cage by sex under pathogen-free conditions. At least 1 week prior to behavioral testing, NPSR1-deficient and WT mice were transferred to conventional housing under a 14 h light/10 h dark cycle (lights on at 600 h) with temperature (19 ± 1°C) and humidity (50 ± 10%) controlled. All procedures were approved by the
Institutional Animal Care and Use Committee of the Cincinnati Children’s
XT Research Foundation. The number of animals used in each test was given in the figure captions and results.
2.2. Reverse Transcription Polymerase Chain Reaction (RT-PCR)
The mRNA expression in the brain from male NPSR1-deficient mice and WT littermates was examined using RT-PCR. Briefly, total RNA was isolated from the tissue, treated with DNAase I and reverse-transcribed using oligo (dT) and
Superscript II reverse-transcriptase (Invitrogen, Carlsbad, CA) in a 20 l reaction.
The cDNA produced by these reactions (2 l) was amplified by PCR using Taq
DNA polymerase and the primers used in the PCR reaction are indicated below: full length NPSR1 sense, 5'-TCGTCAGGCAGAACTCTTCA-3' specific to 5’UTR and antisense, 5'-ATCTGCTAGGTGAGGCAGGA-3' specific to 3’UTR (1300 bp product); LacZ sense, 5'-ATACACTTGCTGATGCGGTGC-3' and antisense, 5'-
AGATGGCGATGGCTGGTTTC-3' (460 bp product).
2.3. LacZ detection
Brains from male NPSR1-deficient and WT littermates were perfused with 4%
PFA and fixed overnight in 4% PFA. Fixed tissues were cryoprotected overnight in cold 30% sucrose in PBS and embedded in OCT embedding medium and frozen on dry ice. These cryostat sections were stored at -80oC until assessment.
XU -Gal staining was performed on 20 μm thick cryostat coronal sections of frozen fixed brain (Steele-Perkins et al., 2005). The slides were allowed to dry at room temperature for 2 h and fixed in 0.2% glutaraldehyde on ice for 10 min, washed in PBS with 2 mM MgCl2 3 times for 10 min, and in PBS with 0.02% NP-40 and 2 mM MgCl2 once for 15 min on ice. Sections were subsequently incubated in the
X-gal staining solution (PBS; pH7.3) containing 5 mM potassium hexacyanoferrate, 5 mM potassium ferrocyanide trihydrate, 2 mM MgCl2, 0.02%
NP-40, 0.01% sodium deoxycholate, and 1 mg/ml X-gal solution (stock 40 mg/ml
X-gal in DMF) at room temperature for 8 h. Stained sections were washed 2 times in PBS with 2 mM MgCl2, cleared in distilled water for 5 min, and then counterstained with 0.1% nuclear fast red in 5% aluminum sulfate for 2 min, washed in running water, dehydrated, and topped with coverslips for analysis.
2.4. Behavioral studies under baseline conditions
The behavioral tests under baseline conditions were carried out between 1300-
1700 h on 2 groups of 8- to 10-week-old NPSR1-deficient mice and WT littermates. Both male and female mice were used. One group of mice was used to do behavioral tests as follows: Week 1: Day 1, elevated zero maze; Day
2, light-dark exploration; Day 3, open-field locomotor activity immediately followed by marble burying; Day 4, acoustic startle with prepulse inhibition; Week
2, Day 1-6, cued version of the Morris water maze (MWM); Week 3, Day 1-7, acquisition phase of MWM spatial version; Week 4, Day 1-7, reversal phase of
MWM spatial version; Week 5, Day 1-7, shift phase of MWM spatial version;
XV Week 6, Day 1, locomotor activity with (+)-methamphetamine challenge. The second group of mice was used to test depression-like behavior in the tail suspension test (TST) and 2 days later in the forced swim test (FST).
2.4.1. Elevated zero maze
Mice were tested in an elevated zero maze as a test of anxiety (Shepherd et al.,
1994). The ring-shaped maze was 105 cm in diameter and 72 cm in height with a 10 cm path width. The maze was equally divided into 4 quadrants, including 2 enclosed areas with black walls 28 cm in height and 2 open areas with a clear acrylic curb 1.3 cm in height. Each mouse was placed in the center of one of the closed areas and behavior was video recorded for 5 min as described previously
(Williams et al., 2003). The room was lit with a single halogen lamp turned to a dim setting. The apparatus was cleaned with 70% ethanol between animals.
The number of head dips, the latency to first open area entry, and time in the open areas in 5 min was scored for each mouse from the video recordings.
2.4.2. Light-dark exploration
Light-dark exploration was measured in Accuscan activity monitors (41 cm L41 cm W30 cm H, Accuscan Instruments, Inc., Columbus, OH) with an inserted box that made half of the chamber dark (Schramm et al., 2001). An opening (5.5 7 cm2) allowed mice to pass freely between the two sides. Mice were placed individually in the corner of the lighted side and tested for 10 min with fluorescent overhead room lights illuminating the chambers. Chambers were cleaned with
XW 70% ethanol between animals. Latency to enter the dark side and total time spent in the light area were recorded.
2.4.3. Open-field locomotor activity
Spontaneous locomotor activity was measured in Accuscan activity monitors (41 cm L41 cm W30 cm H, Accuscan Instruments, Inc., Columbus, OH) in 5 min intervals for 60 min. Movement patterns were scored using VersaMax software
(Pan et al., 2008). Dependent measures included horizontal activity, vertical activity (rearing), total distance, central and margin distances, and time spent in the central and margin areas. Chambers were cleaned with 70% ethanol between animals.
2.4.4. Marble Burying
Each mouse was brought to an adjacent suite and placed in a standard mouse cage with fresh hardwood chip bedding (5 cm deep). On the surface of the bedding there were 15 evenly spaced marbles placed in three rows (4.5 cm apart,
4.5 cm from the long edge, and 3.5 cm from the short end). A filter top was placed over the cage. Testing was conducted for 20 min under fluorescent room lighting. Latency to start marble burying and number of marbles at least 2/3rd buried were counted (Pan et al., 2008).
2.4.5. Acoustic startle reactivity with prepulse inhibition
XX Acoustic startle reactivity was performed in an SR Lab apparatus (San Diego
Instruments, San Diego, CA, USA) as previously described (Brunskill et al., 2005 and Pan et al., 2008). Mice were placed in an acrylic cylindrical test chamber scaled for mice. The test chamber was mounted on a platform with a piezoelectric force transducer attached to the underside. The platform was located inside a sound-attenuated test chamber. A 5 min acclimation period preceded test trials. Each animal received a 12 min test using a 4 × 4 Latin square design that balanced for the 4 trial types: no stimulus, startle stimulus alone, 70 dB or 76 dB prepulse plus startle stimulus. Each set of 16 trials was presented three times for a total of 48 trials. Trials of the same type were averaged together. The intertrial interval was 8 s. The startle signal was a 20 ms, 110 dB sound pressure level mixed-frequency burst and the startle recording window was 100 ms. Pre-pulses preceded the startle-eliciting stimulus by 70 ms from pre-pulse onset to startle signal onset.
2.4.6. Morris water maze (MWM)
Animals were tested for spatial learning and memory using the MWM as described previously (Vorhees et al., 2006). The Morris water maze apparatus was 122 cm in diameter and filled with 17.5 cm depth room temperature water
(21 ± 1°C). For facilitating tracking, the maze interior was painted white and the water was colored with white tempera paint. Proximal cued learning was evaluated using the cued version; in which mice were required to find a platform
(10 cm diameter, submerged 1 cm below the water) marked with a ball (40 mm
XY diameter) affixed to a brass rod mounted 7 cm above the platform. Curtains were drawn around the maze to reduce visibility of extra maze cues, and the mice were given 6 trials on day 1 and 2 trials per day from day 2 to day 6. Both the platform and start position were rotated to different locations on each trial.
The time limit for each trial was 1 min and the intertrial interval (ITI) was 5 s on the platform plus 30 s in the home cage during platform changes. Mice not finding the platform within 1 min were placed on the platform for 5 s. Mice were observed on a closed-circuit television, and latency to reach the platform was recorded for each trial. The cued procedure introduced mice to task requirements of the MWM (swimming, the fact that the platform is not near the perimeter, and that climbing on the platform provides escape from the water).
Subsequent to the cued version, mice were tested in the spatial (hidden platform) version to assess spatial acquisition (Vorhees et al., 2006). In this version, the animal learns to use distal cues to navigate a direct path to the hidden platform when started from different locations around the perimeter of the tank. The wall of the room had a variety of prominent cues, including geometric shapes that were visible from the maze. There were three phases: acquisition, reversal, and shift. In brief, each phase consisted of 4 hidden platform trials/day and 6 training days and a single probe trial given with the platform removed 24 h after the last training day of each phase with the curtains open. In each hidden platform trial there was a 1 min trial limit and a 15 s ITI spent on the platform. If a mouse failed to locate the platform, it was removed from the water and placed on the
XZ platform for 15 s. Throughout each phase the platform that was submerged 1 cm below water surface remained stationary while start positions (i.e., acquisition: N,
E, SE, and NW; reversal: S, W, NW, and SE; shift: S, E, NE, and SW) were quasi-randomized daily with the condition that no position could be used more than once a day. The size and position of the platform in the maze were changed for each phase (i.e., acquisition: 10 cm diameter and SW quadrant; reversal: 7 cm diameter and NE quadrant; and shift: 5 cm diameter and NW quadrant). Mice were tracked using a video tracking system (Anymaze,
Stoelting, Wood Dale, IL). Path length, latency, speed, and cumulative distance to the platform were recorded. On the seventh day of each phase, the platform was removed and mice were given a single 30 s probe trial starting from NE in acquisition, SW in reversal, or SE in shift to determine whether the animals remembered the location of the platform sufficiently to be able to swim back to where it was previously located. For probe trials, the measures recorded were average distance to the platform, time and distance in the target quadrant, initial heading to target error, and number of platform site crossovers.
2.4.7. Locomotor activity with methamphetamine challenge
The week after MWM, mice were reintroduced to the open-field for 30 min of habituation and then injected subcutaneously with 1 mg/kg (+)- methamphetamine (expressed as the freebase) and replaced in the arena.
Locomotor activity was recorded for another 2 h (Brunskill et al., 2005).
Immediately upon removal from the open-field, mice were transferred to an
X[ adjacent suite, quickly decapitated, and blood was collected for later plasma corticosterone determination.
2.4.8. Tail suspension test (TST)
A second group of 8-10 week old mice was used for assessment of depression– like behavior using the tail suspension test (Cryan et al., 2003). The mice were moved to the testing room at least 1 h prior to testing. Mice were individually suspended from a plastic platform (distance from floor = 16 cm) by pulling the tail through a hole in the platform as far as possible and fastening the tail to the top of the platform with adhesive tape. The tail was secured in this fashion to prevent tail grabbing by the mice. Time spent immobile over the entire 6 min testing time was recorded. Mice were disqualified if they grabbed their tail but retained if they held onto one foot.
2.4.9. Forced swim test (FST)
Two days after the TST, the same mice were tested using the Porsolt forced swim test (Porsolt et al., 1977a and Porsolt et al., 1977b). Mice were moved to the test room at least 1 h prior to testing. Each mouse was placed individually in an acrylic cylinder (45 cm in height and 17 cm in diameter) with 30 cm deep water (26°C) for 10 min and time spent immobile in the last 6 min was recorded.
The water was changed between subjects. Immobility was defined as the absence of vigorous movements (small paddling motions used by the animal to remain level or to keep from sinking were not considered escape movements).
YR
2.4.10. Plasma corticosterone levels without NPS stimulation
Immediately following the locomotor activity with challenge or 15 min after the forced swim test, mice were decapitated and blood was collected in polypropylene tubes containing 2% EDTA (0.05 ml) and placed on ice. Samples were centrifuged at 1399 RCF at 4oC for 25 min and plasma was removed and stored at -80oC until assayed. Corticosterone levels were assessed by EIA (IDS
Inc., Fountain Hills, AZ).
2.5. Behavioral tests and plasma corticosterone after intracerebroventricular administration of NPS
Separate groups of mice were used for these experiments. Mice at the age of 8 weeks were anesthetized with isoflurane and placed in a stereotaxic apparatus.
A C313GS-4 guide cannula (Plastic One Inc., Roanoke, VA, USA) was implanted in the right lateral ventricle under aseptic conditions through a hole drilled in the skull at the following coordinates relative to bregma: anterior–posterior, +0.6 mm; lateral, 1.1 mm; vertical, -2.1 mm beneath the surface of the skull, according to coordinates from Paxinos and Franklin (Paxinos and Franklin, 2004). The cannula was fixed to the skull with surgical glue. A C313DCS-4 cannula dummy was inserted into the guide cannula to prevent blockage of the guide cannula prior to intracerebroventricular (ICV) injection. Each mouse was housed individually after cannula implantation. Mice were allowed a 7-day recovery
YS period before experiments were initiated.
For NPS ICV injection, 3 l of artificial cerebrospinal fluid (aCSF at PH 7.4: NaCl
124 mM; KCl 3.0 mM; NaHCO3 26 mM; CaCl2 2.0 mM; MgSO4 1.0 mM; KH2PO4
1.25 mM; D-glucose 10.0 mM), 0.1, 1, or 10 nmol NPS (Phoenix
Pharmaceuticals, Inc. Burlingame, CA USA) in 3 l of aCSF were administered with a C313IS-4 internal cannula (Plastic One Inc., Roanoke, VA USA) connected to a Hamilton syringe with cannula tubing (C313C, Plastic One Inc.,
Roanoke, VA). The injection was administered over a period of 15 s. The injection cannula was left in place for another 60 s before being slowly withdrawn to avoid backflow. In order to reduce the stress from injection, ICV injection was conducted under light isoflurane anesthesia.
Four groups of male C57BL/6 WT mice for NPS dose-response experiments and three groups of male NPSR1-deficient and age matched C57BL/6 WT mice for comparing KO and WT were used in the behavioral tests and assessing plasma corticosterone levels after NPS ICV injection. Wildtype male C57BL/6 mice were transported from their housing room to the testing suite 1 h before the test started. Dose-response effects of ICV NPS were examined in the elevated zero maze, light-dark exploration, and open-field tests between 2000-2400 h. One group of WT mice (n=5-6/dose) was tested in the elevated zero maze for 5 min after receiving a single ICV injection of aCSF, 0.1, 1, or 10 nmol of NPS, and immediately upon removal from the elevated zero maze, the animals were tested
YT for light-dark exploration for 10 min. Another group of WT mice (n=5-6/dose) was tested in the open-field 5 min after receiving an ICV injection of aCSF, 0.1, 1, or
10 nmol of NPS. Following the results with the WT animals, a dose of 1 nmol of
NPS was used to determine if NPSR1 is involved in NPS-induced changes in locomotion and anxiety. Furthermore, the effectiveness of NPS (1 nmol)-induced locomotion in the open-field was tested over a 3 h period in a third group of male
C57BL/6 mice 5 min after NPS ICV injection. One group of male NPSR1- deficient and age matched C57BL/6 WT mice was tested in the elevated zero- maze for 5 min after NPS ICV injection and then tested for light-dark exploration for 10 min. A second group of male NPSR1-deficient and age matched C57BL/6
WT mice was tested for 60 min in the open-field test 5 min after NPS ICV injection.
Plasma corticosterone levels under NPS stimulation were examined 40 min after
NPS ICV injection between 800-1000 h as described by Smith et al. (2006). The dose-response effect of a single ICV injection of NPS (aCSF, 0.1, 1, or 10 nmol) was first examined in WT male C57BL/6 mice. Then to test the role of NPSR1 in
NPS-induced corticosterone release, aCSF or 10 nmol of NPS was applied to
NPSR1-deficient and age matched C57BL/6 WT mice.
After testing, cannula placement was confirmed for each mouse by histological examination of the brain after Evans blue injection. Animals with incorrect placement were not considered in the analysis.
YU
2.6. Statistical analysis
Behavioral data were analyzed using mixed linear ANOVA models (SAS Proc
Mixed, SAS Institute, Cary, NC). For each data set, the covariance matrix was checked using best fit statistics. In most cases the best fit was to the autoregressive-1 (AR (1)) covariance structure. This statistical model calculates adjusted degrees of freedom using the Kenward-Roger method, and therefore do not match those obtained from general linear model ANOVAs and can be fractional. Measures taken repetitively on the same animal, such as trial, interval, or day, were repeated measure factors. For clarity of presentation, only significant main effects of genotype or NPS treatment or the highest order interactions involving these variables are presented. Sex was included as a factor when both sexes were tested. When warranted, separate ANOVAs were performed on each sex. Significance was set at P 0.05. Data are presented as mean ± SEM.
YV 3. Results
3.1. Generation of NPSR1-deficient mice and confirmation of NPSR1 null allele
Genomic organization of the murine NPSR1 gene (Fig. 1A, GenBank accession no. NC_000075) contains 10 exons and 9 introns spanning 218 Kb of genomic sequence. The open reading frame encodes 371 amino acids. Exon 2 encodes the majority of extracellular N terminal part of the NPSR1 gene. The main strategy of NPSR1 gene targeting was replacement of the 3’ 128 bp of exon 2 and the first 1510 bp of intron 2 with a translational-fusion -galactose reporter gene and a neomycin resistance gene (LacZ and Neo, respectively, in Fig. 1B).
PCR genotyping of tail DNA (Fig. 1C) revealed that the NPSR1-deficient allele was inherited to progeny in Mendelian ratios without bias by sex. To confirm that the targeted NPSR1 allele was deleted as described, RT-PCR analysis of brain
RNA, isolated from littermates of each NPSR1 genotype, was performed to amplify the full length NPSR1 using primer pairs specific to the 5’UTR (forward) and 3’UTR (reverse) (Fig. 1D) and LacZ transcripts (Fig. 1E). The full length
NPSR1 transcript was present in the brain of WT and heterozygous mice, but absent in the brain of NPSR1-deficient mice. The expression of LacZ was found in the brain of NPSR1 heterozygous and deficient mice, but not in the brain of
WT mice (Fig. 1D and E). These data indicate that replacement of exon 2 with the fusion protein generates a null allele of NPSR1. NPSR1-deficient mice did not display apparent differences from their WT littermates in growth, weight, or reproduction capability.
YW 3.2. Expression of NPSR1 in the brain by X-gal staining
The homologous integration positioned the LacZ gene under the control of the
NPSR1 endogenous promoter, which allowed us to monitor NPSR1 expression in the brain (Supplemental Fig. 1) by X-galactosidase staining. The brain of
NPSR1-deficient mice did not exhibit obvious structural or morphological abnormalities and X-galactosidase staining was observed in several limbic regions, such as the amygdala, thalamus, and hypothalamus, as well as the motor cortex (Supplemental Fig. 1) (other expression sites not shown). Our X- gal staining results in the NPSR1-deficient mouse brain are consistent with the in situ results of NPSR1 based on the Allen Brain Atlas (http://www.brain- map.org/welcome.do). As a control, no X-galactosidase staining was observed in the brain from NPSR1 WT littermates (not shown).
3.3. Behavioral assessment in NPSR1-deficient mice under basal conditions
In view of the expression pattern of NPSR1 in brain regions involved in locomotion, stress response, and learning and memory (Xu et al., 2007), we assessed the effect of the NPSR1 gene deletion on locomotion, anxiety- and/or depression-like behavior, acoustic startle with prepulse inhibition, and learning and memory in KO and WT mice.
3.3.1. Unaltered anxiety-like behavior and locomotion activity in NPSR1-deficient mice
YX Anxiety-like behavior was examined in the elevated zero maze (Fig. 2A), marble burying test (Fig. 2B), light-dark exploration (Fig. 2C), and open-field (central region entries) (Fig. 2D) (Van Meer et al., 2005). Both male and female NPSR1- deficient mice showed similar performance to sex- and age-matched WT littermates in these tests.
Locomotor activity was assessed in an open-field under baseline conditions and after (+)-methamphetamine challenge. Methamphetamine was used since locomotor activity is strongly modulated by monoamines, especially dopamine, and methamphetamine blocks dopamine reuptake and facilitates dopamine release. NPSR1-deficient mice did not show an alteration in spontaneous locomotor activity during the pre-methamphetamine phase (Fig. 3A).
Methamphetamine markedly increased the activity level of both WT control and
NPSR1-deficient mice, but no significant differential response was observed.
3.3.2. Increased depression-like behavior in the forced swim test, but not the tail suspension test in NPSR1-deficient mice
The FST and TST were employed to evaluate depression-like behavior. In the
FST (Fig. 3B) there was a significant interaction of genotype x sex (F(1, 73.5) =
4.32, P < 0.05). Male NPSR1-deficient mice had significantly increased immobility compared to the male WT littermates (P<0.05), whereas female
NPSR1-deficient mice did not show this difference. Overall, the female animals remained immobile longer than the males (P = 0.01). In the tail suspension test,
YY both male and female NPSR1-deficient mice did not differ from age and sex matched WT littermates in immobility (not shown, n=18-21 per group).
3.3.3. Reduced acoustic startle response in NPSR1-deficient mice
Assessment of acoustic startle response revealed that there were no significant main effects of genotype or sex or the interaction of genotype × sex, but there was a significant prepulse × sex interaction (F(2, 134) = 4.2, P < 0.02).
Examination of males and females separately showed that male NPSR1-deficient mice had decreased acoustic startle responses on all trials compared to male
WT littermates (F(1, 26) = 4.11, P = 0.05)(Fig. 3C). There was no differential change in prepulse inhibition of the startle response as a function of prepulse intensity. Female NPSR1-deficient mice and WT littermates had no differences in the startle response on startle-only or prepulse trials.
3.3.4. Spatial learning and memory in NPSR1-deficient mice
Spatial learning and memory was tested using the Morris water maze (MWM).
Two versions of the MWM test were used: cued platform, which relies on proximal cues, and the hidden platform, which relies on distal cues. No genotype differences were noted in locating the cued platform (not shown). During hidden platform testing, three different phases of learning were used in which the platform position was changed for each phase. In all 3 phases, there were no differences in the path length, latency, speed, or cumulative distance to the platform between NPSR1-deficient and WT littermates on platform trials. After
YZ each learning phase, one probe trial was given with the platform removed, and the animals started from a novel position to assess memory for the position of the platform. In the probe trial after reversal, average heading to target error demonstrated a significant genotype sex effect (F(1, 32) = 4.29, P < 0.05).
Male NPSR1-deficient mice had an increased average heading error compared with WT littermates (mean ± SEM: male NPSR1-deficient mice=82.8 ± 1.5, male
WT mice=78.4 ± 1.4, n=8-9 each group); there were no differences noted among females. No other indexes of probe trial performance, such as platform crossovers, average distance to the target, percent time in target quadrant, percent distance in target quadrant, initial heading to target error after reversal showed genotype effects, nor were genotype differences found on probe trials given after acquisition or shift trials. Figure 3D shows path length per testing day in the acquisition phase (left) and average distance from the platform site (right)
(the best index of memory performance (Maei et al. 2009) in the probe trial after acquisition training).
3.3.5. Plasma corticosterone levels
Immediately on removal from locomotor testing with (+)-methamphetamine challenge (Fig. 4A) or 15 min after forced swim (Fig. 4B), plasma corticosterone was examined. The plasma corticosterone in the NPSR1-deficient mice did not differ from those in WT littermates.
Y[ 3.4. Plasma corticosterone and behavioral assessment in NPSR1-deficient mice following NPS stimulation
3.4.1 NPSR1 is required for increased corticosterone induced by NPS
Smith et al. (2006) reported that ICV NPS could activate the HPA axis and up- regulate plasma corticosterone. Here we first confirmed the dose-response effects of ICV NPS on plasma corticosterone 40 min after NPS ICV injection in
WT mice. In male WT mice, ICV administration of 0.1, 1, or 10 nmol NPS caused a significant dose-dependent increase in plasma corticosterone 40 min after injection compared with aCSF (F(3,39) = 10.7, P<0.0001). Post hoc group comparisons showed that both the 1 and 10 nmol NPS groups had significantly increased corticosterone compared to aCSF controls (mean ± SEM (ng/ml): aCSF = 95.3 ± 13.2; NPS 0.1 nmol = 86.2 ± 13.1; NPS 1 nmol = 136.2 ± 11.6;
NPS 10 nmol = 179.8 ± 13.2; n=10-13 per group; aCSF vs. NPS 0.1 nmol = ns; aCSF vs. NPS 1 nmol = p < 0.05; aCSF vs. NPS 10 nmol = p < 0.001).
Accordingly, the 10 nmol dose of NPS was used for examining if NPSR1 was involved in NPS-induced corticosterone release. There was no significant main effect of genotype on plasma corticosterone; the main effect of NPS was also not significant but showed a trend (p < 0.06), however, there was a significant genotype x NPS interaction (F(1, 34) = 4.3, p < 0.05). As can be seen in Fig. 4C,
10 nmol of NPS induced a significant increase in plasma corticosterone in NPS- treated WT mice (p < 0.01) but not in NPS-treated NPSR1-deficient mice.
3.4.2. NPSR1 is required for NPS-induced hyperlocomotion and exploration
ZR To determine if NPSR1 was a non-redundant receptor for NPS in the brain, male
NPSR1-deficient and WT mice were exposed to NPS by ICV injection and examined for locomotor activity. In order to identify the appropriate dose, a separate group of WT mice was tested first. ICV NPS in WT mice dose- dependently (0.1, 1, and 10 nmol) increased locomotor activity compared to mice receiving aCSF with the 1 nmol dose of NPS having the clearest effect
(horizontal activity (mean ± SEM) over 60 min testing time in open-field test, aCSF=645.9 ± 45.5, NPS 0.1 nmol=1115.9 ± 41.2, NPS 1 nmol=1482.3 ± 44.0,
NPS 10 nmol=1364.3 ± 38.0, n=6 per group). Accordingly, we used 1 nmol of
NPS to examine the role of NPSR1 in NPS-induced hyperlocomotion in NPRS1- deficient and WT mice. Central administration of NPS (1 nmol) induced increased vertical (rearing) and horizontal activity (Fig. 5A, first two panels) and interacted with genotype for both vertical (genotype x NPS, F(1, 36.7) = 25.77, P
< 0.0001) and horizontal activity (genotype x NPS, F(1, 36) = 21.44, P < 0.0001).
In WT animals, ICV NPS (1 nmol) increased vertical and horizontal activity compared to animals given aCSF. In contrast, NPSR1-deficient mice exhibited complete insensitivity to NPS (Fig. 5A) relative to WT mice given NPS and instead performed similarly to NPSR1-deficient mice that were administered aCSF. In order to determine how long the NPS stimulation lasted, a separate group of C57BL/6 WT mice was administered ICV NPS (1 nmol) and observed for 3 h. NPS-stimulated animals showed increased vertical and horizontal activity as previously demonstrated (Fig. 5B). For vertical activity the effect peaked sharply at about 40 min then gradually tapered off showing an inflection
ZS at about 100 min. For horizontal activity the effect peaked almost immediately and then gradually tapered off over the course of the entire 180 min test session.
For both measures, there was an NPS x interval interaction (F(17, 295) =3.60, P
<0.0001 and F(17, 297) = 1.91, P < 0.02, respectively). With the exception of intervals 110 and 180 for vertical activity, NPS continued to significantly stimulate activity greater than aCSF alone at all intervals measured, whereas for horizontal activity all intervals were significantly different between NPS and aCSF.
3.4.3. Impaired anxiolytic effect induced by NPS in NPSR1-deficient mice
To determine if the anxiolytic effect of NPS was NPSR1-dependent, male
NPSR1- deficient mice were analyzed for anxiety related behaviors in the elevated zero maze, light-dark exploration, and the aforementioned open-field test. A separate group of WT mice was used to identify the appropriate dose in the elevated zero maze and light-dark exploration tests. ICV NPS in WT mice dose-dependently (0.1, 1, and 10 nmol) decreased anxiety-like behaviors by increasing time in the open (time in the open area (mean ± SEM) in elevated zero maze, aCSF=34.8 ± 8, NPS 0.1 nmol=34.9 ± 6.6, NPS 1 nmol=108.5 ±
10.1, NPS 10 nmol=108.1 ± 17.1, n=5-6 per group; for horizontal activity in the light area (mean±SEM) in light-dark exploration test, aCSF=1014 ± 157, NPS 0.1 nmol=1179 ± 126, NPS 1 nmol=1912 ± 146, NPS 10 nmol=1491 ± 155, n=5-6 per group); 1 nmol of NPS had the clearest effect. A 1 nmol dose of NPS was applied to NPSR1-deficient and WT mice to examine the role of NPSR1 in the
NPS-induced anxiolytic effect. In the elevated zero maze, no effect of NPS was
ZT observed on latency to enter the open, although there was a genotype effect
(F(1, 39) = 17.06, P < 0.002) that showed the NPSR1-deficient mice took longer to initially enter the open areas (latency to enter the open (s) (mean ± SEM): WT- aCSF=38.1 ± 10.3, KO-aCSF=61.3 ± 8.4, WT-NPS=24.5 ± 8.4, KO-NPS=76.0 ±
8.8, n=8-12 per group) . For time in the open (Fig. 5C, first panel), there was an effect of NPS (F(1, 39) = 28.87, P < 0.0001), genotype (F(1, 39) = 22.85, P <
0.0001), and the interaction of NPS x genotype (F(1, 39) = 20.01, P < 0.0001).
Slice effect ANOVAs by drug showed a genotype effect in NPS (F(1, 39) = 47.01,
P < 0.0001), and slice effect ANOVAs by gene showed a drug effect in WT animals (F(1, 39) = 44.51, P < 0.0001). The WT animals that were administered
NPS had increased time in the open area compared to WT aCSF-administered animals, whereas no differences were noted between the NPSR1-deficient mice receiving NPS versus aCSF. NPS-treated NPSR1-deficient mice were insensitive to NPS stimulation which was seen in WT NPS-treated mice.
Similarly for head dips (Fig. 5C, second panel), there was an NPS (F(1, 39) =
16.65, P < 0.0002), genotype (F(1, 39) = 16.53, P < 0.0002) and NPS x genotype effect (F(1, 39) = 8.90, P < 0.005). Slice effect ANOVAs by drug showed a genotype effect in NPS (F(1, 39) = 27.28, P < 0.0001), and slice effect ANOVAs by gene showed a drug effect in WT animals (F(1, 39) = 22.90, P < 0.0001). The
WT mice given NPS showed increased head dips compared to WT mice given aCSF; whereas the NPSR1-deficient mice given NPS did not respond to NPS stimulation showing similar performance as the NPSR1-deficient mice given aCSF. In the open-field test (Fig. 5A, third panel) for the time in the center, there
ZU was a significant genotype X NPS interaction (F(1, 36.2) = 9.04, P < 0.005). ICV
NPS increased time in the central zone in the WT mice compared with aCSF, but not in the NPSR1-deficient mice. No differences were observed in the NPSR1- deficient mice between those receiving NPS versus aCSF. Margin time was the exact opposite (not shown) showing that NPS-treated NPSR1-deficient mice to be insensitive to NPS compared to NPS-treated WT animals. In the light-dark exploration test for the transition between the light and dark areas (Fig. 5C, third panel), there was an effect of NPS (F(1, 39) = 11.45, P < 0.002) and the interaction of NPS x genotype (F(1, 39) = 7.78, P < 0.008). Slice effect ANOVAs by drug showed a genotype effect in NPS (F(1, 39) = 8.54, P < 0.006), and slice effect ANOVAs by gene showed a drug effect in WT animals (F(1, 39) = 19.45, P
< 0.0001) (mean ± SEM: WT-aCSF=19 ± 5, KO-aCSF=27 ± 5, WT-NPS=50 ± 5,
KO-NPS=30 ± 5, n=8-12 per group); For horizontal activity in the light area, there was only a significant effect of NPS (F(1, 43) = 13.96, P < 0.0006) (mean ±
SEM: WT-aCSF=1228 ± 172, KO-aCSF=1657 ± 172, WT-NPS =2083 ± 158, KO-
NPS=2049 ± 165). For horizontal activity in the dark area, there was an effect of genotype (F(1, 39) = 4.50, P < 0.04), but no effect of NPS or an NPS x genotype interaction (mean ± SEM: WT-aCSF=1857 ± 301, KO-aCSF=1724 ± 301, WT-
NPS=2796 ± 275, KO-NPS=1695 ± 287). For time spent in the light area or in the dark area there was no effect of NPS or genotype or an NPS x genotype interaction (for time (s) in light area (mean ± SEM): WT-aCSF=260 ± 40, KO- aCSF=295 ± 40, WT-NPS=244 ± 37, KO-NPS=348 ± 38; for time(s) in dark area
ZV (mean ± SEM): WT-aCSF=340 ± 40, KO-aCSF=305 ± 40, WT-NPS=356 ± 37,
KO-NPS=252 ± 38).
ZW 4. Discussion
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
ZX 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)
ZY 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
ZZ 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
Z[ 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.
[R
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
[S 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.
Genetic variants of NPSR1 have been linked with inflammatory diseases, such as asthma and inflammatory bowel disease (Laitinen et al., 2004 and D'Amato et al., 2007). Our data suggest that a central mechanism in these inflammatory disorders may be operational. Indeed, stress is a known trigger for asthma and inflammatory bowel disease (Chen et al., 2007 and Santos et al., 2008).
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.
[T Acknowledgments
We wish to thank Drs. Carine Blanchard, Li Yang, Patricia Fulkerson, Eric
Brandt, Kevin Burns and Ying Sun for technical assistance and advice; Dr. Tori L.
Schaefer for behavioral assistance; Mary Moran for assistance in behavioral data analysis; members of my thesis committee, Drs. Simon Hogan, Tim LeCras, and
David Hildeman; and Andrea Lippelman for editorial review.
[U Figure legends
Fig. 1 Generation of NPSR1 gene-targeted mice. (A) Schematic representation
of the genomic organization of murine NPSR1 is shown according to the
GenBank (NC_000075) sequence. Exons are depicted as boxes and introns as
lines. Coding regions of exons are shown as shaded boxes. The length of the
exon and intron are marked. (B) Strategy for disruption of the NPSR1 gene.
Most of exon 2 and part of intron 2 were replaced with the coding sequence of
LacZ and a neo-selection cassette. Primers for PCR genotyping are also shown.
(C) PCR analysis of tail genomic DNA using primers specific for the WT (+/+)
allele (P1 and P2) and for the recombinant mutant allele (P1 and P3). (D and E)
RT-PCR to demonstrate the absence of full length NPSR1 transcripts in the brain
from NPSR1-deficient (KO) mice (D) and the expression of LacZ in the brain of
NPSR1 heterozygous and deficient (KO) mice (E).
Fig. 2 Anxiety-like behaviors in NPSR1-deficient mice. (A) Elevated zero maze: time in open areas and number of head dips in NPSR1-deficient mice (male,
n=17; female, n=21) and WT littermates (male, n=16; female, n=21). (B) Marble
burying test: latency to bury marbles and number of buried marbles in NPSR1-
deficient mice (male, n=17; female, n=21)) and WT littermates (male, n=16;
female, n=21) in defensive object burying test. (C) Light-dark exploration: time in
light area and latency to dark side entry in NPSR1-deficient mice (male, n=17;
female, n=23) and WT littermates (male, n=15; female, n=22). (D) Open-field:
total distance, time in the central area, and time in the margin area of NPSR1-
[V deficient mice (male, n=17; female, n=23) and WT littermates (male, n=15; female, n=22) were analyzed in the locomotor activity test and plotted in 5 min intervals. Values are reported as mean ± SEM.
Fig. 3 Methamphetamine regulated locomotion, depression-like behavior, acoustic startle response, and spatial learning and memory in NPSR1-deficient mice. (A) Open-field locomotor activity of NPSR1-deficient mice (male, n=10; female, n=10) and WT littermates (male, n=11; female, n=10) was evaluated before and after s.c. injection of 1 mg/kg (+)-methamphetamine. NPSR1 gene deletion did not affect methamphetamine modulated locomotor activity. (B)
Forced swim test: immobility duration in NPSR1-deficient mice (male, n=21; female, n=19) compared with WT littermates (male, n=18; female, n=19). Values shown here were the averages of immobility duration per minute for last 6 min of the 10 min test session. Male NPSR1-deficient mice showed significantly increased immobility compared to male WT littermates. (C) Acoustic startle responses on startle-only trials and on trials with 70 or 76 dB prepulse (PP) intensities in NPSR1-deficient mice (male, n=15; female, n=21) compared with
WT littermates (male, n=14; female, n=21). Male NPSR1-deficient mice had decreased acoustic startle responses on all trials compared to male WT littermates, but no change in prepulse inhibition of the startle response. (D)
Morris water maze: Male NPSR1-deficient mice (male, n=9) showed similar path length in the acquisition phase learning trials (left) and average distance from the platform site on the probe trial after acquisition training (right) on hidden platform
[W trials compared to male WT littermates (male, n=8). Values are reported as mean ± SEM. *P 0.05 compared with sex and age matched WT littermates.
Fig. 4 Plasma corticosterone (A) 2 hours after 1 mg/kg (+)-methamphetamine challenge in open-field test, the plasma corticosterone levels in NPSR1-deficient mice (male, n=10; female, n=10) did not differ from those in WT littermates (male, n=10; female, n=10). (B) 15 min after forced swim test, NPSR1-deficient mice
(male, n=10; female, n=10) had similar levels of plasma corticosterone to WT littermates (male, n=10; female, n=10). (C) Plasma corticosterone levels in
NPSR1-deficient and WT mice 40 min after 10 nmol NPS ICV injection (WT-CSF, aCSF treated WT mice, n=8; KO-CSF, aCSF treated NPSR1-deficient mice, n=8;
WT-NPS, NPS treated WT mice, n=13; KO-NPS, NPS treated NPSR1-deficient mice, n=10). NPSR1-deficient mice receiving 10 nmol ICV NPS did not respond to NPS compared to WT mice given NPS. **P < 0.01 for WT-NPS vs. WT-CSF;
#P < 0.05 for WT-NPS vs. KO-NPS. Values are reported as mean ± SEM.
Fig. 5 NPSR1 is required for NPS-induced hyperlocomotion, exploration, and anxiolytic-like effects. (A) Open-field: ICV NPS (1 nmol)-treated WT mice had hyperlocomotion, increased exploratory activity, and increased time spent in central area and NPSR1-deficient mice exhibited complete insensitivity to NPS relative to WT mice given NPS (WT-CSF, aCSF treated WT mice, n=10; KO-
CSF, aCSF treated NPSR1-deficient mice, n=10; WT-NPS, NPS treated WT mice, n=9; KO-NPS, NPS treated NPSR1-deficient mice, n=11). (B) NPS (1
[X nmol)-induced hyperlocomotion and increased exploratory activity in WT mice was observed in open-field test for 3 hours (CSF, n=11; NPS 1 nmol, n=11). (C)
Time in open areas and head dips in the elevated zero maze test (WT-CSF, aCSF treated WT mice, n=8; KO-CSF, aCSF treated NPSR1-deficient mice, n=12; WT-NPS, NPS treated WT mice, n=12; KO-NPS, NPS treated NPSR1- deficient mice, n=11), and transitions between light and dark area in the light- dark exploration test (WT-CSF, aCSF treated WT mice, n=10; KO-CSF, aCSF treated NPSR1-deficient mice, n=10; WT-NPS, NPS treated WT mice, n=12; KO-
NPS, NPS treated NPSR1-deficient mice, n=11) in WT and NPSR1-deficient mice after 1 nmol NPS ICV injection. ***P < 0.001 for WT-NPS vs. WT-CSF; ###P
< 0.001 for WT-NPS vs. KO-NPS. Values are reported as mean ± SEM.
Supplemental figure legend
Supplemental Fig. 1 Expression of –galactosidase in the brain from NPSR1- deficient mice. –galactosidase activity was assessed by the X-Gal staining of frozen coronal sections of brain from NPSR1-deficient mice. X-Gal staining images of primary mortor cortex at bregma 2.22 mm (A-C), accessory basal amygdaloid nucleus at bregma -1.70 mm (D-F) and dorsomedial nucleus of hypothalamus at bregma -1.70 mm (G-I) were shown. (A, D, G) 40× magnification images. (B, E, H) Images of the squared regions in (A), (D) and
(G) respectively by 100× magnification. (C, F, I) Images of the areas indicated by arrows in (B), (E) and (H) respectively by 400× magnification. M1: primary motor cortex; BMA: accessory basal amygdaloid nucleus; BLV: basal amygdaloid
[Y nucleus; LH: lateral hypothalamic area; DM: dorsomedial nucleus of hypothalamus. Scale bar has been marked in individual pictures.
[Z Figure 1
[[ Figure 2
SRR Figure 3
SRS Figure 4
SRT Figure 5
SRU Supplemental figure 1
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SSS Chapter 3
The role of neuropeptide S and neuropeptide S receptor 1 in regulation of respiratory function in mice
Hongyan Zhua, b, Charles Perkinsc, Melissa K. Minglera, Fred D. Finkelmanc, and
Marc E. Rothenberga
aDivision of Allergy and Immunology, bDivision of Developmental Biology,
Graduate Program of Molecular and Developmental Biology, cDivision of
Immunobiology, Cincinnati Children’s Hospital Medical Center, Department of
Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, 45267
USA.
Correspondence and requests for reprints should be addressed to Marc E.
Rothenberg, MD, PhD, Division of Allergy and Immunology, Cincinnati Children’s
Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH, 45229. Tel: 513-636-
7210; fax: 513-636-3310; e-mail: [email protected].
It has been accepted by Peptides.
SST Abstract
Genome-wide screening and positional cloning have linked neuropeptide S receptor 1 (NPSR1) with asthma and airway hyperresponsiveness. However, the mechanism by which NPSR1 regulates pulmonary responses remains elusive.
Because neuropeptide S and its receptor NPSR1 are expressed in brain regions that regulate respiratory rhythm, and Npsr1-deficient mice have impaired stress and anxiety responses, we aimed to investigate whether neuropeptide S and
NPSR1 regulate respiratory function through a central-mediated pathway. After neuropeptide S intracerebroventricular administration, respiratory responses of wildtype and Npsr1-deficient mice were monitored by whole-body or invasive plethysmography with or without serial methacholine inhalation. Airway inflammatory and hyperresponsiveness were assessed in allergen-challenged
(ovalbumin or Aspergillus fumigatus) Npsr1-deficient mice. Analysis of breathing patterns by whole-body plethysmography revealed that intracerebroventricular neuropeptide S, as compared with the artificial cerebral spinal fluid control, increased respiratory frequency and decreased tidal volume in an NPSR1- dependent manner but did not affect enhanced pause. Following serial methacholine inhalation, intracerebroventricular neuropeptide S increased respiratory frequency in wildtype mice, but not Npsr1-deficient mice, and had no effect on tidal volume. Intracerebroventricular neuropeptide S significantly reduced airway responsiveness to methacholine as measured by whole-body plethysmography. Npsr1 deletion had no impact on airway inflammation or hyperresponsiveness in ovalbumin- or Aspergillus fumigatus-induced
SSU experimental asthma. Our results demonstrate that neuropeptide S and NPSR1 regulate respiratory function through a central nervous system-mediated pathway.
Key words: Respiration; brain; neuropeptide S; neuropeptide S receptor 1; panting; stress.
SSV 1. Introduction Asthma is a complex disease involving the interaction of environmental factors and genetic susceptibility. Through genomic scanning and positional cloning, a series of genes including neuropeptide S receptor 1 (NPSR1, also called GPRA or GPR154) have been identified as asthma susceptibility genes
[13, 23]. Single nucleotide polymorphisms (SNPs) and haplotypes of the NPSR1 gene have been associated with asthma or airway hyperresponsiveness (AHR) in Finnish, Canadian, Italian, Chinese, German, European-American, and
Hispanic populations [4, 10, 14, 16, 20, 35]. The NPSR1 gene encodes for a G protein-coupled receptor, and an Asn/Ile107 SNP in the first extracellular loop of
NPSR1 has been associated with increased asthma susceptibility [16]. However, the functional role of NPSR1 in asthma pathogenesis is still unclear.
Mouse NPSR1 shows high amino acid similarity to human NPSR1 and conserves Ile107. Allen et al. used an Npsr1 gene-targeted mouse, in which exon
4 containing Ile107 was deleted, to assess the role of NPSR1 in asthma development [1]. They found no evidence that NPSR1 contributed to asthma development in an ovalbumin (OVA)-induced experimental asthma model or
LPS-mediated airway inflammation model. However, they also did not detect pulmonary expression of NPSR1 in healthy individuals or asthmatic patients or of
Npsr1 in OVA-sensitized, saline-challenged or OVA-challenged mice by real-time
PCR. These results are in contrast to the initial findings by Laitinen et al., who used an OVA sensitization model (OVA in combination with Stachybotrys
SSW chartarum) and found pulmonary upregulation of Npsr1 in mice with allergic lung disease [16]. Although implicated in human and murine models of asthma, the lack of evidence for a functional role of NPSR1 in a known immunological or asthmatic pathway raises questions about its contribution to asthma and the mechanism by which it may operate.
In humans, the Asn/Ile107 SNP in NSPR1 gene is associated with panic disorder in male patients of Japanese ancestry [24]. Interestingly, neuropeptide
S (NPS), the endogenous ligand for NPSR1, has been reported as an important modulator of anxiety in rodents [17, 28, 30, 32]. And our earlier study suggests that the NPS/NPSR1 pathway is involved in stress and anxiety response [34].
Clinical observations have provided evidences that there is strong correlation of respiration change with stress response and emotional diseases. For example, patients with panic disorder display enhanced respiratory variability compared to controls [21] and chronic stress exacerbates the symptoms of asthmatic patients
[3, 31]. Furthermore, Xu et al. have shown that that Npsr1 and Nps are located in the brain stem, limbic system, and/or cerebral cortex, which establish basic respiratory rhythm [32 33]. In light of these findings, we hypothesized that NPS and NPSR1 might operate through a central nervous system (CNS)-mediated pathway to regulate respiratory function at baseline or in response to a cholinergic stimulus (an inducer of airway responses that are typically exaggerated in asthma).
SSX In the present study we use Npsr1-deficient mice in which exon 2 is deleted to investigate whether NPS alters respiratory function at baseline or in response to methacholine challenge by intracerebroventricular (ICV) administration of NPS and whether this process is NPSR1-dependent. In addition, we confirm whether
NPSR1 is directly involved in affecting features of allergen-induced experimental asthma, including AHR using the Npsr1-deficient mice which is different from the mice used in the study from Allen et al. [1]. Experimental asthma is induced by
OVA or Aspergillus fumigatus challenge. Compared with conventional OVA model, repeated intranasal challenge of Aspergillus fumigatus is a relatively chronic model [5].
2. Methods
2.1. Mice
A novel strain of Npsr1-deficient mice in which exon 2 was deleted have been described in our previous paper [34]. Mice with the genetic background of
129Sv/J × C57BL/6 were backcrossed 10 generations with BALB/c mice. In all studies, Npsr1-deficient (knock-out (KO)) mice were compared with sex- and age-matched BALB/c WT mice, which were bred on-site. All of the mice were housed as 4 mice with same sex per cage and maintained under pathogen-free conditions and a 14 h light/10 h dark cycle (lights on at 600 h) with temperature
(19 ± 1 °C) and humidity (50 ± 10%) controlled. The experiments were performed between 0900-1500 h. All procedures were approved by the
Institutional Animal Care and Use Committee of the Cincinnati Children’s
SSY Hospital Research Foundation. The number of mice per group in each test is given in the figure captions.
2.2. ICV administration of NPS
Cannula implantation and ICV injection were done as previously described
[34]. Each mouse was housed individually after cannula implantation.
Experiments were initiated following a 7-day recovery period. For ICV injection,
3 l of artificial cerebrospinal fluid (aCSF at pH 7.4: NaCl 124 mM, KCl 3.0 mM,
NaHCO3 26 mM, CaCl2 2.0 mM, MgSO4 1.0 mM, KH2PO4 1.25 mM, D-glucose
10.0 mM) or 1 nmol of NPS (Phoenix Pharmaceuticals, Inc., Burlingame, USA) in
3 l of aCSF were administered. ICV injection was conducted under light isoflurane anesthesia. The injection was administered over a period of 15 s. To avoid backflow, the injection cannula was left in place for another 60 s before being slowly withdrawn. Mice were returned to their original cage immediately after injection and awoke within 1 minute. For these experiments, 4 mice were processed at the same time.
2.3. Basal respiratory function before and after ICV administration of NPS
Basal respiratory function was measured by whole-body plethysmography
(Buxco Electronics, Inc., Sharon, USA) [25] in conscious, unrestrained mice as described in Fig. 1A. Settings were: Maximal expiration time (Te) = 10 sec, minimal inspiration time (Ti) = 0.04 sec, minimal tidal volume (TV) = 0.04 ml, and value balance = 50%. Baseline measurements were made over a 5-min period before NPS ICV injection. Five minutes after NPS ICV injection, respiratory
SSZ measurements were taken again for a total testing span of 60 min. The average values of respiratory frequency (breath per min, BPM), tidal volume (ml/breath), and enhanced pause (Penh) for each 2-min period were recorded. Minute ventilation (respiratory frequency tidal volume) (ml/min) was calculated, and the values for the above parameters were plotted for every 10 min.
2.4. Methacholine-challenged respiratory function after ICV administration of
NPS
Respiratory response to methacholine challenge was measured by whole- body plethysmography (Buxco Electronics, Inc., Sharon, USA) as described in
Fig. 2A. Baseline measurements were made over a 5-min period before NPS
ICV injection. The measurement of respiratory response to methacholine challenge started 5 minutes after ICV injection of NPS. Mice were serially exposed to increasing concentrations of aerosolized methacholine (Sigma–
Aldrich, St. Louis, USA) in PBS (0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 mg/ml). Each dose of methacholine was delivered for 3 min by inhalation via a DeVilbiss Model
5500D-030 nebulizer (DeVilbiss Healthcare, Somerset, USA), and Penh measurements were made for 5 min, starting 2 min after completion of exposure to the aerosolized methacholine. The average values for the 5-min period of respiratory frequency, tidal volume, minute ventilation, and enhanced pause
(Penh) were calculated.
2.5. Methacholine-challenged airway resistance after ICV administration of NPS
SS[ Seven days after the whole-body plethysmography, methacholine-challenged airway resistance after NPS ICV injection was measured using the flexiVent system (SCIREQ INC., Montreal, Canada) [22]. Briefly, 20 min after ICV injection of 1 nmol NPS, the mice were weighed and anesthetized with 150 mg/kg ketamine mixed with 10 mg/kg xylazine. A tracheotomy was performed, and a cannula was inserted. Mice were then exposed to aerosolized PBS, followed by increasing concentrations of aerosolized methacholine (Sigma–Aldrich, St. Louis,
USA) (6.25, 25, 100 mg/ml). Values for lung resistance were obtained at 5-s intervals for 3 min after each methacholine challenge. The peak responses at each methacholine concentration were used for data analysis.
2.6. Peripheral blood cell counting and flow cytometry
Peripheral blood cells were counted by Hemavet 950 (Drew Scientific Inc.,
Dallas, USA), and CD3+, B220+, CD4+, CD8+ cells in the peripheral blood were analyzed by flow cytometry.
2.7. Experimental asthma models
Asthma-like lung disease was induced by OVA or Aspergillus fumigatus as previously described [36]. Mice were sacrificed 48 h following the last OVA challenge or 18 h following the last Aspergillus challenge. Bronchoalveolar lavage fluid (BALF), plasma, and left lung tissue were harvested for assessments of airway responsiveness. Right lung was harvested for RNA preparation.
STR
2.8. BALF analysis
The mice were sacrificed by an intraperitoneal injection of pentobarbital
(Sodium pentobarbital, 150mg/kg; Vortech Pharmaceuticals, Ltd., Dearborn,
USA). Immediately thereafter, BALF was collected, and differential cell counts were determined as previously described [6].
2.9. Characterization of lung morphology
Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) or Masson’s trichrome staining of the lung was performed as previously described [6].
2.10. Measurement of serum total IgE
Total IgE in the serum from OVA- or Aspergillus-challenged mice were determined by ELISA kit (BD Biosciences Pharmingen, San Diego, USA).
2.11. Methacholine-challenged airway resistance after allergen challenge
At 24 h after the last intranasal challenge with either saline or OVA, or at 18h after the last intranasal challenge with either saline or Aspergillus, airway resistance was measured using the flexiVent system (SCIREQ INC., Montreal,
Canada) with exposed to aerosolized PBS, followed by aerosolized methacholine
(37.5 mg/ml) (Sigma–Aldrich, St. Louis, USA) [22].
2.12. Reverse transcription polymerase chain reaction (RT-PCR)
STS mRNA expression in the lung and brain of saline- or OVA-challenged Npsr1- deficient and WT mice were examined using RT-PCR. The primers used in the
PCR reaction are indicated below: full-length Npsr1 (1300 bp product), sense 5'-
TCGTCAGGCAGAACTCTTCA-3' specific to 5’UTR and antisense 5'-
ATCTGCTAGGTGAGGCAGGA-3' specific to 3’UTR; Npsr1 short fragment (137 bp product), sense 5'-GGCTCATCTCTAAGGCAAAAATCA-3' specific to exon 8 and antisense 5'-ACGCTCCTTGGTGTCTGGAA-3' specific to exon 9.
2.13. Statistical analysis
Values were presented as mean ± SEM. The significance of differences between experimental groups was analyzed using paired Student’s t-tests.
Significance was set at p 0.05.
3. Results
3.1. Role of NPS and NPSR1 in basal respiratory function
Based on the fact that Nps and Npsr1 are expressed in the brain regions that are responsible for establishing respiratory rhythm [32, 33], we examined whether NPS and/or NPSR1 might have a role in regulating basal respiratory function. Respiratory frequency, tidal volume and Penh were evaluated by whole-body plethysmography in WT and Npsr1-deficient mice before and after
ICV administration of NPS or control aCSF (Fig. 1). Before ICV administration, respiratory frequency was similar between genotypes. Mice of both genotypes had a reduction in frequency to approximately 50% of the initial values at 10 min
STT post-ICV aCSF injection under isofluorane anesthesia. The frequency remained dampened during the subsequent 50 min, and there was a greater decrease in frequency in Npsr1-deficient mice than in WT mice at 20 min and 30 min. ICV
NPS (1 nmol) significantly increased the dampened frequency in WT, but not in
Npsr1-deficient mice, with a significant difference between WT and Npsr1- deficient mice treated with NPS at all time points studied (Fig. 1B). No difference was seen in tidal volume between WT and Npsr1-deficent mice before or after aCSF ICV injection. However, ICV NPS decreased tidal volume in WT, but not in
Npsr1-deficient mice, with a significant difference between NPS-treated WT and
Npsr1-deficient mice at 30 and 40 min post-injection (Fig. 1C). Penh (Fig. 1D) and minute ventilation (data not shown) were not affected by ICV NPS.
Collectively, these data demonstrate that Npsr1 is required for respiratory changes showing increased frequency and decreased tidal volume induced by
ICV NPS at baseline.
3.2. Role of NPS and NPSR1 in methacholine-induced changes of respiratory function
Methacholine, a cholinergic stimulus, induces airway constriction that is a typical symptom of asthma. Next, we aimed to define whether ICV administration of NPS attenuates methacholine-induced AHR and whether this process is
NPSR1-dependent. After ICV injection of 1 nmol of NPS, the respiratory function was measured by whole-body plethysmography in Npsr1-deficient and WT mice exposed to methacholine (Fig. 2B). Our results show that Npsr1 deficiency
STU prevented the ability of NPS to suppress methacholine-induced decrease in respiratory frequency (upper panel) and obscured the ability of NPS to decrease the Penh response to methacholine by suppressing that response to the same extent in the presence or absence of NPS (middle panel). In contrast, neither
NPS treatment nor Npsr1 deficiency affected methacholine-induced increase in tidal volume (lower panel). As measured with a flexiVent apparatus, ICV NPS showed the trend of decreasing airway resistance in response to methacholine, but not significantly. Npsr1 deficiency did not have any effect on airway resistance in response to methacholine challenge (Fig. 2C).
3.3. Role of NPSR1 in allergen-induced experimental asthma
Because a previous study showed that Npsr1 was upregulated in the lung of mice subjected to an OVA/Stachybotrys chartarum mold model of asthma [16], we evaluated the role of NPSR1 in standard allergen-induced model of experimental asthma (OVA or Aspergillus). At baseline, no difference in peripheral blood total or CD3+, B220+, CD4+, CD8+ cell numbers was found between WT and Npsr1-deficient mice (Table 1). In the OVA-induced experimental asthma model (Fig. 3A), there was no significant difference in total cell number or specific cell populations in BALF although all cell populations trended lower in Npsr1-deficient mice between OVA-challenged WT and Npsr1- deficient mice. The Npsr1 deficiency did not alter the IgE level in the serum upon
OVA challenge. OVA-induced airway mucus production, examined by PAS staining, and collagen deposition, assessed by trichrome staining, was similar in
STV the Npsr1-deficient and WT mice. Airway resistance in response to 37.5 mg/ml methacholine was not altered in the Npsr1-deficient mice compared with WT mice. Similar results were seen with an Aspergillus fumigatus model of asthma
(Fig. 3B): Npsr1 deficiency had no impact on the BALF cell profile, serum IgE, airway mucus, lung collagen, or methacholine-induced airway hyperreactivity.
These data demonstrate that Npsr1 does not have a dominant direct role in the development of experimental asthma.
3.4. Npsr1 expression in the murine lung and brain
Based on the above results, we examined Npsr1 expression in the lung.
Northern blot analysis of several organs from untreated mice and lung from saline- or OVA-challenged mice revealed the presence of detectable Npsr1 mRNA in the brain, whereas no specific hybridization was noted in untreated lung, spleen, heart, kidney or intestine nor in saline- or OVA-challenged lung
(data not shown). Subsequently, a more sensitive method, RT-PCR, was used to examine Npsr1 mRNA expression. Full-length Npsr1 (5’ UTR forward primer,
3’ UTR reverse primer) was highly expressed in the brain of untreated WT mice, but not in the lung of saline-challenged or OVA-challenged WT mice (Fig. 4A).
Notably, the group who described Npsr1 upregulation in the OVA/Stachybotrys chartarum-challenged lung also showed that the murine RAW 264.7 cell line expressed Npsr1 using PCR primer pair that amplify sequence spanning exon 8 and exon 9 [16, 26]. When we used the same primer pair to examine Npsr1 expression in the lung, a short Npsr1 fragment could be amplified in some, but
STW not in all lung samples from saline- or OVA-challenged WT mice (Fig. 4B).
These results suggest that low levels of Npsr1 transcripts may exist in the lung of saline- or OVA-challenged WT mice.
4. Discussion
In humans, the SNPs in the NPSR1 gene have been associated with asthma phenotypes, including AHR. We examined Npsr1-deficient mice using two models of asthma. Consistent with a previous study [1], we found that there were no differences in the lung inflammation and respiration in response to allergen challenge between WT and Npsr1-deficient mice, suggesting that
NPSR1 is not directly involved in experimental asthma development. Further, our PCR results showed that Npsr1 has a very low level of expression in the basal or allergen-challenged lung, supporting that the direct role of NPS/NPSR1 in the lung may be limited. Our results are in contrast to the study from Laitinen et al. (16) which suggest that NPSR1 is implicated in human asthma. One of possible explanations is that there may be species differences in biological function of NPSR1 because our study is based in mouse.
Studies from Xu et al. [32, 33] showed that Nps and Npsr1 were mainly expressed in the brain stem, limbic system, and/or cerebral cortex. These regions are involved in regulating respiration, suggesting that NPS and NPSR1 have a role in this process. Unrestrained whole-body plethysmography (Buxco system), a noninvasive method, quantifies pulmonary physiological functions,
STX including respiratory frequency, tidal volume, and Penh, in conscious mice repeatedly (12, 18). However, many researchers have criticized that Penh does not accurately measure airway resistance (2, 19). Although the unrestrained whole-body plethysmography is a useful tool to screen for the presence of lung diseases, it is not suitable to discriminate different mechanisms by which altered patterns of breathing come about in the patients with respiratory diseases,(29).
The flexiVent system, an invasive method, allows specific analysis of pulmonary mechanics. In our study, we used these two methods to identify whether ICV
NPS had a direct mechanical effect on the airway or changed the respiratory pattern through the CNS. Measured by whole-body plethysmography, mice treated with ICV NPS showed increased respiratory frequency, decreased Penh and no change in tidal volume following methacholine exposure compared with aCSF-treated mice. However, ICV NPS did not change airway mechanics in response to methacholine as measured by flexiVent, further supporting that the
ICV NPS-induced decrease in Penh in response to methacholine is attributable to increased minute ventilation by ICV NPS.
NPSR1 has been associated with panic disorders in Japanese males [24].
Besides recurrent spontaneous anxiety attacks, panic disorders are dominated by respiratory symptoms such as hyperventilation, a feeling of being smothered, and shortness of breath, and respiratory dysregulation has been identified as a biological marker for panic disorder [9, 11]. Hypersensitivity of brainstem nuclei regulating respiratory activity has been proposed in patients with panic disorder
STY [7]. Our study demonstrated that ICV NPS could increase respiratory frequency in an NPSR1-dependent manner at baseline or in response to methacholine challenge. In addition, our earlier study showed that ICV NPS induced hyperlocomotion, increased exploration, and plasma corticosterone release in
WT mice, not in Npsr1-deficient mice [34]. All these physiological alterations triggered by ICV administration of NPS are part of typical fight-or-flight response, an adaptive response to danger. It is suggested that respiratory change triggered by ICV NPS is a psychophysiological activity in response to stress. As such, we speculate that psychological triggers NPS/NPSR1 mediate changes in respiration and anxiety, possibly modulating asthma symptoms. Further studies are needed to examine the expression of NPS and NPSR1 in the brain in a model of stress. Experimental and clinical evidence has emerged that there is an association of respiration changes with psychological stress and emotional diseases. Experimental exposure to emotional stimuli has been shown to increase respiratory resistance in asthma [27]. Chronic stress can exacerbate the symptoms of asthmatic patients [3, 31]. It will be interesting to define what the role of NPS/NPSR1 is in the exacerbation of asthma by chronic stress in the future as we have shown that NPS/NPSR1 signaling regulates stress and anxiety responses [34].
Our study is limited by testing a dose of ICV NPS (1 nmol) that is indeed a pharmacological dose because our previous paper showed that 1 nmol of ICV
NPS had the best effect on anxiety, locomotion and exploration compared with
STZ 0.1 and 10 nmol of ICV NPS [34]. In addition, the identified effect of NPS on respiratory frequency was conducted using whole-body plethysmography, and the confinement chamber may have anxiety-promoting effects. However, it is notable that the effect of NPS on respiration is NPSR1-dependent, providing evidence that the triggered pathway is specific to NPS/NPSR1.
It has not escaped our attention that ICV NPS induced increase in respiratory frequency and decrease in tidal volume resembles the respiratory panting behavior observed during hyperthermia [8] and mechanical associated high frequency ventilation that is clinically used to treat lung injury [15]. It will be interesting to determine whether natural panting responses may be NPS/NPSR1 dependent. And our results have implications for potentially understanding the responses to mechanical associated high frequency ventilation.
Taken together, our studies have demonstrated that the direct role of NPSR1 in the development of experimental asthma is limited and the effect of
NPS/NPSR1 on respiratory changes is likely mediated via a CNS-mediated pathway, providing a pathway that connects respiratory and stress responses.
ST[ Acknowledgements
We would like to thank Drs. Simon Hogan, Nives Zimmermann, Eric Brandt and
Carine Blanchard for scientific advice; Drs. Charles Vorhees and Michael
Williams for surgery and ICV injection technique assistance; and Dr. Julie
Caldwell and Shawna Hottinger for helpful discussions and review of the manuscript. This work was supported by NIH Grant 564000-100322.
SUR Table 1. Peripheral blood cell number and Percent of T cells, B cells and T cell subsets in the peripheral blood
WBC NE LY MO EO BA RBC
(K/uL) (K/uL) (K/uL) (K/uL) (K/uL) (K/uL) (M/uL)
WT 6.47±0.65 1.51±0.16 4.59±0.53 0.33±0.06 0.03±0.01 0.01±0.01 8.39±0.36
KO 7.94±0.67 1.68±0.20 5.86±0.48 0.36±0.04 0.04±0.02 0.01±0.01 8.87±0.34
HGB PLT CD3 (%) B220 (%) CD4 (%) CD8 (%) (g/dL) (K/uL)
WT 13.96±0.49 647.38±45.79 34±4.73 34±3.06 27±2.96 14±2.40
KO 14.24±0.53 654.13±28.51 30±3.18 28±3.00 26±2.96 11±1.53
Data expressed as mean ± SEM (n=8 for blood cell counting and n=3 for FACS)
Normal blood cell composition was assessed in the Npsr1-deficient (KO) and wildtype (WT) mice (n = 8 for each genotype). WBC, White Blood Cells; NE,
Neutrophils; LY, Lymphocytes; MO, Monocytes; EO, Eosinophils; BA, Basophils;
RBC, Red blood cells; HGB, Hemoglobin; PLT, Platelets. Flow cytometry was used to analyze T cells, B cells and T cell subtypes in the peripheral blood from
WT and KO mice (n = 3 for each genotype) using B220, CD3, CD4, and CD8 markers. These results are representative of three independent experiments.
Data represent the mean ± SEM.
SUS FIGURE LEGENDS
Figure 1. Role of NPS and NPSR1 in basal respiratory function
The protocol for measuring basal respiratory function in response to 1 nmol of
NPS or aCSF ICV injection by whole-body plethysmography in Npsr1-deficient
(KO) and wildtype (WT) mice is outlined in (A). Respiratory frequency (B), tidal volume (C), and enhanced pause (Penh) (D) were recorded every 2 min, and the values were plotted for 10-min intervals (n = 8 - 9 mice for each group). Values are presented as mean ± SEM. * p < 0.05 and ** p < 0.01 indicate the comparison of NPS- and aCSF-treated WT mice; # p <0.05 and ## p < 0.01 indicate the comparison of NPS-treated WT and KO mice; $ p <0.05 and $$ p <
0.01 indicate the comparison of aCSF-treated WT and KO mice.
Figure 2. Role of NPS and NPSR1 in methacholine-challenged respiratory function
The protocol for using barometric plethysmography to measure methacholine- induced changes in respiratory function after 1 nmol of NPS or aCSF ICV injection in Npsr1-deficient (KO) and wildtype (WT) mice is outlined (A).
Respiratory frequency, tidal volume, and enhanced pause (Penh) were assessed in response to methacholine challenge (0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 mg/ml)
5 min after 1 nmol of NPS ICV injection (B). Airway resistance in response to methacholine challenge (6.25, 25, 100 mg/ml) 20 min after 1 nmol of NPS ICV injection was measured using the flexiVent system (n = 11 - 14 mice for each group) (C). Values are presented as mean ± SEM. * p < 0.05 and ** p < 0.01
SUT indicate the comparison of NPS- and aCSF-treated WT mice; # p <0.05 indicates the comparison of NPS-treated WT and KO mice; $ p <0.05 indicates the comparison of aCSF-treated WT and KO mice.
Figure 3. Role of NPSR1 in OVA or Aspergillus -induced experimental asthma
(A) OVA-induced experimental asthma. Forty eight hours after the final challenge, OVA- or saline-challenged Npsr1-deficient (KO) and wildtype (WT) mice were assessed for BALF cell populations (n = 7 - 10 for each group), serum
IgE level (n = 5 - 8 for each group), mucus-secreting cells (n = 4 for each group), and collagen deposition in the lung (n = 4 for each group). Airway responsiveness to 37.5 mg/ml methacholine was assessed by flexiVent 24 hours after the final OVA challenge (n = 6 - 8 mice for each group). Data are representative of one of three experiments. Values are presented as mean ±
SEM. * p < 0.05 and *** p < 0.001 indicate the comparison of OVA-treated and saline-treated WT mice; # p < 0.05 and ### p < 0.001 indicate the comparison of
OVA-treated and saline-treated KO mice.
(B) Aspergillus-induced experimental asthma. Eighteen hours after Npsr1- deficient (KO) and wildtype (WT) mice received a final challenge with Aspergillus or saline, BALF cell populations (n = 7 - 8 for each group), serum IgE level (n = 5
- 6 for each group), mucus-secreting cells (n = 4 for each group), collagen deposition in the lung (n = 4 for each group), and airway responsiveness to 37.5 mg/ml methacholine by flexiVent (n= 8 - 9 mice for each group) were evaluated.
SUU Data are representative of one of three experiments. Values are presented as mean ± SEM. * p < 0.05 and *** p < 0.001 indicate the comparison of
Aspergillus-treated and saline-treated WT mice; # p < 0.05 and ### p < 0.001 indicate the comparison of Aspergillus-treated and saline-treated KO mice.
Figure 4. Npsr1 expression in the mouse lung and brain.
PCR amplification of full-length Npsr1 in the lung of OVA- or saline-challenged
WT mice and the brain of untreated WT mice. Full-length Npsr1 was not found in the OVA- or saline-challenged lung (A). PCR amplification of a short Npsr1 fragment in the lung of OVA- or saline-challenged WT mice using primer pair specific for exon 8 and exon 9. A short fragment of Npsr1 was found in some lung samples of OVA- or saline-challenged WT mice (B). The -actin expression for each set of PCR is shown as control. M indicates standard molecular marker.
These results are representative of one of three experiments.
SUV Figure 1
SUW Figure 2
SUX Figure 3
SUY Figure 4
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SVV Chapter 4
Discussion and future directions
1. Role of NPSR1 in NPS-induced anxiolytic effect
We have identified that NPSR1 is involved in the stress response at baseline and that NPSR1 is required for NPS-induced anxiolytic effect, hyperlocomotion, increased exploration, and plasma corticosterone release. We used elevated zero maze, open-field and light-dark exploration tests to measure anxiety-related behaviors. However, in these behavioral tests, the effects of ICV NPS-induced anxiolytic activity could not be completely separated from the effect of ICV NPS- induced hyperlocomotion because of the limitations that ICV injection could infuse NPS to brain regions related to locomotion and the anxiolytic behaviors in these behavioral tests overlap with hyperlocomotion. Notably, Leonard et al.
(2008) have substantiated the anxiolytic effect of ICV NPS in other behavioral tests, such as the four-plate test and stress-induced hyperthermia test, which are not affected by locomotion. For future experiments, one way to confirm the function of NPSR1 in NPS-induced anxiolytic effect is to test anxiety-related behaviors in Npsr1-deficient mice treated with ICV NPS using the behavior tests that are not affected by locomotion; Leonard et al. only used these tests to examine NPS-induced anxiolytic effect in WT mice, not in Npsr1-deficient mice.
Another way is to use the elevated zero maze, open-field and light-dark exploration tests to measure the anxiety-related behaviors in Npsr1-deficient
SVW mice administrated with NPS in the amygdala because NPS is specifically injected in the amygdala, which is responsible for mood control.
2. Interpretation of sex-biased behavioral phenotypes in WT and Npsr1- deficient mice
In our study we observed sex differences in some behavioral tests in C57BL/6
WT mice. Female C57BL/6 mice showed significantly increased immobility duration in forced swim test and increased plasma corticosterone level after forced swim test compared with male mice. This is consistent with previous reports, showing that the immobilization time in the tail suspension test was greater in female than male WT mice (Gangitano et al., 2009) and female mice displayed higher plasma corticosterone levels than males at baseline or under stressed conditions (Finn et al., 2004). And it is also in line with clinical observations that anxiety and depression symptoms are more frequent in women than in men (Bracke 1998; Olff et al., 2007), which indicates that males and females have different mechanistic responses to stress. The study from Aoki et al. (2010) showed that the higher corticosterone level in females under stressed conditions was an activational effect of the ovaries.
The marble burying test represents a pharmacologically sensitive method to assess defensive responses to anxiogenic stimuli (Broekkamp et al., 1986;
Nicolas et al., 2006). Because low doses of anxiolytic benzodiazepines and other ligands can attenuate buried marbles (Njung'e et al., 1991; Deacon, 2006),
SVX the marble burying test has been used as a model of assessing anxiety-like behaviors. In our study, female WT mice showed decreased number of buried marbles and increased latency to bury compared with male WT mice; however the level of difference did not reach statistical difference. When we interpret the result, we should avoid the temptation to over-anthropomorphize by thinking that female WT mice are more anxiolytic than male WT mice. It is possible that female mice are less defensive than male mice when exposed to new surroundings.
In our study, Npsr1-deficient mice show sex difference in forced swim test and acoustic startle response at baseline (without NPS stimulation). Although we do not know the exact reason leading to this sex-based phenotype, it is consistent with the study from Okamura et al. (2007) indicating NPSR1 gene has been associated with panic disorder in male Japanese. Several groups identified multiple sex-based transcripts by comparing expression levels of individual genes in different tissues (Isensee et al., 2007). The vast majority of sexually dimorphic traits results from the differential expression or translation of genes that are present in both sexes (Rinn et al., 2005). It is still unknown if there is gender differentiation of NPSR1 expression in the brain regions related to the emotion and acoustic startle response pathways. Interpretation of this question will help us understand the sex-based behavioral phenotypes in Npsr1-deficient mice.
SVY 3. Endogenous NPS expression in the brain and interaction with other neurotransmitters under stressful conditions in the WT and Npsr1-deficient mice
The behavioral tests after ICV injection of NPS is a pharmacological strategy to identify the role of NPSR1 in modulation of emotion and stress response, whereas the anxiety and depression-like behavioral tests at baseline reveal physiological reactions under stressful conditions. In anxiety-like behavioral tests at baseline, Npsr1-deficient mice did not show any behavioral changes compared with WT mice. However, male Npsr1-deficient mice showed elevated immobility duration in forced swim testing compared with male WT control. The explanation may be that these behavioral tests induce stress with different strength which results in differential expression of endogenous NPS in WT mice, further leading to the discrepancy in stress response in Npsr1-deficient mice. We reason that anxiety-related behavior tests such as zero maze test, light-dark exploration, locomotor activity and marble burying tests would be less severe stressors than the forced swim test. It is speculated that there are more endogenous NPS expressed in the brain during forced swim test than during anxiety-related behavioral tests in WT mice. In vivo microdialysis sampling system coupled on- line to capillary electrophoresis with a laser-induced fluorescence (CE-LIF) detection apparatus (Presti et al., 2004) wil be used to dynamicaly detect behavior-related endogenous NPS alterations during forced swim test or anxiety- like behavior tests in the brain regions that mediate stress response, such as amygdala. The earlier result suggests that NPS precursor mRNA is co-
SVZ expressed with excitatory or stimulatory transmitters, such as glutamate, acetylcholine, and CRF in the brainstem (Xu et al., 2007). All these neurotransmitters have an important role in emotional regulation and they interact with each other. Changes in one neurotransmitter system elicit changes in another. Thus, it is necessary to monitor these neurotransmitters expression at the corresponding brain regions in WT and Npsr1-deficient mice when detecting endogenous NPS alterations under these behavioral tests; employing in vivo microdialysis sampling is likely to be valuable in this regard.
4. NPS and fight-or-flight response
The behaviors after NPS ICV injection, including respiratory change, hyperlocomotion, increased exploration, and plasma corticosterone release, are part of typical fight-or-flight response to acute stress. The fight or flight response is a beneficial behavioral and physiological strategy in helping the body to "fight" or "flee" from perceived threat to survival. It involves stimulation of the sympathetic nervous system and stress hormones including norepinephrine, epinephrine and corticosterone, which are released into the bloodstream. These hormones facilitate immediate physical reactions associated with preparation for violent muscular action, including increased metabolism, heart and respiratory rate, pupil dilation, intensified awareness, sharpened sight, diminished pain, and increasing bloodflow to skeletal muscle, which require extra energy and fuel for running and fighting (Gleitman et al, 2004). The stress hormones, including norepinephrine, epinephrine and corticosterone should be dynamically examined
SV[ in the circulation after NPS ICV injection to identify the down-stream molecules of
NPS-NPSR1 signaling. The mobilization of the body for survival also has negative consequence. If cumulative buildup of stress hormones is not properly metabolized over time, it can lead to disorders of autonomic nervous system, hormonal and immune systems. The functional changes of physiological systems involved in the fight-or-flight response should be examined in order to understand how ICV NPS affects whole-body homeostasis.
5. Role of NPS and NPSR1 in respiration and asthma
In humans, the SNPs in the NPSR1 gene have been associated with asthma phenotypes, including AHR. We examined Npsr1-deficient mice using two models of asthma. Consistent with a previous study (Allen et al., 2006), we found that there were no differences in the lung inflammation and respiration in response to allergen challenge between WT and Npsr1-deficient mice, suggesting that NPSR1 is not directly involved in experimental asthma. Further, our PCR results showed that Npsr1 has a very low level of expression in the basal or allergen-challenged lung, supporting that NPS/NPSR1’s direct role in the lung may be limited.
Studies from Xu et al. (2004, 2007) showed that Nps and Npsr1 were mainly expressed in the brain stem, limbic system, and/or cerebral cortex. These regions are involved in regulating respiration, suggesting that NPS and NPSR1 have a role in this process. Our study demonstrates that at baseline, ICV NPS
SWR can increase respiratory frequency and decrease TV in WT mice, but not in
Npsr1-deficient mice, supporting the hypothesis that NPS/NPSR1 signaling is involved in regulating respiratory function through a central-mediated pathway. It is speculated that psychological stress up-regulates NPS/NPSR1 expression in the brain, which regulate respiration and anxiety, and futher modulates asthma symptoms.
Unrestrained whole-body plethysmography (Buxco system), a noninvasive method, quantifies pulmonary physiological functions, including respiratory frequency, tidal volume, and enhanced pause (Penh), in conscious mice repeatedly (Lomask et al., 2006; Hoymann et al., 2007). However, many researchers have criticized that the Penh does not accurately measure airway resistance (Lundblad et al., 2002; Bates et al., 2003). Although the unrestrained whole-body plethysmograph is a useful tool to screen the presence of lung diseases, it is not suitable to discriminate different mechanisms by which altered pattern of breathing come about in the patients with respiratory diseases, such as alteration in ventilatory requirements, alteration in respiratory drive from central or peripheral chemoreceptors and alteration in mechanical properties of the airway and/or lung parenchyma (Sly et al., 2004). The flexiVent system, an invasive method, allows specific analysis of pulmonary mechanics. In our study, we used these two methods to identify whether ICV NPS had direct mechanical effect on airway or centrally changed respiratory pattern in response to methacholine challege. Measured by whole-body plethysmography, the mice
SWS treated with ICV NPS showed increased respiratory frequency, decreased Penh and no change in tidal volume following methacholine exposure compared with aCSF-treated mice. However, ICV NPS did not change airway mechanics in response to methacholine as measured by flexiVent, further supporting that the
ICV NPS-induced decrease in Penh in response to methacholine is attributable to increased minute ventilation by ICV NPS. NPSR1 has been associated with panic disorders in Japanese males (Okamura et al., 2007). Besides recurrent spontaneous anxiety attacks, panic disorders are dominated by respiratory symptoms such as hyperventilation, a feeling of being smothered, and shortness of breath. Our study shows that NPS/NPSR1 signaling induces fight-or-flight response, an adaptive response to danger, after NPS central administration. It is suggested that respiratory change triggered by ICV NPS is a psychophysiological response to stress involving the mind and body.
Allergic airway inflammation causes various changes in the morphology and function of structural cells within the lung that contribute to airway hyperreactivity
(de Meer et al., 2004). NPSR1 has been associated with chronic inflammation diseases, such as asthma and inflammatory bowel disease, implying that NPSR1 may be involved in inflammation. Because it has been shown that NPSR1 is not directly involved in the development of airway inflammation, we propose that
NPSR1 may participate in airway inflammation through a central-mediated pathway. The CNS regulates airway inflammation by the generation of glucocorticoids in response to stimulation of the HPA or by autonomic nervous
SWT system through the interaction of CNS, immune system, endocrine system, and autonomic nervous system. Our results show that NPSR1 is required for ICV
NPS-induced increase in plasma corticosterone and anxiolytic effect. Whether
ICV NPS regulates the immune system is still unknown. In the future, it will be interesting to examine whether ICV NPS modulates allergen-specific IgG and IgE production and Th2 cytokine generation after allergen challenge. In addition, in order to understand whether NPS-NPSR1 signaling modulates airway inflammation through the interaction between local immune cells and nerve fiber endings within the lung, it will be of interest to examine the expression pattern of
NPS and NPSR1 in the immune cells and nerve fiber endings within the lung under stress and/or allergen challenge. Some neuropeptides secreted from nerve fiber endings or immune cells can directly affect airway constriction. It would be of interest to examine the direct effect of NPS on airway constriction in vivo by intratracheal challenge with NPS or ex-vivo using isolated tracheal rings incubated with NPS.
Overall, our studies have demonstrated that NPSR1 is not directly involved in experimental asthma development. Rather, NPSR1 is involved in the stress response, and the effect of NPS/NPSR1 on respiratory change is likely mediated via a central-mediated pathway. Although the identified mechanism is yet to be proven under physiological conditions as our study conditions were primarily induced by pharmacological doses of NPS, we propose that a central nervous
SWU system pathway mediates the underlying association of NPSR1 with asthma and
AHR.
6. Limitations of Npsr1-deficient mouse model and asthma mouse model
There are 2 splice variants for human, NPSR1-A and NPSR1-B. Only one mouse ortholog of NPSR1 exists. This indicates that human NPSR1 underwent a significant gene expansion compared with the mouse ortholog during evolution
(Huang et al., 2004; Perez, 2005). NPSR1-A and NPSR1-B differ only in the cytoplasmic C-terminal. Minneman (2001) reviewed that GPCR C-terminal splice variants differently affect G-protein coupling and desensitization of GPCR, and also may remove or add particular motifs resulting in receptors with differential localization and function. Human NPSR1-A and NPSR1-B have different expression patterns in healthy individuals and asthmatic patients. NPSR1-A is consistently expressed in the bronchial smooth muscle cells of both healthy persons and asthmatic patients. However, in asthmatic patients, NPSR1-B is strongly expressed in the bronchial epithelial cells and smooth muscle cells, whereas, in healthy person, NPSR1-B is only faintly positive in airway epithelial cells (Laitinen et al., 2004). The differential localization of NPSR1-B in the lung of healthy person and asthmatic patients suggest that human NPSR1-B may have a role in airway remodeling. Mouse Npsr1 shows high amino acid identity to human NPSR1-A. As an alernative approach, it would be interesting to generate NPSR1-B knock-in mice in which Npsr1 is replaced with NPSR1-B.
Another limitation of our study is the usage of the OVA or Aspergillus models,
SWV which are relatively acute challenge mouse models which replicate many key features of clinical asthma, such as elevated IgE levels, airway inflammation, goblet cell hyperplasia, AHR and epithelial hypertrophy. However, they fail to reproduce a number of chronic manifestations of human asthma, such as chronic airway inflammation and airway remodeling (Nials et al., 2008). In the future, chronic asthma mouse model may be useful to identify the role of NPSR1-B in airway remodeling, especially in the setting of chronic stress.
7. Development of pharmaceutical drugs for NPS/NPSR1 system
The studies from our and other groups suggest that NPS/NPSR1 is a potential target for treatment of some emotional disorders (Xu et al., 2004; Leonard et al.,
2008; Rizzi et al., 2008; Zhu et al., 2010). Before it is available for clinical treatment, there are lots of areas needed to be explored. First, in our study, NPS
ICV injection only involves one dose and the effects of NPS on behaviors are short-term. The chronic effects of NPS on physiological systems involved in fight-or-flight response should be examined to help us understand the safety and side effect of long-term administration of NPS. Second, investigating whether
NPS can cross the blood-brain barrier will help us decide which administration method will be used for treatment. If NPS can cross the blood-brain barrier, we can apply it by systematic administration. If NPS cannot cross the blood-brain barrier, we need to search for the precursor of NPS, or the enzymes that influence NPS synthesis or metabolism. All these precursor or enzymes should cross the blood-brain barrier. Third, currently the half-life of NPS is unlear.
SWW Figuring out the half-life of NPS will help decide the duration between two doses of administration so that the dose-effect maintains. Fourth, although our findings indicate that NPSR1 is the major receptor for NPS, others may exist. Searching for new receptors for NPS will help develop new agonist or antagonist for NPS, which is beneficial for exploring the function and downstream moleculars of NPS system or for disease treatment.
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SXR