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Suppression of Stress-induced nNOS Expression in the Rat Hypothalamus by Biting
N. Hori1,
M.-C. Lee2,*,
K. Sasaguri3,
H. Ishii3,
M. Kamei3,
K. Kimoto1,
M. Toyoda1 and
S. Sato3
1 Oral and Maxillofacial Rehabilitation, Division of Prosthetics,
2 Clinical Care Medicine, Division of Pharmacology and ESR Laboratories, and
3 Craniofacial Growth and Development Dentistry, Kanagawa Dental College, 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan;
Correspondence: * corresponding author, ieeman{at}kdcnet.ac.jp
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ABSTRACT
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Nitric oxide (•NO) modulates the activity of the endocrine system in the behavioral response to stress. The purpose of this study was to investigate the effect of restraining the body of an animal on expression of neuronal nitric oxide synthase (nNOS) in the paraventricular nucleus (PVN) of the hypothalamus, and the inhibitory effect of para-masticatory activity on restraint-induced nNOS expression. We observed an increase in nNOS mRNA expression and nNOS-positive neurons in the rat hypothalamus after 30 or 60 min of restraint. Biting on a wooden stick during bodily restraint decreased nNOS mRNA expression in the hypothalamus. In addition, the number of nNOS-positive neurons was significantly reduced in the PVN of the hypothalamus. These observations clearly suggest a possible anti-stress effect of the masticatory activity of biting, and this mechanism might be unconsciously in operation during exposure to psychological stressors.
Key Words: neuronal nitric oxide synthase (nNOS) • restraint stress • biting • paraventricular nucleus • rat
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INTRODUCTION
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The masticatory activity of biting is a coordinated function that connects peripheral effector organs to the central nervous system (CNS) (Nakata, 1998). Biting behavior has been shown to decrease dopamine metabolism and catecholamine turnover in several brain areas in the rat (Tanaka et al., 1998; Tsuda et al 1988; Gomez et al., 1999). We have previously shown that the expression of corticotropin-releasing factor (CRF) is significantly increased in the paraventricular nucleus (PVN) of the hypothalamus by restraint, and this increase in CRF expression is suppressed by biting (Hori et al., 2004).
Nitric oxide (•NO) is an important signaling molecule in various cell functions (Palmer et al., 1988; Ignarro, 2002). Autonomic or endocrine dissonance is associated with several homeostatic disorders (Selye, 1998; Koob, 1999; Hernandez et al., 1988). These responses are influenced by the effect of •NO on the release of various hormones (Kato, 1992; Goyer et al., 1994; Kadowaki et al., 1994; River and Shen, 1994), including CRF (Costa et al., 1993; Karanth et al., 1993). •NO synthase (NOS) has been observed in several brain areas, particularly the PVN of the hypothalamus. Since these neurons are also the major source of CRF (Costa et al., 1993; Karanth et al., 1993), we speculated that •NO may be involved in the regulation of CRF secretion.
There are three NOS isoforms in the NOS family, termed neuronal Type-I NOS (nNOS), inducible Type-II NOS (iNOS), and endothelial Type-III NOS (eNOS) (Palmer et al., 1988; Ignarro, 2002). Consistent with the involvement of nNOS in various aspects of cellular biology, nNOS mRNA and protein are expressed in numerous tissues (Wang et al., 1999). In the brain, neurons containing nNOS are found in the hypothalamus of rats (McCann et al., 1996) and humans (Thorns et al., 1998). Recently, it has been reported that restraint induces up-regulation of nNOS mRNA in the brain (Calza et al., 1993; Kishimoto et al., 1996). However, there have been no reports to date on the effect of parafunctional masticatory activity (biting) on stress-induced expression of nNOS in the CNS. In this study, we investigated the involvement of nNOS in the suppression of restraint stress induced by biting, and we speculated that biting would suppress the stress-induced expression of nNOS in the PVN of the hypothalamus.
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MATERIALS & METHODS
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Animals
Male Sprague-Dawley rats (7–8 wks old) were group-housed (4 per cage) under controlled temperature (22 ± 3°C) and lighting conditions (12:12-hour light:dark cycle), with free access to food and water. To avoid diurnal variations in nNOS expression in specific brain areas, we conducted all experiments simultaneously between 1030 hrs and 1500 hrs.
Restraint Stress and Biting
Acutely restrained animals were immobilized as described previously (Hori et al., 2004). Control rats were not exposed to restraint and had free access to food and water. The untreated groups were restrained only for either 30 or 60 min. The treated groups were allowed to bite a wooden stick (diameter, 0.5 cm) during restraint for either 30 or 60 min (Hori et al., 2004). The treated and the untreated groups were kept in separate rooms. The experimental procedures used in this study were reviewed and approved by the Committee of Ethics on Animal Experiments of Kanagawa Dental College, and were carried out under the Guidelines for Animal Experimentation of Kanagawa Dental College.
Total RNA Extraction and RT-PCR Analysis
For the mRNA analysis experiment, 5 different groups (n = 3 per group) were used: (1) restraint for 30 min or (2) 60 min, (3) 30 min of restraint with biting, (4) 60 min of restraint with biting, and (5) no restraint (control). At the end of the treatment period, animals were killed by cervical dislocation, the brain was removed, and 100 µg of brain tissue from the hypothalamus, including the PVN, was dissected out. Total RNA was extracted with the use of a Roche 1st-strand cDNA synthesis kit (Roche, Indianapolis, IN, USA) and was precipitated with ethanol. Primers specific for nNOS were used, with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the internal control. The nNOS specific primer for the coding sequence was designed as shown in Table 1 (Hukkanen et al., 1999).
For PCR amplification, 10% of the transcribed cDNA was added to a 50-µL reaction mixture and amplified by a ‘hot start’ method (40 cycles: 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, and 72°C for 5 min) in a Takara PCR Thermal cycler MP (Takara Bio Inc., Shiga, Japan). PCR was carried out in 50 mM KCl, 10 mM Tris-HCl (pH = 9), 0.1% Triton X-100, 50 µM dNTPs, 1.8 mM MgCl2, 2.5 U of Taq DNA polymerase (Takara Bio Inc., Shiga, Japan), and 100 nM nNOS primer. Amplification of GAPDH (35 cycles) provided an internal control for the efficiency of the RT-PCR process. After PCR, equivalent aliquots of each amplification reaction were separated on a 1.0% agarose gel containing ethidium bromide and visualized under UV light.
nNOS Immunohistochemistry
Thirty rats were used for the immunohistochemistry experiment (n = 6 per group). The animals were anesthetized with thiamylal sodium and then perfused with normal saline through the heart, followed by 0.01 M phosphate-buffered saline (PBS) containing 4% paraformaldehyde (pH 7.4). The brain was removed and post-fixed in the same fixative at 4°C for 24 hrs, then placed in PBS with 10% sucrose at 4°C for 24 hrs. Multiple series of frozen coronal sections (40 µm thick) throughout the length of the hypothalamus were collected in PBS. nNOS immunoreactivity was localized in series of sections through the PVN from each animal, with the use of an avidin-biotin-immunoperoxidase method (Vectastain Elite ABC kit; Vector Laboratories, Burlington, CA, USA). The sections were pre-incubated for 1.5 hrs in 0.75% Triton X-100, 0.05% NaN3/0.01 M PBS (PBS-T) containing 1.5% normal goat serum to block non-specific binding and facilitate tissue penetration, followed by reaction with the primary antibody, rabbit polyclonal anti-nNOS protein antiserum (H-299, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), diluted at 1:1000 for 48 hrs at 4°C. The sections were further processed with the secondary antibody, biotinylated anti-rabbit IgG, with PBS-T containing 1.5% NGS (2 hrs at room temperature), and then with the ABC kit. Visualization of the antigen-antibody complex was accomplished by the application of 3,3'-diaminobenzidine tetrahydrochloride (DAB) as the chromogen.
Data Analysis
Expression of nNOS mRNA levels in the hypothalamus was measured in a Science Lab 2002 system coupled to a Windows computer running Multi Gauge software (Fuji film, Tokyo, Japan). nNOS mRNA expression levels were normalized against GAPDH as a control and compared between groups. In the nNOS immunohistochemistry, the anatomical position of the sliced brain sections was determined according to the rat brain map (Paxinos and Watson, 1986). The PVN was located at –1.80 to –2.00 mm from Bregma (5 sections). The numbers of nNOS-positive neurons in the right and left sides of the PVN were counted by Image-Pro Plus (Media Cybernetics, Silver Spring, MD, USA) and corrected for double-counting (Coggeshall and Chung, 1984). In addition, we compared the expression of nNOS and the inhibitory effect of biting among the 4 sub-regions of the PVN: dorsal parvicellular, periventricular, medial parvicellular, and posterior magnocellular.
Data were expressed as mean ± SEM. Differences among groups within an area of interest were statistically evaluated by one-way analysis of variance (ANOVA) and post hoc Fisher’s PLSD test. Probabilities of < 5% (p < 0.05) were considered significant.
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RESULTS
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Expression of nNOS in PVN Neurons
In the present study, nNOS mRNA levels in the hypothalamus were increased by 30 min of restraint as compared with the control group (Fig. 1A). nNOS mRNA levels in the hypothalamus after 60 min approached that in the control group. These results suggest that nNOS mRNA was significantly increased in the hypothalamus with restraint, reaching the maximal level at 30 min (Figs. 1A, 1B). Using immunohistochemical methods, we compared the number of nNOS-positive neurons in the right side of the PVN with that of the control group (Fig. 2A), and found a significant increase after 30 or 60 min of restraint (Fig. 2B). No significant difference in the number of nNOS-positive neurons between the right and left sides in the PVN was observed in the course of this study (data not shown).
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Figure 1. Expression of nNOS mRNA levels in the hypothalamus following the 30- and 60-minute restraint periods, with or without biting (N = 3 per group). (A) nNOS mRNA levels were increased by 30 min of restraint, and were suppressed by biting during restraint. (B) The increase of nNOS mRNA was statistically significant (*p < 0.05, ANOVA/Fisher’s PLSD). Levels in animals allowed to bite during restraint were significantly different ( p < 0.05, ANOVA/Fisher’s PLSD) from those in animals restrained without biting.
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Inhibitory Effect of Biting on nNOS Expression
To examine the effect of biting during restraint, we permitted the animals to bite on a wooden stick during the restraint period. The expression of nNOS mRNA during the 30-minute restraint period, when biting was allowed, was significantly reduced in comparison with that during 30 min of restraint (Figs. 1A , 1B). The numbers of nNOS-positive neurons in the rats treated with 30 or 60 min of restraint with biting were reduced compared with those in the animals without biting (Figs. 2A, 2B). The average numbers of nNOS-positive neurons in the 30-minute restraint groups without and with biting were 152.3 ± 9.8 (n = 3) and 102.3 ± 4.3 (n = 3), respectively, while those in the 60-minute restraint and restraint with biting groups were 210.0 ± 3.5 (n = 3) and 130.3 ± 5.9 (n = 3), respectively (Fig. 2B).
Further, analysis of the effect of biting in sub-regions of the PVN—the dorsal parvicellular, periventricular, medial parvicellular, and posterior magnocellular regions—revealed differences in the expression of nNOS-positive neurons. In the parts of the PVN examined, the highest levels of nNOS expression were observed in the medial parvicellular and posterior magnocellular regions during restraint. In addition, biting significantly decreased the number of nNOS-positive neurons in the medial parvicellular region in the 30- or 60-minute restraint group, and in the posterior magnocellular and dorsal parvicellular regions of the 60-minute restraint group (Table 2).
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DISCUSSION
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In this study, we show that nNOS mRNA levels in the hypothalamus increased after 30 min (Fig. 1), and that the number of nNOS-positive neurons in the PVN of the hypothalamus increased after 30 or 60 min of restraint (Fig. 2). These results appear to support the hypothesis that PVN neurons play an important role in mediating behavioral responses to stressors (Sawchenko et al., 1996; Koob, 1999). The enhancement of nNOS expression was found not only in the medial parvicellular and posterior magnocellular regions, but also in other sub-regions of the PVN (Table 2). However, the expression of nNOS-positive neurons was low in the dorsal parvicellular and periventricular regions (Table 2). Previous studies on animals subjected to restraint suggest that activation of the medial parvicellular and posterior magnocellular regions of the PVN occurs (Sawchenko et al., 1996; Viau and Sawchenko, 2002). There is also a strong positive correlation between PVN activation and other stress stimuli, such as immobilization, hypoglycemia, pain, tissue damage, and injection of physiological saline (Pacak et al., 1995). The medial parvicellular region contains many CRF-producing cells (Ceccatelli et al., 1989; Viau and Sawchenko, 2002; Hori et al., 2004), while the posterior magnocellular region contains vasopressin and oxytocin-producing cells (Goyer et al., 1994; Kadowaki et al., 1994). •NO plays a role in the production of endocrine hormones including CRF—for example, the expression of nNOS in neurons promotes CRF synthesis (Karanth et al., 1993). Biting caused a decrease in the number of nNOS-positive neurons in the medial parvicellular region in the 30-minute and 60-minute restraint groups, and in the posterior magnocellular region in the 60-minute restraint group. These results confirm that biting suppresses the superfluous production of hormones caused by restraint stress, and indicate the critical role of •NO in the activation of CRF gene expression in the medial parvicellular region.
It has been suggested that this induction of the hypothalamic-pituitary-adrenal (HPA) axis by restraint is the first response, causing the release of CRF from the PVN (Cunningham and Sawchenko, 1988; Imaki et al., 1992). In the present study, we demonstrated an increase of nNOS in the PVN after restraint (Figs. 1, 2). Our previous study also showed an increase in CRF expression in the PVN after restraint (Hori et al., 2004). Since the PVN is known to secrete CRF, and it is likely that both nNOS and CRF are increased in response to stress (Costa et al., 1993; Karanth et al., 1993), these results suggest that the generation of •NO by nNOS may be involved in the expression of CRF during the stress response.
In the present study, biting a wooden stick during the restraint period significantly decreased nNOS mRNA levels and the number of nNOS-positive neurons in the hypothalamus. However, both results (Fig. 2, Table 2) show that the effects of biting only partially inhibit the increase in nNOS levels caused by restraint stress, suggesting the possible involvement of other mechanisms. It should be noted that the actual redox-environment of distinct loci within the brain may determine the final function of •NO (Riedel, 2000).
It seems likely that all types of stress influence neuronal excitability by altering the ratio of redox factors, such as thiols and reactive oxygen species (ROS), within the CNS, and that •NO acts either as an amplifier or as a feedback regulator of neuronal excitation or inhibition, which may acutely or chronically alter the homeostasis of a given neurosecretory system. Additional studies are required to evaluate the relationship between redox factors such as thiols and ROS and the expression of nNOS in the PVN.
Our results show that a period of restraint is required for nNOS expression, but that biting a wooden stick during the restraint period suppresses nNOS expression in the PVN. In particular, it was suppressed in the medial parvicellular region of the PVN. The effect of this masticatory activity on the response to psychological stress is very important and leads to suppression of the stress response, which may provide a protective effect on general health.
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ACKNOWLEDGMENTS
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This work was performed in Kanagawa Dental College, Research Center of Advanced Technology for Craniomandibular Function, and supported by grants-in-aid for Bioventure Research from the Japanese Ministry of Education, Science and Culture.
Received for publication April 9, 2004.
Revision received February 17, 2005.
Accepted for publication March 22, 2005.
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Journal of Dental Research, Vol. 84, No. 7,
624-628 (2005)
DOI: 10.1177/154405910508400708
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ABSTRACT
INTRODUCTION