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Biting Suppresses Stress-induced Expression of Corticotropin-releasing Factor (CRF) in the Rat Hypothalamus
1 Departments of Prosthetics, Correspondence: * corresponding author, horin{at}kdcnet.ac.jp
Corticotropin-releasing factor (CRF) expressed in the hypothalamus plays an important role in mediating behavioral responses to stressors. Restraining the body of an animal has been shown to activate and induce an enhanced expression of CRF in paraventricular neurons of the rat hypothalamus. Since aggressive biting behavior is known to suppress stress-induced noradrenaline secretion in the central nervous system and the formation of gastric ulcers, we investigated the effect of biting on restraint-induced CRF expression in the rat hypothalamus. The number of CRF-expressing neurons in the paraventricular nucleus increased significantly after short time restraint (30 or 60 min) followed by a 180-minute post-restraint period. Biting of a wooden stick during the restraint stress significantly suppressed the restraint-induced enhancement of CRF expression in the paraventricular nucleus. These observations suggest a possible anti-stress effect of biting and an important role of para-functional masticatory activity in coping with stressful events.
Key Words: restraint stress • biting • corticotropin-releasing factor (CRF) • paraventricular nucleus
Stress is a collection of physiological and psychological changes that occur in response to environmental changes and to noxious stimuli applied to an animal body (Selye, 1936). Restraint of the animal body, exposure to cold temperature, and noxious stimuli have been shown to induce a variety of disorders, including gastric ulceration, adrenal hypertrophy, and functional disorders in the autonomic nervous and endocrine systems (Chrousos and Gold, 1992; Tanaka et al., 1998; Koob, 1999). There is general agreement that stress-induced disorders are attributed to activation of the hypothalamic-pituitary-adrenal (HPA) axis and/or the sympathetic nervous system. Corticotropin-releasing factor (CRF) is a 41-amino-acid hypophysiotropic peptide secreted from neurons in the paraventricular nucleus (PVN) of the hypothalamus (Swanson et al., 1983; Antoni, 1986; Sawchenko, 1987). CRF activates the anterior lobe of the pituitary gland and releases adrenocorticotrophic hormone (ACTH), which in turn activates the adrenal cortex (Vale et al., 1981). Stress-induced activation of the HPA axis is characterized by enhanced expression of CRF in the PVN and the consequent increases of ACTH and adrenal glucocorticoid in the plasma (Livezey et al., 1985; Koob, 1999; Viau and Sawchenko, 2002). Immobilization or restraint of the animal body, an experimental model for psychological stress, has been shown to activate and induce an enhanced expression of CRF mRNA or hnRNA in the PVN neurons (Imaki et al., 1992; Stout et al., 2002; Viau and Sawchenko, 2002). Acute restraint stress activates noradrenergic neurons in the locus coeruleus (LC) (Abercrombie and Jacobs, 1987) and induces a high level of Fos expression in both the LC and PVN (Chowdhury et al., 2000), confirming the involvement of the sympathetic system as well as the HPA axis in mediating restraint-induced stress responses. Oral parafunctional activity—such as tooth-clenching, nail-biting, or the biting of objects—has been suggested to provide an outlet for emotional tension or stress in humans. It has been shown that aggressive biting behavior during stress exposure suppresses the stress-induced dopamine metabolism in the rat striatum (Gomez et al., 1999) and restraint-induced enhanced turnover of catecholamines in the central nervous system of the same animal (Tsuda et al., 1988; Tanaka et al., 1998). However, no study has reported on the effect of parafunctional masticatory activity on stress-induced expression of CRF in the central nervous system. In this study, therefore, we investigated the effects of physical restraint and biting on the expression of CRF in the rat brain.
We used 57 male Sprague-Dawley rats (7–9 wks old) in this study. They were housed in groups of 4 animals per cage in a room maintained at standardized conditions of light (12:12 hrs light-dark cycle) and temperature (22 ± 3°C). Animals had free access to food pellets and tap water. To avoid diurnal variations in CRF expression, we conducted all experiments between 1000 and 1500 hrs.
Restraint Stress and Biting
CRF Immunohistochemistry
CRF Counting
Statistical Analysis
CRF Expression in the Hypothalamus CRF-expressing neurons in the rat hypothalamus were found in all animals, including those not exposed to restraint stress (Fig. 1B  ). They were localized mainly within the PVN, but some were found scattered around the PVN in the hypothalamus (Figs. 1B, 1C, 1D). The average numbers of CRF-positive PVN neurons found in the control rats (n = 6) were 76.5 ± 4.3 (right side) and 75.0 ± 6.4 (left side) and were comparable (p < 0.05) with those found in the fasting control rats (right, 75.7 ± 7.5; left, 73.3 ± 11.9).
Enhanced Expression of CRF by Restraint Stress During the post-restraint period up to 120 min, no significant change in CRF expression was observed in the rats exposed to 60 min of restraint, and the numbers of CRF-positive neurons remained constant at the same levels as in the control rats (Fig. 2 ). The numbers of CRF-positive neurons increased significantly, however, at 180 min after restraint stress (Figs. 1C , 2A). This strong enhancement in CRF expression occurred predominantly in a small region of PVN between –1.8 and –1.9 mm from the bregma (Fig. 1D). The average numbers of CRF-expressing PVN neurons observed in the right and left sides 180 min after restraint were 405.0 ± 15.1 and 406.0 ± 20.6 (n = 6), respectively, and were comparable with those found at 240 min after restraint (right, 355.0 ± 31.6; left, 370.0 ± 21.8; n = 6) (Fig. 2B). No significant difference in the numbers of CRF-positive neurons between the right and left sides of the PVN was observed in the course of this study.
Effects of Restraint Time and Biting on CRF Expression in PVN When the animals were exposed to 30 min of restraint, an enhancement of CRF expression in the PVN was still induced 180 min post-restraint (Fig. 3A ). The average number of CRF-positive neurons observed after 30 min of restraint (199.0 ± 9.7, n = 6) was significantly (p < 0.05) higher than that in the control (76.6 ± 4.3, n = 6) but less than that induced by 60 min of restraint (Fig. 3B).
Biting of a wooden stick during restraint resulted in a significant reduction in the expression of CRF in the PVN, regardless of the restraint time of 30 or 60 min (Fig. 3A ). The average number of CRF-positive neurons observed after 30 min of restraint decreased significantly (p < 0.05) from 199.0 ± 9.7 (n = 6) to 118.0 ± 8.8 (n = 6) and from 405.0 ± 15.1 (n = 6) to 191.0 ± 18.3 (n = 6) in the rats exposed to 60 min of restraint (Fig. 3A). The suppression of stress-induced expression of CRF occurred mainly in the medial parvicellular part of the PVN, but this was also observed in the dorsal parvicellular and posterior magnocellular parts of the PVN for 30 min and 60 min of restraint-induced CRF expression, respectively (Table).
The present study has shown that acute restraint stress induces enhanced expression of CRF in a small region (between –1.8 and –1.9 mm from the bregma) of the PVN. The detection of CRF expression was possible in this study without pre-treatments of adrenalectomy or colchicine injection, which had been used to enhance CRF expression in the CNS (Swanson et al., 1983; Sawchenko, 1987). A robust increase in the numbers of CRF-positive PVN cells 180 min after the termination of restraint agreed with the results of the previous restraint experiments that showed a maximal expression of CRF mRNA at 120 min after restraint (Imaki et al., 1992). Low levels of CRF expression found in the fasting control rat ruled out the possibility that inaccessibility to food and water during the post-restraint period might have affected the expression of CRF. The strong inhibitory effect of biting on restraint-induced expression of CRF is also in accordance with the previous results on the central noradrenergic transmission (Tsuda et al., 1988; Tanaka et al., 1998), and supports the concept that expression of aggressive parafunctional masticatory activity during the stress exposure attenuates the stress responses. The preferential suppression of CRF-positive neurons in the medial parvicellular part of the PVN by biting and restraint-time-dependent suppression that occurred in the other 2 regions suggests a possible functional differentiation among the 4 sub-regions in responding to restraint stress and inhibitory inputs caused by biting. Activation of paraventricular neurons and increases of the plasma levels of ACTH and glucocorticoid by restraint have been repeatedly confirmed by previous studies (Livezey et al., 1985; Imaki et al., 1992; Chowdhury et al., 2000; Stout et al., 2002; Viau and Sawchenko, 2002). Although there is general agreement that increases of plasma ACTH and corticosterone begin during stress exposure (Livezey et al., 1985; Viau and Sawchenko, 2002), and that maximal induction of c-fos mRNA, a marker for neuronal activation, in PVN neurons occurs as early as at 30 min after the commencement of stress (Imaki et al., 1992; Viau and Sawchenko, 2002), the time course of the CRF marker varies among the studies. A significant enhancement of CRF hnRNA was detected immediately after 30 min of restraint (Imaki et al., 1996), and CRF mRNA in the medial parvicellular part of the PVN reached the maximal level 30 min after the termination of 30 min of restraint stress, while Imaki et al.(1992) reported that maximal expression of CRF mRNA occurred at 120 min after 60 min of restraint. The hypothalamus has reciprocal communications with areas of the cerebral cortex and with the limbic system, integrating autonomic and endocrine functions with behaviors (Herman and Cullinan, 1997; Kandel et al., 2000). There are 2 major afferent pathways consisting of noradrenaline-containing nerve fibers that regulate the function of PVN (Cunningham and Sawchenko, 1988). Noradrenergic afferent fibers from the nucleus ambiguous or from the nucleus solitary tract directly project to the hypothalamus, and control the visceral reflexes for cardiovascular and endocrine functions. The afferent inputs from the locus coeruleus mainly project to the cerebral cortex and the limbic system, and indirectly regulate the activity of the hypothalamus (Herman and Cullinan, 1997). The activation of the former pathway enhanced expression of CRF mRNA in the PVN and increased the plasma level of ACTH (Gartside et al., 1995). Restraint stress of the animal or non-noxious distension of the visceral organs has been shown to increase the neural activity of noradrenergic neurons in the locus coeruleus (Elam et al., 1986; Abercrombie and Jacobs, 1987). Since the method of restraint used in this study (immobilization by taping an animal to a board on its back) has been thought to be a psychological rather than a physical stressor (Chowdhury et al., 2000), we speculate that indirect activation through LC might be the dominant mechanism for CRF expression observed in this study. The inhibitory action of biting on stress-induced expression of CRF observed in this study was in line with the previous studies on the effects of biting that showed suppression of restraint-induced plasma corticosterone level and noradrenaline turnover in the rat brain (Tsuda et al., 1988), tail-pinch-induced dopamine metabolism in the striatum (Gomez et al., 1999), and cold-restraint-induced noradrenaline turnover in the hypothalamus and the limbic system (Tanaka et al., 1998). Therefore, we again speculate that suppression of central noradrenergic transmission, including the afferent pathway mediated by LC, might be the mechanism for suppression of CRF by biting. This anti-stress effect of the motor activity of the masticatory organ appears to be very important, and this mechanism might be unconsciously in operation during an exposure to physical and psychological stressors, reducing adverse effects of stress responses on the animal body.
This work was performed in the 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. The authors thank Dr. M. Kamei for her excellent technical assistance and are grateful to Drs. S. Sato, M. Toyoda, and P.R. Wade and the members of the Department of Oral Physiology, Kanagawa Dental College, for their helpful discussions on this study.
4 present address, World Health Organization, HTM/HIV, 20 Avenue Appia, Geneva 27, CH-1211, Switzerland;  Received for publication January 8, 2003. Revision received October 9, 2003. Accepted for publication November 4, 2003.
Journal of Dental Research, Vol. 83, No. 2,
124-128 (2004) This article has been cited by other articles:
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