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  • Stress is usually comprehended as an


    Stress is usually comprehended as an event affecting mainly the HPA axis and initiating the alarm reaction represented by activation of the adrenal medulla. This means that the levels of related hormones and neurotransmitters are significantly elevated during and after the stress. CRF, following its discovery 30years ago, has been postulated to mediate both hormonal and behavioral responses to stressors. It has been well documented that CRF mRNA was increased after immobilization stress in PVN (Bartanusz et al., 1993, Harbuz and Lightman, 1989, Harbuz et al., 1991, Harbuz et al., 1993, Kiss et al., 1996, Pacak et al., 1996) and amygdala in rats (Kalin et al., 1994, Mamalaki et al., 1992). CRF receptor mRNA expression in rats was also significantly increased after exposure to acute immobilization stress. 120min restraint significantly increased CRF1 receptor and CRF mRNA signals in the PVN examined by in situ hybridization (Imaki et al., 1996). CRF receptor mRNA hybridization in rats became evident 2h after the initiation of acute immobilization, with levels substantially increasing from 2 to 4h, decreasing at 8h and disappearing by 24h (Luo et al., 1994). This is in line with an observation from Bonaz and Rivest (1998) that exposure to a 90-min immobilization stress induced a robust increase in CRF1 receptor mRNA expression in rats, which reached a peak level at 3h after the cessation of immobilization and decreased gradually from 3 to 12h. In contrast to the extensive research on the mRNA expression of CRF and CRF1 receptors after immobilization stress in rats, the expression of CRF and CRF1 receptors in mice has seldom been investigated. Two studies reported an increase in CRH mRNA, not CRF1 mRNA, in the PVN after 2 or 3h restraint in mice by in situ hybridization (Imaki et al., 2003, Makino et al., 2005). By using quantitative real-time RT-PCR, we detected a pronounced increase in CRF mRNA expression in the cortex, hippocampus and hypothalamus after 30-min immobilization and the slight enhancement of the CRF1 mRNA expression in these WEHI-539 hydrochloride and regions, which was confirmed by the Western blot analysis for the CRF1 protein levels. The enhancement of the CRF1 receptor diminished in stress30 group mice. This disagreement of our results with previous reports could be explained by the different mRNA detection technique, stress time and type used. We chose 30-min immobilization as the acute stressor in this experiment instead of restraint. The restraint and immobilization stressors differ in the degree of movement restriction. In immobilization, all limbs are taped to a Plexiglas surface and the animals show increased exercise as they want to escape from that inconvenient position. In restraint, the animals are placed into a tube where their movement is impossible. Although there are clear similarities between these types of stressors, the effects on the animals sometimes are different. CCK is another target neurotransmitter in our experiment. A large body of evidence, both in rodents and in human, supports the idea that central cholecystokininergic neurones play a role in the expression and control of anxiety-related behaviors (van Megen et al., 1996). Nevo et al. (1996) showed that a 30-min immobilization produced a marked but transient increase in cortical CCK-like material release in rats, thus providing the direct biochemical evidence in support of the activation of cortical CCK-ergic neurotransmission in relation with immobilization stress and anxiety-related behavior. Later, Giardino et al. (1999) demonstrated that restraint stress alone increased CCK mRNA levels in rats in the hippocampus, whereas no changes were found in the cerebral cortex, amygdaloid complex and thalamus. A study conducted in jerboas by Barakat et al. (2006) indicated that the number of CCK-immunoreactive neurones within the PVN was significantly increased (138%) in stressed animals compared to controls. Similarly, the number of CRH-containing neurones was higher in stressed jerboas (128%) compared to controls. This evidence suggested that in addition to CRF, CCK is another neuropeptide involved in the response to stress, acting by controlling HPA axis activity.