The dorsal raphe nucleus (DR), contains 60% serotonergic neurons, which appears to be involved in the control of endogenous pain sensation. MK-801, a non-competitive antagonist of N-methyl-D-aspartate receptors, inhibits spinal nociceptive neurons, however, little information is available about the effect of its administration on the DR. To evaluate the distribution of c-Fos and serotonin expression in the DR following experimental tooth movement in the rat upper molar and/or administration of MK-801. Fifty-six Wistar strain male rats underwent experimental tooth movement according to the Waldo’s method. Of all the rats, 28 were administrated MK-801 (3 mg/kg) intraperitoneally before initiating the experimental tooth movement (n=14). These animals were sacrificed 2 and 12 h after the experimental tooth movement. The sections including DR were dissected, and subjected to immunohistochemical staining with c-Fos and serotonin antibodies. In addition, 28 anesthesia control animals with or without administration of MK-801 were used as the controls. In the DR, the numbers of singlelabeled (c-Fos) and double-labeled (c-Fos/serotonin) neurons were increased at 2 h and decreased by 12 h as compared with the controls. MK-801 decreased the numbers of double-labeled neurons at 2 h to the control level. The present results suggest that although blockage of NMDA receptors decreases neuronal activity with in the DR during experimental tooth movement, serotonergic DR neurons are not involved in the MK801-induced analgesia.

NMDA in the DR, we investigated the effect of NMDA receptor antagonist on serotonergic DR neurons using immunohistochemical techniques following a noxious stimulation from experimental tooth movement. The aims of the present study were to evaluate, with a double labeling method, the induction of Fos-LI serotonergic neurons in the DR following experimental tooth movement and to examine the expression of serotonergic DR neurons in-duced by an intraperitoneal MK-801 administration.

For all the groups, the rats were deeply reanesthetized with an overdose of sodium pentobarbital, and perfused intracardially with 200 ml of normal saline, followed by a fixative of 500 ml containing 4% formaldehyde in 0.1 M sodium phosphate buffer (pH 7.2). The mid-brain was dissected, trimmed into blocks and immersed in the same fixative overnight, and then placed in 0.01 M phosphate-buffered saline (PBS) containing 25% sucrose at 4°C. The frozen blocks were cut into serial coronal sections of 50 μm thickness with a cryostat (Leica, Wetzlar, Germany) and stained with a double immunohistochemical staining for c-Fos and 5-HT. For the detection of c-Fos and 5-HT immunoreactivity, sheep polyclonal anti-c-Fos antibody and rabbit polyclonal anti-5-HT antibody were used as primary antibody, respectively. All the sections were rinsed in 0.01 M PBS, pre-incubated in absolute methanol plus 0.3% H2O2 to block endogenous peroxidase activity for 30 min, and then washed with PBS. The sections were incubated with sheep anti-c-fos antibody (Oncogene Research Products, Cambridge, USA) overnight at 4°C. Then, the sections were incubated sequentially with bioti-nylated donkey anti-sheep IgG (1:400; Vector, Burlin-game, USA) and then with avidin-biotin peroxidase complex (Dako, Glastrup, Denmark). The product of peroxidase activity was detected by using 0.04% 3,3’- diamino-benzidine, 0.1% ammonium nickel sulfate, and 0.01% hydrogen peroxidase in 0.05 M Tris-HCl buffer (pH 7.6). After washing in 0.01 M PBS, the sections were incubated with rabbit anti-5-HT antibody (Oncogene Research Products, Cambridge, USA) overnight at 4°C. Then, the sections were sequentially incubated with biotinylated goat anti-rabbit IgG (1:400; Vector, Burlingame, USA) and avidin-biotin peroxidase complex (Dako, Glastrup, Denmark). The product of peroxidase activity was detected by using Vector-VIP® in 0.01 M PBS. For each animal, the numbers of Fos-LI and Fos/5-HT-LI neurons were counted on both sides of the DR on four serial sections at an interval of 0.5 mm distance. These four levels range from 8.8 to 7.3 mm posterior to the Bregma. The stereotaxic coordinates in millimeters were taken from the atlas of Paxinos and Watson using Bregma level. The average numbers of Fos-LI and Fos/5-HT-LI neurons per section were recorded for each Bregma level. ANOVA was used to determine whether statistically significant differences in the numbers of Fos-LI and Fos/5-HT-LI neurons existed between experimental and sham control groups. The differences in these values were examined with a Scheffé’s test.

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