Eight-day-old male Wistar rats were divided into two groups: saline-control (C) and morphine-treated (M). Naive animals were housed in home cages made of Plexiglas (65 cm × 25 cm × 15 cm) find more with sawdust covering the floor. Animals

were maintained on a standard 12-h dark/light cycle (lights on between 0700 h and 1900 h) at room temperature (22 ± 2 °C). The animals had free access to food and water. At birth, the litters were standardized to contain up to 8 pups per dam, and the pups remained with their mothers until 21 days of age. Rats at P8 were chosen because it is accepted that animals of this age are at a similar stage of neurological development to that of a human newborn (Fitzgerald and Anand, 1993). It is also accepted

that they are in a physiologically immature state (Pattinson and Fitzgerald, 2004) since this period is characterized by major developmental changes in the brain and plasticity of the Palbociclib supplier developing pain system (Bishop, 1982, Kim et al., 1996 and Rabinowicz et al., 1996). Animal handling and all experiments were performed in accordance with international guidelines for animal welfare. The protocol of this experimental study was approved by the Ethics Committee of the institution where the work was conducted. Each animal received saline (control group) or morphine (5 μg s.c. in the mid-scapular area; morphine group) starting at P8, then once a day for 7 days. This dose had been chosen based on a previous study by Rozisky et al., 2008 and Rozisky et al., 2010, and Reverse transcriptase it produced analgesia in all animals submitted to the tail-flick test. All treatments were administered at the same time each day (1100 h). One milliliter of morphine sulphate (Dimorf® 10 mg/ml, obtained from Cristália, Porto Alegre, Rio

Grande do Sul, Brazil) was diluted in 9 ml of 0.9% NaCl (saline). The formalin test was performed in 16-, 30-, and 60-day-old rats (Fig. 1). The number of animals used per group was 8 to 15. At the ages where we observed significant differences in the nociceptive behavior in the formalin test, the control and morphine groups were subdivided into four groups, each one designed to evaluate the effect of i.p. administration of an NMDA receptor antagonist or non-steroidal anti-inflammatory drug (NSAID), applied 30 min before the formalin test: (1) non-steroidal anti-inflammatory drug: 10 mg/kg of indomethacin (Indomethacin®, obtained from Sigma-Aldrich, São Paulo, Brazil) (Bastos et al., 2004) diluted in 1.29% sodium bicarbonate solution (control-indomethacin, morphine-indomethacin); (2) vehicle for indomethacin (vehicle I): 10 mg/kg of 1.29% sodium bicarbonate solution (pH = 7.4) (control-vehicle I, morphine-vehicle I); (3) NMDA receptor antagonist: 30 mg/kg of ketamine (Cetamine®, obtained from Hospital de Clínicas de Porto Alegre, Brazil) (Campos et al., 2006) diluted in 0.