The spectra were obtained in
the continuous acquisition mode scanning from m/z 50 to 3000 at a scan time of 10 s. The acquisition and treatment of data were performed with MassLynx software (Micromass, Altrincham). The HRMS analyses were carried out in an ultrOTOF-Q-ESI-TOF (Bruker Daltonics, Billerica, MA, USA). The instrument was externally and internally calibrated using PARP inhibitor trial a 10 mg/mL Na+-TFA solution and by setting the instrument with the following parameters: end plate voltage of 3500 V; capillary voltage of 4000 V; capillary exit voltage of 300 V; skimmer-1 and skimmer-2 voltages of 1:50 V and 1:25 V, respectively. The NMR spectra were recorded at 25 °C on a Bruker DRX 500 operating at 500.11 MHz for 1H and 125.08 MHz for 13C. Measurements were carried out at a probe temperature of 300 K using sample concentrations of 500 μg/cm3 in D2O. Spectra were obtained for about 3 mg of compound in 0.7 mL of Selleckchem PD-L1 inhibitor solution in D2O, which was used as a D lock. The samples were filtered
to obtain better digital resolution. TMS was used as a reference for 1H and 13C. The H2O signal was partially suppressed by applying a presaturation sequence. 1H, 13C, DEPT, and two-dimensional gCOSY, gHSQC and gHMBC spectra were obtained. The signal for the remaining H2O was partially suppressed by applying a presaturation sequence (Braun et al., 1998). The method employed was performed as described previously (Gerfen and Sawchenko, 1984, Shu et al., 1988, Sita et al., 2003 and Cesar et al., 2005). Normal adult male Wistar rats weighing 250–300 g were housed two per cage with food and water ad libitum in a temperature controlled (21 ± 2 °C) room on a 12 h light–dark cycle from one week prior to experimentation to allow them to acclimate to their new environment. All experiments were carried out in accordance with the guidelines of the Institutional Committee for Research and Animal Care of the University of São Paulo and the National Institutes of Health Guide for the Care and Use of Laboratory Animals ( National Academy of Sciences, 1996). The guide cannula was implanted in the lateral ventricle (AP = −0.4; ML = −1.4;
DV = −3.4) under anaesthetic action of a Orotidine 5′-phosphate decarboxylase cocktail (0.2 mL/100 g) containing ketamine (1 mg), xylazine (5 mg), and acepromazine (0.2 mg) seven days before the application of nigriventrine. The animals were manipulated twice a day for 10 min to avoid stress on the day of the experiment. The injection cannula was introduced approximately 2 h before the experiment to acclimate the animals and to minimise stress. Nigriventrine was solubilised in 10 μL of saline (0.9% w/v), and the compound was injected by intracerebroventricular (ICV) administration at a concentration of 1 ng/μL. The control group (n = 6) received only vehicle injection (saline: 0.9% w/v) to compare the effects of nigriventrine ICV administered in vehicle. Two hours were necessary for effective c-Fos induction.