Results Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol879.Issue1-2.2000:

S . Paterson et al. Brain Research 879 2000 148 –155 151 reaction was started by adding 25 ml of the sample supernatant to an assay tube containing 75 ml of 1 mM 3 NADPH, 0.75 mM CaCl and 150 nM [ H]- L -arginine. 2 Following incubation in a water bath for 1 min at 378C, the reaction was stopped by adding 1 ml of 1:1 v v Dowex 1 H O-50W 200–400, 8 cross-linked, Na form and 2 2 ml of 20 mM HEPES buffer containing 2 mM EDTA, pH 5.5 into the tube and transferring the tube onto the ice. After 10 min, 750 ml of the supernatant were removed and 3 added to 4.25 ml of scintillation fluid in a vial. [ H]- citrulline was quantified by liquid scintillation spectros- copy, using a Beckman scintillation counter. The counts- per-minute cpm for duplicate samples were averaged and corrected with respect to the blank control and background radioactivity. NOS activity was expressed as pmol min mg protein. Protein concentrations in the supernatant were measured based on the Bradford method [18] using a Bio-Rad protein assay dye reagent concentrate and spec- tramax microplate reader. Ten microlitres of supernatant were removed from the same tissue sample used for the NOS assay and added to 390 ml of distilled water in a 1.5 ml Eppendorf tube and vortexed. Ten microlitres of standard 0–500 mg ml protein or sample was then added to 190 ml of reagent that consisted of 40 ml of BioRad reagent and 150 ml of water in a 96 well tray. Protein formation was then assessed using spectrophotometry at a wavelength of 595 nm. The counts for duplicate samples were averaged. 2.6. Statistical analysis For SN, YHT and RHT, a single two-factor analysis of variance ANOVA with repeated measures on time was performed [22]. Factor A represented the drug effect on SN frequency, YHT or RHT; factor B, the repeated measure, represented time; and the interaction AB repre- sented the change in the rate of compensation as a result of treatment. As factor B, the repeated measure, was always significant i.e., since vestibular compensation occurred in all cases, it will not be discussed further. Pairwise comparisons were conducted, where appropriate, using the ´ Scheffe F-test; linear and exponential regression analyses were also used on the SN data [22]. The NOS activity data were analysed using one-way ANOVAs and post-hoc student Newman–Keuls multiple comparison tests [22]. The significance level was set at 0.05 for all comparisons.

3. Results

3.1. Behavioural results Post-UVD treatment with L -NAME, at any of the four Fig. 1. Compensation of spontaneous nystagmus, roll head tilt and yaw concentrations used, had no effect on the compensation of head tilt for animals receiving vehicle, 5 mM, 10 mM, 50 mM or 100 mM SN, YHT or RHT Fig. 1. By contrast, pre-UVD treatment L -NAME by s.c. osmotic minipump from the time of the UVD until 50 h with L -NAME resulted in a significant decrease in SN post-UVD. Symbols represent means, bars61 S.D. 152 S frequency P,0.05 and a change in the rate of its compensation P,0.0005 Fig. 2A. Post-hoc analysis showed that these significant effects were due to a large decrease in SN frequency in the 100 mM group at 10 h post-UVD compared to the other drug groups P,0.05 and to a change in the pattern of the decrease in SN over time compared to the vehicle group. Regression analysis revealed that the 100 mM L -NAME group showed a distinct exponential pattern of SN decrease over time compared to the vehicle controls P,0.001 Fig. 3. Pre-UVD L -NAME treatment also resulted in a significant increase in the overall magnitude of YHT using ANOVA P,0.005 Fig. 2C; however, post-hoc analysis failed to detect significant differences between L -NAME and ve- hicle groups at any particular time point. Pre-UVD L - NAME had no significant effect on RHT at any con- centration Fig. 2B. Fig. 3. A Regression analyses of the compensation of spontaneous nystagmus SN for animals receiving 100 mM L -NAME pre-UVD Fig. 2. Compensation of spontaneous nystagmus, roll head tilt and yaw compared to pre-UVD vehicle, showing the acceleration of SN compensa- head tilt for animals receiving vehicle, 5 mM, 10 mM, 50 mM or 100 mM tion relative to the vehicle group. B Mean SN frequency over 50 h L -NAME by s.c. osmotic minipump from 4 h pre-UVD until 50 h post-UVD for animals receiving vehicle, 5, 10, 50 or 100 mM L -NAME post-UVD. Symbols represent means, bars61 S.D. pre-UVD. S . Paterson et al. Brain Research 879 2000 148 –155 153 3.2. NOS assay results and NOS activity in the ipsilateral MVN PH was not significantly affected Fig. 4A. By contrast, in both the Fig. 4 shows that levels of NOS activity were generally contralateral and ipsilateral cerebellum and cortex, 50 and much lower in the MVN PH than in the cerebellum and 100 mM L -NAME resulted in a significant decrease in cortex, and this raised the possibility that even high NOS activity P,0.01 and P,0.001, respectively; Fig. 4B, concentrations of L -NAME might not result in a significant C. inhibition of NOS activity. In fact, amongst the pre-UVD L -NAME treatment groups, only 100 mM L -NAME re- sulted in a significant 72 decrease in NOS activity in

4. Discussion