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To establish whether abnormal behavioral activities were related to neuronal damage, fish treated in the same manner as in the behavioral study with sublethal (n = 3) and MAT (n = 3) concentrations of Cd and endosulfan were decapitated and their brains quickly removed (within 30 sec) and stored at ?cuatro0°C according to common cryostat procedures for unfixed brains (Canonaco et al. 1997). Brains were mounted on a freezing stage of a sliding cryostat (Microm-HM505E; Zeiss, Wallford, Germany), and a serial set of representative coronal sections (30 ?m) was selected at an interval of 240 ?m for amino cupric silver staining procedures according to previously published methods (de Olmos et al. 1994); we adapted the exposition time (25 min) to neutral red for the different brain sections of our fish species. This method, which has been used mainly for mammalian brain studies, proved to be appropriate for cerebral neuronal fields that have undergone degeneration processes. 2O, placed into dishes containing the preimpregnating solution (silver nitrate [AgNO3], distilled H2O, D,L-alanine, copper nitrate [Cu(NO3)2], cadmium nitrate [Cd(NO3)2], lanthanum nitrate [La(NO3)2], neutral red, pyridine triethanolamine, isopropanol), heated in a microwave oven (45–50°C) for 50 min, and cooled at room temperature for 3 hr. The sections were then rinsed in distilled H2O, and after a quick rinse in acetone they were placed in an impregnating solution AgNO3, distilled H2O, ethanol, acetone, lithium hydroxide (LiOH), ammonium hydroxide (NH4OH)] for 50 min, followed by a 25-min fixation in a reducer solution (formalin, citric acid monohydrate, ethanol, distilled H2O) at a temperature range of 32–35°C. These sections were left overnight in distilled H2O, and the next day they were placed in a first bleaching solution [potassium ferricyanide in potassium chlorate solution, lactic acid] for 60 sec at room temperature. Afterward, they were bleached in a second bleaching solution (potassium permanganate, sulfuric acid) for 60 sec and rinsed in distilled H2O. For the stabilization phase, sections were transferred in sodium thiosulfate solution and rinsed again in distilled H2O. Finally, they were immersed in a rapid fixer solution for 5 min and counterstained with 0.5% neutral red solution (Carlo Erba, Milan, Italy) for livelinks promo kodlarÄ± 25 min, dehydrated in ethanol (50–100%) and xylene, and mounted with DPX (p-xylene-bis[N-pyridinium bromide]; Sigma) for observations with a bright-field Dialux EB 20 microscope (Leitz, Stuttgart, Germany).
Effects of Cd and you will endosulfan to the H1R–H3R.
The neurotoxic actions of Cd and endosulfan were also correlated with the type of distribution pattern of the HnR neuronal system. Fish treated with the sublethal (n = 4) and MAT (n = 4) concentrations of Cd and endosulfan along with their control (n = 6) were used. The brains were removed and quickly frozen for storage at ?40°C, and brain sections (14 ?m thick) were thawed, dried at room temperature, and then handled according to in vitro binding studies for mammals (Ryu et al. 1996) that were adapted for fish brain sections (Peitsaro et al. 2000). Briefly, we incubated sections in 150 mM sodium potassium phosphate buffer (Sigma) 2 mM MgCl2 and 100 ?M dithiothreitol pH 7.4 (Roche Diagnostic, Milan, Italy) containing different concentrations (0.5–20 nM) of [ 3 H]-N-?-methyl-HA (NAMH; PerkinElmer Life Sciences, Boston, MA, USA). Some sections were incubated with 10 nM [ 3 H]-NAMH using a wipe assay procedure. This concentration displayed the greatest affinity for HnR in the presence of the different values (1 ?M–1 nM) of the following specific HA antagonists (Sigma): H1R antagonist pyrilamine, H2R antagonist cimetidine, and H3R antagonist thioperamide. Other sections were incubated with 10 nM [ 3 H]-NAMH plus 500 ?M of their corresponding antagonist for nonspecific binding values that proved to be similar to that of the background; subsequently an autoradiographic film (Hyperfilm; Amersham, Piscataway, NJ, USA) was apposed to dried sections and to slides containing plastic standards.