Immediately after the last behavioral test, the animals were placed individually for 10 min in a glass dish containing an ice water immersion (2–4 °C), then rapidly euthanized, as previously reported by Brinza et al. [34 (link)]. Whole brains were carefully excised, weighed, and placed in 0.5 mL tubes, and stored at −20 °C until further use.
The next day, the brains of fish from the same batch of animals were weighed individually (~3–6 mg) and homogenized in extraction phosphate buffer solution (0.1 M potassium phosphate buffer solution, pH 7.4 with KCl 1.15%) at a ratio of 1 to 10 using a ball mill (Mikro-Dismembrator U; Sartorius, NY, USA). The homogenate was centrifuged (15 min at 14,000 rpm) and the supernatant was later used for the determination of biochemical parameters.
The next day, the brains of fish from the same batch of animals were weighed individually (~3–6 mg) and homogenized in extraction phosphate buffer solution (0.1 M potassium phosphate buffer solution, pH 7.4 with KCl 1.15%) at a ratio of 1 to 10 using a ball mill (Mikro-Dismembrator U; Sartorius, NY, USA). The homogenate was centrifuged (15 min at 14,000 rpm) and the supernatant was later used for the determination of biochemical parameters.
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