Hippocampus, striatum and cerebellum were dissected from one hemisphere under microscope between 2 and 4 h after the last cycle of alcohol exposure, weighed and snap frozen at −80°C. Hippocampus and striatum samples were each homogenized in 250 μL 0.1 M perchloric acid using an immersion hand disperser (Polytron PT 1200 E, Kinematica Inc., Keyland Court Bohemia, NY, USA). Cerebellum samples were homogenized with the same method in 500 μL milliQ water. Samples were then centrifuged at 14000 rpm at 4°C for 20 min and then supernatant collected using a 0.22 μm filter (Advantec, Sierra Court, CA, USA, 13CP020AS).
The concentration of the cations sodium (Na+), magnesium (Mg++), potassium (K+), and calcium (Ca++) in the cerebellum were determined by ion chromatography (IC, Dionex Aquion 1100, Thermo Fisher Scientific, Waltham, MA, USA). The cation chromatography consisted of a CS12A 4 mm analytical and a Dionex IonPac CG12A 4 mm guard column set. 20 mmol/L methanesulfonic acid was the eluent and was sonicated for 20 min followed by degassing with nitrogen for an additional 10 min prior to IC. 10 μl of sample were injected, was eluted for 20 min with isocratic 20 mmol/L methanesulfonic acid. Chromeleon Chromatography Data System software (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze and calculate the peaks.
The concentrations of norepinephrine (NE), 3,4-dihydroxy- phenylacetic acid (DOPAC), dopamine (DA), 5-hydroxy- indoleacetic acid (5-HIAA), homovanillic Acid (HVA) and serotonin (5-HT) in the striatum and hippocampus were determined by HPLC with electrochemical detection. The monoamines were separated by reverse-phase liquid chromatography with a Prodigy C18 column (DA 2 × 100 mm, particle size 3 μm, Phenomenex, YMC Europe, Schermbeck, Germany). The mobile phase (55 mM sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na2EDTA and 8% acetonitrile, adjusted to pH 3.7 with 0.1 M acetic acid) was de-gassed with an online de-gasser. 10 μl of sample were injected with a flow rate of 0.15 mL/min. Electrochemical detection was accomplished using an amperometric detector Antec Decade (Antec Scientific, Alphen aan den Rijn, Netherlands) with a glassy carbon electrode set at 0.8 V and an Ag/AgCl reference electrode. The output was recorded, and peak areas were calculated by LC solution software (Shimadzu, Kyoto, Japan).
The concentration of the cations sodium (Na+), magnesium (Mg++), potassium (K+), and calcium (Ca++) in the cerebellum were determined by ion chromatography (IC, Dionex Aquion 1100, Thermo Fisher Scientific, Waltham, MA, USA). The cation chromatography consisted of a CS12A 4 mm analytical and a Dionex IonPac CG12A 4 mm guard column set. 20 mmol/L methanesulfonic acid was the eluent and was sonicated for 20 min followed by degassing with nitrogen for an additional 10 min prior to IC. 10 μl of sample were injected, was eluted for 20 min with isocratic 20 mmol/L methanesulfonic acid. Chromeleon Chromatography Data System software (Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze and calculate the peaks.
The concentrations of norepinephrine (NE), 3,4-dihydroxy- phenylacetic acid (DOPAC), dopamine (DA), 5-hydroxy- indoleacetic acid (5-HIAA), homovanillic Acid (HVA) and serotonin (5-HT) in the striatum and hippocampus were determined by HPLC with electrochemical detection. The monoamines were separated by reverse-phase liquid chromatography with a Prodigy C18 column (DA 2 × 100 mm, particle size 3 μm, Phenomenex, YMC Europe, Schermbeck, Germany). The mobile phase (55 mM sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na2EDTA and 8% acetonitrile, adjusted to pH 3.7 with 0.1 M acetic acid) was de-gassed with an online de-gasser. 10 μl of sample were injected with a flow rate of 0.15 mL/min. Electrochemical detection was accomplished using an amperometric detector Antec Decade (Antec Scientific, Alphen aan den Rijn, Netherlands) with a glassy carbon electrode set at 0.8 V and an Ag/AgCl reference electrode. The output was recorded, and peak areas were calculated by LC solution software (Shimadzu, Kyoto, Japan).
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