3 4 dihydroxybenzylamine

Monobasic sodium phosphate, disodium ethylenediamine tetraacetate (Na₂EDTA), 85% o-phosphoric acid, acetonitrile, methanol, hydrochloric acid (HCl), sodium hydroxide (NaOH), and 0.3 μm alumina powder were obtained from Fisher Scientific (Pittsburgh, PA). Ammonium acetate, 1-octanesulfonic acid [sodium salt] (SOS), sodium tetraborate decahydrate, boric acid, lithium tetraborate, lithium dodecyl sulfate (LDS), tetradecyltrimethylammonium bromide (TTAB), β-alanine (β-Ala), sodium cyanide, 4-hydroxybenzoic acid (4-HBA), L-glutamic acid (Glu), L-aspartic acid (Asp), L-arginine (Arg), γ-amino-n-butyric acid (GABA), DL-2-aminoadipic acid (AAP), sodium cyanide, 3,4-dihydroxybenzylamine (DHBA), 3,4-dihydroxyphenethylamine hydrochloride (DA), L-arterenol (NE), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) were obtained from Sigma-Aldrich (St. Louis, MO). Naphthalene-2,3-dicarboxaldehyde (NDA) was obtained from Invitrogen (Carlsbad, CA). All solutions were prepared in 18.2 MΩ distilled, deionized water (Labconco, Kansas City, MO) and filtered through 0.22 μm pore size membrane filters prior to use unless otherwise noted.

In adolescence and adulthood (PN30, PN45, PN75), animals were decapitated, and the forebrain was separated from the midbrain and brainstem by a cut rostral to the thalamus, after which the hippocampus and striatum were dissected away from the forebrain to isolate the cerebral cortex. For this study, we utilized the cerebral cortex, midbrain, brainstem and, at one age point, cerebellum. Brain regions were frozen in liquid nitrogen and stored at −45°C. We utilized 6 males and 6 females for each treatment group at each age. Based on our earlier work with similar designs, these are adequate sample sizes to detect treatment effects and treatment interactions with the other factors across multiple ages and regions (Kreider et al. 2006 (link); Slotkin et al. 2006 (link)).
For norepinephrine determinations, tissues were thawed on ice and deproteinized by homogenization in 0.1 N perchloric acid containing 3,4-dihydroxybenzylamine (Sigma-Aldrich) as an internal standard. Homogenates were sedimented at 26,000 × g for 20 minutes, the supernatant solutions were decanted, and norepinephrine was then trace-enriched by alumina adsorption, separated by reverse-phase high performance liquid chromatography and quantitated by electrochemical detection (Seidler and Slotkin 1981 (link)); values were corrected for recovery of the internal standard.

The samples were thawed on ice and deproteinized by homogenization in 0.1 N perchloric acid containing 3,4-dihydroxybenzylamine (Sigma) as an internal standard. Homogenates were sedimented at 26,000 × g for 20 minutes, the supernatant solutions were decanted, and norepinephrine was then trace-enriched by alumina adsorption, separated by reverse-phase high performance liquid chromatography and quantitated by electrochemical detection (Seidler and Slotkin, 1981 (link)); values were corrected for recovery of the internal standard.