Tetraisopropyl pyrophosphoramide

This assay is based on a previously described method^{68 (link), 69 (link)} with some alterations. 5,5-Dithiobis-2-nitrobenzoic acid (final concentration 0.6 mM), acetylthiocholine iodide (final concentration 1.5 mM), BW284c51 (final concentration 0.1 mM), tetra-isopropyl pyrophosphoramide (final concentration 0.1 mM) and AChE from Electrophorus electricus were purchased from Sigma-Aldrich. BW284c51 was used for AChE inhibition and tetra-isopropyl pyrophosphoramide for BChE inhibition. The assay was performed in 96-well plates with a total volume of 250 μl. Change in absorbance at 405 nm was measured in a microplate reader (BMG labtech, Ortenberg, Germany) for 8 min. FFs were used in a final dilution of 1/320. Lysates of GCs cells cultured in serum-free medium were frozen at −20 °C, thawed and washed two times in Ellman buffer and used in a 1/8 dilution for activity measurements. Absolute activity values were determined by comparing with an AChE standard (Electrophorus electricus).

To evaluate the enzymatic activity of AChE, the modified version of the method of Ellman et al. was used [38 (link)]. In brief, 20-μl triplicate samples were mixed with a reaction mixture (0.2 mM 5, 5′-dithiobis (2-nitrobenzoic acid) [Sigma-Aldrich], 0.56 mM acetylthiocholine iodide [Sigma-Aldrich], 10 μM tetraisopropyl pyrophosphoramide [Sigma-Aldrich], and 39 mM phosphate buffer; pH 7.2) at 37 °C for 30 min. The quantification of optical density was performed at a wavelength of 405 nm.

Immediately after the behavioral test, 5 out of 8 rats from the normal group, 4 out of 8 rats from the lesion group, 5 out of 9 rats from the pre-stimulation group, 4 out of 9 rats from the training group, and 3 out of 7 rats from the probe group were anesthetized and the brains were quickly removed to acquire proteins. The frontal cortex (FC, including the cingulate cortex and prelimbic cortex), MS, diagonal band (DB) and hippocampus were dissected with fine forceps from 1 mm thick coronal brain slices. The tissues were homogenized in lysis buffer (Intron, Seongnam, Korea) on ice for 30 min and then centrifuged for 20 min at 12,000 rpm. The protein in the supernatant was measured using the bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL, USA). The protein samples were stored at −70 °C until analysis. The activity of AChE was determined using the method of Ellman et al. [12 (link)] with some modifications as previously described. In brief, 20 µl triplicate samples were mixed with the reaction mixture of 0.2 mM dithiobisnitrobenzoic acid (Sigma, Louis, MO, USA), 0.56 mM acetylthiocholine iodide (Sigma), 10 µM tetraisopropylpyrophosphoramide (Sigma), and 39 mM phosphate buffer (pH 7.2) at 37 °C for 30 min. The optical density was measured at 405 nm.

One hour following the final CPF dosing on PND 4, pups were euthanized by decapitation and blood was collected by cardiac puncture into tubes containing EDTA as an anti-coagulant (Becton-Dickinson, Franklin Lakes, NJ). Blood was diluted 1:25 with phosphate buffer with 0.03% Triton X-100 (Fisher Scientific, Pittsburg, PA), vortexed, and snap frozen for later analysis. Brains were collected and snap frozen for later analysis. For the AChE activity assay, brain tissue was thawed on ice, homogenized in phosphate buffer with 1% Triton X-100, and AChE activity quantified using the standard Ellman Assay [39 (link)] with 5,5′-dithio-bis-2-nitrobenzoic acid (DTMB) and acetylthiocholine iodide (ASChI) as the substrates (Sigma-Aldrich, St. Louis, MO). Tetraisopropyl pyrophosphoramide (Sigma) was included to inhibit pseudocholinesterase. Blood AChE activity was normalized to hemoglobin levels, which were determined using a StanBio Laboratory Stat-Site M hemoglobin meter and test strips (Boerne, TX, USA). Brain AChE activity was normalized to protein concentration as determined using the BCA assay kit (Pierce, Rockford, IL).

Drugs and chemicals were obtained from the following sources: chlorpyrifos oxon (CPO; Chem Service, Inc., West Chester, PA, USA), DFP (Sigma‐Aldrich Co., St. Louis, MO, USA), PB (Sigma‐Aldrich Co.), physostigmine (PHY; Sigma‐Aldrich Co.), ethanol (Sigma‐Aldrich Co.), CORT (Steraloids Inc., Newport, RI, USA), 5,5‐dithio‐bis‐(2‐nitrobenzoic acid) (Sigma‐Aldrich Co.), tetraisopropyl pyrophosphoramide (Sigma‐Aldrich Co.), and acetylthiocholine iodide (Sigma‐Aldrich Co.). Rabbit Anti‐phospho STAT3^tyr705 antibodies were obtained from Cell Signaling, Inc. (RRID: AB_621843; Beverly, MA, USA). The materials used in glial fibrillary acidic protein (GFAP) ELISA previously have been described in detail (O'Callaghan et al. 1991; O'Callaghan 2002). Material used for additional tissue analyses were of at least analytical grade and purchased from various commercial sources.

AChE enzymatic activity was determined according to the method of Ellman et al. (1961 (link)) with the modification of adding 0.1 mM tetraiso-propylpyrophosphoramide (Sigma) for 5 min, an inhibitor of butyrylcholinesterase activity, to each reaction. Samples were then added to the reaction mixture containing 0.625 mM acetylthiocholine iodide (Sigma) and 0.5 mM 5,5-dithiobis-2-nitrobenzoic acid (Sigma) in 80 mM Na₂HPO₄, pH 7.4. For determining the catalytic reaction, the increase in absorbance at 412 nm was recorded, and the specific enzyme activity was expressed as absorbance units/min/g of protein. Protein concentrations were measured throughout by the method of Bradford (1976 (link)).