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In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining. In contrast, SC-35- and NeuN-signals (markers of the nuclear speckles) showed a high overlap with DAGLα within specific subdomains of the nuclear matrix. Among the members of the phospholipase C-β (PLCβ) family, PLCβ1, PLCβ2, and PLCβ4 exhibited the same distribution with respect to chromatin, lamin B1, SC-35, and NeuN as that described for DAGLα. Furthermore, by quantifying the basal levels of 2-arachidonoylglycerol (2-AG) by liquid chromatography and mass spectrometry (LC-MS), and by characterizing the pharmacology of its accumulation, we describe the presence of a mechanism for 2-AG production, and its PLCβ/DAGLα-dependent biosynthesis in isolated nuclei. These results extend our knowledge about subcellular distribution of neuronal DAGLα, providing biochemical grounds to hypothesize a role for 2-AG locally produced within the neuronal nucleus.

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A carbon ®ber microelectrode was modi®ed by electrodeposition of a polymeric coating of poly(3-methylthiophene) (P3MT) by cycling the potential between 0.0 and 1.7 V (vs. AgaAgCl). The electrochemical reduction of the herbicide metamitron in this kind of microelectrode was studied by applying several voltammetric techniques like cyclic voltammetry and square wave voltammetry. The cathodic stripping voltammetric response for metamitron was found to be dependent on the ®lm thickness, the accumulation time and on the pH of measurement medium. A good linear relationship between the cathodic stripping peak current and metamitron concentration was obtained over the range 1.0610, using a carbon ®ber microelectrode modi®ed with 15 coatings and 6 min preconcentration time at open circuit. The reproducibility of the measurements was satisfactory, RSD of 0.96 % (n 10) and the detectability achieved was of 2.83610 710 mol L 71.