Microanalysis of amino acids in small regions of the rat brain using dansyl chloride.

Ion exchange chromatographic methods have been used by several investigators to determine the con­ centration of free amino acids in the rat brain1“3. While this method is advantageous in that it allows the concentration of the individual substance to be accurately estimated, it has limited application when it is necessary to determine the concentrations of amino acids in very small regions of the rodent brain. For this reason, the microanalytical proce­ dure for the estimation of amino acids in biological material using dansyl chloride4>5 is of particular interest. The present investigation was undertaken to determine the relative concentrations of the major amino acids in seven regions of the adult rat brain. Male albino rats (180 —200 g) were killed by decapitation. The brains were rapidly removed, placed on ice and dissected into the cortex, hippo­ campus, striatum, thalamus, hypothalamus, pons and “mid-brain” (tegmentum and colliculus). Im­ mediately following dissection, the brain areas were frozen on solid carbon dioxide. Using a metal punch of the type described by Neuhoff °, a small piece of each brain area weighing approximately 1 mg was removed and homogenized in 10 —20//I of 0.05 m sodium bicarbonate solution (pH 10). Following the dansylation of the amino acids in the super­ natant with [14C] dansyl chloride, the amino acids were separated by 2 dimensional chromatography on 3 cm x 3 cm polyamide sheets. After visualization of the dansylated products by ultraviolet light, the spots were marked, scrapped off and transferred to scintillation vials containing 10 ml liquid scintilla­ tor. The details of this method have been published elsewhere 45. In these experiments only those amino acids which were clearly separated were estimated; some 15 dansyl positive substances were determined in this way. The results are shown in Fig. 1. In the two di­ mensional systems, used in this study, glutamine and asparagine, glutamate and aspartate could not be