A sequential double-label 14C- and 3H-2-DG technique: validation by double-dissociation of functional states.

We investigated a double-label 2-DG protocol and method of analysis in which sequential injections of 3H- and 14C-2-DG were used to map brain metabolism during two distinct experimental treatments in the same animal. In initial studies, brain sections from rats given only 3H-2-DG or only 14C-2-DG were exposed on Ultrofilm and on X-ray film with an interposed sheet of mylar (X-ray/mylar). These studies were needed to determine whether, at the 50:1 3H:14C dose ratio used, 3H-2-DG uptake would be revealed only in Ultrofilm images and 14C-2-DG uptake only in X-ray/mylar images. We found that X-ray/mylar images indeed showed only 14C-2-DG uptake as 3H emissions were blocked by the protective coating of the film and the mylar. By contrast, Ultrofilm autoradiograms showed the 2-DG uptake pattern for both the 14C-2-DG and 3H-2-DG cases. We then examined autoradiograms from double-label cases in which 14C-2-DG and 3H-2-DG were sequentially given using a 100:1 3H:14C dose ratio, with a different treatment following each injection. As predicted from the single-label cases, activity in the X-ray/mylar images corresponded to the treatment that followed the 14C-2-DG injection, while the Ultrofilm images reflected both treatments and thus were not veridical representations of 3H label. This paper provides a solution to the contamination of Ultrofilm by 14C label in that we devised a subtraction algorithm using a computerized imaging system which removes the contaminating 14C from the Ultrofilm image, leaving a 'Difference' image of 3H-2-DG uptake. Difference images revealed activity consistent with the treatment that followed the 3H-2-DG injection. Thus, the X-ray/mylar and difference images separately indexed metabolic activity for two different functional states in the same subject. By allowing a subject to serve as its own control, this double-label method greatly increases the applicability and power of the 2-DG method.