Abstract— Freeze-blowing (Veechet al. 1973), focussed microwave irradiation (Stavinohaet al. 1973) and immersion in liquid nitrogen were compared as methods for stopping metabolism in order to assay in vivo levels of intermediary metabolites in developing rat brain. Freeze-blowing was superior at all ages (5. 10, 15 and 20 days post-natal). The differences between this method and immersion in liquid nitrogen were quite small in the youngest rats and increased with age. reflecting the increased time needed to freeze larger brains. Brains frozen by immersion in liquid nitrogen showed evidence of increased anaerobic metabolism, with increased fructose 1.6-diphosphate. dihydroxyacetone phosphate and lactate and decreased glucose 6-phosphate and creatine phosphate concentrations. When brain metabolism was stopped by microwave irradiation there were many differences from freeze-blown brain. Increases in fructose 1.6-diphosphate. dihydroxyacetone phosphate, ADP and AMP, and decreased in ATP and creatine phosphate were especially striking. The differences between microwave irradiation and freeze-blowing were not attributable simply to anoxia. Rather, the changes produced by this method seem to reflect the different thermal characteristics of the various enzymes which must be denatured to stop metabolism of the substrates measured. Unlike freezing in liquid nitrogen, the efficacy of microwave irradiation was not a simple function of head size, in that better results were achieved with 15- and 20-day-old than 5- or 10-day-old rats.

Many glycolytic and Krebs cycle intermediates, as well as glutamate and aspartate, progressively increased over the course of development. The reasons for these increases are uncertain but are probably-related to the concomitant rises in rates of glycolysis and oxidative phosphorylation in brain.