A method is presented for measuring rapid changes in the rate of glucose phosphorylation in mouse brain with nonradioactive 2-deoxyglucose (DG). After times as short as 1 min after DG injection, the mouse is frozen rapidly, and selected brain regions are analyzed enzymatically for DG, 2-deoxyglucose 6-phosphate (DG6P), and glucose. The rate of glucose phosphorylation can be directly calculated from the rate of change in DG6P, the average levels of DG and glucose, and a constant derived from direct comparison of the rate of changes in glucose and DG6P after decapitation. Experiments with large brain samples provided evidence for a 2% per min loss of DG6P and at least two compartments differing in their rates of glucose metabolism, one rapidly entered by DG with glucose phosphorylation almost double that of average brain and another more slowly entered with a much lower phosphorylation rate. The method is illustrated by changes in phosphorylation within 2 min after injection of a convulsant or an anesthetic and over a 48-min time course with and without anesthesia. The sensitivity of the analytical methods can be amplified as much as desired by enzymatic cycling. Consequently, the method is applicable to very small brain samrles. (1) to map patterns of neural activity in a wide variety of physiological and drug-induced states. The number of studies that have used this method shows the great demand for information about regional brain activity. Nevertheless, as useful as the method has proven to be, it has one basic limitation, i.e., the necessary 30-to 45-min lag period between DG injection and brain fixation. The lag is needed to allow free DG to largely dissipate, since it is the 2-deoxyglucose 6-phosphate (DG6P) accumulation that is the index of glucose phosphorylation and hence of its metabolism. Many investigators are interested in brain events that take place in a much shorter time frame. This paper presents a DG procedure with temporal resolution of a minute or less. The method depends upon direct measurement of DG, DG6P, and glucose without physical separation (2). The sensitivity is sufficient to assay samples as small as, or smaller than, the areas resolved in the usual radioautographs. Although a larger than tracer dose of DG is required, this is kept low enough not to significantly distort glucose metabolism. The assessment of glucose phosphorylation from directly observed levels of the primary metabolites concerned (DG, DG6P, and glucose) avoids many of the uncertainties that exist when these metabolites are calculated from plasma DG levels. In working out the method, some features of brain glucose metabolism have become evident that probably must be considered in any study of this kind, whether the time scale is short or long. Analytical Procedures. Measurement of DG, DG6P, and glucose depends on the facts (i) that glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) reacts with DG6P, but at a 2000-fold slower rate than with glucose 6-phosphate and (ii) that hexokinase reacts rapidly with both ...

Eleven regions of mouse brain and twelve layers of monkey retina were assayed for choline acetyl transferase (ChAT), acetylcholine esterase (AChE), and 4 enzymes that synthesize acetyl CoA. The purpose was to seek evidence concerning the source of acetyl CoA for acetylcholine generation. In brain ATP citrate lyase was strongly correlated with ChAT as well as AChE (r = 0.914 in both cases). Weak, but statistically significant correlation, was observed between ChAT and both cytoplasmic and mitochondrial thiolase, whereas there was a significant negative correlation between ChAT and acetyl thiokinase. In retina ChAT was essentially limited to the inner plexiform and ganglion cell layers, whereas substantial AChE activity extended as well into inner nuclear, outer plexiform and fiber layers, but no further. ATP citrate lyase activity was also highest in the inner four retinal layers, but was not strongly correlated with either ChAT or AChE (r = 0.724 and 0.761, respectively). Correlation between ChAT and acetyl thiokinase was at least as strong (r = 0.757), and in the six inner layers of retina, the correlation between ChAT and acetylthiokinase was very strong (r = 0.932).

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.

The activity of glucose-1,6-bisphosphatase and the level of its substrate were measured in 16 gray areas and four fiber areas of mouse brain and 10 layers or sublayers of monkey retina. Because of the low activity of the enzyme and the small sample sizes, it was necessary to develop a method with two different amplification steps (overall amplification about 10(6]. The enzyme ranged in activity 100-fold from a low in monkey retina photoreceptor cells to a high in the pyramidal layer of the hippocampus. However, in gray areas of the brain proper the range was only about fourfold. This, together with its requirement for IMP, suggests that the enzyme has a widespread metabolic function related to states of increased neuronal activity. Glucose-1,6-bisphosphate levels varied from 80 to 960 mu mol/kg dry weight in different areas of mouse brain and from 44 to 200 mu mol/kg dry weight in different layers of monkey retina. In general, the glucose bisphosphate levels correlated positively with the bisphosphatase activities; however, the three areas with the highest enzyme concentrations did not fit this pattern.

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