Recent studies suggest (Schwartz et al., 1979) that local cerebral glucose utilization, measured with the deoxyglucose technique (Sokoloff et al., 1977), correlates most closely with electrical activity in the neuropil in general and synaptic terminals in particular. Presumably, increased glucose utilization associated with increased impulse activity in nervous tissue is, as is oxygen consumption (Ritchie, 1967;Greengard and Ritchie, 1971: De-Weer, 1973, principally due to enhanced activity of the sodium pump. If the increased energy metabolism during impulse activity is used mainly for reconstitution of electrochemical gradients, then it is to be expected that cellular components with larger surface-to-volume ratios will have larger energy demands (Ritchie, 1967;Greengard and Ritchie, 1971;DeWeer, 1975) and, thus, greater rates of glucose utilization. It would be of value for the interpretation of studies that employ the autoradiographic deoxyglucose method to identify the cellular elements in which neural activity and energy metabolism are most closely linked. We have, therefore, studied an in vitro preparation of rat posterior pituitary, which represents a relatively enriched population of axon terminals (Nordmann, 1977) and may serve as a model for synaptic endings in the brain. Because the pituitary is a neurosecretory organ, we have also studied the influence of the secretory process in this system on energy metabolism. As an index of glucose utilization, we have measured the rate at which [ ''C]deoxyglucose is phosphorylated by hexokinase and trapped in the tissue incubated in vitro. This is the in vifro equivalent of the deoxyglucose method in which trapped [ '4C]deoxyglucose-6-phosphate is visualized and measured autoradiographically (Sokoloff et al., 1977). MATERIALS AND METHODSMale Sprague-Dawley rats (180-250 g) were decapitated, and the pituitary glands were removed rapidly and placed in balanced salt solution (BSS) consisting of 10 mM

Physiological stimulation of the hypothalamo-neurohypophysial system by salt loading of rats resulted in a dramatically increased glucose utilization in the posterior pituitary but not in the paraventricular or supraoptic nuclei. The good correlation between glucose utilization and neural activity in the posterior pituitary (that is, nerve terminals) contrasted with the lack of correlation in the paraventricular and supraoptic nuclei (that is, the sites of the cell bodies of the same neurons). This difference in the metabolic response to functional activity between the two regions of these neurons can be explained by the differences in surface-to-volume ratios of these regions.

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The biochemistry and regulation of dual leucine zipper bearing kinase (DLK), a member of the mixed lineage kinase or MLK subfamily of protein kinases, was examined in the nervous system. DLK transcript expression in the nervous system was predominantly neuronal. DLK protein was present in synaptic terminals where it was associated with both plasma membrane and cytosol fractions. Within these two fractions, DLK had differing characteristics. Cytosolic DLK existed in both a phosphorylated and dephosphorylated state; DLK associated with plasma membrane existed in the dephosphorylated state only. On nonreducing SDS-polyacrylamide gel electrophoresis, cytosolic DLK migrated at 130 kDa, while membrane associated DLK migrated with an apparent M r 260,000. Similarly, DLK transiently expressed in COS 7 cells autophosphorylated in vivo and migrated at approximately 260 kDa when separated by nonreducing SDS-polyacrylamide gel electrophoresis. In cotransfection experiments, FLAG-tagged DLK or a FLAG-tagged truncated DLK mutant (F-⌬520) was coimmunoprecipitated with Myc-tagged DLK and formed complexes under nonreducing conditions consistent with the conclusion that DLK formed covalently associated homodimers in overexpressing COS 7 cells. In aggregating neuronal-glial cultures, depolarization of plasma membrane lead to dephosphorylation of DLK. Treatment of aggregates with 5 nM or 200 nM okadaic acid lead to a shift in electrophoretic mobility consistent with phosphorylation of DLK. Treatment with cyclosporin A, a specific inhibitor of the calcium/calmodulindependent protein phosphatase 2B (calcineurin), had no effect on DLK phosphorylation under basal conditions. However, cyclosporin A completely inhibited DLK dephosphorylation upon membrane depolarization.