This is an omnibus project that covers a variety of studies on development, validation, refinement, and applications of methods previously developed in this laboratory to examine basic biochemical and physiological mechanisms underlying the regulation of cerebral blood flow (CBF) and energy metabolism at rest and in response to functional activation. Studies completed and published last year showed that ATP-sensitive K+ ion channels,present in vascular smooth muscle and able to dilate blood vessels, do not contibute to functional activation of CBF. Also published this year was a study that disproved the hypothesis that dopamine receptors are involved in the functional activation of CBF. Studies completed and published this year confirmed that pathways of glucose utilization are to some extent compartmentalized between astroglia and neurons. The astroglia metabolize glucose to lactate and export it to neurons, and the neurons oxidize the lactate to CO2 and H2O. These studies showed, however, that neurons can readily oxidize both glucose and lactate but have a kinetic preference to oxidize lactate derived from the extracellular space over pyruvate/lactate produced intracellularly by glycolysis. Astroglia also can oxidize both glucose and lactate to CO2, but they do so sparingly, and they metabolize glucose mainly to lactate. The limited ability of the astroglia is due to limited pyruvic dehydrogenase (PDH) activity. This enzyme exists in astroglia predominantly in the phosphorylated inactive form, but it can be activated by dichloroacetate, which we found can stimulate astroglia to oxidize lactate and diminish their export of lactate to neurons. When administered in vivo to rats, it produced no obvious changes in gross behavior and led to increases in cerebral glucose utilization, presumably due to increased utilization of glucose to compensate for the diminished import of lactate from the astroglia. The conclusion from these studies is that the compartmentalization of glucose metabolism between neurons and astroglia does exist, but it is neither complete nor obligatory, and the relative rates of neuronal oxidation of glucose and lactate vary with the lactate concentration in the extracellular space. Studies on two strains of mutant mice with either the alpha or the beta thyroid receptor genetically altered so that they could not bind L-triiodothyronine are still in progress. Part of the results were published this year. Cerebral glucose utilization was found to be completely normal in mice with the altered beta thyroid hormone receptor, but markedly and diffusely depressed in mice with the dysfunctional alpha-receptor as in animals made cretinous by radiothyroidectomy at birth. These results indicate that the beta thyroid hormone receptor has little if anything to do with normal brain development and that the effects of thyroid hormone on brain development are mediated by the alpha thyroid hormone receptor. Other studies on these mutant mice that have been published this year showed that although the baseline glucose utilization in cerebral structures is markedly reduced, the percent increases in their rates of glucose utilization evoked by neuronal functional activation reamin the same, indicating that the decreased baseline glucose utilization is due to diminished synaptic density but the remaining synapses are functionally normal. Studies recently completed and submitted for publication also showed marked reductions in heart size and cardiac glucose utilization in the mice with the dysfunctional thyroid alpha-receptors whereas heart size was slightly greater and cardiac glucose utilization were enormously increased in the mice with the mutant beta-receptor. Studies are in progress to examine the hypothesis that increased adenosine levels in brain are responsible for the induction of natural sleep states. There have been reports that brain adenosine levels progressively rise during wakefulness and then decline back to normal levels during sleep. Also, caffeine, which is a potent non-specific blocker of adenosine channels, is known to interfere with natural sleep. In order to raise brain adenosine levels experimentally, we have been infusing into rats drugs that inhibit enzymes which metabolize the adenosine normally produced in brain. These drugs are deoxycoformycin, which inhibits adenosine deaminase, and iodotubericidin, which inhibits adenosine kinase. When administered parenterally, these drugs produce marked falls in arterial blood pressure, undoubtedly due to systemic vasodilatation resulting from activation of adenosine receptors on the vascular smooth muscle. To avoid the complications of this systemic hypotension, we have been administering the drugs intracisternally. Intracisternal infusions of both drugs simultaneusly have thus far been found to produce a sleep-like behavioral state as well as EEG changes similar to those seen in REM and non-REM sleep states. These studies will be continued to assure the reproducibility of these effects, and also local cerebral glucose utilization, which is known to be markedly and diffusely reduced in non-REM sleep, will be measured in rats treated with these drugs.
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