| Mitochondrial function has emerged as a central regulator of normal synaptic physiology and mitochondrial dysfunction has been implicated in the etiology of a number of neurodegenerative disorders. An important goal is to understand the regulation of mitochondrial function in complex brain tissue. One approach to monitor mitochondrial activity exploits redox changes of mitochondrial autofluorescence. The two major electron donors required for mitochondrial ATP synthesis display redox-sensitive intrinsic fluorescence. Following UV excitation, nicotinamide adenine dinucleotide (NADH) displays intrinsic fluorescence, but the oxidized form (NAD+) is non fluorescent. In contrast, FAD+ is fluorescent following excitation with blue light, but the reduced form FADH2 is non fluorescent. NAD(P)H dynamics have been used for over 40 years to assess changes in metabolic state of brain tissue under a wide range of physiological and pathological challenges, but the cell types and metabolic pathways involved remain controversial. The work presented here examines the metabolic pathways and cell types which underlie dynamic autofluorescence signals and considers the potential for these approaches for monitoring ongoing synaptic activity.;Synaptic activation in hippocampal slices results in an initial NAD(P)H decrease (oxidation phase), followed by a longer-lasting transient NAD(P)H increase (reduction phase). It was recently proposed that the reduction phase of NAD(P)H transients is attributable to glycolysis (rather than mitochondrial function) triggered by glial glutamate uptake. Studies here report that different duration stimulus trains have significant differences in the involvement of ionotropic glutamate receptors, suggesting that responses to more intense stimuli may involve astrocytes. Possible contributions of glycolysis were first tested using 2-deoxyglycose and iodoacetic acid. This approach required consideration of (1) the effects of extracellular adenosine accumulation and consequent decreases in synaptic efficacy and (2) effects of supplementation with exogenous pyruvate to sustain mitochondrial metabolism. When these effects were accounted for, responses to all stimuli tested were unaffected by pharmacological inhibition of glycolysis. Flavoprotein autofluorescence transients following extended stimuli matched (with inverted sign) NAD(P)H responses, supporting a role for mitochondrial metabolism in NAD(P)H overshoots. In addition, NAD(P)H responses to synaptic stimuli were not reduced by a non-selective inhibitor of glutamate uptake (TBOA). These results suggest that NAD(P)H transients report mitochondrial dynamics, rather than recruitment of glycolysic metabolism. (Abstract shortened by UMI.)... |
