Original Article
Journal of Cerebral Blood Flow & Metabolism (2006) 26, 1389–1406. doi:10.1038/sj.jcbfm.9600292; published online 15 March 2006
NAD(P)H fluorescence transients after synaptic activity in brain slices: predominant role of mitochondrial function
This work was supported by NIH Grants NS43458 and RR15636.
Angela M Brennan1, John A Connor1 and C William Shuttleworth1
1Department of Neurosciences, School of Medicine, University of New Mexico, Albuquerque, New Mexico, USA
Correspondence: Dr CW Shuttleworth, Department of Neurosciences, School of Medicine, University of New Mexico, Basic Medical Sciences Building, Rm145, 915 Camino de Salud, NE, Albuquerque, New Mexico 87131, USA. E-mail: bshuttleworth@salud.unm.edu
Received 28 January 2005; Revised 6 January 2006; Accepted 30 January 2006; Published online 15 March 2006.
Abstract
Excitatory stimulation in hippocampal slices results in biphasic NAD(P)H fluorescence transients. Previous studies using differing stimulus protocols agreed that the oxidation phase is a consequence of mitochondrial metabolism, but the reduction phase has been attributed to (1) mitochondrial nicotinamide adenine dinucleotide (NADH) generation or (2) astrocytic glycolysis triggered by glutamate uptake. In an attempt to reconcile these two views, the present study examined NAD(P)H signals evoked by a wide range of stimulus durations (40 ms to 20 secs). A combination of ionotropic glutamate receptor (iGluR) antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 2-amino-5-phosphonopentanoic acid (APV)) virtually abolished responses to brief stimuli (40 to 200 ms, 50 Hz), but a significant fraction of the signal elicited by extended stimulation (20 secs, 32 Hz) was resistant to CNQX/APV. Glycolysis was inhibited by removal of glucose and addition of 2-deoxyglucose (2DG) (10 mmol/L) or iodoacetic acid (IAA, 1 mmol/L). Pyruvate was provided as an alternative substrate for oxidative phosphorylation and the A1 receptor antagonist 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) included to prevent decreases in synaptic efficacy. If sufficient pyruvate was supplied, responses to brief and extended stimuli were unaffected by glycolytic inhibition and not significantly reduced by an inhibitor of glucose uptake (3-O-methyl glucose, 3 mmol/L). When timed to arrive at the peak of overshoots generated by extended synaptic stimulation, brief pyruvate applications (10 mmol/L, 2 mins) had little effect on evoked NAD(P)H increases. Flavoprotein autofluorescence transients after extended stimuli matched (with inverted sign) NAD(P)H responses. Responses to extended stimuli were not reduced by a nonselective inhibitor of glutamate uptake DL-Threo-
-benzyloxyaspartic acid (TBOA). These results suggest that NAD(P)H transients report mitochondrial dynamics, rather than recruitment of glycolytic metabolism, over a wide range of stimulus intensities.
Keywords:
astrocyte, CA1, glycolysis, hippocampal slice, mitochondria, NADH fluorescence
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