title: NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices.
abstract: We examined mechanisms contributing to stimulus-evoked changes in NAD(P)H fluorescence as a marker of neuronal activation in area CA1 of murine hippocampal slices. Three types of stimuli (electrical, glutamate iontophoresis, bath-applied kainate) produced biphasic fluorescence changes composed of an initial transient decrease ("initial component," 1-3%), followed by a longer-lasting transient increase ("overshoot," 3-8%). These responses were matched by inverted biphasic flavin adenine dinucleotide (FAD) fluorescence transients, suggesting that these transients reflect mitochondrial function rather than optical artifacts. Both components of NAD(P)H transients were abolished by ionotropic glutamate receptor block, implicating postsynaptic neuronal activation as the primary event involved in generating the signals, and not presynaptic activity or reuptake of synaptically released glutamate. Spatial analysis of the evoked signals indicated that the peak of each component could arise in different locations in the slice, suggesting that there is not always obligatory coupling between the two components. The initial NAD(P)H response showed a strong temporal correspondence to intracellular Ca+ increases and mitochondrial depolarization. However, despite the fact that removal of extracellular Ca2+ abolished neuronal cytosolic Ca2+ transients to exogenous glutamate or kainate, this procedure did not reduce slice NAD(P)H responses evoked by either of these agonists, implying that mechanisms other than neuronal mitochondrial Ca2+ loading underlie slice NAD(P)H transients. These data show that, in contrast to previous proposals, slice NAD(P)H transients in mature slices do not reflect neuronal Ca2+ dynamics and demonstrate that these signals are sensitive indicators of both the spatial and temporal characteristics of postsynaptic neuronal activation in these preparations.
authors.affiliation: Department of Neurosciences, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA. bshuttleworth@salud.unm.edu
authors.forename: C William
authors.initials: CW
authors.lastname: Shuttleworth
authors.forename: Angela M
authors.initials: AM
authors.lastname: Brennan
authors.forename: John A
authors.initials: JA
authors.lastname: Connor
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D004558:Electric Stimulation; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000502:physiology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005453:Fluorescence; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D007478:Iontophoresis; D007608:Kainic Acid / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000378:metabolism; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D055095:Optics and Photonics; D013268:Stimulation, Chemical; D013569:Synapses / Q000187:drug effects / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology
chemical_list: D018691:Excitatory Amino Acid Antagonists; D005456:Fluorescent Dyes; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D009249:NADP; D007608:Kainic Acid; D002118:Calcium