NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices
Open accessAbstract
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.
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Upon stimulation of neurons in a brain slice or individual dissociated neurons, the NAD(P)H signal has a stereotyped response: a rapid dip in fluorescence within the first few seconds that yields in ∼10 s to a prolonged increase above baseline (overshoot) lasting for 2-3 min.
“Upon stimulation of neurons in a brain slice (Kann et al., 2003; Shuttleworth et al., 2003; Kasischke et al., 2004) or individual dissociated neurons (Duchen, 1992), the NAD(P)H signal has a stereotyped response: a rapid dip in fluorescence occurring within the first few seconds that yields in ∼10 s to a prolonged increase above baseline (overshoot) lasting for 2–3 min (Fig. 2 B).”
From Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism (2018) · cites this paper as Shuttleworth et al., 2003
The NAD(P)H overshoot in slices did not disappear with prolonged elimination of extracellular Ca2+, although the mitochondrial Ca2+ transient did disappear.
“However, it is hard to reconcile this with the clear finding by Shuttleworth et al. (2003) that the NAD(P)H overshoot in slices did not disappear with the prolonged elimination of extracellular Ca2+, although the mitochondrial Ca2+ transient did disappear.”
From Fueling thought: Management of glycolysis and oxidative phosphorylation in neuronal metabolism (2018) · cites this paper as Shuttleworth et al., 2003
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“Similar experiments on acute hippocampal slices found little or no reduction in the NAD(P)H signals with removal of Ca2+ (Shuttleworth et al., 2003).”
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- Authors
- Shuttleworth, C. W., Brennan, A. M., & Connor, J. A.
- Affiliations
- University of New Mexico (US)
- Venue
- The Journal of Neuroscience
- Publisher
- Society for Neuroscience
- Published
- 2003-04-15
- Volume
- 23
- Issue
- 8
- Pages
- 3196-3208
- ISSN
- 0270-6474 (ISSN-L)
- 1529-2401
- PMC
- PMC6742314
- OpenAlex
- W2164895349
- Cited by
- 151
- References
- 60
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 3 · 2023: 1 · 2022: 4 · 2021: 3 · 2020: 3 · 2019: 4 · 2018: 6 · 2017: 1 · 2016: 6 · 2015: 3 · 2014: 7 · 2013: 3 · 2012: 8
- FWCI
- 3.47
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Neuroscience (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (RR15636)
- National Institute of Neurological Disorders and Stroke (NS43458, NS35644, R01NS043458, R01NS035644)
- National Center for Research Resources (RR-15636, P20RR015636)
- Topics
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Optical Imaging and Spectroscopy Techniques
- Keywords
- NAD(P)H fluorescence, hippocampal CA1, mitochondrial function, kainic acid, ionotropic glutamate receptors, calcium transients, mitochondrial depolarization, FAD fluorescence, extracellular calcium, electrical stimulation
- MeSH terms
- Animals, Calcium, Electric Stimulation, Flavin-Adenine Dinucleotide, Fluorescence, Fluorescent Dyes, Hippocampus, Iontophoresis, Kainic Acid, Male, Mice, Inbred C57BL, Mitochondria, NAD, NADP, Synaptic Transmission, Neurons, Stimulation, Chemical, Synapses, Excitatory Amino Acid Antagonists, Glutamic Acid, Excitatory Postsynaptic Potentials, Mice, Optics and Photonics, In Vitro Techniques
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