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. 2003 Apr 15;23(8):3196-208.
doi: 10.1523/JNEUROSCI.23-08-03196.2003.

NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices

C William Shuttleworth  1 , Angela M Brennan, John A Connor
Affiliations

NAD(P)H fluorescence imaging of postsynaptic neuronal activation in murine hippocampal slices

C William Shuttleworth et al. J Neurosci. .

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.

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Figures

Fig. 1.
Fig. 1.
Temporal characteristics of NAD(P)H fluorescence changes evoked by electrical stimuli applied to s. radiatum. In all panels the stimuli were applied at the arrow; NAD(P)H fluorescence was monitored in s. pyramidale and expressed as ΔF/Fo. Bicuculline (30 μm) was present in all cases; data shown are from single trials. A, Response to single tetanus (25 pulses, 50 Hz) illustrating the characteristic biphasic NAD(P)H response, comprising an initial NAD(P)H fluorescence decrease (oxidation) followed by a more sustained NAD(P)H fluorescence increase (reduction). Acquisition rate, 3 Hz. B, The onset of the response is shown in more detail in a trial in which the acquisition rate was increased to 18 Hz (stimulus, 25 pulses, 50 Hz). The onset of the response was detected within 100 msec of stimulus onset. C, NAD(P)H transients could be resolved in single trials after more modest presynaptic stimuli. Panels illustrate successive trials (3 min intervals) in which the number of pulses in the stimulus train was decreased from 25 to a single shock. D, Both components of NAD(P)H responses were blocked by a combination of ionotropic glutamate receptor antagonists. Representative trials illustrate responses in normal ACSF and after 10 min exposure to 50 μm CNQX and 100 μm APV.
Fig. 2.
Fig. 2.
Spatial characteristics of NAD(P)H transients evoked by stimulation in s. radiatum. Color panels A–Dillustrate NAD(P)H fluorescence changes at times corresponding to the peak initial component and overshoot (500 msec and 10 sec after stimulus onset, respectively). Scale bar, 200 μm. Filled arrows indicate the position of the stimulating electrode.A, The peak optical signals elicited by a stimulus of five 1 mA pulses at 50 Hz. The optical changes were limited to an ∼150 μm area near the stimulus electrode, centered in s. radiatum. Increasing the stimulus intensity (B; 2 mA) increased the magnitudes of both the initial component and the overshoot and increased the spatial spread of the signal. Near the electrode significant changes became detectable in s. pyramidal and s. oriens.C, Two minute bicuculline exposure. The plume of excitation can be seen to spread further from the stimulus electrode along s. radiatum. Additionally, there are now even larger optical signals detected in s. pyramidal and s. oriens. D, Seven minute bicuculline exposure. The 2 mA stimulus train now triggers large biphasic optical signals in the entire CA1 field within view.E, Bright-field image of the slice showing the position of the stimulating electrode (asterisk) together with the locations of s. oriens (so), s. pyramidale (sp), and s. radiatum (sr).
Fig. 3.
Fig. 3.
Increase in NAD(P)H responses by disinhibition.A, B, Mean biphasic responses to tetani applied at two different stimulus intensities (1 and 4 mA). Control responses are represented by filled circles, and responses in the same preparations after 10 min of bicuculline exposure (30 mm) are in open circles. Arrows indicate stimulus train (25 pulses, 50 Hz).A′, B′, Initial components of responses shown inA and B at an expanded time base.C, Summary of effects on initial and overshoot responses for a range of stimulus intensities. Filled bars represent control responses, and open bars represent bicuculline. Note the large effect on overshoot responses as compared with the more modest increase of initial components. For all panels the data are the mean ± SEM; n = 6.
Fig. 4.
Fig. 4.
Alveus stimulation. A bipolar stimulating electrode was used to deliver a single tetanus (10 pulses, 50 Hz); NAD(P)H fluorescence changes were imaged at 10 Hz. A, Selected frames at 700 msec intervals illustrating a clear spatial difference between the peak initial component and overshoot. Times are indicated in seconds; stimulus onset occurs at 3 sec. The initial component was largest in s. oriens, centered above the stimulus electrode; in s. pyramidale and s. radiatum it was smaller and showed a wider lateral extent. A wide spatial divergence in the responses of the two components developed, beginning 1–2 sec after the stimulus. The initial component remained in the same localized area in s. oriens while a strong overshoot developed in s. pyramidale and s radiatum. This overshoot clearly covers the lateral extent of the field. Scale bar, 200 μm. B, Data from the same trial extracted from the three locations indicated in C. Arrow indicates onset of stimulus train. There is no significant change in fluorescence at location a, almost no overshoot at location c, and a biphasic change in location b. C, Single frame illustrates the location of the pyramidal cell layer (black lines) and stimulating electrode (filled arrow).
Fig. 5.
Fig. 5.
Comparison of NAD(P)H fluorescence transients with other fluorescence signals. Stimuli in all panels were single tetani applied to s. radiatum (10 pulses, 50 Hz) in the presence of bicuculline. A, B, Endogenous FAD fluorescence signals are inverted as compared with NAD(P)H transients. Stimuli were maintained at 5 min intervals, and imaging alternated between the two imaging modalities in each slice. Values are ΔF/Fo, mean ± SEM; n = 5. C, Ca2+ transients peak at similar times to initial NAD(P)H oxidation. Fura-2 ratios were normalized against the peak response in each preparation (mean ± SEM; n = 4). To allow for direct comparison of the three signals, we obtained the data in A–C with identical stimuli and acquisition settings; the stimuli were aligned at the vertical dashed line.D, E, Simultaneous imaging of NAD(P)H and Rh123 fluorescence transients, with the stimulus applied at the vertical dashed line. This is a single trial and illustrates that the Rh123 fluorescence increase is coincident with the initial NAD(P)H fluorescence decrease. In all panels the data were acquired at 3 Hz.
Fig. 6.
Fig. 6.
NAD(P)H responses evoked by iontophoretic application of glutamate (20 μA, 2 sec). A, Pseudocolor images show changes in NAD(P)H fluorescence. Images were acquired at 3 Hz, and selected frames are shown at the times indicated in seconds. The stimulus was applied at t = 6.7 sec. Note the different spatial distributions of the NAD(P)H decrease and overshoot. Scale bar, 200 μm. B, Baseline NAD(P)H fluorescence in the same field, demonstrating that resting levels are relatively uniform over the region of interest. Pyramidal cell layer is indicated by black lines. C, Single frame indicating the position of the glutamate microelectrode (black arrow).D, Data extracted from the three regions (a–c), indicated in B. E, Control for iontophoresis current demonstrating that replacement of glutamate with NaCl produces no discernible NAD(P)H transient with identical stimulus current.
Fig. 7.
Fig. 7.
Inhibition of Ca2+ influx did not reduce NAD(P)H transients. For these experiments the stimulus was glutamate applied via microiontophoresis (20 μA, 2 sec; arrows). Left panels show that in regular ACSF the glutamate stimuli produced biphasic NAD(P)H fluorescence transients and monophasic fura-2 and Rh123 fluorescence increases (A–C;n = 6, 5, 7, respectively). To allow for direct comparison of the three signals, we obtained the data inA–C with identical stimulus and acquisition settings. The initial undershoot was coincident with the rising phase of intracellular Ca2+ increase and with the rising phase of the Rh123 signal. Data in left panels are normalized against peak responses. Right panels plot the effect of the removal of extracellular Ca2+ on these three fluorescence signals (mean ± SEM). Three glutamate trials in regular ACSF (open bars) were followed by three trials in Ca2+-free ACSF/0.5 mm BAPTA (hatched and filled bars; see Materials and Methods). NAD(P)H fluorescence transients were increased consistently 2 min after beginning zero Ca2+perfusion (*p < 0.03); even after 12 min of Ca2+ removal both components were not significantly different from responses in control ACSF (A, right;n = 6). Confirming the effective removal of extracellular Ca2+ from the slice, fura-2 signals virtually were abolished by perfusion with zero Ca2+/BAPTA (B, right; *p < 0.02). Rh123 fluorescence transients were reduced significantly in zero Ca2+ conditions (*p < 0.04; #p < 0.02).
Fig. 8.
Fig. 8.
Extracellular Ca2+ removal had no significant effect on NAD(P)H fluorescence transients evoked by kainate stimulation. A, B, Representative single trials illustrating NAD(P)H fluorescence changes in s. pyramidale after exposure to a single bolus of kainate (at arrows). C, Mean data for six preparations (±SEM) showing the initial deflection and overshoot responses. No significant differences were found for either component.
Fig. 9.
Fig. 9.
Effects of ouabain exposure on spontaneous and evoked NAD(P)H dynamics. A, Exposure to 30 μm ouabain alone (beginning at start of trace) initiated a strong biphasic NAD(P)H response after ∼7.5 min of ouabain exposure (asterisk). B, Illustrated is the progression of this response along the CA1 pyramidal cell layer. The same event as inA is shown at an expanded time base, recorded at three locations along the CA1 pyramidal cell layer. The characteristics of these responses are similar to those described previously for spreading depression (see Results). The onset of spreading depression required that the effects of ouabain on evoked NAD(P)H responses be tested after very brief ouabain exposures. C, NAD(P)H transients after glutamate iontophoresis are shown under control conditions (three trials, top traces) and after 4.5 min of ouabain exposure (bottom trace). Ouabain exposure decreased the overshoot component of the evoked response.
Fig. 10.
Fig. 10.
Reduction of extracellular [Na+] reduced stimulus-evoked NAD(P)H increases.A, Control NAD(P)H transients evoked by glutamate iontophoresis in 153 mm extracellular Na+ are indicated in filled circles. Reduction of extracellular Na+ to 27 mm (open squares) significantly reduced the overshoot component, with no significant effect on the initial component of NAD(P)H transients (n = 9 for each). B, Effect of Na+ reduction on responses to bolus KA exposure. KA applied at the arrows produced large biphasic transients, as shown previously in Figure 8. Na+ reduction for 3 min led to a significant increase in the initial component and decrease in overshoot component of the response. C, Mean data (±SEM) from four slices in normal ACSF and four slices in low Na+ (**p < 0.0005).
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