Gamma oscillations and spontaneous network activity in the hippocampus are highly sensitive to decreases in pO2 and concomitant changes in mitochondrial redox state

Abstract

Gamma oscillations have been implicated in higher cognitive processes and might critically depend on proper mitochondrial function. Using electrophysiology, oxygen sensor microelectrode, and imaging techniques, we investigated the interactions of neuronal activity, interstitial pO2, and mitochondrial redox state [NAD(P)H and FAD (flavin adenine dinucleotide) fluorescence] in the CA3 subfield of organotypic hippocampal slice cultures. We find that gamma oscillations and spontaneous network activity decrease significantly at pO2 levels that do not affect neuronal population responses as elicited by moderate electrical stimuli. Moreover, pO2 and mitochondrial redox states are tightly coupled, and electrical stimuli reveal transient alterations of redox responses when pO2 decreases within the normoxic range. Finally, evoked redox responses are distinct in somatic and synaptic neuronal compartments and show different sensitivity to changes in pO2. We conclude that the threshold of interstitial pO2 for robust CA3 network activities and required mitochondrial function is clearly above the "critical" value, which causes spreading depression as a result of generalized energy failure. Our study highlights the importance of a functional understanding of mitochondria and their implications on activities of individual neurons and neuronal networks.

Figure 1.

Effects of tissue oxygenation on gamma oscillations and associated mitochondrial redox responses in CA3. A, Local field potentials were recorded in stratum pyramidale, and ACh (10 μm) was continuously applied in the presence of cholinesterase inhibitor physostigmine (2 μm) to evoke robust and persistent gamma oscillations. B, From recordings and conditions as illustrated in A, power spectra were calculated from data segments of 60 s. In the histogram, the gamma band power (30–80 Hz) is significantly reduced at 20% O₂ (p < 0.001; n = 15). C, Simultaneous recordings of NAD(P)H fluorescence in stratum radiatum (orange) and pyramidale (light blue) as well as [K⁺]_o (black) and local field potentials (data not shown) in stratum pyramidale were made during application of ACh (10 μm; bottom gray bar). Initially, the increase in [K⁺]_o was associated with a biphasic NAD(P)H fluorescence transient (dip and overshoot component), which transformed into a persistent NAD(P)H elevation (black arrow) when gamma oscillations were fully established (illustrated as top gray bar). Note that persistent NAD(P)H elevations were significantly larger in stratum radiatum. Figure 5A illustrates the selection of regions of interest for fluorescence imaging. *p < 0.05.

Figure 2.

Effects of tissue oxygenation on spontaneous network activity and evoked local field potential responses in CA3. A, Multiunit activity was recorded continuously in stratum pyramidale, and oxygenation was changed according to the protocol as illustrated in B. B, Applying single-unit discrimination in recording periods of 180 s, single units were classified in three groups according to their spike rates (spikes/s), which revealed a distribution of 10% (solid line), 22% (dotted line), and 68% (dashed line) (from n = 69) at 95% O₂ (gray background). Note that the spike rates declined in all groups at 20% O₂ (white background), which was reversible. C, After 15 min under the respective oxygenation condition, local field potential responses (fp) were evoked orthodromically by application of single electrical stimuli to the fiber tracts from dentate gyrus to CA3. Note that there were no differences in shape and amplitude (see Results) of the responses. *p < 0.05.

Figure 3.

Absolute values of interstitial pO₂ in CA3. The oxygen sensor microelectrode was positioned in stratum pyramidale and the pO₂ was continuously measured. Note that “95% O₂” (gray bars) and “20% O₂” (white bars) refer to saturation levels of ACSF in the storage container. A, The pO₂ baseline shift was measured in the slice core (100 μm depth). Each small pO₂ transient corresponds to enhanced O₂ consumption during neuronal activation as evoked by identical electrical stimulus trains (10 s, 20 Hz; black arrows) to the fiber tracts from dentate gyrus to CA3. B, Traces on an expanded time scale illustrate that pO₂ transients were smaller at 20% O₂, although transient increases in [K⁺]_o were similar. C, Histograms summarizing pO₂ baseline values that were determined at the surface (n = 9) and in the core (n = 18) of slice cultures. Note the significantly smaller pO₂ values in the slice core under both oxygenation conditions. D, Rise and decay times of pO₂ transients during stimulation are given for the 10–90% interval (n = 18). Note the significantly slower decay time at 20% O₂. E, pO₂ transients during stimulus trains were significantly smaller at 20% O₂ (n = 18). Note that there is no difference in the amplitudes of [K⁺]_o transients (n = 18), indicating virtually the same degree of neuronal activation under both O₂ conditions. *p < 0.05.

Figure 4.

Changes in NAD(P)H fluorescence and [K⁺]_o in CA3. A, The substantial NAD(P)H baseline elevation at 20% O₂ indicates reduced oxidation of the dinucleotide pools. Brief stimulus trains (10 s, 20 Hz; black arrows and bars) elicited biphasic NAD(P)H transients with different shapes under both oxygenation conditions (gray and white bars). The traces in the middle are from another experiment and displayed on an enlarged time scale. At 20% O₂, the biphasic NAD(P)H transient is characterized by a briefer initial “dip” component (gray rectangles) and a more rapidly developing and pronounced “overshoot” component. Note that the dip component terminates before the end of the stimulus train (right black bar). The bottom trace illustrates a NAD(P)H transient during a stimulus train of 60 s (20 Hz). B, Pairs of stimulus-evoked transient increases in [K⁺]_o (n = 8) (A, [K⁺]_o trace) were analyzed for amplitudes (see Results) and kinetics (illustrated in scheme). Rise and decay times are given for the 10–90% intervals. C, Pairs of stimulus-evoked biphasic NAD(P)H transients (n = 8) [A, NAD(P)H trace] were analyzed for amplitudes of dip (given as positive value) and overshoot components as well as for dip kinetics (illustrated in scheme). Note that NAD(P)H originated as the sum of fluorescence from stratum pyramidale and radiatum and that [K⁺]_o was simultaneously recorded in stratum pyramidale to quantify neuronal activation. *p < 0.05.

Figure 5.

NAD(P)H and FAD fluorescence transients in stratum pyramidale and stratum radiatum in CA3. A, Overlay of NAD(P)H and FAD fluorescence images as recorded with a delay of 130 ms. The K⁺-sensitive electrode was positioned in stratum pyramidale (white asterisk), the bipolar stimulation electrode close to the dentate gyrus (stim) for electrical activation of fiber tracts to CA3. Regions of interests (light blue, stratum pyramidale; orange, stratum radiatum) were selected to determine changes in %ΔF/F₀ from image stacks that were recorded at 0.5 Hz. B, The shapes of biphasic NAD(P)H (top traces) and FAD (middle traces) fluorescence transients as evoked by stimulus trains (10 s, 20 Hz; black bars) are clearly distinct at 20% O₂. Note that FAD transients (peak and undershoot) are inverse to NAD(P)H transients (dip and overshoot) because of different fluorescence properties of the dinucleotides. Transient increases in [K⁺]_o as evoked by stimulus trains were simultaneously recorded (bottom traces) and indicate virtually the same degree of neuronal activation at 95 and 20% O₂ (2.1 ± 0.1 and 2.4 ± 0.2 mm; n = 18; p = 0.26). C, D, Histograms summarizing the analysis of NAD(P)H and FAD transients (n = 18 each) in stratum pyramidale (pyr) and stratum radiatum (rad). Note that differences are most prominent in stratum radiatum. E, Overlay of NAD(P)H and pO₂ transients in stratum pyramidale as evoked by stimulus trains. F, Histogram summarizing the decay time constants of NAD(P)H overshoots and pO₂ transients at 95 and 20% O₂ (n = 18). *p < 0.05.