Graded reductions in oxygenation evoke graded reconfiguration of the isolated respiratory network

Abstract

Neurons depend on aerobic metabolism, yet are very sensitive to oxidative stress and, as a consequence, typically operate in a low O(2) environment. The balance between blood flow and metabolic activity, both of which can vary spatially and dynamically, suggests that local O(2) availability markedly influences network output. Yet the understanding of the underlying O(2)-sensing mechanisms is limited. Are network responses regulated by discrete O(2)-sensing mechanisms or, rather, are they the consequence of inherent O(2) sensitivities of mechanisms that generate the network activity? We hypothesized that a broad range of O(2) tensions progressively modulates network activity of the pre-Bötzinger complex (preBötC), a neuronal network critical to the central control of breathing. Rhythmogenesis was measured from the preBötC in transverse neonatal mouse brain stem slices that were exposed to graded reductions in O(2) between 0 and 95% O(2), producing tissue oxygenation values ranging from 20 ± 18 (mean ± SE) to 440 ± 56 Torr at the slice surface, respectively. The response of the preBötC to graded changes in O(2) is progressive for some metrics and abrupt for others, suggesting that different aspects of the respiratory network have different sensitivities to O(2).

Fig. 1.

Rhythmic bursts of activity were recorded extracellularly from the surface of a slice in an area of ventral respiratory group (VRG) called the pre-Bötzinger complex (preBötC). The top trace is a raw extracellular recording. The bottom trace is a rectified and integrated version of the same voltage signal. The light gray line was integrated with analog circuitry, with a time constant of 50 ms. The superimposed black line is a digitally filtered version of the same signal created with a 2nd-order Butterworth low-pass filter with a cutoff of 1.5 Hz. This twice-filtered signal was used for subsequent data analysis.

Fig. 2.

Reductions in the media O₂ resulted in corresponding reductions in tissue oxygenation. A: average O₂-depth profiles showing differences in tissue oxygenation based on superfusing a brain slice with artificial cerebrospinal fluid (aCSF) bubbled with different levels of O₂ (red = Fo₂ 95%, orange = Fo₂ 75%, yellow = Fo₂ 50%, light blue = Fo₂ 21%, dark blue = Fo₂ 0%, where Fo₂ is the fractional value of oxygen). Inset shows mean tissue partial pressure of O₂ (Po₂) values at the core of the slice (300 μM) with an expanded y-axis. Note that the mean values in 0 and 21% Fo₂ are similar. Depth was measured relative to the top surface of the brain slice. Although slices were cut to a nominal thickness of 600 μm, the average measured slice thickness was 575 ± 10 μm (n = 7). Beneath the bottom of the slice the oxygen tension rose to values similar to those found above the slice, indicating that the flow rate of media below the slice was similar to that above the slice (data not shown). B: plot of media Po₂ (400 μm above the tissue) as a function of Fo₂ in the equilibrating gas mixture. Although theoretically media saturated with 0% O₂ should have a Po₂ of 0 Torr, the actual ambient Po₂ was 38 Torr in the media above the slice, likely due to incomplete saturation of the aCSF in the reservoir that feeds the superfusion bath as well as gas diffusion at the media-ambient atmosphere interface in the superfusion bath. C: plots of tissue Po₂ at the upper surface of the slice (depth = 0 μm) and at the core of the slice (depth = 300 μm) as a function of media Po₂.

Fig. 3.

The response of the in vitro respiratory rhythm is dependent on the degree of reduction in oxygen. Representative preparations were each subjected to a single reduction in O₂. A: Po₂ values measured within the superfusion chamber resulting from a switch between a control reservoir saturated with 95% O₂ and a second reservoir saturated with 0 to 75% O₂. During the 300-s test period there was a rapid decrease in the Po₂ within the superfusion chamber, which eventually stabilized at a new O₂ level. B–E: responses of the in vitro respiratory rhythm to 5 min long reductions in O₂. The dots placed above certain bursts denote sigh-like bursts, which were distinguished by their large amplitude compared with that of normal respiratory bursts (Lieske et al. 2000). F: an averaged burst waveform calculated from the bursts within a 100-s time bin in 95% O₂. In this example, an average waveform was calculated based on 9 individual burst waveforms. The baseline was determined by averaging the voltage immediately prior to and immediately after the averaged burst waveform. The burst amplitude was calculated as the difference between the peak of the burst and the baseline. The burst area was the area bounded by the baseline on the bottom, the averaged waveform on the top and vertical lines at the times corresponding to 20% of the peak amplitude on the rising and falling phases of the waveform. The rise time was calculated as the time interval between 20 and 80% of the peak amplitude on the rising phase of the waveform. The half-width was calculated as the time interval between 50% of the peak amplitude on the rising phase of the waveform and 50% of the peak amplitude on the falling phase.

Fig. 4.

Fictive sighs increase in number during the transition from high O₂ to low O₂. A: fictive sighs occur at a low frequency in 95% O₂ but increase in frequency during the transition to reduced O₂. In this example from a postnatal day 9 mouse, 6 sighs occurred within the first 100 s of exposure to 0% O₂. As in Fig. 3, dots were placed above sigh bursts. The arrows marked B and C denote windows of time that are expanded in B and C, respectively. B: fictive sighs were identified based on amplitude, shape, and by the presence of a longer burst interval immediately after a sigh compared with immediately before (Lieske et al. 2000). The burst area of sighs was on average 136.6 ± 50.3% greater than that of eupneic bursts (n = 6; paired t-test, P = 0.006). C: fictive gasps can be identified based on a lower frequency and faster rise time than eupneic bursts (Lieske et al. 2000). D: a comparison of the number of fictive sighs within the first 100 s of the transition to reduced O₂ revealed that there was a significantly higher number of sighs in response to 0, 21, and 50% compared with the control experiment in which O₂ was maintained at 95%. After an initial increase in the number of sighs during the first 100 s, sighs were very rare during the second 100 s and no sighs were observed in the third 100 s of a 5-min exposure, except in the control experiment in which the level of O₂ was maintained at 95%. Note that empirically derived mean slice surface Po₂ values (20, 73, 177, 341, 440 Torr; see Table 1) are reported on the x-axis rather than the corresponding media Fo₂ values (0, 21, 50, 75, 95%). Error bars represent SD.

Fig. 5.

Changes in burst frequency and burst area of the in vitro respiratory rhythm in response to graded changes in O₂. A: changes in mean normalized burst frequency in response to 3 different test O₂ values: 0% (n = 9), 21% (n = 10), and 95% (n = 11). In response to 0 and 21% O₂, the frequency increased during the augmentation phase and decreased during the depression phase. During the recovery phase the frequency overshot the baseline frequency prior to the reduction in O₂. B: in response to 50% O₂ (n = 12) the frequency increased during the augmentation phase and decreased during the depression phase; however, unlike the responses to 0 and 21% O₂, it did not overshoot during the recovery phase. C: in response to 75% O₂ (n = 9) the frequency declined during the depression phase. D: unlike the biphasic response of frequency, the mean normalized burst area decreased monotonically during exposure to 0 and 21% O₂. During reoxygenation, burst area overshot the baseline prehypoxia value. E: in response to 50% O₂, burst area decreased. On reoxygenation, there was a weak overshoot, followed by a return to the prehypoxia baseline value. F: in response to 75% O₂, there was little difference in the burst area compared with the control experiment (O₂ = 95%).

Fig. 6.

Changes in frequency and shape metrics in response to graded reductions in O₂ tension during the augmentation phase. A: frequency was significantly higher in 0% (P < 0.05), 21% (P < 0.05), and 50% (P < 0.05) O₂ compared with that in 95% O₂. B: burst area was significantly lower in 0% (P < 0.001), 21% (P < 0.05), and 50% (P < 0.05) compared with that in 95% O₂. C: burst amplitude was significantly lower in 0% compared with that in 95% O₂ (P < 0.05). D: half-width was significantly lower in 0% (P < 0.001), 21% (P < 0.01), and 50% (P < 0.01) compared with that in 95% O₂. E: rise time was significantly lower in 0% (P < 0.01) and 21% (P < 0.05) compared with that in 95% O₂. * denotes significance in A–E.

Fig. 7.

Changes in metrics in response to graded reductions in O₂ tension during the depression phase. A: frequency was significantly lower at 0% (P < 0.001), 21% (P < 0.001), 50% (P < 0.001), and 75% (P < 0.05) compared with that at 95% O₂. B: burst area was significantly lower in 0% (P < 0.001), 21% (P < 0.01), and 50% (P < 0.01) compared with that in 95% O₂. C: burst amplitude was significantly lower in 0% (P < 0.01) compared with that in 95% O₂. D: half-width was significantly lower in 0% (P < 0.001), 21% (P < 0.001), and 50% (P < 0.01) compared with that in 95% O₂. E: rise time was significantly lower in 0% (P < 0.01) and 21% (P < 0.01) compared with that in 95%. * denotes significance in A–E. F: differences in both the absolute values of the slopes of linear regressions and r² values of the metrics indicate that they are influenced differentially by O₂.

Fig. 8.

To quantify the ability of the in vitro respiratory network to recover from hypoxia the time to first burst (TTFB) was measured. A: TTFB was measured from the onset of reoxygenation to the first population burst. B: significant differences in TTFB were found at 0% (P < 0.01), 21% (P < 0.01), and 50% (P < 0.01) compared with that at 95% O₂. *, denotes significance. Error bars represent SD.

Fig. 9.

Changes in metrics in response to graded reductions in O₂ tension during the recovery phase. A: frequency was significantly higher after exposure to 0% (P < 0.01) and 21% (P < 0.01) compared with the control experiment (i.e., 95% O₂). B: burst area was significantly greater after exposure to 0% (P < 0.01) and 21% (P < 0.01) compared to 95% O₂. C: burst amplitude was significantly greater after 0% (P < 0.001) and 21% (P < 0.01) compared to 95% O₂. D: half-width was significantly greater after 21% (P < 0.05) compared to 95% O₂. E: rise time did not change significantly compared with 95%. F: differences in both the absolute values of the slopes of linear regressions and r² values of the metrics indicate that they are influenced differentially by O₂.

Fig. 10.

The response of the respiratory network to graded changes in O₂ is different under conditions of blockade of the persistent Na⁺ current (I_NaP) and the blockade of the Ca²⁺-activated nonspecific cation current (I_CAN). A: representative experiment showing that bath application of 10 μM riluzole (RIL) followed by a reduction of the media O₂ to 0% results in a cessation of bursting (n = 4). On reoxygenation the rhythm recovered (data not shown). B: representative experiment showing that bath application of 50 μM flufenamic acid (FFA) does not lead to a cessation of bursting in 0% O₂ (n = 6). On reoxygenation the rhythm recovered its original amplitude and frequency (data not shown). C: representative experiment showing that bath application of FFA followed by RIL in 95% O₂ leads to a cessation of bursting (n = 6). Note that O₂ was maintained at 95% throughout the experiment. Unpaired t-tests were conducted comparing either frequency (D) or burst area (E) in RIL to the respective metrics of untreated rhythms at the same O₂ level. D: in RIL-treated preparations cessation of the rhythm occurred in 0% (n = 4), 21% (n = 5), and 50% (n = 4/11), whereas at 75% O₂ (n = 6) RIL significantly reduced frequency. RIL did not affect frequency at 95% O₂ (n = 8). In the 7 RIL experiments where the rhythm continued to burst at 50% O₂, there were no significant differences in frequency between this group and the untreated group. Frequency during the last 100 s of a 5-min exposure to reduced O₂ was normalized to the mean value in the 100-s time bin immediately preceding the test exposure. E: in RIL-treated preparations cessation of the rhythm occurred in 0% (n = 4), 21% (n = 5), and 50% (n = 4/11), leading to significant differences in burst area. At 75% (n = 6) and 95% (n = 8) O₂ RIL significantly reduced burst area. In the 7 RIL experiments where the rhythm continued to burst at 50% O₂ there were no significant differences between this group and the untreated group. Burst area during the last 100 s of a 5-min exposure to reduced O₂ was normalized to the mean value in the 100-s time bin immediately preceding the test exposure. F: frequency in preparations treated with FFA was significantly reduced at 75% (n = 8) and 95% (n = 11) O₂. FFA did not affect changes in frequency when exposed to 50% (n = 8), 21% (n = 5), and 0% (n = 5) O₂. G: FFA did not affect changes in burst area at 95% (n = 11), 75% (n = 8), 50% (n = 8), 21% (n = 5), or 0% (n = 5) O₂. Unpaired t-tests were conducted comparing either frequency (F) or burst area (G) in FFA to the respective metrics of untreated rhythms at the same O₂. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.