Bicarbonate directly modulates activity of chemosensitive neurons in the retrotrapezoid nucleus

Abstract

Key points: Changes in CO₂ result in corresponding changes in both H⁺ and HCO₃^- and despite evidence that HCO₃^- can function as an independent signalling molecule, there is little evidence suggesting HCO₃^- contributes to respiratory chemoreception. We show that HCO₃^- directly activates chemosensitive retrotrapezoid nucleus (RTN) neurons. Identifying all relevant signalling molecules is essential for understanding how chemoreceptors function, and because HCO₃^- and H⁺ are buffered by separate cellular mechanisms, having the ability to sense both modalities adds additional information regarding changes in CO₂ that are not necessarily reflected by pH alone. HCO₃^- may be particularly important for regulating activity of RTN chemoreceptors during sustained intracellular acidifications when TASK-2 channels, which appear to be the sole intracellular pH sensor, are minimally active.

Abstract: Central chemoreception is the mechanism by which the brain regulates breathing in response to changes in tissue CO₂ /H⁺ . The retrotrapezoid nucleus (RTN) is an important site of respiratory chemoreception. Mechanisms underlying RTN chemoreception involve H⁺ -mediated activation of chemosensitive neurons and CO₂ /H⁺ -evoked ATP-purinergic signalling by local astrocytes, which activates chemosensitive neurons directly and indirectly by maintaining vascular tone when CO₂ /H⁺ levels are high. Although changes in CO₂ result in corresponding changes in both H⁺ and HCO₃^- and despite evidence that HCO₃^- can function as an independent signalling molecule, there is little evidence suggesting HCO₃^- contributes to respiratory chemoreception. Therefore, the goal of this study was to determine whether HCO₃^- regulates activity of chemosensitive RTN neurons independent of pH. Cell-attached recordings were used to monitor activity of chemosensitive RTN neurons in brainstem slices (300 μm thick) isolated from rat pups (postnatal days 7-11) during exposure to low or high concentrations of HCO₃^- . In a subset of experiments, we also included 2',7'-bis(2carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF) in the internal solution to measure pHi under each experimental condition. We found that HCO₃^- activates chemosensitive RTN neurons by mechanisms independent of intracellular or extracellular pH, glutamate, GABA, glycine or purinergic signalling, soluble adenylyl cyclase activity, nitric oxide or KCNQ channels. These results establish HCO₃^- as a novel independent modulator of chemoreceptor activity, and because the levels of HCO₃^- along with H⁺ are buffered by independent cellular mechanisms, these results suggest HCO₃^- chemoreception adds additional information regarding changes in CO₂ that are not necessarily reflected by pH.

Keywords: HCO3; brain slice; chemoreception; pH-independent; pHi.

Figure 1. HCO₃ ⁻‐dependent modulation of chemosensitive RTN neurons

A, trace of firing rate and segments of holding current shows a typical response of an RTN neuron in a slice incubated in normal Ringer's solution containing 26 mM HCO₃ ⁻ to an increase in CO₂ from 5% to 10%. After returning to control conditions, exposure to HCO₃ ⁻‐free Hepes buffer (pH 7.3) strongly inhibited baseline activity. B, summary data (n = 18) show average firing rate under control conditions and during exposure to HCO₃ ⁻‐free Hepes buffer (pH_o 7.3) and 10% CO₂ (pH_o 7.0). C, trace of firing rate and segments of holding current from a chemosensitive RTN neuron shows that exposure to isohydric HCO₃ ⁻ (10% CO₂/52 mM HCO₃ ⁻) caused a robust increase in activity. D, summary data (n = 19) show average firing rate of chemosensitive RTN neurons under control conditions and during exposure to isohydric HCO₃ ⁻ and 10% CO₂ (pH_o 7.0). E, trace of firing rate and segments of holding current from a CO₂/H⁺‐insensitive RTN neuron shows that exposure to isohydric HCO₃ ⁻ (10% CO₂/52 mM HCO₃ ⁻) minimally affected firing behaviour. F, summary data (n = 10) show average firing activity of CO₂/H⁺‐insensitive RTN neurons under control conditions and during exposure to isohydric HCO₃ ⁻ and 10% CO₂ (pH_o 7.0). One‐way ANOVA with Tukey multiple comparison test. ^**** P < 0.0001. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 2. HCO₃ ⁻ directly modulates activity of RTN neurons

A–E, traces of firing rate and segments of holding current from chemosensitive RTN neurons show that exposure to isohydric HCO₃ ⁻ (10% CO₂/52 mM HCO₃ ⁻) stimulated neural activity under control conditions and after 10 min of incubation in a transmitter receptor blocker cocktail containing CNQX (10 μM), gabazine (10 μM) and strychnine (2 μM) (A), KCNQ channels were blocked with XE991 (10 μM) (B), sAC activity was inhibited with KH7 (10 μM) (C), purinergic receptors were blocked with PPADS (5 μM) (D), and when nitric oxide synthase was inhibited with l‐NAME (1 mM). F, summary data show that exposure to HCO₃ ⁻‐free Hepes buffer inhibited activity under control conditions (n = 10), and in XE991 (n = 5) or the blocker cocktail (n = 4). G, summary data plotted as isohydric HCO₃ ⁻‐induced change in activity under control conditions (n = 13) and in the presence of XE991 (n = 5), PPADS (n = 4), blocker cocktail (n = 3), KH7 (n = 4), or l‐NAME (n = 5). //, a 10–20 min break in the recording. ↓, injection of a positive DC current to adjust baseline activity to near control levels. One‐way ANOVA (F _5,33 = 0.5909, P > 0.05). [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 3. HCO₃ ⁻ modulates activity of chemosensitive RTN neurons by a mechanism independent of pH_i

BCECF (100 μM) was included in our pipette internal solution to measure pH_i of chemosensitive RTN neurons incubated in Hepes buffer during exposure to high HCO₃ ⁻ (26 mM) alone and in the presence of acetazolamide (az; 1 mM). A, trace of pH_i (Ai) and fluorescence images (490 nm excitation) (Aii) from a chemosensitive RTN neuron in normal Ringer's solution (26 mM HCO₃ ⁻) equilibrated with 5% CO₂ (pH_o 7.3) shows that exposure to 10% CO₂ decreased pH_i ∼0.1 pH units. After returning to control conditions exposure to HCO₃ ⁻‐free Hepes buffer (pH_o 7.3) increased pH_i ∼0.08 pH units. In the continued presence of Hepes buffer, exposure to HCO₃ ⁻ (26 mM) reversibly decreased pH_i by ∼0.07 pH units under control conditions. Under these conditions exposure to acetazolamide (1 mM) also decreased pH_i by ∼0.4 pH units. However, in acetazolamide subsequent exposure to HCO₃ ⁻ (26 mM) this time increased pH_i ∼0.1 pH units. B, summary data plotted as change in pH_i during exposure to isohydric HCO₃ ^– (10% CO₂ + 52 mM HCO₃ ⁻) in normal Ringer's solution (n = 4), and HCO₃ ⁻ (26 mM) alone (n = 3) or together with acetazolamide under Hepes buffer conditions (n = 3). C, trace of firing rate shows a typical H⁺ response of an RTN neuron in a slice incubated in Hepes buffer. After returning to control conditions (pH– 7.3), exposure to HCO₃ ⁻ (26 mM; pHo 7.3) alone or in the presence of acetazolamide (1 mM) increased ∼0.75 Hz. Note that bath application of acetazolamide minimally affected neuronal activity despite resulting in a strong intracellular acidification. D, trace of firing rate from a chemosensitive RTN neuron in a slice incubated in Hepes buffer shows that exposure to HCO₃ ⁻ (26 mM; pH_o 7.3) increased neural activity by similar amounts under control conditions and in the presence of acetazolamide (1 mM) plus KH7 (10 μM). E, summary data show that the firing response to HCO₃ ⁻ was similar under control conditions (n = 7) and in acetazolamide alone (n = 4) or in combination with KH7 (n = 4) (one‐way ANOVA; F _2,13 = 0.115, P > 0.05). ^*Paired t test, P < 0.05. [Color figure can be viewed at http://wileyonlinelibrary.com]