CO₂chemotransduction in central neurons: role of intracellular pH (pH_i) and extracellular pH (pH_o)

We have been studying the role of changes of pH_i as part of the signaling pathway of hypercapnia in neonatal rat brainstem neurons from both chemosensitive (NTS-nucleus tractus solitarius, VLM-ventrolateral medulla, LC-locus coeruleus) and non-chemosensitive (IO-inferior olive, Hyp-hypoglossal) regions. We are testing the model of chemotransduction that proposes that hypercapnia acidifies neurons, which leads to inhibition of K⁺ channels, resulting in neuronal depolarization and increased firing rate. We measure pH_i in individual neurons within brain slices using fluorescence imaging microscopy [1,2]. We developed a new technique to measure pH_i and V_msimultaneously by loading pH-sensitive fluorescent dyes using perforated patch (amphotericin B) pipettes in LC neurons [3]. All studies were done at 37°C.

Neurons from all brainstem regions have high intrinsic buffering power, around 45 mM/pH unit for NTS, VLM, IO and Hyp neurons [1] and about 90 mM/pH unit for LC neurons [3]. The only pH-regulating transporter available for recovery from an acid load in these neurons is Na⁺/H⁺ exchange (NHE) [1,2]. The only HCO₃-dependent transporter present is the acidifying Cl^-/HCO₃ ^- exchanger, which has been found in VLM, IO, Hyp and some NTS neurons [1]. Interestingly, the NHE in neurons from chemosensitive regions (eg, NTS and VLM) is more sensitive to inhibition by decreased pH_o than neurons from non-chemosensitive regions (eg, IO and Hyp) [1].

We observed different responses to hypercapnia in neurons from chemosensitive vs. non-chemosensitive regions. Hypercapnic acidosis (HA–10% CO₂, pH_o 7.15) resulted in a maintained fall of pH_i in neurons from chemosensitive areas whereas pH_i recovery from acidosis was evident in neurons from non-chemosensitive areas [2]. Under conditions of isohydric hypercapnia (IH–10% CO₂, 52 mM HCO₃-, pH_o 7.45), pH_i recovery was seen in neurons from all regions [2], indicating that pH-regulating mechanisms are present in all these neurons but that they are inhibited more fully by decreased pH_o in neurons from chemosensitive regions. The same pH_i responses to HA (15% CO₂, pH_o 6.8) and IH (15% CO₂, 77 mM HCO₃-, pH_o 7.45) were seen in LC neurons where pH_i and V_m were measured simultaneously. The changes of pH_i ccurred before the changes in neuronal firing rate [3]. In LC neurons, HA resulted in a larger fall of pH_i (0.27 vs. 0.15 pH unit) and a larger increase in firing rate (1.31 vs. 1.06 Hz) than seen with IH. Notably, increased firing rate was accompanied by membrane depolarization (2.5 mV) in response to HA but by membrane hyperpolarization (2.3 mV) in response to IH. Other acid challenges involving decreased pH_o (acidified HEPES; isocapnic acidosis–5% CO₂, 7 mM HCO₃-, pH_o 6.9) resulted in increased firing rate with depolarization while acid challenges involving constant pH_o (50 mM propionate, pH_o 7.45) resulted in slightly increased firing rate with membrane hyperpolarization.

In summary, the response of V_m to a fall of pH_i is dependent on whether changes or not and is not well correlated with pH_o increased firing rate in LC neurons. The parameter that best correlates with increased firing rate in response to an acid challenge is the change of pH_i, indicating that pHi is likely involved as part of the chemosensitive signaling pathway in LC neurons.