Phox2b-expressing retrotrapezoid neurons are intrinsically responsive to H+ and CO2

Abstract

Central respiratory chemoreceptors sense changes in CO2/H(+) and initiate the adjustments to ventilation required to preserve brain and tissue pH. The cellular nature of the sensors (neurons and/or glia) and their CNS location are not conclusively established but the glutamatergic, Phox2b-expressing neurons located in the retrotrapezoid nucleus (RTN) are strong candidates. However, a direct demonstration that RTN neurons are intrinsically sensitive to CO2/H(+), required for designation as a chemosensor, has been lacking. To address this, we tested the pH sensitivity of RTN neurons that were acutely dissociated from two lines of Phox2b-GFP BAC transgenic mice. All GFP-labeled cells assayed by reverse transcriptase-PCR (n = 40) were Phox2b+, VGlut2+, TH-, and ChAT-, the neurochemical phenotype previously defined for chemosensitive RTN neurons in vivo. We found that most dissociated RTN neurons from both lines of mice were CO2/H(+)-sensitive (∼79%), with discharge increasing during acidification and decreasing during alkalization. The pH-sensitive cells could be grouped into two populations characterized by similar pH sensitivity but different basal firing rates, as previously observed in recordings from GFP-labeled RTN neurons in slice preparations. In conclusion, these data indicate that RTN neurons are inherently pH-sensitive, as expected for a respiratory chemoreceptor.

Figure 1.

RTN neurons dissociated from Jx99 mice are sensitive to CO₂. A, Brainstem slice from Jx99 Phox2b-GFP transgenic mouse (left), with magnified fluorescent image of the RTN region (right); the facial nucleus is demarcated (7N). Note the GFP-labeled cells in the RTN region that was microdissected for dissociation. B, Image of a GFP-expressing dissociated RTN neuron under DIC (left) and fluorescence (right). C, Single-cell RT-PCR data revealed expression of Phox2b and VGlut2 in GFP-expressing dissociated RTN neurons (lanes 1–6), including three pH-sensitive neurons examined after recording (4–6); none of those RTN neurons expressed TH, ChAT, or GAD1. Control experiments verified detection of the following: TH (with Phox2b and VGlut2) in C1 neurons (lane 7); ChAT (and Phox2b) in motoneurons (lane 8), and GAD1 in striatal medium spiny cells (lane 9); GAPDH expression was seen in all cells, and negative controls included bath solution (lane 10) and water (lane 11) substituted for cell contents in all reactions. D, Firing activity of a GFP-expressing RTN neuron from Jx99 mouse during changes in pH produced by altering CO₂ and HCO₃⁻. Firing rate histograms (top traces) derived from cell-attached voltage-clamp recordings (bottom traces). Expanded time scale traces of action current waveforms (average of 30–50 spikes). Firing increased during CO₂-induced bath acidification and decreased during HCO₃⁻-induced bath alkalization.

Figure 2.

Type I and type II firing properties are retained in dissociated GFP-expressing RTN neurons. A, A relatively low level of baseline firing in a type I RTN neuron (<2 Hz) was increased by acidification (from pH 7.3 to pH 7.0) and essentially eliminated by alkalization to pH 7.5. B, In a type II cell, a comparatively high initial firing rate (∼5 Hz) was progressively suppressed with stepwise increases in bath alkalization and nearly silenced at pH 8.0. C, Frequency distribution of pH₅₀ values fitted with a biphasic distribution with peaks at pH 7.3 and 7.6; individual neurons were designated as type I (pH₅₀ < 7.42) or type II (pH₅₀ > 7.42). D, Averaged pH sensitivity for dissociated Jx99 RTN neurons classified as type I (open circles) or type II neurons (solid circles). The pH sensitivity (i.e., the slope) was similar for both cell types, but type II neurons displayed higher firing rates at all pH levels.

Figure 3.

Dissociated RTN neurons from B/G and Jx99 mice have identical pH-sensitive firing properties. A, Firing rate histogram (top) and cell-attached recording of firing activity (bottom) from a GFP-expressing RTN neuron from B/G mouse during pH changes evoked by altering CO₂ and HCO₃⁻ levels. B, Type II, pH-dependent firing behavior recorded in an RTN neuron from the B/G mouse line. C, D, Averaged pH sensitivity of firing for all dissociated GFP-labeled RTN neurons from B/G (open circles) and Jx99 (solid circles) mice recorded in CO₂/HCO₃⁻ buffer (C) and HEPES buffer (D). The pH sensitivity was not different in cells from the two lines of mice, regardless of buffer conditions.