Adenosine Signaling through A1 Receptors Inhibits Chemosensitive Neurons in the Retrotrapezoid Nucleus

Adenosine strongly inhibits chemosensitive RTN neurons in slices from rat and mouse pups by an A1 receptor-dependent mechanism. A, Trace of firing rate (Hz) and segments of holding current from a chemosensitive neuron in a brainstem slice from a rat pup shows that exposure to adenosine (1 µM; ado) decreases activity in a reversible and repeatable manner. B, Summary data (n = 6) shows ado inhibits CO₂/H⁺ (15% CO₂)-stimulated activity. C, Trace of firing rate from a chemosensitive RTN neuron in a brainstem slice from a rat pup shows that bath application of a selective A1 receptor blocker (DPCPX, 30 nM) had negligible effect on CO₂/H⁺-stimulated activity but completely eliminated the inhibitory effects of ado (1 µM). D, Summary data shows the inhibitory effects of ado RTN chemoreceptors in slices from rat pups was eliminated by preincubation (∼10 min) in a non-specific ado receptor blocker (8PT, 10 µM; N = 3; Di) or a selective A1 receptor blocker (DPCPX, 30 nM; N = 5; Dii). E, Summary data shows bath application of 8PT or DPCPX alone minimally affected CO₂/H⁺-stimulated activity of RTN chemoreceptors in slices from rat pups, suggesting endogenous ado does not limit chemoreceptor activity under these experimental conditions. F, Firing rate (Hz) trace from a chemosensitive RTN neuron in a brainstem slice from a mouse pup shows that exposure to ado (1 µM) inhibited CO₂/H⁺-stimulated activity under control conditions but not in the presence of DPCPX (30 nM). G, H, Summary data (N = 3) shows in mouse that DPCPX blocked the effect of adenosine, and when applied alone did not affect the CO₂/H⁺-stimulated activity; *p < 0.05; **p < 0.01; ***p < 0.001.

Adenosine inhibition of RTN chemoreceptor activity was blunted by barium. A, Trace of firing rate (Hz) from a chemosensitive RTN neuron in a brainstem slice from a rat pup shows that exposure to adenosine (1 µM; ado) alone caused a near complete suppression of CO₂/H⁺ (15% CO₂)-stimulated activity. Application of Ba²⁺ (100 μM) in the continued presence of high CO₂ caused a modest increase in neuronal activity and blunted subsequent responses to adenosine. Summary data (N = 5) to the right show that 100 μM Ba²⁺ blunted the inhibitory effect of ado by ∼50%. B, Trace of firing rate (Hz) and summary data to the right show that ado (1 µM) inhibition of RTN chemoreceptor activity was retained in the presence of TEA (10 mM) and 4AP (50 µM). These results suggest that ado inhibits activity of RTN chemoreceptors by activation of an inward rectifying K⁺ channel; *p < 0.05; **p < 0.01.

Adenosine activates an inward rectifying K⁺ conductance in chemosensitive RTN neurons. A, Traces of holding current (top) and conductance (bottom; V_hold = –60 mV; TTX) from a chemosensitive RTN neuron in a brainstem slice from a rat pup shows that exposure to 15% CO₂ decreased outward current and conductance. In the continued presence of high CO₂, subsequent exposure to ado (1 µM) increased outward current and conductance. B, Summary data (N = 5) shows the effects of high CO₂ alone and CO₂ plus ado on holding current (Bi) and conductance (Bii). C, Current responses to voltage steps from –60 mV to between –40 and –130 mV during exposure to high CO₂ alone and CO₂ plus ado. D, Average (N = 5) current–voltage (I–V) relationships of the CO₂/H⁺-sensitive (Di) and ado-sensitive (Dii) currents; difference currents were isolated by subtracting I-V relationships recording during exposure to 15% CO₂ or ado from those recorded under control conditions; **p < 0.01.

Adenosine modulation of RTN chemoreceptors was retained when GABA and glycine receptors are blocked. A, B, firing rate trace from a chemosensitive RTN neuron (A) and summary data (B; N = 4) shows the inhibitory effects of adenosine (1 µM) are retained in the presence of bicuculline and strychnine.

Adenosine minimally affects inhibitory synaptic input to RTN chemoreceptors. A, Traces of holding current (Ihold = 0 mV) from a RTN chemoreceptor shows sIPSC events under control conditions and during 15% CO₂ alone and with adenosine or bicuculline and strychnine. B, Traces of holding current at holding potentials ranging from –40 to –80 mV (Δ 10-mV steps) and in the presence of kynurenic acid (block glutamate receptors) show that sIPSCs reverse near –60 mV. This holding potential will be used to isolate EPSCs. C, D, Summary data show effects of ado (1 µM) on IPSC freq (C) and amplitude (D) during high CO₂. E, Plot of IPSC events versus interevent interval shows ado minimally affected the occurrence of IPSCs.