PMC

Model of Putative Mechanism for Hypocapnia-Induced Changes in Extracellular Adenosine and Neuronal Excitability
CO₂ (2%) appears to cause inhibition of ecto-ATPases, which can be mimicked by inhibition with ARL-67156, resulting in increased ATP receptor activation and decreased adenosine A₁ receptor activation. NT, neurotransmitter. Crosses denote site of action of antagonists DPCPX and P-PADS and the ecto-ATPase inhibitor ARL-67156.

Decreased O₂ Levels Do Not Account for Changes Seen during Hypercapnia (A) Inhibition of fEPSP amplitude by hypercapnic buffer was not due to decreased oxygen (n = 7; ***p < 0.001, Student's t test). (B) Average fEPSPs from control (solid line; average of five fEPSPs) and decreased oxygen buffer (dashed line; average of five fEPSPs). Scale bars, 25 ms, 0.5 mV. (C) Adenosine release caused by hypercapnic buffer was not caused by decreased oxygen (n = 5; ***p < 0.001, Student's t test). Error bars indicate standard error.

Adenosine A₁ and ATP Receptors Mediate the Effects of Altered CO₂ Levels
(A) Average inhibition of fEPSPs during 20% CO₂ exposure (n = 33), 20% CO₂ after DPCPX pretreatment (n = 8), 20% CO₂ after P-PADS pretreatment (n = 3), and 20% CO₂ after combined DPCPX and PPADS pretreatment (n = 8). **p < 0.01, ***p < 0.001, and ****p < 0.0001, all compared to 20% hypercapnia, ANOVA, Fisher PLSD. (B) Average excitation of fEPSPs during 2% CO₂ exposure (n = 14), 2% CO₂ after DPCPX pretreatment (n = 6), 2% CO₂ after PPADS pretreatment (n = 8), and 2% CO₂ after combined DPCPX and P-PADS pretreatment (n = 13). *p < 0.05, ***p < 0.001, and ****p < 0.0001, all compared to 2% hypocapnia, ANOVA, Fisher PLSD. Error bars indicate standard error.

Changes in CO₂ Levels Alter Extracellular Adenosine Concentration
(A) Average change in extracellular adenosine levels caused by hypercapnia (20% CO₂, n = 23, ***p < 0.005 compared to baseline sensor measurement, Student's paired t test; and 10% CO₂, n = 10, *p < 0.05 compared to baseline sensor measurement, Student's paired t test) and hypocapnia (2% CO₂, n = 8, *p < 0.05 compared to baseline sensor measurement, Student's paired t test, all groups are significantly different, p < 0.01, ANOVA). (B) Average change in extracellular adenosine levels during GABA_A receptor blockade caused by hypercapnia (20% CO₂, n = 5, ***p < 0.005 compared to baseline sensor measurement, Student's paired t test) and hypocapnia (2% CO₂,n = 7, ***p < 0.005 compared to baseline sensor measurement, Student's paired t test). Error bars indicate standard error.

The Effects of Hypocapnia Are Mediated by Ecto-ATPase, Adenosine A₁ Receptors, and ATP Receptors
(A) Average increase in fEPSP amplitude during 2% CO₂ exposure (n = 14), 2% CO₂ after ARL-67156 pretreatment (n = 10), and 2% CO₂ after combined ARL-67156 and P-PADS pretreatment (n = 5, **p < 0.01 compared to 2% hypocapnia, ANOVA, Fisher PLSD). (B) When ARL-67156 was applied to the slice adenosine levels decreased and the decrease in extracellular adenosine level caused by exposure to 2% CO₂ was significantly attenuated (*n = 8; p < 0.05, Student's t test). Error bars indicate standard error.

Changes in Adenosine and Excitability Correlate with Changes in Buffer pH and Are pH Dependent
(A) fEPSPs are plotted versus calculated pH_i (R² = 0.758). Inset: CA1 pyramidal cell loaded with BCECF-AM dye (black circle = 20% CO₂, n = 6 for pH_i measurement, n = 23 for fEPSP measurement; red circle = 10% CO₂, n = 6 for pH_i measurement, n = 13 for fEPSP measurement; yellow triangle = 20 mM propionic acid, n = 4 for pH_i measurement, n = 5 for fEPSP measurement; green triangle = isohydric hypercapnia, n = 7 for pH_i measurement, n = 7 for fEPSP measurement; blue square = 2% CO₂, n = 8 for pH_i measurement, n = 8 for fEPSP measurement). (B) Changes in extracellular adenosine are plotted versus calculated pH_i (R² = 0.848) (black circle = 20% CO₂, n = 23 for adenosine measurement; red circle = 10% CO₂, n = 13 for adenosine measurement; yellow triangle = 20 mM propionic acid, n = 5 for adenosine measurement; green triangle = isohydric hypercapnia, n = 7 for adenosine measurement; blue square = 2% CO₂, n = 8 for adenosine measurement). (C) fEPSPs are plotted versus buffer pH (R² = 0.992). (D) Changes in extra-cellular adenosine are plotted versus buffer pH (R² = 0.996). (E) Inhibition due to 10% CO₂ requires changes in buffer pH. Isohydric hypercapnia significantly attenuates the inhibition caused by 10% CO₂ (n = 7; **p < 0.01, ANOVA, Fisher PLSD). Intracellular acidification alone (propionic acid exposure) causes significantly less inhibition than 10% CO₂ (n = 5; ***p < 0.001, ANOVA, Fisher PLSD). (F) Increased extracellular adenosine caused by 10% CO₂ requires changes in pH_e. Isohydric hypercapnia completely blocks the adenosine release caused by 10% CO₂ (n = 7; **p < 0.01, ANOVA, Fisher PLSD). Intracellular acidification via propionic acid exposure was not sufficient to cause adenosine release (n = 5; ***p < 0.001 compared to 10% CO₂, ANOVA, Fisher PLSD). Error bars indicate standard error.

Changes in CO₂ Levels Alter Synaptic Transmission and Adenosine Levels in Area CA1
(A) Field EPSPs averaged from a single experiment. Control (solid) and 20% CO₂, 10% CO₂, and 2% CO₂ exposure (dashed). Scale bars, 25 ms, 0.5 mV. (B) Time course of fEPSP inhibition when aCSF CO₂ level was changed from 5% (control) to hypercapnia (20% CO₂, circles, n = 23; 10% CO₂, triangles, n = 11) or hypocapnia (2% CO₂, squares, n = 11) for 15 min (mean ± SEM). Thick line on x axis represents CO₂ manipulation. (C) Time course of fEPSP inhibition during GABA_A receptor blockade with 100 μM picrotoxin when aCSF CO₂ level was changed from 5% (control) to hypercapnia (20% CO₂, circles, n = 8) or hypocapnia (2% CO₂, squares, n = 7) for 15 min (mean ± SEM). Thick line on x axis represents the timing of CO₂ manipulation. (D) Excitatory postsynaptic current (EPSC) from a CA1 pyramidal cell under control conditions (5% CO_2; solid line; average of five EPSCs) and during exposure to 20% CO₂ (dashed line; average of five EPSCs). Scale bars, 25 ms, 200 pA. (E) Excitatory postsynaptic current (EPSC) from a CA1 pyramidal cell under control conditions (5% CO_2; solid line; average of five EPSCs) and during exposure to 2% CO₂ (dashed line; average of five EPSCs). Scale bars, 25 ms, 200 pA. (F) Average EPSC during 5% CO₂ (black bar; n = 5) and during exposure to 20% CO₂ (gray bar; n = 5; ***p < 0.001, Student's t test). (G) Average EPSC amplitude during 5% CO₂ (black bar; n = 5) and during exposure to 2% CO₂ (gray bar; n = 5; **p < 0.01, Student's t test). Error bars indicate standard error.

Hypercapnia and Hypocapnia Modulate Hippocampal Epileptiform Activity in Area CA3 via Adenosine A₁ and ATP Receptors
Extracellular recordings of synchronized bursting in area CA3. (A) Hypercapnia (20% CO₂) reversibly attenuates bursting. Inset, left: (A1) Example of a single burst recorded during control conditions (5% CO₂) (scale bars, 1 mV, 50 ms for all). (A2) Example of extracellular recording during hypercapnia, no bursts present. (Note: exact location of samples indicated on trace below.) Inset, right: Burst frequency during control and hypercapnic conditions. (B) Blocking adenosine A₁ receptors with DPCPX increases tonic firing, and hypercapnia (20% CO₂) did not attenuate bursting during adenosine A₁ receptor blockade. Inset, left: (B1) Example of a single burst recorded during control conditions (5% CO₂) with application of DPCPX. (B2) Example of a single burst recorded during hypercapnia with application of DPCPX, bursts still present. Inset, right: Burst frequency during control/DPCPX and hypercapnic/DPCPX conditions. (C) Hypocapnia (2% CO₂) increases bursting frequency. Inset, left: (C1) Example of a single burst recorded during control conditions (5% CO₂). (C2) Example of extracellular recording during hypocapnia. Inset, right: Burst frequency during control and hypocapnic conditions. (D) Blocking adenosine A₁ receptors and ATP receptors attenuates hypocapnia-induced increases in bursting frequency. Inset, left: (D1) Example of a single burst recorded during control conditions (5% CO₂). (D2) Example of extracellular recording during hypocapnia. Inset, right: Burst frequency does not change during hypocapnia when adenosine A₁ receptors and ATP receptors are blocked. (E) Average percent change in burst frequency. Hypercapnia (20% CO₂; n = 6) attenuates bursting, but burst frequency is not altered by hypercapnia when adenosine A₁ receptors are blocked with DPCPX (n = 4; **p < 0.01, ANOVA, Fisher PLSD). Hypocapnia (2% CO₂) increases average burst frequency from control levels (n = 7). This increase in burst frequency is prevented when DPCPX and suramin are applied prior to exposure to 2% CO₂ (n = 10; ***p < 0.001, ANOVA, Fisher PLSD). (F) Attenuation of bursting by hypercapnic buffer was not due to decreased oxygen (n = 5; ***p < 0.001, Student's t test). Error bars indicate standard error.