Effect of intracellular pH clamping on the response to hypercapnia of locus coeruleus (LC) neurons

Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnia. Currently, pH i is believed to be the major signal in the chemosensitive (CS) response; however, a new model has been proposed suggesting that multiple factors (such as Ca ++ , CO 2 , pH i , and pH o ) contribute to the CS response. While there is evidence for a significant role of pH i in the CS response, we hypothesize that hypercapnic acidosis (HA) can still increase FR even with no change in pH i . To test this hypothesis, we developed a method to clamp pH i using rapid diffusion of the weak acid, acetic acid, through the cell membrane according to the relationship: pH i = pH o + log ([C 2 H 3 O 2 ] i /[C 2 H 3 O 2 ] o ). Pontine brainstem slices (300 μm) were cut from neonatal rat pups (P4‐P16). FR of LC neurons was measured with whole cell patch pipettes, pH i was measured by fluorescence imaging using the dye pyranine, and neurons were loaded from the patch pipette with 50 mM acetate. Extracellular acetate was varied to set pH i at desired values using the weak acid diffusion technique. We eliminated pH i decreases (0.07 ± 0.06 pH unit, p>0.05, n=8) of LC neurons upon exposure to HA (15% CO 2 , pH o 7.0) compared to the normal response (0.25 ± 0.03 pH unit, p<0.05, n=20). Despite pH i clamping, HA induced an increase in FR (CS index = 144 ± 14%, n=7) similar to controls (140 ± 5%, n=17). While a decrease in pH i is sufficient to elicit the chemosensitive response, these data indicate that intracellular acidification is not required. A chemosensitive response to hypercapnia without a change in pH i strongly supports the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].