Release of ACh and ATP on the ventral surface of the medulla oblongata during hypercapnia.

<p>(<b>A</b>) Representative recordings illustrating changes in ACh and ATP concentration on the VMS in response to an increase in the level of inspired CO₂ in anaesthetised, paralyzed and artificially ventilated rats. Note that ATP release precedes ACh release, which occurs after the onset of CO₂-induced enhancement of the respiratory activity. Arrows denote when concentrations of ATP and ACh on the VMS start to increase. PNG–phrenic neurogram (arbitrary units). (<b>B</b>) Representative recordings illustrating changes in ATP and ACh release from the VMS triggered by CO₂ <i>in vitro</i> (horizontal brainstem slice).</p

Principles of operation and performance of acetylcholine (ACh) biosensors.

<p>(<b>A</b>) Schematic of the sensor assembly. (<b>B</b>) Calibration curve of a 2 mm ACh biosensor demonstrating linearity of ACh detection in concentrations between 0.5 and 10 μM. (<b>C</b>) Enzymatic cascade used to detect ACh. In the presence of ACh, the enzymatic cascade generates H₂O₂, which is detected electrochemically. (<b>D</b>) Biosensor placements on the ventral medullary surface. <i>In vivo</i>, 2 mm sensors were placed in direct contact with the ventral surface of the medulla (VMS) overlaying the rostral (R), intermediate (I) and caudal (C) chemosenitive areas. <i>In vitro</i>, 0.5 mm sensors were placed on the VMS within either the rostral or caudal chemosenitive areas. Arrow shows the direction of aCSF flow across the brainstem slice. (<b>E</b>) Representative traces illustrating the responses of ATP, null, ACh, and Ch biosensors to ATP and ACh (calibration of 0.5 mm sensors following <i>in vitro</i> experiments). Subtracting null sensor current from the ATP biosensor current and subtracting Ch biosensor current from ACh biosensor current produce netATP and netACh signals, respectively.</p

ACh release on the ventral surface of the medulla oblongata during hypercapnia is secondary to the release of ATP.

<p>(<b>A</b>) Representative recordings obtained sequentially in the same experiment illustrating the effect of P2 receptor antagonist pyridoxal-5’-phosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS) on changes in ACh concentration on the VMS in response to the increases in inspired CO₂. (<b>B</b>) Summary data illustrating CO₂-induced peak increases in ACh concentration on the VMS in the absence and presence of PPADS and after washout of the drug (n = 8). (<b>C</b>) Calibration of ACh biosensors <i>in vitro</i> demonstrating that ACh (10 μM)-evoked currents are not reduced in the presence of PPADS (200 μM). (<b>D</b>) Representative recordings illustrating lack of changes in ACh release from the VMS in response to bath application of ATP <i>in vitro</i> (horizontal brainstem slice).</p