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

ATP and adenosine release display different sensitivities to blockade of ionotropic glutamate receptors

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> In the presence of 5 mmol/L kynurenic acid, the fEPSP was abolished and the anoxic depolarisation considerably delayed (dashed vertical line). The amount of adenosine release during and following ischaemia (black bar) was somewhat reduced, whereas ATP release was greatly enhanced

Rapid but incomplete conversion of ATP to adenosine in the extracellular space of the hippocampus

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> (a) Bath application of 100, 30 and 10 μmol/L ATP (indicated by black bars) was rapidly detected as adenosine and inosine by biosensors within the slice. (b) About 10% of the applied ATP was converted to adenosine or inosine. However, only around 5% of the applied ATP can be detected within the slice suggesting breakdown and accumulation of ADP/AMP. (c) Measurement, via the malachite green assay, of the time-dependent breakdown of ATP through accumulation of inorganic phosphate (Pi) in a hippocampal slice. Application of ARL 67156 substantially lowered the rate of ATP breakdown but did not abolish it. (d) Summary graph demonstrating that ARL 67156 acts as a competitive inhibitor of ATP breakdown and becomes less effective as the concentration of ATP rises

Inhibition of nucleoside transport enhances the extracellular accumulation of adenosine during ischaemia

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> (a) Combined application of the transport inhibitors dipyridamole (DIPY, 10 μmol/L) and NBTI (5 μmol/L) resulted in a slowly developing inhibition of the fEPSP which coincided with a slowly increasing signal on the Ado/ino sensor, demonstrating that the transport inhibitors were effective in raising extracellular adenosine levels. Magnified examples of the fEPSP are shown taken before (i) and during (ii and iii) the application of NBTI/DIPY at the times indicated. (b) Prolonged incubation with DIPY/NBTI did not prevent release of adenosine during ischaemia (black bar). Instead ado/ino release was enhanced, whilst there was little apparent effect on ATP release. The equilibrative nucleoside transporters are thus highly unlikely to mediate the release of adenosine during ischaemia

Gap junction hemichannels do not appear to mediate ischaemic purine release

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> The hemichannel blocker carbenoxolone (CBX, 100 μmol/L) depressed the fEPSP, but did not prevent occurrence of the anoxic depolarisation (dashed vertical line). Adenosine release was unaltered by CBX. However, ATP release was greatly increased. Black bar denotes period of ischaemia

Real-time measurement of the release of ATP and adenosine during ischaemia

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> (a) Simultaneous recordings from adenosine/inosine (Ado/ino), ATP and null biosensors, the fEPSP (in AC mode) and extracellular DC potential. Ischaemia (black bar) caused the rapid depression of the fEPSP (disappearance of the periodic negative deflections on the traces), which was associated with a rise in extracellular adenosine. Little change is seen in the ATP signal in the early stages of ischaemia. As the ischaemic episode progressed the anoxic depolarisation occurred (large negative deflection on DC trace; dashed vertical line). ATP release followed the anoxic depolarisation. Re-oxygenation resulted in a characteristic surge of adenosine release (the post hypoxic/ischaemic efflux; PPE; arrowhead) and, to a lesser extent, of ATP (arrowhead). Synaptic transmission did not recover after the ischaemic episode in this slice. Magnified examples of the fEPSP shown at the times indicated on the continuous DC trace are shown. The arrow indicates the fibre volley. Note that this has disappeared in trace (iii) (asterisk), obtained shortly after the anoxic depolarisation. On re-oxygenation, the fibre volley was usually enhanced (iv). (b) A short period of ischaemia (black bar), insufficient to evoke the anoxic depolarisation, did not elicit ATP release, but a substantial amount of adenosine was released both during and after the ischaemic episode. Note that full recovery of the fEPSP occurred. The rapid periodic deflections on the biosensor traces are a combination of fEPSP and artefacts from the stimulating electrode used to evoke the fEPSP. The negative deflection on the Ado/ino trace arises from removal of oxygen and does not represent a fall in extracellular adenosine/inosine concentration. In this and subsequent figures the ATP sensor trace reflects net ATP, i.e. the signal after subtraction of the simultaneously-recorded null sensor

Close temporal association between ATP release and the anoxic depolarisation

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> Simultaneous extracellular ATP measurement (upper trace) and whole-cell current-clamp recording of membrane potential in a CA1 pyramidal neurone (lower trace) during ischaemia (black bar). Following a gradual depolarisation, during which there was no change in the signal on the ATP sensor, a rapid membrane depolarisation (the anoxic depolarisation, dashed vertical line) occurred provoking repeated action potential firing. ATP release occurred immediately after the anoxic depolarisation

Adenosine release during ischaemia does not result from the breakdown of ATP

<p><b>Copyright information:</b></p><p>Taken from "Temporal and mechanistic dissociation of ATP and adenosine release during ischaemia in the mammalian hippocampus"</p><p></p><p>Journal of Neurochemistry 2007;101(5):1400-1413.</p><p>Published online Jan 2007</p><p>PMCID:PMC1920548.</p><p>© 2007 The Authors Journal Compilation 2007 International Society for Neurochemistry</p> Application of 100 μmol/L ARL 67156 enhanced ATP release during ischaemia (black bar) but had no effect on adenosine release