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

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

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

Diagrammatic representation of the nervous control of hormone secretion by enteroendocrine cells of the gastrointestinal tract.

<p>Extrinsic vagal parasympathetic nerves either directly or via activation of the enteric neurones trigger release of hormones (hypothesised circulating cardioprotective factors) by releasing acetylcholine (among other transmitters). ACh, acetylcholine; AChR, acetylcholine receptor; BOM, bombesin; CCK, cholecystokinin; CGPR, calcitonin gene-related peptide; GliC, glicentin; GLP-1/2, glucagon-like peptide-1 and 2; OXM, oxyntomodulin; PYY, peptide YY; VIP, vasoactive intestinal peptide.</p

Cardioprotection established by remote ischaemic preconditioning (RPc) requires intact parasympathetic innervation of visceral organs.

<p><b>(a)</b> Illustration of the experimental protocols. RPc was induced by 15 min occlusion of both femoral arteries, followed by 10 min reperfusion. Sham-RPc procedure involved dissection of both femoral arteries without occlusion. Arrows indicate time of total subdiaphragmatic vagotomy, selective sectioning of individual visceral branches or sham surgery. <b>(b)</b> Total subdiaphragmatic vagotomy, bilateral gastric vagotomy and selective sectioning of the posterior gastric branch abolished the cardioprotective effect of RPc, whereas sectioning of the anterior gastric, celiac or hepatic branches had no effect on RPc cardioprotection. The infarct size is presented as the percentage of the area at risk. Individual data and means ± SEM are shown. P-values correspond to the Dunn’s post-hoc tests.</p

Electrical stimulation of the posterior gastric vagal branch mimics RPc cardioprotection.

<p><b>(a)</b> Illustration of the experimental protocols. Electrical stimulation (stim.) of individual vagal branches commenced 25 min before the onset of myocardial ischaemia (MI) and continued 10 min into the period of reperfusion. Sham procedure involved surgical dissection of the nerve and placing it on the electrodes without stimulation. <b>(b)</b> Electrical stimulation of the posterior gastric vagal branch reduced the extent of myocardial ischaemia/reperfusion injury, whereas stimulation of the hepatic vagal branch or sham stimulation of the posterior gastric branch had no effect. The infarct size is presented as the percentage of area at risk. Individual data and means ± SEM are shown. P-values correspond to the Dunn’s post-hoc tests.</p

Experimental interventions.

<p>Previous studies—DVMN silencing, cervical vagotomy. Six types of subdiaphragmatic vagotomy performed in the current study: total, bilateral gastric, anterior gastric, posterior gastric, hepatic and celiac, are shown on a schematic representation of typical distribution of rat abdominal vagal branches. Agb, anterior gastric branch; Avt, anterior vagal trunk; Ccb, common celiac branch; Hb, hepatic branch; Lvn, left vagus nerve; Pgb, posterior gastric branch; Pvt, posterior vagal trunk; Rvn, right vagus nerve. Brain, lungs, heart, diaphragm, liver, stomach, pancreas, small intestine and colon are depicted schematically.</p