Working Heart-brainstem Preparation Of Rat Research Articles

Comparison of the effects of fentanyls and other μ opioid receptor agonists on the electrical activity of respiratory muscles in the rat.

Introduction: Deaths due to overdose of fentanyls result primarily from depression of respiration. These potent opioids can also produce muscle rigidity in the diaphragm and the chest muscles, a phenomenon known as Wooden Chest Syndrome, which further limits ventilation. Methods: We have compared the depression of ventilation by fentanyl and morphine by directly measuring their ability to induce muscle rigidity using EMG recording from diaphragm and external and internal intercostal muscles, in the rat working heart-brainstem preparation. Results: At equipotent bradypnea-inducing concentrations fentanyl produced a greater increase in expiratory EMG amplitude than morphine in all three muscles examined. In order to understand whether this effect of fentanyl was a unique property of the phenylpiperidine chemical structure, or due to fentanyl's high agonist intrinsic efficacy or its lipophilicity, we compared a variety of agonists with different properties at concentrations that were equipotent at producing bradypnea. We compared carfentanil and alfentanil (phenylpiperidines with relatively high efficacy and high to medium lipophilicity, respectively), norbuprenorphine (orvinolmorphinan with high efficacy and lipophilicity) and levorphanol (morphinan with relatively low efficacy and high lipophilicity). Discussion: We observed that, agonists with higher intrinsic efficacy were more likely to increase expiratory EMG amplitude (i.e., produce chest rigidity) than agonists with lower efficacy. Whereas lipophilicity and chemical structure did not appear to correlate with the ability to induce chest rigidity.

Shift of leading pacemaker site during reflex vagal stimulation and altered electrical source-to-sink balance.

Vagal reflexes slow heart rate and can change where the heartbeat originates within the sinoatrial node (SAN). The mechanisms responsible for this process - termed leading pacemaker (LP) shift - have not been investigated fully. We used optical mapping to measure the effects of baroreflex, chemoreflex and carbachol on pacemaker entrainment and electrical conduction across the SAN. All methods of stimulation triggered shifts in LP site from the central SAN to one or two caudal pacemaker regions. These shifts were associated with reduced current generation capacity centrally and increased electrical load caudally. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. However, our findings indicate the LP region is defined by both pacemaker rate and capacity to drive activation. Shifts in LP site provide an important homeostatic mechanism for rapid switches in heart rate. Reflex vagal activity causes abrupt heart rate slowing with concomitant caudal shifts of the leading pacemaker (LP) site within the sinoatrial node (SAN). However, neither the mechanisms responsible nor their dynamics have been investigated fully. Therefore, the objective of this study was to elucidate the mechanisms driving cholinergic LP shift. Optical maps of right atrial activation were acquired in a rat working heart-brainstem preparation during baroreflex and chemoreflex stimulation or with carbachol. All methods of stimulation triggered shifts in LP site from the central SAN to caudal pacemaker regions, which were positive for HCN4 and received uniform cholinergic innervation. During baroreflex onset, the capacity of the central region to drive activation declined with a decrease in amplitude and gradient of optical action potentials (OAPs) in the surrounding myocardium. Accompanying this decline, there was altered entrainment in the caudal SAN as shown by decreased conduction velocity, OAP amplitude, gradient and activation time. Atropine abolished these responses. Chemoreflex stimulation produced similar effects but central capacity to drive activation was preserved before the LP shift. In contrast, carbachol produced a prolonged period of reduced capacity to drive and altered entrainment. Previous studies suggest LP shift is a rate-dependent phenomenon whereby acetylcholine slows central pacemaker rate disproportionately, enabling caudal cells that are less acetylcholine sensitive to assume control. Our findings indicate that cholinergic LP shifts are also determined by altered electrical source-to-sink balance in the SAN. We conclude that the LP region is defined by both rate and capacity to drive atrial activation.

Brainstem sources of cardiac vagal tone and respiratory sinus arrhythmia.

Key points Cardiac vagal tone is a strong predictor of health, although its central origins are unknown.Respiratory‐linked fluctuations in cardiac vagal tone give rise to respiratory sinus arryhthmia (RSA), with maximum tone in the post‐inspiratory phase of respiration.In the present study, we investigated whether respiratory modulation of cardiac vagal tone is intrinsically linked to post‐inspiratory respiratory control using the unanaesthetized working heart‐brainstem preparation of the rat.Abolition of post‐inspiration, achieved by inhibition of the pontine Kolliker‐Fuse nucleus, removed post‐inspiratory peaks in efferent cardiac vagal activity and suppressed RSA, whereas substantial cardiac vagal tone persisted. After transection of the caudal pons, part of the remaining tone was removed by inhibition of nucleus of the solitary tract.We conclude that cardiac vagal tone depends upon at least 3 sites of the pontomedullary brainstem and that a significant proportion arises independently of RSA. Cardiac vagal tone is a strong predictor of health, although its central origins are unknown. The rat working heart‐brainstem preparation shows strong cardiac vagal tone and pronounced respiratory sinus arrhythmia. In this preparation, recordings from the cut left cardiac vagal branch showed efferent activity that peaked in post‐inspiration, ∼0.5 s before the cyclic minimum in heart rate (HR). We hypothesized that respiratory modulation of cardiac vagal tone and HR is intrinsically linked to the generation of post‐inspiration. Neurons in the pontine Kölliker‐Fuse nucleus (KF) were inhibited with bilateral microinjections of isoguvacine (50–70 nl, 10 mm) to remove the post‐inspiratory phase of respiration. This also abolished the post‐inspiratory peak of cardiac vagal discharge (and cyclical HR modulation), although a substantial level of activity remained. In separate preparations with intact cardiac vagal branches but sympathetically denervated by thoracic spinal pithing, cardiac chronotropic vagal tone was quantified by HR compared to its final level after systemic atropine (0.5 μm). Bilateral KF inhibition removed 88% of the cyclical fluctuation in HR but, on average, only 52% of the chronotropic vagal tone. Substantial chronotropic vagal tone also remained after transection of the brainstem through the caudal pons. Subsequent bilateral isoguvacine injections into the nucleus of the solitary tract further reduced vagal tone: remaining sources were untraced. We conclude that cardiac vagal tone depends on neurons in at least three sites of the pontomedullary brainstem, and much of it arises independently of respiratory sinus arrhythmia.

Methylxanthine reversal of opioid-induced respiratory depression in the neonatal rat: Mechanism and location of action

Methylxanthines like caffeine and theophylline have long been used to treat apnea of prematurity. Despite their success in stimulating neonatal breathing, their mechanism of action remains poorly understood. Methylxanthines can act as both non-specific adenosine receptor antagonists and inhibitors of cAMP-dependent phosphodiesterases, sarcoplasmic/endoplasmic reticulum calcium ATPases or receptor-coupled anion channels, depending on the dose used. Though there is evidence for methylxanthine action at the level of the carotid body, the consensus is that methylxanthines stimulate the respiratory centers of the brainstem. Here we used the in situ neonatal rat working heart-brainstem preparation and the ex vivo neonatal rat carotid body preparation to test the hypothesis that methylxanthines act at the level of the carotid body. We conclude that although the neonatal carotid body has active adenosine receptors, the effects of methylxanthine therapy are likely mediated centrally, predominantly via inhibition of cAMP-dependent phosphodiesterase-4.

The anti-malarial drug Mefloquine disrupts central autonomic and respiratory control in the working heart brainstem preparation of the rat

BackgroundMefloquine is an anti-malarial drug that can have neurological side effects. This study examines how mefloquine (MF) influences central nervous control of autonomic and respiratory systems using the arterially perfused working heart brainstem preparation (WHBP) of the rat. Recordings of nerve activity were made from the thoracic sympathetic chain and phrenic nerve, while heart rate (HR) and perfusion pressure were also monitored in the arterially perfused, decerebrate, rat WHBP. MF was added to the perfusate at 1 μM to examine its effects on baseline parameters as well as baroreceptor and chemoreceptor reflexes.ResultsMF caused a significant, atropine resistant, bradycardia and increased phrenic nerve discharge frequency. Chemoreceptor mediated sympathoexcitation (elicited by addition of 0.1 ml of 0.03% sodium cyanide to the aortic cannula) was significantly attenuated by the application of MF to the perfusate. Furthermore MF significantly decreased rate of return to resting HR following chemoreceptor induced bradycardia. An increase in respiratory frequency and attenuated respiratory-related sympathetic nerve discharge during chemoreceptor stimulation was also elicited with MF compared to control. However, MF did not significantly alter baroreceptor reflex sensitivity.ConclusionsThese studies indicate that in the WHBP, MF causes profound alterations in autonomic and respiratory control. The possibility that these effects may be mediated through actions on connexin 36 containing gap junctions in central neurones controlling sympathetic nervous outflow is discussed.

Methylxanthine reversal of opioid‐induced respiratory depression in the in situ neonatal rat working heart‐brainstem preparation

Methylxanthines, such as caffeine and theophylline, are commonly given to premature infants to counter apneas of prematurity, but the mechanism by which these drugs prevent apnea is poorly understood. Methylxanthines act as nonselective adenosine receptor antagonists at lower concentrations and nonselective phosphodiesterase inhibitors at higher concentrations. Previous work in en bloc and slice preparations has demonstrated that >1 mM theophylline can reverse breathing suppressed by the μ‐opioid receptor agonist DAMGO. Using the in situ neonatal rat working heart‐brainstem preparation (a more intact preparation than previously employed) we show that theophylline restores DAMGO‐suppressed respiration in doses <1 mM, starting to effectively restore respiration at 250 μM. Denervation of the carotid bodies did not alter the response, suggesting that the response to theophylline is mediated centrally. These experiments suggest that in vivo, block of adenosine receptors may be involved in countering opioid depression. Funded by CIHR and Hotchkiss Brain Institute (EVF)

Carbachol microinjections into the dorsal pontine reticular formation repeatedly elicit REM sleep‐like depression of hypoglossal (XII) nerve activity in the rat in situ perfused working heart‐brainstem preparation (WHBP)

Obstructive sleep apnea (OSA) is characterized by nocturnal episodes of upper airway collapse and hypoxia, with rapid‐eye‐movement sleep (REMS)‐related apneas being longest and most severe. In OSA patients, upper airway muscle tone is reduced during REMS parallel to the postural atonia characteristic of this state. To assess the central neural mechanisms of REMS, in vivo pharmacological models of REM sleep have been used, including those in which REMS‐like state is elicited by microinjections of a cholinergic agonist, carbachol, into the pontine reticular formation (Kubin, 2001). To test whether upper airway motor tone depression similar to that in in vivo models can be elicited in the rat WHBP, injections of carbachol (25–50 nl, 10mM) were made into the dorsomedial pons in n=33 WHBPs while recording XII and phrenic nerve activities (PNA). In 23 preparations, depression of XII nerve activity lasting 16–24 min occurred within 142 s ± 26(SD). A respiratory rate increase from 8 min−1 ± 4 to 18 ± 9 paralleled the depression, whereas PNA was little affected. The depressant episodes could be elicited repeatedly by injections at the same site at >30 min intervals. Thus, the WHBP offers a novel model with which to pharmacologically manipulate cellular environment and study the mechanisms of REMS‐related respiratory depression. Funded by BCCN Göttingen and HL‐47600.

Are barosensitive cells in the nucleus of the solitary tract (NTS) activated by pressure stimuli that are sub‐threshold for heart rate change?

We have reported a lower pressure threshold for the sympathetic versus parasympathetic component of the arterial baroreflex (Simms et al. 2004; FASEB J. 4, #694.1). We hypothesised that the afferent information for these two limbs is processed separately in the NTS. Using the in situ rat working heart brainstem preparation (n=11), we made extracellular recordings of NTS neurones along with simultaneous recordings of heart rate, perfusion pressure (PP, baseline = 74±3mmHg) and sympathetic chain activity. We employed stimuli to selectively produce baroreflex sympathoinhibition without a change in heart rate using either small flow ramps to increase PP (18±1mmHg over 3–5s), intra-arterial bolus of phenylephrine (PE; 5μg, 19±3mmHg over 4–6s) and graded inflation of an intra-aortic balloon. A population of spontaneously active NTS neurones were identified (7±1.7 Hz, n=9) that showed graded increases in activity in response to PP ramps (33.7±5.7 Hz peak rate). All of these cells that were tested with PE showed increased activity (3.7 fold, n=5) confirming their identification as baroreceptive. Their activity was also decreased by a ramp fall in PP (19±3mmHg, n=6/7). A second set of baroreceptive neurones was identified by aortic balloon inflation (n=6). These were subdivided on the basis of their sensitivity to PP ramps or PE (3/6 excited). These data identify a group of baroreceptive NTS neurones that show graded changes in activity in response to pressure stimuli that alter sympathetic activity but are sub-threshold for heart rate change. Supported by the British Heart Foundation.

Brain stem PO(2) and pH of the working heart-brain stem preparation during vascular perfusion with aqueous medium.

The rat working heart-brain stem preparation (WHBP) is an in situ preparation having many of the advantages associated with in vitro preparations while retaining cardiovascular response functionality and an eupnoeic respiratory motor pattern. The preparation is perfused arterially with an aqueous medium having a much lower oxygen-carrying capacity than blood. To evaluate the efficacy of the artificial perfusion in providing adequate gas exchange within the brain stem, we used polarographic PO(2) and pH microelectrodes to determine the tissue PO(2) and pH of the medulla oblongata at various depths. When the perfusate was equilibrated with 5% CO(2) and 95% O(2), average tissue PO(2) was 294 Torr and no hypoxic areas were encountered. Tissue pH was remarkably uniform throughout the tissue, and on average was only 0.04 +/- 0.02 pH units more acidic than that of the perfusate. Increasing the PCO(2) of the perfusate increased tissue PO(2) and decreased arterial resistance. Decreasing perfusate PCO(2) (while keeping pH constant) decreased tissue PO(2) and reduced the respiratory activity. These results suggest that arterial PCO(2), independent of arterial pH, is an essential variable in determining both respiratory drive and cerebrovascular perfusion. We conclude that the medulla of the WHBP is oxygenated and within a physiological pH, which accounts for the eupneic pattern of respiratory motor activity it generates. Furthermore, this preparation may be a useful model for exploring mechanisms of central chemoreception as well as the dynamics of the cerebral vasculature responses following changes in blood gases.

Post-hypoxic frequency decline characterized in the rat working heart brainstem preparation.

The respiratory response to brief episodes of hypoxia is dynamic, i.e., the breathing pattern changes progressively both during and following an hypoxic episode1. The initial response in breathing with hypoxic exposure includes a sharp and acute increase in breathing frequency (F). Subsequently, F decreases as exposure to hypoxia continues. This decrease in F is a form of short-term depression that is referred to as “hypoxic ventilatory decline.” Immediately after the cessation of hypoxia and return to hyperoxic gas, F decreases further below the baseline frequency; the latter may be a form of short-term depression which is called post-hypoxic frequency decline (PHFD)1,2.