Advancing respiratory-cardiovascular physiology with the working heart-brainstem preparation over 25 years.

Twenty-five years ago, a new physiological preparation called the working heart-brainstem preparation (WHBP) was introduced with the claim it would provide a new platform allowing studies not possible before in cardiovascular, neuroendocrine, autonomic and respiratory research. Herein, we review some of the progress made with the WHBP, some advantages and disadvantages along with potential future applications, and provide photographs and technical drawings of all the customised equipment used for the preparation. Using mice or rats, the WHBP is an in situ experimental model that is perfused via an extracorporeal circuit benefitting from unprecedented surgical access, mechanical stability of the brain for whole cell recording and an uncompromised use of pharmacological agents akin to in vitro approaches. The preparation has revealed novel mechanistic insights into, for example, the generation of distinct respiratory rhythms, the neurogenesis of sympathetic activity, coupling between respiration and the heart and circulation, hypothalamic and spinal control mechanisms, and peripheral and central chemoreceptor mechanisms. Insights have been gleaned into diseases such as hypertension, heart failure and sleep apnoea. Findings from the in situ preparation have been ratified in conscious in vivo animals and when tested have translated to humans. We conclude by discussing potential future applications of the WHBP including two-photon imaging of peripheral and central nervous systems and adoption of pharmacogenetic tools that will improve our understanding of physiological mechanisms and reveal novel mechanisms that may guide new treatment strategies for cardiorespiratory diseases.

Mylohyoid discharge of the in situ rat: a probe of pontile respiratory activities in eupnea and gasping.

Our purpose was to characterize respiratory-modulated activity of the mylohyoid nerve. Since its motoneurons are in the trigeminal motor nucleus, mylohyoid discharge could serve as a probe of the role of pontile mechanisms in the generation of respiratory rhythms. Studies were performed in the decerebrate, perfused in situ preparation of the rat. Phrenic discharge was recorded as the index of the respiratory rhythm. In eupnea, the mylohyoid nerve discharged primarily during neural expiration, in the period between phrenic bursts. This expiratory discharge increased greatly in hypoxia and fell in hypercapnia. The hypoxia-induced increase in mylohyoid discharge was due, at least in part, to a direct influence of hypoxia on the brain stem. In ischemia, phrenic discharge increased, and then declined to apnea, which was succeeded by gasping. The mylohyoid nerve discharged tonically during the apneic period, but still declined during each of the phrenic bursts of gasping. This maintenance of a respiratory-modulation of the mylohyoid discharge in gasping supports the concept that a release of medullary mechanisms, rather than a ubiquitous suppression of pontile influences, underlies the neurogenesis of gasping. Results also provide additional support for our conclusion that activity of any single cranial nerve does not provide an accurate index of the type of respiratory rhythm, be it eupnea or gasping.

Noeud vital for breathing in the brainstem: gasping--yes, eupnoea--doubtful.

For the past 200 years, various regions of the brainstem have been proposed as a 'noeud vital' for breathing-a critical region which, when destroyed, results in an irreversible cessation of breathing and death. Complicating this search for a noeud vital is the extensive network of neurons that comprises the brainstem respiratory control system of pons and medulla. Does a cessation of breathing following ablation of any region reflect the removal of a critical set of neurons whose activity generates the respiratory rhythm or does it reflect the interruption of one component of the neuronal circuit, such that this circuit cannot function, at least temporarily? An additional complication is that in contemporary neuroscience, a number of in vitro preparations have been introduced for the study of the generation of the respiratory rhythms. However, how are the rhythms that these preparations generate related to normal breathing? Are these rhythms similar to those of gasping, which is recruited when normal, eupnoeic breathing fails, or are these rhythms unique to the in vitro preparation and not related to any breathing pattern in vivo?

Location and properties of respiratory neurones with putative intrinsic bursting properties in the rat in situ.

Using the in situ arterially perfused preparations of both neonatal and juvenile rats, we provide the first description of the location, morphology and transmitter content of a population of respiratory neurones that retains a bursting behaviour after ionotropic receptor blockade. All burster neurones exhibited an inspiratory discharge during eupnoeic respiration. These neurones were predominantly glutamatergic, and were located within a region of the ventral respiratory column that encompasses the pre-Bötzinger complex and the more caudally located ventral respiratory group. Bursting behaviour was both voltage and persistent sodium current dependent and could be stimulated by sodium cyanide to activate this persistent sodium current. The population of burster neurones may overlap with that previously described in the neonatal slice in vitro. Based upon the present and previous findings, we hypothesize that this burster discharge may be released when the brain is subject to severe hypoxia or ischaemia, and that this burster discharge could underlie gasping.

Discharge of the hypoglossal nerve cannot distinguish eupnea from gasping, as defined by phrenic discharge, in the in situ mouse.

If normal, eupneic breathing fails, gasping is recruited. Serotonin was proposed as essential for gasping, based on findings using an in vitro mouse preparation. This preparation generates rhythmic activities of the hypoglossal nerve that are considered to be akin to both eupnea and gasping. In previous studies, gasping of in situ rat and mouse preparations continued unabated following blockers of receptors for serotonin. However, hypoglossal activity was not recorded in the mouse, and we hypothesized that its discharge during gasping might be dependent on serotonin. In the in situ mouse preparation, hypoglossal discharge had varying and inconsistent patterns during eupnea, discharging concomitant with the phrenic burst, at varying intervals between phrenic bursts, or was silent in some respiratory cycles. In eupnea, phrenic discharge was incrementing, whereas hypoglossal discharge was decrementing in 15 of 20 preparations. During ischemia-induced gasping, peak phrenic height was reached at 205 +/- 17 ms, compared with 282 +/- 27.9 ms after the start of the eupneic burst (P < 0.002). In contrast, rates of rise of hypoglossal discharge in gasping (peak at 233 +/- 25 ms) and eupnea (peak at 199 +/- 19.2 ms) were the same. The uncoupling of hypoglossal from phrenic discharge in eupnea was exacerbated by methysergide, an antagonist of serotonin receptors. These findings demonstrate that hypoglossal discharge alone cannot distinguish eupnea from gasping nor, in eupnea, can hypoglossal activity be used to differentiate neural inspiration from expiration. These findings have significant negative implications for conclusions drawn from the in vitro medullary slice of mouse.

Genesis of gasping is independent of levels of serotonin in the Pet-1 knockout mouse.

Eupnea is normal breathing. If eupnea fails, as in severe hypoxia or ischemia, gasping is recruited. Gasping can serve as a powerful mechanism for autoresuscitation. A failure of autoresuscitation has been proposed as a basis of the sudden infant death syndrome. In an in vitro preparation, endogenous serotonin is reported to be essential for expression of gasping. Using an in situ preparation of the Pet-1 knockout mouse, we evaluated such a critical role for serotonin. In this mouse, the number of serotonergic neurons is reduced by 85-90% compared with animals without this homozygous genetic defect. Despite this reduction in the number of serotonergic neurons, phrenic discharge in eupnea and gasping of Pet-1 knockout mice was not different from that of wild-type mice. Indeed, gasping continued unabated, even after administration of methysergide, a blocker of many types of receptors for serotonin, to Pet-1 knockout mice. We conclude that serotonin is not critical for expression of gasping. The proposal for such a critical role, on the basis of observations in the in vitro slice preparation, may reflect the minimal functional neuronal tissue and neurotransmitters in this preparation, such that the role of any remaining neurotransmitters is magnified. Also, rhythmic activity of the in vitro slice preparation has been characterized as eupnea or gasping solely on the basis of activity of the hypoglossal nerve or massed neuronal activities of the ventrolateral medulla. The accuracy of this method of classification has not been established.

Eupnea of in situ rats persists following blockers of in vitro pacemaker burster activities.

Two groups of intrinsically bursting neurons, linked to respiration, have been identified using in vitro medullary slice preparations. One group is dependent upon a calcium-activated nonspecific cationic current that is blocked by flufanemic acid. This group is hypothesized as essential for eupnea, but not gasping. The second group is dependent upon conductance through persistent sodium channels that is blocked by riluzole. This group is proposed to underlie both eupnea and gasping. In the decerebrate in situ preparation of the juvenile rat, flufanemic acid caused an increase in frequency and a decrease in peak level of the phrenic and vagus nerve activities in both eupnea and gasping. Similar changes in eupnea followed the simultaneous blockades by flufanemic acid and riluzole. However, gasping was eliminated. These results do not support the hypothesis that conductances through either persistent sodium channels or calcium-activated nonspecific cationic channels are essential for the neurogenesis of eupnea. However, gasping does depend upon a conductance through persistent sodium channels.

Maintenance of gasping and restoration of eupnea after hypoxia is impaired following blockers of alpha1-adrenergic receptors and serotonin 5-HT2 receptors.

In severe hypoxia or ischemia, normal eupneic breathing fails and is replaced by gasping. Gasping serves as part of a process of autoresuscitation by which eupnea is reestablished. Medullary neurons, having a burster, pacemaker discharge, underlie gasping. Conductance through persistent sodium channels is essential for the burster discharge. This conductance is modulated by norepinephrine, acting on alpha 1-adrenergic receptors, and serotonin, acting on 5-HT2 receptors. We hypothesized that blockers of 5-HT2 receptors and alpha 1-adrenergic receptors would alter autoresuscitation. The in situ perfused preparation of the juvenile rat was used. Integrated phrenic discharge was switched from an incrementing pattern, akin to eupnea, to the decrementing pattern comparable to gasping in hypoxic hypercapnia. With a restoration of hyperoxic normocapnia, rhythmic, incrementing phrenic discharge returned within 10 s in most preparations. Following addition of blockers of alpha 1-adrenergic receptors (WB-4101, 0.0625-0.500 microM) and/or blockers of 5-HT2 (ketanserin, 1.25-10 microM) or multiple 5-HT receptors (methysergide, 3.0-10 microM) to the perfusate, incrementing phrenic discharge continued. Fictive gasping was still induced, although it ceased after significantly fewer decrementing bursts than in preparations than received no blockers. Moreover, the time for recovery of rhythmic activity was significantly prolonged. This prolongation was in excess of 100 s in all preparations that received both WB-4101 (above 0.125 microM) and methysergide (above 2.5 microM). We conclude that activation of adrenergic and 5-HT2 receptors is important to sustain gasping and to restore rhythmic respiratory activity after hypoxia-induced depression.

Persistence of eupnea and gasping following blockade of both serotonin type 1 and 2 receptors in the in situ juvenile rat preparation.

In severe hypoxia or ischemia, normal eupneic breathing is replaced by gasping, which can serve as a powerful mechanism for "autoresuscitation." We have proposed that gasping is generated by medullary neurons having intrinsic pacemaker bursting properties dependent on a persistent sodium current. A number of neuromodulators, including serotonin, influence persistent sodium currents. Thus we hypothesized that endogenous serotonin is essential for gasping to be generated. To assess such a critical role for serotonin, a preparation of the perfused, juvenile in situ rat was used. Activities of the phrenic, hypoglossal, and vagal nerves were recorded. We added blockers of type 1 and/or type 2 classes of serotonergic receptors to the perfusate delivered to the preparation. Eupnea continued following additions of any of the blockers. Changes were limited to an increase in the frequency of phrenic bursts and a decline in peak heights of all neural activities. In ischemia, gasping was induced following any of the blockers. Few statistically significant changes in parameters of gasping were found. We thus did not find a differential suppression of gasping, compared with eupnea, following blockers of serotonin receptors. Such a differential suppression had been proposed based on findings using an in vitro preparation. We hypothesize that multiple neurotransmitters/neuromodulators influence medullary mechanisms underlying the neurogenesis of gasping. In greatly reduced in vitro preparations, the importance of any individual neuromodulator, such as serotonin, may be exaggerated compared with its role in more intact preparations.

Maintenance of eupnea of in situ and in vivo rats following riluzole: a blocker of persistent sodium channels.

We have proposed a "switching" concept for the neurogenesis of breathing in which rhythm generation by a pontomedullary neuronal circuit for eupnea may be switched to a medullary pacemaker system for gasping. This switch involves activation of conductances through persistent sodium channels. Based upon this proposal, eupnea should continue following a blockade of persistent sodium channels. In situ preparations of the decerebrate, juvenile rat were studied in normocapnia, hypocapnia and hypercapnia. Regardless of the level of CO(2) drive, riluzole (1-10 microM), a blocker of persistent sodium channels, caused increases in the frequency and reductions in peak integrated phrenic height. Even 20 microM of riluzole, a concentration four-fold higher than that which eliminates gasping, did not cause a cessation of phrenic discharge. In conscious, rats breathing continued unabated following intravenous administrations of 3-9 mgkg(-1) of riluzole. These administrations did cause sedation. We conclude that conductance through persistent sodium channels plays little role in the neurogenesis of eupnea.

High-frequency oscillations in phrenic activity during pontile and medullary respiratory rhythms in rats.

High-frequency oscillations may be signatures of the basic mechanisms underlying the neurogenesis of various patterns of automatic ventilatory activity. These high-frequency oscillations in phrenic activity differ greatly in eupnoea and gasping, implying different mechanisms of neurogenesis. In a decerebrate, in situ preparation of the rat, the peak frequency of high-frequency oscillations fell in apneusis following removal of the rostral pons. Following removal of all pons, phrenic discharge had a mixed pattern of gasps and multiple bursts; some of the latter were incrementing, as in eupnoea. Regardless of pattern, peak frequencies were significantly below those which were found during eupnoea, apneusis or gasping of the decerebrate preparation. Results do not support the concept that 'non-gasping' rhythmic patterns that can be recorded following a removal of pons are generated by the same mechanisms as those generating eupnoea. Indeed, both pons and medulla appear essential for all aspects of eupnoea to be expressed.

Changes in respiratory-modulated neural activities, consistent with obstructive and central apnea, during fictive seizures in an in situ anaesthetized rat preparation.

Sudden unexplained death in epilepsy (SUDEP) has been proposed to result from seizure-induced changes in respiratory and cardiac function. Our purpose was to characterize changes in respiration during seizures. We used a preparation of the anaesthetized, perfused in situ rat. This preparation has the advantage over in vivo preparations in that delivery of oxygen to the brain does not depend upon the lungs or cardiovascular system. Electroencephalographic activity was recorded as were activities of the hypoglossal, vagus and phrenic nerves. The hypoglossal and vagus nerves innervate muscles of the upper airway and larynx while the phrenic nerve innervates the diaphragm. Fictive seizures were elicited by injections of penicillin into the parietal cortex or the carotid artery. Following elicitation of the fictive seizures, activities of the hypoglossal and vagal nerves declined greatly while phrenic activity was little altered. Such a differential depression of activities of nerves to the upper airway and larynx, compared to that to the diaphragm, would predispose to obstructive apnea in intact preparations. With more time, activity of the phrenic nerve also declined or ceased. These changes characterize central apnea. The major conclusion is that seizures may result in recurrent periods of obstructive and central apnea. Thus, seizures can adversely alter respiratory function in a profound manner.

Respiratory rhythm generation during gasping depends on persistent sodium current.

In severe hypoxia, homeostatic mechanisms maintain function of the brainstem respiratory network. We hypothesized that hypoxia involves a transition from neuronal mechanisms of normal breathing (eupnea) to a rudimentary pattern of inspiratory movements (gasping). We provide evidence for hypoxia-driven transformation within the central respiratory oscillator, in which gasping relies on persistent sodium current, whereas eupnea does not depend on this cellular mechanism.

Gap junction blockade does not alter eupnea or gasping in the juvenile rat.

The role of gap junctions in the brainstem respiratory control system is ambiguous. In the present study, we used juvenile rats to determine whether blocking gap junctions altered eupnea or gasping in the in situ, arterially perfused rat preparation. Blockade of gap junctions with 100 microM carbenoxolone or 300 microM octanol did not produce any consistent changes in the timing or amplitude of integrated phrenic discharge or in the peak frequency in the power spectrum of phrenic nerve discharge during eupnea or ischemic gasping beyond those changes seen in time-control animals. These findings do not rule out a role for gap junctions in the expression of eupnea or gasping, but they do demonstrate that these intermembrane channels are not obligatory for either rhythm to occur.

Long-term intracellular recordings of respiratory neuronal activities in situ during eupnea, gasping and blockade of synaptic transmission.

For a definitive evaluation of the hypothesis that different neurophysiological mechanisms underlie the neurogenesis of eupnea and gasping, long-term continuous intracellular recordings of respiratory neuronal activities during both respiratory patterns are required. Such recordings in vivo are technically difficult, especially in small mammals, due to mechanical instability of the brainstem and cardiovascular depression that accompany hypoxia-induced gasping. Respiratory-related rhythmic activities of in vitro preparations are confounded by the lack of a clear correspondence with both eupnea and gasping. Here, we describe new methodologies and report on whole cell patch clamp recordings from the ventrolateral medulla and the hypoglossal motor nucleus in situ during multiple bouts of hypoxia-induced gasping. The longevity of recordings (range 20--35 min) also allowed subsequent analysis of neuronal behaviour after blockade of inhibitory and excitatory synaptic activities. We conclude that whole cell patch clamp recordings in the in situ preparation will allow an analysis of both synaptic and ionic conductances of respiratory neurons during defined eupnea and gasping, providing an additional approach to in vitro preparations.

Phasic pulmonary stretch receptor feedback modulates both eupnea and gasping in an in situ rat preparation.

The perfused in situ juvenile rat preparation produces patterns of phrenic discharge comparable to eupnea and gasping in vivo. These ventilatory patterns differ in multiple aspects, including most prominently the rate of rise of inspiratory activity. Although we have recently demonstrated that both eupnea and gasping are similarly modulated by a Hering-Breuer expiratory-promoting reflex to tonic pulmonary stretch, it has generally been assumed that gasping was unresponsive to afferent stimuli from pulmonary stretch receptors. In the present study, we recorded eupneic and gasplike efferent activity of the phrenic nerve in the in situ juvenile rat perfused brain stem preparation, with and without phrenic-triggered phasic pulmonary inflation. We tested the hypothesis that phasic pulmonary inflation produces reflex responses in situ akin to those in vivo and that both eupnea and gasping are similarly modulated by phasic pulmonary stretch. In eupnea, we found that phasic pulmonary inflation decreases inspiratory burst duration and the period of expiration, thus increasing burst frequency of the phrenic neurogram. Phasic pulmonary inflation also decreases the duration of expiration and increases the burst frequency during gasping. Bilateral vagotomy eliminated these changes. We conclude that the neural substrate mediating the Hering-Breuer reflex is retained in the in situ preparation and that the brain stem circuitry generating the respiratory patterns respond to phasic activation of pulmonary stretch receptors in both eupnea and gasping. These findings support the homology of eupneic phrenic discharge patterns in the reduced in situ preparation and eupnea in vivo and disprove the common supposition that gasping is insensitive to vagal afferent feedback from pulmonary stretch receptor mechanisms.