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1Institute of Arctic Biology, University of Alaska Fairbanks, Alaska; and 2Department of Physiology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
Submitted 16 January 2007 ; accepted in final form 30 March 2007
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ABSTRACT |
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neurotransmitters
Our laboratory has long maintained that different physiological mechanisms are responsible for generating eupnea and gasping. Eupnea is hypothesized to result from a complex interaction of cellular and synaptic properties within a pontomedullary neuronal network, whereas gasping depends on intrinsic cellular mechanisms localized in the medulla (14, 21, 22). Within the medulla, gasping is irreversibly eliminated following ablation of neurons within a ventrolateral "pre-Botzinger" complex and adjoining "gasping center" (4, 8, 20–22). Neuronal activities within this region have the proper discharge pattern for generating the gasp in that their discharge commences just before or concomitant with the onset of the phrenic burst (14, 25). Some of these "preinspiratory" or "inspiratory" medullary neuronal activities acquire an intrinsic burster discharge following a blockade of fast excitatory and inhibitory synaptic transmission. This burster discharge is dependent on conductance through persistent sodium channels because blockers of this conductance eliminate both the burster discharge and also gasping of in situ or in vivo preparations (14). The same concentration of blockers causes little alteration of eupnea of in situ preparations having an intact pontomedullary brain stem or of unanesthetized in vivo preparations (27).
Our laboratory has proposed that gasping is generated by the synchronized activity of excitatory neurons some of which may have intrinsic pacemaker bursting properties dependent on a persistent sodium current (14). Conductances through persistent sodium channels may be markedly influenced by exogenous levels of serotonin (6, 7, 30). Given this linkage of persistent sodium channel, serotonin, and gasping, it is hypothesized that endogenous serotonin is essential for gasping to be generated. Such a critical linkage of gasping and serotonin has been proposed based on results from an in vitro slice preparation in which a blockade of serotonin 2A receptors eliminated one type of rhythmic activity, which is considered to be analogous to gasping (30).
The decerebrate in situ preparations of the juvenile and neonatal rat exhibit patterns of automatic ventilatory activity that are comparable to eupnea and gasping of in vivo preparations (14, 24–27). Using this preparation, we have evaluated the hypothesis that activation of serotonin 5-HT2A subtype is essential for the neurogenesis of gasping. Gasping was still generated and little altered following a blockade of both 5-HT2 and also 5-HT1 receptor subtypes. Eupnea could similarly still be generated. We conclude that serotonin is but one of many neurotransmitters that can influence mechanisms underlying the neurogenesis of gasping.
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METHODS |
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Seventy-four perfused preparations of the decerebrate juvenile rat were used. The preparation was identical to that described previously (24), with surgical procedures being performed under deep halothane anesthesia, as assessed by an absence of a withdrawal response to noxious pinching of a paw. Anesthesia was discontinued following decerebration. Gallamine triethiodide was added to the perfusate to eliminate spontaneous respiratory movements. These procedures were approved by the Institutional Care and Use Committee of Dartmouth College and Dartmouth Medical School
The descending aorta was cannulated, and perfusion was commenced. The perfusate contained the following in distilled water (in mM): 1.25 MgSO4, 1.25 H2PO4, 5.0 KCl, 25 NaHCO3, 125 NaCl, 2.5 CaCl2, 10 dextrose, and 0.1785 Ficoll 70. The temperature of the perfusate as it entered the aorta was 31°C. Under control conditions, the perfusate was equilibrated with a gas mixture of 95% O2-5% CO2.
Efferent activity of the phrenic nerve was recorded in all preparations. Efferent activity was also recorded from the central cut ends of the vagus nerve in 22 preparations, and the hypoglossal nerve was recorded in 29 preparations. Recordings were obtained during eupnea, with the perfusate equilibrated with 95% O2-5% CO2. To alter the pattern to gasping, the perfusion was terminated for a maximum of 70 s, thus producing a condition of ischemia.
Blockade of Serotonin Receptors
We blocked activities of serotonergic neurons using antagonists and agonists of the 5-HT1 and/or 5-HT2 class of receptors. As a mixed 5-HT1 and 5-HT2 receptor antagonist, methysergide maleate was used in concentrations of 5–40 µM. To block activities of neurons having 5-HT1A receptors, the agonist (R)-(+)-8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT) was added to the perfusate in concentrations of 1–3 µM. Activation of the 5-HT1A receptor, which is an autoreceptor located on serotonergic neurons, by 8-OH-DPAT results in hyperpolarization and a decrease in cell firing. Finally, as an antagonist of the 5-HT2 class of receptors, ketanserin tartrate (ketanserin; 5–30 µM) was used. These concentrations of methysergide, 8-OH-DPAT and ketanserin were chosen based on preliminary experiments in which increasing concentrations of drugs were added to the perfusate until a clear alteration of phrenic discharge in eupnea was obtained. Concentrations below and slightly above this level were then used during eupnea and gasping. All drugs were from Tocris and Sigma.
Experimental Protocol
Activities of the phrenic, hypoglossal and vagal nerves were recorded for a minimum of 30 min under control conditions, with the perfusate equilibrated with 95% O2-5% CO2. Neural discharges during this period had an eupneic pattern (14, 24). In one subset of experiments, perfusion was terminated for a maximum of 70 s, and neural discharges of gasping were recorded. Perfusion was then recommenced, and the eupneic pattern of neural discharges was reestablished. One of the blockers of serotoninergic receptors was then added to the perfusate, and neural activities recorded for a minimum of 10 min. Perfusion was again terminated for 70 s. A second round of recovery from ischemia was then performed in some experiments.
In a second subset of experiments, blockers of serotoninergic receptors were introduced during the initial phase of eupnea, with no ischemia before the introduction of drugs. Ten minutes after drug administration, ischemia was produced for 70 s followed by a 30-min recovery with normal perfusion. This second set of experiments was performed to preclude a prior influence of brain stem hypoxia/ischemia on the responses to drugs.
Analyses of Data
Integrated phrenic activity was analyzed as to the duration of the burst [neural inspiratory (TI)], period between bursts [neural expiratory (TE)], peak height, and time to reach peak height, expressed as a percentage of TI. Integrated activities of the hypoglossal and vagal nerves were analyzed as to their peak heights in neural inspiration and expiration. We also defined the difference in time of onset of hypoglossal and vagal activities compared with that of the phrenic nerve. All of these variables were defined in both eupnea and gasping. Statistical evaluations were via an ANOVA with a Bonferroni correction for multiple comparisons. For a few comparisons involving pooled data, a Wilcoxon test was used.
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RESULTS |
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The patterns of phrenic, hypoglossal and vagal activities during eupnea and gasping were as described previously (14, 23–27). In eupnea, integrated phrenic discharge had a sudden onset and then a rise to reach a peak level at a time close to the termination of the burst (Fig. 1). Vagal discharge commenced before that of the phrenic [mean of 471 ± 82 (SE) ms] and had a burst, of equal or greater amplitude, in neural expiration (Fig. 1). Hypoglossal discharge also commenced before the phrenic [mean = 352 ± 46 (SE) ms], but it was largely limited to neural inspiration (Fig. 2).
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Methysergide: Mixed 5-HT1 and 5-HT2 Receptor Antagonist
Alterations in eupnea. Eupnea continued following administrations of 2.5–40 µM of methysergide in 42 of 47 preparations. In the other five, apnea followed administration of 5.0 µM (2 of 14 preparations) or 20 µM (3 of 10 preparations).
For preparations in which rhythmic activities continued, much variability was recorded following methysergide with few significant changes being obtained (see Fig. 4). Peak integrated activity, compared with the same preparation before methysergide, was not altered except at 20 µM at which dose a decline was seen. In addition to data reported in Fig. 4, we also administered methysergide to three preparations at a concentration of 30 µM and one preparation at 40 µM. Eupnea continued, although peak phrenic height was reduced by >50% in each preparation.
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Alterations in Gasping
On a termination of perfusion and induction of ischaemia, gasping was recorded in 41 of 47 preparations that had received methysergide. Five of the other six had exhibited apnea during eupnea, as noted above, and no rhythmic activity returned in ischemia. One other preparation only exhibited tonic phrenic discharge in ischemia following administration of 5.0 µM of methysergide. Responses in gasping were the same whether or not ischemia had been introduced before administering methysergide or whether the drug was administered during the initial period of eupnea. Hence, all data during gasping have been pooled.
Comparison of Figs. 2 and 3 with Fig. 1 reveals that gasping following methysergide was similar to that of preparations that had received no drugs with few statistically significant changes being obtained (Fig. 4). Of interest is that hypoglossal discharge could become uncoupled from the phrenic burst in gasping following methysergide. This separation, evidenced by extra bursts of hypoglossal discharge (Fig. 2) or an absence of hypoglossal discharge, was frequently but not systematically observed.
Preparations that had received 30 µM (n = 3) or 40 µM (n = 1) methysergide also exhibited gasping, but, as in eupnea, peak phrenic height was greatly reduced compared with values before the drug.
8-OH-DPAT: 5-HT1A Receptor Agonist
As for methysergide, eupnea and gasping persisted following administrations of 8-OH-DPAT to 13 preparations. Changes were limited solely to increases in frequency of the phrenic burst in eupnea following 1.5 and 3.0 µM and of gasping following 1.5 µM. These changes were due to diminutions in the interval between bursts (TE) (Fig. 5).
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Alterations in eupnea. Ketanserin, at concentrations of 5–30 µM was administered to a total of 21 rats. Again, much variability in responses was observed between preparations, and, whereas, the frequency of phrenic bursts generally increased and the peak phrenic height generally declined (Figs. 6 and 7), no changes were statistically significant (Fig. 8). No preparation exhibited apnea.
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Alterations in Gasping
In ischemia, multiple gasps were observed in all but two preparations in which only a single gasp, following by a tonic discharge was recorded. Both of these preparations had received 10 µM of ketanserin.
The duration of the phrenic burst (TI), interval between bursts (TE), and frequency of gasping were not significantly different in preparations that received ketanserin compared with control preparation. However, as is shown in Figs. 6 and 7, by comparison with peak height of phrenic discharge in eupnea before administration of ketanserin, this peak height was reduced in gasping after the drug had been administered. Also reduced was the peak height of hypoglossal discharge and, indeed, hypoglossal and phrenic discharges became uncoupled in many preparations following ketanserin (Fig. 7).
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DISCUSSION |
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Differences Between in Vitro and in Situ Findings
We believe that the relationship between rhythmic activities of various in vitro preparations with eupnea and gasping in vivo or in situ continues to be vague (see, e.g., 14, 26, 27). Yet, it is clear that in one type of medullary slice preparation, its rhythmic decrementing discharge and the discharge of one group of burster neurons were both eliminated following a blockade of receptors for serotonin (30). These burster neurons, which are dependent on conductances through persistent sodium channels, are located in the medullary region, which is critical for gasping (14).
Our finding that gasping continued following administration of blockers of serotonin might mean that these blockers did not reach medullary neurons that are critical for the neurogenesis of gasping and/or did not reach these neurons in sufficient concentration. Although changes in peak heights of cranial and spinal nerves and respiratory frequency did occur following administration of blockers, these changes might reflect actions upon multiple components of the pontomedullary and spinal respiratory network (1, 2, 11, 13, 16).
Another explanation for differences between findings from in vitro and in situ preparations is that the importance of neuromodulators such as serotonin may be greatly exaggerated in substantially reduced in vitro preparations. In both the in vitro medullary respiratory system and in many other components of the central nervous system, conductances through persistent sodium channels may be markedly influenced by exogenous levels of serotonin (6, 7, 30). Yet, in addition to serotonin, multiple neuromodulators influence the conductance through persistent sodium channels (e.g., Refs. 6, 7, 30, 31). In in vitro slice preparations, in which neural pathways and endogenous neurotransmitters may be substantially reduced, an alteration in sensitivity to remaining neurotransmitters may be established (6, 7). We submit that in a more intact preparation, norepinephrine and other transmitters that modulate conductance through persistent sodium channels would render endogenous levels of serotonin as of minor importance for the neurogenesis of gasping.
Eupnea and Gasping in Situ: Confirmation of Previous Results
The alterations in the discharge patterns of vagal, hypoglossal and phrenic nerves on exposure to ischemia confirm findings reported in detail previously. Hence, the incrementing pattern of phrenic discharge was altered to a decrementing discharge as were the integrated discharge patterns of the vagus and hypoglossal nerves (21, 24–26). Moreover, the earlier onset of hypoglossal and vagal, compared with phrenic, discharge was eliminated with the change from eupnea to gasping. Also eliminated was the marked discharge of the vagus nerve in early neural expiration (21, 24–26).
Another observation that confirmed previous findings was the uncoupling of hypoglossal from phrenic discharge following administration of blockers of serotonin (23). This uncoupling reinforces the concept, established in many publications, of a differential control of respiratory-modulated activities of cranial compared with spinal neural activities (e.g., Refs. 18, 19, 23). Moreover, this uncoupling is also compatible with the conclusion that serotonin may modulate and coordinate different rhythms but that it is not essential for the generation of any particular rhythm.
Endogenous Serotonin Modulates Eupnea and Gasping
The serotonin system is widespread in the brain stem, and changes in release of serotonin result in multiple alterations in homeostatic functions (1, 2, 11, 13, 16). For the respiratory system, influences on activities of premotor and both cranial and spinal motoneurons are well described. In this context, our results indicate that serotonergic mechanisms may be of importance for the earlier onset of discharge of hypoglossal and vagal than phrenic discharge, because this earlier onset was lessened or eliminated following blockade of serotonin receptors. Yet, the concomitant change in respiratory frequency obscures the specificity of this change in onset of neural activation. Thus serotonin increased the respiratory frequency, which may shorten the lead time between hypoglossal and vagal activities and the onset of phrenic discharge. Finally, an importance of serotonin for "central chemoreception" has also been advanced (17), but this importance is unclear (12, 16). Given these widespread influences of serotonin, the specific factors cannot be defined that are responsible for the changes in both frequency and peak height of activities of cranial and spinal nerves that were observed in some preparations following administration of blockers of the various types of serotonin receptors.
Serotonin, Gasping, and Sudden Infant Death Syndrome
Recently, widespread abnormalities in the serotonergic system within the brain stems of victims of the "sudden infant death syndrome" (SIDS) have been reported (13). Yet, numerous attempts to link an interruption of the serotonergic system with a major failure of the cardiorespiratory system, as would be expected in SIDS, have been unsuccessful (see discussion in Ref. 16). It has long been hypothesized that the SIDS reflects a failure of gasping and/or a failure of gasping to be an effective mechanism of autoresuscitation (3). The importance of endogenous serotonin for the neurogenesis of a rhythm in vitro led to the supposition that serotonin might also be critical for gasping in vivo and, by extension, establish a link between serotonin and SIDS (30). Results reported herein do not confirm that serotonin is an exclusive and necessary factor for the neurogenesis of gasping. Perhaps the lack of significant changes in cardiorespiratory function following interruption of the serotoninergic system in vivo points to the multiplicity of neurotransmitter and neuromodulators that define this function. Hence, interruption of any single neuromodulator/neurotransmitter system alone would not lead to SIDS.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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