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1-adrenergic receptors and serotonin 5-HT2 receptorsDepartment of Physiology, Dartmouth Medical School, Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
Submitted 5 June 2007 ; accepted in final form 24 December 2007
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1-adrenergic receptors, and serotonin, acting on 5-HT2 receptors. We hypothesized that blockers of 5-HT2 receptors and 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 1-adrenergic receptors (WB-4101, 0.0625–0.500 µM) and/or blockers of 5-HT2 (ketanserin, 1.25–10 µM) or multiple 5-HT receptors (methysergide, 3.0–10 µM) 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 µM) and methysergide (above 2.5 µM). 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. control of breathing; sudden infant death syndrome
Our laboratory has proposed that different neurophysiological mechanisms are responsible for generating eupnea and gasping. Eupnea is hypothesized to be generated by interactions within a pontomedullary neuronal network, whereas gasping depends on pacemaker mechanisms localized in the medulla (20, 21). This pacemaker discharge is dependent on conductance through persistent sodium channels (20, 21).
Conductance through persistent sodium channels is influenced by endogenous levels of multiple neuromodulators/neurotransmitters, including, most prominently, serotonin, acting on 5-HT2 receptors, and norepinephrine, acting on 1-adrenergic receptors (8, 9, 33, 34). Activation of 5-HT2 receptors was reported as critical for gasping to be expressed, based on findings using an in vitro medullary slice preparation (33). Our laboratory did not confirm this critical link between activation of 5-HT2 receptors and the generation of "fictive" gasping in an in situ preparation of the juvenile rat having an intact pontomedullary brain stem (32). As opposed to results in vitro, fictive gasping continued following administrations of ketanserin, an antagonist acting primarily of 5-HT2 receptors. Moreover, several other types of 5-HT receptors would also appear not to be critical for the neurogenesis of gasping because administrations of neither methysergide nor (R)-(+)-8-hydroxy-2(di-n-propylamino) tetralin (8-OH-DPAT) eliminated gasping. Methysergide is an mixed antagonist of 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors, as well as a weak agonist of 5-HT1 receptors. 8-OH-DPAT, by activation of the 5-HT1A receptor, which is an autoreceptor located on serotonergic neurons, causes hyperpolarization and a decrease in cell firing (3–6, 11, 12, 27, 32).
The difference in importance of activation of 5-HT2 receptors for the genesis of "gasping rhythms" of in vitro compared with in situ preparations may reflect the substantial reduction of neural pathways and, it is assumed, endogenous neurotransmitters of the in vitro medullary slice, compared with the in situ preparation, which has an intact pontomedullary brain stem. We therefore proposed that in this preparation having an intact pons and medulla, norepinephrine and other neurotransmitters that modulate conductance through persistent sodium channels minimized the alterations in fictive gasping that our laboratory observed follow a blockade of 5-HT2 and the various other classes of 5-HT receptors (32). Implicit to this proposal is that a blockade of 1-adrenergic receptors, although not eliminating fictive gasping per se, might still alter the gasping pattern. Specifically, we hypothesize that this blockade of 1-adrenergic receptors might reduce that number of fictive gasps that are elicited and/or delay the reinstatement of rhythmic, incrementing phrenic discharge following a period of fictive gasping. We further hypothesize that such influence of a blockade of 1-adrenergic receptors would be more pronounced if combined with a simultaneously blockade of 5-HT2 and, possibly, other 5-HT receptors.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Ninety-six perfused preparations of the decerebrate juvenile rat were used. Rats were of age 21–30 days. The preparation was identical to that described previously (20, 30, 31), with surgical procedures being performed under deep halothane or enflurane anesthesia, as assessed by an absence of a withdrawal response to noxious pinching of a paw. Anesthesia was discontinued following decerebration.
The descending aorta was cannulated, and perfusion was commenced. The perfusate contained the following in distilled water: MgSO4 (1.25 mM), KH2PO4 (1.25 mM),KCl (3.0 mM), NaHCO3 (24 mM), NaCl (125 mM), CaCl2 (2.5 mM), dextrose (10 mM), and Ficoll 70 (0.1785 mM). Note that the composition of this perfusate was similar to that used previously (20, 30, 31) except that the total potassium level was reduced to approximate more closely that of the serum of the rat. Gallamine triethiodide was added to the perfusate to block neuromuscular transmission. 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 (hyperoxic normocapnia).
Efferent activity of the phrenic nerve was recorded. This activity was amplified, filtered (0.6–6.0 kHz) and integrated (50-ms time constant).
Eupnea and Gasping
With the perfusate equilibrated with a hyperoxic-normocapnic gas mixture (95% O2-5% CO2), integrated phrenic discharge had an incrementing pattern, which is akin to eupnea (20, 21, 30–32). To alter this phrenic pattern to a decrementing discharge, which our laboratory has concluded is as gasping (20, 21, 30–32), the perfusate was switched to one that was of identical composition, including concentration of drugs (see below), but was equilibrated with a hypoxic-hypercapnic gas mixture (6–7% O2-7–8% CO2). The hypoxic-hypercapnic perfusate was delivered to the preparation for 80 s, after which the hyperoxic-normocapnic (95% O2-5% CO2) perfusate was reintroduced. The time for exposure to the hypoxic-hypercapnic mixture was chosen based on preliminary studies in which we established that, with this exposure, the phrenic pattern was systematically switched from incrementing to decrementing and, yet, the incrementing pattern could be re-established in hyperoxic-normocapnia.
Agonist and Antagonist of Receptors
To block the 1-adrenergic receptor, WB-4101 was used. As an agonist of this class of receptors, we added methoxamine hydrochloride to the perfusate.
We also administered methysergide maleate and ketanserin tautrate. Methysergide is an antagonist acting on multiple types of 5-HT receptors, including 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7. Ketanserin is a more specific blocker primarily of the 5-HT2 class of receptors (3, 4, 5, 6, 11, 12). The 5-HT2 agonist 2,5-dimethoxy-4-iodophenyl (DOI) was also used.
This study with blockers and agonists of 5-HT receptors was performed to extend findings from our laboratory's previous study in which the influence of these blockers on fictive eupnea and ischemia-induced gasping had been assessed (32). In this previous study, the emphasis was on the elicitation of the decrementing phrenic pattern of gasping, per se, and the periods of ischemia were variable. Given these different durations of ischemia in different preparations, any changes in variables such as number of decrementing phrenic bursts and duration of apnea before the reestablishment of rhythmic incrementing phrenic bursts could not reasonably be correlated with administration of various blockers.
In another set of experiments, blockers of both 1-adrenergic receptor and the various classes of 5-HT receptors were administered simultaneously. All drugs were from Sigma or Tocris.
The concentration of agonists and antagonists was based on previous studies (32) and/or preliminary experiments in which drugs were delivered in increasing concentrations until a clear change in the incrementing phrenic discharge of fictive eupnea was observed. Concentrations above and below this level were then tested.
Experimental Protocols
A minimum of 30 min following the start of perfusion, incrementing phrenic discharge was recorded in hyperoxic-normocapnia. During an initial series of control experiments using 15 rats, the decrementing phrenic discharge of fictive gasping was then recorded in hypoxic-hypercapnia. After a return to hyperoxic-normocapnia, the incrementing phrenic pattern was again recorded.
Preparations to receive antagonists or agonists of the 1-adrenergic or 5-HT receptors did not undergo exposure to hypoxia-hypercapnia before administration of drugs. Rather, activities were initially recorded for a minimum of 30 min in hyperoxic-normocapnia. Drugs were then added to the perfusate, and recordings continued for an additional 10 min. The perfusate was then switched to that equilibrated with the hypoxic-hypercapnic mixture; the agonists and/or antagonists had also been added to this second perfusate. Eighty seconds thereafter, the hyperoxic-normocapnia perfusate was reintroduced, and activities were recorded for a minimum of 30 min.
Analyses of Data
Integrated phrenic activity was analyzed as to the duration of the burst (neural inspiratory), period between bursts, peak height, and time to reach peak height, expressed as a percentage of duration of the burst. All of these variables were defined in both fictive eupnea and fictive gasping. In addition, we defined the time from the introduction of the hypoxic-hypercapnic perfusate to the first decrementing phrenic burst of fictive gasping, the number of decrementing bursts, and the period without phrenic discharge that intervened after the reintroduction of the hyperoxic-normocapnic perfusate until the first phrenic burst reappeared. Statistical evaluations were made using t-tests, 
2, or one-way ANOVAs as appropriate. When an ANOVA indicated that significant differences existed between groups, specific preplanned comparisons were made using P values adjusted by the Bonferroni method. Data are presented as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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In hyperoxic normocapnia, phrenic discharge was characterized by a sudden onset and then rise to reach a peak value during the last half of the burst (60 ± 4.9% of neural inspiration). With an alteration to the perfusate equilibrated with a hypoxic-hypercapnic gas mixture, phrenic discharge initially increased. The pattern was then altered such that peak discharge was achieved in the first portion of the burst (39 ± 2.3% of neural inspiration). This decrementing pattern of phrenic discharge is typical of fictive gasping (Fig. 1). The durations of the phrenic burst, period between bursts, frequency, and peak heights were very similar during the incrementing (eupnea) and decrementing (gasping) patterns, with no significant difference between variables during the two patterns. Thus, for all 15 preparations, the frequency of incrementing phrenic bursts averaged 12.4 ± 0.68 (SE)/min and decrementing bursts was 12.7 ± 1.0/min. Peak height in fictive gasping averaged 96.5 ± 4.0% of the value in fictive eupnea.
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1-Norepinephrine Receptor: Antagonist and Agonist (28 Preparations)
Alterations in incrementing phrenic pattern of fictive eupnea. Following administration of WB-4101 to 22 preparations in concentrations of 0.0625 (n = 6), 0.125 (n = 5), 0.250 (n = 5), or 0.500 (n = 6) µM, apnea resulted in three preparations. These preparations had received 0.0625, 0.125, or 0.250 µM. For all others, the phrenic pattern continued with the frequency of bursts having increased (e.g., Fig. 2). This increase in frequency was due to a significant decline in the duration of neural expiration, and the increase in frequency occurred at all doses (P < 0.001), but the size of the increase did not differ among doses. Thus, after administration of WB-4101, mean frequencies at the various concentrations were as follows: 0.0625 µM, 27 ± 8.5/min; 0.125 µM, 15 ±4.0/min; 0.250 µM, 23.5 ± 8.4/min; 0.500 µM, 19.8 ± 5.8/min. The duration of neural inspiration, the time to reach peak height of integrated phrenic discharge, and the peak height per se were not altered compared with controls at any concentration of antagonists. For peak height, mean values, expressed as a percentage of values before drugs, were as follows: 0.0625 µM, 98 ± 10%; 0.125 µM, 104 ± 10%; 0.250 µM, 79 ± 9%; 0.500 µM, 84 ± 9%.
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1-adrenoceptor agonist, was administered to six preparations at a concentration of 10 µM. Changes were again limited to increases in the frequency of phrenic bursts [mean = 187 ± 16 (SE) % of controls, P < 0.001], reflecting a decreased in both neural inspiration and expiration. Peak height was unaltered (96 ± 4.6% of control). Alterations in decrementing phrenic pattern of fictive gasping. On exposure to hypoxic-hypercapnia, a decrementing phrenic pattern was elicited in 18 of the 22 preparations that had received WB-4101 (Fig. 2). Phrenic discharge had ceased in three of the others in hyperoxia and, in one additional preparation, phrenic discharge ceased in hypoxia. For the 18 preparations that exhibited fictive gasping, the time between the onset of hypoxia to the first decrementing burst (54 ± 5.0 s) was the same as control preparations that had not received drugs (53 ± 4.6 s) and did not vary as a function of drug dose. The duration of neural inspiration was similar to control, whereas the duration of neural expiration fell and the frequency rose at the higher concentrations of WB-4101. The increase in frequency reached statistical significance only at the 0.25 µM dose (frequency = 32.5 ± 17.5/min) compared with control (12.7 ± 1.0/min; P < 0.05). At higher concentrations, peak integrated phrenic discharge declined, but this change was not statistically significant (e.g., mean of 77.6 ± 9.5% of control at 0.500 µM).
Another parameter that differed from control preparations was the number of decrementing bursts. Only three of 18 preparations exhibited 10 or more such fictive gasps. This number differed significantly (P < 0.0004, 2) from that of control preparations that received no drugs in which 12 of 15 preparations exhibited 10 or more decrementing bursts. Also significantly different from controls was the period without phrenic activity, which intervened following the reintroduction of hyperoxia. As noted above, for controls, the duration of this apnea was <10 s in 10 of 14 preparations (1 preparation never recovery rhythmic respiratory activity). Following administrations of WB-4101, an apnea of <10 s was found for only 5 of 18 preparations (P < 0.03, 2). However, as shown in Fig. 3, there was much variability in this duration, even at the same concentration of drug. Hence, mean values of duration of apnea following drugs were not significantly different from those of control.
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5-HT Receptors: Antagonists and Agonist (30 Preparations)
Alterations in incrementing phrenic pattern of fictive eupnea. Changes in phrenic discharge were as previously reported (32) with a significant increase in frequency following either methysergide (n = 5) (e.g., Fig. 4) or ketanserin (n = 6) (P < 0.05 for both drugs), but no significant change in peak integrated phrenic activity (methysergide = 98 ± 9.0% of control, ketanserin = 106 ± 6.0% of control). The 5-HT2 agonist DOI (n = 19) also caused an increase in frequency at all concentrations tested (Fig. 5) (0.312 to 10 µM), with the exception of one preparation in which phrenic discharge ceased following administration of 10 µM of DOI. Peak heights were again unaltered, averaging 102 ± 7.9% of control at all concentrations of DOI.
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The time in hypoxic-hypercapnia prior to the onset of the first decrementing bursts was the same following DOI (54 ± 3.0 s) as in controls (53 ± 4.6 s). Changes in the durations of neural expiration and frequency following DOI were variable, even at the same concentration. The number of decrementing bursts was typically less than in control preparations, with only 3 of 19 preparations exhibiting 10 or more decrementing bursts following DOI (P < 0.0003 compared with controls that received no drugs). Also different from controls was the recovery of rhythmic activity after hypoxia, with the time following DOI (96 ± 8.0 s) being longer than in controls (37 ± 18 s, P < 0.01). However, as shown in Fig. 6, there was much variability in this time for rhythmic activity to recommence, even at a single concentration of DOI.
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1-Norepinephrine Receptor and 5-HT Receptors (23 Preparations)Alterations in incrementing phrenic pattern of fictive eupnea. Based on findings from the studies noted above, we added the following to the perfusate: 1) WB-4101 (0.015, 0.03, 0.0625, 0.125 µM), 2) methysergide maleate (1.25, 2.5, 5.0, 10 µM), and 3) ketanserin tautrate (3.0, 5.0, 10 µM). In studies with the combination of WB-4101 and methysergide (n = 15), we increased the concentration of both in individual groups of preparations [i.e., 1) WB-4101 (0.015 µM) + methysergide (1.25 µM), 2) WB-4101 (0.03 µM) + methysergide (2.5 µM), 3) WB-4101 (0.0625 µM) + methysergide (5.0 µM), 4) WB-4101 (0.125 µM) + methysergide (10 µM)]. In studies with the combination of WB-4101 and ketanserin (n = 8), the former was 0.125 µM in all trials.
Following additions of either WB-4101 and methysergide or WB-4101 and ketanserin to the perfusate, incrementing phrenic bursts continued except in two preparations which exhibited apnea after receiving 0.03 µM of WB-4101 and 2.5 µM of methysergide. For all other preparations, the frequency of phrenic bursts increased in each preparation whereas the peak phrenic height was variably altered, with both small increases and decreases being observed in different preparations (e.g., Figs. 7 and 8). Thus peak heights averaged 80.3 ± 9.3% of control values (range = 33–146% of control) for all preparations that received WB-4101 and methysergide and 79 ± 9.3% of control values (range = 36–182% of controls) for all preparations that received WB-4101 and ketanserin. No evidence of an influence of the concentration of the various agonists on the magnitude of the changes could be discerned.
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The time to the first decrementing burst of preparations that have received WB-4101 and methysergide or WB-4101 and ketanserin was not significantly different from that of the group of 15 control preparations, reported above, that had received no blockers.
Following the administrations of WB-4101 and methysergide, the number of decrementing bursts was markedly and significantly reduced. Ten or more decrementing bursts were recorded in 12 of 15 control preparations that had not received blockers but in only 1 of 11 preparations in which any decrementing bursts were observed following WB-4101 and methysergide (P < 0.001). Following WB-4101 and ketanserin, 10 or more decrementing bursts were recorded from 3 of 7 preparations (not significantly different from controls). Again, no correlation was evident between concentration of blockers and the reduction of the number of decrementing bursts.
In those preparations that did exhibit fictive gasping following blockers, frequency, peak height, and rate of rise of phrenic discharge were not significantly different from those of control preparations that had received no drugs.
Following the 80 s of exposure to hypoxic-hypercapnia, hyperoxic-normocapnia was reinstated. The time for recovery of rhythmic phrenic activity was markedly prolonged in preparations that received WB-4101 and methysergide (251 ± 33 s) or WB-4101 and ketanserin (264 ± 112 s), compared with control preparations that had not received drugs (37 ± 18 s). However, as shown in Fig. 9, there was again no relationship between the duration of the apnea and the concentration of blockers that had been administered. What is evident from Fig. 9 is that the duration of apnea was prolonged with more consistency in preparations that received both WB-4101 and methysergide than in preparations that had received no drugs or WB-4101 and methysergide alone. Stated differently, at all concentrations of WB-4101 above 0.125 µM and methysergide above 2.5 µM, the duration of apnea was in excess of 100 s in every preparation. In control preparations, apnea in excess of 100 s was observed in only 3 of 15 preparations, one of which never recovered from apnea. This difference between control preparations and those receiving both WB-4101 and methysergide was significant (P < 0.0005, 2). A similar comparison between preparations receiving WB-4101 alone and those receiving both WB-4101 and methysergide was also significant (P < 0.01, 2) as was the comparison between those receiving methysergide alone and those receiving both drugs (P < 0.01, 2).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1-adrenergic receptors or multiple serotonin receptors, including 5-HT1 5-HT2, 5-HT4, 5-HT6, and 5-HT7. However, activation of both of these groups of adrenergic and serotoninergic may modulate gasping once this pattern is elicited. Thus, with blockade of either these 1-adrenergic or serotoninergic receptors, or especially with a blockade of both groups of receptors, the decrementing phrenic bursts of fictive gasping were not sustained in hypoxia. This failure to sustain fictive gasping was followed in many preparations by a marked prolongation of the apneic period before rhythmic phrenic discharge returned. These results suggest that the processes initiating fictive gasping, which occurred in the majority of preparations, and the processes sustaining decrementing phrenic bursts of fictive gasping, which were deficient after combined blockade of 1-adrenergic and serotoninergic receptors, are distinct and modulated by serotonergic and noradrenergic receptor activation.
Our results lead directly to two primary questions. First, what is the physiological mechanism by which blockade of 1-adrenergic and serotoninergic receptors modulate gasping? Second, after the administration of blockers of 1-adrenergic and serotoninergic receptors, what is the mechanism that can account for the prolongation of the apneic period that intervened after the cessation of hypoxic hypercapnia?
Modulation of Gasping by Blockade of 1-Adrenergic and Serotoninergic Receptors
As noted in the introduction, we and others have proposed that gasping is critically dependent on conductance through persistent sodium channels (20, 21). Significant evidence exists that this conductance is modulated by endogenous levels of serotonin and norepinephrine, specifically acting on 5-HT2 and 1-noradrenergic receptors (8, 9, 22, 33). These two receptors are coupled to the same Gq proteins and are presumed to converge on the same intracellular pathway (8, 9, 11, 12, 16, 18). Such a convergence would explain why blockers of either 1-noradrenergic or 5-HT2 receptors caused a similar modulation of fictive gasping. This convergence would also explain why this alteration in fictive gasping is not severe following a blockade of either receptor alone, because redundancy exists in the system. Finally, this convergence would explain why prolongations of apnea following fictive gasping were more consistently obtained following the simultaneous administration of blockers of 1-noradrenergic and 5-HT receptors than following a single blocker.
From our results, it would appear that 5-HT2 receptors cannot be unique in modifying gasping among receptors for serotonin. Changes in fictive gasping and in the duration of apnea following hypoxic hypercapnia were more marked after administrations of methysergide, which blocks many types of receptors, than ketanserin, which blocks primarily the 5-HT2 receptors (3–6, 11, 12).
Concerning blockade of 1-adrenergic receptors, our results in this study of fictive gasping in the perfused in situ preparation are in general agreement with those from an in vitro preparation in which norepinephrine was reported to increase the frequency of bursts of one class of pacemaker neurons that are proposed to underlie a gasplike rhythm in vitro (34). These "cadmium-insensitive" pacemakers are, like those described in the in situ preparation, dependent on conductance through persistent sodium channels (20, 21, 33, 34).
Failure of Gasping and Autoresuscitation
The potency of gasping as an effective mechanism of autoresuscitation, especially in the newborn, has long been recognized (see Refs. 7, 13, 24, 29 for review). This effectiveness of gasping is linked to the restoration of adequate oxygenation of the brain such that normal cardiovascular function and eupnea are both reestablished. A corollary to this effectiveness of autoresuscitation is that a failure of gasping to reestablish adequate oxygenation leads to terminal apnea and irreversible cardiac failure. Yet, our results demonstrate that a failure of autoresuscitation cannot be linked solely to the reestablishment of adequate oxygenation by gasping. Because the magnitude and duration of hypoxia were identical in control preparations and those that received blockers of 1-noradrenergic or 5-HT receptors, the reduced number of fictive gasps and prolonged apnea must be linked to a direct alteration in the brain stem ventilatory control system rather than a secondary alteration due to hypoxia.
The mechanisms by which a blockade of 1-noradrenergic or 5-HT receptors might hinder the recovery of rhythmic phrenic discharge from a hypoxic-induced depression can only be speculative. A significant complexity is that of 1-noradrenergic and 5-HT receptors are ubiquitous in the brain stem and the sources of norepinephrine and serotonin are similarly ubiquitous (e.g., Refs. 2, 4, 6, 23, 28). Activation of neurons in multiple regions that can release these transmitters results in both augmentations and depressions of respiratory activity. Concerning serotonin, these multiple actions would explain our finding that both relatively high concentrations of the agonist DOI and the antagonist methysergide caused similar changes in fictive gasping and in recovery from hypoxic-hypercapnia. Another possibility is that these similar changes of methysergide and DOI represent a secondary action of methysergide as a weak agonist on 5-HT1 receptor (27). DOI, while primarily an agonist on 5-HT2 receptors, is also a weak agonist acting on 5 HT1 receptors (35). We consider this explanation of the similar responses to methysergide and DOI improbable because these similar responses were observed over a range of concentrations and not at a single high concentration as would be expected if these similar responses were due to secondary actions on 5-HT1 receptors. Also, ketanserin, which is an antagonist at 5-HT2 receptors, resulted in similar changes as methysergide.
Despite lack of information concerning a critical region of action, our findings do indicate that activation of 1-noradrenergic and 5-HT receptors may be more important in restarting eupnea following a hypoxia-induced depression than in a tonic modulation of eupnea, per se. Thus, as shown herein and in a previous study (32), blockade of multiple types of 5-HT receptors or 1-noradrenergic receptors causes little alterations of eupnea. This restoration of eupnea would not appear to represent actions of serotonin or norepinephrine on chemoreceptor mechanisms. Hence, although controversial, a significant role for these neurotransmitters in central chemoreception has been proposed (25 but see 17). Yet a change in central chemoreceptor mechanisms, such as an alteration in the "apneic threshold," cannot account for the sustained apnea that we recorded. Given that the levels of carbon dioxide in the perfusate were fixed, rhythmic phrenic discharge should have never recommenced if the blockers had altered the apneic threshold levels.
Limitations of the Study
While the in situ preparation of the rat exhibits patterns of ventilatory activity comparable to eupnea and gasping of in vivo preparations, the in situ preparation is decerebrate and hypothermic as opposed to intact preparations. Decerebration would remove binding sites for serotonin and norepinephrine in portions of the central nervous system rostral to the mesencephalon. Thus the response of the brain stem respiratory control system to blockers of receptors for 5-HT and norepinephrine might differ in the in situ and in vivo preparations. Compounding this difference in sensitivity would be the reduced temperature of the in situ preparation, which would be expected to alter the binding characteristics of antagonists to receptors. Concerning the binding to receptors, the concentrations of antagonists and agonist that we used in the present study were in the micromolar range, whereas the maximum receptor occupancy is achieved in nanomolar quantities (3–6, 11, 12). However, comparison of this difference in effective concentration is confounded by the difference in diffusion distance to the receptors in various preparations that have been used for analysis of the characteristics of receptor binding.
A final confounding issue of this study is the absence of a direct dose-response relationship between the concentration of antagonists and the diminution in the number of fictive gasps or the augmentation in duration of apnea following the termination of hypoxia. Although we have no firm explanations for this absence of a dose response, the following points would apply. 1) Levels of multiple neurotransmitters are altered in hypoxia (e.g., Ref. 26), and, hence, the levels of any one transmitter might vary in individual preparations. 2) Despite variability, the concentrations of antagonists and agonists that were administered may have produced a maximal response in many preparations even at relatively low doses. 3) In the in situ preparation, viability was maintained by an extracorporeal circuit that removed multiple mechanisms, including continuing hypoxia and hypercapnia, which might have elicited physiological mechanisms to compensate for the prolonged apnea. In fact, the duration of apnea was far in excess of that which would be compatible with survival of an in vivo preparation.
5-HT and Adrenergic Receptors and Sudden Infant Death Syndrome
The sequence of changes following the simultaneous blockade of 1-noradrenergic and 5-HT receptors, including minimal changes in the incrementing phrenic discharge of fictive eupnea, a limited period of the decrementing phrenic discharge of fictive gasping, and failed autoresuscitation is remarkably similar to what has been reported during monitoring of respiration of victims of the sudden infant death syndrome (SIDS) (24, 29). Based on a neurohistochemical evaluation of brain stems of victims of SIDS, a proposal as to its physiological basis has been advanced (19). This proposal, in its most recent form, holds that a deficiency in the brain stem serotonergic system is critical and sufficient to result in a failure of breathing in response to an exogenous stressor, such as hypoxia or hypercapnia. Our findings, although limited to the classes of receptors blocked by methysergide and ketanserin, combined with those of others involving widespread destruction of serotoninergic neurons (23), demonstrate that deficiencies in the serotonin system alone cause but minor changes in cardiorespiratory activity and responses to exogenous stressors of the cardiorespiratory system.
We propose that a deficiency in multiple neurotransmitters/neuromodulators, including serotonin, norepinephrine, and/or others, or a deficiency in receptors for these neurotransmitters within the brain stem respiratory control system may be the physiological basis for SIDS (see also Ref. 13). Thus our findings support the hypothesis that a serotonergic deficiency may contribute to SIDS but probably in the setting of one or more additional deficiencies in neurotransmitter systems (see, e.g., Ref. 10). Moreover, the deficient serotonergic and noradrenergic activities may be more important in preventing the autoresuscitation from apneic events, which may initiate the processes leading to SIDS, than in initiating the apnea itself (13).
Importantly, our proposal that alterations in the serotonergic and noradrenergic systems may contribute to SIDS is based solely on acute responses to administration of drugs. The neurohistochemical changes in brainstems of victims of SIDS imply changes of long duration. Hence, the linkage to a common mechanism for changes in respiratory activity following administration of drugs with changes due to long-term developmental abnormalities must be consisted as tenuous.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1 adrenoreceptors induces protein kinase C-dependent persistent changes in motoneuronal excitability. J Neurosci 27: 4435–4442, 2007.This article has been cited by other articles:
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  J. T. Erickson and B. C. Sposato Autoresuscitation responses to hypoxia-induced apnea are delayed in newborn 5-HT-deficient Pet-1 homozygous mice J Appl Physiol, June 1, 2009; 106(6): 1785 - 1792. [Abstract] [Full Text] [PDF]
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Phr). Recordings in A were obtained in hyperoxic normocapnia. Integrated phrenic discharge had an incrementing pattern (A), as shown in expanded tracing in bottom (A). During period designated by black bar, perfusate was switched to one equilibrated with a hypoxic-hypercapnia mixture. As shown in B, phrenic discharge was altered to a decrementing pattern (B). With reinstatement of hyperoxic normocapnia, the incrementing pattern of phrenic discharge was reestablished. Solid arrow designates first such incrementing burst.
