Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia

Respiration Physiology 129 (2001) 25 – 35 www.elsevier.com/locate/resphysiol

Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia T.L. Baker a,b,*, D.D. Fuller b, A.G. Zabka b, G.S. Mitchell a,b a

b

Center for Neuroscience, Uni6ersity of Wisconsin, 2015 Linden Dri6e West, Madison, WI 53706, USA Department of Comparati6e Biosciences, Uni6ersity of Wisconsin, 2015 Linden Dri6e West, Madison, WI 53706, USA Accepted 19 April 2001

Abstract The objectives of this paper are: (1) to review advances in our understanding of the mechanisms of respiratory plasticity elicited by episodic versus continuous hypoxia in short to intermediate time domains (min to h); and (2) to present new data suggesting that different patterns of hypercapnia also elicit distinct forms of respiratory plasticity. Episodic, but not continuous hypoxia elicits long-term facilitation (LTF) of respiratory motor output. Phrenic LTF is a serotonin-dependent central neural mechanism that requires: (a) activation of spinal serotonin receptors; and (b) spinal protein synthesis. Continuous and episodic hypercapnia also elicit different mechanisms of plasticity. Continuous, severe hypercapnia (25 min of 10% inspired CO2) elicits long-term depression (LTD) of phrenic motor output ( −33 98% at 60 min post-hypercapnia) in anesthetized rats. In contrast, 3, 5 min hypercapnic episodes do not elicit LTD (99 17% at 60 min). We hypothesize that the response of respiratory motoneurons to serotonergic and noradrenergic modulation may contribute to pattern sensitivity to hypoxia and hypercapnia. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Control of breathing, respiratory plasticity; Hypercapnia, episodic vs. continuous Hypoxia, episodic vs. continuous, Short vs. intermediate; Mammals, rat; Mediators, noradrenaline, serotonin; Plasticity, respiratory, long-term facilitation, long-term depression; Receptors, serotonergic

1. Introduction It has long been recognized that the pattern of training has a profound effect on the formation and retention of memories. For example, episodic or spaced training trials elicit long lasting memories more effectively than sustained or massed * Corresponding author. Tel.: + 1-608-263-5013; fax: +1608-263-3926. E-mail address: [email protected] (T.L. Baker).

training trials (Carew et al., 1972; Tully et al., 1994; Yin et al., 1994; Mauelshagen et al., 1998; Hermitte et al., 1999; Baker and Mitchell, 2000a; Freudenthal and Romano, 2000). In the marine invertebrate Aplysia, serotonin-dependent synaptic plasticity differs markedly when serotonin is applied in an episodic versus continuous pattern. Episodic serotonin, when applied to the synapse between sensory and motoneurons mediating the gill-withdrawal reflex, elicits a long-lasting facilitation of synaptic transmission that lasts \ 24 h

0034-5687/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 4 - 5 6 8 7 ( 0 1 ) 0 0 2 8 0 - 8

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(Aplysia long-term facilitation; Montarolo et al., 1986; Clark and Kandel, 1993; Emptage and Carew, 1993; Ghirardi et al., 1995). In contrast, brief continuous (25 min) serotonin exposures do not elicit long-term facilitation (Mauelshagen et al., 1998). On the other hand, if continuous serotonin application is prolonged (\ 1.5 h), longterm facilitation is revealed (Emptage and Carew, 1993; Zhang et al., 1997); thus, although given forms of plasticity are not unique to continuous or episodic activation, the effectiveness of these stimulation patterns can vary considerably. Intracellular signaling pathways associated with synaptic facilitation in Aplysia are also sensitive to the pattern of serotonin application (Mu¨ ller and Carew, 1998; Yanow et al., 1998). Other examples of pattern sensitive neuroplasticity are odoravoidance learning in Drosophila (Yin et al., 1994; Tully et al., 1994) and behavioral training in the crab Chasmagnathus (Hermitte et al., 1999); in these examples, spaced training trials give rise to long-term memory, whereas continuous training does not. In this review, we outline evidence indicating that different patterns of chemoreceptor stimuli (hypoxia and hypercapnia) evoke distinct forms of plasticity in respiratory motor control, with particular focus on short to intermediate stimulation protocols. We begin with a general review of respiratory long-term facilitation (LTF), a longlasting increase in respiratory motor output following episodic, but not continuous hypoxia (for a more detailed review, see Fuller et al., 2000a). We discuss briefly very recent progress in our understanding of the mechanisms giving rise to LTF, and speculate on the cellular and synaptic mechanisms imparting pattern specificity in this form of hypoxia-induced plasticity. Next, we present evidence that hypercapnia-induced plasticity also exhibits pattern sensitivity; continuous hypercapnia is more effective at eliciting longterm depression (LTD) of respiratory mo.tor output than episodic hypercapnia (but see Bach and Mitchell, 1998). Although the mechanisms giving rise to LTD are not well understood, we offer a working model and speculate how this model may be adapted to impart pattern sensitivity in hypercapnia-induced plasticity. Two hypotheses will be

described: (1) different respiratory challenges preferentially activate different neuromodulatory systems; the release pattern of these neuromodulators near respiratory motoneurons is critical in eliciting long-lasting plasticity; or (2) the noradrenergic and serotonergic neuromodulatory systems operate in a ‘push/pull’ manner to elicit plasticity in respiratory motor output.

2. Episodic versus continuous hypoxia Previous studies indicate that different patterns of hypoxia elicit different mechanisms of plasticity (Dwinell et al., 1997; Turner and Mitchell, 1997; Powell et al., 1998; Baker and Mitchell, 2000a; Mitchell et al., 2001). For example, acute intermittent hypoxia elicits a long-lasting (hours), serotonin-dependent increase in respiratory motor output known as long-term facilitation (LTF; Hayashi et al., 1993; Bach and Mitchell, 1996; Turner and Mitchell, 1997; Kinkead and Mitchell, 1999; Baker and Mitchell, 2000a,b, 2001; Fuller et al., 2000a,b,c). LTF generally involves an increased respiratory amplitude following hypoxia, although some studies also report LTF of breathing frequency (Bach and Mitchell, 1996; Turner and Mitchell, 1997; Baker and Mitchell, 2000a,b, 2001). LTF appears to be a central neural mechanism since LTF is elicited by carotid sinus nerve stimulation (Millhorn et al., 1980a,b; Hayashi et al., 1993; Fregosi and Mitchell, 1994), thus bypassing mechanisms of carotid chemosensory transduction. LTF has been reported in a wide variety of experimental preparations, including anesthetized rats (Hayashi et al., 1993; Bach and Mitchell, 1996; Kinkead and Mitchell, 1999; Baker and Mitchell, 2000a,b, 2001; Fuller et al., 2000a,b,c), anesthetized cats (Millhorn et al., 1980a,b), awake ducks (Mitchell et al., 2001), awake dogs (Cao et al., 1992) awake rats (Olson, E.B., Jr. and Mitchell, G.S., unpublished observations) and awake goats (Turner and Mitchell, 1997). Awake humans do not exhibit LTF (McEvoy et al., 1996), although sleeping humans with inspiratory flow limitations may do so (Babcock and Badr, 1998).

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LTF appears to be a unique property of episodic hypoxia, as continuous hypoxia does not elicit LTF (Dwinell et al., 1997; Baker and Mitchell, 2000a; Mitchell et al., 2001). For example, in anesthetized rats, 3, 3 min episodes of hypoxia, separated by 5 min, elicits a phrenic LTF that lasts \ 1 h (Baker and Mitchell, 2000a). When these episodes are massed into a 9 or 20 min continuous bout of hypoxia, LTF is not observed. Although it remains possible that longer bouts of continuous hypoxia elicit LTF, it is clear that episodic hypoxia is more effective at eliciting LTF in similar time domains. In longer time domains, additional forms of plasticity are elicited. For example, following chronic intermittent hypoxia, the short-term hypoxic response and LTF are enhanced (Ling et al., 1998, 1999) by a serotonin-dependent mechanism (Bach et al., 1999a; Ling et al., 1999). Preliminary data suggest enhanced central neural integration of carotid chemoafferent input is sufficient to account for most of these effects (Ling et al., 2000); however, additional effects at the level of peripheral chemoreceptors cannot be ruled out. Chronic sustained hypoxia increases the shortterm hypoxic response and elicits a time-dependent increase in ventilation, known as ventilatory acclimatization (VAH; for review see Powell et al., 1998, 2000; Bisgard and Neubauer, 1995; Bisgard, 2000). VAH is thought to result primarily from carotid body plasticity, at least in its early phases, since sustained hypoxia elicits a progressive increase in carotid body responsiveness to hypoxia (Bisgard and Neubauer, 1995). Further evidence supporting the hypothesis that sustained hypoxia has a greater effect on carotid chemoreceptors are data suggesting that continuous, but not episodic hypoxia increases phosphorylation of the transcription factor cyclic AMP response element binding (CREB) protein in carotid body type 1 cells (Wang et al., 2000). Thus, continuous hypoxia may initiate a cascade of cell signaling and gene expression events in the carotid body that enhances carotid body sensitivity, thereby giving rise to VAH. On the other hand, additional central neural mechanisms may contribute to VAH in longer time domains (Dwinell and Powell, 1999; Powell et al., 2000). The cellular mechanisms dis-

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tinguishing episodic and continuous hypoxia in the CNS and carotid body are not understood at this time.

2.1. Mechanism of LTF We recently reviewed progress on the understanding of the mechanisms giving rise to LTF (Fuller et al., 2000a); however, new developments already warrant a revision of our working model. It is well established that LTF requires 5-HT receptor activation (Millhorn et al., 1980b; Bach and Mitchell, 1996; Kinkead and Mitchell, 1999; Fuller et al., 2000c). New evidence suggests that the relevant serotonin receptors are located on or near respiratory motoneurons, since intrathecal application of the serotonin receptor antagonist methysergide in the cervical spinal cord blocks LTF of phrenic motor output (Baker and Mitchell, 2001). In these same experiments, LTF of hypoglossal motor output was unaffected by spinal methysergide, suggesting that the drug had been restricted at an effective concentration to the spinal cord. Activation of serotonin receptors is required to initiate, but not maintain LTF, since ketanserin blocks LTF when given before, but not after episodic hypoxia (Fuller et al., 2000c). Thus, we propose that serotonin receptor activation initiates an intracellular signaling cascade in the motoneurons that subsequently maintains LTF. This cascade requires spinal protein synthesis, since intrathecal administration of protein synthesis inhibitors selectively blocks phrenic LTF, while leaving hypoglossal LTF intact (Baker and Mitchell, 2000b). Our working model of LTF is that episodic 5-HT2 receptor activation on respiratory motoneurons enhances respiratory amplitude via changes in synaptic efficacy between bulbospinal respiratory neurons and respiratory motoneurons (Fig. 1). Since the 5-HT2A/2C antagonist, ketanserin, abolishes LTF (Kinkead and Mitchell, 1999; Fuller et al., 2000c), and ketanserin has greater selectivity for 5-HT2A versus 5-HT2C receptors (for review, see Hoyer et al., 1994), the relevant serotonin receptor is most likely of the 5-HT2A subtype. This suggestion is further supported by observations that labeled phrenic mo-

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toneurons contain 5-HT2A but little 5-HT2C receptor mRNA (Basura, G. and Goshgarian, H., personal communication). We hypothesize that repetitive activation of 5-HT2A receptors activates dendritic protein synthesis in respiratory motoneurons. These new proteins may act pre- and/ or post-synaptically to increase synaptic efficacy between descending bulbospinal respiratory neurons and their target motoneurons, thereby initiating LTF. After prolonged episodic hypoxia, gene transcription may become more important, consolidating LTF and giving rise to longer lasting forms of plasticity. How then does the model differ during continuous hypoxia? Different patterns of chemoafferent stimulation may be detected on multiple levels in the respiratory control system, and may therefore result in different forms of plasticity. For example, continuous and episodic hypoxia may result in different discharge characteristics of serotonergic neurons in the caudal raphe nuclei, which project to respiratory regions of the brainstem

and spinal cord. At the onset of hypoxia or chemoafferent activation, caudal raphe serotonergic neurons are activated (Erickson and Millhorn, 1994; Morris et al., 1996, 2000; Teppema et al., 1997). Serotonin released during hypoxia may activate auto-inhibitory 5-HT1 receptors on serotonergic cell bodies and terminals, thereby diminishing further serotonin release (for review see Hoyer et al., 1994; Barnes and Sharp, 1999). Thus, prolonged continuous hypoxia could progressively decrease serotonin release due to autoinhibition, resulting in an inadequate activation of 5-HT2 receptors and, therefore, no LTF. This hypothesis is consistent with the observation that a 5-HT1A agonist abolishes LTF (Kinkead and Mitchell, 1999). In contrast, during episodic hypoxia, the inter-stimulus interval would present the target motoneuron with multiple pulses of 5-HT, mitigating the influence of autoinhibitory receptors. An alternative (or additional) explanation is that pattern specificity is encoded in the signal transduction cascade activated by 5-HT2 receptors. Several possibilities exist, among these are differential activation of phosphatases or kinases, sensitization/desensitization of post-synaptic receptors and/or different Ca++ dynamics. Finally, continuous hypoxia may elicit counteracting inhibitory mechanisms that obscure or occlude LTF (see below). Much work remains to be done to further clarify mechanisms leading to stimulus pattern sensitivity in eliciting respiratory plasticity.

3. Episodic versus continuous hypercapnia

Fig. 1. Proposed mechanism of LTF of respiratory amplitude following episodic hypoxia. Episodic hypoxia activates caudal raphe serotonergic neurons, thereby releasing serotonin in respiratory motor nuclei. Serotonin activates 5-HT2A receptors on respiratory motoneurons, eliciting an intracellular signaling cascade that results in local protein synthesis. These newly synthesized proteins may act pre- and/or post-synaptically to enhance the efficacy of synaptic transmission onto respiratory motoneurons, thereby giving rise to LTF.

In contrast to episodic hypoxia, episodic hypercapnia has been reported to elicit long-term depression (LTD; Bach and Mitchell, 1998). LTD is manifested as a long-lasting decrease in the amplitude (\60 min) and frequency (\ 30 min) of respiratory motor output following episodes of severe hypercapnia ( 10% inspired). LTD following episodic hypercapnia requires a2-adrenergic receptors, and has been proposed to result from episodic activation of neurons in the A5 region or locus coeruleus (A6). LTD is not elicited by more moderate levels of hypercapnia ( 5%)

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in anesthetized rats, casting some doubt on its physiological significance. Nevertheless, useful principles of plasticity in respiratory control may be revealed by understanding the mechanistic basis of LTD. To determine if LTD exhibits pattern sensitivity similar to LTF, we exposed urethane-anesthetized, vagotomized, paralyzed and ventilated Charles River Laboratories/Sasco Sprague– Dawley (colony K62) rats to episodic or continuous hypercapnia (10% inspired CO2). Basic procedures have been described previously (Bach and Mitchell, 1998). We quantified the amplitude and frequency of the integrated phrenic discharge as an index of respiratory motor output. Baseline phrenic nerve activity was established 2– 3 mmHg above the CO2 apneic threshold in a background of hyperoxia (FIO2 0.5). Once a stable baseline was reached, we exposed rats to: (1) 3, 5 min episodes of hypercapnia, separated by 5 min normocapnia (n= 8); or (2) 25 min continuous hypercapnia (n=7). Rats were returned to baseline PaCO2 conditions, and phrenic and hypoglossal motor output were monitored for 1 h. Blood samples were taken before, during, and 15, 30 and 60 min following hypercapnia to ensure: (1) PaO2 \120 mmHg at all time points; (2) PaCO2 was between 85 and 95 mmHg during hypercapnia; and (3) PaCO2 was within 1 mmHg of baseline after the hypercapnic episodes. Phrenic discharge corresponding to these time points were expressed as a percent change from baseline. When data were normalized as a percentage of the CO2-stimulated maximum, similar results were obtained (data not shown). In contrast to a previous report (Bach and Mitchell, 1998), we found that episodic hypercapnia did not elicit significant LTD of phrenic amplitude (Fig. 2). There was a trend toward a depression that was evident at 15 and 30 min post-episodic hypercapnia, but this was not statistically significant (− 24 918% and −20 912% at 15 and 30 min post-episodic hypercapnia, respectively; P\0.05). By 60 min post-episodic hypercapnia, phrenic discharge was at baseline levels (99 17%; P\0.05). Unlike phrenic burst amplitude, burst frequency exhibited a significant, transient depression (Fig. 2). Burst frequency was

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Fig. 2. Integrated phrenic amplitude (A) and burst frequency (B) responses at 15, 30 and 60 min following 3, 5 min episodes (filled circles) and 25 min continuous (filled squares) hypercapnia. Only rats exposed to continuous hypercapnia had a significant LTD of phrenic amplitude (expressed as a percent change from baseline) at 15, 30 and 60 min post-hypercapnia (P B0.05). Episodic hypercapnia did not elicit significant LTD of phrenic amplitude at any time point, although a trend towards depression was evident at 15 and 30 min post-hypercapnia. Episodic hypercapnia was significantly different than continuous hypercapnia at 60 min (P B0.05). Rats exposed to episodic or continuous hypercapnia exhibited a depression of burst frequency (expressed as a change from baseline) at 15 min post-hypercapnia. There was no difference between episodic and continuous hypercapnia at any time point posthypercapnia. * significantly different from baseline, c significantly different from continuous hypercapnia.

significantly depressed at 15 min post-episodic hypercapnia (−149 6 bursts/min; PB 0.05), but had recovered by 30 min post-episodic hypercapnia. These results contrast with an earlier report showing that episodic hypercapnia elicits significant LTD of phrenic burst amplitude for \ 60 min and of burst frequency for \ 30 min (Bach and

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Mitchell, 1998). The reasons underlying this discrepancy are unknown. One possibility is rat substrain differences. Bach and Mitchell (1998) used Harlan Sprague–Dawley (colony 205) rats, whereas we used Charles River Laboratories/ Sasco Sprague–Dawley (colony K62). Charles River Laboratories/Sasco and Harlan Sprague– Dawley rats differ in the degree of LTF, even when using the same protocol in a blinded study (Fuller et al., 2000a,b). Charles River Laboratories/Sasco rats (colony K62) have a greater phrenic LTF (  75% at 60 min) than Harlan Sprague– Dawley rats (colony 205;  40% at 60 min). These same substrains also differ in the anatomy of noradrenergic projections to the spinal cord (Clark and Proudfit, 1992). Neurons from the locus coeruleus project primarily to the ventral horn in Charles River Laboratories/Sasco rats, whereas in Harlan Sprague– Dawley rats, locus coeruleus neurons project primarily to the dorsal horn. Thus, it is possible that the disparate results between the present study and that of Bach and Mitchell (1998) are due to genetic differences in the serotonergic or noradrenergic systems. In contrast to episodic hypercapnia, continuous hypercapnia elicited a prolonged LTD. At 60 min post-continuous hypercapnia, phrenic amplitude was significantly depressed from baseline (− 33 9 8%; Fig. 2; P B0.05); this result was significantly different than episodic hypercapnia (P B 0.05). Similar to episodic hypercapnia, burst frequency exhibited only a transient depression (Fig. 2). At 15 min post-continuous hypercapnia, burst frequency was significantly depressed (− 18 9 4, PB 0.05), but had recovered by 30 min. Our data suggest that episodic and continuous hypercapnia elicit different mechanisms of plasticity in the same population of rats. Both stimulation patterns elicit a transient depression of phrenic burst frequency, but only continuous hypercapnia elicits significant LTD of burst amplitude. It is unknown if LTD following continuous hypercapnia is similar to the LTD observed by Bach and Mitchell (1998) in the requirement for a2-receptor activation since we observed different dependencies on the pattern of hypercapnia (i.e. in the present study episodic hypercapnia did not elicit LTD, whereas in Bach and Mitchell (1998),

episodic hypercapnia did elicit LTD). Bach and Mitchell (1998) hypothesized that norepinephrine release due to chemoreceptor activation or direct effects of H+/CO2 on noradrenergic neurons (Coates et al., 1993; Pineda and Aghajanian, 1997; Oyamada et al., 1998, 1999) releases norepinephrine in respiratory regions, thereby activating a2-adrenergic receptors and leading to LTD. The location of the relevant a2-adrenergic receptors is not known, but may be near brainstem respiratory neurons. Studies on the rhythmically active medullary slice preparation from neonatal rats indicate that norepinephrine injected into the pre-Botzinger complex, a region of the brainstem hypothesized to be critical in the generation of respiratory rhythm, results in an a2-receptor dependent depression of respiratory burst activity (Al-Zubaidy et al., 1996; Johnson et al., 1996). In contrast, norepinephrine applied to XII motoneurons in rats or mice results in an a1-receptor augmentation of respiratory burst activity (Parkis et al., 1995; Al-Zubaidy et al., 1996; Funk et al., 1997). Thus, activation of a2-adrenergic receptors on or near brainstem neurons involved in respiratory rhythm generation and pattern formation may be important for respiratory LTD following hypercapnia. Our finding that continuous, but not episodic hypercapnia elicits LTD may be due to cumulative effects of severe, continuous hypercapnia on the noradrenergic system. In this sense, episodic hypercapnia may result in a faster recovery from LTD simply because episodic norepinephrine results in an inadequate stimulation of a2-receptors; i.e. the interstimulus interval may allow enough time for neurons to recover from a2-mediated inhibition, such that the cellular processes giving rise to LTD may not be adequately activated. Alternatively, the lack of apparent LTD following episodic hypercapnia may have been the result of competing mechanisms, i.e. an LTF that counterbalanced LTD. This may occur if: (1) noradrenergic inputs to the respiratory control system exhibit a similar pattern sensitivity as serotonergic inputs; or (2) if episodic hypercapnia elicits a serotonindependent LTF simultaneously with an a2-dependent LTD. These possibilities are discussed below.

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4. Pattern specificity of chemoreceptor stimulation

4.1. Norepinephrine-dependent LTF? Noradrenergic (a1) modulation of respiratory motor output may exhibit a similar pattern dependency as serotonergic neuromodulation. For example, stimulation of noradrenergic neurons by hypercapnia may result in an activation of brainstem a2-adrenergic receptors, giving rise to LTD of burst amplitude and frequency. Simultaneously, excitatory noradrenergic receptors may be activated; when these receptors are stimulated in an episodic pattern, a long-lasting facilitation may result that offsets or counteracts LTD (Fig. 3). It is unknown where in the CNS this facilitation would occur, although a likely site is in the spinal cord on respiratory motoneurons. Indeed, a1-adrenergic receptors on respiratory motoneurons (see Rekling et al., 2000 for review) couple to phospholipase C and activate protein kinase C, similar to 5-HT2 receptors. Norepinephrine-mediated LTF in the spinal cord might also explain the

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apparent substrain difference (Harlan vs. Sasco) in the ability of episodic hypercapnia to elicit LTD. Charles River Laboratories/Sasco Sprague–Dawley rats have a greater number of noradrenergic axons projecting from the locus coeruleus to the ventral cervical spinal cord than do Sprague– Dawley rats from Harlan Inc. (Clark and Proudfit, 1992). Thus, noradrenergic modulation via a1-adrenergic receptors on respiratory motoneurons may be greater in Charles River Laboratories/Sasco rats, thereby counteracting LTD induced by brainstem a2-adrenergic receptor activation. The relative lack of noradrenergic projections from the locus coeruleus to the ventral cervical spinal cord in Harlan Sprague–Dawley rats may not be sufficient to elicit facilitation, accounting for our earlier observation of LTD following episodic hypercapnia (Bach and Mitchell, 1998). On the other hand, since noradrenergic projections from pontine area A5 to the ventral cervical spinal cord are prevalent in Harlan Sprague–Dawley rats (Fritschy and Grzanna, 1990), these projections may provide a pattern of norepinephrine release during episodic hypoxia similar to the locus coeruleus in Sasco Sprague– Dawley rats.

4.2. Serotonergic/noradrenergic counter-balance?

Fig. 3. Possible mechanisms of facilitation following episodic hypercapnia or hypoxia. Episodic severe hypercapnia preferentially activates a1-adrenergic receptors, whereas episodic hypoxia preferentially activates 5-HT2A receptors. Both receptors couple to similar intracellular signaling cascades, and may lead to the synthesis of new proteins, thereby giving rise to LTF. Following severe episodic hypercapnia, the a1-receptor dependent LTF may offset depression caused by brainstem a2-receptor activation.

Another possibility for the pattern dependence of LTD following hypercapnia is that episodic hypercapnia elicits both a norepinephrine-dependent LTD and a serotonin-dependent LTF (Kinkead et al., 2001). In this sense, the serotonergic and noradrenergic systems may interact in a ‘push-pull’ manner to modulate respiratory motor output (Fig. 4). Hypercapnia stimulates the carotid body (Fukuda et al., 1987) and activates raphe serotonergic neurons (Veasey et al., 1995; Morris et al., 1996; Teppema et al., 1997); thus, it is possible that stimulation of the carotid body by episodic hypercapnia is sufficient to induce an LTF-like mechanism similar to that following episodic hypoxia. This serotonin-dependent facilitation would be balanced or offset by noradrenergic inhibitory mechanisms elicited by hypercapnia. In contrast, although continuous hypercapnia also stimulates the carotid body, it would not elicit

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Fig. 4. Possible interaction of the noradrenergic and serotonergic systems during hypercapnia. Hypercapnia stimulates both noradrenergic and serotonergic neurons. Activation of a2adrenergic neurons, perhaps at integrative ( ) brainstem respiratory neurons associated with rhythm generation and burst pattern formation, elicits LTD. When hypercapnia is presented in an episodic pattern, a serotonin-dependent LTF is simultaneously elicited due to episodic activation of 5-HT2 receptors on respiratory motoneurons, thus counterbalaning the norepinephrine-dependent LTD. Bold lines indicate dominant pathways of neuromodulation.

serotonin-dependent LTF, much like continuous hypoxia does not elicit LTF. Without this facilitory mechanism, the inhibition manifested as LTD would be greater. Two lines of circumstantial evidence support this hypothesis: (1) serotonin and norepinephrine have opposing modulatory influences on post-hypoxia frequency decline (PHFD; Bach et al., 1999b; Kinkead and Mitchell, 1999; but see Coles et al., 1998); and (2) the selective a2-adrenergic receptor antagonist, RX-821002, may reveal phrenic LTF following episodic hypercapnia (Bach and Mitchell, 1998), although this trend was not statistically significant due to a small sample size (n = 3).

5. Pattern specificity encoded within respiratory motoneurons? Implicit in these hypotheses is the concept that pattern specificity to chemoreceptor inputs is en-

coded within or near respiratory motoneurons. Thus, episodic chemoreceptor stimulation may facilitate respiratory motoneurons by a 5-HT2-dependent mechanism following episodic hypoxia (Fig. 1), and either an a1-adrenergic (Fig. 3) and/ or 5-HT2 receptor dependent (Fig. 4) mechanism following episodic hypercapnia. Both 5-HT2 and a1-adrenergic receptors initiate similar cell signaling cascades and, thus, the specific intracellular signaling molecules within respiratory motoneurons may impart pattern specificity. a1-Adrenergic and 5-HT2 receptors activate PKC, and prolonged PKC activation downregulates a1-adrenergic (Sugden et al., 1988; Fonseca et al., 1995; GarciaSainz et al., 1999) and 5-HT2 (Kagaya et al., 1990; Rahman and Neuman, 1993; Roth et al., 1995; Vouret-Craviari et al., 1995) receptors; episodic receptor activation might prevent this receptor downregulation. Alternatively, stimulus pattern may be encoded in the pattern of activation of signal transduction molecules, as mentioned above. There are several examples in the literature of pattern sensitive cell signaling processes. For example, in the crab Chasmagnathus, enhancement of DNA binding activity of the transcription factor NFk –B is correlated with the behavioral training pattern conditions required to elicit longterm memory. Intermittent (which induces longterm memory), but not massed (which does not induce long-term memory) behavioral training enhances NFk-B DNA binding activity (Freudenthal and Romano, 2000). Signaling pathways associated with synaptic facilitation in Aplysia are also sensitive to the pattern of serotonin application (Mu¨ ller and Carew, 1998; Yanow et al., 1998). Episodic serotonin (4–5 pulses, 5 min duration) elicits sustained activation of protein kinase A at intermediate (1 h) and long (20 h) time points, whereas continuous (90 min) serotonin application induces persistent protein kinase A activation only at long time points (Mu¨ ller and Carew, 1998). Furthermore, episodic (5, 5 min episodes) but not continuous serotonin (90 min) increases translational protein synthesis 2 h following stimulation in the isolated pleural ganglia of Aplysia (Yanow et al., 1998).

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6. Conclusion A great deal of evidence indicates that the respiratory control system is plastic. Much work remains to be done before we will understand basic mechanisms giving rise to the different forms of respiratory plasticity. One fundamental principle is already beginning to emerge: different stimulus patterns 6ary in their capacity to e6oke respiratory plasticity. Episodic patterns of chemoreceptive stimuli have a greater capacity to elicit facilitory mechanisms of respiratory plasticity, at least in short to intermediate time domains. What role, if any, this plasticity plays in the control of breathing is still unclear. Regardless, understanding basic mechanisms of respiratory plasticity may yield important insights into natural compensatory mechanisms during disease and the rationale for therapeutic intervention. Furthermore, these models of plasticity may reveal fundamental principles of plasticity in the nervous system, particularly in the spinal cord where few examples of plasticity are known.

Acknowledgements These experiments were supported by NIH grants HL 53319 and HL 65383. Tracy Baker was supported by NIH Training Grant HL 07654.

References Al-Zubaidy, Z.A., Erickson, R.L., Greer, J.J., 1996. Serotonergic and noradrenergic effects of respiratory neural discharge in the medullary slice preparation of neonatal rats. Pflu¨ gers Arch. 431, 942 –949. Babcock, M.A., Badr, M.S., 1998. Long-term facilitation of ventilation in humans during NREM sleep. Sleep 21, 709 – 716. Bach, K.B., Mitchell, G.S., 1996. Hypoxia-induced long-term facilitation of respiratory activity is serotonin dependent. Respir. Physiol. 104, 251 –260. Bach, K.B., Mitchell, G.S., 1998. Hypercapnia-induced longterm depression of respiratory activity requires a2-adrenergic receptors. J. Appl. Physiol. 84, 2099 – 2105. Bach, K.B., Kinkead, R., Ling, L., Olson, E.B., Mitchell, G.S., 1999a. Ketanserin blocks the effects of chronic intermittent hypoxia on the short term hypoxic response but not long term facilitation. FASEB J. 13, A166.

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Bach, K.B., Kinkead, R., Mitchell, G.S., 1999b. Post-hypoxia frequency decline in rats: sensitivity to repeated hypoxia and alpha2-adrenoreceptor antagonism. Brain Res. 817, 25 – 33. Baker, T.L., Mitchell, G.S., 2000a. Episodic but not continuous hypoxia elicits long-term facilitation of phrenic motor output in rats. J. Physiol. (Lond.) 529, 215 – 219. Baker, T.L., Mitchell, G.S., 2000b. Spinal protein synthesis inhibition attenuates serotonin dependent long-term facilitation of phrenic motor output. Soc. Neurosci. Abstr. 26, 1458. Baker, T.L., Mitchell, G.S., 2001. Spinal serotonin receptor activation is necessary for phrenic long-term facilitation following episodic hypoxia. FASEB J. A422. Barnes, N.M., Sharp, T., 1999. A review of central 5-HT receptors and their function. Neuropharmacology 38, 1083 – 1152. Bisgard, G.E., Neubauer, J.A., 1995. Peripheral and central effects of hypoxia. In: Dempsey, J.A., Pack, A.I. (Eds.), Regulation of Breathing. Marcel Dekker, New York, pp. 617 – 618. Bisgard, G.E., 2000. Carotid body mechanisms in acclimatization to hypoxia. Respir. Physiol. 121, 237 – 246. Cao, K.Y., Zwillich, C.W., Berthon-Jones, M., Sullivan, C.E., 1992. Increased normoxic ventilation induced by repetitive hypoxia in conscious dogs. J. Appl. Physiol. 74, 2083 – 2088. Carew, T.J., Pinsker, H.M., Kandel, E.R., 1972. Long-term habituation of a defensive withdrawal reflex in Aplysia. Science 175, 451 – 454. Clark, F.M., Proudfit, H.K., 1992. Anatomical evidence for genetic differences in the innervation of the rat spinal cord by noradrenergic locus coeruleus neurons. Brain Res. 591, 44– 53. Clark, G.A., Kandel, E.R., 1993. Induction of long-term facilitation in Aplysia sensory neurons by local application of serotonin to remote synapses. Proc. Natl. Acad. Sci. 90, 11 411 – 11 415. Coates, E.L., Li, A., Nattie, E.E., 1993. Widespread sites of brain stem ventilatory chemoreceptors. J. Appl. Physiol. 75, 5 – 14. Coles, S.K., Ernsberger, P., Dick, T.E., 1998. Post-hypoxic frequency decline does not depend on alpha2-adrenergic receptors in the adult rat. Brain Res. 794, 267 – 273. Dwinell, M.R., Janssen, P.L., Bisgard, G.E., 1997. Lack of long-term facilitation of ventilation after exposure to hypoxia in goats. Respir. Physiol. 108, 1 – 9. Dwinell, M.R., Powell, F.L., 1999. Chronic hypoxia enhances the phrenic nerve response to arterial chemoreceptor stimulation in anesthetized rats. J. Appl. Physiol. 87, 817 – 823. Emptage, N.J., Carew, T.J., 1993. Long-term facilitation in the absence of short-term facilitation in Aplysia neurons. Science 262, 253 – 256. Erickson, J.T., Millhorn, D.E., 1994. Hypoxia and electrical stimulation of the carotid sinus nerve induce fos-like immunoreactivity within catecholaminergic and serotonergic neurons of the rat brainstem. J. Comp. Neurol. 348, 161 – 182.

34

T.L. Baker et al. / Respiration Physiology 129 (2001) 25–35

Fonseca, M.I., Button, D.C., Brown, R.D., 1995. Agonist regulation of alpha 1B-adrenergic receptor subcellular distribution and function. J. Biol. Chem. 270, 8902 –8909. Fregosi, R.F., Mitchell, G.S., 1994. Long-term facilitation of inspiratory intercostal nerve activity following carotid sinus nerve stimulation in cats. J. Physiol. (Lond.) 477, 469 – 479. Freudenthal, R., Romano, A., 2000. Participation of Rel/NFkB transcription factors in long-term memory in the crab Chasmagnathus. Brain Res. 855, 274 –281. Fritschy, J., Grzanna, R., 1990. Demonstration of two separate descending noradrenergic pathways to the rat spinal cord: evidence for an intragriseal trajectory of locus coeruleus axons in the superficial layers of the dorsal horn. J. Comp. Neurol. 291, 553 –582. Fuller, D.D., Bach, K.B., Baker, T.L., Kinkead, R., Mitchell, G.S., 2000a. Long term facilitation of phrenic motor output. Respir. Physiol. 121, 135 –146. Fuller, D.D., Baker, T.L., Behan, M., Mitchell, G.S., 2000b. Expression of hypoglossal long term facilitation differs between sub-strains of Sprague –Dawley rat. Physiol. Genomics 4, 175 – 181. Fuller, D.D., Zabka, A.G., Baker, T.L., Mitchell, G.S., 2000c. Phrenic long-term facilitation in the rat requires 5-HT receptor activation during but not following episodic hypoxia. J. Appl. Physiol. (in press). Fukuda, Y., Sato, A., Trzebski, A., 1987. Carotid chemoreceptor discharge responses to hypoxia and hypercapnia in normotensive and spontaneously hypertensive rats. J. Auton. Nerv. Syst. 19, 1 –11. Funk, G.D., Parkis, M.A., Selvaratnam, S.R., Walsh, C., 1997. Developmental modulation of glutamatergic inspiratory drive to hypoglossal motoneurons. Respir. Physiol. 110, 125 – 137. Garcia-Sainz, J.A., Gottfried-Blackmore, A., Vazquez-Prado, J., Romero-Avila, M.T., 1999. Protein kinase C-mediated phosphorylation and desensitization of human alpha(1b)adrenoceptors. Eur. J. Pharmacol. 385, 263 – 271. Ghirardi, M., Montarolo, P.G., Kandel, E.R., 1995. A novel intermediate stage in the transition between short- and long-term facilitation in the sensory to motor neuron synapse of Aplysia. Neuron 14, 413 – 420. Hayashi, F., Coles, S.K., Bach, K.B., Mitchell, G.S., McCrimmon, D.R., 1993. Time-dependent phrenic nerve responses to carotid afferent activation: intact vs. decerebellate rats. Am. J. Physiol. 265, R811 –R819. Hermitte, G., Pedreira, M.E., Tomsic, D., Maldonado, H., 1999. Context shift and protein synthesis inhibition disrupt long-term habituation after spaced, but not massed, training in the crab Chasmagnathus. Neurobiol. Learn. Mem. 71, 34 – 49. Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R., Humphrey, P.P., 1994. International union of pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). Pharmacol. Rev. 46, 157 – 203.

Johnson, S.M., Smith, J.C., Feldman, J.L., 1996. Modulation of respiratory rhythm in vitro: role of Gi/o protein-mediated mechanisms. J. Appl. Physiol. 80, 2120 – 2133. Kagaya, A., Mikuni, M., Kusumi, I., Yamamoto, H., Takahashi, K., 1990. Serotonin-induced acute desensitization of serotonin2 receptors in human platelets via a mechanism involving protein kinase C. J. Pharmacol. Exp. Ther. 255, 305 – 311. Kinkead, R., Mitchell, G.S., 1999. Time-dependent hypoxic ventilatory responses in rats: effects of ketanserin and 5-carboxyamidotryptamine. Am. J. Physiol. 277, R658 – R666. Kinkead, R., Bach, K.B., Johnson, S.M., Hodgeman, B.A., Mitchell, G.S., 2001. Plasticity in respiratory motor control: intermittent hypoxia and hypercapnia activate opposing serotonergic and noradrenergic modulatory systems. Comp. Biochem. Physiol. in press. Ling, L., Olson, E.B., Mitchell, G.S., 1998. Chronic intermittent hypoxia enhances the hypoxic ventilatory control system. FASEB J. 12, A781. Ling, L., Bach, K.B., Kinkead, R., Olson, E.B., Mitchell, G.S., 1999. Enhancement of the hypoxic ventilatory responses following chronic intermittent hypoxia is serotonin dependent. FASEB J. 13, A166. Ling, L., Olson, E.B., Johnson, S.M., Mitchell, G.S., 2000. Phrenic responses to electrical stimulation of carotid sinus nerve in rats following chronic intermittent hypoxia. FASEB J. 14, A77. Mauelshagen, J., Sherff, C.M., Carew, T.J., 1998. Differential induction of long-term synaptic facilitation by spaced and massed applications of serotonin at sensory neuron synapses of Aplysia californica. Learn. Mem. 5, 246 – 256. McEvoy, R.D., Popovic, R.M., Saunders, N.A., White, D.P., 1996. Effects of sustained and repetitive isocapnic hypoxia on ventilation and genioglossal and diaphragmatic EMGs. J. Appl. Physiol. 81, 866 – 875. Millhorn, D.E., Eldridge, F.L., Waldrop, T.G., 1980a. Prolonged stimulation of respiration by a new central neural mechanism. Respir. Physiol. 42, 87 – 103. Millhorn, D.E., Eldridge, F.L., Waldrop, T.G., 1980b. Prolonged stimulation of respiration by endogenous central serotonin. Respir. Physiol. 41, 171 – 188. Mitchell, G.S., Powell, F.L., Hopkins, S.R., Milsom, W.K., 2001. Time domains of the hypoxic ventilatory response in awake ducks: episodic and continuous hypoxia. Respir. Physiol. 124, 117 – 128. Montarolo, P.G., Goelet, P., Castellucci, V.G., Morgan, J., Kandel, E.R., 1986. A critical period for macromolecule synthesis in long-term heterosynaptic facilitation in Aplysia. Science 234, 1249 – 1254. Morris, K.F., Arata, A., Shannon, R., Lindsey, B.G., 1996. Long-term facilitation of phrenic nerve activity in cats: responses and short time scale correlations of medullary neurones. J. Physiol. (Lond.) 490, 463 – 480. Morris, K.F., Baekey, D.M., Shannon, R., Lindsey, B.G., 2000. Respiratory neural activity during long-term facilitation. Respir. Physiol. 121, 119 – 133.

T.L. Baker et al. / Respiration Physiology 129 (2001) 25–35 Mu¨ ller, U., Carew, T.J., 1998. Serotonin induces temporally and mechanistically distinct phases of persistent PKA activity in Aplysia sensory neurons. Neuron 21, 1423 – 1434. Oyamada, Y., Ballantyne, D., Mu¨ ckenhoff, K., Scheid, P., 1998. Respiration-modulated membrane potential and chemosensitivity of locus coeruleus neurones in the in vitro brainstem-spinal cord of the neonatal rat. J. Physiol. (Lond.) 513, 381 – 398. Oyamada, Y., Andrzejewski, M., Mu¨ ckenhoff, K., Scheid, P., Ballantyne, D., 1999. Locus coeruleus neurones in vitro: pH-sensitive oscillations of membrane potential in an electrically coupled network. Respir. Physiol. 118, 131 –147. Parkis, M.A., Bayliss, D.A., Berger, A.J., 1995. Actions of norepinephrine on rat hypoglossal motoneurons. J. Neurophysiol. 74, 1911 – 1919. Pineda, J., Aghajanian, G.K., 1997. Carbon dioxide regulates the tonic activity of locus coeruleus neurons by modulating a proton- and polyamine-sensitive inward rectifier potassium current. Neuroscience 77, 723 –743. Powell, F.L., Milsom, W.K., Mitchell, G.S., 1998. Time domains of the hypoxic ventilatory response. Respir. Physiol. 112, 123 – 134. Powell, F.L., Huey, K.A., Dwinell, M.R., 2000. Central nervous system mechanisms of ventilatory acclimatization to hypoxia. Respir. Physiol. 121, 223 –236. Rahman, S., Neuman, R.S., 1993. Multiple mechanisms of serotonin 5-HT2 receptor desensitization. Eur. J. Pharmacol. 238, 173 – 180. Rekling, J.C., Funk, G.D., Bayliss, D.A., Dong, X.W., Feldman, J.L., 2000. Synaptic control of motoneuronal excitability. Physiol. Rev. 80, 767 – 852. Roth, B.L., Palvimaki, E.P., Berry, S., Khan, N., Sachs, N., Uluer, A., Choudhary, M.S., 1995. 5-Hydroxytryptamine2A (5-HT2A) receptor desensitization can occur without down-regulation. J. Pharmacol. Exp. Ther. 275, 1638 – 1646.

35

Sugden, D., Ho, A.K., Sugden, A.L., Klein, D.C., 1988. Negative feedback mechanisms: evidence that desensitization of pineal alpha 1-adrenergic responses involves protein kinase-C. Endocrinology 123, 1425 – 1432. Teppema, L.J., Veening, J.G., Kranenburg, A., Dahan, A., Berkenbosch, A., Olievier, C., 1997. Expression of c-fos in the rat brainstem after exposure to hypoxia and to normoxic and hyperoxic hypercapnia. J. Comp. Neurol. 388, 169 – 190. Tully, T., Preat, T., Boynton, S.C., Del Vecchio, M., 1994. Genetic dissection of consolidated memory in Drosophila. Cell 79, 35 – 47. Turner, D.L., Mitchell, G.S., 1997. Long-term facilitation of ventilation following repeated hypoxic episodes in awake goats. J. Physiol. (Lond.) 499, 543 – 550. Veasey, S.C., Fornal, C.A., Metzler, C.W., Jacobs, B.L., 1995. Response of serotonergic caudal raphe neurons in relation to specific motor activities in freely moving cats. J. Neurosci. 15, 5346 – 5359. Vouret-Craviari, V., Auberger, P., Pouyssegur, J., Van Obberghen-Schilling, E., 1995. Distinct mechanisms regulate 5HT2 and thrombin receptor desensitization. J. Biol. Chem. 270, 4813 – 4821. Wang, Z.Y., Baker, T.L., Keith, I.M., Mitchell, G.S., Bisgard, G.E., 2000. Continuous but not episodic hypoxia induces CREB phosphorylation in rat carotid body. Adv. Exp. Med. Biol. 475, 631 – 635. Yanow, S.K., Manseau, F., Hislop, J., Castellucci, V.F., Sossin, W.S., 1998. Biochemical pathways by which serotonin regulates translation in the nervous system of Aplysia. J. Neurochem. 70, 572 – 583. Yin, J.C., Wallach, J.S., Del Vecchio, M., Wilder, E.L., Zhou, H., Quinn, W.G., Tully, T., 1994. Induction of a dominant negative CREB transgene specifically blocks long-term memory in Drosophila. Cell 79, 49 – 58. Zhang, F., Endo, S., Cleary, L.J., Eskin, A., Byrne, J.H., 1997. Role of transforming growth factor-b in long-term synaptic facilitation in Aplysia. Science 275, 1318 – 1320.