Vol. 90, Issue 6, 2466-2475, June 2001
HIGHLIGHTED TOPICS
Physiological and Genomic Consequences of
Intermittent Hypoxia
Invited Review: Intermittent
hypoxia and respiratory plasticity
Gordon S.
Mitchell1,2,
Tracy L.
Baker1,2,
Steven A.
Nanda2,3,
David D.
Fuller1,
Andrea G.
Zabka1,
Brad A.
Hodgeman1,
Ryan W.
Bavis1,
Kenneth J.
Mack2,3, and
E. B.
Olson Jr.4
1 Department of Comparative Biosciences, 3 Department
of Neurology, 4 Department of Preventive Medicine, and
2 Center for Neuroscience, University of Wisconsin, Madison,
Wisconsin 53706
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ABSTRACT |
Intermittent hypoxia elicits long-term facilitation (LTF), a
persistent augmentation (hours) of respiratory motor output. Considerable recent progress has been made toward an understanding of
the mechanisms and manifestations of this potentially important model
of respiratory plasticity. LTF is elicited by intermittent but not
sustained hypoxia, indicating profound pattern sensitivity in its
underlying mechanism. During intermittent hypoxia, episodic spinal
serotonin receptor activation initiates cell signaling events,
increasing spinal protein synthesis. One associated protein is
brain-derived neurotrophic factor, a neurotrophin implicated in several
forms of synaptic plasticity. Our working hypothesis is that increased
brain-derived neurotrophic factor enhances glutamatergic synaptic
currents in phrenic motoneurons, increasing their responsiveness to
bulbospinal inspiratory inputs. LTF is heterogeneous among respiratory
outputs, differs among experimental preparations, and is influenced by
age, gender, and genetics. Furthermore, LTF is enhanced following
chronic intermittent hypoxia, indicating a degree of metaplasticity.
Although the physiological relevance of LTF remains unclear, it may
reflect a general mechanism whereby intermittent serotonin receptor
activation elicits respiratory plasticity, adapting system performance
to the ever-changing requirements of life.
respiratory control; serotonin; neurotrophins; brain-derived
neurotrophic factor; episodic hypoxia
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INTRODUCTION |
IN THIS REVIEW,
EVIDENCE WILL be presented that intermittent hypoxia elicits
unique, serotonin-dependent mechanisms of plasticity in the central
neural control of breathing. These forms of central nervous system
(CNS) plasticity are unique because they are elicited by intermittent
hypoxia but not by an equivalent duration of sustained hypoxia. Recent
literature will be reviewed concerning two specific forms of plasticity
elicited in time domains of many minutes to days by intermittent
hypoxia: 1) long-term facilitation (LTF) of phrenic
motor output as a result of 3-10 hypoxic episodes and 2) enhanced phrenic LTF as a result of chronic intermittent
hypoxia (CIH). A working model has been developed suggesting that,
although these forms of plasticity differ in time course and in their
requirement for gene transcription, they are initiated by the same
sequence of events. The model may have global implications,
representing a mechanism of plasticity common to other neural systems.
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RESPIRATORY RESPONSES TO HYPOXIA |
Recent reviews have emphasized the complex, time-dependent
mechanisms observed during and after hypoxia (16, 88). For example, the response during short-term hypoxia (minutes) consists of
at least three distinct mechanisms in rats: 1) the acute
response, 2) short-term potentiation, and 3)
short-term depression (42, 88). Collectively, these
responses constitute the short-term hypoxic ventilatory (or phrenic)
response. After a single, brief hypoxic exposure (5 min), respiratory
motor activity returns to prehypoxic levels within 10-15 min in
anesthetized rats (6). On the other hand, successive
episodes at 5-min intervals lead to a long-lasting (>1 h) posthypoxia
facilitation of respiratory motor output referred to as LTF (32,
42, 69, 88). Numerous other mechanisms contributing to
ventilation during or after hypoxia of different durations or exposure
patterns can be discerned (88), but an extensive
discussion of these mechanisms is beyond the scope of this review.
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LTF IN DIFFERENT EXPERIMENTAL PREPARATIONS |
LTF was first described in anesthetized cats that received
episodic electrical stimulation of the carotid sinus nerve
(75), thus demonstrating that LTF is a central (vs.
peripheral) neural mechanism. Since that time, a number of reports
utilized episodic carotid sinus nerve stimulation, chemoreceptor
activation, or episodic hypoxia to elicit LTF in a range of
experimental preparations. In anesthetized, vagotomized cats, LTF was
observed after electrical stimulation of the carotid sinus nerve in
phrenic (30, 75, 76) and parasternal inspiratory
intercostal nerve activity (30), as well as hypoglossal
and nasal dilator activity and tidal volume (66). In each
of these studies, LTF was observed for at least 1 h after
stimulation. The protocol used to elicit LTF was high-frequency electrical stimulation of the carotid sinus nerve (25 Hz), a rate exceeding the range of discharge frequencies in carotid chemoafferent neurons during hypoxia (cf. Ref. 29). Phrenic LTF has been
reported to last at least 10 min after episodic activation of carotid
chemoafferent neurons with close, intra-arterial injections of
CO2-saturated solutions (79, 80), but there
have been no complete studies using episodic hypoxia in anesthetized
cats. An additional potential confound in these studies is that the
CO2-apneic threshold differs widely among respiratory motor
outputs in anesthetized cats. Thus, to generate rhythmic, respiratory
activity in the inspiratory intercostal (30) or
hypoglossal motor output (48, 66), baseline CO2 levels were established well above the apneic threshold
for phrenic activity. Thus phrenic LTF was minimized or obscured in studies focused on other respiratory motor outputs (30, 48, 66). There is also evidence that an intact vagus nerve minimizes or delays the manifestation of hypoglossal LTF, although not LTF of
tidal volume, in anesthetized cats (66). Thus a number of experimental details potentially influence the expression of LTF in
anesthetized cats, even when the same stimulation protocol is used.
LTF has been investigated extensively in rats. In anesthetized
rats, electrical stimulation of the carotid sinus nerve elicits a
relatively short and small phrenic LTF (42, 62). Most
studies used episodic hypoxia in anesthetized rats as an experimental model and generally revealed substantial phrenic LTF lasting for at
least 1 h after hypoxia (7-9, 33, 34, 42, 54,
55). Hypoxia-induced LTF has also been reported for hypoglossal
motor output in anesthetized rats (7-9, 33), although
there is considerable variation among rat substrains (32,
33). Again, there are important confounding influences in
different experimental preparations, as exemplified by the inability to
demonstrate phrenic LTF in anesthetized, spontaneously breathing rats
(47) or in anesthetized, ventilated rats with
cerebellectomy (42). Nevertheless, we have recently
demonstrated ventilatory LTF in unanesthetized, spontaneously breathing
rats (83; see Fig. 1), indicating that
LTF is expressed in rats under physiological conditions. Regardless,
the manifestations of LTF in rats are varied, and these differences may
reflect different mechanisms (see WORKING MODEL).
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Fig. 1.
Expression of long-term facilitation (LTF) following
episodic stimulation in 4 experimental rat preparations. All data for
respiratory amplitude (peak integrated phrenic activity or tidal
volume) have been normalized as a percent change from baseline,
allowing for direct comparisons of poststimulation responses. Central
nervous system (CNS) stimulation indicates the phrenic nerve response
after episodic electrical stimulation of the carotid sinus nerve in
anesthetized, vagotomized and ventilated rats (composite of Refs.
42 and 62). Anesth. artificial vent. (H), phrenic nerve
responses following three 5-min hypoxic episodes in anesthetized,
vagotomized and pump-ventilated rats (composite of Refs. 9
and 33). Unanesthetized (H), tidal volume response of unanesthetized
rats following five 5-min hypoxic episodes (data from Ref. 83). Anesth.
spontaneous vent. (H), esophageal pressure response in anesthetized,
vagotomized, spontaneously breathing rats (data from Ref.
47). Arterial PCO2 was held
isocapnic with respect to baseline levels in each case with the
exception of the unanesthetized, spontaneously breathing rats, which
were poikilocapnic. These comparative results indicate that LTF varies
with the experimental protocol in its magnitude and time course, even
within the same species and strain.
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LTF tends to be smaller, shorter, and more difficult to elicit in
unanesthetized animals but has been demonstrated following episodic
hypoxia in awake dogs (21), goats (104),
ducks (78), and rats (83). On the other hand,
ventilatory LTF was not observed in normal awake or sleeping humans
(3, 70), although humans with inspiratory flow limitation
exhibit ventilatory LTF after episodic hypoxia during non-rapid eye
movement sleep (3). Furthermore, a preliminary report
suggested that patients with obstructive sleep apnea exhibit
hypoxia-induced LTF when awake (71). This latter
observation, if confirmed, is consistent with concepts of
"metaplasticity" in LTF (see METAPLASTICITY OF LTF: CHRONIC INTERMITTENT VS. SUSTAINED HYPOXIA) and suggests that
caution should be exercised before ruling out the existence of LTF in any species or experimental preparation. The absence of LTF in one
circumstance does not rule out its expression in another.
Although most species and preparations exhibit LTF to some degree,
there are quantitative differences in magnitude and time course that
raise questions as to whether these reports are in fact manifestations
of the same underlying mechanism. For comparison, the magnitude
and time course of phrenic or ventilatory LTF reported in rats under
several experimental conditions or with different stimulation protocols
are illustrated in Fig. 1. After episodic stimulation of the carotid
sinus nerve (42, 62), phrenic LTF is of modest size and
exhibits a progressively decrementing pattern, suggesting a duration of
<1 h. Similar patterns have been observed in awake dogs
(21), goats (104), and ducks
(78) after episodic hypoxia but not in anesthetized cats
with carotid sinus nerve stimulation (75, 76). In
contrast, phrenic LTF reveals a progressively augmenting pattern after
episodic hypoxia in anesthetized rats (8, 9, 32). Although
a similar pattern has been observed in awake rats exposed to episodic
hypoxia (83), the apparent magnitude of ventilatory LTF
was less in these experiments. The magnitude difference between phrenic
LTF in anesthetized rats and ventilatory LTF in unanesthetized rats may
arise from the nonisocapnic conditions in the latter group. Thus
hypocapnia and the associated CO2-chemoreceptor feedback
may have reduced the apparent ventilatory LTF. When arterial blood
gases were restored to normal 60 min after episodic hypoxia, the
magnitude of LTF in anesthetized and unanesthetized rats was similar.
Another contrast is the report of Janssen and Fregosi
(47), which indicated minimal phrenic LTF in anesthetized
but spontaneously breathing rats. The most likely explanation for the
minimal phrenic LTF in these experiments was the relatively high level
of arterial CO2 in relation to the CO2-apneic
threshold for rhythmic phrenic motor output (47).
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SUSTAINED VS. INTERMITTENT HYPOXIA |
There are fundamental differences between sustained and
intermittent hypoxia in terms of their influence on respiratory
control. For example, although LTF is elicited by episodic hypoxia in
anesthetized rats, it is not observed after equivalent durations of
sustained hypoxia (9). Similarly, acute intermittent
isocapnic hypoxia elicits ventilatory LTF in awake goats
(104), but sustained hypoxia does not, even with longer
cumulative exposures (25, 28).
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GENETIC INFLUENCES ON LTF |
The manifestation of LTF varies widely among genetic populations
of rats, even within the same strain (32, 33). For
example, the magnitude of phrenic LTF varies between Sprague-Dawley
rats from the same supplier but from different colonies (Harlan Sprague Dawley; colony 236 vs. 205), even when the same experimental protocol was used (32). In a blinded experiment, we compared
hypoglossal LTF between Sprague-Dawley rats from two different
suppliers (Harlan Sprague Dawley colony 236 vs. Charles River/Sasco
colony K62) and found that it was present only in rats from Charles
River/Sasco (33). Despite their common origins, years of
genetic drift in these isolated populations of Sprague-Dawley rats
apparently caused significant differences in the anatomy
(22) and physiology of monoaminergic systems. Thus, in
studies of LTF or any form of plasticity, it is essential to consider
genetic influences. There may be overt differences between seemingly
similar genetic strains. Considerable variation is also to be expected
in genetically diverse populations (such as humans). Regardless, the
absence of LTF in an individual does not ensure that it cannot be
expressed under different physiological circumstances (see discussion
of metaplasticity below).
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AGE AND GENDER INFLUENCES ON LTF |
Age and gender affect the expression of phrenic and hypoglossal
LTF in complex ways. Virtually all of the studies on rats reported
above were conducted on young adult (3-4 mo) male rats of the
Sprague-Dawley strain. However, we have recently found that phrenic LTF
is greatly reduced and hypoglossal LTF is virtually eliminated in
middle-aged (>12 mo) male rats when using the same experimental
protocol (109). In female rats, LTF is affected by the
estrus cycle, being larger in diestrus than in estrus
(110). Furthermore, regardless of the estrus cycle,
phrenic and hypoglossal LTF actually increase in middle-aged (>12 mo)
female rats relative to young adults (110). Thus advancing
age, gender, and estrus cycle are important considerations in any study
of LTF, its manifestations, or its significance. The striking
age-gender interaction, particularly in the control of LTF in
hypoglossal motor output, bears a strong similarity to the incidence of
obstructive sleep apnea in the human population (17),
although the lack of menopause in female rats must be accounted for.
Nevertheless, pending specific investigations concerning possible links
between age, gender, LTF (or a lack of LTF), and the incidence of
obstructive sleep apnea in males and females, firm conclusions cannot
be made.
There are no reports concerning the existence of LTF in developing
mammals. However, intermittent hypoxia alters the subsequent short-term
hypoxic response in piglets (81, 107) and neonatal rats
(40, 41). In piglets, episodic hypoxia decreases the short-term hypoxic ventilatory response relative to that observed during continuous hypoxia of equal cumulative duration (81, 107). In neonatal rats, intermittent hypoxia diminishes
ventilatory roll off during the hypoxic episodes by a mechanism
associated with nitric oxide (40, 41). In neither case was
the ventilatory response after intermittent hypoxia reported. Such
posthypoxic measurements would be of considerable interest given the
profound influence of age on the expression of LTF (see above) and the high incidence of intermittent hypoxia in the newborn (see Refs. 40, 81).
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METAPLASTICITY OF LTF: CHRONIC INTERMITTENT VS. SUSTAINED HYPOXIA |
CIH augments the short-term hypoxic ventilatory response in humans
(36, 51, 94, 96, 97) and the short-term hypoxic phrenic
response in rats (60, 61). We recently demonstrated that
enhanced CNS integration of carotid chemoafferent inputs is sufficient
to explain this increase in the short-term hypoxic phrenic response
(59), although an additional effect at the carotid body
chemoreceptors cannot be ruled out. CIH also elicits a form of
metaplasticity (1, 19, 53), manifested as enhanced phrenic
LTF following three subsequent hypoxic episodes (61). Another interesting effect of CIH is the induction of hypoglossal LTF
in a rat substrain that does not ordinarily express significant hypoglossal LTF (Ref. 61 and Baker, Zabka, and Mitchell,
unpublished observations). Additional evidence for metaplasticity in
LTF is observed following cervical dorsal rhizotomy, a procedure that enhances both phrenic and hypoglossal LTF following episodic hypoxia (4, 55). The existence of metaplasticity in respiratory
motor control indicates that an absence of LTF in any given respiratory motor output or experimental circumstance does not constitute evidence
that LTF cannot be induced in that same motor output after suitable
preconditioning (e.g., CIH and dorsal rhizotomy).
There are interesting reports that suggest that metaplasticity may play
a role in circumstances such as disordered breathing during sleep. For
example, as mentioned above, patients with obstructive sleep apnea
expressed ventilatory LTF when awake, whereas normal awake subjects did
not (71). Similarly, when obstructive sleep apnea patients
are treated with continuous positive airway pressure to reduce the
incidence of apneas, the awake hypoxic ventilatory response decreases
(103), an effect one would predict when CIH (apneic
episodes) are discontinued. Regardless of its significance to
disordered breathing during sleep, further investigations are warranted
to elucidate the mechanisms and manifestations of metaplasticity elicited by CIH in the neural control of breathing.
Chronic sustained hypoxia, in contrast to CIH, has been extensively
studied and elicits ventilatory acclimatization, characterized by a
progressive augmentation of ventilation at rest and an increased short-term hypoxic ventilatory response (15, 16, 87). In some models, carotid body hypoxia is both necessary and sufficient to
elicit ventilatory acclimatization (16). Because a
progressive rise in carotid chemoafferent discharge frequency is
observed during sustained hypoxia (14, 16), it appears
that chronic sustained hypoxia predominantly elicits plasticity within
the carotid body (vs. CNS). However, additional effects within the CNS
cannot be ruled out, particularly with more severe or prolonged hypoxia
(87). Indeed, the central integration of carotid
chemoafferent neurons appears to be increased in rats exposed to 7 days
of sustained hypoxia (26). On the other hand, a
preliminary report suggests that phrenic LTF induced by carotid sinus
nerve stimulation is unchanged after 7 days of sustained hypoxia
(27), a clear difference from the enhanced phrenic
LTF following CIH. We suggest that CIH is more effective at
eliciting (serotonin dependent) CNS plasticity, whereas sustained
hypoxia has greater effects on the carotid body, acting via distinct
cellular mechanisms.
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MECHANISM(S) OF LTF |
Since the first demonstrations of LTF by Millhorn and colleagues
(74-76) in anesthetized or decerebrated cats, most
studies concerning its mechanism(s) have been conducted on young adult male Sprague-Dawley rats that were anesthetized, paralyzed,
vagotomized, and artificially ventilated. Thus the following discussion
must be considered largely in the context of hypoxia-induced LTF in anesthetized rats pending verification in other model systems.
LTF is a central neural mechanism.
Because LTF can be elicited by episodic activation of chemoafferent
neurons in the carotid sinus nerve of anesthetized, vagotomized, and
ventilated cats or rats (42, 62, 75, 76), it is a central
neural mechanism that does not require hypoxia per se. On the other
hand, a degree of LTF is still observed following episodic hypoxia in
carotid-denervated cats (35) and rats (Bavis and Mitchell,
unpublished observations). Thus LTF may consist of at least two
distinct mechanisms, one associated with the activation of synaptic
pathways by chemoafferent neurons and another attributable to hypoxic
effects on CNS neurons.
LTF requires serotonin receptor activation.
Millhorn et al. (76) demonstrated that LTF is serotonin
dependent because it could be blocked by a serotonergic neurotoxin (5,7-dihydroxytryptamine), a serotonin depleter
(p-chlorophenylalanine), and a broad-spectrum serotonin
receptor antagonist (methysergide). Furthermore, electrical activation
of raphe pallidus or raphe obscurus elicits a degree of LTF
(74). LTF elicited by episodic hypoxia in rats also
requires the activation of serotonin receptors (8), most
likely of the 5-HT2 receptor subtype (54).
Recent studies demonstrated that serotonin receptor activation is
necessary during but not after hypoxia, indicating that serotonin
receptor activation is necessary for the induction but not maintenance of LTF (34). Furthermore, the relevant receptors for
phrenic LTF have now been localized to the spinal cord because
intrathecal methysergide administration blocks phrenic but not
hypoglossal (i.e., cranial) LTF (11). Thus we suggest that
the relevant serotonin receptors are located within the phrenic motor
nucleus, either on or in the immediate vicinity of phrenic motoneurons. By inference, LTF in other respiratory motor pools, including hypoglossal motoneurons (8) and inspiratory intercostal
motoneurons (30), most likely requires serotonin receptor
activation within these respective motor nuclei.
Because ketanserin pretreatment blocks LTF (34, 54) and
ketanserin has a 30- to 100-fold greater affinity for
5-HT2A vs. 5-HT2C receptors (38, 44, 45,
112), we suggest that 5-HT2A receptors initiate LTF.
This suggestion is supported by the observation that labeled phrenic
motoneurons express 5-HT2A but not 5-HT2C receptors (G. J. Basura and H. G. Goshgarian, personal communication).
The involvement of serotonin in the mechanism of LTF does not rule out
contributions from other neurotransmitter systems. For example, there
are indications that genetically manipulated mice deficient in nitric
oxide synthase are unable to express LTF, suggesting a role for nitric
oxide (56).
LTF requires spinal protein synthesis.
We recently demonstrated that spinal administration of protein
synthesis inhibitors abolishes phrenic LTF (10).
Specifically, intrathecal administration of emetine and cyclohexamide
blocked phrenic LTF but had no effect on hypoglossal LTF in the same
animals. The continued LTF of hypoglossal output suggests that
effective concentrations of the protein synthesis inhibitors were
restricted to the spinal cord. Indeed, if larger doses of emetine were
delivered, both phrenic and hypoglossal LTF were abolished, consistent
with systemic distribution of the higher drug dose (Baker and Mitchell, unpublished observations).
LTF is associated with increased ventral spinal brain-derived
neurotrophic factor synthesis.
Although the relevant spinal proteins in the mechanism of LTF are
unclear, we hypothesize that the neurotrophin brain-derived neurotrophic factor (BDNF) plays a pivotal role. BDNF is a member of
the neurotrophin family (nerve growth factor, neurotrophin-3, and
neurotrophin-4/5). Neurotrophins mediate their effects via receptor
tyrosine kinases (TrK), and the relevant BDNF receptor is TrK-B. BDNF
is necessary and sufficient for several important models of synaptic
plasticity (cf. Refs. 18, 58, 86, 93, 101), such as
hippocampal long-term potentiation.
Complex interactions exist between serotonin and BDNF. For example,
BDNF is a potent neurotrophic factor for serotonergic neurons,
promoting their phenotypic elaboration (63, 64) and increasing serotonin levels and turnover (2, 98) by
increasing tryptophan hydroxylase expression (99).
Conversely, pharmacological manipulations of brain serotonin levels
affect BDNF mRNA expression. Increased serotonin availability increases
BDNF mRNA in some brain regions (frontal cortex), whereas it decreases
it in other regions (dentate gyrus) (111), quite possibly
by the activation of 5-HT2A receptors (105).
Collectively, there is considerable evidence that BDNF has the
potential to play a key role in serotonin-dependent plasticity.
In support of the hypothesis that BDNF plays a key role in
serotonin-dependent LTF, we demonstrated that episodic hypoxia (3 episodes of 5-min duration) increases ventral spinal BDNF protein levels in the cervical segments associated with the phrenic motor nucleus (Baker and Mitchell, unpublished observations). Increased BDNF
concentrations were observed 60 min after the final hypoxic episode,
suggesting that it has an appropriate time course for involvement in
LTF. The rapid time course further suggests the possibility of
increased translation from existing BDNF mRNA vs. transcriptional
regulation (37, 102). Increased ventral spinal BDNF
concentrations were abolished by intrathecal administration of a
serotonin receptor antagonist (methysergide) or protein synthesis inhibitor (emetine), indicating a strong correlation with the incidence
of LTF. Although these results are strictly correlative, they do
suggest that BDNF has the relevant characteristics to play a causal
role in the mechanism of LTF.
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MECHANISM OF CIH-INDUCED PLASTICITY |
Systemic administration of either methysergide (60)
or ketanserin (5) abolished the CIH-induced increase in
the short-term hypoxic phrenic response in anesthetized rats,
suggesting that serotonin receptor activation is necessary in the
underlying mechanism. On the other hand, although systemic methysergide
abolished enhanced phrenic LTF following CIH (60), the
selective 5-HT2 receptor antagonist ketanserin only
partially reversed LTF enhancement (5). Thus, although
serotonin receptor activation is necessary for enhanced phrenic LTF
following CIH, 5-HT2 receptors no longer account for the
entire effect. We suspect that the induction of novel serotonin
receptors and increased BDNF levels via transcriptional regulation play
key roles in the mechanism of enhanced phrenic LTF following CIH.
An additional distinction between the mechanisms of CIH and
ventilatory acclimatization to sustained hypoxia is revealed by the
fact that rats exhibited normal ventilatory acclimatization to
sustained hypoxia after serotonin depletion with
p-chlorophenylalanine (84). In contrast,
methysergide completely blocked the CIH-induced augmentation of the
short-term hypoxic phrenic response and LTF following CIH. Thus it
appears that the effects of CIH require an augmentation of serotonergic
modulation, whereas the effects of chronic sustained hypoxia do not.
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OTHER MODELS OF PATTERN-SENSITIVE PLASTICITY |
The concept that intermittent (spaced) vs. sustained (massed)
stimulation is more effective at eliciting certain forms of neuroplasticity has been established in a number of experimental paradigms, including serotonin-dependent synaptic plasticity in Aplysia (67), conditioned foot contractions in
Hermissenda (82), olfactory conditioning in
Drosophila (13) and honeybees
(92), habituation to danger stimuli in crabs
(31), contextual fear conditioning, spatial learning, and
socially transmitted food preferences in mice (57), and
some forms of hippocampal synaptic plasticity (46). The
concept that some forms of plasticity require prior experience with the
same stimulus has also been established in a number of experimental
models, including stress-induced plasticity in the catecholaminergic
nervous system (91). Conversely, there appear to be other
distinct forms of plasticity that are elicited preferentially by
sustained stimuli. For example, increased carotid body sensitivity to
hypoxia is more effectively elicited by sustained vs. intermittent
hypoxia (14, 16). In association, sustained hypoxia
appears to be a more powerful activator of the transcription factor
cAMP response element binding protein in the carotid body than
intermittent hypoxia (106).
Although the mechanisms underlying profound differences between
intermittent and sustained stimuli in initiating plasticity are not
clear, they may arise from unique cellular properties elicited by
cytosolic calcium oscillations. Calcium oscillations reduce the
effective threshold for the activation of transcription factors and
thus of gene expression (24, 72). Furthermore, calcium-sensitive kinases can become autophosphorylated
(89), leading to elevated and sustained activation
following rapid calcium spikes. Thus intermittent calcium spikes
(triggered by 5-HT2A receptor activation) may be more
efficient than steady levels at inducing plasticity. In addition, the
induction of gene transcription associated with some forms of
neuroplasticity is elicited most effectively when the same stimulus has
been presented at an earlier time (e.g., Ref. 91). Recent
evidence indicates that the transcriptional regulation of BDNF exhibits
just such pattern-sensitive behavior (see Fig. 3).
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WORKING MODEL |
In Fig. 2, a working model is
presented that suggests that common events in phrenic motoneurons
initiate LTF and enhance LTF after CIH. This model may help to explain
some of the apparent differences in LTF observed in different
experimental paradigms (Fig. 1). In our model, LTF and enhanced LTF are
initiated by repeated serotonergic 5-HT2A receptor
activation on phrenic motoneurons, thereby increasing intracellular
kinase activity (largely protein kinase C). As a result, glutamatergic
receptors associated with descending respiratory drive are
phosphorylated (or upregulated), increasing glutamate-induced currents
and phrenic motor output for the same descending respiratory drive
(i.e., LTF). Direct interactions between kinases and glutamate
receptors could underlie shorter versions of LTF, as exhibited with
intermittent carotid sinus nerve stimulation (Fig. 1). Direct kinase
actions on glutamate receptors are expected to be of a shorter
duration, decrementing, and similar in many respects to short-term
facilitation in Aplysia, a model of synaptic facilitation
(minutes) that requires protein kinase C activation (20).
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Fig. 2.
A working model of
serotonin-dependent plasticity in respiratory motoneurons. A
glutamatergic synaptic input from descending bulbospinal respiratory
neurons onto a phrenic motoneuron is depicted. Intermittent hypoxia is
hypothesized to release serotonin within the phrenic motor nucleus,
thereby causing episodic activation of 5-HT2A receptors on
phrenic motoneurons. Activation of these serotonin receptors activates
intracellular signaling molecules, most likely calcium-dependent
kinases including protein kinase C. The active kinases may then
phosphorylate glutamate receptors directly, enhancing
glutamate-dependent currents during respiratory neurotransmission.
However, such an enhancement is expected to be short lived and cannot
explain the protein synthesis dependence of LTF following intermittent
hypoxia. Thus we postulate that the activated kinases increase
translation of existing mRNA, particularly for key proteins such as the
neurotrophin brain-derived neurotrophic factor (BDNF). BDNF is released
in a pattern-sensitive, activity-dependent manner, thereby activating
tyrosine kinase receptor-B (TrK-B) on the phrenic motoneuron and
possibly on the presynaptic terminal. Trk-B activation is expected to
initiate a signaling cascade that further phosphorylates glutamate
receptors, thereby potentiating respiratory motor output (i.e., LTF).
After exposure to chronic intermittent hypoxia, we postulate that
pattern-sensitive BDNF gene transcription is increased, enhancing BDNF
protein and mRNA available for synthesis during subsequent hypoxic
episodes. Thus additional glutamate receptor phosphorylation results
during intermittent hypoxia, further increasing phrenic motoneuron
responses to descending respiratory drive (i.e., enhanced LTF). This
model remains highly speculative at several points and requires
rigorous testing. For example, although we know that intermittent
hypoxia increases BDNF protein levels within the ventral cervical
segments associated with the phrenic nucleus (Baker and Mitchell,
unpublished observations), a causal relationship to LTF has not yet
been demonstrated.
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Direct interactions between the kinases and glutamate receptors cannot
account for longer-lasting forms of LTF, since spinal application of
protein synthesis inhibitors blocks LTF following intermittent hypoxia
(10). The more robust protein synthesis-dependent form(s)
of LTF that occurs as a result of intermittent hypoxia in anesthetized
rats (Fig. 1) is associated with increased ventral spinal BDNF
synthesis. We postulate that increased BDNF synthesis results from
increased translation of preexisting BDNF mRNA because of its
relatively rapid time course (i.e., 60 min after hypoxia). In its time
course and dependence on protein synthesis via translational regulation, phrenic LTF is similar to serotonin-dependent
intermediate-term facilitation in Aplysia (39,
68).
If intermittent hypoxia and the associated 5-HT2A-receptor
activation continue, additional mechanisms are initiated, leading to
enhanced phrenic LTF. We postulate that enhanced phrenic LTF following
CIH requires increased gene transcription and that the resulting
CIH-induced gene transcription increases the expression of at least two
proteins necessary for enhanced phrenic LTF: 1) BDNF and
2) a novel serotonin receptor(s) that accounts for the inability of ketanserin to fully block LTF after CIH. We postulate that
these novel serotonergic receptor(s) and increased BDNF levels in
phrenic motoneurons contribute to further longer-lasting glutamate receptor phosphorylation during episodic hypoxia (via protein kinase A
and TrK-B, respectively), thereby enhancing phrenic LTF.
Recent evidence suggests that BDNF gene transcription exhibits the
pattern sensitivity necessary to play a key role in LTF and enhanced
LTF following CIH. In Fig. 3,
pattern-sensitive transcriptional activation of BDNF genes is
illustrated for cultured cortical neurons transfected with the first
promoter region of the BDNF gene and a luciferase reporter gene (Nanda
and Mack, unpublished observations). In these neurons,
activity-dependent transcriptional activation was investigated 24 h after episodic exposure to glutamate (three 5-min episodes, 15-min
interval) and continuous glutamate exposures of similar cumulative
duration (15 min and 60 min). In each case, data are presented as a
percentage of control, 24 h after the beginning of glutamate
exposure. Continuous glutamate exposures scarcely increased
transcription, whereas episodic glutamate increased BDNF gene
transcription severalfold. Thus BDNF is a neuroactive molecule
exhibiting the pattern-sensitive transcriptional regulation necessary
for a prominent role in neuroplasticity following CIH.
Activity-dependent BDNF release from vagal and petrosal sensory neurons
is also more effectively triggered by episodic vs. continuous stimulation (12). Collectively, these data establish a
powerful precedent for pattern-sensitive gene expression and release of a neuroactive substance that plays a major role in several forms of
synaptic plasticity (18, 58, 86, 93, 101).
View larger version (13K):
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|
Fig. 3.
In dissociated cortical neurons, the BDNF promoter 1 region was transfected along with a luciferase reporter gene. The
cultures were exposed to glutamate in different patterns, presumably
increasing neuronal activity. Glutamate exposures were 15 and 60 min
continuous and episodic (three 5-min exposures with a 15-min washout
interval). All data represent the response 24 h after the onset of
glutamate exposures, expressed as a percentage of controls. Neither 15 nor 60 min continuous exposure significantly increased BDNF gene
transcription. In contrast, episodic glutamate exposure increased BDNF
transcriptional activity severalfold. These data (Nanda and Mack,
unpublished observations) establish a precedent for pattern-sensitive
gene expression in the CNS and demonstrate that the BDNF gene exhibits
the pattern sensitivity necessary to play a key role in respiratory
plasticity following intermittent hypoxia.
|
|
In the final analysis, each time domain of LTF predicted in this
discussion is orchestrated by kinases that were activated by serotonin
receptors on phrenic motoneurons. If intermittent hypoxia and
subsequent serotonin release persists, these kinases initiate
mechanisms leading to increased protein synthesis necessary to
consolidate longer-lasting forms of respiratory plasticity. Although we
focused on relatively few proteins in this model, it is likely that
other proteins will be necessary in the mechanism(s) underlying LTF
and/or enhanced phrenic LTF following CIH. This model is under
continual refinement (e.g., see Ref. 32) and will, we
hope, ultimately lead to a better understanding of these potentially
important mechanisms of respiratory plasticity.
| |
SIGNIFICANCE |
Respiratory plasticity is now recognized in a number of
circumstances, including the effects of hypoxia, exercise, neural injury, and developmental experience. By comparing and contrasting diverse models of plasticity in respiratory motor control, we hope that
fundamental principles will emerge, providing a more comprehensive
picture of respiratory plasticity, its mechanisms, and its importance
in the control of breathing. We have already started to suspect common
elements in different models of plasticity in adults because serotonin
and (we suspect) BDNF appear to play key roles in respiratory
plasticity after intermittent hypoxia, dorsal rhizotomy
(50, 55), and may be critical in long-term modulation of
the exercise ventilatory response (Refs. 49, 65, 77;
R. A. Johnson and G. S. Mitchell, unpublished observations). Common features in these diverse models of plasticity may suggest that
a fundamental mechanism underlies many forms of plasticity in the CNS,
particularly the ventral spinal cord in which few examples of
plasticity are known. We hypothesize that an important but unrecognized
mechanism of CNS plasticity is a change in the capacity for
(serotonergic and BDNF) neuromodulation. To our knowledge, such a
mechanism has not been proposed in any vertebrate neural system,
although there is precedent in the invertebrate literature (52).
By understanding these mechanisms, we may gain insights into natural
compensatory mechanisms during disease and the rationale for
therapeutic intervention (e.g., pharmacological or physical therapy).
Diseases of relevance to respiratory motor control that may involve (or
be treated by utilizing) the capacity for respiratory plasticity in
adult animals include breathing disorders during sleep
(100), the onset of lung disease, sudden infant death
syndrome (90), congenital central hypoventilation syndrome
(73, 108), respiratory impairment after spinal cord
injury, parkinsonism (95), and anxiety hyperventilation
disorders (23, 85).
Although we do not yet know the specific relevance of phrenic LTF or
enhanced phrenic LTF following CIH in any physiological or
pathophysiological state, we nevertheless view intermittent hypoxia as
a suitable model to study the capacity for and mechanisms of
respiration-related plasticity in the CNS. Only by thorough investigations of their mechanisms and manifestations will an appreciation of their biological significance be gained.
| |
ACKNOWLEDGEMENTS |
The principal investigators (G. S. Mitchell and K. J. Mack) were supported by National Institutes of Health Grants HL-53319, HL-36780, HL-65383, and NS-33913.
| |
FOOTNOTES |
Address for reprint requests and other correspondence: G. S. Mitchell, Dept. of Comparative Biosciences, Univ. of Wisconsin, 2015 Linden Drive West, Madison, WI 53706.
| |
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TOP
ABSTRACT
INTRODUCTION
