Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
www.elsevier.com/locate/autneu
Review
Endogenous noradrenaline affects the maturation and function of the
respiratory network: Possible implication for SIDS
Gérard Hilaire *
Groupe d’étude des Réseaux Moteurs, FRE CNRS 2722, 280 boulevard Sainte Marguerite, 13009 Marseille, France
Received 25 October 2005; accepted 30 January 2006
Abstract
Breathing is a vital, rhythmic motor act that is required for blood oxygenation and oxygen delivery to the whole body. Therefore, the
brainstem network responsible for the elaboration of the respiratory rhythm must function from the very first moments of extrauterine life. In
this review, it is shown that the brainstem noradrenergic system plays a pivotal role in both the modulation and the maturation of the
respiratory rhythm generator. Compelling evidence are reported demonstrating that genetically induced alterations of the noradrenergic
system in mice affect the prenatal maturation and the perinatal function of the respiratory rhythm generator and have drastic consequences on
postnatal survival. Sudden Infant Death Syndrome (SIDS), the leader cause of infant death in industrialised countries, may result from
cardiorespiratory disorders during sleep. As several cases of SIDS have been observed in infants having noradrenergic deficits, a possible link
between prenatal alteration of the noradrenergic system, altered maturation and function of the respiratory network and SIDS is suggested.
D 2006 Elsevier B.V. All rights reserved.
Keywords: Noradrenaline; Respiration; Maturation; Mouse; SIDS
Contents
1.
2.
3.
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The maturing respiratory network(s) . . . . . . . . . . . . . . . . . .
The maturing central NA system . . . . . . . . . . . . . . . . . . . .
3.1. Genetic manipulations of NA synthesis . . . . . . . . . . . . .
3.2. Specification of NA phenotype during development . . . . . .
3.3. Location of maturing brainstem NA neurons . . . . . . . . . .
4. Endogenous NA modulates the neonatal RRG activity. . . . . . . . .
4.1. Pontine A5 group . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Pontine A6 group . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Medullary A1/C1 and A2/C2 groups . . . . . . . . . . . . . .
5. Morphological support of the RRG modulation by NA neurons . . . .
6. The brainstem NA groups contribute to the maturation of the neonatal
7. Discussion and conclusion . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
* Tel.: +33 491 75 75 09; fax: +33 491 26 20 38.
E-mail address: hilaire@marseille.inserm.fr.
1566-0702/$ - see front matter D 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.autneu.2006.01.021
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RRG
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G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
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1. Introduction
Respiration is a vital, rhythmic motor act that allows
blood oxygenation in the lungs and oxygen delivery to the
whole body, including the heart and the brain. Therefore
from the very first moments of extrauterine life, the neonatal
mammal must be able to produce rhythmic respiratory
movements and must be able to adapt it to environmental
and behavioral changes. Thus, postnatal survival requires a
normal prenatal maturation of the respiratory rhythm
generator (RRG), i.e. the brainstem neural network responsible for the production of the central rhythmic drive to the
respiratory motoneurons. Postnatal survival also requires a
normal prenatal maturation of the different structures that
are implicated in the modulation of the RRG, i.e. on the one
hand the peripheral receptors that convey messages from the
respiratory apparatus (lungs, trachea, carotid bodies,
muscles, etc.) and on the other hand the central structures
that receive and integrate these peripheral messages to
regulate and modulate the RRG activity. Among the latter
structures, the central bioaminergic neurons have a crucial
role, especially the serotonergic and the noradrenergic ones.
As reviewed below, the brainstem noradrenaline (NA)
system plays a pivotal role in both the modulation and the
maturation of the RRG. This suggests that genetic or
epigenetic alterations of the maturation of the NA system
during the perinatal period may affect the maturation of the
RRG and therefore may have drastic consequences on
postnatal survival. Sudden Infant Death Syndrome (SIDS),
the leader cause of infant death in industrialised countries,
may be due to respiratory disorders (Horne et al., 2004;
Ribas-Salgueiro et al., 2004). SIDS victims frequently
display brainstem NA deficits (Ozawa et al., 1999, 2003;
Cann-Moisan et al., 1999; Obonai et al., 1998; Kopp et al.,
1993; Takashima and Becker, 1991; Denoroy et al., 1987;
Ozand and Tildon, 1983) and frequently present mutations
of genes implicated in specification of NA phenotype
(Weese-Mayer et al., 2004). This is fully in agreement with
a possible link between prenatal NA deficits, respiratory
dysfunction and SIDS.
2. The maturing respiratory network(s)
As first shown by Suzue (1984) in neonatal rats and
thereafter by many others in fetal rats, neonatal mice and
fetal mice (Di Pasquale et al., 1992; Greer et al., 1992,
Hilaire et al., 1997, Viemari et al., 2003), the isolated RRG
may be conveniently studied in brainstem – spinal cord
preparations (‘‘en bloc’’ preparations; Fig. 1A), where it
continues to produce a rhythmic central drive and to induce
rhythmic phrenic bursts for several hours in vitro. In
addition, the RRG also continues to function in medullary
slice preparations from neonatal and young rodents (Smith
et al., 1991; Johnson et al., 1994; Koshiya and Smith,
1999; etc.) as shown by recording the population activity
Fig. 1. In vitro respiratory activity in neonatal mice. (A) Schema of the ‘‘en
bloc’’ preparation from neonatal mice (left part) where the RRG continues
to function in vitro as shown by the recording of integrated rhythmic bursts
of phrenic motoneurons (right part). (B) Schema of the slice preparation
from neonatal mice (left part) where the RRG continues to function in vitro
as shown by the recording of integrated rhythmic bursts of neurons of the
ventral respiratory group (right part). (C) Histograms show the mean
changes in respiratory rhythm (expressed in % of control) in medullary ‘‘en
bloc’’ preparation when the normal artificial cerebrospinal fluid (normal
ACSF, white bar; 100%) was replaced by ACSF containing either the NA
precursor l-tyrosine (light hatched bar, 50AM, 20min) to activate NA
biosynthesis or the a2 adrenoceptor antagonist yohimbine (hatched bar,
10AM, 20min) to block the medullary a2 adrenoceptors. Note the increased
(Tyrosine) and decreased (Yohimbine) respiratory rhythm.
of respiratory neurons from the ventral respiratory group
(Fig. 1B).
The use of these ‘‘en bloc’’ and ‘‘slice’’ in vitro
preparations has considerably improved our knowledge on
the mechanisms through which the RRG produces its
rhythmic drive. It has been shown that the neonatal RRG is
located in a small region of the ventrolateral medulla, the preBotzinger Complex (PBC) where electrolytic lesions disrupt
the in vitro rhythmic drive (Onimaru et al., 1988; Viemari et
al., 2003). In adult mammals, lesion of the PBC also severely
disrupts breathing in vivo (Ramirez et al., 1998; Gray et al.,
2001, Solomon, 2002, 2003, Wenninger et al., 2004a,b;
McKay et al., 2005). Indeed, the PBC contains respiratory
pacemaker neurons that play a crucial role in respiratory
rhythmogenesis (Onimaru et al., 1988; Smith et al., 1991;
Ramirez et al., 1998; Johnson et al., 1994; Koshiya and
Smith, 1999).
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G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
In human and rodent fetuses, respiratory movements
occur in utero, very early during gestation (for a review, see
Achard et al., 2005). Indeed, 3 – 4days before birth, the fetal
RRG is already able to elaborate a rhythmic drive, although
unstable and variable (Di Pasquale et al., 1992; Greer et al.,
1992). However no pacemaker neurons are found in the
fetal PBC during this early developmental period (Di
Pasquale et al., 1994; Pagliardini et al., 2003). Therefore
several questions arise. Are the fetal and neonatal RRG
different networks or the same maturing network? How do
this (these) network(s) functions (pacemaker vs. network
connection properties)? How do pacemaker properties
develop within the maturing PBC? To answer these
questions, a computational model has been developed that
reproduces well the experimental data (Achard et al., 2005).
This computational model shows that the ‘‘pacemaker vs.
network dilemma’’ can be solved simply: fetal conditional
pacemaker neurons only require both a certain amount of
noise and synaptic connections from the other neurons to
express their bursting properties. Therefore, this means that
the prenatal maturation and function of the RRG is at least
in part affected by the inputs it receives. As shown below,
among plenty of inputs, those from NA neurons may have a
pivotal role.
3. The maturing central NA system
As summarized in Fig. 2A, the synthesis of NA from ltyrosine successively requires two main synthetic enzymes,
tyrosine hydroxylase (TH) and dopamine beta-hydroxylase
(DBH). The initial and rate limiting step in NA synthesis is
the hydroxylation of l-tyrosine in l-Dopa through TH.
Then l-Dopa decarboxylation leads to DA, and finally DA
hydroxylation leads to NA through DBH.
3.1. Genetic manipulations of NA synthesis
Different mutant mice have been created in which the
ability to synthesize NA (TH-deficiency and DBH-deficiency), to remove NA from the synapse (NA transporter
deficiency), or to degrade NA are impaired. These mice with
genetically induced alterations of the NA system are
invaluable tools for the study of NA physiology and for
the study of the long-term physiological consequences of
altered NA homeostasis (see the excellent review from
Carson and Robertson, 2002).
TH-deficiency in mice is lethal, with mortality occurring
early during embryonic development, from E11.5 to E15.5,
i.e. few days after the normal appearance of the first NA
neurons (E9 –E10.5). Surgical delivery at E18.5 in mice
reveals that 19% of TH-deficient fetuses survive, some take
a breath but all die within 1day. They could be rescued by
administration of l-Dopa although they failed to thrive and
to survive past 5weeks (Zhou et al., 1995). The presence of
a weak level of DA in TH-deficient mice suggests the
Fig. 2. The maturing NA system in mice. (A) NA biosynthesis chain with
the two main synthetic enzymes, tyrosine hydroxylase (TH) and dopamine
beta-hydroxylase (DBH) and the possible tyrosinase pathway. (B)
Transcription factor cascade controlling the formation of A6 neurons.
Several factors are linked in transcriptional cascade but their precise
positions within the cascade is under debate and vary from an NA group to
another (see text and Goridis and Rohrer, 2002). (C) Drawings of four
transverse brainstem sections from neonatal mice (at P1). TH-immunohistochemistry allows to localize the four main NA groups, with the ventral A5
and dorsal A6 groups in the pons (top sections cut 560 and 1190Am rostral
to the obex) and with the ventral A1/C1 and dorsal A2/C2 in the medulla
(bottom sections cut 490 and 630Am caudal to the obex). In neonatal mice,
the medullary A1/C1 and the pontine A5 ventral groups show a wellmarked dorsal extension, especially with A5 neurons that form quite a
continuum extending towards the A6 group. The grey areas show the
regions where rabies-infected neurons are found 42 h after rabies virus
injection within the diaphragm of neonatal mice (see text and Fig. 4;
unpublished results from G. Hilaire, M. Bevengut and P. Coulon).
existence of an alternative synthesis pathway (Fig. 2A),
probably through tyrosinase (Rios et al., 1999). TH
transcription is mediated by different kinase pathways in
central NA neurons, with possible differences between A1
and A2 neurons (Rusnak and Gainer, 2005).
DBH-deficiency is also lethal in mice (Thomas et al.,
1995) but DBH-deficient fetuses could be rescued though
administration of l-dihydroxyphenylserine (DOPS) that is
G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
323
converted directly to NA. In addition, mobilizing NA to
vesicles is required for mouse survival and complete
removal of the vesicular monoamine transporter is lethal
(Carson and Robertson, 2002).
(Guo et al., 2005). Null mutants for TrkB, the tyrosine
kinase receptor for BDNF display a loss of NA neurons
whereas TrkB ligands increase their number (Holm et al.,
2003).
3.2. Specification of NA phenotype during development
3.3. Location of maturing brainstem NA neurons
During embryonic development, specification of NA
phenotype in central neurons depends on BMP protein
signaling and on four transcription factors at least, Rnx,
Mash1, Phox2a and Phox2b, that can act in different orders
(Fig. 2B), and can interact with distinct factors in different
types of NA cells (Goridis and Rohrer, 2002). Indeed, these
transcription factors are linked in transcriptional cascades
but their respective positions within the cascade differ from
one NA group to another (Tiveron et al., 1996; Hirsch et al.,
1998; Lo et al., 1998; Pattyn et al., 1997, 1999, 2000; Qian
et al., 2001, 2002). Rnx inactivation compromises the
formation of most NA neurons but partly preserved A6
neurons (Qian et al., 2001) whereas Phox2a inactivation
leads to the agenesis of A6 neurons but does not drastically
affect the formation of other NA neurons (Morin et al.,
1997). Both Rnx- and Phox2a-deficient mice die within 24 h
after birth. Phox2b is another genetic factor with an identical
homeodomain-binding site than Phox2a that is required for
TH and DBH transcriptions and its efficiency is comparable
to that of Phox2a (Yang et al., 1998). In the enteric nervous
system and the sympathetic ganglia, Phox2b is needed for
the expression of the GDNF-receptor subunit Ret, for
maintaining Mash1 expression and for the expression of
TH and DBH (Pattyn et al., 1999). Indeed, Phox2b regulates
the NA phenotype in vertebrates since Phox2b is expressed
in all central NA neurons and is required for the
differentiation of all NA centers in the brain, including A6
(Pattyn et al., 2000). The molecular mechanisms underlying
the regulation of Phox2a and Phox2b gene expression and
the relation to NA differentiation are still under debate
(Goridis and Rohrer, 2002; Jong Hong et al., 2004).
Besides Mash1, Rnx, Phox2a, Phox2b, other genes are
implicated in the development of NA neurons, such as Ret
and BDNF. Ret gene encodes a transmembrane tyrosine
kinase receptor and is expressed in presumptive monoaminergic brainstem neurons at E8 – E10 (Pachnis et al., 1993).
Ret contributes to the formation of NA neurons (Dauger et
al., 2001) and is highly expressed in some brainstem NA
areas and in motor nerve nuclei of E18 embryos (Viemari et
al., 2005a). Ret-deficient neonates do not survive past 1 or
2 days (Schuchardt et al., 1994; Aizenfisz et al., 2002), and
Ret-deficient fetuses at E18 have reduced pontine NA
contents and reduced number of A5 and A6 neurons
(Viemari et al., 2005a).
BDNF protein is also crucial for survival and plasticity of
NA neurons (Copray et al., 1999; Akbarian et al., 2002).
The BDNF mRNA is expressed in medullary A1/C1 and
A2/C2 neurons (Cho et al., 1999) and BDNF protein
increases the number and branching of cultured TH-neurons
In adult mice, the NA phenotype is expressed by many
central neurons forming several anatomical groups among
which four main brainstem groups can be distinguished, two
groups in the pons (the compact, dense dorsal A6 group or
locus coeruleus and the more diffuse, ventral A5 group) and
two groups in the medulla (the ventral A1/C1 and the dorsal
A2/C2 groups). These NA groups are implicated in
numerous neurovegetative functions, such as sleep, mood,
thermoregulation, cardiovascular regulations, etc. (see
Guyenet, 1991). During the embryonic development, the
NA neurons from the A6 group that arise in the dorsal
isthmic region of the hindbrain are among the earliest born
neurons, at E9 –E10.5 in mouse embryos (Lauder and
Bloom, 1974). A6 neurons are already well expressed at
gestational week 6 in human embryos (Zecevic and Verney,
1995). In neonatal mice, the four brainstem NA groups are
easily distinguished within the brainstem although a
continuum of scattered NA neurons may be observed in
the pons, between the dorsal A6 and ventral A5 groups (Fig.
2C) and NA neurons of the A1/C1 group may extend
dorsally, towards the A2/C2 group (Fig. 2C). Counting of
TH-expressing neurons in the A1/C1 and A2/C2 areas
reveals a 25% larger number of TH-neurons in neonatal than
in adult mice (Roux, personal communication). In rats aged
from postnatal day 1 to postnatal day 10 (P1 to P10), A6
does not exhibit significant developmental changes but the
number of A5 neurons decreases with age (Ito et al., 2002).
In addition, measuring the NA content in the whole brain of
mice shows 33% lower values in neonates than in adults
(Ide et al., 2005). Thus, the maturation of the NA system
still continues after birth. Indeed, the postnatal development
of TH activity displays two main developmental windows,
the first window within a few days after birth (the intraextrauterine transition) and the second window between 2
and 3weeks of age, corresponding to a deep energetic phase
related to the maturation of cardiorespiratory processes
(Roux et al., 2003). In addition, TH expression during
ventilatory acclimatization shows hypoxia-induced plasticity (Dumas et al., 1996).
4. Endogenous NA modulates the neonatal RRG activity
The use of both ‘‘en bloc’’ and ‘‘slice’’ preparations that
retain the ability to produce a rhythmic central respiratory
drive (Fig. 1) and that contain either the four brainstem NA
groups (ponto-medullary ‘‘en bloc’’ preparations) or only
the two medullary groups (medullary ‘‘en bloc’’ preparations and medullary slices) has considerably improved our
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G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
knowledge on the mechanisms through which endogenous
NA may modulate the RRG activity. As illustrated in Fig. 3,
the four groups of brainstem NA neurons play different roles
in the NA modulation of the RRG activity.
4.1. Pontine A5 group
As reviewed recently (Hilaire et al., 2004), the pontine
A5 and A6 groups exert dual, opposite effects on the RRG,
with A5 inhibitory and A6 facilitatory modulations. In both
rat and mouse neonates, elimination of A5 inputs onto the
medullary RRG by either pons resection or A5 electrolytic
lesions or pharmacological inhibition of A5 neurons
increases the respiratory rhythm. In addition, blockade of
medullary a2 adrenoceptors in ‘‘en bloc’’ preparations
retaining the A5 areas increased the respiratory rhythm. It
has been therefore concluded that the pontine A5 neurons
exert a permanent inhibition onto the medullary RRG
through medullary a2 adrenoceptors (Hilaire et al., 1989;
Errchidi et al., 1990, 1991; Viemari et al., 2003; Hilaire et
al., 2004).
In both rat and mouse fetuses, the A5 inhibitory
modulation of the RRG already exists prior to birth although
it develops rather late during gestation (Di Pasquale et al.,
1992; Viemari et al., 2003). The A5 modulatory process
persists throughout life since in adult animals, (1) activation
of A5 neurons slows down the in vivo respiratory rhythm
(Jodkowski et al., 1997; Dawid-Milner et al., 2001), (2) A5
neurons are synaptically connected to the respiratory
neurons (Dobbins and Feldman, 1994; Gaytan et al.,
2002), (3) A5 neurons display a respiratory-related rhythm
and a chemosensitivity (Guyenet et al., 1993), and (4)
contribute to respiratory responses to hypoxia and hypercapnia (Coles and Dick, 1996; Soulier et al., 1997; Roux et
al., 2000; Peyronnet et al., 2000).
4.2. Pontine A6 group
In neonatal rodents, application of exogenous NA to
medullary preparations alters the RRG rhythm, probably
through the PBC area (Errchidi et al., 1991; Al-Zubaidy et
al., 1996) and the PBC pacemaker neurons (Arata et al.,
1998). However, NA effects are complex and depending on
experimental conditions (ponto-medullary and medullary
preparations) and species (rats or mice), exogenous NA
mainly facilitates or mainly inhibits the neonatal RRG, with
a mixture of a1 facilitatory and a2 inhibitory effects.
Although quantitative differences exist between neonatal
rats and mice (Viemari and Hilaire, 2002), the complexity of
the NA effects on the respiratory rhythm suggested that,
besides the A5 inhibition, other modulatory processes exist.
In ‘‘en bloc’’ preparations retaining the A6 group (but not
the A5 group) from neonatal mice, blockade of medullary
a1 adrenoceptors decreases the respiratory rhythm and the
a1-dependent rhythm depression is abolished after A6
elimination. In addition, blockade of the degradation of
the endogenous NA increases the respiratory rhythm and
this increase is abolished after A6 elimination. As both
effects require A6 groups to be observed, it has been
concluded that A6 neurons exert a facilitatory modulation
onto the RRG via medullary a1 adrenoceptors. The A6
facilitation of the RRG has also been found in neonatal rat
preparations (Hakuno et al., 2004). The A6 facilitation of
the RRG is fully supported by in vitro results showing that
A6 neurons present respiratory-related activity and participate in the central chemosensitivity (Ito et al., 2004;
Ballantyne and Scheid, 2000; Oyamada et al., 1998). In
adult mammals, the influence of the A6 group onto the RRG
is likely to persist since A6 neurons are connected to the
medullary respiratory network (Dobbins and Feldman,
1994) and display respiratory-related firing (Guyenet et
al., 1993).
4.3. Medullary A1/C1 and A2/C2 groups
Fig. 3. Endogenous NA modulates the respiratory rhythm in neonatal mice.
Schema showing the two NA groups in the pons (A5 and A6) and the two
NA groups in the medulla (A1/C1 and A2/C2), as well as the modulatory
effects they exert on the medullary respiratory rhythm generator (RRG).
The A1/C1 and A6 groups exert facilitatory modulations on the RRG (+)
via medullary a2 and a1 adrenoceptors, respectively, whereas the A5 group
exerts an inhibitory modulation ( ) via medullary a2 adrenoceptors. The
medullary A2/C2 group stabilizes the RRG activity but the mechanisms and
the involved adrenoceptors are not identified yet.
The demonstration that the pontine A5 and A6 neurons
exert a dual, opposite control on the medullary RRG (Fig. 3)
does not exclude a possible role of medullary A1/C1 and
A2/C2 NA neurons in RRG modulation (Zanella et al., in
press). In medullary preparations, i.e. in preparations where
the pons and the A5 and A6 groups are absent, activation of
the NA synthesis through application of the NA precursor ltyrosine increases the respiratory rhythm whereas blockade
of the a2 adrenoceptors through application of the a2
adrenoceptor antagonist yohimbine induces a dose-dependent depression of the respiratory rhythm (Figs. 1C and 3).
As the latter effect persists after elimination of the dorsal
A2/C2 group, it is concluded that the A1/C1 group exerts a
permanent facilitation of the RRG through a2 adrenoceptors. In addition, experiments performed in slice preparations from 2-week-old neonatal mice fully confirm the
facilitating role of A1/C1 neurons. As already reported
G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
elimination of the dorsal part of the medulla does not
suppress in vitro respiratory rhythm (Hilaire et al., 1990;
Infante et al., 2003) but it increases the variability of the
respiratory cycle period, suggesting a new, but not yet
explained, role of A2/C2 neurons in stabilizing the RRG
activity (Fig. 3) (Zanella et al., in press).
The schema of Fig. 3 illustrates the complexity of the NA
modulation of the RRG:
– Endogenous NA released from the four NA brainstem
groups may facilitate (A1/C1 and A6) and reduce (A5)
the RRG activity,
– The pontine A5 and A6 groups exert dual, opposite
effects through different subtypes of medullary adrenoceptors, with an A5 break (a2 adrenoceptors) and an A6
accelerator (a1 adrenoceptors),
– The pontine A5 and the medullary A1/C1 groups exert
dual, opposite effects, with an A5 break and an A1/C1
accelerator both acting through medullary a2 adrenoceptors; whether the same a2 adrenoceptors constitute
the final pathway of A5 and A1/C1 modulatory pathways or whether there exist two functional types of a2
adrenoceptors specifically belonging to the two modulatory pathways remain an open question.
– The medullary A2/C2 group might play a stabilizing
role on the RRG through unknown mechanisms and
unknown adrenoceptor subtypes.
Indeed the schema of Fig. 3 is too much simple and
interactions between the different NA groups as well as
interactions between NA neurons and other neurons such as
5HT neurons should be taken into account. However, this
simple schema summarizes rather well the observed results.
5. Morphological support of the RRG modulation by NA
neurons
To know whether anatomical pathways support the A5 –
A6 modulations of the medullary RRG at birth, we are
performing rabies-virus tracing experiments coupled with
immunodetection of TH-neurons (Fig. 2). RV is a
neurotropic virus that retrogradely infects, step by step,
synaptically connected neurons. We inject rabies virus
(RV) in the diaphragm of mice at P1 and sacrifice the
neonates at different times after the diaphragmatic RV
injection. Phrenic motoneurons are infected first within 24
to 30 h post injection, followed by infection of cervical
cord interneurons and brainstem bulbospinal neurons (30 –
36 h post injection) and finally propriobulbar respiratory
neurons, especially those constituting the classical medullary ‘‘respiratory centers’’ such as the ventral respiratory
group (Fig. 4A). In addition, brainstem NA neurons are
also infected (36 – 42 h post injection). In the pons, RVinfection and TH immunoreactivity are co-localized in
some A5 and A6 neurons (arrows in Fig. 4B, C). This
325
neonatal result is consistent with adult results showing
projections from pontine A5 and A6 neurons towards the
medullary respiratory network in rats (Dobbins and Feldman, 1994) and mice (Gaytan et al., 2002). Therefore both
in neonatal and adult rodents, A5 and A6 neurons are
actually connected to the respiratory network and these
connections may support the A5 inhibitory and A6
facilitatory modulations reported above. In the medulla,
TH and RV immunoreactive neurons were sometimes
localized in overlapping areas (Fig. 2) but none of the
TH-neurons of the A1/C1 and A2/C2 groups were RVinfected (Fig. 4D). The lack of RV-infection of the
medullary A1/C1 and A2/C2 neurons may mean either
that these neurons are RV-resistant (Tsiang et al., 1983) or
that they do not modulate the RRG through ‘‘classical’’
synaptic relations but through ‘‘volume transmission’’. This
latter possibility fits with several reports showing that
adrenoceptors are often associated with nonsynaptic
processes and that catecholamine released from axon
terminals plays an important role in modulating the neural
communications without synaptic contacts (Lee et al.,
1998a, 1998b; Vizi, 1999, 2000; Liprando et al., 2004).
6. The brainstem NA groups contribute to the
maturation of the neonatal RRG
As reported above, the differentiation of NA neurons
during CNS development is controlled by several genes
such as Rnx, Phox2a, Phox2b and Ret and the deletion of
either one of these genes produces mutant mice that do not
survive more than P1– P2. The short life span of these
mutants might be indicative of fatal respiratory dysfunction
at birth and might suggest a possible role of endogenous NA
in the prenatal maturation of RRG (Blanchi and Sieweke,
2005). Indeed analysing the in vivo breathing and the in
vitro RRG activity in mutant mice with genetically induced
NA deficits reveal that the proper development of the NA
brainstem groups is required for a normal activity of the
neonatal RRG and survival. Schemas in Fig. 5 summarize
some of the results obtained in mutant mice with NA
deficits.
In Rnx mutants (Fig. 5A), where the formation of most
NA neurons is drastically compromised but that of A6
neurons is partly preserved, the respiratory frequency is
abnormally high at birth, with respiratory cycle of highly
variable duration and frequent respiratory arrests (Shirasawa
et al., 2000). This means that the lack of A5, A1/C1 and A2/
C2 neurons during the gestation has altered the prenatal
maturation of the RRG. Indeed, the loss of A5 break vs. the
persistence of A6 accelerator may contribute to their fast
respiratory rhythm, the lack of A1/C1 facilitation and A2/C2
stabilizing effects to the respiratory arrests and the
variability of cycle duration, respectively.
In Phox2a mutants (Fig. 5B), the formation of most of
NA neurons is preserved (A5, A1/C1 and A2/C2 neurons)
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G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
Fig. 4. Rabies virus labelling of respiratory neurons in neonatal mice. Rabies virus (RV) retrogradely infects, step by step, chains of synaptically related
neurons. After RV injection within the diaphragm of 1-day-old neonatal mice, first the phrenic motoneurons, second their medullary central drivers and
thereafter some NA neurons from the A5 and A6 groups are RV-infected (24 – 30h, 30 – 36 h and 36 – 42h post injection, respectively). Immunohistochemistry
allows to detect TH-expression by NA neurons (green labelling) and to identify RV-infected neurons (red labelling). As some pontine A5 and A6 neurons show
a dual labelling (TH + RV; yellow labelling), it is concluded that these A5 and A6 NA neurons send synaptic projections to the medullary respiratory neurons.
Calibration bars: 100Am in (A), 50 Am in (B), 200Am in (C) and (D) (unpublished results from Hilaire G, Bevengut M. and Coulon P). (A) RV-infected neurons
from the ventral respiratory group, (B) Pontine A5 neuron (arrow) presenting a dual TH RV labelling, (C) pontine A6 neuron (arrow) presenting a dual
TH RV labelling, (D) a dual labelling was never observed in A1/C1 neurons (and A2/C2 neurons, not shown) although RV-infected areas and TH-areas were
often overlapping (see Fig. 2A).
but the formation of A6 neurons is drastically altered. The
agenesis of the A6 neurons resulted in an altered breathing
in fetuses caesarean-delivered by gestational day E18
(Viemari et al., 2004). First plethysmographic recordings
of the in vivo breathing frequency of control and mutant
E18 fetuses reveal an abnormally low breathing frequency
in Phox2a mutants. Second, recording the in vitro rhythm
produced by the isolated RRG in medullary preparations
(pons excluded) revealed central deficits: the in vitro
rhythm was abnormally low in Phox2a mutants (Fig. 5B).
G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
Fig. 5. Deletion of Rnx, Phox2a and Ret transcription factors alter the
respiratory rhythm in mice. Schemas showing the alterations of the NA
system and of the NA modulatory processes of the respiratory rhythm
generator (RRG) resulting from deletion of Rnx, Phox2a and Ret
transcription factors in mice. Retained and altered modulatory NA
processes are represented in black and grey, respectively. Compare with
schema in Fig. 3 drawn for a normal mice. (A) Rnx deletion abolishes the
formation of all the NA groups but A6. Shirasawa et al. (2000) report that
Rnx mutants present a fast respiratory rhythm interrupted by frequent
apnoeas. This deficit probably results from the persistence of the A6
facilitation (a1 adrenoceptors) vs. the loss of the A5, A2/C2 and A1/C1
modulations. (B) Phox2a deletion abolishes the formation of the A6 group
but the other NA groups are retained. Viemari et al. (2004) report that
Phox2a mutants present a slow respiratory rhythm both in vivo and in vitro.
This may result from the persistence of A1/C1, A2/C2 and A5 modulations,
especially the A5 inhibition via a2 adrenoceptors, vs. the loss of the A6
facilitation. (C) Ret deletion alters the formation of both A5 and A6 neurons
but the medullary groups are preserved. Viemari et al. (2005a, b) note that
the respiratory phenotype of Ret mutants was less severe than that of
Phox2a mutants. Ret mutants have a rather normal breathing in vivo but an
abnormally slow respiratory rhythm.
In addition, the response of the isolated RRG to
application of exogenous NA was altered in Phox2a
mutants, with a transient but huge rhythm facilitation by
327
NA. This suggests that a1 adrenoceptors are expressed by
the mutant RRG despite the lack of A6 neurons but that
the responsiveness of the a1 adrenoceptors is exacerbated.
Indeed, the lack of A6 facilitation onto the maturing RRG
has drastic consequence.
In Ret mutants (Fig. 5C), the formation of both A5 and
A6 neurons is altered as shown by measurements of NA
contents in the pons and by counting of TH-neurons in A5
and A6 areas whereas the formation of medullary NA
groups is not significantly altered (Viemari et al., 2005a). In
E18 exteriorized fetuses, plethysmographic recordings
revealed a tendency to a slower breathing frequency in
Ret mutants although not statistically different from control
fetuses. In vitro recordings confirmed central respiratory
deficits in Ret mutants with a slower respiratory rhythm and
an exacerbated response to NA application. Although the
NA and respiratory deficits of Ret mutants (partial loss of
A5 and A6 neurons) were less marked than those of Phox2a
mutants (total loss of A6 neurons), Ret-deficiency has also
altered the prenatal maturation of the RRG.
In Mecp2 mutants, on-going experiments performed in
our laboratory reveal that deletion of this gene also results in
NA and respiratory deficits (Roux et al., 2005; Viemari et
al., 2005b). In Mecp2 mutant neonates, breathing frequency,
NA contents and number of TH-neurons are normal at birth.
However from birth to 1 month of age, NA deficits develop
with a reduction of NA contents (Viemari et al., 2005b; Ide
et al., 2005) and a loss of A1/C1 and A2/C2 neurons.
Meanwhile NA alteration develops in Mecp2 mutants,
respiratory deficits appear, with first an increased variability
of the cycle duration observed both in vivo and in vitro (A2/
C2 alterations?) and second a reduction of breathing
frequency with appearance of recurrent, frightening apnoeas
(A1/C1 alterations?). The respiratory deficits worsen until
death, at about 2months of age. As the pontine NA contents
are not significantly affected in Mecp2 mutants (Viemari et
al., 2005b), it is likely that the respiratory deficits originate
from the medullary NA deficits. The abnormal variability of
cycle duration and the reduction of the breathing frequency
are consistent with A2/C2 and A1/C1 deficits, respectively
(Zanella et al., 2005).
In BNDF mutants, the expression of a normal respiratory
behavior is impaired at birth (Erickson et al., 1996). Both in
vivo and in vitro studies revealed a depressed and highly
variable respiratory rhythm during the postnatal period in
BNDF mutants; indeed BDNF was the first transcription
factor identified that is required for normal respiratory
rhythm and ventilatory control (Balkowiec and Katz, 1998).
As BDNF mRNA is expressed in medullary A1/C1 and A2/
C2 neurons (Cho et al., 1999), A1/C1 and A2/C2 alterations
might explain the depressed and variable respiratory rhythm
of BDNF mutants, respectively. Indeed, BDNF is required
for postnatal survival of some dopaminergic primary
sensory neurons in vivo and BDNF inactivation may alter
peripheral chemoceptor afferent neurons and the respiratory
response to hypoxia (Erickson et al., 2001) as well as the
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G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
long-term facilitation of respiration by intermittent hypoxia
(Baker-Herman et al., 2004). In piglets, intermittent hypoxic
periods markedly increased BDNF mRNA levels in the
entire medulla but only transiently affected the BDNF
protein level (Peiris et al., 2004).
In Phox2b mutants, expression of all NA neurons and
survival is impaired but heterozygous mutant mice for
Phox2b survive; they present a normal respiration when
awake but frequent sleep apnoeas (Durand et al., 2005).
Heterozygous mutations of PHOX2B have been found in a
high proportion of patients suffering from central congenital
hypoventilation syndrome (CCHS or Ondine’s curse), a rare
disorder characterized by an idopathic failure of the
autonomic control of breathing (Sasaki et al., 2003; Gaultier
et al., 2004). Even if mutations of PHOX2A, RET and
BDNF genes have also been reported in some cases of
CCHS, it is more likely that mutations of PHOX2B gene are
responsible for CCHS. Here again the NA deficits induced
by PHOX2B mutations may play a role in the RRG
function.
7. Discussion and conclusion
This review shows that endogenous NA contributes to
both the prenatal maturation and the neonatal function of the
RRG and that maturational NA deficits have deleterious
effects on RRG activity. Briefly, it has been reported that:
(1) The NA neurons develop early during gestation and
NA phenotype specification implicates different genes
such as Rnx, Phox2a, Phox2b and Ret,
(2) The NA neurons from the different brainstem groups
exert complex, specific modulations on the neonatal
RRG activity,
(3) Some of these NA neurons ‘‘belong’’ to the neonatal
respiratory network (i.e. those from the A5 and A6
groups send synaptic connections to the medullary
respiratory neurons), while others may act via
‘‘volume transmission’’ processes (those from A1/C1
and A2/C2 groups),
(4) Finally, deletion of genes implicated in NA phenotype
specification results in lethal respiratory deficits that
develop during either the prenatal (Rnx, Phox2a,
Phox2b, Ret) or the early postnatal periods (Mecp2).
Even if these results have been obtained in animals,
they may be of some relevance with humans. SIDS, the
leader cause of infant death in industrialised countries, is
highly likely due to neurovegetative disorders and more
precisely to cardiorespiratory dysfunction during sleep
(Horne et al., 2004; Ribas-Salgueiro et al., 2004).
Biochemical and histological postmortem analysis of SIDS
victims often reveal alterations of the NA system, with
abnormal levels of NA metabolites, abnormal morphology
of catecholaminergic neurons, abnormal expression of
medullary a2 adrenoceptors and abnormal expression of
catecholamine enzymes (Ozawa et al., 1999, 2003; CannMoisan et al., 1999; Obonai et al., 1998; Kopp et al., 1993;
Takashima and Becker, 1991; Denoroy et al., 1987; Ozand
and Tildon, 1983). Moreover, genetic analysis reveal some
protein-changing rare mutations in 14 of 92 SIDS cases
among the PHOX2a, RET, and RNX genes (Weese-Mayer
et al., 2004). Endogenous NA is known to be implicated in
sleep, cardiovascular and respiratory regulations. The
observations of biochemical, histological and genetic
alterations of the NA systems in some SIDS victims
strengthen the hypothesis that SIDS, or at least some cases
of SIDS, may be at least in part genetically pre-determined.
They also suggest that SIDS might originate at least in part
from NA deficits that in turn facilitate the occurrence of
respiratory deficits. In addition, the rare respiratory
disorder CCHS may also originate from NA deficits
resulting from Phox2b mutations.
During a normal life span, the rhythmic respiratory motor
act occurs about half a milliard of times, whatever the
species. From the very first moment of postnatal life until the
very last, this rhythmic motor act must be continuously
adapted to environmental and behavioral changes in order to
secure a proper oxygen delivery to the whole body, including
the brain. Although long lasting apnoeas with impaired
arousal may be lethal during the neonatal period, possibly
leading to SIDS, less drastic but recurrent respiratory deficits
also exist, such as central or obstructive sleep apnoeas and
CCHS, and may induce a chronic hypoxia. It has been
highlighted that stress during the prenatal and neonatal
periods can affect the programming of NA and other
neurotransmitters and receptors expression, and that this
can lead to long-term behavioral effects (Herlenius and
Lagercrantz, 2004). Thus besides the genetic factors, the
epigenetic factors may also play a role in the maturation of
the NA system whose alteration may lead to respiratory
dysfunction and chronic hypoxia. The latter may in turn
affect the maturation and function of all brain neurons and
functional circuits, with deleterious consequences on sleep,
memory, behaviors, etc. Indeed, it seems highly likely that
normal NA and normal respiratory systems during the
perinatal period are required for a normal life to occur.
Acknowledgements
The author acknowledges the financial supports of the
CNRS, the Université de la Méditerranée, the Rett
Syndrome Research Foundation and the Ministère de
l’Education Nationale, de l’Enseignement Supérieur et de
la Recherche (ACI NIC) while performing the reported
researches and writing this review. He would also like to
thank Dr. Michelle Bevengut and Dr. Patrice Coulon (FRE
CNRS 2722) for sharing unpublished results from common
experiments with rabies virus tracing of the respiratory
network in neonatal mice.
G. Hilaire / Autonomic Neuroscience: Basic and Clinical 126 – 127 (2006) 320 – 331
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