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First published online August 17, 2007
Journal of Experimental Biology 210, 3015-3026 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005983
Developmental changes in central O2 chemoreflex in Rana catesbeiana: the role of noradrenergic modulation
Department of Pediatrics, Université Laval, Centre de Recherche du Centre Hospitalier Universitaire de Québec, Québec City, QC, Canada
* Author for correspondence (e-mail: Richard.Kinkead{at}crsfa.ulaval.ca)
Accepted 18 June 2007
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1-antagonist prazosine (0.5 µmol l–1)
blocked the lung burst response to hypoxia in both groups. Blocking
GABAA/glycine receptors with a bicuculine/strychnine mixture (1.25
µmol l–1/1.5 µmol l–1, respectively)
or activation of GABAB pre-synaptic autoreceptors with baclofen
(0.5 µmol l–1) prevented the lung burst response to
hypoxia and to the 1-agonist phenylephrine (25 µmol
l–1) in both stage groups. We conclude that NA modulation
contributes to the central O2 chemoreflex in bullfrog, which acts
via GABA/glycine pathways. These data suggest that maturation of
GABA/glycine neurotransmission contributes to the developmental changes in
this chemoreflex.
Key words: control of breathing, amphibian, GABA, chloride, bicarbonate
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Moore et al., 1996). However,
the mechanisms linking noradrenergic modulation and hypoxic ventilatory
depression in newborn animals are not well understood
(Bissonnette, 2000).
This ventilatory chemoreflex is well conserved amongst vertebrates, as
exposing adult frogs to hypoxia also leads to ventilatory depression
(Rose and Drotman, 1967).
Furthermore, reducing O2 levels of the artificial cerebrospinal
fluid (aCSF) superfusing in vitro brainstems preparations decreases
fictive lung ventilation frequency in Rana catesbeiana
(Winmill et al., 2005) and
newborn rat (Brockhaus et al.,
1993). As this preparation is completely devoid of peripheral
(sensory) inputs, these data show that this chemoreflex is of central origin.
At this point, little is known about the neural mechanisms underlying the
central O2 chemoreflex in amphibians. While we have recently shown
that, under standard (hyperoxic) conditions, NA bath application onto bullfrog
brainstem preparations elicits fictive lung ventilation responses that are
similar to those observed during hypoxia
(Fournier and Kinkead, 2006),
a direct involvement of noradrenergic modulation in the O2
chemoreflex remains to be demonstrated in this species. With that in mind, the
main objective of the present study was to better understand the mechanisms
underlying the central O2 chemoreflex by testing the hypothesis
that noradrenergic receptor activation is necessary to observe a fictive lung
ventilation response to hypoxia in the brainstem preparation from Rana
catesbeiana. With this aim, we used selective pharmacological agents to
block adrenoceptors prior to exposing brainstems to hypoxic aCSF.
An important aspect of the work of Winmill and colleagues
(Winmill et al., 2005) is that
the response to central hypoxia is stage-dependent, since the frequency change
recorded from brainstems originating from pre-metamorphic tadpoles was not as
strong as the one from adult frogs. Given the lack of knowledge regarding this
intriguing aspect of respiratory control development, our second objective was
to address the mechanisms underlying maturation of the central O2
chemoreflex. In bullfrogs, GABA is an another key modulator of neural activity
that shows important stage-dependent effects on respiratory motor output that
are similar to those observed during hypoxia
(Broch et al., 2002). Based on
the knowledge that (1) noradrenergic modulation of rhythmic motor behaviours
such as locomotion and breathing can occur via indirect GABAergic
pathways (Arata et al., 1998;
Merrywest et al., 2002) and
(2) the post-synaptic response following GABAA receptor activation
changes substantially during development
(Ben-Ari, 2002), we tested the
hypothesis that developmental changes in noradrenergic modulation of fictive
lung ventilation are due to maturation of (indirect) GABAergic pathways. This
hypothesis was addressed using bath application of pharmacological agents
interfering with GABAergic neurotransmission during selective adrenoceptor
activation or hypoxia.
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In vitro brainstem preparations
Animals were anesthetised by immersion in a solution of tricaine methane
sulfonate (MS-222: 0.06 g l–1) buffered to pH 7 with
NaHCO3. For frogs, the beaker containing the MS-222 solution was
placed on ice for 30–60 min to slow metabolism and ensure adequate
anesthesia throughout the dissection
(Winmill and Hedrick, 2003).
Once unresponsive to body pinch, tadpoles and frogs were decerebrated by a
transection just rostral to the eyes. In frogs, a hole was drilled in the
cranium to allow decerebration. Tadpoles were then placed under the dissection
microscope for determination of the developmental stage based on the criteria
of Taylor and Kollros (Taylor and Kollros,
1946), which range between stages I to XXV. According to these
criteria, metamorphosis begins at stage XVIII. Animals between stages V and
XIII were assigned to the pre-metamorphic group (N=57). The cranium
was opened to expose the brainstem and rostral spinal cord and allow
dissection of the cranial nerves. The brain was irrigated with ice-cold
(0–5°C) artificial cerebrospinal fluid (aCSF) to reduce axonal
conductance throughout the dissection procedure. The composition of the aCSF
was identical to the one used in our previous studies
(Kinkead et al., 1994;
Kinkead et al., 2002;
Fournier and Kinkead, 2006).
For tadpoles, the aCSF consisted of (in mmol l–1): 104 NaCl;
4 KCl; 1.4 MgCl2; 10 D-glucose; 25 NaHCO3;
2.4 CaCl2, and for adult bullfrogs: 75 NaCl; 4.5 KCl; 1
MgCl2; 7.5 D-glucose; 40 NaHCO3; 2.5
CaCl2; 1 NaH2PO4. The use of a higher
bicarbonate concentration in the aCSF for adult bullfrogs is common (e.g.
Winmill et al., 2005) and is
based on the fact that (1) during bullfrog development, the transition from
water to air breathing is associated with a respiratory acidosis, which is
compensated by renal HCO –3 retention
(Just et al., 1973), (2) the
blood–brain barrier of frog is permeable to HCO
–3 (Wright,
1972) and (3) increasing aCSF [HCO –3]
results in a more stable respiratory activity in preparations from adult
bullfrogs (Kinkead et al.,
1994). The superfusate was equilibrated with a 98%
O2/2% CO2 gas mixture to pH 7.90±0.15 for
tadpoles and pH 7.8±0.15 for adult bullfrogs
(Kinkead et al., 1994;
Torgerson et al., 1997). The
brainstem was transected between the optic tectum and the forebrain and then
caudal to the hypoglossal nerve before being transferred to a small Petri dish
coated with SylgardTM (Dow Corning, Midland, MI, USA), where it was
immobilised with insect pins. The dura matter and parts of the arachnoid
(where possible) were carefully removed, and the brain was moved to the
recording chamber where it was placed ventral side up.
Electrophysiological recordings
Bursts of respiratory-related motor activity were recorded simultaneously
from the rootlets of cranial nerves V and X using suction electrodes. The
pipettes were constructed from borosilicate glass (0.84 mm i.d.) pulled to a
fine tip with a vertical microelectrode puller (Stoelting Instrument, Wood
Dale, IL, USA). The tip was broken and beveled to achieve appropriate tip
diameter. Neural activity signals recorded from the suction electrodes were
amplified (gain=10 000) and filtered (low cut-off: 10 Hz; high cut-off: 1 kHz)
using a differential AC amplifier (model 1700; A-M Systems, Everett, WA, USA).
Vagal and trigeminal signals were then full-wave rectified and integrated
(time constant: 100 ms) using a moving averager (model MA-821; CWE, Ardmore,
PA, USA). The raw and integrated nerve signals were viewed on an oscilloscope
and digitized for recording with a data acquisition system (model DI-720;
Dataq Instruments, Akron, OH, USA). The sampling rate of the analog to digital
conversion for the raw signal was 2500 Hz.
Experimental protocol
Once the recording electrodes were in place, the brainstem preparation was
superfused with control (drug-free, hyperoxic) aCSF at room temperature
(20–22°C) delivered at a rate ranging between 4 and 6 ml
min–1. The preparation was allowed to return to ambient
temperature and stabilise for 30–45 min, until stable rhythmic neural
activity was recorded from both nerves. Since most of the drugs used are
light-sensitive, drug preparation and experiments were conducted with dim
lights. All drugs were obtained from Sigma/RBI Aldrich (St Louis, MO,
USA).
Series I: the role of -adrenoceptors in the hypoxic chemoreflex in vitro
To demonstrate that our preparations produced a hypoxic response similar to
the one reported previously in this species
(Winmill et al., 2005), these
experiments first compared the effects of acute brainstem superfusion with
hypoxic aCSF on fictive breathing frequencies (both lung and buccal) between
two distinct developmental stages groups: adult bullfrogs and pre-metamorphic
tadpoles. For this series, the protocol began by recording respiratory-related
motor output for 10 min. Meanwhile, a second aCSF reservoir was bubbled with a
hypoxic gas mixture (98% N2, 2% CO2), which was then
delivered to the preparation for 10 min. Hypoxia was followed by a recovery
period, during which the preparation was superfused with drug-free, hyperoxic
aCSF for a period ranging between 50 to 70 min before a final recording of
respiratory-related motor output was made.
To determine whether activation of noradrenergic receptors is necessary for
the central hypoxic chemoreflex, brainstrem preparations were superfused with
a selective -adrenoceptor antagonist prior to hypoxic exposure.
Following 10 min of drug-free baseline recording, brainstem preparations were
superfused with aCSF containing either the 1 receptor
antagonist prazosine (Pr; 0.5 µmol l–1; pre-metamorphic,
N=5; adult, N=6) or the 2 receptor
antagonist RX821002 (RX; 25 µmol l–1; pre-metamorphic,
N=10; adult, N=6) for 20 min to obtain a second baseline
value in the presence of the antagonist. Following this equilibration period,
the preparation was subjected to hypoxia for 10 min in the presence of
antagonist before a 50 min `drug-free' recovery period. Because we were
concerned about potential carry-over effects, each preparation was exposed to
one antagonist only. The choice of these pharmacological agents was based on
our previous work showing that these receptors play a key role in the
modulation of fictive lung ventilation
(Fournier and Kinkead, 2006);
doses were selected from other studies (e.g.
Errchidi et al., 1991) and
preliminary experiments.
Series II: developmental changes in noradrenergic modulation of fictive ventilation: the role of Cl– inhibition
This series of experiments first established the stage-dependent effects of
NA bath application on fictive breathing frequencies, as shown previously
(Fournier and Kinkead, 2006).
The protocol began by recording baseline (drug-free) respiratory-related motor
output for 10 min before the preparation was superfused with aCSF from a
second reservoir containing 5 µmol l–1 NA for 10 min
(pre-metamorphic, N=6; adult, N=6). This procedure was
followed by a 50–70 min wash out period under control conditions.
We then assessed the potential contribution of indirect GABAergic pathways
in the noradrenergic modulation of respiratory activity across developmental
stages. GABA and glycine are commonly co-released
(Jonas et al., 1998;
O'Brien and Berger, 1999),
such that simultaneous application of bicuculline (GABAA
antagonist) and strychnine (glycine antagonist) is necessary for efficient
blockade of this inhibitory pathway (Jonas
et al., 1998). Preliminary experiments confirmed that this was the
case for our system also. Following baseline recording, we applied aCSF with a
bicuculline/strychnine mixture for 30 min (concentration: 1.25 µmol
l–1/1.5 µmol l–1, respectively) and
obtained a second `baseline' recording (in the presence of the antagonist
mixture). The selection of these concentrations was based on other studies
(Broch et al., 2002) and
preliminary experiments, which confirmed that our preparations could still
produce a motor output that was respiratory-like when the drugs were applied
simultaneously. We then added NA (5 µmol l–1; 10 min) to
the aCSF in the presence of the bicuculline/strychnine mixture before a wash
out period of 50–70 min was made with control aCSF.
The involvement of GABAergic/glycinergic pathways in NA modulation of
fictive breathing and hypoxic chemoreflex was also tested using selective
-adrenoceptor agonists and hypoxic aCSF. In those experiments,
preparations were superfused with aCSF containing the bicuculline/strychnine
mixture before the 1 receptor agonist phenylephrine (Phe; 25
µmol l–1), the 2 receptor agonist
clonidine (Clo; 25 µmol l–1) or hypoxia was applied to the
brainstem (pre-metamorphic, N=6; adult, N=6, for all),
according to the protocol described previously. The selection of the agonist
concentration was based on dose–response curves performed previously
(Fournier and Kinkead, 2006).
Although GABAA and glycine receptors are highly selective
Cl– channels, HCO –3 current
(via GABAA receptors) affects GABA responses, especially
in mature neurons (Yamada et al.,
2004). To determine whether the use of a higher [HCO
–3] in the aCSF used in adult frogs affected our
results (especially experiments involving the bicuculline/strychnine mixture),
these experiments were repeated by superfusing adult brainstems with the aCSF
used in tadpoles (low [HCO –3]; N=4).
The effects of the bicuculline/strychnine mixture on the baseline bursting
pattern were minimal (e.g. Fig.
4); however, the potential caveats associated with the fact that
bicuculline blocks voltage-activated K+ currents that help to set
the resting potential and thus control spontaneous cell firing
(Johansson et al., 2001;
Druzin et al., 2004), brought
us to consider an alternate approach. For these experiments, the
GABAB receptor agonist baclofen (0.5 µmol l–1)
was added to the aCSF to activate presynaptic autoreceptors (and thus reduce
endogenous GABA/glycine release). Following baseline measurements, the
preparation was superfused with baclofen for 30 min before preparations were
exposed to phenylephrine or hypoxia for 10 min as described previously
(pre-metamorphic: Phe, N=5; hypoxia, N=6; adults: Phe,
N=4; hypoxia, N=6). Note that because of results obtained in
our previous work (Fournier and Kinkead,
2006) and other data showing that, unlike
2-adrenoceptors, 1-adrenoceptors are
consistently involved in NA modulation of fictive lung ventilation and their
activation is necessary for the O2 chemoreflex in all developmental
stages (Fig. 2), the baclofen
experiments were performed with phenylephrine only. These procedures were
followed by a 50 min recovery period under control (drug free) conditions. For
these experiments, the baclofen concentration was based on the one reported by
Straus et al. (Straus et al.,
2000) and our preliminary experiments.
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;
Torgerson et al., 1998).
Cranial nerve burst amplitude from a single electroneurogram is not always
sufficient to identify fictive lung and buccal bursts adequately
(Sanders and Milsom, 2001).
Thus, two nerve signals were analysed simultaneously; here vagal nerve
activity was used as a sensitive marker of fictive lung activity to
distinguish between lung- and buccal-related signals
(Kogo et al., 1994;
Kogo and Remmers, 1994).
Lung and buccal burst frequencies were obtained by counting the number of
lung- and buccal-related bursting events within the 3-min segment analysed,
and averaged for a 1-min period. Buccal burst frequency could not be
quantified during bicuculline and strychnine application because they abolish
the buccal ventilation (Galante et al.,
1996; Broch et al.,
2002).
All measurements are reported as the mean ± 1 s.e.m. The results were analysed statistically using a two-way analysis of variance (ANOVA; Statview version 5.01; SAS Institute, Cary, NC, USA) followed by Fisher's protected least significant difference (PLSD) test (P<0.05). A repeated-measures design was used when appropriate.
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). Although the sequence for recruiting the glottal and narial
valves are distinct for water and air breathing, the buccal muscles activated
for pumping are essentially the same for gill and lung ventilation; however,
the neural commands driving both types of movements are different. The motor
output driving gill ventilation is characterised by its high frequency and low
amplitude. In contrast, the command driving air breathing is less frequent but
has greater amplitude as lung ventilation requires more forceful contraction
of the buccal pump to push air into the lungs
(Gans et al., 1969;
West and Jones, 1975). These
patterns of neural activity are produced by the in vitro brainstem
preparations from both preparations (brainstems from tadpoles and adult
frogs), even though air breathing is infrequent in pre-metamorphic tadpoles
(McLean et al., 1995;
Liao et al., 1996;
Broch et al., 2002;
Fournier and Kinkead, 2006).
Although adult frogs no longer ventilate their gills, the motor output driving
this activity remains, and the buccal oscillations thus produced are
hypothesised to play a role in olfaction and/or `refreshing' gas contents in
the buccal cavity prior to the next lung inflation. The trigeminal neurograms
shown in Fig. 1 (lower traces)
illustrate the bursting patterns associated with these types of breathing
movements.
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Hypoxia did not change buccal burst frequency in either group stage (hypoxia effect: P=0.79 and P=0.70, in pre-metamorphic and adult group, respectively; Fig. 1B). These data show that, in both groups, hypoxia had no effect on buccal burst frequency. Consequently, only data describing the effects of the pharmacological agents on `baseline' buccal burst frequency are presented for conciseness.
1-adrenoceptor activation is necessary for manifestation of the fictive lung ventilation response to hypoxia
In the pre-metamorphic group, addition of the selective
1-adrenoceptor antagonist prazosine to the aCSF did not
alter baseline fictive lung ventilation frequency (drug effect:
P=0.44; Fig. 2A).
While this drug tended to decrease baseline frequency in the adults, the
effect was not statistically significant (drug effect: P=0.33;
Fig. 2B). During hypoxia,
prazosine prevented the fictive lung burst frequency increase observed in the
pre-metamorphic group (hypoxiaxdrug: P=0.007;
Fig. 2A) whereas in adult
frogs, this antagonist prevented the frequency decrease as this variable
remained unchanged during the hypoxic stimulation period (hypoxiaxdrug:
P=0.02; Fig. 2B).
Statistical analysis confirmed that prazosine had a stage-dependent effect on
the response to hypoxia (stagex hypoxiaxdrug:
P=0.0007).
2-adrenoceptor activation is necessary for manifestation of the fictive lung ventilation response to hypoxia in pre-metamorphic group only
Addition of the selective 2-adrenoceptor antagonist
RX821002 to the aCSF did not alter baseline fictive lung ventilation frequency
in either group (drug effect: P=0.43 and P=0.30, in
pre-metamorphic and adult group respectively;
Fig. 2). In the pre-metamorphic
group, the increase in fictive lung burst frequency by hypoxia was blocked by
application of RX821002 (hypoxiaxdrug: P=0.003;
Fig. 2A). However, the decrease
of fictive lung burst frequency caused by hypoxia was not blocked in the adult
group (hypoxiaxdrug: P=0.60) and statistical analysis confirmed
that this effect is stage-dependent (stagexhypoxiaxdrug:
P<0.001; Fig.
2B).
Series II
GABA/glycine receptor activation is necessary for manifestation of the fictive lung ventilation response to noradrenergic receptor activation
In the pre-metamorphic group, application of NA (5 µmol
l–1) onto brainstem preparations increased fictive lung burst
frequency (drug effect: P=0.003;
Fig. 3A). Conversely, the same
NA concentration applied onto brainstems from adults decreased fictive lung
burst frequency (drug effect: P=0.02). Statistical analysis confirmed
that, as we have shown previously
(Fournier and Kinkead, 2006),
the effects of NA on fictive lung ventilation are stage-dependent
(stagexdrug: P=0.0002). In both groups, the recovery period
restored the fictive lung ventilation frequency values back to their initial
(baseline) values (data not shown).
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Addition of NA to the aCSF in the presence of the bicuculline/strychnine mixture decreased fictive lung ventilation frequency in the pre-metamorphic group (drug effect: P=0.04; Fig. 3B). Conversely, addition of the bicuculline/strychnine mixture prior to NA application prevented the decrease in fictive lung burst frequency normally observed in adults (drug effect: P=0.26; Fig. 3C). For both groups, fictive lung ventilation frequency returned to baseline values during the wash-out period (data not shown).
Addition of the selective 1-adrenoceptor agonist Phe to
the aCSF altered baseline fictive lung ventilation frequency in a
stage-dependent manner (stagexdrug: P=0.0003;
Fig. 4A). In the
pre-metamorphic group, application of Phe (25 µmol l–1)
onto brainstem preparations increased fictive lung burst frequency whereas
application of the same concentration onto adult preparations decreased it
(drug effect: P=0.005 and P=0.016, respectively;
Fig. 4A). Fictive lung
ventilation frequency returned to baseline values during the wash-out period
(data not shown).
Similar to previous experiments (Fig. 3B,C), bath application of the bicuculline/strychnine mixture alone had stage-dependent effects on baseline lung burst frequency (stagexdrug: P<0.0001; Fig. 4B,C). In both groups, this treatment prevented changes in lung burst frequency related to subsequent Phe application (drug effect: P=0.63 and 0.82, in pre-metamorphic and adult groups, respectively; Fig. 4A,B). Fictive lung ventilation frequency returned to baseline values during the wash-out period in the pre-metamorphic group; however, this was not the case for the adults in which a frequency increase was observed (data not shown).
Bath application of the selective 2-adrenoceptor agonist
Clo increased fictive lung burst frequency in both stage groups (drug effect:
P=0.017 and P=0.002, respectively;
Fig. 5A); however, this
response was prevented by pre-treatment with the bicuculline/strychnine
mixture. Addition of Clo to the aCSF containing bicuculline/strychnine
decreased fictive lung burst frequency in pre-metamorphic group, but had no
further effect in preparations from adult bullfrogs (P=0.004 and
P=0.77 respectively; Fig.
5A,B). Fictive lung burst frequency returned to baseline values
during the wash-out period in both groups (data not shown).
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Baclofen blocks lung burst frequency response to 1-adrenoceptor activation and hypoxia
Application of a bicuculline/strychnine mixture often disrupts basal
bursting pattern, which makes data analysis difficult. Although these effects
on the baseline bursting pattern were minimal (e.g.
Fig. 4), the potential caveats
associated with the fact that bicuculline blocks voltage-activated
K+ currents, which help to set the resting potential and thus
control spontaneous cell firing (Johansson
et al., 2001; Druzin et al.,
2004), led us to consider an alternative approach. For these
experiments, the GABAB receptor agonist baclofen (0.5 µmol
l–1) was added to the aCSF to activate presynaptic
autoreceptors (and thus reduce endogenous GABA/glycine release).
Despite suggestive trends, application of low concentrations of the selective GABAB agonist baclofen had no significant effects on lung burst frequency in either stage group (drug effect: P=0.09 and P=0.19, in pre-metamorphic and adult groups, respectively; Fig. 7A,B). However, this drug effectively blocked the lung burst response to Phe application in both groups as preparations pre-treated with baclofen maintained the same fictive lung frequency (Phexdrug: P=0.004 and 0.01, in pre-metamorphic and adult group, respectively; Fig. 7).
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In the next experiment, baclofen application did not change baseline lung burst frequency in either group (drug effect: P=0.82 and P=0.75, in pre-metamorphic and adult groups, respectively) but blocked the lung burst frequency response to hypoxia (hypoxiaxdrug: P=0.001 and P=0.03, in pre-metamorphic and adult group, respectively; Fig. 8).
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), addition of the bicuculline/strychnine mixture to the aCSF
abolished buccal burst frequency in both groups (drug effect:
P=0.0002 and P=0.008, pre-metamorphic and adult
respectively; Fig. 9B), and
subsequent addition of NA had no effect (data not shown). Addition of Phe (25 µmol l–1) to the aCSF did not alter fictive buccal ventilation frequency in either stage group (drug effect: P=0.93 and P=0.49, pre-metamorphic and adult, respectively; Fig. 9C). Fictive buccal ventilation frequency returned to baseline values during the wash out period (data not shown). Moreover, Clo application (25 µmol l–1) to the aCSF decreased fictive buccal ventilation frequency in both stage groups (drug effect: P=0.025 and 0.004, respectively; Fig. 9D). Effects on fictive buccal ventilation were not stage-dependent (stagexdrug: P=0.95) and were reversed in both groups (data not shown). Addition of Clo in the presence of the bicuculline/strychnine mixture did not restore fictive buccal related activity (data not shown). Hypoxia alone had no effect on fictive buccal activity in either group (Fig. 9E); however, addition of the bicuculline/strychnine mixture to the aCSF abolished fictive buccal activity in both groups (drug effect: P=0.0001) and the hypoxia period could not initiate this activity (data not shown).
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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-adrenoceptors) is necessary to central O2 chemoreflex
function (Fig. 2). However, the
modulatory influence that NA exerts onto the neural network generating fictive
lung ventilation likely acts indirectly via GABAergic/glycinergic
pathways, as drugs interfering with this neurotransmission prevent the effects
of noradrenergic agonists as well as the lung burst frequency response to
hypoxia in both stage groups (Figs
3,
4,
5,
6,
7,
8). With development, the
effects of GABA/glycine change from excitatory to inhibitory owing to the
progressive establishment of Cl– gradients in target neurons
(Ben-Ari, 2002), and our data
strongly suggest that this maturation process contributes to the developmental
change of the central hypoxic chemoreflex also. Although the present work
provides new information pertaining to the mechanisms underlying this
chemoreflex and its maturation, it is not possible at this point to determine
whether NA neurons are chemosensors per se or whether they are simply
part of the neural pathway generating this response.
Critique of method
Frogs encounter severe hypoxic conditions, for example during estivation
and overwintering. However, such conditions are infrequent and animals rarely
encounter hypoxia levels similar to the one used in our study. Although the
use of less severe hypoxia may be more physiologically relevant, we chose this
level mainly to reproduce previous in vitro studies
(Brockhaus et al., 1993;
Winmill et al., 2005). Even
so, results obtained in adult bullfrog brainstems were similar to the hypoxic
responses reported in intact frogs (Rose
and Drotman, 1967), newborn lambs
(Dawes et al., 1983;
Moore et al., 1996) and
newborn rats in vitro (Brockhaus
et al., 1993). This protocol allowed us to note that the fictive
breathing response to central hypoxia is restricted to lung ventilation as
fictive buccal movements were not affected by this stimulus. This is a key
observation because this result further distinguishes the mechanisms
regulating these two types of respiratory related motor outputs
(Vasilakos et al., 2005;
Janczewski and Feldman, 2006).
It also indicates that in adults, decrease in fictive lung ventilation is not
related to a non-specific depression of CNS function.
The role of -adrenoceptors in the hypoxic chemoreflex in bullfrog brainstems
Overall, our results are consistent with those reported by Winmill and
collaborators (Winmill et al.,
2005) as we showed that hypoxia affects fictive lung ventilation
in a stage-dependent manner. Hypoxia decreased fictive lung ventilation
frequency in the adult group, but caused a modest increase in lung burst
frequency in pre-metamorphic brainstems. For reasons that are unclear to us,
the latter response differs slightly from the one reported by these authors
who observed no increase in lung burst frequency during hypoxia
(Winmill et al., 2005).
However, the stage-dependent lung burst frequency responses to hypoxia in the
present study were similar to those observed following NA bath application
(Fournier and Kinkead, 2006),
which constitutes circumstantial evidence to support the hypothesis that NA is
involved in the central hypoxic chemoreflex. But given that prazosine or
RX821002 application was sufficient to block the increase in fictive lung
burst frequency in the pre-metamorphic group, the sum of these data lead us to
conclude that manifestation of the central hypoxic chemoreflex requires
-adrenoceptor activation. These results contrast with those observed in
adults in which only prazosine (not RX821002) effectively blocked lung burst
frequency depression during hypoxia, thus indicating that only
1-adrenoceptor activation mediates the hypoxic response in
this group. Reduction in 2-adrenoceptor expression with
maturation may explain why these receptors no longer contribute to this
response in the adults; however, we have no direct evidence in that regard.
These data are nonetheless consistent with the lung burst frequency response
to -adrenoceptor agonist application because only in pre-metamorphic
brainstems can the 2-agonist clonidine mimic the lung burst
frequency change observed during NA application
(Fournier and Kinkead,
2006).
Indirect GABAergic/glycinergic pathways mediate NA modulation of fictive lung ventilation
Our hypothesis that NA modulation of fictive lung ventilation acts
via indirect (GABAergic/glycinergic) pathways is based on previous
work showing that such interaction effectively modulates rhythmic motor
behaviours. For instance, Arata and collaborators
(Arata et al., 1998) showed
that in the medulla-spinal cord preparation from newborn rat, NA depressed
Pre-I rhythm and rhythmic C4 respiratory output in a standard perfusate.
However, the direct effect of NA on Pre-I neuron firings in
Cl–-free solution was excitatory, suggesting that the
respiratory rhythm depression in normal conditions was mediated by another
inhibitory system. This interpretation was confirmed by using a
GABAA antagonist that attenuated the depression observed following
NA application (Arata et al.,
1998). Such organisation seems highly conserved amongst
vertebrates since in Xenopus laevis, blocking GABAA and
glycine receptors prior to -adrenoceptor activation prevents changes in
the spinal locomotor output recorded in vitro. These results indicate
that GABAergic/glycinergic pathways are necessary for NA to modulate fictive
swimming in this species (Merrywest et
al., 2002).
Our results are consistent with those previous reports since GABAA and glycine receptor blockade prior to NA agonist application prevented changes in lung burst frequency. Moreover, the use of baclofen to attenuate endogenous GABA/glycine release corroborated these results. But more importantly in the present context, both approaches were effective in all stage groups. Together, these results support our hypothesis that developmental changes in GABA/glycine neurotransmission are involved in developmental changes in noradrenergic neuromodulation and fictive lung ventilation response to central hypoxia.
GABAergic/glycinergic pathways are involved in central hypoxic chemoreflex
Hypoxia increases GABA concentration in brain tissues as a function of the
severity and duration of hypoxia (Wood et
al., 1968). This response affects ventilatory activity because
GABAA receptor blockade prior to hypoxic exposure attenuated
ventilatory depression (Miller et al.,
2000). Despite species and preparation differences, our results
showing that the lung burst frequency increase observed during hypoxia is
counteracted by the bicuculline/strychnine mixture are consistent with these
studies and the hypothesis that GABAergic/glycinergic neurotransmission is
involved in the central O2 chemoreflex. However, this
interpretation must be made cautiously since in the present study, the hypoxic
response obtained in the adult group was influenced by aCSF [HCO
–3] concentration. Our experiments do not allow us
to explain why in the adult group, application of the bicuculline/strychnine
mixture could block the hypoxic response under low aCSF [HCO
–3] condition only. This result was surprising
because under standard (high [HCO –3]) conditions,
this antagonist mixture effectively blocked all lung burst responses to NA
agonist application. The bicuculline/strychnine concentration used may not
have been sufficient to prevent GABA/glycine receptor activation that occurs
during hypoxia; however, using a higher bicuculline/strychnine concentration
was not possible given the effects on bursting pattern. Incomplete
GABA/glycine receptor blockade, combined with the fact that severe hypoxia
commonly causes intracellular acidosis, would favor inward HCO
–3 current via GABAA receptors
and cell hyperpolarisation. Such a situation would not occur under low
[HCO3] aCSF condition. This explanation is speculative but
consistent with the fact that HCO –3 currents
affect GABA responses, especially in mature neurons
(Yamada et al., 2004).
Accordingly, this suggests that during metamorphosis, the renal compensation
of respiratory acidosis (via HCO –3
retention) provoked by the transition from water to air breathing plays an
important role in respiratory control maturation in this species.
The limitations inherent to the use of bicuculline/strychnine for such
studies are well documented. For instance, bicuculline disrupts lung bursting
pattern, abolishes all buccal-related activity, and blocks voltage-activated
K+ currents (Johansson et al.,
2001; Broch et al.,
2002; Druzin et al.,
2004). Based on this, we used an alternate approach by applying
the GABAB agonist baclofen to activate pre-synaptic autoreceptors
and attenuate GABA (and glycine) release from nerve terminals
(Harrison et al., 1988).
Unlike the bicuculline/strychnine mixture, baclofen did not disrupt baseline
bursting pattern, had minimal effects on buccal activity, and effectively
prevented the hypoxic response in both stage groups under experimental
conditions that mimic physiological CSF [HCO3–].
In light of these results, we conclude that, in this preparation, activation
of GABAergic/glycinergic pathways is necessary to elicit a reflexive lung
burst frequency response to central hypoxia.
Perspectives
Studies using brainstem preparations from newborn mammals have shown that
activation of noradrenergic neurons can exert opposite effects on phrenic
burst frequency, depending on which group of neurons were activated (A5:
inhibitory versus A6: excitatory)
(Hilaire et al., 2004). These
observations are difficult to reconcile given that both groups of NA neurons
converge on the same neural circuits that generate respiratory rhythm
(Dobbins and Feldman, 1994).
There are distinctions in CNS organisations between amphibians and mammals;
however, our demonstration that NA acts via indirect pathways
provides clues to the NA paradox reported in mammals. It is possible that, in
mammals, one pathway (e.g. A5) acts via GABAergic interneurons
whereas A6 neurons act directly. Clearly, more work needs to be done to
address this issue.
The functional significance of the central O2 chemoreflex is not intuitive. In pre-metamorphic tadpoles, for instance, increasing lung ventilation frequency during hypoxia appears futile because lungs are not fully developed. However, lung inflation is a stimulus that facilitates lung development. As such, the central O2 chemoreflex could contribute (albeit indirectly) to lung development. On the other hand, most mature air breathing animals tend to increase lung ventilation during hypoxia, a response that is mainly mediated by peripheral chemoreceptors. Given the energy cost associated with hyperventilation, this response may not be optimal under conditions of reduced O2 availability. It is therefore possible that the central inhibitory response to hypoxia aims to counterbalance the excitatory input from peripheral chemosensory structures to produce a more cost efficient response.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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  Y. Hager TADPOLE TRANSITIONS J. Exp. Biol., September 1, 2007; 210(17): i - i. [Full Text] [PDF]
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V, trigeminal nerve) showing changes
in respiratory-related motor output under baseline and hypoxic conditions in
the presence of the antagonists prazosine or RX 821002 in both stage groups
are shown on the right. Values are means ± s.e.m. *Value
statistically different from baseline at P<0.05.
value statistically different from corresponding mixture
value at P<0.05.
