Regulation of the respiratory central pattern generator by chloride-dependent inhibition during development in the bullfrog (Rana catesbeiana)
Department of Biological Sciences, California State University Hayward, Hayward, CA 94542, USA
* e-mail: mhedrick{at}csuhayward.edu
Accepted 28 January 2002
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Summary |
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Key words: amphibian, bullfrog, Rana catesbeiana, central pattern generator, fictive breathing, GABA, glycine, strychnine, bicuculline
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Introduction |
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In larval amphibians, gas exchange is accomplished primarily by rhythmic
ventilation of the gills, but as development progresses lung ventilation
assumes a greater fraction of overall gas exchange
(Burggren and West, 1982).
Recent evidence suggests that there is a `translocation' of the primary
site(s) for neural generation of lung ventilation from the caudal brainstem to
the rostral brainstem in developing tadpoles
(Torgerson et al., 2001b
).
Upon metamorphic climax, gill ventilation is replaced by rhythmic buccal
movements characteristic of adults, with lung ventilation occurring
episodically. Previous work with tadpole brainstem preparations in
vitro indicates that Cl--dependent synaptic inhibition may be
important for gill ventilation, but not lung ventilation, and that lung
ventilation may be driven by a pacemaker-like mechanism
(Galante et al., 1996
) similar
to that proposed for neonatal mammals
(Smith et al., 1991
).
Furthermore, both GABAergic and glycinergic mechanisms may be important
modulators of the amphibian CPG in tadpoles
(Galante et al., 1996
;
Strauss et al., 2000
).
However, the extent to which GABAA or glycinergic pathways
contribute to age-dependent inhibition of ventilation in amphibians is
unclear.
There is emerging evidence that the mechanisms regulating central
respiratory rhythmogenesis and respiratory motor output in amphibians share
many common features with those of developing mammals
(Gdovin et al., 1999). Thus,
amphibians appear to be excellent models for examining the development of
respiratory rhythm generation. The amphibian brainstem/spinal cord preparation
is particularly well suited as a developmental model for examining the
mechanisms of respiratory rhythm generation because the gamut of stages from
tadpole to adult frog can be studied using identical experimental techniques
at all stages of development.
In this study, we tested the hypothesis that aquatic-to-terrestrial development in amphibians is associated with the development of Cl--dependent synaptic inhibitory mechanisms for respiratory rhythm generation. For this purpose, we used isolated brainstem/spinal cord preparations from larval and adult North American bullfrogs (Rana catesbeiana).
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Materials and methods |
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Surgical procedures
Adult frogs and tadpoles were initially anesthetized in an aqueous solution
of tricaine methanosulfonate (MS222; 0.5 g l-1 for tadpoles and 1.5
g l-1 for adult bullfrogs) buffered to pH 7.4 with
NaHCO3. When breathing movements ceased (2-4 min for tadpoles and
10-20 min for adult frogs), the animals were quickly removed and placed in ice
for 1 h to maintain anesthesia and reduce metabolic rate. Following this, the
brain was exposed and the forebrain rostral to the optic lobes was transected.
Throughout the decerebration and subsequent dissection, the brainstem was
perfused continuously with ice-cold (5-7 °C) artificial cerebrospinal
fluid (aCSF) of the following composition (in mmol l-1): 40
NaHCO3, 1.0 NaH2PO4, 75 NaCl, 4.5 KCl, 1.0
MgCl2.6H2O, 7.5 glucose and 2.5 CaCl2
(Hedrick and Morales, 1999)
equilibrated with 100 % oxygen. The cranial nerves on both sides were
carefully dissected close to the exit from the skull. The entire dissection
required approximately 30 min to complete. The isolated brainstem was
transferred to a Sylgardlined recording chamber (7 ml). The brainstem was
placed ventral side up, and the dura were removed. Throughout this process,
and in all subsequent experiments, the recording chamber was continuously
perfused with aCSF (20-22 °C) equilibrated with 98 % O2 and 2 %
CO2 (pH 7.5-7.6) at a flow rate of 5-10 ml min-1.
Nerve recordings
Nerve recordings were obtained with glass suction electrodes connected to
cranial nerve roots V (trigeminal), X (vagus) and XII (hypoglossal) in the
isolated adult frog brainstem and to cranial nerve roots V, VII (facial) and
XII in the tadpole preparation. The electrodes were connected to a
differential alternating current amplifier (A-M systems, model 1700), and
action potentials were amplified 10 000-fold, filtered (10 Hz to 5 kHz) and
moving time averaged (CWE model MA-821-4). Raw and processed signals were
simultaneously recorded on computer with a data-acquisition system sampling at
2 kHz (MacLab 8S; AD Instruments) and on videotape with pulse code modulation
(Vetter PCM model 402).
Superfusion with Cl--free aCSF
Ten tadpoles and seven adult bullfrogs were used for experiments in which
isolated brainstem preparations were superfused with Cl--free aCSF.
To test the importance of Cl--mediated inhibition for respiratory
rhythmogenesis in the isolated brainstem, Cl- was replaced with the
anion salts of gluconic acid (Cl--free aCSF). The
Cl--free aCSF had the following composition (in mmol
l-1): 40 NaHCO3, 1.0 NaH2PO4, 75
sodium gluconate, 4.5 potassium gluconate, 1.0 magnesium gluconate, 7.5
glucose and 2.5 calcium gluconate.
The recording chamber was connected to two identical parallel reservoirs from which the brainstem was superfused with control aCSF or with Cl--free aCSF by switching between the two reservoirs. CO2 content was adjusted to maintain aCSF pH between 7.5 and 7.6 at all times. In each experiment, the control recording was followed by a 20 min period of superfusion with Cl--free aCSF. The last 10 min of recorded data was used for analysis.
Superfusion with agonists and antagonists of GABA and glycine
receptors
Preliminary experiments were performed to determine the minimal and maximal
effects on respiratory-related activity of the agonists and antagonists of
-aminobutyric acid (GABA) and glycine receptors. The following
concentration ranges were used: GABA and glycine (0.1-5.0 mmol
l-1), bicuculline (1-10 µmol l-1) and strychnine
(2.5-25.0 µmol l-1). The solutions were freshly prepared in aCSF
before each experiment and adjusted to pH 7.5-7.6 while bubbling with 98 %
O2/2 % CO2. The control recording was followed by a 15
min brainstem superfusion with aCSF followed by progressively increasing
concentrations of the agonist or antagonist achieved by switching between the
two reservoirs connected to the recording chamber. The last 10 min of each
drug superfusion period was recorded and used for analysis. After superfusion
with the highest concentration of the drug, a washout period of 30-180 min
with aCSF was necessary to re-establish control conditions, depending on the
drug used. In most cases, a single drug was used per preparation, but in five
tadpole preparations, two different drugs (agonist followed by antagonist)
were used per preparation.
Data analysis and statistics
Previous studies have established the correlation between the mechanical
events and neural activity associated with lung ventilation for adult
(Sakakibara, 1984) and larval
(Gdovin et al., 1998
)
bullfrogs. Thus, neural activity associated with gill and lung ventilation in
tadpoles, and lung ventilation in adults, has been clearly established in
vivo and used to identify fictive gill and lung bursts in vitro
(McLean et al., 1995
;
Galante et al., 1996
;
Torgerson et al., 1998
). In
adult bullfrogs, neural activity associated with lung ventilation occurs as
single breaths or as an episodic series of breaths. Although episodic
breathing does occur in vitro, it is not present in every preparation
and lung bursts often occur as single breaths
(Reid and Milsom, 1998
). For
analysis, we did not distinguish between single breaths or episodic breathing.
Lung burst frequency was measured as the total number of breaths divided by
the total recording period, which was normally 10-15 min. Lung burst duration
was measured from the onset at the baseline to the termination at the baseline
from the integrated neural trace, and lung burst amplitude was measured in
arbitrary units from the integrated neural signal.
In tadpole brainstem preparations, single fictive lung bursts were
distinguished from fictive gill bursts as large-amplitude bursts recorded
simultaneously in cranial nerves VII and XII. Burst activity in cranial nerve
XII in tadpoles serves as a `marker' for lung ventilation in vivo
(Gdovin et al., 1998) and
in vitro (Torgerson et al.,
1998
) because fictive gill ventilation is present in cranial
nerves V, VII and X, but not in cranial nerve XII in pre-metamorphic tadpoles.
In post-metamorphic tadpoles, such as those used in this study, fictive gill
ventilation begins to emerge in cranial nerve XII, but is clearly
distinguishable from fictive lung bursts
(Torgerson et al., 1998
).
Fictive gill ventilation is characterized by low-amplitude, high-frequency
neural activity compared with the high-amplitude, low-frequency lung bursts
(Torgerson et al., 1998
).
For each experiment, at least 10 fictive breaths (if present) associated
with either gill or lung ventilation were analyzed. Mean values for gill and
lung motor output for each preparation were used to calculate the group mean
± S.E.M. A one-way analysis of variance (ANOVA) followed by Dunnett's
multiple-comparison test (Zar,
1974) was used for evaluation of statistical significance between
fictive breaths during drug administration compared with the control within
each experimental group. Percentage data were converted to their arcsine
values prior to statistical analysis (Zar,
1974
). The minimal level of statistical significance was taken as
P<0.05. Statistical analyses were carried out using commercially
available software (GraphPad Prism v. 2.0).
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Results |
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A summary of the effects of Cl--free aCSF superfusion on fictive lung bursts in tadpole and adult brainstem preparations is shown in Table 1. Fictive lung burst frequency in tadpole brainstem preparations was 0.8±0.2 min-1 in aCSF and increased slightly (to 1.1±0.1 min-1; P>0.05) with Cl--free superfusion. Although lung burst frequency in tadpoles was unchanged in Cl--free aCSF, the characteristics of the lung bursts were altered. Individual lung bursts changed from an augmenting burst consisting of several smaller `peaks' superimposed on the lung burst (Fig. 1) to a single, large-amplitude decrementing burst with no small `peaks' (Fig. 2). For the tadpole preparations, there were 5.8±1.6 peaks burst-1, and this decreased significantly (P<0.05) to 1.03±0.03 peaks burst-1 in response to Cl--free superfusion. Following recovery in normal aCSF, the number of peaks per burst returned to control levels. The amplitude of the lung bursts increased nearly fourfold in response to Cl--free aCSF superfusion (Table 1; P<0.01) compared with control bursts. Lung burst duration was 8.3±2.6 s in control conditions, measured from cranial nerve XII, and did not change significantly during Cl--free aCSF superfusion (Table 1).
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Fictive gill burst frequency in tadpoles averaged 41.6±3.3
min-1 with control aCSF, which is similar to that recorded in
vivo for tadpoles at similar developmental stages
(Burggren and West, 1982;
Buggren and Doyle, 1986
;
Gdovin et al., 1998
).
Superfusion with Cl--free aCSF completely abolished gill burst
activity in every preparation (Table
1), but activity recovered when the preparation was re-perfused
with control aCSF, albeit at a slightly lower frequency (32.4±4.4
min-1; P>0.05).
In adult bullfrog preparations, fictive lung bursts were completely abolished in two of seven experiments in response to Cl--free aCSF. In the remaining five preparations, lung-related bursts were infrequent and did not resemble normal lung bursts because of their very long duration (Table 1) compared with control bursts. Lung burst frequency decreased significantly from 3.1±0.7 to 0.4±0.03 min-1 (P<0.01, N=7) in Cl--free aCSF (Table 1), but burst amplitude did not change significantly with Cl- substitution. To determine whether the effects of Cl--free aCSF were species-specific, and unrelated to developmental age, we examined the effects of Cl--free aCSF superfusion on brainstem preparations from six adult grass frogs (Rana pipiens). Similar to the results with adult bullfrogs, Cl--free aCSF abolished or significantly reduced lung burst frequency in adult grass frogs (Fig. 2). Overall, fictive lung burst frequency decreased from 19.4±0.8 to 0.1±0.03 min-1 (P<0.001; N=6) with Cl--free aCSF and recovered fully to control values (15.8±4.0 min-1) with normal aCSF.
Effects of GABA and glycine
Tadpole preparations exhibited a biphasic frequency response to GABA, with
an initial increase in lung burst frequency at 0.5 mmol l-1 GABA
followed by a significant inhibition of burst frequency at 5.0 mmol
l-1 GABA (Fig. 3A).
Overall, lung burst frequency increased significantly from 2.6±0.4 to
6.5±1.1 min-1 at 0.5 mmol l-1 and decreased to
0.1±0.1 min-1 at higher concentrations
(Fig. 3A). At the highest
concentration, lung burst activity was abolished in five of six tadpole
preparations. Tadpole brainstem preparations were relatively insensitive to
glycine, which had no effect on fictive lung burst frequency except at the
highest concentration used (5.0 mmol l-1 glycine), which abolished
lung bursts in every preparation.
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Fictive gill ventilation was more sensitive to the effects of GABA and glycine than were lung bursts. Gill burst frequency was significantly reduced at lower concentrations of both agonists (0.5 mmol l-1) and completely abolished at higher concentrations (Fig. 3A; glycine data not shown). Both gill and lung bursts returned to control levels after recovery in normal aCSF.
Lung burst activity in adult preparations exhibited a greater sensitivity to GABA and glycine compared with tadpole preparations; that is, significant effects on burst frequency occurred at lower concentrations of the agonists compared with tadpole preparations. In response to superfusion with GABA or glycine, lung burst frequency in adult preparations decreased significantly from approximately 5 to 8 min-1 (control) to approximately 1-2 min-1 with 0.5 mmol l-1 agonist (P<0.05, Figs 3B, 4). Increasing the concentration of GABA or glycine to 5.0 mmol l-1 caused further reductions in, or completely abolished, lung burst activity.
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Effects of bicuculline and strychnine
For tadpole preparations, superfusion with bicuculline and strychnine,
antagonists of GABAA and glycine receptors, respectively, caused
significant increases in lung burst frequency at concentrations of 5.0 µmol
l-1 (Figs 5A,
6). By contrast, fictive gill
bursts were abolished at the same concentrations of either antagonist
(Fig. 5A; bicuculline data not
shown). Both antagonists significantly increased lung burst amplitude and
duration at concentrations of 5-10 µmol l-1 in tadpoles:
bicuculline increased lung burst amplitude to 138.1±15.1% of the
control value (P<0.05), while strychnine increased the amplitude
to 264.3±16.6% of the control value (P<0.01).
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In contrast to tadpoles, bicuculline and strychnine significantly decreased burst frequency in adults (Figs 5B, 6). At 5.0 µmol l-1 bicuculline, frequency decreased from 14.0±6.1 to 1.9±0.5 min-1 (P<0.01), with no further decline in lung burst activity at higher concentrations. Strychnine produced similar effects on lung burst frequency, reducing lung bursts from 10.1±3.0 to 4.1±0.8 min-1 (P<0.05) at 5.0 µmol l-1 and further reducing frequency to 2.3±0.5 min-1 at 25.0 µmol l-1 (Fig. 6). Both antagonists had significant effects on burst shape, changing lung bursts from augmenting to decrementing while also increasing burst duration (Fig. 5B). Lung burst duration was typically approximately 1 s in adults, but increased to 5-8 s in the presence of bicuculline or strychnine. Neither bicuculline nor strychnine had a significant effect on lung burst amplitude in adult preparations.
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Discussion |
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Critique of methods
The advantages of in vitro brainstem/spinal cord preparations in
mammals for studying the cellular physiology of breathing are well recognized
(Feldman and Smith, 1989). The
high metabolic rate of mammalian brain tissue, coupled with the lack of
hypoxia-tolerance, has necessitated that studies using in vitro
preparations be limited to neonatal brainstem preparations to avoid diffusion
limitations inherent in these preparations. Mammalian brainstem preparations
must also be maintained at lower than normal temperatures to maintain
viability, and whole brainstem/spinal cord preparations in vitro have
been shown to become severely hypoxic and acidic
(Okada et al., 1993
). Despite
the considerable advances that have been made in our understanding of the
cellular mechanisms of respiratory rhythmogenesis in mammals, the use of
in vivo models and a variety of in vitro models of different
types, and of mammals of different developmental ages, makes comparisons among
studies difficult.
In vitro brainstem models from amphibians offer some advantages
over mammalian models (Luksch et al.,
1996), particularly with respect to developmental regulation of
the brainstem respiratory network. These advantages include a lower tissue
metabolic rate that allows brainstem preparations to be maintained at their
normal temperature, preventing tissue hypoxia. For example,
Po2 measurements within the core of the in vitro
tadpole brainstem have been shown to be normoxic to slightly hyperoxic
(Torgerson et al., 1997a
). In
addition, changes in central chemosensitivity during development have been
measured in bullfrog tadpoles, indicating the viability of the preparation
over a large range of developmental stages (Torgerson et al.,
1997b
,
2001a
). A unique advantage of
the present study compared with similar studies in mammals is that identical
techniques (i.e. whole brainstem preparation in vitro) were used to
examine respiratory rhythmogenesis in both larval and adult animals. Thus, the
effects of development alone were examined in the present study without the
possible confounding effects of different experimental conditions and
models.
In the present study, fictive gill burst frequency in tadpole preparations
was very similar to gill ventilation rates in free-swimming tadpoles
(Burggren and West, 1982;
Burggren and Doyle, 1986
) and
in spontaneously breathing, decerebrate tadpoles
(Gdovin et al., 1998
) at
similar developmental stages. Lung burst frequency, however, was somewhat
higher (0.8 min-1) than the in vivo studies (0.1-0.3
breaths min-1). One possible explanation is that MS-222 facilitates
fictive lung ventilation. MS-222 is a sympathetic stimulant that increases
plasma catecholamine levels for several hours in rainbow trout
Oncorhynchus mykiss (Gingerich
and Drottar, 1989
) and increases heart rate in amphibians
(Smith, 1974
). However, the
direct effects of MS-222 anesthesia on fictive breathing in the amphibian
brainstem preparation have not been examined.
Role of Cl--mediated synaptic inhibition
An important finding of our study is that lung bursts in the adult bullfrog
brainstem are abolished or significantly reduced in the presence of
Cl--free aCSF. This is in contrast to the finding that
Cl--free aCSF does not change lung burst frequency in tadpoles
(this study) (see Galante et al.,
1996). Because of these contrasting results, we examined the
possibility that the effects of Cl- substitution were
species-specific and unrelated to development. Lung burst activity was also
abolished or significantly reduced in adult R. pipiens, suggesting
that removal of Cl- has a greater effect on the respiratory CPG in
adults than in tadpoles. Our studies with tadpole brainstem preparations are
consistent with the results of a previous study
(Galante et al., 1996
) with
tadpoles from similar developmental stages and at similar temperatures to
those used in the present study. The data from tadpoles in the present study,
together with the results of Galante et al.
(1996
), suggest that lung
bursts in tadpoles are driven by `pacemaker'-like neural activity that does
not require Cl--dependent synaptic inhibition. However, because
gill bursts were abolished during perfusion with Cl--free aCSF,
conventional Cl--dependent inhibition may be responsible for gill
bursts. These results are consistent with recent studies indicating that the
gill and lung CPGs in tadpoles are anatomically separable
(Gdovin et al., 1999
;
Torgerson et al., 2001a
). The
results from the present study also extend previous work to show that lung
bursts in adult frogs are abolished, or their frequency significantly reduced,
in the presence of Cl--free aCSF. Cl--dependent
postsynaptic inhibition is required for many network-driven CPGs
(Marder and Calabrese, 1996
),
and lung ventilation in the adult bullfrog appears to rely for rhythmogenesis
on a postsynaptic inhibitory network that is similar to that in mammals
(Ramirez et al., 1997
).
The data from tadpoles and adult frogs are similar to those from studies in
mammals in showing postnatal changes in the mechanism of respiratory
rhythmogenesis. Superfusion of whole brainstem preparations or rhythmically
active brainstem slices from neonatal rats with Cl--free aCSF does
not abolish fictive lung bursts (Feldman
and Smith, 1989) or respiratory-related hypoglossal bursts
(Shao and Feldman, 1997
).
However, perfusion of the adult in situ rat brainstem with a
low-[Cl-] solution reversibly abolishes respiratory activity
(Hayashi and Lipski, 1992
),
which is consistent with the hypothesis that brainstem network interactions
are responsible for driving lung ventilation in adult mammals. Although
removal of extracellular Cl- does not disrupt respiratory rhythm in
the tadpole and neonatal rat brainstem, there are significant effects on lung
burst properties in both preparations. In tadpoles, there was a nearly
fourfold increase in burst amplitude in the presence of Cl--free
aCSF. This is consistent with a similar study with an in vitro
tadpole brainstem preparation (Galante et
al., 1996
) and with increased burst amplitude in the neonatal rat
brainstem (Feldman and Smith,
1989
). These results suggest that Cl--mediated
inhibition is not important for rhythm generation but may affect neurons
involved in pattern formation. It has been suggested that the respiratory
rhythm and pattern are generated by separate mechanisms in mammals
(Feldman and Smith 1989
;
Smith et al., 2000
).
Removal of extracellular Cl- would be expected to cause
depolarization of respiratory neurons as a result of the diffusional efflux of
intracellular Cl-, and this change in membrane potential
(Em) might have different effects on the respiratory CPG
in tadpoles and adults. Changes in Em are important for
modulating respiratory-related pacemaker cell frequency within the
pre-Bötzinger complex in brainstem slices from neonatal rats
(Smith et al., 2000). Thus,
removal of extracellular Cl- might induce changes in tonic
excitation that could drive the cell voltage out of the range for expressing
respiratory activity in our amphibian preparations. We do not believe this is
the case because varying tonic excitation within the entire brainstem by
changing extracellular [K+] results in significant, linear changes
in fictive gill/lung ventilation in tadpoles (R. E. Wade and M. S. Hedrick,
unpublished results). Therefore, it seems unlikely that changes in tonic
excitation induced by Cl- efflux in these experiments would
completely explain the different responses of tadpoles and adults to
Cl--free aCSF.
In the present study, both GABA and glycine produced an overall inhibition
of respiratory activity in tadpoles and in adults. The inhibitory effects of
GABA and glycine are consistent with the study by Galante et al.
(1996) and with the effects of
microinjections of GABA into rostral areas of the adult bullfrog brainstem
(McLean et al., 1995
). Adult
bullfrogs showed a significant reduction in lung burst frequency at an
approximately 10-fold lower concentration of GABA or glycine compared with
tadpoles, suggesting a greater sensitivity to these transmitters in the mature
brainstem. This may imply an increased density of GABA and/or glycine
receptors in adults compared with tadpoles or differences in synaptic uptake
mechanisms, but the development of GABA and/or glycine receptors in the
amphibian brainstem has not been investigated. Differences in diffusion of the
agonists to respiratory neurons might explain these results, but this is
unlikely. Because the brainstem of adults is slightly larger than that of
tadpoles, diffusion distances will probably be greater, and we would expect a
lower sensitivity to GABA and glycine if diffusion alone accounted for these
results.
There were also differences in the response characteristics to GABA in
tadpoles and adults. In tadpoles, GABA produced a biphasic response, with
excitation of lung burst frequency at lower concentrations followed by
inhibition at higher concentrations, whereas GABA produced only inhibition of
lung ventilation in adults. In various regions of the mammalian brain, GABA
and glycine are depolarizing during earlier developmental stages
(Cherubini et al., 1991), but
become hyperpolarizing during maturation
(Garaschuk et al., 1998
). There
is recent evidence for simultaneous excitation and inhibition by GABA in the
developing hippocampus (Ben-Ari,
2001
). The transition from excitation to inhibition is due to a
change in the accumulation of intracellular Cl- and a shift in the
equilibrium potential for Cl-
(Grillner, 1999
).
Developmental differences in cellular Cl- homeostasis might explain
the excitatory effects of GABA on tadpoles, but this possibility has not been
examined in amphibians.
The results of the present study are also similar to those of studies with
mammals in which GABA or glycine receptor blockade augments
respiratory-related burst frequency and amplitude in rhythmically active
brainstem slices from neonates (Shao and
Feldman, 1997), but abolishes or reduces respiratory-related
activity in adults (Hayashi and Lipski,
1992
; Ramirez et al.,
1996
; Pierrefiche et al.,
1998
). In maturing mice, lung burst frequency and pattern becomes
increasingly sensitive to the effects of strychnine during postnatal life
(Paton and Richter, 1995
;
Ramirez et al., 1996
).
However, GABA and/or glycine receptor blockade fails to abolish
respiratory-related activity in neonatal rat brainstem preparations
(Feldman and Smith, 1989
),
rhythmically active brainstem slices (Shao
and Feldman, 1997
) or working heart/brainstem preparations from
adult mice (Büsselberg et al.,
2001
). Bilateral injections of strychnine and bicuculline into the
pre-Bötzinger complex of adult cats in vivo abolishes
respiratory motor activity, illustrating the importance of synaptic inhibition
for respiratory rhythm generation in adult mammals
(Pierrefiche et al., 1998
).
Perfusion of whole rat brainstem with bicuculline and/or strychnine disrupts
normal respiratory rhythm, but breathing is not abolished
(Hayashi and Lipski,
1992
).
The lung burst frequency of tadpoles increased in response to bicuculline
or strychnine, whereas similar concentrations of these drugs decreased lung
burst frequency in adult bullfrog preparations
(Fig. 6). Application of the
GABAB antagonist 2-OH-saclofen produces dose-dependent increases in
lung burst frequency in pre-metamorphic (TaylorKöllros stages
VIX) tadpoles, but inhibits lung bursts in late-stage
(TaylorKöllros stage 24), post-metamorphic tadpoles
(Strauss et al., 2000).
Although the tadpoles in the present study were from intermediate stages, the
similarity between our results and those of Strauss et al.
(2000
) strongly suggests that
GABAergic, and also glycinergic, inputs regulate the respiratory CPG during
development.
Our results indicate that, during development in Rana catesbeiana,
lung ventilation is primarily pacemaker-driven, but becomes increasingly
dependent upon fast Cl- synaptic inhibition mediated by GABA and
glycine receptors. A shift from a pacemaker-driven to a network-dominated
respiratory CPG has also been hypothesized to occur during development in
mammals (Smith et al., 2000).
Thus, there may be common developmental changes in the neural processes
underlying lung ventilation in vertebrates. Our results suggest that
developmental maturation of the bullfrog respiratory CPG may reflect
developmental changes in glycinergic and/or GABAergic synaptic inhibition.
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Acknowledgments |
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