Development of the respiratory response to hypoxia in the isolated brainstem of the bullfrog Rana catesbeiana
Department of Biological Sciences, California State University, Hayward, Hayward, CA 94542, USA
* Author for correspondence (e-mail: mhedrick{at}csuhayward.edu)
Accepted 17 November 2004
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: amphibian, bullfrog, Rana catesbeiana, development, tadpole, respiratory rhythm generation, iodoacetate
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amphibians are clearly less tolerant of severe hypoxia than turtles and
carp, but are capable of surviving bouts of anoxia for 45 h at room
temperature (Knickerbocker and Lutz,
2001) and more than a day at 5°C
(Hermes-Lima and Storey,
1996
). Brain ATP levels in the hypoxic frog gradually decline and
when ATP reaches about 30% of normoxic levels, there is an increase in
extracellular K+, indicative of a loss of ionic homeostasis
signaling the onset of neuronal death
(Lutz and Nilsson, 2004
). The
progressive changes in brain ATP levels and changes in ionic homeostasis are
also likely to compromise respiratory motor activity in amphibians, but this
has not been examined in any detail. In the adult grass frog Rana
pipiens, anoxia results in a progressive decline in respiratory activity
and a complete cessation of breathing in about 30 min
(Rose and Drotman, 1967
).
Most studies in amphibians that have examined the effect of hypoxia on
ventilation have used more moderate levels of hypoxia (1015%
O2). At these levels of oxygen, both larval and adult amphibians
increase ventilation to maintain oxygen homoestasis
(Burggren and Doyle, 1987;
Kruhøffer et al., 1987
;
Smatresk and Smits, 1991
).
Larval (tadpole) amphibians increase gill ventilation and lung ventilation, a
response that changes with development
(West and Burggren, 1982
;
Burggren and Doyle, 1987
). For
example, early stage tadpoles show large increases in gill ventilation in
response to hypoxia, but as development proceeds, larger increases in lung
ventilation occur relative to gill ventilation
(Burggren and Doyle, 1987
).
These shifts in the response to hypoxia presumably reflect the increasing
reliance on lung ventilation for oxygen acquisition prior to
metamorphosis.
Following metamorphosis, when the gills involute and the post-metamorphic
frog becomes an obligate air-breather, the response to hypoxia is an overall
increase in lung ventilation frequency and amplitude, but also a distinct
change in the respiratory pattern to include an increase in episodic breathing
(Torgerson et al., 1998;
Kinkead and Milsom, 1994
).
Preventing oscillations in blood gases does not prevent the production of lung
episodes (Smatresk and Smits,
1991
; Kinkead and Milsom,
1994
), suggesting that episodic breathing is an intrinsic feature
of the amphibian brainstem respiratory oscillator. However, the mechanisms
that stimulate the increase in episodic breathing during hypoxia are
unclear.
The in vitro amphibian brainstem model has recently been used in a
number of studies examining developmental aspects of respiratory rhythm
generation (Gdovin et al.,
1999; Broch et al.,
2002
; Kinkead et al.,
2002
; Winmill and Hedrick,
2003a
,b
).
The amphibian model provides the unique advantage of direct developmental
comparisons, since spontaneous respiratory motor output can be recorded at all
developmental stages. In addition, the presence of spontaneously generated
respiratory activity provides an index of efferent motor output from the
brainstem, thereby allowing the effects of brain hypoxia to be directly
examined without the influence of peripheral oxygen chemoreceptors. Thus, this
model may provide useful insight into the mechanisms and development of the
respiratory response to hypoxia. The aim of the present study was to
characterize the respiratory response to hypoxia in the in vitro
bullfrog brainstem at different stages of development, and to determine the
contribution of anaerobic metabolism to fictive breathing during hypoxia.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In vitro brainstem preparation
Animals were anesthetized prior to surgery. Tadpoles were anesthetized by
submersion in an aqueous solution of ethyl-m-aminobenzoate (MS-222;
Sigma Chemical Co., St Louis, MO, USA; 0.5 g l1) buffered to
pH 7.8 with sodium bicarbonate. Adults were anesthetized by placing them in a
sealed container (1.5 l) with a cotton gauze that held approximately 1 ml of
the volatile anesthetic 2-bromo-2-chloro-1,1,1-trifloroethane (halothane;
Webster Veterinary Supply, Inc., Sterling, MA, USA). In a previous study with
adult bullfrogs, we found that halothane results in the rapid induction of a
surgical plane of anesthesia, and fictive breathing is produced much faster
than with MS-222 anesthesia (Hedrick and
Winmill, 2003). When breathing movements ceased and the withdrawal
and corneal reflexes were abolished (adults: 1520 min; tadpoles:
25 min), animals were removed from anesthesia. Tadpoles were placed in
iced water for 1 h prior to surgery in order to slow metabolism and maintain
anesthesia for subsequent dissection.
A small opening was made in the cranium allowing for the transection of the
brainstem at the rostral border of the optic lobes and subsequent removal of
the forebrain. During decerebration and subsequent dissection, the brainstem
was constantly perfused with cold (510°C) artificial cerebrospinal
fluid (aCSF) with the following composition: (in mmol l1)
adult: NaCl 75.0, KCl 4.0, MgCl2 1.0, NaH2PO4
1.0, NaHCO3 40.0, CaCl2 2.5, glucose 5.0; tadpole: NaCl
104.0, KCl 4.0, MgCl2 1.4, NaHCO3 25.0, CaCl2
2.4, glucose 10.0 (adapted from Kinkead et
al., 1994; Torgerson et al.,
2001
).
The brainstem was removed and placed in a recording chamber where the dura
and arachnoid were removed. Throughout this process and during all subsequent
experiments, the recording chamber was continuously perfused with oxygenated
aCSF (pH 7.8, 20°C) from a gravity-fed reservoir (350 ml) at a flow rate
of 510 ml min1. Glass suction electrodes were
attached to the nerve roots of cranial nerve (CN) V (trigeminal), X (vagus)
and XII (hypoglossal) in the adult preparation and CN V, VII (facial) and XII
in the tadpole, for the recording of respiratory-related neural activity.
These cranial nerves innervate elevator and depressor muscles in the
oropharyngeal region and are responsible for controlling glottal airflow, and
for generating water flow over the gills in tadpoles
(Gradwell, 1972) and airflow
during ventilatory and non-ventilatory behaviors in the adult
(DeJongh and Gans, 1969
).
Previous studies have verified that neural activity from CN V, VII, X and XII
are associated with breathing movements in tadpoles
(Gdovin et al., 1998
) and
adults (Sakakibara, 1984
).
Neural activity was amplified 10,000 times with a differential AC amplifier
(A-M systems model 1700; Everett, WA, USA), filtered (1001 kHz) and
recorded on a computer that interfaced with a data acquisition system sampling
at 2 kHz (Powerlab 8/S; AD Instruments, Milford, MA, USA).
Effects of hypoxia on respiratory-related motor output
Pre-metamorphic tadpole, post-metamorphic tadpole and adult brainstem
preparations were superfused with oxygenated aCSF (20°C; pH 7.8), bubbled
with 98% O2/2% CO2 for approximately 1 h, before a 10
min control recording was made. Following the control recording, superfusate
was switched to a reservoir containing aCSF (20°C; pH 7.8)bubbled with
anoxic, isocapnic gas (98% N2/2% CO2) from an electronic
mixing flowmeter (Cameron Instruments Co., Port Aransas, TX, USA; model
GF-3MP). In early experiments with pre-metamorphic (N=3) and adult
(N=3) brainstems, we monitored the PO2
of the hypoxic aCSF and brain tissue with a polarographic oxygen
microelectrode (30 µm tip diameter; Diamond General Corp., Ann Arbor, MI,
USA). We found that within 5 min of switch to the hypoxic aCSF,
PO2 near the brain surface and within the brain
tissue at any depth was near 0 kPa. Thus, we are confident that the brain
tissue in all experiments underwent sustained, severe hypoxia.
Inhibition of glycolysis with iodoacetate
Pre-metamorphic (N=6) and adult (N=6) preparations were
used to examine the role of anaerobic metabolism during hypoxia. Procedures
were identical to those described above, with the following exceptions. After
recording activity with superfusion of the control aCSF, the superfusate was
switched to hypoxic aCSF containing 100 µmol l1
iodoacetate (IAA) to inhibit anaerobic metabolism. Activity was recorded while
preparations were superfused with IAA in hypoxic aCSF for up to 4 h in
pre-metamorphic tadpoles (average 3 h), and 1.5 h in post-metamorphic tadpoles
and adults. All preparations were allowed to recover in oxygenated aCSF
following IAA in hypoxic aCSF.
Data analysis
Respiratory-related neural discharges were defined by previously described
criteria for fictive gill and lung bursts
(Reid and Milsom, 1998;
Torgerson et al., 1998
;
Hedrick and Winmill, 2003
;
Winmill and Hedrick,
2003a
,b
).
Burst frequency is defined as neural bursts per minute. Lung episodes are
defined as two or more bursts occurring in succession separated by a pause not
longer than the average duration of two ventilatory cycles. Non-respiratory
bursts were defined as bursts with duration longer than 1 s and having a burst
shape not resembling that of normal lung breaths (see
Hedrick and Winmill, 2003
).
Burst duration was measured from the onset of deviation from the baseline to
the return to baseline in the integrated neural trace. Rise time was measured
from the onset of deviation from the baseline to the peak value in the
integrated neural trace and analyzed as a percentage of burst duration. Burst
amplitude was measured in arbitrary units and analyzed as a percentage of
control from the integrated neural trace.
For each experiment a minimum of five bursts were analyzed from the control recording and during 10 minintervals throughout the period of hypoxic exposure until respiratory activity ceased (post-metamorphic and adult preparations). Fictive breathing during hypoxia in postmetamorphic and adult preparations was analyzed during the 515 min period after the switch to hypoxic aCSF. All preparations exhibited respiratory activity during this period and superfusate/tissue PO2 was near 0 kPa (see above).
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 anoxia compared with that in the control period, 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. All values are mean ± S.E.M., unless
otherwise specified. All statistical and graphical analyses were carried out
using commercially available software programs (Graphpad Prism, v. 3.0.1, San
Diego, CA, USA; Igor Pro v. 4.01, Wavemetrics, Inc., Lake Oswego, OR,
USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Fictive breathing in post-metamorphic tadpole and adult preparations
consisted solely of low frequency, high amplitude lung bursts
(Fig. 1B,C). High frequency,
low amplitude bursts, characteristic of non-ventilatory fictive buccal
activity, were present in two of seven adult preparations and were not
analyzed for the purpose of this study. In contrast to the response of
pre-metamorphic tadpoles, post-metamorphic tadpole and adult preparations
exposed to hypoxia within 515 min became very episodic
(Fig. 1B,C), and exhibited an
increase in the number of `non-respiratory' neural bursts
(Fig. 1B) (cf.
Hedrick and Winmill, 2003);
this was followed by a complete cessation of respiratory activity after
2040 min. The cessation of respiratory activity was sustained
throughout the period of hypoxic exposure, but was reversible upon
reoxygenation with control aCSF (Fig.
1B,C).
A summary of the changes in gill/lung burst frequency in pre-metamorphic tadpoles is provided in Fig. 2A. In pre-metamorphic tadpoles, fictive gill frequency was 57.2±1.0 min1 in control aCSF and decreased slightly during hypoxia to 45.2±3.0 min1 at 180 min hypoxic exposure (Fig. 2A; P>0.05). Fictive lung burst frequency decreased significantly from 2.5±0.4 min1 to 0.7±0.1 min1 (P<0.05), but only after 180 min hypoxia (Fig. 2A; P<0.05). Lung frequency returned to control values when preparations were returned to oxygenated aCSF. Gill activity ceased in three of six preparations and continued at a decreased level in the remaining preparations, but was not significantly different from the control recordings (P>0.05).
|
Hypoxic exposure in post-metamorphic tadpoles resulted in a cessation of respiratory-related activity in four of five preparations within 20 min after the onset of hypoxia and neural activity ceased in all preparations by 30 min. The average time to cessation for all five preparations was 18 min in hypoxia. Fictive apnea persisted during prolonged hypoxic exposure of up to 2 h and was reversible in all preparations upon return to oxygenated aCSF. Control lung burst frequency in the post-metamorphic preparation was 5.8±1.8 min1 and did not change significantly during the initial 20 min exposure to hypoxia (Fig. 2B; P>0.05).
In adult preparations, lung burst frequency was 4.6±1.2 min1 in control aCSF (Fig. 2B). Exposure to hypoxia resulted in a complete cessation of neural activity in all seven preparations by 40 min, with an average time of cessation of 24 min. There was no significant change from control until all respiratory activity stopped. Fictive apnea persisted during prolonged hypoxic exposure, in some cases lasting for >2 h, and was fully reversible in six of seven preparations (Fig. 2B).
Contribution of anaerobic metabolism during extreme hypoxia
The role of anaerobic metabolism during extreme hypoxia was investigated by
inhibiting glycolysis with IAA (Fig.
3). IAA inhibits glycolysis by blocking glyceraldehyde-3-phosphate
dehydrogenase (Xia et al.,
1992). These data were compared with the data obtained from
pre-metamorphic and adult brainstems exposed to hypoxia alone
(Fig. 2). In pre-metamorphic
tadpoles, superfusion with hypoxic aCSF containing 100 µmol
l1 IAA significantly reduced the amount of time gill and
lung burst activity could be maintained in hypoxia
(Fig. 3A,B). After 40 min
exposure to IAA in hypoxic aCSF, only three of six pre-metamorphic
preparations exhibited fictive gill ventilation and by 60 min only one of six
preparations showed any gill activity (Fig.
3A,B). After 90 min superfusion with hypoxia-IAA, fictive gill
bursts were abolished in every preparation
(Fig. 3A). Upon reoxygenation
with control aCSF, three of six preparations recovered gill activity. By
contrast, hypoxia alone had no effect on gill activity for 3 h
(Fig. 3A). Lung burst activity
in pre-metamorphic tadpoles (Fig.
3B) showed a similar response to hypoxia-IAA superfusion compared
with gill ventilation. Only three of six preparations exhibited any lung burst
activity after 50 min exposure, and the one preparation that had gill activity
after 60 min also exhibited lung burst activity
(Fig. 3B). All lung burst
activity was abolished by 80 min superfusion with hypoxia-IAA. Upon
reoxygenation with control aCSF, five of six preparations recovered and
produced fictive lung bursts at a frequency that was not significantly
different from control recordings (Fig.
3B; P>0.05).
|
Lung ventilation frequency in adult brainstem preparations showed similar responses whether superfused with hypoxic aCSF alone or with hypoxia-IAA (Fig. 3C). In hypoxia-IAA, four of six preparations ceased activity by 30 min and five of six preparations ceased activity by 40 min; by 50 min, all preparations had ceased activity (Fig. 3C). Upon reoxygenation, five of six preparations recovered to control levels (Fig. 3C).
Hypoxia-induced changes in respiratory pattern formation
Fig. 4 illustrates typical
gill and lung bursts in the pre-metamorphic preparation and typical lung
bursts in the postmetamorphic and adult preparation during control conditions
and hypoxia. Hypoxia had no significant effect on respiratory-related burst
characteristics including burst duration, amplitude or rise time at any of the
developmental stages examined (data not shown). There was no change in rise
time between control and hypoxia at any stage, which would indicate a shift
from the typical `bell-shaped' burst profile, to the rapid onset, rapid offset
burst characteristic of gasping in mammals
(St John and Knuth, 1981).
|
Lung burst episodes were present with control aCSF in three of six pre-metamorphic tadpole preparations. During control superfusion, lung episodes occurred at a frequency of 1.4±0.6 min1 and with a mean of 3.0±0.4 bursts per episode (Fig. 5A,B). There was no significant change in the frequency of episodes or the number of individual bursts per episode during hypoxic exposure in the pre-metamorphic tadpole preparation (Fig. 5A,B).
|
During the initial exposure to hypoxia (515 min), and prior to the cessation of respiratory activity, fictive breathing in postmetamorphic tadpole and adult preparations became more episodic, characterized by an increase in both the frequency of lung episodes and the number of individual bursts per episode in the adult preparations (Fig. 5A,B). Lung episodes were present during control conditions in three of five postmetamorphic tadpoles and two of seven adults. During the initial period of hypoxia, all five post-metamorphic tadpole preparations and all seven adult preparations exhibited lung episodes. In the post-metamorphic tadpoles, episode frequency was 0.6±0.5 min1 with control superfusion and increased to 3.1±0.7 min1 during the initial period of hypoxia (Fig. 5A; P<0.05). The number of individual bursts per episode in the post-metamorphic tadpoles increased from 2.0±0.04 bursts per episode to 3.8±0.6 bursts per episode during hypoxia, but this was not significant (Fig. 5B). In adult preparations, episode frequency increased significantly from 0.2±0.2 min1 to 0.8±0.2 min1 during hypoxia (Fig. 5A; P<0.01). The number of individual bursts per episode also increased significantly during hypoxia in the adult preparation, from a control level of 2.0±0.2 bursts per episode to 5.1±0.8 bursts per episode in hypoxia (Fig. 5B; P<0.05). In both post-metamorphic tadpole and adult preparations, hypoxia-induced changes in episode frequency and the number of individual bursts per episode were fully reversible when preparations were again superfused with oxygenated aCSF, as indicated by the lack of significant difference between control and recovery values (Fig. 5).
We analyzed the frequency of non-respiratory bursts before, during and
following the hypoxia challenge (Fig.
6). Anexample of a non-respiratory lung burst is provided in
Fig. 1 for a post-metamorphic
tadpole (see also Hedrick and Winmill,
2003). Non-respiratory bursts are those that do not conform to the
criteria established for normal lung bursts (see Materials and methods) and
are typically long duration, high amplitude bursts and make up about 10% of
the total bursts (Hedrick and Winmill,
2003
). In this study, pre-metamorphic tadpoles had a
non-respiratory burst rate of 0.2±0.05 min1 and this
did not change significantly during hypoxia or recovery
(Fig. 6). Non-respiratory burst
frequency under control conditions was 0.2±0.1 min1
for post-metamorphic brainstems and 0.03±0.02 min1
for adult brainstems. Both groups showed a significant increase in
non-respiratory burst frequency in hypoxia, increasing to 0.5±0.1
min1 in post-metamorphic tadpoles and to 0.4±0.1
min1 in adult preparations (P<0.05;
Fig. 6). The effect of hypoxia
on non-respiratory bursts was fully reversible upon reoxygenation during
recovery in control aCSF (Fig.
6).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Developmental changes in the respiratory response to hypoxia
The pre-metamorphic tadpole brainstem appears to be very tolerant to severe
hypoxia. This remarkable degree of hypoxia tolerance appears to be due to the
ability of the pre-metamorphic tadpole brain to use anaerobic metabolism to
maintain neural activity. However, these data differ from the in vivo
hypoxic response of tadpoles where hypoxia induces significant increases in
gill and lung frequency at moderate levels of hypoxia
[PO2 100 Torr (100 mmHg)], but is
suppressed when PO2 reaches about 20 mmHg
(West and Burggren, 1982
).
These responses are likely to be mediated by oxygen-sensitive chemoreceptors
in the gills of tadpoles (Straus et al.,
2001
). However, the present study used very severe hypoxia (near 0
kPa), rather than moderate levels of hypoxia used with freely behaving
animals. We are unaware of any studies that have examined the effects of
severe hypoxia/anoxia on respiratory or behavioral activity in tadpoles.
By contrast, the in vitro post-metamorphic tadpole and adult
bullfrog brainstem preparations respond to prolonged hypoxia with a complete
and reversible cessation of all respiratory-related motor activity, occurring
within about 25 min. Fictive apnea in these preparations persists during
prolonged hypoxia of up to 2 h and respiratory rhythm is restored when the
preparations are returned to oxygenated aCSF. The adult brainstem does not
appear to use glycolysis to maintain respiratory activity because the
cessation of respiratory activity was identical in hypoxia with or without
inhibition of anaerobic metabolism (Fig.
3C). Most adult brainstems exposed to hypoxia with IAA recovered
upon reoxygenation, but this differs from the response in vivo where
Rana pipiens given IAA died after 20 min exposure to anoxia
(Rose and Drotman, 1967). This
difference may be due to IAA affecting other organ systems (e.g. heart and
circulation) in the intact animal. Overall, these data are consistent with
natural history observations indicating that Rana tadpoles are more
hypoxia tolerant than adult animals
(Bradford, 1983
).
The mechanisms underlying the cessation of respiratory activity in the
post-metamorphic and adult brainstem in hypoxia are unclear. Respiratory
cessation occurs long before there are measurable changes in extracellular
K+ (Knickerbocker and Lutz,
2001) or excitatory neurotransmitters
(Lutz and Reiners, 1997
)
indicative of acute energy failure in the frog brain. Synaptic depression is a
common feature of the mammalian brain that occurs prior to a loss of ion
homeostasis (Hansen, 1985
),
which could account for the respiratory depression in this study. In the
anoxic turtle brain, reductions in ion channel conductance lead to elevations
of action potential thresholds, thus depressing electrical activity through
`spike arrest' (Sick et al.,
1993
). Spike arrest may be possible in the frog brain because
anoxia results in a decreased rate of K+ leakage into the
extracellular space (Knickerbocker and
Lutz, 2001
). Channel arrest is a characteristic feature of
anoxia-tolerant animals such as turtles
(Hochachka and Lutz, 2001
),
but there is little evidence for such extreme downregulation of ion channels
in the frog brain (Lutz and Nilsson,
2004
).
Other possible mechanisms that explain the respiratory cessation in our
study are the production of lactate in the hypoxic brainstem and/or the
activation of ATP-sensitive K+ (KATP) channels. Blood
lactate increases significantly in toads (Bufo marinus) when inspired
PO2 drops below 10 mmHg
(D'Eon et al., 1978) and in
brain tissue of frogs (Rana temporaria) within 10 min exposure to
anoxia (Wegner and Krause,
1993
). This should cause a significant drop in brain tissue pH and
potentially affect breathing. However, decreased pH increases in fictive
breathing in the adult bullfrog brainstem
(Kinkead et al., 1994
;
Morales and Hedrick, 2002
),
thus making changes in lactate and pH an unlikely mechanism for the
respiratory cessation during hypoxia. Neuronal KATP channels play a
protective role in brain hypoxia in mammals by increasing K+
channel conductance when ATP concentration decreases during severe brain
hypoxia (Ballanyi, 2004
). Brain
ATP levels decrease significantly in Rana temporaria after 20 min
anoxia (Wegner and Krause,
1993
) and drop to approximately 50% of normoxic levels in the
first 30 min of anoxia in Rana pipiens
(Lutz and Reiners, 1997
;
Knickerbocker and Lutz, 2001
).
In preliminary experiments using adult bullfrog brainstems, we examined the
potential role of KATP channels in mediating this response with the
KATP channel blocker glipizide. In some experiments glipizide
blocked the respiratory cessation in hypoxia, consistent with a role for
KATP channels, but glipizide had no effect in other experiments.
This would suggest that the respiratory cessation that occurs in the hypoxic
bullfrog brainstem is not entirely due to reductions in ATP gating the
KATP channel.
Because IAA fails to inhibit breathing with a faster time course than with
hypoxia alone, these data suggest the presence of an oxygen `sensor' in the
mature amphibian brainstem linked to the reversible cessation of respiratory
activity. There is substantial evidence for the presence of an oxygen `sensor'
in the mammalian brainstem from in vivo
(Solomon et al., 2000) and
in vitro (Telgkamp and Ramirez,
1999
) preparations. The oxygen sensing ability of mammalian
neurons may use a variety of cellular mechanisms, including the gating of
several different ion channels (see Acker
and Acker, 2004
). The pre-Bötzinger Complex (PBC), a proposed
site for respiratory rhythm generation in the mammalian brainstem, has been
shown to function as an oxygen sensor that can regulate breathing in
vivo (Solomon et al.,
2000
).
The timing of developmental changes in the respiratory response to hypoxia
may have important ecological implications. Bullfrog tadpoles are facultative
air breathers until T-K stage XXI (Crowder
et al., 1998). All stages of bullfrog tadpoles breathe air, but
the frequency is low in normoxic water and increases in hypoxic water
(West and Burggren, 1982
;
Burggren and Doyle, 1987
;
Crowder et al., 1998
).
Following the loss of the gills between T-K stages XXII-XXIII
(Crowder et al., 1998
), the
post-metamorphic tadpole becomes an obligate air breather. Lacking alternative
means of meeting oxygen demands in a hypoxic environment, the post-metamorphic
tadpole and adult amphibian may tend to shut down metabolic processes,
including respiratory activity, in order to conserve cellular energy. Evidence
for this decrease in metabolic costs in the face of hypoxia has been observed
in vivo. For example, adult Rana pipiens exposed to anoxia
demonstrate a rapid decrease in pulmonary respiratory movements, with
respiratory cessation occurring after approximately 30 min of anoxic exposure
(Rose and Drotman, 1967
).
Interestingly, this time course in vivo is nearly identical with the
time course of respiratory cessation for postmetamorphic tadpoles and adult
bullfrog brainstems in this study (Fig.
2B). We suggest that the reversible cessation of respiratory
activity, by an unknown mechanism, during hypoxia in post-metamorphic tadpoles
and adults may be an adaptive, energy-saving response to periods of severe
hypoxia.
The present data are entirely consistent with the general view that
neonatal mammals survive hypoxic conditions that result in severe neuronal
damage in adult mammals (e.g. Duffy et
al., 1975). The increased hypoxia tolerance of neonatal mammals
involves increased ability to generate ATP anaerobically and to downregulate
ion channels to preserve ion gradients that decrease ATP demand
(Bickler and Hansen, 1998
;
Bickler et al., 2003
). It is
clear from our study that pre-metamorphic tadpole are capable of generating
respiratory neural activity longer in hypoxia and part of this ability
involves increased reliance on anaerobic metabolism compared with
post-metamorphic animals. Taken together, these data point to some common
mechanisms in vertebrates with respect to the development of hypoxia
tolerance.
Hypoxia-induced changes in respiratory pattern generation
The episodic breathing pattern observed during normocarbia in amphibians
and reptiles is clearly an endogenous property of the central nervous system.
In unidirectionally ventilated toads (West
et al., 1987; Smatresk and
Smits, 1991
) and bullfrogs
(Kinkead and Milsom, 1994
),
where the natural oscillations of blood gases associated with periods of
ventilation and apnea were experimentally prevented, breathing is still
episodic. Furthermore, the in vitro bullfrog
(Kinkead et al., 1994
;
Reid and Milsom, 1998
;
Hedrick and Winmill, 2003
) and
turtle (Douse and Mitchell,
1990
) brainstem preparations are capable of generating fictive
lung episodes similar to that seen in the whole animal, despite removal of all
peripheral inputs.
Although episodic breathing is an endogenous feature of the amphibian
brainstem respiratory network, it is not clear if the increase in episodic
breathing during hypoxia (Kruhøffer
et al., 1987; Smatresk and
Smits, 1991
; Kinkead and
Milsom, 1994
) results from stimulation of peripheral oxygen
chemoreceptors or from stimulation of a central oxygen chemosensor.
Chemosensory feedback has been suggested to play a role in the modulation of
the episodic breathing pattern in the decerebrate, unidirectionally ventilated
toad Bufo marinus, since bilateral vagotomy prevents
hypercapnia-induced increases in breathing episodes
(Reid et al., 2000
). Because
our preparation was devoid of feedback from peripheral oxygen-sensitive
chemoreceptors, the data in this study argue that brainstem hypoxia is capable
of stimulating episodic breathing in the bullfrog brainstem.
Severe hypoxia in mammals transforms the breathing pattern from eupnea to
gasping, a form of widespread respiratory excitation
(St John and Knuth, 1981;
Solomon, 2000
). This
transformation is characterized by a shift in the pattern of phrenic nerve
activity from the slowly augmenting, rapidly decrementing eupneic burst, to a
rapid onset, slowly decrementing burst characteristic of gasping
(St John and Knuth, 1981
). The
in vitro medullary slice preparation from neonatal mice has been
demonstrated to be capable of producing multiple respiratory-related burst
patterns resembling eupnea, sighs and gasping and hypoxia changes the pattern
of neural activity recorded from neurons of the ventral respiratory group
(VRG) from eupneic to gasp-like bursts
(Lieske et al., 2000
).
Gasp-like activity does not appear to be a feature of the isolated amphibian brainstem in hypoxia. During superfusion with hypoxic aCSF, there was no significant change in burst characteristics, including burst amplitude, duration, or rise time, which would indicate a change to fictive gasping. These burst characteristics did not change during hypoxic exposure at any of the developmental stages examined; thus, the primary responses to hypoxia in this preparation are associated with changes in respiratory frequency and clustering of breaths into episodes in post-metamorphic tadpoles and adults.
Post-metamorphic tadpoles and adults exhibited a significant increase in
the frequency of non-respiratory bursts in hypoxia
(Fig. 5). Non-respiratory
bursts have been characterized in previous studies with amphibians
(Reid and Milsom, 1998;
Hedrick and Winmill, 2003
) and
lampreys (Thompson, 1985
), but
the function of these bursts remains unclear. In the isolated lamprey
brainstem, non-respiratory bursts have been characterized as `arousal'
breathing (Thompson, 1985
) and
are similar to the non-respiratory bursts in the present study. In adult
amphibians, non-respiratory bursts are typically large amplitude, long
duration bursts that are more resistant to the inhibitory effects of MS-222
anesthesia than typical respiratory bursts
(Hedrick and Winmill, 2003
).
Because non-respiratory bursts increased significantly with hypoxia only in
postmetamorphic and adult brainstem preparations, these breaths may play a
role similar to gasping in adult mammals; that is, a form of `arousal' that is
a last-ditch effort by a brainstem motor network to re-start breathing when
normal respiratory efforts fail (Solomon,
2000
). Another similarity amphibian non-respiratory bursts share
with gasping in mammals is that they are both readily reversible upon
reoxygenation (Solomon, 2000
).
Thus, non-respiratory bursts may be important for generating a widespread
neural activity to resuscitate breathing during severe hypoxia and may be
functionally equivalent to gasping in mammals.
Conclusions
This study demonstrates a developmental change in the central mechanisms
modulating the respiratory response to hypoxia in bullfrogs. The
pre-metamorphic tadpole respiratory CPG appears to be far more tolerant to
hypoxia, primarily owing to the ability to use anaerobic metabolism, while
respiratory activity in the post-metamorphic tadpole and adult is reversibly
abolished shortly after the onset of hypoxia and does not appear to use
glycolysis to maintain respiratory activity. We suggest that the reversible
cessation of respiratory activity in the hypoxic post-metamorphic bullfrog
brain is an adaptive, energy-saving response that may be mediated by a
brainstem oxygen sensor.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acker, T. and Acker, H. (2004). Cellular oxygen
sensing need in CNS function: physiological and pathological implications.
J. Exp. Biol. 207,3171
-3188.
Ballanyi, K. (2004). Protective role of
neuronal KATP channels in brain hypoxia. J. Exp.
Biol. 207,3201
-3212.
Bickler, P. E. and Hansen, B. M. (1998). Hypoxia-tolerant neonatal CA1 neurons: relationship of survival to evoked glutamate release and glutamate receptor-mediatedcalcium changes in hippocampal slices. Dev. Brain Res. 106, 57-69.[Medline]
Bickler, P. E., Donohoe, P. H. and Buck, L. T.
(2000). Hypoxia-induced silencing of NMDA receptors in turtle
neurons. J. Neurosci.
20,3522
-3528.
Bickler, P. E., Fahlman, C. S. and Taylor, D. M. (2003). Oxygen sensitivity of NMDA receptors: relationship to NR2 subunit composition and hypoxia tolerance of neonatalneurons. Neurosci. 118,25 -35.[CrossRef][Medline]
Bradford, D. F. (1983). Winterkill, oxygen relations, and energy metabolism of asubmerged dormant amphibian, Rana muscosa. Ecology 64,1171 -1183.
Broch, L., Morales, R. D., Sandoval, A. V. and Hedrick, M.
S. (2002). Regulation of the respiratory central pattern
generator by chloride-dependent inhibition during development in the bullfrog
(Rana catesbeiana). J. Exp. Biol.
205,1161
-1169.
Burggren, W. W. and Doyle, M. (1987). Ontogeny of regulation of gill and lung ventilation in the bullfrog, Rana catesbeiana. Respir. Physiol. 66,279 -291.[CrossRef]
Crowder, W. C., Nie, M. and Ultsch, G. R. (1998). Oxygen uptake in bullfrog tadpoles (Rana catesbeiana). J. Exp. Zool. 280,121 -134.[CrossRef][Medline]
DeJongh, H. J. and Gans, C. (1969). On the mechanism of respiration in the bullfrog, Rana catesbeiana: a reassessment. J. Morphol. 127,259 -290.
D'Eon, M. E., Boutilier, R. G. and Toews, D. P. (1978). Anaerobic contributions during progressive hypoxia in the toad Bufo marinus. Comp. Biochem. Physiol. 60A, 7-10.[CrossRef]
Douse, M. A. and Mitchell, G. S. (1990). Episodic respiratory related discharge in turtle cranial motoneurons: in vivo and in vitro studies. Brain Res. 536,297 -300.[CrossRef][Medline]
Duffy, T. E., Kohle, S. J. and Vannucci, R. C. (1975). Carbohydrate and energy metabolism in perinatal rat brain: relation to survival in anoxia. J. Neurochem. 24,271 -276.[Medline]
Fernandes, J. A., Lutz, P. L., Tannenbaum, A., Todorov, A. T., Liebovitch, L. and Vertes, R. (1997). Electroencephalogram activity in the anoxic turtle brain. Am. J. Physiol. 273,R911 -R919.[Medline]
Gdovin, M. J., Torgerson, C. S. and Remmers, J. E. (1998). Neurorespiratory pattern of gill and lung ventilation in the decerebrate spontaneously breathing tadpole. Respir. Physiol. 113,135 -146.[CrossRef][Medline]
Gdovin, M. J., Torgerson, C. S. and Remmers, J. E. (1999). The fictively breathing tadpole brainstem preparation as a model for the development of respiratory pattern generation and central chemoreception. Comp. Biochem. Physiol. 124A,275 -286.
Gradwell, N. (1972). Gill irrigation in Rana catesbeiana. Part II. On the musculoskeletal mechanism. Can. J. Zool. 50,501 -521.[Medline]
Hansen, A. J. (1985). Effect of anoxia on ion
distribution in the brain. Physiol. Rev.
65,101
-148.
Hedrick M. S. and Winmill, R. E. (2003). Excitatory and inhibitory effects of tricaine (MS-222) on fictive breathing in the isolated bullfrog brainstem. Am. J. Physiol. 284,R405 -R412.
Hermes-Lima, M. and Storey, K. B. (1996). Relationship between anoxia exposure and antioxidant status in the frog Rana pipiens. Am. J. Physiol. 271,R918 -R925.[Medline]
Hochachka, P. W. and Lutz, P. L. (2001). Mechanism, origin and evolution of anoxia tolerance in animals. Comp. Biochem. Physiol. 130B,435 -459.
Johnson, S. M., Johnson, R. A. and Mitchell, G. S.
(1998). Hypoxia, temperature, and pH/CO2 effects on
respiratory discharge from a turtle brain stem preparation. J.
Appl. Physiol. 84,649
-660.
Kinkead, R. and Milsom, W. K. (1994). Chemoreceptors and control of episodic breathing in the bullfrog (Rana catesbeiana). Respir. Physiol. 95, 81-98.[CrossRef][Medline]
Kinkead, R., Filmyer, W. C., Mitchell, G. S. and Milsom, W.
K. (1994). Vagal input enhances responsiveness of respiratory
discharge to central changes in pH/CO2 in bullfrogs. J.
Appl. Physiol. 77,2048
-2051.
Kinkead, R., Belzile, O. and Gulemetova, R.
(2002). Serotonergic modulation of respiratory motor output
during tadpole development. J. Appl. Physiol.
93,936
-946.
Knickerbocker, D. L. and Lutz, P. L. (2001). Slow ATP loss and the defense of ion homeostasis in the anoxic frog brain. J. Exp. Biol. 204,3547 -3551.[Medline]
Kruhøffer, M., Glass, M. L., Abe, A. S. and Johansen, K. (1987). Control of breathing in an amphibian Bufo paracnemis: effects of temperature and hypoxia. Respir. Physiol. 69,267 -275.[Medline]
Lieske, S. P., Thoby-Brisson, M., Telgkamp, P. and Ramirez, J. M. (2000). Reconfiguration of the neural network controlling multiple breathing patterns: eupnea, sighs and gasps. Nature Neurosci. 3,600 -607.[CrossRef][Medline]
Lutz, P. L. and Nilsson, G. E. (2004). Vertebrate brains at the pilot light. Respir. Physiol. Neurobiol. 141,285 -296.[CrossRef]
Lutz, P. L. and Reiners, R. (1997). Survival of
energy failure in the anoxic frog brain: delayed release of glutamate.
J. Exp. Biol. 200,2913
-2917.
Morales, R. D. and Hedrick, M. S. (2002). Temperature and pH/CO2 modulate respiratory activity in the isolated brainstem of the bullfrog (Rana catesbeiana). Comp. Biochem. Physiol. 132A,477 -487.
Nilsson, G. E. (2001). Surviving anoxia with the brain turned on. News Physiol. Sci. 16,217 -221.[Medline]
Pérez-Pinzón, M., Rosenthal, M., Sick, T., Lutz, P. L. and Marsh, D. (1992). Down-regulation of sodium channels during anoxia: a putative survival strategy of turtle brain. Am. J. Physiol. 31,R712 -R715.
Reid, S. G. and Milsom, W. K. (1998). Respiratory pattern formation in the isolated bullfrog (Rana catesbeiana) brainstem-spinal cord. Respir. Physiol. 114,239 -255.[CrossRef][Medline]
Reid, S. G., Milsom, W. K., Meier, J. T., Munns, S. and West, N. H. (2000). Pulmonary vagal modulation of ventilation in toads (Bufo marinus). Respir Physiol. 120,213 -230.[CrossRef][Medline]
Rose, F. L. and Drotman, R. B. (1967). Anaerobiosis in a frog, Rana pipiens. J. Exp. Zool. 166,427 -431.[Medline]
Sakakibara, Y. (1984). The pattern of respiratory nerve activity in the bullfrog. Jpn. J. Physiol. 34,827 -838.[Medline]
Sick, T. J., Pérez-Pinzón, M., Lutz, P. L. and Rosenthal, M. (1993). Maintaining a coupled metabolism and membrane function in anoxic brain: a comparison between turtle and the rat. In Surviving Hypoxia (ed. P. W. Hochachka, P. L. Lutz, T. J. Sick, M. Rosenthal and G. van den Thillart), pp.351 -364. Boca Raton, FL, USA: CRC Press.
Smatresk, N. J. and Smits, A. W. (1991). Effects of central and peripheral chemoreceptor stimulation on ventilation in the marine toad, Bufo marinus. Respir. Physiol. 83,223 -238.[CrossRef][Medline]
Solomon, I. C. (2000). Excitation of phrenic and sympathetic output during acute hypoxia: contribution of medullary oxygen detectors. Respir. Physiol. 121,101 -117.[CrossRef][Medline]
Solomon, I. C., Edelman, N. H. and Neubauer, J. A.
(2000). Pre-Bötzinger complex functions as a central hypoxia
chemosensor for respiration in vivo. J. Neurophysiol.
83,2854
-2868.
Stecyk, J. A. W. and Farrell, A. P. (2002).
Cardiorespiratory responses of the common carp (Cyprinus carpio) to
severe hypoxia at three acclimation temperatures. J. Exp.
Biol. 205,759
-768.
St John, W. M. and Knuth, K. V. (1981). A
characterization of the respiratory pattern of gasping. J. Appl.
Physiol. 50,984
-993.
Straus, C., Wilson, R. J. and Remmers, J. E. (2001). Oxygen sensitive chemoreceptors in the first gill arch of the tadpole, Rana catesbeiana. Can. J. Physiol. Pharmacol. 79,959 -962.[CrossRef][Medline]
Taylor, A. C. and Köllros, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anat. Rec. 94,7 -24.
Telgkamp, P. and Ramirez, J. M. (1999).
Differential responses of respiratory nuclei to anoxia in rhythmic brain stem
slices of mice. J. Neurophysiol.
82,2163
-2170.
Thompson, K. J. (1985). Organization of inputs to motoneurons during fictive respiration in the isolated lamprey brain. J. Comp. Physiol. 157A,291 -302.[CrossRef]
Torgerson, C. S., Gdovin, M. J. and Remmers, J. E.
(1998). Fictive gill and lung ventilation in the pre- and
postmetamorphic tadpole brain stem. J. Neurophysiol.
80,2015
-2022.
Torgerson, C. S., Gdovin, M. J. and Remmers, J. E. (2001). Sites of respiratory rhythmogenesis during development in the tadpole. Am. J. Physiol. 280,R913 -R920.
Wegner, G. and Krause, U. (1993). Environmental and exercise anaerobiosis in frogs. In Surviving Hypoxia (ed. P. W. Hochachka, P. L. Lutz, T. J. Sick, M. Rosenthal and G. van den Thillart), pp. 217-236. Boca Raton, FL: CRC Press.
West, N. H. and Burggren, W. W. (1982). Gill and lung ventilation responses to steady-state aquatic hypoxia and hyperoxia in the bullfrog tadpole. Respir. Physiol. 47,165 -176.[CrossRef][Medline]
West, N. H., Topor, Z. L. and Van Vliet, B. N. (1987). Hypoxemic threshold for lung ventilation in the toad. Respir. Physiol. 70,377 -390.[CrossRef][Medline]
Winmill, R. E. and Hedrick, M. S. (2003a). Gap junction blockade with carbenoxolone differentially affects fictive breathing in larval and adult bullfrogs. Respir. Physiol. Neurobiol. 138,239 -251.[CrossRef]
Winmill, R. E. and Hedrick, M. S. (2003b). Developmental changes in the modulation of respiratory rhythm generation by extracellular K+ in the isolated bullfrog brainstem. J. Neurobiol. 55,278 -287.[CrossRef][Medline]
Xia, Y., Jiang, C. and Haddad, G. G. (1992). Oxidative and glycolytic pathways in rat (newborn and adult) and turtle brain: role during anoxia. Am. J. Physiol. 262,R595 -R603.[Medline]
Zar, J. H. (1974). Biostatistical Analysis. Englewood Cliffs, New Jersey: Prentice Hall.