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First published online June 11, 2007
Journal of Experimental Biology 210, 2046-2056 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.003202
Effects of medullary Raphé stimulation on fictive lung ventilation during development in Rana catesbeiana
1 Department of Cell Biology, University of Texas Southwestern Medical
Center, Dallas, TX 75390-9039, USA
2 Department of Pediatrics, Laval University, Centre de Recherche
Hôpital St-François d'Assise, Québec City, Québec,
G1L 3L5, Canada
* Author for correspondence (e-mail: richard.kinkead{at}crsfa.ulaval.ca)
Accepted 13 March 2007
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Key words: brainstem, bullfrog, control of breathing, development
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). A
similar developmental pattern has been observed during the transition from
aquatic to aerial breathing in bullfrog tadpoles (Rana catesbeiana).
In this species, lung ventilation occurs infrequently during pre-metamorphic
stages as their contribution towards gas exchange is negligible
(Burggren and West, 1982). Lung
ventilation frequency increases significantly during metamorphosis, and
breathing episodes of more than one breath are observed more regularly
(Burggren and Infantino, 1994).
In adult bullfrogs, breathing is episodic also, but a continuous,
high-frequency, breathing pattern can be observed when respiratory drive is
elevated (Kinkead and Milsom,
1996). Although these data suggest that the ontogeny of the
rhythmic motor activity generating lung ventilation is well preserved amongst
vertebrates, the neural mechanism(s) at the basis of respiratory control
maturation are not well understood.
Results of investigations on the ontogeny of other rhythmic motor
behaviours such as locomotion have shown that, in developing tadpoles, the
onset of serotonergic innervation of spinal motoneurons increases the
intensity and duration of motor bursts necessary to the production of a more
mature swimming pattern (for a review, see
McLean et al., 2000). These
results, combined with the fact that the number of brainstem (Raphé)
serotonergic neurons increases steadily during tadpole development
(van Mier et al., 1986),
brought us to propose that ontogenic changes in serotonergic modulation
facilitates the production of the motor output associated with lung
ventilation during tadpole development.
This hypothesis was first addressed using bath application of
physiologically relevant concentrations of 5-HT onto isolated brainstem
preparations from bullfrog tadpoles of various developmental stages
(Belzile et al., 2002;
Kinkead et al., 2002). Results
from these experiments showed that application of a low 5-HT concentration
(0.5 µmol l–1) to brainstem preparations from
pre-metamorphic tadpoles has little effect on fictive lung ventilation.
Application of the same 5-HT concentration to brainstems from metamorphic
tadpoles increases fictive lung ventilation
(Kinkead et al., 2002). These
results are consistent with the increase in the number 5-HT immunoreactive
neurons in the hypothalamus and brainstem reported in Xenopus laevis
(van Mier et al., 1986) and
suggest that, as for locomotion, the progressive increase in the capacity for
5-HT release near respiratory neurons facilitates the emergence of lung
ventilation over the course of development. However, the fact that bath
application of 5-HT active agents can activate 5-HT receptors anywhere in the
preparation (including pre-synaptic autoreceptors altering endogenous 5-HT
release) combined with potential species-specific differences in the
developmental pattern of the 5-HT system made it difficult to come to a clear
conclusion.
To better understand serotonergic modulation of respiratory motor output
associated with lung ventilation during development in Rana
catesbieana, the present study used a more precise approach (electrical
and chemical stimulation) and tested the hypothesis that rostral medullary
Raphé stimulation increases lung burst frequency in a stage-dependent
fashion. This brainstem area was chosen because it is easily accessible and
corresponds to the Raphé magnus
(Adli et al., 1999), a group of
5-HT neurons that, in mammals, send direct projections onto respiratory
neurons (Manaker and Tischler,
1993; McCrimmon et al.,
1995).
The 5-HT receptor system is highly complex with several receptor types
(5-HT1 up to 5-HT7) and subtypes, many of which can
modulate respiratory activity. Based on previous results suggesting that
receptor subtypes other than 5-HT1A and 5-HT2A/C are
involved in 5-HT modulation of fictive lung ventilation
(Belzile et al., 2002;
Kinkead et al., 2002), and the
fact that 5-HT3 receptors are ligand-gated ion channels which, in
turtles, exert important modulatory effects of ficitive breathing in
vitro (Johnson et al.,
2001), we combined medullary Raphé stimulation with bath
application of a selective 5-HT3 receptor antagonist to determine
whether this receptor subtype contributes to the increase in fictive lung
ventilation. For each stage group, the brainstem region stimulated was
analysed by immunohistochemistry to, (1) demonstrate that 5-HT neurons are
present within the region stimulated, and (2) determine whether the number of
Raphé neurons observed within this brainstem region changes over the
course of development in this species. Parts of this work have been reported
in abstract form (Belzile et al.,
2003).
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In vitro brainstem preparations
Tadpoles were anesthetized by immersion in a solution of tricaine methane
sulfonate (1:10 000) buffered to pH 7.8 with NaHCO3. Once
unresponsive to tail pinch, tadpoles were decerebrated by a transection just
rostral to the eyes. Animals 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), and assigned to one of two groups: pre-metamorphic (stages
VI to XIV; N=27) or metamorphic tadpoles (stages XVI to XXV;
N=14). Adult bullfrogs were anesthetized by immersion in a solution
of tricaine methane sulfonate (1:1 000) for 15 min then they were placed in a
mixture of ice and oxygenated water for 30 min to slow metabolism and maintain
anesthesia for subsequent dissection
(Winmill and Hedrick, 2003). A
hole was drilled in the cranium to allow decerebration. In all animals, the
cranium was opened to expose the brainstem and rostral spinal cord and to
allow dissection of the cranial nerves. To reduce axonal conductance
throughout the dissection procedure, the brain was irrigated with ice-cold
(0–5°C) artificial cerebrospinal fluid (aCSF). The composition of
the aCSF was identical the one used for tadpoles and adult frogs in our
previous studies (Belzile et al.,
2002; Fournier and Kinkead,
2006; Kinkead et al.,
2002) and consisted of (in mmol l–1): 104 NaCl; 4
KCl; 1.4 MgCl2; 10 D-glucose; 25 NaHCO3; 2.4
CaCl2 (tadpoles) or 75 NaCl; 4.5 KCl; 2.5 CaCl2; 1.0
MgCl2; 1.0 NaH2PO4; 7.5 D-glucose;
40 NaHCO3 (adult frogs). The superfusate was equilibrated with a
98% O2/2% CO2 gas mixture to ensure adequate
oxygenation of the preparation and to maintain the pH at physiological levels
(7.8 or 7.9, for tadpoles and adults, respectively)
(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 Sylgard (Dow Corning, Midland, MI, USA) where it was immobilized
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 dorsal side up.
Electrophysiological recordings
Bursts of respiratory-related motor activity were recorded simultaneously
from the rootlets of cranial nerves V (trigeminal) and X (vagus) using suction
electrodes (model 573000; A-M Systems, Everett, WA, USA). 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 bevelled 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 2.5 kHz.
Experimental protocols
Once the recording electrodes were in place, the brainstem preparation was
superfused with control (drug free) aCSF at room temperature
(20–22°C) equilibrated to pH 7.8 or 7.9 for tadpoles or adults,
respectively, delivered at a rate ranging between 4–6 ml
min–1. The preparation was allowed to return to ambient
temperature and stabilize for 45–60 min, until stable rhythmic neural
activity was recorded from both nerves. After the stabilizing period, the
experiments began with a recording of baseline activity for 10 min.
Series I: electrical stimulation
In a first series of experiments, a tungsten electrode (electrode diameter:
125 µmol l–1, tapered tip size: 8°, AC resistance: 5
M
, model 575300; A-M Systems, Everett, WA, USA) was placed in the
rostral portion of the medullary Raphé, according to the description of
Tan and Miletic (Tan and Miletic,
1990) for adult Rana pipiens. These neurons are located
on the floor of the 4th ventricle in the midline at the level of rootlets of
the cranial nerves VII (facial). The presence of 5-HT immunoreactive neurons
in this brain region of Rana catesbeiana tadpoles was confirmed by
immunohistochemistry (Fig. 6).
The tip of the electrode was positioned roughly 25 µm below the surface of
the floor of the ventricle. The tungsten electrode was connected to an
isolated pulse stimulator (model 2100, A-M Systems, Everett, WA, USA).
Stimulation parameters were similar to those used in other studies
(Fuller et al., 2002;
Hilaire et al., 1989;
Ling et al., 2001), and
preliminary experiments conducted on tadpole brainstems confirmed their
effectiveness in evoking a lung ventilation response (pulse duration, 0.2 ms;
burst width, 1 s; frequency, 20 Hz). These parameters were then used for each
preparation (pre-metamorphic, N=8; metamorphic, N=6; adults,
N=11) to determine the minimum current necessary to evoke a response
(typically between 1–2 µA). Once the current threshold was
determined, the stimulator was set to deliver a current 10% greater than
threshold, and the preparation was left undisturbed for another 10 min before
the experiment started. Electrical stimulation (1 s) was then performed every
5 min, and the current frequency was increased progressively (3, 5, 10, 20 and
33 Hz). In preliminary experiments (N=1), this procedure was
performed before and during bath application of a broad spectrum 5-HT receptor
antagonist (methiothepin or methysergide) to confirm that the increase in lung
burst frequency increase observed during electrical stimulation of the
Raphé requires 5-HT receptor activation
(Fig. 1).
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Series II: chemical stimulation
Electrical stimulation of Raphé neurons can elicit non-specific
effects since it is difficult to control current spread and avoid stimulating
axons of passage (Lipski et al.,
1988). Consequently, the results obtained with the electrical
stimulation protocol were validated by performing another series of
experiments in which Raphé neurons were activated using glutamate
microinjections. These experiments were performed on the same three
developmental groups (pre-metamorphic, N=9; metamorphic,
N=8; adults, N=6). In this series, a glass pipette filled
with a glutamate solution (0.5 mol l–1 dissolved in aCSF) was
placed in the medullary Raphé according to the coordinates described
previously. The glutamate concentration and volume were based on those used by
Lipski et al. (Lipski et al.,
1988). The pipettes were constructed from borosilicate glass (0.58
mm i.d.) pulled to the finest possible tip (average tip diameter of
10–15 µm) with a vertical microelectrode puller (Stoelting
Instrument, Wood Dale, IL, USA). This pipette was connected to a pneumatic
picopump (model PV800, WPI Inc., Sarasota, FL, USA) set at roughly 137 kPa.
Before each experiment, the pipette was placed under a microscope to determine
the injection time required to inject specific volumes (0.3 to 10 nl).
Although there was some variation between the experiments, the air pressure
was kept constant for all the injections performed on the same preparation.
The ejected volume was estimated by measuring the diameter of the drop.
Preliminary experiments comparing the volume estimated with the drop diameter
with the volume injected in the tissue calculated from the measured radius of
the pipette and the change in meniscus height, confirmed that this method was
adequate for the volumes used.
The protocol consisted of two glutamate microinjections of increasing volumes (see below), separated by a 5-min recovery period. Following these injections, the 5-HT3 antagonist tropisetron was added to the superfusate and the series of microinjections was repeated. Preliminary experiments showed that, although injections were performed in the same brainstem region, preparations from younger animals responded more vigorously than preparations from older animals. To avoid over-stimulation and a ceiling effect in the response, the glutamate volumes used varied according to the developmental stage of the preparation (pre-metamorphic, 0.3 and 1.0 nl; metamorphic, 1.0 and 2.5 nl; and adults, 5.0 and 10.0 nl). Control injections of glutamate 350 µm lateral to the regular injection site were performed. Control injections of different volumes of drug-free aCSF within the medullary Raphé were performed also. As for electrical stimulation, experiments were performed on the same three developmental groups (pre-metamorphic, N=9; metamorphic, N=8; adults, N=6).
Series III: quantification of 5-HT immunoreactive neurons in the rostral medullary Raphé
Immunohistochemistry was used to quantify the number of serotonergic
neurons present in the brainstem region stimulated in the previous two series
of experiments. Tadpoles and bullfrogs were deeply anesthetised according to
the protocols described in the previous series, and brains were harvested from
animals representing all three developmental groups (pre-metamorphic,
N=7; metamorphic, N=3; adults, N=7). The tissue was
fixed in 4% paraformaldehyde (PFA) in 0.1 mol l–1 phosphate
buffer solution (PB, pH 7.4 at 4°C) overnight. The next day, brains were
placed in 30% sucrose–4% PFA/PB solution until they sank (48 h at
4°C). Brains were then imbedded in Histoprep (Fisher Scientific, Ottawa,
ON, Canada) and frozen on dry ice. The tissue block was mounted in a cryostat
(Leica CM 1900), and cross sections (20 µm) of the medullary region of the
Raphé at the level of nerve VII were cut in series. On average, five to
six sections were obtained from each brain. Sections were first rinsed in 0.1
mol l–1 PB (pH 7.4) and then incubated in 0.3%
H2O2/PB for 10 min. Sections were rinsed in PB and
permeabilized in a solution of 0.2% Triton X-100/0.1 mol l–1
PB for 10 min at room temperature. This was followed by incubation in a
solution of 0.1% bovine serum albumin (BSA fraction V; Sigma, St Louis, MO,
USA) to reduce non-specific activity before being incubated with the primary
antibody, rabbit anti-5-HT (Sigma) at a dilution of 1:4 000 in a solution of
0.1% BSA/PB at 4°C overnight. Tissue sections were washed in 0.1 mol
l–1 PB for 10 min and then incubated in biotinylated goat
anti-rabbit secondary antiserum (Vector laboratories, Burlington, ON, Canada),
diluted 1:200 for 1 h at room temperature. Sections were rinsed and biotin
amplification was performed with the avidin–biotin peroxydase reaction
(Vectastain Elite ABC kit, Vector Laboratories, Burlington, ON, Canada) for 1
h at room temperature. Sections were rinsed in PB and the reaction complex was
stained with the 3-amino-9-ethylcarbazole reaction (AEC substrate kit for
peroxydase; Vector Laboratories, Burlington, ON, Canada) for 10 min at room
temperature. The reaction was stopped by rinsing the sections in PB. Sections
were then mounted onto slides before being dried and sealed under a coverslip
with mounting medium. Control experiments were performed by omitting the
primary antibody, resulting in negative staining.
Data analyses
Frequency values for lung-related activity (large amplitude bursts) were
obtained by analyzing the neurogram for 30 s following electrical or chemical
stimulation of medullary Raphé neurons. Cranial nerve burst amplitude
from a single electroneurogram is not always sufficient to adequately identify
fictive gill and lung bursts (Sanders and
Milsom, 2001). Based upon the criterion proposed by Torgerson and
colleagues (Torgerson et al.,
1998), both nerve signals were analyzed simultaneously, and vagal
nerve activity was used as a sensitive marker of fictive lung activity to
distinguish between gill- and lung-related signals
(Kogo et al., 1994;
Kogo and Remmers, 1994).
Occasionally, Raphé stimulation altered the burst pattern. Under those
circumstances, only bursts that were similar (in terms of amplitude and
duration) to those recorded prior to Raphé stimulation were counted as
respiratory related. Fig. 4
shows neurograms in which glutamate microinjection elicited burst activity
that differs from respiratory-related activity (e.g. prolonged burst
duration). Such bursts were not included in the analysis. Lung burst
frequencies were obtained by counting the number of lung-related burst events
for the 30 sec period analyzed, and calculated for a 1 min period. To
facilitate comparisons between stage groups, frequency data were also
normalised in two ways: (1) as a percentage change from baseline, and (2) as a
percentage change from baseline expressed as the percentage of the maximum
lung burst frequency produced by the preparation. The latter form of
normalisation obviates concerns about expressing date in terms of the
percentage increase above a low baseline value (e.g. pre-metamorphic).
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All measurements are reported as the mean ± 1 s.e.m. The results were analyzed statistically using a two-way analysis of variance (ANOVA) for repeated measures when appropriate. Expressing data as a percentage change can bias data distribution away from normality, which violates a key assumption underlying ANOVA. This issue was addressed by performing the ANOVAs both on normalised data and normalised data following arcsine transformation. The conclusions emerging from both analyses were identical. Results from the immunohistochemistry experiments were analysed using a one-way ANOVA (Statview version 5.01; SAS Institute, Cary, NC, USA). These analyses were followed by Fisher's protected least significant difference (PLSD) test when appropriate (P<0.05).
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Electrical stimulation of the medullary Raphé performed on preparations from adult bullfrogs elicited mixed responses: fictive lung ventilation increased in 45% (5/11) of the preparations, whereas a decrease was observed in 55% (6/11) of the preparations. The increase seen in five adult preparations was smaller than the increases seen in the two other groups. Consequently, the overall (mean) data show no significant effect of electrical stimulation on fictive lung ventilation in this group (Fig. 3C; P=0.69).
Fig. 3D compares the lung burst frequency responses to electrical stimulation (expressed as a percentage change from baseline) recorded at 20 Hz, which elicited the maximum response in all three developmental stages. Expressed this way, the data show that the response to medullary Raphé stimulation was stage dependent (stage effect: P=0.016) and that the increase, when present, tended to be attenuated by tropisetron (drug effect: P=0.07). Expressing these data as a percentage change from maximum response revealed similar effects (Fig. 3E).
II: chemical stimulation
Results obtained in this series of experiments were specific to
Raphé neuron activation because control glutamate injection 350 µm
lateral to the injection site did not affect lung-related neural activity
(Fig. 4C). Whenever observed,
trigeminal bursts were longer than those recorded under any other experimental
condition, and vagal activity was often seizure-like. Thus the activity
recorded could not be interpreted as respiratory related. Furthermore, control
injections of different volumes of drug-free aCSF in the medullary
Raphé had no effect (Fig.
4D).
Overall, data obtained in this series of experiments mainly concur with those reported for electrical stimulation. Only preparations from pre-metamorphic tadpoles showed a lung burst frequency increase following the glutamate microinjections (Fig. 5A; P=0.01). Tropisetron bath application attenuated this response (glutamate effect, P=0.15; drug effect, P=0.02). In metamorphic tadpoles and adult frogs, glutamate microinjections had no significant effect (P>0.05), and in adults an inhibition of fictive lung ventilation was observed in four out of six preparations at 5 and 10 nl (Fig. 5C).
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Expressing these results as a percentage change from baseline indicate that the response to medullary Raphé stimulation was stage dependent (stage effect, P<0.0001) and that the increase, when present, was attenuated by tropisetron (drug effect, P<0.0001). Expressing these data as percent change from maximum response revealed similar effects (Fig. 5E).
Stage-dependent changes in 5-HT immunoreactive neurons in the dorsal medullary Raphé
5-HT immunoreactive neurons are present in the rostral portion of the
medullary Raphé at the level of the VIIth cranial nerve where
electrical and chemical stimulation was performed in all stage groups
(Fig. 6). Micrographs and mean
data show that the number of 5-HT immunoreactive neurons present in this
region increased during development with a peak during metamorphic stages
(stage effect, P=0.0006; Fig.
6B).
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Critique of methods
Various approaches can be used to stimulate a group of neurons located
within a specific CNS area; however, each technique has inherent limitations
that must be considered in our interpretation of the results. For instance,
electrical stimulation can elicit non-specific effects since it is difficult
to control current spread and avoid stimulating axons of passage
(Lipski et al., 1988). In the
case of chemical stimulation, the same glutamate concentration may have
excitotoxic effects that are not constant throughout development.
Another important limitation relates to the difficulty in determining
precisely the area influenced by the stimulation (whether electric or
chemical). For instance, developmental change in the number of glial cell
could affect the spread of the electric stimulus and injection of excitatory
amino acids with volume ranges similar to those we used can elicit effects in
regions quite distant from the injection site, depending on the concentration
used (Lipski et al., 1988;
Nicholson, 1985). Based on the
equations proposed by Nicholson
(Nicholson, 1985) and assuming
that the frog brain tissue has a volume fraction (
) and tortuosity
factor (
) similar to that of mammals (0.21 and 1.6, respectively) the
glutamate volumes used could affect neurons as far as 300 µm away from the
injection site.
To address these issues two different experimental approaches were used to
stimulate medullary Raphé neurons: electrical and chemical stimulation,
and in addition immunohistochemistry was used to evaluate the number of
5-HT-positive neurons located in the immediate vicinity of the stimulation
site (50 µm radius) for each stage group. Results obtained were
essentially similar in both series of experiments as we showed that (1)
brainstems from pre-metamorphic tadpoles were the most responsive to medullary
Raphé neuron stimulation, regardless of the technique used, and (2) for
all developmental stages, the effects of Raphé neuron stimulation (when
they occurred) could be blocked by a 5-HT antagonist. These results differ
from those obtained using bath application of low 5-HT concentrations (0.5
µmol l–1), which enhanced fictive lung ventilation in
preparations from metamorphic but not pre-metamorphic tadpoles
(Kinkead et al., 2002). An
important distinction between the two techniques is that during bath
application, 5-HT activates both post- and pre-synaptic (auto) receptors
located on Raphé neurons to reduce endogenous 5-HT release. In mammals,
5-HT1A receptors located on cell bodies regulate 5-HT release. In
pre-metamorphic tadpoles, 5-HT bath application in the presence of the
selective 5-HT1A antagonist NAN-190 increased fictive lung
ventilation frequency (Kinkead et al.,
2002). These data are consistent with those obtained with focal
Raphé stimulation and suggest that the result obtained with 5-HT bath
application reflects a compromise between two opposing effects.
Although 5-HT immunoreactive neurons are predominant in the medullary Raphé nucleus, this structure is relatively heterogeneous in comparison with other nuclei. Thus, focal stimulation within this region may elicit release of neurotransmitters other than 5-HT. However, the fact that the response observed could be blocked by a low concentration of a selective 5-HT antagonist indicates that 5-HT release was the predominant effect elicited in our experiments.
The role of the 5-HT3 receptor in modulation of lung burst frequency during development
Since application of pharmacological agents inactivating 5-HT1A
or 5-HT2A/C receptor subtypes had little effect on the fictive lung
ventilation response to 5-HT application
(Belzile et al., 2002;
Kinkead et al., 2002), we
proposed that 5-HT3 receptors are involved in serotonergic
modulation of lung burst frequency. Our results showing that, in
pre-metamorphic tadpoles, these receptors participate in the strong excitation
of fictive lung activity that follows Raphé neuron stimulation support
this interpretation. These results concur with those of Johnson et al.
(Johnson et al., 2001) who
reported a 5-HT3-mediated enhancement of fictive lung ventilation
for in vitro preparations from adult turtles, but do not exclude the
possibility that other receptor types are involved also. In that regard,
5-HT4A receptors may contribute to these effects since their
activation stimulates respiratory activity both in vitro and in
vivo in rats (Manzke et al.,
2003).
Results from metamorphic animals show that the decline in the excitatory
influence of medullary Raphé neurons on fictive lung ventilation
becomes apparent with the onset of metamorphosis, thereby suggesting that the
endocrine changes that take place over that period influence modulation of
respiratory activity by 5-HT. It is important to note that, unlike adult
frogs, the modest effect of Raphé neuron stimulation observed in the
metamorphic group was consistent amongst preparations. Experiments on
preparations from adult frogs yielded mixed results even though the animals
used for this study were purchased together and were used in a short period of
time to exclude seasonal changes as a potential confounding factor; however,
frogs' gender was not determined prior to experiments. Knowing that
serotonergic modulation of respiratory activity shows substantial sexual
dimorphism in rats (Behan et al.,
2003), the different responses may reflect the influence of sexual
steroids that becomes apparent only in fully mature adults.
The sum of these data leads us to conclude that the developmental period
during which serotonergic modulation facilitates fictive lung ventilation is
restricted to the early developmental stages. These results are similar to
those observed with noradrenaline bath application
(Fournier and Kinkead, 2006)
but are difficult to reconcile with the overall increase in 5-HT
immunoreactive neurons reported over the course of development in the
brainstem of anurans, Rana (present study) and Xenopus
(van Mier et al., 1986). Thus,
unless the number of serotonergic boutons near respiratory neurons decreases
substantially, the capacity for 5-HT synthesis (and release) by medullary
Raphé neurons likely increases during bullfrog development. Results
from the present experiments do not allow us to provide a clear description of
the mechanisms responsible for this sudden change in the effect of 5-HT
modulation of fictive lung ventilation; however, our data showing the
important role that 5-HT3 receptors play in this function provides
valuable clues in that regard.
The 5-HT3 receptor is a ligand-gated ion channel which, in
mammals, modulates fast synaptic neurotransmission and is located mainly on
GABAergic interneurons (van Hooft and
Vijverberg, 2000). GABA is a neurotransmitter that is excitatory
during early development but inhibitory in the mature nervous system
(Ben-Ari, 2002). This
stage-dependent response to GABA reflects the low expression of chloride
transporters and the inefficiency of neurons in maintaining chloride gradients
across the membrane. Consequently, GABAA receptor activation
results in neuron depolarisation during early life but causes a
hyperpolarization in more mature stages
(Ben-Ari, 2002). In Rana
catesbeiana, in vitro bath application of GABA supports this
interpretation because it enhances fictive lung ventilation in preparations
from tadpoles but inhibits this activity in preparations from adult frogs
(Broch et al., 2002). We
therefore propose that modulation of fictive lung ventilation by medullary
Raphé neurons acts mainly via 5-HT3 receptors
located on GABA interneurons. According to this hypothesis, the developmental
change could reflect the appearance of ion transporters necessary to establish
and maintain chloride gradients in these GABAergic neurons.
Perspectives
In adult mammals, stimulation of the Raphé magnus inhibits, whereas
stimulation of the Raphé pallidus facilitates respiratory activity
(Cao et al., 2006;
Lalley, 1986). Conversely,
electrical stimulation of the Raphé obscurus induces both types of
respiratory responses, depending on the stimulation sites
(Cao et al., 2006). However,
these effects seem more consistent in vitro where Raphé
obscurus stimulation in brainstems preparations from newborn rat augments
fictive breathing frequency (Al-Zubaidy et
al., 1996; Peever et al.,
2001). While comparing our results with those obtained in mammals
must be done cautiously, our data (especially those obtained from more mature
preparations) is nonetheless consistent with our initial assumption that the
medullary Raphé region stimulated corresponds to the Raphé
magnus.
Bath application of selective 5-HT antagonists such as tropisetron
(5-HT3; present study) or NAN-190 (5HT1A)
(Kinkead et al., 2002) have
little effect on `resting' lung burst frequency, indicating that tonic 5-HT
modulation of fictive lung ventilation is minimal in this preparation.
Specific stimuli such as hypoxia may therefore be necessary to trigger 5-HT
release from Raphé neurons. Oxygen-sensitive peripheral chemoreceptors
like those located in the first gill arch of tadpoles
(Straus et al., 2001) relay
their sensory signal to the CNS to increase both gill and lung ventilation
during hypoxia. Since peripheral chemoreceptor stimulation activates
Raphé neurons in mammals (Erickson
and Millhorn, 1994), it is likely that hypoxia elicits such
response in amphibians also. This, in turn, would facilitate air breathing
owing to (at least in part) 5-HT3 receptor activation. Since lungs
of pre-metamorphic tadpoles contribute little to gas exchange, an increase in
air breathing could promote lung development.
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Acknowledgments |
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indicates a value
statistically different from the metamorphic data at P<0.05.
