Download Are pacemaker properties required for respiratory rhythm generation

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1 of 39 in PresS. Am J Physiol Regul Integr Comp Physiol (May 23, 2007). doi:10.1152/ajpregu.00912.2006
Are pacemaker properties required for respiratory rhythm
generation in adult turtle brainstems in vitro?
Stephen M. Johnson, Liana M. Wiegel, David J. Majewski
Department of Comparative Biosciences
School of Veterinary Medicine
University of Wisconsin
Madison, Wisconsin 53706
Abbreviated title: Pacemaker properties and turtle breathing in vitro
Corresponding Author:
Stephen M. Johnson, M.D., Ph.D.
Assistant Professor
Department of Comparative Biosciences
School of Veterinary Medicine
University of Wisconsin
2015 Linden Drive
Madison, Wisconsin 53706
Phone: (608) 261-1104
Fax: (608) 263-3926
e-mail: [email protected]
Copyright © 2007 by the American Physiological Society.
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ABSTRACT
The role of pacemaker properties in vertebrate respiratory rhythm generation is not well
understood. To address this question from a comparative perspective, brainstems from adult
turtles were isolated in vitro and respiratory motor bursts were recorded on hypoglossal (XII)
nerve rootlets. The goal was to test whether burst frequency could be altered by conditions
known to alter respiratory pacemaker neuron activity in mammals (e.g., increased bath KCl or
blockade of specific inward currents). While bathed in artificial cerebrospinal fluid (aCSF),
respiratory burst frequency was not correlated with changes in bath KCl (0.5-10.0 mM). Riluzole
(50 µM; persistent Na+-channel blocker) increased burst frequency by 31 ± 5% (P<0.05) and
decreased burst amplitude by 42 ± 4% (P<0.05). In contrast, flufenamic acid (FFA, 20-500 µM;
Ca2+-activated cation channel blocker) reduced and abolished burst frequency in a dose- and
time-dependent manner (P<0.05). During synaptic inhibition blockade with bicuculline (50 µM;
GABAA channel blocker) and strychnine (50 µM; glycine receptor blocker), rhythmic motor
activity persisted and burst frequency was directly correlated with extracellular KCl (0.5-10.0
mM; P=0.005). During synaptic inhibition blockade, riluzole (50 µM) did not alter burst
frequency while FFA (100 µM) abolished burst frequency (P<0.05). These data are most
consistent with the hypothesis that turtle respiratory rhythm generation requires Ca2+-activated
cation channels but not pacemaker neurons, which thereby favors the group-pacemaker model.
During synaptic inhibition blockade, however, the rhythm generator appears to be transformed
into a pacemaker-driven network that requires Ca2+-activated cation channels.
Key words: control of breathing, respiratory control, reptile, pacemaker, chelonian
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INTRODUCTION
The mechanisms underlying vertebrate respiratory rhythm generation are controversial and
not well understood. Network models propose that respiratory neurons are reciprocally coupled
via inhibitory synaptic connections that work to establish exclusive bursts of activity associated
with inspiration, postinspiration, and expiration (35, 41, 42, 46). The hybrid pacemaker-network
model proposes that pacemaker neurons embedded within a neural network play a necessary role
in rhythm generation (5, 6, 13, 16, 24, 36, 38, 52). A third model is the group-pacemaker model,
which postulates that inspiratory bursts are an emergent property of nonpacemaker neurons that
are interconnected by chemical and electrotonic excitatory synaptic interactions (10, 11, 14, 15,
39). Thus, the role of pacemaker properties (whether in neurons or networks) and synaptic
inhibition are discriminating features of these models.
Most data supporting the hybrid pacemaker-network and group-pacemaker models are
derived from perinatal and juvenile mammals whereas network models are supported primarily
by data from adult mammals. The maturation network-burster hypothesis (43) attempts to
harmonize these findings by stating that the respiratory control system is pacemaker-driven in
young animals, but becomes network-driven in adults due to increasing synaptic inhibition and
activation of specific ionic conductances (17, 18, 31, 32, 33). However, in some nonmammalian
adult vertebrate preparations, there is evidence suggesting that pacemaker properties may be
required for respiratory rhythm generation. For example, in brainstems isolated from adult
lampreys (44) and adult turtles (23), rhythmic motor activity persists during synaptic inhibition
blockade. This persistent rhythmic activity in isolated adult turtle brainstem (and brainstemspinal cord) preparations is hypothesized to be respiratory-related because motor bursts are
produced on a spinal expiratory nerve (23), and abolished by characteristic respiratory
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depressants such as µ-opiate receptor activation (23) and high pH/low CO2 conditions
(unpublished observations). These data suggest that expiratory pacemaker neurons or clusters of
expiratory neurons with pacemaker properties drive the rhythm, rather than non-respiratory
neurons taking over control of the respiratory control system and producing a seizure-like pattern
(4, 43).
To test the hypothesis that pacemaker properties are involved in turtle respiratory rhythm
generation, brainstems from adult turtles were isolated and tested under in vitro conditions.
Specifically, we tested whether depolarization via increased bath KCl increases the frequency of
spontaneous respiratory bursts of motor activity, since the frequency of endogenous bursting in
respiratory pacemaker neurons is voltage-dependent (5, 53). We also tested whether riluzole or
flufenamic acid (FFA) altered respiratory burst frequency produced by turtle brainstems.
Riluzole blocks persistent Na+ currents and FFA blocks Ca2+-activated cation currents in
respiratory neurons in the mammalian medulla (11, 33, 59). Both currents are hypothesized to be
required for pacemaker activity in mammalian inspiratory neurons (33, 59), although their
obligatory role for respiratory rhythm generation has been challenged (11). In separate
experiments under conditions of synaptic inhibition blockade, the effects of altered bath KCl,
riluzole, and FFA were examined to test whether synaptic inhibition blockade transforms the
respiratory rhythm generator into a pacemaker-driven network (45, 47, 48, 49, 50). A
preliminary report of this work was published in abstract form (20).
MATERIALS AND METHODS
All procedures were approved by the Animal Care and Use Committee at the University of
Wisconsin-Madison School of Veterinary Medicine. Adult red-eared slider turtles (Trachemys
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scripta, n = 161, weight = 696 ± 10 g) were obtained from commercial suppliers and kept in a
large open tank where they had access to water for swimming, and heat lamps and dry areas for
basking. Room temperature was set to 27-28°C with light 14 hr/day. Turtles were fed ReptoMin®
floating food sticks (Tetra, Blacksburg, VA) 3-4 times per week.
Turtle brainstem preparations. Turtles were intubated and anesthetized with 5% isoflurane
(balance O2) until limb withdrawal to noxious foot pinch was eliminated. Turtles were rapidly
decapitated and decerebrated. Brainstems were removed and pinned down in a recording
chamber (13 ml volume) with the ventral surface facing upwards (Fig. 1A). Brainstems were
superfused (4-6 ml/min) with artificial cerebrospinal fluid (aCSF) at 23ºC containing HEPES (N[2-hydroxyethyl]piperazine-N’-[2-ethane-sulfonic acid]) buffer as follows (in mM): 100 NaCl,
23 NaHCO3, 10 Glucose, 5 HEPES (sodium salt), 5 HEPES (free acid), 2.5 CaCl2, 2.5 MgCl2,
1.0 K2PO4, and 1.0 KCl (bubbled with 5% CO2-95% O2). The pH of solution in the reservoir was
measured periodically with a calomel glass pH electrode (Digi-Sense , Cole-Parmer Inst. Co.,
Vernon Hills, IL) and averaged 7.30 ± 0.01 during data collection. All brainstems were allowed
to equilibrate for 2-5 hr prior to initiating an experimental protocol. To record respiratory motor
bursts, glass suction electrodes were attached to hypoglossal (XII) nerve rootlets (Fig. 1A).
Signals were amplified (10,000x) and band-pass filtered (1-500 Hz) using a differential AC
amplifier (model 1700, A-M Systems, Everett, WA) before being rectified and integrated (time
constant = 200 ms) using a moving averager (MA-821/RSP, CWE, Inc., Ardmore, PA). Analysis
was performed using Axoscope (Axon Instruments, Foster City, CA) and MiniAnalysis software
(Synaptosoft, Decatur, GA). All drugs used in this study were obtained from Sigma/RBI Aldrich
(St. Louis, MO) and include: (+)-bicuculline (GABAA receptor antagonist), flufenamic acid
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(FFA; blocks Ca2+-activated cation currents), riluzole (blocks persistent Na+ channels), and
strychnine (glycine receptor antagonist). Riluzole and FFA were dissolved in 100 µl of dimethyl
sulfoxide (DMSO) and then dissolved slowly in 0.5-1.0 l of HEPES-buffered solution before
being applied to turtle brainstems. Control experiments with equivalent amounts of DMSO in
HEPES-buffered solution showed that DMSO had no effect on respiratory burst frequency and
amplitude (data not shown).
Experimental protocols. To determine the effects of bath KCl on burst frequency, baseline
data were recorded for 20 min in aCSF (2 mM KCl) before switching to aCSF with altered KCl
for 60 min and then washing out with aCSF (2 mM KCl) for 60 min. To determine the effects of
riluzole and FFA, baseline data were recorded for 20 min before switching to aCSF with riluzole
(50 µM) or FFA (20-500 µM) for 2 hr before washing out with aCSF. To determine the effects
of KCl, riluzole, and FFA during synaptic inhibition blockade, bicuculline (50 µM) and
strychnine (50 µM) were added to the aCSF to block synaptic inhibition. After allowing for
complete transformation of the motor output within 100 min (22), bath KCl was altered for 1 hr,
or riluzole (50 µM) or FFA (100 µM) were added along with bicuculline and strychnine for 2 hr.
Data analysis
XII burst amplitude was measured at the highest point of integrated discharge trajectory in
arbitrary units and normalized to the average amplitude recorded during the baseline period.
Burst frequency was calculated as the number of bursts per min. All measurements were
averaged into 20-min bins and reported as mean ± S.E.M. For statistical inferences, linear
regression, one-way ANOVA, or two-way ANOVA with repeated measures design were used
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(Sigma Stat, Jandel Scientific Software, San Rafael, CA). Post-hoc comparisons were made
using Dunnett’s or Student-Newman-Keul’s test. P-values < 0.05 were considered significant.
RESULTS
Increased bath KCl did not alter burst frequency in aCSF
While bathed in aCSF, increasing bath KCl from 2.0 mM to 4.0-9.0 mM often transiently
increased burst frequency within the first 10-20 min, but burst frequency was highly variable
during the next 40 min (Figs. 2A, 2B, 2D). There were no changes in frequency when bath KCl
was increased from 2.0 mM KCl to 5.0 mM (n=4) or 7.0 mM KCl (n=6; Fig. 2B). There was,
however, a sustained 80-110% frequency increase was observed when bath KCl was increased
from 2.0 mM to 9.0 mM KCl (n=3; P<0.05 for KCl- and time-dependent effects; Fig. 2B). At
other increased bath KCl levels, burst frequency decreased below baseline (Fig. 2D). Increasing
bath KCl to levels greater than 10.0 mM resulted in tonic activity and disruption of the
respiratory rhythm (n=3; data not shown). Decreasing bath KCl from 2.0 to 0.5 mM (n=3) did
not alter burst frequency. A graph of burst frequency after 20-40 min of increased KCl exposure
(i.e., data at the 60-min time point in Fig. 2B) versus bath KCl revealed no correlation (P=0.066;
r2=0.129; Fig. 2D). This time period was chosen for analysis because KCl-induced frequency
changes often reached steady-state without the rhythm being disrupted by prolonged KCl
exposures. Although there was an occasional initial burst frequency increase during the KCl
application, there was no correlation for burst frequency versus bath KCl during the 0-20 min
period (P=0.313; r2=0.041). Burst amplitude was highly variable with time-dependent decreases
(5.0 and 7.0 mM KCl; n=4, 6, respectively) and increases (9 mM KCl; n=3) after 60 min (Fig.
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2C). Overall, there was no correlation between bath KCl and burst amplitude after 60 min of
increased KCl exposure (P=0.296; r2=0.129; Fig. 2E).
Effects of riluzole and FFA in aCSF
Bath-applied riluzole (50 µM; n=18) produced a time-dependent increase in burst frequency
(Figs. 3, 4A, 4B) and decrease in burst amplitude (Figs. 3, 4D). In the presence of riluzole,
frequency increased from 0.71 ± 0.06 bursts/min (baseline) to a maximum of 0.92 ± 0.07
bursts/min (P<0.05) at 80 min before reaching a steady-state frequency of 0.83-0.87 bursts/min
for the next 40 min (P<0.05; Fig. 4A). In contrast, burst frequency in time control experiments
(n=12) did not change; burst frequency was 0.52 ± 0.07 bursts/min at baseline and 0.52 ± 0.07
bursts/min at the 140-min time point (P<0.05; Fig. 4A). When graphed as percent change in
frequency, riluzole steadily increased frequency by 31 ± 5 % within 80-min before reaching a
steady-state increase of 23-26% (P<0.05 for drug and time-dependent effects; Fig. 4B). With
respect to the number of bursts/episode, riluzole had no effect and was constant at 1.4 – 1.6
bursts/episode throughout the riluzole exposure (P>0.05; Figs. 3B; 4C). For burst amplitude,
riluzole produced an immediate, time-dependent decrease such that amplitude was 58 ± 4 % of
baseline after 120 min (P<0.05) compared to an 8 ± 3 % decline (P>0.05) in time control
experiments (Fig. 4D). To rule out the caveat that riluzole was not penetrating the brainstems and
altering rhythm-generating neurons deep within the tissue, riluzole (50 µM) was applied to turtle
hemi-brainstems (i.e., brainstems completely transected along the midline). In these reduced
preparations (n=2), riluzole produced a similar time-dependent frequency increase and amplitude
decrease (data not shown).
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In contrast to riluzole, FFA produced a time- and dose-dependent decrease in burst
frequency with minor decreases in amplitude (Fig. 5A). Although bath-application of 20 µM
FFA (n=6) had little effect on amplitude, FFA at higher concentrations (50, 100, 500 µM; n=5, 7,
8, respectively) decreased burst frequency to new steady-state levels within 40-100 min (P<0.05;
Fig. 5B). The percent decrease in frequency at 140 min post-drug application was significantly
decreased compared to time controls at FFA concentrations between 50-500 µM (Fig. 5C).
Similar to riluzole, FFA had no effect on the number of bursts/episode compared to time controls
(Fig. 5D). With respect to amplitude, biphasic, dose-dependent effects were observed. There was
no change when brainstems were exposed to 20 µM FFA (Fig. 5E). During the 50 µM exposure,
however, there was a transient increase of 25 ± 30% (P<0.05) after 40 min, but amplitude was
decreased by 38-47% during the next 40 min (P<0.05). For the 100 µM exposure, amplitude was
decreased by 17-33% (P<0.05) during 40-80 min of the FFA application before returning to
baseline. The amplitude data after 100-120 min of 50-100 µM FFA application are highly
variable because only 1-3 brainstems produced respiratory bursts at a low frequency.
Increased extracellular KCl increased burst frequency during synaptic inhibition blockade
As demonstrated previously (23), the motor output of the turtle respiratory neural control
system was transformed by blocking fast inhibitory synaptic transmission via bath application of
bicuculline (50 µM) and strychnine (50 µM), resulting in rhythmic bursts that had an increased
frequency and amplitude, and a rapid onset/slow decrementing shape (Fig. 6A). Time control
experiments (n=14) showed that after 100 min of exposure to bicuculline and strychnine,
rhythmic bursts reached a steady-state frequency of 0.86 ± 0.10 bursts/min. After another 80 min
of bicuculline and strychnine, burst frequency was 0.85 ± 0.09 bursts/min and amplitude
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decreased by only 6 ± 4 % (P>0.05; data not shown). Thus, increased bath KCl was applied after
establishing a 20-min baseline the 100-120 min period of bicuculline and strychnine application
(Fig. 6A).
During synaptic inhibition blockade, burst frequency was not altered when bath KCl was
increased from 2.0 mM to 5.0 mM KCl (n=8; Fig. 6B). However, within 60-min of increasing
bath KCl to 7 mM (n=7) or 8 mM KCl (n=7), burst frequency increased to 320 ± 70% and 380 ±
110% of baseline, respectively (P<0.05; Fig. 6B). Decreasing bath KCl to 0.5 mM (n=8)
produced a slight 12 ± 7% decrease in burst frequency. Burst frequency was correlated (P=0.005;
r2=0.167) with bath KCl after 60 min of KCl exposure (i.e., 80-min time point in Fig. 6B) (Fig.
6D). In contrast, burst amplitude generally decreased with time (Fig. 6A) in a dose-dependent
manner with amplitude falling by 18 ± 12% and 27 ± 8% when bath KCl was 7 mM or 8 mM
KCl, respectively (P<0.05 for a time effect after 60 min of increased KCl; Fig. 6C). After 60 min
of KCl exposure, burst amplitude was inversely correlated with bath KCl (P=0.012; r2=0.139;
Fig. 6E).
Effects of riluzole and FFA during synaptic inhibition blockade
To test whether the rhythmic bursts produced during synaptic inhibition blockade were
altered by riluzole or FFA, turtle brainstems were exposed to bicuculline (50 µM) and strychnine
(50 µM) for 100 min and then a 20-min baseline was established. Against a background of
bicuculline and strychnine, either riluzole (50 µM; n=12) or FFA (100 µM; n=7) were applied.
Under these conditions, riluzole had no effect on burst frequency (Fig. 7A, 8A). In contrast, FFA
decreased burst frequency in a time-dependent manner from 0.96 ± 0.10 bursts/min (baseline) to
0.36 ± 0.08 bursts/min after 60 min (P<0.05) and 0.10 ± 0.05 bursts/min after 120 min (P<0.05;
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Figs. 7B, 8A). Burst amplitude was not altered by either riluzole or FFA although there were
significant time effects for all the data during the last 80 min of drug exposure (Fig. 8B).
DISCUSSION
This is the first study to show that FFA, but not riluzole, decreases respiratory burst
frequency in an adult ectothermic vertebrate using in vitro isolated brainstems. This suggests that
riluzole-sensitive persistent Na+ currents in pacemaker neurons are not required for rhythm
generation in aCSF or during synaptic inhibition blockade. In contrast, the finding that FFA
abolished burst frequency in aCSF as well as during synaptic inhibition blockade suggests that
Ca2+-activated cation currents are required for rhythm generation. Since burst frequency was not
correlated with bath KCl in aCSF, it appears unlikely that pacemaker neurons with FFA-sensitive
currents are required for rhythm generation. Following network transformation via synaptic
inhibition blockade, the frequency of the FFA-sensitive rhythmic activity was correlated with
bath KCl, suggesting that synaptic inhibition “releases” pacemaker neurons or properties which
then produce rhythmic motor activity. Taken together, we hypothesize that recurrent excitatory
synaptic transmission involving postsynaptic Ca2+-activated cation currents are responsible for
rhythm generation in turtles (i.e., group-pacemaker model).
Pacemaker properties in vertebrate respiratory rhythm generation
The maturation network-burster hypothesis (43) hypothesizes that the hybrid-pacemaker
network model applies during the early postnatal period in mammals because glycine-mediated
synaptic inhibition is weak, respiratory neurons are relatively depolarized, and persistent Na+
currents in pacemaker neurons are active. During maturation, however, neuronal membrane
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potentials are hypothesized to become more negative and thereby permit a large repertoire of
ionic conductances to contribute to rhythm generation. Furthermore, synaptic inhibition is
hypothesized to become necessary for rhythm generation and the obligatory role of pacemaker
neurons is diminished in adult animals. In support of this model, respiratory-related pacemaker
neurons are found in primarily in vitro preparations from perinatal rodents in key putative
rhythm-generating sites, such as the pre-Bötzinger Complex (preBötC; 22, 53, 57) and the
parafacial respiratory group (pFRG; 1, 26, 27). Consistent with the maturation network-burster
hypothesis, there is little evidence for respiratory-related pacemaker neurons in adult mammals
(although the precise age at which the pacemaker-driven respiratory rhythm presumably
transitions to a network-driven rhythm is not well defined). Interestingly, pre-I neurons in a
perfused in situ preparation from 21-42 day old mice burst rhythmically in low Ca2+/high Mg2+
solution, but it was not clear whether synaptic transmission was completely blocked (28). In
amphibians, the maturation network-burster hypothesis also appears to apply since the lung
rhythm persists during synaptic inhibition blockade in isolated brainstems from tadpoles, but not
from adult frogs (3, 17).
In contrast, evidence suggests that the maturation network-burster hypothesis may not apply
in all vertebrates. For example, respiratory-related motor bursts are produced by isolated adult
lamprey brainstems during bath application of picrotoxin (GABAA antagonist) and strychnine
(44). Also, as shown here (Fig. 6A) and in Johnson et al., (23), rhythmic motor activity persists
in isolated adult turtle brainstems, and this rhythm is abolished by well-known respiratory
depressants, such as µ-opiate receptor agonists (23) and high pH/low CO2 conditions
(unpublished observations). This suggests that pacemaker properties, whether they are expressed
in individual neurons or as an emergent network property, may be involved in respiratory rhythm
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generation in some adult vertebrates.
Since respiratory-related pacemaker neurons (in mammals) have intrinsic, voltagedependent bursting properties, action potential burst frequency is directly related to membrane
potential (5, 53). Thus, under in vitro conditions, increasing bath KCl would be expected to
depolarize pacemaker neurons, increase action potential burst frequency in pacemaker neurons,
and thereby increase respiratory burst frequency within the network. There are several caveats
associated with this approach. For example, networks without pacemaker neurons have voltagedependent conductances whose properties will be altered by depolarization. Also, increased bath
KCl may increase neurotransmitter release, alter intrinsic membrane properties, alter the driving
force through K+-permeable conductances, or alter the function of membrane ion pumps.
Although correlating bath KCl with rhythm frequency is not specific, it is useful when combined
with other experimental approaches, such as synaptic inhibition blockade. For example, the
tadpole lung rhythm frequency was directly related to bath KCl levels (61) and persisted during
synaptic inhibition blockade (3, 17), which is consistent with the network being driven by
pacemaker neurons. In contrast, the adult frog lung rhythm was not correlated with bath KCl (61)
and was abolished during synaptic inhibition blockade (3, 17), which is consistent with a nonobligatory role for pacemaker neurons. In this study, the finding that burst frequency in isolated
turtle brainstems was not correlated with increased bath KCl is consistent with the hypothesis
that pacemaker neurons with voltage-dependent properties are not required for rhythm
generation.
Can the turtle respiratory rhythm generator be transformed into a pacemaker-driven network?
In mammalian in vitro brainstem preparations, the “switching” hypothesis states that the
respiratory network can undergo a non-physiological switch from a network-driven to a
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pacemaker-driven system when there is decreased synaptic inhibition and increased extracellular
K+-ions (45, 47, 48, 49, 50, 55). Under these conditions, pacemaker neurons, which are not
required for normal breathing, are proposed to become rhythmically active and force respiratory
neurons downstream to oscillate rhythmically (4, 45, 47, 48, 49, 50). Accordingly, the precise
nature of the motor output produced by reduced perinatal mammalian preparations is part of an
ongoing debate as to what constitutes normal breathing (7, 12, 29, 34, 40, 54).
In contrast to mammalian preparations, turtle brainstems in vitro are highly resistant to
hypoxia, and have a low metabolic rate at physiologically relevant temperatures (e.g., room
temperature). Thus, turtle brainstems are unlikely to have hypoxia-induced increases in
extracellular H+ and K+ levels (58, 62) that are found in some mammalian in vitro brainstemspinal cord preparations. It should be noted that turtle brainstems used in this study were at
pH=7.30, which is acidic compared to the normal blood pH (~7.60) in red-eared sliders. Whether
or not low pH biases the turtle respiratory rhythm generator towards pacemaker- versus networkbased function is not known. In addition, isolated turtle brainstems produce appropriate phasic
expiratory and inspiratory activity similar to intact turtles, thereby negating the mammalian
“eupnea versus gasping” controversy (21). Nevertheless, blockade of synaptic transmission in
turtle brainstems produces a rhythm that was directly correlated with extracellular KCl levels,
which is consistent with the hypothesis that turtle brainstems, in a manner similar to mammalian
preparations, can undergo a switch to a pacemaker-driven network. It should be noted that this
correlation was statistically significant but only 17% of the variation was due to bath KCl. One
difference is that only synaptic inhibition blockade appears to be necessary to make the switch in
turtles whereas increased extracellular K+ levels may also be necessary in mammals. Thus, this
capacity for switching to a pacemaker-driven network may be a conserved mechanism within the
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respiratory systems of mature vertebrates.
Role of riluzole- and FFA-sensitive conductances in respiratory rhythm generation
In mammals, the ionic conductances hypothesized to be responsible for pacemaker
properties of respiratory neurons include persistent Na+ currents (5, 9, 10, 25, 47, 48), Ca2+activated cation currents (33, 57), and intracellular Ca2+ oscillations (2). For example, persistent
Na+ currents are required for voltage-dependent bursting in a subpopulation of respiratory
pacemaker neurons in the preBötC (10, 33). Riluzole blocks respiratory activity in rhythmically
active rodent slices (37, 48) and in P21 (and older) perfused in situ murine preparations (37),
suggesting that persistent Na+ currents are required for respiratory rhythm generation. In
contrast, riluzole did not change the frequency of respiratory motor output produced by
rhythmically active rodent slices (10) or in 6-week old perfused in situ rat preparations (30, 56),
although positive controls showing the penetrance of riluzole into the tissue were not shown.
These data were interpreted as consistent with the hypothesis that persistent Na+ currents are
required for gasping and not eupnea (30, 56). Another interesting observation is that rhythmic
motor activity (presumed to be respiratory-related) persists during simultaneous blockade of both
persistent Na+ currents (with riluzole) and Ca2+-activated cation currents (with FFA), which
suggests that pacemaker neurons are not required for mammalian respiratory rhythm generation
(11). Although some of the contradictory results may be due to different experimental
conditions, protocols, preparations, and animal strains and species, clearly more work is required
to resolve these questions.
Since both riluzole and FFA block currents other than persistent Na+ and Ca2+-activated
cation currents, respectively, the strongest finding in this paper was that riluzole did not abolish
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respiratory burst frequency in aCSF or during synaptic inhibition blockade. For example, riluzole
blocks NMDA-dependent responses in frog oocytes with an IC50 = 18.2 µM (8). Riluzole
appeared to penetrate the tissue rapidly because the same effects were observed in aCSF for
intact and hemi-brainstems. If persistent Na+ currents are present in turtle respiratory-related
neurons, these currents do not appear to be essential for rhythm generation. On the other hand,
it’s possible that persistent Na+ currents are not present in turtle respiratory neurons. Since
persistent Na+ currents are hypothesized to be necessary for mammalian gasping (30, 56) and
turtles do not appear to gasp during 6 hr of anoxia (unpublished observations), we hypothesize
that the turtle respiratory neurons express low levels, if any, of persistent Na+ channels.
In contrast, FFA blocked turtle respiratory rhythm generation in a time- and dose-dependent
manner with 50 µM FFA producing significant decreases in burst frequency within 40 min of
exposure. One interpretation is that FFA blocked Ca2+-activated cation currents in pacemaker
neurons that are required for respiratory rhythm generation and that increased bath KCl elicits
offsetting complex effects on frequency so that increased bath KCl does not increase burst
frequency in aCSF. If so, intracellular recordings in the absence of synaptic transmission will be
required to identify and characterize candidate pacemaker neurons. On the other hand, the lack of
correlation between burst frequency and bath KCl in aCSF argues against the role of pacemaker
neurons. Instead, Ca2+-activated cation currents may be expressed in non-pacemaker neurons and
the positive feedback interaction of this current with excitatory glutamatergic synaptic currents
may underlie respiratory rhythm generation (i.e., group pacemaker model; 10, 11, 14, 15, 39). In
this case, Ca2+-activated cation currents would play a critical role in initiating and maintaining
inspiratory bursts. However, other interpretations are plausible because FFA blocks other
membrane currents, such as transient receptor potential currents (19) and L-type Ca2+ channels
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(51) that may be necessary for rhythm generation. Also, FFA may inactivate other neurons that
project to the turtle rhythm generator and provide critical neurotransmitters that maintain
respiratory neurons at appropriate levels of excitability.
Acknowledgements
This work was supported by a National Science Foundation Grant (IOB 0517302). The authors
also acknowledge excellent technical assistance provided by Robert Creighton.
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FIGURE LEGENDS
Fig. 1. Spontaneous respiratory motor output produced by isolated in vitro turtle brainstems. A:
Drawing of turtle brainstem preparation with bursts of integrated respiratory motor output
recorded by a suction electrode attached to hypoglossal (XII) nerve rootlets. B: Integrated XII
discharge shows several episodic (i.e., doublet) bursts and one singlet burst.
Fig. 2. Respiratory burst frequency is not correlated with bath [KCl] in aCSF. A: Integrated XII
discharge shows effects of increasing bath [KCl] from 2.0 to 9.0 mM KCl for a brainstem bathed
in aCSF. The dotted lines show expanded traces during baseline (left) and after >30 min of
increased KCl exposure (right). Arrows in the expanded traces show individual XII respiratory
bursts (note that frequency and episodic discharge was not altered). Time-dependent changes in
burst frequency (B) and amplitude (C) are shown in response to increasing bath [KCl] from 2.0
mM (data at 20-min time point) to 5.0, 7.0, or 9.0 mM KCl (closed circles, open circles, and
open squares, respectively). B: Burst frequency increased significantly in a sustained manner
after increasing bath KCl to 9.0 mM, but not after increasing to 5.0 or 7.0 mM KCl. C: Burst
amplitude mostly decreased with increased bath KCl, except for 60 min after increasing to 9.0
mM KCl. D,E: Linear regression analysis of burst frequency (D) and amplitude (E) for data at
the 60-min time point in Figs. 2B and 2C, respectively, shows no correlation with bath [KCl].
Asterisk = P<0.05 compared to 5 mM KCl data; # = P<0.05 compared to baseline; & = P<0.05
for time-dependent effect at that data point; † = P<0.05 for drug effect; ‡ = P<0.05 for timedependent effect.
Fig. 3. Riluzole does not abolish respiratory motor output. Traces of integrated XII motor
Page 29 of 39
Pacemaker properties and turtle breathing in vitro
Johnson, Weigel, Majewski
29
discharge are shown for two different brainstems bathed in aCSF and exposed to riluzole (50
µM, 2 hr). Riluzole increased frequency and decreased amplitude in brainstems producing either
singlet (A) or episodic (B) discharge.
Fig. 4. Effects of riluzole on respiratory variables for brainstems bathed in aCSF. A: Bath-applied
riluzole (50 µM, 2 hr) significantly increased burst frequency (open circles) while frequency
remained constant in time control experiments (closed circles). B: When frequency data were
graphed as percent change from baseline, riluzole increased frequency during the first 80 min of
application until a plateau was reached. C: Riluzole did not alter the number of bursts/episode.
D: Riluzole caused a steady time-dependent decrease in burst amplitude. Asterisk = P<0.05
compared to time control data; # = P<0.05 compared to baseline; † = P<0.05 for drug effect; ‡ =
P<0.05 for time-dependent effect.
Fig. 5. Effects of FFA on respiratory variables for brainstems bathed in aCSF. A: Integrated XII
traces from a brainstem during baseline (left panel), after 1 hr of bath-applied FFA (100 µM;
middle panel), and after 2 hr of bath-applied FFA (right panel). B: FFA produced a dose- and
time-dependent decrease in burst frequency at FFA concentrations between 50-500 µM. C:
Frequency data at the 140-min time point in Fig. 5B are graphed as percent change from baseline
at each dose. D: FFA did not alter the number of bursts/episode (data at all FFA concentrations
were pooled together). The numbers in parentheses above the x-axis indicate the number of
brainstems producing respiratory motor output at that time-point for the FFA experiments. E:
FFA (20 µM) did not alter burst amplitude compared to time controls. FFA (50 µM) produced
complex time-dependent increases and decreases in amplitude, while FFA (100 µM) mainly
Page 30 of 39
Pacemaker properties and turtle breathing in vitro
Johnson, Weigel, Majewski
30
decreased amplitude. Asterisk = P<0.05 compared to time control data; # = P<0.05 compared to
baseline; † = P<0.05 for drug effect; ‡ = P<0.05 for time-dependent effect.
Fig. 6. Burst frequency is correlated with bath [KCl] during synaptic inhibition blockade. A:
Integrated XII discharge for a brainstem bathed in bicuculline and strychnine (50 µM each) and
bath [KCl] was increased from 2.0 to 8.0 mM. Time-dependent changes in burst frequency (B)
and amplitude (C) in response to increasing bath [KCl] from 2.0 mM (data at 20-min time point)
to 5.0, 7.0, or 8.0 mM KCl (closed circles, open circles, and open squares, respectively). B: Burst
frequency increased significantly in a sustained manner for the 7.0 and 8.0 mM KCl data; there
was no change when bath KCl was increased to 5.0 mM. C: Burst amplitude decreased with time
during prolonged KCl application. D,E: Linear regression analysis of burst frequency (D) and
amplitude (E) for data at the 60-min time point in Figs. 6B and 6C, respectively (i.e., 20-40 min
during KCl exposure). D: Burst frequency was directly correlated with bath [KCl], while burst
amplitude (E) was indirectly correlated with bath [KCl]. & = P<0.05 for time-dependent effect at
that data point; ‡ = P<0.05 for time-dependent effect.
Fig. 7. FFA, but not riluzole, alters rhythmic motor bursts during synaptic inhibition blockade
(i.e., bicuculline and strychnine at 50 µM each). (A) Riluzole (50 µM) did not alter burst
amplitude or frequency; (B) FFA (100 µM) decreased burst frequency with little change in
amplitude.
Fig. 8. Effects of riluzole and FFA during synaptic inhibition blockade. A: While bathed in
bicuculline and strychnine (50 µM each), riluzole (50 µM; open circles) did not alter burst
Page 31 of 39
Pacemaker properties and turtle breathing in vitro
Johnson, Weigel, Majewski
31
frequency compared to time controls (closed circles). In contrast, FFA (100 µM; open squares)
decreased burst frequency in a time-dependent manner. B: Riluzole and FFA did not alter burst
amplitude compared to time controls, but there was a time-dependent decrease for all the data.
Asterisk = P<0.05 compared to time controls; # = P<0.05 compared to baseline; & = P<0.05 for
time-dependent effect at that data point; † = P<0.05 for drug effect; ‡ = P<0.05 for timedependent effect.
Page 32 of 39
Fig. 1
A
V
VI
VII
VIII
B
IX
X
XII
C1
XII
60 s
Page 33 of 39
Fig. 2
9 mM KCl
A
5 min
60 s
C
KCl
400
5 mM KCl
7 mM KCl
9 mM KCl
300
&
†‡
200
100
0
0
Frequency change
(% of baseline)
D
20
40
60
Amplitude (normalized)
500
Time (min)
E
600
P = 0.066
r 2= 0.129
200
0
0
2
4
6
Bath [KCl] (mM)
8
*
1.2
1.0
10
‡
0.8
5 mM KCl
7 mM KCl
9 mM KCl
0.6
#
&
0.4
0
800
400
KCl
1.4
80
Amplitude (normalized)
Frequency change
(% of baseline)
B
60 s
20
40
60
80
Time (min)
1.5
P = 0.295
r 2= 0.044
1.0
0.5
0.0
0
2
4
6
Bath [KCl] (mM)
8
10
Page 34 of 39
Fig. 3
baseline
A
Riluzole
(50 µM, 2 hr)
XII
0
B
XII
60 s
Page 35 of 39
Fig. 4
riluzole
1.2
control
riluzole
1.0
*
* *
0.8
B
#* #* #*
#*
†‡
0.6
0.4
0.2
0.0
20
40
60
130
#
#
#
†‡
120
110
100
80 100 120 140
riluzole
2.0
1.5
1.0
0.5
control
riluzole
0.0
0
20
40
0
D
Amplitude (normalized)
Bursts/eposide
#
control
riluzole
90
0
C
riluzole
140
Frequency change
(% of baseline)
Frequency (bursts/min)
A
80 100 120 140
Time (min)
40
60
80 100 120 140
riluzole
1.2
1.0
#* #
*
0.8
0.6
0.4
0.2
#*
#* #
*
#*
control
riluzole
0.0
60
20
0
20
40
60
80 100 120 140
Time (min)
†‡
Page 36 of 39
Fig. 5
FFA
(100 µM, 1 hr)
baseline
A
FFA
(100 µM, 2 hr)
XII
B
1.0
Frequency (bursts/min)
1 min
0.8
FFA
C
Percent change in frequency
at 140 min post-FFA
100
0.6
80
#
0.4
control
20 µM
50 µM
100 µM
500 µM
0.2
0.0
0
#
#
#
*
#*
#*
20
40
#*
60
80
60
#*
*
#*
100
#*
#*
#*
#*
#*
#*
120
†‡
1.5
1.0
control
FFA
0.5
0.0
0
(26)
(26)
20
40
(17) (14)
60
80
(12)
(10)
(10)
100 120 140
Time (min)
Amplitude (normalized)
Bursts/episode
E
FFA
2.0
*
20
0
140
Time (min)
D
*
40
*
ol µM µM µM µM
ntr
co 20 50 100 500
FFA
1.5
*
1.0
*
0.5
control
20 µM
50 µM
100 µM
0.0
0
20
40
60
* *
*
*
80 100 120 140
Time (min)
Page 37 of 39
Fig. 6
8 mM KCl
A
5 min
KCl
500
5 mM KCl
7 mM KCl
9 mM KCl
400
C
&
&
&
300
‡
200
100
0
0
40
60
Time (min)
P = 0.005
r 2= 0.167
600
400
200
0
0
2
4
6
[KCl] (mM)
1.2
8
&
1.0
‡
0.8
5 mM KCl
7 mM KCl
9 mM KCl
0.6
0.4
0
E
800
KCl
1.4
80
Amplitude (normalized)
Frequency change
(% of baseline)
D
20
Amplitude (normalized)
Frequency change
(% of baseline)
B
10
20
40
60
80
Time (min)
1.5
P = 0.012
r 2= 0.139
1.0
0.5
0.0
0
2
4
6
[KCl] (mM)
8
10
Page 38 of 39
Fig. 7
A
Bicuculline/Strychnine
Bicuculline/Strychnine
+
Riluzole (50 µM)
B
Bicuculline/Strychnine
Bicuculline/Strychnine
+
FFA (100 µM)
60 s
Page 39 of 39
Fig. 8
A
Frequency (bursts/min)
Bic/Strych
Bic/Strych + Riluzole (50 µM)
Bic/Strych + FFA (100 µM)
Riluzole or FFA
1.4
1.2
1.0
0.8
#*
0.6
0.4
0.2
Amplitude (normalized)
#*
#*
#* † ‡
120
140
0.0
0
B
#*
20
40
60
80
100
Riluzole or FFA
1.4
1.2
1.0
0.8
&
0.6
‡
&
&
&
&
0.4
0.2
0.0
0
20
40
60
80
Time (min)
100
120
140