Differential modulation of neural network and
pacemaker activity underlying eupnea and sigh
Andrew K. Tryba1*, Fernando Peña2, Steven P. Lieske3,Jean-Charles Viemari4,5, Muriel Thoby-Brisson6
and Jan-Marino Ramirez5
1 Dept of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226
2 Departamento de Farmacobiología, Cinvestav-Sede Sur, Calz. de los Tenorios 235, Col. Granjas Coapa,
14330, México D.F., México
3 Department of Psychiatry, University of California San Francisco, 401 Parnassus Ave, San Francisco, CA,
4Laboratoire Plasticité et Physio-Pathologie de la motricité, CNRS UMR 6196,
31 Chemin Joseph Aiguier, 13402 Marseille cedex 20. France
5Department of Organismal Biology, The University of Chicago, Chicago, IL 60637 USA
6Laboratoire de Neurobiologie Genetique et Integrative, Institut Alfred Fessard, 1 Avenue de la Terrasse,
91190 Gif sur Yvette, France
Running Title: Sigh rhythm generating mechanisms
* Corresponding Author
Andrew K. Tryba, Ph.D.
Medical College of Wisconsin
Department of Physiology
8701 Watertown Plank Road
Milwaukee, WI 53226
FAX: (414) 456-6546
Phone: (414) 456-4975
Abbreviations: pre-Böt – pre-Bötzinger complex – VRG Ventral Respiratory Group –
SIDS – Sudden Infant Death Syndrome
Page 1 of 51
Articles in PresS. J Neurophysiol (February 20, 2008). doi:10.1152/jn.01192.2007
Copyright © 2008 by the American Physiological Society.
Many networks generate distinct rhythms with multiple frequency and amplitude
characteristics. The respiratory network in the pre-Bötzinger complex (pre-Böt) generates
both the low frequency, large amplitude sigh rhythm and a faster, smaller amplitude,
eupneic rhythm. Could the same set of pacemakers generate both rhythms? Here we
used an in vitro respiratory brainslice preparation. We describe a subset of synaptically
isolated pacemakers that spontaneously generates two distinct bursting patterns.These
two patterns resemble network activity including sigh-like bursts that occur at low
frequencies and have large amplitudes and eupneic-like bursts with higher frequency and
smaller amplitudes. Cholinergic neuromodulation altered the network and pacemaker
bursting: fictive sigh frequency is increased dramatically, while fictive eupneic frequency
is drastically lowered. The data suggest that timing and amplitude characteristics of
fictive eupneic and sigh rhythms are set by the same set of pacemakers that are tuned by
changes in the neuromodulatory state.
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Many neuronal networks including those involved in sleep, olfaction, learning and
locomotion generate multiple, context-dependent rhythms (Steriade et al., 1993a; Tryba
and Ritzmann, 2000; Csicsvari et al., 2003; Kay, 2003). Respiratory rhythms are critical
to life and several forms of rhythmic activities are generated by the medullary respiratory
neural network. Under normal conditions, breathing includes eupnea (“normal
respiration”), which transforms into gasping during severe hypoxia (Lieske et al., 2000).
During normal breathing, the low frequency sigh rhythm is superimposed on eupneic
activity and includes larger inspiratory efforts. Each sigh is followed by post-sigh apnea
and phase resetting of the eupneic rhythm.
Sighing is a normal component of breathing and serves to re-open collapsed
alveoli (Issa and Porostocky, 1993); sighs are thought to trigger arousal, failure of which
may contribute to sudden infant death syndrome (SIDS) (Franco et al., 2003). Despite its
clinical relevance, the mechanisms underlying sigh generation remain largely unknown
(Lieske et al., 2000; Lieske and Ramirez, 2006a; Lieske and Ramirez, 2006b).
A better understanding of the mechanisms underlying eupnea and sighs is
facilitated by the medullary slice preparation of mice that contains a critical portion of the
respiratory neural network, the pre-Bötzinger complex (pre-Böt), that simultaneously
generates fictive eupneic and sigh activities. Stereotactic mapping of the population
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activity recorded at the slice surface revealed extensive spatial overlap in the network
active during eupnea and sighs. This suggested that the same network generates different
forms of breathing (Lieske et al., 2000).
Patch-clamp data indicate most pre-Böt inspiratory neurons produce a burst of
action potentials during both fictive eupnea and sighs (Lieske et al., 2000). Thus,
previously described inspiratory neurons typically contribute to both fictive eupneic and
sigh activity (Lieske et al., 2000). However, detailed synaptic analysis revealed distinct
synaptic properties underlie eupneic and sigh rhythm generation (Lieske and Ramirez,
2006a; Lieske and Ramirez, 2006b) and suggest the possibility that distinct populations
of inspiratory neurons underlie sigh rhythm generation. These data pose a very
interesting puzzle that is relevant to many rhythmic neural networks: what types of
cellular mechanisms allow largely overlapping networks to generate two superimposed
and interacting rhythms?
The identification of non-pacemaking, “sigh-only” neurons in the present study is
consistent with the hypothesis that distinct sigh specific neuron populations exist. But
this finding does not resolve the core question: How do two largely overlapping neuronal
networks generate two distinct inspiratory rhythms? Here, we describe the unexpected
finding that some synaptically isolated pacemaker neurons generate two types of bursts
each having distinct amplitude and timing characteristics: fast, low-amplitude bursts are
interrupted by low frequency large-amplitude bursts. Oxotremorine, a muscarinic
agonist, transforms the timing characteristics of this subset of respiratory pacemakers by
increasing the frequency of large- amplitude bursts while simultaneously decreasing low-
amplitude burst frequency. The network responds with similar timing changes: the very
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low frequency (and large-amplitude) fictive sighs increased their frequency dramatically,
while the ordinarily much faster (and lower-amplitude) fictive eupneic rhythm shows the
opposite effect, slowing considerably and in some cases stopping altogether. Thus, the
allegiance to these rhythms depends on the neuromodulatory state which alters
pacemaker and network properties.
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Materials and Methods
All experiments conformed to the guiding principles for the care and use of animals
approved by the National Institutes of Health (U.S.A.) and the Animal Care and Use
Committees at the Medical College of Wisconsin and The University of Chicago
(experimental performance sites).
Medullary brain-slice and pre-Bötzinger complex island preparation
All experiments used the transverse, rhythmic 600-650µm thick medullary brain-slice
obtained from 1-13 day old, CD-1 outbred mice (Charles River Laboratories,
Wilmington, MA) (Thoby-Brisson and Ramirez, 2001). CD-1 mice were deeply
anesthetized with ether (delivered by inhalation) and quickly decapitated at the C3/C4
spinal level (Thoby-Brisson and Ramirez, 2001). The brain-stem was dissected in ice
cold artificial cerebral spinal fluid (ACSF) that was equilibrated with carbogen (95% O2
and 5% CO2, pH=7.4). Rhythmic slice preparations containing the pre-Bötzinger
Complex (pre-Böt) were obtained by slicing the medulla using a microslicer (Leica,
VT1000S, Nussloch, Germany). Slices were submerged in a recording chamber (6 mL)
under circulating ACSF (30°C; flow rate 17 ml/min, total circulating volume = 200mL).
ACSF contained in mM: 118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2*6H2O, 25
NaHCO3, 1 NaH2PO4 and 30 D-glucose, equilibrated with carbogen (95% O2 and 5%
CO2, pH = 7.4). All ACSF chemicals were obtained from Sigma (St. Louis, MO)
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Extracellular KCl was elevated from 3mM to 8mM over a span of 30 minutes before
commencing recordings, to maintain rhythmic population activity (Tryba et al. 2003).
Note that raising ACSF [K+]o does not artificially introduce pacemaker bursting
properties; pacemakers show similar bursting properties in 3mM versus 8mM [K+]o
ACSF (Tryba et al., 2003). Further, it should be noted that [K+]ochanges on a breath by
breath basis and changes in [K+]o do not obviously alter the form of respiratory activity
generated, as both eupneic and gasping activities can be recorded in hypokalemic and
hyperkalemic conditions in situ (St-John et al., 2005) . Bath temperature was monitored
and maintained at 30ºC ± 0.7ºC using a Warner Instrument Corp. (Hamden, CT) TC-
344B temperature regulator with an in-line solution heater (SH-27B); bath temperature at
various locations within the bath was uniform. The VRG-island preparation (Johnson et
al., 2001) was made by taking a brain slice preparation and cutting a wedge shaped piece
of tissue containing the pre-Böt complex out of the transverse brain-slice using a sharp
Electrophysiology- Population activity and identification of inspiratory
Extracellular recordings were obtained with glass suction electrodes positioned on the
slice surface in the ventral respiratory group (VRG) near or on top of the pre-Böt (Figs.
1A, 1B) (Tryba et al., 2003). The VRG population bursting is dominated by inspiratory
neurons such that integrated VRG (∫VRG) activity is in-phase with integrated XII (∫XII)
activity (Tryba et al., 2006). Thus, VRG population bursts serve as a marker of fictive
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inspiration (Tryba et al., 2006). This population activity was rectified and integrated and
the data were digitized with a Digidata acquisition system (Molecular Devices, CA),
stored on an IBM compatible PC using Axoscope 10 (Molecular Devices, CA) software
and analyzed off-line using Igor Pro (WaveMetrics, Lake Oswego, OR). The ∫VRG
population burst amplitude was measured as baseline to peak height, while frequency was
calculated based on the burst intervals. To minimize the potential influence of baseline
fluctuations and differences in burst peak trajectories, the ∫VRG burst duration was
calculated as the duration of the burst at half-maximal burst amplitude.
Intracellular patch-clamp recordings were obtained with a MultiClamp 700B amplifier
(Molecular Devices, CA), applying the blind-patch technique to VRG neurons in 600-
650µm brainstem slice preparations (Thoby-Brisson and Ramirez, 2001). Patch
electrodes were manufactured from filamented borosilicate glass tubes (Clark G150F-4;
Warner Instruments Corp., Hamden, CT, USA) and filled with an intracellular solution
containing (in mM): 140 K-gluconic acid, 1 CaCl2*6H2O, 10 EGTA, 2 MgCl2*6H2O, 4
Na2ATP, 10 HEPES.
Only inspiratory VRG neurons active in-phase with the ∫VRG population burst
were recorded in this study. The discharge pattern of each cell type was first identified in
the cell-attached mode and remained similar in whole-cell configuration (Pena et al.,
2004). Experiments were then performed in the whole cell patch-clamp mode. The Vm
values were corrected for the liquid junction potential as calculated using pClamp 10
software (Molecular Devices). In current-clamp, neurons were isolated from ionotropic
chemical synaptic input using a mixture of glutamatergic, GABAergic and glycinergic
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antagonists. These drugs were bath applied at the final concentrations of: 20 µM 6-
cyano-7-nitroquinoxaline-2,3-dione (CNQX (Tocris, Ellisville MO, USA)), 10 µM (RS)-
3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid ((RS)-CPP)(Tocris), 1 µM
Strychnine (Sigma) and 20 µM bicuculline free-base (Sigma). Note that unlike
bicuculline methiodide, the bicuculline free base derivative is a specific GABA receptor
antagonist that does not block apamin-sensitive Ca2+-activated K+ currents (Johnson and
Seutin, 1997; Debarbieux et al., 1998).
A subset of neurons, considered pacemakers, continued to exhibit voltage-
dependent intrinsic bursting properties following blockade of ionotropic glutamate,
GABA and glycinergic receptors. These pacemakers met several criteria before being
classified as inspiratory pacemaker neurons, the criteria used here are provided in detail
elsewhere (Thoby-Brisson and Ramirez, 2001; Tryba et al., 2003). Briefly, after isolation
of the neuron from chemical synaptic input with bath applied CNQX, CPP, strychnine
and bicuculline, pacemakers continued to burst in absence of VRG population bursts.
Second, isolated pacemakers exhibited voltage-dependent bursting properties. That is,
brief depolarizing current injection could evoke a burst, or hyperpolarizing current could
terminate an ongoing burst; either of these reset the ongoing pacemaker bursting rhythm.
Finally, depolarizing current injection increased, and injected hyperpolarizing current
decreased, the bursting frequency. After synaptic isolation with CNQX, CPP, bicuculline
and strychnine, in some cases, cadmium (Cd2+), a broad-spectrum calcium channel
blocker, was used as a tool to discriminate between pacemaker neurons whose
endogenous bursting mechanism is dependent on calcium versus sodium. Synaptically
isolated pacemakers that continue to burst in the presence of voltage-gated calcium
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channel blockade with 200µM Cd2+ are described as Cd2+-insensitive (CI-) pacemakers
(Thoby-Brisson and Ramirez, 2001) and are typically riluzole-sensitive (Pena et al.,
2004) . Isolated pacemakers that cease bursting in Cd2+, are Cd2+-sensitive (CS-)
pacemakers (Thoby-Brisson and Ramirez, 2001) and have been shown to be FFA-
sensitive (Pena et al., 2004). Note that 200µM Cd2+ ensures blockade of voltage
activated calcium currents in VRG neurons (Elsen and Ramirez, 1997). Thus, following
bath application of 200µM Cd2+ calcium-dependent chemical synaptic transmission
should be blocked.
We describe here a new subset of pacemakers that produce two distinct kinds of
bursts, characterized as eupneic-like vs. sigh-like (see Results). These two burst types
were qualitatively quite distinct, and in light of the small number of sigh-like bursts, were
initially identified by hand. The criteria used to identify sigh-like bursts, included: (1) a
larger amplitude inspiratory drive, (2) lower frequency, (3) longer duration than eupneic-
like bursts; and (4) longer post-burst quiescent period (corresponding to the post-sigh
apnea at the population level). To verify that the two bursts are distinct, we made
statistical comparisons of the eupneic-like and sigh-like bursts so identified which are
presented in the Results.
Working Heart Brainstem Preparation
We used the in situ perfused working heart brainstem preparation (Paton, 1996)
following methods previously described by Ramirez and Viemari (Elsen and Ramirez,
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2005). For these preparations, CD1 mice (P21 and older) were deeply anesthetized with
ether and the brain was transected at the pre-collicular level. The rostral part of the
transaction, including the pons, was removed (Elsen and Ramirez, 2005). A second
transection was performed sub-diaphragmatically and the thorax-brainstem preparation
was submerged in ice-cold ACSF (same ACSF as described for slices preparation) gassed
with carbogen (95% O2 and 5% CO2). The preparation was transferred to a recording
chamber where brain tissue rostral to the medulla was removed under a binocular
dissecting microscope. A cut was made just caudal to the facial nerve (Ramirez and
Viemari, 2005) and the pons was removed from the preparation. The abdominal aorta
was cannulated for retrograde perfusion with ACSF containing 3mM K+. Oxotremorine
(Tocris Cookson, Ballwin, MO) was added to the perfusate at a concentration of 10 µM.
Inspiratory motor output was monitored by placing an extracellular suction electrode on
the phrenic nerve. The signals were amplified 2000 times, filtered (low pass 1.5 KHz,
high pass 250 Hz), rectified and integrated using an electronic filter (time constant of 30-
50 ms). All recordings were stored on a personal computer using Axoscope 10
(Molecular Devices, CA) software and analyzed off-line using analysis software written
with IGOR Pro (Wavemetrics, OR).
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Fictive sighs in vitro
As first described by Lieske et al. (2000), fictive sighs occur periodically in the in
vitro transverse medullary slice preparation from mice, containing the VRG (Figs. 1a-1b).
Each fictive sigh burst is bi-phasic in shape, larger in amplitude (p≤0.001), longer in
duration (p≤0.001) and occurs at a lower frequency (p≤0.001) than isolated eupneic
bursts (Figs. 1c; 1d paired T-Tests of mean values from n=15 preparations, including
n=1840 fictive eupneic and n=61 sigh bursts). Note that the biphasic shape of sigh bursts
typically arises as the result of being triggered by eupneic bursts, giving rise to a longer
inspiratory duration than isolated fictive eupneic bursts (Fig. 1c). Our analysis included
between n=3 and n=6 fictive sigh bursts per slice preparation (average n=4 fictive sigh
bursts / preparation).
We also verified that fictive sigh amplitude, duration and frequency can be
distinguished from those variables in eupneic bursts, by performing an unpaired t-test for
each condition and preparation. In each case, there was a significant difference in
eupneic and sigh amplitude (p≤0.001, unpaired t-test), duration (p≤0.005, Mann Whitney
Rank Sum test) and frequency (p≤0.005, Mann Whitney Rank Sum test).
Sighs are followed by a brief pause in the eupneic rhythm, the so-called “post-
sigh apnea” (Cherniack et al., 1981). A fictive apnea is also observed after in vitro
sighing activity (Figs. 1a, 1b) (Lieske et al., 2000). Thus, the above characteristics of
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fictive sigh-like bursts recorded in vitro (i.e., biphasic shape, longer duration, larger
amplitude inspiratory activity, slower frequency and post-sigh apnea) are consistent with
the definition of sighs in vivo (Glogowska et al., 1972; Cherniack et al., 1981; Orem and
Trotter, 1993; Takeda and Matsumoto, 1998). As is the case for fictive sigh bursts in
vitro (Figs. 1a and 1c), the biphasic shape of the sigh, in vivo, has been interpreted as
consisting of an initial phase that is identical to a normal eupneic breathing activity but
includes a later high-amplitude phase which is coupled to, and triggered by, the initial
Both fictive sighs and fictive eupneic activity were also recorded in VRG-island
preparations (n=4; Figs. 1e-1f), obtained by trimming away tissue outside the ventral
respiratory group (VRG) (Johnson et al., 2001), suggesting that the neurons responsible
for generating both rhythms are contained within this region. Sighs generated in VRG-
islands (Figs. 1e-1g) were qualitatively similar to those recorded from the brainstem slice
preparation as both included fictive eupneic activity and slower, eupneic-triggered fictive
sighs (Figs. 1a-1f) (Lieske et al., 2000).
We additionally quantitatively analyzed fictive sigh bursts in the VRG-islands
(Fig. 1g; 1h). Fictive sigh bursts in the VRG islands were also bi-phasic in shape, larger
in amplitude (p≤0.001), longer in duration (p≤0.001) and occur at a lower frequency
(p≤0.001) than isolated eupneic bursts (Fig. 1g; 1h; paired T-Tests of mean values from
n=4 VRG-island preparations, including n=460 fictive eupneic and n=24 sigh bursts).
Our analysis included between n=3 and n=12 fictive sigh bursts per slice preparation
(average n=6 fictive sigh bursts / preparation; Fig. 1h).
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To additionally verify that in the VRG island preparation, fictive sigh amplitude,
duration and frequency can be distinguished from those variables in fictive eupneic
bursts, we performed an unpaired t-test for each condition and preparation. In each case,
there was a significant difference in eupneic and sigh amplitude (p≤0.001, Mann Whitney
Rank Sum test), duration (p≤0.001, Mann Whitney Rank Sum test) and frequency
(p≤0.001, Mann Whitney Rank Sum test).
As was the case in the slice preparation (Lieske et al., 2000; Lieske and Ramirez,
2006b) fictive sighs generated by the VRG-island could be selectively abolished with
4µM cadmium (Fig. 1e, n=3/3 preparations). These data indicate the isolated VRG
contains sufficient neuronal tissue to generate fictive sighs and also indicates that
bilateral synchronization between pre-Böt nuclei is not required to generate sighs.
Fictive sigh-only neurons
We found a previously undescribed class of inspiratory neurons that are activated
only during sigh bursts and call these ‘sigh-only’ neurons (Fig. 2; n=26 sigh-only cells
recorded in n=26 additional slice preparations; these preparations were different than the
slices used in the above population studies). We refer to these neurons, here, as ‘sigh-
only’ as they are active during sighs, but not eupnea. However, this does not rule-out the
possibility of their participation in other respiratory activities.
Sigh-only neurons represent a minority of inspiratory neurons recorded in current-
clamp, as most inspiratory neurons appear to burst during both fictive eupneic inspiratory
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activity and fictive sighs (Lieske et al., 2000). For example, in a sub-set of all our
inspiratory neuron recordings, a sample including n = 265 inspiratory neurons, we found
4.9% (n=13) were sigh-only neurons. Sigh-only neurons had varied firing patterns, some
spiking sporadically between sighs (Figs. 2ai; 2aii, n=18), others tonically active but
inhibited during eupnea (Fig. 2bi, n=8), yet all were bursting during fictive sighs (Fig. 2,
A concern when using the whole-cell current-clamp recording technique is that
the technique may alter the intracellular milieu and in turn alter neural firing patterns.
Thus, prior to rupturing the cell-attached membrane seal, we identified the discharge
pattern of sigh-only neurons in the cell-attached mode and found that it remained similar
in whole-cell configuration, such that “sigh-only” neurons exhibited a burst of action
potentials coincident only with the population sigh burst, but not during fictive eupneic
activity (Figs. 2ai and 2aii).
To test whether sigh-only neurons required synaptic input to rhythmically trigger
sigh bursts, we bath-applied CNQX (20µM), CPP (10µM), bicuculline (20µM) and
strychnine (1µM). These synaptic antagonists eliminated fictive sigh-only cell bursts as
well as fictive eupnea and sigh bursts at the population level (Tryba et al., 2003). None
of the sigh-only neurons tested were endogenous sigh pacemakers capable of generating
intrinsic rhythmic activity following blockade of glutamatergic synaptic input (n=7) or
following combined blockade of glutamatergic and inhibitory synaptic transmission
(n=6) (Figs. 2bi and 2bii).
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Muscarinic activation suppresses fictive eupneic activity and enhances sighs
Neuromodulators can play a major role in determining respiratory rhythmogenesis
(Pena and Ramirez, 2002, 2004; Viemari et al., 2005; Tryba et al., 2006). Here, we found
that application of the muscarinic agonist oxotremorine (20µM) to the slice preparation
markedly decreased, or abolished, fictive eupneic activity and increased fictive sigh burst
frequency (Figs. 3a-3d; Fig. 4a n=10 slice preparations). In six additional preparations,
bath application of a lower concentration of oxotremorine (10µM) had similar effects, by
increasing the number of fictive sighs from n=25 (control, over 10 mins) to n=120 (in
oxotremorine, 10mins.) and decreasing the number of fictive eupneic bursts from n=421
(control, 10 mins) to n= 162 (in oxotremorine, 10 mins) (Fig. 4b).
Bath application of the M3-preferring mACh-receptor antagonist, 4-
diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP, 1µM), 10 minutes prior to
applying 10µM oxotremorine blocked the effect of oxotremorine application alone,
suggesting that oxotremorine application suppresses fictive eupnea and enhances sighs by
activating M3 mACh receptors (Figs. 4b-4c).
We tested whether oxotremorine also suppresses fictive eupnea and enhances
fictive sighing frequency in a more intact network, using the in situ working heart
brainstem preparation (Paton, 1996) (Figs. 4d-4f). When oxotremorine (10µM) is
perfused in the working heart brainstem preparation, fictive eupneic frequency is
suppressed (p=0.02; Student’s paired t-test) while fictive sigh bursts increase (p=0.029)
(Figs. 4d, n=4 preparations, Student’s paired t-test). As in vitro (Fig. 1c), fictive in situ
eupneic ∫phrenic bursts are distinct from biphasic sigh bursts as the later have a larger
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amplitude (p<0.001), longer duration (p<0.001) and occur at a lower frequency (p<0.001)
(Figs. 4e-4f, n=5 preparations, Student’s paired t-test).
Pacemakers with endogenous sigh-like bursting properties
Having established that neurons contained within the VRG suffice to generate
fictive sighs, in additional experiments, this area was targeted for extracellular and
whole-cell current-clamp recordings in the medullary slice preparation to determine how
fictive sighs are generated. Two basic types of inspiratory pacemaker neurons were
previously described that generate endogenous, voltage-dependent bursting after being
synaptically isolated from ionotropic glutamatergic input (Thoby-Brisson and Ramirez,
2001; Pena et al., 2004). The bursting mechanism of one type of pacemaker is sensitive
to cadmium and flufenamic acid, the other type is cadmium- and flufenamic- insensitive
All inspiratory pacemaker neurons that were previously described burst during
both eupnea and sighs when embedded in the respiratory network (Lieske et al., 2000).
We refer to these cells as pacemakers because following isolation from glutamatergic
synaptic input with CNQX, CPP, and isolation from inhibitory transmission with
bicuculline and strychnine, they exhibited endogenous, voltage-dependent pacemaker
properties (Figs. 5a;5b) (Thoby-Brisson and Ramirez, 2001; Pena et al., 2004). Most
pacemakers do not continue to generate fictive sigh-like bursts following synaptic
isolation (Fig. 5b), but retain their voltage-dependent bursting properties (Fig. 5c) (Lieske
et al. 2000).
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After isolation from chemical synaptic transmission, we found a subset of
pacemaker neurons that differ from those previously described in that they continued to
generate two types of bursts, at two different frequencies (n=5) including a fictive
“eupneic-like” burst and a fictive “sigh-like” burst. It must be emphasized that the terms
“eupneic-like” and “sigh-like” are used here descriptively because the burst pattern
parallels the network bursting fictive eupneic (fast frequency, smaller amplitude bursts)
and sigh activities (lower frequency, large amplitude bursts). Fictive eupneic-like
pacemaker bursts occur at a higher frequency (p<0.03, paired T-test) and are shorter in
duration (p=0.041, paired T-test) than the lower frequency sigh-like bursts; sigh-like
bursts are also followed by a delay before eupneic-like bursts resume (Fig. 6a).
These cells are considered pacemaker neurons by virtue of the persistence of
voltage-dependent rhythmic bursting following synaptic isolation with CNQX, CPP,
bicuculline, and strychnine (Fig. 6a). In addition, all five of these cells remained
rhythmic in cadmium (Figs. 6b, n=4) or flufenamic acid (FFA), Fig. 7, n=1), indicating
that they comprise a subset of the type of pacemaker referred to as cadmium-insensitive
pacemakers (Thoby-Brisson and Ramirez, 2001; Pena et al. 2004).
The absence of VRG population bursting after blocking synaptic transmission
raises the question as to whether or not the two burst types observed in these cells bears
any relation to fictive eupnea and sighs at the population level. To address this question,
we relied on the differential effect of oxotremorine application, which in the intact
network increased fictive sigh network frequency while decreasing fictive eupneic
frequency (Figs. 4a, 4d). When bath applied to these dual-bursting pacemakers, in
synaptic isolation, oxotremorine (20µM) transformed their bursting from generating both
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fictive eupneic-like and sigh-like bursts, to preferentially generating sigh-like bursts at an
increased frequency (Fig. 7a). On average, the frequency of fictive sigh-like bursts
increased from 0.027 Hz (± 0.0056 S.D.) to 0.089 Hz (± 0.037 S.D.; p=0.026, paired t-
test of means). This analysis included 29 sigh-like bursts (average of 5.8 sigh bursts per
neuron) before adding oxotremorine, and n=76 fictive sigh-like bursts after adding
oxotremorine (average of 15.2 sigh-like bursts per neuron) over the same duration
(p=0.02, paired t-test of means). This shift was accompanied by a relative
hyperpolarization during the inter-burst interval and a cessation of interburst spiking (Fig.
In addition to the five pacemakers characterized above, there were an additional
two pacemakers (rhythmic in synaptic isolation) which produced bursts that met the
criteria that we have used here to screen for sigh-like bursts (larger amplitude inspiratory
drive, lower frequency, longer duration, and longer post-burst quiescent period). These
two cells were also cadmium-insensitive, but differed from those characterized above in
that the shape of the putative sigh-like bursts was less qualitatively distinct from the
eupneic-like bursts, and in that they were rendered quiescent after application of 20µM
oxotremorine (Fig. 7b).
Following a sigh, a brief post-sigh apnea occurs before eupnea resumes (Fig. 8a). At the
network level, the post-sigh apnea is significantly reduced by blocking N-type calcium
channels, an effect hypothesized to be due to reduction of a IK(Ca2+) current (Lieske and
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Ramirez, 2006b). The delay in fictive eupnea (that had a mean 2.14 cycles ±0.52 S. D.,
n=39 sighs) following a fictive sigh burst, could also (in part) result from synaptic
inhibition of inspiratory neurons immediately following a sigh. Along these lines,
blockade of glycinergic and GABAergic transmission reduced the post-sigh apnea
duration (n=23 sighs, p=0.004, paired T-test of means), but it still occurred and delayed
eupnea by an average of 1.73 cycles (±0.50 SD, n=10 preparations, Fig. 8b). Because
blocking synaptic inhibition did not eliminate the post-sigh apnea, these experiments
suggest that the post-sigh apnea may result in part from an intrinsic membrane property
of inspiratory neurons.
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The respiratory neural network simultaneously generates two distinct rhythms: a
faster, lower-amplitude rhythm and slower larger-amplitude rhythm, corresponding to
fictive eupnea and fictive sighs, respectively. The muscarinic agonist, oxotremorine alters
the timing of these rhythms: fictive sighs speed up, while fictive eupneic activity slows
considerably. This network effect was likely due to M3-muscarinic acetylcholine
receptor activation, as it was antagonized by 4DAMP.
Embedded in the network are pacemakers that can generate two distinct burst
patterns within the same neuron (or between two such neurons coupled by gap junctions).
These bursts patterns include both fast low-amplitude and slow large-amplitude bursts.
In the presence of oxotremorine the timing of these intracellularly generated bursts was
altered in a manner similar to the entire network. Large amplitude bursts accelerate, while
low amplitude bursts are slowed. There were, however, two pacemakers, with dual-burst
patterns which were silenced in oxotremorine. It is unclear whether they are more appropriately
considered to be a different subclass of dual-bursting pacemakers, or single-bursting pacemakers
with a highly variable bursting pattern.
The ability to generate multiple behavioral rhythms is a common property of
many networks, including the respiratory network. The neocortex, for example, generates
rhythms covering diverse frequency bands that can be associated with differentbrain
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states (Steriade et al., 1993a; Chrobak and Buzsaki, 1994; Buzsáki and Draguhn, 2004;
Steriade, 2006). However, unlike fictive eupnea and sigh bursts, these neocortical
rhythms are not known to occur concurrently. Similar phenomena exist in the olfactory
bulb (Kay, 2003; Tabor et al., 2004). Perhaps a more relevant example of a network
capable of generating multiple rhythms is the stomatogastric ganglion (STG) of
crustaceans, where neurons exists that participate in both the gastric and pyloric rhythms
(Prinz et al., 2004).
Analysis of the STG lead to important insights into the cellular mechanisms that
underlie the generation of multiple rhythms (Prinz et al., 2004). STG neurons can switch
between rhythms depending on neuromodulatory influence (Hooper and Moulins, 1989 ;
Meyrand et al., 1991; Weimann and Marder, 1994; Dickinson, 1995; Goaillard et al.,
2004), which is similar to the oxotremorine response of respiratory neurons described
here. Like mammalian respiratory neurons, many STG neurons simultaneously express
multiple network rhythms (Meyrand et al., 1991; Weimann and Marder, 1994). In doing
so, STG neurons are essentially ‘multiplexing’ by expressing two ongoing rhythms
reflecting influencesof different, yet overlapping networks (Heinzel and Selverston,
1988; Weimann et al., 1991; Thuma and Hooper, 2002) that are characterized by distinct
local synaptic connections (Bartos and Nusbaum, 1997; Clemens et al., 1998) and
synapses derived from upstream neuronal networks (Bartos and Nusbaum, 1997; Marder
et al., 1998; Wood et al., 2004). The situation is reminiscent of the respiratory network as
distinct synaptic properties may also govern eupneic and sigh rhythm generation (Lieske
and Ramirez, 2006a; Lieske and Ramirez, 2006b) that can be modulated by upstream
neuronal networks (Alheid et al., 2004). However, in the STG rhythmic multiplicity is
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lost upon synaptic isolation of these neurons. For example the isolated “AB” neuron
functions as a timing oscillator of the faster pyloric rhythm, but not the gastric rhythm
(Weimann and Marder, 1994). The period of the gastric rhythm is an emergent property
that depends on many time constants in many different neurons (Weimann and Marder,
Within the respiratory network we (and others) described two types of pacemaker
neurons (Thoby-Brisson and Ramirez, 2001; Pena et al., 2004; Del Negro et al., 2005),
the timing of which resembles the eupneic rhythm more closely than the very slow sigh
rhythm. Since these pacemakers express a wide range of timing parameters, it is
conceivable that the period of the eupneic rhythm is an emergent property dependent on
intrinsic time constants in many neurons and their synaptic interactions (Feldman and Del
Negro, 2006). Here we report pacemakers that simultaneously generated bursts with
properties resembling those of the slow and large-amplitude sigh rhythm and fast and
small eupneic rhythm. To the best of our knowledge neurons with intrinsic pyloric and
gastric-like bursting properties have not previously been described in the STG. The
overlap between the two stomatogastric rhythms is generated entirely by synaptic
It may seem surprising that in the mammalian respiratory network, fictive eupneic
and sigh rhythms are apparently also reflected in single neuron activity. But, the
respiratory network may not be an exception. Single neocortical neurons can intrinsically
generate beta activity that transforms into gamma oscillations following membrane
depolarization (Steriade et al., 1993b; Gray and McCormick, 1996). This transformation
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is reminiscent of global EEG changes that fluctuate between distinct rhythmic states
during different mental activities (Steriade et al., 1993b; Steriade, 2006). Similar to the
respiratory network, the expression of two neocortical rhythms in the same neuron does
not mean that both rhythms are associated with the same network mechanisms.
Accordingly, distinct synaptic mechanisms shape gamma and beta activity (Steriade,
2006). Eupnea and sighs are also shaped by distinct synaptic network mechanisms
(Lieske and Ramirez, 2006a; Lieske and Ramirez, 2006b). Thus, irrespective of the
finding that single neurons can generate two types of rhythms and amplitude parameters,
network rhythms occurring in a variety of rhythmic frequencies and patterns are typically
the result of different electrophysiological characteristics and distinct connectivity
features. However, this raises the important question: Is the similarity between the
electrophysiological properties of single neurons and the network output just an
While some suggest that respiratory pacemakers play critical roles in respiratory
rhythmogenesis (Feldman and Smith, 1989; Pena et al., 2004; Tryba et al., 2006) others
suggest that eupneic rhythmogenesis is an emergent property of a synaptically coupled
network (Del Negro et al., 2002). Although the present study cannot answer the question
of whether pacemaker neurons are essential for rhythm generation, this study provides an
interesting finding that pacemakers may synchronize multiple network activities through
different types of burst mechanisms. However, it must be emphasized that this
hypothesis does not negate the critical importance of network mechanisms.
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Indeed, the dual-bursting pacemakers described here were cadmium-insensitive. Yet very
low (4µM) concentrations of Cd2+ abolish sighs at the population level (Lieske et al.,
2000). Our present results are therefore in keeping with the hypothesis that the blockade
of fictive sighs by Cd2+ results from a disruption of a synaptic mechanism (Lieske et al.
2006a; Lieske et al. 2006b) rather than an intrinsic cellular mechanism. In that case, the
generation of sighs is likely very sensitive to mechanisms underlying network
Our data suggest that post-sigh apnea is an expected consequence of pacemakers that
trigger activation of an even larger population of neurons than those activated during
fictive eupnea. Post-sigh apnea results in part from activation of inspiratory neurons to
depolarized levels greater than that which occur during eupneic bursts (Lieske et al.,
2000). For a network dependent on the activation of burst mechanisms, this additional
activation would result in an increased refractory time, causing a delay before the
subsequent eupneic burst. Further, the majority of post-sigh apnea probably does not
result from chemical inhibitory synaptic input to inspiratory neurons immediately
following a sigh. Several lines of evidence support these hypotheses. First, most
inspiratory neurons activated during eupnea receive additional depolarization during the
sigh burst and the currents activated as a result of this depolarization reset the eupneic
rhythm giving rise to apnea following the sigh (Lieske et al., 2000). Second, while
inhibitory synaptic inputs appear to contribute to fictive post-sigh apnea (Carley et al.,
1998) (Fig. 8b), GABAergic and glycinergic inhibition are not necessary for it to occur
Page 25 of 51
The eupneic and sigh rhythms are present even in the absence of sensory
feedback. Fictive sighs were generated not only in slices, where they cannot be triggered
by reflexes (Lieske et al., 2000), but also in the highly reduced VRG-island, indicating
that modulatory and pacemaking influences from outside the VRG region are not
essential for generating sighs. Within the VRG, intracellular recordings confirm an
extensive overlap between neurons active during eupnea and sighs (Lieske et al., 2000).
However, we identified a previously undescribed population of respiratory neurons active
during fictive sighs but not during fictive eupnea. The presence of sigh-only neurons,
provide further support for the notion that there are at least two populations of VRG
neurons that increase their activity during the sigh. These two populations include
inspiratory neurons that burst during both eupneic and sigh bursts (Lieske et al., 2000) as
well as sigh-only neurons. Both of these populations are recruited during fictive sighs
and likely contribute to the characteristic increased amplitude of the sigh burst in VRG
population recordings (Lieske et al., 2000). This increase in VRG inspiratory activity
may contribute to the enhanced amplitude of sighs, as compared to eupneic breaths.
Sensory input plays a role in sighing as vagotomy or cutting the carotid sinus nerves,
abolishes sighs for several hours (Glogowska et al., 1972; Cherniack et al., 1981). When
they return, sighs occur at a reduced frequency (Cherniack et al., 1981; Marshall and
Metcalfe, 1988). These data suggest an important role for sensory feedback in
modulating sigh drive. A very similar pattern can also be evoked by brief inflation pulses
(Cherniack et al., 1981). Thus, the central pattern generator for sighs, while capable of
Page 26 of 51
functioning independently, appears to be integrated into a complex network including
both peripheral feedback and descending inputs from other areas in the intact animal.
The present study has contributed to a better understanding of the central component of
the sigh by: (a) identifying a set of pacemaker neurons that generate two distinct bursts
that respond to modulatory input similarly to the fictive eupneic and sigh activity
produced by the network; and (b) identifying a set of neurons that are specifically
activated during sighs; and (c) proposing that intrinsic mechanisms could in part explain
Page 27 of 51
Acknowledgements - Supported by: NIH R01-HL 079294 and Parker B. Francis
Fellowship (to AKT), Conacyt-42870 (to FP), NIH R01-HL NS60120, HL 68860 to
JMR. Authors thank Bert Forster for editing the manuscript prior to submission.
Correspondence should be addressed to:
Andrew K. Tryba, Ph.D.
Medical College of Wisconsin
Department of Physiology
8701 Watertown Plank Road
Milwaukee, WI 53226
FAX: (414) 456-6546
Phone: (414) 456-4975
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Fig. 1 The medullary brainslice preparation containing the pre-Bötzinger complex
(pre-Böt) generates both fictive eupneic and sigh activity. a) Diagram of the
medullary brain-slice that contains the pre-Bötzinger complex (pre-Böt) within the
ventral respiratory group (VRG). The hypoglossal nuclei (XII) are also within the slice.
Extracellular population recordings are made from the slice surface (VRG). The
integrated trace (∫VRG) is dominated by inspiratory neuron activity as it is in-phase with
inspiratory XII motor neuron activation (Tryba et al., 2006). Fictive eupneic bursts and
sigh bursts are indicated in the ∫VRG trace. b) The ∫VRG trace shows both fictive eupnea
(lower amplitude bursts) and lower frequency fictive sighs (large amplitude bursts) that
are followed by a brief fictive apnea, or cessation of eupneic bursts (Lieske et al., 2000).
c) Averaged fictive eupneic bursts (n=16) and a sigh bursts (n=6) from a single
preparation,illustrating that fictive eupnea and bi-phasic, “eupneic-triggered” sigh bursts,
are clearly different in amplitude and duration. d) Note also that the mean (± S.D.)
fictive sigh burst amplitude and duration are larger than fictive eupneic bursts, while sigh
burst frequency is lower than fictive eupneic bursts (n=14 slices). e) The VRG island
preparation was made by cutting away most of the slice and leaving a wedge-shaped
piece containing the VRG. f) Population recordings were made and integrated (∫) . The
∫VRG island activity always included both fictive eupnea and sighs under control
conditions (n=4/4 preparations). Addition of 4µM Cd2+ selectively blocked sighs. g) An
expanded view of averaged ∫VRG records from a VRG island preparation shows both
fictive eupnea (n=13 averaged from a single preparation) and sighs (n=5 averaged from
the same preparation) are similar to those in the more intact slice preparation (Fig. 1c);
Page 30 of 51
similar results were obtained in n=4/4 preparations). h) As was the case in the more
intact brainslice (Fig. 1a), in the VRG-island preparation, the mean (± S.D.) fictive sigh
burst amplitude and duration are larger than fictive eupneic bursts, while sigh burst
frequency is lower than fictive eupneic bursts (n=4 slices)
Page 31 of 51
Fig. 2 A new class of respiratory neurons are represented here, by “sigh-only”
neurons that burst during fictive sighs (not eupnea); interestingly, no “sigh-only”
pacemakers have been discovered. ai) Extracellular (ETC) recording (top) of a “sigh-
only” neuron, active during fictive sigh bursts (∫VRG), but not fictive eupneic bursts.
aii) Subsequent intracellular (ITC) whole-cell patch recordings of sigh only neurons
revealed they had similar firing patterns during network (∫VRG) activity as when
recorded extracellularly (same cell as in Fig. 2ai). bi) A sigh-only neuron that is tonically
active between fictive eupneic population burst, but inhibited during fictive eupnea. bii)
None of the sigh-only neurons tested were rhythmically active, or showed voltage-
dependent bursting properties upon depolarizing current injection (bottom traces)
following blockade of chemical synaptic transmission, with CNQX, CPP, bicucculline
and strychnine (same cell as in 2bi).
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Fig. 3 Bath applied oxotremorine modulates the respiratory network population
activity (∫ ∫VRG) from generating both fictive eupnea and sighs to preferentially
generating fictive sigh bursting. 3a) Bath-applied oxotremorine (20µM) suppressed in-
vitro fictive eupneic bursts and triggered fictive sighs at an increased frequency (∫VRG,
top). Note the altered pattern of VRG activity in the raw population record (bottom).
Boxed areas are expanded below in 3b-3d) and combined with whole cell current-clamp
records (top), to reveal that oxotremorine alters cellular and population activity.
Page 33 of 51
Fig. 4 Oxotremorine suppresses fictive eupneic bursts while increasing fictive sigh
bursting recorded from the in vitro slice preparation and in situ working heart
brainstem preparation. a) ∫VRG burst histogram reveals in vitro application of
oxotremorine (20µM) suppresses eupneic bursting frequency (p≤0.001) while fictive sigh
frequency increases (p=0.004; n= 10 slice preparations). For both fictive eupnea and
sighs, frequency changes were compared using Wilcoxon signed rank test at indicated (*)
times before (t= -0.5-0 mins) or after (t= 4.5-5.0mins) adding oxotremorine, when the
effect of oxotremorine plateaus (Fig. 4a). The effect of oxotremorine application was
blocked by 4DAMP as shown in Fig. 4b) Oxotremorine (10µM) significantly suppressed
fictive eupnea (Fig. 4b, p≤0.001, Wilcoxon signed rank test) and enhanced fictive sighing
frequency (Fig. 4c, p=0.004, Wilcoxon signed rank test) (n=6 slices, black bars). Bath
applying the M3-preferring mAChR antagonist, 4DAMP (1µM) alone for 10 minutes had
no significant effect on fictive eupneic (Fig. 4b, hashed bar) or fictive sigh bursting
frequencies (Fig. 4c, n=6, hashed bars, p=0.18 eupneic, p=0.53 sighs, Wilcoxon signed
rank test). Fig. 4b-4c) Pre-incubation in 4DAMP (for 10 minutes) prevented an expected
oxotremorine induced suppression of fictive eupnea or enhanced fictive sigh bursting
frequency typically observed following addition of 10µM oxotremorine (white bars,
p=0.52 eupneic, p=0.63 sigh, Wilcoxon signed rank test; n=4/4 slices). 4d-4f) In the in
situ working heart brainstem preparation, oxotremorine also suppresses fictive eupneic
bursts, measured at the phrenic nerve, while increasing fictive sigh bursting. 4d)
histogram showing oxotremorine (10µM) perfused through the working heart brainstem
preparation suppressed fictive eupneic frequency (p=0.02) while fictive sigh bursts
Page 34 of 51
increased. (p=0.029)(n=4 preparations, Student’s paired t-test). For both fictive eupnea
and sighs, frequency changes were compared using student’s T-test at before (t= -0.5-0
mins) or after (t= 5.5-6.0mins) adding oxotremorine, when the effect of oxotremorine
was maximal. 4e-4f) As in vitro, fictive eupnea and sighs are distinguishable in the in
situ preparation, as fictive eupneic bursts are smaller in amplitude, shorter in duration and
occur at a faster frequency than eupneic bursts. Astericks in Fig. 4e denote fictive sigh
Page 35 of 51
Fig. 5 A subset of inspiratory neurons have endogenous voltage-dependent
pacemaker bursting properties. a) Inspiratory neurons burst in-phase with fictive
eupneic and sigh VRG activities and, as in this case, b) a subset of inspiratory neurons
continue to generate voltage-dependent bursting properties, following blockade of
ionotropic receptors with CNQX, CPP, bicucculline and strychnine and loss of network (
∫VRG) rhythmic bursting. Typically, the bursting properties of synaptically isolated
inspiratory pacemakers does not include fictive sigh-like bursts (top panel). Bottom
panel is an expanded view of boxed area shown in the top panel. c) Inspiratory
pacemakers retain voltage-dependent bursting properties following synaptic isolation.
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Fig. 6 Following isolation from ionotropic chemical transmission, some inspiratory
pacemakers continue to generate two different types of bursts at two different
frequencies. a) A subset of previously undescribed isolated pacemakers generate both
shorter, higher frequency bursts and longer bursts at a lower frequency (starred (*)
bursts). Synaptic transmission was blocked with CNQX, CPP, bicuculline and
strychnine, yet this neuron continued to generate both faster bursts and larger slower
bursts (starred), even following (Fig. 6b) subsequent addition of cadmium, that is a non-
specific blocker of calcium mediated synaptic transmission. Note (bottom trace in Fig.
6b) the starred, lower frequency, longer duration bursts are characteristically sigh-like, in
that it is bi-phasic and followed by a phase-reset of shorter, faster bursts.
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Fig. 7 Application of oxotremorine to a subset of synaptically isolated pacemakers
suppressed faster eupneic-like burst activity and triggered large sigh-like bursting
activity. a) As was seen at the network level, oxotremorine application (20µM) also
triggered sigh-like bursts and suppressed shorter eupneic-like bursting activity in some
isolated pacemakers. Shorter eupneic bursts returned with continued application (boxed
area). Lower trace is expansion of area boxed in top trace. Note that sigh-like bursting
activity resulted in a phase-reset of the eupneic-like bursts. Ten minutes prior to adding
oxotremorine, flufenamic acid (FFA, 500µM) was added; FFA-insensitive pacemakers
are typically cadmium-insensitive ( as previously shown (Pena et al., 2004)). b) Not all
cadmium-insensitive pacemakers that intrinsically generated differential bursting
patterns, following synaptic isolation (top traces), continued to generate bursting with
subsequent oxotremorine application (bottom traces, n=2).
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Fig. 8 Intrinsic membrane properties may explain post-sigh apnea. a) Diagram of
time reference points used for definition of delay in fictive eupneic burst period following
a fictive sigh burst. b) Mean delay (± S.D. bars) in fictive eupneic cycle period
following a sigh burst in control saline (sigh) or following bath application of bicuculline
(20µM) and strychnine (1µM) (sigh in Bic + Stry). Cycle periods were determined from
population (∫VRG) records from n=4 preparations.
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