a key unanswered question. We performed whole-cell recordings in the preBo ¨tzinger Complex in slice preparations from neonatal
found Ca2?current (ICa)-dependent bursting in 7.5% of inspiratory neurons in P8–P10 slices, but in P0–P5 slices these cells were
exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 ?M Cd2?or flufenamic acid (FFA) (10–200 ?M),
or coapplication of 20 ?M RIL ? FFA (100–200 ?M) stops the respiratory rhythm, but that adding 2 ?M substance P restarts it. We
by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the
Behaviors like breathing, walking, and eating emanate from cen-
Pacemaker neurons may play a central role in generating these
lian respiratory network.
be examined in neonatal rodent slice preparations that generate
respiratory-related motor output in vitro, where the critical neu-
rons are localized within the preBo ¨tzinger Complex (preBo ¨tC)
(Smith et al., 1991). This rhythm continues after blocking
postsynaptic inhibition, which rules out mechanisms based on
man and Smith, 1989). The pacemaker hypothesis posits that a
kernel of neurons with intrinsic bursting properties are essential
to generate synchronized rhythmic activity that is distributed to
al., 1991, 2000; Rekling and Feldman, 1998; Butera et al., 1999;
Koshiya and Smith, 1999; Del Negro et al., 2001).
If preBo ¨tC pacemaker neurons are essential for rhythmogen-
esis in slices, then removing them from the network should dis-
Johnson et al., 1994; Del Negro et al., 2001, 2002b; Rybak et al.,
2003) and another that depends on Ca2?currents (ICa) and
Ca2?-activated nonspecific cationic current (ICAN) (Thoby-
Brisson and Ramirez, 2001; Pena et al., 2004). Here, we pharma-
cologically abolish INaP-mediated pacemaker activity, and
ratory rhythm persists (with unchanged frequency) in their ap-
parent absence. We also show that pharmacological attenuation
that enhancing neural excitability restarts it. We conclude that
respiratory rhythm in slices is not pacemaker driven but rather
emerges when neurons are sufficiently excitable and there is re-
neurons play in normal rhythmogenesis in intact rodents
(Gray et al., 2001), we suggest that this is also the case in intact
446 • TheJournalofNeuroscience,January12,2005 • 25(2):446–453
Dawley rats (P0–P10) for experiments in vitro. The Office for the Pro-
tection of Research Subjects (University of California Animal Research
College of William and Mary) approved all protocols. Transverse slices
(550 ?m thick) containing the preBo ¨tC (Smith et al., 1991) and hypo-
glossal (XII) motoneurons were dissected in normal artificial CSF
(ACSF) containing the following (in mM): 124 NaCl, 3 KCl, 1.5 CaCl2, 1
MgSO4, 25 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, equilibrated with
mM, and respiratory motor output was recorded from XII nerve roots
using suction electrodes and a differential amplifier (bandpass filtered at
0.3–1 kHz, full wave-rectified, and smoothed for display).
Whole-cell patch electrodes (3–6 M?) contained the following (in
mM): 140 CsMeSO4, 5 NaCl, 0.1 EGTA, 10 HEPES, 2 Mg ATP, and 0.3
Na3GTP, pH 7.25, for voltage-clamp experiments or 140 K gluconate, 1
GTP, pH 7.25, for current clamp. Pipettes were visually guided to the
preBo ¨tC using video microscopy. The z-axis of a Sutter Instruments
(Novato, CA) MP-285 robotic micromanipulator was set to 0 ?m at the
modified ACSF was used in voltage-clamp experiments to isolate Na?
0.5 CaCl2, 2 MgCl2, 25 NaHCO3, and 30 D-glucose. Descending voltage
ramps from ?10 to –90 mV (20 mV/s) were used to measure noninacti-
voltage (I–V) relationship generated by the voltage ramp in the linear
region negative to –60 mV. Cell capacitance (CM) was computed from
was evoked by 15 ms hyperpolarizing voltage step commands (?VC),
using the formula CM? ?IC/?VC. Series (access) resistance (RS) was
acceptable voltage clamp requires RN? 10 * RS. Cells that failed to meet
this criterion were discarded. We applied analog RScompensation with-
out whole-cell capacity compensation to continuously monitor and en-
sure stationary voltage-clamp conditions.
We bath-applied the following drugs obtained from Sigma (St. Louis,
MO) (in ?M): 1–200 riluzole (RIL) (2-amino-6-trifluoromethoxy
benzothiazole), 10–200 flufenamic acid (FFA), 10 6-cyano-7-
nitroquinoxaline-2,3-dione (CNQX), 20 DL-2-amino-5-phosphono-
pentanoic acid (APV), 2–5 bicuculline (BIC) or picrotoxin (PTX), 2–5
strychnine (STR), 0.5–2 substance P (SP), and 0.5 AMPA. In some ex-
from the ACSF to avoid precipitation.
We monitored the effects of RIL on respiratory motor output by plot-
ting the period and amplitude of XII discharge on a cycle-to-cycle basis
(see Fig. 3). We then divided each experiment into contiguous 2 min
segments and computed the mean period during each segment. The
reported in Figure 5.
In control experiments, we found that RIL had very similar effects on
neonatal rats and mice during P0–P5. RIL at 1–200 ?M caused the same
period, regardless of species (n ? 10 mice; n ? 8 rats). RIL at 10–20 ?M
attenuated INaPto the same extent in voltage clamp (n ? 6 mice; n ? 5
rats). Therefore, we pooled systems-level data and INaPvoltage-clamp
experiments (see Figs. 3–5). RIL-sensitive pacemaker neurons were
found in both rats (n ? 46) and mice (n ? 14); this database contains
neurons from previous studies (Del Negro et al., 2002a). Pacemaker
with the first published report of Cd2?-sensitive pacemaker neurons in
juvenile mice (Thoby-Brisson and Ramirez, 2001).
We used the Wilcoxon signed-rank test to assess the effects of RIL on
respiratory frequency (see Fig. 5), and we used the Fisher exact test to
assess the relative prevalence of pacemaker neurons in rodent slices of
different age ranges (see Results for details). We applied the Wilcoxon
signed rank test and the paired t test to evaluate the cellular and network
effects of 20 nM TTX (see Fig. 6), which yielded the same results. We
tested whether RIL caused significant respiratory period fluctuations
(before cessation) using the Kolmogorov–Smirnov test (K–S test). Sig-
nificance was set at p ? 0.05.
INaP-mediated pacemaker neurons
Figure 1 depicts typical INaP-mediated bursting in a mouse pre-
Negro et al., 2002a,b). Here, we used patch solution containing
0.1 mM EGTA. Respiratory drive potentials occurred at mem-
(Rybak et al., 2003). Depolarization evoked INaPand caused ec-
topic bursts in the intervals between XII discharges (Fig. 1A,
middle). Intrinsic bursting was confirmed by its persistence after
blocking synaptic transmission with CNQX, APV, BIC, and STR
in either 9 mM (Fig. 1A, right) or 3 mM (Fig. 1B) K?concentra-
tion in the ACSF. Bursting was voltage dependent. Depolarizing
the baseline membrane potential using bias current (Ia) caused
the cell to move from quiescence to bursting, and further depo-
larization increased burst frequency (Fig. 1B). However, 20 ?M
RIL blocked INaP-mediated bursting within 90 s, and no amount
of depolarizing Iacould restore it (Fig. 1C). All rhythmically ac-
tive preBo ¨tC neurons with stable baseline membrane potentials
and overshooting spikes were tested for pacemaker activity and
are included in our database. INaP-mediated bursting was de-
tected in eight of 178 P0–P5 preBo ¨tC neurons (4.5%) and in
three of 54 P8–P10 preBo ¨tC neurons (5.6%), which was not
significantly different (Fisher exact test, p ? 0.3).
mV baseline membrane potential (VM). ACSF contained 9 mM [K?]. Ectopic bursts emerged
DelNegroetal.•PacemakerNeuronsandRespiratoryRhythmJ.Neurosci.,January12,2005 • 25(2):446–453 • 447
Calcium current-dependent pacemaker neurons
Bursting in preBo ¨tC neurons in mice ?P5 that depends on ICais
a recent discovery (Thoby-Brisson and Ramirez, 2001). In the
past, we never observed this phenotype in P0–P5 rats or mice
(Del Negro et al., 2002a), so here we recorded in juvenile mice
ages P8–P10 to see whether developmental effects could explain
this disparity. After identifying a pacemaker neuron in CNQX,
APV, PTX, and STR (Fig. 2A), we applied Cd2?to test whether
ICawas required. We found four ICa-dependent pacemaker neu-
rons in 54 neurons sampled (7.5%) during P8–P10, whereas we
sampled during P0–P5 (0.6%), which indicates significantly
higher prevalence of bursting cells in the older neonates (Fisher
exact test, p ? 0.01). Of the 54 preBo ¨tC neurons recorded at
P8–P10, 40 were recorded with 10 mM EGTA patch solution
(Thoby-Brisson and Ramirez, 2001) and 14 with 0.1 mM EGTA
patch solution. ICa-dependent bursting neurons in these older
neonates made up 7.5% of the sample regardless of EGTA con-
centration (n ? 3 of 40 and 1 of 14, respectively), which suggests
that slow calcium buffering by 10 mM EGTA does not influence
pacemaker activity (Fisher exact test, p ? 0.43). The ICa-
dependent pacemaker neurons often showed ramp-like trajecto-
ries during the interburst interval, i.e., expiration, and a strong
inspiratory discharge that was enhanced by depolarization (Fig.
2A). Bursting continued in CNQX and RIL but ceased in Cd2?
(Fig. 2A,D), which demonstrates dependence on ICa. Bursting
was also blocked by 10 ?M FFA (Fig. 2B), which suggests that a
Ca2?-activated nonspecific cationic current (ICAN) (Gogelein et
al., 1990; Guinamard et al., 2004) carries significant burst-
generating inward current in addition to ICa.
ICa-dependent bursting was extremely rare in P0–P5 mice (1
we blocked bursting in this one neuron using 200 ?M FFA and
by 2–3 mV and thus suggests that this neuron expressed INaPin
addition to ICAN.
Unlike INaP-mediated bursting, ICa-dependent bursting was
not voltage dependent. For example, the pacemaker neuron in
Figure 2C was subjected to increasing levels of Iabut maintained
a ?4 s burst period (Fig. 2E, filled circles). Figure 2E further
compares this cell with INaP-mediated voltage-dependent burst-
number of action potentials per burst. For example, depolariza-
in Figure 2A (in the context of network activity) and from 4 to 7
in Figure 2C (in the isolated pacemaker neuron after CNQX,
APV, PTX, and STR).
RIL applicationsin vitro
We applied RIL for 30 min and monitored respiratory motor
output in P0–P5 slices from rats and mice (Figs. 3-5). RIL at 10
?M reduced XII amplitude (Fig. 3, black triangles) but did not
iments, Wilcoxon test, p ? 0.6). RIL at 10 ?M also caused cycle-
to-cycle variability in the period that was noticeable but not sta-
tistically significant (K–S test, p ? 0.08).
RIL at 20 ?M decreased XII amplitude monotonically and
caused periodic fluctuation but did not change the mean period
until after the XII amplitude decreased to baseline, which oc-
curred at 40 min. Afterward, the period rapidly increased (Fig. 3,
note the upward thrust of gray circles in the row labeled 20 ?M)
and within a dozen cycles rhythmic output ceased altogether.
Similar results were obtained at RIL concentrations from 50 to
period, which always coincided with XII amplitude reaching
barely detectable levels (Fig. 3). Before the abrupt cessation of
rhythm, the mean period for n ? 3 experiments at each RIL
concentration was not significantly different from control (Wil-
coxon tests, all p ? 0.05). RIL consistently caused periodic fluc-
tuations, which were statistically significant at 50 and 200 ?M
(K–S test, p ? 0.01).
put. The mean time required to abolish XII output decreased
9 min (Fig. 4) (Del Negro et al., 2002a). This suggests that either
experiment, but here the bursting was blocked by 10 ?M FFA. C, Bursting behavior in CNQX,
in C (circles) and the voltage-dependent INaPpacemaker neuron from Figure 1B (squares),
448 • J.Neurosci.,January12,2005 • 25(2):446–453DelNegroetal.•PacemakerNeuronsandRespiratoryRhythm
affected rhythmogenic neurons only at high doses (or in pro-
mice at various depths. We previously reported rapid blockade
deep and applied descending voltage ramps that inactivate the
fast Na?current responsible for action potentials (but do not
rons that satisfied our criteria for adequate voltage clamp, a re-
gion of negative slope was always present in the steady-state I–V
curve because of INaP(Del Negro et al., 2002b). RIL always
I–V curve was monotonic with positive slope between –60 and
–40 mV. Under these conditions, voltage-dependent INaPburst-
ing is impossible (Smith et al., 1975). In three cells, the time
course of INaPblockade could be fitted by an exponential decay
the mean respiratory period computed from three slice experi-
ments like Figure 3, which shows that the mean respiratory
RIL to block INaPat depths ?250 ?m.
Low doses of TTX
Low doses of TTX preferentially antagonize INaPin respiratory
neurons (Koizumi and Smith, 2003). We found that 20 nM TTX
reversibly abolished respiratory rhythm in 20–40 min in 15
to five respiratory bursts showed that XII amplitude decreased
6C). TTX at 20 nM hyperpolarized respiratory neurons by 5–10
tions of RIL plotted from top to bottom. Respiratory period (gray circles) and XII amplitude
from 0–24 s (left ordinate), and XII amplitude is scaled uniformly (right ordinate) for each
DelNegroetal.•PacemakerNeuronsandRespiratoryRhythmJ.Neurosci.,January12,2005 • 25(2):446–453 • 449
determine whether 20 nM TTX also af-
fected spike generation, we applied 20 ms
step currents from a baseline membrane
potential of –60 mV to evoke action po-
tentials (during the expiratory phase).
Rheobase increased from 277 ? 177 pA in
exposure to 20 nM TTX) ( p ? 0.001; n ?
TTX. Changes in rheobase required more
passive charging of the membrane poten-
tial to reach spike threshold, and this pas-
tude in TTX. Therefore, we defined the
active fraction of the evoked spike as the
difference between the maximum spike
amplitude and the peak of the passive
membrane response. Active fraction de-
nM TTX were statistically significant by Wilcoxon signed rank or
paired t tests and were reversible in washout (Fig. 6C). In one
preparation, 20 nM TTX completely abolished spikes (data not
Because 20 nM TTX hyperpolarized preBo ¨tC neurons, in-
creased rheobase and decreased spike amplitude, we posited that
TTX-induced decreases in excitability might be responsible for
the excitatory neuropeptide SP (0.5–2 ?M). SP revived the
rhythm in SP and TTX conditions was stable for 5–10 min and
then gradually became unstable and ceased after 40–60 min ex-
posure to TTX. Respiratory rhythm recovered fully in washout
FFA and RIL coapplication experiments
Although ICa-dependent pacemaker neurons are extremely
sparse in P0–P5 mice, one could argue that during bouts of RIL
(e.g., Figure 3), a small number of RIL-insensitive pacemaker
neurons sustains the rhythm (Pena et al., 2004). We tested this
possibility in FFA and RIL coapplication experiments. In P0–P5
mouse slices, first we applied 100 ?M FFA, which did not affect
XII frequency or amplitude (Fig. 8A,B) but at the cellular level
reversibly reduced (but did not abolish) respiratory drive poten-
tials and spike amplitude (Fig. 8B). After washout, we coapplied
100 ?M FFA and 20 ?M RIL, which blocked respiratory rhythm
FFA and RIL, adding 0.5–2 ?M SP to the bath restored rhythmic
activity: XII output resembled the first FFA washout, but respi-
ratory drive potentials were significantly attenuated (n ? 5
slices). We were also able to rescue the rhythm after it ceased in
100 ?M FFA and 20 ?M RIL using low concentrations of bath-
applied AMPA (500 nM; n ? 2).
Conditions that eliminated bursting in INaP- and ICa-dependent
pacemaker neurons did not prevent respiratory rhythm genera-
ability restores it. We propose that INaPand ICANnormally
enhance excitability and promote inspiratory bursts in all pre-
Bo ¨tC respiratory neurons, regardless of whether or not a small
subset of these neurons (which express INaPand ICANabun-
dantly) support intrinsic bursting.
at low doses (EC50? 3 ?M), whereas it abolishes respiratory
is ?10 ?M (Del Negro et al., 2002a). RIL caused periodic fluctu-
ation at all concentrations, thus INaPand pacemaker neurons
probably help stabilize network rhythmicity. The time to block
35 min for XII discharge to disappear), which is an order of
magnitude higher than the EC50value of 3 ?M to block INaP
within 9 min in neurons recorded at depths up to 350 ?m in
slices. The disparity in dose and time dependence suggests that
the effects of RIL that abolish respiratory rhythm include more
than just blocking INaP.
INaP-mediated bursting in preBo ¨tC neurons does not appear
Pena et al. (2004) reported 16–29% INaPpacemaker neurons in
rons that exhibited pacemaker-like characteristics in the cell-
that discharge many spikes per XII cycle and more frequently
exhibit pacemaker properties after application of CNQX and
other blockers, which significantly overestimates the prevalence
the likelihood of finding pacemaker neurons of any type in a
given slice was ?10%, then the 16–29% figure cannot be repre-
sentative of their actual relative prevalence. In contrast, we sam-
pled preBo ¨tC neurons without regard to activity pattern in cell-
attached mode. All rhythmic neurons were tested for pacemaker
properties in the presence of CNQX, APV, BIC/PTX, and STR.
We avoided using low Ca2?solution to block synaptic transmis-
and can induce silent cells to burst (Pena et al., 2004). Therefore,
450 • J.Neurosci.,January12,2005 • 25(2):446–453DelNegroetal.•PacemakerNeuronsandRespiratoryRhythm
we estimate that the actual fraction of INaPpacemaker neurons in
the preBo ¨tC is closer to 5%.
Calcium-dependent pacemaker neurons
We detected only one ICa-dependent pacemaker neuron of 178
time at P8–P10, which is significantly different. This suggests that
either ICa(Onimaru et al., 1996, 2003; Elsen and Ramirez, 1998) or
ICANare developmentally regulated and that Cd2?-sensitive burst-
in numbers by an order of magnitude after P6. Despite differences
in sampling protocols, these two reports estimate the fraction of
ICANpacemaker neurons to be between 7.5 and ?9% in juvenile
ICais necessary for bursting in these neurons because Cd2?
blocks pacemaker activity (Thoby-Brisson and Ramirez, 2001).
However, the voltage-independent nature of the bursting suggests
that Ca2?activates ICAN, which dominates the burst-generating
mechanism. TRPM4- or TRPM5-like ion channels engender
ICANand are blocked by 30–100 ?M FFA (Teulon, 2000; Launay
et al., 2002; Montell et al., 2002; Guinamard et al., 2004;
Moran et al., 2004). We abolished bursting with 10–200 ?M
FFA, which is consistent with a role for TRPM4/5-mediated
ICAN(Schiller, 2004). FFA also blocks gap junctions (Srinivas
and Spray, 2003; Ye et al., 2003), enhances K?currents
(Stumpff et al., 2001), with EC50? 50 ?m so that doses ?100
?M could have side effects that impact respiratory network
activity (Pena et al., 2004).
Regardless of age, ICa-dependent pacemaker neurons showed
very strong inspiratory bursts in control and FFA always attenu-
ated drive potentials (Figs. 2A, 8B). These data suggest that ICAN
contributes significantly to inspiratory bursts even at young ages
when the actual fraction of cells with ICANpacemaker properties
is extremely low.
Low doses of TTX depress excitability in preBo ¨tC neurons
Low doses of TTX simultaneously diminished the frequency and
XII amplitude. TTX (20 nM) hyperpolarized preBo ¨tC neurons,
increased spike threshold and rheobase, and significantly dimin-
ronal excitability and could ultimately cause the cessation of re-
spiratory rhythm independent of any direct effects on bursting
properties. Unlike TTX, RIL does not significantly alter spike
threshold or rheobase at concentrations that block INaP(Del Ne-
gro et al., 2002a).
Despite the general depression of excitability caused by 20 nM
TTX, SP added to the bath restarted respiratory rhythm in most
linear Na?current, but SP does not induce a region of negative
Bo ¨tC neurons (Gray et al., 1999; Pena and Ramirez, 2004). Here,
SP was applied in the presence of TTX. Therefore, spiking prop-
erties and INaPwould remain attenuated, and we conclude that
for restarting rhythmic activity, and not
the restoration of pacemaker properties.
Rhythm generation in the presence of
FFA and RIL
Similar to 20 nM TTX, coapplication of
RIL and FFA suppressed respiratory
rhythm. Again, the rhythm could be res-
cued with SP or low concentrations of
AMPA, which depolarize cells and boost
excitability but do not directly cause pace-
maker properties (Gray et al., 1999; Pena
and Ramirez, 2004). SP presumably com-
pensates for the hyperpolarization caused
that the ability ofINaPandICANto give rise
to bursting-pacemaker activity in a subset
of isolated neurons is not essential for
rhythmogenesis. Rather, we conclude that
normally INaPand ICANcontribute to
rhythmogenesis by enhancing the general
excitability of the network and promoting
inspiratory burst generation in all respira-
tory preBo ¨tC neurons without requiring
bursting pacemaker activity.
applied to restore baseline VMto ?60 mV (20 nM TTX, 5 min). Additional depolarizing Iawas
required to maintain VMof –60 mV by the time TTX abolished rhythmic activity (20 nM TTX, 15
Rhythm generation in the presence of 100 ?M FFA and 20 ?M RIL. A, Continuous segments of the experiment
DelNegroetal.•PacemakerNeuronsandRespiratoryRhythm J.Neurosci.,January12,2005 • 25(2):446–453 • 451
Emergent network properties
Our data suggest that pacemaker neurons per se are not essential
to generate the respiratory rhythm, but that INaPand ICANcon-
emergent network mechanism of respiratory rhythmogenesis
such as the group pacemaker (Rekling et al., 1996a,b, 2000; Rek-
ling and Feldman, 1998) in which pacemaker neurons can be
embedded but are not obligatory for rhythm generation. We
posit that recurrent synaptic excitation in preBo ¨tC neurons trig-
excitation leads to a lot of neuronal output). INaPand ICANare
present in cells without pacemaker activity and serve to boost
membrane potential from baseline to suprathreshold levels in
response to synaptic activity. Recurrent excitation causes cas-
of cells are not pacemakers because they do not burst unless
synaptically activated. In the absence of INaPand ICAN, we con-
clude that a compensatory boost in excitability depolarizes con-
stituent neurons closer to spike threshold so that excitatory syn-
aptic drive can still evoke the inspiratory burst without the
amplification normally provided by INaPand ICAN.
We propose that inspiratory burst termination results from a
(Del Negro et al., 2002b; Rybak et al., 2003), ICANdeactivation,
recruitment of calcium-dependent potassium channels, or elec-
trogenic ion pumps (Ballerini et al., 1997; Darbon et al., 2003).
These cellular processes can activate based on Na?and Ca2?
accumulation during inspiration. Burst termination does not re-
quire coupling, whereas network burst initiation is impossible
without the excitatory coupling (Rekling et al., 1996b; Rekling
and Feldman, 1998).
Finally, neurons rostral to the preBo ¨tC may contribute to
rhythm generation (Mellen et al., 2003; Onimaru and Homma,
Because these neurons are not contained in slices, we cannot
predict how such cells might contribute to the final pattern of
respiratory activity when the preBo ¨tC is embedded in the more
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