offset the secondary hypoxic depression of breathing. In this study, we used rhythmically active medullary slices from neonatal rat to
map,inrelationtoanatomicalandmolecularmarkersofthepre-Bo ¨tzingercomplex(preBo ¨tC)(aproposedsiteofrhythmgeneration),the
three-dimensional grid within the VLM revealed a “hotspot” where ATP (0.1 mM) evoked a rapid 2.2 ? 0.1-fold increase in inspiratory
P) or DAMGO ([D-Ala2,N-MePhe4,Gly-ol5]-enkephalin), respectively. The relative potency of P2R agonists [2MeSADP (2-
methylthioadenosine 5?-diphosphate) ? 2MeSATP (2-methylthioadenosine 5?-triphosphate) ? ATP?s (adenosine 5?-[?-
ATP response by MRS2179 (2?-deoxy-N6-methyladenosine-3?,5?-bisphosphate) (P2Y1antagonist) indicate that the excitation is medi-
a neurotransmitter that activates ionotropic (P2X1–7) and
metabotropic (P2Y1,2,4,6,11–14) purinergic receptors (P2Rs) (Illes
and Alexandre Ribeiro, 2004). The widespread expression of
P2Rs in the nervous system (Collo et al., 1996; Kanjhan et al.,
1999; Norenberg and Illes, 2000; Yao et al., 2000) and extensive
electrophysiological evidence (Norenberg and Illes, 2000; Hussl
functions for P2R signaling. Recent discoveries have focused at-
tention on the role of P2Rs in respiratory control. First, P2Rs are
expressed in respiratory regions of the PNS and CNS, including
the carotid body, petrosal ganglion (Prasad et al., 2001; Rong et
et al., 2000; Thomas et al., 2001), and respiratory motoneurons
(Funk et al., 1997a; Kanjhan et al., 1999; Miles et al., 2002). Sec-
ond, P2X2Rs in the carotid body are necessary for the hypoxic
data have revealed that ATP is released from discrete regions in
ratory responses (Gourine et al., 2005a,b).
The underlying mechanisms are not known, but distinct spa-
tiotemporal patterns of ATP release in hypercapnia and hypoxia
produce diverse actions. ATP release from the ventral medullary
surface is common to hypoxia and hypercapnia, but ATP release
from the ventral respiratory column (VRC) occurs in hypoxia
only (Gourine et al., 2005a,b). In addition, ATP release in hyp-
initial increase in respiratory frequency when it attenuates the
secondary hypoxic depression of ventilation (Gourine et al.,
ATP is released within the VRC only during hypoxia (Gourine et
al., 2005a), we hypothesize that ATP acts directly in the VRC to
stimulate frequency. Few data address this question. In the adult
rat in vivo, ATP increases the discharge of brainstem respiratory
neurons (Thomas and Spyer, 2000) but blocks phrenic nerve
output causing apnea (Spyer and Thomas, 2000; Gourine et al.,
2005b). In the neonatal rat in vitro, ATP increases respiratory
frequency (Lorier et al., 2002, 2004). However, comparison is
difficult because the site(s) of drug action were poorly defined,
Correspondence should be addressed to Dr. Gregory D. Funk, 7-50 Medical Sciences Building, Department of
TheJournalofNeuroscience,January31,2007 • 27(5):993–1005 • 993
and the experimental conditions and models (adults in vivo vs
neonates in vitro) differed.
The objectives of this study are to explore mechanisms by
which ATP modulates respiratory rhythm. We will (1) map, in
relation to anatomical and molecular markers of medullary re-
spiratory nuclei, the effects of ATP on respiratory rhythm and
identify the site where ATP maximally increases frequency (ATP
“hotspot”); (2) test whether the ATP hotspot corresponds to the
pre-Bo ¨tzingercomplex(preBo ¨tC)(aproposedsiteofinspiratory
rhythm generation); and (3) identify the P2R subtype(s) respon-
sible for the ATP-mediated increase in respiratory frequency.
Rhythmically active medullary slice. Rhythmically active medullary slices
(n ? 145) were obtained from neonatal [postnatal day 0 (P0) to P4]
Wistar rats as described previously (Smith et al., 1991; Funk et al., 1993,
isoflurane and decerebrated. The brainstem–spinal cord was then iso-
lated in cold artificial CSF (aCSF) containing the following (in mM): 120
NaCl, 3 KCl, 1.0 CaCl2, 2.0 MgSO4, 26 NaHCO3, 1.25 NaH2PO4, 20
D-glucose, equilibrated with 95% O2/5% CO2. The brainstem–spinal
in the rostral-to-caudal direction using a vibratome (Pelco-101; Ted
luminated to identify anatomical landmarks. A single 700 ?m slice was
evident and the rostral margin of the inferior olive first appeared in the
gin described above to the obex caudally and contained the preBo ¨tC,
rVRG (rostral ventral respiratory group), most of the XII motor nuclei,
and the rostral XII nerve rootlets. Slices were pinned with the rostral
surface up on Sylgard resin in a recording chamber (volume, 5 or 10 ml)
and perfused with aCSF that was recirculated at a flow rate of 15–20
to 9 mM at least 30 min before the start of data collection (Funk et al.,
1993). Elevated [K?]eis not a necessary condition for rhythm genera-
tion, despite a common misconception to the contrary. Medullary slices
from neonatal Wistar rats that are 700 ?m thick generate stable respira-
tory rhythm in 3 mM [K?]efor 2 h on average, after which rhythm
gradually slows over the next 2 h and then ceases (Ruangkittisakul et al.,
interventions, and therefore required slices that produced stable
inspiratory-related rhythm for extended periods (i.e., ?5 h). Therefore,
the [K?]ewas raised from 3 to 9 mM. It has been hypothesized that the
rundown of rhythmic activity in medullary slices is attributable to the
that normally maintains the excitability of a critical population of neu-
rons at a level necessary for long-term rhythm generation (Smith et al.,
1991). Elevated [K?]eis proposed to compensate for the loss of this
excitatory/modulatory input (for additional discussions, see Funk et al.,
1993; Ruangkittisakul et al., 2006).
All experiments and procedures were approved by the University of
Auckland and/or University of Alberta Animal Ethics Committees and
treatment of experimental animals.
XII (hypoglossal) nerve rootlets and directly from the ventrolateral sur-
face of the slice. Surface field potentials were recorded using a four-axis
manual manipulator to place a suction electrode (120 ?m inside diame-
ter) on the surface of the slice over the approximate region of the ventral
respiratory cell column (Telgkamp and Ramirez, 1999). The pipette was
systematically moved in steps corresponding to one-half of the pipette
diameter until the most robust signal was detected. Signals were ampli-
fied, bandpass filtered (100 Hz to 5 kHz), full-wave rectified, integrated
9.0 (Molecular Devices, Union City, CA). Data were recorded on video-
Rebersburg, PA) or saved to computer using a Digidata 1322 A/D board
and AxoScope software (Molecular Devices) for off-line analysis. All
recordings were conducted at room temperature (22–24°C).
Drugs and their application. Stock solutions of drugs were made in
standard aCSF and frozen in aliquots unless otherwise stated. The final
concentration of K?in the drug solutions was matched to that of the
The following drugs were used: adenosine 5?-triphosphate disodium
salt (ATP) (P2R agonist; 0.01–10 mM; made fresh on the day of experi-
ment); adenosine 5?-[?-thio]triphosphate tetralithium salt (ATP?s)
(nonhydrolyzable ATP analog and P2R agonist; 0.1 mM); ?,?-
methylene-adenosine-5?-triphosphate (??meATP) (P2X1,3R agonist;
0.1 mM); 2-methylthioadenosine 5?-triphosphate (2MeSATP) (P2X2,4,5
and P2Y1R agonist; 0.1 mM); 2-methylthioadenosine 5?-diphosphate
(10–50 ?M; Fisher Scientific, Houston, TX); suramin (10–50 ?M); uri-
dine 5?-triphosphate trisodium salt (UTP) (P2YR agonist; 0.1 mM) (all
agonist; 1 ?M); DAMGO (?-opioid receptor agonist; 50 ?M); 2?-deoxy-
N6-methyladenosine-3?,5?-bisphosphate (MRS2179) (P2Y1R antago-
nist; 50–100 ?M); 2?,3?-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP)
(P2X1,3R antagonist; 10 nM to 10 ?M) (all from Tocris, Ellsville, MO).
Drugs were either bath-applied or microinjected via triple-barreled
capillaries) (World Precision Instruments, Sarasota, FL). Care was taken
to ensure that pipette tip diameter fell within this range, because exami-
nation of Lucifer yellow-filled triple-barreled pipettes under a fluores-
cent microscope (40? objective) revealed that pipettes of this range did
not leak, but they did leak if tip diameter exceeded 6.5 ?m. Avoiding
leakage was essential because of the potential for agonist evoked P2R
desensitization or internalization, and observations in preliminary ex-
periments with larger pipettes showed that ATP responses were either
absent or not repeatable. As an additional step to minimize the con-
founding influence of drug leakage, the pipette was removed from the
injection site to the solution above the slice in the time between consec-
utive injections. Drug microinjections (?10 psi) were controlled via a
programmable stimulator (Master-8; AMPI Instruments, Jerusalem, Is-
rael) linked to a solenoid. Consecutive agonist applications were sepa-
rated by an interval of at least 15 min. We did not systematically assess
whether this was the minimum time interval required for consistent
responses, but it was sufficient for reproducible responses.
the majority of the literature indicating that the concentration of P2R
agonists required to evoke P2R currents in neurons in slices is much
higher than required in dissociated cells or expression systems (North,
2002). This is likely attributable to reduced penetration of agonist
through the tissue, degradation of ATP in slices by ectonucleotidases, or
ing the preparation of the brain slice (North, 2002). An additional factor
is that when drugs are microinjected locally from a drug pipette, the
concentration of drug decreases exponentially with distance from the
pipette tip (Nicholson, 1985). Previous work using in vitro preparations
similar to those used here, indicates that to produce similar effects, the
concentration of drug in the pipette must be ?10-fold higher than the
concentration in the bath (Liu et al., 1990). Therefore, drug concentra-
tions used in our experiments should not be directly compared with
studies in which the same agents are bath-applied to isolated cells.
ATP microinjection mapping studies. Preliminary experiments map-
ping the effects on rhythm of microinjecting ATP into the ventrolateral
medulla (VLM) suggested that the potentiation of frequency by ATP
ventral medullary respiratory group. Subsequent experiments were
therefore performed in reference to a suction electrode placed on the
rostral surface of the slice over the preBo ¨tC region where a large ampli-
tude rhythmic inspiratory-related field potential was recorded.
Mapping studies were performed using 10 s applications of 0.1 mM
ATP. ATP responses were first mapped along the mediolateral (x) axis
994 • J.Neurosci.,January31,2007 • 27(5):993–1005 Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex
starting at the level on the dorsoventral ( y) axis of the surface suction
electrode. The pipette tip was ?150 ?m below the slice surface (depth
corresponds to the z-axis). ATP was injected, and the response was re-
corded. The pipette was then moved in steps of 75 or 150 ?m along the
x-axis. Microinjections were repeated at 15 min intervals until the most
potent frequency response was evoked. The pipette was then returned to
the site of maximum ATP sensitivity (on the x-axis), and the procedure
was repeated (at 75 ?m intervals) along the dorsoventral ( y) axis. Phys-
ical interference between the surface extracellular electrode and the in-
jection pipette prevented microinjections ?150 ?m lateral to the hot-
spot. Once the ATP hotspot was identified along x- and y-axes, ATP was
with x- and y-axes because of the potential for the injected drug to move
up the electrode track (i.e., the deeper drug injections may affect more
tissue). Therefore, to further assess whether the sensitivity of respiratory
networks to ATP varies along the rostrocaudal axis, an additional series
of experiments was conducted in which the ATP hotspot was first
mapped with the rostral surface of the slice mounted upward. The slice
of 15 min) of pontamine sky blue (2%; dissolved in sodium acetate;
Sigma) was used in the mapping experiments to label the site at which
ATP produced the largest increase in frequency. While pontamine sky
inject drugs, placement of the dye pipette in the same site as the drug
ulator axes. After iontophoresis, the slice was fixed in 4% formaldehyde/
phosphate buffer (PB) overnight and cryoprotected in 30% sucrose.
Sections were mounted onto slides, air dried, stained with cresyl violet,
dehydrated, and coverslipped with Permount (Biomeda, Foster City,
CA). Remaining sections were placed in 48-well plates for immunohis-
Although inclusion of dye with the injected drug can be used to esti-
mate the degree of drug diffusion and the region of tissue affected, this
was not done for two reasons. First, marker dyes often do not have the
same diffusion coefficient as the agonist. Second, and most importantly,
extracellular ATP is rapidly hydrolyzed by ectonucleotidases, which
means that measuring the distance over which a marker dye diffuses is
likely to overestimate, perhaps dramatically, the area actually affected by
changed as a function of distance from the hotspot.
Immunohistochemistry. The pattern of NK1R expression in relation to
the pontamine sky blue-labeled injection site was assessed via immuno-
min, incubated in 1% H2O2for 30 min, washed in PBS (three times, 15
min each time), blocked with 2% bovine serum albumin (BSA) (Sigma)
and then incubated overnight at room temperature in rabbit anti-NK1R
antibody (1:1000; Advanced Targeting Systems, San Diego, CA). Sec-
washed in PBS (three times, 30 min each time), incubated in ABC Elite
PBS (15 min each) and once in Tris-HCl (15 min), and reacted with a
wash in PBS, sections were mounted on slides and coverslipped with
To examine P2X2and P2Y1receptor expression in relation to NK1
receptor expression in the neonatal rat preBo ¨tC, animals (P0–P3) were
anesthetized deeply with isoflurane and perfused transcardially with 4%
paraformaldehyde in 0.1 M PB. Brainstems were removed, postfixed
overnight in 4% paraformaldehyde, and sliced into 50 ?m sections on a
Leica VT 1000S vibratome. Sections were stored in 0.01% sodium azide
in PB until processed for immunohistochemisty.
nolabeling for both receptors in relation to NK1 receptor expression in
the preBo ¨tC of neonatal rats. The main exception was that for P2X2
labeling, the rabbit anti-P2X2R antibody was the first primary antibody,
whereas for P2Y1labeling, the rabbit anti-NK1R antibody was used first.
In brief, all sections were initially washed three times with 0.1 M PBS.
Endogenous peroxidase activity was quenched by 20 min incubation
with 1% H2O2. After PBS washes, sections were placed in 0.3% Triton
X-100 in TNB (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5% blocking
reagent, supplied in kit) buffer for 1 h to decrease nonspecific staining
and increase antibody penetration. Sections were then incubated in the
first primary antibody (either rabbit anti-P2X2R; 1:3000; Alomone,
Jerusalem, Israel; or rabbit anti-NK1R antibody; 1:30,000; Advanced
Targeting Systems) and 0.3% Triton-X100 in TNB buffer overnight. Af-
ter this first primary antibody incubation, sections were washed in TNT
(0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.05% Tween 20) buffer and
incubated with biotin-SP-conjugated AffiniPure donkey anti-rabbit IgG
in TNB buffer for 2 h. After washes in TNT buffer, sections were incu-
bated with streptavidin-HRP (1:150) for 1 h, washed in TNT buffer,
incubated for 10 min in Fluorescein Tyramide Reagent (1:75) Amplifi-
cation Diluent, and washed again in TNT buffer. In preparation for the
application of the second primary antibody, sections were again blocked
in TNB buffer. Sections were then incubated overnight in the second
primary antibody (either rabbit anti-NK1R antibody; 1:1000; Advanced
Targeting Systems; or rabbit anti-P2Y1R antibody; 1:100; Alomone),
donkey anti-rabbit IgG (H?L) (1:200; Jackson ImmunoResearch Labo-
ratories), washed again in TNT buffer and PBS, and then mounted on
slides and coverslipped with Calbiochem (La Jolla, CA) Fluorsave
using an Axiovert 100M microscope and the following objectives: Fluar
10? [numerical aperture (NA), 0.5], Fluar 20? (NA, 0.75), or Plan-
Neofluar 40? (NA, 1.3). Images were exported to Adobe Photoshop,
version 7.0, and adjusted for contrast and brightness.
tory bursts recorded via suction electrodes from XII nerve roots and the
preBo ¨tC were assessed off-line using custom-written LabVIEW acquisi-
tion and analysis protocols (Funk et al., 1997a; Miles et al., 2002) or
pClamp 9.0 (Clampfit) and Microsoft Excel software. Values of fre-
quency and amplitude during the drug were compared with the average
value during the 90 s control period that immediately preceded drug
inspiratory frequency (calculation based on the average of four consecu-
tive events) during the first minute after injection. The time course of the
response was obtained by averaging data points in 30 s bins for the 1 min
Parameters are reported in absolute terms, or relative to control (pre-
drug or prestimulus) levels, as mean ? SE. All statistical analyses were
the Student’s t test when only two groups were compared. When more
than two groups were compared, ANOVA with Bonferroni’s or Dun-
nett’s multiple comparison tests was used (GraphPad Prism 4.0). Values
of p ? 0.05 were assumed significant.
Mapping studies identified a circumscribed region within the
VLM where microinjection of ATP evoked a significantly greater
Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplexJ.Neurosci.,January31,2007 • 27(5):993–1005 • 995
increase in frequency compared with surrounding areas. As
shown for an individual slice and population data, responses
evoked by ATP in this hotspot were dose dependent. ATP in-
ATP (n ? 4), respectively (Fig. 1). Responses evoked by ATP (10
s; 0.1 mM) in this hotspot were also remarkably consistent in
magnitude and time course, whether applied repeatedly in a sin-
peaked 4.9 ? 0.4 s after the onset of ATP microinjection at fre-
than control (13.2 ? 1 cycles/min; n ? 18). Frequency remained
one or two cycles) in ?75% preparations and reached a nadir
30–40 s after drug onset before returning to control levels. Ex-
amples of this post-ATP inhibition for individual slice prepara-
tions are shown in Figure 1A (as well as in Figs. 2A, 3A, 5A, 7A,
11). Data from a small subset of these experiments (n ? 18, 4 of
which did not show a post-ATP inhibition) revealed a small but
significant post-ATP decrease in frequency that averaged 0.83 ?
0.02 of control (n ? 18).
The ATP-mediated increase in frequency decreased dramati-
cally when microinjection sites were moved away from the hot-
the hotspot, responses decreased to 0.49 ? 0.06 and 0.17 ? 0.04,
respectively, of the maximum response evoked at the hotspot
(Fig. 2B). Similarly, responses to applications 150 ?m lateral to
the hotspot were 0.52 ? 0.07 of maximum (Fig. 2B). In the
ventral to the hotspot and to 0.62 ? 0.09 and 0.20 ? 0.06 of
maximum when injections were 75 or 150 ?m dorsal to the hot-
spot (Fig. 2B).
Response kinetics was also much slower when ATP was mi-
croinjected away from the hotspot. When microinjected 150 ?m
from the hotspot along either the mediolateral (x)- or dorsoven-
tral ( y)-axes, the time to peak was increased significantly to
10.2 ? 1.4 and 8.6 ? 1.3 s, respectively (Fig. 2C).
The hotspot identified in this first experimental series was
localized only in two dimensions (i.e., in x–y plane at a single
rostrocaudal level). The hotspot corresponded closely to the re-
recorded. Thus, to test whether the high sensitivity of this region
150 ?m below the rostral surface was specific to that locus or a
rostrocaudal (z) axis, 75 and 150 ?m below the rostral surface.
Frequency increased 2.00 ? 0.2-fold (n ? 4) (data not shown)
when ATP was injected 150 ?m below the surface. This response
was significantly greater than at the more rostral injection site
(?75 ?m below the rostral surface) where frequency increased
1.49 ? 0.07-fold.
It remained possible, however, that the greater response
evoked at 150 ?m simply reflected the activation of a greater
volume of neural tissue because of the retrograde movement of
drug up the 150 vs 75 ?m electrode tract. To address this possi-
bility, the ATP-sensitive site was mapped at a depth of 150 ?m
from one surface of the slice, the slice was flipped over and the
other surface mapped. The hotspot on both surfaces mapped
consistently to the region in the x–y plane that gave the largest
inspiratory field potential. However, the increase in frequency
evoked by ATP at the caudal site, which was ?400 ?m caudal to
the rostral site, was significantly less (2.1 ? 0.2-fold; n ? 7) than
Because elevations in [K?]efrom 3 to 9 mM affect ion gradi-
ents, activity of ion pumps, reversal potential for K?currents,
and therefore the actions of some neuromodulators, an addi-
tional series of experiments was performed to test whether the
potentiation of frequency by ATP is dependent on elevated
[K?]e. The ATP-evoked frequency response of slices bathed in 3
and 9 mM [K?]ewere compared. Medullary slices produced ro-
bust inspiratory-related rhythm at a frequency of 5.16 ? 0.4 cy-
9 mM [K?]e(n ? 5) (Fig. 3A). We used 0.05 mM ATP to ensure
that the effect of ATP in 9 mM [K?]ewas not saturating. The
relative effects on frequency of microinjecting ATP (0.05 mM)
into the hotspot were similar in 3 and 9 mM [K?]e, increasing
2.79 ? 0.41- and 2.56 ? 0.33-fold, respectively (Fig. 3B).
the preBo ¨tC
Having mapped the ATP hotspot, we next used anatomical, mo-
effects of ATP (0.01, 0.1, and 1 mM; n ? 5, 6, and 4, respectively) on frequency. Rel. Freq.,
996 • J.Neurosci.,January31,2007 • 27(5):993–1005Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex
hypothesis that the hotspot corresponds to the preBo ¨tC. For an-
atomical and molecular verification, mapping studies were con-
cluded by marking the ATP hotspot via iontophoresis of pon-
tamine sky blue (n ? 10). This tissue was processed to establish
the relationship between the hotspot and cytoarchitectural land-
marks using cresyl violet and NK1R immunolabeling. A repre-
sentative cresyl violet-labeled section and dye spot is shown in
Figure 4, A and B. In the x and y plane of the slice, dye spots were
mapped in relation to the semicompact nucleus ambiguus
axis, dye spots were mapped according to the distance from the
olive (IO) complex. In this axis, dye spots clustered in a region
the facial nucleus, at the level where the dorsal (IOD) and prin-
cipal nuclei of the IO (IOPr) fuse to form one continuous struc-
ture with three loops, and the ventral portion of the medial nu-
cleus of inferior olive (IOM) is apparent.
To further assess whether the ATP hotspot corresponded to
the preBo ¨tC, the spatial relationship between the pontamine sky
blue dye spot and NK1R immunoreactiv-
is not unique to the preBo ¨tC, it is notice-
ably more intense in the preBo ¨tC than in
the adjacent reticular formation and is an
established molecular marker of the pre-
Bo ¨tC (Gray et al., 1999; Guyenet and
Wang, 2001; Wang et al., 2001). The most
intense labeling was detected in the mid-
line (Fig. 4D), consistent with previous
observations that immature glial cells in
the medullary midline express high levels
of NK1-IR (Horie et al., 2000). Moderate
NK1R-IR was also detected in the VLM in
the region of the semicompact division of
nucleus ambiguus (scNA) and regions
ventral to the scNA that overlapped with
pontamine sky blue injection sites (Fig.
4D). This was most obvious in higher
magnification images of the ventrolateral
region of the slice ipsilateral (Fig. 4E) and
et al., 2003) was not apparent.
The hypothesis that the ATP hotspot
corresponds to the preBo ¨tC was then
tested functionally knowing that activa-
tion of NK1R or ?-opioid receptors in the
preBo ¨tC increases or decreases burst fre-
quency, respectively (Johnson et al., 1996;
Gray et al., 1999). Using triple-barreled
drug pipettes, the ATP hotspot was
mapped as before. The NK1R agonist SP
DAMGO were then locally injected into
the ATP hotspot (Fig. 5A) (n ? 6) at 15
min intervals. In this series, ATP (0.1 mM;
10 s) produced a rapid-onset, short-
frequency that lasted for ?20 s. SP pro-
duced a rapid-onset, 2.3 ? 0.18-fold increase in frequency that
peaked ?20 s after onset of SP application and lasted ?30 s.
DAMGO caused a dramatic reduction in frequency that reached
a nadir at 0.37 ? 0.08 of control ?30 s after drug onset (Fig.
5B,C). This inhibitory action often took ?7 min to washout.
Based on immunohistochemical and electrophysiological evi-
dence from the adult rat (Kanjhan et al., 1999; Yao et al., 2000;
Thomas et al., 2001; Gourine et al., 2003) and the hypothesized
importance of pH-sensitive, P2X2R-mediated currents in central
we focused initially on the role of P2XRs, especially P2X2Rs, in
the ATP-mediated frequency response. Consistent with data
from the adult rat (Kanjhan et al., 1999; Yao et al., 2000;
that were, based on local anatomical landmarks and the pres-
ence of NK1R immunolabeling, consistent with the preBo ¨tC.
High magnification revealed preBo ¨tC neurons that were im-
Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex J.Neurosci.,January31,2007 • 27(5):993–1005 • 997
munofluorescent for P2X2(Fig. 6D), NK1 (Fig. 6C), or both
P2X2and NK1Rs (Fig. 6B).
Based on the fact that only P2X2subunit-containing P2XRs
the involvement of P2X2Rs in the ATP-evoked frequency in-
aCSF pH. In the first series, aCSF pH was changed from 7.56 ?
0.06 to 7.25 ? 0.01 by increasing the amount of CO2in the gas
bubbling the aCSF. The effects of 0.1 mM ATP on frequency,
CO2(2.30 ? 0.40-fold increase; n ? 3) (data not shown). In the
response to 0.1 mM ATP was already maximal at 5% CO2. The
effects of ATP on frequency at 5% (2.07 ? 0.003) and 10% CO2
(2.45 ? 0.23; n ? 3) (data not shown) were still similar.
Finally, to address the possibility that the pH change had not
been sufficient to potentiate P2X2R-mediated responses, we
compared the frequency response to ATP (0.1 mM, n ? 5; 0.05
mM, n ? 6; 0.01 mM, n ? 4) over a greater pH range by reducing
the concentration of NaHCO3in the aCSF bubbled with 10%
CO2from 26 to 20 mM (low HCO3
However, the frequency response at each concentration re-
mained insensitive to pH (Fig. 7) (i.e., frequency responses were
insensitive to pH over a tenfold range in ATP concentration).
P2R subtype was also explored by microinjecting from triple-
barreled pipettes a variety of P2R agonists into the preBo ¨tC, as
identified via the ATP mapping protocol. In this series of exper-
iments, application of ATP (0.1 mM; 10 s) into the preBo ¨tC in-
P2X2, P2X3, P2X5, P2X2/3, and P2X1/5receptors (Norenberg and
Illes, 2000; North and Surprenant, 2000), as well as P2Y1, P2Y2,
(2.96 ? 0.39-fold) similar to that of ATP (Fig. 8A) (n ? 4). The
and Illes, 2000) caused only a minor, but significant 1.2 ? 0.04-
fold increase in frequency that reflected a remarkably consistent
response between preparations (Fig. 8A) (n ? 5). UTP, an ago-
nist at P2X3Rs and all P2YRs except P2Y1(von Kugelgen and
increase in frequency (Fig. 8B) when injected at sites where mi-
croinjection of ATP or SP potentiated respiratory frequency
more than twofold (Fig. 8B) (n ? 6). ATP?s, a hydrolysis-
resistant agonist at P2X1–6, P2X2/3, P2X1/5, P2Y1, P2Y2, P2Y4,
0.3-fold increase in frequency in a series in which ATP caused a
3.30 ? 0.3-fold increase (Fig. 11) (n ? 7). The general P2R an-
tagonists, suramin and PPADS, did not significantly affect the
ATP-mediated potentiation of inspiratory frequency. Suramin
antagonizes P2X1, P2X2, P2X3, P2X5, P2X2/3, P2X1/5, P2Y11, and
tions (Norenberg and Illes, 2000; von Kugelgen and Wetter,
2000). Given these broad actions, it was surprising that suramin
(Fig. 8C) (n ? 4). PPADS is generally more potent than suramin
at P2XRs, antagonizing P2X1, P2X2, P2X3, P2X5, P2X2/3, P2X1/5,
Illes, 2000; von Kugelgen and Wetter, 2000). Despite this, bath
application of PPADS (50 ?M) did not inhibit the ATP response.
tion of PPADS was increased to 500 ?M did its local pre-
microinjection into the preBo ¨tC (2 min) partially block the ATP
response from a 2.56 ? 0.2-fold increase in control to 1.79 ? 0.3
in PPADS (Fig. 8E) (n ? 5). This could reflect antagonism of
of PPADS. However, the PPADS-mediated reduction in ATP
sensitivity is more consistent with an inhibition of P2Rs, because
the baseline frequency, which is dependent on glutamatergic
?). This produced a pH of
and?preBo ¨tCrecordingsillustratingtheeffectoflocallyapplying0.05mMATPtothepreBo ¨tCin
998 • J.Neurosci.,January31,2007 • 27(5):993–1005Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex
transmission (Greer et al., 1991; Funk et al., 1993), was not af-
fected by 500 ?M PPADS. The antagonist TNP-ATP (bath ap-
plied, 0.01–10 ?M), which is selective for P2X1, P2X3, and
P2X2/3Rs in the nanomolar range, but can also affect PX2, P2X4,
several orders of magnitude higher (Lam-
The agonist potency profile, in which
ATP ? 2MeSATP (Fig. 8) ? ATP?s (Fig.
11) UTP (Fig. 8) ? ??meATP (Fig. 8)
(Kawamura et al., 2004), and lack of effi-
cacy of the antagonists suramin and
PPADS at low concentrations were most
consistent with a P2Y1-mediated effect.
P2Y1R involvement was then tested by ap-
plying the P2Y1R agonist 2MeSADP (0.1
mM) into the preBo ¨tC. It caused a 2.6 ?
0.2-fold increase in frequency that was
similar in magnitude (2.3 ? 0.1-fold), but
of greater duration, compared with the
ATP effect (Fig. 9A,B) (n ? 12). Fre-
at least 30 s after 2MeSADP application,
but only for 20 s after ATP application
(Fig. 9B). Moreover, local microinjection
of the P2Y1selective antagonist MRS2179
(Camaioni et al., 1998) to the preBo ¨tC for
2 min before agonist microinjection sig-
nificantly attenuated the effects of ATP
and 2MeSADP on burst frequency (Fig.
9C–F). At 50 ?M MRS2179, the ATP po-
tentiation fell from a 2.44 ? 0.2-fold in-
crease to 1.45 ? 0.2 (i.e., MRS2179 de-
creased the response to 0.27 ? 0.10 of the
control ATP increase) (Fig. 9D) (n ? 9).
Increasing the concentration of MRS2179
to 100 ?M in a separate series of experi-
ments did not cause a greater reduction in
the frequency response (the frequency in-
crease was only 0.23 ? 0.07 of the control
ATP increase) (Fig. 9D) (n ? 8). In con-
trast, the MRS2179 inhibition of the
2MeSADP-mediated frequency increase
(n ? 8) was dose dependent. Frequency
of the control 2MeSADP increase in the
presence of 50 and 100 ?M MRS2179, re-
spectively (Fig. 9F) (n ? 6).
The potential for P2Y1R involvement
in the ATP-evoked frequency increase was
also examined using double-labeling fluo-
rescent immunohistochemistry to charac-
terize the pattern of P2Y1R expression rel-
ative to NK1R immunolabeling. The
preBo ¨tC was identified as a cluster of neu-
NK1R (Fig. 10A,C). P2Y1R labeling was
found in the preBo ¨tC region and colocal-
ized with NK1R immunolabeling in some
preBo ¨tC neurons (Fig. 10B,D) (n ? 3).
As described previously, the frequency response to ATP in the
preBo ¨tC is biphasic. An initial increase is followed in most cases
6) illustrating the maximum frequency increase (B) and time course of responses (C) evoked by each agonist. *Significantly
The ATP hotspot is sensitive to NK1 and ?-opioid receptor activation. A, ?XII nerve and ?preBo ¨tC recordings
Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplexJ.Neurosci.,January31,2007 • 27(5):993–1005 • 999
by a short-lasting decrease in frequency
(Fig. 1A). Given that extracellular ATP is
ADP and adenosine (Zimmermann, 2000,
respiratory frequency (Herlenius et al.,
1997; Herlenius and Lagercrantz, 1999),
of frequency results from the hydrolysis of
ATP. We compared the effects on fre-
ATP and ATP?s, a nonhydrolysable ana-
log of ATP. When applied at the same site,
ATP (0.1 mM) and ATP?s (0.1 mM)
evoked similar, rapid increases in respira-
tory frequency that peaked at 3.30 ? 0.3-
fold or 3.10 ? 0.3-fold greater than con-
responses, however, were shorter in dura-
post-ATP decrease in burst frequency
(0.79 ? 0.06-fold) that followed ATP appli-
To determine whether endogenous ATP
ullary slice preparation, we examined the
effects on baseline inspiratory burst fre-
quency of several P2R antagonists or allo-
steric modulators. Most drugs were with-
out effect on frequency (data not shown),
applied at 50 ?M; n ? 5), (2) TNP-ATP (P2X1and P2X3antago-
antagonist; locally microinjected at 100 ?M into the preBo ¨tC for
2 min, n ? 14; or bath-applied at 50 ?M, n ? 4).
frequency by a factor of 0.33 ? 0.06 (from 17.1 ? 0.9 to 11.5 ?
0.6 cycles/min; n ? 4) (Figure 12) when applied to the bath. The
suramin effect did not wash out, but this was unlikely to reflect
nonspecific rundown in activity, because inspiratory frequency
did not change significantly over the same period in time-
matched controls (n ? 6) (Fig. 12). Cu2?, an allosteric modula-
tor of P2X2Rs (Xiong et al., 1999), also evoked a significant in-
crease in frequency at all three concentrations. Frequency
increased 1.63 ? 0.12-fold [from 13.8 ? 0.8 to a maximum of
22.5 ? 2.1 cycles/min at 25 ?M Cu2?(CuCl2)] and recovered to
control levels after washout (n ? 7) (Fig. 12).
for inspiratory rhythm generation (Smith et al., 1991; Feldman
and Del Negro, 2006). Three lines of evidence from this study
support that ATP acts with maximum potency in the preBo ¨tC to
increase frequency. First, the pontamine sky blue-labeled ATP
hotspot maps to a region anatomically consistent with the pre-
regions of moderate NK1R immunolabeling, and NK1-
immunoreactive preBo ¨tC neurons double label for P2X2and
P2Y1receptors. preBo ¨tC markers include NK1, ?-opioid, and
somatostatin receptor labeling (Gray et al., 1999; Stornetta et al.,
2003), but NK1R labeling is used most extensively (Gray et al.,
et al., 2002; Pagliardini et al., 2003, 2005). It is not exclusive to
preBo ¨tCneuronsbutremainsuseful,becausepreBo ¨tCstainingis
greater than in surrounding regions. In addition, NK1R-
expressing preBo ¨tC neurons are essential for rhythm generation
(Gray et al., 2001; Pagliardini et al., 2003; Wenninger et al., 2004;
McKay et al., 2005). Third, activation of NK1 or ?-opioid recep-
tors in the ATP hotspot potently increases or decreases, respec-
tively, respiratory frequency. These responses functionally iden-
tify the preBo ¨tC (Johnson et al., 1996; Gray et al., 1999) and,
combined with histological and immunochemical data, provide
compelling evidence that the ATP hotspot corresponds to the
consistent in neonates in vitro. They were not, however, consis-
tent with in vivo studies that indicate that ATP [at high concen-
excites inspiratory neurons, but blocks phrenic nerve output
causing apnea (Spyer and Thomas, 2000; Thomas et al., 2001;
NK1R (C; red) and P2X1(D; green) immunolabeling in the preBo ¨tC region (boxed area in A) alone (C, D) and overlaid (B). The
1000 • J.Neurosci.,January31,2007 • 27(5):993–1005Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex
Gourine et al., 2005b). Factors underlying these differences are
not known. In vivo effects may represent depolarization block
because 10 mM ATP also arrests rhythm in vitro (Lorier et al.,
2004). However, it may also reflect development of purinergic
signaling (Collo et al., 1996; Kanjhan et al., 1999; Brosenitsch et
al., 2005) in neurons and glia that contribute to ATP responses
2002). Sites of ATP application in vivo and in vitro may also have
of inspiratory neurons and NK1R-labeled preBo ¨tC neurons, and
suramin-sensitive, ATP-evoked increases in inspiratory neuron
discharge (Kanjhan et al., 1999; Yao et al., 2000; Thomas et al.,
support a major role for P2XRs in the ATP-evoked frequency
increase. The P2X1,3R agonist ??meATP had minor effects on
frequency, whereas the P2X1,3R antagonist, TNP-ATP, did not
block the ATP response. P2X2R subunits are also unlikely to play
a dominant role, because P2X2subunit-containing P2XRs are
solution did not affect the ATP response. It is important to em-
cannot exclude the possibility that the pH sensitivity of P2X2
receptors was somehow altered in vitro. However, we are confi-
enhance currents of P2X2subunit-containing receptors. First,
P2X2receptor currents almost double between pH levels of 7.1
and 6.8 (King et al., 1996). Second, tissue pH in medullary slices
and en bloc preparations (Okada et al., 1993; Trapp et al., 1996;
Voipio and Ballanyi, 1997) at a depth in the tissue of 150 ?m,
aCSF. Thus, tissue pH likely decreased from ?7.15 to ?6.75.
Insensitivity of the frequency response to low concentrations of
suramin and PPADS further suggests that P2X2and P2X5recep-
of P2X4and P2X6receptors, which are less sensitive to suramin
and PPADS (Norenberg and Illes, 2000), or heteromeric P2XRs
?preBo ¨tC recordings illustrating the effects on inspiratory-related output in a single slice of
(10% CO2/low HCO3
low (10% CO2/low HCO3
?). B, Histogram of group data illustrating the maximum frequency re-
?) extracellular pH. Error bars indicate SEM. Rel. Freq., Relative
effects on frequency of locally applying ATP (0.1 mM), UTP (0.1 mM), and SP (1 ?M) into the
frequency. *Significantly different from control. Error bars indicate SEM. Rel. Freq., Relative
Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex J.Neurosci.,January31,2007 • 27(5):993–1005 • 1001
with unique pharmacology, remains pos-
are primarily responsible for the ATP-
evoked frequency increase. The agonist
UTP ? ??meATP) (Kawamura et al.,
2004) and antagonist profiles were most
consistent with P2Y1Rs. The ATP fre-
quency increase was highly sensitive only
to the P2Y1R antagonist MRS2179 (Ca-
maioni et al., 1998; von Kugelgen, 2005).
Finally, preBo ¨tC neurons show P2Y1R IR
or double-labeling for P2Y1and NK1R.
The cellular or synaptic mechanisms
underlying the P2Y1R-evoked frequency
increase are not known. It could reflect
activation of P2Y1receptors located on
glia, postsynaptically on preBo ¨tC neu-
rons, or presynaptically on terminals of
glutamatergic, GABAergic, or modula-
tory neurons (Fields and Burnstock,
2006; Hussl and Boehm, 2006) that
project to the preBo ¨tC.
Evidence of P2YR involvement in re-
spiratory or motor control is limited. In
rat, chemosensitive retrotrapezoid nu-
cleus (RTN) cells are excited by P2YR ac-
tivation (Mulkey et al., 2006), whereas in
Xenopus tadpole swimming episodes are
potentiated by P2Y1R-like inhibition of a
K?conductance (Brown and Dale, 2002).
Ours is the first demonstration in the
mammalian CNS of P2Y1R modulation of
respiratory motor networks.
P2R signaling is complicated by a system
of ectonucleotidases that hydrolyze extra-
cellular ATP into adenosine (Dunwiddie
et al., 1997; Zimmermann, 2000), which acts via P1Rs, in partic-
ular the A1R, to inhibit transmitter release (Dunwiddie et al.,
1997; Ralevic and Burnstock, 1998; Haas and Selbach, 2000). In
depresses ventilation (Herlenius et al., 1997; Herlenius and La-
gercrantz, 1999; Mironov et al., 1999), and is implicated in the
hypoxia-induced depression of ventilation (Yan et al., 1995) and
apnea in newborns (Runold et al., 1989; Lopes et al., 1994). Our
observation that the post-ATP decrease in frequency depends on
ATP breakdown suggests that effects of ATP on rhythm will be
determined by an interaction between P2 and P1 receptors. In
other brain regions, P2–P1R interactions influence synaptic
transmission (Kato et al., 2000; Kato and Shigetomi, 2001;
Kawamura et al., 2004) and determine the dynamics of in-
spiratory motor responses to ATP (Funk et al., 1997a; Miles et
al., 2002). Moreover, as seen here for respiratory activity, the
time course of swimming episodes in tadpoles is determined
by an interaction between a P2Y1R-like excitatory mechanism
and the degradation of ATP (Dale and Gilday, 1996; Brown
and Dale, 2002). These data highlight the potential impor-
tance of ectonucleotidases and raise the possibility that regu-
lation of these enzymes may provide an additional mechanism
for controlling network activity.
Although ATP acts as an excitatory neurotransmitter within
many brain regions, including autonomic nuclei (Nieber et al.,
2005a), its physiological significance is only just emerging. In
respiratory networks, ATP is important for peripheral (Rong et
is released in the brainstem during hypoxia or hypercapnia and
stimulates respiratory activity (Gourine et al., 2005a,b). During
medulla (Gourine et al., 2005b) where its stimulation of respira-
tion may reflect an interaction between a P2YR-mediated in-
crease and P2XR-mediated decrease in the excitability of pH-
sensitive RTN neurons (Mulkey et al., 2006). During hypoxia,
ATP is released from the ventrolateral surface of the medulla as
secondary hypoxic depression of respiration (Gourine et al.,
2005a). Our data indicate that this effect could be mediated, in
whole or in part, by the activation of P2Y1Rs in the preBo ¨tC.
It is important to note that the lack of an effect of the P2Y1R
antagonist, MRS2179, on baseline frequency does not exclude
endogenous purinergic modulation of respiratory rhythm. Slices
ergic cell groups (Nieber et al., 1997; Poelchen et al., 2001). It is
preBo ¨tC reduced the response to ATP (C) and 2MeSADP (E). Group data showing attenuation of the ATP (D) or 2MeSADP (F)
1002 • J.Neurosci.,January31,2007 • 27(5):993–1005Lorieretal.•ATPSensitivityofthePre-Bo ¨tzingerComplex
also possible that ATP does not modulate baseline respiratory
activity, but is released in response to specific stimuli such as
hypercapnia or hypoxia (Gourine et al. 2005a, b) that cannot be
reproduced in vitro [e.g., transmitters released during hypoxia in
vitro, which represents a transition from hyperoxia to anoxia
(Ramirez et al., 1997), may differ considerably from those re-
leased during hypoxia in vivo]. Another possibility is that endog-
enous purinergic modulation of rhythmic activity may not be
limited to a direct, P2Y1R-mediated preBo ¨tC effect, but an indi-
is that Cu2?, which potentiates P2X2R currents (Xiong et al.,
1999), reversibly increased baseline frequency. Additionally,
bath-applied suramin did not alter the frequency effects of ATP
in the preBo ¨tC, but significantly reduced baseline rhythm. Al-
though the nonspecific actions of these two agents demand cau-
suggest that important future objectives will be to establish that
the effects observed here in neonates persist into adulthood, and
to identify the full compliment of stimuli that evoke ATP release
from the different compartments of the respiratory network.
In summary, recent data have established an endogenous role
for ATP in central respiratory control; it is released during hyp-
oxia from the brainstem and attenuates the secondary hypoxic
depression of respiratory frequency (Gourine et al., 2005a). The
significance of the present study is that it advances our under-
standing of mechanisms through which purinergic signaling can
modulate respiratory rhythm by establishing that ATP potently
increases respiratory frequency by activating P2Y1Rs in the
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