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Genetic identification of an embryonic parafacial
oscillator coupling to the preBo
¨tzinger complex
Muriel Thoby-Brisson1, Mattias Karle
´n2, Ning Wu1, Patrick Charnay3, Jean Champagnat1& Gilles Fortin1
The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic
nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal
breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic
parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b,
were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing
or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBo
¨tzinger complex, preBo
¨tC), led to an abnormally
slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5,
entrained and then coupled with the preBo
¨tC, which emerged independently at E15.5. These data establish the dual organization
of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.
In mammals, breathing is one of the earliest motor behaviors of the
fetus and it relies on the activity of a brainstem respiratory rhythm
generator (RRG). Recent advances in the neurobiology of breathing in
neonatal mammals suggest that the RRG is located in two prominent
rhythmogenic sites, the preBo
¨tC1and the para-facial respiratory group
(pFRG)2. The genetic regulation of progenitor cell fate and differentia-
tion in the hindbrain, and of the plasticity of the RRG, remains
poorly understood.
The developmental origin and functional nature of the respiratory
rhythm-generating circuits involved in fetal and neonatal breathing
have been studied using mutant mice in which developmental genes
encoding transcription factors have been inactivated, including Egr2,
Phox2b,Hoxa1,Tlx3 and Mafb3–7. In particular, inactivation of Egr2,
the gene for the zinc finger transcription factor Egr2, which controls the
formation of hindbrain rhombomeric segments 3 and 5 (refs. 8,9),
results in defective breathing. This is attributed to a reorganization of
neuronal circuits in the caudal pontine reticular formation that
consequently leads to poor survival at birth6. Further evidence from
chicks shows that Egr2 is involved in the specification of a central
rhythm generator at the level of the facial motor nucleus10. Although
these studies have shown that the importance of hindbrain segmenta-
tion extends beyond modular anatomical organization to the
level of network assembly and function, they have failed to identify a
candidate Egr2-derived cell group that would explain the mutant
respiratory deficit.
Phox2b is a transcription factor that is specifically expressed and
required in neurons that form the visceral reflex circuits controlling
digestive, cardiovascular and respiratory functions11,12. In humans, a
heterozygous mutation in PHOX2B is the main cause of congenital
central hypoventilation syndrome (CCHS)13,14, a genetic disease that
typically manifests itself at birth by respiratory distress during sleep15.
Newborn mice that are heterozygous for the most common human
mutation have a slowed and often irregular breathing pattern, do not
respond to hypercapnia, and die at birth from respiratory failure16.
Anatomically, these mutants selectively lack a group of glutamatergic,
Phox2b-expressing interneurons in the ventro-lateral medulla in the
vicinity of the facial motor nucleus16 called the retrotrapezoid nucleus
(RTN)17. The RTN has previously been identified as a CO2/pH sensor
system in the adult rat18,19. Notably, the rhythmogenic pFRG over-
laps anatomically with the RTN and was recently shown to host
CO2-sensitive Phox2b-positive interneurones in rat neonates20, thus
supporting the view that the pFRG and RTN may correspond to
neonatal and adult forms of the same neuronal population19.
We investigated embryonic stages using knock-in alleles of Egr2 and
identified a population of Phox2b-positive interneurons deriving from
Egr2-expressing cells that forms an e-pF oscillator. The e-pF oscillator,
which is first active at E14.5, couples with the developing preBo
¨tC
oscillator within 24 h, thus establishing the dual organization of the
RRG at the time at which it begins to pace fetal breathing.
RESULTS
The embryonic parafacial oscillator
We used mice carrying a knock-in of the Cre recombinase gene into the
Egr2 locus (Egr2cre)21 and a Cre-responsive indicator allele (R26R-
EYFP)22 to trace the derivatives of Egr2-expressing cells. At E14.5, cells
derived from Egr2-positive progenitors formed two transverse stripes
with little cell dispersal along the anterior-posterior axis; these stripes
correspond to the location of and are likely to be derived from the
Received 14 April; accepted 29 May; published online 5 July 2009; doi:10.1038/nn.2354
1Institut de Neurobiologie Alfred Fessard, Centre National de la Recherche Scientifique UPR2216, Gif sur Yvette, France. 2Department of Cell and Molecular Biology,
Karolinska Institute, Stockholm, Sweden. 3Institut National de la Sante
´et de la Recherche Me
´dicale, U784, Ecole Normale Supe
´rieure, Paris, France. Correspondence
should be addressed to G.F. (gilles.fortin@inaf.cnrs-gif.fr).
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rhombomeric 3 and 5 segmental domains that have been observed
during hindbrain development (see ref. 9, the r5 stripe is shown in
Fig. 1a). Notably, inspecting the ventral surface of the hindbrain, this
restriction along the anterior-posterior axis was clearly disrupted
laterally, resulting in a parafacial stripe of yellow fluorescent protein
(YFP)-expressing cells caudal to r5, flanking and partially capping the
lateral aspect of facial motor nucleus (nVII) and continuing approxi-
mately 200 mm caudal to the nVII (Fig. 1a). Using optical recordings
from brainstem en bloc preparations23 after Calcium Green 1AM
loading, we identified a rhythmic cell population that matched this
parafacial YFP+territory (frequency, 14.3 ± 3.9 bursts min1;range,
8–18 bursts min1;n¼19 preparations; Fig. 1b–f). Occasionally, a
burst of activity in the neighboring nVII (Fig. 1d) was associated with a
burst of activity in the parafacial domain (see below). Low-resolution
imaging revealed bilateral coactivation of parafacial domains and the
absence of activity in other regions of the hindbrain. In transverse facial
slices (n¼8), a comparable rhythm was maintained in cells of the
parafacial region (Supplementar y Fig. 1). These data demonstrate that
the parafacial domain forms an intrinsically active rhythmic network,
which we refer to as the e-pF oscillator.
Imaging at cellular resolution showed synchronized activity of
individual e-pF cells (Fig. 1e,f) and synchronized rhythmic changes
in fluorescence were detected in 95% of the YFP+cells (98 of 103 cells
from three en bloc preparations) that were sampled along the rostro-
caudal extent (B700 mm) of the e-pF (Fig. 1g,h). Because the
proportion of YFPcalcium-loaded cells in the e-pF area was less
than 10%, this suggests that almost all of the e-pF cells expressed YFP
and were therefore presumably derived from rhombomeric segments 3
or 5. Active cells were not detected before E14.5 (data not shown; E12.5,
n¼3; E13.5, n¼5); thus, E14.5 marks the onset of the e-pF.
On the basis of previous work showing that respiratory-related
Phox2b-positive and neurokinin-1 receptor (NK1R)–positive neurons
are located in an area similar to the e-pF at E15.5 (ref. 16), we examined
those markers in the e-pF. We selected rhythmically active e-pF neurons
by loose patch recording, filled them with biocytin and determined
whether the Phox2b and NK1R proteins were present by immuno-
labeling. We found that 15 of 16 e-pF cells expressed Phox2b (Fig. 1i)
and 7 of 10 expressed NK1R (Fig. 1j); thus, a large fraction of e-pF cells
expressed both markers at E14.5. The NK1Rs were functional because
the e-pF rhythm frequency increased twofold in the presence of substance
P, the endogenous ligand for NK1R (0.1 mM, data not shown). In
Egr2cre/+;R26R-EYFPembryos, most, if not all, of the YFP+cells in the
parafacial area expressed Phox2b and could be easily distinguished
from the Phox2b+,YFP
and Islet1/2+nVII cells (Fig. 1k). All Phox2b+
cells in the parafacial region express the type 2 vesicular glutamate
transporter (VGlut2) at birth16, indicating the probable glutamatergic
nature of e-pF cells. The number of e-pF YFP+
,Phox2b
+, Islet1/2
neurons on one side (280 ± 36, n¼3) was similar to the number of
rhythmic cells detected in calcium-loaded preparations (259 ± 54 cells,
n¼5), indicating that the e-pF oscillator is composed of Phox2b+
neurons, which are probably glutamatergic, derived from Egr2-expres-
sing precursors.
Operating principles of the e-pF oscillator
We then investigated the cellular properties underlying rhythm gen-
eration in the e-pF oscillator. In whole-cell recordings performed on
E14.5 whole hindbrain preparations, rhythmic burst discharges of
action potentials in e-pF cells (43 of 43; Fig. 2a) appeared as all-
or-none voltage-dependent events. Spontaneous bursts were curtailed
by short negative-current pulses (Fig. 2b) that were applied during the
burst, whereas burst discharges were evoked in between spontaneous
bursts by comparable current pulses of opposite polarity (Fig. 2c).
Slowly ramping the somatic potential of e-pF cells from 80 to
+40 mV revealed a tetrodotoxin-sensitive (n¼3, data not shown)
and riluzole-sensitive (n¼10) persistent sodium current (INaP;Fig. 2d).
Inthepresenceofriluzole(20mM),rhythmicactivityofthee-pFcell
∆F/F
∆F/F
10%
∆F/F
4%
5 s
5 s
∆F/F50
10
1
0
12
10
1
e-pF
A
r5
YFP IsI1/2
YFP IsI1/2 Phox2b
12
3
4
5
67
9
8
11 10
12
YFP Ca Green1 Merge
Phox2b Biocytin Merge
NK1R Biocytin Merge
1
10
Direct F
M
D
M
abc
e
g
i
j
k
h
f
d
Figure 1 A parafacial oscillator emerges at E14.5 and is derived from Egr2-
positive territories in the mouse embryo hindbrain. (a) Partial ventral view of
a hindbrain from an Egr2cre/+; R26R-EYFP embryo showing the respective
positions of cells derived from rhombomeric segments 3 and 5 (YFP positive,
green) and nVII motoneuronal populations (Islet1/2 positive, red). Note the
stripe of YFP-expressing cells caudal to rhombomeric segment 5, flanking the
lateral aspect of the nVII (blue outline). (b–d) A Calcium Green 1AM–loaded
whole hindbrain preparation (b) showing fluorescence changes, which were
generally restricted to the e-pF oscillator (red outline) in c,andwere
sometimes concomitant to activity of the nVII (d). (e,f) Photomicrograph of
e-pF cells (numbered 1 to 12) during a burst of activity (e) and corresponding
individual (black) and average (red) relative fluorescence changes traces (f).
(g) Same field of an Egr2cre/+; R26R-EYFP preparation showing YFP-
expressing cells (red), Calcium Green 1AM–loaded cells (green) and the
merged image used to derive the individual rhythmic activities of ten double-
labeled (yellow) cells. (h) Fluorescence changes of individual cells (black)
and averaged signal (red) from g.(i,j) Immunolabeling for Phox2b (i) and for
NK1R protein (j) in two biocytin-filled e-pF neurons. (k) Single transverse
section from an E14.5 Egr2cre/+; R26R-EYFP embryo that were triple
immunolabeled with antibodies specific to Islet1/2 (blue), Phox2b (red) and
YFP (green). Cells of the e-pF expressing both Phox2b and YFP are shown in
yellow and nVII motoneurons expressing both Phox2b and Islet1/2 appear in
purple. Scale bars represent 20 mm(e,g,i,j) and 200 mm(a–d,k). A, anterior;
D, dorsal; M, median.
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population was abolished and the all-or-none bursts of action poten-
tials that were evoked by depolarizing current pulses or that occurred
spontaneously were replaced by single spikes (Fig. 2e). Hyperpolariza-
tion-activated and cyclic nucleotide–gated cation channel (HCN)-
mediated currents (Ih), which are often expressed together with INaP
in pacemaking neurons24, were present in 25 of 26 e-pF neurons
(Fig. 2f). Although the hyperpolarization level required to elicit a
somatic Ihcurrent was typically 20 mV below the resting membrane
potential of e-pF cells (Vm¼47 ± 1.5 mV, n¼20), application of
ZD7288 (100 mM) systematically blocked the Ihcurrent present in e-pF
cells and reduced the frequency of the e-pF oscillator from 13.7 ± 1.3
bursts min1to 5.4 ± 0.7 bursts min1(n¼11; Fig. 2g), suggesting a
role for possibly distally located HCN channels in the modulation
of the rhythm.
Rhythm generation and intercellular synchrony of the e-pF oscillator
were preserved in the presence of 10 mMCNQX,whichblocksgluta-
matergic transmission mediated by AMPA/kainate receptors (n¼5;
Fig. 2h), as well as in the presence of either the m-opioid agonist
D-Ala2-N-Me-Phe4-glycol5-enkephalin (DAMGO, 0.3 mM, n¼5) or a
cocktail of bicuculline (10 mM) and strychnine (5 mM), which block
GABAAand glycinergic receptors, respectively (n¼5, data not shown).
Furthermore, the e-pF oscillator rhythm was preserved (although
slower) in mice that do not express VGlut2 (Vglut2f/f;PCre; exons 4–6
of Vglut2 are flanked with loxP sites and cre is driven by the Pgk
promoter)25 (n¼3; Fig. 2i). Moreover, the
e-pF oscillator activity was spared in the pre-
sence of the NMDA-R antagonist D-2-amino-
5-phosphonovaleric acid (AP5, 5 mM, n¼6; Fig. 2j). These data
suggest that glutamatergic synaptic transmission is not essential for
rhythm generation or for intercellular synchrony of the e-pF oscillator.
Application of lanthanum (La3+,100mM, n¼4), which can efficiently
block hemichannels, but not gap junctions permeabilities26,27, failed to
alter the e-pF collective synchronous activity (Fig. 2k). However,
carbenoxolone (CBX, 50 mM, n¼11) blocking gap junctions, in
addition to hemichannels, abolished intercellular synchrony (Fig. 2l).
Under CBX, e-pF cells that maintained rhythmic fluorescence changes
(96 of 229 e-pF cells from ten preparations) were readily silenced by
further application of riluzole (n¼5; Fig. 2l). We observed similar
effects of CBX (n¼2), riluzole (n¼3), CBX and riluzole
(n¼2), and ZD7288 (n¼3) on the activity of e-pF cells in transverse
facial slices (five slices, data not shown), further establishing the e-pF
as the source of rhythmic activities.
Low-resolution imaging on E14.5 whole hindbrain preparations
indicated that application of CNQX (n¼3) spared rhythmic activities
and intercellular synchrony in parafacial domains on either side of
the midline, but caused their left/right de-synchronization. This de-
synchronization was not observed in response to the bicuculline/
strychnine cocktail (n¼3). Independent rhythms in left and right
parafacial domains were also observed in transverse slices from the axial
level of the nVII (n¼4; Supplementary Fig. 2). Thus, on the one hand,
e-pF cells collectively function as an oscillator in a manner that is
Neuron
e-pF int
lh current lh current l/V curve
Vm (mV)
e-pF int
e-pF
12
1
1
1
1
1
e-pF
12
e-pF
e-pF
e-pF
e-pF
11
9
10
1
1
10
9
–60 +20–40 – 20 0 –60 +20–40 –20
–120 – 100
CTL (n = 14)
ZD (n = 5)
–80 –60
10
–10
0
–20
–30
–40
0
Ril 50 pA
Voltage (mV)
0
20
40
CTL–Ril
Current (pA)
lh amplitude (pA)
Control
–60 mV
2 s
i
V
–50 mV
i
i
20 mV
1 s
1 s
–100 V
–50 mV
50 pA
–0.1 nA
–60 mV
∆F/F
10%
∆F/F
10%
∆F/F
2%
∆F/F
4%
∆F/F
4%
∆F/F
4%
∆F/F
4%
–45 mV
Riluzole
CTL
V
V
CTL ZD
13 mV s–1
Neuron
i
Neuron
Control
ZD7288
WT in CNQX
WT
Control
Control
AP5
VGlut2f/f;PCre
CBX + riluzole
CBX
La3+
Control Control
e-pF int
20 mV
20 mV
–50 mV
0.4 nA
20 mV
–50 mV
5 s
20 mV
0.1 nA
20 mV
0.1 nA
1 s
5 s
5 s
5 s
5 s
5 s
5 s
2 s
V
i
a
d
f
hi
jk
l
g
e
bc Figure 2 Operating principles of the e-pF
oscillator at E14.5. (a) Spontaneous rhythmic
depolarization and firing (top trace) of an e-pF cell
synchronous with e-pF population integrated
activity (bottom trace). (b,c) Spontaneous bursts
(top, gray control trace) in phase with the
population activity (bottom traces, e-pF int) could
be prematurely terminated (b, black traces) or
fully evoked (c, black traces) by negative and
positive current pulse injections (i, middle traces),
respectively. (d,e) Bursting activity in e-pF cells
relied on the INaP current. Riluzole (Ril) blocked
the control (CTL) inward current activated in e-pF
cells by slow depolarizing voltage ramps (d, left).
The INaP current-voltage relationship was obtained
by subtracting the riluzole-resistant current from
the control current (d, right, CTL – Ril). Riluzole
also blocked spontaneous and evoked burst of
activity in the e-pF (e). (f,g) The hyperpolarization-
activated current Ihpresent in e-pF cells was
blocked by ZD7288 (ZD, f) and modulated
the e-pF rhythm (g). Error bars represent s.e.m.
(h–j) Glutamatergic transmission was not required
for rhythmic activity of the e-pF oscillator. The
e-pF rhythm was maintained in 10 mMCNQX(h),
in Vglut2f/f;PCre mutant mice25 preparations (i)and
in the presence of 5 mM AP5 (j). (k,l) Intercellular
coactivation in the e-pF oscillator relied on gap
junctions. Intercellular coactivation was maintained
after blockade of hemichannel permeabilities by
100 mM lanthanum (La3+, control not shown, k),
but was lost after CBX (50 mM) treatment and
active cells were silenced on further application
of riluzole (l). Black traces in g–lrepresent
fluorescence changes in individual e-pF cells
captured in the frame and gray traces represent
the average fluorescence changes over the regions
encompassing these cells.
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largely independent of glutamatergic transmission; on the other hand,
e-pF bilateral coactivation appears to rely on glutamatergic commis-
sural projections that are already established at E14.5.
The e-pF oscillator couples with the preBo
¨tC oscillator
In mice, the respiratory preBo
¨tC oscillator, located at the vagal level of
the hindbrain, is first active at E15.5 and can maintain the rhythmic
activity of hypoglossal motoneurons in transverse slices28. Moreover, at
E15.5, fluorescence changes in the e-pF area and the nVII occurred
together with bouts of hypoglossal nerve (XIIn) activity in a conti-
nuous rhythmic and synchronized manner, indicating that there is a
common source of rhythmic activity, which possibly involves the e-pF
oscillator (Fig. 3a–c). To investigate a possible interaction between the
e-pF and the preBo
¨tC, we carried out simultaneous optical recordings
of the e-pF oscillator and nVII and electrophysiological recordings of
XIIn on E15.5 preparations.
We first investigated the consequences on nVII and XIIn activities of
a transection that was just posterior to the facial motor nucleus, in
between the e-pF oscillator and the preBo
¨tC oscillators (n¼4).
Notably, rhythmic activities were maintained on both sides of the
section. Caudal to the section, the XIIn had a lower frequency than
intact preparations (Fig. 3a,d), reflecting the presence of an active
preBo
¨tC oscillator. Indeed, this activity was abolished by bath applica-
tion of DAMGO29 (n¼3, data not shown), but was unaffected by
riluzole (n¼3), which also failed to modify the rhythm observed in
E15.5 preBo
¨tC transverse slices (n¼6; Fig. 3d). Rostral to the section,
the e-pF oscillated faster than in intact preparations, whereas the
adjacent nVII was completely silenced, indicating that the e-pF prob-
ably does not entrain it directly (Fig. 3a). The increased frequency of
the e-pF was consistent with that observed in facial transverse slices,
suggesting that partial e-pF cellular loss and/or severed commissural
fibers are causal factors. At any rate, caudal to the section, the resulting
slower rhythm suggested the elimination of a descending excitatory
input, indicating that the two oscillators interact at E15.5.
Second, in intact E15.5 preparations, bath applications of riluzole
that selectively suppress the e-pF activity (n¼8) led to maintained
rhythmic activities in the nVII and XIIn, although at about half of the
frequency observed in control conditions (Fig. 3b,d). These data
indicate that functional impairment of the e-pF results in a slower
rhythm that is driven by the sole preBo
¨tC oscillator. Finally, silencing
the preBo
¨tC oscillator by application of CNQX (n¼5, Fig. 3c,d)or
DAMGO (n¼7, Fig. 3d) led to a selective cessation of rhythmic
activities of the nVII and XIIn, demonstrating the pre-motor status of
the preBo
¨tC oscillator. Our data support the view that the e-pF
oscillator increases the frequency of rhythmic motor bursts generated
by the preBo
¨tC. Altogether, these data suggest that the dual organiza-
tion of the RRG described at later stages2,30 is already achieved by the
time of its inception at E15.5.
We then investigated, during the E14.5–15.5 period, the establish-
ment of the continuous rhythmic motor output. We confirmed that
spontaneous collective cellular rhythmic behavior was absent at E14.5
in transverse preBo
¨tC slices (n¼5), denoting its immature status28.In
some of the E15.5 and in all of the E14.5 whole hindbrain preparations,
we observed discrete failings of the nVII and XIIn bursts, despite the
continuous generation of e-pF rhythmic bursts (Fig. 4a). This was
reminiscent of skipped respiratory cycles, as opioid-induced reduction
of excitability in one (preBo
¨tC) of the two coupled oscillators causes
transmission failures of the rhythmic drive from the other coupled
oscillator in the neonatal RRG31. Therefore, we investigated, during
E14.5–15.5, the possibility that excitability build-up in the preBo
¨tC in
the presence of an already active e-pF rhythmic drive may conversely
lead to discrete occurrences of motor bursts in phase with e-pF bursts.
To explore this, we first compared the frequency distribution of motor
bursts with that of e-pF bursts between E14.5 and E15.5 prepara-
tions. At E14.5, the frequencies of motor bursts (3.4 ± 1.5 burstsmin1,
n¼11) and e-pF bursts (10.6 ± 1.7 bursts min1,n¼11) were
significantly different (Po0.001), whereas the distributions were
found to partially overlap at E15.5 (Fig. 4b). This resulted from an
increased occurrence of motor bursts (8.6 ± 2.1 bursts min1,n¼9),
whereas the frequency of the e-pF oscillator was unchanged (10.2 ±
2.4 bursts min1,n¼9). At E14.5, each individual burst of motor
activity was generated with a delay after the onset of an e-pF burst,
which was reduced or non-existent by E15.5 (Fig. 4c,d). These data
indicate that the continuous generation of rhythmic motor bursts
Figure 3 Coupled oscillators control the motor
activity at E15.5. Simultaneous optical
recordings of the e-pF oscillator and nVII
activities and electrophysiological recordings of
XIIn were performed at E15.5 in en bloc
preparations. Note that all activities are
synchronized in control conditions (top set of
traces in a–c). (a) A transverse section below the
nVII led to spared independent rhythmic
activities of the e-pF oscillator (top trace) and
the XIIn (bottom trace) and to complete
suppression of activity in the nVII (middle
trace), suggesting that the preBo
¨tC oscillator
drove motoneuronal pools before the section.
(b) Selective silencing of the e-pF oscillator
by 20 mM riluzole resulted in a slower motor
rhythm. (c) CNQX application (preserving
left/right de-synchronized activities of the e-pF)
disrupted the activity of the preBo
¨tC oscillator
and abolished motor activities. (d)Graph
representing the percentage change (mean ±
s.e.m.) of the frequency of rhythmic activities of
the e-pF (black bars), nVII or XIIn (Motor, gray
bars) and preBo
¨tC (white bar) after the transverse section (n¼4), the applications of 10 mMCNQX(n¼11), 0.3 mM DAMGO (n¼7) and riluzole (Ril,
n¼8) in whole-hindbrain preparations (WHB) or transverse slice (slice) preparations (n¼6). * Po0.05.
Control
Control
CNQX
Section 2% 2%
2%
10 s 10 s
10 s
e-pF
e-pF
e-pF
nVII
nVII
nVII
Xlln
Control
Riluzole
e-pF
nVII
Xlln
e-pF
e-pF
Motor
PreBötC
nVII
Xlln
200
Percentage of control
150
Section CNQX DAMGO Ril Ril DAMGO
SliceWHB
Xlln
*
*
***
*
*
100
50
0
Xlln
e-pF
nVII
Xlln
Xlln
ab
dc
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does not appear abruptly at E15.5 together with the preBo
¨tC oscillator,
but instead results from a dynamic process in which the e-pF oscillator
seems to be important. In fact, the frequency of motor activities
increased linearly with the value of transmission ratios; that is, the
number of motor bursts to the number of e-pF bursts (E14.5, n¼11;
E15.5, n¼19) to reach unity at E15.5 in 12 of 19 preparations (two
cases shown in Fig. 4e). Inspection of activities in individual prepara-
tions clearly illustrated the quantal nature of motor burst incorpora-
tion, eventually giving way to a continuous rhythm (Fig. 4c). This
dynamic process reflected an increasing excitability at pre-motor/
motor synapses during E14.5, as the time lags separating the onsets
of e-pF and motor bursts decreased with increasing transmission ratios
and eventually approached zero when the rhythm became continuous
(12 of 19 preparations at E15.5 and 5 of 5 preparations at E16.5;
Fig. 4d,f). Therefore, the motor rhythm became continuous during
the E14.5–15.5 period, as a result of increased efficiency of the e-pF in
transmitting activity to motoneuronal pools via a maturing preBo
¨tC,
which acquires autonomous rhythm generation at the end of the process.
We next investigated the signature of this coupling among oscillators
at the single-cell level by recording the membrane potential of indivi-
dual e-pF cells and the activity of the preBo
¨tC oscillator in E15.5 en
bloc preparations (Fig. 5). We found, using low chloride concentration
(6 mM) pipette solution for whole-cell recordings, that the e-pF oscillator
comprised two distinct sets of neurons. During a burst of activity in
the preBo
¨tC, 16 of 30 e-pF cells discharged a burst of action potentials
(Fig. 5a). In contrast, the remaining 14 e-pF cells showed a pause in
firing that was associated with a barrage of chloride-mediated synaptic
potentials (Fig. 5b). Because these differences could be the results of a
variable effectiveness in changing the intracellular chloride concentra-
tion through whole-cell recordings, we performed voltage-clamp
experiments to check the polarity and kinetics of underlying synaptic
currents. Working at a holding potential of 40 mV, excited (n¼7)
and pausing (n¼10) e-pF cells featured both inward and outward
background synaptic events (Fig. 5b,e), and during preBo
¨tC bursting
activity, they featured barrages of prominently fast (decay, B1.5 ms)
inward (Fig. 5b,c) and slow (decay, B8.0 ms) outward (Fig. 5e,f)
synaptic currents, respectively. Fast inward synaptic currents were
blocked by CNQX (data not shown) and slow outward synaptic
currents by bicuculline (Fig. 5g), indicating their respective mediation
by AMPA/kainate and GABAAreceptors. Thus, pausing cells may
eventually show active inhibition during preBo
¨tC inspiratory
bursts, whereas those discharging in phase with the preBo
¨tC are
candidate neurons through which the e-pF oscillator could contribute
to increase the frequency of the RRG. These data suggest that synaptic
interactions are established at E15.5 between e-pF and preBo
¨tC
oscillators. In sum, these experiments show that the e-pF oscillator is
fated to couple with the preBo
¨tC, the essential oscillatorinvolved in the
control of respiration.
Elimination of the e-pF oscillator in Egr2 null mutants
Our data indicate that the e-pF may be important in increasing
the frequency of fetal breathing. In Egr2lacZ/lacZ mice, which are null
mutants for Egr2, the elimination of populations derived from
rhombomeric segments 3 and 5 leads to abnormally slow breathing
at birth and poor survival6. Calcium imaging in E15.5 Egr2lacZ/lacZ
preparations (Fig. 6) showed rhythmic fluorescence changes of
the nVII in phase with the XIIn activity, although at half of the
frequency of Egr2lacZ/+ or wild-type embryos (pooled Egr2lacZ/+/WT,
f¼8.9 ± 0.7 bursts min1,n¼7; Fig. 6a–c;Egr2lacZ/lacZ,f¼3.8 ± 0.3
bursts min1,n¼9; Fig. 6h–j).
Notably, there was a complete absence of rhythmic fluorescence
changes in the e-pF area in the homozygous mutants (Fig. 6i,j).
Anatomically, the longitudinal stripe of NK1Rexpression lateral to the
nVII (Fig. 6d,e) was absent (Fig. 6k,l). In transverse and parasagittal
(data not shown) sections, NK1R expression (Fig. 6f) and Phox2b+/
Islet1/2cells (Fig. 6g) were lacking at the location of the e-pF
e-pF
e-pF
2%
10 s
10 s 1 s
∆F/F
nVII
nVII
e-pF
nVII
e-pF
15
1
0
3
3
1
1
22
10
0 0.2 0.4 0.6
Transmission ratio
0.8 1.0 0 0.2 0.4 0.6
Transmission ratio
0.8 1.0
5
0
nVII
Xlln
E14.5
E14.5
1%
E14.5
E15.5
E15.5
Freq. (min–1)
Mot. Mot.e-pF e-pF
100
0 5 10 15 0 5 10 15
Nb of preparation (%)
Mot. freq. (min–1)
Lag e-pF-nVII (s)
50
0
a
b
ef
cd
Figure 4 Coupling between the e-pF oscillator and motor activity during
development. (a) Spontaneous calcium changes in the e-pF (red trace) and
nVII (blue trace) with electrophysiological recording of XIIn (black trace)
in an E14.5 preparation. Note the absence of motor bursts with continuous
rhythmic e-pF bursts. (b) Distributions of the frequencies of motor (empty
bars, blue fit line) and e-pF (black bars, red fit line) activities obtained from
E14.5 (left graph, n¼11) and E15.5 (right graph, n¼9) preparations. Grey
bars indicate overlapping bins. (c,d) Calcium changes in the e-pF (red traces
and triangles) and nVII (blue traces and triangles) in two (top and middle)
E14.5 and one (bottom) E15.5 preparation (c). Note the increased
occurrence of motor bursts coupled to the e-pF bursts, associated with the
reduction of the time lag (distance between downward red and blue arrows
and vertical lines) between the sequential onsets of the e-pF and nVII bursts (d).
(e,f) Developmental trends among E14.5 (filled circles) and E15.5 (empty
circles) preparations; motor frequency increased (e) and the e-pF–nVII time
lag decreased (f) with augmenting transmission ratios. Data points 1, 2
and 3 correspond to the top, middle and bottom traces in c.
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oscillator (Fig. 6g,n). Moreover, sectioning the preparation caudal to the
nVII reduced the frequency of rhythmic activity by half in Egr2lacZ/+
preparations, as shown above for wild-type embryos (f¼5.1 ± 0.2 bursts
min1,n¼16 pooled genotypes), but had no significant effect on
the rhythm in Egr2lacZ/lacZ mutants (f¼4.5 ± 0.4 bursts min1,n¼5,
P¼0.3). In fact, the slow rhythm of the homozygous mutants was
comparable with that produced in preBo
¨tC transverse slices prepared
from embryos of either genotypes (wild type, 5.1 ± 0.8 bursts min1,
n¼6; Egr2lacZ/lacZ, 5.6 ± 1.5 bursts min1,n¼5; P¼0.9). Egr2 is thus
required for the development of the NK1R- and Phox2b-expressing
cells that form the e-pF oscillator. This result suggests that the reduced
breathing frequency observed in newborn Egr2 homozygous mutant
pups6may be a result of a lack of entrainment of the preBo
¨tC by
the e-pF.
Figure 6 The e-pF oscillator is absent in Egr2 null
mutant embryos. (a,b) Ventral view of an E15.5
wild-type (WT) hindbrain (a) over the facial area
(inset) imaged at higher magnification (b) during a
burst of activity of the e-pF (red outline) and the
nVII (blue outline). (c) Traces showing the fast
and synchronized rhythmic fluorescence changes
of the e-pF (red) and nVII (blue) with electrical
activity of the XIIn (black). (d,e) Whole-mount
double immunolabeling for NK1R (green) and
Islet1/2 (red) over one facial area (d,NK1Ronly).
The inset (white rectangle) shows the lateral
aspect of the nVII at a higher magnification (e).
(f,g) Single transverse sections, taken at the level
indicated by the arrow in d, were double immuno-
labeled for NK1R (green) and Islet1/2 (red) in f,
and for Phox2b (green) and islet1/2 (red) in g.
(h–n)Egr2 null mutant (data are presented as
in a–g) showing the more rostral position of the
facial area, owing to the absence of rhombomeric
segment 5 (h), and the lacking activity of the
e-pF (i,j) associated with maintained, but slower,
synchronous rhythmic motor activities of the
nVII and XIIn (j). The absence of the e-pF in the
mutant was associated with a loss (arrowheads) of NK1R+and Phox2b+neurons lateral and ventral to the nVII (k,l,n). Dotted lines in aand hindicate the
caudal limit of migrating pontine neurons. Scale bars represent 200 mm.
e-pF cell
20 mV
0.5 s
20 mV
0.5 s 0.2 s
0.2 s
50 pA
50 pA
40 pA
Control
Bic
10 ms
40 pA
10 ms
= 1.72 ± 0.06 ms
= 8.85 ± 0.76 ms
(ms)
= 1.78 ± 0.23 ms
= 7.39 ± 0.23 ms
Vh = –40 mV
Vh = –40 mV
e-pF cell
–50 mV
–50 mV
PreBötC
int
PreBötC
int
25
20
15
Frequency (%)
10
5
0
25
20
15
Frequency (%)
10
5
0
0 2 4 6 8 10 12 14 16
(ms)
0 2 4 6 8 10 12 14 16
abcg
def
Figure 5 The e-pF oscillator at E15.5 comprises two types of neurons. (a,d) Membrane potential trajectories (top traces) of two representative e-pF cells at
E15.5 and preBo
¨tC population activity (bottom traces) reveal that e-pF neurons were either excited (a), discharged a burst of action potential or were inhibited
(d), showing a pause in firing, during preBo
¨tC rhythmic bursts. Red traces show the average membrane potential trajectory and integrated activity of the
preBo
¨tC corresponding to ten superimposed cycles (black traces) locked on the onset of the preBo
¨tC bursts (vertical gray lines and arrowheads). (b,e) In voltage
clamp mode, during preBo
¨tC bursts of activity (gray background), excited (b) and pausing (e) e-pF cells showed a barrage of prominently inward (black
triangles) and outward (white triangles) synaptic currents, respectively. (c) In excited e-pF cells, inward synaptic currents had fast kinetics. The top set of
traces shows five superimposed inward currents (black triangle, black traces) and their average (red trace), which we used to derive the decay time constant (t)
after single exponential fit. The bottom set of traces shows the same analysis for slow outward events collected in between bursts (white triangle). (f)Datafora
pausing e-pF cell are presented as in c.(g) Histograms showing that the bimodal frequency distribution of t’s in one pausing e-pF cell (n¼118 events) in
control (top histogram) transformed under bicuculline into a modal distribution (bottom histogram) owing to preservation of fast (n¼132 events), but not
slow, synaptic events. Whole-cell recordings were performed using a low (6 mM) chloride pipette solution.
∆F/F
A
M
D
M
∆F/F
∆F/F 2%
∆F/F 4%
10 s
10 s
0
4
0
2
Wild type
Egr2
–/–
e-pF
Xlln
nVll
e-pF
Xlln
nVll
cg
h
j
kl m
n
i
abdef
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DISCUSSION
We have identified a neuronal population in the mouse hindbrain that
shows spontaneous rhythmic activity as early as E14.5. The e-pF
oscillator, composed of a small number of neurons (B300 per side),
constitutes, to the best of our knowledge, the earliest hindbrain
neuronal population showing a continuous spontaneous rhythmic
activity (with a period in the second range) that affects the develop-
ment of the RRG. Cells of the e-pFoscillator are confined to the surface
of the hindbrain and are derived from Egr2-expressing cells. We also
found that the e-pF oscillator cells expressed the homeodomain factor
Phox2b, a transcription factor that is expressed and required in
neuronal types that maintain bodily homeostasis through the reflex
control of digestive, cardiovascular and respiratory functions32.Inthe
rat, Phox2b is expressed by the chemosensitive cells of the pFRG in
neonates20 and of the RTN in adults18. Our data now extend the role of
Phox2b in the specification of visceral reflex circuit11,fromfirstand
second order sensory neurons (including the RTN19), noradrenergic
centers or efferent elements, to a set of interneurons linked to the
inception of the fetal respiratory rhythm.
Recurrent glutamatergic synaptic inputs are essential for establish-
ment of the rhythmic inspiratory drive in preBo
¨tC neurons1,25,33,34.
This is not the case in the e-pF, where we found that rhythm generation
was maintained in several conditions that impaired glutamatergic
transmission. We provide pharmacological evidence that the e-pF
rhythm at E14.5 relies on INaP and is modulated by Ih, although the
cellular distribution of HCN channels and the allosteric regulations35
ensuring their activation in e-pF cells remain unknown. Furthermore,
intercellular synchronization in the parafacial region appears to require
communication through gap junctions (CBX sensitive) and probably
not through hemichannels (La3+ insensitive). This contrasts with
synchronization of parafacial regions across the midline that relies on
glutamatergic synaptic transmission mediated by AMPA/kainate recep-
tors. Because bilateral coactivation is absent in facial transverse slices,
the commissural system involved probably resides beyond the slice
limits. One may argue that the preBo
¨tC, which is sensitive to CNQX1,
has built-in commissural connectivity36,37, and is entrained by and
couples to the e-pF, could take part in setting up this synchrony.
However, no functional commissural connectivity was found for the
preBo
¨tC at E14.5, when bilateral coactivation of the e-pF is overt28.
Hence, the possibility remains that there is another unknown com-
missural system or, more simply, that some of the glutamatergic e-pF
cells bear commissural axons coursing outside the anterior-posterior
limits of the slice.
The preBo
¨tC oscillator is spared in Egr2 null mutants, in which the
e-pF oscillator does not form, indicating that the latter is not required
for the emergence of the former. Thus, together with previous evidence
that the preBo
¨tC can develop in an isolated context38, our data indicate
that rhythmogenic circuits in the vicinity of branchiomotor nuclei
(e-pF/nVII, preBo
¨tC/nucleus ambiguous) are deployed independently
at pre- and post-otic levels and connect to form the RRG. The indepen-
dent emergence of the e-pF and preBo
¨tC is consistent with speculations
that they have distinct evolutionary origins; the pFRG would have
appeared first during the evolution of vertebrates, possibly co-opted
when abdominal expiration was combined with buccal pumping in
amphibians39, whereas the preBo
¨tC would have emerged later, with the
mammalian aspiration pump31,40–42.
Three independent lines of evidence (acute riluzole application,
transverse sections and Egr2 invalidation) argue that the inactiva-
tion of the e-pF oscillator slows down the E15.5 respiratory-like
rhythm. We previously proposed that the abnormally slow breathing
phenotype of Egr2 null mutants at birth was a result of the
suppression of a rhythm-promoting system that we tentatively
located in the caudal pontine reticular formation6. The present
data indicate that the e-pF is a prime contender for setting the
neonatal breathing pace at a normal frequency. Hence, at E15.5 in
the mouse, a RRG with a dual organization emerges, in which the
e-pF can be considered to contribute in that it increases the rhythm
frequency2, whereas the preBo
¨tC can be considered essential in that
it entrains the motor output30.
In addition to its anatomical layout of and its expression of Phox2b,
several functional properties indicate that the e-pF may be considered
as a forerunner of the neonatal pFRG. First, similar to the pFRG at
neonatal stages, the e-pF upregulates the respiratory-like rhythm2and
drives the incorporation of motor bursts in a quantal manner31,41 at
E15.5. Second, the e-pF at E15.5 includes cells receiving a barrage of
chloride-mediated synaptic currents in phase with the RRG rhythmic
bursts. These inputs, excitatory during the E14.5–E15.5 period28,43,
may transiently contribute to the phasing of the e-pF to the preBo
¨tC,
but will cause inhibition during inspiration and post-inhibitory
rebound excitation on later maturation of the chloride gradient43,
two features associated with pFRG neurons2,44. Third, our preliminary
results indicate that the frequency of the e-pF (but not that of the
preBo
¨tC) oscillator is increased by a low pH challenge at E14.5
(Supplementary Fig. 3), suggesting a role in chemosensitivity that is
consistent with the demonstration that Phox2b expressing neurons of
the pFRG are CO2-sensitive in the neonatal rat20. Neurons ofthe pFRG,
unlike those of the e-pF, show a pre-inspiratory pattern of discharge
(from E19–20 onward in the rat)44. Even though the e-pF cells can also
be described as being pre-active at the earliest stages (E14.5), when their
associated calcium changes precede those in facial motoneurons, this
delay progressively disappears within 24 h as the e-pF and preBo
¨tC
oscillators couple with one another. Thereafter (at E15.5–16.5), the
rhythmic activities of the e-pF, the preBo
¨tC and the motor nerves are
synchronous. Thus, by E15.5 (that is, the time when it begins pacing
fetal breathing), the RRG produces a respiratory-like rhythm charac-
terized by a single inspiratory-like phase28,45,46. How pre-inspiratory
depolarization later arises in e-pF cells, thereby transforming them into
pFRG neurons, remains obscure, as does the location of the presynaptic
inhibitory interneurons. Nonetheless, these data argue that the e-pF
oscillator is the forerunner of the neonatal pFRG.
A reasonable assumption is that the neonatal pFRG may evolve into
the adult RTN20,47. In this context, the e-pF oscillator could represent
yet another, earlier developmental stage of this same entity. Pacemaking
mechanisms relying on INaP and modulation by Ih, present in the e-pF,
may exist only transiently during the postnatal maturation of rhythmic
neural circuits24. Their partial downregulation in cells destined to form
the adult RTN may account for the advent of their characteristic tonic
firing mode18,19.Egr2 null mutants were reported to preserve a
ventilatory response to hypercapnia at birth6. In contrast, the Phox2-
b27Ala/+ mutant, a mouse model for CCHS, has both a severe and
selective loss of Phox2b/NK1R double-positive neurons at E15.5 in a
region encompassing the e-pF oscillator and a complete lack of
sensitivity to hypercapnia16. These findings, together with ours,
would be best explained if the Egr2-dependent, rhythm-promoting
e-pF represented a subset of a larger chemosensitive Phox2b-positive
population. Examining the contribution to the RTN of progenitor
domains producing Phox2b interneurons48,49 outside of Krox20-
expressing rhombomeres should allow for a better understanding of
this potential heterogeneity.
In conclusion, we propose that a two-step developmental process
establishes fetal breathing in mice. In the first step, an Egr2-dependent
e-pF neuronal population clusters at the ventral surface of the
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hindbrain and functions as an oscillator. In a second step, at around
E15.5, a second oscillator, the preBo
¨tC, emerges independently and the
e-pF couples with and entrains it. In this manner, the dual organization
of the RRG is established at the time of inception of fetal breathing.
Early e-pF rhythms preceding the first breathing movements appear
to be of clinical, developmental and evolutionary relevance, and are
required for the respiratory rhythm generator to pace breathing at a
normal frequency during the perinatal period. The potential implica-
tion of this early stage of respiratory development in breathing
disorders, particularly in CCHS, should be considered.
METHODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
We thank J.F. Brunet and C. Goridis for comments on the manuscript and the
Phox2b antibody, and K. Kullander for VGlut2 mutant mice. This work benefited
from the facilities and expertise of the Imagif Cell Biology Unit of the Gif
campus. This work was supported by the Centre National de la Recherche
Scientifique,Institut National de la Sante
´et de la Recherche Me
´dicale (M.T.-B.)
and ANR grant ANR-07-Neuro-007-01 (G.F.).
AUTHOR CONTRIBUTIONS
M.T.-B. and G.F. designed, performed research and analyzed data, M.K. carried
out immunostaining on Egr2 mutants and N.W. performed pharmacological
treatments at E15.5. J.C. and P.C. helped with interpreting results and G.F. wrote
the manuscript.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprintsandpermissions/.
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NATURE NEUROSCIENCE VOLUME 12
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NUMBER 8
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AUGUST 2009 1035
ARTICLES
© 2009 Nature America, Inc. All rights reserved.
ONLINE METHODS
Mouse l ines. All of the mouse lines used in this study were maintained in a
mixed C57Bl6/DBA2 background. The Egr2lacZ allele carries an in-frame inser-
tion of the lacZ coding sequence in the second exon of Egr2 (ref. 50). In the
Egr2cre/+ allele, the Egr2 coding sequence was substituted by the Cre recombi-
nase coding sequence. The R26R-EYFP mouse line22, which allows Cre-
mediated activation of EYFP expression, was kindly provided by F. Costantini
(Columbia University). The day of the vaginal plug was considered E0.5. All
experiments were carried out in accordance with National (JO 87–848) and
European legislation (86/609/CEE) on animal experimentation.
Hindbrain electrophysiology. Pregnant mice were killed by cervical dislocation
at E14.5–E16.5. Embryos were excised from the uterus and kept in oxygenated
artificial cerebrospinal fluid (aCSF) at 20 1C until they were used in electro-
physiological and optical recordings sessions. aCSF was composed of 120 mM
NaCl, 8 mM KCl, 1.26 mM CaCl2, 1.5 mM MgCl2, 21 mM NaHCO3,0.58mM
Na2HPO4and 30 mM glucose (pH ¼7.4). For low pH aCSF, the NaHCO3
concentration was decreased to 10.5 mM and the NaCl concentration was
increased to 130.5 mM. The pH of the superfusate was measured continuously
in the recording chamber with a microelectrode (MI-410, Microelectrodes)
calibrated with standard buffers. A high external [K+] was purposefully used
to ensure maintenance of the functional mode of the preBo
¨tC, as previously
described28, at the time of its emergence to optimally detect early network
interactions modulating the preBo
¨tC activity, although this may have generally
increased neuronal baseline excitability.
En bloc hindbrain preparations were prepared as described previously28 and
were positioned with the ventral side up in the recording chamber. Transverse
450-mm-thick slices were obtained at the level of the nVII or at the level of
the preBo
¨tC28 using a vibratome and were transferred into a 1-ml recording
chamber that was continuously superfused at 2 ml min1with oxygenated
aCSF at 30 1C. The caudal limit of the facial motor nucleus was used as an
anatomical landmark to generate transverse facial slices and preBo
¨tC slices.
To obtain transverse facial slices, we generated a series of 150-mm-thick slices
progressing from caudal to rostral up to the first planes where the presence of the
facial nucleus could be unambiguously distinguished as a result of its higher
optical refringence compared with that of the neighboring tissue. At this point, a
single 450-mm-transverse slice was cut that had an anterior limit corresponding
approximately to the equatorial transverse plan of the facial motor nucleus.
Facial slices were transferred to the recording chamber and positioned with
the anterior side up. For preBo
¨tC slices, the slicing was carried out as described
above, but progressing from rostral to caudal, up to the last plane of the facial
nucleus. At this point, a 250-mm-thickslicewasmadetoreachtheanteriorlimit
of the preBo
¨tC, and a single 450-mm-thick transverse preBo
¨tc slice was produced.
Hypoglossal nerve root activity and population activity (e-pF or preBo
¨tC)
in whole hindbrain preparations were recorded using glass micropipettes
suction electrodes (150-mm tip diameter). For e-pF recordings in whole hind-
brain preparations, the electrodes were positioned on the brain surface, and
for the recording of the preBo
¨tC, the electrodes were progressively inserted at
depths of about 100–150 mm below the hindbrain surface, sufficient to record
the activity of the preBo
¨tC. The micropipettes filled with aCSF were connected
through silver wires to a high-gain alternating current amplifier (Grass, 7P511),
filtered (bandwidth, 3 Hz through 3 kHz), integrated using an electronic filter
(Neurolog System, time constant of 100 ms), recorded on a computer via a
digitizing interface (Digidata 1322A, Molecular Devices) and analyzed with the
pClamp9 software (Molecular Devices). Whole-cell patch-clamp neuronal
recordings were performed under visual control using differential interference
contrast (DIC) and infrared video microscopy, an Axoclamp2A amplifier
(Molecular Devices), a digitizing interface (Digidata 1322A, Molecular Devices)
and the software program pClamp9 (Molecular Devices). Patch electrodes
(resistance of 4–6 MO) were pulled from borosilicate glass tubes (Clark GC
150TF) and filled with a solution containing 140 mM potassium gluconic acid,
1mMCaCl
2,6mMH
2O, 10 mM EGTA, 2 mM MgCl2,4mMNa
2ATP a nd
10 mM HEPES (pH 7.2). In voltage-clamp mode, we analyzed the persistent
sodium current INaP and the hyperpolarization-activated current Ih.INaP was
activated using a slow depolarizing ramp from 60 mV to +10 mV and blocked
by 5–20 mMriluzole(Fig. 2d). The Ihcurrent was evoked by applying
hyperpolarizing voltage steps (from 50 mV to 120 mV) and was blocked
by 100 mM ZD7288. The Ihcurrent/voltage curve was built by measuring the
difference between current amplitudes values measured at the beginning and
the end of each voltage step (Fig. 2f). In current clamp, the Ihcurrent activation
led to depolarizing sags in response to hyperpolarizing current pulses (Fig. 2f).
Drugs were obtained from Sigma, dissolved in aCSF and bath-applied for
10–15 min a final concentration of 0.1 mM Substance P (SP), 0.3 mM DAMGO,
10 mM CNQX, 10 mMAP5,10mMbicuculline,5mM strychnine, 5–20 mM
Riluzole), 50 mMCBX,100mM ZD7288 and 100 mM lanthanum. To minimize
the risk of nonspecific effects resulting from long-term exposure to riluzole,
ZD7288, La3+ and CBX, we measured their effects during a 2-min period
beginning 5 min after switching on the perfusion source containing the tested
compounds, a delay that is approximately tenfold larger than the time constant
of concentration change kinetics of the chamber. In addition, when examining
rhythm generation in the e-pF at E14.5, nonspecific effects of these compounds
that were linked to interactions with glutamatergic transmission, being dis-
pensable, were probably minimal. We examined whether (e-pF cell recordings,
n¼4, data not shown) 50 mM CBX had an effect on the amplitude and kinetics
of action potential and on the kinetics of synaptic currents. Values are given as
means ± s.e.m. Differences were regarded as significant at Po0.05.
Calcium imaging. Whole hindbrain and slices were incubated for 40 min in
oxygenated aCSF containing the cell-permeable calcium indicator dye Calcium-
Green 1AM (10 mM, Molecular Probes). Whole hindbrain preparations were
positioned in the recording chamber with the ventral side up. After a 30-min
recovery period in the recording chamber to wash out the dye excess, a
standard epi-fluorescent illumination system on an E-600-FN upright micro-
scope (Nikon) equipped with a fluorescein filter block was used to excite the
dye and capture the emitted light. Fluorescence images were captured with a
cooled CCD camera (Coolsnap HQ, Photometrics) using an exposure time of
100 ms in overlapping mode (simultaneous exposure and readout) during
periods of 60–180 s and analyzed using Metamorph software (Universal
Imaging). To perform calcium imaging of YFP-expressing cells, we first
acquired images of EYFP cells with corresponding DIC images. After dye
loading, a careful positioning over the same cellular field was ascertained
through visualizing cellular profiles using DIC images and adjusting their
alignments. The EYFP-labeled cell false-colored red image and the calcium-
loaded cell false-colored green image were overlaid (Fig. 1g)todetermine
double-labeled somas and to position regions of interest for measurements of
fluorescence changes (Fig. 1g). In all cases, the average intensity in a region of
interest was calculated for each frame and the changes in fluorescence were
normalized to their initial value by expression as the ratio of changes in
fluorescence to initial fluorescence (F/F).
Immunofluorescence. Mouse embryos were fixed for 2–3 h in 4% para-
formaldehyde (wt/vol) in phosphate-saline buffer, cryoprotected in 25% sucrose
(wt/vol), embedded in O.C.T. Compound (Tissue-Tek) and sectioned at 14 or
20 mm. Immunohistochemistry was performed on frozen sections as previously
described28. We used antibodies to rabbit NK1R (Sigma, 1:5,000), guinea pig
Islet1/2 (gift from J. Ericson, Karolinska Institute, 1:1500), chick GFP (Aves Lab,
1:2,000) and rabbit Phox2b (gift from C. Goridis, ENS, 1:1,500). Species
specific antibodies conjugated to Alexa 488, (Molecular Probes), Cy3 and
Cy5 (Jackson ImmunoResearch) were used. Biocytin-filled neurons were
labeled using Extra-Avidin-FITC (Sigma, 1:400). Rocking incubation with
primary antibodies was carried out overnight at 4 1C and secondary antibodies
were incubated for 3 h. The material was mounted in Vectashield medium
(Vector Labs). Counts of e-pF cells were made in an area delimited ventrally by
the medullary surface, dorsally by the nVII, rostrally by the rostral end of the
nVII and extending 100 mm caudal to the nVII. Cells were counted in the
transverse plan using all consecutive 20-mmsections(n¼2) or every other
section (n¼1) and in the sagittal plane on every other section (n¼1). In all
cases, cells were counted on both sides; estimation of the total number of cells
included a multiplication factor of 2 when appropriate. Fluorescent labeling
was visualized on a Leica SP2 confocal microscope. All figures were color
corrected and assembled using Adobe Photoshop and Illustrator.
50. Voiculescu, O. et al. Hindbrain patterning: Krox20 couples segmentation and specifica-
tion of regional identity. Development 128, 4967–4978 (2001).
NATURE NEUROSCIENCE doi:10.1038/nn.2354
© 2009 Nature America, Inc. All rights reserved.
