 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 15, 2003, 23(4):1478
A Novel Functional Neuron Group for Respiratory Rhythm Generation
in the Ventral Medulla
Hiroshi
Onimaru and
Ikuo
Homma
Showa University School of Medicine, Shinagawa-ku, Tokyo 142-8555, Japan
 |
ABSTRACT |
We visualized respiratory neuron activity covering the entire
ventral medulla using optical recordings in a newborn rat
brainstem-spinal cord preparation stained with voltage-sensitive dye.
We measured optical signals from several seconds before to several
seconds after the inspiratory phase using the inspiratory motor nerve discharge as the trigger signal; we averaged the optical signals of
50-150 respiratory cycles to obtain an optical image correlating particularly to inspiratory activity. The optical images we obtained from the ventral approach indicated that neuron activity first appeared
during the respiratory cycle in the limited region of the rostral
ventrolateral medulla (RVLM), preceding the onset of inspiratory
activity by ~500 msec. During the inspiratory phase, plateau activity
appeared in the more caudal ventrolateral medulla at the level of
the most rostral roots of the XIIth nerve. Comparison with
electrophysiological recordings from respiratory neurons in the RVLM
suggested that the optical signals preceding the inspiratory burst
reflect preinspiratory neuron activity in this area. This RVLM area was
determined to be ventrolateral to the facial nucleus and close to the
ventral surface. We referred to this functional neuron group as the
para-facial respiratory group (pFRG). Partial, bilateral
electrical lesioning of the pFRG significantly reduced the respiratory
frequency, together with changes in the spatiotemporal pattern of
respiratory neuron activity. Our findings suggest that the pFRG
comprises a neuronal population that is involved in the primary
respiratory rhythm generation in the rostrocaudally extending respiratory neuron network of the medulla.
Key words:
respiratory rhythm; optical imaging; voltage-sensitive dye; ventral medulla; neonatal rat; in
vitro
| |
Introduction |
The neural circuit generating
respiratory rhythm in mammals is located in the medulla of the lower
brainstem. The main groups of medullary respiratory neurons are thought
to be distributed rostrocaudally in the ventrolateral parts of the
medulla, and they play a role in respiratory rhythm and inspiratory
pattern generation (Feldman, 1986 ; von Euler, 1986; Bianchi et al.,
1995; Richter and Spyer, 2001). However, there are still arguments over the fundamental question of which parts of the ventrolateral medulla are essential for primary rhythm generation (Ballanyi et al., 1999;
Richter and Spyer, 2001), and the initiation site of respiratory rhythm
is still unknown. The brainstem-spinal cord preparation isolated from
newborn rats preserves the neuron networks of the respiratory center
essential for respiratory rhythm generation in vitro (Suzue,
1984; Ballanyi et al., 1999). For understanding the macroscopic
behavior of the rostrocaudally extending respiratory neuron network in
the medulla, optical recordings (Cohen and Lesher, 1986; Grinvald et
al., 1988; Momose-Sato et al., 2001) can be very effective. Indeed,
respiratory neuron network activity in the medulla of this preparation
is detectable as an optical image by observation from the ventral
surface after the preparation is stained with a voltage-sensitive dye
(Tokumasu et al., 2001), but detailed analysis of the starting points
of the rhythmic respiratory activity has been difficult because of an
inadequate signal-to-noise ratio of the optical signals. In the present
study, we used a new optical recording system (Tominaga et al., 2000)
that provides for data acquisition by the summation of optical signals
from a certain pretriggering point. The system allows us to analyze neuronal activity correlating to spontaneous activity used as trigger
signals. In brainstem-spinal cord preparations stained with a
voltage-sensitive dye, we measured optical signals from several seconds
before to several seconds after the inspiratory phase using inspiratory
motor nerve discharge as the trigger signal. We found that
preinspiratory population activity occurs in the localized region
ventrolateral to the facial nucleus. Partial bilateral electrical
lesioning of this limited region caused a significant reduction in
respiratory frequency, implying that the neuronal population in this
area may be important in respiratory rhythm generation.
| |
Materials and Methods |
Preparation and staining. Wistar rats
(n = 43), 0-1 d of age, were deeply anesthetized with
ether until the nociceptive reflexes were abolished. Usually,
respiratory movement halted temporarily at this level of anesthesia.
The cerebrum was quickly removed by transection at the intercollicular
level, and the brainstem and spinal cord were isolated according to
methods described previously (Suzue, 1984; Onimaru et al., 1988). The
brainstem was rostrally decerebrated between the VIth cranial nerve
roots and the lower border of the trapezoid body. In some experiments,
the caudal half of the pons was retained, attached to the facial nerve
roots. The brainstem-spinal cord preparation was incubated in a
modified Krebs solution (described below) containing a fluorescent
voltage-sensitive dye (Tominaga et al., 2000), Di-4-ANEPPS or
Di-2-ANEPEQ (Molecular Probes, Eugene, OR). In
preliminary experiments, excessive staining with these dyes decreased
respiratory frequency and changed the spatiotemporal pattern of
respiratory neuron activity. Therefore, the concentration of dye and
the incubation period were carefully adjusted so that there would be no
significant reduction in respiratory frequency: 30-45 min with 0.2 mg/ml Di-4-ANEPPS or 30-55 min with 50 µg/ml Di-2-ANEPEQ. After
being stained, the preparation was placed with the ventral surface up
in a 1 ml perfusion chamber, which was mounted on a fluorescence
microscope (BX50WIF-2; Olympus Optical, Tokyo, Japan). The
preparation was superfused continuously at 2-3 ml/min with a modified
Krebs solution consisting of (in mM): 124 NaCl,
5.0 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgCl2, 26 NaHCO3, and 30 glucose and equilibrated with 95%
O2 and 5% CO2, pH 7.4, at
26-27°C.
Inspiratory motoneuron activity, which was used as the trigger
signal for optical recordings, was monitored at the IVth cervical (C4)
ventral root with a glass capillary suction electrode. This C4 activity
is known to synchronize with discharges of phrenic nerves, which are
derived from the C4 and C5 ventral roots (Suzue, 1984). The electrical
signal was stored through a 0.5 Hz high-pass filter in digital memory
together with the optical imaging data. The onset of inspiratory
activity was defined by visual inspection as the steepest rising point
immediately before a peak of C4 activity, the time at which most
inspiratory neurons start to fire. The frequency of spontaneous
inspiratory activity from the nerve roots (respiratory frequency) was
6.3 ± 1.7 min 1, and the burst
duration was 990 ± 170 msec (n = 32).
For antidromic activation of the facial nucleus, the facial nerve was
stimulated through a glass capillary suction electrode by application
of a 0.1 msec, 15-20 V single pulse. Electrical lesioning was
performed through a tungsten electrode with a 30 µm tip diameter by
applying a 100 msec, 20 µA pulse. The extent of the lesions was
verified histologically in 100 µm transverse sections stained with
neutral red.
Optical recordings. Neuronal activity in the preparation was
detected as a change in fluorescence of the voltage-sensitive dye by
means of an optical recording apparatus (MiCAM01; Brain Vision Inc.,
Tsukuba, Japan; see also Tominaga et al., 2000) through a 510-550 nm
excitation filter, a dichroic mirror, and a 590 nm absorption filter
(U-MWIG2 mirror unit; Olympus Optical) with a
tungsten-halogen lamp (150 W) as the light source. The CCD-based camera head has an 8.4 × 6.5 mm2
imaging area consisting of 180 × 120 pixels, with a maximum time resolution of 3.5 msec. Magnification of the microscope was adjusted to
2× (or 4×) in most experiments so that an area of 4.2 × 3.25 mm2 (or 2.1 × 1.6 mm2) was covered by the image sensor. In
our preliminary experiments, we did not find clear differences in
optical signals from different depths of focus ranging from 0 (surface)
to 300 µm. Therefore, we focused the microscope objective on the
ventral surface plane of the brainstem-spinal cord preparation or on
the surface plane of the rostral cross section.
Most recordings were performed with an acquisition time of 20 msec.
Fluorescence signals for 6.8 sec per trial, including 1.7 sec before
the initiation of the inspiratory burst, were totaled and averaged
50-150 times; C4 inspiratory activity was used as the trigger. During
recordings, the respiratory frequency was not changed significantly, so
the photodynamic toxicity was negligible. The fluorescence changes were
expressed as a ratio (percentage, fractional changes) (i.e., the
fluorescence intensity against that of the reference image). The
differential image, processed with a software-spatial filter for 2 × 2 pixels, was represented by a pseudocolor display in which red
corresponded to a fluorescence decrease, meaning membrane
depolarization. For representation of the time course of the
fluorescence change in the region of interest, optical signals were
inverted. As discussed previously (Tokumasu et al., 2001), optical
signals corresponding to spontaneous respiratory neuron activity were
thought to reflect fluorescence changes primarily accompanying the
subthreshold membrane depolarization (i.e.; respiration-related
excitatory drive potential) in the soma but not each action potential
in the soma or axon during burst activity.
Membrane potential recordings of respiratory neurons.
Membrane potentials of respiratory neurons in the rostral ventrolateral medulla (RVLM) were recorded by means of conventional whole-cell patch-clamp methods (Onimaru and Homma, 1992). The patch pipette solution contained (in mM): 120 K-gluconate, 1 CaCl2, 1 MgCl2, 10 EGTA, 2 Na2-ATP, and 10 HEPES as well as 0.5% Lucifer
yellow (LY), pH 7.3. The membrane potentials were recorded with a
single-electrode voltage-clamp amplifier (CEZ-3100; Nihon
Kohden, Tokyo, Japan) after compensation for the series
resistance (25-50 M ) and capacitance. In the present study, we
recorded membrane potentials from three types of respiratory neurons:
preinspiratory neurons (Pre-I neurons) showing membrane depolarization
leading to the initiation of spike discharge by up to several hundred
milliseconds before the onset of inspiratory C4 activity, type I
inspiratory neurons showing EPSPs before the onset as well as
after the termination of inspiratory-related nerve bursts, and type II
inspiratory neurons showing EPSPs and spike discharge only during the
inspiratory phase (Onimaru and Homma, 1992, Ballanyi et al., 1999).
Pre-I-I neurons, a subtype of type I inspiratory neurons, which
show spike discharge before and during the inspiratory phase, were also
recorded (Smith et al., 1990; Ballanyi et al., 1999). Spike discharge
of the Pre-I neuron is usually inhibited during the inspiratory phase
and appears again after the termination of the inspiratory burst,
although there are subtypes with different burst patterns (Ballanyi et al., 1999). To compare fluorescence changes in the optical recordings with the membrane potential trajectory, we averaged membrane potentials for 20-30 respiratory cycles (with a sampling rate of 0.2 or 1 kHz)
using C4 activity as the trigger. For study of the location and
morphological properties of neurons stained with LY, preparations were
fixed for >48 hr at 4°C in Lillie solution (10% formalin in
phosphate buffer, pH 7.0), rinsed with 15% sucrose-0.1
M phosphate buffer, pH 7.2, and incubated
overnight in sucrose solution. Transverse 70 µm frozen sections were
then cut with a cryostat. The intracellularly LY-marked neurons were
reconstructed with the aid of a camera lucida attached to a
fluorescence microscope (Olympus Optical).
Values are presented as means ± SD. Significance values
(p < 0.05) were determined with a Student's
t test.
| |
Results |
Optical image of respiratory neuron activity in the
ventral medulla
Typical examples of optical images obtained by the
ventral approach are shown in Figures 1
and 2. The optical records showed neuronal activity preceding, by ~500-600 msec, the inspiratory activity that started in the limited region of the RVLM, 1.3-1.6 mm
lateral to the midline and extending rostrally from the level of the
Xth cranial nerve roots (Figs. 1, second frame,
2B,B'). Activity then spread caudally and medially in
the ventrolateral medulla and some also more rostrally during the
preinspiratory phase (Fig. 1, second and third
frames). Activity in the RVLM reached its peak immediately before
a peak of C4 inspiratory activity and then decreased during the
inspiratory phase (Fig. 2B'). During the inspiratory
phase, plateau activity appeared in the more caudal ventrolateral
medulla at the level of the most rostral roots of the XIIth cranial
nerve and in the ventral horns of the cervical cord. Moreover,
fluorescence changes in the caudal medulla and in the ventral horns
were detected 100-300 msec before the onset of inspiratory activity,
with a smaller amplitude than that of the RVLM. This spatiotemporal
pattern of respiratory neuron activity in the medulla was confirmed in
all other preparations examined (n = 31). We also
confirmed this in optical recordings by data acquisition with a longer
pretriggering period (3.4 sec before inspiratory activity).
View larger version (65K):
[in this window]
[in a new window]
|
Figure 1.
Spatiotemporal pattern of respiratory neuron
activity observed on optical images. The image is superimposed on the
ventral surface of the right half of the medulla. Also see Figure 2 for
the orientation of the preparation. The numbers at the
bottom right of each image denote the time from
the start of C4 inspiratory activity. The tracing under
each image is the C4 activity; the inspiratory phase is indicated by
the light-blue bar. The red vertical line
on the C4 tracing shows the time at which the image was obtained.
Results are the average of 100 respiratory cycles triggered by C4
inspiratory activity. The preparation was stained with 50 µg/ml
Di-2-ANEPEQ for 40 min. The sampling clock is 20 msec. Note that
optical signals reflecting respiratory neuron activity appear in the
RVLM during the preinspiratory phase. During the inspiratory phase,
peak activity appears in the more caudal ventrolateral medulla.
|
|
View larger version (53K):
[in this window]
[in a new window]
|
Figure 2.
Fluorescence changes in the optical recording and
electrophysiological recordings of respiratory neuron activity. The
preparation is the same as in Figure 1 except for D.
A, Ventral aspect of the preparation and the optical
image recording area boxed in blue.
B, Optical image of respiratory neuron activity near the
C4 peak (at dark vertical line in B')
superimposed on the ventral surface of the medulla. IX/X,
XII, Cranial nerves; white dotted lines,
approximate area of the RVLM; B', Fluorescence
changes at three different points indicated by dots in
red, blue, and green
circles on B: red, in the RVLM at
the level just rostral to the IX/Xth cranial roots;
blue, in the more caudal ventrolateral medulla at the
level of the rostral roots of the XIIth cranial nerve; and
green, in the ventral horns of the cervical cord.
Fluorescence decrease (i.e., depolarization) is upward.
C4, Electrical record of C4 inspiratory activity. The
red bar and light-blue bar on the C4
tracing denote the preinspiratory and inspiratory phases, respectively.
Note that the fluorescence change precedes the inspiratory phase in all
of the areas indicated, whereas the amplitude is larger in the RVLM
(red tracing). C, Membrane potential
trajectory (MP) of a Pre-I neuron recorded in the
ventrolateral medulla 1.4 mm lateral to the midline and at the level of
the IX/Xth roots of the same preparation (circle with
cross in B). Three membrane potential traces are
shown, one below the other. The averages of the membrane potential
trajectory and C4 activity for 21 respiratory cycles are also shown
(average, C4). The red
bar and light-blue bar on the average C4 tracing
denote the preinspiratory and inspiratory phases, respectively.
D, An example exhibiting clear postinspiratory activity.
Left, Optical image of respiratory neuron activity in
the postinspiratory phase (dark vertical line at
right) superimposed on the ventral surface of the
right-side medulla. Right, Fluorescence changes
at two different points indicated by dots in
red and blue circles on the left
image: red, in the RVLM at the level just
rostral to the IX/Xth cranial roots; and blue, in the
more caudal ventrolateral medulla near the level of the rostral roots
of the XIIth cranial nerve. Results are the average of 100 respiratory
cycles triggered by C4 inspiratory activity. The preparation was
stained with 0.2 mg/ml Di-4-ANEPPS for 30 min. The sampling clock is 25 msec. Note the short (75 msec) preinspiratory activity and long (~2
sec) postinspiratory activity.
|
|
We focused on the analysis of neuron activity, especially in the RVLM.
The average duration of activity in the preinspiratory phase in the
RVLM was 370 ± 170 msec (range, 75-700 msec; n = 32), which was 3.5% of the respiratory cycle period. The
preinspiratory phase may be shorter if activity of more rostral nerve
roots such as C1 or the XIIth cranial nerve is used as the trigger
signal instead of C4, because the onset of the inspiratory activity of these nerves precedes that of phrenic motoneurons (Smith et al., 1990).
The optical signals in the RVLM preceding inspiratory activity were
thought to reflect activity of the Pre-I neurons in this area (Onimaru
et al., 1987, 1988; Arata et al., 1990). Therefore, we compared the
pattern of optical signals with membrane potential trajectories that
were recorded from Pre-I neurons in the RVLM of the same preparation
after the optical recordings (n = 9). The Pre-I neuron
shown in Figure 2C exhibited a long preinspiratory phase and
a short postinspiratory phase in >80% of the respiratory cycles. The
period for which optical signals were detected preceding the
inspiratory phase corresponded well to the period of time for which the
Pre-I neuron depolarized ~4 mV (see below) from the resting potential
level (Fig. 2C, average). In three optical recordings, postinspiratory activity was also clearly detected (Fig.
2D), whereas the activity was less conspicuous than
that during the preinspiratory phase in other preparations. The results indicate that neuron activity during the postinspiratory phase is less
stable than that of the preinspiratory phase.
In nine Pre-I neurons recorded after optical recordings, preinspiratory
membrane depolarization in the averaged trajectory was detectable as
early as 1240 ± 310 msec preceding the inspiratory onset.
Detectable changes in fluorescence during the preinspiratory phase in
the optical recordings of the same preparations appeared as early as
620 ± 70 msec before the inspiratory onset. It is reasonable that
there would be a difference in the period between detectable membrane
potential change and detectable fluorescence change during the
preinspiratory phase, because optical detection may require a certain
threshold voltage change in weak signal intensity (e.g., spontaneous
activity). We estimated this threshold voltage to be ~4 mV, because
the period indicating subthreshold depolarization >4 mV from the
resting level during the preinspiratory phase was 590 ± 150 msec
in the averaged membrane potential trajectory. We also recorded
membrane potentials from seven inspiratory neurons in the RVLM area
similar to the Pre-I neuron recording site after optical recordings.
Figure 3 shows examples of the averaged
membrane potential trajectory of a typical Pre-I-I neuron (as an
example of an inspiratory neuron with prolonged preinspiratory
activity), a Pre-I neuron (Fig. 3A), and type II inspiratory
neuron (Fig. 3B) together with fluorescence changes in the
previously performed optical recordings in the same preparation. These
and other recordings showed that fluorescence changes in the rostral
medulla had fairly good correlation with membrane potential
trajectories of Pre-I neurons, whereas those in the caudal medulla fit
better with those of the inspiratory neurons.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 3.
Comparison between fluorescence changes in optical
recordings and averaged membrane potential trajectories of different
types of respiratory neurons. A, B, top, Fluorescence
changes at two different points similar to Figure
2B or D: thick
line, in the RVLM at the level just rostral to the IX/Xth
cranial roots; thin line, in the more caudal
ventrolateral medulla near the level of the rostral roots of the XIIth
cranial nerve. Results are the average of 50 (in A) or
100 (in B) respiratory cycles triggered by C4
inspiratory activity. Fluorescence decrease (i.e., depolarization) is
upward. The preparation was stained with 50 µg/ml Di-2-ANEPEQ for 45 min in A or 43 min in B. The sampling
clock is 20 msec. Gray bars on the average C4 tracing
(C4) denote the inspiratory phase. A, B,
bottom, Averaged membrane potential trajectories from a Pre-I
neuron, a Pre-I-I type inspiratory neuron, and a type II inspiratory
neuron (Insp-II), recorded in the ventrolateral
medulla 1.3-1.4 mm lateral to the midline and at the level of the
IX/Xth roots. C4, Averaged C4 activity. The
vertical dotted lines in A and
B denote the time of peak C4 activity.
|
|
The RVLM area in which strong preinspiratory optical signals were
detected seems to overlap laterally with the facial nucleus. We
confirmed this anatomically by examining the level of the facial nucleus in 100 µm serial sections dissected from the preparations. Furthermore, we confirmed the location by superimposing the active area
on the optical images of the facial nucleus activated antidromically by
facial nerve stimulation (n = 5). The active area was
thought to correspond to the entire anatomical location of the ventral part of the facial nucleus. The result clearly showed that the main
active area during the preinspiratory phase overlapped to the lateral
edge of the facial nucleus (Fig. 4).
View larger version (98K):
[in this window]
[in a new window]
|
Figure 4.
Optical images of respiratory neuron
activity superimposed on the area of the facial nucleus.
A, The dark blue area in each image
indicates the facial nucleus of the right side of the medulla, which
was activated antidromically by stimulation of the right facial nerve.
Purple denotes a fluorescence change below -0.05%. The
numbers at the bottom left of each
image denote the time from the start of C4 inspiratory activity. The
tracing under each image is the C4 activity; the
inspiratory phase is indicated by the light-blue bar.
The red vertical line on the C4 tracing shows the time
at which the image was obtained. The preparation was stained with 50 µg/ml Di-2-ANEPEQ for 40 min. Results are the average of 50 respiratory cycles triggered by C4 inspiratory activity. The sampling
clock is 30 msec. Note that optical signals during the preinspiratory
phase appear to overlap to the lateral edge of the facial nucleus.
IX/X, XII, Cranial nerves. B, Diagram of
the ventral aspect of the preparation and the optical image recording
area boxed in blue. VII,
Facial nerve.
|
|
Optical signals and respiratory neurons in the ventral superficial
region of the rostral medulla
One major technical problem of this optical measurement method is
how to determine the depth of the location for neurons from which optical signals originate. We approached this problem by first
examining the depth of diffusion of the voltage-sensitive dye into the
tissue by transversely dissecting the rostral medulla after the
preparation was stained (50 µg/ml Di-2-ANEPEQ, 45-50 min). We then
observed the cut surface, monitoring C4 inspiratory activity. In these
experiments (n = 5), optical signals corresponding to
respiratory activity were detected with 0.013% fractional change even
at a depth of 500 µm from the ventral surface, where the dye
concentration was reduced to ~60% of that of the ventral superficial region (data not shown). The results indicated that the dye diffused into the tissue at a concentration sufficient to allow for the detection of spontaneous neuron activity at a depth of 500 µm. This
is considered the maximum depth for the detection of spontaneous neuron
activity in the present system, because the fluorescence from such a
deep region might be weakened because of tissue lying in the emission
light path in the approach from the ventral surface.
Respiratory neurons may also be located in regions of the medulla that
are deeper than the detectable depth by the ventral approach. Thus, to
view respiratory neuron activity in the RVLM from a different
direction, we observed the cut surface of the medulla stained with a
voltage-sensitive dye after transverse sectioning at the rostral
medulla (n = 5). The optical image showed that neuron
activity started at the limited region ventrolateral to the facial
nucleus and close to the ventral surface during the preinspiratory
phase and then propagated medially and dorsally during the inspiratory
phase (Fig. 5A). Major
activity during the inspiratory phase overlapped to the facial nucleus.
Thus, the optical recordings in the region of the facial motor nucleus from the ventral approach (Fig. 4) may in part reflect that of facial
motoneurons, although the signal was rather weak, probably because of
the difficulty in detecting activity from deeper cells. These
observations suggest that the major cluster of neurons active during
the preinspiratory phase is distributed close to the ventral surface
such that the optical signals are detectable by the ventral approach.
This was also supported by the fact that with comparatively weak
staining with voltage-sensitive dye (e.g., for 40 min with 0.1 mg/ml
Di-4-ANEPPS), the optical signals were detected primarily in the RVLM,
in contrast to weak signals coming from the caudal ventrolateral
medulla (data not shown). That is, a longer incubation time (or a
higher dye concentration) for the staining was required for clear
detection of optical signals in the caudal ventrolateral medulla during
the inspiratory phase compared with those in the RVLM during the
preinspiratory phase.
View larger version (86K):
[in this window]
[in a new window]
|
Figure 5.
Optical images of respiratory neuron activity
obtained by observation from the cut surface of a cross section of the
rostral medulla. A, Spatiotemporal pattern of optical
images of respiratory neuron activity superimposed on the cut surface
of the rostral medulla. The numbers at the bottom
right of each image denote the time from the start of C4
inspiratory activity. The tracing under each image is
the C4 activity; the inspiratory phase is indicated by the
light-blue bar. The vertical red line on
the C4 tracing shows the time at which the top image was
obtained. Results are the average of 100 respiratory cycles triggered
by C4 inspiratory activity. The sampling clock is 20 msec. Note that
optical signals reflecting respiratory neuron activity appear in the
part ventrolateral to the facial nucleus (top
left, white dotted line) during the
preinspiratory phase (arrows). B,
Schematic representation of the preparation. The preparation was cut
transversely at the level (indicated by a dotted line)
rostral to the IX/Xth cranial nerve roots and then stained with 0.1 mg/ml Di-4-ANEPPS for 56 min. It was then fixed onto a rubber block
with pins and cut surface up, for observation from the cut surface of
the cross section (arrows). C, Pre-I
neurons in the part ventrolateral to the facial nucleus. The neuron
activities were recorded in the whole-cell configuration in the same
type of preparations as illustrated in B by approaching
from the rostral cut surface. Red dots indicate
locations of recorded neurons. Neurons were found in the reticular
formation ventrolateral to the facial nucleus, and some neurons were
also found in the ventrolateral edge of the facial nucleus.
Dotted lines indicate demarcation between some
anatomical structures. FN, Facial nucleus;
CST, corticospinal tract; STN, spinal
trigeminal nucleus, oral part. D, An example of
reconstruction of a Pre-I neuron stained with LY. The
arrow denotes the site of soma located ventrolateral to
the facial nucleus. The axon could not be traced. The averaged membrane
potential trajectory is shown in the bottom right with
C4 activity.
|
|
To confirm the detailed location of Pre-I neurons in the region
ventrolateral to the facial nucleus, we marked them with LY and plotted
them after whole-cell patch-clamp recordings. Pre-I neurons were found
in the reticular formation ventrolateral to the facial nucleus as well
as in the ventrolateral edge of the facial nucleus (Fig.
5C,D). The resting membrane potential ranged from  47 to
55 mV (49.2 ± 2.5 mV; n = 11), and the input
resistance was 443 ± 328 M. During the recording procedure, we
also found five inspiratory neurons and two expiratory neurons in the
same region, but we did not analyze their electrophysiological
properties in detail.
Effects of partial electrical lesioning in the RVLM
For help in evaluating the functional role of the respiratory
neuron group in the RVLM, we examined the effects of partial electrical
lesioning of the limited area in the RVLM in which the strong
fluorescence change was detected before the onset of inspiratory activity. After unilateral lesioning, the fluorescence change in the contralateral RVLM was still seen clearly during the
preinspiratory phase (Fig.
6B). This optical
signal was markedly depressed (<12% of control) after subsequent
partial lesioning of the remaining side (Fig. 6C). Thus,
after partial, bilateral lesioning of the RVLM, major fluorescence
changes were detected only during the inspiratory phase in the caudal
ventrolateral medulla at the most rostral level of the XIIth cranial
nerve and in the ventral horn of the cervical cord. We confirmed that
these lesioned areas were limited in the superficial ventrolateral
medulla to the facial nucleus (Fig. 6D). The C4 burst
rate did not decrease with unilateral lesioning (5.7 ± 1.6/min in
control; 5.5 ± 1.9/min after unilateral lesioning;
n = 8). In the preparations that indicated a relatively
irregular C4 rhythm in the control (Fig. 6A), the rhythm became more stable after unilateral lesioning. Then subsequent partial lesioning of the other side caused a significant decrease in
the C4 rate (2.1 ± 0.7/min; p < 0.001; 10-15
min after the lesioning). The burst duration of C4 activity was
990 ± 210 msec in control, 760 ± 138 msec after unilateral
lesion, and 1110 ± 298 msec after partial bilateral lesioning.
The decrease in burst duration after unilateral lesioning was not
significant; the increase after bilateral lesioning was significant
(p < 0.05) compared with that after unilateral
lesioning.
View larger version (63K):
[in this window]
[in a new window]
|
Figure 6.
Effects of partial electrical lesioning of the
RVLM on C4 rhythm and spatiotemporal pattern of respiratory neuron
activity. A, C4 activity and optical records in control.
B, C4 activity and optical records after partial
lesioning of three points (arrows) in the right side of
the RVLM. C, C4 activity and optical records after
subsequent partial lesioning of a point (arrow) in the
left side of a similar rostral medulla. C4, Inspiratory
C4 activity. A-C, left, Optical images of respiratory
neuron activity near the C4 onset (dark vertical line at
right) superimposed on the ventral surface of the
medulla. A-C, right, Fluorescence changes at two points
indicated on each right panel: red, in
the RVLM at the level just rostral to the IX/Xth cranial roots;
blue, in the more caudal ventrolateral medulla at the
level of the rostral roots of the XIIth cranial nerve; the inspiratory
phase is indicated by the light-blue bar. Fluorescence
decrease (i.e., depolarization) is upward. The preparation was stained
with 50 µg/ml Di-2-ANEPEQ for 50 min. Results are the average of 50 respiratory cycles triggered by C4 inspiratory activity. The sampling
clock is 20 msec. Note that preinspiratory activity recorded optically
in the intact side is still clearly observed after partial lesioning of
the right side of the rostral medulla (B). After
subsequent partial lesioning of the other side, preinspiratory activity
recorded optically is markedly depressed and the C4 rate is reduced
(arrow on the C4 record), whereas the duration of
inspiratory activity in the caudal medulla as well as that of C4
activity are prolonged. D, Histological verification of
the electrical lesioning; 100 µm transverse section. The lesions
indicated by dotted lines and
arrows are confirmed in the limited area ventrolateral
to the facial nucleus (FN).
|
|
| |
Discussion |
Technical limitations
The basic ability of the present optical recording system to
measure neuron activity is described in detail by Tominaga et al.
(2000). Here, we discuss only specific technical problems for measuring
respiratory neuron activity in the brainstem preparation. The amplitude
of optical signals likely depends on many factors: the type of
preparation (especially its transparency), the concentration of dye in
the tissue, and the density of active (synchronized) neurons (for
review, see Grinvald et al., 1988). In the present study, we examined
the depth of diffusion of the voltage-sensitive dye into the tissue by
observing the cut surface of a transverse section at the rostral
medulla after the preparation was stained. The results suggested that
the spontaneous neuron activity that was detectable in the ventral
approach must be at a depth <500 µm from the ventral surface.
Therefore, our measurement may not detect neuron activity in deeper
regions. Moreover, it should be noted that optical signals from
superficial cells could be detected with larger amplitude and earlier
onset than those from deeper cells, even if these cells were active
over a similar time course. Thus, staining with voltage-sensitive dye
for a longer time or with a higher concentration of dye is required for
the detection of neuron activity in deeper regions (for example,
inspiratory neuron activity in the caudal medulla versus Pre-I neuron
activity in the rostral medulla; see below). However, the staining
process should be carefully controlled, because excessive staining
actually depresses neuron activity (Grinvald et al., 1988; our
unpublished observation). On the basis of comparison between averaged
membrane potential trajectory and optical signals, we presumed that the present system could detect a subthreshold membrane potential change
(of spontaneously bursting-medullary respiratory neurons) of >4 mV as
an optical signal.
Spatiotemporal pattern of respiratory neuron activity in the
ventral medulla
The present optical recording study clearly localized
respiration-related activity to the limited region of the RVLM,
ventrolateral to the facial nucleus, which was characterized by a
change in fluorescence preceding the inspiratory burst. We found that
the fluorescence change during the preinspiratory phase in this area correlated well with the averaged membrane potential trajectory of
Pre-I neurons, suggesting that the optical signal reflects Pre-I neuron
activity in this area. On the contrary, many Pre-I neurons are
repolarized with varied strength during the inspiratory phase (Onimaru
and Homma, 1992). Therefore, the activity of Pre-I neurons exhibiting
only minor repolarization during the inspiratory phase may contribute
to the optical signal in the RVLM during the inspiratory phase; the
signal appeared with a decrease to only a certain level. In addition,
the optical signals may also reflect inspiratory neuron activity in
this area. We confirmed that Pre-I neurons as well as inspiratory
neurons were located in the superficial region ventrolateral to the
facial nucleus. Thus, we propose to refer to this respiratory neuron
group as the para-facial respiratory group (pFRG). Although precise
identification of the anatomical structure of the pFRG remains for
future study, the medial part of the pFRG may overlap to the
retrotrapezoid nucleus (RTN), which has been identified in the cat and
the rat as an area in which neurons with projections to the ventral
respiratory group (VRG) are found (Smith et al., 1989; Ellenberger and
Feldman, 1990; Nunez-Abades et al., 1993) and the presence of
respiratory neurons are reported (Pearce et al., 1989;
Connelly et al., 1990). Because recent studies have suggested that the
RTN plays a role in central chemoreception (Bodineau et al., 2000;
Nattie, 2001), the pFRG may receive tonic drive from central
chemoreceptors. The major population of the pFRG seems to be lateral to
the RTN, although it was originally described to extend more laterally at rostral levels of the facial nucleus (Smith et al., 1989). Therefore, it is important to determine whether the pFRG neurons project directly to the VRG. The caudal part of the pFRG overlaps to
the rostral part of the ventrolateral medulla, which has been examined electrophysiologically in previous studies (i.e., the ventral
part of the retrofacial nucleus near the caudal end of the facial
nucleus) (Onimaru et al., 1987; Arata et al., 1990). Thus, the present
findings show that the respiratory activity in the rostral medulla
extends more rostrally and laterally than known previously.
During the inspiratory phase, corresponding plateau activity appeared
in a region more caudal and medial than the pFRG. This optical signal
may primarily reflect activity of an inspiratory neuron group close to
the ventral surface near this level of the medulla, as shown in Figure
13 of Smith et al. (1990). The caudal inspiratory activity became more
conspicuous with longer voltage-sensitive dye staining than in the case
of pFRG. This suggests that the distribution of neurons active during
the inspiratory phase in the caudal medulla extends to a deeper region
below the ventral surface. Fluorescence changes of small amplitude were
also detected 100-300 msec before the onset of inspiratory activity in
the caudal medulla. Such signals possibly reflect activity of type I
inspiratory neurons (Onimaru and Homma, 1992) and also some Pre-I
neurons in this area (Arata at al., 1990; Smith et al., 1990). This
caudal area may correspond to the level of (or slightly rostral to) the preBötzinger complex, which has attracted attention recently as
the proposed kernel of respiratory rhythm generation (Smith et al.,
1991; Rekling and Feldman, 1998; see below).
Functional considerations
There are a substantial number of studies suggesting that neurons
in the preBötzinger complex are necessary and sufficient for the
generation of respiratory-related motor rhythms (Smith et al., 1991;
Rekling and Feldman, 1998; Koshiya and Smith, 1999). There are also
neurons in the preBötzinger complex that exhibit prominent
preinspiratory depolarization and spike activity in both the presence
and absence of the pFRG (Del Negro et al., 2001). In vivo
selective lesioning of the preBötzinger complex neurons with
neurokinin-1 receptors disrupts the rhythm, indicating that this
area is crucial for rhythm generation in vivo (Gray et al., 2001). It has been shown that respiratory rhythm persists in the brainstem-spinal cord preparation after removal of the pFRG but not
after removal of the preBötzinger complex (Smith et al., 1991).
Moreover, slice preparations from the caudal medulla including the
preBötzinger complex (but not the pFRG) produce respiratory-like neuronal bursts (Smith et al., 1991). However, McLean and Remmers (1994) reported that transection of the medulla just caudal to the Xth
cranial nerve root, corresponding to the caudal border of the pFRG,
significantly decreased the burst frequency. We also confirmed this in
the experiments combined with optical recordings (our
unpublished observations).
The optical images in the present study revealed that respiratory
neuron activity appeared first from the pFRG in the rostrocaudally extending respiratory neuron network in the ventral medulla. However, this finding itself does not necessarily mean that the pFRG functions as the initiation site of respiratory rhythm. For instance, it is
possible that neurons in the pFRG exhibit their activity as a result of
receiving excitatory input from other neuron groups located outside the
area in which the activity could be detected with the present optical
recording. Nevertheless, partial bilateral lesioning of the pFRG area
caused a significant reduction in the respiratory rate together with
changes in the spatiotemporal pattern of the respiratory neuron
activity. This supports the notion that the pFRG plays an important
role in respiratory rhythm generation. Previous studies have shown that
bilateral lesions in the caudal ventrolateral medulla, which seems to
be identical to the active caudal area revealed in the present optical
recording, cause inspiratory activity to disappear, whereas the
rhythmic burst of Pre-I neurons in the more rostral medulla was
preserved (Onimaru et al., 1988). Consequently, it is possible that
rhythmically active Pre-I neurons in the pFRG interact with
preBötzinger complex neurons as a coupled oscillator system to
regulate the rhythm of the intact system.
| |
FOOTNOTES |
Received Sept. 10, 2002; revised Nov. 13, 2002; accepted Dec. 2, 2002.
This work was supported by grants-in-aid for Scientific Research from
the Ministry of Education, Science, and Culture of Japan. We thank F. Kato, Y. Kubo, and K. Ballanyi for comments on this manuscript.
Correspondence should be addressed to Dr. Hiroshi Onimaru, Department
of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai,
Shinagawa-ku, Tokyo 142-8555, Japan. E-mail: oni{at}med.showa-u.ac.jp.
| |
References |
-
Arata A,
Onimaru H,
Homma I
(1990)
Respiration-related neurons in the ventral medulla of newborn rats in vitro.
Brain Res Bull
24:599-604[Web of Science][Medline].
-
Ballanyi K,
Onimaru H,
Homma I
(1999)
Respiratory network function in the isolated brainstem-spinal cord of newborn rats.
Prog Neurobiol
59:583-634[Web of Science][Medline].
-
Bianchi AL,
Denavit-Saubie M,
Champagnat J
(1995)
Central control of breathing in mammals: neuronal circuitry, membrane properties, and neurotransmitters.
Physiol Rev
75:1-45[Free Full Text].
-
Bodineau L,
Cayetanot F,
Frugiere A
(2000)
Possible role of retrotrapezoid nucleus and parapyramidal area in the respiratory response to anoxia: an in vitro study in neonatal rat.
Neurosci Lett
295:67-69[Web of Science][Medline].
-
Cohen LB,
Lesher S
(1986)
Optical monitoring of membrane potential: methods of multisite optical measurement.
In: Optical methods in cell physiology (De Weer P,
Salzberg BM,
eds), pp 71-99. New York: Wiley.
-
Connelly CA,
Ellenberger HH,
Feldman JL
(1990)
Respiratory activity in retrotrapezoid nucleus in cat.
Am J Physiol
258:L33-L44[Abstract/Free Full Text].
-
Del Negro CA,
Johnson SM,
Butera RJ,
Smith JC
(2001)
Models of respiratory rhythm generation in the pre-Bötzinger complex. III. Experimental tests of model predictions.
J Neurophysiol
86:59-74[Abstract/Free Full Text].
-
Ellenberger HH,
Feldman JL
(1990)
Brainstem connections of the rostral ventral respiratory group of the rat.
Brain Res
513:35-42[Web of Science][Medline].
-
Feldman JL
(1986)
Neurophysiology of breathing in mammals.
In: Handbook of physiology. The nervous system. Intrinsic regulatory systems of the brain. Sect 1 (Bloom FE,
ed), pp 463-524. Bethesda, MD: American Physiological Society.
-
Gray PA,
Janczewski WA,
Mellen N,
McCrimmon DR,
Feldman JL
(2001)
Normal breathing requires preBötzinger complex neurokinin-1 receptor-expressing neurons.
Nat Neurosci
4:927-930[Web of Science][Medline].
-
Grinvald A,
Frostig RD,
Lieke E,
Hildesheim R
(1988)
Optical imaging of neuronal activity.
Physiol Rev
68:1285-1366[Free Full Text].
-
Koshiya N,
Smith JC
(1999)
Neuronal pacemaker for breathing visualized in vitro.
Nature
400:360-363[Medline].
-
McLean HA,
Remmers JE
(1994)
Respiratory motor output of the sectioned medulla of the neonatal rat.
Respir Physiol
96:49-60[Web of Science][Medline].
-
Momose-Sato Y,
Sato K,
Kamino K
(2001)
Optical approaches to embryonic development of neural functions in the brainstem.
Prog Neurobiol
63:151-197[Web of Science][Medline].
-
Nattie EE
(2001)
Central chemosensitivity, sleep, and wakefulness.
Respir Physiol
129:257-268[Web of Science][Medline].
-
Nunez-Abades PA,
Morillo AM,
Pasaro R
(1993)
Brainstem connections of the rat ventral respiratory subgroups: afferent projections.
J Auton Nerv Syst
42:99-118[Web of Science][Medline].
-
Onimaru H,
Homma I
(1992)
Whole-cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats.
Pflügers Arch
420:399-406[Web of Science][Medline].
-
Onimaru H,
Arata A,
Homma I
(1987)
Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations from newborn rats.
Neurosci Lett
78:151-155[Web of Science][Medline].
-
Onimaru H,
Arata A,
Homma I
(1988)
Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat.
Brain Res
445:314-324[Web of Science][Medline].
-
Pearce RA,
Stornetta RL,
Guyenet PG
(1989)
Retrotrapezoid nucleus in the rat.
Neurosci Lett
101:138-142[Web of Science][Medline].
-
Rekling JC,
Feldman JL
(1998)
PreBötzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation.
Annu Rev Physiol
60:385-405[Web of Science][Medline].
-
Richter DW,
Spyer KM
(2001)
Studying rhythmogenesis of breathing: comparison of in vivo and in vitro models.
Trends Neurosci
24:464-472[Web of Science][Medline].
-
Smith JC,
Morrison DE,
Ellenberger HH,
Otto MR,
Feldman JL
(1989)
Brainstem projections to the major respiratory neuron populations in the medulla of the cat.
J Comp Neurol
281:69-96[Web of Science][Medline].
-
Smith JC,
Greer JJ,
Liu G,
Feldman JL
(1990)
Neural mechanisms generating respiratory pattern in mammalian brainstem-spinal cord in vitro.
J Neurophysiol
64:1149-1169[Abstract/Free Full Text].
-
Smith JC,
Ellenberger HH,
Ballanyi K,
Richter DW,
Feldman JL
(1991)
Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals.
Science
254:726-729[Abstract/Free Full Text].
-
Suzue T
(1984)
Respiratory rhythm generation in the in vitro brain stemspinal cord preparation of the neonatal rat.
J Physiol (Lond)
354:173-183[Abstract/Free Full Text].
-
Tokumasu M,
Nakazono Y,
Ide H,
Akagawa K,
Onimaru H
(2001)
Optical recording of spontaneous respiratory neuron activity in the rat brain stem.
Jpn J Physiol
51:613-619[Web of Science][Medline].
-
Tominaga T,
Tominaga Y,
Yamada H,
Matsumoto G,
Ichikawa M
(2000)
Quantification of optical signals with electrophysiological signals in neural activities of Di-4-ANEPPS stained rat hippocampal slices.
J Neurosci Methods
102:11-23[Web of Science][Medline].
-
von Euler C
(1986)
Brainstem mechanisms for generation and control of breathing pattern.
In: Handbook of physiology. The respiratory system. Sect 3 (Fishman AP,
ed), pp 1-67. Bethesda, MD: American Physiological Society.
Copyright © 2003 Society for Neuroscience 0270-6474/03/2341478-09$05.00/0
This article has been cited by other articles:
|
|
|
|
 
V. Dubreuil, M. Thoby-Brisson, M. Rallu, K. Persson, A. Pattyn, C. Birchmeier, J.-F. Brunet, G. Fortin, and C. Goridis
Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in Phox2b Mutants Targeting Retrotrapezoid Nucleus Neurons
J. Neurosci.,
November 25, 2009;
29(47):
14836 - 14846.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Champagnat, M. P. Morin-Surun, G. Fortin, and M. Thoby-Brisson
Developmental basis of the rostro-caudal organization of the brainstem respiratory rhythm generator
Phil Trans R Soc B,
September 12, 2009;
364(1529):
2469 - 2476.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Dubreuil, J. Barhanin, C. Goridis, and J.-F. Brunet
Breathing with Phox2b
Phil Trans R Soc B,
September 12, 2009;
364(1529):
2477 - 2483.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Smith, A. P. L. Abdala, I. A. Rybak, and J. F. R. Paton
Structural and functional architecture of respiratory networks in the mammalian brainstem
Phil Trans R Soc B,
September 12, 2009;
364(1529):
2577 - 2587.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. M. Spyer and A. V. Gourine
Chemosensory pathways in the brainstem controlling cardiorespiratory activity
Phil Trans R Soc B,
September 12, 2009;
364(1529):
2603 - 2610.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. L. Abdala, I. A. Rybak, J. C. Smith, and J. F. R. Paton
Abdominal expiratory activity in the rat brainstem\#8211;spinal cord in situ: patterns, origins and implications for respiratory rhythm generation
J. Physiol.,
July 15, 2009;
587(14):
3539 - 3559.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. B. G. Abbott, R. L. Stornetta, M. G. Fortuna, S. D. Depuy, G. H. West, T. E. Harris, and P. G. Guyenet
Photostimulation of Retrotrapezoid Nucleus Phox2b-Expressing Neurons In Vivo Produces Long-Lasting Activation of Breathing in Rats
J. Neurosci.,
May 6, 2009;
29(18):
5806 - 5819.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Amano, M. Fujii, S. Arata, T. Tojima, M. Ogawa, N. Morita, A. Shimohata, T. Furuichi, S. Itohara, H. Kamiguchi, et al.
DSCAM Deficiency Causes Loss of Pre-Inspiratory Neuron Synchroneity and Perinatal Death
J. Neurosci.,
March 4, 2009;
29(9):
2984 - 2996.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Onimaru, K. Ikeda, and K. Kawakami
CO2-Sensitive Preinspiratory Neurons of the Parafacial Respiratory Group Express Phox2b in the Neonatal Rat
J. Neurosci.,
November 26, 2008;
28(48):
12845 - 12850.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Wittmeier, G. Song, J. Duffin, and C.-S. Poon
Pacemakers handshake synchronization mechanism of mammalian respiratory rhythmogenesis
PNAS,
November 18, 2008;
105(46):
18000 - 18005.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pagliardini, J. Ren, P. A. Gray, C. VanDunk, M. Gross, M. Goulding, and J. J. Greer
Central Respiratory Rhythmogenesis Is Abnormal in Lbx1- Deficient Mice
J. Neurosci.,
October 22, 2008;
28(43):
11030 - 11041.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Homma and Y. Masaoka
Breathing rhythms and emotions
Exp Physiol,
September 1, 2008;
93(9):
1011 - 1021.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. G. Guyenet
The 2008 Carl Ludwig Lecture: retrotrapezoid nucleus, CO2 homeostasis, and breathing automaticity
J Appl Physiol,
August 1, 2008;
105(2):
404 - 416.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. Takakura, T. S. Moreira, R. L. Stornetta, G. H. West, J. M. Gwilt, and P. G. Guyenet
Selective lesion of retrotrapezoid Phox2b-expressing neurons raises the apnoeic threshold in rats
J. Physiol.,
June 15, 2008;
586(12):
2975 - 2991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. T. S. Pattinson
Opioids and the control of respiration
Br. J. Anaesth.,
June 1, 2008;
100(6):
747 - 758.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Giraudin, M.-J. Cabirol-Pol, J. Simmers, and D. Morin
Intercostal and Abdominal Respiratory Motoneurons in the Neonatal Rat Spinal Cord: Spatiotemporal Organization and Responses to Limb Afferent Stimulation
J Neurophysiol,
May 1, 2008;
99(5):
2626 - 2640.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. A. Gray
Transcription factors and the genetic organization of brain stem respiratory neurons
J Appl Physiol,
May 1, 2008;
104(5):
1513 - 1521.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. F. Ireland, F. C. Lenal, A. R. Lorier, D. E. Loomes, T. Adachi, T. S. Alvares, J. J. Greer, and G. D. Funk
Distinct receptors underlie glutamatergic signalling in inspiratory rhythm-generating networks and motor output pathways in neonatal rat
J. Physiol.,
May 1, 2008;
586(9):
2357 - 2370.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. G. Guyenet, R. L. Stornetta, and D. A. Bayliss
Retrotrapezoid nucleus and central chemoreception
J. Physiol.,
April 15, 2008;
586(8):
2043 - 2048.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Thoby-Brisson and J. J. Greer
Anatomical and functional development of the pre-Botzinger complex in prenatal rodents
J Appl Physiol,
April 1, 2008;
104(4):
1213 - 1219.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Ruangkittisakul, S. W. Schwarzacher, L. Secchia, Y. Ma, N. Bobocea, B. Y. Poon, G. D. Funk, and K. Ballanyi
Generation of Eupnea and Sighs by a Spatiochemically Organized Inspiratory Network
J. Neurosci.,
March 5, 2008;
28(10):
2447 - 2458.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. G. Fortuna, G. H. West, R. L. Stornetta, and P. G. Guyenet
Botzinger Expiratory-Augmenting Neurons and the Parafacial Respiratory Group
J. Neurosci.,
March 5, 2008;
28(10):
2506 - 2515.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Butler and S. C. Gandevia
The output from human inspiratory motoneurone pools
J. Physiol.,
March 1, 2008;
586(5):
1257 - 1264.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Zanella, F. Watrin, S. Mebarek, F. Marly, M. Roussel, C. Gire, G. Diene, M. Tauber, F. Muscatelli, and G. Hilaire
Necdin Plays a Role in the Serotonergic Modulation of the Mouse Respiratory Network: Implication for Prader-Willi Syndrome
J. Neurosci.,
February 13, 2008;
28(7):
1745 - 1755.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
V. Dubreuil, N. Ramanantsoa, D. Trochet, V. Vaubourg, J. Amiel, J. Gallego, J.-F. Brunet, and C. Goridis
A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons
PNAS,
January 22, 2008;
105(3):
1067 - 1072.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Smith, A. P. L. Abdala, H. Koizumi, I. A. Rybak, and J. F. R. Paton
Spatial and Functional Architecture of the Mammalian Brain Stem Respiratory Network: A Hierarchy of Three Oscillatory Mechanisms
J Neurophysiol,
December 1, 2007;
98(6):
3370 - 3387.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Moreira, A. C. Takakura, E. Colombari, and P. G. Guyenet
Activation of 5-Hydroxytryptamine Type 3 Receptor-Expressing C-Fiber Vagal Afferents Inhibits Retrotrapezoid Nucleus Chemoreceptors in Rats
J Neurophysiol,
December 1, 2007;
98(6):
3627 - 3637.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Taccola, L. Secchia, and K. Ballanyi
Anoxic persistence of lumbar respiratory bursts and block of lumbar locomotion in newborn rat brainstem spinal cords
J. Physiol.,
December 1, 2007;
585(2):
507 - 524.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Oku, H. Masumiya, and Y. Okada
Postnatal developmental changes in activation profiles of the respiratory neuronal network in the rat ventral medulla
J. Physiol.,
November 15, 2007;
585(1):
175 - 186.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Ruangkittisakul, L. Secchia, T. D. Bornes, D. M. Palathinkal, and K. Ballanyi
Dependence on extracellular Ca2+/K+ antagonism of inspiratory centre rhythms in slices and en bloc preparations of newborn rat brainstem
J. Physiol.,
October 15, 2007;
584(2):
489 - 508.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Onimaru, K. Ikeda, and K. Kawakami
Defective interaction between dual oscillators for respiratory rhythm generation in Na+,K+-ATPase {alpha}2 subunit-deficient mice
J. Physiol.,
October 1, 2007;
584(1):
271 - 284.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kepecs, N. Uchida, and Z. F. Mainen
Rapid and Precise Control of Sniffing During Olfactory Discrimination in Rats
J Neurophysiol,
July 1, 2007;
98(1):
205 - 213.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. W. Pace, D. D. Mackay, J. L. Feldman, and C. A. Del Negro
Inspiratory bursts in the preBotzinger complex depend on a calcium-activated non-specific cation current linked to glutamate receptors in neonatal mice
J. Physiol.,
July 1, 2007;
582(1):
113 - 125.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. R. McCrimmon and G. F. Alheid
Reflexively inhibiting respiratory drive
J. Physiol.,
April 1, 2007;
580(1):
3 - 3.
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. S. Moreira, A. C. Takakura, E. Colombari, G. H. West, and P. G. Guyenet
Inhibitory input from slowly adapting lung stretch receptors to retrotrapezoid nucleus chemoreceptors
J. Physiol.,
April 1, 2007;
580(1):
285 - 300.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. M. Stettner, P. Huppke, C. Brendel, D. W. Richter, J. Gartner, and M. Dutschmann
Breathing dysfunctions associated with impaired control of postinspiratory activity in Mecp2-/y knockout mice
J. Physiol.,
March 15, 2007;
579(3):
863 - 876.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. J. Barnes, C.-M. Tuong, and N. M. Mellen
Functional Imaging Reveals Respiratory Network Activity During Hypoxic and Opioid Challenge in the Neonate Rat Tilted Sagittal Slab Preparation
J Neurophysiol,
March 1, 2007;
97(3):
2283 - 2292.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. L. Horner and T. D. Bradley
Update in Sleep and Control of Ventilation 2006
Am. J. Respir. Crit. Care Med.,
March 1, 2007;
175(5):
426 - 431.
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Iigaya, H. Kumagai, H. Onimaru, A. Kawai, N. Oshima, T. Onami, C. Takimoto, T. Kamayachi, K. Hayashi, T. Saruta, et al.
Novel axonal projection from the caudal end of the ventrolateral medulla to the intermediolateral cell column
Am J Physiol Regulatory Integrative Comp Physiol,
February 1, 2007;
292(2):
R927 - R936.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Funke, M. Dutschmann, and M. Muller
Imaging of respiratory-related population activity with single-cell resolution
Am J Physiol Cell Physiol,
January 1, 2007;
292(1):
C508 - C516.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Ruangkittisakul, S. W. Schwarzacher, L. Secchia, B. Y. Poon, Y. Ma, G. D. Funk, and K. Ballanyi
High Sensitivity to Neuromodulator-Activated Signaling Pathways at Physiological [K+] of Confocally Imaged Respiratory Center Neurons in On-Line-Calibrated Newborn Rat Brainstem Slices.
J. Neurosci.,
November 15, 2006;
26(46):
11870 - 11880.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Voituron, A. Frugiere, J. Champagnat, and L. Bodineau
Hypoxia-sensing properties of the newborn rat ventral medullary surface in vitro
J. Physiol.,
November 15, 2006;
577(1):
55 - 68.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. L. Stornetta, T. S. Moreira, A. C. Takakura, B. J. Kang, D. A. Chang, G. H. West, J. F. Brunet, D. K. Mulkey, D. A. Bayliss, and P. G. Guyenet
Expression of Phox2b by Brainstem Neurons Involved in Chemosensory Integration in the Adult Rat
J. Neurosci.,
October 4, 2006;
26(40):
10305 - 10314.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Y. Fong and J. T. Potts
Neurokinin-1 receptor activation in Botzinger complex evokes bradypnoea
J. Physiol.,
September 15, 2006;
575(3):
869 - 885.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Kubin, G. F. Alheid, E. J. Zuperku, and D. R. McCrimmon
Central pathways of pulmonary and lower airway vagal afferents
J Appl Physiol,
August 1, 2006;
101(2):
618 - 627.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. A. Janczewski and J. L. Feldman
Novel Data Supporting the Two Respiratory Rhythm Oscillator Hypothesis. Focus on "Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats"
J Neurophysiol,
July 1, 2006;
96(1):
1 - 2.
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Onimaru, Y. Kumagawa, and I. Homma
Respiration-Related Rhythmic Activity in the Rostral Medulla of Newborn Rats
J Neurophysiol,
July 1, 2006;
96(1):
55 - 61.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Onimaru, I. Homma, J. L. Feldman, and W. A. Janczewski
Point:Counterpoint: The parafacial respiratory group (pFRG)/pre-Botzinger complex (preBotC) is the primary site of respiratory rhythm generation in the mammal
J Appl Physiol,
June 1, 2006;
100(6):
2094 - 2098.
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. T. Takakura, T. S. Moreira, E. Colombari, G. H. West, R. L. Stornetta, and P. G. Guyenet
Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2-sensitive neurons in rats
J. Physiol.,
April 15, 2006;
572(2):
503 - 523.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Kawai, H. Onimaru, and I. Homma
Mechanisms of CO2/H+ chemoreception by respiratory rhythm generator neurons in the medulla from newborn rats in vitro
J. Physiol.,
April 15, 2006;
572(2):
525 - 537.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-C. Viemari and J.-M. Ramirez
Norepinephrine Differentially Modulates Different Types of Respiratory Pacemaker and Nonpacemaker Neurons
J Neurophysiol,
April 1, 2006;
95(4):
2070 - 2082.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. K. Tryba, F. Pena, and J.-M. Ramirez
Gasping activity in vitro: a rhythm dependent on 5-HT2A receptors.
J. Neurosci.,
March 8, 2006;
26(10):
2623 - 2634.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. N. Fisher, V. A. Marchenko, A. G. Yodh, and R. F. Rogers
Spatiotemporal Activity Patterns During Respiratory Rhythmogenesis in the Rat Ventrolateral Medulla
J Neurophysiol,
March 1, 2006;
95(3):
1982 - 1991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. J. Greer, G. D. Funk, and K. Ballanyi
Preparing for the first breath: prenatal maturation of respiratory neural control
J. Physiol.,
February 1, 2006;
570(3):
437 - 444.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. A. Janczewski and J. L. Feldman
Distinct rhythm generators for inspiration and expiration in the juvenile rat
J. Physiol.,
January 15, 2006;
570(2):
407 - 420.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. G. Guyenet
Novel two-rhythm generator theory of breathing in mammals
J. Physiol.,
January 15, 2006;
570(2):
207 - 207.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Soliz, V. Joseph, C. Soulage, C. Becskei, J. Vogel, J. M. Pequignot, O. Ogunshola, and M. Gassmann
Erythropoietin regulates hypoxic ventilation in mice by interacting with brainstem and carotid bodies
J. Physiol.,
October 15, 2005;
568(2):
559 - 571.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. G. Guyenet, D. K. Mulkey, R. L. Stornetta, and D. A. Bayliss
Regulation of Ventral Surface Chemoreceptors by the Central Respiratory Pattern Generator
J. Neurosci.,
September 28, 2005;
25(39):
8938 - 8947.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Y. Masaoka, N. Koiwa, and I. Homma
Inspiratory phase-locked alpha oscillation in human olfaction: source generators estimated by a dipole tracing method
J. Physiol.,
August 1, 2005;
566(3):
979 - 997.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Thoby-Brisson, J.-B. Trinh, J. Champagnat, and G. Fortin
Emergence of the Pre-Botzinger Respiratory Rhythm Generator in the Mouse Embryo
J. Neurosci.,
April 27, 2005;
25(17):
4307 - 4318.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pagliardini, T. Adachi, J. Ren, G. D. Funk, and J. J. Greer
Fluorescent Tagging of Rhythmically Active Respiratory Neurons within the Pre-Botzinger Complex of Rat Medullary Slice Preparations
J. Neurosci.,
March 9, 2005;
25(10):
2591 - 2596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Del Negro, C. Morgado-Valle, J. A. Hayes, D. D. Mackay, R. W. Pace, E. A. Crowder, and J. L. Feldman
Sodium and Calcium Current-Mediated Pacemaker Neurons and Respiratory Rhythm Generation
J. Neurosci.,
January 12, 2005;
25(2):
446 - 453.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Ikeda, H. Onimaru, J. Yamada, K. Inoue, S. Ueno, T. Onaka, H. Toyoda, A. Arata, T.-o Ishikawa, M. M. Taketo, et al.
Malfunction of Respiratory-Related Neuronal Activity in Na+, K+-ATPase {alpha}2 Subunit-Deficient Mice Is Attributable to Abnormal Cl- Homeostasis in Brainstem Neurons
J. Neurosci.,
November 24, 2004;
24(47):
10693 - 10701.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. R. McCrimmon and G. F. Alheid
Capra, eupnea, dyspnea, apnea: respiratory rhythms and the pre-Botzinger complex in the goat
J Appl Physiol,
November 1, 2004;
97(5):
1618 - 1619.
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. M. Wenninger, L. G. Pan, L. Klum, T. Leekley, J. Bastastic, M. R. Hodges, T. R. Feroah, S. Davis, and H. V. Forster
Large lesions in the pre-Botzinger complex area eliminate eupneic respiratory rhythm in awake goats
J Appl Physiol,
November 1, 2004;
97(5):
1629 - 1636.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. P. Coutinho, C. Borday, J. Gilthorpe, S. Jungbluth, J. Champagnat, A. Lumsden, and G. Fortin
Induction of a Parafacial Rhythm Generator by Rhombomere 3 in the Chick Embryo
J. Neurosci.,
October 20, 2004;
24(42):
9383 - 9390.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Duffin
Functional organization of respiratory neurones: a brief review of current questions and speculations
Exp Physiol,
September 1, 2004;
89(5):
517 - 529.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. M. Mellen, M. Roham, and J. L. Feldman
Afferent modulation of neonatal rat respiratory rhythm in vitro: cellular and synaptic mechanisms
J. Physiol.,
May 1, 2004;
556(3):
859 - 874.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-C. Viemari, M. Bevengut, P. Coulon, and G. Hilaire
Nasal Trigeminal Inputs Release the A5 Inhibition Received by the Respiratory Rhythm Generator of the Mouse Neonate
J Neurophysiol,
February 1, 2004;
91(2):
746 - 758.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. R. McCrimmon and G. F. Alheid
On the opiate trail of respiratory depression
Am J Physiol Regulatory Integrative Comp Physiol,
December 1, 2003;
285(6):
R1274 - R1275.
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pagliardini, J. Ren, and J. J. Greer
Ontogeny of the Pre-Botzinger Complex in Perinatal Rats
J. Neurosci.,
October 22, 2003;
23(29):
9575 - 9584.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Iizuka
GABAA and glycine receptors in regulation of intercostal and abdominal expiratory activity in vitro in neonatal rat
J. Physiol.,
September 1, 2003;
551(2):
617 - 633.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
Introduction
Compound via MeSH
