Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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Guyenet, Patrice G. and Hong Wang. Pre-Bötzinger Neurons With Preinspiratory Discharges "In Vivo" Express NK1 Receptors in the Rat. J. Neurophysiol. 86: 438-446, 2001. Substance P stimulates respiration, in part by a direct action on the pre-Bötzinger complex (preBötC). This region of the medulla oblongata contains neurons that are strongly immunoreactive for the neurokinin-1 receptor (NK1R-ir), and a recent theory has postulated that these cells might be the adult form of excitatory interneurons that are essential for respiratory rhythmogenesis in neonates. Here we sought to determine whether preBötC respiratory neurons are indeed NK1R-ir in the adult rat. Preinspiratory (pre-I) neurons were recorded in the preBötC region of halothane-anesthetized rats. Most pre-I cells could be antidromically activated from the contralateral side of the medulla (7 of 10; latency 1.3 ± 0.2 ms), suggesting that most of them were propriomedullary neurons rather than respiratory motoneurons or bulbospinal cells. Thirty-two pre-I neurons including seven cells with contralateral projection were labeled with biotinamide using the juxtacellular method. Eleven cells (34.4%) were NK1R-ir, including three of the seven pre-I cells that were antidromically activated from the contralateral side. In 3 control rats we labeled 20 preBötC neurons with patterns of discharge other than pre-I and found that none were detectably NK1R-ir. In conclusion, some of the intensely NK1R-ir neurons of the adult preBötC region are indeed respiratory interneurons as suggested by Gray et al. The subtype of NK1R identified by the antibody is detectable only in a small minority of preBötC respiratory cells, most notably in pre-I interneurons. Given prior anatomical evidence, these NK1R-ir pre-I interneurons are most likely glutamatergic. The data are consistent with the possibility that the NK1R-ir pre-I interneurons of the adult preBötC could be the adult form of a class of inspiratory neurons that are rhythmogenic in the neonate (either the pacemakers and/or an excitatory subtype of follower neurons).
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INTRODUCTION |
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Transverse slices of the
rostral medulla oblongata cut caudal to the retrofacial nucleus (i.e.,
nucleus ambiguus, pars compacta) are capable of generating a
respiratory-like pattern of discharge in neonate rodents (Smith
et al. 1991). The anatomical area critical for rhythm
generation lies within a segment of the ventral respiratory group (VRG)
named the pre-Bötzinger complex (preBötC) (Smith et
al. 1991
). This site contains a group of inspiratory
interneurons with intrinsic bursting properties (Butera et al.
1999a
; Johnson et al. 1994
; Lieske et al.
2000
). These cells (pacemaker neurons) are coupled via
excitatory amino acid-mediated synaptic connections (Koshiya
and Smith 1999
). A plausible model for rhythmogenesis suggests
that the excitatory connections between pacemaker neurons may be
reciprocal and monosynaptic (Butera et al. 1999b
;
Smith et al. 1995
), which would imply that the pacemaker
cells are glutamatergic. According to the same theory, these neurons
constitute the core of a bilaterally distributed pacemaker network that
is the origin of respiratory rhythm generation in vitro (Butera
et al. 1999b
; Ramirez and Richter 1996
). In the
neonate, GABA and glycine-mediated inhibition appears primarily
involved in shaping the respiratory discharges of expiratory and other
neurons located downstream from the rhythm-generating kernel
(Paton and Richter 1995
; Shao and Feldman
1997
). Neurons with intrinsic bursting properties represent
only a fraction of the inspiratory neurons of the neonate preBötC. Most other inspiratory neurons within this region
discharge purely as a result of synaptic inputs. The general term of
follower neurons sometimes given to this class of inspiratory neurons
(Butera et al. 1999b
; Smith et al. 1995
)
reflects the belief that they are situated one or more synapses
downstream from the pacemaker neurons. Presumed follower neurons have a
wide range of intrinsic properties (Rekling et al.
1996
). Functionally, this class of cell may include both
excitatory and inhibitory interneurons and, possibly, cranial
motoneurons. Except for motoneurons, there is at present no defining
neuroanatomical marker for the various classes of inspiratory neurons
that have been recorded in or close to the preBötC.
The region of the preBötC contains propriobulbar rather than
bulbospinal respiratory cells (Dobbins and Feldman 1994;
Ellenberger and Feldman 1990
). This neuroanatomical
characteristic is in contrast with more rostral (Bötzinger
region) and more caudal regions of the VRG (rVRG). In the adult, the
preBötC region of the VRG can also be distinguished from
surrounding segments of the VRG by a complex but characteristic mix of
neurons with respiration-related discharges typified by the presence of
numerous late expiratory-inspiratory (preinspiratory, pre-I) neurons
(Connelly et al. 1992
; Schwarzacher et al.
1995
; Sun et al. 1998
). The phenotype of these
and most other classes of preBötC cells remains conjectural.
Moreover, it is unclear which of these neurons represent the mature
forms of the various types of putative rhythmogenic neurons found in the neonate preBötC.
The preBötC lies in an area of the ventrolateral medulla that has
long been known to contain a high level of substance P receptors
including the NK1 subtype (Beaujouan et al. 1986;
Nakaya et al. 1994
). The preBötC coincides with
the region of the VRG that contains the highest concentration of
strongly NK1R-ir neurons in the adult rat (Gray et al.
1999
; Wang et al. 2001
). Gray et al. (1999)
have speculated that these NK1R-ir cells may be the adult forms of inspiratory excitatory interneurons that generate or
regulate the respiratory rhythm in neonate slices (Gray et al.
1999
).
The present in vivo experiments sought to determine whether the NK1R-ir
neurons of the adult preBötC include respiratory interneurons
that could be the adult equivalent of the rythmogenic neurons
identified in neonate slices. In anesthetized rats we made
extracellular recordings of preBötC neurons that display a pre-I
(late expiratory-inspiratory) pattern of discharge relative to the
phrenic burst (Connelly et al. 1992; Schwarzacher
et al. 1995
). We focused on these cells because the pre-I
pattern actually corresponds to that of the hypoglossal nerve
(Feldman 1986
; Koshiya and Guyenet
1996
), and we reasoned that the rhythmogenic inspiratory interneurons defined in the neonate brain slice (Butera et al. 1999b
; Gray et al. 1999
; Smith et al.
1995
; Thoby-Brisson and Ramirez 2000
;
Thoby-Brisson et al. 2000
) might remain synchronized with the hypoglossal nerve in the adult. The recorded cells were labeled with biotinamide using a juxtacellular technique
(Pinault 1996
; Schreihofer and Guyenet
1997
), and immunohistochemical methods were then used to
determine whether the biotinamide-labeled cells contained NK1R immunoreactivity.
To determine whether NK1R immunoreactivity was confined to the pre-I
neurons, we also recorded from preBötC neurons with other types
of respiratory patterns. A preliminary account of this work has
appeared in abstract form (Wang and Guyenet
2000).
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METHODS |
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All experiments were performed on male Sprague-Dawley rats (250-350 g; Hilltop Laboratories, Scotsdale, PA) in accordance with National Institutes of Health and Institutional Animal Care and Use Guidelines. All procedures and protocols were approved by the University of Virginia's Animal Research Committee.
Recording and juxtacellular labeling of respiratory neurons
Anesthesia was induced with 5% halothane in 100% oxygen. During surgery, the rats were artificially ventilated with 1.6-1.8% halothane in 100% oxygen via a tracheal cannula (45-60 cycles/min; 1-1.2 ml/100 g). End-expiratory CO2 was monitored using infrared spectroscopy (Columbus Instruments, Columbus, OH) and maintained between 4.5 and 5% during surgery. Rectal temperature was kept between 37.5 and 38.5°C with ventral and dorsal heat sources. A femoral artery and a vein were catheterized to record arterial blood pressure (AP) and to administer drugs, respectively. The rats were placed in a stereotaxic frame (David Kopf Instrument, Tujunga, CA) with the mouthpiece set at 3.5 mm below the interaural line. The right phrenic nerve was dissected via a dorsolateral approach, cut, and placed on a bipolar silver wire electrode. The nerve and electrodes were insulated with polyvinylsiloxane impression material (Impramix, Darby Dental Laboratories, Rockville Center, NY). A 5 × 8-mm window was opened in the interparietal plate caudal to the lambdoid suture, and the dura mater was resected to allow electrode penetration. A concentric bipolar stimulating electrode (Rhodes Medical Instruments, Woodland, CA; diameter 250 µm; tip separation 500 µm) was placed in the fascia surrounding the mandibular branch of the facial nerve on each side of the rat. In some rats a second bipolar electrode was inserted into the right VRG using stereotaxic coordinates that corresponded to the preBötC region (2 mm lateral, 4 mm caudal to lambda, 8.5 mm below cerebellar surface). This second electrode was used to test for contralateral axonal projections of neurons recorded in the left preBötC area (monophasic square pulses, 100 µs). The threshold intensity required to activate the ipsilateral vibrissae hence facial motoneurons from this electrode was 0.7-0.8 mA. To test for antidromic activation of contralateral neurons, we used a maximum intensity of 0.5 mA. Given that facial motoneurons were located 1 mm rostral to the tip of the electrode and needed more than 0.5 mA to be activated, we assume that a current of 0.5 mA could only activate myelinated axons located within a sphere of less than a 1-mm radius centered at the electrode tip.
After completion of surgery the halothane concentration was reduced to 0.9-1%, a level at which a strong pinch of the tail or hind paw produces no retraction. Under paralysis, the same stimuli produced a rise of <10 mmHg in AP and no change in the frequency or amplitude of the phrenic nerve discharge (PND). After 45 min equilibration, the muscle relaxant pancuronium was administered (1 mg/kg iv, with 0.3- to 0.5-mg/kg supplements as required), and electrophysiological recordings were initiated. Ventilation was finally adjusted so that end-expiratory CO2 was about 1% above the threshold of the phrenic discharge. This threshold was found to be high in halothane-anesthetized rats ventilated with 100% O2 (5.5-6.0% end-expiratory CO2) but stable during the course of the experiment. During the recording period, the mean AP of the rats was between 105 and 115 mmHg and remained constant during the remainder of the experiment (3-5 h).
The PND was amplified (Grass model 7P5B AC amplifier), filtered
(200-3,000 Hz), full-wave rectified (Grass model 7P10F), and RC
integrated (6 dB high filter; Grass DC pen driver amplifier). Unit
activity was recorded with glass electrodes filled with 0.5 M sodium
acetate or NaCl containing 1.5% biotinamide (MW 367.3; Molecular
Probes, Eugene, OR; pH 4.5). Recordings were made with an intracellular
amplifier in bridge mode (Axoclamp 2A) so that current could be
injected through the electrode while action potentials were being
monitored. The signal was further processed through an AC amplifier
(X100 gain; 0.2-3 kHz band-pass; 60-Hz notch filter).
The region of the preBötC was determined in each rat with
reference to the location of the caudal pole of the facial motor nucleus. As reported previously (Brown and Guyenet
1985), the caudal end of this field potential is very sharply
defined. However, its stereotaxic coordinates vary from rat to rat by
as much as 600 µm rostrocaudally (from 2.6 to 3.3 mm caudal to the
parietooccipital suture; mean: 3.0 mm). Behind the caudal limit of the
facial field potential and at the level of the bottom of the field
potential in the vertical direction, phase-2 expiration (E2)
incrementing respiratory neurons with properties reminiscent of
Bötzinger neurons were the dominant form of respiratory neurons
detected between 1.8 and 2.1 mm lateral to the midline
(Schreihofer et al. 1999
). This type of neurons remained
dominant for another 600 µm caudal to the facial field and became
increasingly rare between 600 and 800 µm, where increasing numbers of
preinspiratory neurons were detected. Thus in the majority of rats,
after mapping the contour of the facial motor nucleus, the recording
electrode was withdrawn and the search for preBötC units was
performed within a narrow region located 700-1,100 µm behind the
caudal end of the facial motor nucleus and at approximately the same depth and mediolateral coordinates as the putative Bötzinger cells. This specific region was also selected because it corresponds to
where we found the highest concentration of NK1R-ir neurons (Wang et al. 2001
). The classification of
respiratory neurons was based on the timing of their discharge in
relation to that of the phrenic nerve using accepted nomenclature
(Schwarzacher et al. 1995
).
Recorded neurons were individually filled with biotinamide using the
juxtacellular labeling method (Pinault 1996) as
described previously (Schreihofer and Guyenet 1997
;
Schreihofer et al. 1999
). Pulses of positive current
(200 ms, 50% duty cycle) were delivered while the unit was recorded,
and the intensity of the current was slowly raised until the discharge
of the neurons was entrained by the stimulation pulses (1.3-5 nA). The
current pulses trigger the iontophoretic ejection of biotinamide and
cause the uptake of the marker by the recorded cell. The activity of
the unit was monitored during the entire labeling procedure (30 s to 2 min) to ensure that only one recorded cell was being entrained.
All physiological variables (AP, end-expiration
CO2, PND, integrated PND, and unit activity) were
monitored on a chart recorder and simultaneously stored on a video
cassette recorder via a Vetter 3000A interface (frequency range: DC22
kHz). Subsequent processing was made with a Power 1401 interface and
version 3 of the Spike2 software (both from Cambridge Electronics
Design, Cambridge, UK). Analog signals from the combination video
cassette recorder/Vetter 3000A interface were sampled at 9,260 Hz for
spikes, 4,600 Hz for PND, and 100 Hz for AP, integrated PND and
end-expiratory CO2. Instantaneous action
potential frequency was also measured (reciprocal of interval between 2 consecutive spikes). To make the final illustrations, representative
excerpts of the Spike2 data files were exported into a drawing program
(Canvas 6, Deneba, Miami, FL). All results are expressed throughout the
text as means ± SE.
Histology
After juxtacellular labeling of preBötC neurons, the rats
were deeply anesthetized with 4% halothane in 100%
O2. The rats were perfused through the ascending
aorta with 250 ml of phosphate-buffered saline (pH 7.4) followed by 4%
phosphate-buffered (0.1 M; pH 7.35) formaldehyde (Fisher Scientific,
Pittsburgh, PA). The brain stem was removed and stored in the same
fixative overnight at 4°C. Series of coronal sections (30 µm) were
cut through the medulla using a Vibratome (Lancer; Ted Pella, Redding,
CA) and stored in cryoprotectant solution at 20°C for up to
2 wk (20% glycerol plus 30% ethylene glycol in 50 mM phosphate
buffer, pH 7.4).
All immunohistochemical procedures were done using free-floating sections at room temperature. Before any histological protocol, sections were removed from the cryoprotectant mixture and rinsed three times in sodium phosphate buffer (PB, 0.1 M phosphate buffer, pH 7.4). To reveal the presence of biotinamide introduced into single neurons by juxtacellular labeling, the sections were incubated for 2 h with streptavidin Alexa 488 (1:200 dilution, Molecular Probes, Eugene, OR). The sections were then processed for the detection of NK1R-ir using the following protocol. The sections were transferred for 30 min into PB containing 1% normal goat serum and 0.3% Triton X-100. They were incubated for 24-72 h at 4°C with rabbit polyclonal antibody against NK1R (Chemicon International, Temecula, CA, 1:2,000 dilution). The sections were then washed in PB and were incubated for 45 min with a goat anti-rabbit IgG conjugated to Cy3 (1:200 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA). The sections were then rinsed in PB, mounted in sequential order onto gelatin-coated slides, dehydrated and delipidated through graded alcohols and xylenes. Finally, coverslips were affixed with DPX mounting medium (Aldrich, Milwaukee, WI). No staining was observed in the absence of the primary antibody.
Mapping and imaging
The sections were mounted on slides in sequential rostrocaudal
order. They were examined under dark-field illumination to identify the
two sections that contained our chosen diagnostic landmarks. The first
landmark was the very caudal end of the facial motor nucleus, which was
assigned the level 11.7 mm caudal to bregma (Paxinos and Watson
1998). The second landmark was the rostral end of the
lateral reticular nucleus where this structure displays a lateral and a
medial portion as opposed to a single outline. The section
corresponding to that landmark was assigned the level 13.0 mm caudal to
bregma according to the nomenclature of Paxinos and Watson
(1998)
. The theoretical distance between the two landmark
sections (1.3 mm) matched very closely the actual distance represented
by the product of the number of intervening sections time the section
thickness (30 µm). Therefore the bregma level of the section that
contained a biotinamide-labeled cell was determined arithmetically by
its location relative to the two landmark sections.
Under dark-field illumination the outlines and major landmarks of the
sections of interest were drawn using a Lucivid camera (MicroBrightfield, Colchester, VT) and a motor-driven microscope stage
(Ludl Electronic Products, Hawthorne, NY) controlled by the Neurolucida
software (MicroBrightfield) as described previously (Stornetta
and Guyenet 1999). This system was also used to map the precise
location of the biotinamide-labeled neurons. The Neurolucida files were
exported to the Canvas software drawing program for final modifications
and printing.
Some of the fluorescent material stained with Alexa 488 and Cy3 was visualized using a two-color Olympus BX50 WI confocal microscope equipped with a Krypton and Argon laser. The images were scanned through ×40 or ×60 objectives, acquired at a resolution of 1,024 × 1,024 pixels, stored in 24-bit TIFF format and imported into Adobe Photoshop (version 5.0.1; Adobe Systems, Mountain View, CA). Multiple photomicrographs were assembled so that the figure fit the page (minimum resolution of 300 pixels/in.). The output levels of each panel were adjusted against the range of levels containing pixels. Contrast and brightness were also individually adjusted to best reflect the original images. Lettering, scale bars, and arrows were added in Photoshop.
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RESULTS |
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Preinspiratory neurons of the pre-Bötzinger region
Within the narrow region of the ventrolateral medulla that was explored (0.7-1.1 mm caudal to the facial motonucleus), respiratory neurons were by far the dominant type of active neurons (>90%). The respiratory neurons were classified conventionally on the basis of their discharge pattern in relation to the mass discharge of the phrenic nerve (PND). Neurons were considered preinspiratory (pre-I) when the first spike in each respiratory burst occurred more than 20 ms before the start of the integrated PND, and the discharge continued up to the peak of the phrenic burst (Figs. 1, A and C, and 3B). The rate of discharge of these cells was maximal at the onset of the phrenic burst (peak instantaneous frequency between 40 and 170 Hz; Fig. 2) and decreased by 15-60% from this maximum during the rest of the inspiratory period, whereas the PND was always incremental (Figs. 1, A and B, and 3B). Within the region of the brain that we sampled, pre-I neurons were common (about 30% of all respiratory cells encountered). All these cells shared two additional characteristics. First, in response to a brief interruption of the ventilator, they continued to discharge in synchrony with the PND (16 of 16 cells tested; results not illustrated). This test was used to verify that none of these neurons derived their respiratory discharge from respiratory movement artifacts or from pulmonary afferent feedback alone (pump cells). Second, all the cells recorded could be silenced by hyperventilating the rat by increasing the respiratory rate, the tidal volume or both (14 of 14 cells tested; not illustrated). The peak instantaneous discharge rate of the 36 pre-I neurons recorded and the onset of the cell burst relative to PND (pre-I latency; defined in Fig. 1) are shown in Fig. 2. There was no correlation between these two variables. This figure also shows that the pre-I latency covered a wide range of values.
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Contralateral projections of pre-I neurons located in the region of the preBötC
Others have shown previously that the vast majority of the
respiratory neurons located in the preBötC do not have spinal projections (Dobbins and Feldman 1994). This is
especially clear in the case of the preBötC
expiratory-inspiratory neurons of Sun et al. (1998)
,
which most closely resemble our pre-I neurons (see
DISCUSSION). Here we tested whether the pre-I cells
are likely to be medullary interneurons by determining whether they had
axonal projections to the contralateral preBötC (10 pre-I cells
recorded in 2 rats). Constant latency action potentials could be
elicited in seven cells by stimulating the contralateral preBötC
with a current intensity between 50 and 500 µA. In each case the
collision test (Lipski 1981
) was positive, suggesting
that the evoked action potentials were antidromic. More precisely, the
constant latency action potentials were occluded when the stimulus was
delivered within a critical interval following a spontaneous spike.
This critical interval was equal to the antidromic latency plus
0.2-0.4 ms. An example of one pre-I cell that was antidromically
activated from the contralateral preBötC is shown in Fig. 1,
A and B. The average antidromic latency of the
seven pre-I neurons was 1.31 ± 0.18 ms (range 0.65-2.1 ms). The
minimal axonal conduction velocity of these cells calculated from the
straight-line distance between recording and stimulating sites (4 mm)
was 3 m/s.
Phenotype of the pre-I neurons of the pre-Bötzinger complex
Four groups of rats were used as summarized in Table 1. In the first three groups only pre-I cells were entrained by current pulses. In the fourth group (control rats) the entrained neurons did not include any pre-I neurons.
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In group 1 (5 rats), we made a single attempt at labeling a pre-I cell on each side of the brain. Eight cells were successfully entrained, and all were recovered histologically. Juxtacellular stimulation of one of these pre-I cells is illustrated in Fig. 1D, and its resting discharge pattern is shown in Fig. 1C. A decrease in recorded spike amplitude was often observed during the periods of current injection. This decrease is likely the result of the very high rate of discharge of the cell. In two rats, a single cell was labeled per brain, and in the remaining three, one cell per side was labeled. Two of the eight neurons exhibited strong NK1R immunoreactivity. One of the NK1R-negative cells started firing only 28 ms before PND onset and could perhaps have been classified as inspiratory rather pre-I. The remaining five cells without detectable NK1R immunoreactivity were distinctly pre-I (preinspiratory latency: 160 ± 32 ms, mean ± SE), and their discharge pattern was indistinguishable from that of the two NK1R-ir neurons (peak instantaneous discharge of 150 ± 11 spikes/s at onset of PND with 10-40% rate decrement during inspiration).
In a second group of rats (n = 5), we attempted to label one to three pre-I neurons with biotinamide on each side of the brain. Seventeen of 21 neurons entrained by current pulses were recovered histologically. Six of the 17 neurons displayed intense NK1R immunoreactivity.
In the last two animals (group 3), pre-I cells were recorded on the left side only, and only those that could be activated antidromically from the right preBötC were labeled. Seven biotinamide-labeled cells were recovered in these rats, three of which were NK1R-ir.
An example of one NK1R-ir pre-I neuron labeled with biotinamide is
shown in Fig. 3, C and
D. This cell was recorded 900 µm behind the caudal end of
the facial motor nucleus. It had a typical pre-I pattern of discharge
with an average pre-I latency of 80 ms (Fig. 3B). The
low-power dark-field photograph (Fig. 3A) shows the location
of the cell (white dot). This cell was clearly located in the VRG based
on its position in relation to easily identifiable landmarks such as
the inferior olive, the nucleus ambiguus, and the ventral surface of
the medulla oblongata. The section illustrated in Fig. 3A
corresponds approximately to level bregma 12.6 mm (Paxinos and
Watson 1998
).
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Combining the results of the 12 animals (groups 1-3), 11 of 32 pre-I neurons (34.4%) displayed detectable NK1R immunoreactivity. The
location of the 32 biotinamide-labeled pre-I neurons recovered is shown
in Fig. 4. Their mean location was at
12.65 ± 0.19 (SD) mm relative to bregma (range:
12.2 to
13.1 mm). The cells are represented on a single reference plane
(bregma
12.6 mm). All cells were found ventral to the nucleus
ambiguus. All pre-I cells, regardless of whether they were NK1R-ir,
were found within the region containing the highest density of NK1R-ir
neurons. Figure 5, A-C,
illustrates the case of a pre-I neuron without detectable NK1R
immunoreactivity that is surrounded by strongly immunoreactive cells.
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Phenotype of other neurons of the pre-Bötzinger complex
Control experiments were performed in three rats (group 4, Table
1). Recordings were made in the same narrow region as in the other
rats. Numerous pre-I cells were encountered while searching for other
cell types (26 pre-I neurons of 55 cells recorded), but we made no
attempt to label them with biotinamide. Instead we recorded and
entrained 21 neighboring cells that had different discharge patterns.
The sample included the following cell types: 3 tonic (nonrespiratory)
neurons, 12 expiratory neurons (6 throughout expiratory, 3 E1-E2
incrementing, 1 E1-E2 decrementing, 2 E2 incrementing), 3 post-I
neurons, 1 I neuron, 1 phase-spanning E2-I pump cell, and, finally, 1 E2 neuron that also produced 1 or 2 spikes at the I-postI transition.
After histology, 20 biotinamide-labeled neurons were recovered, none of
which contained a detectable level of NK1R immunoreactivity. As
expected from the recording location, the biotinamide-labeled cells
were surrounded by numerous NK1R-ir neuronal somata (Fig. 5,
D-F). The average location of these 20 cells was at
12.73 ± 0.22 (SD) mm relative to bregma (range:
12.4 to
13.0 mm relative to bregma). The location of these cells is shown on
a single reference plane (bregma
12.6 mm) in Fig. 4. The location of
the 20 control cells was indistinguishable from that of the 31 pre-I
neurons recorded in prior experiments (Fig. 4, top and
bottom plots).
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DISCUSSION |
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The major finding of the present study is that approximately
one-third of the preinspiratory neurons of the preBötC region have an intense NK1R immunoreactivity. In contrast, based on a sample
of 20 cells, surrounding neurons with other discharge patterns did not
display detectable levels of NK1R immunoreactivity. The data are
compatible with the possibility that some of the strongly NK1R-ir
neurons of the preBötC could be interneurons involved in
respiratory rhythm generation (either pacemakers or followers as
defined by Smith et al. 1995).
Anatomical boundaries of the pre-Bötzinger complex
The preBötC has been defined as a region of the ventral
respiratory group that contains propriomedullary neurons capable of
generating a respiratory-like activity in slices (Gray et al. 1999; Johnson et al. 1994
; Smith et al.
1991
). In vivo this region contains a complex but
characteristic mix of respiratory neurons, typically including numerous
pre-I and post-I cells, few E2-incrementing neurons of the
Bötzinger type, and few if any I-incrementing neurons
(Connelly et al. 1992
; Schwarzacher et al.
1995
; Sun et al. 1998
). There is no clearly
defined anatomical marker of the pre-Bötzinger region with the
possible exception of the NK1R immunoreactivity investigated presently
(Gray et al. 1999
; Wang et al. 2001
).
Therefore the rostral and caudal anatomical boundaries of the
preBötC are not defined with absolute precision. The anatomical definition adopted presently relies on the general consensus that the
preBötC lies ventral to the nucleus ambiguus, caudal to the retrofacial nucleus (a.k.a. nucleus ambiguus, pars compacta) and rostral to the anterior tip of the lateral reticular nucleus
(Gray et al. 1999
; Smith et al. 1991
).
In the present experiments, we obtained both physiological and
neuroanatomical evidence that our recordings were from neurons located
within the preBötC. First, the pattern of respiratory activities
that we detected in the sampled area conformed to prior descriptions of
the preBötC. Second, the biotinamide-labeled recorded neurons
were always located within the dense cluster of NK1R-ir cells that
defines the region of the preBötC according to Gray et al.
(1999).
Preinspiratory neurons of the pre-Bötzinger complex
Three broad types of respiratory patterns have been defined
previously as preinspiratory (Schwarzacher et al. 1995).
One type defined in vitro consists of cells that discharge before and
after inspiration (postinspiration) (Onimaru et al.
1988
). E2 expiratory neurons with a post-I rebound were found
in our experiments but extremely rarely. The second type of pre-I
neuron described in the literature exhibits a sharply defined peak of
activity at or just before the E/I transition. It has been found in the
cat preBötC in vivo (Schwarzacher et al. 1995
) and
has been called by these authors type 1 pre-I neuron. An apparently
similar type of cell (called in this case type 2 pre-I) has been
described in the adult mouse (Paton 1996
) or in the
neonate rat en bloc preparation (pre-I/I neuron) (Smith et al.
1990
). Although many of our pre-I neurons exhibited a pattern
that was decremental toward the end of the burst, these cells continued
discharging at a high rate until the apex of the phrenic burst, and
consequently their pattern was somewhat different from the type of
pre-I neurons discussed previously. The third type of pre-I neurons
described in the literature consists of neurons that have an
incremental pattern of firing in late E2 and maintain a sustained
though decremental level of discharge throughout inspiration. We refer
specifically to the type 1 pre-I neuron described by Paton in the mouse
(Paton 1996
), to the pre-I cell of the cat preBötC
described by Connelly et al. (1992)
, possibly to type 2 pre-I cells of Schwarzacher et al. (1995)
and to
the EI phase-spanning neurons recorded by Sun et al.
(1998)
in a region of the rat ventral respiratory group identical to ours (see their Figs. 1C and 3A).
The most clearly phase spanning of our pre-I cells (with preinspiratory
latency >100 ms) resembled most closely the type I pre-I cells of the adult mouse (Paton 1996
) and the EI phase-spanning
neurons of the adult rat (Sun et al. 1998
). The latter
authors demonstrated that this type of neuron does not project to the
cervical spinal cord.
Our population of pre-I neurons also included cells whose first spike in a respiratory burst anticipated the mass discharge of the PND by as little as 20 ms. We included those cells because there was no clear demarcation between them and the ones that were more clearly phase spanning. It is possible that this type of neuron might have been classified by others as a form of inspiratory rather than pre-I neuron. In fact within the sampled region we found relatively few neurons (~10%) that were strictly inspiratory by our criteria, i.e., whose first spike anticipated or lagged the onset of PND by 20 ms or less. None of these inspiratory cells had an incremental pattern of discharge.
Phenotype of the pre-I neurons
The juxtacellular labeling method used in the present work relies
on the assumption that the biotinamide-filled cell is the one that was
recorded and entrained by the current pulses. Our prior experience with
this technique supports strongly this assertion (Schreihofer and
Guyenet 1997; Schreihofer et al. 1999
;
Stornetta et al. 1999
). Also, in the present study, when
a single cell was stimulated per side, a single biotinamide-labeled
neuron was recovered after histological procedures. Furthermore NK1R-ir
neurons were labeled only when pre-I neurons were targeted. The
biotinamide-labeled somata were recovered where predicted based on the
recording coordinates in relation to the facial motor nucleus and no
biotinamide-labeled neuron was recovered outside the preBötC.
This last point strongly suggests that most recordings were obtained
from cell somata or from proximal dendrites. In addition, this
observation suggests that the biotinamide-labeled somata are very
unlikely to have been filled by retrograde transport from terminals.
The specificity of the NK1R antibody used presently has been well
characterized by others previously (Brown et al. 1995;
Mantyh et al. 1989
). The antibody is directed against a
15-amino acid segment of the carboxyl terminal end of the classic long
form of the NK1 receptor (amino acids 393-407). Thus this antibody would not be expected to detect the carboxyl terminal-truncated form of
the receptor (Fong et al. 1992
; Li et al.
1997
). The distribution of immunoreactive cells in the medulla
oblongata conformed to prior descriptions using this and another
similar antibody (Gray et al. 1999
; Nakaya et al.
1994
).
Thirty-four percent of the pre-I neurons labeled with biotinamide were strongly NK1R-ir. This results derives its significance from the fact that no NK1R-ir cell was labeled (0/20) when we targeted other types of neurons. These control neurons were also recorded in the preBötC and were in very close proximity to NK1R-ir cell bodies or the dense lattice of NK1R-ir dendrites that traverse the region (Fig. 4). Thus the clearest result of the juxtacellular labeling experiments is that some of the strongly NK1R-ir of the preBötC are indeed respiratory neurons that belong to the general class of pre-I neurons as defined here. However, the fact that only 34% of the pre-I neurons were NK1R-ir indicates that our targeted population was phenotypically and probably functionally heterogeneous.
Functional significance
Is NK1R immunoreactivity a selective marker for the
pre-Bötzinger complex as proposed by Gray et al.
(1999), and does NK1R-immunoreactivity identify a unique type
of preBötC respiratory neuron? According to our prior anatomical
work (Wang et al. 2001
) NK1R-ir neurons are not exclusively
confined to the pre-Bötzinger region of the ventral medulla.
Strongly NK1R-ir neurons are also found in or near nucleus ambiguus
pars compacta, rostral to or dorsal to the preBötC. NK1R-ir
neurons are also found ventromedial to the preBötC. There is no
absolute demarcation line between these cell groups and the NK1R-ir
cells of the preBötC. However, within the VRG, i.e., ventral to
nucleus ambiguus, the largest concentration of NK1R-ir neurons is
indeed found caudal to nucleus ambiguus pars compacta and rostral to
the lateral reticular nucleus, therefore in a region that overlaps
closely with the preBötC (Wang et al. 2001
).
Furthermore, within the region of the preBötC, NK1R
immunoreactivity is not colocalized with choline acetyl-transferase or
tyrosine-hydroxylase (Gray et al. 1999
; Wang et
al. 2001
), and it is very rarely colocalized with GAD67mRNA,
GlyT2 mRNA (Wang et al. 2001
). These data indicate that
NK1R-ir preBötC neurons are neither motoneurons nor C1 cells. This anatomical evidence also suggests that the overwhelming majority of the NK1R-ir preBötC neurons are neither GABAergic nor
glycinergic. Thus NK1R-ir preBötC neurons are most probably
excitatory glutamatergic neurons. The fact that NK1R immunoreactivity
is almost never associated with GABAergic or glycinergic neurons in the
preBötC eliminates a large number of possibilities regarding
which functional classes of respiratory cells might be identified by
the marker. For example, many postinspiratory, inspiratory, or
E2-incrementing interneurons are GABAergic or glycinergic, and
therefore they should not be NK1R-ir. This prediction is in agreement
with the present single-cell labeling data since we did not find any
NK1R-ir neurons in our control sample of 20 nonpre-I neurons that
included 3 post-I and 2 incremental E2 neurons. In short, NK1R
immunoreactivity is indeed confined to a restricted population of
neurons within the preBötC. This population includes many pre-I
cells. However, due to the relatively small number of control cells
that we tested (n = 20), we cannot yet state that
NK1R-ir is a selective marker for pre-I cells.
Could the NK1R-ir pre-I neurons of the adult preBötC be the adult
equivalent of the excitatory interneurons with rhythmogenic properties
identified in neonate slices, and if so which type of rhythmogenic cell
would they be? Clearly, the NK1R-ir pre-I neurons that we recorded
within the preBötC could not have been motoneurons because the
few cholinergic neurons present within this region do not contain
detectable levels of NK1R-ir (Gray et al. 1999;
Wang et al. 2001
). The hybrid pacemaker theory of respiratory rhythm generation in vitro postulates that, in the neonate,
the rhythm is driven by a group of inspiratory neurons with intrinsic
bursting properties that are located in the preBötC (pacemaker
neurons). These neurons are identified by their ability to display a
voltage-dependent bursting behavior under conditions of reduced
synaptic activity produced by a low calcium and high magnesium
incubation medium or by the presence of a blocker of
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (Butera et al. 1999a
; Koshiya and Smith
1999
; Lieske et al. 2000
). The most stringently
characterized inspiratory pacemaker neurons display voltage-dependent
oscillatory bursting behavior when their resting potential is brought
within the range of
65 to
45 mV (Butera et al.
1999a
; Johnson et al. 1994
; Smith et al.
1991
). These cells often have flat interburst membrane
trajectories (Butera et al. 1999a
) or have slowly
depolarizing ramps in which case their discharge may anticipate the
hypoglossal burst to various degrees (Thoby-Brisson and Ramirez
2000
; Thoby-Brisson et al. 2000
). These cells
are most probably glutamatergic, almost certainly neither glycinergic
nor GABAergic, and many of them project to the contralateral
preBötC (Butera et al. 1999b
; Koshiya and
Smith 1999
). The pacemakers display a significant
dispersion of their spike-firing times that can be adequately modeled
using realistic assumptions regarding their intrinsic properties and
the existence of mutual feed-forward excitation (Butera et al.
1999b
). The NK1R-ir pre-I cells that we recorded in vivo have
characteristics that meet many requirements for them to be the adult
equivalent of the pacemaker neurons. As discussed above, these NK1R-ir
cells are almost certainly excitatory interneurons, and many of them project to the contralateral side. They discharge on average 140 ms
before the phrenic onset; therefore their onset coincides approximately with that of the hypoglossal discharge (Koshiya and Guyenet
1996
). Finally they exhibit considerable dispersion in their
spike-firing onset (Fig. 2). However, the above list of characteristics
is equally consistent with the possibility that the NK1R-ir pre-I cells
represent an adult form of excitatory follower cell (Butera et
al. 1999b
). In fact the type-1 cells of Rekling et al.
(1996)
that proved to be especially sensitive to application of
substance P (Gray et al. 1999
) were not shown to have
the voltage-dependent bursting behavior required to establish their
identity as pacemaker neurons (Butera et al. 1999a
).
Furthermore these authors did not demonstrate that the effect of
substance P was produced by or solely by the activation of NK1
receptors (Gray et al. 1999
). An additional complication
comes from the fact that the NK1R antibody used by us and also by
Gray et al. (1999)
can only identify one of two known
forms of the NK1 receptor as discussed above. Thus the responsiveness
of inspiratory neurons to substance P in vitro does not prove that
these cells would have substantial amount of immunoreactivity using the
antibody in question. For instance, ventrolateral medullary neurons
such as C1 neurons respond vigorously to substance P in neonate slices
(Li and Guyenet 1997
), but they do not display
detectable levels of NK1R immunoreactivity in the adult with the
antibody used presently (Gray et al. 1999
; Wang et al. 2001
). Clearly further experimentation is required to
establish the precise nature (pacemaker or follower or both) of the
preBötC neurons that express high level of NK1R immunoreactivity
in vivo and of those inspiratory cells that respond to substance P in vitro.
The range of possibilities regarding the nature of the NK1R-negative
pre-I cells that we recorded from (65% of the cells) is much broader.
A portion could have been cranial motoneurons since cholinergic neurons
of the preBötC are not NK1R-ir and they could have a pre-I
discharge pattern (Gray et al. 1999; Koshiya and
Guyenet 1996
; Rekling et al. 1996
; Wang
et al. 2001
). However, a majority of the pre-I cells that we
recorded from (70%) could be antidromically activated from the
contralateral preBötC, suggesting that most of the recorded
neurons were propriomedullary interneurons rather than cranial
motoneurons. Other NK1R-negative pre-I neurons could be inhibitory
interneurons since the preBötC contains numerous GABAergic or
glycinergic neurons (Wang et al. 2001
). Some of these cells could conceivably be the adult equivalent of inhibitory follower
neurons (Butera et al. 1999b
).
In summary, a third of the pre-I neurons of the preBötC express
NK1R immunoreactivity. Based on prior anatomical data (Wang et
al. 2001), it is probable that most of these cells are
excitatory interneurons. The present results are compatible with the
possibility that these NK1R-ir neurons might represent the adult
equivalent of the inspiratory pacemaker neurons identified "in
vitro" in the neonate, but they could also represent a population of
excitatory follower neurons (Butera et al. 1999b
). In
any case, the presence of NK1 receptors on the pre-I neurons of the
pre-Bötzinger complex is likely to contribute to the well-known
respiratory stimulant effect of substance P administered in this region
of the medulla (Bonham 1995
; Gray et al.
1999
).
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ACKNOWLEDGMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-28785 and HL-60003 to P. G. Guyenet.
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FOOTNOTES |
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Address for reprint requests: P. Guyenet, University of Virginia Health System, PO Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: pgg{at}virginia.edu).
Received 2 November 2000; accepted in final form 14 March 2001.
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REFERENCES |
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