Pre-Bötzinger Neurons With Preinspiratory Discharges "In Vivo" Express NK1 Receptors in the Rat

Patrice G. Guyenet and Hong Wang

Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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: DC-22 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Discharge pattern and contralateral projections of pre-I neurons. A: discharge pattern of a pre-I neuron. From top to bottom: rectified and integrated phrenic nerve discharge (i-PND, arbitrary units), phrenic nerve discharge (PND; arbitrary units), single-unit activity, and instantaneous frequency of discharge of the unit. The pre-I latency of this neuron was 125 ms on average. B: the contralateral preBötC was stimulated (0.1 ms, 50 µA; arrowhead) at various times following a spontaneous action potential (the 1st in the burst). Constant latency action potentials were elicited (a; latency 1.2 ms) except when the stimulus was delivered <1.4 ms after a spontaneous spike (s) in which case collision occurred (sweeps indicated by an asterisk). All 8 sweeps were consecutive. The same neuron is shown in A and B. C: another pre-I neuron. From top to bottom: integrated PND (arbitrary vertical scale), PND, single-unit activity, and instantaneous discharge rate of the unit. D: juxtacellular stimulation of the pre-I neuron shown in C.



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Fig. 2. Preinspiratory neurons of the pre-Bötzinger complex: preinspiratory interval and peak frequency of discharge. Peak instantaneous frequency and pre-I intervals are defined in Fig. 1. Data from 31 cells. Note lack of correlation between these 2 variables.

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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Table 1. Juxtacellular labeling of preBötC neurons: experimental design

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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Fig. 3. Example of an NK1R-ir pre-I neuron located in the preBötC. A: low power dark-field photomicrograph illustrating the location of the biotinamide-labeled cell (white dot). NA, nucleus ambiguus; IO, inferior olive. Scale bar: 300 µm. B: discharge pattern of the recorded unit (from top to bottom: integrated PND, PND, unit activity with 1-mV vertical scale, and discharge rate of unit with 0- to 50-Hz vertical scale. C: biotinamide. D: NK1R immunoreactivity. Note that the labeled neuron is surrounded by a dense mesh of NK1R-ir processes that belong to neighboring NK1R-ir neurons.

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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Fig. 4. Location of juxtacellularly labeled neurons: summary. Top section depicts the location of 32 pre-I neurons that were labeled with biotinamide. black-triangle, NK1R-ir neurons; triangle , neurons without detectable NK1R immunoreactivity. Bottom section (same coronal level) depicts the location of 20 pre-Bötzinger neurons with various respiratory patterns except preinspiratory. open circle , cells other than pre-I without NK1R-ir. Scale bar: 1 mm. The region of the VRG represented in these sections is located 400 µm rostral to the rostral tip of the lateral reticular nucleus. All recordings were made within 200 µm of the level represented. Amb, nucleus ambiguus; IO, inferior olive; Li, nucleus linearis; pyr, pyramidal tract; Sol, nucleus of the solitary tract; sp5, spinal trigeminal tract.



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Fig. 5. Examples of non-NK1R-ir respiratory neurons. A-C: pre-I neuron labeled with biotinamide (arrow) that was not NK1R-ir, although it was surrounded by neurons that are strongly NK1R-ir (B). Superposition of A and B is shown in C. D-F: throughout-expiratory neuron labeled with biotinamide (arrow). This cell was not NK1R-ir, although it was surrounded by neurons that were strongly NK1R-ir (E). F: superposition of images shown in D and E. All images were obtained by confocal microscopy. Scale bar (50 µm in A) applies to all panels.

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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).


    ACKNOWLEDGMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-28785 and HL-60003 to P. G. Guyenet.


    FOOTNOTES

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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ABSTRACT
INTRODUCTION
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DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society