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1Department of Pharmacology, University of Virginia, Charlottesville, Virginia; and 2Department of Physiology, Universidade Federal de São Paulo–Escola Paulista de Medicina, São Paulo, Brazil
Submitted 19 June 2007; accepted in final form 3 October 2007
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ABSTRACT |
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INTRODUCTION |
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; Feldman et al. 2003; Guyenet et al. 2005b; Mulkey et al. 2004; Nattie and Li 2006; Okada et al. 2002; Ritucci et al. 2005; Rosin et al. 2006; Stornetta et al. 2006). RTN neurons are also activated by carotid body stimulation and inhibited by lung inflation, and these effects are probably mediated via direct projections from solitary tract nucleus (NTS) neurons (Moreira et al. 2007; Takakura et al. 2006). This circuitry suggests that the control of the respiratory pattern generator (CPG) by the carotid bodies and by lung mechanoreceptors operates in part by adjusting the excitatory drive that the respiratory network receives from central respiratory chemoreceptors such as RTN neurons (McCrimmon and Alheid 2007; Moreira et al. 2007; Takakura et al. 2006).
In the present study, we test whether this concept can be extended to the control of inspiratory activity by cardiopulmonary C-fiber afferents. Activation of this type of afferent produces protective reflexes characterized by inhibition of the inspiratory motor output, bradycardia, sympathoinhibition, and airway constriction (Coleridge and Coleridge 2001; Verberne et al. 2003). Consistent with our hypothesis that regulation of central chemoreceptors contributes to the respiratory reflexes elicited by cardiopulmonary afferents, we show here that RTN neurons are uniformly inhibited by C-fiber vagal afferent stimulation. In addition, we provide some limited information on the pathway responsible for RTN inhibition by C-fiber vagal afferent stimulation.
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METHODS |
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The experiments were performed on 32 male Sprague-Dawley rats (Taconic; Germantown, NY) weighing 250–350 g. Procedures were in accordance with National Institutes of Health Animal Care and Use Guidelines and were approved by the University of Virginia's Animal Care and Use Committee.
In vivo recordings
General anesthesia was induced with 5% halothane in 100% oxygen. The rats received a tracheostomy. Artificial ventilation with 1.4–1.5% halothane in 100% oxygen was maintained throughout surgery. The surgical procedures (bladder catheterization, arterial cannulation, phrenic nerve dissection, dorsal access to the medulla oblongata through the atlanto-occipital membrane) have been described previously (Guyenet et al. 2005a; Takakura et al. 2006). A bilateral vagotomy was performed in three rats 1–2 cm caudal to the confluence of the superior laryngeal nerve.
On completion of surgical procedures, halothane was reduced to 1% (21 rats) or gradually replaced by urethan (1.2 g/kg iv over 20 min; 11 rats). The initial dose of urethan was adequate for 
1 h and had to be supplemented hourly and at least twice with an injection of 0.1 g/kg. After a total of 1.4–1.5 g/kg, the level of anesthesia was stable for the rest of the experiments (
5 h after the initial anesthetic cross-over). The rats were ventilated with 100% oxygen throughout the experiment. Rectal temperature was maintained at 37°C, and end-tidal CO2 was monitored throughout the experiment with a microcapnometer (Columbus Instruments). After injection of the intravenous anesthetic or reduction of the halothane concentration, the adequacy of anesthesia was monitored during a 30-min stabilization period by testing for absence of withdrawal response, lack of BP change, and lack of change in PND rate or amplitude to firm toe or tail pinch. Then the muscle relaxant pancuronium was administered at the initial dose of 1 mg/kg iv, and the adequacy of anesthesia was thereafter gauged solely by the lack of increase in BP and PND rate or amplitude to firm toe or tail pinch. At the end of the physiological experiment, the rats were deeply anesthetized with halothane (4% until AP reached 40 mmHg). Then they were perfused through the left cardiac ventricle with phosphate-buffered saline (pH 7.4; 150 ml) followed by paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4, 500 ml). The brains were postfixed overnight in the paraformaldehyde solution. The brains were then sectioned in the coronal plane (30 µm), and the sections were kept in cryoprotectant awaiting histological procedures.
Arterial blood pressure (AP), PND, tracheal CO2, and tracheal pressure were recorded as previously described (Moreira et al. 2007; Takakura et al. 2006). Single-unit recording and juxtacellular labeling of RTN neurons with biotinamide were done as described previously (Moreira et al. 2007; Mulkey et al. 2004; Takakura et al. 2006). RTN units were concentrated between 150 and 300 µm below the lower edge of the facial motor nucleus and from 200 µm caudal to 300 µm rostral to the caudal boundary of this nucleus (Mulkey et al. 2004; Stornetta et al. 2006; Takakura et al. 2006). This region lies between coronal planes Bregma –11.8 and –11.3 mm of the Paxinos and Watson atlas (Paxinos and Watson 1998). The defining property of RTN neurons is a strong activation by hypercapnia (discharge threshold at 4–4.5% CO2 and firing rate of 6–14 Hz at 10% CO2). Their high sensitivity to hypercapnia and insensitivity to changes in blood pressure distinguish these neurons from the blood-pressure regulating presympathetic neurons that are the bulk of the other active neurons detected within this limited region of the brain under our experimental conditions (Mulkey et al. 2004). Before searching for RTN neurons, ventilation was adjusted to lower end-expiratory CO2 to 4% at steady-state (60–80 cycles/s; tidal volume 1–1.2 ml/100 g). Variable amounts of pure CO2 were then added to the breathing mixture to adjust end-expiratory CO2 to the desired level without changing ventilation parameters. The barosensitive neurons of the RVLM were identified as described before (Brown and Guyenet 1985). They were recorded 0–0.4 mm caudal to the facial motor nucleus (Bregma levels –11.6 to –12.0 mm after) (Paxinos and Watson 1998). Transient occlusion of the descending aorta just below the diaphragm was used to activate arterial baroreceptors (Brown and Guyenet 1985). Rostral ventral respiratory group (rVRG) respiratory neurons were recorded between Bregma levels –13.0 to –13.4 mm (Schwarzacher et al. 1991; Stornetta et al. 2003). Cell types are named according to standard descriptive terminology referring to the timing of their discharge relative to that of the phrenic nerve (Schwarzacher et al. 1991). We made no attempt to determine whether the recorded respiratory neurons had a motor, premotor, or some other function within the respiratory network.
All analog data (end-expiratory CO2, PND, unit activity, AP) were stored on a microcomputer via a micro-1401 digitizer from Cambridge Electronics Design (CED, Cambridge, UK) and were processed off-line using version 5 of the Spike 2 software (CED) as described before (Moreira et al. 2007; Mulkey et al. 2004; Takakura et al. 2006). Processing included action potential discrimination and binning, neuronal discharge rate measurement. PND "integration" (iPND) consisted of full-wave rectification and smoothing (
: 0.015s). Neural minute x volume (mvPND, a measure of the total phrenic nerve discharge per unit of time) was determined by averaging iPND over a fixed number of lung inflation cycles and normalizing the result by assigning a value of 0 to the dependent variable recorded at low levels of end-expiratory CO2 (below PND threshold) and a value of 1 at the highest level of pCO2 investigated (between 9.5 and 10%) before any injection of muscimol was made. CO2 was set at the beginning of the experiment so that mvPND would be between 0.5 and 0.7 units. The CED software was also used for acquisition of perievent histograms of neuronal activity and peri-event averages of iPND, tracheal CO2, or tracheal pressure. The perievent histograms of neuronal single-unit activity were triggered either on iPND or on the tracheal pressure trace. Each histogram represents the summation of 
100 central respiratory or ventilation cycles (350–800 action potentials per histogram). The steady-state relationship between RTN neuronal activity and end-expiratory CO2 was obtained by stepping the inspired CO2 level to various values for a minimum of 3 and 5 min (Guyenet et al. 2005a; Moreira et al. 2007; Takakura et al. 2006). The mean discharge rate of the neuron was measured during the last 30 s of each step at which time end-expiratory CO2 and the discharge of the neuron appeared to have reached equilibrium. End-expiratory CO2 was measured by averaging the maximum values recorded from 10 consecutive breaths at the midpoint of the time interval sampled.
Stimulation of the carotid bodies was done with bolus injections of sodium cyanide (NaCN) (50 µg/kg iv). Under our experimental conditions, sodium cyanide activates brain stem neurons exclusively via stimulation of peripheral chemoreceptors because denervation of the carotid bodies eliminates the excitatory effect of cyanide on PND and RTN neurons (Takakura et al. 2006). Stimulation of unmyelinated cardiopulmonary receptors was done by intravenous administration of the 5-hydroxytryptamine type 3 (5-HT3) receptor agonist phenylbiguanide (PBG, 50 µg/kg iv). Under our experimental conditions, the inhibitory effect of PBG on breathing depends entirely on the integrity of vagal afferents because the drug was ineffective in three rats with bilateral cervical vagotomy. The cardiorespiratory effects caused by intravenous PBG in the anesthetized rat are mediated by serotonin-3 receptors because they can be antagonized by the selective antagonist MDL-72222 (Verberne and Guyenet 1992). Lung mechanoreceptors were activated by transiently elevating positive end-expiratory pressure (PEEP) (5–20 s) from a resting level of +1 cm H2O to +6 cm H2O (Moreira et al. 2007).
Muscimol injections
The GABA-mimetic drug muscimol (Sigma Chemicals, St. Louis, MO; 1.75 mM in sterile saline pH 7.4) was injected intraparenchymally while a single RTN neuron was being recorded (Moreira et al. 2007; Takakura et al. 2006). The muscimol solution contained a 5% dilution of fluorescent latex microbeads (Lumafluor, New City, NY) for later histological identification of the injection sites (Moreira et al. 2006). Muscimol (30 nl; 50 pmol) was pressure injected over 3–5 s through single-barrel glass pipettes with a 20-µm external tip diameter.
In eight rats, the electrode tip was directed to the rVRG by locating inspiratory-related field potentials (Takakura et al. 2006). These are the same rats in which we examined the effects of muscimol injection into rVRG on the response of RTN neurons to lung inflation (Moreira et al. 2007). In the present study, we report the effect of muscimol on the response of the same eight neurons to intravenous administration of PBG. The eight reported units (1 per rat) were kept for 20 min after the injections of muscimol after which the experiment was terminated.
The protocol used to test the effect of muscimol injection into the commissural or interstitial part of the NTS (commNTS, iNTS) was similar except that surface landmarks were used to identify the insertion point of the pipette. To inhibit commNTS, a single 30 nl injection of muscimol was placed at or near the midline, 0.5 mm below the surface of the medulla oblongata and 200–300 µm caudal to the calamus scriptorius. This experiment was carried out in six rats. The effect produced by muscimol injection into commNTS dissipated after 2 h, allowing the same six animals to be re-used to test the effect of muscimol injection into iNTS. After isolating and characterizing a second RTN chemosensitive neuron, muscimol (30 nl) was injected into iNTS on both sides of the brain (0.5 mm below the surface of the medulla oblongata, 500 µm rostral to the calamus scriptorius and 0.8 mm lateral to the midline) and the same RTN neuron was recorded for another 20 min.
Histology, cell mapping, and imaging
At the end of the experiment the rat was deeply anesthetized with halothane and perfused transcardially with PBS (pH 7.4) followed by paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4). All histochemical procedures were done using 30-µm-thick free-floating sections according to previously described protocols (Stornetta et al. 2006; Takakura et al. 2006). Cells labeled with biotinamide were identified by incubating the sections with streptavidin conjuguated with Alexa-488. The transcription factor Phox2b was detected as previously described using a rabbit polyclonal antibody (1:800 for 48–72 h followed by a Cy3-tagged donkey anti-rabbit IgG at 1:200, Jackson) (Stornetta et al. 2006). The antibody (a gift from J.-F. Brunet, Ecole Normale Supèrieure, Paris, France) was raised against the 14 amino acid C-terminal sequence of the Phox2b protein, and its specificity has been previously established for both mouse and rat (Pattyn et al. 1997; Stornetta et al. 2006).
The relevant photographic techniques and the computer-assisted mapping method used to record the location of drug injection sites, labeled neurons, and nerve terminals have been described in detail previously (Rosin et al. 2006; Stornetta and Guyenet 1999). The neuroanatomical nomenclature and Bregma levels are according to the atlas of Paxinos and Watson (1998).
Statistics
Statistical analysis was done with Sigma Stat version 3.0 (Jandel, Point Richmond, CA). Data are reported as means ± SE. t-test (paired or unpaired) or one-way repeated-measure parametric ANOVA followed by Newman-Keuls multiple comparisons test was used as appropriate. Significance was set at P < 0.05.
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RESULTS |
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Unless specifically mentioned, all experiments were done in rats with intact vagus nerves. According to our prior definition, RTN neurons (24 neurons in 14 rats with vagus nerves intact and 8 neurons in 3 vagotomized rats) were highly active in hypercapnia (6–14 Hz at 10% end-expiratory CO2) and silent in hypocapnia with a threshold between 4 and 5% end-expiratory CO2 (Fig. 1A). The CO2 threshold of RTN neurons was always lower than that of the PND (Fig. 1 A). Prior to testing the responses to NaCN, phenylbiguanide, and lung inflation, end-expiratory CO2 level was set at around 7.5% CO2, concentration at which PND amplitude was 50–70% of its maximal value at 10% CO2. Stimulation of carotid chemoreceptors with NaCN (50 µg/kg iv boluses) increased AP (from 126 ± 3 to 140 ± 3 mmHg, 6 rats, P < 0.05; Figs. 1B and 2, A1 and B), increased PND amplitude (Figs. 1B and 2, A1, F, and G), and vigorously activated every RTN neuron tested (from 7.3 ± 1 to 18.4 ± 2.5 Hz, n = 16, P < 0.01; Figs. 1B and 2, A1 and C). PBG administration decreased AP (from 126 ± 3 to 108 ± 4 mmHg, P < 0.05; Figs. 1B and 2, A1 and B), reduced PND amplitude (Figs. 1B and 2A1, F, and G), and strongly inhibited every RTN neuron tested (n = 16, Figs. 1B and 2, A1 and D). Raising PEEP from a resting level of +1 to +6 cm of H2O eliminated PND (Figs. 1B and 2, A1, F, and G) and inhibited the RTN neurons selected for further study by more than 27% (27–100%; Figs. 1B and 2, A1 and E). The CO2-activated neurons that responded to lung inflation with <25% inhibition (Moreira et al. 2007) were also uniformly inhibited by PBG (from 8.8 ± 0.6 to 4.6 ± 0.6 Hz, n = 5; P < 0.05). These neurons were not studied further because they were unsuitable to accomplish one of the main objectives of the study namely to differentiate the pathways mediating the effect of lung inflation versus pulmonary C-fibers on RTN neurons.
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), PBG produced no detectable effect on tracheal pressure in our experimental conditions (e.g., Fig. 1B). This negative finding is probably due to the vagolytic action of the muscle relaxant pancuronium. To verify that the effects of PBG on RTN neurons were mediated by the activation of vagal afferents, control experiments were performed in three vagotomized rats (n = 8 cells; data not illustrated). As expected, the stimulatory effect of cyanide on PND and RTN neurons was of normal magnitude in these rats, but we did not find any CO2-activated neurons that could be inhibited by lung inflation. The inhibitory effect of PBG on PND and RTN neurons was either absent in vagotomized rats or replaced by a very small and transient excitation. The loss (lung inflation, PBG) or persistence (carotid body stimulation with cyanide) of the phrenic nerve responses following vagotomy are according to expectations. These control experiments also demonstrate that the effect of PBG on RTN neurons is selectively due to the activation of vagal afferents.
Most RTN CO2-sensitive neurons express the transcription factor Phox2b (Moreira et al. 2007; Stornetta et al. 2006). To complement these observations, we determined whether the CO2-sensitive cells that respond vigorously to all three stimuli (NaCN, PBG, and lung inflation; the triple responders) also express this transcription factor. Four such cells were labeled juxtacellularly with biotinamide, and each labeled neuron had a Phox2b-immunoreactive (ir) nucleus (Fig. 1, C and D). As in prior work, the cell bodies of the recorded neurons were small (<15 µm in length) and were located under the caudomedial edge of the facial motor nucleus within 150 µm of the ventral surface (Fig. 1, D and E). One of the biotinamide-filled neurons was labeled with sufficient intensity to allow extensive reconstruction of its dendritic arbor (Fig. 1E). This triple responder had the distinctive morphology of previously examined CO2-sensitive RTN neurons (Mulkey et al. 2004). Its most notable feature was a basal dendrite that bifurcated at right angles within the marginal layer with the two branches spread out in a roughly transverse plane. As is the case of all other RTN neurons examined so far, this cell also had a second dendritic domain located dorsal to the marginal layer within the dorsal cap of the RTN.
Effect of muscimol injections into commissural NTS on RTN neuron responses to NaCN, lung inflation, and PBG
The next experiments were designed to identify which portion of the NTS mediates the inhibitory effect of PBG on RTN neurons and whether the same or different regions mediate the effect of NaCN and lung inflation. We first targeted the commissural portion of the NTS, a region that receives a heavy input from carotid body afferents (Blessing 1997; Boscan et al. 2002; Paton et al. 2001) and rapidly adapting lung mechanoreceptors but is sparsely innervated by the slowly adapting lung mechanoreceptors (Hayashi et al. 1996; Kubin et al. 2006).
As shown before (Takakura et al. 2006), muscimol injection into commNTS (six rats) had no effect on resting blood pressure (Fig. 2, A and B), resting PND (Fig. 2, A, B, and F) nor on the resting discharge rate of RTN neurons (1 triple responder per rat; Fig. 2, A, C, and E). More specifically, PND remained synchronized with ventilation, its amplitude was unchanged (96 ± 4% of control; NS), and its duration (Ti) was unaltered (from 0.33 ± 0.02 to 0.32 ± 0.02 s, NS). These injections eliminated the stimulatory effect of NaCN on blood pressure (Fig. 2, A2 and B), on PND (Fig. 2, A2, F, and G), and on RTN neuron activity (Fig. 2, A2 and C), but they did not change the inhibitory effect of lung inflation on PND (Fig. 2, A2, F, and G) and RTN neurons (Fig. 2, A2 and E). These muscimol injections reduced considerably the hypotension caused by PBG (Fig. 2, A2 and B), but some PND inhibition always persisted (Fig. 2, A2, F, and G). Likewise, RTN neuron inhibition by PBG was reduced but not eliminated by muscimol injection into commNTS (72 ± 4% reduction; P < 0.05; paired t-test; n = 6; Fig. 2, A2 and D).
In summary, muscimol injection into commNTS had no effect on any of the dependent variables measured at rest (blood pressure, PND, RTN). These injections blocked the effects of carotid body stimulation, they had no effect on the responses to lung inflation, and they greatly but incompletely attenuated the effects of PBG.
These muscimol injections were located 200 µm caudal to the calamus scriptorius as indicated in Fig. 3 A. Although muscimol was administered as a single injection in the midline, the injectate spread bilaterally to cover most of the dorsal aspect of commNTS (representative case in Fig. 3B). Based on the area of distribution of the fluorescent microbeads, the injectate appeared to spread somewhat less in the rostrocaudal direction (300 µm from the injection center) than laterally (500 µm from the center). The effective spread of muscimol was not determined and could have been larger than that of the microbeads due to the difference in molecular mass.
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These experiments were done in the six animals described in the preceding text after the rats had completely recovered from the effect of muscimol injection into commNTS (2–2.5 h after the injection into commNTS). In each rat, a second RTN triple-responder was isolated, and its response to NaCN, PBG, and lung inflation was retested after injecting muscimol bilaterally at the level of interstitial NTS (Fig. 3, C and D). Bilateral injection of muscimol into the region of iNTS raised resting AP markedly (from 127 ± 6 to 155 ± 4 mmHg; P < 0.01; Fig. 4, A and B) and increased the resting discharge rate of the selected RTN neurons by 66 ± 5% (P < 0.01; Fig. 4, A, C, and E). As described before (Moreira et al. 2007), muscimol had little effect on PND amplitude (103 ± 5% of control; NS; Fig. 4F), but the drug significantly increased Ti (from 0.32 ± 0.02 to 0.53 ± 0.02 s; P < 0.05; Fig. 4F). Also as previously described (Moreira et al. 2007), muscimol completely desynchronized PND from the ventilation cycle, thereby reproducing a characteristic consequence of interrupting inputs from lung mechanoreceptors. Primarily due to Ti widening, the integrated PND per unit of time (mvPND) was significantly increased by muscimol (Fig. 4F).
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). Despite this initial increase, RTN neurons were still vigorously activated by cyanide (from 11.94 ± 1 to 28 ± 3 Hz, P < 0.01; Fig. 4, A2 and C). In contrast, muscimol injections centered on iNTS eliminated the effects of lung inflation on both PND (Fig. 4, A2, F, and G) and RTN neurons (Fig. 4, A2 and E). These injections also significantly attenuated the effects of PBG including the transient hypotension (Fig. 4, A2 and B), the reduction in PND (Fig. 4, A2, F, and G) and the inhibition of RTN neurons by PBG (53 ± 7% reduction; P < 0.05; paired t-test; n = 6; Fig. 4, A2 and D).
In these six cases, the muscimol injections were centered on or very close to the tractus solitarius at mid-area postrema level (Fig. 3C). The fluorescent microbeads covered the tractus solitarius and the region surrounding it, including the interstitial nucleus and the dorsolateral NTS (Fig. 3D). The latter region contains large concentrations of second-order baroreceptive neurons (Chan and Sawchenko 1998; Weston et al. 2003) whose inhibition by muscimol most likely caused the rise in blood pressure observed in the present experiments.
Effect of PBG and lung inflation on other types of ventrolateral medullary neurons
For comparative purposes, we examined the effect of lung inflation (+6 cm H2O) and PBG on a variety of ventrolateral medullary neurons besides RTN chemoreceptors. The blood pressure-regulating neurons of the RVLM (10 neurons from 6 rats with intact vagus nerves recorded under halothane anesthesia; 2 neurons from 2 rats recorded under urethan anesthesia) were uniformly inhibited by PBG as expected (Verberne and Guyenet 1992) but these neurons were unaffected by lung inflation 6 cm H2O (Fig. 5 A, 1 and 2).
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Every inspiratory or expiratory neuron recorded within the rVRG region was profoundly inhibited by both lung-inflation and PBG (Fig. 5B1). Notably, both expiratory and inspiratory neurons were 100% inhibited by 6 cm H2O PEEP (representative case in Fig. 5B1). Postinspiratory neurons are typically activated by lung inflation (Hayashi et al. 1996). We confirmed this observation and found that the activity of these neurons remained phasic at low levels of PEEP and usually became tonic at 6 cm H2O (Fig. 5C1). Postinspiratory neurons are also activated by administration of PBG in the right atrium (Paton 1997; Wilson and Bonham 1997). Intravenous administration of PBG produced the same effect and we found that rVRG postinspiratory neurons (n = 8) were always activated by both stimuli (Fig. 5C1).
In summary, intravenous PBG changed the discharge of ventrolateral medullary respiratory neurons in the same manner as right atrium injection of a lower dose of the drug assumed to specifically activate bronchiopulmonary receptors (Paton 1997; Wilson and Bonham 1997). The present experiments also confirmed that the postinspiratory neurons of the rVRG region are activated both by vagal C-fiber afferents stimulation and by lung inflation (Hayashi et al. 1996; Wilson and Bonham 1997).
Inhibition of RTN neurons by PBG is attenuated by muscimol injection into rVRG
The postinspiratory interneurons located in the rVRG probably mediate a large part of the inhibitory effect of slowly adapting stretch receptor (SAR) activation on the central respiratory pattern generator (Ezure et al. 2003; Hayashi et al. 1996; Kubin et al. 2006; Okazaki et al. 2001; Richter et al. 1987). Because postinspiratory neurons are also activated by PBG (Wilson and Bonham 1997; present results), these cells presumably also contribute to PND inhibition when PBG-sensitive vagal afferents are activated. We therefore asked whether the activation of rVRG postinspiratory neurons by PBG could also possibly mediate the inhibition of RTN neurons. This hypothesis was tested by determining whether bilateral injection of muscimol into rVRG changes the inhibition of RTN neurons caused by intravenous injection of PBG (8 neurons in 8 rats).
Bilateral injection of muscimol into rVRG eliminated PND and elevated blood pressure, but muscimol did not change the resting discharge rate of RTN neurons (from 6.16 ± 1 to 6.33 ± 0.9 Hz; n = 8; NS; Fig. 6, A and B), and the drug had no effect on their response to raising end-expiratory pressure (data already reported in Moreira et al. 2007). Prior to muscimol injection, PBG inhibited PND as usual in these eight rats (Fig. 6A). As shown before (Verberne and Guyenet 1992), the effect of PBG on arterial pressure was virtually eliminated by injection of muscimol bilaterally into rVRG (Fig. 6, B and C). This result is due to the fact that the rVRG region also contains the neurons that relay the effect of PBG to the blood pressure-regulating neurons of the rostral ventrolateral medulla (RVLM). RTN neuron inhibition by PBG was only reduced by 32 ± 4% after bilateral administration of muscimol into rVRG (n = 8; P < 0.05; Fig. 6, A–C). The same injections had no effect on RTN neuron inhibition by lung inflation (data already reported in Moreira et al. 2007). These results demonstrate that although the rVRG plays no role in transmitting the effects of lung inflation or carotid body stimulation to RTN neurons under our experimental conditions (Moreira et al. 2007; Takakura et al. 2006), the rVRG does contribute to the inhibition of RTN neurons by C-fiber vagal afferent stimulation.
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DISCUSSION |
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Selectivity of phenylbiguanide for 5-HT3 receptor-expressing vagal C-fiber afferents
PBG is a selective 5-HT3receptor agonist that activates a variety of cardiopulmonary vagal C-fiber afferents (Hainsworth 1991; Verberne and Guyenet 1992; Widdicombe 2006). PBG has no effect on myelinated pulmonary stretch afferents or arterial baroreceptors in rats (Ho et al. 2001; Verberne et al. 2003). Under the present experimental conditions, the effects of intravenous PBG on the recorded cardiorespiratory variables were, as expected, predominantly caused by activation of vagal afferents because they were not observed after bilateral section of the vagal nerves just below the level of the superior laryngeal nerve. Thus neither glossopharyngeal (including arterial baroreceptors and chemoreceptors) nor superior laryngeal afferents could have contributed significantly to the effects of PBG examined presently. We presume that bronchio-pulmonary chemosensitive afferents (formerly J receptors) (see historical perspective by Widdicombe 2006) contributed most to the effects of intravenous PBG on PND and on RTN neurons, but the participation of other types of afferents, particularly cardiac receptors, is likely (Hainsworth 1991; Linz and Veelken 2002; Verberne et al. 2003). Activation of these various C-fiber afferents produces a very similar cardiorespiratory reflex characterized by respiratory depression, hypotension, and bronchoconstriction (Coleridge and Coleridge 1994, 2001).
RTN neurons receive convergent inputs from cardiopulmonary C-fiber afferents, SARs, and peripheral chemoreceptors
According to the present study, the CO2-activated neurons of RTN receive convergent inputs from lung mechanoreceptors, peripheral chemoreceptors, and cardiopulmonary C-fiber afferents. Like previously identified CO2-sensitive cells of RTN, neurons responding to all three stimuli expressed the marker Phox2b, and they were located in very close proximity of the ventral medullary surface (Mulkey et al. 2004; Stornetta et al. 2006). Current evidence that these RTN chemosensitive cells function as central respiratory chemoreceptors is based on three types of results: the selectivity of their projection pattern, the consistency between their electrophysiological properties and their postulated role, and the fact that inhibition or stimulation of the brain region in which they are located respectively depresses or activates breathing (Feldman et al. 2003; Guyenet et al. 2005b; Li and Nattie 2002; Mulkey et al. 2004; Nattie and Li 2000; Onimaru and Homma 2003; Rosin et al. 2006). The rest of the discussion presupposes that this interpretation of the role of RTN neurons is correct.
Carotid body afferents and lung mechanoreceptors regulate RTN neurons, apparently via direct neuronal projections from the NTS (Moreira et al. 2007; Takakura et al. 2006). The existence of direct projections from NTS to RTN has led us to propose that the control of the central respiratory pattern generator by the carotid bodies and by lung mechanoreceptors operates in part by regulating the "chemical drive" of respiration i.e., by regulating the excitatory input that the CPG receives from central respiratory chemoreceptors such as RTN neurons (McCrimmon and Alheid 2007; Moreira et al. 2007; Takakura et al. 2006). We interpret the present results along the same lines and suggest that withdrawal of the excitatory drive from RTN chemoreceptors contributes to the inhibition of breathing produced by activation of C-fiber cardiopulmonary afferents.
Further evidence that SARs, not RARs, mediate the inhibitory effect of lung inflation on RTN neurons
Rapidly adapting pulmonary stretch receptors (RARs), carotid chemoreceptors, and SARs innervate largely distinct subnuclei of the intermediate and caudal NTS (Kalia and Richter 1985; Kubin et al. 1991). RARs and carotid body afferents preferentially innervate caudal and medial structures such as the commissural portion of the NTS, whereas SARs target more rostral and lateral regions of the NTS located close to the interstitial nucleus, iNTS (Kubin et al. 2006). The iNTS region contains the greatest density of SAR second-order neurons (the pump cells), which in turn innervate the NTS, the ventrolateral medulla and the pons (Bonham and McCrimmon 1990; Ezure et al. 2002; Kubin et al. 2006). This well-known topography accounts for our observation that bilateral muscimol injection near iNTS blocked selectively the effect of lung inflation on RTN neurons, whereas injection into the commissural nucleus blocked selectively the effect of peripheral chemoreceptor stimulation. Given that most RAR second-order neurons reside in commNTS (Kubin et al. 2006), the fact that muscimol injection into this subnucleus did not change the effect of lung inflation on RTN neurons provides additional evidence that, under anesthesia, RTN chemoreceptors are regulated by SARs rather than RARs (Moreira et al. 2007).
Pathway responsible for RTN inhibition by phenylbiguanide
With rare exceptions, one of which was discussed in the previous paragraph, little functional specificity can be achieved by injecting even very small amounts of muscimol into NTS because of the elongated shape of the subnuclei and the layering and interdigitation of the various vagal afferents and their second-order cells (Altschuler et al. 1989; Kubin et al. 2006). Injections into iNTS, for example, also produced large increases in blood pressure, undoubtedly caused by the close proximity of arterial baroreceptor second-order neurons to iNTS (Weston et al. 2003). Cardiopulmonary C-fiber afferents have extensive projections to midline regions of the NTS both rostral and caudal to the calamus scriptorius (Kubin et al. 2006). Our present results are generally consistent with this anatomical data because the response of RTN neurons to PBG administration was attenuated to a similar extent by injecting muscimol into commNTS or more rostrally in the vicinity of iNTS. Our conclusion also agrees with prior experiments in which cobalt was administered into commNTS and produced attenuation but not elimination of the response to PBG (Bonham and Joad 1991).
Relatively little is known of the properties and axonal projections of the NTS neurons that receive bronchiopulmonary C-fiber input. A frank majority of the commNTS neurons that are activated by cardiopulmonary C-fiber stimulation receive convergent input from the carotid bodies, but a minority have opposite responses to the two inputs, and the region also contains neurons that respond selectively to only one of these stimuli (Paton 1998). The former type of commNTS neuron is unlikely to mediate the effects of PBG on the respiratory network, the RTN, or the sympathetic vasomotor outflow because these targets always respond in opposite directions to stimulation of cardiopulmonary C-fibers versus the carotid bodies. However, the latter two types of commNTS neurons could in theory relay the effect of cardiopulmonary C-fibers to the ventral medullary respiratory network and RTN neurons.
Stimulation of cardiopulmonary C-fibers inhibits ventral medullary sympathoexcitatory neurons by activating GABAergic neurons located in the caudal ventrolateral medulla (CVLM) at rVRG level (Schreihofer and Guyenet 2003). These CVLM cells are also activated by arterial baroreceptors, and they mediate the sympathetic baroreflex (Schreihofer and Guyenet 2003). In theory, information from baroreceptor and cardiopulmonary C-fiber origin could reach CVLM neurons via separate neurons or these inputs could already be integrated at the level of the NTS. The latter possibility is supported to some extent by evidence that the NTS contains neurons that are activated by both types of inputs (Paton 1998).
The way in which information from cardiopulmonary C-fibers is relayed from the NTS to RTN neurons is speculative. A direct inhibitory input from NTS to RTN is plausible but does not fully account for the present data. In support of this hypothesis, monosynaptic transmission of inputs between vagal afferents and NTS neurons with axonal projections to the ventrolateral medulla is extremely common (Bailey et al. 2006), and RTN neurons already receive separate and most likely direct inputs from two other types of presumed second-order cells, i.e., NTS pump cells and carotid body-receptive neurons (Moreira et al. 2007; Takakura et al. 2006). Also in favor of a direct input from NTS to RTN, a large fraction of the effect of PBG (68%) persisted after muscimol injection into the rVRG, a procedure expected to inhibit the neurons located in the injected area and to strongly depress the activity of the CPG. However, the fact that muscimol injection into rVRG attenuated the response of RTN neurons (by 32%) indicates that the pathway between NTS and RTN cannot be entirely direct and that the effect of PBG on RTN neurons must also involve interneurons located in the rVRG region. The present study does not permit to elaborate further on these ventrolateral relay neurons except to suggest that these cells are unlikely to be postinspiratory interneurons. Although such cells are highly represented in the rVRG of the rat (Schwarzacher et al. 1991), consistently activated by stimulating cardiopulmonary C-fibers with PBG (Paton 1997; Wilson and Bonham 1997; present results), and inhibit numerous types of respiratory neurons (Ezure et al. 2003; Richter et al. 1987; Schwarzacher et al. 1991), they are also vigorously activated by lung inflation. Thus if they were implicated in the effect of PBG on RTN neurons, they should also mediate some of the inhibitory effect of lung inflation on these neurons. This hypothesis can be rejected because muscimol injection into rVRG did not change the effect of lung inflation on RTN neurons.
Conclusion
Activation of 5-HT3 receptor-expressing C-fiber vagal afferents inhibits RTN neurons. The pathway relays through a broad region of the NTS and, for its full expression, the response requires the integrity of the rVRG. We interpret the inhibition of RTN neurons by C-fiber vagal afferents as further evidence that respiratory reflexes operate in part by regulating the intensity of the excitatory drive that the respiratory pattern generator receives from central chemoreceptors (McCrimmon and Alheid 2007; Moreira et al. 2007). The present results were obtained under halothane anesthesia in the presence of a high level of CO2, conditions required to overcome the powerful lung stretch reflex present under these experimental conditions. The extent to which RTN contributes to respiratory reflexes in an awake animal under normal levels of CO2 remains to be determined.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. G. Guyenet, Dept. of Pharmacology, University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735 (E-mail: pgg{at}virginia.edu)
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REFERENCES |
|---|
|
Altschuler SM, Bao X, Bieger D, Hopkins DA, Miselis RR. Viscerotopic representation of the upper alimentary tract in the rat: sensory ganglia and nuclei of the solitary and spinal trigeminal tracts. J Comp Neurol 283: 248–268, 1989.[CrossRef][Web of Science][Medline]
Bailey TW, Hermes SM, Andresen MC, Aicher SA. Cranial visceral afferent pathways through the nucleus of the solitary tract to caudal ventrolateral medulla or paraventricular hypothalamus: target-specific synaptic reliability and convergence patterns. J Neurosci 26: 11893–11902, 2006.
Blessing WW. The Lower Brain Stem and Bodily Homeostasis. New York: Oxford, 1997.
Bonham AC, Joad JP. Neurones in commissural nucleus tractus solitarii required for full expression of the pulmonary C fiber reflex in rat. J Physiol 441: 95–112, 1991.
Bonham AC, McCrimmon DR. Neurones in a discrete region of the nucleus tractus solitarius are required for the Breuer-Hering reflex in rat. J Physiol 427: 261–280, 1990.
Boscan P, Pickering AE, Paton JFR. The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents. Exp Physiol 87: 259–266, 2002.[Abstract]
Brown DL, Guyenet PG. Electrophysiological study of cardiovascular neurons in the rostral ventrolateral medulla in rats. Circ Res 56: 359–369, 1985.
Chan RKW, Sawchenko PE. Organization and transmitter specificity of medullary neurons activated by sustained hypertension: implications for understanding baroreceptor reflex circuitry. J Neurosci 18: 371–387, 1998.
Coleridge HM, Coleridge JC. Pulmonary reflexes: neural mechanisms of pulmonary defense. Annu Rev Physiol 56: 69–91, 1994.[CrossRef][Web of Science][Medline]
Coleridge HM, Coleridge JC. Afferent innervation of lungs, airways, and pulmonary artery. In: Reflex Control of the Circulation, edited by Zucker IH, Gilmore JP. Boca Raton, FL: CRC, 2001, p. 579–607.
Ezure K, Tanaka I, Kondo M. Glycine is used as a transmitter by decrementing expiratory neurons of the ventrolateral medulla in the rat. J Neurosci 23: 8941–8948, 2003.
Ezure K, Tanaka I, Saito Y, Otake K. Axonal projections of pulmonary slowly adapting receptor relay neurons in the rat. J Comp Neurol 446: 81–94, 2002.[CrossRef][Web of Science][Medline]
Feldman JL, Mitchell GS, Nattie EE. Breathing: rhythmicity, plasticity, chemosensitivity. Annu Rev Neurosci 26: 239–266, 2003.[CrossRef][Web of Science][Medline]
Guyenet PG, Mulkey DK, Stornetta RL, Bayliss DA. Regulation of ventral surface chemoreceptors by the central respiratory pattern generator. J Neurosci 25: 8938–8947, 2005a.
Guyenet PG, Stornetta RL, Bayliss DA, Mulkey DK. Retrotrapezoid nucleus: a litmus test for the identification of central chemoreceptors. Exp Physiol 90: 247–253, 2005b.
Hainsworth R. Reflexes from the heart. Physiol Rev 71: 617–658, 1991.
Hayashi F, Coles SK, McCrimmon DR. Respiratory neurons mediating the Breuer-Hering reflex prolongation of expiration in rat. J Neurosci 16: 6526–6536, 1996.
Ho CY, Gu Q, Lin YS, Lee LY. Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respir Physiol 127: 113–124, 2001.[CrossRef][Web of Science][Medline]
Kalia M, Richter D. Morphology of physiologically identified slowly adapting lung stretch receptor afferents stained with intra-axonal horseradish peroxidase in the nucleus of the tractus solitarius of the cat. I. A light microscopic analysis. J Comp Neurol 241: 503–520, 1985.[CrossRef][Web of Science][Medline]
Kubin L, Alheid GF, Zuperku EJ, McCrimmon DR. Central pathways of pulmonary and lower airway vagal afferents. J Appl Physiol 101: 618–627, 2006.
Kubin L, Kimura H, Davies RO. The medullary projections of afferent bronchopulmonary C fibers in the cat as shown by antidromic mapping. J Physiol 435: 207–228, 1991.
Li A, Nattie E. CO2 dialysis in one chemoreceptor site, the RTN: stimulus intensity and sensitivity in the awake rat. Respir Physiol Neurobiol 133: 11–22, 2002.[CrossRef][Web of Science][Medline]
Linz P, Veelken R. Serotonin 5-HT(3) receptors on mechanosensitive neurons with cardiac afferents. Am J Physiol Heart Circ Physiol 282: H1828–H1835, 2002.
McCrimmon DR, Alheid GF. Reflexively inhibiting respiratory drive. J Physiol Online jphysiol 2007.
Moreira TS, Takakura AC, Colombari E, Guyenet PG. Central chemoreceptors and sympathetic vasomotor outflow. J Physiol 577: 369–386, 2006.
Moreira TS, Takakura AC, Colombari E, West GH, Guyenet PG. Inhibitory input from slowly adapting lung stretch receptors to retrotrapezoid nucleus chemoreceptors. J Physiol 580: 285–300, 2007.
Mulkey DK, Stornetta RL, Weston MC, Simmons JR, Parker A, Bayliss DA, Guyenet PG. Respiratory control by ventral surface chemoreceptor neurons in rats. Nat Neurosci 7: 1360–1369, 2004.[CrossRef][Web of Science][Medline]
Nattie E, Li A. Muscimol dialysis in the retrotrapezoid nucleus region inhibits breathing in the awake rat. J Appl Physiol 89: 153–162, 2000.
Nattie E, Li A. Central chemoreception 2005: a brief review. Auton Neurosci 126 127: 332–338, 2006.
Okada Y, Chen Z, Jiang W, Kuwana S, Eldridge FL. Anatomical arrangement of hypercapnia-activated cells in the superficial ventral medulla of rats. J Appl Physiol 93: 427–439, 2002.
Okazaki M, Takeda R, Haji A, Yamazaki H. Glutamic acid decarboxylase-immunoreactivity of bulbar respiratory neurons identified by intracellular recording and labeling in rats. Brain Res 914: 34–47, 2001.[CrossRef][Web of Science][Medline]
Onimaru H, Homma I. A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci 23: 1478–1486, 2003.
Paton JF. Rhythmic bursting of pre- and post-inspiratory neurones during central apnoea in mature mice. J Physiol 502: 623–639, 1997.
Paton JF. Pattern of cardiorespiratory afferent convergence to solitary tract neurons driven by pulmonary vagal C-fiber stimulation in the mouse. J Neurophysiol. 79: 2365–2373, 1998.
Paton JF, Deuchars J, Li YW, Kasparov S. Properties of solitary tract neurons responding to peripheral arterial chemoreceptors. Neuroscience 105: 231–248, 2001.[CrossRef][Web of Science][Medline]
Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124: 4065–4075, 1997.[Abstract]
Paxinos G, Watson C. The Rat Brain in Stereotaxic Coordinates. San Diego, CA: Academic, 1998.
Richter DW, Ballantyne D, Remmers JE. The differential organization of medullary post-inspiratory activities. Pfluegers 410: 420–427, 1987.[CrossRef]
Ritucci NA, Erlichman JS, Leiter JC, Putnam RW. The response of membrane potential (Vm) and intracellular pH (pHi) to hypercapnia in neurons and astrocytes from rat retrotrapezoid nucleus (RTN). Am J Physiol Regul Integr Comp Physiol 289: R851–R861, 2005.
Rosin DL, Chang DA, Guyenet PG. Afferent and efferent connections of the rat retrotrapezoid nucleus. J Comp Neurol 499: 64–89, 2006.[CrossRef][Medline]
Schreihofer AM, Guyenet PG. Baroactivated neurons with pulse-modulated activity in the rat caudal ventrolateral medulla express GAD67 mRNA. J Neurophysiol 89: 1265–1277, 2003.
Schwarzacher SW, Wilhelm Z, Anders K, Richter DW. The medullary respiratory network in the rat. J Physiol 435: 631–644, 1991.
Stornetta RL, Guyenet PG. Distribution of glutamic acid decarboxylase mRNA-containing neurons in rat medulla projecting to thoracic spinal cord in relation to monoaminergic brain stem neurons. J Comp Neurol 407: 367–380, 1999.[CrossRef][Web of Science][Medline]
Stornetta RL, Moreira TS, Takakura AC, Kang BJ, Chang DA, West GH, Brunet JF, Mulkey DK, Bayliss DA, Guyenet PG. Selective expression of Phox2b by brain stem neurons involved in chemosensory integration in the adult rat. J Neurosci 26: 10305–10314, 2006.
Stornetta RL, Sevigny CP, Guyenet PG. Inspiratory augmenting bulbospinal neurons express both Glutamatergic and enkephalinergic phenotypes. J Comp Neurol 455: 113–124, 2003.[CrossRef][Web of Science][Medline]
Takakura AC, Moreira TS, Colombari E, West GH, Stornetta RL, Guyenet PG. Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2-sensitive neurons in rats. J Physiol 572: 503–523, 2006.
Verberne AJM, Guyenet PG. Medullary pathway of the Bezold-Jarisch reflex in the rat. Am J Physiol Regulatory Integrative Comp Physiol 263: R1195–R1202, 1992.
Verberne AJ, Saita M, Sartor DM. Chemical stimulation of vagal afferent neurons and sympathetic vasomotor tone. Brain Res Brain Res Rev 41: 288–305, 2003.[CrossRef][Medline]
Weston M, Wang H, Stornetta RL, Sevigny CP, Guyenet PG. Fos expression by glutamatergic neurons of the solitary tract nucleus after phenylephrine-induced hypertension in rats. J Comp Neurol 460: 525–541, 2003.[CrossRef][Web of Science][Medline]
Widdicombe J. Reflexes from the lungs and airways: historical perspective. J Appl Physiol 101: 628–634, 2006.
Wilson CG, Bonham AC. Effect of cardiopulmonary C fiber activation on the firing activity of ventral respiratory group neurons in the rat. J Physiol 504: 453–466, 1997.
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ABSTRACT
INTRODUCTION
) and a primary dendrite pointing toward the marginal layer. C2: biotinamide-labeled cell body at higher magnification. C3: 3 Phox2b-ir nuclei 1 of which (at 
