Abstract
The purpose of these studies is to better understand the nature of the reflex interactions that control the discharge patterns of caudal medullary, expiratory (E) bulbospinal neurons. We examined the effect of central chemodrive inputs measured as arterial CO2 tension (PaCO2) during hyperoxia on the excitatory and inhibitory components of the lung inflation responses of these neurons in thiopental sodium-anesthetized, paralyzed dogs. Data from slow ramp inflation and deflation test patterns, which were separated by several control inflation cycles, were used to produce plots of neuronal discharge frequency (F n) versus transpulmonary pressure (Pt). Pt was used as an index of the activity arising from the slowly adapting pulmonary stretch receptors (PSRs). Changes in inspired CO2 concentrations were used to produce PaCO2 levels that ranged from 20 to 80 mmHg. The data obtained from 41 E neurons were used to derive an empirical model that quantifies the average relationship forF n versus both Pt and PaCO2. This model can be used to predict the time course and magnitude of E neuronal responses to these inputs. These data suggest that the interaction between PaCO2and PSR-mediated excitation and inhibition ofF n is mainly additive, but synergism between PaCO2 and excitatory inputs is also present. The implications of these findings are discussed.
- control of breathing
- central integration
- central chemodrive
- pulmonary stretch receptors
expiratory(E) bulbospinal neurons make up a great majority of the E neurons in the caudal portion of the ventral respiratory group (VRG) in the region of the nucleus retroambigualis (4, 21). E bulbospinal neurons are known to have both monosynaptic and polysynaptic connections with contralateral spinal cord thoracic E motoneurons (9, 16, 17). It also appears that lumbar E motoneurons receive mono- and polysynaptic inputs from the contralateral E bulbospinal neurons (22). E bulbospinal neurons are the major source of drive for thoracic and abdominal E motoneurons (3) and, therefore, are responsible for active expiration. In addition, E bulbospinal neurons provide inhibition (presumably via interneurons) to thoracic inspiratory (I) motoneurons during expiration (29).
The excitability of E bulbospinal neurons is highly dependent on arterial CO2 tension (PaCO2) activation of central chemosensory sources with the greatest sensitivity being found over the hypo- to normocapnic range (2). In addition, these neurons receive excitatory inputs from carotid chemoreceptors (18, 30) and both excitatory and inhibitory inputs from pulmonary mechanoreceptors with vagal afferent fibers (4,9). On the basis of spontaneous discharge patterns and responses to lung inflation, the E bulbospinal neurons in dogs can be divided into two types: type A, augmenting (20–30%), and type D, decrementing (70–80%) (4). Graded inhibition of type A neurons is produced by lung inflation when transpulmonary pressure (Pt) exceeds 3–4 mmHg. For type D neurons, studies using step and slow positive or negative ramp inflations (1–2 mmHg/s) demonstrate that the relationship between neuronal discharge frequency (F n) and Pt is made up of two major, linear components (4). For 1.5 ≤ Pt ≤ 4.5 mmHg, the relation is positive (excitatory), whereas for 4.5 ≤ Pt ≤ 20 mmHg, the relation is negative (inhibitory). The inhibition is strong enough to override the excitation. Because the excitatory and inhibitory responses to step inflations are slowly adapting, it is highly likely that the slowly adapting pulmonary stretch receptors (PSRs) are involved. Two types of canine PSRs have been identified based on the relationship of their discharge frequency to Pt and on their anatomic location in the airways (23, 24). Because the characteristics of these two PSR types coincide with the response characteristics of the type D (decrementing) E bulbospinal neurons, it is possible that these two different types of PSRs mediate the excitatory and inhibitory components.
The activity of E bulbospinal neurons is sensitive to vagal feedback from pulmonary mechanoreceptors and to central and peripheral chemosensory stimulation and thus can influence most of the mechanical properties of ventilation, such as tidal volume, breathing frequency, airflow rate, and end-expiratory lung volume. In response to metabolic demands, they appear to play a significant role in adjusting ventilatory mechanics to provide efficient performance. These studies were undertaken to characterize the interaction between PSR and central chemosensory inputs on the discharge patterns of the type D E neurons of dogs.
METHODS
Experimental preparation.
Experiments were performed on 18 mongrel dogs (10–20 kg) anesthetized with thiopental sodium (induction dose: 15 mg/kg iv; additional doses given as needed during preparation; maintenance dose during data collection, 4–8 mg · kg−1 · h−1 iv continuous infusion). Positive-pressure constant flow ventilation was produced by an alternating two-valve solenoid ventilator through a cuffed endotracheal tube using 100% O2. Airway CO2was measured with an infrared analyzer (Instrumentation Laboratory IL-200), and tracheal pressure was measured from an airway sideport with an air-filled catheter connected to a Gould-Statham P23ID transducer. Arterial pressure was measured from a femoral artery fluid-filled catheter using a Gould-Statham P23ID transducer. Arterial blood samples were obtained hourly for the measurement of pH, PaCO2, and PaO2 using a Radiometer ABL 1 analyzer. When required, metabolic acidosis (base deficit >5 mM) was corrected with an appropriate amount of sodium bicarbonate in saline. The dogs were monitored for signs of inadequate anesthesia, including movement, salivation, lacrimation, and/or increases in blood pressure and heart rate. The anesthetic depth was increased immediately if such signs were present. Esophageal temperature was maintained at 38 ± 1°C using a servocontrolled heating pad.
The dogs were positioned in a Kopf (model 1530) stereotaxic apparatus with the head ventrally flexed by 30°. The right C5phrenic nerve rootlet and both vagi were exposed by dorsolateral neck dissection. The medulla oblongata was exposed by occipital craniotomy, cutting the dura mater along the midline, and retracting the dura with silk sutures. This procedure exposed the dorsal surface of the medulla from 2 mm rostral to 10 mm caudal to the obex and 5 mm bilaterally from the midline. To further stabilize the brain stem before recording unit activity and to minimize feedback from nonvagal, chest wall afferents, the animals were paralyzed with pancuronium bromide (Pavulon), initial dose of 0.1 mg/kg iv, followed by supplemental doses of 0.05 mg/kg as required, and a bilateral pneumothorax was created. Thus in these studies, tracheal and transpulmonary pressure, Pt, are equivalent.
Data recording.
Efferent phrenic activity, spike potentials from the brain stem neurons, airway CO2 concentration, tracheal pressure, and blood pressure were recorded on an FM tape recorder (Vetter, model D). The above-mentioned parameters and time-averaged phrenic activity (PNG), neural spikes/100 ms, I duration (T I), and E duration (T E) were recorded on a Grass model 7 polygraph. Phrenic recordings were obtained with bipolar electrodes from the desheathed central end of the C5rootlet, which was immersed in a mineral oil pool formed from a neck pouch. The phrenic nerve signal was amplified with a band pass of 0.1–3 kHz. The online moving time average of the phrenic activity was obtained by full-wave rectification and low-pass filtering (averaging window = 50 ms). The positive PNG slope at the onset of phrenic activity and the negative PNG slope at the onset of the abrupt decline in phrenic activity were used to generate I and E timing pulses, respectively. These timing pulses were used to compute online values of T I and T E.
Extracellular single-unit recordings from caudal VRG E neurons were obtained using tungsten metal microelectrodes (10–15 MΩ at 1,000 Hz). Locations of recorded neurons relative to obex were in a region 2–4 mm caudal, 2.5–4.5 mm lateral to the midline, and 2–4 mm below the dorsal medullary surface. A time-amplitude window discrimination was used to generate a standard pulse for each spike. Online neuronal spike frequency was determined as spikes per 100 milliseconds, whereas offline data analysis used spikes per 10 milliseconds.
Protocol.
During control cycles, the ventilator frequency was adjusted to be near that of the central respiratory rhythm (as indicated by PNG) and entrained the central pattern via PSR feedback. Once a type D expiratory neuron was located in the caudal VRG at normocapnia, ventilatory tidal volume was increased to produce a hypocapnic PaCO2 level of ∼20 mmHg. After airway end-tidal CO2, F n and peak PNG (if central inspiratory rhythm was still present) had reached a steady state (usually after 3–5 min), four slow positive (+) and four negative (−) test ramp inflations separated by six to ten control ventilator cycles were applied. These test inflations had duration of 6–10 s and produced peak Pt values of 12–20 mmHg. Arterial blood samples were drawn for measurement of PaCO2, pH, and PaO2. To obtain several steady-state PaCO2 levels over the range of 20–80 mmHg, CO2 was added to the inspired O2 via a gas blender. Neuronal responses to the ramp inflations were obtained for one to eight steady PaCO2 levels, averaging four PaCO2 levels per neuron.
Data reduction.
Offline data analyses were carried out on a Hewlett-Packard model 360 computer with a data converter interfaced through an IEEE 488 data port. A conversion rate of 100 Hz was used to enter 5- or 10-min epochs of the number of neuronal spikes per 10 milliseconds, phrenic activity, time averaged for 10 ms, tracheal pressure, and a ventilator I phase indicator into the computer memory and subsequently onto a disk file. Software routines assigned a number to each consecutive ventilation cycle and displayed the signals on the monitor. Ventilator cycles were numbered consecutively, and the numbers corresponding to a given test inflation pattern, (+) or (−) ramps, at a given PaCO2 level were identified and used to generate cycle-triggered histograms (CTHs) of unit activity and ensemble averages of both phrenic activity and tracheal pressure patterns. The temporal alignment of the CTHs and ensemble averages was accurate to within 10 ms of the phase onset indicator signal. CTHs and ensemble averages were saved on disk files for further analyses, which included the generation of F n versus Pt plots for test inflation cycles.
Least-squared-error linear and nonlinear regressions were used to quantify the Pt-dependent effects onF n and the PaCO2-dependent effects on the F n-Pt relationship parameters. Data for these analyses were obtained fromF n-Pt plots using the CTHs forF n and corresponding ensemble averages of Pt. Data are presented as mean values with SEs, unless otherwise stated. Probability levels of P < 0.05 were used to indicate significant differences.
RESULTS
Expiratory neuronal responses to step lung inflations delivered during the expiratory phase.
The E neuronal responses to step inflations during the E phase are slowly adapting and related to the magnitude of the step Ptin a biphasic manner. In the example of Fig.1, the 4-s-long step test inflations (Pt) were delayed 300 ms from the beginning of the E phase and were separated by 8–10 control cycles to minimize changes in PaCO2 during subsequent test cycles. During control and test cycles, 1-s duration inflations were delivered during the I phase to provide steady-state ventilation. For Ptvalues <5 mmHg, step inflations delivered during test respiratory cycles produced reflex increases in T E and increases in F n (Fig. 1 A). A step inflation of 1.3 mmHg prolonged the E phase and the neuronal discharge (Fig. 1 A, S1) of an E neuron with a decrementing control pattern, whereas larger step inflations of 2.6 and 4.8 mmHg increasedF n of the step responses (Fig. 1, Aand B, S2 and S3). For Pt >5 mmHg, step inflations reduced F n below maximum response values and the responses remained relatively independent of time during the step inflation (e.g., Fig. 1 B, S4 = 10.6 mmHg). The neuronal responses are mainly dependent on inflation pressure and only to a small degree on time. This allowed us to quantify the biphasic neuronal response to inflation using plots of F nversus Pt, where Pt is used as an index of slowly adapting PSR activity (Fig. 1 B).
Example of the slowly adapting responses of an expiratory (E) neuron to step inflation patterns. PNG, phrenic neurogram;F n, cycle-triggered histograms (CTHs) of discharge frequency; Pt, ensemble average of transpulmonary pressure, T E0, control E duration. Four test cycles/CTH. A: responses to low-Pt step inflations. con, Control pattern without inflation (thick line). S1: 1.4 mmHg; S2: 2.6 mmHg. B: F n, neuronal responses to higher Pt step patterns. S3: 4.8 mmHg, S4: 10.5 mmHg. B, top: plot ofF n vs. Pt for mean ± SD of CTH data during the step input (between 1.5 and 4.5 s). Note the biphasic nature of the E neuronal response.
E neuronal responses to slow ramp lung inflations delivered at different times during the E phase.
The relationship between F n and Ptcan also be obtained when slow ramp inflations are used to scan the entire Pt range from 0 to 15–20 mmHg in a single test cycle. This is possible because time-dependent effects on these responses are minimal. This is illustrated for an E neuron where slow ramp test inflations were delivered in the E phase with different delays with respect to the onset of the PNG (Fig.2 A). The biphasic nature of the response can be seen for the ramp inflation with the larger delay (Fig. 2 A). This delay was the largest one that could be used, because the control T E was <2 s (T E0, Fig. 2 A) and the next I phase would start before the inflation reflexly prolonged the E phase. Near the beginning of the E phase, the early portion of the control decrementing pattern can be seen before the ramp inflation starts (Fig.2 A, F n). As the ramp Ptincreased, F n increased and reached a maximum at Pt ≈ 5 mmHg, then decreased with increasing Pt. Note that the amount of inhibition is more than able to suppress the Pt-induced excitation. When the ramp inflation was terminated, F n rapidly increased and then decreased as the subsequent I phase began. For those cycles in which the slow ramp inflations began 2 s earlier, the early portion of the response was not seen because I-phase inhibition produced neuronal silence (Fig. 2 A). The lack of time dependence in these neuronal inflation responses, separated by 2 s, is demonstrated by the high degree of overlap in the F n traces when the two Pt traces are superimposed (Fig. 2 B). The plot of F n versus Pt for the more delayed ramp inflation quantifies this biphasic relationship (Fig.2 B). This relationship is similar to the one for step inflations (Fig. 1 B).
Typical E neuronal responses to delayed slow ramp inflations.A: inflations have delays separated by 2 s. Earlier ramp started late in the inspiratory (I) phase (Pt, thin line) and later ramp started just before the end of the E phase indicated by the dashed line at upstroke of control PNG. B,middle and bottom: same data displayed with Pt traces superimposed with time shift. Note the high degree of overlap in the F n CTHs indicative of response dependence on Pt rather than time. Earlier portion of the excitatory response to the earlier ramp is missing due to I phase inhibition of E activity; 6 test cycles/CTH. B,top: plot of F n vs. Ptshows a typical biphasic response similar to that of Fig.1 B, top. F n vs. Pt data were obtained from the 50-ms bin data of the CTH during the more-delayed ramp pattern.
The effects of PaCO2 on E neuronal responses to positive and negative slow ramp inflations.
We used both positive and negative test ramp inflations for the same neuron to better isolate Pt-dependent effects from time-dependent effects. By using “contrasting” pressure-time profiles, the amount of time dependence affecting the neuronal response to inflation will be reflected in the difference betweenF n-Pt plots for each type of inflation pattern. In the absence of time dependence,F n-Pt plots obtained from positive and negative ramps should coincide.
Ventilation patterns synchronized with central rhythm.
Examples of the responses to positive and negative ramps for the same neuron at different levels of PaCO2 are shown in Figs.3 and 4. Both inflation patterns were alternately presented at each PaCO2 level. Increases in PaCO2increased the peak F n of control cycles and enhanced the inflation-induced responses. For the positive ramps (Fig. 3), the biphasic nature of the response is evident and similar to those of Fig. 2. One-second duration control inflations were delivered during the I phase. However, to produce a slower central rhythm, a 1-s delay from the onset of the PNG was used to shift the inflation later into the I phase and subsequent deflation later into the E phase. This prevented too much reflex shortening of T I and produced longerT E values. On test cycles, no delay was used for the I-phase inflation to allow sufficient time for deflation and Pt to return to baseline before the slow test ramp was initiated (1.8-s delay from onset of E phase, Fig. 3) that had to occur early enough to reflexly prolong T E. The negative ramps were produced by an increase in E flow resistance during the test cycle (Pt, Fig. 4). Because Pt is highest at the onset of the E phase,F n is initially reduced and gradually increases as the Pt-induced inhibition decreases, unmasking the Pt-induced excitation. The neuronal response peaks as Pt passes through the inhibitory threshold and then declines as the Pt-induced excitation decreases. The peakF n is again highly dependent on PaCO2 (Fig. 4).
Effect of arterial Pco 2(PaCO2) on E neuronal responses to slow positive ramp inflations. F n: ratemeter output (0.1-s intervals). Vertical dashed lines: onset of the E phase of the test cycle. Slow ramps delayed 1.8 s from onset of E phase.F n of both control and biphasic inflation response patterns increased with increased PaCO2.
Effect of PaCO2 on E neuronal responses to slow negative ramps for the same neuron shown in Fig. 3. Vertical dashed lines: onset of the E phase of the test cycle. E airflow resistance added during test cycle to produce slow negative ramps with no delay. As Pt decreased, the decline in inhibition resulted in increased F n. With further decreases in Pt, excitation was also removed. PeakF n of both control and test cycles increased with increased PaCO2.
Central rhythm entrained by ventilation pattern.
To investigate the effects of PaCO2 both above and below the apneic threshold, the phenomenon of entrainment was used to time lock the central respiratory pattern to the ventilation pattern. For this purpose, the ventilator rate for control cycles is set close to the central respiratory rhythm rate, which can be determined from a few PNG cycles without ventilation. In the example of Fig.5, the control inflation (Pt) can be seen to consistently occur during the early part of the E phase. For the test cycles, slow ramps replaced the control inflation pattern. Slow negative ramps were produced by adding an E flow resistance during test inflations. In addition, the I flow rate was increased to produce greater peak Pt levels during the negative ramp test cycles (15–20 mmHg).
Effect of PaCO2 on inflation responses above and below the apneic threshold. The central respiratory pattern was entrained by the ventilation pattern. Both E neuronal activity (ENA) and PNG decreased as PaCO2 decreased. At a PaCO2 of 35, PNG was abolished but central rhythm remained entrained. In this case, small-amplitude E unit activity was also present. The biphasic characteristic of the inflation response was preserved at all PaCO2 levels.
At all PaCO2 levels, a Pt-induced reduction in spike discharge frequency was seen during both the control and test cycles (Fig. 5). For the three levels of PaCO2 indicated, the traces are time aligned with respect to Pt. As PaCO2 decreased, the peak PNG also decreased and disappeared at the 35 mmHg PaCO2 level (Fig. 5, bottom trace). The E neuronal discharge rate also decreased, but the underlying pattern was preserved. Longer periods of neuronal silence occurred during the test inflation at the lower PaCO2 levels, indicating a baseline shift in discharge activity.
Typical plots of F n versus Pt for positive (+) and negative (−) Pt ramps at several PaCO2 levels are shown in Fig.6, A and B, for two E neurons. Data values for these plots were obtained from CTHs of neuronal activity and ensemble averages of Pt at corresponding times for all 50-ms bins during the test inflation. This analysis demonstrates that the typical biphasic nature of theF n versus Pt relationship (e.g., Figs. 1 B and 2B) is preserved regardless of whether positive or negative test inflations were used. A noticeable difference between positive and negative test inflation plots is the missing data points at low transpulmonary pressures (0–3 mmHg range) for negative test inflations. This is due to the reappearance of inspiratory activity when Pt levels approached 0 mmHg and thus no longer reflexly prolonged the E phase. This effect manifests itself as a sharp fall in F n as Ptdecreases toward zero. Another difference is that theF n-Pt plots for the positive ramp inflations appear to be more skewed to the right, especially at the higher PaCO2 levels. This may be due to a small amount of time dependence in the response. In allF n-Pt plots,F n and the sensitivity of the response to Pt increased with increasing PaCO2 levels. In addition, the Pt value at whichF n is maximal shifted to higher values.
Typical examples of F n vs. Ptplots as a function of PaCO2 for 2 E neurons (A and B). Top: data for positive (+) ramp inflations. Bottom: data for negative (−) ramp inflations. Data obtained from CTHs of F n and ensemble averages of Pt. All CTHs were triggered from the onset of the test Pt patterns, because no PNG is present at low PaCO2 levels.
Pressure-dependent and time-dependent response components.
Although it is clear that the canine E neuronal inflation response is highly dependent on Pt via the PSRs, the response also appears to depend to a minor degree on time (t), i.e.,F n =F(Pt,t). To separate these two effects, we assumed, as a preliminary hypothesis, that the time component was linear or F(t) = αt, where α is the slope, which can be positive or negative and has the units of Hertz per second. This assumption appears reasonable based on the neuronal responses to relatively constant inputs such as those of Fig. 1 and those in which step frequency, electrically induced PSR inputs were used (32). From a practical viewpoint, it would be very difficult to separate the pressure and time-dependent components, if a more complex form of time function is used. ThusF n = F(Pt) + αt, and the pressure-dependent component isF(Pt) = F n − αt. Data for the responses to both the positive and negative ramps were use to calculate an average value of α over the time span of the test inflations (see appendix for details). Figure 7 shows an example ofF n-Pt plots before (Fig. 7,top) and after (Fig. 7, bottom) the removal of the time-dependent component, αt. The latter provides a better estimate of the Pt-dependent relationship. Thus, if the neuronal response is dependent only on Pt, then the same F n-Pt plot should be obtained whether response data from (+) or (−) test ramps are used. To estimate the ability of this method to reduce the differences between theF n-Pt plots for the (+) and (−) ramp inflations, an average error index was calculated before and after the time compensation procedure. The error index was the average of the absolute difference between the two plots for the range of overlap. The average error between plots was reduced by 25.3% from 7.94 ± 0.42 to 5.93 ± 0.34 Hz (P < 0.0001) for the 41 neurons with multiple PaCO2 levels.
Example of F n vs. Ptplots for both positive (P) and negative (N) ramp inflation patterns.Top: before time correction, average error between plots: 11.1 Hz. Bottom: after time correction procedure, average error between plots: 4.0 Hz. PaCO2: 79 mmHg; α: 5.5 Hz/s.
Time-dependent component.
The α values at each PaCO2 level for each neuron were also analyzed for their dependence on PaCO2. The slope and intercept values for plots of α vs. PaCO2were obtained by linear regression for each of the 41 neurons. The mean values of the slopes and intercepts, weighted according to the number of PaCO2 levels per neuron (average no. of levels = 2.8) yielded the following average relationship: α = 0.62 + 0.027 · (PaCO2 − 40) Hz/s, where the slope (i.e., 0.027 ± 0.008) and the intercept at PaCO2 = 40 mmHg (0.62 ± 0.19) were significantly different from zero. This relationship indicates that the time-dependent effect is relatively small. For example, at a PaCO2 of 40 mmHg, α = 0.62 Hz/s and for a 10-s duration inflation, this component would contribute 6.2 Hz at the end of the response. At a PaCO2 of 60, the contribution would be 11.6 Hz.
PaCO2 effect on Fn-Ptrelationship.
To analyze the PaCO2 effect on theF n-Pt relationship, the salient features of the plots were quantified using a piecewise linear approximation of the relationship after the time-dependent component was removed. To facilitate the analysis, theF n-Pt data were sorted according to Pt value and placed in a histogram format and cursors were used to define the analysis ranges. TheF n-Pt relationship was then divided into three linear segments: one with a positive slope (Slp0) and two with negative slopes (Slp1 and Slp2; Fig. 8). Standard linear regression techniques were used to obtain the best-fit lines. The intersection of the positive and negative slope lines was used to determine the Pt value or threshold (Pthr1) where the PSR-mediated inhibition began to reduceF n. The actual F n value, which corresponds to Pthr1 was defined asF max. A second Pt value (Pthr2) was defined by the intersection of the two negative slope line segments. In addition, a third Pt value (Pthr0) was defined as the Pt at which lung inflation began to produce excitation of the E type-D neurons. This value was easiest to obtain at the lower PaCO2 levels when central inspiratory rhythm was slower or absent. However, it was also obtained at higher PaCO2 levels in cases where I-phase inhibition did not coincide in time with the low-Ptportion of the test ramp inflations. Typical plots of these parameters versus PaCO2 for an E neuron are shown in Fig.9. Linear regression was used to quantify the relationship between each parameter and PaCO2(lines through data points). The plots of F maxversus PaCO2 were best fit by a sigmoidal function of the form: F max =F 0[x/(1 + x)], where x = (PaCO2/PaCO2*)k,k is a noninteger exponent related to steepness, PaCO2* is the value of PaCO2at which F max =F 0/2, and F 0 is the asymptotic maximum of the sigmoid function.
Example illustrating method used to quantify the inflation response. A piecewise linear approximation of the relationship was used after time correction. SeePa CO2 effect on Fn-Pt relationship for further details.F max, peak F n.
Typical plots of theF n-Pt relationship parameters vs. PaCO2. Data shown after time correction has been applied. Linear relationships were used to summarize parameter dependencies on PaCO2, except forF max where a sigmoidal relationship was used.Right: data for positive ramps. Left: data for negative ramps. Note the general agreement in parameter responses to PaCO2 for the 2 inflation patterns.
Average PaCO2-dependent parameters of the Fn-Pt relationship.
After correction for time dependence, the corresponding parameters obtained from the best fits of theF n-Pt plots (e.g., Fig. 8) for the (+) and (−) ramp responses were averaged at each PaCO2 level for each neuron. Table1 summarizes the pooled data of 41 neurons. The intercepts have been translated to a PaCO2 value of 40 mmHg. Thirty-nine of forty-one neurons exhibited an Slp2 segment. Data for the sigmoidal type relationship indicates that the average maximumF n at high PaCO2 was 91.9 Hz and that 50% of this value was achieved at a PaCO2 = 37.1 mmHg. Significant linear relationships were found for Slp0, Slp2, Pthr0, Pthr1, and Pthr2, but not for Slp1 (Table 1).
Fn-Pt parameters as function of PaCO2 after time correction and averaged for ± ramp inflations
Average Fn-Pt relationship as a function of PaCO2.
With the use of the mean values of Table 1, it is possible to produce a family of F n-Ptrelationships in which PaCO2 is the family parameter. Similar to the analysis, the empirical model forF n-Pt relationship is comprised of line segments that are functions of Pt(F a, F b,F c, and F d, Fig.10, top). These explicit Pt-dependent functions, which were used to approximate theF n-Pt relationship, are given in Fig. 10, middle. The point Pthr1,F max is the key starting point from which the F n-Pt relationship is constructed. Because the line segments are also functions of PaCO2, the slope, intercept, and intersection point parameters of these line segments are functions of PaCO2 as defined in Fig. 10, bottom. These analytic relations were used to generate a three-dimensional surface plot of F n as a function of both Ptand PaCO2 (Fig. 11). The strong dependence of F n on both of these variables can be appreciated. The inflation-mediated excitation is much more sensitive to PaCO2 than the inflation-mediated inhibition (compare Slp0 with Slp1 and Slp2, Fig. 10, bottom, and Fig. 11). TheF n-Pt relationship is maintained in the absence of central respiratory rhythm at PaCO2 levels typically <30 mmHg. The increases in threshold levels for Pthr0, Pthr1, and Pthr2 with increases of PaCO2 can also be seen.
Description of the empirical model for the E neuronal response to lung inflation and PaCO2. The mathematical description of each segment of theF n-Pt relationship is given below the line plot (box, center). The parameters of the linear functions are themselves functions of PaCO2 as defined by the average relationships in the bottom box, which are based on the data from 41 E neurons. With this model, theF n of an average E neuron can be calculated for any desired values of Pt and PaCO2.
Graphical summary of F n as a function of both Pt and PaCO2 for the average E neuron.
The calculated relationship between F n and PaCO2 at fixed Pt levels is sigmoidal in shape (Fig. 12). Generally,F n increases with PaCO2 at all Pt levels, suggesting that part of the inflation-mediated inhibition of the E neurons can be offset by increases in PaCO2.
Estimated relationship between F nand PaCO2 at various Pt levels, as indicated (parentheses at right of traces). Data computed from the model of Fig. 10. Top, low Pt range;bottom, high Pt range.
DISCUSSION
This study characterizes the integration of mechanosensory and chemosensory inputs by canine E neurons of the caudal VRG. Most of these neurons are presumed to be bulbospinal neurons on the basis of previous studies of E neurons in the same location, where >93% of those neurons were antidromically activated from the cervical spinal cord (5). An empirical model has been developed that can predict the instantaneous discharge frequency,F n, of an average E bulbospinal neuron for given PaCO2 and transpulmonary pressure, Pt, values. In addition, the profile of the discharge pattern is also predictable for a given trajectory of Pt.
Previous studies have investigated the response of these neurons to each input in isolation (2, 4, 9, 31), but not to the combination of both inputs. Open-loop conditions were used to minimize confounding inputs from other sources or secondary effects. In this regard, neuromuscular blockade and bilateral pneumothorax were used to eliminate phasic afferent inputs from areas other than the lung, and test inflation patterns, separated by several control respiratory cycles, were used during hyperoxia (PaO2 >300 mmHg) to minimize the effects of transient changes in PaCO2during test cycles. The delayed responses of neuronal and phrenic activities to step changes in inspired CO2 concentration, which required 3–5 min to reach steady state, suggest that the hyperoxic conditions minimized inputs from the peripheral chemoreceptors that have a fast response time. Thus, in this study, the major source of chemosensory input to the E bulbospinal neurons is of central origin. The PSR-mediated reflex is also controlled by a GABAergic gain-modulating mechanism (20) that may be affected by barbiturates. However, a constant infusion of the thiopental anesthetic was used to maintain a stable blood concentration and hence a relatively constant level of anesthesia. Thus modulation of the GABAergic input by the anesthetic, if present, would be at a constant level and the nature of the interaction between PaCO2 and the PSR input should be unaltered.
On the basis of the sustained E neuronal responses to step inflation patterns, our previous (4) and current study (e.g., Fig.1) suggest that both the excitatory and inhibitory components of the inflation response are mediated by the slowly adapting PSRs. Pt was used as an index of PSR activity, and vagotomy eliminates the inflation response (4). It is possible that the excitatory and inhibitory components are mediated by the two types of PSRs with appropriate characteristics that have been described in dogs (23-25) or by a single set of PSRs in conjunction with a central mechanism (4).
Pressure and time dependence of the E neuronal response.
Although our analysis shows that a time-dependent factor contributes to the discharge pattern of these E neurons, its effect is relatively small and appears to be overridden by inputs from the PSRs, such that the time course of the discharge pattern is highly dependent on the time course of transpulmonary pressure (4). The small contribution of the time related changes in F nis best illustrated by the E neuronal responses to step inflations, delayed ramp inflations, and by overlap of theF n versus Pt plots for data from positive and negative ramp inflations, as demonstrated in Figs. 1, 2, and 7, respectively. When the F n-Ptplots were corrected for time-dependent effects, the average error between plots for the positive and negative ramp inflations for the 41 neurons at the various PaCO2 levels was reduced from 7.9 to 5.9 Hz.
The average coefficient, α, for the assumed time-dependent linear component, αt, at a PaCO2 of 40 mmHg was 0.62 ± 0.19 Hz/s, with 68% of the neurons having values within the range of −0.57 (mean − SD) and 1.81 Hz/s (mean + SD). Thus, at the end of a 10-s test inflation, the time-dependent component could reduce F n by 5.7 Hz for neurons at the low end of the range or increase Fn by 18.1 Hz for neurons at the high end of the range. These contributions would be proportionately less during eupnea, where E durations are 2–4 s. The α coefficient was also dependent to a small degree on PaCO2 with a mean ± SD value of 0.027 ± 0.052 Hz · s−1 · mmHg PaCO2 −1. A 10-mmHg increase in PaCO2 would increase α on average by 0.27 Hz/s.
In contrast to dogs, E bulbospinal neurons in cats exhibit a marked time-dependent component, which manifests itself as an augmenting ramp discharge pattern. Neuronal excitation was observed for the low-Pt range and inhibition for the higher Ptrange in cats (see Fig. 10, top, of Ref. 9); however, the pattern maintained its augmenting profile.
Central chemodrive dependence of the E neuronal response.
The activities of both the control and test inflation cycles increased with increases in PaCO2 (e.g., Figs. 3 and 4). Plots of the peak F n (F max, Fig. 8), as well as F n at various Ptlevels (Fig. 12), versus PaCO2 were sigmoidal in shape (Fig. 9, middle). The steepest part of theF max curve, which occurred at 50% of maximum, was located at an average ± SE PaCO2 of 37.1 ± 1.5 mmHg, and the PaCO2 range for the 20–80% response was 26–62 mmHg. In chloralose-urethane-anesthetized cats, Bainton and Kirkwood (2) also noted that plots of F nversus alveolar Pco 2(Pa CO2) for E bulbospinal neurons were steep and sigmoidal; however, the greatest sensitivity was found to occur at Pa CO2 values from 20 to 30 mmHg. These results suggest that the greatest sensitivity of the caudal VRG E neurons to PaCO2 occurs over a PaCO2range somewhat lower than that for inspiratory (phrenic) activity (13).
Nature of interaction between chemosensory and mechanosensory inputs.
On the basis of the plot of F n vs. Pt and PaCO2 of the average E neuron (Fig.11), the general impression is that these two inputs are mainly additive. That is, an increase in PaCO2 shifts theF n-Pt relationship upward. However, the quantitative relationships used to generate Fig. 11 indicate that there is a synergistic interaction between PaCO2 and the excitatory component of the Pt response. The positive slope, Slp0, increases by 38% for a change in PaCO2 from 30 to 50 mmHg and results in higherF max values. The negative slope, Slp1, is not altered by PaCO2 and can be seen as a parallel shift if theF n-Pt relationship (Fig. 11). The negative slope, Slp2, becomes less negative with increases in PaCO2, and the inhibition of the higher Pt range is less effective at higher PaCO2 levels.
The bidirectional F n-Pt relationship was preserved regardless if ventilation was synchronized with central I activity (e.g., Fig. 3) or if central I activity was entrained by ventilation pattern (e.g., Fig. 5). In addition, for >80% of the cases, central neural apnea (peak PNG decreased to zero) occurred in these barbiturate-anesthetized dogs at PaCO2 <45 mmHg. In some cases, such as Fig. 5, central I inhibition of E neuronal activity was observed. However, with lower PaCO2 levels, the central I inhibition disappears and tonic E activity can be observed when PSR input is prevented by temporarily halting ventilation (data not shown). The transition from rhythm to central neural apnea appears to have no affect on theF n-Pt relationship, suggesting that the functioning of this reflex is not conditional on the presence of rhythm or phasic activities. This is consistent with the finding that the same E bulbospinal neurons are capable of relaying both phasic and tonic excitation to spinal respiratory motoneurons and that rhythmic excitation of E muscles results from a periodic I phase inhibition of the E bulbospinal neurons that are subjected to a graded, tonic, CO2-dependent excitation (2, 3).
Furthermore, the preservation of theF n-Pt relationship below the apneic threshold suggests that more direct neural pathways, which bypass the rhythm generation structures, may mediate integration of the mechano- and chemosensory inputs by the E bulbospinal neurons. PSRs synapse within the nucleus of the solitary tract and second- and possibly higher-order neurons may relay the Pt-related information to the E bulbospinal neurons (8, 32).
Physiological significance of the model parameters.
Although the empirical model for theF n-Pt relationship (Fig. 10) is useful in quantifying the neuronal responses to arbitrary inflation patterns, the model parameters themselves have physiological correlates. Pthr0 indicates the threshold pressure at which the PSR activation begins to excite the E neurons, and Slp0represents the sensitivity of that reflex component. Pthr1appears to represent the point where the Pt-related progressive recruitment of the PSRs and/or PSR activity, which mediate the inhibitory component of the E neuronal response to lung inflation, opposes further neuronal excitation, and actually reverses it. It is not known if there exists a separate group or type of PSRs that mediates the inhibitory response. This inflation-mediated excitatory/inhibitory interaction determinesF max and the sensitivity of the inhibitory component, Slp1, as Pt increases. Because Pthr2 is the Pt value at which the two piecewise linear approximations of the curvilinear inhibitory response component intersect, it represents a point of maximum slope inflection. Slp2 represents the sensitivity of the second subcomponent of the inhibitory portion of theF n-Pt relationship.
Increases in PaCO2 produced small, but statistically significant, increases in the three Pt threshold values, Pthr0, Pthr1, and Pthr2 (Table 1). The upward shift in these parameters with increases in PaCO2 may be due the effects of Pa CO2 on the PSRs, per se, because increases in Pa CO2 have been shown to reduce PSR activity and increase their activation thresholds (10,14, 27, 28).
Correlation of E bulbospinal neuronal with E muscular responses.
The various thoracic and abdominal E muscles respond differentially to changes in PaCO2 and PSR inputs (1). It is possible that these differences may be accounted for by the variability in the sensitivities of the model parameters, although the behavior of the 41 type D neurons of this study was qualitatively similar. A measure of the degree of parameter variation is given by the 10th and 90th percentile values of Table 1. For example, in 80% of these neurons, the slope of the excitatory portion of theF n-Pt relationship ranged from 5 to 30 Hz/mmHg and that of the inhibitory portion ranged from −5.2 to −20.4 Hz/mmHg (Table 1, intercept of Slp0 and Slp1 at a PaCO2 of 40 mmHg). In addition to parameter variability, it is possible that some E muscles do not depend on PSR feedback. For example, during postural changes and use of the rib cage, triangularis sterni E muscles are largely independent of vagal inputs, in contrast to E abdominal muscles, which rely on vagal feedback for this purpose (11).
Perspectives
During spontaneous breathing, E airflow normally is retarded by the combined effects of increased laryngeal airflow resistance, post I activity of the diaphragm, and inhibition of E muscle activity (26, 33). The afferent limb is composed of extrathoracic and intrathoracic pulmonary and tracheal-bronchial stretch receptors with vagal fibers. During expiration, this reflex continuously compensates for changes in upper airway resistance and tends to maintain a normal E flow rate possibly to improve gas exchange and prevent alveolar collapse.
PSR-mediated inhibition of E bulbospinal neurons appears to play an important role in E airflow control. At end-inspiration, elastic recoil pressure and Pt are greatest, and maximum inhibition of E bulbospinal neuronal activity occurs. As deflation proceeds, disinhibition results in an augmenting E neuronal discharge pattern, which would act to maintain E airflow when elastic recoil pressure is decreasing. Hypercapnia increases E neuronal F n; however, tidal volume and peak Pt are also increased and PSR-mediated inhibition of F n would minimize the contribution of E muscles in early expiration. Due to the higher recoil pressure, the lung would empty faster and disinhibition of the PaCO2-elevated E neuronal activity would aid in emptying the lung during the later part of the E phase. In addition, as lung volume and Pt decrease below the Pthr1,F n decreases due to the reduction in the PSR-mediated excitation, effectively braking active expiration and limiting deflation below function residual capacity. Expiratory phase duration is also highly dependent on the E volume trajectory and history via the Hering-Breuer E facilitatory reflex (34), and the discharge pattern of the E bulbospinal neurons would therefore contribute to the control of T E by altering PSR feedback.
E bulbospinal neurons appear to provide a good neural model in which the central integration of different types of information may be studied, such as those arising from mechanosensory (5, 7), chemosensory (12), and central pattern-generating (6, 15, 19) inputs. The model also allows the study of the neurotransmitters and synaptic mechanisms involved.
In summary, this study characterizes the control of the E neurons in the caudal VRG of dogs by the combination of inputs arising from PSRs and central chemosensory sources. The interaction between these two types of input appears to be mainly additive with regard to the PSR-mediated inhibition and synergistic with regard to the PSR-mediated excitation.
Acknowledgments
The authors gratefully thank Jack Tomlinson for technical assistance.
Appendix
Method used to estimate and separate the time-dependent component from the pressure-dependent component.
The response to lung inflation was assumed to be a function of transpulmonary pressure, Pt, and time:F
n(t) = ℱ[Pt(t), t]. As a first approximation, we assumed that the time-dependent component was a linear function of time into the E phase, that is: ℱ(t) = αt, where α is the slope, which can be either positive or negative, and has the units of Hertz per second. The neuronal discharge patterns to a positive ramp inflation (P1, Fig. FA1, left center) and the corresponding negative ramp inflation (P2, Fig. FA1,right center) are
Equation 1
Equation 2For a given Pt level (P), t
1and t
2 were found such that P1(t
1) = P2(t
2) = P [circles on (+) and (−) ramps, Fig. FA1, right and left center]. Under this condition, Eqs. 1
and
2
become
Equation 3
Equation 4Taking the difference between Eqs. 3
and
4
removes the Pt-dependent component that is common to both equations (ℱ[P]) and yields
Thus
Equation 5For the overlapping Pt range of both ramps (Fig. FA1,left bottom), α values are calculated for each P value in 0.25-mmHg increments and then averaged to reduce variation and obtain a better estimate of α.
Method used to calculate the slope, α, of the time-dependent component using hypothetical responses. Left, top andcenter: neuronal response (F 1) to a (+) ramp inflation (P1). Right, top andcenter: neuronal response (F 2) to a (−) ramp inflation (P2). Bottom right andleft: overlaid F n vs. Ptplots before and after time correction, respectively. An average α of 2.0 was used in this example.
The Pt-dependent components were then obtained by rearrangement of Eqs. 1
and
2
Equation 6
Equation 7Plots of F
1 versus P1 andF
2 versus P2 then coincide (Fig. FA1,right bottom), demonstrating independence from time and showing only the pressure-dependent component of the E neuronal response to lung inflation (also see Fig. 7).
Footnotes
This work was supported by the Department of Veterans Affairs Medical Research Funds and the Department of Anesthesiology of the Medical College of Wisconsin, Milwaukee, WI.
J. Bajic and M. Tonkovic-Capin were postgraduate fellows from the University of Zagreb School of Medicine in Split, Croatia. Z. Dogas is currently in the Department of Neuroscience at University of Split Medical School in Split, Croatia.
Address for reprint requests and other correspondence: E. J. Zuperku, Research Service 151, Zablocki V. A. Medical Center, Milwaukee, WI 53295 (E-mail: ezuperku{at}mcw.edu).
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.
- Copyright © 2000 the American Physiological Society
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