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J Appl Physiol 101: 521-530, 2006. First published April 27, 2006; doi:10.1152/japplphysiol.00059.2006
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Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats

Hakan S. Orer,1,2 Gerard L. Gebber,1 and Susan M. Barman1

1Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan; and 2Department of Pharmacology, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Submitted 18 January 2006 ; accepted in final form 14 April 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (TI and TE, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing TI but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing TE and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in TI and TE and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-Bötzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.

central respiratory rate; excitatory and inhibitory amino acid receptors; expiratory duration; inspiratory-burst amplitude; inspiratory duration

THE BASIS FOR RESPIRATORY rhythmogenesis has been debated for many years, and numerous reviews have dealt with this subject as well as the neuronal cell types that comprise the rhythm generator and its follower circuits (6, 13, 16–19, 42). Less has been written about the sources of tonically active inputs that modulate the central respiratory network. Such inputs often play an important, if not critical, role in maintaining the normal breathing pattern. For example, inputs from central and peripheral chemoreceptors are prerequisites for operation of the respiratory rhythm generator in anesthetized and sleeping animals (15). Inputs to the respiratory network from several regions of the reticular formation also play a role in regulating the breathing pattern. These include inspiratory-facilitatory influences of the magnocellular and gigantocellular tegmental fields near the medullopontine border (28) and an influence on respiratory rate mediated by the medullary lateral tegmental field (LTF; 38).

The role played by the medullary LTF in respiratory control is of particular interest to us. The LTF, which lies between the classical dorsal and ventral respiratory neuronal groups, is known to contain neurons with respiratory-related activity patterns (3, 13, 23, 26). Although most such neurons fire predominantly during inspiration, other discharge patterns have also been reported. Cohen (13) proposed that such neurons may serve in an auxiliary capacity for the maintenance of the respiratory rhythm or they may contribute to the respiratory modulation of nonrespiratory neural systems. Recently, our laboratory (38) showed that chemical inactivation of the LTF (muscimol microinjection) in cats often led to an increase in central respiratory rate as reflected by shortening of the interval between eupneic bursts of phrenic nerve activity (PNA). We proposed that muscimol silenced a group of LTF neurons that act on the respiratory oscillator to reduce the rate of breathing. Regarding this proposal, Smith et al. (44) reported direct projections from the LTF to medullary regions involved in the control of respiration, including the dorsal (DRG) and ventral (VRG) respiratory groups, Bötzinger complex, and pre-Bötzinger complex. Pre-Bötzinger complex neurons have been implicated in respiratory rhythmogenesis (17–19, 40, 42, 46).

The present study was initiated to explore further the role of the LTF in modulating the rate and pattern of the central respiratory cycle in vagus-intact and vagotomized cats. We hypothesized that the modulating influences of the LTF on the breathing pattern are dependent on excitatory (EAA)- and inhibitory amino acid (IAA)-mediated synaptic transmission in this region. To test this hypothesis, we monitored changes in PNA in response to microinjection of selective antagonists of EAA and IAA receptors in the LTF. Our data demonstrate distinct patterns of changes in the timing and pattern of PNA after selective blockade of EAA and IAA receptors in the LTF.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
General procedures.   Experiments were performed on 36 adult cats (3.30 ± 0.11 kg) anesthetized with an intraperitoneal injection of a mixture of sodium diallylbarbiturate (60 mg/kg), urethane (240 mg/kg), and monoethylurea (240 mg/kg). The protocols used in these studies were approved by Michigan State University's Institutional Animal Care and Use Committee. A femoral artery and vein were cannulated to measure arterial pressure and to administer drugs, respectively. In 24 of these cats, the vagus nerve was sectioned bilaterally at a cervical level to eliminate the effects of vagal lung inflation afferents on PNA (16, 38). Cats were placed in a stereotaxic apparatus. Mean blood pressure was maintained near 100 mmHg throughout the experiment by adjusting the rate of intravenous infusion of a mixture of saline and dextran.

Cats were paralyzed (gallamine triethiodide, 4 mg/kg iv, initial dose), pneumothoracotomized, and artificially ventilated with room air. The parameters of artificial ventilation (39 ± 2 ml and 21 ± 1 cycles/min) were within the physiological range (14) so that end-tidal CO2 was held near 4.6%. Rectal temperature was kept near 38°C with a heat lamp. Before neuromuscular blockade, the adequacy of anesthesia was indicated by the absence of a palpebral reflex. When cats were paralyzed, an adequate level of anesthesia was indicated by the inability of noxious stimuli (pinch, heat, surgery) to increase blood pressure or change the pattern of PNA.

Phrenic nerve recording.   The right phrenic nerve was isolated in the neck (2–4, 38) and covered with silicone release agent. Potentials were recorded monophasically from the central end of the cut nerve placed on platinum bipolar electrodes with the capacity-coupled preamplifier band pass set at 10–10,000 Hz. The signal was passed through a 50/60-Hz noise eliminator (Hum Bug; Quest Scientific, North Vancouver, BC, Canada) and a moving averager (CWE, model MA-821RSP, Ardmore, PA) with a 100-ms time constant. The moving averager performs a full-wave rectification and uses a third-order Paynter filter (24) to reconstitute the low-frequency components in PNA. This method provides a high degree of smoothing and a better dynamic response compared with simpler first-order filters (integrators).

Data processing.   Data (1-ms resolution) were acquired with a Digidata1322A digitizer (Axon Instruments; Union City, CA). Figure 1 shows how we measured central respiratory rate, the duration of the inspiratory and expiratory phases of PNA (TI and TE, respectively), the TI-to-TE ratio (TI/TE), trough-to-peak inspiratory burst amplitude (I-burst amplitude), and the slope of the ramplike increase in inspiratory activity. Datapac software (Run Technologies; Mission Viejo, CA) was used to create an event whose duration equaled TI. Central respiratory rate (breaths/min) was derived from the interval between the onsets of consecutive events. The time between the offset of one event and the onset of the next was equal to TE. The mean values of these parameters were calculated using a minimum of 15 cycles of PNA. The TI/TE was based on the average values of TI and TE. In addition, the trough-to-peak I-burst amplitudes of at least 15 cycles of PNA were averaged. Values in the text refer to data segments collected before and between 2 and 7 min after the microinjection of an EAA or IAA receptor antagonist.


Figure 1
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  		Fig. 1. Quantification of phrenic nerve activity (PNA). Traces (top to bottom) show raw PNA (10–10,000 Hz band pass), moving averaged PNA (100-ms time constant), and time base (1 s/division). Datapac software was used to create events (shown as square waves) denoting the duration of inspiration. Horizontal dashed lines show the respiratory (Resp) period and inspiratory (TI) and expiratory (TE) durations; vertical dashed line shows the trough-to-peak I-burst amplitude (Amp). Vertical calibration, 350 mV.

 
Cycle-by-cycle measurements of the respiratory period and I-burst amplitude were depicted in the form of a time series by use of software written in our laboratory (22). This allowed us to show the time course of changes in these parameters in response to microinjection of a drug into the LTF.

Microinjections.   All chemicals used for microinjection into the LTF were diluted in phosphate-buffered saline. Solutions were adjusted to a pH of 6–8 (litmus paper test) and placed in a glass micropipette (~40-µm tip diameter) that was glued (cyanoacrylate) to the needle of a 5-µl Hamilton syringe and mounted on a microinjection unit (David Kopf Instruments, model 5000). A 50-nl injection was made slowly (~10 s) at each medullary site by turning the calibrated micrometer on the microinjection unit. The following drugs were injected into the LTF or the pre-Bötzinger complex: the competitive NMDA receptor antagonist D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; 3 mM); the competitive N-methyl-D-aspartate (non-NMDA) receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo-[f]-quinoxaline-7-sulfonamide (NBQX; 1.25 mM); a {gamma}-aminobutyric acid (GABAA) receptor antagonist, either bicuculline (BIC; 1.0 mM) or SR-95531 (1.0 mM); and the glycine receptor antagonist, strychnine (1.0 mM). All drugs were purchased from RBI Sigma (St. Louis, MO). The concentrations of D-AP5, NBQX, BIC, and strychnine were the same as used in past studies from this (4, 36–38) and other laboratories (1, 7, 9–11, 30, 32, 34, 41, 45, 47) and have been shown to block selectively NMDA, non-NMDA, GABAA, and glycine receptors, respectively (9–11, 30, 41, 47).

The dorsal surface of the brain stem was exposed by removing portions of the occipital bone and cerebellum. The midline, obex, and dorsal medullary surface were used as landmarks for placement of the micropipette. Microinjections were made at two sites in the LTF on each side of the medulla over a period of ~3–5 min. The micropipette was positioned in tracks located ~2 and 3 mm rostral to the obex and 2.5–2.8 mm lateral to the midline; microinjections were made bilaterally at a depth of ~3.5 mm (3.2–3.8 mm) from the dorsal surface. This is similar to the region in which microinjection of muscimol led to an increase in central respiratory rate (4, 38). According to investigators who have calculated the spread of injection of a bolus of fluid in the brain (34, 35), the 50-nl volume of injectate used in our experiments should spread over a radius of <0.5 mm. Microinjections were also made into the pre-Bötzinger complex at ~3.3 and 3.6 mm rostral to the obex, 3.5–3.8 mm lateral to the midline, and 4.5 mm below the dorsal surface. These coordinates were based on studies by Pierrefiche et al. (40) and Solomon et al. (46) and extend beyond the limits of the expected spread of the injectate from the LTF.

In two vagotomized cats, NBQX was microinjected into the LTF after recovery from the effects of microinjection of D-AP5 into the LTF (~2 h later). To ensure reproducibility of the responses, in seven cats, we repeated the injection of an EAA or IAA receptor antagonist into the LTF after recovery from the first series of injections. In four cats, blockade of GABAA or glycine receptors in the LTF was done both before and after vagotomy. In 7 of 11 cats in which we microinjected EAA or IAA receptor antagonists into the pre-Bötzinger complex, the same drug had previously been injected into the LTF. In three cats, we microinjected saline into the LTF either before or after microinjection of an EAA or IAA receptor antagonist. PNA was unaffected by the saline injection. In two other cats, injection of NBQX at sites more rostral within the LTF (5–7 mm rostral to the obex) did not affect PNA.

Histology.   At the end of the experiment, the brain stem was removed and fixed in 10% buffered formalin. Frontal sections (40 µm thick) were cut and stained with cresyl violet to locate the levels of microinjection with reference to the stereotaxic planes of Berman (5). Injection sites were identified on the basis of the bottom of the tracks made with the micropipette.

Statistical analysis.   Data are expressed as means ± SE. P ≤ 0.05 indicated statistical significance. A paired t-test was used to compare central respiratory rate, TI, TE, TI/TE, I-burst amplitude, and slope of the ramplike increase in PNA before and after microinjection of a drug into the LTF. An unpaired t-test was used to compare the changes in PNA (expressed as a percentage of control) produced by microinjection of the same drug in vagus-intact and vagotomized cats. After pooling these data from vagotomized and vagus-intact cats, we used an unpaired t-test to compare the effects of different drugs on the same parameter and the effects of a drug on the changes in TI and TE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Microinjection of D-AP5.   We microinjected the NMDA receptor antagonist, D-AP5, bilaterally into the LTF of five vagotomized and five vagus-intact cats. Figure 2A shows superimposed traces of PNA before (black) and ~4 min after microinjection of D-AP5 into the LTF (gray) of one of the vagotomized cats. In this example, both TI and TE were increased after blockade of NMDA receptors, but the increase in TI (~275% of control) was greater than that of TE (~200% of control). Also note the apneustic pattern of PNA after NMDA receptor blockade. The slope of the ramplike phase of PNA was unchanged. Figure 2B shows the time course of the effects of microinjection of D-AP5 into the LTF on respiratory period (top) and I-burst amplitude (bottom) in this cat. As was typically the case, the changes in PNA began to occur during the microinjection of D-AP5 into the LTF (indicated by black line above the time base). Note the marked prolongation of the respiratory period; however, except for an increase in variability, I-burst amplitude was little changed. PNA began to recover ~15 min after completion of the microinjection. Full recovery (not shown) occurred within 1 h.


Figure 2
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  		Fig. 2. Blockade of N-methyl-D-aspartate (NMDA) receptors in the LTF of a vagotomized cat. A: superimposed traces of moving averaged PNA before (black) and ~4 min after (gray) bilateral microinjection of D-(–)-2-amino-5-phosphonopentanoic acid (D-AP5; 150 pmol/injection) into the lateral tegmental field (LTF); time base, 2 s/division. Cont, control. B: cycle-by-cycle measurements of the respiratory period (top) and I-burst amplitude (bottom) showing the time course of changes in these parameters after microinjection of D-AP5 into the LTF. Black line above time base indicates period of injection.

 
Figure 3 summarizes the effects of bilateral microinjection of D-AP5 into the LTF on central respiratory rate, TI, TE, TI/TE, and I-burst amplitude in the five vagotomized (Fig. 3, A–E) and five vagus-intact cats (Fig. 3, F–J). There were no significant differences between the D-AP5-induced changes (expressed as a percent of control) in the two groups of cats. The significant reduction in central respiratory rate reflected significant increases in both TI and TE. However, when expressed as a percentage of control, the increase in TI was significantly greater than the increase in TE. This difference was reflected by a significant increase in TI/TE. I-burst amplitude was not significantly affected by bilateral microinjection of D-AP5 into the LTF. On a group basis, the slope of the ramplike increase in inspiratory activity in the phrenic nerve also was not significantly changed in vagotomized (83 ± 29% of control) or vagus-intact (64 ± 8% of control) cats.


Figure 3
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  		Fig. 3. Summary of the changes in PNA produced by blockade of excitatory and inhibitory amino acid receptors in the LTF of vagotomized (left) and vagus-intact cats (right). Panels (top to bottom) show means ± SE for central respiratory rate (CRR), TI, TE, TI-to-TE (TI/TE) ratio, and I-burst amplitude, respectively, during control (white bars) and 2–7 min after (gray bars) bilateral microinjection of D-AP5, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo-[f]-quinoxaline-7-sulfonamide (NBQX); bicuculline or SR-95531 (BIC/SR), and strychnine (Strych) into the LTF. The number of animals is shown by the insets in A and F. Graphs are based on the average values calculated from at least 15 cycles of PNA. *Significantly different from control, P < 0.05.

 
We microinjected D-AP5 into the pre-Bötzinger complex of two vagotomized cats. In contrast to the effects of blockade of NMDA receptors in the LTF, the central respiratory rate was increased to 112 and 163% of control and I-burst amplitude was reduced to 15 and 55% of control within 5 min after blockade of NMDA receptors in the pre-Bötzinger complex. Figure 4A shows superimposed traces of PNA before (black) and ~4 min after (gray) microinjection of D-AP5 into the pre-Bötzinger complex in one of these cats.


Figure 4
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  		Fig. 4. Blockade of NMDA (A) or GABAA (B) receptors in the pre-Bötzinger complex on PNA in 2 vagotomized cats. A: PNA before (black) and ~4 min after (gray) bilateral microinjection of D-AP5 (150 pmol/injection) into pre-Bötzinger complex; time base, 1 s/division. B: PNA before (black) and ~2 min after (gray) bilateral microinjection of SR-95531 (50 pmol/injection) into pre-Bötzinger complex; time base, 2 s/division.

 
Microinjection of NBQX.   We microinjected the non-NMDA receptor antagonist NBQX bilaterally into the LTF of seven vagotomized and eight vagus-intact cats. A profound reduction in I-burst amplitude without a change in respiratory period was the hallmark of microinjection of NBQX in both groups of cats. Figure 5A shows PNA before (black line) and ~4.5 min after (gray line) microinjection of NBQX into the LTF of a vagotomized cat. The major changes were a marked reduction in I-burst amplitude and slope of the ramplike increase in inspiratory activity. Figure 5B shows cycle-by-cycle measurements of the respiratory period (top) and I-burst amplitude (bottom) in this experiment. Soon after the start of the injection, there was an increase in respiratory period that lasted ~3 min, after which central respiratory rate returned to control. The reduction in I-burst amplitude also began during the injection and progressed to the point of central apnea beginning ~5.5 min after completion of the injection (note the absence of any data points in the graphs between 830 and 960 s); I-burst amplitude began to recover ~5 min later. NBQX often caused a brief (<5 min; n = 4) or prolonged (>30 min; n = 5) period of central apnea (i.e., PNA quiescence) that began during the microinjection or within 6 min after completing the injection.


Figure 5
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  		Fig. 5. Blockade of non-NMDA receptors in the LTF on PNA of a vagotomized cat. A: PNA before (black) and ~4.5 min after (gray) bilateral microinjection of NBQX (62.5 pmol/injection) into the LTF; time base, 2 s/division. B: time course of changes in respiratory period (top) and I-burst amplitude (bottom) after microinjection of NBQX into the LTF. Black line above time base indicates period of injection.

 
Figure 3 compares the various indexes of PNA before and after bilateral microinjection of NBQX into the LTF of the seven vagotomized cats (Fig. 3, A–E) and five of the vagus-intact cats (Fig. 3, F–J). In the other three vagus-intact cats, a prolonged period of apnea began during the microinjection of NBQX; thus we were not able to calculate values for the various parameters of PNA in the 2- to 7-min postinjection time frame used for data analysis. NBQX caused comparable reductions in I-burst amplitude (expressed as percent of control) in vagotomized and vagus-intact cats. Central respiratory rate, TI, and TE were not significantly changed by microinjection of NBQX into the LTF in either group of animals. As a result, TI/TE was also not changed. The slope of the ramplike increase in PNA was significantly reduced to 41 ± 9% of control (P = 0.0042) in vagotomized cats and to 41 ± 13% of control (P = 0.0227) in vagus-intact cats.

In three vagotomized cats, we microinjected NBQX into the pre-Bötzinger complex. In contrast to the effect of blockade of non-NMDA receptors in the LTF, I-burst amplitude was not significantly changed (78 ± 12% of control) by blockade of these receptors in the pre-Bötzinger complex. As was the case with injections into the LTF, central respiratory rate (134 ± 26% of control), TI (84 ± 17% of control), and TE (80 ± 15% of control) were not significantly affected by microinjection of NBQX into the pre-Bötzinger complex.

Microinjection of BIC or SR-95531.   We microinjected a GABAA antagonist bilaterally into the LTF of eight vagotomized and seven vagus-intact cats. The effects produced by microinjection of BIC (n = 9) and SR-95531 (n = 6) were comparable. Figure 6 shows the changes in PNA produced by microinjection of BIC into the LTF of a vagotomized cat. As shown by the superimposed traces of PNA before (black) and ~4 min after (gray) blockade of GABAA receptors (Fig. 6A), central respiratory rate was markedly reduced primarily owing to an increase in TE. I-burst amplitude and slope of the ramplike increase in PNA were also reduced. Figure 6B shows the time course of changes in the respiratory period (top) and I-burst amplitude (bottom) in this experiment in which GABAA receptor blockade led to an ~5-min period of central apnea. Note that these changes were reversible.


Figure 6
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  		Fig. 6. Blockade of GABAA receptors in the LTF on PNA of a vagotomized cat. A: PNA before (black) and ~4 min after (gray) bilateral microinjection of bicuculline (BIC; 50 pmol/injection) into the LTF; time base, 3 s/division. B: time course of changes in respiratory period (top) and I-burst amplitude (bottom) after microinjection of BIC into the LTF. Black line above time base indicates period of injection.

 
Figure 3 shows the changes in PNA produced by GABAA receptor blockade in eight vagotomized (Fig. 3, A–E) and seven vagus-intact cats (Fig. 3, F–J). There were no significant differences between the changes (expressed as a percentage of control) in the two groups of cats. The significant decrease in central respiratory rate was attributable to marked prolongation of TE. In contrast, TI was not significantly affected by microinjection of BIC or SR-95531 (BIC/SR) into the LTF. As a consequence, TI/TE was significantly decreased. Blockade of GABAA receptors in the LTF also led to a significant decrease in I-burst amplitude. In some cases, after a period of marked reduction in I-burst amplitude, there was either a brief (3.5–6.5 min; n = 3) or prolonged (>25 min; n = 5) period of central apnea. In addition, the slope of the ramplike increase in PNA was significantly reduced to 25 ± 4% of control (P = 0.0003) in vagotomized cats and to 38 ± 8% of control (P = 0.0086) in vagus-intact cats. The increase in TE due to blockade of GABAA receptors in the LTF was significantly greater (P = 0.0086) than that occurring after microinjection of D-AP5 into the LTF.

We also microinjected BIC/SR into the pre-Bötzinger complex of three vagotomized cats. In contrast to the changes in PNA seen after blockade of GABAA receptors in the LTF, microinjection of BIC/SR into the pre-Bötzinger complex did not significantly alter central respiratory rate (97 ± 9% of control), TE (80 ± 9% of control), or I-burst amplitude (81 ± 18% of control). TI was also not significantly changed (139 ± 14% of control). In two of the three cats, after microinjection of either BIC or SR-95531 into the pre-Bötzinger complex, the pattern of PNA changed to one of augmented bursts (eupneic bursts ending with a high-amplitude, short-duration burst) that were interspersed with eupneic bursts whose amplitude was unchanged from control. Figure 4B shows superimposed traces of PNA before (black) and ~2 min after (gray) microinjection of SR-95531 into the pre-Bötzinger complex in one of these cats.

Microinjection of strychnine.   We microinjected the glycine receptor antagonist, strychnine, bilaterally into the LTF of six vagotomized and two vagus-intact cats. In the example shown in Fig. 7A, TI and TE were increased proportionately, and I-burst amplitude was reduced to ~75% of control ~5-min after microinjection of strychnine into the LTF of a vagus-intact cat. Figure 7B shows the time course of changes in respiratory period (top) and I-burst amplitude (bottom) after bilateral microinjection of strychnine into the LTF of a vagotomized cat. In this case, the increase in respiratory period was accompanied by a reduction in I-burst amplitude to ~50% of control. Both indexes of PNA approached control values within 40 min after injection of strychnine.


Figure 7
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  		Fig. 7. Blockade of glycine receptors in the LTF on PNA of 2 cats. A: PNA before (black) and ~5 min after (gray) bilateral microinjection of strychnine (50 pmol/injection) into the LTF of a vagus-intact cat; time base, 2 s/division. B: time course of changes in respiratory period (top) and I-burst amplitude (bottom) after microinjection of glycine into the LTF of a vagotomized cat. Black line above time base indicates period of injection.

 
Figure 3, A–E, compares the various parameters of PNA before and after microinjection of strychnine into the LTF of six vagotomized cats. The significant decrease in central respiratory rate was due to increases in TI and TE without a significant change in TI/TE. I-burst amplitude was significantly reduced but to a significantly lesser degree than after blockade of non-NMDA receptors (P = 0.0091) or GABAA receptors in the LTF (P = 0.002). Also, the increase in TE was significantly less (P = 0.0146) than that occurring after blockade of GABAA receptors in the LTF. In addition, the slope of the ramplike increase in PNA was significantly reduced to 44 ± 9% of control (P = 0.0096). Similar changes in PNA occurred in the two vagus-intact cats (see example in Fig. 7A).

We also microinjected strychnine into the pre-Bötzinger complex of three vagotomized cats. Blockade of glycine receptors in this region did not significantly change central respiratory rate (113 ± 15% of control), TI (102 ± 19% of control), or TE (82 ± 3% of control). I-burst amplitude was variably affected (two decreased, one increased). In one cat, blockade of glycine receptors in this region led to low level of activity in the phrenic nerve onto which bursts were superposed.

Histological localization of injection sites.   The histological sections in Fig. 8 show examples of injection sites bilaterally in the LTF (A and B) and pre-Bötzinger complex (C) of three cats. The rostral and caudal limits of the injection sites in the LTF are reflected in the cross sections shown in Fig. 8, A and B. Injection sites in the LTF were located as far caudal as ~1.7 mm anterior to the obex (Fig. 8A, bottom) and as far rostral as ~3.2 mm anterior to the obex (Fig. 8B, top). Injection sites were in the range of 2.5–2.8 mm lateral to the midline and at a depth of 3.2–3.8 mm from the dorsal surface. Injection sites in the pre-Bötzinger complex ranged between 3.3 and 3.6 mm rostral to the obex, 3.5–3.8 mm lateral to the midline, at a depth of 4–4.5 mm below the dorsal surface (Fig. 8C).


Figure 8
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  		Fig. 8. Location of injection sites in the medulla of 3 cats. A: histological sections of the medulla at ~1.7 and 2.7 mm rostral to the obex showing tracks made by the micropipette whose tip was in the LTF. B: sections at ~2.2 and 3.2 mm rostral to the obex with injection sites in the LTF. C: sections at ~3.3 and 3.6 mm rostral to the obex showing injection sites in the pre-Bötzinger complex (PBC). Calibration, 1 mm. 5Sp, spinal nucleus of trigeminal nerve; IO, inferior olive; Py, pyramid; Rgc, nucleus reticularis gigantocellularis; Rpc, nucleus reticularis parvocellularis; Rv, nucleus reticularis ventralis.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The most important finding of the present study was the distinctly different patterns of changes in central respiratory rate, rate of recruitment of inspiratory activity, I-burst amplitude, TI, TE, and TI/TE resulting from selective blockade of NMDA, non-NMDA, GABAA, and glycine receptors in the LTF. Specifically, whereas blockade of NMDA receptors with D-AP5 slowed central respiratory rate but did not alter I-burst amplitude, blockade of non-NMDA receptors with NBQX reduced I-burst amplitude without affecting central respiratory rate. On the other hand, blockade of either GABAA or glycine receptors in the LTF with BIC/SR or strychnine significantly reduced both central respiratory rate and I-burst amplitude.

The results of the present study indicate that the role of the LTF in modulating breathing is more complex than suggested by our earlier work (38), which showed that chemical inactivation of the LTF (muscimol microinjection) often increased respiratory rate. We proposed that muscimol silenced a group of LTF neurons that acted to slow the respiratory rhythm generator. Disinhibition of such neurons by blockade of either GABAA or glycine receptors in the present study could explain the decrease in central respiratory rate produced by microinjection of BIC/SR or strychnine into the LTF. On the other hand, the slowing of respiratory rate produced by blockade of NMDA receptors points to the existence of another group of LTF neurons that acts to facilitate the respiratory rhythm generator. Inactivation of such neurons would explain the reduction in central respiratory rate seen after microinjection of D-AP5 into the LTF. Of the two groups of neurons proposed to exert opposing influences on the respiratory rhythm generator, the group that leads to a slowing of respiratory rate appears to dominate under the conditions of our experiments. This would explain why respiratory rate increased after the nonselective chemical inactivation of the LTF with muscimol microinjection (38).

The changes in TI and TE induced by blockade of EAA and IAA receptors further illustrate the complex role of the LTF in the control of respiratory rate. The reduction in central respiratory rate produced by D-AP5 was due primarily to an increase in TI, whereas microinjection of BIC/SR selectively increased TE and microinjection of strychnine increased TI and TE proportionately. The changes in TI and TE produced by D-AP5 and BIC/SR are consistent with the possibility that LTF neurons influence respiratory phase switching (13, 16, 17, 27, 31). Regarding this possibility, King (25) used anatomic methods to show that LTF neurons project to the pontine parabrachial nucleus, a region known to be involved in phase switching (13, 15). Moreover, King and Knox (26) showed that some LTF neurons could be either antidromically or synaptically activated by electrical stimulation of the parabrachial region.

The reduction in I-burst amplitude without a change in central respiratory rate after microinjection of NBQX further attests to the functional diversity of LTF neurons that influence breathing. This pattern, which often progressed to a brief or sustained period of central apnea (phrenic nerve quiescence), suggests that non-NMDA receptor-mediated excitatory synaptic transmission plays a role in determining the level of activity of LTF neurons that selectively facilitate the recruitment of central inspiratory activity (6, 8, 15). The existence of such neurons is also suggested by the reduction in the slope of the ramplike increase in inspiratory activity that occurred after microinjection of NBQX. Because non-NMDA receptor blockade did not affect central respiratory rate, we presume that the actions of these LTF neurons were mediated on elements of the respiratory network (e.g., inspiratory bulbospinal neurons) not directly involved in rhythm generation.

The reduction in I-burst amplitude without a change in TI or TE leading to central apnea after blockade of non-NMDA receptors in the LTF mimicked the changes in PNA observed by Bruce et al. (8) when PCO2 was lowered to <30 Torr, thereby reducing central chemoreceptor drive to the respiratory network. Whether the LTF contains synaptic stations in the pathway from central chemoreceptors to the respiratory network should be considered in future studies. King and Knox (26) suggested that LTF neurons with respiratory-related activity may be chemosensitive in light of their proximity to vascular sheets running transversely through this region of the reticular formation.

In contrast to central apnea induced by microinjection of NBQX into the LTF, the cessation of PNA after microinjection of BIC/SR may have been more directly linked to changes in the expiratory rather than inspiratory phase of the central respiratory cycle. GABAA receptor blockade induced a dramatic increase in TE in the cycles leading up to the apnea. Thus central apnea induced by BIC/SR injection into the LTF may have involved a steady progression to a state of heightened and sustained expiratory neuronal discharge. The reduction in I-burst amplitude and rate of recruitment of central inspiratory activity produced by GABAA receptor blockade in the LTF might have occurred indirectly as the consequence of heightened expiratory neuronal discharge (12, 13, 20, 27, 31).

It is not surprising that the changes in respiratory rate and pattern produced by each of the four classes of receptor antagonists used in the present study were qualitatively the same in vagus-intact and vagotomized cats (see Fig. 3). These data are consistent with results of our earlier study (38), which showed that coupling of the central respiratory cycle to the artificial ventilation cycle in paralyzed, vagus-intact cats was not prevented by chemical inactivation of the LTF with muscimol. These observations indicate that LTF neurons are not contained in the Hering-Breuer reflex arc.

The effects on PNA produced by microinjection of D-AP5 and NBQX into the LTF are comparable to the results reported by Foutz et al. (21) and Pierrefiche et al. (39) upon intravenous injection of NMDA and non-NMDA receptor antagonists in vagotomized cats. Pierrefiche et al. raised the possibility that the NMDA receptor antagonist acted within the pons to alter the inspiratory off-switch mechanism, whereas the non-NMDA receptor antagonist reduced I-burst amplitude by an action within the bulbar respiratory network or on phrenic motoneurons. However, on the basis of our data, it is possible that these drugs acted within the LTF to alter PNA.

A limitation of the present study is that we did not measure the extent of spread of the injectate from the LTF to adjacent medullary regions with known respiratory function. However, on the basis of work of others (34, 35), it is unlikely that the 50-nl injection volume spread more than 0.5 mm in any direction. The distances between injection sites in the LTF and adjacent portions of the DRG, VRG, and pre-Bötzinger complex were greater than 0.5 mm. Nonetheless, Sasaki et al. (43) have shown that the dendrites of inspiratory-augmenting neurons within the VRG extend to the LTF. This raises the possibility that drugs injected into the LTF exerted their effects on PNA by acting on VRG neurons rather than LTF neurons. This might explain the decreases in PNA burst amplitude and slope after microinjection of NBQX into the LTF because Krolo et al. (29) showed that iontophoresis of this drug onto single inspiratory neurons in the caudal VRG reduced their firing rate as well as the slope of their augmenting discharge pattern. On the other hand, picoejection of BIC onto individual bulbospinal inspiratory neurons in the caudal VRG enhances rather than reduces their discharges (33, 48). Thus it is unlikely that the reduction in I-burst amplitude and slope after blockade of GABAA receptors in the LTF can be explained by an action on the dendrites of VRG inspiratory neurons. Other data also support the view that the changes in PNA seen after microinjection of drugs into the LTF cannot be explained on the basis of spread to the adjacent portion of the VRG. In contrast to the increases in TI and TE after microinjection of D-AP5 into the LTF, Anderson and Speck (1) reported that TE was reduced and TI was unchanged by microinjection of this drug into the VRG ~1.5–2.5 mm rostral to the obex of vagotomized cats. Also, Anderson and Speck reported a marked decrease rather than no change in TE after microinjection of NBQX into this portion of the VRG. Finally, our laboratory (38) has shown that the increase in central respiratory rate produced by microinjection of muscimol into the LTF is not mimicked by microinjection of this drug into adjacent portions of the VRG or, for that matter, the DRG.

It should also be pointed out that the changes in PNA produced by microinjection of D-AP5, NBQX, BIC, or strychnine into the LTF were markedly different than those produced by microinjection of the same drugs into the pre-Bötzinger complex. Although we did not record neuronal activity in the vicinity of the injection site to verify the localization within the pre-Bötzinger complex, the effects of blockade of EAA and IAA receptors in this region were similar to those reported by others (11, 34, 40, 45) who functionally identified this portion of the respiratory network.

Another limitation of our study was the failure to record neuronal activity in the vicinity of the injection sites in the LTF. This might have provided clues regarding the types of neurons whose activity was altered by the EAA and IAA receptor antagonists. We and others (3, 13, 23, 26) have shown that this region of the LTF contains neurons with respiratory-related activity. It is possible that the changes in PNA produced by blockade of EAA and IAA receptors in LTF involved such neurons. If so, the sources of their excitatory and inhibitory inputs may have been derived from the classic respiratory nuclei, including the DRG, VRG, Bötzinger complex, and pre-Bötzinger complex. On the other hand, the changes in PNA reported here might reflect blockade of EAA and IAA receptors on a group of nonrhythmic (tonically firing) LTF neurons that exert a modulating influence on the respiratory rhythm generator and/or its follower circuits. Independent of the types of LTF neurons involved in respiratory control, their targets need to be identified. Regarding this problem, Smith et al. (44) have demonstrated direct projections from the LTF to the DRG, VRG, Bötzinger complex, and pre-Bötzinger complex. Further studies on the inputs and outputs of LTF neurons would help us understand whether this region is part of the respiratory network rather than simply a source of modulating influences on circuits responsible for the timing and pattern of breathing. Yamazaki et al. (49) have identified bulbospinal expiratory-augmenting neurons in the LTF caudal to the obex in cats, suggesting that at least some portions of the LTF should be considered as part of the respiratory network.

In summary, our data support the view that EAA- and IAA-mediated synaptic transmission in the cat medullary LTF influences the timing and pattern of breathing. Each of four receptor types (NMDA, non-NMDA, GABAA, and glycine) in the LTF is involved in controlling one or more critical parameters that, in combination, define the breathing pattern. As such, selective blockade of each receptor type produced a distinct pattern of changes in central respiratory rate, I-burst amplitude, rate of recruitment of central inspiratory activity, TI, TE, and TI/TE. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal breathing pattern in anesthetized cats.


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This study was supported by National Heart, Lung, and Blood Institute Grant HL-33266.


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The authors thank Lindsey Fink for preparation of histological sections.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. M. Barman, Dept. of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI 48824 (e-mail: barman{at}msu.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.


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S. M. Barman and G. L. Gebber
Role of ventrolateral medulla in generating the 10-Hz rhythm in sympathetic nerve discharge
Am J Physiol Regulatory Integrative Comp Physiol, July 1, 2007; 293(1): R223 - R233.
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