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Modulation of reflex and sleep related apnea by pedunculopontine tegmental and intertrigeminal neurons
Respiratory Physiology & Neurobiology 143 (2004) 293–306
Modulation of reflex and sleep related apnea by pedunculopontine tegmental and intertrigeminal neurons Miodrag Radulovackia,∗ , Sasha Pavlovica , Jasna Saponjicb , David W. Carleya,b,c a
Department of Pharmacology, University of Illinois at Chicago, M/C 868, 901 S. Wolcott Avenue, Chicago, IL 60612, USA b Department of Medicine, Section of Respiratory and Critical Care Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA c Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60612, USA Accepted 6 February 2004
Abstract We describe and summarize here our recent findings about the role in respiration of two pontine structures that are not classically included in the pontine respiratory group: the pedunculopontine tegmental nucleus (PPT) and the intertrigeminal region (ITR). We also discuss significant contributions of other workers in the field, especially, S. Datta [Cell. Mol. Neurobiol. 17: 341–365, 1997], R. Lydic and H. Baghdoyan [Sleep, 25: 617–622, 2002], and N. Chamberlin and C. Saper [J. Neurosci. 18: 6048–6056, 1998], who postulated a role for the ITR in modulating reflex apnea. In anesthetized and freely moving rats we have consistently documented that PPT and ITR have a role in respiration. Neurochemical manipulations of each area affected the brainstem respiratory pattern generator and respiratory pattern variability,observed as spontaneous disturbances during sleep or as induced reflex apnea. Although the exact central mechanisms of apnea cannot be determined from our studies to date, we postulate that reflex and sleep-related apneas in rats share some common brainstem pathways, which may include PPT and ITR. © 2004 Elsevier B.V. All rights reserved. Keywords: Control of breathing, pons; Mammals, rat; Pons, pedunculopontine tegmental nucleus, intertrigeminal region; Sleep, reflex apnea
1. Introduction The pons plays a complex and poorly understood role in respiratory rhythmogenesis. Recently, two areas in the rostral pons have been found to modu∗ Corresponding author. Tel.: +1 312 996 3539; fax: +1 312 996 1225. E-mail address: [email protected] (M. Radulovacki).
1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2004.02.012
late respiration: pedunculopontine tegmental nucleus (PPT) (Lydic and Baghdoyan, 1993; Saponjic et al., 2003) and intertrigeminal nucleus (ITR) (Chamberlin and Saper, 1998, 2003; Radulovacki et al., 2003). Lydic and Baghdoyan (1993) first showed in barbiturate anesthetized cats that PPT continuous electrical stimulation leads to increased acetylcholine release in the medial pontine reticular formation and to respiratory depression. However, the respiratory depression was mild
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and transient, diminishing even before cessation of the stimulus. In anesthetized and spontaneously breathing Sprague–Dawley rats we demonstrated the longlasting increase in variability of respiratory parameters following glutamate microinjection into PPT (Carley et al., in press; Saponjic et al., 2003). The induced respiratory perturbations were characterized by intermittent apneas and increased variability of expiratory (TE) and total (TT) breath durations in all animals. Findings of these studies point to a significant impact of the PPT on the brainstem respiratory pattern generator. The other area in the rostral lateral pons that has recently emerged as a contributor to the brainstem respiratory function is the ITR (Chamberlin and Saper, 1998, 2003; Radulovacki et al., 2003). In classical functional anatomical analysis of respiratory circuitry Chamberlin and Saper (1998) suggested that the ITR represents a key relay for several airway protective apneic reflexes via the neural pathways revealed by combining glutamatergic stimulation and tract-tracing methodologies. This idea was supported by findings, which showed that blockade of synaptic transmission with large injections of cobalt or NMDA receptor antagonists aimed at the Kolliker–Fuse (KF) area attenuated the apneic response to ethmoidal nerve stimulation (Dutschmann and Herbert, 1998). Furthermore, injections of gammaaminobutyric acid (GABAA) receptor antagonists potentiated trigeminal apnea (Dutschmann and Herbert, 1998). These findings suggest that the KF mediates trigeminal apnea, because effective injections were centered in this nucleus. However, the control injections were not placed into more ventral regions, and hence it is not clear whether they may have also affected the ITR. Our data in anesthetized freely breathing rats confirmed the ITR role in respiration indicating that one physiological role for the ITR in respiration is to attenuate vagal reflex apneas and to dampen respiratory instability (Radulovacki et al., 2003). In accordance, in freely moving rats a small and well localized unilateral lesion of the ITR produced a lasting sleep-related breathing disturbance by increasing sleep apnea expression over a two week period (Radulovacki et al., in press). These findings are in agreement with the general modulatory role of pontine structures in respiration.
2. A role for PPT in the pontine respiratory network 2.1. Experiments in anesthetized rats REM sleep is uniquely characterized by postural muscle atonia and frequent “phasic” events that are widespread in the brain and are typified by rapid eye movements, for which the behavioral state was named (Aserinsky and Kleitman, 1953). Considerable evidence now indicates that a BPE generator in the pons initiates REM-related eye movements and other transient motor behaviors, and that the PPT is a key site for generation and/or relay of these BPEs (Datta, 1995; Datta et al., 1998). Close electrophysiological BPE markers are readily recorded as large amplitude field potentials in the pons and have been termed pontogeniculo-occipital (PGO) waves in the cat and, more recently, P-waves in the rat (Datta et al., 1999; Mavanji and Datta, 2003). P-waves, the pontine component of PGO waves, represent a close marker of brainstem phasic events and are associated with cardio-respiratory changes, including apnea, in sleeping rats. Numerous studies have demonstrated that cholinergic activation of the medial pontine reticular formation (mPRF) can elicit many features of rapid eye movement (REM) sleep, including increased respiratory variability (Gilbert and Lydic, 1990, 1991, 1994; Kubin, 2001). The PPT provides a significant cholinergic input to the mPRF, but little has been done to examine a specific role for PPT in regulating respiratory pattern (Lydic and Baghdoyan, 1989, 1993). Since local glutamate injections into the PPT of rats have been shown to excite pontine wave (P-wave) neurons (Datta, 1997) we decided to probe the impact of PPT activation and brainstem phasic events (BPE) generation on respiratory pattern by microinjecting glutamate into the PPT of rats. Because the excitability and network properties of brainstem neurons can be grossly altered by general anesthesia, we characterized respiratory responses to PPT stimulation under both a barbiturate agent (nembutal) and a dissociative agent (ketamine). Experiments were performed in spontaneously breathing adult, male, anesthetized Sprague–Dawley rats. Nine rats were anesthetized with nembutal (Abbot Laboratories, North Chicago, IL) with an initial intraperitoneal dose of 50 mg/kg and 13 rats were anesthetized with a combination of 80 mg/kg ketamine (Ab-
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bot Laboratories, North Chicago, IL) and 5 mg/kg xylazine (Phoenix Scientific Inc., St Joseph, MO) given by intraperitoneal injection. After a surgical plane of anesthesia was achieved, rats were placed in the stereotaxic apparatus (David Kopf Inst., model 962 A Tujunga, CA) and bilateral parietal osteotomies were drilled. At the right burr-hole a bipolar twisted wire, teflon-coated electrode was targeted to the PPT (A: 0.5 with respect to lambda; L: 2.0; H: 7.0) for recording P-waves. Using the same coordinates on the left side (Paxinos and Watson, 1986), a multibarrel micropipette was introduced for pressure microinjections of saline and glutamate (10 nM, l-glutamatic acid monosodium salt) into the PPT. We found that local PPT stimulation by glutamate produced sustained respiratory disturbances, including intermittent apnea. Under ketamine anesthesia, glutamate microinjection into the PPT did not evoke an immediate effect on respiration, but respiratory responses lasted more than 1 h and were marked by disturbances of both timing and tidal amplitude (Figs. 1 and 2). Under nembutal anesthesia microinjection of glutamate into the PPT resulted in an immediate apnea, respiratory disturbances were resolved within 15 min and reflected significant disturbances in expiratory timing (Figs. 3 and 4). Spontaneous BPEs were ex-
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pressed as P-waves under ketamine but not nembutal anesthesia, and PPT stimulation at sites associated with respiratory disturbance also reduced BPE expression in these animals. Conversely, at more dorsal sites, PPT stimulation augmented BPE expression but had no respiratory impact. These findings argue that PPT is an important structure for respiratory pattern modulation, and that PPT activation can produce long-lasting respiratory disturbances characterized by both increases and decreases in breathing depth and frequency. The occurrence of significant respiratory disturbances including apnea and lasting at least 15 min after a single glutamate injection under both anesthetic regimens lends robustness to these findings. Still, it is likely that the differences in response latency and duration reflected, at least in part, the differing and complex cellular and network effects of the two anesthetic agents. In addition to its main non-competitive NMDA antagonist action, ketamine significantly reduces firing rates of GABAergic neurons (Lee et al., 2001); increases locus coeruleus neuron firing rates and noradrenaline release in the cerebral cortex (Kubota et al., 1999); disrupts normal sleep-cycle organization, decreases acetylcholine release in the pontine reticular formation, and slows breathing rate (Lydic and Bagh-
Fig. 1. Typical severe respiratory disturbance induced by glutamate injection into the PPT in a ketamine anesthetized rat. (A) Glutamate injection (an arrow marks the time of glutamate injection) did not evoke an immediate effect on respiration. (B) Immediate continuation of tracing presented in (A). After a latency of 2 min glutamate injection produced severe respiratory disturbance.
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Fig. 2. Glutamate-induced changes in the total breath duration (TT) presented as coefficient of variation (CV) in the group of ketamine anesthetized animals (n = 13). Each bar reflects the mean and SE of CV for the group of animals in 5 min bins of each recorded interval except for immediate preinjection and post-injection interval. Baseline (bas) (n = 12), recorded prior to introduction of the pipette into PPT; after saline injection (sal) (n = 4); pre-injection: 30 s epoch immediately preceding glutamate injection (30’pre) (n = 12); initial post-injection: 30 s epoch immediately following glutamate injection (30’post) (n = 12); and every 10 min of overall post-injection interval: up to 70 min following glutamate injection (10 min (n = 12); 20 min (n = 12); 30 min (n = 10); 40 min (n = 9); 50 min (n = 8); 60 min (n = 5); 70 min (n = 5). Single factor ANOVA was significant for each parameter (P < 0.05 for each). t-Tests with Bonferonni correction were used to compare each response interval to baseline: * P < 0.05; ** P < 0.01; *** P < 0.001.
doyan, 2002). In contrast, nembutal functions primarily as a GABAa agonist. The specific contributions of these anesthetic agents to the observed respiratory responses cannot be inferred from the present study. These results strongly support a role for the PPT as an important supramedullary modulator of respiratory pattern generation. Activation of direct or indirect PPT
outputs to the medullary respiratory network induced prolonged destabilization of respiratory drive and genesis of intermittent apnea regardless of the state of the brain under differing anesthetic regimens. Defining the specific pathways and signaling mechanisms as well as the precise impact of BPEs will require further elucidation by future studies.
Fig. 3. Respiratory response to glutamate injection into the PPT of a rat anesthetized by nembutal: immediate apnea is followed by additional apneas of varying duration and irregular spacing. An arrow marks the time of glutamate injection.
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Fig. 4. Changes in the total breath duration induced by glutamate injection in the group of nembutal anesthetized rats (n = 9). Data are presented in the style of Fig. 2 except that the total post-injection recorded period was 20 min rather than 70 min. Each recorded interval was presented as a 5 min bin/per each rat, except for immediate preinjection and post-injection time after glutamate injection: baseline (n = 9); after saline injection (n = 3); immediate preinjection and post-injection 30 s (n = 9); 10 min (n = 9); 15 min (n = 5); 20 min (n = 40) after glutamate injection. Single factor ANOVA demonstrated significant changes in CV of TT (P < 0.05 for each). With respect to baseline, t-tests revealed immediately increased CV for TT upto 15 min after glutamate injection: * P < 0.05.
2.2. Experiments in freely moving rats Long-lasting enhancement of P-wave expression has been demonstrated after cholinergic stimulation of the PPT in cats (Datta et al., 1992). We tested the ability of carbachol microinjections and microinfusions into the PPT of rats to determine, whether in this animal species cholinergic stimulation of the PPT would elicit increased expression of P-waves and sleep related breathing disorder. Fifteen adult male Sprague–Dawley rats (300 g) were anesthetized using a mixture of ketamine (Vetalar 100 mg/ml) and acetylpromazine (10 mg/ml) (4:1, v/v) at a volume of 1 ml/kg body weight for implantation of biparietal cortical electrodes for EEG recording, and neck muscle electrodes for electromyogram (EMG) recording. To record P-waves, a bipolar twisted pair stainless electrode (75 m wires, ∼0.5 mm insulation removed, tips separated by 1 mm) was stereotaxically guided to the region of the locus coeruleus (AP: −0.8, ML: 1.25, DV: 3.0 with respect to the interaural line). In five animals, a stainless steel guide cannula (HTX-22; outside diameter 0.36 in.; Small Parts, Inc., Miami Lakes, FL) was introduced via a burr hole and stereotaxically advanced using a dorsoventral approach. The cannula tip was targeted to a coordinate
1 mm dorsal to the left or right PPT by random assignment (AP: −7.8; ML: 2.0; DV: 6.3 with respect to Bregma). Prior to implantation, a matched and tight fitting needle (HTX-28; outside diameter 0.36 in.; Small Parts, Inc., Miami Lakes, FL) was prepared for each guide cannula so that the needle would extend 1 mm beyond the end of the cannula when positioned for PPT infusions. A positive displacement infusion pump (KD Scientific 210; Fisher, Pittsburgh, PA) was used to deliver 100 nl of saline or carbachol (0.1 g in saline) over a period of 30 s in conscious animals. Each animal received one vehicle infusion and one carbachol infusion. In ten animals, left and right burr-holes were drilled for PPT approach and were closed with bone wax. To allow neurochemical PPT manipulations, these animals were re-anesthetized, the bone wax was removed, and a single barrel micropipette (standard filament glass, outside diameter 1.2 mm, inside diameter 0.68 mm, pulled to a tip diameter of approximately 20 m) was stereotaxically targeted to the PPT. Pressure microinjection was used (Picospritzer II; General Valve Corp., Brookshire, TX) to deliver 100 nl of pontamine blue dye dissolved in saline or carbachol (0.1 g in saline with pontamine blue dye). After removing the pipette, the burr hole was re-closed with bone wax and the animal was
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Fig. 5. P-wave frequency (waves per minute) with no infusion (Control), during the first 6 h after saline infusion to the PPT (Saline), during the first 6 h after carbachol infusion (0.1 g in 100 nl; Day 0), and during the same 6 h circadian phase 3, 7 and 14 days after carbachol infusion (Day 3, Day 7, and Day 14, respectively). Points reflect the mean and SE (n = 5) for REM sleep (squares) and NREM sleep (circles). P values indicate significant differences with respect to the control condition. P-wave frequency was significantly reduced on Day 0 and significantly elevated on Day 7, but was unaffected by saline (sham) infusion. These changes occurred during both REM and NREM sleep.
allowed to recover. Each animal received one vehicle injection and one carbachol injection in random order. One injection was made to the left PPT and one to the right PPT, by random assignment. Before, and at each time point after infusion or injection, we made 6 h polygraphic and respiratory recordings from 10:00 to 16:00 h. Sleep, P-waves and breathing were analyzed using computer algorithms. We found that with respect to control, carbachol infusion initially reduced both P-wave and apnea expression by 70%–90% during REM and non-rapid eye movement (NREM) sleep (P < 0.04 for each). Thereafter, P-wave and apnea frequencies during REM and NREM sleep increased to maximal levels 7 days after infusion (P < 0.005 for each). Similar REM and NREM increases in P-wave and apnea frequencies occurred 3 and 7 days after carbachol microinjections
Fig. 6. Changes in apnea frequency (apneas per hour) after PPT infusions, presented in the format of Fig. 5. Again, apnea expression was acutely diminished (Day 0) and ultimately augmented (Day 7) after carbachol infusion, but was unaffected by saline infusion. These changes occurred during REM sleep and NREM sleep. Fourteen days after carbachol infusion apnea frequency during REM sleep was equivalent to control, but apnea frequency during NREM sleep remained elevated (P = 0.03).
(P < 0.04 for each) (Figs. 5 and 6). These responses evident during both REM and NREM sleep, followed similar time courses, and were not associated with significant changes in sleep architecture, respiratory rate, or minute ventilation (Tables 1 and 2). Overall, the effects of carbachol were equivalent whether the agent was delivered to conscious animals by infusion or to anesthetized animals by microinjection. Our rationale for conducting the initial infusion experiments was two-fold. First, we wished to observe the immediate impact of carbachol on sleep and breathing. Second, we wanted to avoid the confounding impact of anesthesia on both sleep and breathing behaviors. These experiments convincingly demonstrated that in the first 6 h after infusion to the PPT, carbachol reduced expression of both BPEs (detected as P-waves) and apneas (Figs. 5 and 6) in animals that were fully recovered from surgery and well adapted to the monitoring apparatus.
Table 1 Unchanged minute ventilation after carbachol infusion to PPT
NVI-NREM NVI-REM
Saline
Control
Day 0
Day 3
Day 7
Day 14
0.91 ± 0.12 0.89 ± 0.09
0.91 ± 0.15 0.92 ± 0.06
0.94 ± 0.07 0.95 ± 0.07
0.90 ± 0.08 0.99 ± 0.06
1.06 ± 0.11 1.07 ± 0.08
0.98 ± 0.11 1.00 ± 0.08
NVI-NREM = Average (±S.E.) minute ventilation during NREM relative to control wakefulness. NVI-REM = Average (±S.E.) minute ventilation during REM relative to control wakefulness.
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Table 2 ITR Lesion does not change average respiratory pattern Control
Day 2
Day 7
Day 14
P
RR-Wake RR-NREM RR-REM P
119.0 ± 3.4 95.4 ± 2.3 103.4 ± 2.0 0.0001
107.5 ± 4.0 94.6 ± 2.9 98.7 ± 3.9 0.05
108.5 ± 5.5 95.4 ± 3.1 100.8 ± 3.3 0.10
111.9 ± 3.6 93.1 ± 1.2 98.7 ± 4.5 0.004
0.21 0.94 0.73
NRR-Wake NRR-NREM NRR-REM P
1.0 ± 0 0.80 ± 0.2 0.87 ± 0.02 0.0001
0.91 ± 0.03 0.80 ± 0.02 0.84 ± 0.04 0.09
0.91 ± 0.04 0.81 ± 0.03 0.85 ± 0.04 0.18
0.94 ± 0.03 0.79 ± 0.03 0.83 ± 0.05 0.03
0.12 0.96 0.89
NMV-Wake NMV-REM NMV-REM P
1.0 ± 0.0 0.80 ± 0.02 0.79 ± 0.03 0.0001
0.93 ± 0.05 0.79 ± 0.05 0.71 ± 0.07 0.04
0.95 ± 0.11 0.80 ± 0.09 0.77 ± 0.10 0.41
1.05 ± 0.05 0.85 ± 0.06 0.81 ± 0.08 0.03
0.63 0.94 0.82
NRR: Respiratory Rate normalized to the mean value during wakefulness of the control recording. NMV: Minute Ventilation normalized to the mean value during wakefulness of the control recording.
3. A role for ITR in the pontine respiratory network 3.1. Experiments in anesthetized rats Despite many years of study, the central mechanisms of reflex apnea remain incompletely identified. Recent evidence implicates the ITR, described in 1981 by Brodal (Brodal, 1981), as an important pontine area for regulating apneic reflexes in the rat (Chamberlin and Saper, 1998). In their neuroanatomical study Chamberlin and Saper (1998) used glutamate microinjection into the lateral pons of anesthetized rats to identify areas producing an immediate respiratory response. They identified a previously unrecognized group of cells among the fiber bundles between the motor and principal sensory trigeminal nuclei whose stimulation by glutamate produced an immediate apnea. Rostrally, apneic sites stretched up to the ventral border of Kolliker–Fuse (KF) nucleus while, caudally, apneic sites stretched ventrally along the motor trigeminal roots (Chamberlin and Saper, 1994). Using anterograde and retrograde tracers, Chamberlin and Saper (1998) established that these apneic sites in the ITR project to the ventral respiratory group in the medulla. We repeated Chamberlin and Saper’s experiment by microinjecting glutamate to ITR and obtained an instant 3 s apnea (Fig. 7, Panel A). After the animal resumed normal breathing we microinjected kynurenic acid, a glutamate receptor antagonist (Perkins and Stone, 1982), to ITR,
which was followed by a microinjection of glutamate. The result was a complete blockade of the respiratory response to glutamate injection (Fig. 7, Panel B). To determine whether the ITR has a physiological role in mediating or modulating respiratory reflexes and, whether it is functionally involved in the central mechanisms of vagal-reflex apnea, we intravenuously injected 5-HT, which does not cross the blood brain barrier, to 10 anesthetized adult Sprague–Dawley rats to produce apneic responses. This type of reflex apnea arises from stimulation of the vagus nerves and bilateral vagotomy above nodose ganglia in cats (Jacobs and Comroe, 1971; Sampson and Jaffe, 1974; Ginzel, 1975; Sutton, 1981) and rats (Yoshioka et al., 1992a,b; Yoshioka, 1995) was shown to abolish the 5-HT-induced apnea. In addition, 5-HT activates unmyelinated vagal afferents (Coleridge and Coleridge, 1997) and these also may contribute to the observed apnea promoting effect of 5-HT. Since all vagal sensory afferents terminate in the NTS, the NTS could be considered as the principal central relay station for the 5-HT induced reflex apnea. The animals used in this study had a catheter inserted into the femoral vein for administration of 5-HT (0.00375 mg) and respiration was recorded by piezoelectric crystal. Multibarrel pipettes were used to pressure inject glutamate (5–10 nl, 10 mM), kynurenic acid (10 nl, 50 mM), and red dye into the ITR, unilaterally and bilaterally. Intravenous administration of 5HT produced an immediate 3 s apnea (Fig. 8, Panel A). Unilateral glutamatergic blockade at an ITR site with
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Fig. 7. Panel A shows the characteristic apnea evoked by glutamate pressure injection in the ITR. Injection of this site with kynurenic acid applied from a separate barrel of multibarrel pipette, completely blocked the respiratory response to glutamate injection (Panel B). AU – arbitrary units.
kynurenic acid significantly prolonged the 5-HT induced reflex apnea (Fig. 8, Panel B). When kynurenic acid was simultaneously injected to the contraleteral site it further lengthened the reflex apnea (Fig. 8, Panel C). The schematic mapping of kynurenic acid injection sites is presented in Fig. 9. However, the brainstem networks involved in generating reflex apnea have not been well defined. One putative brainstem network for mediating apneic reflexes in the rat includes the NTS, ITR and the ventral medulla (VLM) (Chamberlin and Saper, 1998). In that neuroanatomical pathway of reflex apnea, information from the NTS would reach the ITR, from which it would be transmitted to VLM. Our observations provide a test of this pathway. If the ITR represents a “relay” for reflex apnea, as suggested by Chamberlin and Saper (1998), and because activation of glutamate receptors in small regions of the ITR produces immediate apnea, we initially expected that application of the glutamate receptor antagonist kynurenic acid might atten-
uate the vagally transmitted apnea in this study. Instead, we observed the opposite effect, i.e., that functional glutamatergic blockade of ITR sites by kynurenic acid potentiated vagal reflex apnea (Fig. 8, Panels B and C). We are confident that kynurenic acid microinjections were made into or very near the same location as the glutamate injections because we utilized a multibarrel pipette that was not moved between agonist and antagonist injections. In addition, by leaving the pipette in place, we observed that 1–2 h after kynurenic acid administration, glutamate-induced apnea was again observed and reflex apnea duration returned to baseline. The augmentation of 5-HT-induced apnea by kynurenic acid was demonstrated both with unilateral and bilateral injections of the acid to ITR. In both cases there was a significant increase of apnea duration following the blockade of glutamate receptors. In contrast to unilateral injections of the acid to ITR which produced augmentation of 5-HT-induced apnea of 7.5 s, the bilateral injections of kynurenic acid to
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Fig. 8. Intravenuous administration of 5-HT evoked an apnea (panel A). Unilateral glutamatergic blockade at an ITR site with kynurenic acid significantly prolonged the 5-HT induced reflex apnea (panel B). Kynurenic acid injection of a second, contralateral, site further lengthened the reflex apnea (panel C). AU – arbitrary units.
ITR lengthened the 5-HT-induced apnea upto 10 s and longer (Fig. 8). Thus, it appears from these findings that the ITR exerts a potent dampening influence on vagally-induced reflex apnea. 3.2. Experiments in freely moving rats In the rat model of central sleep apnea, characterized by us and others, (Mendelson et al., 1988; Thomas et al., 1992; Carley et al., 1996; Christon et al., 1996; Radulovacki and Carley, 2003) we have established a putative link between pathways of reflex apnea and
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spontaneous sleep-related apneas (Radulovacki et al., 2003, 2004). Intravenous administration of serotonin to anesthetized cats or rats produced dose-dependent reflex apnea (Jacobs and Comroe, 1971; Yoshioka et al., 1992a,b), whereas in freely moving rats, intraperitoneal administration of serotonin produced dramatic increases in spontaneous sleep-related apneas (Carley and Radulovacki, 1999b). These findings suggested that the central and peripheral pathways of reflex apnea may participate in genesis of sleep apneas. We further explored the potential central link between reflex and sleep apneas. We reported earlier that glutamatergic blockade of the ITR yielded dosedependent lengthening of reflex apnea evoked by intravenous serotonin injection and concluded that one physiological role for the ITR may be to attenuate vagally-induced reflex apneas (Radulovacki et al., 2003). We conducted the next series of experiments to determine whether ibotenic acid lesioning of the ITR would affect spontaneous sleep apneas in freely moving rats. For these series of experiments we implanted rats with EEG and EMG electrodes, recorded them polygraphically for 6 h and monitored their respiration by placing each animal inside a single-chamber plethysmograph. Subsequently, in the next surgical intervention, a respiratory-related ITR site was identified by probing on dorso-ventral tracks with 2–5 nl glutamate (10 nl, 10 mM) injections from a multibarrel glass pipette. This site was then lesioned by injecting ibotenic acid (10 nl, 50 mM) from a second pipette barrel. After recovery period, animals were again recorded for 6 h on days 2, 7 and 14 after the lesion. They were sacrified under anesthesia after recording day 14 and the location of their ITR lesions was histologically verified. The distribution of anatomical localization of the unilateral ibotenic acid lesion is presented in Fig. 10. As in previous investigations (Carley et al., 1996; Christon et al., 1996; Radulovacki et al., 1996, 1998; Carley and Radulovacki, 1999a, 1999b), sleep apneas, defined as cessation of respiratory effort for at least 2.5 s, were scored for each recording session and were associated with the stage in which they occurred: NREM or REM sleep. Apnea index, defined as apneas per hour in a stage, was separately determined for NREM and REM sleep. The timing and volume of each breath were scored by automatic analysis (Experimenter’s Workbench; Datawave Technologies). Sighs
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Fig. 9. Schematic mapping of kynurenic acid injection sites. The injection sites (* ) are well localized to the ITR. Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; Pr5, principal sensory trigeminal nucleus; s5, sensory root of the trigeminal nerve; scp, superior cerebellar peduncle.
were identified by tidal volume more than 150% greater than the overall mean, and post-sigh pauses greater than 2.5 s were excluded from all analysis presented below. For each animal, the mean respiratory rate and inspiratory minute ventilation was computed for wakefulness throughout the 6 h control recording and used as a baseline to normalize respiration during sleep, and following ITR lesioning. The results of the study showed that ITR lesions exerted no impact on mean respiratory pattern during any sleep/wake state, compared to baseline recordings
Fig. 10. Distribution of the anatomical localization of the unilateral ibotenic acid lesion of the ITR. The injection sites (squares and circles) are well localized to the ITR. Mo5, motor trigeminal nucleus; Pr5, principal sensory trigeminal nucleus; s5, sensory root of the trigeminal nerve.
(Table 2) In contrast, apnea frequency during NREM sleep increased following ITR lesion, more than doubling by day 14 (Fig. 11). These findings agree with the experiments in anesthetized rats where the ITR lesion by kynurenic acid lengthened the vagally-induced reflex apnea. Thus, they confirm the inhibitory influence of ITR in the genesis of reflex and sleep-related apneas and point to ITR as a part of pontine inhibitory mechanisms in the central pathways of both types of apneas.
Fig. 11. Group data (mean ± S.E.) of apnea expression during REM and NREM sleep 2, 7 and 14 days following ibotenic acid lesion of the ITR. There was a trend toward increased apnea during REM sleep. Conversely, during NREM sleep, sleep apnea index was significantly elevated on days 2 and 14 following the lesion (P = 0.01 and P = 0.02, respectively).
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4. Discussion Our studies in anesthetized and freely moving rats demonstrated a role for two pontine structures in respiration that are not classically included in the pontine respiratory neuronal network: the PPT and the ITR (Radulovacki et al., 2003, 2004; Saponjic et al., 2003). From a historical point of view, respiratory related functional roles have been assigned to several regions within the pons that, collectively, have been referred to as the pontine respiratory group (see St.-John, 1998 for a review). Within the pons, the parabrachial nuclear complex, including the K–F nucleus, contains many neurons with activity related to various phases of the respiratory cycle (Bertrand and Hugelin, 1971). On balance, available data from cat, rat and rabbit suggest that stimulation of medial parabrachial neurons produces expiratory facilitation including bradypnea or apnea, whereas lateral parabrachial stimulation produces inspiratory facilitation including tachypnea or hyperpnea (Takayama and Miura, 1993; Chamberlin and Saper, 1994; Dick and Coles, 2000). However, unlike the present responses to PPT stimulation, parabrachial stimulation by electrical current (Fung and St-John, 1994; Lara et al., 1994) or glutamate injection (Takayama and Miura, 1993; Chamberlin and Saper, 1994; Lara et al., 1994) produced immediate respiratory responses that did not persist significantly after cessation of the stimulus. It is of interest here that, the behavior of respiratoryrelated parabrachial neurons changes significantly across sleep/wake states. Well-modulated respiratory related activity is readily recorded from pontine units in the conscious cat and the firing rates of these neurons decrease with sleep onset and are lowest during REM sleep (Lydic and Orem, 1979; Sieck and Harper, 1980). A series of more recent studies from Lydic and colleagues (Gilbert and Lydic, 1990, 1994) demonstrates that pontine respiratory neurons may be directly influenced by pontine reticular areas involved with the control of REM sleep. Moreover, the above mentioned study of Lydic and Baghdoyan (1993) first showed in barbiturate anesthetized cats that PPT continuous electrical stimulation leads to increased acetylcholine release in the medial pontine reticular formation and to respiratory depression. However, the respiratory depression was mild and transient – diminishing even
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before cessation of the stimulus. The impact of glutamatergic PPT stimulation, in contrast, included longlasting respiratory disturbance characterized by both increases and decreases of respiratory frequency. Differences between the present findings and those reported previously (Lydic and Baghdoyan, 1993) may reflect differing methods and precise anatomical sites of stimulation, differing anesthetic agents, different species, or other factors. Our data in the freely moving rats demonstrate that neurochemical stimulation of the PPT significantly increases sleep-related respiratory variability – including apnea expression – but do not demonstrate the anatomical pathways for this effect. It is possible that parabrachial nuclei play a role in conveying the influence of PPT neurons to the respiratory pattern generating and premotor integrating areas of the ventrolateral medulla. Because the respiratory-related neurons of the parabrachial complex in the rat are concentrated approximately 1.5 mm caudal to our PPT target, we do not believe these regions were directly destroyed by mechanical introduction of the guide cannula or infusion needle in our experiments. Further, these regions appeared to be intact by light microscopy of the fixed brains. Still, mechanical trauma to the parabrachial complex cannot be ruled out in the animals that received infusions. In addition, we must allow the possibility that these areas, especially the lateral parabrachial region, may have been directly affected by the carbachol following infusion. Chamberlin and Saper conducted a systematic mapping of the parabrachial complex in rats using glutamate pressure injections (Chamberlin and Saper, 1994). They reported hyperpnea following stimulation of the rostral and mid-caudal K–F nucleus as well as most of the lateral parabrachial region. Also, we have reported that non-specific respiratory stimulation can reduce apnea expression in sleeping rats (Christon et al., 1996). Therefore, it is possible that carbachol, acting in the lateral parabrachial region rather than in the PPT, accounted for the reduced apnea expression during the 6 h immediately after infusion. This explanation seems unlikely, however, for two reasons. First, respiratory stimulation via the lateral parabrachial region would be expected to produce hyperpnea, but respiratory rate and minute ventilation remained unchanged from control values (Table 1). Second, P-wave expression and apnea expression followed similar time
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courses throughout the 14 days following carbachol infusion (Figs. 5 and 6). Because the PPT is directly involved in generating and/or relaying P-waves (Datta, 1995; Datta et al., 1992, 1998, 1999) it seems most likely that the actions of carbachol on PPT neurons were responsible for initiating the parallel changes in apnea expression. Perhaps the most striking feature of the P-wave and respiratory responses to cholinergic PPT stimulation was their duration. Following both injections and infusions, carbachol evoked maximal increases in P-wave and apnea expression between 3 and 7 days after administration. This is similar to the time course of increased PGO waves reported in cats after a single dose of carbachol (Datta et al., 1992). Clearly, the responses at 7 days do not reflect the direct pharmacological action of carbachol in the cat (Datta et al., 1992) or the present rat observations. We can only speculate, but it seems probable that the sustained responses observed would require changes in gene expression and protein turnover/localization. In addressing the physiological role of ITR in respiration we performed experiments both in anesthetized and freely moving rats. In anesthetized animals, we tested Chamberlin and Saper’s postulate that ITR is an important structure in the central pathways of reflex apnea (Chamberlin and Saper, 1998). Since reflex apnea may be evoked by a variety of pharmacological interventions we chose the peripheral administration of 5HT to evoke vagally-induced reflex apnea. It has been shown that the triad of bradycardia, hypotension and apnea (the Bezold-Jarisch reflex) can be evoked in anesthetized animals by peripheral administration of several substances including serotonin (5-HT) (Ginzel, 1975). Thus, Jacobs and Comroe (1971) caused reflex apnea in anesthetized cats by injecting 5-HT into the common carotid arteries which was abolished by bilateral section of supranodose vagus. In addition, Yoshioka et al. (1992a, 1992b) produced transient reflex apnea by intravenous injection of 5-HT to anesthetized rats, which again was abolished by bilateral vagotomy above the nodose ganglia. These and other studies (Sampson and Jaffe, 1974; Sutton, 1981) have concluded that the apnea component of the Bezold-Jarisch reflex results from the activation of afferent neurons with somata based in the nodose ganglia in cats and rats and that the primary central synapse of these afferent neurons is in the NTS (Vardhan et al., 1993).
Our results show that in accordance with Chamberlin and Saper’s study (1998), by microinjecting glutamate to ITR, we produced an immediate apneic response (Fig. 7, Panel A) which was prevented by prior microinjection of a broad spectrum glutamate receptor antagonist, kynurenic acid (Fig. 7, Panel B). Following glutamate antagonism, subsequent administration of 5HT produced apneas of much longer duration (Fig. 8, Panels B and C), which was an unexpected finding. As we have mentioned above, we initially expected that application of the glutamate receptor antagonist kynurenic acid might attenuate the vagally transmitted apnea. Because we were surprised by the general augmentation of reflex apnea by functional blockade of the ITR, we sought to test whether the ITR represents an obligatory synapse in the vagal reflex apnea pathway. For this purpose, we conducted acute ponto-medullary transections in 3 additional animals. Following isolation of the medulla, 5-HT-induced apnea was dramatically amplified, with apneas >30 s in duration after a standardized 5-HT injection. This demonstrates that medullary circuits are sufficient to produce vagal reflex apneas, and that the overall effect of more rostral structures is to attenuate the reflex – at least during anesthesia. The observed effects of kynurenic acid support the conclusion that one physiological effect of glutamate in ITR is to dampen vagally-induced apnea. This interpretation does not preclude other respiratory functions for ITR. For example, it may be that ITR is a key relay in apnea evoked by stimulation of the trigeminal nerve (Chamberlin and Saper, 1998). In any case, the physiologically relevant source of glutamatergic input to ITR that results in attenuation of vagal reflex apnea cannot be determined from our studies. In our study of freely moving rats we demonstrated that a small and well localized unilateral lesion of the ITR in the lateral pons can increase the frequency of central apnea during NREM sleep in freely moving rats over a 2 week period (Fig. 11) (Radulovacki et al., 2004). This finding is of interest because it points to a functional role for a pontine group of cells that have not, to date, been implicated in regulation of either sleep or sleep-related apnea. However, previous neuroanatomical studies of the ITR, by documenting a connection of the region both to the NTS and to the ventral medulla, have established a potential neural substrate for this effect (Chamberlin and Saper, 1998, 2003).
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The results of the above experiments are consistent with our previous finding that glutamatergic blockade of the ITR exacerbated vagal reflex apnea (Radulovacki et al., 2004). Thus, impairing the functional integrity of the ITR augmented reflex apnea in anesthetized rats and sleep-related spontaneous apnea in conscious rats. We cannot determine from these data the precise mechanism by which unilateral ITR lesion increased NREM apnea genesis, but our earlier findings (Radulovacki et al., 2004) support the hypothesis that glutamatergic neurotransmission in the ITR may be important. It is unlikely that increased sleep-related apneas resulted indirectly from non-specific alterations in either respiratory drive or sleep architecture. Both respiratory pattern (Table 2) and sleep/wake architecture were equivalent to control recordings 14 days after ITR lesioning, yet apnea frequency was maximal at this same time point (achieving statistical significance only during NREM sleep) (Fig. 11). This implies that the ITR exerts a specific role in dampening respiratory perturbations rather than impacting overall respiratory drive. These observations are in accordance with our previously reported data on the role of ITR in attenuation of reflex apneas. Taken together, the data suggest that both reflex and sleep apneas share some common central pathways. They are also in agreement with the general modulatory role of pontine structures in activities including respiration, heart rate and regulation of blood pressure. In summary, consistent evidence is emerging that two regions in the lateral pons, PPT and ITR, have previously unsuspected role in regulating respiration and respiratory variability. These effects can be demonstrated both in acute and in chronic experiments in rats. Stimulation/lesion of either region affects the brainstem respiratory pattern generator and emergent pattern disturbances. Evidence to date provides strong motivation for further investigations into synaptic, cellular and molecular mechanisms by which the PPT and the ITR influence breathing.
Acknowledgement This work was supported by NIH grant AG16303.
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Modulation of reflex and sleep related apnea by pedunculopontine tegmental and intertrigeminal neurons Miodrag Radulovackia,∗ , Sasha Pavlovica , Jasna Saponjicb , David W. Carleya,b,c a
Department of Pharmacology, University of Illinois at Chicago, M/C 868, 901 S. Wolcott Avenue, Chicago, IL 60612, USA b Department of Medicine, Section of Respiratory and Critical Care Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA c Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60612, USA Accepted 6 February 2004
Abstract We describe and summarize here our recent findings about the role in respiration of two pontine structures that are not classically included in the pontine respiratory group: the pedunculopontine tegmental nucleus (PPT) and the intertrigeminal region (ITR). We also discuss significant contributions of other workers in the field, especially, S. Datta [Cell. Mol. Neurobiol. 17: 341–365, 1997], R. Lydic and H. Baghdoyan [Sleep, 25: 617–622, 2002], and N. Chamberlin and C. Saper [J. Neurosci. 18: 6048–6056, 1998], who postulated a role for the ITR in modulating reflex apnea. In anesthetized and freely moving rats we have consistently documented that PPT and ITR have a role in respiration. Neurochemical manipulations of each area affected the brainstem respiratory pattern generator and respiratory pattern variability,observed as spontaneous disturbances during sleep or as induced reflex apnea. Although the exact central mechanisms of apnea cannot be determined from our studies to date, we postulate that reflex and sleep-related apneas in rats share some common brainstem pathways, which may include PPT and ITR. © 2004 Elsevier B.V. All rights reserved. Keywords: Control of breathing, pons; Mammals, rat; Pons, pedunculopontine tegmental nucleus, intertrigeminal region; Sleep, reflex apnea
1. Introduction The pons plays a complex and poorly understood role in respiratory rhythmogenesis. Recently, two areas in the rostral pons have been found to modu∗ Corresponding author. Tel.: +1 312 996 3539; fax: +1 312 996 1225. E-mail address: [email protected] (M. Radulovacki).
1569-9048/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2004.02.012
late respiration: pedunculopontine tegmental nucleus (PPT) (Lydic and Baghdoyan, 1993; Saponjic et al., 2003) and intertrigeminal nucleus (ITR) (Chamberlin and Saper, 1998, 2003; Radulovacki et al., 2003). Lydic and Baghdoyan (1993) first showed in barbiturate anesthetized cats that PPT continuous electrical stimulation leads to increased acetylcholine release in the medial pontine reticular formation and to respiratory depression. However, the respiratory depression was mild
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and transient, diminishing even before cessation of the stimulus. In anesthetized and spontaneously breathing Sprague–Dawley rats we demonstrated the longlasting increase in variability of respiratory parameters following glutamate microinjection into PPT (Carley et al., in press; Saponjic et al., 2003). The induced respiratory perturbations were characterized by intermittent apneas and increased variability of expiratory (TE) and total (TT) breath durations in all animals. Findings of these studies point to a significant impact of the PPT on the brainstem respiratory pattern generator. The other area in the rostral lateral pons that has recently emerged as a contributor to the brainstem respiratory function is the ITR (Chamberlin and Saper, 1998, 2003; Radulovacki et al., 2003). In classical functional anatomical analysis of respiratory circuitry Chamberlin and Saper (1998) suggested that the ITR represents a key relay for several airway protective apneic reflexes via the neural pathways revealed by combining glutamatergic stimulation and tract-tracing methodologies. This idea was supported by findings, which showed that blockade of synaptic transmission with large injections of cobalt or NMDA receptor antagonists aimed at the Kolliker–Fuse (KF) area attenuated the apneic response to ethmoidal nerve stimulation (Dutschmann and Herbert, 1998). Furthermore, injections of gammaaminobutyric acid (GABAA) receptor antagonists potentiated trigeminal apnea (Dutschmann and Herbert, 1998). These findings suggest that the KF mediates trigeminal apnea, because effective injections were centered in this nucleus. However, the control injections were not placed into more ventral regions, and hence it is not clear whether they may have also affected the ITR. Our data in anesthetized freely breathing rats confirmed the ITR role in respiration indicating that one physiological role for the ITR in respiration is to attenuate vagal reflex apneas and to dampen respiratory instability (Radulovacki et al., 2003). In accordance, in freely moving rats a small and well localized unilateral lesion of the ITR produced a lasting sleep-related breathing disturbance by increasing sleep apnea expression over a two week period (Radulovacki et al., in press). These findings are in agreement with the general modulatory role of pontine structures in respiration.
2. A role for PPT in the pontine respiratory network 2.1. Experiments in anesthetized rats REM sleep is uniquely characterized by postural muscle atonia and frequent “phasic” events that are widespread in the brain and are typified by rapid eye movements, for which the behavioral state was named (Aserinsky and Kleitman, 1953). Considerable evidence now indicates that a BPE generator in the pons initiates REM-related eye movements and other transient motor behaviors, and that the PPT is a key site for generation and/or relay of these BPEs (Datta, 1995; Datta et al., 1998). Close electrophysiological BPE markers are readily recorded as large amplitude field potentials in the pons and have been termed pontogeniculo-occipital (PGO) waves in the cat and, more recently, P-waves in the rat (Datta et al., 1999; Mavanji and Datta, 2003). P-waves, the pontine component of PGO waves, represent a close marker of brainstem phasic events and are associated with cardio-respiratory changes, including apnea, in sleeping rats. Numerous studies have demonstrated that cholinergic activation of the medial pontine reticular formation (mPRF) can elicit many features of rapid eye movement (REM) sleep, including increased respiratory variability (Gilbert and Lydic, 1990, 1991, 1994; Kubin, 2001). The PPT provides a significant cholinergic input to the mPRF, but little has been done to examine a specific role for PPT in regulating respiratory pattern (Lydic and Baghdoyan, 1989, 1993). Since local glutamate injections into the PPT of rats have been shown to excite pontine wave (P-wave) neurons (Datta, 1997) we decided to probe the impact of PPT activation and brainstem phasic events (BPE) generation on respiratory pattern by microinjecting glutamate into the PPT of rats. Because the excitability and network properties of brainstem neurons can be grossly altered by general anesthesia, we characterized respiratory responses to PPT stimulation under both a barbiturate agent (nembutal) and a dissociative agent (ketamine). Experiments were performed in spontaneously breathing adult, male, anesthetized Sprague–Dawley rats. Nine rats were anesthetized with nembutal (Abbot Laboratories, North Chicago, IL) with an initial intraperitoneal dose of 50 mg/kg and 13 rats were anesthetized with a combination of 80 mg/kg ketamine (Ab-
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bot Laboratories, North Chicago, IL) and 5 mg/kg xylazine (Phoenix Scientific Inc., St Joseph, MO) given by intraperitoneal injection. After a surgical plane of anesthesia was achieved, rats were placed in the stereotaxic apparatus (David Kopf Inst., model 962 A Tujunga, CA) and bilateral parietal osteotomies were drilled. At the right burr-hole a bipolar twisted wire, teflon-coated electrode was targeted to the PPT (A: 0.5 with respect to lambda; L: 2.0; H: 7.0) for recording P-waves. Using the same coordinates on the left side (Paxinos and Watson, 1986), a multibarrel micropipette was introduced for pressure microinjections of saline and glutamate (10 nM, l-glutamatic acid monosodium salt) into the PPT. We found that local PPT stimulation by glutamate produced sustained respiratory disturbances, including intermittent apnea. Under ketamine anesthesia, glutamate microinjection into the PPT did not evoke an immediate effect on respiration, but respiratory responses lasted more than 1 h and were marked by disturbances of both timing and tidal amplitude (Figs. 1 and 2). Under nembutal anesthesia microinjection of glutamate into the PPT resulted in an immediate apnea, respiratory disturbances were resolved within 15 min and reflected significant disturbances in expiratory timing (Figs. 3 and 4). Spontaneous BPEs were ex-
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pressed as P-waves under ketamine but not nembutal anesthesia, and PPT stimulation at sites associated with respiratory disturbance also reduced BPE expression in these animals. Conversely, at more dorsal sites, PPT stimulation augmented BPE expression but had no respiratory impact. These findings argue that PPT is an important structure for respiratory pattern modulation, and that PPT activation can produce long-lasting respiratory disturbances characterized by both increases and decreases in breathing depth and frequency. The occurrence of significant respiratory disturbances including apnea and lasting at least 15 min after a single glutamate injection under both anesthetic regimens lends robustness to these findings. Still, it is likely that the differences in response latency and duration reflected, at least in part, the differing and complex cellular and network effects of the two anesthetic agents. In addition to its main non-competitive NMDA antagonist action, ketamine significantly reduces firing rates of GABAergic neurons (Lee et al., 2001); increases locus coeruleus neuron firing rates and noradrenaline release in the cerebral cortex (Kubota et al., 1999); disrupts normal sleep-cycle organization, decreases acetylcholine release in the pontine reticular formation, and slows breathing rate (Lydic and Bagh-
Fig. 1. Typical severe respiratory disturbance induced by glutamate injection into the PPT in a ketamine anesthetized rat. (A) Glutamate injection (an arrow marks the time of glutamate injection) did not evoke an immediate effect on respiration. (B) Immediate continuation of tracing presented in (A). After a latency of 2 min glutamate injection produced severe respiratory disturbance.
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Fig. 2. Glutamate-induced changes in the total breath duration (TT) presented as coefficient of variation (CV) in the group of ketamine anesthetized animals (n = 13). Each bar reflects the mean and SE of CV for the group of animals in 5 min bins of each recorded interval except for immediate preinjection and post-injection interval. Baseline (bas) (n = 12), recorded prior to introduction of the pipette into PPT; after saline injection (sal) (n = 4); pre-injection: 30 s epoch immediately preceding glutamate injection (30’pre) (n = 12); initial post-injection: 30 s epoch immediately following glutamate injection (30’post) (n = 12); and every 10 min of overall post-injection interval: up to 70 min following glutamate injection (10 min (n = 12); 20 min (n = 12); 30 min (n = 10); 40 min (n = 9); 50 min (n = 8); 60 min (n = 5); 70 min (n = 5). Single factor ANOVA was significant for each parameter (P < 0.05 for each). t-Tests with Bonferonni correction were used to compare each response interval to baseline: * P < 0.05; ** P < 0.01; *** P < 0.001.
doyan, 2002). In contrast, nembutal functions primarily as a GABAa agonist. The specific contributions of these anesthetic agents to the observed respiratory responses cannot be inferred from the present study. These results strongly support a role for the PPT as an important supramedullary modulator of respiratory pattern generation. Activation of direct or indirect PPT
outputs to the medullary respiratory network induced prolonged destabilization of respiratory drive and genesis of intermittent apnea regardless of the state of the brain under differing anesthetic regimens. Defining the specific pathways and signaling mechanisms as well as the precise impact of BPEs will require further elucidation by future studies.
Fig. 3. Respiratory response to glutamate injection into the PPT of a rat anesthetized by nembutal: immediate apnea is followed by additional apneas of varying duration and irregular spacing. An arrow marks the time of glutamate injection.
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Fig. 4. Changes in the total breath duration induced by glutamate injection in the group of nembutal anesthetized rats (n = 9). Data are presented in the style of Fig. 2 except that the total post-injection recorded period was 20 min rather than 70 min. Each recorded interval was presented as a 5 min bin/per each rat, except for immediate preinjection and post-injection time after glutamate injection: baseline (n = 9); after saline injection (n = 3); immediate preinjection and post-injection 30 s (n = 9); 10 min (n = 9); 15 min (n = 5); 20 min (n = 40) after glutamate injection. Single factor ANOVA demonstrated significant changes in CV of TT (P < 0.05 for each). With respect to baseline, t-tests revealed immediately increased CV for TT upto 15 min after glutamate injection: * P < 0.05.
2.2. Experiments in freely moving rats Long-lasting enhancement of P-wave expression has been demonstrated after cholinergic stimulation of the PPT in cats (Datta et al., 1992). We tested the ability of carbachol microinjections and microinfusions into the PPT of rats to determine, whether in this animal species cholinergic stimulation of the PPT would elicit increased expression of P-waves and sleep related breathing disorder. Fifteen adult male Sprague–Dawley rats (300 g) were anesthetized using a mixture of ketamine (Vetalar 100 mg/ml) and acetylpromazine (10 mg/ml) (4:1, v/v) at a volume of 1 ml/kg body weight for implantation of biparietal cortical electrodes for EEG recording, and neck muscle electrodes for electromyogram (EMG) recording. To record P-waves, a bipolar twisted pair stainless electrode (75 m wires, ∼0.5 mm insulation removed, tips separated by 1 mm) was stereotaxically guided to the region of the locus coeruleus (AP: −0.8, ML: 1.25, DV: 3.0 with respect to the interaural line). In five animals, a stainless steel guide cannula (HTX-22; outside diameter 0.36 in.; Small Parts, Inc., Miami Lakes, FL) was introduced via a burr hole and stereotaxically advanced using a dorsoventral approach. The cannula tip was targeted to a coordinate
1 mm dorsal to the left or right PPT by random assignment (AP: −7.8; ML: 2.0; DV: 6.3 with respect to Bregma). Prior to implantation, a matched and tight fitting needle (HTX-28; outside diameter 0.36 in.; Small Parts, Inc., Miami Lakes, FL) was prepared for each guide cannula so that the needle would extend 1 mm beyond the end of the cannula when positioned for PPT infusions. A positive displacement infusion pump (KD Scientific 210; Fisher, Pittsburgh, PA) was used to deliver 100 nl of saline or carbachol (0.1 g in saline) over a period of 30 s in conscious animals. Each animal received one vehicle infusion and one carbachol infusion. In ten animals, left and right burr-holes were drilled for PPT approach and were closed with bone wax. To allow neurochemical PPT manipulations, these animals were re-anesthetized, the bone wax was removed, and a single barrel micropipette (standard filament glass, outside diameter 1.2 mm, inside diameter 0.68 mm, pulled to a tip diameter of approximately 20 m) was stereotaxically targeted to the PPT. Pressure microinjection was used (Picospritzer II; General Valve Corp., Brookshire, TX) to deliver 100 nl of pontamine blue dye dissolved in saline or carbachol (0.1 g in saline with pontamine blue dye). After removing the pipette, the burr hole was re-closed with bone wax and the animal was
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Fig. 5. P-wave frequency (waves per minute) with no infusion (Control), during the first 6 h after saline infusion to the PPT (Saline), during the first 6 h after carbachol infusion (0.1 g in 100 nl; Day 0), and during the same 6 h circadian phase 3, 7 and 14 days after carbachol infusion (Day 3, Day 7, and Day 14, respectively). Points reflect the mean and SE (n = 5) for REM sleep (squares) and NREM sleep (circles). P values indicate significant differences with respect to the control condition. P-wave frequency was significantly reduced on Day 0 and significantly elevated on Day 7, but was unaffected by saline (sham) infusion. These changes occurred during both REM and NREM sleep.
allowed to recover. Each animal received one vehicle injection and one carbachol injection in random order. One injection was made to the left PPT and one to the right PPT, by random assignment. Before, and at each time point after infusion or injection, we made 6 h polygraphic and respiratory recordings from 10:00 to 16:00 h. Sleep, P-waves and breathing were analyzed using computer algorithms. We found that with respect to control, carbachol infusion initially reduced both P-wave and apnea expression by 70%–90% during REM and non-rapid eye movement (NREM) sleep (P < 0.04 for each). Thereafter, P-wave and apnea frequencies during REM and NREM sleep increased to maximal levels 7 days after infusion (P < 0.005 for each). Similar REM and NREM increases in P-wave and apnea frequencies occurred 3 and 7 days after carbachol microinjections
Fig. 6. Changes in apnea frequency (apneas per hour) after PPT infusions, presented in the format of Fig. 5. Again, apnea expression was acutely diminished (Day 0) and ultimately augmented (Day 7) after carbachol infusion, but was unaffected by saline infusion. These changes occurred during REM sleep and NREM sleep. Fourteen days after carbachol infusion apnea frequency during REM sleep was equivalent to control, but apnea frequency during NREM sleep remained elevated (P = 0.03).
(P < 0.04 for each) (Figs. 5 and 6). These responses evident during both REM and NREM sleep, followed similar time courses, and were not associated with significant changes in sleep architecture, respiratory rate, or minute ventilation (Tables 1 and 2). Overall, the effects of carbachol were equivalent whether the agent was delivered to conscious animals by infusion or to anesthetized animals by microinjection. Our rationale for conducting the initial infusion experiments was two-fold. First, we wished to observe the immediate impact of carbachol on sleep and breathing. Second, we wanted to avoid the confounding impact of anesthesia on both sleep and breathing behaviors. These experiments convincingly demonstrated that in the first 6 h after infusion to the PPT, carbachol reduced expression of both BPEs (detected as P-waves) and apneas (Figs. 5 and 6) in animals that were fully recovered from surgery and well adapted to the monitoring apparatus.
Table 1 Unchanged minute ventilation after carbachol infusion to PPT
NVI-NREM NVI-REM
Saline
Control
Day 0
Day 3
Day 7
Day 14
0.91 ± 0.12 0.89 ± 0.09
0.91 ± 0.15 0.92 ± 0.06
0.94 ± 0.07 0.95 ± 0.07
0.90 ± 0.08 0.99 ± 0.06
1.06 ± 0.11 1.07 ± 0.08
0.98 ± 0.11 1.00 ± 0.08
NVI-NREM = Average (±S.E.) minute ventilation during NREM relative to control wakefulness. NVI-REM = Average (±S.E.) minute ventilation during REM relative to control wakefulness.
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Table 2 ITR Lesion does not change average respiratory pattern Control
Day 2
Day 7
Day 14
P
RR-Wake RR-NREM RR-REM P
119.0 ± 3.4 95.4 ± 2.3 103.4 ± 2.0 0.0001
107.5 ± 4.0 94.6 ± 2.9 98.7 ± 3.9 0.05
108.5 ± 5.5 95.4 ± 3.1 100.8 ± 3.3 0.10
111.9 ± 3.6 93.1 ± 1.2 98.7 ± 4.5 0.004
0.21 0.94 0.73
NRR-Wake NRR-NREM NRR-REM P
1.0 ± 0 0.80 ± 0.2 0.87 ± 0.02 0.0001
0.91 ± 0.03 0.80 ± 0.02 0.84 ± 0.04 0.09
0.91 ± 0.04 0.81 ± 0.03 0.85 ± 0.04 0.18
0.94 ± 0.03 0.79 ± 0.03 0.83 ± 0.05 0.03
0.12 0.96 0.89
NMV-Wake NMV-REM NMV-REM P
1.0 ± 0.0 0.80 ± 0.02 0.79 ± 0.03 0.0001
0.93 ± 0.05 0.79 ± 0.05 0.71 ± 0.07 0.04
0.95 ± 0.11 0.80 ± 0.09 0.77 ± 0.10 0.41
1.05 ± 0.05 0.85 ± 0.06 0.81 ± 0.08 0.03
0.63 0.94 0.82
NRR: Respiratory Rate normalized to the mean value during wakefulness of the control recording. NMV: Minute Ventilation normalized to the mean value during wakefulness of the control recording.
3. A role for ITR in the pontine respiratory network 3.1. Experiments in anesthetized rats Despite many years of study, the central mechanisms of reflex apnea remain incompletely identified. Recent evidence implicates the ITR, described in 1981 by Brodal (Brodal, 1981), as an important pontine area for regulating apneic reflexes in the rat (Chamberlin and Saper, 1998). In their neuroanatomical study Chamberlin and Saper (1998) used glutamate microinjection into the lateral pons of anesthetized rats to identify areas producing an immediate respiratory response. They identified a previously unrecognized group of cells among the fiber bundles between the motor and principal sensory trigeminal nuclei whose stimulation by glutamate produced an immediate apnea. Rostrally, apneic sites stretched up to the ventral border of Kolliker–Fuse (KF) nucleus while, caudally, apneic sites stretched ventrally along the motor trigeminal roots (Chamberlin and Saper, 1994). Using anterograde and retrograde tracers, Chamberlin and Saper (1998) established that these apneic sites in the ITR project to the ventral respiratory group in the medulla. We repeated Chamberlin and Saper’s experiment by microinjecting glutamate to ITR and obtained an instant 3 s apnea (Fig. 7, Panel A). After the animal resumed normal breathing we microinjected kynurenic acid, a glutamate receptor antagonist (Perkins and Stone, 1982), to ITR,
which was followed by a microinjection of glutamate. The result was a complete blockade of the respiratory response to glutamate injection (Fig. 7, Panel B). To determine whether the ITR has a physiological role in mediating or modulating respiratory reflexes and, whether it is functionally involved in the central mechanisms of vagal-reflex apnea, we intravenuously injected 5-HT, which does not cross the blood brain barrier, to 10 anesthetized adult Sprague–Dawley rats to produce apneic responses. This type of reflex apnea arises from stimulation of the vagus nerves and bilateral vagotomy above nodose ganglia in cats (Jacobs and Comroe, 1971; Sampson and Jaffe, 1974; Ginzel, 1975; Sutton, 1981) and rats (Yoshioka et al., 1992a,b; Yoshioka, 1995) was shown to abolish the 5-HT-induced apnea. In addition, 5-HT activates unmyelinated vagal afferents (Coleridge and Coleridge, 1997) and these also may contribute to the observed apnea promoting effect of 5-HT. Since all vagal sensory afferents terminate in the NTS, the NTS could be considered as the principal central relay station for the 5-HT induced reflex apnea. The animals used in this study had a catheter inserted into the femoral vein for administration of 5-HT (0.00375 mg) and respiration was recorded by piezoelectric crystal. Multibarrel pipettes were used to pressure inject glutamate (5–10 nl, 10 mM), kynurenic acid (10 nl, 50 mM), and red dye into the ITR, unilaterally and bilaterally. Intravenous administration of 5HT produced an immediate 3 s apnea (Fig. 8, Panel A). Unilateral glutamatergic blockade at an ITR site with
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Fig. 7. Panel A shows the characteristic apnea evoked by glutamate pressure injection in the ITR. Injection of this site with kynurenic acid applied from a separate barrel of multibarrel pipette, completely blocked the respiratory response to glutamate injection (Panel B). AU – arbitrary units.
kynurenic acid significantly prolonged the 5-HT induced reflex apnea (Fig. 8, Panel B). When kynurenic acid was simultaneously injected to the contraleteral site it further lengthened the reflex apnea (Fig. 8, Panel C). The schematic mapping of kynurenic acid injection sites is presented in Fig. 9. However, the brainstem networks involved in generating reflex apnea have not been well defined. One putative brainstem network for mediating apneic reflexes in the rat includes the NTS, ITR and the ventral medulla (VLM) (Chamberlin and Saper, 1998). In that neuroanatomical pathway of reflex apnea, information from the NTS would reach the ITR, from which it would be transmitted to VLM. Our observations provide a test of this pathway. If the ITR represents a “relay” for reflex apnea, as suggested by Chamberlin and Saper (1998), and because activation of glutamate receptors in small regions of the ITR produces immediate apnea, we initially expected that application of the glutamate receptor antagonist kynurenic acid might atten-
uate the vagally transmitted apnea in this study. Instead, we observed the opposite effect, i.e., that functional glutamatergic blockade of ITR sites by kynurenic acid potentiated vagal reflex apnea (Fig. 8, Panels B and C). We are confident that kynurenic acid microinjections were made into or very near the same location as the glutamate injections because we utilized a multibarrel pipette that was not moved between agonist and antagonist injections. In addition, by leaving the pipette in place, we observed that 1–2 h after kynurenic acid administration, glutamate-induced apnea was again observed and reflex apnea duration returned to baseline. The augmentation of 5-HT-induced apnea by kynurenic acid was demonstrated both with unilateral and bilateral injections of the acid to ITR. In both cases there was a significant increase of apnea duration following the blockade of glutamate receptors. In contrast to unilateral injections of the acid to ITR which produced augmentation of 5-HT-induced apnea of 7.5 s, the bilateral injections of kynurenic acid to
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Fig. 8. Intravenuous administration of 5-HT evoked an apnea (panel A). Unilateral glutamatergic blockade at an ITR site with kynurenic acid significantly prolonged the 5-HT induced reflex apnea (panel B). Kynurenic acid injection of a second, contralateral, site further lengthened the reflex apnea (panel C). AU – arbitrary units.
ITR lengthened the 5-HT-induced apnea upto 10 s and longer (Fig. 8). Thus, it appears from these findings that the ITR exerts a potent dampening influence on vagally-induced reflex apnea. 3.2. Experiments in freely moving rats In the rat model of central sleep apnea, characterized by us and others, (Mendelson et al., 1988; Thomas et al., 1992; Carley et al., 1996; Christon et al., 1996; Radulovacki and Carley, 2003) we have established a putative link between pathways of reflex apnea and
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spontaneous sleep-related apneas (Radulovacki et al., 2003, 2004). Intravenous administration of serotonin to anesthetized cats or rats produced dose-dependent reflex apnea (Jacobs and Comroe, 1971; Yoshioka et al., 1992a,b), whereas in freely moving rats, intraperitoneal administration of serotonin produced dramatic increases in spontaneous sleep-related apneas (Carley and Radulovacki, 1999b). These findings suggested that the central and peripheral pathways of reflex apnea may participate in genesis of sleep apneas. We further explored the potential central link between reflex and sleep apneas. We reported earlier that glutamatergic blockade of the ITR yielded dosedependent lengthening of reflex apnea evoked by intravenous serotonin injection and concluded that one physiological role for the ITR may be to attenuate vagally-induced reflex apneas (Radulovacki et al., 2003). We conducted the next series of experiments to determine whether ibotenic acid lesioning of the ITR would affect spontaneous sleep apneas in freely moving rats. For these series of experiments we implanted rats with EEG and EMG electrodes, recorded them polygraphically for 6 h and monitored their respiration by placing each animal inside a single-chamber plethysmograph. Subsequently, in the next surgical intervention, a respiratory-related ITR site was identified by probing on dorso-ventral tracks with 2–5 nl glutamate (10 nl, 10 mM) injections from a multibarrel glass pipette. This site was then lesioned by injecting ibotenic acid (10 nl, 50 mM) from a second pipette barrel. After recovery period, animals were again recorded for 6 h on days 2, 7 and 14 after the lesion. They were sacrified under anesthesia after recording day 14 and the location of their ITR lesions was histologically verified. The distribution of anatomical localization of the unilateral ibotenic acid lesion is presented in Fig. 10. As in previous investigations (Carley et al., 1996; Christon et al., 1996; Radulovacki et al., 1996, 1998; Carley and Radulovacki, 1999a, 1999b), sleep apneas, defined as cessation of respiratory effort for at least 2.5 s, were scored for each recording session and were associated with the stage in which they occurred: NREM or REM sleep. Apnea index, defined as apneas per hour in a stage, was separately determined for NREM and REM sleep. The timing and volume of each breath were scored by automatic analysis (Experimenter’s Workbench; Datawave Technologies). Sighs
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Fig. 9. Schematic mapping of kynurenic acid injection sites. The injection sites (* ) are well localized to the ITR. Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; Pr5, principal sensory trigeminal nucleus; s5, sensory root of the trigeminal nerve; scp, superior cerebellar peduncle.
were identified by tidal volume more than 150% greater than the overall mean, and post-sigh pauses greater than 2.5 s were excluded from all analysis presented below. For each animal, the mean respiratory rate and inspiratory minute ventilation was computed for wakefulness throughout the 6 h control recording and used as a baseline to normalize respiration during sleep, and following ITR lesioning. The results of the study showed that ITR lesions exerted no impact on mean respiratory pattern during any sleep/wake state, compared to baseline recordings
Fig. 10. Distribution of the anatomical localization of the unilateral ibotenic acid lesion of the ITR. The injection sites (squares and circles) are well localized to the ITR. Mo5, motor trigeminal nucleus; Pr5, principal sensory trigeminal nucleus; s5, sensory root of the trigeminal nerve.
(Table 2) In contrast, apnea frequency during NREM sleep increased following ITR lesion, more than doubling by day 14 (Fig. 11). These findings agree with the experiments in anesthetized rats where the ITR lesion by kynurenic acid lengthened the vagally-induced reflex apnea. Thus, they confirm the inhibitory influence of ITR in the genesis of reflex and sleep-related apneas and point to ITR as a part of pontine inhibitory mechanisms in the central pathways of both types of apneas.
Fig. 11. Group data (mean ± S.E.) of apnea expression during REM and NREM sleep 2, 7 and 14 days following ibotenic acid lesion of the ITR. There was a trend toward increased apnea during REM sleep. Conversely, during NREM sleep, sleep apnea index was significantly elevated on days 2 and 14 following the lesion (P = 0.01 and P = 0.02, respectively).
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4. Discussion Our studies in anesthetized and freely moving rats demonstrated a role for two pontine structures in respiration that are not classically included in the pontine respiratory neuronal network: the PPT and the ITR (Radulovacki et al., 2003, 2004; Saponjic et al., 2003). From a historical point of view, respiratory related functional roles have been assigned to several regions within the pons that, collectively, have been referred to as the pontine respiratory group (see St.-John, 1998 for a review). Within the pons, the parabrachial nuclear complex, including the K–F nucleus, contains many neurons with activity related to various phases of the respiratory cycle (Bertrand and Hugelin, 1971). On balance, available data from cat, rat and rabbit suggest that stimulation of medial parabrachial neurons produces expiratory facilitation including bradypnea or apnea, whereas lateral parabrachial stimulation produces inspiratory facilitation including tachypnea or hyperpnea (Takayama and Miura, 1993; Chamberlin and Saper, 1994; Dick and Coles, 2000). However, unlike the present responses to PPT stimulation, parabrachial stimulation by electrical current (Fung and St-John, 1994; Lara et al., 1994) or glutamate injection (Takayama and Miura, 1993; Chamberlin and Saper, 1994; Lara et al., 1994) produced immediate respiratory responses that did not persist significantly after cessation of the stimulus. It is of interest here that, the behavior of respiratoryrelated parabrachial neurons changes significantly across sleep/wake states. Well-modulated respiratory related activity is readily recorded from pontine units in the conscious cat and the firing rates of these neurons decrease with sleep onset and are lowest during REM sleep (Lydic and Orem, 1979; Sieck and Harper, 1980). A series of more recent studies from Lydic and colleagues (Gilbert and Lydic, 1990, 1994) demonstrates that pontine respiratory neurons may be directly influenced by pontine reticular areas involved with the control of REM sleep. Moreover, the above mentioned study of Lydic and Baghdoyan (1993) first showed in barbiturate anesthetized cats that PPT continuous electrical stimulation leads to increased acetylcholine release in the medial pontine reticular formation and to respiratory depression. However, the respiratory depression was mild and transient – diminishing even
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before cessation of the stimulus. The impact of glutamatergic PPT stimulation, in contrast, included longlasting respiratory disturbance characterized by both increases and decreases of respiratory frequency. Differences between the present findings and those reported previously (Lydic and Baghdoyan, 1993) may reflect differing methods and precise anatomical sites of stimulation, differing anesthetic agents, different species, or other factors. Our data in the freely moving rats demonstrate that neurochemical stimulation of the PPT significantly increases sleep-related respiratory variability – including apnea expression – but do not demonstrate the anatomical pathways for this effect. It is possible that parabrachial nuclei play a role in conveying the influence of PPT neurons to the respiratory pattern generating and premotor integrating areas of the ventrolateral medulla. Because the respiratory-related neurons of the parabrachial complex in the rat are concentrated approximately 1.5 mm caudal to our PPT target, we do not believe these regions were directly destroyed by mechanical introduction of the guide cannula or infusion needle in our experiments. Further, these regions appeared to be intact by light microscopy of the fixed brains. Still, mechanical trauma to the parabrachial complex cannot be ruled out in the animals that received infusions. In addition, we must allow the possibility that these areas, especially the lateral parabrachial region, may have been directly affected by the carbachol following infusion. Chamberlin and Saper conducted a systematic mapping of the parabrachial complex in rats using glutamate pressure injections (Chamberlin and Saper, 1994). They reported hyperpnea following stimulation of the rostral and mid-caudal K–F nucleus as well as most of the lateral parabrachial region. Also, we have reported that non-specific respiratory stimulation can reduce apnea expression in sleeping rats (Christon et al., 1996). Therefore, it is possible that carbachol, acting in the lateral parabrachial region rather than in the PPT, accounted for the reduced apnea expression during the 6 h immediately after infusion. This explanation seems unlikely, however, for two reasons. First, respiratory stimulation via the lateral parabrachial region would be expected to produce hyperpnea, but respiratory rate and minute ventilation remained unchanged from control values (Table 1). Second, P-wave expression and apnea expression followed similar time
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courses throughout the 14 days following carbachol infusion (Figs. 5 and 6). Because the PPT is directly involved in generating and/or relaying P-waves (Datta, 1995; Datta et al., 1992, 1998, 1999) it seems most likely that the actions of carbachol on PPT neurons were responsible for initiating the parallel changes in apnea expression. Perhaps the most striking feature of the P-wave and respiratory responses to cholinergic PPT stimulation was their duration. Following both injections and infusions, carbachol evoked maximal increases in P-wave and apnea expression between 3 and 7 days after administration. This is similar to the time course of increased PGO waves reported in cats after a single dose of carbachol (Datta et al., 1992). Clearly, the responses at 7 days do not reflect the direct pharmacological action of carbachol in the cat (Datta et al., 1992) or the present rat observations. We can only speculate, but it seems probable that the sustained responses observed would require changes in gene expression and protein turnover/localization. In addressing the physiological role of ITR in respiration we performed experiments both in anesthetized and freely moving rats. In anesthetized animals, we tested Chamberlin and Saper’s postulate that ITR is an important structure in the central pathways of reflex apnea (Chamberlin and Saper, 1998). Since reflex apnea may be evoked by a variety of pharmacological interventions we chose the peripheral administration of 5HT to evoke vagally-induced reflex apnea. It has been shown that the triad of bradycardia, hypotension and apnea (the Bezold-Jarisch reflex) can be evoked in anesthetized animals by peripheral administration of several substances including serotonin (5-HT) (Ginzel, 1975). Thus, Jacobs and Comroe (1971) caused reflex apnea in anesthetized cats by injecting 5-HT into the common carotid arteries which was abolished by bilateral section of supranodose vagus. In addition, Yoshioka et al. (1992a, 1992b) produced transient reflex apnea by intravenous injection of 5-HT to anesthetized rats, which again was abolished by bilateral vagotomy above the nodose ganglia. These and other studies (Sampson and Jaffe, 1974; Sutton, 1981) have concluded that the apnea component of the Bezold-Jarisch reflex results from the activation of afferent neurons with somata based in the nodose ganglia in cats and rats and that the primary central synapse of these afferent neurons is in the NTS (Vardhan et al., 1993).
Our results show that in accordance with Chamberlin and Saper’s study (1998), by microinjecting glutamate to ITR, we produced an immediate apneic response (Fig. 7, Panel A) which was prevented by prior microinjection of a broad spectrum glutamate receptor antagonist, kynurenic acid (Fig. 7, Panel B). Following glutamate antagonism, subsequent administration of 5HT produced apneas of much longer duration (Fig. 8, Panels B and C), which was an unexpected finding. As we have mentioned above, we initially expected that application of the glutamate receptor antagonist kynurenic acid might attenuate the vagally transmitted apnea. Because we were surprised by the general augmentation of reflex apnea by functional blockade of the ITR, we sought to test whether the ITR represents an obligatory synapse in the vagal reflex apnea pathway. For this purpose, we conducted acute ponto-medullary transections in 3 additional animals. Following isolation of the medulla, 5-HT-induced apnea was dramatically amplified, with apneas >30 s in duration after a standardized 5-HT injection. This demonstrates that medullary circuits are sufficient to produce vagal reflex apneas, and that the overall effect of more rostral structures is to attenuate the reflex – at least during anesthesia. The observed effects of kynurenic acid support the conclusion that one physiological effect of glutamate in ITR is to dampen vagally-induced apnea. This interpretation does not preclude other respiratory functions for ITR. For example, it may be that ITR is a key relay in apnea evoked by stimulation of the trigeminal nerve (Chamberlin and Saper, 1998). In any case, the physiologically relevant source of glutamatergic input to ITR that results in attenuation of vagal reflex apnea cannot be determined from our studies. In our study of freely moving rats we demonstrated that a small and well localized unilateral lesion of the ITR in the lateral pons can increase the frequency of central apnea during NREM sleep in freely moving rats over a 2 week period (Fig. 11) (Radulovacki et al., 2004). This finding is of interest because it points to a functional role for a pontine group of cells that have not, to date, been implicated in regulation of either sleep or sleep-related apnea. However, previous neuroanatomical studies of the ITR, by documenting a connection of the region both to the NTS and to the ventral medulla, have established a potential neural substrate for this effect (Chamberlin and Saper, 1998, 2003).
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The results of the above experiments are consistent with our previous finding that glutamatergic blockade of the ITR exacerbated vagal reflex apnea (Radulovacki et al., 2004). Thus, impairing the functional integrity of the ITR augmented reflex apnea in anesthetized rats and sleep-related spontaneous apnea in conscious rats. We cannot determine from these data the precise mechanism by which unilateral ITR lesion increased NREM apnea genesis, but our earlier findings (Radulovacki et al., 2004) support the hypothesis that glutamatergic neurotransmission in the ITR may be important. It is unlikely that increased sleep-related apneas resulted indirectly from non-specific alterations in either respiratory drive or sleep architecture. Both respiratory pattern (Table 2) and sleep/wake architecture were equivalent to control recordings 14 days after ITR lesioning, yet apnea frequency was maximal at this same time point (achieving statistical significance only during NREM sleep) (Fig. 11). This implies that the ITR exerts a specific role in dampening respiratory perturbations rather than impacting overall respiratory drive. These observations are in accordance with our previously reported data on the role of ITR in attenuation of reflex apneas. Taken together, the data suggest that both reflex and sleep apneas share some common central pathways. They are also in agreement with the general modulatory role of pontine structures in activities including respiration, heart rate and regulation of blood pressure. In summary, consistent evidence is emerging that two regions in the lateral pons, PPT and ITR, have previously unsuspected role in regulating respiration and respiratory variability. These effects can be demonstrated both in acute and in chronic experiments in rats. Stimulation/lesion of either region affects the brainstem respiratory pattern generator and emergent pattern disturbances. Evidence to date provides strong motivation for further investigations into synaptic, cellular and molecular mechanisms by which the PPT and the ITR influence breathing.
Acknowledgement This work was supported by NIH grant AG16303.
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