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Inhibition of Trigeminal Respiratory Activity by SucklingThe First Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871 Japan Correspondence: * corresponding author, koizumi{at}dent.osaka-u.ac.jp
The trigeminal motor system is involved in many rhythmic oral-motor behaviors, such as suckling, mastication, swallowing, and breathing. Despite the obvious importance of functional coordination among these rhythmic activities, the system is not well-understood. In the present study, we examined the hypothesis that an interaction between suckling and breathing exists in the brainstem, by studying the respiratory activity in trigeminal motoneurons (TMNs) during fictive suckling using a neonatal rat in vitro brainstem preparation. The results showed that fictive suckling, which was neurochemically induced by bath application of N-methyl-D,L-aspartate and bicuculline-methiodide, or by local micro-injection of the same drugs to the trigeminal motor nucleus, inhibited the inspiratory activities in both respiration TMNs and respiratory rhythm-generating neurons. Under patch-clamp recording, fictive suckling caused membrane potential hyperpolarization of respiration TMNs. We conclude that the brainstem preparation contains an inhibitory circuit for respiratory activity in the trigeminal motor system via the rhythm-generating network for suckling. Abbreviations: BIC, bicuculline methiodide; GABA, gamma aminobutyric acid; NMA, N-methyl-D,L-aspartate; NMDA, N-methyl-D-aspartate; and TMN, trigeminal motoneuron.
Key Words: coordination • rhythm • jaw movement • neonatal rat • in vitro
Rhythmic jaw movement, which is controlled by the trigeminal motor system in the brainstem, is essential for various oral-motor behaviors, such as suckling, mastication, swallowing, and breathing. These complex rhythmic behaviors are regulated by the activation of different rhythm-generating neurons (central pattern generators) in the central nervous system (Goldberg and Chandler, 1990; Smith et al., 1991; Lund et al., 1998; Nakamura et al., 1999; Jean, 2001). The production of these rhythmic movements requires functionally coordinated activities in each rhythm-generating system. It is known that breathing is strongly inhibited during swallowing (McFarland and Lund, 1993), and that the rhythm-generating networks for swallowing and breathing interact in the brainstem (Dick et al., 1993). However, little is known about the brainstem interaction between suckling or mastication and breathing, and there is no general agreement regarding the modulation between these rhythmic behaviors. This controversy is reflected in the literature. One study in humans (Fontana et al., 1992) concluded that breathing was enhanced by mastication, because the frequency of respiratory rhythm increased during chewing. In contrast, other studies in humans (Smith et al., 1989) and rabbits (McFarland and Lund, 1993) showed either that respiratory frequency decreased during mastication, or that respiration stopped completely during mastication. Meanwhile, further studies showed that respiratory frequency increased during chewing, then dropped below baseline as soon as mastication ended (McFarland and Lund, 1995), and that respiration was profoundly perturbed by sucking in infants (Timms et al., 1993). These variable results are probably due to the complicated situation of behaviors in animals or humans. The rhythm of respiration could be affected by several peripheral sensory inputs, such as signals related to hypoxia or hypercapnia and increased upper airway resistance, as well as the central generating network for suckling. These complex modulations make it difficult to analyze the basic control of breathing during suckling in the brainstem. In the present study, we hypothesized that the brainstem contains an inhibitory circuit for breathing by suckling, to prevent aspiration of food into the airway. We investigated the trigeminal respiratory activities during fictive suckling induced by bath application of N-methyl-D,L-aspartate and bicuculline methiodide, using a functionally active in vitro brainstem preparation. This preparation retains a neural circuit adequate for generating both respiration and suckling activity in the trigeminal motor system, and enabled us to analyze the fundamental brainstem modulation without the effects of sensory inputs (Koizumi et al., 1999, 2002).
In vitro Brainstem Preparation Experiments were performed on 0- to three-day-old neonatal Sprague-Dawley rats deeply anesthetized with halothane (Japan SLC Inc., Hamamatsu, Japan). The brainstem preparation was carefully isolated from the rostral pons to the spino-medullary junction, as previously described (Koizumi et al., 2002) (Fig. 1A  ). This preparation captures both the respiratory motor circuit, which involves rhythm-generating neurons in the pre-Bötzinger Complex (the central pattern generator for respiration) (Smith et al., 1991) and active populations of hypoglossal motoneurons, and the suckling motor circuit, which involves rhythm-generating neurons and active populations of trigeminal motoneurons. The preparation was pinned down with the rostral surface upward on Sylgard resin in a recording chamber mounted on the fixed stage of an upright microscope (Eclipse E600-FN: Nikon, Osaka, Japan).
The trigeminal motor nucleus was visually distinguished in the rostral surface as an oval grayish region. The preparation was continuously superfused (flow rate: 5 mL/sec). The control solution contained (in mmol/L) 124 NaCl, 3.0 KCl, 1.5 CaCl2, 1.0 MgSO4, 25 NaHCO3, 0.5 NaH2PO4 and 30 D-glucose, and was equilibrated with 95% O2-5% CO2 at 27°C, pH = 7.4.
Electrophysiological Recordings from Trigeminal Motoneurons
Neurochemical Pharmacology Statistical values were presented as the mean ± SD. Statistical significance was evaluated with a paired t test, and values of p < 0.05 were considered to indicate statistical significance. All animal use procedures were in accordance with the NIH Guidelines for the Care and Use of Laboratory Animals, and were approved by the Osaka University Intramural Animal Care and Use Committee.
Respiratory Activities in Trigeminal Motoneurons The in vitro brainstem preparations were viable for over 8 hrs (n = 32/32). The rhythmic activity in hypoglossal nerves is considered to represent inspiratory activity in respiration (Smith et al., 1991). The respiratory rhythm was stable throughout the experiments. In the majority (n = 30/32) of preparations, trigeminal nerves exhibited spontaneous rhythmic activities (0.06–0.11 Hz) that were synchronous with inspiratory activities in hypoglossal nerves (Fig. 1C  ). Extracellular single-cell recordings from trigeminal motoneurons and pre-Bötzinger Complex neurons exhibited rhythmic spikes synchronized with inspiratory hypoglossal activities. One-quarter of the patch-clamp-examined trigeminal motoneurons (n = 6/24), which we classified as respiration-trigeminal motoneurons (respiration TMNs), showed inspiratory rhythmic drive potential with action potentials at the resting membrane potential (–64 ± 4 mV) under current-clamp conditions (Fig. 1D), and rhythmic synaptic inward currents (–216 ± 62 pA) under voltage-clamp conditions (data not shown).
Inspiratory Activities in Respiration TMNs during Fictive Suckling
Membrane Potential Trajectories in Respiration TMNs and Suckling TMNs during Fictive Suckling We studied respiration TMNs (n = 6) that showed spontaneous rhythmic bursts synchronized with inspiratory hypoglossal activities, and suckling-trigeminal motoneurons (suckling TMNs) (n = 6) that had no respiratory activities, but exhibited rhythmic (5–6 Hz) activities after bath application of NMA and BIC. The neuronal membrane parameters (resting membrane potentials, action potential amplitude, input resistance, and rheobase current) did not differ significantly between respiration TMNs and suckling TMNs. With the exception of input resistance, these parameters were remarkably similar to previous data obtained by intracellular recordings in brainstem slices (Chandler et al., 1994; Kolta, 1997). Bath application of NMA (20 µmol/L) and BIC (10 µmol/L) induced fictive suckling in trigeminal nerves, and caused gradual hyperpolarization (5.8 ± 2.2 mV) of the membrane potentials in respiration TMNs (n = 5/6). The respiratory rhythm also gradually decreased and then stopped (Fig. 3A ). In contrast, the membrane potentials of suckling TMNs (n = 6/6) were gradually depolarized by 6.4 ± 2.6 mV during bath application of NMA and BIC (Fig. 3B).
Effect of Local Micro-injection of NMA and BIC on Respiration TMNs Local micro-injection of NMA (200 µmol/L) and BIC (100 µmol/L) into the trigeminal motor nucleus (Fig. 4A ) augmented the trigeminal nerve discharges (n = 9/10), which were considered to indicate fictive suckling. Inspiratory discharges in respiration TMNs were significantly inhibited (n = 6/10) or completely abolished (n = 2/10). However, inspiratory hypoglossal activities (n = 10/10) were less inhibited than respiration TMN activities. The decreases in amplitude and duration of inspiratory hypoglossal discharges and slowing of the respiratory rhythm were not significant. In contrast, local micro-injection of NMA and BIC into the pre-Bötzinger Complex (Fig. 4B) augmented the inspiratory activities in respiration TMNs (n = 9/10), as well as in trigeminal (n = 9/10) and hypoglossal (n = 10/10) nerves. The amplitude and duration of the inspiratory discharges in all trigeminal motoneurons, trigeminal nerves, and hypoglossal nerves were significantly increased. The frequency of the respiratory rhythm was also significantly increased. Local micro-injection of a control solution into either the trigeminal nucleus (n = 3) or pre-Bötzinger Complex (n = 3) had no effect on the inspiratory activities in respiration TMNs, trigeminal nerves, or hypoglossal nerves.
Many studies have reported strong links between breathing and swallowing or between breathing and locomotion (Bramble and Carrier, 1983; Viala, 1986; Dick et al., 1993; McFarland and Lund, 1995). However, the interaction between suckling and breathing remains unclear. Suckling and breathing occur concurrently, and many of the muscles in the jaws, face, tongue, and upper airway are active in these behaviors. Therefore, functional coordination should be necessary between suckling and breathing. In the present study, using a brainstem preparation, we demonstrated that excitation of a rhythm-generating network for fictive suckling caused inhibition of a rhythm-generating network for respiration and a respiratory network in the trigeminal motor system. The inhibition of respiratory rhythm is required during suckling, and seems to be similar to the inhibition of breathing during swallowing. Previous studies have revealed that rhythmogenesis for rhythmic oral-motor behaviors originates from central pattern-generating circuits within the pons (Goldberg and Chandler, 1990; Lund et al., 1998; Nakamura et al., 1999; Tsuboi et al., 2003; Athanassiadis et al., 2005; Brocard et al., 2006), and that bath application of an NMDA receptor agonist combined with a GABAA receptor antagonist induces fast rhythmic activities that may represent fictive suckling or fictive mastication in the trigeminal motor system in brainstem preparations (Kogo et al., 1996; Koizumi et al., 2002). In the present report, we used the term "fictive suckling" for these rhythmic trigeminal activities induced by NMA and BIC, although it is difficult to verify that these activities are indeed suckling. The present results for fictive suckling could be applicable to mastication, since suckling and mastication are ontogenically continuous behaviors (Westneat and Hall, 1992), and the accompanying masticatory motor pattern is clearly derived from the suckling motor pattern (Langenbach et al., 1992). The trigeminal motoneurons could be classified into two subtypes, namely, jaw-opening TMNs and jaw-closing TMNs. Most of the respiration TMNs were found in the ventral part of the trigeminal motor nucleus. This observation suggests that respiration TMNs are jaw-opening TMNs, since several studies (e.g., Mizuno et al., 1975) on the topography of trigeminal motoneurons within the trigeminal motor nucleus have demonstrated that jaw-opening TMNs are mainly located in the ventromedial part, while jaw-closing TMNs are mainly located in the dorsolateral part. This finding is consistent with the observation that digastric jaw-opening muscles showed inspiratory activity during loaded breathing in dogs, since the oral airway should be open during breathing (Bartlett, 1986). Bath application of NMA and BIC to the pons inhibited respiratory activities in respiratory TMNs, along with membrane potential hyperpolarization, and also inhibited respiratory activities in pre-Bötzinger Complex neurons and hypoglossal nerves. Local injection of NMA and BIC into the trigeminal motor nucleus caused strong inhibition of respiratory activities in respiratory TMNs, and slight inhibition of hypoglossal respiratory activities. These injections caused excitation of rhythm-generating networks for suckling that were located just around the trigeminal motor nucleus (Kolta, 1997; Enomoto et al., 2002), without affecting more distant regions, such as the Kolliker-Fuse and parabrachial area, since the diffusion of drugs administered by local injection in < 10 nL at very low pressure (10–15 psi) would be less than 130 µm (Tehovnik and Sommer, 1997; Scott et al., 2003). These results suggest that activation of rhythm-generating neurons for suckling entirely inhibits rhythm-generating networks for respiration in the pre-Bötzinger Complex, and that the rhythm-generating neurons for suckling have additional inhibitory connections with respiration TMNs at the trigeminal motor nucleus level. The finding that bath application of NMA and BIC hyperpolarized the membrane potentials in respiration TMNs supports this conclusion. Local injection of NMA and BIC into the pre-Bötzinger Complex accelerated the respiratory activities in respiration TMNs and trigeminal nerves, as well as in hypoglossal nerves. These results provide the new insight that respiratory activity in the trigeminal motor system is regulated by excitatory signals arising from rhythm-generating networks in the pre-Bötzinger Complex, while trigeminal respiratory activity has not been studied extensively. In contrast, we previously reported that the pre-Bötzinger Complex sends inhibitory signals to trigeminal motoneurons via serotonergic and noradrenergic receptors (Kogo et al., 2000; Mori et al., 2002). These findings suggest that the pre-Bötzinger Complex differentially controls the trigeminal motor system by excitation of respiratory TMNs and inhibition of suckling TMNs or mastication TMNs.
This study was supported by Research Grants from the Ministry of Education and Science of Japan (No. 18592175 to HK and No. 17390535 to MK). Received for publication December 27, 2006. Revision received May 19, 2007. Accepted for publication July 2, 2007.
Journal of Dental Research, Vol. 86, No. 11,
1073-1077 (2007) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
, were filled with a solution containing 0.5 mol/L sodium acetate (Sigma, Tokyo, Japan) and 2% pontamine sky blue (TCI, Tokyo, Japan) for subsequent histological identification of the recorded cells. Signals were amplified (50,000–100,000 times) and band-pass-filtered (0.3–3 kHz) with a differential amplifier (CyberAmp 380: Axon Instruments Inc., Sunnyvale, CA, USA). Whole-cell recordings from trigeminal motoneurons were obtained by visualized patch-clamp techniques for surface cells (30–60 µm deep), as well as by blind patch-clamp techniques for deeper cells (200–300 µm deep) (
