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Changes in reflex responses of the genioglossus muscle during sleep in rabbits
Brain Research 1065 (2005) 79 – 85 www.elsevier.com/locate/brainres
Research Report
Changes in reflex responses of the genioglossus muscle during sleep in rabbits Yohji Harasawa a, Makoto Inoue a,b,*, Sajjiv Ariyasinghe c, Kensuke Yamamura a, Yoshiaki Yamada a b
a Division of Oral Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan Division of Dysphagia Rehabilitation, Department of Oral Biological Sciences, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan c Department of Basic Sciences, Faculty of Dental Sciences, University of Peradeniya, Peradeniya 20400, Sri Lanka
Accepted 11 October 2005 Available online 28 November 2005
Abstract Changes in reflex responses in the genioglossus (GG) muscle evoked by electrical stimulation of the inferior alveolar nerve and GG muscle tone (background activity, BGA) were investigated during sleep – wakefulness stages in rabbits. The GG muscle showed two types of electromyographic activity patterns: a respiration-related phasic activity and non-respiration-related activity. GG reflex responses and BGA exhibited a stage-dependent decrease as they were constantly suppressed from quiet wakefulness to non-rapid eye movement sleep (NREMS) to rapid eye movement sleep (REMS). Degree of suppression of reflexes was much larger than that of BGA regardless of GG activity patterns. When amplitude of reflex responses was compared between with and without rapid eye movements during REMS, no difference between the conditions was noted. These results suggest that excitability of the GG muscle is affected by sleep stages by not only a modulation of excitability in motoneurons but also in interneurons involved in the reflex arc. D 2005 Elsevier B.V. All rights reserved. Theme: Motor systems and sensorimotor integration Topic: Reflex function Keywords: Sleep; Tongue; Reflex; Genioglossus muscle; Rabbit
1. Introduction The tongue is a highly mobile and unique composite of various muscles that plays a role in functions such as mastication, deglutition, respiration and vocalization [25]. One important role of tongue muscles is to maintain patency of the extrathoracic airways during tidal respiration [38]. It should be noted that tongue muscle activities are suppressed in parallel to atonia of postural muscles during * Corresponding author. Division of Dysphagia Rehabilitation, Department of Oral Biological Sciences, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan. Fax: +81 25 227 2998. E-mail address: [email protected] (M. Inoue). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.013
sleep, particularly during rapid eye movement sleep (REMS) [23]. REMS is quite unique because one of its hallmarks involves desynchronization of the cortical encephalogram similarly to that in wakefulness. Furthermore, strong suppression of postural muscle tone intermittently generated a flurry of activities, manifested by rapid eye movements or muscle twitches [23]. Intracellular recordings of spinal motoneurons in cats also exhibited drastic fluctuations of membrane potentials superimposed on a strong suppression during REMS [8]. Thus, during REMS, excitatory and inhibitory phasic events are superimposed on a tonic and strong muscle suppression. Recently, a number of clinical studies have focused on the pathogenesis of obstructive sleep apnea which may
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involve a combination of reduced upper airway size and loss of tone in upper airway muscles including tongue muscles during sleep [26,28,30,31]. However, the extent to which each muscle contributes to airway patency, and the mechanisms by which this patency is achieved remain uncertain. Difficulties in clarifying the dynamic interplay between combinations of brain stem neural networks that provide respiratory rhythm generation and modulation of the hypoglossal motoneuron discharge may be due to (1) the complexity of anatomical organization of tongue muscles and to (2) the variety of functions of tongue muscles as muscles function not only as respiration-related muscles but also as mastication/swallowing-related or speech/vocalization-related muscles in humans. In a previous study, we reported that there were significant differences in changes of excitability between jaw closer and opener muscles during sleep [14]. Indeed, the jaw closing reflex was inhibited but was occasionally facilitated in association with rapid eye movements while the jaw opening reflex remained inhibited. On the other hand, it is known that the tongue reflex can be evoked by the same stimulus modality as the jaw opening reflex [1,32,40]. Interestingly, there was no difference in changes of responses between the jaw opening and tongue reflexes evoked by the same stimulus modality during swallowing but chewing. The aim of the present study was to investigate how the tongue reflex which can be evoked by the same stimulus modality as the jaw opening reflex is modulated during wakefulness and sleep in freely behaving rabbits.
2. Materials and methods 2.1. Animals Experiments were carried out on 11 adult male Japanese white rabbits weighing between 2.5 and 3.5 kg, in accordance with the Guides for the Care and Use of Laboratory Animal Care (NIH Publication #86-23, revised 1996). The experimental procedure was reviewed and approved by the Niigata University Intramural Animal Care and Use Committee. Animals were initially habituated to the experimental environment. They were accommodated in cages (60 80 122 cm) before surgical intervention. During this period, animals were trained to sit in a small wooden box (40 18 18 cm), where the recording session would be performed. To accustom animals to this circumstance, training sessions were performed for about 1 h per day and were terminated whenever the animal showed any discomfort. 2.2. Surgical procedure The surgical procedure has been extensively reported in previous studies [1,14,16]. Animals were anesthetized with sodium pentobarbital (30 – 35 mg/kg body weight) admin-
istered intravenously through the marginal ear vein. Sodium pentobarbital was further supplemented to maintain anesthesia at a level at which neither an apparent corneal reflex nor spontaneous eye movements occurred. To monitor the sleep – wakefulness stage, bipolar electrodes composed of brass screws were implanted through the frontal bone (5 mm anterior to the bregma, and 2 mm right and left to the midline) for electroencephalographic (EEG) activities, and anterior to the eye socket (10 mm interval between the screws) for electrooculographic (EOG) activities. Pairs of Teflon-coated stainless steel wire electrodes of 0.25-mm diameter and 2 mm long with bared tips for electromyographic (EMG) recordings were inserted into the right clavotrapezius muscle (Trap) with 10 mm of interpolar distance. To record EMG activity of the tongue protractor muscle, a pair of Teflon-coated stainless steel wire electrodes of 0.25-mm diameter was inserted into the genioglossus (GG) muscle with 10 mm of interpolar distance on the left side. To eliminate electrical contamination of the surrounding muscle activity, the digastric nerve was cut on the same side. Tongue reflex was evoked in the GG muscle (GG reflex) by electrical stimulation of the inferior alveolar nerve (IAN) [1]. To access the IAN, the lower edge of the mandibular bone between the anterior margin of the masseter muscle and lateral margin of digastric muscle was exposed, and two small holes (3 mm apart from each other) were drilled. A small socket having a pair of brass electrodes of 1-mm diameter and 2 mm long were connected on the bone, and the tip of electrodes was placed into the mandibular canal alongside the IAN. The socket was then fixed with dental acrylic. The IAN was electrically stimulated (3 train pulses; 0.1 ms in duration; 0.5-ms pulse interval) to evoke the GG reflex at 1 Hz. Current intensity of stimulus was set at 1.5 times the threshold to evoke the GG reflex to evaluate the modulatory mode of reflex responses [1]. Signals from the electrodes were fed subcutaneously to attach the connector fixed on the parietal bone and were conveyed via detachable and flexible cables. 2.3. Data recordings Recordings started after a post-surgical recovery period of at least 1 week. They were carried out for 2 h in the afternoon per day, while the animal was in the wooden box. A recording sequence was divided into four sleep – wakefulness state stages, which were active wakefulness (AW), quiet wakefulness (QW), NREMS and REMS according to criteria determined by our previous study [14]. Stimuli were delivered at through cables, so that the recording and stimulating systems allowed the animal to freely move at least its head and neck. Signals from electrodes amplified with a laboratory-made amplifier (band pass 0.1 –3 kHz for EMGs, and 1 –100 Hz for EEGs and EOGs) were stored on a data recorder (LX-10, TEAC, Japan) and a computer at a sampling rate of 5 kHz. Data
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analysis was performed using the Spike2 analysis package (Cambridge Electronic Design, Cambridge, UK). 2.4. Data analysis In the present study, since our focus was to evaluate GG activity during sleep, recordings during AW were omitted from the analyses. Mean values of peak to peak amplitude of GG reflex responses were obtained for each sleep – wakefulness stage during recording sessions. Values were normalized against those recorded during QW. At the beginning and end of stages, reflex amplitude often fluctuated. Therefore, we did not analyze reflex responses in the first and last 20 consecutive recordings in each stage. Since it is conceivable that GG reflex responses are influenced by background activity (BGA) of the GG muscle, they were also compared among sleep –wakefulness stages. BGAs were obtained as follows: tonic EMGs recorded were selected and rectified for 300 ms, and the mean value was obtained as the BGA for each sleep – wakefulness stage. Data were compared using a non-parametric statistical test (A Kruskal –Wallis ANOVA on Ranks and the Tukey’s HSD as a post hoc test or Mann –Whitney test). Values were expressed as means T SEM, and statistical difference was determined at P < 0.05 (significant) and P < 0.01 (highly significant) level.
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head was held erected, otherwise, it was eating food or sleeping. Recording started when the animal was at rest (i.e., QW), which was normally followed by sleep, and was stopped when the animal woke up. During QW, desynchronized EEG activity was observed. Otherwise, no particular behavioral signs were noted during this stage. During NREMS, cortical spindles appeared in EEG activity, and tonic activity of Trap muscle was maintained (Fig. 1). REMS was characterized by desynchronized EEG activity similar to that in QW and temporal occurrence of REMS. Visual observation of the animal during REMS agreed with previous reports in that eyes were closed and ears were depressed. Sustained EMG activity of Trap muscle was sometimes noted (Fig. 1). Arousal could be behaviorally identified as a sharp and sudden increase in Trap muscle tone and a desynchronized EEG activity. AW was easily noted because of the animal’s active behavior. Mean recording durations were 17 min 53 s T 13 min 6 s for QW (n = 11), 43 min 34 s T 22 min 6 s for NREMS (n = 11) and 50 s T 57 s for REMS (n = 9). Once the animal fell asleep, it always started with NREMS which was often followed by REMS. Although frequency of occurrence and duration of sleep – wakefulness stages varied from animal to animal and/or time by time in each animal, the sequence QW-NREMS-REMS-QW or QW-NREMS-QW was commonly observed.
3. Results
3.2. Changes in GG reflex response during sleep
3.1. Animal’s behavior
An example of GG reflex response during each sleep – wakefulness stage is shown on the top of traces in Fig. 1. Mean latency was 5.1 T 0.2 ms (n = 11) and was obtained during QW. The mean latency was constant throughout the
When the animal was awake during the recording session, it commonly adopted a crouched position with its
Fig. 1. Typical recordings of GG reflex responses during QW, NREMS and REMS. Continuous recordings of EEG, EOG, Trap and Dia EMGs and GG EMG are shown. ECG activities contaminate Dia EMG, and stimulus artifacts contaminate GG EMG. On top of the traces, typical responses of the GG reflex are shown during each sleep – wakefulness stage. Stimulus pulse (4) triggered a display at a faster sweep speed and greater magnification. On the bottom of traces, reflex amplitude changes are also shown. Note that GG reflex responses exhibit a great stage-dependent decrease constantly from QW to NREMS to REMS. Dia, diaphragm; GG, genioglossus muscle; NREMS, non-REM sleep; QW, quiet wakefulness; REMS, REM sleep; Trap, clavotrapezius muscle.
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sleep – wakefulness stages. As shown in our previous study [1], animals never showed discomfort, while 1.5-T stimuli to elicit the GG reflex were delivered during the recording session. During QW, GG reflex amplitude fluctuated. Mean amplitude obtained during QW was 171 T 35 AV (n = 11), ranging from 55 to 435 AV, and the range was the largest of all the three stages. From QW to NREMS, there was a decrease in GG reflex amplitude which paralleled the occurrence of spindle waves in EEG. In this stage, GG reflex amplitude continued to fluctuate. Mean amplitude obtained during NREMS was 131 T 28 AV (n = 11), ranging from 29 to 339 AV. Reduction in amplitude was obviously noted throughout NREMS compared to QW. The most striking change was noted at the transition from NREMS to REMS, in that GG reflex amplitude was drastically reduced. Mean amplitude obtained during REMS was 40 T 8 AV (n = 11), ranging from 0 to 91 AV. Reflex responses were completely suppressed during this stage in R2 and R6. Since amplitude of GG reflex varied with animals, all values were normalized against those obtained during QW to compare among conditions (Fig. 2). GG reflex responses exhibited a constant stage-dependent change from QW to NREMS to REMS. Mean value during REMS (0.36 T 0.06, n = 9) was significantly lower than that during QW (1.00 T 0.00, n = 11) or NREMS (0.76 T 0.04, n = 11) ( P < 0.01), and that during NREMS was significantly lower than that during QW ( P < 0.01). In the case of three animals (R3, R7, R8)
Fig. 3. Regression analysis between the GG reflex amplitude and BGA. Closed and open circles represent the relationship between the GG reflex amplitude and BGA during NREMS and REMS, respectively. GG, genioglossus muscle; BGA, background activity of genioglossus muscle; NREMS, non-REM sleep; REMS, REM sleep.
which showed respiration-related activity of the GG muscle, mean values were 0.88, 0.76 and 0.94 for NREMS and 0.32, 0.27 and 0.41 for REMS. Thus, strong inhibition was observed regardless of activity patterns of the GG muscle. 3.3. BGA BGAs in the three stages were compared using normalized values to those during QW (Fig. 2). Mean value was significantly ( P < 0.05) lower during REMS (0.78 T 0.05, n = 11) than QW, otherwise there was no difference between the stages. A disparity of suppression seemed to exist between amplitudes in BGA and GG reflex. BGAs were moderately suppressed during sleep while strong suppression of GG reflex response was noted particularly during REMS. Furthermore, GG reflex amplitude proportionally changed with BGA during NREMS, but this was not the case during REMS (Fig. 3). 3.4. Effects of REM occurrence on GG activity
Fig. 2. Comparisons between normalized GG reflex amplitudes and BGA during each sleep – wakefulness stage. Mean reflex amplitude during REMS (0.36 T 0.06, n = 9) is significantly lower than that during QW or NREMS while that during NREMS (0.76 T 0.04, n = 11) is significantly ( P < 0.05) lower than that during QW ( P < 0.01). Mean BGA amplitude is significantly lower during REMS (0.78 T 0.05, n = 11) than QW, otherwise there is no difference in BGA between stages. **P < 0.01, *P < 0.05 by comparing among sleep wakefulness stages, .. < 0.01, . < 0.05 by comparing between GG reflex and BGA. BGA, background activity of genioglossus muscle; GG, genioglossus muscle; NREMS, non-REM sleep; QW, quiet wakefulness; REMS, REM sleep.
We have previously showed that the masseteric (jaw closing) monosynaptic reflex was sometimes facilitated in conjunction with occurrence of REMs during REMS while jaw opening reflex remained inhibited regardless of occurrence of REMs [15]. In the present study, relationships between occurrence of REMs and possible fluctuation in suppression of GG reflex were examined. There was no significant difference in the mean normalized value of peak to peak amplitude of the GG reflex between the REM period (with REM, 0.26 T 0.07, n = 9) and non-REM period of REMS (without REM, 0.37 T 0.06, n = 9), correlation variation tended to be higher during the REM period of REMS than non-REM period of REMS (0.27 for with REM vs. 0.16 for without REM).
4. Discussion In the present study, changes in amplitude of the GG reflex were investigated to evaluate changes in excitability
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of hypoglossal motoneurons and neuronal network involved in GG reflex. Decrease in GG muscle tone (observed via BGA) was noted during the transition between wakefulness and sleep, as well as a large decrease in GG reflex amplitude, especially during REMS (78% of control for BGA vs. 36% of control for the GG reflex), suggesting that not only suppression of hypoglossal motoneuron excitability occurred but also that of interneurons responsible for the GG reflex arc must have decreased during REMS. 4.1. GG muscle tone during sleep The present study showed that tonic activity of the GG muscle (observed as BGA) was reduced during the transition from QW, NREMS to REMS. Changes in activity patterns are by no means unique to the GG muscle but not to other muscles or motoneurons [7,9,12,29,34]. It has been suggested that sleep – wakefulness-dependent modulation of serotonergic inputs to hypoglossus motoneurons is involved in changing GG muscle activity [21 – 23]. Medullary raphe neurons provide tonic serotonergic inputs to hypoglossus motoneurons [39] and are known to exhibit discharges which decrease rates from wakefulness to sleep and, in particular, cease during REMS [18]. Furthermore, serotonergic inputs are excitatory inputs to hypoglossus motoneurons [3,19]. Thus, raphe neurons may be responsible for disfacilitation of excitability of hypoglossus motoneurons during sleep. On the other hand, the hypoglossus motor nucleus has also been suggested to heavily receive inputs from noradrenergic neurons [33]. The locus coeruleus complex contains noradrenergic neurons and also exhibits sleep –wakefulness-dependent modulation of discharges like serotonergic neurons [2]. Noradrenergic inputs to hypoglossus motoneurons may also affect excitability of GG muscle tone although we do not have any anatomical evidence of direct inputs. It was proposed that the neurotransmitter mediating the IPSP activity of the trigeminal or spinal motoneurons during REMS is glycine or glycinergic substances but not GABA [10,11,42]. However, this does not seem to be the case in sleep-dependent excitability of hypoglossus motoneurons. Suppression of activity of hypoglossus motoneurons during REMS was not blocked by antagonists of glycine or GABA which are typical inhibitory neurotransmitters [20]. Although hypoglossus motoneurons could be post-synaptically inhibited during REMS [41], it may be conceivable that postsynaptic inhibition of the tongue muscle mediated by glycine or GABA during sleep is somehow minimal. 4.2. Changes in GG reflex responses during sleep Despite that there were a few differences in patterns of the GG muscle activity among animals with regard to respiratory-related activity, GG reflex responses in all animals were strongly suppressed during REMS. Strong suppression of jaw and spinal reflexes [7,14] and a paucity
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of tonic inhibitory influences acting upon motoneurons during REMS [6,9] have been reported. In the present study, suppression of GG reflex amplitude during REMS was much larger than that of BGA. In this regard, the two basic mechanisms that were associated with this suppression of reflex responses during sleep may involve (1) direct disfacilitation/inhibition of motoneurons as mentioned above and (2) suppression of the reflex pathway. In case of moderate inhibition of reflex responses during NREMS, it is likely that the former may be directly involved because degree of inhibition of BGA was almost proportional to that of reflex amplitude. During REMS, inhibitory effects on GG reflex responses were much larger than those on BGAs. Cairns et al. [4] investigated extracellular responses of interneurons to lowintensity stimulation of the tooth pulp and IAN in the rostral trigeminal sensory nuclear complex during wakefulness and sleep in the cat. The authors showed that IAN-evoked orthodromic field potentials or numbers of action potentials of neurons did not differ between wakefulness and NREMS while they were strongly suppressed during REMS. Similar findings were observed in field potentials through lumbar ascending sensory pathways during REMS [36]. Presynaptic inhibition of peripheral inputs from primary afferents of the trigeminal nerve was also observed during sleep [5]. Since some of these afferents may be involved in the reflex pathway for jaw opening and/or GG reflexes [17,37], suppression of the reflex pathway may also be involved in modulation of excitability of central primary endings or secondary neurons. Reflex responses in both digastric (jaw opening) and GG muscles can be evoked disynaptically by electrical stimulation of the low-threshold IAN, which contain Ah fibers in the trigeminal nerve [1,27]. Although both jaw opening and hypoglossus motoneurons showed rhythmical depolarizing potentials which were superimposed by a burst of spikes and no hyperpolarizing potentials between successive depolarizing potentials during mastication [24,29,35], modulatory patterns of those reflexes were reported to be different between each other during mastication [1,13,27]. Considering all these findings, the different normalized amplitude of the reflex as seen in mean normalized values that were 0.43 for the jaw opening reflex [14] and 0.36 for the GG reflex in the present study may be due to differences in excitability of motoneurons or inhibitory inputs to reflex pathways during REMS. 4.3. Functional implications of modulations The functional role of modulations in the GG reflex may be more complex than that in the jaw opening reflex, since the tongue consists of many muscles and neural circuits underlying the reflex are still unknown together with the fact that the tongue plays a critical role in not only respiration but also in orofacial function as we mentioned in the Introduction. It is now noteworthy that BGA was not strongly suppressed during sleep as compared to the GG reflex. These
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results suggest that the neural network involved in hypoglossus motoneuron excitability is minimally maintained to allow patency of the extrathoracic airways, but peripheral inputs should be diminished during sleep in order to avoid unnecessary stimulus which might awake animals.
Acknowledgments The authors would like to thank Mr. Hidetoshi Hirano for his technical support. This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan (#17591934 to M.I., #14207077 to Y.Y. and #116659529 to Y.Y.).
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Research Report
Changes in reflex responses of the genioglossus muscle during sleep in rabbits Yohji Harasawa a, Makoto Inoue a,b,*, Sajjiv Ariyasinghe c, Kensuke Yamamura a, Yoshiaki Yamada a b
a Division of Oral Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan Division of Dysphagia Rehabilitation, Department of Oral Biological Sciences, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan c Department of Basic Sciences, Faculty of Dental Sciences, University of Peradeniya, Peradeniya 20400, Sri Lanka
Accepted 11 October 2005 Available online 28 November 2005
Abstract Changes in reflex responses in the genioglossus (GG) muscle evoked by electrical stimulation of the inferior alveolar nerve and GG muscle tone (background activity, BGA) were investigated during sleep – wakefulness stages in rabbits. The GG muscle showed two types of electromyographic activity patterns: a respiration-related phasic activity and non-respiration-related activity. GG reflex responses and BGA exhibited a stage-dependent decrease as they were constantly suppressed from quiet wakefulness to non-rapid eye movement sleep (NREMS) to rapid eye movement sleep (REMS). Degree of suppression of reflexes was much larger than that of BGA regardless of GG activity patterns. When amplitude of reflex responses was compared between with and without rapid eye movements during REMS, no difference between the conditions was noted. These results suggest that excitability of the GG muscle is affected by sleep stages by not only a modulation of excitability in motoneurons but also in interneurons involved in the reflex arc. D 2005 Elsevier B.V. All rights reserved. Theme: Motor systems and sensorimotor integration Topic: Reflex function Keywords: Sleep; Tongue; Reflex; Genioglossus muscle; Rabbit
1. Introduction The tongue is a highly mobile and unique composite of various muscles that plays a role in functions such as mastication, deglutition, respiration and vocalization [25]. One important role of tongue muscles is to maintain patency of the extrathoracic airways during tidal respiration [38]. It should be noted that tongue muscle activities are suppressed in parallel to atonia of postural muscles during * Corresponding author. Division of Dysphagia Rehabilitation, Department of Oral Biological Sciences, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan. Fax: +81 25 227 2998. E-mail address: [email protected] (M. Inoue). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.10.013
sleep, particularly during rapid eye movement sleep (REMS) [23]. REMS is quite unique because one of its hallmarks involves desynchronization of the cortical encephalogram similarly to that in wakefulness. Furthermore, strong suppression of postural muscle tone intermittently generated a flurry of activities, manifested by rapid eye movements or muscle twitches [23]. Intracellular recordings of spinal motoneurons in cats also exhibited drastic fluctuations of membrane potentials superimposed on a strong suppression during REMS [8]. Thus, during REMS, excitatory and inhibitory phasic events are superimposed on a tonic and strong muscle suppression. Recently, a number of clinical studies have focused on the pathogenesis of obstructive sleep apnea which may
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involve a combination of reduced upper airway size and loss of tone in upper airway muscles including tongue muscles during sleep [26,28,30,31]. However, the extent to which each muscle contributes to airway patency, and the mechanisms by which this patency is achieved remain uncertain. Difficulties in clarifying the dynamic interplay between combinations of brain stem neural networks that provide respiratory rhythm generation and modulation of the hypoglossal motoneuron discharge may be due to (1) the complexity of anatomical organization of tongue muscles and to (2) the variety of functions of tongue muscles as muscles function not only as respiration-related muscles but also as mastication/swallowing-related or speech/vocalization-related muscles in humans. In a previous study, we reported that there were significant differences in changes of excitability between jaw closer and opener muscles during sleep [14]. Indeed, the jaw closing reflex was inhibited but was occasionally facilitated in association with rapid eye movements while the jaw opening reflex remained inhibited. On the other hand, it is known that the tongue reflex can be evoked by the same stimulus modality as the jaw opening reflex [1,32,40]. Interestingly, there was no difference in changes of responses between the jaw opening and tongue reflexes evoked by the same stimulus modality during swallowing but chewing. The aim of the present study was to investigate how the tongue reflex which can be evoked by the same stimulus modality as the jaw opening reflex is modulated during wakefulness and sleep in freely behaving rabbits.
2. Materials and methods 2.1. Animals Experiments were carried out on 11 adult male Japanese white rabbits weighing between 2.5 and 3.5 kg, in accordance with the Guides for the Care and Use of Laboratory Animal Care (NIH Publication #86-23, revised 1996). The experimental procedure was reviewed and approved by the Niigata University Intramural Animal Care and Use Committee. Animals were initially habituated to the experimental environment. They were accommodated in cages (60 80 122 cm) before surgical intervention. During this period, animals were trained to sit in a small wooden box (40 18 18 cm), where the recording session would be performed. To accustom animals to this circumstance, training sessions were performed for about 1 h per day and were terminated whenever the animal showed any discomfort. 2.2. Surgical procedure The surgical procedure has been extensively reported in previous studies [1,14,16]. Animals were anesthetized with sodium pentobarbital (30 – 35 mg/kg body weight) admin-
istered intravenously through the marginal ear vein. Sodium pentobarbital was further supplemented to maintain anesthesia at a level at which neither an apparent corneal reflex nor spontaneous eye movements occurred. To monitor the sleep – wakefulness stage, bipolar electrodes composed of brass screws were implanted through the frontal bone (5 mm anterior to the bregma, and 2 mm right and left to the midline) for electroencephalographic (EEG) activities, and anterior to the eye socket (10 mm interval between the screws) for electrooculographic (EOG) activities. Pairs of Teflon-coated stainless steel wire electrodes of 0.25-mm diameter and 2 mm long with bared tips for electromyographic (EMG) recordings were inserted into the right clavotrapezius muscle (Trap) with 10 mm of interpolar distance. To record EMG activity of the tongue protractor muscle, a pair of Teflon-coated stainless steel wire electrodes of 0.25-mm diameter was inserted into the genioglossus (GG) muscle with 10 mm of interpolar distance on the left side. To eliminate electrical contamination of the surrounding muscle activity, the digastric nerve was cut on the same side. Tongue reflex was evoked in the GG muscle (GG reflex) by electrical stimulation of the inferior alveolar nerve (IAN) [1]. To access the IAN, the lower edge of the mandibular bone between the anterior margin of the masseter muscle and lateral margin of digastric muscle was exposed, and two small holes (3 mm apart from each other) were drilled. A small socket having a pair of brass electrodes of 1-mm diameter and 2 mm long were connected on the bone, and the tip of electrodes was placed into the mandibular canal alongside the IAN. The socket was then fixed with dental acrylic. The IAN was electrically stimulated (3 train pulses; 0.1 ms in duration; 0.5-ms pulse interval) to evoke the GG reflex at 1 Hz. Current intensity of stimulus was set at 1.5 times the threshold to evoke the GG reflex to evaluate the modulatory mode of reflex responses [1]. Signals from the electrodes were fed subcutaneously to attach the connector fixed on the parietal bone and were conveyed via detachable and flexible cables. 2.3. Data recordings Recordings started after a post-surgical recovery period of at least 1 week. They were carried out for 2 h in the afternoon per day, while the animal was in the wooden box. A recording sequence was divided into four sleep – wakefulness state stages, which were active wakefulness (AW), quiet wakefulness (QW), NREMS and REMS according to criteria determined by our previous study [14]. Stimuli were delivered at through cables, so that the recording and stimulating systems allowed the animal to freely move at least its head and neck. Signals from electrodes amplified with a laboratory-made amplifier (band pass 0.1 –3 kHz for EMGs, and 1 –100 Hz for EEGs and EOGs) were stored on a data recorder (LX-10, TEAC, Japan) and a computer at a sampling rate of 5 kHz. Data
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analysis was performed using the Spike2 analysis package (Cambridge Electronic Design, Cambridge, UK). 2.4. Data analysis In the present study, since our focus was to evaluate GG activity during sleep, recordings during AW were omitted from the analyses. Mean values of peak to peak amplitude of GG reflex responses were obtained for each sleep – wakefulness stage during recording sessions. Values were normalized against those recorded during QW. At the beginning and end of stages, reflex amplitude often fluctuated. Therefore, we did not analyze reflex responses in the first and last 20 consecutive recordings in each stage. Since it is conceivable that GG reflex responses are influenced by background activity (BGA) of the GG muscle, they were also compared among sleep –wakefulness stages. BGAs were obtained as follows: tonic EMGs recorded were selected and rectified for 300 ms, and the mean value was obtained as the BGA for each sleep – wakefulness stage. Data were compared using a non-parametric statistical test (A Kruskal –Wallis ANOVA on Ranks and the Tukey’s HSD as a post hoc test or Mann –Whitney test). Values were expressed as means T SEM, and statistical difference was determined at P < 0.05 (significant) and P < 0.01 (highly significant) level.
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head was held erected, otherwise, it was eating food or sleeping. Recording started when the animal was at rest (i.e., QW), which was normally followed by sleep, and was stopped when the animal woke up. During QW, desynchronized EEG activity was observed. Otherwise, no particular behavioral signs were noted during this stage. During NREMS, cortical spindles appeared in EEG activity, and tonic activity of Trap muscle was maintained (Fig. 1). REMS was characterized by desynchronized EEG activity similar to that in QW and temporal occurrence of REMS. Visual observation of the animal during REMS agreed with previous reports in that eyes were closed and ears were depressed. Sustained EMG activity of Trap muscle was sometimes noted (Fig. 1). Arousal could be behaviorally identified as a sharp and sudden increase in Trap muscle tone and a desynchronized EEG activity. AW was easily noted because of the animal’s active behavior. Mean recording durations were 17 min 53 s T 13 min 6 s for QW (n = 11), 43 min 34 s T 22 min 6 s for NREMS (n = 11) and 50 s T 57 s for REMS (n = 9). Once the animal fell asleep, it always started with NREMS which was often followed by REMS. Although frequency of occurrence and duration of sleep – wakefulness stages varied from animal to animal and/or time by time in each animal, the sequence QW-NREMS-REMS-QW or QW-NREMS-QW was commonly observed.
3. Results
3.2. Changes in GG reflex response during sleep
3.1. Animal’s behavior
An example of GG reflex response during each sleep – wakefulness stage is shown on the top of traces in Fig. 1. Mean latency was 5.1 T 0.2 ms (n = 11) and was obtained during QW. The mean latency was constant throughout the
When the animal was awake during the recording session, it commonly adopted a crouched position with its
Fig. 1. Typical recordings of GG reflex responses during QW, NREMS and REMS. Continuous recordings of EEG, EOG, Trap and Dia EMGs and GG EMG are shown. ECG activities contaminate Dia EMG, and stimulus artifacts contaminate GG EMG. On top of the traces, typical responses of the GG reflex are shown during each sleep – wakefulness stage. Stimulus pulse (4) triggered a display at a faster sweep speed and greater magnification. On the bottom of traces, reflex amplitude changes are also shown. Note that GG reflex responses exhibit a great stage-dependent decrease constantly from QW to NREMS to REMS. Dia, diaphragm; GG, genioglossus muscle; NREMS, non-REM sleep; QW, quiet wakefulness; REMS, REM sleep; Trap, clavotrapezius muscle.
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sleep – wakefulness stages. As shown in our previous study [1], animals never showed discomfort, while 1.5-T stimuli to elicit the GG reflex were delivered during the recording session. During QW, GG reflex amplitude fluctuated. Mean amplitude obtained during QW was 171 T 35 AV (n = 11), ranging from 55 to 435 AV, and the range was the largest of all the three stages. From QW to NREMS, there was a decrease in GG reflex amplitude which paralleled the occurrence of spindle waves in EEG. In this stage, GG reflex amplitude continued to fluctuate. Mean amplitude obtained during NREMS was 131 T 28 AV (n = 11), ranging from 29 to 339 AV. Reduction in amplitude was obviously noted throughout NREMS compared to QW. The most striking change was noted at the transition from NREMS to REMS, in that GG reflex amplitude was drastically reduced. Mean amplitude obtained during REMS was 40 T 8 AV (n = 11), ranging from 0 to 91 AV. Reflex responses were completely suppressed during this stage in R2 and R6. Since amplitude of GG reflex varied with animals, all values were normalized against those obtained during QW to compare among conditions (Fig. 2). GG reflex responses exhibited a constant stage-dependent change from QW to NREMS to REMS. Mean value during REMS (0.36 T 0.06, n = 9) was significantly lower than that during QW (1.00 T 0.00, n = 11) or NREMS (0.76 T 0.04, n = 11) ( P < 0.01), and that during NREMS was significantly lower than that during QW ( P < 0.01). In the case of three animals (R3, R7, R8)
Fig. 3. Regression analysis between the GG reflex amplitude and BGA. Closed and open circles represent the relationship between the GG reflex amplitude and BGA during NREMS and REMS, respectively. GG, genioglossus muscle; BGA, background activity of genioglossus muscle; NREMS, non-REM sleep; REMS, REM sleep.
which showed respiration-related activity of the GG muscle, mean values were 0.88, 0.76 and 0.94 for NREMS and 0.32, 0.27 and 0.41 for REMS. Thus, strong inhibition was observed regardless of activity patterns of the GG muscle. 3.3. BGA BGAs in the three stages were compared using normalized values to those during QW (Fig. 2). Mean value was significantly ( P < 0.05) lower during REMS (0.78 T 0.05, n = 11) than QW, otherwise there was no difference between the stages. A disparity of suppression seemed to exist between amplitudes in BGA and GG reflex. BGAs were moderately suppressed during sleep while strong suppression of GG reflex response was noted particularly during REMS. Furthermore, GG reflex amplitude proportionally changed with BGA during NREMS, but this was not the case during REMS (Fig. 3). 3.4. Effects of REM occurrence on GG activity
Fig. 2. Comparisons between normalized GG reflex amplitudes and BGA during each sleep – wakefulness stage. Mean reflex amplitude during REMS (0.36 T 0.06, n = 9) is significantly lower than that during QW or NREMS while that during NREMS (0.76 T 0.04, n = 11) is significantly ( P < 0.05) lower than that during QW ( P < 0.01). Mean BGA amplitude is significantly lower during REMS (0.78 T 0.05, n = 11) than QW, otherwise there is no difference in BGA between stages. **P < 0.01, *P < 0.05 by comparing among sleep wakefulness stages, .. < 0.01, . < 0.05 by comparing between GG reflex and BGA. BGA, background activity of genioglossus muscle; GG, genioglossus muscle; NREMS, non-REM sleep; QW, quiet wakefulness; REMS, REM sleep.
We have previously showed that the masseteric (jaw closing) monosynaptic reflex was sometimes facilitated in conjunction with occurrence of REMs during REMS while jaw opening reflex remained inhibited regardless of occurrence of REMs [15]. In the present study, relationships between occurrence of REMs and possible fluctuation in suppression of GG reflex were examined. There was no significant difference in the mean normalized value of peak to peak amplitude of the GG reflex between the REM period (with REM, 0.26 T 0.07, n = 9) and non-REM period of REMS (without REM, 0.37 T 0.06, n = 9), correlation variation tended to be higher during the REM period of REMS than non-REM period of REMS (0.27 for with REM vs. 0.16 for without REM).
4. Discussion In the present study, changes in amplitude of the GG reflex were investigated to evaluate changes in excitability
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of hypoglossal motoneurons and neuronal network involved in GG reflex. Decrease in GG muscle tone (observed via BGA) was noted during the transition between wakefulness and sleep, as well as a large decrease in GG reflex amplitude, especially during REMS (78% of control for BGA vs. 36% of control for the GG reflex), suggesting that not only suppression of hypoglossal motoneuron excitability occurred but also that of interneurons responsible for the GG reflex arc must have decreased during REMS. 4.1. GG muscle tone during sleep The present study showed that tonic activity of the GG muscle (observed as BGA) was reduced during the transition from QW, NREMS to REMS. Changes in activity patterns are by no means unique to the GG muscle but not to other muscles or motoneurons [7,9,12,29,34]. It has been suggested that sleep – wakefulness-dependent modulation of serotonergic inputs to hypoglossus motoneurons is involved in changing GG muscle activity [21 – 23]. Medullary raphe neurons provide tonic serotonergic inputs to hypoglossus motoneurons [39] and are known to exhibit discharges which decrease rates from wakefulness to sleep and, in particular, cease during REMS [18]. Furthermore, serotonergic inputs are excitatory inputs to hypoglossus motoneurons [3,19]. Thus, raphe neurons may be responsible for disfacilitation of excitability of hypoglossus motoneurons during sleep. On the other hand, the hypoglossus motor nucleus has also been suggested to heavily receive inputs from noradrenergic neurons [33]. The locus coeruleus complex contains noradrenergic neurons and also exhibits sleep –wakefulness-dependent modulation of discharges like serotonergic neurons [2]. Noradrenergic inputs to hypoglossus motoneurons may also affect excitability of GG muscle tone although we do not have any anatomical evidence of direct inputs. It was proposed that the neurotransmitter mediating the IPSP activity of the trigeminal or spinal motoneurons during REMS is glycine or glycinergic substances but not GABA [10,11,42]. However, this does not seem to be the case in sleep-dependent excitability of hypoglossus motoneurons. Suppression of activity of hypoglossus motoneurons during REMS was not blocked by antagonists of glycine or GABA which are typical inhibitory neurotransmitters [20]. Although hypoglossus motoneurons could be post-synaptically inhibited during REMS [41], it may be conceivable that postsynaptic inhibition of the tongue muscle mediated by glycine or GABA during sleep is somehow minimal. 4.2. Changes in GG reflex responses during sleep Despite that there were a few differences in patterns of the GG muscle activity among animals with regard to respiratory-related activity, GG reflex responses in all animals were strongly suppressed during REMS. Strong suppression of jaw and spinal reflexes [7,14] and a paucity
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of tonic inhibitory influences acting upon motoneurons during REMS [6,9] have been reported. In the present study, suppression of GG reflex amplitude during REMS was much larger than that of BGA. In this regard, the two basic mechanisms that were associated with this suppression of reflex responses during sleep may involve (1) direct disfacilitation/inhibition of motoneurons as mentioned above and (2) suppression of the reflex pathway. In case of moderate inhibition of reflex responses during NREMS, it is likely that the former may be directly involved because degree of inhibition of BGA was almost proportional to that of reflex amplitude. During REMS, inhibitory effects on GG reflex responses were much larger than those on BGAs. Cairns et al. [4] investigated extracellular responses of interneurons to lowintensity stimulation of the tooth pulp and IAN in the rostral trigeminal sensory nuclear complex during wakefulness and sleep in the cat. The authors showed that IAN-evoked orthodromic field potentials or numbers of action potentials of neurons did not differ between wakefulness and NREMS while they were strongly suppressed during REMS. Similar findings were observed in field potentials through lumbar ascending sensory pathways during REMS [36]. Presynaptic inhibition of peripheral inputs from primary afferents of the trigeminal nerve was also observed during sleep [5]. Since some of these afferents may be involved in the reflex pathway for jaw opening and/or GG reflexes [17,37], suppression of the reflex pathway may also be involved in modulation of excitability of central primary endings or secondary neurons. Reflex responses in both digastric (jaw opening) and GG muscles can be evoked disynaptically by electrical stimulation of the low-threshold IAN, which contain Ah fibers in the trigeminal nerve [1,27]. Although both jaw opening and hypoglossus motoneurons showed rhythmical depolarizing potentials which were superimposed by a burst of spikes and no hyperpolarizing potentials between successive depolarizing potentials during mastication [24,29,35], modulatory patterns of those reflexes were reported to be different between each other during mastication [1,13,27]. Considering all these findings, the different normalized amplitude of the reflex as seen in mean normalized values that were 0.43 for the jaw opening reflex [14] and 0.36 for the GG reflex in the present study may be due to differences in excitability of motoneurons or inhibitory inputs to reflex pathways during REMS. 4.3. Functional implications of modulations The functional role of modulations in the GG reflex may be more complex than that in the jaw opening reflex, since the tongue consists of many muscles and neural circuits underlying the reflex are still unknown together with the fact that the tongue plays a critical role in not only respiration but also in orofacial function as we mentioned in the Introduction. It is now noteworthy that BGA was not strongly suppressed during sleep as compared to the GG reflex. These
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results suggest that the neural network involved in hypoglossus motoneuron excitability is minimally maintained to allow patency of the extrathoracic airways, but peripheral inputs should be diminished during sleep in order to avoid unnecessary stimulus which might awake animals.
Acknowledgments The authors would like to thank Mr. Hidetoshi Hirano for his technical support. This study was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture and Science of Japan (#17591934 to M.I., #14207077 to Y.Y. and #116659529 to Y.Y.).
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