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Swallowing-like activity elicited in vitro in neonatal rat organ attached brainstem block preparation
Brain Research 955 (2002) 24–33 www.elsevier.com / locate / brainres
Research report
Swallowing-like activity elicited in vitro in neonatal rat organ attached brainstem block preparation Mikihiko Kogo*, Tadashi Yamanishi, Hidehiko Koizumi, Tokuzo Matsuya First Department of Oral and Maxillofacial Surgery, Osaka University, Graduate School of Dentistry, 1 – 8 Yamadaoka, Suita City, Osaka 565 -0871, Japan Accepted 23 July 2002
Abstract The purpose of this study was to induce swallowing in an in vitro neonatal rat brainstem preparation and to analyze the circuit. When we applied GABAA receptor antagonist (bicuculine methiodide, BIC) into the the nucleus tractus solitarius (NTS) in the organ attached brainstem preparation of neonatal (0–3 days after birth) rats, jaw closing movement, palatal lifting, and tongue peristalsis-like movement were seen, subsequent to elevation of the tip of the tongue and anterior movement of the larynx (closure of the trachea). The NTS has been proposed to be a critical locus for swallowing pattern generation in mammals. Electrical stimulation into the NTS or the vagal afferent nerve (X) following an application of BIC (10 mM) to the recording chamber initiated the same organ movement. This movement caused temporary inhibition of respiratory activity that was simultaneously recorded from the fourth cervical ventral nerve (C4). We were also able to elicit this activity in a whole organ (from lip to stomach, midline intact) preparation, whose oral cavity was filled with dye (pontamine sky-blue 3 mM, 50 ml), using each of the three types of stimulation. The esophagus, which was never stained by spontaneous respiratory movements, was stained only after the experimental stimulation. We concluded that the activity elicited was swallowing-like activity and the smallest circuit for swallowing pattern generation exists in this preparation. 2002 Elsevier Science B.V. All rights reserved. Keywords: Swallowing; Neural circuit; Nucleus tractus solitarius; Brainstem; Central pattern generator; Jaw; Tongue; NMDA receptor
1. Introduction Swallowing is a patterned motor activity involving the contraction and relaxation of several muscles in the oropharyngeal and esophageal regions [5,9,10]. Previous studies have shown that there are two main groups of medullary neurons exhibiting neural activity clearly related to swallowing. The first group corresponds to the neurons located within and near the nucleus tractus solitarius (NTS), while the second group includes neurons located within and around the nucleus ambiguus [38]. In both groups, the NTS is thought to play a major role in triggering and patterning swallowing activity [23,38]. Swallowing activity (buccopharyngeal and / or esophageal stage) can be elicited by electrical stimulation to the NTS [22] or the superior laryngeal nerve (SLN) [9,19,21,37], and local application of excitatory amino acids (EAA) *Corresponding author. Tel.: 181-6-6879-2936; fax: 181-6-68765298. E-mail address: [email protected] (M. Kogo).
[15,24–26,45,48]or the GABAA receptor antagonist to the NTS [17,47]. Although many in vivo studies have been conducted to analyze the neural substrates of swallowing activity, the neurotransmission process and synaptic mechanisms underlying swallowing are far from being understood, and in the recent decade scant information has been reported regarding the central mechanisms of swallowing, probably because the neural circuitry for swallowing is extremely complicated and difficult to analyze [22]. By transferring in vivo experimental protocols to in vitro methods, novel investigation of the cellular and synaptic basis for respiratory rhythm generation has been made possible [14,43]. An in vitro brainstem–spinal cord preparation is very useful for analyzing neural circuits in a limited neural region and has played an important role in previous analyses of respiration neural networks [16,30,43]. We have also been able to use such a preparation to analyze rhythmical jaw movements and reported our results in previous studies [27–29,44]. We considered it important to determine whether the neuronal mechanisms responsible for generating the swal-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03339-5
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lowing motor pattern are preserved in an in vitro neonatal rat brainstem preparation. Therefore, we constructed an in vitro experimental condition to further analyze the cellular basis of swallowing activity.
2. Materials and methods
2.1. Preparation Three types of in vitro brainstem preparations were constructed after removal from 0–3-day-old Sprague– Dawley rats (n557). Experiments were performed with sagittal half organ-attached brainstem preparations (n522), brainstem–spinal-cord preparations (n530) and bilateral intact organ-attached brainstem preparations (midline intact) (n55) for this study. We obtained the preparations using a method previously described [27]. Briefly, after deep halothane anesthesia, the rats were decapitated and the cerebellum was removed. The brainstem preparation was pinned with the ventral side down to Sylgard resin in a recording chamber and flexed at 458 to the dorso–ventral axis, to allow insertion of a pipette into NTS and recording of neural activity from XII simultaneously. We constructed the sagittal half organ-attached preparations as follows. The unilateral orofacial and pharyngeal tissues with innervated musculature were retained for in vitro monitoring of organ movements and supra hyoid muscle electromyographs (EMG) after experimental stimulation. Following removal of the skin and underlying epithelia surrounding
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the craniofacial area, a sagittal cut was made, except for the brainstem, and the unilateral craniofacial neck tissue was removed. The other side of the craniofacial neck tissues, connected to the whole brainstem by the cranial nerves, was left intact (Fig. 1). The bilateral intact organattached preparation consisted of the brainstem and bilateral intact organs remaining in the alimentary canal from the oral cavity to the stomach. From that, dorsal tissue and the cerebrum were removed, so that a glass pipette could be inserted into the NTS. For all in vitro preparations, the brainstem was dissected at the superior colliculus and cervicothoracic junction. During dissection as well as recording, the preparations were continuously superfused with a modified Krebs ringer solution of the following composition (in mM): KCl, 3.0; NaCl, 130; MgCl 2 , 1; NaHCO 3 , 26; D-glucose, 30; CaCl 2 , 0.8, and equilibrated with 95% O 2 –5% CO 2 (pH 7.4) at a rate of 5–10 ml / min.
2.2. Stimulation Three types of stimulation were applied to the brainstem and sagittal half organ-attached preparation; drug microinjection (bicuculine methiodide, Sigma, USA; BIC) into the NTS, electrical stimulation to the NTS, and electrical stimulation to the X nerve. For the bilateral intact organattached preparation we only performed a BIC injection into the NTS. Pressure microinjections of BIC (10–20 mM, dissolved in modified Krebs ringer solution, pH 7.4) into the NTS
Fig. 1. Organ-attached isolated brainstem–spinal cord preparation. Photograph of an in vitro brainstem–spinal cord preparation retaining unilateral orofacial, pharyngeal, and laryngeal tissues with an intact cranial nerve innervating the jaw, tongue, supra hyoid, and pharyngolaryngeal musculature. The brain stem was cut by a transection at the superior colliculus and cervicothoracic junction, and then pinned down horizontally in a recording chamber with the dorsal side up at a 458 angle at approximately the midline vertical axis, in order to apply experimental stimulation to the ipsilateral NTS or to contralateral X. We observed organ movements and recorded EMG signals from the supra hyoid muscle and ENG signals (electroneurograms) from the C4 ventral root simultaneously. Prior to stimulation, spontaneous respiratory movements were observed.
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were made through glass micropipettes (15–20 mm in diameter at the tip) using an injection device (Picosprizer II, General Valve, USA). The injection procedure was identical to that used in previous studies [24]. Briefly, the protocol consisted of multiple injections of small volumes of the drug solutions in order to prevent leakage along the pipette track. When used with a fine-tipped micropipette, this procedure has been found to ensure reproducible injection volumes within a nanoliter range. Calibration of amounts injected through the micropipette was performed under microscopic observation, by measuring the diameter of an exuded droplet [34]. Microinjection of BIC was carried out by applying several pressure pulses in order to reach a final volume of 5–20 nl (50–400 pmol). Electrical stimulation (1–5 stimuli, 1–5 mA, 30–50 Hz, 0.5 ms in duration for each) into the NTS was performed with a digital stimulator via an isolation unit (Electronic Stimulator, Nihon Koden, Japan) using a glass micropipette (5–8 MV) filled with the bathing medium [16]. When we applied either BIC microinjection or electrical stimulation into the NTS, stereotaxic positioning of the micropipette at the stimulation site was performed 100– 150 mm rostrally to the obex, 50–80 mm laterally to the midline, and 200–300 mm down from the medullary surface, under microscopic control. The obex was taken at the caudal tip of area postrema, as described by Paxinos and Watson [41]. The location of the injection was checked histologically with unstained frozen sections of the brainstem after labeling by pressure microinjection of dye (3% pontamine sky blue, Sigma) (Fig. 2). The X nerve was transected, drawn into a suction electrode, and electrical stimulation was applied (1 mA, 1 ms in duration for each, 30 Hz, 3–5 times) [35].
2.3. Recording In the experiments using sagittal half organ-attached preparations, we monitored organ movements with a CCD camera (C-003, Sony, Japan) mounted on a microscope (Stem SV6, Carl Zeiss, Germany) and recorded them using an S-VHS video recording system (NV-SX 55w, Panasonic, Japan). The data recorded on S-VHS videotapes was transferred to a personal computer (Macintosh G3, Apple, USA) with movie capturing software (Media 100, Data Translation, USA). From those images, we traced the jaw, tongue, oral palate, larynx and hyoid bone, and were able to analyze their movements in greater detail. Discharges from the motor neurons were recorded extracellularly by attaching suction electrodes to the cut end of XII or the C4 ventral root. EMG images of supra hyoid muscle activity in the sagittal half organ-attached preparations were obtained using nylon suction electrodes (50–60 mm; O.D.) [28]. Signals from the extracellular and EMG recordings were amplified and bandpass filtered (0.3–3 kHz) with a differential amplifier (DAM-50, World Precision Instruments, USA). Signals were recorded on a digital audio tape (DAT) system (PC204Ax, Sony) with an AD board and a thermal array recorder (RTA-1100, Nihon Koden). The obtained data were then analyzed using a personal computer (Macintosh G3, Apple, USA) and data acquisition / analysis software (MacLabs, AD Instruments, USA).
2.4. Pharmacologic agents The GABAA receptor antagonist, BIC, was used in the experiments.
Fig. 2. Localization of injection site. Coronal photo (right) and drawing of the medulla (left) of a neonatal rat 100 mm rostral to the obex. The micropipette tip position in a histologically identified active site is indicated by the darkened circle. Sol, nucleus solitary tract; sol, solitary tract; Cu, cuneate nucleus; Sp5C, spinal trigeminal nu caudal; 10, dorsal motor nucleus vagus; 12, hypoglossal nucleus.
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All experiments were reviewed and approved by the Osaka University Faculty of Dentistry Intramural Animal Care and Use Committee.
3. Results
3.1. Organ movements We applied pressure microinjection of BIC into the unilateral NTS in the sagittal half organ-attached preparations to elicit swallowing activity (n59). In the resting state (before stimulation), the preparations showed spontaneous rhythmical respiratory movements with a cycle
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period of 22.564.7 s, which were characterized by jaw opening, palatal lifting, and tongue retraction. Following pressure microinjection of BIC into the NTS, we could recognize several movements that were quite different from respiratory movements and similar to swallowing. Notable features of these injection-initiated movements were jaw closing, palatal lifting, and tongue peristalsis-like movement subsequent to elevation of the tip of the tongue and an anterior movement of the larynx by which the trachea was closed (Figs. 3 and 4A and B). This organ movement, elicited after local application of BIC into the NTS, always maintained its sequentiality. Once the movement started it continued, that is to say, it was recorded in an all or none fashion. These movements occurred several
Fig. 3. Visual tracings of movements by the jaw, tongue, oral palate, hyoid bone, and larynx after local application of BIC into the NTS (Time 0.00: onset of sequential movement). First, the tip of the tongue moved upward to the hard palate. At the same time or after a short delay, the larynx showed anterior movement and the hyoid bone moved in a posterior direction. Next, the jaw closed and the soft palate was elevated after peristaltic movement of the tongue. When we performed experimental stimulation (except for electrical stimulation to contralateral X) to incorrect areas, this patterned movement was never seen. No other sequential pattern of movements or movements which consisted of some parts were ever seen, in other words, this patterned motor activity was either all or none.
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Fig. 4. (A) Visual tracing of each tissue separately, after local application of BIC into the NTS. (B) Schematic drawing of the activity of each tissue. The height of the line indicates the intensity of the action observed for each tissue, ranging from complete silence to maximum. Contours of the rise and fall of activity are not considered to be accurate.
times with each stimulation. Further, the number of movements and latency from each stimuli were not dependent on the amount of BIC applied to the NTS; this was discovered because of difficulties experienced with positioning the injection pipette into the correct area. The other two types of stimulation also elicited organ movements with the same temporal sequential pattern as those initiated by local application of BIC into the NTS (into the NTS; n54, to X; n56, data not shown). We recorded EMG data from the supra hyoid muscles and C4 neural activity simultaneously in order to study the
relationship between injection-initiated organ movements and respiratory activity (n53) (Fig. 5). When these organ movements occurred, the respiratory activities from C4 were temporarily inhibited. Swallowing is defined as the functional movement involved with taking something into the stomach through the oral cavity, pharynx, and esophagus. Therefore, in the next experiment we elicited this sequential activity in bilateral intact organ attached preparations with dye placed in the oral cavity (n55). After conducting experimental stimulations, we isolated the esophagus and took photos,
Fig. 5. Respiratory activity, recorded from the C4 ventral nerve, was temporarily inhibited by experimental stimuli that induced organ movements. These representative recordings, represented as integrated traces, were obtained simultaneously from the supra hyoid muscle and C4 ventral nerve. When experimental stimuli induced organ movements, the respiratory activities were blocked.
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and found that only after the stimulation was the esophagus stained by the dye from the oral cavity, and without initiating the swallowing-like movements (only the spontaneous respiratory movements—which we recognized) it was never stained (Fig. 6).
3.2. Extracellular activity of motor neurons In order to analyze the neural discharges of swallowinglike activity, we performed three types of stimulation to brainstem preparations (n530). In a resting condition (before stimulation), all of the preparations spontaneously
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generated rhythmical respiratory motor discharges in the XII and C4 nerves. The cycle period and duration of respiratory activities were 1868 and 1.5260.22 s, respectively. Other types of discharges different from respiratory activity were never seen prior to stimulation. When we applied a pressure microinjection of BIC into the NTS, several swallowing-like neural activities were recorded from XII (n513), which were quite different from respiratory activity in both shape and duration. These activities had a mean duration of 2.3660.34 s and were significantly longer than that of the respiratory activities (Student’s t test, P,0.01).
Fig. 6. Esophagus stained by dye from the oral cavity after local injection of BIC unilaterally into the NTS. We performed a local application of BIC unilaterally into the NTS of a bilateral intact organ-attached preparation after dye was injected into the oral cavity. Seen is a representative isolated esophagus. The esophagus was stained after the injection induced movements were elicited.
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Fig. 7. Neural activities from swallowing-like activity obtained from the XII nerve after three types of stimulation. When electrical stimulation was applied to the NTS and X in normal solution, no neural activities from XII were detected. In contrast, when we performed stimulation to both after BIC bath application, neural activities that were similar to those induced by local application of BIC were elicited (left). Integrated swallowing-like activities by three types of stimulation (right). These were quite similar to each other in both duration and shape.
The swallowing-like activities induced by local application of BIC consisted of 1–5 XII activities with progressively decreasing frequencies. When these motor activities were active, respiratory activities seemed to be temporarily inhibited. Fig. 7 shows the activity from XII recorded after local application of BIC into the NTS. The position of peak amplitude was approximately half of the burst duration and showed a slowly augmenting, slowly diminishing pattern. We also attempted to induce swallowing-like activity by electrical stimulation into the NTS (n59) and to the X nerve (n58) (Fig. 7). In normal solution, these two types of stimulation had no effect on the neural activity recorded from the XII nerve, however, when electrical stimulation was applied to X, a prolongation of the respiratory cycle period was seen. In contrast, when we performed either stimulation after application of BIC (10 mM) to the recording chamber, XII motor neuron activities similar to the discharges recorded after microinjection of BIC into the NTS were recorded. The latency from the electrical stimulation into the NTS and to the X nerve to the onset of these neural activities was 12.061.5 and 15.260.9 ms, respectively. The motor neuron discharges obtained from the XII nerve after these three types of stimulation were quite similar to each other in both shape and duration.
animals (i.e. monkeys, sheep, dogs, cats, and rats) will generally initiate deglutition [1,2,6,7,11,9,36,39,40,46]. However, there is no known study in which the induction of swallowing activity in an in vitro experimental model has been attempted, and little is known about the neuropharmacological and synaptic mechanisms underlying swallowing during the perinatal period. In the present experiments, we were able to elicit swallowing-like activity in vitro in neonatal rat brainstem preparations and found that the neural circuit required for generating swallowing activity was preserved within the brainstem preparations. We considered that the neuronal activities and organ movements induced in the present study corresponded to swallowing activity for the following reasons: (1) the organ movements elicited in the present study were quite similar to swallowing activity, (2) the three types of stimulation methods employed are well known to be able to induce swallowing activity in vivo, (3) any inhibition related to GABAA receptors had to be blocked by local or bath application of BIC to elicit the activity, and (4) a temporary inhibition of respiratory activity was recognized during the swallowing-like activity. Moreover, the characteristics of this activity were similar to those of swallowing activity previously generated in vivo and reported [3,9,22,25].
4. Discussion
4.1. Organ movement and nerve discharges
It has been well established that electrical or chemical stimulation to the NTS or SLN of anesthetized adult
In the resting condition, only spontaneous rhythmic organ movements were seen to be synchronized with C4
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inspiratory burst discharges. Features of the respiratory movement were jaw opening, tongue retraction, and elevation of the soft palate in a sequential pattern during the inspiratory phase. Prior to stimulation, movements different from these respiratory activities were never observed. When tactile stimulation with the tips of tweezers were applied to the pharynx, posterior tongue, and soft palate, organ movements similar to swallowing and those elicited by the three types of stimulation were induced. During this stimuli-induced organ movement, respiratory activity from C4 was inhibited. These observations seem to indicate that the organ movements elicited by experimental stimulation constituted swallowing activity. It could also be considered that the organ movement observed was not swallowing activity, but actually another oral reflex such as suckling, coughing, sighing, gasping, yawning, retching, or vomiting. Coughs, gasps, sighs, and yawns are thought to accompany jaw opening movement, and previous reports regarding gasping and sighing concluded that they were the result of a reconfiguration of a single population of neurons that constitute the kernel of the respiratory pattern generator. In other words, they are activities of modulated respiration and can be recorded from the C4 ventral nerve [12,32]. Therefore, we concluded that sighing and gasping were not corresponding activities to those elicited in this study. As for retching and vomiting, it has been shown that the central pattern generator for these activities exists within or near the NTS as well as the swallowing pattern generator [4]. However, when these activities were induced in a previous study, they could also be recorded from C4 and functionally required jaw opening activity [13]. As a result, we concluded that retching and vomiting are different from the activity noted in the present study. The swallowing-like activity elicited in the present study was either the buccopharyngeal stage or complete swallowing, though this could not to be confirmed because it was difficult to record EMG signals from the esophageal muscles in the organ-attached brainstem preparations. However, we believe that we were able to induce at least the buccopharyngeal stage of swallowing in vitro. Moreover, as local application of NMDA (excitatory amino acid) into the NTS induced only jaw closing and tongue peristalsis activity (with or without very slight pharyngeal closure movements) in the in vitro organ-attached brainstem preparations (data not shown), swallowing-like activity induced by BIC was speculated to be complete swallowing, i.e. involving both stages. Motor neuron extracellular discharges of swallowinglike activity from XII were also recorded, and showed a slowly augmenting, slowly diminishing pattern. No statistical differences were found for their duration or timed peak position among the activities induced by the three types of stimulation. These results suggest that the CPG is involved with the organization of swallowing activities.
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4.2. Electrical stimulation Stimulation to the X afferent nerve also initiated swallowing-like activity in the in vitro preparations. It is well known that electrical stimulation to the superior laryngeal nerve (SLN), one of the branches of X, induces swallowing in vivo. In the present study, we did not attempt to stimulate SLN, because its isolation in a neonatal rat is technically difficult, and used the whole X afferent nerve instead, which involves afferent axons from the pharynx, lung, and abdomen. Electrical stimulation to X caused a prolongation of the cycle period of XII respiratory activity in normal solution. This lengthening of the expiratory phase was thought to originate from the Breuer–Hering reflex, and is induced by stimulating the afferent parts of slowly adapting pulmonary mechanoreceptors [35]. It is known that electrical stimulation to the axons from the abdominal area will induce retching and vomiting activities in dogs [13]. However, the swallowing-like activity induced in the present study is thought to be different from those, as has been described above. We could not elicit retching or vomiting activity from the brainstem preparations for unknown reasons, however, a synaptic interaction may occur between inter-neurons that constitutes the retching or vomiting center in the perinatal period of a rat brainstem.
4.3. Inhibition of swallowing-like activity by GABAA receptors Recent immunohistochemical studies have revealed that GABA-ergic neurons are involved in and around the NTS [33]. Pharmacologic results have also demonstrated that GABA-ergic neurons play an important role in the central control of swallowing. BIC, applied to the surface of the dorsomedial medulla, elicited complete swallowing responses in in vivo experiments [8,17,47]. These observations suggest that, in a resting state, swallowing activity is inhibited by GABAA receptors in a tonic manner. In the present study, pressure microinjection of a small amount (50–400 pmol) of BIC into the NTS was enough to initiate swallowing in in vitro isolated brainstem preparations from neonatal rats. It is thought that the GABAA receptor mediated tonic inhibition of the NTS that suppresses the initiation of swallowing also exists in neonates. In vivo studies using electrical stimulation have been able to elicit swallowing activity without local or systemic application of BIC, whereas a bath application of BIC was required in the present experiments to induce swallowinglike activity with electrical stimulation to X and the NTS. Thus, it is assumed that GABA-ergic inhibition to the swallowing center in neonates is stronger than that in adult rats or under in vivo conditions. In conclusion, we were able to elicit swallowing-like activity in an in vitro brainstem preparation. These results
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may be very useful for future experiments on swallowing activity.
5. Uncited references [18]; [20]; [31]; [42]
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tract stimulation or application of N-methyl-D-aspartate, in neurons of the rat nucleus tractus solitarii, Neurosci. Lett. 124 (1991) 221–224. [46] T. Umezaki, T. Matsuse, T. Shin, Medullary swallowing-related neurons in the anesthetized cat, NeuroReport 9 (1998) 1793–1798. [47] Y.T. Wang, D. Bieger, Role of solitarial GABAergic mechanisms in control of swallowing, Am. J. Physiol. 261 (1991) R639–646. [48] Y.T. Wang, D. Bieger, R.S. Neuman, Activation of NMDA receptors is necessary for fast information transfer at brainstem vagal motoneurons, Brain Res. 567 (1991) 260–266.
Research report
Swallowing-like activity elicited in vitro in neonatal rat organ attached brainstem block preparation Mikihiko Kogo*, Tadashi Yamanishi, Hidehiko Koizumi, Tokuzo Matsuya First Department of Oral and Maxillofacial Surgery, Osaka University, Graduate School of Dentistry, 1 – 8 Yamadaoka, Suita City, Osaka 565 -0871, Japan Accepted 23 July 2002
Abstract The purpose of this study was to induce swallowing in an in vitro neonatal rat brainstem preparation and to analyze the circuit. When we applied GABAA receptor antagonist (bicuculine methiodide, BIC) into the the nucleus tractus solitarius (NTS) in the organ attached brainstem preparation of neonatal (0–3 days after birth) rats, jaw closing movement, palatal lifting, and tongue peristalsis-like movement were seen, subsequent to elevation of the tip of the tongue and anterior movement of the larynx (closure of the trachea). The NTS has been proposed to be a critical locus for swallowing pattern generation in mammals. Electrical stimulation into the NTS or the vagal afferent nerve (X) following an application of BIC (10 mM) to the recording chamber initiated the same organ movement. This movement caused temporary inhibition of respiratory activity that was simultaneously recorded from the fourth cervical ventral nerve (C4). We were also able to elicit this activity in a whole organ (from lip to stomach, midline intact) preparation, whose oral cavity was filled with dye (pontamine sky-blue 3 mM, 50 ml), using each of the three types of stimulation. The esophagus, which was never stained by spontaneous respiratory movements, was stained only after the experimental stimulation. We concluded that the activity elicited was swallowing-like activity and the smallest circuit for swallowing pattern generation exists in this preparation. 2002 Elsevier Science B.V. All rights reserved. Keywords: Swallowing; Neural circuit; Nucleus tractus solitarius; Brainstem; Central pattern generator; Jaw; Tongue; NMDA receptor
1. Introduction Swallowing is a patterned motor activity involving the contraction and relaxation of several muscles in the oropharyngeal and esophageal regions [5,9,10]. Previous studies have shown that there are two main groups of medullary neurons exhibiting neural activity clearly related to swallowing. The first group corresponds to the neurons located within and near the nucleus tractus solitarius (NTS), while the second group includes neurons located within and around the nucleus ambiguus [38]. In both groups, the NTS is thought to play a major role in triggering and patterning swallowing activity [23,38]. Swallowing activity (buccopharyngeal and / or esophageal stage) can be elicited by electrical stimulation to the NTS [22] or the superior laryngeal nerve (SLN) [9,19,21,37], and local application of excitatory amino acids (EAA) *Corresponding author. Tel.: 181-6-6879-2936; fax: 181-6-68765298. E-mail address: [email protected] (M. Kogo).
[15,24–26,45,48]or the GABAA receptor antagonist to the NTS [17,47]. Although many in vivo studies have been conducted to analyze the neural substrates of swallowing activity, the neurotransmission process and synaptic mechanisms underlying swallowing are far from being understood, and in the recent decade scant information has been reported regarding the central mechanisms of swallowing, probably because the neural circuitry for swallowing is extremely complicated and difficult to analyze [22]. By transferring in vivo experimental protocols to in vitro methods, novel investigation of the cellular and synaptic basis for respiratory rhythm generation has been made possible [14,43]. An in vitro brainstem–spinal cord preparation is very useful for analyzing neural circuits in a limited neural region and has played an important role in previous analyses of respiration neural networks [16,30,43]. We have also been able to use such a preparation to analyze rhythmical jaw movements and reported our results in previous studies [27–29,44]. We considered it important to determine whether the neuronal mechanisms responsible for generating the swal-
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03339-5
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lowing motor pattern are preserved in an in vitro neonatal rat brainstem preparation. Therefore, we constructed an in vitro experimental condition to further analyze the cellular basis of swallowing activity.
2. Materials and methods
2.1. Preparation Three types of in vitro brainstem preparations were constructed after removal from 0–3-day-old Sprague– Dawley rats (n557). Experiments were performed with sagittal half organ-attached brainstem preparations (n522), brainstem–spinal-cord preparations (n530) and bilateral intact organ-attached brainstem preparations (midline intact) (n55) for this study. We obtained the preparations using a method previously described [27]. Briefly, after deep halothane anesthesia, the rats were decapitated and the cerebellum was removed. The brainstem preparation was pinned with the ventral side down to Sylgard resin in a recording chamber and flexed at 458 to the dorso–ventral axis, to allow insertion of a pipette into NTS and recording of neural activity from XII simultaneously. We constructed the sagittal half organ-attached preparations as follows. The unilateral orofacial and pharyngeal tissues with innervated musculature were retained for in vitro monitoring of organ movements and supra hyoid muscle electromyographs (EMG) after experimental stimulation. Following removal of the skin and underlying epithelia surrounding
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the craniofacial area, a sagittal cut was made, except for the brainstem, and the unilateral craniofacial neck tissue was removed. The other side of the craniofacial neck tissues, connected to the whole brainstem by the cranial nerves, was left intact (Fig. 1). The bilateral intact organattached preparation consisted of the brainstem and bilateral intact organs remaining in the alimentary canal from the oral cavity to the stomach. From that, dorsal tissue and the cerebrum were removed, so that a glass pipette could be inserted into the NTS. For all in vitro preparations, the brainstem was dissected at the superior colliculus and cervicothoracic junction. During dissection as well as recording, the preparations were continuously superfused with a modified Krebs ringer solution of the following composition (in mM): KCl, 3.0; NaCl, 130; MgCl 2 , 1; NaHCO 3 , 26; D-glucose, 30; CaCl 2 , 0.8, and equilibrated with 95% O 2 –5% CO 2 (pH 7.4) at a rate of 5–10 ml / min.
2.2. Stimulation Three types of stimulation were applied to the brainstem and sagittal half organ-attached preparation; drug microinjection (bicuculine methiodide, Sigma, USA; BIC) into the NTS, electrical stimulation to the NTS, and electrical stimulation to the X nerve. For the bilateral intact organattached preparation we only performed a BIC injection into the NTS. Pressure microinjections of BIC (10–20 mM, dissolved in modified Krebs ringer solution, pH 7.4) into the NTS
Fig. 1. Organ-attached isolated brainstem–spinal cord preparation. Photograph of an in vitro brainstem–spinal cord preparation retaining unilateral orofacial, pharyngeal, and laryngeal tissues with an intact cranial nerve innervating the jaw, tongue, supra hyoid, and pharyngolaryngeal musculature. The brain stem was cut by a transection at the superior colliculus and cervicothoracic junction, and then pinned down horizontally in a recording chamber with the dorsal side up at a 458 angle at approximately the midline vertical axis, in order to apply experimental stimulation to the ipsilateral NTS or to contralateral X. We observed organ movements and recorded EMG signals from the supra hyoid muscle and ENG signals (electroneurograms) from the C4 ventral root simultaneously. Prior to stimulation, spontaneous respiratory movements were observed.
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were made through glass micropipettes (15–20 mm in diameter at the tip) using an injection device (Picosprizer II, General Valve, USA). The injection procedure was identical to that used in previous studies [24]. Briefly, the protocol consisted of multiple injections of small volumes of the drug solutions in order to prevent leakage along the pipette track. When used with a fine-tipped micropipette, this procedure has been found to ensure reproducible injection volumes within a nanoliter range. Calibration of amounts injected through the micropipette was performed under microscopic observation, by measuring the diameter of an exuded droplet [34]. Microinjection of BIC was carried out by applying several pressure pulses in order to reach a final volume of 5–20 nl (50–400 pmol). Electrical stimulation (1–5 stimuli, 1–5 mA, 30–50 Hz, 0.5 ms in duration for each) into the NTS was performed with a digital stimulator via an isolation unit (Electronic Stimulator, Nihon Koden, Japan) using a glass micropipette (5–8 MV) filled with the bathing medium [16]. When we applied either BIC microinjection or electrical stimulation into the NTS, stereotaxic positioning of the micropipette at the stimulation site was performed 100– 150 mm rostrally to the obex, 50–80 mm laterally to the midline, and 200–300 mm down from the medullary surface, under microscopic control. The obex was taken at the caudal tip of area postrema, as described by Paxinos and Watson [41]. The location of the injection was checked histologically with unstained frozen sections of the brainstem after labeling by pressure microinjection of dye (3% pontamine sky blue, Sigma) (Fig. 2). The X nerve was transected, drawn into a suction electrode, and electrical stimulation was applied (1 mA, 1 ms in duration for each, 30 Hz, 3–5 times) [35].
2.3. Recording In the experiments using sagittal half organ-attached preparations, we monitored organ movements with a CCD camera (C-003, Sony, Japan) mounted on a microscope (Stem SV6, Carl Zeiss, Germany) and recorded them using an S-VHS video recording system (NV-SX 55w, Panasonic, Japan). The data recorded on S-VHS videotapes was transferred to a personal computer (Macintosh G3, Apple, USA) with movie capturing software (Media 100, Data Translation, USA). From those images, we traced the jaw, tongue, oral palate, larynx and hyoid bone, and were able to analyze their movements in greater detail. Discharges from the motor neurons were recorded extracellularly by attaching suction electrodes to the cut end of XII or the C4 ventral root. EMG images of supra hyoid muscle activity in the sagittal half organ-attached preparations were obtained using nylon suction electrodes (50–60 mm; O.D.) [28]. Signals from the extracellular and EMG recordings were amplified and bandpass filtered (0.3–3 kHz) with a differential amplifier (DAM-50, World Precision Instruments, USA). Signals were recorded on a digital audio tape (DAT) system (PC204Ax, Sony) with an AD board and a thermal array recorder (RTA-1100, Nihon Koden). The obtained data were then analyzed using a personal computer (Macintosh G3, Apple, USA) and data acquisition / analysis software (MacLabs, AD Instruments, USA).
2.4. Pharmacologic agents The GABAA receptor antagonist, BIC, was used in the experiments.
Fig. 2. Localization of injection site. Coronal photo (right) and drawing of the medulla (left) of a neonatal rat 100 mm rostral to the obex. The micropipette tip position in a histologically identified active site is indicated by the darkened circle. Sol, nucleus solitary tract; sol, solitary tract; Cu, cuneate nucleus; Sp5C, spinal trigeminal nu caudal; 10, dorsal motor nucleus vagus; 12, hypoglossal nucleus.
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All experiments were reviewed and approved by the Osaka University Faculty of Dentistry Intramural Animal Care and Use Committee.
3. Results
3.1. Organ movements We applied pressure microinjection of BIC into the unilateral NTS in the sagittal half organ-attached preparations to elicit swallowing activity (n59). In the resting state (before stimulation), the preparations showed spontaneous rhythmical respiratory movements with a cycle
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period of 22.564.7 s, which were characterized by jaw opening, palatal lifting, and tongue retraction. Following pressure microinjection of BIC into the NTS, we could recognize several movements that were quite different from respiratory movements and similar to swallowing. Notable features of these injection-initiated movements were jaw closing, palatal lifting, and tongue peristalsis-like movement subsequent to elevation of the tip of the tongue and an anterior movement of the larynx by which the trachea was closed (Figs. 3 and 4A and B). This organ movement, elicited after local application of BIC into the NTS, always maintained its sequentiality. Once the movement started it continued, that is to say, it was recorded in an all or none fashion. These movements occurred several
Fig. 3. Visual tracings of movements by the jaw, tongue, oral palate, hyoid bone, and larynx after local application of BIC into the NTS (Time 0.00: onset of sequential movement). First, the tip of the tongue moved upward to the hard palate. At the same time or after a short delay, the larynx showed anterior movement and the hyoid bone moved in a posterior direction. Next, the jaw closed and the soft palate was elevated after peristaltic movement of the tongue. When we performed experimental stimulation (except for electrical stimulation to contralateral X) to incorrect areas, this patterned movement was never seen. No other sequential pattern of movements or movements which consisted of some parts were ever seen, in other words, this patterned motor activity was either all or none.
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Fig. 4. (A) Visual tracing of each tissue separately, after local application of BIC into the NTS. (B) Schematic drawing of the activity of each tissue. The height of the line indicates the intensity of the action observed for each tissue, ranging from complete silence to maximum. Contours of the rise and fall of activity are not considered to be accurate.
times with each stimulation. Further, the number of movements and latency from each stimuli were not dependent on the amount of BIC applied to the NTS; this was discovered because of difficulties experienced with positioning the injection pipette into the correct area. The other two types of stimulation also elicited organ movements with the same temporal sequential pattern as those initiated by local application of BIC into the NTS (into the NTS; n54, to X; n56, data not shown). We recorded EMG data from the supra hyoid muscles and C4 neural activity simultaneously in order to study the
relationship between injection-initiated organ movements and respiratory activity (n53) (Fig. 5). When these organ movements occurred, the respiratory activities from C4 were temporarily inhibited. Swallowing is defined as the functional movement involved with taking something into the stomach through the oral cavity, pharynx, and esophagus. Therefore, in the next experiment we elicited this sequential activity in bilateral intact organ attached preparations with dye placed in the oral cavity (n55). After conducting experimental stimulations, we isolated the esophagus and took photos,
Fig. 5. Respiratory activity, recorded from the C4 ventral nerve, was temporarily inhibited by experimental stimuli that induced organ movements. These representative recordings, represented as integrated traces, were obtained simultaneously from the supra hyoid muscle and C4 ventral nerve. When experimental stimuli induced organ movements, the respiratory activities were blocked.
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and found that only after the stimulation was the esophagus stained by the dye from the oral cavity, and without initiating the swallowing-like movements (only the spontaneous respiratory movements—which we recognized) it was never stained (Fig. 6).
3.2. Extracellular activity of motor neurons In order to analyze the neural discharges of swallowinglike activity, we performed three types of stimulation to brainstem preparations (n530). In a resting condition (before stimulation), all of the preparations spontaneously
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generated rhythmical respiratory motor discharges in the XII and C4 nerves. The cycle period and duration of respiratory activities were 1868 and 1.5260.22 s, respectively. Other types of discharges different from respiratory activity were never seen prior to stimulation. When we applied a pressure microinjection of BIC into the NTS, several swallowing-like neural activities were recorded from XII (n513), which were quite different from respiratory activity in both shape and duration. These activities had a mean duration of 2.3660.34 s and were significantly longer than that of the respiratory activities (Student’s t test, P,0.01).
Fig. 6. Esophagus stained by dye from the oral cavity after local injection of BIC unilaterally into the NTS. We performed a local application of BIC unilaterally into the NTS of a bilateral intact organ-attached preparation after dye was injected into the oral cavity. Seen is a representative isolated esophagus. The esophagus was stained after the injection induced movements were elicited.
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Fig. 7. Neural activities from swallowing-like activity obtained from the XII nerve after three types of stimulation. When electrical stimulation was applied to the NTS and X in normal solution, no neural activities from XII were detected. In contrast, when we performed stimulation to both after BIC bath application, neural activities that were similar to those induced by local application of BIC were elicited (left). Integrated swallowing-like activities by three types of stimulation (right). These were quite similar to each other in both duration and shape.
The swallowing-like activities induced by local application of BIC consisted of 1–5 XII activities with progressively decreasing frequencies. When these motor activities were active, respiratory activities seemed to be temporarily inhibited. Fig. 7 shows the activity from XII recorded after local application of BIC into the NTS. The position of peak amplitude was approximately half of the burst duration and showed a slowly augmenting, slowly diminishing pattern. We also attempted to induce swallowing-like activity by electrical stimulation into the NTS (n59) and to the X nerve (n58) (Fig. 7). In normal solution, these two types of stimulation had no effect on the neural activity recorded from the XII nerve, however, when electrical stimulation was applied to X, a prolongation of the respiratory cycle period was seen. In contrast, when we performed either stimulation after application of BIC (10 mM) to the recording chamber, XII motor neuron activities similar to the discharges recorded after microinjection of BIC into the NTS were recorded. The latency from the electrical stimulation into the NTS and to the X nerve to the onset of these neural activities was 12.061.5 and 15.260.9 ms, respectively. The motor neuron discharges obtained from the XII nerve after these three types of stimulation were quite similar to each other in both shape and duration.
animals (i.e. monkeys, sheep, dogs, cats, and rats) will generally initiate deglutition [1,2,6,7,11,9,36,39,40,46]. However, there is no known study in which the induction of swallowing activity in an in vitro experimental model has been attempted, and little is known about the neuropharmacological and synaptic mechanisms underlying swallowing during the perinatal period. In the present experiments, we were able to elicit swallowing-like activity in vitro in neonatal rat brainstem preparations and found that the neural circuit required for generating swallowing activity was preserved within the brainstem preparations. We considered that the neuronal activities and organ movements induced in the present study corresponded to swallowing activity for the following reasons: (1) the organ movements elicited in the present study were quite similar to swallowing activity, (2) the three types of stimulation methods employed are well known to be able to induce swallowing activity in vivo, (3) any inhibition related to GABAA receptors had to be blocked by local or bath application of BIC to elicit the activity, and (4) a temporary inhibition of respiratory activity was recognized during the swallowing-like activity. Moreover, the characteristics of this activity were similar to those of swallowing activity previously generated in vivo and reported [3,9,22,25].
4. Discussion
4.1. Organ movement and nerve discharges
It has been well established that electrical or chemical stimulation to the NTS or SLN of anesthetized adult
In the resting condition, only spontaneous rhythmic organ movements were seen to be synchronized with C4
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inspiratory burst discharges. Features of the respiratory movement were jaw opening, tongue retraction, and elevation of the soft palate in a sequential pattern during the inspiratory phase. Prior to stimulation, movements different from these respiratory activities were never observed. When tactile stimulation with the tips of tweezers were applied to the pharynx, posterior tongue, and soft palate, organ movements similar to swallowing and those elicited by the three types of stimulation were induced. During this stimuli-induced organ movement, respiratory activity from C4 was inhibited. These observations seem to indicate that the organ movements elicited by experimental stimulation constituted swallowing activity. It could also be considered that the organ movement observed was not swallowing activity, but actually another oral reflex such as suckling, coughing, sighing, gasping, yawning, retching, or vomiting. Coughs, gasps, sighs, and yawns are thought to accompany jaw opening movement, and previous reports regarding gasping and sighing concluded that they were the result of a reconfiguration of a single population of neurons that constitute the kernel of the respiratory pattern generator. In other words, they are activities of modulated respiration and can be recorded from the C4 ventral nerve [12,32]. Therefore, we concluded that sighing and gasping were not corresponding activities to those elicited in this study. As for retching and vomiting, it has been shown that the central pattern generator for these activities exists within or near the NTS as well as the swallowing pattern generator [4]. However, when these activities were induced in a previous study, they could also be recorded from C4 and functionally required jaw opening activity [13]. As a result, we concluded that retching and vomiting are different from the activity noted in the present study. The swallowing-like activity elicited in the present study was either the buccopharyngeal stage or complete swallowing, though this could not to be confirmed because it was difficult to record EMG signals from the esophageal muscles in the organ-attached brainstem preparations. However, we believe that we were able to induce at least the buccopharyngeal stage of swallowing in vitro. Moreover, as local application of NMDA (excitatory amino acid) into the NTS induced only jaw closing and tongue peristalsis activity (with or without very slight pharyngeal closure movements) in the in vitro organ-attached brainstem preparations (data not shown), swallowing-like activity induced by BIC was speculated to be complete swallowing, i.e. involving both stages. Motor neuron extracellular discharges of swallowinglike activity from XII were also recorded, and showed a slowly augmenting, slowly diminishing pattern. No statistical differences were found for their duration or timed peak position among the activities induced by the three types of stimulation. These results suggest that the CPG is involved with the organization of swallowing activities.
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4.2. Electrical stimulation Stimulation to the X afferent nerve also initiated swallowing-like activity in the in vitro preparations. It is well known that electrical stimulation to the superior laryngeal nerve (SLN), one of the branches of X, induces swallowing in vivo. In the present study, we did not attempt to stimulate SLN, because its isolation in a neonatal rat is technically difficult, and used the whole X afferent nerve instead, which involves afferent axons from the pharynx, lung, and abdomen. Electrical stimulation to X caused a prolongation of the cycle period of XII respiratory activity in normal solution. This lengthening of the expiratory phase was thought to originate from the Breuer–Hering reflex, and is induced by stimulating the afferent parts of slowly adapting pulmonary mechanoreceptors [35]. It is known that electrical stimulation to the axons from the abdominal area will induce retching and vomiting activities in dogs [13]. However, the swallowing-like activity induced in the present study is thought to be different from those, as has been described above. We could not elicit retching or vomiting activity from the brainstem preparations for unknown reasons, however, a synaptic interaction may occur between inter-neurons that constitutes the retching or vomiting center in the perinatal period of a rat brainstem.
4.3. Inhibition of swallowing-like activity by GABAA receptors Recent immunohistochemical studies have revealed that GABA-ergic neurons are involved in and around the NTS [33]. Pharmacologic results have also demonstrated that GABA-ergic neurons play an important role in the central control of swallowing. BIC, applied to the surface of the dorsomedial medulla, elicited complete swallowing responses in in vivo experiments [8,17,47]. These observations suggest that, in a resting state, swallowing activity is inhibited by GABAA receptors in a tonic manner. In the present study, pressure microinjection of a small amount (50–400 pmol) of BIC into the NTS was enough to initiate swallowing in in vitro isolated brainstem preparations from neonatal rats. It is thought that the GABAA receptor mediated tonic inhibition of the NTS that suppresses the initiation of swallowing also exists in neonates. In vivo studies using electrical stimulation have been able to elicit swallowing activity without local or systemic application of BIC, whereas a bath application of BIC was required in the present experiments to induce swallowinglike activity with electrical stimulation to X and the NTS. Thus, it is assumed that GABA-ergic inhibition to the swallowing center in neonates is stronger than that in adult rats or under in vivo conditions. In conclusion, we were able to elicit swallowing-like activity in an in vitro brainstem preparation. These results
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may be very useful for future experiments on swallowing activity.
5. Uncited references [18]; [20]; [31]; [42]
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