Jpn. J. Oral Biol., 32: 555-562 , 1990.

ORIGINAL

Reflex responses in the infrahyoid muscles by mechanical

stimulation of the tongue in the frog

Kouichi Shiozawa, Niichiro Tanaka, Yasutake Saeki and Keiji Yanagisawa

Department of Physiology, Tsurumi University School of Dental Medicine 2-1-3 Tsurumi, Tsururmi-ku , Yokohama 30, Japan

〔Accepted for publication: June 4, 1990•l

Key words: //reflex tongue/bullfrog

Abstract: In order to investigate the reflex regulation of the infrahyoid muscle activities induced by sensory signals (chemical and mechanical) from the tongue in the frog, excitatory and inhibitory reflex responses in the infrahyoid muscles (sternohyoid muscle-SH muscle, omohyoid muscle-OH muscle) by both chemical and mechanical stimulation of the tongue were studied using electromyography (EMG) recordings in bullfrogs, Rana catesbeiana. The chemical stimulations (1 mM quinine-HCl, 0.5-1.5 M NaCl) evoked no reflex response in either the SH or the OH muscles, and this result suggested that the infrahyoid muscle activities may not be reflexly regulated by chemical signals from the tongue in the bullfrog. Both the mechanical stimulation of the tongue and the electrical stimulation of the glossopharyngeal evoked excitatory responses in the SH and the OH muscles, bilaterally and contralaterally, respectively. The electrical stimulation of the glossopharyngeal nerve evoked no inhibitory response in the SH, while the stimulation evoked the inhibitory response in the ipsilateral OH. These differences may be related to a difference in the role of these muscles during tongue movement in the bullfrog.

by mechanical signals from the tongue7). Introduction However, the reflex regulation of the infra- Sensory signals (chemical and mechanical) hyoid muscle activities has not been fully from the frog tongue are carried only by elucidated since the existence of an inhibi- the glossopharyngeal nerve. Several reports tory reflex regulation of these muscle activi- have revealed that the reflex discharges in ties is still not clear. The aims of present the branches of the to the study are: (1) to determine whether the extrinsic and intrinsic tongue muscles are reflex response is elicited in the infrahyoid elicited by chemical stimulation (gustatory muscles by chemical stimulation of the stimulation) of the frog tongue1-6). Efferent tongue, and (2) to find out whether a reflexly fibers of the hypoglossal nerve also innervate elicited inhibition of these muscle activities the infrahyoid muscles in frogs, but the effect exist in frogs. In pursuit of these objectives, of chemical stimulation of the tongue on the the activities of two infrahyoid muscles infrahyoid muscle activities has not been stud- (sternohyoid muscle-SH muscle, omohyoid ied. Recently, we found that an excitatory muscle-OH muscle) elicited by both chemical response was reflexly elicited in the infra- and mechanical stimulations of the tongue and hyoid muscles by tactile stimulation of the frog electrical stimulation of the glossopharyngeal tongue, and suggested that the infrahyoid nerve were studied with electromyographic muscle activities may be reflexly regulated (EMG) recording. 556 Jpn. J. Oral Biol., 32: 555-562, 1990.

nerve, the right glossopharyngeal nerve bun- Materials and Methods dle was severed peripherally, and the cen-

In the present study, bullfrogs, Rana tral end of the cut nerve was stimulated with catesbeiana were used. Surgery was performed a train of three rectangular pulses. These under anesthesia with ethyl ether. To expose pulses each of 0.5 ms in duration, with a 5 the SH and the OH muscles, the skin of the ms interval, and an intensity of about 30-60 % lower jaw, the submaxillaris muscles and above the threshold level of the excitatory the frontal part of the deltoid muscles were response elicited in the infraphyoid muscles removed. The animal was mounted in a were applied through a pair of silver wire plastic chamber, dorsal side down, and the electrodes (interpolar distance 2 mm). tongue was drawn out and fixed with insect Results pins. For chemical stimulation of the tongue, 1. Chemical and mechanical stimulations

0.5-1.5 M NaCl and 1 mM quinine-HCl of the tongue

(QHCl) were used. NaCl was dissolved in Prior to the application of chemical stimu- deionized water, but the QHCl was dissolved lation, mechanical stimulation of the right in 10 mM NaCl to eliminate the glossopha- side of tongue surface was used to confirm ryngeal nerve response to water8). After rins- that the EMG activities could be elicited in ing the tongue with 10 mM NaCl solution for the two infrahyoid muscles (SH muscle, OH

2 min, each stimulating solution was applied muscle). As can be clearly seen in both to the right side of tongue with a pipette Fig. 1 and Fig. 2, afferent discharges in the

(2 ml) for about 20s (previous to the applica- glossopharyngeal nerve were induced, and tion of the stimulating solution, the left side EMG activities were elicited contralaterally of the dorsal surface of tongue was covered in the OH muscle (left picture in Fig. 1) and by liquid paraffin to protect the left side bilaterally in the SH muscle (left picture in tongue surface from the stimulating solution). Fig. 2). Under these conditions, each stimu-

Mechanical stimulation consist of applying lating solution (1 mM QHCl, 0.5 M NaCl, a tactile stimulus onto the dorsal surface of 1.0 M NaCl and 1.5 M NaCl) was applied to tongue with a small cotton pellet (about 3 mm the right side of the tongue surface. Afferent in diameter). discharges in the glossopharyngeal nerve were

Bipolar electrodes of polyurethane coated evoked by application of these stimulating stainless steel wire (50 ƒÊm in diameter) were solutions as shown in both Fig. 1 and Fig. 2. inserted bilaterally in the SH and the OH The number of afferent spikes was increased muscles (interpolar distance 8 mm) to record with increasing NaCl concentration (lower the EMG activities. The EMG activities records in both Fig. 1 and Fig. 2). However, were amplified (1205 D, NEC-SANEI) and EMG activities were not elicited in either displayed on a storage oscilloscope (5113, the OH (Fig. 1) or the SH muscles (Fig. 2).

TEKTRONIX). The right glossopharyngeal Similar results were obtained in all of experi- nerve bundle was dissected from the ventral mental animals, although the number of side of lower jaw, and afferent discharges afferent spikes evoked by both mechanical and of this nerve were recorded with a pair of chemical stimulations varied from preparation silver electrodes (interpolar distance 2 mm). to preparation. Afferent discharges were amplified (SA 22 N, 2. Electrical stimulation of the glossopha- TEKTRONIX) and displayed on a storage ryngeal nerve oscilloscope together with the EMG. The Excitatory and inhibitory reflex responses number of afferent spikes per second was elicited in both the SH and the OH muscles counted by a spike-counter (DSE-332, DIA by electrical stimulation of the right glosso- MEDICAL SYSTEM), and the output of the pharyngeal nerve were studied. Figure 3 spike-counter was recorded by a pen-recorder shows the effect of electrical stimulation on

(8S, NEC-SANEI). the EMG activities of both the right and In the case of electrical stimulation of the left OH muscles. When electrical stimula- K. Shiozawa et al.: Reflex responses in the frog infrahyoid muscles 557

Fig. 1 Upper photos show simultaneous records of afferent discharges in the right glossopharyngeal nerve (N. glossoph.) and EMG activities in the right (R) and left (L) omohyoid muscles (OH). Lower records indicate the number of afferent spikes per second counted by a spike-counter. Mechanical and chemical stimulations were applied to the right side of tongue. From left to right: M, mechanical stimulation; 1 mM QHCl, 1 mM quinine HCl; and 0.5-1.5 M NaCl. Horizontal bar under M indicates the application period of the mechanical stimulation. Arrows indicate the chemical stimulus onset. Note that the EMG activities were not elicited in the right and left OH muscles by chemical stimulation.

Fig. 2 Upper photos show simultaneous records of afferent discharges in the right glossopharyngeal nerve (N. glossoph.) and EMG activities in the right (R) and left (L) sternohyoid muscles (SH). Lower records indicate the number of afferent spikes per second. Others are same as in Fig. 1. Note that the EMG activities were not elicited in the right and left SH muscles by chemical stimulation. tion was applied to the right nerve, EMG tivity in the right muscle (Fig. 3 b). Under activities were only elicited in the left (con- these conditions, electrical stimulation was ap- tralateral) muscle (Fig. 3 a). Background plied to the right glossopharyngeal nerve with muscle activity is necessary to detect the re- a stimulus intensity the same as shown in flexly evoked inhibitory response. Therefore, Fig. 3a. As can be clearly seen in Fig. 3c, the left side of the tongue surface was me- an apparent inhibition of muscle activity was chanically stimulated continuously by a small elicited in the right (ipsilateral) muscle (the cotton pellet to evoke continuous muscle ac- latency of this inhibitory response was approx 558 Jpn. J. Oral Biol., 32: 555-562, 1990.

Fig. 3 Reflex responses elicited in the right (R) and left (L) omohyoid muscles (OH) by the electrical stimulation of the glossopharyngeal nerve (N. glossoph.). Each record shows 5 superimposed traces. A train of three pulses (0.5 ms duration, 5 ms interval, 0.65 V) was applied to the right nerve (E) as shown in the left diagram. The lowest trace in each photo indicates the electrical stimulation, which was done in a and in c. Continuous mechanical stimulation (M) of the left side of tongue was done in b and c. imately 24 ms). An excitatory response was EMG activities of both the right and left SH also elicited simultaneously in the left muscle muscles. EMG activities were elicited in both (Fig. 3c), but the amplitude of the response the right and left muscles (Fig. 4a). The was smaller than that of the response in Fig. continuous mechanical stimulation of the left 3 a although the stimulus intensity was the side of tongue evoked EMG activities bilat- same. This decrease of the excitatory re- erally (Fig. 4b). When the electrical stimula- sponse amplitude in the left muscle might be tion was applied to the right nerve while due to the reflexly evoked ipsilateral inhibition the left side of tongue was being mechani- elicited by the continuous mechanical stimu- cally stimulated, the inhibitory response lation of the left side of the tongue. The (the suppression of EMG activities) was not inhibition of ipsilateral OH muscle activity seen in either the right or left SH muscles elicited by electrical stimulation of the glosso- (Fig. 4c). No inhibitory response was shown pharyngeal nerve was observed in all of the in the SH muscle by electrical stimulation of experimental animals. Figure 4 shows an the nerve in all the experiments. example of the effect of electrical stimulation 3. The movement of the right glossopharyngeal nerve on the In order to investigate the hyoid bone K. Shiozawa et al.: Reflex responses in the frog infrahyoid muscles 559

Fig. 4 Reflex responses elicited in the right (R) and left (L) sternohyoid muscles (SH) by the electrical stimulation of the glossopharyngeal nerve (N. glossoph.). Each record shows 5 superimposed traces. A train of three pulses (0.5 ms duration, 5 ms interval, 0.9 V). was applied to the right nerve (E). Others are same as in Fig. 3.

movement induced by contraction of the SH in the hypoglossal nerve are reflexly induc- and OH muscles, electrical stimulation was ed6,9-11). The frog hypoglossal nerve inner- applied to directly these muscles through a vates the infrahyoid muscles as well as the pair of electrodes (interpolar distance about 5 extrinsic and intrinsic tongue muscles. Using mm). Experimental contraction of both the the horseradish peroxidase method, Stuesse right and left SH muscles caused a backward et al. have revealed that the motor neurons movement of the hyoid. On the other hand, in the hypoglossal nerve originate in at least the contraction of the right OH muscle and two nuclei, efferents to extrinsic and intrin- the left OH muscle caused a lateral move- sic tongue muscles originate in the medullary ment of the hyoid to the right and to the left, nucleus, and efferents to the infrahyoid respectively. muscles originate in the lateral medullary nucleus. But the latter efferents are thought Discussion to be spinal components and to be homolo- It is known in frogs that, chemical signals gous with the first efferents in (gustatory signals) from the taste organs in mammals12). In the present study, chemical the tongue are sent to the nucleus tractus stimulations of 1 mM QHCl and 1 M NaCl solitarius by the glossopharyngeal nerve, and which were used in the same consentrations these signals are transmitted to the hypoglo- as Nakachi and Ishiko6) could not elicit the ssal motor nucleus, consequently, discharges infrahyoid muscle activities (see Fig. 1 and 560 Jpn. J. Oral Biol., 32: 555-562, 1990.

Fig. 5 Schematic illustration of the proposed role of the tongue-infrahyoid muscle reflex during prey-catching behavior in the frog (see text). Ventral view of the lower jaw. A: a prey attached to the left side of the tongue surface. B: a prey attached to the center of the tongue. Short arrows indicate the muscle contration. Long arrows indicate the move- ment of the prey (black ball). +, reflexly-evoked excitation of the muscle activity ;-, reflexly-evoked inhibition of the muscle activity; SH, sternohyoid muscle; OH, omohyoid muscle; HG, hyoglossal muscle; H, hyoid bone; R, right; L, left.

Fig. 2). Therefore, unlike the extrinsic and a difference in the roles of these infrahyoid intrinsic tongue muscles, the infrahyoid mus- muscles during tongue movement such as cles are not reflexly regulated by the chemical prey-catching behavior, since the frog infra- signals from the tongue. Thus, the chemo- hyoid muscle activities are increased during reflex pathway between the nucleus tractus prey-catching behavior13,14). The contraction solitarius to the infrahyoid muscle moto- of the SH and the OH muscles may cause neurons may be lacking in the frog. This the backward and the lateral movements of could be explained by the idea that efferents the hyoid respectively, since the SH and the to infrahyoid muscles are spinal components. OH muscles directly attach to the postero- The excitatory responses elicited in the SH medial process and the base of posterolateral and the OH muscles by both the mechanical process of the hyoid bone respectively in the stimulation of the tongue and the electrical frog15). To confirm that the movement of stimulation of the glossopharyngeal nerve the hyoid bone was induced by contraction were evoked bilaterally and contralaterally, re- of the infrahyoid muscles, electrical stimula- spectively, as reported in the previous study7). tion was applied to both the SH and the OH On the other hand, an inhibitory response muscles in the present study. As described was elicited in the ipsilateral OH muscle by in the results, experimental contractions of the electrical stimulation of the nerve (Fig. both the right and left SH muscles caused a 3), but it was not elicited in the SH muscle backward movement of the hyoid, while the (Fig. 4). These differences between the SH contraction of the right OH muscle caused and the OH muscles may be associated with a lateral movement of the hyoid to the right. K. Shiozawa et al .: Reflex responses in the frog infrahyoid muscles 561

Therefore, when the reflex responses are contraction of both the right and left OH elicited in the infrahyoid muscles by mechani- muscles may not be sufficient, since reflexly cal stimulation of the left side of the tongue elicited inhibition of the OH muscle activ- surface, such as a prey attached to the left ity occurs simultaneously. While reflexly- side of tongue during tongue retraction during evoked contraction of SH muscle is elicited prey-catching behavior, as illustrated in Fig. bilaterally and it causes the backward move- 5 A, reflexly-evoked bilateral contractions of ment of the hyoid, and may accelerate tongue the SH muscles which cause the backward retraction. Thus, the reflex regulation of movement of the hyoid, and a reflexly-evoked infrahyoid muscle activity elicited by mechani- contraction of the right OH muscle (contra- cal signals from the tongue may contrib- lateral) and reflexly elicited inhibition of the ute to the control of tongue movement dur- ipsilateral OH muscle causes the lateral move- ing prey-catching behavior, and this regu- ment of the hyoid to the right. Since these lation may also contribute to the tongue events are taking place simultaneously, the movements during in the frog. hyoid may move laterally and backwards, and this may move the prey to the center Acknowledgements of the mouth. On the other hand, when We thank Dr. R. Fearnhead, Tsurumi Uni- both sides of tongue are stimulated mechan- versity, for help with the English. ically by the prey (Fig. 5 B), a reflexly-evoked

抄 録:舌 か らの 感 覚 情報 に よ る カ エ ルの 舌 骨 下 筋活 動 の 反 射性 制 御 を調 べ る 為 に,舌 の 化学 的,機 械 的 及 び 舌 咽 神 経 の電 気刺 激 を 行 って,食 用 ガ エ ルの 胸 骨 舌骨 筋(SH)と 肩 甲舌 骨 筋(OH)に 誘 発 され る興 奮性 及 び抑 制 性 反 射 応 答 を 筋電 図 を用 いて 調 べ た 。舌 に化学 的刺 激(1mM塩 酸 キニ ー ネ, 0.5～1.5M食 塩 水) を与 えて もSH, OHに は筋 活 動 が 誘 発 され な か った こ とか ら,カ エ ル の舌 骨 下 筋 の筋 活 動 は 舌 か らの味 覚 情 報 で は 反射 的 に制 御 され て いな い と思 われ る。 舌 の機 械 的 及 び 舌咽 神 経 の電 気 刺 激 で,SHで は両 側 性 に, ま たOHで は対 側 性 に筋 活 動 が誘 発 され た。 一方,舌 咽 神 経 の 電気 刺 激 で,OHで は同 側 の 筋 に筋 活 動 の 抑 制 が 誘 発 され た がSHで は 筋 活動 の抑 制 は認 め られ な か った 。両 筋 で認 め られ た これ らの 反射 応 答 の 差 は, カエ ル の 舌運 動 時 の両 筋 の 機 能 的 役割 の差 に起 因 して い る こ とが 考 え られ る。

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