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Myoneural maturation and function of the foetal rat tongue at the time of secondary palate closure
Archs oral Biol. Vol. 17, pp. 673-682,
1972. Pergamon Press. Printed in Great Britain.
MYONEURAL MATURATION AND FUNCTION OF THE FOETAL RAT TONGUE AT THE TIME OF SECONDARY PALATE CLOSURE L. E. WRAGG,J. A. SMITHand C. S. BORDEN Departments
of Anatomy and Orthodontics, Northwestern Chicago, Illinois 60611, U.S.A.
University,
Summary-Most authors consider the tongue active in secondary palate closure; some consider it passive; but specific information is wanting. We therefore studied the Holtzman rat foetal tongue by direct observation, histologically, under direct and neural stimulation, and when curarized. The tongue has differentiated into myoblasts (longitudinalis and verticalis) and young fibres (transversus, hyoglossus, genioglossus) four hours prior to closure. At this age it responds to both direct electrical and XII nerve nucleus stimulation. Curarization at high levels neither prevents closure nor aboiishes foetal reflexes. The tongue myoneural apparatus is functional at palate closure. INTRODUCTION
IT IS generally accepted that the tongue plays an active and essential role in mammalian secondary palate closure (HIS, 1901; PETER,1924; LAZZARO,1940; MORIARTY, WEINSTEINand GIBSON,1963; HUMPHREY,1969; WALKER,1971). Recently, however, some authors have relegated the tongue to a passive role of no significance (WALKERand FRASER,1956 ; STARKand EHRMANN,1958 ; ‘TRASLERand FRASER,1963; LARSSON,1962; JACOBS,1970). Many of the views expressed are inferences based on fortuitous, isolated, or morphological observations, but there is no scientifically designed study of the tongue itself-its histological differentiation, activity, or neural control at the time of palate closure. We therefore studied these aspects-selecting the rat as it is the animal most widely used in palate closure experiments. MATERIALS
AND
METHODS
From timed pregnancies in Holtzrnan rats, more than 400 foetuses 14-21 days of age were studied. Direct observation With the mother under ether anaesthesia, foetuses were delivered at hysterotomy; then placed supine, head extended, on a saline-moistened mold. After making relaxing incisions at the mouth angles, the mandible was depressed with a soft rubber probe and oral structures observed. Muscle histology Paraffin sections of Zenker-fixed foetuses were stained haematoxylin and examined by light microscopy.
with Mallory’s
phosphotungstic
acid
Muscle stimulation Bipolar electrodes were used to deliver 0.1-50 V stimuli (duration 2 msec, 7 per set) to muscle of tongue and head of isolated foetuses. Response was observed under a dissecting microscope. 673
L. E. WRAGG, J. A. SMITHAND C. S. BORDEN
674 Nerve histology
According to the methods of BODIAN(1937) and DAVENPORT(1960), heads were fixed in (1) 10 per cent formol-saline, (2) alcohol-formaldehyde-glacial acetic acid, or (3) Bouin’s fluid. Paraffin sections 5-15 pm thick were stained with Protargol. Staining qualities varied between specimens and between sections, but best results were obtained using Davenport’s method for young neurofibrils but prolonging the staining time from 1 to 12 hr. Stimulation
of hypoglossal
nucleus with recording from tongue
For operation on pregnant rats, ether was selected as the anaesthetic so that effects on the foetus would be dissipated rapidly. Foetuses were delivered by Caesarean section individually as needed. The abdominal incision of the mother was then clamped and the animal kept warm while the foetus was removed to a copper-mesh isolation unit for electronic isolation. For experimentation, foetuses were placed intact on one side in an alginate mold, relaxing incisions were made at the mouth angles, and bipolar recording electrodes made from 0.014 in. stainless steel wire, electrolytically polished, and insulated to within 1 mm of the tips, were placed in the tongue. Before placement of the stimulating electrode, a strong light was directed through the head of the intact foetus. This makes the obex clearly visible. By means of a micromanipulator, and using the obex as a guide, the stimulating electrode was guided through the floor of the fourth ventricle into the XII nerve nucleus. A Grass stimulator (Model S-4) delivered Faradic impulses. Action potentials were amplified on an oscilloscope (Tektronix 502) and photographed on a Grass Xymograph Camera (C 4-J). Using this method many foetuses responded for a half hour or more. After stimulation, foetuses were fixed in 10 per cent formalin and the brains of a few were serially sectioned to verify electrode placement within the XII nerve nucleus. Denervation
of foetal
rat tongue
Denervation of the foetal rat tongue was attempted by administering d-tubocurarine. Saline solutions of various strengths were made from the powdered form (Tubocurarine chloride, Nutritional Biochemicals Corporation, Cleveland, Ohio). For direct foetal injection, volume was limited to 0.02 ml. Several methods were tried: (1) _, -,, (2) lethal dosage . , curarization of the mother (0.186 m&k& of curare but with maintenance of the mother on a Baird small animal respirator, (3) anaesthetization of the mother (Diabuta14 mg/lOO g I.P.) then, at laparotomy, injection of curare directly into the foetus in situ or into amniotic fluid, (4) paralysis of the mother by cervical cord section followed by injection of curare directly into the foetus or into amniotic fluid. OBSERVATIONS
Direct observation of living foetuses In the Holtzman strain of rat, at 14 days, the tongue is an appreciable structure and the shelves are just beginning to form. The shelves become horizontal between 16 days and 16 days 8 hr. Our studies centred around this age range. From 14/14 (14 days 14 hr) to 15/8 (l-1.5 days before palate closure), when the jaws were gently opened with a soft rubber probe, the ventral surface of the tongue was seen between the edges of vertical palatal shelves. As the mouth was opened further, the tongue was withdrawn and the vertical shelves assumed a horizontal position by a slow flowing motion. As reported by WALKER and FRASER (1956), when the mouth was subsequently closed, the tongue resumed its previous position. When re-opened, the tongue was again withdrawn from between the shelves which, once more, slowly flowed to a horizontal position. Later (15/2&-only a few hours before closure), if the mandible were depressed the tongue remained in the nasal cavity until the mouth was opened wide and the lingual frenulum stretched. With continued extension the tongue was pulled free, usually in a
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FOETALRAT TONGUE
posterior direction where it came through the wider gap there between the shelves. The shelves moved from vertical to horizontal in less than a second. At this age the palatine artery appeared as a fine bright line, and rugae were visible on the palate. When the mouth was closed and re-opened at this later age, it was apparent that the tongue had not re-entered the nasal cavity. Furthermore, the tongue flattened upon leaving the nasal cavity and it then maintained a low flat outline. At 16/S all shelves were horizontal. At this age a shallow median sulcus, similar to that in the adult, was first observed on the dorsum of the tongue. Muscle diferentiation Developing muscle was classified according to the traditional stages : mesenchyme, myoblast, young muscle fibre, and adult muscle cell. Four to eight foetuses were examined at each of 7 ages between 14/14 and 17/S. There were no marked variations in muscle histology among specimens within a Muscle Exfrinsic Genioglossus
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FIG. 1. Tongue muscle differentiation in the foetal rat. M-mesenchyme-undifferentiated, rounded cells. --cell moderately elongate and multinucleate, fibrils in periphery. I%-myoblast elongate cell, nuclei still centrally located; myofibrils have Y-young fibre -markedly cross striations and fill sarcoplasm.
group. At 14/14 all muscle sites in the tongue were represented by mesenchyme, and at 17/8 both intrinsic and extrinsic muscles had reached the young muscle fibre stage. However, there were differences between specific muscles in the onset and duration of the various stages of differentiation (Fig. 1). Extrinsic muscles (hyoglossus and genioglossus) and the transversus muscle of the tongue began to differentiate into myoblasts first, and reached the young muscle fibre stage before palate closure. Longitudinal and vertical muscles of the tongue were slightly slower to show the features of the myoblast, remained in this stage longer, and did not reach the young muscle fibre stage until well after palate closure. Histologically tongue muscle appeared to be functional. We therefore examined its response to direct electrical stimulation.
676
L.
E. WRAGG, J. A. SMITH AND C. S. BORDEN
Tongue muscle response to direct stimulation On the seventeenth day, when all muscle is in the young muscle fibre stage, tongue muscle responded repeatedly to electrical stimulation with a twitch which was comparable to that elicited in the newborn. At 16/S (immediately after palate closure) contractions were weaker and slower, and at 15120 (prior to closure) the tongue gave only slight slow contractions, resembling those of smooth muscle; nevertheless the response could be elicited repeatedly. It was difficult to see responses in specimens younger than 15 days. Suprahyoid muscles were also stimulated. Their response prior to palate closure (15/20) produced opening movements of the mouth. A few hours after palate closure (16/14), mouth opening in response to stimulation resembled that of the newborn. Histology-hypoglossal
nerve
In serial sections of younger specimens, the XII nerve was traced easily from the medulla to its entrance into the tongue near the hyoid anlage. To distinguish hypoglossal fibres from lingual and glossopharyngeal fibres, it was helpful to note that motor fibres had characteristic endings in the vicinity of muscle fibre nuclei; in older foetuses, they characteristically crossed muscle fibres at right angles before breaking up into their final branches to muscle cells (CAJAL 1909). In late 14-day specimens, fine nerve fibres curved near muscle nuclei, and delicate networks of anastomosing neurofibrils were present. The morphology of the neurofibrils remained simple, while muscles differentiated into the myoblast or young fibre stage (days 15-17). By day 21 (5 days after palate closure), some elongate muscle nuclei were peripheral in location and a primitive type of neural end plate had developed. Tongue response to stimulation of hypoglossal newe nucleus Older foetuses show obvious neural control of sucking and swallowing actions. Recordings from the tongue of such older foetuses were made first for comparison with younger foetuses in which tongue action has not been seen. At each age too, tongue muscle was stimulated directly so that its response to the electrode could be compared with that to the nerve stimulus. Direct stimulation of tongue muscle, 19 days of age, produced an immediate response. This is shown in Fig. 2 where it is partially obscured by stimulus artifact. A one volt stimulus to the hypoglossal nucleus was followed by a typical triphasic response with a large negative spike at 22 msec latency (Fig. 3). When the stimulus was increased (5 V), a multiphasic response occurred (Fig. 4). At a younger age of 1619, only a few hours after palate closure, similar responses occurred. The tongue responded immediately to direct stimulation (Fig. 5) and, after hypoglossal nucleus stimulation, the response was triphasic with the negative spike at a latency of about 22 msec (Fig. 6). Stimulations were then carried out in foetuses 6-10 hr prior to palate closure. A stimulus of O-12 V to the hypoglossal nucleus was followed by a triphasic response (Figs. 8 and 9). The response occurred in about one of ten sweeps of the oscilloscope and the latency was variable. When the stimulus voltage was raised to 50 V, the response
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EXPLANATION OF FIGS. 2-10
The oscilloscope tracing is labelled in Fig. 3. It shows on the upper sweep, the time base (T.B.) in 10 msec intervals and the stimulus (St.) to the tongue or to the hypoglossal nerve nucleus. The lower sweep is the recording from the tongue muscle. It shows a stimulus artifact (St.A.) which, owing to rapid oscillation, was only partially recorded in some cases; and tongue muscle activity (T.M.) in the remainder of the tracing. The scale in each case represents the indicated number of millivolts. FIGS. 24. Age 19 days, 14 hr. This age is about 3.25 days after palate closure, and the animal shows spontaneous and reflex movements of the head and neck. FIG. 2. Stimulus to excised tongue, 5 V, O-5 msec duration. The tongue twitched with each stimulus but the recording is obscured by the superimposed stimulus. FIG. 3. Stimulus to XII nerve nucleus, 1 V, 1 msec duration. There is a triphasic response with a large negative spike at 22 msec latency. FIG. 4. Stimulus to XII nerve nucleus, 5 V, 0.5 msec duration. The stimulus of increased voltage was followed by a multiphasic recording from the tongue. At each stimulus the tongue protruded from the mouth. FIGS. 5-7. Age 16 days, 9 hr (about 4 hr after palate closure). FIG. 5. Stimulus to tongue in situ, 5 V, 1 msec duration. The stimulus artifact is only partially recorded, however, it is superimposed on the short latency positive spike recorded from the tongue. Note the most prominent component of the triphasic response is negative. FIG. 6. Stimulus to XII nerve nucleus, 50 V, 0.5 msec duration. The response is triphasic at a latency of 22 msec. FIG. 7. Stimulus to XII nerve nucleus, 50 V, 5 msec duration. This is one of 40 consecutive responses showing the typical triphasic curve with the large negative spike at a latency of about 22 msec. FIGS. S-10. Age 15 days, 16 hr (about 8 hr prior to palate closure). FIGS. 8, 9. Stimulus to XII nerve nuc!eus, 0.12 V, 1 msec duration. Tongue responded about once every 10 sweeps of the oscilloscope. The response is triphasic with the major deflection negative. The latency of the negative spike is variable-50 msec in Fig. 7, and 18 msec in Fig. 8. FIG. 10. Stimulus to XII nerve nucleus, 50 V, 1 msec duration. At this increased voltage stimulus there was a response at each sweep of the oscilloscope and the latency was constant at 12 msec.
678
L. E. WRAGG.J. A.
SMITH
AND
C. S. BORDEN
occurred consistently and at a uniform latency (Fig. 10). Note that the scale in Fig. 10 is different from that in Figs. 8 and 9, but the negative spike is about 10 MV in all three figures. During the course of these recordings, spontaneous activity was seen occasionally on the oscilloscope at all ages-even 15 days. Tracings were not obtained from all specimens. The older the foetus the easier it was to place the electrode within the XII nerve nucleus. In 21-day-old foetuses, all of five tongues responded to stimulation of the nerve nucleus but, at 15 days, recordings were obtained from only three of 18 specimens. Serial sections showed that, in two specimens which responded, the electrode path was in the XII nerve nucleus but, in two specimens which did not respond, the electrode path had missed the nucleus. Effect of curare on palate closure Two hundred of the 400 foetuses in this study were used in this phase of the experiment but, since the results show no specific and conclusive effects of curare on palate closure, they will be given only briefly. The curarizing dose for three of the pregnant females (0.186 mg/kg, GALLAGHER and KOCH, 1962) was lethal to six others. In the survivors, curarizing doses at 7-hr or at 8-hr intervals from 15/8 to 17/7 did not prevent palate closure, nor did this treatment abolish foetal reflex response to a stroke stimulus on the snout. A small animal respirator was then used in an attempt to maintain the mothers while under at least 12 hr of constant curarization, but only 2 of 4 survived that length of time. In the survivors, foetuses delivered at intervals showed the palate open at first, but it later closed even when the mother was given five times the LD~~ dose at each hour from 15/24 to 16/14. Under this consistently high dosage to the mother, foetuses responded to stroke stimulus with sluggish head and neck movements. To enable the administration of even higher doses to the foetus but avoid maternal deaths due to curare or to prolonged artificial respiration, another approach was tried. Pregnant rats were anaesthetized (Diabutal 4 mg/lOO g) or the cervical spinal cord was severed. At laparotomy, prior to palate closure, 20-45 times the adult LD~~ dose of curare was injected directly into the foetus in situ or into the amniotic fluid. Control foetuses in the same mother were injected with peanut oil or saline only, and were delivered periodically to determine time of closure. In four experiments, the shelves of curarized foetuses remained open after the controls closed; in one, the shelves of curarized foetuses closed before the controls, but in most cases death of the mother, inconsistency in closure, collapse of membranes, deformed or haemorrhaging foetuses made observations irrelevant. Foetuses given 20 x adult LD,, dose of curare were tested by stroke stimulus. They appeared flaccid but they still responded with slow sluggish movements of snout and neck, thus demonstrating that paralysis was not complete. Heart rate of curarized foetuses was 30 per cent less than that of controls, and tissues appeared less turgid. DISCUSSION Our observations on tongue muscle differentiation correlate well with observations on living foetuses. About 28 hr prior to palate closure, muscle differentiation had not
FOETAL
RAT
TONGUE
619
begun, and no movement was seen in response to direct stimulation. The first muscles to begin differentiation were the extrinsic and the transversus muscles. By the time of palate closure, these muscles were all in the young fibre stage but the remaining intrinsic muscles were myoblasts. Even myoblasts are contractile (LEWIS 1915; RENYI and HOGUE, 1934) but the resistance which they can overcome is directly related to the degree of development of the cross-striated myofibrillar apparatus (HOLTZER and ABBOTT,1958). Since the genioglossus, hyoglossus and transversus muscles have developed cross striations, they not only have the potential, but are ideally located to narrow and depress the arched tongue prior to closure. To observe actual contraction, muscles were electrically stimulated directly. Myoblast stages responded with slow slight contractions, but the young muscle fibres responded with a definite twitch. Stimulation of suprahyoids elicited mouth-opening movements. This was unexpected because the rat temporomandibular joint does not begin to form until 19 days (BHASKAR,1953) and it has been thought that without a joint the mouth could not open. Musculature may respond to electrical stimulation before the neural mechanism is functional (WINDLE,1950). We therefore looked for hypoglossal nerve fibres and found them at the earliest stage studied-14 days. At palate closure (16 days), only fine fibres and anastomosing networks were found. This early stage in nerve development is functional in the 17-day foetal rat diaphragm (DIAMONDand MILEDI, 1962). Such primitive endings functioned at older ages of 17-19 days, for then the tongue showed coordination in swallowing movements, but motor end plates were not found. These findings correlate well with the early presence of the hypoglossal nerve in the tongue of man at 10 mm CRL (STREETER,1904), and at 17 mm CRL (PEARSON,1939). Neither of these authors described terminal arborizations within the foetal tongue. Having determined that, prior to palate closure, tongue muscle was contractile and that the motor neurons were present, we felt that the anatomic basis had been established for studying muscle response to nerve stimulation. We chose to stimulate the hypoglossal nucleus rather than the peripheral nerve; thus avoiding both dissection and the risk of exciting the tongue extraneurally. Recordings showed that a nerve stimulus evoked muscle activity. That impulses were transmitted neurally is confirmed by the delay between nucleus stimulation and tongue contraction, and by the absence of response, even to high voltage input, when the stimulating electrode missed the XII nerve nucleus, verified histologically. The tracings must reflect muscle action because intact tongue responds to XII nerve nucleus stimulation with a triphasic curve resembling that of isolated tongue stimulated directly. Had it been from nerve, the recording would have been a single spike. A precise delineation of the appearance of contractility in various muscles may be fundamental to the understanding of developmental mechanisms. The earliest reflex observed in man is a complex of mouth opening, trunk and limb movements (HUMPHREY, 1969). In the rat, electrical stimulation of part of the innervation of this complex, the brachial plexus, produces no response until the age of 16 days (STRAUSS and WEDDEL, 1940); whereas the tongue responded a full day earlier. The striated
680
L. E. WRAGG,J. A. SMITHAND C. S. BORDEN
muscles of the tongue then, must be among the first to become contractile, and are functional at the time of palate closure. Studies claiming palatal closure in paralysed foetuses would be informative if they had demonstrated paralysis of tongue musculature. In JACOBS studies (1970, 1971) the essential condition fundamental to the experiment and the conclusion was paralysis of the foetal tongue, but no such paralysis was shown. Jacobs admits that a non-lethal paralysing dose of curare to the mother does not paralyse the foetus, yet he reports these cases in support of his conclusion. To produce higher curare concentrations in the foetus he gave the mother eight times the apnea-producing dose and maintained respiration by a rodent respirator. Even this dose did not paralyse the foetus but it “diminished” intercostal activities. Furthermore, the return of reflexes in the mother was used as the indication that more paralytic agents should be given. When the titre of curare decreases to a level which permits maternal reflexes, both he and I have shown that it also permits even greater motor activity in the foctus. Activity in the foetal tongue was not studied directly and none of Jacobs observations rule it out. Jacobs states that in experimental mice no spontaneous movements were observed and that all reflexes “seemed” to be totally abolished. Furthermore, there is no indication of what reflexes he tested-if any. We tested rat foetuses by lightly stroking the snout and found face and neck reflexes slow but nevertheless present when the mother was (1) anaesthetized, (2) curarized, (3) given 20 times the lethal dose, and even when 20 times the adult LD~~ dose was injected directly into the foetus. Our conclusions are that paralysis of the foetal tongue has not yet been achieved; and where we showed delay in closure, it was not consistent between litters, and side effects clouded the role of the curare. Many animals on the respirator died-we assume owing to acid-base imbalance. Foetal heart rate was slowed, and tissues appeared not only flaccid but less turgid. Since these factors have a role in foetal physiology, it is clear that more sophisticated methods must be used. It will be necessary to determine the foetal paralysing dose, to monitor physiological parameters such as acid-base balance, blood pressure and tissue fluid pressure; and to obtain electromyographic proof of specific tongue muscle paralysis. Indeed the insertion of fine electrodes in the tongue throughout the period of closure does not seem a difficult feat, and the records would provide data of real significance. It is probable that tongue muscle is active in palate closure. Direct observation shows that, to extract the tongue, this flaccid pliable mass must be stretched far beyond that range possible by the head extension or mandibular depression of an intact animal, so that muscular activity of the tongue must be involved. Another indication of developing muscle function is the significant change in tongue morphology from an arched tongue on day 14 which is easily remoulded and replaced into the nasal cavity, to the low flattened tongue of day 16 which retains this shape and, after withdrawal, cannot be readily replaced between the shelves. Muscular control of the form and position of the tongue itself appears essential, and the myoneural apparatus for such control has now been shown functional.
681
FOETALRAT TONGUE
Acknowledgements-Some of this material is from theses submitted by J. A. SMITH and C. S. BORDEN to Northwestern University in partial fulfillment of the requirements for the M.S. degree. This work was supported by Grant NIH 5 SO1 FR 0531106. R&sum&-La plupart des auteurs consid&rent la langue comme &ant active dans la fermeture secondaire du palais; certains la considtrent passive; mais des informations spkcifiques manquent. Par conskquent, nous avons CtudiC la langue du foetus de rat Holtzman par observation directe, histologiquement, sous stimulation directe et neurale et par curarisation. La langue a ct& diff&enciee en myoblastes (longitudinaux et verticaux) et fibres jeunes (transversus, hyglossus, genioglossus) quatre heures avant la fermeture. A cet Bge elle rtagit &la stimulation electrique directe et g celle du noyau du XII nerf. La curarisation & un haut niveau ne prCvient pas la fermeture, et n’abolit pas les reflexes foetaux. L’appareil myoneural de la langue est fonctionnel ii la fermeture du palais. Zusammenfassung-Viele Verfasser meinen, dass die Zunge bei sekundarem Gaumenschliessen aktiv ist, einige andere, dass sie passiv ist, aber prazise Kenntnis ist nicht vorhanden. Wir untersuchten daher die Fijtuszunge der Holtzman Ratte bei direkter Beobachtung, histologisch, unter direktem und neutralen Stimulus und, wenn kurarisiert. Die Zunge hatte sich vier Stunden vor dem Schliessen in Myoblaste (longitudinalis und verticalis) und junge Fasem (transversus, hyoglossus, genioglossus) abgesondert. Bei diesem Alter reagiert sie sowohl auf direkte elektrische wie XII Nervnukleus Stimulation. Kurarisierung in hohem Grade verhindert das Schliessen nicht, noch hebt sie Reflexe des FZitus auf. Der myoneurale Zungenapparat funktioniert bei Gaumenschliessen. REFERENCES BHASKAR,S. N. 1953. Growth pattern of the rat mandible from thirteen days insemination age to thirty days after birth. Am. J. Anat. 92, l-54. BODIAN,D. 1937. The staining of paraffin sections of nervous tissue with activated protargol. Annt. Rec. 69, 153-162. BORDEN,C. S. 1968. Motor innervation of the fetal rat tongue: Its development, function, and significance in the mechanism of palate closure. Master’s Thesis, Northwestern Univ. Dental School. CAJAL, R. S. 1909. Histologic du Systeme Nerveux de I’Homme et des Vertebres. Maloine, Paris. DAVENPORT,H. A. 1960. Histological and Histochemical Techniques. Saunders, Philadelphia. DL~MOND,J. and MILEDI, R. 1962. A study of foetal and new-born rat muscle fibres. J. Physiol. 162, 393408.
GALLAGHER,C. H. and KOCH, J. H. 1962. The toxicity of d-tubocurarine
to rats. Aust. J. exp. Biol.
40,515-522.
HIS, W. 1901. Beobachtungen zur Geschichte der Nasen-und Gaumen-bildung beim men&lichen Embryo. Abhand. Math. Phys. Classe Koenig. Suech. Gesell. Wissenschaft. 27,347-387. HOLTZER, H. and ABBOTT,J. 1958. Contraction of glycerinated embryonic myoblasts. Anat. Rec. 131,417-428. HUMPHREY,T. 1969. The prenatal development of mouth opening and mouth closure reflexes. Pediatrics Digest 11, 2840. JACOBS,R. M. 1970. Normal closure of secondary palate in paralyzed mouse embryos. J. dent. Res. 49, 1495-1497.
JACOBS,R. M. 1971. Failure of muscle relaxants to produce cleft palate in mice. Teratology 4,25-30. LARSSON,K. S. 1962. Studies on the closure of the secondary palate III: Autoradiographic and histochemical studies in the normal mouse embryo. Acta morphol. neerl. &and. IV, 349-367. LEWIS, M. R. 1915. Rhythmical contraction of the skeletal muscle tissue observed in tissue cultures. J. Physiol. 38, 153-161. MORIARTY,T. M., WEINSTEIN,S. and GIBSON,R. D. 1963. The development in vitro and in vivo of fusion of the palatal processes of rat embryos. J. Embryol. exp. Morph. 2, 605-619. A.O.B. 17/4--E
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PEARSON,A. A. 1939. The hypoglossal nerve in human embryos. J. cotnp. Neuvol. 71,21-29. DERENYI, G. S. and HOGUE. M. J. 1934. Studies on skeletal muscle grown in tissue culture. Arch exD. ZelljI Bd. 16, S.167.
SMITH, J. 1967. Histodifferentiation and contractility of rat tongue musculature and its relation to secondary palate closure. Master’s Thesis, Northwestern Univ. Dental School. STARK, R. B. and EHRMANN,N. A. 1958. The development of the center of the face with particular reference to surgical correction of bilateral cleft lip. Plasl. reconstr. Surg. 21,177-192. STRAUSS,W. L. and WEDDEL,G. 1940. Nature of the first visible contractions of the forelimb musculature in rat fetuses. J. Neurophysiol. 3, 358-369. STREETER,G. L. 1904. The development of the cranial and spinal nerves in the occipital region of the human embryo. Am. J. Amt. 4, 83-116. TRASLER,D. G. and FRASER,C. F. 1963. Role of the tongue in producing cleft palate in mice with spontaneous cleft lip. Develop. Biol. 6, 45-60. WALKER,B. E. 1971. Palate morphogenesis in the rabbit. Archs oral Biol. 16,275-286. WALKER, B. E. and FRAZER, F. C. 1956. Closure of the secondary palate in three strains of mice. J. EmbryoI. exp. Morph. 4, 176-189. WINDLE, W. F. 1950. Reflexes of mammalian embryos and fetuses. In: Genetic Neurology (Edited by WEISS, P.) pp. 214-222. The University of Chicago Press, Chicago.
1972. Pergamon Press. Printed in Great Britain.
MYONEURAL MATURATION AND FUNCTION OF THE FOETAL RAT TONGUE AT THE TIME OF SECONDARY PALATE CLOSURE L. E. WRAGG,J. A. SMITHand C. S. BORDEN Departments
of Anatomy and Orthodontics, Northwestern Chicago, Illinois 60611, U.S.A.
University,
Summary-Most authors consider the tongue active in secondary palate closure; some consider it passive; but specific information is wanting. We therefore studied the Holtzman rat foetal tongue by direct observation, histologically, under direct and neural stimulation, and when curarized. The tongue has differentiated into myoblasts (longitudinalis and verticalis) and young fibres (transversus, hyoglossus, genioglossus) four hours prior to closure. At this age it responds to both direct electrical and XII nerve nucleus stimulation. Curarization at high levels neither prevents closure nor aboiishes foetal reflexes. The tongue myoneural apparatus is functional at palate closure. INTRODUCTION
IT IS generally accepted that the tongue plays an active and essential role in mammalian secondary palate closure (HIS, 1901; PETER,1924; LAZZARO,1940; MORIARTY, WEINSTEINand GIBSON,1963; HUMPHREY,1969; WALKER,1971). Recently, however, some authors have relegated the tongue to a passive role of no significance (WALKERand FRASER,1956 ; STARKand EHRMANN,1958 ; ‘TRASLERand FRASER,1963; LARSSON,1962; JACOBS,1970). Many of the views expressed are inferences based on fortuitous, isolated, or morphological observations, but there is no scientifically designed study of the tongue itself-its histological differentiation, activity, or neural control at the time of palate closure. We therefore studied these aspects-selecting the rat as it is the animal most widely used in palate closure experiments. MATERIALS
AND
METHODS
From timed pregnancies in Holtzrnan rats, more than 400 foetuses 14-21 days of age were studied. Direct observation With the mother under ether anaesthesia, foetuses were delivered at hysterotomy; then placed supine, head extended, on a saline-moistened mold. After making relaxing incisions at the mouth angles, the mandible was depressed with a soft rubber probe and oral structures observed. Muscle histology Paraffin sections of Zenker-fixed foetuses were stained haematoxylin and examined by light microscopy.
with Mallory’s
phosphotungstic
acid
Muscle stimulation Bipolar electrodes were used to deliver 0.1-50 V stimuli (duration 2 msec, 7 per set) to muscle of tongue and head of isolated foetuses. Response was observed under a dissecting microscope. 673
L. E. WRAGG, J. A. SMITHAND C. S. BORDEN
674 Nerve histology
According to the methods of BODIAN(1937) and DAVENPORT(1960), heads were fixed in (1) 10 per cent formol-saline, (2) alcohol-formaldehyde-glacial acetic acid, or (3) Bouin’s fluid. Paraffin sections 5-15 pm thick were stained with Protargol. Staining qualities varied between specimens and between sections, but best results were obtained using Davenport’s method for young neurofibrils but prolonging the staining time from 1 to 12 hr. Stimulation
of hypoglossal
nucleus with recording from tongue
For operation on pregnant rats, ether was selected as the anaesthetic so that effects on the foetus would be dissipated rapidly. Foetuses were delivered by Caesarean section individually as needed. The abdominal incision of the mother was then clamped and the animal kept warm while the foetus was removed to a copper-mesh isolation unit for electronic isolation. For experimentation, foetuses were placed intact on one side in an alginate mold, relaxing incisions were made at the mouth angles, and bipolar recording electrodes made from 0.014 in. stainless steel wire, electrolytically polished, and insulated to within 1 mm of the tips, were placed in the tongue. Before placement of the stimulating electrode, a strong light was directed through the head of the intact foetus. This makes the obex clearly visible. By means of a micromanipulator, and using the obex as a guide, the stimulating electrode was guided through the floor of the fourth ventricle into the XII nerve nucleus. A Grass stimulator (Model S-4) delivered Faradic impulses. Action potentials were amplified on an oscilloscope (Tektronix 502) and photographed on a Grass Xymograph Camera (C 4-J). Using this method many foetuses responded for a half hour or more. After stimulation, foetuses were fixed in 10 per cent formalin and the brains of a few were serially sectioned to verify electrode placement within the XII nerve nucleus. Denervation
of foetal
rat tongue
Denervation of the foetal rat tongue was attempted by administering d-tubocurarine. Saline solutions of various strengths were made from the powdered form (Tubocurarine chloride, Nutritional Biochemicals Corporation, Cleveland, Ohio). For direct foetal injection, volume was limited to 0.02 ml. Several methods were tried: (1) _, -,, (2) lethal dosage . , curarization of the mother (0.186 m&k& of curare but with maintenance of the mother on a Baird small animal respirator, (3) anaesthetization of the mother (Diabuta14 mg/lOO g I.P.) then, at laparotomy, injection of curare directly into the foetus in situ or into amniotic fluid, (4) paralysis of the mother by cervical cord section followed by injection of curare directly into the foetus or into amniotic fluid. OBSERVATIONS
Direct observation of living foetuses In the Holtzman strain of rat, at 14 days, the tongue is an appreciable structure and the shelves are just beginning to form. The shelves become horizontal between 16 days and 16 days 8 hr. Our studies centred around this age range. From 14/14 (14 days 14 hr) to 15/8 (l-1.5 days before palate closure), when the jaws were gently opened with a soft rubber probe, the ventral surface of the tongue was seen between the edges of vertical palatal shelves. As the mouth was opened further, the tongue was withdrawn and the vertical shelves assumed a horizontal position by a slow flowing motion. As reported by WALKER and FRASER (1956), when the mouth was subsequently closed, the tongue resumed its previous position. When re-opened, the tongue was again withdrawn from between the shelves which, once more, slowly flowed to a horizontal position. Later (15/2&-only a few hours before closure), if the mandible were depressed the tongue remained in the nasal cavity until the mouth was opened wide and the lingual frenulum stretched. With continued extension the tongue was pulled free, usually in a
675
FOETALRAT TONGUE
posterior direction where it came through the wider gap there between the shelves. The shelves moved from vertical to horizontal in less than a second. At this age the palatine artery appeared as a fine bright line, and rugae were visible on the palate. When the mouth was closed and re-opened at this later age, it was apparent that the tongue had not re-entered the nasal cavity. Furthermore, the tongue flattened upon leaving the nasal cavity and it then maintained a low flat outline. At 16/S all shelves were horizontal. At this age a shallow median sulcus, similar to that in the adult, was first observed on the dorsum of the tongue. Muscle diferentiation Developing muscle was classified according to the traditional stages : mesenchyme, myoblast, young muscle fibre, and adult muscle cell. Four to eight foetuses were examined at each of 7 ages between 14/14 and 17/S. There were no marked variations in muscle histology among specimens within a Muscle Exfrinsic Genioglossus
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FIG. 1. Tongue muscle differentiation in the foetal rat. M-mesenchyme-undifferentiated, rounded cells. --cell moderately elongate and multinucleate, fibrils in periphery. I%-myoblast elongate cell, nuclei still centrally located; myofibrils have Y-young fibre -markedly cross striations and fill sarcoplasm.
group. At 14/14 all muscle sites in the tongue were represented by mesenchyme, and at 17/8 both intrinsic and extrinsic muscles had reached the young muscle fibre stage. However, there were differences between specific muscles in the onset and duration of the various stages of differentiation (Fig. 1). Extrinsic muscles (hyoglossus and genioglossus) and the transversus muscle of the tongue began to differentiate into myoblasts first, and reached the young muscle fibre stage before palate closure. Longitudinal and vertical muscles of the tongue were slightly slower to show the features of the myoblast, remained in this stage longer, and did not reach the young muscle fibre stage until well after palate closure. Histologically tongue muscle appeared to be functional. We therefore examined its response to direct electrical stimulation.
676
L.
E. WRAGG, J. A. SMITH AND C. S. BORDEN
Tongue muscle response to direct stimulation On the seventeenth day, when all muscle is in the young muscle fibre stage, tongue muscle responded repeatedly to electrical stimulation with a twitch which was comparable to that elicited in the newborn. At 16/S (immediately after palate closure) contractions were weaker and slower, and at 15120 (prior to closure) the tongue gave only slight slow contractions, resembling those of smooth muscle; nevertheless the response could be elicited repeatedly. It was difficult to see responses in specimens younger than 15 days. Suprahyoid muscles were also stimulated. Their response prior to palate closure (15/20) produced opening movements of the mouth. A few hours after palate closure (16/14), mouth opening in response to stimulation resembled that of the newborn. Histology-hypoglossal
nerve
In serial sections of younger specimens, the XII nerve was traced easily from the medulla to its entrance into the tongue near the hyoid anlage. To distinguish hypoglossal fibres from lingual and glossopharyngeal fibres, it was helpful to note that motor fibres had characteristic endings in the vicinity of muscle fibre nuclei; in older foetuses, they characteristically crossed muscle fibres at right angles before breaking up into their final branches to muscle cells (CAJAL 1909). In late 14-day specimens, fine nerve fibres curved near muscle nuclei, and delicate networks of anastomosing neurofibrils were present. The morphology of the neurofibrils remained simple, while muscles differentiated into the myoblast or young fibre stage (days 15-17). By day 21 (5 days after palate closure), some elongate muscle nuclei were peripheral in location and a primitive type of neural end plate had developed. Tongue response to stimulation of hypoglossal newe nucleus Older foetuses show obvious neural control of sucking and swallowing actions. Recordings from the tongue of such older foetuses were made first for comparison with younger foetuses in which tongue action has not been seen. At each age too, tongue muscle was stimulated directly so that its response to the electrode could be compared with that to the nerve stimulus. Direct stimulation of tongue muscle, 19 days of age, produced an immediate response. This is shown in Fig. 2 where it is partially obscured by stimulus artifact. A one volt stimulus to the hypoglossal nucleus was followed by a typical triphasic response with a large negative spike at 22 msec latency (Fig. 3). When the stimulus was increased (5 V), a multiphasic response occurred (Fig. 4). At a younger age of 1619, only a few hours after palate closure, similar responses occurred. The tongue responded immediately to direct stimulation (Fig. 5) and, after hypoglossal nucleus stimulation, the response was triphasic with the negative spike at a latency of about 22 msec (Fig. 6). Stimulations were then carried out in foetuses 6-10 hr prior to palate closure. A stimulus of O-12 V to the hypoglossal nucleus was followed by a triphasic response (Figs. 8 and 9). The response occurred in about one of ten sweeps of the oscilloscope and the latency was variable. When the stimulus voltage was raised to 50 V, the response
FOETAL RAT TONGUE
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EXPLANATION OF FIGS. 2-10
The oscilloscope tracing is labelled in Fig. 3. It shows on the upper sweep, the time base (T.B.) in 10 msec intervals and the stimulus (St.) to the tongue or to the hypoglossal nerve nucleus. The lower sweep is the recording from the tongue muscle. It shows a stimulus artifact (St.A.) which, owing to rapid oscillation, was only partially recorded in some cases; and tongue muscle activity (T.M.) in the remainder of the tracing. The scale in each case represents the indicated number of millivolts. FIGS. 24. Age 19 days, 14 hr. This age is about 3.25 days after palate closure, and the animal shows spontaneous and reflex movements of the head and neck. FIG. 2. Stimulus to excised tongue, 5 V, O-5 msec duration. The tongue twitched with each stimulus but the recording is obscured by the superimposed stimulus. FIG. 3. Stimulus to XII nerve nucleus, 1 V, 1 msec duration. There is a triphasic response with a large negative spike at 22 msec latency. FIG. 4. Stimulus to XII nerve nucleus, 5 V, 0.5 msec duration. The stimulus of increased voltage was followed by a multiphasic recording from the tongue. At each stimulus the tongue protruded from the mouth. FIGS. 5-7. Age 16 days, 9 hr (about 4 hr after palate closure). FIG. 5. Stimulus to tongue in situ, 5 V, 1 msec duration. The stimulus artifact is only partially recorded, however, it is superimposed on the short latency positive spike recorded from the tongue. Note the most prominent component of the triphasic response is negative. FIG. 6. Stimulus to XII nerve nucleus, 50 V, 0.5 msec duration. The response is triphasic at a latency of 22 msec. FIG. 7. Stimulus to XII nerve nucleus, 50 V, 5 msec duration. This is one of 40 consecutive responses showing the typical triphasic curve with the large negative spike at a latency of about 22 msec. FIGS. S-10. Age 15 days, 16 hr (about 8 hr prior to palate closure). FIGS. 8, 9. Stimulus to XII nerve nuc!eus, 0.12 V, 1 msec duration. Tongue responded about once every 10 sweeps of the oscilloscope. The response is triphasic with the major deflection negative. The latency of the negative spike is variable-50 msec in Fig. 7, and 18 msec in Fig. 8. FIG. 10. Stimulus to XII nerve nucleus, 50 V, 1 msec duration. At this increased voltage stimulus there was a response at each sweep of the oscilloscope and the latency was constant at 12 msec.
678
L. E. WRAGG.J. A.
SMITH
AND
C. S. BORDEN
occurred consistently and at a uniform latency (Fig. 10). Note that the scale in Fig. 10 is different from that in Figs. 8 and 9, but the negative spike is about 10 MV in all three figures. During the course of these recordings, spontaneous activity was seen occasionally on the oscilloscope at all ages-even 15 days. Tracings were not obtained from all specimens. The older the foetus the easier it was to place the electrode within the XII nerve nucleus. In 21-day-old foetuses, all of five tongues responded to stimulation of the nerve nucleus but, at 15 days, recordings were obtained from only three of 18 specimens. Serial sections showed that, in two specimens which responded, the electrode path was in the XII nerve nucleus but, in two specimens which did not respond, the electrode path had missed the nucleus. Effect of curare on palate closure Two hundred of the 400 foetuses in this study were used in this phase of the experiment but, since the results show no specific and conclusive effects of curare on palate closure, they will be given only briefly. The curarizing dose for three of the pregnant females (0.186 mg/kg, GALLAGHER and KOCH, 1962) was lethal to six others. In the survivors, curarizing doses at 7-hr or at 8-hr intervals from 15/8 to 17/7 did not prevent palate closure, nor did this treatment abolish foetal reflex response to a stroke stimulus on the snout. A small animal respirator was then used in an attempt to maintain the mothers while under at least 12 hr of constant curarization, but only 2 of 4 survived that length of time. In the survivors, foetuses delivered at intervals showed the palate open at first, but it later closed even when the mother was given five times the LD~~ dose at each hour from 15/24 to 16/14. Under this consistently high dosage to the mother, foetuses responded to stroke stimulus with sluggish head and neck movements. To enable the administration of even higher doses to the foetus but avoid maternal deaths due to curare or to prolonged artificial respiration, another approach was tried. Pregnant rats were anaesthetized (Diabutal 4 mg/lOO g) or the cervical spinal cord was severed. At laparotomy, prior to palate closure, 20-45 times the adult LD~~ dose of curare was injected directly into the foetus in situ or into the amniotic fluid. Control foetuses in the same mother were injected with peanut oil or saline only, and were delivered periodically to determine time of closure. In four experiments, the shelves of curarized foetuses remained open after the controls closed; in one, the shelves of curarized foetuses closed before the controls, but in most cases death of the mother, inconsistency in closure, collapse of membranes, deformed or haemorrhaging foetuses made observations irrelevant. Foetuses given 20 x adult LD,, dose of curare were tested by stroke stimulus. They appeared flaccid but they still responded with slow sluggish movements of snout and neck, thus demonstrating that paralysis was not complete. Heart rate of curarized foetuses was 30 per cent less than that of controls, and tissues appeared less turgid. DISCUSSION Our observations on tongue muscle differentiation correlate well with observations on living foetuses. About 28 hr prior to palate closure, muscle differentiation had not
FOETAL
RAT
TONGUE
619
begun, and no movement was seen in response to direct stimulation. The first muscles to begin differentiation were the extrinsic and the transversus muscles. By the time of palate closure, these muscles were all in the young fibre stage but the remaining intrinsic muscles were myoblasts. Even myoblasts are contractile (LEWIS 1915; RENYI and HOGUE, 1934) but the resistance which they can overcome is directly related to the degree of development of the cross-striated myofibrillar apparatus (HOLTZER and ABBOTT,1958). Since the genioglossus, hyoglossus and transversus muscles have developed cross striations, they not only have the potential, but are ideally located to narrow and depress the arched tongue prior to closure. To observe actual contraction, muscles were electrically stimulated directly. Myoblast stages responded with slow slight contractions, but the young muscle fibres responded with a definite twitch. Stimulation of suprahyoids elicited mouth-opening movements. This was unexpected because the rat temporomandibular joint does not begin to form until 19 days (BHASKAR,1953) and it has been thought that without a joint the mouth could not open. Musculature may respond to electrical stimulation before the neural mechanism is functional (WINDLE,1950). We therefore looked for hypoglossal nerve fibres and found them at the earliest stage studied-14 days. At palate closure (16 days), only fine fibres and anastomosing networks were found. This early stage in nerve development is functional in the 17-day foetal rat diaphragm (DIAMONDand MILEDI, 1962). Such primitive endings functioned at older ages of 17-19 days, for then the tongue showed coordination in swallowing movements, but motor end plates were not found. These findings correlate well with the early presence of the hypoglossal nerve in the tongue of man at 10 mm CRL (STREETER,1904), and at 17 mm CRL (PEARSON,1939). Neither of these authors described terminal arborizations within the foetal tongue. Having determined that, prior to palate closure, tongue muscle was contractile and that the motor neurons were present, we felt that the anatomic basis had been established for studying muscle response to nerve stimulation. We chose to stimulate the hypoglossal nucleus rather than the peripheral nerve; thus avoiding both dissection and the risk of exciting the tongue extraneurally. Recordings showed that a nerve stimulus evoked muscle activity. That impulses were transmitted neurally is confirmed by the delay between nucleus stimulation and tongue contraction, and by the absence of response, even to high voltage input, when the stimulating electrode missed the XII nerve nucleus, verified histologically. The tracings must reflect muscle action because intact tongue responds to XII nerve nucleus stimulation with a triphasic curve resembling that of isolated tongue stimulated directly. Had it been from nerve, the recording would have been a single spike. A precise delineation of the appearance of contractility in various muscles may be fundamental to the understanding of developmental mechanisms. The earliest reflex observed in man is a complex of mouth opening, trunk and limb movements (HUMPHREY, 1969). In the rat, electrical stimulation of part of the innervation of this complex, the brachial plexus, produces no response until the age of 16 days (STRAUSS and WEDDEL, 1940); whereas the tongue responded a full day earlier. The striated
680
L. E. WRAGG,J. A. SMITHAND C. S. BORDEN
muscles of the tongue then, must be among the first to become contractile, and are functional at the time of palate closure. Studies claiming palatal closure in paralysed foetuses would be informative if they had demonstrated paralysis of tongue musculature. In JACOBS studies (1970, 1971) the essential condition fundamental to the experiment and the conclusion was paralysis of the foetal tongue, but no such paralysis was shown. Jacobs admits that a non-lethal paralysing dose of curare to the mother does not paralyse the foetus, yet he reports these cases in support of his conclusion. To produce higher curare concentrations in the foetus he gave the mother eight times the apnea-producing dose and maintained respiration by a rodent respirator. Even this dose did not paralyse the foetus but it “diminished” intercostal activities. Furthermore, the return of reflexes in the mother was used as the indication that more paralytic agents should be given. When the titre of curare decreases to a level which permits maternal reflexes, both he and I have shown that it also permits even greater motor activity in the foctus. Activity in the foetal tongue was not studied directly and none of Jacobs observations rule it out. Jacobs states that in experimental mice no spontaneous movements were observed and that all reflexes “seemed” to be totally abolished. Furthermore, there is no indication of what reflexes he tested-if any. We tested rat foetuses by lightly stroking the snout and found face and neck reflexes slow but nevertheless present when the mother was (1) anaesthetized, (2) curarized, (3) given 20 times the lethal dose, and even when 20 times the adult LD~~ dose was injected directly into the foetus. Our conclusions are that paralysis of the foetal tongue has not yet been achieved; and where we showed delay in closure, it was not consistent between litters, and side effects clouded the role of the curare. Many animals on the respirator died-we assume owing to acid-base imbalance. Foetal heart rate was slowed, and tissues appeared not only flaccid but less turgid. Since these factors have a role in foetal physiology, it is clear that more sophisticated methods must be used. It will be necessary to determine the foetal paralysing dose, to monitor physiological parameters such as acid-base balance, blood pressure and tissue fluid pressure; and to obtain electromyographic proof of specific tongue muscle paralysis. Indeed the insertion of fine electrodes in the tongue throughout the period of closure does not seem a difficult feat, and the records would provide data of real significance. It is probable that tongue muscle is active in palate closure. Direct observation shows that, to extract the tongue, this flaccid pliable mass must be stretched far beyond that range possible by the head extension or mandibular depression of an intact animal, so that muscular activity of the tongue must be involved. Another indication of developing muscle function is the significant change in tongue morphology from an arched tongue on day 14 which is easily remoulded and replaced into the nasal cavity, to the low flattened tongue of day 16 which retains this shape and, after withdrawal, cannot be readily replaced between the shelves. Muscular control of the form and position of the tongue itself appears essential, and the myoneural apparatus for such control has now been shown functional.
681
FOETALRAT TONGUE
Acknowledgements-Some of this material is from theses submitted by J. A. SMITH and C. S. BORDEN to Northwestern University in partial fulfillment of the requirements for the M.S. degree. This work was supported by Grant NIH 5 SO1 FR 0531106. R&sum&-La plupart des auteurs consid&rent la langue comme &ant active dans la fermeture secondaire du palais; certains la considtrent passive; mais des informations spkcifiques manquent. Par conskquent, nous avons CtudiC la langue du foetus de rat Holtzman par observation directe, histologiquement, sous stimulation directe et neurale et par curarisation. La langue a ct& diff&enciee en myoblastes (longitudinaux et verticaux) et fibres jeunes (transversus, hyglossus, genioglossus) quatre heures avant la fermeture. A cet Bge elle rtagit &la stimulation electrique directe et g celle du noyau du XII nerf. La curarisation & un haut niveau ne prCvient pas la fermeture, et n’abolit pas les reflexes foetaux. L’appareil myoneural de la langue est fonctionnel ii la fermeture du palais. Zusammenfassung-Viele Verfasser meinen, dass die Zunge bei sekundarem Gaumenschliessen aktiv ist, einige andere, dass sie passiv ist, aber prazise Kenntnis ist nicht vorhanden. Wir untersuchten daher die Fijtuszunge der Holtzman Ratte bei direkter Beobachtung, histologisch, unter direktem und neutralen Stimulus und, wenn kurarisiert. Die Zunge hatte sich vier Stunden vor dem Schliessen in Myoblaste (longitudinalis und verticalis) und junge Fasem (transversus, hyoglossus, genioglossus) abgesondert. Bei diesem Alter reagiert sie sowohl auf direkte elektrische wie XII Nervnukleus Stimulation. Kurarisierung in hohem Grade verhindert das Schliessen nicht, noch hebt sie Reflexe des FZitus auf. Der myoneurale Zungenapparat funktioniert bei Gaumenschliessen. REFERENCES BHASKAR,S. N. 1953. Growth pattern of the rat mandible from thirteen days insemination age to thirty days after birth. Am. J. Anat. 92, l-54. BODIAN,D. 1937. The staining of paraffin sections of nervous tissue with activated protargol. Annt. Rec. 69, 153-162. BORDEN,C. S. 1968. Motor innervation of the fetal rat tongue: Its development, function, and significance in the mechanism of palate closure. Master’s Thesis, Northwestern Univ. Dental School. CAJAL, R. S. 1909. Histologic du Systeme Nerveux de I’Homme et des Vertebres. Maloine, Paris. DAVENPORT,H. A. 1960. Histological and Histochemical Techniques. Saunders, Philadelphia. DL~MOND,J. and MILEDI, R. 1962. A study of foetal and new-born rat muscle fibres. J. Physiol. 162, 393408.
GALLAGHER,C. H. and KOCH, J. H. 1962. The toxicity of d-tubocurarine
to rats. Aust. J. exp. Biol.
40,515-522.
HIS, W. 1901. Beobachtungen zur Geschichte der Nasen-und Gaumen-bildung beim men&lichen Embryo. Abhand. Math. Phys. Classe Koenig. Suech. Gesell. Wissenschaft. 27,347-387. HOLTZER, H. and ABBOTT,J. 1958. Contraction of glycerinated embryonic myoblasts. Anat. Rec. 131,417-428. HUMPHREY,T. 1969. The prenatal development of mouth opening and mouth closure reflexes. Pediatrics Digest 11, 2840. JACOBS,R. M. 1970. Normal closure of secondary palate in paralyzed mouse embryos. J. dent. Res. 49, 1495-1497.
JACOBS,R. M. 1971. Failure of muscle relaxants to produce cleft palate in mice. Teratology 4,25-30. LARSSON,K. S. 1962. Studies on the closure of the secondary palate III: Autoradiographic and histochemical studies in the normal mouse embryo. Acta morphol. neerl. &and. IV, 349-367. LEWIS, M. R. 1915. Rhythmical contraction of the skeletal muscle tissue observed in tissue cultures. J. Physiol. 38, 153-161. MORIARTY,T. M., WEINSTEIN,S. and GIBSON,R. D. 1963. The development in vitro and in vivo of fusion of the palatal processes of rat embryos. J. Embryol. exp. Morph. 2, 605-619. A.O.B. 17/4--E
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PEARSON,A. A. 1939. The hypoglossal nerve in human embryos. J. cotnp. Neuvol. 71,21-29. DERENYI, G. S. and HOGUE. M. J. 1934. Studies on skeletal muscle grown in tissue culture. Arch exD. ZelljI Bd. 16, S.167.
SMITH, J. 1967. Histodifferentiation and contractility of rat tongue musculature and its relation to secondary palate closure. Master’s Thesis, Northwestern Univ. Dental School. STARK, R. B. and EHRMANN,N. A. 1958. The development of the center of the face with particular reference to surgical correction of bilateral cleft lip. Plasl. reconstr. Surg. 21,177-192. STRAUSS,W. L. and WEDDEL,G. 1940. Nature of the first visible contractions of the forelimb musculature in rat fetuses. J. Neurophysiol. 3, 358-369. STREETER,G. L. 1904. The development of the cranial and spinal nerves in the occipital region of the human embryo. Am. J. Amt. 4, 83-116. TRASLER,D. G. and FRASER,C. F. 1963. Role of the tongue in producing cleft palate in mice with spontaneous cleft lip. Develop. Biol. 6, 45-60. WALKER,B. E. 1971. Palate morphogenesis in the rabbit. Archs oral Biol. 16,275-286. WALKER, B. E. and FRAZER, F. C. 1956. Closure of the secondary palate in three strains of mice. J. EmbryoI. exp. Morph. 4, 176-189. WINDLE, W. F. 1950. Reflexes of mammalian embryos and fetuses. In: Genetic Neurology (Edited by WEISS, P.) pp. 214-222. The University of Chicago Press, Chicago.