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Topographic segregation of genioglossus motoneurons in the neonatal rat
102
Neuroscience Letters, 155 (1993) 102-106 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL 09545
Topographic segregation of genioglossus motoneurons in the neonatal rat Alan J. Sokoloff Section of Population Biology, Morphology, and Genetics, Brown University, Providence, RI 02912 (USA) (Received 22 January 1993; Accepted 2 March 1993)
Key words: Genioglossus; WGA-HRP; Motoneuron; Development; Musculotopy; Brainstem; Tongue; Hypoglossal The location of the motoneurons that innervate the main tongue protrusor muscle, genioglossus, was studied in the 1-2 day old, 10 14 day old and adult rat. Following injection of the retrograde axonal tracer WGA-HRP into the genioglossus muscle, labeled neurons were localized to a discrete column within the ventral division of the hypoglossal nucleus in both the neonatal and adult rats. These results demonstrate an adult pattern of genioglossus innervation in the neonatal rat.
Despite recent studies of the physiological and anatomical maturation of hypoglossal nucleus motoneurons in the rat [5-7], the innervation of individual tongue muscles has not been investigated in the developing animal. The hypoglossal nucleus (HGN) of the adult rat is composed of three subdivisions: a dorsal subdivision which innervates the tongue retrusor muscles, hyoglossus and styloglossus; a ventral subdivision which innervates the tongue protrusor muscle, genioglossus; and a ventrolateral subdivision, separated ventrally from the main portion of the HGN, which innervates the suprahyoid muscle, geniohyoid [10, 17]. A similar discrete musculotopic innervation of retrusor, protrusor and geniohyoid muscles has been reported for the adult cat and macaque [14-16]. Musculotopic organization of the hypoglossal nucleus of mammals may be less discrete in the neonate than in the adult. Chibuzo and Cummings [3] reported overlap in the location of the HGN motoneurons which innervate retrusor and protrusor muscles in the neonatal dog. They also reported that the geniohyoid and genioglossus muscles each receive innervation from both ventral and ventrolateral HGN subdivisions. A ventral and ventrolateral HGN innervation of the genioglossus muscle was also observed in the kitten [2]. These studies, however, did not explore possible ontogenetic, species-specific or
Correspondence." A. Sokoloff, Department of Physiology, MS 409, Hahnemann University, Broad and Vine, Philadelphia, PA 19102-1192, USA.
methodological factors that might explain reported differences in neonatal and adult HGN musculotopic organization. To investigate the development of HGN musculotopy, the innervation of the genioglossus muscle was studied in the 1-2 day old, 10-14 day old and adult rat. The genioglossus muscle was chosen for study because of its important function in the maintenance of upper airway patency [1, 4, 13]. Under metofane (1-2 day old rats) or a combination of ketamine (40-74 mg/kg, i.p.) and xylazine (5-8 mg/kg, i.p.) anaesthesia, the genioglossus muscle was exposed by pulling the tongue anteriorly and superiorly from the mouth. A single injection of 0.2~.5/ll of the retrograde axonal tracer wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP, Sigma, 5% in distilled water) was made into the genioglossus muscle in ten 1-2 day old rats with a glass micropipette attached to a 1/.tl Hamilton syringe. A single injection of 0.5-1.5 /11 of 5% WGA-HRP was made into the genioglossus muscle of six 10-14 day old rats and 4 adult rats with a 10 1ll Hamilton syringe. Eighteen to 36 h following WGA-HRP injection, the animals were euthanized with pentobarbital sodium (5055 mg/kg, i.p.) and perfused through the left ventricle with 100-300 ml of 0.9% saline, followed first by 100-300 ml of a fixative containing 0.5% paraformaldehyde, 2.0% glutaraldehyde and 2.0% sucrose in a 0.1 M phosphate buffer, and second by 100-300 ml of 10% sucrose in 0.1 M phosphate buffer. The brainstem, tongue and geniohyoid muscle were removed and stored for 1-3 days in a 20% sucrose solution of 0.1 M phosphate buffer at 4 ° C. The brainstem was cut serially in 75 or 100ktm transverse
103
sections on a freezing microtome. Frozen 100 or 125/2m sections of the tongue and geniohyoid muscle were cut in sagittal or transverse planes in order to determine the injection site. Brainstem and tongue tissue was reacted with tetramethylbenzidine (TMB; following Mesulam [12]) for 2~, h, mounted on gelatin-subbed slides, counterstained in neutral red and coverslipped with Permount. Slides were examined under brightfield, darkfield and polarized light microscopy to determine the extent of the injection site and resulting brainstem label. Brainstem and tongue sections with TMB label were photographed and traced. In each case, the WGA-HRP injection was centered in the genioglossus muscle (Table I). In some cases, the injection spread ventrally to involve fibers of the geniohyoid muscle and/or laterally to involve fibers of the hyoglossus muscle. The pattern of HGN label was similar in all age groups. Following exclusive injection of the genioglossus muscle, labeled neurons formed an uninterrupted column in the dorsolateral corner of the ventral HGN subdivision (Table I; Figs. 1 and 2). Following injections which involved geniohyoid in addition to genioglossus fibers, labeled neurons were additionally located in the ventrolateral HGN subdivision. All cases with geniohyoid injection resulted in ventrolateral subdivision label, and ventrolateral subdivision label was only observed in cases with geniohyoid involvement (Table I). In three cases, labeled neurons were also clearly present in the dorsal HGN subdivision. These cases were distinguished by the involvement of hyoglossus fibers (Table I). The results of the present study demonstrate that genioglossus motoneurons are localized to a discrete column in the ventral HGN subdivision in neonatal and adult rats. These results indicate that an adult pattern of genioglossus motoneuron innervation is established by day 1 in the neonatal rat. This finding is similar to the report of an adult musculotopic organization of the facial nucleus in the newborn rat [9]. The location of genioglossus motoneurons reported in the present study is in agreement with the results of previous studies of the adult rat. Both Krammer et al. [10] and Uemura-Sumi et al. [17] localized genioglossus motoneurons to the ventral HGN subdivision in the adult rat. These studies also demonstrated that motoneurons in the ventrolateral HGN subdivision innervate the geniohyoid muscle and motoneurons in the rostral levels of the dorsal HGN subdivision innervate the hyoglossus muscle [8, 10, 17]. The association of ventrolateral HGN label with geniohyoid injection and dorsolateral HGN label with hyoglossus injection in the present study (Table I), suggests that an adult musculotopic organization of the HGN is formed by day 2 in the rat.
The exclusive ventral HGN subdivision innervation of the genioglossus muscle in the neonatal rat is in contrast to reports of both ventral and ventrolateral subdivision label following genioglossus injection in the neonatal kitten and dog [2, 3; but see 17]. Differences in the findings of the present study from those previously reported may reflect different techniques, different amounts of tracer injected (e.g. 40-50/21 in [3] versus 0.2-1.5/11 in the present study) and/or species differences in the organization or development of HGN musculotopy. Given the current interest in the development and control of respiration and upper airway patency in humans [4, 11, 13], further studies of the development of hypoglossal nucleus organization and function are warranted.
TABLE I LOCATION OF TONGUE INJECTION SITES AND CORRESPONDING HYPOGLOSSAL M O T O N E U R O N LABEL IN THE NEONATAL AND ADULT RAT D, dorsal subdivision of the hypoglossal nucleus; GG, genioglossus muscle; GH, geniohyoid muscle; HG, hyoglossus muscle, V, ventral subdivision of the hypoglossal nucleus; VL, ventrolateral subdivision of the hypoglossal nucleus. Case
Days 1 2 1 2 3 4 5 6 7 8 9 10 Days 10-14 11 12 13 14 15 16
Adult 17 18 19 20
Muscles injected
Location of motoneuron label
GH
VL
GH
GH GH
GH
GH GH
GH GH GH
GG
GG GG GG GG GG GG GG GG GG GG
GG GG GG GG GG GG
GG GG GG GG
HG
VL
VL VL HG
VL HG
:
HG
VL VL
VL VL VL
V
D
V V V V V V V V V V
D
V V V V V V
V V V V
D
D
104
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Fig. 1. Photomicrograph showing labeled motoneurons in the caudal hypoglossal nucleus following injection of W G A - H R P into the gcnioglossus muscle in (a) the 1 2 day old (case 5) and (b) adult (case 20) rat. In both age groups, labeled motoneurons are located in a discrete region of the ventral hypoglossal nucleus subdivision. Filled arrow demarcates the separation of genioglossus motoneurons from unlabeled neurons in the dorsal region of the hypoglossal nucleus. Unfilled arrow demarcates the separation of genioglossus motoneurons from unlabeled neurons in the ventromedial region of the hypoglossal nucleus. CC, central canal. Bar = 100 ,um.
105
Fig. 2. Photomicrograph showing genioglossus motoneurons in middle levels of the hypoglossal nucleus in the (a) 10-14 day old (case 15) and (b) adult (case 18) rat. In both age groups, labeled motoneurons are located in a discrete region of the ventral hypoglossal nucleus. Arrow demarcates separation of genioglossus motoneurons from unlabeled neurons in the dorsal region of the hypoglossal nucleus. IV, fourth ventricle. Bar = 100 ,um.
106
I thank Mary D. Kramer for comments on the manuscript and Jennifer Gray for technical assistance. This research was supported by the Rhode Island Foundation. 1 Brouilette, R.T. and Thach, B,T., A neuromuscular mechanism maintaining extrathoracic airway patency, J. Appl. Physiol. Resp. Environ. Exercise Physiol., 46 (1979) 772-779. 2 Brozanski, B.S., Guthrie, R.D., Volk, E.A. and Cameron, W.E., Postnatal growth of genioglossal motoneurons, Ped. Pulmonol., 7 (1989) 133-139. 3 Chibuzo, G.A. and Cummings, J.F., An enzyme tracer study of the organization of the somatic motor center for the innervation of different muscles of the tongue: evidence for two sources, J. Comp. Neurol., 205 (1982) 273-281. 4 Gauda, E.B., Miller, M.J., Carlo, W.A., Difiore, J.M., Johnsen, D.C. and Martin, R.J., Genioglossus response to airway occlusion in apneic versus nonapneic infants, Ped. Res., 22(6) (1987) 683-687. 5 Haddad, G.G. and Donnelly, D.F., 0 2 deprivation induces a major depolarization in the brainstem neurons in the adult but not the neonatal rat, J. Physiol., 429 (1990) 411-428. 6 Haddad, G.G., Donnelly, D.F. and Getting, RA., Biophysical properties of hypoglossal neurons in vitro: intracellular studies in adult and neonatal rats, J. Appl. Physiol., 69 (1990) 1509-1517. 7 He, F., Nunez-Abades, P.A. and Cameron, W,E., Morphology of developing hypoglossal motoneurons studied in slice preparations of the rat brainstem, Soc. Neurosci. Abstr., 18(2) (1992) 1049. 8 Kitamura, S., Nishiguchi, T. and Sakai, A., Location of cell somata and the peripheral course of axons of the geniohyoid and thyrohyoid motoneurons: a horseradish peroxidase study in the rat, Exp. Neurol., 79 (1983) 87 96.
9 Klein, B.G., Rhoades, R.W. and Jacquin, M.F., Topography of the facial musculature within the facial (VII) motor nucleus of the neonatal rat, Exp. Brain Res., 81 (1990) 649-653. 10 Krammer, E.B., Rath, T. and Lischka, M.F., Somatotopic organization of the hypoglossal nucleus: a HRP study in the rat, Brain Res., 170 (1979) 533 537. 11 Lawson, E.E., Respiratory control after birth. In V. Chernick and R. Mellins (Eds.), Basic Mechanisms of Pediatric Respiratory Disease, B.C. Decker, Philadelphia, 1991, pp. 288-302. 12 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem, 26 (1978) 106-117. 13 Praud, J.E, D'Allest, A.M., Delaperche, M.F., Bobin, S. and Gaultier, C., Diaphragmatic and genioglossus electromyographic activity at the onset and at the end of obstructive apnea in children with obstructive sleep apnea syndrome, Ped. Res., 23 (1988) 1 4. 14 Sokoloff, A.J. and Deacon, T.W., Musculotopic organization of the hypoglossal nucleus in the cynomolgus monkey, Macaca jhscicularis, J. Comp. Neurol., 324 (1992) 81-93. 15 Uemura, M., Matsuda, K., Kume, M., Takeuchi, Y., Matsushima, R. and Mizuno, N., Topographical arrangement of hypoglossal motoneurons: an HRP study in the cat, Neurosci. Lett., 13 (1979) 99 104. 16 Uemura-Sumi, M., Mizuno, N., Nomura, S., lwahori, N., Takeuchi, Y. and Matsushima, R., Topographical representation of the hypoglossal nerve branches and tongue muscles in the hypoglossal nucleus of macaque monkeys, Neurosci. Lett,, 22 (1981) 31 35. 17 Uemura-Sumi, M., Itoh, M. and Mizuno, N., The distribution of hypoglossal motoneurons in the dog, rabbit and rat, Anat. Embryol., 177 (1988) 389 394.
Neuroscience Letters, 155 (1993) 102-106 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL 09545
Topographic segregation of genioglossus motoneurons in the neonatal rat Alan J. Sokoloff Section of Population Biology, Morphology, and Genetics, Brown University, Providence, RI 02912 (USA) (Received 22 January 1993; Accepted 2 March 1993)
Key words: Genioglossus; WGA-HRP; Motoneuron; Development; Musculotopy; Brainstem; Tongue; Hypoglossal The location of the motoneurons that innervate the main tongue protrusor muscle, genioglossus, was studied in the 1-2 day old, 10 14 day old and adult rat. Following injection of the retrograde axonal tracer WGA-HRP into the genioglossus muscle, labeled neurons were localized to a discrete column within the ventral division of the hypoglossal nucleus in both the neonatal and adult rats. These results demonstrate an adult pattern of genioglossus innervation in the neonatal rat.
Despite recent studies of the physiological and anatomical maturation of hypoglossal nucleus motoneurons in the rat [5-7], the innervation of individual tongue muscles has not been investigated in the developing animal. The hypoglossal nucleus (HGN) of the adult rat is composed of three subdivisions: a dorsal subdivision which innervates the tongue retrusor muscles, hyoglossus and styloglossus; a ventral subdivision which innervates the tongue protrusor muscle, genioglossus; and a ventrolateral subdivision, separated ventrally from the main portion of the HGN, which innervates the suprahyoid muscle, geniohyoid [10, 17]. A similar discrete musculotopic innervation of retrusor, protrusor and geniohyoid muscles has been reported for the adult cat and macaque [14-16]. Musculotopic organization of the hypoglossal nucleus of mammals may be less discrete in the neonate than in the adult. Chibuzo and Cummings [3] reported overlap in the location of the HGN motoneurons which innervate retrusor and protrusor muscles in the neonatal dog. They also reported that the geniohyoid and genioglossus muscles each receive innervation from both ventral and ventrolateral HGN subdivisions. A ventral and ventrolateral HGN innervation of the genioglossus muscle was also observed in the kitten [2]. These studies, however, did not explore possible ontogenetic, species-specific or
Correspondence." A. Sokoloff, Department of Physiology, MS 409, Hahnemann University, Broad and Vine, Philadelphia, PA 19102-1192, USA.
methodological factors that might explain reported differences in neonatal and adult HGN musculotopic organization. To investigate the development of HGN musculotopy, the innervation of the genioglossus muscle was studied in the 1-2 day old, 10-14 day old and adult rat. The genioglossus muscle was chosen for study because of its important function in the maintenance of upper airway patency [1, 4, 13]. Under metofane (1-2 day old rats) or a combination of ketamine (40-74 mg/kg, i.p.) and xylazine (5-8 mg/kg, i.p.) anaesthesia, the genioglossus muscle was exposed by pulling the tongue anteriorly and superiorly from the mouth. A single injection of 0.2~.5/ll of the retrograde axonal tracer wheatgerm agglutinin-conjugated horseradish peroxidase (WGA-HRP, Sigma, 5% in distilled water) was made into the genioglossus muscle in ten 1-2 day old rats with a glass micropipette attached to a 1/.tl Hamilton syringe. A single injection of 0.5-1.5 /11 of 5% WGA-HRP was made into the genioglossus muscle of six 10-14 day old rats and 4 adult rats with a 10 1ll Hamilton syringe. Eighteen to 36 h following WGA-HRP injection, the animals were euthanized with pentobarbital sodium (5055 mg/kg, i.p.) and perfused through the left ventricle with 100-300 ml of 0.9% saline, followed first by 100-300 ml of a fixative containing 0.5% paraformaldehyde, 2.0% glutaraldehyde and 2.0% sucrose in a 0.1 M phosphate buffer, and second by 100-300 ml of 10% sucrose in 0.1 M phosphate buffer. The brainstem, tongue and geniohyoid muscle were removed and stored for 1-3 days in a 20% sucrose solution of 0.1 M phosphate buffer at 4 ° C. The brainstem was cut serially in 75 or 100ktm transverse
103
sections on a freezing microtome. Frozen 100 or 125/2m sections of the tongue and geniohyoid muscle were cut in sagittal or transverse planes in order to determine the injection site. Brainstem and tongue tissue was reacted with tetramethylbenzidine (TMB; following Mesulam [12]) for 2~, h, mounted on gelatin-subbed slides, counterstained in neutral red and coverslipped with Permount. Slides were examined under brightfield, darkfield and polarized light microscopy to determine the extent of the injection site and resulting brainstem label. Brainstem and tongue sections with TMB label were photographed and traced. In each case, the WGA-HRP injection was centered in the genioglossus muscle (Table I). In some cases, the injection spread ventrally to involve fibers of the geniohyoid muscle and/or laterally to involve fibers of the hyoglossus muscle. The pattern of HGN label was similar in all age groups. Following exclusive injection of the genioglossus muscle, labeled neurons formed an uninterrupted column in the dorsolateral corner of the ventral HGN subdivision (Table I; Figs. 1 and 2). Following injections which involved geniohyoid in addition to genioglossus fibers, labeled neurons were additionally located in the ventrolateral HGN subdivision. All cases with geniohyoid injection resulted in ventrolateral subdivision label, and ventrolateral subdivision label was only observed in cases with geniohyoid involvement (Table I). In three cases, labeled neurons were also clearly present in the dorsal HGN subdivision. These cases were distinguished by the involvement of hyoglossus fibers (Table I). The results of the present study demonstrate that genioglossus motoneurons are localized to a discrete column in the ventral HGN subdivision in neonatal and adult rats. These results indicate that an adult pattern of genioglossus motoneuron innervation is established by day 1 in the neonatal rat. This finding is similar to the report of an adult musculotopic organization of the facial nucleus in the newborn rat [9]. The location of genioglossus motoneurons reported in the present study is in agreement with the results of previous studies of the adult rat. Both Krammer et al. [10] and Uemura-Sumi et al. [17] localized genioglossus motoneurons to the ventral HGN subdivision in the adult rat. These studies also demonstrated that motoneurons in the ventrolateral HGN subdivision innervate the geniohyoid muscle and motoneurons in the rostral levels of the dorsal HGN subdivision innervate the hyoglossus muscle [8, 10, 17]. The association of ventrolateral HGN label with geniohyoid injection and dorsolateral HGN label with hyoglossus injection in the present study (Table I), suggests that an adult musculotopic organization of the HGN is formed by day 2 in the rat.
The exclusive ventral HGN subdivision innervation of the genioglossus muscle in the neonatal rat is in contrast to reports of both ventral and ventrolateral subdivision label following genioglossus injection in the neonatal kitten and dog [2, 3; but see 17]. Differences in the findings of the present study from those previously reported may reflect different techniques, different amounts of tracer injected (e.g. 40-50/21 in [3] versus 0.2-1.5/11 in the present study) and/or species differences in the organization or development of HGN musculotopy. Given the current interest in the development and control of respiration and upper airway patency in humans [4, 11, 13], further studies of the development of hypoglossal nucleus organization and function are warranted.
TABLE I LOCATION OF TONGUE INJECTION SITES AND CORRESPONDING HYPOGLOSSAL M O T O N E U R O N LABEL IN THE NEONATAL AND ADULT RAT D, dorsal subdivision of the hypoglossal nucleus; GG, genioglossus muscle; GH, geniohyoid muscle; HG, hyoglossus muscle, V, ventral subdivision of the hypoglossal nucleus; VL, ventrolateral subdivision of the hypoglossal nucleus. Case
Days 1 2 1 2 3 4 5 6 7 8 9 10 Days 10-14 11 12 13 14 15 16
Adult 17 18 19 20
Muscles injected
Location of motoneuron label
GH
VL
GH
GH GH
GH
GH GH
GH GH GH
GG
GG GG GG GG GG GG GG GG GG GG
GG GG GG GG GG GG
GG GG GG GG
HG
VL
VL VL HG
VL HG
:
HG
VL VL
VL VL VL
V
D
V V V V V V V V V V
D
V V V V V V
V V V V
D
D
104
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~
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Fig. 1. Photomicrograph showing labeled motoneurons in the caudal hypoglossal nucleus following injection of W G A - H R P into the gcnioglossus muscle in (a) the 1 2 day old (case 5) and (b) adult (case 20) rat. In both age groups, labeled motoneurons are located in a discrete region of the ventral hypoglossal nucleus subdivision. Filled arrow demarcates the separation of genioglossus motoneurons from unlabeled neurons in the dorsal region of the hypoglossal nucleus. Unfilled arrow demarcates the separation of genioglossus motoneurons from unlabeled neurons in the ventromedial region of the hypoglossal nucleus. CC, central canal. Bar = 100 ,um.
105
Fig. 2. Photomicrograph showing genioglossus motoneurons in middle levels of the hypoglossal nucleus in the (a) 10-14 day old (case 15) and (b) adult (case 18) rat. In both age groups, labeled motoneurons are located in a discrete region of the ventral hypoglossal nucleus. Arrow demarcates separation of genioglossus motoneurons from unlabeled neurons in the dorsal region of the hypoglossal nucleus. IV, fourth ventricle. Bar = 100 ,um.
106
I thank Mary D. Kramer for comments on the manuscript and Jennifer Gray for technical assistance. This research was supported by the Rhode Island Foundation. 1 Brouilette, R.T. and Thach, B,T., A neuromuscular mechanism maintaining extrathoracic airway patency, J. Appl. Physiol. Resp. Environ. Exercise Physiol., 46 (1979) 772-779. 2 Brozanski, B.S., Guthrie, R.D., Volk, E.A. and Cameron, W.E., Postnatal growth of genioglossal motoneurons, Ped. Pulmonol., 7 (1989) 133-139. 3 Chibuzo, G.A. and Cummings, J.F., An enzyme tracer study of the organization of the somatic motor center for the innervation of different muscles of the tongue: evidence for two sources, J. Comp. Neurol., 205 (1982) 273-281. 4 Gauda, E.B., Miller, M.J., Carlo, W.A., Difiore, J.M., Johnsen, D.C. and Martin, R.J., Genioglossus response to airway occlusion in apneic versus nonapneic infants, Ped. Res., 22(6) (1987) 683-687. 5 Haddad, G.G. and Donnelly, D.F., 0 2 deprivation induces a major depolarization in the brainstem neurons in the adult but not the neonatal rat, J. Physiol., 429 (1990) 411-428. 6 Haddad, G.G., Donnelly, D.F. and Getting, RA., Biophysical properties of hypoglossal neurons in vitro: intracellular studies in adult and neonatal rats, J. Appl. Physiol., 69 (1990) 1509-1517. 7 He, F., Nunez-Abades, P.A. and Cameron, W,E., Morphology of developing hypoglossal motoneurons studied in slice preparations of the rat brainstem, Soc. Neurosci. Abstr., 18(2) (1992) 1049. 8 Kitamura, S., Nishiguchi, T. and Sakai, A., Location of cell somata and the peripheral course of axons of the geniohyoid and thyrohyoid motoneurons: a horseradish peroxidase study in the rat, Exp. Neurol., 79 (1983) 87 96.
9 Klein, B.G., Rhoades, R.W. and Jacquin, M.F., Topography of the facial musculature within the facial (VII) motor nucleus of the neonatal rat, Exp. Brain Res., 81 (1990) 649-653. 10 Krammer, E.B., Rath, T. and Lischka, M.F., Somatotopic organization of the hypoglossal nucleus: a HRP study in the rat, Brain Res., 170 (1979) 533 537. 11 Lawson, E.E., Respiratory control after birth. In V. Chernick and R. Mellins (Eds.), Basic Mechanisms of Pediatric Respiratory Disease, B.C. Decker, Philadelphia, 1991, pp. 288-302. 12 Mesulam, M.-M., Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a noncarcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem, 26 (1978) 106-117. 13 Praud, J.E, D'Allest, A.M., Delaperche, M.F., Bobin, S. and Gaultier, C., Diaphragmatic and genioglossus electromyographic activity at the onset and at the end of obstructive apnea in children with obstructive sleep apnea syndrome, Ped. Res., 23 (1988) 1 4. 14 Sokoloff, A.J. and Deacon, T.W., Musculotopic organization of the hypoglossal nucleus in the cynomolgus monkey, Macaca jhscicularis, J. Comp. Neurol., 324 (1992) 81-93. 15 Uemura, M., Matsuda, K., Kume, M., Takeuchi, Y., Matsushima, R. and Mizuno, N., Topographical arrangement of hypoglossal motoneurons: an HRP study in the cat, Neurosci. Lett., 13 (1979) 99 104. 16 Uemura-Sumi, M., Mizuno, N., Nomura, S., lwahori, N., Takeuchi, Y. and Matsushima, R., Topographical representation of the hypoglossal nerve branches and tongue muscles in the hypoglossal nucleus of macaque monkeys, Neurosci. Lett,, 22 (1981) 31 35. 17 Uemura-Sumi, M., Itoh, M. and Mizuno, N., The distribution of hypoglossal motoneurons in the dog, rabbit and rat, Anat. Embryol., 177 (1988) 389 394.