Development of the human hypoglossal nucleus from mid-gestation to the perinatal period: A morphological study.

INTRODUCTION: The human hypoglossal nucleus (nXII) was morphologically examined from mid-gestation to the perinatal period. MATERIALS/METHODS: Serial brain sections from 6 preterm and 4 perinatal infants aged 21-43 postmenstrual weeks (PW) were stained with the Klüver-Barrera method. Following microscopic observation, morphometric parameters (volume, neuronal number, and neuronal profile area [PA]) were analysed. RESULTS: Two types of neurons, motor and non-motor neurons, were observed at 21 PW. The motor neurons were distributed into clusters, which were not completely separated. The non-motor neurons were dispersed among the motor neurons. Myelination of the hypoglossal nerve roots was noted at 21 PW, when degenerated neurons were sporadically encountered. To a lesser extent, they were seen until 35 PW. The nXII volume increased exponentially with age. Conversely, the neuronal numerical density decreased exponentially, while the total number remained relatively stable. The neuronal PA increased gradually, with a greater rate of increase measured in the caudal part. CONCLUSIONS: In the human nXII, motor and non-motor neurons are distinguishable from mid-gestation. Then, while the nXII expands exponentially in volume, the two types of neurons change in number and PA almost in parallel during the second half of gestation. Natural neuronal death may also occur.

Developmental changes in the morphology of mouse hypoglossal motor neurons.

Hypoglossal motor neurons (XII MNs) innervate tongue muscles important in breathing, suckling and vocalization. Morphological properties of 103 XII MNs were studied using Neurobiotin™ filling in transverse brainstem slices from C57/Bl6 mice (n = 34) from embryonic day (E) 17 to postnatal day (P) 28. XII MNs from areas thought to innervate different tongue muscles showed similar morphology in most, but not all, features. Morphological properties of XII MNs were established prior to birth, not differing between E17-18 and P0. MN somatic volume gradually increased for the first 2 weeks post-birth. The complexity of dendritic branching and dendrite length of XII MNs increased throughout development (E17-P28). MNs in the ventromedial XII motor nucleus, likely to innervate the genioglossus, frequently (42 %) had dendrites crossing to the contralateral side at all ages, but their number declined with postnatal development. Unexpectedly, putative dendritic spines were found in all XII MNs at all ages, and were primarily localized to XII MN somata and primary dendrites at E18-P4, increased in distal dendrites by P5-P8, and were later predominantly found in distal dendrites. Dye-coupling between XII MNs was common from E18 to P7, but declined strongly with maturation after P7. Axon collaterals were found in 20 % (6 of 28) of XII MNs with filled axons; collaterals terminated widely outside and, in one case, within the XII motor nucleus. These results reveal new morphological features of mouse XII MNs, and suggest that dendritic projection patterns, spine density and distribution, and dye-coupling patterns show specific developmental changes in mice.

Transcriptional regulation of mouse hypoglossal motor neuron somatotopic map formation.

Somatic motor neurons in the hypoglossal nucleus innervate tongue muscles controlling vital functions such as chewing, swallowing and respiration. Formation of functional hypoglossal nerve circuits depends on the establishment of precise hypoglossal motor neuron maps correlating with specific tongue muscle innervations. Little is known about the molecular mechanisms controlling mammalian hypoglossal motor neuron topographic map formation. Here we show that combinatorial expression of transcription factors Runx1, SCIP and FoxP1 defines separate mouse hypoglossal motor neuron groups with different topological, neurotransmitter and calcium-buffering phenotypes. Runx1 and SCIP are coexpressed in ventromedial hypoglossal motor neurons involved in control of tongue protrusion whereas FoxP1 is expressed in dorsomedial motor neurons associated with tongue retraction. Establishment of separate hypoglossal motor neuron maps depends in part on Runx1-mediated suppression of ventrolateral and dorsomedial motor neuron phenotypes and regulation of FoxP1 expression pattern. These findings suggest that combinatorial actions of Runx1, SCIP and FoxP1 are important for mouse hypoglossal nucleus somatotopic map formation.

Contribution of the Runx1 transcription factor to axonal pathfinding and muscle innervation by hypoglossal motoneurons.

The runt-related transcription factor Runx1 contributes to cell type specification and axonal targeting projections of the nociceptive dorsal root ganglion neurons. Runx1 is also expressed in the central nervous system, but little is known of its functions in brain development. At mouse embryonic day (E) 17.5, Runx1-positive neurons were detected in the ventrocaudal subdivision of the hypoglossal nucleus. Runx1-positive neurons lacked calcitonin gene-related peptide (CGRP) expression, whereas Runx1-negative neurons expressed CGRP. Expression of CGRP was not changed in Runx1-deficient mice at E17.5, suggesting that Runx1 alone does not suppress CGRP expression. Hypoglossal axon projections to the intrinsic vertical (V) and transverse (T) tongue muscles were sparser in Runx1-deficient mice at E17.5 compared to age-matched wild-type littermates. Concomitantly, vesicular acetylcholine transporter-positive axon terminals and acetylcholine receptor clusters were less dense in the V and T tongue muscles of Runx1-deficient mice. These abnormalities in axonal projection were not caused by a reduction in the total number hypoglossal neurons, failed synaptogenesis, or tongue muscles deficits. Our results implicate Runx1 in the targeting of ventrocaudal hypoglossal axons to specific tongue muscles. However, Runx1 deficiency did not alter neuronal survival or the expression of multiple motoneuron markers as in other neuronal populations. Thus, Runx1 appears to have distinct developmental functions in different brain regions.

Influence of developmental nicotine exposure on spike-timing precision and reliability in hypoglossal motoneurons.

Smoothly graded muscle contractions depend in part on the precision and reliability of motoneuron action potential generation. Whether or not a motoneuron generates spikes precisely and reliably depends on both its intrinsic membrane properties and the nature of the synaptic input that it receives. Factors that perturb neuronal intrinsic properties and/or synaptic drive may compromise the temporal precision and the reliability of action potential generation. We have previously shown that developmental nicotine exposure (DNE) alters intrinsic properties and synaptic transmission in hypoglossal motoneurons (XIIMNs). Here we show that the effects of DNE also include alterations in spike-timing precision and reliability, and spike-frequency adaptation, in response to sinusoidal current injection. Current-clamp experiments in brainstem slices from neonatal rats show that DNE lowers the threshold for spike generation but increases the variability of spike-timing mechanisms. DNE is also associated with an increase in spike-frequency adaptation and reductions in both peak and steady-state firing rate in response to brief, square wave current injections. Taken together, our data indicate that DNE causes significant alterations in the input-output efficiency of XIIMNs. These alterations may play a role in the increased frequency of obstructive apneas and altered suckling strength and coordination observed in nicotine-exposed neonatal humans.

Nerve terminal distribution in the human tongue intrinsic muscles: An immunohistochemical study using midterm fetuses.

Intrinsic tongue muscles, especially the transverse and vertical (T&V) muscles, regulate the shape of the tongue. However, little information is available on the nerve distribution pattern in human T&V muscles. Using S100 protein immunohistochemistry for paraffin-embedded histology, we investigated semiserial sagittal or frontal sections of eight human fetal tongues (180-240 mm crown-rump length: CRL). The height of the T&V muscle bundle showed a threefold difference between specimens with a small and a large CRL. Thus, the T&V muscles were still growing at the stages examined. In the intrinsic longitudinal muscles and all extrinsic tongue muscles, we observed the typical motor endplate band. In lower-magnification views, the T&V muscles also appeared to carry the band in the lateral part of the tongue, where the genioglossus muscle fibers did not cross these muscles. However, in higher magnification views, the nerve terminal distribution in the T&V muscles showed a unique rule: the nerve terminal for the transverse muscle bundle was located distantly from that of the adjacent vertical muscle bundle. This pattern seemed to be established during the stages examined. To provide such "distantly separated nerve terminals," thin nerve twigs took a highly curved course oblique to the T&V muscle bundles. We hypothesize that the unique nerve course and terminal distribution in the T&V muscles are a result of sorting to provide a good functional match between the nerve fiber and the muscle bundle. After sorting, the T&V muscle cells may initiate proliferation to increase the muscle bundle.

Cranial nerve fasciculation and Schwann cell migration are impaired after loss of Npn-1.

Interaction of the axon guidance receptor Neuropilin-1 (Npn-1) with its repulsive ligand Semaphorin 3A (Sema3A) is crucial for guidance decisions, fasciculation, timing of growth and axon-axon interactions of sensory and motor projections in the embryonic limb. At cranial levels, Npn-1 is expressed in motor neurons and sensory ganglia and loss of Sema3A-Npn-1 signaling leads to defasciculation of the superficial projections to the head and neck. The molecular mechanisms that govern the initial fasciculation and growth of the purely motor projections of the hypoglossal and abducens nerves in general, and the role of Npn-1 during these events in particular are, however, not well understood. We show here that selective removal of Npn-1 from somatic motor neurons impairs initial fasciculation and assembly of hypoglossal rootlets and leads to reduced numbers of abducens and hypoglossal fibers. Ablation of Npn-1 specifically from cranial neural crest and placodally derived sensory tissues recapitulates the distal defasciculation of mixed sensory-motor nerves of trigeminal, facial, glossopharyngeal and vagal projections, which was observed in Npn-1(-/-) and Npn-1(Sema-) mutants. Surprisingly, the assembly and fasciculation of the purely motor hypoglossal nerve are also impaired and the number of Schwann cells migrating along the defasciculated axonal projections is reduced. These findings are corroborated by partial genetic elimination of cranial neural crest and embryonic placodes, where loss of Schwann cell precursors leads to aberrant growth patterns of the hypoglossal nerve. Interestingly, rostral turning of hypoglossal axons is not perturbed in any of the investigated genotypes. Thus, initial hypoglossal nerve assembly and fasciculation, but not later guidance decisions depend on Npn-1 expression and axon-Schwann cell interactions.

Embryonic anastomosis between hypoglossal nerves.

This article presents two cases of anastomosis of hypoglossal nerves in the suprahyoid region in human embryos of CR length 10.75 and 17.5 mm. This variation was studied in two human specimens at this stage of development and compared with the normal arrangement of the hypoglossal nerves in embryos at the same stage. The anastomotic branches were of similar caliber to the main trunks. In both cases the anastomosis was located dorsal to the origin of the geniohyoid muscles and caudal to the genioglossus muscles, lying transversally over the cranial face of the body of the hyoid bone anlage. The anastomosis formed a suprahyoid nerve chiasm on the midline in the embryo of 10.75 mm CR length.

Vagus, hypoglossal, and median nerves in human development.

In spite of the wealth of literature on the changes of the neurons in development of the brainstem and the spinal cord in vertebrates, the alterations of the cranial nerves and somatic nerves during the prenatal period was largely neglected. Particularly in humans, little information was available. The article reports the changes in the vagus, hypoglossal, and median nerves in the fetus and term babies. The changes of proportion of different-sized nerve fibers are documented. The patterns were different in the three nerves and the hypoglossal nerve seemed to show "pruning" of fibers during this period.

Time-specific effects of ethanol exposure on cranial nerve nuclei: gastrulation and neuronogenesis.

During the development of the central nervous system, neurons pass through critical periods or periods of vulnerability. We explored periods of vulnerability for cranial nerve nuclei by determining the effects of acute exposure to ethanol during development on the number of neurons in mature brainstem. Long-Evans rats were injected with 2.9 g ethanol/kg body weight on one day between gestational day (G) 7 and G13, inclusive. Two hours later, animals received a second injection of 1.45 g/kg. Controls were injected with equivalent volumes of saline. Brainstems of 31-day-old offspring were cryosectioned and stained with cresyl violet. Stereological methods were used to determine the volume and numerical density of neurons in three trigeminal sensory nuclei (the principal sensory nucleus of the trigeminal nerve, and the oral and interpolar subnuclei of the spinal trigeminal nuclear complex) and three motor nuclei (the trigeminal, facial, and hypoglossal nuclei). The numbers of neurons in most nuclei were lower following early (on G7 and/or G8) or later (on G12 and/or G13) exposure. Only the trigeminal interpolar nucleus was affected by neither early nor late ethanol exposure. Thus, prenatal exposure to ethanol affects the number of neurons in brainstem nuclei in a time-dependent manner. Windows of vulnerability coincide with gastrulation (G7/G8) and the period of neuronal generation (G12/G13).

[Anatomy of the hypoglossal nerve and the hypoglossal ansa cervicalis]. / Anatomie du couple nerf hypoglosse, anse cervicale.

The hypoglossal nerve is the motor nerve of the tongue and the ansa cervicalis is a motor nerve for the sub-hyoid muscles. The hypoglossal nerve seems to give the innervation of the thyrohyoid although it is a sub-hyoid muscle. Most of axons in the ansa cervicalis arise from the three first cervical nerves. These nerves are in close contact because of the cervical ontogeny of the tongue and the hypoglossal nerve. Nerve impulse in the superior root of the ansa cervicalis runs caudally to rostrally. This is why neurotization techniques using the superior root of the ansa cervicalis produce poor results in the treatment of facial palsy sequelae.

Persistent carotid-vertebrobasilar anastomoses: how and why differentiating them?

The persistent carotid-vertebrobasilar anastomoses (PCVBA) can be explained by an interruption of the vertebrobasilar system (VBS) embryogenesis. We present two very rare cases of persistent anastomoses: a hypoglossal artery and a type I proatlantal artery, insisting on the angiographic criteria allowing differentiation. After a brief review of the embryogenesis of the VBS, we describe the different types of persistent anastomoses (hypoglossal, type I and II proatlantal, trigeminal and otic arteries). We will insist on the potential risks, not well-known, but typical of each anastomosis. PCVBA usually are incidental findings but imaging follow-up may be required since aneurysms may develop.

Androgen receptors in the embryonic zebra finch hindbrain suggest a function for maternal androgens in perihatching survival.

Bird embryos are exposed to maternal androgens deposited in the egg, but the role of these hormones in embryonic development and hatchling survival is unclear. To identify possible target organs, we used in situ hybridization to study the distribution of androgen receptor (AR) RNA in the developing zebra finch brain. The first brain expression domain of AR mRNA is in the hindbrain. From embryonic day 7 (E7) onward, when the hypoglossal motor nucleus (nXII) has just formed, there was AR mRNA expression in both its lingual (nXIIl) and its tracheosyringeal (nXIIts) parts, and this was the major site of hindbrain expression at all embryonic stages and in both sexes. From E8 onward, we also found AR mRNA in the supraspinal motor nucleus (nSSp), which innervates neck muscles. Furthermore, the syrinx, the target of the nXIIts, contained AR mRNA by E10, localized principally in the perichondria. Muscle was first evident in the syringeal region at E9, but no AR was detected in syringeal muscles until after hatching. The expression pattern of AR in the zebra finch embryo suggests that maternal androgens act via AR in the brainstem and syrinx to influence hatching as well as acoustic and visual components of food-begging behavior. Maternal androgens seem unlikely to function in the development of sexual dimorphisms in the zebra finch nXIIts and syrinx, insofar as these are not evident until between 10 and 20 days posthatching.

Cranial nerve XII: the hypoglossal nerve.

The hypoglossal nerve, cranial nerve XII, is the motor supply of the tongue. An understanding of the intracranial and extracranial components is fundamental in the evaluation of hypoglossal pathology. The following discussion of the evaluation of the hypoglossal nerve will involve the embryology, anatomy, clinical basis, and imaging techniques with pathologic correlations.

Macrophage-stimulating protein is a neurotrophic factor for embryonic chicken hypoglossal motoneurons.

Macrophage-stimulating protein (MSP) exerts a variety of biological actions on many cell types, but has no known functions in the brain. MSP is structurally related to hepatocyte growth factor (HGF), another pleiotropic factor whose many functions include promoting neuronal survival and growth. To investigate whether MSP is also capable of acting as a neurotrophic factor, we purified hypoglossal motoneurons from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. MSP promoted the in vitro survival of these neurons during the period of naturally occurring neuronal death and enhanced the growth of neurites from these neurons. MSP mRNA was detected in the developing tongue whose musculature is innervated by hypoglossal neurons. Our study demonstrates that MSP is a neurotrophic factor for a population of developing motoneurons.

The effects of prenatal X-irradiation on hypoglossal nucleus: a GFAP immunohistochemical study.

The effects of prenatal X-irradiation on hypoglossal (XII) nucleus were investigated in the rat. Pregnant animals were exposed to a single whole body X-irradiation on day 11 and 16 of gestation at a does of 1.3 Gy. The offspring were killed at 7-14 days of age for the histological and immunohistochemical observations. Nissl staining revealed no significant changes of XII motoneurons in these experimental animals. In the control case it was of interest that expression of glial fibrillary acidic protein-immunoreactivity (GFAP-IR) is largely confined to the dorsomedial region including the XII nucleus at the level caudal to the obex. Exposure of X-irradiation on day 16 of gestation led to similar expression of GFAP-IR in the nucleus at the same level. However, exposure on day 11 of gestation apparently led to strong expression of GFAP-IR in the XII nucleus at the level caudal to the obex and the expression was observed to extend rostrally. The GFAP-IR cells showed hypertrophy of cell bodies and longer cytoplasmic processes. Horse-radish peroxidase (HRP) injection into the tip of the tongue including the intrinsic muscles resulted in retrograde labeling in the ventromedial portion of the XII nucleus bilaterally from +0.30 to -1.25 mm. The present study would indicate that motoneurons of the XII nucleus supplying mainly the intrinsic and partly the extrinsic tongue muscles are more sensitive to X-ray exposure before the formation of the XII nucleus.

Migration of hypoglossal myoblast precursors.

The intrinsic hypoglossal musculature develops from precursor myoblasts which undergo long-range migration from the occipital somites to the tongue. Little detail is known about the precise spatiotemporal pathway taken by these cells or the factors controlling migration. In this study, chick/quail chimeras in which the occipital paraxial mesoderm is quail derived, reveal that the pathway taken by the tongue muscle progenitors is both complex and highly specific. Precursor myoblasts are Pax-3 positive cells which descend from the somite and migrate around the pharyngeal endoderm. They then course rostrally, following the base of the pharynx, remaining in a tight strand. We have examined a number of factors implicated in the control of migration of the hypoglossal precursors. Replacement of the occipital somites with those originating in the flank reveals that intrinsic differences do not exist between these somites with respect to their capacity to respond to migratory cues. The lack of high level HGF/SF expression along the pathway of the migrating hypoglossal precursors suggests that this factor is not involved in the actual process of migration of the hypoglossal precursors to the tongue. The pathway followed by the migrating precursors is identical to that of both the developing hypoglossal nerve and the circumpharyngeal crest--a subpopulation of the cranial neural crest, and importantly these populations utilize this pathway before the myoblast precursors. However, ablation neither of the hypoglossal nerve nor of the neural crest results in a perturbation in the ability of this Pax-3 positive population to migrate. This demonstrates that migration of the precursors is independent of both of these cell populations, and that it is controlled by the peripheral tissues.

Chronic placental insufficiency in the fetal guinea pig affects neurochemical and neuroglial development but not neuronal numbers in the brainstem: a new method for combined stereology and immunohistochemistry.

This study has examined the development of the brainstem in a suboptimal intrauterine environment induced via chronic placental insufficiency in the fetal guinea pig. Placental insufficiency was produced by unilateral ligation of the maternal uterine artery at mid-gestation (term = 66-68 days) resulting in the production of growth-retarded fetuses that are chronically hypoxic and malnourished. The structural and neurochemical development of brainstem nuclei either directly or indirectly related to cardiorespiratory control were analysed by using new stereological methods and immunohistochemistry. A technique was devised to enable the procedures to be performed on alternate frozen sections. There were no significant differences between control and growth-retarded fetuses in the total number of neurons, area of neuronal somata or volume of the hypoglossal nucleus. Quantitative densitometry was used to measure immunohistochemical staining in the brainstem of growth-retarded fetuses compared to controls and revealed a significant (P < 0.02) decrease in substance P(SP)-immunoreactivity in the spinal trigeminal nucleus and a significant (P < 0.05) increase in met-enkephalin-immunoreactivity in the hypoglossal nucleus. Counts of stained neurons demonstrated a significant increase in the density of SP-positive neurons in the nucleus tractus solitarius (P < 0.05) and of met-enkephalin-positive neurons in the ventral medullary reticular formation (P < 0.05). There was also a proliferation of astrocytes, as determined by immunoreactivity to glial fibrillary acidic protein in the dorsal motor nucleus of the vagus, nucleus tractus solitarius and more generally around blood vessels throughout the brainstem. Thus, these results have been shown that although chronic intrauterine deprivation does not alter neuronal numbers, at least in the hypoglossal nucleus, there is a proliferation of astrocytes, and the expression of neurotransmitters/neuromodulators is markedly effected in some of the nuclei involved with cardiorespiratory control.

Initial innervation of embryonic rat tongue and developing taste papillae: nerves follow distinctive and spatially restricted pathways.

The rat tongue has an extensive, complex innervation from four cranial nerves. However, the precise developmental time course and spatial routes of these nerves into the embryonic tongue are not known, although this knowledge is crucial for studying mechanisms that regulate development and innervation of the lingual taste organs, gustatory papillae and resident taste buds. We determined the initial spatial course of nerves in the developing tongue and papillae, and tested the hypothesis that sensory nerves first innervate the tongue homogeneously and then retract to more densely innervate papillae and taste buds. Antibodies to GAP-43 and neurofilaments were used to label nerve fibers in rat embryo heads from gestational day 11 through 16 (E11-E16). Serial sagittal sections were traced and reconstructed to follow paths of each nerve. In E11 rat, geniculate, trigeminal and petrosal ganglia were labeled and fibers left the ganglia and extended toward respective branchial arches. At E13 when the developing tongue is still a set of tissue swellings, the combined chorda/lingual, hypoglossal and petrosal nerves approached the lingual swellings from separate positions. Only the chorda/lingual entered the tongue base at this stage. At E14 and E15, the well-developed tongue was innervated by all four cranial nerves. However, the nerves maintained distinctive entry points and relatively restricted mesenchymal territories within the tongue, and did not follow one another in common early pathways. Furthermore, the chorda/lingual and glossopharyngeal nerves did not set up an obvious prepattern for gustatory papilla development, but rather seemed attracted to developing papillae which became very densely innervated compared to surrounding epithelium at E15. To effect this dense papilla innervation, sensory nerves did not first innervate the tongue in a homogeneous manner with subsequent retraction and/or extensive redirection of fibers into the taste organs. Results contribute to a set of working principles for development of tongue innervation. Points of entry and initial neural pathways are restricted from time of tongue formation through morphogenesis, suggesting distinctive lingual territories for each nerve. Thus, sensory and motor nerves distribute independently of each other, and sensory innervation to anterior and posterior tongue remains discrete. For taste organ innervation, gustatory papillae are not induced by a prepatterned nerve distribution. In fact, papillae might attract dense sensory innervation because neither chorda/lingual nor glossopharyngeal nerve grows homogeneously to the lingual epithelium and then redistributes to individual papillae.