Fetal facial nerve course in the ear region revisited.

PURPOSE: The aim of this study was to re-examine the structures that determine course of the facial nerve (FN) in the fetal ear region. MATERIALS AND METHODS: We used sagittal or horizontal sections of 28 human fetuses at 7-8, 12-16, and 25-37 weeks. RESULTS: The FN and the chorda tympani nerve ran almost parallel until 7 weeks. The greater petrosal nerve (GPN) ran vertical to the distal FN course due to the trigeminal nerve ganglion being medial to the geniculate ganglion at 7 weeks. Afterwards, due to the radical growth of the former ganglion, the GPN became an anterior continuation of the FN. The lesser petrosal nerve ran straight, parallel to the FN at 7 weeks, but later, it started to wind along the otic capsule, possibly due to the upward invasion of the tympanic cavity epithelium. Notably, the chorda tympanic nerve origin from the FN, and the crossing between the vagus nerve branch and the FN, was located outside of the temporal bone even at 37 weeks. The second knee of the FN was not evident, in contrast to the acute anterior turn below the chorda tympanic nerve origin. In all examined fetuses, the apex of the cochlea did not face the middle cranial fossa, but the tympanic cavity. CONCLUSION: Topographical relation among the FN and related nerves in the ear region seemed not to be established in the fetal age but after birth depending on growth of the cranial fossa.

Blood supply to the chorda tympani: A review and clinical applications.

The chorda tympani (CT) is important in gustatory sensation from the anterior two-thirds of the tongue and in secretomotor innervation to the submandibular and sublingual glands. Although the blood supply to the CT is not well delineated in the literature, some studies have shown that a posterior tympanic branch of the stylomastoid artery supplies CT at its origin from the mastoid segment of the facial nerve. We review the blood supply to the CT comprehensively. A better understanding of the vasculature involved is required to prevent iatrogenic injury during middle ear surgery and complications secondary to ischemia.

Extensive reorganization of primary afferent projections into the gustatory brainstem induced by feeding a sodium-restricted diet during development: less is more.

Neural development is especially vulnerable to environmental influences during periods of neurogenesis and rapid maturation. In fact, short periods of environmental manipulations confined to embryonic development lead to significant changes in morphology and function. A guiding principal emerging from studies of sensory systems is that experimentally induced effects are most dramatic in higher neural levels (e.g., cortex) and primarily involve postnatal synaptic refinements. In contrast to other sensory systems, the gustatory system is particularly susceptible to the effects of deprivation much earlier and with profound changes evident in the brainstem. Here we show that feeding pregnant rats a custom diet featuring a low-sodium content for 9 d before the tongue appears in the fetus produces extensive restructuring of the gustatory brainstem. Rats born to mothers fed the custom diet from embryonic day 3 (E3) to E12 have terminal field volumes of the greater superficial petrosal, chorda tympani, and glossopharyngeal nerves at adulthood that are expanded as much as 10 times beyond that found in rats fed a standard rat chow. The widespread alterations are not attributable to increased numbers of nerve cells, increased target size, or obvious changes in peripheral taste function. Moreover, we show that the limited period of feeding the custom diet has much larger effects than if rats were fed the diet to postweaning ages. Our results suggest that early periods of altered experience, especially during nucleus of the solitary tract neurogenesis, leads to a restructuring of the gustatory brainstem, which in turn may impact the control of sensory and homeostatic processes.

Early prenatal critical period for chorda tympani nerve terminal field development.

In order to determine whether the developing central gustatory system responds to dietary manipulation during restricted developmental periods, terminal fields of the chorda tympani nerve within the nucleus of the solitary tract were investigated via anterograde transport of horseradish peroxidase in control rats and in rats in which a low sodium diet was systematically fed during specific periods of development. Rats fed a low sodium diet (0.03% NaCl) from embryonic day 3 (E3) to day E12 and then fed a sodium replete diet to at least 60 days postnatal exhibited enlarged and irregularly shaped chorda tympani terminal fields. Specifically, the dorsal zone of the field was the smallest in controls, whereas it was the largest in restricted rats, occupying more territory within the nucleus. This alteration in the terminal field was apparent in all groups of rats fed the low-NaCl diet beginning at E3, and continuing beyond E12. In contrast, no effects of the dietary manipulation on the developing chorda tympani field was evident when it occurred from E3 to day E9, from E0 to day E9 or when it occurred at adulthood only. Therefore, only 9 days of maternal exposure to a sodium-restricted diet is required for a permanent expansion of the chorda tympani terminal field in the offspring. Moreover, a brief period from E9 to E12 must be included within the 9-day dietary restriction to yield the expanded field. Since this period is before taste receptors appear on the tongue, it is likely that nonactivity-dependent factors determine the formation of the chorda tympani terminal field during later development.

Development of fungiform papillae, taste buds, and their innervation in the hamster.

Fungiform taste buds in mature hamsters are less subject to neurotrophic influences than those of other species. This study evaluates taste-bud neurotrophism during development in hamsters by examining the relation between growing nerves and differentiating fungiform papillae. Chorda tympani (CT) or lingual (trigeminal) nerve (LN) fibers were labelled with Lucifer Yellow as they grew into (CT fibers) or around (LN fibers) developing taste buds. Developing fungiform papillae and taste pores were counted with the aid of a topical tongue stain. The tongue forms on embryonic days (E) 10.5-11 and contains deeply placed CT and LN fibers but no papillae. By E12, the tongue epithelium develops scattered elevations. These "eminences" selectively become innervated by LN fibers that grow to the epithelium earlier and in larger numbers than CT fibers. Definitive fungiform papillae form rapidly during E13-14 and become heavily innervated by LN fibers. Intraepithelial CT fibers, rare at E13, invariably innervate fungiform papillae containing nascent taste buds at E14. During E14-15 (birth = E15-16), most papillae contain taste buds with pores, extensive perigemmal LN innervation, and extensive intragemmal CT innervation. At birth, numbers of fungiform papillae and taste pores are adultlike. The results show that fungiform eminences begin forming in the absence of innervation. The subsequent differentiation of definitive fungiform papillae and their innervation by LN fibers occur synchronously, prior to the differentiation of taste buds and their CT innervation. The hamster is precocious (e.g., compared to rat) in terms of LN development and the structural maturity of the anterior tongue at birth.

Embryonic geniculate ganglion neurons in culture have neurotrophin-specific electrophysiological properties.

Geniculate ganglion neurons provide a major source of innervation to mammalian taste organs, including taste buds in the soft palate and in fungiform papillae on the anterior two thirds of the tongue. In and around the fungiform papillae, before taste buds form, neurotrophin mRNAs are expressed in selective spatial and temporal patterns. We hypothesized that neurotrophins would affect electrophysiological properties in embryonic geniculate neurons. Ganglia were explanted from rats at gestational day 16, when growing neurites have entered the papilla core, and maintained in culture with added brain-derived neurotrophic factor (BDNF), neurotrophin 4 (NT4), nerve growth factor (NGF) or neurotrophin 3 (NT3). Neuron survival with BDNF or NT4 was about 80%, whereas with NGF or NT3 less than 15% of neurons survived over 6 days in culture. Whole cell recordings from neurons in ganglion explants with each neurotrophin condition demonstrated distinctive neurophysiological properties related to specific neurotrophins. Geniculate neurons cultured with either BDNF or NT4 had similar passive-membrane and action potential properties, but these characteristics were significantly different from those of neurons cultured with NGF or NT3. NGF-maintained neurons had features of increased excitability including a higher resting membrane potential and a lower current threshold for the action potential. About 70% of neurons produced repetitive action potentials at threshold. Furthermore, compared with neurons cultured with other neurotrophins, a decreased proportion had an inflection on the falling phase of the action potential. NT3-maintained neurons had action potentials that were of relatively large amplitude and short duration, with steep rising and falling slopes. In addition, about 20% responded with a repetitive train of action potentials at threshold. In contrast, with BDNF or NT4 repetitive action potential trains were not observed. The data demonstrate different neurophysiological properties in developing geniculate ganglion neurons maintained with specific neurotrophins. Therefore, we suggest that neurotrophins might influence acquisition of distinctive neurophysiological properties in embryonic geniculate neurons that are fundamental to the formation of peripheral taste circuits and a functioning taste system.

The innervation of the primate fungiform papilla--development, distribution and changes following selective ablation.

The development of the terminal parts of the chorda tympani nerve, lingual nerve and cranial sympathetics in the macaque fungiform papillae were studied by light- and electron microscopy. Their respective distributions in the intra- and extragemmal compartments of papillae from adult macaques were examined following selective ablation of each nerve. Prior to midgestation, a single bundle of unmyelinated axons which contained numerous axoaxonic synapses passed through the subepithelial connective tissue and ramified in the single nascent chemosensory corpuscle and surrounding non-gustatory epithelium. Following midgestation, additional chemosensory corpuscles appeared, possibly by division of existing corpuscles, myelination of axons was begun, axoaxonic synapses were eliminated, and nerve terminals appeared in the subepithelial connective tissue as free nerve endings and coiled simple nerve endings. In the perinatal period, coiled simple endings, corpuscular receptors and Meissner corpuscles were present in the papilla core. Large numbers of intra-epithelial nerve endings were present in the extragemmal epithelium throughout development. Tonofilament collars ensheathed intra-epithelial axons and 80-100 nm dense core granules, occupying adjacent epithelial cells, appeared to be sequestered near such axons. Experimental selective ablation indicated that the terminal parts of chorda tympani fibers were present only within chemosensory corpuscles. In contrast, lingual nerve endings were present both in the extragemmal epithelium and chemosensory corpuscles and also were the sole supply of corpuscular receptors. Sympathetics appeared to be sparsely distributed in the papilla core. Intra-epithelial axons degenerated within 24 h following transection, while axons with Schwann or lamellar cell sheaths or myelin persisted for at least 3 days.

Refinement of innervation accuracy following initial targeting of peripheral gustatory fibers.

During development, axons of the chorda tympani nerve navigate to fungiform papillae where they penetrate the lingual epithelium, forming a neural bud. It is not known whether or not all chorda tympani axons initially innervate fungiform papillae correctly or if mistakes are made. Using a novel approach, we quantified the accuracy with which gustatory fibers successfully innervate fungiform papillae. Immediately following initial targeting (E14.5), innervation was found to be incredibly accurate: specifically, 94% of the fungiform papillae on the tongue are innervated. A mean of five papillae per tongue were uninnervated at E14.5, and the lingual tongue surface was innervated in 17 places that lack fungiform papillae. To determine if these initial errors in papillae innervation were later refined, innervation accuracy was quantified at E16.5 and E18.5. By E16.5 only two papillae per tongue remained uninnervated. Innervation to inappropriate regions was also removed, but not until later, between E16.5 and E18.5 of development. Therefore, even though gustatory fibers initially innervate fungiform papillae accurately, some errors in targeting do occur that are then refined during later embryonic periods. It is likely that trophic interactions between gustatory neurons and developing taste epithelium allow appropriate connections to be maintained and inappropriate ones to be eliminated.

Early development of the facial nerve in the chick embryo with special reference to the development of the chorda tympani.

The development of the facial nerve from Hamburger and Hamilton stage 17 to stage 28 is described in chick embryos by means of a new immunochemical nerve staining method that uses an antineurofilament protein (NFP) antibody. A postspiracular branch and an unknown transient posterior branch beneath the ostocyst were observed at stage 17. At stage 19, the primordia of the r. palatinus were observed. A prespiracular branch appeared at stage 21, and with the postspiracular nerve, it made a loop encircling the spiracle (spiracular loop). The first primordium of the ramus (r) hyoideus and transient rami (rr) dorsales appeared around stage 23. At stage 25, the chorda tympani was first observed to arise from the ventral end of the spiracular loop. At stage 26, a communicating branch, connexus cum nervo glossopharyngeo, was found along with the vena (v) capitis lateralis. The rr. dorsales seemed to represent the r. supratemporalis in lower animals. The communicating branches around the v. capitis lateralis seemed to correspond to the cutaneous nerve communications between the branchial nerves frequently encountered in Amphibia. It was found that the chorda tympani becomes a prespiracular nerve for the most part in the chick by the reduction of the postspiracular component of the spiracular loop. Thus, the nerve differs markedly from that in other animals, which is postspiracular. This difference explains the different passage of this nerve in the chick as compared with other amniotes.

The distribution of the chorda tympani in the middle ear area in man and two other primates.

A serial section study of the distribution of the chorda tympani in the middle ear area was carried out in man, baboon and monkey. The tissues innervated by the chorda tympani could be related to a branchiomeric pattern. The early branches distributed post-trematic facial nerve fibres to hyoid arch tissues, where they were joined by elements from glossopharyngeal and vagus nerves. The rest of the distribution was to structures derived from mandibular arch tissue where branches of the auriculotemporal nerve were also present. Contributions to perivascular plexuses were noted as well as a connexion with the otic ganglion.

Target pioneering and early morphology of the murine chorda tympani.

Many studies demonstrate that differentiation of certain sensory receptors during development is induced by their nerve supply. Thus the navigational accuracy of pioneering fibres to their targets is crucial to this process. The special gustatory elements of the facial and glossopharyngeal nerves are used extensively as model systems in this field. We examined the chorda tympani, the gustatory component of the facial nerve, to determine the precise time course of its development in mice. The transganglionic fluorescent tracer DiI was injected into the anterior aspect of the mandibular arch of fixed embryos aged between 30 and 50 somites (E10-E12). It was allowed to diffuse retrogradely via the geniculate ganglion to the brainstem for 4 wk, before the distribution of DiI was determined using confocal laser scanning microscopy. Geniculate ganglion cells were first labelled at the 34 somite stage (E10). Pioneering chorda tympani fibres that arise from these cells passed peripherally and followed an oblique course as they grew towards the mandibular arch. At the 36 somite stage (E10.5), the peripheral component followed an intricate postspiracular course and passed anteriorly to arch over the primitive tympanic cavity, en route to the lingual epithelium. From the 36 to 50 somite stages (E10.5-E12), it consistently traced in the fashion of a 'U' bend. The central fascicle also traced at the 36 somite stage (E10.5) and just made contact with the brainstem. At the 40 somite stage (E11), the central fibres clearly chose a route of descent into the spinal trigeminal tract and branched into the solitary tract. Pioneering chorda tympani fibres contact the lingual epithelium when the target is primordial. The lingual epithelium may be a source of a neurotropic factor that attracts peripheral chorda tympani fibres to the sites of putative papillae. However, the chorda tympani is probably not a vital influence on the subsequent differentiation of gustatory papillae, since the papillae are elaborated 5 d later at E15 in murine embryos. The early morphology of the nerve is true to the amniote vertebrate phenotype.

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.

Building sensory receptors on the tongue.

Neurotrophins, neurotrophin receptors and sensory neurons are required for the development of lingual sense organs. For example, neurotrophin 3 sustains lingual somatosensory neurons. In the traditional view, sensory axons will terminate where neurotrophin expression is most pronounced. Yet, lingual somatosensory axons characteristically terminate in each filiform papilla and in each somatosensory prominence within a cluster of cells expressing the p75 neurotrophin receptor (p75NTR), rather than terminating among the adjacent cells that secrete neurotrophin 3. The p75NTR on special specialized clusters of epithelial cells may promote axonal arborization in vivo since its over-expression by fibroblasts enhances neurite outgrowth from overlying somatosensory neurons in vitro. Two classical observations have implicated gustatory neurons in the development and maintenance of mammalian taste buds--the early arrival times of embryonic innervation and the loss of taste buds after their denervation in adults. In the modern era more than a dozen experimental studies have used early denervation or neurotrophin gene mutations to evaluate mammalian gustatory organ development. Necessary for taste organ development, brain-derived neurotrophic factor sustains developing gustatory neurons. The cardinal conclusion is readily summarized: taste buds in the palate and tongue are induced by innervation. Taste buds are unstable: the death and birth of taste receptor cells relentlessly remodels synaptic connections. As receptor cells turn over, the sensory code for taste quality is probably stabilized by selective synapse formation between each type of gustatory axon and its matching taste receptor cell. We anticipate important new discoveries of molecular interactions among the epithelium, the underlying mesenchyme and gustatory innervation that build the gustatory papillae, their specialized epithelial cells, and the resulting taste buds.

Taste placodes are primary targets of geniculate but not trigeminal sensory axons in mouse developing tongue.

Tongue embryonic taste buds begin to differentiate before the onset of gustatory papilla formation in murine. In light of this previous finding, we sought to reexamine the developing sensory innervation as it extends toward the lingual epithelium between E 11.5 and 14.5. Nerve tracings with fluorescent lipophilic dyes followed by confocal microscope examination were used to study the terminal branching of chorda tympani and lingual nerves. At E11.5, we confirmed that the chorda tympani nerve provided for most of the nerve branching in the tongue swellings. At E12.5, we show that the lingual nerve contribution to the overall innervation of the lingual swellings increased to the extent that its ramifications matched those of the chorda tympani nerve. At E13.0, the chorda tympani nerve terminal arborizations appeared more complex than those of the lingual nerve. While the chorda tympani nerve terminal branching appeared close to the lingual epithelium that of the trigeminal nerve remained rather confined to the subepithelial mesenchymal tissue. At E13.5, chorda tympani nerve terminals projected specifically to an ordered set of loci on the tongue dorsum corresponding to the epithelial placodes. In contrast, the lingual nerve terminals remained subepithelial with no branches directed towards the placodes. At E14.5, chorda tympani nerve filopodia first entered the apical epithelium of the developing fungiform papilla. The results suggest that there may be no significant delay between the differentiation of embryonic taste buds and their initial innervation.