Hmx1 is required for the normal development of somatosensory neurons in the geniculate ganglion.

Hmx1 is a variant homeodomain transcription factor expressed in the developing sensory nervous system, retina, and craniofacial mesenchyme. Recently, mutations at the Hmx1 locus have been linked to craniofacial defects in humans, rats, and mice, but its role in nervous system development is largely unknown. Here we show that Hmx1 is expressed in a subset of sensory neurons in the cranial and dorsal root ganglia which does not correspond to any specific sensory modality. Sensory neurons in the dorsal root and trigeminal ganglia of Hmx1dm/dm mouse embryos have no detectable Hmx1 protein, yet they undergo neurogenesis and express sensory subtype markers normally, demonstrating that Hmx1 is not globally required for the specification of sensory neurons from neural crest precursors. Loss of Hmx1 expression has no obvious effect on the early development of the trigeminal (V), superior (IX/X), or dorsal root ganglia neurons in which it is expressed, but results in marked defects in the geniculate (VII) ganglion. Hmx1dm/dm mouse embryos possess only a vestigial posterior auricular nerve, and general somatosensory neurons in the geniculate ganglion are greatly reduced by mid-gestation. Although Hmx1 is expressed in geniculate neurons prior to cell cycle exit, it does not appear to be required for neurogenesis, and the loss of geniculate neurons is likely to be the result of increased cell death. Fate mapping of neural crest-derived tissues indicates that Hmx1-expressing somatosensory neurons at different axial levels may be derived from either the neural crest or the neurogenic placodes.

Neurotrophin-4 regulates the survival of gustatory neurons earlier in development using a different mechanism than brain-derived neurotrophic factor.

The number of neurons in the geniculate ganglion that are available to innervate taste buds is regulated by neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF). Our goal for the current study was to examine the timing and mechanism of NT-4-mediated regulation of geniculate neuron number during development. We discovered that NT-4 mutant mice lose 33% of their geniculate neuronal cells between E10.5 and E11.5. By E11.5, geniculate axons have just reached the tongue and do not yet innervate their gustatory targets; thus, NT-4 does not function as a target-derived growth factor. At E11.5, no difference was observed in proliferating cells or the rate at which cells exit the cell cycle between NT-4 mutant and wild type ganglia. Instead, there was an increase in TUNEL-labeling, indicating an increase in cell death in Ntf4(-/-) mice compared with wild types. However, activated caspase-3, which is up-regulated in the absence of BDNF, was not increased. This finding indicates that cell death initiated by NT-4-removal occurs through a different cell death pathway than BDNF-removal. We observed no additional postnatal loss of taste buds or neurons in Ntf4(-/-) mice. Thus, during early embryonic development, NT-4 produced in the ganglion and along the projection pathway inhibits cell death through an activated caspase-3 independent mechanism. Therefore, compared to BDNF, NT-4 plays distinct roles in gustatory development; differences include timing, source of neurotrophin, and mechanism of action.

Developmental expression of Bdnf, Ntf4/5, and TrkB in the mouse peripheral taste system.

Brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4), and their TrkB receptor regulate taste system development. To determine where and when gustatory neurons come in contact with these important factors, temporospatial expression patterns of Bdnf, Ntf4/5, and TrkB in the peripheral taste system were examined using RT-PCR. In the lingual epithelium, Ntf4/5 mRNA expression was higher than that of Bdnf at embryonic day 12.5 (E12.5), and the expression of both factors decreased afterwards. However, Ntf4/5 expression decreased at an earlier age than Bdnf. Bdnf and Ntf4/5 are expressed in equal amounts at E12.5 in geniculate ganglion, but Bdnf expression increased from E14.5 to birth, whereas Ntf4/5 expression decreased. These findings indicate that NT4 functions at early embryonic stages and is derived from different sources than Bdnf. TrkB expression in the geniculate ganglion is robust throughout development and is not a limiting factor for neurotrophin function in this system.

Differentiation of the facio-vestibulocochlear ganglionic complex in human embryos of developmental stages 13-15.

A study was made on 18 embryos of developmental stages 13-15 (5(th) week). Serial sections made in horizontal, frontal, and sagittal planes were stained with routine histological methods and some of them were treated with silver. In embryos of stage 13, the otic vesicle is at the rhombomere 5, and close to the vesicle is the facial-vestibulocochlear ganglionic complex in which the geniculate, vestibular, and cochlear ganglion may be discerned. These ganglia are well demarcated in embryos of stage 14. In the last investigated stage (15(th)) the nerve fibres of the ganglia reach the common afferent tract.

TrkB expression and dependence divides gustatory neurons into three subpopulations.

BACKGROUND: During development, gustatory (taste) neurons likely undergo numerous changes in morphology and expression prior to differentiation into maturity, but little is known this process or the factors that regulate it. Neuron differentiation is likely regulated by a combination of transcription and growth factors. Embryonically, most geniculate neuron development is regulated by the growth factor brain derived neurotrophic factor (BDNF). Postnatally, however, BDNF expression becomes restricted to subpopulations of taste receptor cells with specific functions. We hypothesized that during development, the receptor for BDNF, tropomyosin kinase B receptor (TrkB), may also become developmentally restricted to a subset of taste neurons and could be one factor that is differentially expressed across taste neuron subsets. METHODS: We used transgenic mouse models to label both geniculate neurons innervating the oral cavity (Phox2b+), which are primarily taste, from those projecting to the outer ear (auricular neurons) to label TrkB expressing neurons (TrkBGFP). We also compared neuron number, taste bud number, and taste receptor cell types in wild-type animals and conditional TrkB knockouts. RESULTS: Between E15.5-E17.5, TrkB receptor expression becomes restricted to half of the Phox2b + neurons. This TrkB downregulation was specific to oral cavity projecting neurons, since TrkB expression remained constant throughout development in the auricular geniculate neurons (Phox2b-). Conditional TrkB removal from oral sensory neurons (Phox2b+) reduced this population to 92% of control levels, indicating that only 8% of these neurons do not depend on TrkB for survival during development. The remaining neurons failed to innervate any remaining taste buds, 14% of which remained despite the complete loss of innervation. Finally, some types of taste receptor cells (Car4+) were more dependent on innervation than others (PLCß2+). CONCLUSIONS: Together, these findings indicate that TrkB expression and dependence divides gustatory neurons into three subpopulations: 1) neurons that always express TrkB and are TrkB-dependent during development (50%), 2) neurons dependent on TrkB during development but that downregulate TrkB expression between E15.5 and E17.5 (41%), and 3) neurons that never express or depend on TrkB (9%). These TrkB-independent neurons are likely non-gustatory, as they do not innervate taste buds.

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.

Localization of congenital tegmen tympani defects.

OBJECTIVE: This study sets out to demonstrate the normal developmental steps of the tegmen tympani and thus explains the typical localization of congenital tegmental defects. SPECIMENS: For this study, 79 macerated and formalin-fixed human temporal bones from 14th fetal week to adults were observed and prepared. INTERVENTION: Macroscopic and microscopic examination of the prenatal and postnatal changes of the tegmen tympani during its development. MAIN OUTCOME MEASURE: Temporal bones from 14th fetal week to adults underwent descriptive anatomic studies to understand the normal development of the tegmen tympani and to find a possible cause of its congenital defects. RESULTS: The medial part of the tegmen tympani develops from the otic capsule during chondral ossification, thus forming the tegmental process of the petrous part. The lateral part shows membranous ossification. The tegmental process cases a temporary bony dehiscence lateral to the geniculate ganglion between the 23rd and 25th fetal week. CONCLUSION: Congenital defects develop near the geniculate ganglion and seem to be due to an incomplete development of tegmental process of otic capsule. Because of that, congenital lesion of the tegmen tympani can be defined as an inner ear defect.

A comparative morphologic and morphometric study about geniculate ganglion development in man and in rat.

CONCLUSIONS: Human-rat geniculate ganglion (GG) have multiple origins: (1) An initial proximity (20 µm) to the endocranial foramen of the IAM, suggests neural crest induction; and (2) The influence of epibranchial placodes: the tensor tympani muscle (TTM) and the otic apical coil. OBJECTIVES: This study was undertaken to determine the comparative development of human-rat GG. MATERIALS AND METHODS: A light microscopic study of the GG in human material obtained from spontaneous abortions at 9, 13, 14, 17, 18, and 30 weeks, and one neonate was done. This study examined Webster rat embryos and a post-natal series. Specimens were fixed in Bouin fluid, embedded in paraffin, cut, and stained with H&E. The histomorphometric data were obtained with image analysis software. RESULTS: The human fetus of 9 weeks presents two neuronal groups in the VII nerve: one near (20 µm) the IAM endocranial foramen, foraminal, and the other, tympanic. Neonate GG is located between the TTM and the cochlear apex (inwards). In the 16 day old rat embryo GG is placed within a canal containing the stapedial artery. In the adult rat the GG and the stapedial artery are placed within the IAM.

Neuronal cell death and population dynamics in the developing rat geniculate ganglion.

In contrast to many neuronal systems, the pattern of developmental neuronal degeneration in the rat geniculate ganglion has remained undefined. To address this issue sectioned geniculate ganglia from embryonic day 13 to postnatal day 3 have been examined using standard histological techniques, TdT-mediated dUTP-digoxigenin nick end labeling to verify apoptotic activity, bromo-deoxyuridine incorporation to monitor neuronal precursor proliferation, and anti-beta-neurotubulin III to verify the neuronal identity of pycnotic cells. Results summed from alternate (embryonic day 13) or every third (embryonic day 14-postnatal day 3) section show that neuronal degeneration occurs as early as embryonic day 13 (6.8% of neurons counted), well before geniculate innervation of lingual taste buds at embryonic day 16. A degenerative peak occurs at embryonic day 17 (9.5%) followed by a decline (1.7% at embryonic day 18) and leveling off (0.1%-0.2% at embryonic day 22-postnatal day 3). Thus, geniculate neuronal degenerative pattern includes both innervation-associated histogenetic and morphogenetic cell death. Corresponding counts of mean neuronal numbers in the sections showed a continual rise from embryonic day 13 through embryonic day 18 (approx. 330-760) followed by a slight decline at embryonic day 19 (to approx. 630) and then a final leveling off at 800-825 by embryonic day 20. This pattern differs from many other developing neural systems which show a major population crash during initial target contact. It likely reflects different but slightly overlapping neuronal precursor proliferation and degeneration patterns in multiple geniculate neuronal subpopulations.

Comparison of neurotrophin and repellent sensitivities of early embryonic geniculate and trigeminal axons.

Geniculate (gustatory) and trigeminal (somatosensory) afferents take different routes to the tongue during rat embryonic development. To learn more about the mechanisms controlling neurite outgrowth and axon guidance, we are studying the roles of diffusible factors. We previously profiled the in vitro sensitivity of trigeminal axons to neurotrophins and target-derived diffusible factors and now report on these properties for geniculate axons. GDNF, BDNF, and NT-4, but not NT-3 or NGF, stimulate geniculate axon outgrowth during the ages investigated, embryonic days 12-14. Sensitivity to effective neurotrophins is developmentally regulated and different from that of the trigeminal ganglion. In vitro coculture studies revealed that geniculate axons were repelled by branchial arch explants that were previously shown to be repellent to trigeminal axons (Rochlin and Farbman [1998] J Neurosci 18:6840-6852). In addition, some branchial arch explants and untransfected COS7 cells repelled geniculate but not trigeminal axons. Sema3A, a ligand for neuropilin-1, is effective in repelling geniculate and trigeminal axons, and antineuropilin-1, but not antineuropilin-2, completely blocks the repulsion by arch explants that repel axon outgrowth from both ganglia. Sema3A mRNA is concentrated in branchial arch epithelium at the appropriate time to mediate the repulsion. In Sema3A knockout mice, geniculate and trigeminal afferents explore medial regions of the immature tongue and surrounding territories not explored in heterozygotes, supporting our previous hypothesis that Sema3A-based repulsion mediates the early restriction of sensory afferents away from midline structures.

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.

Dehiscence of the facial canal: developmental aspects.

In a series of human fetuses, the course of the facial canal in the temporal bone was investigated by the use of light and scanning electron microscopy. The normal development of the facial canal was correlated to clinical aspects of facial nerve dehiscences. Our observations demonstrate a more complex way of facial canal development not limited to the 'simple' ossification of the otic capsule. Endochondral ossification of the otic capsule does not virtually change the shape of the primitive facial sulcus. The fibrous layers surrounding the facial nerve seem to be responsible for the final architecture of the facial canal and not the otic capsule ossification by itself. The time sequence of their histological development is equally important and permitted us to distinguish three phases in facial canal development. The role of disturbances in epigenetic control for the initiation of dehiscences is discussed.

Labyrinthine segment and geniculate ganglion of facial nerve in fetal and adult human temporal bones.

The later stages of development (15-40 weeks in utero) of the geniculate ganglion and labyrinthine segment of the facial nerve in the human fetus demonstrate minimal neuronal growth. The vascular supply is well established. The major changes occur in the perineural ossification pattern. The canal of the labyrinthine facial nerve segment ossifies first via the petrous apex and periotic capsule. The narrowest portion of the canal is at the geniculate ganglion in the earlier stages and at the fundus of the internal auditory canal at term. The geniculate ganglion area ossifies by means of two bony plates. The medial plate is a derivate of the periosteal growth of the petrous apex and the lateral plate is an extension of membranous bone from the squama. The major relationships to the middle ear do not change. The hiatus of the facial canal diminishes in size during gestation, but remains patent at birth.

[Experimental study on early development of rat ear in vitro using whole embryo culture].

Rat embryos were explanted on late 11 day of gestation and cultured for 24 hours in rotating bottles with the yolk sac opened. Rat serum was used as culture medium and culture bottles were filled with 5% CO2 + 95% O2 gas mixture as gas phase. At the time of explantation and the end of the culture period, differentiation and growth of the embryos were monitored by counting somites and measuring crown rump length. About part of the embryos, protein determinations were made to measure growth. These data were compared with values found for 12 and 13 day embryos. The results for cultured embryos showed slight retardation in their differentiation and slight depression in the growth. At the same time to study early inner ear development in vitro, 9 of the other cultured embryos were serially sectioned and observed by light microscopy. In the cultured embryos endolymphatic duct elongated and acoustico-facial ganglion enlarged remarkably. Otocysts became flattened and elongated ventrally. Vestibular and cochlear portion were identifiable in the otocysts. Nerves arose from the ganglion reached to brain centrally and wall of pharynx peripherally. There was no significant difference in inner ear development between 13 day and the cultured embryos. This culture system should prove useful for studies on early inner ear development of mammalian embryos.

Genetic dissection of TrkB activated signalling pathways required for specific aspects of the taste system.

BACKGROUND: Neurotrophin-4 (NT-4) and brain derived neurotrophic factor (BDNF) bind to the same receptor, Ntrk2/TrkB, but play distinct roles in the development of the rodent gustatory system. However, the mechanisms underlying these processes are lacking. RESULTS: Here, we demonstrate, in vivo, that single or combined point mutations in major adaptor protein docking sites on TrkB receptor affect specific aspects of the mouse gustatory development, known to be dependent on BDNF or NT-4. In particular, mice with a mutation in the TrkB-SHC docking site had reduced gustatory neuron survival at both early and later stages of development, when survival is dependent on NT-4 and BDNF, respectively. In addition, lingual innervation and taste bud morphology, both BDNF-dependent functions, were altered in these mutants. In contrast, mutation of the TrkB-PLCγ docking site alone did not affect gustatory neuron survival. Moreover, innervation to the tongue was delayed in these mutants and taste receptor expression was altered. CONCLUSIONS: We have genetically dissected pathways activated downstream of the TrkB receptor that are required for specific aspects of the taste system controlled by the two neurotrophins NT-4 and BDNF. In addition, our results indicate that TrkB also regulate the expression of specific taste receptors by distinct signalling pathways. These results advance our knowledge of the biology of the taste system, one of the fundamental sensory systems crucial for an organism to relate to the environment.

Taste neurons consist of both a large TrkB-receptor-dependent and a small TrkB-receptor-independent subpopulation.

Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) are two neurotrophins that play distinct roles in geniculate (taste) neuron survival, target innervation, and taste bud formation. These two neurotrophins both activate the tropomyosin-related kinase B (TrkB) receptor and the pan-neurotrophin receptor p75. Although the roles of these neurotrophins have been well studied, the degree to which BDNF and NT-4 act via TrkB to regulate taste development in vivo remains unclear. In this study, we compared taste development in TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice to determine if these deficits were similar. If so, this would indicate that the functions of both BDNF and NT-4 can be accounted for by TrkB-signaling. We found that TrkB(-/-) and Bdnf(-/-)/Ntf4(-/-) mice lose a similar number of geniculate neurons by E13.5, which indicates that both BDNF and NT-4 act primarily via TrkB to regulate geniculate neuron survival. Surprisingly, the few geniculate neurons that remain in TrkB(-/-) mice are more successful at innervating the tongue and taste buds compared with those neurons that remain in Bdnf(-/-)/Ntf4(-/-) mice. The remaining neurons in TrkB(-/-) mice support a significant number of taste buds. In addition, these remaining neurons do not express the TrkB receptor, which indicates that either BDNF or NT-4 must act via additional receptors to influence tongue innervation and/or targeting.

Restored brain perfusion after non-invasive stimulation of the facial nerve in a canine stroke model.

Ischemic stroke affects over 15 million patients per year and is a leading cause of death worldwide. Currently available treatments are indicated for less than 5% of patients. Stimulation of the facial nerve has been proposed as a possible new treatment of ischemic stroke that acts by increasing blood flow to the brain and thereby restoring perfusion through collateral vessels. The objective of this project was to evaluate the changes in brain perfusion, following facial nerve stimulation in an animal stroke model using MRI measures of cerebral blood flow. Autologous blood clot was injected in the internal carotid artery to occlude the middle cerebral artery (MCA) in 17 mongrel dogs. Occlusion in the MCA was verified using fluoroscopy and MRI angiography. Following baseline and post-stroke MRI images, the facial nerve at the site of the geniculate ganglion was located and then stimulated using a transcranial magnetic stimulator and a neuro-navigation system in 11 animals. Six animals followed the same procedure but were not stimulated (control group). The perfusion index of both sides of the brain was measured using gadolinium contrast MRI before and after stroke, and at 30 minute intervals after stimulation. Results show a significant and persistent increase in perfusion in the stroke side of the brain relative to the non-stroke / contralateral side, after stimulation, when compared to the control group. These results strongly support the future development and evaluation of a non-invasive facial nerve stimulator device for the early treatment of ischemic stroke.

Lingual and palatal gustatory afferents each depend on both BDNF and NT-4, but the dependence is greater for lingual than palatal afferents.

Neurons of the geniculate ganglion innervate taste buds located in two spatially distinct targets, the tongue and palate. About 50% of these neurons die in Bdnf(-/-) mice and Ntf4/5(-/-) mice. Bdnf(-/-)/Ntf4/5(-/-) double mutants lose 90-95% of geniculate ganglion neurons. To determine whether different subpopulations are differentially influenced by neurotrophins, we quantified neurons from two ganglion subpopulations separately and remaining taste buds at birth within each target field in wild-type, Bdnf(-/-), Ntf4/5(-/-), and Bdnf(-/-)/Ntf4/5(-/-) mice. In wild-type mice the same number of neurons innervated the anterior tongue and soft palate and each target contained the same number of taste buds. Compared to wild-type mice, Bdnf(-/-) mice showed a 50% reduction in geniculate neurons innervating the tongue and a 28% loss in neurons innervating the soft palate. Ntf4/5(-/-) mice lost 58% of the neurons innervating the tongue and 41% of the neurons innervating the soft palate. Taste bud loss was not as profound in the NT-4 null mice compared to BDNF-null mice. Tongues of Bdnf(-/-)/Ntf4/5(-/-) mice were innervated by 0 to 4 gustatory neurons and contained 3 to 16 taste buds at birth, indicating that some taste buds remain even when all innervation is lost. Thus, gustatory neurons are equally dependent on BDNF and NT-4 expression for survival, regardless of what peripheral target they innervate. However, taste buds are more sensitive to BDNF than NT-4 removal.

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.

Distinctive neurophysiological properties of embryonic trigeminal and geniculate neurons in culture.

Neurons in trigeminal and geniculate ganglia extend neurites that share contiguous target tissue fields in the fungiform papillae and taste buds of the mammalian tongue and thereby have principal roles in lingual somatosensation and gustation. Although functional differentiation of these neurons is central to formation of lingual sensory circuits, there is little known about electrophysiological properties of developing trigeminal and geniculate ganglia or the extrinsic factors that might regulate neural development. We used whole cell recordings from embryonic day 16 rat ganglia, maintained in culture as explants for 3-10 days with neurotrophin support to characterize basic properties of trigeminal and geniculate neurons over time in vitro and in comparison to each other. Each ganglion was cultured with the neurotrophin that supports maximal neuron survival and that would be encountered by growing neurites at highest concentration in target fields. Resting membrane potential and time constant did not alter over days in culture, whereas membrane resistance decreased and capacitance increased in association with small increases in trigeminal and geniculate soma size. Small gradual differences in action potential properties were observed for both ganglion types, including an increase in threshold current to elicit an action potential and a decrease in duration and increase in rise and fall slopes so that action potentials became shorter and sharper with time in culture. Using a period of 5-8 days in culture when neural properties are generally stable, we compared trigeminal and geniculate ganglia and revealed major differences between these embryonic ganglia in passive membrane and action potential characteristics. Geniculate neurons had lower resting membrane potential and higher input resistance and smaller, shorter, and sharper action potentials with lower thresholds than trigeminal neurons. Whereas all trigeminal neurons produced a single action potential at threshold depolarization, 35% of geniculate neurons fired repetitively. Furthermore, all trigeminal neurons produced TTX-resistant action potentials, but geniculate action potentials were abolished in the presence of low concentrations of TTX. Both trigeminal and geniculate neurons had inflections on the falling phase of the action potential that were reduced in the presence of various pharmacological blockers of calcium channel activation. Use of nifedipine, omega-conotoxin-MVIIA and GVIA, and omega-agatoxin-TK indicated that currents through L-, N-, and P/Q- type calcium channels participate in the action potential inflection in embryonic trigeminal and geniculate neurons. The data on passive membrane, action potential, and ion channel characteristics demonstrate clear differences between trigeminal and geniculate ganglion neurons at an embryonic stage when target tissues are innervated but receptor organs have not developed or are still immature. Therefore these electrophysiological distinctions between embryonic ganglia are present before neural activity from differentiated receptive fields can influence functional phenotype.