Sensory Neuroblast Quiescence Depends on Vascular Cytoneme Contacts and Sensory Neuronal Differentiation Requires Initiation of Blood Flow.

In many organs, stem cell function depends on communication with their niche partners. Cranial sensory neurons develop in close proximity to blood vessels; however, whether vasculature is an integral component of their niches is yet unknown. Here, two separate roles for vasculature in cranial sensory neurogenesis in zebrafish are uncovered. The first involves precise spatiotemporal endothelial-neuroblast cytoneme contacts and Dll4-Notch signaling to restrain neuroblast proliferation. The second, instead, requires blood flow to trigger a transcriptional response that modifies neuroblast metabolic status and induces sensory neuron differentiation. In contrast, no role of sensory neurogenesis in vascular development is found, suggesting unidirectional signaling from vasculature to sensory neuroblasts. Altogether, we demonstrate that the cranial vasculature constitutes a niche component of the sensory ganglia that regulates the pace of their growth and differentiation dynamics.

Reducing Current Spread by Use of a Novel Pulse Shape for Electrical Stimulation of the Auditory Nerve.

Improving the electrode-neuron interface to reduce current spread between individual electrodes has been identified as one of the main objectives in the search for future improvements in cochlear-implant performance. Here, we address this problem by presenting a novel stimulation strategy that takes account of the biophysical properties of the auditory neurons (spiral ganglion neurons, SGNs) stimulated in electrical hearing. This new strategy employs a ramped pulse shape, where the maximum amplitude is achieved through a linear slope in the injected current. We present the theoretical framework that supports this new strategy and that suggests it will improve the modulation of SGNs' activity by exploiting their sensitivity to the rising slope of current pulses. The theoretical consequence of this sensitivity to the slope is a reduction in the spread of excitation within the cochlea and, consequently, an increase in the neural dynamic range. To explore the impact of the novel stimulation method on neural activity, we performed in vitro recordings of SGNs in culture. We show that the stimulus efficacy required to evoke action potentials in SGNs falls as the stimulus slope decreases. This work lays the foundation for a novel, and more biomimetic, stimulation strategy with considerable potential for implementation in cochlear-implant technology.

Differential roles for EphA and EphB signaling in segregation and patterning of central vestibulocochlear nerve projections.

Auditory and vestibular afferents enter the brainstem through the VIIIth cranial nerve and find targets in distinct brain regions. We previously reported that the axon guidance molecules EphA4 and EphB2 have largely complementary expression patterns in the developing avian VIIIth nerve. Here, we tested whether inhibition of Eph signaling alters central targeting of VIIIth nerve axons. We first identified the central compartments through which auditory and vestibular axons travel. We then manipulated Eph-ephrin signaling using pharmacological inhibition of Eph receptors and in ovo electroporation to misexpress EphA4 and EphB2. Anterograde labeling of auditory afferents showed that inhibition of Eph signaling did not misroute axons to non-auditory target regions. Similarly, we did not find vestibular axons within auditory projection regions. However, we found that pharmacologic inhibition of Eph receptors reduced the volume of the vestibular projection compartment. Inhibition of EphB signaling alone did not affect auditory or vestibular central projection volumes, but it significantly increased the area of the auditory sensory epithelium. Misexpression of EphA4 and EphB2 in VIIIth nerve axons resulted in a significant shift of dorsoventral spacing between the axon tracts, suggesting a cell-autonomous role for the partitioning of projection areas along this axis. Cochlear ganglion volumes did not differ among treatment groups, indicating the changes seen were not due to a gain or loss of cochlear ganglion cells. These results suggest that Eph-ephrin signaling does not specify auditory versus vestibular targets but rather contributes to formation of boundaries for patterning of inner ear projections in the hindbrain.

Regional differences in myelination of chick vestibulocochlear ganglion cells.

In vertebrates, vestibular and cochlear ganglion (VG and CG, respectively) cells are bipolar neurons with myelinated axons and perikarya. The time course of the myelination of the VG and CG cells during development of chick embryos was investigated. Chick VG and CG from embryonic day at 7-20 (E7-20) were prepared for a transmission electron microscopy, myelin basic protein immunohistochemistry, and real-time quantitative RT-PCR. In the VG cells, myelination was first observed on the peripheral axons of the ampullar nerves at E10, on the utricular and saccular nerves at E12, and on the lagenar and neglecta nerves at E13. In the VG central axons, myelination was first seen on the ampullar nerves at E11, on the utricular and saccular nerves at E13, and on the lagenar nerves at E13. In the CG cells, the myelination was first observed on the peripheral and central axons at E14. In both VG and CG, myelination was observed on the perikarya at E17. These results suggest that the onset of the axonal myelination on the VG cells occurred earlier than that on the CG cells, whereas the perikaryal myelination occurred at about the same time on the both types of ganglion cells. Moreover, the myelination on the ampullar nerves occurred earlier than that on the utricular and saccular nerves. The myelination on the peripheral axons occurred earlier than that on the central axons of the VG cells, whereas that on the central and peripheral axons of the CG cells occurred at about the same time. The regional differences in myelination in relation to the onset of functional activities in the VG and CG cells are discussed.

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.

[Structural anatomy of cranial nerves (V, VII, VIII, IX, X)]. / Anatomie structurelle des nerfs crâniens (V, VII, VIII, IX, X).

This study reports a review of the literature on the structural anatomy of the Vth, VIIth, VIIIth, IXth, and Xth cranial nerves, known to harbor dysfunction syndromes in humans. Because these dysfunctions are hypothesized to be caused by neurovascular conflicts at the root entry/exit zone and the transitional zone between central and peripheral myelinization, this investigation focused on the study and description of this junction. All the cranial nerves, except the optic and olfactory nerves, which are considered to be more a direct expansion of the central nervous system, have a transitional zone between central myelin (coming from oligodendrocytes) and peripheral myelin (produced by Schwann cells). The human studies reported in the literature argue in favor of a dome-shaped transitional zone directed to the periphery. It seems that this junctional region is situated more peripherally in sensory nerves than in motor nerves. The transitional zone is situated very peripherally for the cochlear and vestibular nerves, and on the contrary very close to its exit from the brain stem for the facial nerve.

Subthreshold sodium current underlies essential functional specializations at primary auditory afferents.

Primary auditory afferents are generally perceived as passive, timing-preserving lines of communication. Contrasting this view, identifiable auditory afferents to the goldfish Mauthner cell undergo potentiation of their mixed--electrical and chemical--synapses in response to high-frequency bursts of activity. This property likely represents a mechanism of input sensitization because they provide the Mauthner cell with essential information for the initiation of an escape response. Consistent with this synaptic specialization, we show here that these afferents exhibit an intrinsic ability to respond with bursts of 200-600 Hz and this property critically relies on the activation of a persistent sodium current, which is counterbalanced by the delayed activation of an A-type potassium current. Furthermore, the interaction between these conductances with the membrane passive properties supports the presence of electrical resonance, whose frequency preference is consistent with both the effective range of hearing in goldfish and the firing frequencies required for synaptic facilitation, an obligatory requisite for the induction of activity-dependent changes. Thus our data show that the presence of a persistent sodium current is functionally essential and allows these afferents to translate behaviorally relevant auditory signals into patterns of activity that match the requirements of their fast and highly modifiable synapses. The functional specializations of these neurons suggest that auditory afferents might be capable of more sophisticated contributions to auditory processing than has been generally recognized.

Otolith endorgan input to the Mauthner neuron in the goldfish.

The Mauthner (M-) cell of the goldfish, Carassius auratus, triggers the rapid escape response of the fish in response to various stimuli, including visual and auditory. The large size and accessibility of the M-cell make it an ideal model system for the study of synaptic transmission, membrane properties, and sensory-motor gating. Although physiological recordings have suggested that afferents from all three of the inner ear endorgans (the saccule, lagena, and utricle) synapse directly on the ipsilateral M-cell, the specific contacts and anatomical distributions of these inputs along the M-cell lateral dendrite remain unknown. We traced specific branches of the auditory (VIIIth) nerve from the three otolith organs of the fish inner ear to the M-cell. The goldfish sacculus gives rise to the vast majority of inputs that contact a large portion of the M-cell lateral dendrite, and these inputs vary greatly in size. In contrast to the ubiquitous distribution of saccular inputs, those from the lagena are segregated to distal regions of the M-cell and synapse on the distal dorsal branch of the lateral dendrite. Similarly, inputs from the utricle are also segregated to distal regions, synapsing on the ventral branch of the lateral dendrite. These results demonstrate that nerves from all three endorgans contact the M-cell, with input-specific segregation of synapses along the M-cell lateral dendrite.

Localization of vesicular glutamate transporters in the peripheral vestibular system of rat.

OBJECTIVE: To examine the vesicular glutamate transporters (VGluTs: VGluT1-VGluT3) in the peripheral vestibular system. METHODS: The vestibular structures, including Scarpa's ganglion (vestibular ganglion, VG), maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs, by avidin-biotinylated peroxidase complex method, with 3-3'-diaminobenzidine (DAB) as chromogen. RESULTS: (1) VGluT1 was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs. (2) Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3-positive afferent fibers was in the maculae and ampullary cristae. (3) No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. CONCLUSION: These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.

T-Box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration.

Members of the T-box transcription factor family (Tbx) are associated with several human syndromes during embryogenesis. Nevertheless, their functions within the developing CNS remain poorly characterized. Tbx20 is expressed by migrating branchiomotor/visceromotor (BM/VM) neurons within the hindbrain during neuronal circuit formation. We examined Tbx20 function in BM/VM cells using conditional Tbx20-null mutant mice to delete the gene in neurons. Hindbrain rhombomere patterning and the initial generation of post-mitotic BM/VM neurons were normal in Tbx20 mutants. However, Tbx20 was required for the tangential (caudal) migration of facial neurons, the lateral migration of trigeminal cells and the trans-median movement of vestibuloacoustic neurons. Facial cell soma migration defects were associated with the coordinate downregulation of multiple components of the planar cell polarity pathway including Fzd7, Wnt11, Prickle1, Vang1 and Vang2. Our study suggests that Tbx20 programs a variety of hindbrain motor neurons for migration, independent of directionality, and in facial neurons is a positive regulator of the non-canonical Wnt signaling pathway.

AML1/Runx1 is important for the development of hindbrain cholinergic branchiovisceral motor neurons and selected cranial sensory neurons.

The mechanisms that regulate the acquisition of distinctive neuronal traits in the developing nervous system are poorly defined. It is shown here that the mammalian runt-related gene Runx1 is expressed in selected populations of postmitotic neurons of the embryonic central and peripheral nervous systems. These include cholinergic branchial and visceral motor neurons in the hindbrain, restricted populations of somatic motor neurons of the median and lateral motor columns in the spinal cord, as well as nociceptive and mechanoreceptor neurons in trigeminal and vestibulocochlear ganglia. In mouse embryos lacking Runx1 activity, hindbrain branchiovisceral motor neuron precursors of the cholinergic lineage are correctly specified but then fail to progress to a more differentiated state and undergo increased cell death, resulting in a neuronal loss in the mantle layer. In contrast, the development of cholinergic somatic motor neurons is unaffected. Runx1 inactivation also leads to a loss of selected sensory neurons in trigeminal and vestibulocochlear ganglia. These findings uncover previously unrecognized roles for Runx1 in the regulation of mammalian neuronal subtype development.

A procedure to label inner ear afferent nerve endings for calcium imaging.

Characterization of synaptic transmission between the inner ear sensory cells and primary neuron dendrites has been hampered by the limited access to the postsynaptic terminals. Because direct physiological recording of postsynaptic currents are difficult to achieve, no information regarding the synaptic and dendritic events are available. This is due to the small size of the postsynaptic afferent nerve endings that do not allow a clear identification, and thus compromise direct electrophysiological recordings of the buttons. To study the physiology of afferent nerve endings, we have developed a two-photon imaging technique in cochlear and vestibular slice preparations from neonatal rats and turtles. This technique is based on a retrograde labeling of afferent nerve endings with high-affinity calcium-sensitive dyes. Dye filling was achieved by 6 h application of the dextran-amine conjugate of calcium green-1. Calcium changes were measured in afferent nerve endings in line scan and time lap mode. To address recording in a near-physiological situation, iontophoretic application of K+ was performed in the area of the stereocilia whereas glutamate was applied at the basal pole of sensory hair cells. Both types of application cause a reversible and sustained increase of Ca2+ in the button of afferent nerve fibers. Typical recordings are presented and potential interests for pharmacological studies of inner ear sensory cell synapses are discussed.

Optical detection of developmental origin of synaptic function in the embryonic chick vestibulocochlear nuclei.

Functional organization of the brain stem vestibulocochlear nuclei during embryogenesis was investigated using a multiple-site optical recording technique with a fast voltage-sensitive dye. Brain stem slices with the cochlear and/or vestibular nerves attached were dissected from 6- to 8-day-old (E6-E8) chick embryos. Electrical responses evoked by cochlear or vestibular nerve stimulation were optically recorded simultaneously from many loci of the preparations. In E7 and E8 preparations, we identified two components of the optical response with cochlear or vestibular nerve stimulation; one was a fast spike-like signal related to the action potential, and the other was a slow signal related to the glutamate-mediated excitatory postsynaptic potential. The location of the cochlear nerve response area was mainly located on the dorsolateral region, while that of the vestibular nerve was deviated ventromedially. At E6, cochlear nerve stimulation evoked only the fast spike-like signals in normal Ringer solution. However, when we removed Mg2+ from the extracellular solution, significant slow signals were elicited in the E6 preparation. The present results demonstrated that in the chick vestibulocochlear nuclei, functional synapses are already generated by the E7 embryonic stage and that postsynaptic activity related to N-methyl-d-aspartate receptors emerges latently, at least in the cochlear nerve-related nucleus, at the E6 embryonic stage. This chronological sequence of the emergence of postsynaptic function is different from that reported previously (E10-E11), suggesting that the developmental origin of sensory information transfer in the auditory pathway is much earlier than has been anticipated.

Increased cell death in the developing vestibulocochlear ganglion complex of the mouse after prenatal ethanol exposure.

BACKGROUND: Previous studies have demonstrated that excessive prenatal alcohol exposure can damage the auditory and vestibular systems, in particular, cochlear hair cells. However, the direct effect of ethanol on the peripheral neurons in these pathways has not been examined. To study the effects of prenatal ethanol exposure on the developing vestibulocochlear ganglion (VCG) complex and the peripheral sensory organs, we exposed pregnant mice to ethanol and examined the levels of cell death in the inner ear. METHODS: Pregnant C57BL/6J mice were administered one of three doses of either ethanol (3.0, 4.5, and 5.5 g/kg) or isocaloric maltose/dextrin via intragastric intubation on gestational day (GD) 12.5. Embryos were dissected out of the uterus 8 hr after the intubation. Dying cells in the inner ear were stained with Nissl stain and labeled by in situ terminal dUTP nick-end labeling (TUNEL), and the percentage of dying cells was quantified. RESULTS: Ethanol exposure produced region-specific effects, with ethanol-exposed embryos exhibiting enhanced cell death only in the VCG complex, and not in the primitive saccule, cochlea, semicircular canal, or endolymphatic sac. The effects of ethanol on cell death in the VCG are dose dependent, with a significant increase in the level of cell death found only at the higher doses. CONCLUSIONS: Ethanol has a selective cytotoxic dose-dependent effect on the VCG at GD 12.5 suggesting that loss of VCG neurons may contribute to hearing and /or vestibular abnormalities in FAS children. Furthermore, the presence of TUNEL-positive cells and DNA laddering is consistent with the cells undergoing apoptotic cell death.

Lineage analysis in the chicken inner ear shows differences in clonal dispersion for epithelial, neuronal, and mesenchymal cells.

The epithelial components of the vertebrate inner ear and its associated ganglion arise from the otic placode. The cell types formed include neurons, hair-cell mechanoreceptors, supporting cells, secretory cells that make endolymphatic fluid or otolithic membranes, and simple epithelial cells lining the fluid-filled cavities. The epithelial sheet is surrounded by an inner layer of connective and vascular tissues and an outer capsule of bone. To explore the mechanisms of cell fate specification in the ear, retrovirus-mediated lineage analysis was performed after injecting virus into the chicken otocyst on embryonic days 2.5-5.5. Because lineage analysis might reveal developmental compartments, an effort was made to study clonal dispersion by sampling infected cells from different parts of the same ear, including the auditory ganglion, cochlea, saccule, utricle, and semicircular canals. Lineage relationships were confirmed for 75 clones by amplification and sequencing of a variable DNA tag carried by each virus. While mesenchymal clones could span different structural parts of the ear, epithelial clones did not. The circumscribed epithelial clones indicated that their progenitors were not highly migratory. Ganglion cell clones, in contrast, were more dispersed. There was no evidence for a common lineage between sensory cells and their associated neurons, a prediction based on a proposal that the ear sensory organs and fly mechanosensory organs are evolutionarily homologous. As expected, placodal derivatives were unrelated to adjacent mesenchymal cells or to nonneuronal cells of the ganglion. Within the otic capsule, fibroblasts and cartilage cells could be related by lineage.

Afferents of cranial sensory ganglia pathfind to their target independent of the site of entry into the hindbrain.

In vertebrates, sensory neurons interconnect a variety of peripheral tissues and central targets, conveying sensory information from different types of sensory receptors to appropriate second-order neurons in the central nervous system (CNS). To explore the possibility that the different rhombomere environments where sensory neurons enter into the hindbrain affect the pathfinding capability of growth cones, we studied the development of the VIIIth ganglion afferent both in vivo and in vitro. We focused on the vestibular nerve because it is the only cranial nerve projecting to the cerebellum, allowing for ready identification from its pattern of projection. Embryonic rat brain was cut along the dorsal midline and, with the VIIIth and Vth ganglia still attached, flat mounted and visualized with antibodies specific for sensory ganglia. Axons reached the cerebellar primordium at embryonic day (E) 13, then splayed out towards the edges of the rhombic lip of rostral hindbrain. In vitro, the VIIIth ganglion showed development similar to that in vivo and innervated the cerebellum, an appropriate target, indicating that mechanisms for axon guidance and target recognition are preserved in vitro. When the VIIIth ganglion was transplanted to the position of the Vth ganglion, axons from the transplanted ganglion entered the cerebellar primordium with a trajectory characteristic of the VIIIth nerve. These results indicate that the central projection pattern of the VIIIth nerve is not affected by the environment of nerve entry into the brainstem, suggesting that axons of sensory cranial ganglion intrinsically possess the capacity to find their target correctly.

Role for basic fibroblast growth factor (FGF-2) in tyrosine kinase (TrkB) expression in the early development and innervation of the auditory receptor: in vitro and in situ studies.

A previous study showed that basic fibroblast growth factor (FGF-2) promotes the effects of brain-derived neurotrophic factor (BDNF) on migration and neurite outgrowth from the cochleovestibular ganglion (CVG). This suggests that FGF-2 may up-regulate the receptor for BDNF. Thus we have examined TrkB expression during CVG formation and otic innervation in vitro and in the chicken embryo using immunohistochemistry. Following anatomical staging according to Hamburger-Hamilton, results were compared with mRNA expression in vitro using in situ hybridization. In the embryo at stage 16 (E2+) clusters of either lightly stained or immunonegative cells occurred within the otocyst and among those migrating to the CVG. By stage 22 (E3.5), immunostaining was concentrated in the CVG perikarya and invaded the processes growing into the otic epithelium but not into the rhombencephalon. Subsequently TrkB expression decreased in the perikarya and became localized in the leading processes of the fibers invading the epithelium and in the structures participating in synapse formation with the hair cells. In vitro there was moderate immunostaining and modest in situ hybridization for trkB in the neuroblasts migrating from the otocyst under control conditions. In contrast, neuroblasts previously exposed to FGF-2 exhibited accelerated migration and differentiation, with increased trkB mRNA expression. Morphological differentiation was associated with more intense immunostaining of processes than cell bodies. Evidently TrkB shifts its expression sequentially from sites engaged in migration, ganglion cell differentiation, axonal outgrowth, epithelial innervation, and synapse formation. FGF-2 may promote the role of BDNF in these developmental events by upregulating the TrkB receptor.

Trimetazidine prevents cochlear lesions induced by intraperitoneal and perilymphatic administration of kainic acid.

The protective activity of trimetazidine (TMZ) against cochlear neurotoxicity induced by intraperitoneal and intracochlear administration of kainic acid (KA) has been analyzed. The amplitude of the CAP N1 wave was significantly higher in KA rats pretreated with TMZ, independently of the administration route, than in those only treated with KA. However, CAP N1 amplitude of both TMZ pretreated and non-pretreated animals was always lesser than the N1 wave amplitude observed in the control group. The CAP N1 latency did not show any significant difference between KA and TMZ+KA groups except at high intensities of 8 and 12 kHz. As a complementary control, we have demonstrated that the intraperitoneal administration of TMZ (5 mg/kg) alone did not affect either the electrophysiological activity or the morphology of the auditory nerve. Morphological results fit well with electrophysiology. Some isolated swollen afferent fibers were observed in TMZ+KA cochleae, the swollen dendrites being sparser than in the KA only treated animals. In TMZ+KA animals, the cochlear apical coils were less affected than the basal coils. Our results are in agreement with recent clinical studies and suggest that TMZ could be an active drug on cochlear impairment linked to hypoxic-ischaemic syndromes.

The basic mechanism for the electrical stimulation of the nervous system.

Neural signals can be generated or blocked by extracellular electrodes or magnetic coils. New results about artificial excitation are based on a compartmental model of a target neuron and its equivalent electrical network, as well as on the theory of the generalized activating function. The analysis shows that: (i) in most cases, the origin of artificial excitation is within the axon and the soma is much more difficult to excite; (ii) within the central nervous system, positive and negative threshold currents essentially depend on the position and orientation of the neurons relative to the applied electric field; (iii) in several cases, stimulation with positive currents is easier; and (iv) it should be possible to excite synaptic activity without the generation of propagating action potentials. Furthermore, the theory of the generalized activating function gives hints to understanding the blockage of neural activity.

Ventromedial focus of cell death is absent during development of Xenopus and zebrafish inner ears.

We present the normal patterns of programmed cell death in the developing inner ears of a primitive anuran, Xenopus laevis, and an ostariophysan fish, Danio rerio. A prominent ventromedial focus of cell death was described previously in the developing chicken and mouse otocysts. We hypothesize that this focus of cell death might be associated with a signaling center that directs morphogenesis of the surrounding tissue. Amphibian and fish ear anatomies differ considerably from those of birds and mammals, particularly in the structures derived from the ventral part (pars inferior) of the otic vesicle. We reasoned that these anatomical differences between species might result from a difference in the size, location, or presence of a putative morphogenetic signaling center. Using in situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) to detect apoptotic cells, we show that developing Xenopus and zebrafish ears have apoptotic cells in the eighth cranial ganglia, the developing sensory patches, and in various positions in the otocyst wall. However, both species lack the persistent ventromedial hot spot of cell death that is prominently situated between the pars superior and pars inferior in the chicken and mouse otocysts.