MicroRNA-194 Regulates the Development and Differentiation of Sensory Patches and Statoacoustic Ganglion of Inner Ear by Fgf4.

BACKGROUND MicroRNA 194 is involved in the differentiation of various types of cells, such as adipose derived stem cells, human embryonic stem cells, and bone marrow mesenchymal stem cells. Previously, we found that miR-194 was highly expressed in the inner ear sensory patch and neurons in mice embryos. However, the role of miR-194 in the development of the inner ear and its underlying mechanism have not been elucidated yet. MATERIAL AND METHODS The expression level of miR-194 has been altered by using antisense morpholino oligonucleotides (MO) and synthesized miRNAs in zebrafish. RESULTS We found that miR-194 was vastly expressed in the inner ear and central nervous system (CNS) in zebrafish. Loss of function of miR-194 could strongly affected the development of zebrafish embryos, including delayed embryonic development, edema of the pericardium, small head, axial deviation, delayed development of inner ear, closer location of two otoliths, delayed fusion of the semicircular canals, and abnormal otolith number in some cases. In addition, the behavior of zebrafish was also adversely affected with impaired balance and biased swimming route. Misexpression of miR-194 could strongly affected the development and differentiation of spiral ganglion neuron (SGN) in inner ear through Fgf4 in vitro. Similar results have also been observed that the overexpression and knockdown of miR-194 strongly disturbed the development and differentiation of the sensory patches and Statoacoustic ganglion (SAG) through Fgf4 in zebrafish in vivo. Our results indicated that miR-194 may regulate the development and differentiation of sensory patches and SAG through Fgf4. CONCLUSIONS Our data revealed a vital role of miR-194 in regulating the development and differentiation of the inner ear.

Spemann organizer gene Goosecoid promotes delamination of neuroblasts from the otic vesicle.

Neurons of the Statoacoustic Ganglion (SAG), which innervate the inner ear, originate as neuroblasts in the floor of the otic vesicle and subsequently delaminate and migrate toward the hindbrain before completing differentiation. In all vertebrates, locally expressed Fgf initiates SAG development by inducing expression of Neurogenin1 (Ngn1) in the floor of the otic vesicle. However, not all Ngn1-positive cells undergo delamination, nor has the mechanism controlling SAG delamination been elucidated. Here we report that Goosecoid (Gsc), best known for regulating cellular dynamics in the Spemann organizer, regulates delamination of neuroblasts in the otic vesicle. In zebrafish, Fgf coregulates expression of Gsc and Ngn1 in partially overlapping domains, with delamination occurring primarily in the zone of overlap. Loss of Gsc severely inhibits delamination, whereas overexpression of Gsc greatly increases delamination. Comisexpression of Ngn1 and Gsc induces ectopic delamination of some cells from the medial wall of the otic vesicle but with a low incidence, suggesting the action of a local inhibitor. The medial marker Pax2a is required to restrict the domain of gsc expression, and misexpression of Pax2a is sufficient to block delamination and fully suppress the effects of Gsc The opposing activities of Gsc and Pax2a correlate with repression or up-regulation, respectively, of E-cadherin (cdh1). These data resolve a genetic mechanism controlling delamination of otic neuroblasts. The data also elucidate a developmental role for Gsc consistent with a general function in promoting epithelial-to-mesenchymal transition (EMT).

Vestibular-mediated synaptic inputs and pathways to sympathetic preganglionic neurons in the neonatal mouse.

To assess when vestibulosympathetic projections become functional postnatally, and to establish a preparation in which vestibulosympathetic circuitry can be characterized more precisely, we used an optical approach to record VIIIth nerve-evoked synaptic inputs to thoracic sympathetic preganglionic neurons (SPNs) in newborn mice. Stimulation of the VIIIth nerve was performed in an isolated brainstem-spinal cord preparation after retrogradely labelling with the fluorescent calcium indicator Calcium Green 1-conjugated dextran amine, the SPNs and the somatic motoneurons (MNs) in the thoracic (T) segments T2, 4, 6, 8, 10 and 12. Synaptically mediated calcium responses could be visualized and recorded in individual SPNs and MNs, and analysed with respect to latency, temporal pattern, magnitude and synaptic pharmacology. VIIIth nerve stimulation evoked responses in all SPNs and MNs investigated. The SPN responses had onset latencies from 90 to 200 ms, compared with much shorter latencies in MNs, and were completely abolished by mephenesin, a drug that preferentially reduces polysynaptic over monosynaptic transmission. Bicuculline and picrotoxin, but not strychnine, increased the magnitudes of the SPN responses without changing the onset latencies, suggesting a convergence of concomitant excitatory and inhibitory synaptic inputs. Lesions strategically placed to test the involvement of direct vestibulospinal pathways versus indirect pathways within the brainstem showed that vestibulosympathetic inputs in the neonate are mediated predominantly, if not exclusively, by the latter. Thus, already at birth, synaptic connections in the vestibulosympathetic reflex are functional and require the involvement of the ventrolateral medulla as in adult mammals.

Auditory input to CNS is acquired coincidentally with development of inner ear after formation of functional afferent pathway in zebrafish.

Auditory perception in vertebrates depends on transduction of sound into neural signals in the inner ear hair cells (HCs) and on transmission of these signals to the brain through auditory (VIIIth) nerve afferents. To investigate the developmental acquisition of auditory inputs by the CNS, we have electrophysiologically and morphologically examined the process of acquisition of auditory responsiveness by zebrafish macular HCs and the Mauthner cells (M-cells) in vivo. The M-cells are a paired large reticulospinal neurons in the hindbrain; they receive direct inputs from the VIIIth nerve afferents and initiate an acoustic startle response. Whole-cell recordings from the M-cells showed that sound-evoked postsynaptic currents were first observed around 40 h postfertilization (hpf); during subsequent development, onset latency decreased and amplitude increased. The appearance and development of microphonic potentials in the inner ear coincided with those of the acoustic responses of the M-cell, whereas the functional auditory circuits from the macular HCs to the M-cell were already formed at 27 hpf. These results suggest that the functional maturation of inner ear after formation of the auditory pathway is a critical process in the acquisition of auditory inputs by CNS neurons.

Morphological study on the rootlets comprising the root of the intermediate nerve.

The number and arising sites of the rootlet comprising the intermediate nerve root were investigated in 100 sides of human brains. The arising sites of the rootlets were the pons halfway between the facial and vestibulocochlear nerves (31.9%), the arising portion of the vestibulocochlear nerve on the pons (28.8%) and the stem of the vestibulocochlear nerve (15.9%). The number of roots varied from one to five, with the most common being one root (58%). The number of rootlets per root also varied from one to five. Most of the roots had one rootlet (63%), while 2% of the roots had five rootlets. The variation of the arising sites of the intermediate nerve rootlets and its clinical significance were also 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.

Thyroid hormone affects Schwann cell and oligodendrocyte gene expression at the glial transition zone of the VIIIth nerve prior to cochlea function.

All cranial nerves, as well as the VIIIth nerve which invades the cochlea, have a proximal end in which myelin is formed by Schwann cells and a distal end which is surrounded by oligodendrocytes. The question which arises in this context is whether peripheral and central parts of these nerves myelinate simultaneously or subsequently and whether the myelination of either of the parts occurs simultaneously at the onset of the cochlea function and under the control of neuronal activity. In the present paper, we examined the relative time course of the myelinogenesis of the distal part of the VIIIth nerve by analyzing the expression of peripheral protein P0, proteolipid protein and myelin basic protein. To our surprise, we observed that the expression of myelin markers in the peripheral and central part of the intradural part of the VIIIth nerve started simultaneously, from postnatal day 2 onwards, long before the onset of cochlea function. The expression rapidly achieved saturation levels on the approach to postnatal day 12, the day on which the cochlea function commenced. Because of its importance for the neuronal and morphological maturation of the cochlea during this time, an additional role of thyroid hormone in cochlear myelinogenesis was considered. Indeed, it transpires that this hormone ensures the rapid accomplishment of glial gene expression, not only in the central but also in the peripheral part of the cochlea. Furthermore, an analysis of the thyroid hormone receptors, TRaplha and TRbeta, indicates that TRbeta is necessary for myelinogenesis of the VIIIth nerve. Rapid thyroid hormone-dependent saturation of myelin marker gene expression in Schwann cells and oligodendrocytes of the VIIIth nerve may guarantee nerve conduction and synchronized impulse transmission at the onset of hearing. The thyroid hormone-dependent commencement of nerve conduction is discussed in connection with the patterning refinement of central auditory pathways and the acquisition of deafness.

Development and differentiation of the anuran auditory brainstem during metamorphosis: an acetylcholinesterase histochemical study.

The time course of cell differentiation and the presence of histochemically defined areas in brainstem auditory nuclei were examined in developing bullfrogs, Rana catesbeiana, using cresyl violet staining and acetylcholinesterase (AChE) histochemistry. In the medulla, the dorsolateral nucleus (DLN) can be seen as a distinct structure in its adult location only at Gosner stage 40 and beyond. The majority of cells in the DLN are not fully differentiated until late metamorphic climax (stages 45-46) and early postmetamorphosis. The more ventral vestibular nucleus differentiates earlier (stage 37) than the DLN. Adult-like organization of auditory nuclei in the torus semicircularis (TS) of the midbrain cannot be reliably discerned until metamorphic climax stages. Cellular masses in the brainstem reveal AChE from the earliest stage examined (stage 27) but the intensity of staining differs among cell groups. Staining intensity in the DLN is at a peak in recently metamorphosed froglets. The time course of cell differentiation in the DLN precedes slightly or is coincident with the increased, transient presence of AChE. Staining of the superior olive stabilizes at a moderate level in early postmetamorphic stages. Ventral regions of the principal nucleus in the TS stain more intensely than dorsal regions beginning at stage 40. This dorsal-ventral gradient in staining persists in adult stages. There is a transient decline in staining of the laminar nucleus in metamorphic climax stages. Staining intensity in the magnocellular nucleus peaks during stages 40-46 and in early postmetamorphic froglets and then declines in adults, paralleling the pattern seen in the DLN. These data suggest that metamorphic climax and early froglet periods are an important developmental window for major differentiation and maturational events in the auditory brainstem.

Long-term effects of sectioning the olivocochlear bundle in neonatal cats.

The olivocochlear bundle (OCB) was cut in neonatal cats to evaluate its role in the development of normal cochlear function. Approximately 1 year after deefferentation, acute auditory nerve fiber (ANF) recordings were made from lesioned animals, lesion shams, and normal controls. The degree of deefferentation was quantified via light microscopic evaluation of the density of OCB fascicles in the tunnel of Corti, and selected cases were analyzed via electron microscopy. In the most successful cases, the deefferentation was virtually complete. ANFs from successfully lesioned animals exhibited significant pathophysiology compared with normals and with other animals in which the surgery failed to interrupt the OCB. Thresholds at the characteristic frequency (CF), the frequency at which ANFs are most sensitive, were elevated across the CF range, with maximal effects for CFs in the 10 kHz region. Frequency threshold or tuning curves displayed reduction of tip-to-tail ratios (the difference between CF and low-frequency "tail" thresholds) and decreased sharpness of tuning. These pathological changes are generally associated with outer hair cell (OHC) damage. However, light microscopic histological analysis showed minimal hair cell loss and no significant differences between normal and deefferented groups. Spontaneous discharge rates (SRs) were lower than normal; however, those fibers with the highest SRs remained more sensitive than those with lower SRs. Findings suggest that the interaction between OC efferents and OHCs early in development may be critical for full expression of active mechanical processes.

ARIA is heavily expressed in rat peripheral auditory and vestibular ganglia.

The ARIA (acetylcholine receptor inducing activity) polypeptide is a member of the neuregulin gene family. It was originally purified on the basis of its ability to induce skeletal muscle nicotinic acetylcholine receptors (nAChRs). ARIA mRNA is expressed in ventral horn motor neurons and brain cholinergic neurons. We report here that ARIA mRNA is heavily expressed in the embryonic, developing, and adult peripheral auditory and vestibular ganglia, the spiral ganglion and Scarpa's ganglion. Neither ganglion is cholinergic, but both express mRNAs for nicotinic and muscarinic receptors. The expression of ARIA in these ganglia may be related to the regulation of cholinergic receptors or a more general role for ARIA in growth and development.

Development of dynorphin-like immunoreactive auditory nerve terminals in the chick.

The novel discovery that auditory nerve terminals in the chick cochlear nucleus magnocellularis (NM) are immunoreactive for the opioid peptide dynorphin (DYN) was recently reported [3]. The present study examines the development of DYN-immunoreactivity (DYN-I) in auditory nerve terminals in NM from embryos through young post-hatch chicks. No DYN-I was observed in NM at embryonic day 13 (E13). DYN-I first appeared at E16 as short flat structures partially surrounding NM cell bodies. Around post-hatch day 1 (P1), these structures had a more rounded, chalice-type of morphology reminiscent of the specialized auditory nerve terminals found in birds, the end-bulbs of Held. At P6, most NM neurons were circumscribed by a prominent DYN-I calyceal-type of ending. By P13, fewer NM cells were ringed by this DYN-I and by the third post-hatch week, there was very little DYN-I in NM. There were no obvious differences in the density of DYN-I terminals across either the rostrocaudal length or the mediolateral width of NM at any age examined. These results suggest that during a restricted time of development, end-bulbs of Held in the chick NM contain DYN.

Development of the Xenopus laevis VIIIth cranial nerve: increase in number and area of axons of the saccular and papillar branches.

Development of three branches of the VIIIth cranial nerve was examined in the anuran, Xenopus laevis. Sectioned tissue from the saccular, amphibian papillar, and basilar papillar branches of stage 52 larvae, 1 day postmetamorphosis juveniles, and 2-year adult animals was analyzed under the light microscope with a digital image analysis system. Numbers and cross-sectional areas of myelinated axons were measured in five to six nerve sections at each developmental age for each of the three branches. In all three branches, results show a significant increase in axon numbers between larval stage 52 and juvenile ages and negligible increase in axon number between the juvenile and adult ages. There were differences in the average number of axons between the saccular (704.4 +/- 39.5; n = 5), amphibian papillar (508.4 +/- 35.0; n = 5), and basilar papillar (316.0 +/- 7.0; n = 5) branches of adult animals. Myelinated axons increase at an estimated rate of 11.7, 15.1, and 6.2 axons per day for the saccular, amphibian papillar, and basilar papillar branches, respectively. Axonal cross-sectional areas increased throughout the developmental ages of this study, with the greatest increase taking place between juvenile and adult ages. In adult animals, 98% of axons in all three branches have diameters between 2-10 microns. Ratios of axons to hair cells in adult animals were estimated at 0.3, 1.1, and 5.3 for the sacculus, amphibian papilla, and basilar papilla, respectively. The higher axon to hair cell ratio correlates with the increasing acoustical frequency sensitivity of the end organ.

Distortion generated by the ear: its emergence and evolution during development.

Physiologically vulnerable active mechanics associated with the transduction of sounds in adults distort cochlear output. Specifically, frequencies not present in the incoming acoustic signal are represented in its output (ie., the spike trains of auditory nerve fibers). The purpose of this investigation was to study the appearance and evolution of intermodulation distortion during development. Established surgical and electrophysiologic techniques were used to record the extracellular electrical activity of individual auditory nerve fibers from developing kittens. Discharge-rate or synchrony-based responses to two tones (f1 and f2) presented simultaneously at various ratios of f2/f1 were recorded. The cubic distortion product (CDP; 2f1-f2, where f1

Responses of peripheral auditory neurons to two-tone stimuli during development: I. Correlation with frequency selectivity.

The responses of peripheral auditory neurons to two-tone stimuli were used to inferentially examine the nature of cochlear processing during development. Rate suppression was not seen in the youngest animals, and was first observed at 77 gestational days, in units exhibiting adultlike frequency selectivity. Suppression was highly correlated with the degree of tuning, and neurons were segregated into three classes based on these responses. Broadly tuned neurons (type IB) with low characteristic frequencies (CFs) did not exhibit suppression, and were observed early in postnatal life. Sharply tuned, but still immature neurons (type IS) exhibited suppression, but to a lesser degree than mature neurons (type M). One interpretation of these results is that basilar membrane mechanics are linear during the final stages of cochlear development, indicating that the immature signal transduction process is fundamentally different from that of adults.

Responses of peripheral auditory neurons to two-tone stimuli during development: III. Rate facilitation.

Approximately 25% of peripheral auditory neurons having low characteristic frequencies (CFs) and broad tuning, and recorded from immature animals, responded to two-tone stimuli with increases in discharge rate greater than predicted by linear summation of the responses to probe tones (facilitation), in contrast to the two-tone suppression observed in adult animals and in more sharply tuned immature neurons. Facilitation was not seen after 81 gestational days and was not observed when test tones produced a substantial increase in rate when presented alone. The fact that some neural responses were facilitated under conditions of two-tone stimulation during the final stages of cochlear differentiation provides additional evidence that signal transduction is fundamentally different in neonatal kittens than in adults. A linear model of basilar membrane mechanics coupled to nonlinear neural processes is proposed which can account for the production of facilitation.

Responses of peripheral auditory neurons to two-tone stimuli during development: II. Factors related to neural responsiveness.

In an accompanying paper (Fitzakerley et al., 1994), it was demonstrated that there is a significant developmental correlation between the appearance of tuning and two-tone suppression. However, it was also found that some sharply tuned neurons meeting minimal adult standards did not exhibit suppression. Therefore, in order to investigate other factors that may be related to the demonstration of suppression in peripheral auditory neurons, the relationship of two-tone suppression with various parameters related to neural responsiveness was studied in perinatal kittens. A positive correlation was made between the observance of suppression and driven discharge rate, characteristic frequency (CF), and threshold, all properties which change significantly during the final stages of cochlear differentiation. In older animals, suppression was also observed in higher percentages of neurons having low spontaneous rates (< 1 spk/s). Suppression was evoked less often by test tones placed below CF than above CF in neurons recorded from younger animals, and was generally produced by a narrower range of test tone intensities than those recorded from adults. As a result, the conventional description of suppression as observed in peripheral auditory neurons in mature animals must be extended to include these factors when responses of immature neurons are considered.

Patterned neural activity in brain stem auditory areas of a prehearing mammal, the tammar wallaby (Macropus eugenii).

Is patterned neural activity in immature, prefunctioning sensory systems a general phenomenon? Such patterning has been found in the prenatal visual and somatosensory systems. We have now identified patterning in the immature auditory system of a prehearing mammal, the tammar wallaby. Neurones recorded in vivo from the eighth nerve and cochlear nucleus at pouch days 94-122 discharged in bursts with rhythmic inter-spike intervals. Our findings are applied to the argument that neural activity is vital to sensory development.

Modes of neuronal arbor enlargement in the ear of a postembryonic fish, Astronotus ocellatus.

New hair cells are added during postembryonic life in several species of fishes and birds. The production of new hair cells appears to require enlargement of eighth nerve arbors during growth since, at least in fish, eighth nerve neurons are added more slowly than hair cells or not at all. This situation provides an intriguing opportunity to study the mechanisms of growth of the neuronal arbors. In this paper, we report the results of studies on the postembryonic growth of eighth nerve dendritic arbors in the saccular epithelium of the cichlid fish Astronotus ocellatus. Arbor sizes and shapes were compared in small and large fish using the axonal tracer cobaltous lysine. Our data suggest that postembryonic eighth nerve arbors enlarge in 2 ways. First, arbors add new terminal endings to their distal ends. Second, whole new branches appear to be added at locations up to hundreds of micrometers proximal to the terminal endings. These 2 modes of growth suggest that more than one mechanism may be operative in controlling arbor enlargement.

Peripheral and central target-derived trophic factor(s) effects on auditory neurons.

In the developing inner ear, a naturally occurring programmed cell death of cochleovestibular ganglion (CVG) neurons as well as peripheral and central target-derived trophic effects on survival of embryonic CVG neurons are known. To further analyze these target derived trophic interactions, spiral ganglion explants obtained from 5 day postpartum (P5) rat pups were cultured with an intact organ of Corti and in the absence of Corti's organ. Both neuronal survival and neurite extension were influenced by the presence of this peripheral target tissue. Local destruction of Corti's organ caused both neuritic retraction and neuronal cell death to occur in a corresponding portion of the spiral ganglion. This peripheral target-derived neurotrophic effect may be mediated by a diffusible factor(s) since organ of Corti conditioned medium also had a neurotrophic effect on the survival of auditory neurons in cell cultures of dissociated spiral ganglia from P5 rat pups. A component of central target tissue, i.e. astrocytes, was also shown to release a diffusible factor(s) that supported the survival of dissociated P5 rat spiral ganglion neurons. The neurotrophic effects on the in vitro survival of spiral ganglion neurons by both of these conditioned medium factors were concentration dependent.