Hanna M. Sobkowicz

Organotypic development of the organ of Corti in culture.

SummaryThe preservation and development of the innervation pattern in the organ of Corti have been studied in culture up to 27 daysin vitro. The explants were obtained from the newborn mouse. Segments of the cochlear duct dissected together with the appropriate sectors of the spiral ganglion may retain their structural organization for about two weeks. Maturation of some nonneuronal elements which occurs during that time is followed by a subsequent regression of the organ.Only a fraction of the explanted neurons survive. However, the surviving neurons, if connected with the hair cell region, maintain a complex peripheral innervation pattern that contains all the major fibre components which characterize the normal pattern in a young mouse. The peripheral innervation pattern in culture seems largely composed of preserved fibres, that is, of fibres which at the time of explantation have already ramified within the organ of Corti. Nonetheless, there is evidence for growth or maturation, in culture, of at least some peripheral processes of the spiral neurons. Thus, only in older cultures is the innervation of the apical tip established. Likewise, it is only in older expiants that the inner spiral bundle becomes prominent.Spiral neurons survive in culture in several modes. Most frequently, the central process is altogether absent and the neuron is effectively a unipolar cell which maintains only the peripheral process. A distinct minority of neurons is bipolar possessing both the peripheral process and a central axon which grows freely, though no central target is present. A neuron may survive also as a unipolar or, rarely, as a bipolar cell with no processes entering the organ of Corti.The observations imply that (1) most or all major fibre systems in the organ of Corti carry components of spiral neuron origin; (2) a small population of spiral neurons innervating a short segment of the organ contributes importantly not only to the radial but also to the spiral innervation of the segment.

Distribution of synaptic ribbons in the developing organ of Corti

SummaryStudies of synaptogenesis in the developing organ of Corti in the intact mouse and in culture indicate that the inner and outer hair cells contain three populations of synaptic ribbons, i.e. ribbons adjacent to nerve fibres, free intracellular ribbons and misplaced ribbons apposed to non-neuronal elements. Ribbons adjacent to nerve fibres can be further classified into: ribbons synaptically engaged, ribbons participating in formation of presynaptic complexes only and ribbons that are not engaged to the hair cell membrane. In the developing innervated cultures the ribbon distributions are similar to those in the normal animal. Inner and outer hair cells differ in distribution of the ribbons. In the inner hair cells the ribbons adjacent to the nerve fibres are dominant (over 90%) and most of them (88%) are synaptically engaged. In the outer hair cells the presynaptic ribbons dominate the population (up to 60%) during the first postnatal week when the cells acquire afferent synaptic connections. This stage is followed by a marked reduction in the number of all ribbons. In the intact animal the rapid decrease results in a relative increase of misplaced and free ribbons. These changes are presumably due to the loss of some of the afferents. In the denervated hair cells the distribution of ribbons indicated the presence of conspicuous scatter. In the areas of incomplete denervation, however, the ribbons are apposed to the preserved fibres. Despite denervation, most of the ribbons develop the entire presynaptic complex in apposition to non-neuronal structures.The different populations of synaptic ribbons appear to reflect different stages in synapse formation. Possibly, the synaptic body originates in the interior of the hair cell and subsequently migrates to the cell membrane. In any case, a nerve fibre appears critical in influencing the location of the synaptic ribbon. At the apposition of the ribbon to the hair cell membrane, presynaptic densities are formed and the ribbon appears to become anchored. Typically, the nerve fibre membrane apposed to the presynaptic complex responds with the formation of postsynaptic densities.

The kinocilium of auditory hair cells and evidence for its morphogenetic role during the regeneration of stereocilia and cuticular plates

SummaryAuditory hair cells that survive mechanical injury in culture begin their recovery by reforming the kinocilium. This study is based on cultures of the organ of Corti of newborn mice and two control animals. The axonemal patterns were examined in 165 kinocilia in cross-section. In the immature and regenerating kinocilium, one of the normally peripheral doublets is frequently located inward, forming the modified 8 + 1 (double) form; the distribution of the remainingmicrotubules is irregular. As the cell matures, the 9 + 0 form predominates. Overall, 34–61% of auditory kinocilia consist of 9 + 0 microtubules. The 9+2 (single) form, previously thought to characterize the organelle, occurs only in about 3–14%, whereas the remaining population comprises the modified 8 + 1 (double) form. Normally, the kinocilium lasts only about 10 postnatal days; however, post-traumatic hair cells reform their kinocilia regardless of age. Concomitant with the regrowth of the kinocilium, the basal body and its cilium take a central location in the cuticular plate, stereocilia regrow, and the cytoplasmic area adjacent to the basal body displays pericentriolar fibrous densities, growth vesicles, and microtubules, all surrounded by actin filaments. Pericentriolar bodies nucleate microtubules. Involvement of microtubules is seen in the alignment of actin filaments and in the formation of the filamentous matrix of the cuticular plate. We propose that reformation of the kinocilium in recovering post-traumatic hair cells indicates the possible role of its basal body in the morphogenesis and differentiation of cuticular plates and stereocilia.

Development of acetylcholinesterase-positive neuronal pathways in the cochlea of the mouse.

SummaryDevelopment of the cholinergic enzymes, choline acetyltransferase (ChAT) and AChE, and of the AChE-positive innervation in the cochlea was studied biochemically and morphologically in the postnatal mouse up to 26 days. Both ChAT and AChE are already present at birth in levels comparable to 50 and 20% of near-adult values, respectively. Increases in the enzymatic activities occur mainly during the second postnatal week. ChAT increases primarily in the basal turn; the specific activities in the basal and mid turns become about equal and at least twice of the values found in the apex. AChE increase continues throughout the entire cochlea; at all times its activity is highest in the base and lowest in the apex.In the light microscope, AChE-positive fibres are seen to enter the organ in the intraganglionic bundle during late foetal development and travel upwards via radial bundles. The fibres destined for outer hair cells usually differentiate first and take a separate route. They either cross the prospective tunnel of Corti directly or take a spiral course in front of inner pillar cells to form the inner pillar bundle. The tunnel fibres are radially oriented and provide the innervation to outer hair cells in narrow vertical sectors. In most cases, the outer hair cells are being innervated by the 4th day. Between the 4th and the 6th day, the tunnel fibres reach the outer hair cells in the third row; the first and second outer spiral bundles are formed. The AChE-positive innervation of the inner spiral bundle and plexus forms in short segments, and the bundle may be still discontinuous even by the 6th day. By the 12th day the innervation is complete. In the electron microscope, the stain for AChE may allow identification of growing efferent fibres before their ultrastructural differentiation.Both ChAT and AChE activities are early markers of the differentiating efferent system. An ingrowth of the cholinergic fibres to the entire cochlea occurs before birth. The greatest increase of AChE occurs between the 4th and 10th day, relating in time to efferent synaptogenesis.

GABA-like immunoreactivity in the cochlea of the developing mouse

SummaryGABA-like immunoreactivity was studied in surface preparations of cochleas from postnatal developing mice, and GAD-like immunoreactivity was studied in the adult. GABA-positive fibres are already present at birth; they innervate both the inner and outer hair cells and some spiral ganglion cells. The GABA-positive fibres that enter via the intraganglionic bundle send collaterals to the spiral ganglion and to the hair cell region. Fibres that enter along the central processes of spiral neurons end predominantly among the spiral ganglion cells. A few spiral neurons display pericellular rings of GABA-positive boutons at birth. In older animals, the endings occur on a small number of spiral ganglion cells either as rings or as brush formations. The early GABA-positive fibres reach the inner hair cells around the second day and the outer hair cells (of the upper turns only) around the seventh day. In 12-day animals, tunnel fibres arborize in the outer hair cell region; their collaterals make contacts with the outer hair cells within four to eight cell-wide segments, distributing the endings high, up to the reticular plate. In older animals, fibres (both GABA- and GAD-positive) may innervate single vertical rows of outer hair cells. In the maturing and the adult cochlea, the GABA-positive component of the inner spiral bundle is conspicuous and extends along the entire cochlear length, but the innervation of the outer hair cells comprises only the mid and apical turns.GABA-positive nerve cells occur among the small vestibular neurons, occasionally among the cells of eighth nerve nucleus and only exceptionally in the spiral ganglion. In the adult animal, GAD-positive cells, although uncommon, were observed among the spiral neurons.In the developing animal, GABA-positive fibres give rise to transitory formations: (1) a convoluted plexus running beneath and among the radial bundles and (2) a sparse plexus, continuous with the inner spiral bundle and running in the upper plane of the inner spiral sulcus. GABA-like immunoreactivity was also observed in neuronal growth cones and in some fibres running along blood vessels.In conclusion, GABA immunoreactive fibres appear to reach the cochlea by two routes: via the intraganglionic bundle and to a much lesser extent via the central bundles of the spiral ganglion. The fibres innervate sensory cells and also some spiral neurons. The occasional presence of GABA-positive neurons in the vestibular ganglion, in the VIIIth nerve nucleus, and exceptionally among the spiral neurons raises the possibility of a local GABAergic circuitry within the inner ear.

Patterns of neurofilament stain in the spiral ganglion of the developing and adult mouse

The objective of the study was to identify neurofilament-positive cells and their projections in the intact spiral ganglia of the mouse. One polyclonal and three monoclonal antibodies against neurofilament triplet subunits NF 68 K, 160 K and 200 K were used. In the newborn mouse most of the spiral neurons and their processes stain positively, although the perikaryal stain is very light. During early postnatal development, some cells show a selective intense stain. The progressive myelination of the neuronal processes further restricts the stain to a small neuronal population of positive perikarya and to their nonmyelinated fibers. This pattern of stainability implies that the neurofilament-positive cells are compatible with the type II spiral neurons. The stain reveals two populations of spiral neurons: 1) the cells which are scattered within the ganglion and show a bipolar distribution of fibers; and 2) the cells that form an interrupted chain along the intraganglionic bundle. The latter cells are also bipolar, but their peripheral processes join the intraganglionic bundle for varying distances before reaching the radial bundles. The identification of selective groupings of filamentous nonmyelinated cells in the corresponding location in different mammals is discussed. In conclusion, the use of neurofilament antibodies in staining of the intact spiral neurons permitted us to identify a distinct cell population of neurofilament-positive nonmyelinated nerve cells located along and projecting (at least partly) into the intraganglionic bundle.

The efferents interconnecting auditory inner hair cells

The work describes the system of efferent terminals that interconnect inner hair cells through a chain of direct somatic synapses organized in repetitive patterns. The efferent boutons were discovered in the apical turns of 12-day-old (hearing) mice. Clusters or short rows of vesiculated boutons are located between adjoining hair cells at the lower half of the receptors, close to their modiolar side. The individual endings, about 1.2 microns in diameter, adjoin inner hair cells and form one synapse per hair cell. On the hair cell side, the synaptic contact is apposed by a classical postsynaptic cisterna. Within a cluster of endings, some synapse simultaneously with either or both neighbouring inner hair cells. The efferent boutons also connect synaptically with each other and with other--different in type--vesiculated and nonvesiculated endings. These endings seem to derive from the climbing collaterals of the inner spiral bundle, and we believe them to be GABAergic.

Influence of neurotrophins on the synaptogenesis of inner hair cells in the deaf Bronx waltzer (bv) mouse organ of Corti in culture.

The Bronx waltzer (bv) deaf mouse is characterized by massive degeneration of the primary auditory receptors, the inner hair cells, which occurs during the time of expected afferent synaptogenesis. The process is associated with degeneration and protracted division of the normally postmitotic afferent spiral ganglion neurons. To investigate the potential role of neurotrophins in the afferent synaptogenesis of inner hair cells, we exposed bv newborn cochleas in organotypic culture to brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF), and also to gamma aminobutyric acid (GABA), for up to 8 days. The study was done using light and electron microscopy. Only about 20% of the inner hair cells survived in culture, regardless of the treatment, similar to the number in the intact mutant in our colony. Depending on the exogenous treatment, this population consisted of either innervated ultrastructurally normal cells or denervated dedifferentiated cells wrapped—in lieu of nerve endings—by the supporting inner phalangeal and border cells. In the control and GABA cultures, inner hair cells were mostly denervated. BDNF and NT‐3 alone or combined increased synaptogenesis and hair cell survival only during the first 3 days (by about 10%); however, the cells became denervated by 8 postnatal (PN). Only NGF induced stable innervation and differentiation of neurosensory relationships, including supernumerary innervation characteristic of the intact bv. Denervation among the remaining 20% of inner hair cells induced a reactive wrapping by inner phalangeal and border cells which evidently extended inner hair cell survival. Immunocytochemical studies of these reactive supporting cells were done in the intact (8 PN) mutant cochlea. The supporting cells that provide sustenance to the denervated inner hair cells displayed strong BDNF (and possibly NT‐3) immunoreactivity. Subsequently, we revealed the presence of all three neurotrophins in the inner hair cell region of the developing (1–8 PN) cochlea of the normal ICR mouse. The inner hair cells expressed all three neurotrophins; BDNF prevailed in the inner phalangeal cells, NT‐3 in the pillar cells and inner phalangeal cells, and NGF in the pillar cells. In conclusion: initially, the 80% loss of inner hair cells is apparently caused by their failed afferent synaptogenesis. Exogenous neurotrophins influence synaptogenesis in the bv in culture, but NGF alone is successful in promoting stable neurosensory relationships. The presence of neurotrophins in supporting cells in the normal and degenerating cochlea indicates their role in the sustenance of inner hair cells.

Differentiation and distribution of acetylcholinesterase molecular forms in the mouse cochlea

The activities of the globular and asymmetric forms of acetylcholinesterase (AChE) were measured in the whole cochlea and cochlear turns of the developing postnatal mouse. The globular AChE forms (G4, G2 and G1) were present in each cochlear turn at birth. An asymmetric AChE form (A12) was detected in the midturn on the 4th postnatal day, and in the base and apex on the 7th postnatal day. The activities of all AChE molecular forms increased rapidly during the second postnatal week and reached a plateau by the 19th postnatal day. In the 26-day old mouse, G4 constitutes the largest proportion of total cochlear AChE (57%), G2/G1 being 37% and A12 being 6%. The distribution of the AChE forms among the different turns is as follows: the combined value of the activities of G2 and G1 AChE was the same in each turn; G4 was the major form in the base and midturn; and A12, the least abundant AChE form of all, was localized mainly in the base. Our results indicate that in the cochlea (1) the content of molecular forms is similar to that of other neuronal systems, (2) the expression of AChE molecular forms is developmentally regulated, and (3) the AChE isoenzymes develop and are distributed differentially along the cochlear length; resulting near maturity in the greater proportional expression of G4 and A12 in the base and midturn and G2/G1 in the apex.

COMPOUND SYNAPSES WITHIN THE GABAERGIC INNERVATION OF THE AUDITORY INNER HAIR CELLS IN THE ADOLESCENT MOUSE

Ultrastructural investigation of the γ‐aminobutyric acid (GABA) component of the inner spiral bundle in adolescent mice revealed a pathway of glutamic acid decarboxylase (GAD)‐positive and ‐negative fibers and vesiculated endings that contact inner hair cells and their afferents through a complex of axosomatic and axodendritic synapses. Ultrastructural details were investigated by using conventional electron microscopy. Several synaptic arrangements were observed: Main axosomatic synapses form between vesiculated endings and individual or adjoining inner hair cells (interreceptor synapses). Spinous synapses form on long, spinelike processes that protrude from inner hair cells to reach distant efferent endings. The efferent endings associate with inner hair cells and their synaptic afferents through compound synapses—serial, “converging,” and triadic—otherwise characteristic of sensory relay nuclei. Serial synapses form by the sequential presynaptic alignment of the efferent→receptor→afferent components. Converging synapses result from the simultaneous apposition of a receptor ribbon synapse and a presynaptic efferent terminal on a recipient afferent dendrite. Triadic synapses comprise a vesiculated efferent ending in contact with an inner hair cell and with its synaptic afferent. Additionally, efferent endings may form simple axodendritic and axoaxonal synapses with GAD‐negative vesiculated endings. The combination of different synaptic arrangements leads to short chains of compound synapses. It is assumed that these synaptic patterns seen in the adolescent mouse represent adult synaptology. The patterns of synaptic connectivity suggest an integrative role for the GABA/GAD lateral efferent system, and imply its involvement in the pre‐ and postsynaptic modulation of auditory signals. J. Comp. Neurol. 377:423–442, 1997.