Susan M. Slapnick

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.

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.

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.

Neuronal sprouting and synapse formation in response to injury in the mouse organ of corti in culture

The effect of mechanical injury on induction of regenerative phenomena within the neurosensory epithelium was investigated in cultures of neonatal mouse cochlea. The oldest examined culture in which new neuronal growth followed insult, was injured at 13 days in vitro and fixed 24 h later. By far, the most vigorous regenerative reaction was observed in a 3‐day culture 4 h post‐injury. The reaction included sprouting of nerve fibers injured directly, synapse formation between the surviving hair cells and sprouting neuronal growth cones, wrapping of growing nerve fibers by extending processes of hair cell cytoplasm, and collateral sprouting of synaptically‐engaged nerve endings and of nerve fibers in passage.

Presynaptic fibres of spiral neurons and reciprocal synapses in the organ of Corti in culture

SummaryIsolated segments of the newborn mouse organ of Corti were explanted together with the spiral ganglion components. Within the innervation provided by the spiral neurons, we observed presynaptic vesiculated nerve endings that form reciprocal ribbon-afferent/efferent synapses with inner hair cells. These intracochlear presynaptic fibres are characteristically located between adjoining inner hair cells, on the modiolar side, low and close to the supporting cells. The presynaptic fibres display different modes of synaptic connectivity, forming repetitive reciprocal synapses on single inner hair cells or on adjoining hair cells, or connecting adjoining inner hair cells through simultaneous efferent synapses. Many presynaptic fibres exhibit a distinctive ultrastructure: defined clusters of synaptic vesicles, dense core vesicles, coated vesicles, and mitochondria. These organelles occur focally at the synaptic sites; beyond the efferent synaptic specializations, the endings appear quite nondescript and afferent-like.We believe that the reciprocal synapses, although observed in cultures of the organ of Corti, represent real intracochlear synaptic arrangements providing a feedback mechanism between the primary sensory receptors and a special class of spiral ganglion cells that have yet to be recognized in the organin situ.

Ryanodine receptor localisation in the mammalian cochlea: an ultrastructural study.

Calcium-induced calcium release (CICR) in the mammalian cochlea has been suggested to enhance neurotransmitter release from inner hair cells and facilitate the efferent response in outer hair cells. Light microscopic evidence exists for the presence of ryanodine receptors in the organ of Corti but there is so far no information about their ultrastructural localisation. We have therefore used post-embedding immunogold labeling with antibodies that predominantly recognise ryanodine receptor isoforms 1 (RyR1) and 2 (RyR2) to investigate their distribution in rat cochleae. In inner hair cells, the highest levels of labeling were observed over an area of rough endoplasmic reticulum that lies in the cytoplasmic region beneath the nucleus; in outer hair cells, the cytoplasmic region above the nucleus displayed most labeling. Labeling was also associated with the subsurface cisternae adjacent to the lateral membranes of both types of hair cell, with the efferent terminals on the outer hair cells and was observed in adjacent supporting cells. Labeling in outer hair cells was significantly higher than that in inner hair cells or in the supporting cells. Our results support the presence of RyR1 in the cochlea but do not rule out the presence of other isoforms. CICR may be involved in the control of calcium levels in the base of the inner hair cells and supporting cells, and in the cholinergic efferent response and motile behaviour of the outer hair cells.

Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse

The bronx waltzer (vb) mutation in the mouse results in the degeneration of most but not all of the primary auditory receptors, the inner hair cells, and their afferent neurons. We analyzed the ultrastructure of 94 inner hair cells in the intact postnatal mutant mouse and in neonatal cochleas in culture to understand the pathogenesis of hair cell death and to detect factors that may prevent it. The vb spiral neurons of the bronx waltzer display two distinctive features: some of them continue to divide mitotically for at least seven postnatal days, and the type I radial fibers that innervate inner hair cells display a deficiency in immunoexpression of GAD. The growing endings of spiral neurons converge around the inner hair cells or, in their absence, invade the outer hair cell region. Their profuse sprouting among inner spiral sulcus cells contributes to the characteristic ultrastructural picture of the bv cochlea. During the first three days after birth, 40% of the inner hair cells appear normal and innervated, 40% are mostly denervated and degenerating, and 20% are immature, with minimal or no neuronal appositions. However, in mutants 6 days and older only a few inner hair cells survive, and these show either normal or superfluous afferent innervation and axosomatic GABAergic efferent innervation. Degeneration of inner hair cells begins with a distention of the nuclear envelope and the ribosomal endoplasmic reticulum. The outer nuclear membrane eventually breaks, and exudate fills the cell interior. The cellular edema leads to cell death. We propose that success or failure in synaptic acquisition is a decisive factor in the survival or decline of the mutant inner hair cells. We also suggest that the developmental delay in maturation of the spiral ganglion neurons (type I) and the failure in their synaptogenesis may be caused by an impairment in neurotrophin (NT3/BDNF) synthesis by their mutant receptor cells.

Apoptosis of inner hair cells caused by laser ablation of their spiral ganglion neurons in cultures of the mouse organ of Corti.

Laser beam ablation of spiral ganglion neurons was performed in seven organotypic cultures of the newborn mouse cochlea between 5 and 8 days in vitro, with a recovery period of from 18 hours to 3 days. Direct somatic injury (laser or mechanical) inflicted on hair cells does not necessarily cause their death; many of them survive, repair damage and re-establish their neurosensory connections. By contrast, laser irradiation and ablation of their afferent spiral ganglion neurons causes a most spectacular degeneration of sensory cells within 18–48 hours after the insult. Ultrastructurally, the degenerated hair cells—characteristically the inner hair cells—display “dark-cell vacuolar degeneration” that combines the signs of apoptotic death (the peripheral condensation of nuclear chromatin and nuclear pyknosis) with signs of cell edema, vacuolization and necrosis. The ultimate condensation of the cytoplasm gives the dead cells a jet black appearance. The irradiated spiral ganglion neurons die displaying similar pathological characteristics. The extent and locus of inner hair cell degeneration correspond to that of ablated spiral ganglion neurons: ultimately the ablation of one neuron causes degeneration of a single inner hair cell within the closest radial segment of the afferent innervation. The elimination of spiral ganglion neurons by mechanical means does not affect hair cell survival.It is inferred that the laser pulse acts as a stimulus depolarizing the neuronal membrane of the spiral ganglion neurons and their radial fibers and causing the excitotoxic death of their synaptic sensory cells through excessive stimulation of the glutamatergic receptors. Reciprocal pre-and postsynaptic synapses between the afferent dendrites and inner hair cells in culture could possibly serve as entryways of the stimulus. The pathogenesis of this apparent transsynaptically-induced apoptotic death of inner hair cells will be further examined in culture.

Terminal dendritic sprouting and reactive synaptogenesis in the postnatal organ of Corti in culture

Synaptogenesis in the organ of Corti between the primary receptors, the inner hair cells, and the peripheral processes of their afferent spiral ganglion neurons in the mouse lasts for 5 days postnatally (Sobkowicz et al. [1986] J. Neurocytol. 15:693–714). The transplantation of the organ into culture at the fifth postnatal day induces a reactive sprouting of dendritic terminals and an extensive formation of new ribbon synapses within 24 hours. This reactive synaptogenesis differs strikingly from the primary synaptogenesis and has been seen thus far only in the inner hair cells. The synaptically engaged neuronal endings sprout a multitude of filopodia that intussuscept the inner hair cells. The filopodial tips contain a heavy electron‐dense matter that appears to attract the synaptic ribbons, which form new synaptic contacts with the growing processes. The intensity of the filopodial growth and synaptogenesis subsides in about 3 days; the filopodia undergo resorption, leaving behind fibrous cytoplasmic plaques mostly stored in the supranuclear part of the hair cells. However, occasional filopodial growth and formation of new synaptic connections continued. The data demonstrate that any disruption or disturbance of the initial synaptic contacts between the inner hair cells and their afferent neurons caused by transplantation results in prompt synaptic reacquisition. Furthermore, we suggest that the transitory phase of terminal sprouting and multiribbon synapse formation manifests a trophic dependence that develops postnatally between the synaptic cells. J. Comp. Neurol. 397:213–230, 1998.