Doubt and certainty in counting.

Some of the methods used for counting objects in histological sections are discussed. The method best suited for any particular counting program depends on many variables, which include the level of accuracy required, the type of preparation available for study, the size of the objects to be counted, the thickness of the sections that can be used, the equipment available and the amount of labor that can reasonably be invested. For light and electron microscopy, profile counts are simple and quick for objects that are small relative to section thickness and whose dimensions are readily defined. The 'physical disector' is particularly useful where objects to be counted are large relative to sections thickness, or where their dimensions are unknown or highly variable. For light microscopy, the optical disector is often easier to use. However, it makes more assumptions than the physical disector; some of these can introduce serious bias in the counts, and they are explored. Electron microscopy raises some special problems that relate to the depth of focus, the relatively very thin sections, and the tendency for thin structures that do not span the full thickness of a section to be lost or unrecognizable in some section planes. The importance of recognizing the assumptions that underlie any method of counting and its interpretation is stressed.

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.

Tunnel crossing fibers and their synaptic connections within the inner hair cell region in the organ of corti in the maturing mouse.

Combined ultrastructural and immunocytochemical studies reveal that in the adolescent 12- to 17-day-old mouse the afferent tunnel crossing fibers that innervate outer hair cells receive synaptic contacts from three distinct sources: the GABAergic fibers (GABA = gamma-aminobutyric acid) of the lateral olivocochlear bundle, the non-GABAergic efferent tunnel crossing fibers, and the inner hair cells themselves. The GABAergic fibers give off collaterals that synapse with the afferent tunnel fibers as they cross the inner hair cell region. These collaterals also form synapses with afferent radial dendrites that are synaptically engaged with the inner hair cells. Vesiculated varicosities of non-GABAergic efferent tunnel fibers also synapse upon the outer spiral afferents. Most of this synaptic activity occurs within the inner pillar bundle. Distinctive for this region are synaptic aggregations in which several neuronal elements and inner hair cells are sequentially interconnected. Finally, most unexpected were the afferent ribbon synapses that inner hair cells-formed en passant on the shafts of the apparent afferent tunnel fibers. The findings indicate that: (1) the afferent tunnel (i.e., outer spiral) fibers may be postsynaptic to both the inner and the outer hair cells; (2) the non-GABAergic efferent and the afferent tunnel fibers form extensive synaptic connections before exiting the inner pillar bundle; (3) the GABAergic component of the lateral olivocochlear system modulates synaptically both radial and outer spiral afferents.

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.

Cellular interactions as a response to injury in the organ of Corti in culture.

We discovered and described ultrastructurally the intricate relationships between the sensory cells and their supporting cells in cultures of the organ of Corti following laser beam irradiation. Injury was performed using a 440 nm nitrogen-dye pulse laser aimed at the cuticular plates of inner hair cells. Laser injury is compared with mechanical injury inflicted on the hair cell region by a pulled-glass pipette. Regardless of the type of injury, but depending on its severity, the surviving hair cells may: (1) lose their stereocilia but subsist at the surface of the organ; (2) retain contact with the reticular lamina but be overgrown by the processes of the supporting cells; or (3) become sequestered from the reticular lamina and internalized among the supporting cells, where they either remain dedifferentiated or regrow an apical process which regains contact with the surface of the organ. All supporting cells, including pillar and Deiters cells take part in wrapping their respective inner or outer hair cells. The supporting cells not only cover the injured sensory cells, but also invert their villi toward the maimed cuticular plates and release an extracellular matrix around them. We suggest that the supporting cells play a protective and trophic role in the recovery of injured hair cells.

Compound synapses within the GABAergic innervation of the auditory inner hair cells in the adolescent mouse.

Ultrastructural investigation of the gamma-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.

Post-traumatic survival and recovery of the auditory sensory cells in culture.

Following mechanical injury in organotypic cultures, auditory hair cells show the ability to survive and to initially reform their apical specializations, cuticular plates and stereocilia, but none show incorporation of tritiated thymidine, the mitotic marker. Disruption of the reticular lamina and local injury to hair cell cuticular plates induces proliferation of supporting cells. The regenerating apices of inner hair cells are wrapped by the cells of the inner spiral sulcus and the inner phalangeal cells, while those of outer hair cells are wrapped by the phalangeal processes of Deiters' cells and outer spiral sulcus cells. Some of these hair cells subsequently resurface with newly formed tops. Hair cells that lose contact with the surface of the organ remain buried--but alive--deep within the epithelium. Our study provides evidence that the mammalian organ of Corti responds to injury not by the formation of new sensory cells but by the recovery of the pre-existing postmitotic hair cells.

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

Auditory 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 remaining microtubules 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.

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

Isolated 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 organ in situ.