D. Kent Morest

βIV Spectrins Are Essential for Membrane Stability and the Molecular Organization of Nodes of Ranvier

High densities of sodium channels at nodes of Ranvier permit action potential conduction and depend on βIV spectrins, a family of scaffolding proteins linked to the cortical actin cytoskeleton. To investigate the molecular organization of nodes, we analyzed qv3J“quivering” mice, whose βIV spectrins have a truncated proline-rich “specific” domain (SD) and lack the pleckstrin homology (PH) domain. Central nodes of qv3J mice, which lack βIV spectrins, are significantly broader and have prominent vesicle-filled nodal membrane protrusions, whereas axon shape and neurofilament density are dramatically altered. PNS qv3J nodes, some with detectable βIV spectrins, are less affected. In contrast, a larger truncation of βIV spectrins in qv4J mice, deleting the SD, PH, and ankyrinG binding domains, causes βIV spectrins to be undetectable and causes dramatic changes, even in peripheral nodes. These results show that quivering mutations disrupt βIV spectrin retention and stability at nodes and that distinct protein domains regulate nodal structural integrity and molecular organization.

Noise-induced degeneration in the brain and representation of inner and outer hair cells

Noise that destroys receptor cells in the chinchilla cochlea also results in degeneration of axonal endings in the brain from the cochlear nerve fibers and the auditory pathways ascending to the superior olivary complex and inferior colliculus. The patterns of degeneration provide experimental evidence for differential representation of inner and outer hair cells and their functions.

Where Is the Spike Generator of the Cochlear Nerve? Voltage-Gated Sodium Channels in the Mouse Cochlea

The origin of the action potential in the cochlea has been a long-standing puzzle. Because voltage-dependent Na+ (Nav) channels are essential for action potential generation, we investigated the detailed distribution of Nav1.6 and Nav1.2 in the cochlear ganglion, cochlear nerve, and organ of Corti, including the type I and type II ganglion cells. In most type I ganglion cells, Nav1.6 was present at the first nodes flanking the myelinated bipolar cell body and at subsequent nodes of Ranvier. In the other ganglion cells, including type II, Nav1.6 clustered in the initial segments of both of the axons that flank the unmyelinated bipolar ganglion cell bodies. In the organ of Corti, Nav1.6 was localized in the short segments of the afferent axons and their sensory endings beneath each inner hair cell. Surprisingly, the outer spiral fibers and their sensory endings were well labeled beneath the outer hair cells over their entire trajectory. In contrast, Nav1.2 in the organ of Corti was localized to the unmyelinated efferent axons and their endings on the inner and outer hair cells. We present a computational model illustrating the potential role of the Nav channel distribution described here. In the deaf mutant quivering mouse, the localization of Nav1.6 was disrupted in the sensory epithelium and ganglion. Together, these results suggest that distinct Nav channels generate and regenerate action potentials at multiple sites along the cochlear ganglion cells and nerve fibers, including the afferent endings, ganglionic initial segments, and nodes of Ranvier.

Precursors of neurons, neuroglia, and ependymal cells in the CNS: What are they? Where are they from? How do they get where they are going?

Neurons, neuroglia (astrocytes and oligodendrocytes), and ependymal cells are three distinct categories of neural cells in the central nervous system. In the mature brain and spinal cord, the classical histological criteria define these cells by their microscopic structure very well. During development, the precursors for all of these cells reside within the epithelium of the neural plate and its successor, the neural tube. These precursor cells are the undifferentiated, primitive neuroepithelium of the classical literature. As the cerebral vesicles enlarge and their walls thicken, the primitive neuroepithelial cells elongate, maintaining a radial orientation until they migrate. Although many, but not all, of these cells span the extent of the ventricular wall, they are the precursors of neurons, neuroglia, and ependymal cells. Thus, it is useful to retain their classical designation as primitive neuroepithelial cells and to treat them as neural precursor cells. Neural precursor cells are neither neuroglia nor neurons. It is not appropriate to call them radial glial cells anymore than it is to call them radial neuronal cells. The term “radial glia” has long been used to describe the mature, elongated astrocytes, represented by Bergmann cells in the cerebellum and Müller cells in the retina. Inevitably, during development, transitional forms between neural precursor cells and the neurons, neuroglia, and ependymal cells will occur. Such transitional cells are known as neuroblasts, glioblasts, or ependymoblasts, even though they may be postmitotic. Alternative terms are “immature neurons,” “immature neuroglia,” and “immature ependymal cells.” The migration of many neural precursor cells is accomplished by translocation rather than free cellular locomotion. There is both direct and indirect evidence to document the translocation of the nuclear/perikaryal/somal complex through the leading process of primitive neuroepithelial cells. This is conspicuous in the neocortex, where the discrete radial arrangement of pyramidal cells may result from translocation of neuroblasts, while their leading processes still contact the pial surface. Migration by translocation occurs throughout the CNS. GLIA 43:6–18, 2003.

Degeneration of axons in the brainstem of the chinchilla after auditory overstimulation

The patterns of axonal degeneration following acoustic overstimulation of the cochlea were traced in the brainstem of adult chinchillas. The Nauta-Rasmussen method for axonal degeneration was used following survivals of 1-32 days after a 105 min exposure to an octave-band noise with a center frequency of 4 kHz and a sound pressure level of 108 dB. Hair-cell and myelinated nerve-fiber loss were assessed in the cochlea. The cochleotopic pattern of terminal degeneration in the ventral cochlear nucleus correlated with the sites of myelinated fiber and inner-hair-cell loss: this correlation was less rigorous with outer-hair-cell loss, especially in the dorsal cochlear nucleus. These results are consistent with a dystrophic process with a slow time course depending on hair-cell loss and/or direct cochlear nerve-fiber damage. However, in a number of cases with no damage in the apical cochlea, fine fiber degeneration occurred with a faster course in low-frequency regions in the dorsal cochlear nucleus and, transynaptically, in a non-cochleotopic pattern in the superior olive and inferior colliculus. These findings suggest that neuronal hyperactivity plays a role in the central degeneration following acoustic overstimulation, possibly by an excitotoxic process.

Cisplatin-Induced Ototoxicity: Effect of Intratympanic Dexamethasone Injections

Hypothesis: Intratympanic (IT) application of dexamethasone will reduce ototoxicity associated with systemic cisplatin therapy. Background: Cisplatin is a common chemotherapeutic drug often dose-limited by ototoxicity attributed to the formation of reactive oxygen and nitrogen species damaging critical inner ear structures. Steroids have been shown to reduce formation of reactive oxygen species and thus may reduce ototoxicity. In the present pilot study, we test this hypothesis by IT administration of dexamethasone in a novel murine model of cisplatin ototoxicity. Methods: Click- and pure-tone-evoked auditory brainstem responses (ABRs) in young CBA/J mice were measured. The first phase consisted of a dosing study to identify the optimal cisplatin dose for ototoxicity. In the next phase, ABR thresholds were measured in cisplatin-treated mice after 5 days of IT injection of 24 mg/ml of dexamethasone in 1 ear and normal saline in the opposite ear to serve as controls. Results: Intraperitoneal injection of 14 mg/kg of cisplatin induces significant hearing loss (click-evoked ABR threshold elevation = 12 ± 7 dB, &mgr; ± standard error of the mean) with acceptable mortality (20%). The ears that received IT dexamethasone in cisplatin-treated mice had minimal ABR threshold shifts with the click, 8 and 16 kHz of stimuli. There was no significant difference between IT dexamethasone and IT saline ears at 32 kHz. Conclusion: IT dexamethasone protected the mouse ear against cisplatin-induced ototoxicity in a frequency-dependent manner. The present results suggest that IT dexamethasone may be a safe, simple, and effective intervention that minimizes cisplatin ototoxicity without interfering with the chemotherapeutic actions of cisplatin.

The form and distribution of GABAergic synapses on the principal cell types of the ventral cochlear nucleus of the cat

The distribution of GABAergic endings was examined histochemically in the ventral cochlear nucleus (VCN) of the cat using an antibody to glutamate decarboxylase (GAD), the synthetic enzyme for GABA. Immunoreactive (GAD+) endings appeared in all subdivisions of the cat VCN. Each of the principal cell types had a characteristic labeling pattern, based on the size, concentration, and distribution of GAD+ endings on its soma. Spherical bushy cell somata were typically contacted by many small (less than 1.5 microns in diameter) and medium-sized (1.5-2 microns in diameter) endings, many of which aggregated into tight clusters. Globular bushy cells had a similar pattern, but the clusters of GAD+ endings were less tightly packed. Reactive endings on stellate cells were more evenly distributed. GAD+ endings on octopus cells were larger (up to 2.5 microns in diameter) than those on the bushy cells and tended to aggregate into small clusters or rows on the somata and dendrites. Reactive endings contained small pleomorphic vesicles and formed symmetrical synaptic contacts on each of the cell types examined. The patterns formed by GAD+ endings on each type of neuron resemble those of certain types of non-cochlear axons previously described with the Golgi methods as projecting from the dorsal cochlear nucleus and the trapezoid body.

Fine structure of cochlear innervation in the cat

Examination of adult and juvenile cat cochleas by electron microscopy and semi-serial sections permitted identification of the cytological features characteristic of the afferent and efferent nerve fiber populations identified in Golgi impregnations of the cochlea. This study demonstrated the distribution of synaptic contacts made by these fiber populations. As in the Golgi findings, radial and outer spiral afferent fibers were identified in well separated zones of the inner spiral bundle. The trunks of the outer spiral fibers, containing many microtubules and few neurofilaments, at first coursed spirally below the inner hair cells on the proximal face of the inner pillar, turned abruptly between adjacent pillar cells and entered the tunnel without branching. Radial afferents, containing many neurofilaments and a few microtubules, coursed through the inner spiral bundle, maintaining a radial or oblique orientation and proceeded directly toward the inner hair cells. Efferent fibers in the region of the inner spiral bundle were distinguishable by size, by orientation, and, to a lesser extent, by cytology. Small (1 micron) efferent fibers, containing few neurofilaments, an occasional microtubule, and mitochondria, occurred in the inner and tunnel spiral bundles and formed large varicosities, which contacted radial afferents. A separate population of much thicker efferents, containing many neurofilaments, mitochondria and dense-cored vesicles, but no microtubules, did not enter the inner spiral bundle but coursed directly to the level of the tunnel spiral bundle on the proximal face of the inner pillar cells. These fibers crossed the tunnel at the level of the tunnel spiral bundle and, upon reaching the outer hair cells, formed large synaptic contacts on outer hair cells and on outer spiral fibers as well. Some of these efferent fibers also synapse on afferent fibers while crossing the tunnel. The findings agree with previous observations with the Golgi method showing that entirely separate populations of spiral ganglion cells innervate the inner and outer hair cells. Likewise, there are efferent fibers innervating only inner or outer hair cells, but the probability of efferent fibers to both inner and outer hair cells cannot be ruled out.

Cell death during development of the cochlear and vestibular ganglia of the chick.

This report documents and quantitates the naturally occurring death of neurons in the cochlear and vestibular ganglia during the normal development of the chick embryo. The data are compared with the amount of cell loss in the cochlear and vestibular nuclei to evaluate the possibility that the death of the neurons in the same sensory pathway may be related to the formation of their connections. Neurons of the cochlear and vestibular ganglia of the chick were counted and their cell bodies measured with light microscopic methods at the beginning of their period of synapse formation, embryonic day 8 (E8), at the end of the period of synapse formation (E14), and again at a more mature stage, post‐hatching day 14 (P14).

A degenerative disorder of the central auditory system of the gerbil

A previously unidentified disorder which affects primarily the cochlear nucleus was observed in two species of gerbils, Meriones unguiculatus and M. libycus. Unusual lesions were observed in the cochlear nucleus bilaterally in all animals examined. In light and electron microscopic specimens these lesions were characterized by the presence of microcysts and vacuolar neuronal degeneration. The microcysts resembled large holes, containing trabeculae, organelles and cellular remnants. Also observed were light and dark degeneration of neuronal perikarya and degenerated axons, dendrites, and synapses, accompanied by phagocytosis. Astrocytosis was not conspicuous. In the one cochlea examined, no microcysts were observed. In young animals the microcysts were prevalent in the cochlear nerve root region and the posteroventral cochlear nucleus. In older animals the microcysts increased in number and area. In the oldest animals, the microcysts had spread to other central auditory structures, including the superior olivary complex, the nuclei of the lateral lemniscus, and the inferior colliculus. Other regions of the brain were largely free of microcysts. The etiology and behavioral manifestations of this disorder are unknown, although it is clearly neurodegenerative and perhaps genetically determined.