David K. Ryugo

Widespread expression of Huntington's disease gene (IT15) protein product

Huntingtons Disease (HD) is caused by expansion of a CAG repeat within a putative open reading frame of a recently identified gene, IT15. We have examined the expression of the genes protein product using antibodies developed against the N-terminus and an internal epitope. Both antisera recognize a 350 kDa protein, the predicted size, indicating that the CAG repeat is translated into polyglutamine. The HD protein product is widely expressed, most highly in neurons in the brain. There is no enrichment in the striatum, the site of greatest pathology in HD. Within neurons, the protein is diminished in nuclei and mitochondria and is present in the soluble cytoplasmic compartment, as well as loosely associated with membranes or cytoskeleton, in cell bodies, dendrites, and axons. It is concentrated in nerve terminals, including terminals within the caudate and putamen. Thus, the normal HD gene product may be involved in common intracellular functions, and possibly in regulation of nerve terminal function. The product of the expanded allele is expressed, consistent with a gain of function mechanism for HD at the protein level.

Mossy fiber projections from the cuneate nucleus to the cochlear nucleus in the rat

A reciprocal connection is known to exist between the cuneate nucleus, which is a first‐order somatosensory nucleus, and the cochlear nucleus, which is a first‐order auditory nucleus. We continued this line of study by investigating the fiber endings of this projection in the cochlear nucleus of rats using the neuronal tracer Phaseolus vulgaris leucoagglutinin in combination with ultrastructural and immunocytochemical analyses. In the cochlear nucleus, mossy fiber terminals had been described and named for their morphologic similarity to those in the cerebellum, but their origins had not been discovered. In the present study, we determined that the axonal projections from the cuneate region gave rise to mossy fiber terminals in the granule cell regions of the ipsilateral cochlear nucleus. The cuneate mossy fibers appear to be excitatory in nature, because they are filled with round synaptic vesicles, they make asymmetric synapses with postsynaptic targets, and they are labeled with an antibody to glutamate. The postsynaptic targets of the mossy fibers include dendrites of granule cells. This projection onto the granule cell interneuron circuit of the cochlear nucleus indicates that somatosensory cues are intimately involved with information processing at this early stage of the auditory system.

ULTRASTRUCTURAL ANALYSIS OF PRIMARY ENDINGS IN DEAF WHITE CATS : MORPHOLOGIC ALTERATIONS IN ENDBULBS OF HELD

Changes in structure and function of the auditory system can be produced by experimentally manipulating the sensory environment, and especially dramatic effects result from deprivation procedures. An alternative deprivation strategy utilizes naturally occurring lesions. The congenitally deaf white cat represents an animal model of sensory deprivation because it mimics a form of human deafness called the Scheibe deformity and permits studies of how central neurons react to early‐onset cochlear degeneration. We studied the synaptic characteristics of the endbulb of Held, a prominent auditory nerve terminal in the cochlear nucleus. Endbulbs arise from the ascending branch of the auditory nerve fiber and contact the cell body of spherical bushy cells. After 6 months, endbulbs of deaf white cats exhibit alterations in structure that are clearly distinguishable from those of normal hearing cats, including a diminution in terminal branching, a reduction in synaptic vesicle density, structural abnormalities in mitochondria, thickening of the pre‐ and postsynaptic densities, and enlargement of synapse size. The hypertrophied membrane densities are suggestive of a compensatory response to diminished transmitter release. These data reveal that early‐onset, long‐term deafness produces unambiguous alterations in synaptic structure and may be relevant to rehabilitation strategies that promote aural/oral communication. J. Comp. Neurol. 385:230–244, 1997.

Differential afferent projections to the inferior colliculus from the cochlear nucleus in the albino mouse

The axonal projections from the cochlear nuclear complex to the inferior colliculus (IC) were examined using the retrograde transport of horseradish peroxidase. Thin sheets of neurons in the dorsal and ventral cochlear nuclei were found to project axons in a topographic fashion to restricted laminae of the central nucleus of the IC; the dorsal cochlear nucleus was also found to project axons to the external cortex. No projections were detected from the cochlear nuclear complex to the dorsal cortex of the IC.

Projections from the ventral cochlear nucleus to the dorsal cochlear nucleus in rats.

Local circuit interactions between the dorsal and ventral divisions of the cochlear nucleus are known to influence the evoked responses of the resident neurons to sound. In the present study, we examined the projections of neurons in the ventral cochlear nucleus to the dorsal cochlear nucleus by using retrograde transport of biotinylated dextran amine injected into restricted but different regions of the dorsal cochlear nucleus. In all cases, we found retrogradely labeled granule, unipolar brush, and chestnut cells in the granule cell domain, and retrogradely labeled multipolar cells in the magnocellular core of the ventral cochlear nucleus. A small number of the labeled multipolar cells were found along the margins of the ventral cochlear nucleus, usually near the boundaries of the granule cell domain. Spherical bushy, globular bushy, and octopus cells were not labeled. Retrogradely‐labeled auditory nerve fibers and the majority of labeled multipolar neurons formed a narrow sheet extending across the medial‐to‐lateral extent of the ventral cochlear nucleus whose dorsoventral position was topographically related to the injection site. Labeled multipolar cells within the core of the ventral cochlear nucleus could be divided into at least two distinct groups. Planar neurons were most numerous, their somata found within the associated band of labeled fibers, and their dendrites oriented within this band. This arrangement mimics the organization of isofrequency contours and implies that planar neurons respond best to a narrow range of frequencies. In contrast, radiate neurons were infrequent, found scattered throughout the ventral cochlear nucleus, and had long dendrites oriented perpendicular to the isofrequency contours. This dendritic orientation suggests that radiate neurons are sensitive to a broad range of frequencies. These structural differences between planar and radiate neurons suggest that they subserve separate functions in acoustic processing. J. Comp. Neurol. 385:245–264, 1997.

Projections From Auditory Cortex to the Cochlear Nucleus in Rats: Synapses on Granule Cell Dendrites

Previous work has demonstrated that layer V pyramidal cells of primary auditory cortex project directly to the cochlear nucleus. The postsynaptic targets of these centrifugal projections, however, are not known. For the present study, biotinylated dextran amine, an anterograde tracer, was injected into the auditory cortex of rats, and labeled terminals were examined with light and electron microscopy. Labeled corticobulbar axons and terminals in the cochlear nucleus are found almost exclusively in the granule cell domain, and the terminals appear as boutons (1–2 μm in diameter) or as small mossy fiber endings (2–5 μm in diameter). These cortical endings contain round synaptic vesicles and form asymmetric synapses on hairy dendritic profiles, from which thin (0.1 μm in diameter), nonsynaptic “hairs” protrude deep into the labeled endings. These postsynaptic dendrites, which are typical of granule cells, surround and receive synapses from large, unlabeled mossy fiber endings containing round synaptic vesicles and are also postsynaptic to unlabeled axon terminals containing pleomorphic synaptic vesicles. No labeled fibers were observed synapsing on profiles that did not fit the characteristics of granule cell dendrites. We describe a circuit in the auditory system by which ascending information in the cochlear nucleus can be modified directly by descending cortical influences.

Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats: differentiation and structural integration into the segmental motor circuitry

Cell replacement strategies for degenerative and traumatic diseases of the nervous system depend on the functional integration of grafted cells into host neural circuitry, a condition necessary for the propagation of physiological signals and, perhaps, targeting of trophic support to injured neurons. We have recently shown that human neural stem cell (NSC) grafts ameliorate motor neuron disease in SOD1 transgenic rodents. Here we study structural aspects of integration of neuronally differentiated human NSCs in the motor circuitry of SOD1 G93A rats. Human NSCs were grafted into the lumbar protuberance of 8‐week‐old SOD1 G93A rats; the results were compared to those on control Sprague‐Dawley rats. Using pre‐embedding immuno‐electron microscopy, we found human synaptophysin (+) terminals contacting the perikarya and proximal dendrites of host α motor neurons. Synaptophysin (+) terminals had well‐formed synaptic vesicles and were associated with membrane specializations primarily in the form of symmetrical synapses. To analyze the anatomy of motor circuits engaging differentiated NSCs, we injected the retrograde transneuronal tracer Bartha‐pseudorabies virus (PRV) or the retrograde marker cholera toxin B (CTB) into the gastrocnemius muscle/sciatic nerve of SOD1 rats before disease onset and also into control rats. With this tracing, NSC‐derived neurons were labeled with PRV but not CTB, a pattern suggesting that PRV entered NSC‐derived neurons via transneuronal transfer from host motor neurons but not via direct transport from the host musculature. Our results indicate an advanced degree of structural integration, via functional synapses, of differentiated human NSCs into the segmental motor circuitry of SOD1‐G93A rats. J. Comp. Neurol. 514:297–309, 2009.

Restoration of auditory nerve synapses in cats by cochlear implants.

Congenital deafness results in abnormal synaptic structure in endings of the auditory nerve. If these abnormalities persist after restoration of auditory nerve activity by a cochlear implant, the processing of time-varying signals such as speech would likely be impaired. We stimulated congenitally deaf cats for 3 months with a six-channel cochlear implant. The device used human speech-processing programs, and cats responded to environmental sounds. Auditory nerve fibers exhibited a recovery of normal synaptic structure in these cats. This rescue of synapses is attributed to a return of spike activity in the auditory nerve and may help explain cochlear implant benefits in childhood deafness.

Single Unit Recordings in the Auditory Nerve of Congenitally Deaf White Cats: Morphological Correlates in the Cochlea and Cochlear Nucleus

It is well known that experimentally induced cochlear damage produces structural, physiological, and biochemical alterations in neurons of the cochlear nucleus. In contrast, much less is known with respect to the naturally occurring cochlear pathology presented by congenital deafness. The present study attempts to relate organ of Corti structure and auditory nerve activity to the morphology of primary synaptic endings in the cochlear nucleus of congenitally deaf white cats. Our observations reveal that the amount of sound‐evoked spike activity in auditory nerve fibers influences terminal morphology and synaptic structure in the anteroventral cochlear nucleus. Some white cats had no hearing. They exhibited severely reduced spontaneous activity and no sound‐evoked activity in auditory nerve fibers. They had no recognizable organ of Corti, presented >90% loss of spiral ganglion cells, and displayed marked structural abnormalities of endbulbs of Held and their synapses. Other white cats had partial hearing and possessed auditory nerve fibers with a wide range of spontaneous activity but elevated sound‐evoked thresholds (60–70 dB SPL). They also exhibited obvious abnormalities in the tectorial membrane, supporting cells, and Reissners membrane throughout the cochlear duct and had complete inner and outer hair cell loss in the base. The spatial distribution of spiral ganglion cell loss correlated with the pattern of hair cell loss. Primary neurons of hearing‐impaired cats displayed structural abnormalities of their endbulbs and synapses in the cochlear nucleus which were intermediate in form compared to normal and totally deaf cats. Changes in endbulb structure appear to correspond to relative levels of deafness. These data suggest that endbulb structure is significantly influenced by sound‐evoked auditory nerve activity. J. Comp. Neurol. 397:532–548, 1998.

The Auditory Nerve: Peripheral Innervation, Cell Body Morphology, and Central Projections

In mammals, all known auditory information enters the brain by way of the cochlear division of the vestibulocochlear nerve, hereafter referred to as the auditory nerve. Primary neurons, whose cell bodies reside in the spiral ganglion of the cochlea, send peripheral processes out to the organ of Corti to contact the acoustic receptor cells; the central processes or axons bundle together to form the auditory nerve. The terminus of the auditory nerve is the cochlear nucleus. In this way, primary neurons convey the output of the receptors to neurons of the cochlear nucleus. There arc two types of receptors, inner hair cells and outer hair cells (Retzius 1884; Ramon y Cajal 1909), two populations of primary neurons (Munzer 1931; Spoendlin 1973), and many neuron classes in the cochlear nucleus (Lorente de No 1933; Osen 1969; Brawer, Morest, and Kane 1974). In turn, the cells of the cochlear nucleus give rise to all central auditory pathways. In a general way, the role of the cochlear nucleus is to receive incoming auditory nerve discharges, to preserve or transform the signals, and to distribute outgoing activity to higher brain centers. In order to understand the earliest stages of stimulus coding in the auditory system, we need to know (1) the nature of the signals conveyed by auditory nerve fibers, (2) their source in the periphery, and (3) their destination in the brain. This report shall review the progress that has been made along these lines of investigation.