Tan Pongstaporn

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.

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.

Ultrastructural Study of the Granule Cell Domain of the Cochlear Nucleus in Rats: Mossy Fiber Endings and Their Targets

The principal projection neurons of the cochlear nucleus receive the bulk of their input from the auditory nerve. These projection neurons reside in the core of the nucleus and are surrounded by an external shell, which is called the granule cell domain. Interneurons of the cochlear granule cell domain are the target for nonprimary auditory inputs, including projections from the superior olivary complex, inferior colliculus, and auditory cortex. The granule cell domain also receives projections from the cuneate and trigeminal nuclei, which are first‐order nuclei of the somatosensory system. The cellular targets of the nonprimary projections are mostly unknown due to a lack of information regarding postsynaptic profiles in the granule cell areas. In the present paper, we examined the synaptic relationships between a heterogeneous class of large synaptic terminals called mossy fibers and their targets within subdivisions of the granule cell domain known as the lamina and superficial layer. By using light and electron microscopic methods in these subdivisions, we provide evidence for three different neuron classes that receive input from the mossy fibers: granule cells, unipolar brush cells, and a previously undescribed class called chestnut cells. The distinct synaptic relations between mossy fibers and members of each neuron class further imply fundamentally separate roles for processing acoustic signals.

Activity-related features of synapse morphology: a study of endbulbs of held.

The myelinated fibers of the auditory nerve can be divided into two separate populations on the basis of sensitivity to sound, average levels of spike activity, and central branching patterns. The synaptic endings of these populations were investigated for the presence of structural specializations that might correlate with levels of neural activity. We applied intracellular recording and staining methods in cats to analyze directly the relationship between spike activity and the structure of synapses using endbulbs of Held, the large synaptic endings in the anteroventral cochlear nucleus. Endbulbs from fibers having low or high levels of activity were examined and compared using light and electron microscopic methods. All endbulbs exhibited relatively large but incomplete coverage by one‐to‐several lamellae of glial processes. Endbulbs of high activity fibers were large and contained larger mitochondria than endbulbs of low activity fibers. Furthermore, the synapses of high activity endbulbs were on average smaller but more numerous, possessed greater numbers of associated synaptic vesicles, and exhibited greater curvature of their postsynaptic densities. These structural features are hypothesized to reflect specializations that optimize synaptic transmission.

Projections from the spinal trigeminal nucleus to the cochlear nucleus in the rat

The integration of information across sensory modalities enables sound to be processed in the context of position, movement, and object identity. Inputs to the granule cell domain (GCD) of the cochlear nucleus have been shown to arise from somatosensory brain stem structures, but the nature of the projection from the spinal trigeminal nucleus is unknown. In the present study, we labeled spinal trigeminal neurons projecting to the cochlear nucleus using the retrograde tracer, Fast Blue, and mapped their distribution. In a second set of experiments, we injected the anterograde tracer biotinylated dextran amine into the spinal trigeminal nucleus and studied the resulting anterograde projections with light and electron microscopy. Spinal trigeminal neurons were distributed primarily in pars caudalis and interpolaris and provided inputs to the cochlear nucleus. Their axons gave rise to small (1–3 μm in diameter) en passant swellings and terminal boutons in the GCD and deep layers of the dorsal cochlear nucleus. Less frequently, larger (3–15 μm in diameter) lobulated endings known as mossy fibers were distributed within the GCD. Ventrally placed injections had an additional projection into the anteroventral cochlear nucleus, whereas dorsally placed injections had an additional projection into the posteroventral cochlear nucleus. All endings were filled with round synaptic vesicles and formed asymmetric specializations with postsynaptic targets, implying that they are excitatory in nature. The postsynaptic targets of these terminals included dendrites of granule cells. These projections provide a structural substrate for somatosensory information to influence auditory processing at the earliest level of the central auditory pathways. J. Comp. Neurol. 484:191–205, 2005.

The effects of congenital deafness on auditory nerve synapses and globular bushy cells in cats

It is well known that auditory deprivation affects the structure and function of the central nervous system. Congenital deafness represents one form of deprivation, and in the adult white cat, it has been shown to have a clear effect upon the synaptic interface between endbulbs of Held and spherical bushy cells. It is not known, however, whether all primary synapses are affected and/or whether they are affected in the same way and to the same extent. Thus, we studied a second neuronal circuit in the deaf white cat involving modified (small) endbulbs and globular bushy cells. Compared to normal hearing cats, modified endbulbs of congenitally deaf cats were 52.2% smaller but unchanged in structural complexity. There was also a striking loss of extracellular space between ending and cell body. The somata of postsynaptic globular bushy cells were 13.4% smaller and had enlarged postsynaptic densities. These data reveal that axosomatic synapses demonstrate abnormal structure as a consequence of deafness and that the extent of the abnormalities can vary with respect to the circuits involved. The implication of these observations is that synaptic anomalies would introduce differential delays within separate circuits, thereby desynchronizing neural activity from sound stimuli. This loss of synchronization could in turn disrupt temporal processing and compromise a host of related functions, including language comprehension.

Projections of the second cervical dorsal root ganglion to the cochlear nucleus in rats

Physiological, anatomical, and clinical data have demonstrated interactions between somatosensory and auditory brainstem structures. Spinal nerve projections influence auditory responses, although the nature of the pathway(s) is not known. To address this issue, we injected biotinylated dextran amine into the cochlear nucleus or dorsal root ganglion (DRG) at the second cervical segment (C2). Cochlear nucleus injections retrogradely labeled small ganglion cells in C2 DRG. C2 DRG injections produced anterograde labeling in the external cuneate nucleus, cuneate nucleus, nucleus X, central cervical nucleus, dorsal horn of upper cervical spinal segments, and cochlear nucleus. The terminal field in the cochlear nucleus was concentrated in the subpeduncular corner and lamina of the granule cell domain, where endings of various size and shapes appeared. Examination under an electron microscope revealed that the C2 DRG terminals contained numerous round synaptic vesicles and formed asymmetric synapses, implying depolarizing influences on the target cell. Labeled endings synapsed with the stalk of the primary dendrite of unipolar brush cells, distal dendrites of presumptive granule cells, and endings containing pleomorphic synaptic vesicles. These primary somatosensory projections contribute to circuits that are hypothesized to mediate integrative functions of hearing. J. Comp. Neurol. 496:335–348, 2006.

Postnatal development of a large auditory nerve terminal: The endbulb of Held in cats

The endbulbs of Held are formed by the ascending branches of myelinated auditory nerve fibers and represent one of the largest synaptic endings in the brain. Most of the developmental changes in structure occur during the first 30 postnatal days of age. The neonatal endbulb begins as a flattened expansion with many filopodia, resembling a growth cone and characterized by numerous puncta adherentia and synapses associated with small postsynaptic densities; the most impressive feature of the ending at this age is its highly irregular plasma membrane that interdigitates with that of the postsynaptic spherical bushy cell. During these first 30 days, the number of puncta adherentia diminishes, postsynaptic densities nearly double in size, intermembraneous cisternae emerge, and plasma membranes flatten. These features endow the endbulb with an adult-like appearance. On the other hand, synaptic vesicle density increases progressively from approximately 50/microm2 at birth to 100/microm2 at adulthood. Mitochondria size remains constant over this developmental period but mitochondrial volume fraction increases until 60 days postnatal. Although many features of endbulb morphology stabilize by 30 days, other features suggest that endbulb development continues into the third month of age. Many of these observations correlate with the maturation of physiological response properties and suggest issues for further study.

Postnatal development of the endbulb of Held in congenitally deaf cats

The endbulbs of Held are formed by the ascending branches of myelinated auditory nerve fibers and represent one of the largest synaptic endings in the brain. Normally, these endings are highly branched and each can form up to 1000 dome-shaped synapses. The deaf white cat is a model of congenital deafness involving a type of cochleosaccular degeneration that mimics the Scheibe deformity in humans. Endbulbs of mature deaf white cats exhibit reduced branching, hypertrophy of postsynaptic densities (PSDs), and changes in synaptic vesicle density. Because cats are essentially deaf at birth, we sought to determine if the progression of brain abnormalities was linked in time to the failure of normal hearing development. The rationale was that the lack of sound-evoked activity would trigger pathologic change in deaf kittens. The cochleae of deaf cats did not exhibit abnormal morphology at birth. After the first postnatal week, however, the presence of a collapsed scala media signaled the difference between deaf and hearing cats. By working backwards in age, endbulbs of deaf cats expressed flattened and elongated PSDs and increased synaptic vesicle density as compared to normal endbulbs. These differences are present at birth in some white kittens, presaging deafness despite their normal cochlear histology. We speculate that hearing pathology is signaled by a perinatal loss of spontaneous bursting activity in auditory nerve fibers or perhaps by some factor released by hair cell synapses before obliteration of the organ of Corti.

The effects of congenital deafness on auditory nerve synapses: Type I and Type II multipolar cells in the anteroventral cochlear nucleus of cats.

Sensory deprivation has been shown to exert detrimental effects on the structure and function of central sensory systems. Congenital deafness represents an extreme form of auditory deprivation, and in the adult white cat, synapses between auditory nerve endings and resident cells of the anteroventral cochlear nucleus exhibited abnormal structure. Endbulbs of Held were reduced in branching and displayed striking hypertrophy of their postsynaptic densities. So-called modified endbulbs showed no change in branching complexity but did exhibit hypertrophy of their postsynaptic densities. These differential pre- and postsynaptic effects prompted the question of how deafness might affect other primary endings and synapses. Thus, we studied type I and type II multipolar cells that receive bouton endings from auditory nerve fibers. Type I multipolar cells project to the contralateral inferior colliculus and have relatively few axosomatic endings; type II multipolar cells project to the contralateral cochlear nucleus and have many axosomatic endings. Compared with normal-hearing cats, bouton endings of congenitally deaf cats were smaller but there was no difference in synaptic vesicle density or size of postsynaptic densities. These data reveal that different classes of primary endings and second-order neurons exhibit different degrees of synaptic anomalies to deafness.