Kenna D. Peusner - US grants

The long-term goal of the projects is to identify the rules that govern the cellular interactions between afferent axons and their target cells under the changing conditions of neurogenesis, maturation, aging, and sensory deprivation resulting from deafferentation. The interactions between the PNS and the CNS that affect synapse formation between sensory neurons will be focused on, as studied in the chick vestibular system. One class of vestibular ganglion cells and their target cells, in the tangential vestibular nucleus and in the vestibular epithelium, will be studied. These ganglion cells produce colossal vestibular fibers which form spoon endings on the principal cells of the tangential nucleus and calyces with the type I and type II hair cells in the vestibular epithelium. In the preceding project, the normal synaptic interactions of the spoon endings and the calycine endings were quantified in embryos, hatchlings, and young adult chickens using ultrastructural techniques. These findings provide a basis to evaluate future projects on what happens to the same target neurons deprived of vestibular inputs in embryos and hatchlings. Moreover, the previous findings have generated a need to expand the scope of the proposal to include ultrastructural studies of the spoon endings in aging animals, to employ the freeze-fracture technique for the study of gap junction at the spoon endings, to study early synaptogenesis in the vestibular epithelium, to determine the synaptic arrangements of vestibular type II hair cells, to replace the otocyst with an optic cup in chick embryos, and to use horseradish peroxidase and tritiated amino acids for tracing the afferent and efferent connections of the tangential nucleus. The work has both clinical and basic science relevance. When animals undergo deafferentation, the response of the target neurons is variable, depending in part on age. Knowledge of the rules that govern the formation of synaptic organization and its reorganization should be useful in future studies concerned with the prevention or treatment of various conditions that cause failure in normal processing of vestibular information, or sensory information in general. Structural analysis of the development of synaptic connections in the vestibular pathways should lead to a greater appreciation of the development of the neural coding mechanism and the role of afferent fibers on the development of neural networks.

The major goal of this work is to better understand the fundamental mechanisms involved in the development of signal processing by vestibular nuclei neurons, and how this excitability changes after peripheral vestibular injury. The chick tangential nucleus (lateral vestibular nucleus) will be investigated using brain slices and whole animals at embryonic and newborn ages. This nucleus was chosen due to its rather simple and well-known anatomy, including the fact that the two main neuron populations of this nucleus function in two of the vestibular reflexes. Already, there is a wealth of knowledge existing on neuron morphogenesis and synaptogenesis by the primary vestibular fibers in this system. Further, there is a high degree of plasticity in young animals and it is relatively easy to perform vestibular deafferentations in the chicken. The techniques applied will include whole-cell patch-clamp recordings in current- and voltage-clamp modes, outside-out patch recordings, pharmacology, and light and immunogold electron- microscopic immunocytochemistry. The specific aims include to characterize and quantify the voltage-gated outward potassium currents, vestibular synaptic currents, excitability, and glutamate neurotransmission in tangential nucleus neurons during development and after vestibular-nerve transection in the newborn. By applying multiple approaches to analyze the structure-function relationships of identified vestibular nuclei neurons, we will clarify the roles that different neuron classes play in vestibular signal processing. In addition, as we learn more about the basic mechanisms involved in excitability and synaptic transmission by vestibular nuclei neurons, we can apply this to improve drug treatments for central vestibular disorders resulting from disease, injury or aging.

DESCRIPTION (provided by applicant): In certain sensory systems, natural sensory input has been to shown to play a crucial role in the formation of connections and functional activity of CNS neurons. However, this has not been demonstrated for the otolithic pathways since there is no simple way to deprive the system of gravitational stimulation on Earth. Thus, the effects of microgravity on central vestibular development can be studied uniquely in space flight. Here we will perform ground-based studies which will be the controls for our future space flight experiments. For this work, a simple and established avian model will be studied, the chick tangential vestibular nucleus. The principal cells of this nucleus are second-order vestibular neurons participating in the three-neuron vestibule-ocular and vestibulocollic reflexes, which are involved in coordinating head and eye movements. This laboratory has a long history performing combined structure/function studies to investigate neuronal excitability in identified vestibular nucleus neurons of older chick embryos and hatchlings. More is known about principal cell development than for any other class of vestibular nucleus neuron. However, little is established on their earliest functional development, and how it may be influenced by microgravity. The techniques include whole-cell patch clamp recording, pharmacological testing, immunocytochemistry combined with quantitative analysis of fluorescent confocal images, and low molecular weight tracer injections in brain slices of chick embryos (E5-E12) and intact hatching chicks. The specific aims include: (1) Describe the pathways, distribution, and terminals formed by otolithic afferents in the chick vestibular nuclei and their relationship to ampullary nerve inputs in hatchlings; (2) Study the onset and emergence of action potential generation in response to depolarizing current pulses and identify the underlying membrane conductance's, focusing on potassium channels; (3) Investigate gap-junction mediated intercellular communication among vestibular neurons and among glial cells; (4) Identify the source of axons forming the first non-vestibular inputs in the tangential nucleus (lateral vestibular nucleus).