The experiments described in this proposal will investigate the function of ryanodine-sensitive intracellular Ca2+ stores in peripheral neurons, including their role in the differentiation and maturation of these cells from neural crest precursors. Our approach to this problem is fourfold. First, antibodies specific for isoforms of the avian ryanodine receptor and immunohistochemical techniques will be used to localize ryanodine receptors in neural crest cells and cells in developing peripheral ganglia. This work will identify cell types and times during development in which Ca2+ release from intracellular Ca2+ stores may be important. We will examine cells in sections of intact embryonic chicks to monitor the development of ryanodine receptors in situ. Similar observations made on cells differentiating and maturing in culture will characterize the development of ryanodine receptors under more defined conditions in vitro. Second, functional measurements of Ca2~+ using will be made using fluorescent Ca2+ indicator dyes to monitor bulk cytoplasmic Ca2+, and Ca2+ activated membrane conductances to monitor Ca2+ activity at the cell membrane. Functional Ca2+ stores will be identified by their caffeine- and ryanodine-sensitivity. These studies will be done using primary cultured cells from peripheral ganglia of E4 through post-hatch chicks in order to characterize the functional properties of Ca2+ stores in morphologically mature neurons. Third, cell survival assays will be used to investigate the role of intracellular Ca2+ stores, Ca2+-induced Ca2+ release, ryanodine receptors, and Ca2+ oscillations in the prevention of natural cell death of peripheral neurons by NGF or high K+. Ca2+ has been implicated in the cell saving action of both of these treatments and the existence of active intracellular Ca2+ stores raises the possibility that dynamic Ca2+ oscillations may contribute to this action or mimic it. Pharmacological agents, including ryanodine, thapsigargin, and caffeine will be used to manipulate intracellular Ca2+ stores in order to examine their effects on cell survival. Fourth, we will investigate the development of peripheral neurons in crooked neck dwarf mutant (cn/cn) chicks where the alpha isoform of the ryanodine receptor is not expressed normally in skeletal muscle. The goal of these experiments will be to establish whether or not this mutation affects peripheral neurons, to characterize the functional consequences of the mutation, and to investigate the suitability of cn/cn mutant birds as a system for future investigations of the role of neuronal ryanodine receptors in cell development and function.
| Kenyon, J L; Goff, H R (1998) Temperature dependencies of Ca2+ current, Ca(2+)-activated Cl- current and Ca2+ transients in sensory neurones. Cell Calcium 24:35-48 |
| Bauer, R J; Carl, A; Kapicka, C L et al. (1996) Determination of channel open probabilities from multichannel data. J Neurosci Methods 68:101-11 |
| Ward, S M; Shuttleworth, C W; Kenyon, J L (1994) Dorsal root ganglion neurons of embryonic chicks contain nitric oxide synthase and respond to nitric oxide. Brain Res 648:249-58 |
