Research reportDifferential distribution and subcellular localization of ryanodine receptor isoforms in the chicken cerebellum during development
Introduction
Recent work on the distribution of proteins involved in the uptake, sequestration and release of calcium from internal stores has provided strong evidence that neurons, like muscle cells, possess intracellular calcium pools localized within components of the internal membrane system. Previous work from this laboratory and others has shown that the ryanodine receptor (RyR), a calcium channel first isolated from the muscle sarcoplasmic reticulum 11, 16, 26, 32, is also found in the nervous system where it is concentrated in the endoplasmic reticulum of cerebellar Purkinje neurons and other cell types 12, 34, 44, 59. The RyR is a high conductance calcium channel that releases calcium from intracellular stores in response to calcium and caffeine 16, 24, 32, 33, 35, 50, 56. The role of the RyR in muscle excitation–contraction coupling has been well studied 18, 41. In contrast, little is known about the functional significance of the neuronal RyRs. Preliminary evidence suggests that the neuronal receptor may operate as a calcium-induced calcium release channel 28, 52, 54. As such, it may play a role in the spreading and amplification of intracellular calcium responses within neurons.
Several isoforms of the RyR have been distinguished in both mammalian and avian tissue 3, 4, 27. In mammals, three genes encoding RyRs have been reported: a skeletal muscle form, a cardiac muscle form 39, 42, 49, 58, 60, and a third form found in brain and smooth muscle 19, 21. Although they are products of different genes, the cardiac and skeletal muscle RyRs have many structural and functional similarities. However, they display differences in ryanodine binding kinetics 15, 25, 26, 37, in their sensitivities to regulatory ligands 6, 35, 36, and in their phosphorylation [57], suggesting functional and regulatory differences between isoforms. Avian tissues express forms of the RyR distinct from mammalian tissue. Three isoforms of the RyR have been identified in avian striated muscle [4]. Two of them isolated from skeletal muscle were first referred to as α- and β-based on their differential migration on SDS–PAGE [3]. These isoforms differ in their extent of phosphorylation by calcium/calmodulin-dependent protein kinase II and also in their ability to bind calmodulin [4]. A cardiac form of the avian RyR has also been described 3, 13.
Several studies have indicated that the brain expresses multiple isoforms of the RyR 14, 21, 31, 45. In a previous study, we provided biochemical and immunocytochemical evidence that different isoforms of the RyR are differentially distributed within the brain of hatchling chicks. Using antibodies that recognize either the α-skeletal muscle form or the β- and cardiac forms of the RyRs, a Western blot analysis indicated that the α-form is predominately expressed in cerebellum while the β/cardiac form is found throughout the brain [45]. Immunocytochemical evidence indicated that the α-form was mainly in Purkinje cells while the β/cardiac form was widely distributed throughout the brain. These studies are consistent with studies in mammalian brain indicating that the various isoforms of the RyR are found within distinct populations of neurons 21, 31.
In addition to the differential distribution of the α- and β/cardiac isoforms in the chick brain, we noted that there was a change in both the amount and in the pattern of distribution of these isoforms during development. Since these isoforms appear to differ in their functional properties, a detailed analysis of their relative distribution during development may yield clues as to their function in both the developing and the adult brain. Therefore, in the present study, the localization of different isoforms of the RyR was investigated in the chick brain during development and adulthood using immunocytochemical techniques. Two monoclonal antibodies were used: 110F which recognized the α-skeletal muscle form exclusively and 110E which recognizes both the β-skeletal muscle and the cardiac isoform, but not the α-skeletal muscle form 3, 4. In addition to differences in distribution between the two isoforms at all stages of development, we noted a dramatic change in the localization of the β/cardiac form immunoreactivity in the cerebellum during the first two postnatal weeks. A brief account of this work has appeared in abstract form [44].
Section snippets
Materials and methods
Tissue was obtained from chick embryos at age E18 and from post-hatching White Leghorn chicks (eggs obtained from McIntyre Poultry Farm, Lakeside, CA) ranging in age from 1 day to adult. Embryonic tissue was fixed by immersion of the whole embryo in 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.2 (PBS) for 30 min to 2 h at 4°C. The brain was then removed and further fixed for 1 h in the same fixative. Post-hatching chickens were first anesthetized deeply with Nembutal then
Terminology
Because the monoclonal antibody 110E does not distinguish between the β-skeletal muscle and the cardiac form of the RyR, we use the term `β/cardiac form' when referring to labeling with 110E. The term `α-form' refers to immunolabeling with antibody 110F.
A differential distribution of the major isoforms of RyR discriminated by antibodies 110E and 110F was observed in chicken CNS
The pattern of immunoreactivity observed for the α- and β/cardiac form of the RyR was quite different in the central nervous system of chicken at all developmental times examined. A similar pattern of labeling was observed in tissue sections
Discussion
The results of this study provide additional evidence that ryanodine receptor isoforms possess distinct cellular and subcellular distributions in the avian brain. The distribution of the α- and β/cardiac form of the RyR within chicken brain is consistent with biochemical results indicating that the α-form is expressed predominately within the cerebellum while the β/cardiac form is widespread throughout the brain 43, 45. Labeling for the β/cardiac form was found in neurons of many nuclei
Acknowledgements
We thank Drs. Harvey Karten and Enrico Mugnaini for helpful discussions, and Ms. Victoria M. Edelman for technical assistance. This research was supported by NIH Grants NS14718 and RR04050 to M.H.E.; NIH Grant HL27470, and NSF Grants DCB9108091, IBN9306850 to J.L.S.
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