Expression of G_D3 ganglioside by developing rat cerebellar purkinje cells in situ

Abstract

GD3 is a major ganglioside of the immature vertebrate CNS, and its expression is suggested to be characteristic of immature neuroectodermal cells. Using immunocytochemistry on cryostat sections of developing rat cerebellum with a monoclonal antibody specific for GD3, we have found that GD3 begins to be expressed on the plasma membrane of Purkinje cell bodies and dendrites beginning at postnatal day 7. Staining became brighter as the dendritic tree of the cells enlarged. As the Purkinje cells began to mature in different folia, they became GD3+, until by 15 days postnatal all Purkinje cells were GD3+. Positive staining of the dendritic tree was still present in the adult cerebellum. Using a monoclonal antibody 7-8D2, which recognizes cerebellar granule cells and their axons (the parallel fibres), and polyclonal antibodies against a synaptic vesicle component synaptophysin, double-immunofluorescence staining together with anti-GD3 antibodies suggested that the appearance of GD3 immunoreactivity did not correlate either with the ingrowth of parallel fibres or the presence of their synapses on Purkinje cell dendrites. However, comparison with earlier morphological studies showed that the appearance of GD3 immunoreactivity correlated well with the formation of climbing fibre synapses on Purkinje cell dendrites and the onset of the rapid expansion of the dendritic tree. These results are in keeping with the idea that elevated GD3 concentrations are found in certain cell types during periods of rapid growth or high metabolic activity but also show that this is not only restricted to immature cells.

Immunostaining of ganglioside GD1b, GD3 and GM1 in rat cerebellum: Cellular layer and cell type specific associations

“…In the PL, we found anti-GD3 staining in the cytoplasm of the Purkinje cell bodies. Although previously regarded mainly as a marker of astrocytes, microglia and neuroectodermal cells (Goldman and Reynolds, 1996), ganglioside GD3 expression in Purkinje cells has also been reported previously (Reynolds and Wilkin, 1988;Kawai et al, 1994;Yamamoto et al, 1994). Kotani et al (1995) reported no anti-GD3 staining in the PL.…”

Determination of the Localization of Gangliosides Using Anti-ganglioside Antibodies: Comparison of Fixation Methods

“…It has been suggested that gangliosides become antigenic only after acetone treatment, because acetone may act to promote the accessibility of the ganglioside (Graus et al 1994) or may unmask membrane-bound carbohydrates (Thorpe and Kerr 1994). However, no GM3 was detected with an anti-GM3 antibody in prefixed cryostat sections after acetone treatment (Daniotti et al 1994) or after tratment of formaldehyde-fixed tissue sections with methanol, acetone, or ethanol/acetic acid (Reynolds and Wilkin 1988).…”

“…For example, using two different antibodies to ganglioside GD3, opposite conclusions were reached about the presence of GD3 in rat cerebellar Purkinje cells. With monoclonal antibody (MAb) R24 no labeling of Purkinje cells was observed (Goldman et al 1984), but labeling was observed with the LB1 antibody (Reynolds and Wilkin 1988). Although these differences could be caused by each antibody recognizing different epitopes on ganglioside GD3, a more likely explanation is that the different fixation methods used in each study (Reynolds and Wilkin 1988) are responsible for the different results.…”

A reappraisal of ganglioside GD3 expression in the CNS

“…However, this reactivity is lost as the cells begin to migrate through the molecular layer. In a study of LB1 binding in the developing cerebellum Purkinje cells were found to express GD3 in the cell membranes of the dendritic tree (Reynolds and Wilkin, 1988b). GD3+ Purkinje cells were first seen at 7 days postnatal and a n increase in the intensity of the immunostaining was observed as the dendritic tree increased in complexity, correlating well with climbing fibre synaptogenesis.…”