Localization of axonally transported 125I-wheat germ agglutinin beneath the plasma membrane of chick retinal ganglion cells.

Abstract

The distribution of 12Sl-wheat germ agglutinin (WGA) transported by axons of chick retinal ganglion cells to layer d of the optic tectum was studied by electron microscopic autoradiography. We found that 52% of the radioactivity was located in axons and axon terminals in the contralateral optic tectum 22 h after intravitreal injection of affinity-purified t2sl-WGA. Axons comprised 43% of the volume of layer d. Dendrites, glial ceils, and neuron cell bodies contained 20%, 17%, and 3% of the label, whereas these structures comprised 24%, 21%, and 2% of the tissue volume, respectively. We also measured the distances between the autoradiographic silver grains and the plasma membranes of these profiles, and compared observed distributions of grains to theoretical distributions computed for band-shaped sources at various distances from the plasma membranes. This analysis revealed that the radioactive source within axons was distributed in a band of cytoplasm extending in from the plasma membrane a distance of 63 nm. Because WGA is known to bind to specific membrane glycoconjugates, we infer that at least some glycoconjugates may be concentrated within an annular region of cytoplasm just beneath the axonal plasma membrane after axoplasmic transport from the neuron cell body.In light of the specific affinity of wheat germ agglutinin (WGA) for N-acetylglucosamine and sialic acid, WGA has been widely used as a probe for membrane glycoconjugates containing these sugar groups (17, 49). For example, the binding of WGA to neuronal membranes has been used to study differences in the carbohydrate composition of neuron cell surfaces during development (14,28,34). We recently found that 12sI-WGA was selectively taken up by chick retinal ganglion cells and transported intact in their axons to the optic tectum (31). The rate at which the iodinated lectin was transported by these cells (30) was within the range of rates of anterograde transport of newly synthesized, endogenous proteins and glycoproteins (l 9, 24, 25).The objective of the present study was to determine the location of the radio-labeled lectin within the tectum after anterograde axonal transport by chick retinal ganglion cells. To achieve this goal, we adapted the methods of analysis of electron microscopic autoradiography developed by Salpeter and others (see reference 56). We present evidence that (a) 52% of the radioactive label is found in axons and axon terminals; (b) the label within the axons is restricted to a narrow band of cytoplasm beneath the plasma membrane; and (c) nearly half of the radioactivity is present in dendrites and gila and therefore the lectin apparently is transferred from cell to cell after transport to the optic tectum. MATERIALS AND METHODS Preparation of Tissues:We used six l-to 2-d-old cockerels (Feather Hill Farms Hatchery, Petaluma, CA). The WGA was iodinated then purified by affinity chromatography as previously described (31). Within 2 d of preparation, the 1251-WGA (sp. act. 12-16 ~tCi/~tg) was'injected in 10-30/tl of 0.1 ...

Genetic and Molecular In Vivo Analysis of Herpes Simplex Virus Assembly in Murine Visual System Neurons

“…After uptake at the dendritic or cell soma membrane, these macromolecules can be moved into and down the axon. For example, wheat germ agglutinin is transcytosed by chick retinal ganglion cells (10). Subsequent release after anterograde transport results in transcytosis across the neuron and release at the synapse (for a review, see reference 40).…”

Multivesicular bodies in neurons: Distribution, protein content, and trafficking functions

“…C. The location of silver grains was quantified in a histogram for a total of 119 silver grains representing either radiolabeled BDNF or GDNF that were injected in the tongue muscle and transported retrogradely to the soma and dendrites of hypoglossal motoneurons (Rind et al ., 2005). The method (LaVail et al ., 1983) relies on measuring the distance of the center of silver grain from the outer membrane, plotted separately for dendritic and somatic MVBs. The data for BDNF and GDNF was combined, as there was no significant difference between the two data sets.…”

Connecting the dots: trafficking of neurotrophins, lectins and diverse pathogens by binding to the neurotrophin receptor p75NTR

“…Lectins are widely used as probes for membrane glycoconjugates. Wheat germ agglutinin (WGA) has specific affinity for N ‐acetyl‐ d ‐glucosamine and sialic acid, and the binding of WGA and WGA–tracer conjugates to neuronal membranes has been utilized for axonal and transneuronal tracer studies (LaVail et al ., 1983; Fabian & Coulter, 1985). The first indication that WGA may bind to the p75 receptor came from a comparison of NGF and lectin transport studies (Dumas et al ., 1979).…”