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When Xenopus spinal cord (SC) neurons are grown on an appropriate substrate of basal lamina molecules, the agrin they externalize along their neuritic outgrowth remains bound to the substrate even after the neurons are removed. Here we demonstrate that these former neuritic pathways containing substrate-bound, neural agrin cause an accumulation of acetylcholine receptors (AChR) and cholinesterase (ChE) at sites of contact with muscle cells and inhibit AChR aggregation over the rest of the muscle cell surface. These local and global synaptogenic effects were not triggered by former neuritic pathways that were agrin-negative. The length of AChR accumulation along the agrin pathways contacted by individual muscle cells corresponded to a saturation process, in agreement with the notion that muscle cells have a limited capacity to cluster AChR. The AChR accumulation caused by the agrin pathways was almost twice as extensive as that induced by living neurites. It is concluded that agrin and possibly other synaptogenic molecules externalized by competent SC neurons bind to the culture substrate in quantities which are more than sufficient to account fully for the local and global changes in AChR and ChE distribution associated with embryonic nerve-muscle synaptogenesis.
FIG. 1. AChR accumulation at sites of muscle contact with former neuritic pathways containing substrate-bound neural agrin. (A) Muscle
cells after 1 day in culture. (B) Agrin immunofluorescence. (C) AChR fluorescence. Similarly numbered arrows in B and C point to the same
s i t.es. AChR accumulated at sites where muscle cells contacted the agrin path ways. Arrow heads point to characteristic AChR patches on muscle
cells which did not contact agrin pathways. Bar, 25;;m.
FIG. 2. AChR accumulation confined to the borders of the agrin pathway. (A) Muscle cells after 1 day in culture. (B) Agrin immunofluorescence.
(C) AChR fluorescence. Almost all of the AChR accumulation is limited to the width of the agrin pathway. Bar, 5 .um.
F IG. 3. Length of AChR accumulation along ag!'in pathways con tacted
by individual musc.le cells. Contact lengths were grouped in 20-
l'm bins up to 100 .unt and in 50-,..m bins between 101 and 200 .urn. Means
and SE bars are plotted at the mean contact length for the muscle cells
in each bin. The number of muscle cells is indicated abO\•e theSE bar.
The smooth curve was drawn according to the equation shown. The
dashoo curve and its corresponding equation are from a previous study
and indicate the relationship between length af AChR accumulation
and length of neurite- mtlscle contact on muscle cells which were innervated
by competent SC neurons (see F ig. 4 in Cohen et al, 19$7).
FIG. 4. ChE accumulation along agrin pathways. (A) Agrin immunofluorescence. (B) AChR fluorescence . (C) ChE reaction product. The muscle
cells were cultured fo•· 2 days. ChE, like AChR, accumulated at sites where muscle cells contacted agrin pathways. Some ChE reaction product
i.s also seen elsewhere on the muscle ~ells. Bar, 1Q I'm.
FIG. 5. AChR accumulation along prestained agrin pathiVays. (Ai .Muscle cells after 1 day in culture. {B) Agrin immunofluorescence. The
immunofluorescent staining was carried out before the muscle cells were plated. (C) AChR fluorescence. Note the extensive accumulation of
AChR at sites where muscle cells contacted the prcstaincd agrin pathways. Ba r, 25J'm.
FIG. 6. AChR accumulation induced by ag•·in-positive, but not by agrin-negative, former neurit ic pathways. (A) Living SC neurons. After this
photograph was taken, the neurons were eliminated and muscle cells were plated. (B) F ixed muscle cells after 1 day in culture. (C) Agrin
immunofluor escence. Note that agrin was associated with only two of the former neuri tic pathways. (D) AChR fluorescence. The position of the
neurites before they were eliminated is indicated by the hlack lines. Note the extensive accumulation of AChR at sites wh<!re muscle cells
contacted agrin-positive pathways and the almost complete absence of AChR fluorescence where muscle cells contacted agri n-negative pathways.
Bar, 25 pm.
FIG. 7. Lack of AChR accumulation along former DRG neuritic pathways. (A) Living DRG neurons. Pa r t of an ink dot on the outside of the
culture chamber is seen out of focus in the upper left portion of the field . The ink dots, in combination with the X-Y coordinates on the microscope,
served as reference points. After this photograph was taken, the neurons were eliminated and muscle cells were plated. (B) Fixed muscle
cells after 1 day in culture. (C) Agrin immunofluorescence. None was apparent. (D) AChR fluorescence. Very little was seen along the former
neuritic pathways which arc indicated by the black lines. Instead there were patc.hes of AChR elsewhere on th~ muscle cells. Bar, 251'm.
