Aggregates of acetylcholine receptors are not observed under anti‐agrin staining Schwann cell processes at the frog neuromuscular junction

Abstract

The frog neuromuscular junction offers a unique structure in which to observe fine details in the relationship between the motor neuron, muscle, and Schwann cell, which together comprise the neuromuscular junction. Schwann cell processes that extended from the synapse stained positively with anti-agrin antibodies. Immunocytochemistry revealed strong anti-agrin staining of the extracellular matrix surrounding the entire Schwann cell and the Schwann cell processes come in close contact with the muscle fiber. Dual-labeling experiments revealed a lack of acetylcholine receptor (AChR) aggregates on the surface of the muscle fiber directly under these anti-agrin-staining Schwann cell processes. The cDNA that codes for the C-terminal portion of agrin in frog (Rana pipiens) was cloned and sequenced. Polymerase chain reaction of frog brain, nerve, and muscle cDNA revealed that like other species, agrin transcripts that contain the B8, B11, or B19 inserts were observed only in brain tissue. Unlike other species, the exact site of the B inserts in frog was slightly altered, with the B insertion site occurring within a codon sequence. Our data are consistent with the hypothesis that Schwann cells produce agrin which lacks inserts at the B region, and that agrin lacking B inserts does not direct the aggregation of AChRs in vivo under physiological conditions and concentrations.

Schwann Cells Express Active Agrin and Enhance Aggregation of Acetylcholine Receptors on Muscle Fibers

“…Although the predominant cell type in the sciatic nerve is the Schwann cell, the epineurial sheath, which is rich in fibroblasts, was routinely removed to ensure that we used primarily Schwann cells for the RT-PCR study. We found that frog Schwann cells along the sciatic nerve expressed not only the inactive isoform B0, as shown previously (Werle et al, 1999), but also the active isoforms B11 and B19 ( Fig. 1 A, lane 6 ).…”

“…To confirm the identity of agrin transcripts in Schwann cells, each PCR fragment was subcloned and sequenced. PCR with nested primers (see Materials and Methods) were also used to further confirm that these PCR products were frog agrin gene fragments (Werle et al, 1999). Our results showed that agrin expression in adult Schwann cells was similar to that in the spinal cord neurons, except for the absence of the B8 isoform.…”

“…Receptor aggregation by glia may complement the role of neurons and may be particularly important in ensuring rapid restoration of synaptic function during regeneration. Werle et al (1999) have reported that Schwann cells in the frog sciatic nerve express only the inactive B0 isoform. However, using the same preparation and the same primers for RT-PCR, we found that frog Schwann cells do express the active agrin isoforms B11 and B19, besides the inactive B0.…”

“…Previous molecular cloning studies have shown that motor neurons express four isoforms of agrin that contain either 0, 8, 11, or 19 amino acids at the B (for chick and frog) or Z (for rat) site (Ferns et al, 1992;Ruegg et al, 1992;Tsim et al, 1992;Werle et al, 1999). Only B8, B11, and B19 isoforms are capable of inducing AChR aggregation at the NMJ, whereas B0 is inactive in inducing AChR aggregation (Ferns et al, 1992(Ferns et al, , 1993Ruegg et al, 1992;Gesemann et al, 1995;Daggett et al, 1996).…”

Schwann Cells Promote Synaptogenesis at the Neuromuscular Junction via Transforming Growth Factor-β1

“…Gels were visualized using a UV transilluminator, and images were taken using a digital camera. For agrin, primers flanking the frog agrin alternative splicing site B were designed based on the GenBank sequence AF096690 (Werle et al, 1999): forward, 5Ј-TCT CTG GAG GAC AAT GTG AGA AAG C-3Ј; and reverse, 5Ј-CCT GAG TGG CTT CAG TCT TTA TGC-3Ј. The housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) was used to confirm the equal loading of samples for the reverse transcription and PCRs.…”