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.

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Electrical stimulation is a therapeutic modality available for the preservation of muscle function following peripheral nerve injury. Agrin, a synaptic basal lamina protein, induces accumulation of acetylcholine receptors (AChRs) and other molecules at the neuromuscular junction. Electrical stimulation of denervated muscle does not alter agrin and AChR distribution at abandoned synaptic sites, supporting the hypothesis that the existing aggregation of synaptic molecules, which may be necessary for successful reinnervation, is unaltered by electrical stimulation of denervated muscle.

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs.

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