Anterograde synaptic transport of neuronal tracer enzyme (WGA-HRP): Further studies with Rab3A-siRNA in rats

Neuronal tracer enzyme of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was employed to elucidate the detailed morphology of anterograde synaptic transport. After injection of WGA-HRP into the vagus nerve, sensory terminals in the nucleus of solitary tract (NST) were observed at the electron microscopic level using the tetramethyl benzidine and diaminobenzidine methods. In neuropil of the NST, electron-dense HRP-reaction product (HRP-RP) showed various types of lysosomal-like structures. The RP characterized by containing membranous substance crossed synapses forming a mass without membrane surrounding the RP. Additionally, phenomena of anterograde synaptic transport of the RP and exocytosis of synaptic vesicles never occurred simultaneously. These findings raised the possibility of inducing synaptic transport of WGA-HRP at the stage of no neurotransmitter release, i.e. no activation of neuron. Therefore, further experiments were performed after co-injection of Rab3A-siRNA with WGA-HRP into the vagus nerve. This co-injection frequently resulted in not only suppression of vesicle trafficking to active zones and docking to pre-synaptic membranes but also abnormal aggregation of synaptic vesicles at terminals. Furthermore, synaptic transport of WGA-HRP, including secretion, followed by endocytosis of post-synaptic neurons was better seen in the experiments.