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

Abstract

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 lysosomallike 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.

Alteration of the Expression Levels of Rab3A Affects the Extent of Transcytosis of HRP-Labeled Marker Proteins in Rat CNS Neurons

“…Furthermore, it was of particular interest that the neuronal transcytosis of tracer enzyme appeared to have occurred randomly at various portions of the axodendritic membranes. The appearance of the HRP-RP in relation to the membranes was characterized as either the "passing type" (Figure 3(a)) or, because of the indentation of axodendritic membranes into the dendrites, as the "secretion type" (Figures 3(b)-(d)) [9]. = 0.1 μm in (a,b,e), 0.2 μm in (c,d) and 1.0 μm in (f).…”

“…In a recent study using Rab3A gene-deleted mice, synaptic vesicle docking to the presynaptic membrane and regulation of synaptic vesicle exocytosis were shown to be impaired at the neuromuscular synapses [7,8]. Furthermore, our previous study in-dicated that Rab3A-siRNA injected into the rat vagus nerve is involved in modulating the level of transcytosis of the tracer enzyme, wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP), at synaptic junctions [9]. However, the mechanisms involved in this effect of Rab3A-siRNA on transcytosis remain to be elucidated.…”

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

“…They suggested its mechanism to be "diacrine secretion" which was characterized by passing through cell membrane. With respect to the non-vesicular transport, our recent studies in Rab3A-siRNA-and ethanol-treated rats also indicated alteration of membrane of the vagus nerve terminals in the nucleus of solitary tract and it was called "apocrine-like structure" which was quite different from normal vesicular exocytosis (5)(6)(7). On the basis of its morphological characteristic, it was hypothesized that this structure possibly plays an important role in the process of synaptic transport of materials including proteins.…”