Male Wistar rats weighing 180-236 g were anesthetized with intraperitoneal injection of chloral hydrate (490 mg/kg) for all surgical procedures. The experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. The Kagawa University Animal Care and Use Committee approved the procedures, and all efforts were made to minimize the number of animals used and their suffering.
A 4% solution of WGA-HRP (0.4-2.0 μl) or a working solution of 1 nM Rab3A-siRNA containing 4% WGA-HRP was injected into the vagus nerve on one side using a 10-μl Hamilton microsyringe (Reno, Nevada, U.S.A.) . After a survival period of 12-72 h, the animals were sacrificed by perfusion with 0.1 M phosphate buffer (pH 7.4) followed by a fixative of 1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 M phosphate buffer. The blocks containing the nucleus of the solitary tract (NST) and dorsal motor nucleus (DMV) of the vagus nerve were processed for visualization of HRP-reaction products (RP) according to the heavy metal-intensified DAB methods. The blocks were then postfixed in buffered 1% osmium tetroxide for 2 h, block-stained in saturated uranyl acetate for 1 h, dehydrated in a graded acetone or an alcohol series and embedded in an epoxy resin mixture. The NST region was identified by the examination of toluidine blue-stained or unstained 1-μm-thick sections. Ultrathin sections of the region were cut and observed without further lead staining using a JEM 200 CX electron microscope.
A solution of fluorescent (F) WGA (1.5-2.0 μl) (Alexa 594-conjugated WGA; Invitrogen, USA), FAβ (1.5-2.0 μl) (Alexa 488-conjugated Aβ; Invitrogen, USA) or FWGA containing FAβ was injected into the vagus nerve on one side using a Hamilton microsyringe fitted with a 33-gauge needle in a volume of 5 µL. Further experiments involved the injection of FAβ into the vagus nerve and FWGA into the common carotid artery. For tissue cryosection analysis, the brain stem and nodose ganglions of the animals were processed with 0.1 M phosphate buffer perfusion and a 4% PFA fixative solution, dehydrated in a solution of graded sucrose in 0.1 M phosphate buffer and embedded in an O.C.T. compound. The sliced NST region and nodose ganglions were placed onto slide glasses and processed for nuclear counter staining with 50 ng/ml Hoechst 33258 (Sigma-Aldrich) for 10 min. All slides were mounted in fluorescence mounting medium (DAKO); the localization of molecules was then detected by an epi-illumination fluorescence microscope (DP-72, Olympus) or a confocal laser scanning microscope (Radiance 2100 rainbow system, Zeiss) and photographed. All images were analyzed using NIS-elements software (Nikon).
In the terminals of the NST, electron-dense HRP-RP showed various types of lysosomal-like structures and was characterized by the presence of membranous substance crossing synapses (Figure 1). Neuronal transcytosis of WGA-HRP was induced at the stage when no neurotransmitter was released. Based on neurotransmitter release suppression by siRNA interference methods, Rab3A-siRNA was co-injected with WGA-HRP into the vagus nerve. Furthermore this experiments were performed neuronal transcytosis of WGA-HRP including secretion followed by endocytosis of postsynaptic neurons (Figure 2).