Sprague-Dawley and Fischer 344 rats (male; three- to twelve-months-old) were obtained from Harlan Industries (Indianapolis, IN; n = 18). Upon arrival, rats were single housed at 22–24°C and 40–60% humidity on a 12:12 h light:dark cycle. Solid chow (laboratory diet no. 5001; PMI Feeds, Inc., Brentwood, MO) and tap water were available ad libitum. All procedures were conducted in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996), and were approved by the Purdue University Animal Care and Use Committee.
Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and then mounted in a stereotaxic frame. The medulla was exposed and the obex was used as the reference point for coordinates for multiple (8 to 13) injection sites into the dorsal motor nucleus of the vagus nerve (dmnX). In separate rats, one of four different 5% solutions of lysine fixable, 10000 MW dextran conjugates in H2O was pressure injected through a glass micropipette (ID 25 μm) with a picospritzer (80 psi; General Valve Corporation, Fairfield, NJ) into the dmnX. Several 4 msec applications of the conjugate were made in each location and the pipette was left in each site for 2–3 min following the last application to allow the solution to disperse, and prevent excess leakage from the line of penetration. The four conjugates of dextran used were Texas Red (D-TR; D1863; Invitrogen, Carlsbad, CA), Rhodamine Green (D-RG; D7153; Invitrogen), tetramethylrhodamine and biotin (D-TMR-B; D3312; Invitrogen), or biotin (D-B; D1956; Invitrogen).
To evaluate the feasibility of double-labeling vagal efferents and afferents in the same rat, a subset of rats were re-anesthetized five days after dmnX injections of D-B and the left and right nodose ganglia were exposed by blunt dissection. The ganglia were then pressure injected with D-TR through a glass micropipette (ID 25 μm).
Following dmnX injections, a time course of 19 days was allowed for the dextrans to transport to the GI tract. Rats were then weighed and euthanized with a lethal dose of sodium pentobarbital (180 mg/kg, i.p.); once unresponsive to paw pinch, the abdomen and chest cavity were opened. Rats were then injected in the left ventricle of the heart with heparin (0.5 ml; 1,000 units/ml; Baxter Healthcare Corporation, Deerfield, IL) followed by transcardial perfusion with 200 ml of 0.01 M sodium phosphate-buffered saline (PBS; pH 7.4; 38°C). The stomach was distended with approximately 10 to 15 ml of PBS to provide uniformity in organ size. Tissue fixation was then achieved by intracardiac perfusion of 500 ml of 4% paraformaldehyde (PF) in 0.1 M PBS (pH 7.4; 4°C).
The stomach and small intestine from the pyloric sphincter to the ileocaecal junction were dissected out of each rat. Whole mounts of the small intestine, including the duodenum (first 8 cm anal to the pyloric sphincter), jejunum (8 cm taken from the middle of the small intestine), and ileum (first 8 cm oral to the ileocaecal junction) were designated according to the criteria of Hebel and Stromberg (1976) and saved. The stomach was separated into ventral and dorsal halves, with cuts along the greater and lesser curvatures. Intestinal segments were opened with a longitudinal cut along the mesenteric attachment and rinsed out with tap water. Whole mounts were stored for an additional 2 hours in 4 % PF prior to the removal of the mucosa, submucosa, and circular muscle by dissection with fine-tip forceps.
For those rats that received dmnX injections of D-TR, D-RG, or D-TMR-B and no other labeling or staining, the removal of the mucosal, submucosal, and circular muscle layers was followed by three five-minute PBS rinses. Whole mounts were then mounted on gelatin-coated slides, air-dried overnight, dehydrated in a series of ascending alcohol rinses, cleared in xylene, and coverslipped in DPX neutral mounting medium (Aldrich Chemical Company, Inc, Milwaukee, WI).
For double-tracer-labeling observations, GI tissue from rats injected with D-B in the dmnX and D-TR in the nodose ganglia were prepared as whole mounts, rinsed in PBS, and then soaked for 3 days at room temperature in PBS containing 0.5% Triton X-100, and 0.08% Sodium Azide. Whole mounts were then rinsed in PBS and incubated for 1 h at room temperature in streptavidin ALEXA Fluor 488 (1:500; S32354; Invitrogen) diluted with PBS. Finally, labeled whole mounts were rinsed in PBS, mounted on gelatin-coated slides, air-dried overnight, dehydrated in alcohol, cleared in xylene, and cover-slipped with DePeX (13514; Electron Microscopy Sciences, Hatfield, PA).
For triple-labeling trials, whole mounts from rats injected with D-TR in the dmnX were rinsed in PBS and then soaked for 5 to 6 days at room temperature in PBS containing 5% normal goat serum, 2% bovine serum albumin, 0.5% Triton X-100, and 0.08% Sodium Azide, followed by incubation for 24 h at room temperature in a cocktail consisting of the primary antibodies mouse alpha-synuclein (1:2,500; 610787; BD Biosciences, San Jose, CA) and rabbit NOS (1:2,500; SC-648; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted with PBS containing 2% normal goat serum, 2% bovine serum albumin, 0.3% Triton X-100, and 0.08% Na Azide. Whole mounts were then rinsed in PBS and 0.3% Triton X-100 (PBST), and incubated for 2 h at room temperature in a cocktail consisting of goat anti-mouse ALEXA Fluor 488 (1:500; A11029; Invitrogen) and biotinylated anti-rabbit IgG raised in goat (1:500; 111-065-144; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted with PBST. Next, whole mounts were further rinsed in PBS and then incubated for 1 h with streptavidin ALEXA Fluor 350 (1:500; S11249; Invitrogen). Finally, labeled whole mounts were rinsed in PBS, mounted on gelatin-coated slides, air-dried overnight, dehydrated in alcohol, cleared in xylene, and cover-slipped with DePeX (Electron Microscopy Sciences).
Whole mounts were processed for double labeling by DAB staining of the vagal efferents following D-B injections into the dmnX and NBT staining of the subpopulation of nitrergic neurons in the myenteric plexus with nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd; Phillips et al 2003
) protocol. Briefly, following the post-fixation in PF, the whole mount tissue was rinsed with 0.1 M Tris–HCl (pH 7.9) and then incubated for 1 h in 0.1 M Tris–HCl (pH 7.6) containing 1.0 mg/ml b
-NADPH (Sigma, St Louis, MO), 0.33 mg/ml nitroblue tetrazolium (NBT; Sigma) and 0.5% Triton X-100 at 37°C, followed by rinses in Tris–HCl (pH 7.9). Then whole mounts were rinsed in PBS followed by a 30 min soak in an endogenous peroxidase block (methanol:3% H2
; 4:1). After additional rinses in PBS, the tissue was soaked overnight in PBS with 0.5% Triton X-100 and 0.8% Sodium Azide followed by a 60 min soak in ABC (prepared as per the directions provided with the Vectastain Elite ABC kit; Vector Laboratories, Inc., Burlingame, CA). The tissue was than rinsed in PBS, reacted with DAB for 5 min, rinsed in distilled water and mounted on slides. The slides were air-dried overnight then dehydrated in an ascending series of alcohol rinses, cleared in xylene, and coverslipped in Cytoseal mounting medium (8312; Richard-Allen Scientific, Kalamazoo, MI).
Another double labeling procedure was done by DAB staining the vagal efferents that were labeled with D-B injected into the dmnX and then counterstaining the neuronal population of the myenteric plexus with the pan-neuronal marker Cuprolinic Blue (Phillips and Powley, 2001; Phillips et al 2004
). Briefly, the whole mounts were rinsed in PBS followed by a 30 min soak in an endogenous peroxidase block (methanol:3% H2
; 4:1). After additional rinses in PBS, the tissue was rinsed in distilled water and stained for 4 h in a humidified slide warmer (38°C) with0.5% Cuprolinic blue (quinolinic phthalocyanine; Polysciences, Inc., Warrington, PA) in 0.05 M sodium acetate buffer containing 1.0 M MgCl2
, pH 4.9. The whole mounts were then rinsed in distilled water, differentiated for 1 min in 0.05 M sodium acetate buffer containing 1.0 M MgCl2
(pH 4.9), and rinsed again in distilled water followed by another series of PBS rinses. The whole mounts were then soaked overnight in PBS with 0.5% Triton X-100 and 0.8% Sodium Azide followed by a 60 min soak in ABC (prepared as per the directions provided with the Vectastain Elite ABC kit; Vector Laboratories). The tissue was than rinsed in PBS, reacted with DAB for 5 min, rinsed in distilled water and mounted on slides. The tissue was allowed to air-dry overnight then dehydrated in an ascending series of alcohol rinses, cleared in xylene, and coverslipped with Cytoseal.
Analysis and Photography
Tissue visualization and qualitative analysis was made using a Leica DMRE widefield microscope (Leitz, Wetzlar, Germany). Photomicrographs were acquired using a Spot RT Slider camera (Diagnostic Instruments, Sterling Heights, MI, USA). Adobe Photoshop CS3 (version 10; Adobe Systems, San Jose, CA, USA) was used to apply the text and scale bars, make minor adjustments to the color, brightness, contrast, and sharpness of the images to match as closely as possible to the appearance of the original material viewed under the microscope, and to organize the final layout of the figures.