Fast dii

Three to four-week-old mice were anesthetized (1.5 % isoflurane) and then placed on a rodent stereotactic apparatus supplied with a mouse adapter (Stoelting, Wood Dale, IL). To identify STTn, a retrograde tracer (1% FAST-DiI in 10% ethanol; Invitrogen, Carlsbad, CA) was injected into the ventrobasal thalamus using a 5 μL Hamilton syringe needle. Coordinates of the injection site relative to bregma were: 1.48 mm posteriorly, 1.37 mm laterally, and 3.3 mm deep into the brain using the adjusted ratio of bregma-lambda distance specific for young mice (4.5 mm)^{14 (link)}. Dye was allowed 5-7 days post injection to be transported to the lumbar spinal cord, after which the mice were euthanized for spinal cord slicing and patch clamp recording.

STTn were labeled by a retrogradely transported dye which was injected into the thalamus. Two to 3-week old mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and placed on a rodent stereotactic apparatus fitted with a mouse adaptor (Stoelting, IL). A retrograde tracer, FAST-DiI (1 µl, 5% in ethanol, Invitrogen), was injected into the ventrobasal (VB) complex of the thalamus, using a micropipette attached to a 5 µl Hamilton syringe needle [25 (link),46 (link)]. For VB injection, the stereotactic coordinates [41 ] were calculated and adjusted with respect to the bregma, by using the ratio of the bregma-lambda distance of young mice to that of adult mice (4.5 mm). The coordinates of the injection site were 1.48 mm posterior from the bregma, 1.37 mm lateral from the mid-sagittal plane and 3.3 mm deep from the surface. Three to 4 days were allowed for the dye to be transported to the lumbar spinal cord.

For rats in which DRGs were to be examined histologically or electrophysiologically, an additional skin incision was made, on the right side, seven days prior to the experimental skin incision to allow for tracer injection and transport. This skin incision to enable tracer injection was placed on the right side to prevent damage to the axons of interest on the left side. Following incision on the right side, the skin was reflected to expose the underside of the contralateral (left) skin. 0.5% DiI (1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; 5 mg FastDiI dissolved in 1 ml methanol; Invitrogen) was injected into the subdermal layer using a Hamilton syringe. Ten injections of 1 μl each, were used to target the terminal field as described previously (Jiang et al., 2006 (link); Rau et al., 2007 (link)). The Experimental Incision was approximately 5 mm distal to, and extended at least two dermatomes rostral and caudal from the DiI injected region (see Fig. 1). Preliminary studies indicated that this method maximized the percentage of DiI-labeled cells that were injured by incision, as indicated by ATF3/DiI co-localization.

To verify the origins of CGRP-IR axons in the stomach, mice (n = 4) were anesthetized with isoflurane (2%–3%). The depth of anesthesia was determined by the lack of the hind paw and tail pinch withdrawal reflex. Tracer Fast DiI (Invitrogen, Catalog# D3899) was injected into the stomach wall using a Nanofil syringe microinjection system (NANOFIL-100, World Precision Instrument). Each region, that is, fundus, corpus, and antrum-pylorus received 3–4 injections (2 μL/each, 9–12 injections per surface) to cover the whole ventral or dorsal walls. The injection areas were cleaned thoroughly with cotton swabs, the displaced stomach was returned to the animal’s abdominal cavity in its original position, followed by the closure of the abdominal muscle using interrupted sutures and the skin using a single continuous suture. The animals were returned to their cages for 14–16 days, then they were perfused. The left and right DRG (T1–T12) and left and right vagal nodose-petrosal ganglia complex (VNG) were removed for IHC labeling of CGRP.

Cell membranes were stained with 5 μg/ml of FAST-DiO, FAST-DiI or DiD (Invitrogen), respectively, green, red or far-red fluorescent lipid dyes that strongly partition to disordered phases ^{42 (link), 43 (link)}. Following staining, GPMVs were isolated from transfected RBLs, HEK-293, or HeLa cells as described ^{19 (link)} (cell type had no effect on results). Briefly, GPMV formation was induced by 2 mM N-ethylmaleimide (NEM) in hypotonic buffer containing 100 mM NaCl, 10 mM HEPES, and 2 mM CaCl2, pH 7.4. To quantify protein partitioning, GPMVs were observed on an inverted epifluorescence microscope (Nikon) at 4°C after treatment with 200 μM DCA to stabilize phase separation; this treatment has been previously demonstrated not to affect raft affinity of various proteins ^{44 (link)}. The partition coefficient (K_p,raft) for each protein construct was calculated from fluorescence intensity of the construct in the raft and non-raft phase for >10 vesicles/trial (Fig. 1), with multiple independent experiments for each construct. The results are displayed as log (K_p,raft), in analogy with log P values oil-water partitioning, to avoid appearance of compression for values less than 1.