A total of 51 adult male rats (Sprague-Dawley) that weighed between 280 and 320 g (China SH, Xi’an, People’s Republic of China) were used in the current study. Animal use and care was approved by the Animal Care and Use Committee at the Fourth Military Medical University. Of these rats, 30 rats were used for dual retrograde tract-tracing combined with FISH histochemistry or immunofluorescence histochemistry, 15 rats were used for anterograde tract-tracing combined with immunofluorescence histochemistry, and 6 rats were used for electron microscopy.
Microinjection of TMR and FG solution into the thalamus and PBN for retrograde tract-tracing
Following an intraperitoneal injection of sodium pentobarbital (40 mg/kg body weight), the anaesthetized rats were placed in a stereotaxic frame (NARISHIGE, Japan). Using a glass micropipette (internal tip diameter: 15–25 μm) that was attached to a 1 μl Hamilton microsyringe, 0.6–0.8 μl of 10% TMR (D-3308, 3000 MW; Molecular Probes, Eugene, OR, USA) dissolved in 0.1 M of citrate-NaOH (pH 3.0) was injected into the right thalamus, and 0.2 μl of 4% FG (80,014, Biotium, Hayward, CA, USA) dissolved in normal saline was injected into the left PBN. After each injection, the glass micropipette was maintained in place for 15 min. All 30 rats injected with TMR and FG were allowed to survive for 7 days. Furthermore, the rats were equally divided into two groups. While lightly anaesthetized with ethyl ether, 0.1 ml of normal saline was injected into the upper lip ipsilateral to the FG injection site of the 15 rats in the first group, whereas the rats in the second group were subcutaneously injected with 0.1 ml of 4% formalin dissolved in normal saline into the upper lip ipsilateral to the FG injection site. The animals subsequently survived for 2 h prior to euthanasia. The results of the tract tracing were obtained from 6 rats in which the tracer was injected properly into the two target areas; the remaining 24 rats were discarded because of inappropriate injection sites.
FISH histochemistry combined with FG and TMR retrograde tract tracing
The riboprobes for VGLUT1 mRNA and VGLUT2 mRNA have previously been described . A cDNA fragment of VGLUT1 (nucleotides 855–1788; GenBank accession number XM_133432.2) or VGLUT2 (nucleotides 848–2044; GenBank accession number NM_080853.2) was cloned into a vector pBluescript II KS (+) (Stratagene, La Jolla, CA, USA). Using the linearized plasmids as templates, we subsequently synthesized the digoxigenin (DIG)-labeled antisense single-strand RNA probes with a DIG RNA labeling kit (Roche Diagnostic, Basel, Switzerland).
Seven days after the injection of FG and TMR, the 15 rats in the first group were re-anaesthetized intraperitoneally with an overdose of sodium pentobarbital (60 mg/kg); the rats were then transcardially perfused with 0.01 M of sodium phosphate-buffered 0.9% (w/v) saline (PBS, pH 7.3), followed by 500 ml of 4% (w/v) paraformaldehyde in 0.1 M of phosphate buffer (PB, pH 7.3). After perfusion, the brains were further postfixed in 4% paraformaldehyde for 3 days at 4 °C. Cryoprotected with 30% (w/v) sucrose in 0.1 M of PB for 2 days, the whole brains were serially cut into 20-μm-thick transverse sections using a freezing microtome (Leica CM1950; Leica, Germany).
The sections were divided into 7 series of alternate serial sections. One series of sections was directly mounted onto clean glass slides and air dried. In these sections, the location and extent of the TMR and FG injection sites, as well as the distribution of TMR- and FG-labeled neurons in the Vsp, were observed with an epifluorescence microscope (BX60; Olympus, Tokyo, Japan) under an appropriate filter for TMR (excitation 540–552 nm; emission 575–625 nm) and FG (excitation 360–370 nm; emission≥395 nm).
An additional three series of the sections were used for FISH combined with immunofluorescence histochemistry. In brief, free-floating sections were treated with 2% H2O2 in 0.1 M of PB for 10 min at room temperature (RT). After rinsing with 0.1 M of PB, the sections were incubated in 0.3% Triton-X100 in 0.1 M of PB at RT for 20 min and 10 min in acetylation solution, which consisted of 0.25% (v/v) acetic anhydride in 0.1 M of triethanolamine. After rinsing for 10 min twice, the sections were pre-hybridized for 1 h at 58 °C in a hybridization buffer, which contained 50% (v/v) formamide, 5 × saline sodium citrate (SSC; 1×SSC = 0.15 M of NaCl and 0.015 M of sodium citrate, pH 7.0), 2% (w/v) blocking reagent (Roche Diagnostics), 0.1% (w/v) N-lauroylsarcosine (NLS) and 0.1% (w/v) sodium dodecyl sulfate (SDS). VGLUT1 or VGLUT2 riboprobes were subsequently added into the hybridization system with a final concentration of 1 μg/ml and hybridized at 58 °C for 20 h. After two washes for 20 min at 55 °C with wash buffer, which contained 2 × SSC, 50% (v/v) formamide and 0.1% (w/v) NLS, the hybridized sections were incubated with 20 μg/ml ribonuclease A for 30 min at 37 °C in a mixture of 10 mM of Tris–HCl (pH 8.0), 1 mM of EDTA and 0.5 M of NaCl, followed by 2 washes for 20 min at 37 °C in 0.2 × SSC that contained 0.1% (w/v) NLS. The sections were subsequently incubated overnight at room temperature with a mixture of 0.5 μg/ml peroxidase-conjugated anti-digoxigenin sheep antibody (11–207–733-910; Roche Diagnostics, Basel, Switzerland), 1 μg/ml guinea pig anti-FG antibody (NM-101, Protos Biotech Corporation, NY, USA) and 1 μg/ml rabbit anti-TMR antibody (A-6397, Invitrogen, Eugene, OR, USA) in 0.1 M of Tris–HCl (pH 7.5)-buffered 0.9% (w/v) saline (TS 7.5) that contained 1% blocking reagent (TSB). To amplify the VGLUT1 or VGLUT2 mRNA hybridization signals, we performed the biotinylated tyramine (BT)-glucose oxidase (GO) amplification method with a reaction mixture that consisted of 1.25 μM of BT, 3 μg/ml GO, 2 mg/ml β-D-glucose, and 1% bovine serum albumin (BSA) in 0.1 M of PB for 30 min. The sections were subsequently treated with a mixture of 10 μg/ml Fluorescein Avidin D (A-2001; Vector, Burlingame, CA, USA), 10 μg/ml Alexa 647-conjugated goat anti-guinea pig IgG antibody (A-21450; Invitrogen) and 10 μg/ml Alexa 594-conjugated donkey anti-rabbit antibody (A-21207; Invitrogen) in TSB for 4 h.
The FISH was also performed using the sense probe (the third and fourth series of sections); however, no hybridization signals were detected in these sections.
CGRP-immunoreactive axonal varicosities in the Vc were detected in apposition to FG- and TMR-labeled neuronal profiles by triple-immunofluorescence histochemistry
The fifth series of the sections through the Vc was incubated overnight with a mixture of 1 μg/ml guinea pig anti-FG antibody (Protos Biotech Corporation), 0.5 μg/ml rabbit anti-TMR antibody(Invitrogen), and 1 μg/ml goat anti-CGRP antibody (ab36001; Abcam) in PBS-XCD. After three washes with PBS, the sections were treated with 10 μg/ml biotinylated donkey anti-goat IgG (AP180B, Millipore, Temecula, CA, USA). The sections were then further incubated overnight with a mixture of 10 μg/ml Alexa 647-conjugated goat anti-guinea pig IgG antibody (Invitrogen), 10 μg/ml Alexa 594-conjugated donkey anti-rabbit IgG antibody (Invitrogen), and 10 μg/ml Fluorescein Avidin D (Vector) in PBS that contained 5% (v/v) normal donkey serum.
The sixth sections, which contained the Vsp of the rats injected with FG and TMR, were used to conduct the control experiments for immunofluorescence histochemistry, in which CGRP antibody was omitted. Under these conditions, no immunoreactivity for the omitted antibody was observed.
Detection of Fos immunoreactivity in Vc neurons labeled with FG and TMR by triple-immunofluorescence histochemistry
The last sections through the Vc of the rats in the first group and the sections through the Vc from the second group injected with FG, TMR, and formalin were also processed for triple-immunofluorescence histochemistry for Fos, TMR and FG. Briefly, the sections were incubated with (1) a mixture of 1 μg/ml mouse anti-Fos antibody (ab 208,942, Abcam, Cambridge, MA, USA), 1 μg/ml guinea pig anti-FG antibody (NM-101, Protos Biotech Corporation) and 0.5 μg/ml rabbit anti-TMR antibody (A-6397, Invitrogen) in PBS that contained 0.3% (v/v) Triton X-100, 0.25% (w/v) λ-carrageenan, and 3% (v/v) donkey serum (PBS-XCD) overnight at room temperature; (2) 10 μg/ml biotinylated donkey anti-mouse IgG (AP192B, Millipore, Temecula, CA, USA); and (3) a mixture of 10 μg/ml Alexa 647-conjugated goat anti-guinea pig IgG antibody (A-21450, Invitrogen), 10 μg/ml Alexa594-conjugated donkey anti-rabbit IgG antibody (A-21207, Invitrogen), and 10 μg/ml Fluorescein Avidin D (A-2001; Vector) in PBS that contained 3% (v/v) normal donkey serum.
Another series of sections from the second group was used to conduct the control experiments for immunofluorescence histochemistry, in which the Fos antibody was omitted. Under these conditions, no immunoreactivity for the omitted antibody was observed.
Immunofluorescence histochemistry combined with biotinylated dextran amine (BDA) anterograde tract tracing
Anterograde tract tracing from the Vsp to the PBN regions was performed using BDA (D1956, Invitrogen) in 15 rats. In each rat, 0.2 μl of 2% (w/v) BDA in distilled water was injected into the Vo (5 rats), Vi (5 rats) or Vc (5 rats). After a period of 5 days, the rats injected with BDA were sacrificed. Similar to the procedure of the FG and TMR injection experiments, the brainstem of the rats was cut into transverse sections in series, and one series of sections through Vo, Vi or Vc was directly incubated with Fluorescein Avidin D to detect the location and extent of the BDA injection site.
The sections through the PBN from the rats that had a proper BDA injection site were subsequently incubated overnight at room temperature with a mixture of 1 μg/ml mouse anti-NeuN antibody (MAB377; Millipore, Billerica, MA) and 2 μg/ml guinea pig anti-VGLUT2 (135,404, Synaptic Systems, Goettingen, German) in PBS that contained PBS-XCD, followed by 6 h at RT with a mixture of 5 μg/ml Fluorescein Avidin D (Vector), 5 μg/ml Alexa 594-conjugated goat anti-guinea pig IgG antibody (A-11076, Invitrogen) and 5 μg/ml Alexa 647-conjugated donkey anti-mouse IgG antibody (A-31571, Invitrogen).
Confocal laser scanning microscopy for the immunofluorescence stained sections
After incubation, all immunofluorescence stained sections were observed under a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan) with appropriate laser beams and filter sets for fluorescein (excitation 488 nm, emission 510–530 nm), TMR and Alexa 594 (excitation 543 nm, emission 590–615 nm) or Alexa 647 (excitation 633 nm, emission 650 nm). We captured the digital images with an FV10-ASW 1.6 from Olympus; following modifications (15–20% contrast enhancement) in Photoshop CS4 (Adobe Systems, San Jose, CA), these images were saved as TIFF files.
Cell counting and statistics
In the 3 rats in which the FG injection into the PBN and TMR injection into the thalamus were successful, the TMR- and FG-labeled cell bodies of neurons that expressed VGLUT1 or VGLUT2 mRNA were identified by FISH combined with retrograde tract tracing. For these counts, in each rat, we selected 15 sections that covered the whole rostral-caudal axis of the Vsp, 5 sections of the Vo, 5 sections of the Vi and 5 sections of the Vc. In the other 3 rats, after the TMR and FG were administered and the formalin was subcutaneously injected into the upper lip ipsilateral to the FG injection site, the TMR- or FG-labeled cell bodies of neurons that expressed Fos immunoreactivity were identified by immunohistochemistry combined with retrograde tract tracing. For these counts, in each rat, 10 sections that covered the whole rostral-caudal axis of the Vc were selected. The target areas (Vo, Vi or Vc) were subsequently photographed with a confocal laser microscope under a 10× objective, and the number of neurons double-labeled with TMR/FG, TMR/VGLUT1 mRNA, TMR/VGLUT2 mRNA, FG/VGLUT1 mRNA, or FG/VGLUT2 mRNA, and the neurons triple-labeled with TMR/FG/VGLUT1, TMR/FG/VGLUT2 or TMR/FG/Fos were counted based on these photographs. In all experiments, only cells with a clear nucleus were counted.
For statistics, the numbers of the counted cells of each rat were initially summed and then averaged among the three rats. All data are presented as the mean ± standard deviation (SD). Student’s t test was used to determine whether the number of Fos immuno-reactive cells in the Vc of the rats after formalin injection was significantly different from that in the rats injected with normal saline. For cell counting in the Vc, the identification of each lamina of the Vc referred to the rat brain atlas by Paxinos  and the book “The Rat Nervous System” by Paxinos . The layers consist of a marginal layer (lamina I) and substantia gelatinosa (lamina II), which together comprise the superficial laminae, and a deeper magnocellular layer (laminae III and IV; a separate lamina III is not obvious in the rat; however, it is typically included in the magnocellular layer).
Triple immune-electron microscopy showed CGRP-immunoreactive terminals in synaptic contact with WGA-HRP- and FG-labeled neuronal profiles in Vc
Six rats were injected with WGA-HRP and FG for electron microscopy. In each rat, 0.2 μl of FG was injected by pressure into the left PBN as previously described. After 4 days, the rats were re-anaesthetized with sodium pentobarbital (40 mg/kg body weight) and stereotaxically injected with 0.6 μl of 1% WGA-HRP (PL-1026, Vector) into the right thalamus of the FG injected rats. The procedures for the stereotaxic microinjection of the WGA-HRP solution were the same as described for FG. After the injection of WGA-HRP, the rats were allowed to survive for 3 days.
The rats were deeply anaesthetized and transcardially perfused with 200 ml of 4% (w/v) paraformaldehyde, 0.1% (w/v) glutaraldehyde, and 15% (v/v) saturated picric acid in 0.1 M of PB. The brainstems were serially cut into 50-μm-thick transverse sections with a Vibratome (Microslicer DTM-1000; Dosaka EM, Kyoto, Japan). The staining of WGA-HRP was processed using tetramethylbenzidine (TMB) with sodium tungstate as a stabilizer , and the WGA-HRP reaction products were further intensified with DAB/cobalt/H2O2 solution. The sections that contained the injection sites were subsequently mounted onto glass slides for the conformation of the injection site. The sections through the Vc from 3 animals with both WGA-HRP and FG injection sites restrained in the target area were selected and incubated in a mixture of 25% (w/v) sucrose and 10% (v/v) glycerol in 0.05 M of PB for 1 h. The sections were transiently frozen and thawed with liquid nitrogen. Following incubation at room temperature with 0.05 M of Tris-HCl-buffered saline (TBS; pH 7.4) that contained 20% (v/v) normal donkey serum for 1 h, the sections were processed for double immunolabeling of FG and CGRP. In brief, the sections were incubated at room temperature overnight with 1 μg/ml rabbit anti-FG antibody (A153-I, Millipore) and 1 μg/ml mouse anti-CGRP antibody (ab81887, Abcam) in TBS that contained 2% (v/v) normal donkey serum (TBS-D). After rinsing with TBS, the sections were incubated in TBS-D with 10 μg/ml 1.4-nm gold-particle-conjugated goat anti-rabbit IgG antibody (2004; Nanoprobes, Stony Brook, NY, USA) and 10 μg/ml biotin-conjugated donkey anti-mouse IgG antibody (Millipore) overnight. The sections were subsequently treated with 1% (w/v) glutaraldehyde in 0.1 M of PB (pH 7.4) for 10 min and rinsed with distilled water. An HQ Silver Kit (2012; Nanoprobes) was subsequently employed to perform silver enhancement. The sections were then incubated with a 1:50-diluted Elite ABC Kit (PK-2101, Vector) in 0.05 M of TBS for 6 h and further treated with 0.02% (w/v) 3,3-diaminobenzidine tetrahydrochloride (DAB; D5637, Sigma, St. Louis, MO, USA) and 0.3% (v/v) H2O2 in 0.05 M of Tris–HCl (pH 7.6) for 30 min. The sections were subsequently incubated in 1% (w/v) OsO4 in 0.1 M of PB (pH 7.4) for 35 min and counterstained in 70% ethanol that contained 1% (w/v) uranyl acetate for 1 h. With dehydration, the sections were mounted onto silicon-coated glass slides, and 1 was embedded in epoxy resin (Durcupan; Fluka, Buchs, Switzerland). Once the resin polymerized, section fragments that contained the superficial layer of the Vc were removed from the resin. The selected tissue fragments were further cut into 60-nm-thick sections using an ultramicrotome (Reichert-Nissei Ultracut S; Leica). The ultrathin sections were then mounted onto single-slot grids coated with pioloform membranes and detected with a JEM-1400 electron microscope (JEM, Tokyo, Japan).