Altered Expression of D 1 and D 2 Dopamine Receptors in Vagal Neurons Innervating the Gastric Muscularis Externa in a Parkinson’s Disease Rat Model

Animals

Adult male Sprague-Dawley rats weighting 250-300 g were used for this study. All surgical procedures were carried out under chloral hydrate (i.p., 0.1–0.15 g/kg). All procedures were approved by the Animal Care and Use Committee of Capital Medical University (Beijing, China).

Retrograde tracing

After food and water were withheld overnight, the abdominal wall of the anesthetized rats was opened and the liver was pushed aside to expose the stomach. Five shots of 1μl of 4% retrograde tracer Fluoro-Gold (FG) (dissolved in distilled water at pH 7.4) were microinjected into the anterior wall of the body of the stomach. Cotton swabs were used to prevent the tracer from spreading to adjacent structures during the injection process. For control experiments, 10μl of 4% FG was poured into the abdominal cavity [12]. One week later, the DMV was examined for the presence of tracer.

Preparation of rat brains

The anesthetized rats were perfused transcardially. Following a brief rinse with 0.9% saline, 4% paraformaldehyde in 0.01 M phosphate buffer solution (PBS) at pH 7.4 was perfused for 30 min at room temperature. The brain was removed immediately and placed into the same fixative overnight at 4°C and then immersed in 30% sucrose overnight at 4°C. After dehydration with 30% sucrose, serial coronal sections were cut at a thickness of 20μm using a cryostat (Leica CM1850).

Nissl staining

The method has been described elsewhere [9]. Briefly, serial frontal sections of the DMV were stained with 0.5% cresyl violet, dehydrated using graded alcohols, and then immersed in xylene and coverslipped.

Observation of FG-labeled neurons

FG-labeled neurons in the DMV were observed directly through a fluorescence microscope (Nikon e80i, Japan), and photomicrographs were obtained using a Leica Image acquisition system (Leica, Germany).

Immunofluorescence staining

The brain slices were permeabilized with PBS containing 0.3% Triton X-100, then blocked for unspecific binding with 5% goat serum for 30 min. The sections were incubated overnight in a mixture of two primary antibodies derived from different host species for 16 h at 4°C (ChAT/D₁, ChAT/D₂, TH/D₁ and TH/D₂, see Table 1) and were then incubated with the secondary antibodies for 1 h at room temperature. DAPI was used to counterstain the cellular nuclei. Images were taken using the same equipment as well as the observation of FG-labeled neurons.

6-OHDA PD model

The methods used have been previously described [13, 14]. Briefly, anesthetized rats were placed on a Kopf stereotaxic instrument. Two small areas of the skull were exposed (coordinates: AP, –5.6 mm; ML, ±2.0 mm; DV, –7.5 mm) and 6-OHDA (4μg in 2μl of 0.9% saline containing 0.05% ascorbic acid) was injected using a 10μl Hamilton syringe. 0.2% ascorbic acid/saline was injected in the control group. Six weeks later, the 6-OHDA rats showed relatively uniform nigrostriatal lesions.

Western blotting

As in our previous reports [15], the dorsal medulla, including the DMV, was collected on ice from the brains of control and 6-OHDA rats. Tissues were homogenized in cold lysis buffer supplemented with protease inhibitors for protein extraction. Proteins (100μg) were separated using a 10% SDS-PAGE gel and transferred onto a nitrocellulose membrane. After blocking with 5% skim milk in PBS for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against D1, D2 and GAPDH, followed by incubation in appropriate secondary antibodies for 1 h at room temperature. The membranes were then washed in PBS. Finally, the membranes were scanned using an Odyssey Infrared Imager (LI-COR, Nebraska USA) and analyzed using Odyssey software (version 1.2).

Statistical analysis

Results are presented as the mean±SD. Statistical analyses were performed using unpaired Student’s t tests. Differences were considered significant at p < 0.05.

Distribution of FG-labeled gastric-projecting neurons in the DMV

Nissl staining showed that the DMV is located in the medulla oblongata. In the caudal region of medulla (Fig. 1A), the DMV was distributed bilaterally to the central canal (CC), oriented from medial to lateral. In the rostral region (Fig. 1B), the CC was open and became the fourth ventricle (4V). The DMV was located on both sides of the lateral wall of 4V, from mediosuperior to lateroinferior. Compared to the hypoglossal nucleus (HN) and the nucleus of the solitary tract (NTS), the DMV was located in the dorsomedial region of the medulla, dorsolateral to HN and ventromedial to NTS.

After injection of FG into the anterior wall of the gastric corpus (Fig. 1C, D), many retrogradely FG-labeled gastric-projecting neurons were observed throughout the DMV from the caudal region (Fig. 1E, F) to rostral region (Fig. 1G, H). FG-labeled neurons were predominantly distributed in the left side of the dorsal medulla (Fig. 1E, G), which was consistent with our previous study [12].

Colocalization of FG-labeled gastric-projecting neurons with D1/D2 and TH/ChAT in the DMV

After FG-labeled gastric-projecting neurons in the DMV were identified in successfully prepared brain slices, the same FG-labeled sections were further used to determine whether the FG-labeled neurons expressed ChAT/D₁, ChAT/D₂, TH/D₁ and TH/D₂ by means of double immunofluorescence staining. The results showed that the FG-labeled neurons displayed immunoreactivity (IR) for D₁, D₂, TH and ChAT. Nearly all the FG-labeled neurons were D₁- and ChAT-IR, whereas not all the D₁- and ChAT-IR neurons were FG positive (Fig. 2A). Similar results were observed in the colocalization of D₂ with ChAT in FG-labeled neurons (Fig. 2B). All the FG-labeled neurons were D₁-IR, but only a few of FG-labeled neurons were TH-IR (Fig. 2C). Similar results were found in the colocalization of D₂ with TH in FG-labeled neurons (Fig. 2D).

Increased D₂ and decreased D₁ expression in the DMV of 6-OHDA rats

As shown in Fig. 3A and C, a large number of D₁-IR or D₂-IR neurons were surrounded by a large number of TH-IR fibers in the DMV, while only a few TH-IR neurons colocalized with D₁ or D₂. In 6-OHDA rats, the number and intensity of D₁-IR neurons in the DMV was significantly decreased (Fig. 3Bb). In contrast, the number and intensity of D₂-IR neurons in the DMV was visibly increased in 6-OHDA rats (Fig. 3Db). Meanwhile, increased TH-IR expression was observed in 6-OHDA rats, consistent with our previous study [7].

Altered expression of D₁ and D₂ in the dorsal medulla of 6-OHDA rats was detected by western blot (Fig. 3E, F). The results showed a significant decrease in the level of D₁ from 0.55±0.06 to 0.22±0.07 (n = 6, p < 0.01) but a significant increase in the level of D₂ from 0.28±0.04 to 0.34±0.04 (n = 6, p < 0.01).