Abstract
Keywords
INTRODUCTION
Gastroparesis and constipation are the most common non-motor system symptoms of Parkinson’s disease (PD) and result from gastrointestinal movement disorder [1]. The dorsal motor nucleus of the vagus (DMV) plays an important role in the regulation of gastrointestinal function, including gastric motility [2, 3].
Idiopathic PD manifests dopaminergic neuron degeneration in the substantia nigra (SN) and is always accompanied by extensive extra-nigral pathology, including in the DMV [4–6]. There are many tyrosine hydroxylase (TH) immunoreactive (IR) fibers surrounding DMV neurons [7, 8], which are dopaminergic [9, 10]. Dopamine receptors (D1 and D2) are extensively expressed in DMV neurons [8], and rats that were subjected to bilateral SN destruction (PD rats) manifested a typical gastroparesis accompanied with increased catecholaminergic and decreased cholinergic expression in the DMV [7]. In addition, activating D1 or D2 in the DMV can hyperpolarize or depolarize the membrane potential of DMV neurons innervating the stomach [11]. Therefore, we hypothesize that the destruction of bilateral SN may affect dopamine receptor expression in DMV neurons innervating the stomach, decreasing acetylcholine (ACh) release from cholinergic neurons in the DMV and leading to impaired gastric motor function via the vagus nerve. However, it is unclear whether DMV neurons innervating the stomach express dopamine receptors (D1 and D2) and whether their expression is altered in the DMV of PD rats.
To address our questions, the retrograde tracer Fluoro-Gold (FG) was injected into the gastric muscular layer of rats and sequential double immunofluorescent labeling procedures were used to detect the relationship between D1/D2 and gastric-projecting vagal neurons. The PD rat model (6-OHDA rats) was established by bilaterally microinjecting 6-hydroxydopamine (6-OHDA) into the SN, and alterations in dopamine receptor expression were evaluated using western blot.
MATERIALS AND METHODS
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.
RESULTS
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).
DISCUSSION
The present study demonstrated for the first time that nearly all gastric-projecting neurons in the DMV express D1 or D2 and that the destruction of SN dopaminergic neurons in 6-OHDA rat leads to a D1 decrease and D2 increase in the DMV, which may contribute to the formation of impaired gastric motor function in PD patients.
Using electrophysiological techniques, Zheng et al. reported that 28% of gastric-projecting neurons in the DMV are depolarized by D1-like selective agonists, whereas 43% of gastric-projecting neurons are hyperpolarized by D2-like receptor selective agonists; this activity was blocked by selective antagonists of D1- and D2-like receptors, respectively [11], indicating that dopamine receptors play an important role in the activity of DMV motoneurons that innervate gastric muscle. Although our previous study demonstrated that motoneurons (ChAT-IR) in the DMV colocalized with D1/D2 [8], direct morphological evidence of the distribution of dopamine receptors in gastric-projecting DMV motoneurons is still lacking. Our present study demonstrates colocalization of gastric-projecting neurons with D1 and/or D2 using a neural tracing technique combined with double immunofluorescence. Our results suggest that dopamine receptors might affect the activity of gastric-projecting neurons in the DMV.
Dopamine receptors have been divided into two families, D1-like (D1 and D5) and D2-like (D2, D3 and D4). D1-like receptors increase cellular cAMP by activating adenylyl cyclase, and D2-like receptors decrease cellular cAMP by inhibiting adenylyl cyclase. D1 and D2 are generally considered representative of D1-like and D2-like receptors [16]. Behavioral and electrophysiological studies have also observed cooperative effects of D1 and D2 in dopamine target areas [17, 18], especially in the basal ganglion [19]. In the present study, both D1- and D2-IR neurons projected to the stomach and a cooperative effect on the neurons there is theoretically possible. Moreover, 6-OHDA PD rats showed decreased D1 and enhanced D2 expression in the DMV. In addition, increased TH and decreased ChAT in the DMV was observed in 6-OHDA rats in our previous study [7]. Therefore, it is possible that the decreased expression of D1 and increased expression of D2 might play a role in the expression alteration of TH and ChAT in PD rats.
The altered expression of dopamine receptors in the DMV of 6-OHDA rats might influence the function of gastric-projecting neurons innervating gastric smooth muscle and this adjustment may be derived from the nuclei bridging the DMV and the SN. Using retrograde and anterograde neural tracing techniques, our group demonstrated that neurons in the SN do not directly innervate the DMV in rats. However, the DMV might be indirectly modulated by the SN through the lateral hypothalamus (LH), paraventricular nucleus (PVN) and/or the locus coeruleus (LC) [20]. Therefore, identification of the role of LH/PVN/LC on impaired gastric motor function in PD patients will be the focus of our next study.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.
Footnotes
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (31200897, ZY Wang; 31400991, LF Zheng; 81370482, JX Zhu) and the Doctor Scientific Research Foundation of Xinxiang Medical University (XXBSKYZZ201501, ZY Wang).