Blandini F, Balestra B, Levandis G, Cervio M, Greco R, Tassorelli C, Colucci M, Faniglione M, Bazzini E, Nappi G, Clavenzani P, Vigneri S, De Giorgio R, Tonini M. Functional and neurochemical changes of the gastrointestinal tract inside a rodent model of Parkinson’s disease. DVC neurons but an increase in dopamine–hydroxylase-IR in the A2 area. Within the myenteric plexus of the esophagus, belly, and duodenum, there were no changes in the total quantity of neurons; however, the percentage of NOS-IR neurons improved, whereas that of ChAT-IR decreased. Our data suggest that the delayed gastric emptying inside a 6-OHDA rat model of PD may be caused by neurochemical and neurophysiological alterations in the brain-gut axis. = 5) received a microinjection of PBS in the DVC, and these microinjections per se did not vary either gastric firmness or motility index (observe below). The catecholaminergic A2 area plays a relevant part in the modulation of vago-vagal reflexes (46, 54). To assess the effects of endogenous catecholamines, the indirect sympatomimetic tyramine (4.5 nmol/60 nl) was microinjected in the DVC at (in mm): 0.2C0.3 RC from calamus scriptorius, 0.1C0.3 ML from midline, and ?0.5 DV from your Peficitinib (ASP015K, JNJ-54781532) brainstem surface. To assess the effects of direct activation of vagal efferent motoneurons, thyrotropin-releasing hormone (TRH, 1C100 pmol/60 nl) was injected in the DVC because it is well recognized that TRH effects are mediated by vagal efferent materials (34, 50, 53). Thirty minutes after the 1st tyramine microinjection, either a combination of 1- and the 2-adrenergic receptor antagonists, prazosin (100 pmol/2 l) and yohimbine (500 pmol/2 l), or a combination of D1 and D2 dopamine receptor antagonists, SCH 23390 (45 nmol/2 l) and L-741,626 (45 nmol/2 l), were applied to the surface of the fourth ventricle at the level of obex, adopted two 5 min later on by a second tyramine microinjection; all drugs were dissolved in PBS. The strain Peficitinib (ASP015K, JNJ-54781532) gauge output was monitored for any changes for at least 10 min following drug infusion. Gastric motility was determined using the following formula, as explained previously (8): Motility index percent = [(equals the number of peaks in a particular pressure range, and equals the interval time in which the gastric motility was measured. Presuming that a 0-mV transmission is definitely indicative of no gastric motility, the grouping of peak-to-peak sinusoidal signals reflected 25C50 mg for = 5) received a microinjection of PBS in the DVC; these microinjections per se did not vary either gastric firmness or motility index. The results are displayed as the means SE. Data were evaluated within each group by ANOVA and the appropriate 0.05. Immunohistochemistry Control Tissue preparation. Immunohistochemical analyses were carried out on rats 5 wk after 6-OHDA or vehicle microinjections into SNpc. At the conclusion of measurements of corpus firmness and motility experiments, rats were perfused transcardially with heparinized saline followed by paraformaldehyde fixative (4% PFA in PBS). Brains were eliminated and postfixed for 4 days at room heat with 4% PFA comprising 20% sucrose before becoming transferred to a solution comprising PBS and 20% sucrose at 4C for at least 1 day. The entire rostrocaudal extension of SNpc at the level of the midbrain and of nucleus ambiguus (NAmb) and DVC at the level of brainstem were sliced, using a microtome, into four series of 50-m transverse sections and maintained in long-term storage buffer (Phosphate buffer 0.1 M, sucrose 30%, RYBP ethylene glycol 30%). Segments of the GI tract comprising the esophagus, belly, and duodenum were extracted before PFA perfusion and immersed in PBS. Cells were opened along the mesenteric border, washed, and pinned under pressure to the bottom of silicon-coated dishes. Specimens were fixed 1C2 days in 4% PFA at 4C, washed in PBS, and stored in PBS + 0.05% sodium azide until used, generally within 2C5 days. Specimens of esophagus, fundus, and corpus Peficitinib (ASP015K, JNJ-54781532) of the belly and duodenum were then processed as longitudinal muscle-myenteric plexus whole-mount preparation by peeling aside the mucosa, submucosa, and circular muscle. All methods were performed at space temperature on a shaker. The primary antibodies used [mouse anti-tyrosine hydroxylase (TH), 1:10,000 dilution for brainstem and SNpc, 1:500 for Peficitinib (ASP015K, JNJ-54781532) myenteric plexus (Immunostar, Hudson, WI); mouse anti-dopamine–hydroxylase (DH), 1:30,000 dilution (Millipore, Bedford, MA); mouse anti-neuronal nitric oxide synthase (nNOS), 1:3,000 dilution for brainstem, 1:500 for myenteric plexus (Sigma); goat anti-choline acetyl transferase (ChAT), 1:5,000 dilution for brainstem, 1:250 for myenteric plexus (Millipore); and rabbit anti-protein gene product (PGP9.5), 1:5,000 dilution (Millipore)] and their dilutions were determined by titration in cells fixed and processed in the same ways.