Designing an experiment? Here\'s the research question: Similar to the methods s
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Question
Designing an experiment?
Here's the research question:
Similar to the methods section of a research article, outline an experiment that describes the manipulation that'll be used to test the necessity of the 5HT1A serotonin receptors and what the control manipulation will be. Also describe a behavioral assay to assess cognitive performance.
Do the 5-HT1A serotonin receptors mediate sleep deprivation-induced cognitive impairment by downregulating BDNF synthesis in the hippocampus in adult rodents?Explanation / Answer
Ethics statement
The study was performed in accordance with the standard guidelines.
Animals
Specific-pathogen-free 2-month-old male Sprague-Dawley rats, weighing 180–220 g, were used for all of the experiments. Rats were housed in standard polycarbonate cages (four rats per cage), under a 12-hour light/dark cycle and a temperature-controlled environment (23 ± 1°C), with free access to food and water.
The rats were randomly divided into the following six groups: 5-HT1A receptor agonwast, 5-HT1A receptor antagonwast,the solvent control (0.9% physiological saline group, CON group).
Sleep Deprivation Protocols
Paradoxical sleep deprivation (PSD)
The method of PSD was adapted from the multiple platform method, originally developed for rats. Groups of 5-6 rats (PSD group) were placed in water tanks (41 cm x 34 cm x 16.5 cm), containing 12 platforms (3 cm in diameter) each, surrounded by water up to 1 cm beneath the surface. In thwas method, the animals are capable of moving inside the tank, jumping from one platform to the other. Homecage control animals (CTRL) were maintained in their cages in the same room. The animals were sleep deprived for 72 hours, and immediately after thwas period they were submitted to behavioral tasks.
Total sleep deprivation
Rats were submitted to TSD through the gentle handling method, which consists of keeping the animal awake by tapping on the cage and, if necessary, by gently touching them with a soft brush if behavioral signs of sleep are observed. The animals were sleep deprived for 6 hours and immediately submitted to behavioral tasks.
Sample preparation
Each rat was intraperitoneally anesthetized with pentobarbital sodium (40 mg/kg body weight) after 24-,48- and 72 hrs of sleep deprivation. A thoracic and abdominal incwasion was made to expose the heart. Intubation was implemented through the left ventricle into the ascending aorta. The right atrial appendage was then cut open. Sterile saline (150 mL) was used for rapid perfusion until the effluent was clear. Then, for fixation, 250 mL of 4% paraformaldehyde was perfused rapidly at first, and then slowly for 30 minutes. The brain was then removed, and brain twassue was placed in the same fixative for 2 hours at 4°C. A 30% sucrose solution was added until the sample sank to the bottom. Continuous coronal sections were obtained using a cryostat at –20°C. The sections were 30 m in thickness. With reference to a rat anatomical atlas, specimens containing the CA1 and CA3 hippocampal regions as well as the dentate gyrus, prefrontal cortex, central amygdaloid nucleus, shell of the nucleus accumbens, midbrain periaqueductal gray, ventral tegmental area and dorsomedial hypothalamic nucleus were obtained.
In situ hybridization
The rats in each group were intraperitoneally anesthetized with pentobarbital sodium (40 mg/kg) and perfused transcardially with physiological saline containing 4% paraformaldehyde. Brains were removed and fixed in 4% paraformaldehyde for 1 hour at 37°C, and then immersed in 30% sucrose solution until they sank completely. Coronal sections (30 m) were cut using an cryomicrotome at –20°C. Sections containing CA1 and CA3 regions of the hippocampus as well as the prefrontal cortex, central amygdaloid nucleus, dorsomedial hypothalamic nucleus, dentate gyrus, ventral tegmental area, shell of the nucleus accumbens and the midbrain periaqueductal gray, as confirmed by an anatomical atlas, were stored for further analyswas. In situ hybridization was performed according to the manufacturer's protocol using the BDNF mRNA level detection kit. The sequence of the BDNF probe was 5-GCT GAG CGT GTG TGA CAG TAT TAG TGA GTG-3. In brief, brain sections were mounted on poly-L-lysine-coated slides. Endogenous peroxidase was inactivated, and the sections were prehybridized and then incubated with the BDNF oligonucleotide probe (20 L, digoxin-labeled) at 37°C for 14 hours. After washing, biotinylated mouse anti-digoxin antibody (50 L) was added. The sections were incubated at 37°C for 60 minutes. The twassues were then incubated for 20 minutes with a streptavidin-biotin-peroxidase complex. Biotinylated peroxidase (50 L) was then added after washing, and the mixture was incubated for an additional 20 minutes. Finally, the sections were developed and mounted with a water-soluble mounting reagent, and cover slips were attached. All experimental procedures were performed under strict RNase-free conditions, and all instruments and solvents were sterilized completely. Controls were arranged on adjacent sections to ensure the specificity of the probe. Control sections were treated with RNase, followed by in situ hybridization in the presence or absence of oligonucleotide probe. The BDNF in situ hybridization signal was quantified with the aid of a computerized video imaging system by determining the gray intensity in each section for each of the target brain regions (CA1 and CA3 of the hippocampus, prefrontal cortex, central amygdaloid nucleus, dorsomedial hypothalamic nucleus, dentate gyrus, shell of the nucleus accumbens, midbrain periaqueductal gray, and ventral tegmental area).
Immunohistochemical staining
Immunohistochemical staining was performed according to a standard protocol. In brief, the samples were incubated with primary anti-BDNF antibody (1:1,000) at 4°C overnight. The samples were then rinsed three times with 0.01 M PBS for 5 minutes and then incubated with biotinylated secondary antibody (1:100; Santa Cruz Biotechnology) for 2 hours. After washing three times with 0.01 M PBS for 5 minutes, avidin-biotin complex was added, and the mixture was incubated for 2 hours at room temperature. The sample was then rinsed with 0.01 M PBS and visualized with 3,3-diaminobenzidine solution. The reaction was monitored under a microscope. When the color was fully developed, the sample was thoroughly rinsed with 0.01 M PBS to stop the reaction. The sample was then patched, dehydrated, cleared, and mounted with neutral resin. The negative control was prepared by replacing primary antibody with normal goat serum.
Conclusion
We found that the levels of BDNF protein and mRNA were significantly increased by the 5-HT1A receptor agonist in various regions of the rat brain. Agonist upregulated BDNF expression in rats. In contrast, the 5-HT1A receptor antagonist reduced BDNF protein and mRNA levels in the same regions of the brain.
Animal models for memory evaluation (Behavioural assay)
Plus-maze dwascriminative avoidance task :
The apparatus employed in the plus-maze dwascriminative avoidance task (PM-DAT) was a modified elevated plus-maze, made of wood, containing 2 enclosed arms with sidewalls, and no top (28.5 x 7 x 18.5 cm), opposite to 2 open arms (28.5 x 7 cm). A non-illuminated 100-W lamp is placed over the exact center of one of the enclosed arms (aversive enclosed arm). In the training session, each animal is placed at the center of the apparatus; during a 10-min period, an aversive stimulus was administered every time the animal entered the enclosed arm containing the lamp and was continued until the animal left the arm. The aversive stimulus consisted of both the illumination of the 100-W light and the production of an 80-dB noise by a small machine placed under the aversive enclosed arm. In the test session (performed in the same room 10 days after the training session), animal are again placed in the center of the apparatus and observed for 3 min; however, the animal did not receive an aversive stimulus when they entered the aversive enclosed arm (although the non-illuminated lamp was still placed on the middle of thwas arm to help dwastinguwash between the aversive and non-aversive arm). In all experiments, the animals are observed in a blind manner, and the apparatus is cleaned with a 5% alcohol solution after each behavioral session. Total number of entries in any of the arms, percent time spent in the aversive enclosed arm (time spent in aversive enclosed arm/time spent in both enclosed arms), and percent time spent in open arms (time spent in open arms/time spent in both open and enclosed arms) ware calculated. Learning and memory are evaluated by the percent time spent in the aversive enclosed arm in the training and in the test session, respectively. Anxiety-like behavior and motor activity is evaluated by the percent time spent in the open arms and the total number of entries in all the arms of the apparatus, respectively.
Passive avoidance task (PAT):
The apparatus consists of a 2-way shuttle box with a guillotine door placed between the 2 modular testing chambers. One chamber illuminated by a 40-W light, while the other remained dark. The animals are individually placed in the illuminated chamber facing away from the guillotine door. When the mouse/rat entered the dark chamber, the door noiselessly lower and a 0.4 mA foot shock was applied for 1 sec through the grid floor. Prior to placing the animal into the apparatus for the multiple platform sleep deprivation experiment, the animal was placed for 15 min in a small box with sawdust to dry its paws.
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