What techniques and methods are used?
Learning and memory assessment in animals is based on behavioral tests. Pharmacological approaches are used to test the effects of neuroprotective drugs on memory deficits. After behavioral testing animals are euthanized for isolation of brain structures used for molecular analysis.
Object recognition and object placement tasks
Object recognition and object placement take place in an open field apparatus (45 x 40 x 60 cm) with sawdust covering its floor. On the first day, rats are submitted to a habituation session during which they are placed in the empty open field for 5 min. On the following day, rats are given one 5-min training trial in which they are exposed to two identical objects (A1 and A2). The objects are positioned in two adjacent corners, 9 cm from the walls. On the long-term memory (LTM) testing trial (24 h after the training session), rats are allowed to explore the open field for 5 min in the presence of two objects: the familiar object A and a novel object B, placed in the same locations as in the training session, for object recognition. For object placement, in the retention test session one of the familiar objects is moved to the southeast or southwest corner of the arena. All objects are made of plastic Duplo Lego Toys and have a height of about 10 cm. For object recognition, objects present similar textures, colors, and sizes, but distinctive shapes. Different objects are used in object recognition and object placement. Between trials the objects are washed with 10% ethanol solution. Trials are videotaped and object exploration is measured by an experimenter blind to group treatment assignments, using two stopwatches to record the time spent exploring the objects. Exploration is defined as follows: sniffing or touching the object with the nose or forepaws while sniffing. Sitting on the object is not considered as exploration. A recognition index calculated for each animal is expressed by the ratio TN/(TF+TN) [TF= time spent exploring the familiar object (A) or the object placed on an unchanged location; TN= time spent exploring the novel object (B) or object moved to a new location]. The object recognition task is performed as previously described (Silva et al., 2012; Figueiredo et al., 2016).
Behavior during habituation to the open field prior to object recognition training is evaluated, as previously described (Figueiredo et al., 2016). The open field is a 40 X 45 X 60 cm arena surrounded by 50 cm high walls, made of plywood with a frontal glass wall. The floor of the arena is divided into 12 equal squares by black lines. Animals are placed in the rear left corner and left to explore the field freely for 5 min. Latency to start locomotion and line crossings, are registered. The number of crossings and the number of rears are used as measures of locomotor and exploratory activity, whereas the latency to start locomotion is used as a measure of anxiety.
Inhibitory Avoidance Task
We use the single-trial, step-down inhibitory avoidance (IA) conditioning as an established model of fear-motivated memory. In IA training, animals learn to associate a location in the training apparatus with an aversive stimulus (footshock). The IA behavioral training and retention test procedures are described in previous reports (Schröder et al., 2001; Silva et al., 2012; Figueiredo et al., 2016). The IA apparatus is a 50x25x25-cm3 acrylic box (Albarsch, Porto Alegre, Brazil) whose floor consist of parallel caliber stainless steel bars (1-mm diameter) spaced 1 cm apart. A 7-cm wide, 2.5-cm high platform is placed on the floor of the box against the left wall. On the training trial, rats are placed on the platform and their latency to step-down on the grid with all four paws is measured with an automatic device. Immediately after stepping down on the grid, rats receive a mild footshock (0.4 mA) and are removed from the apparatus immediately afterwards. A retention test trial is carried out 24 h after the training trial. The retention test trial is procedurally identical to training, except that no footshock is presented. Step-down latencies (in seconds) on the retention test trial (maximum 180 s) are used as a measure of IA retention.
Western Blot Analysis
Hippocampi obtained from rats are homogenized in 0.2 mL ice-cold lysis buffer (50 mM Tris, pH 7.5, 50 mM NaCl, 5 mM ethylene glycol tetraacetic acid, 5 mM Ethylenediaminetetraacetic acid, 2 mM SodiumPyrophosphate, 4 mM Para-Nitrophenylphosphate, 1 mM Na3VO4, 1.1 mM Phenyl-methyl-sulphonyl fluoride, 20 µg/µL leupeptin, 50 µg/µL aprotinin, protease inhibitor cocktail, 0.1% SDS) using a pestle, sonicated briefly, and centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant is collected and protein concentration is determined using Bradford assay (Bradford, 1976). Aliquots are stored at - 20 °C.
Samples are diluted in a mix of lysis buffer and loading buffer 2× (50 mM Tris, pH 6.8, β-mercaptoethanol, 10% glycerol, 1% bromophenol blue, and 2% sodium dodecyl sulphate (SDS)) and boiled for 10 minutes at 95°C. Forty µg of protein are separated on a 10% SDS polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane.
Membranes are blocked in Tris-buffered saline, pH 7.6, containing 0.1% of Tween 20 (TBST) and 5% skimmed milk for 2 hours at room temperature and then incubated overnight, with the following antibodies: anti-β-actin (1:2500, Abcam, Cambridge, UK), anti-caspase 3 (1:1000, Abcam, Cambridge, UK), anti-GluA1 (1:2000, Abcam, Cambridge, UK), anti-GluA2 (1:500, Abcam, Cambridge, UK), anti-pS845 GluA1 (1:500, PhosphoSolutions, Aurora), anti-pS880 GluA2 (1:500, PhosphoSolutions, Aurora) e anti-NCS-1 (Abcam, Cambridge, UK), which are dissolved in TBS-T with 5% bovine serum albumin. Membranes are then washed 3 times with TBS-T and incubated for 120 minutes at room temperature in TBS-T with 1% skimmed milk containing anti-rabbit IgG H&L (HPR) (Abcam, Cambridge, UK) secondary antibody, detected using ECL Western Blotting Substrate Kit (Abcam, Cambridge, UK). Pre-stained molecular weight protein markers (SuperSignal Molecular Weight Protein Ladder, Thermo Scientific, Rockford, USA) are used to determine the detected bands molecular weight and confirm antibodies target specificity. The densitometric quantification will be performed using Chemiluminescent photo finder (Kodak/Carestream, model GL2200).
Data from recognition indexes, total time exploring both objects in the training session, latencies to step-down and data from the experiments evaluating open field behavior, as well western blot are expressed as mean ± standard deviation (S.D.). Comparisons among experimental groups will be performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc tests when necessary, using GraphPad Prism® software version 5.01. In all comparisons, p values less than 0.05 were considered to indicate statistical significance.