Federal University of Rio Grande do Sul (UFRGS)
Department of Physiology - Universidade Federal do Rio Grande do Sul Rua Sarmento Leite, 500 – ZIP CODE 90050-170 Porto Alegre, RS, Brazil.
Dr Anapaula Sommer Vinagre
Dr Nadja Schröder
English, Spanish, Portuguese
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
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Type of Research Project
- Basic science
What is the background of the project?
Neurodegenerative disorders selectively affect different neuronal populations, and are characterized by an insidious onset and chronic progression. Age-related iron accumulation in brain regions can be viewed as an important factor underlying neurodegeneration, since its accumulation leads to oxidative and mitochondrial damages and persistent changes in learning and memory. The cognitive dysfunction induced by iron overload during the neonatal period in rats has been used in our laboratory as an animal model that reproduces an important feature of neurodegenerative diseases – memory impairment. This model of memory dysfunction has been characterized in rats and has been used to investigate the effects of novel drugs for the treatment of memory dysfunctions associated to aging and neurodegenerative processes. THIS PROJECT IS BOTH 4 OR 8 WEEK, THE STUDENT CAN CHOOSE HOW LONG HE/SHE WANTS TO STAY.
What is the aim of the project?
The present research project aims to investigate the neuroprotective and memory-ameliorating effects of perampanel (PER), a recently developed AMPA receptor antagonist, in rats submitted to iron overload. Specific aims are: To evaluate the effects of subchronic (21 days) PER on memory deficits induced by iron overload, using memory tasks described below; To evaluate the effects of subchronic (21 days) PER on AMPA receptor subunits GLUA1 and GLUA2 expression and phosphorylation, as well as expression of proteins involved in AMPA receptor signaling, using western blot.
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. Behavioral procedures 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). Open-field behavior 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). Statistical Analysis 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.
What is the role of the student?
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Students with interest in basic research and neuroscience. During his/her internship in our laboratory, he/she will be invited to join our weekly meetings for discussion of scientific papers as well will take part in the experimental procedures mentioned above (animal manipulation, behavioral tasks, pharmacological administration, euthanasia and brain dissection, sample preparation for molecular analysis). It is important to note that all the students will be always under supervision of master/doctoral students, laboratory technicians, or faculty professors. Our experiments are based in animal models, so it is important that the student feels comfortable working with laboratory rats. No previous experience is requested. All the projects are approved by the local ethics committee in research and animal use (CEUA/UFRGS).
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes. Preliminary readings will be required and a comprehensive explanation of the line of research will be provided by the tutor. The student will also be invited to participate in weekly lab meetings.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- No specific outcome is expected
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Personal skills such as interest, creativity, empathy, and good team spirit are welcome. Our experiments are based in animal models, so it is important that the student feels comfortable with animal experimentation. No previous experience is requested. Subjects passed: Neuroscience or Neurophysiology
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts:
- Medical students
- Pre-Medical students from the American-British system
- Students in biomedical fields
- Schröder; N; Figueiredo; L.S.; de Lima; MNM. (2013) Role of brain iron accumulation in cognitive dysfunction: Evidence from animal models and human studies. Journal of Alzheimer's Disease; 34: 797-812
- da Silva VK; de Freitas BS; da Silva Dornelles A; Nery LR; Falavigna L; Ferreira RD; Bogo MR; Hallak JE; Zuardi AW; Crippa JA; Schröder N. (2014) Cannabidiol normalizes caspase 3; synaptophysin; and mitochondrial fission protein DNM1L expression levels in rats with brain iron overload: implications for neuroprotection. Molecular Neurobiology; 49(1):222-33.
- Figueiredo LS; de Freitas BS; Garcia VA; Dargél VA; Köbe LM; Kist LW; Bogo MR; Schröder N. (2016) Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory. Molecular Neurobiology 53(9):6228-6239. doi: 10.1007/s12035-015-9514-6.
- da Silva VK; de Freitas BS; Garcia RCL; Monteiro RT; Hallak JE; Zuardi AW; Crippa JAS; Schröder N. (2018) Antiapoptotic effects of cannabidiol in an experimental model of cognitive decline induced by brain iron overload. Translational Psychiatry. 8(1):176. doi: 10.1038/s41398-018-0232-5.
- Alcalde LA; de Freitas BS; Machado GDB; de Freitas Crivelaro PC; Dornelles VC; Gus H; Monteiro RT; Kist LW; Bogo MR; Schröder N. (2018) Iron chelator deferiprone rescues memory deficits; hippocampal BDNF levels and antioxidant defenses in an experimental model of memory impairment. Biometals. Aug 16. doi: 10.1007/s10534-018-0135-1.
- da Silva VK; de Freitas BS; Dornelles VC; Kist LW; Bogo MR; Silva MC; Streck EL; Hallak JE; Zuardi AW; Crippa JAS; Schröder N. (2018) Novel insights into mitochondrial molecular targets of iron-induced neurodegeneration: Reversal by cannabidiol. Brain Research Bulletin 139:1-8. doi: 10.1016/j.brainresbull.2018.01.014