Novel object recognition study; methods and important information

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Novel object recognition study; methods and important information:

Method:

Animals used totalled 17 male and 13 female, adult Wistar rats, housed in groups of 4 and supplied by Central Biomedical Services, University of Leeds. The weight range was approximately 250-350 g. Room parameters were temp: 21 °C ± 2, humidity: 30 % ± 2, lights on a 12 hour daily cycle, lights on at 8 am.

All animals were habituated, individually, to the enclosed test arena (black Plexiglass circular arena 50 cm diameter, 40 cm high) for 2 minutes each over two days. On the third day the ‘training phase’ commenced which consisted of rats placed individually in the arena with two identical objects (golf ball tower) for 6 minutes. On day 4, the ‘testing phase’, the animals were recorded for 2 minutes with a familiar object (golf ball tower) and a novel object (sealed green plastic drinks bottle). All objects were secured to the bottom of the arena to avoid movement upon any investigation of them and in the centre of a 10 cm diameter circle marked upon the floor of the arena). Each object was equidistant from each other and the walls of the arena.

All activity was recording using a webcam (Netgear WC) connected to a laptop to capture the video with the webcam connected positioned directly above the arena using a tall adjustable tripod (Manfrotto tripod, MF600). All recordings of animals in the testing phase were subsequently scored according to the following ethogram using a behavioural scoring package (Hindsight, Scott Weiss, University of Leeds):

Table 1. Novel object recognition test ethogram/scoring sheet.

Frequency counts were made of each of the behaviours i.e. how many times each were observed to occur along with time of interaction with the presented objects. Investigation of the objects was determined by physical touching and sniffing of the object or an approach towards the object with the nose orientated towards it to within the circle marked upon the arena floor.

All objects and the arena were wiped with 70% ethanol between each animal and faecal boli were removed. The order of animals tested was randomly assigned to avoid any order effects.

Data analysis:

First thing you need to do is calculate the discrimination ratio (DR) for all animals. Then I suggest you complete a mean for the DR and the standard error- without holding your hands too much you may want to do a quick analysis (t-test) to see if there was a difference in time/frequency of interactions with the objects on the raw data and then on the DR between sexes of animals. As part of your write up you may want to look at the literature as to what it says about the relative differences in sexes of rodents on their performance in different object discrimination tasks- might be worth having a look: does your data support this or not?

You use the other variables to compare against each other also to help explain any differences if found. To do this you can use a series of t-tests or ANOVA- the latter being preferable due to the more stringent nature of it, but either way, I won’t mark anyone down as long as the statistical tests are applied correctly i.e. if you do use a t-test it’ll be non-paired, two-tailed etc.

The analysis is quite simple and will not require anything more complex than a t-test that you can perform in the Excel spreadsheet provided and also the calculation of the discrimination ratio which is simple and given for you at the top right of the spreadsheet. There are a few variations on the way NOR data is analysed but the discrimination ratio is the most objective and gives you a nice number- the closer to 1 it is, the more accurate the discrimination is between the objects.

Focus of the write up:

You should introduce the work in the context of cognitive testing and animal models with particular reference to the established models and short reviews of each with a build up to covering principles of the NOR test.

The aim of the study was primarily to investigate the reproducibility of the rat novel object recognition and to investigate any effects of the sex of the animals on performance in the test. Please use the literature to interpret and discuss your results and make sure to NOT make reference to the current study in your introduction until the end when you state the aims/hypotheses.  Make sure you write the abstract as the last part of the overall report write up-

ABSTRACT TIP: when writing an abstract you can put in subheadings of intro, methods, results, conclusion and then write the relevant elements from those sections. Once completed, DELETE the subheadings and any spaces because you don’t have subheadings or separate paragraphs in an abstract (should be one continuous paragraph), but by taking this approach it makes sure you include all the relevant information from all the sections of the write up.

General information:

For the report I would like it to be presented in the style of a paper with an abstract, intro, methods, results and discussion. It must be 5 pages maximum from the start of the abstract to the end of the discussion/conclusion- the references section and title page are not included in the page limit. The font should be 11 point Arial, 1.5 line spacing and 2.5 cm margins. The abstract should be no more than 250 words.