TITLE: Novel Object Recognition test for rats, AJA, 10/18/22
AUTHORS: Alicia Avelar

[DESCRIPTION]
Purpose: to study animal memory using recognition of a familiar object vs. a novel object

[BEFORE_START]
Need to establish that protocol fully works with naive animals.  Potentially accounting for side of chamber and object shape biases and use a larger room, with less clutter, and more soundproof walls will help the test work better with easier-to-interpret results. 

[GUIDELINES]
Ideas for future experimental troubleshooting/optimizing:
Ennaceur & Delacour, 1988 BehavBrainRes- excluded NOR data from rats with no exploratory behavior of objects during the tests
need to counterbalance objects used for familiar and novel and position in open field to account for object or location of object biases that could confound results
rat placed on side of chamber opposite the objects and facing away from the objects
white noise 70 dB above human threshhold and 40 lux light from one lightbulb above the apparatus
    


[STEPS]
SECTION: 3D printing
1. Objects were printed on Prusa 3D printers using ultimaker cura software at the UCSD digital media lab in the Geisel library. 
  
    

SECTION: 3D printing
2. The object sizes  were standardized as much as possible depending on the objects shape.  

SECTION: 3D printing
3. 2 of the object shapes were from Inayat et al., 2021 publication that supported rats would interact with those 3D printed shapes.  

SECTION: 3D printing
4. Additional object shapes were chosen based on having differing shapes and being printable in similar dimensions to the published objects for the NOR test.
SECTION: 3D printing
5. Multiple sets of objects (5-7 copies each shape) were printed to allow for testing multiple retention intervals (RIs- the time between the sample and test phases of the NOR test)
SECTION: 3D printing
6. Object shapes 3D printed are vase (Inayat et al., 2021), twizzler (Inayat et al., 2021), spiral vase, bishop, king, plumbob.  All 3D files used were found on thingiverse.com. https://www.thingiverse.com/thing:4964541
SECTION: 3D printing
1. Objects were printed on Prusa 3D printers using ultimaker cura software at the UCSD digital media lab in the Geisel library.
SECTION: 3D printing
2. The objects size was standardized as much as possible depending on the objects shape.
SECTION: 3D printing
3. 2 of the object shapes were from Inayat et al., 2021 publication that supported rats would interact with those 3D printed shapes.
SECTION: 3D printing
4. Additional object shapes were chosen based on having differing shapes and being printable in similar dimensions to the published objects for the NOR test.
SECTION: 3D printing
5. Multiple sets of objects (5-7 copies each shape) were printed to allow for testing multiple retention intervals (RIs- the time between the sample and test phases of the NOR test)
SECTION: 3D printing
6. Object shapes 3D printed are vase (Inayat et al., 2021), twizzler (Inayat et al., 2021), spiral vase, bishop, king, plumbob.  All 3D files used were found on thingiverse.com. https://www.thingiverse.com/thing:4964541
SECTION: Set up
7. Open field chambers were set on a work table in MTF behavior room.
SECTION: Set up
8. Velcro squares were applied to the floor of each open field chamber towards the back wall. (12 cm from the back and side walls so that objects adhered to the Velcro would be equidistant from each other and leave space around the object for the rats to explore)  

SECTION: Set up
9. a video camera was mounted to the ceiling above the open field chamber and a long power cord was used to reach the wall outlet. 
SECTION: Set up
10. The power cord was taped in place to the wall near the outlet and the ceiling near the video camera to reduce shaking of the camera during recording from cord weight. 
SECTION: NOR test procedure
11. All rats were handled prior to tests.
SECTION: NOR test procedure
12. Rats were brought into behavior room and given 1 hr habituation time in the room in home cages. For collection of NOR test intoxication data rats spent at least 3 hours in the ethanol vapor self-administration (EVSA) chambers immediately before NOR testing began.
SECTION: NOR test procedure
13. 2 identical objects are placed in the open field chamber, attached to the Velcro on the chamber floor.
SECTION: NOR test procedure
14. First rat is placed in the chamber, centered and close to the front wall (opposite the objects), facing the objects.
SECTION: NOR test procedure
15. This is the sample phase and the rat is given 10 minutes to freely explore the open field chamber and objects. Vase shaped objects were used for the sample phase.  

SECTION: NOR test procedure
16. Next, is the retention interval (RI).  The retention interval is the time between the end of the sample phase and the beginning of the test phase. We have used 2 retention intervals for the NOR test: 1 minute (to test short term memory) and 24 hours (to test long term memory).
SECTION: NOR test procedure
17. At the end of the sample phase, the rat is removed from the open field chamber.
SECTION: NOR test procedure
18. The objects are replaced with one object identical to those present in the sample phase (left side) and a novel object (right side). One vase and one twizzler object were used during the RI 1 minute test phase.  One vase and one plumbob object were used during the RI 24 hours test phase.
SECTION: NOR test procedure
19. After the RI, the rat is placed into the open field chamber for the test phase.  There is one familiar object and one novel object in the chamber during the test phase.  The test phase lasts for 10 minutes.
SECTION: NOR test procedure
20. When the test phase ends the rat is place back into their home cage and testing continues with other rats in the cohort until complete for each RI.
SECTION: NOR test procedure
21. Objects and open field chambers were cleaned and dried with Airx44 disinfectant cleaner and paper towels between tests and sexes.
SECTION: Data processing
22. Use anymaze for video behavior tracking and data collection. (I made an anymaze protocol to analyze NOR data from videos)
SECTION: Data processing
23. Use excel to create an electronic record of human tallied data and anymaze data and do further data analysis.
SECTION: Data processing
24. Use graphpad prism for graphing and statistical tests.
SECTION: Data processing
25. Noldus ebook behavior basics has information on NOR test data to graph. 
SECTION: Data processing
25.1. “The most important results from the novel object or novel location test include the exploration times of the objects and the frequency of each object exploration. These are generally expressed as the percentage of exploration time spent on novel object:
SECTION: Data processing
25.2. Time spent exploring novel object (x 100)/ Time spent exploring any object
SECTION: Data processing
25.3. The preference for novelty is a positive value if there is preference for novelty, and is zero if there is no preference:
SECTION: Data processing
25.4. Time novel - Time familiar (x 100)/ Time novel + Time familiar”
SECTION: Data processing
26. Danışman B, Akçay G, Gökçek-Saraç Ç, Kantar D, Aslan M, Derin N. The Role of Acetylcholine on the Effects of Different Doses of Sulfite in Learning and Memory. Neurochem Res. 2022 Jul 27. doi: 10.1007/s11064-022-03684-z. Epub ahead of print. PMID: 35895153.  

SECTION: Data processing
27. Example data presentation from George lab ethanol vapor grant.