Oct 19, 2022
Novel Object Recognition test for rats, AJA, 10/18/22
This protocol is a draft, published without a DOI.
- 1UCSD, Scripps
Protocol Citation: Alicia Avelar 2022. Novel Object Recognition test for rats, AJA, 10/18/22. protocols.io https://protocols.io/view/novel-object-recognition-test-for-rats-aja-10-18-2-ch34t8qw
Manuscript citation:
References: Cruz-Sanchez A, Wilkin J, Arruda-Carvalho M. Ontogeny of spontaneous recognition memory in rodents. Neurobiol Learn Mem. 2021 Jan;177:107361. doi: 10.1016/j.nlm.2020.107361. Epub 2020 Dec 8. PMID: 33307181. 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. Ennaceur A, Delacour J. A new one-trial test for neurobiological studies of memory in rats. 1: Behavioral data. Behav Brain Res. 1988 Nov 1;31(1):47-59. doi: 10.1016/0166-4328(88)90157-x. PMID: 3228475. George lab ethanol vapor binge grant Inayat M, Cruz-Sanchez A, Thorpe HHA, Frie JA, Richards BA, Khokhar JY, Arruda-Carvalho M. Promoting and Optimizing the Use of 3D-Printed Objects in Spontaneous Recognition Memory Tasks in Rodents: A Method for Improving Rigor and Reproducibility. eNeuro. 2021 Sep 30;8(5):ENEURO.0319-21.2021. doi: 10.1523/ENEURO.0319-21.2021. PMID: 34503967; PMCID: PMC8489023. https://www.noldus.com/ethovision-xt/resources
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 19, 2022
Last Modified: October 19, 2022
Protocol Integer ID: 71516
Keywords: Novel object recognition, NOR, memory, rat, behavior
Disclaimer
This protocol requires additional troubleshooting with naive rats to ensure interpretable data. If you have suggestions for improvement please comment or email: aavelar@health.uscd.edu
Abstract
Purpose: to study animal memory using recognition of a familiar object vs. a novel object
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
Materials
Facilities and supplies needed:
A quiet room where the test will be performed (MTF behavior room at UCSD was the location of these tests)
Access to a 3D printer, software, and filament (USCD Geisel library digital media lab was used by Alicia Avelar)
Red light (red light bulbs in metal lamps on tripods were used)
Open field chambers (dimensions: 42.5 width X 42.5 length X 30 cm wall height)
Multiple sets of identical objects (3D printed objects similar in size to adult Wistar rats.Objects chosen based on Inayat et al., 2021 using files found on thingiverse.com from the Khokhar lab.)
Velcrow tape/adhesives to attach objects to floor of open field chamber
Cleaning supplies (Airx44 disinfectant cleaner and paper towels)
Worksheet for tallying rat behavior during the test. (created in excel by Alicia Avelar)
Video camera for recording the tests (Zohulu 4K video camera with infrared night vision was used- recordings done in red lighted room using infrared night vision mode)
Very long video camera power cord to reach wall outlets (not always easily accessible)
Camera mount to secure camera to the ceiling directly above the open field chamber where NOR testing occurs.
Memory cards for collecting and transferring videos (32 and 64 GB SD cards were used.64 GB SD cards are better since videos don’t have to be transferred as frequently throughout a testing day)
A safe, stable ladder to climb up to allow for mounting of the video camera and insertion and retrieval of the memory card each test day.
a computer that has a SD memory card slot or a SD memory card to USB adaptor (which is what we needed to transfer videos to the computer for anymaze analysis)
Anymaze software for tracking and data analysis of the videos
Excel for further data analysis of human tallied and anymaze data.
Graphpad prism for graphing the data and statistical analysis.
Computer(s) to run the necessary software on for data collection with anymaze and data analysis, graphing, and statistical analysis.
Worksheet for human tallied data collection (made in excel by Alicia Avelar).
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.
3D printing
3D printing
Objects were printed on Prusa 3D printers using ultimaker cura software at the UCSD digital media lab in the Geisel library.
Printing took about 1-3 hours per object.
3D printed object (vase) compared to adult Wistar rat size. (Pictures and 3D prints by Alicia Avelar)
The object sizes were standardized as much as possible depending on the objects shape.
Bishop: https://www.thingiverse.com/thing:40605, Spiral Vase: https://www.thingiverse.com/thing:2421704
2 of the object shapes were from Inayat et al., 2021 publication that supported rats would interact with those 3D printed shapes.
Sprague-Dawley rat exploration of 3D printed objects. Rats preferred interacting with the vase and twizzler objects closer to their height than with the smaller castle object. (Inayat et al., 2021)
Additional object shapes were chosen based on having differing shapes and being printable in similar dimensions to the published objects for the NOR test.
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)
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
Set up
Set up
Open field chambers were set on a work table in MTF behavior room.
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)
NOR set up image created in BioRender (2/15/22) by Alicia Avelar
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.
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.
NOR test procedure
NOR test procedure
1h 10m
1h 10m
All rats were handled prior to tests.
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.
1h
2 identical objects are placed in the open field chamber, attached to the Velcro on the chamber floor.
First rat is placed in the chamber, centered and close to the front wall (opposite the objects), facing the objects.
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.
NOR test experimental design. (Cruz-Sanchez et al., 2020)
10m
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).
At the end of the sample phase, the rat is removed from the open field chamber.
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.
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.
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.
Objects and open field chambers were cleaned and dried with Airx44 disinfectant cleaner and paper towels between tests and sexes.
Data processing
Data processing
Use anymaze for video behavior tracking and data collection. (I made an anymaze protocol to analyze NOR data from videos)
Use excel to create an electronic record of human tallied data and anymaze data and do further data analysis.
Use graphpad prism for graphing and statistical tests.
Noldus ebook behavior basics has information on NOR test data to graph.
“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:
Time spent exploring novel object (x 100)/ Time spent exploring any object
The preference for novelty is a positive value if there is preference for novelty, and is zero if there is no preference:
Time novel - Time familiar (x 100)/ Time novel + Time familiar”
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.
Example data presentation from George lab ethanol vapor grant.