(K-411) Methodology in Novel Object Recognition: Object and Arena Validation

Friday, October 13, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 411

Presenting Author(s)

IP

Isabella Phillips (she/her/hers)

Undergraduate Researcher
Mercer University
Macon, Georgia, United States

Primary Investigator(s)

FK

Forrest Kievit

Associate Professor
University of Nebraska - Lincoln, United States

Co-Author(s)

CG

Connor Gee

Graduate Student
University of Nebraska - Lincoln, United States

Introduction::

Sub-track: Neural Disease and Injury: Modeling and Therapeutics

The Central Nervous System (CNS) is susceptible to various issues, including disorders such as strokes, neurodegenerative diseases, and traumatic brain injuries (TBIs). TBIs prove to be a major cause of death and disability in the United States, making it a key area of interest in biomedical research. While discovering biomarkers is a crucial part of TBI research, behavioral testing helps provide a more thorough understanding of biochemical changes and their effects on an individual's functional abilities. Biomedical research labs can accomplish this by implementing behavioral testing into their work, such as the Novel Object Recognition Test. The Novel Object Recognition Test (NORT) is dependent on the experimenter using object pairs that the rodents have an equal attraction to. If the rodent has a preferential bias towards one of the objects or sections of the arena, the results of the test will be invalid. Additionally, the rodent must also experience enough anxiety to feel the need to explore the arena, while still remaining calm enough to handle efficiently. Our results suggest that using different methods for the handling and habituation of the mice can help control their anxiety levels. Because of the numerous opportunities for variability in the methodology of NORT, our results also strongly suggest a need for a standardized protocol in rodent behavioral testing.

Materials and Methods:: In order to determine an ideal object pair, we compared objects of varying width, height, and color over a series of nine experiments. The experimental apparatus consisted of four 40 cm x 40 cm x 40 cm arenas and used objects measuring 10 to 13 cm tall and 2 to 3 cm wide. The objects used are as follows: 50 mL Falcon tube, 15 mL Falcon tube, Lego tower, syringe plunger, blue bowling pin, and a carrot shaped easter egg. Each experiment consisted of eight C57BL/6 mice, divided into two groups of four mice to ensure the mice would not see the same object twice. In experiments one through eight, Group One was tested against the syringe plunger vs. 50 mL tube and the 15 mL tube vs. the Lego tower, while Group 2 was tested against the syringe plunger vs. the Lego tower and the 15 mL tube vs. the 50 mL tube. Experiment nine was used to validate the arena setup and cleaning method for the objects. Group One was tested against a Bowling Pin vs. 50 mL tube and two identical unused bowling pins, while Group Two was tested against the carrot easter egg vs. 50 mL tube and an unused carrot easter egg vs. a previously used clean carrot easter egg. Each experiment’s habituation and handling methods are as follows: 5 habituation days (1, 2), 3 habituation days (3, 5, 7), 0 habituation days (4, 6), and 2 handling days (8, 9).

Results, Conclusions, and Discussions:: To determine the preferences of the mice, we calculated a bias ratio (BR) for all of the object pairs by dividing the time spent at Object One by the time spent at Object Two. If a mouse had an equal preference towards both objects, the resulting BR should be 1 (± 0.2). The 15 mL tube vs. 50 mL tube and 50 mL tube vs. Carrot Easter Egg object pairs did not reach this window, with averages of 0.721 and 0.553 respectively. Though the remaining object pairs had averages within the desired window, when the individual data points were examined we discovered that the majority of data points clustered outside of the boundaries, suggesting that the mice did have preferential bias towards the objects. Only the Bowling Pin vs. 50 mL tube showed clustering within the desired ranges. While this implies that the mice show no preferential bias towards this object pair, only one experiment has been run with the Bowling Pin vs. 50 mL pairing. Future testing will be performed with this object pairing to confirm these results.

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