Rigor and reproducibility in rodent behavioral research

Abstract

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

Keywords: Behavior; Behavioral assays; Best practices; Cognitive; Experimental design; Mice; Novel object recognition; Rats; Replication; Reproducibility; Rigor; Statistical analysis.

Figure 1

Examples of object pairs used in novel object recognition testing by IDDR Rodent Behavior Cores. A) Left: Orange traffic cone, 7 cm high × 5 cm wide (source unknown), and green cylindrical magnet, 7 cm high × 3 cm wide (source: Magneatos, Guide Craft, Amazon.com); photo by Michael Pride, UC Davis MIND Institute Rodent Behavior Core). B) Premium Big Briks, 3 staggered reclining bricks, ~ 6 cm high × 3 cm wide (source: Amazon.com # B01N5FGUHB), coral, 5 cm high × 3 cm wide (source unknown) and treasure chest 2 cm high × 4 cm wide (source unknown); photo by Melanie Schaffler, UC Davis MIND Institute. Unpublished photos in A and B were contributed by Jacqueline Crawley, UC Davis MIND Institute IDDRC Rodent Behavior Core. C) Binder clip (source: Office Depot, Washington DC, USA), Open field chamber (source: Accuscan, Columbus, OH, USA); decorated binder clip. Unpublished photos contributed by Li Wang, Children’s National Health System (CNHS) Center for Neuroscience IDDRC Neurobehavioral Core. D) R2D2 toy, plastic Easter egg, gold oval, metal toy car (source: Target), and Lego block (source unknown). Unpublished photos contributed by Heather Mitchell, University of Wisconsin-Madison, Waisman Center, IDD Models Core. E) Upper left: Plastic shapes (source: Toys”R”Us); photo by Brett Mommer and Zhengui Xia. Upper right: Gloss-painted wooden blocks (source unknown); photo by Christine Cheah and William Catterall. Lower left: small sand-filled and water-filled glass jars (source unknown), photo by Brett Mommer and Zhengui Xia. Lower right: Plastic train whistles and mini color 3×3 cube puzzles (source: Amazon.com), photos provided by Melissa Barker-Haliski and H. Steve White. Assembled photos contributed by Toby Cole, University of Washington IDDRC Mouse Behavior Laboratory. F) Left: child’s sippy cup, right, baby bottle – middle- ruler for scale. Unpublished photos contributed by Maria Gulinello, Albert Einstein College of Medicine IDDRC Animal Phenotyping Core. G) Plastic pipe fittings (source: ACE hardware, Porter Square, Cambridge, MA) 7 cm × 2 cm, and glass jar 10 cm × 3.5 cm. Unpublished photo contributed by Nick Andrews, Boston Children’s Hospital IDDRC Neurodevelopmental Behavior Core. H) LEGO Classic Medium Creative Brick Box and LEGO Duplo Deluxe Box (source: Amazon.com #10696 and #10580). Orange, black and yellow tower dimensions: 15 cm tall × 6.5 cm wide × 6.5 cm long. White, red, blue, yellow tower dimensions: 15.5 cm × 10 cm at the base, 7.5 cm wide along the rest of the body. Brown, yellow, green, orange tower dimensions: 16.5 cm tall × 6.5 cm wide × 9.5 cm long. Unpublished photo contributed by Surabi Veeraragavan, Baylor College of Medicine IDDRC Neurobehavioral Core. I) Bottle, 4.5 cm diam × 16.5 cm tall, clear, filled with white sand and capped (source: Michaels.com). Metal bar, 3.75 cm × 3.75 cm × 15 cm tall, thin aluminum sheet cut and fabricated with glue, filled with white sand (source: in-house fabrications shop). Objects are mounted on a 6.5 cm × 6.5 cm base. Unpublished photos contributed by Tim O’Brien, University of Pennsylvania, Children’s Hospital of Philadelphia IDDRC Neurobehavior Testing Core.

Figure 2

Common methods for analyzing and illustrating novel object recognition data. (A) Total object exploration of both objects (in seconds) during the familiarization phase (training, Trial 1, sample phase) scored and illustrated separately for each of four litters (X axis- designated A, B, C and D) (B) Total object exploration of both objects (in seconds) during the novel object recognition phase (Test, Trial 2). (C) The absolute exploration of the new (novel) object (red, circle) and the old (familiar) object (blue, triangle), in seconds, during the novel object recognition test phase. (D) Preference score = [(exploration novel object in sec)/(exploration novel + exploration familiar)] X 100 during the novel object recognition test phase. A 50% preference score = the same exploration both the novel (new) and the familiar (old) object). (E) Pass/Fail rate indicates the % of subjects with and without a preference for the novel object, with a preference designated as > 55% preference score during the novel object recognition test phase. (F): Difference score = (exploration of novel object in sec)-(exploration of familiar object in sec) during the novel object recognition test phase. A difference score of 0 = the same exploration both the novel and the familiar object. Data shown are from 4 individual litters of C57Bl/6 mice wherein dams were treated during pregnancy with either normal drinking water or a drug in the drinking water. Offspring were tested at 5 mos. Litters A and B are control treated (open bars) and litters C and D are drug treated (green bars). Unpublished data by Gulinello, Einstein, IDDRC core.

Figure 3

High degree of inter-experimenter reliability is dependent on sufficient training. (A). The % difference in total exploration in seconds was scored by a novice and a trained experimenter (circles, open bars) when scoring identical movies of the same subjects vs the % difference between 2 trained experimenters also scoring identical movies (triangles, closed bars. % difference = [ABS value (trained-novice)/trained %100] or [ABS value (trained1-trained2)/trained2 %100] (B) The Pass/Fail Rate for a novice vs a trained experimenter (left panel) and for 2 trained experimenters (right panel). N for each condition is shown in panel A as individual points in and within the bars in panel B. Note – the trained and novice in panel A and viewed the same movies as did the trained experimenters. The two trained experimenter in panel B viewed identical movies for each comparison, but these experiments were conducted separately, hence the different sample size. Unpublished data by Gulinello, Einstein, IDDRC core.

Figure 4

The size of the zones (X Axis) in automated tracking software can affect apparent object exploration scores. Very small differences of 2 cm in the size of the zone around the object can result in significant differences in recorded exploration (F (_2,71)=5.4, p<0.01). N=24. Results obtained by a trained human scorer are provided for comparison only. Unpublished data by Gulinello, Einstein, IDDRC core

Figure 5

The effect of isolation on performance in the novel object recognition test (3 min familiarization, 3 min recognition, 30 min retention interval). Female Long-Evans rats were housed in either grouped (2–3 per cage) or isolated conditions for 4–6 weeks and then assessed in the novel object recognition test. Sample sizes are shown in the bars in the pass/fail graph at right. Unpublished data by Gulinello, Einstein, IDDRC core

Figure 6

Absence of sex differences in the novel object recognition test. The graph depicts the performance of male and female mice (total n=551) on a C57Bl6 background, tested between 3–10 months old. Sex differences in performance were not detected at any of the retention intervals. Unpublished data by Gulinello, Einstein, IDDRC core