1. John Hershey, VMD, PhD ACLAM Forum April 25, 2017
The Effect of Sanitation on a Common Rodent Behavioral Assay of Anxiety
John Hershey, VMD, PhD
ACLAM Forum
April 25, 2017

2. Additional Authors…
Shoreh Miller, DVM, PhD, DACLAM: Director, Research Animal Facility, Assistant Professor, Cell Biology and Neuroscience, Rowan University, NJ
Lauren Zizza, MS: IACUC Manager, Office of Research Regulatory Affairs, Rutgers University, NJ
George Wagner, PhD: Professor, Graduate Vice Chair, Department of Psychology, Rutgers University, NJ
Janace Gifford: PhD Candidate, Department of Psychology, Rutgers University, NJ
Darya Pavlenko: Undergraduate, Rutgers University, NJ
Darya is undergrad in Psychology, working on honors thesis

3. Begin with discussion of how idea started – after IACUC meeting

4. What is the value of cleaning a behavior device between animals, cohorts, investigators?
Main question that developed during discussion

5. “The smell of the agent will be aversive to the test subject”
Clean
“The smell of the agent will be aversive to the test subject”
Two sides of discussion
“It’s the best way to prevent disease transmission”
“I’ve never done it before”
“What about residual odors/pheromones from previous subjects?”

6. Exposure to sanitizing agents Exposure to odors, pheromones

7. How can we test this idea?

8. Elevated Plus Maze (EPM)
One of the most common assays currently used in behavioral research
PubMed currently yields 6449 hits
Used for screening and phenotyping transgenic and knockout mice
Strong predictive validity for screening anxiolytic drugs

9. Less anxious
More anxious

10. Hypothesis
Animals tested on an EPM apparatus soiled with animal excretions would score higher in anxiety parameters due to exposure to residual pheromones produced from the previous test subjects
More entries into closed arms, more time spent in closed arms, more distance in closed arms

11. Hypothesis
Animals tested on an EPM apparatus soiled with animal excretions would score higher in anxiety parameters due to exposure to residual pheromones produced from the previous test subjects
More entries into closed arms, more time spent in closed arms, more distance in closed arms
More anxious state when EPM not cleaned

12. Experimental design:
C57Bl/6Crl mice, 8 weeks old, acclimated 1 week to facility
4 groups (10M, 10F per group), all mice naïve to EPM
MB-10 (100ppm)
70% isopropyl alcohol
Bleach (600ppm)
No sanitation – ‘dirty’

13. ‘Dirty’ EPM
Created soiled EPM prior to first ‘no sanitation’ mouse trial
Ran 20 mice in pairs (5min per pair) on EPM prior to ‘no sanitation’ group – did not clean EPM between mice

14. Experimental design:
C57Bl/6Crl mice, 8 weeks old, acclimated 1 week to facility
4 groups (10M, 10F per group), all mice naïve
MB-10 (made to manufacturer’s specifications)
70% isopropyl alcohol
Bleach (600ppm)
No sanitation
Five minute trials, start at intersection
30s after cleaning to all vapors to dissipate
Parameters measured - # entries, distance, time
ANY-maze software

15. Results - Males

16. Results - Females *

17. Results - Summary
Significant differences between closed/open arms for all conditions (experimental validation)
-HOWEVER-
No significant differences between dirty or clean EPM
No significant difference between cleaning agents

18. Now what?

19. Validate observations with another test…

20. Light/Dark Box Two dark chambers connected by illuminated center
Place potential aversive stimulus on one side, water on other side

21. Light/Dark Box - protocol
Step 1 - determine preferred side for each mouse
Step 2 - place agent in the preferred side
Step 3 - reassess side preference 

22. Light/Dark Box - results

23. Conclusions
Sanitation of EPM did not affect performance in 8 week old C57 mice (compared to other agents and compared to no sanitation)
Mice in this study did have an aversion to the cleaning agents based on the Light/Dark experiments

24. Future Studies Strain differences Age Other agents
Other behavioral assays

25. Thank you!
Shoreh, George, Lauren, Janace, Darya
ACLAM Foundation