The document summarizes an experiment comparing neuronal activation in mice bred for high, normal, and low anxiety levels after exposure to stress. Mice were exposed to an elevated plus maze to induce stress and brain tissue was analyzed for expression of c-Fos, a marker of neuronal activation. Preliminary results showed differential c-Fos expression in the amygdala, hippocampus, and hypothalamus between normal and low anxiety mice. The researchers aim to quantify c-Fos positive cells in these brain regions and investigate the cellular mechanisms underlying the anxiety-related differences in c-Fos expression.

1. Open-Arm Plus Maze Results
Image 1:
Picture of an elevated plus maze used for stress-induction on the left. Sample pictures of the 3
brain regions identified from left to right: amygdala, hippocampus, hypothalamus.
Differential Neuronal Activation of Stress-Induced c-Fos
Transcription Factor in Mice Bred for High and Low Anxiety
INTRODUCTION
 Selective Breeding of Mice for High and Low anxiety (Figure 1)
 Used normal CD-1 mice to start with as generation 1
 Mice that appeared to have the highest or lowest anxiety levels mated
with each other for subsequent generations
 Mice were exposed to elevated plus maze for 5 min
− Spent average of 100 sec on open arms
Figure 1: Significant difference in time spent in open arms shown in second and
subsequent generations of selective breeding; less anxious mice spent more time on
open arm, more anxious mice spent less time on open arm.
Eric Lai, Mary Anne Hutchison, Wei Lu. NINDS
National Institutes of Health: Bethesda, MD 2015
METHODS
 Stress Induction
 4 Experimental Groups:
− Normal CD-1 Mice Stressed (n=4)
− Normal CD-1 Mice Control (n=4)
− Low Anxiety Mice Stressed (n=4)
− Low Anxiety Mice Control (n=3)
 Elevated Plus Maze
− Put up barrier so only exposed to open arm for 5 min
− Open arm divided up in 3 zones: Proximal, Medial, Distal
− Measurements taken on open arm using camera (Figure 2)
 Perfusion & Cryostat Slicing
 DAB Staining
 Work in progress of optimizing ideal conditions
− Concentrations of primary and secondary antibodies; blocking solution
− Pre mounted (free floating) vs. mounted slides
 Rinsed in PBS, TBST, .03% H2O2, Blocking Solution
− Incubated overnight in rabbit anti-c-Fos primary antibody
− Incubated in Vectastatin ABC
 All sections incubated with DAB-solution for 3 min
 Mounting
 Imaging & Analysis
Figure 2:
Open-arm results of the stress-induction test. Significant
differences found in head dip entries and time, in which the
low anxiety mice spent a significant amount of time head
dipping more than the normal anxiety mice. Both groups of
mice spent about the same amount of time travelling on the
open arm.
RESULTS
 Imaging (Figure 3):
 Work in progress for next 2 weeks
 Used bright field microscope to take images of 3 brain regions in all
experimental groups
 All images taken under 10x magnification, from left to right: amygdala,
hippocampus, hypothalamus
DISCUSSION
REFERENCES
Muigg P, Scheiber S, Salchner P, Bunck M, Landgraf R, et al. (2009) Differential Stress-Induced Neuronal
Activation Patterns in Mouse Lines Selectively Bred for High, Normal or Low Anxiety.
ACKNOWLEDGEMENTS
 Members of Dr. Lu’s Laboratory
 Animal Care taker professionals
 OITE and the Summer Internship Program
 Research support from Division of Intramural Research at NINDS
ABSTRACT
 Background:
 c-Fos is a proto-oncogene, and more specifically a transcription factor
 c-Fos cited as reliable marker of neuronal activation in key brain areas for anxiety
(Muigg, 2009)
 Research question:
 Will mice bred for normal and low anxiety show different levels of c-Fos expression
after stress induction?
 Brain Region Overview
 Specific regions of the brain identified to see if genetics play a role in anxiety
 3 regions expressed significantly different levels of c-Fos
− Amygdala
− Hippocampus
− Hypothalamus
Future Directions
 Take pictures of all slides
 Analysis of number of c-Fos positive cells in different brain regions with ImageJ
computer software
 Results and conclusion
 Include high anxiety mice in experiment
 Electrophysiology to investigate cellular and synaptic mechanisms behind c-Fos levels
and anxiety
Normal CD-1 Mice Stressed
Low Anxiety Mice Control
Low Anxiety Mice Stressed
Normal CD-1 Mice Control