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. NINDSNational 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

REFERENCESMuigg 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

Breeding of High and Low Anxiety Mice

Generation

Tim

e in

Ope

n A

rms

(s)

1 Norm

al

2 Low

2 High

3 Low

3 High

4 Low

4 High

5 Low

5 High

6 Low

6 High

7 Low

7 High

8 Low

8 High

0

50

100

150

200

* * ** *

* *

Normal CD-1 Mice Stressed

Low Anxiety Mice Control

Low Anxiety Mice Stressed

Normal CD-1 Mice Control