Differential Neuronal Activation of Stress-Induced c-Fos Transcription Factor in Mice

Bred for High and Low Anxiety

Eric Lai8/3/2015

c-Fos Overview

• Proto-oncogene; Transcription factor

• 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?

Mice Strain

• Used normal CD-1 mice to start with and selectively bred for high and low anxiety mice

• Mice that appeared to express high anxiety mated with each other; mice that appeared to express low anxiety mated with each other

Elevated Plus Maze

• Mice were exposed toelevated plus maze for 5 min

• Spent average of 100 sec on open arms

• Less anxious mice spent moretime on open arm, more anxiousmice spent less time on open arm

Selectively Bred Mice

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

* * ** *

* *

Brain Region Overview

• In order to see if genetics play a role in anxiety, specific regions of the brain needed to be identified

• Research paper looked at whole brain for c-Fos expression (Muigg, 2009) in high, normal and low anxiety mice after stress-induction– 3 regions expressed significantly different levels

• Amygdala• Hippocampus• Hypothalamus

Amygdala

• Very obvious c-Fos expression

• Located in temporal lobe of the brain

• Part of limbic system, which is responsible for emotions, survival, instincts, and memory

• Involved in anger, fear, and anxiety

Hippocampus

• Somewhat obvious in c-Fos expression

• Located in medial temporal lobe

• Involved in learning and storing long-term memory

• Also involved in stress and anxiety

Hypothalamus

• Significant expression of c-Fos• Pituitary gland responsible for producing

essential hormones and chemical substances• Govern temperature regulation, thirst, hunger, sleep,

mood, etc.

• Maintain homeostasis• Stress hormones produced when anxious

Methods

• Stress-Induction

• Perfusion

• DAB Staining

• Mounting

• Imaging & Analysis

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)

Stress-Induction

• Elevated Plus Maze– Put up barrier so only exposed to open arm for 5

minutes– Open arm divided up in 3 zones: Proximal, Medial, Distal

• Measurements– Time & number of entries spent in each zone– Distance travelled– Number of head dips– Time spent head dipping– Latency until first head dip

Open-Arm Plus Maze Results

Open-Arm Plus Maze Results

DAB Staining

• Work in progress of optimizing ideal conditions– Concentrations of primary and secondary

antibodies– Concentrations of blocking solutions– Pre-mounted (free-floating) vs. mounted slides

Staining Protocols

• Rinsed in PBS, TBST, .03% H2O2, Blocking Solution– Incubated overnight in rabbit anti-c-Fos primary antibody

• Rinsed and placed in biotinylated goat anti-rabbit secondary antibody– Incubated in Vectastatin ABC– Slides mounted

• All sections incubated with DAB-solution for 3 min

Imaging

• Work in progress for next 2 weeks– Used bright field microscope to take images of all

3 brain regions: amygdala, hippocampus, hypothalamus

– Took pictures of Normal CD-1 Control mice brain regions

Amygdala Imaging

Amygdala (5x) Amygdala (10x)

Hippocampus Imaging

Hippocampus (5x) Hippocampus (10x)

Hypothalamus Imaging

Hypothalamus (5x) Hypothalamus (10x)

Future Works

• Take pictures of all slides

• Analysis of number of c-Fos positive cells in different brain regions with ImageJ

• Results and conclusion

• Include high anxiety mice in experiment

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.

Questions?

Thank you for your time!