c-fos labmeeting presentation
TRANSCRIPT
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!