Download +ΔFosB

Neuron
2007
Speaker: Yu-han Lee
Advisor: Yn-ho Huang Ph.D
Dr. Chen-Jee Hong
Escape deficit
( learned helplessness)
repeated stress
(eg. inescapable footshock)
escape test
No escape deficit
(Non learned helplessness)
ΔFosB
( brain: emotional regions)
﹖
role
Introduction
Learned Helplessness
Animals exposed to inescapable shock present with a series of symptoms similar to
those observed in depressed patients.

-Willner et al.,1984
Seligman,1967
escapable shocks : ES
Shuttle box
Inescapable shocks : IS
home-cage control : HC
ΔFosB : a truncated splice variant of FosB,
which lacks 101 amino acids from
the C-terminal end of the full-length fosB
AP-1 (activator protein - 1) transcription factor
stimuli
AP-1 : rapidly in response to a variety of stimuli
AP-1 site
Fos family protein : c-Fos, FosB, ΔFosB ,Fra-1, Fra-2
1. The nature of the biochemical modification
that converts the unstable 33-kDa
isoform into the stable 35- to 37-kDa
isoforms has remained obscure.It has
been speculated that phosphorylation
may be responsible.
2. The 35- to 37-kDa isoforms of
ΔFosB dimerize predominantly
with JunD to form an active and
long-lasting AP-1 complex
within these brain regions.
Eric J. Nestler et al, 2001
stimuli
stimuli
Colleen A. McClung et al, 2004
Escape deficit
( learned helplessness)
repeated stress
(inescapable footshock)
escape test
No escape deficit
(Non learned helplessness)
ΔFosB
( brain: emotional regions)
﹖
role
Results and Methods
C57BL/6 mice , 8 wks
Shuttle box
120 inescapable footshock (IS) / day
2 days , 0.45mA
No IS , 1hr / day , 2days
Escape test (Shuttle box) : 15 trials , 25 sec / trial
24hr
No IS
IS - Escape deficit (LH : learned helplessness)
IS – no escape deficit (NLH : non learned helplessness)
24hr
Heterogeneous Escape Deficits Induced by IS in mice
LH
＃
★
NLH
Define 2
subgroups
C57BL/6 mice , 8 wks
inescapable footshock (IS)
24hr
escape test :
No IS
IS
No IS - Yoked
Immunohistochemistry ΔFosB
(monoaminergic regions: dorsal raphe nucleus (DR)
ventral tegmental area (VTA)
locus coeruleus (LC)
Full-length fosB Only
ΔFosB
only
Middle region of both
full-length fosB and
ΔFosB
ΔFosB Accumulates in the DR and LC, but Not in the VTA,
after Repeated IS
TPH : tryptophan hydroxylase
vIPAG : ventrolateral
periaqueductal gray
(lateral margins of the rostral DR)
ΔFosB Levels in the Ventrolateral Periaqueductal Gray Correlate
Negatively with the Severity of the Behavioral Deficit
ΔFosB
NLH
LH
Rostral
portion of DR
vIPAG
Repeated stress (IS)
ΔFosB
ΔFosB
In “No escape deficit ” NLH
In “escape deficit ” LH
The level of ΔFosB induction by IS in this brain region
predicts the individual degree of resilience to IS.
(Also in repeated social defeat paradigm)
- The vlPAG has been characterized previously as an important neural substrate
for passive coping responses.
- IS, which induces primarily passive behavioral responses, engages the
vlPAG.
Bandler and Shipley, 1994
Hypothesis : IS
ΔFosB
In vlPAG
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
PPT-A (preprotachykinin A) promoter :
PPT-A
enhancer
or
repressor
PPT-A mRNA
Alternative splicing
substance P
AP-1
Paterson et al,1995
substance P
vlPAG
ascending to
nucleus accumbens(NAc), amygdala
It has been shown that emotional stressors triggers the release of substance P
in the NAc,
Karl Ebner et al, 2004
IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
？
AP-1(ΔFosB)
substance P in vlPAG
regulate
ΔFosB Accumulates in Substance P-Positive Neurons after IS
B6 after IS
immunohistochemistry (ΔFosB)
In situ hybridization
ΔFosB Accumulates in Substance P-Positive Neurons after IS
GABA
5-HT
63%
25%
10%
ΔFosB Decreases Substance P Gene Expression In Vivo
-ΔFosB
+ΔFosB
Substance P
Hypothesis : IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
In vivo :
AP-1(ΔFosB)
substance P
regulate
In vitro ????
Luciferase assay
PPT-A promoter
-865 ~ +447
pGL3basic
HSV-ΔFOSB
or HSV-LacZ
PC12
(substrate)
ΔFosB Decreases Substance P Gene Expression In Vitro
Dose-dependent
HSV-LacZ(control)
Hypothesis : IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
AP-1(ΔFosB)
substance P
regulate
In vitro ????
YES
PPT-A
repressor
ΔFosB
PPT-A mRNA
Alternative splicing
substance P
AP-1
Hypothesis : IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
AP-1(ΔFosB)
substance P
regulate
Question:
Whether this regulation represents a direct effect of ΔFosB on the PPT-A
gene???
Mice - IS: 2hr , 24hr
ChIP(chromatin immunoprecipitation) assay
control : 2hr , 24hr
vlPAG of 3~4 mice
Crosslink with 1% formaldehyde
sonication
Anti-fosB Ab (c-terminal Ab)
(full-length fosB only)
Anti-fosB Ab
(middle region of both fulllength fosB and ΔFosB)
Reverse crosslink
Real-time PCR
PCR analysis(164bp)
ΔFosB accumulating in substance P-positive neurons in the vlPAG after IS
binds directly to the promoter region of the PPT-A gene in vivo.
IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
AP-1(ΔFosB)
substance P
regulate
Question:
Whether the regulation of substance P gene expression in vlPAG neurons by ΔFosB
may contribute to functional alterations of forebrain substance P neurotransmission??
Microdialysis with simultaneous behavioral analysis
to mimic the endogenous pattern of ΔFosB induction after IS
baseline
Rats: HSV- LacZ
HSV- ΔFosB
In NAc
4 samples for baseline
(every 30min,homecage )
In vlPAG
Swim tank 10 min
3 samples
(every 30 min,
homecage)
IS, 20min
5 samples,
(every 30 min,
homecage)
After viral Overexpression of ΔFosB in the vlPAG
After IS
Fig.1B
Hypothesis : IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
AP-1(ΔFosB)
In vlPAG
substance P
regulate
In NAc
Hypothesis : IS
ΔFosB
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
desensitize substance P input to the NAc
Substance P
vIPAG
NAc
NK1(substance P receptor)
drug
Function ?
D1
D2
D3
D4
IS
IS
rest
test
Drug
drug
drug
drug 30min before test
NK1 antagonist
antidepressant
Decreased substance P signaling in the NAc exerts an antidepressant-like effect
and promotes active escape responses in the learned helplessness paradigm.
ΔFosB
Hypothesis : IS
Antidepressant-like effect ???
alterations in AP-1 transcription
desensitize passive coping-related circuits in the vlPAG.
desensitize substance P input to the NAc
Substance P
vIPAG
NAc
NK1(substance P receptor)
drug
antidepressant-like effect
Overexpression of ΔFosB in the vlPAG Opposes Behavioral Despair
ΔJunD, a truncated isoform
lacking 48 amino acids at the
N terminus , is a truncated
variant of JunD, the major in
vivo partner for ΔFosB
(Chen et al., 1995; Hiroi et al., 1998)
Forced swim test
Antidepressant-like effect
No change
sensitivity to foot shocks
Rotarod test of motor coordination
Locomotor activity
conclusion
ΔFosB
Repeated stress
stress
substance P
accumulate in vlPAG
release in NAc
In resilient animals
Repeated stress
ΔFosB
accumulate in vlPAG
repress
substance P gene in vlPAG
substance P
release in NAc
Promoting active coping responses
ascending to
In resilient mice
ΔFosB
substance P
accumulate
sufficiently to repress
substance P signaling
In helpless mice
substance P
Not accumulate
sufficiently to repress
substance P signaling
ΔFosB
Rotarod
• A reliable test to study motor function and learning
• Bar is rotated at constant or increasing speed
• Time to fall is recorded manually or automatically
RT Series滾筒式跑步機主要是應用於研究藥物對動作協調性和抗疲勞特性的實驗
△Ct = (Nacute,chronic - Navecontrol) x Ctavecontrol,
(= CtPPT / Ct whole lysate - Ctavesynaptophysin / Ct whole lysate ) x Ctavesynaptophysin
- N is the normalized Ct value of PPT [Ct(PPT)/Ct(Input)]
- Nave is the mean N value for the control
- Ctave is the mean Ct value for the control.
- Fold differences (stress ChIP relative to control ChIP) were then determined by raising 2 to the Ct power.
Input : One hundred microliters of the pre-immunoprecipitated lysate
Control ChIP : samples were immunoprecipitated with 5 µg nonimmune rabbit IgG