Transcript Induction of Inducible cAMP Early Repressor Expression in Nucleus

Induction of Inducible cAMP
Early Repressor Expression
in Nucleus Accumbens by
Stress or Amphetamine
Increases Behavioral
Responses to Emotional
Stimuli
Introduction
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cAMP response element (CRE) is
sensitive in many brain areas to
drugs, stress, and reward
increased CRE-mediated
transcription in NAc (by drugs or
stress) decreases behavioral
response to +/- stimuli
Blocking CRE transcription via CREB
(CRE-binding protein) generally does
the opposite
ICER
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Inducible cAMP early repressor
(ICER) protein represses CREmediated transcription
ICER = alternative to CREM (CREmodulator gene)
ICER opposes CREB in behavior
regulation to aid in adapting to
aversive & rewarding stimuli
ICER
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ICER = collective name for a group
of proteins produced from the
CREM/ICER gene
1st described as the major CREM
transcript in pineal gland during daynight flux of CREM expression
Studies suggest ICER is involved in
returning stimulated cells to the
steady-state level
Why Amphetamines?
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Amphetamines induce behavioral
response by releasing DA from
synapse
DA release after AMPH administration
is Ca2+ independent
Relies on cytosolic DA & vesicular DA
permeating to neuronal cytoplasm
from synaptic vesicles
Why Amphetamines?
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Repeated, intermittent Tx of rats w/
AMPH or cocaine enhances DA
release from striatum & NAc
Previously, AMPH induces CREmediated transcription w/in striatum
Materials & Methods
M&M
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Male Sprague Dawley rats
Pair-housed
12h light/dark cycle w/ dark @ 7pm
Procedures during the day
Rats killed by rapid decapitation
mRNA isolated & purified
mRNA rev transcribed into cDNA
ICER quantified using real-time PCR
M&M
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Amphetamine administered
intraperitoneally @ 0.5, 2, or 4 mg/kg
7 daily injections either saline or
AMPH
Acute = 6 days of saline + 1 day
AMPH
7d = 7 days of AMPH
Acute stressed rats in conical sleeve
for <60 min
M&M
Unpredictable stressed rats:
 Day 1: cage rotation 50 min @
12pm
 Day 2: wet cage 60 min @ 11pm,
lights overnight
 Day 3: lights off 180 min @ 12pm,
wet cage 15 min @ 3 pm
 Day 4: cage rotation 50 min @ 7pm,
food/H2O deprivation overnight
M&M
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Day 5: swim stress 3 min @ 1 pm,
isolation housing overnight
Day 6: restraint stress 60 min @
11 am, lights off 120 min @ 3 pm
Day 7: swim stress 4 min @ 10 am,
restraint stress 60 min @ 12 pm
M&M
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ICER mRNA levels in NAc measured
to acute stress
Killed 4 h after stresses
ICER & CREM cDNAs amplified from
rev transcribed mRNA
HSV (Herpes Simplex Virus)
mediated CREM & ICER transcription
was driven by a viral promoter
Enhanced green fluorescent protein
(GFP) used as control
M&M
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HSV vectors injected bilaterally in NAc
Behavioral measures taken 48-96h
after surgery
48h after injection, rats given 4mg/kg
AMPH
Brains harvested 3h later & frozen on
dry ice
M&M
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GFP used as injection site marker &
infected tissue micro dissected
RNA purified, amplified, and rev
transcribed
CREB target = brain-derived
neurotrophic factor (BDNF)
measured using real-time PCR
HSV-ICER vector was injected into
rat NAc shell
Expression verified using fluorescent
immunohistochemistry
M&M
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PC12 cells were infected w/ HSVLacZ or HSV-ICER & protein
harvested 12h after
ICER protein ID via Western Blot
90% HSV-LacZ or HSV-ICER & 10%
HCR-GFP, 2 separate groups of rats
injected into NAc
Chromatin immunoprecipitation
(ChIP) assays used to study ICER
binding to promoter
M&M
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Locomotor activity measured in
circular corridor via 4 photoelectric
cells in every 90° of circle
Spontaneous activity measured for
120 min in circular corridor chambers
AMPH-stim rats given 2 - 60 min
sessions 48h after surgery before
testing
Test day: 60 min in corridor before ip
injections of 2 or 4 mg/kg AMPH
M&M
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H2O removed w/ 1% sucrose in its
place but removed 48h before
surgery
Testing includes:
• Isolation @ 5pm w/ just food
• @ 7pm H2O & 1% sucrose bottles
for 10 min
• Neophobia: rats not pre-exposed &
30 min session
M&M
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Viral vectors injected into NAc
Post op day 3: 24h isolation
Post op day 4: measured social
behavior
Cage mates together in 50x50cm for
30 min
• Watched for social grooming,
exploring, playing, resting
M&M
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Force swim test rats in 25° H2O tubs
for 15 min then 5 min
Immobility: no struggling for 1s
(anti-depressive)
Elevated plus maze for 5 min
measured time in open arms
Peanut butter chip neophobia:
• Isolated rats 16h before testing
• PB chips in cup for 2h in home
cage during light phase
RESULTS
Figure 1
Results – Figure 1
AMPH on ICER overexpression
 AMPH increased ICER mRNA in
striatum (3h peak & 12h normal)
 AMPH regulation= dose dependent
decreasing w/ repeated use
• 1st dose= 3x increase
• 7th dose= 50% increase
• Shown over 24h
Figure 2
Results – Figure 2
Stress Tx
 Increased ICER mRNA 75% (1-4h
peak after stress & 12h normal)
 Smaller compared to AMPH
 Peak duration longer w/ acute
restraint stress
 Chronic restraint stress= no increase
 10 min forced swim or open arm
confinement increased ICER mRNA
4h after stress
Results
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Why HSV-ICER vector?
• Expresses ICER under viral promoter
control
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Compare mRNA levels of BDNF, CRE
target gene, in NAc w/ HSV-ICER or
HSV-LacZ
3h after 4mg/kg AMPH ICER
decreased BDNF mRNA
ChIP experiment: CREM/ICER
binding to BDNF promoter (ICER
induced)
Results
Further study:
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Injected HSV-ICER into NAc of CRELacZ reporter mice
3rd day (@ optimum HSV
expression): 1mg/kg AMPH
• Boost LacZ expression
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Observed 4h later
Figure 3
Results – Figure 3
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ICER caused decreased GFP
CRE activity not affected by viral
infection
HSV-ICER inhibits CRE transcription
in NAc
No significance in CREM
Figure 4
Results – Figure 4
Locomotor activity
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ICER overexpression in NAc:
Decreased spontaneous activity
during middle of test
ICER enhanced AMPH-induced
locomotor activity in entire test after
2 mg/kg dose
20-60min after AMPH= increase
Higher dosage? Not significant
Figure 5
Results – Figure 5
Natural reward
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ICER compared to LacZ expression
ICER increased sucrose preference in
familiarized animals
ICER increased social reward or grooming
time
Depression-related behavior
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ICER created an anti-depressant effect
Longer latency to immobility in forced
swim
Figure 6
Results – Figure 6
Anxiety-related behavior
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ICER increased anxiousness
More time spent on closed arms of
elevated plus maze
Animals avoided unfamiliar tastes or
neophobia (sucrose & PB chips)
Rats frequently turned away from sucrose
as a novel taste
But…enhanced preference when familiar
Neophobia is regulated by the NAc causing
increased anxiety
Discussion
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Stress & AMPH increase ICER
expression in striatum
Mimic effect by viral-mediated gene
transfer
Gradual peaks in behavior response
d/t delay in ICER induction so CREB
can induce transcription in target
genes - ICER translation
ICER overexpression in NAc =
increase response to AMPH
Discussion
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ICER affects other protein expression
which affect neuronal function –
cannot do it alone
CREB-ICER competition @ CRE sites
create behavioral response to stimuli
ICER = negative feedback shutting
down transcription normally induced
by CREB
Rats not as affected w/ repeated
stimulation – CREB contributes to
pathologic behavior d/t stress or
chronic AMPH
Thank You!