Induction of Inducible cAMP Early Repressor Expression in Nucleus Accumbens by Stress or Amphetamine Increases Behavioral Responses to Emotional Stimuli

1
Induction of Inducible cAMP Early Repressor
Expression in Nucleus Accumbens by Stress or
Amphetamine Increases Behavioral Responses to
Emotional Stimuli
2
Introduction

• 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

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ICER

• Inducible cAMP early repressor (ICER) protein
  represses CRE-mediated transcription
• ICER alternative to CREM (CRE-modulator gene)
• ICER opposes CREB in behavior regulation to aid
  in adapting to aversive rewarding stimuli

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ICER

• 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 day-night flux of CREM
  expression
• Studies suggest ICER is involved in returning
  stimulated cells to the steady-state level

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Why Amphetamines?

• 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

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Why Amphetamines?

• Repeated, intermittent Tx of rats w/ AMPH or
  cocaine enhances DA release from striatum NAc
• Previously, AMPH induces CRE-mediated
  transcription w/in striatum

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Materials Methods
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M M

• Male Sprague Dawley rats
• Pair-housed
• 12h light/dark cycle w/ dark _at_ 7pm
• Procedures during the day
• Rats killed by rapid decapitation
• mRNA isolated purified
• mRNA rev transcribed into cDNA
• ICER quantified using real-time PCR

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M M

• Amphetamine administered intraperitoneally _at_ 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 lt60 min

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M M

• Unpredictable stressed rats
• Day 1 cage rotation 50 min _at_ 12pm
• Day 2 wet cage 60 min _at_ 11pm, lights overnight
• Day 3 lights off 180 min _at_ 12pm, wet cage 15 min
  _at_ 3 pm
• Day 4 cage rotation 50 min _at_ 7pm, food/H2O
  deprivation overnight

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M M

• Day 5 swim stress 3 min _at_ 1 pm, isolation
  housing overnight
• Day 6 restraint stress 60 min _at_ 11 am, lights
  off 120 min _at_ 3 pm
• Day 7 swim stress 4 min _at_ 10 am, restraint
  stress 60 min _at_ 12 pm

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M M

• 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

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M M

• 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

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M M

• 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

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M M

• PC12 cells were infected w/ HSV-LacZ 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

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M M

• 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

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M M

• H2O removed w/ 1 sucrose in its place but
  removed 48h before surgery
• Testing includes
• Isolation _at_ 5pm w/ just food
• _at_ 7pm H2O 1 sucrose bottles for 10 min
• Neophobia rats not pre-exposed
• 30 min session

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M M

• 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

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M M

• 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

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RESULTS
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Figure 1
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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

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Figure 2
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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

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Results

• Why HSV-ICER vector?
• Expresses ICER under viral promoter control
• 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)

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Results

• Further study
• Injected HSV-ICER into NAc of CRE-LacZ reporter
  mice
• 3rd day (_at_ optimum HSV expression) 1mg/kg AMPH
• Boost LacZ expression
• Observed 4h later

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Figure 3
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Results Figure 3

• ICER caused decreased GFP
• CRE activity not affected by viral infection
• HSV-ICER inhibits CRE transcription in NAc
• No significance in CREM

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Figure 4
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Results Figure 4

• Locomotor activity
• 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

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Figure 5
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Results Figure 5

• Natural reward
• ICER compared to LacZ expression
• ICER increased sucrose preference in familiarized
  animals
• ICER increased social reward or grooming time
• Depression-related behavior
• ICER created an anti-depressant effect
• Longer latency to immobility in forced swim

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Figure 6
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Results Figure 6

• Anxiety-related behavior
• 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
• Butenhanced preference when familiar
• Neophobia is regulated by the NAc causing
  increased anxiety

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Discussion

• 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

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Discussion

• ICER affects other protein expression which
  affect neuronal function cannot do it alone
• CREB-ICER competition _at_ 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

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Thank You!