1 / 37

Introduction

Induction of Inducible cAMP Early Repressor Expression in Nucleus Accumbens by Stress or Amphetamine Increases Behavioral Responses to Emotional Stimuli. Introduction. cAMP response element (CRE) is sensitive in many brain areas to drugs, stress, and reward

tamika
Download Presentation

Introduction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

  3. 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

  4. 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

  5. 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

  6. 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

  7. Materials & Methods

  8. M & M • 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

  9. M & M • 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

  10. 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

  11. M & M • 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. M & M • 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

  18. 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

  19. 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

  20. RESULTS

  21. Figure 1

  22. 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

  23. Figure 2

  24. 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

  25. 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)

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

  27. Figure 3

  28. 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

  29. Figure 4

  30. 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

  31. Figure 5

  32. 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

  33. Figure 6

  34. 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 • But…enhanced preference when familiar • Neophobia is regulated by the NAc causing increased anxiety

  35. 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

  36. Discussion • 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

  37. Thank You!

More Related