Funk et al The Journal of Neuroscience November 1 2006

1. Corticotropin-Releasing Factor within the Central Nucleus of the Amygdala Mediates Enhanced Ethanol Self-Administration in Withdrawn, Ethanol Dependent Rats Funk et al The Journal of Neuroscience November 1 2006

2. Operant Conditioning • Defined • Use of consequences to modify the occurrence and form of behavior • Distinguished from Pavlovian Conditioning in that OC deals with the modification of voluntary behavior by using consequences • Reinforcement & Punishment

3. Operant Conditioning • Positive reinforcement • Adding a stimulus to reinforce a behavior • Negative reinforcement • Removing a stimulus to reinforce a behavior • Pavlovian Conditioning • deals with the conditioning of behavior so that it occurs under new conditions

4. Alcoholism • Chronic relapsing disorder characterized by compulsive use of alcohol, and loss of control over intake • Dependence develops, shift from controlled use to uncontrolled, excessive consumption • Characterized by a shift from positive to negative reinforcement which ultimately drives continued alcohol use • Removing withdrawal symptoms by continued EtOH use

5. Cessation of chronic use leads to: • Negative emotional symptoms like increased anxiety • Alleviation of negative emotional states hypothesized to be major factor for continued alcohol consumption • Similar to humans, ethanol dependent animals exhibit anxiety-like behaviors and excessive ethanol self-administration during periods of withdrawal

6. Homeostasis is the holding constant of internal parameters within the normal range • Allostasis is maintenance of stability at any level outside the normal range • Achieved by varying the internal environment to match perceived and anticipated environmental demands • So as certain demands/conditions become chronic the set point for functioning is altered and may be maintained

7. Excessive drug use is hypothesized to involve such a change in internal mechanisms or set points • Chronic drug or alcohol exposure may elicit allostasis w/in the brain’s reward mechanisms as a means to maintain stability in the face of chronic demand • This change may play role in vulnerability to relapse • Roberts et al 2000

8. Stress increases CRF release from PVN • CRF increases ACTH release from PIT • ACTH increases Glucocorticoid release • High Gluco levels produce feedback on several different levels • Koob and Le Moal 2000

9. Stress also activates CRF systems in basal forebrain • BNST • CeA • To mediate sympathetic activation • Koob and Le Moal 2000

10. Glucocorticoids instead of inducing inhibition of CRF synthesis actually increase synthesis of CRF • Provides means for contribution of brain stress systems to allostasis • Koob and Le Moal 2000

11. EtOH Dependency (brief overview) • Induced in rats • Placing in operant chambers • Two bowls w levers on either side • Levers operate on FR1 to deliver water or sugar solution w/ % of alcohol • Rats allowed to respond for time period/day for 4-6 weeks • At this point rats trained for operant EtOH self-administration

12. Chronic exposure • Rats now placed in vapor chamber and chronically administered EtOH • Able to closely monitor BAL • Or placed in vapor chamber and chronically administered water

13. Roberts et al 2000 • Previously shown that EtOH dependent rats increased operant responding for EtOH when tested during first 12 hr after withdrawal • This study indicated that operant responding for EtOH was enhanced during protracted abstinence by 30-100% and remained elevated for 4-8 weeks

15. Roberts et al 2000 • The principal result of this experiment was that rats w/ history of chronic EtOH exposure, sufficient to produce signs of physical dependence, showed persistent increases in operant EtOH self-administration during withdrawal and protracted abstinence

16. Alcohol and CRF • Previously shown that increased anxiety-like behaviors during ethanol withdrawal are believed to result, in part, from increased extracellular CRF within the extended amygdala (from the extrahypothalamic CRF system) • So…central administration of CRF antagonists can attenuate these behaviors

17. Extended Amygdala • BNST • NAcSh • CeA

19. Purpose • Explore role of CRF with extended amygdala • In mediating excessive ethanol self-administration during acute withdrawal

20. Methods and Materials • 68 male Wistar rats • Housed 2 or 3 per cage • Food and water ad libitum • 12 hour light/dark cycle

21. Drugs • Ethanol 10% for oral administration • CRF antagonist D-Phe-CRF12-41 • Immediately before use dissolved in 0.5xPBS pH 7.4, kept on ice • Varying concentrations • Injection given intracranially, 5 mins before operant self-administration testing

22. Operant EtOH Self-Administration • Established in standard operant chambers • Housed in sound-attenuated ventilated cubicles • Animals trained to self administer ethanol or water in concurrent, 2 lever, free choice contingency • Syringe pumps dispensed ethanol/water into drinking cups

23. Operant EtOH Self-Administration • 2 retractable levers located on either side of drinking cups • Fluid delivery & recording of operant self-administration were controlled by microcomputer • Lever presses not recorded during 0.5s in which pumps were active • FR1 resulted in 0.1mL of fluid

24. Operant EtOH Self-Administration • Rats trained to press a lever for ethanol using a modification of sweetened solution fading procedure • Start with very sweet and proceed to less sweet ethanol solution • Culminates in rats consuming unsweetened 10% EtOH to produce pharmacologically relevant BALs

25. Operant EtOH Self-Administration • During training rats allowed to press for water on the opposite lever • Whether the lever dispensed water or EtOH was switched daily • Daily 30 min access to EtOH for 20-25 days until stable rates of intake observed • +/- 20% across three consecutive sessions

26. EtOH Vapor Exposure Procedure • To induce EtOH dependence, 2 standard cages house in separate, sealed, clear plastic chambers into which EtOH vapor was released intermittently • Chambers activated by a timer that turns on vapor on 4:00pm and off 6:00am • 14 hrs of vapor

27. EtOH Vapor Exposure Procedure • Tail blood samples taken, target BALs were 150-200mg% across 4 week exposure • This paradigm shown to produce physical dependence • Appearance of somatic withdrawal signs after removal from chambers

28. Cannulation & Infusion • Rats anesthetized with isoflurane • Cannulas implanted bilaterally • Rats allowed 5 days recovery • Injections were 0.5µL per slide over 1 min • After 5 mins the animals place back in self-administration chambers for testing

29. Histology • At experiment completion, animals killed w pentobarbitol and transfused transcardially • 1st with saline • 2nd with 4%paraformaldehyde • Brains removed and frozen, sectioned in 60µL slices, mounted and stained with cresyl violet • Animals with incorrect placement not used

30. CRF Immunochemistry • Goat anti-CRF polyclonal antibody

31. Timeline

32. Effects of CRF receptor antagonist administered intra-CeA on EtOH and water self-administration in Dependent and Nondependent rats

33. Results • Levels of EtOH lever responding b/f chronic vapors 20.7+/- 1.6 (dependent) • 19.3+/-1.9 (nondependent) • Prevapor levels of water lever responding were 7.5+/- 1.3 (dependent) • 8.4+/- 1.3 (nondependent)

34. Results • No difference in prevapor responding b/w dependent and nondependent groups • Mean BAL for entire EtOH vapor exposure was 180 +/- 36.6 mg%

35. Results • When CRFr ant injected directly into CeA at varying concentrations • Vehicle injection of PBS • Dependent 67 presses • Nondependent 20.2 presses • BAL of 25-50mg% to 125-150mg%

37. Results • Statistics • Significant effect of EtOH exposure • Significant effect of CRFr Antagonist • Revealed a significant reduction in EtOH self-administration in Dependent rats at 0.5µg/µL compared with 0µg/µL

39. Results • 0.5µg/µL of CRFr Ant dose showed NO difference b/w the Dependent and Nondependent groups • No dose of CRFr Ant was effective in altering EtOH self-administration in Nondependent rats

41. Results • For water self-administration • no effect of EtOH vapor exposure • No effect of CRFr Ant • No interaction b/w EtOH exposure and CRFr Ant

44. Summary • EtOH Dependence was induced by intermittent exposure to EtOH vapors for 4 weeks • Withdrawn, EtOH dependent animals displayed a significant increase in EtOH lever pressing compared with Nondependent animals

45. Summary • CRFr Ant decreased EtOH self-administration in withdrawn, Dependent but not Nondependent animals • Administered directly into CeA

46. Effects of CRF receptor antagonist administered intra-lateral BNST on EtOH and water self-administration in dependent and nondependent rats

47. Results • Levels of EtOH lever responding b/f exposure to chronic vapors • 17.5+/-0.9 presses (Dependent) • 15.7+/-0.9 presses (Nondependent) • For water lever responding prevapor • 5.6+/-1.2 (Dependent) • 6.8+/-1.9 (Nondependent)

48. Results • So there was no difference in prevapor responding b/w the dependent and nondependent groups • Mean BAL for entire EtOH vapor exposure was 179.2 +/- 32.7 mg%

49. Results • When CRFr ant injected directly into BNST at varying concentrations • After vehicle Dependents pressed 65.2 times for 10%EtOH • 19 times for Nondependents