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Factor within the Central Nucleus of the Amygdala Mediates Enhanced Ethanol Self- Administration in Withdrawn, Ethanol Dependent Rats Funk et al The Journal of Neuroscience November

Operant Conditioning Defined 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 Distinguished from Pavlovian Conditioning in that OC deals with the modification of voluntary behavior by using consequences –Reinforcement & Punishment

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

Alcoholism 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 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 Characterized by a shift from positive to negative reinforcement which ultimately drives continued alcohol use –Removing withdrawal symptoms by continued EtOH use

Cessation of chronic use leads to: 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 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 Similar to humans, ethanol dependent animals exhibit anxiety-like behaviors and excessive ethanol self-administration during periods of withdrawal

Homeostasis is the holding constant of internal parameters within the normal range Homeostasis is the holding constant of internal parameters within the normal range Allostasis is maintenance of stability at any level outside 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

Excessive drug use is hypothesized to involve such a change in internal mechanisms or set points 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 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 This change may play role in vulnerability to relapse Roberts et al 2000 Roberts et al 2000

 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

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

 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

EtOH Dependency (brief overview) Induced in rats Induced in rats Placing in operant chambers Placing in operant chambers Two bowls w levers on either side Two bowls w levers on either side Levers operate on FR1 to deliver water or sugar solution w/ % of alcohol 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 At this point rats trained for operant EtOH self-administration

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

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

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 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

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) 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 So…central administration of CRF antagonists can attenuate these behaviors

Extended Amygdala BNST BNST NAcSh NAcSh CeA CeA

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

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

Drugs Ethanol 10% for oral administration Ethanol 10% for oral administration CRF antagonist D-Phe-CRF CRF antagonist D-Phe-CRF –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 Injection given intracranially, 5 mins before operant self-administration testing

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

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

Operant EtOH Self-Administration Rats trained to press a lever for ethanol using a modification of sweetened solution fading procedure 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

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

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 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 Chambers activated by a timer that turns on vapor on 4:00pm and off 6:00am –14 hrs of vapor

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

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

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

CRF Immunochemistry Goat anti-CRF polyclonal antibody Goat anti-CRF polyclonal antibody

Timeline

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

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

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

Results When CRFr ant injected directly into CeA at varying concentrations 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 mg%

Results Statistics 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

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

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

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

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

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

Results Levels of EtOH lever responding b/f exposure to chronic vapors 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 For water lever responding prevapor –5.6+/-1.2 (Dependent) –6.8+/-1.9 (Nondependent)

Results So there was no difference in prevapor responding b/w the dependent and nondependent groups So there was no difference in prevapor responding b/w the dependent and nondependent groups Mean BAL for entire EtOH vapor exposure was / mg% Mean BAL for entire EtOH vapor exposure was / mg%

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

Results Statistics revealed Statistics revealed –Significant effect of EtOH exposure –No effect of CRFr Ant –No interaction b/w EtOH exposure and CRFr Ant dose

Results For water self-administration statistics showed For water self-administration statistics showed –No effect of EtOH vapor exposure –No effect of CRFr Ant dose –No interaction b/w EtOH exposure and CRFr Ant

Summary EtOH Dependence induced by intermittent exposure to EtOH vapors for 4 weeks EtOH Dependence induced by intermittent exposure to EtOH vapors for 4 weeks Animals test for EtOH & water self- administration after 2 h or acute withdrawal Animals test for EtOH & water self- administration after 2 h or acute withdrawal Withdrawn EtOH dependent animals displayed significant increase in EtOH lever pressing compared with Nondependents Withdrawn EtOH dependent animals displayed significant increase in EtOH lever pressing compared with Nondependents

Summary –CRFr Ant had no effect in either group on ethanol or water responding –Release into the BNST

Effects of CRF receptor antagonist administered intra-NAcSh on EtOH and water self-administration in dependent and nondependent rats

Results Levels of EtOH lever responding pre-vapor Levels of EtOH lever responding pre-vapor –18.2+/-1.3 presses (Dependent) –17.7+/-1.4 presses (Nondependent) Levels of water lever responding pre- vapor Levels of water lever responding pre- vapor –5.3+/-0.5 presses (Dependent) –3.4+/-0.4 presses (Independent)

Results No difference in prevapor responding b/w Dependent and Nondependent groups No difference in prevapor responding b/w Dependent and Nondependent groups Mean BLA across entire period of vapor exposure was /-30.8mg% Mean BLA across entire period of vapor exposure was /-30.8mg%

Results When CRFr ant injected directly into NAcSh at varying concentrations When CRFr ant injected directly into NAcSh at varying concentrations –After vehicle injection Dependent animals pressed ~61 times for 10% EtOH –Nondependents pressed 16 times

Results Significant effect of EtOH vapor exposure Significant effect of EtOH vapor exposure No effect of CRFr Ant dose No effect of CRFr Ant dose No interaction b/w EtOH exposure and CRFr Ant No interaction b/w EtOH exposure and CRFr Ant

Results Water self-administration Water self-administration –No effect of EtOH exposure –No effect of CRFr Ant dose –No interaction b/w EtOH exposure and CRFr Ant

Summary EtOH Dependence induced by intermittent exposure to EtOH vapors for 4 weeks EtOH Dependence induced by intermittent exposure to EtOH vapors for 4 weeks Animals test for EtOH & water self- administration after 2 h or acute withdrawal Animals test for EtOH & water self- administration after 2 h or acute withdrawal Withdrawn EtOH dependent animals displayed significant increase in EtOH lever pressing compared with Nondependents Withdrawn EtOH dependent animals displayed significant increase in EtOH lever pressing compared with Nondependents

Summary –CRFr Ant had no effect in either group on ethanol or water responding –Release into NAcSh

CRF Immunoreactivity in CeA Five 40µm serial sections through the lateral CeA for each animal used to analyze CRF immunoreactivity Five 40µm serial sections through the lateral CeA for each animal used to analyze CRF immunoreactivity Significant decrease in CRF immunoreactivity in CeA at points in withdrawn dependent animals Significant decrease in CRF immunoreactivity in CeA at points in withdrawn dependent animals

CRF Immunoreactivity in CeA Total CRF immunoreactivity was also significantly decreased in CeA Total CRF immunoreactivity was also significantly decreased in CeA

CRF Immunoreactivity in CeA Total number of CRF positive cells in CeA was 1580+/ for Nondependent Total number of CRF positive cells in CeA was 1580+/ for Nondependent –1295+/-80 for Dependent No significant difference between the two groups suggest the decrease in total CRF immunoreactivity w/in CeA reflects decrease in CRF w/in fibers No significant difference between the two groups suggest the decrease in total CRF immunoreactivity w/in CeA reflects decrease in CRF w/in fibers

CRF immunoreactivity in lateral BNST Was a trend towards a decrease in dependent animals Was a trend towards a decrease in dependent animals –No significant change in CRF immunoreactivity in BNST in Dependent Total CRF immunoreactivity was also not significantly altered in lateral BNST Total CRF immunoreactivity was also not significantly altered in lateral BNST

CRF immunoreactivity in the NAcSh No detectable CRF immunoreactivity within NAcSh of Dependent or Nondependent rats No detectable CRF immunoreactivity within NAcSh of Dependent or Nondependent rats –Data not shown

Discussion Data reported in paper showed that CRF antagonist, administered into the CeA significantly reduces EtOH self- administration in EtOH dependent rats Data reported in paper showed that CRF antagonist, administered into the CeA significantly reduces EtOH self- administration in EtOH dependent rats Suggests that after chronic EtOH, during withdrawal, dysregulation (allostasis) of brain CRF systems that appears to mediate negative reinforcement (withdrawals symptoms) associated with EtOH withdrawal Suggests that after chronic EtOH, during withdrawal, dysregulation (allostasis) of brain CRF systems that appears to mediate negative reinforcement (withdrawals symptoms) associated with EtOH withdrawal

Discussion Provides key information about the functional organization of CRF systems in mediating critical motivational aspects of alcohol dependence Provides key information about the functional organization of CRF systems in mediating critical motivational aspects of alcohol dependence