Corticotropin-Releasing Factor Corticotropin-Releasing Factor within the Central Nucleus of the within the Central Nucleus of the

Amygdala Mediates Enhanced Amygdala Mediates Enhanced Ethanol Self-Administration in Ethanol Self-Administration in

Withdrawn, Ethanol Dependent Withdrawn, Ethanol Dependent RatsRats

Funk et alFunk et alThe Journal of NeuroscienceThe Journal of Neuroscience

November 1 2006November 1 2006

Operant ConditioningOperant Conditioning

DefinedDefined– Use of consequences to modify the Use of consequences to modify the

occurrence and form of behavioroccurrence and form of behavior Distinguished from Pavlovian Distinguished from Pavlovian

Conditioning in that OC deals with Conditioning in that OC deals with the modification of voluntary the modification of voluntary behavior by using consequencesbehavior by using consequences– Reinforcement & PunishmentReinforcement & Punishment

Operant ConditioningOperant Conditioning

Positive reinforcementPositive reinforcement– Adding a stimulus to reinforce a behaviorAdding a stimulus to reinforce a behavior

Negative reinforcementNegative reinforcement– Removing a stimulus to reinforce a Removing a stimulus to reinforce a

behaviorbehavior

Pavlovian ConditioningPavlovian Conditioning– deals with the conditioning of behavior so deals with the conditioning of behavior so

that it occurs under new conditions that it occurs under new conditions

AlcoholismAlcoholism– Chronic relapsing disorder characterized Chronic relapsing disorder characterized

by compulsive use of alcohol, and loss of by compulsive use of alcohol, and loss of control over intakecontrol over intake

Dependence develops, shift from Dependence develops, shift from controlled use to uncontrolled, controlled use to uncontrolled, excessive consumptionexcessive consumption

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

continued EtOH usecontinued EtOH use

Cessation of chronic use leads to:Cessation of chronic use leads to:– Negative emotional symptoms like Negative emotional symptoms like

increased anxietyincreased anxiety Alleviation of negative emotional Alleviation of negative emotional

states hypothesized to be major factor states hypothesized to be major factor for continued alcohol consumptionfor continued alcohol consumption

Similar to humans, ethanol dependent Similar to humans, ethanol dependent animals exhibit anxiety-like behaviors animals exhibit anxiety-like behaviors and excessive ethanol self-and excessive ethanol self-administration during periods of administration during periods of withdrawalwithdrawal

HomeostasisHomeostasis is the holding constant is the holding constant of internal parameters within the of internal parameters within the normal rangenormal range

AllostasisAllostasis is maintenance of stability is maintenance of stability at any level at any level outsideoutside the normal range the normal range– Achieved by varying the internal Achieved by varying the internal

environment to match perceived and environment to match perceived and anticipated environmental demandsanticipated environmental demands

– So as certain demands/conditions become So as certain demands/conditions become chronic the set point for functioning is chronic the set point for functioning is altered and may be maintainedaltered and may be maintained

Excessive drug use is hypothesized Excessive drug use is hypothesized to involve such a change in internal to involve such a change in internal mechanisms or set pointsmechanisms or set points

Chronic drug or alcohol exposure Chronic drug or alcohol exposure may elicit allostasis w/in the brain’s may elicit allostasis w/in the brain’s reward mechanisms as a means to reward mechanisms as a means to maintain stability in the face of maintain stability in the face of chronic demandchronic demand

This change may play role in This change may play role in vulnerability to relapsevulnerability to relapse

Roberts et al 2000Roberts 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 activationKoob 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 allostasisKoob and Le Moal 2000

EtOH Dependency (brief EtOH Dependency (brief overview)overview)

Induced in rats Induced in rats Placing in operant chambersPlacing in operant chambers Two bowls w levers on either sideTwo bowls w levers on either side Levers operate on FR1 to deliver water Levers operate on FR1 to deliver water

or sugar solution w/ % of alcoholor sugar solution w/ % of alcohol– Rats allowed to respond for time Rats allowed to respond for time

period/day for 4-6 weeksperiod/day for 4-6 weeks At this point rats trained for operant At this point rats trained for operant

EtOH self-administrationEtOH self-administration

Chronic exposure Chronic exposure

Rats now placed in vapor chamber Rats now placed in vapor chamber and chronically administered EtOH and chronically administered EtOH – Able to closely monitor BALAble to closely monitor BAL

Or placed in vapor chamber and Or placed in vapor chamber and chronically administered waterchronically administered water

Roberts et al 2000Roberts et al 2000

Previously shown that EtOH Previously shown that EtOH dependent rats increased operant dependent rats increased operant responding for EtOH when tested responding for EtOH when tested during first 12 hr after withdrawalduring first 12 hr after withdrawal

This study indicated that operant This study indicated that operant responding for EtOH was enhanced responding for EtOH was enhanced during protracted abstinence by 30-during protracted abstinence by 30-100% and remained elevated for 4-8 100% and remained elevated for 4-8 weeksweeks

Roberts et al 2000Roberts et al 2000

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

Alcohol and CRFAlcohol and CRF

Previously shown that increased Previously shown that increased anxiety-like behaviors during ethanol anxiety-like behaviors during ethanol withdrawal are believed to result, in withdrawal are believed to result, in part, from increased extracellular CRF part, from increased extracellular CRF within the extended amygdala (from within the extended amygdala (from the extrahypothalamic CRF system)the extrahypothalamic CRF system)

So…central administration of CRF So…central administration of CRF antagonists can attenuate these antagonists can attenuate these behaviorsbehaviors

Extended AmygdalaExtended Amygdala

BNSTBNST NAcShNAcSh CeACeA

PurposePurpose

Explore role of CRF with extended Explore role of CRF with extended amygdalaamygdala– In mediating excessive ethanol self-In mediating excessive ethanol self-

administration during acute withdrawaladministration during acute withdrawal

Methods and MaterialsMethods 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 Food and water ad libitumad libitum 12 hour light/dark cycle12 hour light/dark cycle

DrugsDrugs

Ethanol 10% for oral administrationEthanol 10% for oral administration CRF antagonist D-Phe-CRFCRF antagonist D-Phe-CRF12-4112-41

– Immediately before use dissolved in Immediately before use dissolved in 0.5xPBS pH 7.4, kept on ice0.5xPBS pH 7.4, kept on ice

– Varying concentrationsVarying concentrations Injection given intracranially, 5 mins Injection given intracranially, 5 mins

before operant self-administration before operant self-administration testingtesting

Operant EtOH Self-Operant EtOH Self-AdministrationAdministration

Established in standard operant Established in standard operant chamberschambers– Housed in sound-attenuated ventilated Housed in sound-attenuated ventilated

cubiclescubicles Animals trained to self administer Animals trained to self administer

ethanol or water in concurrent, 2 ethanol or water in concurrent, 2 lever, free choice contingencylever, free choice contingency

Syringe pumps dispensed Syringe pumps dispensed ethanol/water into drinking cupsethanol/water into drinking cups

Operant EtOH Self-Operant EtOH Self-AdministrationAdministration

2 retractable levers located on either 2 retractable levers located on either side of drinking cupsside of drinking cups

Fluid delivery & recording of operant Fluid delivery & recording of operant self-administration were controlled self-administration were controlled by microcomputerby microcomputer

Lever presses not recorded during Lever presses not recorded during 0.5s in which pumps were active0.5s in which pumps were active– FR1 resulted in 0.1mL of fluidFR1 resulted in 0.1mL of fluid

Operant EtOH Self-Operant EtOH Self-AdministrationAdministration

Rats trained to press a lever for Rats trained to press a lever for ethanol using a modification of ethanol using a modification of sweetened solution fading proceduresweetened solution fading procedure– Start with very sweet and proceed to Start with very sweet and proceed to

less sweet ethanol solutionless sweet ethanol solution– Culminates in rats consuming Culminates in rats consuming

unsweetened 10% EtOH to produce unsweetened 10% EtOH to produce pharmacologically relevant BALspharmacologically relevant BALs

Operant EtOH Self-Operant EtOH Self-AdministrationAdministration

During training rats allowed to press During training rats allowed to press for water on the opposite leverfor water on the opposite lever

Whether the lever dispensed water Whether the lever dispensed water or EtOH was switched dailyor EtOH was switched daily

Daily 30 min access to EtOH for 20-Daily 30 min access to EtOH for 20-25 days until stable rates of intake 25 days until stable rates of intake observedobserved– +/- 20% across three consecutive +/- 20% across three consecutive

sessionssessions

EtOH Vapor Exposure EtOH Vapor Exposure ProcedureProcedure

To induce EtOH dependence, 2 To induce EtOH dependence, 2 standard cages house in separate, standard cages house in separate, sealed, clear plastic chambers into sealed, clear plastic chambers into which EtOH vapor was released which EtOH vapor was released intermittentlyintermittently

Chambers activated by a timer that Chambers activated by a timer that turns on vapor on 4:00pm and off turns on vapor on 4:00pm and off 6:00am6:00am– 14 hrs of vapor14 hrs of vapor

EtOH Vapor Exposure EtOH Vapor Exposure ProcedureProcedure

Tail blood samples taken, target Tail blood samples taken, target BALs were 150-200mg% across 4 BALs were 150-200mg% across 4 week exposureweek exposure

This paradigm shown to produce This paradigm shown to produce physical dependence physical dependence – Appearance of somatic withdrawal signs Appearance of somatic withdrawal signs

after removal from chambersafter removal from chambers

Cannulation & InfusionCannulation & Infusion

Rats anesthetized with isofluraneRats anesthetized with isoflurane Cannulas implanted bilaterallyCannulas implanted bilaterally

– Rats allowed 5 days recoveryRats allowed 5 days recovery Injections were 0.5Injections were 0.5µL per slide over 1 µL per slide over 1

minmin After 5 mins the animals place back After 5 mins the animals place back

in self-administration chambers for in self-administration chambers for testingtesting

HistologyHistology

At experiment completion, animals killed At experiment completion, animals killed w pentobarbitol and transfused w pentobarbitol and transfused transcardiallytranscardially– 11stst with saline with saline– 22ndnd with 4%paraformaldehyde with 4%paraformaldehyde

Brains removed and frozen, sectioned in Brains removed and frozen, sectioned in 6060µL slices, mounted and stained with µL slices, mounted and stained with cresyl violetcresyl violet

Animals with incorrect placement not Animals with incorrect placement not usedused

CRF ImmunochemistryCRF Immunochemistry

Goat anti-CRF polyclonal antibodyGoat anti-CRF polyclonal antibody

TimelineTimeline

Operant Conditioning 4-5 days4-5 days4-5 days4-5 days

Total ~20 daysCannulation 5 days

Reestablish Baseline 2 weeks Dependent NondependentVapor Cage 4 weeks Dependent Nondependent

Retest of Conditioning 2 hours withdrawal Dependent Nondependent or Terminal Sampling

Effects of CRF receptor antagonist administered intra-CeA on EtOH and water self-

administration in Dependent and Nondependent rats

ResultsResults

Levels of EtOH lever responding b/f Levels of EtOH lever responding b/f chronic vapors 20.7+/- 1.6 chronic vapors 20.7+/- 1.6 (dependent)(dependent)– 19.3+/-1.9 (nondependent)19.3+/-1.9 (nondependent)

Prevapor levels of water lever Prevapor levels of water lever responding were 7.5+/- 1.3 responding were 7.5+/- 1.3 (dependent)(dependent)– 8.4+/- 1.3 (nondependent)8.4+/- 1.3 (nondependent)

ResultsResults

No difference in prevapor responding No difference in prevapor responding b/w dependent and nondependent b/w dependent and nondependent groupsgroups

Mean BAL for entire EtOH vapor Mean BAL for entire EtOH vapor exposure was 180 +/- 36.6 mg%exposure was 180 +/- 36.6 mg%

ResultsResults

When CRFr ant injected directly into When CRFr ant injected directly into CeA at varying concentrationsCeA at varying concentrations– Vehicle injection of PBSVehicle injection of PBS

Dependent 67 pressesDependent 67 presses Nondependent 20.2 pressesNondependent 20.2 presses

– BAL of 25-50mg% to 125-150mg%BAL of 25-50mg% to 125-150mg%

ResultsResults

Statistics Statistics – Significant effect of EtOH exposureSignificant effect of EtOH exposure– Significant effect of CRFr AntagonistSignificant effect of CRFr Antagonist– Revealed a significant reduction in EtOH Revealed a significant reduction in EtOH

self-administration in Dependent rats at self-administration in Dependent rats at 0.50.5µg/µL compared with 0µg/µL µg/µL compared with 0µg/µL

ResultsResults

0.50.5µg/µL of CRFr Ant dose showed µg/µL of CRFr Ant dose showed NO difference b/w the Dependent NO difference b/w the Dependent and Nondependent groupsand Nondependent groups

No dose of CRFr Ant was effective in No dose of CRFr Ant was effective in altering EtOH self-administration in altering EtOH self-administration in Nondependent ratsNondependent rats

ResultsResults

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

CRFr AntCRFr Ant

SummarySummary

EtOH Dependence was induced by EtOH Dependence was induced by intermittent exposure to EtOH vapors intermittent exposure to EtOH vapors for 4 weeksfor 4 weeks

Withdrawn, EtOH dependent animals Withdrawn, EtOH dependent animals displayed a significant increase in displayed a significant increase in EtOH lever pressing compared with EtOH lever pressing compared with Nondependent animalsNondependent animals

SummarySummary

CRFr Ant decreased EtOH self-CRFr Ant decreased EtOH self-administration in withdrawn, administration in withdrawn, Dependent but not Nondependent Dependent but not Nondependent animalsanimals

Administered directly into CeAAdministered directly into CeA

Effects of CRF receptor antagonist administered intra-

lateral BNST on EtOH and water self-administration in

dependent and nondependent rats

ResultsResults

Levels of EtOH lever responding b/f Levels of EtOH lever responding b/f exposure to chronic vaporsexposure to chronic vapors– 17.5+/-0.9 presses (Dependent)17.5+/-0.9 presses (Dependent)– 15.7+/-0.9 presses (Nondependent)15.7+/-0.9 presses (Nondependent)

For water lever responding prevaporFor water lever responding prevapor– 5.6+/-1.2 (Dependent)5.6+/-1.2 (Dependent)– 6.8+/-1.9 (Nondependent)6.8+/-1.9 (Nondependent)

ResultsResults

So there was no difference in So there was no difference in prevapor responding b/w the prevapor responding b/w the dependent and nondependent dependent and nondependent groupsgroups

Mean BAL for entire EtOH vapor Mean BAL for entire EtOH vapor exposure was 179.2 +/- 32.7 mg%exposure was 179.2 +/- 32.7 mg%

ResultsResults

When CRFr ant injected directly into When CRFr ant injected directly into BNST at varying concentrations BNST at varying concentrations – After vehicle Dependents pressed 65.2 After vehicle Dependents pressed 65.2

times for 10%EtOHtimes for 10%EtOH– 19 times for Nondependents19 times for Nondependents

ResultsResults

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

CRFr Ant doseCRFr Ant dose

ResultsResults

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

CRFr AntCRFr Ant

SummarySummary

EtOH Dependence induced by EtOH Dependence induced by intermittent exposure to EtOH vapors intermittent exposure to EtOH vapors for 4 weeksfor 4 weeks

Animals test for EtOH & water self-Animals test for EtOH & water self-administration after 2 h or acute administration after 2 h or acute withdrawalwithdrawal

Withdrawn EtOH dependent animals Withdrawn EtOH dependent animals displayed significant increase in EtOH displayed significant increase in EtOH lever pressing compared with lever pressing compared with NondependentsNondependents

SummarySummary

– CRFr Ant had no effect in either group CRFr Ant had no effect in either group on ethanol or water respondingon ethanol or water responding

– Release into the BNSTRelease into the BNST

Effects of CRF receptor antagonist administered intra-NAcSh on EtOH

and water self-administration in dependent and nondependent rats

ResultsResults

Levels of EtOH lever responding pre-Levels of EtOH lever responding pre-vaporvapor– 18.2+/-1.3 presses (Dependent)18.2+/-1.3 presses (Dependent)– 17.7+/-1.4 presses (Nondependent)17.7+/-1.4 presses (Nondependent)

Levels of water lever responding pre-Levels of water lever responding pre-vaporvapor– 5.3+/-0.5 presses (Dependent)5.3+/-0.5 presses (Dependent)– 3.4+/-0.4 presses (Independent)3.4+/-0.4 presses (Independent)

ResultsResults

No difference in prevapor No difference in prevapor responding b/w Dependent and responding b/w Dependent and Nondependent groupsNondependent groups

Mean BLA across entire period of Mean BLA across entire period of vapor exposure was 184.5+/-30.8mgvapor exposure was 184.5+/-30.8mg%%

Results Results

When CRFr ant injected directly into When CRFr ant injected directly into NAcSh at varying concentrationsNAcSh at varying concentrations– After vehicle injection Dependent After vehicle injection Dependent

animals pressed ~61 times for 10% animals pressed ~61 times for 10% EtOHEtOH

– Nondependents pressed 16 timesNondependents pressed 16 times

ResultsResults

Significant effect of EtOH vapor Significant effect of EtOH vapor exposureexposure

No effect of CRFr Ant dose No effect of CRFr Ant dose No interaction b/w EtOH exposure No interaction b/w EtOH exposure

and CRFr Antand CRFr Ant

ResultsResults

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

CRFr AntCRFr Ant

SummarySummary

EtOH Dependence induced by EtOH Dependence induced by intermittent exposure to EtOH vapors intermittent exposure to EtOH vapors for 4 weeksfor 4 weeks

Animals test for EtOH & water self-Animals test for EtOH & water self-administration after 2 h or acute administration after 2 h or acute withdrawalwithdrawal

Withdrawn EtOH dependent animals Withdrawn EtOH dependent animals displayed significant increase in EtOH displayed significant increase in EtOH lever pressing compared with lever pressing compared with NondependentsNondependents

SummarySummary

– CRFr Ant had no effect in either group CRFr Ant had no effect in either group on ethanol or water respondingon ethanol or water responding

– Release into NAcShRelease into NAcSh

CRF Immunoreactivity in CRF Immunoreactivity in CeACeA

Five 40Five 40µm serial sections through µm serial sections through the lateral CeA for each animal used the lateral CeA for each animal used to analyze CRF immunoreactivityto analyze CRF immunoreactivity

Significant decrease in CRF Significant decrease in CRF immunoreactivity in CeA at points in immunoreactivity in CeA at points in withdrawn dependent animalswithdrawn dependent animals

CRF Immunoreactivity in CRF Immunoreactivity in CeACeA

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

CRF Immunoreactivity in CRF Immunoreactivity in CeACeA

Total number of CRF positive cells in Total number of CRF positive cells in CeA was 1580+/-125.3 for CeA was 1580+/-125.3 for NondependentNondependent– 1295+/-80 for Dependent 1295+/-80 for Dependent

No significant difference between the No significant difference between the two groups suggest the decrease in two groups suggest the decrease in total CRF immunoreactivity w/in CeA total CRF immunoreactivity w/in CeA reflects decrease in CRF w/in fibersreflects decrease in CRF w/in fibers

CRF immunoreactivity in CRF immunoreactivity in lateral BNSTlateral BNST

Was a trend towards a decrease in Was a trend towards a decrease in dependent animalsdependent animals– No significant change in CRF No significant change in CRF

immunoreactivity in BNST in Dependentimmunoreactivity in BNST in Dependent Total CRF immunoreactivity was also Total CRF immunoreactivity was also

not significantly altered in lateral not significantly altered in lateral BNSTBNST

CRF immunoreactivity in the CRF immunoreactivity in the NAcShNAcSh

No detectable CRF immunoreactivity No detectable CRF immunoreactivity within NAcSh of Dependent or within NAcSh of Dependent or Nondependent ratsNondependent rats– Data not shownData not shown

DiscussionDiscussion

Data reported in paper showed that Data reported in paper showed that CRF antagonist, administered into the CRF antagonist, administered into the CeA significantly reduces EtOH self-CeA significantly reduces EtOH self-administration in EtOH dependent ratsadministration in EtOH dependent rats

Suggests that after chronic EtOH, Suggests that after chronic EtOH, during withdrawal, dysregulation during withdrawal, dysregulation (allostasis) of brain CRF systems that (allostasis) of brain CRF systems that appears to mediate negative appears to mediate negative reinforcement (withdrawals reinforcement (withdrawals symptoms) associated with EtOH symptoms) associated with EtOH withdrawalwithdrawal

DiscussionDiscussion

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