corticotropin-releasing factor within the central nucleus of the amygdala mediates enhanced ethanol...
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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Withdrawn, Ethanol Dependent Withdrawn, Ethanol Dependent RatsRats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
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