Transcript Paul Tschetter

Corticotropin-Releasing Factor within
the Central Nucleus of the Amygdala
Mediates Enhanced Ethanol SelfAdministration in Withdrawn, Ethanol
Dependent Rats
Funk et al
The Journal of Neuroscience
November 1 2006
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
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

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

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

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
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
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
 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
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
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

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 selfadministration during withdrawal and
protracted abstinence
Alcohol and CRF
Previously shown that increased anxietylike 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

Extended Amygdala
BNST
 NAcSh
 CeA

Purpose

Explore role of CRF with extended
amygdala
– In mediating excessive ethanol selfadministration during acute withdrawal
Methods and Materials
68 male Wistar rats
 Housed 2 or 3 per cage

– Food and water ad libitum

12 hour light/dark cycle
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
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

Operant EtOH Self-Administration
2 retractable levers located on either side
of drinking cups
 Fluid delivery & recording of operant selfadministration were controlled by
microcomputer
 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
– 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
 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
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
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
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

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

CRF Immunochemistry

Goat anti-CRF polyclonal antibody
Timeline
Operant Conditioning
4-5 days
4-5 days
4-5 days
4-5 days
Total
~20 days
Cannulation 5 days
Reestablish Baseline 2 weeks
Vapor Cage
4 weeks
Retest of Conditioning 2 hours withdrawal
or Terminal Sampling
Dependent Nondependent
Dependent Nondependent
Dependent Nondependent
Effects of CRF receptor
antagonist administered intra-CeA
on EtOH and water selfadministration in Dependent and
Nondependent rats
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)
Results
No difference in prevapor responding b/w
dependent and nondependent groups
 Mean BAL for entire EtOH vapor exposure
was 180 +/- 36.6 mg%

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%
Results

Statistics
– Significant effect of EtOH exposure
– Significant effect of CRFr Antagonist
– Revealed a significant reduction in EtOH selfadministration 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
 No dose of CRFr Ant was effective in
altering EtOH self-administration in
Nondependent rats

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
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

Summary
CRFr Ant decreased EtOH selfadministration in withdrawn, Dependent
but not Nondependent animals
 Administered directly into CeA

Effects of CRF receptor
antagonist administered intralateral BNST on EtOH and
water self-administration in
dependent and nondependent
rats
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)
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%

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
Results

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
– 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
 Animals test for EtOH & water selfadministration after 2 h or acute
withdrawal
 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
– 18.2+/-1.3 presses (Dependent)
– 17.7+/-1.4 presses (Nondependent)

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
 Mean BLA across entire period of vapor
exposure was 184.5+/-30.8mg%

Results

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
 No effect of CRFr Ant dose
 No interaction b/w EtOH exposure and
CRFr Ant

Results

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
 Animals test for EtOH & water selfadministration after 2 h or acute
withdrawal
 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
 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
CRF Immunoreactivity in CeA

Total number of CRF positive cells in CeA
was 1580+/-125.3 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
CRF immunoreactivity in lateral
BNST

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
CRF immunoreactivity in the NAcSh

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 selfadministration 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

Discussion

Provides key information about the
functional organization of CRF systems in
mediating critical motivational aspects of
alcohol dependence