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Role for Hypocretin in Mediating
Stress-Induced Reinstatement of
Cocaine-Seeking Behavior
Investigating the effects of Hypocretin-1+2
(Hcrt-1 / Hcrt-2)
on reinstatement of drug seeking
behavior through stress pathways
Hypocretin-1 and -2
(Hcrt-1 & -2)

Recently discovered Lateral Hypothalmic Neuropeptides
(1996) also known as Orexins

1+2 bind equally at Hcrt-R

Projections

Locus Ceruleus (Major) - NE

Dorsal Raphe Nuclei - 5-HT

Amygdala

Suprachiasmatic Nucleus – biological clock

Basal Forebrain

Cholinergic Brainstem

Spinal Cord
Hypocretin

Evidence points to excitatory function

↑energy expenditure, ↑feeding behavior, ↑locomotor activity.

Evidence indicates that Hcrt neurons drive hyper-arousal
through modulation of stress


Stress→ ↑CRF→ ↑Hypocretin
Role for Hcrt in reward seeking?
Hypocretin
(background)


Foot shock (FS) and
Restraint (RS) induce cFos expression in
Hypocretin neurons
Less so in neurons with
CRF-R knockouts
Materials and Methods
Animals

Male Wistar rats

12 hr light/dark cycle (lights off 10AM)

250-350 grams
Testing during dark cycle except during
intracranial-self stimulation testing.
1
2
3
4
(1)
Cocaine self-administration training


Two lever system

Active lever: light + .25 mg cocaine in saline iv. over 4s

20s timeout – pushes recorded but no cocaine delivered

7days 1hr sessions, then 5-7days 2hr session
When there was ≤20% variation in cocaine use
for three days the rats were considered “trained”
(1)
Cocaine extinction

Active lever


Light but no cocaine
2h sessions for minimum of 14 days
(1)
Drugs administered
1.
2.
After extinction, rats were given various
amounts of hypocretin icv
Various drugs that interfere with the stress
pathway were then given and active levers
were again introduced.

Clonidine – α2 agonists (NE agonist) inhibits CRF
by neg feed back

D-Phe-CRF12-41 – CRF antagonist
(2)
Footshock


Some rats from the previous groups were given
another extinction session similar to the first.(!)
Rats were given a Hcrt-Receptor antagonist,
SB-334867, then shocked


0.5mA for 0.5s intermittent for 15 min
Cocaine administration levers were then
introduced
(3)
Food Reinforcement





Similar to first experiment except with active lever
dispensing food pellets instead of cocaine.
During testing rats were food restricted to 14g of food
pellets/day
Self administered pellets were 45g
Training until stable intake, extinction, then
reinstatement with Hcrt.
Experiment was done with one and two levers.
(3)
Food Reinforcement

A similar group was brought up without food
reinforcement.

Active lever pushed turned light on but did not
deliver food.
(4)
Intra cerebral self stimulation





Electrode was implanted in medial fore brain bundle.

Stimulation causes Nucleus accumbens activity

Turns on measolimbic system
Three trials were preformed for each current intensity
Stimulus was applied in 5 micro Amp steps, in for
alternating and descending series.
Rat had 7.5s to respond on wheel to get an equal
stimulus.
If rat responded to two out of the three stimuli, it was
counted as the threshold
Results
(1)
Varied amount of Hypocretin and
reinstatement
(1)
Amount of Hypocretin



Increased in dose dependent fashion
0.3 nmol did not produce significantly different
results from saline control.
Pulls on inactive lever were never significantly
different suggesting increased locomotor
activity had to do with increasing active lever
pushes.
(1)
Stress Pathway Antagonists
(1)
Stress Pathway Antagonists

Evidence Suggests Hcrt + CRF interact during
stress response



Stress pathway is a major cause of drug relapse
Both Clonidine and D-Phe-CRF12-41 reduced
active lever hits in Hcrt treated mice.
When combined, drug seeking behavior was
extinguished
(2)
Foot shock
Hypocretin receptor blocker (SB 334867)
No added Hcrt
(2)
Foot shock



Used to test endogenous Hcrt systems and
their role in stress-induced drug relapse
Control rats display strong drug seeking relapse
after footshock
Rats treated with HcrtR-antagonist showed a
marked decrease in relapse proportional with
amount of inhibitor.
(3)
Food Training
(3)
Food Training


Hcrt increased lever responses in extinguished
rats previously trained to respond to food
reinforces
Hcrt only reinstated lever pushing in rats that
previously had the active lever paired with food
reward

Inactive levers were insignificant

Parallel results to drug seeking experiment
(4)
Inter Cerebral Self Stimulation
(4)
Inter Cerebral Self Stimulation



Mean thresholds

Saline 104.5 +/- 11.4 μA

Hcrt-1 129.9 +/- 13.6 μA
Hcrt produced long lasting increase in response
thresholds (between 24-36h)
Shows that the response Hcrt has on brain
reward center is negative unlike priming
Conclusions






Icv Infusions of Hcrt reinstated extinguished cocaine-seeking
behavior.
Hcrt-R antagonist blocks relapse.
Hcrt increases ICSS stimulation threshold, suppressing the
brain reward system. Cocaine priming typically lowers this
threshold
These data suggest that Hcrt reinstates cocaine seeking
through stress pathways and not dopamine release
Similar to: glucocoticoids, 5-HT3, Egr1, CRF
Because the CRF antagonist can also block relapse in Hcrt
treated mice, the system must work in conjunction with the
stress pathways.