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 seekingbehavior 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 c-Fos expression in Hypocretin neurons

Less so in neurons with CRF-R knockouts

Materials and Methods

Animals

Male Wistar rats 250-350 grams

12 hr light/dark cycle (lights off 10AM) Testing during dark cycle except during

intracranial-self stimulation testing.

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(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. After extinction, rats were given various amounts of hypocretin icv

2. 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.