Role for Hypocretin in Mediating Stress-Induced Reinstatement of Cocaine-Seeking Behavior

1. Investigating the effects of Hypocretin-1+2 (Hcrt-1 / Hcrt-2)‏ on reinstatement of drug seeking behavior through stress pathways Role for Hypocretin in Mediating Stress-Induced Reinstatement of Cocaine-Seeking Behavior

2. 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

3. 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?

4. Hypocretin (background) • Foot shock (FS) and Restraint (RS) induce c-Fos expression in Hypocretin neurons • Less so in neurons with CRF-R knockouts

5. Materials and Methods

6. 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.

7. 1 2 3 4

8. (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”

9. (1)Cocaine extinction • Active lever • Light but no cocaine • 2h sessions for minimum of 14 days

10. (1)Drugs administered • 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

11. (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

12. (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.

13. (3)Food Reinforcement • A similar group was brought up without food reinforcement. • Active lever pushed turned light on but did not deliver food.

14. (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

15. Results

16. (1)Varied amount of Hypocretin and reinstatement

17. (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.

18. (1)Stress Pathway Antagonists

19. (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

20. (2)Foot shock Hypocretin receptor blocker (SB 334867) No added Hcrt

21. (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.

22. (3)Food Training

23. (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

24. (4)Inter Cerebral Self Stimulation

25. (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

26. 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.