1. 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

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 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 – α 2 agonists (NE agonist) inhibits CRF by neg feed back  D-Phe-CRF 12-41 – 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. 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. 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. 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-HT 3, Egr 1, CRF Because the CRF antagonist can also block relapse in Hcrt treated mice, the system must work in conjunction with the stress pathways.