2. M.Sabrina Spano, Maria Ellgren, X. Wang, Yasmin L. Hurd
Prenatal Cannabis Exposure Increases Heroin Seeking with Allostatic Changes in Limbic Enkephalin Systems in Adulthood
M.Sabrina Spano, Maria Ellgren, X. Wang, Yasmin L. Hurd

3. THC exposure in prenatal development:
Higher rates of fetal distress
Growth retardation
Transferred from mother to offspring via placental blood during gestation and via maternal milk during lactation

4. Clinical studies indicate in utero exposure is associated
CB1 receptors mediate neural actions of cannabinoids & emerge early in development
synaptogenesis, proliferation and migration of neuronal cells
Clinical studies indicate in utero exposure is associated
impulsive behavior, cognitive impairment, psychiatric disorders (schizophrenia, anxiety) in later life

5. Kandel 03 suggested potential for cannabis to increase the risk of consumption of other drugs of abuse
hypothesized that long lasting neurobiological changes in neuronal systems linked with limbic function might be affected by in utero THC exposure
Gateway….?

6. Opioids
Endogenous opioid system shares neuroanatomical and neurochemical characteristics with the cannabinoid system; tightly linked
3 families
Enkephalin from preproenkephalin (PENK)
Dynorphin from preprodynorphin
Endorphin from proopiomelanocortin (POMC)

7. Cannabinoids stimulate release of enkephalin in the NAc and VTA
NAc is part of the mesocorticolimbic circuit
VTA is origin of DA mesocorticolimbic circuit
PENK widely dist. (NAc, amygdala, PFC)

8. Previous human studies show sig
Previous human studies show sig. impairment of PENK and meso-limbic DA genes in association with in utero CB exposure
Enduring effects into adulthood?
Influence adult behavior relative to addiction disorders?

9. treated with THC through gestation & entire lactation period
In rats, perinatal THC exposure alters opioid gene expression, opioid receptor binding and morphine self-administration
Differs between males and females
treated with THC through gestation & entire lactation period
Dams treated with THC orally, different pharmacokinetic property than alternate routes of ingestion e.g. smoking, iv

10. Methods Fear Leads to Anger---Anger Leads to Stress
Stress Leads to Doobies—Doobies lead to Twinkies

11. Test animals Female Long-Evans rats Reversed light/dark cycle
Permanent right jugular catheter for IV
Following surgery were mated with a male to induce pregnancy

12. Admin of drug Drug exposure limited to prenatal period
Gestational day5 to post-natal day 2
Corresponds with mid-gestation period in humans ~ week 20
THC given IV to better mimic the pharmacokinetics of smoking cannabis in pregnant human females
THC injected .15mg/kg daily

13. During Drug Treatment
Maternal weight gain, gestational length and fetal weights were recorded
PND 2 pups from both groups cross fostered; some brains taken at this time
PND 21 males weaned; housed 4/cage
PND 55 iv catheter implanted
Housed individually, 7 day recovery
These are the test animals
More brains taken PND 62 prior to behav studies

14. Heroin Self Admin Active lever press = 15ug/kg in 85 ml fluid
Fixed Ratio 1, 10s timeout, 3 hour sessions during dark cycle
After 6 days, dose increased to 30 mg/kg
Baseline= <15% change in # bar presses for 3 consecutive days
During training food=20g/day
Ad libitum after stable response reached

15. Dose Response Between session dose response test
Rats now receive either higher 60,100mg or lower 7.5, 15 mg doses
Self admin as normal for 3 days, order of presentation was random
Following test, back to 30mg maintenance for 5 more days

16. Food Stress
Food deprived for 24 hours
Response measured following day

17. Extinction and Reinstatement
First week of extinction = saline
Following days = no fluid injected
Decrease in bar presses of 85% baseline for 3 days = extinction
Priming:
saline
heroin 0.25 mg/kg sc 10 min prior
CB1 antagonist 3 mg/kg ip + heroin

18. Results No significant change between groups in
% weight gain in pregnant dams
Gestational length
Pup length at PND 2
Pup weight
Weight between groups at PND 62
Start of heroin self adminstration training

19. 15 mg/kg/infusion
30 mg/kg/infusion

20. No self admin at 15 mg/kg dose
Both THC and Vehicle groups self admin at 30 mg/kg dose
THC animals show shorter latency to first active response 34 ± 0.84s vs 115 ± 0.91s

22. Dose Response Test
Dose-dependant decrease in responding with increasing heroin dose
THC animals show higher responding at lower doses (7.5 & 15 mg/kg)
No diff in responding at 30, 60 or 100 mg doses between groups

24. Response after Stress Test
THC animals respond to food deprivation stress with more active lever presses

26. Extinction, Priming, SR Treatment
THC group responded to active bar more during first day of extinction (more than maintenance, more than vehicle group)
Heroin priming reinstated active bar presses
THC group responded to active bar sig more than they had during maintenance
SR treatment abolished priming induced reinstatment

28. Locomotion Response
THC group showed less locomotion during acquisition and maintenance phases
No diff during extinction phase
Differences are due to heroin intake

31. CB1 and m Opioid Receptor G-Protein Coupling
Agonist-stimulated GTPgS binding
No change in m or CB1 binding at PND2
In adults, prenatal THC exposure significantly associated with
decreased m opioid binding in NAc shell
Increased binding in SN
No change in CB1 receptor coupling

32. 1=NAc shell, 2=NAc core, 3=CP, 4=VTA, 5=SN

33. PENK mRNA Expression
Prenatal THC Exposure decreased PENK mRNA expression in NAc at PND2
No reliable measurements from amygdala
No change in preprodynorphin which is colocalized in the NAc
HOWEVER…
Prenatal THC exposure increased PENK mRNA in Adults at PND 62 in
NAc core and shell
Central and medial amygdala nuclei
Compensation?

34. 1=NAc shell
2=NAc core
3=CP
4=m.amy
5=c.amy
Increased
PENK mRNA in
cAmy

36. Discussion
Hyperactivity of the mesocorticolimbic enkephalinergic system in adult animals may be due to the blunted PENK gene expression during early development

37. Self Administration
No diff in self admin of heroin between groups in adulthood
THC group showed
Shorter latency to first active lever press
Higher response to lower doses of heroin
Increased response following food stress
Higher level of seeking during extinction
Suggests long term vulnerability in the motivation to self-administer heroin

38. Relapse! Following 21 days of drug extinction
Heroin primed THC group responded with higher number of bar presses than maintenance (increased seeking)
CB1 antagonist SR141716A completely blocked heroin primed relapse in both groups
Cross talk between opioid and cannabinoid systems

39. m opioid receptor binding
THC group show less mOR binding in NAc
Key region in reward processing
also modulates locomotion
THC group showed less loco when on heroin
Similar to mOR deficient animals

40. Take-Home Message
THC administered during prenatal period significant affects PENK mRNA expression during prenatal period and adulthood
Alterations of the opioid system last into adulthood and enhance vulnerability to opiate-seeking behavior