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

• 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

• 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

• 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….?

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)

• 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)

• 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?

• 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

Methods

Fear Leads to Anger---Anger Leads to StressStress Leads to Doobies—Doobies lead to Twinkies

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

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

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

Heroin Self Admin

• Active lever press = 15ug/kg in 85 l fluid

• Fixed Ratio 1, 10s timeout, 3 hour sessions during dark cycle

• After 6 days, dose increased to 30 g/kg

• Baseline= <15% change in # bar presses for 3 consecutive days

• During training food=20g/day– Ad libitum after stable response reached

Dose Response

• Between session dose response test

• Rats now receive either higher 60,100g or lower 7.5, 15 g doses– Self admin as normal for 3 days, order of

presentation was random

• Following test, back to 30g maintenance for 5 more days

Food Stress

• Food deprived for 24 hours

• Response measured following day

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

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

15 g/kg/infusion 30 g/kg/infusion

• No self admin at 15 g/kg dose

• Both THC and Vehicle groups self admin at 30 g/kg dose

• THC animals show shorter latency to first active response 34 ± 0.84s vs 115 ± 0.91s

Dose Response Test

• Dose-dependant decrease in responding with increasing heroin dose

• THC animals show higher responding at lower doses (7.5 & 15 g/kg)

• No diff in responding at 30, 60 or 100 g doses between groups

Response after Stress Test

• THC animals respond to food deprivation stress with more active lever presses

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

Locomotion Response

• THC group showed less locomotion during acquisition and maintenance phases– No diff during extinction phase

• Differences are due to heroin intake

CB1 and Opioid Receptor G-Protein Coupling

• Agonist-stimulated GTPS binding

• No change in or CB1 binding at PND2

• In adults, prenatal THC exposure significantly associated with – decreased opioid binding in NAc shell– Increased binding in SN

– No change in CB1 receptor coupling

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

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?

1=NAc shell2=NAc core3=CP4=m.amy5=c.amy

IncreasedPENK mRNA incAmy

Discussion

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

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

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

opioid receptor binding

• THC group show less OR binding in NAc – Key region in reward processing– also modulates locomotion

• THC group showed less loco when on heroin

• Similar toOR deficient animals

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