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

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

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

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 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?

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

Methods Fear Leads to Anger---Anger Leads to Stress Stress 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 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

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

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 mg/kg/infusion 30 mg/kg/infusion

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

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

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 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

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

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

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

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