Modelling the effects of cannabis in adolescent mice

1. Modelling the effects of cannabis in adolescent mice Dr Cathy Fernandes RCUK Academic Fellow Psychological Medicine & Psychiatry Institute of PsychiatryKing’s College London

2. Introduction • cannabis is generally considered to be a safe drug • side-effects include cognitive impairment (Sitskoorn et al, 2004), anxiety attacks and paranoia (Ames, 1958) • increased risk of developing schizophrenia (Arseneault et al, 2004) • worrying trends.... • increase in the concentration of the main active ingredient of cannabis, ∆9 -tetrahydrocannabinol (δ-THC) • age of first-time cannabis users is rapidly decreasing with 40% of 15 year olds in the UK having experienced the drug (European School Survey Project on Alcohol and Other Drugs)

3. Introduction • effects of cannabis during development • neuronal networks are still under development (Romeo, 2003) • role of the endocannabinoid system (Fernandez-Ruiz et al, 2000) • focus on prenatal/perinatal periods (Viveros et al, 2005) • impaired executive function (Fried et al, 1998), impaired social behaviours and emotional reactivity (Trezza et al, 2008) • fewer studies on the effects of postnatal (adolescent) exposure • long-lasting cognitive effects (Stiglick & Kalant, 1985), sensory motor gating deficits and anhedonia (Schneider and Koch, 2003) • synthetic cannabinoid compounds used, incremental or irregular dosing regimens [pharmacokinetic analyses?]

4. What are we modelling? Model=‘small measure’ a simplified representation used to explain the workings of a real system • behavioural endophenotypes • genetic perturbation • environmental perturbation

5. Endophenotype concept Kas et al (2007) • association with the psychiatric disorder in the population • heritable and primarily state-independent • endophenotype and disorder co-segregate within families • occur more in non-affected family members than in general population • (Gottesman & Gould, 2003)

6. What are we modelling? • knock them out (one) at a time ? • one perturbation (knock-out/transgenics) • make use of (natural) variation • multiple perturbations (inbreds, outbreds, crosses)

7. Mouse - a model organism diverse range of genetic manipulations physiological & anatomical similarity numerous behavioural models available

8. Developmental stages http://jaxmice.jax.org/images/literature/pupsposter-large.jpg childhood adolescence adulthood (> pd 7-28) (pd 28-49) (> pd 56) puberty (pd 35-56) • critical postnatal ontogenetic stages • neurophysiological and hormonal processes (Spear, 2000)

9. Modelling the effects of cannabis • ∆9 -tetrahydrocannabinol (δ-THC) administration • 20 mg/kg once daily by oral gavage for 14 days • two different inbred strains of mice (males only) • developmental stages • juvenile (7-21 days old) versus adolescent (puberty; 30-44 days old) • behavioural phenotypes (drug-free, 10 wks old) • working/episodic-like memory (delayed matching to place task) • molecular phenotypes • synaptic markers • epigenetic profiling of candidate genes • genomewide transcription profiling C57BL/6J DBA/2J

10. Behavioural effects of δ-THC in mouse DBA/2J DMP task C57BL/6J * juvenile adolescent saving time (Zeng et al) * STRAIN x DRUG x AGE: F(1,72)=13.9, p < 0.001 δ-THC exposure in early life but not during late adolescence causes a working/ episodic-like deficit in adulthood dependent on genetic background 8 trials/day, 8 platform locations

11. Molecular effects of δ-THC in mouse synaptophysin prefrontal cortex (PFC) hippocampus (CA3) • immunohistochemical staining for synaptic marker synaptophysin * * DBA/2J (* p <0.05) C57BL/6J • increased synaptophysin in PFC and CA3 after δ-THC exposure in • juvenile but not adolescent mice dependent on genetic background but synaptophysin is reduced in prefrontal cortex in schizophrenic brains

12. Genetic influence on the effects of δ-THC • individual susceptibility to the harmful effects of cannabis • genetic factors (Harrison & Weinberger, 2005) • COMT (catechol-O-methyltransferase) • Val158Met polymorphism associated with the increased risk of psychosis incurred by adolescent-onset cannabis use (Caspi et al, 2005) • Val158 carriers appear to be more sensitive to the psychotic experiences and cognitive impairments following administration of δ-THC (Henquet et al, 2006)

13. Genetic influence on the effects of δ-THC • mutation in Comt between DBA/2J and C57BL/6J mice (Fernandes et al) • mRNA expression level (current data confounded by the mutation) • protein level and activity of COMT (in collaboration with Elizabeth Tunbridge, Oxford) • DNA methylationof Comt promoter (in collaboration with Jon Mill, IoP)

14. Summary • early life exposure to δ-THC in DBA/2J but not C57BL/6J mice resulted in a long-lasting, mild impairment in cognition and changes in synaptic density • genetic background and developmental stage influence the effects of δ-THC • evidence for a genetically mediated vulnerability (resilience) to the effects of cannabis exposure in early life • is Comt mediating this gene by environment interaction?

15. What next . . . • extend the behavioural characterisation of the effects of δ-THC • anxiety, social behaviours, cognitive abilities • pharmacokinetic analyses to translate levels of δ-THC • broaden the molecular characterisation of the effects of δ-THC • genomewide gene expression profiling (hippocampus and PFC) • genomewide DNA methylation profiling (PFC) • DNA methylation of specific candidate genes (Comt and Nrg1 across several brain regions)

16. Elke Binder Rachel Kember Leo Schalkwyk Maria Teresa Ortin Paul Morrison Jon Cooper JelleMostert Jon Mill David Collier Robin Murray Acknowledgements Institute of Psychiatry • Funding • Research Councils UK