Marina E. Wolf Chicago Medical School Rosalind Franklin University of Medicine & Science

1. How do drugs of abuse rewire the motivational circuitry? Marina E. Wolf Chicago Medical School Rosalind Franklin University of Medicine & Science

2. Acute effects of cocaine and amphetamine:interference with monoamine reuptake Addiction:long-lasting adaptations triggered by increased monoamine levels These adaptations “rewire” the motivational circuitry, facilitating the formation of new habits that center around drug-seeking, usually at the expense of more appropriate behaviors.

3. HypothesisAddiction is a form of neuronal plasticity. The adaptations leading to addiction involve the same glutamate-dependent cellular mechanisms that enable learning and memory.

4. Glutamate-dependent plasticity and addiction • Animal model of addiction (behavioral sensitization) • Behavioral changes blocked by glutamate antagonists or lesions of glutamate pathways • Behavioral changes are associated with changes in glutamate receptor function and expression • Drugs of abuse and plasticity activate common signal transduction cascades (kinases & phosphatases, transcriptional regulators, neurotrophins, etc) • Drugs of abuse influence LTP and LTD in reward-related pathways

5. Glutamate and glutamate receptors • Glutamate: major excitatory transmitter in the brain, activates ionotropic receptors and GPCRs • Ionotropic receptors • AMPA receptors – bulk of fast excitatory transmission • NMDA receptors – slower synaptic potentials, enable plasticity (and excitotoxicity) • Kainate receptors – less understood • Metabotropic receptors (GPCRs) • at least 8 subtypes (mGluR1-8) • important in normal synaptic transmission and plasticity

6. Long-term potentiation (LTP) stimulate record LTP • Strong NMDAR activation • Large postsynaptic Ca increase • Preferential activation of kinases • Potentiation of AMPAR currents LTD • More modest NMDAR activation • Less robust postsynaptic Ca increase • Preferential activation of phosphatases • Depression of AMPAR currents LTP in NAc (Julie Kauer, Brown Univ)

7. Functional significance of LTP in addiction-related pathways? Wolf, Molecular Interventions 2002

8. How do drugs that initially target monoamine transporters influence glutamate transmission and glutamate-dependent processes such as LTP and LTD?

9. Drugs may modulate LTP via actions on neuronal circuits Everitt and Wolf, J Neurosci 2002

10. Drugs may modulate LTP at the single cell level Glutamate Dendritic spine (postsynaptic) Dopamine

11. GluR1 is phosphorylated during LTP, and dephosphorylated during LTD • GluR1-containing AMPA receptors are inserted into synapses during LTP and removed during LTD Two inter-related mechanisms for regulating AMPA receptor transmission during LTP and LTD Song & Huganir, TINS 2002

12. AMPA receptor trafficking Bredt & Nicoll, Neuron 2003

13. D1 receptors are coupled to PKA PKA is involved in both mechanisms for regulating AMPA receptor function during LTP Hypothesis: D1 receptors modulate AMPA receptor phosphorylation and trafficking through PKA-dependent mechanisms, and thereby influence LTP and LTD. Link to dopamine? Song & Huganir, TINS 2002

14. Do D1 receptors regulate AMPA receptor trafficking in NAc neurons? Nucleus accumbens Glutamate afferents (cortex, hippocampus, amygdala) Medium spiny projection neuron Dopamine afferents

15. Postnatal NAc cultures reproduce many features of the intact NAc ~80% medium spiny neurons and 20% interneurons Almost all neurons are GABAergic GluR1 is expressed by all neurons D1 receptors are expressed by ~80% of neurons D2 receptors are expressed by ~80% of neurons Chao et al, J Neurochem 83:704-12, 2002

16. D1 receptor stimulation increases GluR1 surface expression in NAc neurons Control 1mM SKF Chao et al, J Neurochem 83:704-12, 2002

17. Pre-blocking protocol for selectively detecting newly externalized GluR1 • Lu, Man, Ju, Trimble, MacDonald, & Wang, Neuron, 2001 • Pre-block GluR1 on the surface of live cells with N-GluR1 antibody and unlabeled secondary antibody • Incubate at room temperature to allow GluR1 externalization • Second round of immunostaining, under non-permeant conditions, with N-GluR1 antibody and Cy3 secondary antibody 1o Ab 2o Ab Cy3 2oAb

18. D1 receptor stimulation increases the rate of GluR1 externalization Control Media 1uM SKF 20um 10uM SCH + SKF SCH Mangiavacchi & Wolf J Neurochem 88:1261-71, 2004

19. Requires PKA activation (Mangiavacchi & Wolf, J Neurochem 88:1261-71, 2004) • GluR1 may be the relevant substrate because D1 agonists also stimulate phosphorylation of GluR1 at the PKA site in NAc cultures (Chao et al, J Neurochem 81:984-992, 2002) • Consistent with results in hippocampus indicating that PKA phosphorylation of GluR1 promotes its trafficking to the cell surface (Ehlers, Neuron 28:511-25, 2000; Esteban et al, Nat Neurosci 6:136-43,2003)

20. Glutamate agonists produce rapid internalization of GluR1 Medium spiny neurons Interneurons Control 50µM Glu Control 50μM Glu Mangiavacchi & Wolf, Eur J Neurosci, submitted

21. Prefrontal cortex, BLA, HPC Glu Dopamine inputs increase rate of AMPAR externalization Glutamate inputs increase rate of AMPAR internalization Changes in afferent activity result in minute-by-minute modulation of AMPAR surface expression on NAc neurons? NAc AMPA D1 DA VTA

22. Do D1 receptors drive GluR1 all the way into the synapse? ??? D1R PKA Modified from Bredt & Nicoll, Neuron 2003

23. NMDAR Prefrontal Cortex D1R CB1R

24. Synaptic and extrasynaptic AMPA receptors in PFC cultures Red = GluR1 Green = SB Yellow = overlay Sun & Wolf, unpublished

25. Summary of unpublished results presented at APA meeting but not provided on NIDA website • D1 receptor stimulation increases GluR1 externalization at extrasynaptic sites, facilitating synaptic insertion as a result of subsequent NMDA receptor stimulation (Sun et al., Soc Neurosci Abstr 30, in press)

26. D1 receptor stimulation externalizes AMPAR at extrasynaptic sites, increasing the pool available for synaptic insertion during LTP Glutamate input DA input NMDAR D1R (PKA) Modified from Bredt & Nicoll, Neuron 2003

27. Our results are consistent with studies showing that: • DA facilitates LTP induction in prefrontal cortex (Gurden et al, Neurosci 94:1019-1027, 1999; Blond et al, Eur J Pharmacol 438:115-116, 2002) • The facilitation is mediated by D1 receptors and PKA (Jay et al, Eur J Neurosci 10:3302-3306,1998; Gurden et al, J Neurosci 20:RC106, 2000)

28. Summary • D1 receptor stimulation increases GluR1 surface expression through a PKA-dependent pathway at extrasynaptic sites. • By increasing extrasynaptic GluR1, D1 receptors increase the pool available for synaptic insertion as a result of NMDA receptor stimulation. This may facilitate the induction of LTP. • These findings suggest a mechanism by which drugs of abuse can directly “tap into” fundamental mechanisms for regulating synaptic strength.

29. Relevance to addiction? Prefrontal cortex, BLA, HPC D1 and glutamate inputs normally regulate AMPA receptor trafficking Chronic drug exposure: adaptations in D1 receptor and PKA signaling Compensatory changes in AMPA receptor trafficking Inappropriate synaptic plasticity, rewiring of motivational circuitry, and formation of new habits focused on drug seeking Glu NAc AMPA Cocaine Amphetamine D1 DA VTA

30. Proposed sequence of mechanisms for long-term plasticity Luscher, Nicoll, Malenka & Muller (Nature Neurosci 2000)

31. Steven Chao Amy Bluestein Marjorie Ariano Dan Peterson Kathy Steece-Collier Robert Malenka Reed Carroll Richard Huganir Hey-Kyoung Lee Simona Mangiavacchi Xiu Sun National Institute on Drug Abuse