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

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.

Hypothesis Addiction is a form of neuronal plasticity Hypothesis Addiction is a form of neuronal plasticity. The adaptations leading to addiction involve the same glutamate-dependent cellular mechanisms that enable learning and memory. The hypothesis that drives work in my laboratory is that the adaptations leading to addiction involve the same glutamate-

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 This slide outlines some of the key lines of evidence supporting a role for glutamate-dependent plasticity in addiction. The first was published 15 years ago by RK lab and then by mine. Both of us showed that the development of sensitization could be prevented by coadministering an NMDA antagonist with cocaine or amphetamine, providing the first evidence that an LTP-like step might be required for the development of sensitization.

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

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 way to increase the strength of glutamate synapses. Leading candidate for mediating neuronal plasticity, therefore focus of addiction research. Phenomenon itself is simple. Explain LTP and LTD. Why important? Provides a way for the amount of use a pathway receives to regulate the strength of the pathway, providing a cellular basis of use-dependent or experience-dependent plasticity so important during development and learning and memory. If there weren’t such a mechanism, our brains would be hard wired and it would be hard to imagine how developmental changes or latning would occur. LTP in NAc (Julie Kauer, Brown Univ)

Functional significance of LTP in addiction-related pathways? What is the functional significance of this sort of finding? Well we know that certain excitatory pathways are very important in particular aspects of addiction, for example, BLA-ANAc, PFC- NAC. Plasticity in these pathways could account for ….. Wolf, Molecular Interventions 2002

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

Drugs may modulate LTP via actions on neuronal circuits Everitt and Wolf, J Neurosci 2002 I think there are two levels at which DA may be acting. First is the circuit level. This is a schematic of circuits important in addiction. Very complex, but can use it to make some simple points. Blue pathways are glutamate pathways. Second is that all the regions in boxes receive DA inputs from midbrain and DA’s role is generally to serve as neuromodulator. So, by changing neuronal activity in HPC, for example, a cocaine induced increase in DA in HPC could lead to LTP in its target PFC or even downstream.

Drugs may modulate LTP at the single cell level Glutamate Dendritic spine (postsynaptic) Another way to think about this problem is at the single cell level. This is reasonable because in many addiction related brain regions – eg striatal complex, PFC – one finds this synaptic arrangement in which postsynaptic spine receives convergent DA and glutamate inputs, raising the possibility that DA released by coc or amp might modulate L TP or LTD generation at this nearby synapse onto the same spine. Dopamine

Two inter-related mechanisms for regulating AMPA receptor transmission during LTP and LTD GluR1 is phosphorylated during LTP, and dephosphorylated during LTD GluR1-containing AMPA receptors are inserted into synapses during LTP and removed during LTD Before I describe our approach to answering this question, I’d like to talk briefly about mechanisms for regulating excitatory strength at glutamate synapses. Recent work suggests that there are two major mechanisms both of which involve the AMPA R subunit GluR1. Both involving GluR1 and protein phosphorylation. Song & Huganir, TINS 2002

AMPA receptor trafficking The next slide summarized the regulation of AMPAR trafficking in a little more detail. In most forebrain regions, AMPARs are either made up of GluR1 and GluR2 subunits or GluR2/3 subunits. Because of important role of GluR1 in trafficking, we’ve focused on this subunit in our studies. Bredt & Nicoll, Neuron 2003

Link to dopamine? 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. Before I describe our approach to answering this question, I’d like to talk briefly about mechanisms for regulating excitatory strength at glutamate synapses. Recent work suggests that there are two major mechanisms both of which involve the AMPA R subunit GluR1. Both involving GluR1 and protein phosphorylation. PKA increases peak open probability. Song & Huganir, TINS 2002

Do D1 receptors regulate AMPA receptor trafficking in NAc neurons? Nucleus accumbens Glutamate afferents (cortex, hippocampus, amygdala) Medium spiny projection neuron NAc is important because it is an interface between limbic and motor systems. It receives inputs from cortical and limbic regions that are important in generating motivated behaviors. Medium spiny neurons integrate this information and send it on to motor regions that are important in the execution of goal directed behaviors. Ultimately, drug seeking behavior depends on glutamate transmission in the NAc. Dopamine afferents Do D1 receptors regulate AMPA receptor trafficking in NAc neurons?

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

D1 receptor stimulation increases GluR1 surface expression in NAc neurons Simplest way is to just count before and after a treatment. Control 1mM SKF Chao et al, J Neurochem 83:704-12, 2002

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 But not sensitive enough.

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

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)

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

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 I emphasize this because it is such a far cry from the very static view of postsynaptic Rs that existed when I was being trained, and in fact up until just a few years ago DA VTA

Do D1 receptors drive GluR1 all the way into the synapse? ??? D1R Cant pursue this in NAc cultures because they lack glutamate synapses. Can restore glutamate synapses by co-culturing with cortical glutamate-containing neurons but then have the problem of telling who is who. PKA Modified from Bredt & Nicoll, Neuron 2003

NMDAR Prefrontal Cortex D1R CB1R

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

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)

D1 receptor stimulation externalizes AMPAR at extrasynaptic sites, increasing the pool available for synaptic insertion during LTP Glutamate input DA input NMDAR The next slide summarizes the regulation of AMPAR trafficking in a little more detail. In most forebrain regions, AMPARs are either made up of GluR1 and GluR2 subunits or GluR2/3 subunits. D1R (PKA) Modified from Bredt & Nicoll, Neuron 2003

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) Studies in the rat have shown that….. So our hypothesis is that…..

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. Completely novel mechanism for DA/Glu interactions and the first example of a non-glu GPCR modulating AMPAR trafficking

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 D1 receptors, by influencing GluR1 phosphorylation and surface expression, normally mediate a regulatory influence on AMPA receptor transmission in the NAC, and perhaps on synaptic plasticity. As a result of repeated drug exposure and withdrawal, changes in D1 receptor sensitivity and PKA signaling occur in the NAC. We hypothesize that this leads to compensatory changes in AMPA receptor phosphorylation and trafficking, and perhaps to the production of inappropriate forms of synaptic plasticity. Supporting this, a behaviorally sensitizing regimen of cocaine produced long-lasting LTD at excitatory synapses in NAc shell (Thomas et al., 2001). Furthermore while acute cocaine increases GluR1 phosphorylation, striatal neurons from chronic cocaine treated rats exhibit decreased GluR1 phosphorylation and reduced AMPA receptor currents (Bibb et al., 2001). DA VTA

Proposed sequence of mechanisms for long-term plasticity I’d like to transition to Terry’s talk by making the point that LTP doesn’t end with AMPAR trafficking but ultimately involves structural remodeling of synapses. This slide shows a proposed sequence for these events taken from an excellent review by ……. Luscher, Nicoll, Malenka & Muller (Nature Neurosci 2000)

National Institute on Drug Abuse 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