Acetylcholine (ACh)

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
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.
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

• Two inter-related mechanisms for regulating AMPA
  receptor transmission during LTP and LTD

1. GluR1 is phosphorylated during LTP, and
   dephosphorylated during LTD
2. GluR1-containing AMPA receptors are inserted into
   synapses during LTP and removed during LTD

Song Huganir, TINS 2002
12
AMPA receptor trafficking
Bredt Nicoll, Neuron 2003
13

• 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.

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 83704-12, 2002
16
D1 receptor stimulation increases GluR1 surface
expression in NAc neurons
Control
1mM SKF
Chao et al, J Neurochem 83704-12, 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
Media
1uM SKF
Control
20um
SCH
10uM SCH SKF
Mangiavacchi Wolf J Neurochem 881261-71,
2004
19

• Requires PKA activation (Mangiavacchi Wolf, J
  Neurochem 881261-71, 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 81984-992, 2002)
• Consistent with results in hippocampus indicating
  that PKA phosphorylation of GluR1 promotes its
  trafficking to the cell surface (Ehlers, Neuron
  28511-25, 2000 Esteban et al, Nat Neurosci
  6136-43,2003)

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 941019-1027, 1999 Blond
  et al, Eur J Pharmacol 438115-116, 2002)
• The facilitation is mediated by D1 receptors and
  PKA (Jay et al, Eur J Neurosci 103302-3306,1998
  Gurden et al, J Neurosci 20RC106, 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
D1
Cocaine Amphetamine
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