The Thorny Side of Addiction: Adaptive Plasticity and Dendritic Spines
Judson Chandler
Department of NeurosciencesMedical University of South Carolina
Peter W Kalivas and Charles O’Brien
NMDA Receptors: Subunits and membrane trafficking
EtOH
EtOH
Prolonged ethanol(Homeostatic plasticity restores
stability to neuronal circuits; underlies tolerance development)
EtOH
EtOH
Acute ethanol(Instability of neuronal circuits)
Normal balance
Ethanol withdrawal(Hyper-excitability;
promotes aberrant plasticity)
Inhibitory ExcitatoryGABAergic Glutamatergic
Alcohol Tolerance and Homeostatic Plasticity
Primary rat hippocampal neurons
Used for experimentation when fully mature (2-3 weeks in vitro)
Chronic ethanol exposure in sealed vapor chambers
Evaluated by IHC/confocal imaging & electrophysiology
In Vitro Model of Chronic Alcohol Exposure
EtOH NR1 syn overlay
NR1 syn overlayControl
NR10
2
4
6
8
10
12
14
16 *
#
NR1 Cluster Size
(# Pixels/Cluster)
Synaptic Extrasynaptic
ControlEtOHEtOH+NMDA
Size
# NR1 Cluster/10 µM
Synaptic Extrasynaptic0
2
4
6
8
10
12
ControlEtOHEtOH+NMDA
*
*#
Density
Control EtOH
Synaptic Extrasynaptic0
2
4
6
8
10
12
14
16
18
20
Control
EtOH
*
*NR2B Cluster Size
(# Pixels/Cluster)
Synaptic Extrasynaptic0
2
4
6
8
10
12
Control
EtOH
*
*
# NR2B
Clusters/10 µm
0102030405060708090
100
0
2
4
6
8
10
12
14
NR2B
GluR1
Control EtOH
Size Density
Control Ethanol Control Ethanol
Percent Colocalization
GluR1 Cluster Size
(# pixels/cluster
SizeColocalization
Amplitude Frequency
Control
EtOH
02468101214161820
02468101214161820
Amplitude, pA
Frequency, Hz
AMPA mEPSC
*
Control EtOH Withdrawn0.00.10.20.30.40.50.60.70.80.91.0
NMDA/AMPA
Current Ratio
NMDA mEPSC
0.0
0.1
0.2
0.3
0.4**
0
250
500
750
1000
FrequencyAmplitude
Amplitude, pA
Frequency, Hz
ControlEtOH
Spontaneous NMDA EPSC
10-6 10-5 10-4 10-3
0
20
40
60
Control
EtOH
[NMDA], M
Current, pA/pF
Extrasynaptic NMDA Currents
•Prolonged ethanol exposure results in the enhancement of NR2B-containing NMDA receptors selectively at the synapse.
•No changes in AMPA receptors.
•Activity and PKA-dependent.
•Slowly reverses upon ethanol removal.
•Electrophysiological observations correlated with confocal image analysis confirming functional plasticity.
Summary of findings
Is there a corresponding structural component of this homeostatic response to chronic alcohol exposure?
Phalloidin stained F-actin
Actin/NR1
Matus, Science 290:754, 2000
Dendritic Spines and Structural Plasticity
Con EtOH NMDAEtOH+NMDA
Con EtOH NMDA EtOH+NMDA0
10
20
30
40
50
60
*##
**
EtOH+NMDA
*
*#
Con EtOH NMDA EtOH+NMDA0123456789
101112
EtOH+NMDAActin
EtOHControl
actin Con 25 50 100 N 50+N0
10
20
30
40
50
60
*
**
**
@
EtOH (mM)
Size Density
% N
euro
ns
wit
h la
rge
spin
es
F-A
ctin
Clu
ster
s(#
pix
els.
clu
ster
)
# F
-act
in C
lust
ers/
10 u
M
EtOH
Control
PSD-95
PSD-95/synapsin
Cont EtOH NMDAEtOH+NMDA
*
#
Synaptic Extrasynaptic0
5
10
15
20
25
30Con
EtOH
NMDA
EtOH+NMDA
*
* ##
Synaptic Extrasynaptic0
1
2
3
4
5
6
7 Con
EtOH
NMDA
EtOH+NMDA
PS
D-9
5 C
lust
er S
ize
(# p
ixel
s/cl
ust
er)
#PS
D-9
5 C
lust
ers/
10 u
MSynaptic Extrasynaptic Synaptic Extrasynaptic
Size Density
Synaptic Extrasynaptic Synaptic Extrasynaptic
**
**
With PSD-95 No PSD-950
1
2
3
4
5
Con
EtOH
Control EtOH
With PSD-95 No PSD-950
102030405060708090
100
Con
EtOH
Actin
PSD-95
2um
F-A
ctin
Clu
ster
Siz
e(#
pix
els/
clu
ster
)
# F
-Act
in C
lust
ers/
10 u
M
With PSD-95 Without PSD-95 With PSD-95 Without PSD-95
Size Density
*
Con EtOH0
10
20
30
40
50
F-A
ctin
/PS
D-9
5%
co
loca
lizat
ion
Control EtOH
EtOH
EtOH+2Pal
Control
Actin
2Pal
2um
PSD-95
Control
2Pal
**
Cont EtOH 2Pal EtOH+2Pal
05101520253035404550
#
Size
# pixels/cluster
Density
0123456789
***
#
Cont EtOH 2Pal EtOH+2Pal
# clusters/10um
Control EtOHEtOH+2Pal
NR1/synapsin
2Pal
*
Synaptic Extrasynaptic0123456789
10
EtOH
Con
2Pal
EtOH+2Pal
EtOH
2Pal
EtOH + 2Pal
Control
##
Density
Synaptic Extrasynaptic
NR2A NR2B
PSD-95
Molecular model of ethanol-induce plasticity
Chronic activity blockade
PSD Signaling Complex
PSD Signaling Complex
NR2BPSD-95
Spine enlargement
Actincycling
PSD Signaling Complex
recruitment
“learning spines” “memory spines”(more plastic, but less stable) (more stable, but less plastic)
Repeated EtOH
exposure and
withdrawal
experiences
PSD Signaling Complex
recruitment
•Does this model have in vivo validity?
Control Ethanol0.0
0.1
0.2
0.3
0.4
0.5
0.6 *
NM
DA
/AM
PA
rat
io
•How do these homeostatic changes impact synaptic plasticity in the context of the addiction neurocircuitry?
Everitt and Robbins, 2005
Novel object recognition
Chronic Ethanol-induced Plasticity
versusToxicity
Ezekiel Carpenter-HylandNick Luong
Patrick Mulholland John Woodward
Sven KroenerHoward Becker
Kenneth AbernathyLuca Pellicoro
Jeremy Seamans Chris Lapish
NIAAANIHABMRF
Acknowledgements