1. The Thorny Side of Addiction: Adaptive Plasticity and Dendritic Spines Judson Chandler Department of Neurosciences Medical University of South Carolina

2. Peter W Kalivas and Charles O’Brien

3. NMDA Receptors: Subunits and membrane trafficking

4. EtOH Prolonged ethanol (Homeostatic plasticity restores stability to neuronal circuits; underlies tolerance development) EtOH Acute ethanol (Instability of neuronal circuits) Normal balance Ethanol withdrawal (Hyper-excitability; promotes aberrant plasticity) InhibitoryExcitatory GABAergic Glutamatergic Alcohol Tolerance and Homeostatic Plasticity

5. 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

6. EtOH NR1syn overlay NR1syn overlay Control NR1 0 2 4 6 8 10 12 14 16 * # NR1 Cluster Size (# Pixels/Cluster) SynapticExtrasynaptic Control EtOH EtOH+NMDA Size # NR1 Cluster/10 µM SynapticExtrasynaptic 0 2 4 6 8 10 12 Control EtOH EtOH+NMDA * *# Density

7. Control EtOH Synaptic Extrasynaptic 0 2 4 6 8 10 12 14 16 18 20 Control EtOH * * NR2B Cluster Size (# Pixels/Cluster) SynapticExtrasynaptic 0 2 4 6 8 10 12 Control EtOH * * # NR2B Clusters/10 µm 0 10 20 30 40 50 60 70 80 90 100 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 Size Colocalization

8. AmplitudeFrequency Control EtOH 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 Amplitude, pA Frequency, Hz AMPA mEPSC * ControlEtOH Withdrawn 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 NMDA/AMPA Current Ratio NMDA mEPSC 0.0 0.1 0.2 0.3 0.4 * * 0 250 500 750 1000 Frequency Amplitude Amplitude, pA Frequency, Hz Control EtOH Spontaneous NMDA EPSC Extrasynaptic NMDA Currents

9. 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?

10. Phalloidin stained F-actin Actin/NR1 Matus, Science 290:754, 2000 Dendritic Spines and Structural Plasticity

11. Con EtOHNMDA EtOH+ NMDA * ## ** EtOH+ NMDA * * # EtOH+ NMDA Actin EtOH Control actin * ** @ EtOH (mM) Size Density % Neurons with large spines F-Actin Clusters (# pixels.cluster) # F-actin Clusters/10 uM

12. EtOH Control PSD-95 PSD-95/synapsin Cont EtOH NMDA EtOH+ NMDA * # * * # # PSD-95 Cluster Size (# pixels/cluster) #PSD-95 Clusters/10 uM SynapticExtrasynaptic SynapticExtrasynaptic Size Density Synaptic Extrasynaptic Synaptic Extrasynaptic

13. ** Control EtOH Actin PSD-95 2um F-Actin Cluster Size (# pixels/cluster) # F-Actin Clusters/10 uM With PSD-95 Without PSD-95 With PSD-95 Without PSD-95 Size Density * F-Actin/PSD-95 % colocalization Control EtOH

14. EtOH+2Pal Control Actin 2Pal 2um PSD-95 Control 2Pal ** Size # pixels/cluster Density # clusters/10um

15. Control EtOH +2Pal NR1/synapsin 2Pal * EtOH 2Pal EtOH + 2Pal Control ## Density Synaptic Extrasynaptic

16. NR2A NR2B PSD-95 Molecular model of ethanol-induce plasticity Chronic activity blockade PSD Signaling Complex NR2B PSD-95 Spine enlargement Actin cycling PSD Signaling Complex recruitment “learning spines” “memory spines” (more plastic, but less stable) (more stable, but less plastic) Repeated EtOH exposure and withdrawal experiences

17. PSD Signaling Complex recruitment Does this model have in vivo validity? ControlEthanol 0.0 0.1 0.2 0.3 0.4 0.5 0.6 * NMDA/AMPA ratio How do these homeostatic changes impact synaptic plasticity in the context of the addiction neurocircuitry? Everitt and Robbins, 2005

18. Novel object recognition Chronic Ethanol-induced Plasticity versus Toxicity

19. Ezekiel Carpenter-Hyland Nick Luong Patrick Mulholland John Woodward Sven Kroener Howard Becker Kenneth Abernathy Luca Pellicoro Jeremy Seamans Chris Lapish NIAAA NIH ABMRF Acknowledgements