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Research Articles: Behavioral/Cognitive

Cascades of Homeostatic Dysregulation Promote Incubation of CocaineCraving

Junshi Wang1, Masago Ishikawa1, Yue Yang1, Mami Otaka1, James Y. Kim1, George R. Gardner1, Michael

T. Stefanik4, Mike Milovanovic4, Yanhua H. Huang2, Johannes W. Hell3, Marina E. Wolf4, Oliver M.

Schlüter1,5 and Yan Dong1,2

1Departments of Neuroscience2Psychiatry, University of Pittsburgh, Pittsburgh, PA 152603Department of Pharmacology, University of California, Davis, Davis, CA 956154Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, IL600645Department of Psychiatry and Psychotherapy, University Medical Center, 37075 Göttingen, Germany

DOI: 10.1523/JNEUROSCI.3291-17.2018

Received: 20 November 2017

Revised: 18 January 2018

Accepted: 22 January 2018

Published: 6 April 2018

Author contributions: J.W., M.I., Y.Y., M.O., M.T.S., Y.H.H., J.W.H., M.E.W., O.M.S., and Y.D. designedresearch; J.W., M.I., Y.Y., M.O., J.Y.K., G.R.G., M.T.S., and M.M. performed research; J.W., M.I., M.T.S., andM.M. analyzed data; J.W., Y.H.H., J.W.H., M.E.W., O.M.S., and Y.D. wrote the paper.

Conflict of Interest: The authors declare no competing financial interests.

We thank Dr. A. Barria for providing GluN2B constructs, Kevin Tang for excellent technical support, andRobert Malenka for helpful suggestions on the manuscript. The study was supported by NIH NIDA DA029565,DA028020, DA023206, DA024570, DA031551, DA35805, MH101147, NS978792, and grants from theGerman Research Foundation through the Collaborative Research Center 889 “Cellular Mechanisms ofSensory Processing” and the Cluster of Excellence “Nanoscale Microscopy and Molecular Physiology of theBrain” (O.M.S.). Cocaine was provided by the NIH NIDA drug supply program.

Corresponding Author: Dr. Yan Dong, Corresponding Address: Dept. Neuroscience, Univ. of Pittsburgh, A210Langley Hall/5th & Ruskin Ave, Pittsburgh, PA 15260, Email: yandong@pitt.edu, Phone: 412-624-3140

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3291-17.2018

Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formattedversion of this article is published.

1

1 Cascades of Homeostatic Dysregulation Promote Incubation of Cocaine Craving 2

3 4

Junshi Wang1, Masago Ishikawa1, Yue Yang1, Mami Otaka1, James Y. Kim1, 5 George R. Gardner1, Michael T. Stefanik4, Mike Milovanovic4, 6

Yanhua H. Huang2, Johannes W. Hell3, Marina E. Wolf4, Oliver M. Schlüter1,5, Yan Dong1,2 7 8

Departments of 1Neuroscience and 2Psychiatry, University of Pittsburgh, Pittsburgh, PA 15260 9 3Department of Pharmacology, University of California, Davis, Davis, CA 95615 10 4Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North 11 Chicago, IL60064 12 5Department of Psychiatry and Psychotherapy, University Medical Center, 37075 Göttingen, 13 Germany 14 15 Running title: Homeostatic Dysregulation by Cocaine 16 17 Corresponding Author: Dr. Yan Dong 18 Corresponding Address: Dept. Neuroscience, Univ. of Pittsburgh, A210 Langley Hall / 5th & 19 Ruskin Ave, Pittsburgh, PA 15260 20 Email: yandong@pitt.edu 21 Phone: 412-624-3140 22 23 Number of text pages: 32 24 Number of figures: 3 25 Word counts: Abstract: 250 Significance: 119 Introduction: 645 Methods: 1670 26 Results: 3292 Discussion: 1500 27 28 Key words: cocaine incubation; homeostatic plasticity; nucleus accumbens; membrane 29 excitability; addiction; relapse. 30 31

Acknowledgement: We thank Dr. A. Barria for providing GluN2B constructs, Kevin Tang for 32 excellent technical support, and Robert Malenka for helpful suggestions on the manuscript. The 33 study was supported by NIH NIDA DA029565, DA028020, DA023206, DA024570, DA031551, 34 DA35805, MH101147, NS978792, and grants from the German Research Foundation through 35 the Collaborative Research Center 889 “Cellular Mechanisms of Sensory Processing” and the 36 Cluster of Excellence ‘‘Nanoscale Microscopy and Molecular Physiology of the Brain’’ (O.M.S.). 37 Cocaine was provided by the NIH NIDA drug supply program. 38

2

Abstract 39

In human drug users, cue-induced drug craving progressively intensifies after drug abstinence, 40

promoting drug relapse. This time-dependent progression of drug craving is recapitulated in 41

rodent models, in which rats exhibit progressive intensification of cue-induced drug seeking after 42

withdrawal from drug self-administration, a phenomenon termed incubation of drug craving. 43

While recent results suggest that functional alterations of the nucleus accumbens (NAc) 44

contribute to incubation of drug craving, it remains poorly understood how NAc function evolves 45

after drug withdrawal to progressively intensify drug seeking. The functional output of NAc relies 46

on how the membrane excitability of its principal medium spiny neurons (MSNs) translates 47

excitatory synaptic inputs into action potential firing. Here, we report a synapse-membrane 48

homeostatic crosstalk (SMHC) in male rats, through which an increase or decrease in the 49

excitatory synaptic strength induces a homeostatic decrease or increase in the intrinsic 50

membrane excitability of NAc MSNs, and vice versa. After short-term withdrawal from cocaine 51

self-administration, despite no actual change in the AMPA receptor-mediated excitatory synaptic 52

strength, GluN2B NMDA receptors, the SMHC sensors of synaptic strength, are upregulated. 53

This may create false SMHC signals, leading to a decrease in the membrane excitability of NAc 54

MSNs. The decreased membrane excitability subsequently induces another round of SMHC, 55

leading to synaptic accumulation of calcium-permeable AMPA receptors and upregulation of 56

excitatory synaptic strength after long-term withdrawal from cocaine. Disrupting SMHC-based 57

dysregulation cascades after cocaine exposure prevents incubation of cocaine craving. Thus, 58

cocaine triggers cascades of SMHC-based dysregulation in NAc MSNs, promoting incubated 59

cocaine seeking after drug withdrawal. 60

61

62

3

Significance 63

Here, we report a bidirectional homeostatic plasticity between the excitatory synaptic input and 64

membrane excitability of nucleus accumbens (NAc) medium spiny neurons (MSNs), through 65

which an increase or decrease in the excitatory synaptic strength induces a homeostatic 66

decrease or increase in the membrane excitability, and vice versa. Cocaine self-administration 67

creates a false homeostatic signal that engages this synapse-membrane homeostatic crosstalk 68

mechanism, and produces cascades of alterations in excitatory synapses and membrane 69

properties of NAc MSNs after withdrawal from cocaine. Experimentally preventing this 70

homeostatic dysregulation cascade prevents the progressive intensification of cocaine seeking 71

after drug withdrawal. These results provide a novel mechanism through which drug-induced 72

homeostatic dysregulation cascades progressively alter the functional output of NAc MSNs and 73

promote drug relapse. 74

75

4

Introduction 76

Drug addiction is a pathological emotional and motivational state resulting from neural 77

alterations after exposure to drugs of abuse (Nestler, 2001). Whereas many drug-induced 78

cellular alterations have a signature of Hebbian plasticity (White, 1996; Hyman et al., 2006), 79

others are thought to be homeostatic responses, i.e., they are induced to compensate for 80

altered functional output of neurons or neural circuits (Aghajanian, 1978; Nestler and 81

Aghajanian, 1997; Kalivas, 2005; Huang et al., 2011; Dong et al., 2017). Homeostatic 82

responses are important in maintaining a stable function of the brain, but they can also go awry 83

(Huang et al., 2011; Dong et al., 2017). Indeed, homeostatic dysregulation has long been 84

hypothesized as a key mechanism underlying the progression of the addictive state (Koob and 85

Le Moal, 1997). However, how addiction-related homeostatic alterations are formed and evolve 86

remains poorly understood. 87

Human drug users can experience progressive intensification of cue-induced drug 88

craving, a risk factor for relapse, after drug abstinence (Parvaz et al., 2016; Wolf, 2016). This 89

time-dependent change is recapitulated in rodent models, in which rats exhibit progressive 90

intensification of cue-induced cocaine seeking after withdrawal from cocaine self-administration, 91

a phenomenon termed incubation of cocaine craving (Grimm et al., 2001). A prominent feature 92

of incubation is that it occurs when drug withdrawal takes place in the home cage, with no 93

apparent Hebbian stimulation present. This prompted us to explore the role of homeostatic 94

plasticity in incubation of cocaine craving. 95

While incubation of cocaine craving involves many cellular mechanisms, increasing 96

evidence suggests that functional changes in medium spiny neurons (MSNs) in the nucleus 97

accumbens (NAc) play a critical role (Wolf, 2016). The functional output of NAc MSNs relies on 98

the integration of excitatory synaptic activity and membrane excitability. By definition, the 99

5

functional output of a neuron is action potential firing. Lacking endogenous pace-making 100

mechanisms, NAc MSNs rely on excitatory synaptic drive to climb toward the threshold of action 101

potentials and, around the threshold, the membrane excitability determines how many action 102

potentials will fire (O'Donnell et al., 1999). The excitatory postsynaptic strength is primarily 103

determined by AMPA receptors (AMPARs), and the membrane excitability is determined by a 104

variety of ion channels. 105

Incubation of cocaine craving begins in the first week of withdrawal and plateaus after a 106

month or so (Lu et al., 2004). After short-term (e.g., 1-2 d) withdrawal from cocaine self-107

administration, while the AMPAR-mediated excitatory synaptic strength of NAc MSNs remains 108

largely unchanged, the membrane excitability is decreased (Conrad et al., 2008; Mu et al., 109

2010; Wolf, 2016). After long-term (e.g., 45 d) withdrawal, the excitatory synaptic strength of 110

NAc MSNs is increased through synaptic accumulation of calcium-permeable (CP) AMPARs 111

(Conrad et al., 2008; Wolf, 2016). Thus, membrane excitability and excitatory synaptic strength 112

of NAc MSNs are altered sequentially after withdrawal from cocaine. In the present study, 113

focused on NAc shell (NAcSh) MSNs, we that these sequential changes are mechanistically 114

linked through homeostatic dysregulation cascades. 115

Specifically, our results show that the excitatory synaptic activity and membrane 116

excitability of NAcSh MSNs are coordinated via a synapse-membrane homeostatic crosstalk 117

(SMHC), through which an increase or decrease in excitatory synaptic strength induces a 118

homeostatic decrease or increase in the membrane excitability. Furthermore, we show that 119

GluN2B-containing NMDARs function as synaptic sensors, which detect alterations in synaptic 120

AMPARs. During cocaine self-administration and early withdrawal, although the AMPAR-121

mediated synaptic strength in NAcSh MSNs is not altered, synaptic GluN2B NMDAR-signaling 122

is unregulated. This triggers the first round of SMHC to decrease the membrane excitability of 123

NAcSh MSNs. Subsequently, the decreased membrane excitability initiates the second round of 124

6

SMHC, resulting in synaptic accumulation of CP-AMPARs and strengthening of NAc excitatory 125

synapses after long-term withdrawal from cocaine. Preventing the progression of these 126

homeostatic dysregulation cascades in NAcSh MSNs after withdrawal from cocaine prevents 127

incubation of cocaine craving. These results delineate a cellular mechanism through which 128

cocaine-induced homeostatic dysregulation promotes drug relapse after withdrawal. 129

Materials and methods 130

Subjects 131

Male Sprague-Dawley rats (Simonsen, CA; Harlan Lab, MD) of 35-45 days (d) old upon arrival 132

were used in most experiments. They were given ~1 week for acclimation to the housing 133

environment before experimental procedures. Some rats were used at postnatal d 6 for slice 134

cultures (Fig. 1). Male C57BL/6J GluN2B knock-in mice (Halt et al., 2012) of 4-8 weeks old 135

were used in experiments presented in Fig. 2J,K. Rats and mice were housed on a regular 12-h 136

light/dark cycle (light on at 07:00 AM) with food and water available ad libitum. 137

Repeated i.p. injections of cocaine 138

We used a 5-d cocaine procedure, similar to earlier studies (Dong et al., 2005; Dong et al., 139

2006). Briefly, once per day for 5d, rats were taken out of the home cage for an i.p. injection of 140

either cocaine HCl (15 mg/kg in saline) or the same volume of saline (control), and placed back 141

in the home cages immediately. 142

Virus preparation and in vivo delivery 143

The wild type (wt) and mutant (m) GluN2B (RS/QD) constructs were described previously 144

(Barria and Malinow, 2005). The cDNA for wtGluN2B-GFP or mGluN2B-GFP was cloned into 145

the recombinant, replication-defective sindbis virus backbone vector (pSINrep2S726). The 146

protocol for making sindbis virus was similar to that used previously (Marie et al., 2005; Dong et 147

7

al., 2006; Huang et al., 2008), except that the toxicity was further minimized by using a new 148

sindbis virus-based vector, pSINrep (nsP2S726). The GluN2B-, SK2, and luciferase control-RNAi 149

(bu2Bd: GCTGGTGATAATCCTTCTGAA; suSKc: GCCAGAGTCATGCTATTACAT; shLC: 150

CCTAAGGTTAAGTCGCCCTCG) were packaged into a recombinant AAV2 vector pseudotyped 151

with AAV8 capsids (referred to as AAV2/8 in the text) as previously described (Huang et al., 152

2015). 153

To infect in vivo NAcSh MSNs, a stereotaxic microinjection technique was utilized. 154

Briefly, rats were anesthetized with 1-3% isoflurane gas and a stainless steel cannula (31-155

gauge) was implanted bilaterally into the NAcSh (in mm: A, +1.65; L, ±0.8; D, -7.65). 156

Concentrated viral solutions (1 μl/side) were infused into the NAcSh through a pump at a flow 157

rate of 0.2 μl/min. The injection cannula was left in place for 5 min and then slowly withdrawn. 158

After the skin was sutured, the rats were placed on the heating pad for post-surgical recovery. 159

After waking up, rats were transferred to regular housing cages, where they were individually 160

housed. For experiments involving sindbis virus, the electrophysiological recordings of infected 161

MSNs were performed 1-2 d following the viral injection. For experiments involving AAV2/8, 162

recordings were performed >3 weeks after viral injection, and viral injection was often combined 163

with the self-administration surgery using a multi-surgical procedure. Infected neurons were 164

identified in living slices by their fluorescent signals using epifluorescence microscopy. 165

Intravenous surgery 166

Rats were anesthetized with a xylazine-ketamine mixture (5-10/50-100 mg/kg, i.p.). Ketoprofen 167

(5 mg/kg, s.c.) was given as an additional analgesic agent when need. The silicone catheter 168

was inserted into the jugular vein passing subcutaneously to the midscapular region and 169

connected to a vascular access button (Instech, PA) implanted under the skin. The rats were 170

then single-housed for 5-7 d to recover before the training sessions. Catheters were flushed 171

8

with sterile saline containing gentamycin (5 mg/ml) and heparin (10 units/ml) every 24 h during 172

recovery and training. 173

174

Drug self-administration 175

Each self-administration chamber, controlled by a Med Associates (Georgia, VT) system, was 176

equipped with 2 nose poke holes located 6 cm above the grid floor, but only one hole (active 177

hole) activated the infusion pump. The vascular access button with a quick connecting luer was 178

connected with dual-luer spring tether to a liquid swivel, which was connected to the syringe of 179

the infusion pump. A nose poke to the active hole resulted in a cocaine infusion (0.75 mg/kg per 180

infusion), accompanied by switch-on of a light within the holes, which served as conditioned 181

stimulus (CS), and a background light. The CS stayed on for 6 sec, and the background light 182

stayed on for 20 sec to signal the timeout period. During this 20-sec timeout period, additional 183

nose pokes were counted but did not result in cocaine infusion. Poking in the inactive hole did 184

not activate the infusion pump or elicit contingent cues, but was recorded. To avoid overdose, 185

we set the ceiling number of drug infusions at 100 for the 12-h overnight sessions as well as the 186

2-h daily sessions. After one overnight and then 5 d of self-administration training (>20 187

infusion/2-h session/day), rats were placed into their home cages for withdrawal. Rats that self-188

administered saline (adjusted to the same volume used for cocaine infusions) were used as 189

controls. 190

Preparation of NAc slice cultures and acute slices 191

Detailed procedures for obtaining and culturing NAc slices can be found in our previous 192

publications (Dong et al., 2006; Huang et al., 2008). Briefly, for slice cultures, rats were deeply 193

anesthetized with isoflurane and decapitated. Coronal NAc slices (200-μm thick; normally 2-3 194

slices were obtained from each rat) were collected in ice-cold sterile low Ca2+ solution 195

9

containing (in mM: 126 NaCl, 1.6 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 0.625 CaCl2, 18 NaHCO3, and 196

11 glucose), and then placed on Millicell Millipore culture plate inserts in wells containing 197

Neurobasal-A Media with 4% B-27 and 1% Glutamax-I Supplements (Invitrogen) for ~24 h until 198

they were transferred to the recording media for electrophysiological recordings. 199

For acute slices, rats were decapitated following isoflurane anesthesia. Coronal slices 200

(250-300 μm) containing the NAc were prepared on a VT1200S vibratome (Leica, Germany) in 201

4˚C cutting solution (in mM: 135 N-methyl-D-glucamine, 1 KCl, 1.2 KH2PO4, 0.5 CaCl2, 1.5 202

MgCl2, 20 choline-HCO3, and 11 glucose, saturated with 95% O2 / 5% CO2, pH adjusted to 7.4 203

with HCl). Slices were incubated in artificial cerebrospinal fluid (aCSF, in mM: 119 NaCl, 2.5 204

KCl, 2.5 CaCl2, 1.3 MgCl2, 1 NaH2PO4, 26.2 NaHCO3, and 11 glucose), saturated with 95% O2 / 205

5% CO2 at 37°C for 30 min and then allowed to recover for >30 min at room temperature before 206

experimentation. 207

Electrophysiological recordings 208

Electrophysiological recordings were preferentially made from MSNs located in the ventral-209

medial subregion of the NAcSh. MSNs are oval-shaped and middle-sized, and therefore can be 210

visually differentiated from local interneurons. Passive membrane properties were used as 211

another criterion for selecting MSNs (Cm 50-150pF, Rm 100-400mΩ). Interneurons could be 212

readily distinguished by their signature electrophysiological properties, such as a relatively 213

depolarized resting membrane potential (~-72 mV), higher frequency of action potential firing 214

upon current injection, or presence of large voltage sag at hyperpolarized potentials. 215

Interneurons were excluded from data analysis. 216

During recordings, slices were superfused with aCSF that was heated to 30-32°C by 217

passing the solution through a feedback-controlled in-line heater (Warner, CT) before it entered 218

the chamber. To measure the membrane excitability, electrodes (2-5 MΩ) were filled with a 219

10

potassium-based internal solution (in mM: 130 KMeSO3, 10 KCl, 0.4 EGTA, 10 HEPES, 2.5 Mg-220

ATP, 0.25 Na-GTP, 2 MgCl2-6H2O, pH 7.3). To record EPSCs, electrodes were filled with a 221

cesium-based internal solution (in mM: 140 CsCH3O3S, 5 TEA-Cl, 0.4 Cs-EGTA, 20 Hepes, 2.5 222

Mg-ATP, 0.25 Na-GTP, 1 QX-314, pH 7.3). To record AMPAR EPSCs, picrotoxin (100 μM) was 223

included in the recording bath to inhibit GABAA receptor-mediated currents. To record NMDAR 224

EPSCs, both picrotoxin and NBQX (5 μM) were included to inhibit GABAA receptor- and 225

AMPAR-mediated currents. Presynaptic afferents were stimulated by a constant-current isolated 226

stimulator (Digitimer, UK), using a monopolar electrode. Series resistance was 9-20 MΩ, left 227

uncompensated but monitored continuously during recording. Cells with a change in series 228

resistance beyond 15% were not accepted for data analysis. Synaptic currents were recorded 229

with a MultiClamp 700B amplifier, filtered at 2.6–3 kHz, amplified 5 times, and then digitized at 230

20 kHz. 231

Only electrophysiologically “stable” neurons were included for data analysis. In 232

experiments measuring the membrane excitability, after forming the whole-cell current-clamp 233

configuration, the recorded cells were given ~5 min for stabilization (oscillations of<5 mV) of 234

their resting membrane potentials. Recordings were discontinued if cells failed to meet this 235

criterion. After the stabilization period, the resting membrane potential was adjusted to -80 mV 236

by injecting a small current (usually <30 pA). A current step protocol (from -200 to +450 pA, with 237

a 50-pA increment; inter-pulse interval, 15 sec) was then repeated at least three times. 238

Immediately after the recording of a particular cell, the number of evoked action potentials was 239

compared across all three runs. Cells with a run-up or run-down of >15% were excluded from 240

further analysis. Thus, only MSNs with stable resting membrane potential and stable evoked 241

action potential firing were included in the final analysis. The afterhyperpolarization potential 242

(AHP) was sampled following the first action potential elicited by the 300-pA current injection. 243

The threshold of action potential was defined as the membrane potential for which the dV/dt 244

11

reached 10 before the spike. The amplitude of the fAHP was defined as the voltage difference 245

between the action potential threshold and the lowest hyperpolarization point after the action 246

potential. The mAHP was defined as the difference of the membrane potentials between the 247

action potential threshold and 10 ms after the action potential threshold. 248

Drugs 249

D-APV and NBQX were purchased from Tocris (Bristol, UK). All other chemicals were 250

purchased from Sigma-Aldrich. 251

Data acquisition and statistics 252

All data were analyzed off-line. >75% of the data were analyzed blind to experimental group. 253

Normal distribution was assumed for all data collected. All descriptive results were presented as 254

mean ± SEM. A two-tail t-test, paired t-test, or ANOVA was used for statistical comparisons as 255

specified in the text. Bonferroni posttest was performed if ANOVA indicated significance. No 256

data points were excluded unless specified in the text. For experiments in which the endpoints 257

were behavioral results (Fig. 7), animal-based statistics were presented. For experiments in 258

which the endpoints were from individual cells, both animal- and cell-based statistical analyses 259

were performed, and these results were highly consistent. For animal-based statistics, we used 260

the averaged value of a parameter from all cells recorded from an animal to represent the 261

parameter of this animal. In cell-based analyses, sample sizes were presented as n/m, in which 262

n represented the number of cells, and m represented the number of animals from which cells 263

were collected. 264

Results 265

GluN2B-CaMKII signaling as the synaptic sensor of SMHC 266

12

AMPARs mediate the majority of excitatory synaptic transmission and AMPAR-mediated 267

currents are often used as a measure of excitatory synaptic strength. We previously 268

demonstrated a form of synapse-to-membrane homeostatic plasticity in NAcSh MSNs in which 269

an increase or decrease in the AMPAR-mediated excitatory synaptic strength induced a 270

homeostatic decrease or increase in the membrane excitability, respectively (Ishikawa et al., 271

2009) (Fig. 1A). We termed this and the potential reverse form (i.e., membrane-to-synapse) of 272

homeostatic plasticity SMHC. 273

For an effective homeostatic feedback loop, the minimum requirements are a detector of 274

alterations, an effector to make compensatory changes, and a signaling pathway that links the 275

detector to the effector. Our previous results identified synaptic NMDA receptors (NMDARs) as 276

the detectors of excitatory synaptic strength, based on evidence that inhibiting NMDARs 277

prevents synapse-to-membrane homeostatic plasticity, while persistently increasing or 278

decreasing NMDAR function without affecting synaptic AMPARs is sufficient to induce 279

homeostatic decreases or increases in the membrane excitability of NAcSh MSNs (Ishikawa et 280

al., 2009). However, it remains unclear which subtypes of NMDARs and which signaling 281

pathways are involved. 282

In the rodent forebrain, NMDARs can be largely divided into two categories, GluN2B-283

containing vs. GluN2B-lacking NMDARs, each with unique intracellular signaling properties 284

(Monyer et al., 1994; Cull-Candy and Leszkiewicz, 2004). To evaluate the roles of these two 285

NMDAR subtypes in synapse-to-membrane homeostatic plasticity, we used a set of 286

pharmacological manipulations in NAcSh slice cultures (Fig. 1B-D). In our experimental 287

condition, NMDAR-mediated EPSCs were evoked at -40 mV in the presence of the AMPAR-288

selective antagonist NBQX (5 μM); these EPSCs are presumably mediated by both GluN2B-289

containing and GluN2B-lacking NMDARs. Application of the GluN2B-selective antagonist 290

Ro256981 (20 nM) decreased the amplitude and shortened the decay kinetics of NMDAR 291

13

EPSCs, as expected based on properties of GluN2B NMDARs (Fig. 1B-D). D-cycloserine 292

(DCS) is an NMDAR glycine-site agonist, and therefore enhances the function of both GluN2B-293

containing and GluN2B-lacking NMDARs. Application of DSC (100 nM) alone increased the 294

amplitudes of NMDAR EPSCs (Fig. 1B-D). When DCS and Ro256981 were co-applied, the 295

overall amplitude of NMDAR EPSCs returned to the control level. This “lack” of change in the 296

overall amplitude of NMDAR EPSCs likely resulted because the DCS-mediated increase in the 297

GluN2B-lacking NMDAR component was offset by the Ro256981-mediated decrease in the 298

GluN2B-containing NMDAR component (F2,14=21.0, p<0.01, one-way ANOVA repeated 299

measure; p=0.02, control vs. DCS; p=0.03, DCS vs. DCS+Ro256981, Bonferroni posttest; Fig. 300

1B-D). Thus, co-application of DCS and Ro256981 creates an experimental condition in which 301

NMDARs are simultaneously manipulated in three different ways: 1) no change in the overall 302

NMDAR activity, 2) upregulation of GluN2B-lacking NMDAR function, and 3) downregulation of 303

GluN2B-containing NMDAR function (Fig. 1D). 304

We used this co-application strategy to gain insight into which NMDAR subtype acts as 305

the sensor of synapse-to-membrane homeostatic plasticity. If the total synaptic NMDAR activity 306

is the key regardless of the contributing NMDAR subtypes, co-incubation of DCS and 307

Ro256981, which did not significantly affect the total NMDAR activity, should not induce any 308

homeostatic change in membrane excitability. If GluN2B-lacking NMDARs are the key, an 309

increase in this component through co-application of DCS and Ro256981 should induce a 310

homeostatic decrease in the membrane excitability. Conversely, if GluN2B-containing NMDARs 311

are the key, a decrease in this component by co-incubation of DCS and Ro256981 should 312

induce a homeostatic increase in the membrane excitability. Similar to previous results, after an 313

overnight (~12 h) incubation of the NAcSh slice cultures with DCS alone, we observed a 314

decrease in the membrane excitability of NAcSh MSNs (Fig. 1E,F; see statistics below). In 315

contrast, overnight incubation with either Ro 256981 alone or co-incubation of DCS and 316

14

Ro256981 induced a similar increase in the membrane excitability of NAcSh MSNs (F2,75=45.7, 317

p<0.01, two-way ANOVA; p<0.01, control vs. DCS, control vs. Ro256981, or control vs. DCS + 318

Ro256981, Bonferroni posttest; Fig. 1E,F). These results support the scenario in which GluN2B, 319

but not GluN2A, NMDARs mediate the synapse-to-membrane homeostatic plasticity in NAcSh 320

MSNs. Note that acute pharmacological manipulations of NMDARs did not change the 321

membrane excitability of NAcSh MSNs (Ishikawa et al., 2009), suggesting that this NMDAR-322

mediated synapse-to-membrane homeostatic plasticity happens gradually. 323

Enlightened by the above pharmacological results, we next used molecular tools to 324

scrutinize synaptic GluN2B and its coupled signaling during synapse-to-membrane homeostatic 325

plasticity. At the signaling level, GluN2B differs from GluN2A in its ability to bind and recruit 326

activated calcium-calmodulin dependent protein kinase II (CaMKII), thereby promoting the 327

maintenance of CaMKII activation (Strack and Colbran, 1998; Leonard et al., 1999; Strack et al., 328

2000; Mayadevi et al., 2002; Halt et al., 2012). We used viral-mediated gene transfer to express 329

a mutant form of GluN2B (mGluN2B). This mGluN2B shares all other biophysical properties with 330

wild type (wt) GluN2B, including slow decay kinetics, but its CaMKII interacting site is 331

compromised (Barria and Malinow, 2005). Expression of wtGluN2B was the control condition. 332

One day after virus injection, many NAcSh MSNs were infected to express targeted genes, as 333

indicated by GFP expression (Fig. 1G). NMDAR EPSCs in both wtGluN2B- and mGluN2B-334

expressing MSNs exhibited increased decay kinetics, indicating that virally expressed wtGluN2B 335

or mGluN2B subunits were assembled into functional synaptic NMDARs (decay kinetics was 336

measured as the time required for NMDAR-mediated EPSCs to decrease from peak amplitude 337

to ½ peak amplitude: F2,35=6.2, p=0.01, one-way ANOVA; p<0.05 control vs. wtGluN2B or 338

mGluN2B, Bonferroni posttest; Fig. 1H). Note that due to the complicated decay properties of 339

NMDAR EPSCs in NAcSh MSNs, we chose to use the time elapsed from the peak EPSCs to 340

2/3 or 1/2 of the peak amplitude of EPSCs to assess the decay kinetics (Huang et al., 2009). 341

15

Importantly, the membrane excitability of wtGluN2B-expressing NAcSh MSNs was decreased 342

compared to MSNs that expressed GFP alone (F2,33=7.5, p<0.01, two-way ANOVA; p<0.01 343

control vs. wtGluN2B, Bonferroni posttest; Fig. 1I,J). These molecular results, together with 344

above pharmacological results, indicate that upregulation of GluN2B NMDARs is sufficient to 345

induce a homeostatic decrease in the membrane excitability. In contrast, the membrane 346

excitability was not altered in mGluN2B-expressing MSNs (p>0.99, control vs. mGluN2B, 347

Bonferroni posttest; Fig. 1H), suggesting that loss of CaMKII-signaling abrogates GluN2B 348

NMDAR-mediated synapse-to-membrane homeostatic plasticity. 349

GluN2B-CaMKII signaling initiates synapse-to-membrane homeostatic dysregulation after 350

cocaine 351

After short-term (1-2d) withdrawal from either repeated i.p. injections of cocaine or cocaine self-352

administration, the overall excitatory synaptic strength in NAc MSNs, which is primarily 353

mediated by synaptic AMPARs, is not significantly altered (Boudreau and Wolf, 2005; Kourrich 354

et al., 2007; Conrad et al., 2008). Our previous results show that repeated i.p. injections of 355

cocaine induce an upregulation of synaptic GluN2B NMDARs in NAcSh MSNs, which results 356

from activation of CREB, one of the earliest molecular effects of cocaine (Huang et al., 2009; 357

Brown et al., 2011). Similarly, our present study detected prolonged decay kinetics of NMDAR 358

EPSCs in NAcSh MSNs 1 d after discontinuing cocaine self-administration, suggesting a similar 359

increase in synaptic GluN2B NMDARs (Fig. 2A-G; see below for statistics). Thus, although the 360

actual AMPAR-mediated excitatory synaptic strength is not altered at this withdrawal time point, 361

cocaine-induced upregulation of GluN2B NMDARs may create a false homeostatic signal, 362

triggering synapse-to-membrane homeostatic plasticity to decrease the membrane excitability of 363

NAcSh MSNs. Consistent with this scenario, the membrane excitability of NAcSh MSNs is 364

decreased after short-term (1-2d) withdrawal from either noncontingent or contingent cocaine 365

16

administration (Zhang et al., 1998; Dong et al., 2006; Ishikawa et al., 2009; Mu et al., 2010; 366

Kourrich et al., 2013). 367

To examine whether upregulation of GluN2B NMDARs plays a causal role in decreasing 368

the membrane excitability of NAcSh MSNs in cocaine-exposed rats, we designed a GluN2B-369

RNAi, which, after viral-mediated expression, shortened the decay kinetics of NMDAR EPSCs in 370

NAcSh MSNs in saline-exposed rats, demonstrating in vivo knockdown efficacy (Fig. 2D-G; see 371

statistics below). Knocking-down GluN2B prevented the cocaine-induced increase in decay 372

kinetics of NMDAR EPSCs, thus validating this RNAi as an effective tool preventing cocaine-373

induced GluN2B upregulation (time to 2/3 and 1/2 peak: F1,85=6.9 and 6.8, p=0.01 and 0.01, 374

cell-based two-way ANOVA; p<0.01 and 0.01 saline-noninfected vs. saline GluN2B-RNAi, 375

p<0.01 and 0.01 cocaine-noninfected vs. cocaine GluN2B-RNAi, Bonferroni posttest; F1, 27=4.5 376

and 4.2, p=0.042 and 0.049, animal-based ANOVA; p<0.01 and 0.01 cocaine-noninfected vs. 377

saline-noninfected, p<0.01 and 0.01 cocaine-noninfected vs. cocaine-GluN2B-RNAi, p=0.03 and 378

0.13 saline-noninfected vs. saline-GluN2B-RNAi, Bonferroni posttest; Fig. 2D-G). We then 379

examined the membrane excitability. In control (non-infected) MSNs, the membrane excitability 380

was decreased after 1 d of withdrawal from cocaine self-administration (Fig. 2H,I; see statistics 381

below), similar to previous results (Mu et al., 2010). This cocaine-induced membrane adaptation 382

was prevented in NAcSh MSNs expressing GluN2B-RNAi (F1,86=11.0, p<0.01, cell-based three-383

way mixed ANOVA with repeated measures injected current 200, 250, 300, 350, 400 and 450 384

pA; p<0.01 saline-noninfected vs. cocaine-noninfected p<0.01 cocaine-noninfected vs. cocaine-385

GluN2B-RNAi, Bonferroni posttest; F1,35=10.1, p<0.01, animal-based ANOVA; p,0.01 cocaine-386

noninfected vs. cocaine-GluN2B-RNAi, p>0.99 saline-noninfected vs. saline-GluN2B-RNAi, 387

p>0.99 saline-GluN2B-RNAi vs. cocaine-GluN2B-RNAi, Bonferroni posttest; Fig. 2H,I). These 388

results suggest that synaptic upregulation of GluN2B NMDARs is essential for the cocaine-389

induced decrease in membrane excitability of NAcSh MSNs. 390

17

To delineate the role of GluN2B-coupled CaMKII-signaling, we employed a knock-in (KI) 391

mouse line, in which the endogenous GluN2B was replaced by a mutant with reduced ability to 392

activate CaMKII signaling, and thus, conceivably, with reduced basal GluN2B-CaMKII signaling 393

(Halt et al., 2012). Due to the technical difficulties associated with self-administration in mice, we 394

used the 5 d i.p. injection procedure, which also results in synaptic upregulation of GluN2B and 395

decreased membrane excitability of NAcSh MSNs (Huang et al., 2009; Ishikawa et al., 2009; Mu 396

et al., 2010; Brown et al., 2011). Compared to WT mice, NAcSh MSNs in KI mice exhibited 397

increased membrane excitability after saline (control) administration, suggesting that decreased 398

basal GluN2B-CaMKII signaling is sufficient to induce synapse-to-membrane SMHC (F1, 42=4.3, 399

p=0.04, cell-based three-way mixed ANOVA with repeated measures over injected current-200, 400

225, 250, 275 and 300 pA; p=0.04 WT-saline vs. WT-cocaine, Bonferroni posttest; F1,11=5.3, 401

p=0.04, animal-based ANOVA; p=0.02 WT-cocaine vs. WT-saline, p<0.01 wt-cocaine vs. 402

GluN2B KI-cocaine, p=0.64 WT-saline vs. GluN2B KI-saline, Bonferroni posttest; Fig. 2J,K). 403

Furthermore, in these mutant mice, exposure to cocaine could no longer induce a decrease in 404

membrane excitability of NAcSh MSNs (p=0.21 WT-saline vs. KI-cocaine, p>0.99 KI-saline vs. 405

KI-cocaine, Bonferroni posttest; Fig. 2J,K). 406

Collectively, these results support the hypothesis that cocaine-induced upregulation of 407

synaptic GluN2B NMDARs creates as a false signal that inappropriately engages SMHC, 408

resulting in decreased membrane excitability of NAcSh MSNs. 409

GluN2B-to-SK2 channels in cocaine-induced SMHC 410

The SK2 type of calcium-activated potassium channel mediates the medium component of the 411

afterhyperpolarization potential (mAHP), which transiently hyperpolarizes the cell membrane 412

after action potentials to delay the generation of subsequent action potentials. The SK2-413

mediated mAHP in NAcSh MSNs is low in naïve or saline-exposed rats, but is increased after 414

18

repeated i.p. injections of cocaine, contributing to the decreased membrane excitability of 415

NAcSh MSNs (Ishikawa et al., 2009). Similarly, 1 d after discontinuing cocaine self-416

administration (0.75mg/kg per infusion, 1 overnight followed by 2 h/d x 5 d; Fig. 2A-C), control 417

(non-infected) NAcSh MSNs exhibited increased mAHP amplitudes (Fig. 3A,B; see statistics 418

below). 419

To determine whether SK2 is the target for GluN2B-mediated depression of membrane 420

excitability after cocaine, we virally expressed GluN2B-RNAi ~2 weeks before the self-421

administration training. Cocaine-induced upregulation of mAHP was prevented in NAcSh MSNs 422

expressing GluN2B-RNAi, tested 1 d after discontinuing cocaine self-administration (F1,86=10.6, 423

p<0.01, cell-based two-way ANOVA; p<0.01 cocaine-noninfected vs. saline-noninfected, p<0.01 424

cocaine-noninfected vs. cocaine-GluN2B-RNAi, Bonferroni posttest; F1,35=6.8, p=0.01, animal-425

based ANOVA; p<0.01 cocaine-noninfected vs. saline-noninfected, p<0.01 cocaine-noninfected 426

vs. cocaine-GluN2B-RNAi, p>0.99 saline-noninfected vs. saline-GluN2B-RNAi, p>0.99 saline-427

GluN2B-RNAi vs. cocaine-GluN2B-RNAi, Bonferroni posttest; Fig. 3A,B). Furthermore, cocaine-428

induced upregulation of the mAHP in NAcSh MSNs persisted after 45 d of drug withdrawal, and 429

this long-lasting effect was also prevented by pre-training expression of GluN2B-RNAi (F1, 97 430

=4.4, p=0.04, cell-based two-way ANOVA; p=0.03 saline-noninfected vs. cocaine-noninfected, 431

p<0.01 cocaine-noninfected vs. cocaine-GluN2B-RNAi, Bonferroni posttest; F1,35=5.4, p=0.03 432

animal based ANOVA; p=0.02 cocaine-noninfected vs. saline-noninfected, p<0.01 cocaine-433

noninfected vs. cocaine-GluN2B-RNAi, Bonferroni posttest; Fig. 3C-E). Consistently, the 434

membrane excitability of NAcSh MSNs remained low after 45 d of withdrawal from cocaine, and 435

this long-lasting membrane effect was prevented by pre-expression of GluN2B-RNAi (F1, 97 436

=11.2, p<0.01, cell-based three-way mixed ANOVA with repeated measures; p<0.01 saline-437

noninfected vs. cocaine-noninfected, p<0.01 cocaine-noninfected vs. cocaine-GluN2B-RNAi, 438

Bonferroni posttest; F1,35=11.6, p<0.01, animal-based ANOVA; p<0.01 cocaine-noninfected vs. 439

19

saline-noninfected, p<0.01 cocaine-noninfected vs. cocaine-GluN2B-RNAi, p>0.99 saline-440

noninfected vs. saline-GluN2B-RNAi, p>0.99 saline-GluN2B-RNAi vs. cocaine-GluN2B-RNAi, 441

Bonferroni posttest; Fig. 3F,G). Thus, SK2 channels are potential downstream effectors of 442

GluN2B/CaMKII-signaling and key for the expression of SMHC-mediated decreases in 443

membrane excitability after cocaine exposure. 444

To manipulate SK2 channels, we made an adeno-associated virus 2 pseudotyped with 445

AAV8 capsids (AAV2/8) that expressed SK2-RNAi. Because of the low basal levels of SK2 446

channels in NAcSh MSNs (Ishikawa et al., 2009), we functionally validated this virus using 447

dorsal striatal MSNs, which expressed higher basal levels of SK2 and a more robust mAHP. 448

The mAHP amplitude was significantly decreased in striatal MSNs that expressed SK2-RNAi 449

(t19=2.4, p=0.03, cell-based two-tailed t-test; Fig 4 A-D), while the fAHP was unaffected 450

(t19=0.52, p=0.61, cell-based two-tailed t-test), suggesting an effective and selective knockdown 451

of SK2. We then virally expressed this SK2-RNAi in the NAcSh before self-administration 452

training. One day after discontinuing saline self-administration, SK2-RNAi-expressing and non-453

infected NAcSh MSNs exhibited similarly low mAHP amplitudes, confirming that the basal level 454

of SK2 was low in NAcSh MSNs and could not be further decreased (Fig. 4F,G; see statistics 455

below). On the other hand, expression of SK2-RNAi effectively prevented the upregulation of 456

mAHP in cocaine-exposed rats on the first day of withdrawal (F1,63=4.5, p=0.04, cell-based two-457

way ANOVA; p<0.01 cocaine-noninfected vs. saline-noninfected, p<0.01 cocaine-noninfected 458

vs. cocaine-SK2-RNAi, p>0.99 saline-noninfected vs. saline-SK2-RNAi, Bonferroni posttest; 459

F1,30=7.6, p=0.01, animal based ANOVA; p<0.01 cocaine-noninfected vs. saline-noninfected, 460

p<0.01 cocaine-noninfected vs. cocaine-SK2-RNAi, p=0.17 saline-noninfected vs. saline-SK2-461

RNAi, p=0.33 saline-SK2-RNAi vs. cocaine-SK2-RNAi, Bonferroni posttest Fig. 4F,G). 462

Furthermore, expression of SK2-RNAi did not affect basal membrane excitability of NAcSh 463

MSNs, as shown in saline-exposed rats, but prevented the cocaine-induced decrease in their 464

20

membrane excitability (F1,63=4.5, p=0.04, cell-based three-way mixed ANOVA with repeated 465

measures over injected current of 200, 250, 300, 350, 400 and 450 pA; p<0.01 cocaine-466

noninfected vs. saline-noninfected, p<0.01 cocaine-noninfected vs. cocaine-SK2-RNAi, p>0.99 467

saline-noninfected vs. saline-SK2-RNAi, Bonferroni posttest; F1,30=6.7, p=0.02 animal based 468

ANOVA; p<0.01 cocaine-noninfected vs. saline-noninfected, p<0.01 cocaine-noninfected vs. 469

cocaine-SK2-RNAi, p=0.48 saline-noninfected vs. saline-SK2-RNAi, p>0.99 saline-SK2-RNAi 470

vs. cocaine-SK2-RNAi, Bonferroni posttest; Fig. 4H,I). Thus, the SK2 channel is likely one of the 471

key ion channels that are changed after cocaine administration to decrease the membrane 472

excitability. 473

Membrane-to-synapse SMHC during withdrawal from cocaine 474

The above results demonstrate a synapse-to-membrane SMHC and suggest that it is hijacked 475

by cocaine to decrease the membrane excitability of NAcSh MSNs. Next, we asked whether 476

SMHC also exists in the membrane-to-synapse direction and contributes to synaptic alterations 477

after withdrawal from cocaine. A critical adaptation at NAcSh excitatory synapses after long-478

term withdrawal from cocaine self-administration is the delayed upregulation of synaptic 479

strength (Wolf, 2016). Specifically, synaptic AMPARs in NAc MSNs largely remain at the basal 480

level (saline controls) 1 d after cocaine self-administration. When cocaine withdrawal proceeds, 481

excitatory synapses in both the shell and core subregions of the NAc are gradually strengthened 482

by recruiting calcium-permeable (CP) AMPARs (Wolf, 2016). A long-held question is what 483

mechanisms drive AMPARs toward NAc excitatory synapses during cocaine withdrawal, during 484

which no apparent external stimuli are present to induce Hebbian plasticity. We hypothesize that 485

the decreased membrane excitability in early withdrawal from cocaine self-administration 486

initiates a membrane-to-synapse SMHC process, which gradually develops as withdrawal 487

continues, leading to the strengthening of excitatory synapses after long-term withdrawal (Fig. 488

5A). 489

21

The above hypothesis predicts that preventing the cocaine-induced decrease in the 490

membrane excitability will prevent upregulation of synaptic AMPARs after long-term withdrawal 491

from cocaine. To test this prediction, we performed a pairwise recording to simultaneously 492

measure EPSCs in a non-infected NAcSh MSN and a neighboring SK2-RNAi-expressing MSN 493

after cocaine self-administration (Fig. 5B,C). We started with two important control experiments. 494

First, 1 d after the self-administration procedure (Fig. 5B), the amplitudes of EPSCs were 495

similar between non-infected versus SK2-RNAi-expressing MSNs in saline-exposed rats 496

(t13=0.61, p=0.55, cell-based paired t-test; Fig. 5D-H). Because the basal level of SK2 in NAcSh 497

MSNs was low (Fig. 4F-I), expression of SK2-RNAi did not affect the membrane excitability in 498

control rats. These results also indicate that neither the AAV system nor expression of SK2-499

RNAi per se directly affects excitatory synapses. Second, 1 d after cocaine self-administration, 500

the amplitudes of EPSCs were similar between non-infected versus SK2-RNAi-expressing 501

NAcSh MSNs (t24=1.19, p=0.25, cell-based paired t-test; Fig. 5D-H). At this withdrawal time-502

point, although the SK2-mediated membrane adaptation in NAcSh MSNs is accomplished (Fig. 503

4F-I), the excitatory synaptic strength has not been altered yet (Wolf, 2016). Thus, if the 504

potential membrane-to-synapse SMHC is triggered after cocaine exposure, it is still in the initial 505

stage without a detectable synaptic effect. As such, preventing the SK2-mediated membrane 506

adaptation at this time-point does not affect excitatory synapses on NAcSh MSNs in cocaine-507

exposed rats. Consistent with these results, perfusion of the selective CP-AMPAR antagonist 508

Naspm did not significantly alter AMPAR EPSC amplitudes in either non-infected MSNs or SK2-509

expressing MSNs from saline- or cocaine-exposed rats on the first day of withdrawal (F1, 510

52=0.63, p=0.43, cell-based two-way ANOVA; F1, 27=0.38, p=0.54, animal-based ANOVA; Fig. 511

5I-M). Therefore, viral-mediated expression of SK2-RNAi does not affect the subunit 512

composition of synaptic AMPARs in early withdrawal. 513

22

Forty-five d after discontinuing cocaine self-administration, there is an overall 514

strengthening of excitatory synapses in the NAc, primarily mediated by synaptic insertion of CP-515

AMPARs (Wolf, 2016). To test whether this synaptic adaptation is achieved through SMHC 516

triggered by the decreased membrane excitability, we expressed SK2-RNAi in the NAcSh 3 517

weeks before cocaine self-administration, and made pairwise recordings after 45 d of 518

withdrawal. In saline-exposed control rats, no difference in EPSC amplitudes between SK2-519

RNAi-expressing MSNs and their neighboring non-infected MSNs was detected, indicating that 520

expression of SK2-RNAi does not change basal excitatory synaptic strength with this time 521

course of viral expression (t14=0.36, p=0.72, cell-based paired t-test; Fig. 6B,C). In cocaine-522

exposed rats, the EPSC amplitudes in SK2-RNAi-expressing NAcSh MSNs were significantly 523

smaller compared to their neighboring non-infected MSNs (t17=4.03, p<0.01, cell-based paired t-524

test; Fig. 6D-F). Given that strengthening of NAcSh excitatory synapses in non-infected MSNs 525

has been accomplished at this withdrawal time-point, this result can be interpreted to indicate 526

that the withdrawal-associated synaptic strengthening is prevented in SK2-RNAi-expressing 527

MSNs. Consistently, application of Naspm significantly inhibited EPSCs in non-infected MSNs in 528

cocaine-exposed rats, reflecting withdrawal-associated CP-AMPAR accumulation, and this CP-529

AMPAR accumulation was prevented in SK2-RNAi-expressing MSNs (F1,46=8.0, p<0.01, cell-530

based two-way ANOVA; p<0.01 saline-noninfected vs. cocaine-noninfected, p<0.01 cocaine-531

noninfected vs. cocaine-SK2-RNAi, p>0.99 saline-noninfected vs. saline-SK2-RNAi, Bonferroni 532

posttest; F1,27=8.2, p<0.01, animal-based two-way ANOVA; p<0.01 cocaine-noninfected vs. 533

saline-noninfected, p<0.01 cocaine-noninfected vs. cocaine-SK2-RNAi, p>0.99 saline-534

noninfected vs. saline-SK2-RNAi, Bonferroni posttest; Fig. 6G-K). Thus, membrane-to-synapse 535

SMHC-based dysregulation occurs over long-term withdrawal from cocaine self-administration, 536

promoting synaptic accumulation of CP-AMPARs in NAc MSNs. 537

Preventing SMHC dysregulation prevents incubation of cocaine craving 538

23

To test the role of SMHC in the incubation of cocaine craving, we bilaterally injected AAV2/8 539

expressing either GluN2B-RNAi or SK2-RNAi into the NAcSh of rats, thus preventing the SMHC 540

cascade after cocaine through two different routes. Rats with intra-NAcSh injection of luciferase-541

RNAi-expressing AAV2/8 served as controls. Three weeks later, these rats were trained with the 542

5 d cocaine self-administration procedure, followed by either 1 or 45 d of withdrawal. Control 543

rats assessed after 45 d of withdrawal exhibited higher levels of cue-induced cocaine seeking in 544

a 1-h extinction test compared to rats assessed after only 1 d of withdrawal, indicating 545

incubation of cocaine craving (Fig. 7A-E). Intra-NAcSh knockdown of GluN2B prevented 546

incubation of cocaine craving (F1,35=9.6, p<0.01, two-way ANOVA; p<0.01 luciferase-d1 vs. 547

luciferase-d45, p=0.02 luciferase-d45 vs. GluN2B-RNAi-d45, p>0.99 GluN2B-RNAi-d1 vs. 548

GluN2B-RNAi-d45, Bonferroni posttest; Fig. 7A-E). Similarly, intra-NAc expression of SK2-RNAi 549

also prevented the incubation of cocaine craving (F1, 36=9.6, p<0.01, two-way ANOVA; p<0.01 550

luciferase-d1 vs. luciferase-d45, p=0.02 luciferase-d45 vs. SK2-RNAi d45, p>0.99 SK2-RNAi-d1 551

vs. SK2-RNAi-d45, Bonferroni posttest; Fig. 7F-J). Thus, cocaine-induced SMHC cascades in 552

NAcSh MSNs promote incubation of cocaine craving. 553

Discussion 554

Our current results demonstrate a bi-directional SMHC (Fig. 7K), through which two critical 555

physiological parameters interact to coordinate the functional output of NAcSh MSNs. 556

Furthermore, upregulation of GluN2B NMDARs after cocaine self-administration initiates 557

cascades of SMHC to sequentially change the membrane excitability and AMPAR-mediated 558

excitatory strength of NAcSh MSNs (Fig. 7L). 559

GluN2B NMDAR signaling 560

Our previous results showed that up- or downregulation of AMPAR-mediated excitatory synaptic 561

strength induces homeostatic down- or upregulation of membrane excitability in NAcSh MSNs 562

24

(Ishikawa et al., 2009). Our present results identify the GluN2B NMDAR subtype as the sensor 563

of excitatory synaptic strength that subsequently engages SMHC. 564

In vivo, striatal MSNs often reside at upstate membrane potentials (~-55 mV) (Mahon et 565

al., 2006), where the Mg2+-mediated blockade of NMDARs is partially relieved (Jahr and 566

Stevens, 1990). The Mg2+ blockade curve rises/declines sharply around upstate membrane 567

potentials (Jahr and Stevens, 1990), such that small changes of the membrane potential by 568

altered excitatory inputs may be translated into substantial changes in basal NMDAR activity, 569

rendering NMDARs highly sensitive in monitoring excitatory synaptic strength. 570

NMDARs in the adult forebrain are thought to exist as tetramers primarily consisting of 571

GluN1 and GluN2 subunits (Monyer et al., 1994; Standaert et al., 1994). In NAcSh MSNs, ~25% 572

of synaptic NMDARs contain GluN2B (Huang et al., 2011; Graziane et al., 2016). Compared to 573

GluN2A NMDARs, GluN2B NMDARs exhibit slower decay kinetics, and thus can remain active 574

during trains of synchronous synaptic activation, which is typical for in vivo NAcSh MSNs (Vicini 575

et al., 1998; O'Donnell et al., 1999). Furthermore, GluN2B NMDARs exhibit a high ability to 576

augment and prolong postsynaptic CaMKII signaling (Strack and Colbran, 1998; Leonard et al., 577

1999; Strack et al., 2000; Mayadevi et al., 2002; Halt et al., 2012). These properties endow 578

GluN2B NMDARs with a unique role in synaptic plasticity (Tang et al., 1999; Tang et al., 2001; 579

Wong et al., 2002; Liu et al., 2004; Massey et al., 2004; Barria and Malinow, 2005; Berberich et 580

al., 2005; Halt et al., 2012). Our current results suggest that, with these properties, upregulated 581

GluN2B NMDARs engage SMHC for cascades of cellular adaptations in NAcSh MSNs after 582

drug withdrawal. It is worth noting that our results cannot rule out the possibility that the 583

increased GluN2B-signaling may also operates independently of MSHC to regulate synaptic 584

and membrane substrates. 585

25

While synaptic transmission is likely the main source of glutamate that activates 586

GluN2B-signaling, glial source should not be ignored. Glial cells are critically important for brain 587

glutamate homeostasis. After cocaine self-administration, certain glial-mediated glutamate 588

reuptake mechanisms are compromised, resulting in elevated levels of extracellular glutamate 589

(Scofield and Kalivas, 2014). This ambient glutamate may operate together with synaptically 590

released glutamate to argument excitatory tone and promote SMHC. 591

Synaptic expression of GluN2B NMDARs is sensitively regulated by development. In our 592

experiments, animals ranging in age from 6 d (slice cultures) to 20 weeks (after 45 d withdrawal) 593

were used. Although results from these animals consistently suggest the critical role of GluN2B 594

NMDARs in SMHC, we cannot rule out the possibility that developmental regulation of GluN2B 595

NMDARs influences the magnitude or pattern of SMHC. It is also important to note that NAc 596

MSNs are highly heterogeneous. For example, they can be divided into dopamine receptor D1- 597

vs. D2-expressing neurons that can play distinct functional roles (Smith et al., 2013). Future 598

studies should conduct parallel examinations of membrane excitability and synaptic strength in 599

each population following cocaine self-administration. 600

SMHC in early withdrawal from cocaine 601

We showed previously that elevation of GluN2B expression in NAcSh MSNs during 602

noncontingent cocaine injections corresponded to the generation of GluN2B-enriched silent 603

synapses (Huang et al., 2009; Brown et al., 2011). After similar non-contingent procedures, the 604

AMPAR/NMDAR ratio at NAcSh synapses is decreased after 1 d of withdrawal from cocaine 605

(Kourrich and Thomas, 2009), whereas surface AMPARs are unchanged (Boudreau and Wolf, 606

2005). Thus, upregulation of GluN2B NMDARs may underlie the decreased AMPAR/NMDAR 607

ratio. 608

26

During the first round of SMHC, we show that one of the targeted membrane substrates 609

is the SK2 channel, and that preventing cocaine-induced upregulation of synaptic GluN2B 610

NMDARs prevents SK2 upregulation (Figs. 3, 4F,G). Importantly, SK2 is likely not the only 611

membrane substrate responding to synaptic signals. It is conceivable that, in most homeostatic 612

responses including SMHC, cells will mobilize a wide range of adaptations to maintain overall 613

functional stabilization. Consistent with this scenario, it has also been observed that voltage-614

gated potassium currents are increased, while voltage-gated sodium and calcium currents are 615

decreased in NAcSh MSNs after cocaine exposure, all likely contributing to decreased 616

membrane excitability (Zhang et al., 1998; Zhang et al., 2002; Hu et al., 2004; Dong et al., 2006; 617

Kourrich et al., 2013). 618

SMHC in late withdrawal from cocaine 619

After prolonged withdrawal from cocaine self-administration, CP-AMPARs accumulate in both 620

NAc shell and core (Conrad et al., 2008; Lee et al., 2013; Ma et al., 2014). Thereafter, 621

manipulations that remove or block CP-AMPARs in either region inhibit expression of incubated 622

craving (Conrad et al., 2008; Lee et al., 2013; Loweth et al., 2014; Ma et al., 2014). The 623

requirement for activation of both core and shell may be explained by interconnections between 624

these regions (Haber et al., 2000; Ikemoto, 2007). 625

The present findings identify one mechanism contributing to CP-AMPAR accumulation, 626

namely a second round of SMHC. We demonstrated this by developing an RNAi approach to 627

prevent cocaine-induced upregulation of SK2, and showing that this manipulation prevents 628

synaptic accumulation of CP-AMPARs assessed after 45-d withdrawal from cocaine (Fig. 6). 629

While these results indicate that upregulation of SK2 is essential for withdrawal-associated CP-630

AMPAR accumulation, other mechanisms may also be required, as reviewed recently (Dong et 631

al., 2017), and discussed below. 632

27

Early and late SMHC: relationship 633

Mechanisms leading to upregulation of AMPARs in classical synaptic scaling have been 634

extensively studied in cortical and hippocampal neurons (Turrigiano and Nelson, 2004; Yee et 635

al., 2017) and one study has characterized mechanisms in cultured NAc MSNs (Sun and Wolf, 636

2009). In contrast, the mechanisms that mediate CP-AMPAR accumulation during membrane-637

to-synapse SMHC during cocaine withdrawal are less clear, and may differ between NAc core 638

and shell. 639

After cocaine self-administration, an increase in GluN2B NMDARs in NAcSh MSNs was 640

observed after 1 d of withdrawal (Fig. 2), corresponding to an increase in silent synapses, which 641

matured partially by recruiting CP-AMPARs as withdrawal proceeded (Lee et al., 2013; Ma et al., 642

2014). In late withdrawal, optogenetic LTD to remove CP-AMPARs restored high levels of silent 643

synapses (Lee et al., 2013; Ma et al., 2014). The timing of these events is largely consistent 644

with the idea that silent synapses gradually mature through the addition of CP-AMPARs, which 645

subsequently contribute to incubated cocaine craving. In theory, this maturation could resemble 646

a developmental process in hippocampus whereby silent synapses, through a Hebbian process, 647

which preferentially recruit CP-AMPARs (Shi et al., 2001). However, there are no obvious 648

Hebbian stimuli during drug withdrawal for animals in their home-cages. Thus, SMHC may 649

provide a major driving force for synaptic accumulation of CP-AMPARs. Furthermore, as 650

withdrawal proceeds, we cannot rule out the possibility that CP-AMPARs also move into non-651

silent synapses. 652

In NAc core, an extended-access cocaine self-administration regimen (6 h/d x 10 d) 653

leads to CP-AMPAR accumulation only after ~30 d of withdrawal (Wolf and Tseng, 2012). 654

Preliminary results suggest that NMDAR plasticity also occurs in NAc core MSNs during 655

incubation (Christian et al., 2017). In addition, a withdrawal-dependent reduction in mGluR1 656

28

expression, leading to impaired mGluR1-mediated removal of synaptic CP-AMPARs, precedes 657

and enables the synaptic accumulation of CP-AMPARs (Loweth et al., 2014). It will be important 658

to determine the extent to which this mechanism contributes in NAcSh MSNs and, conversely, 659

the extent to which SMHC contributes in NAc core. 660

Significance of homeostatic dysregulation cascades 661

In NAcSh MSNs, excitatory synaptic input serves as the main driving force for action potential 662

firing, an output gated by the membrane excitability. SMHC functions to maintain stability of this 663

input-output relationship. Our present results depict two elements in a cascade of SMHC-based 664

dysregulation over the 45-d cocaine withdrawal period, and it is possible that this homeostatic 665

dysregulation continues cascading thereafter (Fig. 7L). If a single NAcSh MSN is 666

conceptualized as a linear electrical system with an input, a gain, and an output, cocaine-667

induced SMHC cascades may progressively increase the input and reduce the gain 668

simultaneously, which keeps the input-output balanced at the check point (e.g., the middle point 669

in Fig. 7M), but increases the output variability anywhere else, polarizing the input-output 670

relationship. Specifically, after long-term withdrawal from cocaine, low intensity excitatory 671

synaptic inputs, which otherwise would trigger action potential firing in drug-naïve rats, may fail 672

to do so due to the substantially reduced membrane excitability. On the other hand, high 673

intensity inputs can pass the hurdle of the reduced membrane excitability, and possibly 674

predominate in triggering action potential firing. As such, normal, low intensity inputs to NAcSh 675

MSNs may be suppressed after withdrawal from cocaine, while high intensity inputs, some of 676

which may convey cocaine-related information, are prioritized for functional output. This may 677

bias NAc output towards cocaine-related behaviors. 678

Figure Legends 679

29

Figure 1 GluN2B NMDARs triggers synapse-to-membrane SMHC. A Diagram summarizing 680

our hypothesis that synaptic NMDARs detect increases or decreases in the excitatory synaptic 681

strength, and regulate membrane-located ion channels through NMDAR-coupled signaling to 682

decrease or increase the membrane excitability of NAcSh MSNs, respectively. B,C Example 683

traces (B) and summary (C) showing that the peak amplitude of NMDAR EPSCs in NAcSh 684

MSNs in slice cultures was inhibited by perfusion of Ro256981 alone, increased by perfusion of 685

DCS alone, and exhibited no net change in response to co-perfusion of DCS + Ro256981. Inset 686

in B showing that the decay kinetics of NMDAR EPSCs (normalized) were not affected by 687

perfusion of DCS, but became faster during co-perfusion of DCS with Ro256981. NMDAR 688

EPSCs were recorded in this and all subsequent experiments at membrane potentials of -30 to -689

40 mV in the presence of NBQX (5μM) and picrotoxin (100 μM). D Diagram showing putative 690

impact of the pharmacological manipulations on different NMDAR subtypes. In control 691

conditions, NMDAR EPSCs are mediated by both GluN2A- and GluN2B-containing NMDARs. 692

Perfusion of DCS enhances both GluN2A- and GluN2B-containing NMDARs, resulting in an 693

overall increase in NMDAR EPSCs. During co-perfusion of DCS and Ro256981, GluN2B 694

NMDARs are inhibited by Ro256981, while GluN2A NMDARs are enhanced by DCS, with no 695

net change in the overall amplitude of NMDAR EPSCs. E,F Example traces (E) and summary 696

(F) showing that the frequency of evoked action potentials was decreased after incubation of 697

DCS, and increased after incubation with either Ro256981 alone, or DCS and Ro256981. G 698

Example images showing virally infected NAcSh MSNs in acutely prepared slices. H Example 699

traces (upper) and summary (lower right) showing that expression of wtGluN2B or mGluN2B 700

prolonged the decay kinetics of NMDAR EPSCs. I,J Example traces (I) and summary (J) 701

showing that the frequency of evoked action potentials was lower in NAcSh MSNs expressing 702

wtGluN2B, but not in MSNs expressing mGluN2B. *p<0.05, **p<0.01. 703

30

Figure 2 Preventing cocaine-induced GluN2B upregulation prevents the cocaine-induced 704

decrease in membrane excitability of NAcSh MSNs. A-C Summary of saline and cocaine 705

self-administration training data for rats used in Fig. 2D-I and 3A,B. D-G Example traces (D, E) 706

and summaries (F, G) showing that the normally observed cocaine-induced increase in the 707

decay kinetics of NMDAR EPSCs was prevented in GluN2B-RNAi-expressing NAcSh MSNs). 708

H,I Example traces (H) and I summary (I) showing that the membrane excitability of NAcSh 709

MSNs was decreased after 1 d of withdrawal from cocaine self-administration, while preventing 710

cocaine-induced upregulation of synaptic GluN2B-containing NMDARs by viral-mediated 711

expression of GluN2B-RNAi prevented this membrane adaptation. J,K Example traces (J) and 712

summary (K) showing that in GluN2B-KI mice in which the interaction between GluN2B and 713

CaMKII was disrupted, repeated cocaine administration no longer decreased the membrane 714

excitability of NAcSh MSNs. *p<0.05, **p<0.01. 715

716

Figure 3 SK2 is the effector of cocaine-induced synapse-to-membrane homeostatic 717

dysregulation. A,B Example traces (A) and summary (B) showing that the mAHP in NAcSh 718

MSNs was increased after 1 d of withdrawal from cocaine self-administration, and this cocaine-719

induced membrane adaptation was prevented in MSNs expressing GluN2B-RNAi. C, Summary 720

of saline and cocaine self-administration training data for rats used for D-G. D,E Example traces 721

(D) and summary (E) showing that the mAHP in NAcSh MSNs was increased after 45 d of 722

withdrawal from cocaine self-administration, and this cocaine-induced membrane adaptation 723

was prevented in MSNs expressing GluN2B-RNAi. F,G Example traces (F) and summary (G) 724

showing that the cocaine-induced decrease in membrane excitability persisted after 45 d of 725

withdrawal from cocaine self-administration, and this cocaine-induced membrane adaptation 726

was prevented in NAcSh MSNs infected by GluN2B-RNAi-expressing virus. *p<0.05, **p<0.01. 727

728

31

Figure 4 Preventing upregulation of SK2 prevents the cocaine-induced decrease in 729

membrane excitability. A,B Example traces showing evoked action potential firing and mAHP 730

in SK2-RNAi-expressing MSNs in the dorsal striatum. C,D Summaries showing that expression 731

of SK2-RNAi increased the frequency of evoked action potentials and decreased the amplitude 732

of the mAHP without affecting the fAHP. E Summary of the self-administration results of rats 733

used in F-I. F,G Example traces (F) and summary (G) showing that the cocaine-induced 734

increase in the mAHP was prevented in NAcSh MSNs infected by SK2-RNAi-expressing virus 735

tested 1 d after self-administration. H,I Example traces (H) and summary (I) showing that the 736

cocaine-induced decrease in membrane excitability was prevented in NAcSh MSNs infected by 737

SK2-RNAi-expressing virus. *p<0.05, **p<0.01. 738

Figure 5 Preventing the cocaine-induced upregulation of SK2 does not affect excitatory 739

synaptic strength in NAcSh MSNs after 1 d of withdrawal. A Diagram showing the 740

hypothetical membrane-to-synapse homeostatic crosstalk: changes in the membrane excitability 741

induce compensatory changes in the overall strength of excitatory synapses. B Summary of 742

self-administration training data for rats used in D-M. C Images showing the pairwise recording 743

setup, in which a virally infected MSN and its neighboring non-infected MSN are simultaneously 744

recorded in response to the same presynaptic stimulation. D Example traces simultaneously 745

recorded from an SK2-RNAi-expressing and a non-infected neighboring MSN from a saline-746

exposed rat. E Amplitudes of AMPAR EPSCs of MSNs expressing SK2-RNAi versus those of 747

simultaneously recorded non-infected MSNs in saline-exposed rats. F Example traces 748

simultaneously recorded from an SK2-RNAi-expressing and a non-infected neighboring MSN 749

from a cocaine-exposed rat. G Amplitudes of AMPAR EPSCs of MSNs expressing SK2-RNAi 750

versus those of simultaneously recorded non-infected MSNs in cocaine-exposed rats. H 751

Summary showing that after 1 d of withdrawal from self-administration, preventing the cocaine-752

induced decrease in membrane excitability did not affect excitatory synaptic strength in NAcSh 753

MSNs. I-M Example trials (I-L) and (M) summary showing that 1 d after self-administration, the 754

32

Naspm sensitivity of AMPAR EPSCs is not different between non-infected MSNs and SK2-RNAi 755

expressing MSNs in both saline- and cocaine-exposed rats. 756

Figure 6 Preventing cocaine-induced upregulation of SK2 prevents CP-AMPAR 757

accumulation in NAcSh MSNs after long-term withdrawal. A Summary of self-administration 758

training data for rats used in B-K. B Example traces simultaneously recorded from an SK2-759

RNAi-expressing and a non-infected neighboring MSN from a saline-exposed rat. C Amplitudes 760

of AMPAR EPSCs of MSNs expressing SK2-RNAi vs. those of simultaneously recorded non-761

infected MSNs in saline-exposed rats. D Example traces simultaneously recorded from an SK2-762

RNAi-expressing and a non-infected neighboring MSN from a cocaine-exposed rat. E 763

Amplitudes of AMPAR EPSCs of MSNs expressing SK2-RNAi vs. those of simultaneously 764

recorded non-infected MSNs in cocaine-exposed rats. F Summary showing that after 45 d of 765

withdrawal from cocaine self-administration, EPSC amplitudes in SK2-RNAi-expressing NAcSh 766

MSNs were significantly lower than those in non-infected MSNs. G-K Example trials (G-J) and 767

summary (K) showing that after 45 d of withdrawal from cocaine self-administration, Naspm 768

significantly inhibited AMPAR EPSCs in NAc MSNs, and this effect was prevented in MSNs 769

expressing SK2-RNAi. *p<0.05, **p<0.01. 770

Figure 7 Preventing SMHC-mediated homeostatic dysregulation prevents incubation of 771

cocaine craving. A,B Summary showing nose pokes to active vs. inactive holes of rats with 772

intra-NAcSh expression of GluN2B-RNAi or luciferase-RNAi (controls) during 5 d of cocaine(A) 773

or saline (B) self-administration training (2 h/d) and during the 1 h extinction test on withdrawal d 774

1. C,D Summary showing nose pokes to active vs. inactive holes of rats with intra-NAcSh 775

expression of GluN2B-RNAi or with intra-NAcSh expression of luciferase-RNAi (controls) during 776

5 d of cocaine (C) or saline (D) self-administration training (2 h/d) and during the 1h extinction 777

test on withdrawal d 45. E Summary showing that preventing cocaine-induced upregulation of 778

GluN2B NMDARs prevented incubation of cocaine craving after drug withdrawal. F,G Summary 779

33

showing nose pokes to active vs. inactive holes of rats with intra-NAcSh expression of SK2-780

RNAi or with intra-NAcSh expression of luciferase-RNAi (controls) during 5 d of cocaine (F) or 781

saline (G) self-administration training (2 h/d) and during the 1 h extinction test on withdrawal d 1. 782

H,I Summary showing nose pokes to active vs. inactive holes of rats with intra-NAcSh 783

expression of SK2-RNAi or luciferase-RNAi (controls) during 5 d of cocaine(H) or saline(I) self-784

administration training (2 h/d) and during the 1 h of extinction test on withdrawal d 45. J 785

Summary showing that preventing cocaine-induced upregulation of SK2 prevented incubation of 786

cocaine craving after drug withdrawal. K Diagram showing two-way SMHC in NAcSh MSNs, in 787

which the membrane excitability and excitatory synaptic strength are homeostatically 788

coordinated. L Hypothetical diagram showing that, after cocaine self-administration, cascades of 789

SMHC-mediated dysregulation lead to progressive increases in excitatory synaptic strength and 790

decreases in membrane excitability in NAcSh MSNs. M Diagram depicting the hypothesis that, 791

after cocaine-induced SMHC dysregulation, NAcSh MSNs become preferentially responsive to 792

relatively strong excitatory synaptic inputs but less responsive to weak inputs. **p<0.01. 793

794

Cited Reference 795

Aghajanian GK (1978) Tolerance of locus coeruleus neurons to morphine and suppression of withdrawal 796 response by clonidine. Nature 276:186-187. 797

Barria A, Malinow R (2005) NMDA receptor subunit composition controls synaptic plasticity by 798 regulating binding to CaMKII. Neuron 48:289-301. 799

Berberich S, Punnakkal P, Jensen V, Pawlak V, Seeburg PH, Hvalby O, Kohr G (2005) Lack of NMDA 800 receptor subtype selectivity for hippocampal long-term potentiation. J Neurosci 25:6907-6910. 801

Boudreau AC, Wolf ME (2005) Behavioral sensitization to cocaine is associated with increased AMPA 802 receptor surface expression in the nucleus accumbens. J Neurosci 25:9144-9151. 803

Brown TE, Lee BR, Mu P, Ferguson D, Dietz D, Ohnishi YN, Lin Y, Suska A, Ishikawa M, Huang YH, Shen H, 804 Kalivas PW, Sorg BA, Zukin RS, Nestler EJ, Dong Y, Schlueter OM (2011) A Silent Synapse-Based 805 Mechanism for Cocaine-Induced Locomotor Sensitization. Journal of Neuroscience 31:8163-806 8174. 807

Christian D, Tseng KY, Wolf M (2017) Extended access cocaine self-administration leads to increased 808 GluN3-containing NMDA receptor function in the rat nucleus accumbens. Soc Neurosci Abstr 43, 809 334.15. 810

34

Conrad KL, Tseng KY, Uejima JL, Reimers JM, Heng LJ, Shaham Y, Marinelli M, Wolf ME (2008) Formation 811 of accumbens GluR2-lacking AMPA receptors mediates incubation of cocaine craving. Nature 812 454:118-121. 813

Cull-Candy SG, Leszkiewicz DN (2004) Role of distinct NMDA receptor subtypes at central synapses. Sci 814 STKE 2004:re16. 815

Dong Y, Taylor JR, Wolf ME, Shaham Y (2017) Circuit and Synaptic Plasticity Mechanisms of Drug 816 Relapse. J Neurosci 37:10867-10876. 817

Dong Y, Green T, Saal D, Marie H, Neve R, Nestler EJ, Malenka RC (2006) CREB modulates excitability of 818 nucleus accumbens neurons. Nat Neurosci 9:475-477. 819

Dong Y, Nasif FJ, Tsui JJ, Ju WY, Cooper DC, Hu XT, Malenka RC, White FJ (2005) Cocaine-induced 820 plasticity of intrinsic membrane properties in prefrontal cortex pyramidal neurons: adaptations 821 in potassium currents. J Neurosci 25:936-940. 822

Graziane NM, Sun S, Wright WJ, Jang D, Liu Z, Huang YH, Nestler EJ, Wang YT, Schluter OM, Dong Y 823 (2016) Opposing mechanisms mediate morphine- and cocaine-induced generation of silent 824 synapses. Nat Neurosci 19:915-925. 825

Grimm JW, Hope BT, Wise RA, Shaham Y (2001) Neuroadaptation. Incubation of cocaine craving after 826 withdrawal. Nature 412:141-142. 827

Haber SN, Fudge JL, McFarland NR (2000) Striatonigrostriatal pathways in primates form an ascending 828 spiral from the shell to the dorsolateral striatum. J Neurosci 20:2369-2382. 829

Halt AR, Dallapiazza RF, Zhou Y, Stein IS, Qian H, Juntti S, Wojcik S, Brose N, Silva AJ, Hell JW (2012) 830 CaMKII binding to GluN2B is critical during memory consolidation. The EMBO journal 31:1203-831 1216. 832

Hu XT, Basu S, White FJ (2004) Repeated cocaine administration suppresses HVA-Ca2+ potentials and 833 enhances activity of K+ channels in rat nucleus accumbens neurons. J Neurophysiol 92:1597-834 1607. 835

Huang X, Stodieck SK, Goetze B, Cui L, Wong MH, Wenzel C, Hosang L, Dong Y, Lowel S, Schluter OM 836 (2015) Progressive maturation of silent synapses governs the duration of a critical period. 837 Proceedings of the National Academy of Sciences of the United States of America 112:E3131-838 3140. 839

Huang YH, Schluter OM, Dong Y (2011) Cocaine-induced homeostatic regulation and dysregulation of 840 nucleus accumbens neurons. Behavioural brain research 216:9-18. 841

Huang YH, Lin Y, Brown TE, Han MH, Saal DB, Neve RL, Zukin RS, Sorg BA, Nestler EJ, Malenka RC, Dong Y 842 (2008) CREB modulates the functional output of nucleus accumbens neurons: a critical role of N-843 methyl-D-aspartate glutamate receptor (NMDAR) receptors. J Biol Chem 283:2751-2760. 844

Huang YH, Lin Y, Mu P, Lee BR, Brown TE, Wayman G, Marie H, Liu W, Yan Z, Sorg BA, Schluter OM, Zukin 845 RS, Dong Y (2009) In vivo cocaine experience generates silent synapses. Neuron 63:40-47. 846

Hyman SE, Malenka RC, Nestler EJ (2006) Neural mechanisms of addiction: the role of reward-related 847 learning and memory. Annu Rev Neurosci 29:565-598. 848

Ikemoto S (2007) Dopamine reward circuitry: two projection systems from the ventral midbrain to the 849 nucleus accumbens-olfactory tubercle complex. Brain Res Rev 56:27-78. 850

Ishikawa M, Mu P, Moyer JT, Wolf JA, Quock RM, Davies NM, Hu XT, Schluter OM, Dong Y (2009) 851 Homeostatic synapse-driven membrane plasticity in nucleus accumbens neurons. J Neurosci 852 29:5820-5831. 853

Jahr CE, Stevens CF (1990) Voltage dependence of NMDA-activated macroscopic conductances 854 predicted by single-channel kinetics. J Neurosci 10:3178-3182. 855

Kalivas PW (2005) How do we determine which drug-induced neuroplastic changes are important? Nat 856 Neurosci 8:1440-1441. 857

Koob GF, Le Moal M (1997) Drug abuse: hedonic homeostatic dysregulation. Science 278:52-58. 858

35

Kourrich S, Thomas MJ (2009) Similar neurons, opposite adaptations: psychostimulant experience 859 differentially alters firing properties in accumbens core versus shell. J Neurosci 29:12275-12283. 860

Kourrich S, Rothwell PE, Klug JR, Thomas MJ (2007) Cocaine experience controls bidirectional synaptic 861 plasticity in the nucleus accumbens. J Neurosci 27:7921-7928. 862

Kourrich S, Hayashi T, Chuang JY, Tsai SY, Su TP, Bonci A (2013) Dynamic interaction between sigma-1 863 receptor and Kv1.2 shapes neuronal and behavioral responses to cocaine. Cell 152:236-247. 864

Lee BR, Ma Y-Y, Huang YH, Wang X, Otaka M, Ishikawa M, Neumann PA, Graziane NM, Brown TE, Suska 865 A, Guo C, Lobo MK, Sesack SR, Wolf ME, Nestler EJ, Shaham Y, Schlueter OM, Dong Y (2013) 866 Maturation of silent synapses in amygdala-accumbens projection contributes to incubation of 867 cocaine craving. Nature Neuroscience 16:1644-1651. 868

Leonard AS, Lim IA, Hemsworth DE, Horne MC, Hell JW (1999) Calcium/calmodulin-dependent protein 869 kinase II is associated with the N-methyl-D-aspartate receptor. Proceedings of the National 870 Academy of Sciences of the United States of America 96:3239-3244. 871

Liu L, Wong TP, Pozza MF, Lingenhoehl K, Wang Y, Sheng M, Auberson YP, Wang YT (2004) Role of 872 NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 873 304:1021-1024. 874

Loweth JA, Scheyer AF, Milovanovic M, LaCrosse AL, Flores-Barrera E, Werner CT, Li X, Ford KA, Le T, 875 Olive MF, Szumlinski KK, Tseng KY, Wolf ME (2014) Synaptic depression via mGluR1 positive 876 allosteric modulation suppresses cue-induced cocaine craving. Nat Neurosci 17:73-80. 877

Lu L, Grimm JW, Hope BT, Shaham Y (2004) Incubation of cocaine craving after withdrawal: a review of 878 preclinical data. Neuropharmacology 47 Suppl 1:214-226. 879

Ma Y-Y, Lee BR, Wang X, Guo C, Liu L, Cui R, Lan Y, Balcita-Pedicino JJ, Wolf ME, Sesack SR, Shaham Y, 880 Schlueter OM, Huang YH, Dong Y (2014) Bidirectional Modulation of Incubation of Cocaine 881 Craving by Silent Synapse-Based Remodeling of Prefrontal Cortex to Accumbens Projections. 882 Neuron 83:1453-1467. 883

Mahon S, Vautrelle N, Pezard L, Slaght SJ, Deniau JM, Chouvet G, Charpier S (2006) Distinct patterns of 884 striatal medium spiny neuron activity during the natural sleep-wake cycle. J Neurosci 26:12587-885 12595. 886

Marie H, Morishita W, Yu X, Calakos N, Malenka RC (2005) Generation of silent synapses by acute in vivo 887 expression of CaMKIV and CREB. Neuron 45:741-752. 888

Massey PV, Johnson BE, Moult PR, Auberson YP, Brown MW, Molnar E, Collingridge GL, Bashir ZI (2004) 889 Differential roles of NR2A and NR2B-containing NMDA receptors in cortical long-term 890 potentiation and long-term depression. J Neurosci 24:7821-7828. 891

Mayadevi M, Praseeda M, Kumar KS, Omkumar RV (2002) Sequence determinants on the NR2A and 892 NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII. 893 Biochimica et biophysica acta 1598:40-45. 894

Monyer H, Burnashev N, Laurie DJ, Sakmann B, Seeburg PH (1994) Developmental and regional 895 expression in the rat brain and functional properties of four NMDA receptors. Neuron 12:529-896 540. 897

Mu P, Moyer JT, Ishikawa M, Zhang Y, Panksepp J, Sorg BA, Schluter OM, Dong Y (2010) Exposure to 898 cocaine dynamically regulates the intrinsic membrane excitability of nucleus accumbens 899 neurons. J Neurosci 30:3689-3699. 900

Nestler EJ (2001) Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2:119-901 128. 902

Nestler EJ, Aghajanian GK (1997) Molecular and cellular basis of addiction. Science 278:58-63. 903 O'Donnell P, Greene J, Pabello N, Lewis BL, Grace AA (1999) Modulation of cell firing in the nucleus 904

accumbens. Ann N Y Acad Sci 877:157-175. 905

36

Parvaz MA, Moeller SJ, Goldstein RZ (2016) Incubation of cue-induced craving in adults addicted to 906 cocaine measured by electroencephalography. JAMA Psychiatry 73:1127-1134. 907

Scofield MD, Kalivas PW (2014) Astrocytic Dysfunction and Addiction: Consequences of Impaired 908 Glutamate Homeostasis. The Neuroscientist : a review journal bringing neurobiology, neurology 909 and psychiatry 20:610-622. 910

Shi S, Hayashi Y, Esteban JA, Malinow R (2001) Subunit-specific rules governing AMPA receptor 911 trafficking to synapses in hippocampal pyramidal neurons. Cell 105:331-343. 912

Smith RJ, Lobo MK, Spencer S, Kalivas PW (2013) Cocaine-induced adaptations in D1 and D2 accumbens 913 projection neurons (a dichotomy not necessarily synonymous with direct and indirect 914 pathways). Current opinion in neurobiology 23:546-552. 915

Standaert DG, Testa CM, Young AB, Penney JB, Jr. (1994) Organization of N-methyl-D-aspartate 916 glutamate receptor gene expression in the basal ganglia of the rat. J Comp Neurol 343:1-16. 917

Strack S, Colbran RJ (1998) Autophosphorylation-dependent targeting of calcium/ calmodulin-918 dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor. The 919 Journal of biological chemistry 273:20689-20692. 920

Strack S, McNeill RB, Colbran RJ (2000) Mechanism and regulation of calcium/calmodulin-dependent 921 protein kinase II targeting to the NR2B subunit of the N-methyl-D-aspartate receptor. The 922 Journal of biological chemistry 275:23798-23806. 923

Sun X, Wolf ME (2009) Nucleus accumbens neurons exhibit synaptic scaling that is occluded by repeated 924 dopamine pre-exposure. Eur J Neurosci 30:539-550. 925

Tang YP, Wang H, Feng R, Kyin M, Tsien JZ (2001) Differential effects of enrichment on learning and 926 memory function in NR2B transgenic mice. Neuropharmacology 41:779-790. 927

Tang YP, Shimizu E, Dube GR, Rampon C, Kerchner GA, Zhuo M, Liu G, Tsien JZ (1999) Genetic 928 enhancement of learning and memory in mice. Nature 401:63-69. 929

Turrigiano GG, Nelson SB (2004) Homeostatic plasticity in the developing nervous system. Nat Rev 930 Neurosci 5:97-107. 931

Vicini S, Wang JF, Li JH, Zhu WJ, Wang YH, Luo JH, Wolfe BB, Grayson DR (1998) Functional and 932 pharmacological differences between recombinant N-methyl-D-aspartate receptors. J 933 Neurophysiol 79:555-566. 934

White NM (1996) Addictive drugs as reinforcers: multiple partial actions on memory systems. Addiction 935 91:921-949; discussion 951-965. 936

Wolf ME (2016) Synaptic mechanisms underlying persistent cocaine craving. Nat Rev Neurosci 17:351-937 365. 938

Wolf ME, Tseng KY (2012) Calcium-permeable AMPA receptors in the VTA and nucleus accumbens after 939 cocaine exposure: when, how, and why? Frontiers in molecular neuroscience 5:72. 940

Wong RW, Setou M, Teng J, Takei Y, Hirokawa N (2002) Overexpression of motor protein KIF17 enhances 941 spatial and working memory in transgenic mice. Proc Natl Acad Sci U S A 99:14500-14505. 942

Yee AX, Hsu YT, Chen L (2017) A metaplasticity view of the interaction between homeostatic and 943 Hebbian plasticity. Philos Trans R Soc Lond B Biol Sci 372. 944

Zhang XF, Hu XT, White FJ (1998) Whole-cell plasticity in cocaine withdrawal: reduced sodium currents 945 in nucleus accumbens neurons. J Neurosci 18:488-498. 946

Zhang XF, Cooper DC, White FJ (2002) Repeated cocaine treatment decreases whole-cell calcium current 947 in rat nucleus accumbens neurons. J Pharmacol Exp Ther 301:1119-1125. 948

949

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cted

coca

ine

GluN2B

-KD

*

12 pA

10 pA

10 ms

*

*

**

**

*

E F

G I K

ime

ela

pse

d (

ms)

Ove

rnight

A B CSaline (17)Cocaine (17)

Ove

rnight

Ove

rnight

0

20

40

60

80

100

120

Num

ber

of in

fusio

ns

0

100

200

300

400

500

Active n

ose p

oke

0

50

100

150

1 2 3 4 5Training day

0

50

100

150

200

250

Inactive n

ose p

oke

0

20

40

60

80

100

Training day1 2 3 4 5

0

10

20

30

40

50

1 2 3 4 5Training day

**

Fig. 3

8 mV

2.5 ms

non-infected

saline

cocaine

mAHP

threshold

10 ms

GluN2B-RNAi

saline

cocaine

Am

plit

ude o

f m

AH

P(m

V)

A B **

coca

ine

non-

infe

cted

coca

ine

GluN2B

-RNAi

salin

e

non-

infe

cted sa

line

GluN2B

-RNAi

10 ms

mAHP

Non-infected GluN2B-RNAi

saline

cocaine

saline

cocaine

Am

plit

ude o

f m

AH

P(m

V)

threshold

8 mV

2.5 ms

100 200 300 400

5

10

15

saline non-infected 22/11

saline GluN2B-RNAi 25/10

cocaine non-infected 29/10

cocaine GluN2B-RNAi 25/8

Num

ber

of action p

ote

ntials

Injected current (pA)

50 ms

20 mV

saline non-infected saline GluN2B-RNAi

cocaine non-infected cocaine GluN2B-RNAi

300 pA

0

E

D

GF

27/12 28/12 15/6 20/9

2

6

10

14

18

2

6

10

14

18

22/11 29/10 25/10 25/8

coca

ine

non-

infe

cted

coca

ine

GluN2B

-RNAi

salin

e

non-

infe

cted sa

line

GluN2B

-RNAi

**

*

**

*

*

0

20

40

60

1 2 3 4 5

Training day

0

20

40

60

80

100

120num

ber

of in

fusio

ns

C

Saline (11)Cocaine (10)

Ove

rnight

1d after self-administration 45d after self-administration

Fig. 4

H IGF

300 pA

20 ms

25 mV

saline, non-infected saline, SK2-RNAi

cocaine, non-infected cocaine, SK2-RNAi

cocaine, non-infected (23/9)

saline, non-infected (12/9)

saline, SK2-RNAi (13/9)

cocaine, SK2-RNAi (19/7)

Injected current (pA)

5

10

15

Nu

mb

er

of a

ctio

n p

ote

ntia

ls

100 200 300 400

0

mA

HP

am

plit

ud

e (

mV

)

threshold

8 mV

2 ms

mAHP

10 ms

cocaine, non-infected

cocaine, SK2-RNAi

saline, non-infected

saline, SK2-RNAi

**

2

6

10

14

18

13/9 11/9 20/723/9

coca

ine

non-

infe

cted

coca

ine

SK2-

RNAi

salin

e

non-

infe

cted sa

line

SK2-

RNAi

*

* *

*

20 mV

50 ms

non-infected

SK2-RNAi

300 pA

fAHP mAHP

SK2-RNAi

non-infected

20 mV

10 ms

A B

200 400

0

5

10

15

non- infected (9/3)

SK2-RNAi (12/3)

Current (pA)

-12

-14

-16

-18

fAHP mAHP

mA

HP

am

plit

ud

e (

mV

)

*

Non-In

f

SK2-

KD

Non-In

f

SK2-

KD

9/3 12/3 9/3 12/3Nu

mb

er

of a

ctio

n p

ote

ntia

lsC D E

0

20

40

60

80

100

120

Num

ber

of in

fusio

ns

0

20

40

60

1 2 3 4 5Training day

* *

cocaine (10)

saline (9)

Fig. 5

B

25 μm

A

AMPAR

C

60

70

80

90

100

110

Num

ber

of in

fusio

ns

0

20

40

60

80

1 2 3 4 5Training day

overn

ight

1d after cocaine

0200 400 600

200

400

600

EP

SC

am

plit

ude (

pA

)

non-infe

cte

d

EPSC amplitude (pA)

saline cocaine

non-infected

non-infected

SK2-RNAi

(14/6) (25/9)EP

SC

am

plit

ud

e r

atio

0.0

0.5

1.0

1.5

2.0

2.5

3.0

(25/9)

1d after saline

EP

SC

am

plit

ude (

pA

)

non-inf e

cte

d

EPSC amplitude (pA)

SK2-RNAi

(14/6)

0 200 400 600

200

400

600

50 pA

10 ms50 pA

10 ms

aCSF

Am

plit

ud

e (

x1

00

pA

)

-8

-6

-4

-2

0

Time (sec)200 400 600

Naspm

saline, non-infected

50 pA

10 ms

200 400 600

-8

-6

-4

-2

0

Time (sec)

aCSF Naspm

Am

plit

ud

e (

x1

00

pA

)

saline, SK2-RNAi

100 pA

10 ms

Time (sec)

200 400 600

-8

-6

-4

-2

aCSF Naspm

Am

plit

ud

e (

x1

00

pA

)

cocaine, non-infected

10 ms

100 pA

Time (sec)200 400 600

-8

-6

-4

-2

aCSF Naspm

Am

plit

ud

e (

x1

00

pA

)

100 pA

10 ms

D F H

I J K L

Re

lative

am

plit

ud

e

0.6

0.8

1.0

1.2

1.4

8/615/9 11/624/10

SK2-RNAiSK2-R

NAi

cocaine, SK2-RNAi

non-infected

non-infected

SK2-RNAi

SK2-RNAi

1d after saline 1d after cocaineM

E Gnoninfected SK2-RNAi noninfected SK2-RNAi

cocaine (12)

saline (9)

Fig. 6

B F

G H J

EP

SC

am

plit

ud

e (

pA

)

non-infe

cte

d

EPSC amplitude (pA)

100 200 300 4000

100

200

300

400

45d after saline 45d after cocaine

EPSC amplitude (pA)

0 200 400 600

200

400

600

EP

SC

am

plit

ud

e (

pA

)

no

n-in

fecte

d

(15/7) (18/6) EP

SC

am

plit

ud

e r

atio

(18/6)(15/7)

non-infected

non-infected

saline cocaine

D

50 pA

10 ms 50 pA10 ms

ITime (sec)

-5

-4

-3

-2

200 400 600

Am

plit

ud

e (

x1

00

pA

)

aCSF Naspm

100 pA

10 ms

Time (sec)200 400 600

-8

-6

-4

-2

-10Am

plit

ud

e (

x1

00

pA

)

aCSF Naspm

10 ms200 pA

Time (sec)200 400 600

-8

-6

-4

-2

-10Am

plit

ud

e (

x1

00

pA

)

aCSF Naspm

100 pA

10 ms

0.20.40.60.81.01.21.41.6

*

SK2-RNAi SK2-RNAi

saline, SK2-RNAi cocaine, SK2-RNAi

SK2-RNAi

SK2-RNAi

cocaine, non-infected

10/7 14/8 10/716/9

**

1.2

1.0

0.8

0.6

Re

lative

am

plit

ud

e

non-infected

non-infected

SK2-RNAi

SK2-RNAi

45d after saline 45d after cocaineTime (sec)

200 400 600

-8

-6

-4

-2

Am

plit

ud

e (

x1

00

pA

)

aCSF Naspm

saline, non-infected

10 ms100 pA

K

C EA

0

20

40

60

80

100

120

nu

mb

er

of in

fusio

ns

0

10

20

30

40

50

1 2 3 4 5Training day

overn

ight

noninfected SK2-RNAi noninfected SK2-RNAicocaine (9)

saline (11)

A

Fig. 7

C

withdrawalexposure

SMHC

sensor

CP-AMPARGluN2B-

NMDAR

HSMC

Ion channels

(SK2, etc.)

Presynaptic

membrane excitability

Membrane excitability

Excitatory synaptic strength

Intensity of synaptic input

Actio

n p

ote

ntia

l o

utp

ut

baselin

e

short-

term

long-te

rm

K L M

increase

decrease

further increase

further decrease

control, active (10)

GluN2B-RNAi, active (10)control, inactive

GluN2B-RNAi, inactive

control, active (10)

GluN2B-RNAi, active (9)

control, inactive

GluN2B-RNAi, inactive

0

20

40

60

80

100

120Active Inactive

10 10 10 9

controlGluN2B-RNAi

*

control, active (13)

SK2-RNAi, active (9)control, inactive

SK2-RNAi, inactive

control, active (9)

SK2-RNAi, active (9)control, inactive

SK2-RNAi, inactive

Nose p

oke

E

F H J

Withdrawal days

1 45 1 45 1 45 1 45

Active Inactive

controlSK2-RNAi

*

Nose p

oke

Withdrawal days

1 45 1 45 1 45 1 450

20

40

60

80

100

120

13 9 9 9

1 2 3 4 5

0

50

100

150

Training day

Nose p

oke

51 2 3 4

Training day

0

50

100

150

Nose p

oke

withdrawal

d1

withdrawal

d45

0

50

100

150

200

Nose p

oke

1 2 3 4 5Training day

0

50

100

150

Nose p

oke

1 2 3 4 5Training day

withdrawal

d1

withdrawal

d45

*

*

B D

G I

saline

Nose p

oke

cocaine

1 2 3 4 50

20

40

60

80

100

Training day

withdrawal

d1

control, active (7)

GluN2B-RNAi, active (7)control, inactive

GluN2B-RNAi, inactive

Nose p

oke

1 2 3 4 50

20

40

60

80

100

Training day

control, active (7)

GluN2B-RNAi, active (7)control, inactive

GluN2B-RNAi, inactive

withdrawal

d45

salinecocaine

Nose p

oke

1 2 3 4 50

20

40

60

80

100

Training daywithdrawal

d1

control, active (7)

SK2-RNAi, active (8)control, inactive

SK2-RNAi, inactive

Nose p

oke

1 2 3 4 50

20

40

60

80

100

Training daywithdrawal

d45

control, active (7)

SK2-RNAi, active (7)control, inactive

SK2-RNAi, inactive

salinecocaine salinecocaine

AMPAR