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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: [email protected], 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.
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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: [email protected] 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
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
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949
100 pA
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overlay Ro256981
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