Cocaine-induced Generation of Silent Synapses Is Initiated by Activation of CREB
To identify the molecular mechanism underlying cocaine-induced generation of silent synapses, we focused on CREB, a transcription factor activated in response to cocaine exposure (reviewed by (Carlezon et al., 2005
)) and implicated in synaptogenesis (reviewed by (Lonze and Ginty, 2002
)). To test a requirement for CREB in generation of silent synapses, we expressed constitutively active CREB (caCREB) or a dominant negative form of CREB (dnCREB) by means of the sindbis viral expression system by stereotaxic injection directly into the NAc in vivo
We examined cocaine-generated silent synapses using two independent functional electrophysiological assays, coefficient of variation analysis (CV) and minimal stimulation assay (Huang et al., 2009
). The CV is dependent on the number of recruited synapses during trials of evoked EPSCs (Kullmann, 1994
). Comparing the CV of the AMPAR component (measured at −80 mV) and the CV of the NMDAR (measured at +40 mV) elicited by the same stimulation strength provides a relative measure of changes in the receptor content of synapses. We compared the variability of evoked NMDAR EPSCs to that of AMPAR EPSCs as the ratio CV(NMDAR)/CV(AMPAR) in different experimental groups. Expression of caCREB for 24 h decreased the CV of NMDAR EPSCs (F5,33
= 4.65, p < 0.01, one-way ANOVA; p < 0.05, caCREB vs. uninf-saline or GFP-saline; Bonferroni posttest; n/m (total animals/total cells) = 6/10 for caCREB, 12/29 for uninf-saline, and 5/9 for GFP-saline) and CV(NMDAR)/CV(AMPAR) (F5,33
= 13.57, p < 0.01, one-way ANOVA; p < 0.01, caCREB vs. uninf-saline or GFP-saline, Bonferroni posttest; ) with no significant effect on the CV of AMPAR EPSCs (F5,33
= 1.81, p = 0.14, one-way ANOVA), consistent with an increase in silent synapses. A similar reduction in the CV of NMDAR EPSCs (p < 0.01, uninf-saline vs. uninf-cocaine, Bonferroni posttest; n/m = 12/29 for uninf-saline and 5/9 for uninf-cocaine) and CV(NMDAR)/CV(AMPAR) (p < 0.01, uninf-saline vs. uninf-cocaine, Bonferroni posttest) was observed when rats were treated for 2.5 days with cocaine, a regimen adapted for the time course of the viral manipulations. Importantly, the changes in CV elicited by this shortened cocaine procedure were comparable to those of our previous study with a five-day procedure (Huang et al., 2009
Figure 1 Activation of CREB mediated cocaine-induced generation of silent synapses in NAc MSNs. CV analysis of AMPAR and NMDAR EPSCs in control, caCREB- (24 h) or dnCREB-(72 h) expressing MSNs from saline- or cocaine- (2.5 d procedure; analysis 16 h after last (more ...)
Whereas expression of dnCREB alone did not affect the CV of NMDAR EPSCs (p = 1.0, dnCREB-saline vs. GFP-saline or uninf-saline, Bonferroni posttest; n/m = 5/9 for dnCREB-saline, 5/9 for GFP-saline, and 12/29 for uninf-saline) and the ratio of CV(NMDAR):CV(AMPAR) (p = 1.0, dnCREB vs. GFP-saline or uninf-saline, Bonferroni posttest; ) in saline-treated rats, it prevented the effects of cocaine on the CV(NMDAR)/CV(AMPAR) in the 2.5-day procedure (p = 0.49, dnCREB-cocaine vs. uninf-saline, p < 0.05, dnCREB-cocaine vs. uninf-cocaine, Bonferroni posttest; n/m = 6/9 for dnCREB-cocaine; ). Note that expression of GFP alone did not affect either of the above parameters (p = 1.0, GFP vs. uninf-saline for CV-AMPAR, CV-NMDA, and CV(NMDAR)/CV(AMPAR), Bonferroni posttests; ). The findings that both GFP and dnCREB did not by itself alter the NMDA or AMPA CV, demonstrate that delivery of viral vectors under our experimental conditions did not themselves affect these parameters. As such, the manipulated groups (virally infected cells) were directly compared with uninfected cells in most comparisons.
In the minimal stimulation assay (see Material and Methods), we adjusted the stimulation strength of the AMPAR EPSCs such that successful trials with a measurable EPSC were interleaved with trials with no detectable EPSC (failures) (). We calculated the estimated percentage of AMPAR-silent synapses from the failure rates of AMPAR EPSCs and NMDAR EPSCs (see Material and Methods for details) (Kullmann, 1994
; Marie et al., 2005
; Huang et al., 2009
). Expression of caCREB for 24 h induced an increase in the percentage of failures, a predictor of the percentage of silent synapses in cells of saline-treated animals, compared to GFP-expressing control cells (F6, 32
= 12.06, p < 0.01, one-way ANOVA; p < 0.01, caCREB vs. uninf-saline or GFP-saline Bonferroni posttest; n/m = 6/12 for caCREB-saline, 5/8 for uninf-saline, and 5/10 for GFP-saline; ). Similarly, cocaine administration in the 2.5-day procedure increased the percentage of failures, consistent with the generation of silent synapses in control and GFP-expressing neurons, confirming our previous results with a five-day procedure (p < 0.05, uninf-saline vs. uninf-cocaine, p < 0.05 GFP-saline vs. GFP-cocaine, Bonferroni posttest; n/m = 5/8 for uninf-cocaine and 5/10 for GFP-cocaine; ) (Huang et al., 2009
). Whereas dnCREB expression did not significantly alter the percentage of silent synapses in saline treated rats (p = 1.0, dnCREB-saline vs. uninf-saline or GFP-saline; Bonferroni posttest; n/m = 5/12), expression of dnCREB prevented the generation of silent synapses in cocaine treated rats (p < 0.05 dnCREB-cocaine vs. uninf-cocaine, p < 0.01, dnCREB-cocaine vs. GFP-cocaine, p = 1.0 dnCREB-cocaine vs. uninf-saline or GFP-saline, Bonferroni posttest; n/m = 8/16 for dnCREB-cocaine; ).
In the above experiments, we used relatively young (~30 d) rats. Young rats exhibit higher basal level of silent synapses (Durand et al., 1996
; Hsia et al., 1998
; Kerchner and Nicoll, 2008
), which may lead to different behavioral responses to drugs of abuse compared with more matured rats (Andersen et al., 2002
; Tirelli et al., 2003
). However, it has been shown that silent synapses are generated in the NAc of both young and adult rats upon cocaine exposure (Huang et al., 2009
), and similar to adult rats (~65 d), young rats used in this study exhibited robust cocaine-induced locomotor sensitization (see below). These findings suggest that activation of CREB in NAc neurons is both necessary and sufficient for cocaine-induced generation of silent synapses.
Exposure to cocaine generates silent synapses across different neuronal subtypes in the NAc
Neurons in the NAc are highly heterogeneous and can be divided into different subpopulations depending on different criteria. For example, based on receptor/transmitter subtypes, the NAc neurons can be divided into two subpopulations, one (~50% of total NAc neurons) co-expressing dopamine D1-class receptors and substance P, and the other (~50% of total NAc neurons) co-expressing dopamine D2-class receptors and enkephalin (Le Moine and Bloch, 1995
; Gong et al., 2003
; Lee et al., 2006a
). This distribution includes a heterogenous population of 5–10% interneurons. Based on electrophysiological properties, NAc neurons can be categorized as neurons with (~24%) or without gap junctions with their neighboring NAc neurons (O'Donnell and Grace, 1993
). Based on behavioral correlates, it has been shown that ~10% (43 out of 420 cells) of NAc neurons respond to a rewarding stimulus (Apicella et al., 1992
; Schultz et al., 1992
). Thus, a key question is that whether cocaine-induced generation of silent synapses occurs in all NAc neurons, or just in a subtype of NAc neurons. To detect potential differences in cocaine responses among these various neuronal subpopulations (e.g., responders vs. non-responders), we characterized the distribution of the electrophysiological parameters, including percentage of silent synapses (see methods for calculation) and decay time constant of NMDAR-mediated EPSC across the recorded NAc neurons. As shown in , the NAc neurons from saline-treated rats (n = 53) exhibited a mono-modal pseudo-normal distribution with a median at ~0.1 (i.e., % of silent synapses is ~10%). On the other hand, NAc neurons from cocaine-treated rats (n = 47) also exhibited a mono-modal distribution with a median at ~0.4 (% of silent synapses is ~40%) (). These results suggest that exposure to cocaine changed the median (of the portion of silent synapses) but not the distribution (e.g., no sign of bi-modal distribution was observed).
Figure 2 Monomodal distribution of portion of silent synapses and decay kinetics of NMDAR responses in control and cocaine treated rats indicate a non-selective induction of silent synapses and NR2B expression in MSNs. A The distribution of portion of silent synapses (more ...)
To further focus on a hypothetical situation in which ~50% of NAc neurons were the responders whereas the other ~50% were not, we built a hypothetical distribution model, in which we operationally defined the distribution of responders as that in and the distribution of non-responders as that in . We pooled the same number of cells (~45) from each group, and the pooled distribution exhibited a typical bi-modal property. This bi-modal distribution (solid curve) can be computationally peeled into two mono-modal distributions (dashed curves) corresponding to the distribution curves ().
Thus, results from the above analyses suggest that it is likely that following cocaine exposure, silent/nascent synapses are generated across different neuronal subtypes in NAc neurons.
Cocaine induced NR2B expression is mediated through CREB activation
Cocaine-induced generation of silent synapses appears to arise from insertion of NR2B subunit-enriched NMDARs (Huang et al., 2009
). We examined whether this increase is mediated by CREB activation. We first treated rats with a 5 day cocaine procedure and immunoprecipitated chromatin-bound activated CREB (phopho-CREB, pCREB) from NAc extracts. The levels of co-precipitated NR2A and NR2B promoter fragments were quantified by PCR and levels in cocaine treated animals were normalized to levels in saline treated control rats (). Whereas the pCREB bound levels of NR2A promoter fragments did not change upon cocaine exposure, the pCREB bound levels of NR2B increased more than twofold, suggesting a specific activation of the NR2B gene by CREB (saline NR2A, 1.00 ± 0.16, n = 7 vs. cocaine NR2A, 1.25 ± 0.30, n = 6, p > 0.05; saline NR2B, 1.00 ± 0.16, n = 5 vs. cocaine NR2B, 3.46 ± 0.70, n = 6, p < 0.05).
We next tested whether CREB activation is sufficient for increasing NR2B protein levels. We incubated NAc tissue with sindbis viruses expressing caCREB or dnCREB and examined the protein level of NR2B subunits 24 h later. When normalized to the loading control actin, our results show that expression of caCREB increased total cellular NR2B protein abundance in the NAc more than threefold, whereas expression of dnCREB or GFP had little or no effect (relative level to uninfected tissues at total level: GFP 0.89 ± 020, n = 4; caCREB, 3.58 ± 0.56, n = 5; dnCREB, 1.10 ± 0.18, n = 5; F3, 16 = 11.6, p < 0.01, one-way ANOVA; p < 0.01 caCREB vs. uninf, GFP, or dnCREB, Bonferroni posttest; ). Although the use of caCREB does not mimic the endogenous time course of CREB activation, the results show, that CREB activation by itself is sufficient to induce upregulation of NR2B.
A selective up-regulation of NR2B-containing NMDARs, as demonstrated here as well as in our previous study (Huang et al., 2009
), would increase the relative portion of NR2B-containing NMDARs across all synaptic NMDARs. This scenario predicts that the overall (evoked) NMDAR EPSCs should be shaped by NR2B subunits. To test this prediction, we combined cocaine treatment with CREB manipulations and measured the time course of the decay of the NMDAR-mediated EPSC, a sensitive biophysical measurement of the relative contribution of synaptic NR2B-containing NMDARs, but may also reflect a small (< 15%) contribution of NR2D-containing NMDARs, known to be expressed in mouse striatum (Logan et al., 2007
). Exposure to cocaine (the 2.5 cocaine procesure) induced a marked increase in the decay kinetics of NMDAR EPSCs (assessed by the time decayed from the peak to ½ peak amplitude, or T1/2
) in NAc neurons (F5, 35
= 9.25, p < 0.01,one-way ANOVA; p < 0.05, uninf-saline vs. uninf-cocaine; Bonferroni posttest; n/m = 15/29 for uninf-saline and 5/9 for uninf-cocaine; ). Furthermore, this effect of cocaine was mimicked by expression of caCREB (p < 0.01 caCREB-saline vs. uninf-saline or GFP-saline, Bonferroni posttest; n/m = 6/11 for caCREB-saline and 5/10 for GFP-saline; ), and was prevented by expression of dnCREB (p < 0.05, dnCREB-cocaine vs. uninf-cocaine, p = 1.0, dnCREB-cocaine vs. uninf-saline or GFP-saline, Bonferroni posttest; n/m = 5/16 for dnCREB-cocaine; ).
Taken together, our results suggest that activation of CREB is required for the cocaine-induced increase in synaptic incorporation of NR2B-containing NMDARs and that activation of CREB alone is sufficient to trigger NR2B-containing receptors in NAc MSNs following exposure to cocaine.
Cocaine-induced increase in spine number is mediated through CREB
Chronic exposure to cocaine increases the number of spines in NAc neurons, which persists for weeks after drug discontinuation (Kolb et al., 2003
; Robinson and Kolb, 2004
). We examined the number of dendritic spines in NAc neurons following the 5-day cocaine procedure used in our previous electrophysiology experiments (Huang et al., 2009
). To quantify the spine density, we labeled neurons in lightly fixed tissues with the lipophilic fluorescent dye 1,1‘-diotadecyl-3,3,3’,3‘-tetramethylindocarbocyanine perchlorate (DiI) and examined dendritic spines using confocal microscopy. Two days after the 5-day cocaine protocol, the numbers of filopodia-like (t6
= −2.74, p < 0.05, t-test; n/m = 4/74 for saline and 4/55 for cocaine), mushroom-like protrusions (t6
= −2.89, p < 0.05, t-test), as well as the total (filopodia + mushroom) protrusions (t6
= −3.45, p < 0.05, t-test) on the distal dendrites of NAc MSNs were significantly increased (). Furthermore, when pooling examined dendrites from all animals together, the spine densities in the cumulative plot exhibited a mono-modal distribution in saline-treated rats, and this mono-modal distribution was not altered by cocaine treatment (). This distribution pattern suggests that dendrites within the NAc were evenly affected following exposure to cocaine, supporting our electrophysiological data on the mono-modal distribution of the effects on individual neurons ().
In agreement with the electrophysiological data, the increase in spine number after repeated cocaine administration was prevented by viral-mediated dnCREB expression compared to expression of GFP alone (F1, 13
= 19.70, p < 0.01, drug factor; F1, 13
= 13.59, p < 0.01, virus factor; F1, 13
= 19.11, p < 0.01, drug × virus, two-way ANOVA; p < 0.05, GFP-cocaine vs. GFP-saline, or dnCREB-saline, or dnCREB-cocaine, p > 0.05, dnCREB-cocaine vs. GFP-saline or dnCREB-saline, Tukey’s Multiple Comparison posttest; ). In these experiments, we counted spines visualized by the GFP fluorescence of the virally-transduced neurons according to published protocols (Russo et al., 2009
; Maze et al., 2010
). While this method reveals a smaller total number of spines compared to the DiI stained neurons, the relative increase caused by repeated cocaine administration is consistent between the two methods. In conclusion, these results indicate a requirement of CREB activation for the cocaine-induced increase in spine number.
Potential role of silent synapses in cocaine-induced locomotor sensitization
Finally, we explored the potential role of silent synapses in cocaine-induced locomotor sensitization. Silent synapses are generated gradually during early exposure (3–4 days) over the 5-day cocaine procedure (Huang et al., 2009
), with a time course similar to that of the development of cocaine-induced locomotor sensitization (Wolf, 1998
; Kalivas, 2007
; Kauer and Malenka, 2007
). Without highly selective approaches to manipulate silent synapses, we focused on NR2B-containing NMDARs within the NAc. Our experimental design was to bilaterally and chronically perfuse the NR2B-selective antagonist Ro256981 into the NAc shell () through pre-implanted osmotic mini-pumps. As controls, rats with intra-NAc sham surgery and rats without surgery were tested with the 5-day cocaine procedure. Both control groups exhibited similar patterns of cocaine-induced locomotor sensitization upon repeated cocaine injections (control-cocaine × sham-cocaine: F1, 14
= 0.27, p = 0.95, two-way ANOVA; ) and similar basal locomotor activity upon saline injection (control-saline × sham-saline: F1, 12
= 0.28, p = 0.94, two-way ANOVA with repeated measure over day; ). This result indicates that the intra-NAc surgery did not affect basal and cocaine-induced locomotor responses. We next verified a concentration of Ro256981 that could effectively inhibit NR2B-containing NMDARs with minimal side effects. We examined the inhibitory effect of Ro256981 on NMDAR EPSCs at different concentrations and observed that at 20 – 200 nM, NMDAR EPSCs exhibited the first high affinity inhibition that was likely due to NR2B-selective inhibition (% inhibition: 2 nM, 4.7 ± 4.2, n = 3; 20 nM, 33.9 ± 5.5, n = 10; 200 nM, 35.4 ± 6.1, n = 6, ). One caveat of this pharmacological approach is that, if considered at the overall NMDAR level, application of 20 nM Ro256981 would cause a partial inhibition of total NMDARs by ~35%. This decrease in total NMDAR activity may produce some behavioral effect and thus complicate the interpretation of Ro256981-induced behavioral alteration. To address this potential caveat, we also verified that the non-selective NMDAR antagonist APV at the low concentration of 0.5 µM inhibited total NMDAR EPSCs to a similar degree (39.4 ± 6.2 %, n = 6) as 20 nM Ro256981 (). However, application of APV did not alter the decay kinetics of NMDAR EPSCs as found for Ro256981 (% decrease in T1/2
: 20 nM Ro256981, 11.2 ± 1.5, n = 10; 0.5 µM APV, 3.3 ± 2.5, n = 6, ). Thus, using APV at 0.5 µM in parallel with Ro256981 would allow us to clarify the potential behavioral alterations resulting from partial inhibition of total NMDARs.
Figure 5 Intra-NAc inhibition of NR2B-containing NMDARs prevented cocaine-induced locomotor sensitization. A Representative diagram showing the installation sites of intra-NAc mini pumps in Ro-cocaine (red), Ro-saline (green), APV-cocaine (blue), and APV-saline (more ...)
We then examined the potential effects of intra-NAc perfusion of APV (500 nM) and Ro256981 (20 nM) on basal locomotor activity. Rats receiving intra-NAc perfusion of APV or Ro256981 exhibited similar basal activity in control rats or rats receiving sham surgery (p > 0.2 between any two groups, two-way ANOVA with repeated measure over day; ). To verify that Ro256981 via our intra-NAc perfusion setup could rapidly achieve maximal pharmacological and behavioral effects, we used the identical experimental procedure to perfuse the AMPAR-selective antagonist NBQX at a low concentration (1 µM). It has been demonstrated that inhibiting the NAc AMPARs prevents drug-induced locomotor response (Kaddis et al., 1993
; Burns et al., 1994
; Li et al., 1999
; David et al., 2004
). In the identical experimental setup that we used below, intra-NAc perfusion of a low concentration of NBQX blocked the cocaine-induced acute increases in locomotor activity (). This result verifies that a brief preceding period (~15 min) of intra-NAc perfusion is sufficient for loaded pharmacological agents to achieve effective concentrations.
After verifying the experimental approaches, we started the core experiments. Shortly after the intra-NAc perfusion started, rats received one injection of saline (on day 0), followed by a 5-day cocaine regimen starting from day 1. A challenge injection of cocaine (12 mg/kg) was given on day 8, following a 2-day withdrawal period (day 6 to 7). The intra-NAc perfusion of Ro256981 (20 nM) or APV (0.5 µM) was applied from day -1 until day 8. Rats receiving only sham treatment developed steady cocaine-induced locomotor sensitization (p < 0.01, day 5 vs. day 1; p < 0.01, day 8 vs. day 1; ). The development of cocaine-induced locomotor sensitization was not affected by intra-NAc perfusion of a low concentration of APV (p < 0.01, day 5 vs. day 1; p < 0.01, day 8 vs. day 1; ). However, the development of locomotor sensitization was prevented by intra-NAc perfusion of Ro256981; indeed, in Ro256981-perfused rats, cocaine-induced locomotor activity decreased along the course of cocaine injection (p < 0.05, day 5 vs. day 1; p < 0.01, day 8 vs. day 1; F1, 12 = 14.22, p < 0.01 interaction between Ro-cocaine and Sham-cocaine, two-way ANOVA; ). This behavioral effect of Ro256981 was unlikely to be a general suppressive effect on locomotor activity because chronic intra-NAc perfusion of Ro256981 did not alter the basal locomotor activity in saline-treated rats ().
We next examined whether intra-NAc inhibition of NR2B-containing NMDARs affected the expression of cocaine-induced locomotor sensitization. Rats with similar intra-NAc surgery were treated with the same 5-day cocaine procedure. On day 8, the intra-NAc perfusion of Ro256981 was turned on. Upon a challenge injection of cocaine (12 mg/kg), rats with intra-NAc perfusion of Ro256981 exhibited similar locomotor activities as observed in the sham controls (p > 0.5, t-test, ). Therefore, intra-NAc inhibition of NR2B-containing NMDARs and thus cocaine-generated silent synapses disrupted the development, but not the expression, of cocaine-induced locomotor sensitization. Taken together, these behavioral results suggest that NR2B-signaling, which is enriched in cocaine-generated silent synapses, is importantly implicated in addiction-related behaviors.