A silent synapse-based mechanism for cocaine-induced locomotor sensitization

Abstract

Locomotor sensitization is a common and robust behavioral alteration in rodents whereby following exposure to abused drugs such as cocaine, the animal becomes significantly more hyperactive in response to an acute drug challenge. Here, we further analyzed the role of cocaine-induced silent synapses in the nucleus accumbens (NAc) shell and their contribution to the development of locomotor sensitization. Using a combination of viral vector-mediated genetic manipulations, biochemistry, and electrophysiology in a locomotor sensitization paradigm with repeated, daily, noncontingent cocaine (15 mg/kg) injections, we show that dominant-negative cAMP-element binding protein (CREB) prevents cocaine-induced generation of silent synapses of young (30 d old) rats, whereas constitutively active CREB is sufficient to increase the number of NR2B-containing NMDA receptors (NMDARs) at synapses and to generate silent synapses. We further show that occupancy of CREB at the NR2B promoter increases and is causally related to the increase in synaptic NR2B levels. Blockade of NR2B-containing NMDARs by administration of the NR2B-selective antagonist Ro256981 directly into the NAc, under conditions that inhibit cocaine-induced silent synapses, prevents the development of cocaine-elicited locomotor sensitization. Our data are consistent with a cellular cascade whereby cocaine-induced activation of CREB promotes CREB-dependent transcription of NR2B and synaptic incorporation of NR2B-containing NMDARs, which generates new silent synapses within the NAc. We propose that cocaine-induced activation of CREB and generation of new silent synapses may serve as key cellular events mediating cocaine-induced locomotor sensitization. These findings provide a novel cellular mechanism that may contribute to cocaine-induced behavioral alterations.

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 injection; see Material and Methods) treated rats. A, Example traces and amplitude plots were from a caCREB–saline and a dnCREB–cocaine MSN, showing the variability of multiple sweeps at each holding potential. B, A summary shows that repeated exposure to cocaine decreased the ratio of CV–NMDAR:CV–AMPAR in NAc MSNs, expression of GFP alone did not affect this ratio, expression of caCREB alone decreased this ratio, expression of dnCREB in saline-treated rats did not cause changes in this ratio, and expression of dnCREB prevented the cocaine-induced decrease in this ratio. C, Minimal stimulation assays in control, caCREB- or dnCREB-expressing MSNs from saline- or cocaine-treated rats. Example traces and amplitude plots were from a caCREB–saline and a dnCREB–cocaine MSN, showing multiple sweeps at each holding potential, where failures are marked in gray and successes in black. D, A summary shows that repeated exposure to cocaine increased the estimated percentage of silent synapses in NAc MSNs, expression of GFP alone did not affect this percentage, expression of caCREB was sufficient to increase this percentage in saline-treated rats, expression of dnCREB in saline-treated rats did not cause changes in this percentage, and expression of dnCREB prevented cocaine-induced increase in this percentage. Numbers in parentheses indicate number of cells; *p < 0.05; **p < 0.01.

Figure 2.

Monomodal distribution of portion of silent synapses and decay kinetics of NMDAR responses in control and cocaine-treated rats indicates a nonselective induction of silent synapses and NR2B expression in MSNs. A, The distribution of portion of silent synapses in NAc MSNs from saline-treated rats was consistent with a single-peak normal modal distribution. B, The distribution of portion of silent synapses in NAc MSNs from cocaine-treated rats was consistent with a single-peak normal distribution. Data in A and B were each best fit by a monomodal distribution. C, A hypothetical distribution that contained ∼50% of responders and ∼50% of nonresponders. This hypothetical distribution was constructed based on the data in A and B, which was operationally used to represent responders and nonresponders, respectively. The distribution was best fit by bimodal distributions (dashed lines for two monomodal distributions that make up the overall bimodal distribution).

Figure 3.

Cocaine induces CREB-mediated increases in NR2B expression. A, ChIP analysis with real-time PCR of phospho-CREB binding to NR2A and NR2B gene promoters in saline- and cocaine- (5 d procedure) treated NAc samples. B, C, Sample (B) and quantification (C) of Western blots of NAc extracts for NR2B levels after viral-mediated gene expression of dnCREB and caCREB. D, Scaled example EPSCs recorded at +40 mV from uninf–saline (Sal), caCREB–saline, uninf–cocaine (Coc), and dnCREB–cocaine. E, A summary shows that repeated exposure to cocaine significantly prolonged the decay kinetics of NMDAR EPSCs in NAc MSNs, expression of GFP alone did not affect the decay kinetics, expression of caCREB alone mimicked the effect of cocaine on the decay kinetics of NMDAR EPSCs in NAc MSNs, expression of dnCREB alone did not affect the decay kinetics of NDMAR EPSCs, and expression of dnCREB prevented the effect of cocaine on the decay kinetics of NMDAR EPSCs in NAc MSNs. Numbers in parentheses indicate number of cells; *p < 0.05; **p < 0.01.

Figure 4.

Generation of new spines in cocaine- (5 d procedure) treated rats can be prevented with dnCREB expression. A, Example dendrites of NAc MSNs from saline- (left) and cocaine- (right) treated rats, imaged after DiI labeling with a confocal microscope and two-dimensional projection. B, Summarized data showing that the numbers of filopodia, mushroom-like spines, and the total protrusions are all increased in dendrites of NAc MSNs from cocaine-treated rats. C, Cumulative plots showing that the cocaine-induced increase in spine density is evenly distributed across examined dendrites. D, Example dendrites of GFP-infected MSNs from saline- (top left) or cocaine- (top right) treated rats and dnCREP-expressing MSNs from saline- (bottom left) or cocaine- (bottom right) treated rats. E, Summarized data showing the increased spine density of cocaine-treated, GFP-positive MSNs compared with saline-treated animals, and a blockade of the cocaine-induced increase in dnCREB-expressing neurons. Numbers in parentheses indicate number of cells. *p < 0.05.

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 Ro256981–cocaine (black), Ro256981–saline (open circle), APV–cocaine (light gray), and APV–saline (dark gray) rats. B, Cocaine-induced locomotor sensitization (tested in cocaine-treated rats) as well as the basal locomotor activity (tested in saline-treated rats) were not affected by the surgical installation of intra-NAc mini pumps. C, Examples showing the inhibition of NMDAR EPSCs by Ro256981 (20 nm) and APV (500 nm). D, Dose–response curve showing a concentration range (20–200 nm) in which Ro256981 exhibited a high affinity inhibition of NMDAR EPSCs. E, Grouped data showing that application of 20 nm Ro256981, but not 500 nm APV, significantly decreased the T_1/2 (decay kinetics) of NMDAR EPSCs in NAc MSNs. F, Basal/spontaneous locomotor activity was not affected by chronic intra-NAc perfusion of Ro256981 or APV. G, Control for rapid intra-NAc perfusion. With the identical experimental procedure, intra-NAc perfusion of 1 μm NBQX 15 min before cocaine injection prevented cocaine-induced increase in locomotor responses. H, Grouped data showing that intra-NAc inhibition of NR2B-containing NMDARs (by Ro256981), but not a partial, nonselective inhibition of NAc NMDARs, prevented the development of locomotor sensitization. I, Grouped data showing that intra-NAc inhibition of NR2B-containing NMDARs did not affect the expression of cocaine-induced locomotor sensitization. Numbers in parentheses indicate number of cells or number of rats; *p < 0.05; **p < 0.01. Contl, Control; Ro, Ro256981; Sal, saline; Coc, cocaine.