CREST in the Nucleus Accumbens Core Regulates Cocaine Conditioned Place Preference, Cocaine-Seeking Behavior, and Synaptic Plasticity

Abstract

Epigenetic mechanisms result in persistent changes at the cellular level that can lead to long-lasting behavioral adaptations. Nucleosome remodeling is a major epigenetic mechanism that has not been well explored with regards to drug-seeking behaviors. Nucleosome remodeling is performed by multi-subunit complexes that interact with DNA or chromatin structure and possess an ATP-dependent enzyme to disrupt nucleosome-DNA contacts and ultimately regulate gene expression. Calcium responsive transactivator (CREST) is a transcriptional activator that interacts with enzymes involved in both histone acetylation and nucleosome remodeling. Here, we examined the effects of knocking down CREST in the nucleus accumbens (NAc) core on drug-seeking behavior and synaptic plasticity in male mice as well as drug-seeking in male rats. Knocking down CREST in the NAc core results in impaired cocaine-induced conditioned place preference (CPP) as well as theta-induced long-term potentiation in the NAc core. Further, similar to the CPP findings, using a self-administration procedure, we found that CREST knockdown in the NAc core of male rats had no effect on instrumental responding for cocaine itself on a first-order schedule, but did significantly attenuate responding on a second-order chain schedule, in which responding has a weaker association with cocaine. Together, these results suggest that CREST in the NAc core is required for cocaine-induced CPP, synaptic plasticity, as well as cocaine-seeking behavior.SIGNIFICANCE STATEMENT This study demonstrates a key role for the role of Calcium responsive transactivator (CREST), a transcriptional activator, in the nucleus accumbens (NAc) core with regard to cocaine-induced conditioned place preference (CPP), self-administration (SA), and synaptic plasticity. CREST is a unique transcriptional regulator that can recruit enzymes from two different major epigenetic mechanisms: histone acetylation and nucleosome remodeling. In this study we also found that the level of potentiation in the NAc core correlated with whether or not animals formed a CPP. Together the results indicate that CREST is a key downstream regulator of cocaine action in the NAc.

Keywords: CREST; LTP; cocaine; epigenetics; nucleosome remodeling; nucleus accumbens.

Figure 1.

CREST expression. A, Mouse atlas image illustrates the region of the NAc where the immunohistochemistry staining image was taken. B, Immunoreactivity for CREST protein expression and its overlap with DAPI and NeuroTrace in NAc core is shown. CREST protein expression overlapping with DAPI and NeuroTrace staining. Scale bars: (in Merged) 7 μm; enlarged inset, 70 μm. C, A schematic of the experimental design showing that data were collected from mice killed after either saline or 5, 10, or 20 mg/kg cocaine-HCl conditioning in a single cocaine-CPP trial (see Materials and Methods). D, Gene expression was analyzed from tissue punches collected from the NAc. No changes in CREST mRNA after CPP conditioning with 5, 10, or 20 mg/kg cocaine-HCl relative to conditioning with saline were observed. E, Protein expression from tissue punches collected from the NAc of the same animals was quantified using Western blot. No changes in CREST protein after CPP conditioning with 5, 10, or 20 mg/kg cocaine-HCl relative to conditioning with saline were found. For CREST mRNA expression experiment, n = 7 for Saline, n = 7 for 5 mg/kg cocaine-HCl, n = 6 for 10 mg/kg cocaine-HCl, and n = 6 for 20 mg/kg cocaine-HCl. For CREST protein expression experiment, n = 10 for Saline, n = 7 for 5 mg/kg cocaine-HCl, n = 6 for 10 mg/kg cocaine-HCl, and n = 9 for 20 mg/kg cocaine-HCl.

Figure 2.

Anti-CREST siRNA in the NAc impairs cocaine CPP. A, Quantified Western blot data showing CREST protein levels following intra-NAc core siRNA infusion. siRNA anti-CREST in the NAc core results in a significant knockdown of CREST protein 2–6 d following infusion. B, Schematic of the cocaine CPP procedure. C, Cocaine CPP expression indicated by mean PS (CS+ − CS−) ± SEM. At 5 mg/kg cocaine-HCl training dose, siRNA anti-CREST mice exhibited a trend toward significantly attenuated CPP PSs compared with Control siRNA. D, At 5 mg/kg cocaine-HCl training dose, anti-CREST siRNA animals showed similar locomotion compared with nontargeting siRNA. For time course experiment, n = 3–4 per hemisphere per day. For cocaine CPP experiment, n = 9 for Control siRNA and n = 12 for anti-CREST siRNA. ****p < 0.0001.

Figure 3.

CREST knockdown in the NAc core using morpholino impairs cocaine CPP. A, Quantified Western blot data showing CREST protein levels following intra-NAc core morpholino infusion. Morpholino against CREST in the NAc core results in a significant knockdown of CREST protein 2 and 6 d postinfusion. B, Cocaine-CPP expression indicated by mean PS (CS+ − CS−) ± SEM. At 5 mg/kg cocaine-HCl training dose, anti-CREST morpholino mice (depicted as Anti-CREST in figure) show significantly attenuated PS compared with Scrambled morpholino control (depicted as Control in figure). C, At 5 mg/kg cocaine-HCl training dose, anti-CREST animals showed similar locomotion compared with Controls. D, Left, Representative image of CREST (red) expression in the 5 mg/kg cocaine-HCl CPP mice that had received intra-NAc infusion of Control or anti-CREST. Nuclei (blue) were counterstained with DAPI. Right, Mean intensity of CREST immunofluorescence from NAc core. There was a significant knock down in CREST in anti-CREST animals compared with Control. E, Left, Representative Western blots showing reduced levels of CREST protein (55 kDa) with GAPDH as a loading/transfer control in the same animals. Right, Quantification of anti-CREST Western blot data. CREST protein was significantly knocked down in anti-CREST animals compared with Control. F, At 10 mg/kg cocaine-HCl training dose, no differences in PS were seen between groups. G, At 10 mg/kg cocaine-HCl training dose, anti-CREST animals showed similar locomotion compared with Controls. For time course experiment, n = 6 per hemisphere per day. For 5 mg/kg cocaine CPP experiment, n = 12 for Control and n = 11 for anti-CREST. For 10 mg/kg cocaine CPP experiment, n = 11 for Control and n = 10 for anti-CREST. *p < 0.05, **p < 0.01.

Figure 4.

Anti-CREST morpholino blocks theta-burst-induced LTP in the NAc core. A, Left, Schematic describing the cocaine-CPP and image of an acute slice containing the NAc. Animals underwent handling, a pre-test, intra-NAc infusion surgeries, and then 2 d off. The next day, mice received either saline or 5 mg/kg cocaine-HCl immediately before a single 30 min exposure to one compartment. There was a 30 min lag period between the end of animal testing and sacrificing the animal for electrophysiology studies (horizontal black arrow). Right, Image illustrates the placement of the recording (green arrow) and stimulating (stim) electrode (red) in a NAc core slice. B, Theta burst-induced LTP (3 small upward arrows) produced reliable stable potentiation in slices from control mice that received an infusion of control + injection of saline (Control+SAL; white circles, n = 12). LTP was significantly enhanced in slices from Scrambled morpholino control (depicted as Control in figure) mice that received an intraperitoneal injection of 5 mg/kg cocaine-HCl (Control+COC; gray circles, n = 10) compared with saline-injected mice. Infusions of anti-CREST morpholino (depicted as Anti-CREST in figure) into the NAc significantly impaired theta burst-induced LTP in this area in mice that received saline injections (light blue circles; n = 8) relative to control mice; however, a 5 mg/kg dose of cocaine-HCl could not overcome anti-CREST-induced impairment in LTP (anti-CREST+COC; dark blue circles; n = 7). There was a marked reduction in the level of potentiation 60 min post-TBS in these slices relative to control mice. Right, Representative traces collected during baseline (solid line) and 50 min post-TBS (dotted line) for each group. C, Summary graph representing average change in fEPSP slope as percentage of baseline (BL) for each group. Control animals that received 5 mg/kg cocaine-HCl showed facilitated potentiation compared with Control animals that received saline. The level of potentiation during 50–60 min post-TBS period showed a significant reduction in anti-CREST + saline compared with Control + saline. anti-CREST + cocaine-HCl (5 mg/kg) also showed significantly reduced potentiation compared with Control + cocaine-HCl (5 mg/kg). D, Input (stimulation current)/output (slope of fEPSP) relationships were not detectably different between groups. E, Transmitter release kinetics, as assessed with paired pulse facilitation (PPF), were also comparable for the four groups of slices. Scale: 0.4 mV/5 ms. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.

Higher dose of cocaine overcomes anti-CREST-induced LTP impairment. A, Left, Conditions were the same as described in Figure 4A with the exception that animals received 10 mg/kg cocaine-HCl before the single 30 min exposure to one compartment. In figure, anti-CREST morpholino mice is depicted as Anti-CREST and Scrambled morpholino control is depicted as Control. Slices from control animals injected with the higher dose of cocaine (Control + COC; gray circles; n = 6) produced LTP in the NAc that was indistinguishable from the LTP measured in slices from control animals injected with saline (Control+SAL; white circles; n = 6). The 10 mg/kg dose of cocaine was effective in rescuing anti-CREST-induced LTP (anti-CREST+COC; dark green circles; n = 7), which was found to produce a similar level of potentiation as in slices from control mice injected with saline. NAc slices from mice infused with anti-CREST and injected with saline (anti-CREST+SAL; light green circles; n = 6) produced the predicted deficit in LTP relative to control mice. Right, Representative traces collected during baseline (solid line) and 50 min post-TBS (dotted line) for each group. B, The average level of potentiation during 50–60 min post-TBS period showed a significant enhancement in potentiation in anti-CREST + cocaine (10 mg/kg cocaine-HCl) animals versus anti-CREST+ saline. There was a significant reduction in anti-CREST + saline compared with Control + saline. C, The 10 mg/kg dose did not affect the input/out curves measured before delivery of TBS. D, Paired pulse facilitation (PPF) curves were equivalent in slices from each group tested. Scale: 0.4 mV/5 ms. ****p < 0.0001.

Figure 6.

CREST knockdown in the NAc core initially impairs the acquisition of a chain schedule in a rat self-administration model. A, B, Lever pressing behavior (A) and time to completion (B) during the final two sessions of take responding (Days 1 and 2), and the eight sessions of seek–take responding with increasing VI requirements on the seek lever (Days 3–10; see Materials and Methods). Anti-CREST and Control animals total lever presses were similar (A), but introduction of the seek–take chain schedule significantly increased the time to complete session with the introduction of the seek–take chain schedule (Days 3 and 4), which was attenuated with the increasing VI schedule (B). C, D, Cumulative lever press plots for Scrambled morpholino control (Control) and anti-CREST morpholino (Anti-CREST) animals. C, Control and anti-CREST groups displayed similar lever pressing frequency during the final day of the FR1 take procedure (Day 2; shaded region = SE; p > 0.1). D, An introduction of a seek–take chain schedule of reinforcement significantly reduced lever pressing frequency for the anti-CREST but not the Control group during the first day of seek–take training (Day 3). E, Representative Western blot comparing the expression of CREST protein (55 kDa) in Control and anti-CREST rats. F, CREST Protein was significantly knocked down in the anti-CREST group compared with Control. n = 5 for Control and n = 5 for anti-CREST. *p < 0.05, **p < 0.01.