Chronic cocaine-induced H3 acetylation and transcriptional activation of CaMKIIalpha in the nucleus accumbens is critical for motivation for drug reinforcement

Abstract

The regulation of gene expression in the brain reward regions is known to contribute to the pathogenesis and persistence of drug addiction. Increasing evidence suggests that the regulation of gene transcription is mediated by epigenetic mechanisms that alter the chromatin structure at specific gene promoters. To better understand the involvement of epigenetic regulation in drug reinforcement properties, rats were subjected to cocaine self-administration paradigm. Daily histone deacetylase (HDAC) inhibitor infusions in the shell of the nucleus accumbens (NAc) caused an upward shift in the dose-response curve under fixed-ratio schedule and increased the break point under progressive-ratio schedule, indicating enhanced motivation for self-administered drug. The effect of the HDAC inhibitor is attributed to the increased elevation of histone acetylation induced by chronic, but not acute, cocaine experience. In contrast, neutralizing the chronic cocaine-induced increase in histone modification by the bilateral overexpression of HDAC4 in the NAc shell reduced drug motivation. The association between the motivation for cocaine and the transcriptional activation of addiction-related genes by H3 acetylation in the NAc shell was analyzed. Among the genes activated by chronic cocaine experiences, the expression of CaMKIIalpha, but not CaMKIIbeta, correlated positively with motivation for the drug. Lentivirus-mediated shRNA knockdown experiments showed that CaMKIIalpha, but not CaMKIIbeta, in the NAc shell is essential for the maintenance of motivation to self-administered cocaine. These findings suggest that chronic drug-use-induced transcriptional activation of genes, such as CaMKIIalpha, modulated by H3 acetylation in the NAc is a critical regulatory mechanism underlying motivation for drug reinforcement.

Figure 1

HDAC inhibitor infusions into the NAc shell increase the motivation for cocaine self-administration. (a, b) Daily infusions of TSA into the shell (a), but not the core (b) of NAc induced an upward shift in dose-response and dose-intake curves under fixed-ratio 5 (FR5) schedule of cocaine self-administration. (c, d) Daily infusions of TSA into the shell (c), but not the core (d) of NAc increased break point under progressive-ratio schedule. Injection sites in the shell or core are illustrated on the right panel. Daily infusions of SAHA (e) into the shell of NAc increased responding in dose-response curve and break point (N=8–12 per group). Data are expressed as mean±SEM. ^*P<0.05, ^**P<0.01, compared with vehicle-chronic cocaine control.

Figure 2

HDAC inhibitor augments histone acetylation elevation induced by chronic cocaine experience in the NAc shell. (a, b) Immunoblots of histone acetylation of H3 (AcH3) (a) and H4 (AcH4) (b) in the shell or core of NAc 30 min after 1 day (acute) or 18 days (chronic) cocaine yoked (Coc Yoke) or self-administration (Coc SA). (c, d) Immunohistochemistry data of AcH3 (c) and AcH4 (d) in the shell or core of NAc after chronic Coc Yoke or Coc SA. AC, anterior commissure; LV, lateral ventricle. (e, f) AcH3 (e) and AcH4 (f) levels after daily HDAC inhibitor infusions in the NAc shell or core. Immunoblot and immunohistochemistry data from cocaine-treated groups (N=8–12 per group) are expressed as mean±SEM percentage of saline self-administration control (N=8–12 per group). ^*P<0.05, ^**P<0.01, compared with saline self-administration control or vehicle-chronic saline control; ^#P<0.05, compared with vehicle-chronic cocaine control.

Figure 2

HDAC inhibitor augments histone acetylation elevation induced by chronic cocaine experience in the NAc shell. (a, b) Immunoblots of histone acetylation of H3 (AcH3) (a) and H4 (AcH4) (b) in the shell or core of NAc 30 min after 1 day (acute) or 18 days (chronic) cocaine yoked (Coc Yoke) or self-administration (Coc SA). (c, d) Immunohistochemistry data of AcH3 (c) and AcH4 (d) in the shell or core of NAc after chronic Coc Yoke or Coc SA. AC, anterior commissure; LV, lateral ventricle. (e, f) AcH3 (e) and AcH4 (f) levels after daily HDAC inhibitor infusions in the NAc shell or core. Immunoblot and immunohistochemistry data from cocaine-treated groups (N=8–12 per group) are expressed as mean±SEM percentage of saline self-administration control (N=8–12 per group). ^*P<0.05, ^**P<0.01, compared with saline self-administration control or vehicle-chronic saline control; ^#P<0.05, compared with vehicle-chronic cocaine control.

Figure 3

HDAC4 overexpression in the NAc shell downregulates histone acetylation elevation and reduces the motivation for cocaine self-administration. (a) HDAC4 domain structure, depicting the CaMKII docking site and the catalytic HDAC domain. The HDAC4 truncation (ΔHDAC) without the catalytic HDAC domain used for the behavioral experiments is also shown. (b) Representative staining of AV-HDAC4 in the NAc shell (left) and core (right). Neurons infected with HDAC4 were labeled using Flag immunofluorescence. (c–e) Immunoblots of AcH3 (c) and AcH4 (d) in the NAc shell of rats that underwent HDAC4 overexpression. Densitometric quantification of immunoblot in c, d (e) of NAc shell (N=8–12 per group). Data are presented as mean±SEM. ^*P<0.05, compared with gal-chronic saline control, ^#P<0.05, compared with gal-chronic cocaine control. (f–i) HDAC4 overexpression in the NAc shell induced a downward shift in dose-response, dose-intake curves (f) and decreased break point (h), but not in the NAc core (g, i). N=8–12 per group. Data are expressed as mean±SEM. ^*P<0.05, ^**P<0.01, compared with gal-chronic cocaine control.

Figure 3

HDAC4 overexpression in the NAc shell downregulates histone acetylation elevation and reduces the motivation for cocaine self-administration. (a) HDAC4 domain structure, depicting the CaMKII docking site and the catalytic HDAC domain. The HDAC4 truncation (ΔHDAC) without the catalytic HDAC domain used for the behavioral experiments is also shown. (b) Representative staining of AV-HDAC4 in the NAc shell (left) and core (right). Neurons infected with HDAC4 were labeled using Flag immunofluorescence. (c–e) Immunoblots of AcH3 (c) and AcH4 (d) in the NAc shell of rats that underwent HDAC4 overexpression. Densitometric quantification of immunoblot in c, d (e) of NAc shell (N=8–12 per group). Data are presented as mean±SEM. ^*P<0.05, compared with gal-chronic saline control, ^#P<0.05, compared with gal-chronic cocaine control. (f–i) HDAC4 overexpression in the NAc shell induced a downward shift in dose-response, dose-intake curves (f) and decreased break point (h), but not in the NAc core (g, i). N=8–12 per group. Data are expressed as mean±SEM. ^*P<0.05, ^**P<0.01, compared with gal-chronic cocaine control.

Figure 4

Motivation for cocaine is associated with H3 acetylation in the NAc shell. (a, b) The break point was positively correlated with global level of AcH3 (a), but not AcH4 (b) in the NAc shell. Diagonal solid lines indicate linear regression fit of data points (N=18). (c, d) Changes of histone acetylation in specific gene promoter regions in the NAc shell under progressive-ratio schedule (N=6–8 per group). Data are presented as mean±SEM. ^*P<0.05, compared with saline self-administration control, ND, acetylation not detected. (e, f) The effect of HDAC inhibitor (e) or HDAC4 overexpression (f) on AcH3 at the indicated gene promoters in the NAc shell under progressive-ratio schedule (N=8–10 per group). Data are presented as mean±SEM. ^*P<0.05, ^**P<0.01, ^***P<0.001, compared with vehicle- or gal-chronic saline control, ^#P<0.05, compared with vehicle- or gal-chronic cocaine control.

Figure 5

Localized CaMKIIα knockdown in the NAc shell decreases the motivation for cocaine. (a) CaMKIIα but not CaMKIIβ mRNA levels were increased in the NAc shell 30 min after chronic cocaine yoked or self-administration (N=8–12 per group). Data are expressed as mean±SEM. ^**P<0.01, compared with chronic saline self-administration control. (b, c) The break point was correlated with level of CaMKIIα mRNA (b), but not CaMKIIβ mRNA (c) in the NAc shell. Diagonal solid lines indicate linear regression fit of data points (N=14). (d) GFP-tagged CaMKIIα or CaMKIIβ were cotransfected into HEK293T cells with or without plasmid encodes shRNA for CaMKIIα (αshRNA) or CaMKIIβ (βshRNA) expression and the cell lysate was immunoblotted with anti-GFP antibody. pBS-hU6 (vector) was used as a control. (e) Localized Lenti–GFP expression in the NAc shell. (f, g) Immunoblot from the NAc shell infected with Lenti–αshRNA or Lenti–βshRNA showed a reduction in protein level of CaMKIIα (f) or CaMKIIβ (g), compared with Lenti–GFP control (N=8–10/ group). Data are expressed as mean±SEM. ^*P<0.05, compared with Lenti–GFP control. (h) Intra-NAc-shell Lenti–αshRNA infusions decreased the motivation for cocaine (N=8–12 per group). Data are expressed as mean±SEM. ^*P<0.05, compared with Lenti–GFP control.