Neuropsychopharmacology (2012) 37, 321–337 & 2012 American College of Neuropsychopharmacology. All rights reserved 0893-133X/12 www.neuropsychopharmacology.org Differential Regulation of MeCP2 Phosphorylation in the CNS by Dopamine and Serotonin 1 1 2 1,2,3,4 ,1 Ashley N Hutchinson , Jie V Deng , Dipendra K Aryal , William C Wetsel and Anne E West* 1 2 Department of Neurobiology, Duke University Medical Center, Durham, NC, USA; Department of Psychiatry and Behavioral Sciences, Duke 3 University Medical Center, Durham, NC, USA; Mouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University Medical Center, 4 Durham, NC, USA; Department of Cell Biology, Duke University Medical Center, Durham, NC, USA Systemic administration of amphetamine (AMPH) induces phosphorylation of MeCP2 at Ser421 (pMeCP2) in select populations of neurons in the mesolimbocortical brain regions. Because AMPH simultaneously activates multiple monoamine neurotransmitter systems, here we examined the ability of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) to induce pMeCP2. Selective blockade of the DA transporter (DAT) or the 5-HT transporter (SERT), but not the NE transporter (NET), was sufficient to induce pMeCP2 in the CNS. DAT blockade induced pMeCP2 in the prelimbic cortex (PLC) and nucleus accumbens (NAc), whereas SERT blockade induced pMeCP2 only in the NAc. Administration of selective DA and 5-HT receptor agonists was also sufficient to induce pMeCP2; however, the specific combination of DA and 5-HT receptors activated determined the regional- and cell-type specificity of pMeCP2 induction. The D1-class DA receptor agonist SKF81297 induced pMeCP2 widely; however, coadministration of the D2-class agonist quinpirole restricted the induction of pMeCP2 to GABAergic interneurons of the NAc. Intra-striatal injection of the adenylate cyclase activator forskolin was sufficient to induce pMeCP2 in medium-spiny neurons, suggesting that the combinatorial regulation of cAMP by different classes of DA and 5-HT receptors may contribute to the cell-type specificity of pMeCP2 induction. Consistent with the regulation of pMeCP2 by multiple monoamine neurotransmitters, genetic disruption of any single monoamine transporter in DAT-, SERT-, and NET- knockout mice failed to eliminate AMPH-induced pMeCP2 in the NAc. Together, these studies indicate that combinatorial signaling through DA and 5-HT receptors can regulate the brain region- and cell-type specific pMeCP2 in the CNS. Neuropsychopharmacology (2012) 37, 321–337; doi:10.1038/npp.2011.190; published online 28 September 2011 Keywords: MeCP2; monoamine transporters; signal transduction; dopamine; serotonin; nucleus accumbens INTRODUCTION A substantial body of evidence indicates that psychostim- ulant-induced changes in gene expression contribute to the Psychostimulant-induced activation of monoamine recep- long-lasting effects of these drugs on behavior (Nestler, tors is essential to the mechanism by which these drugs 2008; Kumar et al, 2005). Examination of the molecular induce changes in behavior (Gainetdinov and Caron, 2003; processes underlying these changes in gene expression has Missale et al, 1998; Xu et al, 1997). Cocaine and revealed a significant role for epigenetic mechanisms of amphetamine (AMPH) serve as indirect monoamine transcription, which are processes that mediate changes receptor agonists by inhibiting and/or reversing the in gene expression by regulating chromatin structure function of monoamine transporters. However, despite the (Maze et al, 2010; Renthal et al, 2009). Therefore, chromatin fact that these drugs rapidly increase extracellular levels of regulatory proteins that are targets of modulation by dopamine (DA), serotonin (5-HT), and norepinephrine monoaminergic signaling pathways are strong candidates (NE), repeated exposure is necessary to produce long- to link psychostimulant exposure to persistent changes in lasting alterations in behavior (Hyman et al, 2006). This gene expression and behavior. Recently, we and others have observation suggests that molecular mechanisms down- demonstrated that the methyl-CpG-binding protein-2 stream of monoamine receptor activation are required to (MeCP2) contributes to both neural and behavioral adapta- mediate the observed behavioral adaptations. tions induced by repeated psychostimulant exposure (Deng et al, 2010; Im et al, 2010). We have shown that lentiviral shRNA-mediated knockdown of MeCP2 in the nucleus *Correspondence: Dr AE West, Department of Neurobiology, Duke accumbens (NAc) increases sensitivity to AMPH-induced University Medical Center, Box 3209, Durham, NC 27710, USA, conditioned place-preference (CPP) whereas overexpression Tel: + 1 919 681 1909, Fax: + 919 684 4431, of MeCP2 decreases CPP (Deng et al, 2010). These data E-mail: [email protected] suggest that MeCP2 acts in the NAc to limit the rewarding Received 29 March 2011; revised 29 July 2011; accepted 29 July 2011 properties of psychostimulants. Differential regulation of MeCP2 phosphorylation AN Hutchinson et al 322 Psychostimulants induce rapid and robust phosphoryla- 21–30 days of age, housed in groups of 3–5, and segregated tion of MeCP2 at Ser421 (pMeCP2), a site that is thought to by sex and genotype. All animals were given free access to modulate MeCP2-dependent chromatin regulation (Deng standard laboratory chow and water and were housed in a et al, 2010; Zhou et al, 2006; Chen et al, 2003). MeCP2 humidity- and temperature-controlled room on a 14-h/10-h regulates gene transcription by binding to methylated DNA light/dark cycle (lights on at 0700 hours). All experiments and recruiting enzymes that modify histones (Nan et al, were conducted with an approved protocol from the Duke 1998). Although the specific mechanisms by which Ser421 University Institutional Animal Care and Use Committee in phosphorylation affects MeCP2 function remain poorly accordance with the guidelines from the National Institutes understood, overexpression of a Ser421Ala mutant MeCP2 of Health for the Care and Use of Laboratory Animals. has been shown to inhibit neuronal activity-induced Bdnf expression, suggesting that Ser421 phosphorylation could Immunofluorescent Staining of the Brain Sections contribute to gene derepression (Zhou et al, 2006). Interestingly, systemic administration of AMPH induces a Two hours after vehicle or drug injection, mice were perfused restricted pattern of pMeCP2 induction in select neurons transcardially with 4% paraformaldehyde in 0.1 M PBS. The of the NAc and prelimbic cortex (PLC) (Deng et al, 2010). brains were post-fixed in 4% paraformaldehyde/PBS over- D1-class DA receptor activation is required for AMPH- night, then sunk into 20% (wt/vol) sucrose/PBS overnight. dependent induction of pMeCP2 in the NAc, and admin- Coronal sections (40 mm) were cut on a freezing microtome, istration of a D1-class DA receptor agonist is sufficient to and the brain regions were identified by anatomical land- induce pMeCP2 (Deng et al, 2010). However, AMPH marks. One section from each brain region of interest was induces pMeCP2 selectively in GAD67- and parvalbumin- selected for each mouse, based upon anatomical structures to positive GABAergic interneurons of the NAc whereas it fails represent the closest approximation of identical sections to induce pMeCP2 in medium-spiny neurons (MSNs), between individual mice. To minimize technical variations in despite the fact that these cells express high levels of DA immunostaining across genotypes, sections from different receptors. In addition to its effects on DA neurotransmis- individual mice were first photographed for visual identifica- sion, AMPH also impacts 5-HT and NE neurotransmission. tion. The sections were then pooled and incubated with Thus, we considered the possibility that the selective pattern antibodies in a single chamber, and finally they were of pMeCP2 induced by AMPH might arise as a result of separated after processing for image analysis (see Deng combinatorial signaling through one or more of these et al, 2010). For immunostaining, tissue sections were monoaminergic systems. permeabilized with either 1% (vol/vol) (for the pMeCP2 Here, we use both pharmacological and genetic tech- antibody) or 0.3% (vol/vol) (all other antibodies) Triton X- niques to characterize the differential roles of DA, 5-HT, 100 for 1 h and then sections were blocked with 3% (wt/vol) and NE signaling in the regulation of the regional BSA (for the DARPP-32 antibody) or 16% (vol/vol) goat distribution and cell-type specificity of pMeCP2 induction. serum (all other antibodies) in PBS. Sections were incubated We find that selective inhibition of either the DA with the following primary antibodies overnight at 4 1C: rabbit transporter (DAT) or 5-HT transporter (SERT) but not anti-phospho-Ser421 MeCP2 1:15 000 (Deng et al, 2010), the NE transporter (NET) is sufficient to induce pMeCP2 in mouse anti-GAD67 1:500 (MAB5406; Chemicon/Millipore, the NAc. However, only DAT inhibition induces pMeCP2 in Billerica, MA), rabbit anti-c-Fos 1:15 000 (PC38; Calbiochem, the PLC. We also find that differential activation of specific San Diego, CA), goat anti-DARPP-32 1:50 (sc31519; Santa classes of DA and 5-HT receptors induces pMeCP2 in Cruz Biotechnology, Santa Cruz, CA), and mouse anti-GFAP distinct cell types and brain regions, suggesting that 1:100 (Clone G-A-5; Sigma, St Louis, MO). Whenever possible, combinatorial signaling through these receptors sculpts double immunostaining with primary antibodies raised in the specific pattern