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. Author manuscript; available in PMC: 2019 Nov 1.
Published in final edited form as: Exp Neurol. 2018 Aug 10;309:160–168. doi: 10.1016/j.expneurol.2018.08.002

Targeting histone deacetylation for recovery of maternal deprivation-induced changes in BDNF and AKAP150 expression in the VTA

Ryan D Shepard 1, Shawn Gouty 1, Haifa Kassis 1, Aylar Berenji 1, William Zhu 1, Brian M Cox 1, Fereshteh S Nugent 1,†
PMCID: PMC6139260  NIHMSID: NIHMS1504395  PMID: 30102916

Abstract

Severe early life stressors increase the probability of developing psychiatric disorders later in life through modifications in neuronal circuits controlling brain monoaminergic signaling. Our previous work demonstrated that 24h maternal deprivation (MD) in male Sprague Dawley rats modifies dopamine (DA) signaling from the ventral tegmental area (VTA) through changes at GABAergic synapses that were reversible by in vitro histone deacetylase (HDAC) inhibition which led to restoration of the scaffold A-kinase anchoring protein (AKAP150) signaling and subsequently recovered GABAergic plasticity (Authement et al., 2015). Using a combination of in situ hybridization, Western blots and immunohistochemistry, we confirmed that MD-induced epigenetic modifications at the level of histone acetylation were associated with an upregulation of HDAC2. MD also increased Akap5 mRNA levels in the VTA. Western blot analysis of AKAP150 protein expression showed an increase in synaptic levels of AKAP150 protein in the VTA with an accompanying decrease in synaptic levels of protein kinase A (PKA). Moreover, the abundance of mature brain-derived neurotrophic factor (BDNF) protein of VTA tissues from MD rats was significantly lower than in control groups. In vivo systemic injection with a selective class I HDAC inhibitor (CI-994) was sufficient to reverse MD-induced histone hypoacetylation in the VTA for 24h after the injection. Furthermore, HDAC inhibition normalized the levels of mBDNF and AKAP150 proteins at 24h. Our data suggest that HDAC-mediated targeting of BDNF and AKAP-dependent local signaling within VTA could provide novel therapeutics for prevention of later-life psychopathology.

Keywords: A-kinase anchoring protein (AKAP), dopamine, histone deacetylase (HDAC), histone deacetylase inhibitor (HDAC inhibitor), brain derived neurotrophic factor (BDNF), ventral tegmental area (VTA), early life stress, maternal deprivation (MD), epigenetics, histone acetylation

Introduction

Child abuse and neglect are shown to increase the risk of developing stress-related disorders and substance abuse. This increased vulnerability seems to be related to brain monoaminergic dysfunction which includes an altered dopamine (DA) signaling from the ventral tegmental area (VTA) (Authement et al., 2015; Heim and Nemeroff, 2002; Pena et al., 2017; Sinha, 2008; Strathearn, 2011). Early life stressors also have a significant impact on epigenome, including histone modifications that may underlie subsequent changes in gene expression, synaptic plasticity and behavior (Abel and Zukin, 2008; Heim and Binder, 2012; Klengel and Binder, 2015; Levine et al., 2012; Palmisano and Pandey, 2017; Tesone-Coelho et al., 2015). One of the robust long-lasting histone modifications associated with severe early life stress are histone deacetylase (HDAC)-mediated changes in histone acetylation (Adler and Schmauss, 2016; Levine et al., 2012; Tesone-Coelho et al., 2015; Xie et al., 2013). In general, histone deacetylation by HDACs is associated with chromatin condensation and gene repression. On the other hand, blocking histone deacetylation by HDAC inhibitors can increase histone acetylation to possibly promote gene expression through chromatic relaxation. HDAC inhibitors have shown great potential for treatment of age-associated cognitive and memory impairments by improving synaptic plasticity (Penney and Tsai, 2014). Moreover, HDAC inhibitors have also been shown to have antidepressant properties and ameliorate symptoms of post-traumatic stress disorder, depression, and addiction (Covington et al., 2009; Klengel and Binder, 2015; Palmisano and Pandey, 2017).

We have also shown that acute morphine-induced synaptic plasticities in VTA DA neurons involved HDAC-mediated changes in histone acetylation and were also reversible by in vitro application of an HDAC inhibitor through increases in histone acetylation (Authement et al., 2016; Langlois and Nugent, 2017). We demonstrated that a 24h early maternal deprivation (MD, an animal model of child abuse), on postnatal day 9 (P9) induces synaptic abnormalities at GABAergic synapses onto VTA DA neurons through disruption of AKAP79/150 (human 79/rodent 150; also known as AKAP5) signaling in juvenile rats that might also be targeted by HDACs during MD (Authement et al., 2015). AKAPs were first discovered as the scaffold proteins that principally mediate the crosstalk of cAMP/PKA signaling with other signaling pathways. Although AKAPs identified to bind to the type II regulatory subunit of PKA (RII); it is now known that AKAPs have several binding sites for other signaling molecules including protein kinase C, protein phosphatases including calcineurin (CaN), G-protein coupled receptors, adenylyl cyclases and phosphodiesterases. AKAPs tether these signaling enzymes with their substrates (for example, synaptic AMPA, NMDA and GABAA receptors and ion channels) within distinct subcellular compartments for specific spatial and temporal interplay of postsynaptic signaling molecules in synaptic plasticity and neuronal function, as well as in synaptic and neuronal dysfunction associated with disease (Esseltine and Scott, 2013; Wild and Dell’Acqua, 2017; Woolfrey and Dell’Acqua, 2015). Therefore, therapeutic targeting of AKAP-directed signaling has become an emerging and novel concept in selective normalization of dysfunctional signaling pathways assembled by AKAPs in neurological disorders (Wild and Dell’Acqua, 2017). We found that MD-induced GABAergic metaplasticity (an increased susceptibility of GABAergic synapses to induction of AKAP150-dependent long-term depression, LTD) and dysregulated AKAP signaling could be reversed with local in vitro HDAC inhibition in the VTA, suggesting the potential clinical benefits of targeting of AKAP signaling within the VTA by HDAC inhibitors soon after the stress. Given that MD-induced disruption of AKAP signaling was associated with significant increases in the levels of AKAP150 expression in VTA DA neurons (Authement et al., 2015), this suggested that MD may induce HDAC-mediated transcriptional changes in specific signaling molecules that directly interact with AKAPs or act upstream from AKAP signaling. In fact, activity-dependent alterations of brain-derived neurotrophic factor (BDNF) transcriptional levels and BDNF expression act upstream to regulate proteasome-dependent synapse remodeling and synaptic protein concentrations including synaptic levels of AKAP150 (Jia et al., 2008). Moreover, early life adversity results in epigenetic changes in BDNF gene expression and signaling that are critical for synaptic plasticity (Adler and Schmauss, 2016; Daskalakis et al., 2015; Palmisano and Pandey, 2017; Roth et al., 2009).

Here, we investigated whether reversible HDAC-mediated histone modifications were associated with MD in the VTA and tested the effects of a single in vivo injection of a cell permeable potent selective class I HDAC inhibitor (CI-994, also called N-acetyldinaline or tacedinaline) on MD-induced changes at the level of histone acetylation, BDNF protein levels and AKAP gene expression within the VTA. We found that MD indeed increased HDAC2 (a class I HDAC) expression specifically in VTA DA neurons and is associated with a reduction of histone H3 acetylation at lysine 9 (Ac-H3K9). MD also reduced levels of BDNF protein while increased synaptic levels of AKAP150 protein in the VTA, and these changes were reversible by the in vivo HDAC inhibition 24h after the injection (see our model in Figure 7). Taken together, our results suggest that a single in vivo HDAC inhibition soon after the stress may be sufficient to epigenetically ameliorate MD-induced changes in BDNF signaling that may act upstream to regulate subcellular organization of AKAP150 complexes in VTA synapses.

Figure 7: Proposed epigenetic mechanisms supporting MD-induced synaptic modifications in the VTA.

Figure 7:

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MD induces GABAergic metaplasticity in VTA DA neurons through disruption of AKAP150 signaling that renders GABAergic synapses more susceptible to LTD. Our recent findings show that MD increases HDAC2 activity in VTA DA neurons and reduces Ac-H3K9 in the VTA. MD also increases synaptic levels of AKAP150 protein in the VTA with accompanying decreases in synaptic levels of PKA and the levels of mBDNF protein in the VTA. In vivo systemic injection with a selective class I HDAC inhibitor is sufficient to reverse MD-induced histone hypoacetylation and to normalize the levels of mBDNF and AKAP150 proteins in the VTA at 24h after the injection. Our model suggests that HDAC2-mediated targeting of mBDNF could decrease mBDNF signaling through its receptor tropomyosin receptor kinase B (TrkB). We hypothesize that the reduced local BDNF signaling in turn decreases AKAP150-PKA association and AKAP-dependent PKA anchoring at GABAergic synapses while enhancing AKAP150-CaN association that favors the induction of LTD in VTA DA neurons. These epigenetic and synaptic modifications induced by MD could then affect DA neuronal excitability and DA release in VTA projection areas.→ means excitation and ┤ means inhibition.

Materials and Methods

All experiments were carried out in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals and were approved by the Uniformed Services University Institutional Animal Care and Use Committee. All efforts were made to minimize animal suffering, and to reduce the number of animals used.

Maternal Deprivation Procedure

Half of the male pups in litters of Sprague–Dawley rats (Taconic Farms) at P9 were isolated at 10:00 a.m. from the dam and their siblings for 24h (MD group). The isolated rats were placed in a separate quiet room and kept on a heating pad (34° C) and not disturbed until being returned to their home cage 24h later. The remaining non-separated male rat pups received the same amount of handling but were kept with the dam serving as the non-maternally deprived control group (non-MD group). Rats were maintained on a 12 hour light/dark cycle and provided food and water ad libitum. The animals were taken for study during the light period, between 3 and 5 h after light was turned on. Each day, two MD and non-MD rats (age-matched) from the same litter were sacrificed over days P14–21 for electrophysiology recordings, immunohistochemistry and Western Blotting. We blindly performed the analysis with respect to the treatment of the rats to reduce the potential for investigator bias.

Slice preparation for Western blotting

Rats were anesthetized with isoflurane and immediately decapitated. The brains were quickly dissected and placed into ice-cold artificial cerebrospinal fluid (ACSF) containing (in mM): 126 NaCl, 21.4 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 2.4 CaCl2, 1.00 MgSO4, 11.1 glucose, 0.4 ascorbic acid, saturated with 95% O2–5% CO2. Horizontal slices were cut at 300 μm for Western blot experiments.

HDAC inhibitor treatment

Sprague–Dawley male rats (P14-P21) (non-MD or MD) received either one intraperitoneal (i.p.) injection of CI-994 (10mg/kg) dissolved in 1% Tween80 (vehicle) or an injection of comparable volumes of 1% Tween80 (vehicle) 24h prior to sacrifice for immunohistochemical or Western blot studies.

Western Blotting

The VTA was dissected bilaterally from horizontal slices (300 μm) of non-MD or MD rats in ACSF and then snap frozen in liquid nitrogen and stored at −80°C. Tissues were thawed, washed in ice-cold PBS and lysed in RIPA buffer containing protease inhibitors (Sigma). Samples were then sonicated, incubated on ice for 30 minutes and centrifuged at 10,000g for 20 min at 4°C. Protein concentration in the supernatant was determined by Pierce BCA Protein Assay Kit (Life Technologies). Equal amounts of protein (20 μg) were combined with loading buffer, boiled for 5 min, and loaded onto 4–20% precast polyacrylamide gel (Bio-Rad Laboratories). Separated proteins were transferred onto nitrocellulose membranes, blocked with casein-based blocking reagent (I-Block, Life Technologies) for 60 minutes at room temperature and then incubated overnight at 4°C with antibodies recognizing HH3 (1:10,000, Abcam ab1791), antibody against PKA regulatory β2 subunit (1:5000, Abcam, ab75993), antibody against PSD95 (1:500, Cell signaling 362333), antibody against ac-H3K9 (1:1,000 cell signaling 3649), antibody against calcineurin subunit A (1:2,000, Abcam ab3673), antibody against AKAP150 (1:500, Santa Cruz Sc-6445), antibody against mature BDNF(mBDNF, 1:1000 ab108319), antibody against vinculin (1:1,000, Abcam ab129002) and antibody against β-actin (1:10,000, Abcam, ab6276). Secondary antibodies used were HRP-linked specific for rabbit (1:2000, Cell signaling), mouse (1:2000, Cell signaling) and goat (1:5000, Abcam ab97110) IgG. After incubation, the membranes were washed with PBS-T and exposed to the appropriate horseradish peroxidase-linked secondary antibody (Cell Signaling). Blots were developed with Clarity Western ECL Substrate (Bio-Rad Laboratories) and detected using a BioRad ChemiDoc Touch image acquisition system (BioRad Laboratories, Hercules, CA, USA). Data was analyzed using ImageJ software. Total abundance of target protein was normalized to appropriate endogenous control. All data were normalized to non-MD group with summary data reported as fold change.

Subcellular Fractionation

The subcellular fractionation method was modified from (Hallett et al., 2008). In brief, VTA tissues were collected from non-MD and MD rats via horizontal sectioning (300 μm). Tissues were homogenized and lysed using TEVP buffer (1 mM Na3VO4, 1mM EDTA, 1mM EGTA, 1M Tris pH 7.5, phosphotase inhibitor, protease inhibitor) containing sucrose (320mM). Samples were then centrifuged at 800g for 10 min at 4°C to yield both P1 and S1 segments. TEVP buffer was then added to S1 segments and centrifuged at 9,200g for 15 min at 4°C to yield S2 and P2 segments. P2 represents the crude synaptosomal membranes. P2 fractions were suspended with TEVP buffer containing sucrose, and transferred to polycarbonate microcentrifuge tubes (Beckman Coulter). Samples were centrifugated using Optima Max Ultracentrifuge (Beckman Coulter, Indianapolis, IN, USA). Samples were ultracentrifugated at 25,000g for 20 min at 4°C to yield LS1 and LP1. The LP1 fractions contained the synaptosomal membranes and contents; this fraction was then used to perform western blot for protein expression specifically in the synaptic compartment, as described above.

Immunohistochemistry and image analysis

Non-MD and MD rats were anesthetized with an intraperitoneal injection containing ketamine (85 mg/kg) and xylazine (10 mg/kg) and perfused through the aorta with 300ml of heparinized 1x phosphate buffered saline (PBS) followed by 250ml of 4% paraformaldehyde (PFA, USB, Cleveland, OH). The brains were dissected and placed in 4% PFA for 24 hr and then cryoprotected by submersion in 20% sucrose for 3 days, frozen on dry ice and stored at −70 C until sectioned. Sections of the VTA were cut using a cryostat (Leica CM1900) and mounted on slides. Serial coronal sections (20 μm) of the midbrain containing the VTA (from −4.92 to −6.72 mm caudal to bregma; Paxinos and Watson, 2007) were fixed in 4 % PFA for 5 minutes and washed in 1x PBS then blocked in 10% normal horse serum (NHS) containing 0.3 % Triton X-100 in 1x PBS for 1 hr. Sections were incubated in rabbit anti-tyrosine hydroxylase (anti-TH) (1:1000, Calbiochem, San Diego, CA.), goat anti-AKAP150 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA.) in carrier solution (0.5 % NHS in 0.1 % Triton X-100 in 1x PBS) overnight at room temperature. After rinsing in 1x PBS, sections were incubated for 2 hours in Alexa Fluor® 488 labeled chicken anti-goat IgG and Alexa Fluor® 568 labeled donkey anti-rabbit IgG (both diluted 1:200). For double immunofluorescence of TH and HDAC2, serial coronal sections of the midbrain containing the VTA were fixed in 4% PFA for 5 min, washed in 1x PBS, and then blocked in 10% normal goat serum (NGS) containing 0.3% Triton X-100 in 1x PBS for 1 h. Sections were then incubated in rabbit anti-TH (1:1000; Calbiochem) and mouse anti-HDAC2 (1:1000; Abcam) in carrier solution (0.5% NGS in 0.1% Triton X-100 in 1x PBS) overnight at room temperature. After rinsing in 1x PBS, sections were incubated for 2 h in Alexa Flour 488-labeled goat anti-mouse IgG and Alexa Flour 568-labeled goat anti-rabbit IgG (both diluted 1:200). Finally sections were rinsed in 1x PBS, dried, and cover slipped with Prolong mounting medium containing DAPI to permit visualization of nuclei. Background staining was assessed by omission of primary antibody in the immunolabeling procedure (negative control). VTA tissue sections of rats with previously established presence of TH/AKAP150 or TH/HDAC2 immunoreactive neurons were processed as positive control tissue. Images were captured using a Zeiss Confocal Inverted Microscope System (Carl Zeiss Inc.) 40x/1.4 n.a. oil immersion objective. For HDAC2 density quantification three AP locations (−5.4, −5.7, and −6.0 relative to bregma) were studied. At three AP locations, total of eighteen TH positive neuron were identified within the VTA. From each TH positive neuron two HDAC2 density readings (3 μm × 3 μm) were taken from the somatic region (clearly labeled with TH) and nuclear region (clearly labeled with DAPI). Three background density readings were taken from an area clearly not labeled with HDAC2. All density readings were normalized to background. For each AP location normalized density readings were averaged across the neurons.

In situ Hybridization procedure

An Akap5 probe corresponding to nucleotides 949–1367 of rat Akap5 cDNA (accession number NM_133515) was used for all in situ hybridization procedures. 35S-UTP–labeled riboprobes were synthesized using T7 (antisense) RNA polymerase. After incubation at 37 °C for 1 h, probes were treated with DNase I, precipitated and re-suspended. Sections were fixed in 4% formaldehyde followed by two 5 min washes in 1x PBS. They were then placed in 0.25% acetic anhydride/triethanolamine (1.5%) for 10 min and rinsed in 2x SSC twice for 5 minutes each. Sections were dehydrated in ascending ethanol concentrations (70, 80, 95, and 100%) and then air-dried. Antisense-labeled probes (2.04 × 106 dpm/100 ul) were hybridized to tissue sections overnight at 55 °C in hybridization buffer (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate,1x Denhardt’s). Slide-mounted sections including VTA (from – 5.4 to −6.0 mm caudal to bregma) were rinsed in 4x SSC at room temperature to remove coverslips and then washed four times for 5 min each in 4x SSC containing 1 mM dithiothreitol (DTT). Free probe was removed using 20 mg/mL RNase (Sigma, St. Louis,MO, USA) in buffer (0.5 M NaCl, 0.01 M Tris-HCl, 0.25 mM EDTA) at 37 °C for 30 min. After rinsing twice in 2x SSC/1 mM DTT, then once in 1 × SSC/1 mM DTT and once in 0.5 × SSC/1 mM DTT (all rinses for 5 min), sections were washed twice for 30 min in 0.1x SSC, 1 mM DTT at 65 °C. Sections were cooled in 0.1x SSC, 1 mM DTT at room temperature and then dehydrated in ascending ethanol concentrations (70, 80, 95, and 100%) and then air-dried. Hybridized slides were then exposed to Hyblot CL film (Denville Scientific, Holliston, Ma.) for 5 days. A slide containing 14C microscale standards was also exposed to the film (ARC, St Louis, MO). The autoradiogram was digitized and optical density readings were taken from the VTA at – 5.4, −5.7 and −6.0 mm caudal to bregma. Density readings were also taken from the 14C micro scale standards at 0, 0.63, 0.193, 0.324, 0.453, 0.573, and 0.7 uCi/g in order to generate a standard curve. Data was generated by comparing the sample readings to the standard curve.

Data analysis

Values are presented as means ± SEM. Statistical significance was determined using unpaired or paired two tailed Student’s t-test or two-way ANOVA with Bonferroni post hoc analysis. The threshold for significance was set at *p < 0.05 for all analyses. All statistical analyses were performed using GraphPad Prism 7.

Results

MD increased HDAC2 expression in VTA DA neurons and induced histone hypoacetylation in the VTA.

Here, we performed HDAC2 double immunofluorescence using antibodies against TH (a marker for DA neurons) and HDAC2 as previously described (Authement et al., 2016) to examine whether MD induces changes in HDAC2 expression that are limited to VTA DA neurons. We found higher levels of nuclear, but not somatic HDAC2 immunoreactivity in TH-positive cells of MD compared to non-MD rats at three AP levels within the VTA (−5.4, −5.7 and −6 mm caudal to bregma, Paxinos and Watson, 2007, Figure 1A, n=6–7 per group, Somatic HDAC2: F(1,33)=0.2709, p=0.6062; Nuclear HDAC2: F(1,33)=8.107, p= 0.0075, two-way ANOVA). To test whether increased HDAC2 expression translates to histone hypoacetylation that is normally associated with transcriptional repression of genes, we performed Western blot assays of VTA homogenates isolated from non-MD and MD rats to quantify acetylated histone H3 (histone H3 acetylation at lysine 9 using an antibody against Ac-H3K9). Consistently, MD-induced increases in HDAC2 expression was associated with significant reduction of Ac-H3K9 (Figure 1B, n=6 per group, t(10)=2.588, p=0.0135, unpaired Student’s t test). We also confirmed that the total level of histone 3 did not change following MD (n=3 per group, non-MD: 1.054 ± 0.1534; MD: 0.9884 ± 0.06021, t(4)=0.4005, p=0.7093, unpaired Student’s t test).

Figure 1. MD increased HDAC2 expression in VTA DA neurons and decreased histone acetylation at H3K9 in the VTA.

Figure 1.

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(A) Examples of brain sections stained with antibodies to TH (green) and HDAC2 (red), and DAPI (blue) with the merged panels, which show the expression of HDAC2 in TH+ neurons in the VTA of non-MD (left column) and MD (right column) rats. Scale bar, 20μm. Figure also shows the averaged levels of nuclear and somatic HDAC2 expression at three AP levels from non-MD and MD rats. (B) Representative Western blots and quantitative data of total levels of Ac-H3K9 and β-actin (control) in VTA homogenates from non-MD and MD rats. In this and all subsequent figures * denotes statistical significance and average graphs show means ± SEM from biological replicates or rats for each condition. Also in all western blots, fold-change quantification was pre-normalized to endogenous control.

MD increased synaptic levels of AKAP150 protein with significant alterations in synaptic expression of PKA but not CaN.

MD is associated with higher levels of AKAP150 immunoreactivity in VTA DA cells following MD (Authement et al., 2015), however it is unknown whether the levels of AKAP150 are increased at synapses. To test this, we used biochemical fractionation to examine the synaptic levels of AKAP150, postsynaptic density protein 95 (PSD95, that is shown to colocalize with AKAP150 in complexes with AMPA receptors, AMPARs, at glutamatergic synapses), PKA regulatory subunit IIβ (PKA—RIIβ) and CaN A subunit levels in synaptosomal membrane fractions (LP1) from VTA tissue extracts. We found that MD was associated with higher synaptic levels of AKAP150 and a significant decrease in the synaptic levels of PKA-RIIβ (the total levels of PKA-RIIβ were unchanged). No significant changes were detected in the total or synaptic levels of the CaN A subunit in fractions of VTA tissue extracts from MD rats compared to those from non-MD rats (Figure 2 A-C, AKAP-LP1: n=4 per group, t(6)=2.837, p=0.0148; total PKA-RIIβ: n=8 per group, t(14)=1.538, p=0.0732; PKA-RIIβ LP1: n=4 per group, t(6)=2.094, p=0.0406; total CaN A: n=8 per group, t(14)=0.03334, p=0.4869, CaN A LP1: n=3–4 per group, t(5)=0.4414, p=0.3387, unpaired Student’s t test). We also did not detect any significant change in levels of PSD95 in synaptosomal membrane fractions following MD (Figure 2A, n=4 per group, t(6)=0.0866, p=0.9338, unpaired Student’s t test). Quantitative in situ hybridization also revealed that MD significantly increased Akap5 mRNA expression in the VTA at three AP levels within the VTA (−5.4, −5.7 and −6 mm caudal to bregma, Paxinos and Watson, 2007, Figure 3, n=6–7 per group, F(1,33)= 13.38, p= 0.0009, two-way ANOVA).

Figure 2. MD increased AKAP150 abundance and decreased PKA abundance in synaptic fractions of the VTA.

Figure 2.

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(A) Representative Western blots and quantitative data of AKAP150, PSD95 (postsynaptic marker) and β actin (control) in synaptosomal membrane fractions (LP1) of VTA homogenates from non-MD and MD rats. (B) Representative Western blots and quantitative data of synaptic (LP1) and total levels of PKA-RIIβ in LP1 fractions or total homogenates of VTA from non-MD and MD rats. (C) Representative Western blots and quantitative data of synaptic (LP1) and total levels of CaN A subunit in LP1 fractions or total homogenates of VTA from non-MD and MD rats.

Figure 3. MD was associated with higher levels of Akap5 mRNA in the VTA.

Figure 3.

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Relative expression of Akap5 mRNA with representative images in the VTA from non-MD and MD rats is shown.

HDAC inhibition reversed histone hypoacetylation in the VTA.

To determine whether decreases in Ac-H3K9 levels were reversible by a single in vivo i.p. injection of CI-994 similar to in vitro application of CI-994 (Authement et al., 2016), we performed Western blots of VTA tissue extracts from non-MD and MD rats with i.p. injection of either vehicle or CI-994. We found that the abundance of Ac-H3K9 was significantly increased in VTA tissues from MD rats treated with CI-994 at 3h and 24h post-injection compared to those from MD rats injected with vehicle. Consistently, histone hypoacetylation was still detectible in Western blots of VTA tissue extracts from vehicle-treated MD rats compared to those from vehicle-treated non-MD rats (Figure 4A-C, 4C represents the quantification of the 24h-post injection Western blot data: n=11–12 per group, F(1,27)=8.563, p=0.0069, two-way ANOVA).

Figure 4: MD-induced histone hypoacetylation at H3K9 was reversible by HDAC inhibition for 24h after the injection with CI-994.

Figure 4:

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(A) Representative Western blots of Ac-H3K9 and β actin (control) in VTA homogenates 3h after the injection of non-MD and MD rats with either vehicle or CI-994 (i.p. injection of 10mg/kg). (B and C) Representative Western blots and quantitative data of Ac-H3K9 and β actin in VTA homogenates from non-MD and MD rats 24h after the injection with either vehicle or CI-994.

In vivo HDAC inhibition reversed MD-induced AKAP150 upregulation in VTA DA neurons.

Given that MD triggered reversible changes in histone acetylation for 24h post-injection, we next tested whether MD-induced changes in AKAP150 expression were also normalized at 24h after HDAC inhibitor injection. We performed a double-immunofluorescence staining technique using antibodies against TH and AKAP150 allowing us to visualize DA neurons expressing AKAP150 from non-MD and MD rats that were injected with either vehicle or CI-994 and then sacrificed 24h after the injection. Consistent with our previous result (Authement et al., 2015), we detected higher levels of AKAP150 immunoreactivity in TH+ cells of MD+vehicle compared to non-MD+vehicle rats within the VTA. Moreover, we found that AKAP150 expression in TH+ neurons was returned to normal levels in MD rats injected with CI-994 compared to those from MD rats injected with vehicle (Figure 5, n=7–8 per group, F(1,27)=9.902, P= 0.004, two-way ANOVA). Neither MD nor CI-994 altered the levels of expression of TH in the same sections (data not shown).

Figure 5: MD-induced upregulation of AKAP 150 was reversible by in vivo HDAC inhibition.

Figure 5:

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Top: Examples of brain sections stained with antibodies to TH (red), and AKAP150 (green) with the merged panels, which show the expression of AKAP150 in TH+ neurons in the VTA of non-MD and MD rats injected with either vehicle or CI-994 (i.p. injection of 10mg/kg) at 24h post-injection. Scale bar, 20μm. Bottom: Graph shows the averaged levels of AKAP150 expression (pooled and averaged data at three AP levels for each group) from non-MD and MD rats in each group.

HDAC inhibition reversed MD-induced decreases in the levels of mBDNF protein in the VTA.

Consistently, we observed that the levels of mBDNF (mature BDNF cleaved from pro-BDNF which preferentially binds and signals through TrkB) in Western blots of VTA tissue extracts from MD rats were lower than controls. Moreover, we found that MD-induced decreases in mBDNF were reversible 24h after a single injection of CI-994 (Figure 6, A: n=5 per group, t(8)=2.55, p=0.0171, unpaired Student’s t test, B: represents the quantification of the 24h-post injection Western blot data: n=7–13 per group, F(1,34)=3.028, p=0.0427, two-way ANOVA).

Figure 6: MD-induced decreases in BDNF were reversible by HDAC inhibition for 24h after the injection with CI-994.

Figure 6:

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(A) Representative Western blots and quantitative data of mBDNF and β actin (control) in VTA homogenates of non-MD and MD rats. (B) Representative Western blots and quantitative data of mBDNF and β actin in VTA homogenates from non-MD and MD rats 24h after the injection with either vehicle or CI-994 (10mg/kg, i.p.).

Conclusions

Early childhood adversities are associated with impaired DA function and signaling which underlie the increased risks of developing drug addiction and other stress-related disorders. The epigenetics of early life stress in relation to DA dysfunction and later-life health outcomes is of great interest considering that DA cell responses to early life stress can be epigenetically modulated during selective and sensitive windows of development. Epigenetic changes significantly impact the expression of genes controlling VTA DA neuronal function and signaling, which subsequently alter motivation and reward-related behaviors (Klengel and Binder, 2015; Palmisano and Pandey, 2017). Previously, we found that MD triggered an AKAP-dependent metaplasticity at GABAergic synapses onto VTA DA neurons in response to spike-timing-dependent plasticity (STDP) protocols that rendered GABAergic synapses more susceptible to LTD (Authement et al., 2015). These GABAergic synaptic modifications were reversed to normal STDP by short-term in vitro HDAC inhibition using a pan-HDAC inhibitor (sodium butyrate) or a selective class I HDAC inhibitor (CI-994) (Figure 7). Indeed, adverse experiences in early life, such as MD, are associated with long-lasting changes in the expression of critical synaptic plasticity-associated genes through chromatin remodeling (Levine et al., 2012; Palmisano and Pandey, 2017; Pena et al., 2017; Rodenas-Ruano et al., 2012; Roth et al., 2009; Tesone-Coelho et al., 2013; Zhang et al., 2010). Based on this and our finding of normalization of STDP by acute in vitro HDAC inhibition, we hypothesized that MD-induced modifications in histone acetylation and HDAC2 expression in the VTA mediated MD-induced alterations in GABAergic STDP and AKAP signaling (Authement et al., 2015). Here, we demonstrate that epigenetic mechanisms involved in MD-induced changes in VTA DA function may include HDAC2-mediated decreases in levels of histone acetylation at H3K9 in the VTA which could be restored to normal levels as soon as 3h and up to 24h after a single in vivo injection of MD animals with a selective class I HDAC inhibitor, CI-994. We also found that MD increased the expression of the scaffold Akap5 gene (elevated Akap5 mRNA levels) which translated to increases in synaptic levels of AKAP150 protein, suggesting MD-induced HDAC-independent transcriptional activation of this gene in the VTA. This increase in synaptic levels of AKA150 by MD was associated with lower synaptic levels of PKA-RIIβ suggesting an MD-induced reduction in postsynaptic PKA-RII localization and probably reduced availability of AKAP150-anchored PKA at the synapse. This was opposite to our expectation as there is a positive correlation between synaptic AKAP and PKA expression. On the other hand, total and synaptic levels of CaN A, as well as synaptic levels of PSD95 in the VTA, were unaltered by MD. Consistent with this, recently generated AKAP150-deficient mice that lack PKA anchoring to AKAP150 (by deletion of ten amino acids within the PKA-RII subunit binding near the AKAP C terminus, the AKAP150ΔPKA knockin mice) have reduced PKA-RII levels in hippocampal PSD fractions and AKAP150-anchored PKA signaling. This mutation specifically reduces postsynaptic PKA-RII localization without alterations in the expression levels and localization of other AKAP-associated proteins including CaN A, GluA1 subunit of AMPARs and PSD-95 in the hippocampal synapses (Sanderson et al., 2016). In the light of our earlier findings of the functional impairment of AKAP signaling and induction of GABAergic metaplasticity towards a calcineurin-dependent LTD, we suggest a possible dysfunctional association between AKAP150 and PKA affecting proper anchoring of PKA at the synapse which could result in the less availability of AKAP150-anchored pool of PKA at GABAergic synapses. The impaired association of AKAP150 and PKA may also lead to a biased association of AKAP150 with CaN and AKAP targeting of CaN to GABAARs to promote CaN-dependent GABAergic LTD (MD-induced GABAergic metaplasticity as previously reported by our team)(Authement et al., 2015). Indeed, AKAP150 interaction with CaN is found to be necessary for hippocampal LTD (Jurado et al., 2011; Sanderson et al., 2016). Interestingly, PKA anchoring to AKAP150 also plays a critical role in induction of hippocampal LTD (Lu et al., 2008; Sanderson et al., 2016). In a recent elegant study using knockin mice that were deficient in AKAP-anchoring of either PKA or CaN (AKAP150ΔPKA knockin mice and AKAP150ΔPIX mice), AKAP150-anchored PKA was shown to transiently augment NMDAR Ca2+ signaling during NMDAR-dependent hippocampal LTD by recruiting calcium-permeable AMPARs (CP-AMPARs). Once LTD was induced, AKAP150-anchored CaN rapidly removed CP-AMPARs from hippocampal synapses (Sanderson et al., 2016). Whether recruitment and insertion of CP-AMPARs by the PKA-AKAP complex play a transient role in boosting Ca2+ signaling during induction of NMDAR-dependent GABAergic STDP, and or in induction of MD-induced GABAergic metaplasticity are open questions that merit further investigation.

Since AKAP150 was upregulated by MD, we hypothesized that the rescue of normal AKAP150 signaling in MD rats by HDAC inhibitors may occur through the reversal of HDAC2-mediated transcriptional changes in the expression of AKAP150-interacting proteins (such as PKA) or signaling molecules upstream to AKAP150 that are necessary for proper functioning, interaction or assembly of AKAP150-anchored complexes of signaling molecules together with their targets at the synapse.

BDNF signaling through its receptor tyrosine receptor kinase B (TrkB) affects neuronal excitability, synaptic transmission and synaptic and structural plasticity. In addition, its expression is regulated by neuronal activity (Jia et al., 2008; Russo et al., 2009). Upregulation of BDNF expression is a common finding after the administration of many drugs of abuse in the mesolimbic system particularly during prolonged drug withdrawal (Kauer and Malenka, 2007; Thomas and Malenka, 2003; Vargas-Perez et al., 2014; Vargas-Perez et al., 2009). In contrast, a counteractive and opposite role for BDNF is also proposed where suppression of BDNF signaling in the VTA promotes the rewarding effects of opioids (Koo et al., 2012). Early life stresses can induce long-lasting alterations in BDNF signaling through epigenetic modifications of BDNF expression in the brain. Both increases and decreases of BDNF gene expression in different brain regions through such mechanisms have been shown following early life adversity (Daskalakis et al., 2015; Roth et al., 2009) so we hypothesized that HDAC-mediated transcriptional repression of genes important for synaptic plasticity, such as BDNF, in the VTA might occur in MD rats. We also found that MD decreased the levels of mBDNF protein in the VTA and this decrease was reversed by in vivo HDAC inhibition. Recent evidence also emphasizes the transcriptional regulation of the Bdnf gene through histone modifications including Ac-H3K9 that also involves HDAC2 (Chen and Chen, 2017; Chen et al., 2003; Wang et al., 2014). Therefore, we assume that MD increased-HDAC2 expression could lead to transcriptional repression of Bdnf gene through increased HDAC2 occupancy at Bdnf promoters and histone H3K9 deacetylation in the promoter region of specific Bdnf exon/s thus mitigating the synaptic dysfunction induced by MD in the VTA (Figure 7). Activity-dependent changes in synaptic concentrations of AKAP150 is shown through BDNF-TrkB signaling regulation of ubiquitination of AKAP150 (Jia et al., 2008). Although it is possible that decreases in BDNF expression following MD-induced changes in VTA neuronal activity dysregulates the ubiquitin proteasome system to increase synaptic levels of AKAP150, given the complexity of mBDNF signaling and the myriad of actions of mBDNF it is more likely that mBDNF signaling engages signaling molecules and scaffold proteins interacting with AKAP150 to mediate MD-induced metaplasticity in a complex manner. MD also increased Akap5 expression in the VTA; we assume that the increased expression of AKAP150 and altered AKAP150-anchored signaling in VTA DA neurons by MD may also be related to HDAC2-mediated epigenetic modifications leading to downregulation of a transcriptional repressor that prevents transcription of Akap5 gene upon activation. In summary, possible HDAC-mediated epigenetic regulation of BDNF- and AKAP-dependent signaling within VTA highlights the potential power of HDAC inhibitors for prevention of VTA DA dysfunction associated with later-life psychopathology.

Highlights.

  • MD increases HDAC2 while decreases AC-H3K9 levels in the VTA.

  • MD increases Akap5 mRNA levels in the VTA.

  • MD increases synaptic levels of AKAP150 protein in the VTA.

  • MD decreases synaptic levels of PKA and mBDNF levels in VTA.

  • In vivo HDAC inhibition normalizes the levels of mBDNF and AKAP150 proteins at 24h.

Acknowledgements:

The opinions and assertions contained herein are the private opinions of the authors and are not to be construed as official or reflecting the views of the Uniformed Services University of the Health Sciences or the Department of Defense or the Government of the United States. We thank Dr. Frank Shewmaker for advice and technical assistance with ultracentrifugation.

Funding:

This work was supported by the National Institutes of Health (NIH)–National Institute of Drugs of Abuse (NIDA) Grant#R01 DA039533 to FSN. The funding agency did not contribute to writing this article or deciding to submit it.

Abbreviations:

Ac-H3K9

Histone H3 acetylation at lysine 9

AKAP

A-kinase anchoring protein

CaN

Calcineurin

DA

Dopamine

HDAC

Histone deacetylase

LTD

Long-term depression

(mBDNF)

mature brain derived neurotrophic factor

CP-AMPARs

Calcium permeable AMPARs

MD

Maternal deprivation

NAc

Nucleus accumbens

NMDAR

NMDA receptor

non-MD

Non-maternally deprived

PFC

Prefrontal cortex

PKA

Protein kinase A

STDP

Spike-timing-dependent plasticity

RII

Type II regulatory subunit of PKA

VTA

Ventral tegmental area

Footnotes

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Declarations of interest:

none.

References:

  1. Abel T, Zukin RS, 2008. Epigenetic targets of HDAC inhibition in neurodegenerative and psychiatric disorders. Current opinion in pharmacology 8, 57–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adler SM, Schmauss C, 2016. Cognitive deficits triggered by early life stress: The role of histone deacetylase 1. Neurobiol Dis 94, 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Authement ME, Kodangattil JN, Gouty S, Rusnak M, Symes AJ, Cox BM, Nugent FS, 2015. Histone Deacetylase Inhibition Rescues Maternal Deprivation-Induced GABAergic Metaplasticity through Restoration of AKAP Signaling. Neuron 86, 1240–1252. [DOI] [PubMed] [Google Scholar]
  4. Authement ME, Langlois LD, Kassis H, Gouty S, Dacher M, Shepard RD, Cox BM, Nugent FS, 2016. Morphine-induced synaptic plasticity in the VTA is reversed by HDAC inhibition. Journal of neurophysiology 116, 1093–1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen KW, Chen L, 2017. Epigenetic Regulation of BDNF Gene during Development and Diseases. Int J Mol Sci 18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen WG, Chang Q, Lin Y, Meissner A, West AE, Griffith EC, Jaenisch R, Greenberg ME, 2003. Derepression of BDNF transcription involves calcium-dependent phosphorylation of MeCP2. Science (New York, N.Y) 302, 885–889. [DOI] [PubMed] [Google Scholar]
  7. Covington HE 3rd, Maze I, LaPlant QC, Vialou VF, Ohnishi YN, Berton O, Fass DM, Renthal W, Rush AJ 3rd, Wu EY, Ghose S, Krishnan V, Russo SJ, Tamminga C, Haggarty SJ, Nestler EJ, 2009. Antidepressant actions of histone deacetylase inhibitors. J Neurosci 29, 11451–11460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daskalakis NP, De Kloet ER, Yehuda R, Malaspina D, Kranz TM, 2015. Early Life Stress Effects on Glucocorticoid-BDNF Interplay in the Hippocampus. Frontiers in molecular neuroscience 8, 68. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Esseltine JL, Scott JD, 2013. AKAP signaling complexes: pointing towards the next generation of therapeutic targets? Trends Pharmacol Sci 34, 648–655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hallett PJ, Collins TL, Standaert DG, Dunah AW, 2008. Biochemical fractionation of brain tissue for studies of receptor distribution and trafficking. Curr Protoc Neurosci Chapter 1, Unit 1 16. [DOI] [PubMed] [Google Scholar]
  11. Heim C, Binder EB, 2012. Current research trends in early life stress and depression: review of human studies on sensitive periods, gene-environment interactions, and epigenetics. Experimental neurology 233, 102–111. [DOI] [PubMed] [Google Scholar]
  12. Heim C, Nemeroff CB, 2002. Neurobiology of early life stress: clinical studies. Seminars in clinical neuropsychiatry 7, 147–159. [DOI] [PubMed] [Google Scholar]
  13. Jia JM, Chen Q, Zhou Y, Miao S, Zheng J, Zhang C, Xiong ZQ, 2008. Brain-derived neurotrophic factor-tropomyosin-related kinase B signaling contributes to activity-dependent changes in synaptic proteins. J Biol Chem 283, 21242–21250. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jurado S, Biou V, Malenka RC, 2011. A calcineurin/AKAP complex is required for NMDA receptor-dependent long-term depression. Nature neuroscience 13, 1053–1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kauer JA, Malenka RC, 2007. Synaptic plasticity and addiction. Nat Rev Neurosci 8, 844–858. [DOI] [PubMed] [Google Scholar]
  16. Klengel T, Binder EB, 2015. Epigenetics of Stress-Related Psychiatric Disorders and Gene x Environment Interactions. Neuron 86, 1343–1357. [DOI] [PubMed] [Google Scholar]
  17. Koo JW, Mazei-Robison MS, Chaudhury D, Juarez B, LaPlant Q, Ferguson D, Feng J, Sun H, Scobie KN, Damez-Werno D, Crumiller M, Ohnishi YN, Ohnishi YH, Mouzon E, Dietz DM, Lobo MK, Neve RL, Russo SJ, Han MH, Nestler EJ, 2012. BDNF is a negative modulator of morphine action. Science (New York, N.Y) 338, 124–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Langlois LD, Nugent FS, 2017. Opiates and Plasticity in the Ventral Tegmental Area. ACS Chem Neurosci 8, 1830–1838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Levine A, Worrell TR, Zimnisky R, Schmauss C, 2012. Early life stress triggers sustained changes in histone deacetylase expression and histone H4 modifications that alter responsiveness to adolescent antidepressant treatment. Neurobiol Dis 45, 488–498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lu Y, Zhang M, Lim IA, Hall DD, Allen M, Medvedeva Y, McKnight GS, Usachev YM, Hell JW, 2008. AKAP150-anchored PKA activity is important for LTD during its induction phase. The Journal of physiology 586, 4155–4164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Palmisano M, Pandey SC, 2017. Epigenetic mechanisms of alcoholism and stress-related disorders. Alcohol 60, 7–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pena CJ, Kronman HG, Walker DM, Cates HM, Bagot RC, Purushothaman I, Issler O, Loh YE, Leong T, Kiraly DD, Goodman E, Neve RL, Shen L, Nestler EJ, 2017. Early life stress confers lifelong stress susceptibility in mice via ventral tegmental area OTX2. Science (New York, N.Y) 356, 1185–1188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Penney J, Tsai LH, 2014. Histone deacetylases in memory and cognition. Science signaling 7, re12. [DOI] [PubMed] [Google Scholar]
  24. Rodenas-Ruano A, Chavez AE, Cossio MJ, Castillo PE, Zukin RS, 2012. REST-dependent epigenetic remodeling promotes the developmental switch in synaptic NMDA receptors. Nature neuroscience 15, 1382–1390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Roth TL, Lubin FD, Funk AJ, Sweatt JD, 2009. Lasting epigenetic influence of early-life adversity on the BDNF gene. Biological psychiatry 65, 760–769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Russo SJ, Mazei-Robison MS, Ables JL, Nestler EJ, 2009. Neurotrophic factors and structural plasticity in addiction. Neuropharmacology 56 Suppl 1, 73–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sanderson JL, Gorski JA, Dell’Acqua ML, 2016. NMDA Receptor-Dependent LTD Requires Transient Synaptic Incorporation of Ca(2)(+)-Permeable AMPARs Mediated by AKAP150-Anchored PKA and Calcineurin. Neuron 89, 1000–1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sinha R, 2008. Chronic stress, drug use, and vulnerability to addiction. Annals of the New York Academy of Sciences 1141, 105–130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Strathearn L, 2011. Maternal neglect: oxytocin, dopamine and the neurobiology of attachment. J Neuroendocrinol 23, 1054–1065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tesone-Coelho C, Morel LJ, Bhatt J, Estevez L, Naudon L, Giros B, Zwiller J, Dauge V, 2013. Vulnerability to opiate intake in maternally deprived rats: implication of MeCP2 and of histone acetylation. Addict Biol. [DOI] [PubMed] [Google Scholar]
  31. Tesone-Coelho C, Morel LJ, Bhatt J, Estevez L, Naudon L, Giros B, Zwiller J, Dauge V, 2015. Vulnerability to opiate intake in maternally deprived rats: implication of MeCP2 and of histone acetylation. Addict Biol 20, 120–131. [DOI] [PubMed] [Google Scholar]
  32. Thomas MJ, Malenka RC, 2003. Synaptic plasticity in the mesolimbic dopamine system. Philosophical transactions of the Royal Society of London 358, 815–819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vargas-Perez H, Bahi A, Bufalino MR, Ting AKR, Maal-Bared G, Lam J, Fahmy A, Clarke L, Blanchard JK, Larsen BR, Steffensen S, Dreyer JL, van der Kooy D, 2014. BDNF Signaling in the VTA Links the Drug-Dependent State to Drug Withdrawal Aversions. J Neurosci 34, 7899–7909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vargas-Perez H, Kee RT, Walton CH, Hansen DM, Razavi R, Clarke L, Bufalino MR, Allison DW, Steffensen SC, van der Kooy D, 2009. Ventral tegmental area BDNF induces an opiate-dependent-like reward state in naive rats. Science (New York, N.Y) 324, 1732–1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wang BY, Zhong Y, Zhao Z, Miao Y, 2014. Epigenetic suppression of hippocampal BDNF mediates the memory deficiency induced by amyloid fibrils. Pharmacology, biochemistry, and behavior 126, 83–89. [DOI] [PubMed] [Google Scholar]
  36. Wild AR, Dell’Acqua ML, 2017. Potential for therapeutic targeting of AKAP signaling complexes in nervous system disorders. Pharmacol Ther. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Woolfrey KM, Dell’Acqua ML, 2015. Coordination of Protein Phosphorylation and Dephosphorylation in Synaptic Plasticity. J Biol Chem 290, 28604–28612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xie L, Korkmaz KS, Braun K, Bock J, 2013. Early life stress-induced histone acetylations correlate with activation of the synaptic plasticity genes Arc and Egr1 in the mouse hippocampus. Journal of neurochemistry 125, 457–464. [DOI] [PubMed] [Google Scholar]
  39. Zhang TY, Hellstrom IC, Bagot RC, Wen X, Diorio J, Meaney MJ, 2010. Maternal care and DNA methylation of a glutamic acid decarboxylase 1 promoter in rat hippocampus. J Neurosci 30, 13130–13137. [DOI] [PMC free article] [PubMed] [Google Scholar]

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