Maternal Separation And Handling Procedures

Published Date: 02 Nov 2017

All experiments were performed in accordance with EU guidelines (directive 86/609/EEC) on the ethical use of animals using the experimental protocol approved by the Ethics committee of the University of Tartu Medical Faculty. Male C57BL/6 mice, 4-5 months old, weight 25–30g were obtained from Scanbur BK, Sweden (Papers I, II) and 20-25 pairs of male and female Wistar rats were obtained from Harlan Laboratories, Netherlands (Paper III). All animals were housed in standard polypropylene cages under temperature and humidity-controlled rooms with 12 h light–dark cycle (light from 7:00 a.m.) and were allowed access to rodent chow and water ad libitum. Animals were allowed to acclimate to laboratory conditions and were handled at least 4-7 days before use in behavioural testing. All behavioural experiments were conducted during the light phase in an isolated experimental room

2. Maternal separation and handling procedures (Paper III)

As the pups were born (total 46, from 3-5 litters) female offspring were removed and male offspring were randomly assigned into one of three groups. During the procedures every dam had 8 pups. The experimental groups are as follows: (1) animal facility reared (AFR), the mothers and pups were handled only on postnatal day 10 (PND10) for a cage change and not separated; (2) handled group (MS15), pups were handled and separated to new cage on PND 2-15 for 15 minutes, then returned to home cage; (3) maternally-separated group (MS180), pups were removed on PND 2-15 to 180 minutes from the home cage and placed individually in a new cage. At the end of separation, pups were placed back in their home cage, where the mother was. The manipulation of the pups in the MS15 and MS180 was initiated at 10:00 a.m. and after the manipulation, we monitored mothers and pups behaviour in home cage for 10 min. We carried out 7 separation procedures to collect pups from different dams for behavioural and neurochemical tests. We consider AFR as control group for both handling (MS15) and separation (MS180) groups. A comparison of MS15 and MS180 groups will provide ‘‘time course’’ data for maternal separation. Cocaine-induced locomotor activity was evaluated on PND 130-140. Animals were allowed to acclimate to laboratory conditions and were handled at least 7 days before use in behavioural experiments. For the evaluation of neurochemical changes, rats were sacrificed on PND15 and PND 120.

3. Drug administration (Papers I-III)

Cocaine hydrochloride (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in sterile saline (0.9% NaCl) and administered intraperitoneally (i.p.) in the dose of 15 mg/kg (Paper I) and in the dose of 10 mg/kg (i.p.) (Papers II, III) before locomotor activity training. For experiments investigating the effect of inhibition of DNMTs, zebularine (Tocris Bioscience, UK) was dissolved in sterile saline and administered intracerebroventricularly (icv) 300 ng/0.5 l 20 min before cocaine or saline (0.1 ml/10 g body weight, ip) treatment (Paper I). SAM (Sigma-Aldrich Co, St. Louis, MO, USA) was dissolved in sterile saline and administered (i.p.) in the dose of 4520 mg/kg, 10 mM/kg 20 minutes prior to cocaine hydrochloride treatment (Paper II).

3.1. Intracerebroventricular cannula implantation (Paper I)

For implantation of the intracerebroventricular (icv) injection cannula, mice were anesthetized with Hypnorm (VetaPharma, Leeds, UK) and Dormicum (Roche, Basel, Switzerland). During surgery, each mouse was implanted with a 26-gauge bilateral stainless steel guide cannula (Plastics One, Roankoke, USA) from which the injector extended 0.5 mm to end in the ventricle. Stereotaxic coordinates used were as follows: antero-posterior, -0.4 mm from bregma, -1.25 mm lateral from the midline, and -2.0 mm from bregma measured from the tip of the cannula guide (Paxinos and Franklin, 2001). Animals were habituated to dummy cannula removal and given 4 days of recovery and handling before the start of the experimental procedure. At the beginning of the study, target coordinates and proper cannula placement were verified by slowly injecting 1 μl of methylene blue dye into the cannulas of the first four mice, while they were under anaesthesia. After waiting 5 min for the dye to circulate through the ventricles, the animals were sacrificed. Brains were removed, sliced at the point of cannula entry and target coordinates were verified.

4. Behavioral experiments

4.1. Locomotor activity measurement (Papers I-III)

Horizontal locomotor activity was assessed in standard polypropylene cages (36 x 20 x 15 cm) uniformly illuminated with dim lighting. The light-sensitive video camera, connected to the computer, was mounted about 1.5 m above the observation cage and locomotor activity of 8 animals in time was monitored and analysed using VideoMot2 software (TSE Systems, Germany) (Papers I, II). In Paper I, for experiments investigating the effect of acute (AC) and repeated cocaine (RC) treatment, mice (n = 11) were randomly assigned to the following treatment groups: (1) "SAL", mice were treated for 7 days with saline 0.1 ml/10 g body weigh (i.p.); (2) "AC", mice were treated for 6 days with saline and on the 7th day with cocaine hydrochloride; (3) "RC", mice were treated for 7 days with cocaine hydrochloride. On the 1st and 7th treatment days, locomotor activity was recorded for 60 min after the last injection. On the 2nd through to the 6th treatment days, mice were injected and placed in the test cages for 60 min without locomotion recording. To investigate the effect of zebularine, intracerebroventricular (icv) infusions of zebularine (300 ng/0.5 l) or saline (0.5 l) were performed 20 min before cocaine or saline (0.1 ml/10 g body weight, ip) treatment. Mice (n = 7-12) were randomly assigned to the following treatment groups: (1) saline (icv) + saline (ip) "S+S"; (2) zebularine (icv) + saline (ip) "Z+S"; (3) saline (icv) + cocaine (ip) "S + C" and (4) zebularine (icv) + cocaine (ip) "Z + C". Locomotor activity was recorded for 60 min after i.p. injection, daily for 7 days.

In Paper II, mice were treated for 7 days intraperitoneally (i.p.) with sterile saline (0.1 mL/10 g body weight) or SAM (4520 mg/kg, 10 mM/kg, Sigma-Aldrich Co, St. Louis, MO, USA) 20 minutes prior to cocaine hydrochloride administration (10 mg/kg, i.p., Oriola Oy, Espoo, Finland). Animals (n = 17-22) were randomly assigned into one of the following treatment groups: (1) Saline + Saline "S+S"; (2) SAM + Saline "M+S"; (3) Saline + Cocaine "S+C" and (4) SAM + Cocaine "M+C". To reduce interindividual variability, mice (4 mice in S+C and 5 mice in M+C groups) whose cocaine-induced ambulation was less than 2000 cm/90 minutes on the first day were excluded. Locomotor activity was recorded for 90 minutes after the second injection on days 1, 3, 5, and 7, and analyzed using VideoMot2 software (TSE Systems, Bad Homburg, Germany). On the days 14 and 28, all groups were tested for locomotor activity for 90 minutes after cocaine challenge (7 mg/kg, i.p., n = 8). On the days 8 to 13 and 15 to 27 mice did not receive any treatment. Mice were sacrificed 24 h after the end of the repeated treatment.

In Paper III, cocaine hydrochloride administered (i.p.) immediately before locomotor activity training. Locomotor activity was monitored in standard polypropylene cages (54 x 32 x 20 cm) uniformly illuminated with dim lighting. The light-sensitive video camera, connected to the computer, was mounted about 1.5 m above the observation cage and locomotor activity of 4 animals in time was monitored and analysed using VideoMot2 software. In experiments of AC treatment, adult male rats (PND 130-140) from AFR, MS15 and MS180 groups (n = 13 animals per group; weight 485-520g) were treated for 4 days with saline (2.5 ml/kg, i.p.) and on the 5th day with cocaine hydrochloride. On the 1st, 3rd and 5th treatment days, locomotor activity was recorded for 60 min after the treatment. On the 2nd and 4th treatment days, rats were treated and placed in the test cages for 60 min without locomotion recording.

4.2. Exploration box test (Paper III)

At PND 120, adult males from AFR, MS15 and MS180 groups (n = 8 animals in each group) were tested for exploratory behaviour. The test apparatus (modified from Matto et al., 1996 and Mällo et al., 2007) was made of metal and consisted of an open area 50 (width) × 100 (length) × 40 (height) cm with a small compartment (20×20×20 cm) attached to one of the shorter sides of the open area. The open area was divided into eight squares of equal size (25×25 cm) and four objects were situated in certain positions. Three objects were unfamiliar (a glass jar, a cardboard box, a wooden handle) and one familiar (food pellet), which remained the same throughout the experiment. The floor of the small compartment was covered with wood shavings and was directly linked to the open area through an opening (size 20 cm × 20 cm). The rat was placed into the small compartment, which was then covered with a lid and during the 15 min test session the following measures were taken by the observer: (a) latency of entering the open area with all four paws on it; (b) entries into the open area; (c) line crossings; (d) rearings; (e) exploration of the three unfamiliar objects in the open area and (f) the time spent exploring the open area. After each animal, the exploration box test was cleaned with dampened laboratory tissue. To provide an index of exploration (considering both the elements of inquisitive and inspective exploration), the scores of line crossing, rearing and object investigation were summed for each animal.

5. Tissue isolation (Papers I-III)

Mice and rats (on PND15 and PND120) were sacrified with decapitation. The NAc, hippocampus, cerebellum and PFC were rapidly dissected and immediately frozen in liquid nitrogen and stored at -80 °C until RNA and/or DNA extracts were prepared. Dissection of the NAc was performed using a round-shape puncher (Papers I-III).

6. PC12 cells and DNMT activity measurement (Paper II)

Rat pheochromocytoma cells (PC12) purchased from German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) were cultured in a humidified 5% CO2/95% air atmosphere at 37 C. To differentiate cells, RPMI 1640 medium (Gibco CO, USA) was supplemented with 10% heat-inactivated horse serum and 5% fetal bovine serum (Gibco CO, USA) on polyethyleneimine (Sigma-Aldrich, St. Louis, MO, USA) precoated plastic dishes (Nunc, Thermo Fisher Scientific, MA, USA) at a density of 106 cells/ml (2 ml of cell suspension per dish). The cells from the 4th to 7th passages were treated with 0.5 mM SAM and used for the Dnmt activity, gene expression, and DNA methylation experiments. For single SAM treatment, 0.5 mM SAM solution (prepared in RPMI medium) was added to growing cells at time points 0, 2, 6, 12 and 24 hours. For repeated SAM experiment, 0.5 mM SAM was added to the cells once a day for 7 days and nuclear extract was prepared 0, 2, 6, 12 and 24 hours after last treatment. Control measurements were performed with vehicle (RPMI medium) treated cells. Nuclear extract was isolated from cells using EpiQuik Nuclear Extraction kit (Epigentek Group, Brooklyn, USA). Total Dnmt activity was determined using an EpiQuik Dnmt activity assay kit (Epigentek Group, Brooklyn, USA). Dnmt activity (OD/h/mg) was calculated according to the formula: (sample OD − blank OD)/(protein amount × hour)×1000. Two dishes combined as a sample, 4 samples per group were used. Experiments were repeated twice.

7. Primary culture of rat cortical neurons and drug exposure (Paper III)

Primary cultures of rat cortical neurons were prepared from newborn (1st postnatal day) Wistar rats. Briefly, cortices were dissected in ice-cold Krebs-Ringer solution (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 0.4 mM K2HPO2, 15 mM glucose, 20 mM HEPES, pH 7.4) containing 0.3% bovine serum albumin (BSA) and trypsinized in 0.8% trypsin for 10 min at 37 °C. This was followed by trituration in a 0.008% DNase solution containing 0.05% soybean trypsin inhibitor. Neurons were resuspended in basal medium Eagle with Earle’s salts (Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum, 25 mM KCl, 2 mM glutamine, and 100 µg/ml gentamicin and plated onto 35-mm plastic dishes (Nunc, Thermo Fisher Scientific, MA, USA) precoated with poly-L-lysine at a density of 106 cells/ml (2 ml of cell suspension per dish). Three hours later, medium was changed to NeurobasalTM-A medium containing B-27 supplement, 2 mM GlutaMAXTM-I, and 100 µg/ml gentamicin. The cells were cultured in a humidified 5% CO2/95% air atmosphere at 37 C for 6 days before starting with the corticosterone (Sigma-Aldrich, St. Louis, MO) and mifepristone (Sigma-Aldrich) treatment.

Corticosterone (1 mM) and mifepristone (0.4 mM) stock solutions were prepared by dissolving them in dimethyl sulfoxide (DMSO) followed by dilution in Neurobasal medium. Drug exposures were started at day 6 in culture. Corticosterone 1, 5, 10 and 50 µM (final concentration in the medium) alone or in combination (50 μM corticosterone) with mifepristone 20 µM (final concentration in the medium) were added to the cell cultures and cells were incubated for 3 or 24 h. Cells were harvested after washing with fresh, ice-cold phosphate buffered saline (PBS) and immediately lysed to obtain total RNA or protein. Two dishes were combined as a sample, 6 samples per group were used. Experiments were repeated twice.

8. Gene expression analyses (Papers I-III)

8.1. Measuring mRNA levels by real-time PCR (Papers I-III)

Total RNA was extracted from the mouse and/or rat NAc, hippocampus, cerebellum, prefrontal cortex (PFC) and from cells using RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Oligo-dT first-strand cDNA was synthesized from 400 ng or 750 ng of total RNA using First Strand cDNA Synthesis Kit (Fermentas Inc, Burlington, Canada). Real-time PCR was performed using an ABI PRISM 7000 Sequence Detection System equipped with ABI Prism 7000 SDS Software (Applied Biosystems Inc, USA).

Primers for mouse and/or for rat DNMT1, DNMT3A, DNMT3B, A2AR, fosB, Reln were designed using Primer3 with BLAST sequence verification. Primers were synthesised by TAG Copenhagen AS (Copenhagen, Denmark) and were listed in table 1 (primer sequences table). Commercial assays were from SABiosciences (Qiagen, Hilden, Germany) for measuring mouse PP1c (Cat. No. PPM37272B), Cck (Cat. No. PPM24836G), Gal (Cat. No. PPM25148F), Slc17a7 (Cat. No. PPM35361A) and rat PP1c (Cat. No. PPR42515B) mRNA expression. PCR amplification was performed in a total reaction volume of 25 l in three parallels. The reaction mixture consisted of 1 l First Strand cDNA diluted template, 12.5 l 2x Master SYBR Green RT-PCR Master Mix (Applied Biosystems Inc, USA), 10.5 l H20 and 1 l gene-specific 10 M PCR primer pair stock. Amplification specificity was controlled by a melting curve analysis and a gel electrophoresis of the PCR product. Serial dilutions (5-fold) from one wild type sample total RNA were analysed for each target gene and allowed to construct linear standard curves from which the concentrations of the test sample and efficiency of PCR reaction were calculated. Results were normalized to -actin or GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) using the CT method (Livak and Schmittgen, 2001).

8.2. Gene expression profiling (Paper II)

Total RNA was extracted from the mouse NAc as previously described. Two tissues were combined to a sample, 4 samples per group used. RNA quantity and quality were assessed using the NanoDrop-1000 spectrophotometer and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Labeled cRNA was prepared using Illumina TotalPrep RNA amplification Kit according to the manufacturer’s instructions using 750 ng of total RNA as a template (Ambion Inc, Austin, TX, USA). The Illumina BeadChip platform (Illumina, San Diego, USA) and the corresponding whole-genome Mouse Ref-8 v2.0 BeadChip (approximately 25,698 transcripts; over 19,100 genes) were used for the gene expression analysis. The raw data was analyzed with Illumina BeadStudio Gene Expression Module v3.3.7 (Illumina, San Diego, USA). Further data analysis was performed with R version 2.13.0 (http://www.r-project.org)/Bioconductor software (www.bioconductor.com) using lumi (Du et al., 2008) and limma packages (Smyth, 2005). The 'fdr' method to adjust the p values for multiple testing was used to control the false discovery rate (Benjamini and Hochberg, 1995). With a statistical discrimination p value set at less than 0.05, limma software and B-statistics analyses were used to identify up- and downregulated genes and filtered for 1.5-fold or greater differences in expression in accordance with standards for microarray analysis (Allison et al., 2006). Gene ontology analysis was conducted using DAVID Bioinformatics Resources (Huang et al., 2009). Microarray data were submitted in the Gene Expression Omnibus (GEO) repository (http://www.ncbi.nlm.nih.gov/geo/).

9. DNA Methylation Assays

9.1. Methylated DNA Immunoprecipitation (Papers I-III)

Methylated DNA immunoprecipitation (MeDIP) was performed using EpiQuik Methylated DNA (Paper III) and Tissue Methylated DNA Immunoprecipitation kit (Epigentek Group Inc, USA) according to the manufacturer protocol. Genomic DNA was extracted from the frozen mouse and/or from the rat (PND15 and PND120) NAc, cerebellum, PFC and sonicated into fragments ranging in size from 200-1000 bp and divided into immunoprecipitated (IP) and input (IN) portions. IP DNA incubated with anti-5-methylcytosine monoclonal antibody to bind methylated DNA, negative control was normal mouse IgG from manufacturers EpiQuik MeDIP kit. Methylated DNA (750 ng) was subjected to quantitative real-time PCR using commercial assays from SABiosciences (Qiagen, Hilden, Germany) for mouse DNMT3A (Cat. No. EPMM102350-1A), DNMT3B (Cat. No. EPMM106719-1A), PP1c (Cat. No. EPMM108835-1A), fosB (Cat. No. EPMM109802-1A), Cck (Cat. No. EPMM111951-1A), Gal (Cat. No. EPMM105518-1A), Slc17a7 (Cat. No. EPMM110039-1A) (Papers I, II) and for rat DNMT3A (Cat. No. EPRN107117-1A), PP1c (Cat. No. EPRN102434-1A), A2AR (Cat. No. EPMM100909-1A) and Reln (Cat. No. EPRN105980-1A) (Paper III). To evaluate the relative enrichment of target sequences after MeDIP, the ratios of the signals in the IP DNA vs IN DNA was calculated. The resulting values were standardized against the unmethylated control sequence GADPH and fold changes were calculated.

9.2. Methylation-specific real-time PCR analysis (Paper I, III)

DNA was isolated from the mouse and rat NAc using QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany) and processed for bisulphite modification using Epitect Bisulfite Kit (QIAGEN, Hilden, Germany). Quantitative real-time PCR was used to determine the DNA methylation status of the mouse and rat PP1c and of the mouse fosB genes. Methylation-specific real-time PCR primers were designed using Methprimer software (www.urogene.org/methprimer). Methylation-specific and unmethylated PCR primers were designed to target putative CpG islands detected in silico in promoter or non-promoter regions of the PP1c and fosB genes and were synthesised by TAG Copenhagen AS (Copenhagen, Denmark). Primer sequences are listed in table 1. PCR reactions were performed as described earlier. Ct values were chosen within the linear range and the comparative Ct method was used to calculate differences in methylation between samples.

10. Global DNA methylation analysis (Paper III)

Total DNA was isolated from the rat NAc and PFC using QIAmp DNA Mini Kit (QIAGEN, Hilden, Germany). Global DNA methylation was performed using a MethylFlash Methylated DNA Quantification kit (Epigentek Group Inc., Farmingdale, NY, USA) according to the manufacturer instructions. Briefly, sample DNA (100 ng) was bound to high DNA affinity strip wells. The methylated fraction of DNA is detected using capture and detection antibodies and then quantified colorimetrical by reading the absorbance at 450 nm using a Tecan Sunrise microplate reader (Tecan Group Ltd., Switzerland). The amount of methylated DNA is proportional to the measured OD intensity. The percentage of methylated DNA (5-mC) in total DNA was quantified according to the manufacturer’s protocol and formula.

11. Chromatin immunoprecipitation assay (Paper I)

Chromatin immunoprecipitation (ChIP) of genomic DNA associated with a methyl CpG binding protein 2 (MeCP2) was carried out according to the manufacturers protocol (Millipore Inc, USA). Mouse NAc was minced to small pieces and cross-linked in 1% formaldehyde (10 l/mg) for 15 min at 37 C. Adding glycine stopped the cross-linking reaction and the tissue was washed twice in ice-cold PBS containing a protease inhibitor cocktail. The minced, fixed tissue was homogenized in SDS lysis buffer and the homogenate was sonicated to produce 200-1000 bp genomic fragments. Following this the resulting homogenate was centrifuged for 15 min at 13,000 x g and the supernatant was used for ChIP assay. Immunoprecipitations were carried out at 4 C overnight with 5 g of rabbit polyclonal MeCP2 (Abcam plc, Cambridge, UK), negative control was anti-Rat IgG (Vector Laboratories Inc, Burlingname, USA) and positive control was anti-RNA Polymerase II from manufacturers EZ-ChIP kit. A portion of the sonicated DNA was left untreated to serve as input control. Immune complexes were collected with protein A beads and, according to the manufacturers protocol, sequentially washed two times with low salt buffer, high salt buffer, LiCl immune complex buffer and TE buffer. Immunoprecipitated DNA was subjected to quantitative real-time PCR using ChIP commercial assays from SABiosciences for mouse PP1c (Cat. No. GPM1037458(-)01A) and fosB (Cat. No. GPM1052791(-)01A) mRNA expression.

12. Western immunoblotting (Papers I, III)

The NAc was lysed in 10 vol (w/vol) of RIP-A lyses buffer: 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X 100 and protease inhibitors and homogenized with ultrasound homogenizer, incubated 1 h at +4 °C and centrifuged 13,000 x g for 10 min at 4 °C (Paper I). In paper II, NAc was lysed in 10 vol RIP-A lysis buffer, which consisted of 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA containing protease and phosphatase inhibitors, homogenised manually, incubated for 20 min on ice and centrifuged (13000 g for 20 min at 4 ºC). The supernatants were resolved by electrophoresis on 8% SDS-polyacrylamide gel. Proteins were transferred onto Hybond-P PVDF Transfer Membranes (Amersham Biosciences, UK) in 0.1 M Tris-base, 0.192 M glycine and 20% (w/w) methanol using an electrophoretic transfer system. The membranes were blocked with 0.1% (w/w) Tween 20/TBS (T-TBS) containing 5% (w/w) non-fat dried milk at room temperature for 1 h. After blocking the membranes were incubated overnight with one of the following antibodies: the anti-PP11 polyclonal antibody (1:1,000, Millipore, Temecula, CA, USA) followed by incubation with biotinylated anti-rabbit IgM secondary antibody (1:1,000) for 2 h and ABC solution for 30 min (Paper I); chicken anti-DNMT3A (1:1000; ab14291; AbCam, USA) followed by incubation with secondary antibody anti-chicken HRP conjugate (1:2000; Pierce, US). The membranes were incubated with ECL detection reagent (ECL, Amersham, UK) for 5 min to visualise proteins, and then exposed to autoradiography X-ray film (Amersham hyperfilm ECL, UK). To normalise immunoreactivity of the proteins, the β-actin protein was measured on the same blot with a mouse monoclonal anti-β-actin antibody (1:10000; Sigma, St. Louis, US) followed by anti-mouse HRP secondary antibody (1:2000; Pierce, US). The blots probed for proteins of interest were densitometrically analysed using a QuantityOne 710 System (BioRad). The ratio of proteins of interest and β-actin were calculated and expressed as mean OD ratio in arbitrary units ± SEM.

13. Statistical analysis (Papers I-III)

Behavioral data of all experiments were analysed using one-way or two-way ANOVA for repeated measures, followed by Bonferroni’s post-test, as appropriate. Expression level of genes, protein levels, global DNA methylation, MeDIP and ChIP data were analysed using one-way ANOVA, followed Bonferroni’s post-test or t-test. Differences in methylation-specific real-time PCR data were analysed by two-way ANOVA with Bonferroni’s post-test. GraphPad Prism software (GraphPad, San Diego, CA, USA) was used for statistical analysis of the data and all data expressed as mean±SEM, significance levels were set at p < 0.05.