Animals and housing

All of the procedures used in this study were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care, the institutional animal welfare committee, in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals. Adult male C57black6 mice (Taconic Farm, Germantown, NY) (n ≥ 3 litters at a time), 2 to 3 months of age and weighing 23 ± 2 g, or transgenic mice expressing green fluorescent protein (GFP) directed by Fos promoter [B5;DBA-Tg(Fos-tPA, fos-EGFP*)1Mmay Tg(terO-lacZ, tPA*)1Mmay/J] (Jackson Lab, ME) were kept in cages with sawdust bedding, in a room with controlled light cycles (12 h light/12 h dark). All animals had free access to water, and were fed standard lab chow. Mice were trained, operated on, and tested in a random manner; a blinded observer performed the behavioral testing. All contrast agents and aptamers were delivered using BBB bypass to mice with icv puncture [23, 24], through which the distribution of agents has been shown to be more uniform than by icv alone.

For each series of MRI experiments, we started with eight mice (n = 4 each for acute and chronic paradigms, Fig. 1). Mice in each paradigm received amphetamine or saline (n = 2 each); we repeated the series of experiments until we had gathered data on the appropriate number of animals, as determined by power analysis. We used a double-blind design for all experiments, in which sample preparation and data acquisition were blinded, and the samples were given coded identifiers. Following MRI and photography the coded datasets were delivered to and decoded by the Principal Investigator.

Fig. 1 Panel a Summary of amphetamine sensitization using chronic exposures to amphetamine or saline with abstinence. Panel b Summary of protocol for MRI acquisition and amphetamine (acute or challenge) administrations Full size image

Amphetamine exposure paradigms

For the chronic exposure paradigm, eight age-matched, amphetamine-naïve, male C57black6 mice received a single dose of amphetamine in their home cage every other day, for a total of seven injections of amphetamine (4 mg/kg, by injection intraperitoneally [i.p.] A7) [18, 25–28]; this was followed by 2 weeks with no drug exposure (abstinence, A7W in Fig. 1a). A final dose of amphetamine or saline was given on the day of post-SPION MRI, such that we could compare the effect of a challenge dose of amphetamine following chronic exposure and abstinence in our A7WA group to control groups without challenge dose (A7WS or S7WS). For the acute exposure paradigm (A1), age-matched, amphetamine-naïve, male C57black6 mice received a single dose of amphetamine (4 mg/kg, i.p.); the control group received a single dose of saline (S1, vehicle, 10 ml/kg, i.p.).

Immunohistochemistry of total and phosphorylated HDAC5 antigens in amphetamine exposure paradigms

For ex vivo assays we examined two C57black mice in the A7W group, as well as four normal naïve mice with saline or acute amphetamine exposure (n = 4 each); these six mice received no icv puncture, nor were they given contrast agent. We administered saline (100 μl, i.p.) or amphetamine (4 mg per kg, i.p.) 3 h before the mice (n ≥ 2, each group) were put under general anesthesia and retrograde-perfused with ice-cold saline. Isolating brain tissue as described previously [21], we stained slices of brain tissue (25 μm in thickness) with total or phosphorylated HDAC5 antigens with ab1439 (Abcam) and ab192339 (phosphor S259, detects HDAC5 phosphorylated at Serine 259), respectively. We then co-stained Cy2-labeled rabbit polyclonal IgG against glial fibrillary acidic protein (GFAP, Z0334, Dako) or Cy3-labeled polyclonal IgG against ionized calcium-binding adaptor molecule 1 (IBA1, ab5076, Abcam); we used Cy2-labeled monoclonal IgG against NeuN (A60, Millipore) or Cy3-labeled goat IgG against GFAP (clone C-19, Santa Cruz) for progenitor cells. Nucleic acids were stained with DAPI (1:500 dilution) [18]. To examine HDAC5 mRNA expression using Cy3-sODN (see below), transgenic mice (n = 3) underwent icv puncture 1 week before they were administered Cy3-sODN (120 pmol in 0.1 ml, i.p.) and a dose of amphetamine, as in the acute paradigm (Fig. 1b). All histological images were acquired using the same exposure time and gain, using a Retiga EXi camera on an Olympus microscope and cellSens Dimension software (Olympus America Corp, Nashua, NH).

Biotinylated sODN for HDAC5 transcripts

We designed two sODNs with antisense sequence to HDAC5 mRNA; for all sODNs we used nucleotide BLAST to validate target mRNA with potential binding (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences were obtained from GenBank (AF207748): sODN of HDAC5 mRNA or miR-2861 binding site (hdac5) = 5′-aggctgagaggcaggccctt-3′ (forward to 1188–1169); miD2861 = 5′ aagggcctgcctctcagcct-3′ (antisense to 1188–1169) and its upstream primer (USP)1 = 5′-acggctttactggctcagtc-3′ (forward to 971–980); HDAC5AS2 = 5′-atctcattccacaccgtgtc-3′ (antisense to 2341–2360) and its USP2 = 5′- tcaaggatgaggatggcgag-3′ (forward to 1781–1800); passenger of matured miR-2861 = cuccggcucccccuggccucccgg (passenger); hairpin and miR-2861 = gaacuacaagucccagggggccuggcggcgggcggag (premiR-2861); antisense to premir-2861 = ccctgggacttgtagttc (mpremiR-2861a). All primers were phosphorothioated by replacing non-bridging oxygen with sulfur on the phosphate linkages. We attached one biotin to the 3′ termini. We also synthesized sODNs for pre-miR-2861 and matured miR-2861 [15]. We stored all of these sODNs in aliquots of 0.05 ml (0.1 μM), at −20 °C.

Target binding and cellular retention

We mixed Cy3-miD2861 with one sODN of four different sequences (the passenger of miR-2861, matured miR-2861, Cy3-miD2861 and cDNA of HDAC5 mRNA at the miR-2861 binding site) at a 1:1 ratio (100 pmol each in 50 μl) at room temperature. For ex vivo hybridization, we heated the mixture at 65 °C for 5 min, then slowly cooled it on a thermocycler (at a rate of 1° drop per minute) to 20 °C, where it was maintained for 30 min. We resolved the hybrids in agarose gel (1.5%) using gel electrophoresis, and obtained photographs at 495 nm/521 nm (excitation and emission spectrum peak wavelengths) using an Imager FluorChem Q (Alpha Innotech, CA). To test binding ability in vivo, we transfected Cy2-miD2861 (30 pmol) to PC-12 cells in a 35 mm (dia) culture dish on a microscope stage incubator chamber with humidified air comtaining 5% CO2, at 37 °C (model INU-UK-F1; Tokai Hit, Shizuoka, Japan) as described [29]. We washed and changed DNA-free medium 3 h later, then transfected Cy3-sODNhdac5 to both plates. We acquired live cell images at 18 and 42 min and 2 h after Cy3-sODNhdac5 using automatic time-lapse photography with constant exposure time and image gain (CellSense Imaging Software, Olumpus). Representative cell images were processed using Adobe Photoshop CS2 (Adobe Systems, San Jose, CA).

Modular contrast agent using SPION-NA

We synthesized NeutrAvidin (NA)-labeled Molday Ion (CL-30Q02-2, BIOPAL, Worcester MA) using the protocol previously published [20]. This commercially available Molday Ion contains 6 k–9 k molecules of iron oxide (dextran-coated superparamagnetic iron oxide nanoparticles, or SPION): it has a unique Zeta potential (−5 mV) with an effective size of 25 nm (dia) and relaxivities of R1 = 15.4 and R2 = 33.9 s−1mM-1. Briefly. we conjugated SPION-NA with biotinylated aptamer (3 nmol biotinylated sODN per mg SPION-NA) by mixing. The resulting SPION-sODN remained at 4 °C for 16 h before it was administered to animals. Before delivery we added 1 μg of lipofectamine (Lipofectamine 2000, Invitrogen) to the mix.

Intracerebroventricular (icv) puncture to create a BBB-bypass port

One week before SPION-sODN or sODN delivery we anesthetized the mice with pure O 2 plus 2% halothane at a flow rate of 800 ml/min. We performed icv puncture (LR −1; AP −0.4 and DV: −3 mm, bregma) using a 28G needle to create a BBB-bypass port, and afterward sealed the skull with bone wax, then sutured and disinfected the wound (Povidone-Iodine, Medline Ind, Mundelein, IL). The BBB remained open for approximately 21 days after the icv procedure; we utilized this 21-day window to deliver NPs or sODN by injection intraperitoneally (i.p.) [23, 24]. On the day of MRI, we acquired baseline MRI in a group of four mice (30 min each). We eliminated any mice that exhibited baseline R2* values more than one standard deviation of the average R2* values in normal brains (n >500) we had examined in previous studies (stratification). However, we note that the need to eliminate animals based on this criterion is rare.

Dynamics of SPION-sODN uptake

Before SPION delivery, we acquired baseline T2*-weighted (T2*W) MRI scans (30 min each scan) on four mice identified individually as mice A, B, C, and D, and immediately afterward delivered SPION-hdac5AS2 or SPION-miD2861 (4 mg Fe or 12 nmol sODN per kg, i.p.) (Fig. 1b). Each mouse remained in awake in its home cage. We acquired MRI at 2-h intervals following SPION-sODN from mice A to D, discontinuing MRI acquisition at 6 h. We repeated the evaluation until we had gathered data from enough mice (n = 4 at each time point, or as determined by power analysis).

Cy3-sODN-hdac5AS2 uptake using in vivo hybridization

We examined the distribution of Cy3-sODN-hdac5AS2 using in vivo hybridization and ex vivo assay [23]. We delivered Cy3-sODN-hdac5AS2 (12 nmol per kg, i.p) to transgenic mice with icv port, and 3 h later administered amphetamine (4 mg per kg, i.p.). Four hours after amphetamine administration we obtained brain samples and froze them by slow cooling with liquid nitrogen. After staining slices of brain tissue (25 μm in thickness) with 0.5% Hoechst for nucleic acid, we obtained photographs using the same technique and equipment described above [18].

Molecular contrast-enhanced (MCE) MRI in vivo

We acquired background T2*W MRI and delivered SPION-hdac5AS2 or SPION-miD2861 to four mice, as shown in Fig. 1b; 3 h later we injected amphetamine (4 mg per kg) or saline (100 μl, i.p.). We acquired MRI 3 h after amphetamine, and statistically analyzed SPION retention in various regions of interest (ROIs). We used the mean and SD from the first pair (n = 2) in each paradigm to compute the sample size needed to avoid type II error for SPION-actin uptake at each time point. This MRI acquisition was repeated in enough mice to achieve adequate sample size, according to power analysis (see Statistical analysis below).

We performed MCE MRI using a 9.4 T MRI system (Bruker Avance system, Bruker Biospin MRI, Inc., Billerica, MA). We measured R2* changes before and after NP delivery, and evaluated SPION-labeled gene expression in the R2* maps acquired using multi-echo gradient echo sequences; TR 800 ms, six echoes (TE = 1.94, 3.41, 4.88, 6.35, 7.82, 9.29 ms) with spatial resolution of 0.1 mm × 0.1 mm × 0.25 mm. We followed the same protocol described in our previous publications [21] for stratification before MCE MRI, data acquisition, data analysis and examinations of within- and between-litter differences.

MCE MRI data analysis

We have used subtraction R2* maps of the chronic paradigm and acute paradigm and found ROI with neurogliosis based on elevated GFAP mRNA expression [29]. Because R2* values above baseline are positively proportional to iron concentration [22], where R2* is the rate of signal reduction (R2* = 1/T2*, s−1), we compared the R2* maps from all T2*W MRI scans in the regions contralateral to the hemisphere with icv port. We aligned T2*W MRI using the “jip analysis toolkit” (URL: http://www.nmr.mgh.harvard.edu/~jbm/jip/). Any R2* values greater than three standard error of the mean (SEM) of the average pre-SPION R2* values were considered significantly different from the background. We computed the ΔR2* as the increase in R2* SPION-sODN brain above the background: i.e., R2* post SPION-sODN - (R2* baseline + R2* 3 SEM of baseline ), or as a percent elevation above the baseline (ΔR2*/average baseline R2*) ×100%. Any R2* values above the baseline R2* were shown in the color scale.

Contrast-to-noise ratio (CNR)

The noise in the ROI comes from the background before contrast agent delivery. CNR is defined as the ratio of the difference between two image signals to the square root of the standard deviation of the background noise. For our purposes, baseline R2* maps showing endogenous iron levels serve as ‘background,’ and their standard deviations are the ‘noise’ to R2* maps of brains containing SPION-sODN. Therefore, we defined the CNR representative of SPION-sODN uptake in each ROI, and at any given time point, as the change in contrast, i.e., ΔR2* divided by noise (the square root of the standard deviation of R2* within the same ROI in baseline brains).

Validation of SPION-sODN delivery using transmission electron microscopy (TEM)

We collected tissue samples immediately after MRI; the left NAc of S1 and A1 mice was immersed in 2.5% PBS-buffered glutaldehyde at 4 °C and sent to the TEM laboratory of the Histology Core Facility at the MGH Center for Systems Biology for preparation and double-blinded examination. After tissue was dehydrated in ascending concentrations of ethanol, immersed in propylene oxide, and embedded in Epon 812 resin (Agar Scientific Ltd., Standstead, England), samples were cut into ultrathin sections (~60 nm). The Core prepared tissue with and without standard TEM stain using osmium tetroxide (1%, 2 h), uranyl acetate (Ua, 2%, 5 min) and Reynold’s lead citrate [20]. We found that standard staining masked NP identification; we modified TEM staining by omitting all stains, unless indicated, to reduce the background of membrane structure and enable visualization of SPION. The neuronal nucleus was identified as a smooth, round nuclear body with diameter of ~7 μm. We defined microglia (MG) by the presence of irregular euchromatic nucleus, a peripheral rim of heterochromatin, and various empty and partially filled lysosomes/exosomes (Ly/Ex).

Ex vivo RT-qPCR methods

For TaqMan® analysis we extracted the total RNA from the brain tissue of each mouse using the RNeasy Lipid Tissue Mini Kit (Qiagen), which supplied all required buffers. For RT-qPCR, we obtained total RNA from striatal or hippocampal tissue from three groups of mice that were administered saline (S), or acute (A1) or chronic paradigms of amphetamine. The total RNA from each mouse was reverse-transcribed using oligo (dT) 25 , and the SuperScript III First-Strand Synthesis System (Invitrogen Life Technologies, Carlsbad, CA, CA). The initial RNA concentration in each sample was determined by OD260 reading and converted to the total amount of RNA. Preparation of striatal tissue from one side of each mouse brain yielded 2.8 ± 0.9 μg total RNA in 40 μl solution. From each sample, we used 280 ng, or 4 μl total RNA for cDNA synthesis in a 20 μl total volume of buffer solution; 1 μl of this solution was used for qPCR. The qPCR was performed using a TaqMan® probe-based assay (Applied Biosystems) for fosB (Assay ID: Mm00500401_m1); beta Actin (Assay ID: Mm02619580_g1) served as the internal control. We carried out relative quantification of the mRNA amount using standard SDS software, which is based on ΔΔCt models [30]. For FosB, HDAC5, and GFAP mRNA in normal brains, we measured copy number using an internal control of Actin mRNA. We did not calculate the copy number of HDAC5 mRNA in amphetamine exposure paradigms because the exposure to amphetamine might damage the brain and alter the copy number of the internal control.

Locomotor assessments

We measured locomotor behavior according to published drug sensitization protocols [18, 25–28]. To measure horizontal locomotion and fine motor activity, we used an automated recording device (San Diego Instrument, San Diego, CA) located in the same room in which the animals were individually housed. The system has eight chambers, each of which is composed of frames equipped with five infrared photocell beams (spaced 5 cm apart) in one polypropylene cage (15 × 25 cm). The photocell beams traverse each cage in a plane above the floor. We recorded the frequency of locomotion (ambulation) as the number of sequential breaks in two adjacent beams, and measured fine motor activities (such as grooming or other stereotyped motions) by counting the number of sequential breaks in a single beam. Recordings were made every minute for at least 60 min. We reported the distance traveled as the product of 5 cm and the summation of frequencies of beam break during the time interval.

Mice were individually housed and tested in their own home cages. We pre-conditioned the mice by removing each mouse from and returning it to its cage daily for 5 days prior to behavior assessment. To examine the effect of HDAC5 knockdown, we pretreated mice with a dose of miD2861 or placebo (sODN with random sequence, or sODN-Ran) at 1.2 mmol/kg (i.p./icv) 3 h before administering amphetamine to naïve (A1) mice or mice that had been previously exposed to one dose of amphetamine (A2) or A7W), as previously described [2, 18]. We performed locomotor assessment immediately, as described above. We obtained data from twice the number of mice calculated by power analysis; the results were compared using two-way analysis of variance.

Statistical analysis

Once we had obtained the first MRI dataset, we calculated the number of animals needed in each group to achieve at least 85% power for an α value of 0.05, to avoid type II error (a post hoc power analysis). We computed the mean and SEM from the average values in each group of animals, and compared the statistical significance of these values using a t test (two tail, type II or equal variant) or two-way ANOVA (GraphPad Prism IV, GraphPad Software, Inc., San Diego, CA). A p value of ≤ 0.05 was statistically significant [18].