Epigenetics of amphetamine-induced sensitization: HDAC5 expression and microRNA in neural remodeling
Liu and Liu Journal of Biomedical Science
Epigenetics of amphetamine-induced sensitization: HDAC5 expression and microRNA in neural remodeling
Philip K. Liu 0 1
Christina H. Liu 0 1
0 Department of Radiology, Molecular Contrast-Enhanced MRI Laboratory at the Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital and the Harvard Medical School , CNY149 (2301) Thirteenth Street, Charlestown, MA 02129 , USA
1 Authors' information Philip Liu, PhD Associate Professor of Radiology, Director of Molecular Contrast- Enhance MRI Laboratory at Athinoula A. Martinos Center for Biomedical Imaging
Background: Histone deacetylase (HDAC) activities modify chromatin structure and play a role in learning and memory during developmental processes. Studies of adult mice suggest HDACs are involved in neural network remodeling in brain repair, but its function in drug addiction is less understood. We aimed to examine in vivo HDAC5 expression in a preclinical model of amphetamine-induced sensitization (AIS) of behavior. We generated specific contrast agents to measure HDAC5 levels by in vivo molecular contrast-enhanced (MCE) magnetic resonance imaging (MRI) in amphetamine-naïve mice as well as in mice with AIS. To validate the MRI results we used ex vivo methods including in situ hybridization, RT-PCR, immunohistochemistry, and transmision electron microscopy. Methods: We compared the expression of HDAC5 mRNA in an acute exposure paradigm (in which animals experienced a single drug exposure [A1]) and in a chronic-abstinence-challenge paradigm (in which animals were exposed to the drug once every other day for seven doses, then underwent 2 weeks of abstinence followed by a challenge dose [A7WA]). Control groups for each of these exposure paradigms were given saline. To delineate how HDAC5 expression was related to AIS, we compared the expression of HDAC5 mRNA at sequences where no known microRNA (miR) binds (hdac5AS2) and at sequences where miR-2861 is known to bind (miD2861). We synthesized and labeled phosphorothioated oligonucleic acids (sODN) of hdac5AS2 or miD2861 linked to superparamagentic iron oxide nanoparticles (SPION), and generated HDAC5specific contrast agents (30 ± 20 nm, diameter) for MCE MRI; the same sequences were used for primers for TaqMan® analysis (RT-qPCR) in ex vivo validation. In addition, we used subtraction R2* maps to identify regional HDAC5 expression. (Continued on next page)
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Results: Naïve C57black6 mice that experience acute exposure to amphetamine (4 mg/kg, by injection intraperitoneally)
show expression of both total and phosphorylated (S259) HDAC5 antigens in GFAP+ and GFAP− cells, but the appearance
of these cells was attenuated in the chronic paradigm. We found that MCE MRI reports HDAC5 mRNA with precision in
physiological conditions because the HDAC5 mRNA copy number reported by TaqMan analysis was positively correlated
(with a linear coefficient of 1.0) to the ΔR2* values (the frequency of signal reduction above background, 1/s) measured
by MRI. We observed SPION-mid2861 as electron dense nanoparticles (EDNs) of less than 30 nm in the nucleus of
the neurons, macrophages, and microglia, but not in glia and endothelia. We found no preferential distribution in
any particular type of neural cells, but observed scattered EDNs of 60–150 nm (dia) in lysosomes. In the acute
paradigm, mice pretreated with miD2861 (1.2 mmol/kg, i.p./icv) exhibited AIS similar to that exibited by mice in
the chronic exposure group, which exhibited null response to mid2861 pretreatment. Moreover, SPION-miD2861
identified enhanced HDAC5 expression in the lateral septum and the striatum after amphetamine, where we
found neurprogenitor cells coexpressing NeuN and GFAP.
Conclusions: We conclude that miD2681 targets HDAC5 mRNA with precision similar to that of RT-PCR. Our MCE
MRI detects RNA-bound nanoparticles (NPs) in vivo, and ex vivo validation methods confirm that EDNs do not
accumulate in any particular cell type. As HDAC5 expression may help nullify AIS and identify progenitor cells, the
precise delivery of miD2861 may serve as a vehicle for monitoring network remodeling with target specificity and
signal sensitivity after drug exposure that identifies brain repair processes in adult animals.
Amphetamine-type stimulants are associated with
aggressive behavior as well as behaviors like those
characteristically seen in schizophrenia and bipolar
syndrome in humans . One of the pathways that
rewards these behavioral manifestations involves the
brain’s ventral tegmental area (VTA), the origin of the
dopaminergic cell bodies of the mesocorticolimbic
dopaminergic pathway . GABAergic neurons in the
VTA send projections to brain regions including the
nucleus accumbens (NAc), striatum (Cpu), and
prefrontal cortex (PFC) in the forebrain, and to the
hippocampus through the lateral septum (LS). The LS
lies below the rostrum of the corpus callosum; neurons
of the LS receive reciprocal connections from the olfactory
bulb, hippocampus, amygdala, hypothalamus, midbrain,
habenula, cingulate gyrus and thalamus . The LS is
considered a pleasure/fear zone in animals, and plays a role
in reward and reinforcement along with the NAc [4–6].
Epigenetic regulatory events have been shown to mediate
the lasting effects of drugs of abuse; one possible means of
such epigenetic regulation is chromatin remodeling via
histone modifications. Acetylation by histone acelytransferases
(HATs) promotes transcriptional activity by relaxing
chromatin through the insertion of an acetyl group to the lysine
residue of histone. However, HDACs catalyze deacetylation
of acetylated chromatin, removing the modification by
HATs in chromatin. One of the class IIa HDACs that have
cell-type-restricted patterns of expression, HDAC5
associates with HDAC3 in vivo to shuttle between the nucleus
and cytoplasm [7, 8]. HDAC5 is known to be involved in
learning and memory function, as well as playing a role in
axon regeneration in repair [9–11]. Other reports have
suggested, as observed in various models of addictive drugs,
that reduced HDAC activity may be involved in neural
plasticity [12–14]. However, the mechanism by which HDAC5
may act in neural repair in adults remains unclear.
Recent studies have suggested that HDAC5 activity
may be regulated by miR-2861 [15, 16]. Endogenous
miRNAs are noncoding RNAs that bind to specific gene
transcripts, modulate gene expression and play key roles
in the regulation of developmental processes . The
possible associations between miR-2861 and HDAC5
expression prompted us to investigate the role of
endogenous miR-2861 and HDAC5 in the context of
Our goal is to investigate how AIS is associated with
endogenous miR-2861 and HDAC5 expression after chronic
amphetamine exposure. We have developed NPs that
specifically target two segments of HDAC5 mRNA: miD2861
and hdac5AS2. We conjugated SPION (core diameter
5–10 nm, hydrodynamic diameter 30 ± 20 nm) via
Schiffbase reduction with biotinylated miD2861 or hdac5AS2
. Upon binding to gene transcripts, these NPs
produced target-specific signal reduction in T2*-weighted
MR images [19–21], which we converted to R2* (1/s) for
quantitation. As R2* values above background (ΔR2*) are
positively proportional to iron concentration , our data
demonstrate that together SPION-miD2861 and MCE
MRI measure HDAC5 mRNA in vivo with accuracy
similar to that of reverse transcription PCR. Using
noninvasive MRI in a preclinical mouse model of chronic
amphetamine exposure, we demonstrated that
SPIONmiD2861 monitors regional HDAC5 expression in the LS,
where we found endogenous progenitor cells. These
results suggest miD2861 may monitor neural repair and
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.
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
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
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
previously , 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
Cy2labeled 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) . 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′-atctcattccacaccg
tgtc-3′ (antisense to 2341–2360) and its USP2 = 5′- tcaagga
tgaggatggcgag-3′ (forward to 1781–1800); passenger of
matured miR-2861 = cuccggcucccccuggccucccgg (passenger);
hairpin and miR-2861 = gaacuacaagucccagggggccuggcggcgg
gcggag (premiR-2861); antisense to premir-2861 = ccctggg
acttgtagttc (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 . 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 . 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 . 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
One week before SPION-sODN or sODN delivery we
anesthetized the mice with pure O2 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
SPIONmiD2861 (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 . 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 .
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
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
multiecho 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  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 . Because
R2* values above baseline are positively proportional to iron
concentration , 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
SPIONsODN. 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%
PBSbuffered 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
doubleblinded 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 . 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 . 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.
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.
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 .
We compared total and phosphorylated HDAC5 antigens
[ab1439 and ab192339, respectively] in the NAc of mice
that experienced either acute or chronic amphetamine
exposure (Fig. 1). As Fig. 2a shows, there was some
expression of total HDAC5 antigen around microvessels in
saline-treated mice (GFAP−/HDAC5+, arrowhead); we
observed spotty areas of HDAC5 antigen in non-astroglia
(GFAP−/HDAC5+), which showed no blending yellow
stains (arrows). Figure 2b shows near null expression of
S259-HDAC5 antigen in the NAc without amphetamine.
Detailed photographs can be found in supplemental figures
(Additional file 1 and Additional file 2: Figure S1). After
one exposure to amphetamine (A1) we observed both
antigens of total (red, GFAP−/HDAC5+, Fig. 2c) and
phosphorylated S259 HDAC5 (blending yellow, GFAP+/HDAC5+,
Fig 2d, arrows) in neural cells. In addition, phosphorylated
HDAC5 was visible in the soma and axons of GFAP+ cells
(Additional file 3: Figure S2A & S2B). On the other
hand, both total and phosphorylated S259 HDAC5
antigens were scarce after chronic amphetamine
exposure (Fig. 2e, f; Additional file 4: Figure S3A &
S3B), compared to the acute (A1) paradigm (Fig. 2,
Additional file 3: Figure S2A & S2B). We observed
Fig. 2 Expression of HDAC5 antigens in naïve (saline), acute and chronic exposure to amphetamine. We compared total (cy3-ab1439,
Abcam) or phosphorylated HDAC5 at S259 (cy3-ab192339) in the nucleus accumbens (NAc) of mice that experienced either saline
(a & b), acute amphetamine (c & d) or chronic amphetamine (e & f) exposure. Brain tissues were obtained 1 h after amphetamine or
saline (i.p.) in isopropanol on dry ice. Brain slides were stained with Cy3-IgG then Cy2-gfap (Z0334, Daka), and DAPI for nucleus
that phosphorylated S259 antigens remained in
enlarged nuclei of GFAP+ cells (Fig. 2f, arrows).
We designed three additional primers (miR-2861 binding
site on HDAC5 mRNA, a passenger of matured miR-2861,
and premiR-2861) to demonstrate target specificity
(Fig. 3a). We found that miD2861 hybridized only to the
sequence of HADC5 mRNA but not to other sequences of
passenger or pre-miR-2861 in an ex vivo hybridization test
(Fig. 3b, Lane 4). We further demonstrated that sODNs
with antisense sequences were stable in PC12 cells after
transfection (Fig. 3c). Labeled sODNs with sense sequence
was excluded from PC12 cells within 2 h after transfection
(Fig. 3c). We then investigated precise binding of miD2861
to total cDNA that had been reverse-transcribed from
HDAC5 mRNA; PCR with USP1 and miD2861 primers
amplified only one fragment of 212 basepairs (bp) in
samples from the hippocampus and striatum (Fig. 4a). The
same was true for USP2 and hdac5AS2 (Fig. 3a) for
amplification of cDNA of 580 bp (data not shown). We noted a
slight variation in the intensity of the HDAC5-fragment
that had been amplified from mice in the chronic
paradigm. Therefore, we employed SPION-hdac5AS2 or
SPION-miD2861 to measure the expression of HDAC5
mRNA in vivo.
To determine the best time to obtain optimal ΔR2* after
SPION delivery, we monitored SPION-miD2861 retention
in normal mouse brains (n = 4). Figure 5a shows R2*
above baseline R2* (ΔR2*) at 4 h after delivery. We found
that the average ΔR2* values in the somatosensory cortex
from these mice (Fig. 5b, arrows) reached a plateau at 4 h
and remained elevated at 6 h post-delivery (Fig. 5c). The
CNR in the ROI had an average of 3.2 ± 0.1 from the
beginning of ΔR2* elevation (at 2 h) to the peak ΔR2*
elevation (at 4 & 6 h) after delivery. We found copy
numbers of mRNAs from normal mouse brains correlated with
the peak ΔR2* values of HDAC5, GFAP and FosB [18, 29],
with a linear coefficient (r2) of 1.0 (Fig. 4b). The same was
found for ΔR2* values using SPION-hdac5AS2 in normal
mice (r2 = 0.97). It appears SPION-miD2861 or
SPIONhadc5AS2 reported HDAC5 mRNA of normal mouse
brains with the same mechanism of RT-PCR. The uptake
plateau of 6 h would be used in future MRI (Fig. 1b).
We validated the variations of HDAC5 mRNA in
acute and chronic paradigms using MRI in vivo. We
started with SPION-hdac5AS2, because the HADC5
sequence on the binding site of hdac5AS2 has no
known interference. Figure 6 shows changes in HDAC5
mRNA expression of seven ROIs in both the acute and
chronic paradigms. Compared to the control group
(S1), the A1 group showed no significant differences in
the medial prefrontal cortex (mPFC), NAc, and caudate
putamen (CPu) of the mesolimbic pathway (S1 of Fig. 6a
versus A1 of Fig. 6b). We found that animals in the
chronic paradigm showed higher ΔR2* values in all
ROIs compared to animals in the control and A1
groups, although these values were not significantly
higher except in the NAc and motor cortex (Fig. 6a).
However, the data illustrated in Fig. 6a did not support
Fig. 3 Antisense sODN binds the target sequence. Panel a Relative location of four primers on HDAC5 mRNA. Both miD2861 and hdac5AS2 are in AS
orientation. Panel b Modified gel shift assay. We mixed Rhd-miD2861 with one sODN of four different primers (lane 1: the passenger of miR-2861, lane
2: matured miR-2861, lane 3: Rhd-miD2861, and lane 4: cDNA of HDAC5 mRNA at the miR-2861 binding site [sODNhdac5]). Binding of miD2861 slowed
mobility during gel electrophoresis. Panel c Differential retention assay. We transfected Cy2-AS first for 3 h, washed, then transfected
Rhd-sODN. Eighteen minutes later we washed and changed medium again, then took photography immediately and at 2 h later.
Sense sODN was excluded preferentially at 2 h
those from immunohistochemistry and RT-PCR (Fig. 2c
versus 2E; 4A).
To investigate whether or not sODN was stable in vivo,
we transfected rhd-labeled sODN-hdac5AS2 to transgenic
mice expressing green fluorescent protein (GFP) directed
by Fos promoter (according to the A1 paradigm) to induce
cFos and hDAC5. We found rhd-sODN-hdac5AS2 was
retained in the cytoplasm (GFP+/DAPI−, arrows) and nuclei
(GFP−/DAPI+, arrowhead) or the nuclei of neural cells
expressing GFP in cytoplasm (Fig. 7A–C, GFP+/DAPI+,
broken arrows). We observed several
Cy5.5-SPIONmiD2861 in IBA1+ cells in separate experiments (Fig. 7E).
We employed TEM to resolve the location of
SPIONsODN retention in samples from the NAc.
Upon comparing electron dense nanoparticles (EDN) in
mouse brains with TEM stain we found no significant
difference in the EDNs of normal mice (Additional file 5:
Figure S4A–C) compared to mice that received one
optimal dose of SPION-sODN (Additional file 5: Figure S4E).
Partial TEM stains (uranyl acetate and lead phosphate)
showed pinocytosis of SPION-sODNs (Additional file 5:
Figure S4D). We observed phagocytes with more
EDNfilled Ly/Exs in mice that had been given high doses of iron
(Additional file 5: Figure S4F) compared to mice given no
SPION (Additional file 5: Figure S4A–C) or one optimal
dose of SPION-sODN (Additional file 5: Figure S4E, G and
H). The high doses we tested were 120 μg/kg (3× of
optimal dose, icv delivery), or 4 mg/kg weekly (i.p) for 8
weeks in mice with large BBB opening. Although we could
clearly identify EDNs of 100–500 nm (dia) in Ly/Ex in all
neurons, microglia, and phagocytes of fully stained tissue,
not all EDNs could be discerned; the exception was
cytoplasmic EDNs of 60–150 nm in samples stained only with
uranyl acetate (Additional file 5: Figure S4G1 & H1). It was
Fig. 4 Quantitative validation of HDAC5 mRNA expression using RT-PCR. The frequencies of signal reduction above baseline (ΔR2* values) by MCE MRI
positively correlate with mRNA copy numbers determined by TaqMan® analysis. Panel a miD2861 and upstream primer 1 (UPS1) amplified one single
fragment of 212 bp of HDAC5 cDNA from brains of normal mice (saline, n = 3) and mice in the A7WA group (n = 2). Panel b The mRNA copy numbers of
three genes and the changes in their ΔR2* values measured by MRI show positive correlation. MR data of GFAP and FosB mRNAs were from published
results [18, 29]. HDAC5 data were from Fig. 5c (6 h) and Fig. 6a (S1). Panels c & d Correlations of copy numbers of HDAC5 mRNA and
ΔR2* in three paradigms; MR data from Figs 6a, b and 9a., b. R2 or LCorr are linear correlation
only in unstained tissue that we identified EDN, of ≤ 30 nm
(dia), being transported in the endoplasmic reticulum (ER)
(Additional file 6: Figure S5A1), distributed in the nucleus
of microglia and neurons (Additional file 6: Figure S5B1 &
B2) in the same mice presented in Additional file 5: Figure
S4D & E. Indeed, TEM with no stain revealed
SPIONmiD2861, observed as EDNs, in the nucleus of neurons
(Fig. 7F & F1), macrophages (Fig. 7G & G2), and microglia
(Fig. 7H & H1). We observed several individual EDNs in
Ly/Ex of microglia; there were no EDNs in the glia, either
partially stained or unstained (Additional file 5: Figure S4H
or Fig. 7I). The EDNs near the endothelia, identified by
uranyl stain, were within the Ly/Ex of nearby cells (Fig. 7J
& J1). The control (non-targeting SPION only) shows EDN
scattered in the Ly/Ex (Fig. 7K). Importantly, we have no
evidence showing preferential retention in any one type of
cells when SPION-sODN is administered at the optimal
quantities we used in MEC MRI. We investigated whether
EDN retention was related to HDAC5 mRNA binding. The
correlation (linear coefficiency) of HADC5 mRNA copy
number and ΔR2* values by SPION-hdac5AS2 in the
striatum of mice in the control (S1), acute, and chronic
paradigms was 1.0 (Fig. 4c).
After receiving SPION-miD2861, A1 (n = 5) animals
showed no significant differences in all ROIs of the brain
compared to normal mice that received saline (the S1)
(data not shown). Figure 8a, b show ΔR2* maps in the
control (A7WS) and challenge (A7WA) mice of the
chronic paradigm, respectively. Compared to A7WS
group, mice in the challenge group (A7WA) exhibited
regional reduction in HDAC5 mRNA levels. We
observed significant reductions of HDAC5 mRNA in the
mPFC, Cpu, and MC, but no significant attenuation in
the NAc, Hippo and SSC regions (Fig. 9a). TaqMan®
analysis of HDAC5 mRNA copy number in the striatum
showed a decrease in the chronic exposure paradigm
(Fig. 9b); there was no significant change in the control
Actin mRNA in any of the three conditions (not shown).
The attenuation of HDAC5 mRNA in the striatum was
confirmed by a corresponding decrease in total HDAC5
antigen (Fig. 9c, b).
Having demonstrated that SPION-miD2861 targets and
reports HDAC5 expression, we monitored changes in
behavior after amphetamine. The appearance of AIS in the
A1 control (sODN-Ran + A1) had a delay of onset during
the first 20 min (no significant elevation) then a gradual
increase during the second 20 min after amphetamine
(p < 0.04, activities at 20 min versus 40 min). This delay
was not significantly different from that seen in mice
treated with saline before amphetamine . In the A1
control animals AIS returned toward normal 60 min after
amphetamine (not shown). On the other hand, we
observed no delay to AIS in the chronic paradigm. In the
first 5-min interval the A7WA group showed immediate
Fig. 5 Contrast-to-noise ratio (CNR). Panel a Representative ΔR2* maps of 0.5 mm brain slices from the cerebellum (upper left slice) in percent
increase from baseline (color scale was 0–250%) from one mouse (no amphetamine, SPION-miD2861). ΔR2* Maps [(Post R2*−bR2*)/bR2*) ×100%].
Panel b ROI (arrows) of the somatosensory cortex (SSC, contralateral to icv port) for data analysis [18, 20]. Panel c Elevations of ΔR2* values of the
SSC by longitudinal MRI in vivo; mean ΔR2* values of four mice are shown. Contrast-to-noise ratio was calculated for R2* data at 2, 4, and 6 h.
CNR = (R2* of mean/[SD of baseline]1/2)
AIS, which lasted for at least 60 min. We did not observe
AIS in the A7WS and S7WS control groups (Additional
file 7: Figure S6). The total distance (meters) traveled in
the 40 min after amphetamine was 25 ± 9 for A1 control
mice versus 90 ± 13 for A7WA mice; resulting in p < 0.004
per two-tailed t-test. Figure 10 shows the effect of
sODNmiD2861 pretreatment on AIS during the first 20 min.
Pretreatment of sODN-miD2861 in the A1 paradigm
slightly but non-significantly elevated AIS at earlier time
than A1 control but the attenuation in the delay was not
significantly different (not shown). Pretreatment of
sODN-miD2861 in the A2 paradigm attenuated the delay
from 40 to 15 min. The same pretreatment to the chronic
paradigm did not significantly change already sensitized
locomotor behavior (not shown). Statistical analysis by
two-way ANOVA shows significant difference in AIS by
amphetamine and miD2861 treatments (F = 29.78, df = 3,
p < 0.0001), with significant difference in the time of AIS
delay (F = 2.585, DF = 7, p = 0.043).
Based on subtraction R2* maps that identified
regional repair/remodeling [23, 29, 31], we are able to
also identify changes in HDAC5 expression in the
chronic paradigm. Figure 11a shows R2* elevation in
the LS of three consecutive MR sections (a total of
1.5 mm in the dorsal LS); however, we observed
several ROIs in the NAc/Striatal (arrows). We found the
LS contained progenitor cells that exhibited NeuN+
(neuronal biomarker) and GFAP+ phenotype (Fig. 11b). We also
Fig. 6 Summary of HDAC5 expression. Panels a & b Elevation of R2* values in various ROIs using SPION-hdac5AS2 and MCE MRI from mice of
paradigms in Fig. 1. Panel c ROIs (arrows) of mPFC, NAc, CPu and hippocampus (HP) for data analysis
Fig. 7 Modified in situ hybridization using Rhd-sODNhdeac5AS2 (A–D, A1) and Cy5.5-miD2861 (E–J, S1) or SPION (K) for HDAC5 mRNA expression.
We transfected Rhd-hdac5AS2 (120 pmol) to 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] according to Fig. 1b, except no MR acquisition. After we obtained brain tissues (Fig. 2), frozen slices
were prepared and stained with DAPI only. Panels A–C show NAc cells with HADC5 mRNA by Rhd-sODNhdeac5AS2 (A), GFP (B) and DAPI (C); panel D
shows the merged photographs of A–C. These panels show neural cells of HADC5+ in GFP+/DAPI+ (broken arrows in A–C), HDAC5+ in GFP+ (arrows in
A & B, or yellow in D) and HADC5+ in GFP− (arrowheads in A & C, purple in D). Panel E shows the NAc stained with goat poly-IgG against IBA1
(Ab5076, Abcam) for microglia (MG) in mice after Cy5.5-SPION-miD2861. HDAC5 mRNA is expressed in one of two MG (arrow, E1). Panel F Electron
dense nanoparticles (EDN) of SPION-miD2861 were retained in the nucleus (23 nm, dia) of a neuron (N, F1 arrowhead). Panel G shows
macrophage (M) and endothelium (E); we observed nuclear EDN (17 nm) in mcrophage (arrowheads, G2). Panel H shows two nuclear EDNs
(28 nm each) in a microglia (H1) although other MG and glia (G) do not contain any EDN in the nucleus except in the lysosomes/exosomes
(I, arrows). We observed several EDNs (ranging 20 to 80 nm) in Ly/Ex in close proximity of an endothelium (J1, arrows). Panel K shows EDN
in the control (mice received non-targeting SPION). Bars (microns) = 20 (E), 6 (A–D), 2 (F, G & I), 0.5 (G1, H, J & K) or 0.1 (G2). TEM samples
were without stains (F–I) or stained with uranyl acetate (2%, 5 min, J & K). See also Additional file 2: Figure S1 for our rationale for using
the no stains
found ventral striatum (Cpu) contained few progenitor
cells except on the ventricular wall (Fig. 11b, black/white
panel); limited progenitor cells on microvascular walls in
the NAc/Striatum (arrows, Fig. 11c). Immunohistology of
progenitor cells in panels B & C matched elevated ΔR2*
maps in panel A. In control mouse brains, we found glial
cells do not express NeuN and have long axons
(Fig. 11d). We concluded that SPION-miD2861 could
reliably monitor HDAC5 expression and locate
neuroprogenitors based on HDAC5 expression in preclinical
models of neural network remodeling.
We have reported here that miD2861 targets HDAC5
mRNA in living brains; our data support that a reduction
in HDAC5 expression involves AIS in a preclinical model
of amphetamine exposure. Moreover, SPION-miD2861
and MCE MRI identify regional repair/remodeling in the
network that involves the pleasure, aggression, and reward
pathways in the LS and NAc . These conclusions
come from studies of semi-quantitative MCE MRI in vivo,
validated by ex vivo assays. We have shown that MCE
MRI of the miR-2861 binding site on HDAC5 mRNA is
Fig. 8 Representative ΔR2* maps (in percent increase from baseline (color scale was 0–250%) for saline control (a) and chronic paradigm (b),
according to Fig. 1
consistent with both immunohistochemistry and
RTqPCR results. However, results from MCE MRI with a
primer that does not target the miR-2861-binding site do
not agree with immunohistochemistry. The differences
between these results could be related to intact versus
degraded HDAC5 transcripts; our HDAC5 mRNA
measurements using SPION-miD2861 (MCE MRI),
sODNmiD2861 (RT-qPCR) and IgG against HDAC5 agree with
attenuation of intact and translatable HDAC5 transcripts.
Therefore, the evidence we show here provides solid
support to the hypotheses that SPION-miD2861 reports
HDAC5 expression, which leads to mechanistic
investigation of AIS and neuroprogenitor monitoring.
Acetylation patterns in chromatin affect gene
transcription and induce drug sensitization following chronic
exposure to drugs of abuse. Among the mechanisms that
have been proposed to mediate exposure to drugs of abuse
are increases in immediate early genes and HDAC5
shuttling [33, 34], as well as a reduction in monoamine oxidase
A by proteins of immediate early genes . HDAC
inhibitors are used therapeutically in the psychiatry and
neurology fields as mood stabilizers, antidepressants, and
anti-epileptics [35, 36], for neuroprotection from ischemic
injury and anti-inflammatory responses [37–42], as well as
for neural remodeling [6, 11, 43]. Transport of
phosphorylated HDAC3 protein from nucleus to cytoplasm is a
known mechanism of drug sensitization [13, 44, 45].
However, we have presented evidence here that attenuation of
HDAC5 (mRNA and antigen) mediates early appearance
of AIS in the acute paradigm with miD2861 pretreatment
and chronic paradigm. The results thus support the
proposal that miR-2861 may reduce translatable HDAC5
mRNA . In this study, we identified neuroprogenitors
in the LS. Neuroprogenitor cells may be associated with
neural plasticity , and therefore, may be active in
neural network remodeling in brain repair after stroke
. Amphetamine has been shown to induce neuronal
division (neurogenesis) in the hippocampus . We have
not measured neurogenesis using S-phase markers;
however, we have previously monitored neuroprogenitors
Fig. 9 Quantitative HDAC5 expression using SPION-mid2861, MCE MRI, RT-PCR and immunohistochemistry. Panel a Chronic versus Control. There
was a general reduction in HDAC5 mRNA measured using SPION-miD2861. The p values are listed above each ROI. Panel b The copy number of
HDAC5 mRNA on the 212 region was measured from total striatal RNA of S1 (SS), A1 (A1s) and A7Wa (A8s) paradigms (n = 4 each) using TaqMan®
analysis. Panel c & d HDAC5 protein (red) shows antigen around the nuclei (purple, DAPI) in the striatum; the intensity of total HDAC5 protein was
similar in both S1 and A1 paradigms but it was null in the chronic (A7WA) paradigm
Fig. 10 Pretreatment of miD-2861 and AIS in the acute paradigm. The distance traveled at each 5-min interval is the product of 5 cm and the
summation of frequencies every 5 min
Fig. 11 (See legend on next page.)
(See figure on previous page.)
Fig. 11 SPION-miD2861 identifies neuroprogenitor cells. Subtraction R2* maps (chronic minus acute) identify ROIs in the LS in vivo (panel a). We
stained brain tissue using Cy3-IgG-GFAP and Cy-2-IgG-NeuN; neuroprogenitor cells were identified by co-expression of both antigens (arrows,
Panels b & c). Panel c contains image spectrum (purple) for Cy5.5-SPION-miD2861. The arrow in Panel c shows ordinary GFAP+/NeuN− astroglia.
Corpus callosum (CC) and lateral ventricle (LV) are shown
using SPION-nestin and SPION-gfap. Here we expanded
the functional applications of MCE MRI by precise
delivery of SPION-miD2861. Target specificity of small
molecules with signal sensitivity will further enable
monitoring of cerebral repair and remodeling in vivo.
We have applied SPION to label miD2861. Iron oxide
NPs are commonly used for stem cell labeling ; the
SPION we have used is at much lower dose than that used
in stem cell labeling. Our earlier studies used SPION with
a hydrodynamic diameter (dia) of 20 ± 10 nm and
molecular weight (as SPION-cfos for Fos mRNA) of 5.6 × 10−19 g
Fe per NP. Each SPION-cfos contains ~6000 iron atoms,
based on the relative viscosity of SPION in sodium citrate
buffer at pH8 . For our current studies we use
commercially available dextran-coated iron oxide, which
provides strong T2 contrast, with a similar number of Fe
molecules per particle (1.5 × 10−20 mol Fe per NP).
Molday Ion has an average hydrodynamic size of 30 ± 20 nm
(Zetasizer, Malvern Instruments) and a core size of 8 ±
5 nm. Long-term use of these NPs does not change liver
enzyme levels or induce oxidative stress [49, 50].
Moreover, we have observed transient passage of iron through
the hepatic system; there is no hepatic retention of iron
until 10 h after i.p. delivery to mice with an icv port. 
Whereas our previous studies used the icv route alone to
deliver NPs, which was associated with an accumulation
of excess macrophages at the icv puncture site , we
have modified our methods in rodents to use a small icv
port for BBB bypass. This new delivery route is
noninvasive, and yields more uniform R2* values as well as
eliminating macrophage accumulation.
We have observed retention of NPs of approximately
30 nm (dia) in the nuclei of neurons, microglia,
macrophages, and/or phagocytes when uptake plateaus
between 4 and 6 h after SPION delivery. Once the NP
reaches neural cells, the cytoplasmic EDNs in our
studies could be transported via a membrane-bound
mechanism associated with the ER (Additional file 6:
Figure S5A1), and then to the nucleus, perhaps through
the nuclear pore complex associated with the ER [52, 53].
Because we identified SPION-miD2861 in TEM without
staining, we have not identified endosomes before reaching
the ER. The presence of scattered EDNs in Ly/Ex after
SPION-miD2861 delivery suggests these EDNs are being
degraded or excluded in Ly/Ex to microvessels
(Fig. 7J1 & K). Most important, we have no evidence
of preferential retention in any type of neural cells
when SPION-sODN is administered at the optimal
quantities we use in MEC MRI.
Cell typing using NeuN and GFAP for progenitor cells
that are identified using miD2861 show the NeuN+ and
GFAP+ phenotypes in progenitor cells (Fig. 11). Our
results show that HDAC5 mRNA may be present in cells
that express NeuN+ (neurons), GFAP+ (astroglia), or
IBA1+ (activated microglia). The TEM data in Fig. 7F
and Additional file 5: Figure S4G, showing EDNs in
neurons, do not contradict this conclusion. Our observation
differs from others’ reports that non-targeting iron oxide
NPs are preferentially retained in phagocytes or
macrophages of hepatic RES within one hr of delivery [54, 55].
Non-targeting iron NPs are retained in the Lys and
eventually degraded .
Our HDAC5 mRNA-targeting NPs reports HDAC5
expression by binding to its target, as demonstrated by
the linear coefficiency between MR-derived ΔR2* values
and mRNA copy number quantified by TaqMan analysis
(Fig. 4). There are several important factors that inform
and provide rationale for using correlation studies of
ΔR2* values and TaqMan analysis. First, the peak (or
perhaps more precisely, plateau) ΔR2* values are
positively proportional to regional iron content . Second,
the CNR in all ROIs of the brain is high and uniform at
the plateau . Third, in preparing SPION-sODN we
conjugated the targeting sODN and SPION reporting
agent at a constant ratio of 3 nmols sODN per mg iron.
Fourth, the linkage between SPION and sODN is strong
and biologically stable (via NeutrAvidin and biotin), and
the use of a common transfection reagent
(Lipofectamine 2000) facilitates and protects SPION-sODN in
body fluids during delivery. Some SPION-sODN may
enter the nuclei where the target RNA transcripts are
present for binding (Fig. 7). Fifth, we use ΔR2* values at
plateau retention, or 6 h after SPION-sODN delivery; the
plateau ΔR2* represents strong CNR, and a steady state of
RNA-bound SPION-sODN at physiological conditions
during MRI. Sixth, the mRNA copy number measured
under normal conditions represents the equilibrium
between synthesis and degradation in the physiological
condition at the time of RNA isolation. We have shown
SPION and antisense sODN are intact in vivo, as
SPIONsODN can be used to produce cDNA for amplification by
in situ RT-PCR . The correlation coefficiency between
the plateau ΔR2* of cerebral FosB, HDAC5, and GFAP
mRNA and copy numbers by TaqMan® Gene Expression
Assay (with Actin mRNA as reference) supports precision
hybridization as the mechanism of MCE MRI reporting
mRNA, as validated by RT-PCR. Last but not the least,
ΔR2* values of SPION-sODN representing a steady state
of RNA-bound iron oxide in the nucleus is supported by
the static dephasing regime theory of iron oxide
Some studies have reported advanced fluorescent in
situ hybridization (FISH) of miRNA . Short
interfering nucleic acids (siDNA or siRNA) can be used in
targeted molecular imaging for theranostic
applications [18, 61]. The core size of SPION-sODN of 8 ±
5 nm (dia) is within the pore size of the nuclear
envelope for passage . Although the mechanism of
precision translocation of SPION-sODN to nuclear
HDAC5 mRNA in the neural environment is not yet
well understood, neural cells may have intrinsic repair
mechanisms to meet the required transport and
exclusion of unbound or excess SPION-sODN, and
preferential protection of bound SPION-sODN under
normal physiologic temperature. These mechanisms
must work in concert in vivo for various physiological
functions. The positive correlation between
MRderived ΔR2* values and mRNA quantitation by
RTPCR suggests that ΔR2* values from MCE MRI report
iron, predominately, hybridized to target mRNA; this
in turn supports the view that one optimal dose of
targetspecific NPs does not yield more EDNs in Ly/Ex than seen
in the control (Additional file 2: Figure S1). Therefore, the
exclusion of unbound targeting NPs is rapid, within 4 h
after delivery to living mouse brains. However, we must
note that we cannot estimate copy number from MRI
because the current technology does not provide an
internal control for copy number estimation.
We have also examined for possible change in
miR2861 level. When comparing across the three
paradigms we detected no significant differences in
the level of miR2861 (not shown). This is in agreement
with reports that no detectable miR2861 is found in
adult brains . The main function of the class of
small single-stranded noncoding RNAs known as
miRNA is to regulate mRNA translation; this occurs
in normal developmental as well as in pathological
conditions across the general population, in all ages,
genders, and races. By regulating mRNA translation,
miRNA affects differentiation of normal biological
development, resistance to environmental changes,
cellular proliferation and apoptosis; it may also have
dire consequences, causing an array of health
problems. Although they were once mistakenly termed
“junk” RNA, we now know miRNAs play an important
role in epigenetic pathways. The human genome has
thousands of miRNAs that target about 600 genes.
The availability of miRNA can also help stabilize and
protect cytoplasmic enzymes. All cells express
miRNA, starting with precursor miRNAs, which are
transcribed as part of one arm of a ~80 nucleotide
RNA stem-loop by RNA polymerase II or III. These
precursors are post-transcriptionally processed to
premiRNA by Digeorge Syndrome Critical Region 8
(DGCR8), a nuclear splicesome with a de-branching
enzyme, and the catalytic ribonuclease (RNase) III
domain, which liberates a hairpin loop structure of about
70 nucleotides each of precursor miRNA. Mirtrons
directly splice out introns of some pre-miRNAs, which
are composed of a matured sequence and a passenger
sequence on the hairpin and a two-nucleotide
overhang at the 3′-hydroxyl end. Exportin-5, a
nucleocytoplasmic shuttle, exports pre-miRNA from the nucleus
by an energy-dependent process. The Dicer enzyme
with RNase III activity in the cytoplasm produces an
imperfect RNA: RNA duplex of about 22 nucleotides
in length, by cutting away the loop in the hairpin of
pre-miRNA. The RNA-induced silencing complex
(RISC) incorporates one miRNA strand in the duplex
based on thermodynamic instability and weaker base
pairing relative to the other strand. The passenger
strand can be incorporated into another RISC for a
different RNA target, but is generally preferentially
degraded. The Argonaute (Ago) family of proteins provide
the active part of miRISC’s (RISC with miRNA) silencing
function to block translation. Because extensive ex vivo
purification is used to detect miRNA, how RISC may
protect miRNA in vivo for detection is not well
understood. What is known is that the Ago family encodes four
functional domains: the N-terminal, PAZ, Mid, and
Cterminal PIWI domains. The PAZ domain (a conserved
domain of PIWI, Ago and Zwille proteins) binds to the
single-stranded 3′ end, while the RNase-H activities of the
PIWI (P-element induced wimpy testis) domain cleave the
target mRNA. Gene silencing occurs within miRISC,
between the matured strand of miRNA and its target
mRNA. Perfect complementarity in the seed region, at
nucleotides 2–7 of miRNA, will speed up degradation of
the target mRNA and allow transient gene silencing,
manifesting as a gene knockdown phenotype; therefore, a
perfect match up between matured miRNA and target
mRNA is not required to inhibit translation. The addition
of a methyl moiety (in plants) or an adenine residue to the
3′ end of the miRNA protects or stabilizes the matured
Amphetamine-type stimulants (e.g., methamphetamine
or 3-4-methylenedioxy methamphetamine (MDMA), also
known as Ecstasy or MOLLY) are highly addictive Schedule
I drugs, and widely abused worldwide (2011 Global ATS
Assessment, a United Nations report). Those who
habitually use MDMA are at high risk for learning impairment
and aggressive behavior , as well as other symptoms
that mimic those seen in schizophrenia and depression
[64, 65]. Further compounding the health issues associated
with drug addiction is that, given the propensity for needle
sharing, drug addicts face a highly increased risk of
infection from blood-borne pathogens such as human
immunodeficiency virus type 1 (HIV-1). Significant
variations in brain structure are associated with both HIV
infection and methamphetamine dependence . This
relationship is all the more significant because
deacetylation of histone within the HIV-1 long terminal repeat by
HDACs helps to maintain viral latency, allowing the virus
to evade both immune detection and antiviral drugs .
Methamphetamine has been found to induce regional
variations in oxidative stress and behavioral modifications
in HIV-1Tg rats [68–70]. Moreover, non-selective
inhibitors of HDAC may induce HIV-1 expression from the
HIV-1 reservoir in resting CD4(+) T cells, without new
infection . As such reservoirs are also known to be
present in the microglia of the CNS, the technology we
have developed to monitor transcripts in microglia
promises additional significance as a tool to monitor viral latency
and drug addiction longitudinally. Understanding
mechanisms of HDAC expression and neural remodeling in vivo
are of great importance, with broad implication for public
We designed sODN-miD2861 to target HDAC5 mRNA;
our binding assays and primer are specific to the location
of miD2861 binding ex vivo and in vivo. Our MCE MRI
reports RNA-bound NPs. The linear regression of coefficient
of MRI and TaqMan® analysis for copy number was near
1.0 at different conditions, demonstrating that the
mechanism of gene targeting for SPION-sODN and MRI is similar
to that of RT-PCR. Because HDACs or cells that harbor
them are involved in cerebral repair/remodeling processes,
changes in HDAC5 expression, and the availability of tools
to monitor such change, may have still broader implications
than we have explored in the scope of this work.
Additional file 1: Supplemental Material. (DOCX 85 kb)
Additional file 2: Expression of HDAC5 antigens in the nucleus
accumbens (NAc) of naïve mice mice. We compared total (cy3-ab1439,
Abcam) or phosphorylated HDAC5 (cy3-ab192339) in the nucleus
accumbens (NAc). (PDF 1 kb)
Additional file 3: Expression of HDAC5 antigens in mice of acute
amphetamine exposure groups. We compared total (file 2) or
phosphorylated (file 3) HDAC5 in the NAc. (PDF 3461 kb)
Additional file 4: Expression of HDAC5 antigens in mice in the chronic
amphetamine exposure groups. We compared total (file 4) or
phosphorylated (file 5) HDAC5 in the NAc. (PDF 2600 kb)
Additional file 5: We observed electron dense nanoparticles (EDN) in
mice with (D, E, G & H) or without (A-C) SPION-sODN at the optimal dose
(0.04 mg/kg, intracerebroventricular [icv] injection); there is no preferential
accumulation of EDNs in mice with optimal dose of SPION-sODN.
Accumulation of EDNs are observed in mice with 3X optimal dose (0.12
mg/kg, intracerebroventricular injection, not shown) or 8 optimal doses at
one injection every week (4 mg/kg, intraperitoneal [i.p] injection, F). We
attempted to identify SPION-sODN of 30 nm (dia) in Fig S4E; TEM stains
masked our ability to identify SPION-sODN as EDN at ~30 nm (dia).
(PDF 5888 kb)
Additional file 6: We observed several nuclear EDNs with a uniform
diameter of 30 nm (A1, B1 & B2, arrowheads); three of these EDNs
appeared on the membrane in tandem near the rough ER (A1). Only
EDN larger than 60 nm appeared to be in the cytoplasm (B & B1, arrows).
These unstained samples had reduced background noise, and we found
EDNs (arrows) in the cytoplasm and nuclei. Although EDNs were visible,
we cannot identify Ly/Ex, but can identify MG and N from the outline of
their nuclei. Bars (microns) = 500 (A, A1 & B). (PDF 2836 kb)
Additional file 7: We conducted a gross comparison of the total
locomotor activities, and compared changes in locomotion between
AMPH-treated and saline-treated animals in various exposure paradigms
(SAL vs. AMPH groups in acute exposure, SAL7/W/S vs. A7/W/A and SAL7/
W/S vs A7/W/S [placebo] in chronic exposure groups). Using one-way
ANOVA followed by Newman-Keuls Multiple Comparison test, we found
that AMPH induced a significant main effect (p < 0.001), with an exception
between the SAL7/W/S and A7/W/S groups (p > 0.05). The average rate of
locomotion (in meters per hour) was 57 + 6 and 105 + 6 for A1 and A7/W/
A, respectively. (PDF 107 kb)
A: Amphetamine; AIS: Amphetamine-induced sensitization; B0: External
magnetic field; bp: base pairs; CC: Corpus callosum; Cpu: Caudate putamen;
EDN: Electron-dense nanoparticles; Ex: Exosome; GFAP: Glial fibrillary acidic
protein; GFP: Green fluorescent protein; HDACs: Histone deacetylases;
hipp: hippocampus; i.p.: intraperitoneal; IBA1: Ionized calcium-binding
adaptor molecule 1; icv: intracerebroventricular; LS: Lateral septum;
LV: Lateral ventricles; Ly: Lysosome; M: Monocytes; MC: Motor cortex;
MG: Microglia; mPFC: medial prefrontal cortex; MRI: Magnetic resonance
imaging; N: neuron; NA: NeutrAvidin; NAc: Nucleus accumbens;
NP: Nanoparticles; P: Phagocytes; R2*: The effective rate of transverse
relaxivity; ROI: Region(s) of interest; RT-PCR: Reverse transcription polymerase
chain reaction; SPION: Superparamagnetic iron oxide nanoparticle;
SSC: Somatosensory cortex; ΔR2*: Above the background transverse relaxivity
We thank Ms. Nichole Eusemann for reading and making suggestions during
writing of the manuscript, Drs. C-M Liu for sODN synthesis, J Ren for delivery
of nanoparticles, behavior testing and histology, HF Wang for MRI, and JS
Yang for RT-qPCR; Mrs. M Mckee & D Capen of the Center for System Biology
and Program in Membrane Biology/Division of Nephrology, MGH for
excellent work of TEM.
Availability of data and materials
Current studies are supported by the NIH of USA, therefore, we follow the
policies of funding agency for rigor authentication of key biologicals,
chemical resources and transparency and material sharing.
Ethics approval and consent to participate
This study does not involve patients. 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
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