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Decreased Proliferation of Adult Hippocampal Stem Cells
During Cocaine Withdrawal: Possible Role of the
Cell Fate Regulator FADD
M Julia Garcı
´a-Fuster*
,1
, Shelly B Flagel
1
, S Taha Mahmood
1
, Leah M Mayo
1
, Robert C Thompson
1
,
Stanley J Watson
1
and Huda Akil
1
1
Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI, USA
The current study uses an extended access rat model of cocaine self-administration (5-h session per day, 14 days), which elicits several
features manifested during the transition to human addiction, to study the neural adaptations associated with cocaine withdrawal. Given
that the hippocampus is thought to have an important role in maintaining addictive behavior and appears to be especially relevant to
mechanisms associated with withdrawal, this study attempted to understand how extended access to cocaine impacts the hippocampus
at the cellular and molecular levels, and how these alterations change over the course of withdrawal (1, 14, and 28 days). Therefore, at
the cellular level, we examined the effects of cocaine withdrawal on cell proliferation (Ki-67 + and NeuroD + cells) in the DG. At the
molecular level, we employed a ‘discovery’ approach with gene expression profiling in the DG to uncover novel molecules possibly
implicated in the neural adaptations that take place during cocaine withdrawal. Our results suggest that decreased hippocampal cell
proliferation might participate in the adaptations associated with drug removal and identifies 14 days as a critical time-point of cocaine
withdrawal. At the 14-day time-point, gene expression profiling of the DG revealed the dysregulation of several genes associated with
cell fate regulation, highlighting two new neurobiological correlates (Ascl-1 and Dnmt3b) that accompany cessation of drug exposure.
Moreover, the results point to Fas-Associated protein with Death Domain (FADD), a molecular marker previously associated with the
propensity to substance abuse and cocaine sensitization, as a key cell fate regulator during cocaine withdrawal. Identifying molecules that
may have a role in the restructuring of the hippocampus following substance abuse provides a better understanding of the adaptations
associated with cocaine withdrawal and identifies novel targets for therapeutic intervention.
Neuropsychopharmacology (2011) 36, 2303–2317; doi:10.1038/npp.2011.119; published online 27 July 2011
Keywords: extended access cocaine self-administration; withdrawal; cell fate regulation; rat brain hippocampus; microarray analysis
INTRODUCTION
Cocaine is a highly abused drug that imposes an enormous
health, social, and economic toll on society. One of the main
problems in the treatment of substance abuse is the
propensity of addicts to relapse even long after the
discontinuation of drug use. It is therefore critical to
achieve a better understanding of the neurobiological
mechanisms as well as the neural adaptations that occur
during the course of withdrawal, in order to develop better-
tailored treatments. To accurately study cocaine withdrawal
in the laboratory, it is essential to use an animal model of
drug exposure that captures some of the key features
of human cocaine addiction. Previous studies have shown
that rats exposed to extended daily access cocaine self-
administration (SA) procedures result in a pattern of drug-
intake (ie, escalation of drug intake with session day)
similar to that seen in human addicts (Bozarth and Wise,
1985; Ahmed and Koob, 1998) as well as a number of other
symptoms characteristic of human addiction, including
increased motivation for cocaine (Paterson and Markou,
2003) and deficits in fronto-cortical function and cognition
(Briand et al, 2008). Moreover, extended daily access to
drugs of abuse produces changes in neural plasticity in
several brain regions implicated in addictive behavior
(Ferrario et al, 2005) and therefore, represents an excellent
rat model to study the neural adaptations that accompany
the cessation of cocaine exposure.
A great deal is known about the impact of cocaine on the
reward circuitry in the brain, which contains cell bodies in
the ventral tegmental area that project to the nucleus
accumbens and the prefrontal cortex (Gold et al, 1989).
Recently, however, much interest has been generated in
understanding the role of neural structures outside of the
primary reward pathway, as these regions can critically
modulate aspects of the cocaine experience beyond its
Received 22 November 2010; revised 31 May 2011; accepted 31 May
2011
*Correspondence: Dr MJ Garcı
´a-Fuster, IUNICS, University of the
Balearic Islands, Spain, Tel: + 34 971 25 99 92, Fax: + 34 971 25 99 35,
E-mail: j.garcia@uib.es
Neuropsychopharmacology (2011) 36, 2303 –2317
&
2011 American College of Neuropsychopharmacology. All rights reserved 0893-133X/11
www.neuropsychopharmacology.org
immediate rewarding effects, such as contextual learning,
memory, and the motivational value associated with drug
stimuli. One region of interest, the hippocampus, is highly
interconnected with the reward system (Everitt and
Robbins, 2005: Dietz et al, 2009) and can influence the
learning component of addiction. Indeed, previous work
has associated different facets of hippocampal plasticity
with psychostimulant effects on contextual conditioning
(Shen et al, 2006) and behavioral sensitization (Lodge and
Grace, 2008; Garcı
´a-Fuster et al, 2010). In a recent study,
reduced hippocampal neurogenesis, a novel drug-induced
hippocampal neuroadaptation, was shown to confer vulner-
ability in an animal model of cocaine addiction (Noonan
et al, 2010).
The hippocampus is one of two known brain regions
that continue to produce new neurons during adulthood
(Altman and Das, 1965; Eriksson et al, 1998), representing a
dramatic form of structural plasticity. Adult hippocampal
neurogenesis is a highly dynamic process whereby mature,
functional granular neurons are generated from newly born
progenitor cells in the subgranular zone (SGZ) of the
dentate gyrus (DG) (Ming and Song, 2005). These granular
neurons are believed to integrate into existing circuitry (van
Praag et al, 2002). Each stage of neurogenesis (ie,
proliferation, differentiation and migration, maturation
and survival) is demarcated by distinct molecular markers
(von Bohlen Und Halbach, 2007) and is differentially
regulated by a variety of factors, including adverse
environmental conditions (Schmidt and Duman, 2007)
and exposure to addictive substances (Eisch and Harburg,
2006). Previous reports have shown that experimenter-
delivered cocaine decreased SGZ proliferation (Yamaguchi
et al, 2004) in a dose- and time-dependent manner (Eisch
and Nestler, 2002), without altering the survival and growth
of immature cells (Domı
´nguez-Escriba
`et al, 2006). More-
over, the decrease in cell proliferation after repeated
administration of cocaine returned to normal levels
following one week of withdrawal (Yamaguchi et al, 2005).
When following a more clinically relevant paradigm of
intravenous cocaine SA, similar to the one used in this
study, 3 weeks of cocaine SA (4-h session per day) resulted
in decreased cell proliferation in the adult SGZ (Noonan
et al, 2008), without affecting cell death. Interestingly,
following 4 weeks of withdrawal these deficits were
normalized and the maturity of adult-generated hippocam-
pal neurons was increased (Noonan et al, 2008). However, it
is worth noting that the same study showed that a longer
chronic paradigm of cocaine SA (7 weeks, 4-h session per
day) did not change proliferation of Ki-67 + cells. Taken
together, these data demonstrate that cocaine differentially
modulates hippocampal cells at different stages of neuro-
genesis and highlights the importance of distinguishing
between proliferation and neurogenesis. Therefore, a better
understanding of the cellular and molecular changes that
contribute to the restructuring of the hippocampusFsuch
as proliferation of neural progenitor cells or hippocampal
cell fate regulationFwill be key to better understanding the
neural adaptations that occur during the course of cocaine
withdrawal.
The aim of this study was to understand how extended
daily access to cocaine SA impacts the hippocampus at the
cellular and molecular levels, and to ask how these
alterations change over the course of withdrawal. To this
end, we relied on two approaches: (1) to study the self-
renewing capacity of the hippocampus by immunohisto-
chemistry (IHC) analysis (Ki-67 + and NeuroD + cells in
the DG); and, (2) to ascertain new candidate molecules
likely involved in these processes by gene expression profile
analysis in the DG.
MATERIALS AND METHODS
Rats
Forty-eight adult male Sprague–Dawley rats (Charles River
Laboratories, Wilmington, MA) weighing 225–250 g at the
start of the experiment were used. Rats were housed in pairs
and allowed to acclimatize to housing conditions for 1 week
before any manipulations. Rats were housed in a tempera-
ture and humidity controlled room with a 12 : 12-h light–
dark cycle, with water and food available ad libitum.
Following catheterization surgeries, rats were individually
housed for the remainder of the experiment. Rats were
treated in accordance with the ethical guidelines of the
University of Michigan Committee on the Use and Care of
Animals.
Surgical Procedures
Rats were anesthetized with a mixture of ketamine (50mg/kg)
and xylazine (10 mg/kg) administered i.p. A silicone
catheter was implanted in the jugular vein and passed
subcutaneously to exit from the midscapular region of the
animal’s back. One rat died during surgery. The remaining
47 rats were allowed to recover from surgery for 1 week
before drug SA. Catheters were flushed daily with 0.1 ml of
gentamicin (10 mg/ml in 0.9% sterile bacteriostatic saline).
The day before SA testing, catheter patency was assessed in
the morning (around 1000 hours) by injecting 0.1–0.2 ml
of thiopental sodium (20 mg/ml in sterile water) into
the catheter. Rats that did not lose muscle tone within
2–3 s were excluded from the cocaine SA (LgA) group
and utilized as no drug (ND) controls. Out of the 47 rats,
30 underwent cocaine SA procedures (LgA groups) and
the remaining 17 were used as ND control groups (see
experimental design, Figure 1a).
Extended Access Cocaine SA Procedures
During the training sessions, rats from the LgA groups
(30 rats) were transported from their home cage to an
operant chamber for 6 consecutive days, where they were
allowed to nose-poke for cocaine (0.5 mg/kg per infusion in
50 ml of saline administered over 1.6 s) for 1-h sessions on a
fixed ratio schedule of reinforcement (FR1) with a time-out
of 20 s. One response in the ‘active’ nose-poke hole resulted
in delivery of a single drug infusion. Responding in the
‘inactive’ hole had no consequences, but was recorded.
Given the dose of cocaine used, the fact that these animals
were not food-deprived or pre-trained for food responding,
and that they were trained and tested during their light cycle,
33% of the rats tested did not acquire stable SA behavior
(ie, at least five infusions each day for 3 consecutive days)
and were removed from the study. After the initial training
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2304
Neuropsychopharmacology
period, rats from the LgA groups were switched to extended
access (5-h session per day) cocaine SA at 0.5 mg/kg per
infusion of cocaine for a total of 14 sessions (5 days per
week). Rats were separated into two waves, which were
balanced according to the amount of drug intake during the
initial training period. The first wave was run from 0700 to
1200 hours and the second wave from 1300 to 1800 hours.
Rats were checked for catheter patency once a week (1000
hours on a day with no LgA session) throughout the SA
paradigm and immediately following the last day of testing.
Only those rats that passed the catheter patency tests were
included in the analyses. Twenty rats successfully completed
the experiment for the LgA groups. Following the last SA
session, LgA rats were counterbalanced by drug intake and
wave and underwent withdrawal for 1, 14, or 28 days (see
experimental design, Figure 1a).
ND control rats (n¼17) were also divided into two waves,
transported daily to the same testing room, and placed in
holding chambers similar to the SA chambers for the same
amount of time as LgA rats on each day of training or
extended access exposure. Following the last session, these
rats were also divided into three groups ND-Day 1, ND-Day
14, ND-Day 28 and were left in their home cage for 1, 14, or
28 days, respectively.
Tissue Collection
Rats were killed by rapid decapitation and their brains
removed at the conclusion of the assigned withdrawal
period: 1, 14, or 28 days following the final SA session.
Brains were processed in such a way as to allow the
investigation of hippocampal cell fate regulation using
different approaches. The left half-brain was quickly frozen
in a 30 1C isopentane solution and stored at 80 1C until
further processing. For each animal, 30 mm sections were
cryostat cut and slide-mounted throughout the entire extent
of the HC (1.72 to 6.80 mm from Bregma; Paxinos and
Watson, 1997) and kept at 80 1C until further analysis. The
following analyses were conducted on this tissue from the
left hemisphere: (1) cell proliferation markers (ie, Ki-67 +
and NeuroD + cells in the DG) by IHC; (2) laser capture
microdissection (LCM) of the DG followed by gene
expression profile analysis using Illumina microarray plat-
form and RT-PCR validation; and (3) mRNA analysis in the
DG by in situ hybridization (ISH). The right half of the
brain was used to dissect the hippocampus, which was fast
frozen on dry ice, and kept at 80 1C until use for western
blot (WB) experiments (ie, protein content of cell fate
markers). Proliferation of adult hippocampal stem cells (ie,
Ki-67 + and NeuroD + cells) was assessed in ND and LgA
groups at the three time points of withdrawal (Day 1, Day
14, and Day 28). However, given that the main changes in
cell proliferation occurred following 14 days of withdrawal,
further analysis (ie, microarray gene expression and
validation) were ascertained only at this time-point (ND-
Day 14 vs LgA-Day 14).
Immunohistochemistry Analysis
The rate of cell proliferation was determined in the left
hemisphere DG by performing IHC labeling of Ki-67, an
intrinsic marker of ongoing cell proliferation, which is expressed
n=47 rats Sprague-Dawley
ND controls (n=17 rats)
Pretraining (FR-1)
1h,5 days, 0.5 mg/kg/infusion
2 waves of animals
(n=30 rats)
55
50
45
35
Cocaine lntake (mg/kg)
25
20
15
10
12345678
LgA SA Session Day
9 1011121314
30
40
LgA cocaine SA
5h, 14 days, 0.5 mg/kg/infusion
Withdrawal Time-Course
ND LgA
10 rats did not learn
(n=20 rats)
Day 1
Day 14
Day 28 n=6
n=5
n=6 n=7
n=7
n=6
Jugular vein
catheterization
sugery
Figure 1 Escalation of cocaine intake during LgA cocaine SA procedures. (a) Experimental design (see Materials and methods section). (b) Data represent
mean±SEM amount of cocaine intake (0.5 mg/kg cocaine per infusion) over the entire daily sessions (5-h session per day of cocaine SA) for 14 days in adult
male Sprague–Dawley rats (LgA, n¼20). Rats progressively increased their daily cocaine intake starting at an average of 15 mg/kg on the first day of SA and
finishing at an average intake of about 50 mg/kg on the final SA session day (day 1 vs day 14; ***po0.0001).
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2305
Neuropsychopharmacology
in all dividing cells (Scholzen and Gerdes, 2000; Kee et al,
2002). Following a standardized protocol (see Perez
et al, 2009; Garcı
´a-Fuster et al, 2010), hippocampal tissue
sections (30 mm) were post-fixed for 1 h in 4% paraformal-
dehyde (PFA) and incubated for epitope retrieval
(10% sodium citrate pH 6.0 at 90 1C for 1 h). Sections were
then rinsed with PBS, washed in 0.3% peroxide, blocked
with BSA containing 1% goat serum and 0.05% Triton
X-100, and incubated overnight with polyclonal rabbit
anti-Ki-67 (1 : 40 000; University of Michigan). After PBS
washes, sections were incubated in biotinylated anti-
rabbit secondary antibody 1 : 1000 (Vector Laboratories,
Burlingame, CA) followed by avidin–biotin complex
(Vectastain Elite ABC kit; Vectors Laboratories) and
diaminobenzidine (DAB) as chromogen. Sections were
counterstained with cresyl violet before dehydration
through graded alcohols, xylene immersion and cover-
sliping (Permount mounting medium).
The proliferation of neural progenitors was analyzed in
the left hemisphere DG by performing IHC labeling of
NeuroD, a transient transcription factor restricted to
developing neurons (Lee, 1997; Miyata et al, 1999; Hevner
et al, 2006; Gao et al, 2009). As previously described
(Garcı
´a-Fuster et al, 2010), sections were post-fixed for 1 h
in 4% PFA and incubated in endogenous peroxidase
blocking reagent (0.1% peroxide). Sections were then
blocked with BSA (1% donkey serum and 0.05% Triton
X-100) and incubated overnight with goat polyclonal anti-
NeuroD (1 : 2500; Santa Cruz Biotechnology). The next day,
sections were incubated in biotinylated anti-goat secondary
antibody 1 : 1000 (Vector Labs) followed by avidin–biotin
complex and DAB reaction with nickel chloride for
visualization of signal. Finally, tissue was dehydrated in
graded alcohols, immersed in xylene and cover-slipped
using Permount mounting medium.
To ensure an unbiased count, all slides were randomly
coded by a non-experimenter before quantification. Codes
were broken after all slides were quantified and analyzed.
The number of immunostained positive ( + ) cells was
counted unilaterally in every eighth section throughout the
extent of the left DG (1.72 to 6.80 mm from Bregma)
with a Leica DMR light microscope using a 63 oil
objective lens and focusing through the thickness of the
section (approximately 30 mm). For each rat, multiplication
of raw cell counts by eight provided a final estimate of the
total number of positive cells for one hippocampi. This
method is based on a modified unbiased stereological
procedure (Malberg and Duman, 2003) and has been used
previously in our lab (see Perez et al, 2009; Garcı
´a-Fuster
et al, 2010).
In addition to estimating the total number of Ki67 + and
NeuroD + cells, the number of cells was also quantified
within the anterior (180 to 4.52 mm from Bregma) and
posterior (4.52 to 6.80 mm from Bregma) hippocampal
demarcation (see Guzma
´n-Marı
´net al, 2003; Noonan et al,
2008). A more detailed anatomical analysis in relation to the
distance from Bregma was conducted for the groups in
which the overall number of Ki-67 + and NeuroD + cells
was decreased (ND-Day 14 and LgA-Day 14). For this
analysis, data are presented as total number of positive cells
in the DG per region at both the anterior and posterior
levels (Paxinos and Watson, 1997).
Gene Expression Profile Analysis
To ascertain the possible molecular adaptations that might
accompany the cellular changes observed at the level of cell
proliferation in the DG (ie, decreased Ki-67 + and NeuroD
+ cells), we conducted gene expression profile analysis of
the left hemisphere DG taken after 14 days of withdrawal
(ND-Day 14 vs LgA-Day 14). The tissue used for this
experiment was taken from consecutive slides adjacent to
those used to assess proliferating markers. Moreover, it is
important to note that while proliferation mainly occurs in
a small percentage of cells in the SGZ of the DG, for the gene
profiling analysis all cells in the DG were dissected.
Following a standardized lab protocol (see Kerman et al,
2006), four DGs (SGZ and GCL) were laser capture
microdissected (LCM) from selected slides for each rat.
Each slide contained a total of eight cryostat-cut sections
(approximately 30 mm thick) from the left half of the brain.
Given the changes in Ki-67 + and NeuroD + cells were
observed throughout the whole hippocampal extent, both
within the anterior (1.80 to 4.52 mm from Bregma) and
the posterior (4.52 to 6.80 mm from Bregma) demarca-
tion, the samples for this analysis were taken from tissue
between 3.60 and 5.80 mm to Bregma. Before LCM, the
slides were removed from 801C, thawed for 30 s at room
temperature, dehydrated, and defatted (Kerman et al, 2006).
After LCM, during which tissue was captured and placed
onto caps, all samples underwent RNA isolation (PicoPure
RNA Isolation Kit; Molecular Devices, Sunnyvale, CA) as
previously described (Bernard et al, 2010), and RNA
concentration and quality was assessed by Agilent BioAna-
lyzer (Agilent Technologies, Palo Alto, CA). Total cellular
RNA samples were subjected to two rounds of amplification
(RiboAmp Plus). Before biotinylation, a portion of ampli-
fied double-stranded complementary DNA (cDNA) was
saved for validation of candidate genes by RT-PCR
(ie, TaqMan Array analysis, see below). Biotinylated
amplified RNA concentration was calculated for each rat
using a Nanodrop ND-1000 spectrophotometer (Thermo-
Scientific, Wilmington, DE), yielding amounts in the range
of 800–1600 ng/ml for most of the samples, except for three
samples: 3-ND (265 ng/ml), 12-LgA (162 ng/ml), and 48-LgA
(351 ng/ml). Equal amounts of biotinylated amplified RNA
was hybridized to Illumina microarrays (RatRef-12 Bead-
chips) and processed according to the manufacturer’s
instructions.
Gene Expression Profile Validation
Gene expression microarrays yielded a list of candidate
genes altered in our samples when comparing LgA-Day 14
with their respective controls (ND-Day 14). In an attempt
to validate some of these changes through RT-PCR, we
performed gene quantification with custom ordered
TaqMan Gene Expression Assays (Applied Biosystems,
Carlsbad, CA). A portion of previously saved amplified
cDNA from ND-Day 14 and LgA-Day 14 samples obtained
from the LCM dissected left DG (see above) were prepared
in a 50 ml volume containing 30 ng of cDNA. Following
mixture with 50 ml of TaqMan Universal PCR Master
Mix 2 , samples were centrifuged twice at 1200 r.p.m.
for 1 min and then loaded to the Array containing the
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2306
Neuropsychopharmacology
pre-designed probes and primer sets (based on 50nuclease
chemistry). Samples were then run on the Applied
Biosystems 7900HT Fast Real-Time PCR System (University
of Michigan Core Facility). Relative gene expression
was determined using the 2
DDCt
method (Livak and
Schmittgen, 2001), in which 2
DDCt
yields fold change in
gene expression of the gene of interest normalized to the
internal control gene expression and relative to a cali-
brator. The threshold cycle (Ct) is defined by the number of
cycles to reach threshold of detection. DDCt ¼(Ct gene–Ct
GAPDH)–(mean Ct gene control group–mean Ct GAPDH
control) was determined for each reaction. Normalization
to a housekeeping gene (GAPDH) is valid under experi-
mental conditions where the housekeeping gene is not
altered by the experimental treatments, as occurs with
GAPDH in the present experimental treatments (data not
shown).
In Situ Hybridization Analysis
Since Fas-Associated protein with Death Domain (FADD)
gene expression was seen as altered by microarray but was
not detectable by the TaqMan PCR procedure, ISH was used
to monitor the changes in FADD mRNA levels in the DG
(Bregma 1.80 to 5.80 mm) of rats exposed to 14 days
of withdrawal following cocaine SA (LgA-Day 14) and
their respective controls (ND-Day 14). FADD mRNA
analysis was conducted for each rat in two slides contain-
ing eight tissue-sections each, starting from the begin-
ning of the DG (Bregma 1.80 mm) until approximately
Bregma 5.80 mm. Briefly, before probe hybridization,
slide-mounted tissue sections (approximately 30 mm) from
the left-half brain were fixed for 1 h in 4% PFA at room
temperature, rinsed with salt buffers, and dehydrated with
graded alcohols. After air-drying, the sections were
hybridized with a
35
S-labeled cRNA probe previously cloned
from cDNA fragments with specific primers using standard
in vitro transcription methodology (FADD: 542 nucleotide
fragment directed against the rat FADD mRNA coding
region, nucleotides 355–898; see Garcı
´a-Fuster et al, 2009).
The probes were labeled by incorporation of
35
S-UTP and
35
S-CTP and hybridized to tissue overnight at 551C. The
next day, sections were washed with increasing stringency,
dehydrated with graded alcohols, air-dried, and exposed to
film for 2 weeks. The specificity of the hybridization signal
was confirmed with a sense probe control (data not shown).
Digital images were scanned and integrated optical density
(IOD) was measured for all the DGs per rat using an image
analysis system (Image J, Version 1.43u). For the overall
analysis, percent changes in LgA-Day 14 IOD with respect to
control samples (ND-Day 14, 100%) were calculated. In
addition to estimating the overall change in FADD mRNA,
quantitative analysis was shown in relation to distance to
Bregma in ND- and LgA-Day 14 rats. For this analysis, data
are presented for each group as the mean IOD in the DG at
the specific Bregma level.
Western Blot Analysis
The regulation of FADD protein was assessed in the right-
half hippocampus of ND- and LgA- (Day 1, 14, and 28) rats
by WB analysis. To do so, brain tissue samples were
prepared in the presence of various protease inhibitors.
Aliquots of total homogenate of known protein concentra-
tion (BCA Protein Assay, Pierce, IL) were mixed with equal
volumes of electrophoresis loading buffer, denatured, and
stored at 20 1C until use. Hippocampal proteins (40 mg)
were separated under non-reducing conditions on 10%
SDS-PAGE minigels (Bio-Rad Laboratories, Hercules, CA),
followed by standard immunoblotting procedures (Garcı
´a-
Fuster et al, 2003). The primary antibodies used (overnight
incubation at 4 1C) were selected based on previous
experiments and characterization in rat brain (see Garcı
´a-
Fuster et al, 2009): anti-FADD (dilution 1 : 2500; sc-5559;
Santa Cruz); and anti-b-actin (dilution 1 : 10 000; clone AC-
15; Sigma). The secondary antibody (anti-rabbit or -mouse)
was incubated for 1 h at room temperature at a 1 : 5000
dilution in blocking solution. The immunoreactivity was
detected with an ECL detection system (Amersham Inter-
national, Buckinghamshire, UK) and visualized by exposure
to Hyperfilm ECL (Amersham) for 30 s to 60 min. The films
were quantified by densitometric scanning (IOD) using
Image J software. The amount of hippocampal target
proteins was compared in the same gel between the control
group (ND) and the drug group (LgA) at each specific time
point of withdrawal. This procedure was repeated several
times in different gels (each gel with different samples from
ND-control and LgA rats). Percent changes in immuno-
reactivity with respect to control samples ND (100%) were
calculated in various gels and the mean value was used as a
final estimate. The content of b-actin was quantified as a
loading control.
Assay for Apoptotic Cell Death
The induction of abnormal cell death was monitored in
the right-half hippocampus (ND-Day 14 vs LgA-Day 14)
by measuring the cleavage of nuclear enzyme poly(ADP-
ribose)-polymerase (PARP) mediated by executioner cas-
pase-3 (116 kDa PARP to 85 kDa fragment), which is a
hallmark of apoptosis (see Garcı
´a-Fuster et al, 2007, 2009).
The kit is designed to detect PARP cleavage by WB ana-
lysis (anti-PARP: dilution 1 : 800; Calbiochem) as described
above.
Data and Statistical Analysis
SA behavior was analyzed with linear mixed effects model
analysis (PASW 18.0) followed by Bonferroni corrected post
hoc comparisons. Microarray data were analyzed using
Illumina BeadStudio Software (version 3.1). Two samples
from LgA-Day 14 group were excluded from further analysis
because of poor quality (BeadStudio Gene Expression
Module, version 3). Ingenuity Pathway Analysis (Ingenuity
Systems, version 8.0, Redwood City, CA) was used to filter
microarray data as described in Bernard et al (2010) to only
include genes with Student’s t-test p-values of p0.1. These
relaxed parameters were used to maximize the number of
candidate genes for downstream analysis. A specific gene
was considered significantly altered if (1) the p-value was
p0.05, and (2) the fold-change was X1.2. The rest of the
data were analyzed with GraphPad Prism, version 5. Results
are expressed as mean values±SEM. One-way or two-way
ANOVAs followed by a multiple comparison test and
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2307
Neuropsychopharmacology
Student’s one or two-tailed t-test were used for statistical
evaluations. Pearson’s correlation coefficients were calcu-
lated to test for possible association between variables. The
level of significance was chosen as pp0.05.
Drugs and Chemicals
Cocaine–HCl was obtained from Mallinckrodt Inc (St Louis,
MO). PARP Cleavage Detection Kit was obtained from
Calbiochem (Darmstadt, Germany). Other materials were
purchased from Santa Cruz Biotechnology, Sigma-Aldrich,
and Vector Laboratories.
RESULTS
Escalation of Cocaine Intake during Cocaine SA
Procedures
Before the experiments reported in this paper, we con-
ducted a pilot dose-finding study using a separate group of
purchased Sprague–Dawley rats to determine the optimal
dose of cocaine to elicit escalation of drug intake during
LgA cocaine SA procedures (three doses tested: 0.5, 1, and
2 mg/kg per infusion, 5-h session per day, 14 days; data not
shown). Our preliminary studies showed that a dose of
0.5 mg/kg per infusion resulted in a profound increase in
drug intake from the first 5-h SA session (day 1) to the last
(day 14) (data not shown). Consequently, for the present
results, we followed a similar paradigm (0.5 mg/kg per
infusion, 5-h session per day, 14 days; see Figure 1a for
experimental design) with a new batch of rats. As shown in
Figure 1b, LgA cocaine SA procedures (0.5 mg/kg per
infusion, 5-h session per day, 14 days) elicited a profound
increase in drug intake from the first SA session (day 1) to
the last (day 14) (effect of session: F(13, 100) ¼26.09,
po0.0001). Rats progressively increased their daily
cocaine intake starting at an average of 15 mg/kg on the
first day of SA and finishing at an average intake of about
50 mg/kg on the final SA session day (Bonferroni post hoc
comparison session 1 vs session 14, ***po0.0001). When
examining the motivational aspects of cocaine intake by
analyzing the first hour of each of the 5-h sessions there
was no difference in the pattern of intake when compared
with the entire sessionFaverage intake of 3.5 mg/kg
and 12.5 mg/kg on the first and last day of SA respective-
lyF(data not shown). The present behavioral data provided
a good animal model to further ascertain the cellular
and molecular adaptations that occur in the hippocampus
following cocaine exposure.
Decreased Proliferation of Adult Hippocampal Stem
Cells
As depicted earlier, the brains from rats that underwent
cocaine SA procedures (LgA) and their respective controls
(ND, see Figure 1a) were used to examine hippocampal
proliferation with different cell markers. Figure 2 shows the
effects of the time-course of withdrawal (ie, Day 1, Day 14,
and Day 28) following cocaine SA on: (1) Ki-67 + cells
(proliferation of all dividing cells, within a cell cycle time of
25 h; Cameron and McKay, 2001), and (2) NeuroD + cells
(proliferation of neural progenitor cells) in the DG (see
Garcı
´a-Fuster et al, 2010).
Measures of Ki-67 + (Figure 2a) and NeuroD +
(Figure 2b) cells at the different time points of analysis
revealed that: (1) chronic exposure to cocaine, as measured
24 h following the last 5-h SA session (LgA-Day 1), did not
alter the number of Ki-67 + or NeuroD + cells in the DG
relative to ND-Day 1 control group (Ki-67: p¼0.95, NS;
NeuroD: p¼0.31, NS); (2) 14 days of withdrawal following
the last 5-h SA session led to a significant decrease in the
number of Ki-67 + and NeuroD + cells in the DG relative to
ND-Day 14 control group (Ki-67: **po0.01; NeuroD:
*po0.05); and (3) 28 days of withdrawal following the last
5-h SA session did not change the number of Ki-67+ or
NeuroD + cells in the DG when compared with the ND-Day
28 control group (Ki-67: p¼0.65, NS, NeuroD: p¼0.90, NS).
Interestingly, if only comparing the LgA groups (LgA-
Day 1, LgA-Day 14, and LgA-Day 28), Day 28 samples have
similar levels of Ki-67 + and NeuroD + cells compared with
those observed at Day 14 (see groups LgA-Day 28 in Figures
2a and b). However, as the control groups also had lower
values on these measures (see ND-Day 28 both for Ki-67 +
and NeuroD + cells), these comparisons did not reach
statistical significance. In fact, there were no significant
differences between the control groups at any time point
of withdrawal (ND-Day 1, ND-Day 14, and ND-Day 28)
as measured by a one-way ANOVA followed by Tukey’s
multiple comparison test (Ki-67: F(2, 14) ¼1.861, p¼0.19, NS;
NeuroD: F(2, 14) ¼1.474, p¼0.26, NS).
When splitting the analysis by anterior (1.80 to 4.52 mm
from Bregma) and posterior (4.52 to 6.80 mm from
Bregma) hippocampal demarcation (Guzma
´n-Marı
´net al,
2003; Noonan et al, 2008), the posterior had more positive
cells than the anterior part (Ki-67: ***po0.001, Figure 2c;
NeuroD: **po0.01, Figure 2d). The results showed a similar
pattern as the overall cell quantification, with decreased Ki-67
+ (Figure 2c) and NeuroD + (Figure 2d) cells in LgA-Day 14
both in the anterior and posterior demarcation (Ki-67:
*po0.05; NeuroD: *po0.05) when compared with ND-Day
14. Moreover, if the analysis was split by anatomical
level (Figures 2e and f), a two-way ANOVA followed
by Bonferroni’s post hoc test revealed an effect of group
(ND-day 14 vs LgA-Day 14; Ki-67, F(1, 120) ¼15.82,
p¼0.0001; NeuroD, F(1, 119) ¼14.77, p¼0.0002) and an
effect of anatomical level (Bregma 1.80 to 6.80; Ki-67,
F(11, 120) ¼13.09, po0.0001; NeuroD, F(11, 119) ¼14.82,
po0.0001). However, no interaction between group and
anatomical level was found (Ki-67, F(11, 120) ¼1.41,
p¼0.175; NeuroD, F(11, 119) ¼0.88, p¼0.566). Post hoc
analysis revealed that changes in Ki-67 + cells occurred at
the more posterior level of the hippocampus (distance from
Bregma: -6.30 mm, **po0.01).
In an attempt to ascertain the relationships between
the different variables that account for the changes in
Ki-67 + and NeuroD + cells during cocaine withdrawal we
carried out several correlational analyses. There was a
significant positive correlation between Ki-67 + and
NeuroD + cells (r¼0.76; n¼37; ***po0.00001) in the DG
when all treatment and control groups were collapsed
(Figure 3a). If the analysis was conducted separately
for each treatment group (ie, basal effect in the ND controls
and drug effect in the LgA groups), the correlation
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2308
Neuropsychopharmacology
Figure 2 Decreased proliferation of adult hippocampal stem cells. (a, b) Quantitative analysis of Ki-67 + (a) and NeuroD + (b) cells in the left
hemisphere DG revealed a decrease in cell proliferation at Day 14 of withdrawal following LgA cocaine SA. **po0.01, *po0.05 when compared with their
respective control (ND-Day 14). (c, d) Analysis split by anterior (1.80 to 4.52 mm from Bregma) and posterior (4.52 to 6.80 mm from Bregma)
hippocampal demarcation. The posterior DG had more Ki-67 + and NeuroD + cells than the anterior DG. ***po0.001, **po0.01. Ki-67 + and
NeuroD + cells were decreased both in the anterior and posterior demarcation (Ki-67: *po0.05; NeuroD: *po0.05) in LgA-Day 14 when compared with
ND-Day 14. (e, f) Quantitative analysis of Ki-67 + (e) and NeuroD + (f) cells in relation to distance from Bregma in ND-Day 14 and LgA-Day 14 groups. (g,
h) Representative IHC of Ki-67 + (g, brown labeling) and NeuroD + (h, dark blue labeling) comparing ND vs LgA at Day 14. The bigger images were taken
in a light microscope using a 20 objective lens to illustrate the anatomy of the DG. For each image, the left-bottom corner shows a representative
IHC image taken in a light microscope using a 63 oil objective lens. Scale bar: 25 mm. The color reproduction of this figure is available at the
Neuropsychopharmacology journal online.
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2309
Neuropsychopharmacology
remained for both groups (ND groups, r¼0.84; n¼17;
***po0.0001; LgA groups: r¼0.60; n¼20; **p¼0.005).
However, the area under the curve for the LgA groups is
smaller, indicating that cocaine exposure reduced the
number of Ki-67 + and NeuroD + cells in the DG of
drug-exposed animals.
Figure 3b illustrates that the magnitude of decrease in
cell proliferation as measured by Ki-67 + cells in the
DG was correlated with the total amount of cocaine intake
(mg/kg) (r¼0.51, n¼20, *p¼0.02). However, there was
no correlation when comparing the number of NeuroD +
cells with the amount of cocaine intake (r¼0.03, n¼20,
p¼0.91, NS).
Thus, this study provides a specific time-point of cell
proliferation regulation during withdrawal (ie, 14 days) at
which both Ki-67 + (ie, proliferation of all dividing cells)
and NeuroD + cells (ie, proliferation of cells that are fated
to become neurons) were decreased. Since the only
significant effects were found at Day 14 of withdrawal, the
remaining experiments and analyses were conducted at this
time-point.
Gene Expression Profile Analysis
The analysis comparing the left DG of ND vs LgA at Day 14 of
withdrawal revealed the top networks by rank order and their
associated network functions. The results uncovered differ-
ential regulation of several genes associated with cell fate
regulation. In particular, the top bio functions associated with
gene expression changes included molecular and cellular
functions such as cell death, cell-to-cell signaling and inter-
action, cell morphology, cellular assembly and organization,
and cellular development (see Table 1). Given the decrease in
Ki-67 + and NeuroD + cells at 14 days of withdrawal following
LgA cocaine SA, we decided to further analyze the list of
candidate genes rendered in the top bio function (ie, cell
death). The list provided 28 genes, 10 of which were genes of
known sequences. A complete list of altered genes for the
associated top three network functions can be found in the
Supplementary Material (Supplementary Figure I).
As mentioned above, given that cell proliferation occurs
mainly in the SGZ, which represents a fraction of the total DG,
evaluation of gene expression profiles in the entire DG
may dilute the effects taking place in the SGZ (Ki-67 + and
NeuroD + cell counts). Indeed, NeuroD gene expression was
not found altered in the microarray analysis when comparing
ND and LgA groups at Day 14 of withdrawal. Moreover, among
the genes found to be altered in a gene expression profile
analysis some will be false positives and some false negatives.
Gene Expression Profile Validation
To validate some of the changes observed in gene
expression by RT-PCR, we performed gene quantification
in samples from the left hemisphere DG with custom
ordered TaqMan Arrays. The results showed that two genes
reached statistical significance, Achaete-scute complex
homolog 1 (Ascl-1) and DNA (cytosine-5)-methyltransfer-
ase 3 beta (Dnmt3b), both previously described to have a
role in cell fate regulation. However, FADD gene expression
fell below the gene-card threshold level, and thus was
undetectable by this method. Two factors could account for
LgA-Day 1
LgA-Day 14
LgA-Day 28
ND-Day 1
ND-Day 28
ND-Day 14
2500 5000 7500 10000 12500
10000
15000
20000
25000
30000
35000 r=0.76; n=37; p<0.0001
Ki-67+ Cells
NeuroD+ Cells
35000 r=0.84; n=17; p<0.0001
r=0.60; n=20; p=0.005
r=-0.51; n=20; p=0.02 r=0.03; n=20; p=0.91, n.s.
30000
25000
20000
NeuroD+ Cells
15000
10000
2500
LgA Groups
35000
30000
25000
20000
NeuroD+ Cells
15000
10000
35000 LgA-Day 1
LgA-Day 14
LgA-Day 28
30000
25000
20000
15000
10000
0 200 400
Cocaine lntake (mg/kg)
600 800 1000
NeuroD+ Cells
2500
LgA Groups
5000 7500 10000 12500
Ki-67+ Cells
12500
10000
7500
5000
Ki-67+ Cells
2500
0
0200 400
Cocaine lntake (mg/kg)
600 800 1000
ND-Day 1
ND-Day 14
ND-Day 28
5000 7500
Ki-67+ Cells
LgA-Day 1
LgA-Day 14
LgA-Day 28
10000 12500
Figure 3 Correlation analysis. (a) Scatterplot depicting a significant positive correlation between the number of Ki-67 + and Neuro + cells in the DG of
rats independently of their treatment. Each symbol represents a different rat. The solid line is the best fit of the correlation (r¼0.76, n¼37, po0.0001). The
dotted curves indicate the 95% confidence interval for the regression line. To note that this correlation is still there for each treatment group individually
(ND groups: r¼0.84, n¼17, po0.0001; LgA groups: r¼0.60, n¼20, p¼0.005). (b) Scatterplot depicting a significant negative correlation between the
number of Ki-67 + cells and the amount of cocaine intake (mg/kg) in the DG of LgA groups rats. Each symbol represents a different rat. The solid line is the
best fit of the correlation (r¼0.51, n¼20, p¼0.02). The dotted curves indicate the 95% confidence interval for the regression line. To note that there was
no correlation when comparing the number of NeuroD + cells with the amount of cocaine intake (r¼0.03, n¼20, p¼0.91).
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2310
Neuropsychopharmacology
this issue: (1) the initial limited amount of cDNA material in
our samples, and (2) the fact that this transcript might have
relatively low abundance in the DG. Therefore, considering
FADD has been described as a key molecule regula-
ting apoptotic and/or proliferative events (Tourneur and
Chiocchia, 2010) involved in cocaine addiction (Garcı
´a-
Fuster et al, 2009), and was significant in the microarray
analysis (F-change ¼1.31, *p¼0.022), further validation
was done at the mRNA level by ISH and at the protein level
by WB analysis.
Possible Role of the Cell Fate Regulator FADD
As depicted in Figure 4a, FADD mRNA was increased in the
left hemisphere DG of rats exposed to 14 days of withdrawal
following LgA cocaine SA procedures when compared with
their respective controls (*p¼0.014). Moreover, if the
analysis was split by anatomical level (see Figure 4b), a
two-way ANOVA followed by Bonferroni’s post hoc test
revealed an effect of group (ND-Day 14 vs LgA-Day 14;
F(1, 145) ¼20.62, po0.0001), an effect of anatomical level
Table 1 This Table Illustrates the Results of the Microarray Experiment and Further Validation by RT-PCR or ISH Performed in ND- and
LgA-Day 14 Samples from the Left Hemisphere DG
Top networks
Associated network functions by rank order (po0.001) # Molecules Score
1. Cellular growth and proliferation, embryonic development, molecular transport 22 42
2. Cellular assembly and organization, behavior, organ morphology 23 40
3. Neurological disease, genetic disorder, cancer 17 30
4. Cellular development, nutritional disease, organismal injury and abnormalities 15 25
5. Cellular compromise, cell signaling, molecular transport 14 22
Top bio functions
Molecular and cellular functions # Molecules p-Value
Cell death 28 0.0003–0.0472
Cellular development 28 0.0015–0.0498
Cellular assembly and organization 20 0.0015–0.0498
Cell-to-cell signaling and interaction 14 0.0011–0.0498
Cell morphology 14 0.0015–0.0498
Cell death # Molecules p-Value
ABCC5, ASCL1, CD200, DICER1, DMD, DNASE1, DNMT3B, F2R, FADD, FAF1,
JAK3, KIAA1967, LDHA, LGALS1, MAOB, MT1F, NTRK3, NUF2, PHIP, PLCD1,
PPP2R2B, PPT1, PTPRE, RECK SCG5, SMAD2, SNCG, SOD2
28 0.0361
Microarray
results
Gene expression
validation
Gene name Symbol F-change p-Value F-change p-Value
Achaete-scute complex homolog 1 (Drosophila) ASCL1 1.35 0.036 3.03 0.010
Deoxyribonuclease I DNASE1 1.32 0.036
DNA (cytosine-5-)-methyltransferase 3 beta DNMT3B 1.57 0.030 4.01 0.059
Coagulation factor II (thrombin) receptor F2R 2.36 o0.001
Fas (TNFRSF6)-associated via death domain FADD 1.31 0.022 1.35 0.014
Janus kinase 3 JAK3 1.47 0.020
Phospholipase C, delta 1 PLCD1 1.35 0.047
Protein phosphatase 2 (formerly 2A), regulatory subunit B, beta isoform PPP2R2B 1.34 0.018
Palmitoyl-protein thioesterase 1 PPT1 1.30 0.046
Synuclein, gamma (breast cancer-specific protein 1) SNCG 1.68 0.022
Only genes with known sequences
F-change41.2,po0.05
The microarray data were filtered to only include genes with Student’s t-test p-values of p0.1. These relaxed parameters were used to maximize the number of
candidate genes for downstream analysis. The table includes the results from the analysis and includes the top network functions and the top bio functions. Cell death
was the function with more molecules dysregulated (28 genes). For further analysis, a specific gene was considered significantly altered if (1) the p-value was p0.05,
and (2) the fold-change was X1.2.
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2311
Neuropsychopharmacology
(Bregma 1.80 to 5.80 mm; F(16, 145) ¼38.52, po0.0001),
and an interaction between group and anatomical level
(F(16, 145) ¼2.31, po0.01). Post hoc analysis revealed that
changes in FADD mRNA occurred at the most posterior
level of the DG (distance from Bregma: 4.55 mm, *po0.05;
4.80 mm, *po0.05; 5.05 mm, ***po0.001). These results
validated the data observed in the microarray analysis and
pointed out the importance of the abundance of the gene
and tissue limitations when choosing a method to validate
the results.
Moreover, the results showed that, although it did not
reach statistical significance, FADD protein was increased
(20%, p¼0.09) in the right hemisphere hippocampus of
LgA-Day 14 rats when compared with ND-Day 14 group (see
Figure 4d). At the other withdrawal time-points, there was
no change in FADD protein content (ND- vs LgA-Day 1 and
ND- vs LgA-Day 28) (data not shown). b-Actin was used as a
loading control and therefore was not altered by the
treatment. To note that for WB analysis we freshly dissected
the whole right hippocampus, while for gene expression
analysis we focused on the left hemisphere DG and did so in
an anatomical context. Thus, some of the protein changes
occurring at the level of the DG could be masked or diluted
when examining the whole hippocampus. Moreover, some
studies suggested that cell proliferation (Cze
´het al, 2007) and
gene expression profiles (Stansberg et al, 2007) in rat brain
are different and differently regulated between the left and
right hemisphere and along front and back of the DG.
Assay for Apoptotic Cell Death
PARP enzyme, which is involved in DNA damage following
DNA nicks, was investigated in the right hippocampus as a
molecular marker of cell death (Cagnol et al, 2006). As
shown in Figure 4e, ND-Day 14 and LgA-Day 14 showed
similar levels of basal 116 kDa PARP cleavage (B85 kDa
fragment) indicating similar rates of basal induction of
apoptotic cell death.
DISCUSSION
This study examined changes in hippocampal cell fate
regulation in an animal model that mimics some of the key
features manifested during the transition to cocaine
addiction in humans (eg, escalation of drug intake).
Previous work from our lab had examined the effects
associated with experimenter-administered cocaine on
alterations in neural plasticity (see Garcı
´a-Fuster et al,
2009, 2010). In this study, we utilized a rat model with better
Figure 4 Possible role of the cell fate regulator FADD. (a) ISH analysis: data represents mean±SEM mRNA level of LgA-Day 14 rats when compared
with the percentage of the ND-control group (100%) at 14 days of withdrawal (ND-Day 14) in the left hemisphere DG determined by ISH analysis
(*p¼0.014). (b) ISH analysis: FADD mRNA across the anatomical level of the DG analyzed (1.80 to 5.80 mm from Bregma) (*po0.05, ***po0.001 vs
ND-Day 14). (c) ISH analysis: representative X-ray images of FADD mRNA for each treatment group at two levels of analysis (anterior DG: 2.30 mm and,
posterior DG: 4.80 mm from Bregma) (d) WB analysis: data represent mean±SEM protein level (LgA-Day 14), and are expressed as percentage of the
ND-control group (100%) at 14 days of withdrawal (ND-Day 14) in dissected right hippocampus. Student’s t-test did not detect significant changes between
groups. FADD: nonsignificant 19% increase, p¼0.09. The figure also illustrates representative immunoblots of the corresponding proteins. b-Actin was used
as a loading control. (e) Assay for apoptotic cell death. ND-Day 14 and LgA-Day 14 showed similar levels of basal 116 kDa PARP cleavage (B85 kDa
fragment) indicating similar rates of basal induction of apoptotic cell death in the right hemisphere hippocampus. The figure also illustrates a representative
immunoblot of the corresponding protein.
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2312
Neuropsychopharmacology
clinical validity in which to study the hippocampal
adaptations associated with withdrawal after cocaine abuse.
Using Sprague–Dawley rats we demonstrated that 5-h of
extended daily access to cocaine for 14 days (LgA) can elicit
a profound increase in drug intake from the first SA session
to the last, providing an excellent model to study the neural
adaptations that accompany cessation of cocaine exposure.
Our findings demonstrate that LgA cocaine SA leads to
alterations at various levels of hippocampal cell fate
regulation at a specific time point during the course of
withdrawal (ie, Day 14). Specifically, we showed that: (1)
proliferation of adult hippocampal stem cells was decrea-
sedFKi-67 + mitotic progenitor cells, and NeuroD +
neural progenitor cells; (2) several ensembles of genes were
significantly altered, generally revolving around various
facets of cellular morphology, cell development or cell
death; (3) specific genes associated with cell fate regulation
were validated as being dysregulated during withdrawal,
including Ascl-1 and Dnmt3b, two potential novel targets
for the treatment of cocaine withdrawal; and (4) FADD, a
molecular marker previously associated with the propensity
to substance abuse and cocaine sensitization (Garcı
´a-Fuster
et al, 2009), was upregulated, which suggests this is a key
molecular candidate in mediating hippocampal cell fate
regulation during cocaine withdrawal.
Decreased Proliferation of Adult Hippocampal
Stem Cells
The chronic administration of cocaine following LgA SA
procedures (Day 1) did not alter proliferation of adult
hippocampal stem cells, as measured by the number of Ki-
67 + cells 24 h after the last SA session. Using a similar
experimental design, Noonan et al (2008) reported a
decrease in cell proliferation as measured by BrdU + cells
24 h following the last cocaine SA session (0.5 mg/kg per
infusion, 4-h session per day, 3 weeks). However, differ-
ences in the experimental design (ie, food-restriction during
pre-training, amount of cocaine intake in 4- vs 5-h of daily
SA session) and in the marker used to quantify cell
proliferation (ie, BrdU only labels the % of proliferating
cells that entered S-phase) could account for this discrep-
ancy and adds to the complexity of comparing data across
labs. Even though BrdU + and Ki-67 + cell numbers are
often used as interchangeable indices of proliferation
(Kee et al, 2002; Wojtowicz and Kee, 2006), cells in S-phase
may be particularly susceptible to certain stimuli (Arguello
et al, 2008). The same group (Lagace et al, 2010) recently
described discrepancies in how a specific treatment in
mice affected cells that entered S-phase (BrdU labeling) vs
all proliferating cells (Ki-67 labeling), as they found a
decrease in BrdU + and no change in Ki-67 + cell number
24 h after treatment. This interpretation is limited
because of the fact that cell cycle length is about half as
long in the mouse than in the rat. However, in another study
conducted in rats and using BrdU and Ki-67 as mitotic
makers to measure changes in cell proliferation (Domı
´n-
guez-Escriba
`et al, 2006), Ki-67 + cell counts failed to reach
statistical significance between the groups because of
variability of the data. In any case, Noonan et al (2008)
have also shown that a longer chronic paradigm of cocaine
SA (7 weeks, 4-h session per day) did not alter cell
proliferation as measured by Ki-67 + cells. Moreover, the
present results show that chronic administration of cocaine
(ND- vs LgA-Day 1) did not have an impact on the number
of the proliferating cells that are fated to become neurons
(NeuroD + cells). Similarly, cocaine SA did not alter the
number DCX + cells (immature neurons) (Noonan et al,
2008).
During the course of withdrawal (Day 14), proliferation of
mitotic (Ki-67 + ) and neural progenitor (NeuroD + ) cells
was decreased. To our knowledge there are no studies
looking at these measures at Day 14 of withdrawal after
extended daily access to cocaine SA in rats. Following a
similar anterior–posterior cell proliferation pattern as the
one presented in this study, Lagace et al (2006) showed that
the posterior part of the DG had more proliferating cells
than the anterior part. The same pattern was described for
DCX + cells (Noonan et al, 2010). The decrease in Ki-67 +
and NeuroD + cells occurred throughout the DG, both at
the anterior and posterior demarcations (Figures 2c and d).
However, when comparing ND- and LgA-Day 14 by
anatomical level (Figures 2e and f), the decrease in Ki-67 +
cells was observed only at the most posterior part of the DG.
Similarly, previous studies showed differences in prolifera-
tion across the entire longitudinal axis; yet the more
posterior part of the hippocampus seems to be the
preferential site of regulation of proliferation by restraint
stress (Kim et al, 2005) and by the antidepressant drug
agomelatine (Banasr et al, 2006). Decreased cell prolifer-
ation might be a reflection of enhanced cell death. However,
the present results showed similar levels of basal induction
of apoptotic cell death when comparing ND-Day 14 and
LgA-Day 14.
At Day 28 of withdrawal there was no effect on the
number of Ki-67 + and NeuroD + cells (ND- vs LgA-Day
28). Previous results found that withdrawal from cocaine
normalized the deficits in cell proliferation and increased
the number of immature neurons (DCX + cells) in the DG
(Noonan et al, 2008). The normalization seen at Day 28
could be due to a lower rate of cell proliferation in the ND-
Day 28 control groups. However, there were no significant
differences in the number of positive cells between the
control groups (Day 1, Day 14, and Day 28) for Ki-67
(F(2, 14) ¼1.861, p¼0.192) or for NeuroD (F(2, 14) ¼1.474,
p¼0.262). Moreover, basal corticosterone levels were
measured in trunk blood at the time of killing and there
were no differences when comparing the control groups
(one-way ANOVA followed by Tukey’s test: F(2, 14) ¼1.812,
p¼0.120, data not shown). Nevertheless, we question
whether differences at Day 28 could be masked because of
a floor effect and whether a number of variables inherent in
the experimental design might obfuscate the results. For
example, the age of the animals at this later time-point
(ie, approximately 140 days old when killed) and the fact
that the rats were socially isolated (ie, individually housed)
for a prolonged period of time and withdrawn from
experimental handling could all be potential stressors
impacting interpretation of the results.
Changes in Ki-67 + and NeuroD + cells were positively
correlated when all treatment groups were collapsed. Our
results indicate that 14 days of cocaine withdrawal
decreased cell proliferation (Ki-67 + cells) and the prolifer-
ation of cells that are fated to become neurons (NeuroD +
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2313
Neuropsychopharmacology
cells). There appears to be some controversy as to whether
Ki-67 and NeuroD colocalize in the same population of
cells. It has been described that about 75% of all prolifer-
ating type-2 cells co-express NeuroD (type 2b/3 progenitors;
see Steiner et al, 2006). In mice NeuroD has been described
to colocalize with Ki-67 only in a small subset of progenitors
(Gao et al, 2009). In contrast, another study showed that
Ki-67 and NeuroD do not colocalize in the same population
of cells (Larsen et al, 2007), and therefore label distinct
groups of cells. In any case new cells are born every day
throughout life, leading to the situation where many
different maturation stages exist in parallel and with close
proximity to each other. It seem reasonable to hypothesize,
therefore, that at least some of the reduction in NeuroD +
cells could be a consequence of the decreased prolifer-
ation of Ki-67 + cells. Further, our data show that both
markers label different number of cell pools: Ki-67 labels all
dividing cells during the 25-h length of the cell cycle, about
6000–9000 cells (see Figure 2a); while NeuroD labels neural
precursors from different pools of proliferating cells, about
20 000–25 000 cells (Figure 2b). This could explain why our
findings linked decreased number of Ki-67 + cells to the
increased amount of drug administered and no relationship
between NeuroD + cells and drug intake was observed.
Similarly, previous data reported that decreases in Ki-67
were attributable to the total amount of drug administered
based on use of differential paradigms (ie, intermittent
short-access SA, short-access SA, and long-access SA)
(Mandyam et al, 2008). Moreover, the same study reported
that doublecortin (DCX), a marker of young neurons, was
not correlated with the amount of drug intake.
What might be the functional significance of these
observed changes in the hippocampus? A reduction of
adult hippocampal neurogenesis (DCX + cells) has recently
been shown to enhance vulnerability to addiction and
relapse (Noonan et al, 2010). The present results support
the view that decreased cell proliferation of neural
precursors (NeuroD + cells) might participate in the
restructuring of the hippocampus during cocaine with-
drawal and identifies 14 days as a critical time-point of
regulation. Withdrawal to drugs of abuse in general, and
cocaine in particular, has been associated with increased
propensity to develop a depression-like phenotype. Since
alterations in hippocampal cellular composition have been
implicated in depression and the mode of action of
antidepressants (Malberg et al, 2000; Jacobs et al, 2000),
one could speculate that the decreased number of mitotic
(Ki-67 + ) and neural progenitors (NeuroD + ) cells ob-
served at 14 days of withdrawal might contribute to
increased propensity for negative affect during withdrawal.
For example, withdrawal from chronic SA enhances startle-
induced ultrasonic distress vocalizations in the rat (Barros
and Miczek, 1996; Mutschler and Miczek, 1998). A recent
study using mice reported a significant negative correlation
between rates of cell proliferation and cell survival and
depression-like behavior at 14 days of withdrawal from
alcohol. Moreover, the behavior was reversed by chronic
antidepressant treatment during withdrawal (Stevenson
et al, 2009). Therefore, changes in the cellular composition
(ie, proliferation) of the DG could be involved in some of
the alterations underlying cocaine withdrawal (ie, affective
components) and warrants further investigation.
Gene Expression Profile Analysis and Validation
A fundamental understanding of the DG cellular adapta-
tions induced by cocaine withdrawal requires investigating
its molecular basis in order to develop novel targets for its
regulation. To this end, we relied on a discovery approach
by using gene expression profile analysis in the DG of ND-
Day 14 vs LgA-Day 14 rats where the greatest changes in Ki-
67 + and Neuro + cells were observed. Given that cell
proliferation mainly occurs in the SGZ, which represents a
fraction of the total DG, evaluation of gene expression
profiles in the entire DG may dilute the effects taking place
in the SGZ (Ki-67 + and NeuroD + cell counts). Therefore,
given the number of cells analyzed (SGZ vs the whole DG)
and the dilution factor derived from the analysis, unraveling
effects on cell proliferation from transsynaptic effects on
other hippocampal cells should be evaluated carefully. The
results revealed highly significant dysregulation in a
number of networks and molecular and celullar functions
(Table 1). Interestingly, the dysregulation of ‘cell death’
emerged as the top bio function with highly significant
changes in 28 genes. We went on to validate 3 of the 10
transcripts by RT-PCR or ISH.
One such gene, Dnmt3b, was downregulated in the DG
of rats that underwent 14 days of withdrawal following
LgA cocaine SA. Epigenetic mechanisms, such as DNA
methylation, were initially described for their ability to
promote differentiation and drive cell fate in embryonic
and early postnatal development (reviewed in Hsieh and
Eisch, 2010). In particular, Dnmt3b is a cellular methyl-
transferase that adds methyl groups de novo to unmethyl-
ated DNA and has been previously shown to participate
in various stages of neural fate (Feng et al, 2005) including
the regulation of synaptic plasticity in the hippocampus
(Levenson et al, 2006). Dnmt3a and Dnmt1, which are two
other key methyltransferases, were not found altered in
the microarray analysis (data not shown). Thus, these
data suggest a specific role for Dnmt3b on cell fate
during cocaine withdrawal and one that deserves further
investigation.
A second candidate gene, Ascl-1, also called as Mash-1,
was decreased in the DG of rats with decreased number of
Ki-67 + and NeuroD + cells. Mash-1 is a member of the
basic helix-loop-helix transcription factor group and
contributes to the production of neuronal precursor cells
(Johnson et al, 1990; Jessberger et al, 2008), thus having a
role in the regulation of the restructuring of the hippo-
campus. Therefore, a decrease in the pro-neural gene Ascl1
goes along with the decrease observed at the level of
proliferation of adult hippocampal stem cells (Ki-67 + and
NeuroD + cells), and also deserves further investigation.
Possible Role of the Cell Fate Regulator FADD
The present data showed an increase in FADD by gene
expression profile analysis in the DG during the course of
cocaine withdrawal (Day 14). This change was confirmed by
ISH, with increased gene expression at the more posterior
level of the DG. Interestingly, the differential response of the
anterior and posterior demarcation, also observed by
changes in cell proliferation, might be related to the distinct
anatomic connections and functions of these regions. The
Cell fate regulation during cocaine withdrawal
MJ Garcı
´a-Fuster et al
2314
Neuropsychopharmacology
ventral and posterior DG receives many limbic projections,
and is more implicated in emotion-like behaviors relative to
the dorsal and anterior DG, which has a greater role in
spatial processing (Bannerman et al, 2004; Sahay and Hen,
2007). Therefore, it is reasonable to hypothesize that
changes in the cellular/molecular composition (ie, prolif-
eration, FADD gene expression) of the more posterior part
of the DG during cocaine withdrawal may be critical in
understanding the neural adaptations that accompany drug
cessation. Moreover, the hypothesis that FADD regulation
in the DG parallels changes in cell proliferation (Ki-67 +
and NeuroD + cells) at Day 14 of withdrawal is strength-
ened by the fact that there was no change in FADD protein
content nor in cell proliferation markers either at Day 1 or
Day 28 between ND and LgA groups.
FADD, among the many cell fate mediators, is a unique
regulator of cell life and death and has a critical role in
many essential cellular processes (Tourneur and Chiocchia,
2010). FADD, initially described to be part of the apoptotic
machinery, has since been shown to control a variety of
intracellular processes that regulate cell fate toward cell
proliferation, cell growth, cell survival, and cell death. Thus,
this molecule is pivotal for maintaining cellular function
and homeostasis. Importantly, FADD has also been
implicated in the neural adaptations associated with cocaine
(Garcı
´a-Fuster et al, 2009) and morphine sensitization
(Ramos-Miguel et al, 2010) as well as with responsiveness to
opiate (Garcı
´a-Fuster et al, 2007) and cannabinoid drugs
(Alvaro-Bartolome et al, 2010). Moreover, animals selec-
tively bred for differential propensity to substance abuse
(high- vs low-responder rats) showed basal differences in
the expression of FADD (Garcı
´a-Fuster et al, 2009),
suggesting this gene might be involved in conferring
vulnerability to developing addiction. However, to our
knowledge, its role in an animal model that better mimics
the transition to cocaine addiction and resembles some of
the key features of human addictive behavior has not
previously been described. The fact that FADD emerged as
one of the most altered transcripts in the context of gene
expression profiling converges with the previous hypoth-
esis-driven studies of its role, and underscores its im-
portance in the sequelae of drugs of abuse and withdrawal
on brain structure and function. Further experiments will
attempt to better understand the molecular mechanisms
behind FADD activation during cocaine administration and
withdrawal and its specific role in hippocampal cell fate
regulation. Moreover, the specific role of FADD in the
regulation of newly proliferating cells in the SGZ will be
evaluated in detail.
CONCLUSION
This study identified specific hippocampal adaptations
during cocaine withdrawal at the cellular and molecular
levels in an animal model that mimics some aspects of
human addiction. In particular, the proliferation of adult
hippocampal stem cells was decreased in the SGZ of the DG
at several stages of regulation (Ki-67 + cells and NeuroD +
cells) following 14 days of cocaine withdrawal. Moreover,
several molecules were dysregulated in the DG of rats with
impaired proliferation rates. FADD, a protein previously
linked to cell fate dysregulation (balance proliferation/
apoptosis) in response to cocaine abuse (Garcı
´a-Fuster et al,
2009), was increased following 14 days of cocaine with-
drawal. Moreover, this study identified two additional key
molecules, Ascl-1 and Dnmt3b, which appear to be involved
in the structural changes that accompany cocaine with-
drawal. We suggest that the decrease in the self-renewal
capacity of the DG together with the altered expression of
some cell fate markers are key to better understanding the
restructuring of the hippocampus during cocaine with-
drawal. These discoveries, therefore, offer novel candidate
targets for further investigation into the neural adaptations
underlying cocaine withdrawal.
ACKNOWLEDGEMENTS
This study was supported by NIDA 5P01DA021633-02;
NIMH Conte Center Grant #L99MH60398; Office of Naval
Research (ONR) N00014-09-1-0598 to HA and SJW; and The
Pritzker Neuropsychiatric Research Foundation. The
authors would like to thank Sharon Burke, Jennifer
Fitzpatrick, James Beals, and Tracy Simmons for excellent
technical assistance. Moreover, the authors would like to
thank Suzanne Smith for her help in the microarray analysis
as well as Stephanie Cooke and Ellen Pedersen for their help
in performing the TaqMan Arrays. MJGF is a ‘Ramo
´ny
Cajal’ Researcher (MICINN-UIB).
DISCLOSURE
The authors declare no conflict of interest.
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Cell fate regulation during cocaine withdrawal
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