Addiction Biology

PRECLINICAL STUDY doi:10.1111/j.1369-1600.2010.00241.x

High cocaine dosage decreases neurogenesis in the

hippocampus and impairs working memory

Einav Sudai1, Ofri Croitoru1, Alona Shaldubina2, Lital Abraham1, Iris Gispan1,
Yakov Flaumenhaft1, Ilana Roth-Deri1, Noa Kinor1, Shai Aharoni1, Moshe Ben-Tzion3 &
Gal Yadid1,2
Neuropharmacology Laboratory, The Mina & Everard Goodman Faculty of Life Sciences, Israel1, The Leslie and Susan Gonda (Goldschmied) Multidisciplinary
Brain Research Center, Israel2 and Department of Chemistry, Bar-Ilan University, Israel3

ABSTRACT adb_241 251..260

Drug addiction is a chronic brain disorder, characterized by the loss of the ability to control drug consumption. The
neurobiology of addiction is traditionally thought to involve the mesocorticolimbic system of the brain. However, the
hippocampus has received renewed interest for its potential role in addiction. Part of this attention is because of the fact
that drugs of abuse are potent negative regulators of neurogenesis in the adult hippocampus and may as a result impair
learning and memory. We investigated the effects of different dosages of contingent cocaine on cell proliferation and
neurogenesis in the dentate gyrus of the hippocampus and on working memory during abstinence, using the water
T-maze test, in adult rats. We found that cocaine, in addition to the changes it produces in the reward system, if taken
in high doses, can attenuate the production and development of new neurons in the hippocampus, and reduce working
memory.

Keywords Cocaine, hippocampus, neurogenesis, self-administration, water T-maze, working memory.

Correspondence to: Gal Yadid, Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail:
yadidg@mail.biu.ac.il

INTRODUCTION and reciprocally innervates the hippocampal formation

It has been demonstrated that repeated exposure to sub-
stances of abuse results in brain region-specific neuroad-
aptations that correlate with the loss of the ability to
control drug use (Everitt, Dickinson & Robbins 2001).
This evidence indicates a prominent role for limbic brain
regions, such as the prefrontal cortex (PFC; Everitt &
Robbins 2005) and the hippocampus in drug addiction
(Berke & Eichenbaum 2001). Drug-induced neuroadap-
tations in the hippocampus are of particular interest,
because of the fact that this structure is anatomically well
positioned to influence brain reward circuitry. The hip-
pocampus receives input from both the nucleus accum-
bens (NAc) and ventral tegmental area (VTA) and sends
output to the NAc (Kelley & Domesick 1982; Totterdell &
Smith 1989; Blood & Zatorre 2001). Furthermore, neu-
roadaptations in the PFC were recently shown to impair
working memory in rats trained to self-administer
cocaine (George et al. 2008). Given that the PFC densely
(Swanson & Kohler 1986; Jay, Glowinski & Thierry 1989;
Jay & Witter 1991), it is essential to study the hippocam-
pus’s contribution to this task.
The sub-granular zone (SGZ) of the hippocampal
dentate gyrus (DG) and the subventricular zone of the
lateral ventricle are the two known neurogenic sites of
the adult brain. In the hippocampus, contingents of
newly generated cells born in the SGZ of the DG travel
short distances to become incorporated as granular
neurons in a dynamic process that continues throughout
life (Alvarez-Buylla & Lim 2004; Kempermann, Wiskott
& Gage 2004). The role of adult neurogenesis in the hip-
pocampus is of present unclear. In light of the critical role
of the hippocampus in the process of forming and recov-
ering certain types of memory (Squire, Stark & Clark
2004), the integrity of the neurogenesis process may be
necessary for the acquisition and consolidation of memo-
ries (Shors et al. 2001; Cao et al. 2004; Crandall et al.
2004). Only a limited number of studies have examined

Conflict of interest: All the authors state that they have no conflict of interest to declare.

© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction Addiction Biology, 16, 251–260

252 Einav Sudai et al.
the role of working memory that involves both the hip-
pocampus and the PFC (Wall & Messier 2001; Jones
2002). Winocur et al. (2006) suggest that the suppres-
sion of hippocampal neurogenesis interferes with
working memory at long intra-trial delays. Others argue
that it might not fulfill a unitary function in memory and
may have opposite roles in distinct types of memory (Saxe
et al. 2007).
Accrued experimental evidence shows that addictive
substances, such as alcohol (Herrera et al. 2003), opiates
(Eisch et al. 2000; Eisch & Harburg 2006) and amphet-
amines (Teuchert-Noodt, Dawirs & Hildebrandt 2000)
can negatively affect the self-renewal capacity of the hip-
pocampus by diminishing the rate of proliferation of
neural progenitors or by impairing the long-term survival
of neural precursors, or both.
A number of studies have demonstrated that chronic
cocaine exposure (non-contingent or contingent drug
administration) decreases the rate of proliferation
(Yamaguchi et al. 2004, 2005; Dominguez-Escriba et al.
2006; Noonan et al. 2008) of neural progenitor cells
in the DG without altering their rate of survival
(Dominguez-Escriba et al. 2006; Noonan et al. 2008).
There remains, however, considerable uncertainty over
the functional significance of this rate of proliferation in
relation to cocaine abuse. One study (Del Olmo et al.
2006) demonstrated that cocaine self-administration
affects long-term potentiating (LTP) but does not notably
affect performance in the Morris water maze test. In con-
trast to these findings, in a different study, results showed
an improvement in the rats’ performance in a difficult
Morris water maze task following cocaine self-
administration (Del Olmo et al. 2007).
We used the self-administration method combined
with the water T-maze test to examine the effect of with-
drawal of dose-dependent chronic cocaine intake on neu-
rogenesis in the DG of the hippocampus, and on working
memory during abstinence in adult rats.
MATERIALS AND METHODS
Subjects
Male Sprague–Dawley rats (250–280 g) were main-
tained on a 12–12 hour dark-light cycle with free access
to food and water. All experimental procedures were
approved by the Animal Care and Use Committee of the
University and were performed in accordance with the
guidelines of the National Institutes of Health.
Behavioral procedures
Cocaine self-administration
The rats were implanted with intravenous silastic
catheters under anesthesia (ketamine 100 mg/kg and
© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction
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xylazine 10 mg/kg) into the right jugular vein (Roth-Deri
et al. 2003). The catheter was secured to the vein with
silk sutures and was passed subcutaneously to the top of
the skull where it exited into a connector (a modified
22-gauge cannula; Plastics One, Roanoke, VA) mounted
to the skull with MX-80 screws (Small Parts, Inc., Miami
Lakes, FL) and dental cement (Yates & Bird, Chicago, IL).
Catheters were flushed every 24 hours with sterile saline
and the antibiotic gentamicine (0.08 mg/ml) to prevent
infections.
Rats were trained to self-administer cocaine or saline
as previously described (Roth-Deri et al. 2003). Briefly, 5
days after catheterization, rats were transferred to
operant conditioning chambers (Medical-Associates,
Inc.; St. Albans, VT; hardware and software of interface
were designed by Dr M. Ben Tzion using LabVIEW,
National Instruments) for 1 hour daily until stable main-
tenance levels were attained (at least 3 days of 20% devia-
tion from the mean) during their dark cycle, and were
allowed to self-administer saline (0.13 ml/infusion) or
cocaine (0.13 ml/infusion; 0.5 mg/kg/20 seconds; or
1.5 mg/kg/20 seconds; obtained from the National Insti-
tute of Drug Abuse, Research Technology Branch, Rock-
ville, MD) via a lever press under an FR-1 schedule of
reinforcement. The group defined as 0.5 mg/kg cocaine,
received 1 mg/kg cocaine per infusion during the first 5
days of training and then switched to 0.5 mg/kg cocaine
for the rest of the experiment. During the infusion, a light
located above the active lever was lit for 20 seconds.
During the 20-second intervals of cocaine infusion, active
lever presses were recorded, but no additional cocaine
reinforcement was provided. Presses on the inactive lever
were recorded, but did not activate the infusion pump.
Water T-maze
General procedure. The water escape T-maze consisted of
grey Plexiglas T-maze pool (1-cm thick) filled with water
(23 ⫾ 1°C). The main alley (100 cm, 20 cm, 40 cm) was
connected to two side arms (right and left) (45 cm,
20 cm, 40 cm) by two sliding doors manually operated to
close off either arm. At the end of each arm there was a
platform (Plexiglas, 15 cm, 18 cm) submerged 2 cm
below the surface of the water. The apparatus was set on
a 75-cm high table, in a room without visual cues that
could be used by the animals to guide their response. In
addition, the behavioral task was performed under red
light, during the dark period of the circadian light/dark
cycle. The delayed alternation task in the water escape
T-maze consisted of a pseudorandom sequence of 10 dis-
crete trial pairs of forced-choice runs. Briefly, each trial
pair consisted of a forced run in which animals were
given access to only one arm (right or left), where a sub-
merged platform to escape from the water was located,
Addiction Biology, 16, 251–260

followed by a choice run in which animals have access to
both arms, but the platform was found in the arm oppo-
site the one they had just entered on the previous forced
run. Therefore, rats had to learn to alternate in order to
find the submerged platform during the choice run. If the
animal chose the arm where the platform is not present
(i.e. the same arm where it was forced to enter in the
forced run), the sliding door of that arm was closed and
the animal was maintained 10 seconds in the water
(failed choice). Afterwards the sliding door was opened to
allow the animal to find the submerged platform located
in the opposite arm. Once the animal reached the plat-
form after either forced or choice runs, the sliding door
was closed and it was maintained on the platform for 10
seconds. The retention intra-trial interval (delay)
between the forced run and the choice run was set at 10
seconds and the intertrial interval between trial pairs was
set at 30 seconds. During intratrial and intertrial inter-
vals, animals were placed in a plastic holding cage
(27 cm, 27 cm, 23 cm) that was placed adjacent to the
maze.
Training procedure. On the first day, animals were gently
immersed in the water escape T-maze for 1 minute,
without platforms. On the second day, they were sub-
jected to 10 trial pairs of forced-forced alternation runs in
which during the second run of the pair, the animals had
access only to the opposite arm they had visited previ-
ously. From the third day on, animals were subjected to
10 trial pairs of forced-choice alternation runs. A differ-
ent, pseudorandom sequence of forced runs was used
every day (e.g. L-R-L-L-R-L-R-R-L-R), for all animals
tested. Animals were trained until they performed cor-
rectly 7 or more out of 10 trials (> 70% correctness) for 3
consecutive days (Del Arco et al. 2007).
Testing brain cell- proliferation and neurogenesis
Administration of BrdU and tissue preparations
Rats housed under standard conditions were injected i.p.
with BrdU (Sigma-Aldrich; 50 mg per kg body weight)
three times at 4-hour intervals, after reaching stable
maintenance levels. Twenty-four hours (proliferation
test) or 28 days (neurogenesis test) after the first BrdU
injection, rats were euthanized and perfused transcar-
dially, first with phosphate buffered saline (PBS) and then
with 4% paraformaldehyde. Their brains were removed,
postfixed overnight and equilibrated in phosphate-
buffered 30% sucrose. Coronal hippocampal sections
(40-mm thick) were collected on a freezing cryostat. Every
ninth section (total of nine sections, 360 mm apart) was
taken and stored free-floating in PBS containing sodium
azide (1%) at 4°C for immunohistochemical analysis.
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Cocaine, neurogenesis and working memory 253
Immunohistochemistry
For BrdU staining of the rats’ specimens’ tissue, sections
were washed with PBS, incubated in 2N HCl at 37°C for
30 minutes and then blocked for 1 hour with blocking
solution (PBS containing 20% normal horse serum and
0.5% Triton X-100). The tissue sections were stained
overnight with specified combinations of the following
primary antibodies: rat anti-BrdU (1:200; Oxford Bio-
technology, Kidlington, Oxfordshire, UK) and mouse anti-
NeuN (1:200; Chemicon, Temecula, CA). Secondary
antibodies used for both rat tissues were Cy-3-conjugated
donkey anti-rat (1:200; Jackson ImmunoResearch, West
Grove, PA) and Cy-2 donkey anti-mouse (1:200; Jackson
ImmunoResearch).
Quantification
Proliferation in the DG was measured by counting manu-
ally the cells that were labeled for BrdU. Neurogenesis was
evaluated by counting the cells that were double labeled
with BrdU and NeuN (using fluorescent microscope
Nikon Eclipse E400, Yokohama, Japan; magnification
¥400). We counted the number of labeled cells in nine
coronal sections per rat brain that were stained and
mounted on coded slides (blind to the observer). To obtain
an estimate of the total number of labeled cells per DG,
the total number of cells counted in the selected coronal
sections from each brain was multiplied by the volume
index (the ratio between the volume of the DG and the
total combined volume of the selected sections). Cellular
co-labeling of BrdU and NeuN was confirmed by confocal
microscopy (Zeiss LSM 510 laser scanning microscope,
Jena, Germany).
Experiment 1a: effect of cocaine self-administration on
hippocampal proliferation
A catheter was surgically implanted i.v. as described pre-
viously. Rats were randomly divided into three groups,
trained to self-administer either cocaine (0.5 mg/kg,
n = 5 or 1.5 mg/kg, n = 11) or saline (n = 7). On day 15 of
the experiment (1 day after cocaine-trained rats reached
maintenance), all three groups were injected with BrdU
as described previously. Twenty-four hours after the first
BrdU injection, rats were euthanized and brains were
labeled for BrdU (Fig. 1).
Experiment 1b: effect of cocaine self-administration on
hippocampal neurogenesis
The same procedure as in experiment 1a was used, albeit
that rats were injected with BrdU and 28 days later
(day 43 of the experiment), rats were euthanized and
brains were double labeled for BrdU and NeuN. Rats were
trained to self-administer cocaine (0.5 mg/kg, n = 5 or
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254 Einav Sudai et al.

Figure 1 Flow chart of experimental procedures. Rats were trained to self-administer cocaine (0.5 or 1.5 mg/kg) or saline for 14 days.When
the rats achieved stable maintenance, they were injected i.p. with BrdU (50 mg/kg) three times, at 4-hour intervals for 1 day.Twenty-four hours
or 28 days after BrdU injection, rats were euthanized and their brains were excised. Cells that were double positive for BrdU, and the mature
neuronal marker, NeuN, were counted in the hippocampi (Experiment 1a, 1b). In a following experiment, rats were trained to self-administer
cocaine (1.5 mg/kg) or saline for 14 days. After reaching stable maintenance levels, they started a water escape T-maze training procedure. On
the first day of training, animals were immersed in the maze for 1 minute, without platforms. On the second day, they were subjected to 10
trial pairs of forced-forced alternation runs. From the third day on, rats were subjected to 10 trial pairs of forced-choice alternation runs. At
the cessation of the last trial, rats were euthanized and their brains were excised. Cells that were double positive for BrdU, and the mature
neuronal marker, NeuN, were counted in the hippocampi (Experiment 2)

© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction Addiction Biology, 16, 251–260

1.5 mg/kg, n = 15) or saline (n = 12). On day 15 of the
experiment (1 day after cocaine-trained rats reached
maintenance), all three groups were injected with BrdU
as described previously. Twenty-eight days later (day 43
of the experiment), the rats were euthanized and brains
were double labeled for BrdU and NeuN (Fig. 1).
Experiment 2: association of the performance of rats in
a water T-maze and neurogenesis after cocaine
self-administration
An i.v. catheter was surgically implanted as described pre-
viously. Rats were randomly divided into two groups,
trained to self-administer either cocaine (1.5 mg/kg,
n = 15) or saline (n = 15). On day 15 of the experiment (1
day after the cocaine-trained rats reached maintenance),
both groups were injected with BrdU (as described previ-
ously) and on the next day rats were trained in the water
T-maze. At the secession of the water T-maze training
(day 36 of the experiment), rats were euthanized and
brains were double labeled for BrdU and NeuN (Fig. 1).
Statistical analysis
For the behavioral data (cocaine self-administration and
water T-maze test), we applied one way analysis of vari-
ance (ANOVA) with repeated measures followed by a post
hoc test Student Newman–Keuls. The between groups
variable was the factor of treatment (1.5 mg/kg cocaine,
0.5 mg/kg cocaine or saline) and the within subjects’
variable was factor of days.
The histological data (cell proliferation and survival)
were analyzed by ANOVA followed by Student Newman–
Keuls post hoc test. For comparison between cell prolifera-
tion and cell survival, we used two-way ANOVA followed
by Student Newman–Keuls post hoc test.
To analyze how cell proliferation and neurogenesis in
the hippocampus is affected by cocaine consumption, we
used linear regression analysis.
Data are presented as mean ⫾ SEM. Results were con-
sidered significantly different if P < 0.05.
RESULTS
Cocaine self-administration
Rats were trained to self-administer cocaine (0.5 or
1.5 mg/kg, respectively) or saline over 14 days. A one-way
ANOVA with repeated measures for the active lever presses
revealed a main effect of treatment [F(2, 840) = 76.14,
P < 0.001], time [F(13, 840) = 14.32, P < 0.001]
and treatment ¥ time interaction [F(26, 840) = 12.6,
P < 0.001]. For number of inactive lever presses, there
was a main effect of treatment [F(2, 840) = 192.5,
P < 0.001], no main effect of time [F(13, 840) = 1.18,
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Cocaine, neurogenesis and working memory 255
P > 0.05] and treatment ¥ time interaction [F(26,
840) = 1.48, P = 0.05]. The difference between inactive
lever presses between treatment groups stems from rela-
tive high inactive lever responses of the saline treated rats
compared with cocaine self-administered rats (0.5 and
1.5 mg/kg cocaine, P < 0.05, the Student Newman–
Keuls post hoc test). However, the lack of distinction, dis-
played by saline-treated rats, between active and inactive
levers indicates that rats were not rewarded by saline.
Also, in the groups of cocaine self-administration (0.5 or
1.5 mg/kg), the number of inactive lever responses was
low and did not differ significantly throughout the days of
the experiment (P > 0.05, the Student Newman–Keuls
post hoc test) (Fig. 2).
Experiment 1a: effect of cocaine self-administration on
cell proliferation
When the rats achieved stable maintenance of cocaine
administration, they were injected i.p. with BrdU three
times, at 4-hour intervals. Twenty-four hours later, rats
were euthanized and brains were sliced and labeled for
BrdU. Slices were counter-stained with NeuN. A one-way
ANOVA showed a significant decrease in newly formed
cells (BrdU+; F(2, 19) = 4.1, Student Newman–Keuls post
hoc analysis P < 0.05) in the dentate gyri of rats trained
to self-administer cocaine at dose of 1.5 mg/kg compared
with control (saline treated), whereas cocaine at dose of
0.5 mg/kg did not affect the number of newly formed
cells in comparison with control (Fig. 3a).
Experiment 1b: Effect of cocaine self-administration on
hippocampal neurogenesis
Some of the rats that achieved stable maintenance of
cocaine self-administration were injected i.p. with BrdU
three times at 4-hour intervals, and their brains were
removed after 28 days. These brains were analyzed for
BrdU and NeuN. A one-way ANOVA of BrdU+ cells in the
DG showed a main effect of group [F(2,29) = 21.57;
P < 0.001]. Student Newman–Keuls post hoc test showed
a significant decrease in newly formed cells in the dentate
gyri of rats trained to self-administer cocaine at
1.5 mg/kg compared with control (saline treated),
whereas 0.5 mg/kg cocaine did not affect the number of
newly formed cells in comparison with control
(P < 0.001).
A one-way ANOVA of BrdU+ NeuN+ cells in the DG
showed a main effect of group [F(2,32) = 16.95;
P < 0.001]. The post hoc test showed a significant decrease
in newly formed neurons in the dentate gyri of rats trained
to self-administer cocaine at 1.5 mg/kg compared with
control (saline treated), whereas cocaine 0.5 mg/kg did
not affect the number of newly formed neurons in com-
parison with control (P < 0.001; Figure 3b).
Addiction Biology, 16, 251–260

256 Einav Sudai et al.
Figure 2 Cocaine or saline self-administration. Rats were trained to
self-administer saline (0.13 ml/infusion; panel a) or cocaine (0.13 ml/
infusion; 0.5 mg/kg/20 seconds, panel b; or 1.5 mg/kg/20 seconds,
panel c), on a fixed ratio 1 schedule of reinforcement in a daily
1-hour session, until stable maintenance levels were attained
A two-way ANOVA of total BrdU+ cells in the DG of
rats showed a main effect of group [F(2,36) = 23.4;
P < 0.001], a main effect of proliferation and survival
[F(5,36) = 2.4; P < 0.01] and no main effect in
© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction
13691600, 2011, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1369-1600.2010.00241.x, Wiley Online Library on [22/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
interaction [F(10,36) = 0.8; P > 0.05]. A post hoc test
showed a significant decrease in cell proliferation and cell
survival in the dentate gyri of rats trained to self-
administer cocaine 1.5 mg/kg compared with the saline
self-administered group and to the 0.5 mg/kg cocaine
group (P < 0.05, P < 0.01, respectively; Student
Newman–Keuls). Moreover, the extent of decrease in the
number of BrdU+ cells was significantly greater when
tested 28 days after BrdU injection than the decrease in
number of BrdU+ cells when tested 24 hours after BrdU
injection, only when rats were allowed to self administer
1.5 mg/kg cocaine (P < 0.001, Student Newman–Keuls;
Figure 3c).
Experiment 2: association of the performance of rats in
a water T-maze and neurogenesis after cocaine
self-administration
Because Experiment 1b indicated a decrease in neurogen-
esis only when rats were allowed to self-administer
1.5 mg/kg, we conducted another experiment to test
learning and memory abilities, after reaching stable main-
tenance, using the water T-maze in parallel to neurogen-
esis in the same subjects.The results indicate that from day
10 of the test and onward, cocaine-administered rats
showed significantly lower levels of performance com-
pared with saline-administered rats and did not reach the
criterion for learning the task [performing correctly in at
least 7 out of 10 trials (70%) for three consecutive days
throughout all tested days (Del Arco et al. 2007)]. ANOVA
with repeated measures of the correct choices revealed
a significant effect of treatment F[(1, 448) = 63.08,
P < 0.0001], time [F(15,448) = 33.58, P < 0.001]
and treatment ¥ time interaction [F(15,448) = 2.51,
P < 0.005] (Fig. 4a).
Results of behavioral performance were associated
with the number of newly formed matured neurons. A
one-way ANOVA of BrdU+ cells in the DG of rats showed
a main effect of group [F(1,28) = 45.15; P < 0.001]. A
post hoc test showed a significant decrease in newly
formed cells in the dentate gyri of rats trained to self-
administer cocaine compared with saline-treated rats
(P < 0.001).
A one-way ANOVA of BrdU+ NeuN+ cells in the DG of
rats showed a main effect of group [F(1,28) = 32.54;
P < 0.001]. A post hoc test showed a significant decrease
in newly formed neurons in the dentate gyri of rats
trained to self-administer cocaine compared with saline-
treated rats (P < 0.001; Fig. 4b).
DISCUSSION
Our results indicate a significant decrease in cell prolifera-
tion and in newly generated neurons in the DG of the
hippocampus in rats trained to self-administer cocaine.
Addiction Biology, 16, 251–260
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Cocaine, neurogenesis and working memory 257

a b c

d e f

g h l

Figure 3 Cocaine self-administration decreases cell proliferation and neurogenesis in the hippocampus. Adult hippocampi slices were
prepared as described in Methods. Panel a: Quantification of BrdU+ cells in the dentate gyrus (DG) of rats showed significantly less newly
formed cells (proliferation) in the dentate gyri of rats trained to self-administer cocaine 1.5 mg/kg compared with control (saline treated),
whereas cocaine 0.5 mg/kg did not affect the number of newly formed cells compared with control (*P < 0.05 versus 0.5 mg/kg cocaine and
saline groups). Panel b: Quantification of BrdU+ and NeuN+ cells in the DG of rats showed significantly less newly formed cells (BrdU+) and
neurons (BrdU+ NeuN+; differentiation) in the dentate gyri of rats trained to self-administer cocaine 1.5 mg/kg compared with control (saline
treated), whereas cocaine 0.5 mg/kg did not affect the number of newly formed cells and neurons compared with control (panel b; *P < 0.001
versus the correspondence measure in the 0.5 mg/kg cocaine and saline groups). Panel c: Comparison of total BrdU+ cells in the DG of rats
showed a significant decrease in cell proliferation and in cell survival in the dentate gyri of rats trained to self-administer cocaine 1.5 mg/kg
compared with saline self-administeed group and to 0.5 mg/kg cocaine group (P < 0.05, Student Newman–Keuls). Panels d–f: Representative
micrographs of newly generated cells, BrdU+ (red) summed in panel a are depicted; in saline self-administration (d), 0.5 mg/kg cocaine
self-administration (e), 1.5 mg/kg cocaine self-administration (f). Slices were counter stained with NeuN+ (green) for anatomical demonstra-
tion. Panel g–i: A representative micrograph (double labeling) of newly generated neurons NeuN+ (green) and BrdU+ (red) summed in panel
b are depicted; in saline self-administration (g); 0.5 mg/kg cocaine self-administration (h); 1.5 mg/kg cocaine self-administration (i)

This effect was concomitant with impairment in the per-
formance of working memory during withdrawal from
cocaine.
This decrease was only identified when a 1.5 mg/kg/
injection cocaine was allowed, but not at a lower dose.
The extent of decrease in the number of BrdU+ cells was
significantly greater when tested 28 days after BrdU
injection than the decrease in the number of BrdU+ cells
when tested 24 hours after BrdU injection. Therefore, we
postulate that not only cell proliferation was affected by
this specific dose, but also cell survival.
In contrast to our results, others have reported that
chronic cocaine administration does not alter the sur-
vival of neural progenitor cells in the DG (Dominguez-
Escriba et al. 2006; Noonan et al. 2008). However, in one
study (Dominguez-Escriba et al. 2006), BrdU was injected
prior to non-contingent cocaine administration and
assayed 24 days later, while in another study, BrdU was

© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction Addiction Biology, 16, 251–260

258 Einav Sudai et al.
Figure 4 Association of the performance of rats in a water T-maze
and neurogenesis after cocaine self-administration. Panel a: Rats were
trained to self-administer cocaine (1.5 mg/kg/infusion) or saline. After
reaching stable maintenance, they were tested for learning and
memory abilities in the water T-maze. From day 10 of the test and
onward, cocaine-administrated rats showed significantly lower levels
of performance compared with saline administrated rats and did not
reach the learning criterion throughout all tested days (P < 0.005,
Student Newman–Keuls). Panel b: Adult hippocampi slices were
prepared as described in Methods. Quantification of BrdU+ and
NeuN+ cells in the dentate gyrus of rats showed significantly less
newly formed cells (BrdU+) and neurons (BrdU+ NeuN+; differentia-
tion) in the dentate gyri of rats trained to self-administer cocaine
1.5 mg/kg compared with saline treated rats (*P < 0.001, Student
Newman–Keuls)
injected at maintenance (Noonan et al. 2008) and the
self-administration protocol was used only with a rather
low dose of cocaine (0.5 mg/kg/per injection).
Cocaine at a dose of 0.5 mg/kg/injection did not affect
cell proliferation or survival of newly formed neurons,
but results of a threefold higher dose (1.5 mg/kg/
injection) showed a decrease in proliferation and to
higher extent in the survival of newly generated neurons
in the DG. The total amount of cocaine injected, during
the 5 days of maintenance in rats trained on 1.5 mg/kg/
injection, did not significantly differ from the total
© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction
13691600, 2011, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1369-1600.2010.00241.x, Wiley Online Library on [22/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
amount of cocaine injected in rats trained on 0.5 mg/kg/
injection (51 ⫾ 3 mg/kg and 42 ⫾ 4 mg/kg, respec-
tively). Because of cocaine’s rapid half-life, and the fact
that BrdU labels divide progenitors, it is likely that any
effect of cocaine on the progenitor cells at the time of
labeling is a result of the amount of cocaine present that
day. In the final cocaine session before BrdU injection,
rats in both the 1.5 and 0.5 mg/kg/injection groups con-
sumed about 7.5 mg/kg/session. Thus, the progenitor
cells should have been exposed to equal amounts of
cocaine in each group. However, neurogenesis is a
process rather than a single time event and therefore,
although the amount of cocaine was equal at the time of
BrdU injection, the total longitudinal cocaine consump-
tion during the entire experiment was higher in 1.5
than in 0.5 mg/kg-trained rats (122 ⫾ 6 mg/kg and
90 ⫾ 5 mg/kg, respectively; P < 0.05). The amount of
cocaine intake negatively correlated with the number of
cell proliferation and neurogenesis (r2 = 0.75, P < 0.001
and r2 = 0.76, P < 0.0005, respectively). It has been pre-
viously suggested that high versus low doses and pro-
longed versus short access to cocaine result in a different
pattern of cocaine intake that reflects changes in the
brain receptors (Ben Shahar et al. 2007; Wee, Specio &
Koob 2007). Based on these reports and on our results, it
seems that the intensity and duration of exposure to the
substance relate to the decrease in neurogenesis.
Integrated theories regarding the role of the hippoc-
ampus in learning and memory have been suggested
(Rolls et al. 1998). However, the literature on the function
of adult hippocampal neurogenesis leaves room for
debate (Kempermann 2002; Kempermann et al. 2004;
Lledo, Alonso & Grubb 2006; Jessberger et al. 2009).
Experiments aimed at knocking out neurogenesis have
proven to be inconclusive regarding the effect on memory.
A significant reduction in the number of new neurons in
the adult hippocampus was associated with impaired
memory performance in some tasks, but not with others.
Specifically, treatment with an anti-mitotic agent reduced
the amount of fear acquired after exposure to trace fear
conditioning paradigm, but did not affect contextual fear
conditioning or spatial navigation learning in the Morris
water maze (Shors et al. 2002). These results suggest that
neurogenesis may be associated with the formation of
some types of hippocampal-dependent memories. Simi-
larly, the effects of cocaine self-administration on
memory are unclear. On the one hand, cocaine self-
administration has been shown not to affect spatial
memory (Del Olmo et al. 2006) while on the other hand,
a different study showed an improvement in performance
in a difficult Morris water maze task following cocaine
self-administration (Del Olmo et al. 2007).
It has been suggested that cocaine seeking as a goal-
directed behavior involves long-term adaptations in the
Addiction Biology, 16, 251–260

PFC that can serve to explain the impairment of working
memory seen only in rats exposed to prolonged excess of
cocaine (George et al. 2008). It has been postulated that
these alterations serve as the mechanism underlying
the compulsivity aspect of drug seeking. Nonetheless,
it is important to note that working memory involves
both the hippocampus and PFC; the hippocampus–
orbitomedial PFC circuit activates, maintains, monitors
and modifies current and recent past cognitive and emo-
tional representations and enables the integration of cog-
nition, emotion and behavior (Wall & Messier 2001).
Our aim in this study was to test working memory
performance of previously addicted rats during absti-
nence in relation to the formation of new hippocampal
neurons (BrdU+/NueN+). A study by George et al. (2008)
that examined working memory following cocaine self-
administration did not discover changes when using
0.5 mg/kg/injection. Accordingly, previously reported
data (Noonan et al. 2008) as well as the present
data showed that 0.5 mg/kg/injection cocaine self-
administration did not affect neurogenesis. In light of
these findings, we assumed that if a connection between
working memory and neurogenesis exists, it may be found
in rats trained to inject 1.5 mg/kg cocaine, the dose we
found to have an effect on newly formed neurons. Indeed,
a decrease in neurogenesis was associated with a deficit in
working memory performance at this dose. Research has
shown (Wang, Scott & Wojtowicz 2000) that young
neurons are completely unaffected by GABA-A inhibition
and produce LTP more readily than mature neurons. As
LTP is considered to be a neural basis of learning, even a
minor decrease in the number of young neurons may
exert a significant impact on hippocampal dependent
learning. Several computational theories have recently
been suggested to explain the functional role of neuro-
genesis, including the idea that memory capacity
increases with the number of dentate granule cells, while
neuronal turnover with a fixed dentate layer size improves
recall by minimizing interference between highly similar
items (Becker 2005). Another hypothesis is that newly
formed neurons assist adaptation to new situations. The
assumption behind this hypothesis is that old neurons are
rather stable and preserve an optimal encoding of known
environments, while the plasticity of new neurons
enables them to adapt and encode new features present in
new environments. A simple network simulation has
demonstrated that the addition of new plastic neurons
proved to be a successful strategy for adaptation, with
limited interference between different memories (Wiskott,
Rasch & Kempermann 2006).
Based on some of the existing literature (Simon et al.
2002) and on our own study’s results, it is tempting to
speculate that cocaine attenuates the development and
survival of new neurons, which in turn leads to a
© 2010 The Authors, Addiction Biology © 2010 Society for the Study of Addiction
13691600, 2011, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1369-1600.2010.00241.x, Wiley Online Library on [22/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Cocaine, neurogenesis and working memory 259
reduction in cognitive performance. Furthermore, we
believe that the difficulty in creating new memories could
eventually evoke memories of past drug-induced behavior
and as a result increase the propensity for drug relapse.
Acknowledgements
This study was supported by a grant from The Israel Min-
istry of Health to GY and with a fellowship by the Israel
Anti-Drug Authority to ES. The study is part of Einav
Sudai’s PhD dissertation, in the Mina & Everard
Goodman Faculty of Life Sciences, Bar-Ilan University,
Ramat-Gan 52900, Israel. The authors wish to thank Dr
D. Har-Even for his critical help with the statistics analy-
ses and Dr Y. Ziv for consultation and helpful advice with
neurogenesis data.
Authors Contribution
ES performed the experiments, conducted data acquisi-
tion and analyses, and wrote the manuscript; OC per-
formed the experiments that constitute the main body of
this work; AS performed the water T maze experiments
and data analysis; LA and IRD performed the in-vivo
experiments; IG technical support and surgery; YF paper
preparation and technical support; NK carried out the
histological and immunofluorescence studies; SA con-
structed the figures; MBZ instrumental and program
design and support; GY supervised the project, designed
the experiments and helped write the manuscript.
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