Neurodegeneration of lateral habenula efferent bers after

intermittent cocaine administration: Implications for deep

brain stimulation
Elad Lax
a
, Alexander Friedman
b
, Ofri Croitoru
a
, Einav Sudai
a
, Hila Ben-Moshe
a
,
Lior Redlus
b
, Efrat Sasson
c
, Tamar Blumenfeld-Katzir
c
, Yaniv Assaf
c
, Gal Yadid
a, b,
*
a
The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
b
The Leslie & Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel
c
Department of Neurobiology, George S. Wise Faculty of Life Science, Tel Aviv University, Tel Aviv, Israel
a r t i c l e i n f o
Article history:
Received 19 December 2012
Received in revised form
29 May 2013
Accepted 3 June 2013
Keywords:
Lateral habenula
Cocaine addiction
Neurodegeneration
Diffusion tensor imaging
Deep brain stimulation
Magnetic resonance imaging
Retrograde labeling
a b s t r a c t
Deep brain stimulation (DBS) is an emerging technique for effective, non-pharmacological intervention
in the course of neurological and neuropsychiatric diseases. Several brain targets have been suggested as
suitable for DBS treatment of drug addiction. Previously, we showed that DBS of the lateral habenula
(LHb) can reduce cocaine intake, facilitate extinction and attenuate drug-induced relapse in rats trained
to self-administrate cocaine. Herein, we demonstrated that cocaine self-administration dose-depen-
dently decreased connectivity between the LHb and midbrain, as shown by neurodegeneration of the
main LHb efferent ber, the fasciculus retroexus (FR). FR degeneration, in turn, may have caused lack of
response to LHb stimulation in rats trained to self-administer high-dose cocaine (1.5 mg/kg; i.v.).
Furthermore, we show that the micro-structural changes caused by cocaine can be non-invasively
detected using magnetic resonance imaging and diffusion tensor imaging. Detection of cocaine-
induced alterations in FR anatomy can aid the selection of potential responders to LHb stimulation for
treatment of drug addiction.
2013 Elsevier Ltd. All rights reserved.
1. Introduction
Cocaine addiction is a serious clinical and social problem, for
which there is no currently approved pharmacological treatment.
Addiction is characterized by progressive increases in drug intake
over time, causing maladaptive changes in motivational and reward
systems (Everitt et al., 2001; Kauer and Malenka, 2007).
The lateral habenula (LHb), part of the epithalamus, plays a
critical role in the reward system as a source of negative reward-
related signals (Hikosaka, 2010). The LHb is connected to various
reward-related midbrain nuclei such as the rostro-medial
tegmentum (RMTg) (Jhou et al., 2009) and the ventral tegmental
area (VTA) via its main efferent ber, the fascicules retroexus (FR)
(Herkenham and Nauta, 1979). The LHb inhibits midbrain dopa-
minergic neurons both directly and via a bi-synaptic connection
through RMTg GABAergic neurons (Geisler and Zahm, 2005)
(Brinschwitz et al., 2010; Stamatakis and Stuber, 2012). Inhibition of
midbrain nuclei reduces dopaminergic cell ring (Ji and Shepard,
2007), which lowers motivation and reward.
LHb abnormalities have been shown in several psychiatric dis-
orders such as depression(Li et al., 2011; Sartorius et al., 2010; Savitz
et al., 2010), psychosis (Ellison, 1994; Shepard et al., 2006) and drug
addiction (Brown et al., 2011; Zhang et al., 2005). LHb activity was
shown to be elevated followingexposure toexperimenter-delivered
cocaine- and cocaine-associated cues (Brown et al., 1992; Franklin
and Druhan, 2000). Others have shown that LHb over-activation
following continuous subcutaneous cocaine exposure (103 mg/5
days), but not experimenter-delivered cocaine, leads to axonal
neurodegeneration of the FR (Ellison, 2002). It was suggested that
this cocaine-induced neurodegeneration compromises descending
control pathways, enabling an increase in dopaminergic tone which
can further lead to psychosis (Ellison et al., 1996).
Deep brain stimulation (DBS) is an emerging technique for
effective, non-pharmacological intervention in the course of
neurological and neuropsychiatric diseases, including Parkinsons
disease, major depression and obsessive compulsive disorder
* Corresponding author. The Leslie & Susan Gonda (Goldschmied) Multidisci-
plinary Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel. Tel.: 972 3
531 8123; fax: 972 3 635 4965. yadidg@mail.biu.ac.il
E-mail address: yadidg@mail.biu.ac.il (G. Yadid).
Contents lists available at ScienceDirect
Neuropharmacology
j ournal homepage: www. el sevi er. com/ l ocat e/ neuropharm
0028-3908/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuropharm.2013.06.034
Neuropharmacology 75 (2013) 246e254
(Kern and Kumar, 2007; Sartorius and Henn, 2007). Recently,
we demonstrated that DBS of the LHb can modulate reward-related
behaviors, reducing sucrose-seeking and cocaine-seeking behavior
(up to 1 mg/kg) in the self-administration paradigm (Friedman
et al., 2010a, 2011).
In an effort to advance existing knowledge on DBS treatment for
drug addiction, herein we endeavored to determine the relation-
ship between cocaine-seeking behavior, brain structure, and DBS
efcacy. Therefore, we rst examined to what extent cocaine self-
administration, particularly at high doses, can affect FR micro-
structure and LHb activity. Using retrograde labeling of LHb neu-
rons via intra-VTA injection of Fluoro-Gold (FG), we found that
cocaine self-administration (0.5e1.5 mg/kg) induced dose-
dependent FR degeneration. Next, we demonstrated that DBS of
the LHb during maintenance, extinction and reinstatement reduced
cocaine-seeking behavior at the lower and intermediate cocaine
doses (0.5 and 1 mg/kg), but not at the highest dose (1.5 mg/kg).
This nding highlights the fundamental difculty in selecting po-
tential human responders to DBS treatment; it is even more chal-
lenging because the underlying mechanism of DBS is still vague.
Thus, before establishing a consensus for routine usage of DBS,
more neuro-functional data are needed. Herein, we used non-
invasive diffusion tensor imaging (DTI), which provides measures
of white-matter micro-structure in the human brain, revealing
abnormalities in white matter ber structure and supplying models
of brain connectivity (Blumenfeld-Katzir et al., 2011; Scholz et al.,
2009). The DTI analysis of magnetic resonance imaging (MRI)
scans detected neurodegeneration of the FR and other elements of
the LHb-midbrain circuit, in rats which self-administered high-
dose cocaine.
2. Materials and methods
2.1. Animals
Male (250e300 g) SpragueeDawley rats (Harlan, Jerusalem, Israel) were
maintained under conditions of unvarying temperature (23

C) and humidity (50%),
in a 12:12 h reversed light/dark cycle (light off at 07:00 am), with free access to food
and water. All animal procedures were approved by the Bar-Ilan University Animal
Care Committee and were carried out in accordance with the U.S. National Research
Council Guide for the Care and Use of Laboratory Animals.
2.2. Surgical procedures
2.2.1. Jugular vein catheterization for cocaine self-administration
Rats were anesthetized with ketamine hydrochloride and xylazine (100 mg/kg
and 10 mg/kg, respectively; i.p.), and were implanted with intravenous silastic
catheters (Dow Corning, Midland, MI) in 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 modied
22 gauge cannula; Plastics One, Roanoke, VA). For the DTI experiment, the catheter
was secured to the rats back where it exited through a small incision and was
afxed to a plastic pedestal inside a harness system (SAI, Libertyville, IL) as
described before (Covington and Miczek, 2005), in order to avoid disturbance to the
magnetic eld.
2.2.2. DBS electrode implantation
After catheterization, rats designated for DBS treatment were placed in a ste-
reotaxic apparatus (Kopf Instruments; Tujunga, CA) and a bipolar stimulating
electrode (consisting of 2 stainless steel electrodes, 0.01 mm diameter, 1 mm be-
tween cathode and anode, non-isolated electrode tip 1 mm, Plastic One, Va, USA)
was inserted unilaterally into the brain, in such a way that the LHb was situated
between the cathode and anode electrodes (cathode/anode anterior 3.8/3.8,
lateral 1.4/2.4, ventral 4.74/4.74 mm from Bregma, with 14 angle). The connector
and electrode were secured to the skull with MX-80 screws (Small Parts, Inc., Miami
Lakes, FL) and dental cement (Yates and Bird, Chicago, IL). Carprofen (2 mg/kg, i.p.)
was injected post-surgery.
2.2.3. Guide cannula implantation for Fluoro-Gold microinjection
After catheterization, rats designated for FG microinjectionwere implanted with
a unilateral guide cannula (22 gauge, Plastics One), inserted into the VTA in close
proximity to the FR (anterior 5.3; lateral 0.5; ventral 7.2). The guide cannula
was sealed by a cannula obstructer (Plastic One) and secured to the skull with screws
and dental acrylic cement. Carprofen (2 mg/kg, i.p.) was injected post-surgery.
2.3. Behavioral measurements
2.3.1. Cocaine self-administration
Rats were allowed a 10-day recovery from surgery. Rats were then transferred
daily into the operant conditioning (self-administration) chambers (Med-Associates,
Inc.; St Albans, VT) during their dark cycle, and trained to self-administer cocaine
(obtained from the National Institute on Drug Abuse, Research Technology Branch,
Rockville, MD, USA) in 60 min sessions, on an FR-1 schedule. Each self-
administration chamber (30 25 22 cm) had two levers, one active and one
inactive, located 5 cm above the oor of the chamber. An active lever press gener-
ated a cocaine infusion (i.v., 0.13 ml/0. 5e1.5 mg/kg/5 s) through the catheter, which
was connected to an injection device. In addition, a light located above the active
lever was lit for 20 s, 15 s beyond the cocaine infusion period which lasted only 5 s.
During the 15-s intervals, active lever presses were recorded, but no additional
cocaine reinforcement was provided. Rats were trained for 11 days until stable
maintenance levels were attained (dened as at least 3 days with <20% variation in
the number of active lever presses). Presses on the inactive lever were also recorded,
but did not activate the infusion pump and light. Rats were returned to their home
cages at the end of the daily session. The self-administration chambers and the
computer interface were built locally and were controlled by a computer program
written by Steve Cabilio (Concordia University, Montreal, PQ, Canada).
After reaching stable maintenance levels (at least 3 days with less than 20%
variation in number of active lever presses), rats were again placed in the self-
administration chambers for 1 h. DBS (as detailed in Section 2.5) was adminis-
tered during the rst 15 min of the session.
Rats subjected to saline self-administration underwent the same protocol,
except that cocaine was substituted with saline.
2.3.2. Extinction procedure
After maintenance, rats were placed in the self-administration chambers for 1 h
with no cocaine available, and DBS (Section 2.5) was administered during the rst
15 min of the session. Rats then continued daily extinction sessions until reaching
the extinction criterion, a 20% decrease in active lever presses as compared to the
rst day of cocaine extinction.
2.3.3. Reinstatement procedure
Three days after reaching the extinction criterion and stable extinction
responding, rats were placed in the self-administration chambers, in which they
received a priming injection of cocaine (10 mg/kg, 0.4 ml, i.p.) combined with a light
cue. Cocaine was not available during the session.
2.4. Retrograde labeling of LHb cells
2.4.1. FG injection and brain slicing
Retrograde labeling using the uorescent dye Fluoro-Gold was performed based
on (Schmued and Fallon, 1986). Briey, twenty-four hours after the last cocaine self-
administration session, rats were anesthetized with ketamine hydrochloride and
xylazine (100 mg/kg and 10 mg/kg, respectively; i.p.). An internal cannula (28 gauge)
with 1 mm projection was inserted into the guide cannula, through which FG
(100 nl, 2% in saline; SigmaeAldrich, Rehovot, Israel) was then injected over 10 min.
The internal cannula was kept in place for at least 5 min after the injection, to avoid
reux.
After a 6-day recovery in their home cages, rats were again anesthetized with
ketamine hydrochloride and xylazine as above, and were then transcardially
perfused with 1 PBS followed by 4% paraformaldehyde. Brains were removed and
immersed in 4% paraformaldehyde for 24 h, and then in a phosphate buffer with 30%
sucrose for 48 h. The brains were subsequently frozen on dry ice and sliced in the
coronal plane (40 mm sections) using a cryostat microtome. Each 6th serial brain
section was collected from approximately 2.56 mm to 4.16 mm posterior from
Bregma, and mounted on glass slides (coated with 2% gelatin). LHb cells were then
counted as detailed below, in brain sections of animals in which the VTA was suc-
cessfully targeted.
2.4.2. FG-positive cell counting
The cell count was conducted manually according to Greenwell et al. (2002) and
Sredni et al. (2007), using a uorescence microscope (Olympus AX-70) at 10 40
magnication, by two experimenters who were blind to the experimental condi-
tions. Each section was counted twice by each experimenter and the results were
averaged for each section. Results are presented as the average sum of cells from 6
LHb sections.
2.5. Electrical stimulation procedure
Stimulation parameters were identical to those used in previous studies
(Friedman et al., 2010a, 2011). Animals were placed in the self-administration
chambers, and then received DBS during the rst 15 min of the session. The
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 247
parameters for DBS were 200 m-amperes, with a spike-duration of 0.5 msec. In a
previous study (Friedman et al., 2010a) we continually adjusted the stimulation
pattern, in order to achieve a reduction in the number of active lever presses within
the sucrose self-administration task. We found that low frequency stimulation
(10 Hz) for 15 min elevated the number of presses (120% over baseline), while high
frequency stimulation (100 Hz) for 15 min had no effect at all. We then found that a
specic combination of alternating low (10 Hz) and high (100 Hz) frequency stim-
ulations achieved an optimal reductive effect on lever presses. Therefore we decided
to employ a combined stimulation pattern which consisted of four different sets of
patterns: 1) low frequency stimulation of 10 Hz, 2) high frequency stimulation of
100 Hz, 3) low-frequency-burst stimulation (180 msec intervals between bursts;
within each burst there were 5 spikes with 80 msec intervals between spikes. Total
stimulation was 10 Hz), and 4) high-frequency-burst stimulation (300 msec in-
tervals between bursts; within each burst there were 30 spikes with 3 msec intervals
between spikes. Total stimulation was 60 Hz). Spike duration was 1 msec. Alterna-
tions between high and low frequency patterns were constantly performed. The
duration of high frequency stimulation (pattern 2 and 4) was 4 s per stimulation,
with 20 s-long pauses between stimulations (in order to avoid tissue damage). The
duration of low frequency stimulation (patterns 1 and 3) was 60 s (Fig. 1AeC).
At the conclusion of the DBS experiments, animals were anesthetized and
transcardially perfused with PBS 1 followed by 4% paraformaldehyde. Brains were
removed and treated as in Section 2.4.1, then examined under a microscope to verify
the placement of the electrode. Data was analyzed only for rats in which the elec-
trode was accurately implanted (Fig. 1D).
2.6. Diffusion tensor imaging (DTI)
2.6.1. Imaging
MRI was performed using a 7T MRI scanner (Bruker, Karlsruhe, Germany) with a
30-cm bore and a gradient strength of up to 400 mT/m. The MRI protocol included
diffusion tensor imaging (DTI) acquisition with a diffusion-weighted (DW) spin-
echo echo-planar-imaging (EPI) pulse sequence with the following parameters:
TR/TE 4000/25 ms, D/d 10/4.5 ms, four EPI segments, and 15 noncollinear
gradient directions with a single b-value shell at 1000 s/mm
2
and one image with a
b-value of 0 s/mm
2
(referred to as b0). Geometrical parameters were: 20 slices, each
0.48 mmthick (brain volume) and with in-plane resolution of 0.2 0.2 mm
2
(matrix
size 128 128; FOV 25.6 mm
2
). The imaging protocol was repeated three times for
signal averaging and to compensate for acquisition in which signicant head motion
was observed. Each DTI acquisition lasted 4.5 min and the entire MRI protocol lasted
about 20 min.
2.6.2. MRI image analysis
Image analysis included DTI analysis of the DW-EPI images to produce the
fractional anisotropy (FA), apparent diffusion coefcient (ADC), and radial and axial
diffusivity indexed maps. The DTI analysis was implemented in Matlab (Mathworks,
USA) using in-house software (Pasternak et al., 2009). For statistical comparisons
between rats, each rat brain volume was normalized with a template rat atlas
allowing voxel-based statistics. All image transformations and statistical analyses
described below were carried out using SPM2 software (Wellcome Trust Centre for
Neuroimaging, London, UK). Each rat data set was normalized to the template im-
ages (registered to a digitized version of Paxinos Rat Brain Atlas (Paxinos and
Watson, 2005)). The normalization procedure included the following steps: (a) all
b0 images initially underwent bias correction. (b) For each rat, the b0 image was co-
registered with the b0 template (using a 6-parameter rigid-body transformation).
The co-registration parameters were then applied on the different DTI indexed maps
(FA, ADC, radial and axial diffusivities). (c) The registered FA maps were normalized
to the FA template (having spatial resolution of 0.066 0.066 0.48 mm
3
) using a
12-parameter afne nonlinear transformation and 0.2 mm smoothing (which was
used only for the parameter estimation procedure and not smoothing of the output
image). The normalization parameters were then applied on all DTI indexed maps,
including the FA, ADC, and radial and axial diffusivities. (d) The normalized indexed
maps were smoothed with a 0.3-mm Gaussian kernel.
3. Experimental design
3.1. Experiment 1: dose-dependent neurodegeneration of the FR
A guide cannula was implanted into the VTA, and three groups
of rats were trained to self-administer 0.5, 1 or 1.5 mg/kg cocaine
(n 6 per group). One day after reaching stable maintenance levels,
15 sec 4 sec
15 sec 4 sec
180
180
180
mSec
mSec
1 Sec
80 80
(B)
300
mSec
300
mSec
(C)
1 Sec
(D)
-3.8 from bregma
Fig. 1. Stimulation scheme. (A) Combined pattern stimulation scheme. (B) Low frequency burst stimulation. (C) High frequency burst stimulation. (D) Schematic illustration of
electrode placement. Dots indicate approximate electrode placements in the LHb (gure adapted from Paxinos and Watson (2005)).
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 248
experimental rats received an intra-VTA injection of FG (0.1 ml over
10 min). As sham-controls served drug-naive rats (n 6), which
were not exposed to cocaine self-administration but received the
same intra-VTA FG injection. In previous work, we found no dif-
ferences in FG measures between the trained and drug-naive rats.
Therefore, according to the Animal Care Committee guidelines to
minimize the animals suffering, the jugular catheterization was
excluded from the experimental protocol. Six days later, rats were
sacriced and brains removed and dissected. Labeled LHb neurons
from brains in which the guide-cannula was accurately implanted
(n 3e5 per group) were counted using a uorescence microscope.
3.2. Experiment 2: effect of DBS of the LHb on cocaine-seeking
behaviors
An electrode was implanted into the LHb and six groups of rats
were trained to self-administer 0.5, 1, or 1.5 mg/kg cocaine (n 15, 6,
and 9, respectively). One day after reaching stable maintenance, rats
of all three groups were again placed in the operant chambers and
received either combined pattern DBS or sham stimulation (con-
nectedtothestimulator but withnoelectric pulse transferred) during
the rst 15minof self-administration. Onthenext day, rats of all three
groups underwent an extinction session during which they received
combined pattern DBS (15 min), and extinction responding was
further measuredfor thenext 6days. Once rats reachedthe extinction
criteria they were reinstated with cocaine (10 mg/kg, i.p.), and
cocaine-seeking behavior was monitored. No additional stimulation
was applied after the rst extinction session or during reinstatement.
3.3. Experiment 3: high dose cocaine alters diffusion tensor imaging
parameters of the LHb-midbrain circuit
Rats were trained to self-administer cocaine (1.5 mg/kg, n 6) or
saline (n 5) for 11 days. Two days later, rats were anesthetized and
transferred to the MRI scanner for DTI acquisition. The regions of in-
terest (ROI) were the habenula, FR, VTA and the habenular commis-
sure that was recently found to connect between contralateral LHb
cells (Kim, 2009). After scanning, rats were euthanized with CO
2
.
3.4. Statistical analysis
All data are expressed as mean standard error of the mean
(SEM). Signicance of behavioral data was determined by analyses
of variance (ANOVA); repeated measures (days) were used to
examine the longitudinal effect of the experimental procedures on
cocaine self-administration. ANOVAs were followed by Studente
NewmaneKeuls (SNK) post-hoc tests to dene the altered treat-
ment groups. Signicance of DTI for each region of interest was
determined by unpaired Students t-tests. Correlation between to-
tal cocaine intake and histochemistry results (FG) was calculated
using Pearsons correlation coefcient. For all statistical analyses,
p < 0.05 was considered signicant. One result from the LHb cell
count experiment was detected as an outlier (p < 0.05) by Grubbs
test and excluded from the data analysis.
4. Results
4.1. Dose-dependent neurodegeneration of the FR
First, we examined the effect of different doses of cocaine on FR
integrity, using retrograde labeling. Rats were trained to self-
administer cocaine (0.5, 1 or 1.5 mg/kg; i.v.) for 11 days, until
reaching stable maintenance levels (Fig. 2A). One-way ANOVA
followed by SNK post-hoc revealed a signicant, dose-dependent
increase in the overall amount of cocaine intake over the experi-
mental period (F [2, 18] 3.48; p < 0.0001; p <0.005 for 0.5 mg/kg
vs. 1 mg/kg, and p < 0.0005 for 0.5 mg/kg vs. 1.5 mg/kg; Fig. 2B).
After reaching stable maintenance, rats received intra-VTA injec-
tion of FG (Fig. 3A). Cell count analysis showed a signicant, dose-
dependent reduction in the number of FG-labeled LHb neurons of
cocaine-trained rats (One way ANOVA, F [3, 13] 4.32, p < 0.03)
(Fig. 3B and C). SNK post-hoc test showed signicant differences
between naive and 0.5 mg/kg trained rats vs. 1.5 mg/kg trained rats
(p < 0.001 and p < 0.002, respectively) (Fig. 3D). In addition,
Pearsons correlation coefcient demonstrated a signicant,
reverse correlation between total cocaine intake and the number of
FG-labeled LHb neurons (r 0.894; p < 0.0003) (Fig. 3E).
4.2. Effect of combined pattern DBS of the LHb on cocaine-seeking
behavior
After nding a marked, dose-dependent decrease in midbrain-
projecting LHb neurons following intermittent cocaine, we
compared the effect of LHb DBS on cocaine-seeking behavior at
these doses. Rats were trained to self-administer cocaine (0.5, 1 or
1.5 mg/kg) until stable maintenance levels were reached (Fig. 4A,
***
(A) (B)
30
1.5 mg/kg 1 mg/kg 0.5 mg/kg
150
200
n
t
a
k
e
***
**
20
25
t
i
o
n
s
50
100
a
l

c
o
c
a
i
n
e

i
n
(
m
g
/
k
g
)
5
10
15
I
n
j
e
c
t
0
50
T
o
t
a
0
5
1 2 3 4 5 6 7 8 9 10 11
Days y
Fig. 2. (A) Dose-dependent cocaine self-administration. Rats were trained to self-administer different cocaine doses until reaching stable maintenance. (B) Dose-dependent in-
crease in total cocaine intake. Rats that self-administered higher cocaine doses consumed signicantly more drug during the overall experimental period. ***p < 0.0005, 1.5 mg/kg
vs. 0.5 mg/kg; **p < 0.005, 1 mg/kg vs. 0.5 mg/kg (One-way ANOVA followed by SNK post-hoc; n 6 per group).
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 249
M), and were either DBS-treated or sham-operated during the last
day of maintenance (Fig. 4A, M DBS). We found that DBS
reduced active lever responding at the low doses, but not at the
high cocaine dose. Two-way ANOVA revealed a main effect of dose
[F(2,26) 18.85; p < 0.0001] and an interaction between dose and
group [F(2,26) 37.09; p < 0.0001], but no main effect of group
[F(1,26) 3.47; p > 0.05]. SNK post-hoc test showed a signicant
reduction in cocaine-seeking behavior of DBS-treated rats which
self-administered 0.5 mg/kg or 1 mg/kg cocaine, as compared to
DBS-treated rats which self-administered 1.5 mg/kg (p < 0.001 for
both cases) (Fig. 4A). There were no signicant differences between
the three sham groups at all doses (p > 0.05).
One day later, rats began extinction training and received
combined pattern DBS (during the rst 15 min of the session), with
no cocaine available (Fig. 4A, E1 DBS). Extinction responding
was examined up to day E6. DBS-treated rats trained to self-
administer 0.5 mg/kg or 1 mg/kg cocaine showed signicantly
reduced cocaine-seeking behavior, compared to their respective
sham-treated groups. One-way ANOVA with repeated measures for
0.5 mg/kg revealed a main effect of group [F(1,112) 70;
p <0.0001], a main effect of days [F(6,112) 10.91; p <0.0001], and
an interaction between group and days [F(6,112) 5.12; p < 0.04]
during extinction. StudenteNewmaneKeuls post-hoc showed sig-
nicant differences between the sham-operated and DBS-treated
group (p < 0.001, for days E1eE2). One-way ANOVA with
repeated measures for 1 mg/kg cocaine showed a main effect of
group [F(1,112) 14; p < 0.0001], a main effect of days
[F(6,112) 10.91; p < 0.0001], and an interaction between group
and days [F(6,112) 5.12; p < 0.04] during the extinction process.
StudenteNewmaneKeuls post-hoc showed signicant differences
between the sham-operated and DBS-treated group (p 0.001, for
days E1eE3). In contrast, no effect of DBS was seen on cocaine-
seeking behavior of 1.5 mg/kg trained rats throughout the entire
extinction process (days E1eE6), when compared to their respec-
tive sham-operated control (p > 0.05) (Fig. 4A).
One day after reaching the extinction criterion, rats were rein-
stated with a priming cocaine injection (10 mg/kg, i.p.) combined
with a light cue. DBS-treated rats trained to self-administer 0.5 or
1 mg/kg cocaine, but not 1.5 mg/kg cocaine, demonstrated signi-
cantly decreased cocaine-seeking behavior as compared to
respective controls. Two-way ANOVA for the number of active lever
presses revealed a main effect of group [F(1,21) 8.1; p < 0.01] and
Fig. 3. Dose-dependent neurodegeneration of the FR. (A) Left: Schematic viewof a coronal section. Dots indicate approximate cannulae placements into the VTA (gure adapted from
Paxinos and Watson (2005)). Right: Representative uorescence microphotograph of FG injection into the VTA. (B, C) Fluorescence microphotographs of habenular neurons labeled
with FG in a coronal section, taken from naive (B) and 1.5 mg/kg cocaine-treated (C) rats. Arrows indicate labeled neuronal cells. (D) Labeled LHb neurons in cocaine treated vs. naive
rats. Amount of labeled LHb neurons in the 1.5 mg/kg cocaine-treated group signicantly decreased as compared to 0.5 mg/kg cocaine-treated (*p < 0.002) and naive groups
(**p < 0.001) (One-way ANOVA followed by SNK post-hoc test; n 3e5 per group). (E) Correlation between total cocaine intake and labeled LHb neurons. A substantial, reverse
correlation was found between the amount of labeled LHb neurons and total cocaine intake (mg/kg) (Pearsons correlation coefcient, r 0.894; p < 0.0003). Scale bars: 100 mm.
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 250
dose [F(2,21) 20.63; p < 0.0001], and an interaction between
group and dose [F(2,21) 3.49; p < 0.05]. SNK post-hoc test
showed signicant differences between DBS-treated and sham-
operated rats in the 0.5 mg/kg (p < 0.005) and 1 mg/kg
(p < 0.02) groups, but no difference between 1.5 mg/kg and con-
trols (p > 0.05; Fig. 4B).
4.3. High-dose cocaine alters DTI parameters of the LHb-midbrain
circuit
The above results imply that intermittent high-dose cocaine
intake, as opposed to lower doses, caused substantial neuro-
degeneration of the FR. This may have reduced the efciency of LHb
DBS treatment at the high dose. Therefore, we used MRI followed
by DTI analysis to examine whether FR neurodegeneration
following high-dose cocaine can be non-invasively detected. If so,
future DBS outcomes could be improved.
DTI analysis of rats that self-administered 1.5 mg/kg cocaine
showed a signicant increase in FA values in the habenula
(p < 0.004), FR (p < 0.0005), habenular commissure (p < 0.05) and
in the anterior VTA (p < 0.03), compared to counterparts rats that
self-administered saline (unpaired Students t-test, Fig. 5A and B).
Nonetheless, the analysis of FAvalues of the lateral habenula cannot
exclude minor contamination from the medial habenula. As a
control for brain site specicity, FA values of unrelated ROI, namely,
the ventral posterior thalamus and substantia nigra, were also
analyzed and showed no signicant differences between groups
(p > 0.05, Students t-test, Supplementary Fig. 1 and B). ADC values
did not change signicantly between groups in any of the observed
brain regions (p > 0.05, Students t-test, Fig. 5C). Analysis of sepa-
rate vectors of the calculated FA revealed that axial diffusivity (l
ax
)
signicantly increased in the FR of cocaine-trained rats (p < 0.03,
Students t-test, Fig. 6 A and B).
5. Discussion
Herein, we found that intermittent, 1-h daily, high-dose (1.5 mg/
kg) cocaine intake via self-administration induced a signicant
reduction in FG-labeled LHb neurons, indicating considerable
neurodegeneration of the LHb-midbrain circuit. In addition, DBS of
the LHb showed reduced efcacy at the high dose of cocaine. The
imaging techniques used herein provided a useful diagnostic tool
for neurodegeneration, and as such may aid in future selection of
potential responders to DBS, for treatment of addiction in humans.
A line of evidence demonstrates toxic effects of cocaine on the
nervous system. The main mechanism by which cocaine causes
damage to brain tissue is via oxidative stress and mitochondrial
impairment (Cunha-Oliveira et al., 2008; Numa et al., 2010; Poon
et al., 2007). Though several studies have shown cocaine-induced
apoptosis (Imam et al., 2005; Xiao and Zhang, 2008), others have
not found such an effect either in vitro (Oliveira et al., 2002) or
in vivo (Alvaro-Bartolome et al., 2011; Dietrich et al., 2005).
Considerable cocaine neurotoxicity was observed herein, though
this nding is in disagreement with the common phenomenon of
titration of cocaine intake during self-administration (Zimmer
et al., 2012). However, neurotoxicity is probably due to the accu-
mulating effect of exposure to the drug (consumption) throughout
the entire experiment, as previously demonstrated (Sudai et al.,
2011). Indeed, FG measures were inversely correlated with total
drug intake. In addition to neurotoxicity, high-dose cocaine com-
bined with extended access to the drug causes various behavioral
and neurochemical changes in the brain, including hormonal
changes and alterations in gene expression levels (Mantsch et al.,
2003), decreased hippocampal neurogenesis and impaired work-
ing memory (Sudai et al., 2011). Thus, it seems that at high cocaine
doses, both behavioral manifestations as well as neural correlates
undergo major disruption.
To the best of our knowledge, the present study is the rst to
show that FR atrophy occurs not only after continuous, subcu-
taneous cocaine administration (Ellison, 1992), but also after
intermittent cocaine exposure via self-administration. It is notable
that as compared to constant, passive administration, the self-
administration model more accurately simulates cocaine intake
patterns of human addicts (i.e., drug intake intensity (dose time)),
emphasizing the relevance of our ndings to drug addiction (Myers
et al., 1995).
The LHb receives high frequency excitatory inputs from the in-
ternal part of the globus pallidus, which encodes for aversion
(Shabel et al., 2012). In addition, the LHb receives low frequency
inputs from dopaminergic midbrain neurons, which inhibit LHb
activity when positive reward is expected (Fiorillo et al., 2003). We
believe that the combination of low and high stimulation patterns
0
20
40
60
80
100

A
c
t
i
v
e

l
e
v
e
r

p
r
e
s
s
e
s

0.5 mg/kg 1 mg/kg 1.5 mg/kg
Sham DBS
0
20
40
60
80
100
120
A
c
t
i
v
e

l
e
v
e
r

p
r
e
s
s
e
s

1.5 mg/kg Sham
1.5 mg/kg DBS
1 mg/kg Sham
1 mg/kg DBS
0.5 mg/kg Sham
0.5 mg/kg DBS *
*
**
^
#
(A) (B)
Fig. 4. (A) Effect of LHb DBS on cocaine-seeking behavior. Six groups of rats were allowed to self-administer cocaine (0.5, 1 or 1.5 mg/kg; n 15, 6, and 9, respectively). After
reaching stable maintenance levels (day M), rats were either DBS treated or sham-operated (day M DBS). DBS-treated, 0.5 mg/kg and 1 mg/kg cocaine-exposed groups showed
decreased active lever presses compared to the 1.5 mg/kg group (*p < 0.001 for both; two-way ANOVA). DBS treatment was also given during the rst extinction session, and
extinction responding was measured for 6 days (E1DBS through E6). A signicantly accelerated rate of extinction was found for the 0.5 and 1 mg/kg DBS-treated groups compared
to respective controls (^
, #
p < 0.001, one-way ANOVA repeated measures). No changes were found between DBS-treated and sham operated rats trained to self-administer 1.5 mg/kg
cocaine (p > 0.05). (B) Effect of LHb DBS on reinstatement. After E6, rats were reinstated to cocaine by a priming cocaine injection (10 mg/kg, i.p.) and light-cue. Rats which received
DBS treatment on E1 and which self-administered either 0.5 or 1 mg/kg cocaine showed signicantly reduced active lever presses compared to controls (**p < 0.005, *p < 0.05
respectively; two-way ANOVA). However, no difference was found between DBS-treated and sham-operated rats trained for 1.5 mg/kg cocaine (p > 0.05; two-way ANOVA).
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 251
mimics these LHb inputs, and resembles a condition in which a
reward (i.e., cocaine) is expected (due to the low frequency stim-
ulation), and expectation is then immediately eliminated (due to
high frequency stimulation). Thus, at least at the two lower doses,
the process of extinction learning is hastened by the combined
stimulation.
Cocaine exposure and cocaine-related cues increase c-Fos
expression in the LHb, indicating that the drug increases LHb ac-
tivity (Brown et al., 2011; James et al., 2011; Mahler and Aston-
Jones, 2012). Moreover, neurodegeneration of the FR following
continuous administration of cocaine (Ellison, 1992; Lipton et al.,
1991) is associated with a long-lasting decrease in LHb GABA ter-
minal immunolabeling. This suggests decreased synaptic inhibitory
activity, and therefore an increase in excitatory LHb activity.
Excitatory LHb inputs from the basal ganglia as well as LHb outputs
to the midbrain mostly encode negative reward (Hikosaka et al.,
2008; Shabel et al., 2012); thus, excitatory activity of LHb neurons
and their afferents may be elevated as a form of homeostatic
regulation, to counteract the robust increase in dopaminergic ring
following intense cocaine exposure (Jhou et al., 2013; Zahm et al.,
2009). Yet, prolonged excitation of LHb neurons after intensive
cocaine exposure can eventually induce neurotoxicity followed by
neurodegeneration of the FR (Meshul et al., 1998), and a possible
decrease in LHb inhibition of midbrain neurons. We postulate that
this reduced inhibition may have abolished the effect of LHb DBS on
cocaine extinction and relapse at the high dose of cocaine, as
opposed to its benecial effect on the lower doses of cocaine. This
suggests that the efcacy of DBS treatment is dependent on
addiction intensity. It is additionally puzzling that during the
maintenance phase of high-dose cocaine, DBS was not only less
effective, but cocaine-seeking behavior was actually increased.
Herein, DBS was utilized to promote the LHbs homeostatic regu-
lation of the VTA, i.e. serve as an additional braking control to
further inhibit the dopaminergic reward system. We propose that
the summation of high-dose cocaine and DBS resulted in transient
over-activation of the LHb, leading to a rapid shut-down response
in LHb ring. This, in turn, enabled an increase in dopamine neuron
ring, ultimately expressed as elevated drug self-administration.
DTI analysis, though not applied at the low and medium doses,
nonetheless shows that high-dose cocaine can increase FAvalues in
the LHb, FR, habenular commissure and anterior VTA. Although
neurodegeneration usually causes decreases in FA values, the bio-
logical interpretation of DTI indices is still not clear; thus any given
effect could be equally expected. Future studies should be directed
towards better understanding of the association between DTI
indices and the underlying neural mechanisms (Jones, 2010).
However, according to previous studies, FA increases may indicate
morphological changes in the axons towards diminished axonal
size (Barazany et al., 2009). Alternatively, these changes may
correspond either with abnormal axonal migration and dense
Habenula
FR
FA
Cocaine > Saline
(A)
FR
Habenular
commisure
Anterior
VTA
(B)
(C)
#
1 2
( )
0 2
0.25
0.3
0.35
Saline
Cocaine
**
***
*
#
0.8
1
1.2
C
0.05
0.1
0.15
0.2
F

A
0.2
0.4
0.6
A
D
C
0 0
Fig. 5. (A) Statistical parametric maps of FA values for cocaine- vs. saline-treated rats. (B) FA values of cocaine- vs. saline-treated rats. A signicant increase in FA values was
observed in all four ROI (unpaired t-tests;
#
p < 0.03; *p < 0.05; **p < 0.004; ***p < 0.0005). (C) ADC values of cocaine- vs. saline-treated rats. No signicant differences were
observed between the two groups (unpaired t-tests, p > 0.05 for all cases; n 5e6 per group).
E. Lax et al. / Neuropharmacology 75 (2013) 246e254 252
packing of myelinated bers (Sierra et al., 2011; Spoletini et al.,
2009), or with cytotoxic edema (Ling et al., 2012).
In addition, high-dose cocaine induced increases in axial diffu-
sivity (l
ax
), a parameter which represents the longitudinal aspects
of a given ber. This correlates with the nding that FR bers tend
to become shortened, segmented and disintegrated after contin-
uous cocaine administration (Ellison, 2002). A recent DTI study of
cocaine-dependent patients revealed that white matter integrity at
treatment onset is associated with treatment outcome measures
(Xu et al., 2010). Although specic regional alterations are not
indicated in this report, the ndings strengthen the notion that
abnormal brain connectivity in addicts may result in inferior
treatment outcomes. This supports the use of brain imaging as a
non-invasive tool for monitoring cocaine-induced brain alterations
prior to DBS treatment.
In conclusion, our ndings indicate that exposure to intermit-
tent, high-dose cocaine causes FR deterioration. This implies
decreased LHb inhibition of mesolimbic activity, which may abolish
the positive effect of LHb DBS on maintenance, extinction and
relapse stages. Lack of DBS effect can be predicted by non-invasive
discernment of the integrity of the LHb-midbrain circuit. Thus,
careful screening of patients in the clinic can predict habenular DBS
efcacy, and further promote this technique as a valuable tool for
treatment of cocaine addiction. Future studies can conrm the
usefulness of this tool in mirroring brain plasticity and its correla-
tion with behavioral outcome.
Financial disclosures
All the authors state that they have no conict of interest to
declare.
GY (corresponding author) certies that all authors have agreed
to all the content in the manuscript, including the data as
presented.
Acknowledgments
This study was supported by the NIH (grant #R21DA027776) to
GY. EL was supported by a Presidents Fellowship, Bar-Ilan Univer-
sity, and fellowships from the Israel Anti-Drug Authority and the
Wolf Foundation, Israel. The research reported in this article was
completed as part of ELs Ph.D. dissertation. The authors wish to
thank Michal Goldberg (Technion, Haifa, Israel) for her valuable
contribution to the rst stages of this research and to Dr. Tamar
Sadan for critically reading and editing the manuscript.
We wish to thank the Strauss Center for computational neuro-
imaging, the Sackler institute for biophysics and the Israel science
foundation for the purchase of the MRI system.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.neuropharm.2013.06.034.
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