The Journal of Neuroscience, January 30, 2013 • 33(5):1797–1803 • 1797

Neurobiology of Disease

Adolescent Cocaine Exposure Causes Enduring Macroscale
Changes in Mouse Brain Structure
Anne L. Wheeler,1,2 Jason P. Lerch,1,3,4 M. Mallar Chakravarty,5,6,7 Miriam Friedel,1,3 John G. Sled,1,3,4 Paul J. Fletcher,8,9
Sheena A. Josselyn,1,2,9,10 and Paul W. Frankland1,2,9,10
Program in Neurosciences and Mental Health, The Hospital for Sick Children, Toronto, Canada, M5G 1X8, 2Institute of Medical Science, University ofToronto, Toronto, Canada, M5S 1A8, 3Mouse Imaging Centre, The Hospital for Sick Children, Toronto Centre for Phenogenomics, Toronto, Canada, M5T
3H7, 4Department of Medical Biophysics, University of Toronto, Toronto, Canada, M5G 2M9, 5Kimel Family Translational Imaging Genetics Laboratory,
Research Imaging Centre, Centre for Addiction and Mental Health, Toronto, Canada, M5T 1R8, 6Department of Psychiatry, University of Toronto, Toronto,
Canada, M5T 1R8, 7Rotman Research Institute, Baycrest, Toronto, Canada, M6A 2E1, 8Biopsychology Section, Neuroscience Research Department, Centre
for Addiction and Mental Health, Toronto, Canada, M5T 1R8, 9Department of Psychology, University of Toronto, Toronto, Canada, M5S 3G3, and10Department of Physiology, University of Toronto, Toronto, Canada M5S 1A8
1

Cocaine dependence is associated with abnormalities in brain structure in humans. However, it is unclear whether these differences inbrain structure predispose an individual to drug use or are a result of cocaine’s action on the brain. This study investigates the impact ofchronic cocaine exposure on brain structure and drug-related behavior in mice. Specifically, mice received daily cocaine or saline
injections for 20 d during two developmental time periods: adolescence (27– 46 d old) and young adulthood (60 –79 d old). Following 30 dof abstinence, either fixed brain T2 weighted magnetic resonance images were acquired on a 7 T scanner at 32 ␮m isotropic voxel
dimensions or mice were assessed for sensitization to the locomotor stimulant effects of cocaine. Three automated techniques(deformation-based morphometry, striatum shape analysis, and cortical thickness assessment) were used to identify population differences in brain structure in cocaine-exposed versus saline-exposed mice. We found that cocaine induced changes in brain structure, andthese were most pronounced in mice exposed to cocaine during adolescence. Many of these changes occurred in brain regions previously
implicated in addiction including the nucleus accumbens, striatum, insular cortex, orbitofrontal cortex, and medial forebrain bundle.
Furthermore, exposure to the same cocaine regimen caused sensitization to the locomotor stimulant effects of cocaine, and these effects
were again more pronounced in mice exposed to cocaine during adolescence. These results suggest that altered brain structure following
1 month of abstinence may contribute to these persistent drug-related behaviors, and identify cocaine exposure as the cause of thesemorphological changes.

Introduction
Cocaine is a psychostimulant drug that acts acutely by inhibiting
monoamine reuptake at synapses in the brain, producing a feeling of euphoria (Gawin, 1991). However, repeated cocaine use
can cause persistent alterations in brain regions important for
reward and learning (Nestler, 2005; Thomas et al., 2008), leading
to a long-lasting psychological dependence with detrimental effects for the addicts, those close to them, and society.
Previous magnetic resonance imaging (MRI) studies haveshown that cocaine dependence is associated with structural abnormalities in the brain, predominantly in the frontal cortex andtemporal lobe (Bartzokis et al., 2000; Franklin et al., 2002; Sim etReceived Aug. 8, 2012; revised Nov. 7, 2012; accepted Nov. 21, 2012.
Author contributions: A.L.W., J.P.L., J.G.S., P.J.F., S.A.J., and P.W.F. designed research; A.L.W. performed research; A.L.W., M.M.C., and M.F. analyzed data; A.L.W. and P.W.F. wrote the paper.
This work was supported by a grant from the Canadian Institutes of Health Research and the Ontario Mental
Health Foundation to P.W.F. (MOP-77561). A.L.W. received support from the Ontario Mental Health Foundation.
Correspondence should be addressed to Paul Frankland, Program in Neurosciences and Mental Health, The
Hospital for Sick Children, 555 University Avenue, Toronto, Canada, M5G 1X8. E-mail: paul.frankland@sickkids.ca.
DOI:10.1523/JNEUROSCI.3830-12.2013
Copyright © 2013 the authors 0270-6474/13/331797-07$15.00/0

al., 2007; Makris et al., 2008; Ersche et al., 2011). While, in somecases, structural differences may predate onset of cocaine use andtherefore predispose an individual toward cocaine dependence(Ersche et al., 2012), other structural differences likely reflect the
impact of repeated cocaine use on the brain. However, isolating
the impact of cocaine on brain structure in human imaging studies can be challenging. First, alcohol, nicotine, and caffeine consumption are characteristic of chronic drug users (Compton etal., 2007) and so concurrent drug use or other lifestyle factors
may contribute to structural variability in the brain. Second, comorbid diagnoses of mood, anxiety, and disruptive behavior disorders are common in cocaine-dependent subjects (Swendsen etal., 2010), making it difficult to attribute morphological differences to cocaine use specifically. Third, typically there is variability in subjects’ history of cocaine exposure (e.g., pattern andduration of cocaine use, drug quality, route of administration,
periods of abstinence, and total lifetime intake) and in the accuracy of self-reports concerning these factors.
Experimental animal models allow for careful control of drug
exposure and therefore may be used to study the impact of cocaine exposure on brain structure under controlled conditions.

1798 • J. Neurosci., January 30, 2013 • 33(5):1797–1803

Indeed, previous studies have used animal models to investigate
cocaine-related changes in brain structure at the microscopic
level, focusing, for example, on changes in neuronal morphology
in reward-related brain regions (Robinson and Kolb, 2004). Similar to the human imaging studies, here we used high-resolution
mouse MRI to comprehensively map cocaine-associated changes
in brain structure at the macroscopic level (Nieman et al., 2007).
As drug use is often initiated during adolescence—a time point
where the brain is still developing (Giedd, 2008; Casey and Jones,
2010) and therefore thought to be particularly vulnerable to druginduced alterations in structure and function—we compared theimpact of cocaine exposure in adolescent and young adult mice.
Accordingly, mice were exposed to cocaine (or saline) daily for 20 dfollowed by a 30 d period of drug abstinence and then either structure of the whole brain was assessed with MRI or the mice were tested
for cocaine locomotor sensitization to provide a functional measureof the effects of chronic cocaine exposure.

Materials and Methods
Mice
Male offspring from a cross between C57BL/6NTacfBr (C57B6) and
129Svev (129) mice (Taconic) were used in all experiments. All mice werebred in our colony at The Hospital for Sick Children. Mice were weanedon postnatal day (PND) 21, group housed (3–5 mice per cage), andmaintained on a 12 h light/dark cycle with ad libitum access to food andwater. In these studies we used both adolescent (PND 27) and young
adult (PND 60) mice. PND27 was chosen as this age coincides with thestart of puberty and growth spurt, and is associated with the onset ofadolescent-typical behaviors including increased sociability, risk taking,and novelty seeking (Spear, 2000). Behavioral procedures were conducted during the light phase of the cycle. Experiments were conductedaccording to protocols approved by the Animal Care Committee at The
Hospital for Sick Children.

Cocaine administration
Cocaine hydrochloride (Medisca Pharmaceutique) was dissolved in saline and injected intraperitoneally. Adolescent and young adult micereceived daily injections of cocaine (20 mg/kg) or saline for 20 consecutive days (adolescent: PND 27– 46; adult: PND 60 –79). This dose waschosen because it falls within the range of doses (15–30 mg/kg per day)that cause persistent increases in dendritic branching and spine density inthe nucleus accumbens and prefrontal cortex of rodents (Robinson and
Kolb, 2004; Pulipparacharuvil et al., 2008). Following 30 d of abstinence,
mouse brains were either fixed for MRI or behavioral sensitization wastested. Imaging and testing occurred at a time when both groups wereconsidered adults (adolescent exposure: PND 77; adult exposure: PND110) to examine the long-term impact of cocaine exposure during theadolescent and young adult period.

MRI

Brain preparation. Mice (adolescent cocaine exposure, n ⫽ 7; adolescentsaline exposure, n ⫽ 8; adult cocaine exposure, n ⫽ 9; adult saline exposure, n ⫽ 8) were deeply anesthetized with chloral hydrate (400 mg/kg,
i.p.), and perfused through the heart with PBS followed by paraformaldehyde (4% PFA, 4°C). Bodies, along with the skin, lower jaw, ears, andthe cartilaginous nose tip were removed. The remaining skull structurescontaining the brain were allowed to postfix in 4% PFA at 4°C for 12 h.
Following a washout period of 5 d in PBS and 0.01% sodium azide at
15°C, the skulls were transferred to a PBS and 2 mM ProHance (Bracco
Diagnostics Inc.) solution for at least 7 d at 15°C before imaging.
MR acquisition. A multichannel 7.0 T MRI scanner (Varian) with a 6 cm
inner bore diameter insert gradient was used to acquire anatomical images ofbrains. Brains were imaged within skulls to minimize geometric distortion.
Before imaging, the samples were removed from the contrast agent solution,
blotted, and placed into plastic tubes (13 mm in diameter) filled with aproton-free susceptibility-matching fluid (Fluorinert FC-77; 3 M). Threecustom-built, solenoid coils (14 mm in diameter, 18.3 cm in length) withover wound ends were used to image three brains in parallel. Parameters

Wheeler et al. • Cocaine Exposure Changes Brain Structure in Miceused in the scans were optimized for gray/white matter contrast: a T2weighted, 3D fast spin-echo sequence with 6 echoes, with TR/TE ⫽ 325/32
ms, four averages, field-of-view 14 ⫻ 14 ⫻ 25 mm 3, and matrix size ⫽ 432 ⫻432 ⫻ 780 giving an image with 32 ␮m isotropic voxels. Geometric distortion due to position of the three coils inside the magnet was calibrated using
a precision machined MR phantom.
Volume analysis. We used an image registration-based approach toassess anatomical differences related to cocaine exposure. Image registration finds a smooth spatial transformation that best aligns one image toanother such that corresponding anatomical features are superimposed.
We used an automated intensity-based groupwise registration approach(Lerch et al., 2011) to align all brains in the study into a common coordinate system, yielding an average image of the 32 MRI scans. The deformation that brings the images into alignment becomes a summary of
how they differ. To assess volume differences between groups
deformation-based morphometry was used as it provides a continuous
voxel by voxel definition of volume changes (expansion/contraction)
related to drug and age of exposure. Deformations were mapped fromthe individual scans back to the average image. The final deformation
fields were computed with a greedy symmetric diffeomorphic registration (the SyN algorithm in ANTS; Avants et al., 2008; Klein et al., 2009),
then inverted and blurred with a 100 ␮m Gaussian smoothing kernel.
The Jacobian determinants of these deformations were extracted, givinga measure of local volume expansion/contraction at every voxel in thebrain (Chung et al., 2001). Log-transformed Jacobian determinants wereused to assess differences between groups because they better estimate anormal distribution (Leow et al., 2007). In this analysis individual voxel
measurements were assessed by comparing the log-transformed Jacobian
determinants at each voxel across the brain in the four groups with a
two-way ANOVA. Cocaine-treated and saline-treated groups were subsequently compared at each age of exposure using a t statistic. Multiple
comparisons were controlled for using the False Discovery Rate (FDR;
Genovese et al., 2002).
Striatum shape analysis. Changes in striatum shape and size may bedescribed by computing inward and outward displacement of the surfaceof the structure (Lerch et al., 2008b). Accordingly, a pre-existing expertsegmented MRI atlas with striatum labels (Dorr et al., 2008) was warped
to match the population average of the mice imaged in this study. A surface
representation of the striatum was generated from the final nonlinear atlas.
The dot product of the surface normal of the atlas striatum with the inverted
deformation field from each mouse was calculated to estimate the inward
and outward displacement of the surface at each vertex of the striatum. These
displacement values were then blurred along the surface using a 300 ␮mdiffusion smoothing kernel (Chung et al., 2003). The displacement distanceswere analyzed for group differences with a two-way ANOVA followed by t
tests to compare saline and cocaine exposure at each age. Multiple comparisons were controlled for using the FDR.
Cortical thickness analysis. Cortical thickness was assessed as previously
described (Lerch et al., 2008a). Briefly, cortical labels were transformed toeach mouse brain that defined the cortex boundaries and stored them on
a rasterized grid. Laplace’s equation was used to create streamlines between the inside and outside cortical surfaces and the length of thesestreamlines was used to measure cortical thickness. A surface coordinate
system was mapped to each subject to compare cortical thickness acrossgroups of mice. The thickness of each of the 18,000 vertices was related todrug and age of drug exposure with a two-way ANOVA. Cocaine-treatedand saline-treated groups were subsequently compared at each age of
exposure using a t statistic. Multiple comparisons were controlled forusing the FDR.

Behavioral sensitization
Locomotor sensitization was assessed in a dimly lit square-shaped Plexiglas arena (45 ⫻ 45 ⫻ 20 cm height). Locomotion was tracked by acamera located above the arena, and total distance traveled computedwith Limelight2 (Actimetrics). Mice (adolescent cocaine exposure, n ⫽ 9;adolescent saline exposure, n ⫽ 7; adult cocaine exposure, n ⫽ 7; adultsaline exposure, n ⫽ 7) were initially habituated to the arena for 45 min
on 2 d before cocaine/saline exposure. To habituate mice to the injection
procedure, all mice received an injection of saline before being placed inWheeler et al. • Cocaine Exposure Changes Brain Structure in MiceJ. Neurosci., January 30, 2013 • 33(5):1797–1803 • 1799

Table 1. Regional changes in mice exposed to cocaine during adolescence
Brain region

Direction

Hemisphere

Orbital frontal cortex
Striatum (anterior)
Striatum (posterior)
Nucleus accumbens
Anterior cingulate cortex
Ventral pallidum
Medial forebrain bundle
Insular cortex
Cortical amygdaloid area
Substantia nigra
Anterior olfactory area
Corpus callosum
Rhinal cortex
Piriform cortex
MedullaIncrease
Increase
DeceaseDeceaseDecrease
Decrease
Decrease
Decrease
Decrease
Increase
Increase
Increase
Decrease
Decrease
Increase

Right
Right
Bilateral
Bilateral
Bilateral
Bilateral
Bilateral
Bilateral
Bilateral
Bilateral
Right
Bilateral
Bilateral
Right
Bilateral

Brain regions where volumetric differences were observed following cocaine exposure during adolescence. Directionof change refers to cocaine exposed mice relative to saline exposed mice. Changes were observed at the voxel level
after correcting for multiple comparisons with 5% FDR correction. See Figure 1 for visualization of these data.

the chamber on the second habituation day. The mice then received 20
consecutive daily injections of cocaine or saline. On days 1, 2, and 20 ofcocaine/saline exposure, locomotor activity was assessed in the arena for
45 min following injection. On the 17 remaining drug-exposure days, mice
were returned to their homecage following cocaine/saline injection. Following cocaine/saline exposure, mice then remained in their homecage, undisturbed for 28 drug-free days. All mice, regardless of drug-exposure group,
were tested identically. Locomotor testing consisted of three testing sessions
on consecutive days where mice were placed in the arena for 45 min following injection. On test days 1–3 mice were injected with saline, 10 mg/kg
cocaine, and 20 mg/kg cocaine, respectively. For each test, data were analyzed
using a three-way ANOVA with age of exposure (adult or adolescent) anddrug pre-exposure (cocaine or saline) as between-subject variables and time(0 –15, 16 –30, and 31– 45 min) as a within-subject variable. Significant main
effects and interactions were followed up by two-way ANOVAs within test
time intervals with age of exposure and drug pre-exposure as betweensubject variables and t tests to compare the effect of drug pre-exposure ineach age group.

Results
MRI
Volume analysis provided an assessment of voxel level volumetric change across the entire brain allowing for the precise localization of cocaine-induced alterations within brain structures.
We observed significant interactions between age of exposureand drug in several brain regions. Subsequent direct comparisonof cocaine-exposed versus saline-exposed mice in each age groupindicated that these interactions were primarily driven by structural changes in mice exposed to cocaine during adolescence.
Strikingly, many of these volumetric alterations were localized tobrain regions previously implicated in reward processing andaddiction (Fig. 1; Table 1). For example, there were robust, bilateral volumetric reductions in the nucleus accumbens [(Fig. 1c.
Two-way ANOVA: drug by age interaction F(1,28) ⫽ 55.75 p ⬍4Figure 1. Coronal slices showing the localization of voxelwise differences between micetreated with cocaine versus saline during adolescence. Slices arranged from anterior (top) to

posterior (bottom). These maps show voxels where there was an interaction between age anddrug (t ⬎ 10.95, 5% FDR) and a difference between cocaine and saline exposure during adolescence (t ⬎ 3.98, 5% FDR). Colors indicate regions where cocaine-exposed mice showed
increased (red) and decreased (blue) volume relative to saline exposed mice. L, Left hemisphere; R,right hemisphere. Relative voxel size in the adolescent-exposed mice at selected highlighted voxels(yellow crosshairs) are displayed in the box plots to the right (a–j) where the midline represents
the median of the data, the box shows the first and third quartiles, and the vertical line represents the range. There were no significant differences between animals exposed to saline andcocaine during adulthood.

1800 • J. Neurosci., January 30, 2013 • 33(5):1797–1803

Wheeler et al. • Cocaine Exposure Changes Brain Structure in Mice

Figure 2. Striatum shape was altered by cocaine exposure. Group differences in striatum surface position show that the lateral surface of the striatum was displaced outward (away from thecenter of the structure) and the medial surface of the striatum was displaced inward (toward the center of the structure) in mice exposed to cocaine during adolescence relative to saline-exposed mice(a). Maps show surface points where there is an interaction between age and drug (right hemisphere, t ⬎ 2.35; left hemisphere, t ⬎ 2.79; 5% FDR) and a difference between cocaine and saline
exposure during adolescence (right hemisphere, t ⬎ 2.57; left hemisphere, t ⬎ 2.79; 5% FDR) reflecting outward (b) and inward (c) displacement of the striatum surface. A, Anterior; P, posterior;
L, left; R, right. Surface position in the adolescent-exposed mice at selected highlighted vertices (yellow crosshairs) are displayed in the box plots below. In these box plots the midline represents themedian of the data, the box shows the first and third quartiles, and the vertical line represents the range.

0.01, t test (cocaine vs saline in adolescent-treated groups): t(13) ⫽8.86, p ⬍ 0.01)], anterior cingulate cortex [Fig. 1d; two-way
ANOVA: drug by age interaction F(1,28) ⫽ 26.01, p ⬍ 0.01; t test
(cocaine vs saline in adolescent-treated groups): t(13) ⫽ 5.81, p ⬍0.05)], ventral pallidum [(Fig. 1e; two-way ANOVA: drug by age
interaction F(1,28) ⫽ 22.19, p ⬍ 0.05; t test (cocaine vs saline inadolescent-treated groups): t(13) ⫽ 5.02, p ⬍ 0.05)], medial forebrain bundle [(Fig. 1f; two-way ANOVA: drug by age interaction
F(1,28) ⫽ 21.61, p ⬍ 0.05; t test (cocaine vs saline in adolescenttreated groups): t(13) ⫽ 5.74, p ⬍ 0.05)], insular cortex [(Fig. 1 g;
two-way ANOVA: drug by age interaction F(1,28) ⫽ 16.12, p ⬍0.05; t test (cocaine vs saline in adolescent-treated groups): t(13) ⫽4.84, p ⬍ 0.05)], and cortical amygdaloid area [(Fig. 1i, two-way
ANOVA: drug by age interaction F(1,28) ⫽ 20.79, p ⬍ 0.05; t test
(cocaine vs saline in adolescent-treated groups): t(13) ⫽ 6.19, p ⬍0.05)] in mice exposed to cocaine during adolescence comparedwith saline-exposed controls. Volumetric increases caused by cocaine were observed in the right orbitofrontal cortex [(Fig. 1a;
two-way ANOVA: drug by age interaction F(1,28) ⫽ 23.35, p ⬍0.05; t test (cocaine vs saline in adolescent-treated groups): t(13) ⫽5.86, p ⬍ 0.05)] and substantia nigra [(Fig. 1j; two-way ANOVA:
drug by age interaction F(1,28) ⫽ 19.10, p ⬍ 0.05; t test (cocaine vssaline in adolescent-treated groups): t(13) ⫽ 6.11, p ⬍ 0.05)] inmice exposed during adolescence. Additionally there was bothvolumetric expansion and reduction in the striatum as a result of
cocaine exposure in adolescent mice. The anterior dorsal striatum showed volumetric expansion [(Fig. 1b; two-way ANOVA

drug by age interaction F(1,28) ⫽ 20.89, p ⬍ 0.05; t test (cocaine vssaline in adolescent-treated groups): t(13) ⫽ 5.79, p ⬍ 0.05)] while
the posterior striatum showed volumetric reduction [(Fig. 1h;
two-way ANOVA drug by age interaction F(1,28) ⫽ 17.48, p ⬍0.05; t test (cocaine vs saline in adolescent-treated groups): t(13) ⫽5.18, p ⬍ 0.05)]. All p values were adjusted to account for multiple comparisons with the FDR (with the FDR corrected value
corresponding to the expected false positive rate). Beyond
changes in these reward-related regions, significant changes wereadditionally observed in the anterior olfactory area, piriform cortex, corpus callosum, rhinal cortex, and medulla in mice treatedwith cocaine during adolescence (Table 1). We found no significant regional changes in volume following cocaine exposure during adulthood.
The local bidirectional changes in striatum volume suggestthat cocaine exposure caused the shape of the striatum to change,and detailed striatal shape analysis supported this conclusion.
The lateral surface of the striatum was displaced outward (i.e.,away from the center of the structure) and the medial surface ofthe striatum was displaced inward (i.e., toward the center of thestructure) bilaterally in adolescent cocaine-exposed mice relative
to saline-exposed mice (Fig. 2a). For example, these shape alterations were reflected in significant changes in striatum surface
position for points on the right lateral [(Fig. 2b; two-way
ANOVA: drug by age interaction F(1,28) ⫽ 3.18, p ⬍ 0.05; t test
(cocaine vs saline in adolescent-treated groups): t(13) ⫽ 5.16, p ⬍0.05)], left lateral [(Fig. 2b; two-way ANOVA: drug by age interac-Wheeler et al. • Cocaine Exposure Changes Brain Structure in MiceJ. Neurosci., January 30, 2013 • 33(5):1797–1803 • 1801groups): t(13) ⫽ 4.83, p ⬍ 0.10)], the piriform cortex [(Fig. 3c; two-way ANOVA:
drug by age interaction F(1,28) ⫽ 3.49, p ⬍0.10; t test (cocaine vs saline in adolescenttreated groups): t(13) ⫽ 3.06, p ⬍ 0.10)],and the insular cortex [(Fig. 3d; two-way
ANOVA: drug by age interaction F(1,28) ⫽5.38, p ⬍ 0.05; t test (cocaine vs saline inadolescent-treated groups): t(13) ⫽ 4.51,
p ⬍ 0.10)]. All p values were adjusted with
the FDR.
Behavioral sensitization
We assessed the impact of adolescent andadult cocaine exposure on subsequent
responses to cocaine using a cocaine
sensitization paradigm (Robinson andBerridge, 1993). Following cocaine orsaline pre-exposure and abstinence, allmice were challenged with an injectionof saline (0 mg/kg), low-dose cocaine
Figure 3. Cortical surface shows localization of cortical thinning in mice exposed to cocaine during adolescence. Blue (10 mg/kg), and high-dose cocaine (20color indicates vertices where there is an interaction between age and drug (t ⬎ 2.6, 10% FDR) and a difference between mg/kg) before locomotor testing on
cocaine and saline exposure during adolescence (t ⬎ 2.6, 10% FDR). The letters A, P, L and R refer to the anterior, posterior, successive days (Fig. 4). To assess theleft and right directions in the brain, respectively. Cortical thickness in the adolescent-exposed mice at selected highlighted impact of cocaine exposure and age ofvertices (yellow crosshairs) are displayed in the box plots (a– d) where the midline represents the median of the data, thecocaine exposure on sensitization webox shows the first and third quartiles, and the vertical line represents the range.
conducted three separate three-way
ANOVAs for the test data for each challengedose (0, 10, and 20 mg/kg cocaine). For each
three-way ANOVA, age at time of exposure(adolescence vs adult) and drug (cocaine vssaline) were between-subject factors andtime (0 –15, 16 –30, and 31– 45 min) was a
within-subject factor.
In all three test sessions mice with a hisFigure 4. Locomotor sensitization in mice pre-exposed to cocaine during adolescence and adulthood. Distance traveled in tory of cocaine exposure exhibited alteredresponse to challenge injections of 0 mg/kg (a), 10 mg/kg (b), and 20 mg/kg (c) of cocaine following pre-exposure to cocaine or levels of locomotion following challenge insaline during adolescence or adulthood. Mice pre-exposed to cocaine during adolescence showed an enhanced response to 10 and jections compared with those that had pre20 mg/kg doses of cocaine during the first 15 min of the test session compared with mice exposed to cocaine during adulthood viously been exposed to only saline, and(*p ⬍ 0.05). Values are means ⫾SEM.
these effects depended on both age of exposure and test time (three-way ANOVA
age ⫻ drug ⫻ time interaction: 0 mg/kg
tion F(1,28) ⫽ 4.69, p ⬍ 0.05; t test (cocaine vs saline in adolescentF(2,52) ⫽ 3.24, p ⬍ 0.05; 10 mg/kg F(2,52) ⫽ 10.60, p ⬍ 0.001; 20
treated groups): t(13) ⫽ 4.97, p ⬍ 0.05)], right medial [(Fig. 2c;
mg/kg F(2,52) ⫽ 5.32, p ⬍ 0.01). Mice with a history of cocaine
two-way ANOVA: drug by age interaction F(1,28) ⫽ 4.07, p ⬍exposure exhibited an enhanced locomotor response to an injec0.01; t test (cocaine vs saline in adolescent-treated groups): t(13) ⫽tion of saline, reflecting sensitization to the previously drug6.37, p ⬍ 0.05)], and left medial [(Fig. 2c; two-way ANOVA: drug
paired environment (three-way ANOVA: main effect of drugby age interaction F(1,28) ⫽ 3.50, p ⬍ 0.05; t test (cocaine vs salineF(1,26) ⫽ 14.72, p ⬍ 0.001). Mice that had been pre-exposed toin adolescent-treated groups): t(13) ⫽ 4.46, p ⬍ 0.05)] surfaces of
cocaine also exhibited an enhanced locomotor response followthe striatum. All p values were adjusted with the FDR.
ing 10 mg/kg (three-way ANOVA: main effect of drug F(1,26) ⫽In addition to these regional changes, we found that adoles31.60, p ⬍ 0.0001) and 20 mg/kg (three-way ANOVA: main effectcent but not adult cocaine exposure produced cortical thinningof drug F(1,26) ⫽ 27.28, p ⬍ 0.0001) cocaine, indicating sensitithat was significant in regions of the right hemisphere. Thesezation to the locomotor stimulant effects of cocaine.
changes are consistent with the more pronounced voxel level
Exposure to cocaine during adolescence, but not adulthood,differences in the right hemisphere, and supported by significant
altered brain structure. Therefore we next evaluated whether
age of exposure by drug interactions in several cortical regions.
mice pre-exposed to cocaine during adolescence showed an enFor example, comparison of mice treated with saline and cocaine
hanced response to cocaine compared with the mice pre-exposed
during adolescence revealed thinning of the cortex in the primaryduring adulthood. Since changes in locomotor activity were mostsomatosensory cortex [(Fig. 3a; two-way ANOVA: drug by age
pronounced in the first 15 min, we focused on this test period.
interaction F(1,28) ⫽ 3.83, p ⬍ 0.05; t test (cocaine vs saline in
Strikingly, adolescence-exposed mice exhibited enhanced sensiadolescent-treated groups): t(13) ⫽ 4.55, p ⬍ 0.10)], the rhinaltization following the 10 mg/kg (two-way ANOVA: age ⫻ drug
cortex [(Fig. 3b; two-way ANOVA: drug by age interaction F(1,28) ⫽interaction F(1,26) ⫽ 6.57, p ⬍ 0.05) and 20 mg/kg (two-way
4.73, p ⬍ 0.05; t test (cocaine vs saline in adolescent-treated
ANOVA: age ⫻ drug interaction F(1,26) ⫽ 4.18, p ⫽ 0.051) co-1802 • J. Neurosci., January 30, 2013 • 33(5):1797–1803

caine challenge dose but not following the 0 mg/kg dose (two-way
ANOVA: age ⫻ drug interaction F(1,26) ⫽ 1.13, p ⫽ 0.81). Post hoccomparisons confirmed that locomotor activity was greater inmice exposed to cocaine during adolescence than during adulthood following the 10 mg/kg (t test: t(14) ⫽ 2.7, p ⬍ 0.05) and 20
mg/kg (t test: t(14) ⫽ 2.3, p ⬍ 0.05) cocaine challenge dose.

Discussion
Using unbiased whole-brain analysis of high-resolution MR images in
mice, we examined the impact of chronic cocaine exposure on brain
structure. There were two main findings. First, cocaine exposure produced persistent structural alterations that were, in many cases, localized
to brain regions involved in addiction, including the nucleus accumbens, striatum, medial forebrain bundle, ventral pallidum, substantia
nigra, and orbitofrontal and insular cortices. Second, these changes
were most pronounced in mice exposed to cocaine during adolescence. A recent study reported volumetric alterations in several graymatter regions including the striatum and insular cortex in humancocaine addicts and their nonstimulant-dependent siblings, suggesting that certain brain abnormalities precede drug use and predispose
these individuals to addiction (Ersche et al., 2012). Our results further indicate that cocaine itself impacts the development of many of
the same regions associated with drug dependence.
Perhaps the most striking finding was that many of the cocaineinduced structural abnormalities were identified in regions involved
in mediating cocaine’s acute and chronic effects that are associatedwith reward and sensitization. For example, the nucleus accumbens, medial forebrain bundle, and ventral pallidum were reduced in volume, consistent with cocaine’s primary action on themesolimbic dopaminergic pathway. Long-lasting neuroadaptation within this system likely contributes to locomotor sensitization (Vanderschuren and Pierce, 2010). Additionally, cocaine
exposure led to a decrease in the volume of the anterior cingulate
cortex and cortical amygdaloid area, regions where decreased
metabolic activity has been associated with cocaine craving inhuman subjects (Childress et al., 1999). The insular cortex was
likewise reduced in volume, and the structural alterations in theinsular have previously been associated with dependence on cocaine (as well as other drugs of abuse) in human addicts (Franklin
et al., 2002; Ersche et al., 2011; Gardini and Venneri, 2012). Following cocaine exposure the substantia nigra—a dopamine richarea of the midbrain—and the orbitofrontal cortex were increased in volume. Previous human imaging and rodent studies
implicate orbital frontal dysfunction in cocaine addiction
through its impact on conditioned reinforcement and drug craving (Everitt et al., 2007). Finally, we found volume and shapedifferences in the striatum following cocaine exposure in adolescent mice. Volume analysis revealed that cocaine exposure
caused portions of the anterior dorsal striatum to increase in size
and the posterior striatum to reduce in size, while shape analysis
revealed lateral outward displacement and medial inward displacement of the striatum bilaterally as a result of cocaine exposure. These bidirectional changes may account for previous
discrepant results from human imaging studies, with some reporting reduced (Barrós-Loscertales et al., 2011; Hanlon et al.,2011) and others increased (Jacobsen et al., 2001; Ersche et al.,2011) striatal volume in cocaine-dependent subjects. These results are consistent with an emerging literature suggesting that
reorganization of circuitry within the striatum leads to loss ofcontrol of voluntary behavior, which is a hallmark of addiction(Everitt and Robbins, 2005). Therefore, together these analyses revealthat adolescent cocaine exposure impacts a network of regions that are
directlyinnervatedbydopaminergicneurons.Thissuggeststhatthevol-

Wheeler et al. • Cocaine Exposure Changes Brain Structure in Miceumetric changes may be mediated by the direct actions of cocaine inthese regions, and raises the possibility that structural alterations in thisnetwork of regions might contribute to altered subsequent responding
to drugs of abuse. However, it is important to note that not all regions
innervated by dopaminergic neurons were impacted by adolescent cocaine exposure (e.g., medial prefrontal cortex). Moreover, there werevolumetric alterations in regions that do not receive dopaminergic innervation. This latter observation indicates that chronic cocaine exposure additionally causes secondary and likely tertiary effects on brain
morphology.
We used an MRI-based approach to systematically track cocaineinduced alterations in brain structure. These analyses provided information about the precise localization of cortical thickness andvolume change within structures and revealed shifts in the overall
shape of brain structures. What is responsible for these structuralchanges? Previous studies focusing on individual regions of the brain
have shown that chronic cocaine increases spine number and dendritic branching in the nucleus accumbens and prefrontal cortex(Robinson and Kolb, 1999) and that astrocyte number increases
following cocaine exposure and 3 weeks of abstinence in the nucleus
accumbens (Bowers and Kalivas, 2003). Since we observed a decrease, rather than an increase, in accumbens volume here this suggests that factors other than neuronal morphology and gliosis must
contribute to volumetric alterations. These factors may include altered axon sprouting, neurogenesis, fiber reorganization, myelin
formation, and remodeling and angiogenesis (Zatorre et al., 2012).
Most likely combinations of these factors interact to contribute to
the changes in MR signal described here.
The effects of cocaine exposure on brain structure and locomotor
sensitization were more pronounced during adolescence comparedwith young adulthood. Although adolescence is sometimes perceived to be a developmental phase that is unique to humans, allmammals undergo a similar transition from dependence to independence, and adolescence has been widely modeled in rodent studies (Adriani and Laviola, 2004). Cocaine use is often initiated duringadolescence when the brain is still developing (Lenroot and Giedd,
2006; Blakemore, 2012). The adolescent brain has been characterized by an imbalance between an early developing subcortical striatal
system that is sensitive to motivational stimuli and a late developing
prefrontal cognitive control system (Casey and Jones, 2010). Given
that the brain, and specifically its reward circuitry, is undergoing
dramatic changes throughout adolescence, this might suggest thatthe adolescent brain is more vulnerable to drugs of abuse like cocaine. This is supported by epidemiological studies that show thatwhen drug use is initiated during adolescence there are higher lifetime rates of drug use and faster progression to dependency than inpeople who begin in adulthood (Anthony and Petronis, 1995; Grant
and Dawson, 1998; O’Brien and Anthony, 2005). Here we observed
enhanced sensitization to the locomotor stimulant effects of cocaine
following cocaine exposure during adolescence. These behavioral
results parallel our structural analyses and indicate that cocaine exposure has a more pronounced impact on the developing brain. In
our experiments the direct relationship between brain anatomy andbehavior cannot be explored as the two outcomes were assessed inseparate groups of mice. However, the absence of significant structural differences in the mice treated during adulthood suggests that
forms of plasticity that are not associated with volumetric changes(e.g., modified strength and/or rearrangement of synapses) likely
contribute to the observed behavioral sensitization following
chronic cocaine exposure in these mice.
In human studies it is often challenging to isolate the effect ofage of onset of drug use on brain structure while controlling forthe total amount of drug exposure in an individual. By making

Wheeler et al. • Cocaine Exposure Changes Brain Structure in Micecocaine exposure equivalent, our experiments indicate that an
earlier age of onset is associated with more pronounced structural
alterations in reward-related regions of the brain. These results
also show that structural alterations seen in the brains of cocaine
users can be caused by the drug and do not just reflect preexisting anatomical differences. These cocaine-induced effects
may aggravate or interact with pre-existing abnormalities inbrain structure leading to behaviors related to drug dependence.

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