Pharmacology, Biochemistry and Behavior 203 (2021) 173117

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Pharmacology, Biochemistry and Behavior

journal homepage: www.elsevier.com/locate/pharmbiochembeh

Novelty preferences and cocaine-associated cues influence regions

associated with the salience network in juvenile female rats
Michael L. Rohan, Steven B. Lowen, Anna Rock, Susan L. Andersen *
McLean Hospital, Department of Psychiatry, Harvard Medical School, 115 Mill Street, Belmont, MA 02478, United States of America

A R T I C L E I N F O A B S T R A C T

Keywords: Preferences for novel environments (novelty-seeking) is a risk factor for addiction, with little known about its
Addiction underlying circuitry. Exposure to drug cues facilitates addiction maintenance, leading us to hypothesize that
Adolescence exposure to a novel environment activates a shared neural circuitry. Stimulation of the D1 receptor in the
Cocaine
prelimbic cortex increases responsivity to drug-associated environments. Here, we use D1 receptor over­
D1
Juvenile
expression in the prelimbic cortex to probe brain responses to novelty-preferences (in a free-choice paradigm)
Novelty and cocaine-associated odors following place conditioning. These same cocaine-conditioned odors were used to
Salience study neural circuitry with Blood Oxygen Level Dependent (BOLD) activity. D1 overexpressing females had
Sex deactivated BOLD signals related to novelty-preferences within the insula cortex and amygdala and activation in
the frontal cortex and dopamine cell bodies. BOLD responses to cocaine cues were also sensitive to D1. Control
females demonstrated a place preference for cocaine environments with no significant BOLD response, while D1
overexpressing females demonstrated a place aversion and weak BOLD responses to cocaine-conditioned odor
cues within the insula cortex. For comparison, we provide data from an earlier study with juvenile males
overexpressing D1 that show a strong preference for cocaine and elevated BOLD responses. The results support
the use of a pharmacological manipulation (e.g., D1 overexpression) to probe the neural circuitry downstream
from the prelimbic cortex.

1. Introduction
Early initiation of drug use is a significant risk factor for lifelong
addiction, making identification of high-risk teenagers imperative
(Grant and Dawson, 1998; Jordan and Andersen, 2016). The presence of
high-risk behaviors, including preferences for novelty, increases the
susceptibility to early drug use (Khurana et al., 2015; Palmer et al.,
2013; Wills et al., 1994). While novelty preferences can play a causal
role in addiction (Foulds et al., 2017), not everyone with elevated
preferences for novelty develops an addiction (SAMHSA, 2014). Addi­
tional measures are needed to identify high risk individuals.
In animals, novelty preferences are assessed by allowing an animal to
freely choose to enter a novel space (Beckmann et al., 2011; Belin et al.,
2011; Flagel et al., 2009; Leyton and Vezina, 2014; Molander et al.,
2011). A preference for a novel environment occurs when the value
placed on environment is rewarding (Puglisi-Allegra and Ventura,
2012). Similarly, preferences can be developed for drug cues, whereby
repeated pairings of a drug and the environment result in the formation
of conditioned cues that motivate later drug-seeking (Carlezon Jr., 2003;
Carr and White, 1983; Tzschentke, 1998). Unbiased placed conditioning
is a method used to detect the salience (either rewarding or aversive) of
cocaine-associated environmental cues (Carlezon Jr., 2003; Carr and
White, 1983; Tzschentke, 1998). A greater understanding of the inter­
section of the processing of novelty and drug cues that are associated
with addiction could help identify those most vulnerable to addiction.
Here, we used functional magnetic imaging (fMRI) to offer insight into
the role of the prelimbic cortex and the salience network.
The salience network processes information from the environment,
including drug cues, that is used to modulate future behavior (Bowers
et al., 2004; Sun and Rebec, 2005; Zavala et al., 2003). Recent evidence
in rats shows that the salience network includes the prelimbic cortex, as
well as the ventral insular cortex, and anterior cingulate cortex (Tsai
et al., 2020). Activity within the prelimbic cortex is at its greatest
developmentally during adolescence (Brenhouse et al., 2008; Galvan,
2010), when both novelty preferences (Adriani et al., 1998; Steinberg,
2008) and drug-seeking also reach their peak (Brenhouse et al., 2008).

Abbreviations: (BOLD), Blood Oxygen Level Dependent; (vmPFC), ventromedial prefrontal cortex.
* Corresponding author at: Mailstop 333, McLean Hospital, 115 Mill St., Belmont, MA 02478, United States of America.
E-mail address: sandersen@mclean.harvard.edu (S.L. Andersen).

https://doi.org/10.1016/j.pbb.2021.173117
Received 26 August 2020; Received in revised form 18 January 2021; Accepted 19 January 2021
Available online 6 February 2021
0091-3057/© 2021 Elsevier Inc. All rights reserved.
M.L. Rohan et al.
Studies in humans further show elevated activity in this prefrontal re­
gion in individuals that use or are dependent on substances (Franklin
et al., 2002; Grant et al., 1996; Leyton and Vezina, 2014; Maas et al.,
1998; Volkow, 2005; Volkow et al., 2002; Volkow et al., 2001).
Pharmacologically, increased dopamine D1 receptor expression in
the prelimbic cortex is associated with greater novelty preferences,
sensitivity to cocaine-associated cues, the motivation to take cocaine,
and elevated cocaine reinstatement to drug cues in male rats (Cicco­
cioppo et al., 2001; Kalivas et al., 2005; Sonntag et al., 2014; Sun and
Rebec, 2005; Weiss et al., 2001). Dopamine D1 receptor expression in
the prelimbic cortex parallels the heightened sensitivity to preferences
for cocaine-associated environments that emerges between juvenility
and adolescence in male rats (Brenhouse et al., 2008). To determine the
role of D1 in adolescence, we have manipulated D1 activity by micro­
injection of the D1 agonist SKF-23390 (Brenhouse et al., 2010) or
overexpression the D1 receptor by lentivirus (on glutamate neurons via
a CamKinase IIa promotor; CK⋅D1) (Brenhouse et al., 2015) in juvenile
rats. Both of these manipulations increased cocaine place preferences,
suggesting that increased prelimbic D1 (or activity in the salience
network) could increase risk for novelty preferences and drug-seeking
behavior. However, we know little about the role of D1 receptors on
2
Pharmacology, Biochemistry and Behavior 203 (2021) 173117
novelty-seeking and the salience of drug cues in juvenile females.
In the current study, female juvenile rats received prelimbic in­
jections of the CK⋅D1 or CK.GFP virus to elucidate the role of D1 on
novelty preferences and drug-seeking. Increased D1 expression in the
prelimbic cortex produces a pharmacological “hub” to probe the
salience network. Other studies have seeded the ventral insula cortex
and show prelimbic involvement in drug effects (Tsai et al., 2020).
Previously, we determined that Blood Oxygenation Level Dependent
(BOLD) responses to cocaine-associated cues in juvenile male rats with
D1 overexpression (CK⋅D1, but not CK.GFP) in the prelimbic cortex
(Lowen et al., 2015) were nearly identical to BOLD response in human
addicts (Garavan et al., 2000; Vaquero et al., 2017) and in male rats
chronically exposed to cocaine (Johnson et al., 2013; Liu et al., 2013;
Marota et al., 2000). Here, the inter-relationship between novelty
preferences and drug cues and whether they share neural circuitry was
further explored through the use of fMRI. For comparison, blood flow
data from the juvenile males are re-presented in a more extensive data
set to facilitate comparison between males and females. The experi­
mental timeline is shown in Fig. 1a. Our goal is to determine how spe­
cific CK⋅D1 overexpression influences activity in other brain regions
associated with cocaine cue reactivity.
Fig. 1. Determination of behavioral effects of D1
overexpression on novelty and place conditioning. a)
Timeline of the behavioral and fMRI paradigm.
Following viral transduction with CK.GFP (n = 7) or
CK⋅D1 (n = 7), female subjects were assessed for
novelty preferences and then underwent place con­
ditioning to 10 mg/kg cocaine. After place condi­
tioning testing on P27, BOLD responses to CS+ and
CS− were determined on P28 using a block design.
The location of the virus injection was confirmed to
the prelimbic cortex, with staining for CK.GFP and
CK⋅D1 are shown at 5×. b) Preference for novelty was
not different between virus conditions, with the mean
score shown by the horizontal bar for each group. c)
Using an unbiased paradigm, where rats had no
preference for either side, the subject underwent two
conditioning days at postnatal day (P)25 and 26, a
previously neutral odor was paired with cocaine
(CS+ odor) and second odor (CS− ) was paired with
saline for 60 min each. On day four, subjects had
access to all three chambers. CK.GFP animals
demonstrated a preference for CS+ environments
(gray bar: post-conditioning), whereas CK⋅D1 sub­
jects show an aversion (black bar: post-conditioning);
white bars: pre-conditioning. Circles show individual
data points.
M.L. Rohan et al.
2. Materials and methods
2.1. Subjects
Female Sprague Dawley rat litters were obtained from Charles River
Laboratories (Boston, MA) at P12. Pups were weaned at postnatal day 21
(P21) and housed with same-sex littermates with three rats per cage.
One pup from each litter/condition was used to avoid litter effects,
resulting in n = 7 for CK.GFP and 7 for CK⋅D1 subjects. Rats were housed
with food and water available ad libitum in constant temperature and
humidity conditions on a 12-h light/dark cycle (lights on 0700). The
experiments were conducted in accordance with the National Institutes
of Health guide for the care and use of laboratory animals (NIH Publi­
cations No. 8023, revised 1978) and approved by the Institutional An­
imal Care and Use Committee at McLean Hospital. Females were too
young (<P28) to achieve pubertal status, and therefore there was no
need to determine the estrous state.
2.2. Lentiviral injections
At P16 (45–50 g), rats were anesthetized with a ketamine/xylazine
mixture (80/12 mg/kg, respectively). Subjects were randomly assigned
to receive a lentiviral vector that targets glutamate neurons (via the
CamKinase IIa [CK] promotor) and expresses either green fluorescent
protein (CK.GFP) or drd1 (CK⋅D1). The Massachusetts General Viral core
made the viruses with a concentration of 108–109 transducing units per
μl. Subjects received 0.6 μl of the virus bilaterally into the prelimbic
cortex at stereotaxic coordinates (AP +2.7, ML: 0.5; DV: − 2.7) following
our methods (Sonntag et al., 2014) and behavioral assessment did not
commence for five days post-surgery, an interval that we have deter­
mined is sufficient for stable expression (Sonntag et al., 2014). After
completing the study, subjects were deeply anesthetized with sodium
pentobarbital and perfused transcardially with phosphate-buffered sa­
line and 4% paraformaldehyde. The brains were cryoprotected and
sliced into 40 μm sections. Following standard histological procedures,
CK⋅D1 was confirmed by staining for the D1 receptor (D1 antibody;
Sigma-Aldrich, St. Louis, MO: 1:250; made in rat with an Alexa-Flour
568-coupled IgG secondary antibody [1:400; Molecular Probes]). Sec­
tions were mounted on slides and with Stereo Investigator Image
Analysis System (MBF Bioscience, Williston, VT) to confirm location.
Only subjects with histological confirmation of placement within the
prelimbic cortex were used (Fig. 1b). Novelty preference habituation
was initiated three days later to allow for viral expression.
2.3. Novelty preferences
At P20, each subject was placed on one side of a two-sided
24 × 18 × 33 cm dimly lit chamber (MedAssociates Inc., St Albans,
VT), and the number of beam breaks recorded for 20 min (Macri et al.,
2010; Sonntag et al., 2014). Subjects were habituated two additional
and consecutive days then remained in the home cage for 24 h. On day
five, the door connecting to the second chamber was opened, and the
total time spent on the novel side was used as an index of novelty
preference.
2.4. Place conditioning
Place conditioning is well suited for developmental studies in young
subjects due to minimal training time. Here, we used an unbiased place
conditioning protocol to establish behavioral preferences or aversions to
cocaine cues (Andersen et al., 2002). A three-compartment chamber was
used, with the two side compartments differing in floor texture, lighting,
and coloring. A custom-built olfactometer (Lowen and Lukas, 2006)
delivered either rose (phenethyl alcohol) or almond (acetophenone)
odor at a flow rate of 10 mL/min to the corner of each side that was
vented through the opposite corner via a vacuum.
3
Pharmacology, Biochemistry and Behavior 203 (2021) 173117
Negative pressure prevented odor mixing. The odors enhanced as­
sociations between drug and the environment (Lowen et al., 2015).
Repeated pairing of the environment with a rewarding drug permits the
environment to develop conditioned motivational properties (Everitt
and Robbins, 2005). Juvenile rats (P24) were habituated to the 3-cham­
bered apparatus for 30 min to establish the absence of baseline prefer­
ences for odor-associated environments (defined as spending >18 of
30 min on one side). Sixty-minute conditioning sessions on days 2 and 3
paired one odor with saline (a conditioned stimulus that does not predict
drug [CS− ]) and a second odor paired with 10 mg/kg cocaine in the
afternoon (a CS odor that predicts drug [CS+]). Odors and side of odor
presentation were counterbalanced within groups. The 10 mg/kg dose of
cocaine was selected because juvenile male rats typically do not show
place preferences with this dose (Brenhouse et al., 2008) and is a
threshold dose to detect D1/GFP overexpression and age differences
(Andersen et al., 2002; Sonntag et al., 2014). On day 4, rats could freely
explore the entire apparatus for 30 min in a drug-free state. Time spent
on each side was measured to determine behavioral preferences to drug-
conditioned, odor-paired environments. Animals were imaged the
following day.
2.5. MRI
2.5.1. Odor delivery (MRI)
Odors were presented to the animals using a second, similar olfac­
tometer that passed filtered room air at 0.3 L/min through one of three
flasks that contained solutions of “rose,” “almond,” or neutral (water)
odors. A separate odor tube exited each flask in tubes (PTFE) in a design
that minimized dead space and provided discrete periods of odor
exposure (verified by (Lowen and Lukas, 2006)). An electronic trigger
programmed into the pulse sequenced selected the odor. During the rest
periods between odors, the system was purged with the neutral (water)
selection.
2.5.2. fMRI acquisition
At P28, odor-cued fMRI data were acquired on a 9.4 T Agilent (Palo
Alto, CA) MRI system with a 120 mm diameter gradient system. The rats
were anesthetized with 2% isoflurane and maintained on 1.5% mixed
with 1.5 L/min breathing quality air during image acquisition following
the methods of Lowen et al., 2015. Prior studies have shown that
anesthetized animals respond to drug-conditioned cues in a manner
consistent with their responses in awake studies (Boyett-Anderson et al.,
2003; Liu et al., 2013). Respiration and EKG were monitored (Small
Animal Instruments, Inc.), and a recirculating water blanket (Gaymar T/
Pump) was used for temperature.
A tight-fitting birdcage RF coil (fabricated in-house) acquired fMRI
images using a single-shot Echo-Planar acquisition, TE/TR = 17 ms/3 s;
matrix = 64 × 64 on 40 mm FOV with 38 × 0.625 mm slices for 10 min.
fMRI had a one minute baseline period followed by a repeated block of
15 s almond, 15 s no odor, 15 s rose, 15 s no odor for the remaining nine
minutes. All animals received the same order of odor presentation,
illustrated in Fig. 1a. As a result, half of the animals were exposed to
their cocaine-conditioned odor first. This block design produces
temporally linked activation in the amygdala following cue presentation
(Lowen et al., 2015), similar to human studies (Garavan et al., 2000,
Lukas et al., 2013). In our previous study (Lowen et al., 2015), we show
that odor-induced increases in activation return to baseline simulta­
neously during the 15-sec odor presentation and remains at baseline
levels for the remainder of the 45-sec of the 60-sec block. For these
reasons, we conclude that there is minimal to no odor mixing.
2.6. Statistical analyses
2.6.1. Behavioral
Novelty preferences were tested between virus conditions by an in­
dependent samples t-test between GFP and D1 groups. Place
M.L. Rohan et al.
conditioning data were analyzed by a mixed ANOVA (SPSS v 20) with
the virus as a between-subjects factor and pre- and post-conditioning as
a repeated-measures factor. Regression analysis was used to determine
the relationship between novelty preferences (time spent on the novel
side of the chamber) and the individual place conditioning scores
(defined as time spent on the drug side – time spent on the saline side).
Significance was set at p < 0.05.
2.6.2. MR image processing
2.6.2.1. The BOLD fMRI images were analyzed with an in-house approach,
following our previous methods (Lowen et al., 2015).. Nyquist ghosting
was minimized using a parameterized phase correction for spatially
constant and linear terms (Kutter et al., 1994). Magnitude and phase
images were obtained. Standard preprocessing included motion
correction, slice timing correction, spatial smoothing to 10 mm FWHM
(1 mm in native space), and high pass filtering with a cutoff of 100 s.
2.6.2.2. fMRI subject analysis. Subject-wise BOLD analysis was per­
formed using two passes of a standard general linear model (GLM)
processing (FSL). This analysis used a whole-brain voxel-wise approach;
the model included two odor stimuli (convolved with the standard he­
modynamic response function and high-pass filtered) and their time
derivatives, and six detected motion regressors. The first pass analysis
was performed to derive confound regressors, and the image phase
change from the first image formed an additional voxel-wise nuisance
regressor. The residuals from the first pass were reduced using a prin­
cipal component analysis to the first eight components. The component
time courses were used as 8 × 1D global nuisance regressors (Madsen
and Lund, 2006) for the second pass. The residuals were also averaged
over each slice within each volume and then replicated throughout that
slice, forming an additional, voxel-wise nuisance regressor. A GLM
analysis included all of the regressors from the first model and the nine
listed regressors above derived from the residuals in the second pass.
The “almond – rose” contrast was used for the group analysis.
2.6.2.3. Registration. Anatomic registration was performed iteratively
to a group average for both anatomic and fMRI data. A standard
anatomic brain was constructed from anatomic images by scaling the
voxel size entries in the header to clinical values (×10) to use FSL
software (FLIRT, using 12 DOF; FMRIB, Oxford University). The
anatomic images were then registered to the MNI standard (human)
brain with 6 DOF initially, and the results then averaged to form a first
template. The brains were then registered to this template and averaged
again. The process was repeated two more times; the converged result
was the standard anatomic rat brain for this study, and the original voxel
size entries were restored.
The BOLD scans were used to construct a standard BOLD brain to be
used as a registration target. The same iterative methods were used as
for the anatomic images, with four iterations. The resulting standard
BOLD brain was aligned to the standard anatomic rat brain and used to
register the subject-wise statistical images.
2.6.3. fMRI group analysis
Subject-wise results were combined in a mixed-effects group analysis
for the almond–rose contrast (Chase et al., 2011), following our previous
approach (Lowen et al., 2015). This group analysis was performed as a
voxel-wise analysis, relying on the registered statistical images. Four
regressors were used. The first (“D1”) was +1 for D1 rats trained with
almond as the active odor, − 1 for D1 rats trained with rose as the active
odor, and 0 for GFP rats. The second (“GF”) was +1 for GFP rats trained
with almond as the active odor, − 1 for GFP rats trained with rose as the
active odor, and 0 for D1 rats. The third regressor (“NovD”) was the
novelty score demeaned within D1 rats and then multiplied by D1 (and
therefore equal to zero for GFP rats). The last (“NovG”) was the novelty
4
Pharmacology, Biochemistry and Behavior 203 (2021) 173117
score demeaned within GFP rats and then multiplied by GF. Contrasts
comprised positive and negative activation for D1, GF, D1-GF, NovD,
NovG, NovD+NovG, NovD-NovG. Voxel-wise results for these contrasts
were converted to z scores, and a threshold of z > 2.3 was applied.
Second, Gaussian random field theory was used to find clusters of
contiguous voxels over the threshold with a cluster significance level of
p < 0.05, family-wise error corrected for multiple comparisons over the
whole brain.
2.6.4. Statistical analysis (regional)
A post-hoc analysis was performed on the subject-wise effects of
behavioral measures on the BOLD activation. Mean BOLD activation was
identified in regions of interest (ROI) defined by anatomic localization
by the atlas of (Sherwood and Timeras, 1970). Using correlational an­
alyses (Pearson’s r), individual BOLD levels were correlated with
behavioral scores for novelty preferences. The a priori defined anatomic
regions examined were the dorsal striatum, prelimbic cortex, amygdala,
and substantia nigra. All correlation data were subject to bootstrap
analysis with 10,000 random resampling analyses; data were considered
significant following this analysis if they met the adjusted p values
(p < 0.05). The relationships between CK.GFP and CK⋅D1 groups were
statistically compared with Fisher’s z transformation, followed by
Bonferroni correction to determine significant differences.
3. Results
3.1. Novelty preferences
Data from n = 7 CK⋅D1 and n = 7 CK.GFP subjects were used in the
final data analysis for imaging; data for one CK.GFP subject was not
available due to MRI difficulties. Virus condition (CK⋅D1 versus CK.GFP)
had no significant influence on novelty preference scores (Virus:
F1,13 = 0.49, p > 0.05). Fig. 1b shows the individual scores presented in
a scatter plot of the two groups.
3.2. Place conditioning
The same n = 7/7 subjects assessed for novelty preferences under­
went place conditioning. Neither the D1 nor the GFP virus significantly
influenced place conditioning to cocaine: (Virus × [pre/post]: p = 0.17;
Fig. 1c). However, the place conditioning score (post – pre-conditioning
values within each animal) that was used in the BOLD analysis revealed
a significant difference between CK.GFP and CK⋅D1 (independent sam­
ples t-test: t12 = 2.4, p < 0.05; Fig. 3c left panel). Specifically, CK.GFP
females demonstrated a place preference for cocaine-associated envi­
ronments, whereas CK⋅D1 females demonstrated an aversion. These
same subjects underwent imaging.
3.3. fMRI whole-brain and regional brain associations
The main findings of the study are that novelty-preferring modulates
CK⋅D1 animals’ BOLD responses to cocaine-associated odors; BOLD re­
sponses to cocaine-associated odor cues in CK⋅D1 females reflect an
aversive response. First, the influence of novelty-seeking on cocaine
cued CS+ BOLD responses are found in Fig. 2. In the whole-brain
analysis, only the CK⋅D1 group showed significantly different re­
sponses to the CS+ (relative to the CS− ) odors. Based on the whole-brain
analysis, regions with negative BOLD responses included those related to
the salience network (the prefrontal and insula cortices, amygdala, and
dopamine cell bodies; (Menon, 2015)). The contrast generated by
correlating novelty scores with odor-cued brain activation within the
CK⋅D1 group alone yielded several significant changes in regional BOLD
responses (Fig. 2). Regions of interest were then a priori defined focusing
on regions associated with salience. Individual novelty-seeking scores
for CK⋅D1 and CK.GFP rats correlated with individual differences in
BOLD responses within the selected regions (medial prefrontal cortex,
M.L. Rohan et al. Pharmacology, Biochemistry and Behavior 203 (2021) 173117

Fig. 2. Modulation of whole-brain analysis of the BOLD

response to CS+ versus CS− associated by novelty-seeking in
CK⋅D1 females (n = 7). a) Significant responses were observed
in the CK⋅D1 group, whereas no significant activation was
observed in the CK.GFP group. Scale bar hue indicates z-score
with increases in red/yellow areas, decreases in blue. Clusters
shown are significant (p < 0.05) after correction for multiple
comparisons across the whole brain. Increased BOLD responses
are associated with novelty scores in the anterior cingulate
cortex, thalamic and cerebellar regions, and negative responses
in the frontal cortex, dorsal striatum, insula, amygdala, and the
dopamine cell body region. b) References plates from Sher­
wood and Timeras (1970) highlighting affected regions that
are part of the salience network. Key figures are identified:
medial prefrontal cortex (mPFC); right amygdala (AMY), dor­
sal striatum (DSTR); dopamine cell bodies (SN/VTA) with
Bregma coordinates shown.

right amygdala, left dorsal striatum, and substantia nigra; Table 1). shown in Fig. 3d.
Novelty preference scores did not significantly correlate with BOLD
scores in these regions in CK.GFP rats. Activity as a covariate signifi­ 4. Discussion
cantly influenced BOLD responses in the left substantia nigra (z = − 1.72;
p < 0.05). The CK⋅D1 virus did not significantly affect novelty-seeking but
Second, the relationship between regional responses to the CS+ odor resulted in a place aversion for cocaine-associated environments in ju­
was significant when expressed as a function of place conditioning score venile females. Cocaine-associated odors produce BOLD signaling
in CK⋅D1 animals, but not in CK.GFP animals (Fig. 3a and c). Positive changes in anesthetized rats (Johnson et al., 2013; Liu et al., 2013;
BOLD regions associated with placed conditioning included the so­ Lowen et al., 2015). This study’s first goal was to examine novelty-
matosensory region, the insula cortex, and the amygdala. To facilitate seeking as a risk factor for cocaine sensitivity in young female rats.
comparison between female and male juvenile rats, we provide an The results show that increased novelty-seeking is associated with
expanded data set from our (Lowen et al., 2015) paper where juvenile deactivated BOLD responses to cocaine-associated odors at this early age
males expressing the CK⋅D1 virus underwent place conditioning, and in CK⋅D1 females. Specifically, a deactivation of regions found within
their responses to cocaine-associated odors were imaged. Subjects were the salience network was observed in female subjects that were related
treated identically and analyzed relative to their same-sex CK.GFP to novelty-seeking. Resting-state connectivity in fMRI studies identifies
subjects are shown in Fig. 3b for males. Responses to the CS+ stimulus in the salience network (Menon and Uddin, 2010) with similarities found
the CK⋅D1 subjects are either aversion or approach (preference) and are between humans (imaging) and non-human primates (anatomy; (Haber

Table 1
Correlations between regional BOLD responses to cocaine cues and behavior.

Bregma ROI Fisher's Fisher's Novelty Fisher's

+5 0 -5 -10 -15
Bregma 0

5 M2 M1
Figure 2
n=7/7 Activity Z Place Conditioning Z Preference Z
S1HL
RSA
nteraural 10
S1FL

D1 GFP D1 GFP D1 GFP

+ 15 + 10 +5 0 -5
S1DZ
cg RSGb

IG
cc S1BF
df
TS
LV

S1
fi DG
D3V LDVL
sm MHb AD
st
ec
MD

-1.6
PVA AVDM
PC
PT PC S2
ic
AVVL
CM
eml AM
IAD Rt

Left 0.04 -0.29 -- -0.10 0.18 -- -0.36 -0.04 --

VA CPu
IAM GI
mt AMV
Rh DI
LGP
Re VM

Amygdala
rf Xi LaDL
Sub
IPAC Cl AIP
VRe B B AStr
ns
ZI
al 3VPaDC
PaLM CeL CeC DEn
PaMP CeM
f LH SI
mfb SPa BLA
PaV
cst
AHP I VEn
Pe
AHC MeAD BMA

Right 0.41 -0.18 -- 0.28 0.23 -- -0.86 0.32 P<0.01

LOT opt SO BAOT ACo
sox RCh CxA

2.2 Left 0.12 -0.47 -- 0.45 0.53 -- -0.68 -0.01 --

Cingulate
Right 0.08 -0.03 -- 0.47 0.18 -- -0.63 0.28 P<0.05
1.2 Left -0.13 0.40 -- 0.50 -0.11 -- -0.67 0.30 P<0.1
Striatum
Right 0.03 0.20 -- -0.01 -0.09 -- -0.80 0.27 P<0.05
1.4
Insula Left 0.68 0.16 -- 0.08 -0.68 P<0.1 -0.13 -0.05 --
Right 0.32 0.06 -- 0.08 -0.30 -0.18 0.60 P<0.05
0.4 Left 0.01 0.62 -- -0.13 -0.53 -- -0.12 0.36 --
Accumbens
Right 0.15 -0.10 -- -0.36 -0.11 -- -0.25 0.22 --

-5.3 Left 0.00 -0.34 -- 0.36 0.19 -- -0.74 0.41 P<0.05

Substantia nigra
Right -0.21 -0.26 -- 0.16 -0.08 -- -0.53 -0.26 --

5
M.L. Rohan et al.
and Knutson, 2010)). The rat salience network comprises the dorsal/
ventral agranular insular cortex as well as the prelimbic, infralimbic,
and primary/secondary cingulate cortices (Tsai et al., 2020), with
downstream activity in the dopamine cell bodies, amygdala, and thal­
amus. Novelty-seeking in the females was associated with a significant
BOLD response within a number of these regions. Elevated BOLD re­
sponses in individuals self-identified as novelty seekers are found in the
ventral striatum (Wittmann et al., 2008), medial prefrontal cortex
(Bermpohl et al., 2008), and the dopamine cell bodies in humans
(Bunzeck et al., 2007; Krebs et al., 2011). Our study further shows that
novelty-seeking is associated with decreased BOLD responses to cocaine-
associated cues in dorsal and ventral striatal regions, the dopamine cell
bodies, and the dorsal agranular insular cortex. No activity was observed
in the ventral insula cortex, which is associated with the salience
network (Tsai et al., 2020), when novelty was the regressor. Therefore,
whether our results reflect the observations in humans that novelty-
seeking is strongly associated with a negative BOLD response in the
insula (Wang et al., 2015) that is part of the salience network is unclear.
The insula cortex is involved in interoceptive functions, including self-
awareness of emotions (Decety et al., 2014), body function (i.e., heart
rate), and drug addiction (Breiter et al., 1997; Naqvi and Bechara,
6
Pharmacology, Biochemistry and Behavior 203 (2021) 173117
Fig. 3. Whole-brain analysis of the BOLD response to
CS+ versus CS− associated with place conditioning in
CK⋅D1 females (n = 7). a) Regional BOLD signal re­
sponses to CS+ versus CS− are shown on top for fe­
males. Scale bar hue indicates z-score with increases
in red/yellow areas, decreases in blue. Clusters
shown are significant (p < 0.05) after correction for
multiple comparisons across the whole brain. b)
Extended data set from Lowen et al. (2015), demon­
strating BOLD changes from the identical experiment
in males (published with permission). c) Preference
scores (post-conditioning –pre-conditioning effect)
for females and d) males that were used to determine
the effects of cocaine cues on BOLD responses.
2010). The insula’s negative BOLD response related to novelty suggests
that less salience is given to interoceptive cues (Naqvi and Bechara,
2010). Consequently, reduced activity in the insula would facilitate
more attention to external, environmental factors, including novel en­
vironments or drug cues. The insula is the only area influenced by both
novelty preferences and cocaine cues in the CK⋅D1 animals. Opposing
BOLD activity, for reasons we do not yet understand, could contribute to
a limited role of novelty preferences as a risk factor for drug use at this
stage. To this end, novelty preferences only predicted cocaine condi­
tioning scores in a subset of animals. While this relationship was sig­
nificant, it was based on a small number of animals and should be
investigated in a larger cohort.
Place conditioning data show similar effects of cocaine cues on BOLD
activity despite sex differences in behavioral responses. We hypothe­
sized that conditioned cocaine cues would produce a place preference
and activate the same regions in the CK⋅D1 males, which include the
prefrontal regions, ventral striatum, amygdala, dopamine cell bodies
(Liu et al., 2013; Lowen et al., 2015; Zhou et al., 2014). The aversion to
cocaine-associated environments in CK⋅D1 females was unexpected.
Female CK⋅D1 rats show increased BOLD signals in the ventral insular
cortex (e.g., salience network; (Tsai et al., 2020)), whereas the males
M.L. Rohan et al.
activated the dorsal insular cortex, which is associated with drug cues
during reinstatement (Cosme et al., 2015). The sex difference observa­
tion is consistent with higher c-fos responses following exposure to drug
cues in the ventral insula in females relative to males (Zhou et al., 2014)
Increased activity in the ventral insular cortex is consistent with a
negative state (Moschak et al., 2018) and could underlie the place
aversion. The ventrolateral and the caudal ventrolateral caudate puta­
men were also significantly activated in females, but not males. Earlier
studies with c-fos mRNA show that cocaine sensitivity is emerging by
P28, but cocaine-induced increases in extracellular dopamine are absent
(Cao et al., 2007). Females have higher levels of cortical dopamine than
males during periadolescence (Staiti et al., 2011) that would increase
the effectiveness of cocaine. Together, cocaine-associated cues activate
different parts of neural circuitry in juvenile females than males.
Whether age-related changes in the dopamine system drive this sex
difference is likely but requires further investigation.
Cocaine-associated cues activated BOLD signals in the ventral striatal
area when the behavioral response is aversive to cocaine and when the
response is positive to cocaine cues (Flagel et al., 2011; Rebec and Sun,
2005; Schultz, 2006). Drug cues are associated with prelimbic activity,
as reflected in the different behavioral outcomes between females and
males. The anticipation of a positive effect activates the medial pre­
frontal cortex in juvenile CK⋅D1 males (Lowen et al., 2015), rats
chronically exposed to cocaine (Liu et al., 2013), or exposed to cocaine
cues (Johnson et al., 2013). Juvenile CK⋅D1 males show strong BOLD
activity in this region. In contrast, CK⋅D1 females show greater activa­
tion in the insula cortex, suggesting an internal focus on cocaine cues.
Both sexes show activation of the amygdala. These data confirm that the
placement of the CK⋅D1 virus in the prelimbic cortex produces a salience
hub.
Comparison across juvenile females and males in Fig. 3 should take
into account a few factors. First, males and females experience puberty
at different ages (Juraska and Willing, 2017). The age used here, P28 at
the time of imaging, is an early age for adolescence with neither sex
showing strong hormonal changes (Juraska and Willing, 2017). Second,
differences in the degree of activation could reflect different virus titers,
although this is highly unlikely. The virus is injected at a high concen­
tration designed to produce a maximal effect. Third, males and females
have different cocaine responses. Nonetheless, it was surprising to
observe different patterns of BOLD activity in response to cocaine-
associated cues. The observation that the CK.GFP control females
demonstrated a place preference raises the possibility that too much
stimulation of the salience hub produced a place aversion and a decrease
in the salience network activity. Established literature shows that some,
not all, D1 related behaviors demonstrate an inverted U-shaped dose-
response curve (Floresco, 2013). Both CK.GFP females and CK⋅D1
males (Lowen et al., 2015) show a place preference for cocaine at the
10 mg/kg dose used here, suggesting that CK⋅D1 females were
overstimulated.
Together, these data show that the behavioral responses to condi­
tioned cocaine cues are reflected in regions associated with the salience
network and other regions as well. The CK⋅D1 in the prelimbic cortex
produced a pharmacological hub that may be useful tool similar to an­
alyses for resting connectivity (Ash et al., 2016). Behaviorally, D1
overexpression selectively is associated with increased drug-seeking in
response to conditioned drug cues (Ciccocioppo et al., 2001; Sanchez
et al., 2003; Sun and Rebec, 2005). The use of the CK⋅D1 virus increases
the cortical activity to increase drug-seeking in adults (Sonntag et al.,
2014). However, juvenile females already have elevated levels of drug-
seeking and extra prelimbic D1 stimulation may have been too much,
leading to an aversion. Further investigation is needed in control females
(or CK.GFP) to determine how novelty and drug-seeking interact to
modulate the immature neural circuitry. The preliminary data presented
here show regions of activation involved in both behaviors.
Using a pharmacological seed with fMRI to detect the effects of
cocaine-conditioned cues provides an approach to assess the extensive,
7
Pharmacology, Biochemistry and Behavior 203 (2021) 173117
and possibly transient, changes in the salience network that may reflect
different drug use stages. In the current study, increases in the D1 re­
ceptor in the prelimbic cortex selectively decreased activity in the dorsal
insular cortex, striatal regions and the cell bodies when novelty served as
a regressor. The clear delineation between the dorsal and ventral insular
cortex further validates the utility of this approach to parse the role of
specific brain regions that in turn can be translated to human pop­
ulations (Tsai et al., 2020). Similarly, conditioned place preferences can
be used as a rapid screen for determining drugs that can reduce craving
(Napier et al., 2013) or its underlying neural circuitry. Place condi­
tioning paradigms are translatable and have been used in children
(Childs and de Wit, 2009). Alternatively, a map of target regions asso­
ciated with the place aversion in females can guide addiction inter­
vention. Together, this pharmacological seed approach can provide
insight into addiction-related behaviors early in life to facilitate the
identification of the 8–15% of the population at risk for substance use
disorder (Grant and Dawson, 1998; Jordan and Andersen, 2016). More
research is needed to assess behavioral risk factors’ role and scope to
identify such individuals and sex differences.
Declaration of competing interest
None.
Acknowledgment
The NIH had no involvement in the design, execution, data analysis,
or presentation of the final results.
Funding
This work was supported by the National Institutes of Health DA-
10543 and DA-026485 (to SLA). The NIDA Drug Supply Program sup­
plied cocaine.
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