Hierarchical recruitment of phasic dopamine signaling in the striatum during the progression of cocaine use.
ABSTRACT Drug addiction is a neuropsychiatric disorder that marks the end stage of a progression beginning with recreational drug taking but culminating in habitual and compulsive drug use. This progression is considered to reflect transitions among multiple neural loci. Dopamine neurotransmission in the ventromedial striatum (VMS) is pivotal in the control of initial drug use, but emerging evidence indicates that once drug use is well established, its control is dominated by the dorsolateral striatum (DLS). In the current work, we conducted longitudinal neurochemical recordings to ascertain the spatiotemporal profile of striatal dopamine release and to investigate how it changes during the period from initial to established drug use. Dopamine release was detected using fast-scan cyclic voltammetry simultaneously in the VMS and DLS of rats bearing indwelling i.v. catheters over the course of 3 wk of cocaine self-administration. We found that phasic dopamine release in DLS emerged progressively during drug taking over the course of weeks, a period during which VMS dopamine signaling declined. This emergent dopamine signaling in the DLS mediated discriminated behavior to obtain drug but did not promote escalated or compulsive drug use. We also demonstrate that this recruitment of dopamine signaling in the DLS is dependent on antecedent activity in VMS circuitry. Thus, the current findings identify a striatal hierarchy that is instantiated during the expression of established responses to obtain cocaine.
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ABSTRACT: Rats emit ultrasonic vocalizations (USVs) that are thought to serve as situation-dependent affective signals and accomplish important communicative functions. In appetitive situations, rats produce 50 kHz USVs, whereas 22 kHz USVs occur in aversive situations. Reception of 50 kHz USVs induces social approach behavior, while 22 kHz USVs lead to freezing behavior. These opposite behavioral responses are paralleled by distinct brain activation patterns, with 50 kHz USVs, but not 22 kHz USVs, activating neurons in the nucleus accumbens (NAcc). The NAcc mediates appetitive behavior and is critically modulated by dopaminergic afferents that are known to encode the value of reward. Therefore, we hypothesized that 50 kHz USVs, but not 22 kHz USVs, elicit NAcc dopamine release. While recording dopamine signaling with fast-scan cyclic voltammetry, freely moving rats were exposed to playback of four acoustic stimuli via an ultrasonic speaker in random order: (1) 50 kHz USVs, (2) 22 kHz USVs, (3) time- and amplitude-matched white noise, and (4) background noise. Only presentation of 50 kHz USVs induced phasic dopamine release and elicited approach behavior toward the speaker. Both of these effects, neurochemical and behavioral, were most pronounced during initial playback, but then declined rapidly with subsequent presentations, indicating a close temporal relationship between the two measures. Moreover, the magnitudes of these effects during initial playback were significantly correlated. Collectively, our findings show that NAcc dopamine release encodes pro-social 50 kHz USVs, but not alarming 22 kHz USVs. Thus, our results support the hypothesis that these call types are processed in distinct neuroanatomical regions and establish a functional link between pro-social communicative signals and reward-related neurotransmission.Journal of Neuroscience 08/2014; 34(32):10616-23. · 6.75 Impact Factor
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ABSTRACT: Brief fluctuations in dopamine concentration (dopamine transients) play a key role in behavior towards rewards, including drugs of abuse. Drug-evoked dopamine transients may result from actions at both dopamine cell bodies and dopamine terminals. Inhibitory opsins can be targeted to dopamine neurons permitting their firing activity to be suppressed. However, as dopamine transients can become uncoupled from firing, it is unknown whether optogenetic hyperpolarization at the level of the soma is able to suppress dopamine transients. Here, we used in vivo fast-scan cyclic voltammetry to record transients evoked by cocaine and raclopride in nucleus accumbens (NAc) of urethane-anesthetized rats. We targeted halorhodopsin (NpHR) specifically to dopamine cells by injecting Cre-inducible virus into ventral tegmental area (VTA) of transgenic rats that expressed Cre recombinase under control of the tyrosine hydroxylase promoter (TH-Cre(+) rats). Consistent with previous work, co-administration of cocaine and raclopride led to the generation of dopamine transients in NAc shell. Illumination of VTA with laser strongly suppressed the frequency of transients in NpHR-expressing rats, but not in control rats. Laser did not have any effect on amplitude of transients. Thus, optogenetics can effectively reduce the occurrence of drug-evoked transients and is therefore a suitable approach for studying the functional role of such transients in drug-associated behavior.Frontiers in Neural Circuits 09/2014; 8:114. · 2.95 Impact Factor
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ABSTRACT: While most drugs of abuse increase dopamine neurotransmission, rapid neurochemical measurements show that different drugs evoke distinct dopamine release patterns within the nucleus accumbens. Rapid changes in dopamine concentration following psy-chostimulant administration have been well studied; however, such changes have never been examined following opioid delivery. Here, we provide novel measures of rapid dopamine release following intravenous infusion of two opioids, morphine and oxyco-done, in drug-na€ ıve rats using fast-scan cyclic voltammetry and rapid (1 min) microdialysis coupled with high-performance liquid chromatography -tandem mass spectrometry (HPLC-MS). In addition to measuring rapid dopamine transmission, microdialysis HPLC-MS measures changes in GABA, glutamate, monoamines, monoamine metabolites and several other neurotransmitters. Although both opioids increased dopamine release in the nucleus accumbens, their patterns of drug-evoked dopamine transmis-sion differed dramatically. Oxycodone evoked a robust and stable increase in dopamine concentration and a robust increase in the frequency and amplitude of phasic dopamine release events. Conversely, morphine evoked a brief (~ 1 min) increase in dopamine that was coincident with a surge in GABA concentration and then both transmitters returned to baseline levels. Thus, by providing rapid measures of neurotransmission, this study reveals previously unknown differences in opioid-induced neuro-transmitter signaling. Investigating these differences may be essential for understanding how these two drugs of abuse could differentially usurp motivational circuitry and powerfully influence behavior.European Journal of Neuroscience 11/2014; · 3.67 Impact Factor
Hierarchical recruitment of phasic dopamine signaling
in the striatum during the progression of cocaine use
Ingo Willuhna,b,1, Lauren M. Burgenoa,b, Barry J. Everittc,d, and Paul E. M. Phillipsa,b
Departments ofaPsychiatry and Behavioral Sciences andbPharmacology, University of Washington, Seattle, WA 98195; andcDepartment of Psychology
anddBehavioural and Clinical Neuroscience Institute, University of Cambridge, Cambridge CB2 3EB, United Kingdom
Edited by Robert C. Malenka, Stanford University School of Medicine, Stanford, CA, and approved October 19, 2012 (received for review August 3, 2012)
Drug addiction is a neuropsychiatric disorder that marks the end
stage of a progression beginning with recreational drug taking but
culminating in habitual and compulsive drug use. This progression is
considered to reflect transitions among multiple neural loci. Dopa-
mine neurotransmission in the ventromedial striatum (VMS) is
pivotal in the control of initial drug use, but emerging evidence
indicates that once drug use is well established, its control is
dominated by the dorsolateral striatum (DLS). In the current work,
spatiotemporal profile of striatal dopamine release and to investi-
use. Dopamine release was detected using fast-scan cyclic voltam-
metry simultaneously in the VMS and DLS of rats bearing indwelling
i.v. catheters over the course of 3 wk of cocaine self-administration.
We found that phasic dopamine release in DLS emerged progres-
sively during drug taking over the course of weeks, a period during
which VMS dopamine signaling declined. This emergent dopamine
signaling in the DLS mediated discriminated behavior to obtain drug
but did not promote escalated or compulsive drug use. We also
dependent on antecedent activity in VMS circuitry. Thus, the current
findings identify a striatal hierarchy that is instantiated during the
expression of established responses to obtain cocaine.
is characterized by habitual and compulsive drug use in which
other factors, such as withdrawal symptoms, stress, and drug-as-
sociated conditioned stimuli (CS), also contribute to the motiva-
tion to consume drugs, and drug taking becomes increasingly
prioritized over other behaviors (1). A wealth of evidence shows
that the mesolimbic dopamine projection from the ventral teg-
mental area to the ventromedial striatum (VMS) is central to drug
reinforcement (2). The ambient concentration of dopamine in the
VMS is increased when animals self-administer drugs of abuse,
including cocaine (3), and animals maintain this elevated dopa-
mine level by regulating their rate of responding for drug (4). In
addition, with repeated pairing of environmental stimuli with the
drug, these CS also gain the propensity to elicit changes in do-
pamine concentration in the VMS (5–8); and even though these
phasic neurochemical responses last only a few seconds, they are
capable of controlling drug-seeking and -taking behavior (5).
Together, these results implicate dopamine release in the VMS as
a critical substrate in the control of drug use (2, 3, 9).
However, the progression of drug taking beyond recreational
use is thought to reflect the engagement of different psychological
processes mediated within several neural loci (10, 11), with a par-
ticular emphasis on the incorporation of the sensorimotor (dor-
solateral) striatum (DLS) in the control of established drug-
seeking behavior (10, 12). Specifically, dopamine transmission in
the DLS has been linked to habitual CS-elicited reward seeking
(13) and therefore may play an important role in the development
of habitual and compulsive seeking of drugs (14–16). However, it is
not known whether the encoding of drug-related actions or stimuli
by phasic dopamine changes as drug-taking behavior advances
from recreational drug use or whether this coding extends beyond
the VMS to other parts of the striatum. In support of generalized
signaling properties of dopamine across striatal regions, reward-
rug use often begins as a recreational behavior driven by the
rewarding properties of the abused drug. However, addiction
associated cues produce transient increases in the firing rate of
dopamine neurons throughout midbrain nuclei where the pro-
jection targets collectively encompass the entire striatum (17).
However, evidence for this “global” signaling scheme from neu-
rochemical recordings within the striatum itself is lacking. In fact,
recent studies with natural rewards have challenged the concept of
uniform phasic dopamine signaling throughout the striatum, in-
stead reporting dopamine release in the VMS in response to
natural rewards and associated cues but little or no dopamine
release in the DLS (18, 19).
Therefore, to gain a fuller comprehension of the neural sub-
strates underlying the development of drug abuse, we assessed the
spatiotemporal dynamics of phasic dopamine release across the
striatum during the progression of the early stages of drug taking
by conducting neurochemical recordings in the VMS and DLS
simultaneously and repeatedly over multiple sessions of cocaine
self-administration (3 wk) in rats. We complemented these meas-
urements with pharmacological and lesion approaches to in-
vestigate the behavioral function of DLS dopamine signaling and
its relationship to that in the VMS, respectively.
Male Wistar rats with chronically implanted microsensors (20) in
the VMS and DLS (see Fig. S1 for histological verification of
electrode placement) and indwelling i.v. catheters were trained to
self-administer cocaine during daily 1-h sessions in a chamber
equipped with two nose-poke ports (Fig. 1A). A nose poke into the
active port elicited an infusion of cocaine (0.5 mg/kg body weight
per infusion) and a 20-s presentation of a light/tone CS on a FR-1
schedule of reinforcement (Fig. 1B). Responses in the second (in-
active) nose-poke port or in the active port during CS presentation
(time-out) were without programmed consequence. Cocaine-rein-
forced responding remained relatively stable over 3 wk with only
a modest increase in intake that did not reach significance [n = 18;
F(2, 34)= 1.682, P = 0.201; Fig. 1 C and D], whereas inactive and
time-out responding (i.e., nonreinforced responding) diminished
significantly [F(2, 34)= 5.075, P = 0.012; Fig. 1 C and E]. Conse-
quently, the ratio of reinforced to total responses (the efficiency of
responding) was significantly greater in the second and third weeks
than in the first week [F(2, 34)= 16.803, P < 0.001; Fig. 1F].
Drug Cue-Induced Phasic Dopamine Release in the VMS Is Present
Early in Cocaine Self-Administration. To characterize the long-term
dynamics of dopamine transmission, longitudinal neurochemical
recordings were carried out using fast-scan cyclic voltammetry. In
the first week of self-administration, there was a significant phasic
increase in extracellular dopamine concentration in the VMS fol-
lowing active responses (P = 0.002; Fig. 2A and Fig. S2). This in-
crease produced an average change in dopamine concentration
over the 7 s following the response of 7.77 ± 1.69 nM, with a mean
Author contributions: I.W., B.J.E., and P.E.M.P. designed research; I.W. and L.M.B. per-
formed research; I.W. analyzed data; and I.W. and P.E.M.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| December 11, 2012
| vol. 109
| no. 50
peak of 13.47 ± 2.16 nM occurring 2.45 ± 0.26 s after the response
andreturningtobaselineat7.41 ±0.28s. Thesekinetics aresimilar
to those reported in previous studies following a comparable
amount of training (5–8), and the concentration matches those
from recordings in the VMS with unbiased recording site selection
(8), as in the current study (SI Discussion and Fig. S3). This pattern
of activation continued into the second and third weeks (P < 0.01;
Fig. 2A and Fig. S2) but diminished in amplitude with an average
change in dopamine concentration of 5.96 ± 0.84 in the second
week and 2.99 ± 0.85 nM in the third week [main effect of week:
in dopamine concentration was detected following inactive nose
inactive poke: F(1,160)= 1.392, P = 0.240; Fig. 2C], indicating that
the neurochemical signal was not simply a result of the motor re-
sponse. However, noncontingent CS presentation alone was suf-
ficient to elicit a significant VMS dopamine signal [t(17)= −2.361,
P = 0.030; Fig. 2D] that was similar in magnitude and duration to
Phasic Dopamine Signaling in the DLS Develops over the Course of
Weeks. Measurements in DLS revealed phasic dopamine release,
similar to that in the VMS, in the second and third weeks of self-
administration, with an average change in dopamine concentration
of 3.10 ± 0.70 and 2.24 ± 0.38 nM, respectively (P < 0.001; Fig.
3A). However, such signaling was absent in the DLS during the
first week (0.14 ± 0.50 nM; P = 0.298; Fig. 3A), demonstrating
that phasic dopamine release in the DLS emerges over the course
of drug taking [main effect of week: F(2,62)= 8.843, P < 0.001;
active poke × week interaction: F(2,62)= 6.468, P = 0.003; Fig. 3B
and Fig. S2]; that is, the long-term dynamics are in the opposite
direction of those in the VMS [nose poke × week × region in-
teraction: F(2, 106)= 5.505, P = 0.005; Figs. S2 and S4]. None-
theless, as in the VMS, the signal in the DLS was not elicited by
the motor response [main effect of inactive poke: F(1,193)= 2.238,
P = 0.136; Fig. 3C] but increased following CS presentation [t(17)=
-3.083, P = 0.007; Fig. 3D; R = 0.91; P < 0.001]. These data dem-
onstrate that phasic dopamine signalsin the DLS and theVMS are
elicited by the same drug-associated stimuli, but the signals emerge
at a later stage of drug taking in the DLS, at a time when the VMS
dopamine signal actually is decreasing.
Dopamine Receptors in the DLS Are Necessary for Discriminated
Responses to Obtain Cocaine. To test the causal relationship be-
tween these neurochemical and behavioral observations, dopa-
mine signaling was manipulated by bilateral infusion (see Fig. S5
for histological verification of cannula placement) of the non-
specific dopamine receptor antagonist alpha-flupenthixol into the
DLS ofadditional groups ofanimals(n = 32; Fig. 4).In one group,
flupenthixol and vehicle were infused on counterbalanced days in
the first week of cocaine self-administration, corresponding to an
early time point before the onset of CS-associated DLS signaling.
A second group of animals was infused in the third week, corre-
sponding to the later time point when DLS dopamine signals were
present. The temporal pattern of the responses assessed in these
animals (Fig. 4 A–C) was similar to that observed in the previous
cohort (Fig. 1 D–F). Specifically, the rate of reinforced nose pokes
remained stable over time [F(2, 141)= 1.092, P = 0.338; Fig. 4A)],
but the rate of nonreinforced nose pokes decreased significantly
overthe weeksofself-administration[F(2, 141)=4.155, P=0.018;
Fig. 4B], producing an increase in response efficiency across this
period [F(2, 141)=7.843,P<0.001;Fig. 4C].Intra-DLS infusion of
flupenthixol resulted in an increase in cocaine intake (reinforced
nose pokes) at both the early and late time points (P < 0.05 vs.
vehicle; Fig. 4D), suggesting that DLS dopamine may contribute
to the reinforcing properties of cocaine, as is consistent with
previous reports (21, 22). Importantly, this effect therefore is not
attributable to the CS-associated phasic dopamine signal, which
was present at the late time point but not the early time point. In
contrast to the effect on reinforced responding at both time
points, the average number of nonreinforced responses was in-
creased after the late infusion (P = 0.024; Fig. 4E) but not after
the early infusion (P = 0.970). Accordingly, the nose-poke effi-
ciency was decreased after the intracerebral administration of
flupenthixol at the late (P = 0.004; Fig. 4F) but not the early
a rat connected to voltammetric recording equipment and infusion pump
for i.v. delivery of cocaine during an approach to the active nose-poke port
in the operant chamber. (B) A nose poke (dashed line) into the active port
elicits an infusion of cocaine (0.5 mg/kg per infusion) and the presentation of
a CS (yellow box) during a 20-s time-out. (C) Nose pokes into the active port,
inactive port, and during the time-out period over 20 d of self-administra-
tion (n = 18). (D) The number of reinforced nose pokes did not change
significantly across weeks, whereas the number of nonreinforced responses
decreased (E), and the ratio of reinforced over total number of nose pokes
(efficiency) increased (F) in the second and third weeks compared with the
first week. *P < 0.05, ***P < 0.001; n.s., not significant.
Drug-taking behavior over the course of weeks. (A) Depiction of
dopamine release in the VMS following responses into the active nose-poke
port was observed during all 3 wk of cocaine self-administration (n = 10). (B)
Dopamine signals decreased in amplitude over the course of 3 wk. (C) Do-
pamine signals following responses into the active nose-poke port were
larger than signals following inactive responses. (D) Noncontingent delivery
of the CS induced dopamine release. *P < 0.05, **P < 0.01, ***P < 0.001.
Dopamine signaling in the VMS over the course of weeks. (A) Phasic
| www.pnas.org/cgi/doi/10.1073/pnas.1213460109 Willuhn et al.
(P = 0.762) time point [drug × time-point interaction: F(1, 27)=
7.482, P = 0.011]. These data show that the gain in efficiency as
measured by discriminated drug-taking responses between the
first and third weeks of cocaine self-administration was re-
versed by the infusion of flupenthixol into the DLS, indicating
that emergent dopamine signaling in the DLS is necessary for
the improved action selection of drug-taking behavior.
Development of Phasic Dopamine Signaling in the DLS Depends on
the VMS. A salient feature of the current findings and those of
others (21) is the progressive onset of function in the DLS during
drug use. This progressive involvement of the DLS in drug seeking
has been linked to circuitry that connects the VMS to the DLS by
a serial disconnection study that demonstrated that the de-
velopment of advanced cue-controlled drug-seeking behavior is
dependent on intact VMS circuitry (23). Therefore, to test whether
the later-emerging phasic dopamine signal in DLS reported in the
present study was dependent upon antecedent activity in the VMS
circuitry, we mimicked a disconnection of the VMS from DLS on
one side of the brain with a unilateral excitotoxic lesion of the
nucleus accumbens core (VMS) by infusing quinolinic acid before
training (23), leaving the other side intact. Voltammetric micro-
sensors were implanted bilaterally in the DLS (n = 17), permitting
within-subject comparison of emergent DLS dopamine trans-
mission between hemispheres, one hemisphere having an intact
and the other a lesioned VMS (see Fig. S6 for histological verifi-
cation of lesion and electrode placement). Cocaine intake was
similar to that in nonlesioned animals (Fig. S7), as is consistent
with previous findings (23). Also, similar to nonlesioned animals
(Fig. 3), active nose-poke responses evoked significant dopamine
release in the DLS contralateral to the lesion in the second and
third weeks (1.81 ± 0.23 and 1.77 ± 0.22 nM; P < 0.01; Fig. 5A) but
not in the first week (−0.19 ± 0.49; P = 0.778; Fig. 5A). However,
in the hemisphere ipsilateral to the VMS lesion, there were no
significant changes in dopamine release compared with baseline at
any time point of cocaine self-administration, with an average
change in dopamine concentration of 0.85 ± 0.38, 0.84 ± 0.22, and
0.92 ± 0.23 nM in weeks 1–3, respectively (P > 0.05; Fig. 5B).
Thus, phasic dopamine signals evolved over the 3 wk of self-ad-
ministration contralateral [main effect of active poke: F(1, 63)=
19.386, P < 0.001; main effect of week: F(2, 63)= 15.294, P < 0.001;
active poke × week interaction: F(2, 63)= 19.386, P = 0.048; Fig. 5C
and Fig. S8] but not ipsilateral [main effect of week: F(2, 43)= 0.001,
P = 0.999; Fig. 5C] to the lesion, conferring significantly different
patterns of dopamine release in the two hemispheres [brain
region × week interaction: F(2, 106)= 7.204, P < 0.001; Fig. 5C)]
Similarly, noncontingent delivery of the CS induced significant
dopamine release (P = 0.040; Fig. 5D) contralateral but not
ipsilateral to the VMS lesion (P = 0.761; Fig. 5E and Fig. S9).
Importantly, during periods in the recording sessions that were
free of operant behavior and CS presentations, the magnitude
of “spontaneous” dopamine release in the DLS was similar
ipsilateral and contralateral to VMS lesion (Fig. S10). Fur-
thermore, the magnitude of DLS signals measured in the first
study (Fig. 3A) and in the DLS contralateral to the lesion (Fig.
5A) were not significantly different [main effect of brain region:
F(1,125)= 0.851; P = 0.358]. Therefore, the VMS lesion did not
produce a general suppression of dopamine transmission in the
DLS but had a selective effect on task-related signaling. These
results demonstrate that neural activity in VMS is required for the
development of CS-elicited dopamine signaling in the DLS that
regulates the efficiency, or automaticity, of drug-taking responses.
Spatiotemporal Changes in Striatal Dopamine Signaling. Drug self-
administration studies in animals have revealed neuroadaptations
in functional markers that progress from the VMS to encompass
complementary changes in phasic dopamine transmission, we
carried out longitudinal subsecond dopamine measurements si-
multaneously in the VMS and DLS during the establishment of
drug taking in rats. We observed phasic dopamine release in both
the VMS and DLS following the operant response for drug during
the course of our study in which the VMS signal declined and the
DLS signal emerged during the progression of drug taking. De-
spite these differences in temporal profiles, phasic dopamine re-
lease encoded similar information in the VMS and DLS. In both
pokes, indicating that the signal was not simply related to the
motoric action of making a response. Instead, we hypothesized
that dopamine release was a result of successful completion of the
response to obtain cocaine (signaled by the CS). This notion was
dopamine release in the DLS following responses into the active nose-poke
port was observed during the second and third weeks of cocaine self-ad-
ministration (n = 15). (B) Dopamine signals in the second and third weeks
were greater in amplitude than those in the first week. (C) Dopamine signals
following responses into the active nose-poke port were larger than signals
following inactive responses during the second and third weeks but not
during the first week. (D) Noncontingent delivery of the CS induced dopa-
mine release. *P < 0.05, **P < 0.01, ***P < 0.001.
Dopamine signaling in DLS over the course of weeks. (A) Phasic
drug-taking behavior. (A–C) The rate of reinforced nose pokes remained
stable across weeks (A), but the rate of nonreinforced nose pokes was de-
creased (B), and response efficiency increased (C) during the second and third
weeks compared with the first week. (D) Infusion of flupenthixol (FLU) into
the DLS produced an increase in reinforced nose pokes in both the first (n =
16) and the third weeks (n = 16). (E) The average number of nonreinforced
responses was increased after flupenthixol only during the third but not
during the first week. (F) Consequently, response efficiency was decreased
after flupenthixol at the late but not at the early time point. *P < 0.05, **P <
0.01, ***P < 0.001; VEH, vehicle.
Blockade of dopamine receptors in the DLS disrupts discriminated
Willuhn et al.PNAS
| December 11, 2012
| vol. 109
| no. 50
supported, because noncontingent presentation of the CS alone
was sufficient to recapitulate dopamine release following an active
a time point equivalent to the first week of training in the present
study. Drug-associated CS are integral to drug use, guide the ac-
quisitionand maintenanceofdrug taking, and increasingly assume
control over behavior to the extent of triggering the resumption of
drug taking even after long periods of abstinence (24, 25). Thus,
the current findings reveal a process by which drug-associated
stimuli gain access to sensorimotor circuitry with repeated drug
use. Interestingly, the emergent sensorimotor signal generally was
smaller thanthat in theVMS, even whendrug usewasestablished.
This observation is notable because the density of dopamine ter-
minals (26), tissue content (27), and capacity for release (27, 28)
dopamine responses use less of the available “bandwidth” for
encoding of drug cues in the DLS than in the VMS. Similarly, the
long-term effect of prior cocaine exposure on the processing of
stimuli associated with natural reinforcers is not uniform across
these two regions. Instead of increasing processing in both the
VMS and DLS, cocaine reduces the degree and flexibility of cue-
effects in the DLS being relatively weak compared with those in
the VMS (29).
Overall, our data identify the spatiotemporal pattern of phasic
taking behavior. The gradual decline in VMS dopamine signaling
is somewhat surprising in the context of models postulating that
the amount of dopamine release in response to drug cues, spe-
cifically in the nucleus accumbens, increases over repeated drug
contrast, the emergence of phasic dopamine signaling in the DLS
provides further empirical support forcurrent theories postulating
the engagement of an increasing number of brain regions with
prolonged drug use (10–12, 16).
Dopamine Signaling in the Sensorimotor Striatum Emerges Before
Compulsive Drug Abuse. The observed spatiotemporal dynamics
of striatal dopamine signaling illustrate the progressive engage-
ment of brain systems with persistent drug self-administration. It
has been suggested that each of the stages in the series of tran-
responding for drug is associated with specific brain systems that
are recruited progressively (10). Indeed, the DLS comes to exert
more dominant control over drug seeking during the course of
drug use (21, 30) as drug taking becomes maintained by drug-as-
sociated stimuli (10, 12, 16). Although we have demonstrated that
phasic dopamine release does indeed develop at a later stage of
drug use in the DLS than in the VMS, the training regimen used
typically is not sufficient to produce compulsive responding or the
significant escalation of drug intake that emerges following ex-
tended or long-access training in drug self-administration (31, 32).
Thus, our data demonstrate that the engagement of DLS dopa-
mine, which is thought to be linked closely to stimulus–response
processing (13), is not sufficient to account for the loss of control
over drug intake characteristic of addiction, underlining the im-
drug taking and their neural substrates (33).In fact,thebehavioral
measure that most closelycorrelated with theemergence ofphasic
the number of active nose-poke responses as a proportion of the
total number of responses (including time-out responses and
responses in theinactiveport).This increasein responseefficiency
between the first and third weeks of self-administration was re-
treatment had no effect on efficiency in the first week, before
phasic dopamine signaling in the DLS had emerged. In contrast,
cocaine intake (reinforced nose pokes) was increased by the an-
tagonistinboththefirst andthirdweeks, suggestingthat this effect
likely is not associated with the phasic modality of dopamine sig-
naling time-locked to drug taking, and therefore tonic dopamine
signaling may be implicated. This notion is consistent with the
work of others indicating a role for DLS dopamine in mediating
the reinforcing properties of cocaine (21, 22). Therefore, rather
than contributing to escalated or compulsive responding, the
progressive recruitment of DLS phasic dopamine promotes the
refinement of behavior toward reinforced actions, as operant
responding for the drug becomes more reliably discriminated over
the course of weeks in the absence of escalated drug intake.
Although DLS dopamine appears to suppress nonreinforced
responses, it was not observed around these actions. Instead, it
seems that the feedback collected from reinforced responses
promotes exclusivity (i.e., actions that are not associated with
a DLS dopamine signal are not maintained). Although this in-
striatum does not generate movement itself but rather promotes
the DLS. (A) Phasic dopamine release was observed in the DLS contralateral
to the unilateral lesion of the VMS following responses into the active nose-
poke port during the second and third weeks of cocaine self-administration
(n = 17). (B) Dopamine release in the ipsilateral DLS was not significantly
increased in any week (n = 11). (C) In the contralateral DLS, phasic signaling
in the second and third weeks was larger in amplitude than signals detected
in the first week (Left), whereas signals did not change in amplitude across
weeks in the ipsilateral DLS (Right). Emergence of such signaling had sig-
nificantly different patterns of dopamine release between hemispheres. A
direct post hoc comparison between ipsilateral and contralateral hemi-
spheres showed greater dopamine release in the contralateral hemisphere in
the second and third weeks of training but not in the first week (#P < 0.05).
(D and E) Noncontingent delivery of the CS consistently induced DLS do-
pamine release contralateral (D) but not ipsilateral (E) to the VMS lesion.
*P < 0.05, **P < 0.01, ***P < 0.001.
VMS lesion prevents development of phasic dopamine signaling in
| www.pnas.org/cgi/doi/10.1073/pnas.1213460109Willuhn et al.
removing the inhibition of specific actions and acting broadly by
inhibiting rivaling/conflicting motor mechanisms that otherwise
would interfere with the desired action (34). Consistent with our
findings, dorsal striatal circuits serve to evaluate behavior and to
exploit optimal behaviors following initial behavioral variability
during trial-and-error learning (exploration) (35) as an integral
part of the sensorimotor domain of the basal-ganglia network
mediating action sequencing as well as selection/inhibition of
competing motor programs (34, 36). Thus, our findings suggest
that the observed changes in DLS dopamine signaling (i.e., task
representation in DLS circuits) might facilitate a switch from ex-
ploring the availability of drug rewards present in theenvironment
to exploiting this environment.
Addiction often is described as a disorder of brain memory
learning (37), with dopamine acting as a neurotransmitter that
induces plasticity to enable the formation of long-lasting network
changes. Brain regions that mediate the evolving discrimination of
drug cues and drug taking are potentially of great interest in the
identification of neural systems underlying addiction. Our data
suggest that the observed behavioral refinement may represent an
amplified focus on drug-related behaviors that causes the priori-
tization of drug taking over behavior not reinforced by drug,
a development also observedin drug addiction (1). Thus, although
the efficiency of drug taking does not itself imply compulsive or
addiction-like behavior, monitoring response discrimination may
prove useful in the investigation of abuse-related behaviors com-
parable to a period when drug abusers narrow their behavioral
repertoire to actions that prioritize the intake of drugs over other
actions. Taken together, these data demonstrate a mechanism
involving sequential recruitment of phasic dopamine transmission
in the striatum in the dynamically changing neural control over
drug intake even before compulsive use emerges.
A Hierarchy for Recruiting Dopamine in Different Striatal Modules.
affect and enable sensorimotor circuits, thus functioning as a gate-
way for limbic structures to reach motor systems (38). Sensorimotor
aspects of the striatum are thought to contribute to facilitating au-
tomatic execution of motor acts or to implementing habits by
units (36). We investigated interactions between motivational
and sensorimotor networks within the striatum during drug self-
bilateral electrochemical recordings in DLS. This approach enabled
the study of dopamine neurotransmission simultaneously in intact
animal and thus in the same motivational state. Our data provide
functional evidence supporting an interaction between limbic and
motor networks in the development of discriminated responses to
obtain cocaine, in which the VMS, which receives limbic inputs,
enables dopamine signaling in the sensorimotor DLS.
Previous support for a role of serial circuitry that connects the
VMS and DLS comes from a study that combined lesioning of the
VMS on one side of the brain and antagonism of dopamine
receptors in the contralateral DLS, thereby functionally dis-
connecting serial interactions between these striatal domains on
both sides of the brain (23). Although either manipulation on its
own was without effect, the combined procedures selectively de-
creased cocaine seeking in extensively trained rats but not in rats
that had undergone only moderate training (23). Together with
our study, these findings underline the functional significance of
the network interaction between the VMS and the DLS in drug-
related behavior. Specifically, they indicate that this circuit is used
prioritization of drug-taking behavior and the exploitation of
a drug environment in animals with a moderate drug history
(present study) and in energizing and driving drug-seeking be-
havior in an environment where the drug is not readily available in
animals with an extended drug-taking history (33). Therefore, the
hierarchical recruitment of striatal subregions for dopamine-
mediated control of behavior may signify an overarching orga-
nizing principle throughout the stages of drug use to enable rep-
resentation of drug cues in DLS.
There has been a long-standing debate on how interactions be-
tween limbic and motor systems are implemented. On the level of
basal ganglia circuitry, a potential anatomical substrate for this in-
projection neurons and the dopaminergic midbrain. Nauta et al.
(39) discovered that VMS neurons, which receive dopaminergic
afferents from the ventral tegmental area, send axons to the sub-
stantia nigra, which provides a dopaminergic projection to the
dorsal striatum. This connectivity later was found to display an
elaborate spiraling organization with several striato-nigro-striatal
However, other pathways also channel information from VMS to
DLS via the midbrain (41–43). Irrespective of anatomical pathway,
within neural circuits regulating behavior is shaped over prolonged
Overall, the present data offer insight into neurobiological pro-
cesses that establish drug-taking behavior. It demonstrates that
phasic dopamine signaling in the striatum is dynamic and region
specific, emerging sequentially in the VMS and then in the DLS in
the early stages of drug use. We ascertained that the progression
from limbic to sensorimotor regions of the striatum requires intact
VMS circuitry. This hierarchical control enables drug-associated
stimuli to access the brain systems implicated in the development
of a drug-taking habit.
Surgical Procedures. Stereotaxic surgery was performed as described pre-
viously (20). The target coordinates were 1.2 mm anterior, 3.1 mm lateral,
and 4.8 mm ventral to bregma for the DLS and 1.3 mm anterior, 1.3 mm
lateral, and 7.2 mm ventral to bregma for the nucleus accumbens core of the
VMS. For the pharmacological experiment, guide cannulas were implanted
bilaterally into the DLS. For the lesion experiment, quinolinic acid (0.09 M;
0.5 μL) was infused unilaterally into the VMS to induce an excitotoxic lesion
(23). The i.v. catheters were implanted in a separate surgery.
Cocaine Self-Administration. Rats were trained to obtain cocaine following an
operant response on a continuous reinforcement (FR-1) schedule in an op-
erant chamber equipped with two nose-poke response devices. Nose-poking
intheactiveport resultedinan i.v. infusion ofcocaine (0.5mg/kg) paired with
a 20-s presentation of an audiovisual stimulus (CS). During CS presentation,
a 20-s time-out was imposed during which nose poking did not result in any
programmed consequences. To control for response specificity, nose-poking
for 1 h/d, 6 d/wk, for 3 wk.
Infusion of Flupenthixol into the DLS. The effects of the dopamine receptor
antagonist flupenthixol (5 μg dissolved in 0.5 μL vehicle into each side; 0.5 μL/
min) or vehicle on drug-taking behavior were examined in single sessions
during the first or third weeks of self-administration. One group of rats
received flupenthixol or vehicle in the first week of cocaine self-adminis-
tration, counterbalanced on 2 d, and a separate group received counter-
balanced infusions in the third week.
Voltammetric Measurements and Analysis. Electrochemical recordings (2 d/wk)
using chronically implanted carbon-fiber microsensors and data analysis were
carried out as described previously (20) and are described in more detail in SI
Experimental Procedures. In brief, during each voltammetric scan (every 100
ms), the potential at the carbon-fiber electrode was ramped linearly from
−0.4 V versus Ag/AgCl to +1.3 V and back at 400 V/s (total scan time, 8.5 ms).
Dopamine at the surface of the electrode is oxidized during the anodic
sweep to form dopamine-o-quinone which is reduced back to dopamine in
the cathodic sweep. The ensuing flux of electrons is measured as current and
is directly proportional to the number of molecules that undergo electrol-
ysis. The background-subtracted, time-resolved current obtained provided
a chemical signature characteristic of the analyte, allowing resolution of
dopamine from other substances. Dopamine was isolated from the
Willuhn et al.PNAS
| December 11, 2012
| vol. 109
| no. 50
voltammetric signal using chemometric analysis using a standard training set
(20) based on electrically stimulated dopamine release detected at chroni-
cally implanted electrodes. Dopamine concentration was estimated based on
the average postimplantation sensitivity of electrodes (20), averaged over
the 7 s following the operant response (postresponse) or noncontingent
presentation of the CS and compared with the average concentration over
the 2 s prior to the response or CS (baseline).
Statistical Analysis. Individual voltammetric recordings were averaged across
session, animals, and weeks. These means then were compared using one-,
two-, and three-way ANOVAs with postresponse, brain region, and week as
factors. For comparison with voltammetric data, behavioral data also were
binned into weeks. For the flupenthixol-infusion experiment, mean baseline
values for weeks 1 and 3 during which flupenthixol was infused were com-
puted by averaging the data over 3 d in the week during which no infusions
were administered. Behavioral data were analyzed using one- and two-way
ANOVAswithdrug andweeks as factors.When appropriate,post hoc analyses
were conducted, and P values were adjusted according to the Holm–
Bonferroni correction method for multiple testing (44). Plots were made
using Prism (GraphPad Software). All statistical analyses were carried out
using SPSS, version 17.0. All data are presented as mean plus SEM.
Histological Verification of Recording Sites. On completion of experimenta-
tion, recording sites were marked with an electrolytic lesion and verified
using cresyl violet staining.
ACKNOWLEDGMENTS. We thank Christina Akers, Lauren Haggerty, and Scott
Ng-Evans for technical support and Michela Marinelli for technical advice. This
work was supported by German Research Foundation (Deutsche Forschungsge-
meinschaft, D.F.G.) Grant WI 3643/1-1 (to I.W.), Medical Research Council Pro-
grammeGrant G1002231 (to B.J.E.), a grant from the Alcohol and Drug Institute
(to P.E.M.P.), and National Institutes of Health Grants T32-DA027858 (to L.M.B.)
and P01-DA015916, R21-DA021793, and R01-DA027858 (all to P.E.M.P.).
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