Article

Striatal dopamine signals are region specific and

temporally stable across action-sequence habit
formation
Graphical abstract Authors
Wouter van Elzelingen,
Pascal Warnaar, João Matos, ...,
Damiaan Denys, Tara Arbab,
Ingo Willuhn

Correspondence
i.willuhn@nin.knaw.nl

In brief
Van Elzelingen et al. simultaneously track
striatal dopamine signaling and
development of habitual behavior. In the
dorsomedial striatum, previously
associated with non-habitual behavior,
dopamine release increases during action
initiation in habitual rats and decreases
during action completion, whereas non-
habitual rats show opposite effects.

Highlights
d Validation of a novel test that monitors habit development
individually across time

d Dopamine release during habit development is stable across

relevant striatal regions

d Only dopamine release in dorsomedial striatum correlates

with habit development

d Optogenetic stimulation of dorsomedial striatal dopamine

accelerates habit formation

van Elzelingen et al., 2022, Current Biology 32, 1163–1174

March 14, 2022 ª 2022 The Authors. Published by Elsevier Inc.
https://doi.org/10.1016/j.cub.2021.12.027 ll
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OPEN ACCESS

Article
Striatal dopamine signals
are region specific and temporally stable
across action-sequence habit formation
Wouter van Elzelingen,1,2 Pascal Warnaar,1,2 João Matos,1,2 Wieneke Bastet,1,2 Roos Jonkman,1,2 Dyonne Smulders,1,2
Jessica Goedhoop,1,2 Damiaan Denys,1,2 Tara Arbab,1,2 and Ingo Willuhn1,2,3,4,*
1Netherlands Institute for Neuroscience, Royal Netherlands Academy of Arts and Sciences, Meibergdreef 47, 1105 BA Amsterdam, the

Netherlands
2Department of Psychiatry, Amsterdam University Medical Centers, University of Amsterdam, Meibergdreef 5, 1105 AZ Amsterdam, the

Netherlands
3Twitter: @_the_ing
4Lead contact

*Correspondence: i.willuhn@nin.knaw.nl
https://doi.org/10.1016/j.cub.2021.12.027

SUMMARY

Habits are automatic, inflexible behaviors that develop slowly with repeated performance. Striatal dopamine
signaling instantiates this habit-formation process, presumably region specifically and via ventral-to-dorsal
and medial-to-lateral signal shifts. Here, we quantify dopamine release in regions implicated in these pre-
sumed shifts (ventromedial striatum [VMS], dorsomedial striatum [DMS], and dorsolateral striatum [DLS])
in rats performing an action-sequence task and characterize habit development throughout a 10-week
training. Surprisingly, all regions exhibited stable dopamine dynamics throughout habit development. VMS
and DLS signals did not differ between habitual and non-habitual animals, but DMS dopamine release
increased during action-sequence initiation and decreased during action-sequence completion in habitual
rats, whereas non-habitual rats showed opposite effects. Consistently, optogenetic stimulation of DMS
dopamine release accelerated habit formation. Thus, we demonstrate that dopamine signals do not shift
regionally during habit formation and that dopamine in DMS, but not VMS or DLS, determines habit bias,
attributing ‘‘habit functions’’ to a region previously associated exclusively with non-habitual behavior.

INTRODUCTION and pre-programmed appetitive behaviors.17,18 The behavioral

Our daily lives are governed by learned routines, exemplified by
many actions we perform automatically without paying attention,
such as pulling out our smartphone to seek rewarding input by
perpetually scrolling down the screen. Such habits are thought
to gradually develop from initially goal-directed, conscious ac-
tions (e.g., using our smartphone to contact a friend) that reliably
resulted in desired outcomes. Habit formation is often tied to
detection of stimuli predictive of these outcomes and is facili-
tated by both extended high-rate action repetition (referred to
as ‘‘overtraining’’1) and relative uncertainty as to when precisely
the desired outcome will be achieved.2,3
The striatum has long been implicated in habit formation,4–6
and its subregions are assumed to fulfill distinct functions in
this process. The dorsolateral striatum (DLS) is hypothesized
to actively mediate habit learning, supported by ample evidence
from rodent experiments which link DLS activity with (overtrain-
ing-induced) habit formation and DLS lesion or inactivation with
attenuated habitual responding.7–14 The (posterior) dorsomedial
striatum (DMS) is hypothesized to control goal-directed behavior
and, thus, oppose habitual strategies.15,16 A third functional
domain heavily investigated in the context of reward learning is
the ventromedial striatum (VMS), which is involved in motivation
impact of these three regions is thought to evolve with the
gradual development of a habit, reflecting an inter-regional shift
of the locus of behavioral control: a dorsal shift away from VMS
after early stages of instrumental learning9,17–20 and a relative
transfer from DMS to DLS as behavior becomes more ingrained
and habitual.7,9,13,16,21–27
These presumed intra-striatal shifts of the locus of behavioral
control are thought to depend on striatal-dopaminergic neuro-
transmission, which is in line with the anatomy of the dopamine
system28 and is corroborated by studies using dopaminergic
drugs as reinforcers.29–31 Consistently, ample evidence implicates
striatal dopamine in habit formation,6,9,23,24,32 and performance of
ingrained action sequences is reflected in action-chunking activity
patterns both in striatal and dopamine neurons.33 Furthermore,
genetic deletion of dopamine-neuron NMDA receptors impairs
habit learning,34 as does lesion of nigrostriatal dopamine projec-
tions,35 whereas pharmacological enhancement of dopamine
transmission biases behavior toward habit formation.36 In hu-
mans, dopamine-depleted Parkinson’s patients exhibit deficits
in habitual control.6,37,38 However, although dopamine is indubi-
tably involved in habit formation, its regional specificity and its
involvement in the widely assumed regional shift of behavioral
control during the transition from goal-directed to habitual

Current Biology 32, 1163–1174, March 14, 2022 ª 2022 The Authors. Published by Elsevier Inc. 1163
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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OPEN ACCESS
behavior is understudied. Conclusions were advanced on the
grounds of anatomical studies and sparse functional evidence:
one study tracked regionally shifting striatal dopamine signaling
across development of a presumed habit reinforced by cocaine,31
whereas another study reported no such shift using a natural rein-
forcer.39 Thus, the central questions that remain are how region-
specific and regionally shifting dopamine signals regulate habit
formation.
In experimental animals, habit formation is best achieved by
either overtraining or employing variable-interval (VI) reinforcement
schedules that lead to high response rates and reward-timing un-
certainty.1–3,40–43 Dickinson and collaborators developed a widely
applied operational definition that became the gold standard for
habit classification:9,40,41,44 an insensitivity to (short-term) changes
in value of action outcomes and/or action-outcome contingency.42
However, this definition identifies habits on the failure to satisfy
criteria for goal-directed action (negative definition), including the
possibility that unforeseen factors compromise integration of
changes in action-outcome conditioning.45,46 Besides, tradition-
ally used habit measures can only be applied once in a given indi-
vidual and are not suited to detect individual differences, both of
which are critical features, since habits develop slowly and recruit-
ment of habitual strategies vary significantly between individuals.
To address these limitations, we set out to develop a paradigm
that ‘‘positively’’ identifies habits, accounts for individual differ-
ences, and may be repeated within a subject to track gradual habit
development.
We delineate the differential contributions of relevant striatal
subregions in habit formation by measuring real-time fluctua-
tions in extracellular dopamine concentrations in VMS, DMS,
and DLS of rats performing in an action-sequence reinforcement
task (a ‘‘seeking-taking’’ chain task, which combines VI and
fixed-ratio (FR) schedules and overtraining) and subsequently
validating the role of region-specific dopamine signaling using
optogenetic stimulation. Our findings challenge the traditionally
assumed shifting of regional dopamine dynamics and surpris-
ingly indicate that DMS, instead of DLS, dopamine determines
the transition to habitual behavior, thereby questioning the idea
that DMS opposes habit formation.
RESULTS
Habitual behavior is induced by excessive behavioral repetition
(overtraining)9,11,24,47 or by reinforcing behavior on a VI
schedule.3,48–50 Here, we overtrained animals under a chained
VI60-FR1 reinforcement schedule, thereby combining the two
most prominent paradigms. Rats learned to press a seeking
lever (distal to reward) on a VI60 schedule (followed by a 3-s
delay in which no levers were extended) and, subsequently, an
FR1 taking lever (proximal to reward) that produced a food pellet
(Figure 1A). The number of seeking presses under VI reinforce-
ment increased over 10 weeks of training (week 1 versus week
10; t35 = 12.21, p < 0.0001; Figure 1B, left), whereas the number
of food-magazine head entries did not (n = 36, Z = 0.440, p =
0.660; Figure 1B, middle). We assessed the probability with
which rats checked the magazine for food during the 3-s delay
period between retraction of the seeking lever and extension of
the taking lever. This probability decreased dramatically across
weeks (week 1 versus week 10: n = 36, Z = 5.185, p <
1164 Current Biology 32, 1163–1174, March 14, 2022
Article
0.0001; Figure 1B, right), demonstrating that animals learned
the task structure quickly.
We tested whether behavior persists despite reward devalua-
tion by sensory-specific satiety via a 1-h pre-feeding of regular
training pellets (devalued condition) or alternative pellets (valued
condition) after 10 weeks of seeking-taking training (Figure 1C,
left). Subsequent presentation of the seeking lever under extinc-
tion conditions generated no significant difference in the number
of seeking-lever presses between valued and devalued condi-
tions (n = 14, Z = 0.785, p = 0.433; Figure 1C, middle), demon-
strating that seeking-taking training induced a habit. A ‘‘pellet
choice test’’ verified that satiety was specific to the pre-fed pellet
type, i.e., each pellet type was devalued effectively (n = 14,
valued condition: Z = 3.296, p < 0.001; devalued condition:
Z = 3.297, p < 0.001; Figure 1C, right).
We characterized dopamine release longitudinally across
weeks, using fast-scan cyclic voltammetry in VMS, DMS, and
DLS. Electrode implantation induced minimal brain damage,
and placement was verified (Figure 1E). Dopamine release in
response to unpredicted delivery of a food pellet was measured
as a proxy for electrode sensitivity, which remained stable
across weeks in all regions (VMS: t24 = 0.7162, p = 0.4808;
DMS: t10 = 1.355, p = 0.2052; DLS: t15 = 1.046, p = 0.3121; Fig-
ures 1F and S1). Besides modestly diminished DMS dopamine
release upon seeking-lever extension (distal; t10 = 2.267, p =
0.0468), no longitudinal differences were found (distal VMS:
t24 = 1.186, p = 0.2473; distal DLS: t18 = 0.1541, p = 0.8793; prox-
imal VMS: t24 = 1.798, p = 0.0848; proximal DMS: t10 = 1.041, p =
0.3223; proximal DLS: t18 = 0.3994, p = 0.6943; Figures 1G and
1H). Thus, regional dopamine release is relatively stable across
training, with the only task-relevant changes occurring in DMS.
To overcome limitations of traditional habit testing, we devel-
oped a test during which both seeking and taking levers were
extended simultaneously for 1 min under extinction conditions
(Figure 2A, left), allowing rats the choice to engage with the lever
proximal to reward delivery (taking; goal-directed choice) or the
lever at the distal to reward delivery (seeking; habitual choice). In
week 1, rats exhibited a preference for the taking over the
seeking lever, but by week 10, this preference had shifted to
the seeking lever (n = 36; week 1: Z = 3.540, p = 0.0004;
week 10: Z = 4.357, p < 0.0001; Figure 2A, right). This temporal
evolution of lever preference was highly reliable between
different rat cohorts (Figure S2A). Extinction effects due to
repeated testing are unlikely because (1) the time spent in regular
training exceeds preference testing almost 500 times and (2) the
frequency of preference tests did not influence the habit readout
(Figures S2A and S2B).
To capitalize on individual differences, we classified animals
(same data as in Figures 1C–1H) into two equal-sized groups
based on their relative seeking-taking preference in week 10 (i.e.,
number of seeking minus number of taking presses, divided by to-
tal number of presses, as shown in Figure 2F), which is identical to
a median split. There were no significant changes in number of
head entries into the food magazine over time in either group (Fig-
ures 2B and 2C [top]). Both groups displayed a taking-lever prefer-
ence in week 1 (habit: n = 18, Z = 1.810, p = 0.0370; no habit: n =
18, Z = 3.312, p = 0.0002; Figure 2C), but only the habitual ani-
mals developed a steadily increasing seeking-lever preference
(habit group: lever, F1,44 = 58.17, p < 0.0001; lever x week
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A Distal to reward Intermediate Proximal to reward

D
+ 2.04 + 1.80 + 1.56 + 1.20 + 0.84 + 0.60 + 0.36 0.0
Final press & Delay Taking Taking
Seeking lever Pellet
seeking lever period lever press &
extension delivery
retraction (no levers) extension retraction

Seeking (VI60 s) 3s ~1 s 3s ITI

VI60 FR1
Seeking

Seeking

Taking
E F
lever

lever

lever
Delivery of an unpredicted food pellet (control)

VMS DMS DLS

Food Food ns ns ns
magazine pellet
120 30 30

[Dopamine] (nM)
B Distal Intermediate 100
80 20 20

Seeking (VI60 s) Head entries (VI60 s) Head entries before 60

taking-lever extension
VI<60 VI60 VI<60 VI60 VI<60 VI60
# Seeking presses / session

40 10 10
# Head entries / session

7000 1000 1.0

probability / session

6000 20
800 0.8
Head entry

5000 0 0 0
600 0.6
4000 Week 1 Week 10 Week 1 Week 10 Week 1 Week 10
3000 400 0.4
2000
1000
200 0.2 G Distal H Intermediate and proximal

ns ns ns ns ns
*
0 0 0.0
-1 0 1 2 3 4 5 6 7 8 9 10 -1 0 1 2 3 4 5 6 7 8 9 10 -1 0 1 2 3 4 5 6 7 8 9 10 12 6 6 25 10 10
Week Week Week
8 8
C Validation of habit induction by seeking-taking paradigm at week 10 8 4 4 20
1-h ad-libitum access Extinction test: 5-min seeking-lever exposure Pellet choice test: 5-min access 6 6
= Alternative pellets
Extinction test:
*** 4 2 2 15
[Dopamine] (nM)

[Dopamine] (nM)
Seeking

100
Consumption

seeking presses 4 4
(% of max)

Feeding cage Feeding cage 75

lever

300
ns 50 0 0 0 10
25 2 2
0
# Presses

200 Alt Reg

Regular pellets = valued -4 -2 -2 5 0 0
= Regular pellets 100
100 ***
Consumption
Seeking

-2 -2
(% of max)

Feeding cage Feeding cage 75 -8 -4 -4 0

lever

0 50
25
d
ed

-4 -4
ue
lu

0
al

-12 -6 -6 -5
Va

Alt Reg
ev

Regular pellets = devalued Week 1 Week 10 Week 1 Week 10 Week 1 Week 10 Week 1 Week 10 Week 1 Week 10 Week 1 Week 10
D

Figure 1. A seeking-taking chain task to monitor dopamine signaling during habit formation
(A) A VI60:FR1 trial begins with seeking-lever extension. Seeking-lever presses (‘‘distal’’) are registered until the final VI60 press (‘‘intermediate’’), which triggers
seeking-lever retraction, followed by taking-lever extension 3 s later. Approximately 1 s later, animals press the taking lever (‘‘proximal’’), it is retracted, and the
food-pellet reward is delivered 3 s later, marking the start of the variable inter-trial interval (ITI).
(B) Number of seeking-lever presses (left) and food-magazine head entries (middle) during the VI60 segment. The probability of a food-magazine head entry
during the 3-s period following seeking-lever retraction and prior to taking-lever extension (right) decreases rapidly with training, demonstrating task acquisition.
Data are mean ± SEM (black) with individual animals (gray).
(C) Rats that were trained for 10 weeks underwent outcome devaluation via sensory-specific satiety (pre-feeding), in which, on two separate days, rats had 1-h ad
libitum access to either regular pellets (bottom) or alternative (grain-based) pellets (top) before they were exposed to the seeking-lever (extinction) test. Seeking
was unaffected by outcome devaluation, demonstrating that a habit was induced. Subsequently, rats were exposed to both pellets (‘‘choice test’’) to demonstrate
that outcome-specific devaluation was successful (non-pre-fed pellets preferred; p < 0.001).
(D) Coronal brain sections with recording sites in VMS (blue), DMS (green), and DLS (red), relative to bregma.51
(E) Representative verification of electrode-tip placement in VMS (blue circle), DMS (green circle), and DLS (red circle) and corresponding electrode tracks
(arrows).
(F) Dopamine release to an unpredicted food pellet demonstrates stable electrode sensitivity. Data are median with range in quartiles and mean (+); ns, not
significant.
(G) DMS dopamine following seeking-lever extension (distal) was modestly diminished after 10 weeks. No significant differences (ns) were observed in VMS or DLS.
(H) During proximal actions, no significant differences (ns) were detected in dopamine release between weeks 1 and 10. *p < 0.05, ***p < 0.001.
See also Figure S1.

interaction, F4,98 = 42.82, p < 0.0001; no-habit group: lever, F1,46 = not week 1 (U(nhabit = 18, nno-habit = 18) = 115.000, Z = 1.487,
1.206, p = 0.2779; Figures 2B and 2C [middle]) and exhibited this p = 0.1370;). Across weeks 1 and 10, seeking-lever presses (n =
preference in week 10 (habit: n = 18, Z = 3.724, p < 0.0001; no 18, Z = 3.724, p = 0.0006; Figure 2D) and seeking-preference in-
habit: n = 18, Z = 1.491, p = 0.1454; Figures 2B and 2C [bottom]). dex (n = 18, Z = 3.724, p = 0.0006; Figure 2F) increased in habitual
Habitual animals showed more seeking presses (U(nhabit = 18, animals, whereas taking responses decreased (n = 18, Z = 3.725,
nno-habit = 18) = 11.500, Z = 4.765, p < 0.0001; Figure 2D) and p = 0.0006; Figure 2E). In non-habitual animals, seeking responses
fewer taking presses (U(nhabit = 18, nno-habit = 18) = 1.500, Z = (n = 18, Z = 3.680, p = 0.0005; Figure 2D) and seeking-preference
5.098, p < 0.0001; Figure 2E) compared to non-habitual rats in index (n = 18, Z = 3.636, p = 0.0005; Figure 2F) also increased,
week 10, but not week 1 (seeking: U(nhabit = 18, nno-habit = 18) = albeit to a lesser extent than in habitual rats, whereas taking re-
106.500, Z = 1.758, p = 0.0790; Figure 2D; taking: U(nhabit = 18, sponses did not change (n = 18, Z = 0.104, p = 0.9175; Figure 2E).
nno-habit = 18) = 153.500, Z = 0.269, p = 1.0000; Figure 2E). During lever-preference tests, habitual and non-habitual rats
Consistently, grouping the rats based on week-10 performance made more head entries into the food magazine after pressing
(Figure 2F) separated habit from no-habit animals in week 10 the taking lever than the seeking lever (Figure 2G) in weeks 1
(U(nhabit = 18, nno-habit = 18) = 0.000, Z = 5.144, p < 0.0001), but (habit group: t32 = 7.542, p < 0.0001; non-habit group:

Current Biology 32, 1163–1174, March 14, 2022 1165

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A D 180 E 80 F 1.0
Habit

(# Seeking- # Taking) / # total

160 70 0.8
Lever preference (LP) test Lever presses during LP test No habit

# Seeking-lever presses

# Taking-lever presses
140 0.6
120
Seeking lever 120
60
0.4 ***
Taking lever 50
Seeking
100 0.2
100

# Lever presses
Taking

*** 40 0.0
lever

lever
80 80
-0.2
60 60 *** 30
-0.4
Group
classification
40 *** 40 20
10 *** -0.6
20 -0.8
20
0 0 -1.0
0 Week 1 Week 10 Week 1 Week 10 Week 1 Week 10
Simultaneous presentation of both levers for 0 1 2 3 4 5 6 7 8 9 10
one minute (under extinction condition) Week
B C G 20 20
Week 1 Week 10
Habit No habit
# Head entries

12 12
# Head entries

15 Habit 15
10 10

# Rats
No habit

# Rats
8 8
6 6 10 10
4 4
2 2 5 5
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 0
0 2 4 6 8 0 2 4 6 8
120 120
100
Seeking lever
Taking lever *** 100
# Seeking-press Head entry # Seeking-press Head entry
# Lever presses
# Lever presses

8 8
80 80 Week 1 Week 10
6 6
60 60
ns

# Rats

# Rats
40
* 40 *** 4 4

20 20 2 2
0 0
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10 0 2 4 6 8 0 2 4 6 8
Week Week # Taking-press Head entry # Taking-press Head entry

180 Week 10 180 Week 10

H Seeking-lever stickiness I Head entries into food magazine
Probability to repeat seeking press

160 160
1.0 0.4

Probability of head entry

140 140 Habit R-value: -0.4301
No habit P-value: 0.0088
120 120 0.9
# Lever presses
# Lever presses

0.3
100 100
0.8
80 80 0.2
60 60 0.7

40 40 0.1
0.6 R-value: 0.6797
20 20 P-value: <0.0001
0.5 0.0
0 0 -1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
Taking Seeking Taking Seeking [#Seeking - #Taking] / total # presses [#Seeking - #Taking] / total # presses

Figure 2. A novel preference test to measure the development of habits

(A) Habit formation was tested by presenting both levers simultaneously for 1 min under extinction conditions (left). In week 1 of VI60:FR1 seeking-taking training,
rats preferentially pressed the taking lever, but by week 10, preference had shifted to the seeking lever (right).
(B) In the habit group, the number of food-magazine head entries remained stable (top) across weeks, but rats switched from a taking- to a strong seeking-lever
preference (middle) and exhibited more seeking than taking presses in week 10 (bottom).
(C) Food-magazine head entries remained stable in the no-habit group (top), but rats switched from a taking-lever preference to no preference (middle) and
exhibited no significant difference between seeking and taking presses in week 10 (bottom).
(D and E) Habitual rats performed more seeking-lever presses (D) and fewer taking-lever presses (E) than non-habitual rats in week 10, but not in week 1.
(F) The seeking-taking preference index demonstrates a higher seeking-lever preference in habitual rats compared to non-habitual rats in week 10, but not in
week 1.
(G) The conditioning ‘‘logic’’ translated from training to preference test: food-magazine head entries were executed more frequently after taking-lever presses
than after seeking-lever presses, both in habitual and non-habitual rats. Histogrammed data with mean ± SEM shown separately.
(H and I) Linear regression analyses demonstrate associations of the seeking-lever index with other habit-like behavior during the preference test, i.e., (H) a
positive correlation with seeking-lever stickiness and (I) a negative correlation with food-magazine head-entry probability. Data are mean ± SEM, in some panels
combined with individual animals (open circles). *p < 0.05, ***p < 0.001.
See also Figures S2 and S3.

t34 = 8.488, p < 0.0001) and 10 (habit group: t27 = 2.510, p = [Figure S3B] and habit rats on the seeking lever [Figure S3C])
0.0184; non-habit group: t34 = 6.202, p < 0.0001). Thus, both rather than performing action sequences involving both levers
groups learned the task structure and translated it from training (Figure S3A).
sessions (levers presented sequentially) to the test situation (le- We validated the preference test by carrying out regression
vers presented simultaneously). Habit and no-habit rats did not analyses, testing for a relationship between the seeking-taking
differ in number of head entries that followed seeking- or tak- preference index (shown in Figure 2F) and other ‘‘habit-like’’ be-
ing-lever presses in weeks 1 (seeking: t32 = 1.506, p = 0.4257; haviors displayed during the test. The preference index corre-
taking: t34 = 1.430, p = 0.3238; Figure 2G, left) or 10 (seeking: lated positively with the probability to repeat seeking-lever
t34 = 0.5440, p = 0.5900; taking: t27 = 2.393, p = 0.0956; Fig- presses (R value = 0.6797, p < 0.0001; Figure 2H) and negatively
ure 2G, right). Notably, repeated presses on the same lever with the probability to make food-magazine head entries (R
were much more prevalent than seeking-taking action se- value = 0.4301, p = 0.0088; Figure 2I).
quences in both habit and no-habit animals in both weeks 1 Classifying rats as habitual or non-habitual based on week-
and 10 (Figures S3A–S3C). Thus, simultaneous lever presenta- 10 preference-test performance, revealed that the two groups
tion during the preference test is marked by rats ‘‘getting stuck’’ perform differently during seeking-taking training (Figures 3A
on one of the levers (no-habit rats on the taking lever and S4): the frequency of ‘‘distal’’ seeking lever presses across

1166 Current Biology 32, 1163–1174, March 14, 2022

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A Distal Intermediate
Figure 3. Diametrically opposing DMS dopamine
signals in habitual and non-habitual rats during distal
Head entries outside VI Seeking (VI60s) Head entries (VI60s) Head entries before and proximal reward seeking
taking-lever extension
VI<60 VI60 VI<60 VI60 VI<60 VI60 VI<60 VI60 (A) Habitual animals showed a higher frequency of distal
frequency / session (Hz)
frequency / session (Hz)

frequency / session (Hz)

0.20 2.0 0.20 1.0
*

probability / session
Seeking-lever press

0.8 seeking-lever presses during the VI60 trial segment

0.15 1.5 ** 0.15
* *

Head entry
Head entry

Head entry
*** *** 0.6 compared to non-habitual rats (middle left). Conversely,
0.10 1.0 0.10
0.4 habitual animals exhibited fewer food-magazine head en-
Habit
0.05 0.5 0.05 tries during VI60 (middle right). However, this difference in
No habit
** 0.2

0.00 0.0 0.00 * 0.0 head entries was not present outside VI60, when no levers
-1 0 1 2 5 10 -1 0 1 2 5 10 -1 0 1 2 5 10 -1 0 1 2 5 10
Week Week Week Week
were extended (left). Similarly, no group differences were
B Delivery of an unpredicted food pellet (control) observed for the head-entry probability during the 3-s period
Week 1 Week 10 following seeking-lever retraction and prior to taking-lever
VMS DMS DLS VMS DMS DLS extension (right). Data are mean ± SEM.
ns ns ns ns ns ns
120 30 30 120 30 30 (B) Dopamine release to an unpredicted food-pellet delivery
[Dopamine] (nM)
[Dopamine] (nM)

100 25 25 100 25 25
(proxy for electrode sensitivity) did not differ between
80 20 20 80 20 20
groups. Data are median with range in quartiles and mean
60 15 15 60 15 15
(+); ns, not signifcant.
40 10 10 40 10 10
(C) During distal actions (start of VI60), no significant dopa-
20 5 5 20 5 5
mine group differences were observed in VMS or DLS.
0 0 0 0 0 0
No Habit No Habit No Habit No Habit No Habit No Habit However, DMS dopamine was greater in habitual rats in both
habit habit habit habit habit habit
Distal weeks 1 and 10. Data are mean + SEM; traces are aligned to
C
seeking-lever extension.
VMS DMS DLS
No habit No habit No habit (D) During proximal actions, no significant group differences
Seeking Habit Week1 Seeking Habit Week1 Seeking Habit Week1
ns 3 ns
*
[Dopamine] (nM)

[Dopamine] (nM)
[Dopamine] (nM)

lever lever lever

extension 6 extension 3 extension in dopamine were observed in VMS or DLS. However, again,
l 4 l 2 l 2
l 2 l 1 l 1 DMS dopamine differed between habitual and non-habitual
l l l
5 nM 0 2.5 nM 0 2.5 nM 0
l
l -2
l
l -1
l
l -1
rats, but in contrast to distal actions, proximal events
[Dopamine] (nM)

l l l
l -4 l -2 l -2 significantly decreased dopamine in habitual animals in both
l -6 l -3 l -3
l
Week l1
No Habit
habit
l
Week l1
No Habit
habit
l
Week l1
No Habit
habit weeks 1 and 10. Data are mean + SEM; gray-shaded areas
l l l
l Week10 l Week10 l Week10 indicate approximate final seeking-press timing.
[Dopamine] (nM)
[Dopamine] (nM)

[Dopamine] (nM)

l
l
l
6 ns
4
l
l
l
3
2
*** l
l
l
3 ns
2 (E) To evaluate the association of dopamine with reward
Week l10 2 Week l10 1 Week l10 1
l l l seeking (distal actions: lever presses early in the VI60
l 0 l 0 l 0
l
l
-2 l
l
-1 l
l
-1 segment), trial-by-trial correlations were calculated for week
l -4 l -2 l -2
0 5 10 -6 0 5 10 -3 0 5 10 -3 1 (top) and week 10 (bottom). Gray bars display the animals’
Time (s) No Habit Time (s) No Habit Time (s) No Habit
habit habit habit R values distributed over given histogram brackets. Each
Intermediate and proximal circle represents a single animal (habitual in orange, non-
D
VMS No habit DMS No habit DLS No habit
habitual in brown), and significant correlations are depicted
Habit Week1 Habit Week1 Habit Week1
al Taking d Pe al Taking d Pe al Taking d Pe as filled circles and non-significant correlations as empty
Finkinsg ns Finkinsg
*** Finkinsg ns
[Dopamine] (nM)

el lle el lle el lle

[Dopamine] (nM)

[Dopamine] (nM)

e press iv t 10 e press iv t 5 e press iv t 5

sepres er sepres er sepres er circles. VMS demonstrated the closest relationship between
l l y 8 l l y 4 l l y 4
l l 6 l l 3 l l 3
l l 4 l l 2 l l 2 dopamine and seeking-lever presses. *p < 0.05, **p < 0.01,
l l 10 nM 2 l l 5 nM 1 l l 5 nM 1
l l 0 l l 0 l l 0 ***p < 0.001.
[Dopamine] (nM)

l l -2 l l -1 l l -1
l l l l l l
l l
-4 l l
-2 l l
-2 See also Figure S4.
l l -6 l l -3 l l -3
Week 1 No Habit Week 1 No Habit Week 1 No Habit
l l habit l l habit l l habit
l l l l l l
l l Week10 l l Week10 l l Week10
[Dopamine] (nM)

[Dopamine] (nM)

[Dopamine] (nM)

10 ns
**
l l l l l l
l l l l
5 l l 5 ns
l l 8 l l 4 l l 4
Week 10 l l 6 Week 10 l l 3 Week 10 l l 3
l l 4 l l 2 l l 2
l l 2 l l 1 l l 1
l l 0 l l 0 l l 0
l l -2 l l -1 l l -1
l l -4 l l l l
-2 -2
-10 -5 0 5 -6 - 10 -5 0 5 -3 -10 -5 0 5 -3
Time (s) No Habit Time (s) No Habit Time (s) No Habit
habit habit habit

E 60 Week 1
VMS: dopamine & seeking presses
significant

Habit
% Rats

40 10 Week 1 No habit
# Rats

11 / 25 rats P < 0.05

20 5
P < 0.05
0 0
VMS DMS DLS -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
R-value
60 Week 10
Week 10
significant

10
% Rats

40
# Rats

5 13 / 28 rats
20 P < 0.05
0
0 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
VMS DMS DLS R-value

DMS: dopamine & seeking presses DLS: dopamine & seeking presses
10 Week 1 10 Week 1
# Rats

# Rats

5 3 / 12 rats 5 2 / 22 rats
P < 0.05 P < 0.05
0 0
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
R-value R-value

10 Week 10 10 Week 10
# Rats

# Rats

5 4 / 13 rats 5 5 / 19 rats
P < 0.05 P < 0.05
0 0
-0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
R-value R-value

Current Biology 32, 1163–1174, March 14, 2022 1167

 ll
OPEN ACCESS Article

A Optic C Real-time
D
fiber
Intracranial self-stimulation
place preference
ns ns
100 150
ChR2 Active Nose Pokes

in active quadrant (%)

Nose pokes in 120 min

DMS DMS
MS DM
DMS ChR2 Inactive Nose Pokes
75

Time spent
100 EYFP Active Nose Pokes
pc EYFP Inactive Nose Pokes
SN VTA 50
Th::Cre rat 50
25
B GFP TH Merge
0 0
Cortex ChR2 EYFP Day 1 Day 2 Day 3
Session day
Optic E
fiber
15 15

Week 1 Week 3
DMS
10 10
ChR2

# Rats

# Rats
EYFP

Striatum 5 5

SNpc 0 0
0 2 4 6 8 0 2 4 6 8
# Seeking-press Head entry # Seeking-press Head entry
VTA
SNR
Midbrain
6 6
Week 1 Week 3

4 4
# Rats

# Rats
2 2

0 0
0 2 4 6 8 0 2 4 6 8
Midbrain # Taking-press Head entry # Taking-press Head entry
60 μm

Distal Intermediate
F G H
Head entries before
Head entries outside VI Seeking (VI60s) Head entries (VI60s) taking-lever extension
VI<60 VI60 VI<60 VI60 VI<60 VI60 VI<60 VI60
0.4 2.0 0.3 1.0
**
frequency / session (Hz)

frequency / session (Hz)

frequency / session (Hz)

ChR2
Seeking-lever press

probability / session

0.3 EYFP
1.5 ** 0.2
0.8
Head entry

Head entry
Head entry

0.6
0.2 1.0 **
0.4
0.1
0.1 0.5
0.2

0.0 0.0 0.0 0.0

-1 0 1 2 3 -1 0 1 2 3 -1 0 1 2 3 -1 0 1 2 3
Week Week Week Week

I ChR2: Lever presses EYFP: Lever presses J Seeking presses Taking presses
during LP test during LP test during LP test during LP test
120 120 120 120
Seeking lever ChR2
# Seeking-lever presses

# Taking-lever presses

100 Taking lever 100 100 EYFP 100

80 ** 80 80 ** 80
# Presses

# Presses

60 60 60 60
40 * 40 * 40 40
20 20 20 20
0 0 0 0
1 2 3 1 2 3 1 2 3 1 2 3
Week Week Week Week

Figure 4. Optogenetic stimulation of DMS dopamine during seeking accelerates habit formation
(A) AAV injection into substantia-nigra pars-compacta (SNpc) of Th::Cre rats induced ChR2-EYFP or EYFP (control) expression in dopamine neurons. DMS
dopamine terminals were optogenetically stimulated bilaterally.
(B) Immunostaining for tyrosine hydroxylase (TH) and EYFP indicates selective virus expression in midbrain dopamine neurons (bottom) and their striatal terminals
(top). SNR, substantia nigra pars reticulata; VTA, ventral tegmental area.
(C) Percentage of time spent in the DMS-photo-stimulated (active) quadrant of an open-field arena. No significant difference (ns) was observed between ChR2
and EYFP rats.
(D) Nose pokes into the active port triggered DMS dopamine photo stimulation. We observed no significant differences (ns) within or between ChR2 and EYFP
animals in the number of active and inactive nose pokes.
(E) During seeking-taking training, seeking presses triggered DMS dopamine photo stimulation. The conditioning ‘‘logic’’ translated from training to preference
test: food-magazine head entries were executed more frequently after taking-lever presses than after seeking-lever presses, both in habitual and non-habitual
rats. Histogrammed data with mean ± SEM shown separately.

(legend continued on next page)

1168 Current Biology 32, 1163–1174, March 14, 2022

Article
VI training weeks was higher in habitual rats (group, F5,34 =
18.48, p = 0.0001; group x week interaction, F5,170 = 3.154,
p = 0.0095). Conversely, the frequency of distal food-magazine
head entries during VI was lower in habitual rats (group, F1,32 =
10.66, p = 0.0026). In contrast, we observed no differences in
head-entry frequency outside VI when levers were retracted
(group, F1,32 = 0.0003168, p = 0.9859) and in ‘‘intermediate’’
head-entry probability during the 3-s period following
seeking-lever retraction, prior to taking-lever extension (group,
F1,34 = 1.463, p = 0.2348). Together, these data suggest that
habit animals exhibit a greater affinity to the seeking lever
(habit-like behavior) paired with less outcome checking (goal-
directed behavior) and that group differences are specific to
task-relevant actions and do not reflect discrepancies in gen-
eral activity.
Dopamine release to an unpredicted food pellet (proxy for elec-
trode sensitivity) did not differ significantly between habitual and
non-habitual animals (Figure 3B) in VMS (week 1: t23 = 0.3175,
p = 0.7537; week 10: t26 = 1.146, p = 0.2623), DMS (week 1:
t10 = 0.7898, p = 0.4480; week 10: t11 = 0.5837, p = 0.5712), and
DLS (week 1: t17 = 1.541, p = 0.1417; week 10: t18 = 0.4060, p =
0.6895). Dopamine measurements throughout the 10-week
training (Figures 3C and 3D) did not reveal any differences between
habitual and non-habitual rats during ‘‘distal,’’ ‘‘intermediate,’’ or
‘‘proximal’’ actions in VMS (distal week 1: t23 = 0.8278, p =
0.4163; distal week 10: t26 = 1.648, p = 0.2228; proximal week 1:
t23 = 0.6261, p = 1.0000; proximal week 10: t26 = 0.3088, p =
0.7600) and DLS (distal week 1: t20 = 0.6727, p = 1.0000; distal
week 10: t17 = 0.09775, p = 0.9233; proximal week 1: t20 =
0.6011, p = 0.5545; proximal week 10: t18 = 1.922, p = 0.1412).
However, distal DMS dopamine concentration was higher in
habitual animals in week 10 (t11 = 5.213, p = 0.0006), a surprising
effect that was already detectable in week 1 (t10 = 2.438, p =
0.0350). Remarkably, this effect was reversed during intermediate
and proximal actions: habitual animals had lower DMS dopamine
in both week 1 (t10 = 5.428, p = 0.0006) and week 10 (t11 = 3.663, p =
0.0037). Within-animal trial-by-trial correlations (Figure 3E) re-
vealed that reward seeking in itself, irrespective of training amount,
was strongly associated with VMS, but not DMS or DLS dopamine;
seeking-lever pressing in itself, therefore, does not bring about the
changes in DMS dopamine we observe during habit development.
In summary, habitual behavior was correlated with increased DMS
dopamine release during distal behavioral responses, which was
decreased during responses proximal to reward delivery. More-
over, DMS dopamine differences were already apparent early in
training (before habits developed) and were not involved in (motor)
performance of distal seeking.
To determine whether augmented DMS dopamine signaling is
sufficient in itself to induce habitual behavior, we bilaterally opto-
genetically stimulated the terminals of DMS-projecting
(F) No significant differences were found in frequency of food-magazine head entries outside VI60, when no levers were extended.
(G) ChR2 animals made more distal VI60 seeking-lever presses (left). No significant difference was found for food-magazine head entries during VI60 (right).
(H) Similarly, no group differences were observed for the head-entry probability during the 3-s period following seeking-lever retraction and prior to taking-lever
extension (right).
(I) In week 1, both ChR2 and EYFP rats preferentially pressed the taking lever during the habit test. By week 3, this preference had shifted to the seeking lever for
the ChR2 rats, but not the EYFP rats.
(J) The ChR2 group made significantly more seeking-lever responses, but groups did not differ in taking-lever presses. Data in (E) and (F) are mean ± SEM. *p <
0.05, **p < 0.01.
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OPEN ACCESS
dopamine neurons during seeking presses (Figures 4A and
4B). We controlled for possible reinforcing effects by exposing
rats to an open-field arena where presence of the animal in a
specific quadrant triggered photo-stimulation (real-time place
preference [RT-PP] assay) and observed no significant differ-
ence between ChR2 and control rats (expressing EYFP only) in
time spent in the active quadrant (t15 = 1.745, p = 0.1014; Fig-
ure 4C). In comparison, other studies applying RT-PP via dopa-
mine photo stimulation have reported animals spending about
80% of their time in the photo-stimulated position,52–56 whereas
we observed only 45%. Similarly, when rats were given the op-
portunity to self-stimulate DMS dopamine terminals optogeneti-
cally by poking their noses into a device (over the course of
3 days), there was no significant difference in the number of
active or inactive nose pokes between ChR2 and EYFP rats
(active-port group, F1,9 = 4.476, p = 0.0635, time x group interac-
tion: F2,18 = 2.566, p = 0.105; inactive-port group, F1,9 = 2.127,
p = 0.1787, time x group interaction: F2,18 = 1.046, p = 0.372; Fig-
ure 4D), and there was no difference in active and inactive nose
pokes within either of these groups (ChR2: poke-port, F1,12 =
4.514, p = 0.0551, time x poke-port interaction: F2,24 = 1.395,
p = 0.267; EYFP: poke-port, F1,6 = 0.06194, p = 0.8118, time x
poke-port interaction: F2,12 = 0.016, p = 0.984; Figure 4D).
Furthermore, for ChR2 animals, no difference between active
and inactive pokes was detected on day 3 (t9 = 1.576, p =
0.150). In comparison, overall, other studies applying optoge-
netic self-stimulation of dopamine have reported about 25–40
responses/min,52,55,57,58 whereas we observed only about 1
response/min on day 1 and 0.15/min on day 3. Taken together,
stimulation of dopaminergic DMS terminals was without signifi-
cant reinforcing effects.
During seeking-taking training, each (distal) seeking press trig-
gered a 1 s DMS photo stimulation. The lever-preference test
showed that both groups made more food-magazine head en-
tries after taking compared to seeking responses (ChR2: lever,
F1,18 = 22.49, p = 0.0002; EYFP: lever, F1,12 = 20.10, p =
0.0007; Figure 4E). Furthermore, we detected no differences
over time (between weeks 1 or 3) between the number of
ChR2 and EYFP rats that performed taking-lever-to-head-entry
sequences (group, F1,15 = 0.003275, p = 0.9551) or seeking-
lever-to-head-entry sequences (group, F1,15 = 0.1307, p =
0.7228), indicating that task-structure representation was stable
across training sessions.
During seeking-taking training, ChR2 animals increased their
seeking-lever presses during VI60 compared to EYFP controls
(group, F1,15 = 18.27, p = 0.0007, group x week interaction,
F4,60 = 15.20, p < 0.0001; Figure 4G, left). Post hoc analysis re-
vealed that the number of seeking-lever presses was higher in
weeks 1–3 (respectively: t15 = 4.546, p = 0.0012; t15 = 4.521,
p = 0.0016; t15 = 4.654, p = 0.0015), indicating that optogenetic
Current Biology 32, 1163–1174, March 14, 2022 1169
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OPEN ACCESS
DMS dopamine stimulation promoted habit formation. In
contrast, no differences in frequency of magazine head entries
were found between ChR2 and EYFP rats, either during VI (group,
F1,15 = 0.3029, p = 0.5901; Figure 4G, right) or outside VI (group,
F1,15 = 1.088, p = 0.3134; Figure 4F). Therefore, DMS dopamine
did not simply invigorate general responding but specifically
enhanced habitual responding. No group differences were found
for head-entry probability during the 3-s period following
seeking-lever retraction and prior to taking-lever extension
(group, F1,15 = 0.4022, p = 0.5355; Figure 4H), indicating that
photo stimulation did not affect task-structure comprehension.
In comparison, optogenetic effects are not robust in the self-stim-
ulation condition (Figure 4D) but are very robust when applied in
conjunction with seeking-lever presses during seeking-taking
training (Figure 4G, left). Thus, we conclude that optogenetic ef-
fects on seeking-lever preference cannot be explained by rein-
forcing properties of DMS photo stimulation alone.
DMS photo-stimulation effects also translated into behavior
during the lever-preference test. ChR2 animals pressed the
seeking lever more than the taking lever across 3 weeks of
training (lever, F1,15 = 7.840, p = 0.0135; Figure 4I, left), whereas
EYFP animals did not (lever, F1,12 = 0.6623, p = 0.4316; Figure 4I,
right). When compared to taking-lever presses, post hoc analysis
revealed that both groups made fewer seeking-lever presses in
week 1 (EYFP: n = 7, Z = 2.366, p = 0.036; ChR2: n = 10, Z =
2.397, p = 0.017), but only ChR2 rats made significantly more
seeking- than taking-lever presses in week 3 (EYFP: n = 7, Z =
1.609, p = 0.1077; ChR2:n = 10, Z = 2.805, p = 0.010; Figure 4I).
The number of taking-lever presses did not differ between groups
(group, F1,15 = 0.2355, p = 0.6345; Figure 4J, right), but ChR2 rats
made more seeking-lever responses than EYFP animals (group,
F1,15 = 7.840, p = 0.0135; Figure 4J, left), where post hoc analysis
revealed that the number of seeking-lever presses was higher in
week 3 (U(nChR2 = 10, nEYFP = 7) = 1.500, Z = 3.273, p = 0.001).
This suggests that augmented DMS dopamine during training
contributes to (1) an increased occurrence of seeking, but not
taking responses and (2) accelerating habit formation assessed
in the lever-preference test.
DISCUSSION
We set out to delineate the role of the striatal dopamine system in
habit formation (i.e., automation of reward seeking) and as-
sessed dopamine release in three striatal regions implicated
heavily in acquisition and execution of reward seeking: VMS,
DMS, and DLS. A 10-week (over)training in a seeking-taking
chain task resulted in habitual seeking, as evidenced by reward
devaluation via sensory-specific satiety, traditionally employed
to identify habits. The sequential nature of the task enabled us
to dissect dopamine signaling during distal (seeking initiation)
and proximal (seeking completion and reward taking) epochs.
Surprisingly, dopamine release was stable between training
weeks 1 and 10, the only exception being a modestly decreased
distal DMS signal in week 10. To better interrogate the devel-
oping habit, we designed a novel lever-preference test. In
week 1, animals displayed a taking-lever preference (goal-
directed), but with continued training, rats began either to sample
both levers equally (no-habit rats) or to develop a seeking-lever
preference (habit rats). Habit rats displayed increased DMS
1170 Current Biology 32, 1163–1174, March 14, 2022
Article
dopamine during distal reward seeking and decreased DMS
dopamine during actions more proximal to reward delivery
(compared to no-habit rats), whereas VMS and DLS dopamine
did not differ between groups. Although this DMS ‘‘habit signa-
ture’’ was not related to seeking actions themselves (these
were more associated with VMS dopamine), we hypothesized
it to be causally related to the gradually developing seeking-lever
bias. Thus, we optogenetically stimulated DMS dopamine
neuron terminals during seeking (during training), which resulted
in a nearly instant selective promotion of seeking behavior during
training, and accelerated the shift toward seeking-lever prefer-
ence (in the test). Together, our findings demonstrate that dopa-
mine release in DMS, but not VMS or DLS, predicts future
reward-seeking strategies, where augmented distal DMS dopa-
mine promotes the development of habitual reward-seeking (and
more pronounced proximal DMS dopamine reflects the propen-
sity toward a non-habitual strategy).
To study neural mechanisms of a gradually developing habit,
we introduced a novel (lever-preference) test that, unlike tradi-
tionally employed indicators of habits (invoked via satiety-spe-
cific devaluation, contingency degradation, or taste aversion),
was designed to allow for repeated testing and analysis of indi-
vidual differences. We validated that seeking-taking training
conditions translated to preference-test conditions by demon-
strating that taking-lever presses are followed more often by
reward-magazine entries than seeking-lever presses, indi-
cating that rats expect reward after taking-lever, but not
seeking-lever, presses, and thus learned and generalized the
task structure. That training-induced seeking-lever preference
is a valid indicator of habit formation was underlined by the
fact that it correlated positively with seeking-lever stickiness
(another habit-like feature) and negatively with food-magazine
entries (presumably a goal-directed action). Our test enables
positive identification of habitual performance by measuring
an active-choice preference, as opposed to the ‘‘traditional’’
negative identification via exclusion of a goal-directed strategy.
Notably, the test consistently and reliably exposed the gradual
nature of the change in lever preference across training weeks
in several cohorts of animals (Figure S2A). The slow speed at
which the preference shift develops is another validating
feature (as reported in other seeking-taking studies59,60), as
habits in humans are thought to form slowly via extensive repe-
tition.3,42,61,62 Moreover, the test demonstrated that within the
same animals, via overtraining, habit-like performance can
arise out of a behavior that was initially under goal-directed
control;61,62 overtraining has been shown to effectively pro-
duce habits, both in single-action and action-sequence tasks,
but aspects of action sequences (e.g., initiation or termination)
may remain under goal-directed or mixed control (the latter may
explain that no-habit rats do not exhibit a lever preference in
week 10).1,63–65 Crucially, by chaining two operant behaviors,
we temporally separated initiation (distal) and termination
(proximal) of seeking (followed by reward consumption: taking),
which allowed clear distinction between underlying neuronal
mechanisms. Finally, our approach offers additional measures
(reward-magazine head entries and different action sequences)
that further qualify behavioral strategy (goal-directed or
habitual). Taken together, we propose that ‘‘habits’’ are not a
unitary concept and can be detected in different ways (see

Article
STAR Methods for more discussion). Here, we introduce a valu-
able addition to traditionally employed habit paradigms.
Although dopamine neurons are well known to contribute to
habit formation,6,9,10,23,32–36,66–68 little is known about their pro-
jection-specific activity of the dopamine system therein. Based
on the presumed functions of VMS (motivation/Pavlovian
behavior), DMS (goal-directed behavioral control), and DLS
(sensorimotor/habit learning), we recorded dopamine transients
in these striatal domains, hypothesizing that regional dopamine
signals throughout habit development would align to these func-
tions. Additionally, based on the generally presumed dopamine-
dependent inter-regional shift in locus of behavioral control during
habit development (see introduction), we expected task-relevant
dopamine signals to shift from VMS to the dorsal striatum and/or
from DMS to DLS. An alternative hypothesis suggests that the in-
fluence of dopamine subsides altogether with increasing training/
automaticity.23,69,70 However, surprisingly, our results demon-
strate that dopamine signaling (1) does not shift inter-regionally
(neither across the animal population as a whole nor between in-
dividual habit and non-habitual animals) and (2) does not
decrease with increasing behavioral automaticity in any of the
three regions. In fact, the stability of dopamine signals across
10 weeks of training is remarkable given dopamine’s role as a
‘‘learning/teaching’’ signal (the task is fully acquired within first
few weeks). Even more surprisingly, DMS, a region traditionally
associated with the control of goal-directed reward seeking, ex-
hibited the most critical features, where an increase in dopamine
during reward-seeking initiation was predictive of future habit-like
behavior. This habitual distal DMS dopamine surge was paralleled
by a depression of proximal DMS dopamine. In contrast, DMS
dopamine signals in non-habitual animals were not simply absent
but opposite to those of habitual animals: they decreased distally
and increased proximally. This result also underlined that DMS
dopamine signaling was not compromised in non-habitual rats.
Furthermore, although ‘‘action vigor’’ is a potential contributor to
DMS dopamine signals, the number of seeking presses in training
(i.e., a proxy for vigor) increases over time in both habit and no-
habit animals (Figure 3A). Furthermore, DMS dopamine signals
are opposite between these groups, which is the case from the
beginning of training, and the signals do not correlate with
seeking-press frequency (Figure 3E). Thus, it seems unlikely that
vigor is itself a major contributor. Together, our data indicate
that the temporal distribution (distal/proximal) of dopamine
signaling in DMS, but not VMS or DLS, governs whether a
behavior becomes habitual or not.
Since the DMS is traditionally not associated with habit forma-
tion directly,8,13,16,25,27,29 the question is what function dopamine
released into the DMS fulfills during habit formation. From the
beginning of training, DMS dopamine signals were present during
seeking initiation in animals that displayed an affinity to the seeking
lever, reflecting a predisposition toward habit formation, which
manifested in the preference test only after weeks of training.
This suggests that DMS dopamine signals may strengthen the
seeking preference incrementally over many repetitions, which
then translates into other situations (i.e., the lever-preference
test) later. This is corroborated by our optogenetic-stimulation
study: optogenetic DMS dopamine stimulation applied during
training is reflected in a significant seeking-lever preference only
after 2 weeks. Notably, repetition of seeking presses (overtraining)
ll
OPEN ACCESS
alone was not sufficient to establish a lever preference in itself,
since non-habitual rats were also overtrained, suggesting that
DMS dopamine is essential to habit formation.
Enhanced engagement with the seeking lever is reminiscent of
‘‘sign tracking,’’ but sign tracking is a Pavlovian trait that does
not develop gradually and, thus, does not explain the gradual
shift in lever preference across weeks. Furthermore, sign
tracking is strongly linked to VMS dopamine. Consistently,
VMS dopamine was correlated with seeking-press frequency,
whereas DMS dopamine was less so. This suggests that DMS
dopamine release does not directly induce seeking behavior
(although it does exacerbate it, as evidenced by our optogenetic
study), which is supported by the finding that optogenetic
manipulation of DMS dopamine (outside of seeking-taking
training) did not suffice to robustly reinforce behavior during
intracranial self-stimulation and RT-PP.
An intriguing, yet speculative, idea is that in habit animals, the
dopamine response to the earliest predictor of reward ‘‘propa-
gated back in time’’ to the extension of the seeking lever (distal),
whereas in no-habit animals, this dopamine response ‘‘re-
mained’’ at seeking-lever retraction (a more conservative and
precise predictor of reward [proximal]). This way, DMS dopa-
mine stimulates development of the habit-like reward pursuit,
since DMS dopamine increased in habit rats and decreased in
no-habit rats at the time of seeking-lever presentation and coin-
cided with seeking initiation without causing this behavior
directly. In other words, DMS dopamine may be the effector
that, during repeated performance, slowly strengthens the asso-
ciation between seeking stimulus/lever and response and
thereby may reflect a proclivity to habit formation, consistent
with our finding that such DMS dopamine was already enhanced
prior to habit development.
Our results challenge the reigning hypotheses of regionally
shifting dopamine involvement during habit formation, dopamine
independence of habits, and exclusive DLS governance of habits
and thereby break with the idea that the DMS simply counterbal-
ances a DLS habit system. Notably, without our in-depth analysis
of individual animal behavior across habit development, our
dopamine data (averaged across the entire population) would
have supported the idea of a weakened goal-directed DMS sys-
tem that biases animals toward habitual performance and
masked the intricate contribution of the DMS dopamine system.
Instead, we were able to delineate two distinct rat populations
and demonstrate that DMS-dopamine release during seeking
initiation critically contributes to the formation of a habit. Intrigu-
ingly, DMS dopamine exhibited a double dissociation, where
proximal events were accompanied by a signal opposite to distal
events (in both habit and no-habit rats). In consideration of the
fact that dopamine signaling did not vanish in, or shift between,
any of the measured striatal regions, we suggest that habit forma-
tion is a decentralized, concerted effort of many collaborating
striatal domains including the DMS.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:
d KEY RESOURCES TABLE
Current Biology 32, 1163–1174, March 14, 2022 1171
 ll
OPEN ACCESS Article
d RESOURCE AVAILABILITY 3. Dickinson, A. (1985). Actions and habits: the development of behavioural
B Lead contact autonomy. Philos. Trans. R. Soc. Lond. B. 308, 67–78.
B Materials availability 4. Packard, M.G., Hirsh, R., and White, N.M. (1989). Differential effects of
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B Animals 5. Packard, M.G., and McGaugh, J.L. (1992). Double dissociation of fornix
d METHOD DETAILS and caudate nucleus lesions on acquisition of two water maze tasks:
further evidence for multiple memory systems. Behav. Neurosci. 106,
B Stereotactic surgery procedures
439–446.
B Viral vectors and optogenetic stimulation
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B Operant chambers
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d QUANTIFICATION AND STATISTICAL ANALYSIS lateral striatum enhances sensitivity to changes in the action-outcome
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SUPPLEMENTAL INFORMATION 10. Amaya, K.A., and Smith, K.S. (2018). Neurobiology of habit formation.
Curr. Opin. Behav. Sci. 20, 145–152.
Supplemental information can be found online at https://doi.org/10.1016/j.
11. Jog, M.S., Kubota, Y., Connolly, C.I., Hillegaart, V., and Graybiel, A.M.
cub.2021.12.027.
(1999). Building neural representations of habits. Science 286, 1745–1749.

ACKNOWLEDGMENTS
We thank Ralph Hamelink and Nicole Yee for technical support, Matthijs Feen-
stra for input on the manuscript, and Lucia Economico for illustrations. This
research was funded by the following organizations: H2020 European
Research Council (ERC) (ERC-2014-STG 638013 to I.W.), Netherlands Organi-
sation for Scientific Research (NWO) (VIDI 864.14.010,2015/06367/ALW to
I.W.), and Netherlands Organisation for Scientific Research (NWO) (Gravitation
program, BRAINSCAPES 024.004.012 to I.W.). The funders had no role in
study design, data collection and interpretation, or the decision to submit
the work for publication.
AUTHOR CONTRIBUTIONS
Conceptualization, W.v.E., J.G., T.A., and I.W.; resources, I.W.; data curation,
W.v.E., P.W., and I.W.; formal analysis, W.v.E. and P.W.; investigation, W.v.E.,
J.M., W.B., R.J., D.S., and J.G.; visualization, W.v.E., and P.W.; supervision,
I.W.; funding acquisition, I.W.; methodology, W.v.E. and I.W.; writing – original
draft, W.v.E., T.A., and I.W.; writing – review & editing, W.v.E., D.D., T.A., and
I.W.; project administration, I.W.
DECLARATION OF INTERESTS
The authors declare no competing interests.
INCLUSION AND DIVERSITY
One or more of the authors of this paper self-identifies as an underrepresented
ethnic minority in science.
Received: July 31, 2021
Revised: November 3, 2021
Accepted: December 9, 2021
Published: February 7, 2022
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Activity of striatal neurons reflects dynamic encoding and recoding of pro-
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Article OPEN ACCESS

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit anti-GFP (primary antibodies) Thermo Fisher Scientific Cat#A-6455; RRID: AB_221570
Chicken anti-TH (primary antibodies) Aves Labs RRID: AB_10013440
Donkey anti-rabbit Alexa 488 Jackson Immunoresearch RRID: AB_2340619; 711-546-152
Donkey anti-chicken Alexa 647 Jackson Immunoresearch RRID: AB_2340380; 703-606-155
Bacterial and virus strains
AAV5.EF1a -DIO-ChR2-EYFP Viral Vector Facility, University N/A
of Zurich
AAV5.EF1a -DIO-EYFP Viral Vector Facility, University N/A
of Zurich
Chemicals, peptides, and recombinant proteins
Phosphate-buffered saline Thermo Fisher Scientific Cat#18912014
Normal donkey serum Jackson ImmunoResearch RRID: AB_2337258
Bovine serum albumin Sigma-Aldrich Cat#A9647-1kg
Sodium azide Sigma-Aldrich Cat#S2002-100 g
Triton X-100 Sigma-Aldrich Cat#X-100-500ml
4% Paraformaldehyde (37% formalin) VWR Cat#97064-606
Deposited data
Code This paper https://osf.io/j3zaq/
Experimental models: Organisms/strains
Long-Evans rats Janvier Labs, France RjOrl:LE
TH-Cre rats on Long-Evans background Rat Resource & Research LE-Tg(TH-Cre)3.1Deis / RRID:RRRC_00659
Center https://www.rrrc.us/
Software and algorithms
MATLAB version R2018b MathWorks https://www.mathworks.com/products/new_
products/release2018b.html
IBM SPSS statistics 25 IBM https://www.ibm.com/nl-en/products/spss-statistics
Prism version 9.2.0 GraphPad https://www.graphpad.com/scientific-software/prism/
LabVIEW version 2014 National Instruments https://www.ni.com/nl-nl/shop/labview.html
Med-PC version IV Med-associates https://www.med-associates.com/med-pc-v/
QuPath version 0.2.3 Open source https://qupath.github.io/
Other
45mg Dustless Precision Pellets Rodent, Purified Bio-Serv Cat#: F0021
45mg Dustless Precision Pellets Rodent, Bio-Serv Cat#: F0165
Grain-Based

RESOURCE AVAILABILITY

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Ingo Will-
uhn (i.willuhn@nin.knaw.nl).

Materials availability
This study did not generate new unique reagents.

Data and code availability

d All data reported in this paper will be shared by the lead contact upon request.

Current Biology 32, 1163–1174.e1–e6, March 14, 2022 e1

 ll
OPEN ACCESS Article
d The code used for this study is available at https://osf.io/j3zaq/ and, if necessary, more detailed information is available from the
corresponding author upon request.
d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

Animals
Adult male Long-Evans rats from Janvier Labs were used for voltammetry and behavior-only studies. For the optogenetic study, adult
male TH-Cre rats (on Long-Evans background71) were used. Rats (320-410 g) were housed individually and kept on a reversed 12 h
light/dark cycle (lights off from 08:00 to 20:00) with controlled temperature and humidity. Rats were food-restricted to 85% of their
free-feeding body weight, and water was provided ad libitum. Regular laboratory chow was available in the home cage two h after the
end of daily behavioral training (executed during the dark phase), supplementing food intake during training. All animal procedures
were conducted in accordance with Dutch and European law and approved by the Animal Experimentation Committee of the Royal
Netherlands Academy of Arts and Sciences. For dopamine recordings, 35 animals had at least one functional and histologically veri-
fied recording electrode. 16 rats were used to verify whether reward-driven behavior was habitual after 10 weeks of training by tradi-
tional satiety-specific outcome devaluation, of which two rats were excluded from analysis, because outcome devaluation failed. 17
rats were used for optogenetic stimulation of dopaminergic neuron-terminals in DMS that had histologically verified virus expression
in both hemispheres and placement of optical fibers bilaterally in DMS.

METHOD DETAILS

Stereotactic surgery procedures

Stereotactic surgery was executed as described previously.31,72 Before surgery, all surgical equipment was sterilized and the imme-
diate surroundings were wiped with 70% ethanol. Rats were anesthetized with 1%–3% isoflurane and placed in a stereotactic frame.
Heart rate and breathing frequency where monitored to assess surgical anesthetic depth. Body temperature was monitored and
maintained with a heating pad. Following subcutaneous injection with the analgesic Metacam (0.2mg meloxicam/100 g), the
scalp was shaved, disinfected with 70% alcohol, and incised to expose the cranium. The site of incision was treated with lidocaine
(100mg/mL). For the voltammetric experiment, holes were drilled for three anchor surgical screws, a Ag/AgCl reference electrode,
and two custom-made carbon-fiber micro-electrodes73 unilaterally targeting two of three regions: VMS (1.2mm rostral, 1.5mm
lateral, and 7.1mm ventral from Bregma51), DMS ( 0.2mm rostral, 2.5mm lateral, and 4.5mm ventral), and DLS (1.2mm rostral,
3.6mm lateral, and 4.5mm ventral). Electrodes were secured to the skull with dental acrylic cement anchored to the surgical screws.
For the optogenetic experiment, holes were drilled for three anchor surgical screws, two optic fibers targeting the DMS bilaterally
( 0.2mm rostral, +/ 2.7mm lateral, and 3.7mm ventral), and four virus injections in the ventral midbrain across both hemispheres,
targeting the substantia nigra pars compacta ( 5.2mm rostral, +/ 2.4mm lateral, and 7.8mm ventral, and 6.0mm rostral,
+/ 2.4mm lateral, and 7.6mm ventral). After slowly lowering the microinjector into position, virus was infused at a rate of 0.1ml/min
using a microsyringe pump. After infusion, injectors were left in place for 10min and then removed slowly. Optic fibers (diameter of
230mm, custom-made) targeted at the DMS (the dopamine-neuron projection target of interest) were secured to the skull with dental
acrylic cement anchored to the surgical screws.
Following surgery, rats received 0.5ml of saline s.c for rehydration and were placed in an incubator for at least 1 h. To monitor any
possibility of discomfort or pain and to make sure that the animals properly recovered, we monitored their overall appearance,
weight, behavior, and the state of the incision (wound healing) daily for 3 days post-surgery. Surgery and anesthesia duration was
shorter than 2 h.

Viral vectors and optogenetic stimulation

For optogenetic stimulation, AAV5.Ef1a-DIO-ChR2-EYFP (0.9ml/injection site, for a total of 3.6ml/rat, titer 3x1012 particles/mL)
was injected into the ventral midbrain for Cre-dependent expression of channelrhodopsin in dopamine neurons of TH-Cre rats.
AAV5.Ef1a-DIO-EYFP (0.9ml/injection site, for a total of 3.6ml/rat, titer 3x1012 particles/mL) was used as a Cre-dependent fluoro-
phore control. For optogenetic stimulation, we used laser light with a wavelength of 470 nm, a pulse width of 10ms, a frequency
of 20Hz, and an intensity of 20mW at the tip of the optic-fiber patch cable (constant illumination).

Operant chambers
All behavioral experiments were conducted in modified modular operant chambers (32x30x29cm, Med Associates Inc., VT, USA),
equipped with a food magazine with an integrated light (connected to an automated food-pellet dispenser) flanked by two retractable
operant levers, a house light, multiple tone generators, a white-noise generator, and metal grid floors. The wall opposite the food
magazine was outfitted with two nose-poke response devices (port with integrated cue lights) located on adjacent panels. Each op-
erant box was surveilled by a video camera. The boxes were housed in metal Faraday cages, that were insulated with sound-
absorbing polyurethane foam and ventilated by a fan.

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Training on seeking-taking chain schedule of reinforcement

The seeking-taking chain schedule of reinforcement was adapted from Olmstead and colleagues74 and Balleine and colleagues.75
After rats were placed in the operant chamber, the start of each daily behavioral-training session was marked by illumination of the
house light and the sound of the white noise, which both remained on for the duration of the session. During each trial, rats could earn
one ‘‘purified’’ food pellet (Bio. Serv. Inc., USA), delivered into the tray of the food magazine, accompanied by a 2 s-illumination of the
magazine light. Behavioral training commenced when rats reached 85% of free-feeding body weight. On the first day, animals were
habituated to the operant chamber and 30 food pellets were delivered on a variable-interval (VI) schedule of 90 s. On the second day,
rats learned to press the taking lever, the proximal link of the chain (i.e., proximal to the food reward), on a fixed ratio 1 (FR1) schedule
for an immediate delivery of a pellet, with a maximum of 40 pellets. The following three days, taking-lever presses triggered pellet
delivery (FR1) plus lever retraction, and an inter-trial interval (ITI) of 25 s was imposed, followed by re-extension of the lever into
the operant box. In the next session, rats learned to press the seeking lever, the distal link of the chain (i.e., distal to the food reward),
in order to gain access to the taking lever. Following its extension into the box (marking the start of each trial), one press on the
seeking lever (FR1) triggered retraction of this lever, and after a delay of 3 s, the taking lever was extended. One press on the taking
lever (FR1) triggered retraction of the taking lever and delivery of a food pellet 3 s thereafter (FR1:FR1 schedule). During the variable ITI
of 25 s, both levers remained retracted. In the following sessions, rats were trained on increasing VIs (ranging from 2 s to eventually
60 s) for the seeking lever: VI2:FR1, VI7:FR1, VI15:FR1, VI30:FR1, VI60:FR1. Each VI started with the rat’s first response on the
seeking lever, once extended. Subsequent lever presses went without programmed consequences, until the first press after the
VI ended, which triggered seeking-lever retraction and taking-lever extension 3 s thereafter. Figure 2A depicts a schematic overview
of a VI60:FR1 trial. Sessions ended once rats earned 40 rewards. Rats were trained daily on the VI60:FR1 for 10 weeks. Lever position
(seeking/taking) was counterbalanced between rats.

Lever-preference test
Behavioral automaticity was probed regularly (no more than once per week) immediately preceding the daily training session, using a
novel ‘‘lever-preference’’ test: After a variable interval of 25 s with only house-light and white noise turned on, animals were exposed
to both seeking and taking levers simultaneously for one minute under extinction conditions (recording lever presses and head entries
into the food magazine without programmed consequences). During this minute, a preference for the taking lever (i.e., more presses
on the taking than seeking lever) was interpreted as a goal-directed behavioral strategy, whereas a seeking-lever preference was
taken as evidence for habitual responding.
We aim to establish the lever-preference test as a new way to assess the development of habitual behavior. Our intention is not to
emulate another version of an outcome-devaluation procedure or to design a proxy for it. Instead, we introduce an alternative way to
track progressive automation of behavior and present a number of points that validate and/or support this test as a tool that identifies
a habit:

1) Across the 10 weeks of training, the animals’ initial test preference to interact with the taking lever (that is associated with
immediate (FR1) food delivery) switches to the seeking lever (that never directly leads to food delivery and never leads to
food quickly, but instead only leads to the extension of the taking lever).
2) We demonstrate that this preference switch occurs within individual animals.
3) The taking preference at the beginning of training is present in animals from both groups (habit and no-habit; Figures 2B and 2C).
4) This switch only occurs after a very high rate of action and session repetition (1000s of repetitions in 10 weeks of daily training
sessions; Figure 1B). It is widely accepted that habit learning materializes through high repetition of responding. Our paradigm
requires many repetitions of the behavior before the habit is detectable (long after the motor skill to lever press had been
acquired).
5) The slow speed of this switch is comparable to the speed of habit development in human everyday life (Figures 2B and 2C),
establishing face validity, in contrast to some other habit tasks that report habits developing as quickly as after 1-4 days of
behavioral training.
6) Induction of sensory-specific satiety after 10 weeks of seeking-taking training demonstrates that our behavioral training
schedule induces a habit as defined and tested by traditional outcome devaluation (and in the traditional way: as a singular
group, as opposed to considering individual differences; Figure 1C), establishing construct validity and fulfilling the traditional
requirement to classify a behavior as habitual.
7) Habitual animals exhibit a higher probability to repeat seeking-lever presses (lever stickiness) during the preference test (Fig-
ure 2H), a measure associated with habitual behavior.
8) Habitual animals commit fewer head entries into the food magazine during the preference test (Figure 2I), a measure asso-
ciated with goal-oriented behavior (non-habitual).
9) The measures mentioned under points 7 and 8 correlate with seeking-lever preference (Figure 2H and 2I).
10) The preference test detects a similar preference progression across weeks independent of the frequency with which the test
is applied (Figure S2).
11) Rats do not perform head entries into the food magazine during the 3-s waiting period before extension of the taking lever,
indicating that the animals learn the ‘‘task logic’’ (i.e., the taking lever needs to be pressed in order to receive food (Figure 1B
(right)).

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12) The ‘‘task logic’’ translates well from the training situation (VI60/FR1) to the preference-test situation (both levers out), as an-
imals in both groups (habit and no-habit) perform substantially more food-magazine entries after taking-lever presses (the
lever associated with food delivery; Figure 2G).

We do not provide an extensive cross-validation of our preference test with the sensory-specific satiety data presented in Figure 1C
for several reasons: 1) We employed behavioral training under a VI60 reinforcement schedule, which has in fact already been widely
validated and widely accepted to induce habits, for both single-action and action-sequence tasks. 2) Data acquired using the reward-
devaluation approach is relatively ‘‘noisy’’ and provides little clustering of the population data, as exemplified in Figure 1C (center). 3)
We believe that what is commonly referred to as a ‘‘habit’’ is not a unitary concept and has a number of different aspects and that,
therefore, there are different ways to approach its detection. An approach that focuses on devaluation-insensitivity criteria is a widely
accepted approach, originally put forward by Dickinson and colleagues, and has led to invaluable research findings. However, it does
not capture all aspects of a (human) habit. For example, using its underlying logic, a habit can be induced in a single, relatively short
behavioral training session in humans, and no more than 4-5 sessions in rodents. Yet, the human behavior to be modeled by this
research, is behavior that has slowly developed across weeks or months, or even years. Our paradigm requires weeks of daily training
to induce a habit. 4) To our knowledge, sensory-specific satiety devaluation has never been reported as a way to assess individual
differences in habit formation. In addition to this type of devaluation being very sensitive to disturbances in general, we think that this
approach is actually not suitable to assess individual differences: Following the ad-lib pre-feeding, rats were exposed to the seeking
lever for five minutes under extinction conditions during which lever presses were recorded. Each animal was subjected to both
valued (pre-feeding with pellets different from training pellets) and devalued (pre-feeding with training pellets) conditions (on separate
days); the sequence of conditions was counterbalanced between animals. An important observation is that during the first pre-
feeding session, animals eat more than after the second. Thus, whichever condition comes first, valued or devalued, the first session
elicits more lever presses relative to the same condition when it occurs second in temporal sequence (in other animals). We have
observed this phenomenon reliably in several cohorts of animals, and believe it to be a general phenomenon, despite the fact
that, to our knowledge, it has not been reported in the literature. This phenomenon complicates looking at individual differences,
as individuals are strongly affected by the sequence of pre-feeding, which restricts the utility of pre-feeding devaluation to group
comparisons, where an equal number of animals have undergone each condition first (due to counterbalancing).

Experimental procedures
Experiment 1: Week 10 validation of habit induction by seeking-taking paradigm
One group of rats (n = 14) was tested for habitual responding using a traditional satiety-specific outcome devaluation, after 10 weeks
of daily training on the VI60:FR1 seeking-taking chain schedule of reinforcement. Outcome devaluation (sensory-specific satiety) was
achieved by giving rats one-h ad-libitum access to either grain-based pellets (used to remove hunger as a motivating factor for lever
pressing; the pellets that are used for behavioral training retain their ‘‘rewarding’’ value, this is the ‘‘valued condition’’), or regular (pu-
rified) pellets (used to decrease both hunger and value of the behavioral-training pellets, this is the ‘‘devalued condition’’) prior to
behavioral testing. Food pellets were placed in custom-made metal cups in an empty, otherwise unused home cage (‘‘feeding
cage’’; water freely available). Following one h of this pre-feeding, rats were exposed to the seeking lever in the operant chamber
for five minutes under extinction conditions during which lever presses were recorded. This was subsequently followed by a ‘‘pel-
let-choice’’ test to determine the efficacy of the pre-feeding procedure, measured by the preference for either pellet type during
two minutes of simultaneous ad-libitum access to both types in the feeding cage. Animals were excluded from analyses if they
consumed any of the prefed-type pellets during this test (insufficient sensory-specific satiety). Each animal was subjected to both
valued and devalued conditions (on separate days); the sequence of conditions was counterbalanced between animals.
Experiment 2: Dopamine measurement during seeking-taking chain training
In a second group of rats (n = 35), we employed fast-scan cyclic voltammetry (FSCV) using chronically implanted carbon-fiber micro-elec-
trodes73 to record rapid changes in dopamine release in different domains of the striatum, during week 1 and week 10 of training on the
seeking-taking chain of reinforcement schedule. On recording days, before the start of behavioral training, electrodes were connected to
a head-mounted voltammetric amplifier that was interfaced through an electrical swivel above the test chamber (allowing animals free
movement; Crist Instrument, MD, USA) with a PC-driven data-acquisition and analysis system (National Instruments, TX, USA).73
Voltammetric scans were repeated every 100ms (10Hz sampling rate). During each scan, the alternating potential at the carbon-
fiber electrode tip was ramped linearly from –0.4V versus Ag/AgCl to +1.3V (anodic sweep) and back to 0.4V (cathodic sweep) at
400V/s (total scan time of 8.5ms) and held at 0.4V between scans. Waveform generation, data acquisition, and analysis were carried
out on a PC-based system using two PCI multifunction data acquisition cards and software written in LabVIEW (National Instru-
ments). Dopamine is oxidized during the anodic sweep, if present at the surface of the electrode, forming dopamine-o-quinone
(peak reaction detected around +0.7V), which is reduced back to dopamine in the cathodic sweep (peak reaction detected around
0.3V). The ensuing flux of electrons is measured as current and is directly proportional to the number of molecules that undergo
electrolysis. The background-subtracted, time-resolved current obtained from each scan provides a chemical signature character-
istic of the analyte, allowing resolution of dopamine from other substances.76
Experiment 3: Optogenetic stimulation of DMS dopamine-neuron terminals
In a third group of rats (n = 17), we optogenetically stimulated DMS dopamine-neuron terminals (bilaterally) in response to seeking-
lever presses for three weeks during daily seeking-taking chain training sessions starting under VI30:FR1 and progressing to

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VI60:FR1. A seeking-lever press triggered a 1 s laser photo-stimulation (20mW, 20Hz, 20x10ms light pulses) followed by a 1 s timeout.
Starting 10 s after the first seeking-lever press, only every third seeking-lever press triggered stimulation. Rats were exposed to the
lever-preference test (without optogenetic stimulation) regularly to assess whether an approach habit was developing. Laser-stim-
ulation visibility was masked using a custom-made LED mounted on the wall of the operant box emitting blue light pulses (20Hz,
20x10ms).
Experiment 4: Real-time place preference (RT-PP) and intracranial self-stimulation
Prior to experiment 3 (after 8-9 week post-surgery period to allow for sufficient virus expression), rats were exposed to an open-field
arena in which an RT-PP assay was implemented. Following a 5min baseline period without photo-stimulation, the presence of the rat
in one of the quadrants of the open-field (i.e., active quadrant) triggered photo-stimulation (20mW, 20Hz, 20x10ms light pulses) of
DMS dopamine-neuron terminals during a 15min assay. The position of the active quadrant was assigned randomly. Time spent
in the active quadrant was recorded. Laser-stimulation visibility was masked using a custom-made LED mounted on the wall of
the open-field emitting blue light pulses (20Hz, 20x10ms).
After Experiment 3, rats were tested in the same operant box for three days in an operant self-stimulation experiment, where
nose pokes into the active nose-poke device triggered a 1 s photo-stimulation (20mW, 20Hz, 20x10ms light pulses) of DMS dopa-
mine-neuron terminals, followed by a 1 s timeout. Nose pokes into the inactive nose-poke device were recorded without pro-
grammed consequence. The location of active and inactive nose-poke devices was counterbalanced between rats. Laser-stimula-
tion visibility was masked using a custom-made LED mounted on the wall of the operant box emitting blue light pulses (20Hz,
20x10ms).
Histological verification of recording sites, optic-fiber placement, and virus expression
After completion of experiments, rats were deeply anesthetized using a lethal dose of pentobarbital (14mg/100 g), and transcardially
perfused with saline followed by 4%-paraformaldehyde (PFA). In animals with electrode implants, recording sites were marked
with an electrolytic lesion before perfusion. Brains were removed and post-fixed in PFA for one day after which they were placed
in 30% sucrose for cryoprotection. The brains were rapidly frozen using an isopentane bath, sliced on a cryostat (50-mm coronal sec-
tions, 20 C), and stained with Cresyl violet to aid visualization of anatomical structures, electrode-induced lesion or virus-infusion
sites, and optic-fiber placement.
For the optogenetic experiment, sliced serial coronal sections (50mm) were stored in phosphate-buffered saline (PBS) with 0.02%-
sodium azide at 4 C until further use. For immunohistochemical stainings, sections were incubated in blocking buffer (5% bovine
serum albumin (BSA), 5% normal donkey serum (NDS), and 0.2%-triton-X in PBS) for 1 h, followed by overnight incubation in primary
antibodies rabbit anti-GFP (1:1000, Invitrogen A6455, Thermo Fisher) and chicken anti-TH (1:1000, Aves TYH) in blocking buffer. All
sections were washed 4x (10min each) in PBS and then incubated for 1 h in blocking buffer containing fluorescent Alexa-conjugated
secondary antibodies (1:1000, donkey anti-chicken Alexa 647 and donkey anti-rabbit Alexa 488; Jackson Immunoresearch 711-546-
152 and 703-606-155). Sections were washed again 4x in PBS, with the second wash containing DAPI (nuclei stain; Sigma Aldrich
D9542), then mounted onto slides and coverslipped with Mowiol mounting medium. Slides were imaged using three fluorescent
channels of the ZEISS Axio Scan.z1 slide-scanner at 10x magnification for qualitative expression and targeting verification. Sections
were subsequently imaged in z stacks with a confocal microscope (Leica TCS SP5) to verify specificity of viral expression.

QUANTIFICATION AND STATISTICAL ANALYSIS

Data analysis
Dopamine-related current was isolated from the voltammetric signal by chemometric analysis using a standard training set, based on
electrically stimulated dopamine release detected with chronically implanted electrodes; resulting dopamine concentration was esti-
mated based on average post-implantation sensitivity of electrodes.73 Resulting dopamine traces were smoothed with a moving 10-
point median filter prior to analysis of average concentration. Dopamine concentration was averaged over 10 s (approximate duration
of the observed phasic signal) following seeking-lever extension (distal link of the chain) and over 7.5 s before and 2.5 s after reward
delivery (proximal link of the chain); resulting averages were compared to the average concentration over the 2 s before each trial
(baseline). Prior to each FSCV recording session, two unpredicted food-pellet deliveries (spaced apart by two minutes) confirmed
electrode viability to detect dopamine, as well as electrode sensitivity over time. Data analysis was performed using MATLAB
R2018b (The Mathworks, Inc., MA, USA).

Statistical analysis
Individual electrochemical signals were averaged across sessions and then across animals. For comparison of electrochemical data,
individual behavioral data were binned into weeks and then averaged across animals. Corresponding electrochemical and behavioral
data from week 1 were compared to week 10 using paired Student’s t test. When separated into groups, behavioral data were
analyzed using multivariate ANOVAs with group, lever, nose-poke port, and week as factors. When main effects or interactions
were significant, post hoc analyses were carried out and P values were adjusted according to the Holm-Bonferroni correction method
for multiple comparisons. For the optogenetic self-stimulation experiment (Figure 4D), we used two-way ANOVAs, with nose-poke
port (active versus inactive) or group (ChR2 versus EYFP) as factors, and time (day) as repeated-measurement. Non-parametric
testing was conducted when data was not normally distributed. Graphical representations were made using Prism (GraphPad Soft-
ware, La Jolla, CA, USA) and MATLAB. Statistical analysis was carried out using Prism and SPSS (version 25; Chicago, IL, USA) and

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MATLAB. Data collection and analysis were not performed blind to the conditions of the experiments. Statistical significance was set
to p < 0.05. All data are presented as mean or median ± SEM.
Sample size was 35 rats for dopamine recordings (Figures 1 and 3), 16 for testing habitual behavior by traditional outcome deval-
uation via sensory-specific satiety (Figure 1), and 17 for optogenetic stimulation of dopaminergic neuron-terminals in the DMS (Fig-
ure 4), as described under ‘‘Animals.’’ To demonstrate reliability of the lever-preference test, we used seven cohorts of rats with
different sample sizes (ncohort1 = 12, ncohort2 = 12, ncohort3 = 13, ncohort4 = 16, ncohort5 = 6, ncohort6 = 14 ncohort7 = 6;
Figure S2).

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