The Journal of Neuroscience, Month XX, 2017 • 37(XX):XXXX–XXXX • 1

Systems/Circuits

Ventrolateral Striatal Medium Spiny Neurons Positively
Regulate Food-Incentive, Goal-Directed Behavior
Independently of D1 and D2 Selectivity
Akiyo Natsubori,1,2* Iku Tsutsui-Kimura,1* Hiroshi Nishida,1 Youcef Bouchekioua,1 Hiroshi Sekiya,3Motokazu Uchigashima,4 X Masahiko Watanabe,4 X Alban de Kerchove d’Exaerde,5 Masaru Mimura,1 Norio Takata,1and X Kenji F. Tanaka1
1

Department of Neuropsychiatry, Keio University School of Medicine, Tokyo 160-8582, Japan, 2Sleep Disorders Project, Tokyo Metropolitan Institute ofMedical Science, Tokyo 156-8506, Japan, 3Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan,
4Department of Anatomy, University of Hokkaido Graduate School of Medicine, Sapporo 060-8638 Japan, and 5Laboratory of Neurophysiology, ULB
Neuroscience Institute, Université Libre de Bruxelles (ULB), 1050 Brussels, Belgium

The ventral striatum is involved in motivated behavior. Akin to the dorsal striatum, the ventral striatum contains two parallel pathways:
the striatomesencephalic pathway consisting of dopamine receptor Type 1-expressing medium spiny neurons (D1-MSNs) and the striatopallidal pathway consisting of D2-MSNs. These two genetically identified pathways are thought to encode opposing functions inmotivated behavior. It has also been reported that D1/D2 genetic selectivity is not attributed to the anatomical discrimination of two
pathways. We wanted to determine whether D1- and D2-MSNs in the ventral striatum functioned in an opposing manner as previous
observations claimed, and whether D1/D2 selectivity corresponded to a functional segregation in motivated behavior of mice. To address
this question, we focused on the lateral portion of ventral striatum as a region implicated in food-incentive, goal-directed behavior, andrecorded D1 or D2-MSN activity by using a gene-encoded ratiometric Ca2⫹ indicator and by constructing a fiberphotometry system, andmanipulated their activities via optogenetic inhibition during ongoing behaviors. We observed concurrent event-related compound Ca2⫹
elevations in ventrolateral D1- and D2-MSNs, especially at trial start cue-related and first lever press-related times. D1 or D2 selective
optogenetic inhibition just after the trial start cue resulted in a reduction of goal-directed behavior, indicating a shared coding of
motivated behavior by both populations at this time. Only D1-selective inhibition just after the first lever press resulted in the reductionof behavior, indicating D1-MSN-specific coding at that specific time. Our data did not support opposing encoding by both populations infood-incentive, goal-directed behavior.
Key words: fiberphotometry; food-incentive goal-directed behavior; motivation; optogenetic inhibition; ventrolateral striatum; yellow
cameleon

Significance Statement
An opposing role of dopamine receptor Type 1 or Type 2-expressing medium spiny neurons (D1-MSNs or D2-MSNs) on striatummediated behaviors has been widely accepted. However, this idea has been questioned by recent reports. In the present study, wemeasured concurrent Ca2⫹ activity patterns of D1- and D2-MSNs in the ventrolateral striatum during food-incentive, goaldirected behavior in mice. According to Ca2⫹ activity patterns, we conducted timing-specific optogenetic inhibition of each typeof MSN. We demonstrated that both D1- and D2-MSNs in the ventrolateral striatum commonly and positively encoded action
initiation, whereas only D1-MSNs positively encoded sustained motivated behavior. These findings led us to reconsider theprevailing notion of a functional segregation of MSN activity in the ventral striatum.

Introduction
The projection neurons of the striatum are GABAergic medium
spiny neurons (MSNs), which are subdivided into two populaReceived Oct. 30, 2016; revised Jan. 16, 2017; accepted Jan. 29, 2017.
Author contributions: A.N., I.T.-K., and K.F.T. designed research; A.N., I.T.-K., Y.B., M.U., and M.W. performed
research; A.N., I.T.-K., H.S., A.K.E., M.M., and N.T. contributed unpublished reagents/analytic tools; A.N., I.T.-K., and
H.N. analyzed data; A.N., I.T.-K., and K.F.T. wrote the paper. All authors edited the paper.

tions: the dopamine receptor Type 1-expressing (D1)- and the
dopamine receptor Type 2-expressing (D2)-MSNs. These MSNs
are critically involved in motor control and goal-directed motivational processes (Kreitzer and Malenka, 2008; Kravitz et al.,This work was supported by Japan Society for the Promotion of Science Grant for Research Fellow 15J06790 to
A.N. and 2640100 to I.T.-K., Ministry of Education, Culture, Sports, Science and Technology Grant-in-Aid for Scientific
Research (B) 15H03123 Grant-in-Aid for Scientific Research on Innovative Area “Oscillology” (16H01621), and2 • J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX2010). D1- and D2-MSNs in the dorsal striatum (DS) project todistinct targets within the basal ganglia. D1-MSNs project to output nuclei of basal ganglia (substantia nigra/ventral tegmentalarea) directly and D2-MSNs project to the external globus pallidus, which is a relay nucleus to the output nuclei. Based on theseconnections, DS D1-MSNs are regarded as the striatomesencephalic pathway or direct pathway, and DS D2-MSNs are regarded as the striatopallidal pathway or indirect pathway. This
clear separation of striatal circuitry may account for the neuralbasis of the opposing D1 vs D2 roles in the DS, initially proposedby Albin et al. (1989) in terms of motor control. That is, activation of the direct pathway facilitates movement and activation of
the indirect pathway inhibits movement. Studies using D1- andD2-type-specific loss- and gain of function have supported theview of opposing roles of D1- and D2-MSNs in the DS on motor
function (Kravitz et al., 2010; Durieux et al., 2012), reinforcement learning (Kravitz et al., 2012), and drug sensitization (Ferguson et al., 2011; Durieux et al., 2012).
This view has been extended to studies on the ventral striatum(VS), especially on the nucleus accumbens (NAc; the rostral VS)
in a series of drug-seeking behavioral studies (Hikida et al., 2010;
Lobo et al., 2010; Stefanik et al., 2013). Such an extension was
supported in part by anatomical similarity; in the VS, D1-MSNs
project to the ventral tegmental area (ventral mesencephalon[VM]) directly and D2-MSNs project the ventral pallidum (VP),a relay nucleus to the VM. Akin to the DS, VS D1-MSNs andD2-MSNs can be regarded as direct and indirect pathways, respectively. However, further anatomical evidence questions an
exclusive D1 versus D2 separation of direct/indirect pathways inthe VS (Gangarossa et al., 2013; Kupchik et al., 2015). First, half of
VP neurons receive NAc D1-MSNs input, and the VP acts as an
output nucleus of the basal ganglia. The former evidence contradicts the idea that D1-MSNs do not project to a relay nucleus. The
latter resolved the above contradiction but gave rise to anothercontradiction (i.e., D2-MSNs projecting to an output nucleus
directly). In addition, the former evidence points out that D1and D2-MSNs share the same pathway from the NAc to the VP.
Therefore, it is unknown whether VS-mediated behaviors, aside
from drug-seeking behaviors, are encoded by D1- and D2-MSNs
in an opposing or cooperative manner.
We recently demonstrated that ventrolateral striatal (VLS) D2MSNs control motivated behavior through experiments combining
cell type-specific ablation/inhibition with food-incentive, goaldirected behavior (Tsutsui-Kimura et al., 2017); toxin-mediated lossof function of the entire (from the rostral to the caudal) VLS D2MSNs impaired motivation, and optogenetics-mediated loss offunction of D2-MSNs in the middle portion of the VLS impaired
motivation as well. Although the VLS is not identical to the NAc, ourrecent data highlighted that the VLS regulates motivated behavior.
These results encouraged us to tackle the above question. We firstdeveloped a fiber photometric system to observe the activity of VLS
D1- or D2-MSNs in freely moving mice and examined whether eachcell type signaled in an opposing or coordinated manner duringmotivated behavior. We next examined the functional readout of
D1- or D2-selective, activity-oriented optogenetic inhibition to deTakeda Science Foundation to K.F.T. A.K.E. is a Senior Research Associate of the FRS-FNRS (Belgium) and an investigator of WELBIO. We thank H. Sano for the gift of Pde10a2-tTA transgenic mice.
The authors declare no competing financial interests.
*A.N. and I.T.-K. contributed equally to this work.
Correspondence should be addressed to Dr. Kenji F. Tanaka, Department of Neuropsychiatry, Keio University
School of Medicine, Tokyo 160-8582, Japan. E-mail: kftanaka@keio.jp.
DOI:10.1523/JNEUROSCI.3377-16.2017
Copyright © 2017 the authors 0270-6474/17/370002-11$15.00/0

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

termine whether D1 or D2-MSNs encode motivated behavior in an
opposing or a cooperative manner.

Materials and Methods
Animals. All animal procedures were conducted in accordance with the
National Institutes of Health Guide for the care and use of laboratory
animals and approved by the Animal Research Committee of Keio University School of Medicine (approval 14027-(1)). Experiments were performed using 3- to 12-month-old male and female mice. D2-YC mice
(Drd2-tTA::tetO-YCnano50 double-transgenic mice) were obtained bycrossing between Drd2-tTA mice (Tsutsui-Kimura et al., 2017) andtetO-YCnano50 mice (Kanemaru et al., 2014). D2-ArchT mice (Drd2tTA::tetO-ArchT-EGFP double-transgenic mice) were obtained bycrossing between Drd2-tTA mice and tetO-ArchT-EGFP mice (Tsunematsu et al., 2013). D1-YC mice (Pde10a2-tTA::tetO-YCnano50;
Adora2a-Cre triple-transgenic mice) were obtained by crossing between
Pde10a2-tTA mice (Sano et al., 2008), tetO-YCnano50 mice, andAdora2a-Cre mice (Durieux et al., 2009) (see Fig. 1). D1-ArchT mice(Pde10a2-tTA::tetO-ArchT-EGFP; Adora2a-Cre triple-transgenic mice)
were obtained by crossing Pde10a2-tTA mice, tetO-ArchT-EGFP mice, andAdora2a-Cre mice. The genetic background of all transgenic mice was mixed
C57BL6 and 129SvEvTac.
The following primer sets were used in mouse genotyping: D2R-582U(5⬘-GCGTTTGACTAAGTTGCCAAGCTG-3⬘) and mtTA24L (5⬘-CGGAGTTGATCACCTTGGACTTGT-3⬘) were used for Drd2-tTA andyielded a 610 bp band; tTAup (5⬘-GCTGCTTAATGAGGTCGG-3⬘) andtTAlow (5⬘-CTCTGCACCTTGGTGATC-3⬘) were used for Pde10a2-tTA
and yielded a 500 bp band; CreP1 (5⬘-GCCTGCATTACCGGTCGATGCAACG-3⬘) and CreP2 (5⬘-AAATCCATCGCTCGACCAGTTTAGTTACCC-3⬘) were used for Adora2a-Cre and yielded a band of 640 bp. Wildtype mice were negative for the above PCR products. Genotyping for
tetO-YCnano50 and tetO-ArchT-EGFP was previously described (Tsunematsu et al., 2013; Kanemaru et al., 2014). While transgene induction can beturned off by administration of doxycycline, we used the system solely toachieve cell-type specificity.
Immunohistochemistry. The method for immunohistochemistry has
been described previously (Kanemaru et al., 2014). The following
antibodies were used: anti-dopamine receptor Type 1 (D1) (1:1000,
goat polyclonal, Frontier Institute), anti-D2 (1:1000, guinea pig polyclonal, Frontier Institute), anti-adenosine receptor Type 2a (A2a)(1:1000, guinea pig polyclonal, Frontier Institute), and anti-GFP (1:1000, rabbit polyclonal, Frontier Institute). For fluorescence microscopy, sections were treated with a mixture of species-specific secondary
antibodies conjugated to AlexaFluor-488, -555, or -633 (1:1000, Invitrogen) for 2 h at room temperature. Fluorescent images were obtained witha confocal microscope (LSM710, Zeiss).
Surgery. Stereotaxic surgery was performed under anesthesia with aketamine-xylazine mixture (100 and 10 mg/kg, respectively, i.p.). For the
optical measurement, animals were unilaterally (right side) implantedwith an optical fiber cannula (CFMC14L05, ␾ 400 ␮m, 0.39 NA; Thorlabs) into the VLS (0.4 mm anteroposterior, 2.0 mm mediolateral frombregma, ⫺4.0 mm dorsoventral from the skull surface), according to theatlas of Paxinos and Franklin (2004). For the optogenetic inhibition,
mice were bilaterally implanted with 200 ␮m core diameter optical fiber
cannula (0.39 NA, Thorlabs) into the VLS. For microinjection studies,
mice were unilaterally (right side) implanted with 400 ␮m core diameter
optical fiber and microinjection guide cannula (Eicom) into the VLS,
with a respective 15 degree angle of insertion in the coronal plane.
Fiberphotometry. We used a fiber photometric system to detect compound neuronal calcium fluctuations. The system was designed by
Olympus Engineering (see Fig. 2A). The input light (435 nm; silver-LED,
Prizmatix) was reflected off a dichroic mirror (DM455CFP; Olympus),
coupled into an optical fiber (M41L01, ␾ 600 ␮m, 0.48 NA; Thorlabs)
linked to a 400 ␮m 0.39 NA optical fiber (M79L01; Thorlabs) through apinhole (␾ 600 ␮m), and then delivered to an optical fiber cannula
implanted into the mouse brain. LED power was ⬍200 ␮W at the fiber
tip. Emitted cyan and yellow fluorescence from YCnano50 was collected
via an optical fiber cannula, divided by a dichroic mirror (DM515YFP;
Olympus) into cyan (483/32 nm band-path filters, Semrock) and yellow

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation(542/27 nm), and detected by each photomultiplier tube (H10722–210;
Hamamatsu Photonics). The fluorescence signals in addition to TTL
signals from behavioral settings were digitized by a data acquisition module (NI USB-6211; National Instruments) and simultaneously recordedby using a custom-made LabVIEW program (National Instruments).
Signals were collected at a sampling frequency of 1000 Hz. The fluorescence signals were smoothed using a 100-point moving-average method.
Fiberphotometry with pharmacological intervention in freely moving
mice. One week after guide cannula and optical fiber implantation targeted at the VLS, a stainless-steel microinjection cannula (CXMI-4T;
Eicom) with a length of 1 mm longer than the guide cannula was inserted.
The microinjection cannula was connected to a 25 ␮l Hamilton syringe
operated by an infusion pump (Eicom). The following drugs were used:
0.64 ng/␮l TTX (Wako), 1 ␮g/␮l muscimol (MUS; Tocris Bioscience), 1
␮g/␮l NBQX (Tocris Bioscience), and 4 ␮g/␮l AP-5 (Tocris Bioscience),
which were dissolved in the vehicle (HEPES-Ringer’s solution): 125 mM
NaCl, 5 mM KCl, 10 mM HEPES, 2 mM CaCl2, 2 mM MgSO4, 10 mM
glucose, pH 7.4. The microinjection volume was always 0.4 ␮l, given at a
rate of 0.05 ␮l/min. After a 5 min postinjection period, the optical recording resumed.
Fixed ratio (FR) operant task. Mice were housed individually underconditions of food restriction. Their body weights were maintained at
85% of initial body weight. Behavioral trainings and tests were performed in an aluminum operant chamber measuring W22 ⫻ D26 ⫻ H18
cm (Med Associates) under constant darkness. A food dispenser flanked
by two retractable levers was located on the floor. The lever on the left
side is designated as “active” (triggering delivery of a food reward), andthe one on the right is “inactive” (no relation to food reward). Each trial
began with the presentation of two levers (trial start [TS]). After micepressed the active lever (lever press [LP]), the levers were retracted and
one food pellet (20 mg each, dustless precision pellets, Bio-serv) was
delivered as a reward (RW). After the food delivery, 30 s of intertrial
interval was added, during which levers were retracted, followed by theautomatic starting of the next trial. The intertrial interval allows time formice to consume the food pellet. The session lasted 60 min or until micereceived 100 food rewards. The training started with a FR-1 schedule, inwhich the animals obtained one food reward after each active LP. Oncethe animals were able to obtain 50 rewards within 60 min, the training
progressed to an FR-2 schedule, in which two active LPs were needed foreach RW. The training moved on to an FR-3 schedule when the animals
could obtain 50 rewards in 60 min. The animals were then trained on an
FR-10 schedule after they could obtain 50 rewards in 60 min for a coupleof sessions. Then mice were implanted with an optic fiber cannula andwere left to recover for a week. After the recovery, the mice connectedwith an optic fiber were trained up from the FR-1 schedule until the mice
performed the FR-10 operant task under the optical measurement. On
average, it took 28 d for the entire behavioral procedure, consisting oftraining, surgery and recovery, and retraining.
Progressive ratio (PR) operant task. The same apparatus used in the FR
task was used in the PR task. The training procedure and task sequenceswere the same as previously described (Tsutsui-Kimura et al., 2017).
After the training was completed, the mice were implanted with optic
fiber cannulas bilaterally and allowed to recover for a week. After recovery, the mice connected with a bifurcated optic fiber were trained again,
and when their performance in the PR task stabilized for three consecutive sessions, we moved to the optical manipulation phase of the experiment. On average, it took 28 d for the entire behavioral procedure. The
PR breakpoint was defined as an index of instrumental motivation. The
time spent to complete the PR (the mean time from the first active LP toachieving the required number of active LP) was defined as an index of
instrumental motivation. Percent inactive lever pressing [inactive LP/(inactive and active LP) ⫻ 100] was calculated and was defined as anindex of associative learning. The latency to collect reward was defined as
an index of appetite.
In vivo optical measurement of MSN activity during the FR operanttasks. Four D2-YC and five D1-YC mice were subjected to the FR task. YC
ratio (a ratio of yellow to cyan fluorescence intensity; R) in one session
was detrended using cubic spline, and normalized within each trial cal-J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX • 3culating the Z score as (R ⫺ Rmean)/RSD, where Rmean and RSD were themean and SD of the YC ratio for 5 s just before each TS.
Optogenetic inhibition during the PR operant tasks. Six D2-ArchT andthree D1-ArchT mice were subjected to the PR task under the influenceof optogenetic inhibition. Before each experiment, optical fibers (DoricLenses) were connected bilaterally into the optical cannula. Yellow (575
nm, 3 mW each at the fiber tip) or blue (475 nm, 2 mW each) light was
generated by a SPECTRA 2-LCR-XA light engine (Lumencor).
Data analysis. YC ratio and all statistics were analyzed using MATLAB(The MathWorks). The power spectral data of YC ratio was obtained bya wavelet analysis. In the pharmacological study, a power spectral profile
over a 0.05–1.0 Hz window was used for analysis.
Data for all experiments were analyzed using parametric statistics with
Student’s t test, Student’s t test with Bonferroni correction, paired t test,or paired t test with Bonferroni correction.

Results
Optical detection of compound Ca2ⴙ signals of
D1- or D2-MSNs in the VLS
To understand the temporal patterns of VLS D1- or D2-MSN
activity during goal-directed, motivated behavior, we generated
transgenic mice harboring a Ca2⫹ indicator in a D1- or D2-MSNspecific manner (Fig. 1) and developed a fiber photometric system to detect compound Ca2⫹ signals from the targeted cells (Fig.
2A). We used a Förster resonance energy transfer-based ratiometric gene-encoded Ca2⫹ indicator, called yellow cameleon(YC) nano50 (Horikawa et al., 2010), which allowed us to reducenoise levels originated from motion artifacts under physiologicalconditions (Kanemaru et al., 2014).
To sufficiently express the Ca2⫹ indicator in the targeted
cell population, we took advantage of an improved tetracycline-controllable gene induction system (KENGE-tet system,e.g., tetO-YCnano50 mice) (Tanaka et al., 2012). To express
YCnano50 selectively in D2-MSNs, we generated Drd2-tTA::
tetO-YCnano50 double-transgenic mice (D2-YC; Fig. 1 B, D). In
the D2-YC striatum, 93% of D2-MSNs expressed YCnano50 andnone of D1-MSNs expressed it. Striatal cholinergic interneurons
expressed Drd2 mRNA; however, Drd2-tTA mice failed to express tTA in these cells (Tsutsui-Kimura et al., 2017), resulting in
no YCnano50 induction in cholinergic interneurons. Outside thestriatum, Drd2 mRNA positive-dopamine neurons in the midbrain expressed tTA and ⬍1% of dopamine neurons induced
YCnano50, but the direct fluorescence of YCnano50 was invisible(data not shown). Drd2 mRNA-positive neurons in the insularcortex did not express tTA and none of insular neurons induced
YCnano50 (Tsutsui-Kimura et al., 2017). Among the VLS, although there might be YCnano50 positive axons of dopamine
neurons, their levels were negligible.
To express it in D1-MSNs, we generated triple-transgenic mice(Pde10a2-tTA::tetO-YCnano50; Adora2a-Cre) in which Pde10a2tTA expressed tTA in both D1- and D2-MSNs and drove tTAmediated YCnano induction, but with D2-MSN-specific Cre
expression under the control of an Adora2a promoter excised tetOcassette, resulting in D1-MSN-specific YCnano50 expression (D1YC; Fig. 1C,D). YCnano50 was expressed by 98.1% of D1-MSNs andnone of D2-MSNs (Fig. 1D). Pde10a2 gene exclusively expressed
within the striatum; therefore, none of projection neurons to thestriatum expressed YCnano50 in D1-YC mice.
Although fiberoptic intracellular Ca2⫹ recording systems havebeen previously developed for the use of GCaMPs (Cui et al., 2013;
Gunaydin et al., 2014), we designed a new optical recording systemusing a ratiometric calcium indicator. Our fiber photometric system
includes one light source (435 nm LED) and two photomultipliertubes for the detection of cyan and yellow fluorescence derived from

4 • J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX

YCnano50. The compound intracellularCa2⫹ signals in the VLS were measured as aratio of yellow to cyan fluorescenceintensity.
To estimate the effective detection rangeof our assay system, we used fixed D2-YC
mouse brains. We carefully inserted the optical fiber cannula (␾ 400 ␮m) into the VLS
in a fixed coronal brain slice (⬎1.0 mm
thickness) that was put on a black shadingplate. We recorded the cyan fluorescence intensity at every sequential 100 ␮m depth
from the top to the bottom. Because we observed more than a 10% reduction of fluorescence at a distance of 600 ␮m from thebottom, we estimated that the range of detection limit was at ⬃700 ␮m depth (⬎600␮m) (n ⫽ 4 slices from 2 brains; Fig. 2B).
We counted the number of fluorescent cells
in the column (400 ␮m in diameter and 700
␮m in height) and inferred that our systemcollected compound signals from ⬃300
cells in D2-YC mice.
We observed a fluctuation of the yellow/cyan fluorescence intensity ratio (YCratio) in the VLS in both freely moving
D2-YC and D1-YC mice. The convex
wave of YC ratio corresponded to fluctuations of anti-parallel yellow (convex) andcyan (concave) fluorescence intensitiesin D2-YC mice, but we did not observe
the wave of YC ratio in control wildtype mice (Fig. 2C). The frequency ofthese YC ratios ranged mainly from 0.1
Hz to ⬍1.0 Hz, and the duration of convex waves was typically 10 –20 s (0.05–
0.1 Hz) (Fig. 2D).
To confirm whether the waves of YC ratio correspond to compound neuronal activities within the VLS, we manipulated
neuronal activity and measured expected
outcomes. We observed that the YC ratio in
the VLS was gradually diminished by sevoflurane anesthesia in D2-YC mice (Fig.
2E,F; n ⫽ 4, Pre [from ⫺90 s to ⫺30 s] vssevoflurane [from 110 s to 170 s], t(3) ⫽ 4.03,
p ⫽ 0.03, paired t test). In addition, the YC
ratio was significantly diminished after microinjection of several inhibitors of neuronal activity, such as the action potential
blocker TTX (n ⫽ 4, t(3) ⫽ 6.34, p ⫽ 0.03,
paired t test with Bonferroni correction vscontrol of HEPES-Ringer’s solution), the
GABAA receptor agonist MUS (n ⫽ 4, t(3) ⫽9.26, p ⫽ 0.008), and the mixture of the
AMPA receptor antagonist NBQX and the
NMDA receptor antagonist AP-5 (NBQX⫹ AP-5; n ⫽ 4, t(3) ⫽ 6.79, p ⫽ 0.01) (Fig.
2G). These findings indicate that the waves
of YC ratio correspond to compound neuronal activities of MSNs. Hereafter, we referto the YC ratio waves as a compound Ca2⫹
signal.

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

Figure 1. Engineering of D1- or D2-MSN-specific YCnano50 or ArchT-EGFP mice. A, Gene manipulation strategies. Top,
tetO cassette was inserted downstream of the Actb polyA signal in tetO knockin mice, but with no gene induction. Middle,
tTA-mediated gene induction. tTA binds to the tTA-dependent promoter (tetO) and transactivates the transcription of
cDNA in double-transgenic mice. Bottom, The tetO cassette was removed by crossing with Cre mice, yielding Cre recombination. tetO, Tetracycline operator; tTA, tetracycline-controlled transcriptional activator; pA, polyadenylation signal; loxP,
locus of X-over P1. B, Strategy for the generation of D2-cDNA (Drd2-tTA::tetO double-transgenic) mice. Drd2-tTA induced
the expression of cDNA (YCnano50 or ArchT-EGFP) in D2-MSNs, but not in D1-MSNs. C, Strategy for the generation of
D1-cDNA (Pde10a2-tTA::tetO-cDNA; Adora2a-Cre triple-transgenic) mice. Pde10a2-tTA induced the expression of cDNA (YC
nano50 or ArchT-EGFP) in D1-MSNs, but not in D2-MSNs by Cre-mediated removal of tetO cassette. D, Left panels,
YCnano50 expression in the striatum of adult D2-YC mice. Green represents GFP indirect fluorescence of YCnano50 (left).
Blue and red represent D1 and D2 receptors, respectively (middle). Right, Merged image represents that YCnano50 was
selectively expressed in D2-MSNs. Right panels, YCnano50 expression in the striatum of adult D1-YC mice. Green represents
GFP fluorescence of YCnano50 (left). Red and blue represent D1 and A2a receptor, respectively (middle). Right, Merged
image represents that YCnano50 was selectively expressed in D1-MSNs.

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX • 5

Figure 2. Optical measurement of neuronal activity in D2- and D1-MSNs in freely moving mice. A, Schematic of fiber-photometric system. Light path for fluorescence excitation and emission isthrough a single multimode fiber connected to the optical fiber cannula implanted in the VLS. The fluorescence emission is separated in two by a dichroic mirror, and cyan and yellow fluorescenceare corrected through bandpass filters and enhanced by photomultiplier tubes. B, Estimating the effective detection range of the fiberphotometry. When we carefully inserted the optical fiber
cannula into the VLS in the fixed brain slice of a D2-YC mouse put on a black shading plate, ⬎10% reduction of fluorescence was observed beyond the distance of 700 ␮m from the bottom. n ⫽ 4slices from 2 brains. C, Example of yellow/cyan fluorescence intensity ratio (YC ratio; top) and corresponding fluorescence intensity changes of yellow and cyan fluorescence (bottom) recorded in the
VLS in D2-YC (left) and wild-type control (right) mice, respectively. D, Examples of YC ratio (top) and corresponding power spectral data (bottom) in the VLS in D2-YC (left) and D1-YC (right) mice,
respectively. YC ratio was recorded from a freely moving mouse in the home cage. E, The diminishment of YC ratio in the VLS of D2-YC mice under sevoflurane anesthesia. F, The effect of sevoflurane
on YC ratio (power value) in the VLS of D2-YC mice. *p ⬍ 0.05, compared with pretreatment (paired t test). n ⫽ 4. G, The response of YC ratio (power value) in the VLS of D2-YC mice to microinjectionof HEPES-Ringer’s solution (HR), TTX, MUS, NBQX, and AP-5. *p ⬍ 0.05, compared with HR as a control (Student’s t test with Bonferroni correlation). **p ⬍ 0.01, compared with HR as a control(Student’s t test with Bonferroni correlation). n ⫽ 4 for each drug. Error bars indicate ⫾ SE.

Similar temporal patterns of VLS D2- and D1-MSN activities
during an operant task
We previously demonstrated that dysfunction of VLS D2MSNs led to decreased motivation during an operant task
(Tsutsui-Kimura et al., 2017). It remains unclear at what specific time these neurons activated during the task and whether
the counterpart of these cells in the same region (VLS D1MSNs) behaved in an opposite manner or not. To verify thetiming of targeted cell activity during a task, we chose an
FR-10 schedule as a food-incentive, operant task (Fig. 3A) and
we recorded compound Ca2⫹ signals from VLS D2- or D1MSNs separately using our optical recording setup (Fig. 3).
The position of the VLS ranged from the rostral to the caudal
part of the striatum. We selected the middle portion of the VLS
for the recording because the optogenetic ablation of the middle portion of the VLS sufficiently produced impaired motivation (Tsutsui-Kimura et al., 2017).
Apart from Ca2⫹ signal dynamics seen in a freely moving
situation, compound Ca2⫹ signals from VLS D2-MSNs exhibited a periodic pattern during the task. Transient increases ofcompound Ca2⫹ signals were observed at the initiation of
every trial (TS) when both levers were automatically presented(Fig. 3Ea; n ⫽ 12 sessions, mean Z score during 5 s just before
TS [Pre] vs the mean during each 1 s period for 5 s after TS,
paired t test with Bonferroni correction, t(11) ⫽ 3.36 – 6.92, p ⬍0.01 [red] and p ⬍ 0.05 [blue]). After TS, the level of compound Ca2⫹ signals gradually increased, reached a peak at thetime of the first LP (median value of TS-LP latency was 11.6 ⫾2.3 s) (Fig. 3Eb; Pre vs the mean during each 1 s period forrespective 5 s before and after LP, paired t test with Bonferroni
correction, t(11) ⫽ 3.59 – 4.89, p ⬍ 0.01 [red] and p ⬍ 0.05
[blue]), and then decreased by the 10th LP. The last (10th) LP
was contingent with the RW, and LP-RW duration was 10.0 ⫾1.1 s (median value). A 30 second intertrial interval (RW to
next TS) started at the last LP during which time mice collected a food pellet after RW. A Ca2⫹ surge (averaged peaktime was 4.4 s after RW) was observed when mice consumed apellet (Fig. 3Ec; Pre vs the mean during each 1 s period for 20 safter RW, paired t test with Bonferroni correction, t(11) ⫽6 • J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

Figure 3. Temporal changes of compound intracellular Ca2⫹ signals in VLS D2- and D1-MSNs during an operant task. A, Schematic illustration of the fixed ratio (FR)-10 operant task sequence.
B, Recording site of Ca2⫹ signal of D2- or D1-MSNs in the VLS (cyan-dotted region). Scale bar, 1 mm. C, D, Representative trace of compound intracellular Ca2⫹ transients in VLS D2- (C) and D1-MSNs(D), respectively. Vertical ticks indicate time stamps of TS, each LP, and RW. E, F, Temporal changes of Ca2⫹ signals in VLS D2- (E) and D1-MSNs (F ) aligned to the timing of TS (a), first LP (b), andRW (c), respectively. Top row, Color-coded compound Ca2⫹ signals in one representative session (85 trials, E; and 79 trials, F ) sorted by TS-LP latency or LP-RW duration. Middle row, Averaged
signals from the same session in the top row. Boxplot represents the timing of LP (a), RW (b), and LP (c), respectively. Bottom row, Averaged signals from all sessions of all mice (n ⫽ 12 sessions from4 mice). Black bar represents the statistical period. Blue bar represents p ⬍ 0.05. Red bar represents p ⬍ 0.01. Paired t test with Bonferroni correction: mean Z score during 5 s just before TS vs thatduring each 1 s period for 5 s after TS (a), respective 5 s before and after LP (b), or 20 s after RW(c). Boxplots represent the peak of Ca2⫹ signal within the statistical period. G, H, Trace of averaged
VLS D2- (G) and D1-MSN (H ) Ca2⫹ signals from all sessions in which the duration between trigger points was normalized. E, G, n ⫽ 12 sessions from 4 D2-YC mice. F, H, n ⫽ 15 sessions from 5 D1-YC
mice. Shadow areas represent ⫾ SE.

4.86 –12.09, p ⬍ 0.01 [red]). An averaged trace (n ⫽ 12 sessions) normalized by events clearly summarized the temporal
pattern of compound VLS D2-MSN Ca2⫹ signals: elevation at
the TS, gradual increase to the first LP, gradual decrease to thelast LP, and surge right after RW (Fig. 3G).
Interestingly, we observed a similar temporal pattern of compound Ca2⫹ signals in VLS D1-MSNs (Fig. 3 D, F,H ). Ca2⫹ signal
changes were observed at the time of TS (Fig. 3Fa; n ⫽ 15 ses-

sions, Pre vs the mean during each 1 s period for 5 s after TS,
paired t test with Bonferroni correction), and small and large
peaks were observed at the first LP (median value of TS-LP latency was 4.8 ⫾ 1.0 s) and after RW (median value of LP-RW
duration was 10.3 ⫾ 0.9 s), respectively (Fig. 3Fb,Fc; Pre vs themean during each 1 s period for respective 5 s before and after LP,
or 20 s after RW, paired t test with Bonferroni correction, t(14) ⫽3.22– 6.70, p ⬍ 0.01 [red] and p ⬍ 0.05 [blue]). The shape of theNatsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX • 7

D1-MSN Ca2⫹ surge seen after RW (averaged peak time was 6.2 s after RW) was
different from that of D2-MSNs: the period of Ca2⫹ surge of D1-MSNs (10.7 ⫾1.4 s, n ⫽ 15 sessions) was longer than thatof D2-MSNs (9.1 ⫾ 1.8 s, n ⫽ 12 sessions)but not statistically significant (t(25) ⫽0.72, p ⫽ 0.48, Student’s t test). A normalized averaged trace (n ⫽ 15 sessions)demonstrated similar event-related Ca2⫹
dynamics in VLS D1-MSNs to those in
D2-MSNs, except for a longer Ca2⫹ surge
after RW (Fig. 3H ).
Higher D2-MSN Ca2ⴙ elevation after
TS cue correlated to a higher motivatedstate
In our FR-10 schedule, the intertrial interval (RW to next TS) was fixed, but thelatency from TS cue to first LP (TS-LP
latency; Fig. 4A, blue arrow) and the duration of 10 LPs (LP-RW duration; Fig.
4A, red arrow) varied in trials. These periods can be considered as indices ofmotivation; mice with higher motivation displayed a shorter TS-LP latency
and shorter LP-RW duration, and viceversa. We hypothesized that trials conducted under a higher motivation would
be accompanied with a higher neuronal
activation.
To address the above, we first definedand extracted trials with relatively motivated or unmotivated conditions bysorting TS-LP latency. We selected the
top 10% of trials with short TS-LP as
motivated conditions and the bottom
10% of trials with long TS-LP as unmotivated conditions from each session(Fig. 4B), and collected trials from allsessions in terms of short and longgroups. We analyzed the degrees ofCa2⫹ elevation at different time points(a-d inFig. 4A) and compared Ca2⫹ levels among groups. We also ranked trials
with LP-RW duration, extracted shortand long groups, and compared Ca2⫹levels. We did these groupings with bothD2- and D1-MSN data.
We found that the short TS-LP groupexhibited higher D2-MSN Ca2⫹ levels
4

Figure 4. Relationship between Ca2⫹ signals of D2-MSNs or D1-MSNs in the VLS and goal-directed behavior of mice.
A, Illustration of normalized Ca2⫹ signal trace (same as Fig. 3G) with four time-points (a– d) and two behavioral parameters (blueand red arrows). B, Definition of short and long latency/duration in color-coded Ca2⫹ signals from one session consisting of 85
trials (same as Fig. 3D, E). Trials with the shortest and longest 10% were extracted and analyzed as “short” and “long” groups,
respectively. C, Comparison of Ca2⫹ levels with two behavioral parameters in D2-YC mice. Ca, Averaged Ca2⫹ levels (0.1 s) after TS
were compared between short and long groups (left). Short TS-LP latency group displayed higher Ca2⫹ elevations compared withthe long group. LP-RW duration did not influence Ca2⫹ elevation after TS (right). Cb, Averaged Ca2⫹ levels (0.1 s) before LP werecompared between short and long groups. Neither TS-LP latency nor LP-RW duration affected Ca2⫹ elevation around thefirst LP. Cc, Peak Ca2⫹ levels after RW were compared between both groups. None of parameters influenced peak Ca2⫹levels after RW. Cd, Peak Ca2⫹ levels after previous RW werecompared between both groups. None of parameters influenced levels of previous Ca2⫹ surges. Line graphs represent
averaged latency/duration in short and long groups. Top, a– c.
Bottom, d. D, Same analyses in D1-YC mice. *p ⬍ 0.05 (pairedt test). **p ⬍ 0.01 (paired t test). n ⫽ 12 sessions from 4
D2-YC mice; n ⫽ 15 sessions from 5 D1-YC mice. Error bars
indicate ⫹ SE.

8 • J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX

just after TS compared with the long TS-LP group (Fig. 4Ca;n ⫽ 12 sessions, t(11) ⫽ 3.68, p ⫽ 0.05, paired t test), suggestingthat the degree of Ca2⫹ elevation after the TS cue correlated to
the level of motivation. From other comparisons (short vslong latency/duration at different time points), we did not findany significant differences. Among these, the following negative data should be noted: (1) Time lengths of TS-LP latency
and LP-RW duration did not correlate to Ca2⫹ levels at thefirst LP (time b). (2) Time lengths of TS-LP latency and leverpressing did not correlate to Ca2⫹ levels following reward collection (time c). (3) Ca2⫹ levels at reward collection (time d)
did not correlate to either time length related to future events.
Short TS-LP and short LP-RW groups exhibited higher
D1-MSN Ca2⫹ levels just after TS compared with long groups,
but there was no significant difference (Fig. 4Da; n ⫽ 15 sessions, t(14) ⫽ 1.10, p ⫽ 0.10, paired t test). Time lengths of
TS-LP latency and LP-RW duration did not correlate to D1MSN Ca2⫹ levels at the first LP (time b), at the time following
reward collection (time c), or at the time of previous reward
collection (time d).
Temporally specific optogenetic inhibition of
D2- and D1-MSNs in the VLS induces similar effects togoal-directed behavior
To examine the causal relationship between the event-related
activities of D2- or D1-MSNs and the goal-directed behavior ofmice, we used an optogenetic inhibition of VLS D2- or D1-MSNs
and evaluated the effect on motivated behaviors. In this experiment, we used the PR schedule task (Richardson and Roberts,
1996) to address effort-related motivation. We used transgenic
mice in which D2-MSNs or D1-MSNs expressed a light-driven
proton pump (archaerhodopsin [ArchT]; Fig. 1), referred to as
D2-ArchT and D1-ArchT, respectively, and we targeted thesecells in the bilateral VLS (Fig. 5A). After PR training, surgery, andretraining, we conducted the PR task combined with event targeted illumination.
We first selected the TS. D2- and D1-ArchT mice were illuminated with yellow (sensitive to ArchT) or blue (insensitive) lightfor 2 s at the time of TS in every trial of the PR schedule (Fig. 5B)(Tsutsui-Kimura et al., 2017). With yellow light illumination at
the TS, both D2- and D1-ArchT mice showed quantitative reductions in goal-directed behavior in terms of breakpoint (1) (n ⫽ 12
sessions, t(11) ⫽ 3.61, p ⬍ 0.05 for D2-ArchT; n ⫽ 8 sessions,t(7) ⫽ 3.83, p ⬍ 0.05 for D1-ArchT, paired t test), first LP latency(2) (t(11) ⫽ 2.90, p ⬍ 0.05 for D2-ArchT; t(7) ⫽ 3.86, p ⬍ 0.05 forD1-ArchT), and time spent to complete the PR (3) (t(11) ⫽ 2.72,
p ⬍ 0.05 for D2-ArchT; t(7) ⫽ 2.70, p ⬍ 0.05 for D1-ArchT), but
no significant differences in active LP rate (4) (t(11) ⫽ 1.90, p ⫽0.08 for D2-ArchT; t(7) ⫽ 1.97, p ⫽ 0.09 for D1-ArchT), percentage inactive LPs (5) (t(11) ⫽ 0.57, p ⫽ 0.58 for D2-ArchT; t(7) ⫽1.24, p ⫽ 0.24 for D1-ArchT), or collect reward latency (6)(t(11) ⫽ 0.89, p ⫽ 0.40 for D2-ArchT; t(7) ⫽ 1.84, p ⫽ 0.10 for
D1-ArchT) compared with the blue light session (Fig. 5D). These
data indicate that VLS D2- and D1-MSN inhibition immediately
after the TS cue resulted in decreased motivation without affecting cognition and appetite, and more importantly, both TSrelated VLS D2- and D1-MSN activation encodes motivatedbehavior.
We next tested the effect of inhibition at the LP time (Fig. 5C).
In D2-ArchT mice, 2 s of VLS D2-MSN inhibition after the first
LP did not change any index of goal-directed behavior (1– 6:
breakpoint: n ⫽ 12 sessions, t(11) ⫽ 0.31, p ⫽ 0.76, first LP latency: t(11) ⫽ 0.71, p ⫽ 0.49, time spent to complete the PR:

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivationt(11) ⫽ 1.68, p ⫽ 0.12, active LP rate: t(11) ⫽ 0.32, p ⫽ 0.76,
percentage inactive LPs: t(11) ⫽ 0.03, p ⫽ 0.97, collect reward
latency: t(11) ⫽ 0.01, p ⫽ 0.99). Whereas in D1-ArchT mice, VLS
D1-MSN inhibition at the same time point reduced goal-directedbehavior in terms of breakpoint (1) (n ⫽ 8 sessions, t(7) ⫽ 3.56,
p ⬍ 0.05) and active LP rate (4) (t(7) ⫽ 4.26, p ⬍ 0.05), but not infirst LP latency (2) (t(7) ⫽ 1.96, p ⫽ 0.09), time spent to complete
the PR (3) (t(7) ⫽ 1.93, p ⫽ 0.10), percentage inactive LPs (5)(t(7) ⫽ 0.42, p ⫽ 0.69), and collect reward latency (6) (t(7) ⫽ 1.91,
p ⫽ 0.10) did not significantly change (Fig. 5E). These data indicate that LP-related VLS D1-MSN activation encodes motivatedbehavior but VLS D2-MSN activation does not. In addition, thedata indicate that the functional readout was distinct between
VLS D1 versus D2-MSN activation at the LP time point, but thereadout was not exactly opposite.

Discussion
The present study demonstrates concurrent Ca2⫹ activity
within D1- and D2-MSNs in the VLS during a food-incentive,
goal-directed behavior. Based on this observation, we conducted a timing- and cell-type-specific optical inhibition ofeach type of MSN. We showed that the inhibition of both typesof MSN at the TS diminished motivated behavior. These results indicate that both D1- and D2-MSN subpopulations in
the VLS positively encode food-incentive behavior and do notsupport the opposing functional roles of D1 versus D2-MSNs.
One caveat is that previous other motivation related studies
were conducted in the NAc (i.e., the ventromedial striatum);
therefore, it is possible that our findings are simply due to thepositional difference.
When we discuss “opposing roles” in general, we have to consider the activity of each population on one hand, and the readout
after a population-specific manipulation on the other hand. In
cases of psychostimulant-related behaviors, the application of a
psychostimulant (cocaine) results in increased protein kinase A(PKA) activity in VS D1-MSNs and decreased PKA activity in VS
D2-MSNs (Goto et al., 2015), demonstrating a clear opposite
reaction. A recent study using Ca2⫹ fiberphotometry also demonstrated an opposite reaction during the application of cocaine(Calipari et al., 2016). Enforced activation of D1-MSNs increases
drug-incentive behavior, whereas activation of D2-MSNs decreases it (Hikida et al., 2010; Lobo et al., 2010; Lobo and Nestler,2011) and vice versa (Augur et al., 2016), supporting the notionof an antagonistic readout. Accordingly, the scenario of drugincentive behavior supports opposing roles of VS D1- versus
D2-MSNs.
Our study on food-incentive, goal-directed behavior does notsupport opposing roles of VS D1- versus D2-MSNs in either activity or readout. In terms of activity, we measured compound
intracellular Ca2⫹ signals from each population, whose signals
corresponded to neuronal activities (Cui et al., 2013), and foundconcurrent changes of compound intracellular Ca2⫹ signals inboth populations during behavior. It is known that extracellular
dopamine in the VS increases at the time of operant cue exposureand response in food-incentive operant behaviors (Roitman etal., 2004). These results with our findings imply that dopamine
input to either population is not enough to induce a detectable,
opposing change of compound intracellular Ca2⫹ signal levels. In
this case, we have to note that the increase of extracellular dopamine is the response or the outcome to the natural reward, andthe degree of dopamine increase would be more physiologicalthan that in drug-related behaviors including psychostimulant
exposure and drug seeking in previously exposed situation. Thus,

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX • 9

Figure 5. Effect of optogenetic inhibition of D2-MSNs or D1-MSNs in the VLS on goal-directed behavior in mice. A, Illumination site in the bilateral VLS. Scale bar, 1 mm. B, C, The timing of
illumination in the PR task. B, Yellow or blue light was applied at the time of every TS with the lever presentation for 2 s (yellow bar). C, Yellow or blue light was applied at the time of LP behavior
for 2 s (yellow bar). D, The effect of yellow-light illumination at the time of TS (B) in D2-ArchT (top row) and D1-ArchT (bottom row) mice, respectively. Yellow-light illumination decreased breakpoint(1), prolonged first LP latency (2), and time spent to complete the PR (3) in both D2 and D1-ArchT mice, whereas it did not affect the active LPs (4), percentage inactive LPs (5), or collect reward latency(6). E, The effect of yellow-light illumination at the timing of LP (C) in D2-ArchT (top row) and D1-ArchT (bottom row) mice, respectively. Yellow-light illumination had no effect in D2-ArchT mice,
whereas it decreased breakpoint (1) and active LPs (4) in D1-ArchT mice. Other behavioral parameters were not affected by yellow-light illumination in D1-ArchT mice. *p ⬍ 0.05, compared withthe blue light control (paired t test). n ⫽ 12 sessions from 6 D2-ArchT mice; n ⫽ 8 sessions from 3 D1-ArchT mice. Error bars indicate ⫹ SE.

10 • J. Neurosci., Month XX, 2017 • 37(XX):XXXX–XXXX

it is possible that the pathological massive dopamine release seenin drug-related behavior would induce opposing Ca2⫹ signals ineither population.
What triggered changes in compound intracellular Ca2⫹
signal levels? We assume that excitatory/inhibitory inputs to
MSNs dominantly control Ca2⫹ levels. Glutamatergic neurons
from the cortex and thalamus innervate both D1- and D2MSNs in the striatum. In particular, VLS MSNs receive glutamatergic afferents from the insular cortex (Berendse et al.,1992). The insular cortex activates at the time of cue presentation and the blockade of this activation causes decreased
food-incentive behavior (Kusumoto-Yoshida et al., 2015).
This report may mean that concurrent D1- and D2-MSN activation is mediated by insular cortex activation during behavior. We also have to consider other excitatory inputs (e.g.,
projections from the thalamus) as well as inhibitory inputs(e.g., projections from striatal interneurons) as candidate factors that determine compound intracellular Ca2⫹ signal levels.
However, there has not been evidence that excitatory/inhibitory inputs to MSNs are biased to D1- or D2-MSNs. Indeed,
concurrent activities of D1- and D2-MSNs during operanttasks are accepted even in the DS (Cui et al., 2013; Isomura etal., 2013; Tecuapetla et al., 2014, 2016). Together, theories of
opposition regarding dopamine signals (e.g., PKA) are evident, but opposing neuronal activity (e.g., electrical membrane properties) no longer appears appropriate in terms ofdescribing D1- and D2-MSN function.
Regarding opposing readouts, our data do not correspond tostudies using psychostimulant exposure (Creed et al., 2016). Optogenetic inhibition revealed that the activities of both VLS
D1- and D2-MSNs at the TS cue positively encoded the foodincentive motivation, whereas at the first LP, that of only D1MSNs positively encoded the motivation (Fig. 5). We would liketo address the behavioral readout according to anatomical characteristics of the VS. Recently, Kupchik et al. (2015) showed that
50% of VP neurons receive input from NAc D1-MSNs, whereas
89% of VP neurons receive input from D2-MSNs and that 100%of VM neurons receive input from D1-MSNs and 0% of VM
neurons receive input from D2-MSNs. The VP projects to theVM; therefore, VP-projecting D1-MSNs can be regarded as anindirect pathway. The VP also sends axons outside the basal ganglia as an output nucleus. In this case, D2-MSNs can be a direct
pathway. The sharing of pathways between VS D1- and D2MSNs may attribute a common readout after a manipulation.
According to these anatomical findings and our optogenetic silencing data, we hypothesize that the neural projections from the
VS to the VP, which is composed of both D1- and D2-MSNs,
including potential collaterals of D1-MSNs (Cazorla et al., 2014),
can be responsible for promoting motivation at the TS time,
whereas the direct projection from the VS to the VM, which iscomposed of only D1-MSNs, may be responsible for promoting
motivation at the time of LP (Fig. 6). This hypothesis can explain
the roles of MSNs in timing- and pathway-specific manners infood-incentive, goal-directed behavior. One caveat is that ourtargeted region, the middle portion of the VLS, is not identical to
the NAc where Kupchik et al. (2015) studied. The VLS receives
the input from the insular cortex and projects to the VP (TsutsuiKimura et al., 2017); however, the proportion of D1-MSN to VP
projection is unknown. Another effort is needed to generalize theKupchik et al. (2015) findings in the entire VS.
In trying to propose a conceptual scheme for the interpretation of the roles of VLS MSNs in motivated behavior, it isimportant to consider the distinction among the activational

Natsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

Figure 6. Proposed encoding of VLS MSNs. Schematic illustration of basal ganglia circuitry(Kupchik et al., 2015) and proposed VLS MSN encoding. Red represents unique connections (VLS
D1-MSNs to VP, output from the VP). VLS D1-MSN to VM pathway encodes action sustainment(red shading), whereas VLS D1- and D2-MSN to VP pathway encodes action initiation(blue-shading).

aspects of motivation (Salamone, 1988). Motivation can be
defined as the activation of goal-directed behavior. It involves
a directional component that enables subjects to select a behavior leading to an optimal outcome and an activational
component that initiates and maintains the actions directed togoals (Salamone, 1988; Bailey et al., 2015). In our study, weused FR and PR schedules of lever pressing tasks as a motivational assessment. Both tasks required mice to initiate goaldirected action at various times from the TS to the first LP, andto continue lever pressing until achieving the required ratio.
Thus, the period from the TS to the first LP was related toaction initiation, and the period from the first LP to RW was
related to the sustainment of goal-directed action. Interestingly, during the above periods, concurrent Ca2⫹ activities
were observed in VLS D1- and D2-MSNs (Fig. 3). Unlike thesecommon activity patterns, our optogenetic silencing revealed
timing- and population-specific contributions to the activational component of motivation: both D2- and D1-MSNs
encode initiation, but only D1-MSNs encode sustainment ofgoal-directed action (Fig. 5). In turn, supposing our above
hypothesis is correct, the initiation of action is encoded by the
VLS-VP mixed pathway and the sustainment of action is encoded by the VLS-VM direct pathway. This study is the first toclearly dissociate the contribution of the VLS subpopulations
to activational components of motivation, although a previous study observed dissociable patterns of neural firing in the
DS regarding action initiation and maintenance (Jin et al.,2014).
In conclusion, our data indicate that D1- and D2-MSNs in
the VLS exhibit event-related concurrent Ca2⫹ activities during
food-incentive, goal-directed behavior in mice. Both types of
MSNs in the VLS positively encode the action initiation motivational component, whereas only D1-MSNs positively encode thesustainment of motivation. These results allow us to reconsider
the conventional thinking of opposing functions of MSNs in the
striatum, and propose the possibility of new pathway-specificNatsubori, Tsutsui-Kimura et al. • D1- and D2-MSNs Behave Likewise in Motivation

coding for action initiation and sustainment of motivational
processes.

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