Neuron

ArticleWhole-Brain Mapping of Direct Inputs
to Midbrain Dopamine Neurons
Mitsuko Watabe-Uchida,1 Lisa Zhu,1 Sachie K. Ogawa,1 Archana Vamanrao,1 and Naoshige Uchida1,*
1Center for Brain Science, Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA*Correspondence: uchida@mcb.harvard.edu
DOI 10.1016/j.neuron.2012.03.017

SUMMARY

Recent studies indicate that dopamine neurons in
the ventral tegmental area (VTA) and substantia nigra
pars compacta (SNc) convey distinct signals. To
explore this difference, we comprehensively identified each area’s monosynaptic inputs using therabies virus. We show that dopamine neurons inboth areas integrate inputs from a more diverse
collection of areas than previously thought, including
autonomic, motor, and somatosensory areas. SNc
and VTA dopamine neurons receive contrasting
excitatory inputs: the former from the somatosensory/motor cortex and subthalamic nucleus, which
may explain their short-latency responses to salient
events; and the latter from the lateral hypothalamus,
which may explain their involvement in value coding.
We demonstrate that neurons in the striatum thatproject directly to dopamine neurons form patches
in both the dorsal and ventral striatum, whereas
those projecting to GABAergic neurons are distributed in the matrix compartment. Neuron-typespecific connectivity lays a foundation for studying
how dopamine neurons compute outputs.

INTRODUCTION
A central goal of neuroscience is to understand brain function interms of interactions among a network of diverse types ofneurons. A critical step is to understand the inputs and outputsof a given type of neuron in an intact network. Electrophysiological and optical imaging techniques have advanced our understanding of outputs, but our progress in understanding thenature of inputs has been slow. Establishing methods to efficiently identify inputs to a given type of neuron will facilitate
our understanding of how neurons communicate.
Dopamine neurons in the ventral tegmental area (VTA) andsubstantia nigra pars compacta (SNc) play pivotal roles invarious brain functions including motivation, reinforcement
learning, and motor control (Cohen et al., 2012; Ikemoto, 2007;
Redgrave and Gurney, 2006; Schultz, 2007; Wise, 2004). Electrophysiological studies have shown that dopamine neurons
are activated phasically (100–500 ms) by unpredicted reward
or sensory cues that predict reward (Bromberg-Martin et al.,858 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

2010; Schultz et al., 1997). In contrast, they do not respond to
fully predicted reward, and their activity is transiently suppressed by negative outcomes (e.g., when a predicted reward
is omitted or the animal expects or receives negative outcomes).
Thus, dopamine neurons appear to calculate the difference
between the expected and actual reward (i.e., reward prediction
errors).
Reward prediction error may not be the only function of dopamine neurons, however. For example, several studies have suggested that dopamine neurons are activated by noxious stimuli(Brischoux et al., 2009; Joshua et al., 2008; Redgrave andGurney, 2006). Indeed, a recent study in nonhuman primatesfound at least two types of dopamine neurons, saliency coding
and value coding, that are activated and inhibited, respectively,by aversive events (Matsumoto and Hikosaka, 2009). Importantly, saliency-coding dopamine neurons were found preferentially in the dorsolateral part of the midbrain dopamine nuclei (i.e.,
mainly SNc) while reward-value-coding dopamine neurons were
found in the more ventromedial part (i.e., mainly VTA). Furthermore, responses in SNc were generally earlier than those in
VTA. These findings raise the possibility that inputs encoding
noxious stimuli or saliency specifically innervate SNc dopamine
neurons. Although efforts have been made to identify the sources of such inputs, they remain unidentified (Bromberg-Martin
et al., 2010; Coizet et al., 2010; Dommett et al., 2005; Jhouet al., 2009; Matsumoto and Hikosaka, 2007). More generally,
although the aforementioned findings indicate that dopamine
neurons integrate diverse kinds of information, the mechanisms
by which the firing of dopamine neurons is regulated in a behavioral context remain largely unknown (Bromberg-Martin et al.,2010; Lee and Tepper, 2009; Sesack and Grace, 2010).
A critical step toward understanding the aforementioned
questions is to know what kinds of inputs dopamine neurons in
the VTA and SNc receive. Circuit-tracing experiments havebeen performed to address this question (Geisler et al., 2007;
Geisler and Zahm, 2005; Graybiel and Ragsdale, 1979; Phillipson, 1979; Sesack and Grace, 2010; Swanson, 2000; Zahm
et al., 2011), but limitations of conventional tracing methods
have hampered a full understanding of inputs to dopamine
neurons. For example, conventional tracing cannot distinguish between dopaminergic and nondopaminergic cells (e.g.,
GABAergic neurons). Furthermore, SNc dopamine neurons
form a thin layer and are heavily interconnected with the neighboring substantia nigra pars reticulata (SNr), and conventional
tracing might label inputs to SNr in addition to SNc. Finally, there
are many axons of passage through or near these structures,
which may take up tracers nonspecifically. Thus, it is unclear

NeuronDirect Inputs to Midbrain Dopamine Neurons

whether neurons in a given area project to VTA or SNc andwhether they actually make synaptic contacts with dopamine
neurons.
Electron microscopy can resolve several of these issues (e.g.,
Bolam and Smith, 1990; Carr and Sesack, 2000; Omelchenko
et al., 2009; Omelchenko and Sesack, 2010; Somogyi et al.,1981), but this technique is not suitable for a comprehensive
identification of inputs. Another approach is to combine anatomical methods with electrophysiological or optogenetic techniques (Chuhma et al., 2011; Collingridge and Davies, 1981;
Grace and Bunney, 1985; Lee and Tepper, 2009; Xia et al.,2011). However, the validity of this approach has been called
into question after these studies (Chuhma et al., 2011; Xiaet al., 2011) failed to demonstrate well-accepted direct projections from striatum to dopamine neurons in the VTA and SNc(Bolam and Smith, 1990; Collingridge and Davies, 1981; Grace
and Bunney, 1985; Lee and Tepper, 2009; Somogyi et al., 1981).
To resolve these methodological issues, we combined the
Cre/loxP gene expression system (Gong et al., 2007) withrabies-virus-based transsynaptic retrograde tracing (Wickersham et al., 2007b) to comprehensively identify monosynaptic
inputs to a genetically defined neural population (Haubensak
et al., 2010; Miyamichi et al., 2011; Wall et al., 2010). This technique allowed us to identify the sources of monosynaptic inputs
to VTA and SNc dopamine neurons in the entire brain. We thenasked whether we can identify different sources of candidate
excitatory inputs that may account for rapid activation of SNc
dopamine neurons by salient events, in contrast to activationof VTA dopamine neurons by reward values, and whether there
are indeed direct projections from the striatum to dopamine
neurons. We show that SNc dopamine neurons receive relatively
strong excitatory inputs from the somatosensory and motor
cortices, as well as subthalamic nucleus (STh), whereas VTA
dopamine neurons receive strong inputs from the lateral hypothalamus (LH). Furthermore, we show that neurons in the striatum project directly to VTA and SNc dopamine neurons, forming‘‘patch’’ compartments in both the ventral striatum (VS) anddorsal striatum (DS).
RESULTS
Visualization of Monosynaptic Inputs to a GeneticallyDefined Population of Neurons Using Rabies Virus andcre/loxP Recombination System
We used the modified rabies virus SADDG-GFP(EnvA), which
has two key modifications that determine the specificity of its
initial infection and transsynaptic spread (Wickersham et al.,2007b). First, this virus is pseudotyped with an avian virus envelope protein (EnvA) and therefore cannot infect mammalian cells.
In mammalian brains, the initial infection thus occurs only whena host neuron is engineered to express a cognate receptor(e.g., TVA). Second, the gene for the rabies virus envelope glycoprotein (RG), which is required for transsynaptic spread, is replaced by the gene for a fluorescent marker (enhanced green
fluorescent protein; EGFP). Transsynaptic transfer thus occurs
only from neurons that exogenously express RG.
Our strategy was to express TVA and RG only in a geneticallydefined cell population (Haubensak et al., 2010; Miyamichi et al.,2011; Wall et al., 2010). Thus, we generated adeno-associatedviruses (AAVs) that express either TVA or RG (AAV5-FLEXTVA-mCherry and AAV8-FLEX-RG, respectively). We used thetransmembrane type of the TVA receptor protein (TVA950) togenerate a fusion protein with a red fluorescent protein(mCherry). TVA and RG proteins were expressed under thecontrol of a high-specificity Cre/loxP recombination system (amodified Flex switch) and different promoters (EF-1a and CAG,
respectively) (Figure 1A).
To visualize monosynaptic inputs to dopamine neurons, we
injected AAV5-FLEX-TVA-mCherry and AAV8-FLEX-RG stereotaxically into VTA or SNc of transgenic mice that express Cre in
dopamine neurons (dopamine transporter-Cre or DAT-Cre)(Bäckman et al., 2006). After 14 days, SADDG-GFP(EnvA) was
injected into the same area and the brain was analyzed after
7 days (Figure 1B). The whole brain was sectioned at 100 mm,
and every third section was processed for further analysis.
The starter cells were identified based on the coexpression of
TVA-mCherry and EGFP (Figures 1C and 1H; Figure S1 available online). Coexpressing neurons were found only in the injected area, while EGFP-positive neurons outside the injectedarea did not express TVA-mCherry, indicating that they aretranssynaptically labeled neurons. We found a large numberof these transsynaptically labeled neurons (Figure 1D;6.1 3
103 ± 4.2 3 103 neurons; mean ± SD, n = 12 mice), although
the number of labeled neurons varied across animals, in partdue to different injection volumes (Figures 1E and 1F). Nevertheless, the numbers of transsynaptically labeled neurons wereroughly proportional to the numbers of starter neurons(Figure 1G).
To examine the specificity of tracing, we first repeated theaforementioned procedure in mice with no Cre expression (Figure 1D, right). This resulted in much smaller numbers of EGFPlabeled neurons both outside and near the injection site (87 ±61 neurons outside VTA or SNc and 31 ± 21 neurons in VTA
or SNc; mean ± SD) compared to the aforementioned result.
This small degree of labeling was likely due to inevitablecontamination of the unpseudotyped rabies virus that occurred
during the viral preparation. Note that these numbers should be
regarded as the upper bounds of nonspecific labeling, as someof the labeled neurons are likely dopamine neurons and their
inputs. Next, to examine the specificity of the initial infection
and to verify that the transsynaptic spread is under the tightcontrol of RG expression, we repeated the experiment without
AAV8-FLEX-RG in DAT-Cre mice. A larger number of labeledneurons were found at the injection site, and 97% of thelabeled neurons coexpressed tyrosine hydroxylase, a marker
for dopamine neurons. Furthermore, very few neurons were
found outside the injection site. This result confirms that the
TVA proteins were expressed specifically in Cre-expressing
neurons and that transsynaptic spread did not occur without
RG protein. Together, these results suggest that labeledneurons outside the injection site represent monosynaptic
inputs to dopamine neurons, while the injection site contains
a small number of nonspecifically labeled neurons that contributed very little labeling outside the injection site (1.3%). In thefollowing analysis, we will focus on labeled neurons outside theinjection site.
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NeuronDirect Inputs to Midbrain Dopamine Neurons

AC

AAV-FLEX-TVA-mCherry

EF-1

TVA950-mCherry

WPRE

mCherry

AAV-FLEX-RG

CAG

RG

WPRE

SAD G-EGFP(EnvA)(Rabies virus)

EGFP

lox2272

VTA-targeted

SNc-targeted

loxP

EGFP

B
Day 1
AAV-FLEX-TVA-mCherry
AAV-FLEX-RG

DAT-CreTH

Day 14
SAD G-EGFP(EnvA)

VTA/SNc
Input

D

SNc

SNc

VTA

VTA

Day 21
Observe

Starter

SNc-targeted

VTA-targeted
contraControl

ipsi

Nissl
EGFP

v009
v008
v001
v007
v010
v004
v003
s002
s001
s004
s003
s006

Cell count (Input)

v009
v008
v001
v007
v010
v004
v003
s002
s001
s004
s003
s006

Input = 7.0 x Starter

G
10000

5000

0
0 500 1000 1500
Cell count (Starter)

0

5000
10000
Cell count (Input)

H

Mouse

F

Mouse

Mouse

E

r: 0 86-3(P<10 )

01000 2000
Cell count (Starter)

VTA

RRF

SNc

v009
v008
v001
v007
v010
v004
v003
s002
s001
s004
s003
s006

0

20

40

60

80 100Starter (%)

Figure 1. Identification of Monosynaptic Inputs to Midbrain Dopamine Neurons Using the Rabies Virus and Cre-Transgenic Mice(A) Recombinant AAV strains and rabies virus.
(B) Experimental design.
(C) Characterization of the injection site at the ventral midbrain. Scale bar, 0.5 mm.
(D) Transsynaptically labeled neurons at the forebrain areas. The location of the coronal sections is indicated by the black arrowhead in (B). Left, v004; Middle,s002; Right, control with wild-type mouse. Scale bar, 1 mm. In all images, the right side corresponds to the side ipsilateral to the injected side.
(E) Numbers of starter neurons.
(F) Numbers of transsynaptically labeled neurons (‘‘input neurons’’).
(G) Relationship between numbers of starter and input neurons.
(H) Proportions of labeled neurons in each of the midbrain dopamine nuclei. RRF, retrorubural field (A8).

Distinct Areas Project to Dopamine Neurons in VTA
and SNc
Figure 2 shows the sections obtained from two mice that wereadministered the selective injections into VTA and SNc (v001
and s003; see Figure 1H). Using custom software, we identified
860 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

anatomical areas based on a standard mouse atlas (Franklin and
Paxinos, 2008) using fluorescent Nissl staining; the location ofeach neuron was registered on the anatomical coordinate. We
then counted the number of neurons in each area. To correct
for the variability in the total number of neurons, the numbers

NeuronDirect Inputs to Midbrain Dopamine Neurons

Figure 2. Distinct Areas Project to VTA and SNc Dopamine Neurons
Series of coronal sections for VTA- and SNc-targeted cases (v001 and s003, respectively). Only the side ipsilateral to the injection site is shown. Scale bar, 1 mm.
LO, lateral orbital cortex; M1, primary motor cortex; M2, secondary motor cortex; S1, primary somatosensory cortex; Acb, nucleus accumbens; Tu, olfactory
tubercle; DS, dorsal striatum; VP, ventral pallidum; Pa, paraventricular hypothalamic nucleus; EA, extended amygdala; GP, globus pallidus; LH, lateral hypothalamus; Ce, central nucleus of the amygdala; PSTh, parasubthalamic nucleus; STh, subthalamic nucleus.

were normalized by the total number of input neurons (Figure 3,
left; Figure S2). We further computed the density of labeledneurons in each area by dividing the number by the area (mm2)
on each section (Figure 3, right). For each group, four animals
that had preferential injections into VTA or SNc were used togenerate Figure 3 (v009, v001, v010, and v004 for VTA; ands001, s004, s003, and s006 for SNc; note that v008 and v007
were not included because these mice had a small number oflabeled neurons). Consistent results were obtained even when
we restricted our analysis to three animals with higher specificity
for each group. Furthermore, we have verified that the patterns oflabeling are similar at 5 days (n = 3 mice, VTA) and 9 days (n = 2
mice, VTA) after the injection of SADDG-GFP(EnvA) compared tothe main data set obtained at 7 days after injection (Pearson
correlations for the mean numbers of labeled neurons acrossareas were r = 0.82 and 0.95 between 5 versus 7 days and 7
versus 9 days, respectively; p < 107 for both). This suggeststhat the results we report here are temporally stable and notaffected by gross cell death over time.
Basal Ganglia and Hypothalamus: Global Shift of Input
Areas
Across the whole brain, the most abundant labeling was found in
the basal ganglia (striatum and pallidum) (Figure 3). In theseareas, labeled neurons are predominantly found ipsilateral tothe injection site (e.g., Figure 1D). Both for VTA- and SNctargeted animals, labeled neurons formed continuous bands
that ran from the striatum to specific hypothalamic areas (Figure 2). The densely labeled bands for VTA and SNc dopamine
neurons showed rough segregation such that the areas projecting to SNc dopamine neurons were found in the more dorsal
and lateral parts in this continuum, relative to those projecting
to VTA dopamine neurons (Figures 2–4). These bands often did
not reflect the boundaries of anatomically identified areas(Franklin and Paxinos, 2008), but the densely labeled regions
included various areas in the striatum and pallidum and,more posteriorly, the basal forebrain and hypothalamus (Figures2–4). In terms of numbers, the most prominent labeling wasobserved in the striatum partly due to its large volume, withgreater emphasis on the ventral portion (nucleus accumbens[Acb] and olfactory tubercle [Tu]) in VTA-targeted mice and on
the DS in SNc-targeted mice. In the amygdala, the central
nucleus of the amygdala (Ce; in particular, the lateral central
nucleus of amygdala [CeL]) was found to project to both VTA
and SNc dopamine neurons (e.g., Figures 4D and 4E) while otheramygdala regions, including the cortical amygdala, did notproject much to dopamine neurons in either area.
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NeuronDirect Inputs to Midbrain Dopamine Neurons

NumberDensity

SNc

VTA

SNc
Frontal association cortex
Dorsal peduncular cortex
Infralimbic cortex
Prelimbic cortex
Cingulate cortex
Orbital cortex, lateral
Orbital cortex, ventral/medial
Orbital cortex, dorsal
Motor cortex, primary
Motor cortex, secondary
Agranular insular cortex
Dysgranular/granular insular cortex
Somatosensory cortex
Auditory cortex
Visual cortex
Entorhinal cortex
Retrosplenial cortex
Perirhinal cortex
Ectorhinal cortex
Parietal cortex
Olfactory bulb
Anterior olfactory nucleus
Piriform cortex
Tenia tecta
Claustrium
Endopiriform nucleus
N.Accumbens, Shell
N.Accumbens, Core
Olfactory tubercle
Dorsal striatum
Ventral pallidum
Globus pallidus
IPAC
BNST
Sublenticular extended amygdala
Endopeduncular nucleus
Anterior amygdaloid area
Medial nucleus
Central nucleus
Cortical amygdaloid transition zone
Basolateral nucleus
Hippocampus (CA1, CA2, CA3)
Hippocampus (dentate gyrus)
Dorsal Subiculum
SeptumDiagonal band of Broca
Median preoptic are
Medial preoptic area
Lateral preoptic area
Suprachiasmatic nucleus
Arcuate nucleus
Magnocellular preoptic area
Anterior hypothalamic area
Paraventricular hypothalamic nucleus
Ventromedial hypothalamic nucleus
Lateral hypothalamic area
Posterior hypothalamic area
Dorsal hypothalamic area
Tuber cinereum area
Perifornical nuclues
Mammilary nucleus
Premammilary nucleus
Subthalmic nucleus
Parasubthalamic nucleus
Zona incerta
Supraoptic nucleus
Field of Forel, Prerubral field
Retrochiasmatic nucleus
Lateral habenula
Medial habenula
Parafascicular thalamic nucleus
Paraventricular thalamic nucleus
Lateral geniculate nucleus
Medial genicuulate nuclues
Mediodorsal thalamic nucleus
Central medial thalamic nucleus
Ventromedial thalamic nucleus
Microcell tegmental nucleus
Parageminal nucleus
Retrorubral nucleus
Reticular formation
Nucleus of posterior commissure
Subpeduncular tegmental nucleus
Retrorubral field
Anterior pretectal nucleus
Superior colliculus
Inferior colliculus
Periaqueductal gray
Nucleus of the optic tract
Olivary pretectal nucleus
Subtantia nigra pars reticulata
Dorsal tegmental nucleus
Anterotegmental nucleus
Red nucleus
Pontine reticular nucleus
Dorsal raphe nucleus
Median raphe nucleus
Pontine raphe nucleus
Pedunculopontine tegmental nucleus
Laterodorsal tegmental nucleus
Cuneiform nucleus
Locus coeruleus
Inferior olive
Parabrachial nucleus
Raphe magnus nucleus

LO
M1
M2
S1

AcbSh
AcbC
Tu

DS

VP

GP
IPAC
BNST
SLE

EP

Ce

DB
MPA
LPO

Pa
LH

STh
PSTh
ZI

mRt
RRf
SC
PAG
SNr

DR
PTg

PB

4000

3000

2000

1000

0

1000

400

2000

VTA
CortexOlfactory areas
Claustrium/endopiriform
AcbSh
AcbC
Tu

DS

Striatum
Pallidum

GP
IPAC
BNST
SLE

EP

Amygdala
Ce

Hippocampus/Septum
DB

Hypothalamus

MPA
LPO

Pa
LH

STh
PSTh
ZI

Thalamus/Epithalamus

Midbrain
mRt
RRf
SC
PAG
SNr

DR
PTg

Hindbrain
PB

200

0

200

400

600

Inputs / 10,000 Inputs mm2

Inputs / 10,000 Inputs

Figure 3. Comparison of Input Areas between VTA and SNc Dopamine Neurons(Left) Number of input neurons in each area. Numbers are normalized by the total number of input neurons. Mean ± SEM (n = 4 mice).
(Right) Density of input neurons in each area. Mean ± SEM. BNST, bed nucleus of stria terminalis; IPAC, interstitial nucleus of the posterior limb of the anterior
commissure; Extended amygdala and substantia innominata are included in sublenticular extended amygdala (SLE).

In pallidal areas, more ventral and medial structures suchas the ventral pallidum (VP) and sublenticular extended amygdala (EA) project predominantly to VTA dopamine neurons,
whereas more dorsal and lateral structures such as the globus
pallidus (GP) and entopeduncular nucleus (EP) project
predominantly to SNc dopamine neurons (Figures 4A–4C).
The bed nucleus of stria terminalis (BNST; in particular, its
dorsal lateral division [STLD]) projects to both VTA and SNc(Figure S6A).
862 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

From the basal forebrain and hypothalamic areas, VTA dopamine neurons receive the greatest input from the LH (including
the peduncular part of the lateral hypothalamus [PLH]). VTA
dopamine neurons also receive inputs from scattered neurons
in the diagonal band of Broca (DB) and medial and lateral preoptic areas (MPA and LPO) (Figures 3, 4A, 4D, and S3C). In theseareas, the paraventricular hypothalamic nucleus (Pa) is unique inthat it contains densely labeled neurons, for both VTA- and SNctargeted cases (Figure S6B). In contrast, in SNc-targeted cases,

NeuronDirect Inputs to Midbrain Dopamine Neurons

A

Figure 4. Input Areas for VTA and SNc
Dopamine Neurons Are Spatially Shifted in
the Basal Ganglia and Hypothalamus

D

VTA-targeted

ZI
PSTh

CeL
PLH

PLH

B

E
DS
GP

SNc-targeted

cp

STh

(A–C) Striatal and pallidal area. (A) VTA-targeted.
(B) SNc-targeted. Green, EGFP; Red, fluorescent
Nissl staining. (C) The center of mass of input
neurons. Circles indicate the centers of mass fromindividual animals. The blue and red crosses
indicate the mean ± SEM across animals (n = 4
mice for each group). PLH, peduncular part of
the LH; DS, dorsal striatum; GP, globus pallidus;
EP, entopeduncular nucleus. Right, lateral. Scalebar, 1 mm.
(D–F) Hypothalamic areas. Same conventions as(A) through (C). PSTh, parasubthalmic nucleus; ZI:
zona incerta; ZID, dorsal zona incerta; ZIV, ventral
zona incerta; cp, cortical peduncle; CeL, lateraldivision of the central nucleus of the amygdala(Ce); STh, subthalamic nucleus.

cp

EP
CeL

we generated ‘‘unrolled maps’’ of the
neocortex. For each section, we projected labeled cortical neurons on toa line running through the middle of thecortical sheet (Figures 5C, 5F–5H). The
same method was applied to a standard
atlas to generate a reference map (Figure 5I). This analysis revealed that labeled
fi
OPC
VPM
LDDM LDVL
CM
VPL
neurons are localized mostly in the rostral
stCF
half of the cortical sheet encompassing
AV
VPPC
RRe
motor, somatosensory, medial prefrontal,
Rt
DS
VL
scp
PHD
VPL
and orbitofrontal areas. In contrast, very
ZID
opt
ZIV
cp
ic
CeL
GP
sparse labeling was found in the caudal
STh
mt
VM
STIA
half, the parietal, visual, auditory, and enPH
BLA
VCI
EP
ns
ASt Latorhinal cortices. In SNc-targeted cases,
MePD
PSTh
CeL
the most dense labeling was found
DEn
BMP
PLH
CeM
AHiAL
ns
BLA
DMC
MePV
EA
in the primary and secondary motorloCenter
of
mass
f
Te
f
mfb PLH
st
CeC
DM
v009
PMCo
cortices (M1 and M2) (Figures 5B, 5E,
MTu
I BLV
v001
MeAD
BMA
PLCo
v010
5H, and S4). Somatosensory cortex (S1)
opt
VMH
Pir
v004
MeAV
sox
CxA
has moderate labeling, but, due to its
s001
ACo
s004
large size, it provides the largest number
s003
s006
of inputs among cortical areas (Figure 3).
VTA dopamine neurons receive fewer
fewer neurons were labeled in hypothalamic areas except Pa, cortical inputs than SNc dopamine neurons, but the lateral orbiwhile the STh contained a dense collection of neurons that tofrontal cortex (LO) is the major sources of cortical inputs to VTA
project preferentially to SNc dopamine neurons (Figures dopamine neurons (Figures 3, 5A, and 5G). Areas encompassing
4D–4F). Para-STh (PSTh) and zona incerta project both to VTA the medial prefrontal cortex (PrL, IL, DP, and MO) and the cinguand SNc dopamine neurons with a slight bias to VTA. Together, late cortex (Cg1 and Cg2) have moderate labeling. These resultsthese results show that VTA and SNc dopamine neurons receive demonstrate that dopamine neurons in the VTA and SNc receive
input from largely segregated, continuous ‘‘bands’’ in the basal significant numbers of cortical inputs from overlapping but
ganglia and hypothalamus. Interestingly, LH and STh provide distinct areas.
contrasting preferential inputs to VTA and SNc, respectively.
Midbrain and Hindbrain: Discrete Foci
Cortical Projections: Direct Inputs from Rostral
At more caudal regions, the intermediate layer of the superior
Neocortex
colliculus (SC) and supraoculomotor (ventrolateral) periaqueWe found significant monosynaptic input from cortical areas ductal gray (PAG) contained large numbers of labeled neurons(Figures 3 and 5). In the neocortex, labeled neurons are widely in both VTA- and SNc-targeted cases (Figure S6C). The dorsal
distributed across cortical areas (Figures 5A–5F). To visualize raphe (DR) contained the densest population of labeled neurons
the distributions of labeled neurons across entire cortical areas, both for VTA- and SNc-targeted cases, with slightly stronger
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A

DG
Bregma (mm)

VTA-targeted

2

-2

-4

LO

M2

B

0

E

M2

-2

0
2
4
Unrolled cortex (mm)

6

8

-2

0
2
4
Unrolled cortex (mm)

6

8

H

M1

SNc-targeted2
Bregma (mm)

M1
S1

0

-2

-4

C

F

M1

2

PrL

M1
Fr3
VO

LO

AI

S1

Cg2
IL

DI/GI

DP

AI

Bregma (mm)

Cg1

Cg1

MO

I

M2

M2

rf/rs
DLO
FrA
MO
LO VO
M2
PrL
Fr3
DP IL
M1
Cg1
AI
Cg2

DI/GI

0

S1
S2
PRh
Ect

Pt

-2

Pir

RS

Pir

rostoral

-4

Au
V

Ent
TeA

caudalBregma 2.34 mm
Bregma 1.4 mm

-2

0
2
4
Unrolled cortex (mm)68Figure 5. Widespread Cortical Neurons Project Directly to VTA and SNc Dopamine Neurons(A and B) Distributions of input neurons for (A) VTA- and (B) SNc dopamine neurons. LO, lateral orbital cortex; M1, primary motor cortex; M2, secondary motor
cortex. Scale bar, 1 mm.
(C) Schematic of cortical areas at the similar bregma level (2.34 mm) as defined by Franklin and Paxinos (2008). Unrolled maps in (G) and (H) were generated byprojecting labeled neurons on to the gray line that runs through the middle of the cortical sheet. Two reference points on the gray line (black dot and red cross)were defined by projecting the most dorsomedial point on the hemisphere (black cross) and the rhinal fissure (red arrowhead).
(D–F) Another example from a more posterior section (Bregma: + 1.4 mm). S1, primary somatosensory cortex. Scale bar, 1 mm.
(G) Unrolled map of input neurons obtained in one VTA-targeted animal (v004, same as in A and D). Each green dot represents a single EGFP-labeled neuron.
(H) Unrolled map of input neurons obtained in one SNc-targeted animal (s001, same as in B and E).
(I) Unrolled representation of cortical areas as defined in a standard mouse atlas (Franklin and Paxinos, 2008). AI, agranular insular cortex; AIP, agranular insularcortex, posterior part; Au, auditory cortex; Cg1, cingulate cortex area 1; Cg2, cingulate cortex area 2; DI, dysgranular insular cortex; DLO, dorsolateral orbital
cortex; DP, dorsal peduncular cortex; Ect, ectorhinal cortex; Ent, entorhinal cortex; FrA, frontal association cortex; Fr3, frontal cortex area 3; GI, granular insularcortex; IL, infralimbic cortex; MO, medial orbital cortex; PRh, perirhinal cortex; PrL, prelimbic cortex; Pt, parietal cortex; RS, retrosplenial cortex; S2, secondary
somatosensory cortex; TeA, temporal association cortex; V, visual cortex; VO, ventral orbital cortex.

projections to VTA (Figure S6D; also see Figure 3). The pedunculotegmental nucleus (PTg) and cuneiform nucleus (CnF) preferentially project to SNc dopamine neurons, whereas laterodorsal
tegmental nucleus (LDTg) preferentially projects to VTA dopamine neurons (Figure S6D). The parabrachial nucleus (PB),
both ipsilateral and contralateral to the injection side, projects
to both VTA and SNc dopamine neurons (Figure S6E). We also
864 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

found that cerebellar nuclei project to dopamine neurons(Figure S6F).
The aforementioned results are, to a large degree, consistent
with previous results using conventional tracers (Geisler andZahm, 2005) but differ in some critical ways. For example,
some areas such as the septum and mHb were not labeledheavily in our experiment, despite being strongly labeled inNeuronDirect Inputs to Midbrain Dopamine Neurons

previous experiments involving injection of a retrograde tracer(Fluoro-gold) in VTA (Geisler and Zahm, 2005). Furthermore,even in the areas that were labeled both in our and in otherprevious experiments, our methods resulted in labeling of
more specific subsets of neurons (see below). To test whether
these differences are due to the greater specificity of ourlabeling methods, we performed a control experiment usingrabies virus that was not pseudotyped with EnvA but still lacks
RG (SADDG-GFP) (therefore, this virus can infect mammaliancells but cannot spread transsynaptically). In these experiments, injection of the virus into VTA resulted in a significant
number of retrogradely labeled neurons in the septum andmHb (Figures S3A, S3B, S3D, and S3E). Furthermore, in the
hypothalamus, labeled neurons were scattered widely with
the nonpseudotyped virus, although the pseudotyped virus
labeled more confined populations, resulting in densely labeled
Pa and LH surrounded by largely negative areas (Figures S3C
and S3F). This implies that previous results may be explainedby inputs to nondopaminergic neurons in (or near) the VTA
and SNc.
In short, we demonstrate various connections that have been
largely overlooked in previous studies (e.g., M1, M2, S1, andSTh). Furthermore, these results allowed for comprehensive
and direct comparisons of the inputs to VTA and SNc dopamine
neurons.
Specific Populations of Striatal Neurons Project to
Dopaminergic and GABAergic Neurons in SN
The aforementioned observation that a large number of striatal
neurons project directly to dopamine neurons appears tocontradict recent optogenetic studies indicating that striatal
neurons form synapses almost exclusively on to nondopaminergic neurons (presumed GABAergic neurons) in VTA or SN(Chuhma et al., 2011; Xia et al., 2011). To address this issue,
we performed transsynaptic tracing from GABAergic neurons
in the SN using transgenic mice that express Cre in
GABAergic neurons (vesicular GABA transporter-Cre or Vgatires-Cre) (Vong et al., 2011).
The DS is divided into subregions, so-called patch and matrix
compartments, that can be defined by the expression of molecular markers such as calbindin D-28k (Gerfen, 1992; Graybiel,1990). Previous studies have suggested that the medium spiny
neurons in the patch compartments project to SNc while thosein the matrix project to SNr (Fujiyama et al., 2011; Gerfen,
1984), although this idea was later cast into doubt (Lévesqueand Parent, 2005). More importantly, cell-type specificity oftarget neurons has not been demonstrated. We therefore sought
to test the hypothesis that the patch and matrix separately
project to dopaminergic and GABAergic neurons, respectively.
We reasoned that, given the close proximity of dopaminergic
and GABAergic neurons in SN, such separation would support
the specificity of our transsynaptic tracing.
A closer look at the distribution of labeled neurons in the
striatum showed that neurons labeled in DAT-Cre mice tended
to form clusters (Figure 6A). These clusters were found in areasthat correspond to the patches (including the ‘‘subcallosalstreak’’), defined by low calbindin D-28k levels (Figures 6B
and 6C), although the boundary of patches and matrices is

not always clear and some labeled neurons were observed
outside of the boundary. In contrast, neurons labeled in
Vgat-ires-Cre mice showed little clustering and were found in
the matrix defined by higher calbindin D-28k levels (Figures
6E–6G). Quantification of fluorescent levels in cell bodies
showed that most of the neurons projecting to dopamine
neurons expressed calbindin D-28k to a much lower degree,compared to neurons projecting to GABAergic neurons(Figure 6I).
Furthermore, we found that labeled neurons in the two
conditions showed different morphologies (Figures 6D, 6H,
6J, and 6K). In neurons projecting to GABAergic neurons,
dendrites spread radially outward. In contrast, in neurons projecting to dopamine neurons, dendrites curved and coursed
circuitously or turned inward toward the soma (Figure 6K).
Furthermore, spines of inputs to GABAergic neurons were
evenly spaced and were of similar size. In contrast, inputs to
dopamine neurons had uneven spines and varicosities, andtheir dendrites were irregular in contour (Figures 6D and 6H,
inset). These results suggest that, whereas neurons projecting
to GABAergic neurons are consistent with typical medium
spiny neurons, neurons projecting to dopaminergic neurons
have significantly different morphologies. We make two
conclusions from these data: First, striatal neurons do projectmonosynaptically to dopamine neurons; and second, our technique is capable of revealing exquisite, cell-type-specificconnectivity.
Dopamine-Projecting Neurons in the Acb Form Patches
Whereas SNc dopamine neurons receive the most input from theDS, VTA dopamine neurons receive the most input from the Acb(Figure 3). Although heterogeneity of the Acb was reported previously with different molecular markers (Zahm and Brog, 1992),
a patch/matrix organization has not been documented consistently. We found that neurons that project to dopamine neurons
form patches in the VS, albeit much larger than the patches
found in the DS (Figure 7). These ‘‘ventral patches’’ containextremely dense groups of labeled neurons (Figure 7A). Stainingof calbindin D-28k showed that EGFP-positive neurons were
found preferentially where calbindin D-28k expressions arelower, although dopamine-neuron-projecting patches were
smaller than areas defined by weak staining of calbindin D-28k(Figures 7B–7D). Comparison across animals indicates stereotypical patterns of dopamine neuron-projecting patches (Figures
7E–7J; Figure S5). For this, we first identified regions with highdensity of labeled neurons (‘‘predicted patches’’) using four offive animals tested (v009, v001, v010, v004, and v003). In the
one remaining animal, we then obtained the proportion oflabeled neurons that fell into the contour of the predicted
patches. This proportion was then compared against that expected from a random distribution (i.e., percentage of the Acb
contained within the predicted contours). This analysis showedthat neurons tended to localize within the contours obtained
from other animals (Figure 7J; p < 0.02, paired t test). These
results support the idea that Acb neurons indeed project todopamine neurons and that most of these neurons are clusteredin stereotypical locations, or ‘‘ventral patches,’’ which were overlooked in previous studies.
Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc. 865

NeuronDirect Inputs to Midbrain Dopamine Neurons

B

Starter: Dopamine neurons

A

Figure 6. Dopamine and GABA neurons in
SN Receive Inputs from Striatal Neurons in
Patch and Matrix, Respectively

DDS
calbindin

C

calbindin
EGFP

calbindin
EGFPF

Starter: GABAergic neurons

E

H

DS

G

I

* P<10

-13

J

K
15

0.8
0.6
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* P<0.05
Radial distance/dendritic length1Soma diameter (µm)

Calbindin immunofluorescence(Normalized intensity)

EGFP

10

5

1
d
dendrite

0.8(A) Distribution of neurons projecting to SNc
dopamine neurons. A low-magnification view ofthe dorsal striatum (DS) in a DAT-Cre mouse. Red,
calbindin D-28k immunostaining. Green, EGFP
(input neurons). Subcallosal streak is indicated byan arrow. Representative patches are indicated byarrowheads.
(B) Higher magnification view stained for calbindin
D-28k.
(C) Higher magnification view showing the locations of input neurons with respect to calbindin
D-28k staining.
(D) Morphology of a labeled neuron. The inset
shows a high-magnification view of a dendrite.
(E–H) Distribution and morphology of neurons
projecting to SN GABAergic neurons. The results
were obtained using Vgat-ires-Cre mice. Scalebars, 1 mm in (E), 80 mm in (G), and 20 mm in (H).
(I) Quantification of calbindin D-28k expression
levels. p < 1013, t test (n = 126, 68 neurons). In box
plots, the central mark represents the median, andthe edges of the box are the 25th and 75th
percentiles.
(J) Quantification of the cell body size (diameter).
p > 0.05, t test (n = 23, 25 neurons).
(K) Quantification of the complexity of thedendrites. r, radial distance (straight line distance
between the cell body and the tip of dendrites);d, dendritic length. p < 0.03, t test (n = 20, 20
neurons).

somar
r: radial distanced: dendritic length

0.6High Specificity of Transsynaptic
Tracing by Cre-Transgenics with0
Rabies Virus
Rabies-virus-based transneuronal tracing
is expected to play an important role
StarterStarterStarter
in elucidating neuronal connectivity (Callaway, 2008; Ugolini, 2011). Interpretationof the results, however, critically depends on the specificity
DISCUSSION
and generality of the tracing (that is whether rabies can propaIn the present study, we developed a technique to obtain gate to all synaptically connected neurons). We successfully
a comprehensive list of monosynaptic inputs to midbrain dopa- labeled diverse cortical and subcortical areas that appear
mine neurons. Our direct comparison of inputs to VTA and SNc to differ in their neurotransmitter types, modes of firing, anddopamine neurons resolves several outstanding questions that functions. Although most of our findings matched conventional
previous methodologies lacked the specificity to address. We tracing experiments, there were several important exceptions,demonstrate that SNc dopamine neurons receive direct input in which we failed to observe labeling in regions previously
from the somatosensory and motor cortices and from the STh. thought to project to VTA and/or SNc. Most of these areas
In contrast, VTA dopamine neurons receive input from the LH (septum, mHb, striatal neurons in the matrix compartment)
and, to a lesser extent, the LO. Furthermore, we show that the were labeled by nonspecific rabies virus or were fromDS and VS project directly to SNc and VTA dopamine neurons, GABAergic neurons, indicating that these structures project
respectively, thus resolving a recent dispute over whether to nondopaminergic neurons in VTA and/or SNc or that theirneurons in the striatum project directly to dopamine neurons, axons pass through these areas. Most importantly, we wereas was long assumed. The results also reveal that striatal able to label largely separate neuronal populations in the strianeurons that project to dopamine neurons form patches both tum, those in patch and matrix compartments, which projectin the DS and VS. These results thus provide foundational knowl- to dopaminergic and GABAergic neurons respectively, inedge on the different inputs to VTA and SNc dopamine neurons the SN. Given that dendrites of SNc dopaminergic neurons
extend to the SNr where GABAergic neurons reside, the result
as well as the basic organization of the basal ganglia circuit.
A

G

op

D

866 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

AB

e

in

am

AB

e

A

0.4

G

in

am

op

G

AB

D

D

op

am

in

e

A

0

NeuronDirect Inputs to Midbrain Dopamine Neurons

calbindin B
EGFP

A

EGFP C

calbindin D

calbindin
EGFPJ* P<0.02

ac

DS

AcbC

ac

AcbSh

F

G

Average (n=5)

H

I

max
min

Predicted patches

30
20
10
0

D
at
a
R
(% and
ar om
ea
)

Nissl
EGFP

% neurons
in predicted contrours

E

Figure 7. Dopamine-Projecting Neurons in the Acb Form Patches(A) Coronal section containing the Acb of a VTA-targeted animal (v003). Red, calbindin D-28k immunostaining; green, EGFP (input neurons); ac, anterior
commissure; AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell; DS, dorsal striatum. Plus sign indicates the center of the ac. Scale bar, 1 mm.
(B–D) Medium magnification views of the Acb in (A). The same section as in (A), but the images were obtained using a confocal microscope resulting in a thin
optical section. Scale bar, 80 mm.
(E and F) Example distributions of input neurons in two animals. In (E), v010; in (F), v004. Scale bar, 1 mm.
(G) Average density map obtained using the five VTA-targeted animals (v009, v001, v010, v004, and v003).
(H and I) ‘‘Predicted patches’’ (orange contours) superimposed on the distribution of input neurons in two animals (same as in E and F). The contours of predicted
patches were obtained using data from the four other animals, not the animal’s own data.
(J) Percentage of neurons located in the predicted patches (‘‘Data’’) compared to the percentage of the Acb taken up by patches (‘‘Random’’). The latter
represents the percentage expected when neurons are randomly distributed. Mean ± SEM, p < 0.02, paired t test (n = 5 mice).

suggests that transneuronal spread does not occur throughmere proximity.
One caveat of the present method (common to other retrograde tracing methods) is that a small amount of labeling does
not necessarily indicate functionally weak connectivity. For
example, one input neuron may form synapses on to many postsynaptic neurons, and a small number of synapses may nonetheless be strong. Therefore, some of the discrepancies
between the present and previous studies may be, at least inpart, explained by these limitations. These issues need to beaddressed using anterograde tracing or electrophysiologicalexaminations. Nevertheless, although future experiments needto validate the method further, our results together with existing
literature (Callaway, 2008; Ugolini, 2011) support the utility ofrabies virus-mediated transsynaptic tracing.
Our methods have further technical advantages over conventional methods. First, the ability to target the tracer (initial infection of the virus) was greatly aided by the use of Cre-transgenic
mice. This is in contrast with conventional tracing experiments inwhich the accuracy of targeting largely depends on the precise

positioning of the injection pipet and proper injection parameters. A similar approach was first introduced using pseudorabies(DeFalco et al., 2001; Yoon et al., 2005), but the transsynaptic
spread was not restricted to monosynaptic inputs. Second, ourability to directly identify starter neurons by fluorescent markers
is useful for quantitative analyses. With conventional methods, it
is often difficult to distinguish between direct depositions andtransported tracers. Our use of a fusion protein between a transmembrane type of TVA (TVA950) and mCherry allowed us todirectly identify starter neurons and appears to be a viableapproach. Third, the high efficiency of the tracing enables
comprehensive mapping that consistently covers most areas ineach animal. Fourth, extremely high expressions of fluorescentmarkers with rabies virus allowed for observations of detailed
morphologies of individual neurons (Wickersham et al., 2007a).
Due to the strong signal, low magnification images obtainedusing semiautomatic acquisitions were sufficient for identifying
labeled neurons. These characteristics are useful for systematicand quantitative mapping of neuronal connectivity and will facilitate future high-throughput efforts.
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NeuronDirect Inputs to Midbrain Dopamine Neurons

Distinct Inputs to SNc and VTA May Explain Their
Saliency- and Value-Related Activity
Our data show that VTA and SNc dopamine neurons receive
distinct excitatory inputs. This may help explain recent electrophysiological data from nonhuman primates. Matsumoto and Hikosaka (2009) found that, whereas VTA dopamine neurons are
excited and inhibited by appetitive and aversive events, respectively, dopamine neurons in the lateral SNc are excited by both.
Furthermore, response latencies were generally shorter in dopamine neurons in the lateral SNc. Our data suggest that distinct
excitatory inputs to VTA and SNc dopamine neurons may
provide value- and saliency-related information differently tothese neurons. Note, however, that there are important anatomical differences between dopamine neurons in rodents andprimates (Berger et al., 1991; Joel and Weiner, 2000). For
example, dopamine neurons that project to the NAc are contained not only in VTA but also the medial part of SNc in primates,
whereas they are more confined to VTA in rodents, suggestingthat the position of the VTA/SNc boundary might be shifted
between primates and rodents (Brog et al., 1993; Joel andWeiner, 2000; Lynd-Balta and Haber, 1994). Therefore, comparisons between species need to be done carefully.
Previous studies proposed that inputs from the Ce, PB, SC,
and the basal forebrain may account for short-latency activations of SNc dopamine neurons (Bromberg-Martin et al., 2010;
Coizet et al., 2010; Dommett et al., 2005; Matsumoto and Hikosaka, 2009). Contrary to these proposals, however, our data
showed that the Ce, PB, and SC project strongly to both VTA
and SNc dopamine neurons (although SC has a slight preference for the SNc). The basal forebrain (originating mainly fromthe DB) was found to project preferentially to VTA dopamine
neurons.
Instead, our data showed that there are specific projections
from S1 and motor cortices (M1 and M2) to SNc dopamine
neurons. Whether the neocortex directly projects to the SNc,
and where in the cortex these inputs originate, have received
less attention partly due to inconsistent results in previous
studies (Bunney and Aghajanian, 1976; Graybiel and Ragsdale,
1979; Naito and Kita, 1994; Zahm et al., 2011). Although therole of somatosensory and motor inputs in dopamine regulation
has not been fully explored previously, somatosensation constitutes an important component of rewarding and noxious stimuli.
Furthermore, dopamine neurons increase their firing when an
animal initiates reward-oriented behavior (Jin and Costa, 2010).
Given that these cortical inputs are most likely excitatory, they
may play a role in short-latency activation of SNc dopamine
neurons in response to stimuli predicting salient events or thesalient stimulus itself.
We also found that the STh provides specific and relatively
strong inputs to SNc dopamine neurons. Previous studies foundonly sparse projections from the STh to the SNc using anterograde tracers (Groenewegen and Berendse, 1990; Kita and Kitai,
1987; Smith et al., 1990). One possible reason for this discrepancy is that dopamine neurons receive STh inputs at theirdendrites that elongate into SNr. STh neurons respond to various
motor events and rewards as well as a sudden change in theenvironment (Isoda and Hikosaka, 2008; Matsumura et al.,1992). Anatomically, STh constitutes the ‘‘hyperdirect pathway’’
868 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

as well as the ‘‘indirect’’ pathway of the corticobasal ganglia
loops (Nambu et al., 2002) (Figure 8C). The former term emphasizes the high conduction velocity of this pathway. On the other
hand, the LH is a major input for VTA dopamine neurons. LH
neurons are known to process reward information (Ono et al.,1986), and these responses are modulated by internal states of
the animal such as hunger (Burton et al., 1976), indicating that
LH responses reflect subjective values. Our results together
with previous findings raise the possibility that STh and LH
provide contrasting excitatory inputs encoding saliency- andvalue-related information, respectively.
Direct Projections from the Striatum to Dopamine
Neurons: Patch/Matrix Projection Systems andComputation of Reward Prediction Errors
The striatum has received much attention as an important input
to dopamine neurons. For example, various computationalmodels of reinforcement learning posit an important role fordirect projections of striatal neurons to dopamine neurons in
calculating reward prediction error (Doya, 1999; Houk et al.,
1996; Joel et al., 2002; Suri, 2002). Recent studies have indicated, however, that direct projections from striatal neurons to
dopamine neurons are weak or nonexistent (Chuhma et al.,2011; Xia et al., 2011). Contrary to these recent results, our study
demonstrates that, in terms of numbers, the striatum is the mostprominent input to both VTA and SNc dopamine neurons.
Although we cannot rule out the possibility that the striatumdopamine neuron synapses are mostly ‘‘silent,’’ the prominent
labeling and exquisite specificity indicate that this connection
exists. Our result indicates that only a very specific, small subsetof striatal neurons project to dopamine neurons. This raises thequestion as to whether channelrhodopsin was expressed inthis particular population in the previous experiments. Anotherpossible explanation is that these synapses use a different
neurotransmitter than GABA.
Our results have implications for the basic organizing principleof the basal ganglia circuit. Corticobasal ganglia circuits form
multiple, parallel pathways between the cortex and the output
structures of the basal ganglia (i.e., EP and SNr) (Figure 8). The
DS can be parceled into patch and matrix compartments thatmay define distinct projection systems (Gerfen, 1992; Graybiel,1990). Previous studies have indicated that striatal neurons in
the patches project to SNc, whereas those in the matrix projectto SNr (Fujiyama et al., 2011; Gerfen, 1984), although a recent
study indicated that these projections are not as specific aspreviously thought, at least in primates (Lévesque and Parent,2005), and the cell-type specificity of postsynaptic neurons has
not been established. We extend the previous findings byshowing that the patch-matrix system represents segregatedneural pathways that comprises distinct types of neurons bothpre- and postsynaptically (Figure 8C). Importantly, dopamineneuron-projecting striatal neurons differ from GABAergicneuron-projecting medium spiny neurons in their morphology
and calbindin D-28k expression, suggesting that these neurons
are a new class of medium spiny neurons. Furthermore, we
showed that the Acb also has dopamine-neuron-projecting
patch structures, which are smaller than the shell/core divisions
defined by molecular markers (Figure S5). A recent study foundNeuronDirect Inputs to Midbrain Dopamine Neurons

A

B

M2
LDTg

>70
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S1

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EA
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SNc
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Dopamine

GP
EP

SNr
/EP

SNc

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/EP

Dopamine
GABAergic
Glutamatergic

SNr
Dopamine

Figure 8. Summary of the Connectivity(A) Direct comparison of the distributions of monosynaptic inputs to VTA and SNc dopamine neurons. SADDG-EGFP(EnvA) and SADDG-mCherry(EnvA)
were injected into VTA and SNc, respectively. Cyan: VTA targeted. Red: SNc targeted. Top, medial. Bottom, lateral. Scale bar, 1 mm.
(B) Flatmap summary of monosynaptic inputs to VTA and SNc dopamine neurons. Blue indicates inputs to VTA. Red indicates inputs to SNc dopamine neurons.
The thickness of each line indicates the number of input neurons in each area (inputs per 10,000 total inputs) as defined at the top right corner. The flatmap
representation is after Swanson (2000). Connections from DS to VTA and Acb to SNc were omitted for clarity.
(C) Models of the corticalbasal ganglia circuit. Left, conventional model; right, new model based on the present result. Some connections are omitted forsimplicity.

a ‘‘hedonic hotspot,’’ a potential microdomain defined by thehedonic (or ‘‘liking’’) effect of opioids (Peciña and Berridge,2005). Based on the available data, the hedonic hotspot (inrats) appears to lie just dorsal to one of the ‘‘ventral patches’’
we found (in mice). These results indicate that the VS also forms
parallel channels for information flow.
Taken together, these results suggest that the corticobasal
ganglia inputs to dopamine neurons form multiple pathways,
akin to the corticobasal ganglia output pathways via EP and
SNr: Dopamine neurons receive direct and indirect inputs fromthe striatum, inputs from the cortex via STh, and direct inputs
from the cortex (Figure 8).
Inputs from Motor and Autonomic Areas
The comprehensive identification of inputs revealed that one
common feature for both VTA and SNc is that many of the areasthat project directly to dopamine neurons have been characterized as autonomic (Ce, lateral BNST, Pa, LH, PAG, and PB)(Saper, 2004). As mentioned earlier, SNc also receives inputs
from motor areas (M1, M2, and STh). The central autonomic
system receives taste and visceral information and regulates
various autonomic responses (Saper, 2002; Yamamoto, 2006).
Highly overlapping structures are also identified for pain processing (Gauriau and Bernard, 2002; Saper, 2002). Autonomic
and motor responses are tightly coupled to rewarding as well
as aversive events (and their expectations) or the saliency of
sensory cues. In this sense, efferent copies of autonomic or
motor signals may serve as a surrogate of important information
for dopamine neurons, such as reward expectation and motivational saliency, in addition to general states of the animal.
Although the role of these motor and autonomic inputs in theregulation of dopamine neuron activities is unclear, our finding
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NeuronDirect Inputs to Midbrain Dopamine Neurons

provides a framework with which to explore the mechanisms of
dopamine neuron regulation.
Inputs from the PTg
It has been proposed that PTg plays an important role in reward
prediction error computations (Kawato and Samejima, 2007;
Okada et al., 2009). Previous studies have shown that electricalstimulation of PTg produced monosynaptic activation of dopamine neurons (Futami et al., 1995; Lokwan et al., 1999; Scarnati
et al., 1984). Some anatomical studies have also indicated thatPTg projects to both VTA and SNc using anterograde and retrograde tracing methods (Jackson and Crossman, 1983; Oakman
et al., 1995; Zahm et al., 2011). These results appear to differ
from our data indicating relatively sparse labeling of PTg fromthe VTA compared to SNc dopamine neurons. This difference
may be explained if single PTg neurons make many synapses
onto VTA dopamine neurons or synapses transmissions arestrong. The aforementioned results may also be confoundedby nonspecific electrical stimulation of passing fibers or uptakeof tracers. Whether VTA receives strong direct inputs from PTg
neurons remains to be clarified. Our method allowed us to avoid
limitations of previous methods (i.e., cell-type specificity andlabeling axons of passage), and the difference from other studies
may come, at least in part, from the specificity achieved using
our method although the exact reasons need to be clarified in
the future. It should also be noted that other anatomical studies
have indicated that VTA does not receive strong inputs from PTg(Geisler and Zahm, 2005; Phillipson, 1979).
Implications for Deep Brain Stimulation for Parkinson’s
DiseaseDegeneration of SNc dopamine neurons leads to the severe
motor impairments of Parkinson’s disease. Symptoms of thisdisease can be ameliorated by high-frequency electrical stimulation of specific brain areas (deep brain stimulation [DBS]) (Benabid et al., 2009; Wichmann and Delong, 2006). Despite the wide
use and success of DBS, its mechanisms remain highly debated,
and it is unknown why specific targets are more effective thanothers.
The most popular target of DBS is the STh. As describedearlier, we found relatively strong direct projections from the
STh to SNc dopamine neurons. Interestingly, when the densityof labeled neurons was calculated, the STh emerged as one of
the highest density areas due to its small size. It is well known
that symptoms improved by STh DBS coincide with those
improved by levodopa (dopamine precursor) treatment, andpatients’ response to levodopa is the best outcome predictorof DBS (Benabid et al., 2009; Wichmann and Delong, 2006; but
see Zaidel et al., 2010). Furthermore, one of the major effects
of STh DBS is the reduction in required levodopa dose. Considering these observations and the relatively strong direct connections found earlier, one simple idea for the mechanism of DBS is
the direct stimulation of residual dopamine neurons throughdirect activation of STh neuron axons, which, in turn, leads to
the restoration of dopamine concentrations in target areas of
SNc dopamine neurons (e.g., DS). Although earlier studies suggested ‘‘inhibition’’ of STh neurons by high-frequency stimulation may be the mechanism, recent studies have indicated that
870 Neuron 74, 858–873, June 7, 2012 ª2012 Elsevier Inc.

direct electrical stimulation of axons of STh neurons may actuallycause an increase in the transmitter release at their target (Deniau et al., 2010; Johnson et al., 2008). Although whether STh
DBS causes an increase in dopamine concentration remains
controversial (Benazzouz et al., 2000; Hilker et al., 2003; Iribe
et al., 1999; Nakajima et al., 2003; Pazo et al., 2010; Smith andGrace, 1992; Strafella et al., 2003), our study provides anatomical support for this model.
Interestingly, our results demonstrate that other targets of
DBS also predominantly project directly to SNc dopamine
neurons. These include the EP (homologous to the internalsegment of the globus pallidus in humans), PTg, and motor
cortex (Benabid et al., 2009; Wichmann and Delong, 2006).
Although the relevance of these direct connections in DBS
remains to be examined, cell-type-specific connectivity
diagrams will aid future studies of the mechanisms as well as
the search for new targets for DBS.
Future Directions
In the present study, we have focused on gross differences in
inputs to VTA versus SNc dopamine neurons. Recent studies,
however, have demonstrated more diversity in dopamine
neurons than previously assumed (Ikemoto, 2007). For example,
VTA dopamine neurons are composed of different subgroups
that project to distinct areas, have distinct physiological properties, and involve distinct synaptic plasticity in response to cocaine
and pain (Lammel et al., 2008; Lammel et al., 2011). It is thus ofgreat interest to examine inputs to these subgroups separately.
Although VTA and SNc dopamine neurons have long been
associated with different functions (e.g., reward and motor functions), it is only recently that the differences in firing patterns of
VTA versus SNc dopamine neurons have been revealed (Matsumoto and Hikosaka, 2009). It is, therefore, important to replicate
these results in different animals, including mice. Although
response properties of dopamine neurons in awake-behaving
mice have been relatively understudied, the availability ofgenetic and molecular tools can greatly facilitate detailed characterizations of midbrain dopamine neurons (Cohen et al.,2012). Furthermore, the present study revealed various direct
inputs to dopamine neurons from relatively underappreciatedareas such as motor, somatosensory, and autonomic areas.
This knowledge will be useful in designing future recording
experiments to probe further differences between VTA andSNc dopamine neurons.
EXPERIMENTAL PROCEDURES
Viral Injections
All procedures were approved by Harvard University Institutional Animal Careand Use Committee. Adult male mice (2 to 6 months old) were used. DAT-Cre(Bäckman et al., 2006) and Vgat-ires-Cre (Vong et al., 2011) lines were backcrossed with C57BL6. For some control experiments, C57BL6 mice wereused. For cell-type-specific tracing, 0.1–0.5 ml of AAV8-FLEX-RG (2 3 1012
particles/ml) and AAV5-FLEX-TVA-mCherry (4 3 1012 particles/ml) werestereotaxically injected into the targeting areas using a micromanipulator
with a pulled glass needle. Two weeks later, 4 ml of pseudotyped rabies virus,
SADDG-GFP(EnvA) (1.0 3 107 plaque-forming units [pfu] per milliliter; Wickersham et al., 2007b), was injected into the same area. For tracing using the nonpseudotyped rabies virus, 4 ml of SADDG-GFP (2 3 108 pfu/ml) (Wickersham
et al., 2007a) was injected into VTA. To directly compare the distributions ofNeuronDirect Inputs to Midbrain Dopamine Neurons

neurons projecting to VTA versus SNc dopamine neurons, SADDG-GFP(EnvA)(5 3 107 pfu/ml) and SADDG-mCherry(EnvA) (1.0 3 106 pfu/ml) (Marshel et al.,2010) were injected at 3.0 mm posterior to Bregma, 4.2 mm deep from dura,
0.5 mm and 1.5 mm lateral to the midline, respectively.
Histology and Image Analysis
One week after injection of rabies virus, mice were perfused with PBS followed
by 4% paraformaldehyde (PFA) in PBS. After 2 days of postfixation in 4% PFA,
coronal brain slices at 100 mm thickness were prepared using a vibratome.
Every third section was counterstained with NeuroTrace Fluorescent Nissl
Stains (Molecular Probes, Eugene, OR, USA). Immunohistochemistry was performed using the anti-calbindin rabbit polyclonal antibodies (Calbiochem,
Darmstadt, Germany), anti-tyrosine hydroxylase AB152 (Millipore, Billerica,
MA, USA), the biotinylated goat anti-rabbit secondary antibodies (Jackson
ImmunoResearch, West Grove, PA, USA), streptoavidin-conjugated Alexa
Fluor 405, and Alexa Fluoro 594 goat anti-rabbit secondary antibodies(Molecular Probes). Slices were permeabilized with 0.5% Triton X-100, andincubation with antibodies and washing was done with 0.05% Triton X-100.
Whole-section mosaics of high-magnification images were taken semiautomatically with AxioImager Z2 or LSM 510 Inverted Confocal microscopes(Zeiss) and assembled using software (Axiovision or LSM, Zeiss). The locationsof labeled neurons and the outlines of brain areas were manually registeredusing custom software written in MATLAB (Mathworks, Natic, MA, USA).
Further data analyses were performed using custom software written in
MATLAB (see Supplemental Experimental Procedures).
SUPPLEMENTAL INFORMATION
Supplemental Information includes six figures and Supplemental Experimental
Procedures and can be found with this article online at doi:10.1016/j.neuron.
2012.03.017.
ACKNOWLEDGMENTS
We are grateful to Dr. Edward Callaway for providing us with the rabies virus
and other reagents and his advice. We thank Dr. John A.T. Young for
pCMMP-TVA950. We thank Dr. Catherine Dulac for her support, providing
us with various reagents, and her comments on the manuscript. We thankDr. Joshua Sanes for comments on the manuscript, Drs. Linh Vong and Bradford Lowell for Vgat-ires-Cre mouse, Dr. Edward Boyden for AAV8-CAGArchT-GFP, and Dr. Shenqin Yao for pCDNA-mCherry and her technicaladvice. We thank Neir Eshel, Jeremiah Cohen, and other members of the
Uchida lab for critical comments on the manuscript and discussions. This
work was supported by a Howard Hughes Medical Institute Collaborative
Innovation Award, a Smith Family New Investigator Award, the Alfred Sloan
Foundation, and the Milton Fund (N.U.).
Accepted: March 10, 2012
Published: June 6, 2012
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Neuron, Volume 74
Supplemental Information

Whole-Brain Mapping of Direct Inputs
to Midbrain Dopamine Neurons
Mitsuko Watabe-Uchida, Lisa Zhu, Sachie K. Ogawa, Archana Vamanrao, and Naoshige Uchida

INVENTORY OF SUPPLEMENTAL INFORMATION

Figure S1 (injection sites) is related to Figure 1
Figure S2 (cell counts from individual animals) is related to Figure 3
Figure S3 (control injections) is related to Figure 4
Figure S4 (anterograde labeling from the motor cortex) is related to Figure 5
Figure S5 (location of the ventral patches) is related to Figure 7
Figure S6 (other major input areas) is related to Figure 8

Page | 1

SUPPLEMENTAL FIGURES

Page | 2

Figure S1. Distribution of starter neurons
Starter neurons (magenta) are plotted on five coronal sections. Sections were immunostained for TH (blue) except
for v001. The reference atlas (Left) is from Franklin and Paxinos (2008). White crosses, midline. Cyan crosses,1
mm lateral to midline.
IF: interfascicular nucleus, PBP: parabrachial pigmented nucleus of VTA, PFR: parafasciclus retroflexus area,
PIF: parainterfascicular nucleus of VTA, PN: paranigral nucleus of VTA, RLi: rostral linear nucleus, RRF:
retrorubral field (A8).
Note that the IF, PBP, PFR, PIF, PN and RLi constitute the VTA or A10 (Ikemoto, 2007). In Franklin and Paxinos(2008), the VTA refers to PFR only.

Page | 3

Figure S2. Cell counts from individual animals
LO, lateral orbital cortex; M1, primary motor cortex; M2, secondary motor cortex; S1, primary somatosensory
cortex; PC, piriform cortex; AcbSh, nucleus accumbens shell; AcbC, nucleus accumbens core; Tu, olfactory
tubercle; DS, dorsal striatum; VP, ventral pallidum; GP, globus pallidus; EP, entopeduncular nucleus; Ce, central
nucleus of the amygdala; LH, lateral hypothalamus; STh, subthalamic nucleus; PTg, pedunculotegmental nucleus.

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Figure S3. Comparison between tracing using Cre-dependent, transsynaptic tracing and Cre-independent,
non-transsynaptic tracing.
(A, D) Lateral septum (LS)(B, E) Medial and lateral habenula (mHb and lHb).
(C, F) Hypothalamic areas. Pa, paraventricular hypothalamic nucleus; PLH, peduncular part of the lateralhypothalamus (LH).
The EGFP patterns with selective tracing using SADdG-GFP(envA) in DAT-Cre mice (A-C) are compared withthose with nonselective tracing using unpseudotyped SADdG-GFP (D-F). Green, EGFP expressed by rabies virus;
red, fluorescent Nissl staining. Insets show low magnification views of sections. The white rectangles indicate themagnified regions. Scale bars, 250 μm and 1 mm (insets). Significantly more labeling is observed with nonspecifictracing in LS (D) and mHb (E) compared to the tracing with DAT-Cre mice. In the hypothalamus, Pa and LH
showed prominent labeling with DAT-Cre mice (C) while more widespread labeling was observed with nonspecifictracing (F).

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Figure S4. Anterograde tracing from the motor cortex.
(A) A low magnification view of a sagittal section. AAV8-CAG-ArchT-GFP was injected into the motor cortex.
The mouse expresses tdTomato in dopamine neurons. Green: AAV8-CAG-ArchT-GFP. Red: tdTomato. Strong
signals in the olfactory bulb (OB) reflect dopaminergic interneurons. Scales bar: 1 mm.
(B) A medium magnification view near SNc. Scale bar: 500 μm.
(C) A high magnification view of SNc. Arrowheads indicate buttons.
(D) A high magnification view of VTA. Scale bar: 20 μm.
cp: cortical peduncle. ic: internal capusule. OB: olfactory bulb. M1: primary motor cortex. M2: secondary motor
cortex.
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Figure S5. Patterns of dopamine neuron-projecting “ventral patches” in the nucleus accumbens.
The average patch patterns are obtained from 5 VTA-targeted mice. The atlas is after Franklin and Paxinos (2008).
MS, medial septal nucleus; aca, anterior part of anterior commissure; AcbC, nucleus accumbens core; AcbSh,
nucleus accumbens shell; LAcbSh, lateral accumbens shell; DS, dorsal striatum; VP, ventral pallidum; Tu,
olfactory tubercle.

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Figure S6. Comparisons of other major input areas(A) Bed nucleus of stria terminalis (BNST). STLD, dorsal lateral division of the bed nucleus of stria terminalis.
(B) Paraventricular hypothalamic nucleus (Pa).
(C) Superior colliculus (SC) and periaqueductal gray (PAG). Dopamine projecting neurons are clustered in theventrolateral part of the PAG (vlPAG).
(D) Dorsal raphe (DR) and pedunculotegmental nucleus (PTg).
(E) Parabrachial nucleus (PB). Both lateral and medial PB contains labeled neurons.
(F) Cerebellar nuclei. Lat, lateral (dentate) cerebellar nucleus.
Insets show low magnification views of sections. The white rectangles indicate the magnified regions. Scale bar, 1
mm (C, D) and 250 μm (A, B, E, F).
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SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Production of viral vectors
Different serotypes of AAVs and different promoters were used to express RG and TVA under the control of a
FLEX switch (Atasoy et al., 2008), to avoid any possible competition. AAV8 and AAV5 were chosen due to thehighest expression efficiency in midbrain dopamine neurons in the pilot experiments. To maximize the expression
efficiency after Cre recombination, a FLEX switch was designed to contain lox2272 and loxP sites, oriented to haveno false ATG start, with point mutations in the inverted repeats to destabilize hairpin structures in transcribed RNA
as below. To generate a mammalian expression vector with the FLEX switch under chicken actin promoter,annealed oligonucleotides (Integrated DNA Technologies, Coralville, IA) were ligated between EcoRI and NheI
sites of pCA-MCS (pCA-FLEX). CA-FLEX fragment, digested with SmaI and NheI and blunted by T4 DNApolymerase, was inserted at HindIII site, blunted and BamHI site into pAAV-DIO-hChR2EYFP (pAAV-CAFLEX) (Gunaydin et al., 2010). Rabies glycoprotein ORF was obtained from pHCMV-RG (Sena-Esteves et al.,2004) by AseI/EcoRV digestion, blunted, and inserted at EcoRV site of pAAV-CA-FLEX (pAAV-CA-FLEX-RG).
FLEX:
GAATTCATtACcTCGTATAGGATACTTTATACGAAGTTATGCAGAATGGTAGCTGGATTGTAAGTACTATTAGCAATATGAAACCTCTTAATAACcTCGTATAGCATACATTATACGAAGTaATAGATCTAAGCTTGATATCGTCGACATAACTTCGTATAAAGTATCCTATACGAgGTaATTTGCCTTAACCCAGAAATTATCAGTACTATTCTTTAGAATGGTGCAAAGAATtACTTCGTATAATGTATGCTATACGAgGTTATCGCATGCTAGCA transmembrane version of TVA, TVA950 (Qin and Luo, 2009), was shuttled from pCMMP-TVA950 by
PmlI/BamHI digestion at BamHI and EcoRV sites of pBluescriptII SK (pB-TVA950). mCherry open reading
frame was amplified by PCR using pCDNA-mCherry and primers below, and inserted at StyI site blunted, and
BamHI site of pB-TVA950 (pB-TVA950-mCherry). TVA950-mCherry fragment, digested with SalI and EcoRVand blunted, was inserted at NheI and AscI sites, blunted, of pAAV-DIO-hChR2EYFP (pAAV-EF1α-FLEX-TVAmCherry).
mCherry-s: TTTTT CCC GGG GGT GGG GTG AGC AAG GGC GAG GAG G
mCherry-a: TTTTT GGATCC GAT ATC TTA CTT GTA CAG CTC GTC CA
AAV8-FLEX-RG and AAV5-FLEX-TVA-mCherry were produced by Virus Vector Core Facility of the Universityof North Carolina (Chapel Hill, NC). Pseudotyped and unpseudotyped rabies viruses were produced as described
previously (Wickersham et al., 2010). Contamination of unpseudotyped virus was <1 in 104 pseudotyped virus
using 293T cells.
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Image analysis
The locations of labeled neurons and the outlines of brain areas were manually registered using custom software
written in MATLAB (Mathworks, Natic, MA). In some cases, images were low-pass filtered using a Gaussian
kernel before analysis. This reduced diffuse fluorescent signals originating from axon fibers and emphasized
signals from somata. The nomenclatures and outlines of brain areas are according to a standard mouse atlas(Franklin and Paxinos, 2008). Abbreviations are also according to Franklin and Paxinos (2008) except we used thedorsal striatum (DS) instead of caudate/putamen (CPu). Note that the pedunculotegmental nucleus (PTg) is often
called the pedunculopontine tegmental nucleus, and that the orbital cortex is often called the orbitofrontal cortex inother literature. The globus pallidus and entopeduncular nucleus in rodents are thought to be equivalent to theexternal segments and internal segments of the globus pallidus in primates, respectively. Numbers of neurons werenormalized by the total number of input neurons in the entire brain of each animal. Because the numbers of input
neurons are roughly proportional to those of starter neurons, both produce similar results. Because v008 and v007
contained relatively small numbers of transsynaptically labeled neurons (Figure 1F), the results from these mice
were excluded from quantitative analysis. However, except some areas that contained a small number of input
neurons, the results from these mice were consistent with other animals (Figure S2).

Center of mass analysis
To compare global shift of EGFP-positive neurons, the center of mass of a brain section was obtained by averaging
positions of neurons. To superimpose results from different animals onto a standard atlas, neuron’s positions werenormalized by anatomical landmarks. For Figure 4A-C, we used the midline and the lateral most part of the dorsal
striatum (DS). For Figure 4D-F, we used the midline and the medial most position of the cerebral peduncle (cp).

Analysis of the neocortex
Various efforts have been made to identify cortical areas in rodents. However, resulting maps differ significantly
across studies (Plalomero-Gallagher and Zilles, 2004). We therefore examined distributions of the labeled neurons
in the entire neocortex using an “unrolled map”. To make an unrolled map, we first drew a line running at the
middle of the cortical sheet (the gray lines in Figure 5C and F). For reference points, we used the most dorsomedial
corner of the hemisphere (the black crosses in Figure 5C and F) and rhenal fissure or sulcus (red triangles). These
reference points were projected onto the line (black dots and red crosses). These lines were “unrolled” and aligned
according to the dorsomedial reference point and the rostro-caudal position in reference to the bregma (Figure 5G
and H). This unrolled map allows one to compare results across studies independent from the discrepancies incortical maps. For comparison, we generated a reference unrolled map according to a mouse standard atlas (Figure5I) (Franklin and Paxinos, 2008).
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For cell counting (Figure 3), areas in the neocortex were parceled mostly according to the proportions of corticalareas on the coronal slices in the standard atlas (Franklin and Paxinos, 2008). Slight changes in the locations of theboundaries of cortical areas do not significantly affect the results.

Analysis of striatal neurons
For the quantification of calbindin D-28k immunofluorescence, we normalized the pixel intensities by the
maximum and minimum of the area of interest. For each neuron, intensities were obtained in a circular area withdiameter 6.8 μm. For measuring soma sizes, a stack of confocal images was projected along the z-axis. The somaof each neuron was outlined using custom software written in MATLAB and the area within the contour was
calculated. We then calculated the diameter based on the area assuming a circle.
For a measure of dendritic complexities (Figure 6K), dendrites were traced using custom software in MATLAB.
Dendritic length is defined as the total length along the dendrite. Radial distance is defined as the straight line
distance between the most proximal point of the dendrite and the tip of the dendrite.
To obtain standard “ventral patches”, locations of labeled neurons from 5 VTA-targeted mice were pooled. The
data from different animals were combined using a normalized coordinate using the midline and the anterior
commissure (the cross in Figure 7) as the reference points. The information about the locations of labeled neurons
was “smoothed” by a circular averaging filter of the radius of 0.08 mm to obtain a density map. A contour line wasdrawn at half of the maximum density. To test the conservation of the location of the patches, “predicted” contour
maps were obtained for each animal using data from the other 4 animals (one-leave-out cross-validation method)
and the proportion of the neurons within the contour lines (patches) against the total number in Acb was calculated(“% neurons in predicted contours”). We then tested whether this value is larger than the proportion of the areas ofthe patches compared to that of the Acb (paired t-test).

Anterograde labeling from the motor cortex
To confirm projections from the motor cortex to SNc dopamine neurons, AAV8-CAG-ArchT-GFP (1.3 x 1011
particles/ml, a gift from Dr. Edward Boyden, generated by UNC Virus Vector Core Facility, Chapel Hill, NC) was
injected into the motor cortex (bregma: +2.0 mm, 1.3 mm lateral from the midline) in two mice. We used DATCre/Gt(ROSA)26 Sortm(CAG-tdTomato)Hze (Jackson Lab, Bar Harbor, ME) to label dopamine neurons with a fluorescentmarker, tdTomato. After 17 days from injection, mice were perfused with 4% paraformaldehyde, and 100 μm
sagittal sections were cut for observation (Figure S4).

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SUPPLEMENTAL REFERENCES

Atasoy, D., Aponte, Y., Su, H.H., and Sternson, S.M. (2008). A FLEX switch targets Channelrhodopsin-2 tomultiple cell types for imaging and long-range circuit mapping. J Neurosci 28, 7025-7030.
Franklin, B.J., and Paxinos, G. (2008). The mouse brain in stereotaxic coordinates. Compact third eddition. (San
Diego, Academic Press).
Gunaydin, L.A., Yizhar, O., Berndt, A., Sohal, V.S., Deisseroth, K., and Hegemann, P. (2010). Ultrafast
optogenetic control. Nat Neurosci 13, 387-392.
Plalomero-Gallagher, N., and Zilles, K. (2004). Isocortex. In The rat nervous system, Third edition, G. Paxinos, ed.
(San Diego, Academic Press), pp. 729-757.
Qin, C., and Luo, M. (2009). Neurochemical phenotypes of the afferent and efferent projections of the mouse
medial habenula. Neuroscience 161, 827-837.
Sena-Esteves, M., Tebbets, J.C., Steffens, S., Crombleholme, T., and Flake, A.W. (2004). Optimized large-scale
production of high titer lentivirus vector pseudotypes. J Virol Methods 122, 131-139.
Wickersham, I.R., Sullivan, H.A., and Seung, H.S. (2010). Production of glycoprotein-deleted rabies viruses formonosynaptic tracing and high-level gene expression in neurons. Nat Protoc 5, 595-606.

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