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Comple, Soares, 2019
Comple, Soares, 2019
Comple, Soares, 2019
a b c d 100
100 500 500 100
80 80
(eYFP+/D1R+ cells)
400 400
70 70
60 60 60
Time (s)
Time (s)
300 300 50 50
40 40 40
30 30
200 200
20 20 20
10 10
D1-eYFP D2-eYFP D1-eYFP D2-eYFP
0 100 100 0 0
OFF ON OFF ON OFF ON OFF ON
FP
2
FP
2
1- FP
D R2
2- P
hR
hR
hR
D YF
eY
eY
eY
C
C
C
C
e
2-
2-
1-
1-
1-
2-
D
D
D
D
D
Supplementary Figure 1. Brief and prolonged D1- and D2-MSN stimulation oppositely regulate CPP. (a)
Quantification of immunofluorescence for dopamine receptor D1 or D2 and YFP. Around 60% of neurons were
transfected with ChR2 or eYFP (n=4 animals). (b) Total time spent in the stimulus-associated chamber (ON) and
the no-stimulus-associates chamber (OFF) by D1-eYFP and D2-eYFP mice during the post-test session of the
CPP with brief optical stimulation (left, light colors) and total time spent in the stimulus-associated chamber (ON)
and the no-stimulus-associates chamber (OFF) by D1-eYFP and D2-eYFP mice during the post-test session of
the CPP with prolonged optical stimulation (right, dark colors). (c-d) Time spent in each entry in the RTPP test.
(nD1-eYFP=7; nD2-eYFP=8).
Supplementary Figure 2
a b c d
Conditioned Place Preference (CPP) 100 500 500
in no-stimulus chamber
0 400 400
Day 1 Days 2, 3 Day 4
Pre-test Conditioning Post-test **
Time (s)
Time (s)
-100 300 ** 300
OFF ON OFF OFF
Optical inhibition:
10s of constant light every min -200 200 200
**
D1-eNpHR D2-eNpHR D1-eYFP D2-eYFP
-300 * 100
OFF ON OFF ON
100
OFF ON OFF ON
FP
FP
R
pH
pH
eY
eY
eN
eN
1-
2-
1-
2-
D
D
D
D
Supplementary Figure 2. Optogenetic inhibition of D1- or D2-MSNs induces aversion. (a) Schematic
representation of optogenetic inhibition parameters in the CPP test. (b) Optical inhibition of D1- or D2-MSNs
causes significant aversion to the stimulus-associated chamber (nD1-eYFP=5, nD1-eNpHR=5; nD2-eYFP=5, nD2-
eNpHR=5). (c) All D1-eNpHR and D2-eNpHR mice spend less time in the ON side. (d) No differences were found
between time spent in the ON side and the OFF side of the CPP apparatus of both D1-eYFP and D2-eYFP
mice. *p<0.05, **p<0.01. Data is represented as mean ± SEM.
Supplementary Figure 3
CPP with cocaine + blue light stimulation (10Hz, 10ms, 3 min ON, 5 min OFF)
a b
Time in cocaine/blue light chamber -
400 800
**
time in saline chamber
300 * 600 **
Time (s)
200 400
***
100 200
D1-ChR2 D2-ChR2
0 0
saline cocaine saline cocaine
FP
FP
2
hR
hR
eY
eY
C
C
1-
2-
1-
2-
D
D
D
CPP with blue light stimulation (10Hz, 10ms, 3 min ON, 5 min OFF)
c d
60 550
Time in stimulus chamber - time
40
in no-stimulus chamber
500
20
Time (s)
0 450
-20
400
-40
D1-ChR2 D2-ChR2
-60 350
OFF ON OFF ON
FP
FP
2
hR
hR
eY
eY
C
C
1-
2-
1-
2-
D
D
D
Supplementary Figure 3. Replication of the CPP protocol described by Lobo et al, 2010. Optogenetic
activation (10Hz, 10ms, 3 min ON, 5 min OFF) of D1- or D2-MSNs has no effect itself, and oppositely regulates
cocaine reward. (a-b) Optical activation of D1-MSNs enhances cocaine (5 mg/kg) conditioning in D1-ChR2 mice
when compared with D1-eYFP controls (nD1-eYFP=5, nD1-ChR2=6; Student’s t test, t9=3.2, p=0.01). In contrast,
optical activation of D2-MSNs decreased cocaine (5 mg/kg) conditioning in D2-ChR2 mice when compared with
D2-eYFP controls (nD2-eYFP=5, nD2-ChR2=6; Student’s t test, t9=6.2, p=0.0002). (c,d) No preference was observed
for D1- or D2-MSN stimulation in the absence of cocaine (nD1-eYFP=4, nD1-ChR2=4; nD2-eYFP=4, nD2-ChR2=4).
*p<0.05, **p<0.01, ***p<0.001. Data is represented as mean ± SEM.
Supplementary Figure 4
D1-MSN brief optical stimulation (1s) D2-MSN brief optical stimulation (1s)
a b c d 80
e
Optical stimulation 100 600 500
in no-stimulus chamber
in no-stimulus chamber
500
50 400
Pre-test Conditioning Post-test
40
15 minutes 30 minutes 15 minutes 400
Time (s)
Time (s)
OFF ON OFF OFF 0 20 300
300
Conditioned place preference
0
-50 200
200
-20
Brief stimulation:
10 Hz stimulation
FP
FP
2
hR
at 10Hz, every min
hR
eY
eY
C
C
1-
1-
2-
2-
D
D
D1-MSN prolonged optical stimulation (60s) D2-MSN prolonged optical stimulation (60s)
Optical stimulation f 50
g 500
h 100
i
500
in no-stimulus chamber
in no-stimulus chamber
Time (s)
Time (s)
OFF ON OFF OFF 300 300
-50 0
200 200
Prolonged stimulation:
60s, 50 ms pulses, -100 100
OFF ON
-50 100
OFF ON
FP
2
FP
hR
hR
eY
eY
C
C
2-
2-
1-
1-
D
D
D1-MSN brief optical stimulation (1s) D2-MSN brief optical stimulation (1s)
j k l m n
Optical stimulation 150 ** 600 200 500
in no-stimulus chamber
in no-stimulus chamber
500 150
100
Pre-test Conditioning Post-test *** 400
15 minutes 30 minutes 15 minutes 400 100
Time (s)
Time (s)
OFF ON OFF OFF 50 300
300 50
Conditioned place preference
0 200
200 0
Brief stimulation:
20 Hz stimulation
100 -50
1s, 25 ms pulses, -50
OFF ON
100
OFF ON
FP
2
FP
hR
hR
C
C
2-
2-
1-
1-
D
D
D1-MSN prolonged optical stimulation (60s) D2-MSN prolonged optical stimulation (60s)
Optical stimulation o p q r
100 500 100 500
Time in stimulus chamber - time
in no-stimulus chamber
-100
200 -200 200
Prolonged stimulation: -150
60s, 25 ms pulses, *** *
-200 100 -300 100
at 20Hz, every 2 min OFF ON OFF ON
FP
2
FP
hR
hR
eY
eY
C
C
2-
1-
2-
1-
D
D
D
D
Supplementary Figure 4. Behavioral effect of different protocols of D1- and D2-MSN stimulation. (a)
Schematic representation of brief and prolonged optogenetic stimulation in the CPP test, using 10Hz frequency.
(b-c) Brief (10Hz, 1s) optical stimulation of NAc D1-MSNs did not induce preference nor aversion (nD1-eYFP=5,
nD1-ChR2=5; Mann-Whitney test, U=10.0, p=0.6905). (d-e) Brief (10Hz, 1s) optical stimulation of NAc D2-MSNs
did not induce preference (nD2-eYFP=4, nD2-ChR2=4; Student’s t test, t6=0.5, p=0.6467). (f-g) Prolonged (10Hz, 60s)
optical stimulation of NAc D1-MSNs does not induce place preference or aversion (nD1-eYFP=6, nD1-ChR2=5;
t9=0.3, p=0.7565). (h-i) Prolonged (10Hz, 60s) optical stimulation of NAc D2-MSNs does not induce place
preference or aversion (nD2-eYFP=5, nD2-ChR2=5; t8=0.3, p=7395). (j) Schematic representation of brief and
prolonged optogenetic stimulation in the CPP test, using 20Hz frequency. (k-l) Brief (20Hz, 1s) optical
stimulation of NAc D1-MSNs induces place preference (nD1-eYFP=5, nD1-ChR2=5; Mann-Whitney test, U=0.0,
p=0.0079; l- Paired t test, t4=9.4, p=0.0007). (m-n) Brief (20Hz, 1s) optical stimulation of NAc D2-MSNs induces
place preference (nD2-eYFP=5, nD2-ChR2=5; Student’s t test, t8=10.6, p<0.0000; n- Paired t test, t4=11.5, p=0.0003).
(o-p) Prolonged (20Hz, 60s) optical stimulation of NAc D1-MSNs induces place aversion (nD1-eYFP=6, nD1-ChR2=5;
t9=9.6, p<0.0000; p- Paired t test, t4=17.5, p<0.0000). (q-r) Prolonged (20Hz, 60s) optical stimulation of NAc D2-
MSNs induces place aversion (nD2-eYFP=6, nD2-ChR2=5; t9=4.6, p=0.0014; r- Paired t test, t4=4.4, p=0.0120).
*p<0.05, **p<0.01, ***p<0.001. Data is represented as mean ± SEM.
Supplementary Figure 5
a b 2500
c 2500
D1-ChR2 D1-eYFP
D1-ChR2 D1-ChR2
2000 D1-eYFP 2000 D1-eYFP
Distance (cm)
Distance (cm)
1500 1500
1000 1000
500 500
0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time (min) Time (min)
d e 2000
f 2500
D2-ChR2 D2-eYFP
D2-ChR2
D2-eYFP 2000 D2-ChR2
1500 D2-eYFP
Distance (cm)
Distance (cm)
1500
1000
1000
500
500
0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time (min) Time (min)
g h 2500
D1-eNpHR D1-eYFP
D1-eNpHR
2000
D1-eYFP
Distance (cm)
1500
1000
500
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time (min)
i j
2500
D2-eNpHR D2-eYFP
D2-eNpHR
D2-eYFP
2000
Distance (cm)
1500
1000
500
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29
Time (min)
Supplementary Figure 5. Histological representation of optic fiber placement and locomotor activity of
D1-cre and D2-cre mice. Optic fiber placement of (a) D1-ChR2 and D1-eYFP mice (nD1-ChR2=7, neYFP=8), (d)
D2-ChR2 and D2-eYFP mice (nD2-ChR2=9, neYFP=8), (g) D1-eNpHR and D1-eYFP (nD1-eNpHR=5, neYFP=5), (i) D2-
eNpHR and D1-eYFP (nD2-eNpHR=5, neYFP=5). No differences in locomotor activity of stimulated animals were
found (b, c, e, f, h, j). Data are represented as mean ± SEM. Blue stripes correspond to optogenetic stimulus.
Supplementary Figure 6
a
8000
6000
Distance (cm)
4000
2000
0
FP
FP
ng
ie
or
ge
br
lo
sh
eY
eY
on
2
2
1-
2-
hR
hR
ol
hR
D
D
pr
C
C
C
2
2-
1-
2-
hR
D
D
D
C
1-
D
Normalized activity
Normalized activity
4
6 pMSN
2
4
0
D1-MSN brief optical stimulation
2
-2
0 -4
-2 -6
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
01
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
10
10
11
12
12
13
13
14
15
15
16
16
17
18
10
10
11
12
12
13
13
14
15
15
16
16
17
Time (s) Time (s)
c VTA - pDAergic
d VTA - pGABAergic
8 6
pDAergic
6 4
2
Normalized activity
Normalized activity
4
0
2
-2
0
-4
-2
-6
-4 -8 pGABAergic
-6 -10
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
10
10
11
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12
13
13
14
15
15
16
16
17
10
10
11
12
12
13
13
14
15
15
16
16
17
Time (s) Time (s)
e NAc
f VP
10 8
6 pGABAergic
8 pMSN
Normalized activity
Normalized activity
4
6
2
4
0
D2-MSN brief optical stimulation
2
-2
0 -4
-2 -6
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
10
10
11
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15
15
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16
17
10
10
11
12
12
13
13
14
15
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16
16
17
g VTA - pDAergic
h VTA - pGABAergic
8 6
6 pDAergic 4
Normalized activity
2
Normalized activity
4
0
2
-2
0
-4
-2
-6
-4 -8 pGABAergic
-6 -10
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10 1
21
81
12 1
12 1
13 1
13 1
14 1
15 1
15 1
16 1
16 1
17 1
41
12
18
24
30
36
42
48
54
60
66
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78
84
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12
18
24
30
36
42
48
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60
66
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78
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4
0
6
2
8
4
0
6
2
8
10
10
11
12
12
13
13
14
15
15
16
16
17
10
11
Supplementary Figure 7. Electrophysiological responses of NAc, VP and VTA during 30 minute protocol
of brief stimulation. A 30 minutes session with 1s stimulation (12.5 ms pulses at 40 Hz) every min (which is
equivalent to CPP test) was recorded, in order to evaluate the neuronal responses to repetitive stimulations. (a)
Temporal variation of NAc pMSNs activity with D1-MSN brief stimulation; note the increase in activity during
optical stimulation (blue; stim vs no stim, p<0.0000, n=12 cells/6mice). (b) Temporal variation of VP pGABAergic
neuronal activity with D1-MSN brief stimulation; note the decrease in activity during optical stimulation (blue;
stim vs no stim, p<0.0000, n=9 cells/5mice). (c) Temporal variation of VTA pDAergic neuronal activity with D1-
MSN brief stimulation; note the increase in activity during optical stimulation (blue; stim vs no stim, p<0.0000,
n=6 cells/5mice). (d) Temporal variation of VTA pGABAergic neuronal activity with D1-MSN brief stimulation;
note the decrease in activity during optical stimulation (blue; stim vs no stim, p<0.0000, n=3 cells/5mice). (e)
Temporal variation of NAc pMSNs activity with D2-MSN brief stimulation; note the increase in activity during
optical stimulation (blue; stim vs no stim, p<0.0000, n=12 cells/5mice). (f) Temporal variation of VP pGABAergic
neuronal activity with D2-MSN brief stimulation; note the decrease in activity during optical stimulation (blue;
stim vs no stim, p<0.0000, n=10 cells/5mice). (g) Temporal variation of VTA pDAergic neuronal activity with D2-
MSN brief stimulation; note the increase in activity during optical stimulation (blue; stim vs no stim, p<0.0000,
n=6 cells/5mice). (h) Temporal variation of VTA pGABAergic neuronal activity with D2-MSN brief stimulation;
note the decrease in activity during optical stimulation (blue; stim vs no stim, p<0.0000, n=3 cells/5mice). Data
are represented as mean ± SEM.
Supplementary Figure 8
a b
NAc VP
10 8
pMSN
pGABAergic
8 6
Normalized activity
Normalized activity
4
6
2
4
D1-MSN prolonged optical stimulation
0
2
-2
0 -4
-2 -6
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
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10
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17
10
10
11
12
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13
13
14
15
15
16
16
17
Time (s) Time (s)
c VTA - pDAergic
d
VTA - pGABAergic
8 6
pDAergic
6 4
Normalized activity
Normalized activity
4
0
2
-2
0
-4
-2
-6
-4 -8 pGABAergic
-6 -10
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
01
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
10
10
11
12
12
13
13
14
15
15
16
16
17
18
10
10
11
12
12
13
13
14
15
15
16
16
17
Time (s) Time (s)
e NAc
f
VP
10 8
pMSN
6 pGABAergic
8
Normalized activity
Normalized activity
4
6
2
4
D2-MSN prolonged optical stimulation
0
2
-2
0 -4
-2 -6
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
12
18
24
30
36
42
48
54
60
66
72
78
84
90
96
10
10
11
12
12
13
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14
15
15
16
16
17
10
10
11
12
12
13
13
14
15
15
16
16
17
Time (s) Time (s)
g VTA - pDAergic
h VTA - pGABAergic
8 6
6 pDAergic 4
2
Normalized activity
Normalized activity
4
0
2
-2
0
-4
-2
-6
-4 -8 pGABAergic
-6 -10
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
10 1
21
81
12 1
12 1
13 1
13 1
14 1
15 1
15 1
16 1
16 1
17 1
41
1
61
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
21
81
41
01
61
21
81
41
01
61
21
81
41
12
18
24
30
36
42
48
54
60
66
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78
84
90
96
12
18
24
30
36
42
48
54
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66
72
78
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90
96
4
0
6
2
8
4
0
6
2
8
10
10
11
12
12
13
13
14
15
15
16
16
17
10
11
Supplementary Figure 8. Electrophysiological responses of NAc, VP and VTA during 30 minute protocol
of prolonged stimulation. A 30min session with 60s stimulation (12.5ms pulses at 40 Hz) every 2min (which is
equivalent to CPP test) was recorded, in order to evaluate the neuronal responses to repetitive stimulations. (a)
Temporal variation of NAc pMSNs activity with D1-MSN prolonged stimulation; note the increase in activity
during optical stimulation (n=9 cells/5mice). (b) Temporal variation of VP neuronal activity with D1-MSN brief
stimulation; note the decrease in activity in the first 1-3s of optical stimulation and normalization thereafter (n=9
cells/5mice). (c) Temporal variation of VTA pDAergic neuronal activity with D1-MSN prolonged stimulation; note
the increase in activity in the first second of optical stimulation and normalization thereafter (n=10 cells/6mice).
(d) Temporal variation of VTA pGABAergic neuronal activity with D1-MSN prolonged stimulation; note the
decrease in activity in the first 1-3s of optical stimulation and normalization thereafter (n=3 cells/6mice). (e)
Temporal variation of NAc pMSNs activity with D2-MSN prolonged stimulation showing the increase in activity
during optical stimulation (n=10 cells/5mice). (f) Temporal variation of VP pGABAergic neuronal activity with D2-
MSN prolonged stimulation; note the decrease in activity during the first 2s of optical stimulation, and significant
increase in the remaining stimulation period (n=10 cells/5mice). (g) Temporal variation of VTA pDAergic
neuronal activity with D2-MSN prolonged stimulation. pDAergic neurons increase activity during the first 2s of
optical stimulation, and then present a substantial decrease in activity in the remaining stimulation period (n=9
cells/5mice). (h) Temporal variation of VTA pGABAergic neuronal activity with D2-MSN prolonged stimulation.
No significant effect of optical stimulation was found (n=3 cells/5mice).
Statistical significance is provided in Table 1 and Table 2 in Supp. Material.
Supplementary Figure 9
D1-MSN brief optical stimulation
a pCINs (4 cells)
b c d
pFSs (3 cells)
activity activity
20 25 10 10
5
20
activityNormalized
15 150 8
Firing rate (Hz)
Latency (ms)
pCINs A
15 5 pCINs B 6
Normalized
-100
10
-5
20 Time
10 1 6 11 (s) 16pFSs 21
15
-10 4
10
-15
5 5 1 6 11 16 21
Normalized
5 0 2
-5 Time (s)
pFSs
-10
0 0 -15 0
1 6 11 16 21
e
re
Ss
te
te
r
in
in
SN
IN
fo
fo
Af
Af
pF
ur
Time (s)
ur
pC
Be
Be
pM
D
-5
10
Latency (ms)
pCINs A
15 -105 pCINs B 6
-150
10
-5 1
35 21 41 61 81 101 121
10 30 pFSs 4
-10
25
-15
20 1 pFSs A
5 15 6 11 16 21
5 pFSs B 2
10 Time (s)
5
0
0 0 -5 0
1 21 41 61 81 101 121 141
Ss
e
1s
1s
r
te
te
SN
IN
59
59
r
Time (s)
fo
fo
pF
Af
Af
pC
Be
Be
pM
D2-MSN brief optical stimulation
i j pFSs (4 cells)
k l
pCINs (6 cells)
25
activity activity
25 10 14
5
20 12
20
activityNormalized
150
10
Firing rate (Hz)
Firing rate (Hz)
10
-5
Latency (ms)
15 5 pCINs A
15 pCINs B
NormalizedNormalized
-100 8
-5 1 6 11 16 21
20 Time (s) 6
10 10 -10
15 pFSs
10
-15
5 1 6 11 16 21 4
5 5 0 Time (s) pFSs A
2
-5 pFSs B
-10
0 0 -15 0
1 3 5 7 9 11 13 15 17 19 21
e
r
e
Ss
te
te
in
SN
IN
r
in
fo
fo
Af
pF
Af
ur
ur
Time (s)
pC
Be
Be
pM
D
D
25 25 20 14
15
10 12
20 20 5
Normalized
150
10
Firing rate (Hz)
-5
10 pCINs A
rate (spikes/s)
Latency (ms)
15 15 -105 pCINs B
-15 8
Normalized firingNormalized
0
-5 1
35 21 41 61 81 101 121 141
6
10 10 30 pFSs
-10
25
-15
20 1 pFSs 4
15 6 11 16 21
5 5 10
5 Time (s) 2
0
0 0 -5 0
1 21 41 61 81 101 121 141
s
Ss
e
1s
1s
r
te
te
SN
IN
59
59
r
Time (s)
fo
fo
pF
Af
Af
pC
Be
Be
pM
Latency (msec)
10
AP: +1.3
VP 5
Animal 1 Animal 2 Animal 3 Animal 4
VP
(p A)
A)
VP
(p A)
A)
D
AB
AB
(p
(p
G
G
A
A
VT
VT
A
A
AP: -0.1
VT
VT
VTA
Animal 1 Animal 2 Animal 3 Animal 4
D1-MSN brief D1-MSN prolonged
optical stimulation optical stimulation
AP: -3.2
c d
NAc
Animal 1 Animal 2 Animal 3 Animal 4
200
150
100
Latency (msec)
50
10
AP: +1.3
8
VP
Animal 1 Animal 2 Animal 3 Animal 4 6
4
2
0
VP
A)
A)
VP
A)
A)
D
AB
AB
(p
(p
G
G
A
A
(p
(p
VT
VT
A
A
VT
VT
AP: -0.1
AP: -3.2
Supplementary Figure 10. Electrophysiological correlates of ventral pallidum and ventral tegmental
area. (a) Placement of recording electrodes in the NAc, VP and VTA of D1-cre mice. (b) Latency to fire of VP
and VTA neurons in response to brief (1s) or prolonged (60s) NAc D1-MSNs stimulation. (c) Placement of
recording electrodes in the NAc, VP and VTA of D2-cre mice. (d) Latency to fire of VP and VTA neurons in
response to brief (1s) or prolonged (60s) NAc D2-MSNs stimulation. In b and c, depicted in green are the
neurons that increase activity in response to D1-MSN stimulation; in red are those that are inhibited (20%
difference from baseline activity, as described in the methods). nD1-ChR2=4 mice (npGABA VP=40 cells, npDA VTA= 35
cells, npGABA VTA=12 cells); nD2-ChR2=4 mice (npGABA VP=62 cells, npDA VTA= 42 cells, npGABA VTA=20 cells). Data are
represented as mean ± SEM.
Supplementary Figure 11
a b c
Optical stimulation 50 600
in no-stimulus chamber
500
Pre-test Conditioning Post-test -50
15 minutes 30 minutes 15 minutes
400
Time (s)
-100
No stim ON OFF No stim **
Prolonged stimulation -150 300
+ -200
Naltrindole (Nal) 200
-250 ***
-300 100
OFF ON
al
al
N
N
FP
2
hR
eY
C
1-
1-
D
D
d e f
Optical stimulation 50 600
in no-stimulus chamber
500
Pre-test Conditioning Post-test -50
15 minutes 30 minutes 15 minutes
400
Time (s)
-100
No stim ON OFF No stim
-150
***
Prolonged stimulation 300
+ -200
***
norbinaltorphimine (nor-BNI) 200
-250
-300 100
OFF ON
I
I
N
N
r-B
r-B
no
no
FP
2
hR
eY
C
2-
2-
D
Supplementary Figure 11. Control experiments for optogenetic modulation with opioid receptors
antagonists delivery. (a-c) Injection of DOR antagonist (naltrindole, nal, 0.1µg/0.5µl) in the VTA of D1-cre
mice, 20 minutes prior to CPP conditioning, had no effect on the D1-MSN prolonged stimulation-induced
aversive behaviour (nD1-eYFPveh=5, nD1-eYFPnal=5, nD1-ChR2veh=6, nD1-ChR2nal=6). (d-f) Injection of KOR antagonist
(norbinaltorphimine, Nor-BNI, 1µg/0.5µl) in the VP of D2-cre mice, 20 minutes prior to CPP conditioning, had no
effect on the D2-MSN prolonged stimulation-induced aversive behaviour (nD2-eYFPveh=5, nD2-eYFPnor-BNI=5, nD2-
ChR2veh=6, nD2-ChR2veh=6). **p<0.01, ***p<0.001. Data is represented as mean ± SEM.