of 49
www.sciencemag.org/
cgi/
content/
full/
science.
aat4422
/DC1
Supplementary
Material
s for
Ultrafast neuronal imaging of dopamine dynamics with designed
genetically encoded sensors
Tommaso Patriarchi, Jounhong Ryan Cho, Katharina Merten, Mark W. Howe,
Aaron Marley, Wei
-Hong Xiong, Robert W. Folk, Gerard Joey Broussard,
Ruqiang Liang, Min Jee Jang, Haining Zhong, Daniel Dombeck, Mark von Zastrow,
Axel Nimmerjahn, Viviana Gradinaru, John T. Williams, Lin Tian*
*Corresponding author. Email:
lintian@ucdav
is.edu
Published
31 May
2018 on
Science
First Release
DOI:
10.1126/science.
aat4422
This PDF file includes:
Materials and Methods
Figs. S1 to S16
Captions for data S1 to S3
References
Other supplementary material for this manuscript includes:
Data S1 to S3
2
Materials and Methods
Molecular Cloning
All constructs were designed using circular polymerase extension cloning (CPEC)
(
37
)
,
restriction cloning, and gBlock gene fragments (Integrated DNA Technologies). Sequences
coding for a hemagglutinin (HA) cleavable secretion motif and a FLAG epitope were placed at
the 5’
-
end of the construct as in
(
23
)
. HindIII and NotI cut sites were placed at the 5’
-
and 3’
-
end, respectively, for cloning into pEGFP
-
N1
(Addgene)
to generate
all p
CMV
con
structs
.
BamHI and HindIII sites were introduced via PCR for final subcloning onto pAAV.
hSynapsin
1
and pAAV.
CAG
vectors.
To maximize coupling between conformational changes and chromophore fluorescence, we
chose to use a cpGFP module (LSS
-
LE
-
cpGFP
-
LP
-
DQL)
from GCaMP6
(
18
)
for insertion into
DRD1
via CPEC
.
For screening linker variants, we generated a linker library by first creating an
insert DNA carrying a 2 aminoacid
-
long randomized linker on each side of cpGFP (LSS
-
xx
-
cpGFP
-
xx
-
DQL). Single colonies were manually picked and grown overnight as described in
(
38
)
. All
sensor
sequences
generated
in this study are listed in the
Supplementary
Data
S1
file.
Structural modeling and sequence alignments
Sequence alignments were performed using Jalview s
oftware (UK)
(
39
)
using a
percentage
identity color map
.
Inactive conformation of the sensor was predicted with rosetta_cm protocol
of rosetta 3 (v
ersion 2015.31)
(
40
)
. Primary sequence of sensor design was threaded with partial
thread ro
utine onto template PDB structures of inactive
β
2
adrenergic receptor
(
41
)
(ID: 2RH1)
and unbound state of GCaMP3
(
42
)
(ID: 4IK3). The threaded structures were then hybridized
together with rosetta_cm protocol for membrane protein
(
43
)
. A total of 374 PDB structures
3
were generated by rosetta_script routine. Structure with lowe
st total score was considered the
final model.
Cell culture
,
i
maging and quantification
HEK293T cells (ATCC #1573) and U2OS cells (ATCC #HTB
-
96) used in this study we
cultured and transfected as in
(
23, 44
)
. Primary hippocampal neurons were freshly isolated as
previously described
(
45
)
. Neurons were infected using AAVs (1 x 10
9
GC/ml) at DIV5, two
weeks prior to imaging. Prior to imaging
,
cells were washed with HBSS (Life Technologies).
Cell i
maging was performed
using
a 40X oil
-
based objective on an inverted Zeiss Observer
LSN710 confocal microscope with 488/513 ex/em wavelengths. For testing sensor responses,
neurotransmitters/drugs were directly applied to the bath during the imaging session. For
neurotransmitter tit
rations, a dual buffer gravity
-
driven perfusion system was used to exchange
buffers between different drug concentrations. Surface labeling was achieved as described
previously
(
23, 44
)
. One
-
photon emission spectrum for the sensors was d
ertermined
using the
lambda
-
scan function
of the
confocal
microscope
. Two
-
photon emission spectr
um
w
as
obtained
with a 40X water
-
based object
ive on a SliceScope (Scientifica) and w
as
used to obtain the
normalized two
-
photon cross
-
section using a custom
-
made script on M
ATLAB
.
For ROIs
selection, masks were generated either on the cell membrane or around the cytosol, depending on
the experiment,
using the threshold function in Fiji. We calculated spatial movies and images of
Δ
F/F
in response to an applied ligand as
(
)
'
()*+,,-.+
'
()*+,-.+
/
with
(
)
the pixel
-
wise
fluorescence value at each time,
t
, and mean fluorescence in t
ime points prior to ligand
application,
'
()*+,-.+
. To avoid the possibility of infinite pixel values, we added a small offset to
each pixel in
'
()*+,-.+
. Based on the
Δ
F/F maps, we calculated a corresponding SNR map as
4
×
2
'
()*+
,
-.+
.
Δ
FF heatmaps were generated using a
custom
M
ATLAB
script. Surface
expression of sensors was quantified as the ratio of membrane fluorescence over cytoplasm
fluorescence. For titration curves, Kd values were obtained by fitting the data with a Hill
function on Igor Pro
(WaveMetrics)
.
Dopamine
-
uncaging
Uncaging experiments on neuronal dendrites were performed in the presence of 100 μM
caged
-
dopamine (carboxynitroveratryl
-
DA, CNV
-
DA
(
46
)
) in the
HBSS
. Optical recordings
were performed at 153 ms/frame scan rate. Uncaging was achieved by shining 405 nm light
(40% laser power) on a 2 μm wide circular region selected 10 μm away from the dendrite
surface.
Internalization assay with flow
cytometry
24 hours post transfection, cells were re
-
plated onto 6 well dishes. The following day,
surface levels of receptors were assayed by addition of Alexa
-
647
-
conjugated M1 antibody
(Sigma) for 45 minutes, as described previously
(
23
)
. Fluorescence intensity profiles of cells
populations (>5000 cells) were measured using a FACS
-
Calibur instrument (BD Biosciences
).
Each condition was performed in duplicate. Internalization was calculated by measuring the
fraction of surface fluorescence remaining after 30 minutes of 1
μ
M SKF81297 (Tocris) and
divided by the non
-
treated condition.
5
Luminescence
-
based cAMP assay
E
xperiments were conducted as previously described
(
23
)
. Briefly, cells were transfected
with the cyclic
-
permuted lucif
erase pGLOSensor
-
20F plasmid (Promega) and then treated with
luciferin (GoldBio) in phenol and serum free media for 1 hour in a 24 well dish. Luminescence
values for SKF81297 (Tocris) treated conditions were measured at their peaks and normalized
with refe
rence to 10
μ
M forskolin (Sigma) at its peak.
Total Internal Reflection Fluorescence Microscopy (TIRF
-
FM) Live Imaging.
Live cell TIRF
-
FM was conducted with a Nikon Ti
-
E inverted microscope at 37°C
in
a
controlled humidity and CO
2
controlled chamber as de
scribed previously
(
23
)
. D1 specific
agonist S
KF81297 (Tocris) was added at 1
μ
M while D1 antagonist SCH23390 (Tocris)
was
added at 10
μ
M.
Preparation and transfection of cortical organotypic slice cultures
Rat hippocampal slice cultures were prepared from P6
P7 pups as previously described
(
47, 48
)
. pCMV
-
dLight
1.
2 (12
μ
g) and pCMV
-
mCherry (3
μ
g) were mixed with single cell
electroporation solu
tion (160 mM NaCl , 5.4 mM KCl, 12mM MgCl
2
, 2 mM CaCl
2
, 5 mM
HEPES, pH 7.4 ) to total volume of 36
μ
l. One
-
week old hippocampal cultured slices were
transfected with single cell electroporation
(
49
)
. 5
7 days after transfection, single
hippocampal culture
d slice was transferred to the imaging chamber in a custom built two
-
photon imaging setup. Slices were perfused in 10ml gassed artificial cerebral spinal fluid
(ACSF) containing 4mM Ca
2+
, 4mM Mg
2+
, 0.5
μ
M TTX and 10
μ
M cocaine (DA transporter
blocker) durin
g imaging.
6
AAV viral production
All dLight
1
AAV constructs were cloned in the laboratory, and viruses were produced by the UC
Davis Vision Center Vector Design and Packaging Core facility. The viral titers of the viruses
used in this study were: AAV1.
CAG
.f
lex.tdTomato, ~8 x 10
12
genome copies (GC)
/mL
(University of Pennsylvania); AAV5.
hSyn
apsin
1
.
flex
.ChrimsonR.tdTomato, ~4 x 10
12
GC
/mL
(University of North Carolina); AAV1.
hSynapsin
1
.NES
-
jRGECO1a, ~3 x 10
13
GC
/mL
(University of Pennsylvania); AAV1.
hSynapsin
1
.dLight
1.
1, ~1 x 10
12
GC/mL; AAV1.
hSynapsin
1
.dLight
1.
2, ~2 x 10
12
GC/mL; AAV9.
hSynapsin
1
.dLight
1.
2, ~4
×
10
12
GC/mL;
AAV9.
CAG
.dLight
1.
1, ~7
×
10
11
GC/mL; AAV9.
CAG
.
control_sensor
, ~1 x 10
12
GC/mL.
Animals
Animal studies were conducted in compli
ance with
the
Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal
Care and Use Committee (IACUC) at the University of California, Davis or the relevant
institutional regulatory body
. Wild type rats were used in this study (0
-
2 months old) for
neuronal and organotypic slice culture preparation and two
-
photon imaging in brain slice.
Dopamine receptor
-
D1 (Drd1)
-
Cre mice (4
-
6 months old, Jackson Labs, Strain B6; 129
-
Tg(Drd1
-
cre)120Mxu/MM
Jax) were used for two
-
photon imaging in the dorsal striatum.
Vesicular GABA transporter (VGAT)::IRES
-
Cre
(
50
)
(Jackson Labs, Slc32a1tm2(cre)Lowl/J)
and Tyrosine hydroxylase (TH)::IRES
-
Cre
(
51
)
(Jackson Labs, B6.129X1
-
Thtm1(cre)Te/Kieg)
knock
-
in mice between 3 and 5 m
onths old were used for in vivo imaging using fiber photometry
and for optogenetic manipulations. Wild type
male
mice (2 to 5 months old,
C57BL6/J, Jackson
Laboratories)
were used for two
-
photon in vivo imaging in the cortex. Sample sizes (number of
7
mice)
for each experiment are stated in main text. All animals were group housed in standard
plastic cages on a 12
-
hour light/dark cycle with food and water available
ad libitum
, except for
mice trained on the visuomotor association task (see below). These mice
were house individually
on a reverse 12
-
hour light/dark cycle.
Anesthesia was performed with 4.5% isoflurane for
induction and 2%
for
maintenance.
Viral injections
Injection procedures were essentially identical to those described in
(
10, 52
-
54
)
with a few
exceptions.
For dopamine imaging in brain slice,
male and female Sprague
-
Dawley rats (p23
-
2
7) were
used for intracerebral microinjections. Briefly, anesthetized rats were immobilized in a
Stereotaxic Alignment System (Kopf Instruments). AAV1.
hSyn
apsin1
.dLight
1.
2 (200 nl) was
injected uni
-
laterally into the dorsal striatum using the following coo
rdinates from bregma (in
mm): 0.48 anteroposterior (AP),
±
2.19 mediolateral (ML), 4.69 dorsoventral (DV).
For dopamine imaging in the dorsal striatum, mice were injected in the dorsal striatum at
two caudal locations (+0.6 and +0.2 AP and 2.1mm ML to bregm
a) and 3 depths below the
surface (
-
1.6mm,
-
1.9mm, and
-
2.2mm; 100 nL at each depth, total 0.6uL) with either AAV1.
hSynapsin1
.dLight
1.
1 or AAV1.
hSynapsin1
.dLight
1.
2 in combination with
AAV1.
CAG
.flex.tdTomato (diluted 1:100 in PBS).
The flex
-
tdTomato in o
ur experiments is
expressed in roughly half of the SPNs (Drd1
-
cre mice) to avoid potential issues, such as
competition of expression when two AAVs are co
-
expressed in the same neurons.
For dopamine imaging using fiber photometry,
AAV9.
CAG
.dLight
1.
1 or
AAV9.
CAG
.
control_sensor
were injected into the NAc (+1.3 mm AP,
-
1.25 mm ML,
-
4.25
8
mm DV) and cre
-
dependent AAV encoding ChrimsonR
-
tdTomato was injected into the VTA
(
-
3.3 mm AP,
-
0.5 mm ML,
-
4.3 mm DV). For dual color photometry experiments, AAV
encoding
jRGECO1a was 1:1 mixed with dLight
1.
1 virus and injected into the NAc region
(+1.3 mm AP,
-
1.
5 mm ML,
-
4.25 mm DV). A 10 μL NanoFil microsyringe (World Precision
Instruments) with a blunt 35
-
gauge needle was used for viral injection and manipulated by a
mi
crosyringe pump (UMP3, World Precision Instruments) and a controller (Micro4, World
Precision Instruments). 500 nL of AAV was slowly injected into the target coordinates over
10 minutes, and additional 10 minutes were waited to allow diffusion. The needle
was slowly
withdrawn over 10
-
15 minutes.
For dopamine imaging in the cortex,
anesthetized animals (4% isoflurane for induction;
~1.5% during surgery) were positioned in a computer
-
assisted stereotactic frame with digital
coordinate readout and atlas target
ing (Angle Two, Leica Biosystems Inc., Buffalo Grove,
IL)
.
M
icropipettes were loaded with virus solution and slowly lowered into the brain under a
~32º injection angle until the target depth (~0.2 mm) was reached. Manual pressure was applied
to a 30 mL syr
inge connected to the injection pipette. Virus solution was slowly injected over a
period of 5
10 min. Once desired injection volume (200
-
400 nl) was delivered, the syringe’s
pressure valve was locked and position maintained for approximately 10 min to all
ow virus to
spread and to avoid backflow upon needle retraction. Each mouse received two injections of
AAV9.
hSynapsin1
.dLight
1.
2 (1:50 dilution). Injection coordinates were (AP 3
-
3.2 mm, ML 1
-
1.2 mm) and (AP 1.5 mm, ML 1
-
1.5 mm).
9
Slice preparation
5%
isoflurane was used to deeply anesthetize rats prior to decapitation. Brains were rapidly
removed and placed in modified Krebs buffer containing (in mM) 126 NaCl, 2.5 KCl, 1.2
MgCl
2
, 2.4 CaCl
2
, 1.4 NaH
2
PO
4
, 25 NaHCO
3
, 11 D
-
glucose, and with 10
μ
M MK
-
801.
H
orizontal slices (245
μ
m) were taken using a Leica vibratome and allowed to recover for at
least 30 minutes prior to use in Krebs with MK
-
801 being continuously bubbled with 95/5%
O
2
/CO
2
. Slicing and recovery was done at elevated temperatures (30
-
34
°
C). Following recovery,
slices were secured in a recording chamber maintained at 34
°
C and perfused with modified
Krebs at a rate of 3 ml/min.
Surgical procedures for in vivo imaging
Cannula implant for in vivo imaging in dorsal striatum
Two weeks post
-
i
njection, a chronic imaging window was implanted over the external
capsule fibers above the striatal injection site as described previously
(
10
)
.
Briefly, a 2.75mm
craniotomy was performed on anaesthetized mice, and cortical tissue was slowly aspirated until
the white matter of the external capsule was exposed. A thin layer of Kwik
-
Sil (WPI) was
applied over the imaging region, and a metal cannula
covered at one end by a glass coverslip was
inserted into the aspiration site down to the fiber surface.
Optical fiber implantation for fiber photometry and optogenetics
Mice were anesthetized with isoflurane and carbogen mixture (4
-
5% for induction, 1.5
-
2% for maintenance) and carefully placed to a stereotaxic frame (Kopf Instruments) after
shaving their hair. Head skin was sterilized with chlorohexidine and a midline incision was
10
made using a sterile scalpel. The skull surface was exposed and cleaned wi
th sterilized cotton
swabs. Bregma and lambda points were identified and leveled to be at the same dorsal
-
ventral
axis. Small craniotomy holes were made with drill bits (#73 size, Kyocera) over the nucleus
accumbens (NAc) and ventral tegmental area (VTA).
After viral delivery, optical ferrule/fiber
(for VTA/optogenetics, 300 μm diameter, 5
-
6 mm cut length, NA 0.37, home
-
made; for
NAc/photometry, 400 μm diameter, 5 mm cut length, NA 0.48, Doric lenses) was mounted to
a stereotaxic cannula holder (Doric lense
s). First, a 400 μm fiber was lowered to the NAc
through a craniotomy hole and stopped at 200 μm above the virus injection target. A layer of
adhesive cement (C&B Metabond, Parkell Inc.) was applied to the anterior portion of skull
surface to strongly hold
the implanted ferrule. Care was taken not to apply Metabond near the
posterior craniotomy sites for VTA. Next, a 300 μm fiber was inserted to the VTA and
stopped at 500 μm above the injection site. Another layer of adhesive cement was applied to
hold the
second fiber. After adhesive cement was fully dried, a thick layer of dental cement
(Lang Dental) was applied to build a head cap.
Head
plate
and cranial window
implantation for in vivo imaging in the cortex
.
A few weeks after viral injections,
mice were
implanted with a head plate and cranial
window on a custom surgical bed (Thorlabs Inc., Newton, NJ).
Body
temperature was
maintained at 36
37 °C with a DC temperature control system and ophthalmic ointment was
used to prevent eyes from drying. Skin was cl
eaned and disinfected with 70% ethanol and
Betadine. A small (~10 mm) incision was performed along the midline. The scalp was pulled
open and periosteum cleaned. First, a portion of the scalp was surgically removed to expose
frontal, parietal, and interpar
ietal skull segments. Then, the metal plate was affixed to the
11
bone with C&B Metabond Quick Adhesive Cement (Parkell Inc., Edgewood, NY). Next, to
enable chronic two
-
photon imaging, a custom
-
made cranial window was implanted, similar to
(
55
)
. The craniotomy (2.5 mm diameter) was centered around (AP 2 mm and ML 1.5 mm) and
sealed with a custom
three
-
layered cover glass assembly (each No.1 thickness) with the two
layers closest to the corte
x consisting of two pieces of circular 2.5 mm
-
diameter cover glass
and the outermost layer consisting of a circular 4 mm
-
diameter cover glass that rested on the
thinned skull. The dura mater was kept intact.
Behavioral experiments
Sucrose consumption
Mice were handled daily for 5
-
10 minutes to reduce anxiety associated with
experimenter’s handling. Following recovery from surgery (~7 days) mice were water
-
restricted to 1.5 mL per day and maintained at 85
-
90 % of ad libitum weight. All behavioral
experi
ments were carried out in an operant chamber within a sound
-
attenuating box (Lafayette
Instruments). Behavioral tasks were implemented and controlled by ABET II software
(Lafayette Instruments). TTL pulses were used to synchronize with fiber photometry
rec
ordings. After at least 7 days of water deprivation, mice were introduced to an operant
chamber and allowed to freely explore with a patch cord connected. 50 μL of 5 % sucrose
water was delivered every 60 seconds (total 10 times) so that mice can learn the
position of a
lick port. Lick was detected as a break of infrared beam at the lick port. This habituation
session was repeated two to three times for each mouse until they showed robust lick activity
(>400 licks per session). At the recording day, mice un
derwent the same sessions while
12
recording dLight1 fluorescence with fiber photometry.
For analysis, mean fluorescence values
were obtained from baseline (
-
10~
-
5 s from lick onset) and during consumption (0~5 s).
Unpredictable footshock delivery
Mice were
placed into an operating chamber with a patch cord connected for photometric
recordings. Five electric footshocks (0.6 mA for 1 second) were delivered with variable
intervals (randomly chosen from uniform distribution between 45 and 75 seconds) without
pr
edictive cues. This was performed after all other experiments, since such aversive stimuli
can induce sustained fear and anxiety to the context.
For analysis, mean fluorescence values
were obtained from baseline (
-
5~
-
1 s from lick onset) and during footsho
ck (0~2 s).
Cue
-
reward learning and extinction
In cue
-
reward learning sessions, CS was turned on for 10 seconds and US was available 7
seconds after CS onset, after variable inter
-
trial intervals (ITI). In cue
-
reward extinction sessions,
same CS was turne
d on for 10 seconds without US delivery after variable ITI.
In “expected reward delivery” trials, US was delivered after CS presentation, as in previous
learning sessions.
After sucrose consumption experiments, mice started appetitive Pavlovian
condition
ing, or cue
-
reward learning,
in
the same operant chamber. Conditioned stimuli (CS)
consisted of house
-
light and 70 dB 5kHz tone, and were turned on for 10 seconds with
variable intervals (randomly drawn from uniform distribution between 75 and 105 seconds)
.
Unconditioned stimulus (US) was 50 μL of 5 % sucrose water, available at the 7th second
after each CS onset. Pump sound from liquid dispenser was audible from 6th to 7th seconds.
13
CS
-
US pairings were repeated 20 times per session (therefore ~30 minutes fo
r each session).
Mice underwent total of 12 sessions for cue
-
reward learning. Chambers and lick port were
sanitized with Accel and 70% ethanol between animals to remove any odor. For all sessions,
lick data and photometry recordings were simultaneously obt
ained. Two to three days after
the last cue
-
reward learning sessions mice began cue
-
reward extinction. Identical CS were
given 30 times per session with variable intervals (between 45 and 75 seconds), now without
US delivery. Mice underwent total of 5 exti
nction sessions. To quantify CS
-
triggered
behavior, number of licks was counted during CS presentation. For photometry data, peak
fluorescence was obtained for CS (0~3 s after CS onset) and US (
-
2~5 s around US
consumption onset
).
US consumption onset for
each trial was defined as the lick bout onset
after US is available, where lick bout onset was detected with a threshold of 2 Hz. To examine
fluorescence response shift from US to CS (or vice versa), we calculated CS
-
US index,
defined as (CS response
US
response) / (CS response + US response).
Unexpected reward delivery and omission
After 12 cue
-
reward learning sessions, mice underwent reward prediction error
experiments. In “unexpected reward delivery” session, animals were exposed to normal CS
-
US pairing trials; but in 25% of trials, US was delivered without CS, so that mice can exp
lore
to the lick port without predictive cues and consume reward in an unexpected manner. Peak
fluorescence values after US consumption onset was obtained and compared between
expected and unexpected conditions. In “unexpected reward omission” sessions, US
delivery
was omitted in 4 out of 20 trials (4th, 8th, 13th, and 16th trials) after predictive CS onset. For
14
analysis, mean fluorescence values were obtained from baseline (
-
10~0 s from CS onset) and
after CS offset (10.5~11.5 s) and compared.
Visuomotor
learning task
Prior to surgical preparation and behavioral training, mice were handled/tamed on two
consecutive days to reduce stress during experiments. Following recovery from surgery (~7
days) mice were water
-
restricted to 25 ml kg
-
1
per day and mainta
ined at 80
-
85% of ad libitum
weight. Training was performed in a custom
-
built setup that included a color LCD monitor
(12.1" LCD Display Kit/500cd/VGA, ICP Deutschland GmbH) on which visual stimuli were
presented. To reduce noise in optical recordings, the
monitor was covered with a color filter
(R342 Rose Pink, Rosco Laboratories Inc.). During training, mice were head
-
fixed with a
custom
-
build head holder and placed on a spherical treadmill (Habitrail Mini Exercise Ball,
Animal World Network) facing the LC
D display. An optical encoder (E7P OEM, US Digital)
attached to the treadmill allowed measurement of both speed and direction of ball movement.
Water reward was delivered with a programmable syringe pump (NE
-
500 OEM Syringe Pump,
New Era Pump Systems, Inc.
). Behavioral signals were acquired through a data acquisition board
(PCI
-
6221, National Instruments) connected to a breakout box (BNC
-
2110, National
Instruments) and interfaced to MATLAB using the Data Acquisition Toolbox (Version
R2010bSP2, The MathWorks
Inc.). Behavioral task sequence was controlled by the MATLAB
-
based software MonkeyLogic (www.monkeylogic.net)
(
56, 57
)
. Custom
-
written functions were
added to MonkeyLogic to enable analysis
and control of ball rotation parameters. Treadmill
encoder signals, trial marker codes (generated by MonkeyLogic), and imaging data were
acquired in synchrony allowing running parameters, behavioral task events, and image frames to
15
be linked.
Once the mou
se stopped moving on the ball, a sequence of task events was initiated.
First, a blue square frame was displayed on the monitor and required the animal to continue to
stand still for a period of 10 s (ball rotation/velocity
2 mm/s). If the mouse continue
d to stand
still for the entire stand
-
still phase, a second stimulus, a filled blue square, was presented for 3 s,
instructing the mouse to initiate a run. If the mouse initiated sustained movement (ball
rotation/velocity >10 mm/s for >1s duration) during
the 3s stimulus phase, a water reward was
delivered. In 20% of pseudo
-
randomly selected trials the reward was withheld (“Unexpected
reward omissions”; light green traces in Fig. 5,
fig.
S1
5
and S1
6
). If no running occurred, the
trials counted as a miss trial. If the animal began to move during the 10 s stand
-
still phase (ball
rotation/velocity > 2 mm/s), the trial was aborted and no water reward was delivered
(“Spontaneous run trials”; pink traces).
The mouse was able to initiate a new trial after an
intertrial interval (ITI) of 5 s.
During the first two days of training, mice spent ~15
-
30 min/day in the setup to become
accustomed to head restraint. Mice were then trained daily for 60
-
90 min during which they
performed ~300
-
700 trials. Task parameters were adjusted depending on individ
ual animal’s
performance. Initially, the duration of the stand
-
still phase was set to 3 s, the stimulus phase to
20 s, and the running threshold to 2 mm/s. This increased the chance for the mouse to receive the
task
-
dependent reward. The stand
-
still phase
was then progressively extended and the stimulus
phase shortened to establish the association between stimulus onset and running onset. Training
continued until median reaction times (RT) had dropped below 1500 ms, which indicated that
mice had learned to
associate the visual cues with the desired behavior. Task proficiency was
typically reached within 5
-
7 days. During the initial training phase all successful “Go” trials
16
were rewarded. Unexpected reward omission trials were introduced during imaging sessio
ns
only.
In vivo optogenetic and pharmacological manipulations
For optogenetic experiments, a patch cord (1 m length, 1.25 mm zirconia ferrule, 300 μm
diameter fiber, Doric lenses) was used to connect to a ferrule on an animal’s head cap and a
swivel com
mutator (fiber
-
optic rotary joints, Doric lenses) to allow free movement. Another
patch cord was connected from the commutator and to a 635 nm diode
-
pumped solid
-
state
laser (Changchun New Industries Optoelectronics Technology). The intensity of laser was
measured with a power meter (PM100D, Thorlabs) and calibrated to be 10 mW at the fiber
tip. An external TTL pulse generator (OTPG4, Doric Lenses) was used to control the laser
output. For activating VTA DA neurons, 5 Hz, 10 Hz, and 20 Hz of 10 ms pulses we
re
applied for 2 seconds. For activating VTA GABA neurons, 40 Hz pulses were used
(
58
)
.
These pulse trains were repeated 40 times per animal with intervals of 30 seconds.
For
pharmacological manipulations, the following drugs were used: DRD1 antagonist (SCH
-
23390, 0.25 mg/kg) and selective DA reuptake inhibitor (GBR
-
12909, 10 mg/kg)
(
59
)
.
Animals were injected with these drugs or saline (0.9% sodium chloride) through
intraperitoneal delivery 30 minutes before optogenetics/photometry experiments
. Mean or
peak fluorescence values were obtained before (
-
5~
-
1 s from onset), during (0.5~2 s) or after
(4~10 s) photoactivation.
17
Data acquisition and analysis for ex vivo and in vivo imaging
Ex vivo imaging in rat brain slice
Imaging was carried out i
n a custom built 2
-
photon microscope. Data were acquired and
collected using ScanImage software
(
60
)
. Slices were scanned at different frequencies,
depending on the experiment, using 920 nm light. Electrical stimulation through a glass mono
-
polar electrode placed within the slice near the area of imaging was used to evoke dopamine
release. Experiments w
ere carried out with a scan rate of 2 (128x128 pixels) or 15 (32x32 pixels)
Hz. The fluorescence over the entire field (20x20
μ
m) was measured for each frame and plotted
against time. Drugs were applied by superfusion. Line scans were taken at a 500 Hz fra
me rate.
To measure the dopamine response curve, two
-
photon images were taken every minute. Image
analysis was performed using custom software written in MATLAB.
In vivo imaging in mouse dorsal striatum
Treadmill velocity and acceleration were sampled at
1000Hz by a rotary encoder (E2
-
5000,
US Digital) attached to the axl
e
of the treadmill. Two
-
photon imaging was performed as
described previously
(
61
)
, using the same collection optics, but without the electric lens. 920 nm
laser light was used for excitation and 1024x512 pixels time series datasets were acquired at 30
Hz. Imaging sessions began after mice were acclimated to head fixation and ran fr
equently on
the treadmill (~1
-
3 days). Imaging data was collected over 1
-
2 days from dorsal striatum fields
ranging from 250 to 450
μ
m in diameter. All analyses were carried out using custom software
written in MATLAB. Time series movies were motion corre
cted on the static red channel using
algorithms described previously
(
62
)
and x and y shifts from the red were used to correct the
green channel. Mean whole field fluorescence was calculated from a large, hand
-
selected ROI
18
con
taining all regions of the field with visible cellular structure in the red td
-
Tomato channel.
Fluorescence time series were converted to
Δ
F/F by normalizing signals to an 8th percentile
baseline
(
62
)
within a sliding window (
+/
-
15s around each point) to correct for slow drift and
bleaching. Significant positive
-
going transients were calculated as previously described
(
62
)
.
In vivo imaging with fiber photometry
Fiber photometry was performed as in
(
28, 63, 64
)
. For dual color imaging a three LED
system was used (FMC7, Doric Lenses): 490 nm for dLight1, 565 nm for jRGECO1a, and 405
nm to be used as isosbestic wavelength for both indicators.
The following dichroic filters and
excitation/emi
ssion filters were used (all from Semrock): Di02
-
R405, Di02
-
R442, FF495
-
Di03,
FF552
-
Di02, FF593
-
Di03, FF01
-
405/10, FF01
-
433/24, FF01
-
475/28, FF02
-
520/28, FF01
-
565/24 and FF02
-
641/75
(
28, 63, 64
)
.
Acquired photometry data were processed with custom
-
written codes in MATLAB. Raw data
from each channel were low
-
pass filtered at 25 Hz (for
single color imaging) or 12 Hz (for dual color imaging) using a 2nd order Butterworth filter
with zero
-
phase distortion. To calculate
Δ
F/F time series, a linear fit was applied to
the
405
nm
signals
a
nd aligned to the 490 and 565 nm signals. The fitted 405 nm signal was
subtracted from 490 and 565 nm channels, and then divided by the fitted 405 nm signal to
yield
Δ
F/F values. Dual color imaging data were down
-
sampled to 100 Hz. In Pavlovian
conditionin
g,
Δ
F/F time
-
series signal is further normalized using robust Z
-
score (subtraction
of median and division by median absolute deviation, calculated from the entire session) to
account for potential differences in signal variance across animals and sessions.
Photometry
signals were then extracted around relevant behavioral events (e.g. lick onset, footshock
delivery, CS onset) and averaged. Power spectral density was estimated for averaged
19
dLight1/
control sensor
fluorescence data upon optogenetic stimulation
of different
frequencies. We used Welch’s method with 2
-
second window size and 50% overlap to
estimate power from 0.5 to 25 Hz in 0.1 Hz step.
In vivo two
-
photon imaging in the cortex.
Once mice had reached proficiency on the task, they were imaged daily for up to 9 days.
Imaging was
performed using a resonant scanning two
-
photon microscope (Sutter Instrument)
equipped with a pulsed femtosecond Ti:Sapphire laser (Chameleon Ultra II, Cohe
rent). dLight
1
fluorescence was
excited with
910
nm
light, and
detected using a ET525/70M emission filter
(
Chroma Technology Corp.
) and H7422
-
40 GaAsP photomultiplier tube (Hamamatsu
Photonics)
.
Average excitation power was 40
-
130
mW depending on imaging d
epth and dLight
1
expression levels/duration. Typical recording depth was 80
-
150
μm
below the pia. Data were
acquired using a Nikon 16x 0.8
-
NA water immersion objective.
A custom
-
made blackout curtain
around the microscope’s detector was used to reduce ligh
t contamination by the LCD monitor.
Images (512
×
512 pixels) were acquired at 30.8 frames/sec. Recording sessions consisted of five
to twelve ~10 min recordings, separated by short imaging breaks (3
-
5 min). Recordings within a
given session were performed a
t the exact same location to maximize the number of trial
repetitions for analysis. Recordings from different sessions were performed at the same injection
sites but offset either laterally or axially to maximize tissue volume being sampled
.
Analysis of
cortical in vivo imaging data
All data were analyzed using custom code written in MATLAB (MathWorks). Data from
four
animals were included in the analysis. Mouse #1 underwent 4 recording sessions starting 8
20
weeks after virus injection. Mouse #2 underwent 9
recording sessions starting 3 weeks after
injection
. M
ouse #3 underwent 2 recording sessions starting 2 weeks after injection
and Mouse
#4 underwent 4 recording sessions starting 6 weeks after injection.
Recording locations
(M1/M2/FrA) are indicated in
f
i
g. S1
6
E.
To confirm the animals’ responsiveness to the “Go” stimulus, we compared proportions
of trials in which runs were triggered by “Go” stimulus presentation (“Hit trials”) and
proportions of trials in which no such response occurred (“Miss trials”)
(
f
ig. S1
6
A and C). A
steep increase in “Miss trials” at the end of the session indicated that mice had lost interest in the
water reward. Trials beyond that point (black vertical line in
f
ig. S1
6
A) were excluded from
analysis. To confirm that mice had lear
ned the task, we analyzed the animals’ RTs (defined as
the time interval between “Go” stimulus onset and movement onset/ball velocity >10 mm/s) (
f
ig.
S1
6
B
and D). RTs below 1500 ms indicated that mice had learned to associate the visual cues
with the desir
ed behavior.
To reduce random noise in our time
-
lapse recordings a sliding average filter was applied
(8 frames or 260 ms). This filtering largely retained the temporal dynamics of dLight1
fluorescence signals. Lateral image motion (e.g., due to mouse mov
emen
t) was corrected using a
cross
-
correlation registration algorithm, with an average image of 50 consecutive frames from
the time
-
lapse recording serving as the reference image. The same reference image was used to
correct image motion of other recordings from the same location. Noise in
motion
-
corrected
image data was further reduced using a Kalman filter.
To quantify/classify dLight1 transients, we tiled the field of view (FOV) with equally
sized (~17x17μm) regions of interest (ROIs). To exclude ROIs with little or no dLight1
expression
(e.g., on blood vessels) we first generated a mean fluorescence projection image from
21
the corresponding time
-
lapse recording. We then calculated the projection image’s pixel intensity
distribution. ROIs with mean pixel intensity values below the 55
th
perc
entile of this distribution
were excluded from analysis. To extract fluorescence time traces F(t) from all remaining ROIs,
pixel intensities of each ROI were averaged.
Δ
F(t)/F was calculated as (F(t)
mean F) / mean F.
ROIs that showed transients with neg
ative amplitudes (predominantly located near blood
vessels) were excluded from further analysis.
Each ROI’s time trace included multiple trials/task repetitions. Based on the animal’s
performance on the trials, time traces were subdivided into “Expected r
eward”, “Unexpected
reward omission”, and “Spontaneous run” trial traces. This was done for all recordings from the
same location using the same ROIs. Extracted trial traces were then sorted by trial type, aligned
to running onset, and averaged (
f
ig. S1
6
G
,
single ROIs
). To ensure that cued and spontaneous
running bouts were comparable, only traces from spontaneous runs starting > 5 s after stand
-
still
cue onset and with > 5 mm/s ball rotation/velocity were included. To calculate population
responses, “Expec
ted reward”, “Unexpected reward omission”, and “Spontaneous run” trial
traces from all animals, all sessions, and all significant ROIs were averaged (Fig. 5E
, group
average
).
To identify ROIs with significant increases in dLight1 fluorescence in response
to the
task, three analysis intervals were defined (
f
ig. S1
5
A):
(I
)
Baseline (from
-
7 to
-
2 s prior to run
onset),
(II)
reward expectation (from run onset to 3.3 s after run onset), and
(III)
reward interval
(from 4.3 s to 10.8 s after run onset).
Δ
F/F tra
ces for each trial were averaged during the three
trial intervals respectively. The distributions of the averaged
Δ
F/F traces for particular trial types
were then statistically compared among each other and between intervals. ROIs that showed
significant f
luorescence increases during the reward expectation but not the reward interval, and
22
for all trial types compared to the baseline interval, were defined as “ROI active during
locomotion” (white squares in Fig. 5 and
fig.
S1
6
G).
ROIs that showed significant
fluorescence
increases during the reward expectation but not reward interval for cued/triggered but not
spontaneous run trials were classified as “ROI active during reward expectation” (black squares).
Finally, ROIs that showed significant fluorescence in
creases during the reward interval for
rewarded but not un
-
rewarded or spontaneous run trials were defined as “ROI active during
reward” (red squares). Significance was determined using the Wilcoxon’s rank
-
sum test
(p<0.05), Bonferroni corrected for multip
le comparisons.
All ROI analysis was performed on motion corrected image data (see above) averaged
across trials of the same type and from the same session. To investigate whether this averaging
introduces artifacts in the DA transients (e.g., due to ster
eotypic running
-
related image
movements) we inspected our data more closely at cellular and population levels. Close
observation of a 40x45 pixel detail of the motion corrected image data revealed stable and sharp
images even during the most vigorous runni
ng periods (
f
ig. S1
5
A).
Next, we plotted the full field
of view (FOV) intensity changes for the average data of each trial type aligned at run onset (
f
ig.
S1
5
B).
This revealed that the main characteristics of the single ROI transients are retained in the
f
ull FOV average, arguing against a pronounced contamination by motion artifacts. Next, to
investigate residual lateral shifts in the average image data for each trial type, we applied our
motion correction algorithm. The detected residual image motion was
<2 pixels for all sessions
and mice (
f
ig. S1
5
C and D). To verify that our motion correction algorithm reliably detects sub
-
and supra
-
pixel image displacements we artificially introduced random lateral shifts of up to 5
pixels to the same average image dat
a (
f
ig. S1
5
E).
The error between the introduced shifts and
the shifts detected by the algorithm across 100 repetitions was 0.089 ± 0.001 pixels (mean ±
23
SEM). Together, this suggests that DA transients extracted from our 17x17μm ROIs are unlikely
an artifac
t of image motion (
f
ig. S1
5
F).
Histology
Histological verification of proper sensor expression was as described previously
(
64
)
.
Primary antibodies used were: chicken anti
-
GFP (1:500; GFP
-
1020, Aves Labs), chicken anti
-
TH (1:500; TYH, Aves Labs) and rabbit anti
-
RFP (1:500; 600
-
401
-
379, Rockland A&A).
Hybrid chain reaction comb
ined with immunohistochemistry
To
assess the expression pattern of ChrimsonR
-
tdTomato injected into the VTA of
VGAT::IRES
-
Cre mice, we performed combined in situ hybridization for labeling
vgat
mRNA
and immunohistochemistry (IHC) for detecting DAerg
ic neurons (with TH). To achieve hig
h
-
sensitivity
in situ hybridization of
vgat
in tissue slices, we used hybridization chain reaction
(HCR
)
(65,
6
6
).
First, we designed
22
probes
for targeting
vgat
using a custom
written software
(available at https://github.com/GradinaruLab/HCRprobe). Each probe consists of 20
-
nt target
sequence, 2
-
nt spacer, and 18
-
nt initiator
. We
selected the target sequences that
has
(1)
the GC
content of
45
-
60%, (2) no nucleotide repeats more than 3, (3) no
more than 20 hits when blasted,
(4) higher than a
Δ
G of
-
9 kcal/mol to avoid self
-
dimers. Then we blasted full sequences (target
sequences with spacer and initiator)
and
calculate Smith
-
Waterman alignment scores between all
possible pair
s
to exclude probes
forming cross
-
dimers. The designed probes were synthesized by
Integrated DNA Technologies.
HCR was performed on
50
μ
m
-
thick slices and kept in RNAlater solution
(ThermoFisher
Scientific).
Slices
were permeabilized in PBST (1xPBS with 0.1% Triton X
-
100) for 1 hour at
24
room temperature
(RT)
and pre
-
hybridized in hybridization buffer (2x saline
-
sodium citrate
(SSC) , 10% ethylene carbonate, 10% dextran sulfate) for another hour at 37
°C
.
Then the
samples were incubated in pre
-
warmed hybridization buffer i
ncluding probes (2 nM for each) at
37
°C
overnight.
After hybridization, w
e performed stringent washes with wash buffer (2xSSC,
10% ethylene carbonate) for 30 min at 37
°C
and washes with 2xSSC for 30 min at
RT
(
twice
for
each)
.
The amplification step was performed as described in
(
67
)
overnight.
For IHC labeling of TH on HCR
-
labeled tissue slices, we used IHC buffer consist
ing
of
2xSSC, 1% donkey serum, and 0.1% Triton X
-
100.
We
incubate
d
the samples in IHC buffer for
1 hour at
RT
for blocking
and added primary antibody (anti
-
TH, AB152, Millipore
, 1:200
).
Primary antibody reaction was performed for overnight at
RT
. Following the wash steps with
2xSSC at
RT
for 30 min twice, the samples were incubated in IHC buffer including secondary
antibodies
(1:200)
for overnight at
RT
. The samples were rinsed with 2xSSC a few times and
mounted on a glass slide with mounting media (Prolong Diamond, ThermoFisher Scientific).
Probe sequences
(5’
-
>3’)
1
CACTTCATATCACTCACTaaGACACGGAGGTGGCCACATT
2
CACTTCATATCACTCACTaaGCGATGCTCAAAGTCGAGAT
3
CACTTCATATCACTCACTaaCCTGAATGGCATTTGTCACG
4
CACTTCATATCACTCACTaaACCAGGACTTCTGCGACACG
5
CACTTCATATCACTCACTaaTTCTTCAGGAAGGCGCAGGG
6
CACTTCATATCACTCACTaaTTCTCCCAGGCCCAATCACG
7
CACTTCATATCACTCACTaaGTGTAGCTGAACACGA
TGAT
8
CACTTCATATCACTCACTaaGCGGCGATGTGTGTCCAGTT
9
CACTTCATATCACTCACTaaACTTCCTTGGTCTCGTCGGC
10
CACTTCATATCACTCACTaaCGCGAAGAAGGGCAACGGAT
11
CACTTCATATCACTCACTaaAGGCGCAAGTGGAAGAGGCT
12
CCTTGGCCTGGGACTTGTTGaaCCCAATCTCTATCTACCC
13
ATGTCCATCTGCAGGCCCTGaaCCCAATCTCTATCTACCC
25
14
TAGGCCCAGCACGAACATGCaaCCCAATCTCTATCTACCC
15
CACCGCTGTGGCTATGATGGaaCCCAATCTCTATCTACCC
16
ACTTGGACACGGCCTTGAGAaaCCCAATCTCTATCTACCC
17
CGTCGATGTAGAACTTCACCaaCCCAATCTCTATCTACCC
18
AGGGCAGGAAGATCTGCGACaaCCCAATCTCTATCTACCC
19
AGAGACCCTTGAGCACGCAGaaCCCAATCTCTATCTACCC
20
CGGGCAGGTTATCCGTGATGaaCCCAATCTCTATCTACCC
21
TTCTCCAGCACTTCGACGGCaaCCCAATCTCTATCTACCC
22
ACAGCAGCTTGCGCCAGAGAaaCCCAATCTCTATCTACCC
Statistical Analysis
All
statistical analyses were performed in M
ATLAB (MathW
orks)
, Igor Pro (WaveMetrics)
or Prism (GraphPad). We used both parametric (t
-
test,
one
-
way
ANOVA
, two
-
way ANOVA
)
and non
-
parametric (Wilcoxon’s rank
-
sum) tests.
Pearson’s correlation coefficients and their p
-
values were calculated to assess how behavior or dLight1 fluorescence evolved across reward
learning or extinction.
All tests were two
-
tailed. Error bars are standard error of the mean or
standard deviation as
indicated in figure legends and main text. No statistical methods were used
to predetermine sample size.
26
Fig. S1.
Screening dLight1 variants
.
(
A
) Membrane expression and response of original dopamine sensor built by
inserting the original GCaMP6 cpG
FP module (LSS
-
LE
-
cpGF
-
LP
-
DQL) into DRD1. Peak response (
-
19.4 ± 0.02 %
F/F, mean ± SEM, n=12 cells). Inset shows membrane intensity profile. Scale bar, 10 μm. (
B
) Resting fluorescence for
all screened variants that showed expression. P
roportion of respon
se type in horizontal bar
.
Color
-
coding indicates
significance value (n=3). (
C
-
G
) Optimizing insertion site of cpGFP in DRD1. (
C
) Sequence alignment of DRD1 and
β
2
adrenergic receptor encompassing TM5
-
TM6 (replacement sites was indicated as 0 aa in each TM
region) is shown. (
D,
E
) Representative images showing membrane expression of dLight1 variants. Scale bar, 10 μm. (
F
) Quantification of
F/F and surface expression from variants shown in
D
.
Δ
F/F:
-
3 aa, 70.1 ± 10.8 %;
-
2 aa,
-
1.3 ± 6.7 %;
-
1 aa, 199.4 ±
1
3.1 %; 0 aa, 223.2 ± 8.2 %; +1 aa, 652.3 ± 9.8 %. Surface expression values quantified in arbitrary units and
normalized to 0 aa control (
-
3 aa, 33.6 ± 4 %;
-
2 aa, 30 ± 1.8 %;
-
1 aa, 88.8 ± 6.4 %; +1 aa, 87 ± 2.7 %). (
G
)
Quantification of fluorescent signa
l changes and surface expression from variants shown in e.
Δ
F/F values:
-
2 aa,
-
9.2 ±
2.3 %; 0 aa, 223.2 ± 8.2 %; +2 aa, 205 ± 21 %). Surface expression values quantified in arbitrary units and normalized
to 0 aa control:
-
2 aa, 36.4 ± 2.9 %; +2 aa, 25.9 ±
0.86 %. All values are shown as mean ± SEM. *p<0.05, **p<0.01,
****p<0.0001 versus 0 aa position control, One
-
way ANOVA with Dunnett’s post
-
hoc test.
27
F
ig. S2. Characterization of dLight1.3a and dLight1.5.
(
A
) Top, aminoacid sequence of DRD2 insertion site for the
cpGFP module. Bottom, representative images of dLight1.3a and dLight1.5 expressed on HEK293 cells before and after
addition of DA (10 μM) and corresponding SNR heatmaps. Scale bars, 10 μm. (
B
) Quanti
fication of fluorescence
response of dLight1.3a (n=12) and dLight1.5 (n=4) to DA titrations on HEK293 cells.
Data were fit with Hill equation
(EC50: dLight1.3a, 2300
±
20
nM; dLight1.5, 110
±
10
nM). (
C
) Representative images of dLight1.5 under basal
condi
tions, after addition of quinpirol
e
(10 μM) and haloperidol (50 μM), and trace showing
quantification of
fluorescence
response during bath application of drugs
(n=15). All data are shown as mean ±
SEM
.
28
F
ig. S3. One photon emission spectrum and two
-
photon cross
-
section of
dLight
1 expressed in HEK293T cells.
Emission spectrum of both dLight1.1 and dLight1.2 peaks at 517 nm. Under 2P illumination, emission peaks are driven
at 920 nm.