RecV recombinase
system
for
in vivo
targeted optogenomic modifications of
single cells or cell
populations
Shenqin Yao
,
*
1
P
eng
Yuan
,
*
2
Ben Ouellette
,
1
Thomas Zhou
,
1
Marty Mortrud
,
1
Pooja Balaram
,
1
Soumya
Chatterjee
,
1
Yun Wang
,
1
Tanya L. Daigle
,
1
Bosiljka
Tasic
,
1
Xiuli Kuang
,
3
Hui Gong
,
4
Qingming Luo
,
4
Shaoqun
Zeng
,
4
Andrew Curtright,
5
Ajay Dhaka,
5
Anat Kahan
,
6
Viviana Gradinaru
,
6
Radosław Chrapkiewicz
,
2
Mark
Schnitzer
,
2
Hongkui Zeng
,
1
and
Ali Cetin
1
,
5
1
Allen Institute for Brain Science, Seattle, Washington, USA.
2
CNC program, Stanford University,
Palo
Alto,
California, USA.
3
School of Optometry and Ophthalmology, Wenzhou Medical
College, Wenzhou, Zhejiang, China.
4
Britton Chance Center for Biomedical Photonics, Wuhan
National Lab for Optoelectronics, Huazhong
University of Science and Technology, Wuhan, Hubei,
China.
5
Department of Biological structure, UW, Seattle, WA, USA.
6
Division of Biology and Biological Engineering
,
California Institute of Technology, Pasadena, California, USA.
*These authors contributed equally
to this work
.
Correspondence should be
addressed to A.C. (
alic@alleninstitute.org
).
Keywords:
Light
-
inducible,
optogenomic,
recombinase, recombination, RecV, Vivid, Cre, Dre, Flp
ABSTRACT
Brain circuits are composed of vast numbers of intricately interconnected neurons with diverse molecular,
anatomical and p
hysiological properties. To allow
highly specific “user
-
defined”
targeting of individual neurons
for structural and functional studies
, we modified three site
-
specific DNA recombinases, Cre, Dre and Flp, by
combining them with a fungal light
-
inducible prot
ein, Vivid,
to create light
-
inducible recombinases (named RecV)
.
We generated viral vectors to express these light
-
inducible recombinases and demonstrated that they can induce
genomic modifications in dense or sparse populations of neurons in
superficial a
s well as deep brain areas of
live mouse brains by one
-
photon o
r two
-
photon light induction.
These l
ight
-
inducible recombinases can produce
highly targeted, sparse and strong labeling of individual neurons
in multiple loci and species.
They
can be used
in
combination with other genetic strategies
to achieve
specific i
ntersectional targeting of
mouse cortical
layer
5
or
inhibitory somatostatin neurons.
I
n mouse cortex sparse light
-
induced recombination
allows whole
-
brain
morphological reconstruction
s
to iden
tify axonal projection specificity.
Furthermore these enzymes
allow
single
cell targeted
genetic modifications
via
soma
restricted
two
-
photon
light
stimulation
in individual cortical neurons
and can be used in combination with functional optical indicators
with minimal interference
.
In summary
,
RecVs
enable spatiotempo
rally
-
precise, targeted
opto
genomic modifications that
could
greatly facilitate detailed
analysis of
n
eural circuits at
the
single
cell level
by
linking genetic identity, morphology, connecti
vity and function.
INTRODUCTION
To understand how a biological system works it is often useful to
define
its building blocks and
understand
how
those building blocks work
together to generate its function. Mammalian brain is one of the most complex
biolo
gical systems. It is composed of millions to billions of cells
1
with diverse
characteristics. To understand this
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
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extraordinary complexity, it will be essential
to define cell types based on properties such as gene expression,
morphology and physiology at
the
single cell level. Furthermore, the unique properties of individual cells
need to
be related to their connectivity patterns and their activities in a behavio
ral context. Anatomical information
combined with genetic identity and functional properties at the single cell level will enable
better
analysis of brain
circuitry underlyi
ng complex behaviors in health and disease.
One of the most powerful approaches to
characterizing cell types and studying their functions relies on mouse
genetics
2
.
S
trategies that rely on transgenic or viral expression of recombinases allow a highly specific level of
genetic modification
3
-
5
.
A m
ultitude of cell type
-
specific Cre recombinase mouse lines has led to many
discoveries in neuroscience over the last few decades.
Further
improvements on
spatiotemporal control
can
allow much higher resolution
manipulations and
studies of biological systems
.
Current
ly
finding
individual
cells/neurons
in vivo
;
characterizing
and
genetically manipulat
ing
them
for targeted
genomic modifications
as well as
express
ing
genes
in a targeted manner
is challenging
to execute in
an efficient
manner
. The state
-
of
-
the
-
art
approach
for introducing an exogenous gene to a specific neuron is eit
her by the
patch clamp
technique
6
or via
single
-
cell electroporation
7,8
.
T
he
s
e
technique
s
are
highly challenging and usually
result in low and variable yields.
Sparse neuro
nal labeling or manipulation can be achieved by controlling
transgene
recombination by lowering the dose of inducers (
e.g.
, tamoxifen, in the case of CreER) or
by employing
‘inefficient’ recombinase reporters
(
e.g.
, MADM
9
). However, the sparse genetic modification achieved using
these methods is random and cannot be easily directed to specific
or individual
cells of interest.
Light is a particu
larly powerful and versatile regulator with its tremendous adjustabil
ity in the dimensions of
spectrum, intensity, space (location and size) and time (timing and duration). Using photons to access and
genetically modify individual neurons will offer an imp
rovement over the current state
-
of
-
the
-
art. Multi
-
photon
additive cha
racteristics of light that can generate a spatiotemporally
-
restricted excitation
are
well suited
for
achieving fine
spatial control
10
. Thus, modifying current genomic manipulation enzymes to make them light
inducible could be an ideal approac
h to reach a high spatiotemporal resolution for targeted
single cell
manipulations.
S
everal light inducible
protein
-
based spatiotemporal control
systems
have been
develope
d to date
11
-
33
.
These
systems depend on various light sensing modules to co
ntrol protein states, protein localization, transcription
and genetic alterations.
So far optical manipulation of
genomes
-
optogenomics
-
of individually targeted single
cells within live intact tissues
has
not
been
demonstrated using such methods.
To leave
a permanent genetic
mark in individual cells in this study, we developed and validated light inducible
site
-
specific
recombinase
systems and its associated set of viral tools via comparing and optimizing various light inducible systems with
the cr
iteria t
hat they induce robust genomic modifications with no or minimally detectable background under
no
-
light conditions at different genomic locations and species. Our work resulted in the generation of highly
efficient light inducible versions of the mo
st commo
nly used site
-
specific recombinases
-
Cre, Dre and Flp
-
that
allow tight population level or target
-
specific single cell level optogenomic modifications
in vivo
.
RESULTS
A Split Vivid
-
Cre enables efficient light
-
inducible
site
-
specific DNA modification
s
To spatiotemporally regulate site
-
specific recombination, we generated a light
-
inducible genetic switch based on
a fungal light sensitive protein
–
Vivid (VVD)
34
.
We
chose VVD mainly because it is the smallest (450 bp) of all
Light Oxygen or Voltage (LOV) domain
-
containing proteins, and therefore suitabl
e for fitting into viral vectors
with limited genomic capacity. In addition, the spectral proper
ties of VVD
-
its excitation and emission drops
sharply to
near
0
with light
above ~520 nm
-
would allow us to use other fluorescent proteins, activity indicators
or optogenetic molecules that work at longer wavelengths
20,35
.
Upon light illumination, VVD forms a homodimer
35
due to
the
conformational changes induced by Flavin Adenine
Dinucleotide (FAD) cofactor within the LOV domain. The FAD cofactor within the LOV domain has
a peak single
-
photon (1P)
excitation at 450 nm and
a
peak two
-
photon (2P) activation at 900 nm wavelength
36
.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
To
generate
a precise spatiotemporal
control of gene expression, we used a split
-
Cre recombinase system
20,37
-
39
and
converted
it into a light
-
inducible system using the VVD protein (
Fig. 1
a
). For Cre recombinase to function
p
roperly
,
the N
-
termin
al
and the C
-
termin
al portion
s
of the protein need to come in close proximity
to
form the
correct tertiary structure. The crystal structure of Cre
is known
40
and so
of
VVD
35,41
. In the dimer form, the N
-
termin
u
s
of one VVD
monomer
gets into close proximity to the C
-
terminus of the other (
Fig. 1a
).
Guided by this
information o
ur
design
attempted to bring together the N
-
and C
-
portions
of Cre in the correct orientation upon
light
-
induced conformational change and
dimerization of VVD. To split the Cre protein, we relied on
a previous
split Cre system, since
it
was shown to generate stable dimers
37
.
We
fused the inactive N
-
terminal segment of
Cre to the N terminus of one VVD
mon
omer
and inactive C
-
terminal
segment of Cre to the C terminus of another
VVD monomer, which we codon diversified to reduce the sequence similarity
between the two VVD monomers
and
minimize the risk of recombination.
We
cloned each
of these
into recomb
inan
t adeno
-
associated virus
(rAAV) expression vector
s
(
Fig. 1
c
)
42
.
The resulting NCreV and CCreV constructs along with
fluo
rescent
Cre
reporter construct
s
were co
-
transfected into
mammalian
cells and robust light
-
inducible recombination
was
observed with 458 nm
LED
light induction compared to the no
-
light condition
s
(
Fig.
1
d, e
).
We named these new
proteins CreV, and the
general light
-
inducible Vivid
-
recombinase system RecV.
Vivid
-
based light
-
inducible system
for Dre recombinase
To
broaden the capabilities of the RecV approach
we modified
another site
-
specific DNA recombinase
,
Dre
43
,
which is a relative of Cre, and is likewise derived from a bacterial phage
. Dre
recombinase recognizes a
sequence
called Rox
44
, which is different from LoxP.
This is especially important since it would allow us to utilize
already existing Cre driver mouse lines to study Cre
-
defined popu
lations
and to develop intersectional strategies
to further refine cell type
-
specific genetic manipulation.
We reasoned that the sequences surrounding the homology region within Dre N
-
terminus, where we split Cre
into two non
-
functional
compartments
,
could also
be targeted to generate a split Dre
(
Fig.
1b
)
.
After generation
of the DreV constructs based on CreV designs
we tested
the
light inducibility of DreV constructs using fluorescent
Dre reporter plasmids
(
Fig.
1
c
and
Supp.
Fig. 1
). Our results indicate that t
he light
-
induced DreV recombination
is efficient
using 1P
excitation
and is similar to CreV (
Fig.
1
d, e
).
Spatiotemporal regulation of gene expression can be further fine
-
tuned using 2P excitation since it has a much
narrower
point spread function in the
axial direction. We tested if the VVD based recombination can take place
using 2P light under cell culture conditions. Our results indicate that DreV
-
mediated light
-
inducible recombination
can be achieved by application of 900
-
nm wavelength of 2P light (
Su
pp.
Fig.
2
).
Single
RecV
expression constructs
and comparison to other existing light inducible
recombinase
systems
To implement the RecV strategy efficiently and reduce the number of viruses or transgenic mouse lines
needed
to
use this system
, it is pre
ferable to co
-
express the two halves of RecV
s
in a single construct. To achieve this,
we tested a variety of ways to link the N and C components of CreV or DreV that would provide the highest
efficiency of recombination
with the least amount of background
recombination
:
IRES, 2A, single open reading
frame with permutations of the open reading frames
; see methods section for more details
(
Supp. Fig. 3
a
)
.
A
mong these constructs
highest
efficienc
ies
w
ere
observed
when
optimized
elements from
Cre
-
Magnet
s
(
mutant
VVD
s
)
13
,
33
were used
,
which
we
named
iCreV and iDreV
.
iCreV and iDreV are composed of
N
-
and C
-
terminal
portions of the Cre and Dre split recombinases
fused to nuclear localized
wild
-
type
VVD and co
-
expressed with
optimized linkers and
2A elements that wer
e used in Cre
-
Mag
nets
(
Supp. Fig. 1
)
.
We
next
compared
iCreV and iDreV,
with
Magnet
b
ased
v
ersions
.
In cell culture
Magnet
based constructs
induced
significant amount of background recombination in the absence of li
ght
.
i
D
reV and i
C
reV exhibited
control levels of
recombination in the absence of light and retained high levels of inducibility
–
~1.6 fold higher
for iCreV as compared to split CreV and ~0.6 fold lower for iDreV compa
red to
split DreV in relative fluorescence
intensities
(
Supp. Fig. 3
b
)
.
For both iCreV and Cre
-
Magnet
s
efficient
recombination was observed
in vivo
after
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
light stimulation
-
please see the details of the
in vivo
experiments below
.
However,
in no ligh
t control cases with
Cre
-
Magnet
s
,
back
ground
recombination was observed
throughout the
brain structures
-
~50 cells per 100
μm
coronal slice
,
whereas
i
CreV
did not lead to
significant
recombination
(
Supp. Fig. 4
)
.
We also
compared
the
cryptochrome
-
ba
sed light inducible Cre recombination system
-
CRY2
32
,
with iCreV.
The
c
omparison
in cell culture conditions
revealed that
iCreV
performs ~4 fold better after 60 minutes of light
stimulation than
the
C
RY
2 system
,
w
hereas
the
C
RY
2 based Cre recombination system allows only a slight
increase over baseline recombination
(
~1.2 fold
)
(
Supp.
Fig.
3c
).
We were not able to test the Cry2 based
constructs
in vivo
since the
se
genes
were too
large
to fit into the
rAAV
viral vectors
we used.
Our results
suggest
that under the assayed conditions,
VVD
-
based optogenomic control
is
tighter and more
suitable for
certain
downstream applications
compared to the previously described systems
.
Design and screening
of
light
-
inducible Flp rec
ombinase
s
To
increase intersectional genomic modification
possibilities,
we turned into a third wi
dely used site
-
specific DNA
recombinase: Flp
45
.
T
here
isn’t enough
protein
sequence
similarity
between Flp,
a recombinase from yeast, and
Cre or Dre, recombinases from bacterial phages
to guide a split based on homology
.
Thus, we resorted to
structure
-
based
de novo
design
and screening for split
sites based on c
rystal structure of the Holliday junction
bound Fl
p
46
. Using
a more efficient
,
codon
-
optimized variant of Flp
(FlpO)
47
; we made Flp splits at 21 loop
locations that
correspond to
transitions
between
alpha helices and
/or
beta sheets with hopes to not alter overall
functionality within the dimer form
.
We generated
these
VVD
-
fused split
i
Flp
V constructs using the iCreV
backbone by replacing the N
-
and C
-
termini of Cre with the 21 split Flp variants (
Supp.
Fig.
5
a
).
After gene
synthesis,
these constructs were cloned into CMV promoter
-
driven mammalian expression plasmids
and tested for light
-
induced recombination in
cell culture
via transient transfections
together
with
a Flp
-
dependent
fluorescent
reporter plasmid. Amo
ng the variants,
i
FlpV2, 19 and 20 gave the most significant light
-
induced
recombination
(
Supp. Fig. 5b
).
D
ark conditions
for
a
ll these cases were almost identical to the controls indicating
that,
like
for
iCreV and iDreV, the background recombination
was
almost non
-
existent.
I
n an effort to
further improve the efficiency of light
-
inducible
Flpase
activity
a
fter
th
is
initial scre
en, we
designed
62 more iFlpV
variants
to scan
Flp
split sites surrounding those of iFlpV2, 19 and 20
(
Supp. Fig. 5c
)
.
Among all these
constructs
FlpV2 still yielded the highest
efficiency.
L
ight
-
inducible recombination with RecVs
with
whole
-
brain infect
ions
in vi
v
o
Recently
,
a new blood brain barrier permeable rAAV serotype, PHP.eB, was reported to be effectively infecting
large proportions of the entire nervous system in mice after retroorbital delivery
48
.
Thus,
we reasoned
this
technique
could be a
good and
rapid
approach
for ex
amining RecV constructs in the entire mouse brain
.
We
examined background
recombination in the absence of light in
any part of
the brain
,
as well as
spatial restriction
of
light inducib
le
recombination.
For this
purpose,
we generated
a
n
EF1
α
promoter
-
driven Cre
-
expressing rAAV as well as RecV rAAVs
of the
PHP.eB serotype.
We
found
that
th
e
PHP.eB EF1a
-
Cre virus, when injected
either
intracerebroventricularly (ICV)
or
retroorbitally into the
fluorescence Cre
-
reporter mice
,
efficiently
infect
s
the entire brain
relatively
homogenously
(
Fig.
2
a
, Supp. Fig. 6a
).
To our knowledge
,
this is the first demonstration of effective PHP.eB
mediated gene delivery into the
brain
using the ICV route.
This
method may help
to
further
restrict
the infection
to
t
he
nervous system and may
help
overcome obstacles related to intravenous delivery of
genes for both basic
research as well as gene therapy field applications.
We next tested the light
-
inducible recombination using RecVs with whole brain infections. To do
so
first we
injected a mixture of
PHP.eB
NDreV and CDreV virus
es
into the right ventricle of
the
Dre
-
dependent
fluorescent
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
reporter
mouse
, Ai66R.
A
fter
an incubation period of two weeks,
the left hemisphere
w
as
exposed to light.
A
gradient of recombination
could be observed from the top of the left hemisphere to deeper
structures
(
Fig.
2
b
).
D
e
eper brain structures and sites far
away
from the light stimulation did not have any fluorescently labeled cell
s
-
except for
ICV needle tract
where
recombination took
place
likely due to multiple copies of infection.
In addition,
we tested the iCreV
construct
with PHP.eB serotype
,
delivered by ICV or RO routes,
in a Cre
-
dependent nuclear
reporter mouse line
, Ai75
. Very similarly
,
we observed substantial recombination in
the hemisphere
exposed to
light regardless of the
viral delivery route
(
Fig.
2
c
and Supp. Fig. 6b
).
Equivalent tests of the
iDreV
construct
in
a Dre reporter line yielded a gradient of recombination from the light stimulated hemisphere
(
Fig.
2
d
).
As an
ad
ditional control iCreV
-
injected Cre
-
dependent repo
rter mice were maintained without light stimulation for four
weeks after initial injection. No significant amount of recombination was observed, indicating tight optogenomic
control under these conditions (
Supp. Fig. 6
d
). Finally, equivalent tests of
the iFlpV construct in the Flp reporter
mouse line resulted in efficient and specific light
-
induced recombination
(
Fig.
2
e
).
These results
indicat
e
that
the technique allows highly specific light
-
inducible reco
mbination.
Thus far all the recombinase repor
ter mouse lines we used were generated via targeted insertions into the
Rosa26
locus
49
. To test if a different locus
could be modified by the RecV system, we RO injected PHP.eB iCreV
rAAVs into the Ai167 ChrimsonR reporter mouse line, which is inserted into the Tigre
locus
50
. We
again
observe
d
efficient recombination
shaped
as a gradient
from the
light
-
stimulated
left
hemisphere
confirming that other
genomic
loci
in mice
can be modified by the RecV system
(
Supp. Fig. 6
c
).
W
e
also
tested
the
feasibility of light inducible recombination within a
deeper brain area
,
striatum
. Deep
brain
imaging
or recording is
more challenging due to tissue damage and blood clotting
;
both create
obstacles
for
light
passing
through
the tissue
.
By utilizing 1P
-
light through an optical fiber we were
able
to induce loc
al
recombination
via
iCreV in
the
striatum of
Ai162
GCaMP6s Cre
reporter mice
50
,
and record
changes in
fluorescence due to calcium concentration
dynamics
befo
re and after light stimulation (
Supp.
Fig
.
7
a
-
d
)
.
D
ue to
the localized
illumination
,
GCaMP
6s
was expressed
directly
under the
fiber
,
in comparison
to the
wide
expression of
red fluorescence coming from the control virus
(
Supp
.
Fig
.
7
b
)
.
This
expression pattern
is
beneficial
for
calcium
imaging and recording, as
it allows expression directly at the desired location
.
Our results
show
that light
-
mediated genomic modifications can be efficiently spatiotemporally regulated
in vivo
also
with
in
deep
brain
targets
therefore
providing a tool for
restricted
reporter expre
ssio
n under the optical device
.
Cell
-
class specific targeting by intersection of viral RecV
s
and transgenic recombinases
Versatile and refined cell type targeting can be achieved by combined use of two or more
recombinases with
distinct
recombination ac
tivit
ies
50
.
For example,
new RecV tools could be combined with existing transgenic
recombinase lines and intersectional reporters.
To test the feasibility of this approach
,
we
co
-
injected PHP.eB iFlpV viruse
s
toge
ther with a
PHP.eB
Cre/Flp
dependent
fluorescent
reporter
virus
into
the cortex of mice containing the
Rbp4
-
Cre
-
KL
-
100 transgene, which
drives Cre expression mostly in L5 excitatory cortical cells
. After unilateral light stimulation we observe
d
layer
5
spe
cific reporter gene
expression
within the light
-
stimulated hemisphere
in these mice
(
Fig.
2
f
)
, suggesting high
degree of intersectional specificity under the tested conditions
.
To provide further evidence that light
-
mediated intersectional targeting can b
e achieved in other
neuron types,
we used the Sst
-
IRES
-
FlpO
mouse line, which expresses FlpO recombinase selectively in inhibitory somatostatin
(Sst) neurons, in conjunction with iCreV. We crossed this mouse line to a Cre/Flp double dependent fluorescent
r
eporter mouse line
Ai65. The resulting double
-
positive mice were retro
-
orbitally injected with PHP.eB iCreV
viruses to infect the entire brain relatively homogenously. Reporter expression was observed in sparsely
distributed neurons close to the light stim
ulation site across
multiple cortical layers (
Fig
.
2
g
). To ascertain that
the recombination we observed within the light induced hemisphere is indeed specific to the Sst
-
positive neurons,
we performed immunohistochemistry and found that all the fluorescent
ly labeled neurons
were also immune
-
reactive to the SST antibody (
Fig
.
2
g
)
. These results confirm that tight intersectional control can be achieved by
combining virally delivered iFlpV or iCreV with transgenic class
-
specific Cre or FlpO, respectively.
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The copyright holder for this preprint
.
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doi:
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Cr
eV induces
tight
light
-
dependent recombination in zebrafish
embryos
in man
y tissue types.
To determine if RecVs could effectively induce light
-
dependent recombination in
other loci and organisms other
than mice we
tested it in
another model organism: Dani
o rerio
(zebrafish)
. W
e co
-
injected plasmids containing
either
N
-
CreV or C
-
CreV under the control of the ubiquitous ubiquitin (
ubi
) promoter into single cell
zebrafish
embryos of transgenic zebrabow strain tg
(
ubi:Zebrabow
)
51
and reared embryos either in the light or the dark
for 72 hours. In
this line within
the default state, RFP is exp
ressed in all cells. After Cre
-
dependent
recombination
,
YFP and/or CFP
are expected to be
expressed. Of the 24 viable embryos reared in the dark
1/24 displayed any recombination as measured by
the presence of YFP or CFP. Of the 37 viable embryos
reared in
the light nearly all
(
32/37
)
had YFP or CFP expression, confirming that
CreV
-
induced recombination
was light dependent. We observed recombination in many tissues including muscle, skin, heart,
spinal cord
neurons and non
-
neuronal cells,
hindbrain, trigeminal ganglion, Rohon
-
Beard sensory neurons
and
hair cells of
the lateral line (
Fig
. 3
a
-
f
), suggesting that Cre
V
is effective at inducing recombination across many cell types
and tissues
.
RecV
-
me
diated sparse labeling enables
whole
-
brain reconstruction of single neuron morphologies
The development and
in vivo
validation of the light
-
inducible recombinase system (RecV)
opens
doors to many
applications. Here we present
several of
such application
s
,
mainly using
light to control the precise location and
number of neurons labeled to achieve the desired sparsity of targeted neuronal populations for the
ir
visualization
,
functional imaging
,
as well as
reconstruction of single neuron morphologies across th
e entire bra
in to
understand
their axonal projection specificity.
Cre
-
mediated
in vivo
reporter expression using a Cre reporter mouse line Ai139 result
s
in strong expression of
GFP in multiple cortical layers. Ai139 is a
TIGRE
2.0
-
reporter line expressing
very
high level of GFP (via tTA
-
mediated transcriptional amplification) allowing visualization
of detailed cellular morphologies
50
.
We tested
if light
applied to the tissue could
sparsely
induce
CreV, to result in
sparse yet strong expression
of the reporter at the
single cell level, which would enable
whole
-
brain reconstruction of single neuron morphologies.
For this purpose, we injected the cortex of Ai139 mice with the 1:1 mixture of NCreV and CCreV rAAVs in ser
ial
dilutions and applied various durations of light across the skull
to
induce recombinat
ion in a sparse population of
cortical neurons. We then imaged the whole brains using the fluorescence micro
-
optical sectioning tomography
(fMOST) technique
52
.
Lower dose of CreV virus
es and shorter duration of light induction led to sparse and strong
labeling of
individual neurons (
Supp.
Fig.
8
). Sparseness was low enough that axons from many neurons could
be traced. In the example brain shown in
Figure
4
(also see
Supp.
Movies 1 and 2
), 8 primary somatosensory
cortical neurons were manually reconstructed. These n
eurons include 3
L
ayer 2/3 pyramidal cells (PCs) with
ipsilateral cortico
-
cortical projections, 2
L
ayer 2/3 PCs with contralateral cortico
-
cortical projections, and 3
L
ayer
5 t
hick tufted PCs with ipsilateral cortico
-
subcortical projections, revealing dist
inct axonal projection patterns.
In
many
cases
where
restricted
genetic access is not possible
with conventional methods
,
it
is
desirable to further
confine
the region of r
eco
mbination by precise
induction
through localized
illumination
.
To
demonstrate this
ability
,
we performed
headpost craniotomy and
two
-
photon (
2P
)
a
ssisted local laser stimulation experiments
in
trained head
-
fixed
Ai139 reporter mice
.
To achieve sparseness,
we
co
-
injected
a 1:5 mixture of rAAV iCreV and
rAAV tdTomato
-
expressing viruses
into the
primary visual cortex.
The
tdTomato
virus
was
included
to guide our
light
stimulation
,
as well as to serve as a control of overall infection
breadth
. Our results indic
ate that local
and
sparse labeling of neurons is achievable using this method. Furthermore, due to the strength of the
Ai139 reporter
expression
,
individual axons and boutons can be readily visualized without immunohistochemical enhancement
at sites far aw
ay from neuron
al cell
bodies
, suggesting that
these brains could be subjected to
whole
-
neuron
reconstruction
(
Supp.
Fig.
9
)
.
RecVs
enable
2P
-
mediated single cell specific targeted
optogenomic modifications in combination with
functional imaging
in vivo
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.
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A
powerful application for the light
-
inducible iCreV system is to identify
-
e.g.
functionally
or
genetically
-
individual
cells of interests and then activate the Cre
-
dependent gene expression in these
defined cells at will. However,
1P
illumination is not s
uitable for this application, since it inevitably activates the iCreV in cells
within
the
light
path in addi
tion to the target. One approach to achieve single
-
cell specific targeting is
through 2P
activat
ion
of
iCreV
which minimizes
unspecific targeting. T
o investigate the feasibility of this approach
in vivo
at single cell
level
, we next tested the iCreV system
using
2P
induction in m
ouse
somatosensory
cortex (
Fig
.
5
a
-
b
).
We co
-
injected iCre
V
and GFP
-
expressing viruses in the
somatosensory
cortices
of a
Cr
e
-
dependent red
fluorescent reporter mouse line (Ai14)
. The GFP fluorescence served as a landmark for target cells. After
identifying the target cell, we focused the laser in the middle of the cell soma (potentially nuclear region), and
then
laser
-
scanned
within this small region
-
as a
window of
~6x8
μ
m
-
under various conditions of
laser power
and time. Seven to ten days after this initial induction session, we re
-
imaged the target cells to examine the
expression of tdTomato. In order to reveal all the ind
ucible cells in the region, we then exposed the neu
rons
to
1P
LED
light
for 30 minutes, and then counted all the tdTomato
-
exp
ressing cells seven days later.
We showed that
2P
induction of iCreV led to
C
re
-
dependent gene expression in target cell with singl
e
-
cell
precision. In many cases, we observed potent
tdTomato expression in the target cell but not in the inducible cell
right
next to it (
Fig
.
5
c
and Supp
.
Fig.
10
), demonstrating the high spatial specificity. We tested various
2P
induction protocols and
plotted the probability of iCreV activation against the distance to the target cell in each
condition (
Fig
.
5
d
). The results showed that target cell induction rate increased as more laser power and scan
time was applied during the
induction. However, power
ful induction in some cases also led to
no
nspecific
induction within 10
μ
m
radius of the target cell (as high as 18%), which may
result
from the activation of the
iCreV constructs in the neural processes surrounding the target cell
, or from micron
-
level mo
vement of the mouse
brain during the induction window. Cells within
the
10
-
50
μ
m
radius from the target cell showed a baseline
induction rate of 6
-
7%. Since the induction rate observed in session 1 was very low (~3% on average,
Supp
.
Fig.
1
1
), this non
-
tar
geted induction was likely due to the non
-
specific activation of iCreV by ambient
light during
mouse handling, by the two
-
photon stimulation during acquiring the Z
-
stack images
or by spurious recombination
due to
very
high multiplicity of infection with th
e viral vectors
.
We next tested the feasibility of combining calcium imaging with
the iCreV system, by measuring the induction
probability of iCreV after 30 minutes
of
calcium imaging. We co
-
injected iCreV and GCaMP7f
53
viral constructs
in
to
Ai14 mice cortex. 920 nm excitation provided good quality jGCaMP7f signal, however
,
was also potent in
activating the iCreV system (close to 10
0%).
To
reduce
iCreV
induction during the imaging session, we tested
longer wavelengt
h excitation of jGCaMP7f at 1000, 1010 and 1040 nm. At these wavelengths, the induction of
iCreV was significantly reduced, with moderately reduced jGCaMP7f signals (
Fig.
5e
-
g
).
DISCUSSION
In
this
study, we
used
wild
-
type VVD
to create three light
-
induci
ble recombinases Cre,
Dre, and Flp, thus
expanding the repertoire of
optogenomic
manipulation tools even further. We also showed that these light
-
inducible recombinases work highly efficiently and intersectionally in the
mouse
brain
to label specific cell
classes
or types. We further demonstrated that RecVs allow effective light induc
ed optogenomic modifications in multiple
loci within the mouse genome and zebrafish.
We
provide
d
proof
-
of
-
principle
experiments showing
that RecV mediated light
-
inducible site
-
specific DNA
modification
s
are possible
in the mammalian nervous system at sing
le cell level.
W
e provide
d
examples of single
-
cell
soma
-
targeted
2P
-
mediated
optogenomic modifications
and established imaging and conversion parameters
to induce
such
modifica
tions with unprecedented
spatial
resolution.
Our data also provide a quantitativ
e
description of the induction specificity of iCreV at different 2P wavelengths and support the feasibility of
combining calcium imaging with iCreV system in mice
in vivo
.
Ligh
t
-
inducible Cre
recombinases were
reported previously
32,33,54
, and they were mainly based on two types of
light
-
inducible protein dimerization systems: CRY2
-
CIB1
and
Magnets
.
A direct comparison of
an
improved
CRY2
based
32
and the
VVD
based system presented in this study shows that the CreV system
may be preferable
d
ue to its higher level of inducibility under the conditions tested
in vitro
.
Additionally, our results indica
te that
the
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
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heterodimeric
Magnet
-
based light
-
inducible Cre recombination is efficient, but it is also relatively leaky compared
to
our
VVD
-
based counterpart
both
in vitro
and
in vivo
.
By
incorporating the improved designs reported
in the
Magnet
system
33
we were able to produce efficient RecV co
-
expression constructs
, thereby decreasing the
number of independent components that need to be delivered to the experimental animal
.
The demonstration
of
the
ICV route for efficient whole
-
brain infection
usin
g PHP
.eB AAV viruses
could
be
valuable
in many scenarios. For example, it could
infect the brain
s
of embry
o
s in which RO
vein
is too small to
inject
,
or to avoid damage to the eye of the
embryo since in mice RO route requires penetrating a needle next to
t
he
eye
.
This approach
may
further
help
focus infection
to
the nervous system and
overcome obstacles related
to intravenous delivery of genes for basic research
in
a multitude of mammalian species. Genetic modifications
of larger animal models such as prima
tes
are expensive. Intravenous delivery in the adult primate may require
substantial number of viral particles.
Thus,
if
the PHP.eB
viruses are
delivered by the ICV route during adulthood
or development, it may help
infect majority of the nervous system
wi
th much less virus
. This
approach
may also
overcome immune
-
response related issues
that interfere with infections
. Finally
,
this
method
may be useful in
clinical
applications
for gene therapy purposes
.
The spatiotemporally selective targeting of cell popul
ations or individual cells using light could allow fine
-
scale
combinatorial functional, anatomical and circuit
-
level studies of cell type
-
specific networks.
In addition to
combining
opsin expression with
i
CreV,
and
achieving
efficient GCaMP expression usin
g
i
CreV
, t
he RecV viral
vectors and Cre/Flp
-
dependent
i
DreV or
i
FlpV mouse lines can be combined with existing cell type
-
specific
genetic tools, and RecVs can also be used to generat
e new driver lines.
New RecV versions incorporating drug
-
inducible (e.g. t
amoxifen
55
or trimethoprim (TMP) inducible
56
) recombinases can be developed to further reduce
background
recombination
and enhance tempor
al specificity.
With this approach
,
one can envision performing
a variety of combinatorial experiments including
single
-
cell activity imaging using genetically encoded voltage
57,58
or calcium indicators
59
; single cell monosynaptic rabies traci
ng
6,7
or targeted optogenetic
and chemogenetic
protein expression
60
, all in a noninvasive or minimally invasive manner. The sp
ectral and temporal separation
as
well as drug
-
inducible versions could enable sequential investigations of funct
ionally relevant cell populations
,
followed by light
mediated DNA recombination to selectively activate effector gene expression in specific cells of
interest
,
to examine their structure and connectivity and/or to perturb their function.
This technology c
ould also enable
loss/gain
-
of
-
function studies
by swi
tching genes off or on with much refined
regional and cell type specificity
, followed by monitoring of
effects on physiology or animal behavior
. One
could
also
employ this method to
study development
by
switching genes
on or off
with
high
temporal precisio
n during
the rapid cascade of events throughout the proliferation and differentiation processes. The half
-
life of the VVD
homodimer is ~2 hours after brief light activation
13
. One
can
try to apply targeted light at very precise time points
using standard or gentler
in utero
techniqu
es. This
may be
advantageous over the current CreER
-
based genetic
fate mapping studies
61
as it would eliminate
the
tamoxifen
side effects
as well as provide
spatially and temporally
more precise
recombination
. Fur
thermore, this approach
could
allow highly targeted
neuronal
network
reconstructions by selectively labeling neurons in particular locations or having
particular activity patterns or
functional properties (
e.g.
, using 2P induction). Sparse expression of re
porter genes using diluted Cre viruses or
low doses of tamoxifen in CreER mice already exist. However, with RecV
s
one
can
perform even more
spatiotempo
rally restricted induction, and it can be combined with prior functional characterization using 2P
imagin
g
and
optical indicators in an intersectional manner.
The RecV approach can also be used in other species of interest in which light could be used to
access individual
cells of interest. For model systems in which germline genetic modification is feasible
, the experiments can be
designed to integrate multiple components of the described systems into the germline
62
. In model systems in
which germline modification is not available
or is prohibitively expensive
, these
experiments can be performed
by using viral vectors in which a certain level of cell
-
type specificity may be achieved by using short cell type
specific promoters or enhancers
63,64
, or target
-
defined specificity ma
y be achieved with highly efficient
retrogradely infecting designer viruses
65
.
Overall, the broad range of potential applications show that the light
-
inducible recombinase system should
enable much improved spatio
temporal precision and multiple combinatorial st
rategies
for
the micro
-
and macro
-
level analys
e
s of neural circuits
a
s well as many other biological systems
in a multitude of organisms
.
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
METHODS
Plasmid and virus construction for
RecVs
Sequences of NCre
-
5G
-
VVD, VVD
-
5G
-
CCre, NCre
-
5G
-
VVD
-
IRES
-
VVD
-
5G
-
CCre, VVD
-
5G
-
CCre
-
IRES
-
NCre
-
5G
-
VVD, NCre
-
5G
-
VVD
-
PQR
-
VVD
-
5G
-
CCre, VVD
-
5G
-
CCre
-
PQR
-
NCre
-
5G
-
VVD, VVD
-
5G
-
CDre
-
IRES
-
NDre
-
5G
-
VVD, NCre
-
Magnets
-
NLS
-
P2A
-
NLS
-
Magnets
-
CCre, NCre
-
VVD
-
NLS
-
P2A
-
NLS
-
VVD
-
CCre,
NDre
-
Magnets
-
NLS
-
P2A
-
NLS
-
Magnets
-
CDre, NDre
-
VVD
-
NLS
-
P2A
-
NLS
-
VVD
-
CDre, and all
i
FlpV versions were chemically
synthesized (GenScript, Piscataway, NJ).
19
-
59 amino acid N terminus
and
60
-
343
C terminus of Cre were used
in all CreV cases.
To screen poly
-
cist
ronic cassettes wi
th the best light
-
inducible recombinase activity, N and C
parts of RecV, as well as IRES
-
mediated, PQR
-
mediated and P2A
-
mediated RecV poly
-
cistronic cassettes were
cloned into pCDNA3.1 with the CMV promoter (
Supp.
Fig. 1
).
To generate re
combinant AAV viru
ses expressing split VVD
-
Cre (CreV) or VVD
-
Dre (DreV), the N or C part of
CreV and DreV were cloned after the human EF1a promoter, followed by WPRE and hGH
-
polyA signal (
Supp.
Fig. 1
). The Cre reporters, pAAV
-
EF1a
-
Flex
-
dTomato or EGFP
-
WPR
E
-
hGHpA, used pair
s of double inverted
LoxP and Lox2272 sites to flank the reporter dTomato or EGFP sequence. The Dre reporter, pAAV
-
EF1a
-
Frex
-
dTomato
-
WPRE
-
hGHpA, was generated by inserting an inverted dTomato sequence flanked with Rox sites after
the huma
n EF1a promoter, f
ollowed by WPRE and hGH
-
polyA signal (
Supp.
Fig. 1
).
21
i
FlpV variants were generated with custom gene synthesis as follows:
i
FlpV1: 11 amino acids (aa) N and 412
aa C;
i
FlpV2: 27 aa N and 396 aa C;
i
FlpV3: 49 aa N and 374 aa C;
i
FlpV4: 67 aa N and 356 aa
C;
i
FlpV5 72 aa
N and 351 aa C;
i
FlpV6: 85 aa N and 338 aa C;
i
FlpV7: 95 aa N and 328 aa C;
i
FlpV8: 114 aa N and 309 aa C;
i
FlpV9: 129 aa N and 294 aa C;
i
FlpV10: 151 aa N and 272 aa C;
i
FlpV11: 169 a
a N and 254 aa C;
i
FlpV12: 197
aa N and 226 aa C;
i
FlpV1
3: 208 aa N and 215 aa C;
i
FlpV14: 237 aa N and 186 aa C;
i
FlpV15: 251 aa N and
172 aa C;
i
FlpV16: 290 aa N and 133 aa C;
i
FlpV17: 318 aa N and 105 aa C;
i
FlpV18: 343 aa N and 80 aa C;
i
FlpV19: 374 aa
N and 49 aa C;
i
FlpV20: 388 aa N and 35 aa C;
i
FlpV21:
408 aa N and 15 aa C.
Additional
iFlpV2 variants were generated spanning amino acids
16
-
39
and 366
-
405
covering the entire region
leading to
61 additional constructs
.
C
onstruct
62
was generated based
on iFlpV2 with an addition of the linker GGSGG
present b
etween the C terminus VVD and FlpV to also N terminus FlpV and VVD.
These constructs were cloned
in pcDNA3.1 mammalian expression plasmids.
AAV1, AAV DJ and AAV PHP.eB serotype viruses were
produced in house with titers of AAV1
-
EF1a
-
NCreV,
1.05 x 10
12
gen
ome copies
(GC)
; AAV1
-
EF1a
-
CCreV, 5.16 x 10
12
; AAV1
-
EF1a
-
NDreV, 4.20 x 10
13
; AAV1
-
EF1a
-
CDreV, 5.40 x 10
13
; AAV
-
DJ
-
EF1a
-
Cre, 2.00 x 10
13
; AAV1
-
CAG
-
Flex
-
EGFP, 1.34 x 10
13
; AAV
-
DJ
-
EF1a
-
Frex
-
dTom
ato, 1.90 x 10
12
; 7.7 x 10
11
; 1.6 x 10
13
; AAV
-
PHP.eB
-
EF1a
-
Cre, 5.
8 x10
13
; AAV
-
PHP.eB
-
Syn
-
NDreV, 4.2 x
10
13
; AAV
-
PHP.eB
-
EF1a
-
CDreV, 3.9 x 10
13
;
PHP.eB iCreV, 2.6 x 10
13
;
PHP.eB iDreV, 3.3 x 10
13
,
PHP.eB iFlpV,
2.7 x 10
13
,
AAV
-
PHP.eB
-
EF1a
eGFP
2.03E+13
and
AAV
-
Cre
-
Magnets
3.00E+13
per ml.
AAV5.CAG.tdTomato
(1.0 x 10
13
G
C/mL) were purchased
from
UNC Vector core.
Light activation in cultured cells
HEK293T cells were seeded into 6
-
well plates one day before transfection and reached 80% confluency on the
day of transfection. Cells were co
-
transfected with a reporter express
ing dTomato
–
for Cre and Flp
-
or dTomato
-
for Dre
-
for Cre, Dre or Flp mediated
recombination and various constructs of split
RecVs
. Cells in the control
groups were transfected with reporters alone. Each condition contained 4 replicates. Plates were kept i
n dark
immediately after transfection. Twenty
-
four hours later, cells were expo
sed to blue light, and were then kept in
dark immediately after light exposure. Cells were imaged for fluorescent reporter expression 48 hours after light
induction, using an in
verted fluorescence microscope.
RecV
activated dTomato expression in each condi
tion
was quantified using Image J.
Surface/Cortical
In vivo
1P optogenomic modifications
Stereotaxic injections were made into adult C57BL/6J (stock no. 00064, The Jackson Lab
oratory, Bar Harbour,
ME) or transgenic reporter mice with a 1:1:1 or 1:1 mixtu
re of three or two different rAAVs. For all experiments,
animals were anesthetized with isoflurane (5% induction, 1.5% maintenance) and placed on a stereotaxic frame
(model no.
1900, David Kopf Instruments, Tujunga, CA). An incision was made to expose the
skull, including
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
b
regma,
l
am
b
da, and the target sites. Stereotaxic coordinates were measured from Bregma and were based on
The Mouse Brain in Stereotaxic Coordinates
66,67
. A hole was made above the target by thinning the skull using
a small drill bit until only a very thin layer remained. An opening was then made using a micro
probe, and the
remaining thinned skull was gently pulled away
. All animals were injected at each target with 500 nl of virus at
a rate of ~150 nl/min using a Nanoject II microinjector (Drummond Scientific, Broomall, PA). Intraventricular
injection of AAV
-
PHP.eB viruses was conducted by injecting 2 μl of virus into
the lateral ventricle using a Nanoject
II microinjectior. The glass pipettes had inner diameters between 10
-
20 μm.
Two weeks following AAV injection, animals were anesthetized and returned to the
stereotaxic frame. An incision
was made in the previous location to once again reveal the location of the injection sites. An LED light source
(LED
-
64s, Amscope, Irvine, CA) was mounted to the surgical microscope and positioned 3
-
4 inches directly
above
the animal’s skull. The amount of time the animal was exposed to light was varied by experiments. Small
amounts of sterile PBS were periodically applied to the scalp and skull to prevent drying.
Two weeks following light exposure, animals were perfused
with 4% paraformaldehyde (PFA). Brains were
dissected and post
-
fixed in 4% PFA at room temperature for 3
–
6 h and then overnight at 4°C. Brains were then
rinsed briefly with PBS and stored in PBS with 10% sucrose solution. Brains were then sectioned at a t
hickness
of 100 μm while frozen on a sliding microtome (Leica SM2010 R, Nussloch, Germany). Brain sections were
mounted on 1x3 in. Plus slides and coverslipped with Vectashield with DAPI (H
-
1500, Vector Laboratories,
Burlingame, CA). Slides were then image
d using a 10x objective on a Leica TCS SP8 confocal microscope
(Leica Microsystems, Buffalo Grove, IL).
For fMOST imaging, two weeks or longer following light exposure, animals were perfused with 4%
paraformaldehyde (PFA). Brains were dissected and post
-
f
ixed in 4% PFA at room temperature for 3
–
6 h and
then overnight at 4°C. Brains were then transferred to PBS with 0.1% sodium azide for storage at 4°C until
embedding.
Surface/Cortical
i
n vivo
population
2P
optogenomic modifications
A titanium head
plate
was attached to the skull of mice to allow positioning and restraint of the animal during
imaging. The hole of the head plate was positioned over visual cortical areas, approximately 2.9 mm posterior
and 2.7 mm lateral from Bregma. A 5 mm craniotomy
was cu
t into the skull using a dental drill. The dura was
then removed, and a multilayer glass coverslip was positioned above the craniotomy. The head plate and
coverslips were secured using cyanoacrylate glue and metabond. After a period of at least one w
eek, a
dental
drill was used to remove the cement and metabond holding the coverslip in place, and the coverslip was
removed. A Dumont Nanoject II was then
used to inject 500 nL of viruses
into visual cortex. A new coverslip was
placed and adhered. The ar
ea abo
ve the coverslip was blocked from light using a combination of dental cement
and
Kwik
-
cast, both mixed with black acrylic paint powder.
After at least 3 weeks following viral injection, the animal received two
-
photon laser stimulation. Under dark
conditi
ons, the
K
wik
-
cast was
removed,
and the animal’s head plate was mounted in position. The injection area
was identified by the presence of
the EGFP labelled cells. Laser output was set to 900 nm to optimally induce
recombination. A 600 x 600 μm area was sti
mulated at three depths (100 μm, 150 μm, and 200 μm) for 15
minutes each. After stimulation, black
Kwik
-
cast was reapplied. Two weeks foll
owing stimulation, mice were
perfused.
D
eep
brain
in vivo
stimulation/imaging experiments
For deep brain optogenomic m
odification and imaging experiments stereotaxic
injections were made into
Ai162
-
GC6s (Stock No. 031562, The Jackson Laboratory, Bar Harbour, ME)
Cre dependent
GCaMP6s reporter
mice with a 1:1 mixture of PHP
.
eB.iCreV and a
control
AAV
unconditionally expres
sing red fluorescent protein
(AAV5.CAG.tdTomato)
into the striatum
. For all experiments, animals were anesthetized with isoflurane (5%
induction, 1.5% maintenance) and placed on a
stereotaxic
frame
(
942
, David Kopf Instruments, CA, USA). An
incision was ma
de to expose the skull, including
b
regma,
l
am
b
da, and the target sites. Stereotaxic coordinates
were measured from Bregma and were based on The Mouse Brain Atlas
66,67
.
A
full
hole was made above the
target. All ani
mals were injected with 400 nl X 2 of virus mixture, at two
dorsoventral positions
, 300um apart,
at a rate of ~80 nl/min using
UltraMicroPump
(
UMP3
-
4, World Precision Instrument
,
Sarasota
,
FL)
.
Following
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
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virus
injecti
on
, an optical fiber with cut length of
5 mm and diameter of 400
μ
m (NA 0.48, Doric lenses, Quebec,
QC, Canada) for
1P
stimulation
was firmly mounted to a stereotaxic holder.
The
optical fiber
was then inserted
to the
s
triatum (AP
+1.0
mm, ML ± 1.3 mm, DV
-
3.5 mm, from either left or right sid
e) through a
craniotomy
and
positioned
300
μ
m above the deeper viral injection site. A thin layer of
metabond
was applied on the skull
surface to
secure
the fiber.
In addition,
a thick layer of black dental cement was applied
to secure
fiber implant
for 1P
illumination
to allow positioning and restraint of the animal.
One week following AAV injection of 1:1 virus mixture of PHP.eB.iCreV
and
AAV5.CAG.tdTomato into
GCaMP6s reporter
mice, animals’ baseline signals were recorded
with a
fiberphotometry rig for 1
0 minutes
in
the
home
cage
. Fiberphotometry is a method
for
measuring population
calcium
dependent
fluorescence from
genetically defined cell types in deep brain structures usin
g a single optical fiber for both excitation and emission
in freely moving mice
. Detailed description of the system can be found elsewher
e
68
.
After recording, mice were
connected to a 447nm laser (Opto Engine LLC, UT) using a 200
μ
m optical fiber, illuminated with 5m
W
, 100ms
pulses, 1Hz for 3
0 min
utes
(
TTL
-
controlled by
OTPG_4, Doric lenses, Qu
ebec, QC, Canada)
, in the home
-
cage
. A week following light exposure, fiberphotometry signal was recorded again
for 10 minutes
.
Fiberphotometry peak detection was performed with MATLAB (R2018a), using ‘f
indpeaks’ function, using a
prominence of 2.5.
Mice were perfused 4 weeks after illumination.
Animals were perfused with 4% paraformaldehyde (PFA). Brains were dissected and post
-
fixed in 4% PFA
overnight at 4°C. Brains were then rinsed briefly with PBS a
nd then sectioned at a thickness of 100 μm on
a
vibr
a
tome (VT1200 Leica Biosystems, IL, USA). Brain sections were incubated in a blocking solution, containing
1x PBS solution
with
0.1% Triton X
-
100 and 10% normal donkey serum (NDS; Jackson ImmunoResearch,
PA,
USA) for at least an hour, washed, and fur
ther incubated with blocking buffer containing primary antibody (see
below for details) at 4C overnight. Afterward, sections were thoroughly washed three times (15 min each) in 1x
PBS and then transferred into
the
blocking solution with secondary antibody
(see below for details) for 2h at
room temperature.
Finally,
sections were again washed by 1x PBS solution four times (15 min each), mounted
on glass microscope slides (Adhesion Superfrost Plus Glass Slides, #5
075
-
Plus, Brain Research Laboratories,
MA, USA
), dried, and coverslipped with mounting media (Prolong Diamond, P36965, Thermo
-
Fischer, CA, USA).
For primary antibody: chicken
anti
-
m
C
herry (1:1000; ab205402, Abcam, Cambridge, UK
)
was used
. For
secondary ant
ibody, anti
-
chicken Alexa Fluor 594 (1:500; 70
3
-
585
-
155, Jackson ImmunoResearch) was used.
Fluorescent images from brain tissue were acquired
with
a
n
LSM 880 confocal microscope (Carl Zeiss, Jena,
Germany). We used a 10x Plan Apochromat air objective (NA
0.45), 25x Plan Apochromat water immersion
obj
ective (NA 1.2) and
three
laser wavelengths (488 nm, 561 nm, and 633 nm). Image acquisition was
controlled by Zen 2011 software (Zeiss), which also allowed automated tiling, and maximum intensity
projection. Im
ages were not further processed.
Expression co
unts were done by summation of the values of
the fluorescence within 1mmX1mm below the fiber tip subtracted with the same area at the opposite
hemisphere, line by line, and normalized to the maximal value.
Ze
brafish experiments
N
-
CreV and C
-
CreV were
PCR amplified from pAAV
-
Ef1a NCreV and pAAV
-
Ef1a CCreV, and cloned into the
pDest
-
ubi vector (Addgene plasmid # 27323) by Gibson assembly with the following primers: 5' N
-
Cre overlap
pdest
-
UBI: attcgacccaagtttgtac
aaaaaagcaggctggacgccaccatgacgagtgatgaggtt;
5' C
-
Cre overlap pdest
-
UBI:
tcgacccaagtttgtacaaaaaagcaggctggacgccaccatgcatacactgtatgcccc; 3' polyA (used for both constructs):
actgctcccttccctgtccttctgcatcg
atgatgatccagacatgataagatacattga
The pDest
-
ubi:N
-
vCre
-
pDes
t and pDest
-
ubi:C
-
vCre mixture containing e
qual amounts of each plasmid (25pg
each) and tol2 transposase RNA (25 pg) was injected into one
-
cell stage tg(ubi:Zebrabow
-
M) zebrafish embryos.
Embryos were either light or dark reared for 72 hrs. At 3dpf injecte
d embryos were anesthetized with Mesab,
mou
nted in 2% agarose, and imaged on a Zeiss LSM 880 confocal microscope.
fMOST imaging
All tissue preparation has been described previously
69
. Following fixation, each intact brain was rinsed three
times (6 h for two washes and 12 h for the third wash) at 4°C in a 0.01 M PBS solution (Sigma
-
Aldrich Inc
., St.
Louis, US). Then the brain was subse
quently dehydrated via immersion in a graded series of ethanol mixtures
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
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(50%, 70%, and 95% (vol/vol) ethanol solutions in distilled water) and the absolute ethanol solution three times
for 2 h each at 4°C. After d
ehydration, the whole brain was impregnated
with Lowicryl HM20 Resin Kits (Electron
Microscopy Sciences, cat.no. 14340) by sequential immersions in 50, 75, 100 and 100% embedding medium in
ethanol, 2 h each for the first three solutions and 72 h for the fi
nal solution. Finally, each whole brain was
embedded in a gelatin capsule that had been filled with HM20 and polymerized at 50°C for 24 h.
The whole brain imaging is
performed
using a fluorescence microscopic optical sectioning tomography (fMOST)
system. T
he basic structure of the imaging
system is the combination of a wide
-
field upright epi
-
fluorescence
microscopy with a mechanic sectioning system. This system runs in a wide
-
field block
-
face mode but updated
with a new principle to get better image contras
t and speed and thus enables high
throughput imaging of the
fluorescence protein labeled sample (manuscript in preparation). Each time we do a block
-
face fluorescence
imaging across the whole coronal plane (X
-
Y axes), then remove the top layer (Z axis) by
a diamond knife, and
then expose n
ext layer, and image again. The thickness of each layer is 1.0 micron. In each layer imaging, we
used a strip scanning (X axis) model combined with a montage in Y axis to cover the whole coronal plane
70
. The
fluorescence, collected using a microscope o
bjective, passes a bandpass filter
and is recorded with a TDI
-
CCD
camera. We repeat these procedures across the whole sample volume to get the required dataset.
The objective used is 40X WI with numerical aperture (NA) 0.8 to provide a designed optical res
olution (at 520
nm) of 0.37 μm in
XY axes. The imaging gives a sample voxel of 0.30 x 0.30 x 1.0 μm to provide proper resolution
to trace the neural process. The voxel size can be varied upon difference Objective. Other imaging parameters
for GFP imaging i
nclude an excitation wavelength of
488 nm, and emission filter with passing band 510
-
550 nm.
Cortical targeted in vivo 2P stimulation of single cells
For
in vivo
targeted single cell optogenomic modifications as well as
simultaneous
GCaMP
7
f
imaging
experim
ents Stanford
Administrative Panel on Laboratory Animal Care (APLAC) approved all animal procedures.
Ai14 mice (The Jackson Laboratory, 07908) of two to four months age were used for experiments. For viral
vector injection, mice were anesthetized with isof
lurane. iCreV
virus used: AAV2/PHP.eB
-
EF1a
-
i
Cre
V
; GFP virus
used: AAV2/PHP.B
-
CAG
-
GFP; GCaMP virus used: AAV2/9
-
CamkIIa
-
jGCaMP7f. Viral vectors were loaded into
a glass pipette and injected into cortex with picospritzer (Paker Hannifin). ~500 nL was deliver
ed over 15 min
utes
and then a 4 mm craniotomy was made with the injection site at the center, 30 minutes after the injection. Dura
was removed before cover glass was installed and sealed. A custom
-
made head bar and cover were secured
with dental cement on
mouse skull. I
maging experiments started one month after the surgery to allow gene
expression.
Mouse was mounted on a running wheel with head fixation and was remained awake during the whole
experiment. Before imaging, the head mount cover was removed. In
order to opera
te the mouse and the
microscope, a red LED light was used for illumination (WAYLLSHINE). Ultrasound gel (Parker, Aquasonic) was
put on the cover glass for the water immersion objective lens. The mouse was aligned manually without checking
the
focal plane w
ith eye piece to avoid iCreV induction during this process.
For induction experiment, a femtosecond Ti:sapphire laser (Spectra
-
Physics, Mai Tai) was tuned to 920 nm
wavelength. The scanning and image acquisition were achieved with a Prairie (
Bruker) two
-
ph
oton microscope,
through a 20X 0.95 N.A. water immersion lens (Olympus XLUMPLFLN
-
W 0.95 NA 20×). For all the imaging
sessions, laser power at a specimen was kept at 25 mW and it was monitored (Thorlabs, PM100D and S130C)
at an additional outp
ut of the opti
cal path before entering the microscope, whose splitting ratio was calibrated
prior the measurements. During the first imaging session, a starting point with unique vessel pattern was
identified and recorded as the starting position. All targ
et cells’ rela
tive coordinates to the starting position were
recorded and used for relocation in later sessions. Images were acquired with 4 μs pixel dwell time at 1024 x
1024 pixel frame size (field of view 450 micron x 450 micron), with 3 μm Z
-
axis step
size. After id
entifying a target
cell, an induction scanning was carried out using the ROI function to limit the scanning the middle of the target
cell’s somata region. The scanning pixel dwell time is increased to 10 μs and various scanning conditions wer
e
used in each
mouse tested. After the first induction session, the head cover was re
-
installed to seal the
craniotomy from ambient light. Seven to ten days after the first session, all the cells were re
-
imaged to check
tdTomato expression and at the end u
nderwent a 30
minutes blue LED exposure (5 mW, 470 nm, Thorlabs,
M470L3). And the third imaging session was carried out seven to ten days after the LED exposure.
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;
For calcium imaging experiment, a custom
-
made two
-
photon microscope was used with an objective
lens
Olympus
XLUMPLFLN
-
W 0.95 NA 20×, 8 kHz resonant scanner head (Cambridge Technologies,
CRS8K/
6215H). Fluorescence light have been captured using H11706
-
40 and H10770PA
-
40 photomultiplier
tubes equipped with low noise amplifiers (Femto, DHPCA
-
100), wh
ose analog sig
nal was subsequently digitized
(National Instruments, NI
-
5732) and formed into images using field programmable gate array (National
Instruments, FPGA NI
-
7961R) and ScanImage software (Vidrio Technologies) [Pologruto et al., Biomedical
Enginee
ring Online 2:
13, 2003]. GCaMP signal was collected at 30 Hz frame repetition rate for 30 minutes in
each mouse.
Data availability
All relevant plasmids will be deposited to Addgene. Data are available from the corresponding author upon
request.
ACKNOWLE
DGMENTS
We are grateful to the Structured Science teams at the Allen Institute for their technical support in stereotaxic
injections and mouse colony management. The
work was funded by the Allen Institute for Brain Science, NIMH
BRAIN
Initiative grant
RF1M
H114106
to A.C.,
NSFC Science Fund for Creative Research Group of China
(Grant No. 61421064) to H.G., Q.L. and S.Z
., NIH Director's New Innovator award IDP20D017782
and
NIH/NIA 1R01AG047664
to V.G.
,
and
Colvin divisional fellowship
of
Division of Biology a
nd
Biological
Engineering
,
California Institute of Technology
,
to
A.K.
NIH Brain Initiative
U01NS107610
grant to Mark
Schnitzer.
I
mmunohistochemistry
experiments
in
F
igure 8
w
ere
performed in the Biological Imaging Facility,
with the support of the
Califor
nia Institute of Technology
Beckman
Institute and the Arnold and Mabel Beckman
Foundation.
The cre
ation of Ai139
mouse line was supported by the NIH grant R01DA036909 to B.T.
The
authors thank Sevi Durdu
, Bilal Kerman
and Keisuke Yonehara for critical read
ing and feedback.
The authors
wish
to thank the Allen Institute founder, Paul G. Allen, for his vision, encourag
ement, and support.
AUTHOR CONTRIBUTIONS
A.C. concept
ualized
the light
-
inducible recombinase system. S.Y. performed cloning and characterizati
on of the
construct
s
as well
as
participated in image acquisition. B.O. performed all the surgeries and image acquisition.
T.Z. Performed cloning. M.M. performed some of the surgeries and light stimulations. T.D. performed some of
the initial cloning exper
iments. B.T. and H.Z. contribute
d to the generation of the Ai139 transgenic mice. H.G.,
Q.L. and S.Z. acquired fMOST data. X.K. and Y.W. performed Neurolucida reconstructions. V.G. and A.K.
performed deep brain imaging experiments.
S.C. and P.B. performed
2P induced
recombination experim
ents.
A.CT. and A.D. performed zebrafish experiments.
R.C., P.Y and M.S. performed the targeted single cell 2P
experiments and combinatorial
cortical
GCamp
7
F
calcium imaging experiments.
A.C. and H.Z. designed and
coordinate
d the study as well as wrote the manuscript, with inputs from all coauthors.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
All rights reserved. No reuse allowed without permission.
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/553271
doi:
bioRxiv preprint first posted online Feb. 18, 2019;