of 33
1
Identification
of
peripheral
neural circuit
s
that regulate heart rate
using optogenetic
and viral vector strategies
Pradeep S. Rajendran
,
1
Rosemary
C.
Challis
,
2
Charless
C.
Fowlkes
,
3
Peter Hanna
,
1
John
D. Tompkins
,
1
Maria C. Jordan
,
1
Sarah Hiyari
,
1
Beth
A. Gabris
-
Weber
,
4
Alon Greenbaum
,
2
Ken Y. Chan,
2
Benjamin E. Deverman,
2
Heike M
ü
nzberg
,
5
Jeffrey L. Ardell
,
1
Guy Salama
,
4
Viviana Gradinaru
,
2
*
and
Kalyanam
Shivkumar
1
*
1
Cardiac Arrhythmia Center and Neurocardiology Research
Program
of Excellence
, David
Geffen School of Medicine,
UCLA
, Los Angeles, CA
2
Division of Biology and Biological Engineering, California Institute of Technology
,
Pasadena,
CA
3
Department of Computer Science, University of California
-
Irvine, Irvine, CA
4
Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA
5
Neurobiology of Nutrition and Metabolism Department
, Louisiana State University, Baton
Rouge, LA
K.Y.C and B.
E.D. are present
ly affiliated with
Stanley Center for Psychiatric Research, Broad
Institute, Massachusetts Institute of Technology, Cambridge, MA
.
*
To whom correspondence should be a
ddress
ed
:
Kalyanam Shivkumar, M
.
D
.
, Ph
.
D
.
UCLA
Cardiac Arrhythmia Center
100 Medical Pl
aza
S
uite
660
Los Angeles, CA 90095
Phone:
1
-
310
-
206
-
6433
Email:
ks
hivkumar@mednet.ucla.edu
Viviana Gradinaru,
Ph
.
D
.
Division of Biology and Biological Engineering
California
Institute
of
Technology
1200 East California
B
oulevard
M
ail Code
156
-
29
Pasadena, CA 91125
Phone: 1
-
626
-
395
-
6813
Email:
viviana@caltech.edu
The authors have declared that no conflict of interest exists.
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(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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/10.1101/456483
doi:
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2
Abstract
H
eart
rate
is under the precise control of the autonomic nervous system
.
However,
the wiring of
peripheral
neural circuits
that regulate
heart rate is poorly understood
.
Here
,
w
e
developed a
clearing
-
imaging
-
analysis pipeline to visualize innervation of
intact
hearts in 3D
and
employed a
multi
-
technique approach to map
parasympathetic and sympathetic
neural
circuits
that control
heart rate
in mice
.
We
anatomically and functionally identify
cholin
ergic
neurons
and
noradrenergic neurons
in an intrinsic cardiac ganglion and
the
stellate ganglia
, respectively,
that
project to the
sinoatrial node
. We also
report
that
the heart rate response to
optogenetic
versus
electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal
afferents
enhance parasy
mpathetic
and reduce
sympath
etic
tone
to the heart
via central
mechanisms
.
O
ur findings
provide
new insights into neural
regulation
of heart rate
, and our
methodology to study cardiac circuits can be readily used to interrogate neural control of other
visceral organs.
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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/10.1101/456483
doi:
bioRxiv preprint first posted online Oct. 29, 2018;
3
Situation
-
dependent changes in heart rate are essential for survival and
are
under the
precise
control of the autonomic nervous system (ANS)
1
.
Heart rate r
eduction during sleep
2
and elevation
during
exercise
3
result from
changes in parasympathetic and sympathetic tone
.
In fact, h
eart rate
variability has
been
utilized
extensively as an index
of ANS function
4
-
6
.
Although it
i
s well known
that the parasympathetic and sympathetic nervous system
s innervate the sinoatrial (SA) node
7,8
and
regul
ate heart rate
9
-
12
, the wiring of the
se
neural
circuits
in the periphery
is
not well
characterized
.
Ana
tomical
and functional maps of
these
fundamental
cardiac
circuits
are needed
to
understand
physiology, characterize
remodeling in disease
(e.g., sick sinus syndrome
13
)
, and
develop
novel therapeutics
.
However,
these efforts have been hindered by
a
shortage
of
tools
that target
the
peripheral nervous system (
PNS
)
with specificity and precision.
Cardiac
circuit
anatomy has
traditionally been studied
using
thin
sections
14
and whole
-
mount preparations
15
. However, these methods
do not preserve t
he structure of intact circuits and
only
provide 2D information
.
In contrast,
tissue
clearing
methods
render
tissues
optically
transparent while preserving
their
molecular and cellular architecture and can be combined with
a variety of
labeling strategies
to enable 3D visualization of intact circuits
16,17
.
To trace cardiac
circuits, d
yes and proteins have historically been used
. However, achieving cell type
-
specificity
and/or sparse labeling needed for
singe
-
cell tracing and
delineating
circuit
connectivity is
difficult
or
not possible with these methods
1
8
.
Additionally, many
peripheral neuronal populations such as
intrinsic cardiac ganglia
are c
hallenging to access surgically
for
tracer
delivery
.
A
d
eno
-
associated
viruses (AAVs)
can address these limitations
because they can be
utilized
for efficient
in
vivo
gene
expression
in
defined cell populations when used in Cre transgenic animals
or
with cell
-
type
specific regulatory elements
19
-
21
.
In addition, i
ntersectional strategies can
be used to titrate gene
expression to achieve sparse labeling
20
.
AAVs can
also
be delivered systemically to target
difficult
-
to
-
reach populations
19
-
21
.
F
unctional
mapping
of
cardiac circuits
has
rel
ie
d
on
electrical
or pharmacological
manipulation
of
the
ANS
with simultaneous physiological measurements
22,23
.
However,
each of
these methods ha
s
disadvantages. Electrical
techniques
lack spatial precision and specificity.
A
utonomic nerves
such as the vagus
contain motor and sensory fibers
24
,
and electrical
stimulation
typically
activates
both
fiber types
25,26
as well as surrounding tissues
27
.
P
harmacological
techniques
exhibit improved sele
ctivity but
lack temporal resolution.
In contrast, o
ptogenetics
,
which
us
e
s
light
-
sensitive ion channels
(e.g.,
channelrhodopsin
-
2
(ChR2)
,
halorhodopsin
)
,
enables
precise
spatiotemporal
control of defined cell
populations
.
Here
,
w
e
develop a clearing
-
imaging
-
analysis pipeline to visualize innervation of whole
hearts
in 3D
and
employ
a multi
-
technique approach, which includes
AAV
-
based
sparse
labeling
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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.
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.
http://dx.doi.org/10.1101/456483
doi:
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4
and
tract
-
tracing,
retrograde n
eur
ona
l tracing with cholera toxin subunit B (CTB)
,
a
nd
optogenetics
with simultaneous
physiological
measurements
,
to
map
peripheral
parasympathetic
and
sympathetic
neural
circuits
that regulate
heart rate
in
mice
.
RESULTS
Tissue clearing and computational pipeline to
assess
global
i
nnervation
of
whole
heart
s
To
characterize
global
innervation
of the mouse heart in 3D
,
we developed a
clearing
-
imaging
-
analysis
pipeline (Figure 1a). W
e stained
whole hearts
with an antibody against
the
pan
-
neuronal
marker protein gene product 9.5 (PGP9.5)
and rendered
them
optically
transparent
using a
n
immunolabeling
-
enabled three
-
D
imensional
I
maging of
S
olvent
-
C
leared
O
rgans
(iDISCO)
protocol
(Figure 1
b)
28
.
We used c
onfocal microscopy to image large
tissue
volumes (
Figure 1c
and
Supplemental Movie
1
)
and lightsheet microscopy to image entire
hearts (Supplemental
Movie 2
)
29,30
.
W
e
observe
d
cardiac ganglia
surrounding the pulmonary vein
s
and a dense
network
of
nerve fibers
coursing through
the
atrial and ventricular
myocardium
(Figure 1c
). Innervation
was
seen throughout the entire thickness of the myocardium, with large
-
diameter
nerve
fiber
bundles
located
near
the epicardium
and smaller fiber bundles in the mid
-
myocardium and
endocardium
(Fi
gure 1c
and Supplemental Movie 3
).
T
o analyze these data
,
w
e created a
semiautomated
computational pipeline to detect nerve fibers
over large tiled
volumes and
to
measure
microanatomical
features of
fibers
such as
diameter and or
ientation
(Figure 1, d and e
)
.
Large
-
diameter nerve fiber
bundles
typically
enter
ed
near the base of the heart
. These bundles
coursed
perpendicular to the atrioventricular (AV) groove
and
branch
ed
into smaller fiber bundles
as they progress
ed
towards the apex.
These
data
from healthy hearts will be
important
for
future
characterization of
neural remodeling
in
cardiovascular
diseases
such as myocardial infarction
(MI)
in which
innervation patterns
are disrupted
and nerve sprouting occurs
31,32
.
AAV
-
based
sparse
labeling
and tract
-
tracing
of cholinergic neurons
in
intrinsic cardiac
ganglia
After visualizing
global
cardiac
innervation, w
e
assessed whether we
could
identify
a subset
of
cholinergic
neurons
that form a
n
anatomical
circuit with the
SA
node
to potentially regulate heart
rate
.
W
e
used
cell type
-
restricted sparse viral labeling
to trace
cholinergic
neurons
in intrinsic
cardiac ganglia
to their regions of innervation
.
W
e systemically co
-
administered Cre
-
dependent
vectors expressing
fluorescent proteins (
XFPs) from
the
tetracycline
-
responsive element
(TRE)
-
containing promoter at a high dose and
the
tetracycline
transactivator
(
tTA
)
from the human
synapsin I promoter (hSyn1)
at a low
er
dose in ChAT
-
IRES
-
Cre transgenic
mice
(Figure 2a)
20
.
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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/10.1101/456483
doi:
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5
Compared to
dense multicolor
labeling
(Figure 2b
)
, sparse
multicolor
labeling resulted i
n a
labeling density
in intrinsic cardiac ganglia
that was lower
and would more easily allow for tracing
(Figure 2c
)
20
. For
tract
-
tracing
of cholinergic neurons in intrinsic cardiac ganglia
, we
utilized
sparse
s
ingle
-
color labeling
with tdTomato
.
Three weeks after viral delivery
, h
earts were
collected and
stained
with an antibody
for
hyperpolarization
-
activated cyclic nucleotide
-
gated potassium
channel 4 (HCN4)
to identify the
SA
node
,
the
AV
node,
and
the
conduction system
33
.
We
observed
cholinergic
fibers
from
a
cardiac ganglion
,
located
below
the
junction of the
right and
left
inferior pulmonary veins
(inferior pulmonary veins
-
ganglionated plexus, IPV
-
GP)
,
projecting
towards
the
SA
node
, the AV node,
and
the
ventricles
(Figure 2d
)
, identifying
cholinergic
neurons
that are
potentially
involved in c
hronotropic
, dromotropic, and
ventricular control
, respectively
.
Ex vivo
o
ptogenetic stimulation of cholinergic neurons
in
the
IPV
-
GP
Next, t
o
functionally
assess whether
cholinergic neurons in the
IPV
-
GP
regulate
heart rat
e,
AV
conduction,
and ventricular electrophysiology
,
we used
an
optogenetic
approach
.
We expressed
ChR2 in
choline
rgic neurons
by crossing
transgenic
ChAT
-
IRES
-
Cre
mice
with
reporter mice
containing a Cre
-
dependent ChR2
-
enha
nced yellow fluorescent protein
allele
(ChR2
-
e
YFP
;
offspring from
this cross are subse
quently referred to as
ChAT
-
ChR2
-
e
YFP
mice
)
.
ChR2
-
e
YFP
expression
in
intrinsic cardiac neurons was
confirmed by staining
hearts
for
GFP
(
to amplify
ChR2
-
e
YFP detection
)
and PGP9.5 (Figure 3, a and b
).
A
lmost all PGP9
.
5
+
neurons
in cardiac
ganglia
were
GFP
+
(96.4
±
1.2%) (Figure 3
c).
GFP staining
was
also present in PGP9.5
+
nerve
fibers
in the atria and ventricles (Figure 3b).
These data are consistent with previous studies
reporting
that the
majority of intrinsic car
diac neurons are
cholinergic
34
and that the ve
ntricles as
well as atria receive
cholinergic
innervation
34
-
36
.
After verifying ChR2 expression, we
next
assessed whether selective stimulation
of
cholinergic neurons
in
the
IPV
-
GP
modulated
heart rate
,
AV
nodal
conduction
, and ventricular
electrophysiology
using optogenetics
in
ex vivo
Langendorff
-
perfused hearts
.
A blue laser
-
coupled optical fiber was positioned for focal illumination of
the
IPV
-
GP
while cardiac electrical
activity was simultaneously recorded (Figure 3
,
c and d
).
Optogenetic
stimulation resulted
in a
decrease in heart rate that
was
dependent on light pulse power, frequency, and pulse width
(Figure 3,
e
-
g
) but
did not change
the
AV
interval
(35.7
±
1.2 ms versus 35.9
±
1.0 ms;
P
= 0.88)
(Figure 3
h
).
T
he lack of change in
AV nodal conduction
suggests that fibers from this ganglion
may pass through the AV node without
synapsing
.
In addition, s
timulation
prolong
ed
the
P wave
duration (9.3
±
1.0
ms
versus 12.3
±
2.4 ms
;
P
< 0.05
) and caused
P wave frac
tionation (
Figure
3,
i and j
).
P wave fractionation has
been reported in human
s
following
administration
of
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.
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6
adenosine
37
, which mimics the effects of acetylcholine
released from
cholinergic nerve terminals
38,39
.
D
uring
stimulation
s at higher frequencies
,
we
occasionally
observ
e
d
ectopic atrial rhythms
(
n
=
3
/5 mice
)
and
even asystole
(
n
=
1
/5 mice
)
(Figure 3d
), demonstrating the profound effect of
the IPV
-
GP
on
the
SA
node
and atrial function.
The response to stimulation
was
abo
lished by
administration of the
muscarinic receptor antagonist
atropine (
-
33.5
±
11% versus
-
0.3
±
0.2%
;
P
< 0.05
) (Figure 3
k
)
, indicating that the bradycardic response
was
indeed
mediated by selective
stim
ulation of cholinergic neurons.
Since
ChR2 is expressed in both
preganglionic
cholinergic inputs to
and
postganglionic
cholinergic
neurons
in the IPV
-
GP in ChAT
-
ChR2
-
eYFP mice, we assessed whether we
could
stimulate
only
postganglionic cholinergic neurons in the IPV
-
GP using optogenetic
s
and
s
till
modulate
heart rate
. W
e
first
evaluated
whether we could
preferentially
deliver
transgenes
to
peripheral
cholinergic neurons in intrinsic cardiac ganglia
rather than central cholinergic neurons
in the medulla
using systemic AAVs.
We used
AAV
-
PHP.S,
a
capsid variant
that more
efficiently
transduces
the PNS and many
visceral organs including the heart,
as compared to AAV9
20
.
We
packaged a
Cre
-
depen
dent genome that expresses
e
YFP
from
the ubiquitous
CAG
promoter
into
AAV
-
PHP.S
and systemically
administered the virus to
ChAT
-
IRES
-
Cre transgenic mice.
Three
weeks later, we
evaluated
eYFP
expression
in
the medulla, the vagus nerve
, and intrinsic cardiac
ganglia with
GFP
staining
.
C
entral cholinergic neurons
in the
dorsal motor nucleus of the vagus
nerve did not
express detectab
le levels of eYFP
and cholinergic fibers in the vagus nerve were
weakly
t
ransduced (Supplemental Figure 1
, a
-
c).
In contrast
, we observed
robust
transduction of
peripheral cholinergic neurons
in
intrinsic cardiac ganglia (
80.1
±
1.5%
PGP9.5
+
cells expressed
GFP
)
(Supplemental Fi
gure 1
, d
and e)
,
likely due to
the strong tropism
AAV
-
PHP.S
displays for
the PNS
over the CNS
.
For functional
studies
, w
e
packaged
a Cre
-
dependent genome that
expresses ChR2
-
e
YFP
from
the ubiquitous CAG promoter in
AAV
-
PHP.
S,
systemically
administered the virus t
o ChAT
-
IRES
-
Cre transgenic mice
, and evaluated expression
5
weeks
later
.
In Langendorff
-
perfused hearts,
w
e
were able to
optogenetically
stimulate
postganglionic
cholinergic neurons in
the
IPV
-
GP
and
decrease heart
rate (Supplemental Figure 2
).
Taken
together,
our anatomical
and
functional
data
establish an IPV
-
GP
-
SA node circuit
involved in heart
rate regulation
(Figure 3j)
. Furthermore,
the
engineered
AAV
, AAV
-
PHP.S
20
,
can
be a powerful
tool to dissect out the roles of peripheral versus central circuits on organ control.
In vivo
o
ptogenetic versus
electrical stimulation of the vagus nerve
Electrical
vagus nerve stimulation (VNS)
has been used
in numerous preclinical and clinical
studies
for the
treatment of
cardiovascular
disease
s
40
and other conditions
(e.g., rheumatoid
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.
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doi:
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7
arthritis
41
)
.
However,
the
relative contributions
of vagal effer
ent and afferent fibers on
cardiac
function
are
not
well understood
because conventional techniques do not allow for
fiber type
-
specific
stimulation.
To address th
is
limitation
,
w
e
examined
whether
we could selectively
stimulate
efferent
fibers
in
the vagus nerve
using optogenetics
.
We
also
assessed
whether
there
was a difference between
optogenetic
and
electrical
VNS
on
heart rate
(Figure 4
a
), as a
previous
study showed that electrical stimulation of motor
nerve
s
results in a non
-
orderly,
non
-
physiological
recru
itment
of fibers
, wi
th large
r
fibers activated
first
42
.
The vagus and other autonomic nerves
contain both motor and sensory fibers that vary in diameter and myelination
27
and non
-
electrical
techniques such as optogenetic
s are needed to study their physiological role.
To confirm
that
ChR2
-
e
YFP
expression was limited to
vagal
efferents
in ChAT
-
ChR2
-
e
YFP
mice
,
we
stained
for
GFP and PGP9.5 in
the
nodose
-
petrosal
ganglion
complex
, which
contains the cell bodies of vagal sensory neurons,
and the cervical vagus nerve
(Figure 4
b
)
.
e
YFP
was not detected
in PGP9.5
+
cell bodies in
the
nodose
-
petrosal ganglion complex
and
was
only
present
in a subset of
PGP9.5
+
vagal fibers
(
n =
5 mice)
.
After verifying expression
,
w
e
next
performed function
al studies in anesthetized mice
in
which we
positioned
a
laser
-
coupled optical
fiber
for focal illumination above
and a
hook electrode underneath
the right vagus nerve
.
In this
context,
optogenetic
VNS
activate
s
only
efferent fibers (GFP
+
), whereas electrical VNS
presumably
activates
subsets of
efferent and afferent fibers (PGP9.5
+
)
in
the vagu
s nerve (Figure
4
b
).
With
both
vagi
intact
, optogenetic and electrical
right
VNS
resulted
in
a
similar
decrease
in
heart rate
(Figure 4
d
)
; h
owever, the
slope
s
of the
responses
were
dramatically
different (
n =
6/8
mice;
Figure 4
c
),
likely
due to differential
fiber
recruitment
42
.
The he
art rate response to
optogenetic
versus electrical stimulation of the caudal end of the right vagus nerve
was
also
similar following either right or bilateral
vagotomy
(Figure 4
, e and g)
.
In contrast, optogenetic
stimulation of the rostral end of the right vagus nerve followi
ng
either
right or bilateral
vagotomy
did
not
affect
heart rate, whereas electrical stimulation
surprisingly
result
ed
in a
decrease in
heart
rate
at
10
Hz
(
-
6.5
±
1.8% versus
-
0.1
±
0.1%
following right vagotomy for
electrical versus
optogenetic
stimulation
;
P
<
0.05
)
and 20 Hz
(
-
9.8
±
1.9% versus
-
0.2
±
0.1% following right
vagotomy and
-
3.2
±
0.4% versus
-
0.2
±
0.1% following bilateral vagotomy for electrical versus
optogenetic
stimulation
;
P
< 0.01 for both
)
(Figure 4
, f and h)
.
The
decrease in
heart
rate to
electrical stimulation of the rostral end of the right vagus nerve following right vagotomy (Figure
4f, red line) was greater than that following bilateral vagotomy (Figure 4h, red line) (
P
< 0.035),
suggesting that the response following right vagotomy was
in part
due to v
agal afferent
-
mediated
increase
in parasympathetic efferent outflow
through
the
intact contralateral vagus nerve.
In
addition, there
was
an
increased latency
t
o peak heart
rate response with electrical stimulation of
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8
the rostral right vagus nerve following bilateral vagotomy
compared to
that of
the intact right vagus
nerve (8.7
±
0.4 ms versus 3.9
±
0.5 ms
;
P
< 0.01
) (Figure 4
i)
, indicating that the response post
-
bilateral
vagotomy was
likely
due to vagal afferent
-
mediated withdrawal of sympathetic tone
43
.
Taken together,
our data
suggest that
optogenetic stimulation selectively activates vagal effere
nt
s
in ChAT
-
C
hR2
-
e
Y
FP mice and that vagal afferents
act centrally to 1) increase parasympathetic
efferent outflow and 2) decrease sympathetic efferent outflow to the heart.
Localization and
in vivo
optogenetic stimulation of
cardiac
-
projecting
nor
adrenergic
neurons
in
the
stellate ganglia
The s
ympathetic
nervous system
,
along with the parasympathetic nervous system,
precise
ly
regulates
heart rate
in normal physiology
.
To
anatomically and functionally
dissect
noradrenergic
neurons that form a circuit with the SA node
,
we used a
retrograde
n
euronal tracer and an
optogenetic approach
.
Noradrenergic
nerve
fibers
densely innervate
the
heart as
shown
by
staining
for
tyrosine
hydroxylase
(TH)
, the rate
-
limiting enzyme in norepinephrine
synthesis
(Figure 5
a).
T
o identi
fy the location of cardiac
-
projecting
sympathetic
neurons, w
e injected
the
retrograde neurona
l tracer
CTB
conjugated to Alexa Fluor 488
into
the heart
(Figure 5
b).
The
majority of
CTB
+
neurons
were
located
in
the stellate gangli
a
of
the paravertebral chain
, with
fewer labeled neurons in
the middle cervical and
second thoracic
(T2)
ganglia
(Figure 5
c).
On
average, 236
±
39 neurons
were
labeled in the right and 261
±
34 neurons
labeled
in the left
stellate ganglion
(
n
=
5 mice)
.
Heat maps of the right and
left st
ellate ganglia
show that
cardiac
-
projecting
sympathetic
neurons
are
clu
stered in the craniomedial
aspect
(Figure 5
d)
and
suggesting
that
these ganglia may have a viscerotopic organization.
Next
,
we
assessed whether we could
selectively
stimulate
noradrenergic neurons
in
the
paravertebral chain
using
optogenetics
and modulate heart rate
.
We expressed ChR2 in
noradrenergic
neurons by crossing transgenic TH
-
IRES
-
Cre mice with reporter mice c
ontaining a
Cre
-
dependent ChR2
-
e
YFP allele
(
offspring from this
cross are subsequently referred to as
TH
-
ChR2
-
e
YFP mice)
.
ChR2
-
e
YFP
exp
ression
in
stellate ganglion
neurons was confirmed by
staining
for
GFP
and TH
(Figure 6
b).
A
lmost all TH
+
neurons
were
also
GFP
+
(
95.9
±
1.6%
;
n
=
4
mice
)
.
After verifying expression, w
e
performed function
al studies in
open
-
chest
anesthetized
mice
in
which
we positioned a laser
-
coupled optical fiber for focal illumination above the
craniomedial
right stellate
ganglion
(RSG)
or
right
T2
ganglion
(Figure 6
a)
. Optogenetic
stimulation of the
craniomedial
RSG
resulted
in
a
frequency
-
and pulse
width
-
de
pendent
increase
in heart rate
(Figure 6
, d and e)
.
Although a small number of cardiac
-
projecting neurons
were
loca
ted in the T2 ganglion (Figure 5
c),
there
was
no
heart rate
response to stimulation
of this
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9
ganglion
(Figure 6
f).
T
he response to
craniomedial
RSG
stimulation
was
abo
lished by
administration of the
b
-
adrenergic
receptor antagonist propranolol (
5.6
±
1.4% versus 0.2
±
0.1%
;
P
<
0.05
)
(Figure 6
g),
indicating that the
tachycardic
response was
indeed
mediated by selective
stimulation of
nor
adrenergic
neurons.
Taken together, o
ur
anatomical and functional
data
establish
a
craniomedial RSG
-
SA node
circuit
involved in heart rate regulation
.
DISCUSSION
W
e
developed a clearing
-
imaging
-
analysis pipeline to
visualize
innervation
of whole hearts
and
employed
a multi
-
technique approach
to dissect
fundamental
parasympathetic and sympathetic
neural circuits
involved in heart rate regulation
. We
report
several
novel findings
:
1)
cholinergic
neurons in the IPV
-
GP
and noradrenergic neurons in the craniomedial RSG
project to the SA
node
and
modulate
its function
; 2)
the
evoked cardiac
response to optogenetic versus electrical
stimulation of the vagus nerve
displays
different temporal characteristics;
and
3
)
vagal afferents
enhance parasympathetic
and reduce
sympathetic efferent outflow
to the heart
via central
mechanisms
.
Despite advances in
tissue clearing
32,44
and imaging techniques
29,30
,
h
igh
-
resolution
,
3D
datasets of global cardiac innervation
do not
exist
.
We show, for the first time, innervation of an
entire cleared
mouse
heart with
cardiac ganglia
located
around the pulmonary veins
and a dense
network of
nerve fibers
throughout the myocardium
.
To analyze these data
,
we developed
a
semiautomated
computational
pipeline
to
detect nerve fibers
and
to
measure
microanatomic
al
features
such as diameter and orientation
.
These analytical tools and resulting me
asurements
are
needed
to build a reference atlas of cardiac innervation and
for
quantitative descriptions of
innervation
in
healthy versus diseased
states
such as
MI
.
Following
MI
,
innervation around the
infarct
scar and
of
remote regions of the heart
is altered
31,32,45
, and this
neural remodeling
can
modulate
the arrhythmia substrate
46
.
U
nderstanding
changes in
innervation
post
-
MI
can
provide
new
insights into arrhythmia mechanisms.
Furthermore
,
our
clearing
-
imaging
-
analysis pipeline
can
be
readily
applied to
assess innervat
ion of other visceral organs.
It
is well known that the parasympathetic and sym
pathetic nervous system
s
are cri
tical for
heart rate regulation. The SA node and
conduction system are
densely innervated
7,8
, and
stimulat
ion of the vagus nerve
9
,
stellate ganglia
10
, and
noradrenergic fibers
11,12
modulate
s
heart
rate. However,
t
he precise
wiring of
the underlying
neural circuits
has not been delineated
.
W
e
used a novel sparse
AAV
labeling system
20
and an optogenetic approach
to anatomically and
functionally characterize cholinergic neurons that regulate heart rate
. We
identified
cholinergic
neurons in the
IPV
-
GP t
hat project to
the SA nod
e,
the
AV node,
and the ventricles.
Selective
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10
optogenetic
stimulation of cholinergic neurons in the IPV
-
GP modulate
d
heart rate and ventricular
electrophysiology but
, interestingly,
not AV
nodal
conduction.
Previous studies
showed
that
electrical stimulation of
pulmonary vein
ganglia
results in
biphasic heart rate response
s
(initial
bradycardia followed by tachycardia)
22,23
. However, since electrical stimulation is non
-
specific,
it
is difficult to
interpret
whether the biphasic response
was
due to
activation of
a
mixed
population
of neuron
s
contained
in
intrinsic
cardiac ganglia (
i.e.,
parasympathetic, sympathetic, and
senso
ry)
14
and/or
pass through fibers. Our findings
demonstrate an IPV
-
GP
-
SA node circuit
and
highlight the
importance
of
using
techniques
such as optogenetics
, which
confer
cell type
-
specificity
,
to
dissect
cardiac
neural circuity.
Furthermore,
electrical and optogenetic techniques
(using traditional transgenic and AAV
-
based approaches for ChR2 delivery) stimulate both central
preganglionic inputs to and postganglionic neurons in intrinsic cardiac ganglia.
Therefore,
we
used
a novel
engineered AA
V,
AAV
-
PHP.S
20
,
that has
a strong tropism for the PNS
to
preferentially
deliver ChR2 to
postganglionic
cholinergic neurons
on the heart
rather than
preganglionic cholinergic neurons in the
medulla
. Future studie
s
of peripheral neural circuits
should
use AAV
-
PHP.S and other
engineered AAVs
19,21
to
dissect
the role of c
entral versus
peripheral neuronal populations
on organ function.
To
map
noradrenergic neurons that
regulate
heart rate, we used
a
retrograde neuronal
trac
er
and optogenetic
approach
. A
previous
study
in canines
using horseradish peroxidase
showed
that sympathetic postganglionic neurons that innervate the heart are
primarily
located
in
the middle cervical
ganglia
of
the paravertebral chain
47
.
However
, we report
,
using CTB and
confirm with optogenetic
stimulation
,
that
the stellate ganglia ha
ve
a viscerotopic organization
with cardiac
-
projecting neurons clustered in the craniomedial aspect
, consistent with a
study in
cats
48
.
In addition to the heart, the stellate ganglia project to many other thoracic structures,
including the sweat glands in the forepaw
49
, the lung and trachea
50
, the esophagus
48
, and brown
fat
51
.
Characterizing
stellate ganglia
targ
et innervation
and cell
-
type specification
is an area of
ongoing
investigation
52,53
that is of interest
from a development
al, physiological,
and therapeutic
perspective.
Current
understanding of the
role of vagal efferent and afferent
fibers
on cardiac function
is
largely
based on studies using electrical stimulation,
which
is non
-
specific and
results in
non
-
orderly
,
non
-
physiological recruitment of fibers
42
.
Electrical
stimulation of the vagus nerve
typically
activates large
-
diameter
myelinated A fibers, followed by medium
-
diameter
myelinated B fibers
and
then
small
-
diameter
unmyelinated C fibers
27
.
I
n vivo
w
e report that
optogenetic stimulation
of
motor fibers in the vagus nerve results in a heart rate response
that has a
slower onset
than
electrical stimulation of motor and sensory fibers, likely due to differential fiber recruitment
42
.
Our
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.
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11
findings
suggest
that non
-
electrical techniques such as optogenetics are needed to characte
rize
neural control of
cardiac
physiology
.
W
e
also
show that
activation of vagal afferents decreases
heart rate by enhancing parasympathetic efferent outflow and reducing sympathetic efferent
outflow centrally, consistent with a prior study showing that gl
obal activation of vagal afferents in
Vglut2
-
ChR2 mice and selective activation of Npy2r
-
ChR2 vagal afferents
causes
a profound
bradycardia
54
. In support of our functional data, anatomical tracing studies have previously shown
that cardiac vagal afferent neurons in the nodose
-
petrosal ganglion complex project to neurons
in the
nucleus tractus solitarii
of the
medulla
55
. These neurons then project to the nucleus
ambiguus and the dorsal motor nu
cleus of the vagus nerve in the medulla to modulate
parasympathetic efferent outflow
55
and to the paraventricular nucleus of the hypothalamus to
modulate sympathetic efferent outflow
56
.
Future
studies
aimed at identifying cardiac
-
specific vagal
efferent and afferent fibers
, similar to those performed in the lungs
54
and
the
gastrointestinal
system
57
,
are needed to
better
understand vagal control of
cardiac
physiology and
to
design next
-
generation VNS therapie
s.
Overall, our data highlight the complexity of cardiac neural circuit
ry
and demonstrate that
a multi
-
technique approach
is needed to
delineate circuit wiring. Understanding the neural control
of organ function
in greater detail
is critical as neuromodulation therapies are emerging as
promising approaches to treat a wide range
of diseases. Tools such as optogenetics
and AAVs
are already providing new scientific insights into the structure and function of peripheral neural
circuits
54,57
.
A
combination of these approaches will help disentangle neural control of autonomic
physiology and enable a new era of targeted neuromodulat
ion approaches.
METHOD
S
Animals
All animal
experiments were conducted in accordance with the NIH Guide for the Care and Use
of Laboratory Animals and
were approved by the
UCLA
and California Institute of Technology
Institutional Animal Care and Use
Committee
.
ChAT
-
IRES
-
Cre
(028861)
58
, Ai32 (024109)
59
, and
C57BL/6J mice (000664) were purchased from the Jackson Laboratory. TH
-
IRES
-
Cre mice
(254)
60
were purchased from
t
he European Mutant Mouse Archive. ChAT
-
ChR2
-
e
YFP
and TH
-
ChR2
-
e
YFP
mice were created by
crossing
Ai32 mice
wit
h
ChAT
-
IRES
-
Cre or TH
-
IRES
-
Cre
mic
e
, respectively.
All
animals
were kept on a 12 h
light/dark
cycle
with ad libitum access to food
and water.
Data for all experiments were collected from male and female adult
mice
(greater than
8
weeks
old).
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The copyright holder for this preprint
.
http://dx.doi.org/10.1101/456483
doi:
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12
Plasmids
Plasmids used for AAV production include pUCmini
-
iCAP
-
PHP.S (Addgene, 103006), pAAV
-
CAG
-
DIO
-
mRuby2 (Addgene, 104058), pAAV
-
CAG
-
DIO
-
mNeonGreen
20
,
pAAV
-
CAG
-
DIO
-
mTurquoise2 (Addgene, 104059), pAAV
-
ihSyn1
-
DIO
-
tTA (Addgene, 99121), pAAV
-
TRE
-
DIO
-
mRuby
2
(Addgene, 99117),
pAAV
-
TRE
-
DIO
-
mNeonGreen
20
, pAAV
-
TRE
-
DIO
-
mTurquoise2
(Addgene, 99115), pAAV
-
TRE
-
DIO
-
tdTomato (Add
gene, 99116), and pAAV
-
CAG
-
DIO
-
e
YFP
(Addgene, 104
052). pAAV
-
CAG
-
DIO
-
ChR2(H134R)
-
e
YFP was generated by replacing the Ef1a
promoter in pAAV
-
Ef1a
-
DIO
-
ChR2(H134R)
-
e
YFP (a gift from Karl Dei
sseroth, Addgene, 20298)
with the CAG promoter from pAAV
-
CAG
-
mNeonGreen
20
.
The pHelper plasmid was obtained from
Agilent’s AAV helper
-
free kit (Agilent, 240071).
CAG,
Cytomegalovirus early enhancer element
chicken
b
-
actin promoter
; DIO = double
-
floxed inverted
open reading frame
.
Virus production and purification
AAVs were produced and purified as previously described
61
. Briefly, AAVs were generated by
triple transient transfection of HEK293T cells (AT
TC
, CRL
-
3216
) using polyethylenimine
(
Polysciences
, 23966
-
2
)
62
. Viral particles were harvested from the media at 72 h post
-
transfection
and from the cells and media at 120 h post
-
transfection. Virus from the m
edia was precipitated at
4°C with 40% polyethylene glycol (
Sigma
-
Aldrich
, 89510
)
63
in 2.5 M NaCl and combined with cell
pellets suspended at 37°C in 500 mM NaCl, 40 mM Tris, 10 mM MgCl
2
,
and 100 U/ml of salt
-
active nuclease (
ArcticZymes
, 70910
-
202
). Clarified lysates were purified over iodixanol (
Cosmo
Bio USA
, Opt
iPrep,
AXS
-
1114542
) step gradients (15%, 25%, 40%, and 60%)
64
. Purified viruses
were concentrated, washed in sterile
phosphate buff
ered saline (
PBS
)
, sterile filtered, and
tittered
using quantitative
PCR
65
.
Systemic
delivery
of
viruses
Intravenous administration of AAV vectors was performed by
retro
-
orbital in
jection
with a 31
-
gauge needle
in
6
-
8
w
eek
old
mice
as previously described
61
.
Following injection, 1
-
2 drops of
prop
aracaine (Akorn Pharmaceuticals, 17478
-
263
-
12) were applied to the cornea to provide local
analgesia.
Optogenetic s
timulation
and physiological
measurements
Langendorff
-
perfused heart
an
d cardiac ganglion
stimulation
.
Mice
were
given
heparin (100U,
i.p.
)
to prevent blood clotting
and
euthanized
with
sodium pentobarbital
(150
mg
/kg
, i.p.
).
Once
all reflexes subsided and
following a midsternal incision,
h
earts were rapidly exci
se
d and
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13
Langendorff
-
perfused with
37
°
C
modified
Tyrode’s solution
containing the
following
(in m
M
):
112
NaCl
,
1.8
CaCl
2
,
5 KCl
,
1.2
MgSO
4
,
1
KH
2
PO
4
,
25
NaHCO
3
, and
50 D
-
glucose at pH 7.4
.
The
solution was
continuously bubbled with 95% O
2
and 5% CO
2
. Flow rate was adjusted to maintain
a pe
rfusion pressure of 60
-
80 mmHg.
An
optic
al
fiber (400 μm core, Doric Lens)
coupled to a
diode
-
pumped solid
-
state
laser light source (473 nm, 400
mW,
Optic Engine
)
was positioned
for
focal illumination of
the IPV
-
GP
under
the
guidance
of a
fluorescent stereomicroscope (Leica
,
M205 FA
) fitted with
a
1x Plan
-
Apochromat.
A
n
octa
polar electrophysiology catheter (
1.1F,
Transonic) was inserted
into the left atrium and ventricle
via a small incision in the left atrium
and
2
platinum electrodes were positioned in the bath
to obtain intracardiac electrogram
s and
a bath
electrocardiogram,
respectively.
Intracardiac electrograms and
electrocardiogram
were
amplified
with a differential alternating current amplifier
(
A
-
M Systems
, Model 1700
and
Grass,
P511
,
respectively) and
continuously
acquired
(AD Instrumen
ts
,
PowerLab 8/35
).
Dose response curves
were performed to evaluate the effects of altering
light pulse power
(at 10 Hz
and
10 ms),
frequency (at 10 ms
and
221
mW), and pulse width (at 10 Hz
and
221
mW) on heart rate
and AV
interval
.
At
the end of the experiment
, atropine (10 uM) was administered into the perfusate
and
stimulation
(10 Hz
,
10 ms
, and
221 mW
)
was repeated.
All stimulations were performed
for 10 s
with 5 min
between stimulations for heart rate to return to baseline values.
In vivo vagus nerve and paravertebral ganglia stimulation.
Mice
were anesthetized with isoflurane
(
induction at
3
-
5%, maintenance at
1
-
3%
vol/vol
,
inhalation
), intubated, and
mechanical
ly
ventilated (CWE
, SAR
-
830
)
.
Core body temperature was measured and maintained at 37
°
C.
For
VNS
, a midline neck incision was performed and the left and right cervic
al vagus nerves were
exposed. A
laser
-
coupled
optic
al
fiber
was positioned above and bipolar platinum hook electrodes
co
upled to a constant current stimulator (Grass
, PSIU6 and
Model S88
) below the right vagus
nerve for focal optical or electrical stimulation, respectively.
Irradiance
and current were
titrated
to
a
chieve a
10% decrease in heart rate
with
both vagi intact
at 10 Hz
and
10 ms
, which was
defined as threshold intensity
.
Threshold intensity fo
r optogenetic stimulation was 77
±
6
mW
and
for
electrical stimulation was 33
±
13 μA.
Frequency response curves were
performed at
threshold
intensity and 10 ms.
All stimulations we
re performed for 5 s with 5 min
between stimulations for
heart rate to return to baseline values.
For paravertebral ganglia stimulation,
a right lateral
thoracotomy was performed at the second intercostal space and the paravertebral cha
in from the
stellate to
T2
ganglion was exposed.
A laser
-
coupled optic
al
fiber
was positioned for focal
illumination
above
the
craniomedial
RSG
or
right
T2
ganglion
.
Dose response curves
were
performed
to evaluate the effect of altering
RSG
sti
mulation frequency (at 10 ms
and
126
mW)
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.
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14
and pulse width (
at 10 Hz
and
126
mW) on heart rate.
The effect of
RSG
versus
right
T2
ganglion
stimulation
(
10 Hz
,
10 ms,
and
126
mW)
on heart rate
was also
evaluated
.
At
the end of the
experiment
, propranolol (2 mg/kg, i.v.) was administered via
a
femoral vein and
craniomedial
RSG
stimulation
(10 Hz
,
10 ms
,
and
126
mW)
was repeated.
All stimulations were performed for 10 s
with 5 m
in
between stimulations for heart rate to return to baseline values. For vagus nerve and
paravertebral ganglia stimulation
experiments
, a
lead II
electrocardiogram
was
obtained
by
2
needle electrodes inserted subcutaneously into the righ
t forepaw and the lef
t hindpaw.
Electrophysiology data analysis.
A minimum of 3 beats were averaged at baseline and during
stimulation
for all electrophysiological data.
Cholera toxin subunit B heart
i
njections
Mice
were
given
carprofen
(5 mg/kp, s.c.
)
and
buprenorphine
(0.05 mg/kg, s.c.)
1 h
before
surgery.
Animals
were
anesthetized with
isoflurane (
induction at 5%, maintenance at
1
-
3%,
inhalation
), intubated,
and
mechanically ventil
ated. Core body temperature was measured and
maintained at 37
°
C.
The
surgical incision site was cleaned
3
times with 10% povi
done iodine and
70% ethanol in H
2
O (v
ol
/v
ol
).
A left lateral thoracotomy was performed at the
4th
intercostal space,
the pericardium open
ed
, and the heart was exposed.
Ten microliters of
CTB
conjugated to Alex
a
Fluor 488 (
0.1%
in 0.
0
1 M
PBS
(v
ol
/v
ol
)
,
ThermoFischer Scientific
,
C22841
)
was
subepicardially
injected
in the heart
with
a 31
-
gauge
needle.
The surgical wounds were closed with 6
-
0 sutures.
Buprenorphine
(0.05 mg/kg, s.c.)
was administered
once daily
for up to 2
d
after surgery.
Animals
were sacrificed 6
d
later for tissue harvest.
Tissue preparation, i
mmunohistochemistry
, and imaging
Transcardial perfusion.
Mice
were
given heparin (100U,
i.p.) to prevent blood clotting and
euthanized with
sodium pentobarbital
(
15
0 mg
/kg, i.p.
)
.
Once all reflexes subsided,
a midsternal
incision
was made and
animals
were
transcardially perfused with
50 mL
ice
-
cold 0.
0
1 M
PBS
containing 100
U heparin followed by
50 mL freshly prepared,
ice
-
cold 4% paraformaldehyde
(
PFA;
EMS,
RT
15714
)
in PBS. Tissues were postfixed in 4%
PFA
overnight
at 4
°
C
,
washed, and
stored
in
PBS with 0.01% sodium azide.
Langendorff
-
perfused hearts were
immersion fixed
in 4%
PFA
overnight
at 4
°
C
at the end
of the functional experiments
Heart
clearing
.
W
hole
mouse
hearts
were
stained
and cleared
using
a modified
iDISCO protocol
28
.
F
ixed
heart
s
were
dehydrated
with a graded methanol series
(
20%, 40%, 60%,
and
80%
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/10.1101/456483
doi:
bioRxiv preprint first posted online Oct. 29, 2018;
15
methanol
in H
2
O (v
ol
/v
ol
)
, each for 1 h
at room temperature),
wash
ed twice
with
100% methanol
for
1 h
at
room temperature
,
and
chilled at
4
°
C.
Hearts were
then
incubated
in 66%
d
ichloromethane
/33% methanol
overnight
at room temperature with
agitation,
washed
twice
in
100% methanol for 1 h
at room temperature,
and
chilled to 4
°
C.
Next, h
earts were
bleached
with
5%
H
2
O
2
in methanol
(v
ol
/v
ol
)
overnight at
4
°
C.
After bleaching, h
earts were rehydrated with a
graded methanol series
, followed
by one wash
with 0.
0
1 M P
BS and
2
washes
with
0.
0
1
M PBS
w
ith 0.2% Triton X
-
100, each for
1 h
at
room temper
ature.
For staining,
h
earts were
permeabilized
with
0.
0
1 M
PBS with
0.2% Triton X
-
100, 20%
DMSO
, and
0.3 M glycine
and blocked with
0.
0
1
M PBS with
0.2% Triton X
-
100
, 10% DMSO
, and
5
% normal
d
o
nkey
s
erum
(NDS)
, each for
2
d
at 37
°
C with agitation
.
Hearts were incubated in
primary
antibody
rabbit anti
-
PGP9.5 (Abcam
,
ab108986
, 1:200
)
diluted in
0.
0
1 M PBS with
0.2% Tween
-
20
and
10 mg/ml heparin (PTwH) for
1
w
eek
at 37
°
C with agitation.
Hearts were then washed several times in
PTwH
overnight at room
temperature befor
e
secondary
antibody
donkey anti
-
sheep Cy3 (Jackson ImmunoResearch
,
713
-
165
-
003
, 1:300
)
incubation
in
PTwH for
1
w
eek
at 37
°
C with agitation.
P
rimary and
secondary
Ab
were replenished half way through staining.
Hearts were then wash
ed
several times in PTwH
overnight at room temperature.
For clearing, s
tained hearts were dehydrated with a graded
methanol series
and
incubated in 66% d
ichloromethane
/33% methanol for 3 h
a
t room
temperature with agitation. Hearts were then washed twice in 100% d
ichloromethane
for 15 min
at room temperature. Hearts were stored
and imaged
in benzyl ether (
Millipore Sigma
,
108014
ALDRICH
; refractive index
: 1.5
5).
Immunohistochemistry
.
Fixed h
eart
s
and ganglia
were block
ed
in 0.
0
1 M PBS with
10%
NDS
and
0.2% Triton X
-
100 PBS
for 6 h
at room temperature
with agitation. Tissues were then incubated
in primary
Ab
diluted in 0.
0
1 M PBS with 0.2% Triton X
-
100 and 0.01% sodium azide for 3 nights
at r
o
om temperature with agitation.
The following primary
antibodies
were used:
rabbit anti
-
HCN4
(Alomone Labs,
APC
-
052
, 1:100),
rabbit anti
-
PGP9.5 (Abcam
,
ab108986
, 1:500
), chicken anti
-
GFP (Aves
,
GFP
-
1020
, 1:1000
), and sheep anti
-
TH (Millipore Sigma
,
AB1542
,
1:200
).
T
issues
were
washed several times in 0.
0
1 M
PBS overnight before incubation
in secondary
Ab
diluted in
0.
0
1 M PBS with 0.2% Tr
iton X
-
100
and 0.01% sodium azide for 2 nights at room temperature
with agitation
. The following secondary
antibodies
were used: donkey anti
-
rabbit Cy3 (Jackson
ImmunoResearch
,
711
-
165
-
152
, 1:400
), donkey anti
-
chicken 647 (Jackson ImmunoResearch
,
703
-
605
-
155
, 1:400
), and donkey anti
-
sheep Cy3 (Jackson ImmunoResearch
,
713
-
165
-
003
,
1:400
). Tissues were washed
several times
in 0.
0
1 M PBS overnight before being mounted on
microscope
slides in refractive index matching solution (
refractive index
= 1.46)
29
.
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/10.1101/456483
doi:
bioRxiv preprint first posted online Oct. 29, 2018;