Reviewers' comments:
Reviewer #1 (Remarks to the Author):
This is an interesting manuscript that describes how to use optogenetic
approaches to image the
anatomy of the cardiac nervous system as well as how to use those approaches to dissect functional
circuits within living heart tissue. There is a nice mix of technology development and mechanistic
insights via an anatomic/function
al analysis of the role of the cardiac IPV
-GP. Very nice work is
presented yet there are multiple areas where the manuscript can be improved, as explained below.
The clearing of mouse hearts using iDISCO is a new development and the results shown in Figu
re 1
are impressive. In Figure 1a it is interesting that PGP9.5 staining was done before clearing. Can the
authors provide data regarding the loss of PGP9.5 immunofluorescence that results from iDISCO (ie,
fluorescence signals for PFA
-fixed then stained ti
ssue compared to stained then iDISCO
-cleared
tissue). It is important to quantify how clearing approaches do or do not affect fluorescent probes.
It is interesting that secondary staining with GFP was required to amplify ChR2
-eYFP fluorescence (ie,
Figur
e 3a). Is this because iDISCO attenuated the fluorescence of ChR2
-eYFP? Please consider recent
work of Moreno, et al., available on bioRxiv (doi: https://doi.org/10.1101/451450) that reports high
fluorescence of ChR2
-eYFP (without GFP amplification) in a m
ouse model that is almost identical to
that used by the authors.
In addition to the Yokoyama 2017 publication please also consider an additional publication that shows
CLARITY clearing of a mouse heart (Figure 6i in Hsueh, Scientific Reports 2017). Figur
e 1 of Hsueh
2017 also reports the amount of protein loss after clearing. How does this compare to iDISCO?
Nearly
-selective expression of ChR2 in peripheral neurons was accomplished using AAV
-PHP.S to
provide for specific stimulation of postganglionic ca
rdiac neurons. Nearly
-selective expression was
confirmed via MIP confocal imaging (Supp Figure 2). Additional evidence for the selectivity of this viral
approach would be to repeat the functional experiments (Supp Figure 2) after severing the cardiac
vagal
inputs, as was done for the experiments shown in Figure 4. Can data from such studies be
provided?
A large portion of the manuscript is devoted to investigations of cholinergic neurons and the IPV
-GP.
The selectivity of the Cre
-Lox system (transgenic mo
del and the viral approach) for expression of
ChR2 in cholinergic neurons is not assessed in a direct manner. The primary assessment is indirect, in
that the authors report an overlay of PGP9.5 fluorescence and GFP
-amplified ChR2-
eYFP fluorescence.
This is
generally convincing yet quantitative assessment of co-
location of ChR2
-eYFP with ChAT in the
heart would seal the deal. For example, the Moreno, et al., preprint shows co-
localization of ChR2
-
eYFP with ChAT in cardiac neurons in a mouse model almost iden
tical to that used by the authors.
Indeed, the authors used ChAT immunohistochemistry for the dorsal motor nucleus in the medulla
(Supp Figure 1a) yet there are no ChAT images shown for the cardiac ganglia. Such definitive proof of
selective expression is particularly important for the ChAT
-IRES
-Cre transgenic mice transfected with
AAV-
PHP.S
Figure 3c: heart rate appears to be lower after cessation of illumination. Why? At 20Hz stimulation the
R wave amplitude also drops after cessation of illumination. T
his is also true for 10Hz in Figure 3i. Is
this typical? Please report the average response of the hearts after cessation of illumination. Is the
post
-illumination response dependent upon the duration of illumination or other illumination
parameters?
Fig
ure 6a: heart rate does not return to baseline levels. How long before it does?
For all experiments please report the illumination area. Irradiance values in mW/mm^2 will be more
informative than the currently reported power levels in mW. What might be the expected levels
(increase in degC) of tissue warming if the power was directed onto a small area (ie, high irradiance)?
Could slight warming above physiologic temp increase neuron firing rate and release of
neurotransmitter?
How does the heart rate response to optical stimulation of the paravertebral ganglia compare to
electrical stimulation of the ganglia? TH
-ChR2
-eYFP mice were used for the stellate experiments. It is
possible that preganglionic neurons in the stellate were also stimulated? Such syna
pses are
presumably nicotinic, yet is it safe to assume that there is no expression at all of ChR2 in those
preganglionic neurons? Could ChAT + TH co
-staining reveal the synaptic maps (re: Figure 5) and
possibly indicate potential cross
-over expression of ChR2?
Figures 3e, 3f, 4d, 4e, 4g, 6e, Supp 2b: none of these “dose-
response” plots show a plateau or dose
saturation. Can additional data be added to indicate maximal response levels?
Reviewer #2 (Remarks to the Author):
This is an interesting and technically impressive study using opticogenetic stimulation and genetic
labeling to map autonomic innervation of the heart in mice. Inquiry into the anatomy and physiology
of heart rate and conduction control by the autonomic nervous systems represents some of the oldest
questions in ‘modern’ biology, and the current study adds some interesting new data. It also offers a
new twist on these investigations using modern tools that, though technically challenging, may be
used by others in the future. I have so
me questions, outlined below, on the fidelity of the systems,
and whether the differences in optical studies and standard electrical stimulation studies provide true
insights into the biology of the autonomic system, or may have less exciting, perhaps triv
ial,
explanations due to imperfect expression or optical capture in the mouse models. Further comments
follow:
why is HCN4 immunodetection adequate for defining the SAN/AVN? HCN4 is expressed in other
cardiac tissues, including atrial myocardium. Further
more, HCN4 expression may increase in response
to injury, suggesting this approach could be misleading in the future studies foreshadowed in the
Results section (e.g. MI and nerve sprouting) describing findings in Fig 1.
Were some neurons PGP9.5-
? if so,
why, given this is a pan
-neuronal marker? if this marker has
incomplete fidelity, what are the implications for your subsequent conclusions?
Last paragraph, page 5 -
Could the lack of AVN conduction effect also mean that labeling and/or
optical capture were incomplete? How can you rule out these possibilities?
Please show absolute heart rates in addition to percent change
Why is mixed recruitment of efferents/afferents by electrical stimulation more effective at rapidly
increasing HR compared to opti
cal pacing that is designed to only stimulate a pure population of
nerves? Fig 4c -
Given the relative delay in the optical verus electrical stimulation response, I wonder
what is the evidence that the slower HR change with optical stimulation is not due to incomplete
expression of the optically activated channel or a phased or delayed pattern of recruitment? How do
your results unravel the previous findings re electrical stimulation of the PV ganglia (i.e. bradycardia
followed by tachycardia), as mentioned
in the Discussion?
The post vagotomy experiments with a different response to optical (none) and electrical (HR slowing)
is fascinating. Nevertheless, i wonder how you can be sure it is not due to lack of adequate
expression/capture/recruitment using th
e optical method? For example, an electrode recording
showing an impulse with optical stimulation in the rostral limb of the severed vagus would strengthen
your conclusion that the biology, rather than technical aspects of your approach, was driving your
findings.
Paragraph 2, page 10 -
The contrast between findings on sympathetic neurons in canines (post
ganglionic neurons mostly in the middle cervical ganglia of the PV chain) versus mice (showing a
craniomedial localization) might be due to species diff
erences.
Minor
a minimum of 3 beats is excessively vague, arbitrary and, perhaps, insufficient to represent actual
rates (i.e. 3 is a small number).
Reviewer #3 (Remarks to the Author):
In this well-
written manuscript the authors present a novel tool for studying the role of
parasympathetic and sympathetic nervous system in murine cardiac electrophysiology using
optogenetics. They use parasympathetic and sympathetic specific Cre lines in combination with AAV
driven fluorescent proteins to visualize the cardiac autonomic nervous system. Moreover, using tissue
-
specific ChR2 expression they specifically stimulate either the parasympathetic or sympathetic nervous
system and investigated the effect on heart rate. The authors conclude to have provided novel insight
into neural regulation of heart rate and tools for studying neuronal cardiac circuits or neural control of
other organs.
comments
• The 3D visualizations of the neural circuit anatomy are beautiful but passive (movies showing pre
-
defined angl
es and magnifications), it would be great if the authors could provide files where the user
can decide at what angle and magnification the 3D reconstructions are visualized. Is this possible? 3D
pdf for example?
• Figure 2 and labeling of the cholinergic
neurons. This part is confusing. Please explain the function of
the human Synapsin I promotor. What is the reason for using the two component expression system?
Please elaborate. Why not placing Cre directly under control of the Synapsin I promotor? Figur
e 2c
shows results of an entirely different expression system. Please explain. How did the authors verify
that labeling was indeed restricted to the cholinergic neurons? Low levels of Cre expression outside
these neurons would generate aspecific labeling. Please provide additional data verifying specificity
using an independent marker.
• The authors use multiple fluorescent proteins to visualize the parasympathetic nervous system.
Please explain why.
• Figure 2D. It is unclear how the sections are orien
ted. I suggest to add an overview section
indicating the location of the sections shown. Moreover, Hcn4 is used to mark the Sinus node and AV
node. To me, however, the Hcn4 staining seems stronger outside the areas marked with the dashed
white lines than inside. The AV node does not seem to be labeled. Please address this issue.
• Page 5. The authors use ChR2
-enhanced yellow fluorescent protein but switch to GFP in their
descriptions, which is confusing. “rostral” is archaic, cranial or anterior is more c
ommonly used.
• Figure 4 C. The authors compare optical stimulation of the right vagal nerve with electrical
stimulation of the right vagal nerve. The approaches differ in 2 ways. 1) Only a part of the RVNS is
stimulated optically whereas the whole RVNS is stimulated du
ring electrical stimulation, and 2) optical
stimulation in itself differs from electrical stimulation. Why didn’t the authors compared electrical
stimulation with optical stimulation of both the efferent and afferent fibers in the vagal nerve? This
allows
for discriminating the different responses to the different approaches.
• The authors report p wave fractionation during IPV
-GP stimulation. Do the authors mean a biphasic
p wave? It is known that the right and left atrium activation with a delay of abou
t 5 ms. The activation
wave propagates from the right atrium towards the left atrium via a dorsal myocardial connection. Do
the authors think this delay between right and left atrium is increased during IPV
-GP stimulation? Or
does IPV
-GP stimulation affect
atrial conduction?
• Page 5 line 31. The authors mention that the nerve fiber passes through the AV node but does not
synapse. Where is the nerve going? Was ventricular conduction affected?
• On page 7 Figure 4G is discussed before Figure 4F. Please a
djust the figure order to the text.
• On page 8 and Figure 6, please provide validation of the specificity of the TH Cre driver.
• Discussion conclusion 3, it is not clear how the authors arrive at this conclusion. Please better
explain. “central mecha
nisms”?
• References: In some cases the author list is abbreviated and in other case it is not. Please adjust to
the correct style.
1
NCOMMS
-
18
-
29234A
-
Identification of peripheral neural circuits that regulate heart rate
1
using optogenetic and viral vector strategies
2
3
4
Reviewers' comments:
5
6
We thank the editor and reviewers for their thoughtful
review
of our manuscript and
for
the
7
opportunity to revise our wor
k.
We feel that in addressing the comments raised by the
reviewers
8
the manuscript has
been
significantly improved.
9
10
11
Reviewer #1 (Remarks to the Author):
12
13
This is an interesting manuscript that describes how to use optogen
etic approaches to image the
14
anatomy of the cardiac nervous system as well as how to use those approaches to dissect
15
functional circuits within living heart tissue. There is a nice mix of technology development and
16
mechanistic insights via an anatomic/func
tional analysis of the role of the cardiac IPV
-
GP. Very
17
nice work is presented yet there are multiple areas where the manuscript can be improved, as
18
explained below.
19
20
We thank the reviewe
r for
careful
reading
of the
manuscript and
providing
constructive comments.
21
We have addressed the reviewer’s comments point
-
by
-
point below.
22
23
The clearing of mouse hearts using iDISCO is a new development and the results shown in Figure
24
1 are impressive. In Figure 1a it is interesting that PGP9.5 staining wa
s done before clearing. Can
25
the authors provide data regarding the loss of PGP9.5 immunofluorescence that results from
26
iDISCO (ie, fluorescence signals for PFA
-
fixed then stained tissue compared to stained then
27
iDISCO
-
cleared tissue). It is important to qu
antify how clearing approaches do or do not affect
28
fluorescent probes.
29
30
We performed additional experiments to compare hearts that were whole
-
mount stained (fixation
31
followed by staining)
and
hearts that underwent the iDISCO protocol
.
The whole
-
mount
stained
32
hearts
and iDISCO
-
cleared hearts were stained
identically
with the same concentrations of
33
antibodies and for the same duration of time.
In both whole
-
mount stained
hearts
and iDISCO
34
cleared hearts,
we
observed
dense innervation and
similar patterns
of
PGP9.5
labeling
on the
35
epicardial surface
(
Supplemental
Figure
1b and d
). Importantly, following the iDISCO protocol,
36
small nerve fiber bundles were preserved
.
The advantage of the iDISCO protocol versus whole
-
37
mount staining is the ability to label and
visualize molecular and cellular structures in large tissue
38
volumes
1
. In whole
-
mount stained hearts we were able to visualize fibe
rs
only
within the first 100
-
39
200 μm of the
epicardium
(
Supplemental
Figure
1
b)
, whereas
in iDISCO
-
cleared hearts we were
40
able to visualize fibers throughout the entire thickness of the myocardium
from epi
-
to
41
endocardium
(Supplemental Figure 1
d
and Supplemental Movie 3
).
42
2
1
Supplemental Figure 1. Whole
-
mount stained heart versus iDISCO
-
cleared heart. (a)
A heart
2
before (top) and after (bottom) whole
-
mount staining. (
b
)
3D confocal projection of the dorsal side
3
of a heart (2000 μm z
-
stack) whole
-
m
ount stained with PGP9.5 (gray). Insets show a MIP image
4
of the left ventricular wall (top right) and a 1000 μm
-
thick 3D projection of the left ventricular wall
5
(bottom right). (
c
) A whole heart (top) was rendered transparent (bottom) using the iDISCO
6
prot
ocol. (
d
) 3D confocal projection of the dorsal side of an iDISCO
-
cleared heart (2000 μm z
-
7
stack) stained with PGP9.5 (gray). Insets show a MIP image of the left ventricular wall (top right)
8
and a 1000 μm
-
thick 3D projection of the left ventricular wall (bo
ttom right). In contrast to whole
-
9
mount stained hearts, nerve fibers could be visualized throughout the entire thickness of
10
myocardium in iDISCO
-
cleared hearts. Scale bars are 2 mm (
a
,
c
), 1 mm (
b
(left),
d
(left)), and
11
100 μm (
b
(right),
d
(right)).
12
13
Fluorescence intensity was lower in the iDISCO
-
cleared hearts as compared to the whole
-
mount
14
stained hearts
(Response Figure
1
)
.
However
, we
cannot
comment on
whether this is due to
a
15
loss of protein or
the
fact that antibodies penetrate much
deeper
(and
,
therefore
,
do not saturate
16
antigen binding sites on the surface)
in iDISCO
-
cleared tissues.
Regardless, we do not see this
17
as a major limitation
of using iDISCO for characterizing innervation of the heart.
18
19
3
1
Response Figure
1
.
Fluorescence intensity in whole
-
mount stained hearts versus iDISCO
-
2
cleared hearts.
(
a
)
Representative
MIP confocal images from
the base of a whole
-
mount stained
3
heart and an iDISCO
-
cleared heart with PGP9.5 staining (gray). (
b
) Mean PGP9.5 intensity from
4
the
base of
whole
-
mount stained hearts versus iDISCO
-
cleared hearts (
t
6
=
3.119
;
*
P
= 0.02
06
).
5
Note staining conditions were identical across all hearts and microscope laser setting
s
remained
6
constant across all images.
n
= 4 hearts (
b
).
mean ± s.e.m.; unpaired, two
-
tailed
t
-
test.
Scale bar
7
is 100 μm (
a
).
8
9
It is interesting that secondary staining with GFP was required to amplify ChR2
-
eYFP
10
fluorescence (ie, Figure 3a). Is this because iDISCO attenuated the fluorescence of ChR2
-
eYFP?
11
Please
consider
recent
work
of
Moreno,
et
al.,
available
on
bioRxiv
12
(doi:
https://doi.org/10.1101/451450
) that reports high fluorescence of ChR2
-
eYFP (without GFP
13
amplification) in a mouse model that is almost identica
l to that used by the authors.
14
15
T
he
iDISCO
protocol
was developed
for
immunostaining
applications
1
, in contrast to other tissue
16
cle
aring techniques such as
ScaleA2
2
, 3DISCO
3
, SeeDB
4
, CLARITY
5,6
(Supplemental Figure 2)
,
17
and CUBIC
7
that are better suited for imaging endogenous fluorescence. The
dehydration
18
processes
and
use of
dibenzyl ether as an optical clearing agent in the iDISCO protocol
are
known
19
to
attenuate
endogenous fluorescence
1
.
20
21
In our manuscript, the heart
s
in Figure 1
, Supplemental Figure 1,
and Supplemental Movies 1
-
3
22
were
immunostained
with the pan
-
neuronal marker PGP9.5
and cleared with the iDISCO protocol
23
as
we were interested in characterizing innervation of entire hearts in 3D
.
However,
the tissues in
24
Figures 2
-
6
and Suppl
emental Figures
3
and
4
were whole
-
mount stained (as described in the
25
Methods section under Immunohistochemistry)
and not cleared using the iDISCO protocol as our
26
goal was to
confirm ChR2
-
eYFP expression in defined cell types (i.e. ChAT and TH neurons
)
.
We
27
have clarified
in the figure legends
whether tissues were iDISCO
-
cleared or whole
-
mount stained.
28
The
tissue
s
from
ChAT
-
ChR2
-
eYFP and TH
-
ChR2
-
eYFP mice
were
stained with a
widely
-
used
29
antibody against
GFP
(Aves Labs, GFP
-
1020
; 545 citations on CiteAb
)
to amplify the detection
of
30
the endogenous
ChR2
-
eYFP protein
and improve the signal
-
to
-
noise ratio
. As shown in Response
31
Figure
2
, endogenous ChR2
-
eYFP signals
were
weak compared to
those
of GFP, even when
32
using much higher laser power (100% laser power for imaging
endogenous
ChR2
-
eYFP v
ersus
33
6% laser power for imaging GFP). Additionally,
the
use of high laser
power
results in more
34
background autofluorescence.
35
36
4
1
Response Figure
2
.
ChR2
-
eYFP versus GFP
signals
in cardiac ganglia
.
Confocal
image
s
2
showing
native
ChR2
-
eYFP
signal
versus GFP
staining, along with PGP9.5 staining, from
the
3
cardiac ganglia of a ChAT
-
ChR2
-
eYFP mouse. Inset shows higher magnification image of
a
4
cardiac ganglion.
Note ChR2
-
eYFP signal
was
imaged at 100% laser power and GFP was imaged
5
at 6% laser power
for all images
.
B
ackground autofluorescence
from
myocardial tissue is
6
observed in
upper left ChR2
-
eYFP panel and weak signals
are observed in
lower
left ChR2
-
eYFP
7
panel
likely due to photobleaching
.
Scale bars are 100 μm (upper panels) and 50 μm (lower
8
panels).
9
10
We thank the reviewer for bring to our attention the manuscript by Moreno and colleagues.
I
n the
11
manuscript by M
oreno
et al
.
,
endogenous ChR2
-
eYFP fluorescence was imaged from
a small
12
volume
of the a
tria. However, in our manuscript, we were concerned about
background
13
autofluorescence and
photobleaching
resulting from
use of
high laser
power
to image
14
endogenous
ChR2
-
eYFP signals
over
large tissues volumes
such as entire hearts
; thus, tissues
15
were stained with an antibody against GFP.
16
17
In addition to the Yokoyama 2017 publication please also consider an additional publication that
18
shows CLARITY clearing of a mouse heart (Figure 6i
in Hsueh, Scientific Reports 2017). Figure
19
1 of Hsueh 2017 also reports the amount of protein loss after clearing. How does this compare to
20
iDISCO?
21
22
Although protein loss
with
iDISCO has not been
previously
reported
1,8,9
,
Reiner
et al.
compared
23
FoxP2 expression in the inferior olive of an iDISCO
-
cleared adult mouse brain with tissue sections
24
from the same level
in the original iDISCO manuscript
1
. The Fox2P expression pattern and the
25
number of Fox2P
-
positive cells was consistent between the two methods
and other publications
.
26
In addition, our data show s
imilar patterns of PGP9.5 expression between whole
-
mount stained
27
hearts and iDISCO
-
cleared hearts, including preservation of smaller fiber bundles
(Supplemental
28
Figure 1)
. Taken together, this
suggests that protein loss is not a major limitation of using
t
he
29
iDISCO
protocol
for
characterizing innervation of the heart.
30
31
32
5
Nearly
-
selective expression of ChR2 in peripheral neurons was accomplished using AAV
-
PHP.S
1
to provide for specific stimulation of postganglionic cardiac neurons. Nearly
-
selective expression
2
was confirmed via MIP confocal imaging (Supp Figure 2). Additional evidence for the selectivity
3
of this viral approach would be to repeat the functional experiments (Supp Figure 2) after severing
4
the cardiac vagal inputs, as was done for the experiments sh
own in Figure 4. Can data from such
5
studies be provided?
6
7
In the mouse heart, cardiac ganglia are located on the dorsal surface around the pulmonary veins
8
(Figures 1
c
and 3
a
)
10
-
12
. I
t is not possible to expose them
surgically
in vivo
.
Therefore
,
the
9
experiments in Figure 3 and Supplemental Figure
4
(old Supplemental Figure 2)
in which the
10
inferior pulmonary vein
-
ganglionated plexus
(IPV
-
GP)
was optogenetically stimulated
were
11
performed in
ex vivo
Langendorff
-
perfused hearts
(as indicated in the figure legends and
12
described in the Methods section unde
r Optogenetic stimulation and physiological
13
measurements),
whereas the experiments in Figure 4 in which the cervical vagus nerve was
14
optogenetically stimulated were performed
in vivo.
In Figure 3 and Supplemental Figure
4
(old
15
Supplemental Figure 2)
, when
excising the heart from the thorax for the Langendorff preparation,
16
vagal preganglionic
fibers originating from the medulla in the brainstem
were
indeed
transected.
17
However,
even after transection, the caudal end of the
vag
us nerve, which contains pregangl
ionic
18
fibers,
can still be stimulated
in vivo
(Figure
4
e) and in Langendorff
-
perfused hearts
13,14
.
19
20
In addition to viral transduction efficiency,
another
potential
reason for the smaller bradycardic
21
response
observed
with optogenetic stimulation of the IPV
-
GP
in Supplemental Figure
4
(old
22
Supplemental Figure 2)
compared to that in Figure 3 is
that in
ChAT
-
IRES
-
Cre mice injected with
23
AAV
-
PHP.S:CAG
-
DIO
-
ChR2
-
eYFP only postganglionic cholinergic neurons
were transduced
24
(Supplemental Figure
3
(old Supplemental Figure 1)
) and optogenetically stimulated,
whereas in
25
ChAT
-
ChR2
-
eYFP mice both
vagal
preganglionic
fibers
and postganglionic cholinergic neurons
26
expressed ChR2
-
eYFP and
were stimulated
. Future studies will focus on
leveraging engineered
27
AAVs
such as AAV
-
PHP.S
15
-
17
to selectively
transduce defined cell typ
es in the peripheral nervous
28
system.
29
30
A large portion of the manuscript is devoted to investigations of cholinergic neurons and the IPV
-
31
GP. The selectivity of the Cre
-
Lox system (transgenic model and the viral approach) for
32
expression of ChR2 in cholinergi
c neurons is not assessed in a direct manner. The primary
33
assessment is indirect, in that the authors report an overlay of PGP9.5 fluorescence and GFP
-
34
amplified ChR2
-
eYFP fluorescence. This is generally convincing yet quantitative assessment of
35
co
-
location
of ChR2
-
eYFP with ChAT in the heart would seal the deal. For example, the Moreno,
36
et al., preprint shows co
-
localization of ChR2
-
eYFP with ChAT in cardiac neurons in a mouse
37
model almost identical to that used by the authors. Indeed, the authors used ChAT
38
immunohistochemistry for the dorsal motor nucleus in the medulla (Supp Figure 1a) yet there are
39
no ChAT images shown for the cardiac ganglia. Such definitive proof of selective expression is
40
particularly important for the ChAT
-
IRES
-
Cre transgenic mice tra
nsfected
with AAV
-
PHP.S
41
42
Most, if not all, commercially available antibodies
against
ChAT recognize a central rather than
43
peripheral isoform of ChAT,
labeling peripheral cholinergic neurons
weakly
18
.
An antibody has
44
been developed against the peripheral
isoform of ChAT
19
; however, it is not
yet
comm
ercially
45
available.
Therefore, we used
an
antibody against ChAT in the medulla in the central nervous
46
system and an antibody against PGP9.5 as a
surrogate for ChAT in cardiac ganglia in the
47
peripheral nervous syst
em
due to the weak labeling and high background associated with ChAT
48
staining in the periphery.
49
50
6
W
e performed additional
immunohistochemistry
to address the reviewer’s comment
. Please see
1
Response Figure 3 and
Supplemental Figure
3
(old Supplemental Figure 1)
.
In ChAT
-
IRES
-
Cre
2
mice injected with ssAAV
-
PHP.S:CAG
-
DIO
-
eYFP, we
observed strong
co
-
localization of
3
endogenous ChR2
-
eYFP with
GFP and ChAT staining
in cardiac ganglion neurons
(Response
4
Figure 3).
The virus
was highly
efficient
at transducing
peripheral neurons in cardiac ganglia (91.0
5
1.5) versus central neurons in the dorsal motor nucleus of the vagus (1.5
0.9%) (Supplemental
6
Figure
3
b and
e).
E
xpression was also
highly
specific for cholinergic neurons in cardiac
ganglia
7
(
100
0%
of GFP+ neurons were
ChAT
+) (Supplemental Figure
3
d and
e).
8
9
10
Response Figure 3.
ssAAV
-
PHP.S:CAG
-
DIO
-
eYFP was systemically administered to ChAT
-
11
IRES
-
Cre mice at 1 x 10
12
vg per mouse. Three weeks later, eYFP fluorescence was assessed.
12
Representative single
-
plane confocal images of
a
cardiac ganglion with
native
eYFP signal
13
(green)
and
ChAT (red) and GFP (magenta) staining. Scale bar is 50 μm.
14
15
7
1
Supplemental Figure 3. AAV
-
PHP.S preferentially transduces peripheral versus central
2
cholinergic neurons.
ssAAV
-
PHP.S:CAG
-
DIO
-
eYFP was systemically administered to ChAT
-
3
IRES
-
Cre mice at 1 x 10
12
vg per mouse. Three weeks later, eYFP fluorescence was assessed
4
using an antibody for GFP. (
a
) Single
-
plane confocal images of the medulla (left) and dorsal motor
5
nucleus of the vagus nerve (DMV) (right) whole
-
mount stained with ChAT (red) and GFP (green)
.
6
White dashed ovals in the medulla show the location of the DMV. White dashed boxes in the DMV
7
images indicate location of higher magnification images in white boxes. (
b
) Percentage of DMV
8
neurons expressing GFP and ChAT over those expressing ChAT. (
c
) MI
P images of the
9
nodose/jugular ganglion complex whole
-
mount stained with PGP9.5 (red) and GFP (green). (
d
)
10
MIP images of a cardiac ganglion from a heart whole
-
mount stained with PGP9.5 (red) and GFP
11
(green). White dashed boxes indicate location of higher m
agnification images in white boxes. (
e
)
12
Percentage of cardiac ganglion neurons expressing GFP and ChAT over those expressing GFP
13
or ChAT, indicating specificity or efficiency of viral transduction, respectively.
n
= 4 mice (
b
) and
14
8
5 mice (
e
); mean ± s.e.m.
. Scale bars are 500 μm (
a
(left)), 100 μm (
a
(right),
c
,
d
(right), and 1
1
mm (
d
(left)).
2
3
Figure 3c: heart rate appears to be lower after cessation of illumination. Why? At 20Hz stimulation
4
the R wave amplitude also drops after cessation of illumination.
This is also true for 10Hz in Figure
5
3i. Is this typical? Please report the average response of the hearts after cessation of illumination.
6
Is the post
-
illumination response dependent upon the duration of illumination or other illumination
7
parameters?
8
9
Following
optogenetic stimulation of the IPV
-
GP
,
heart rate returned to baseline
within 1
6
.
6
±
4
.
4
10
s at 10 Hz and within 13.
1
± 2.
7
s
at 20 Hz (Response Figure
4
c
).
These recovery times are in
11
line with those reported by Moreno
et al.
(5
-
10 s; doi:
https://doi.org/10.1101/451450
).
The heart
12
rate recovery
was not
dependent on frequency or pulse width of stimulation. The duration of
13
stimulation was 10 s and was not varied during the experiments.
The absolute an
d
relative
change
14
in heart rate at baseline, during stimulation, and following stimulation are shown in Response
15
Figure
4
a
,
b
,
d
and
e
.
16
17
18
Response Figure
4
.
Heart rate recovery following optogenetic stimulation of cholinergic
19
neurons in the IPV
-
GP.
(
a
,
b
)
Summary of the effects of altering stimulation frequency on the
20
absolute (
a
) and delta heart rate response (
b
) before, during, and following
optogenetic
21
stimulation
of the IPV
-
GP
at 10 ms and 221 mW. (
c
) Summary of the time to recovery to baseline
22
heart r
ate
from
a
and
b
(
F
(1.470, 5.881)
= 4.503
,
P
= 0.07
16
)
. (
d
,
e
) Summary of the effects of
23
altering stimulation pulse width on the absolute (
d
) and delta heart rate response (
e
) before,
24
during, and following stimulation at 10 Hz and 221 mW. (
f
) Summary of the time to recovery to
25
baseline heart rate
from
d
and
e
(
F
(
1.245, 4.981) = 0.3002
,
P
= 0.65
57
)
.
n
=
6
mice (
a
-
f
).
mean
26
± s.e.m.; mixed
-
effects
ANOVA.
27
28
We used
two electrodes in the bath to record a pseudo
-
electrocardiogram (ECG)
and a
29
quadripolar
electrophysiology
(EP)
catheter in the left atrium and ventricle to record intracardiac
30
electrograms. The change in R wave amplitude following stimulation is likely due to cardiac
31
9
motion
—
movement of the heart between the ECG electrodes a
nd movement of the EP catheter
1
within the heart. The change in R wave amplitude had no effect on any of our
measurements.
2
3
Figure 6a: heart rate does not return to baseline levels. How long before it does?
4
5
Following optogenetic stimulation of the right st
ellate ganglion, heart rate returned to baseline
6
within 18.9 ± 7.6 s at 10 Hz and 31.9 ± 13.7 s at 20 Hz
(Response Figure
5
c)
. Heart rate did not
7
return to baseline in 2/7 mice at 10 Hz and 1/7 mice at 20 Hz
.
H
owever,
we waited for heart rate
8
to stabilize
before continuing
the
experimental protocol.
The absolute and
relative
change in heart
9
rate at baseline, during stimulation, and following stimulation are shown in Response Figure
5
a,
10
b, d and e.
11
12
13
Response Figure
5
.
Heart rate recovery following optogenetic stimulation of noradrenergic
14
neurons in the right stellate ganglion (RSG).
(
a
,
b
) Summary of the effects of altering
15
stimulation frequency on the absolute (
a
) and delta heart rate response (
b
) before, during, and
16
following optogenetic stimulation of the
RSG
at 10 ms and 126 mW. (
c
) Summary of the time to
17
recovery to baseline heart rate
from
a
and
b
(
F
(1.673, 7.945) = 2.607
,
P
= 0.13
86
)
. (
d
,
e
)
18
Summary of the effects of altering stimulation
pulse width on the absolute (
d
) and delta heart rate
19
response (
e
) before, during, and following stimulation at 10 Hz and 126 mW. (
f
) Summary of the
20
time to recovery to baseline heart rate
from
d
and
e
(
F
(1.029, 3.088) = 3.591
,
P
=
0.
152
1
)
.
n
= 7
21
mice (
a
-
c
) and 5 mice (
d
-
f
).
mean ± s.e.m.; mixed
-
effects
ANOVA.
22
23
24
25
10
For all experiments please report the illumination area. Irradiance values in mW/mm^2 will be
1
more informative than the currently reported power levels in mW. What might be the expected
2
levels (inc
rease in degC) of tissue warming if the power was directed onto a small area (ie, high
3
irradiance)? Could slight warming above physiologic temp increase neuron firing rate and release
4
of neurotransmitter?
5
6
Laser power was reported rather than irradiance be
cause it is difficult to accurately measure the
7
area of tissue illuminated. For all experiments, the optical fiber was positioned
directly
above (
<1
-
8
2 millimeters
) the tissue of interest. Laser power at the tip of the optical fiber was measured with
9
an
optical power meter.
10
11
In
our
Langendorff
experiments
, the temperature increase at the
tip
of the laser
-
coupled optical
12
fiber at 221 mW, 20 Hz, and 10 ms was 1.1 ± 0.0
C, which is well below the
6
-
10
C
increase
in
13
temperature
that has been reported to be
necessary
for
peripheral
nerve
stimulation
20
.
We
14
performed
add
itional experiments
to determine
whether
this
increase in temperature
could result
15
in
neuronal firing
. I
n Langendorff
-
perfused
wild
-
type
mouse hearts
in which there was no
16
expression of ChR2
, an
optical fiber was positioned
for focal illumination of the
IPV
-
GP
. The IPV
-
17
GP was
stimulated at
221 mW,
20 Hz,
and 10 ms
. There was no change in heart rate with
18
stimulation
(Response Figure
6
a and b)
.
Using
the same stimulation parameters in ChAT
-
ChR2
-
19
eYFP mice, heart rate decreased by
50.0
± 14.
9
%
.
These data
in
dicate that
the
slight
increase in
20
temperature
was not responsible for
neuronal firing in our preparation.
21
22
23
Response Figure
6
.
Heart rate response to optogenetic stimulation of the IPV
-
GP in wild
-
24
type and ChAT
-
ChR2
-
eYFP mouse hearts.
A laser
-
coupled
optical fiber was positioned for focal
25
illumination of the IPV
-
GP in Langendorff
-
perfused wild
-
type versus ChAT
-
ChR2
-
eYFP mouse
26
hearts. (
a
,
b
) Summary of the absolute (
a
) and delta heart rate response (
b
) to optogenetic
27
stimulation of wild
-
type versus ChAT
-
ChR2
-
eYFP mouse hearts at 20 Hz, 10 ms, and 221 mW
28
(
t
4
= 0.5979,
P
= 0.582
1
for wild
-
type baseline versus stimulation;
t
4
= 3.033,
*
P
= 0.03
87
for
29
ChAT
-
ChR2
-
eYFP baseline versus stimulation;
t
8
= 3.470,
**
P
=
0.008
4
for wild
-
type versus
30
ChAT
-
ChR2
-
eYFP)
.
n
= 5 mice (
a
,
b
).
mean ± s.e.m.; paired, two
-
tailed
t
-
test (
a
) and unpaired,
31
two
-
tailed
t
-
test (
b
).
32
33
How does the heart rate response to optical stimulation of the paravertebral ganglia compare to
34
electrical stimulation of the ganglia?
35
36
We performed a
dditional experiments to compare electrical stimulation of the
RSG
in wild
-
type
37
mice
to optical stimulation in TH
-
ChR2
-
eYFP mice (
Response
Figure
7
).
In TH
-
ChR2
-
eYFP
mice,
38
11
a laser
-
coupled optical fiber
was positioned above
the
RSG
for focal optical stimula
tion
(as
1
described in the Methods under Optogenetic stimulation and physiological measurements)
.
In
2
wild
-
type mice, a concentric bipolar electrode (FHC, 30324) coupled to a constant current
3
stimulator (Grass, PSIU6 and Model S88) was positioned above the R
SG for electrical stimulation.
4
The
current
for electrical stimulation
in wild
-
type mice
was
titrated to achieve a
n increase
in heart
5
rate
at 10 Hz and 10 ms equivalent to that of optical stimulation at 126 mW, 10 Hz, and 10 ms
in
6
TH
-
ChR2
-
eYFP mice
, which w
as defined as threshold current
.
The t
hreshold
current
was 7
2
9
7
μA.
The frequency and pulse width response curves
for optical versus electrical stimulation of the
8
RSG
were not significantly different.
9
10
11
Response Figure
7
.
In vivo
optogenetic versus electrical stimulation of the RSG.
(
a
) Cartoon
12
depicting optogenetic and electrical RSG stimulation strategy in TH
-
ChR2
-
eYFP and wild
-
type
13
mice, respectively.
The right paravertebral chain was surgically exposed in anesthetized mice and
14
either light or electricity was used for RSG stimulation. (
b
) Frequency response curve
s
15
summarizing the effects of optogenetic versus electrical RSG stimulation at 10 ms
(
t
11
= 0.2395,
16
P
=
0.815
1
at 1 Hz
;
t
11
= 0.06439,
P
= 0.9
498
at 2 Hz
;
t
11
= 0.1467,
P
= 0.886
0
at 5 Hz
;
t
11
=
17
0.4398,
P
= 0.66
86
at 10 Hz
;
and
t
11
= 2.050,
P
= 0.0650 at 20 Hz for optical versus electrical)
.
18
(
c
) Pulse width response curve
s
summarizing the effects of optogenetic versus electrical RSG
19
stimulation at 10 Hz
(
t
8
= 1.047,
P
=
0.32
55
at 1 ms
;
t
9
=
0.6464
,
P
= 0.534
2
at 2 ms
;
t
9
= 0.3787,
20
P
= 0.71
37
at 5 ms
;
and
t
9
= 0.4506,
P
= 0.66
29
at 10 ms for optical versus electrical)
.
n
= 7 mice
21
(
b
optical), 6 mice (
b
electrical,
c
optical), and 5 mice (
c
electrical).
mean ± s.e.m.
; paired, two
-
22
tailed
t
-
test.
T2G, second thoracic ganglion.
23
24
TH
-
ChR2
-
eYFP mice were used for the stellate experiments. It is possible that preganglionic
25
neurons in the stellate were also stimulated? Such synapses are presumably nicotinic, yet is it
26
safe to
assume that there is no expression at all of ChR2 in those preganglionic neurons? Could
27
ChAT + TH co
-
staining reveal the synaptic maps (re: Figure 5) and possibly indicate potential
28
cross
-
over expression of ChR2?
29
30
While it is
possible that there was
non
-
s
pecific expression of ChR2
-
eYFP
in TH
-
ChR2
-
eYFP mice,
31
our
functional
data suggest that
sympathetic preganglionic fibers
within the paravertebral chain
32
did not express ChR2
-
eYFP
.
As the reviewer indicates, sympathetic preganglionic neurons are
33
cholinergic
21
and ChR2
-
eYFP expression in TH
-
ChR2
-
eYFP mice should be restricted to neurons
34
that express TH.
Sympathetic preganglionic neur
ons
that
project to
sympathetic
postganglionic
35
neurons innervating
the
heart are located in the T1
-
T6 segments of th
e spinal cord
22
. Axons from
36
these sympathetic preganglionic
neurons exit the ventral rami
of the spinal cord
and course up
37
and down the paravertebral chain to synapse
predominately
on neurons in the
craniomedial
38
stellate ganglia
(Figure
5
c and d)
, which then
project to the heart.
Previous studies
in large
39