Supplementary Information
Identification of peripheral neural circuits that regulate heart rate
using optogenetic and viral vector strategies
Rajendran et al.
2
Supplementary
Table 1.
Ex vivo
optogenetic stimulation of cholinergic neurons in the IPV
-
GP.
Table
corresponds to
Figure 3.
Dose response curves summarizing the effects of altering
light pulse power, frequency, and pulse width on heart rate. Summary of the heart rate response
to stimul
ation before versus after atropine administration (
t
4
= 2.993
,
*
P
= 0.0402
). mean ± s.e.m.;
paired, two
-
tailed
t
-
test.
Power (mW)
(
n
= 6)
Baseline heart
rate (bpm)
Stimulation
heart rate (bpm)
Δ Heart rate (%)
0
355.8 ± 37.7
355.1 ± 38.0
-0.2 ± 0.2
38
365.8 ± 34.1
307.2 ± 46.1
-16.0 ± 8.7
115
353.0 ± 35.4
228.3 ± 23.7
-31.8 ± 11.0
226
379.2 ± 35.6
200.3 ± 30.0
-45.9 ± 9.7
Frequency (Hz)
(
n
= 6)
1
353.5 ± 27.2
324.6 ± 13.2
-7.2 ± 3.3
5
357.8 ± 25.7
282.4 ± 18.3
-19.7 ± 7.1
10
347.9 ± 23.0
219.7 ± 21.0
-35.2 ± 8.0
20
351.2 ± 29.0
164.5 ± 42.9
-50.0 ± 14.9
Pulse width (ms)
(
n
= 6)
1
355.6 ± 28.5
278.1 ± 23.8
-20.4 ± 8.0
2
362.1 ± 30.4
244.8 ± 25.4
-29.7 ± 10.3
10
356.6 ± 25.5
227.1 ± 21.6
-34.4 ± 8.2
Atropine
(
n
= 5)
Pre
354.9 ± 35.3
224.2 ± 25.0
-33.5 ± 11.0*
Post
333.8 ± 37.7
333.0 ± 37.9
-0.3 ± 0.2
3
Supplementary
Table 2.
In vivo
optogenetic versus electrical stimulation of the vagus
nerve.
Table
corresponds to
Figure 4.
Frequency response curves summarizing the effects of
optogenetic versus electrical
stimulation of the right vagus nerve
on heart rate in the intact state,
of the c
audal end following RVNx and BVNx, and of the cranial end following RVNx
(
t
4
= 3.576,
*
P
= 0.0232 at 10 Hz;
t
4
= 5.229,
**
P
= 0.0064 at 20 Hz)
and BVNx
(
t
4
= 8.588,
**
P
= 0.0010 at 20
Hz).
mean ± s.e.m.; paired, two
-
tailed
t
-
test.
Frequency (Hz)
(
n
= 8)
Baseline heart
rate (bpm)
Stimulation
heart rate (bpm)
Δ Heart rate (%)
Baseline heart
rate (bpm)
Stimulation
heart rate (bpm)
Δ Heart rate (%)
1
560.7 ± 5.5
552.5 ± 5.9
-1.5 ± 0.2
594.1 ± 4.4
587.9 ± 4.5
-1.0 ± 0.2
5
561.6 ± 6.6
533.6 ± 5.3
-5.0 ± 0.3
593.4 ± 5.5
556.0 ± 8.7
-6.3 ± 0.8
10
552.0 ± 8.0
481.6 ± 8.7
-12.8 ± 0.4
590.7 ± 6.1
509.5 ± 6.3
-13.8 ± 0.6
20
558.3 ± 6.3
401.3 ± 13.0
-28.1 ± 2.2
591.6 ± 5.7
370.3 ± 32.0
-37.7 ± 5.0
Frequency (Hz)
(n
= 3)
1
545.8 ± 12.9
535.9 ± 13.1
-1.8 ± 0.6
564.2 ± 8.9
555.3 ± 8.0
-1.6 ± 0.5
5
550.9 ± 11.5
501.5 ± 9.2
-9.0 ± 1.0
567.7 ± 7.9
519.9 ± 13.3
-8.4 ± 1.9
10
562.2 ± 8.2
459.6 ± 19.5
-18.3 ± 2.5
568.2 ± 4.6
449.2 ± 33.0
-21.0 ± 5.7
20
549.3 ± 11.5
302.9 ± 46.0
-44.9 ± 8.1
564.2 ± 7.4
276.2 ± 47.6
-51.0 ± 8.5
Frequency (Hz)
(
n
= 3)
1
530.0 ± 6.6
516.4 ± 5.9
-2.6 ± 0.4
558.9 ± 7.1
548.3 ± 10.6
-1.9 ± 0.7
5
534.0 ± 2.7
493.6 ± 5.7
-7.5 ± 1.5
549.8 ± 10.6
508.3 ± 28.5
-7.7 ± 3.4
10
560.6 ± 6.4
478.2 ± 16.7
-14.7 ± 2.3
546.9 ± 9.5
457.2 ± 33.8
-16.5 ± 5.1
20
545.6 ± 3.0
370.8 ± 44.5
-32.0 ± 8.3
551.5 ± 8.1
354.4 ± 69.3
-36.1 ± 11.5
Frequency (Hz)
(
n
= 5)
1
533.4 ± 10.6
531.2 ± 10.7
-0.4 ± 0.1
569.5 ± 13.0
568.2 ± 13.5
-0.2 ± 0.2
5
539.3 ± 11.0
536.9 ± 10.5
-0.4 ± 0.2
569.6 ± 14.1
559.0 ± 19.5
-2.0 ± 1.3
10
556.6 ± 12.9
556.0 ± 12.7
-0.1 ± 0.1*
576.1 ± 10.8
539.5 ± 18.8
-6.5 ± 1.8*
20
550.1 ± 12.1
549.3 ± 11.7
-0.1 ± 0.1**
576.1 ± 11.2
519.5 ± 17.3
-9.9 ± 1.8**
Frequency (Hz)
(
n
= 5)
1
518.5 ± 7.4
519.7 ± 8.5
0.2 ± 0.2
546.7 ± 7.5
546.9 ± 8.0
0.0 ± 0.2
5
526.1 ± 9.0
525.5 ± 9.4
-0.1 ± 0.1
545.2 ± 9.1
545.9 ± 8.9
0.1 ± 0.1
10
547.4 ± 5.9
545.5 ± 5.5
-0.3 ± 0.2
542.4 ± 8.5
539.7 ± 11.0
-0.5 ± 0.5
20
535.7 ± 6.9
534.9 ± 7.2
-0.2 ± 0.1**
545.6 ± 7.8
528.3 ± 8.7
-3.2 ± 0.4**
BVNx cranial RVNS
Intact RVNS
Optical
Electrical
RVNx caudal RVNS
BVNx caudal RVNS
RVNx cranial RVNS
4
Supplementary
Table 3.
In vivo
optogenetic stimulation of noradrenergic neurons in the
RSG.
Table
corresponds to
Figure 6.
Dose response curves summarizing the effects of altering
frequency and pulse width on heart rate.
Summary of the heart rate response to stimulation of the
RSG versus RT2G (
t
6
= 5.435,
**
P
= 0.0016
).
Summary of the heart rate response to RSG
stimulation before versus after propranolol administration (
t
3
= 3.951,
*
P
= 0.0289
). mean ± s.e.m.;
paired, two
-
tailed
t
-
test.
Frequency (Hz)
(
n
= 7)
Baseline heart
rate (bpm)
Stimulation
heart rate (bpm)
Δ Heart rate (%)
1
464.8 ± 22.6
471.7 ± 22.1
1.5 ± 0.4
2
458.6 ± 21.3
476.2 ± 22.6
3.8 ± 0.6
5
459.6 ± 22.1
486.5 ± 20.6
6.1 ± 1.1
10
455.7 ± 20.8
499.5 ± 20.4
9.9 ± 1.8
20
462.4 ± 20.2
499.8 ± 18.3
8.3 ± 1.1
Pulse width (ms)
(
n
= 6)
1
469.5 ± 37.5
482.9 ± 33.8
3.4 ± 1.6
2
475.0 ± 38.8
492.0 ± 35.3
4.1 ± 1.8
5
475.3 ± 36.3
500.4 ± 33.0
5.9 ± 2.4
10
464.2 ± 32.0
499.0 ± 31.2
7.9 ± 2.2
RSG versus RT2G
(
n
= 7)
RSG
461.7 ± 24.2
504.1 ± 23.1
9.5 ± 1.8**
RT2G
486.1 ± 39.4
487.6 ± 39.3
0.3 ± 0.2
Propranolol
(
n
= 4)
Pre
471.5 ± 23.4
498.0 ± 26.0
5.6 ± 1.4*
Post
364.1 ± 20.1
364.7 ± 20.5
0.1 ± 0.1
5
Supplementary
Figure 1. Whole
-
mount stained heart versus iDISCO
-
cleared heart.
(
a
)
A
heart before (top) and after (bottom) whole
-
mount staining. (
b
)
3D confocal projection of the dorsal
side of a heart (2000 μm z
-
stack) whole
-
mount stained with PGP9.5 (gray). Insets sh
ow a MIP
image of the left ventricular wall (top right) and a 1000 μm
-
thick 3D projection of the left ventricular
wall (bottom right). (
c
) A whole heart (top) was rendered transparent (bottom) using the iDISCO
protocol. (
d
) 3D confocal projection of the do
rsal side of an iDISCO
-
cleared heart (2000 μm z
-
stack) stained with PGP9.5 (gray). Insets show a MIP image of the left ventricular wall (top right)
and a 1000 μm
-
thick 3D projection of the left ventricular wall (bottom right).
In contrast to whole
-
mount st
ained hearts, nerve fibers could be visualized throughout the entire thickness of
myocardium in iDISCO
-
cleared hearts.
Scale bars are 2 mm (
a
,
c
), 1 mm (
b
(left),
d
(left)), and
100 μm (
b
(right),
d
(right)).
6
Supplementary
Figure 2. PACT clearing preserves fluorescence in virally and
endogenously labeled hearts.
(
a
)
Example of a
whole heart (top)
that was
rendered transparent
(bottom) using the PACT protocol.
(
b
)
ssAAV
-
PHP.S:TRE
-
DIO
-
tdTomato
-
farnesylated (1 x 10
12
vg) and s
sAAV
-
PHP.S:CAG
-
DIO
-
tTA (1 x 10
11
vg) were systemically administered to
a
ChAT
-
IRES
-
Cre
mouse
. Four weeks later,
the
whole
heart
w
as
cleared using the PACT protocol. A
402
μm
-
thick 3D projection
of the
left
atrium
showing cholinergic nerve fibers
with native tdTomato
fluorescence (gray). (
c
) tdTomato was expressed in
nor
adrenergic neurons by crossing
transgenic TH
-
IRES
-
Cre
mice
with Ai14 reporter
mice
containing a Cre
-
dependent tdTomato
fluorescent protein allele (T
H
-
tdTomato).
The dorsal half of a
heart from a TH
-
tdTomato mouse
was cleared
using the PACT
protocol
.
A
1000
μm
-
thick 3D projection of the dorsal
atrial wall
showing
nor
adrenergic neurons in
cardiac ganglia
with native tdTomato fluorescence (gray)
(left).
An
85
μm
-
thick 3D projection of the ventricle showing
nor
adrenergic
nerves and
nerve
terminals
(right). Scale bar is 2 mm (
a
), 100 μm (
b
), 500 μm (
c
, left), and 50 μm (
c
, right).
7
Supplementary
Figure
3
. AAV
-
PHP.S preferentially transduces peripheral versus central
cholinergic neurons.
ssAAV
-
PHP.S:CAG
-
DIO
-
eYFP was systemically administered to ChAT
-
IRES
-
Cre mice at 1 x 10
12
vg per mouse. Three weeks later, eYFP fluorescence was asses
sed
using an antibody for GFP. (
a
) Single
-
plane confocal images of the medulla (left) and dorsal motor
nucleus of the vagus nerve
(DMV)
(right)
whole
-
mount stained with
ChAT (red) and GFP (green).
White dashed
ovals
in the medulla show the location of the
DMV
.
White dashed boxes in the DMV
images indicate location of higher magnification images in white boxes.
(
b
) Percentage of
DMV
neurons
expressing GFP
and ChAT over those expressing ChAT
. (
c
) MIP images of the
nodose
/jugular
ganglion complex
whole
-
mount stained with
PGP9.5 (red) and GFP (green). (
d
)
MIP images of a cardiac ganglion
from a heart whole
-
mount stained with PGP9.5 (red) and GFP
(green)
.
White dashed boxes indicate location of higher magnification images in white b
oxes.
(
e
)
Percentage of
cardiac ganglion neurons expressing GFP and ChAT over those
expressing GFP
or ChAT
, indicating specificity or efficiency of viral transduction, respectively
.
n
= 4 mice (
b
)
and
8
5 mice (
e
)
; mean ± s.e.m.. Scale bars are 500 μm (
a
(le
ft)), 100 μm (
a
(right),
c
,
d
(right), and 1
mm (
d
(left)).