Microsoft Word - Cardiac_ANS-Structure-Function-Manuscript-Nature_Communications(FINAL).docx

Identification

peripheral

neural circuit

that regulate heart rate

using optogenetic

and viral vector strategies

Pradeep S. Rajendran

Rosemary

Challis

Charless

Fowlkes

Peter Hanna

John

D. Tompkins

Maria C. Jordan

Sarah Hiyari

Beth

A. Gabris

Weber

Alon Greenbaum

Ken Y. Chan,

Benjamin E. Deverman,

Heike M

nzberg

Jeffrey L. Ardell

Guy Salama

Viviana Gradinaru

and

Kalyanam

Shivkumar

Cardiac Arrhythmia Center and Neurocardiology Research

Program

of Excellence

, David

Geffen School of Medicine,

UCLA

, Los Angeles, CA

Division of Biology and Biological Engineering, California Institute of Technology

Pasadena,

Department of Computer Science, University of California

Irvine, Irvine, CA

Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA

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

Kalyanam Shivkumar, M

, Ph

UCLA

Cardiac Arrhythmia Center

100 Medical Pl

aza

uite

660

Los Angeles, CA 90095

Phone:

310

206

6433

Email:

hivkumar@mednet.ucla.edu

Viviana Gradinaru,

Division of Biology and Biological Engineering

California

Institute

Technology

1200 East California

oulevard

ail Code

156

Pasadena, CA 91125

Phone: 1

626

395

6813

Email:

viviana@caltech.edu

The authors have declared that no conflict of interest exists.

(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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;

Abstract

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

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

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

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.

(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;

Situation

dependent changes in heart rate are essential for survival and

are

under the

precise

control of the autonomic nervous system (ANS)

Heart rate r

eduction during sleep

and elevation

during

exercise

result from

changes in parasympathetic and sympathetic tone

In fact, h

eart rate

variability has

been

utilized

extensively as an index

of ANS function

Although it

s well known

that the parasympathetic and sympathetic nervous system

s innervate the sinoatrial (SA) node

7,8

and

regul

ate heart rate

, the wiring of the

neural

circuits

in the periphery

not well

characterized

Ana

tomical

and functional maps of

these

fundamental

cardiac

circuits

are needed

understand

physiology, characterize

remodeling in disease

(e.g., sick sinus syndrome

)

, and

develop

novel therapeutics

However,

these efforts have been hindered by

shortage

tools

that target

the

peripheral nervous system (

PNS

)

with specificity and precision.

Cardiac

circuit

anatomy has

traditionally been studied

using

thin

sections

and whole

mount preparations

. 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

not possible with these methods

Additionally, many

peripheral neuronal populations such as

intrinsic cardiac ganglia

are c

hallenging to access surgically

for

tracer

delivery

eno

associated

viruses (AAVs)

can address these limitations

because they can be

utilized

for efficient

vivo

gene

expression

defined cell populations when used in Cre transgenic animals

with cell

type

specific regulatory elements

In addition, i

ntersectional strategies can

be used to titrate gene

expression to achieve sparse labeling

AAVs can

also

be delivered systemically to target

difficult

reach populations

unctional

mapping

cardiac circuits

has

rel

electrical

or pharmacological

manipulation

the

ANS

with simultaneous physiological measurements

22,23

However,

each of

these methods ha

disadvantages. Electrical

techniques

lack spatial precision and specificity.

utonomic nerves

such as the vagus

contain motor and sensory fibers

and electrical

stimulation

typically

activates

both

fiber types

25,26

as well as surrounding tissues

harmacological

techniques

exhibit improved sele

ctivity but

lack temporal resolution.

In contrast, o

ptogenetics

which

light

sensitive ion channels

(e.g.,

channelrhodopsin

(ChR2)

halorhodopsin

)

enables

precise

spatiotemporal

control of defined cell

populations

Here

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

(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;

and

tract

tracing,

retrograde n

eur

ona

l tracing with cholera toxin subunit B (CTB)

optogenetics

with simultaneous

physiological

measurements

map

peripheral

parasympathetic

and

sympathetic

neural

circuits

that regulate

heart rate

mice

RESULTS

Tissue clearing and computational pipeline to

assess

global

nnervation

whole

heart

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

immunolabeling

enabled three

imensional

maging of

olvent

leared

rgans

(iDISCO)

protocol

(Figure 1

We used c

onfocal microscopy to image large

tissue

volumes (

Figure 1c

and

Supplemental Movie

)

and lightsheet microscopy to image entire

hearts (Supplemental

Movie 2

)

29,30

observe

cardiac ganglia

surrounding the pulmonary vein

and a dense

network

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

o analyze these data

e created a

semiautomated

computational pipeline to detect nerve fibers

over large tiled

volumes and

measure

microanatomical

features of

fibers

such as

diameter and or

ientation

(Figure 1, d and e

)

Large

diameter nerve fiber

bundles

typically

enter

near the base of the heart

. These bundles

coursed

perpendicular to the atrioventricular (AV) groove

and

branch

into smaller fiber bundles

as they progress

towards the apex.

These

data

from healthy hearts will be

important

for

future

characterization of

neural remodeling

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

intrinsic cardiac

ganglia

After visualizing

global

cardiac

innervation, w

assessed whether we

could

identify

a subset

cholinergic

neurons

that form a

anatomical

circuit with the

node

to potentially regulate heart

rate

used

cell type

restricted sparse viral labeling

to trace

cholinergic

neurons

in intrinsic

cardiac ganglia

to their regions of innervation

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

dose in ChAT

IRES

Cre transgenic

mice

(Figure 2a)

(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;

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

)

. For

tract

tracing

of cholinergic neurons in intrinsic cardiac ganglia

, we

utilized

sparse

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

node

the

node,

and

the

conduction system

observed

cholinergic

fibers

from

cardiac ganglion

located

below

the

junction of the

right and

left

inferior pulmonary veins

(inferior pulmonary veins

ganglionated plexus, IPV

GP)

projecting

towards

the

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

ptogenetic stimulation of cholinergic neurons

the

IPV

Next, t

functionally

assess whether

cholinergic neurons in the

IPV

regulate

heart rat

conduction,

and ventricular electrophysiology

we used

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

YFP

;

offspring from

this cross are subse

quently referred to as

ChAT

ChR2

YFP

mice

)

ChR2

YFP

expression

intrinsic cardiac neurons was

confirmed by staining

hearts

for

GFP

(

to amplify

ChR2

YFP detection

)

and PGP9.5 (Figure 3, a and b

lmost all PGP9

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

and that the ve

ntricles as

well as atria receive

cholinergic

innervation

After verifying ChR2 expression, we

assessed whether selective stimulation

cholinergic neurons

the

IPV

modulated

heart rate

nodal

conduction

, and ventricular

electrophysiology

using optogenetics

ex vivo

Langendorff

perfused hearts

A blue laser

coupled optical fiber was positioned for focal illumination of

the

IPV

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,

) but

did not change

the

interval

(35.7

1.2 ms versus 35.9

1.0 ms;

= 0.88)

(Figure 3

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

the

P wave

duration (9.3

1.0

versus 12.3

2.4 ms

;

< 0.05

) and caused

P wave frac

tionation (

Figure

i and j

P wave fractionation has

been reported in human

following

administration

(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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doi:

bioRxiv preprint first posted online Oct. 29, 2018;

adenosine

, which mimics the effects of acetylcholine

released from

cholinergic nerve terminals

38,39

uring

stimulation

s at higher frequencies

occasionally

observ

ectopic atrial rhythms

(

/5 mice

)

and

even asystole

(

/5 mice

)

(Figure 3d

), demonstrating the profound effect of

the IPV

the

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%

;

< 0.05

) (Figure 3

)

, 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

and

till

modulate

heart rate

. W

first

evaluated

whether we could

preferentially

deliver

transgenes

peripheral

cholinergic neurons in intrinsic cardiac ganglia

rather than central cholinergic neurons

in the medulla

using systemic AAVs.

We used

AAV

PHP.S,

capsid variant

that more

efficiently

transduces

the PNS and many

visceral organs including the heart,

as compared to AAV9

packaged a

Cre

depen

dent genome that expresses

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

the medulla, the vagus nerve

, and intrinsic cardiac

ganglia with

GFP

staining

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

ransduced (Supplemental Figure 1

, a

c).

In contrast

, we observed

robust

transduction of

peripheral cholinergic neurons

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

packaged

a Cre

dependent genome that

expresses ChR2

YFP

from

the ubiquitous CAG promoter in

AAV

PHP.

systemically

administered the virus t

o ChAT

IRES

Cre transgenic mice

, and evaluated expression

weeks

later

In Langendorff

perfused hearts,

were able to

optogenetically

stimulate

postganglionic

cholinergic neurons in

the

IPV

and

decrease heart

rate (Supplemental Figure 2

Taken

together,

our anatomical

and

functional

data

establish an IPV

SA node circuit

involved in heart

rate regulation

(Figure 3j)

. Furthermore,

the

engineered

AAV

, AAV

PHP.S

can

be a powerful

tool to dissect out the roles of peripheral versus central circuits on organ control.

In vivo

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

and other conditions

(e.g., rheumatoid

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doi:

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arthritis

)

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

limitation

examined

whether

we could selectively

stimulate

efferent

fibers

the vagus nerve

using optogenetics

also

assessed

whether

there

was a difference between

optogenetic

and

electrical

VNS

heart rate

(Figure 4

), as a

study showed that electrical stimulation of motor

nerve

results in a non

orderly,

non

physiological

recru

itment

of fibers

, wi

th large

fibers activated

first

The vagus and other autonomic nerves

contain both motor and sensory fibers that vary in diameter and myelination

and non

electrical

techniques such as optogenetic

s are needed to study their physiological role.

To confirm

that

ChR2

YFP

expression was limited to

vagal

efferents

in ChAT

ChR2

YFP

mice

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

)

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

performed function

al studies in anesthetized mice

which we

positioned

laser

coupled optical

fiber

for focal illumination above

and a

hook electrode underneath

the right vagus nerve

In this

context,

optogenetic

VNS

activate

only

efferent fibers (GFP

), whereas electrical VNS

presumably

activates

subsets of

efferent and afferent fibers (PGP9.5

)

the vagu

s nerve (Figure

With

both

vagi

intact

, optogenetic and electrical

right

VNS

resulted

similar

decrease

heart rate

(Figure 4

)

; h

owever, the

slope

of the

responses

were

dramatically

different (

n =

6/8

mice;

Figure 4

likely

due to differential

fiber

recruitment

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

either

right or bilateral

vagotomy

did

not

affect

heart rate, whereas electrical stimulation

surprisingly

result

in a

decrease in

heart

rate

(

6.5

1.8% versus

0.1

0.1%

following right vagotomy for

electrical versus

optogenetic

stimulation

;

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

;

< 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) (

< 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.

addition, there

was

increased latency

o peak heart

rate response with electrical stimulation of

(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;

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

;

< 0.01

) (Figure 4

, indicating that the response post

bilateral

vagotomy was

likely

due to vagal afferent

mediated withdrawal of sympathetic tone

Taken together,

our data

suggest that

optogenetic stimulation selectively activates vagal effere

in ChAT

hR2

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

the

stellate ganglia

The s

ympathetic

nervous system

along with the parasympathetic nervous system,

precise

regulates

heart rate

in normal physiology

anatomically and functionally

dissect

noradrenergic

neurons that form a circuit with the SA node

we used a

retrograde

euronal tracer and an

optogenetic approach

Noradrenergic

nerve

fibers

densely innervate

the

heart as

shown

staining

for

tyrosine

hydroxylase

(TH)

, the rate

limiting enzyme in norepinephrine

synthesis

(Figure 5

a).

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

the stellate gangli

the paravertebral chain

, with

fewer labeled neurons in

the middle cervical and

second thoracic

(T2)

ganglia

(Figure 5

c).

average, 236

39 neurons

were

labeled in the right and 261

34 neurons

labeled

in the left

stellate ganglion

(

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

and

suggesting

that

these ganglia may have a viscerotopic organization.

assessed whether we could

selectively

stimulate

noradrenergic neurons

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

YFP allele

(

offspring from this

cross are subsequently referred to as

ChR2

YFP mice)

ChR2

YFP

exp

ression

stellate ganglion

neurons was confirmed by

staining

for

GFP

and TH

(Figure 6

b).

lmost all TH

neurons

were

also

GFP

(

95.9

1.6%

;

mice

)

After verifying expression, w

performed function

al studies in

open

chest

anesthetized

mice

which

we positioned a laser

coupled optical fiber for focal illumination above the

craniomedial

right stellate

ganglion

(RSG)

right

ganglion

(Figure 6

. Optogenetic

stimulation of the

craniomedial

RSG

resulted

frequency

and pulse

width

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

heart rate

response to stimulation

of this

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The copyright holder for this preprint

http://dx.doi.org/10.1101/456483

doi:

bioRxiv preprint first posted online Oct. 29, 2018;

ganglion

(Figure 6

f).

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%

;

0.05

)

(Figure 6

g),

indicating that the

tachycardic

response was

indeed

mediated by selective

stimulation of

nor

adrenergic

neurons.

Taken together, o

anatomical and functional

data

establish

craniomedial RSG

SA node

circuit

involved in heart rate regulation

DISCUSSION

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

cholinergic

neurons in the IPV

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

)

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

igh

resolution

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

semiautomated

computational

pipeline

detect nerve fibers

and

measure

microanatomic

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

healthy versus diseased

states

such as

Following

innervation around the

infarct

scar and

remote regions of the heart

is altered

31,32,45

, and this

neural remodeling

can

modulate

the arrhythmia substrate

nderstanding

changes in

innervation

post

can

provide

new

insights into arrhythmia mechanisms.

Furthermore

our

clearing

imaging

analysis pipeline

can

readily

applied to

assess innervat

ion of other visceral organs.

is well known that the parasympathetic and sym

pathetic nervous system

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

stellate ganglia

, and

noradrenergic fibers

11,12

modulate

heart

rate. However,

he precise

wiring of

the underlying

neural circuits

has not been delineated

used a novel sparse

AAV

labeling system

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

the

AV node,

and the ventricles.

Selective

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optogenetic

stimulation of cholinergic neurons in the IPV

GP modulate

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

(initial

bradycardia followed by tachycardia)

22,23

. However, since electrical stimulation is non

specific,

is difficult to

interpret

whether the biphasic response

was

due to

activation of

mixed

population

of neuron

contained

intrinsic

cardiac ganglia (

i.e.,

parasympathetic, sympathetic, and

senso

ry)

and/or

pass through fibers. Our findings

demonstrate an IPV

SA node circuit

and

highlight the

importance

using

techniques

such as optogenetics

, which

confer

cell type

specificity

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,

used

a novel

engineered AA

AAV

PHP.S

that has

a strong tropism for the PNS

preferentially

deliver ChR2 to

postganglionic

cholinergic neurons

on the heart

rather than

preganglionic cholinergic neurons in the

medulla

. Future studie

of peripheral neural circuits

should

use AAV

PHP.S and other

engineered AAVs

19,21

dissect

the role of c

entral versus

peripheral neuronal populations

on organ function.

map

noradrenergic neurons that

regulate

heart rate, we used

retrograde neuronal

trac

and optogenetic

approach

. A

study

in canines

using horseradish peroxidase

showed

that sympathetic postganglionic neurons that innervate the heart are

primarily

located

the middle cervical

ganglia

the paravertebral chain

However

, we report

using CTB and

confirm with optogenetic

stimulation

that

the stellate ganglia ha

a viscerotopic organization

with cardiac

projecting neurons clustered in the craniomedial aspect

, consistent with a

study in

cats

In addition to the heart, the stellate ganglia project to many other thoracic structures,

including the sweat glands in the forepaw

, the lung and trachea

, the esophagus

, and brown

fat

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

largely

based on studies using electrical stimulation,

which

is non

specific and

results in

non

orderly

non

physiological recruitment of fibers

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

n vivo

e report that

optogenetic stimulation

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

Our

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findings

suggest

that non

electrical techniques such as optogenetics are needed to characte

rize

neural control of

cardiac

physiology

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

. 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

. 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

and to the paraventricular nucleus of the hypothalamus to

modulate sympathetic efferent outflow

Future

studies

aimed at identifying cardiac

specific vagal

efferent and afferent fibers

, similar to those performed in the lungs

and

the

gastrointestinal

system

are needed to

better

understand vagal control of

cardiac

physiology and

design next

generation VNS therapie

Overall, our data highlight the complexity of cardiac neural circuit

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

combination of these approaches will help disentangle neural control of autonomic

physiology and enable a new era of targeted neuromodulat

ion approaches.

METHOD

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)

, Ai32 (024109)

, and

C57BL/6J mice (000664) were purchased from the Jackson Laboratory. TH

IRES

Cre mice

(254)

were purchased from

he European Mutant Mouse Archive. ChAT

ChR2

YFP

and TH

ChR2

YFP

mice were created by

crossing

Ai32 mice

wit

ChAT

IRES

Cre or TH

IRES

Cre

mic

, 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

weeks

old).

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Plasmids

Plasmids used for AAV production include pUCmini

iCAP

PHP.S (Addgene, 103006), pAAV

CAG

DIO

mRuby2 (Addgene, 104058), pAAV

CAG

DIO

mNeonGreen

pAAV

CAG

DIO

mTurquoise2 (Addgene, 104059), pAAV

ihSyn1

DIO

tTA (Addgene, 99121), pAAV

TRE

DIO

mRuby

(Addgene, 99117),

pAAV

TRE

DIO

mNeonGreen

, pAAV

TRE

DIO

mTurquoise2

(Addgene, 99115), pAAV

TRE

DIO

tdTomato (Add

gene, 99116), and pAAV

CAG

DIO

YFP

(Addgene, 104

052). pAAV

CAG

DIO

ChR2(H134R)

YFP was generated by replacing the Ef1a

promoter in pAAV

Ef1a

DIO

ChR2(H134R)

YFP (a gift from Karl Dei

sseroth, Addgene, 20298)

with the CAG promoter from pAAV

CAG

mNeonGreen

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

. Briefly, AAVs were generated by

triple transient transfection of HEK293T cells (AT

, CRL

3216

) using polyethylenimine

(

Polysciences

, 23966

)

. 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

)

in 2.5 M NaCl and combined with cell

pellets suspended at 37°C in 500 mM NaCl, 40 mM Tris, 10 mM MgCl

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%)

. Purified viruses

were concentrated, washed in sterile

phosphate buff

ered saline (

PBS

)

, sterile filtered, and

tittered

using quantitative

PCR

Systemic

delivery

viruses

Intravenous administration of AAV vectors was performed by

retro

orbital in

jection

with a 31

gauge needle

eek

old

mice

as previously described

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

d cardiac ganglion

stimulation

Mice

were

given

heparin (100U,

i.p.

)

to prevent blood clotting

and

euthanized

with

sodium pentobarbital

(150

/kg

, i.p.

Once

all reflexes subsided and

following a midsternal incision,

earts were rapidly exci

d and

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doi:

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Langendorff

perfused with

modified

Tyrode’s solution

containing the

following

(in m

112

NaCl

1.8

CaCl

5 KCl

1.2

MgSO

NaHCO

, and

50 D

glucose at pH 7.4

The

solution was

continuously bubbled with 95% O

and 5% CO

. Flow rate was adjusted to maintain

a pe

rfusion pressure of 60

80 mmHg.

optic

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

under

the

guidance

of a

fluorescent stereomicroscope (Leica

M205 FA

) fitted with

1x Plan

Apochromat.

octa

polar electrophysiology catheter (

1.1F,

Transonic) was inserted

into the left atrium and ventricle

via a small incision in the left atrium

and

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

(

M Systems

, Model 1700

and

Grass,

P511

respectively) and

continuously

acquired

(AD Instrumen

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

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

5%, maintenance at

vol/vol

inhalation

), intubated, and

mechanical

ventilated (CWE

, SAR

830

)

Core body temperature was measured and maintained at 37

For

VNS

, a midline neck incision was performed and the left and right cervic

al vagus nerves were

exposed. A

laser

coupled

optic

fiber

was positioned above and bipolar platinum hook electrodes

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

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

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

ganglion was exposed.

A laser

coupled optic

fiber

was positioned for focal

illumination

above

the

craniomedial

RSG

right

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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and pulse width (

at 10 Hz

and

126

mW) on heart rate.

The effect of

RSG

versus

right

ganglion

stimulation

(

10 Hz

10 ms,

and

126

mW)

on heart rate

was also

evaluated

the end of the

experiment

, propranolol (2 mg/kg, i.v.) was administered via

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

between stimulations for heart rate to return to baseline values. For vagus nerve and

paravertebral ganglia stimulation

experiments

, a

lead II

electrocardiogram

was

obtained

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

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

3%,

inhalation

), intubated,

and

mechanically ventil

ated. Core body temperature was measured and

maintained at 37

The

surgical incision site was cleaned

times with 10% povi

done iodine and

70% ethanol in H

O (v

A left lateral thoracotomy was performed at the

4th

intercostal space,

the pericardium open

, and the heart was exposed.

Ten microliters of

CTB

conjugated to Alex

Fluor 488 (

0.1%

in 0.

1 M

PBS

)

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

after surgery.

Animals

were sacrificed 6

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

(

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.

1 M

PBS

containing 100

U heparin followed by

50 mL freshly prepared,

ice

cold 4% paraformaldehyde

(

PFA;

EMS,

15714

)

in PBS. Tissues were postfixed in 4%

PFA

overnight

at 4

washed, and

stored

PBS with 0.01% sodium azide.

Langendorff

perfused hearts were

immersion fixed

in 4%

PFA

overnight

at 4

at the end

of the functional experiments

Heart

clearing

hole

mouse

hearts

were

stained

and cleared

using

a modified

iDISCO protocol

ixed

heart

were

dehydrated

with a graded methanol series

(

20%, 40%, 60%,

and

80%

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doi:

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methanol

in H

O (v

)

, each for 1 h

at room temperature),

wash

ed twice

with

100% methanol

for

1 h

room temperature

and

chilled at

Hearts were

then

incubated

in 66%

ichloromethane

/33% methanol

overnight

at room temperature with

agitation,

washed

twice

100% methanol for 1 h

at room temperature,

and

chilled to 4

Next, h

earts were

bleached

with

in methanol

)

overnight at

After bleaching, h

earts were rehydrated with a

graded methanol series

, followed

by one wash

with 0.

1 M P

BS and

washes

with

M PBS

ith 0.2% Triton X

100, each for

1 h

room temper

ature.

For staining,

earts were

permeabilized

with

1 M

PBS with

0.2% Triton X

100, 20%

DMSO

, and

0.3 M glycine

and blocked with

M PBS with

0.2% Triton X

100

, 10% DMSO

, and

% normal

nkey

erum

(NDS)

, each for

at 37

C with agitation

Hearts were incubated in

primary

antibody

rabbit anti

PGP9.5 (Abcam

ab108986

, 1:200

)

diluted in

1 M PBS with

0.2% Tween

and

10 mg/ml heparin (PTwH) for

eek

at 37

C with agitation.

Hearts were then washed several times in

PTwH

overnight at room

temperature befor

secondary

antibody

donkey anti

sheep Cy3 (Jackson ImmunoResearch

713

165

003

, 1:300

)

incubation

PTwH for

eek

at 37

C with agitation.

rimary and

secondary

were replenished half way through staining.

Hearts were then wash

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

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

and ganglia

were block

in 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

diluted in 0.

1 M PBS with 0.2% Triton X

100 and 0.01% sodium azide for 3 nights

at r

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

issues

were

washed several times in 0.

1 M

PBS overnight before incubation

in secondary

diluted in

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.

1 M PBS overnight before being mounted on

microscope

slides in refractive index matching solution (

refractive index

= 1.46)

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The copyright holder for this preprint

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doi:

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