With m

uch gratitude to Sarah Friskeyfor

her outstanding technical expertise!

Funding: RO1 HL-98602 and IDEXX-BioResearch

CR

H in

the n

TS

(nu

cleus tractu

sso

litarii) con

tribu

tes to

cardio

respirato

ry respo

nses to

hyp

oxia

Gab

rielle F. Callan

an, B

rian C

. Ru

yle, Eileen

M. H

asserC

ollege of Veterinary Medicine and D

alton Cardiovascular R

esearch Center, U

niversity of Missouri, C

olumbia, M

O 65211

INT

RO

DU

CT

ION

HY

PO

TH

ES

IS

RE

SU

LTS

SU

MM

AR

Y A

ND

CO

NC

LU

SIO

NS

AC

KN

OW

LE

DG

EM

EN

TS

.

0 50

100

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100% O

295%

O2/5%

CO2

Breaths Per Minute

Pre-CRH Saporin

Post-CRH Saporin

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295%

O2/5%

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295%

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Minute Ventilation

CR

H p

un

cta co

nsis

tent w

ith te

rmin

als

are

pre

se

nt in

th

e n

TS

an

d s

urro

un

d T

H c

ells

Effe

cts

of le

sio

n o

f nT

S n

eu

ron

s w

ith C

RH

recep

tors o

n ve

ntilato

ryresp

on

ses to

hyp

oxia

BL

AN

K S

AP

OR

INC

RH

SA

PO

RIN

Peripheral chemoreceptors located in the carotid body and

aortic body monitor blood oxygen levels and send this inform

ation via afferent projections to the nucleus tractussolitarii(nTS) in the brainstem

. The nTS then modulates and integrates this inform

ation before relaying it to other brain regions im

portant in cardiorespiratory control. The m

ajor chemoreflex pathw

ay that produces increases in respiration and m

ean arterial pressure in response to hypoxia involves activation of neurons in the nTS that project to the rostral ventrolateral m

edulla (RVLM). How

ever, the paraventricular nucleus (PVN

) of the hypothalamus is also activated by hypoxia (Berquin

et. al, 2000) and cardiorespiratory responses to chem

oreflex activation are blunted w

hen the PVN is inhibited or lesioned—

evidence that suggests the PVN

is necessary for full expression of the chemoreflex

(Reddy et. al, 2005, Olivan

et. al, 2000). The PVN

receives input from m

ultiple brainstem regions,

including the nTS and caudal ventrolateral medulla (CVLM

), and has projections to the RVLM

, nTS and intermediolateralcell colum

n (IML).

Interestingly, PVN neurons that project to the RVLM

or IML –w

hich w

ould be expected to contribute to cardiorespiratory responses—are

not activated by hypoxia (Coldrenet. al, 2013). How

ever, nTS projecting PVN

neurons are activated by hypoxia and nearly 90% of

these neurons express corticotropin-releasing hormone (CRH) (Figure

1B;Ruyleet. al, 2015). Based on these results, w

e hypothesized that CRH neurons located w

ithin the PVN m

odulate cardiorespiratory responses to chem

oreflex activation via a projection to the nTS. The purpose of this study w

as to further characterize the pathw

ay between the PVN

and nTS and elucidate the importance of

CRH in the nTS in increasing ventilation during chemoreflex activation.

Imm

unohistochemistry w

as used to verify CRH terminals and CRH

receptors in the nTS. Labeling with Synaptophysin

(synaptic marker)

and MAP2 (dendritic m

arker) was used to further exam

ine the location of these receptors. Tyrosine hydroxylase (TH; catecholam

inergicmarker) w

as investigated as prior studies have show

n that nTS catecholaminergiccells are activated by hypoxia and

are required for full expression of the chemoreflex (King et. al, 2015).

In order to evaluate ventilatoryresponses before and after lesion of nTS cells w

ith CRH receptors, plethysmography experim

ents were

conducted on rats injected with a CRH saporin into the nTS.

Effe

cts

of le

sio

n o

f nT

S n

eu

ron

s w

ith C

RH

recep

tors o

n ve

ntilato

ry respo

ns

es to h

ypercap

nia

CR

H S

AP

OR

IN

BL

AN

K S

AP

OR

IN

CRH released from PVN neurons acts at CRH receptors located in the nTS

to modulate cardiorespiratory responses to chem

oreflex activation.If true, w

e also predict the following (Figure 1C):

•CRH is located at term

inals in the nTS•

CRH receptors (CRHRs) are present in the nTS•

Lesion of nTS cells expressing CRHRs using a CRH-specific saporin will

lead to a decreased respiratory response to acute hypoxia

PVN M

icroinjection: Male Sprague Daw

ley rats (240-310g) were anesthetized and placed in a stereotaxic apparatus. The brainstem

at the level of the nTS was

exposed and 4 (30-60nL) microinjections of Blank saporin (controls) or CRH saporin w

ere injected bilaterally into the nTS. Rats were allow

ed 14 days to recover.

Imm

unohistochemistry (IHC):Standard IHC procedures w

ere performed on 30µm

hindbrain sections from naïve rats.

Chemoreflex challenges:Prior to m

icroinjection with CRH saporin or Blank saporin (controls), respiratory responses w

ere assessed via plethysmographyw

hile the rats w

ere conscious and unrestrained. Rats were conditioned to w

hole body plethysmography cham

bers (Data Sciences International) for two hour periods

under room air over three days. O

n the day of the experiment, rats w

ere exposed to normoxic(21%

O2 ), a range of hypoxic (14, 12, 10, 8%

O2 ), and hypercapnic

(95% O

2 /5% CO

2 )conditions. A pressure transducer connected to a PowerLab

(ADInstruments) data acquisition system

was used to m

easure changes in chamber

pressure and calculate respiratory rate (f), tidal volume (V

T ) and minute ventilation (V

E =VT x f). O

xygen saturation was m

easured using a pulse oximeter

(MouseO

x, StarrLifeSciences Corp.). Plethysm

ography studies were repeated after a tw

o week recovery period follow

ing microinjection.

Analysis:An epifluorescentO

lympus BX51 m

icroscope was used to visualize rostral-caudal sections of the nTS. Im

ageJsoftware w

as used to compile im

ages w

ith positive labeling for CRF, CRFR2, TH, MAP2, and Synaptophysin. Plethysm

ography data were com

piled and analyzed using PowerLab

software and M

icrosoft Excel.

•CRH puncta consistent w

ith terminals are located in the nTS

and surround TH cells•

CRHR2 receptors are present in the nTS•

CRHR2 receptors can be found in close apposition to terminals and dendrites

•Rats exhibited decreased respiratory responses w

hen cells with CRH receptors w

ere lesioned

with a CRH saporin in both hypoxic and hypercapnicconditions

Anatomic and functional data support a role for CRH and CRH receptors in the nTS

in m

ediating cardiorespiratory responses to hypoxia

Figure 6. Effects of lesion in the nTS on ventilatoryresponses to peripheral chem

oreceptor stim

ulation. Injection of CRH saporin (n=3) into the nTS reduced tidal volume and m

inute ventilation responses to hypoxia to a greater extent than Blank saporin (n=4).

Lesion of nTS cells expressing CRHRs using a CRH-specific saporin may lead to a decreased

respiratory response under acute hypoxic conditions.

Figure 7. Effects of CRH saporin in the nTS on ventilatory responses to central chem

oreceptor stimulation. Group data (n=7) show

ing depressed respiratory rate, tidal volum

e and minute ventilation in CRH saporin anim

als. Data suggest that CRH and CRHRs in the nTS play a role in m

ediating the response to central chem

oreceptor stimulation.

FU

TU

RE

DIR

EC

TIO

NS

•Verify efficacy of CRH saporin injection in nTS

•Investigate if glial cells w

ithin the nTScontain CRH receptors

•Exam

ine whether CRH receptors are expressed in RVLM

projecting nTSneurons

•O

ptimize CRHR1 antibody for use in future studies

0 20 40 60 80100120140160

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295%

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CO2

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Figure 2. CRH is present in the nTS. A) 10XCRH (green), TH (red) B)60X m

erged image CRH, TH

Legend: Central canal (CC), dorsal motor nucleus of the vagus(DM

V)

Figure 4. Determining the location of CRHR2 in the nTS. 60X photom

icrographs of A) CRHR2 (red)B) CRHR2 and Synaptophysin (green; synaptic). C) CRHR2 and M

AP2 (blue; dendritic). D) M

erged image w

ith CRHR2, Synaptophysin, and MAP2.

Figure 3. CRHRs are present in the nTS. A)Schematic representations of the nTS (Paxinos and

Watson, 6

thedition). B)Corresponding 4X photomicrographs show

ing fluorescent images of

CRHR2 in the nTS. Scale bar: 1mm

. Legend: Area Postrema (AP), 4

thventricle (4V), Solitary tract (Sol)

ME

TH

OD

S

Figure 5. Plethysmography raw

data (10s traces). Ventilatory responses to normoxia and

hypoxiaA)before and B) after adm

inistration of CRH saporin

A) P

RE

-CR

H S

AP

OR

IN

B) P

OS

T-C

RH

SA

PO

RIN

CR

H sap

orin

alters respirato

ry fun

ction

un

der acu

te h

ypo

xic

co

nd

ition

s

21% O

214%

O2

12% O

210%

O2

8% O

2

21% O

214%

O2

12% O

210%

O2

8% O

2

TH

CR

HP

VN

RV

LM

nT

S

Pa O

2

PR

OP

OS

ED

PA

TH

WA

YS

INV

OLV

ED

:

Pa O

2R

VL

M

RE

SP

IRA

TIO

NB

LO

OD

PR

ES

SU

RE

HE

AR

T R

AT

ES

NS

AC

TIV

ITY

nT

SP

VN

1°AntibodiesRabbit anti-CRFR2 (1:5000)

Mouse anti-M

AP2 (1:500)

Guinea pig anti-Synaptophysin

(1:500)2°Antibodies

Cy2 donkey anti-rabbit IgG (1:200)

Cy5 donkey anti-m

ouse IgG (1:200)Cy3 donkey anti-guinea pig IgG (1:200)

1°AntibodiesM

ouseanti-TH (1:1000)

Guinea pig anti-CRF (1:1000)

2°Antibodies Cy2 Donkey anti-m

ouseIgG

(1:200)Cy3 donkey anti-guinea pig IgG (1:200)

Figure 1: Experimental M

odel. A) Hypoxia activates a reciprocal nTS-PVN

pathway to increase ventilation and arterial pressure B) CRH neurons located

within the PVN

that project to the nTS are activated by hypoxia. Photom

icrograph of the PVN show

s Fos(red),CRH (green),and nTS-projecting (w

hite) cells. C) CRH projections from the PVN

may influence nTS neurons to

modulate cardiorespiratory responses

A)

B)

Breg

ma

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nTSnTS

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