Mapping of NPR-B immunoreactivity in the brainstem of Macaca fascicularis

ORIGINAL ARTICLE

Mapping of NPR-B immunoreactivity in the brainstemof Macaca fascicularis

Essam M. Abdelalim • Ikuo Tooyama

Received: 17 November 2010 / Accepted: 21 March 2011 / Published online: 1 April 2011

� Springer-Verlag 2011

Abstract C-type natriuretic peptide (CNP), the most

abundant natriuretic peptide hormone in the brain, plays an

important role in neuroendocrine function. The physio-

logical effects of CNP are mediated by the natriuretic

peptide receptor-B (NPR-B). Although CNP and NPR-B

have been detected in several brain regions, little is known

about the neuroanatomical localization of NPR-B protein

in the brainstem. In the present study, we investigated the

topographical distribution of NPR-B immunoreactivity in

the monkey brainstem. The data demonstrate widespread

NPR-B immunoreactivity throughout the brainstem. NPR-B

immunoreactivity was located in the superior colliculus,

inferior colliculus, periaqueductal gray, oculomotor

nucleus, red nucleus, ventral tegmental area, substantia

nigra, and cerebral peduncle of the midbrain, as well as in

the abducens nucleus, medial vestibular nucleus, lateral

vestibular nucleus, parabrachial nucleus, locus coeruleus,

trigeminal motor nucleus, pontine reticular nucleus, facial

nucleus, oral part of the spinal trigeminal nucleus, cochlear

nucleus, raphe magnus nucleus, raphe pallidus nucleus,

pontine nucleus of the pons, the dorsal motor nucleus of the

vagus, hypoglossal nucleus, nucleus tractus solitarius,

gracile nucleus, cuneate nucleus, medial vestibular nucleus,

spinal trigeminal nucleus, nucleus ambiguus, lateral para-

gigantocellular nucleus, lateral reticular nucleus, and the

inferior olivary nucleus of the medulla oblongata. The

widespread distribution of NPR-B-immunoreactive struc-

tures throughout the monkey brainstem indicates that CNP

may be involved in several physiological mechanisms,

acting as a neurotransmitter and/or neuromodulator.

Keywords Neuropeptide � NPR-B � Midbrain � Pons �Medulla � Primates

Introduction

The natriuretic peptide receptor-B (NPR-B), also known as

guanylyl cyclase (GC)-B, is one of the natriuretic peptide

receptors that bind natriuretic peptide hormones. The

natriuretic peptides are a family of three structurally related

hormones, namely atrial natriuretic peptide (ANP), B-type

natriuretic peptide (BNP), and C-type natriuretic peptide

(CNP) (Nakao et al. 1992; Anand-Srivastava and Trachte

1993; Espiner et al. 1995). The actions of these natriuretic

peptides are mediated by binding to the natriuretic peptide

receptors. ANP and BNP elicit their effects by binding and

activating the cell surface NPR-A receptor, whereas CNP

activates the homologous NPR-B receptor (Koller et al.

1991; Kuhun 2003; Garbers et al. 2006; Potter et al. 2006).

All three natriuretic peptides also bind the natriuretic

peptide clearance receptor NPR-C, which chiefly regulates

local concentrations of the natriuretic peptides through

receptor-mediated internalization and degradation (Maack

1992; Matsukawa et al. 1999).

In addition to established roles for natriuretic peptides in

regulating neuroendocrine and cardiovascular function (see

Electronic supplementary material The online version of thisarticle (doi:10.1007/s00429-011-0313-1) contains supplementarymaterial, which is available to authorized users.

E. M. Abdelalim (&) � I. Tooyama

Molecular Neuroscience Research Center,

Shiga University of Medical Science, Setatsukinowa-cho,

Otsu, Shiga 520-2192, Japan

e-mail: [email protected]

E. M. Abdelalim

Department of Cytology and Histology,

Faculty of Veterinary Medicine, Suez Canal University,

Ismailia, Egypt

123

Brain Struct Funct (2011) 216:387–402

DOI 10.1007/s00429-011-0313-1

http://dx.doi.org/10.1007/s00429-011-0313-1

Imura et al. 1992; Gutkowska et al. 1997; Levin et al.

1998), these peptides have other functions. For example,

natriuretic peptides enhance the survival of PC12 and

embryonic basal forebrain cells (Fiscus et al. 2001). In

addition, activation of the CNP/NPR-B system in chon-

drocytes of the growth plate can be used as a therapy

for achondroplasia in humans (Yasoda et al. 2004) and

Sabbatini et al. (2005) found that centrally applied CNP

enhances pancreatic secretion by activating its receptor.

Recently, the CNP/NPR-B system was found to be

involved in the regulation of bidirectional plasticity in the

CA1 area of the hippocampus (Decker et al. 2010).

CNP predominates in the central nervous system, with

highest levels of CNP mRNA found in the olfactory nuclei,

limbic cortex, hippocampus, amygdala, hypothalamus, and

brainstem (Garbers 1992). The presence of natriuretic

peptides and their receptors has been reported in monkey

brain (Wilcox et al. 1991; Abdelalim et al. 2006, 2007),

indicating a central role for natriuretic peptides in this

species. A previous study in the monkey reported that

NPR-B mRNA expression was confined to the adrenal

medulla, pituitary, and cerebellum (Wilcox et al. 1991).

In the rat, NPR-B mRNA is expressed in the limbic

cortex, neocortex, olfactory bulb, hippocampus, amygdala,

preoptic–hypothalamic neuroendocrine circuits, ventral

tegmental area (VTA), substantia nigra, and in the motor

nuclei of the cranial nerves. Intermediate expression of

NPR-B mRNA has been found in brainstem nuclei con-

trolling autonomic function (Langub et al. 1995; Herman

et al. 1996; Thiriet et al. 2001). Although NPR-B mRNA

has been demonstrated in astrocyte cultures (Deschepper

and Picard 1994), an in situ hybridization study in the rat

brain failed to detect NPR-B signals in the glia (Herman

et al. 1996). In the rat brain, NPR-B protein immunore-

activity has been found in the VTA, substantia nigra,

caudate–putman, nucleus accumbens, frontal cortex, hip-

pocampus, cortex, and cerebellum (Thiriet et al. 2001).

The physiological effects of CNP in the brain depend on

the distribution of its receptors. To our knowledge, the

expression of NPR-B protein has not been investigated in

detail in the brainstem of any species. To further under-

stand the central actions of CNP in the brainstem, we

examined NPR-B immunoreactivity in the brainstem of the

cynomologus monkey (Macaca fascicularis) in the present

study using immunohistochemical techniques.

Materials and methods

Tissue preparation

Samples were obtained from the brains of cynomologus

monkeys (M. fascicularis), after animals that had been used

for other research purposes by other investigators had

been killed. The animal use protocols in the present study

were approved by the Institutional Animal Care and Use

Committee (IACUC) of Shiga University of Medical

Science.

The brains were collected at different times and handled

individually. All brains from the three monkeys were

subjected to the same procedures. The brain was removed

from two males and one female of cynomologus monkeys

(3, 10, and 12 years) and fixed immediately in 4% para-

formaldehyde in 0.1 M phosphate buffer (pH 7.4) for 2

days at 4�C before being immersed in 15% sucrose in

0.1 M phosphate buffer (pH 7.4) with 0.1% sodium azide

for cryoprotection. The sucrose solution was changed every

day for 4 days, after which time the brains were stored in

sucrose at 4�C until sectioning. Fixed tissues were sub-

jected to cryostat sectioning at –20�C.

Western blotting

Western blot analysis was performed to confirm the spec-

ificity of the NPR-B antibody. Briefly, rat brain and mon-

key heart samples were homogenized in ice-cold 50 mM

Tris–HCl buffer (pH 7.4) containing protease inhibitors

(Complete Mini; Roche Diagnostics, Mannheim, Ger-

many). After initial centrifugation at 800g for 5 min at 4�C

to remove tissue fragments, the supernatant fraction was

centrifuged at 100,000g for 1 h at 4�C. The supernatant

was collected and considered as the cytosolic fraction,

whereas the pellets (i.e. the membrane fraction) were

resuspended in RIPA buffer (50 mM Tris–HCl, 150 mM

NaCl, 1% Nonidet P-40, 0.25% Na-deoxycholate, 1 mM

phenylmethylsulfonyl fluoride, and 19 Complete Mini

protease inhibitor cocktail).

Protein samples (40 lg) from the cytosolic and mem-

brane fractions were electrophoresed by sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS–PAGE;

7% gels) and transferred onto polyvinylidene difluoride

membranes. Non-specific protein binding sites were

blocked by incubating membranes for 2 h at room tem-

perature in 10% skim milk in 25 mM Tris-buffered saline

containing 0.1% Tween-20 (TBST; pH 7.4), followed by

4 h incubation at 4�C with a rabbit polyclonal antibody

against NPR-B (Santa Cruz Biotechnology) at a dilution of

1:1000 in TBST. Blots were washed twice for 10 min each

time with 25 mM TBST before being incubated for 1 h at

room temperature with peroxidase-conjugated anti-rabbit

IgG (1:10000 dilution; Jackson ImmunoResearch Labora-

tories, Inc.). After extensive washing with 25 mM TBST,

blots were developed using SuperSignal West Pico

Chemiluminescent substrate (Pierce) and visualized using

an LAS-3000 FujiFilm Lumino-Image Analyzer (FujiFilm,

Tokyo, Japan).

388 Brain Struct Funct (2011) 216:387–402

123

Immunohistochemistry

Serial cryostat sections (20 lm) were floated in 0.1 M

phosphate-buffered saline with 0.3% Triton X-100, pH 7.4

(PBST), for 4 days at 4�C and treated in a free-floating state.

Endogenous peroxidase activity was blocked by incubating

the sections in 0.5% H2O2 in PBST for 30 min. After sections

had been rinsed three times with PBST for 10 min each time,

they were incubated in 4% normal horse serum (Invitrogen)

for 40 min at room temperature, and then they were incu-

bated for 72 h at 4�C with a rabbit polyclonal antibody

against human NPR-B (1:400 dilution; Santa Cruz Bio-

technology) or a polyclonal rabbit anti-NPR-B antibody

(1:300 dilution; Abgent, Sant Diego, CA, USA). The primary

antibody was diluted in 1% normal horse serum in PBST.

After sections had been rinsed three times with PBST

(10 min each time), they were incubated for 1 h at room

temperature with biotinylated goat anti-rabbit IgG (1:3000

dilution; Vector Laboratories, Burlingame, CA, USA)

before being rinsed another three time with PBST. The

sections were finally incubated for 1 h at room temperature

with an avidin–biotin–peroxidase complex (1:4000 dilution;

ABC Elite; Vector Laboratories) and the peroxidase-labeled

sections were developed in 0.02% 3,3-diamine-benzidine

tetrahydrochloride with 0.07% nickel ammonium sulfate in

50 mM Tris–HCl (pH 7.6), with 0.005% hydrogen peroxide.

Double immunohistochemistry

For double immunostaining we used 4% normal horse

serum as a blocking solution. After the first NPR-B

immunostaining which produced a purple color, the sec-

tions were treated for 30 min at room temperature with

0.5% H2O2 in PBST to eliminate residual horseradish

peroxidase activity. The sections were then washed in

PBST and incubated with mouse monoclonal anti-tyrosine

hydroxylase (TH) antibody (1:3000; Chemicon). After

three rinses with PBST, the sections were incubated for 2 h

at room temperature with histofine anti-mouse IgG com-

plex (1:100; Nichirei, Japan). After thee further rinses with

PBST, the sections were developed in 0.02% 3,3-diamine-

benzidine tetrahydrochloride in 50 mM Tris–HCl (pH 7.6),

with 0.005% hydrogen peroxide, to yield a brown color.

Mapping

Mapping was performed using a camera lucida, and diagrams

of the midbrain, pons, and medulla oblongata were prepared.

Data analysis

The major divisions and nuclei of the brainstem were

identified according to Szabo and Cowan (1984). Images of

selected immunostained sections were captured and orga-

nized into photographic panels. Adobe Photoshop was used

to view the images. To improve visualization of the results,

only the brightness and contrast of the images were

adjusted using Adobe Photograph, without any further

manipulation of the images.

Results

Specificity of the NPR-B antibody

Since the Western blotting has been reported as a strong

tool to determine the antibody specificity (Burry 2000), the

specificity of the NPR-B antibody was examined by wes-

tern blotting. The antibody recognized a single band at

approximately 120 kDa in the membrane fractions of the

rat brain and monkey heart which corresponded to NPR-B

protein (Fig. 1a, b).

Also, we compared the sequence of the immunogen used

to produce the NPR-B antibody with the corresponding

sequences of the monkey and rat (Fig. 1c). The antibody

used in this study is a polyclonal antibody raised against

the amino acids 171–250 of NPR-B of human origin. The

comparison of these 80 amino acids sequences of NPR-B

between human and monkey showed that only one amino

acid is different between human and monkey (98.75%

identity). Also, comparing this region between human and

rat showed 98.75% identity, indicating that the antibody

used in the current study can recognize NPR-B in monkey

and rat tissues (Fig. 1c).

Additionally, we examined the immunolabeling of NPR-

B in monkey brainstem using another antibody (from dif-

ferent company) raised against NPR-B. This antibody has

been previously used in other studies (Abdelalim et al.

2008b). The antibody showed identical staining patterns in

the brainstem of monkey (supplementary Fig. 1). Collec-

tively, these findings suggest that the protein recognized by

the NPR-B antibody in the present study is indeed, NPR-B.

Distribution of NPR-B-immunoreactive structures

in the monkey brainstem

NPR-B immunoreactivity was distributed extensively in

neuronal structures throughout the brainstem of the cyno-

mologus monkey. Examination of the immunostained

sections from the three monkeys showed no difference in

the pattern of the distribution of NPR-B immunoreactivity.

Figure 2 summarizes the distribution of NPR-B immuno-

reactivity in the midbrain (Fig. 2a, b), pons (Fig. 2c, d),

and medulla oblongata (Fig. 2e–g), as drawn using the

camera lucida. As shown in Fig. 2, NPR-B-positive neu-

rons were observed in specific brain regions. The highest

Brain Struct Funct (2011) 216:387–402 389

123

density of NPR-B-immunoreactive structures was observed

in the pons, followed by the medulla oblongata.


in the midbrain

In the midbrain, NPR-B immunoreactivity was observed in

the superior and inferior colliculi, periaqueductal gray

(PAG), medial lemniscus, oculomotor nucleus, red nucleus,

cerebral peduncle, VTA, and substantia nigra (Fig. 2a, b).

In the PAG, several NPR-B-immunoreactive neurons

were seen surrounding the aqueduct of Sylvius (Fig. 3a, b).

High-intensity NPR-B immunoreactivity was observed in

different-shaped neurons in the oculomotor nucleus, with

several neurons having immunoreactive processes (Fig. 3c,

d). The highest intensity of NPR-B immunoreactivity in the

midbrain was seen in the large neurons of the red nucleus

(Fig. 3e, f).

NPR-B immunoreactivity was observed in the VTA

(Fig. 4a, b). At the level of the substantia nigra, there was a

large number of closely packed, moderately NPR-B-

immunoreactive neurons (Fig. 4c, d). In the cerebral

peduncle, intense NPR-B immunoreactivity appeared in

elongated perikarya and their processes (Fig. 4e, f). Some

immunoreactive primary dendrites divided into secondary,

then tertiary dendrites (Fig. 4e). Intensely NPR-B-immu-

noreactive axons were found in the cerebral peduncle area

(Fig. 4e, f).


in the pons

The highest density of NPR-B immunoreactivity was found

in the pons. NPR-B immunoreactivity was seen in neurons

in several regions, including the abducens nucleus, medial

vestibular nucleus, parabrachial nucleus, lateral vestibular

nucleus, facial nucleus, oral part of the spinal trigeminal

nucleus, cochlear nucleus, locus coeruleus, trigeminal

motor nucleus, pontine reticular formation, pontine

nuclei, raphe magnus nucleus, and raphe pallidus nucleus

(Fig. 2c, d).

Strongly stained NPR-B-immunoreactive neurons were

detected just under the fourth ventricle in the abducens

nucleus (Fig. 5a, b). In the medial vestibular nucleus, oval

to fusiform immunoreactive neurons were observed

(Fig. 5c, d). Immunoreactive neurons with different shapes

were observed scattered throughout the parabrachial

nucleus (Fig. 5e, f) and immunoreactive neuronal pro-

cesses were seen in some neurons (Fig. 5f). High-intensity

NPR-B immunoreactivity was observed in the trigeminal

motor nucleus (Fig. 5g, h).

NPR-B-immunoreactive perikarya were detected in the

locus coeruleus (Fig. 6a, b). Diffuse distribution of dif-

ferent-sized, intensely NPR-B-immunoreactive neurons

were observed in the pontine reticular nucleus (Fig. 6c, d).

In the pontine nucleus, a high density of NPR-B-

immunoreactive neurons was observed (Fig. 6e, f). In

Fig. 1 Antibody specificity for

the NPR-B receptor. Western

blots were performed on

extracts from rat brain (a) and

monkey heart (b). Adjacent

lanes were loaded with

equivalent amounts of protein

from membrane and cytosolic

fractions. The antibody

recognized a single band at

approximately 120 kDa in the

membrane fraction only in rat

brain and monkey heart.

c Comparison of the amino acid

sequences of the human

immunogen (from 171 to 250)

used to produce NPR-B

antibody with those of monkey

and rat.The GeneBank

accession numbers are human

NPR-B: NM_003995, monkey

NPR-B: XM_001085221, and

rat (NM_053838). Dots (.) show

identical amino acids


123

Fig. 2 Distribution of NPR-B

receptor immunoreactivity in

the brainstem of cynomologus

monkeys, drawn from

representative sections using a

camera lucida. 3V third

ventricle, 4V fourth ventricle,

6N abducens nucleus, 7N facial

nucleus, Aq aqueduct of

Sylvius, CC central canal, CNcochlear nucleus, Cp cerebral

peduncle, CuN cuneate nucleus,

DMNv dorsal motor nucleus of

the vagus, GrN gracile nucleus,

HN hypoglossal nucleus, ICinferior colliculus, IO inferior

olivary nucleus, LC locus

coeruleus, LRN lateral reticular

nucleus, LVe lateral vestibular

nucleus, ml medial lemniscus,

MO5 trigeminal motor nucleus,

MVe medial vestibular nucleus,

NA nucleus ambiguus, NOoculomotor nucleus, NTSnucleus tractus solitarius, PAGperiaqueductal gray, PBNparabrachial nucleus, PGiparagigantocellular nucleus,

Pn pontine nuclei, Pnc caudal

pontine reticular formation,

RMg raphe magnus nucleus, RNred nucleus, Rpa raphe pallidus

nucleus, SC superior colliculus,

SN substantia nigra, SP5Ccaudal part of the spinal

trigeminal nucleus, SP5Iinterpolar part of the spinal

trigeminal nucleus, SP5O oral

part of the spinal trigeminal

nucleus, VTA ventral tegmental

area


123

addition, several neurons in the raphe magnus nucleus

and raphe pallidus nucleus were NPR-B positive

(Fig. 6g, h, i)

A cluster of immunoreactive polygonal to ovoid peri-

karya with immunoreactive processes was seen in the facial

nucleus (Fig. 7a, b). NPR-B immunoreactivity was

observed in oval neurons of the principal sensory nucleus

of the trigeminal nerve, as well as in neuronal processes

(Fig. 7c, d). The highest density of NPR-B immunoreac-

tivity was detected in the dorsal and ventral cochlear

nucleus; the immunoreactive perikarya here appeared

spherical and of a uniform size (Fig. 7e, f).


in the medulla oblongata

Several regions in the medulla oblongata were NPR-B

immunoreactive, including the dorsal motor nucleus of the

vagus, hypoglossal nucleus, nucleus tractus solitarius,

gracile nucleus, cuneate nucleus, nucleus ambiguus, retic-

ular formation, paragigantocellular nucleus, lateral reticu-

lar nucleus, spinal trigeminal nucleus, and the inferior

olivary nucleus (Fig. 2e, f, g).

In the dorsal motor nucleus of the vagus, ovoid- to

fusiform-shaped perikarya with immunoreactive processes

Fig. 3 NPR-B receptor

immunoreactivity in the

midbrain of cynomologus

monkeys. a NPR-B-

immunoreactive perikarya and

fibers in the periaqueductal gray

(PAG). aq aqueduct of Sylvius.

b At higher magnification, small

immunoreactive neurons are

apparent in the PAG. c NPR-B-

immunoreactive neurons in the

oculomotor nucleus (NO).

d Immunoreactive neurons in

the NO seen at higher

magnification. e NPR-B

immunoreactivity in the red

nucleus (RN). f Large NPR-B-

immunoreactive perikarya seen

at higher magnification.

Bars = 100 lm (a, d, e);

50 lm (b, c, f)


123

were positive for NPR-B (Fig. 8a, b). In the hypoglossal

nucleus, NPR-B immunoreactivity was seen in elongated

and fusiform perikarya (Fig. 8c, d). In the dorsal portion of

the caudal medulla, many immunoreactive perikarya were

distributed in the cuneate nucleus (Fig. 8e, f). Considerable

NPR-B immunoreactivity was seen in the spinal trigeminal

nucleus (Fig. 8g, h).

In the paragigantocellular nucleus, different-shaped

neurons, with long immunoreactive processes, were

positive for NPR-B (Fig. 9a, b). Nerve fibers were dis-

tributed among the immunoreactive perikarya (Fig. 9a, b).

A cluster of strongly stained NPR-B neurons was

observed in the lateral reticular nucleus (Fig. 9c, d). In

addition, neurons in the nucleus ambiguus with high-

intensity NPR-B immunoreactivity appeared as a cluster

(Fig. 9e, f). Several immunoreactive neurons were seen

scattered in the reticular formation (Fig. 9e). A large

number of NPR-B-immunoreactive neurons were distrib-

uted uniformly throughout the inferior olivary nucleus

(Fig. 9g, h).

Localization of NPR-B in the dopamine neurons

in the monkey brainstem

To investigate whether dopamine neurons express NPR-B

receptors, we performed double immunostaining for NPR-B



midbrain of cynomologus

monkeys. a NPR-B-

immunoreactive neurons in the

ventral tegmental area (VTA).

b Immunoreactive neurons in

the VTA seen at higher

magnification. c NPR-B-

immunoreactive perikarya in the

substantia nigra (SN). d At

higher magnification,

immunoreactive neurons are

apparent in the SN. e NPR-B-


fibers in the cerebral peduncle

(CP). Note the immunoreactive

dendritic arborization

(arrowhead). f Higher

magnification image showing


fibers. Bars = 100 lm (a, d, e);

50 lm (b, c, f)


123

and TH in the monkey brainstem. It has been reported that

in the CNS, nearly 75% of all dopamine neurons reside in

the ventral part of the midbrain (Reiner 1994). Double

immunostaing showed the co-localization of NPR-B

immunoreactivity with the TH-immunoreactive perikarya

in the ventral tegmental area and the substantia nigra and

(Fig. 10a–d). The co-localization was mainly in the

perikarya.


immunoreactivity in the pons of

cynomologus monkeys.

a Immunoreactive perikarya in

the abducens nucleus (6N).

4V, fourth ventricle.

b Immunoreactive perikarya in

the 6N at higher magnification.

c NPR-B-immunoreactive

neurons in the medial vestibular

nucleus (MVe).

d Immunoreactive neurons in

the MVe seen at higher

magnification. e NPR-B


parabrachial nucleus (PBN).

f NPR-B-immunoreactive

perikarya in the PBN seen at

higher magnification. g NPR-B


trigeminal motor nucleus

(MO5). h Higher magnification

image showing NPR-B-


MO5. Note the

immunoreactivity in neuronal

processes (arrowhead).

Bars = 100 lm (a, c, e, g);

50 lm (b, d, f, h)


123

Discussion

The present study is the first to map the distribution of

NPR-B protein throughout the brainstem. The results

demonstrate the widespread distribution of NPR-B immu-

noreactivity in this region, which may highlight the

potential importance of natriuretic peptides within the

brainstem.




a Immunoreactive neurons are

apparent in the locus coeruleus

(LC) at low magnification.

b Higher magnification view of


LC. c Immunoreactive neurons

in the caudal pontine reticular

formation (Pnc). d At higher

magnification, immunoreactive

perikarya in the Pnc can be

seen. e NPR-B

immunoreactivity in the pontine

nuclei (Pn). f Immunoreactive

perikarya in the Pnc at higher

magnification.

g Immunoreactive neurons in

the raphe magnus nucleus

(RMg) and raphe pallidus

nucleus (RPa). h At higher

magnification, immunoreactive

perikarya in the RMg can be

seen. i Higher magnification

view of immunoreactive

perikarya in the RPa.

Bars = 100 lm (a, c, e, g);

50 lm (b, d, f, h, i)


123

Distribution of NPR-B immunoreactivity

in the brainstem

The extensive distribution of NPR-B immunoreactivity in

the brainstem regions examined in the present study is in

general agreement with previous results described in the rat

brainstem obtained using in situ hybridization (Herman

et al. 1996). However, some differences are apparent.

Specifically, in the monkey but not rat brainstem, NPR-B

was found in the inferior colliculus, locus coeruleus,

gracile nucleus, and dorsal motor nucleus of the vagus.

Conversely, in the rat but not monkey brainstem, NPR-B

was found in the trochlear nucleus, dorsal raphe nucleus,

Barrington’s nucleus, prepositus, and area postrema (Her-

man et al. 1996). These differences in the distribution of

NPR-B between monkey and rat could be due to either

species or technical considerations differences, because, in

the monkey, we used immunohistochemical analysis to

detect NPR-B.

NPR-B immunoreactivity was observed in the VTA and

substantia nigra and is expressed by the dopamine neurons.

These data are in agreement with the results of a previous

study in rat, which reported abundant CNP and NPR-B

mRNA and protein expression in the VTA and substantia

nigra (Thiriet et al. 2001). Moreover, NPR-B has been

found in the dopaminergic neurons themselves in the VTA

(Jouvert et al. 2004).

The results of the present study demonstrate strong NPR-

B immunoreactivity in the cochlear nucleus. This is in

agreement with previous results reported for the rat, in which

extremely high levels of NPR-B mRNA were detected in the

anteroventral cochlear nucleus (Herman et al. 1996).




a Immunoreactive neurons in

a the facial nucleus (7N), (c) the

oral part of the spinal trigeminal

nucleus (SP5O), and (e) the

cochlear nucleus (CN). Higher

magnification view of


7N (b), SP5O (d), and CN (f).Bars = 100 lm (a, c, e, g);

50 lm (b, d, f, h, i)


123

Comparing the distribution of NPR-B immunoreactivity

with that of other natriuretic peptide receptors in the

brainstem seems to indicate a close anatomical relationship

between these receptors, because in both the monkey and

rat, NPR-A and NPR-C receptors were found in some

regions in which NPR-B receptors were also found, sug-

gesting the possible coexistence of natriuretic peptide

receptors. For example, in the monkey, NPR-A



medulla oblongata of


Immunoreactive neurons in

(a) the dorsal motor nucleus of

the vagus (DMNv),

(c) hypoglossal nucleus (HN),

(e) cuneate nucleus (CuN), and

(g) the caudal part of the spinal

trigeminal nucleus (SP5C).

Higher magnification images of


DMNv b, HN d, CuN f, and

SP5C h. CC central canal.

Bars = 100 lm (a, c, e, g);

50 lm (b, d, f, h)


123

immunoreactivity was found in the oculomotor nucleus,

red nucleus, parabrachial nucleus, locus coeruleus, gracile

nucleus, cuneate nucleus, dorsal motor nucleus of the

vagus, hypoglossal nucleus, lateral reticular nucleus,

nucleus ambiguus, and inferior olivary nucleus (Abdelalim

et al. 2007). In the rat, NPR-C immunoreactivity was found

in the PAG, oculomotor nucleus, substantia nigra, VTA,

facial nucleus parabrachial nucleus, locus coeruleus, dorsal

motor nucleus of the vagus, hypoglossal nucleus, lateral

reticular nucleus, and the inferior olivary nucleus



medulla oblongata of


Immunoreactive neurons in the

(a) paragigantocellular nucleus

(PGi), (c) lateral reticular

nucleus (LRN), (e) nucleus

ambiguus (NA), and (g) the

inferior olivary nucleus (IO).

Higher magnification views of


b PGi (note the long

immunoreactive neuronal

processes; arrowhead), d LRN,

f NA, and h IO. Bars = 100 lm

(a, c, e, g); 50 lm (b, d, f, h)


123

(Abdelalim et al. 2008a). The presence of both guanylyl

cyclase receptors (NPR-A and NPR-B) in the same

regions suggests an action of ANP, BNP, and CNP in

these regions. On the other hand, the presence of NPR-B

and NPR-C receptors in the same regions may suggest a

role for NPR-C in limiting the local action of CNP in

these areas.

Overall, NPR-B immunoreactivity detected in the pres-

ent study was more widely distributed in the brainstem

than the other natriuretic peptide receptors (NPR-A and

NPR-C). This finding is in agreement with the previous

hypothesis that CNP is the predominant natriuretic peptide

in the brain (Garbers 1992).

Possible function of the NPR-B receptor

in cardiovascular control

In addition to their peripheral role in controlling water

balance and cardiovascular tone, natriuretic peptides

appear to regulate blood pressure centrally (Ermirio et al.

1989, 1991; Dev et al. 1998), with the central administra-

tion of natriuretic peptides and natriuretic peptide anti-

bodies modulating heart rate and blood pressure. For

example, intracerebroventricular injection of CNP has been

reported to decrease blood pressure in conscious sheep

(Charles et al. 1992). Furthermore, a physiological role for

the NPR-B receptor in homeostasis is supported by studies

Fig. 10 Double

immunostaining for NPR-B

with TH in the brainstem of


a Colocalization of NPR-B

immunoreactivity with TH-

immunoreactivity in the ventral

tegmental area (VTA).

b Colocalization of NPR-B



substantia nigra (SN)

(arrowheads). Higher

magnification images of

colocalization of NPR-B


immunoreactivity in the VTA

(c) and SN (d). Note a NPR-B-

positive neuron which is

negative for TH (arrow).

Bars = 100 lm (a, c);

50 lm (b, d)


123

demonstrating that CNP expression in the olfactory region

is modulated by water and salt balance (Cameron et al.

2001). These effects may be mediated through receptors

located in the brainstem because the PAG (Carrive et al.

1989; Inui et al. 1994), parabrachial nucleus (Chamberlin

and Saper 1992), locus coeruleus (Anselmo-Francil et al.

1999), dorsal motor nucleus of the vagus, nucleus tractus

solitarius (Paton 1999), paragigantocellular nucleus

(Brown and Guyenet 1984; Schreihofer and Guyenet

1997), and nucleus ambiguus (Machado and Brody 1988)

are thought to be involved in the central control of car-

diovascular homeostasis. Consistent with this, we found

abundant NPR-B immunoreactivity in these regions in the

monkey brainstem, which is highly indicative of an

important role for CNP in central cardiovascular

regulation.

Other possible functions of the NPR-B receptor

in the brainstem

CNP has been postulated to have diverse functional roles in

different regions of the central nervous system (Thiriet

et al. 2001; Jouvert et al. 2004; Sabbatini et al. 2005),

including many of the brainstem structures examined in the

present study. The presence of the NPR-B receptor in many

regions of the monkey brainstem implies that CNP may

have different functions.

The presence of NPR-B immunoreactivity in the supe-

rior colliculus, oculomotor nucleus, vestibular nuclei,

abducens nucleus, facial nucleus, and hypoglossal nucleus

indicates that CNP could be involved in the control of eye

and head movements. The neural organization of the

pathways from the superior colliculus to motoneurons in

the horizontal oculomotor system has been analyzed

extensively (Sparks 1999; Scudder et al. 2002). Further-

more, the natriuretic peptides and their receptors have been

detected in eye structures (Fernandez-Durango et al. 1995;

Kuribayashi et al. 2006; Abdelalim et al. 2008b).

Centrally applied CNP enhances pancreatic secretion

through a vagal pathway by activating central natriuretic

peptide guanylyl cyclase-coupled receptors (Sabbatini et al.

2005). mRNA expression for the natriuretic peptide

receptor and CNP has been localized in the paraventricular

nucleus and the dorsal motor nucleus of the vagus, both of

which are important sites for the regulation of gastroin-

testinal function (Langub et al. 1995; Herman et al. 1996).

Together with the results of the present study, these find-

ings suggest a role for NPR-B receptors located in the

dorsal motor nucleus of the vagus of the monkey brainstem

in the central control of pancreatic function.

In the present study, NPR-B immunoreactivity was

detected in the dopamine neurons in the substantia nigra

and VTA, which suggests the involvement of NPR-B in

dopamine neurons functions. Recently, we found that the

NPR-B receptor is expressed by dopaminergic cells in the

retina (Abdelalim et al. 2008b). It has been reported pre-

viously that CNP inhibits dopamine release by stimulating

NPR-B receptors and increasing intracellular GMP con-

centrations (Jouvert et al. 2004). In addition, CNP has been

shown to regulate cocaine-induced dopamine release and

the expression of immediate early genes in brain neurons

(Thiriet et al. 2001). CNP has been found to improve

learning and consolidation of learning in a passive avoid-

ance paradigm (Telegdy et al. 1999). Interestingly, dopa-

mine has been shown to be one of the mediating

neurotransmitters in the effect of CNP on learning effects

(Telegdy et al. 1999).

In addition, in the present study NPR-B immunoreac-

tivity was observed in brain regions related to general

somatic afferents, including the principal sensory trigemi-

nal nucleus, gracile nucleus, and cuneate nucleus. Although

previous studies have reported the involvement of natri-

uretic peptides in the baroreflex (Thomas et al. 1997), little

information is available regarding the role of CNP/NPR-B

system in general somatic afferents. Further studies are

required to identify the potential physiological relevance of

the NPR-B receptor.

Also, the presence of the NPR-B receptor in the locus

coeruleus suggests that CNP could be involved in the

control of neurons in the dorsal horn in the monkey,

because neurons in the locus coeruleus project to the spinal

cord.

The widespread distribution of NPR-B immunoreactiv-

ity observed in the current study may help establish the

possible functions of the NPR-B receptor in brainstem

nuclei. Further studies are needed to establish the physio-

logical functions of the NPR-B receptor in the brainstem

because the role of CNP as a neurotransmitter and/or

neuromodulator in these nuclei remains to be elucidated.

Conclusion

The present study provides the first detailed map of NPR-B

immunoreactivity in the monkey brainstem. Combined

with previous functional studies, our data support a role for

natriuretic peptides in the central control of cardiovascular

homeostasis. Furthermore, we demonstrated that the NPR-B

receptor is widely distributed throughout brainstem regions

related to other functions, suggesting the involvement of

CNP and the NPR-B receptor in mediating a wide range of

brainstem functions.

Acknowledgments This work was supported by a Grant-in-Aid for

Scientific Research (no. 21-09133) from the Japan Society for the

Promotion of Science.


123

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Documents

Mapping of NPR-B immunoreactivity in the brainstem of Macaca fascicularis