expression and distribution of gaba and gaba b -receptor in the rat...

Expression and distribution of GABA and GABAB-receptor in the rat adrenal glandKanae Kato, Chieko Nakagawa, Hiroshi Murabayashi and Yukio Oomori

Division of Anatomy and Physiology, Japanese Red Cross Hokkaido College of Nursing, Kitami, Japan

Abstract

The inhibitory effects of gamma-aminobutyric acid (GABA) in the central and peripheral nervous systems and the

endocrine system are mediated by two different GABA receptors: GABAA-receptor (GABAA-R) and GABAB-

receptor (GABAB-R). GABAA-R, but not GABAB-R, has been observed in the rat adrenal gland, where GABA is

known to be released. This study sought to determine whether both GABA and GABAB-R are present in the

endocrine and neuronal elements of the rat adrenal gland, and to investigate whether GABAB-R may play a role

in mediating the effects of GABA in secretory activity of these cells. GABA-immunoreactive nerve fibers were

observed in the superficial cortex. Some GABA-immunoreactive nerve fibers were found to be associated with

blood vessels. Double-immunostaining revealed GABA-immunoreactive nerve fibers in the cortex were choline

acetyltransferase (ChAT)-immunonegative. Some GABA-immunoreactive nerve fibers ran through the cortex

toward the medulla. In the medulla, GABA-immunoreactivity was seen in some large ganglion cells, but not in

the chromaffin cells. Double-immunostaining also showed GABA-immunoreactive ganglion cells were nitric oxide

synthase (NOS)-immunopositive. However, neither immunohistochemistry combined with fluorescent microscopy

nor double-immunostaining revealed GABA-immunoreactivity in the noradrenaline cells with blue-white

fluorescence or in the adrenaline cells with phenylethanolamine N-methyltransferase (PNMT)-immunoreactivity.

Furthermore, GABA-immunoreactive nerve fibers were observed in close contact with ganglion cells, but not

chromaffin cells. Double-immunostaining also showed that the GABA-immunoreactive nerve fibers were in close

contact with NOS- or neuropeptide tyrosine (NPY)-immunoreactive ganglion cells. A few of the GABA-

immunoreactive nerve fibers were ChAT-immunopositive, while most of the GABA-immunoreactive nerve fibers

were ChAT-immunonegative. Numerous ChAT-immunoreactive nerve fibers were observed in close contact with

the ganglion cells and chromaffin cells in the medulla. The GABAB-R-immunoreactivity was found only in

ganglion cells in the medulla and not at all in the cortex. Immunohistochemistry combined with fluorescent

microscopy and double-immunostaining showed no GABAB-R-immunoreactivity in noradrenaline cells with blue-

white fluorescence or in adrenaline cells with PNMT-immunoreactivity. These immunoreactive ganglion cells were

NOS- or NPY-immunopositive on double-immunostaining. These findings suggest that GABA from the intra-

adrenal nerve fibers may have an inhibitory effect on the secretory activity of ganglion cells and cortical cells, and

on the motility of blood vessels in the rat adrenal gland, mediated by GABA-Rs.

Key words: adrenal gland; GABA; GABAB-receptor; ganglion cells; rat.

Introduction

Gamma-aminobutyric acid (GABA) exerts its inhibitory

actions through two distinct types of receptors. The GABAA-

receptor (GABAA-R) is an ionotropic receptor, permeable to

chloride ions, at which the action of GABA is antagonized

by bicuculline (Macdonald & Olsen, 1994). The GABAB-

receptor (GABAB-R) is a metabotropic receptor, blocked by

baclofen, which mediates neuronal responses via the sec-

ond messenger systems regulating calcium and potassium

channels (Bowery, 1989; Bettler et al. 1998).

The adrenal medulla comprises two types of chromaffin

cells, adrenaline and noradrenaline cells, and these cells

secrete large amounts of catecholamines containing adren-

aline and noradrenaline (Ungar & Phillips, 1983). Further-

more, a few large ganglion cells are also present in the

medulla (Oomori et al. 1994; Holgert et al. 1996a,b).

Previous immunohistochemical studies have demon-

strated GABA or glutamate decarboxylase (GAD)-immuno-

reactivity in the chromaffin cells and nerve fibers of the

Correspondence

Yukio Oomori, Division of Anatomy and Physiology, Japanese Red

Cross Hokkaido College of Nursing, Kitami, 090-0011 Japan.

E: [email protected]

Accepted for publication 17 October 2013

Article published online 20 November 2013

© 2013 Anatomical Society

J. Anat. (2014) 224, pp207--215 doi: 10.1111/joa.12144

Journal of Anatomy

adrenal gland (Kataoka et al. 1984; Alho et al. 1986;

Ahonen et al. 1989; Oomori et al. 1993; Iwasa et al. 1998,

1999).

Pharmacological, physiological and molecular data all

point to the presence of GABAA-R and/or GABAB-R in the

adrenal chromaffin cells (Kataoka et al. 1986; Castro et al.

1989, 2003; Ymer et al. 1989). However, although previous

physiological studies have reported the presence of GABAA-

R in rat adrenal chromaffin cells (Busik et al. 1996;

Matsuoka et al. 2008), it remains unclear whether the

GABAB-R exists in the cells of the rat adrenal gland and, if

so, what functional significance it plays.

To clarify these issues, we examined the cellular and neu-

ronal GABA- and GABAB-R-immunoreactive elements and

the co-localization of other bioactive substances and

enzymes, such as neuropeptide tyrosine (NPY), nitric oxide

synthase (NOS) and choline acetyltransferase (ChAT) in the

immunoreactive cells of the rat adrenal gland by light

microscopy.

Materials and methods

Five male Wistar rats (Japan SLC, Shizuoka, Japan; 8 weeks old;

body weight 180–200 g) were used in this study. The animals

received commercial food pellets and water ad libitum. They were

kept under constant conditions (temperature 22 °C, relative humid-

ity 45%, LD 14 h light from 05:00 to 19:00 hours). All experimental

procedures were performed according to the Guidelines for Animal

Care by the Japanese Red Cross Hokkaido College of Nursing.

The animals were anesthetized with ether, and perfused through

the heart with 200 mL of physiological saline and 200 mL of 4%

paraformaldehyde or 0.1% glutaraldehyde plus 4% paraformalde-

hyde in 0.1 M phosphate buffer (PB) pH 7.3. The adrenal gland was

then removed and immersed in the same fixative for 2 h at 4 °C.

After rinsing in PB, the adrenal gland was left overnight in phos-

phate-buffered saline (PBS) containing 30% sucrose at 4 °C. The

adrenal gland was cut at a thickness of 12 lm using a cryostat, and

mounted on glass slides coated with poly-L-lysine (Sigma; St Louis,

MO, USA).

For immunohistochemistry, the sections were incubated with

primary antibodies (Table 1) overnight at 4 °C, followed by

incubation for 2 h with a secondary antibody conjugated with

indocarbocyanine (Cy3) or cyanine (Cy2; Table 1). To identify

noradrenaline cells in the medulla, the cryostat sections were

examined and photographed using a Zeiss fluorescent microscope

equipped with a filter for noradrenaline fluorescence. Fixation

containing 4% paraformaldehyde is suitable for demonstrating

noradrenaline fluorescence in these tissues (Falck & Torp, 1961). In

order to confirm the distribution of noradrenaline cells or adrena-

line cells, we used both the formaldehyde-induced fluorescence

(FIF) method for noradrenaline cells and phenylethanolamine N-

methyltransferase (PNMT) immunohistochemistry for adrenaline

cells in the same sections of the rat adrenal medulla. For immuno-

histochemistry and FIF, the cryostat sections were photographed

by fluorescence microscope and then immunostained by the

primary antibodies.

For double-immunostaining, the sections were incubated with a

mixture of two primary antisera (GABA/ChAT, GABAB-R/NOS,

GABAB-R/NPY)-raised different species for 12 h at 4 °C. The immuno-

reacted sections were rinsed in PBS and then incubated with a mix-

ture of secondary antibodies conjugated with Cy3 or Cy2. In order

to show double-staining of GABA/NPY, GABA/NOS, the elution

technique of Nakane (1968) was used. The sections were first incu-

bated with GABA antiserum and photographed; the antibody was

then eluted and finally incubated with NPY or NOS antiserum,

respectively.

The specificity of the immunohistochemical staining was con-

firmed by replacing the primary antibodies with normal rabbit

serum, and by using diluted antiserum pretreated with adequate

Table 1 List of primary antisera and secondary fluorescence conjugated antisera used for immunohistochemistry in the present study.

Host animals Dilution Catalogue no. Source

Primary antisera

ChAT Goat 1 : 250 AB144p Chemicon International, Temecula,

CA, USA

GABA Rabbit 1 : 5000 A-2052 SIGMA BIO SCIENCES, MO, USA

GABAB-R Guinea pig 1 : 4000 AB1531 Chemicon International

NOS Rabbit 1 : 4000 B220-1 EURO-DIAGNOSTICA, Beijerinckweg,

the Netherlands

NPY Rabbit 1 : 5000 6730-0204 Biogenesis, England, UK

PNMT Sheep 1 : 5000 AB146 Chemicon

Secondary antisera

Anti-goat IgG Cy2 1 : 100 705-225-147 Jackson ImmunoResearch,

West Grove, PA, USA

Anti-guinea pig IgG Cy3 1 : 250 706-165-148 Jackson ImmunoResearch

Anti-rabbit IgG Cy2 1 : 100 711-225-152 Jackson ImmunoResearch

Anti-rabbit IgG Cy3 1 : 250 711-165-152 Jackson ImmunoResearch

Anti-sheep IgG Cy3 1 : 250 713-165-147 Jackson ImmunoResearch

ChAT, choline acetyltransferase; Cy2, cyanine; Cy3, indocarbocyanine; GABA, gamma-aminobutyric acid; GABAB-R, GABAB-receptor;

NOS, nitric oxide synthase; NPY, neuropeptide tyrosine; PNMT, phenylethanolamine N-methyltransferase.


GABA- and GABAB-receptor-immunoreactivities in the rat adrenal gland, K. Kato et al.208

antigen (5–5.7 lg mL�1) for 24 h at 4 °C. No immunostaining was

observed under this condition.

Results

In the cortex, no GABA-immunoreactivity was seen in the

cortical cells. GABA-immunoreactive nerve fibers were seen

in the blood vessels under the capsule and in high numbers

in the superficial cortex (zona glomerulosa) compared with

other cortex areas. GABA-immunoreactive nerve fibers were

both associated and not associated with the blood vessels

(Fig. 1). In addition, some GABA-immunoreactive nerve

fibers ran through the cortical cells. Double-immunostain-

ing with GABA and ChAT antibodies revealed the GABA-

immunoreactive nerve fibers in the cortex were ChAT-im-

munonegative (Fig. 2A,B). Some GABA-immunoreactive

nerve fibers and bundles ran through the cortex and

divided into thinner nerve fibers in the medulla.

Numerous ganglion cells in the medulla were GABA-im-

munonegative and had long cytoplasmic processes and a

large nucleus in the cytoplasm. Ganglion cells were located

mainly in the periphery or in the center of the medulla, and

sometimes in the juxtamedullary cortex (zona reticularis).

Double-immunostaining with GABA and NOS antibodies

showed some of the GABA-immunoreactive ganglion cells

to be NOS-immunopositive (Fig. 3A,B), but no GABA-

immunoreactivity was found in the chromaffin cells.

Immunohistochemistry combined with FIF showed no

GABA-immunoreactivity in noradrenaline cells with blue-

white fluorescence (Fig. 4A,B). Double-immunostaining

revealed no GABA-immunoreactivity in adrenaline cells

with PNMT-immunoreactivity (Fig. 4C,D). In some cases, a

few GABA-immunoreactive nerve fibers were seen running

into the medulla. However, these GABA-immunoreactive

nerve fibers did not closely appose the chromaffin cells and

finally reached the ganglion cells in the medulla. GABA-

immunoreactive nerve bundles and nerve fibers were found

in clusters of the large ganglion cells or in single ganglion

cells in the medulla, and were in close contact with the gan-

glion cells. Double-immunostaining with GABA, NOS and

NPY antibodies revealed the GABA-immunoreactive nerve

fibers in the medulla were in close contact with the NPY-

immunoreactive ganglion cells, and in even closer contact

with NOS-immunoreactive ganglion cells (Fig. 5A,B). In the

medulla, NPY-immunoreactive ganglion cells (40–60 lm in

diameter) were present in large clusters, while NOS-immu-

noreactive ganglion cells (30–40 lm in diameter) were pres-

ent as single cells or in small clusters. Furthermore, a few

GABA-immunoreactive nerve fibers were ChAT-immuno-

positive, while most of the GABA-immunoreactive nerve

fibers were ChAT-immunonegative (Fig. 6A,B). Numerous

ChAT-immunoreactive nerve fibers without GABA-immuno-

Fig. 1 Fluorescent micrograph of gamma-aminobutylic acid (GABA)

immunoreactivity in the rat adrenal cortex. GABA-immunoreactive

nerve fibers are seen along the blood vessels (V) and among the corti-

cal cells. C, capsule; Co, cortex; V, blood vessel. Scale bar: 30 lm.

A B

Fig. 2 Fluorescent micrographs of double-

immunostaining with gamma-aminobutyric

acid (GABA) (A) and choline acetyltransferase

(ChAT) (B) antibodies in the same section of

the rat adrenal cortex. GABA-immunoreactive

nerve fiber (single arrow) is ChAT-

immunonegative, while ChAT-immunoreactive

nerve fiber (double arrows) is GABA-

immunonegative. Scale bar: 60 lm.


GABA- and GABAB-receptor-immunoreactivities in the rat adrenal gland, K. Kato et al. 209

reactivity were in close contact with the ganglion cells and

chromaffin cells in the medulla.

In the cortex, no GABAB-R-immunoreactivity was seen in

the cortical cells, whereas in the medulla, GABAB-R-immu-

noreactivity was observed in both large and small ganglion

cells, but not in chromaffin cells or nerve fibers (Fig. 7). In

the medulla, immunohistochemistry combined with FIF

showed no GABAB-R-immunoreactivity in the noradrenaline

cells with blue-white fluorescence (Fig. 8A,B). Double-

immunostaining showed no GABAB-R-immunoreactivity in

the adrenaline cells with PNMT-immunoreactivity (Fig. 8C,

D). Immunoreactivity was observed as fine dots on the

A B


immunostaining of gamma-aminobutyric acid

(GABA) (A) and nitric oxide synthase (NOS) (B)

antibodies in the same section of the rat

adrenal juxtamedullary cortex. GABA-

immunoreactive ganglion cells (asterisks) are

NOS-immunopositive. Scale bar: 30 lm.

A B

C D

Fig. 4 Fluorescent micrographs of

formaldehyde-induced fluorescence (FIF) (A)

and immunostaining of gamma-aminobutyric

acid (GABA) (B) antibody, double-

immunostaining of phenylethanolamine

N-methyltransferase (PNMT) (C) and GABA

(D) antibodies in the same section (A and B,

C and D) of the rat adrenal medulla. A few

GABA-immunoreactive nerve fibers (arrows)

are found in the medulla (B, D). However, no

GABA-immunoreactivity is seen in

noradrenaline cells (NA) showing blue-white

fluorescence and in adrenaline cells (A)

demonstrating PNMT-immunoreactivity (A

and B, C and D). Scale bar: 40 lm.



membrane of the ganglion cells. Double-immunostaining

with GABAB-R and NPY antibodies showed the GABAB-R-

immunoreactive ganglion cells were NPY-immunopositive

(~50% of ganglion cells; Fig. 9A–D). Large NPY-immunore-

active ganglion cells were present as clusters in the medulla.

Similarly, some GABAB-R-immunoreactive ganglion cells

were also NOS-immunopositive, while others were NOS-im-

munonegative (Fig. 9E,F). In the medulla, the NOS-

immunopositive ganglion cells were smaller than the

NPY-immunoreactive ganglion cells.

In the control, immunohistochemical staining was con-

firmed by replacing the primary antibody with normal rab-

bit serum. No immunostaining was observed in the control

sections of the rat cortex and medulla (Fig. 10A,B).

Discussion

In the present study, no GABA- or GABAB-R-immunoreactiv-

ity was observed in the adrenaline cells or noradrenaline

cells, and no GABA-immunoreactive nerve fibers were

observed in close contact with the chromaffin cells in the

rat adrenal medulla. It is therefore likely that neither GABA

nor GABAB-R is expressed in the chromaffin cells of the rat

adrenal medulla. In contrast, previous studies have shown

the presence of GABA- or GAD-immunoreactive chromaffin

cells in various mammals (Alho et al. 1986), including mice

(Oomori et al. 1993; Iwasa et al. 1998, 1999), and close

apposition of GABA-immunoreactive nerve fibers to chro-

maffin cells in the adrenal medulla of both dogs and mice

(Alho et al. 1986; Oomori et al. 1993; Iwasa et al. 1998,

1999). Furthermore, GABA-R agonists bound to the plasma

membranes of bovine chromaffin cells have also been

detected (Alho et al. 1986). A GABA-R agonist has been

shown to inhibit the release of catecholamines from canine

adrenal chromaffin cells elicited by nicotinic receptor stimu-

lation (Kataoka et al. 1986). These results suggest that

GABA exists in adrenal chromaffin cells and that

A B



(GABA) (A) and nitric oxide synthase (NOS) (B)

antibodies in the same section of the rat

adrenal medulla. Numerous GABA-

immunoreactive nerve fibers are in close

contact with GABA-immunonegative ganglion

cells (A) (asterisks). NOS-immunoreactivity is

seen in these ganglion cells (B) (asterisks).

Scale bar: 30 lm.

A B



(GABA) (A) and choline acetyltransferase

(ChAT) (B) antibodies in the same section of

the rat adrenal medulla. Few GABA-

immunoreactive nerve fibers (arrowhead) are

ChAT-immunopositive, while some GABA-

immunoreactive nerve fibers (arrows) are

ChAT-immunonegative. Numerous ChAT-

immunoreactive nerve fibers are GABA-

immunonegative. Scale bar: 50 lm.

Fig. 7 Fluorescent micrograph of gamma-aminobutyric acidB-receptor

(GABAB-R)-immunoreactivity in the rat adrenal medulla. A cluster of

GABAB-R-immunoreactive ganglion cells is found in the medulla. Scale

bar: 50 lm.



intra-adrenal nerve fibers may mediate the secretory activity

of the chromaffin cells via the GABA-R. On the other hand,

previous physiological studies have indicated that GABAA-R

but not GABAB-R exists in the chromaffin cells of the rat

adrenal medulla (Busik et al. 1996). Analysis of physiologi-

cal, molecular and immunohistochemical data has revealed

the expression of GAD, vesicular GABA transporter, and

GABAA-R mRNA and proteins in rat adrenal chromaffin

cells, but no GAD-immunoreactive nerve fibers in the

medulla (Matsuoka et al. 2008). Adrenal chromaffin cells

are generally regarded as homologs of sympathetic gan-

glion cells and are innervated by preganglionic neurons

(Ungar & Phillips, 1983). In fact, our previous studies have

demonstrated the presence of dense GABA-immunoreactive

nerve fibers in adrenaline cells, but not noradrenaline cells,

and in ganglion cells of the mouse adrenal medulla

(Oomori et al. 1993; Iwasa et al. 1998, 1999). Thus, although

chromaffin cells secretion is regulated by neuronal ele-

ments, it is enigmatic that chromaffin cell secretion in the

rat is controlled via the GABAA-R alone, with no GABA

innervation.

The present study revealed that GABA-immunoreactive

nerve fibers were in close contact with GABAB-R-immunore-

active ganglion cells in the rat adrenal medulla. These

results suggest that GABA from intra-adrenal nerve fibers

may have an inhibitory effect on the ganglion cells via the

GABAB-R. Our previous electron microscopic study revealed

that GABA-immunoreactive nerve fibers were in close

apposition to the intra-adrenal ganglion cells and the post-

synaptic membrane specialization of the ganglion cells in

the mouse adrenal gland (Oomori et al. 1993). Other

physiological studies have shown that GABA-produced

depolarization depends on the resting membrane potential,

and that GABA-induced depolarization inhibited synaptic

transmission in the ganglion cells of the autonomic ganglia

(DeGroat, 1970; Adams & Brown, 1975). Furthermore, inhi-

bition of the GABAB-R in neurons was achieved mainly via

modulating the release of neurotransmitters from presyn-

aptic terminals and hyperpolarizing the postsynaptic mem-

branes (Bowery et al. 2002). It is probable, therefore, that

GABA from the intra-adrenal nerve fibers exerts an inhibi-

tory effect on the secretory activity of the ganglion cells via

pre- and postsynaptic GABAB-R.

In the present study, numerous GABA-immunoreactive

nerve fibers without ChAT-immunoreactivity were in close

contact with the ganglion cells in the medulla, while numer-

ous ChAT-immunoreactive nerve fibers were in close contact

with the ganglion cells in general. In contrast, a previous

study revealed the co-localization of GABA and acetylcholin-

esterase in the intra-adrenal nerve fibers of the mouse, sug-

gesting that GABA and acetylcholine are co-localized in the

same nerve fibers (Iwasa et al. 1999). Another previous

study reported differences in the frequency and distribution

of GAD-immunoreactive nerve fibers in the adrenal gland

of various mammals (Alho et al. 1986). Thus, this discrep-

ancy in the co-localization of GABA and acetylcholine in the

intra-adrenal nerve fibers might be due to interspecies dif-

ferences. Previous studies have also described acetylcholines-

terase activity, ChAT and vesicular acetylcholine transporter

immunoreactivity in the intra-adrenal nerve fibers around

the ganglion cells and chromaffin cells in the adrenal gland

of both rats and mice (Oomori et al. 1994; Holgert et al.

A B

C D

Fig. 8 Fluorescent micrographs of

formaldehyde-induced fluorescence (FIF) (A)

and immunostaining of gamma-aminobutyric

acidB-receptor (GABAB-R) (B) antibody,

double-immunostaining of

phenylethanolamine N-methyltransferase

(PNMT) (C) and GABAB-R (D) antibodies in the

same section (A and B, C and D) of the rat

adrenal medulla. GABAB-R-immunoreactivity

is seen in the ganglion cells (asterisks) in the

medulla (B, D). No GABAB-R-immunoreactivity

is observed in noradrenaline cells (NA)

demonstrating blue-white fluorescence and in

adrenaline cells (A) demonstrating PNMT-

immunoreactivity (A and B, C and D). Scale

bar: 40 lm.



1996b; Iwasa et al. 1999; Murabayashi et al. 2009). GABA

from the intra-adrenal nerve fibers may have an inhibitory

effect on cholinergic transmission via postsynaptic GABAB-R,

and on GABA release from the nerve fibers via the presyn-

aptic GABAB-R as an autoreceptor. In the present study,

some GABAB-R-immunoreactive ganglion cells were NOS-

immunopositive, while others were NPY-immunopositive.

Studies involving the rat adrenal gland have reported two

types of ganglion cell: large ganglion cells showing NPY-,

tyrosine hydroxylase- and dopamine b-hydroxylase-immuno-

reactivity; and small ganglion cells exhibiting vasoactive

intestinal polypeptide- and NOS-immunoreactivity (Oomori

et al. 1994; Holgert et al. 1996a,b). These findings imply

that GABA from the nerve fibers may also inhibit the

A B

C D

E F


immunostaining of gamma-aminobutyric

acidB-receptor (GABAB-R) (A, C, E), and

neuropeptide tyrosine (NPY) (B, D) and nitric

oxide synthase (NOS) (F) antibodies in the

same section (A and B, C and D, E and F) of

the rat adrenal medulla. GABAB-R-

immunoreactive ganglion cells (A) (asterisks)

are all NPY-immunopositive (B) (asterisks).

GABAB-R-immunoreactive ganglion cells (C)

(asterisks) are all NPY-immunonegative (D)

(arrows). GABAB-R-immunoreactive ganglion

cells (asterisks) are all NOS-immunopositive (F)

(asterisks). Scale bar: 50 lm.

A B

Fig. 10 Fluorescent micrographs showing

replacement of the primary antibody with

normal rabbit serum in the sections of the rat

adrenal medulla (A) and cortex (B). No

immunoreactivity is seen in the cortex (A) and

in the medulla (B). C, capsule; Co, cortex; M,

medulla; V, blood vessel. Scale bar: 60 lm.



release of nitric oxide and vasoactive intestinal polypep-

tide or of NPY and catecholamines from the terminals of

the ganglion cells via the GABAB-R.

In this study, GABA-immunoreactive nerve fibers were

both associated and not associated with blood vessels in the

rat adrenal cortex, while no GABAB-R-immunoreactivity was

found in the vessels or in the cortical cells. These findings

suggest that GABA from the intra-adrenal nerve fibers may

mediate both the vasodilation and the secretion of cortical

hormones from cells in the rat adrenal cortex via GABA-Rs

other than GABAB-R. However, the direct effect of GABA

on smooth muscles is not known. In fact, previous studies

have reported that GABA exerts an inhibitory effect

through the GABA-R located on the adrenergic nerve termi-

nals innervating peripheral blood vessels (Starke & Weitzell,

1980) and that GABA nerve fibers innervated blood vessels

(Imai et al. 1991). Furthermore, the GABAA-R in the glome-

rulosa cells of the adrenal cortex has been shown to inhibit

aldosterone secretion (Kenyon et al. 1999).

Because the present study revealed the GABA-immunore-

active ganglion cells in the rat adrenal gland, GABA-immu-

noreactive nerve fibers in the rat adrenal gland may be of

both intrinsic and extrinsic origin. This study also showed

that intra-adrenal GABA-immunoreactive nerve fibers were

mainly ChAT-immunonegative. It is well known that the

neurons that innervate the adrenal medulla are located in

the intermediolateral horn of the spinal cord (Kesse et al.

1988). Thus, GABA nerve fibers in the rat adrenal gland

may originate mainly from all other neurons except those

in the intermediolateral horn of the spinal cord. However,

the precise origin of the extrinsic immunoreactive nerve

fibers in the rat adrenal gland has not yet been established.

There have been reports of GAD- or GABA-immunoreactive

neurons in the spinal cord (McLaughlin et al. 1975; Hunt

et al. 1981; Barber et al. 1982; Fuji et al. 1985; Ito et al.

2007) and the intestine (Jessen et al. 1986; Saito & Tanaka,

1986; Davanger et al. 1987; Hills et al. 1987; Furness et al.

1989; Sang & Young, 1998). Taken together, GABA-

immunoreactive nerve fibers in the rat adrenal gland may

originate from the extra-adrenal neurons running along

blood vessels and partly from intrinsic ganglion cells.

Acknowledgement

The authors would like to thank Dr Sharon J.B. Hanley (Department

of Reproductive Endocrinology and Oncology, Hokkaido University

Graduate School of Medicine) for helpful suggestions and manu-

script corrections.

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expression and distribution of gaba and gaba b -receptor in the rat...

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