Serotonin in the Rat Sympathetic Gangl on: Microdetermination of Monoamines and Their Metabolites by High-Performance Liquid Chromatographic Electrochemical Detection

Japan. J. Pharmacol, 35, 237-245

Serotonin

in the

Rat

of Monoamines Liquid

237

(1984)

Sympathetic

and

Their

Ganglion:

Metabolites

Chromatographic

Microdetermination by High-Performance

Electrochemical

Detection

Masami NIWA, Keiko KUNISADA, Akihiko HIMENO and Masayori OZAKI Departmentof Pharmacology2, NagasakiUniversitySchool of Medicine, 12-4 Sakamoto-machi,Nagasaki852, Japan Accepted March26, 1984

Abstract•\•\We

attempted

small

fluorescent

intensely

(SCG)

by

lation in

on

evaluating the

the

Wistar

1.19•}0.11

and

performance amount n=8)

SCG

of

of

The

increase

of

capsule

5-HT.

The

mode

decrease

of

was

the

thus of direct the

that

5- HT

connection serotonergic

in

slow 5-HIAA

electrical

the

5-HIAA in the

in

the

prewas

in

n=8,

not

rat and

to

the

case

our

injection

observed

to

content,

whereas

stimulation

SCG,

postganglionic

that

if so,

then

ganglion sympathetic

the

adult were high-

relatively large ng/capsule,

increased

the

(DOPAC)

the of

changes

DA

in

content and

DOPAC. content

increased

support

the

It for

be

would

existence

obtained.

neurons

the and

DOPAC

observed.

can

DA

content

5-HT

the

not

dopamine

(5-HIAA)

significantly were

and

in

without in

compared

sympathetic

of

changes

reverse-phase

acid

was

stimu-

assess

method. A SCG (0.62•}0.16

pargyline

content rat

was

acid by

the

ganglion

electrical

average•}S.E.)

induced-increase

was the

is produced

cell

The

content

Although

between

findings

electrochemical capsule of

pargyline

alter

cell

objective

determined

the

serotonergic

sympathetic

5-hydroxyindoleacetic

5-HT

content not

SIF

SIF

in

functional

and

3,4-dihydroxyphenylacetic

of

changes

serotonergic

with

5-HIAA.

the

reduced.

content,

appear a

5-HIAA did

markedly

DOPAC

and

a

cervical

The

(ng/ganglion,

the

rapidly.

Decentralization

fibers.

respectively,

decreased

of

superior

decentralization

compared

5-HT

detection

and

existence rat

pargyline,

contents

liquid chromatographic 5-HT was detected

of

in the

of

metabolism

0.11•}0.01,

without

content

effects

The rat

the

F) cell

sympathetic

(5-HT)

metabolism.

male

determine

(SI

preganglionic

serotonin

(DA)

to

A

through

demonstrated.

Mammalian sympathetic ganglia seem to possess various types of small intensely fluorescent (SIF) cells, among which the dopaminergic SIF cell is best understood (1, 2). The SIF cells in the sympathetic ganglia are composed of not only dopaminergic cells, but also other types of cells (3). The possible presence of a serotonergic SIF cell system in addition to the catecholaminergic cells in the rat SCG was suggested by the recent morphological studies of Verhofstad et al. (4). Taken in conjunction with earlier data that exogenous serotonin (5-HT) and its analogues stimulate ganglion cells and

modify neuronal transmission (5-7), the question arises as to whether the serotonergic SIF cell is functional in trans-synaptic neuronal transmission in the SCG. We compared the biochemical characteristics of 5-HT metabolism in the rat SCG with that of dopamine (DA) metabolism. The present study was designed 1) to confirm the existence of a 5-HT production system in the rat SCG and 2) to determine whether the activity of the ganglional 5-HT cell depends on preganglionic sympathetic neurons. In addition, our newly developed and sensitive quantitative analytical method of

238

M. Niwa et al.

catecholamines (CAs), 5-HT and their acidic metabolites, 3,4-di hydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5hydroxyindoleacetic acid (5-H IAA), based on high-performance liquid chromatography (LC) with electrochemical detection (EC) is descri bed.

end

of

this

dissected

stimulation, out

plate

on

tissues

residual

pre-

off

the

the

an

(Data

and is

are

amines frozen of

few

hr

metabolites were

10%

(50

and for

were

assayed. in

an

CAs

ice-water

Branson,

KHz,

4

nate,

NaOH and

10

the

of

5-HIAA.

HCIO4

the

five of

added

15

min), ZnSO4-

injected

into

quantitation

of

the 5-HT

ZnSO4-HC104 into

quantitation

to and

the

of

of

homoge-

4•Ž,

injected

of id

vortexing

from

Fifty ƒÊl

an I FI ER

setting

was

rpm,

the

using

a

50 ƒÊl

After

was for

at

was for

homogenates

sec

Thirty

to

3%

CAs,

performed

(SON

supernatant

system

system

30

sec).

homogenates

LCEC and

30

(15,000

of

NaOH

for

added

id

acidic and

was

homogenate.

centrifugation 70 ƒÊl

IAA), HVA)

U.S.A.)

W,

was

residual

standards

homogenizer

185,

the

DOPPA

5-H

bath

probe

(20

(20

internal

Homogenization

5

N

the

(DOPAC,

Model

0.5

as

(5-HT, of

ultrasonic

3,4-

(DOPPA)

N-methyl-5-HT,

indoles

in

solution

3,4-dihydroxybenzylamine

used

respectively.

The 100 ƒÊl

ng/ml),

acid

were

metabolites

metabolites:

(10

ng/ml).

DHB

5-HT

preparations,

homogenized

dihydroxyphenylpropionic

(DHB)

capsule

EDTA-2Na

and

The

of

and

N-methyl-5-HT

ng/ml)

the

amounts

sample

ZnSO4-1.0%

containing

an

ice.

1).

after

and

SCG in

dry

because

amines

tissues

under

isolated

on

Table

of

carefully

washed

some

in

the were

plate

The

critical

given

a

was glass

frozen

contained

Determination Within

capsule

extensively

bath

SCG

and fibers

ice-cooled

were

decapsulation

connective

removed

(•~20).

capsule

the

glass

postganglionic

SCG, on

ice-water

of

were

cooled

the

been

a stereomicroscope and

ganglia a

After

had and

desheathed

Materials and Methods Animals, drug treatments and surgical procedures: Male Wistar rats weighing 250-300 g were fed a standard diet (F-2, Funabashi Farm Co., Chiba, Japan) and water ad libitum and housed at 24°C. These rats were given a 10% sucrose solution orally 24 hr before decapitation because the cereal chow contained tyrosine and DOPA which modify the level of the endogenous metabolites of biogenic amines (8). To confirm the existence of the 5-HT production system in the SCG, the experimental group and the control group were given pargyline, a monoamine oxidase inhibitor (75 mg/kg, i.p.) or the vehicle (0.9% NaCI) alone, respectively. Three groups of five experimental animals were decapitated at 5, 15 and 30 min after pargyline had been given i.p. Increases in the DA and 5-HT contents were measured within a few hr after the decapitation. To determine whether or not the activity of the ganglional 5-HT cell depends on the input from preganglionic neurons, two experiments were done. In the first, the SCG was decentralized by aseptic removal of several millimeters of the right sympathetic chain caudal to the ganglion. The decentralized and sham operated rats were decapitated 7 days after this surgery and ganglia were rapidly removed and placed on a cooled glass plate on ice. Ptosis in the right eye was taken as evidence of decentralization. In the second experiment, electrical monophasic stimulation (10 V, 10 Hz, 1 msec duration) to the right preganglionic nerves was performed after the bilateral SCG and preganglionic nerve were exposed, under conditions of urethane (1.2 g/kg, s.c.) anaesthetization. The stimulation was provided by a bipolar platinum electrode, a stimulator (3F-46, Sanei, Tokyo, Japan) and an isolator (5361, San-ei) for 3 and 10 min, respectively. At the

on

preparations:

fatty

cut

both

placed

ice.

Sample and

and

of

the

LCEC

DOPAC

and

HVA. Isolation natant

of was

alumina

(Wolem

activated

by

Sayre

(9).

pH 8.6 by HCI buffer conical

the

from

the

The

by

Neutral the

of was

of 0.5 transferred

vial The

alumina

by

superon

activity

supernatant

alumina.

residual absorption

method

the addition and then

reaction

activated into

CAs achieved

ml

containing CAs vortexing

Grade

I)

Anton

and

adjusted of to

to

3

M Trisa 0.5 ml

20 were

mg

of

absorbed for

5

min,

5-HT in Rat Sympathetic After

washing

were

eluted

with by

Eighty ƒÊl

of

into

LCEC

the

distilled

water,

100 ƒÊl

the

of

HCIO4

eluent

system

the

0.1

N was

for

CAs

HCI04. injected

quantitation

of

CAs. The

LCEC

6000A

system

pump,

Bondapak 0.39

C18 cm)

reverse

System,

the

the

case

of

5-HIAA

the

and

mobile

buffer,

pH

DOPAC in

were

HVA

and

case

of

pumped

at

temperature.

15.3 tration

20.6

internal

the

Drugs

and

the

0.6

others:

times DA

DA were

HVA

Chemical

carbon the

HCI,

creatinine

NA

bitartrate, from

U.S.A.

Co.)

using

the 3,4-

(DOPPA)

by

catalytic

temperature

and

10%

palladium

on

are

expressed

as

Values The

using

U.S.A.

HT

MO,

room

a catalyst.

differences

obtained WI,

purchased

Chemical

pressure

analyzed

was

acid

average±S.E.

of

run

3,4-dihydroxycinnamic

at

as

with were

5-

Co.,

from

atmospheric

9.0,

respective

Co.,

Dihydroxyphenylpropionic

(Aldrich

nor-

calculated

the

H Br

oxalate,

and

hydrogenation

of

compared

DHB

5-HIAA,

acid

room

concen-

was to

CAs

at

were

The

Chemical

DOPAC

prepared

octyl of

which

sulfate,

was

sodium

and

ratio

in

procedure.

5- HT

Sigma

In

5-HIAA,

ml/min

and

Aldrich

N -methyl-

the

methanol

respectively.

whole

of

5-HT,

mM

of

height

and

HVA.

quantitation

compounds

through

from

0.5 the

standard

authentic

case

9%

compound

peak

in

5-HT

and

retention

min,

+0.55

monochloroacetate

DHB

each

the

at

the

of

rate

The

of

from

a

(NA).

and

M

glassy

electrode

DOPAC

quantitation

the

adrenaline

in

an

working

of

containing

the

and

sulfate

V

0.15

3.5,

of

a

maintained

CAs,

phase,

case

with

quantitation

of

(30•~ and

The

reference

+0.70

quantitation

the

was

Ag/AgCI

a

Bioanaly-

equipped (TL-5).

potential

versus

column U.S.A.)

(LC-4A,

U.S.A.)

electrode

the

phase

electrode

of

injector, ƒÊ-

Assoc., detector

carbon

V

composed

universe

(Waters

electrochemical tical

was

U6K

statistical

between

mean

Student's

t-test.

significance values

was

Results Our 5-H of SCG

IAA

quantitation used

Lackovic was

method

in this et

al.

deproteinized

for

5-HT

study

is based

upon

(10).

First,

the

by

10%

and that

isolated ZnSO4

Ganglion

239

solutions and the homogenate alkalinized by the addition of NaOH. The advantage of this method is that a minute amount of 5-HIAA can be measured because 5-HIAA is more stable in alkaline than in acidic solution. The CAs, DOPAC and HVA are stable in acidic solution; therefore, we divided the ZnSO4 homogenate into two parts, and 0.5 N NaOH was added to one part for the quantitation of the 5-HT and 5-HIAA, and 3% HC104 was added to the other part for the quantitation of the CAs, DOPAC and HVA. Figure 1 and 2 show typical chromatograms obtained from rat SCGs (A) and capsule (B). The DOPAC (No. 1 peak in chromatograms), 5-HT (No. 2), N-methyl-5-HT (No. 3), DHB (No. 4), 5-HIAA (No. 5) and HVA (No. 6) were eluted from a reverse phase ii-Bondapak C18 column in the LCEC system with retention times of 15.0, 16.5, 21.5, 27.0 and 36.0 min, respectively. Under the conditions described in the section on Materials and Methods, a good separation of each compound was obtained. Chromatograms of Fig. 1 were obtained from ZnSO4-HCIO4 homogenates from rat SCGs (A) and capsule (B). The decrease of the peak height of DOPAC (No. 1) and HVA (No. 6) in a chromatogram (A2) obtained from the SCGs of a rat decapitated 15 min after pargyline (75 mg/kg, i.p.) treatment was observed when compared with that (Al) from SCGs of a control rat. The peaks of DOPAC and HVA were not observed in chromatogram (B) obtained from the capsule of the SCG. Although all peaks of the object compounds were present on the chromatogram (Al), calibration curves illustrated in the upper portion of the figure indicate the lack of linearity in 5-HT and 5-HIAA (the dotted lines), unlike the strict linearity of DOPAC and HVA. Standard curves for DOPAC and HVA were linear between 50 pg to 2 ng and 100 pg to 2 ng, respectively. A detection limit of 10-20 pg of DOPAC and 50 pg of HVA was obtained when it was defined as the amount giving a peak twice as high as the fluctuation from the baseline. As mentioned above, 5-HT and 5-HIAA could not be quantified under the conditions for measurement of DOPAC and HVA. Chromatograms for the quantitation of

240

M. Niwa et al.

5-HT and 5-HIAA obtained from ZnSO4NaOH homogenates of rat SCGs are shown in Fig. 2. The pargyline (75 mg/kg, i.p.) treatment increased the peak height of 5-HT

Fig.

1.

Typical

DOPAC combined gram (75

the

mg/kg,

i.p.,

in

15.0,

24.6

curves

of

line)

and upper

and

(P,

portion

of the

DOPAC of

observed,

and

HVA. (1)

chromatograms

treatment. observed

No in

HVA

the

peaks

No unlike

The and

of

chromatogram

times

5-HT

(•Z,

are

illustrated

linearities the

strict

decrease HVA

(6)

A2)

after

DOPAC of

of are the

capsule

were (B).

(B)

the

Peak

and

with

peak height

and

(•£) in of

times

and the

upper

of

was 5-HT

chromatogram

of

and of

of 5-HIAA

5-HT (5).

A2, (2)

The

of dotted

of

not

observed,

from in

21.5

portion

LCEC

and

27.0

5-HT line) figure.

As

illustrated

unlike

decreased

a

a

the

pargyline and

5-HT,

eluted

16.5,

5-HIAA.

The

conditions

column

(•Z,

4,

5-HIAA. the

a 2,

standard);

curves

DOPAC

DOPAC

linearity

height

of

Calibration

of

DOPAC;

5,

were C18

i.p.,

capsule

5-HIAA.

5-HIAA

retention

respectively.

the

under

5-HT

the

mg/kg,

1,

and

phase ƒÊ-Bondapak

min,

the

of

identities:

obtained of

of

(75

(internal

were

5-HT combined

chromatogram

standard);

quantitation

two

pargyline

chromatogram

SCG.

N-methyl-5-HT

strict

(A2)

with

(internal

linearity in

pargyline

and

illustrated

peak

HVA

for

rat,

ganglional of

N-methyl-5-HT

DOPPA

5-HIAA

5-HT

the

control treated

3,

of

Chromatogram

a rat

rat

system in

illustrated

a

min),

reverse

linearities

and the

of

dotted

of

of

control

chromatograms (Al)

chromatograms

Calibration

line)

figure.

(Al,

HVA

Typical

of

5-HT; of

C18

retention

(•£),

SCGs

were

respectively.

dotted

were

DOPAC

with

SCGs

5-

quantitation and

5-HIAA.

15

5,

DOPPA

2.

1,

(internal

phase ƒÊ-Bondapak

Fig. and of

identities:

the

DOPAC,

min,

(•œ),

pargyline

chromatograms

system

5-HIAA

two

chromatogram Peak

for

reverse

DOPAC

of

standard);

These

36.0

chromato-

N-methyl-5-HT

The a

LCEC

and

5- H IAA

heights the

a

(B)

SCG.

conditions

HVA. from

(A2) with

(internal

HVA.

ganglional

treated

and rat

3,

the

and

column

of

6,

rat,

rat

DOPPA

under

eluted

a

min),

5-HT;

and

DOPAC

control

of 15

4,

obtained

a

of Chromatogram

a control

2,

HIAA;

were

of

of

standard);

(Al)

SCGs

capsule

DOPAC;

the

HVA.

SCGs of

the

chromatograms

and

and decreased that of 5-HIAA, 15 min after injection (A2 in Fig. 2). The peak of HVA was not observed as HVA cannot be oxidized at

increased the

(•œ), are The the in the peak

5-HT in Rat Sympathetic

the potential of +0.55 V of a carbon electrode vs. Ag/AgCI reference electrode (11). As in Fig. 1, although we can observe the peak of DOPAC, the calibratory linearity of DOPAC was not observed, as indicated by the dotted line in the calibration curves cited in the upper portion of Fig. 2. Standard curves for 5-HT and 5-HIAA were linear between 50 pg to 2 ng. A detection limit of 20 pg of 5-HT and 5-H IAA was obtained. The CAs, DOPAC, HVA, 5-HT, 5-HIAA contents of SCG are summarized in Table 1. A minute amount of HVA was detected. The 5-HT was found both in the capsule of SCG and the SCG itself. As shown in Table 1, although the 5-HIAA, a main metabolite of 5-HT, was detected in the SCG, 5-HIAA was not measurable or was a smaller value than our minimum detection limit of 20 pg/sample

Ganglion

241

in the rat SCG capsule. Additionally, when we calculated the molecular ratio of DOPAC to DA and 5-HIAA to 5-HT, the former was 0.57•}0.05 (Each value is the average•}S.E. of 8 determinations.) and the latter was 0.10•}0.01. We found that the DOPAC/DA ratio was significantly higher than the 5HIAA/5-HT ratio (P<0.001, Student's ttest). The treatment with pargyline (75 mg/kg, i.p.), a monoamine oxidase inhibitor, in creased the content of DA and 5-HT with a decline of DOPAC, HVA and 5-H IAA. Figure 3A shows that the DA content rapidly increased, and the DOPAC content decreased exponentially. The increase of DA reached a plateau 5 min after pargyline treatment. The decline of HVA was slower than that of DOPAC. HVA was not detected 30 min after

Table 1. Biogenic amines (5-HT. DA and NA) and their acidic metabolites contents in rat SCG and capsule

Results

are the average•}S.E.

of 8 determinations.

(A)

Fig.

3.

Changes

pargyline. The figure. content **P<0

in

amines

Pargyline

contents

of

5-HT (A) .01

DA

(•œ) are and

and

was (•œ),

and indicated

***P<0.001,

(5-HIAA, DOPAC and HVA)

metabolites

injected DOPAC

i.p., (•œ,

5-HIAA after

(B)

(•œ,

dotted

dotted

treatment when

contents and

compared

the line)

line) with

(ng/ganglion) rats

in

were

and (B)

HVA of

pargyline. with

0 time.

in

decapitated

the

(•£,

dotted

figure.

Results

rat

SCG

at

the

time

line) In

are

the

the

(B),

are no

after

illustrated

changes

average•}S.E.

treatment

intervals in

(A)

of capsule of

with

indicated.

5 rats.

of

the

5-HT *P<0.05,

242

M. Niwa et al.

Table 2. Effect of decentralization on biogenic amines (5-HT and DA) and their acidic metabolites (5-HIAA, DOPAC and HVA) contents (ng/ganglion) in rat SCG

Decentralization was achieved by aseptic removal of theright sympathetic chain caudal to the ganglion (RS-decentralized SCG). The decentralized and sham operated rats were decapitated 7 days after surgery. RS: right side, LS: left side. Results are the average•}S.E. of 5 determinations. *P<0.05 when compared with the RS-sham operated SCG.

Table 3.

Effect of electrical stimulation on 5-HIAA, DOPAC and HVA contents (ng/ganglion)

in rat SCG

Electrical stimulation (10 V. 10 Hz, 1 msec duration) to the right preganglionic nerves was performed after the bilateral SCG and preganglionic nerves were exposed following anaesthetization with urethane (1.2 g/kg, s.c.) for 3 and 10 min. RS: right side, LS: left side. Results are the average•}S.E. of 5 determinations. *P<0.02, **P<0.001 when compared with the LS-nonstimulated (control) SCG.

the pargyline treatment. Although the increment of DA was rapid, the 5-HT content increased gradually (Fig. 3B). There were no significant differences between the values at 0, 5, and 15 min. Interestingly, the content of 5-HT in the capsule remained the same at all times after the pargyline treatment. Decentralization of the right side SCG induced a significant decrease of DOPAC content (RS-decentralized SCG) with a small increase in the left, sham-operated side (LSsham operated SCG). Unexpectedly, we observed no changes in the content of 5H IAA both in the decentralized side and the sham-operated side of the SCG (Table 2). This observation is noteworthy compared to the findings on the change of DOPAC content after decentralization. The effects of preganglionic stimulation on the contents of DOPAC, HVA and 5-H IAA are presented in Table 3. Whereas the content of DOPAC increased by 91% and 666%, respectively, at 3 and 10 min after stimulation (10 V.

10 Hz, 1 msec duration) we observed no increase in 5-H IAA content in the stimulatedright side of the SCG (RS-stimulated SCG.) Discussion We attempted to determine whether the serotonergic SI F cell is functional in ganglionic transmission between the preand postganglionic sympathetic neurons. Efforts were made to quantitate trace amounts of 5-HT and 5-HIAA. As described in Figs. 1 and 2, our modifications of the quantitative analysis of biogenic amines and their metabolites led to investigations of endogenous 5-HT systems in the rat SCG. This LCEC method is equally matched in sensitivity to the gas chromatographic-mass spectrometric method (12) and to the radioenzymatic microassay method (13). We measured the 5-HT and 5-HIAA contents in the rat SCG and its capsule separately because the capsule was presumed to have 5-HT storage cells, i.e., the mast cells, and it was technically

5-HT in Rat Sympathetic

difficult to avoid contamination from surfaceagglutinating blood which contains thrombocytic 5-HT, even when the capsulated SCG was extensively washed in an icewater bath. In fact, a relatively large amount of 5-HT existed in the capsule without detection of 5-HIAA. The low value of the ratio of 5-HIAA to 5-HT in the SCG is noteworthy. This is of particular interest in view of the finding that a high ratio of the central serotonergic neuron system was noted in a study concerning the regional brain contents of 5-HT and 5-H IAA (14). According to the data that the ratio is an index of changes in the activity of the monoaminergic system (15), this result suggests that the rat SCG has a low 5-HT neuronal activity. The effect of pargyline (75 mg/kg, i.p.) on the content of DA and DOPAC in this study is in complete accordance with findings in previous reports (16, 17). It has been thought that the turnover rate of DA in the dopaminergic SIF cell of the sympathetic ganglion is the same as the central dopaminergic neuron. The mode of the pargyline-induced 5-HT accumulation and 5-HIAA decrease may indicate low neuronal activity of the 5-HT system in the rat SCG compared with findings in the case of the central serotonergic neuron system (18). However, until more data are obtained with regard to the rate of formation of 5-HT and 5-HIAA calculated from the steady-state of kinetics, the low neuronal activity of the rat ganglionic 5-HT system remains only speculative. When we compared the decreased rate of the DOPAC content induced by pargyline, the ganglional 5-HT content increased gradually. This discrepancy may be explained by the suggestion that the nerve activity of the dopaminergic SIF cell is higher than that of the serotonergic SIF cell. The resulting high DOPAC/DA ratio and the low 5-HIAA/5-HT ratio support this idea, but one might question whether such a difference between dopaminergic and serotonergic metabolism reflects the deamination process of DA and 5-HT. Actually, 5-HT seems to be mainly deaminated by monoamine oxidase (MAO)-A, and the DA is deaminated by MAO-A and MAO-B in the rat brain (19). Moreover, pargyline is an

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inhibitor of MAO-B (20). If such is indeed the case, then the different increases of DA and 5-HT may be based on neuronal activity, since a high dose (75 mg/kg) of pargyline can block both types of MAO (21). However, the present finding that pargyline did not increase 5-HT content in the SCG capsule, an area considered to contain mast cells and/or a small amount of blood, but did increase the 5-HT content of the SCG, supports the idea that 5-HT is produced by the neuronal structure in the rat SCG. We assumed that the 5-HT detected in the capsule was mainly derived from platelets of agglutinated blood, because platelets have only a small amount of tryptophan-hydroxylase (22), the ratelimiting enzyme of the biosynthesis of 5-HT, and take up the 5-HT from the blood and store it in the granules (23). As rat mast cells contain 5-HT (24), these cells are candidates for the origin of capsule 5-HT. The presence of specific receptors for 5-HT on ganglion cells in the SCG (25, 26), and the existence of a 5-HT cell indicated by immunohistochemical studies (4) led to the hypotheses that a serotonergic SIF cell may lie between the pre- and postganglionic sympathetic neurons. We did not detect any decrease of 5-H IAA content in the SCG after decentralization or any increase by preganglionic electrical stimulation (Tables 2 and 3). The serotonergic SIF cell does not seem to be stimulated by acetylcholine from the nerve endings of preganglionic sympathetic neurons. The serotonergic SIF cell may not influence the ganglionic neuronal transmission in the rat SCG. Similarly, unique and isolated serotonergic neurons were found in the rat pancreas (27) and in lamina X of the monkey spinal cord (28). Particularly, the latter spinal interneurons are considered to be one of the cerebrospinalcontacting neurons and to control spinal blood flow through vessel innervation (28). These studies suggest the physiological significance of the serotonergic SIF cell in the rat SCG. The possibility that the serotonergic SIF cell is innervated by the recurrent collateral from the postganglionic noradrenergic neuron cannot be entirely excluded since the ganglion cells have specific receptors for 5-HT (25, 26).

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The changes in DOPAC presented in this study are in accord with data of other workers (17, 29). Electrical stimulation induced a dramatic increase in the DOPAC content, and the elevation was dependent on the time length of stimulation. Thus, measuring the DOPAC content in sympathetic ganglia may be a useful approach to assess the preganglionic sympathetic activity which is controlled by the descending bulbospinal monoaminergic neurons (30). Acknowledgement: We thank M. Ohara for adviceon the manuscriptand K.Katofor manuscript preparation.

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