Evidence for the localization of 5HT1A binding sites on serotonin containing neurons in the raphe dorsalis and raphe centralis nuclei of the rat brain

Neurochem. Int. Vol. 7, No. 6, pp. 1061-1072, 1985 Printed in Great Britain

0197-0186/85 $3.00 +0.00 Pergamon Press Ltd

RAPID IMPORTANT PAPER EVIDENCE

FOR THE LOCALIZATION

CONTAINING

NEURONS

OF 5HTIA BINDING

IN THE RAPHE DORSALIS

SITES (~ SEROTONIN

AND RAPHE CENTRALIS

NUCLEI

OF THE RAT BRAIN Dinah Weissmann-Nanopoulos, Yannick D6partement

Demassey

de Pharmacologie,

Centre 111,

Evelyne

Mach,

Jocelyne

and Jean-Franqoie

Unit6 de Neuropharmacologie

de Recherches

route de Noisy,

Magre,

Pujol

Roussel

Uclaf

93230 Romainville,

FRANCE

(Received 12 July 1985! accepted 17 July 1985) Abstract

:

i Precise anatomical distribution of 5HTI binding sites has been nvestlgated in the nuclel rapne dorsalis, rathe. ~gnt~alls and locul caeruleus or the rat braln. An orlg~nal pattern or alsnrlounlon was oomervea in the raDhe nuclei, closely correlated %o the already known distribution of 5HT gqn%alning elements%.Thls pat%ernL more pronqunceB w h e n 5H~l%.sitgs we.re lahellea, qompletely ulsappear~ after leslonlp 4 b x ~ , T D M T . ~ n ~ c a n l n g une pre@ence oz ~nls subtype of 5HT1 binalng sines o n 3~T onnnalnlng neurons. Zt is postulated that these 5HTIA sites corresponu in these rapne nuc&el to 5HT aunoreceptors. INTRODUCTION Previous of

studies have already

5HT1 binding

after

established

sites could be investigated

incubation

of rat brain

slices with tritiated

8-hydroxy-2(diNl3H]propylamino)-tetralin range

(Biegon

1983 of

several

by

previous

allowing

of

little

is

coupling

this

structures perikarya, been

these about

if

of

study the precise

shown to occur after

tryptamine

(5,7 EHT)

1974 ; BjSrklund

distribution

because

and/or

et el.,

and the existence

differences

in the

of selective

ligands

1984

; Pedigo

et al.,

of

putative

5HTI receptors

localization

and their

1981).

functional

to correspond

of 5HT1 binding

of frontal caeruleus of

fibers,

brain

their high density

to 5HT

1061

raphe

These

three

1972

of 5HT of

analy-

in the

containing

denervation

administration

and Lachenmayer,

1974).

sites was

sections

of the rat brain.

and the selective

intraventricular

(Baumgarten

et al.,

; Deshmukh

1983).

and locus

chosen

et al.,

or

nanomolar

sites were also documented

~xistence

subtypes

cellular

radioautography

raphe centralis were

(Pazos

1984

anatomical

the

(3H-5HT)

the

labelling

them have been postulated

(Gozlan et al.,

dendrites

and

different their

some

by quantitative

dorsalis,

subtypes

distribution

radioautography

serotonin in

et al.,

of this

demonstrating

differentiation

known even

autoreceptors In

~ Marcinkiewicz

(A, B and C) of 5HT1 binding

these

concerning

(3H-PAT)

The selectivity

observations,

their

However

1982

1983).

subtypes

distribution

sed

et al.,

; Palacios,

that the regional by quantitative

which has

5,7dihydroxy-

; Baumgarten

et al.,

1062

Rapid Important Paper

EXPERIMENTAL I. Q u a n t i t a t i v e In

vitro

developed

radioautogra~h[

binding

by

Unnerstall

and

al.,

1982).

E a c h b r a i n was

et

-40°C,

then

-15°C.

Sections

for

24

cut hours

the

strips section

were

placed

each

to

taneous

manipulation

designed

one

tanks

minutes

Tris-HCl 20°C

(0.01%),

(CEA,

for

minutes

specific (I~M)

84

posed

buffer

Ci/mole was

for

a 3H-sensitive

Exposure

3 and 6 w e e k s

of

carefully the

were

film.

used

were

to

using

The the

interest

sections to

the

tions

slice.

Comparisons

parametric 2.

anatomical Results

and

system

505 A M E R S H A M ) Radioautographs

(IBAS II. K O N T R O N

axis

for a l i g n i n g

were

+

the serial

for e a c h b r a i n by r e f e r i n g on

estimated

tissue

For

the o r i g i n of the s t r u c t u r e

observed as

(BOSCH)

(Polyvar-Reichert-Jung).

selected

parameters

values

durations

to a v o i d o v e r e x p o s u r e

SEM

the c r e s y l - s t a i n e d mean

site c o n c e n t r a -

(4 to 5 b r a i n s

statistically

in each

a n a l y s e d by u s i n g

t test.

study

were

and glutaraldehyde same b u f f e r

containing

mean

then ap-

incubated with

These

(RPA 501

anterior

was

of

for

determinations.

containing

expressed

ligand/mg

of

Immunohistochemical

binding the

are

in fmoles of f i x e d

group).

structure)

5HT

s l i c e was u s e d

performed with a video camera

slice

Non

in K o d a k X - o m a t i c

3H-5HT.

image analysis

of the p o s t e r o

the

times air. cold

for s e c t i o n s

l u c i d a of a m i c r o s c o p e

(zero p o i n t

three

sections were

in o r d e r

3H-micro-scales

acid

; 2 nM) or

staining.

: dried

and 6 w e e k s

was

for 60 mi-

s e c t i o n s by a d d i n g

(LKB) and e x p o s e d

of r a d i o a c t i v e

the i n i t i a l

23 C i / m m o l e

of

for 30

ascorbic

then washed

incubated with

getting

camera

classical

adjacent

2

a computerized

image

containing

violet

after pre-experiments

calibration

e a c h r e g i o n examined, of

were

Autoradiographic

analysed by

ultrofilm

in s p e c i a l l y

and d r i e d by c o l d p u l s e d

radioauto~ra~h~

for t h o s e

chosen

for

electronics). coupled

times

(NEN,

third consecutive

after a cresyl

quantitative

3H-PAT

A

corres-

the s i m u l -

and w a s h i n g

incubated

C a C I 2 (4mM),

consecutive

medium.

immersed

of and

By s e l e c t i n g

then a l l o w e d

and then

slices w e r e

on

Series

II0 s e c t i o n s

incubation

;

microtome

slices w e r e p r e - i n c u b a t e d

3H-5HT buffer,

1981

and s t o r e d at

(24x30cm).

the

frames w e r e

; pH 7.6)

The

estimated

cassettes. were

; 2nM).

The

cryostat incubation.

frame

containing

and

incubating

against and

(170 mM

(10~M),

characterisation

Procedure

a

of that

al.,

in i s o p e n t a n e

coversiips

for

to p l a c e

studies,

the same b u f f e r

et

l e n g t h s of a d h e s i v e p o l y m e r

pre-incubation,

in ice cold T r i s - H C l

the

anatomical

use

steel

was p o s s i b l e

For the b i n d i n g

in

binding

in

until

of 220 c o v e r s l i p s .

pargyline,

3H-PAT

coated

on a d e q u a t e

for the s i m u l t a n e o u s

slices.

in

of 20~m w i t h

a stainless

it

Palacios

b r a i n on one side of the frame w h i c h

the a t t a c h e d at

on

200~m,

-80°C

is a m o d i f i c a t i o n

;

f r o z e n by i m m e r s i o n

onto gelatin

fixed

used

1979

sections

at

were

ponding

5

then

coverslips

one

nutes

in s e r i a l

Kuhar,

were mounted

sites.

: the m e t h o d

(Young

at

-20°C

experiments

Kuhar

at

adjacent

PROCEDURES

of 5HTI b i n d i n g

staining

fixed

(0.1%)

they were

neurons

with

:

sections

for 12 h o u r s in p h o s p h a t e treated

adjacent

to t h o s e u s e d

buffer

(0.2M,

p H 7.4).

for i m m u n o h i s t o c h e m i c a l

antibodies

for the

in a s o l u t i o n of p a r a f o r m a l d e h y d e

directed against

5HT

After

(4%)

rinsing

localization (Steinbusch

1978 ; S t e i n b u s c h , 1981) and s u b s e q u e n t s t a i n i n g b y the S t e r n b e r g e r ' s (DAKO PAP KIT) ( S t e r n b e r g e r et al., 1970).

in

of 5HT et al., method

Rapid Important Paper

3. Lesions of 5HT containing neurons, were performed by intraventricular injection of 5,7 DHT (creatinine sulfate, SIGMA ; 75~g of base) dissolved in 10pl of Merles' solution containing 0.1% of ascorbic acid injected in each lateral ventricle (coordinates t At 7 mm ; L t 3 mm~ Ht 1.4 mm in the K6nig and Klippel references (K~nig and Klippe1,1967 ~ The rats were pretreated with nomifensine (HOECHST, 10 mg/kg i.p.) 30 minutes before administration of the cytotoxic. Sham operated animals were injected with the same volume of the vehicle. All the rats were sacrificed by decapitation 15 days after lesioning. RESULTS Nucleus Rathe Dorsalis (NRD) t the analysis of the density of 5HT1 binding sites along a postero anterior axis revealed an original pattern of distribution in the NRD of control animals. When detected by incubation with 3H-5HT or 3H-PAT the density of sites was the lowest in the posterior portion of the nucleus, then increased to a maximum at a level corresponding to the anterior third of the nucleus (Fig. 1). The mean absolute difference between the extreme densities of sites were highly significant (p < 0.001). However the relative increase in the density of sites measured in the anterior portion as compared to the posterior portion was more pronounced when slices were incubated with 3H-PAT (412 % + 48 as compared to the lowest value) than when incubated with 3H-5HT (185 % + 15). After 5,7 DHT treatment both 3H-5HT and 3H-PAT labelled sites were significantly reduced (Fig. 1). Three main observations appeared characteristic t (1) the density of sites labelled by 3H-5HT was only partially reduced(from -37% to -60% of the corresponding sham values) ~ (2) the 3H-5HT binding sites remaining after 5,7 DHT lesion did not present the postero-anterior differential distribution observed in sham operated animals (3) the reduction of 3H-PAT labelled sites (from -70% to -90% of the control values) was much more pronounced than that of 3H-5HT labelled sites in all sections examined. As shown in figure 2 this significant decrease in the density of 5HT1 binding sites was associated with an almost complete disappearance of 5HT immunoreactive fibers or perikarya in the NRD. Nucleus Raphe Centralis (NRC) t as shown in figure 3 the three major results observed in the NRD were also seen but to a lesser extent in the NRC z the existence of a postero-antsrior distribution of 5HTI sites with a higher density in the anterior third of the nucleus which was more pronounced when sites were labelled with 3H-PAT (202% + 13 as compared to the lowest value) than after 3H-5HT labelling (132% + 15) I the almost total disappearance of this postero-anterior distribution after lesion by 5,7 DHT ; a greater decrease in the binding sites labelled by 3H-PAT than by 3H-5HT in lesioned animals. Finally the calculated mean density of 5HT1 sites labelled by either ligand in the NRC was inferior to that measured in the NRD (the mean 3H-5HT density was 119.66 + 5.25 fmolee/mg of tissue in the NRD and 50.66 + 2.2 fmoles/mg of tissue in the NRC 7 the mean 3H-PAT density was 99.78 ~ 10.2 fmoles/mg of tissue in the NRD and 23.26 + 1.65 fmoles/mg of tissue in the NRC).

1063

1064

Rapid Important Paper

LOCUS that

Caeruleus

observed

incubation tissue).

with

the

reduction

3H-5HT

In c o n t r a s t

incubation of

(LC)

(13.81

density of

: a high

in the p o s t e r i o r

5HTI

of

(calculated

a very

+ 1.28

was

sites

of

of

5HTI

binding

of the N R D w a s mean

low density

fmoles/mg

sites

binding

density part

of

value

observed

in this

was

in

seen

:

labelling

tissue).

sites

measured 82.62 was

comparable

in the + 5.06

found

structure

5,7 DHT

treated

and

fmoles/mg

after

No postero-anterior

to.

LC a f t e r

3H-PAT

variation

no s i g n i f i c a n t

animals

(Fig.

Figure 1 : Postero-anterior d t s t r f b u t i o n of 3H-gHT and 3H-PAT bindtng sttes in the nucleus raphe dorsalis. Each bar represents the mean + SEN (4 animals) of the density of 3H-SHT or 3H-PAT binding sites measured at the corresponding level of a p o s t e r o - a n t e r i o r dist r t b u t t o n . The d e f i n i t i o n of anatomical sampling is given in the upper part of the figure with reference to the Paxinos' rat brain ATLAS (Paxtnos and WatSon, 1982). Open bars correspond to sham animals; hatched bars correspond to 5,7 OHT lesioned rats. *p < 0 . 0 5 ; **p <0.01. Ft~ure 2 : Radtoautographtc l o c a l i z a t i o n of 3H-SHT and 3H-PAT high afflntty binding sttes in the mesencephalic raphe nuc]ei of sham operated animals and 5,7 DHT treated rats. I n t e n s i t y of l a b e l l i n g is indicated by ustng a color code in which grey values of corresponding radloautographs are transformed according to a gtven color scale. The same color code was used to t r a n s f o m the Images obtained in the nucleus raphe dorsalts by imunohistechemlcal staining of 5HT containing neurons with a n t t - ~ T antibodies. The color scale corresponds from black to white to the following upper concentration l i m i t s : for 3H-PAT incubated sections : 1.0 ; 11.7 ; 21.1 ; 32.9 ; 44.7 ; 63.5 ; 87.0 ; 123.5 fmoles/mgt ; for 3H-gHT Incubated sections : 10.8 ; 30.4 ; 43.4 ; 56.0 ; 74.0 ; 100.0 ; 130.0 ; 182.6 fmoles/nKjt. Figure 3 : Postero-anterlor d i s t r i b u t i o n of 3H-gdT and 3H-PAT btndtng sites tn the nucleus raphe c e n t r a l t s . Open bars correspond to sham operated animals; hatched bars correspond to 5,7DHT treated rats. Results ace expressed as in flguce 1. Figure 4 : Postero-antertor d i s t r i b u t i o n of 3H-SHT and 3H-PAT blndtng sttes In the rat locus caeruleus (LC). Results, are expressed as In ftguce 1 and typtcal t ~ g e s observed a f t e r 3H-9~T are represented tn the upper part of the flguce by ustng the sam color code as In ftgure 2. Mo5 : motor trtgemtnal nucleus.

4).

Rapid Important Paper

I

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I

I

1065

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:: 2oo~

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I sham

E

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o

E

1: im

_I.

v

F-

• 1" 1 0 0

ul

'o c o m 0

I

I

I

I

,,I

I

I--

<[

!

!

L

200

i

iJ

shim lesioned

_L

I 100

a.

m

i Z

II c o m I

i

Postero-anterior

I axis

I

I

i

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|

i 2.1021jm

1066

Rapid lmporl~mt Paper

sham

lesioned

I-u~ i ,v. ¢o

I,-, a, i

m u,= 0 m I.,. z

I-, u) i I-. 2

Fig.2

Rapid Important Paper

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l

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i

1067

[

60,

i

] T e

J ,~

,

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li

:c

i

m I

i

i

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\

eO,

•

Ii GI

4C

i ,i s h a m =--~ ,~ lesioned 3©

.

o

:

I

Posters-anterior

:

I

I

axis

:

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I

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,,

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Rapid Important I:'aper

:='OOI' ;ham esloned

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Rapid Important Paper

1069

DISCUSSION The interest of the methodology developed in this study was to allow the simultaneous treatment of a great number of brain slices together with a minimum of manipulations from the incubating tanks to the exposure upon the 3H-sensitive

films.

It was thus possible

~ (1) to respect

a

precise

serial

sampling of the brain which allowed the adjustment of the alignement for each structure examined from one brain to the other ; (2) the simultaneous treatment of one control and one lesioned brain in exactly the same conditions ; (3) the use of coverslips as supports for brain sections which gave the advantage of a better application of samples onto the film and also a better anatomical resolution and less background around the radioautographs. These technical improvments were well illustrated by reproducible quantitative results

obtained

in each anatomical plane chosen.

In the raphe nuclei the original

distribution

of 5HT1 binding

sites can

be correlated with p r e v i o u s l y published anatomical observations of the postero anterior distribution of 5HT containing neuronal elements (cell bodies and fibers). It was clearly demonstrated that 5HT Containing fibers and perikarya were less densely localized in the posterior part of the raphe dorsalis nucleus as compared to the anterior third of this structure (Steinbusch, 1981). It is thus possible to suggest that the p o s t e r o - a n t e r i o r increase in 5HT1 binding sites in this nucleus reflects, at least partially, a

specific

ments. marked

localization

of some of these sites on the 5HT containing

ele-

The observation that the p o s t e r o - a n t e r i o r distribution was more when sections were incubated with 3H-PAT suggests that 5HTIA sub-

types of b i n d i n g sites ( PaZos et ai.1984) are specifically localized on 5HT neurons. The results observed after 5,7 DHT treatment strongly support this hypothesis since : (I) the labelling with 3H-PAT p r a c t i c a l l y disappeared together with 5HT containing cells ; (2) the postero-anterior increase in labelling also observed after 3H-5HT incubation was

eradicated

after

5,7 DHT treatment. In this latter case the significant amount of sites remaining after the lesion of the majority of the 5HT contaihing cells and fibers, could correspond to sites of the 5HTIB subtype localized in non serotoninergic elements. In the nucleus raphe centralis similar interpretation can reasonably be made since : (1) the density of 5HT containing cells is lower in this structure (Steinbusch, 1981) as was the density of 5HTIA binding sites ; (2) a similar p o s t e r o - a n t e r i o r correlation can be suggested b e t w e e n 5HT containing elements (Steinbusch, 1981) and 5HTI binding sites ; (3) as observed in the NRD the same greater density in binding sites was observed at the anterior level when 3H-PAT was used to reveal 5HTIA sites ; (4) a similar more pronounced decrease in these sites was observed in 5,7 DHT treated animals with a complete disappearance of the typical postero-anterior profile of 3H-5HT or 3H-PAT binding in this nucleus. The fact that the lesion induced a decrease in 3 H - S H T a n d 3H-PAT binding which was less pronounced in the NRC as compared to the NRD seems to be due rather to a lower proportion of 5HTIA sites localimed on 5HT containing cells and/or fibers than to

107(I

Rapid [nap,.,riant Paper

a

lesser

degree

of

specific

lesion which in i m m u n o h i s t o l o g i c a l controls

a p p e a r e d to affect both nuclei to the same extent. The

locus

caeruleus,

densely

innervated

already

been

2

weeks

by

an

important

5HT

d e m o n s t r a t e d that this

after

et

al.,

1982).

(Leger et al.,

1980).

is

It has

5HT innervation was totally suppressed

5,6 or 5,7 DHT treatment

McRae-Degueurce

n o r a d r e n a l i n e containing group,

containing fibers

In

(McRae-Degueurce and Pujol,

our

1979

;

study this structure p r e s e n t e d a

r e l a t i v e l y high density of 5HTI sites labelled w i t h 3H-5HT and only a poor labelling was

with

observed

sites,

After

5,7 DHT treatment

no

s i g n i f i c a n t change

that these sites, p r o b a b l y c o r r e s p o n d i n g to 5HTIB

are not localized on 5HT terminals at the p r e - s y n a p t i c level.

Finally NRD

3H-PAT. indicating

an

original

and the NRC.

d i s t r i b u t i o n of 5HTI sites was d e m o n s t r a t e d in the

This d i s t r i b u t i o n p r o b a b l y corresponds to the l o c a l i z a t i o n

of 5HTIA sites on the 5HT cell bodies, present

dendrites and/or fibers and terminals

in these nuclei. They could represent the sites c o r r e s p o n d i n g to the

5HTI a u t o r e c e p t o r s w h i c h have already been p o s t u l a t e d Other

5HTI

correspond

sites to

have

been

identified

5HTI receptors,

in

these

(Gozlan et al.,

1983).

nuclei. They p r o b a b l y

mainly of type B localized post s y n a p t i c a l l y

as in the LC in w h i c h no p r e - s y n a p t i c 5HTI sites have been identified. Acknowledgements

:

review

manuscript,

help

of in

this the

typewriting

the

authors are very grateful to M. Peter HUNT for the

preparation of

the

to M. J.Y. MAURICE and Ch. BOULINGRE for their

of

the

figures

and

to

A.

V E R C A M B R E for the

manuscript. REFERENCES

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H.G.,

Bj~rklund,

A., Lachenmayer,

L. and Nobin,

A.

(1974) Evalua-

tion of the effects of 5,7 DHT on serotonin and c a t e c h o l a m i n e neurons in the rat CNS. Acta Physiol. Scand. Baumgarten,

H.G.

and Lachenmayer,

vement in chemical brain. Biegon,

(1972)

1-19.

5,7 d i h y d r o x y t r y p t a m i n e : impro-

lesioning of indoleamine neurons in the m a m m a l i a n

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A.,

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B.S.

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phy of serotonin receptors in the rat brain. B r a i n Res. 242,197-204. Bj~rklund, A., Baumgarten, H.G. and Nobin, A. (1974) Chemical lesioning of central m o n o a m i n e axons by m e a n s of 5,6 DHT and 5,7 DHT. In E. Costa, G°L. Gessa and M. S a n d l e r Press, New Y o r k p. 13-33. Deshmukh,

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L. and Nelson,

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Raven

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338-343.

Rapid Important Paper

1071

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Sternberger, L.A., Hardy, P.H., Cuculis, J.J. and Meyer, H.G. (1970) The unlabeled antibody enzyme method in immunohistochemistry. P r e p a r a t i o n and properties of soluble antigen antibody complex and its use in identification of spirochetes. J. Histochem. Cytochem. 19, 315-333. Unnerstall, titative

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