0022- 1554/79/2712-1577$02.00/0 THE JOURNAL OF HISTOCHEMISTRY Copyright © 1979 by The Histochemical

Effect

Vol.

AND CYTOCHEMISTRY Society, Inc.

of Coichicine on the in the Rat Brain:

Immunohistochemical Light and Electron

DONALD MRC

Group

in Molecular

Received

Somatostatin the

injection nucleus

of

The

words:

isolation

study

the

the fully staining

hypothalamus,

an

immunohistochemical

level.

The

effect

cellular

of

Lava!,

1979 U.S.A.

of Somatostatin Studies GI V 4G2,

27, 1979 (MS

of

increased

growth

hormone

after

intracerebral

in the

eminence

treatment.

increased

secretory

in

granules

and

treated

intensity

of

that

brain;

technics;

The

the

in agreement with the hypothesis into the median eminence.

immunoenzyme

by

periventricular

after

was

Canada

79-130)

hypothalamus

bodies

specific

Quebec

secretion

rat

cell

are axons

hormone;

to see

in

to the

cessive diluted

the

brain

and

see

15).

bodies

SRIF

localization;

could

of the

was

with

similarly

treatment to

was detect

the also

possible

has

thyrotropin Specificity

primary

AND

7.4)

and

(19)

embedded

involved

in

the

suc-

thyroglobulin

by the

(1, 6).

The

glutaraldehyde

specificity

technique of the

antiserum

(6). It did not cross-react with different LHRH, adrenocorticotropin (ACTH), releasing hormone (TRH), oxytocin and vasopressin. of the staining reaction was tested by incubating the and

pothalamic

of rabbit anti-SRIF serum, ‘y-globulin serum diluted at (PAP) complex used at 1:80 serum (503) was produced in our SRIF (Ayerst Labs, New York, N.Y.)

rabbits

reported

peptides,

antiserum

ACTH’24

such

as:

with

10”

vasopressin.

sections

M

synthetic

SRIF,

Immunoabsorption

from

both

LHRH,

performed colchicine-treated was

untreated

and

TRH,

in hyrats.

RESULTS At the technique

of the

median

applied

light microscopic performed on eminence

by

much less the normal

in

level, frontal

revealed

containing nerve fibers in the colchicine-treated

microscopic

organelles. MATERIALS

(pH

technique

anti-SRIF

into

been

known

et

success-

changes

buffer

synthetic

to bovine

already

of

cells. a drug inhibit

investigated

rabbit

In brief, injection

other

enhance and

light

before

bodies,

nucleus

colchicine at

used

The

was coupled

by

led Barry staining cell

periventricular

technique microscopy

the

hypothalamic colchicine, (21) and

(22),

if colchicine

of been

M cacodylate

immunohistochemical

application on tissue sections at 1:500 to 1:2000, goat anti-rabbit and the peroxidase-anti-peroxidase

laboratory.

that has

in 0.1 The

dilution.

brain,

of immunoreactive difficult to detect

(LHRH)

of proteins

rat

before

cell

1:10

and

In the

found

hormone

rats

peripheral

of radioimmunoassay

that levels be low and

in the

aldehyde

subsequently

(7). A similar argument treatments to enhance

we injected

immunoelectron

number

observations send their

the

the

nerve

and

was in localizing

delay

SRIF-cells

rat

the

Araldite.

a review,

transport

of the

in

June

for SRIF was in the median

the

group of neuropeptide-secreting those treatments, injection with has been shown to bind to microtubules In order

staining decreased

to

sera

hormone-releasing

(3).

form

inhibits

of somatotropin-release(4) has led

fibers

This

cytoplasmic

l’Universit#{233}

in

the

These nucleus

SRIF)

SRIF

SRIF-nerve

immunohistochemistry al. (3) to use various another Among which

which

SRIF

between

periventricular

attributed to the possibility SRIF in the cell bodies might

luteinizing

containing reaction.

by means (for

bodies.

was

or

of

animals

of

cell

bodies

fibers

nerve

a correlation

anti-SRIF

immunohistochemistry nerve

ofcell

of

characterization

distribution

distribution

The number granules

(somatostatin

of various

de

immunohistoehemically

Somatotropin-release-inhibiting

of specific

of

tissues

hormone

12, pp. 1577-1581, Printed in

PELLETIER

Hospitalier

19, 1979, and in revised

hypothalmic

No.

microscopy.

and

factor

production

secretory

GEORGES

AND

Centre

March

localized

number

suggesting

electron

inhibiting

the

immunohistochemical bodies in the

Key

the

been

ofcolchicine.

number animals, the cell

has

while

Le

for publication

(SRW),

pituitary,

DUBE

Endocrinology,

Localization Microscopic

27,

the

in the rats,

presence

of

external SRIF

numerous (Fig. 2). In the rats, small SRIF-positive

m in diameter could be seen the number of SRIF-containing

METhODS

the immunohistochemical sections of the normal zone fibers

numerous

rat SRIF-

(Fig. 1) whereas appeared to be

penventricular cell bodies

(Fig. 3). In the treated cells was strikingly

nucleus of 10 to

of 15

animals, increased

over controls (Fig. 4). Interestingly, this increase in number of cell bodies was coincident with a stronger staining of the cytoplasm of these cells which appeared larger than the control ones. This increase in staining was consistently observed

Four adult male rats (200-225 g) from Sprague-Dawley strain were injected in the lateral ventricle with 30 tg of colchicine (Merck and Co., Rahway, N.J.) dissolved in 30 zl ofsaline with a Hamilton syringe over 5 mm. Four controls were injected similarly with saline only. After 2 days, all the animals were fixed by intracardiac perfusion of Bouin’s fluid and brains were excised and kept in the same fixative for 24 hr. After dehydration and embedding in paraffin, they were cut in sections (7 m) and mounted on glass slides. For electron microscopy, brains were perfused with 4% paraformaldehyde or 1% glutar-

in all the

In both

treated

animals.

normal

and

sorption prevented

with excess immunostaining

peptides

were

completely

colchicine-treated of

synthetic of fibers ineffective

1577

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animals, somatostatin and cell bodies. in

blocking

immunoabcompletely The other reaction.

1578

DUBE

AND

PELLETIER

I

#{188}i.

V !‘= .

-

-

,

,:

:4,--

.,‘,..

‘.

.;

‘,:....i.

4-’,

I .:

.

‘

V

‘--

-

.

,.,

-

.

!.

1±

-

‘

-

-

-

:

I

I.

,.;,.

:

--

.

.

:rT:

4

‘--

.

--

‘

.

-

#{248}:.’

: -*

-

,

-

-

-,Sfr -

.

;ir

‘

: !

:

-...-

-.

)_

-

#{149}..p

-

:

:

1 and 2. Cross-sections at approximately the same level in the rat median eminence reacted with anti-somatostatin (SRIF) serum. In staining of a section of a control rat shows a dark reaction (-*) in the external zone of the median eminence; V, third ventricle; bar represents 100 tm. In Figure 2, section of a colchicine treated-rat shows a strongly decreased staining (-+); V, third ventricle; same magnification as in Figure 1. FIGS. 3 and 4. Cross-sections to rat periventricular nucleus reacted with anti-SRIF serum. In Figure 3, two cell bodies are stained (long arrows) as well as some nerve fibers (short arrow) in a section of a control animal; V, third ventricle; bar represents 25 m. In Figure 4, note the stronger staining and the apparent increase in diameter of cell bodies (-+) in the colchicine-treated rat. Nerve fibers stained for SRIF are less numerous; same magnification as in Figure 3. FIGS.

Figure

1,

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SOMATOSTATIN

At the localized

plasm A few

electron on

small

microscope

level,

secretory

granules

of some

neurons

granules

very

of the similar

the

PAP scattered

periventricular to

those

which

IN

THE

RAT

molecules were in the cytonucleus (Fig. 5). were

BRAIN

stained

other about small.

strongly

COLCHICINE

AFI’ER

were

1579

consistently

weakly

positive

organelles were immunostained. 80-110 nm in diameter and their In the

treated

rats,

the

stained

The number

or

negative.

No

granules were was relatively

cytoplasmic

granules

;:‘ -

I-

-

e

4”..

.

-

‘

,

,-

_*.;, ‘

. FIGs. 5 and 6. Immunohistochemical detection of SRIF at the ultrastructural level in neurons of the periventricular nucleus. In Figure 5, a positive reaction is present in control rat section in most secretory granules (long arrows) whereas a few granules (short arrows) are unstained. Bar represents 500 nra; N, nucleus. In Figure 6, SRIF-positive (long arrow) and SRIF-negative (short arrow) secretory granules are still evident in a nerve cell body of a colchicine treated rat. Note the increase in number of secretory granules compared to Figure 5; N, nucleus; L, lysosome; same magnification as in Figure 5.

Downloaded from jhc.sagepub.com at USD & Wegner Health Science Information Center on April 11, 2015

DUBE

1580

were

AND

storage

of similar

size and much more numerous than those control animals (Fig. 6). The SRIF-positive in the periventricular nucleus seemed to be less numer-

observed ous

in the

colchicine

the

positive

axons

treated

rats

contained

than

fewer

in controls.

able

an

granules.

thalamus

SRIF

of normal

microscopic

level,

treatment

induces

staining

with

in nerve cells and and colchicine-treated

our

for SRIF

results

in the

a decrease

clearly

number

detection

show

of a larger

number

substance

enkephalins

P

(13,

14),

to accumulate after colchicine treatment. At the ultra.structural level, secretory hypothalamus

in the

with other brain where and

to

was

determine

nately,

sections

processed

fine morphological as microtubules.

tory (12)

granules in cell and paraventricular

have

already

level.

increased Injection

bules, only

noted

contain

this

technique

of other

Although bodies

cells

ACTH

on (3),

(16,

were

18)

seen

after

colchicine

the correlation at the electron

such

Some

other

one,

of secre-

9.

ganglia (10) this

report

light

of the

treatment

hypothalamus

and

hypophysectomy

(9, 20),

the

but

treatment

(9, 20)

have

with

10.

J,

bodies

bodies

R, Schally

rat

hypothalamus,

Am

JO,

Brazeau

P.

Martin

JB:

Fernandez-Durango R, Arimura pothalamic somatostatin and

or hypothyroidism

11.

and

Flamant-Durand

157:235, 1978 J, Dustin

tory

granules

in

Action

of

Hoffman

the

colchicine

DL,

BL:

during

P: Studies

on

the

transport

flow

Z Zellforsch

and

(2) after

rats

with

pituitary

growth

20)

and

SRIF-fibers

immunohistochemically

removal.

Treatment

hormone

seemed

of

12.

the

to partially

increase

the

Proc

Soc

neurons.

Mikrosk

oftreatment

Anat with

1.

in

130:440,

growth

the

1972

hormone

H#{246}kfelt T, Dahlstrom A: Effects of two chicine and vinbiastine) on the distribution of noradrenaline storage particles, studied

mitosis and by

inhibitors (colaxonal transport fluorescence and 119:460, 1971

Z Zellforsch Mikrosk Anat JO, Nilsson G, Pernow B: Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Res 100:235, 1975

Parsons

JA,

Erlandsen

SL,

Central

and

peripheral

localization

zyme

median

of hypophysectomized

Hyin

neurotubules

electron microscope, 13. H#{246}kfeltT, Kellerth

shown

to be depleted

AV:

of secre-

hypothalamic

axonal

Effect

in rats.

15.

(9,

of

on somatostatin in the median eminence of hypophysectomized rats. Proc Soc Exp Biol Med 156:265, 1977

the

in the rat hypothalamus. Alcause a diminution in SRIF, content with the intensity of has only been established in the case of In fact, SRIF was shown to be decreased in

shown

144:

Effects

J, Schally hypophysectomy

anesthesia

magnocellular on

nuclei. Baker

A, Fishback LHRH after

staining

the hypothalamus eminence were

AV,

J Anat

14. H#{246}kfeltT, Ljungdahl A, Terenius L, Elde R, Nilsson nohistochemical analysis of peptide pathways possibly pain and analgesia: enkephalin and substance P. Proc Sci 74:3081, 1977

reaction

of

deafferentation Endocrinology 100:

to lower the content of SRIF though these treatments may direct correlation of the SRIF hypophysectomy.

of

zone

external

CITED

of the

Willoughby

paraventricular in

anes-

all been

in cell

Epelbaum

hyper

micro-

be involved

of

basal

to the

hypothesis

cell

1975

Exp Biol Med

staining. functions of microtu-

increase

up

the

brain lesions and hypothalamic deafferentation on somatost.atin distribution in the rat brain. Endocrinology 101:1495, 1977

de-

(8),

to

transported

the

MJ, Arimura A, Fernandez-Durango M, Kizer JS: The effect ofhypothalamic

matostatin 8.

allow

number

thetics

able

support

within

of

effect

eminence.

77, 1973 Brownstein Palkovits

541,

SRIF-containing granules in the cell bodies and possibly crease it in the nerve fibers. Brain lesions (8), deafferentation of medial

knowledge,

not

number level and

at the

is a nonspecific

also

marked

on somatostatin-like activity in the rat brain. 246, 1977 6. Dupont A, Coy DH, Alvardo-Urbina G, Cote J, Meyers CA, McManus J, Barden N, De Lean A, Labrie F: Sensitive radioimmunoassay for somatostatin using N-[’I]-Tyr-somatoststin as labelled antigen. Clin Endocrinol 10:47, 1979 7. Elde RP, Parsons JA: Immunocytochemical localization of so-

organelles

the

however

intensity of the cytoplasmic of drugs which inhibit the to our

did

treatment,

might

5.

unfortu-

peripheral hypothalamus

staining

factors

as colchicine,

SRIF;

between microscopic

They

any

somatostatin in the hypothalamus and pituitary stem. Proc Soc Exp Biol Med 151:599, 1976 3. Barry J, Dubois MP, Poulain P: LRF-producing cells of the mammalian hypothalamus: a fluorescent antibody study, Z Zellforsch Mikrosk Anat 146:351, 1973 4. Brazeau P, Vale W, Burgus R, Ling N, Butcher M, Rivier J, Guillemin R: Hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone, Science 179:

on

normal rat in number

in number

of sympathetic nucelus of the

be of

physiological

of

cell bodies

important

changes

of immunohistochemical

scopic

the

been

establishes granules

intensity

using

observations

such

clearly secretory

nerve

median

without

is synthesized being

this

1. Arimura A, Sato H, Coy DH, Schally, AV: Radioimmunoassay of GH-release inhibiting hormone. Proc Soc Exp Biol Med 148:784, 1975 2. Baker BL, Yen SCC: The influence of hypophysectomy on stores

the

which

ofgranules.

before

by

of SRIF in the nerve cell bodies. strongly suggest that short-term can inhibit the axonal migration

LITERATURE

periven-

reports LHRH

nerve

molecules

formation

(1 1). It would

induced

colchicine

principal change noted in the cytoplasm of SRIF cells after treatment. It may be suggested that the increase in number of secretory granules could be partly responsible for the darker immunohistochemical staining and the apparent larger diameters of these cells as seen with the light microscope. Immunohistochemistry is the only way

granules

the

eminence

changes

granules

cell bodies

granules of a few SRIF cells in the as already reported (17). An increase

of secretory

median

the

somatostatin

neurons

of SRIF-containing

in specific PAP

that

The stronger staincould be responsi-

(16)

shown

been

concomitantly

fibers

eminence. coichicine

are consistent systems in the

been

that

nucleus

median after

have

of nerve

of nerve

cells. These observations different neuropeptide

have

we

fibers of the hyporats. At the light

in number

periventricular

in the

tricular nucleus and the ing observed in SRIF-cells

ble for the

on the technique,

an increase

in the

know

somatostatin-containing

immunohistochemical

to localize

to

treatment on the storage The present observations treatment with colchicine

Generally,

DISCUSSION Using

of SRIF

interest

in the

axons

PELLETIER

872,

16.

Pelletier

immunocytochemical

Hegre

OD,

McEvoy

RC,

of somatostatin:

studies,

G: Irnrnurelated to NatI Acad Elde

RP:

immunoen-

J Histochem

Cytochem

24:

1976

G:

Immunohistochemical

hormones and Central Nervous

other peptides System. Effects

Downloaded from jhc.sagepub.com at USD & Wegner Health Science Information Center on April 11, 2015

localization

in the central of Hypothalamic

of

hypothalamic

nervous system, Hormones and

SALIVARY Other

Peptides.

Edited

by R Collu.

Raven

Press,

GLAND New

York,

AND

20.

Pelletier G, Dub#{233}D, scope immunohistochemical

rat hypothalamus. 18.

Pelletier docrine

nology. 1979,

19.

G, Leclerc functions

Edited p. 15-28

Sternberger

R: Somatostatin: electron microlocalization in secretory neurons of Science 196:1469, 1977 R, Dub#{233},D: Morphological basis of neuroenin the hypothalamus, Clinical Neuroendocri-

LA:

F Labrie.

Immunocytochemistry,

Raven

Press,

Prentice-Hall

New Inc.,

York, En-

Cliffs,

Wakabayashi

New

1581 Jersey,

I, Demura

Effect of hypophysectomy in rats, Endocrinol Jap

Puviani

by G Tolis,

KALLIKREIN

glewood

1979,

p. 331-344 17.

KIDNEY

21.

Weisenberg protein of Biochemistry

22. Wooten blastine thetic

Kanda

Ann

M,

on hypothalamic 23:439, 1976

RC, Borisy GG, Taylor mammalian brain and 7:4466, 1968

GF, Kopin on axonal nerves,

1974, 246 pp R,

EW:

its

Demura

H,

The

relation

K:

content

colchicine-binding

to microtubules,

IJ, Axelrod J: Effects of colchicine transport and transmitter release NY Acad Sci 253:528, 1975

Downloaded from jhc.sagepub.com at USD & Wegner Health Science Information Center on April 11, 2015

Shizume

somatostatin

and yinin sympa-