0022-

1554/78/2607-0000/$02.00/0

THE

JOURNAL

Copyright

OF

HISTOCHEMISTRY

© 1978 by The

AND

Histochemical

Inc.

LOCALIZATION DAVID Department

OF

and

WALTER

Surgery,

ofPharmacology,

(W.B.S.)

ANGIOTENSIN

G. CHANGARIS,

ofNeurological

Department

Medical

Hershey

Biomedical

Vol. 26, No. 7, pp. 593-607, 1978 Printed in U S. A.

CYTOCHEMISTRY

Society,

Research

IN

B. SEVERS University

RAT

Center,

Pennsylvania

Division,

NASA,

Ames

LANNY

AND

ofSouth

Medical

BRAIN’ C. KEIL

Carolina,

Charleston,

State

Research

S.C.

University,

Center,

29401;

Hershey,

Moffett

Field,

Pa. Ca.

17033

94035

(L.C.K.) (MS

78-139)

localization of angiotensin II (All) by the unlabeled antibody when rats were cardiac perfused with sub-zero propylene glycol containing phenylmethyl sulfonyl fluoride, a potent protease inhibitor. Neurons within the deep cerebellar nuclei and brain stem were studded with dense synaptic A!! immunoprecipitate. Afferent projections to the hippocampus showed All immunoprecipitate Immunocytochemical

method

was

within and

successful

fibers

ofthe

Ammon’s

cipitate

nucleus

Horn

include

(CA

the

illotegmental

ofthe 1-3).

alveus,

tract.

All

diagonal

band

Efferent cingulate

reaction

ofBroca,

hippocampal gyrus,

product

the

olfactory

rich

precommissural was

tractus

projections

fornix,

demonstrated

in

lateralis,

in All

immunopre-

fimbria,

and

pericapillary

mam-

pinealo-

posterior pituicytes, third ventricular tanycytes, and some cells within the spinal trigeminal tract. Preincubating the All antiserum with excess angiotensin I and antidiuretic hormone did not affect this distribution while pre-incubation with All abolished it. Cross-reactivity of All antiserum with angiotensin I, dog renin substrate, and synthetic renin substrate, tetradecapeptide was less than 1%. However, the antiserum showed 70% cross reactivity with angiotensin III (Al!!); therefore this immunohistochemical map could also correlate with the presence of Al!!. This morphology of the All/All! localization in the rat brain when correlated to the known patterns of All/All! cytes,

receptors by

within

both

brain

neurons

and

suggests

angiotensin

vascular

may

be

transported

to

sensitive

brain

The brain is now recognized as a target tissue of many peptide hormones (15). When injected or infused into the central nervous system, one

in the tomic

of these

angiotensin II (All) and/or (AlIT) in many cells and cell

sodium excretion, adrenocorticotrophic

and

after

Angiotensin

angiotensin

havior

injections

modify

retards

be-

stereotyped

angiotensin’s hormone

were

work

was

present supported

from the National Institutes from the National Aeronautics tion. DGC was supported 1F32N55177-01 from NINDS,

Health and Neurological Carolina.

the

it would

participate

by grants

HLB

results

of The

presented

multiple neuroanaimmunohistochem-

in this

article

demonstrate

angiotensin parts within

endogenous at sites where they act. Angiotensin is a circulating

behavior and (19). Central

threshold (17). In encephale after intraventricular andesynchronization of the occurred (7). The diversity of effects in the brain suggests that if

electrocortigram

‘This

injections

avoidance processes

neurophysiology compartments.

III many

brain regions. A crucial question concerning all peptide hormones acting on the brain is whether they are

and 30).

(3) and pain immediately injections,

isol#{233}cats

giotensin

release of antidiuretic hormones (1, 9, 18,

neostriatal

retention of passive could affect mnemonic

this

ical

hormones, angiotensin II (All), promultiple biologic effects including elevated pressure, drinking behavior, altered renal

duces blood

sites

channels.

may

also

11).

Therefore,

be synthesized

locally

a brief

chemical

studies

giotensin

in

brain,

are

by the

summary

describing along

the with

presumed hormone

to and

brain

(8,

of non-histopresence

of an-

associated

prob-

lems of interpretation, are presented here. Intravenous injection of radioactive angiotensin to determine if blood-borne angiotensin enters brain tissue sults. Intravenous

19320

followed 12, 36)

of Health and NSG-2122 and Space Administraby fellowship no. National Institutes of

terial

has not tritiated

by accumulation but re-identification as

angiotensin

yielded conclusive or ‘4C angiotensin of isotope of the

is difficult.

reis

in brain (5, isotopic maThis

task

(re-

identification) is difficult because angiotensin is a reactive molecule; after homogenization of brain it probably binds extensively to tissue

Departments of General Surgery and Surgery Medical University of South

593

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594

CHANGARIS,

SEVERS

AND

KEIL

4

: 1e

/J.

I

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

#{149}

‘

ANGIOTENSIN

components angiotensinases

or is degraded (13). This

illustrated

by

angiotensin (2).

It

not

been

data

showing

receptors is

that

re-extracted

tested

for biologic

ies

involving

i.v.

ters

activity.

not

the

has

in a high

yield

Therefore, of

allowed

of whether

affinity

material

brain

administration

have

pretation

high

‘251-angiotensin

isotopic

from

and

amounts of is further

that

catabolize

noteworthy

angiotensin

by large problem

AND

studinter-

hormone

of the

renin-angiotensin

system

increase

extravasation

fect may showed

apply to angiotensin. Anatomic that circulating angiotensin

ultrastructural widening

of proteins

endothelial of intercellular

(13);

changes spaces (28).

this

These

and/or sites

that affect

where

angiotensin cerebral

the

structures;

“blood-brain

do to

angiotensin has also hormone vations

at

remn

activity

tease

activity in the existence

especially

barrier”

that

a renin

renin D, a

question raised by this enzyme could

Ganten

(12)

found

renin

conditions where cathto be active. Printz et

human

cerebrospinal

content

of about

fluid

0.2

mU/nil

activated by proteases (22). Cathepsin D has been shown to activate “inactive” renin (20). The ability of brain tissue to transform labeled

angiotensin again suggest, but may have access

observations

prove,

reported has

that brain to cathepsin

including Penetra-

with

(24).

(26)

been

concept Whether a controver-

(WHO standard renin). Inactive renin species are present in plasma and may be endogenously

hanced not

in vivo.

has

the

studies produces

into brain after injection was en-

co-administered

(27) showed similarities

protease. The is whether

as a “renin”

(CSF)

system

(8, 11) raising is locally synthesized. in brain is currently

activity in brain under epsin D does not appear al.

ef-

tion of horseradish peroxidase i.v. or intracerebroventricular when

renin-angiotensin brain

subject. Reid enzymological

act

en-

brain.

Components

A complete described in that angiotensin “renin” exists sial has

595

CNS

lysosomal acid this observation

radioactive

unequivocal

blood-borne

THE

is deficient

during immunoassay measurements made difficult the quantification of the in brain (27). However recent obser(la) made by Osman et al. show that can

be

separated

from

brain extracts. of a specific

acid

pro-

While this afbrain iso-renin,

or made deficient by endogenous renin/angiotensin during physiologic or pathologic events. An interesting concept, not yet tested, is whether hypernatremia affects entry of angio-

firms

tensin

and/or locally synthesized angiotensin exists at angiotensin-sensitive brain sites still remains an enigma. But this histochemical study to clarify angiotensin’s presence and distribution, utilizing

into

brain.

Hyperosmolality

opens

the

blood-brain-barrier (23); and iv. administration of small amounts of angiotensin reduced threshold for drinking and vasopressin release

the in

response to hypertonic saline infusions (16, 32). These physiological observations suggest that hypernatremia, by opening the blood-brain-barrier, could allow access of circulating to brain structures when it might excluded. Currently, there that endogenous circulating responsive

sites

a biologically does

not

in the

active exclude

outside

since the

FIG. roxidase higher neuron

la. Darkfield technique magnification within the

cially

this

is no rigorous angiotensin

central

form.

angiotensin normally

nervous

The

possibility

lack

known regions ysis.

acid

of evidence espelie

thetized

remains

whether

method, in the

enig-

blood-borne

documents the brain in regions

to have receptors for angiotensin too small to do adequate receptor

Animal drated

protease

to resolve

MATERIALS in

sites

with

the unlabeled antibody presence of angiotensin

proof enters system

however,

some angiotensin-sensitive blood-brain-barrier (30).

be

the interaction matic. The inability

and male

AND

tissue

with

sodium

median

sternotomy,

clamped

above

rats

pentobarbital the

the

METHODS

preparation:

Sprague-Dawley

and in anal-

(n

=

Normally 6) were

40mg/kg

descending diaphragm.

thoracic A

photomicrograph of cerebellar fastigial nucleus stained for AII/AIII shows dense white perineuronal immunoprecipitate. The arrow shows in Figure ib; x1250. (ib) Oil immersion phase contrast photomicrograph cerebellar fastigial nucleus stained for AII/AIII by the immunoperoxidase

blunt

hyanes-

i.p. After aorta

was

16-gauge

by the

the immunopeneuron seen at of an isolated technique shows hematoxylin; x4000. (ic)

dark immunoprecipitate within synaptic boutons. The section is counterstained with Darkfield photomicrograph of adjacent locus coeruleus from the same coronal section of brainstem as la shows essentially no perineuronal immunoprecipitate; xl000. The arrow shows a single unstained neuron. (id) Low power darkfield photomicrograph of the cerebellum stained for AII/AIII by the immunoperoxidase technique shows arching white fibers contiguous to the granular Purkinje cells (arrows); x300. (le) Nemarsky interference shows perineuronal fibers rich with dark immunoprecipitate, birefringence outlines the Purkinje cells (P) x400.

layer.

Numerous white fibers are interspersed amidst photomicrograph of the cerebellar Purkinje layer small arrows. The section has no counterstain;

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596

CHANGARIS,

SEVERS

AND

KEIL

r

-

_;...,,

.

.)jp.

.\ .2d

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

‘ #{149}

ANGIOTENSIN

needle was threaded puncturing the left then perfused with solution

into the ascending aorta after ventricle. The rostral torso was 150-200 ml 3.7% formaldehyde

(-10#{176}C) containing

mM

Na2EDTA

oride

(PMSF).

AND

and

10%

1 mM

PMSF

propylene

glycol,

was

prepared

Evidence

flu-

at a stock

concen-

in 100% propylene

were

noreactive

col

tibody

of 10 mM

containing

washed

10 mM

in two

24 hr each.

cleared

blocks

were

overnight.

at

-10#{176}C for

of 100% ethanol

blocks

were

in xylene

brought

and

sectioned

at

The

sections in 100% ethanol,

washed Staining with

changes

The

ature,

PMSF

were and

protocol:

to room

deparaffinized air dried.

Al

serum

which

had

in xylene,

with

vasopressin and 10 jzg angiotensin 4#{176}C. Control slides were exposed sorbed antiserum, i.e., preincubated (10

tg/ml),

or

to

the

antibody

ten

not

affect

this

Tissue

tissue

in the

young

injections

New

ington,

Pa.

benzidine

After and

albumin

and

mented

and

bright

field,

and

dark

of

5 mg/mi

killed

than

0.01%.

0.2%,

des-Asp’

and

Nemarsky

70%

cross

background

contrast

with

show

a bright

high

The

with

contrast.

bers.

increasing

FIG.

2a.

Darkfield

amidst

nucleus accumbens sure (AC) shows interspersed within

micrograph Nemarsky (arrows)

nuclear

photomicrograph

the

perikarya

dark

contrast

of the

of the

which

are

scattered

gives

amidst

the

the appearance

and

was

synthetic

an-

angiotensin

vasotocin

with

Al,

was

less

less

than

tetradecapeptide

of being

immunoreactive

in synaptic deep cerebellar e.g.,

giant

boutons of the nuclei and neurons

structures

which

a high The

comprise

density

of

the

results

the

of the

study

anatomically presented

do

shows

(2b)

immunoprecipitate

Darkfield

of this

layer.

above

the cytoplasm

The

faint

outlines

and

system

imor the

in cells

within

photomicrograph

immunoprecipitate within fibers. The in this section. The adjacent intemal All/AIII immunoprecipitate; x400. (2c)

fibers within the medial horn CA 2 shows All/AlIl

regions

distinct comnot provide

is present

x600.

lobe fi-

of these

neuronal compartment. Immunoreactive AII/AIII

lateralis

limbic

of All/AIII-positive

the limbic vascular-related

(circle);

All/AIlI-rich of Ammon’s perikarya

tentorium

to link with the

olfactory

cortex

septi (ACB) shows dense AII/AIII essentially no immunoprecipitate the caudoputamen also contains

of the hippocampus shows interference photomicrograph membranes

tractus

pyriform

the

enough evidence munoprecipitate

methas

resolution.

scattered

with

reactivity

are discussed as an partment. The data

Bright

interference

with

harvested

1.1%.

contain

bright

immunoprecipitate

background

mycobacte-

the

tegmental grey matter. This is defined as the neuronal compartment. Included are cells within the zone of Lissauer, spinal trigeminal tract and spinal white matter which are densely positive.

examined

appears

and Nemarsky

supple-

Cross-reactivity

P,

Al, 0.7% and

serum

adjuvant

reaction II).

repeated

bovine

RESULTS

interference,

immunoprecipitate

of

data

to All were by

to

with

Neurotensin

Below

Down-

counterstaining

contrast,

be

of the

tuberculosis

studies

revealed

substrate

anti-rabbit

were

may

rabbits

complete

III (des-Asp’-angiotensin All, substance

microscopy.

microscopy

against

sections

represents

This

coupled

Sar’-Ala8

in 0.05

The

possible

anti-

Laboratories,

hematoxylin

field,

a dark

phase

goat

and

Antibodies

All

Cross-reactivity

tiserum

in 0.05% 3,3’ diaminoM Tris acetate, pH in 0.1 M Tris, pH 7.4, and dipped

H2O2

phase

dark

against

field, ods

after

field

With

The

All;

hormone

interpretation

Freund’s

with

rium.

developing

0.03%

7.6 slides were rinsed in 1.0 N HC1 for 30 sec. before

(35). Cappel

below).

Zealand

of synthetic

neurons,

al.

of the anexcess

It is entirely

characteristics:

in

brainstem

et

binding

mapping (see

final

(6)

from

is a high

with

binding.

of AIII

is localized of the

purchased

there

radioimmunoassay

with anti-diuretic

All/AIII perikarya

Sternberger

All, by

by preincubation

(PAP) bridges. The PAP solution was a gift from Dr. Ludwig Sternberger, Edgewood Arsenal, Md. Initial incubations of the All antiserum were at 4#{176}C for 18-24 hr. Subsequent incubations and development were performed as described by Burns was

is not en-

herein.

raised

generous

and

of immu-

antibody

octapeptide

peroxidase

IgG

presence

the

immunohistochemical

Antibody

units

peroxidase

angio-

the

determined

preincubation

importance

I (A-I) for 24 hr at to either All adwith excess All and

the

blocked

presented

preincubated

for

Although

as

distribution

Antiserum

been

All

was

did

the

8-OH ton, Mass.), 10 l/mi dimercaprol (New England Nuclear) 0.1 mM PMSF and 3% normal goat serum. This solution will be referred to as Tris diluent. Angiotensin antiserum was diluted 1:100 with Tris diluent. The experimental slides were exposed to the diluted anti10 tl/ml

criteria

for

for

that

dilutions were made ofO.1 M Tris acetate pH 7.4, containing quinoline (New England Nuclear, Bos-

a solution

the

specific

whereas

The dry

to

of immunoreactive

and immunohistochemistry.

temper-

permitted

presence

material.

affmity

and

embedded.

and

t

tirely

at -10#{176}Cfor

paraffin

5

24 hr

for the

sections

glycol. The brains and cervical cords were removed, blocked in 1 mM coronal slabs and post-fixed for i hr at -10#{176}C.The tissue blocks were dehydrated in 100% propylene glytration

597

CNS

tensin or cross reacting material is presented. Either brown reaction product or bright dark field positive material in the experimental slides coupled with the absence of the same product or material in the control

15

phenylmethylsulfonyl

THE

of

fibers

of the

anterior commiscapsule fibers Darkfield photoalveus (arrows); x400. (2d) immunoprecipitate in fibers of the granular layer cell and

nucleoplasm;

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

x3500.

CHANGARIS,

598 those barrier stretch

regions devoid (14). These between the

dymal blood Also included such These

pineal,

cells

by

related

are

are They

visualized

of

in the the

rat

synaptic

boutons

neurons

are

brain.

neurons unstained Fibers

within (Fig. coursing

diverge within bers are also Purkinje cells.

represent

neurons

of white

light

With higher microscopy

are

discerned

studded.

the

ib).

of an

unstained

C,

lum.

all

shows is densely

tive All/Alli round nuclei,

(Fig. 3-5

a collection positive i

for

Pur-

of Lis-

of cells whose immunoreac-

6b). The cells have oval to in diameter, and pale cyto-

Limbic

substantia fibers.

Fibers

lateralis which

are is best

within

densely

microscopy.

Scattered

karya

of the

pyriform

cortex

fibers

the

anterior

commissure

by

While

no immunoprecipitate,

the

alveus overlying a dense collection (Fig. 3b). It are projections fimbria

CA2

2d). hip-

is visinduseum

are seen

a number (Fig. 3a).

on coronal of dark-field

remains from

uncertain the subicu-

hippocampae fibers. midline,

are

a number

The greatest density forming a “V” (Fig.

mammillotegmental for AII/AIII appear larger

localized

tract

(Fig.

3d)

immunoprecipitate. than the positive

is also

These fibers of

for field

elements.

such as the thalamic commissure (Fig. 6c) and fornix have capillaries devoid of All/AIII-rich fibers while the larger blood vessels show a dense perivascular dark field positivity which is not clearly localized to cells or cellular elements. Dark

the

dark

or cellular

the anterolateral and farlateral hypothalamus. Larger blood vessels in these regions have only scattered All/AIlI-positive fibers in the perivascular space. By contrast, white matter structures

the

tractus

to cells

matter of the immunopreis not clearly

Vascular related compartment: Capillaries of the anterolateral hypothalamus show AII/AIII-positive fibers surrounding the perivascular space (Fig. 4a). These are found only in

through

cle,

field the shows

microscopy caudal

of of the

coronal

plate,

sections

median

immunoreactive

subependymal and the Numberous

limb

eminence

material the

dorsal

within

third

ventri-

median eminence (Fig. 5a). tanycytes within the posterior

amidst

the

peri-

limb

are

also

noted

(Fig.

transition zone show dense collections munoprecipitate. These cells stretch ventricular lumen to capillaries within

of from the

the

nucleus

pothalamus.

extremes

2a).

tially

positive fibers

are numer-

positive

visualized

(Fig. of the

3c).

(ME)

structures:

olfactory AII/AIII

gelatinosa

horn

funbria hippocampae. The ventral periaqueductal grey Aqueduct of Sylvius shows diffuse cipitate (Fig. 6a). The precipitate

plasm on routine hematoxylin-eosin staining. Scattered throughout white matter tracts such as the dorsal columns are similar cells which are slightly more spindle shaped (Fig. 6d). Scattered throughout the ous All-positive

angiotensin hippocanipus are scattered

the

shows

zone

that

to the fibers

these structures no synapses were

of AII/AIII-positive appears to be in the The

section

the

Within

we

great

section

Within again

the

the granular layer (Fig. id). Fipresent within the plane of the High power Nemarsky interfer-

microscopy

griseum. of fibers,

fibers these

the locus coeruleus remained ic). in the cerebellar white matter

a dense collection of fibers surrounding kinje cells (Fig. le). The spinal trigeminal tract and

suggest

projections, immunoprecipitate within the cingulate gyrus and

positive whether

Not

same

pocampal ualized

imon

magnification the individual (Fig.

On

fornix

amidst the perikarya ofAmmon’s Within the anatomic framework

lateral shows

is outlined dark field

on

of the

The section

that

of the

limb

of syn-

All/AIII visualized

Because

of these la).

so

with

with best

These

border

microscopy (Fig. and phase-contrast

neurons

immunoprecipitate

surface

a confluent

sauer at cytoplasm

surpositive. of the

studded

rich are

examination. density

density

The

nuclei

which

field

an-

capillaries

KEIL

peptides are transported (Fig. 2c). Similar positive

com-

as the

which are All/AIlI within the category

cerebellar

aptic boutons munoprecipitate. greatest

related

have

ent

postrema.

such

Compartment:

deep

zone. organs

compartment.

Neuronal

ence

vascular

area

regions

fibers included

are

vascular

by

the

hypothalamus

rounded These

dark

and

diencephalic

terolateral

the

pituitary

comprise

partment. Certain

the contact neurohemal

AND

bens septi shows a dense collection of A!I/AIIIrich fibers (Fig. 2b). These do not appear to form synaptic boutons. Immunoprecipitate within fibers of the affer-

of the classical blood-brain include tanycytes which III ventricle and subepen-

vessels within are cells of the

as the

SEVERS

shows

essenaccum-

of the

median

The

eminence

cytoplasm

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

at

at

or

both

below

the imthe hy-

ANGIOTENSIN

AND

THE

599

CNS

FIG. 3a. Darkfield photomicrograph of supracallosal region just caudal to the genu shows coarse All/AIII immunoprecipitate within the cingulate gyrus (C). The induseum griseum (IG) shows symmetrical collections of fibers containing All/AIII immunoprecipitate; xl000. (3b) Darkfield photomicrograph of lateral alveus overlying CA2 on coronal section shows numerous fibers (arrows) which contain immunoprecipitate in sections immunostained for All/AIII; x i500. (3c) Darkfield photomicrograph of the midline fimbrae hippocampae shows numerous

fibers which

are positive

for All/Alil

direction; x 1000. (3d) Darkfield photomicrograph fibers positive for All/AIII immunoprecipitate;

immunoprecipitate.

of the mammillo-tegmental x3000.

These

fibers course

in a rostro-ventral

tract

showing

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

(arrows)

numerous

600

CHANGARIS,

SEVERS

AND

KEIL

(

,0

at

-..v. .

4b

a

..it’

-

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

ANGIOTENSIN show

immunoprecipitate.

This

dense

population

of tanycytes

is restricted to a portion hypothalamus (Figs. 4b,c).

caudal Both supraoptic

paraventricular neurons

neurons (SON; 4e)

munoprecipitate. deep

Indeed,

cerebellar

nuclei,

and

SON

bers with

are seen neurons.

The

by

appear and

dorsal

the

these

third

do

not

ventricular

the

and im-

tricular

with the infundibulum

ependyma

cipitate laminae

AII/AIlI shows

at the junction of the median

precipitate

is not

cellular

elements.

The

capillaries

show

antibody dense

of internal eminence

localized

shows

of

perivascular

the

lateral

fibers

are

cells

brane adjacent to a capillary postrema (Fig. Se). The pineal has an extensive alicular network. Within this apparent

terminal

All-positive

clubs

(Figs.

spaces, particles seen (Fig. 7a).

or

intra-cellular Lining the

the

limit this

staining remains cleft between the

uncertain. anterior

are be or

pituitary

FIG. around

below

4a. Darkfield small blood

the transition

capillaries; surrounding

X4000;

a blood

vessel

(BV);

chiasm

(SON)

(OC)

shows

however

little

shows

x7000.

inhibition between However, found

We

to the vascular study (10) does

did

cular regions

Darkfield

reaction

product

numerous

staining

fibers

which

conjucompar-

apparently

compartment. not record

study absent

re-

One fluoresobservation

this

commented (21). Fuxe

et

product neurons

that vascular al. (10) re-

not indicate localWe observed intraonly within the vas-

compartment discrete synapses.

Neither ported on

of the earlier the tanycyte,

in cell bodies and Purkinje

did

and

relatively

few

fluorescein studies repineal, area postrema

immunoprecipitate pituitary immunoprecipitate

which

we ob(angio-

fibers;

suggests these cells (with electrophysiologidentified receptors, (29) may be stimuby angiotensin from the vascular compart-

hypothalamus x2000. (4b)

shows AII/AIII Phase contrast

photomicrograph

near

contain

the

neurons

All/AlIl

immunoreactivity of the third ventricle

from the third ventricle to subependymal shown in 4b shows a tanycyte foot process of the

paraventricular

immunoreactive fibers. The neurons of the paraventricular shows an unstained neuron. (4e) Darkfield photomicrograph immunoreactive

on im-

study are not warranted in antibody characters(fixed vs frozen sections,

find reaction supraoptic

related with

tanycytes (T) stretching contrast of the region “T” (4d)

reports

etc.) as well as intrinsic diithe PAP and fluorescein techsome comparisons are of inter-

est.

other was

brief

fluores-

with fluorescein (10, 21). Detailed

lated cein

rons ically lated

are den-

of the antero-lateral as white perivascular

zone shows numerous (4c) High power phase

shows only scattered All/AIII unstained; x 1200. The arrow nucleus

of the pituitary (Fig. 7d). The

photomicrograph vessels (circle)

yenimmu-

tensin) may be associated with angiotensin’s known vasopressin releasing effects (30). The lack of reaction product in the supraoptic neu-

particulate.

posterior lobe pituicytes

of

70. The choroidal immunoreactivity.

published

antibodies

and pituitary served. The

and the pars intermedia are a number of pituicytes which are densely positive for All/AIlI. The cells in Figure 7c show an intracytoplasmic distribution of All/AIII which is not clearly Within the All/AIII-positive

All/AIII

of PAP and immunohistochemistry:

cells) as well as fibers but ization in nerve terminals. cellular immunoprecipitate

these can extra-

All

al. (21) (including

canspace,

Within

of resolution represents

junction

ported most immunoprecipitate was in nerve terminals; no nerve cell bodies were observed and angiotensin-positive axons were found only in the lateral retrochiasmatic area. Nahmod et

area

pinealocytes

7a,b).

laboratories

and the staining

surface mem-

perivascular perivascular

of light

6e,f,

at the Whether

within

shows

munohistochemistry

protease ferences niques.

dark-field

are cuboidal but have one ventricle and a basement

plexus

isons with the present because of differences tics, tissue preparation

positive (Fig. Sd). The pattern is symmetrical with the capillaries of the opposite side. The ependymal surface of the area postrema has cells which contain immunoreactive material. These cells on the fourth

the

DISCUSSION

gated

infundibulum

which

choroid

Two of

and external (Fig. 5c). This to

near

and posterior lobe. membrane of the fourth

General comparison cein angiotensin

(Fig. 5b,d). immunopre-

clearly

increases

fi-

synapses

an infolding associated with a proliferation vascular channels. These ciliated ependymal cells rest upon a matrix which shows immunoreactivity The

cells

intermedia basement

pars

PVN

scattered form

601

noreactivity (Fig. 7e; Control cells themselves do not show

to the

of the

Only

CNS

of these

the

The

(PVN; 4d) show little

neurons

THE

sity

of

comparison

unstained.

AND

of this

nucleus

structure.

immunoreactivity;

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

(PVN)

nucleus are essentially of the supraoptic The X

1200.

adjacent

optic

602

CHANGARIS,

SEVERS

AND

KEIL

.a

F

-

?

-

..:

:tuI

‘.Td.

:J -

‘4

:-_

1 -,

Id’

.,J.,,..

.-

I’

-

#{149}1’

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,

ANGIOTENSIN

ment.

Surprisingly,

fluorescein

the

emphasize

cerebellum, was easily

dense

especially seen by the

cerebellum,

to our

reaction

AND

studies

did

not

(2)

product

in

the

brain

the deep nuclei, which methods used herein. The

knowledge,

has

not

been

pos-

tulated to be a central site of action of angiotensin. However, angiotensin receptors are present in the greatest density within the cerebellum in one

mammal

(2). There

appears

to be agreement

among the available histochemical observations that angiotensin II is present in spinal, mesencephalic grey, tegmental, hypothalamic and himbic regions. The the positive cell been sufficiently High

source of angiotensin types and organelles clarified.

affinity

Investigations

AU

receptors

on high

as well as have not

in

affinity

rat

binding

brain: of All

in

THE

603

CNS

that

was

was

high

bellum;

especially

binding

was

amount gions.

affinity

almost

the

observed

was not In contrast,

cortex.

Although

of

sitive to the into a vertebral

angiotensin

hormone: artery

unit discharge mone-elevated

reof

relative

to

showed

a

reasons binding should

for the studies be of

regions

sen-

in

Angiotensin increased area

and direct injection blood pressure (38).

infusions postrema

of the horWe observed

rat brain showed a broad distribution of “receptors” (2, 34). The thalamus, hypothalamus, septum, midbrain and brainstem had relatively high

immunoprecipitate

binding capacity compared to cortex, striatum, hippocampus and cerebellum. Correlations of the immunohistochemical data with these observations are not feasible but it is noteworthy that “receptors” and AII/AIII both have a broad distribution in brain and encompass areas re-

the hormone requires further clarification since the hormone does not raise blood pressure when applied to the fourth ventricle (31). The subnucleus medialis has been identified as a site of angiotensin’s pressor activity when administered

garded as sites of action of the high affinity binding studies have ruled

out

contributions

of

Such receptors may be regions and may or may sity of vascularization. to the eventual solution

hormone. The not completely

vascular

receptors.

dense in discrete brain not correlate with denThis point of receptor

is important location. For

example, data reported herein show vascularrelated immunoprecipitate around capillaries in some regions (i.e., anterolateral hypothalamus) whereas others (thalamic commissure, fornix) have mainly large vessels surrounded by dense precipitate. An

interesting

finding

by Bennett

and

the area correlates

on the

the

other density

often

diffuse immunoprecipitate. The different observations by specific and PAP immunohistochemistry great interest. Presence

specific

with a high

which

calf cere-

tissue,

cerebellum

structures

in

to the

cerebellar

great compared we observed in the

brain

binding

restricted

in rat

immunoprecipitate

other

All

entirely

postrema. to the

intracerebroventricularly munoprecipitate which was not

surface

of

observation effect

of

in ventral localized

Although this specific fined well, we believe close proximity The region

(4). We observed imperiaqueductal gray to cellular elements.

nuclear zone the hormone

was not dewas in, or in

to, the subnucleus medialis. of the anterior ventral third

tricle and anterior quently implicated effects (1, 25). to iontophoretic observe discrete and pericapillary

ven-

hypothalamus have been freas sites of central angiotensin Some cells in this region respond angiotensin (37). We did not cells, but diffuse subependymal immunoprecipitate was evi-

dent in this region. The supraoptic

Snyder

ependymal

Whether this central hypertensive

nucleus

responds

to

ionto-

FIG. 5a. Low power darkfield photomicrograph of the rat third ventricle shows periventricular immunoreactive All/AIII which is not clearly localized to cells or cell processes. Within the median eminence a band of immunoreactive All/All! is present at the junction of the intemal and external layers; x 120. The triangle and square are shown at higher magnification in Sc and Sd, respectively. The arrows denote diffuse acellular immunoprecipitate in periventricular gray matter. (Sb) Phase contrast of the dorsal third ventricle shows a subependymal collection of channels (arrow); there is a diffuse perilumenal collection of immunoreactive

All/Alli; magnification

x2500.

(Sc) Darkfield

shows

the

dense

photomicrograph immunoprecipitate

of the infundibulum at the

junction

of the section of internal

and

shown external

in 5a taken layers

of the

at higher median

eminence (large arrows). Several fibers course the external border of the median eminance (small arrows); x750. (Sd) Darkfield of the blood vessels of the lateral infundibulum of Sa (square) shows numerous fibers rich in All/AIlI immunoprecipitate. These fibers (arrow) course in close proximity to blood vessels of this region; x750. (Se) Phase contrast of the ependymal surface of the area postrema shows dark All/AIII immunoprecipitate in particulate form (arrow). The slide is counterstained with hematoxylin; x6000. (50 Phase contrast of the area postrema ependyma stained with a solution of AII/AIII antibody preincubated with excess All shows the ependymal cells devoid of brown immunoprecipitate. The section is counterstained with hematoxylin; x6000.

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

604

CHANGARIS,

SEVERS

AND

KEIL

‘“t

‘S

-,

?

:-

61

‘

-

-,

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_,j__’

ANGIOTENSIN

AND

THE

605

CNS

“

4#{231} ,4%,,

-

4’

,i

2

-

.-‘

..,,

7e

..4

iL.

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

CHANGARIS,

606 phoretic is

angiotensin

and

antagonized

by

drugs (29). in supraoptic sults

We

with

et al.

Therefore, stimulates putative

blocking

(21)

who

present

found

positive

of angiotensin

But

the

II

uncertain cells to

hormone

of other regions devoid of a “blood-brainsuch as the area postrema, pituitary

pineal

glands.

tanycytes

The

presence

the

possibility

opens

of

in

present

in cells barrier”

the

where

the

cation

of the

may reach ous system nels. The

the

lag

time

for

contributions

release.

many

disciplines

is a potent have been angiotensin,

high-affinity

been

receptors

munoassay

FIG.

have

of angiotensin

6a.

Darkfield

of the

aqueduct

here,

showed in brain.

pre-

methdemonwhen

brain

that the horThe peptide

sensitive sites within the central nervby both vascular and neuronal chanprecise anatomic pathways to defme

multiple

effects

further

raphy

of angiotensin

on

the

brain

investigation.

presented

Timothy of the

Fitzharris microscopy

in this

for his guidand photog-

paper.

LITERATURE

CITED

1. Andersson B: Regulation Physiol 39:185, 1977

of body

fluids.

Ann

Rev

Osman MY, Sen 5, Smeby RR: Separation of renin activity from acid protease activity in brain extracts. Fed Proc 37:3S4, 1978 2. Bennett JP, Snyder SH: Angiotensin II binding to mammalian brain membranes. J Biol Chem 2S1:7423,

1976

3. Braszko J, Wisniewski K: Effect the central action of dopamine. Pharm

Im-

28:667,

shows

a diffuse

AII/AIII

of angiotensin on Pol J Pharmacol

1976

4. Buckley JP, Smookler HH, RR: A central site of action

to measure

of Sylvius

presented

inhibited, distributed

We thank Dr. ance and support

neuroshown and

isolated.

in brain

the

la.

ACTH

from

Identifi-

lacks

ACKNOWLEDGMENTS

CONCLUSIONS Research

in brain.

by bioassay

(All!)

in

hormone

have shown that angiotensin peptide. Specific cells in brain to respond to iontophoretic

activity if and

and

properties; CSF injections of the hormone have a lag time relative to direct pituitary infusions (18). Tanycyte transport of the peptide could explain

is present

peptide

ofAlI

await

discharges specific neurons within the contact zone and/or is translocated into the portal circulation. Angiotensin is known to have CRF

easily

hormone

renin about

cision of identification that immunologic ods confer. The immunohistochemical

the

angiotensin

that

hormone levels and conflicting results

proteases were mone is widely

i.e.,

vasculosum included was

KEIL

stration

how release

action,

organ and the organ (25, 33) were not

study.

hormone the re-

with

AND

endogenous has generated

angiotensin

it remains these

sites

the subfornical lamina terminalis the

discharge

receptor

fluorescein-coupled

antibodies. angiotensin vasopressin. Other

electrical

specific

did not find endogenous cells. This contrasts

of Nahmod

cells

the

SEVERS

immunoprecipitate

Severs WB, of angiotensin

within

Deuben II and

the

ventral

periaqueductal grey matter; x200. (6b) Phase contrast photomicrograph of the spinal trigeminal tract at the level of C, shows dark All/All! immunoprecipitate. The slide is counterstained with hematoxyhin; x2600. (6c) Dark field photomicrograph of large blood vessels (arrow) within the thalamic commissure, shows dense perivascular immunoreactive All/Alli; x200. (6d) Phase contrast photomicrograph of the dorsal column at C45 seen on sagittal section with capillaries; x2600. method of Sternberger

The

slide

shows scattered (6e) Brightfield et al. (1970)

is counterstained

with

AII/AIlI rich cells (arrow). low power photomicrograph shows immunoreactive AII/AlIl

hematoxylin;

x175.

section stained FIG.

stained with AII/AIII adsorbed with hematoxylin; xl7S. 7a. Phase contrast photomicrograph

antibody

within

the

bleb

pencapillary

granules within the pineal shows particles cannot photomicrograph

(arrow).

The

posterior counterstained

space.

section

pituitary

adjacent

(61) Brightfield shows

no

of a capillary

(B)

photomicrograph

uptake within

is densely

within the

positive

pineal

the

of adjacent pineal.

shows

The

All/AIII

for AII/AIII

control

section

pineal

is counter-

immunoreactivity

immunoreactivity;

smaller

the oval are equally positive; x9000. (7b) Phase contrast photomicrograph of a capillary within All/AIII immunoreactivity within the pericapillary area (P). Small densely AII/AIII-positive be clearly localized to intracellular or extracellular compartments; x9000. (7c) Phase contrast of anterior pituitary shows densely AII/AIII positive pituicytes along the pituitary cleft

with

is counterstained shows intracytoplasmic hematoxylin; x7500.

plexus stained for angiotensin epithelium; x4000. (70 Phase section X4000.

A single

These cells have an infrequent juxtaposition of pineal stained for All/Alli by the PAP within the pericapillary regions (arrow).

to 7e shows

with (7e)

hematoxylin; All/AIII Phase

shows immunoprecipitate contrast photomicrograph no immunoreactivity

x7S00.

(7d)

within a posterior contrast photomicrograph

Phase

contrast

photomicrograph

pituicyte of the

(arrow). The fourth ventricular

of the section choroid

along the basement membrane (arrows) of the choroidal of the fourth ventricular choroid from a control serial within

the

basement

membrane

of the

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on June 5, 2015

choroidal

epithelium;

is

ANGIOTENSIN its

possible

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cardiovascular

tensin ley

in

the

central

system,

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of the

Actions

Hormones.

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by JP Buck-

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wich

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(PAP)

peroxidase

formalin

fixed

Histochemistry

com-

antibody

sand-

paraffin

embed-

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