Histochemical and ultrastructural features of neuronal pigment in some encephalic nuclei of ruminants

(1)

;

Exp Biol (1985)44: 109-121

Experimental

Biology

Histochemical and ultrastructural features of neuronal

pigment in some encephalic nuclei of ruminants

A. Merighi and

S.

M. Peirone

lnstitute of Yeterinary Systematic and Comparative Anatomy, Via Nizza 52, 10126 Torino,

ltaly (Director: Prof. A. Gobetto)

Summary.

Neurona

l

pigment

i

n

red nucleus,

loc

u

s

coeruleus and

sub-stantia nigra from

catt

l

e, sheep a

nd

goat was examined with the

aid

of

li

ght and e

l

ectron

microsco

py.

In th

e

red nucleus

a

nd l

ocus coe

rul

e

u

s

n

euro

n

s of a

li

th

e

species

examined

the

pi

g

ment

sbows tbe bis

t

ocbem

i

-ca

l

and

ultrastructural features

typical

o

f

lipofuscins.

Th

e

ultra

structu-r

a

l

observations

indi

cated

tbat tbe

m

orpho

l

ogy of

pigment granules

i

s

r

e

lat

ed

to

age a

nd

permitted

id

e

ntific

ation of various stages

of pigment

evo

luti

on, whicb

suggested

a

l

ysoso

mal

or

i

gi

n

o

f

the pigment bodies.

I

n

bovine

and

shee

p the substantia

ni

g

ra

is

very reduced

and

con-tains

n

o

detectable pigment; while in

goat,

some ne

u

rons a

pp

ear

to

contain di

sc

r

ete amounts

of neuromelanin. Ultras

tructurally this

kind

of

pigment

appears c

h

aracte

ri

zed

by

t

he

features

of

a

lipofuscin-like

granule which

stores

highl

y

electron dense materia!

id

entifiab

l

e as

mel-a

nin

.

Key words:

Centra

i ner

vous syste

m

-

Lipofusc

in-

Neurome

l

a

nin-

Ru-min

ants-

Light

and

e

l

ectron

m

i

c

r

oscopy

Introduction

Many

a

uth

ors

showed that neuromelanin

and

lip

o

fu

scin pigments

co

n

-tained in the neurons

of

the

ce

ntr

a

i

ne

r

vous system

share the sa

me di

stribu-tion

in

ali the species

so far studied. Melanin was found in

substant

i

a

ni

gra,

Offprint requests to: A. Merighi

(2)

110 A. Merighi and S. M. Peirone

l

oc

u

s coeruleus and dorsal

vagus

nucleu

s

in

carnivores (Brown 1943;

Levi

et a

l.

1977)

,

primate

s

(Sc

h

erer

1939

,

Barden 1970)

and

man

(Adler

1939

;

Foley

and

Baxter 1956

,

19 58;

D'Ago

s

tino

and

Lu

se

1 964; Feniche! and

Bazelon 1968

;

Barden

19 69;

L

ev

i 1974

)

. The di

st

ribution of lipofuscin

pig-ment in the

centra

i

nervous

system

has been

st

udied in

carnivores (Wolf

an

d Pappenheimer 1945

;

Sulkin

1955)

,

rodent

s (A

lpert et a

l.

1960; Duncan

e

t al.

1960

;

Samorajsky et al.

1965; Brizzee et al.

1969

;

Nand

y

1971

;

Brunk

and

Erics

so

n 1972

;

Hein

se

n 1979

,

1981)

and man

(Zeglio

1935; Wolf and

Pappenheimer

1 945; Bondareff 1959

,

1964; Brody 1960

;

F

rie

de 1962

,

1966

;

Timiras

1 972;

Braak

1 974,

1 977;

Braak 1976

;

Braak

and

Braak

1 976).

Lipofu

sci

n i

s

present in almost ali

e

ncepòalic nuclei.

Many h

y

pothe

ses

hav

e

been put forward

on the function a

n

d t

h

e or

i

gin

of neuronal pigment

s

. Lipofu

scin

pigment

,

which i

s

u

sua

lly regarded

as

a

wear and tear pigment

,

r

e

present

s,

in the opinion of many autho

rs,

a waste

product

of cellular

m

etabo

lis

m. Almo

s

t

ali

th

e

celi organelles are thought

to be involved in the genesis

of lipofu

sci

n

.

Among the different theories

two

are

mo

s

t

widely accepted.

Th

e

fir

s

t i

s

b

.ase

d upon the

concept

that

swo

llen degen

e

rated mito

c

hondria tran

s

form into pigment particle

s

(Hess

1955; Dun

ca

n

et

al. 1960

;

Hein

se

n

1 979). Th

e seco

nd,

s

upported by many

hi

s

toch

e

mical and

UV light microscopica

!

observations, point

s o

ut the

con-cept that pigment granu

l

es are derived from ly

soso

mal bodie

s

and are to be

co

n

s

id

ere

d

as

teloly

soso

m

es,

i.

e.

t

e

rminai nonfunctional ly

sosomes,

whic

h

are no

l

onger recycled by the celi (Essner and

Novikoff

1960

;

Malkoff

and

Stre

hle

r

19 63; Jami

eso

n and

Palade 1964).

Even

le

ss

data are available on

the origin of

n

euromelanin; mo

s

t

st

udi

es o

n thi

s s

ubject ha ve dealt with the

difference

s

between neuronal and extraneuronal melanin

s

(Foley and

Bax-t

e

r 1958

;

Kennedy an

d Zelick

so

n 1963)

,

but the fun

c

tion of

thi

s

pigment i

s

tili

completely unknown.

A review of existing literature

s

how

s

a

lack of avai

l

able data about

neu-ronal pigment di

s

tribution and

c

haracteristic

s

in h

e

rbivor

es,

particul

a

rly in

ruminant

s.

In

two preliminary

s

tudi

es

th

e exis

tence

of lipofu

scin

pigment

was reported for the neuron

s

of the red nucleu

s

and the locu

s coer

uleu

s

in

adu

lt

cattle (Peirone 1980 a, b). In order to d

esc

rib

e

the morphological fea

-tures and the hi

s

tochemical prop

e

rtie

s, s

uch a

s

ti me of appearance and age

related changes

in

neuronal pigment, a thoro

u

gh study was

carri

ed out on

so

me

encephalic

nuclei

of

ruminant

s

with the

aid of light

and electron

micro

s

copy

.

Materials and methods

For light microscopy, eight bovine brains ranging in age from 8 months to 15 years, eight ovine brains from new born to IO years and six goat brains form 30 days to 12 years were examined. Gross sections of the cranial portion of the medulla oblongata an d of the mesence phalon were fixed in 100/o formalin or in Zenker formalin, paraffin embedded and sectioned in toto at 7 ~m. Red nucleus, substantia nigra and locus coeruleus were examined with tbe fol-lowing tecbniques: Haematoxylin-Eosin and Toluidine Blue to determine tbe topography of the nuclei; periodi c aci d Schiff; Suda n Black 8 an d O il Red O for tbe demonstration of Jipids; Ziehl-Nielsen for acid fastness; Perls for ferric ion and Lillie for ferrous ion uptake. Some

(3)

Neuronal pigment in ruminants Ili unstained sections were observed by a blue light fluorescence microscope. The light source consisted of a high pressure mercury vapour burner (Osram H 80-200); the light bea m was fil-tered through a BG 12 exciter filter to obtain a wide band emission ranging from 300 nm to 500 nm.

For electron microscopy, eight bovine brains from animals ranging in age from the new

born t o 15 years, si x ovine brains from the new born to l O years an d four goat brains from 6 months to 12 years were examined. Tissue fragments of l mm3 _{obtained from the}_red_nucleus,

the locus coeruleus and the substantia nigra of ali the species were fixed in 2.5% glutaralde-hyde buffered at pH 7.4 with 0.1 M phosphate buffer, postfixed in l% osmium tetroxide, blocked stained with 2% uranyl acetate and embedded in Durcupan. Sections (l Jlm thick)

were stained with Toluidine Blue to identify the pigment containing cells. The ultrathin sec-tions were stained with Reynolds lead citrate and examined in a Siemens Elmiskop lA elec-tron microscope.

Results

Li

ght

microscopy

R

ed

n

uc/eus.

In

ali the species examine

d the

red nucleus appeared as a

con-s

pi

c

uou

s

mass o

f neur

ons deeply

embedde

d in th

e

me

se

n

ce

ph

a

li

c

tegmen-tum at

t

h

e

l

eve

!

o

f

the

r

os

tr

a

l

co

lli

c

ulu

s.

Th

is

nudeus

conta

in

ed two

d

is-t

i

nct

ce

llul

a

r

populations: a ca

ud

a

l

p

o

r

t

i

o

n

(pars

m

ag

n

oce

llul

a

ri

s)

formed

b

y a great

numb

e

r

of

l

arge

n

eu

r

ons and a rostral

p

ort

i

on (pars

parvocellu-l

a

ri

s) fo

rm

ed by

f

ew s

m

a

ll

ce

ll

s.

Th

e

l

arge

n

e

ur

o

n

s of

t

h

e pars

magnocellu-laris

va

ri

e

d in

size and sha

p

e,

so

m

e

b

e

ing r

ou

nded

o

r

ova

l

s

h

aped, some

elongated

or with

irr

egu

l

ar polygonal outlines.

Th

e axon

hillo

ck was

usu-a

ll

y we

ll

evide

nt. In

Toluidine Blue

or

H

aematoxy

lin

stai

n

ed sections t

h

e

ce

li

nucleus a

pp

eared cent

r

a

ll

y

lo

cate

d

w

ithin

the ce

li

body, the nucleolus

stai

n

ed darkly

and

Nissl

bodie

s were abu

ndant. In

t

h

e a

dult

a

nimai th

ese

ce

ll

s co

ntain

ed var

iabl

e a

m

o

unt

s

of

pi

gme

nt

w

hi

c

h

gene

r

a

ll

y

formed

conspicuo

u

s clusters within the ce

li b

ody.

The

peri

odic acid Sc

b

iff

rea

ct

i

o

n

gave the best

r

es

ult

s a

mon

g

t

he

histo-chem

i

ca

l u

sed: the

pi

g

m

e

nt

was sta

i

n

e

d

a

n int

e

n

se

purpli

sh

red

co

l

o

r

a

n

d

s

t

oo

d

out

f

ro

m the

pa

l

e

cyto

pl

asm

which

s

urr

o

unded i

t

(F

i

g

.

l

)

.

In

a

ddi

-tion, the pigment s

t

a

in

e

d d

ee

pl

y w

itb

lipophilic s

t

ai

n

s s

u

c

h

as

Oil

R

ed

O

and Sudan

Bla

ck B. The

P

e

rl

s an

d Lilli

e

r

eactions were

h

oweve

r

a

l

ways

n

e-ga

ti

ve

. Th

e

pi

g

ment

, o

n

the other band, sta

ined

stro

n

gly

with Ziehl carbo

l

-fu

scin, thus s

howing it

s acid fastness (Tab

l

e

l).

UV

li

g

ht

microscopy demonstrated that the pigment in the red

nucl

e

u

s

n

e

ur

o

n

s was a

l

ways

auto

fluor

esce

nt,

with

a

li

g

ht

ye

llow

co

l

o

r. In

a

li

the

species

examined, the

amou

nt

of

neur

onal

pigment appea

r

ed

to

b

e

corre-l

ate

d t

o the age of

th

e a

ni m

a

l

s.

The

fir

st pigment gra

nul

es a

pp

ea

r

e

d

, at

th

e

nd

o

f

the

fir

s

t

year

of

!i

fe,

as

brilliant

b

o

di

es scattered

throughout the

cyto-plasm.

With

age,

there

was a

n

increase

in

the

amo

unt

of pigment

a

nd th

e

g

r

a

nul

es

t

e

nd t

o co

lle

c

t

into

groups

lo

ca

ted in

o

n

e or

both

n

e

ur

o

n

a

l

poles.

The

pi

gme

nt

g

r

anu

l

es

in

6 to

8-year

-

o

l

d a

nimal

s were

l

a

r

ge

r

than

in

the

yo

un

ge

r

animals and they were clumped

in

cons

pi

c

uou

s

clu

sters;

neverthe-l

ess

we observed

minut

e g

r

a

nule

s scatte

r

ed

throughout the

cytop

l

asm in

th

ese aged a

nim

a

l

s too. In o

l

d an

i

ma

l

s (IO years a

nd

over)

th

e pigment may

fili

a

lm

ost ent

ir

e

l

y

th

e

perikaryon, it

was somet

im

es o

b

served w

ithin

the

(4)

axon

h

i

llock

but

never

wit

hi

n t

he d

en

dri

tes. The most

co

nspicuous

p

i

gme

n

t

clus

ters were

observed i

n

bov

i

ne; i

n

the

ot

her

species

t

h

e

amount o

f

(5)

Neuronal pigment in ruminants 113

Table l. Histochemical features of neuronal pigment in the bra in stem nuclei examined Autofluo- P.A.S. Aci d Sudan O il Perls Fe++ rese e n ce fastness Black B Red

o

uptake . .

+

bovine Re d

₊

_s_h_ee_p nucleus

+

go a t

+

bovine Locus

+

sheep coeruleus

+

go a t

no demonstrable pigment bovine Substantia _no _demo_nst_rab_l_e _p_i_g_m_en_t _s_h_eep

nigra

_'

+

go a t

**

Lillie reaction

S

u

bst

a

nt

i

a

n

igr

a

.

I

n

ru

mi

n

a

nt

s

we fo

un

d

th

a

t

h

e

f

eatu

r

es o

f

t

h

e s

u

b-sta

nt

ia n

i

gra we

r

e

so

m

ew

h

at

di

ffe

r

e

nt f

ro

m

t

h

ose

d

escribe

d

for

m

a

n

a

nd

o

th

er species.

B

ovi

n

e s

ub

s

t

antia

n

ig

r

a was

not

eas

il

y

d

e

t

ectab

l

e beca

u

se of

t

h

e ex

i

g

u

o

u

s

num

be

r

o

f li

tt

l

e ne

u

ro

n

s

r

an

d

o

ml

y scattered t

h

roug

h

t

h

e

b

asa

i

port

i

o

n

o

f m

ese

n

ce

ph

a

l

o

n

. S

h

ee

p

s

ub

s

t

a

ntia

ni

g

r

a was eve

n l

ess

d

e

fin

e

d

; we

fo

un

d o

nl

y

f

ew

littl

e ce

ll

s

sca

tt

e

r

e

d h

e

r

e a

nd th

e

r

e o

n

the

b

o

rd

e

r b

e

t

wee

n

th

e

m

ese

n

ce

ph

a

l

o

n t

eg

m

e

ntum

a

nd th

e c

ru

s ce

r

e

bri

.

On th

e co

ntr

a

r

y, go

at

s

ub

s

t

a

n

t

i

a

n

ig

r

a was

m

ore

d

etec

t

a

bl

e,

it

co

n

s

i

s

t

e

d

o

f num

e

r

o

u

s

l

a

r

ge

p

o

l

y-gona

l

n

e

ur

o

n

s w

hi

c

h

f

o

rm

e

d

a co

m

pac

t

c

ellul

a

r

ba

nd

.

W

e obse

r

ve

d

n

o

pi

g-ment

i

n bov

in

e a

nd

s

h

eep ne

u

ro

n

s, w

hil

e i

n un

s

t

a

i

ned o

r

in

To

luid

i

n

e

Blu

e

s

t

a

in

e

d

sec

t

ions of goat s

u

bs

t

an

t

ia

n

ig

r

a some ce

ll

s

we

r

e

f

ou

n

d

t

o co

nt

a

in

dar

k

brow

n

o

r

bl

ack

pi

gme

n

t (

Fi

g. 2). Ne

ur

o

n

a

l p

ig

m

ent

i

n goa

t

subs

t

a

n

t

i

a

n

i

g

r

a

ce

ll

s

was

n

ot a

ut

onuo

r

esce

nt

;

i

t

was a

l

so

n

ot

s

t

ai

n

e

d

with pe

r

io

d

i c

aci

d

Sc

hi

ff, l

i

pop

hili

c sta

in

s o

r

Z

i

e

hl

ca

rb

o

lfu

s

in

;

m

oreove

r i t

was nega

ti

ve

for

P

er

l

s reac

ti

o

n

b

ut p

os

iti

ve fo

r Lilli

e

f

e

rrou

s

i

o

n u

p

t

a

k

e (

T

ab

l

e

1 ) .

..___

Fig. l. 12 year-old cow. Red Nucleus. Lipofuscin pigment showing a strong positive PAS cu-mulutes in great amounts in most of the celi s. Zenker-formalin. Periodic Aci d Schiff. x 625

Fig. 2. 6 year-old goal. Substantia nigra. Dark brown or blackish pigment granules can be ap-preciated. Zenker-formalin. T o Juidine blue. x 160

Fig. 3. 2 year-old sheep. Locus coeruleus. Lipofuscin shows a strong affinity for lipophilic

stains. Zenker-formalin. Oil Red O.

x

190

Fig. 4. 8 year-old sheep. Locus coeruleus. Lipofuscin pigment appears clearly autofluorescent

(6)

Locus

coeru

l

eus.

The nucleus of

the locus coeruleus showed the sa

me

to-pographical a

nd

structura

l features

in

a

li

the

species

examined.

lt

was

lo-cated

beneath

th

e

floor

of th

e

fo

urth

ve

ntricle

in the

latera!

recess

at

the

transverse level

immediately

rostral

to the

colliculu

s

facia

lis. The

nucleus

of locus coreuleus consisted of ovo

id

neurons; the celi

nucl

eus was

round

(7)

a

n

d the

nu

cleolus was qu

i

te evide

n

t.

I

n t

h

e

p

er

i

karyon, at o

ne

or bo

t

h ce

l-lu

l

ar poles, we observe

d

some

pigme

nt

wi

t

h

istoche

m

ica

l

f

eatu

r

es ide

nt

i-ca! t

o t

h

ose

d

esc

r

ibed

f

o

r

the

r

ed n

u

cle

us

ce

lls

(Table

l

;

Figs. 3 and

4);

whe

r

eas UV ligh

t

m

i

croscopy

sh

owed t

h

at i

n

t

he

loc

us coeru

leus t

h

e

pig-me

n

t gran

u

les we

r

e larger a

n

d

l

ess

nu

me

r

o

us.

In

this

site

too t

h

e

r

e

i

s a

n

i

n

c

rease,

w

i

t

h

age,

i

n t

h

e n

u

mbe

r

o

f

p

i

g

m

e

n

ted

cells

and

i

n t

h

e a

m

o

un

t o

f

p

igmen

t i

n

eac

h n

e

ur

o

n.

Electron

microscopy

Neuro

nal

p

i

gment

gran

ule

s

in red nucleus

ce

lls were easi

ly

detected

be-cause of

t

heir

hi

gh

electron

de

nsity (Fi

g.

5);

hig

her

magnifications

r

evea

l

ed

a complex

ultrastructure

whose

featu

res are closely

related to

age. Unti!

t

he

t

h

ird mo

n

th of postnata

ll

ife

only small

dense

rou

nd part

icles, co

nsisting of

a

homogeneous

matrix of

a

fine

granu

la

r

membrane bounded mate

r

ia

!

,

are

present i

n

t

h

e neu

r

o

n

al perikaryo

n.

These pa

rt

icles

st

rongly rese

m

ble

lyso-so

mal

bod

i

es. F

r

om t

he

thi

r

d

month on,

i

n

a

d

i

tion

t

o these partic

l

es ot

h

er

s

phe

r

oida

l

bod

i

es

,

w

i

t

h

a few

l

ame

ll

ar

st

ructures a

n

d

one

o

r

more

li

g

ht

areas i

n t

h

e

gran

u

lar

mat

rix,

were also

observed (

· Fig.

6).

The p

igmen

t

p

ar-ticles of adul

t

animals appeared as large

irregu

l

ar me

m

bra

nes-bound

dense

compl

exes (Fig. 7). Fu

lly

deve

loped

p

ig

m

ent g

ranules co

nsisted

of: l)

a

fi

ne gra

n

ular matrix formed by 5

n

m gra

n

ules;

2)

o

ne or

mo

re

lig

ht

areas

of varyin

g

size,

w

h

ic

h

were identi

fi

ed

as

lip

i

d conta

i

ning

vacuoles

by

m

any

authors (Hess

1 955; Ess

ner

an

d Novikoff

1 960);

3) a

period

ica!

la

m

ella

r

system

w

h

ic

h

was

not always clearly

evide

n

t due to masking by the gran

u

-lar mat

r

ix; t

h

e f

u

nda

menta

l u

n

i

t

of t

h

is system was

f

o

r

med by two t

h

in

lam

e

ll

a e, sepa

r

a

ted b

y a

l

O

n

m s

p

ace,

in

t

h

e ce

nt

er of w

h

ic

h

a t

hird

t

hi

ck-er

a

nd

da

r

ker

l

ame

ll

a was p

resen

t; the la

m

e

ll

ae ofte

n

fo

rmed

a si

nu

o

u

s

patte

rn lik

e a

fin

ger p

rin

t

(Fig. 8).

I

n the

r

e

d nu

cle

us

n

eu

rons

o

f

a

n

i

m

als 10 yea

rs

o

l

d or ove

r,

we

ob-serve

d

many

pa

le gran

u

les wi

th

sca

nty granu

lar

matrix

and very

promine

n

t

lame

ll

ae

toget

h

er

wit

h

a

progre

ss

ive disap

p

earing

of

the limiti

n

g

mem-brane (Fig. 9).

+

-Fig. 5. 8 year-old sheep. Red nucleus. The neuronal cytoplasm contains many pigment gra-nules, appearing as dense bodies. Dark (arrow lleads) and pale (arrows) granules are evident.

x 10,200

Fig. 6. 3 month-old calf. Red nucleus. lnitial stages of pigment granules deposition. Spherical shaped homogeneous granules about 0,5 ~m in diameter are surrounded by a single limiting membrane; in two ofthese a lipid vacuole has formed arrow heads). x 32,000

Fig. 7. 6 year-old goaL Red nucleus. Pigment granules typical of adult animals. Note the pres-e n ce of many lipid vacuoles (arrows) and the irregular outlines of the granules due to the fusion of smaller pigment bodies resembling the protopigment described in young animals

(arrow heads). x 18,000

Fig. 8. IO year-old cow. Red nucleus. A higher magnification shows the lamellar system; the fundamental unit of this system is formed by a thick lamella with two thin lamellae on both sides. Thick lamellae are well evident (arrows). x 130,000

(8)

Neuronal

pigment g

ra

nul

es

in the

locus

coe

rul

e

u

s ce

ll

s

have

the

sa

me

ultra

st

ructural

characteristics as in

the red

nucle

u

s,

but they we

r

e

larger

a

nd more regu

l

ar in

s

h

a

p

e;

the gran

ul

a

r m

atrix was more d

ense (

Fig.

IO

).

Electro

n

micro

sco

pi

ca

l observations confir

m

ed th

e lack

of

pigment

gra-nul

es

in

the

neur

ons o

f

bo

vin

e

a

nd

s

heep

s

ub

s

tantia nigra. On th

e

other

h

a

nd

we co

lud

eas

il

y d

e

tect

n

eurona

l

pigment in

goa

t

s

ub

s

tantia

nigra.

Pi

(9)

t

he three

ult

rastru

c

tura

l

compon

ents

descri

bed ab

ove

in

pigment

granul

es

of red nucleus a

nd

locus coerul

eus

(i. e.

th

e

matri

x, th

e

la

m

ell

ar system

a

nd

t

h

e

tipi

d

vacoules), the presence o

f a

hi

g

hl

y electron d

en

se

materia

! s

tored in

t

h

e matrix (Fig.

Il

).

The a

mount o

f

this

m

ateria!

was extremel

y

variable,

som

eti

mes bei

ng very sca

nty a

nd

so

metimes filling a

l

most a

li tbe matrix.

In

the

l

atter

case the other

structures o

f the granu

les were a

l

most

co

mp

l

etely

masked b

y t

he electro

n

den

se

mater

ia! and the g

ran

ules a

ppeared

as highly

electron d

ense bod

ies in

w

hich no

s

tructures was ev

ident

,

with the

ex-ception

of

a scarce

number

of li

pid

vacuol

es (Fig.

12).

In

yo

ung a

nim

a

ls,

pigment granules we

re sma

ll

in

s

ize a

nd their stru

cture was

quite

s

im

ple.

Th

ey consisted of a granul

ar

matrix w

itb

a few

la

mell

ae and

some

li

pid

vacoules; the

matrix a

lways contained a

little a

mount of

the electr

o

n

dense

materia

l.

Discuss

ion

l n

the

rumina

nt

s

exam

in

ed

the features an d the d

i

s

tr

i

bution

of n

e

u

ron

a

l

pigment in

t

h

e

red n

ucleus and

locus

coeru

l

eus

cell

s

are almost

s

imilar;

thus they wi

ll

be di

sc

ussed

t

oge

ther whi

l

e t

h

e

s

ub

sta

ntia n

igra

w

ill

be

treat-ed sepa

rately

.

R

ed

n

uc/e

u

s and focus

coeru

/

eus

In

t

h

e red nucleu

s

a

nd

in the

l

ocu

s coer

uleus

,

t

h

e neuro

na

l

pigment

is

char-act

e

rized

by bright

yellow a

utoflu

orescence,

positiveness

to

periodic aci

d

Schiff,

acid fa

s

tne

ss,

s

trong

a

ffi

n

it

y

for

lipopbili

c sta

ins

and

negativity

to

P

erls

and Lilli

e

reacti

o

n

s.

Th

e ultra

s

t

ructural

observatio

n

s

o

n neu

rona

l

p

ig-m

ent

in

the

se

nu

cle

i

s

howed the exi

s

tence of a tripha

s

i

c subs

tructure in

th

e

pigment bod

ies. T

h

ese l

ight a

nd electron

microsco

pica! f

eat

ures are d

i

s

tinc-tive of l

ipofuscin

pigment

s (

W

o

l

f and Pa

ppenh

eimer 1945, Sulkin 1

953,

1955; D'Ange

l

o et a

l. 1

956 ;

Bo

nd

areff 1

95

9 ;

Pear

se

1 960; Samorajsky et a

l.

+

-Fig. 9. 15 year-old cow. Red Nucleus. Pigment granules show a difTerent electron density: pale granules show more evi de n t lamellar systems.

x 33,000

Fig. IO. IO year-old ox. Locus coeruleus. Pigment granules are formed of a fine granular ma-trix from which one or more lipidic vacuoles stand out. The limiting membrane is clearly ap-preciable at some point of the granule periphery (arrows heads}, whilst the lamellar system is basely detectable.

x

33,000

Fig. Il. 12 year-old goat. Substantia nigra. Pigment granules are characterized by the presence of an highly electron dense materia l stored in the matrix. A lipidic vacuole bounded by a rim

of the electron dense materia! is evident (arrow~ A protopigment-like panicle is present in the center of the micrograph (arrow head~ Note the irregular outlines of the granules and the

granular appearance of the electron dense materia!. x 30,000

Fig. 12. 12 year-old goat. Substantia nigra. Another aspect of neuromelanin pigment. Note the regular outlines of the granules and the homogenous appearance of the electron dense materia! which fills almost completely the granular matrix. x 24,000

(10)

1964, 1965; Strehler 1964). The

assessment

of lipofuscin pigments in

rumi-nant red nucleus is in accordance

with

the

reports in man

(Friede

1962,

1966),

Macacus rhesus (Barden 1970), dog

(Sulkin

1955), pig

(Nanda and

Getty 1971) and rat

(Na

ndy

1971). On the contrary, the

existence

of

lipo-fuscin pigment

in the

locus coeruleus points out remarkable differences

be-tween these

and

other

species such

as

carnivores,

primates

and

man where

the

pigmented

cell

s appear

to

contai

n neuromelanin

(Adler

1939; Brown

1943;

Barden

1970;

Levi 197 4; Levi

et a

l.

1977).

UV

light

microscopy pointed out a

similarity

in the autofluorescent

pro berti es of the pigment granules in both the nuclei, but at the

same

ti me

the existance of age

differences among the

species

examined. Our

observations suggest

that

the ultrastructure of the pigment granules

un-dergoes progressive modification with age. Small

spherically shaped

masses

of homogeneous granular

content,

which are morphologically

similar

to

lysosomal

bodies, represent the first stage of formation of the

lipofuscin

pigment. These

small

masses could therefore be considered as

a

protopig-ment. The

lipid

vacuoles appear in these

granules

in the

second stage;

to-gether

with the appearance of the

lipid

component, the lamellar

system

dif-ferentiates to form the first pigment

granule~.

In a

subsequent

stage

the

masses of pigment

merge

into

large

complexes which constitute the

charac-teristic elements of the pigment in the adult ani mals; indeed the

obser-vation of

some incompletely

merged granules, bounded by a

single

limiting

membrane, is quite common. In old animals, pigment granules with a pale

matrix and a well defined

lamellar system bave constantly been

detected.

This

suggests

that

lipofuscin

granules could

undergo further

morphological

changes

as a consequence of metabolic processes leading to

modification

of some of the

structures

considered metabolically inertial enti ti es by many

authors.

W

e

do not

share entirely

the view of the mitochondrial

origin

of

lipo-fuscin granules,

in

fact we

seld

om observed

pigment

granules

in

a spatial

relationship

with

degenerated

swollen

mitochondria

or

celi

membrane

fragments. On the

contrary

the close morphological

similarity

between

pro-topigment

granules and

lysosomes

is

highly

suggestive of

the

possibility

that at least a

part

of the

lipofuscin pigment is derived from

lysosoma

l

ma-teria!. Under this

view,

the morphological changes observed

in

the

ultra-structure

of the pigment granules

could

be related to

the

continuous

supply

of metabolic waste products which are no

longer recycled

by

lysosomal

enzymes.

Substantia nigra

Contrary

t

o

the

observations of

H uber

et al.

(Quoted

in Levi et

al.

1 977),

the

substantia

nigra of the ruminants

exam

ined showed

different

morpho-logical

features with respect

to

other

mammalian

species.

The

sections

in

toto of the brain

stem

demonstrate that it is

impossible

to find

a

typical

substantia

nigra

in cattle and

sheep,

because of the exiguous number of

neurons an

d

the

lack

of pigment in them.

(11)

• On the

contrary, goats show a definite substantia

nigra

with a

larger

number of cells;

bere

some

neurons do

contai

n

a

brownish

-

black pigment

which

is

characterized

by the histochemical properties

specific of

neuromel-anin (Lillie

1955,

1957).

The

electron micrographs

attest the light

microscop-ical

observations.

the

existence of a

highly

electron

dense materia!

,

identi-fiable

as

melanin

,

besides the ultrastructural

components typical of

lipo-fuscin granules

is held to be

characteristic

of the

neuromelanic

pigment

(Moses

et

al.

1966;

Forno and

Alvord 1974). Our

observations show the

similarity between

lipofuscin

and

neuromelanin

at the

ultrastructurallevel.

They

support

the hypothesi

s

of

Barden

and other (Barden

1981)

,

based on

histochemical ob

se

rvation

s,

that neuromelanin is

to

be regarded

as a

mela-nized lipofu

sci

n.

In

conclusion, remarkable differences exist

in

the

di

strib

ution

of

n

e

u

-ronal pigment in

the brain

ste

m nu

c

lei

even

among

species

which are

close-ly

related taxonomic entities

s

uch

as the

s

pecie

s

examined in this

s

tudy.

The ultra

st

ructural

observations

clearly

demonstrate

that the pigment

fea-tures

undergo

progressive changes with age and that

in

a

s

ingl

e s

pe

c

ie

s

these

ultra

st

ructural

features vary among the

differ

ent

nuclei

;

moreover

th

ese

observations

s

ugge

st

a

ly

soso

m

al

origin of"lipofuscin. Nevertheless

futher

obse

r

va

tion

s

are to be undertaken to clarify better the relationship

between lipofu

sci

n

and

n

eu

romelanin pigm

e

nt.

Acknowledgements. This work was supported by a grant of the ltalian Consiglio Nazionale delle Ricerche and M.P.I. We wish to thank Prof. G. Godina for his advice and support during the course ofthis study.

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