Exp Biol (1986) 46: 89-99
Experimental
Biology
@ SprinAcr-Verlag 1986
The relationship between lipofuscin and neuromelanin in some sites
of the nervous system of the borse*
Marisa Bianchi and Adalberto Merigbi
l nstitute of Systematic an d Comparative Veterinary Anatomy. Faculty of Veterinary Medicine, Via Nizza 52, 1-1 O 126 Torino, ltaly
Summary.
Hi
stochemical a
nd
cytochemical
fea-t
ur
es of neuronal
pigment in
t
h
e
sp
inal
co
rd
, a
n
d
in
the
sym
patheti
c
and
s
pinal ga
n
g
li
a of t
h
e adu
l
t
borse have been
studied.
Light
a
n
d
e
le
ct
ron
mi-crosco
pi
ca!
obse
rvations
revealed
th
at
lipofuscin
pigm
ent is
pr
ese
nt in
t
h
e
spi
na
l
ganglia
a
nd
in the
sp
in
a
l
co
rd,
whe
r
eas
in
t
h
e
sy
mpath
e
tic
ga
ngli
a
both
lip
of
uscin and
neurom
ela
nio
are
prese
n
t.
The ult
r
astructural
s
tudi
es s
ho
wed
t
hat
lip
o-fu
sci
n
gra
nul
es exhib
it
ed a
tripha
sic o
rga
ni
zation
consist
in
g of a
m
atr
i
x, a
system
of lamellae
and
one or
mor
e
l
ipid vacuoles. Neurome
la
nin
gra-nules
have
stru
ctural
f
eatu
r
es
in
common w
ith
lip
o
fusci
n-
l
ike
gran
ul
es, except
th
e
forme
r
types
s
t
o
re
a
highl
y e
le
c
tron
dense materia! on their
matr
ix.
The
e
l
ectron
de
nse
m
ateria! has
b
een
i
de
n
tified as m
ela
n
i
n
b
y a
modification of the Li
l-l
ie
ferro
u
s sul
fate react
io
n
a
pplied
a
t
the
ultra-struct
ural lev
el.
From the
ultra
str
uctura
l
appear-ance
and
cytoche
mis
try of the
neuronal
pigme
nts
in horse it
is concluded
that neuromelanin
sh
ould
be
considered as
me
l
anized l
i
pof
usci
n
.
Th
e
possibility that the d
i
fferent
r
a
t
io
of
lip
o-fuscin to melan
i
n within
the same
gra
n
u
l
e is
re-spons
ibl
e
fo
r
t
he
pec
uli
ar
reaction
of
ce
rtain
neu-r
omel
anin
gra
n
ules to l
i
pop
hi
lic dyes,
the PAS
reactio
n
,
a
histochemica
l
assay fo
r
me
l
anin
and
exposure to
ultra
vio
le
t
l
ight, is disc
ussed.
K
ey
w
o
rds:
Neu
rons -
Pi
gme
nt
s
-
Spi
nal
cord
-
Ganglia -
Histoc
h
e
mi
s
tr
y
• This work was supported by a grant of the Italia n Consiglio Nazionale delle Ricerche (C. N. R.) and by a grant of the ltalian Ministero della Pubblica Istruzione.
Offprint requests to: M. Bianchi
lntr
o
duct
io
n
Litt
le
inform
at
i
o
n i
s
availab
le
which documents
t
h
e
r
elatio
n
shi
p
between lipof
usci
n
and
neuro-melanin, t
he
tw
o
p
i
gme
nt
t
ypes
which
accu
m
ulate
within the
cytoplasm
of ne
u
rons
in
ce
rtain
areas
of th
e
n
ervo
us system
.
An
ex
ten
sive
literature
exists on
the distr
i
bu-t
i
o
n,
o
rigin
and c
h
e
mi
ca
l natur
e of
lipofuscin.
Most investigat
ions were carried o
ut i
n
rodent
tis-sue (
H
ess
1
955; Su
lkin
and Scriva
ni
j
1
960;
Ku-mamoto a
n
d Bourne,
1
963;
Samorajski
et al.
1
965; Nandy and
B
ourne
1
966;
Miyagishi
et al.
1
967;
Sekhon
et al.
1969; Nandy 1971),
but
some
reports we
r
e on
ca
rniv
or
es
(Pi
ca
r
d et a
l.
19
62),
subhu
m
a
n
p
ri
mates
a
nd
man
(Zeglio
1
935
;
An-drew 1956; Oya 1959;
H
yde
n
1960;
Sm
irn
ova
1960;
Fr
iede 1962;
Bard
e
n
1970).
Hi
stochem
i
cal
a
nd
u
ltrastructu
r
al c
h
aracte
r
ist
ics of
li
pofuscin
are we
ll
known
.
It demonstrates
a strong
affinity
f
or
lipoph
i
lic dyes,
and
can
be
stained w
ith
N
il
e
B
l
ue su
l
fate. Lipofusci
n
depos
its
ex
hibit acid
fast-ness and
P
AS pos
iti
vity
a
n
d an a
d
dit
io
n
al c
h
arac-teri
s
ti
c
of th
is
kind of pigm
e
nt is it
s
bright
ye
ll
ow
orange fluorescence
when
irradiated by
ultr
a-violet
l
i
ght.
A nu
m
ber of ultrast
r
uctural investigations
h
ave
indi
cated the
s
imil
ar
it
y
b
etwee
n neuron
al
a
nd
ext
ran
eu
ro
nal
l
ipof
uscins
(Essner
and
Novi-koff
1
960;
R
adnot
and Follma
n 1974;
l
ves et al.
1975;
H
a
nzlik
owa
and Gutman
n 1
975; Koobs
et
a
l. 1
978;
Miqu
el
et a
l.
1978; Nicke
rso
n
,
1
979).
I
n
both
categories a
triphasic
o
rgani
zat
i
on
was
d
e-mo
n
strated, co
n
sisti
ng
of a
n
electron dense
ma-trix,
an electron
lu
cent
co
mpon
e
nt
a
nd
severa!
geomet
ri
ca
ll
y a
rranged
s
ub
s
tructure
s.
lt
i
s
w
i
dely
accepted that
lipofu
scin
,
which is formed
wit
hin
90
the
neuron
s
of
many
reg
ions of t
he
cen
tra
i
ner-vous syste
m
and
in
a
li
mammali
an
species,
re-present
s
a progressively
increasing
by-product
of
the aging
proce
ss.
Converse
l
y
the nature
of neu
-rome
l
anin
,
which
i
s
characteri
s
tic
of
neuron
s
found
in
pa
rticular brain
regions
i
n man
and
some, though not a
li
,
mammal
ian
s
pecies
,
ha
s
re-ma
ined as yet poorly
defi
ned. The claim
that
neu-rona
l mela
n
in
pigment first appears in man by the
5th year, it
s
amount
increasing
up to as
late as 20
years,
t
hereafter
remai
ning
quantitativel
y
a
nd
qualitati
ve
l
y
unchanged
is controve
rs
ial
(
Levi
1946).
I
n
man,
s
ubhuman primates a
nd
ca
rni-vo
res,
m
e
lan
in pigment occurs only
in the n
e
u-ron
s
of
th
e s
ub
s
tantia nigra
,
red nucleus, locus
co-erule
us,
dorsa
l motor
nucleus of
vagus
and
some
hypotha
lam
ic nuclei. Acco
rdi
ng
to
Hyden (1960),
o
nly
m
ela
n in occur
s
in the
sy
mpat
hetic gang
l
ia in
man
,
however
,
other
workers also
reported the
pre
se
nc
e
of
melanin mixed with lipofuscin in
both
sy
m
patheti
c (
Kuntz 1934
,
1945
)
and
spinal
gang-lia
(Oya
1
959;
Sm
irn
ova
1
960)
as well
as in the
Gasseri
an
ga
ng
li
on
(S
mirnova
1
960) o
f man.
W
it
hi
n
other
mam
ma
lian
s
pecies,
mela
nin
pig-ment
i
s
fou
nd
on
ly
i
n the
s
ubsta
ntia n
igra
of
gi-raffe
(Sc
herer
1944
)
a
nd in
the
s
ubsta
ntia
n
igra
an
d lo
c
u
s
coeruleus of hor
se (Sc
herer
1
944;
Ru
s-se
li
1
955).
Hi
s
tochemical investigations carried out at the
ultrastruct
ural
l
evel
by Foley a
n
d
Baxter
(t958)
an
d Ken
nedy a
n
d Zelick
so
n
(t
963) suggested t
hat
the melani
n
pigment
found in
hu
man
brain
dif-fers
from
the
me
l
ani
n
occurri
ng
in the skin,
hair
s
a
nd
vascular
membrane
s
of the eye.
The
se
find-ings
have
been corroborated
by more
recent
ul-trastructural results
(
D
uffy
and
T
ennyso
n
1965
;
Moses et a
l.
1966;
Hirosawa
1
968; R
oy a
nd
Wol-man
1
969: Schwyn et a
l.
1970; Forno and Alvord
1974
)
which have pointed to a close s
im
ilarity
be-tween
neuromelani
n
a
nd lipofu
sci
n
,
rather
than
ext
raneurona
l
mela
nin. Electron
micrographs
presented
by
these
workers
showe
d that be
s
ide
s
the typical
triphasic structu
re
of li
pofuscin
,
neu-rome
l
a
nin d
i
s
plays
an
add
itional highly electron
dense granul
ar
component.
On
the basis
of th
ese
findi
ngs
the concept of a
n
intrinsic differen
ce
be
-tween
extra
neu
ronal
and
neuronal
melan
in wa
s
established a
n
d the term
"
neu
romelani
n
"
coi
n
ed.
Accordi
ng to histochemical
investigation
s
in-tegrated w
ith
spectro
photometric
and
d
iffrac-tometric obse
r
vatio
n
s at the optical level (Barde
n
t
98 t)
neurome
lanin
is
characterized by the
pres-ence of both lipofuscin
and
melanin com
po
nents.
P
re
l
iminar
y
morphologica
l
and histochemical
s
tudies
carried out
at
t
he optica
l
(
Bianc
h
i and
M. Bianchi and A. Merighi: curonal pigment in the horse
God
i
n
a
l
982,
1
983) an d
u
ltra
s
tructura
l level
(M
er
i
ghi and
Bianchi
t
984)
in
the
h
o
rse
s
howed
the presence of lipofusc
in
alone in the
neurons of
the
s
pinal ga
nglia
and
s
pinal
cord; wh
ile th
e
pig-ment in neuro
n
s
of the ganglia of
the sympathetic
t
runk
was
found to compri
se
both lipofu
sc
in
and
melanin.
l
t
see
med therefo
re of intere
st
to
extend
our research
to a closer characterization of
intran-euro
nal
pigments.
Material
s
and
metbod
s
Examinations at the optical level were carried out on tissue from 30 horses ranging from 3 months to 20 years of age. Spi-nal ganglia, sevcral segments from thc spinal cord and ganglia of the sympathetic trunk were collected. Fresh materia! was fixed in Oouin nuid, 10% formalin, Zenker-formol and bi
-chromate-formol and Carnoy solution with minimal delay <lf-ter dea t h. Morphological characteristics an d tinctorial propert-ies of the pigment were visualized by the following proce-dures: unstained paraffin sections were mounted in a low nu-orescence mounting medium for exaÌnination under uhravio-let light; para
m
n or frozen sections were subjected t o the fol-lowing treatments (Pearse 1985): staining with lipophilic dyes (Oil Red, Sudan 111 and Sudan black R); Lillie's ferrous sul -fate reaction; Perl's method for ferri c ions: Lillic's sul furie N ile blue reaction; PAS reaction, oftcn after diasrasc trcat-ment.
In addition, bleaching with hydrogen peroxide (36 voi%). potassium permanganate, peracetic acid or oxalic acid was performed (Barden 1981).
Electron microscopical examination was conducted on spinal ganglia and ganglia of the sympathetic trunk from IO horses aged from 5 to 20 years. Uhrastructural studies of the pigment occurring in the neurons of the spina l cord were omit -ted, since at the optical level thcir characteristics appeared quite comparable to those recorded in the spinal ganglia. Small samples. promptly removed upon the dea t h of the ani· mal were fixcd in O. l M phosphatc-buffered 2.5°t glutaraldc
-hyde (p H 7.4). post-fixed in l~o Os04 in the sa me buffer. dch
y-drated, stained en bloc in 3% uranyl acetate in absolute ace-tone and embcdded in Epon-Araldite. Ultrathin scctions,
countcrstained with lead citra te or Reynolds solution, were ex-amined with a Siemens lA Elmiskop microscope. The follow-ing histochemical methods were also used: (a) periodic acid
-bismuth (PA-Ri) reaction after Ainsworth et al. (1972) modi
-fied by us t o use aqueous periodic aci d as oxidant: (b) Lillic'l> ferrous sulfate reaction modified for electron microscopy as follows:
(l) fixation in 0.1 M phosphate-bun·ered Karnovsky tìxa-tive (pH 7.4; Karnovsky 1965) at roorn temperature for 5 h; (2) treatment with 2.5% ferrous sulfate aqueous solution for 2 h followed by rinses in distilled water: (3) treatment with l% po
-tassium ferricyanide in l% aqueous acetic acid for l h: (4) wash in l o/o aqueous aceti c aci d: (5) dehydration an d embed-ding as usual. The preparations obtained by means of (a) an d (b) were observed without any further contrast reaction.
Re
s
ult
s
S
ize and location of pi
gment granules
Pigment granules w
i
thin
the neurons of the
s
pi
na
l
M. Bianchi an d A. Merighi: Neuronal pigment in the horse
Fig. l. Spina) cord, 15-year-old horse. Sudan Black x 742
Fig. 2. Spina) ganglion, 15-year-old horse. Suda n Black x 715
Fig. 3. Cranial cervical ganglion, 9-year-old horse. Sudan
Black x730
ranged
in
size
between 0.3
a
n
d 1.2 IJ.m.
In
t
h
e fo
r-mer neurons they
t
end to
accumulate into den
se
cl
ump
s a
t
o
n
e
poi
e of
the
ce
li
(Fig. l),
w
bile
in
the
latter they
appeared eithe
r
wide
l
y
dispersed
or
91
clustered into large clump
s
polarized to
t
h
e
peri-pheral
cytoplasm (Fig. 2).
In
so
me neuron
s
th
e
g
r
an
ul
es were so
tightly
packed
that
they
masked
the
celi
nucleus. Within the neuron
s of
the
gangli a
of
the
sy
mpath
e
tic trunk the
granules appeared
to
form
l
arger
masse
s,
globular or ovoid
in
shape, or
they
ex
hi
bited
irr
egular outli
n
es. They were
parti-c
ul
arly
prominent in n
e
uron
s of
the
crania
l
cervi-ca! gang
l
ion
,
in
whic
h
they atta
in
ed and
occasion-a
ll
y exceeded 5 IJ.m
in
diameter
(Fig
.
3)
.
The
gra-nules
were largely
di
s
tributed in
a
perinuclear
pattern. In the
other ganglia of t
h
e
sy
mpathetic
trunk they were less prominent
,
and appeared
to
be
r
ando
ml
y dispersed
throughout the
cytoplasm.
ln
se
ria!
sectio
n
s of
botb
s
pina!
and
autonom-i
e ga
n
gl
i
a from two
horses
,
aged 5 and
15
yea
r
s
respective
l
y,
s
tained by PAS reaction
after
dias-ta
se
tr
eatment, p
i
gment g
r
an
ul
es were found
un
-equivocally prese
nt
in
ali of t
h
e ce
ll
s,
even
though
the
amounts
differed from
o
n
e
neuron
to a
n
other
neuro
n
.
Tinctorial properties and cy
tochemical
characteristics
Light
microscopica/ ex
amination.
Under
transmit-ted light
a
n
d w
i
t
h
out s
taining
,
the pigment
occ
ur
-ring
wit
hin
the
n
eu
r
ons of t
h
e spi
n
a
!
card, s
pinal
ga
n
g
li
a a
n
d
sy
m
pathet
i
c ga
n
glia
from
young
speci
men
s exhibited a
l
ight yellow colo
r
.
However
in
you
n
g adu
l
t
an
imal
s
(4-5 year old) the
pig-ment
granu
l
es occurring
in
sy
mpathetic
gang
li
o
n
neurons
exhibited a variety
of
shades s
u
ch
a
s
dark
brown
a
nd
sometimes b
la
ck; others were pa
l
e
yel-low
and
indeed
so
me
gra
nu
les were almost
entire-ly
color
l
ess.
The pigment
granules
de
scr
ibed below
related
to
adu
lt
individual
s.
Lipophili
c
dyes.
Sudan
bla
c
k
B
elic
i
ted
m
ore
se
lec-tive
granule sta
inin
g and was
us
e
d pr
e
f
e
rentially.
We
obse
r
ved
that in the neuron
s
of the spi
n
a!
cord and spina
)
ganglia p
i
gment granules were
generally greyish ye
ll
ow, sometimes
tending
to
brown.
Converse
l
y,
in the
sympathetic ganglia,
m
ost obvious
l
y
in
t
h
e cranial cervical ganglion,
neuron
s
co
ntainin
g co
l
orless or
faintly
s
tained
granules were
found
adjace
n
t to
neuron
s
exhibit-in
g a
r
ange of shades
from dark
grey
to brown
and
black.
I
t
is
int
eresting
that
paraftin and
cryo-stat sec
tions
gave
the
sa
me result
and a
l
so
that the
affinity of
the
granu
l
es for
lipidic dye
s
was
unaf-fected
by treatment
wit
h lipid
solvents ( ethanol,
benzene,
xylene)
.
Pa
s
reaction. Ali of the
intr
aneurona
l
pigm
e
nt
granules
in the
spina
!
ganglia a
nd
the
s
pina!
cord
92
exh
ibit
ed a
rather uniform reaction
,
wherea
s
with-in the sympathetic ga
n
glion neuron
s,
in particular
in
the cra
ni
al cervica
l
gang
l
ion
,
the granules di
s
-played a variety of
s
had
es
from bright
red
to bri
c
k
r
e
d
and
brown-yellow
(the
latt
e
r being rather
s
imilar to th
e
natura! color). On
ce
again
so
m
e
granules
remained colorless.
Exposure
to ultraviol
e
t light. A bright
golden
yel-low fluore
sce
nce
,
typical
of
lip
ofuscin, was e
mit
-ted by the granules within the
s
pin
a)
cord and
s
pi-na!
ga
n
glion
n
e
uron
s (F
ig.
4)
.
Conversely,
in
the
sy
mpathetic ganglia, w
hil
e golden
ye
ll
ow
autoflu-o
r
escent granules were present withi
n
mo
s
t n
e
u-r
o
n
s
(Fig.
5), a
t
the ve
r
y s
tart of th
e
i rradiation
a
unif
o
rm d
ee
p brickred
tinge was
s
hown by th
e
pigm
e
nt granu
l
es which were pre
se
nt in
so
m
e
ran-domly
sca
tt
e
r
e
d n
e
uron
s
(Fig. 6)
.
In the latter,
howev
e
r
, a
fter
ex
po
s
ur
e
to ultra
v
iol
et
irradiation
(few
min to
2
4 h
according to the materi
a
!
und
e
r
analysis)
a
pro
g,ress
i
ve
l
y
incr
eas
ing fluore
sce
n
ce
was
s
hown b
y
th
e cy
t
oplasmic
g
ranul
es,
the
o
pti-ca)
properti
es
of which
eve
ntuall
y
became
co
m
-p
a
r
a
ble to th
ose
characteristic of t
h
e s
urroundin
g
a
utotluor
esce
nt
g
ranule
s (
Fi
g.
7).
Thi
s
ph
e
nom
e-non
was
undimini
s
hed
even whe
n
t
h
e sa
m
e
preparations
,
s
ub
se
qu
e
ntl
y
kept in
the
dark for
var
y
in
g
time interv
a
l
s
up to 3
month
s,
were again
ex
po
se
d to ultr
av
i
o
l
et light; t
h
e
pigment granules
ex
hibited
a
di
s
tinct fluor
esce
n
ce
from the
begin-ning
of
thi
s
additiona
l
ex
po
s
ure to ultra
v
iolet
light.
Melanin hi
s
to
c
hemical a
ss
ay. After treatment with
Lilli
e's
ferrou
s s
ulfate r
eac
tion no response was
ex
hibit
e
d by the neuron
s
in the
s
pina
)
cor
d
and
s
pina!
ganglia. Converse
l
y,
in the
sy
mp
at
h
etic
gang
li
a,
while pigment granul
es
occurring
within
seve
ra! n
e
uron
s
remained co
l
orless, in oth
e
rs they
appeared
faintl
y grey
and
in
a
f
ew cell
s
the
gran-ule
s
w
e
r
e di
s
tinctly positive to ferrou
s
ion uptake
,
ex
hibitin
g a
more
or
l
ess
inten
se
gree
n
co
l
or
(F
ig.
8)
. In
the
sa
me preparation
s
we
observed
that under ultraviol
et
light the intense
l
y green
pig-ment was
not fluores
ce
nt, a faint
fluore
scence w
as
Fig. 4. Spina l ganglion, l S·year-old horse. Ultraviolet light. x 790
M. Bianchi an d A. Merighi: Neuronal pigment i.n the horse
s
h
own
b
y
th~weakly
po
s
itive
ce
ll
s,
whe
r
eas
th
e
pigment th
a
t
was
e
ntir
ely
n
egat
i
ve
to
th
e
f
e
rrou
s
i
o
n
uptak
e
m
e
thod was highly fluore
sce
nt.
Th
e
pr
e
paration
s s
ubj
ec
t
ed
to
bleaching w
ith
hydrogen peroxide (36
vo
l
o/o)
for 24
h
yielded the
mo
s
t
reliab
l
e
r
esults
.
After thi
s
lap
se
of time
,
th
e
pigment n
at
urall
y
brownisb
in
visible
light
ap-pea
r
ed entirely co
l
orless.
In
th
e s
ame pr
e
para-ti
o
n
s
tr
ea
ted with the Lillie
's
ferrou
s s
ulfate
meth-od the bindin
g c
apacity of
th
e
pigm
e
nt f
o
r
ferrous
ion
uptake was
s
uppre
sse
d. Th
ese
r
es
ult
s
indicate
th
at
th
e
pigm
e
nt
g
ranule
s co
n
s
i
s
tent
l
y found
with-in
the
n
eurons of
th
e s
pina!
gang
li
a and
s
pinal
cord
,
as well
in
sy
mpathetic ganglia in hor
ses
un-d
e
r 4
yea
r
s
of a
ge,
ma
y
be cons
id
ered as
typical
lipofu
sc
in.
Co
nv
e
r
se
ly
,
withi
n
t
h
e c
ranial cervical
gangl
ion
and to a !e
sse
r extent in the ot
h
er gangl
'
i
a
of th
e sy
mpath
e
tic trunk from pre
-a
dult or adult
spec
imen
s,
other granules be
s
id
es
tho
se ex
hibiting
typical
lipofu
s
cin
feature
s,
di
s
pla
ye
d
characteris-tics
s
ugge
st
i
ve
of m
e
l
a
nin
or an
int
e
rm
e
diat
e
be-tween
lipofu
sc
in
a
nd m
e
l
a
nin.
Electron
mi
c
r
oscopy
G
e
n
e
ra/
morphology.
Within the
neuron
s
o
f the
s
pina!
gangl
i
a,
numerou
s
r
e
l
atively
s
mall
gran-ule
s,
genera
ll
y
clu
s
tered into
co
n
s
pi
c
u
ous
c
lump
s
at th
e
celi
p
e
riphery
,
were
found. They were
m
ore
or
le
ss
g
l
obular or ovoid
in
s
h
a
p
e,
th
e
ir
out
lin
e
b
eco
ming irr
eg
u
l
ar due to th
e
fu
s
ion of finer
gra
-nule
s
.
They appeared
to contain a
r
el
atively
hom-oge
n
eo
u
s
or g
r
anular
,
mod
e
rat
el
y
e
l
ec
tron
opaq
u
e
m
atrix
,
and were
bound by
a
limiting
membrane which
,
how
eve
r
,
was not always
c
l
ea
r-l
y
di
sce
rnible. Within th
e
m
a
trix
,
one or
mor
e
el
ectron lucent a
re
as and a
numb
e
r
of
l
a
m
e
llar
sys
tems
were
v
i
sua
li
ze
d
(
Fig.
9)
.
The
electron
lu
-cent areas
identifiable
as
lipid
vacuo
l
es,
ma
y
be
ce
ntrally or
ecce
ntrically embedded
in the m
atri x;
frequ
e
ntl
y
they pr
o
truded from th
e
granu
l
e
p
e
ri-phery
,
though
s
tili bound b
y
the limiting
mem-brane
.
The l
a
mellar
sys
t
ems s
howed a
s
traight
or
c
urvilinear
configuration
,
r
e
mini
sce
nt
of
"
finger-print
s".
Each
J
ame
ll
ar
unit
was co
mpo
se
d
of a
s
lend
e
r
(22
nm) dark
ce
ntrai
l
ame
ll
a
flanked
on
Fig. 5. Cranial cervical ganglion, 12-year-old horse. Ultraviolet light. x 790
Fig. 6. Cranial cervical ganglion, 12-year-old horse. Ultraviolet light, at the very start of irradiation, x 790
Fig. 7. The same celi of Fig. 6, 2 h after irradiation with ultraviolet light. x 790
94 M. Bianchi an d A. Merighi: Ncuronal pigment in the horsc
Fig. 9. Low magnification of the lipofuscin pigment in a neuron of the spinal_ganglia. n nucleus of a satellite celi. Arrowhead.r indi-cate the border between the satellite celi and the neuron. x 7,750
Fig. IO. High magnification of the lipofuscin pigment in a spina) ganglion neuron. Note the lamellar system. Arrow.r indicate the lamellae in which the fundamental uni t of the system is clearly appreciable. m matrix: v vacuo le. x 72,000
M. Bianchi and A. Merighi: Neuronal pigment in the horse 95
Fig. t t. Neuromelanin pigment in the cranial cervical ganglion. Different morphological aspects of the deposition of the highly
electron dense materia l are shown. In the granule of b the limiting membrane is well evident (arrows). x 32,000
Fig. 12. Neuromelanin pigment in a neuron of a thoracic sympathetic trunk ganglion. Note the similarity with the granule of Figs. Il b an d c. x 25,000
96
M. Bianchi and A. Merighi: Neuronal pigment in the horseM. Bianchi an d A. Merighi: Neuronal pigment in the borse
e
ith
er s
id
e
b
y a
thi
cker (
45 nm
)
l
ess e
l
ectron
dense
l
a
m
e
ll
a (Fig.
l
0).
Lam
e
ll
a
r
syste
m
s
d
iffered
in
exte
nt
w
ithin
the
same
speci
m
e
n
,
r
e
pl
ac
in
g
the matrix
a
lm
os
t
e
n
-tirely
in
so
m
e cases, o
r
consis
tin
g of a
few
i
nd
i-vid
uai unit
s scatte
r
ed
within tbe
matri
x.
In
the
c
r
a
ni
a
l
cervica
l
ga
n
g
li
o
n
,
pigment
gra
nul
es were
les
s num
e
r
o
u
s, coa
r
se
r
, and
m
o
r
e
irregular
i
n
out-lin
e
th
an
those
occu
rrin
g
intr
a
n
e
uron
a
ll
y
in
the
sp
in
a
!
gang
li
a. Not
in
f
r
e
qu
en
tl
y gra
n
u
l
e
coales-ce
nce int
o
m
o
r
e
pr
o
min
e
nt
,
irr
egu
l
a
r
, o
ft
e
n
lobu-l
a
r
clu
mp
s was
noted
.
Genera
ll
y,
th
e gra
nul
e
li
-m
iti
ng m
embra
ne
.
wa
s
more readily
d
emonstrab
l
e.
In
a
dditi
o
n t
o
th
e g
r
a
nule
subst
ru
c
tur
es
d
escr
ib
e
d
i
n t
he
sp
i
nal ga
n
gl
i
a
(i. e.,
the
mat
rix,
l
ame
ll
a
r
sys-t
ems
and
lipid
vac
u
o
l
es), t
h
ese gran
ul
es co
n
s
i
s-tently di
spl
ayed
a
highly
e
l
ectro
n
dens
e
particu
-late
co
m
po
n
e
nt.
Thi
s
materia!
appeared
i
n
th
e
form
of
s
mall
c
l
usters randomly
scatte
r
ed
thr
o
u
gh
t
h
e
m
a
t
rix
in
so
me
gra
nul
es,
in
othe
r
s as a
fine
gra
nul
a
rit
y
d
isse
min
ate
d
u
p
o
n th
e
l
ame
ll
ae, a
nd
yet
in
ot
her
granules it
form
e
d
a
n
e
l
ec
tr
o
n d
e
n
se
rin
g
a
r
ound
the
li
pid
vac
u
oles.
Th
ese va
r
io
u
s
di
s-tribution
patt
e
rn
s
also
coex
i
ste
d
wi
thin
s
in
g
l
e
gra-nul
es (Fig.
Il
).
Si
mil
ar
features
to
th
ose j
u
st desc
r
ibed i
n
the
c
r
a
nial
ce
r
vica
l
ga
n
g
li
o
n
were exhibited by the
pi
gme
nt
occ
urr
i
n
g
i
n the neuron
s
o
f
ot
h
er a
ut
o-n
o
mi
e ga
n
glia.
H
owever,
in
t
h
ese gra
nul
es
th
e
e
l
ect
ron den
se
mat
e
ri
a
!
wa
s
l
ess ab
und
a
nt
,
and
t
h
e
granu
l
e
t
ype
pertaining
to
t
h
e
cra
ni
al cervical
ga
nglion
,
represe
n
ted in Fig.
Il
a, was
n
ever
f
o
un
d
(see
Fig.
1
2).
Ultrastru
c
tural
cy
t
oche
mi
stry
Periodi
c
acid-bi
s
muth
(
PA
-Bi)
reaction
.
In
t
h
e
sp
i
-n
a
!
ga
n
glia,
th
e a
re
as occ
upi
ed
b
y the
m
atrix and
l
a
m
e
ll
ar
sys
tem
s
were
s
een
to
be
coated
b
y t
h
e
electron de
n
se
bi
s
muth
pr
ecipitate which
s
harply
d
e
mar
cate
d
the
zo
n
es occ
upi
e
d b
y
the
l
i
pid
vacu-oles (Fig.
1
3). Except for t
h
e occas
i
o
n
a
i ri
boso
m
e,
97
n
o ot
h
e
r
cyto
pl
as
mi
c st
ru
c
tur
e yie
ld
ed a
reaction
de
p
osit.
I
n gang
l
ia of
the
sy
m
pathetic
trunk
,
n
ot-ably
in the
c
r
a
n
ia
l ce
rvica
l
gang
li
o
n
,
th
e
matrix of
int
raneuro
n
al
pigm
e
nt
g
r
an
ul
es was
n
ot
so
h
omo-geneously
loa
ded
with t
h
e
bi
smut
h pr
ec
ipitate
;
so
m
e
negativ
e
a
r
eas were c
l
ea
rl
y detec
t
able
(Fig
. 14
).
The
se
areas appea
r
ed
to
cor
r
es
pond
to
t
h
ose occu
p
ied by t
h
e
addit
i
onal
highl
y
e
l
ectron
d
e
n
se
particu
l
ate
m
ater
ia!
typical
of
neuro
m
e
l
a
n
-i
n g
r
anu
l
es.
.
Th
e
reaction
pr
oduct was co
n
s
i
ste
ntl
y
re
st
ri
ct-e
d to
t
h
e
matrix
a
n
d t
h
e
l
a
m
e
ll
ar
syste
m
s
(i. e. to
PAS posit
i
ve
co
m
pone
n
ts), while
the
response
o
f
lipid
vac
u
o
l
es
to
th
e b
i
s
muth
reaction
was
n
ega-t
i
ve.
Lillie
's
ferrous
s
u/fate
re
a
c
ti
o
n
.
Lilli
e's
ferrous
s
u
l
-f
ate
r
eac
tion
was never
p
osit
i
ve i
n
the
n
eu
r
ons
o
f
the
spina
!
ga
n
g
li
a
(F
i
g
.
1
5).
Whereas
in
t
h
ose of
t
h
e crania
l
ce
r
vica
l
ga
n
glion and
in
ot
h
er ga
n
gl
i
a
of
the
sy
mpathet
ic
trunk, uptake
o
f
ferrous ions
by the p
i
gme
nt
g
r
a
nul
es occ
urr
e
d
,
ferrous
i
ons
de
m
arcati
n
g the
l
ipid
vacuoles and
in
some
cases
bei
n
g
di
st
r
ibuted over t
h
e
matr
i
x (Fig.
1
6).
The
l
ipid vacuo
l
es were
n
egative.
The
pr
ec
i
p
it
a
t
e ex
hi
-b
it
e
d
a
l
ocat
i
o
n
cor
r
espo
ndin
g
to
t
h
e
s
it
es
occup-i
e
d b
y t
h
e
hi
g
hl
y e
l
ec
tr
o
n
dense particu
l
ate
mat
e-ri
a!
in
co
n
ventiona
l m
ic
r
ogra
ph
s.
Non
s
pe
c
i
fic
rea
ct
i
o
n
s
were
n
ot
s
hown
by any
ot
h
er ce
li
compo
n
ents.
Discussion
F
u
rt
h
e
r
ev
id
ence is p
r
esented bere for the
d
iffer-ent
ial
intraneurona
l
pigmentation between the
s
p
i
n
a) ga
n
g
li
a a
nd
those
o
f
the
sym
path
etic
trunk
in th
e
h
orse,
prev
i
ously o
utlin
ed
b
y
li
g
ht
a
nd
e
l
ec-tro
n
microscopic
in
ves
tig
at
i
o
n
s
(B
i
anchi and
Go-d
i
na
1
982,
1
983;
M
er
i
ghi a
n
d Bianchi
1
984).
Within
th
e
n
e
ur
ons of
the
spi
n
a
!
ga
n
gl
i
a a
nd
s
pina!
co
rd
,
pigment
granu
l
es
ex
hibit
a
m
o
r
e o
• Fig. 13. P A-Bi. reaction performed on the lipofuscin pigment of a spina l ganglion neuron. a contro l section (treatment: periodi c acid
3 mi n, no counterstain); b positive reaction. The electrondense precipitate fills almost completely the matrix, while the lipid vacuoles
rcmain negative (treatment: periodic acid 3 min, bismuth subnitrate 60 min, no counterstain). x 25,000
Fig. 14. P A-Bi. reacti<\n performed on the neuromelanin pigment of a cranial cervical ganglion neuron. a control section. Note the residua! electrondensity of the melanin component of the granules (treatment: periodi c aci d 3 m in no counterstain). b positive reac·
tion. Note the beterogeneous deposition of ù1e bismuth precipitate on the granules (treatment: periodic acid 3 min, bismuth
subni-trate 60 m in, no counterstain). x 35,000
Fig. IS. Lillie ferrous sul fate reaction modified for electron microscopy performed on the lipofuscin pigment of a spina l ganglion neuron. Negative reaction (compare with Fig. 16). x 54,000
Fig. 16. Lillie ferrous sul fate reaction modified for electron microscopy performed on t be neuromelanin pigment of a cranial cervi-ca! ganglion neuron. Positive reaction (compare with Fig. 15). Arrow indicates the electroudeuse rim in the lipid vacuole in materia! not fixed in osmium x 40,000
