Autoradiographic localization of putative melatonin receptors in the brains of two old world primates: Cercopithecus aethiops and Papio ursinus


Stankov, B.; Capsoni, S.; Lucini, V.; Fauteck, J.; Gatti, S.; Gridelli, B.; Biella, G.; Cozzi, B.; Fraschini, F.

Neuroscience 52(2): 459-468

1993


The distribution of putative melatonin receptors in the brains of two Old World primates of the superfamily Catarrhina, Cercopithecus aethiops and Papio ursinus, was characterized using 2-[125I]iodomelatonin autoradiography. The specific binding demonstrated a discrete distribution pattern. The median eminence was intensely labelled, and examination at the light microscopic level demonstrated that the binding was confined to the small layer of cells comprising the pars tuberalis of the pituitary gland. The collar of pars distalis, present in the baboon (Papio ursinus), was diffusely labelled. No binding was detected in the pars distalis proper or the neural lobe of the pituitary gland. The binding in the suprachiasmatic nuclei was weaker, but well discernible. Diffuse faint specific binding was found in the frontal cortex and the dentate gyrus of the hippocampus. Two non-neural sites expressed strong, well-delineated binding: the walls of some brain blood vessels (the vertebral and spinal arteries, the inferior cerebellar and acoustic arteries, the basilar, pericallosal, internal carotid arteries, the arteries forming the circle of Willis) and the choroid plexuses. Binding in the arteries of the circle of Willis, the pars tuberalis and the suprachiasmatic nuclei was readily displaceable. Addition of 1 microM unlabelled 2-iodomelatonin following 45 min of preincubation with the radioactive ligand completely abrogated the binding. Co-incubation with guanosine 5'-O-(3-thiotriphosphate) led to a significant decrease in the apparent binding density in the pars tuberalis and abolished binding in the suprachiasmatic nuclei, but was without effect on the binding in the walls of the adjacent arteries, forming the circle of Willis, in the cortex and in the hippocampus. This qualitative distribution pattern demonstrates that in the two primate species studied, melatonin high-affinity, G-protein-linked binding sites are present in the pars tuberalis and the hypothalamic suprachiasmatic nuclei, and that melatonin may be acting as a synchronizer of the endogenous pacemakers' circadian activity, apart from its possible reproductive effects at the level of pars tuberalis, where the highest receptor density was observed. The strongly labelled arterial walls, and the flimsy labelled cortex and hippocampus, expressed different characteristics: though the binding was readily reversible, it was apparently not regulated by a guanine nucleotide-binding protein.

Neuroscience
Vol.
52,
No.
2,
pp.
459-468,
1993
0306-4522/93
$6.00
+
0.00
Printed
in
Great
Britain
Pergamon
Press
Ltd
C
1992
IBRO
AUTORADIOGRAPHIC
LOCALIZATION
OF
PUTATIVE
MELATONIN
RECEPTORS
IN
THE
BRAINS
OF
TWO
OLD
WORLD
PRIMATES:
CERCOPITHECUS
AETHIOPS
AND
PAPIO
URSINUS
B.
STANKOV,*t
S.
CAPSONI,
*
V.
LucINI,I
J.
FAUTECK,*
S.
GATT1,§
B.
GRIDELLI,§
G.
BIELLA,11
B.
Cozzi11
and
F.
FRASCHIN1*
*Chair
of
Chemotherapy,
Department
of
Pharmacology,
Institute
of
Human
Anatomy,
§Center
for
Liver
Transplantation,
II
Institute
of
Physiopathology
and
Therapy
of
Pain,
¶Institute
of
Anatomy
of
Domestic
Animals,
University
of
Milan,
via
Vanvitelli
32,
20129
Milano,
Italy
Abstract—The
distribution
of
putative
melatonin
receptors
in
the
brains
of
two
Old
World
primates
of
the
superfamily
Catarrhina,
Cercopithecus
aethiops
and
Papio
ursinus,
was
characterized
using
2-
[
I25
1]iodomelatonin
autoradiography.
The
specific
binding
demonstrated
a
discrete
distribution
pattern.
The
median
eminence
was
intensely
labelled,
and
examination
at
the
light
microscopic
level
demonstrated
that
the
binding
was
confined
to
the
small
layer
of
cells
comprising
the
pars
tuberalis
of
the
pituitary
gland.
The
collar
of
pars
distalis,
present
in
the
baboon
(Papio
ursinus),
was
diffusely
labelled.
No
binding
was
detected
in
the
pars
distalis
proper
or
the
neural
lobe
of
the
pituitary
gland.
The
binding
in
the
suprachiasmatic
nuclei
was
weaker,
but
well
discernible.
Diffuse
faint
specific
binding
was
found
in
the
frontal
cortex
and
the
dentate
gyrus
of
the
hippocampus.
Two
non-neural
sites
expressed
strong,
well-delineated
binding:
the
walls
of
some
brain
blood
vessels
(the
vertebral
and
spinal
arteries,
the
inferior
cerebellar
and
acoustic
arteries,
the
basilar,
pericallosal,
internal
carotid
arteries,
the
arteries
forming
the
circle
of
Willis)
and
the
choroid
plexuses.
Binding
in
the
arteries
of
the
circle
of
Willis,
the
pars
tuberalis
and
the
suprachiasmatic
nuclei
was
readily
displaceable.
Addition
of
1µM
unlabelled
2-iodomelatonin
following
45
min
of
preincubation
with
the
radioactive
ligand
completely
abrogated
the
binding.
Co-incubation
with
guanosine
5'-0
-(3-thiotriphosphate)
led
to
a
significant
decrease
in
the
apparent
binding
density
in
the
pars
tuberalis
and
abolished
binding
in
the
suprachiasmatic
nuclei,
but
was
without
effect
on
the
binding
in
the
walls
of
the
adjacent
arteries,
forming
the
circle
of
Willis,
in
the
cortex
and
in
the
hippocampus.
This
qualitative
distribution
pattern
demonstrates
that
in
the
two
primate
species
studied,
melatonin
high-affinity,
G-protein-linked
binding
sites
are
present
in
the
pars
tuberalis
and
the
hypothalamic
suprachiasmatic
nuclei,
and
that
melatonin
may
be
acting
as
a
synchronizer
of
the
endogenous
pacemakers'
circadian
activity,
apart
from
its
possible
reproductive
effects
at
the
level
of
pars
tuberalis,
where
the
highest
receptor
density
was
observed.
The
strongly
labelled
arterial
walls,
and
the
flimsy
labelled
cortex
and
hippocampus,
expressed
different
characteristics:
though
the
binding
was
readily
reversible,
it
was
apparently
not
regulated
by
a
guanine
nucleotide-binding
protein.
Melatonin
(N-acetyl-5-methoxytryptamine)
is
now
considered
the
most
important
hormonal
product
of
the
vertebrate
pineal
gland.
The
synthesis
and
release
of
melatonin
in
the
peripheral
blood
and
the
cere-
brospinal
fluid
(CSF)
is
controlled
by
an
endogenous
biological
clock(s)
and
is
synchronized
to
the
preva-
lent
photoperiod
by
light.'
The
peripheral
blood
and
the
CSF
levels
of
this
indole
are
extremely
high
at
night
and
low
to
undetectable
during
daytime.
In
most
species,
the
length
of
melatonin
peak
augments
in
proportion
to
the
preponderant
period
of
dark-
tTo
whom
correspondence
should
be
addressed.
Abbreviations:
CP,
choroid
plexus;
CSF,
cerebrospinal
fluid;
GTP
7
S,
guanosine
5'-0
-(3-thiotriphosphate);
ME,
median
eminence;
PCA,
posterior
communicating
artery;
PT,
pars
tuberalis
of
the
pituitary
gland;
SCN,
suprachiasmatic
nuclei;
Tris,
Tris[hydroxymethyl]-
aminomethane.
ness.
Melatonin
is
now
thought
to
serve
as
a
funda-
mental
biochemical
transducer
of
the
photoperiodic
information
from
the
environment,
the
"chemical
expression
of
darkness".
26,38
In
recent
years
the
presence,
distribution,
kinetic
properties
12,20,25
and
in
some
cases
the
signal-
transduction
mechanism(s)
19
.
283
"
of
the
melatonin
receptor
in
the
CNS
of
different
vertebrates,
includ-
ing
mammals,
have
been
described
(for
reviews
see
Refs
10,
31,
35
and
36),
by
using
24
125
fliodomelatonin.
This
high-specific
activity
ligane
(c.
1800
Ci/mmol)
proved
to
be
an
extremely
effective
agonist
probe,
having
an
enhanced
affinity
for
the
receptor
and
is
now
considered
a
safe
tool
for
mapping
of
melatonin
receptors
in
the
CNS
(for
reviews
see
Refs
10,
31,
35
and
36).
From
the
studies
performed
so
far
with
mam-
malian
brains
it
appeared
that
the
melatonin
receptor
was
expressed
with
a
discrete
distribution,
and
the
459
460
B.
STANKOV
et
al.
pars
tuberalis
(PT)
of
the
pituitary
gland
and
the
suprachiasmatic
nuclei
of
the
hypothalamus
(SCN)
have
been
most
frequently
mentioned
as
potential
melatonin
targets.
9
'
2
"'
3744
'
45
It
should
be
noted,
how-
ever,
that
there
seem
to
exist
subtle
species
differences
in
the
general
distribution
pattern.
In
some
mammals,
high-affinity
melatonin
receptors
were
discovered
in
other
neural
and
non-neural
brain
structures,
like
certain
thalamic
nuclei,'
area
postrema
in
the
rat,
19
the
retina,"
the
cortex
and
hippocampus,"'"."
and
the
choroid
plexuses
of
some
rodents'
and
the
rabbit'
(for
recent
review
see
Ref.
36).
Interestingly,
melatonin
receptors
were
seemingly
absent
in
the
ferret'
and
sheep
9
SCN,
and
were
not
reported
in
the
human
PT.
In
the
latter
species,
specific
binding
was
described
in
the
SCN.
27
A
recent
report'
showed
that
in
the
rat,
melatonin
receptors
were
expressed
in
the
arteries
involved
in
thermoregulation
(the
basal
brain
arteries,
forming
the
circle
of
Willis
and
the
caudal
arteries).
Thus,
melatonin
is
now
thought
to
be
acting
at
the
level
of
non-neural
tissues
as
well
(arterial
vasculature
in
certain
blood
vessels
and
the
choroid
plexuses),
at
least
in
some
species.
Little
is
known
about
the
melatonin
receptor
distri-
bution
in
the
brain
of
primates.
Apart
from
one
study
that
examined
the
human
hypothalamus,'
a
recent
short
communication
reported
the
existence
of
2-
[
125
I]iodomelatonin
binding
in
the
SCN
and
the
PT
of
the
Rhesus
monkey.'
We
report
here
the
apparent
distribution
of
the
putative
melatonin
receptors
in
the
brains
of
two
Old
World
primate
species,
Cercopithecus
aethiops
and
Papio
ursinus.
EXPERIMENTAL
PROCEDURES
Materials
21
125
Illodomelatonin
(sp.
act.
1600-2000
Ci/mmol)
was
purchased
from
Amersham
(Buckinghamshire,
U.K.).
Drugs
and
chemicals
were
obtained
from
Sigma
Chemical
Company
(St
Louis,
MO)
unless
otherwise
stated.
2-
FIPodomelatonin
was
acquired
from
RBI
(Natick,
MA).
The
anaesthetics
and
the
sterile
solutions
used
during
the
surgery
were
from
Farmitalia-Carlo
Erba,
Milano,
Italy.
The
autoradiographic
film
X-OMAT
S
was
from
Kodak,
Rochester,
NY.
Hyperfilm
MP
was
from
Amersham.
Animals
The
brains
of
five
(three
females
and
two
males)
vervet
monkeys
(Cercopithecus
aethiops)
and
four
(three
females,
one
male)
baboons
(Papio
ursinus)
were
obtained
from
the
Center
for
Liver
Transplantations,
University
of
Milan.
The
animals
employed
were
healthy,
sexually
mature
individuals,
used
as
donors
in
the
course
of
a
combined
lung—liver
transplantation
experimental
surgery.
The
brains
were
collected
4-6
h
after
the
beginning
of
the
surgery,
when,
following
removal
of
the
organs
needed
for
transplantation
purposes,
euthanasia
was
effectuated
by
exsanguination.
The
anaesthesia
consisted
of
initial
intra-
muscular
Ketamine
(10
mg/kg
body
weight),
followed
by
intravenous
Ketamine
(2
mg/kg),
and
continued
with
1
ml
intravenous
Droperidol
plus
Fentanyl
(50:1).
If
necessary,
this
was
followed
by
Fentayl
in
the
course
of
the
surgical
intervention.
The
brains
were
rapidly
removed,
and
the
dissected
areas
frozen
by
immersion
in
cold
isopentane
at
30°C.
The
samples
were
then
transferred
and
stored
at
—70°C
before
analysis.
Autoradiography
Sections
(20µm)
were
cut
in
a
cryostat
at
—20°C
and
thaw-mounted
on
chrome—alum
gelatin-coated
slides.
The
sections
were
left
overnight
at
4°C
in
a
desiccator
and
kept
thereafter
at
—70°C
for
no
longer
than
two
weeks.
The
whole
hypothalamus
and
the
overlaying
thalamus,
up
to
the
ventral
border
of
corpus
callosum
were
examined
at
inter-
vals
of
100-800
pm.
Additionally,
samples
of
the
occipital,
parietal
and frontal
cortices
and
medulla
oblongata
were
inspected.
Slide
mounted
sections
were
allowed
to
warm
at
room
temperature,
passed
sequentially
in
preincubation
Tris—bovine
serum
albumin
buffer
(50
mM
Tris—HCI,
pH
7.4
at
25°C,
4
mM
CaC1
2
,
0.1%
bovine
serum
albumin),
then
incubated
with
100-120
pM
2-VIDodomelatonin
in
the
same
buffer
for
1
h
at
24°C,
followed
by
two
15-min
washes
at
0°C
in
Tris—bovine
serum
albumin
and
Tris—CaC1
2
,
respectively,
and
dipping
for
30
s
in
bidistilled
water.
The
non-specific
binding
was
determined
in
adjacent
sections
in
the
presence
of
1µM
2-iodomelatonin.
In
a
separate
series
of
displacement
studies,
following
initial
incubation
(45
min)
with
the
radioactive
ligand,
1µM
2-
iodomelatonin
was
added
to
the
incubation
medium
and
the
incubation
was
continued
for
another
45
min.
When
the
effects
of
100µM
guanosine
5'-O-(3-thiotriphosphate)
(GTR,S)
were
evaluated,
the
quantity
of
the
labelled
2-
[
125
I]iodomelatonin
was
30
pM.
The
slides
were
apposed
to
the
autoradiographic
film
in
light-proof
autoradiographic
cassettes.
Autoradiograms
were
visually
inspected,
and,
subsequently,
a
semi-quantitat-
ive
analysis
was
performed
on
a
computerized
image
analy-
sis
system.
The
optical
densities
of
the
film
images
generated
from
sets
of
slides
processed
under
identical
experimental
conditions
were
compared
to
express
the
results.
Adjacent
sections
were
stained
with
Cresyl
Violet
or
haematoxylin—eosin
and
examined
by
light
microscopy
to
verify
the
location
of
the
structures.
In
certain
cases
micro-
manipulating
apposition
of
the
autoradiographic
film
over
the
same
section
slide,
following
staining,
was
employed
to
pinpoint
the
exact
location
of
the
binding.
RESULTS
The
specific
binding
on
the
autoradiograms
was
detected
in
discrete
brain
regions,
as
dark
spots
and
areas,
in
contrast
to
the
non-specific
binding,
which
was
usually
extremely
low,
homogeneous
and
equal
to
the
background
of
the
sections.
Figures
1
and
2
show
that
the
general
distribution
pattern
observed
in
brains
of
both
species
under
study
was
very
similar.
The
region
of
the
median
eminence
was
the
most
intensely
labelled.
However,
further
examination
of
the
binding
at
the
light
micro-
scopic
level
clearly
demonstrated
that
the
binding
in
this
area
was
located
exclusively
in
the
tiny
layer
of
cells
comprising
the
pars
tuberalis
of
the
pituitary
gland
(Fig.
3).
Clearly,
in
the
course
of
specimen
collection
the
PT
was
divided
in
two,
and
binding
was
found
in
both
the
hypothalamic
PT
and
pituitary
PT
(Figs
IA
and
2A,
upper
and
lower
panels).
The
median
eminence
(ME),
even
in
its
external
zone,
which
is
close
to
the
PT,
was
devoid
of
binding
(Fig.
3).
The
collar
of
pars
distalis,
present
in
the
Melatonin
receptors
in
the
primate
brain
461
4
S
C
N
OC
SA
Fig.
1.
Distribution
of
24
125
1liodomelatonin
binding
in
coronal
sections
(A—D)
of
vervet
monkey
brain
and
pituitary.
The
dark
zones
in
the
autoradiographs
indicate
the
areas
of
specific
2-[
125
I]iodomelatonin
binding.
The
non-specific
binding
(B,
adjacent
section
to
A)
was
homogeneous
and
equal
to
the
background.
(E,
F)
Parasagittal
sections.
Scale
bar
=
2.5
mm.
Abbreviations
used
in
the
figures
AA
inferior
cerebellar
and
inferior
acoustic
arteries
AC
anterior
commissure
ACA
anterior
cerebral
artery
AH
Ammon's
horn
BA
basilar
artery
CP
choroid
plexus
DG
dentate
gyrus
of
the
hippocampus
FLM
fasciculus
longitudinalis
medialis
G
gray
matter
ICA
internal
carotid
artery
MCA
middle
cerebral
artery
ME
median
eminence
NL
neurolobe
of
the
pituitary
gland
OC
optic
chiasm
PCA
posterior
communicating
artery
PcB
pericallosal
branch
of
the
anterior
cerebral
artery
PD
pars
distalis
of
the
pituitary
gland
PI
pars
intermedia
of
the
pituitary
gland
SA
spinal
artery
SCN
suprachiasmatic
nuclei
V
vein
VA
vertebral
artery
VP
venous
plexus
Illy
(3v)
third
ventricle
462
B.
STANKOV
et
al.
A
3v
PT
11
,4111
PD
NL
pi
DG
CP
SA
l/
AA
I
A
H
C
P
A
Fig.
2.
Distribution
of
24
12
1podomelatonin
binding
in
coronal
sections
of
baboon
brain
and
pituitary.
The
non-specific
binding
(B,
adjacent
section
to
A)
was
homogeneous
and
equal
to
the
background.
Scale
bar
=
2.5
mm.
P
PT
T
44
0-
-
~
ME
re
Lf
,
17
1
11
."
PT
v
4
,
1 t
PT
M
E
41#
Fig.
3.
Higher
magnifications
of
the
autoradiographs
shown
in
the
upper
panels
of
Figs
IA
and
2A,
i.e.
the
region
of
the
median
eminence
of
the
vervet
monkey
(a)
and
baboon
(d).
The
autoradiographic
films
(a,
d)
were
apposed
by
micromanipulation
over
the
microscopic
slide
(b,
e,
respectively)
following
staining
with
Cresyl
Violet,
to
produce
the
composite
images
of
c
and
f,
respectively.
Note
that
the
binding
is
confined
to
the
layer
of
pars
tuberalis
cells,
and
that
the
median
eminence
is
devoid
of
binding
even
in
its
external
zone,
close
to
the
PT.
Scale
bar
=
1
mm.
Melatonin
receptors
in
the
primate
brain
463
baboon,
was
diffusely
labelled
(Fig.
1A,
lower
panel)
but
no
binding
was
detected
in
pars
distalis
proper
or
the
neurolobe
of
both
species.
The
suprachiasmatic
nuclei
were
labelled
diffusely,
but
the
level
of
the
binding
was
higher
than
that
seen
in
the
gray
of
the
frontal
cortex
and
the
dentate
gyrus
of
the
hippocampus.
Diffuse
weak
binding
was
recorded
in
the
anterior
hypothalamic
area
(Figs
1D—F,
2D
and
4D;
see
Table
1
for
semi-
quantitative
comparison).
Another
interesting
finding
in
both
species
was
the
extremely
strong
binding
in
two
non-neural
sites
of
the
brain,
the
choroid
plexuses
(CPs)
and
the
basal
brain
vessels.
Figures
1C
and
2C
show
the
heavily
labelled
CP
in
the
vicinity
of
the
fourth
ventricle
and
the
hippocampus
(Fig.
2D).
The
inferior
cerebellar
and
inferior
acoustic
artery
and
a
number
of
basal
brain
blood
vessels,
that
were
confidently
identified
as
the
internal
carotid
artery,
posterior
communicating
artery
(PCA),
basilar
artery,
spinal
artery
and
the
vertebral
artery
(Figs
1
C
and
2C),
showed
strong
binding.
In
an
attempt
to
verify
if
the
binding
observed
in
the
above-mentioned
brain
arteries
was
present
in
other
brain
arteries,
including
the
circle
of
Willis,
as
demonstrated
in
the
rat,'
we
carefully
examined
the
binding
pattern
over
a
wide
range
of
medial
sagittal
sections.
An
example
of
autoradiographs
from
serial
sections
(800
gm
apart)
of
the
vervet
monkey
preoptic
area
is
shown
in
Fig.
4.
Clearly,
the
specific
binding
was
expressed
over
a
long
dis-
tance
of
the
arterial
vasculature,
starting
anteriorly
from
the
pericallosal
branch
of
the
anterior
cerebral
artery.
The
anterior
cerebral
arteries
and
middle
cerebral
arteries
also
expressed
strong,
well-
delineated
binding.
Higher
magnification
of
the
autoradiographs,
gen-
erated
from
sections
of
different
arteries,
revealed
that
the
binding
was
located
in
the
arterial
walls.
Micromanipulation
apposition
of
the
autoradio-
graphic
film
over
the
same
section
slide
following
Cresyl
Violet
staining
showed
that
the
veins
in
the
vicinity
of
the
labelled
arteries
did
not
express
any
binding
(Fig.
5).
Higher
magnification
and
micromanipulation
apposition
of
the
autoradiographic
film
over
the
stained
sections
containing
the
CPs
demonstrated
that
binding
was
confined
to
the
CP
(Fig.
6a,
b)
A
\
AC
As
MCA
E
O"
ACA
S
ACA
.1
MCA
ra.
L
ACAS
Fig.
4.
Autoradiographs
from
coronal
sections
of
the
basal
part
of
the
preoptic
area
of
the
vervet
monkey
brain,
showing
strong
binding
in
the
arteries
forming
the
circle
of
Willis.
Sections
A—E
are
800µm
apart.
(F)
Section
adjacent
to
C,
showing
the
level
of
non-specific
binding.
Scale
bar
=
2.5
mm.
464
B.
STANKOV
et
al.
Table
1.
Distribution
of
24
123
I]iodomelatonin
binding
in
the
brains
of
the
vervet
monkey
and
the
baboon,
and
the
effects
of
GTP
y
S
and
1µM
2-iodemelatonin
in
the
displacement
studies
Papio
ursinus
Cercopithecus
aethiops
Area
Control
+
GTP,
St
2IMI
Control
+
GTP,
St
2IMI
PT
0.86
0.042*
ND
0.93
0.051*
ND
SCN
0.23
NT
NT
0.25
ND
ND
AHy
0.16
NT
NT
0.18
NT
NT
DG
0.11
0.10
NT
0.10
0.10
NT
AH
0.06
NT
NT
0.08
NT
NT
FC
0.08
0.07
NT
0.10
0.12
NT
PCA
0.91
0.89
0.16**
0.88
0.86
0.19
1
*
CP
0.56
NT
NT
0.42
NT
NT
The
data
are
given
as
mean
comparative
optical
densities
of
the
autoradiographic
film.
The
S.D.
values,
were
homogeneously
distributed
and
amounted
to
less
than
20%
of
the
mean
values.
See
text
for
details.
tGTP
Y
S
was
used
at
a
final
concentration
of
100µM
and
2-['
ZS
I]iodomelatonin
(2IM)
concentration
was
30
pM.
Unlabelled
2-iodomelatonin
(1µM)
added
45
min
following
the
beginning
of
incubation
of
the
sections
with
100
pM
24
1
"Thodemelatonin.
AHy,
anterior
hypothalamic
area;
DG,
dentate
gyrus
of
the
hippocampus;
AH,
Ammon's
horn;
FC,
frontal
cortex;
CP,
choroid
plexus
of
the
IVth
ventricle;
NT,
not
tested;
ND,
not
detectable
above
the
background.
**P
<
0.001;
*P
<
0.05
vs
control
(ANOVA).
and
that
the
surrounding
tissue
was
devoid
of
binding.
Addition
of
unlabelled
2-iodomelatonin
to
the
incubation
medium
following
initial
incubation
with
the
radioactive
ligand,
in
the
displacement
studies,
abrogated
the
binding
in
the
arterial
walls
and
abol-
ished
the
binding
in
the
PT
and
the
SCN.
Co-incubation
with
100
µ
M
GTP
y
S
significantly
reduced
the
intensity
of
binding
in
the
PT
and
the
SCN,
but
in
the
walls
of
the
arterial
vessels
in
the
immediate
vicinity,
binding
was
not
affected
to
a
significant
extent.
Similarly,
the
diffuse
binding
in
the
frontal
cortex
and
the
dentate
gyrus
of
the
hippo-
campus
was
not
influenced
by
GTP
y
S.
A
summary
of
the
binding
distribution
in
the
brains
of
the
two
species
studied,
the
effects
of
GTP
y
S,
or
addition
of
1
µM
2-iodomelatonin
in
the
displacement
exper-
iments
is
presented
in
Table
1.
b
VP
e
c
Fig.
5.
Higher
magnifications
of
the
autoradiographs
shown
in
Fig.
4A
and
C,
i.e.
the
pericallosal
branch
of
the
anterior
cerebral
artery
(a)
and
the
anterior
cerebral
arteries
(d).
The
autoradiographic
films
(a,
d)
were
apposed
by
micromanipulation
over
the
same
microscopic
slides
(b,
e,
respectively)
following
staining
with
Cresyl
Violet,
to
produce
the
composite
image
of
c
and
f,
respectively.
Note
that
the
binding
is
confined
to
the
arterial
walls
and
the
small
vein
(V)
and
the
venous
plexus
are
devoid
of
binding.
Scale
bar
=
500µM.
Melatonin
receptors
in
the
primate
brain
465
a
b
Fig.
6.
Higher
mangifications
of
the
autoradiographic
image
of
the
CP
of
the
baboon
(a).
The
picture
in
b
was
generated
by
micromanipulating
apposition
of
a
over
the
same
section
after
staining
with
Cresyl
Violet.
Scale
bar
=
500
µM.
DISCUSSION
In
the
present
study,
we
used
24
125
I]iodomelatonin
to
examine
the
distribution
of
putative
melatonin
receptors
in
the
brain
of
two
Old
World
primates.
Description
of
the
kinetic
properties
and
the
pharma-
cological
profile
of
the
binding
site
was
not
our
goal
in
this
case
for
two
reasons:
first,
24
125
I]iodomelatonin
has
been
convincingly
demonstrated
to
specifically
label
the
melatonin
receptor
in
all
species
and
tissues
studied
so
far
(for
reviews
see
Refs
10,
31,
35
and
36)
and,
secondly,
the
limited
number
of
animals
avail-
able
precluded
collection
of
sufficient
quantities
of
tissue
for
a
large
enough
series
of
in
vitro
ligand-bind-
ing
and
signal-transduction
experiments
with
isolated
membrane
preparations
or
tissue
samples.
On
the
other
hand,
the
washing
procedure
used
in
the
pre-
sent
experiments
was
aimed
at
detection
of
high-
affinity
binding
sites
(see
also
Refs
19,
33
and
41).
Additionally,
we
investigated
the
existence
of
a
signal-transduction
mechanism,
linked
to
guanine
nucleotide-binding
regulatory
proteins,
as
described
for
other
high-affinity
melatonin
receptors
in
the
PT,
SCN
or
cortex
of
other
species
(for
review
see
Ref.
36).
The
results
reported
herein
describe
for
the
first
time
the
presence
of
melatonin
binding
sites
in
several
discrete
areas
of
the
primate
brain.
High-
affinity
melatonin
binding
sites
have
been
convinc-
ingly
demonstrated
before
in
the
human
SCN,"
but
surprisingly,
no
binding
was
reported
in
the
PT
of
this
species.
Our
results
leave
no
doubts
about
the
presence
of
melatonin
receptors
in
the
PT
of
the
vervet
monkey
and
the
baboon.
High-affinity
melatonin
receptors
are
present
in
the
PT
of
all
the
other
mammals
studied
so
far,
with
the
possible
exception
of
the
human.
In
most
cases
the
kinetic
parameters
of
the
receptor
have
been
described,
and,
in
some
reports,
physiological
responses
to
melatonin
have
been
demonstrated
(for
review
see
Ref.
36).
The
consistent
presence
of
melatonin
receptors
in
the
PT
of
the
non-human
primates
would
suggest
that
as
with
most
of
the
other
mammalian
species
studied
to
date,
the
PT
plays
an
important
role
in
mediating
melatonin
effects
on
the
hypothalamo-pituitary-go-
nadal
axis.
Melatonin
exhibits
well-defined
diurnal
rhythm
in
monkeys
and
man,
7
"
and
has
been
able
to
produce
biological
effects,
related
to
puberty
and
seasonal
reproduction,
in
the
Rhesus
monkey.'
However,
both
primates
studied
by
us
show
a
con-
tinuous
menstrual
cyclicity,
and
in
the
laboratory
can
breed
throughout
the
year.
In
nature,
these
species
express
somewhat
restricted
seasonal
conception
(birth)
rates.
°
'"
However,
these
rates
coincide
with
the
availability
of
food,
and
the
conception
time
seemingly
does
not
depend
on
the
photoperiod.
The
vervet
monkeys
breed
successfully
in
sub-Saharan
Africa,
on
the
islands
of
lake
Victoria,
Uganda,
at
latitude,
where
there
is
practically
no
annual
change
in
the
length
of
the
day."
Moreover,
two
populations
from
areas
with
similar
rainfall
patterns,
separated
by
less
than
300
miles
and
approximately
of
longi-
tude,
showed
a
completely
reversed
phase
in
the
birth
seasons.
15
The
local
breed
populations,
which
pro-
vided
the
animals
of
both
species
used
in
the
present
study,
do
not
display
true
seasonality,
manifested
by
anovulatory
phase
(De
Klerk,
personal
communi-
cation).
Thus,
our
present
results
strongly
suggest
that
melatonin
receptors
in
the
primate
PT
may
express
an
action
beyond
that
supposedly
related
to
changes
in
the
photoperiod.
Virtually
nothing
is
known
about
the
function
of
the
PT
in
primates.
Anatomical
evidence
from
several
other
species
demonstrates
the
interaction
of
luteinizing
hormone-releasing
hormone
axons
from
different
populations
of
luteinizing
hormone-
releasing
hormone
neuronal
cell
bodies,
with
the
external
zone
of
the
ME.
22
Melatonin
receptors,
however,
are
absent
in
the
ME.
9
.
33
'
37,44
''stu
d
y
Existence
of
a
paracrine
mechanism
for
control
of
gonado-
tropin
hormone-releasing
hormone
involving
PT
is
possible,
though
it
might
comprise
very
complex
mechanisms.'
Evidence
from
the
rat
suggests
that
melatonin
or
its
potent
analogues
are
able
to
suppress
the
preovulatory
luteinizing
hormone
surge
and
the
ovulation.'
4,35
'
36
It
is
not
known
466
B.
STANKOV
et
al.
to
what
extent
the
rat
data
can
be
extrapolated
to
other
species,
and
primates
in
particular,
but
humans
living
in
regions
with
a
strong
seasonal
contrast
in
luminosity
seem
to
express
an
inverse
seasonal
re-
lationship
between
melatonin
and
ovarian
activity.'
Recently,
melatonin,
and
a
combination
of
melatonin
with
a
progestin
agonist,
were
reported
to
have
a
suppressive
effect
on
the
phasic
preovulatory
luteiniz-
ing
hormone
release,
thus
expressing
antiovulatory
and
potentially
contraceptive
properties
in
human
females.'
Evidence
of
2-r
5
Iliodomelatonin
binding
in
the
SCN
was
present
in
all
mammalian
species
studied
to
date
(with
the
exception
of
the
ferret'
and
the
sheep
9
),
and
thus,
our
present
findings
in
both
pri-
mates
were
not
totally
surprising.
On
the
other
hand,
we
found
no
binding
in
the
paraventricular
thalamic
nuclei,
a
feature
common
to
most
rodent
species
studied
to
date.
The
SCNs
are
now
considered
to
be
the
site
of
an
endogenous
pacemaker
and
are
targets
of
direct
and
indirect
retinal
projections
necessary
for
the
entrain-
ment
of
the
circadian
rhythms
to
the
environmental
cycles.
They
have
been
proposed
as
a
site
for
mela-
tonin
action
well
before
the
development
of
reliable
methods
for
characterization
and
localization
of
melatonin
receptors.
It
is
well
documented
now
that
melatonin
is
very
efficient
in
influencing
the
circadian
rhythmicity
of
reptiles,
birds
and
mammals,
pre-
sumably
acting
at
the
level
of
the
SCN
(for
reviews
see
Refs
5
and
39).
In
humans
with
disturbed
sleep—waking
cycles
due
to
blindness
or
jet-lag,
re-en-
trainment
of
the
circadian
rhythmicity
has
been
achieved
by
giving
melatonin."."
The
presence
of
high-affinity
24
125
Iliodomelatonin
binding
sites
in
the
cortex
and
hippocampus
seems
to
be
species
dependent."'"''
In
the
brains
of
both
primates
studies
in
the
present
experiments,
the
bind-
ing
in
these
two
areas
was
rather
weak
and
did
not
show
preference
for
any
of
the
cortical
or
hippocam-
pal
layers,
contrary
to
the
findings
in
the
rabbit
or
sheep
parietal
cortices
or
hippocampi.
In
that
sense,
the
binding
to
the
frontal
cortex
in
both
vervet
monkey
and
baboon
was
similar
to
what
was
ob-
served
in
the
rabbit
frontal
cortex.'
Moreover,
as
in
the
human
frontal
cortex,'
binding
in
those
two
areas
could
not
be
modulated
by
coincubation
with
GTP,
S.
It
is
still
difficult
to
assign
a
function
for
melatonin
in
the
neocortex,
though
significant
progress
in
the
field
has
been
achieved
recently.
It
has
been
reported
that
melatonin
is
able
to
interact
with
GABA,
the
major
inhibitory
neurotransmitter
in
the
central
ner-
vous
system,
to
regulate
the
neuronal
activity.
1,6,21,29
Using
the
rabbit
cortex
as
a
model
system,
we
demonstrated
that
melatonin
was
able
to
exercise
benzodiazepine-like
effects
on
the
firing
activities
of
single
neurons
and
to
augment
significantly
the
in-
hibitory
action
of
GABA,
when
administered
simul-
taneously
by
iontophoresis
into
the
immediate
vicinity
of
the
neurons.'
The
effect
of
melatonin
in
various
rat
thalamic
nuclei
was
similar.
6
Moreover,
melatonin,
in
combination
with
short-lived
benzo-
diazepines,
was
able
to
improve
the
quality
of
sleep
in
healthy
human
volunteers,
mainly
in
terms
of
sleep
microstructure."
All
this
could
explain
to
a
certain
extent
the
hypnotic
properties
of
pharmacological
doses
of
melatonin,
reported
years
ago.
The
CPs
have
been
demonstrated
as
potential
melatonin
targets
in
a
rodent"
and
a
lagomorph."
Our
present
data
show
that
this
may
be
the
case
in
primates
as
well.
Melatonin
diurnal
rhythm
in
the
CSF
has
been
described
in
a
number
of
species,
including
man'
(for
review,
see
Ref.
36).
In
the
CP,
melatonin
may
be
functionally
involved
in
the
control
of
the
secretion
of
the
CSF
and,
therefore,
in
the
control
of
the
intraventricular
pressure.
Interestingly,
a
recent
preliminary
report
demonstrated
the
absence
of
melatonin
receptors
in
the
brain
of
severely
hydro-
cephalic
rats,
2
while
a
recent
study
demonstrated
the
presence
of
circadian
variations
in
human
CSF
pro-
duction
with
significantly
higher
levels
at
night.
24
Further
investigation
is
needed
to
clarify
whether
the
presence
of
melatonin
receptors
in
these
areas
is
linked
to
a
direct
effect
of
the
indole
on
the
regulation
dynamics
of
CSF
composition
and
volume.
Expression
of
melatonin
receptors
in
the
arteries
involved
in
thermoregulation
in
the
rat
was
reported
recently.'
To
our
knowledge,
our
present
report
is
the
first
description
of
24
125
Iliodomelatonin
binding
in
the
brain
arteries
of
primates.
Several
lines
of
investigation
have
implicated
melatonin
in
the
con-
trol
of
arterial
pressure.
Pinealectomy
has
been
shown
to
enhance
the
vascular
reactivity
to
vasocon-
strictive
agents"
and
to
cause
a
transient
hyperten-
sion
that
could
be
reversed
by
melatonin.
I6
Recently,
a
decrease
with
age
in
melatonin
binding,
down
to
undetectable
levels,
was
described
in
the
anterior
cerebral
arteries
of
spontaneously
hypertensive
rats.'"
Moreover,
melatonin
was
seemingly
able
to
augment
the
effect
of
adrenergic
analogues
on
the
muscular
arterial
tone,
having
vasoconstriction
properties
in
the
periphery'
and
vasodilatory
action
centrally.'
However,
24
125
Illodomelatonin
binding
in
the
basal
brain
arteries
of
both
primates
that
we
studied
expressed
peculiar
characteristics.
Though
binding
was
readily
reversible
upon
addition
of
1
µ
M
2-
iodomelatonin,
the
binding
site
did
not
appear
to
be
linked
to
a
guanine
nucleotide-binding
protein.
Co-
incubation
with
GT1
3
7
S
did
not
induce
a
loss
of
binding.
Therefore,
it
is
difficult
to
assign
meaningful
significance
of
the
binding
site
in
the
arterial
vascula-
ture
of
these
two
species,
in
the
absence
of
functional
data.
It
is
becoming
clear
that
melatonin
can
exert
biological
effects,
not
necessarily
acting
through
the
mechanism
described
in
the
PT,
SCN
or
the
cortex
(in
certain
species),
which
involves
G-protein-mediated
inhibition
of
adenylyl
cyclase
and
cAMP.
In
the
rat
caudal
arteries,
melatonin
was
found
to
influence
the
muscular
tone
through
a
cAMP-independent
Melatonin
receptors
in
the
primate
brain
467
mechanism
of
action.'
In
fact,
a
dual
mode
of
action
for
melatonin
was
proposed
in
certain
areas,
like
the
rat
cortex
and
thalamus.
b
34
High-affinity
21
125
I]iodo-
melatonin
binding
that
is
not
regulated
by
guanine
nucleotides
was
recently
reported
in
the
chicken
neuronal
retina.'
Though
binding
in
the
human
carotid
arteries
and
anterior
cerebral
arteries
was
specific,
it
was
also
not
affected
by
co-incubation
with
guanine
nucleotides
(Stankov
B.,
unpublished
data).
CONCLUSIONS
In
essence,
the
results
presented
here
show
that
in
the
two
primates
studied,
high-affinity,
G-protein-
coupled
melatonin
receptors
apparently
have
limited
distribution
in
the
primate
CNS.
Most
importantly,
they
demonstrate
that
apart
from
the
SCN,
the
PT
of
two
species
that
are
not
clearly
photoperiodic
in
terms
of
their
reproductive
competence
expresses
a
high
density
of
melatonin
receptors.
The
present
data
should
not
preclude
the
existence
of
melatonin
recep-
tors
in
other
areas,
which
could
possibly
have
been
omitted
because
of
the
limits
of
resolution
or
not
enough
high
section
frequency.
Low
levels
of
diffuse
binding
were
recorded
in
the
anterior
hypothalamus.
Weak
binding
that
could
not
be
modulated
by
GTP,S
was
recorded
in
the
hippocampus
and
the
frontal
cortex.
The
binding
in
the
walls
of
the
arterial
vasculature
forming
the
circle
of
Willis,
as
in
some
human
arteries,
differed
from
those
in
the
PT
and
the
SCN
in
that
the
binding
site
was
seemingly
not
linked
to
a
regulatory
guanine
necleotide-binding
protein.
Acknowledgements-We
acknowledge
with
thanks
the
col-
laboration
of
Dr
W.
A.
De
Klerk
from
the
Laboratory
Animal
Center,
The
Medical
University
of
South
Africa.
Some
of
this
work
was
supported
by
grants
from
the
Italian
National
Research
Council
(CNR,
Rome,
Italy).
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