Bipolar cells in the turtle retina are strongly immunoreactive for glutamate. Proceedings

ArticleinProceedings of the National Academy of Sciences 85(21):8321-5 · December 1988with19 Reads
Impact Factor: 9.67 · DOI: 10.1073/pnas.85.21.8321 · Source: PubMed
Abstract

Strong glutamate immunoreactivity was observed by both light and electron microscopy in bipolar cells of the turtle (Pseudemys scripta elegans) retina after postembedding immunohistochemistry. Virtually all bipolar cells showed strong labeling, on average 18 times that of the Müller (glial) cells. The data suggest that both on- and off-center bipolar cells are glutamatergic. Photoreceptors were also labeled, but with a labeling intensity about half that of the bipolar cells. Other types of retinal neurons showed less immunoreactivity, except for a small population of strongly labeled amacrine cells.

Full-text

Available from: Jon Storm-Mathisen, Sep 28, 2014
Proc.
Natl.
Acad.
Sci.
USA
Vol.
85,
pp.
8321-8325,
November
1988
Neurobiology
Bipolar
cells
in
the
turtle
retina
are
strongly
immunoreactive
for
glutamate
B.
EHINGER*t,
0.
P.
OTTERSENt,
J.
STORM-MATHISEN*,
AND
J.
E.
DOWLING*
*Department
of
Cellular
and
Developmental
Biology,
The
Biological
Laboratories,
Harvard
University,
Cambridge,
MA
02138;
tDepartment
of
Ophthalmology,
University
of
Lund,
S-22185
Lund,
Sweden;
and
tDepartment
of
Anatomy,
University
of
Oslo,
N-0162
Oslo
1,
Norway
Contributed
by
J.
E.
Dowling,
July
21,
1988
ABSTRACT
Strong
glutamate
immunoreactivity
was
ob-
served
by
both
light
and
electron
microscopy
in
bipolar
cells
of
the
turtle
(Pseudemys
scripta
elegans)
retina
after
postembed-
ding
immunohistochemistry.
Virtually
all
bipolar
cells
showed
strong
labeling,
on
average
18
times
that
of
the
Muller
(glial)
cells.
The
data
suggest
that
both
on-
and
off-center
bipolar
cells
are
glutamatergic.
Photoreceptors
were
also
labeled,
but
with
a
labeling
intensity
about
half
that
of
the
bipolar
cells.
Other
types
of
retinal
neurons
showed
less
immunoreactivity,
except
for
a
small
population
of
strongly
labeled
amacrine
cells.
Bipolar
cells
carry
visual
information
from
the
outer
to
the
inner
retina
and
are
the
first
cells
along
the
visual
pathway
to
be
divided
into
separate
on-
and
off-channels.
Furthermore,
they
show
a
center-surround
organization
similar
to
that
observed
in
retinal
ganglion
cells
and
other
neurons
in
the
visual
system
(1,
2).
Surprisingly,
little
is
known
about
the
transmitters
employed
by
the
bipolar
cells.
Physiological
evidence
has
indicated
that
bipolar
cells
are
excitatory
to
ganglion
cells
(3-6),
but
firm
evidence
for
the
presence
of
an
excitatory
transmitter
in
bipolar
cells
has
not
been
forthcom-
ing.
Both
amacrine
and
ganglion
cells
possess
receptors
spe-
cific
for
the
acidic
amino
acids,
and
therefore
it
has
been
proposed
that
L-glutamate
or
a
similar
excitatory
amino
acid
is
a
neurotransmitter
in
bipolar
cells
(7-11).
Attempts
at
localizing
endogenous
glutamate
in
the
retina
have
been
only
partially
successful,
and
at
times
contradictory,
most
likely
because
of
the
indirect
nature
of
the
methods
available
(12).
Recently,
a
technique
has
been
developed
to
localize
glutamate
immunohistochemically
by
applying
a
purified
antibody
to
etched
plastic
sections
and
demonstrating
the
binding
site
of
the
first
antibody
with
a
second
antibody
tagged
with
small
(15-nm)
colloidal
gold
particles
(13,
14).
The
method
gives
a
good
signal-to-noise
ratio,
and
since
only
a
small
fraction
of
the
glutamate
of
the
cell
is
available
for
detection
by
the
primary
antibody
(i.e.,
that
at
the
section
surface),
the
gold
particles
do
not
obscure
the
cytological
characteristics
of
the
labeled
neurons.
We
have
used
this
technique
to
localize
glutamate
in
the
turtle
retina.
MATERIALS
AND
METHODS
For
light
microscopy,
small
pieces
from
the
posterior
pole
of
light-adapted
eyes
of
the
turtle
Pseudemys
scripta
elegans
were
fixed
in
2%
glutaraldehyde
in
0.1
M
phosphate
buffer
(pH
7.4)
for
2
hr
at
room
temperature,
dehydrated,
and
embedded
in
Durcupan
ACM
(Fluka).
For
electron
micros-
copy,
small
pieces
of
retina
were
fixed
at
room
temperature
for
90
min
with
4%
glutaraldehyde,
1%
formaldehyde,
and
0.2
mM
CaCl2
in
0.1
M
phosphate
buffer
(pH
7.4).
After
washing
and
postfixation
for
1
hr
in
1%
OS04
in
the
same
buffer,
the
specimens
were
dehydrated,
embedded
in
Durcupan
ACM,
and
cured
at
520C.
For
light
microscopy,
0.5-
to
1-gm
tissue
sections
were
processed
according
to
Somogyi
et
al.
(13,
15),
using
the
peroxidase-antiperoxidase
(PAP)
technique
(13-16).
For
electron
microscopy
,
a
modification
(14)
of
the
immunogold
procedure
of
Somogyi
and
Hodgson
(17)
was
used
that
employed
Janssen
AuroProbe
15-nm
gold
particles
coated
with
goat
anti-rabbit
antibodies.
The
antiserum,
13
Glu,
was
purified
by
immunoadsorption
on
three
different
Sepharose
columns,
one
bearing
bovine
serum
albumin
treated
with
glutaraldehyde
and
the
others
bearing
the
same
protein
to
which
glutamine
or
y-aminobutyrate
had
been
conjugated
with
glutaraldehyde
(14,
18,
19).
The
antiserum,
which
has
been
characterized
(14,
17,
20),
was
diluted
1:800
for
both
light
and
electron
microscopy.
Three
kinds
of
controls
were
undertaken.
First,
the
tissue
was
processed
as
described
above,
but
the
primary
antibody
was
omitted
from
the
reaction
mixture.
As
expected,
no
specific
labeling
was
subsequently
observed.
Second,
the
antiserum
was
absorbed
with
glutamate
(200
AM)
that
had
been
treated
with
glutaraldehyde
(19).
This
also
abolished
the
labeling.
On
the
other
hand,
absorbing
the
antiserum
with
glutaraldehyde-treated
glycine,
aspartate,
taurine,
p-alanine,
or
glutamine
did
not
appreciably
diminish
the
reaction
(19).
Third,
various
amino
acids
were
added
to
brain
tissue
homogenates
that
had
been
extensively
dialyzed
to
remove
all
free
amino
acids.
The
resulting
mixtures
were
fixed
with
glutaraldehyde
and
embedded
in
Durcupan
(14).
Electron
microscopy
of
this
material
showed
a
95-fold
higher
density
of
label
when
glutamate
was
added
to
the
homogenates
as
compared
with
glutamine
and
even
higher
density
ratios
in
comparison
with
the
other
substances.
This
agrees
well
with
previously
published
data
on
this
antibody;
for
example,
it
does
not
react
with
glutathione
or
a
number
of
other
small
molecules
(14).
The
degree
of
labeling
was
assessed
by
counting
the
number
of
grains
in
identified
cell
processes
in
a
large
number
of
electron
micrographs.
Process
area
was
measured
with
the
aid
of
a
digitizing
pad
connected
to
a
small
computer.
Because
the
grain
density
with
the
colloidal
gold
procedure
is
low,
small
processes
often
showed
no
or
only
a
few
particles.
The
number
of
small
processes
was
usually
con-
siderable,
and
statistics
based
on
observed
grain
densities
are
therefore
quantized
and
not
distributed
normally.
Conse-
quently,
nonparametric
confidence
and
tolerance
limits
were
calculated
with
the
quantile
test,
and
probabilities
of
differ-
ences
in
median
values
were
obtained
with
the
nonparametric
median
test
(21).
Background
labeling
of
pure
plastic
was
negligible
(<0.05
grain
per
jum2),
and
the
grain
count
in
Muller
cells
was
the
lowest
of
all
cells
in
the
retina.
The
labeling
index
used
was
the
labeling
for
the
different
retinal
neurons
relative
to
the
Muller
cells
and
is
introduced
to
take
into
account
the
fact
that
some
glutamate
is
likely
to
be
found
in
all
cells.
The
upper
95%
tolerance
limit
of
the
90th
percentile
of
the
Muller
8321
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
Page 1
8322
Neurobiology:
Ehinger
et
al.
cell
grain
density
was
found
to
be
3.3
grains
per
Aum'
(a
labeling
index
of
4.1).
Only
profiles
with
higher
grain
densi-
ties
were
considered
significantly
labeled.
RESULTS
In
the
light
microscope,
bipolar
cells
were
the
most
promi-
nently
labeled
of
all
the
retinal
neurons
(Fig.
1).
Bipolar
cell
perikarya
were
found
mainly
in
the
middle
or
outer
half
of
the
inner
nuclear
layer.
They
were
often
readily
identified
by
their
Landolt
club
processes
(Fig.
1,
arrows),
which
extend
from
the
dendritic
arborization
of
the
bipolar
cells
to
between
the
photoreceptor
inner
segments
(22,
23).
Many
of
the
bipolar
cells
also
showed
an
axonal
process
that
typically
took
a
very
oblique
course
through
the
inner
nuclear
and
inner
plexiform
layers
(Fig.
1,
arrowhead)
(23).
Numerous
strongly
immunoreactive
profiles
were
also
observed
in
the
inner
plexiform
layer;
many
of
these
were
likely
to
be
bipolar
cell
processes
or
terminals
(see
below).
Photoreceptor
terminals
were
also
consistently
labeled,
although
they
were
never
as
strongly
labeled
as
the
bipolar
cells.
Most
amacrine
cells
showed
no
or
only
moderate
labeling,
but
a
few
strongly
labeled
cells
were
observed
(Fig.
1,
open
arrowhead).
Most
of
the
cells
in
the
ganglion
cell
layer
exhibited
modest
staining,
and
an
occasional
horizontal
cell
showed
some
immunoreactivity.
Some
staining
in
the
optic
nerve
fiber
layer
was
seen.
>
t X ;
| _ e
~~~~~~I
-
L
FIG.
1.
Glutamate
immunoreactivity
in
the
turtle
retina,
shown
by
the
peroxidase-antiperoxidase
procedure.
Strong
labeling
is
seen
in
bipolar
cells
(B)
and
their
Landolt
club
processes
(arrows).
The
centripetal
processes
of
the
bipolar
cells
have
a
characteristic
oblique
course
through
the
inner
nuclear
layer
(large
arrowheads).
There
are
numerous
strongly
labeled
processes
in
the
inner
plexiform
layer
(IPL).
Photoreceptor
terminals
are
labeled
in
the
outer
plexiform
layer
(small
arrowheads).
There
is
also
a
well-labeled
amacrine
cell
(open
arrow),
whereas
most
other
cells
at
the
same
level
(A)
are
only
moderately
or
weakly
labeled.
Ganglion
cells
(G)
show
some
label,
as
do
the
bundles
of
optic
nerve
fibers
(NF)
embedded
in
unlabeled
Muller
cell
processes
(M).
The
small
white
spots
in
the
inner
plexiform
layer
are
holes
in
the
section
caused
by
the
etching
combined
with
the
vigorous
peroxidase
reaction.
Ph,
photoreceptors;
OPL,
outer
plexiform
layer.
(Phase-contrast
micrograph;
x
540.)
In
the
electron
microscope,
the
bipolar
cells
and
their
processes
were
the
most
prominently
labeled
of
any
of
the
retinal
elements.
Relative
to
the
Muller
cells,
they
had
an
average
labeling
index
of
18
(Table
1),
and
individual
bipolar
cell
processes
or
terminals
had
indexes
as
high
as
40-45.
Fig.
2a
shows
a
heavily
labeled
bipolar
cell
terminal
in
the
inner
plexiform
layer.
This
process
can
be
confidently
identified
as
a
bipolar
cell
terminal
because
of
the
synaptic
ribbon
it
contains
(arrow)
(24).
Fig.
2b
shows
another
bipolar
cell
terminal
(B)
in
the
inner
plexiform
layer,
showing
a
more
average
labeling
density.
Gold
grains
are
clearly
seen
in
this
terminal
(arrowheads),
but
they
are
not
nearly
as
numerous
as
in
the
terminal
shown
in
Fig.
2a.
Furthermore,
the
label
is
not
evenly
distributed
throughout
the
terminal.
A
Muller
cell
process
(M)
of
approximately
the
same
area
as
the
bipolar
cell
terminal
is
also
present
in
this
micrograph.
It
is
devoid
of
gold
grains.
All
parts
of
the
bipolar
cells
appeared
to
be
labeled
to
the
same
extent.
Displaced
bipolar
cells
were
observed
occa-
sionally
lying
among
the
photoreceptor
cell
perikarya
and
terminals,
and
the
glutamate
immunoreactivity
was
analyzed
in
five
such
cells.
The
labeling
of
the
displaced
bipolar
cells
could
not
be
distinguished
from
that
of
other
bipolar
cells.
Some
bipolar
cell
processes
showed
no
gold
grains
or
only
one
or
two
grains.
In
most
of
these
cases
the
bipolar
cell
profiles
were
small,
and
thus
these
may
represent
labeled
processes
in
which
the
label
was
not
distributed
evenly
and
the
section
passed
through
an
unlabeled
region
(see
Fig.
2b).
However,
a
few
larger
bipolar
cell
processes
with
no
or
only
a
few
grains
were
noted.
We
cannot
exclude,
therefore,
that
there
may
be
a
small
percentage
of
bipolar
cells
with
no
or
very
low
glutamate
immunoreactivity.
The
density
of
labeling
in
-25O
bipolar
and
Muller
cell
profiles
is
shown
in
Fig.
3.
The
average
sizes
of
the
bipolar
and
Muller
cell
profiles
whose
grain
densities
were
analyzed
for
this
figure
were
approximately
the
same.
The
figure
shows
clearly
that
the
two
cell
types
are
distinctly
different
in
terms
of
grain
density
(median
test,
P
<
0.001).
The
overwhelming
majority
of
the
Muller
cell
processes
had
a
<4
grains
per
,um',
whereas
the
grain
density
of
all
but
four
of
the
bipolar
cell
profiles
was
between
4
and
38
grains
per
1Xm2.
The
distribution
of
grain
density
in
the
bipolar
cell
profiles
did
not
appear
Gaussian;
indeed
the
distribution
appeared
to
fit
better
a
bimodal
distribution
with
one
peak
at
about
12-16
grains
per
Am2
and
the
other
at
about
22-26
grains
per
Am2.
Photoreceptor
terminals
were
also
consistently
labeled.
Fig.
4
shows
a
typically
labeled
cone
photoreceptor
terminal,
identified
by
its
position
in
the
outer
plexiform
layer,
char-
acteristic
shape,
and
long
synaptic
ribbon.
Although
the
average
labeling
density
in
the
photoreceptors
was
about
half
that
of
the
bipolar
cells
(Table
1),
it
was
comparable
to
that
of
labeled
amacrine
cells
in
the
retina.
Variation
in
labeling
Table
1.
Glutamate
immunoreactivity
(grain
counts)
in
turtle
retina
99%
rank
grain
density,
confidence
No.
of
grains
per
limits
Labeling
Profile
profiles
,um2
Lower
Upper
index
Bipolar
cell
processes
121
14.7
12.8
17.6
18.1
Photoreceptor
terminals
46
7.6
5.5
7.2
9.4
Muller
cell
processes
133
0.81
0.48
0.90
1.0
Data
were
obtained
from
three
sections
on
a
single
grid.
The
degree
of
labeling
of
the
three
cell
types
was
significantly
different
in
all
cases
(P
<
0.001).
The
background
(<0.05
grain
per
Am2)
was
not
subtracted.
Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
Page 2
Proc.
Natl.
Acad.
Sci.
USA
85
(1988)
8323
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FIG.
2.
Glutamate
immunoreactivity
in
terminals
of
bipolar
cells
(B)
in
the
inner
plexiform
layer
of
turtle
retina,
densely
labeled
in
a
and
with
average
label
density
(arrowheads)
in
b.
For
clarity,
the
cell
boundaries
have
been
outlined
in
b.
The
synaptic
ribbons
(arrow
in
a)
identify
the
processes
as
bipolar
cell
terminals.
Note
the
absence
of
gold
particles
in
the
Muller
cell
process
(M)
in
b.
(a,
x
45,000;
b,
x
34,000.)
density
was
observed
in
photoreceptor
terminals,
but
signif-
icant
labeling
was
seen
in
both rod
and
cone
photoreceptor
terminals
(25-28).
Most
amacrine
cell
perikarya
had
a
low
density
of
label,
but
a
few
amacrine
cells
showed
strong
labeling.
Of
45
amacrine
cell
perikarya
analyzed
in
the
electron
microscope,
24
had
a
labeling
index
of
<4
(i.e.,
were
unlabeled),
13
had
an
index
in
the
range
5-11
and
8
had
a
labeling
index
averaging
about
14.
The
strongly
labeled
amacrine
cell
perikarya
most
often
had
a
relatively
clear
and
voluminous
cytoplasm,
and
their
nuclei
were
indented.
Most
amacrine
cell
processes
identified
in
the
inner
plexi-
form
layer
had
either
no
grains
or
only
one
grain,
showing
that
there
is
a
large
population
of
unlabeled
amacrine
cell
processes.
However,
a
number
of
clearly
labeled
amacrine
cell
processes
were
also
observed,
and
an
example
is
shown
in
Fig.
5.
Amacrine
cell
processes
in
the
inner
plexiform
layer
typically
show
scattered
synaptic
vesicles
and
little
cytoplas-
mic
density.
Furthermore,
they
make
conventional-type
synaptic
contacts.
The
synapse
made
in
Fig.
5
is
onto
a
labeled
process
containing
numerous
synaptic
vesicles
and
showing
significant
cytoplasmic
density,
suggesting
that
this
postsynaptic
profile
is
likely
to
be
a
bipolar
cell
terminal.
Labeled
amacrine
cell
processes
were
observed
to
contact
many
different
elements
in
the
inner
plexiform
layer.
They
were
presynaptic
to
ganglion
cell
dendrites,
pre-
and
postsy-
naptic
to
labeled
or
unlabeled
amacrine
cell
processes,
and
pre-
and
postsynaptic
to
labeled
bipolar
cell
terminals.
100
90
80
CA
0
-
4._
2
a
4-4
0
ce
70
60
50
40
30
20
10
0
U
D
11
11~~~T
Muller
cells
Bipolar
cells
rmg
ru
m
0.0
4.0
8.0
12.0
16.0
20.0
24.0
28.0
32.0
36.0
to
3.9
to
7.9
to
11.9
to
15.9
to
19.9
to
23.9
to
27.9
to
31.9
to
35.9
to
39.9
Gold
grains
per
,uM2
FIG.
3.
Percentages
of
133
Muller
cell
and
121
bipolar
cell
process
profiles
in
the
inner
plexiform
layer
with
different
grain
densities.
The
distributions
of
profile
sizes
of
the
two
cell
types
were
sufficiently
similar
to
allow
a
comparison
to
be
made
in
the
plot.
Note
that
very
nearly
all
bipolar
cell
profiles
had
grain
densities
higher
than
that
of
the
Muller
cells
and
that
they
tend
to
form
more
than
one
peak.
Neurobiology:
Ehinger
et
al.
Page 3
8324
Neurobiology:
Ehinger
et
al.
1
,A
V
4
FG
4
.
i
vit
y
v
a
p
i
ntd
P~
~~
s
VV
V
.....
:t
;
t:
.
!?
,
.'
.''::
'
by
its
characteristic
morphology
and
position
in
the
outer
plexiform
layer.
The
colloidal
gold
grains
are
indicated
by
arrowheads.
(
x
18,750.)
Reciprocal
synapses
between
labeled
amacrine
cell
pro-
cesses
and
bipolar
cell
terminals
were
not
seen,
however.
Most
horizontal
cell
profiles
showed
little
labeling,
with
an
average
labeling
index of
3.2
and
2.6
in
30
and
49
cell
bodies
and
axon
terminals,
respectively.
However,
a
few
cell
bodies
and
axon
terminals
did
have
a
somewhat
higher
grain
density
(labeling
index
around
8).
At
least
some
of
these
labeled
cell
bodies
appeared
to
have
cytoplasmic
characteristics
of
the
H1
horizontal
cell
type
(26).
Many
of
the
perikarya
in
the
ganglion
cell
layer
showed
a
moderate
amount
of
label
(average
labeling
index
of
6.5),
although
some
cells
showed
very
little.
Since
many
of
the
cells
in
the
ganglion
cell
layer
may
be
displaced
amacrine
cells,
it
is
difficult
to
decide
the
type
of
cell
showing
label.
However,
of
97
ganglion
cell
nerve
fibers
examined,
about
60%
had
a
labeling
index
above
4.
1,
indicating
that
a
significant
number
of
ganglion
cells
were
moderately
labeled.
DISCUSSION
The
most
striking
observation
in this
study
was
the
strong
glutamate
immunoreactivity
observed
in
bipolar
cells.
These
results
suggest
that
there
is
a
significant
amount
of
endoge-
toS
zdr*
a
a
~
,,
A
' ^ 5
jt
~
.4
A.
FIG.
5.
Glutamate
immunoreactivity
in
an
amacrine
cell
process
that
makes
a
synapse
onto
a
labeled
process,
most
likely
a
bipolar
cell
terminal.
The
gold
grains
in
the
amacrine
cell
process
are
indicated
by
arrows.
As
can
be
seen
here,
clusters
of
gold
grains
were
occasionally
observed.
Clustering
may
be
artifactual
(42);
thus,
each
cluster
was
counted
as
a
single
grain.
(
x
50,000.)
nous
glutamate
in
bipolar
cells,
and
thus
they
support
the
notion
that
glutamate
may
be
a
bipolar
cell
neurotransmitter.
That
the
immunoreactivity
demonstrated
in
the
present
study
does
represent
tissue
glutamate
is
established
beyond
rea-
sonable
doubt
by
the
controls
performed
and
by
previous
investigations
(13,
14,
18,
20).
Furthermore,
recent
studies
(29)
with
the
postembedding
electron
microscopic
immuno-
gold
method
have
shown
that
the
grain
density
over
gluta-
mate-glutaraldehyde-brain
protein
conjugate
particles
is
ap-
proximately
proportional
to
the
concentration
of
glutamate
in
the
conjugate,
at
least
in
the
higher
biologically
relevant
concentration
range.
Thus,
the
relative
labeling
densities
reported
here
are
likely
to
reflect
real
differences
is
endog-
enous
glutamate
concentrations.
Slaughter
and
Miller
(30)
provided
evidence
a
few
years
ago
that
on-center
bipolar
cells
use
an
excitatory
amino
acid
as
their
transmitter,
and
a
number
of
other
studies
(7-11)
have
provided
indirect
evidence
that
both
on-
and
off-center
bipolar
cells
are
likely
to
employ
an
excitatory
amino
acid
as
a
neurotransmitter.
The
present
study
provides
direct
evi-
dence
for
high
levels
of
glutamate
in
bipolar