Localization

of

metabotropic

glutamate

receptors

in

the

outer

plexiform

layer

of

the

goldfish

retina

We

studied

the

localization

of

metabotropic

glutamate

receptors

-LRB-

mGluRs

-RRB-

in

the

goldfish

outer

plexiform

layer

by

light-and

electron-microscopical

immunohistochemistry

The

antibody

labeled

putative

ON-type

bipolar

cell

dendrites

and

horizontal

cell

processes

in

both

rod

spherules

and

cone

triads

Immunolabeling

for

was

absent

in

the

rod

synaptic

complex

but

was

found

at

horizontal

cell

dendrites

directly

opposing

the

cone

synaptic

ribbon

The

antibody

labeled

Müller

cell

processes

wrapping

rod

terminals

and

horizontal

cell

somata

The

antibody

labeled

mainly

horizontal

cell

dendrites

invaginating

rods

and

cones

and

some

putative

bipolar

cell

dendrites

in

the

cone

synaptic

complex

The

finding

of

abundant

expression

of

various

mGluRs

in

bipolar

and

horizontal

cell

dendrites

suggests

multiple

sites

of

glutamatergic

modulation

in

the

outer

retina

Introduction

Stimulation

of

vertebrate

photoreceptors

by

light

decreases

the

release

of

glutamate

-LRB-

Cervetto

and

MacNichol

Murakami

et

al.

Kaneko

and

Shimazaki

-RRB-

which

is

sensed

by

glutamate

receptors

-LRB-

GluRs

-RRB-

in

the

dendrites

of

second-order

neurons

Glutamate

receptors

are

roughly

divided

into

ionotropic

and

metabotropic

receptors

-LRB-

Eccles

and

McGeer

-RRB-

Ionotropic

receptors

-LRB-

iGluRs

-RRB-

form

an

integral

ion

channel

whereas

metabotropic

receptors

-LRB-

mGluRs

-RRB-

mediate

responses

through

indirect

mechanisms

involving

signal

transduction

cascades

So

far

eight

different

mGluRs

have

been

cloned

and

classified

into

three

groups

based

on

their

sequence

similarities

second-messenger

cascade

and

pharmacology

group

I

comprises

-LRB-

Houamed

et

al.

Masu

et

al.

-RRB-

and

-LRB-

Abe

et

al.

-RRB-

and

their

splice

variants

-LRB-

d

and

b

-RRB-

group

II

contains

and

-LRB-

Tanabe

et

al.

Tanabe

et

al.

-RRB-

and

group

III

includes

-LRB-

Tanabe

et

al.

-RRB-

-LRB-

Nakajima

et

al.

-RRB-

-LRB-

Okamoto

et

al.

Saugstad

et

al.

-RRB-

and

-LRB-

Duvoisin

et

al.

-RRB-

and

their

splice

variants

-LRB-

b

b

b

and

b

-RRB-

The

lack

of

specific

pharmacological

tools

to

distinguish

between

mGluRs

within

a

group

makes

it

difficult

to

link

a

particular

receptor

type

to

a

certain

response

property

-LRB-

for

reviews

see

Thoreson

and

Ulphani

Schoepp

et

al.

-RRB-

In

the

retina

mGluRs

play

an

important

role

in

the

transmission

and

modulation

of

visual

signals

Light

responses

of

ON-type

bipolar

cells

-LRB-

ON

BCs

-RRB-

of

a

number

of

species

for

instance

are

sensitive

to

substances/drugs

that

target

group

III

mGluRs

-LRB-

Shiells

et

al.

Slaughter

and

Miller

Thoreson

and

Miller

-RRB-

The

receptor

activated

by

these

drugs

is

most

likely

-LRB-

Nakajima

et

al.

Akazawa

et

al.

Masu

et

al.

Ueda

et

al.

Vardi

and

Morigiwa

Laurie

et

al.

Vardi

et

al.

-RRB-

In

fish

amphibians

and

mammals

group

III

mGluRs

modulate

the

glutamate

release

of

cones

-LRB-

Koulen

et

al.

Hirasawa

et

al.

Hosoi

et

al.

-RRB-

Furthermore

pharmacological

activation

of

both

group

I

and

group

III

mGluRs

modulates

horizontal

cell

-LRB-

HC

-RRB-

responses

-LRB-

Nawy

et

al.

Takahashi

and

Copenhagen

Linn

and

Gafka

-RRB-

suggesting

that

multiple

mGluRs

must

be

present

in

the

fish

outer

plexiform

layer

-LRB-

OPL

-RRB-

At

the

moment

however

data

available

on

the

identity

and

distribution

of

the

various

mGluRs

in

the

teleost

outer

retina

are

scarce

-LRB-

Yazulla

et

al.

Klooster

et

al.

-RRB-

We

have

therefore

performed

a

light

and

electron

microscopy

study

of

the

mGluRs

present

in

the

goldfish

OPL

An

antibody

against

the

ON

BC

marker

protein

kinase

C

-LRB-

PKC

Negishi

et

al.

Suzuki

and

Kaneko

Yazulla

and

Studholme

-RRB-

has

been

used

in

order

to

identify

possible

localization

in

mixed-input

ON

BCs

-LRB-

ON

MBCs

-RRB-

Materials

and

methods

Subjects

Goldfish

-LRB-

Carassius

auratus

-RRB-

cm

standard

body

length

were

obtained

from

a

commercial

supplier

and

maintained

at

°C

in

aerated

tanks

filled

with

tap

water

circulating

through

a

biological

filter

system

The

fish

were

fed

and

kept

on

a

h

light-dark

cycle

All

animal

experiments

were

approved

by

the

ethical

committee

of

the

Royal

Netherlands

Academy

of

Arts

and

Sciences

acting

in

accordance

with

the

European

Communities

Council

Directive

of

November

-LRB-

-RRB-

Preparation

Dissections

took

place

early

in

the

morning

-LRB-

between

a.m.

and

a.m.

-RRB-

under

ambient

light

The

fish

used

in

this

study

were

therefore

all

in

the

light

phase

of

their

circadian

regime

After

cervical

transection

the

cornea

and

lens

of

light-adapted

animals

were

removed

and

the

remaining

eyecups

were

cut

in

half

through

the

equator

Half-eyecups

were

placed

vitreous-side-down

on

a

Millipore

filter

-LRB-

mm

diameter

μm

pore

size

Millipore

Amsterdam

The

Netherlands

-RRB-

placed

on

a

filter

holder

Suction

was

applied

to

remove

the

vitreous

the

sclera

and

retinal

pigment

epithelium

were

peeled

away

Light

microscopy

Retinas

were

fixed

at

room

temperature

for

min

in

M

phosphate-buffered

paraformaldehyde

-LRB-

pH

-RRB-

and

for

another

min

in

M

sodium

carbonate-buffered

paraformaldehyde

-LRB-

pH

-RRB-

These

short

fixation

times

were

chosen

to

prevent

loss

of

antigenicity

After

being

rinsed

in

M

phosphate

buffer

-LRB-

PB

pH

-RRB-

the

tissue

was

cryoprotected

at

room

temperature

for

min

in

PB

containing

sucrose

and

for

h

in

PB

containing

sucrose

The

pieces

of

retina

still

attached

to

the

filter

were

embedded

in

Tissue

Tek

-LRB-

Sakura

Finetek

Europe

Zouterwoude

The

Netherlands

-RRB-

in

an

aluminum

boat

and

frozen

in

liquid

nitrogen

Sections

-LRB-

μm

thick

-RRB-

were

cut

in

a

cryostat

mounted

on

poly-L-lysine-coated

slides

-LRB-

Menzel-Gläser

Braunschweig

Germany

-RRB-

dried

and

stored

in

a

non-frost-free

freezer

at

°C

until

use

Retinal

sections

were

washed

-LRB-

min

-RRB-

in

phosphate-buffered

saline

-LRB-

PBS

-RRB-

blocked

for

min

in

PBS

with

normal

goat

serum

-LRB-

NGS

Jackson

ImmunoResearch

Lab

West

Grove

USA

-RRB-

incubated

for

h

with

the

primary

antibody

-LRB-

for

dilutions

see

Table

-RRB-

in

PBS

containing

Triton

and

NGS

at

°C

After

several

washes

-LRB-

min

-RRB-

in

PBS

sections

were

incubated

in

the

secondary

antibodies

diluted

in

PBS

containing

Triton

for

min

at

°C

-LRB-

antibody

Alexa-conjugated

antibody

-RRB-

Table

antibodies

-LRB-

LM

light

microscopy

EM

electron

microscopy

aas

amino

acids

PKC

protein

kinase

C

mGluR

metabotropic

glutamate

receptor

-RRB-

AntibodySourceCatalog

numberLotTypeHostPurificationAntigenDilution

for

LMDilution

for

EMBlot

-LRB-

kDa

-RRB-

aPKCbSigma-Aldrich

St.

Louis

ABovine

within

aas

of

brain

Temecula

C-terminal

aas

-LRB-

PNVTYASVILRDYKQSSSTL

-RRB-

C-terminal

aas

-LRB-

NGREVVDSTTSSL

-RRB-

and

C-terminal

aas

-LRB-

LIIRDYTQSSSSL

-RRB-

Lake

Placid

AHuman

C-terminal

aas

-LRB-

NSPAAKKKYVSYNN

-RRB-

aWestern

blots

performed

by

external

groups

relative

mass

values

as

reported

in

manufacturer

's

data

sheetsbProduced

after

Parker

et

al.

and

Young

et

al.

Sections

were

cover-slipped

with

Vectashield

-LRB-

Vector

Labs

Burlingame

USA

-RRB-

and

stored

at

°C

Slides

were

observed

on

a

Leica

DMRD

-LRB-

Leica

Microsystems

Wetzlar

Germany

-RRB-

fluorescence

microscope

equipped

with

filter

sets

that

were

designed

for

fluorescein

isothiocyanate

and

Sections

from

double-label

experiments

were

observed

on

an

inverted

Zeiss

Axiovert

M

microscope

equipped

with

the

LSM

META

laser

scanning

confocal

module

-LRB-

Carl

Zeiss

Jena

Jena

Germany

-RRB-

Two

types

of

controls

were

performed

To

control

for

unspecific

labeling

attributable

to

secondary

antibodies

experiments

were

performed

by

omitting

the

primary

antibody

This

eliminated

all

labeling

To

control

for

unspecific

labeling

attributable

to

the

primary

antibodies

preadsorption

experiments

were

performed

by

mixing

in

a

molar

excess

a

synthetic

peptide

having

the

amino

acid

sequence

against

which

each

mGluR

antibody

had

been

raised

-LRB-

see

below

-RRB-

with

the

corresponding

primary

antibody

overnight

at

°C

Incubation

proceeded

as

previously

described

with

the

same

concentrations

for

the

primary

antisera

Labeling

was

also

completely

eliminated

in

these

preadsorption

controls

Electron

microscopy

The

same

fixation

and

cryoprotection

procedures

were

used

as

described

for

light

microscopy

The

retinas

were

peeled

off

the

filter

and

embedded

in

Tissue

Tek

-LRB-

Sakura

Finetek

Europe

Zouterwoude

The

Netherlands

-RRB-

in

an

aluminum

boat

The

boat

was

frozen

in

liquid

nitrogen

Transverse

frozen

sections

-LRB-

μm

thick

-RRB-

were

obtained

on

a

freezing

microtome

and

collected

in

PB

at

room

temperature

-LRB-

equivalent

to

freeze-thawing

-RRB-

The

retinal

sections

were

incubated

for

h

with

diluted

antisera

-LRB-

for

dilutions

see

Table

-RRB-

After

being

rinsed

the

tissue

was

incubated

in

a

poly-horseradish-peroxidase

-LRB-

HRP

-RRB-

conjugated

goat

anti-mouse

or

anti-rabbit

IgG

-LRB-

PowerVision

ImmunoVision

Technologies

Daly

City

USA

-RRB-

To

visualize

the

peroxidase

the

material

was

incubated

in

a

TRIS-HCl

diaminobenzidine

-LRB-

DAB

-RRB-

solution

containing

The

DAB

reaction

product

was

subsequently

intensified

by

the

gold-substituted

silver

peroxidase

method

-LRB-

Gorcs

et

al.

-RRB-

Sections

were

rinsed

in

sodium

cacodylate

buffer

M

-LRB-

pH

-RRB-

and

post-fixed

for

min

in

supplemented

with

potassium

ferricyanide

in

sodium

cacodylate

buffer

M

-LRB-

pH

-RRB-

After

being

rinsed

in

sodium

cacodylate

buffer

the

material

was

dehydrated

and

embedded

in

epoxy

resin

Ultrathin

sections

were

observed

in

a

Philips

electron

microscope

-LRB-

Philips

Eindhoven

The

Netherlands

-RRB-

and/or

Technai

-LRB-

CM

-RRB-

electron

microscope

-LRB-

FEI

Company

Eindhoven

The

Netherlands

-RRB-

Sections

examined

with

the

Philips

electron

microscope

were

counter-stained

with

uranyl

acetate

and

lead

citrate

Sampling

and

production

of

photomicrographs

At

least

retinal

sections

obtained

from

a

minimum

of

five

animals

were

used

for

a

given

light

microscopy

or

electron-microscopy

experiment

-LRB-

i.e.

single-immunolabeling

at

the

light-microscopy

level

for

-RRB-

About

five

photomicrographs

were

taken

from

the

most

representative

sections

in

each

experiment

Light

micrographs

of

the

single-label

experiments

were

acquired

as

TIFF

files

directly

from

the

Leica

microscope

by

using

a

Leica

F

digital

B/W

camera

-LRB-

pixels

ppi

-RRB-

Light

micrographs

of

the

double-label

experiments

were

acquired

as

TIFF

files

from

the

Zeiss

LSM

META

-LRB-

pixels

ppi

-RRB-

For

electron-microscopy

experiments

the

same

number

of

retinal

sections/animals

as

described

above

was

examined

at

the

light-microscopical

level

labeled

sections

were

chosen

for

ultra-thin

sectioning

A

minimum

of

photoreceptor

terminals

were

observed

per

ultra-thin

section

Micrographs

-LRB-

n

-RRB-

were

taken

from

these

sections

depending

on

the

presence

and

amount

of

labeling

Electron

micrographs

obtained

with

the

FEI

electron

microscope

were

directly

acquired

as

TIFF

files

-LRB-

pixels

ppi

-RRB-

Electron

micrographs

obtained

from

the

Philips

electron

microscope

were

first

printed

from

the

negatives

for

analysis

The

negatives

of

the

prints

that

were

selected

for

publication

were

scanned

on

a

sprint

scan

scanner

-LRB-

Polaroid

Nederland

Breda

The

Netherlands

-RRB-

and

acquired

as

TIFF

files

at

ppi

All

TIFF

files

were

resampled

at

ppi

and

subsequently

re-sized

and

optimized

for

brightness

and

contrast

by

using

Photoshop

-LRB-

Adobe

Systems

San

Jose

USA

-RRB-

Immunochemicals

The

primary

antibodies

used

in

this

study

were

purchased

from

Chemicon

International

-LRB-

Temecula

USA

-RRB-

Sigma-Aldrich

-LRB-

St.

Louis

USA

-RRB-

and

Upstate

-LRB-

Lake

Placid

USA

-RRB-

Details

of

these

antibodies

are

listed

in

Table

Goat

anti-mouse

Alexa

was

obtained

from

Molecular

Probes

-LRB-

Breda

The

Netherlands

-RRB-

and

goat

anti-rabbit

from

Jackson

ImmunoResearch

Lab

-LRB-

West

Grove

USA

-RRB-

For

preadsorption

controls

custom-made

peptides

were

obtained

from

Eurogentec

-LRB-

Seraing

Belgium

-RRB-

Western

blotting

Goldfish

retinas

and

brains

were

homogenized

with

a

Teflon

pestle

in

ice-cold

PBS

-LRB-

pH

-RRB-

containing

a

tablet

of

protease

inhibitor

cocktail

-LRB-

Boehringer

Mannheim

Mannheim

Germany

-RRB-

per

ml

PBS

After

homogenization

a

TRIS-buffered

sample

-LRB-

pH

-RRB-

solution

was

added

with

final

concentrations

of

SDS

β-mercaptoethanol

glycerol

bromophenol

blue

The

samples

were

boiled

for

min

and

centrifuged

for

min

at

g

supernatants

were

stored

at

°C

until

use

The

diluted

samples

were

boiled

for

min

-LRB-

-RRB-

cooled

and

then

spun

in

a

microfuge

at

g.

For

sodium

dodecyl-sulfate

polyacrylamide

gel

electrophoresis

samples

were

run

through

a

polyacrylamide

stacking

gel

at

mA

and

through

a

polyacrylamide

gel

at

mA

Protein

standards

-LRB-

Bio-Rad

Laboratories

Veenendaal

The

Netherlands

-RRB-

were

run

in

adjacent

lanes

Gels

were

electroblotted

on

a

poly-vinylidene-di-fluoride

blot

membrane

-LRB-

Millipore

-RRB-

overnight

at

a

constant

current

of

mA

After

being

rinsed

with

a

M

TRIS

buffer

containing

M

NaCl

and

Tween

the

membrane

was

blocked

in

the

same

buffer

containing

dry

milk

for

h.

Subsequently

the

membrane

was

incubated

for

h

in

the

primary

antibodies

-LRB-

-RRB-

washed

in

the

TRIS

buffer

incubated

in

an

HRP-conjugated

goat

anti-rabbit

IgG

-LRB-

Santa

Cruz

Biotechnology

Santa

Cruz

USA

-RRB-

and

washed

in

PBS

The

immunoreaction

was

visualized

by

enhanced

chemiluminescence

-LRB-

ECL

Amersham

Arlington

Heights

USA

-RRB-

by

using

Kodak

film

Exposures

times

were

between

and

min

Results

In

this

study

we

aimed

at

a

qualitative

description

of

the

mGluR

composition

in

the

goldfish

OPL

To

have

a

parameter

for

comparisons

with

regard

to

ON

BC

labeling

with

the

antibodies

against

the

various

mGluRs

we

first

investigated

the

immunoreactivity

pattern

yielded

by

the

PKC

antibody

a

widely

used

ON

BC

marker

Therefore

the

labeling

for

PKC

will

be

described

first

followed

by

each

of

the

mGluRs

studied

Immunolabeling

for

PKC

The

PKC

antibody

was

characterized

by

Western

blot

previously

in

a

number

of

studies

-LRB-

Negishi

et

al.

Suzuki

and

Kaneko

Yazulla

and

Studholme

-RRB-

and

yielded

similar

results

in

various

species

-LRB-

Table

-RRB-

Light

microscopy

Immunolabeling

for

PKC

in

the

goldfish

retina

at

the

light-microscopical

level

has

been

described

in

detail

elsewhere

-LRB-

Suzuki

and

Kaneko

-RRB-

Briefly

two

populations

of

ON

BCs

were

labeled

cells

with

small

somata

in

the

middle

of

the

inner

nuclear

layer

-LRB-

INL

-RRB-

and

small

axonal

terminations

-LRB-

cone-driven

ON

BCs

-RRB-

and

cells

with

large

flask-shaped

somata

and

bulbous

axon

terminals

-LRB-

ON

MBCs

-RRB-

Electron

microscopy

rod

synapses

In

the

rod

synaptic

complex

the

antibody

against

PKC

labeled

invaginating

dendrites

at

the

central

position

and

processes

at

some

distance

from

the

ribbon

-LRB-

Fig.

c

arrowheads

-RRB-

The

labeled

structures

were

elongated

-LRB-

Fig.

-RRB-

u-shaped

-LRB-

Fig.

-RRB-

or

round

-LRB-

Fig.

-RRB-

and

were

found

either

directly

opposite

to

the

synaptic

ribbon

-LRB-

Fig.

b

-RRB-

or

a

little

distance

away

from

it

-LRB-

Fig.

-RRB-

Dendrites

with

these

characteristics

and

at

these

locations

in

the

cyprinid

retina

were

previously

shown

to

belong

to

ON

MBCs

-LRB-

Scholes

Stell

Saito

et

al.

Klooster

et

al.

Yazulla

and

Studholme

-RRB-

Fig.

localization

of

protein

kinase

C

-LRB-

PKC

-RRB-

in

the

outer

plexiform

layer

In

the

rod

spherules

-LRB-

a

c

-RRB-

invaginating

dendrites

of

putative

mixed-input

ON-type

bipolar

cells

-LRB-

ON

MBCs

-RRB-

were

positively

labeled

-LRB-

arrowheads

-RRB-

Lateral

elements

of

horizontal

cells

-LRB-

HCs

-RRB-

were

unlabeled

-LRB-

arrows

-RRB-

as

were

some

invaginating

processes

that

possibly

belonged

to

OFF-type

MBCs

-LRB-

asterisks

-RRB-

In

the

cone

pedicles

-LRB-

d

e

-RRB-

PKC

immunoreactivity

was

observed

in

small

invaginating

profiles

at

the

base

of

the

pedicles

-LRB-

arrowheads

-RRB-

These

structures

were

never

associated

with

the

synaptic

ribbons

-LRB-

SR

-RRB-

HC

lateral

elements

were

unlabeled

-LRB-

arrows

-RRB-

Bars

μmLateral

processes

immediately

adjacent

to

the

pre-synaptic

ridge

previously

classified

as

dendrites

of

rod-driven

HCs

-LRB-

Stell

Scholes

-RRB-

were

not

labeled

-LRB-

Fig.

c

arrows

-RRB-

Some

invaginating

processes

were

also

not

labeled

-LRB-

Fig.

c

asterisks

-RRB-

their

position

and

fine

structure

in

combination

with

the

lack

of

labeling

suggested

that

they

were

either

dendrites

of

OFF-type

MBCs

or

rod-driven

HCs

-LRB-

Stell

Saito

et

al.

-RRB-

Electron

microscopy

cone

synapses

PKC-immunoreactive

structures

were

not

abundant

in

the

vicinity

of

the

cone

pedicles

When

present

the

labeled

structures

were

never

associated

with

the

synaptic

ribbon

but

were

instead

localized

at

the

base

of

the

cone

pedicle

-LRB-

Fig.

e

arrowheads

-RRB-

These

were

probably

ON

MBC

processes

approaching

the

cone

pedicle

In

the

closely

related

carp

dendrites

of

horseradish

peroxidase-injected

ON

MBCs

have

been

found

at

similar

sites

in

the

cone

synaptic

complex

-LRB-

Saito

et

al.

-RRB-

Lateral

elements

were

unlabeled

-LRB-

Fig.

e

arrows

-RRB-

Immunolabeling

for

Western

blotting

The

antibody

did

not

recognize

any

proteins

in

the

goldfish

retina

possibly

because

of

the

low

concentration

of

the

receptor

in

this

tissue

The

antibody

produced

a

diffuse

band

at

approximately

kDa

in

goldfish

brain

and

rat

samples

-LRB-

Fig.

-RRB-

close

to

the

predicted

molecular

mass

of

-LRB-

Tables

-RRB-

The

immunoreactive

protein

found

in

the

rat

retina

was

faint

probably

reflecting

the

restricted

expression

of

this

receptor

in

retinal

tissue

Fig.

for

-LRB-

ONL

outer

nuclear

layer

OPL

outer

plexiform

layer

INL

inner

nuclear

layer

IPL

inner

plexiform

layer

-RRB-

a

Immunoreactivity

was

concentrated

at

the

OPL

and

outer

half

of

the

INL

in

which

some

BC

somata

were

partially

labeled

-LRB-

arrowheads

-RRB-

b

Preadsorption

with

the

control

peptide

-LRB-

pread

-RRB-

abolished

all

labeling

c

Western

blot

of

total

protein

from

goldfish

retina

-LRB-

ret

-RRB-

goldfish

brain

-LRB-

br

-RRB-

rat

retina

-LRB-

ret

-RRB-

and

rat

cerebrum

-LRB-

cer

-RRB-

A

protein

at

around

kDa

close

to

the

predicted

molecular

mass

of

was

detected

in

goldfish

brain

rat

retina

and

rat

cerebrum

d

eRed

and

green

channel

confocal

images

of

the

same

tissue

section

double-labeled

with

antibodies

against

-LRB-

d

-RRB-

and

PKC

-LRB-

Alexa

e

-RRB-

Some

structures

were

positive

for

but

not

for

PKC

-LRB-

circles

-RRB-

Some

small

BCs

were

positive

for

the

ON

BC

marker

but

not

-LRB-

asterisks

-RRB-

f

Merged

image

of

the

double-labeling

experiment

A

band

of

immunoreactivity

in

the

OPL

partially

overlapped

with

the

dendritic

processes

of

PKC-positive

cells

Areas

of

overlap

between

the

two

proteins

appear

yellow

Bars

μmTable

-LRB-

basic

local

alignment

search

tool

-RRB-

results

and

relative

molecular

mass

-LRB-

Mr

-RRB-

of

fish

mGluRs

-LRB-

aa

amino

acids

-RRB-

Source

proteinZebrafish

matchesSequence

comparisonLength

sourceLength

zebrafishMr

sourceMr

zebrafishThis

studyRat

-LRB-

-RRB-

Similar

to

bifunctional

mGluR

forextracellular

Ca

and

Glu

-LRB-

previouslysimilar

to

-RRB-

kDaConsensusP

VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLSimilar

to

bifunctional

mGluR

forextracellular

Ca

and

Glu

partial

-LRB-

-RRB-

kDaConsensusP

VTYASVILRDYKQSSSTLZebrafishPSVTYASVILRDYKQSSSTLRat

-LRB-

-RRB-

Hypothetical

protein

-LRB-

-RRB-

kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLHypothetical

protein

-LRB-

-RRB-

kDaConsensusNGREVVDSTTSSLZebrafishNGREVVDSTTSSLNovel

protein

similar

to

-LRB-

-RRB-

kDaConsensusNGRE

VDSTTSSLZebrafishNGREIVDSTTSSLHuman

-LRB-

-RRB-

Similar

to

GluR

-LRB-

previously

similar

-RRB-

kDaConsensusNSPAAKKKYVSYN

ZebrafishNSPAAKKKYVSYNDRat

-LRB-

-RRB-

Similar

to

bifunctional

mGluR

forextracellular

Ca

and

Glu

-LRB-

previouslysimilar

to

-RRB-

kDaConsensus

I+RDY

QSSS+LZ

ebrafishVILRDYKQSSSTLSimilar

to

bifunctional

mGluR

forextracellular

Ca

and

Glu

partial

-LRB-

-RRB-

kDaConsensus

I+RDY

QSSS+LZ

ebrafishVILRDYKQSSSTLSimilar

to

-LRB-

-RRB-

kDaConsensusL

R

Y+QSSSSLZ

ebrafishLVLRHYSQSSSSLAccession

numbers

listed

below

protein

names

Codes

in

parentheses

indicate

accession

numbers

in

the

NCBI

protein

database

a

Proteins

whose

DNA

coding

sequences

are

almost

identical

-LRB-

only

nucleotide

substitutions

-RRB-

Light

microscopy

immunoreactivity

in

the

goldfish

retina

was

concentrated

in

the

OPL

-LRB-

Fig.

-RRB-

and

outer

half

of

the

INL

in

which

the

somata

of

some

bipolar

cells

were

labeled

-LRB-

Fig.

arrowheads

-RRB-

A

confocal

examination

of

the

immunofluorescence

pattern

yielded

by

this

antibody

revealed

diffuse

labeling

in

the

INL

inner

plexiform

layer

-LRB-

IPL

-RRB-

and

ganglion

cell

layer

-LRB-

GCL

Fig.

-RRB-

Red

and

green

images

of

a

confocal

view

were

taken

from

a

retinal

slice

double-labeled

for

-LRB-

Fig.

-RRB-

and

PKC

-LRB-

Fig.

-RRB-

the

yellow

areas

in

the

merged

image

-LRB-

Fig.

-RRB-

indicated

the

strong

colocalization

in

ON

BC

processes

in

the

OPL

However

immunoreactivity

in

this

layer

was

not

restricted

to

PKC-positive

processes

Furthermore

the

small

PKC-positive

ON

BCs

were

not

positive

for

-LRB-

Fig.

e

asterisks

-RRB-

Conversely

the

labeled

structures

-LRB-

presumably

amacrine

cell

somata

-RRB-

in

the

inner

border

of

the

INL

that

were

not

PKC-positive

-LRB-

Fig.

e

circles

-RRB-

Electron

microscopy

rod

synapses

In

the

rod

spherule

immunoreactivity

was

present

at

invaginating

dendrites

in

close

apposition

to

the

synaptic

ribbons

-LRB-

Fig.

d

arrowheads

-RRB-

These

processes

were

elongated

-LRB-

Fig.

-RRB-

ellipsoidal

-LRB-

Fig.

-RRB-

goblet-shaped

-LRB-

Fig.

-RRB-

or

round

-LRB-

Fig.

-RRB-

consistent

with

ON

MBC

dendrites

Roughly

two

invaginating

dendrites

were

stained

per

rod

spherule

the

remaining

invaginating

dendrites

-LRB-

Fig.

b

asterisks

-RRB-

were

not

labeled

These

were

possibly

OFF

MBCs

rod-driven

HCs

or

a

subtype

of

ON

MBC

negative

for

Fig.

localization

of

in

the

OPL

In

the

rod

terminals

-LRB-

a

d

-RRB-

ON

MBC

invaginating

processes

at

the

summit

of

the

synaptic

ribbon

-LRB-

SR

-RRB-

were

positively

labeled

-LRB-

arrowheads

-RRB-

Some

invaginating

processes

were

not

labeled

-LRB-

asterisks

-RRB-

these

might

belong

to

OFF

MBCs

to

rod-driven

HCs

or

to

a

subtype

of

ON

MBC

negative

for

Occasionally

a

laterally

positioned

dendrite

showed

immunoreactivity

-LRB-

thick

arrow

in

d

-RRB-

In

the

cone

pedicles

-LRB-

e

f

-RRB-

immunoreactivity

was

found

in

small

invaginating

profiles

that

lay

at

the

base

of

the

pedicles

or

close

to

the

ribbon

-LRB-

arrowheads

-RRB-

and

that

might

have

originated

in

BCs

In

addition

a

HC

dendrite

occupying

the

lateral

position

of

the

triads

was

positively

labeled

-LRB-

thick

arrows

in

e

-RRB-

whereas

the

other

lateral

element

was

unlabeled

-LRB-

arrows

-RRB-

The

antibody

also

labeled

HC

spinules

in

the

cone

terminals

-LRB-

thick

arrows

in

f

-RRB-

Bar

μmThe

antibody

also

labeled

single

lateral

elements

facing

the

synaptic

ribbon

-LRB-

Fig.

thick

arrow

-RRB-

Whenever

a

lateral

element

was

labeled

the

other

one

was

unlabeled

-LRB-

Fig.

arrow

-RRB-

Although

processes

in

the

lateral

position

are

classically

attributed

as

belonging

to

HCs

-LRB-

Stell

-RRB-

we

could

not

fully

exclude

the

possibility

that

some

of

the

labeled

processes

were

aberrant

ON

MBC

dendrites

Electron

microscopy

cone

synapses

In

the

cone

pedicles

various

types

of

post-synaptic

structures

were

labeled

In

addition

to

small

invaginating

processes

-LRB-

Fig.

f

arrowheads

-RRB-

which

could

belong

to

BCs

immunoreactivity

was

also

present

at

one

lateral

dendrite

of

some

cone

triads

-LRB-

Fig.

thick

arrows

-RRB-

The

position

and

fine

structure

of

these

dendrites

were

consistent

with

those

of

cone-driven

HCs

-LRB-

Stell

Sakai

and

Naka

-RRB-

When

one

lateral

HC

dendrite

in

a

triad

was

positive

for

the

opposite

HC

dendrite

was

not

-LRB-

Fig.

thick

and

thin

arrows

respectively

-RRB-

A

few

invaginations

in

the

cone

pedicle

containing

discontinuous

patches

of

electron-dense

material

in

the

membrane

and

resembling

HC

spinules

-LRB-

Wagner

-RRB-

exhibited

immunoreactivity

-LRB-

Fig.

thick

arrows

-RRB-

Immunolabeling

for

Western

blotting

The

antibody

against

reacted

with

proteins

of

approximately

kDa

in

goldfish

brain

rat

retina

and

rat

cerebrum

-LRB-

Fig.

-RRB-

This

is

the

predicted

molecular

mass

of

and

-LRB-

Tables

-RRB-

In

the

goldfish

retina

only

a

protein

at

about

kDa

was

detected

which

also

appeared

in

the

other

three

tissue

samples

This

probably

represents

receptor

dimers

-LRB-

Poli

et

al.

-RRB-

which

occur

naturally

in

many

G-protein-coupled

receptors

including

mGluRs

-LRB-

Bouvier

Kubo

and

Tateyama

Pin

et

al.

Poli

et

al.

Beraudi

et

al.

-RRB-

In

goldfish

and

rat

retinas

a

smaller

band

at

around

kDa

was

detected

Fig.

for

-LRB-

ONL

outer

nuclear

layer

OPL

outer

plexiform

layer

INL

inner

nuclear

layer

IPL

inner

plexiform

layer

-RRB-

a

Immunoreactivity

was

seen

as

tiny

bands

in

the

OPL

-LRB-

thick

arrows

-RRB-

irregular

patches

in

the

INL

and

diffuse

labeling

in

the

IPL

b

Preadsorption

with

the

control

peptide

-LRB-

pread

-RRB-

abolished

all

labeling

c

Western

blot

of

total

protein

from

goldfish

retina

-LRB-

ret

-RRB-

goldfish

brain

-LRB-

br

-RRB-

rat

retina

-LRB-

ret

-RRB-

and

rat

cerebrum

-LRB-

cer

-RRB-

A

protein

at

around

kDa

close

to

the

predicted

molecular

mass

of

and

was

detected

in

goldfish

brain

rat

retina

and

rat

cerebrum

The

antibody

also

detected

a

protein

at

kDa

-LRB-

probably

receptor

dimers

-RRB-

in

all

four

tissues

d

eRed

and

green

channel

confocal

images

of

the

same

tissue

section

double-labeled

with

antibodies

against

-LRB-

d

-RRB-

and

PKC

-LRB-

Alexa

e

-RRB-

f

Merged

image

of

the

double-labeling

experiment

No

colocalization

with

ON

BCs

was

found

Bars

μm

g

h

In

a

few

cone

pedicles

immunoreactivity

was

found

in

HC

central

and

lateral

elements

of

the

cone

triad

-LRB-

thick

arrows

-RRB-

Note

the

unlabeled

lateral

processes

-LRB-

thin

arrows

-RRB-

and

synaptic

ribbons

-LRB-

SR

-RRB-

Bars

μm

Light

microscopy

Immunocytochemistry

with

the

antibody

revealed

signal

in

the

OPL

and

INL

and

strong

labeling

in

the

IPL

Tiny

discontinuous

immunoreactive

bands

were

visible

in

the

OPL

-LRB-

Fig.

thick

arrows

-RRB-

Confocal

images

showed

a

similar

pattern

with

a

slightly

stronger

OPL

labeling

-LRB-

Fig.

thick

arrows

-RRB-

Clearly

labeled

somata

could

not

be

distinguished

although

immunolabeling

in

the

INL

was

concentrated

in

patches

Immunoreactivity

for

PKC

-LRB-

Fig.

-RRB-

did

not

colocalize

with

-LRB-

Fig.

-RRB-

as

can

be

seen

by

the

absence

of

yellow

areas

in

the

merged

confocal

image

-LRB-

Fig.

-RRB-

Electron

microscopy

rod

synapses

No

immunoreactive

processes

could

be

identified

in

the

rod

synaptic

terminals

Electron

microscopy

cone

synapses

HC

dendrites

occupying

both

the

lateral

and

central

position

were

positive

for

in

only

a

few

cone

pedicles

-LRB-

Fig.

h

thick

arrows

-RRB-

The

fine

structure

of

the

central

processes

was

consistent

with

that

of

HC

dendrites

because

of

their

large

dimensions

compared

with

BC

dendrites

cytoplasm

devoid

of

organelles

some

electron-dense

amorphous

material

applied

to

the

cytoplasmic

surface

and

concavity

of

the

surface

facing

the

synaptic

ribbon

-LRB-

Stell

Scholes

-RRB-

No

immunoreactivity

was

seen

in

structures

resembling

BC

dendrites

in

agreement

with

our

light-microscopical

findings

Immunolabeling

for

Western

blotting

Immunoblots

for

revealed

a

protein

at

around

kDa

in

goldfish

retina

and

brain

and

in

rat

cerebrum

-LRB-

Fig.

-RRB-

An

additional

band

at

kDa

was

detected

in

the

goldfish

samples

and

in

the

rat

retina

A

heavier

protein

of

around

kDa

was

detected

in

rat

retina

and

cerebrum

roughly

corresponding

to

the

deduced

molecular

mass

of

rat

-LRB-

Koulen

et

al.

-RRB-

Fig.

for

-LRB-

ONL

outer

nuclear

layer

OPL

outer

plexiform

layer

INL

inner

nuclear

layer

IPL

inner

plexiform

layer

-RRB-

a

Immunoreactivity

was

concentrated

in

the

ONL

-LRB-

open

arrows

-RRB-

with

some

granular

labeling

in

the

OPL

INL

and

IPL

-LRB-

thick

arrow

labeled

HC

body

-RRB-

b

Preadsorption

with

the

control

peptide

-LRB-

pread

-RRB-

abolished

all

labeling

c

Western

blot

of

total

protein

from

goldfish

retina

-LRB-

ret

-RRB-

goldfish

brain

-LRB-

br

-RRB-

rat

retina

-LRB-

ret

-RRB-

and

rat

cerebrum

-LRB-

cer

-RRB-

A

protein

around

kDa

was

detected

in

the

goldfish

tissue

and

rat

brain

The

antibody

also

a

smaller

band

-LRB-

kDa

-RRB-

in

the

goldfish

samples

and

in

the

rat

retina

and

a

heavier

band

-LRB-

kDa

-RRB-

in

the

rat

retina

and

cerebrum

d

eRed

and

green

channel

confocal

images

of

the

same

tissue

section

double-labeled

with

antibodies

against

-LRB-

d

-RRB-

and

PKC

-LRB-

Alexa

e

-RRB-

f

Merged

image

of

the

double-labeling

experiment

showing

no

colocalization

Bars

μm

g

Labeling

is

restricted

to

processes

ensheathing

photoreceptor

terminals

in

the

ONL

-LRB-

open

arrows

-RRB-

h

A

similar

labeling

pattern

-LRB-

open

arrows

-RRB-

is

obtained

with

the

monoclonal

antibody

against

glial

fibrilary

acidic

protein

-LRB-

GFAP

clone

Sigma-Aldrich

-RRB-

which

labels

exclusively

Müller

cells

-LRB-

SR

synaptic

ribbon

-RRB-

Bars

μm

Light

microscopy

The

antibody

labeled

vertical

bands

at

the

outer

nuclear

layer

-LRB-

ONL

-RRB-

in

both

conventional

-LRB-

Fig.

open

arrows

-RRB-

and

confocal

microscopy

-LRB-

Fig.

open

arrows

-RRB-

and

yielded

granular

labeling

in

the

OPL

INL

and

IPL

Some

HC

somata

were

positively

labeled

at

the

outer

margin

of

the

INL

-LRB-

Fig.

d

thick

arrows

-RRB-

There

was

no

colocalization

with

PKC

-LRB-

Fig.

f

-RRB-

Electron

microscopy

Ultrastructurally

immunoreactivity

was

seen

in

longitudinally

oriented

structures

between

photoreceptor

terminals

-LRB-

Fig.

open

arrows

-RRB-

This

pattern

was

consistent

with

previous

reports

concerning

the

localization

and

ultrastructural

appearance

of

Müller

cell

processes

-LRB-

Villegas

Lasansky

Pedler

Miller

and

Dowling

-RRB-

Furthermore

it

resembled

closely

that

of

immunolabeling

for

glial

fibrilary

acidic

protein

-LRB-

Fig.

open

arrows

-RRB-

which

is

exclusively

expressed

by

glial

cells

-LRB-

Bignami

and

Dahl

Liepe

et

al.

-RRB-

Immunolabeling

for

Western

blotting

A

protein

of

approximately

kDa

was

labeled

in

goldfish

and

rat

samples

with

an

additional

band

of

around

kDa

in

the

goldfish

brain

In

the

rat

retina

a

protein

of

kDa

was

also

recognized

A

clear

band

could

not

be

distinguished

in

rat

cerebrum

although

there

was

a

larger

precipitation

around

kDa

-LRB-

Fig.

-RRB-

at

some

distance

from

the

value

reported

by

the

producer

-LRB-

Table

-RRB-

Fig.

for

-LRB-

ONL

outer

nuclear

layer

OPL

outer

plexiform

layer

INL

inner

nuclear

layer

IPL

inner

plexiform

layer

-RRB-

a

Immunoreactivity

was

concentrated

in

the

OPL

-LRB-

arrowheads

-RRB-

with

some

granular

labeling

in

the

INL

IPL

and

GCL

b

Preadsorption

with

the

control

peptide

-LRB-

pread

-RRB-

abolished

all

labeling

c

Western

blot

of

total

protein

from

goldfish

retina

-LRB-

ret

-RRB-

goldfish

brain

-LRB-

br

-RRB-

rat

retina

-LRB-

ret

-RRB-

and

rat

cerebrum

-LRB-

cer

-RRB-

A

pronounced

band

at

around

kDa

was

produced

in

all

four

samples

Additional

immunoreactive

proteins

were

found

at

kDa

in

the

goldfish

tissue

and

at

kDa

in

the

rat

retina

d

eRed

and

green

channel

confocal

images

of

the

same

tissue

section

double-labeled

with

antibodies

against

-LRB-

d

-RRB-

and

PKC

-LRB-

Alexa

e

-RRB-

A

BC

soma

-LRB-

arrowheads

-RRB-

and

an

HC

soma

-LRB-

thick

arrows

-RRB-

were

positive

for

but

negative

for

PKC

f

Merged

image

of

the

double-labeling

experiment

showing

no

colocalization

in

ON

BCs

g

In

rod

spherules

lateral

elements

were

labeled

-LRB-

thick

arrows

-RRB-

h

In

the

cone

pedicles

small

profiles

not

related

to

the

synaptic

ribbons

-LRB-

SR

-RRB-

were

positive

-LRB-

arrowheads

-RRB-

as

were

lateral

elements

of

the

triads

-LRB-

thick

arrow

-RRB-

Note

the

unlabeled

lateral

elements

-LRB-

thin

arrows

-RRB-

Bars

μm

Light

microscopy

The

antibody

against

immunoreactivity

yielded

strong

labeling

in

the

OPL

-LRB-

Fig.

arrowheads

-RRB-

and

granular

labeling

at

the

INL

IPL

and

GCL

Occasionally

positive

amacrine

and

ganglion

cell

somata

were

seen

Confocal

images

of

double-labeling

with

PKC

-LRB-

Fig.

f

-RRB-

did

not

show

colocalization

areas

were

concentrated

close

to

the

outer

border

of

the

INL

immediately

below

the

band

of

PKC-positive

ON

BC

dendrites

Some

somata

that

were

not

labeled

for

PKC

could

be

identified

in

the

outer

half

of

the

INL

their

form

and

position

was

consistent

with

that

of

HCs

-LRB-

Fig.

e

thick

arrow

-RRB-

and

OFF

BCs

-LRB-

Fig.

e

arrowhead

-RRB-

Electron

microscopy

rod

synapses

Lateral

elements

of

the

rod

triads

were

labeled

consistent

with

the

labeling

of

putative

HCs

reported

above

-LRB-

Fig.

thick

arrows

-RRB-

We

found

no

evidence

of

MBC

labeling

in

rod

spherules

Electron

microscopy

cone

synapses

A

few

small

processes

away

from

the

ribbons

were

labeled

by

the

antibody

-LRB-

Fig.

arrowheads

-RRB-

Their

small

profiles

resembled

those

of

BCs

This

observation

added

to

the

finding

that

the

antibody

labeled

PKC-negative

BC

somata

-LRB-

Fig.

e

arrow

-RRB-

suggested

that

these

were

OFF

BC

profiles

Lateral

elements

-LRB-

Fig.

thick

arrow

-RRB-

and

spinules

were

also

labeled

consistent

with

HC

labeling

at

the

light-microscopical

level

-LRB-

Fig.

e

thick

arrows

-RRB-

These

labeled

HC

processes

were

however

rare

most

of

the

lateral

elements

were

not

positive

-LRB-

Fig.

thin

arrows

-RRB-

Discussion

Summarizing

scheme

This

study

shows

that

mGluRs

of

all

three

groups

can

be

found

in

the

goldfish

OPL

The

main

findings

are

summarized

in

Fig.

Cell

types

have

been

identified

based

on

the

results

obtained

at

both

light-microscopy

and

electron-microscopy

levels

Fig.

of

a

rod

spherule

a

Müller

cell

process

and

a

cone

pedicle

depicting

the

localization

of

PKC

and

the

patterns

yielded

by

the

various

mGluR

antibodies

-LRB-

red

group

I

mGluRs

green

group

II

mGluRs

blue

group

III

mGluRs

SR

synaptic

ribbon

-RRB-

In

the

rod

spherule

PKC

labels

invaginating

dendrites

of

putative

ON

MBCs

The

antibody

labels

similar

structures

and

some

lateral

elements

Lateral

elements

of

rod

triads

are

also

positive

for

Müller

cell

processes

between

the

rod

spherules

are

positive

for

We

have

found

no

evidence

for

in

the

rod

synaptic

complex

In

the

cone

pedicles

PKC

labels

fine

dendrites

at

the

base

of

the

terminal

or

invaginating

structures

away

from

the

ribbons

Elements

at

similar

positions

and

with

similar

fine

structure

are

positive

for

and

BC

central

processes

in

the

cone

triads

are

positive

for

Spinules

are

positive

for

and

and

lateral

elements

are

labeled

by

the

and

antibodies

Finally

HC

central

elements

are

positive

for

Specificity

of

the

antibodies

No

data

are

available

on

the

amino

acid

and/or

nucleotide

sequences

of

goldfish

mGluRs

The

closest

available

information

is

from

the

zebrafish

This

database

is

however

not

fully

completed

and

the

various

zebrafish

mGluRs

have

not

been

unambiguously

identified

-LRB-

Table

-RRB-

The

finding

of

candidates

for

zebrafish

and

has

been

reported

but

the

actual

nucleotide

and

amino

acid

sequences

have

not

been

disclosed

-LRB-

Bjarnadottir

et

al.

-RRB-

We

therefore

used

multiple

data

sources

to

infer

the

specificity

of

the

antibodies

used

in

this

study

Western

blots

and

BLAST

searches

in

the

zebrafish

genome

for

the

amino

acid

sequences

in

Table

-LRB-

at

-RRB-

the

sequence

alignments

-LRB-

with

and

-RRB-

and

the

molecular

mass

calculations

-LRB-

at

-RRB-

We

also

BLASTed

the

proteins

retrieved

from

the

zebrafish

database

against

all

other

species

available

in

order

to

check

for

protein

similarities

among

species

To

choose

good

candidates

for

fish

mGluRs

among

the

BLAST

hits

we

relied

on

the

scores

and

E-values

the

cutoff

was

E

We

adopted

a

stringent

significance

level

because

the

chance

of

obtaining

random

matches

with

short

amino

acid

sequences

was

relatively

large

All

scores

used

for

these

comparisons

were

higher

than

For

the

C-terminal

peptide

the

cutoff

was

lowered

to

E

since

no

candidates

could

be

found

with

the

same

stringency

as

for

the

remainder

of

the

peptide

sequences

As

summarized

in

Table

all

of

the

anti-mGluR

antibodies

used

in

this

study

were

raised

against

polypeptides

that

yielded

mGluR-like

hits

when

BLASTed

against

the

zebrafish

database

Two

homologs

were

found

for

but

one

of

these

-LRB-

-RRB-

was

an

incomplete

sequence

that

might

have

corresponded

to

a

fragment

of

the

other

-LRB-

-RRB-

since

they

only

differed

by

one

amino

acid

and

their

respective

coding

sequences

by

two

nucleotides

For

three

putative

mGluRs

were

identified

-LRB-

Table

-RRB-

but

one

of

them

had

a

slightly

smaller

molecular

mass

than

the

other

two

-LRB-

kDa

-RRB-

We

did

not

however

detect

multiple

proteins

around

kDa

in

our

immunoblots

which

suggested

that

was

not

present

in

the

goldfish

retina

According

to

the

database

search

the

C-terminal

peptides

of

and

have

one

fish

homolog

each

The

candidate

shows

lowest

amino

acid

sequence

similarity

at

the

C-terminus

but

the

lack

of

overlapping

expression

patterns

between

and

in

the

OPL

of

the

goldfish

retina

excludes

cross-reactivity

of

and

In

summary

we

feel

confident

about

the

specificity

of

the

commercially

available

anti-mGluR

peptide

antibodies

since

the

carboxytermini

of

mammalian

and

fish

mGluR

homologs

are

nearly

identical

and

the

molecular

masses

of

fish

and

mammalian

homologs

are

similar

-LRB-

Table

-RRB-

mGluRs

of

Müller

cells

The

finding

of

in

Müller

cells

is

in

agreement

with

two

previous

studies

of

isolated

Müller

cells

from

the

salamander

these

studies

have

shown

that

activation

of

group

I

mGluRs

has

two

major

effects

First

it

leads

to

the

closure

of

the

K

conductance

of

these

cells

which

is

by

far

the

largest

in

Müller

cells

-LRB-

Schwartz

-RRB-

Since

their

glutamate

uptake

is

both

voltage-dependent

-LRB-

Brew

and

Attwell

Schwartz

and

Tachibana

-RRB-

and

regulated

by

the

internal

K

concentration

-LRB-

Barbour

et

al.

-RRB-

one

can

imagine

that

by

modulating

this

K

conductance

indirectly

modulates

glutamate

uptake

by

changing

the

membrane

potential

and/or

internal

K

concentration

The

second

effect

of

group

I

mGluR

activation

in

Müller

cells

is

the

generation

of

waves

by

triggering

the

release

of

from

internal

stores

-LRB-

Keirstead

and

Miller

-RRB-

The

physiological

consequences

of

this

effect

are

not

completely

clear

but

they

could

contribute

to

lateral

signaling

among

Müller

cells

as

in

astrocytes

-LRB-

Kim

et

al.

-RRB-

mGluRs

of

HCs

Here

we

show

that

rod

and

cone-driven

HCs

of

the

goldfish

express

mGluRs

from

all

three

groups

although

the

exact

mGluR

composition

of

these

cells

differs

slightly

from

reports

in

other

species

-LRB-

i.e.

no

in

cat

HCs

Cai

and

Pourcho

no

in

catfish

HCs

Gafka

et

al.

-RRB-

We

have

found

that

lateral

elements

in

the

rod

triads

are

positively

labeled

for

and

Since

the

labeling

for

is

rare

we

can

not

at

this

point

positively

identify

these

processes

as

belonging

to

either

rod-driven

HCs

or

MBCs

In

the

cone

pedicle

mGluRs

seem

to

be

expressed

in

a

highly

organized

manner

and

tend

to

be

localized

to

spinules

and

to

dendrites

occupying

the

lateral

position

in

the

cone

triad

whereas

has

been

found

at

both

lateral

and

central

processes

directly

opposing

the

synaptic

ribbon

This

extremely

ordered

pattern

indicates

that

either

a

single

cone-driven

HC

type

expresses

distinct

mGluRs

in

different

dendrites

or

that

the

expression

of

a

given

mGluR

is

restricted

to

a

certain

subpopulation

of

HCs

-LRB-

i.e.

the

distribution

is

cell-type-specific

-RRB-

In

cyprinid

retinae

three

physiological

and

morphological

HC

types

have

been

described

-LRB-

MacNichol

and

Svaetichin

Norton

et

al.

Stell

et

al.

-RRB-

one

could

easily

imagine

that

each

HC

class

expresses

only

one

or

two

mGluRs

at

their

dendritic

tips

The

light

responses

of

HCs

are

mediated

by

AMPA

-LRB-

acid

-RRB-

kainic

acid

receptors

-LRB-

Lasater

and

Dowling

Slaughter

and

Miller

Zhou

et

al.

-RRB-

and

are

not

abolished

by

mGluR

agonists

or

antagonists

-LRB-

Yang

and

Wu

Luo

and

Liang

-RRB-

showing

that

mGluRs

are

not

implicated

in

the

direct

generation

of

light

responses

They

are

most

likely

involved

in

the

modulation

of

the

light-driven

conductance

of

these

cells

In

the

fish

retina

for

instance

mGluR

agonists

have

been

shown

to

modify

both

light-modulated

-LRB-

Nawy

et

al.

Yasui

et

al.

Takahashi

and

Copenhagen

-RRB-

and

voltage-gated

conductances

of

cone-driven

HCs

-LRB-

Dixon

and

Copenhagen

Linn

and

Gafka

Gafka

et

al.

-RRB-

mGluRs

of

ON

MBCs

The

labeling

of

ON

MBC

processes

in

the

rod

spherule

with

the

antibody

against

corroborates

previous

studies

in

fish

-LRB-

Klooster

et

al.

Yazulla

and

Studholme

Yazulla

et

al.

-RRB-

and

mammalian

-LRB-

Koulen

et

al.

-RRB-

species

although

it

differs

from

the

situation

in

the

cat

retina

in

which

is

pre-synaptic

to

rod-driven

BCs

-LRB-

Cai

and

Pourcho

-RRB-

Since

no

evidence

has

as

yet

been

presented

for

the

participation

of

group

I

mGluRs

in

the

generation

of

the

light

responses

of

ON

BCs

one

can

only

speculate

that

this

protein

plays

a

modulatory

role

in

the

first

synapse

In

many

parts

of

the

central

nervous

system

group

I

mGluRs

are

involved

in

controlling

the

intracellular

calcium

concentration

and

membrane

channel

activity

-LRB-

for

a

review

see

Fagni

et

al.

-RRB-

In

the

spinal

cord

neurons

of

the

lamprey

for

instance

has

been

shown

to

regulate

the

resting

membrane

potential

and

thus

the

excitability

of

the

cells

by

controlling

a

leak

current

-LRB-

Kettunen

et

al.

-RRB-

Although

there

is

physiological

evidence

for

the

existence

of

a

group

III

mGluR

in

fish

ON

MBCs

-LRB-

Shiells

et

al.

Nawy

and

Copenhagen

Kamermans

et

al.

-RRB-

we

have

not

been

successful

in

obtaining

specific

immunoreactivity

with

the

antibodies

commercially

available

against

-LRB-

mammalian

-RRB-

and

Furthermore

there

is

as

yet

no

candidate

for

in

the

zebrafish

database

which

makes

the

production

of

custom-made

antibodies

against

this

protein

difficult

One

would

however

expect

these

cells

to

express

an

receptor

given

the

many

physiological

similarities

between

the

rod-driven

conductance

in

fish

and

mammalian

ON

BCs

Whether

and

in

what

manner

this

receptor

and

the

protein

interact

in

ON

MBCs

is

still

to

be

resolved

G-protein-coupled

receptors

can

interact

at

many

levels

For

instance

gamma-aminobutyric

acidB

receptors

form

complexes

with

dimers

and

thereby

regulate

the

sensitivity

of

the

latter

to

glutamate

-LRB-

for

a

review

see

Kubo

and

Tateyama

-RRB-

Interactions

between

mGluRs

however

have

so

far

not

been

described

Another

level

of

interaction

lies

within

the

second-messenger

cascade

Until

recently

only

one

G-protein

-LRB-

Goα

-RRB-

had

been

described

in

ON

BCs

-LRB-

Vardi

et

al.

-RRB-

which

suggests

that

and

share

-LRB-

part

of

-RRB-

the

same

intracellular

machinery

Nonetheless

a

second

G-protein

-LRB-

-RRB-

has

been

shown

to

coexist

with

Goα

in

rat

ON

BCs

and

not

to

couple

to

-LRB-

Tian

and

Kammermeier

-RRB-

providing

an

independent

G-protein

signaling

pathway

within

these

cells

this

pathway

might

involve

O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O B I O O B I O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

metabotropic glutamate receptor
retina
electron microscopy
immunohistochemistry
bipolar cell
horizontal cell