Dopaminergic neuromodulation in the retinas of lower vertebrates

Neuroscience Research, Suppl. 8 (1988) S183-$196 Elsevier Scientific Publishers Ireland Ltd.

DOPAMINERGIC

RETO

NEUROMODULATION

WEILER

GERTRUD

° , KONRAD

KURZ-ISLER

IN

KOHLER

^,

S183

THE

°,

RETINAS

WALTER

HANS-JOACHIM

OF

LOWER

KOLBINGER

VERTEBRATES

° , HARTWIG

WOLBURG

,

WAGNER"

"Department of Neurobiology, University of Oldenburg, Oldenburg; ^Department of Pathology, University of T~bingen, T~bingen; "Department of Anatomy & Cell Biology, Philipps University, Marburg

INTRODUCTION

Many lower vertebrates light

conditions

live in habitats

occur.

mechanisms have evolved to visual

Several

where

neuronal

overcome

rapidly and

changing

non-neuronal

the resulting problems for the

system. Since most of these mechanisms are localized

retina,

this

reversible

neural

in

the

tissue is well suited to study short-term and

neuronal

processes

such

as

adaptation

and

their

underlying neuromodulatory actions. There is ample evidence that within amacrine

cells

sensitivity

play

an

(5, 18).

In

the

retina

horizontal

addition

to

these intrinsic retinal neu-

rons, efferent fibres projecting to the retina, might volved

in

sensitivity

lation has found

control

support

tides in these fibres

by

also

be

the

degree

the

recent localization of neuropep-

(12, 16).

just

uncovered

xity of the underlying retinal circuit cells

ticity is localized at pedicles.

parts of the comple-

(8). Moreover,

teleost

In

the

spinules with membrane densities Several reports

have

light

dendritic

light-adapted

During dark adaptation,

processes

retinas, into

(II, 13, 15). This plas-

the

invaginating

from

membrane

the

these dendrites extend cavity of the pedicles.

these spinules are degraded and disappear. assigned

the neurotransmitter dopamine in

the retina a key role in the regulation of adaptational changes ranging

cone-

change their synaptic connections with the photo-

receptors depending on the available

cone

modi-

of their electrical coupling through gap junctions.

Intensive recent studies have horizontal

in-

(3). The idea of an efferent modu-

Horizontal cells in the fish and turtle retina are able to fy

and

important role in the neuronal control of

conductance

(5)

changes to morphological plas-

ticity. Presented at the 10th Taniguchi International Symposium on Visual Science, November 23-27, 1987 0168-0102/88/$03.50 © Elsevier Scientific Publishers Ireland Ltd.

S184

Here we s u m m a r i z e dopamine

in c o n t r o l l i n g

spinule

dynamics

SPINULE

PLASTICITY

the

cone

reveals

invaginate

cytoplasm.

These

membrane

originate section also

in a

at

from a single spinules

isolated

In dark a d a p t e d the

synaptic

order

retinas,

spinules

within

(spr) w i t h i n

light a d a p t e d

the

retina

retina

has the

raises

the q u e s t i o n to

that g l u t a m a t e

retina

3.

retinal

cell and

an

have

spinules ultrathin

are found but

dendrites

next

bear no spinules. related

the

the n u m b e r

to In of

number

of

synaptic

Typically

the

spr for a

of s p i n u l e s

in a d a r k

45

min.

between

Interestingly,

horizontal

cone

experiments

spinule

transmit

We have

cells

and

relevant

demonstrated

and

its a g o n i s t s

of s p i n u l e s

on the other

plasticity

the

recently

neurotransmitter

the number

upon

of cAMP

triggered

pathways

projecting

effects

increase

about

cell.

Dopamine

cells

prominent

reconstructions

In

250

(15).

in r e d u c i n g

(17).

into the cone

i).

Formation

light-dark

horizontal

additional

mine-stimulated

a

which

not evoke c o n s i s t e n t

to

interactions

the p u t a t i v e

by i n t e r p l e x i f o r m

conducted

about

of

the

w e r e very e f f e c t i v e adapted

pedicle

same time c o u r s e

observation

(Fig.

we have

to light takes

of f e e d b a c k

The

information

is

horizontal

exhibit

dendrite.

the h o r i z o n t a l

cone

of

f r o m the d e n d r i t e

inconspicuous

at

i).

length of a b o u t

they

the same cone pedicle.

exposed

formation

cones

a given

by

sections

retina up to t w e n t y

these observations,

ribbons

adapted

are

reflected

processes

Computer

cell

arising

(Fig.

average

the cone c y t o p l a s m a

ribbon

to q u a n t i f y

an

adapted

horizontal

directly

within

finger-like

tips.

light

role of

light or d a r k a d a p t e d

dendrites

Typically,

their

fully

either

the t e r m i n a l

nm.

as

the

from tangential

differences

(13) have

i00

densities

shown that

from

with numerous

of

plasticity

micrographs

retina,

concerning

morphology.

pedicles

spinules

nm and a d i a m e t e r

cell

characteristic

In a light a d a p t e d cells

experiments

horizontal

of e l e c t r o n

level of the retinas

recent

and gap junction

A comparison

carp

our

in

hand w h i c h

a

light

is r e l e a s e d

onto

horizontal

cells

spinule

formation.

We

focusing

on d o p a m i n e

and the d o p a -

(14).

(4), did therefore

S185

spr I-

T I

05

L

D

Fig. I. Effect of light (L)and dark (D)-adaptation on spinule formation, a,b) Electron micrographs of tangential sections. The bar on these and all subsequent e l e c t r o n m i c r o g r a p h s indicates 1 ~m. H o r i z o n t a l cell d e n d r i t e s (large, pale structures) invaginate into the c o n e c y t o p l a s m a (cp). Several spinules w i t h m e m b r a n e densities (arrows) are v i s i b l e in the light a d a p t e d retina (a) but not in the d a r k a d a p t e d retina (b). c) Histogramms. spr = normalized v a l u e of the number of s p i n u l e s per s y n a p t i c ribbon.

S186 For

this

purpose

electron

microscopic

r e t i n a s w h i c h w e r e kept for 40-60 m i n NaCI,

5

mM

KCI,

HEPES;

pH 7.5)

1

to

mM

MgCI=,

which

the

analysis

in a R i n g e r

2.5 m M CaCI=,

substance

w e r e done w i t h c a r p of a b o u t

10

were

under

cycle.

kept

a

12-hour

w e r e at least 4 hours darkness The same

left eye of e a c h animal treatment

during

of

except

the e x p e r i m e n t . destroyed

prior

to e n u c l e a t i o n .

vations,

by an i n j e c t i o n

spinules

cone

normalized

The number

Ringer

of s p i n u l e s the

(Fig.

been d e s t r o y e d (Fig.

values

of

were

a

adaptation,

Ringer

2a&d). it

investigated

has been

in a

neurons 12 days obser-

minimum

same

of

responsible

the control

the

retina

retina, Only

to

synaptic

the

of the

~M) to the

during

light

light-adapted to the

did

not

were observed

(17)

stimulates

an a d e n y l a t e -

horizontal

cells

incubated

complex

values

however,

with

forskolin

did not s i g n i f i c a n t l y

forskolin,

to

in 3 out of 14 e x p e r i -

formation

or

had

light adap-

of d o p a m i n e

of cAMP on s p i n u l e

(500 ~M)

(50-250

neurons

(50-250

spinules

within

either

phosphate

of

of

spinule

effect

during

concentrations

cAMP

not a f f e c -

were c l o s e

of d o p a m i n e

formation.

of

were

formation,

spinules

spr-values

dark adapted

of 8 - b r o m o - c A M P

morphology

the d o p a m i n e r g i c

shown that d o p a m i n e

a possible

3':5'-cyclicmono-

number

increases

retinas

the a d d i t i o n

activity

was m a i n t a i n e d

retina was

a dopamine-depleted

spinule

stages

s y s t e m and

where

addition

the

a normal

(Fig.

all

f r o m the Ringer.

dopamine-containing

to d e v e l o p

of the

induce

transitional

in

retina w h e r e a s

The

containing

ments

on spinule

the

and the spr ratios w e r e

light a d a p t e d

Retinas

dark adapted

containing

adapted

underwent

dopaminergic

ribbon w e r e c o u n t e d

a

not able

increased

consistently

cyclase

in c o m p l e t e

the e l e c t r o n m i c r o s c o p i c

The c o r r e s p o n d i n g

The a d d i t i o n

Since

animals

of 100 ~g 6 - h y d r o x y d o p a m i n e

3 to 4 r e t i n a s

in

2a&b).

2a&c).

solution

values.

and

experiment

experiments

preparation

left eyes w e r e normal. Ringer

from

All

which

for c o m p a r i s o n .

ted by i n c u b a t i n g

tation

taken

Dark adapted

as control

the

quantify

per s y n a p t i c

pedicles

length

and s u r g e r y was d o n e

to

some

To

body

mM 2 mM

test was added.

cm

the drug was o m i t t e d

prior

For

(116

for

system. served

that

adaptation

were

~M)

adapted

w i t h an i n f r a r e d v i d e o

The state

50

dark

light-dark

solution

10 mM Glucose,

under

experiments

was done

(5), we

plasticity.

Dark

8-bromoadenosine (50 ~M). W h e r e a s change

the

over-

(Fig.

3) and had no e f f e c t

an i n h i b i t o r

of p h o s p h o d i e s t e r a s e

for the b r e a k d o w n

of cAMP,

had s i g n i f i c a n t

$187

DA 6-0HO

DA

spr

,,m,,,

1-

1

::::::: -~..., ::::::: :.:-:-:

!i!

0.5

......,

I

!!!!!!i

~!i!iii I :i:i:i:i

a

L

D

L

Fig. 2. Effects of d o p a m i n e (DA) and d o p a m i n e - d e p l e t i o n (6-OHD) on spinule formation during dark (D) and light a d a p t a t i o n (L). a) Histogramms; b-c) E l e c t r o n m i c r o g r a p h s of the t r e a t e d retinas: LDA (b), DDA (c) and L 6 - O H D (d). effects

(Fig.

overall

morphology

ribbons, seemed

were that

magnification was v i s i b l e of

cone p e d i c l e s 4e).

of the

affected.

no

spinule

synaptic

The

were

(Fig.

4d) a t e r m i n a l

was never

seemed were

digested

to

seen be

affected;

formation

complex

ribbons

they

which

forskolin

(Fig.

4). A l t h o u g h

and

became f r o m their patch

nearby

much

specific, ribbons

induced, the

end.

since only in

and

it

At higher

dense

retinas.

the

synaptic

shorter

distal

of e l e c t r o n

in u n t r e a t e d very

was

mainly

material

The

effect

ribbons

rods w e r e

in

intact

S188

spr I-

0.5

DcAMP Fig. 3. Effect of 8-bromo-cyclic s p i n u l e f o r m a t i o n in a d a r k adapted b) i n c u b a t e d retina; c) h i s t o g r a m m

a d e n o s i n e m o n o p h o s p h a t (cAMP) on (D) retina, a) control retina;

$189

i ¸ i~,~ ,~i!i~i ~i~<~ ii ¸¸¸

spr 1 -

0.5

o

OForsk

Fig. 4. E f f e c t of f o r s k o l i n (Forsk) on s p i n u l e f o r m a t i o n in a dark adapted (D) retina, a-c) see Fig. 3. d) short r i b b o n (arrow) w i t h e l e c t r o n d e n s e m a t e r i a l at its edge (thick arrow), e) c o n e p e d i c l e w i t h short r i b b o n s (arrows) and rod s p h e r u l e with unaffected, long r i b b o n (thick arrow).

S190 Preliminary components

experiments,

on

the n u m b e r of s p i n u l e s kolin

selectively

neurons

of spinules to d e p e n d these

is

cone

transfer

on a d i r e c t

neurons.

retina

decreased

synaptic

an e s s e n t i a l cells.

elevation

two

indicate

that

and that a g a i n

show that

information

fors-

however, level

the s e n s i t i v i t y

dopamin-

for the f o r m a t i o n

The formation,

of the c e l l u l a r

During darkness

is d r a s t i c a l l y

of t h e s e

ribbons.

the data of these e x p e r i m e n t s

to h o r i z o n t a l

to d o p a m i n e

the e f f e c t

of a light a d a p t e d slightly

affected

T a k e n together, ergic

w h e r e we t e s t e d

the s p i n u l e s

seems

not

cAMP

in

of

of h o r i z o n t a l

cells

reduced.

GAP J U N C T I O N S The e l e c t r i c a l retina

is

established

to

increased

of

and

of the

horizontal

cells

by gap

by c o n n e x o n s these

(2).

In f r e e z e

a possible

connexon

aggregates.

retina

(6,

on two s u c c e s s i v e

Lucifer

from

the

of

the c o u p l i n g

This m e t h o d was of

microscopy

on

upon

carp is

formed

prepara-

on the P - f a c e

the

recently

adaptation

for

channels

therefore

dopamine

cells.

this c o u p l i n g

as p a r t i c l e s We

increase

horizontal

of m e m b r a n e

E-face.

receptive

an

should affect

electron

Yellow.

dye transfer,

the overall

(7). M o r p h o l o g i c a l l y ,

three

decided

or

to in-

morphology

of

u s e d by two of us to

gap

junctions

in c a r p

(6-OHD)

days,

respectively.

(19).

pieces

using

marks.

One p i e c e

eyes

from the

comprising

dopamine

dose of

The a n i m a l s w e r e Both

were d e t a c h e d

three

Pseudemys scripta w e r e i n j e c t e d

turtles

days w i t h a total

mine

endogenous

dopamine

was

21).

The right eyes of

retinas

dye

neighbouring

fracture

turtle

the r e c e p t i v e

decreased

decreased

aggregates

influence

influence

the

as r e s u l t i n g

are v i s u a l i z e d

pits on

the

as shown e l e c t r o p h y s i o l o g i c a l l y

in c u l t u r e

connexons

of

between

of

measuring

dopamine

but

is correct,

junction

vestigate

the

response

cells

This type of m o d u l a t i o n

methods

were explained

junctions,

as c o r r e s p o n d i n g

examine

(i0).

transfer

resistance

If this e x p l a n a t i o n resistance

tions

horizontal

experiments

effects

coupling

mediated

the

these

the central

These

the

between

by d o p a m i n e

by e l e c t r o p h y s i o l o g i c a l

field p r o p e r t i e s According

field.

coupling

modulated

release

were

the visual the

100

sacrificed enucleated

pigment

~g

6-hydroxydopa-

after

5, 12 and 15

and h e m i s e c t e d .

epithelium

and

cut

The into

s t r e a k and the optic disc as landnasal

and total

half content.

was u s e d to d e t e r m i n e The

second

piece,

SI91

the

ventro-temporal

chemistry

quarter

was

and the d o r s o - t e m p o r a l

used

for

subsequent

immunohisto-

p a r t was u s e d for f r e e z e - f r a c t u r e -

EM e x a m i n a t i o n . The nasal chamber

half of

the

retina

and c o n t i n u o u s l y

containing

0.1 M

m i n and r e l e a s e

(1 ml/min)

ascorbic

acid.

of d o p a m i n e

was

potassium

concentration

of

extraction

was p e r f o r m e d

using

fractioning system

a

dopamine

was measured.

dopamine

content

Release

after

induced

a

Ringer

all

of e n d o g e n o u s the

second

diffusion

superfusion

Twelve

right

days

eye

5

the

release

This m i g h t

prevent

after

from

concentration

The basal

which

total

obtained

fractions.

°

a

rapid

injection,

~

.,_6

8~

a,i t,_

\4.

6' '~ 4.L,.

I::m

~2-

2-

1::3

I

a

the

of

fraction.

from the

dopamine.

the

content

of

potassium

Ringer

and HPLC

the r e l e a s e

final

5a w e r e

and

barriers

medium.

After

the

5

the

this m e t h o d

as p e r c e n t a g e

the

every

Catecholamine

of the c o r r e s p o n d i n g

10

<

With

and

animals

solution

increasing

absorbance

of i0 pg dopamine.

Increasing

during

be due to the retinal of the

of

release

higher

oxide

like the one of Figure

injection.

marked

by

standard.

is given

superfusion

with Ringer

up to 25 mM.

homogenized

Release

a

were collected

twice

aluminium

at the b e g i n n i n g

left eyes

the

was t y p i c a l l y

change

was

protocols

the u n t r e a t e d days

the

into

superfused

initiated

sensitivity

the retina

placed

Fractions

w i t h D H B A as an internal

reached

experiments

was

R

K

I

R

o

K

b

A 6-OHD

Fig. 5. a) R e l e a s e of e n d o g e n o u s d o p a m i n e from a control retina;= R= S u p e r f u s i o n with Ringer; K= Superfusion with high p o t a s s i u m Ringer. The release from a retina w i t h depleted dopaminergic neurons was not m e a s u r a b l e , b) Total c o n t e n t of d o p a m i n e in the same control retina (blank) and in the corresponding d e p l e t e d right retina (6-OHD).

S192

Fig. 6. a) T y r o s i n e h y d r o x y l a s e - l i k e i m m u n o r e a c t i v i t y in a control retina. Frozen section, b) Depleted retina. Only a few w e a k l y labeled somata were discernible. There is no b a n d i n g p a t t e r n in the IPL. S c a l e 25 ~m the total d o p a m i n e c o n t e n t was ng

in

the

control

c o u l d be d e t e c t e d however,

retina

r e d u c e d to 1.6 ng as o p p o s e d (Fig.

in the i n j e c t e d

5b). A f t e r eye

treated

r e l e a s e nor a

retina

(12

days)

potassium

To c o n f i r m the

cellular

no d o p a m i n e

8.2 ng) which,

the r e t i n a since s e c t i o n s of cut

ringer).

is a r e l i a b l e m a r k e r

this

tissue

and

incubated

Thus,

the

the i n j e c t i o n of

lacks

of

tyrosine

noradrenergic ventro-temporal

revealed

hydroxylase

for d o p a m i n e r g i c n e u r o n s

with a primary monoclonal

I m m u n o r e a c t i v i t y was

from

of d o p a m i n e and its d e p l e t i o n

localization

paraformaldehyde-fixed

release

its d o p a m i n e c o n t e n t .

location

we u s e d i m m u n o h i s t o c h e m i c a l This e n z y m e

evoked

was d i s c e r n i b l e .

6 - O H D d e p l e t e d the r e t i n a of 80 % of

were

(control v a l u e

6.7

s h o w e d signs of d e t e r i o r a t i o n .

N e i t h e r a basal

(TOH).

15 days,

to

using

in

neurons.

Frozen

retina

pieces

antiserum

(B6h-

an FITC- or H R P - c o u p -

led s e c o n d a n t i b o d y . TOH-like described

i m m u n o r e a c t i v i t y was o n l y f o u n d elsewhere

responded perfectly with the c o n t r o l 6-OHD,

as well

the

as in the

l a b e l l e d a m a c r i n e cell

in,er p l e x i f o r m

in

(9, 20). The o c c u r r e n c e of

layer y i e l d e d

f r o m the t h r e e r a m i f i c a t i o n

findings retina

of 5

amacrine

pattern

as

the r e l e a s e studies.

days

after

somata were visible a

cells

labelled cells cor-

injection

(Fig.

of t h r e e

levels of t h e s e neurons.

In of

6a) and the

layers

resulting

12 and 15

days

$193

after

the

sections, somata

injection roughly

were

this p a t t e r n

covering

encountered

retina

ensured,

that the d e p l e t i o n

was q u i t e ergic

complete

mm =

only

4

The c o r r e s p o n d i n g

somata. of

and that

comparison

between

outer

of these

of

The

data

they

were

weakly

value

neurons

In i0

labelled

for

f r o m these

dopaminergic

two a n i m a l s

control

scattered

were

connexons

this

towards

other. ergic

an

un-

experiments

in two retinas

therefore

lacking

dopamin-

replicas

f r o m gap

junctions

These

were

and

are

good

retina

(I).

direct

measurement

drawn

gap

differ

7).

In and

contrast,

densely

distribution

junctions

control

with

in the a b s e n c e from

resistance

whether coupling

They

Corresponding

work

correlate.

a modulatory

experiments,

(i0)

allow

This

action

i.e.

a

will

of a sub-

or for example,

are p r e s e n t l y

sup-

in the c a r p

subsequently

resistance

each

by the d o p a m i n -

observations

will

and

confirmed

of gap junctions.

recent

replicas

in the

packed

significantly

a modulatory

and f r e e z e - f r a c t u r e - E M

laboratories.

By

retinas

arranged

f r o m the e l e c t r o p h y s i o l o g i c a l

distinction

conductance.

electrophysiology in our

packed

(Fig.

loosely

were

The f r e q u e n c y

of the c o u p l i n g

the

difference

found.

formation

fracture

in fact c h a n g e s

membrane

the

and u n t r e a t e d

rather

connexons

rare.

agreement

Freeze

the

on

were

frequently the

data d e m o n s t r a t e cells

the c o n c l u s i o n s

facilitate

a remarkable

more densely

port

stance

the t r e a t e d

The two d i s t r i b u t i o n s

amacrine

in

of

the c o n n e x o n s

retinas

scattered

of dopamine.

cells

revealed

retinas

connexons

shift

freeze-fracture

horizontal

dopamine-depleted

taken

66

1

disappeared.

activity.

A

the

was

about

(6b).

treated

had c o m p l e t e l y

the

combining being

under-

$194

0,6

J~ 6-0H-DA

[-]CONTROL ~= 4320 s=606 n=9

U= 6270 s=315 n:9 ,111, r//l~ r///j r///~

0,4

0,2

,[ s

C

6

7

8

CONNEXONS/.urn2

Fig. 7. F r e e z e f r a c t u r e e l e c t r o n m i c r o g r a p h s of g a p j u n c t i o n s f r o m a c o n t r o l r e t i n a (a) and a dopamine-depleted r e t i n a (b). c) F r e q u e n c y d i s t r i b u t i o n of t h e c o n n e x o n d e n s i t i e s f r o m a) a n d b). Scale 0.i ~m

S195

CONCLUSIONS Neuromodulation neural ding

principles

within giving

and a s t o n i s h i n g

trated

on

activity these

some

actions

our recent

horizontal

perfect

is

the

morphological

structures

possible

pathways

as

as

We have

amenable

used

report

of

electron

this

have

concen-

What makes

neuromodulatory its traces

within

microscopy.

of

It is

different

retinal

of i n t r a c e l l u l a r

morphological the

outstan-

neuromodulatory

of

leaves

influence

contribution

retina

we

the retina.

analysis

to

the

one of the

enormous,

concerning

level an

is c e r t a i n l y their

neuromodulation

the

in the fish and t u r t l e

In this findings

for

to a n a l y s e

well

circuit

networks

cell

targets

fact that

therefore

components.

neuronal

capabilities.

of

at the

cells

a local

approach

metabolic

to e l u c i d a t e

possible

role

of

dopaminergic

affects

both

analyzed

neuromodulation. The

loss of d o p a m i n e r g i c

nents

of

synaptic

horizontal gap

cell

junctions

followed does

no

tics.

plasticity:

dendrites

of these

by

the

longer

In terms

neurons

and

the the

neurons.

produce

density

Light

these

which

morphological

features

the

at the

within

is

the

normally

and a low c o n n e x o n

synaptic

morphological

of spinules

of c o n n e x o n s

adaptation

of s p i n u l e s

formation

of these

formation

compo-

density

characteris-

retina

remains

d a r k adapted. Surprisingly a

dark

the neuronal great

the f o r m a t i o n

adapted

activity

extend

underlying

retina

the

of s p i n u l e s

by e x o g e n o u s

within

the

retina

dopamine-sensitivity

mechanisms

ty of

neuromodulation

certainly

system

information

changing

photic

during of

be induced

or cAMP.

It seems

darkness

horizontal

are not yet u n d e r s t o o d .

visual

to e n s u r e

can h a r d l y

dopamine

reflects

blocks

in a

cells.

The

The o b v i o u s the

processing

complexi-

difficulty in

a

in that

of the

complex

and

environement.

ACKNOWLEDGEMENT The p r o j e c t s

were

ungsgemeinschaft. to

give

tenth

one

Taniguchi

financically We w o u l d

of

us

like to thank

(R.W.)

Symposium

supported

the c h a n c e

on Visual

by the

the D e u t s c h e Taniguchi

to report

Science.

these

Forsch-

Foundation data at the

S196

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