Dopamine release induced by electrical field stimulation of rat hypothalamus in vitro : Inhibition by melatonin

Vol. 104, No. 4, 1982 February 26, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNlCATlONS Pages 1610-1616

DOPAMINE RELEASE INDUCED BY ELECTRICAL STIMULATION

OF RAT HYPOTHALAMUS --IN VITRO:

INHIBITION

BY MELATONIN.

Nava Zisapel Department The George Tel-Aviv Received

January

12,

FIELD

and Moshe Laudon of Biochemistry

S. Wise Faculty University,

of Life

Tel-Aviv

Sciences

69978,Israel.

1982

Microdissected slices of rat hypothalamus were incubated with 3H -dopamine and then subjected to two successive sets of electrical field stimulation in a superfusion chamber. Neurotransmitter release was found to be calcium dependent and the amount of release was determined by scintillation counting of the effluent buffer. The release obtained following the first train of stimuli served as an internal reference. The samples were exposed to drugs during the interval between the two sets of stimuli. Using this technique, as well as K+-evoked depolarization, we were able to show that subnanomolar concentrations of melatonin, the hormone secreted from the pineal gland, inhibits dopamine release from hypothalamic slices. The possibility that melatonin modulates neurotransmission in the brain is therefore indicated. Melatonin,

a hormone

by the pineal

gland

of all

regulated

predominantly

endocrine

organs,

system.

It

namely,

also

protein

especially affects

uptake

review,

1).

has been

the hippocampus demonstrated lo-*M

It

a variety serotonin

The presence

demonstrated

in

and the striatum (3).

was found

to modulate

pituitary

release

affects

the

(3).

secretion

reportedly

from

stimulated

0006-291X/82/041610-07$01.00/0 Cop.vright AN rights

0 1982 by Academic Press, Inc. of reproduction in any form reserved.

1610

which

is

of a variety

of the

central

of

nervous

in the brain,

butyric

acid

(GABA)

and tubulin binding

sites

at concentrations

mela(2),

of 10m7 to (GH) by Since

(4). (5,6),

(for

have been

hormone

the hypothalamus by dopamine

for

membranes

binding

of growth

content,

levels

sites

hypothalamic

Low affinity

of somatostatin is

affinity

Melatonin,

rhythm

functioning

synaptosomes,

of high

and secreted

a circadian

functions

and 4-amino from

synthesized

systems

the medial-basal

the midbrain

somatostatin

in

of metabollic

and release

the release

species

the neuroendocrine

in

stimulating

is

serotonin,

vertebrate

synthesis,

see ref.

from

by light.

neurotransmitter

tonin

derived

it

was per-

vol. 104, No. 4, 1982 tinent

to ask whether

ted through

release

were

in

field

stimulation.

shown

to occur

concentration which

in

in turn

organ modified

field of Torpedo

the external an

endings

stimulation (9)

the role

b-induced

depolarization

evoked

--in vivo

and --in vitro

medium brings

about

in neurotransmitter for

techniques

(7). membrane

slices

brain

release

(10)

to measure

(7). release

neurotransmitter

from rat

is

exer-

neurotransmitter of melatonin

the

has been effect

hypothalamic

in

and electrical

by depolarization

Ca2' permeability

and from brain

release

modes of inducing

release

increased results

and used these

the neurotransmitter

both

release

release.

to elucidate

the hypothalamus:

synapses

RESEARCH COMMUNICATIONS

on somatostatin

different

in the study

causes

the nerve

electrical

with

Transmitter in

effect

of dopamine

systems

employed

neurosecretion

AND BIOPHYSICAL

the melatonin

the,modulation

Two superfusion

into

BIOCHEMICAL

has been Increasing

the Kt

depolarization, Introducing (8).

Ca2+

The use of

from the electric reported. of m.elatonin

We have on

sections.

METHODS Preparation and labeling of the tissue: Adult, female rats (CD strain), at the estrous stage, were decapitated and their brains rapidly removed and placed on ice. The hypothalami were dissected out and cut into two halves correspondin to the contralateral sides. Tissue sections were incubated with 0.4uM [ 5H]-dopamine (New England Nuclear, Chicago) in Krebs-Ringer buffer, pH 7.4, (123 mM NaCl, 0.5 mM NaHzPOb, 0.4 mM MgC12, 0.25 mM NazHPOb, 3.0 mM KCl, 1 mg/ml glucose, 0.75 mM CaC12) for 20 min at 37'C. Using opthalmic tweezers, the labeled hypothalamic sections were gently placed on a small piece of nylon cloth between two platinum electrodes, lying parallel to each other and 5 mm apart, in a 0.5 ml superfusion chamber. The electrodes were connected to a stimulator (Grass Model SDS). Prior to stimulation, the tissue was superfused for 3G min with a continuous flow (1.2 ml/min) of the KrebsRinger buffer, which was constantly aerated and kept at pH 7.2 - 7.4 and 37°C. The effluent buffer was collected in a fraction collector. A set (train) of stimuli was then applied to the tissue (75V, 20H2, 10 msec pulse duration, for 25 set). The perfusated buffer was collected at 25 set intervals (0.5 ml/ fraction) into scintillation counting vials. Hydroluma scintillation cocktail (4.5 ml) was then added to each vial, and the samples were counted by liquid scintillation spectrometry. After the first train of stimuli, the tissue was superfused with the Krebs-Ringer solution in the absence of CaC12, in the presence of CaC12 (O-l mM) together with lo-l5 - lo-' M of melatonin (Sigma Corp), or in the presence of CaC12 and 0.5 nM benztropine mesylate (Merck, Sharp and Dohme). After 25 min, a second train of stimuli was applied (75V, 20H2, 15 msec pulse duration, for 25 set) and the released t3H]-dopamine was collected by superfusion as The results were recorded either as counts per min ofi3H]described above. -dopamine in the effluent fractions, or the quantity of [3H]-dopamine released after the second train of stimulation, expressed as a percentage of the quantity released after the first train of stimulation. Release by elevation of extracellular K+ concentration: e-evoked depolarization of the labeled slices was carried out by partially substituting Na+ for K+ in the superfusing buffer (43 mM KCl, 33 mM NaCl) which also contained 1611

Vol. 104, No. 4, 1982

Figure

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1

/

I

1

I

I

2

4

6

2

4

6

1 Release of radioactive dopamine from dissected rat brain hypothalamus in the absence (A) and in the oresence (B) of 0.5 UM benztropine. The curves.are mean counts in the superfusion medium, from two different slices after the electrical stimulation (25 set, 75V, 2OHz, 10 msec peak duration) at the times indicated by arrows.

0 - 10m5 M melatonin. One side of a hypothalamus was subjected to melatonin and its contrlateral side served as an untreated control. treatment, Before the depolarizing buffer was applied, the latter tissue was exposed to superfusion with normal Krebs-Ringer solution containing 0 - 10s5 M of melatonin at 37'C for 15 min. RESULTS AND DISCUSSION The time

course

electrical

stimulation

mitter level

increased only

inhibitor

is

essentially

uptake

ted by two exponentials

thus

take

to the

system The release

probably

having represents

decrease

in the

of the neurotransmitter

to be calcium

dependent,

since,

of labeled

persisted

neurotrans-

to the background

for

by 20-25%

and

(Fig.

How-

1R).

following

stimulation

and in the

absence

of benztropine.

of benztropine

a time

of 4.8 constant

the component total

amount from in

1612

could

of 8.6 min. contributed

was

and in its The faster

by the reup-

of neurotransmitter

the absence

The

be approxima-

and 7.8 min,

the hypothalamic

an

even longer

released

constants

upon

of 0.5 PM benztropine,

increased

in the absence time

hypothalamus

and returned

the efflux

initially

having

by one exponential

exponential

shown

(111,

released

decay

from rat

The efflux

1A.

stimulation

same in the presence

of radioactive

[3H]-dopamine

In the presence

minutes.

of dopamine

the

presence,

after

of neurotransmitter

the amount

of

shown in Fig.

several

of dopamine

extent

release

immediately

after

the amount ever,

of the

slices

of CaC12 in the

released. was super-

Vol. 104, No. 4, 1982

Figure

fusing of

2

the

observed

observed

in

in the

cium retained When the

tissue

pulse

duration

train

of

the

presence

within

of radioactivity

first

amount

CaClp-free

lease

a release

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Release of radioactive dopamine from rat hypothalamus --in vitro in the absence (A) and presence (B) of melatonin (1 fl). The tissue was subjected twice to stimulation at the times indicated by arrows. The curves are mean counts from two different slices in the perfusion medium after electrical stimulation. The first stimulation (25 sec. 75V, 20&z, 10 msec peak duration) was performed under standard conditions and serves as a reference. The second stimulation (25 sec. 75V, 20 Hz, 15 msec peak duration) was performed after the tissue was equilibrated without (A) or with (B) 10 UM melatonin.

buffer,

that

BIOCHEMICAL

of

at

upon stimulation 0.4 mM CaC12.

least

medium may reflect

the presence

was 30-40% The basal

release

of endogenous

cal-

the tissue. was restimulated was 65-70%

of the second

of neurotransmitter (Fig.

released

dopamine

train equal

after of that

an interval of the

of stimuli in amount

2A). 1613

from

of 15-25

first

peak.

min,

Increasing

10 to 15 msec,

to that

released

the

the

resulted

after

re-

the

in

BIOCHEMICAL

Vol. 104, No. 4, 1982 The slices series

could

be reloaded

of stimulations

thus

indicating

RESEARCH COMMUNICATIONS

with[3H]-dopamine

without

that

AND BIOPHYSICAL

and exposed

any significant

the electrical

shock

loss did

to another

of releasing

not

cause

capacity, irreversible

any

inactivation, When different often

yielded

tissue decay

slices

curves

was presumably

due to the

from different

depths

evoked

by the

condition,

fact

set

of the

set,

shape

the

blocks.

as an internal

the

reference

released

first

was

release

of the

standard

release.

drugs

was carried

of stimuli,

beginning

radioactivity

This

released

used the

of the experimental

two trains

they

constants.

radioactivity

We therefore

to the different the

experiments,

and time

diffusing

as indicative

tissue

after

different

of stimuli

between

i.e.,

used in successive

that

of tissue

first

25 min interval first

having

and the second

Exposure

were

returned

out

during

the

10 min after

the

to background

level. Figures

2A and 2B show the effect

of dopamine ZA),

the

from the hypothalamic

amount

92% of that amount that

of dopamine following

Figure dopamine

release

is of

tissue

In the absence

This

in the

effect were

not

suggests

contributing

released

during

presence

of melatonin the second

of melatonin

the second

to which

the

on the

(Fig.

stimulation

the

absence

inhibition

is

(Fig.

peak is ZB),

only

the 30% of

amount

0.6 nM.

to an increase

also

the melatonin

also

in the

inhibits

amount 1614

shows

was caused

increasing

of dopamine

was only

The concentration

Fig.3

release

stimulated At melatonin

released

benztropine

inhibition

the melatonin

of dopamine

experiments,

of benztropine, the

of melatonin.

of the hormone.

when the dopamine In these

of electrically

concentration

was approximately

obtained

increase that

extent

1 UM and above,

depolarization.

1 nM did

following

dependent

released

a half-maximal results

In the

first.

released

3 shows the

40% of that

dopamine

release

the first.

concentrations

lar

the

(1 uM) on the

In the absence

slices.

of radioactive

during

of melatonin

release the reuptake

of neurotransmitter

causing that

simi-

by e-induced

was also

present.

concentration (data

35-

not

above

shown).

of dopamine, retained

thus in

the

vol. 104, No. 4, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

613-

3-

II-

log

Figure

buffer,

centration

on the

Electric

and reducing amount

field

advantages

over

slices k-

arising

from

zation;

2) it

causes

trolled

conditions.

on a single

ric

tissue

avoiding

seen

In conclusion, lamus

raises

secretion

1

for

less

extensive

enables

depletion

in different

in the brain.

that Since

this

re-

has certain

the technical

environment

diffi-

during

of neurotransmitter

depolari-

under

neurosecretion

con-

experiments

in size

peak,

and geomet-

slices.

by melatonin

the possibility

manner

from the heterogeneity

brain

con-

neurotransmitter

the use of one of them as a reference

arising

inhibition

obviates

the ionic

The use of double-stimulation slice

studying

1) it

to change

melatonin

released.

calcium-dependent

depolarization:

the need

of the increased

dopamine

as a technique

complications

structure

effect

in a reproducible,

evoked

culties

the net

of radioactive

stimulation

from brain

thus

M

3 Inhibition of dopamine release from hypothalamic slices by inThe curves are mean values creasing concentrations of melatonin. showing the percentage of inhibition from two different slices, of radioactive dopamine release from slices subjected to electrical stimulation (o-o) or to I@-induced depolarization (o-o) by meIatonin. The amount of inhibition is expressed as a percentage, with 0% indicating the amount of inhibition after the first train Of electrical stimulation (0-s) or after the K+-induced depolarization of the contralateral side of the same h pothalamus (o-o). The superfusing buffer contained benztropine ( d .5 PM).

superfused

lease

(melatonln.

of dopamine hormone

the concentration 1615

may act

release

from

as a modulator

of melatonin

within

the hypothaof neurothe rat

vol. 104, No. 4, 1982 hypothalamus melatonin logical

--in vivo effect

has been

it

effect

and not releasing

estimated

Melatonin

seems likely on the responses

directly

AND BIOPHYSICAL

on neurotransmitter

significance.

therefore tory

BIOCHEMICAL

from

the

that

release has not this

1 )JM (12),

processes

been identified

inhibitory

of the activity

at around

RESEARCH COMMUNICATIONS

activity

dopaminergic of "melatoninergic"

neuron

is

the observed

therefore

of physio-

as a neurotransmitter, stems

from its

to external neurons

signals,

on dopamine-

neurons.

Acknowledgement:

We would like to thank Mr. Y. Egoai for excellent maninulation of the microdissections and to Prof. M. Sokolovsky for fruitful discussions.

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

modula-

Cardinali, D.P.(1981) Endocr. Rev. 2: 327-346. Cardinali, D.P., Vacas, M.I. and Boyer, E.E. (1979) Endocrinology 105: 437-441. Niles, P.L., Wong, Y.W., Mishra, R.K. and Brown, G.M. (1979) Eur. J. Pharmacol. 55: 219-220. Richardson, S.z, Hollander, D.S., Prasad, J.A. and Hirooka, Y. (1981) Endocrinology 109: 602-606. Negro-Villar, A., Ojeda, S.E., Arimora, A. and McCann, S.M. (1978) Life Sci. 23: 493-497. RichardsonTS.B., Hollander, C.S., and Greenleaf, P.W. (1980) Clin. Res. 28: 544-548. Blaustein, EP. (1975) J. Physiol. (Lond) 247: 617-655. Katz, B. and Miledi, R. (1967) Proc. Roy. Sot. B. 167: 23-28. Dunant, Y., Edin, L. and Serveriadis-Hirt L. (1980) J. Physiol. 298: 185-203. Beani, L., Bianchi, C., Giacomelli, A. and Tamberi, F. (1978) Eur. J. Pharmacol. 48: 179-193. Iversen, L.L. -975) Handbook of Psychopharmacology, Vol. 3 (L.L. Iversen, S.D. Iversen and S.H. Snyder, eds.) p. 421, Plenum Press. Koslow, S.H. and Green, A.R. (1973) Adv. Biochem. Psychopharmacol. -7: 33.

1616