EUROPEAN JOURNAL OF PHARMACOLOGY 5 (1968) 107-110.

NORTH-HOLLAND PUBL COMP, AMSTERDAM

6-HYDROXY-DOPAMINE
CENTRAL

INDUCED

DEGENERATION

OF

MONOAMINE NEURONS

Urban UNGERSTEDT
Department of Htstology, Karohnska Instituter, 10401 Stockholm, Sweden

Received 21 October 1968

Accepted 19 November 1968

U UNGERSTEDT, 6-hydroxy-dopamme reduced degeneratton of central monoamme neurons, European J. Pharmacol 5 (1968) 107-110
Intracerebral injection of 6-hydroxy-dopamme induced degeneration of central dopamme and noradrenahne
neurons. InJection into the substantaa mgra produced an anterograde degeneration of the whole mgro-neostnatal
dopamme neuron system. This was associated with marked motor disturbances. It is suggested that lntracerebral administration of 6-hydroxy-dopamine may be used as a tool for anatomical and functional studies on central monoamine

neurons.

6-hydroxy-dopamme
central monoamme neurons

1 INTRODUCTION
6-Hydroxy-dopamme (6-OH-DA) has been found to
deplete peripheral organs o f noradrenahne (NA) (Porter et al., 1963, Laverty et al., 1965, Thoenen et al.,
1968). This may be explained by recent studies which
reported that 6-OH-DA caused selective, acute degeneration of sympathetic nerve terlmnals in the peripheral
nervous system (Tranzer and Thoenen, 1968, Malmfors and Sachs, 1968). This study is concerned with the
use of 6-OH-DA as a tool for producing degenerations
of monoammnerglc neurons m the central nervous
system.

2. MATERIAL AND METHODS

6-OH-DA-hydrobromlde was dissolved in Ranger-solution with added ascorblc acid (0.2 mg/ml). Unilateral
stereotaxic rejections o f 6-OH-DA (2/ag m 1/A or 8 lag
m 4 lul, calculated as the base; speed 1/A/nun) were
performed on rats (Sprague-Dawley b wt. 150 g) during

mgro-neostnatal DA system
degeneration

halothane-O2-N20 anaesthesia InJections were made

into the nucleus caudatus putamen (NCP), mto the
zona compacta of the substantla mgra and into the
area dorso-lateral to the nucleus mterpedunculans.
The rats were killed after one, sevenand twelve days
and their brains analyzed usmg the Falck and Hlllarp
hlstochemlcal fluorescence method for monoamlnes
(for references see Hfllarp, Fuxe and Dahlstrom,
1966, Corroda and Jonsson, 1967). The animals were
killed by decapitation during light chloroform anaesthesia. The brains were rapidly dissected out, cut m
2 - 3 mm ttuck frontal sections, frozen in propane
cooled by hquld mtrogen, freeze-dned, reacted with
formaldehyde gas, generated from paraformaldehyde
embedded m paraffin, sectioned and studied m the
fluorescence mmroscope (Dahlstrom and Fuxe, 1964a,
Fuxe and Jonsson, 1967, Ungerstedt, to be pubhshed).
The formaldehyde treatment was ormtted m the substantla nigra region from one seven-day antmal.

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U UNGERSTEDT

3 RESULTS

3 1. hzlectzon znto the nucleus caudatus putamen

When wewed in the fluorescence microscope the
normal NCP contains an extensive plexus of fluorescent
DA nerve terminals (Fuxe, 1965).
One day after the mjection of 6-OH-DA no fluorescent nerve terminals were seen in the central area of the
NCP where the injection had been made. This area was
abou~l 1 5 m m in diameter after the injection of 2/2g
6-OH-DA in 1/21 and about 2 mm in diameter after
8/2g in 4 t21 At the border of the depleted area strongly
green fluorescent axons appeared that had accumulated
monoammes These axons could be followed in a caudal direction from the site of the injection
In rats killed twelve days after the InIectmn the depleted area was of about the same size as after one day
but its borderhne was less well defined No axons
with accumulated fluorescence were visible.
3 2 lnlectton into the substantza mgra
The zona compacta of the substantla nigra contains
DA cell bodies that normally display a green fluorescence in the fluorescence microscope (Dahlstrom and
Fuxe, 1964a)
One day after the rejection ot 6-OH-DA (8/2g in
4/21) practically all cell bodies had acquired a strong
orange-yellow fluorescence (fig. 1) At the very periphery of the group a few green fluorescent cell bodies
persisted Weakly yellow fluorescent fibres were seen
among the cell bodies In some cells the nucleus
showed a "dense" yellow fluorescence and after twelve
days the surrounding cytoplasm in such cells was hardly
visible No signs of regeneration in the cell bodies were
observed after twelve days The affected area had a
diameter of about 2 mm The same results were found
after seven days and twelve days After 2/2g in 1 /21 the
affected area was smaller in diameter (1 and 12 days}
The yellow fluorescence was found to be due to autofluorescence, 1 e it persisted when the formaldehyde
treatment was omitted (7 days).
Both NCP showed normal fluorescence one day after
the injection of 6-OH-DA into the substantia nigra
After seven and twelve days, however, the NCP on the
s~de lpsilateral to the injection was devoid of fluorescence The nucleus accumbens shcwed normal fluorescence on both sides (figs 2 and 3)

The injection of 6-OH-DA into the substantla nigra

produced a marked motor asymmetry in the animals,
1 e when the animals moved they turned to the side
ipsllateral to the injection The same symptom, but
considerably weaker, was observed after 6-OH-DA rejection into the NCP

3 3 hzlectzon mto the area dorso-lateral to the nucleus mterpedunculans

This area contains fluorescent monoamlne neurons
(Dahlstrom and Fuxe, 1964a) and is traversed by ascending NA and 5-hydroxytryptamlne (5-HT) axons
(Ungerstedt, unpublished observations. Anden et al ,
1966a, b)
One, seven and twelve days after the injection of
6-OH-DA most of the cell bodies showed a strong
orange-yellow fluorescence Yellow fluorescent fibers
were observed between the cell bodies Strong accumulation of green fluorescence appeared in the NA
axons ascending through the area Seven days after
the rejection the accumulated axons could be traced
several mllhmeters caudal from the site of injection,
i e from the area dorsal to the nucleus interpedunculiris to an area just caudal to the R i d nervl faclalls
The accumulation was less prominent one day after
the injection and had partly disappeared after twelve
days

4 DISCUSSION
In order to test the effect of 6-OH-DA on central
monoamlne neurons, lntracerebral injections were
made into areas k n o w n to contain monoamme cell
bodies, axons or terminals (Dahlstrom and Fuxe,
1964a, Fuxe, 1965) 6-OH-DA was found to deplete
the transmitter content of DA nerve terminals and
cell bodies, when injected into the NCP and the substantla nigra, respectively Both NA and DA axons accumulated transzmtter proximal to the site of injection
Injection of 6-OH-DA into the substantla nlgra not
only caused a depletion of the DA cell bodies but also
a depletion of the DA terminals in the NCP This was
a well localized eftect and e g the nuc accumbens,
bordering on the NCP, showed normal fluorescence
The depletion of the NCP occurred somewhere between one and seven days after the rejection These
effects correlate well with the existing strong evidence

6-HYDROXY-DOPAMINE INDUCED DEGENERATION OF CENTRAL MONOAMINE NEURONS

109

Fig 1. Yellow fluorescent fibers and DA cell bodws m the substantm mgra one day after mtracerebral InJectionof 6-OH-DA 160 X
Fig 2 Nucleus caudatus putamen (A) and nuc accumbens (B) twelve days after rejection of 6-OH-DA into the substantm nlg~a
No fluorescence is visible m the NCP indicating a degeneration of DA terminals. Nuc. accumbens appears normal 100 X
h g 3 Same ammal as m fig 2 Control side. Nucleus caudatus putamen (A) and nuc accumbens (B) show normal fluorescence
100 X
for a nlgro-neostrlatal DA neuron system (And6n et al.,
1964, 1965; Polrier and Sourkes, 1965, Hokfelt and
Ungerstedt, 1968), and suggest an anterograde degeneration o f the whole DA neuron
In the peripheral nervous system 6-OH-DA is
known to Induce specific degeneration o f sympathetic
nerve terminals (Tranzer and Thoenen, 1968, Malmfors and Sachs, 1968) It is thus probable that the depletion resulting from 6-OH-DA injection into the
NCP is a sign o f local degeneration o f DA terminals.
The axonal accumulation o f transmitter which is a well
known phenomenon in severed axons (Dahlstrom and
Fuxe, 1964b) and the yellow "autofluorescence" in
DA cell bodies are probably further indications o f degenerative changes in the catecholamme neurons after
6-OH-DA
The injection o f 6-OH-DA into the substantia nigra
and the subsequent depletion o f the nlgro-neostnatal
DA system was assocmted with m a r k e d m o t o r asymmetry m the ammals This correlates well with the
results o f Pottier et al. (1966) They found a hypoklnesla o f the lunbs lpsflateral to the depletion o f striatal

amines after lesions in monkeys The same type o f

electrothermal lesion in rats also depleted stnatal DA
(Hokfelt and Ungerstedt, 1968) and produced lpsilateral turning behavlour (Ungerstedt, unpublished)
which IS very similar to that found after nlgral injection o f 6-OH-DA Motor asymmetries were also reported b y And6n et al (1966c) after removal of the
corpus striatum and treatment with various drugs
Interfering with catecholamlne neurotransmlssion.
Similar results to these were found after the unilateral
injection o f DA Into the NCP which produced contralateral turning behavlour (Ungerstedt et a l , 1968)
F r o m the above results it seems probable that lntracerebrally injected 6-OH-DA is able to induce degeneration o f catecholamine cell bodies, axons and terminals m the vicinity o f the site o f Injection. It thus
offers the possibility o f tracing these neuron systems
in the brain b y studying the accumulation and disappearance o f the transmitter after local InJections of
6-OH-DA into various cell groups, axon bundles or
terminal areas The degeneration of the nlgro-neostriatal DA neuron system, obtained after 6-OH-DA,

110

U UNGERSTEDT

produced marked motor d~sturbances k n o w n to be associated with this system. This re&cares that 6-OH-DA
can also serve as a valuable tool for functional studies m
the central nervous system

ACKNOWLEDGEMENTS
This work has been supported by grants from the
Swedish Mechcal Research Councd (B69-14X-715-04A)
and "Olhe och Elof Encssons Stlftelse". For generous
supplies of 6-OH-DA I am mdebted to Dr. H.Thoenen,
Hoffmann-La Roche. The assistance of Miss Kerstm
Sterner is gratefully acknowledged.

REFERENCES
And6n, N -E, A Carlsson, A Dahlstrom, K Fuxe, N -~ Hdlarp
and K Larsson, 1964, Demonstration and mapping out of
mgro-neostnatal dopamlne neurons, Life Scl 3, 523
And6n, N - E , A Dahlstrom, K Fuxe and K Larsson, 1965,
Further evidence for the presence of mgro-neostnatal
dopamme neurons m the rat, Am J Anat 116, 329
And6n, N -E, A Dahlstrom, K Fuxe, K Larsson, L Olson and
U Ungerstedt, 1966a, Ascending m o n o a m m e neurons to
the telencephalon and &encephalon, Acta Physlol Scand
67,313
And6n, N.-E, A Dahlstrom, K Fuxe, L Olson and U Ungerstedt, 1966b, Ascending noradrenahne neurons from the
pons and the medulla oblongata, Expenentla 22, 44
And6n, N -E, A Dahlstrom, K Fuxe and K Larsson, 1966c,
Funclaonal role of the nlgro-neostrlatal dopamme neurons,
Actapharmacol toxlcol 24,263
Corrodl, H and G Jonsson, 1967, The formaldehyde fluorescence method for the hlstochemlcal demonstration of
blogenlc m o n o a m m e s A review on the methodology,
J Hlstochem Cytochem 15, 65
Dahlstrom, A and K Fuxe, 1964a, Evidence for the existence
of monoamme-eontammg neurons m the central nervous
system. I Demonstration of monoammes in the cell bodies of brain stem neurons, Acta physlol scand 62, suppl
232, 1

Dahlstrom, A and K Fuxe, 1964b, A method for the demonstralaon of m o n o a m m e contmmng nerve fibres in the central nervous system, Acta physlol scand 60, 293
Fuxe, K , 1965, Evidence for the existence of m o n o a m m e
neurons m the central nervous system IV Distribution of
monoamlne nerve terminals m the central nervous system,
Acta physlol scand 64, Suppl 247, 39
Fuxe, K and G Jonsson, 1967, A modification of the hlstochemical fluorescence method for the Improved locahzataon of 5-HT, Hlstochemle 11, 161
Hdlarp, N . - ~ , K Fuxe and A.Dahlstrom, 1966, Central monoamine neurons, m Mechamsms on release of central blogemc amines (Pergamon Press) p. 31
Hokfelt, T and U Ungerstedt, 1968, Electron and fluorescence
microscopical studies on the nucleus caudatus putamen of
the rat after unilateral lesions of ascending mgroneostrlatal dopamlne neurons, Acta physlol scand, m press
Laverty, R , D F Sharman and M Vogt, 1965, Action of 2,4,5trthydroxyphenylethylamme on the storage and release of
noradrenalme, Bnt J Pharmacol Chemother 2 4 , 5 4 9
Malmfors, T and Ch Sachs, 1968, Degeneration of adrenerglc
nerves produced by 6-OH-DA, European J Pharmacol 3.
89
Pomer, L J and T L Sourkes, 1965, Influence of the substantm mgra on the catecholamme content of the stnatum,
Brain 88, 181
Pomer, L J , T L Sourkes, G Bouvler, R Bourker and S Carabin, 1966, Stnatal amines, experimental tremor and the
effect of harmahne m the monkey, Brmn 89, 37
Porter, C C , J H Totaro and C H Stone, 1963, Effect of
6-hydroxydopamlne and some other compounds on the
concentration of norepmephnne m the hearts of mice,
J Pharmacol Exp Ther 140, 308
Thoenen, H , A Hurhmann and W Haefely, 1968, Mechanisms
of amphetamine accumulation in the Isolated perfused
heart of the rat, J Pharm Pharmacol 20, 1
Tranzer, J P and H Thoenen, 1968, An electron microscopic
study of selectwe, acute degeneration of sympathetic
nerve terminals after administration of 6-hydroxydopamme, Experlentaa 24, 155
Ungerstedt, U , L L Butcher, G Butcher, N -E And6n and K
Fuxe, 1968, Dtrect chemical stimulation of dopamlnerglc
mechanisms in the neostnatum of the rat, European J
Pharmacol, In press