Stereotype hyperactive behaviour produced by dopamine in the absence of noradrenaline

LIFE SCIENCES Vol . 6, pp . 1389-1398, 1967 . Printed in Great Britain .

Pergamon Presa Ltd .

STEREOTYPE HYPERACTNE BEHAVIOUR PRODUCED BY DOPAMINE IN THE ABS~A?CE OF NORADRr.NALII`:E J . Scheel-Krüger and A . Randrup St . Hans Hospital, Dept . E, Roskilde, Denmark

(Received 9 March 1967 ; in final form 7 April 1967) TWO phases of behavioural excitation result from injection of DOPA (physiological precursor of dopamine and noradrenaline)into rats pretreated with a monoamineoxidase (MAO) inhibitor (1) .

The first phase, starting 20 to 35 min . after the

injection, is characterized by rage : hissing and spitting, some rapid movements, fighting ü animals were put together .

During this period the head is kept elevated

in a characteristic fashion, and there is no sniffing . 1 to 2 hrs . after the injection there is a gradual transition into the second phase, where the behaviour is completely different, characterized by continuous stereotype sniffing, licking or biting of the cage wire netting .

This behaviour is

similar to that elicited by medium doses of amphetamine or apomorphine (1, 2) . Since it is generally agreed that the behavioural excitation produced by DOPA is due to the formation of the catechol amines dopamine and noradrenaline in the brain (3, see also "Discussion" below), we thought that determination of these brain amines might help to explain the behavioural observations .

In the following

w? present the results obtained by this approach . Methods All rats were young white males weighing about 250 g .

The experiments

shown in Tables 1 and 2 were made with `rJistar rats, those shown in Fig . 1 with rats bred at the State Serum Institute, Copenhagen .

The animals were kept in

individual cages with a few exceptions which are indicated in Table 1 . In the experiments shown in Tables 1 and 2 rèserpine was always given 19 hrs . prior to the catEChol amine precursors and pargyline (Mo 911, eutonyl, Table 1)

f

hr . prior to the precursors .

Diethyldithiocarbamate (DDC) as sodium salt was 1389

1390

HYPERACTIVE BEHAVIOUR

Vol .

6, No .

13

given 1 hr . prior to the precursors, additional doses (Table 2) were given 4 and 16 hrs . earlier.

The catecholamine precursors were DOPA (1-form) and DOPS

(dl-threo dihydroxyphenylserine) .

In the experiments recorded in Table 1 DOPA

was given 1-~ hrs . and DOPS 2~ hrs . before sacrifice with the exception of the experiment shown in the bottom line, in which both precursors were given 2 hrs . before sacrifice . sacrifice .

In the experiments of Table 2, DOPA was given 2 hrs . before

Doses are shown in the tables .

Brain catechol amines were determined by the methods of Hä.ggendal (4,5) . Two to four whole brains (minus olfactory bulbs) were extracted by 0 .4 N perchloric acid and passed through a 12 cm long column (0 .34 cm in diameter) of Amberlite CG-120, type II, Na+ , pH 6 .5 . overlap of the four amines :

This allows a clear séparation without

noradrenaline, dopamine and their 0-methylated

metabolites, which are normetanephrine and 3-methoxytyramine, respectively . After separation the four amines were determined fluorimetrically on an Aminco-Bowman spectrophotofluorometer .

The fractions were first measured

directly, and in cases of low amine concentration the measurement was repeated after enhancement of the fluorescence by the trihydroxyindole methods (5,6) . Tae modified the methods of Häggendal slightly, i .a . by including more extensive washing of the column prior to elution :

25 ml water, 15 ml 0 .1 M phos-

phate buffer pH 6 .5 and again 25 ml water all at a rate of 25 ml per hour . then followed with 1 N HCl at a rate of 9 ml per hour .

Çlution

The methods give results of

high specificity as proved by the test experiments in the publications referred to (4,5,6) .

This was further confirmed by control experiments of our own, i .a . an

experiment in which the rats were first treated with the specific inhibitor of catechol amine synthesis, d-methyltyrosine (100 mg/kg i .p . every third hour during 30 hrs .) and then with a monoamineoxidase inhibitor (100 mg/kg s .c . pargyline 5 hrs . before decapitation) .

This treatment would lower the level of

catechol amines and increase the level of other monoamines in brain ; by our analysis no fluorescence was found except a trace at the usual place of noradrenaline .

The specificity was also confirmed by paper chromatography (sing

Vol . 6, No . 13

HYPERACTIVE BEHAVIOUR

1391

u-butanole-acetic acid-water 4:1.25:1) of the fractions from the Amberlite column . Our modifications and control experiments will be published in detail elsewhere . In model experiments we found the following recoveries : noradrenaline 59 ± 2 96 normetanephrine 94 ± 3 ~, dopamine 69 ± 1 96 and 3-methoxytyramine 76 ± 496 . The figures in the tables are corrected for these recoveries . Results Fig . 1 shows the time course of catechol amines in the brain after doses of MAO inhibitor and DOPA which were previously found to produce very strong rage reaction followed in most animals by stereotype sniffing - licking - biting activity (1) . It may be seen that all four amines are increased during the behavioural reactions . The early rise in normetanephrine was unexpected, and shows that although there is only a late and insignificant rise in the level of noradrenaline, the turnover of this amine must be elevàted already at the time when the rage reaction begins . According to presently prevailing opinions the 0-methylated metabolites reflect the pathway of catecholamine turnover, which is related to the physiological action of the amines . A differentiation between the behavioural effects of norad=enaline and dopamine was obtained, when reserpine and diethyldithiocarbamate (DDC) were added to the treatment of the rats . Reserpine empties the endogenous stores of both amines, and DDC blocks the synthesis of noradrenaline from injected DOPA but not that of dopamine (7,19) . The biochemical analyses presented in Table 1 show that both level and turnover of noradrenaline is very effectively reduced by reserpine and DDC, while the formation of dopamine from injected DOPA remains high . Since the stereotype activity also remains unaffected, it is indicated that this behaviour can be produced by dopamine alone without any noradrenaline . The experiments with DOPS shown in Tablè 1 support this interpretation . DOPS is an artificial precursor which by decarboxylation is transformed directly to noradrenaline . The animals given DOPS instead of DOPA performed no ster eotype activity, they sat quiet, but the eye openings became large and circular,

139 2

HYPERACTIVE BEHAVIOUR

Vol .

6, No .

FIGUIZ~ 1 Time course of catechol amines in rat brain after an injection of 1-DOFA 25 mg/kg s .c . All rats were pretreated with a MAO inhibitor (pargyline, MO 911, eutonyl, 150 mg/kg s .c ., 5 hrs . before DOPA) . The bars represent,ag amine per g brain tissue . A . dopamine left and the O-methylated metabolite of dopamine, 3 methoxytyramine, right (hatched) . B . noradrenaline left and its O-methylated metabolite, normetanephrine, right . All the bars are based on two to four determinations each made on a pool of three to four brains .

13

Vol . 6,

No .

13

HYPERACTIVE BEHAVIOUR

1393

m ac~

+~ o

m m

r-I O U m

00

m . +~ m

""

0? J~

m

m

U

m m ,-I

Y" I

q

O O ~ ~ O O . .. ~ ~ I OO

..

1~1 +~

m

cd

N aa

..

""

L

" "

O

m m . .

ô0

,i s

a

b

U am ô e~ m~ ti~

rm"I m

~o

m

r°I >

~~

~

~

ôôôo

m

.~

z q

m a

~~ ~~ ~ ~~ ~ o0 00 00 00 0~ or"I v

ô0

v v v

ô0

v

ô0

v

ôô

oI oo ~,~

3 r~-I

aol ~

ro

,""

d

â .. +~ q

m

m rl

I

O m Fi m m of 0 >~ 0 m ti N O U

v .

b ~ cd,~ ô cà z ~

N N NN N O N 00 00 O~ O

~

o0 00 00 00 00 0o

r., S +~ I-I

m

Fa "rl

~

N O N ON ON

00 00 00 00 00 00 v v v v " v

m~

g

o m

°°

SS SS 5~~ SS SS °° S r~ r"I r" I r-I ra r-I rl rl rl r""I rl

S°S°S°~°~°S°S ° :

~ m

~ !ti

m O _ +~ m~ rl *i m m ,~ ~b q N

m m q

rl m m m m m m m

139 4

HYPERACTIVE BEHAVIOUR

Vol . 6, No . 13

the eyeballs protruding .

Lack of substance prevented us from repeating the

experiments with DOPS .

The rats given the pretreatments alone but neither DOFA

nor DOFS (first two lines in Table 1) were quiet with DDC, without nDC locomotion and some tremor occurred . Although stereotypy was unaffected by the pretreatment with reserpine the first behavioural phase elicited by DOPA after MAO inhibition (the rage phase) was only faint and after reserpine + DDC it was completely lacking .

The rats

started their activity by sporadic sniffing and some locomotion, then the sniffing gradually increased and became accompanied by licking and biting, concurrently locomotion decreased .

Although there was no distinct initial rage phase, spells

of rage behaviour (vocalization and defense postures) were, however, seen now and then during the whole period, also after the stereotype activity had become prominent . Most of the rage behaviour was seen in the rats pretreated with reserpine only, but a few definite defense postures with vocalizations were observed also in rats pretreated with both reserpine and DD ;~ . (These behavioural observations are based or_ the rats included in Table 1 + twenty four other rats which were seen both in individual cages and two in each cage) . The effects of the pretreatments on rage behaviour was, therefore, not completely clear but the indication is that noradrenaline is most important for the production of this behaviour, arhile dopamine may play a minor role .

This is in

agreement with the taming effect of DDC on mice and rats, which we have observed in other experiments, where this drug was given alone (unpublished) .

In our

experiments with DOPS (Table 1), however, no rage was elicited in spite of a high level and turnover of noradrenaline . The interpretation of the results shown in Table 1 is somewhat obscured by the application of so many drugs - four to each rat .

` ..re, therefore, controlled our

interpretation by the simplified experiments of Table 2, in which only two drugs were given to each rat .

It may be seen from the table that the results ronfirm the

association of dopamine with stereotype activity .

'I'hesA simplified experiments

give no information on rage, since without MAO inhibition there is only little r~ige

Vol . 6, No .

13

HYPERACTIVE BEHAVIOUR

m s~ ,~ m c! ,~

+~ o o ~.~1 m D iy m

ôq

m m

m ,~ +~

w .cj m

O F+ m W m D~ 1~ xi +~

H

4-i O Ô ,~ +~ U

b 0

ti ~d a1 Ô z

m~

~ ~, rn +~

à

q

.n

a ~°

ao

r~1 ~ m ri q

Ô m d m

1 m ~ rpl m~ ~ w

x~

,i Fa m a N W ~d m ,~ 9-i rl ri a v~

o0 -: o~ a~

ô

0 ôô 0 ôô

ô0

~O r


rl O r-1 .-1

r-1 rl O 00 ô 0 ô v v v v

r1 r~ 00

ô

r~ O

m Cd ,~-1 O~ z

m P a

O O v

Ô ÔN O O  .r

~

ô0

ôô

v v

OO

sss N ~N

W

rl O W W i~

+ +

1

0

O m m

+

m~

0 A r~ p

N

+

ti

r-1 a ,o r~i

Li

41 w b

Ô m ri

m m Fi ti O O W

m ~

N N ~ r-1

m

~

A

m m ~ ~ ti 1~ m m m m m a. W A A

ti cm,

L~

d

>

~i

Ô ,~ â

3

â

O

m +~ 14 w° b m o 0 a m m

O

q

m O ,i cà +~

CO i~

m

ao m m +~ m ~ Fi CI 1-I m

,r7 Û

1395

Vol . 6, No . 13

HYPERACTIVE BEHAVIOUR

1396

after DOPA in any case (1) . Discussion The association of dopamine with the stereotype behaviour agrees with recent findings on amphetamine excitation .

It was found that while DDC decreases

locomotor activity (both spontaneous and amphetamine-induced) it does not inhibit the amphetamine-induced stereotype activity (8) .

a .-methyltyrosine, however,

which inhibits the synthesis of DOPA and thus of both dopamine and noradrenaline, does prevent stereotypy as well as locomotion after amphetamine (9,10) .

It thus

seems that dopamine alone is responsible for the stereotype activity, while noradrenaline (perhaps supported by dopamine) is involved in other forms of activity, such as locomotion and aggressive behaviour . This role of dopamine also agrees with other experiments in which we have found corpus striatum involved in the mechanism underlying the stereotype behaviour (11,12) .

Corpus striatum is the part of the brain which contains by far the highest

concentration of dopamine .

In these experiments the stereotype behaviour was

elicited by microinjection of dopamine in corpus striatum, thus confirming that the effect is exerted by the amine, not by the precursor, DOPA . The experiments in the present report also have relevance to the muchdiscussed problem of the roles of various brain amines in reserpine sedation .

The

fact that this sedation can be reversed by DOPA indicated already many years ago Our results, as presented in Tables 1 and

the importance of catechol amines (13) .

2,show that the reversal bf reserpine sedation can be brought about by dopamine even when the level and turnover of noradrenaline are extremely low .

This low

formation of noradrenaline, even without DDC, may appear surprising, possibly reserpine inhibits the synthesis of noradrenaline from dopamine by preventing contact of dopamine with the granula, to which the enzyme dopamine-S-oxidase is bound (14, 15, 16) . Summary Injection of a monoamineoxidase inhibitor and DOPA (the physiological

Vol . 6, No . 13

HYPERACTIVE BEHAVIOUR

1397

precursor of dopamine and noradrenaline) produces in rats a rage reaction followed by a stereotype behaviour, which is characterized by continuous sniffing, licking or biting . Pretreatment with reserpine and diethyldithiocarbamate (DDC) strongly reduces the formation of noradrenaline but not that of dopamine . Behaviourally the rage reaction is reduced, while the stereotype behaviour remains unaffected . The experiments show that the stereotypy can be produced by dopamine in the absence of noradrenaline, other evidence supporting this conclusion is cited in the "Discussion" . The experiments also show that formation of dopamine in brain leads tq reversal of reserpine sedation . Acknowledgements The authors are indebted to Dr . I. Munkvad, director, for continuing encouragement and to the Knud Hajgaard Foundation, Copenhagen, for financial support. References A . Randrup and I . Munkvad, Acta Psychiatric Scandinavica suppl . 191 (ad volume 42), 193 (1966) . 2.

A . M. Ernst, Psychopharmacologia 7 , 391 (1965) .

3.

A . Carlsson in H . Himwich and 'rl . Himwich, Biogenic Amines, Progress in Brain Research vol. 8, Elsevier, Amsterdam (1964) .

4.

J. Häggendal, Scandinav. J. Clin . Lab.Invest. 14, 537 (1962) .

5.

J. Häggendal, A cta physiol .scand . ~, 261(1963).

6.

A . Carlsson and B . Waldeck, Scandinav .J .C1in.Lab .Invest . 16, 133 (1964) .

7.

M . Goldstein and K . Nakajima, Life Sciences ~, 1133 (1966) .

8.

A . Randrup and J. Scheel-Krüger, J .Pharm .Pha.rmac . 18 , 752 (1966) .

9.

A . Randrup and I . Munkvad, Nature 211, 540 (1966) .

10 .

A . ?~Jeissman, B . Koe and S . Tenen, J.Pharmacol . Exper .Therap. 1~7, 339 (1966) . R . L . Fog, A . Randrup and H. Pakkenberg . IV World Congress of Psychiatry, Madrid (1966) .

12 .

R . I, . Fog, A . Randrup and H. Fakkenberg, Psychopharmacologia, in press .

13 :

A . Carlsson, Margit Lindquist and Tor Magnusson, Nature 1180, 1200 (1957) .

HYPERACTIVE BEHAVIOUR

1398

Vol . 6, No . 13

14 .

S . Udenfriend, Harvey Lectures , Series 60, 57 (1966} .

15 "

L. Stjârne, Acta physiol.scandina.v .

16 .

S . Kaufrran and S . Friedman, Pharmacol. Rev . 17 , 71 (1965) .

67, 411 (1966) .

17 .

h.

18 .

A . Carlsson and M. Lindquist, Acta physiol . ~4, 83 (1962) .

19 .

A . Carlsson, M . Lindquist, K . Fuxe and T. HSkfelt, Fharmac . 18, 60 (196F) .

Bertler and E . Rosengren, Acta physiol .scand . 47, 350(1959) "