Brab~ Research Bulletin, Vol. 6, pp. 165-170. Printed in the U.S.A.

The Influence of Unilateral 6-OH-Dopamine Lesions of the Substantia Nigra in the

Absence of the Telencephalon I

G E O R G E S P A P A D O P O U L O S , 2 J O S E P H P. H U S T O N a A N D B A R B A R A N E F

Institute o f Psychology III, University o f Diisseldorf, Universitdtsstrasse 1, D-4000 Diisseldorf, Federal Republic o f Germany

Rece ived 5 June 1980

PAPADOPOULOS, G., J. P. HUSTON AND B. NEF. The influence of unilateral 6-OH-dopamine lesions of the substan- tia nigra in the absence of the telencephalon. BRAIN RES. BULL. 6(2) 165-170, 1981.--Rats were injected unilaterally into the rostral substantia nigra pars compacta with 8/zg of 6-OHDA. Those animals that conformed to the behavioral criteria for an effective nigrostriatal dopamine lesion by turning ipsilaterally to the injected side upon IP amphetamine, and contralaterally upon apomorphine injection were bilaterally detelencephalized (neocortex, hippocampus, striatum, septum, amygdaloid complex were removed). Most detelencephalized animals exhibited spontaneous turning to the contralaterai side, i.e. they no longer behaved like 6-OHDA-lesioned rats, but, instead, acted like animals with nigral kainic acid or electrolytic lesions. Amphetamine (2-3 mg/kg) increased general activity but no longer influenced turning. Apomorphine in doses of 2-3 mg/kg completely suppressed all motility. In small doses (0.1-0.2 mg/kg) it reversed the spontaneous con- traversive turning. This effect could be blocked by haloperidol (0.1 mg/kg) pretreatment. High doses of haloperidol (5.0-7.5 mg/kg) reversed the direction of circling from contraversive to ipsiversive. Arecoline (10-12 mg/kg) induced tremor as in normal animals. Atropine (50-100 mg/kg) did not affect turning, but increased activity level in the thalamic rats.

6-OHDA Thalamic rats Substantia nigra Turning Telencephaion ablation Apomorphine Amphetamine Haloperidol Arecoline

AN injection of 6-OH-dopamine into the rostral end of the substantia nigra pars compacta, or still more rostrally, into the area of the ascending axons of the dopaminergic nigro- striatal neurons, causes degeneration of both axons and terminals of these neurons in the striatum and of the perikarya in the substantia nigra. After some time a super- sensitivity of the "denervated" postsynaptic dopamine recep- tors in the striatum probably develops. Such a unilateral 6-OH-dopamine lesion of these neurons leads to postural asymmetries and turning in the direction ipsilateral to the injected substantia nigra. The injection of drugs that act on dopamine systems induces vigorous rotation in such a prep- aration..For example, amphetamine, which releases dopa- mine from the intact nerve terminals in the striatum of the nonlesioned hemisphere, induces ipsiversive circling (towards the lesioned side) while apomorphine, a dopamine agonist that acts more strongly on the supersensitive dopamine receptors on the lesioned side, causes contraver- sive circling (away from the lesioned substantia nigra). This model, which is based mainly on the work of Ungerstedt

[29,30], has provided an explanation for both spontaneous and drug-induced turning in animals with unilateral lesions of the dopaminergic nigrostriatal system in terms of events happening at the dopamine nerve endings and/or the postsynaptic dopamine receptors in the striatum.

However, a lesion of the dopaminergic nigrostriatal neurons not only causes alterations at the level of the striat- um, but must also have a profound influence on the function of the substantia nigra, which is known to have a complex synaptic organization [7]. Furthermore, recent research has pointed to the role of the substantia nigra as an "output station" for behaviors that have been studied only or mainly in relation to striatal function, such as circling and stereotypies [4,5].

Since circling behavior after unilateral 6-OH-dopamine lesions of the substantia nigra of otherwise intact animals seems to be dominated by the phenomena taking place in the striatum, we found it interesting to study such behavior in rats with unilateral lesion of the nigrostriatal neurons after ablation of the telencephalon, i.e. in "thalamic rats" [14].

~Supported by a grant from the Ministry of Sciences of Northrhine-Westphalia. 2Present address: Institute of Experimental Pharmacology, University of Athens, Goudi, Athens, Greece. Supported in part by the

European Training Program in Brain and Behavioral Research. aSend reprint requests to: Joseph P. Huston, Institute of Psychology III, Universityof DiJsseldorf, Universit/itsstrasse l, D-4000 Diisseldorf,

Federal Republic of Germany.

Copyr ight 1981 A N K H O In t e rna t iona l Inc.--0361-9230/81/020165-06501.10/0

166 PAPADOPOULOS, HUSTON AND N E F

METHOD

Animals

Male Sprague-Dawley rats were used. Their weight at the time of the 6-OH-dopamine lesion was 300 +- 30 g. Both preoperatively and until the second operation they were housed under normal laboratory conditions under a 12 hr light/12 hr dark cycle with free access to food and tap water.

6-OH-Dopamine Lesion

Six-OH-dopamine hydrochloride (Fluka) was dissolved in cold physiological saline which contained 0.1% ascorbic acid at a concentration of 4 mg 6-OH-dopamine base per ml solu- tion. The solution was prepared freshly for each experi- mental day and was kept in the refrigerator until the moment of the injection. The rats were anesthesized with Equithesin (about 0.3 ml/100 g body weight IP) and placed into a stereotaxic frame. After incision of the scalp and trephining of the skull an injection of 6-OH-dopamine solution was made with a 28 gauge (0.36 mm outer diameter) injection nee- dle connected through a polyethylene tube to a hand-driven I0/~1 Hamilton microsyringe. The tip of the injection needle was aimed to the rostral end of the substantia nigra pars compacta. The injection coordinates were, using the Hurt, Hanaway and Netsky stereotaxic atlas [13]: 3.0-3.5 mm anterior, 1.8-2.3 mm lateral and 2.1-2.4 mm dorsal to the interaural line. The dose was 8/zg 6-OH-dopamine base (2 g.l solution) injected at a rate of 1 /~l/min. The injection needle was left in place for another 3 rain after termination of the injection. Then the scalp skin was sutured and the rats were returned to their cages.

Within 8--12 days after 6-OH-dopamine injection the rats were tested for successful lesion of the dopaminergic nigro- striatal neurons with IP injections of amphetamine (2 mg/kg body weight) and apomorphine (0.5 mg/kg). Rats were selected for further experimentation only if amphetamine in- duced clear-cut ipsiversive circling (at least 3 turns/min for at least 45 min) and if apomorphine elicited clear-cut con- traversive circling (at least 2 turns/min for at least 20 min).

Removal of the Telencephalon

Those rats which satisfied the criterion for a successful lesion of the nigro-striatal dopamine system by virtue of their response to amphetamine and apomorphine were again operated within 15-25 days after the first operation. Under pentobarbital anesthesia the skull was opened and cerebral tissue was carefully aspirated with a Pasteur pipette con- nected to a vacuum pump. Details of the operation are de- scribed elsewhere [14,22]. On the whole, the following struc- tures were removed: neocortex, hippocampus, bulbus olfac- torius, the amygdaloid complex, striatum, globus pallidus and the septum, including all or most of the nucleus accum- bens (at least as far caudally as the commissura anterior). After completion of the suction the cranial cavity was filled with pieces of a gelatine sponge (Gelfoam, Upjohn) soaked in physiological saline and the skull was covered with dental acrylic.

Maintenance of the Thalamic Rats

The thalamic rats were fed intragastrically with Ovomal- tihe (Wander) (15-25%) in a Tyrode solution. In general, they received 3 meals of 10 ml each per day but the quantity of feeding was frequently adjusted to compensate for weight

loss. Diarrhoea was controlled with Kaopectate (Upjohn) or a suspension of aluminium hydroxide given with the meals.

Testi~g of the Thalamic Rats

The thalamic rats were observed for 10 min in their cages at least twice per day (morning and afternoon) for spontane- ous circling or turning tendency. At various time intervals they were put into a rectangular box (50x50x60 cm height) and connected through a rubber band to a rotometer by which circling was recorded (see [22]).

At various times postlesion the animals were tested with IP injections of a series of drugs. For this purpose they were placed into the rotometer box and a base-line turning behav- ior was recorded for at least 30 min preceding the IP injec- tion. The animal was left connected to the rotometer for at least I hour or until the effect of the drug had subsided. The following substances were used:

Apomorphine hydrochloride: 0.1-3.0 mg/kg (20 rats, 54 trials) I-Amphetamine sulphate: 0.5-3.0 mg/kg (21 rats, 41 trials)

Arecoline: Atropine sulphate: Haloperidol: Picrotoxin:

10-20 mg/kg (4 rats, 4 trials) 50-100 mg/kg (3 rats, 3 trials) 0.1-7.5 mg/kg (8 rats, 22 trials)

4 mg/kg (2 rats, 2 trials)

All drugs were dissolved in physiological saline, except for haloperidol which was dissolved in a few drops of glacial acetic acid and the volume made up with isotonic dextrose solution.

Histological Examination

The brains were embedded in paraffin, sectioned and stained with toluidine blue. They were examined microscop- ically to verify the extent of the removal of the telencephalic structures.

RESULTS

A total of 32 rats satisfied the criteria for a successful unilateral 6-OHDA-dopamine lesion and underwent removal of the telencephalon. Of these animals 5 died during the first 12 hours postoperatively. Twenty-five rats survived for 3 or more days and of these 19 survived for at least 5 days. Re- constructions of representative detelencephalized brains are depicted in Fig. I.

Spontaneous Behavior

At the time of the second operation all 6-OH-dopamine lesioned rats had ceased to show a preference in the direc- tion of turning. After removal of the telencephalon most rats exhibited spontaneous circling or at least a turning tendency. As can be seen from Table 1 most of the thalamic rats that exhibited nonambiguous preference in direction of turning rotated to the side opposite to the 6-OH-dopamine lesion (p<0.012 for days 1 and 7; Binomial Test, 2-tailed). This tendency was weakest on the 3 ra postoperative day.

In addition to turning, the thalamic rats exhibited the usual behaviors observed in such animals, i.e., periodic ac- tivity, obstinate progression, grooming, rearing and other stereotypies, etc. (see [14]).

Amphetamine

In the doses used amphetamine had no effect on turning.

NIGRO-STRIATAL LESIONS IN THALAMIC RATS

A 39 A t+0

A 620 p

• J

r

• J

A 2790 p

TABLE 1 NUMBER OF THALAMIC RATS SHOWING SPONTANEOUS

TURNING CONTRA- OR IPSILATERAL TO 6-OH-DA'LESIONED SUBSTANTIA NIGRA

Postoperative Day

Ist 2nd 3rd 5th 7th

Contraversive Turning 16 14 8 9 10

Ipsiversive Turning 4 6 7 3 1 Ambiguous 4 4 6 6 2 Total Rats 24 24 21 18 13

167

A /,620 p

A 6570 .u

A 8920 p

A 10300 p

FIG. 1. Reconstruction of two representative telencephalized rat brains with unilaterally 6-OHDA lesioned substantia nigrae on plates from Krnig and Klippel [19]. The blackened area indicates the ab- lated tissue.

In doses of 2.0-3.0 mg/kg an increase of general activity and motility was observed (which sometimes increased the speed of the already present circling behavior).

Apomorphine

In the usual doses (0.5-3.0 mg/kg) apomorphine had no effect on turning. With a dose of 2.0-3.0 mg/kg it induced an almost complete suppression of motility, beginning almost immediately after injection and lasting for 10-30 min. During this period the rat remained immobile in a crouched position and showed no turning tendency.

In 5 rats, which showed spontaneous contraversive cir- cling, apomorphine was given in a low dosage (0.1-0.2

mg/kg) during the 6 m postoperative day. In three of them this dose of apomorphine reversed the mean direction of turning (see Fig. 2). The overall level of activity (forward locomo- tion, various stereotyped limb and head movements, rearing, grooming, etc.) was not influenced by this dosage. This ef- fect could be blocked by a low dose (0.1 mg/kg) of haloperi- dol given 30 min before apomorphine.

Haloperidol

Doses of haloperidol lower or equal to 2.5 mg/kg had no effect on these animals. The activity of the thalamic rats remained practically unchanged with these dosages of halo- peridol. Four rats which showed spontaneous contraversive circling were given high doses of haloperidol (5.0 or 7.5 mg/kg IP). These doses reversed the direction of circling from contraversive to ipsiversive in all rats for at least 3 hours (Fig. 3).

Arecoline

In the doses used (10-20 mg/kg) arecoline induced inten- sive tremor of the large amplitude variety, beginning shortly after injection and lasting for about 10 min. The tremor af- fected the whole body, especially the four limbs, and was accompanied by dacryorrhoea and piloerection. It was indis- tinguishable from the tremor induced by the same doses of arecoline in the control rats, except that it was of larger amplitude in the thalamic rats. Furthermore, in the thalamic rats, after the tremor had subsided, there ensued a period of akinesia during which the animals lay fiat on the abdomen. This period lasted less. than 10 min, and 20-25 min after injection the rats again appeared quite normal. Arecoline did not elicit turning behavior, and the existing turning tenden- cies were not influenced during the tremor period.

Atropine

In the doses used (50-100 mg/kg) atropine did not influ- ence turning behavior, but induced behavioral "act ivat ion" and increased the motility of the thalamic rats.

Picrotoxhz

In the two animals tested 4 mg/kg picrotoxin did not in- duce turning behavior. About 10 min after injection general- ized seizures appeared, which led to the death of one animal about 10 min after injection, while the other one recovered after the seizures had lasted for about 1 hour.

168 PAPADOPOULOS, HUSTON AND NEF

A 37 day 6

~ t t t 4' -~ APO APO HAL APO

0.1mglkg 0.1mg/kg

i=

,E = I

t - "

2 -

1 0 o ~

I-

2 3

4' 5

6" 7

A38

t APO APO .___

0.1 mg/k g

A40 ~ 0 . . . . . . . . . . . . . . . . . . . . .

¢--

~/, APO 0.1mg/kg AP0 0.2mg/kg

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%+ t1' t HAL APO

0.1 mglkg

day6

o Cs ~o ,:s ~o ¢s 9'0 1~s 6 ~'s ~o ,:s ~o 7's ".~o los time -rain.

FIG. 2. Left: Effects of apomorphine (APO) on direction of turning in 3 rats 6 days after detelencepha- lization. Right: The effects of apomorphine on turning after treatment 30 min earlier with haloperidol (HAL).

DISCUSSION

The above findings show that after unilateral 6- OH-dopamine lesion of the nigrostriatal dopaminergic neurons and subsequent removal of the telencephalon there remain asymmetries in posture and turning behavior, which appear either spontaneously or are elicited by drug adminis- tration.

However, the direction of the spontaneous turning tend- ency is opposite to that seen in telencephalon-intact rats with

unilateral nigrostriatal 6-OHDA lesions, i.e., after bilateral removal of the telencephalon, rats with unilateral 6- OH-dopamine lesions rotated spontaneously to the side of the intact substantia nigra. (That this was not seen in all cases can be due to the fact that bilateral removal of the telencephalon can cause spontaneous circling in either direction, probably due to unintended minor asymmetries in brain tissue removal (see [14,22].) This reversal in the direc- tion of turning can be accounted for in the following way: We

NIGRO-STRIATAL LESIONS IN THALAMIC RATS 169

.E

~1.2 0.8

" 0./+ .=- 0 ~ 0.t+ g 0.8 ~1.2

A 33 day I/,

HAL HAL 2.5 mg/kg

A 39 day8

• -= 0] . . . . . . . . . . 0.~ +

if_, HAL 7.5 mg/kg

.E

~1.2 0.8

' r o.~. o

= 0.t~ ~= 0.8 -~ 1.2

1.6

doy8

+ HAL 7.5mg/kg

FIG. 3. Reversal of direction of turning from contralateral (C) to ipsilateral (I) side by high doses of haloperidol (HAL) in 3 rats on days 8 or 14 postdetelencephalization.

may assume that, in addition to the lesioning of the catechol- aminergic neurons, the 6-OHDA injection also caused non- specific damage to the substantia nigra, as pointed out by several authors [1,16]. In the telencephalon-intact animal ip- siversive turning caused by a unilateral nigral 6-OHDA le- sion of nigro-striatal catecholaminergic neurons predomi- nates over the tendency towards contraversive turning in- duced by either nonspecific damage to non-catecholaminer- gic neurons or damage to as of yet unknown non-nigrostriatal catecholamine neurons. Therefore, after bilateral removal of the teloncephalon, including the striatum, the animals be- have as if they had unilateral electrolytic or kainic-acid le- sions of the substantia nigra, which cause turning in the con- tralateral direction to the lesioned side in forebrain-intact as well as detelencephalized animals [21, 23, 26].

Small non-specific lesions of the rostral part of the sub- stantia nigra were detected in most animals. It is difficult to assess whether contraversive circling should be attributed to such small lesions, particularly if quite a long time has passed after the initial operation. On the other hand, we have found in rats with unilateral radiofrequency lesions of the substantia nigra in which the spontaneous contraversive cir- cling had already ceased, that removal of the telencephalon caused a reappearance of the contraversive circling (unpub- lished observations).

Independently of the mechanism of this spontaneous con- tralateral turning in the thalamic rats, it must be noted that it was modifiable by the dopamine agonist apomorphine and the dopamine antagonist haloperidol. The active doses of apomorphine were so small as to remind of doses acting on (presynaptic) autoreceptors [2,28] or supersensitive dopa- mine receptors [30]. On the o ther hand, the doses of haloperidol required to reverse the spontaneous contraver- sive circling were so large as to arouse doubts as to whether this effect was due to a specific dopamine-biocking action.

In any case it is obvious that non-telencephalic substantia nigra connections must be invoked in relationship to these phenomena. It is known from several studies [4, 6, 10, 12] that the major non-telencephalic projections of substantia nigra cells are those to the thalamus (mainly ventromedial nucleus), to the superior colliculus and to the (brain stem) reticular formation. The nigrothalamic and nigrocollicular neurons seem to be GABAergic [6].

So far, there is no known non-telencephalic dopaminergic projection from substantia nigra, although dopaminergic ceils have been found with histofluorescence methods in the substantia nigra pars reticulata [7,9], the source of the most important non-telencephalic substantia nigra projections. Such cells could play a role in the contraversive turning and the drug-action we have described in detelencephalized rats (the effect of their lesioning in non-detelencephalized animals being overshadowed by damage to nigro-telen- cephal ic influences). A large number of dopaminergic synapses (dendrodendritic or dendroaxonic) are assumed to exist in the substantia nigra between dendrites of the dopaminergic pars compacta cells and other neurons [7, 11, 20, 31]. It is nonetheless unlikely that these synapses play any role in the observed phenomena, since the pars com- pacta cells should undergo retrograde degeneration also in the intact substantia nigra after destruction of their axons during removal of the telencephalon [25]. In fact, we ob- served histologically an absence of pars compacta cells bilat- erally in all animals that survived for at least several days after removal of the telencephalon.

It is also possible that a long-standing unilateral absence of dopaminergic pars compacta cells leads to a "functional asymmetry" of (possibly non-dopaminergic) non-telen- cephalic neuronal systems originating in the substant ia nigra. This asymmetry might not be reversible by a restora- tion of a "ba lance" between the two substantiae nigrae after retrograde degeneration of the dopaminergic pars compacta cells in the intact substantia nigra caused by removal of the telencephalon. In this case the observed action of the dopamine-receptor-related substances apomorphine and haloperidol would not be on the substantia nigra or on sys- tems directly originating from substantia nigra, but rather on dopaminergic systems topographically remote, yet con- nected to these postulated systems originating from the sub- stantia nigra and rendered functionally asymmetric after the 6-OH-dopamine lesion. Besides the well-known tuberoin- fundibular system, other probable dopaminergic, non- telencephalic systems have been found in the caudal thalamus, the zona incerta and the dorsal and anterior hypo- thalamus [27].

A further finding of some interest is that haloperidoi failed to reduce motility in the thalamic rats even when adminis- tered in quite large doses. This would be expected on the assumption that the " s i t e" of the cataleptogenic action of dopamine receptor blockers is the striatum dopamine recep- tors [3, 8, 18]. Also of interest is the "immobil izing" action

170

o f apomorph i ne . S u c h - a n ac t ion could also be o b s e r v e d in tha lamic ra ts wi thou t subs t an t i a nigra lesion.

F u r t h e r m o r e , we have found tha t t r e m o r af te r a recol ine admin i s t ra t ion appear s in qui te the same m a n n e r in bo th tha lamic and in tac t rats . This suggests tha t t r e m o r af te r a recol ine is not c o n n e c t e d to ac t iva t ion of the we l l -known chol inergic sys t em of the s t r ia tum. It is in a g r e e m e n t wi th

P A P A D O P O U L O S , H U S T O N A N D N E F

prev ious work on the local izat ion of the ac t ion of chol inerg ic t r emorogen ic agents , mainly o x o t r e m o r i n e [ 17]. On the o the r hand ne i the r a reco l ine no r a t rop ine had any ac t ion on tu rn ing behax;ior, a l though such ac t ion has been repor ted for bo th chol inergic agonis ts and an tagon i s t s in rats with 6- O H - d o p a m i n e les ion in the subs t an t i a nigra but in tac t te len- cepha lon [15,24].

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