Drug-induced rotational behaviour following unilateral intracerebral injection of saline-ascorbate solution: neurotoxicity of ascorbic acid and monoamine-independent circling

Brain Research, 161 (1979) 371-376 © Elsevier/North-Holland BiomedicalPress

371

Drug-induced rotational behaviour following unilateral intracerebral injection of saline-ascorbate solution: neurotoxicity of ascorbic acid and monoamineindependent circling

JOHN L. WADDINGTON and TIMOTHY J. CROW Division of Psychiatry, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex, HA1 3UJ (U.K.)

(Accepted October 19th, 1978)

In the classical rotating rat model of Ungerstedt 2s,26, the nigrostriatal dopamine (DA) system of the rat brain is unilaterally destroyed by intracerebral application of the catecholamine neurotoxin 6-hydroxydopamine (6-OHDA) onto DA neurons. Rotational responses contralateral and ipsilateral to the lesioned hemisphere are induced by subsequent peripheral administration of directly- and indirectly-acting dopaminergic drugs respectively5,16,25-27. More recently, a similar effect has been described for serotonergic mechanisms, where unilateral destruction of ascending serotonin (5-HT) pathways by intracerebral injection of the indoleamine neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) results in similarly-directed rotational responses to challenge with direct and indirect serotonergic drugs t,17,29. A systematic examination of the effects of control procedures in these unilaterally lesioned rat models is made necessary by debate as to the specificity of action of monoamine neurotoxins 2,11, 32. This study reports the rotational responses to challenge with the DA agonist, apomorphine 4, and the 5-HT agonist 1%13, 5-methoxy-N,N-dimethyltryptamine, (5MeODMT) in rats subjected only to differing control procedures for the 6-OHDA and 5,7-DHT lesion techniques. It is found that under certain circumstances druginduced rotational behaviour following intracerebral injections of the vehicle for these neurotoxins (i.e. saline-ascorbate solution) is indistinguishable from that occurring following lesion with the neurotoxin itself. The results demonstrate the neurotoxicity of saline-ascorbate solution, indicate monoamine-independent rotation and emphasize the necessity of valid control procedures for the neurotoxin lesion technique. Male Sprague-Dawley rats of 150-200 g were anaesthetized with nembutal (60 mg/kg, i.p.) and given unilateral lesions of ascending monoamine pathways 14,24 in the medial forebrain bundle (MFB) by stereotaxic intracerebral injection of 6-OHDA (Sigma). The injection volume was 4 #114,24-26, containing 8 #g 6-OHDA (expressed as base) dissolved in 0.9 % physiological saline containing 1 mg/ml ascorbic acid as antioxidant. Injections were made by slow motion infusion at a rate of 1/~l/min with the injection unit retained in position for a further minute following deposition to allow diffusion. Coordinates were: A, 3.4; V, --3.0; L, 1.519. Behavioural responses of 6-

372 OHDA-lesioned animals ( n = 9 ) were compared with those of groups of animals receiving one of the following control procedures: (a) intact animals, no surgical intervention ( n = 5 ) ; (b) injection needle lowered to appropriate coordinate but no injection made ( n = 5 ) ; (c) 4 #1 saline injected ( n = 15); (d) 4 #1 saline containing 0.2 mg/ml ascorbate injected ( n = 5); and (e) 4/~1 saline containing 1.0 mg/ml ascorbate injected (n=5). Eight days after surgery, rats were placed in automated rotometer bowls 31 and challenged with apomorphine (Evans Medical; 1 mg/kg, i.p.). Rotational behaviour was continuously quantified for 1 h. After a further 3 days, rats were similarly tested after challenge with 5MeODMT (Sigma; 2 mg/kg, i.p. after a 45 min pretreatment with the monoamine oxidase inhibitor, nialamide, 75 mg/kg, i.p.). After a further 3 days (i.e. 14 days postsurgery) rats were decapitated, their brains removed and dissected on ice into striatal and cortical samples for assay of striatal DA and striatal and cortical 5-HT by a fluorimetric method 22, and of cortical noradrenaline (NA) by a radioenzymatic technique 6. In some animals residual tissue was frozen and the substantia nigra dissected from 500 # m sections for assay of the 7-aminobutyric acid (GABA) synthesizing enzyme glutamic acid decarboxylase (GAD) 3°. Animals in each of the control groups showed no significant rotational response to apomorphine. Only animals receiving 6-OHDA showed contralateral rotational responses to apomorphine (Fig. 1). However, when challenged with 5MeODMT, control groups showed ipsilateral rotational responses that increased in magnitude proportionally with increasing severity of control procedure. For animals receiving the full control procedure (4 #1 saline containing 1 mg/ml ascorbate), the response to 5MeODMT was indistinguishable from that of animals receiving 6-OHDA, while the rotational responses of these two groups each differed significantly from those of intact Turns per minute ipsilateral -I

0

I

2

3

4

Turns per minute contralateral -8 [

-6 I

I

-4 I

I

-2 I

I

2 I

I

I

None Needle only Saline Saline + Asc. 0.2 Saline + Asc. 1.0 6-OHDA 5NeODMT 12 mg/kg)

Apomorphine 11 mglkg)

Fig. 1. Rotational responses to intraperitoneally administered apomorphine and 5MeODMT in rats given unilateral infusions at 1/d/min of 6-OHDA or varying control procedures (see text). Results are mean turning rates/min -k S.E.M. of 5-15 animals/group determined over a 5 rain interval 20 min after apomorphine or 25 min after 5MeODMT administration. The monoamine oxidaseinhibitor, nialamide (75 mg/kg i.p.), was administered 45 min prior to 5MeODMT, and produced no rotation when given alone.

373 TABLE I Regional concentrations of DA, N.4, 5-HT and GAD activity following control procedures and 6-0HD.4, expressed as percentage change in injected compared with non-injected hemisphere

Mean concentrations in non-injected hemisphere: striatal DA, 4.842 4- 0.178/,g/g; striatal 5-HT, 0.853 i 0.103/~g/g; cortical NA 0.119 ± 0.008 #g/g; cortical 5-HT, 0.180 q- 0.015 #g/g; mean nigral GAD activity, 413 ± 46 pmol/pg protein/h. Values are the mean of 3-9 samples. Procedure

D.4(striatum)

5-HT(striatum) NA(cortex) 5-HT(cortex)GAD(nigra)

None Needle only Saline Saline + Ascorbate0.2 Saline + Ascorbate 1.0 6-OHDA

+ 17.9 --10.7 --18.5 --22.1" --21.4"* --83.7***

+6.4 --2.8 --6.9 --13.8 --11.7 --23.7**

----4.9 --5.1 --10.0 --92.3***

--+7.9 +0.6 +1.1 --8.7

----+14.3 --

Significant differences between injected and non-injected hemispheres: * P < 0.05; ** P < 0.01; • ** P < 0.001 (2-tailed t-test).

animals not subjected to any control procedure (P < 0.05, 2-tailed t-test) (Fig. 1). Regional neurochemical assays (Table I) revealed that concentration of striatal and cortical 5-HT and of cortical N A were unaffected by any control procedure. Both concentrations of saline-ascorbate, however, induced small but significant depletions of striatal DA. 6 - O H D A induced a small (24 ~ ) but significant depletion of striatal 5H T in addition to the expected large depletions of striatal D A and cortical NA. Rats subjected to control procedures failed to show any rotational response to the D A agonist, apomorphine. Only 6 - O H D A lesions resulted in the established 16,25 vigorous contralateral rotation to challenge with apomorphine. This is presumed to involve its action on postsynaptic striatal D A receptors in the lesioned hemisphere rendered supersensitive by denervation 7,8,25. The absence of rotational response to apomorphine in saline-ascorbate-lesioned rats, with significant 21-22 ~ depletions of striatal DA, is consistent with our recent demonstration s that D A depletions of at least 60 ~ are necessary for denervation supersensitivity to occur, as assessed by correlated rotational/receptor binding indices using [3H]spiroperidol as a D A receptor ligand. Control animals showed ipsilateral rotation to 5 M e O D M T , the magnitude of which increased with increasing severity of control procedure. For rats receiving intracerebral saline containing 1 mg/ml ascorbate, the vehicle for 6-OHDA, the rotational response to 5 M e O D M T exceeded that of intact animals and was indistinguishable from that in 6-OHDA-lesioned animals. An ipsilateral rotation to 5MeO D M T in 6-OHDA-lesioned rats has been previously reported by ourselves 1° and by others 23. The present data indicate that, at least in our own studies, this response is independant of D A depletion induced by 6 - O H D A as it occurred with equal magnitude in rats with 21 ~ and 8 4 ~ depletions of striatal D A (Fig. 1, Table I). Though 5 M e O D M T has been shown to be a serotonergic agent 12,13, the minor changes in striatal and cortical 5-HT concentrations found in the present study after control and 6 - O H D A lesions do not indicate a serotonergic basis for this rotational

374 response. Also, rats with more substantial and specific unilateral depletions of forebrain 5-HT, induced with 5,7-DHT, show rotational responses to 5-HT agonists that are in the opposite direction to those seen after such vehicle injections1,17, 29. It is unlikely that procedures that did not damage either striatal or cortical 5-HT innervations could damage serotonergic projections to other forebrain areas 14,z4. The biochemical data also fail to indicate a noradrenergic basis for this effect, and we conclude that this rotational response is not a result of destruction of monoamine pathways. The striatonigral GABA pathway has been shown to constitute the striatal output system 15,21 and apomorphine-induced ipsilateral rotation following its unilateral destruction has been described 15,21. In this study, control rats failed to circle to apomorphine and were shown to have an intact striatonigral GABA pathway by assays of nigral G A D activity that have previously been used to assess striatonigral lesions15,80. Though rotational behaviour has often been equated with asymmetric activity in dopaminergic mechanisms 16, it has lately become clear that not all circling is DAdependent3,gAs, zS. 5MeODMT is an obvious agent for investigating the role of serotonergic mechanisms in rotation 10 but the present results suggest that circling responses to 5MeODMT may sometimes be unrelated to asymmetries of monoamine neurones, and are presumably a result of destruction of an unidentified pathway. Whether or not in these circumstances the turning is dependent upon an interaction with 5-HT receptors remains to be demonstrated. This study systematically demonstrates a neurotoxic action of intracerebrallyadministered saline-ascorbate solution on non-monoaminergic and dopaminergic neurons. As behavioural responses to 5MeODMT are indistinguishable between vehicle and 6-OHDA-lesioned animals, we conclude that in this test situation the addition of 6-OHDA to saline-ascorbate vehicle serves only to severely deplete catecholamines, and to a considerably smaller extent 5-HT, but does not increase the destruction of non-monoaminergic neurons induced by the saline-ascorbate vehicle alone. The results of some other studies are consistent with a non-selective action of the saline-ascorbate vehicle used for 6-OHDA H,2°,32. Wolfarth et al. z2 have recently described an ipsilateral circling response to amphetamine in the cat after a unilateral intranigral injection of saline-ascorbate, with no change in striatal DA content, but did not delineate which component of the control procedure was responsible for the observed effect. It would therefore seem that rats injected intracerebrally with saline-ascorbate solution as a control for neurotoxic lesions may not be considered 'normal' controls unless shown in the test situation to be indistinguishable from intact animals. The use of monoamine neurotoxins would seem to be capable of both erroneously implicating monoamines and failing to indicate non-monoaminergic mechanisms in behavioural control unless such a double control procedure is used. J.L.W. is an M R C Scholar and is grateful to Prof. D. W. Straughan for discussion on these experiments. We are grateful to Mr. A. Cross and Mr. A. Longden for radioisotopic assays of G A D and noradrenaline.

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