Ibotenic acid lesions of the striatum reduce drug-induced rotation in the 6-hydroxydopamine-lesioned rat

Exp Brain Res (1994) 101:365-374 �9 Springer-Verlag 1994

O R I G I N A L P A P E R

Roger Barker �9 Stephen B. Dunnett

Ibotenic acid lesions of the striatum reduce drug-induced rotation in the 6-hydroxydopamine-lesioned rat

Received: 11 March 1994/Accepted: 13 June 1994

Abstract Lesions of the dopaminergic nigrostriatal tract produce a range of motor and sensorimotor deficits. One of the simplest and most reliable is the rotational response of the animal following activation with drugs that stimulate the dopaminergic network, most notably amphetamine and apomorphine. Conse- quently, the rotation test has been extensively used in assessing the success of treatments designed to restore dopaminergic function, including neural transplants. The present study investigates whether rotation induced by 6-hydroxydopamine lesions of the nigrostriatal bundle in rats is modified by additional lesions in the neostriatum. It was found that apomorphine-induced rotation can be reduced by ibotenic acid lesions of the dopamine-deafferented striatum, and that the extent of the reduction was proportional to the size of the lesions. In contrast, such lesions produced a non-significant reduction in amphetamine-induced rotation, although the correlation between the extent of the reduction and the size of the lesion was again apparent. Since the pattern of change was similar in direction, albeit smaller in magnitude, than the previously reported effects of intrastriatal transplantation in rats with similar nigrostriatal lesions, rotation tests alone do not provide an unequivo- cal test of graft survival and function.

Key words 6-Hydroxydopamine �9 Ibotenic acid Rota t ion. Amphetamine �9 Apomorphine �9 Rat

R. Barker �9 S.B. Dunnett ([~) MRC Cambridge Centre for Brain Repair and Department of Experimental Psychology, Cambridge University, Downing Street, Cambridge CB2 3EB, UK; FAX no: +44-223-333564

Introduction

Degeneration of the dopaminergic nigrostriatal pathway is the central pathological event in Parkinson's disease (PD), a condition unique to the human CNS and characterised by a progressive movement disorder (Clough 1991; Forno 1990). It can be mimicked experi- mentally in animals using selective neurotoxins for the central catecholaminergic neurons such as 6-hydroxydopamine (6-OHDA) (Zigmond and Stricker 1989). When 6-OHDA is infused unilaterally into the nigrostriatal bundle in rats, it results in a number of deficits, of which the easiest to quantify is the rotational response of the animal to stimulant drugs that provide a dopaminergic activation of the neostriatum. The two most commonly used agents are amphetamine, which stimulates the release of dopamine from the intact nigrostriatal terminals, and apomorphine, which at low doses selectively activates the supersensitive dopamine receptors on the lesioned side (Ungerstedt 1971a, b). Although the way in which asymmetric activation of the neostriatum is translated into rotational behaviour is far from clear, the test is simple, stable over time and reproducible, and has been widely used to assess the extent of damage of the nigrostriatal dopaminergic lesions and the effects of alternative treatments, including neural grafts of embryonic nigral or adrenal tissues (Barker and Dunnett 1992; Emerich et al. 1992; Freed 1991).

The model system for neural transplantation that has been most extensively studied involves implantation of embryonic ventral mesencephalon containing the dopamine neurons of the developing substantia nigra into the dopamine-denervated host neostriatum (Bj6rklund et al. 1987; Dunnett 1991; Freed 1991). The ability of nigral grafts to reduce rotation has been equated with their proven ability to release dopamine (Dunnett et al. 1988a; Freed etal. 1980; Zetterstrom etal. 1986), whereas it remains a matter of debate whether the synaptic connections formed by outgrowing dopaminergic axons with host striatal targets are an essential

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component of the functional response (Bj6rklund et al. 1987). Several different graft tissues have been found effective in alleviating the rotation response (Barker and Dunnett 1992; Bing etal. 1988; Brown and Dunnett 1989; Dunnett 1991; Emerich et al. 1992; Ewing et al. 1992; Fisher et al. 1991; Freed 1991; Kamo et al. 1986; Pezzoli et al. 1988; Winn et al. 1989), although this has usually been assessed only in terms of down-regulation of the supersensitive response to apomorphine. Howev- er, there is some doubt whether a similar mechanism necessarily applies to all graft types. Thus, adrenal tissues were originally considered as an alternative source of catecholamine-secreting tissue for grafting, but subse- quent studies have indicated that they may exert their functional effects even in the absence of surviving chromaffin cells (Barker and Dunnett 1992, 1993; Bohn et al. 1987), and non-neural as well as degenerating grafts can both reduce rotation (Pezzoli et al. 1988; Plunkett et al. 1990). Indeed, these latter studies, along with earlier neurosurgical results (Meyers 1951), suggest that striatal damage alone may alleviate some of the experimental and clinical features of parkinsonism. Furthermore, the possibility that the influence of some grafts may involve rather non-specific consequences of striatal damage is suggested by previous observations that excitotoxic lesions of the striatum can on their own produce drug-induced, rotational behaviour opposite to that seen with the 6-OHDA-lesioned rat (Dunnett et al. 1988b; San- berg et al. 1986; Schwartz et al. 1979).

In the present study, we therefore investigated explic- itly the effects of intrinsic striatal lesions on the rotational asymmetry induced by unilateral nigrostriatal damage. We report that excitotoxic lesions of the neostriatum made with ibotenic acid can reduce, albeit to different degrees, both apomorphine- and amphetamine-induced rotation to an extent that was dependent on the volume of striatum damaged.

Materials and methods

Animals

A total of 54 female Sprague-Dawley rats (Interfauna, Hunting- don) weighing 180-200 g at the start of the experiment were used. Animals were housed throughout the experiment in groups of six rats per cage, under a natural light-dark cycle and with free access to food and water.

Surgery

All animals initially received unilateral 6-OHDA lesions of the right nigrostriatal bundle performed under equithesin anaesthesia (0.3 ml/100 g). The animals were pretreated 1 h before surgery with pargylene (Sigma, 37.5 mg/kg per ml of 0.9% saline), anaes- thetised with equithesin (0.3 ml/100 g), positioned in a stereotaxic frame, and injected with 8 ~tg/4 pl of 6-OHDA. HBr (Sigma, free base dissolved in 0.9% saline/0.02% ascorbate). The injections were made using a 30~gauge needle, connected by polyethylene tubing to a 10 ~tl Hamilton syringe in a micro-drive pump and positioned into the right nigrostriatal bundle at coordinates: -4 .4 mm anterior to the bregma (A), -1 .0 mm lateral to the

bregma (L) and vertically (V) -7 .8 m from the dura with the incisor bar set 2.3 mm below the interaural line. The infusions were delivered over 4 min, and then the cannula was left in situ for 3 min following infusion of the neurotoxin before removal.

Six weeks after the original 6-OHDA lesioning, 26 animals received excitotoxic lesions of the dopamine-deafferented striatum. Under equithesin anaesthesia, each animal received two 0.75 ~tl infusions of 0.06 M ibotenic acid (Sigma) at co-ordinates A +0.6mm, L -3 .2mm, V -4 .5 ram and A + l .8mm, L --3.0 mm, V -4 .5 mm, with the incisor bar set 2.3 mm below the interaural line. The infusions were made via the same 30-gauge cannula connected to a micro-drive pump delivered at a rate of 0.25 Ixl per min over 3 min, after which the cannulae was left in situ for a further 3 min before retraction and closure of the wound.

Behavioural tests

Rotation was assessed in a bank of automated rotometer bowls modelled after the design of Ungerstedt and Arbuthnott (1970). Amphetamine-induced rotation was recorded as the number of full turns over 90 min following an intraperitoneal injection of 2.5 mg/kg metamphetamine sulphate (Sigma) dissolved in 0.9% saline; apomorphine-induced rotation was recorded over 60 min following a subcutaneous injection of 0.05 mg/kg apomorphine hydrochloride (Sigma) dissolved in 0.02% ascorbate-saline.

Amphetamine tests were conducted 2 and 4 weeks and apomorphine tests 3 and 5 weeks after the 6-OHDA lesions. Only animals with rotational scores of greater than 5 turns/min and 3 turns/min with amphetamine and apomorphine, respectively, on both occasions were included in the experiment and the rats were matched on these scores for allocation to the control or striatal lesion groups. The animals were then tested under apomorphine at 1, 4 and 10 weeks, and under amphetamine at 2, 5 and 11 weeks after receiving ibotenic striatal lesions. All behavioural data were analysed using two-factor split-plot analysis of variance (Genstat, Rothamsted Experimental Research Station).

Histology

All animals were sacrificed for histology 12 weeks after the ibotenic acid lesion. Each animal received a lethal dose of Sagatal (May and Baker), and was then perfused with intra-cardiac deliv- ery of 150 ml of pre-wash (0.1 M phosphate buffered saline, PBS) followed by 200 ml of 10% formalin in 0.1 M PBS. The brains were post-fixed in 10% formalin overnight and then placed in 25% sucrose in 0.1 M PBS at 4 ~ C until they sank. Sixty micron sections were taken using a sledge microtome from the genu of the corpus callosum to the pons. Every sixth section from each series was taken and stained for cresyl violet, tyrosine hydroxylase (TH), parvalbumin and acetylcholinesterase (ACHE).

Nissl staining of cell bodies with cresyl violet was performed using a conventional protocol. The immunocytochemical staining for TH and parvalbumin used a standard protocol. The sections were quenched with methanol and hydrogen peroxide and then placed in 0.1 M Tris-saline containing 0.2% Triton and 3% normal goat serum (NGS) for 90 min. This was then replaced with the primary antibody to TH (Jacques Boy, 1:4000, polyclonal antibody) or parvalbumin (Sigma, 1:1000, monoclonal antibody) in Tris-saline containing 0.2% Triton and 1% NGS and left for 72 h. The sections were then washed three times in Tris-saline and the respective secondary antibodies of anti-mouse IgG (DAKO, 1:200) or anti-rabbit IgG (DAKO, 1:200) in Tris-saline containing 1% NGS added. After 3 h, the secondary antibody was removed, the sections washed three times in Tris-saline and streptavidin- biotin complex in Tris-saline/l% NGS added (Streptavidin ABC kit, DAKO, 1:200) for 90 min. The sections were washed and developed in diaminobenzidine (DAB) in 0.05 M Tris. The sections were then mounted, dehydrated in alcohols to xylene and cover-slipped with DPX. Histochemical staining for acetylcholinesterase was made using a modified version of the Koelle

(1955) method employing 0.01% ethroproprazine as the inhibitor of non-specific esterase activity and 0.25% silver nitrate to enhance the sulphide reaction product.

The extent of the lesions was analysed using an image analysis package (Seescan, Cambridge, UK). The volume of the striata on the lesioned and control sides were estimated by outlining the areas of normal acetylcholinesterase staining on each section, summing over all sections containing the striatum, and scaling for section thickness and sampling frequency (1:6). In order to ac- commodate for any variation in shrinkage during processing, the data were analysed in terms of the ratio of striatal volumes on the ibotenic acid-lesioned and non-lesioned sides.

The extent of the lesions was also assessed by quantifying the 2loss of a representative group of cells within the striatum - the �9 parvalbumin containing neurons. The numbers of parvalbumin

positive neurons were counted within the striatum in individual sections at two levels; one in the anterior striatum, where the lateral ventricles first appear, and the second at the point of fusion of the anterior commissures (see Fig. 1). In order to compensate for any staining variation between animals, the cell loss was also analysed in terms of the ratio of the counts in the lesioned and intact striata. An analysis of medial-lateral effects of the lesions

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was not attempted, since the expansion of the ventricles and associated collapse of striatal tissue on the lesioned side made this unreliable. The histological and behavioural data were compared using linear regression and partial correlations.

Results

Rota t ion

All experimental animals were selected on the basis of exhibiting effective apomorphine- and amphetamine-induced rota t ion following the initial 6 -OHDA lesions of the nigrostriatal bundle, and the rotat ional behaviour of the two groups was well matched on both tests prior to their receiving ibotenic acid lesions of the striatum (Fig. 2).

Following ibotenic acid lesions of the neostriatum, made on the same side as the previous dopamine-de- pleting 6 -OHDA lesions, there was a significant reduction in apomorphine- induced rotat ion compared to control animals with unilateral 6 -OHDA nigrostriatal tract lesions only (group x weeks interaction, F4,1s 6 = 8.22, P < 0.001). This reduction in rota t ion was seen at the first time of testing after lesioning and re- mained stable th roughout the durat ion of the study (see Fig. 2). Although several of the individual animals (12 of 26) with ibotenic acid lesions showed a reduction in their ro ta t ion by greater than 10% under amphetamine, as a group, this reduction approached but did not achieve significance (groups F1,54-3.53 , P = 0 . 0 7 ; groups x weeks interaction, F4,1s 9 =0.83, n.s.).

Fig. 1 Schematic figure representing areas of analysis for acetylcholinesterase staining of the normal rat brain at the level of the anterior margin of the lateral ventricles, at the level of the mid- portion of the striatal complex and at the level of fusion of the anterior commissures. The first and last levels correspond to the anterior and posterior parts of the striatum used in parvalbumin cell counts, represented on the right of the figure

Histology

In agreement with the screening by rotat ion after the initial 6 -OHDA lesion, all animals had severe unilateral nigrostriatal lesions, as assessed by T H immunoreact iv- ity, and no clear differences were apparent between the two experimental groups.

The ibotenic acid lesions were apparent in all stains and in all animals: (1) as a loss of large pale neuronal cell staining and modest gliosis in the cresyl violet stained sections; (2) as a lightening of neuropil staining and compact ing of the fibre bundles in the acetylcholinesterase stain; (3) as a dramatic reduction in the number of parvalbumin-stained neurons over a large area in the lesioned striatum; and (4) as an expansion of the ventricular area (see Figs. 3, 4).

The extent of the striatal collapse due to lesions was quantified by estimating the volume reduction (based on summation over acetylcholinesterase-stained series of sections) and by the loss of an easily identified sub- popula t ion of striatal neurons (in two representative sections through the striatum).

In the control animals, the 6 -OHDA lesions alone caused no significant drop in striatal volume or decline in the number of parvalbumin-stained cells (see Fig. 5 a, b). By contrast, the striatal lesions caused a highly signifi-

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cant (50%) reduction in the mean striatal volume (groups x side interaction, F1,41 = 51.83, P < 0.001) and a highly significant (60%) decline in the number of parvalbumin-stained neurons (groups x side interaction, F1,4o = 16.09, P<0.001). Although apparent in all animals, the extent of the lesions differed within the ibotenic-acid lesioned group, and the two measures of the lesion were highly correlated (Pearson's correlation coefficient between the ratio of volume loss and ratio of decline in the numbers of parvalbumin-stained neurons r = 0 . 8 2 , t24=7.01, P<0.001, see Fig. 6).

Histology-behaviour correlations

The change in apomorphine-induced rotation following the ibotenic acid striatal lesion was highly correlated to the extent of striatal damage, both in terms of the volume loss (r=0.79, t24=6.22, P<0.001) and the reduction in the number of parvalbumin neurons within the striata (r = 0.67, t24 = 4.46, P < 0.001, Fig. 7). Further analysis using partial correlations demonstrated that the major determinant was the loss in striatal volume rather than the loss of parvalbumin cells (partial correlation of volume with rotation r=0.57, t24=3.43, P < 0 . 0 5 with the variable parvalbumin neuronal cell loss held constant; partial correlation of parvalbumin cell counts with rotation r = 0.063, t24 = 0.31, ns with the volume held constant). A reduction in rotation was, however, only seen when the damage resulted in about a 50% loss of either volume or parvalbumin-positive neurons, with smaller lesions tending to exacerbate the rotational response to apomorphine.

Fig. 3 a, b Acetylcholinesterase staining demonstrating the extent of the lesions, a The smallest lesions produced only minimal changes in a localised area of the striatum, b In contrast, the larger lesions produced a dramatic and widespread reduction in acetylcholinesterase staining, which, in some cases, involved structures outside the dorsal striatum. Scale bar 1 mm

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Fig. 5 The absolute striatal volumes and density of parvalbumin- positive neurons in both striata of control animals and in animals receiving excitotoxic striatal lesions. The striatum receiving the ibotenic acid lesions was of less volume and contained a greatly reduced number of parvalbumin-positive neurons

Fig. 4a-e Parvalbumin immunoreactivity in the control striatum selectively stains a subpopulation of neurons (a). In the ibotenic acid-lesioned striatum, these neurons are lost (b), although occa- sionally animals with large striatal lesions still had parvalbumin- positive neurons adjacent to the margin of the lesion (e). (* lesion site, Cx cerebral cortex) Scale bar 100 pm

The correlat ion between this change in rotat ional behaviour to apomorphine and the extent of the striatal damage induced by the ibotenic acid in this study was not critically dependent on the antero-poster ior site of the lesion. Both anterior and posterior volumes and the corresponding cell counts all correlated significantly with the changes in apomorphine- induced rotat ion ( r = 0 . 6 8 , t 2 4 = 4 . 5 5 , P<0 .001 for anterior volume

changes; r = 0.59, [ 2 4 ~ - 3.60, P < 0.005 for anterior cell count changes; r = 0.79, t24 = 6.29, P < 0.001 for posteri- or volume changes; r = 0.57, t24 = 3.43, P < 0.005 for posterior cell count changes). This tendency may, however, reflect differential tissue collapse following the ibotenic acid lesion rather than the true site of greatest damage.

Although, as a group, there was no significant change in amphetamine- induced rota t ion following ibotenic acid striatal lesions, there was great variat ion with this measure (Fig. 8). Furthermore, there was a significant correlation between the changes in amphetamine-induced rota t ion and the extent of the lesion, both in terms of the volume and the number of parvalbumin cells lost ( r=0.47, t24=2.60 and r=0.49, t24=2.72 respectively, both P<0.05) . However, unlike the relationship with apomorphine, striatal volume loss and parvalbumin cell loss were equal in their contribu- tion to the rotat ional changes seen with amphetamine,

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with neither being a dominant factor on partial correlation analysis (r = 0.137 and r = 0.207 when the cell count and volume changes are held constant, respectively, both ns). Moreover, this correlation was less significant than those seen with apomorphine-induced rotation, but again the correlation existed for all but one of the antero-posterior sites studied, the exception being for volume changes in the anterior striatum (anterior volume r=0.37, t24 = 1.95, ns; anterior cell counts r=0.39, t24 = 2.07, P < 0.05; posterior volume r = 0.47, t24-- 2.57, P<0.05; posterior cell counts r--0.44, t24=2.38, P<0.05).

Discussion

This study has demonstrated that ibotenic acid lesions of the dopamine-deafferented striatum can reduce apomorphine- and to a lesser extent amphetamine-induced rotation in the 6-OHDA-lesioned rat. These reductions in drug-induced rotational behaviour seen in this study occur soon after the ibotenic acid lesioning and are dependent more on the extent of the striatal damage than on its site within the striatum (but see below). Further- more, although there is an excellent correlation between the degree of damage as assessed by striatal volume changes and parvalbumin-positive neuronal cell loss, apomorphine rotational changes are most specifically associated with the volume changes, whilst changes in amphetamine-induced rotation are smaller in magnitude and associated to a similar degree with volume reduction and cell losses.

The changes seen with apomorphine-induced rotation can best be explained by the loss of the neurons

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bearing the supersensitive dopamine receptors in the dopamine-deafferented striatum. Apomorphime is a relatively non-selective dopaminergic receptor agonist which can activate both the D1 and D2 dopaminergic receptors that are found on striatal neurons (Paul et al. 1992; Surmeier et al. 1992). In this study, the use of a low dose of apomorphine (0.05 mg/kg) results in the prefer- ential activation of supersensitive dopamine receptors in the striatum on the side of the 6-OHDA lesion (Ungerstedt 1971b). This in turn produces a rotational turning of the animal away from this lesioned side in the

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contralateral direction, a response which is dependent to some extent on an intact corticostriatal input (Cenci and Bj6rklund 1993; Paul et al. 1992). The striatal neuronal cell loss that follows ibotenic acid lesions of the striatum results in a reduction in the number of supersensitive DA receptors, which results in a reduced rotational response to low-dose apomorphine (see Fig. 2). However, in this study, there had to be a considerable degree of damage for this reduction in apomorphine-induced rotation to be apparent, with smaller lesions ac- tually leading to increased rotational responses. There

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are several possible explanations for this. Firstly, the full expression of the rotational behaviour seen in response to low-dose apomorphine requires only a fraction of the supersensitive receptors to be activated, implying that up to 40% of them may be acting as 'spare' receptors in an analogous fashion to other receptor systems (Williams 1991). Alternatively, the positive effect of small lesions is simply a reflection of the sensitisation effect of repeated apomorphine administration (Mat- tingly and Gotsick 1989). Sensitisation is seen in our control group (Fig. 2), although the mechanism is still unknown (Rowlett et al. 1993). A final explanation for the change in magnitude of rotation with apomorphine seen after striatal lesions relates to the antero-posterior location of the lesion focus (Norman et al. 1992). Al- though there are no apparent correlations with rotational changes and site of damage, this may relate to the effects of perfusion in damaged structures, causing tissue collapse and thus distorting the true anatomical relationship of the striatum, and so obscuring any anterior- posterior relationship. However, we consider this final explanation to be less plausible than the interpretation based on direct disruption by lesions of striatal connections.

The changes in amphetamine-induced rotation following ibotenic striatal lesions are more variable, so that, as a group, there is no significant change against control, although the two groups were tending to sepa- rate as the time from striatal lesioning increased, due in part to a sensitisation effect with amphetamine in the control group (Wolf et al. 1993). However, within the group, there were animals that showed significant reductions in amphetamine-induced rotation, a reduction which was equally well correlated with striatal volume and parvalbumin cell loss. The explanation for this is not clear, as the mechanism of action for amphetamine is thought to involve potentiation of the release of dopamine presynaptically on the intact side, leading to ipsilateral rotation (Ungerstedt 1971a). If this was the only mechanism underlying amphetamine-induced rotation, then striatal lesions of the dopamine-deafferented striatum would be expected to have no effect or even, in the presence of incomplete lesions, to increase the animals, rotational response.

We cannot explain the correlation between the changes in amphetamine-induced rotation and the extent of the striatal lesion, although the following factors may have an influence, Firstly, striatal damage, when extensive enough, could lead to retrograde death or dys- function of some dopaminergic neurons in the contralateral substantia nigra, as the pathway has a small, but significant, contralateral projection (Bj6rklund and Lindvall 1986). As the area of striatal damage increases so does the degree of contralateral nigral cell death, as well as the loss of any remaining nigrostriatal neurons on the lesioned side (Pasinetti et al. 1991). However, these crossed and ipsilateral nigrostriatal projections originate from discrete populations of nigral neurons, and thus the retrograde loss of the contralateral projec-

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tion will be without effect on the ipsilateral striatum (Loughlin and Fallon 1982). This argues against any role in the reduction in rotation which we observed, although changes in the contralateral striatum have been reported with unilateral striatal ibotenic acid lesions (Narang et al. 1993). Furthermore, the reduction in rotation, when present, was seen at the first time of testing and did not take time to develop, arguing against a delayed cell death. Finally, there were no obvious differences in the terminal staining for TH within the striatum of controls and animals with striatal lesions.

An alternative and more likely explanation is that the excitotoxic lesion damaged structures outside the striatum either directly or indirectly by interfering with their afferent projections into the striatum. Lesions to the ventral striatum as well as disruption of the corticostriatal input can both reduce the drug-induced rotational responses of animals with nigrostriatal 6-OHDA lesions (Cenci and Bj6rklund 1993; Kelly and Moore 1976; Paul et al. 1992; Reading 1992). The corticostriatal projection to the intact striatum has been shown to be important in the rotational responses of animals with unilateral 6-OHDA lesions of the nigrostriatal tract (Cenci and Bj6rklund 1993). This process is therefore unlikely to be underlying the reduction in rotation seen in this study. However, damage to the ventral extension of the striatum ipsilateral to the side of the 6-OHDA lesion has been shown to affect the rotational behaviour of the animal in response to drug challenges (Reading 1992). In its simplest form, the rotational behaviour of an animal has two components: a direction for rotation determined by the dorsal striatum and the degree of rotation determined by the ventral striatum (Kelly and Moore 1976; Pycock and Marsden 1978). Thus, damage to the ventral striatum will reduce rotation by a loss of this magnification factor, although others have claimed that the ventral striatum itself can dictate the direction of rotation (Koshikawa et al. 1990; Reading 1992). In this study, therefore, damage to the dorsal striatum may be having minimal effects on amphetamine-induced rotation, but large lesions, by involving the ventral striatum, may be expected to decrease rotation by reducing the gain of the system or inducing a rotational bias in the opposite direction. It is clear that there is some evidence to corroborate this, as some of the animals with extensive lesions causing reductions in amphetamine- induced rotation did have damage involving the ventral striatum. Although we were not able to evaluate this aspect of the lesion quantitatively, there was no evidence that damage to this structure affected amphetamine-induced rotation more than damage to the dorsal striatum. In this respect, it would be of interest to study the effects of ibotenic acid lesions of the ventral striatum in the 6-OHDA-lesioned rat on drug-induced rotation, as it may also be involved in the reduction in apomorphine-induced rotation which we observed.

A final possibility is that amphetamine at the dose used in this study can stimulate the release of other amines (see, for example, Kuczenski and Segal 1989)

which may have their own effects on rotation, as many neurotransmitters have been implicated in the rotational behaviour of animals (Pycock 1980).

Few other studies have specifically addressed the question of the effects of striatal damage on rotational behaviour in 6-OHDA-lesioned animals, in contrast to the large number of studies that have specifically looked at the rotational behaviour of animals with striatal lesions only (see, for example, Dunnett et al. 1988b). Freed and colleagues) have recently reported that kainic acid lesions of the unilaterally 6-OHDA-lesioned rat did not reduce apomorphine-induced rotation, although the size, location and extent of the lesions were not com- mented on (Takashima et al. 1993). In contrast, Friehs et al. (1991) reported that radiofrequency lesions of the striatum in 6-OHDA-lesioned rats dramatically reduced apomorphine-induced rotation, although, again, the site and extent of the lesions were not correlated with the reduction in rotation. In this latter study, radiofrequency lesions were compared with intrastriatal adrenal medulla grafts, which produced a smaller, but still significant, reduction in apomorphine-induced rotation, in line with other studies on adrenal medullary grafts (Freed et al. 1990).

In many studies, the long-term survival of the adrenal medullary tissue has been shown to be poor, and yet a reduction in drug-induced rotation, especially with apomorphine, is common (Barker and Dunnett 1992; Bing et al. 1988; Freed et al. 1990). This, coupled to the success of non-neural grafts, especially in partially lesioned animals, has lead to a re-evaluation of the mechanism of action of these grafts (Barker and Dun- nett 1993; Freed et al. 1990; Riopelle 1988). Although there is compelling evidence that embryonic nigral grafts ameliorate the rotational asymmetry of 6- OHDA-lesioned rats by restoring dopaminergic inner- vation in the anterolateral striatum (Dunnett et al. 1981, 1988a), the effects of striatal damage as an indirect me- diator of rotational changes seen with other grafts, especially adrenal medullary transplants, cannot be exclud- ed. Indeed, in advanced Parkinson's disease, surgery leading to striatal damage has been used with some success in treating some of the patients' motor disabilities (Meyers 1951; Motti et al. 1988). Although this study emphasises that this is possible, in that striatal damage can reduce both apomorphine- and amphetamine-induced rotation, it does so only when at least 40% of the striatum is damaged or lost. Therefore, although intrastriatal grafts may reduce drug-induced rotation, it is probable that striatal damage contributes little to this effect, unless the area of damage following grafting is quite extensive.

In conclusion, ibotenic acid lesions of the ipsilateral striatum in the 6-OHDA-lesioned rat can reduce both apomorphine- and amphetamine-induced rotation, and this may contribute to the effects of intrastriatal grafts of tissue in animal models of Parkinson's disease. Howev- er, the lesions need to produce relatively extensive striatal damage before reductions in rotation are seen, and

this mechanismalone cannot account for the full effects of the graRs.

Acknowledgements This work was supported by the MRC. R.A.B. is an MRC training fellow.

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Ibotenic acid lesions of the striatum reduce drug-induced rotation in the 6-hydroxydopamine-lesioned rat