[3H]Dihydrotetrabenazine, a new marker for the visualization of dopaminergic denervation in the rat striatum

Neuroscience Letters, 114 (1990) 45-50

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Elsevier Scientific Publishers Ireland Ltd. NSL 06930

[3H]Dihydrotetrabenazine, a new marker for the visualization of dopaminergic denervation in the rat striatum Yoshinori Masuo 1, Didier P61aprat l, Daniel Scherman 2 and William Rost+ne l qNSERM U.55, H6pital Saint-Antoine, Paris (France) and 21nstitut de Biologie Physieo-Chimique, Paris ( France )

(Received 6 November 1989; Revised version received 12 February 1990; Accepted 21 February 1990) Keywords:

Striatum; Nigral lesion; 6-Hydroxydopamine; Dihydrotetrabenazine; Autoradiography; Dopamine; Tyrosine hydroxylase

[3H]Dihydrotetrabenazine ([3H]TBZOH), a derivative of the monoamine depleting agent, tetrabenazine, has been shown to bind to the vesicular monoamine transporter. We studied the effect of unilateral nigral lesion with 6-hydroxydopamine on [3HITBZOH binding in the striatum by means of quantitative autoradiography. Topographical analysis of striatal [3H]TBZOH binding showed in control rats a pattern similar to the known distribution of dopaminergic innervation, and allowed to visualize dopaminergic denervation in lesioned rats. A good correlation was found between striatal [3HITBZOH binding levels and tyrosine hydroxylase activities in similar areas on adjacent slices following 6-OHDA lesions with varying severity. These data reveal that autoradiography of [3H]TBZOH binding is a good index for the visualization and quantitation of dopaminergic nerve terminals integrity in the striatum.

T h e s t r i a t u m is k n o w n to be highly i n n e r v a t e d by d o p a m i n e ( D A ) terminals o f the n i g r o s t r i a t a l D A p a t h w a y [3]. U p to now, the striatal D A e r g i c i n n e r v a t i o n has been studied by w e l l - k n o w n b i o c h e m i c a l m a r k e r s , such as D A c o n t e n t a n d tyrosine hyd r o x y l a s e ( T H ) activity. It was d e m o n s t r a t e d t h a t ~ - d i h y d r o t e t r a b e n a z i n e ( T B Z O H ) b l o c k e d vesicular m o n o a m i n e u p t a k e [1 1], a n d [3H]TBZOH was recently i n t r o d u c e d as a specific high affinity ligand for the vesicular m o n o a m i n e t r a n s p o r t e r in chromaffin granules or a d r e n a l medulla, b r a i n synaptic vesicles, a n d crude tissue h o m o g e n a t e s [7]. The density o f [3H]TBZOH b i n d i n g in h o m o g e n a t e s from v a r i o u s regions o f the m o u s e b r a i n was r e p o r t e d to be p r o p o r t i o n a l to the total a m o u n t o f e n d o g e n o u s m o n o a m i n e s [12].

Correspondence." Y. Masuo, INSERM U.55, H6pital Saint-Antoine, 184, rue du Faubourg Saint-Antoine,

F-75771 Paris Cedex 12, France. 0304-3940/90/$ 03.50 ~_~1990 Elsevier Scientific Publishers Ireland Ltd.

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[zig, I Autoradiograms ofpIt]dihydroletrabenazine ([3H]TBZOH) binding from rat brain frontal sections at the level of the slriatum. A: Sham-operated control. B-D: Three typical slices following 6 - O H D A lesions of the left substantia nigra with increasing severity (B: slightly lesioned to D: most severely lesioned). 6O H D A (8 iLg) was injected according to three different coordinates at the lateral level. B: 2. I ram; C: 1.8 ram: 1): 1.5 mm from the midline.

Quite recently, we applied [3H]TBZOH to in vitro autoradiography [4], and demonstrated that the regional distribution of [3H]TBZOH binding site densities in the rat brain showed a pattern similar to that of monoaminergic neurons. The purpose of the present study was to investigate whether this new ligand could be used for the topographic and quantitative analysis of DA depletion in the striatum following intranigral injection of 6-hydroxydopamine (6-OHDA). Male Wistar rats weighing approximately 200g were anaesthetized with ketamine ( 150 175 mg/kg), and 6-OHDA hydrobromide (Sigma, 8/2g in 2/21 of ice-cold Ringer solution containing 0.02% l,-ascorbic acid) was stereotaxically injected into the left substantia nigra for 2 min. In order to produce varying DAergic damage, 3 different coordinates at the lateral level in 3 rats for each group were used as follows: L 2.1, 1.8, 1.5 mm from the midline with AP + 3.0 mm and H + 2. I mm from the interaural line, calculated according to the atlases of K6nig and Klippel [8] and of Paxinos and Watson [10]. For the sham operation. 2/21 of vehicle were injected under identical conditions. The animals were killed 14 days following the operation and the brains immediately removed, frozen and cut into 10/2m and 300/2m thick serial sections alternately. Striatal tissue samples were punched out (3.0 mm diameter) from four 300/2m thick sections rostral to the anterior commissure (see Fig. 2), and TH activity

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Fig. 2. Linear correlation between TH activity and [3H]TBZOHbinding site density in the ipsilateral striaturn following unilateral nigral lesion with 6-OHDA. The regression line was drawn by means of values in the lesioned animals (n=9, r=0.973, P<0.001 by Student's t-test). The open circle represents means _+ S.E.M. of 6 sham operated controls (TH activity: 3.42 + 0.23 nmol/h/mg protein, [3H]TBZOHbinding: 638 _+49 fmol/mg protein). Densitometric measurementsfor [3H]TBZOHbinding sites were performed on 4 serial sections in each animal. Schematic drawing shows striatum "punched area" in which TH activities and [3H]TBZOHbinding site densities were measured.

was determined by the method of Nagatsu et al. [9]. Protein content was estimated by the method of Bradford [2]. [3H]TBZOH binding and autoradiography were carried out on corresponding alternate sections (10/tin thickness) as previously described [4]. Briefly, striatal sections were incubated for 40 min at room temperature with 12 nM [3H]TBZOH (15 Ci/ mmol, CEA, France) in 10 m M HEPES, p H 8.0, containing 0.3 M sucrose. Nonspecific binding was determined by 5 / t M unlabeled tetrabenazine. After incubation, sections were washed twice for 3 rain at 4°C in 40 m M Tris-HC1 buffer, pH 8.0, and dipped in distilled water. Autoradiographs were generated by apposition of dried radiolabeled sections to Hyperfilm-3H (Amersham, U.K.) for 8 weeks at room temperature. Quantitative optical density measurements were performed using an image analyzer (Biocom 200, Les Ulis, France), and converted into femtomoles of [3H]TBZOH bound per mg protein [14]. Optical densities of non-specific binding (3 % of total binding in the unlesioned striatum) were subtracted from total binding. Unilateral injection of 6 - O H D A into the substantia nigra produced individual depletion in TH activities ranging from 39 to 100% in the ipsilateral striatum when compared to the corresponding side of sham-operated controls. No change was found in the contralateral structure. In agreement with previous studies [4], high densities of [3H]TBZOH binding were observed in the unlesioned striatum, nucleus accumbens, olfactory tubercle and lateral septum (Fig. 1A). We first realized an 'optical dissection' with the image analyzer by selecting an area corresponding to that punched out for TH activity mea-

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Fig. 3. Topographical changes in [3H]TBZOH binding site densities in the ipsilateral striatum following unilateral nigral lesion with 6-OHDA. Schematic drawing represents the ipsilateral striatum divided into 18 areas in which densitometric measurements were carried out. A D correspond to autoradiograms represented in Fig. I. A ( /): Means +_ S.E.M. of sham operated controls (n=6). B D(II): Mean values of4serial sections from 6-OHDA-lesioned animals with increasing severity.

sured on adjacent 300 ,urn thick section. As shown in Fig. 2, when [3H]TBZOH binding site densities in this optically 'punched area' were compared to TH activities in the striatum ipsilateral to the lesion with varying severity, a highly significant linear correlation was found between the 2 sets of data (r=0.973, P<0.001 by Student's t-test). In the striatum, in addition to the dotty appearance due to the presence of fiber tracts, lateral and dorsomedial zones of higher [3H]TBZOH labeling can be observed in control rats (Fig. 1A). A topographical analysis was then performed by measuring [3H]TBZOH binding in 18 areas dividing the striatum into 6 lateromedial portions at 3 dorsoventral levels (Fig. 3). Heterogeneous distribution of [3H]TBZOH binding sites was clearly revealed in control rats (Fig. 3A). Higher densities were observed in the lateral parts throughout the dorsoventral distribution. Central portions showed much lower labeling (area numbers 3- 5, 9 11) with the exception of the ventral part (area numbers 15, 16). Finally, regions close to the midline contained the lowest amount of [3H]TBZOH binding sites, except the dorsal part (area number 6). For low TH depletion ( - 3 9 % ) by 6-OHDA injection into the lateral substantia nigra, the decrease in [3H]TBZOH binding (Figs. IB and 3B) occurred only in the lateral portions of the striatum (area numbers I, 2, 7, 8, 13, 14). In the more severely lesioned rat corresponding to 67% decrease in TH activity (Figs. IC and 3C), the decrease in the binding extended to medial parts of the structure in dorsal and central portions and to central parts in ventral portions (area number 15). In the rat which showed the most pronounced decrease ( - 95 %) in TH activity (Figs. I D and 3D) due to 6-OHDA injected into the medial substantia nigra, the decreases in [3H]TBZOH binding site densities were similar in all parts of the structure. These data are in agreement with the topography of the nigrostriatal projection [1]. It should be noted that

49

in rats presenting a 90% decrease in striatal [3H]TBZOH binding sites, concomitant decreases were also observed in the nucleus accumbens ( - 4 3 %) and olfactory tubercle (-46%), which were statistically significant (n = 3, P < 0.05 by Student's t-test), indicating diffusion of the toxin into the ventral tegmental area [1]. The present results clearly indicate that [3H]TBZOH binding is a good index for the integrity of striatal DA innervation. Firstly, topographical analysis of striatal [3H]TBZOH binding revealed a heterogenous pattern of distribution with high labeling in the lateral and dorsomedial parts. This distribution is consistent with that obtained in quantitative studies on TH immunohistochemistry [5]. Secondly, after various nigral 6-OHDA lesions producing different extents of DA denervation, the observed decrease in [3H]TBZOH binding was well correlated with the loss in TH activity. This study, performed by both mechanical (for biochemical analysis) and optical (for densitometric measurement) punching out of a large striatal zone, revealed a linear correlation between the 2 sets of data obtained on alternate sections (Fig. 2). However, when TH activity was extrapolated to zero on the curve, a remnant [3H]TBZOH binding still appeared, representing around 5 % of the initial value. It is interesting to note that this percentage is similar to the relative amount of serotonin [12] which is unaffected in the striatum following 6-OHDA lesion [15]. Although 6-OHDA induces depletion not only of DA but also of noradrenaline [15], striatal noradrenaline levels are negligibly low [12]. Indeed, Hefti et al. [6] reported a good correlation between TH activities and DA contents in the striatum after nigral lesion with 6-OHDA. Taken together, the present data suggest that around 95 % of striatal [3H]TBZOH binding sites effectively result from a labeling of DA innervation. This finding in the rat is in agreement with a recent study [13] showing that [3H]TBZOH binding was decreased in the caudate-putamen of patients with Parkinson's disease. [3H]TBZOH binding may thus be a good index for the loss of striatal DA terminals in both the rodent and human [4, 13]. The present study demonstrates the suitability of [3H]TBZOH for the visualization and quantitative evaluation of the extent of DAergic denervation in the rat striatum. This ligand may thus be considered as a new tool presenting complementary advantages toward the 2 commonly used markers, TH activity and DA content. The fact that [3H]TBZOH binding can be performed on unfixed tissues allows receptor autoradiography with other ligands on successive thin sections. This work was supported by grants from la Fondation de France and the Institut National de la Sant6 et de la Recherche M6dicale, France. Y.M. is a recipient of a fellowship from the Canon Foundation in Europe. 1 Bj6rklund, A. and Lindval, O., Dopamine-containing systems in the CNS. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2 (Classical Transmitters in the CNS, Part 1), Elsevier, Amsterdam, 1984, pp. 55 122. 2 Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72 (1976) 248 254. 3 Dahlstr6m, A., and Fuxe, F., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand. 62 (Suppl. 232) (1964) 1-55.

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