Long-term behavioral and biochemical effects of 6-hydroxydopamine injections in rat caudate-putamen

Brain Research

Bulletin,

0361-9230191 53.00 + .oo

Vol. 26, pp. 707-713. 0 PergamonPress pk. 1991.Printedin the U.S.A.

Long-Term Behavioral and Biochemical Effects of 6-Hydroxydopamine Injections in Rat Caudate-stamen JEAN LUD CADET,’ REUBEN LAST, VLADIMIR KOSTIC, SERGE PRZEDBORSKI AND VERNICE JACKSON-LEWIS Laboratory of Preclinical Neurosciences, Department of Neurology Columbia University, College of Physicians and Surgeons, New York, NY 10032 Received 22 October 1990 CADET, J. L., R. LAST, V. KOSTIC, S. PRZEDBORSKI AND V. JACKSON-LEWIS. Long-term behavioral and biochemical effects of Ghydroxydopamine injections in rat caudate-putamen. BRAIN RES BULL 26(5) 707-713, 1991.-Unilateral injections of 6-hydroxydopamine into the rat striatum result in amphetamine-induced circling behavior. This rotational behavior was associated with an almost complete disappearance of desmethylimipramine-insensitive [‘Hlmazindol binding sites-which represent dopamine uptake sites-in the ipsilateral caudate-putamen (CPU), the substantia nigra pars compacta (SNpc), and in the ventral tegmental area (VTA). There were significant increases in [3H]spiperone-labeled dopamine (DA) D2 receptors in specific subdivisions of the ipsilateral CPU, with the dorsolateral (DL) and ventrolateral (VL) regions showing significant increases in DA D2 receptors. There were nonsignificant increases in the dorsomedial (DM) aspects of the ipsilateral CPU whereas there were no changes in the ventromedial (VM) aspects of that structure. In contrast, there were no significant changes in [‘H]SCH 23390labeled DA Dl receptors in any of the sub~v~sions of the CPU ipsifateral to the 6-OHDA-induced lesions. These results provide evidence that intrastriatal injections of 6-OHDA result in biochemical changes in rat brain which are almost identical to those observed after 6-OHDA-induced lesions of the substantia nigra. These long-term biochemical effects caused by intrastriatal 6OHDA injections provide further support for the idea that the nigral DA cell loss observed in the brains of parkinsonian patients could be secondary to retrograde changes due to oxyradicals generated during the metabolism of catecholamines within the caudate-putamen. 6-Hydroxydopamine Dopamine receptors

Striatnm

Rotation

Parkinson’s disease

THE caudate-put~en integrates information from the cerebral cortex and the substantia nigra which it translates into both motor and cognitive behaviors. These integrative mec~nisms depend on a functional mosaicism which is biochemically and neuroanatomically evident in the mammalian central nervous system (CNS) (14, 15, 34). Dysfunctions of these systems result in

clinically identifiable syndromes in humans (4,21). For example, lesion of the nigrostriatal dopamine (DA) system is accompanied by depletion of striatal DA and in the signs of Parkinson’s disease (4,21). It has been proposed that this neurodegenerative disorder might be secondary to deleterious effects of exogenous or endogenous toxins on specific subpopulations of neurons in the CNS (7,9). These neurotoxin-based theories are of special interest because they provide frameworks which might help to clarify the etiology of these disorders. 6-Hydroxydopamine is a neurotoxin that destroys catecholaminergic neurons selectively (6). Infusion of the drug into the cerebrospinal fluid of rodents results in significant depletion of dopamine, norepinephrine and of their metabolites in various re-

Quantitative autoradiography

gions of the brain (6). However, its injection into the DA-rich subst~tia nigra pars compacta (SNpc) results in almost complete loss of DA cell bodies in the SNpc and conco~~t depletion of DA in rat striatum (33,35). This loss of DA is accompanied by a specific behavioral pattern, namely dopamine agonist-induced rotation behavior. Since its first description, this model of hemiparkinsonism has been the subject of a large number of behavioral and biochemical studies. More recently, it has been used to assess the behavioral and biochemical effects of transplantation of various tissues (13,26) in rat brain. Although the use of this model has been very productive, it does not allow for studies of the regenerative properties of the nigrostriatal DA system ipsilateral to the side of 6-OHDA injection because of the almost immediate and complete destruction of the cell bodies of the SNpc and in the ventral tegmental area (VTA) which results after the administration of 6OHDA in that region. Although it is known that intrastriatal injections of 6-OHDA can also result in moderate DA depletion in the basal ganglia and in circling behavior in rat (8, 11, 17), that rotational model has

‘Requestsfor reprints should be addressed to Jean Lud Cadet, M.D., Department of Neurology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032.

CADET ET AL.

Therefore, we undertook the present study to evaluate the long-term effects of intrastriatal 6-OHDA on rotational behavior and on indices of the DA system in rat brain. We thus used quantitative receptor autoradiographic techniques to assess the effects of these lesions on the distribution of dopamine uptake sites. and of Dl and D2 receptors in the rat basal ganglia. These studies are parts of our continuing efforts investigating the biochemical mechanisms involved in the specific degeneration of the nigrostriatal dopaminergic system (3,s). /

_

Rotation

at 2 wks METHOD

Animals

0

20

60

40

Time

60

100

120

Adult male Sprague-Dawley rats weighing 220-2.50 g at the beginning of the experiments were used. The animals were housed in a temperature-controlled environment with a 12-h light-dark cycle and were given free access to food and water.

(minl On the day of surgery, the rats were anesthetized with an intraperitoneal injection of ketamine (15 mg/kg) plus xylazine (15 mg/kg) and then placed in a stereotaxic instrument for the performance of unilateral intrastriatal injection of 6-OHDA. The animals were injected at two sites of the left striatum. The coordinates were: AP 1.6 ML 2.4 DV 4.2 and AP 0.2 ML 2.6 DV 7.0 from Bregma. Each animal received two infusions of 6-OHDA (20 kg in 5 ~1 of normal saline containing 0.02% ascorbic acid). The drug was infused at a rate of 1 pJ/min through a Hamilton syringe. At the end of the infusion. the needle was left in place for another 5 min in order to allow for diffusion of the drug away from the tip of the needle. The animals were then tested for amphetamine-induced circling. Rotation Studies

0

;

0 Weeks

I

I

!

I

1

2

4

6

8

10

Post

6-OHDA-induced

Lesions

FIG. 1. Ipsilateral rotation induced by 2 mg/kg of amphetamine in rats after intrastriatal injections of 6-OHDA. (A) Rotation two and eight weeks after the 6-OHDA injections. (B) Time course of the increase in total rotation per 120 min over the two-month period of observation. The values represent means 2 SEM of the 5 animals used in the receptor autoradiographic studies. Note the differences in the scales on the Y-axis.

not been investigated as extensively as the one induced by SNpc lesions. Experiments related to the biochemical and behavioral effects of intrastriatal injection of 6-OHDA are of special interest for a number of reasons. For example, recent hypotheses regarding Parkinson’s disease suggest that this neurodegenerative disorder might be secondary to the production of oxyradicals during the metabolism of DA by monoamine oxidase (MAO) (8,9). These ideas thus inferred the possibility that striatal DA nerve terminals might be the first sites to disintegrate and that the loss of nigral cell bodies might be secondary to retrograde degeneration. Thus, neurochemical studies of the alterations which might occur in the rodent nigrostriatal system after intrastriatal injections of 6-OHDA might help to evaluate, in the long-term, the response of the substantia nigra to an oxidative load which might have occurred some distance away.

On the day of testing, the animals were placed in cages and left to adapt for 60 min. At that time, the injected with amphetamine (2 mg/kg, subcutaneously) cling behavior was monitored at lo-min intervals for The animals were monitored weekly for a period of starting on the second week after surgery. Receptor Autoradiographic

individual rats were and cir120 min. 8 weeks,

Studies

The animals were sacrificed by decapitation with a guillotine. The brains were removed, rapidly frozen on dry ice, and then kept at - 70°C until further use. Sections (12 PM) thick were cut at - 18°C and thaw-mounted on gelatin-coated glass slides for the performance of the receptor autoradiographic studies. The slides were then desiccated and stored at -20°C until used in various assays. Dopamine uptake sites were labeled with [‘Hlmazindol (15 Ci/mmol, New England Nuclear, Boston, MA) according to published protocols (18) with minor modifications. For autoradiographic assays, the slides were warmed up to room temperature for 30 min and the preincubated at 4°C for 5 min in a buffer consisting of 50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, pH 7.9. The slide-mounted sections were then incubated with 15 nM [3H]mazindol in 50 mM Tris-HCl containing 300 mM NaCl, 5 mM KCl, pH 7.9 for 40 min at 4°C. Desmethylimipramine (DMI) (0.3 PM) was used to block binding to norepinephrine uptake sites. Specific binding was defined using 30 p,M benztropine and represented 80% of total binding. At the end of the incubation period, the slides were put through

6-OHDA, STRIATUM,

BEHAVIOR

709

AND BIOCHEMISTRY

FIG. 2. Representative dark-field photomicrographs of the effects of 6-OHDA on [3H]mazindol binding at the levels of the rat caudate-putamen (A) and of the substantia nigra (B). The lightest colour represents higher concentration of binding. Injection of 6-OHDA caused almost total disappearance of [sH]mazindol binding sites in both the caudate and the nigra on the side of the lesions. The quantitative data are listed in Table 1.

two 3-min washes, dipped in deionized water, and dried under a stream of cold air. Dried sections were placed in a X-ray cassette with plastic standards ([3H]microscales, Amersham) and were apposed to t&urn-sensitive Hyperfilm (Amersham) for 3 weeks at 4°C. The developed autoradiograms were quantitated using a computer-based analysis system (Loats, Amersham). The film optical densities were converted to fmol/mg of tissue usin a standard curve generated by the 3H microscales. [9H]SCH 23390 (71.3 Ci/mmol; NBN, MA) was used to measure the density of Dl receptor in rat brain (28). Briefly, after removal from the freezer, the slide-mounted tissues were warmed up to room temperature for 30 min. The tissues were

preincubated in a buffer containing 120 mM NaCl, 5 mM KCl, 1 mM MgCl,, and 2 mM Ca,Cl, pH 7.4 at 24°C. The slides were then incubated in a similar buffer containing 2.5 nM of [3H]SCH 23390 for 60 min. Nonspecific binding was determined in the presence of 1 PM of unlabeled SCH 23390. Specific binding represented 95% of total binding. At the end of the incubation, the slides were washed in fresh ice-cold buffer for two consecutive 5-min periods, dipped in ice-cold deionized water, and then dried under a stream of cool dry air. The sections are then apposed to Amersham Hyperfilms for 10 days. The autoradiographic films were developed and quantified as described above. D2 receptors were labeled with 0.6 nM [3H]spiperone

710

CADET ET AL.

FIG. 3. Representative dark-field photomicrographs of [jH]SCH 23390 (A) and of [3H]spiperone (B) binding in the striatum after intrastriatal injections of 6-OHDA. (A) There were no signi~c~t changes in 13H]SCH 23390 binding in the CPU. (B) There were visually noticeabfe increases in [‘Hlspiperone binding in the dorso- and ventrolateral aspects on the side of the 6-OHDA-induced lesions. The quantitative data are given in Table 2.

(32.1 Ci/mmol; NEN, MA) (19,20) using a buffer identical to the one described for t3H]SCH 23390, except for the addition of 0.001% ascorbic acid. Binding of spiroperidoi to serotonin (5I-U-2) receptors was prevented by incubation in the presence of 100 nM of ketanserin. Nonspecific binding was determined by 10 PM of unlabeled ( - )-sulpiride. Specific binding was 70% of total binding. The exposure time was for 4 weeks. The developed autoradio~ms were analyzed as described above. In order to obtain regional concentration of binding for the three binding sites, the CPU was subdivided into four quadrants: dorsolateral (DL), dorsomedial (DM), ventrolateral (VL), and

ven~medi~ (VM). Mean values were obtained from 5 animals. Differences between the two sides of the brain were analyzed by Student’s r-test. The null hypothesis was rejected at the 0.05 level. RESULTS

Amphetamine (2 mg/kg) caused significant ipsilateral rotation in the rats with unilateral 6-OHDA-induced lesions of the stri-

6-OHDA,

STRIATUM,

BEHAVIOR

AND

BIOCHEMISTRY

711

1

TABLE

the rat (18). DMI-insensitive [3H]mazindol binding sites which represent dopamine uptake sites are concentrated mainly in the basal ganglionic structures of the rat. Within the striatum proper, we found significant high-to-low lateromedial gradients but no dorsoventral gradients in the concentration of DA uptake sites. As shown in Fig. 2, there was almost total disappearance of specific DMI-insensitive [3H]mazindol sites in the caudateputamen ( - 94%) and in the substantia nigra ( - 85%) ipsilateral to the side of 6-OHDA injections. Table 1 shows the quantitative data from these experiments. The mean decreases in [3H]mazindol binding in the four striatal quadrants varied from 90% to 96% of controls. There were also decreases in the ventral tegmental area (VTA) (- 80%) which were almost as marked as the changes in the SN ( - 85%) ipsilateral to the lesions (Table 1). [3H]SCH 23390. Figure 3A shows the autoradiographic distribution of [3H]SCH 23390-labeled Dl dopamine receptors in the rat striatum. All four quadrants of the CPU show similar binding of [3H]SCH23390. Intrastriatal injections of 6-OHDA cause no differences between the 6-OHDA-injected side and the control side in the CPU (Table 2). [3H]Spiperone. Figure 3B shows the autoradiographic distribution of [3H]spiperone-labeled D2 dopamine receptors in the striatum. Within the normal caudate there was a high-to-low lateromedial gradient of D2 receptors. There was also a small lowto-high dorsoventral gradient in on the lateral hemisphere of the CPU. Intrastriatal injections of 6-OHDA resulted in increases in D2 receptors on the ventrolateral (+46%) and in the dorsolateral (+65%) striatal subdivision. There were nonsignificant increases in [3H]spiperone observed in the DM subdivision whereas there was a very slight nonsignificant decrease in the VM. in

EFFECTS OF INTRASTRIATAL

INJECTIONS OF 6-OHDA ON L3HlMAZINDOL BINDING IN RATS

[3H]Mazindol

Binding (fmohmg

Contralateral

Brain Areas

Striatum Total

tissue)

Ipsilateral

1110 2 25

86 k 11*

Dorsolateral

1156 2 46

(7.8) 84 2 18*

Dorsomedial

1045 k 52

(7.2) 44 k 23*

Ventrolateral

1190 t

30

(4.2) 113 2 19*

Ventromedial

1052 + 47

(9.5) 104 k 13*

SN

443 k 17

VTA

523 k 27

(9.9) 65 2 26* (14.7) 105 2 23* (20.1)

The values represent means k SEM of 5 animals. The numbers in parentheses represent percentages of the contralateral side. *p
atal DA terminals (Fig. 1). The mean ipsilateral rotation reached 11 rotations/min by the seventh week of testing (Fig. 1B). These values are comparable of those of other authors who have used similar or slightly higher doses of amphetamine (5 mglkg) in the rat model of 6-OHDA-induced nigral lesions (33) or after chronic infusion of the 1-methyl-phenylpyridinium species (MPP + ) into the rat striatum (31).

DISCUSSION

Autoradiographic Receptor Studies

Significance of the Circling Behavior and the Loss of [3H]Mazindol Binding Observed After Intrastriatal Injection of 6-OHDA

[3H]Mazindol binding. The autoradiographic distribution of [3H]mazindol is similar to what has been described previously

Intrastriatal injections of 6-OHDA result in ipsilateral amphetamine-induced circling behavior in rats. Since amphet-

TABLE 2 EFFECTS OF INTRASTRIATAL

INJECTIONS

OF 6-OHDA ON [‘HISCH 23390 AND ON [3HlSPIPERONE

BINDING IN THE

RAT CAUDATEPUTAMEN [3H]SCH 23390 Binding (fmol/mg tissue)

[3H]Spiperone Binding (fmol/mg tissue)

Striatum

Contralateral

Total

361.4

+ 4.6

367.4

” 3.8

117.4 k

Dorsolateral

365.2

2 3.7

364.2

2 3.5

119.4 * 10.5

Dorsomedial

364.2

k 4.4

360.4

” 2.2

89.4 2

4.9

Ventrolateral

377.2

f

3.2

372.4

+ 3.2

151.6 2

6.6

Ventromedial

357.8

2 7.0

354.0

k 8.9

104.2 +

6.0

Ipsilateral

Contralateral

7.5

Ipsilateral

169.4 + 11.6* (144.2) 196.6 k 13.1* (164.7) 111.4 k 10.6 (124.6) 221.6 k 12.7* (146.2) 98.4 2 10.4 (94.4)

The values represent mean f SEM of 5 animals. There were no significant differences in Dl between the two sides. However, 6-OHDA caused significant increases in DA D2 receptors in the dorsolateral and ventrolateral aspects of the CPU of these animals. Contalateral and ipsilateral refer to the side of the 6-OHDA injections. *p
CADET ET AL.

712

amine-induced behavioral excitation is thought to be related to increases in the release of dopamine at the levels of the caudateputamen (35), the time course of the development of amphetamine-induced rotational behavior suggests that there might have developed progressive differences in DA terminals between the two striata in animals treated with intrastriatal injections of 6-OHDA. Thus, the circling rate might have stabilized at a time where there were maximal differences between the two sides of the brain. This interpretation is consistent with the demonstration, using intracerebral dialysis, that amphetamine-induced circling was closely linked to the concentration of DA released in the intact striatum (35). When taken together with the uniform and nearly complete disappearance of [3H]mazindol-labeled dopamine uptake sites in the caudate-putamen and the marked depletion of dopamine uptake sites in the SNpc observed on the side of the lesions, the behavioral data indicate that intrastriatal injections of 6-OHDA could indeed serve as a model for the assessment of phenomenological and biochemical effects of this dopaminotoxic agent. In addition, this approach could be used to investigate the effects of trophic substances on the time course of degeneration and regeneration of the nigrostriatal DA system. Changes in Dopamine Receptors in Response to Striatal Denervation in Relationship to Their Subtypes and Their Localization Dl receptors. The lack of changes in DA Dl receptors is not surprising since other investigators have failed to report any increases in these receptors when they were measured several weeks after lesions of nigral cell bodies with 6-OHDA (1, 12, 28). Subchronic infusion of MPP+ which causes marked reduction of DA in rat striata also failed to cause any changes in the concentration of striatal Dl receptors (2). Nevertheless, our present findings are in contrast with those of other investigators who had reported decreases (22) or increases (27) in striatal Dl receptors after nigral lesions. Filloux et al. (12) had also reported that Dl receptors increased only in the area adjacent to an intrastriatal injection of a much smaller dose (8 kg) of 6-OHDA than the one (40 pg) used in the present study. Although the reason for these discrepancies is not clear, they might be related to differences in length of animal survival after injection of 6-OHDA because we had found significant decreases in striatal Dl receptors in animals sacrificed two weeks after intracaudate injection of a similar dose of 6-OHDA (3). It is interesting to note that, despite the lack of DA depletion-related increases in the number of striatal DA Dl receptors, significant increases in Dl-stimulated adenyl cyclase have been found subsequent to lesions of the nigrostriatal system (16,23). These increases might be related to alteration in the receptorcoupling mechanisms in the basal ganglia. 02 receptors. The autoradiographic distribution of striatal D2 receptors is similar to that reported by other authors (2, 19, 20, 25, 28, 29). There are consistent high-to-low lateromedial gradients in the concentration of DA D2 receptors. In addition, the concentration of D2 receptors is higher in the ventral than in the dorsal subdivision of the lateral aspects of rat striata. This is the case in the medial hemisphere in the CPU. The distribu-

tion of the increases in D2 receptor is almost identical to that reported by other investigators (28,29) who had previously shown that lesions of the nigrostriatal DA system cause increases in DA D2 receptors mainly in the lateral aspects of the striatum. The present study also found that the percentage of increases in DA D2 was somewhat higher in the dorsolateral ( + 65%) than in the ventrolateral ( + 46%) subdivision of the lesioned striata. These changes are of special interest since they indicate that the lateral aspects of the caudate nucleus, which contain higher concentrations of DA uptake sites and DA D2 receptors under normal conditions, can undergo greater postsynaptic compensation than the medial aspects of the striatum. It is to be noted that DA D2 antagonists affect regional striatal dopamine turnover differentially, with the lateral aspects of the CPU showing greater DA turnover than the medial hemisphere (34). Although the reason for these compartmentalized reactions of DA D2 receptors to DA depletion or to DA receptor blockade is not clear, it is of interest that the dorsolateral aspects of the caudate nucleus which show the highest percentage of increases in D2 receptors are known to receive bilateral innervation of glutamatergic afferents from the motor cortex (10). It is thus possible that these pathways might have significant modulatory effects on D2 receptors located on striatal cell bodies or dendrites. Although this conjecture is not consistent with reports that DA D2 receptors are not localized on corticostriatal neurons (20,32), a somewhat regionally specific modulatory phenomenon has been reported in the supersensitivity observed in Dl sensitive adenyl cyclase activity which occurs after SNpc lesions ( 16). Furthermore, the findings that striatal afferents from the cortex and from the substantia nigra synapse on the same dendrites of intrastriatal neurons (5) provide partial support for this argument. In any case, the elucidation of these interesting phenomena will depend on future experiments. Conclusion In summary, the present experiments present both behavioral and biochemical data on the effects of intrastriatal injection of 6-OHDA into rat striata. First, we show that intrastriatal lesions cause stable rotational behavior. We also demonstrate that intrastriatal injection of 6-OHDA results in depletion of dopamine uptake sites and in changes in DA Dl and D2 receptors which are similar to those observed after intranigral lesions. The use of intrastriatal lesions might be of special interest in the study of retrograde changes in the SNpc resulting from lesions of the caudate-putamen. This may be of clinical relevance because one hypothesis regarding the etiology of Parkinson’s disease suggests that free radicals formed during the metabolism of dopamine in nerve terminals might play a causative role in the degenerative process observed in the brains of these patients (9). ACKNOWLEDGEMENTS

Reuben Last was supported by a Student Summer Fellowship

under

a NIH Short-term Training Grant HL 07616 to Columbia University. Vladimir Kostic is supporteh by a Fulbright Fellowship. Serge Przedborski is a Research Assistant of the National Fund for Scientific Research (Belgium) and is also supported by the Parkinson’s disease Foundation.

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6-OHDA, STRIATUM, BEHAVIOR AND BIOCHEMISTRY

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21.

22.

23.

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28.

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