Cobalt infections into the substantia nigra of the rat: Effects on behavior and dopamine metabolism in the striatum

EXPERIMENTAL

58, 486-499 (1978)

NEUROLOGY

Cobalt Injections on Behavior

into and

the Substantia Dopamine

Nigra

Metabolism

of the Rat:

Effects

in the Striatum

MASATO SHIBUYA, RUCGERO FARIELLO, IRENE J. FARLEY, KATHLEEN S. PRICE, KENNETH G. LLOYD, AND OLEH HORNYKIEWICZ 1 Department

of Psychopharmacology, Clarke Institute of Psychiatry Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada Received

August

and

1,1977

Small amounts of cobalt unilaterally injected into the substantia nigra of rats caused a strong, spontaneous contralateral turning which was suppressed by haloperidol. One to 2 days after intranigral cobalt microinjection, damphetamine decreased the intensity of the contralateral turning; 5 days after cobalt application, d-amphetamine changed the direction of the turning to the ipsilateral side. Intranigral cobalt microinjection initially caused a significant increase in the concentration of dopamine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid in the ipsilateral striatum. Thereafter, dopamine and its metabolites declined progressively to values significantly below control values. Similar biochemical changes in the striatum were also seen after partial electrolytic lesions of the substantia nigra. However, such lesions produced only mild contralateral turning. Frontal hemisection did not result in any increase of striatal metabolite values, and produced mild ipsilateral turning. Thus, there was no parallelism between the intensity and time course of the turning behavior induced by the different techniques and the changes in dopamine metabolism in the striatum. Within the cobaltinjected substantia nigra, y-aminobutyric acid (GABA) and glutamic acid decarboxylase were decreased to less than 60% of the contralateral control Abbreviations : DA-dopamine ; SN-substantia nigra ; HVA-homovanillic acid ; DOPAC-3,4-dihydroxyphenylacetic acid; GABA--r-aminobutyric acid; ip-intraperitoneal. 1 Drs. Shibuya and Fariello are Fellows of the Medical Research Council of Canada. The present address of Dr. Shibuya is Department of Neurosurgery, Nagoya University School of Medicine, Nagoya, Japan; of Dr. Fariello is EEG Laboratory, Department of Neurology, University of Wisconsin, Madison, WI 53704; and of Dr. Hornykiewicz is Institute of Biochemical Pharmacology, University of Vienna, Vienna, Austria. 486 0014-4886/78/0583-0486$02.00/0 Copyright All rights

Q 1978 by Academic Press, Inc. of reproduction in any form reserved.

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values. The possibility is considered that the depression of a GABA-mediated inhibitory influence on the nigrostriatal or mesolimbic dopamine neurons may play a role in the cobalt-induced contralateral turning and altered striatal dopamine metabolism.

INTRODUCTION There is considerable experimental evidence suggesting that an imbalance of nigrostriatal dopaminergic activity causespostural asymmetry and turning in experimental animals. When dopamine (DA), or its agonist apomorphine, is unilaterally applied to the striatum in rodents, the animals assumean asymmetrical posture and turn to the contralateral side. On the other hand, injected intrastriatally, DA antagonists such as chlorpromazine and haloperidol cause ipsilateral turning (11, 4.5). Electrical stimulation of the nigrostriatal DA system causes contralateral turning (7), whereas its chemical or mechanical lesions cause ipsilateral turning either spontaneously,

upon

handling

of the

animal,

or after

systenlic

injection

of

amphetamine (4, 29, 44, 46, 51). J$‘hen degeneration of the nigrostriatal DA neurons is established, drugs which stimulate postsynaptic DA receptors (e.g., apomorphine or L-dopa) cause contralateral turning; this has been related to the development of DA receptor supersensitivity (47). Cobalt is an effective experimental epileptogenic agent when introduced in nervous structures with low convulsant threshold (10, 14, 25). However, when topically applied to seizure-resistant brain regions, cobalt mimics the effects of electrocal stimulation without inducing epileptiform phenomena (33, 36). While studying the possible influence of substantia nigra (SN) on generalized epileptiform discharges (Fariello and Hornykiewicz, unpublished data), it was observed that unilateral injection of a small amount of cobalt into the SN caused strong spontaneous contralateral turning in rats. The present report describes the characteristics of this cobalt-induced turning and the concomitant changes in the DA metabolism in the rat striatum. hIATERIALS

AND

METHODS

Operative Procedures. Male 200-g Wistar rats were pretreated with atropine methyl nitrate [O.l mg/kg, intraperitoneal (ip) ] and placed in a stereotaxic device while under sodium pentobarbital (60 “g/kg, ip) anesthesia. Cobalt chloride (CoC13.6 HZO) was dissolved in 0.9% sodium chloride. This solution (0.6 ~1) containing various amounts (0.4 to 25 pg; 2.8 to 175 111~) of cobalt (as salt) was injected into the left SN during 30 s using a lo-p1 Hamilton syringe. Stereotaxic coordinates according to

488

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ET

AL.

the atlas of Pellegrino and Cushman (37) (anterior-posterior, lateral, depth) were, for SN, -3, 2.2, 7.8, and, for corpus striatum, $2.0, 2.2, 5.0. Hemisections were done according to Bedard et al. (9) at the level of the posterior hypothalamus. Electrolytic lesions were made unilaterally in the SN at the same coordinates used for cobalt injection. A current of 2 mA was passed for 30 s through a stainless-steel electrode insulated except for 0.5 mm from the tip, using the ear bar as cathode. Behavioral and Pharwaucological Studies. At different time periods (6 and 12 h, 1, 2, 4, and 8 days) after the operative procedure rats were placed in a white rectangular observation box (35 X 45 x 16 cm) and the number of rotations in a 2-min interval was counted in duplicate. Those rats which showed less than 10 contralateral rotations per 2 min within the first 2 days after 25 pg cobalt were removed from the group. Haloperidol (Haldol, McNeil), 0.5 mg/kg, was injected ip in four rats, 6 h after injection of 25 pg cobalt. Rotations were counted at 0.5, 1, 2, 3, 4, and 24 h after haloperidol. d-Amphetamine was dissolved in water and injected (4 mg/kg, ip) 1, 2, 5, and 60 days after cobalt implantation in separate groups of four rats each. Rotations were counted 30 min after d-amphetamine. Biochemical Assays. Rats were killed by decapitation and the brains were removed quickly and frozen immediately on crushed dry ice. Placement of lesions was verified either at the time of brain removal or upon dissection of the frozen brain. Only those brains with satisfactory lesions were used for biochemical assays. Corpus striatum and SN were dissected from the frontal sections with the aid of the stereotaxic atlas. DA was measured in homogenates of whole striatum by a radio enzymatic assay (12) using S-adenosyl-L- [ wzethyZ-3H] methionine (specific activity 10 Ci/mmol, New England Nuclear). Sensitivity for DA (twice the blank value) was 50 pg. Homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured spectrofluorometrically in homogenates of whole striatum using modifications of the method of Murphy et al. (32) and And& et al. (3). Recovery for both HVA and DOPAC was approximately 70%. Results were corrected for the recovery. L-Glutamic acid decarboxylase was assayed from homogenates of each SN by a slight modification of the method of Albers and Brady (2) using DL- [ l-%1 glutamic acid (50 mCi/mmol, New England Nuclear). Substrate concentration of L-glutamic acid was was measured in homogenates of 11.5 mM. y-Aminobutyric acid (GABA) SN by the method of Enna and Snyder (16) using [2,3-3H(N) ] GABA (40 Ci/mmol, New England Nuclear). Protein was measured according to Lowry et al. (28). The statistical evaluation was done by the two-tailed t-test.

COBALT

EFFECT

IN

SUBSTANTIA

NIGRA

489

RESULTS Turning Behuvior Strong spontaneous contralateral turning in a tight circle was observed after unilateral cobalt injection into the SN. The dose-response curve is shown in Fig. 1. No effect was seen with 0.4 or 2 pg (2.8 or 14mM) but with 4pg (< 28 mM) or more all animals showed contralateral turning. The number of rotations increased in a dose-dependent manner up to 25 pg. At the maximal dose of 25 pg the animals showed a repetitious flexing of the neck to the intact side during recovery and started to turn to the intact side as soon as they recovered from anesthesia. At about 6 h after cobalt injection the number of rotations reached its maximum, remaining at this level for 48 h. During this period rats showed increased sniffing with constant nodding of the head and mild gnawing on the intact side. Flexion of the contralateral forelimb [as described by Arbuthnott and Ungerstedt (8) ] was a regular feature. After 2 days following intranigral cobalt (Fig. 2), the number of the contralateral rotations started to decline progressively. At no time period during the experiments was spontaneousipsilateral turning observed. In contrast to marked contralateral turning after cobalt implantation, only mild contralateral turning was seen 6 h after electrolytic lesion in the unilateral SN (contralateral turning = 5.2 * 1.3/2 min vs. ipsilateral turning = 1.0 -+ 0.4/2 min, mean f SE, N = 17). Whereas 6

40

Cobaltpg Injected Unilaterally into the Substantia nigra I?IG. 1. Dose-response curve for cobalt on contralateral turning; 0.6 ~1 0.9% NaCl solution containing different amounts of cobalt (salt) was injected into the left substantia nigra and turning was counted 6 h later. Mean f SE of four rats at each dose.

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ET

AL.

0

days Time After Cobalt injection

FIG. 2. Time course of contralateral SE of at least four rats at each time.

turning after injection

of 25 pg cobalt. Mean -t

h after frontal hemisection rats showed mild ipsilateral turning (ipsilateral turning = 7.0 * 0.3/Z min vs. contralateral turning =O, N = 4). Efect of Dogs on Cobailt-Induced Turning One day after cobalt injection, d-amphetamine slightly decreased contraturning (Fig. 3). Two days after cobalt, d-amphetamine markedly reduced the contralateral rotation and a few ipsilateral rotations appeared in three of the four rats. At 5 days after cobalt injection, d-amphetamine changed the main direction of turning to the ipsilateral side in all four rats (separate groups for each time period). The intensity of the ipsilateral turning following d-amphetamine increased with the length of time after cobalt injection (longest observation period, 2 months). The effect of haloperidol on cobalt-induced turning is shown in Table 1. One hour after administration, haloperidol significantly decreased the number of contralateral rotations. The number of rotations returned to control values 24 h after baloperidol. To study cobalt’s effects on the striatal terminals of DA neurons, a larger dose of cobalt (7.5 fig in 1.5 pl 0.9% NaCl) was injected unilaterally into the corpus striatum. All of five rats showed very mild ipsilateral turning, which was apparent only during the 24-h period immediately following the intrastriatal injeclateral

COBALT

EFFECT

IN

SUBSTANTIA

491

NIGRA

2

2 lom &I f;; .= z

i-J-----J+ 11

2

5

60 day;

Time After Cobalt Injection FIG. 3. Effects of ~-amphetamine (4 mg/kg, ip) at different times after cobalt injection. Turning was counted before (open bars) and 30 min after administration of amphetamine (hatched bars). Mean * SE of separate groups of four rats each.

tion (ipsilateral turning, 0.5 to 3/2 min ; contralateral turning, 0 to I/2 mill). Metabolic Changes Dopamine and Mefabolites. Six hours after cobalt injection into the substantia nigra, DA, DOPAC, and HVA concentrations increased signiticantly (I’ < 0.01, < 0.05, and < 0.001, respectively) to 150, 2.50, and 440% of the control side, respectively (Table 2). These increases were still significant at 12 h. However, 24 h after cobalt injection DA concentrations had declined to the range of control values. Two days after cobalt TABLE Effect of Haloperidol

on Cobalt-Induced

ControI period

Hours 0.5

Number of contralateral turns/2 min

28 f4

a Results expressed as mean 3s~. * P values: p)
1

15 * 4 (NSF

p)

<0.005;

after 1

11 f.5”

(NS)

Turning H~o~ridDl, 2

(N = 4)a 0.5 mg/kg, 3

9 f4b

7 *2c

not significantly

different

ip 24

4

7 f2G

from control.

29 f

2 (NS)

(8) (4) (3) (3) (4) (3)

DA

TABLE

f f f f f zt

Left

0.91 (9) 1.09 (6) 1.64 (3) 1.34 (4) 0.25 (4) 0.36 (3)

0.97 rt 0.11 1.28 f 0.04 1.27 f 0.08 nd 1.23 f 0.16 nd

Right

Not

determined.

0.33a

(4)

2

0.40 0.45 0.29 nd f 0.02 nd

Left

2.40’1 f 2.76= f 2.130 f

ha/g)

(4) (5) (9)

DOPAC

and HVA in the Striatum and L-Glutamic Substantia Nigra after Intranigral

Number of rats in parentheses. nd, (9
12.76~.* 13.23* 11.65 7.45s 2.50’ 1.72~

be/d

DOPAC,

a Results expressed as mean &SE. *P values: (9
0.31 0.67 0.31 0.24 0.72 2.38

f f f f f f

6h 12 h 24 h 2 Days 4 Days 8 Days

8.43 8.50 11.47 12.21 11.44 12.11

Right

Time after cobalt injection

DA,

(4)

(4) (5) (9)

0.57 0.67 0.60 0.57 0.39 0.37

f 0.06 3~0.05 f 0.05 f0.12 f 0.06 * 0.11

Right

(12) (13) (15) (5) (8) (5)

HVA

Acid Decarboxylase (Left) Cobalt Injection’

2.50’ 2.48’ 1.49’ 0.54 0.220 0.23

(rrg/d

(GAD)

* f f f * zt

0.23 0.25 0.18 0.21 0.03 0.08

Left

Activity

(12) (13) (15) (5) (8) (5)

175 173 243 214 262 246

f f f f zk f

Right 16 14 23 23 44 23

GAD Activity of protein/30

in the

f

l * * f f

100

100 79’ 59d 520 4ld

Left

19 14 14 11

40

30

(nmol/mg min, N = 3)

z

2

8

8

i tf rt: f

0.85 0.49 0.53 1.53

10.09 IO.82

6.38 8.21 9.72 9.44

Control

(3) (5) (4) (3)

0.89 0.83 0.55 1.11

13.32 (NS) j, 1.40

f f f; *

Lesion

12.0.5b** 11.06” 9.46 (NS) 3.68*

DA b%w

(3) (6) (44) (3)

f 0.09 (3) nd f 0.61 (4) f 0.1‘5 (3)

Lesion

1.33 (NS) f 0.04

(N = 4)

B. Hemisection 1.37 zk 0.09

2.95= 0.53”

3.w

lesion

(w’d

0.74 f 0.08 (3) nd 1.08 f 0.09 (4) 1.15 f 0.13 (3)

A. Electrolytic

Control

DX’AC

f * f $;

0.07 0.55 0.04 0.06 0.60 f 0.09

0.50 0.44 0.51 0.42

Control

* Results expressed as mean &SE. Number of rats in parentheses. nd, Not determined. * P values: (a) < 0.05; (&) < 0.02; (“1 < 0.005 ; (3 < 0.001; (NS) not significantly different from control.

6h

6h 12 h 24 h 4 Days

Time after surgicai procedure

(3) (4) (5) (3)

f f * i

0.38 0.29 0.26 0.03 OS3 (NS) f 0.07

3.590 2.31c 2.06d 0.17”

Lesion

Nigra and

E-EVA ha/g)

TABLE 3 DA, DOPAC, and HVA in the Striatum after (A) Partial Xi&lateral Electrolytic Lesions of the Substantia (B) Hemisection at the Level of the Posterior Hypothalamus”

(3) (5) (6) (3)

2 6” m $ z :! * z n” $

8 Mr E:

8 5r $

494

SHIBUYA

ET

AL.

implantation the DA had decreased significantly (P < 0.05) to less than normal values. In contrast to DA, the increase in striatal HVA and DOPAC persisted markedly longer, at least 24 h after cobalt; at 2 days the concentrations of HVA had declined to control range. Both HVA and DOPAC were below normal values only by Day 4. The ratios DOPAC: DA and HVA: DA in the striatum were at all times higher on the cobalt-treated side. Analogous but greater increases in striatal DA and its metabolites were seen 6 to 12 h after electrolytic lesion of the SN. Also the time course of the delayed (1 to 4 days) decline in these parameters was similar between the cobalt-injected and electrolytic-lesion animals (Tables 2 and 3). The electrolytic lesion was considered to be partial or incomplete, as both (i) 40% of striatal DA was still present 4 days postlesion ; and (ii) morphological examination of the SN indicated incomplete destruction. Striatal DA, DOPAC, and HVA concentrations were measured 6 h after complete hemisection, as shown in Table 3. In contrast to the increase in DA metabolites after cobalt injection into, or a partial electrolytic lesion of, the SN, after hemisection there was no increase in either DOPAC or HVA. DA increased to 130% of the control side without reaching statistical significance. GABA. L-Glutamic acid decarboxylase activity in the ipsilateral SN decreased to about 60% of the intact side at 6 and 12 h after cobalt injection. When the results of these two time periods were combined the decrease was statistically significant (P < 0.02). After 1 day, L-glutamic acid decarboxylase activity in the cobalt side was significantly lower and remained so until at least the eighth day (Table 2). The GABA concentrations in the SN of rats injected with cobalt 6 h to 4 days previously were combined as a group, as the number of rats in each time period was small. GABA decreased significantly from 62.39 f 8.07 nmolJmg of protein (control side, 12 rats)to 17.44 -C 7.9lnmol/mg of protein (ipsilateral SN, 9 rats,

P < 0.005). DISCUSSION Relatively small amounts of cobalt (4 to 25 pg, 28 to 175 mM) unilaterally injected into the SN caused spontaneous contralateral turning in rats. The intensity of the cobalt-induced turning (Fig. 1) is the strongest ever reported in the literature. Turning is commonly explained as due to predominance of DA activity in one striatum, the direction of turning being away from the absolutely or relatively hyperfunctioning side. The extraordinary increase in DA and its metabolites in the first 6 to 24 h after cobalt is in agreement with this hypothesis. However, lesions in the ascending DA system also induce early increase in DA in the ipsilateral neostriatum (5). DA synthesis in the striatum is in fact, at least partially,

COBALT

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modulated by nerve impulse flow in the ascending DA pathway. When impulse flow is stopped by either hemisection (5, 22), y-hydroxybutyrate (20, 42, 48), or electrothermic lesions (49)) initially there are both increased endogenous activities and synthesis of striatal DA. However, DA catabolism does not increase, as evidenced by the finding that DOPAC decreased 30 min after lesion (40). Striatal DA synthesis can also be activated by electrical stimulation of the DA ascending pathway. The two mechanisms of activation are different, as shown by the facts that only the activation following cessation of impulse flow can be reversed by addition of calcium to the incubation medium (31) and that striatal DOPAC (40) and HVA (26) are increased in a frequency- and/or intensity-dependent manner after electrical stimulation. Other major differences exist between our model and lesions of the DA ascending system as induced, for instance, by intranigral injection of 6-hydroxydopamine ; in the latter no stereotypy occurs and contralateral turning is only temporarily seen upon nialamide (a monoaminoxidase inhibitor) administration (44, 46). Further, the effects of haloperidol and d-amphetamine injection are different in the two experimental situations, and in our experiment would be rather consistent with the hypothesis of DA hyperactivity in the ipsilateral striatum. These data further support the postulate that turning in cobalt-treated rats is due to chemical stimulation of the ascending DA system by cobalt. The increase in DA metabolites after cobalt might not be due exclusively to a direct stimulating effect but could also be caused by hyperactivity of the remaining DA neurons; in this respect, partial degeneration in the nigral neurons was histologically observed after 48 h (Fariello and Hornykiewicz, unpublished data) and functionally might have taken place at an earlier time. This possibility is supported by the increase in striatal DOPAC and HVA after partial electrolytic lesion of the SN (Table 3) and also by the reported increase in DA synthesis and turnover in the remaining neurons after application of small amounts of 6-hydroxydopamine in the SN (1). The possibility of the cobalt-induced changes being the result of a combination of its stimulating and damaging effects on the SN seems to offer a more complete explanation for the quantitative aspects of both the observed biochemical changes and the turning behavior. In this respect it may be relevant that contralateral turning behavior has been reported after partial electrolytic lesions of the SN (41). Our results, especially those with cobalt, show that there was no close correlation between the degree of increase in DA metabolites (or the ratio of these to DA) in the striatum and the rate of contralateral turning: Actually contralateral turning persisted long after DA, HVA, and DOPAC values

496

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were well below control values. The above considerations make it doubtful that the turning behavior elicited by intranigral cobalt is due solely to an imbalance of striatal DA. In this respect other factors such as the participation of the mesolimbic DA (13, 30, 38) and/or the GABA system should be considered. There is extensive documentation in the literature on the existence of a GABA-mediated inhibition of the nigrostriatal (6, 17, 18, 21, 23, 35, 39, 53) an d mesolimbic (19) DA systems. The observed decrease in L-glutamic acid decarboxylase activity and GABA concentrations in the SN after cobalt implantation may therefore, at least partially, account for the increased neostriatal DA activity. There is additional evidence that GABA neurons are involved in the rotation phenomenon: After systemic amphetamine or apomorphine, intranigral application of ethanolamine-osulfate (which increases GABA concentrations) causes ipsilateral turning (15) ; furthermore picrotoxin (a GABA antagonist) induces contralateral turning in rats (43). Having used a cobalt concentration from 28 to 175 mM, we postulate that at the periphery of the injection site there was a large adjacent region in which, due to diffusion gradient, cobalt was still able to inhibit L-glutamic acid decarboxylase activity. In this respect Wu and Roberts (52) showed that 10 mM cobalt inhibited 8070 of L-glutamic acid decarboxylase activity in vitro. In addition to the above-proposed mechanisms of action, other effects of cobalt on neuronal activity should be considered. Cobalt is known to change peripheral release of acetylcholine (24, 50)) and glutamic acid, glycine, and taurine are altered, in cobalt-induced cortical epileptic foci (27). Nichols and Nakajima (34) have reported that cobalt inhibited the generation of inhibitory postsynaptic potentials in crustacean stretch receptor neurons, probably affecting the coupling between excitation and GABA secretion at presynaptic level. Relevance of these effects in the mechanisms of contralateral turning after cobalt is not clear at present but they might have contributed to the complexity of the observed biochemical and behavioral alterations. In conclusion, we demonstrated that unilateral injection of small amounts of cobalt into the SN produces a typical contralateral turning behavior in rats that is accompanied by an initial increase and, subsequently, a decrease in DA, DOPAC, and HVA concentrations in the ipsilateral striatum. However, our results show that the hypothesis of turning behavior as a simple consequence of striatal DA imbalance does not account for all the features, especially the quantitative aspects, of this complex motor response. REFERENCES 1. Acm, Y., F. JAVOY, AND J. GL~WINSKI. 1973. Hyperactivity of remaining dopaminergic newones after partial destruction of the nigrostriatal dopaminergic system in the rat. Nature (London) New Bid. 245: 150-151.

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