- Email: [email protected]
Effect of bilateral 6-OHDA lesions of the substantia nigra on locomotor activity in the rat
BRAIN RESEARCH ELSEVIER
Brain Research 633 (1994) 144-150
Research Report
Effect of bilateral 6 - O H D A lesions of the substantia nigra on locomotor activity in the rat Kyoji Sakai a,b, Don M. Gash a,, '~ Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA h Department of Neurological Surgery, Okayama University Medical School, Okayama 700, Japan
(Accepted 27 August 1993)
Abstract Previous parkinsonian rat models have utilized stereotactic 6-OHDA injections to completely lesion the dopaminergic mesostriatal system on one side. Recently, hemiparkinsonian rat models in which the mesolimbic system is left intact have been developed. The selective, partial lesion models better mimic the neuropathology of human parkinsonism in which there is usually an incomplete destruction of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and a relative sparing of ventral tegmental area (VTA) cell groups. However, such hemiparkinsonian models which possess dopaminergic asymmetry cannot demonstrate bradykinesia, one of the main symptoms in human parkinsonism. Meanwhile, bilateral lesions of the ascending forebrain dopaminergic system have been reported to induce severe aphagia, adipsia and akinesia. We, therefore, undertook development of a bilateral partial SNpc lesion model which also spares the VTA on both sides. We have investigated spontaneous locomotor activities as well as amphetamine, apomorphine and levodopa induced activities during a subchronic period of up to 27 days after the bilateral lesion. Three activity parameters, i.e. horizontal activity, vertical activity and distance traveled, were measured. The relationship between dopamine neuron loss in the SNpc and changes in the locomotor activity was analyzed. Spontaneous activity was significantly decreased in animals with extensive (> 80%) SNpc lesions on both sides, Animals with a > 95% lesion were severely aphagia and adipsia. Responses to amphetamine and apomorphine were variable. It is possible that in some cases the bilateral SNpc neurons were not equally damaged, which could cause the enhanced rotational behavior. Bradykinetic rats displayed on the average, a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled after the lesion. These bradykinetic rats consistently showed a marked increase in activity in response to levodopa therapy as observed in human parkinsonian patients. The results indicate that rats with bilateral partial lesions of the SNpc may be useful for the evaluation of new therapeutic approaches for treating Parkinson's disease. Key words: Parkinsonism; Bilateral lesion; 6-Hydroxydopamine; Substantia nigra; Activity; Rat
I. Introduction Parkinson's disease is a progressive n e u r o d e g e n e r a tive disorder characterized by the m o t o r disturbances such as bradykinesia, muscular rigidity and resting tremor. T h e principal lesion is the progressive loss of dopaminergic neurons in the substantia nigra pars c o m p a c t a (SNpc), that results in a p r o f o u n d d o p a m i n e deficiency in the nigrostriatal system [2,13]. T h e clinical
* Corresponding author. Present address: Department of Anatomy and Neurobiology, University of Kentucky, College of Medicine, MN 224 Chandler Medical Center, Lexington, KY 40536-0084, USA. Fax: (1) (606) 258-5946. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E1174-2
parkinsonian symptoms typically are not manifest until 60 to 80% of the d o p a m i n e input to the striatum is lost [2,13-15]. Previous parkinsonian rat models have been characterized by stereotactic 6-hydroxydopamine ( 6 - O H D A ) injections into the unilateral dopaminergic mesostriatal system, which destroy virtually all nigral dopaminergic neurons. Perese et al. [18] have developed a hemiparkinsonian rat model in which the mesolimbic system is left intact. C a r m a n et al. [4] have also developed a similar partial 6 - O H D A lesion model. Both selective, partial lesion models better mimic the n e u r o p a t h o l o g y of h u m a n parkinsonism in which there is usually an incomplete destruction of dopaminergic neurons in the SNpc and a relative sparing of the ventral tegmental
K. Sakai, D.M. G a s h / B r a i n Research 633 (1994) 144-150
area (VTA) cell groups [10,11]. Moreover, such selective, partial lesion models may prove to be useful for examining the reported sprouting phenomenon of host dopaminergic neurons in response to neural or paraneural grafting [1,3,7]. While the inherent dopaminergic asymmetry in hemiparkinsonian models allows lesion quantification by drug induced rotation behavior, animals with unilateral lesions do not demonstrate bradykinesia, one of the main symptoms in human parkinsonism. The parkinsonian tremor and rigidity are considered to require extrapyramidal lesions in addition to the striatal dopamine depletion [16,21]. Although some patients with idiopathic parkinsonism may first present with unilateral symptoms, the disease essentially progresses to bilateral motor disturbances. We, therefore, undertook development of a bilateral parkinsonian model in the rat using 6-OHDA to lesion the SNpc on both sides.
2. Materials and methods 2.1. Animals Animals used in this experiment were 28 adult male Fisher-344 rats (Charles River, MA) weighing 245-285 g at the start of experiment. They were randomly divided into 2 groups: twenty animals which received partial bilateral 6-OHDA lesions and eight control (sham-operated) rats. They were maintained in a 12-h light/dark cycle (light on 06.00 h; light off 18.00 h) with food and water available ad libitum. Our vivarium was fully accredited by the American Association for Accrediation of Laboratory Animal Care, and in compliance with the state law, federal statutes and the policy of National Institutes of Health.
2.2. Partial, bilateral 6-hydroxydopamine lesion Animals were deeply anesthetized with chloral hydrate-sodium pentobarbital (0.35 ml/100 g b.wt., i.p.) and placed in a stereotactic frame (David Kopf Instruments, CA) with the tooth bar 3.3 mm below horizontal. Lesioned animals received an injection of 4 /zg 6-hydroxydopamine hydrobromide (6-OHDA, Sigma, MO) diluted in 1 /zl saline containing 0.2% ascorbic acid into each SNpc, using a Hamilton 10 /zl syringe with a 26-gauge blunt tapered needle, at a rate of 0.2 ~l/min. Both injections were made with the needle bevel directed rostrally at the following coordinates: anterior - 5 . 4 mm, lateral 2.2 mm, dorsoventral 7.5 mm with respect to bregma and dura, according to Paxinos and Watson [17]. At the completion of each injection, the needle was left in place for 5 min and then withdrawn at a rate of 1 mm/min. All surgeries were carried out under sterile conditions. Animals in the control group received 1 /~1 injections of normal saline instead of 6-OHDA in the manner described above.
2.3. Postoperative care Following the bilateral 6-OHDA lesion, the rats were provided daily with a moist food supplement consisting of rat chow, glucose and water. Animals that were aphagic and adipsic following surgery received an additional supplement via intragastric tube of a high energy diet (Nutri-Cal, Evsco Pharmaceuticals, NJ) diluted 1:5 in water until they could eat on their own.
145
2.4. Spontaneous activity test Spontaneous locomotor activity was measured during the animal's normal nocturnal period (18.00 to 06.00 h). The activity tests were conducted at 8 days and 1 day prior to surgery. Animals were tested again at 20 days and 27 days after surgery. We used an animal activity monitoring system (Digiscan-16, Omnitech Electronics, OH) to quantify the locomotor activity. Two pairs of 16x16 arrayed infrared horizontal photosensors were attached to the animal cage (42 x 42 × 30 cm) equally spaced 2 cm above the floor, and one pair of 16 vertical sensors were attached 12 cm above the floor. The lower row of photosensors was used to measure the horizontal locomotor activity and the upper row to detect the vertical activity such as rearing or jumping behavior. 2.5. Induced activity test All animals were also tested for locomotor activity induced by 5 mg/kg De-amphetamine sulfate (dissolved to 5 mg/ml in sterile saline, i.p.; Sigma) and 0.05 mg/kg apomorphine hydrochloride (dissolved to 0.05 mg/ml in sterile saline, s.c.; Sigma), respectively, at the first week of this experiment (presurgery) and at the 4th week following surgery. Moreover, representative animals received methyl levodopa (dissolved in sterile saline, 0.1 ml/100 g b.wt., i.p.; Sigma). Three dose levels (0 (placebo), 50 and 100 mg/kg) were tested after the last 12-h spontaneous activity test had been completed. These induced activity tests were conducted only between 09.30 and 14.30 h with a minimum of 48 h between drugs. These induced activities were counted for a 90-min period after the drug injection. All activity tests, i.e. spontaneous activity tests, amphetamine, apomorphine and levodopa induced activity tests, were performed in a dark and quiet isolated room. Animals had free access to water but not food during activity testing. 2.6. Tyrosine hydroxylase immunocytochemistry Animals were sacrificed 28 or 36 days after surgery. Rats were anesthetized with an overdose of sodium pentobarbital and perfused with heparinized saline followed by 4% paraformaldehyde solution in 50 mM potassium phosphate buffered saline (KPBS). Brains were removed and postfixed for 24 h in the perfusion fixative at 4°C and transferred to 30% sucrose in 50 mM KPBS for an additional 72 h at 4°C. Serial coronal frozen sections of 30 ~m thickness were cut on a sliding microtome. Six sets of slices were collected in cryoprotective solution and stored at -20°C until immunocytochemically stained. Every sixth section was processed for the visualization of tyrosine hydroxylase (TH)-immunoreactive (IR) profiles using procedures previously described with minor modifications [7]. Sections were exposed to the primary anti-TH antibodies (1:1,000; BoehringerMannheim, Germany) and incubated in biotinylated horse anti-mouse secondary antibodies (1:1,500; Vector, CA). Sections were then incubated in the avidin-biotin-peroxidase complex using the Elite ABC Vectastain Kit (Vector, CA). TH-immunoreactivity was visualized using 3,3'-diaminobenzidine as the chromogen with nickel enhancement.
2. 7. Analysis of TH-IR neurons and fibers The total number of TH-IR neurons in the SNpc and VTA was counted on both sides at 3 levels: - 5.0 mm, - 5.4 mm and - 5.8 mm with respect to bregma. The degree of dopamine depletion was determined by the loss of TH-IR SNpc neurons in individual lesioned animal relative to the average number of TH-IR SNpc neurons in sham-operated (control) animals, i.e. (control-lesion)/ control X 100. The percentage area of the striatum occupied with TH-IR fibers was measured on both sides at 3 levels: + 1.2 mm, +0.5 mm and - 0 . 2 mm with respect to bregma. The sections were analyzed at 20 x magnification with an Olympus Cue 2 system (Olympus, NY).
146
KI Sakai, D.MI Gash/Brain Research 633 (1994) 144-1.50
The measured striatum was delinated by the corpus callosum, lateral ventricle and the horizontal line above anterior commissure.
reals and two o t h e r bilaterally lesioned rats out of a total of 19 surviving bilaterally lesioned rats (47%) exhibited bradykinesia, flexed posture a n d gait disturbance. T h e affected rats m a i n t a i n e d a h u n c h e d back p o s t u r e and walked in short steps with evident limb rigidity, especially in the hind legs. N o n e of control a n i m a l s d e m o n s t r a t e d hypophagia, hypodipsia or bradykinesia.
2. 8. Statistical analysis The experimental data were statistically analyzed by means of an analysis of variance (ANOVA) and t-test. Results were provided as means _+S.E.M. Correlations were determined by linear regression analysis and determination of Pearson's correlation coefficient.
3.2. T H i m m u n o c y t o c h e m i s t r y I n the control rats (n = 8), d a m a g e to SNpc n e u r o n s a p p e a r e d to be m i n i m a l a n d primarily consisted of m a c r o p h a g e infiltration in a n d a r o u n d the saline injection sites. T h e m e a n n u m b e r of T H - I R n e u r o n s c o u n t e d at the t h r e e levels in the SNpc and V T A on both sides was 1,138 + 52 and 1,315 + 25, respectively. In the bilaterally lesioned rats (n = 19), T H - I R n e u r o n s in the V T A were relatively spared ( d e p l e t i o n s of 10-50%). A subtle dorsolateral to v e n t r o m e d i a l g r a d i e n t loss of
3. Results 3.1. General condition T e n out of 20 bilateral 6 - O H D A lesioned rats (50%) showed hypophagia a n d hypodipsia to various degrees. T h e s e a n i m a l s were tube fed 5 - 2 0 ml of high energy liquid per day for 3 - 8 days following surgery. Except for o n e a n i m a l that was severely aphagic a n d adipsic a n d died, all affected rats gradually r e s u m e d eating a n d drinking. Seven of the hypophagic, hypodipsic ani-
I.
Fig. 1. TH immunocytochemistry.A: the mesencephalon of a sham-operated (control) rat. Minimal damage was seen to substantia nigra pars compacta (SNpc) neurons. Macrophage infiltration was evident around the saline injection sites. B: the striatum of a control rat showing normal TH immunoreactivity. C: the mesencephalon of a rat with a 90% lesion of SNpc; the ventral tegmental area (VTA) and the medial SNpc is relatively spared. D: the striatum of a rat with a 90% lesion of SNpc. The nucleus accumbens and olfactory tubercle, which receive dopaminergic fibers from the VTA, were relatively left intact on both sides.
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150 Table 1 Control animals and 4 subgroups of animals with bilateral 6 - O H D A lesions into the SNpc Group
n
Control 20-80% 80-90% 90-95% > 95 %
8 5 3 8 3
SNpc SNpc SNpc SNpc
lesion lesion lesion lesion
200 ontal Activity cal Activity Lnce Traveled
No. of T H - I R neurons SNpc
VTA
1138 + 52 622_+59 180 + 28 96+ 5 30 _+ 12
1315 + 25 1229_+99 939 + 63 837+66 693 _+21
Values shown represent the m e a n s +_S.E.M. The n u m b e r of T H - I R neurons in the SNpc and V T A was counted on both sides at 3 levels as described in the text. T h e degree of dopamine depletion was determined by the loss of T H - I R SNpc neurons in the individual lesioned animal relative to the average n u m b e r of T H - I R SNpc neurons in controls.
T H - I R fibers was observed in the denervated striatum. The nucleus accumbens and olfactory tubercle were left relatively intact (Fig. 1). In both the lesioned rats and controls, the percetage of striatum innervated by T H - I R fibers was strongly correlated with the number of T H - I R neurons in the SNpc (r 2 = 0.81, P < 0.0001). The lesioned rats were subdivided into 4 subgroups: 20-80% depletion in the SNpc on both sides (n = 5); 80-90% depletion (n = 3); 90-95% depletion (n = 8); and > 95% depletion (n = 3) as shown in Table 1.
a00
4
147
~~ Q"
150
oe
o e
O =-
e~
100
o
i
"
¢:m
==; o~.
50
Control 20-80% 80-90% 90-95%
>95%
Group (% Depletion of the SNp¢) Fig. 3. Change of spontaneous locomotor activity between pre- and postsurgery in the control group and 4 lesion subgroups. Three activity parameters, i.e. horizontal activity, vertical activity and distance traveled were measured for 12 h at 8 days and 1 day presurgery and at 20 days and 27 days postsurgery. In animals with a > 80% lesion, the percent changes of 3 activity parameters were significantly lower than in controls. In animals with a > 95% lesion, the percent change of vertical activity was greatly reduced relative to in controls. * P < 0.05, ** P < 0.01 and * * * P < 0.001, significantly different from the control group.
3.3. Body weight The mean body weights of each group during this experiment are shown in Fig. 2. Animals with a > 95% lesion were severely aphagic and adipsic, and exhibited a more marked weight loss. However, even animals with a > 95% lesion were able to increase their body weight from 2 weeks postsurgery.
200
-t •~
0 m
150
lesion -o- 80-90% lesion -*-90-95% lesion -o- >95% lesion
-i-
100 -
50
Control
-~-
3.4. Spontaneous locomotor activity
20-80%
--
i
0
I -10
0
T
I
'
10
I 20
30
Days Poateurgery Fig. 2. M e a n body weights of the control group and 4 lesion subgroups during pre- and postsurgery periods. Animals with a > 95% lesion were severely aphagic and adipsic, and showed a more marked weight loss. * P < 0.05, significantly different from the control group and 3 other lesion subgroups.
The lesioned animals showed a moderate correlation between the degree of SNpc lesion and the percent changes of horizontal activity, vertical activity and distance traveled as measured by comparing between pre- and postsurgery (r 2 = 0.42, 0.51, 0.45, P < 0.005, 0.001, 0.005, respectively). In most animals with a > 80% lesion, horizontal activity, vertical activity and distance traveled were significantly reduced compared to controls. In animals with a > 95% lesion, the percent change of vertical activity was greatly reduced relative to in controls ( P < 0.001) (Fig. 3). All 3 animals with a > 95% lesion, 5 out of 8 animals with a 90-95% lesion and 1 out of 3 animals with an 80-90% lesion showed much less spontaneous activity than controls.
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150
148
Twenty to 27 days following the bilateral lesions, these bradykinetic animals (n = 9) exhibited the levels of < 80% in holizontal activity, < 60% in vertical activity and < 70% in distance traveled relative to preoperative values.
25000
f
' : Control animals Nonbradykinetic animals
[] Bradykinatic animals 20000
-i
15000
i
10000
i
5000
-
A .n
3.5. Amphetamine and apomorphine induced actiuities The lesioned animals showed a p o o r correlation between the degree of SNpc lesion and the percent changes of a m p h e t a m i n e and a p o m o r p h i n e induced activities (r 2 = 0.0015, 0.10, P = 0.87, 0.18, respectively). In the groups with 2 0 - 8 0 % lesions and 9 0 - 9 5 % lesions, the percent c h a n g e of a m p h e t a m i n e induced activity was significantly higher than in controls ( P < 0.05. P < 0.001, respectively). Only in 2 animals with a 98% lesion, was a m p h e t a m i n e induced activity remarkably reduced ( - 8 8 % , - 7 9 % ) c o m p a r e d to in all other animals including controls. Meanwhile, no statistical difference from controls in the percent change of apom o r p h i n e induced activity was found in any of the lesion subgroups (Fig. 4).
3.6. Levodopa response T h e dose response to levodopa was m e a s u r e d in 7 bradykinetic lesioned animals, 5 non-bradykinetic le-
1400 t 1200
,
Amphetamine
[] Apomorphlne 1
"L°~ 1000
E g v
_e 0 m I--
0
0
- ~
0
-
~
...... 50
100
Methyl Levodope (mg/kg)
Fig. 5. Response to levodopa administration in the bradykinetic lesioned animals, the nonbradykinetic lesioned animals and controls. Bradykinetic rats are the animals which meet the criteria "'a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled as measured in the spontaneous activity test following surgery". They received methyl levodopa at 3 dose levels (0, 50, 100 mg/kg) 30-34 days postsurgery. The distance traveled, one of 3 activity parameters, was measured for 90 min. The baseline activity to saline injection was significantly lower in the bradykinetic animals than in both the nonbradykinetic animals and controls. Only the bradykinetic animals consistently demonstrated a marked increase in activity in response to levodopa administration. * P < 0.05, significantly different from the other 2 groups. P < 0.05 significantly different from the saline injection.
800 600
,
O~ID ilIO
o g
400
ae~
!
200 -
* i
T
J-
Control 20-80% 80-90% 90-95%
>95%
Group (% DepletIon of the $Npc)
Fig. 4. Changes of a m p h e t a m i n e and apomorphine induced activities between pre- and postsurgery in the control group and 4 lesion subgroups. The distance traveled, one of 3 activity parameters, was measured for 90 min pre- and postsurgery. Percent change of amphetamine induced activity was significantly higher in animals with a 20-80% lesion and ones with a 9 0 - 9 5 % lesion than in controls. Each lesion subgroups had no statistical difference from controls in the percent change of apomorphine induced activity. * P < 0.05, * * P < 0.001, significantly different from the control group.
sioned animals and 5 controls 3 0 - 3 4 days after surgery. T h e baseline activity to saline injection was significantly lower in the bradykinetic animals than in both the nonbradykinetic animals and controls ( P < 0.05). Only the bradykinetic animals consistently demonstrated a m a r k e d increase in activity in response to levodopa administration. This hyperactivity e n h a n c e d by levodopa in the bradykinetic animals was far above the baseline activities in the non-bradykinetic animals and controls. Meanwhile, levodopa had a tendency to suppress activity in the non-bradykinetic animals and controls (Fig. 5).
4. Discussion A bilateral 6 - O H D A lesion of the ascending forebrain dopaminergic neurons has b e e n reported to induce severe aphagia and adipsia in addition to akinesia [8,9,16,19,22,25]. Such bilateral lesion rats are depen-
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150
dent on the maintenance of intragastric tube feeding for survival. Marshall et al. [16] suggested there was a close relationship between striatal dopamine depletion and feeding impairments. In the present study, animals with > 95% nigral lesions were severely aphagic and adipsic and showed marked weight loss. The present study demonstrated that the spontaneous locomotor activity remained within the usual range until neuronal loss in the SNpc lesion exceeded 80%. All 3 activity parameters, especially vertical activity, were greatly reduced after the bilateral lesion. The percent change of vertical activity was more significantly correlated with the degree of SNpc lesion than those of the other 2 activity parameters, i.e. horizontal activity and distance traveled. Amphetamine acts presynaptically by releasing dopamine from nigrostriatal terminals [23]. Apomorphine, a dopamine receptor agonist, stimulates the hypersensitive postsynaptic receptors in the dopamine depleted striatum [24]. In unilaterally lesioned rats, amphetamine causes ipsilateral rotation when the striatal dopamine is depleted by at least 50% [12], while apomorphine causes contralateral rotation when more than 90% of striatal dopamine is destroyed [4,12]. In bilaterally lesioned rats, locomotor responses to amphetamine are enhanced when more than 5% [5] or 10% [6] of striatal dopamine remains. Amphetamine stimulated dopamine release in the striatum remains within the control range until the lesion size exceeds 95%, but it is remarkably depressed only in animals with a more than 95% depletion [5]. Therefore, it is considered that 5% of the normal dopaminergic innervation to the striatum must be critical for any significant recovery of function. Meanwhile, it has been reported that more than 80% of striatal dopamine depletion increases the locomotor responses to apomorphine [20]. In the present study, responses to amphetamine and apomorphine were variable. It is possible that in some cases the bilateral SNpc neurons were not equally damaged, which could cause the rotational behavior enhanced by amphetamine and apomorphine. Interestingly, only 2 animals with a 98% lesion showed much lower activity in response to amphetamine than other lesion animals and controls. It is thought because after very extensive (98%) lesions the remaining dopaminergic terminals in the striatum cannot release further sufficient amount of dopamine in spite of pronounced dopamine receptor supersensitivity. Finally, all bradykinetic rats displayed on the average a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled as measured in the spontaneous activity test following the bilateral lesion. Bilaterally lesioned animals which met this criteria consistently demonstrated a marked increase in activity in response to levodopa therapy as observed in human parkinso-
149
nian patients. Previous studies on bilaterally lesioned rats [6,8,9,19,22,25] have generally been characterized by near complete bilateral destruction of the nigrostriatal pathway. The near complete bilateral lesions produce long lasting adipsia, aphagia and akinesia. Moreover, it is difficult to keep such severely sick animals alive. The completely lesioned animals exhibit more extensive depletion of dopaminergic nigrostriatal system than human parkinsonian patients. In the present study, we undertook development of bilateral partial lesion animals in which there is an incomplete destruction of dopaminergic neurons in the SNpc and a relative sparing of VTA cell groups. We suggest the bradykinetic animals with an 80-95% lesion of SNpc are appropriate for bilateral parkinsonian rat models. However, we have examined the change in locomotor activity only during a subchronic period of up to 27 days following the bilateral lesion. The next step is to determine if these reduced activity levels remain stable for longer periods postlesion. Acknowledgements. This study was supported by National Institutes of Health Grant NS25778.
References [1] Bankiewicz, K.S., Plunkett, R.J., Jacobowitz, D.M., Porrino, L., di Porzio, U., London, W.T., Kopin, I.J. and Oldfield, E.H., The effect of fetal mesencephalon implants on primate MPTP-induced parkinsonism, J. Neurosurg., 72 (1990) 231-244. [2] Bernheimer, H., Birkmayer, W., Hornykiewicz, O., Jellinger, K. and Seitelberger, F., Brain dopamine and the syndromes of Parkinson and Huntington: clinical, morphological and neurochemical correlations, J. Neurol. Sci., 20 (1973) 415-455. [3] Bohn, M.C., Cupit, L., Marciano, F. and Gash, D.M., Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers, Science, 237 (1987) 913-916. [4] Carman, kS., Gage, F.H. and Shults, C.W., Partial lesion of the substantia nigra: relation between extent of lesion and rotational behavior, Brain Res., 553 (1991) 275-283. [5] Castafieda, E., Whishaw, I.Q. and Robinson, T.E., Changes in striatal dopamine neurotransmission assessed with microdialysis following recovery from a bilateral 6-OHDA lesion: variation as a function of lesion size, J. Neurosci., 10 (1990) 1847-1854. [6] Creese, I. and Iversen, S.D., The pharmacological and anatomical substrates of the amphetamine response in the rat, Brain Res., 83 (1975) 419-436. [7] Date, I., Felten, S.Y. and Felten, D.L, Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice, Brain Res., 537 (1990) 33-39. [8] Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D,, Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. II. Bilateral lesions, Brain Res., 229 (1981) 457-470. [9] Dunnett, S.B., Bj6rklund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., lntracerebral grafting of neuronal cell suspensions. V. Behavioural recovery in rats with bilateral 6-OHDA lesions following implantation of nigral cell suspensions, Acta. Physiol. Scand. Suppl., 522 (1983) 39-47.
150
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144 150
[10] German, D.C., Manaye, K., Smith, W.K., Woodward, D.J. and Saper, C.B., Midbrain dopaminergic cell loss in Parkinson's disease: computer visualization, Ann. Neurol., 26 (1989) 507-514. [11] Goto, S., Hirano, A. and Matsumoto, S., Subdivisional involvement of nigrostriatal loop in idiopathic Parkinson's disease and striaton]gral degeneration, Ann. Neurol, 26 (1989) 766-770. [12] Hefti, F., Melamed, E., Sahakian, B.J. and Wurtman, R.J., Circling behavior in rats with partial, unilateral nigro-striatal lesions: effect of amphetamine, apomorphine, and DOPA, Pharm. Biochem. Behac., 12 (1980) 185-188. [13] Hornykiewicz, O., Dopamine and extrapyramidal motor function and dysfunction, Res. Publ. Assoc. Res. Nero. Ment. Dis., 50 (1972) 390-415. [14] Jellinger, K., Overview of morphological changes in Parkinson's disease, Ado. Neurol. 45 (1986) 1-18. [15] Leenders, K.L., Salmon, E.P., Tyrrell, P., Perani, D., Brooks, D.J., Sager, H., Jones, T., Marsden, C.D. and Frackowiak, R.S.J., The nigrostriatal dopaminergic system assessed in vivo by positron emission tomography in healthy volunteer subjects and patients with Parkinson's disease, Arch. Neurol., 47 (1990) 1290-1298. [16] Marshall, J.F,, Richardson, J.S. and Teitelbaum, P., Nigrostriatal bundle damage and the lateral hypothalamic syndrome, J. Comp. Physiol. PsychoL, 87 (1974) 808-830. [17] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Second edition, Academic Press, Australia, 1986.
[18] Perese, D.A., Ulman, J., Viola, J., Ewing, S.t.I. and Bankicwicz, K.S., A 6-hydroxydopamine-induced selective parkinsonian ral model, Brain Res., 494 (1989) 285-293. [19] Rogers, D.C., Martel, F.L and Dunnelt, S.B., Nigral grafts in neonatal rats protect from aphagia induced by subsequent adult 6-OHDA lesions: the importance of striatal location, Exp. Brai~z Res., 80 (1990) 172-176. [20] Schoenfeld, R. and Uretsky, N., Altered response to apomorphine in 6-hydroxydopaminc-treated rats, Ear. J. Pharmac., 19 (1972) ll5-118. [21] Schultz, W., Depletion of dopamine in the striatum as an experimental model of parkinsonism: direct effects and adaptive mechanisms, Prog. Neurobiol., 18 (1982) 121-166. [22] Schwarz, S.S. and Freed, W.J., Brain tissue transplantation in neonatal rats prevents a lesion-induced syndrome of adipsia, aphagia and akinesia, E~p. Brain Res., 65 (1987) 449-454. [23] Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour. Acta. Physiol. Scand. Suppl., 367 (1971) 49-68. [24] Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta. Physiol. Scand. Suppl., 367 (1971) 69-93. [25] Ungerstedt, U., Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta. Physiol. Scand. Suppl., 367 (197/)95-122.
Brain Research 633 (1994) 144-150
Research Report
Effect of bilateral 6 - O H D A lesions of the substantia nigra on locomotor activity in the rat Kyoji Sakai a,b, Don M. Gash a,, '~ Department of Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA h Department of Neurological Surgery, Okayama University Medical School, Okayama 700, Japan
(Accepted 27 August 1993)
Abstract Previous parkinsonian rat models have utilized stereotactic 6-OHDA injections to completely lesion the dopaminergic mesostriatal system on one side. Recently, hemiparkinsonian rat models in which the mesolimbic system is left intact have been developed. The selective, partial lesion models better mimic the neuropathology of human parkinsonism in which there is usually an incomplete destruction of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and a relative sparing of ventral tegmental area (VTA) cell groups. However, such hemiparkinsonian models which possess dopaminergic asymmetry cannot demonstrate bradykinesia, one of the main symptoms in human parkinsonism. Meanwhile, bilateral lesions of the ascending forebrain dopaminergic system have been reported to induce severe aphagia, adipsia and akinesia. We, therefore, undertook development of a bilateral partial SNpc lesion model which also spares the VTA on both sides. We have investigated spontaneous locomotor activities as well as amphetamine, apomorphine and levodopa induced activities during a subchronic period of up to 27 days after the bilateral lesion. Three activity parameters, i.e. horizontal activity, vertical activity and distance traveled, were measured. The relationship between dopamine neuron loss in the SNpc and changes in the locomotor activity was analyzed. Spontaneous activity was significantly decreased in animals with extensive (> 80%) SNpc lesions on both sides, Animals with a > 95% lesion were severely aphagia and adipsia. Responses to amphetamine and apomorphine were variable. It is possible that in some cases the bilateral SNpc neurons were not equally damaged, which could cause the enhanced rotational behavior. Bradykinetic rats displayed on the average, a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled after the lesion. These bradykinetic rats consistently showed a marked increase in activity in response to levodopa therapy as observed in human parkinsonian patients. The results indicate that rats with bilateral partial lesions of the SNpc may be useful for the evaluation of new therapeutic approaches for treating Parkinson's disease. Key words: Parkinsonism; Bilateral lesion; 6-Hydroxydopamine; Substantia nigra; Activity; Rat
I. Introduction Parkinson's disease is a progressive n e u r o d e g e n e r a tive disorder characterized by the m o t o r disturbances such as bradykinesia, muscular rigidity and resting tremor. T h e principal lesion is the progressive loss of dopaminergic neurons in the substantia nigra pars c o m p a c t a (SNpc), that results in a p r o f o u n d d o p a m i n e deficiency in the nigrostriatal system [2,13]. T h e clinical
* Corresponding author. Present address: Department of Anatomy and Neurobiology, University of Kentucky, College of Medicine, MN 224 Chandler Medical Center, Lexington, KY 40536-0084, USA. Fax: (1) (606) 258-5946. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E1174-2
parkinsonian symptoms typically are not manifest until 60 to 80% of the d o p a m i n e input to the striatum is lost [2,13-15]. Previous parkinsonian rat models have been characterized by stereotactic 6-hydroxydopamine ( 6 - O H D A ) injections into the unilateral dopaminergic mesostriatal system, which destroy virtually all nigral dopaminergic neurons. Perese et al. [18] have developed a hemiparkinsonian rat model in which the mesolimbic system is left intact. C a r m a n et al. [4] have also developed a similar partial 6 - O H D A lesion model. Both selective, partial lesion models better mimic the n e u r o p a t h o l o g y of h u m a n parkinsonism in which there is usually an incomplete destruction of dopaminergic neurons in the SNpc and a relative sparing of the ventral tegmental
K. Sakai, D.M. G a s h / B r a i n Research 633 (1994) 144-150
area (VTA) cell groups [10,11]. Moreover, such selective, partial lesion models may prove to be useful for examining the reported sprouting phenomenon of host dopaminergic neurons in response to neural or paraneural grafting [1,3,7]. While the inherent dopaminergic asymmetry in hemiparkinsonian models allows lesion quantification by drug induced rotation behavior, animals with unilateral lesions do not demonstrate bradykinesia, one of the main symptoms in human parkinsonism. The parkinsonian tremor and rigidity are considered to require extrapyramidal lesions in addition to the striatal dopamine depletion [16,21]. Although some patients with idiopathic parkinsonism may first present with unilateral symptoms, the disease essentially progresses to bilateral motor disturbances. We, therefore, undertook development of a bilateral parkinsonian model in the rat using 6-OHDA to lesion the SNpc on both sides.
2. Materials and methods 2.1. Animals Animals used in this experiment were 28 adult male Fisher-344 rats (Charles River, MA) weighing 245-285 g at the start of experiment. They were randomly divided into 2 groups: twenty animals which received partial bilateral 6-OHDA lesions and eight control (sham-operated) rats. They were maintained in a 12-h light/dark cycle (light on 06.00 h; light off 18.00 h) with food and water available ad libitum. Our vivarium was fully accredited by the American Association for Accrediation of Laboratory Animal Care, and in compliance with the state law, federal statutes and the policy of National Institutes of Health.
2.2. Partial, bilateral 6-hydroxydopamine lesion Animals were deeply anesthetized with chloral hydrate-sodium pentobarbital (0.35 ml/100 g b.wt., i.p.) and placed in a stereotactic frame (David Kopf Instruments, CA) with the tooth bar 3.3 mm below horizontal. Lesioned animals received an injection of 4 /zg 6-hydroxydopamine hydrobromide (6-OHDA, Sigma, MO) diluted in 1 /zl saline containing 0.2% ascorbic acid into each SNpc, using a Hamilton 10 /zl syringe with a 26-gauge blunt tapered needle, at a rate of 0.2 ~l/min. Both injections were made with the needle bevel directed rostrally at the following coordinates: anterior - 5 . 4 mm, lateral 2.2 mm, dorsoventral 7.5 mm with respect to bregma and dura, according to Paxinos and Watson [17]. At the completion of each injection, the needle was left in place for 5 min and then withdrawn at a rate of 1 mm/min. All surgeries were carried out under sterile conditions. Animals in the control group received 1 /~1 injections of normal saline instead of 6-OHDA in the manner described above.
2.3. Postoperative care Following the bilateral 6-OHDA lesion, the rats were provided daily with a moist food supplement consisting of rat chow, glucose and water. Animals that were aphagic and adipsic following surgery received an additional supplement via intragastric tube of a high energy diet (Nutri-Cal, Evsco Pharmaceuticals, NJ) diluted 1:5 in water until they could eat on their own.
145
2.4. Spontaneous activity test Spontaneous locomotor activity was measured during the animal's normal nocturnal period (18.00 to 06.00 h). The activity tests were conducted at 8 days and 1 day prior to surgery. Animals were tested again at 20 days and 27 days after surgery. We used an animal activity monitoring system (Digiscan-16, Omnitech Electronics, OH) to quantify the locomotor activity. Two pairs of 16x16 arrayed infrared horizontal photosensors were attached to the animal cage (42 x 42 × 30 cm) equally spaced 2 cm above the floor, and one pair of 16 vertical sensors were attached 12 cm above the floor. The lower row of photosensors was used to measure the horizontal locomotor activity and the upper row to detect the vertical activity such as rearing or jumping behavior. 2.5. Induced activity test All animals were also tested for locomotor activity induced by 5 mg/kg De-amphetamine sulfate (dissolved to 5 mg/ml in sterile saline, i.p.; Sigma) and 0.05 mg/kg apomorphine hydrochloride (dissolved to 0.05 mg/ml in sterile saline, s.c.; Sigma), respectively, at the first week of this experiment (presurgery) and at the 4th week following surgery. Moreover, representative animals received methyl levodopa (dissolved in sterile saline, 0.1 ml/100 g b.wt., i.p.; Sigma). Three dose levels (0 (placebo), 50 and 100 mg/kg) were tested after the last 12-h spontaneous activity test had been completed. These induced activity tests were conducted only between 09.30 and 14.30 h with a minimum of 48 h between drugs. These induced activities were counted for a 90-min period after the drug injection. All activity tests, i.e. spontaneous activity tests, amphetamine, apomorphine and levodopa induced activity tests, were performed in a dark and quiet isolated room. Animals had free access to water but not food during activity testing. 2.6. Tyrosine hydroxylase immunocytochemistry Animals were sacrificed 28 or 36 days after surgery. Rats were anesthetized with an overdose of sodium pentobarbital and perfused with heparinized saline followed by 4% paraformaldehyde solution in 50 mM potassium phosphate buffered saline (KPBS). Brains were removed and postfixed for 24 h in the perfusion fixative at 4°C and transferred to 30% sucrose in 50 mM KPBS for an additional 72 h at 4°C. Serial coronal frozen sections of 30 ~m thickness were cut on a sliding microtome. Six sets of slices were collected in cryoprotective solution and stored at -20°C until immunocytochemically stained. Every sixth section was processed for the visualization of tyrosine hydroxylase (TH)-immunoreactive (IR) profiles using procedures previously described with minor modifications [7]. Sections were exposed to the primary anti-TH antibodies (1:1,000; BoehringerMannheim, Germany) and incubated in biotinylated horse anti-mouse secondary antibodies (1:1,500; Vector, CA). Sections were then incubated in the avidin-biotin-peroxidase complex using the Elite ABC Vectastain Kit (Vector, CA). TH-immunoreactivity was visualized using 3,3'-diaminobenzidine as the chromogen with nickel enhancement.
2. 7. Analysis of TH-IR neurons and fibers The total number of TH-IR neurons in the SNpc and VTA was counted on both sides at 3 levels: - 5.0 mm, - 5.4 mm and - 5.8 mm with respect to bregma. The degree of dopamine depletion was determined by the loss of TH-IR SNpc neurons in individual lesioned animal relative to the average number of TH-IR SNpc neurons in sham-operated (control) animals, i.e. (control-lesion)/ control X 100. The percentage area of the striatum occupied with TH-IR fibers was measured on both sides at 3 levels: + 1.2 mm, +0.5 mm and - 0 . 2 mm with respect to bregma. The sections were analyzed at 20 x magnification with an Olympus Cue 2 system (Olympus, NY).
146
KI Sakai, D.MI Gash/Brain Research 633 (1994) 144-1.50
The measured striatum was delinated by the corpus callosum, lateral ventricle and the horizontal line above anterior commissure.
reals and two o t h e r bilaterally lesioned rats out of a total of 19 surviving bilaterally lesioned rats (47%) exhibited bradykinesia, flexed posture a n d gait disturbance. T h e affected rats m a i n t a i n e d a h u n c h e d back p o s t u r e and walked in short steps with evident limb rigidity, especially in the hind legs. N o n e of control a n i m a l s d e m o n s t r a t e d hypophagia, hypodipsia or bradykinesia.
2. 8. Statistical analysis The experimental data were statistically analyzed by means of an analysis of variance (ANOVA) and t-test. Results were provided as means _+S.E.M. Correlations were determined by linear regression analysis and determination of Pearson's correlation coefficient.
3.2. T H i m m u n o c y t o c h e m i s t r y I n the control rats (n = 8), d a m a g e to SNpc n e u r o n s a p p e a r e d to be m i n i m a l a n d primarily consisted of m a c r o p h a g e infiltration in a n d a r o u n d the saline injection sites. T h e m e a n n u m b e r of T H - I R n e u r o n s c o u n t e d at the t h r e e levels in the SNpc and V T A on both sides was 1,138 + 52 and 1,315 + 25, respectively. In the bilaterally lesioned rats (n = 19), T H - I R n e u r o n s in the V T A were relatively spared ( d e p l e t i o n s of 10-50%). A subtle dorsolateral to v e n t r o m e d i a l g r a d i e n t loss of
3. Results 3.1. General condition T e n out of 20 bilateral 6 - O H D A lesioned rats (50%) showed hypophagia a n d hypodipsia to various degrees. T h e s e a n i m a l s were tube fed 5 - 2 0 ml of high energy liquid per day for 3 - 8 days following surgery. Except for o n e a n i m a l that was severely aphagic a n d adipsic a n d died, all affected rats gradually r e s u m e d eating a n d drinking. Seven of the hypophagic, hypodipsic ani-
I.
Fig. 1. TH immunocytochemistry.A: the mesencephalon of a sham-operated (control) rat. Minimal damage was seen to substantia nigra pars compacta (SNpc) neurons. Macrophage infiltration was evident around the saline injection sites. B: the striatum of a control rat showing normal TH immunoreactivity. C: the mesencephalon of a rat with a 90% lesion of SNpc; the ventral tegmental area (VTA) and the medial SNpc is relatively spared. D: the striatum of a rat with a 90% lesion of SNpc. The nucleus accumbens and olfactory tubercle, which receive dopaminergic fibers from the VTA, were relatively left intact on both sides.
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150 Table 1 Control animals and 4 subgroups of animals with bilateral 6 - O H D A lesions into the SNpc Group
n
Control 20-80% 80-90% 90-95% > 95 %
8 5 3 8 3
SNpc SNpc SNpc SNpc
lesion lesion lesion lesion
200 ontal Activity cal Activity Lnce Traveled
No. of T H - I R neurons SNpc
VTA
1138 + 52 622_+59 180 + 28 96+ 5 30 _+ 12
1315 + 25 1229_+99 939 + 63 837+66 693 _+21
Values shown represent the m e a n s +_S.E.M. The n u m b e r of T H - I R neurons in the SNpc and V T A was counted on both sides at 3 levels as described in the text. T h e degree of dopamine depletion was determined by the loss of T H - I R SNpc neurons in the individual lesioned animal relative to the average n u m b e r of T H - I R SNpc neurons in controls.
T H - I R fibers was observed in the denervated striatum. The nucleus accumbens and olfactory tubercle were left relatively intact (Fig. 1). In both the lesioned rats and controls, the percetage of striatum innervated by T H - I R fibers was strongly correlated with the number of T H - I R neurons in the SNpc (r 2 = 0.81, P < 0.0001). The lesioned rats were subdivided into 4 subgroups: 20-80% depletion in the SNpc on both sides (n = 5); 80-90% depletion (n = 3); 90-95% depletion (n = 8); and > 95% depletion (n = 3) as shown in Table 1.
a00
4
147
~~ Q"
150
oe
o e
O =-
e~
100
o
i
"
¢:m
==; o~.
50
Control 20-80% 80-90% 90-95%
>95%
Group (% Depletion of the SNp¢) Fig. 3. Change of spontaneous locomotor activity between pre- and postsurgery in the control group and 4 lesion subgroups. Three activity parameters, i.e. horizontal activity, vertical activity and distance traveled were measured for 12 h at 8 days and 1 day presurgery and at 20 days and 27 days postsurgery. In animals with a > 80% lesion, the percent changes of 3 activity parameters were significantly lower than in controls. In animals with a > 95% lesion, the percent change of vertical activity was greatly reduced relative to in controls. * P < 0.05, ** P < 0.01 and * * * P < 0.001, significantly different from the control group.
3.3. Body weight The mean body weights of each group during this experiment are shown in Fig. 2. Animals with a > 95% lesion were severely aphagic and adipsic, and exhibited a more marked weight loss. However, even animals with a > 95% lesion were able to increase their body weight from 2 weeks postsurgery.
200
-t •~
0 m
150
lesion -o- 80-90% lesion -*-90-95% lesion -o- >95% lesion
-i-
100 -
50
Control
-~-
3.4. Spontaneous locomotor activity
20-80%
--
i
0
I -10
0
T
I
'
10
I 20
30
Days Poateurgery Fig. 2. M e a n body weights of the control group and 4 lesion subgroups during pre- and postsurgery periods. Animals with a > 95% lesion were severely aphagic and adipsic, and showed a more marked weight loss. * P < 0.05, significantly different from the control group and 3 other lesion subgroups.
The lesioned animals showed a moderate correlation between the degree of SNpc lesion and the percent changes of horizontal activity, vertical activity and distance traveled as measured by comparing between pre- and postsurgery (r 2 = 0.42, 0.51, 0.45, P < 0.005, 0.001, 0.005, respectively). In most animals with a > 80% lesion, horizontal activity, vertical activity and distance traveled were significantly reduced compared to controls. In animals with a > 95% lesion, the percent change of vertical activity was greatly reduced relative to in controls ( P < 0.001) (Fig. 3). All 3 animals with a > 95% lesion, 5 out of 8 animals with a 90-95% lesion and 1 out of 3 animals with an 80-90% lesion showed much less spontaneous activity than controls.
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150
148
Twenty to 27 days following the bilateral lesions, these bradykinetic animals (n = 9) exhibited the levels of < 80% in holizontal activity, < 60% in vertical activity and < 70% in distance traveled relative to preoperative values.
25000
f
' : Control animals Nonbradykinetic animals
[] Bradykinatic animals 20000
-i
15000
i
10000
i
5000
-
A .n
3.5. Amphetamine and apomorphine induced actiuities The lesioned animals showed a p o o r correlation between the degree of SNpc lesion and the percent changes of a m p h e t a m i n e and a p o m o r p h i n e induced activities (r 2 = 0.0015, 0.10, P = 0.87, 0.18, respectively). In the groups with 2 0 - 8 0 % lesions and 9 0 - 9 5 % lesions, the percent c h a n g e of a m p h e t a m i n e induced activity was significantly higher than in controls ( P < 0.05. P < 0.001, respectively). Only in 2 animals with a 98% lesion, was a m p h e t a m i n e induced activity remarkably reduced ( - 8 8 % , - 7 9 % ) c o m p a r e d to in all other animals including controls. Meanwhile, no statistical difference from controls in the percent change of apom o r p h i n e induced activity was found in any of the lesion subgroups (Fig. 4).
3.6. Levodopa response T h e dose response to levodopa was m e a s u r e d in 7 bradykinetic lesioned animals, 5 non-bradykinetic le-
1400 t 1200
,
Amphetamine
[] Apomorphlne 1
"L°~ 1000
E g v
_e 0 m I--
0
0
- ~
0
-
~
...... 50
100
Methyl Levodope (mg/kg)
Fig. 5. Response to levodopa administration in the bradykinetic lesioned animals, the nonbradykinetic lesioned animals and controls. Bradykinetic rats are the animals which meet the criteria "'a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled as measured in the spontaneous activity test following surgery". They received methyl levodopa at 3 dose levels (0, 50, 100 mg/kg) 30-34 days postsurgery. The distance traveled, one of 3 activity parameters, was measured for 90 min. The baseline activity to saline injection was significantly lower in the bradykinetic animals than in both the nonbradykinetic animals and controls. Only the bradykinetic animals consistently demonstrated a marked increase in activity in response to levodopa administration. * P < 0.05, significantly different from the other 2 groups. P < 0.05 significantly different from the saline injection.
800 600
,
O~ID ilIO
o g
400
ae~
!
200 -
* i
T
J-
Control 20-80% 80-90% 90-95%
>95%
Group (% DepletIon of the $Npc)
Fig. 4. Changes of a m p h e t a m i n e and apomorphine induced activities between pre- and postsurgery in the control group and 4 lesion subgroups. The distance traveled, one of 3 activity parameters, was measured for 90 min pre- and postsurgery. Percent change of amphetamine induced activity was significantly higher in animals with a 20-80% lesion and ones with a 9 0 - 9 5 % lesion than in controls. Each lesion subgroups had no statistical difference from controls in the percent change of apomorphine induced activity. * P < 0.05, * * P < 0.001, significantly different from the control group.
sioned animals and 5 controls 3 0 - 3 4 days after surgery. T h e baseline activity to saline injection was significantly lower in the bradykinetic animals than in both the nonbradykinetic animals and controls ( P < 0.05). Only the bradykinetic animals consistently demonstrated a m a r k e d increase in activity in response to levodopa administration. This hyperactivity e n h a n c e d by levodopa in the bradykinetic animals was far above the baseline activities in the non-bradykinetic animals and controls. Meanwhile, levodopa had a tendency to suppress activity in the non-bradykinetic animals and controls (Fig. 5).
4. Discussion A bilateral 6 - O H D A lesion of the ascending forebrain dopaminergic neurons has b e e n reported to induce severe aphagia and adipsia in addition to akinesia [8,9,16,19,22,25]. Such bilateral lesion rats are depen-
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144-150
dent on the maintenance of intragastric tube feeding for survival. Marshall et al. [16] suggested there was a close relationship between striatal dopamine depletion and feeding impairments. In the present study, animals with > 95% nigral lesions were severely aphagic and adipsic and showed marked weight loss. The present study demonstrated that the spontaneous locomotor activity remained within the usual range until neuronal loss in the SNpc lesion exceeded 80%. All 3 activity parameters, especially vertical activity, were greatly reduced after the bilateral lesion. The percent change of vertical activity was more significantly correlated with the degree of SNpc lesion than those of the other 2 activity parameters, i.e. horizontal activity and distance traveled. Amphetamine acts presynaptically by releasing dopamine from nigrostriatal terminals [23]. Apomorphine, a dopamine receptor agonist, stimulates the hypersensitive postsynaptic receptors in the dopamine depleted striatum [24]. In unilaterally lesioned rats, amphetamine causes ipsilateral rotation when the striatal dopamine is depleted by at least 50% [12], while apomorphine causes contralateral rotation when more than 90% of striatal dopamine is destroyed [4,12]. In bilaterally lesioned rats, locomotor responses to amphetamine are enhanced when more than 5% [5] or 10% [6] of striatal dopamine remains. Amphetamine stimulated dopamine release in the striatum remains within the control range until the lesion size exceeds 95%, but it is remarkably depressed only in animals with a more than 95% depletion [5]. Therefore, it is considered that 5% of the normal dopaminergic innervation to the striatum must be critical for any significant recovery of function. Meanwhile, it has been reported that more than 80% of striatal dopamine depletion increases the locomotor responses to apomorphine [20]. In the present study, responses to amphetamine and apomorphine were variable. It is possible that in some cases the bilateral SNpc neurons were not equally damaged, which could cause the rotational behavior enhanced by amphetamine and apomorphine. Interestingly, only 2 animals with a 98% lesion showed much lower activity in response to amphetamine than other lesion animals and controls. It is thought because after very extensive (98%) lesions the remaining dopaminergic terminals in the striatum cannot release further sufficient amount of dopamine in spite of pronounced dopamine receptor supersensitivity. Finally, all bradykinetic rats displayed on the average a > 20% decline in horizontal activity, a > 40% decline in vertical activity and a > 30% decline in distance traveled as measured in the spontaneous activity test following the bilateral lesion. Bilaterally lesioned animals which met this criteria consistently demonstrated a marked increase in activity in response to levodopa therapy as observed in human parkinso-
149
nian patients. Previous studies on bilaterally lesioned rats [6,8,9,19,22,25] have generally been characterized by near complete bilateral destruction of the nigrostriatal pathway. The near complete bilateral lesions produce long lasting adipsia, aphagia and akinesia. Moreover, it is difficult to keep such severely sick animals alive. The completely lesioned animals exhibit more extensive depletion of dopaminergic nigrostriatal system than human parkinsonian patients. In the present study, we undertook development of bilateral partial lesion animals in which there is an incomplete destruction of dopaminergic neurons in the SNpc and a relative sparing of VTA cell groups. We suggest the bradykinetic animals with an 80-95% lesion of SNpc are appropriate for bilateral parkinsonian rat models. However, we have examined the change in locomotor activity only during a subchronic period of up to 27 days following the bilateral lesion. The next step is to determine if these reduced activity levels remain stable for longer periods postlesion. Acknowledgements. This study was supported by National Institutes of Health Grant NS25778.
References [1] Bankiewicz, K.S., Plunkett, R.J., Jacobowitz, D.M., Porrino, L., di Porzio, U., London, W.T., Kopin, I.J. and Oldfield, E.H., The effect of fetal mesencephalon implants on primate MPTP-induced parkinsonism, J. Neurosurg., 72 (1990) 231-244. [2] Bernheimer, H., Birkmayer, W., Hornykiewicz, O., Jellinger, K. and Seitelberger, F., Brain dopamine and the syndromes of Parkinson and Huntington: clinical, morphological and neurochemical correlations, J. Neurol. Sci., 20 (1973) 415-455. [3] Bohn, M.C., Cupit, L., Marciano, F. and Gash, D.M., Adrenal medulla grafts enhance recovery of striatal dopaminergic fibers, Science, 237 (1987) 913-916. [4] Carman, kS., Gage, F.H. and Shults, C.W., Partial lesion of the substantia nigra: relation between extent of lesion and rotational behavior, Brain Res., 553 (1991) 275-283. [5] Castafieda, E., Whishaw, I.Q. and Robinson, T.E., Changes in striatal dopamine neurotransmission assessed with microdialysis following recovery from a bilateral 6-OHDA lesion: variation as a function of lesion size, J. Neurosci., 10 (1990) 1847-1854. [6] Creese, I. and Iversen, S.D., The pharmacological and anatomical substrates of the amphetamine response in the rat, Brain Res., 83 (1975) 419-436. [7] Date, I., Felten, S.Y. and Felten, D.L, Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice, Brain Res., 537 (1990) 33-39. [8] Dunnett, S.B., Bj6rklund, A., Stenevi, U. and Iversen, S.D,, Behavioural recovery following transplantation of substantia nigra in rats subjected to 6-OHDA lesions of the nigrostriatal pathway. II. Bilateral lesions, Brain Res., 229 (1981) 457-470. [9] Dunnett, S.B., Bj6rklund, A., Schmidt, R.H., Stenevi, U. and Iversen, S.D., lntracerebral grafting of neuronal cell suspensions. V. Behavioural recovery in rats with bilateral 6-OHDA lesions following implantation of nigral cell suspensions, Acta. Physiol. Scand. Suppl., 522 (1983) 39-47.
150
K. Sakai, D.M. Gash/Brain Research 633 (1994) 144 150
[10] German, D.C., Manaye, K., Smith, W.K., Woodward, D.J. and Saper, C.B., Midbrain dopaminergic cell loss in Parkinson's disease: computer visualization, Ann. Neurol., 26 (1989) 507-514. [11] Goto, S., Hirano, A. and Matsumoto, S., Subdivisional involvement of nigrostriatal loop in idiopathic Parkinson's disease and striaton]gral degeneration, Ann. Neurol, 26 (1989) 766-770. [12] Hefti, F., Melamed, E., Sahakian, B.J. and Wurtman, R.J., Circling behavior in rats with partial, unilateral nigro-striatal lesions: effect of amphetamine, apomorphine, and DOPA, Pharm. Biochem. Behac., 12 (1980) 185-188. [13] Hornykiewicz, O., Dopamine and extrapyramidal motor function and dysfunction, Res. Publ. Assoc. Res. Nero. Ment. Dis., 50 (1972) 390-415. [14] Jellinger, K., Overview of morphological changes in Parkinson's disease, Ado. Neurol. 45 (1986) 1-18. [15] Leenders, K.L., Salmon, E.P., Tyrrell, P., Perani, D., Brooks, D.J., Sager, H., Jones, T., Marsden, C.D. and Frackowiak, R.S.J., The nigrostriatal dopaminergic system assessed in vivo by positron emission tomography in healthy volunteer subjects and patients with Parkinson's disease, Arch. Neurol., 47 (1990) 1290-1298. [16] Marshall, J.F,, Richardson, J.S. and Teitelbaum, P., Nigrostriatal bundle damage and the lateral hypothalamic syndrome, J. Comp. Physiol. PsychoL, 87 (1974) 808-830. [17] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Second edition, Academic Press, Australia, 1986.
[18] Perese, D.A., Ulman, J., Viola, J., Ewing, S.t.I. and Bankicwicz, K.S., A 6-hydroxydopamine-induced selective parkinsonian ral model, Brain Res., 494 (1989) 285-293. [19] Rogers, D.C., Martel, F.L and Dunnelt, S.B., Nigral grafts in neonatal rats protect from aphagia induced by subsequent adult 6-OHDA lesions: the importance of striatal location, Exp. Brai~z Res., 80 (1990) 172-176. [20] Schoenfeld, R. and Uretsky, N., Altered response to apomorphine in 6-hydroxydopaminc-treated rats, Ear. J. Pharmac., 19 (1972) ll5-118. [21] Schultz, W., Depletion of dopamine in the striatum as an experimental model of parkinsonism: direct effects and adaptive mechanisms, Prog. Neurobiol., 18 (1982) 121-166. [22] Schwarz, S.S. and Freed, W.J., Brain tissue transplantation in neonatal rats prevents a lesion-induced syndrome of adipsia, aphagia and akinesia, E~p. Brain Res., 65 (1987) 449-454. [23] Ungerstedt, U., Striatal dopamine release after amphetamine or nerve degeneration revealed by rotational behaviour. Acta. Physiol. Scand. Suppl., 367 (1971) 49-68. [24] Ungerstedt, U., Postsynaptic supersensitivity after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta. Physiol. Scand. Suppl., 367 (1971) 69-93. [25] Ungerstedt, U., Adipsia and aphagia after 6-hydroxydopamine induced degeneration of the nigro-striatal dopamine system, Acta. Physiol. Scand. Suppl., 367 (197/)95-122.