Brain
Rewwch
Bullrtrn.
Vol. 26, pp. 317-320. Q Pergamon Presr plc, 199 I. Printed in the U.S.A
036
I ~Y23O/YI $3 00 t .OO
BRIEF COMMUNICATION
Hypokinesia, Rigidity, and Tremor Induced by Hypothalamic 6-OHDA Lesions in the Rat FRANCOIS
Department
B. JOLICOEUR,’
of Psychiatry,
ROBERT
Faculty of Medicirle,
Received
RIVEST
Universiv
AND ANDREA
of Sherbrooke,
26 September
DRUMHELLER
Quebec.
Canada JIH 5N4
1990
JOLICOEUR, F. B., R. RIVEST AND A. DRUMHELLER. H ~po 1 k’rnesiu, rigidity and tremor induced bx hypothalnmic~ 6.OHDA Irsims in the rat. BRAIN RES BULL 26(2) 317-320, 1991. -Bilateral administration of 6-hydroxydopamine in the medial forebrain bundle at the level of the posterolateral hypothalamus in rats resulted in hypokinesia, muscular rigidity and tremor as determined by various behavioral assessment procedures. These neurological signs were accompanied by marked decreases in the concentrations of dopamine and its main metabolites dihydroxyphenylacetic acid and homovanillic acid in both striatum and nucleus accumbens. Administration of apomorphine (I mg/kg) or L-Dopa (60 mgikg) reversed or totally abolished the hypokinesia. rigidity and tremor in lesioned animals. Together, the present findings demonstrate that bilateral intrahypothalamic administration of 6. OHDA results in the appearance of the three cardinal symptoms of Parkinson’s disease in rats. This model should prove to be valuable for both the study of the neuropathological processes underlying the neurological signa of this disease and the screening of potential antiparkinson agents. 6-OHD 4
Hypokinesia
Muscular
rigidity
Tremor
Parkinson’s
Animal model
bundle at the level of the posterolateral hypothalamus have been shown to induce a marked hypokinesia in rats and produce neurochemical changes very similar to those seen in PD (4.9). Furthermore, the hypokinesia could be reversed selectively with known antiparkinson drugs (4). However, the possible presence of the two other neurological signs of PD. tremors and muscle rigidity, were not verified in these studies. We hereby report that, in addition to hypokinesia, bilateral administration of 6-OHDA into the posterolateral hypothalamus results in detectable and measurable body tremors and muscular rigidity and propose that this procedure provides a reliable and valid model of PD in rats.
THE principal neurological signs of Parkinson’s disease (PD) are akinesia. muscle rigidity and tremors (20). Although many animal models of PD have been utilized, a valid and reliable model in rats is still lacking. This is particularly true in view of the fact that this species appears to be impervious to the neurotoxic action of N-methyl-4-phenyl- I ,2,3,6-tetra-hydropyridine (MPTP) which induces. particularly in primates, neurochemical and neurological changes similar to those seen in PD (14). The rotation model following unilateral lesion of the substantia nigra with 6-hydroxydopamine (6-OHDA) has been widely utilized in rats and is useful for detecting clinically effective antiparkinson drugs (18). However, rats with unilateral nigral lesions do not display akinesia or muscular rigidity although they do manifest sporadic bursts of head and neck tremors (3). Tremors in rats can also be induced by a variety of cholinomimetic agents such as carbachol and oxotremorine, but akinesia or rigidity are not seen in these animals (12,16). Furthermore, the tremors induced by cholinergic stimulation can be reversed by a variety of nonantiparkinson agents (6). The administration of reserpine has been shown to induce all three neurological symptoms of PD in rats (5). However. the fact that reserpine depletes indiscriminantly a multiplicity of amines, both in brain and in periphery. raises doubts on the neurochemical validity of this model. Bilateral microinjections of 6-OHDA in the medial forebrain
‘Requests for reprints should be addressed
disease
METHOD
Animals Male hooded rats obtained from Canadian Breeding Farm (StConstant, Quebec) and weighing between 250-300 g were used. They were housed in individual cages situated in a temperaturecontrolled room having a 12-h light/dark cycle. Animals were anaesthetised with a xylazineketamine mixture and 4 ~1 of a 6OHDA solution (6.5 pg per IJ-Iof distilled water containing 0.04% ascorbic acid) was injected bilaterally into the hypothalamus according to the following coordinates: A.P.: 5.0 mm, L: 2.0 mm and V: 8.0 mm from dura (7). 6-OHDA solutions were prepared
to Dr. F. B. Jolicoeur, Department of Psychiatry. CHUS, Sherbrooke.
317
Quebec. Canada JIH 5N4
JOLICOEUR.
RIVESI
AND DRUMHELLER
TABLE 1 SUMMARY OF NEUROBEHAVIORAL EFFECTS OBTAINED WITH THE VARIOUS TREATMENTS
Groups
Sham-operated 6-OHDA + Saline 6-OHDA + Apomorphine 6-OHDA + L-Dopa
Motor
Grasping
Activity
Time
23.1 +- 6.1 228.6 ? 16.2*
171.0 ? 24.5 21.5 r s.o*
64.3 -t 11.1 363.1 ? 12.4*
o+
201.3 2 28.4t
96.8 -t- 17.lt
0+
268.5 -c 39.3t
39.3 +
Catalepsy
Tail
Tremor Intensity
Rigidity
14.2t
10.2 t 4.3 104.6 t S.6*
0 36*
8.8 2 3.6:
13t
6.7 t
1.81
ot
*Significant differences from sham-operated animals. TSignificant differences from 6-OHDA animals injected with saline.
fresh immediately prior to each injection in order to minimize oxidation. Sham-operated animals received isovolumetric solutions of ascorbic acid. Solutions were administered by means of 30-ga needles at a rate of 1 ~1 per min after which the injection needles were kept in place for 4 min to allow for complete diffusion. Following surgery, because 6-OHDA results in aphagia and adipsia (9), animals were intubated daily with 8 ml of a liquid diet containing 25 g sucrose, 1.8 ml Polyvisol vitamins, 2 eggs, 30 ml Kaopectate, 125 ml water and 400 ml evaporated milk. Procedure Forty-eight h following surgery, behavioral testing was initiated. Spontaneous motor activity was measured for 1 min by means of a photocell activity apparatus (Lehigh Valley Electronics). The presence and intensity of catalepsy were determined by placing an animal’s front paws on a horizontal wooden bar (1 cm in width) suspended 10 cm above the testing table surface. Time spent in that position, up to a maximum of 1 min, was recorded. Muscular rigidity was assessed in two tests. In the grasping test, a rat was suspended by its front paws grasping a metal rod (diameter: 0.5 cm) which was held by the experimenter about 50 cm above the table. The time the animal remained on the bar (maximum 1 min) was noted. A prolonged grasping response has been correlated with direct measures of muscle rigidity (17). In the tail rigidity test, the animal’s tail is raised approximately 5 cm from the table with a metal rod (diameter: 0.5 cm) positioned 2 cm from the end of the tail. The time the tail stayed on the rod was recorded (maximum 30 s). For tremors, an animal was lifted by the tail so that the hind quarters were suspended approximately 10 cm above the table, with the forelimbs still resting on the surface. The animal was kept in that position for a period of 10 s. When present, the intensity of body or hindleg tremors was evaluated with the following scores: 0 for absence of tremors; 1 for relatively weak and/or discontinuous tremors; and 2 for vigorous and/ or continuous tremors. Among animals displaying all three symptoms of PD, as revealed by the above tests, 3 groups of 6 animals each were constituted. One group received a subcutaneous injection of 0.9% NaCl, another 1 mg/kg apomorphine hydrochloride (Research Biochemicals Inc.). These first two groups were then submitted to the battery of behavioral tests at 15, 30, 45 and 60 min following injections. The third group was preadministered 50 mg/kg of the peripheral decarboxylase inhibitor Ro 4-4602 (Hoffmann-La Roche) and, 30 min later, 60 mg/kg of L-Dopa (Sigma Chemicals) via the intraperitoneal route. This group was then tested at 30, 45, 60 and 75 min following L-Dopa administration. The time schedule for these pharmacological experiments was based
on the reported time courses for these drugs (10). Finally, for comparison purposes, behavioral data were collected in sham-operated animals injected subcutaneously with 0.9% NaCl. For biochemical analyses, a separate group of 6 animals, also displaying all symptoms of PD, were sacrificed and their brains rapidly removed and placed on a frozen dissection block. The nucleus accumbens, corpus striatum and substantia nigra were excised according to a previously published procedure (10). Separation and quantification of dopamine (DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were then performed using HPLC coupled with electrochemical detection according to a previously described method from this laboratory (8). Results obtained on activity, catalepsy, grasping and tail rigidity were analysed by means of ANOVAs for independent samples. When significant main effects were found, comparisons between individual groups were carried out by means of Tukey tests (19). Intensities of tremors constituted nonparametric data and were analysed by Mann-Whitney tests (15). Differences between sham-operated and 6-OHDA-treated animals in regional concentrations of dopamine or a given metabolite were analyzed by means of t-tests for independent samples (19). For all statistical analyses, a difference between groups was considered significant if it had a probability of random occurrence of less than 5 percent. RESULTS
Bilateral intrahypothalamic administration of 6-OHDA resulted in a significant increase in catalepsy scores. This catalepsy was entirely abolished by the administration of apomorphine and LDopa. Motor activity was significantly reduced in lesioned animals and this hypokinesia was reversed by both pharmacological treatments. Both grasping time and tail suspension time were significantly increased in 6-OHDA-treated animals. Both indices of muscular tone returned to normal following apomorphine and LDopa. Finally, tremors, which were not seen in sham-operated animals, were detected in lesioned animals, with an intensity score of 46 out of a maximum possible score of 72. Again, tremors were significantly reduced and totally abolished by apomorphine and L-Dopa respectively. These results are presented in Table 1 where the neurobehavioral effects obtained during the 4 test periods, with the various treatments, are summarized. Neurochemically , intrahypothalamic administration of 6-OHDA resulted in significant decreases in dopamine, DOPAC and HVA in both the striatum and nucleus accumbens. No significant changes were observed in the substantia nigra. These results are presented in Table 2.
6-OHDA-INDUCED
PARKINSON
TABLE REGIONAL
BRAIN
SYMPTOMS
2
CONCENTRATIONS
EXPRESSED
IN
ngimg
319
OF DA, WET
DOPAC
AND
HVA
WEIGHT
DA
DOPAC
HVA
7.02 + 0.52 0.85 ? 0.20*
1.66 i- 0.25 0.27 ? 0.06*
0.63 t 0.09 0.06 + 0.02*
5.80 + 0.56 1.61 2 0.52*
2.62 2 0.52 0.62 2 0.13*
0.82 t 0.15 0.15 21 0.08*
Sham
0.66
0.30 2 0.09
0.17
6-OHDA
1.72 t 1.65
0.32 + 0.16
0.15 i 0.05
Regions
Corpus striatum Sham 6-OHDA Nucleus accumbens Sham 6-OHDA Substantia nigra
% 0.15
i 0.06
Values represent means 2 SD of each group (n = 6). Significant differences as revealed by t-tests for independent samples are indicated by asterisks (*p
Rewwch
Bullrtrn.
Vol. 26, pp. 317-320. Q Pergamon Presr plc, 199 I. Printed in the U.S.A
036
I ~Y23O/YI $3 00 t .OO
BRIEF COMMUNICATION
Hypokinesia, Rigidity, and Tremor Induced by Hypothalamic 6-OHDA Lesions in the Rat FRANCOIS
Department
B. JOLICOEUR,’
of Psychiatry,
ROBERT
Faculty of Medicirle,
Received
RIVEST
Universiv
AND ANDREA
of Sherbrooke,
26 September
DRUMHELLER
Quebec.
Canada JIH 5N4
1990
JOLICOEUR, F. B., R. RIVEST AND A. DRUMHELLER. H ~po 1 k’rnesiu, rigidity and tremor induced bx hypothalnmic~ 6.OHDA Irsims in the rat. BRAIN RES BULL 26(2) 317-320, 1991. -Bilateral administration of 6-hydroxydopamine in the medial forebrain bundle at the level of the posterolateral hypothalamus in rats resulted in hypokinesia, muscular rigidity and tremor as determined by various behavioral assessment procedures. These neurological signs were accompanied by marked decreases in the concentrations of dopamine and its main metabolites dihydroxyphenylacetic acid and homovanillic acid in both striatum and nucleus accumbens. Administration of apomorphine (I mg/kg) or L-Dopa (60 mgikg) reversed or totally abolished the hypokinesia. rigidity and tremor in lesioned animals. Together, the present findings demonstrate that bilateral intrahypothalamic administration of 6. OHDA results in the appearance of the three cardinal symptoms of Parkinson’s disease in rats. This model should prove to be valuable for both the study of the neuropathological processes underlying the neurological signa of this disease and the screening of potential antiparkinson agents. 6-OHD 4
Hypokinesia
Muscular
rigidity
Tremor
Parkinson’s
Animal model
bundle at the level of the posterolateral hypothalamus have been shown to induce a marked hypokinesia in rats and produce neurochemical changes very similar to those seen in PD (4.9). Furthermore, the hypokinesia could be reversed selectively with known antiparkinson drugs (4). However, the possible presence of the two other neurological signs of PD. tremors and muscle rigidity, were not verified in these studies. We hereby report that, in addition to hypokinesia, bilateral administration of 6-OHDA into the posterolateral hypothalamus results in detectable and measurable body tremors and muscular rigidity and propose that this procedure provides a reliable and valid model of PD in rats.
THE principal neurological signs of Parkinson’s disease (PD) are akinesia. muscle rigidity and tremors (20). Although many animal models of PD have been utilized, a valid and reliable model in rats is still lacking. This is particularly true in view of the fact that this species appears to be impervious to the neurotoxic action of N-methyl-4-phenyl- I ,2,3,6-tetra-hydropyridine (MPTP) which induces. particularly in primates, neurochemical and neurological changes similar to those seen in PD (14). The rotation model following unilateral lesion of the substantia nigra with 6-hydroxydopamine (6-OHDA) has been widely utilized in rats and is useful for detecting clinically effective antiparkinson drugs (18). However, rats with unilateral nigral lesions do not display akinesia or muscular rigidity although they do manifest sporadic bursts of head and neck tremors (3). Tremors in rats can also be induced by a variety of cholinomimetic agents such as carbachol and oxotremorine, but akinesia or rigidity are not seen in these animals (12,16). Furthermore, the tremors induced by cholinergic stimulation can be reversed by a variety of nonantiparkinson agents (6). The administration of reserpine has been shown to induce all three neurological symptoms of PD in rats (5). However. the fact that reserpine depletes indiscriminantly a multiplicity of amines, both in brain and in periphery. raises doubts on the neurochemical validity of this model. Bilateral microinjections of 6-OHDA in the medial forebrain
‘Requests for reprints should be addressed
disease
METHOD
Animals Male hooded rats obtained from Canadian Breeding Farm (StConstant, Quebec) and weighing between 250-300 g were used. They were housed in individual cages situated in a temperaturecontrolled room having a 12-h light/dark cycle. Animals were anaesthetised with a xylazineketamine mixture and 4 ~1 of a 6OHDA solution (6.5 pg per IJ-Iof distilled water containing 0.04% ascorbic acid) was injected bilaterally into the hypothalamus according to the following coordinates: A.P.: 5.0 mm, L: 2.0 mm and V: 8.0 mm from dura (7). 6-OHDA solutions were prepared
to Dr. F. B. Jolicoeur, Department of Psychiatry. CHUS, Sherbrooke.
317
Quebec. Canada JIH 5N4
JOLICOEUR.
RIVESI
AND DRUMHELLER
TABLE 1 SUMMARY OF NEUROBEHAVIORAL EFFECTS OBTAINED WITH THE VARIOUS TREATMENTS
Groups
Sham-operated 6-OHDA + Saline 6-OHDA + Apomorphine 6-OHDA + L-Dopa
Motor
Grasping
Activity
Time
23.1 +- 6.1 228.6 ? 16.2*
171.0 ? 24.5 21.5 r s.o*
64.3 -t 11.1 363.1 ? 12.4*
o+
201.3 2 28.4t
96.8 -t- 17.lt
0+
268.5 -c 39.3t
39.3 +
Catalepsy
Tail
Tremor Intensity
Rigidity
14.2t
10.2 t 4.3 104.6 t S.6*
0 36*
8.8 2 3.6:
13t
6.7 t
1.81
ot
*Significant differences from sham-operated animals. TSignificant differences from 6-OHDA animals injected with saline.
fresh immediately prior to each injection in order to minimize oxidation. Sham-operated animals received isovolumetric solutions of ascorbic acid. Solutions were administered by means of 30-ga needles at a rate of 1 ~1 per min after which the injection needles were kept in place for 4 min to allow for complete diffusion. Following surgery, because 6-OHDA results in aphagia and adipsia (9), animals were intubated daily with 8 ml of a liquid diet containing 25 g sucrose, 1.8 ml Polyvisol vitamins, 2 eggs, 30 ml Kaopectate, 125 ml water and 400 ml evaporated milk. Procedure Forty-eight h following surgery, behavioral testing was initiated. Spontaneous motor activity was measured for 1 min by means of a photocell activity apparatus (Lehigh Valley Electronics). The presence and intensity of catalepsy were determined by placing an animal’s front paws on a horizontal wooden bar (1 cm in width) suspended 10 cm above the testing table surface. Time spent in that position, up to a maximum of 1 min, was recorded. Muscular rigidity was assessed in two tests. In the grasping test, a rat was suspended by its front paws grasping a metal rod (diameter: 0.5 cm) which was held by the experimenter about 50 cm above the table. The time the animal remained on the bar (maximum 1 min) was noted. A prolonged grasping response has been correlated with direct measures of muscle rigidity (17). In the tail rigidity test, the animal’s tail is raised approximately 5 cm from the table with a metal rod (diameter: 0.5 cm) positioned 2 cm from the end of the tail. The time the tail stayed on the rod was recorded (maximum 30 s). For tremors, an animal was lifted by the tail so that the hind quarters were suspended approximately 10 cm above the table, with the forelimbs still resting on the surface. The animal was kept in that position for a period of 10 s. When present, the intensity of body or hindleg tremors was evaluated with the following scores: 0 for absence of tremors; 1 for relatively weak and/or discontinuous tremors; and 2 for vigorous and/ or continuous tremors. Among animals displaying all three symptoms of PD, as revealed by the above tests, 3 groups of 6 animals each were constituted. One group received a subcutaneous injection of 0.9% NaCl, another 1 mg/kg apomorphine hydrochloride (Research Biochemicals Inc.). These first two groups were then submitted to the battery of behavioral tests at 15, 30, 45 and 60 min following injections. The third group was preadministered 50 mg/kg of the peripheral decarboxylase inhibitor Ro 4-4602 (Hoffmann-La Roche) and, 30 min later, 60 mg/kg of L-Dopa (Sigma Chemicals) via the intraperitoneal route. This group was then tested at 30, 45, 60 and 75 min following L-Dopa administration. The time schedule for these pharmacological experiments was based
on the reported time courses for these drugs (10). Finally, for comparison purposes, behavioral data were collected in sham-operated animals injected subcutaneously with 0.9% NaCl. For biochemical analyses, a separate group of 6 animals, also displaying all symptoms of PD, were sacrificed and their brains rapidly removed and placed on a frozen dissection block. The nucleus accumbens, corpus striatum and substantia nigra were excised according to a previously published procedure (10). Separation and quantification of dopamine (DA) and its major metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were then performed using HPLC coupled with electrochemical detection according to a previously described method from this laboratory (8). Results obtained on activity, catalepsy, grasping and tail rigidity were analysed by means of ANOVAs for independent samples. When significant main effects were found, comparisons between individual groups were carried out by means of Tukey tests (19). Intensities of tremors constituted nonparametric data and were analysed by Mann-Whitney tests (15). Differences between sham-operated and 6-OHDA-treated animals in regional concentrations of dopamine or a given metabolite were analyzed by means of t-tests for independent samples (19). For all statistical analyses, a difference between groups was considered significant if it had a probability of random occurrence of less than 5 percent. RESULTS
Bilateral intrahypothalamic administration of 6-OHDA resulted in a significant increase in catalepsy scores. This catalepsy was entirely abolished by the administration of apomorphine and LDopa. Motor activity was significantly reduced in lesioned animals and this hypokinesia was reversed by both pharmacological treatments. Both grasping time and tail suspension time were significantly increased in 6-OHDA-treated animals. Both indices of muscular tone returned to normal following apomorphine and LDopa. Finally, tremors, which were not seen in sham-operated animals, were detected in lesioned animals, with an intensity score of 46 out of a maximum possible score of 72. Again, tremors were significantly reduced and totally abolished by apomorphine and L-Dopa respectively. These results are presented in Table 1 where the neurobehavioral effects obtained during the 4 test periods, with the various treatments, are summarized. Neurochemically , intrahypothalamic administration of 6-OHDA resulted in significant decreases in dopamine, DOPAC and HVA in both the striatum and nucleus accumbens. No significant changes were observed in the substantia nigra. These results are presented in Table 2.
6-OHDA-INDUCED
PARKINSON
TABLE REGIONAL
BRAIN
SYMPTOMS
2
CONCENTRATIONS
EXPRESSED
IN
ngimg
319
OF DA, WET
DOPAC
AND
HVA
WEIGHT
DA
DOPAC
HVA
7.02 + 0.52 0.85 ? 0.20*
1.66 i- 0.25 0.27 ? 0.06*
0.63 t 0.09 0.06 + 0.02*
5.80 + 0.56 1.61 2 0.52*
2.62 2 0.52 0.62 2 0.13*
0.82 t 0.15 0.15 21 0.08*
Sham
0.66
0.30 2 0.09
0.17
6-OHDA
1.72 t 1.65
0.32 + 0.16
0.15 i 0.05
Regions
Corpus striatum Sham 6-OHDA Nucleus accumbens Sham 6-OHDA Substantia nigra
% 0.15
i 0.06
Values represent means 2 SD of each group (n = 6). Significant differences as revealed by t-tests for independent samples are indicated by asterisks (*p
