[1] 17w Pole test is a useful method for evaluating the mouse movement disorder caused by striatal dopamine depletion

[1] 146w We evaluated the behavioral recovery of mice with 6-hydroxydopamine (6-OHDA)-induced lesions using a pole test. T LA (locomotor activity time) 1, 2, and 3 days after intracerebroventricular 6-OHDA injection (T LA(1-3D) ) was correlated significantly with the levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum 7 days after the injection of 6-OHDA, but 5-hydroxyindoleacetic acid (5-HIAA) and serotonin (5-HT) had no correlation with T LA(1-3D) . The mice whose T LA(1-3D) was more than the median showed about 60% depletion of striatal DA and increased DA turnover, and recovered from movement disorders 4 days after injection. These results show that presynaptic neuroadaptations and behavioral recovery exist in this animal model. Thus, the pole test appears to be useful in predicting the extent of the lesion to select a mouse in which the receptive fields of the dopaminergic cells are denervated.

[1] 248w Parkinson's disease (PD) is a chronic, progressive, neurodegenerative disorder characterized by resting tremor, rigidity, stooped posture, bradykinesia and akinesia or hypokinesia. In rats with unilateral 6-hydroxydopamine (6-OHDA) injection along the nigrostriatal pathway, degeneration of the dopaminergic neurons does not result in motor behavior impairment until 80 -90% of neurons are lost (Zigmond and Stricker, 1984), and the animals gradually recover from such movement disorders if destruction of the nigrostriatal dopamine system is less than 95% (Robinson et al., 1994a). This behavioral recovery depends on presynaptic and postsynaptic adaptations (Marshall, 1979;Neve et al., 1982;Robinson and Whishaw, 1988;Altar et al., 1989;Robinson et al., 1994a,b). In rats, the apomorphine or amphetamine rotation test is commonly used to assess the degree of striatal DA denervation (Hudson et al., 1993;Robinson et al., 1994b). In mice, a pole test is used to assess the effects of an injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Ogawa et al., 1985(Ogawa et al., , 1987;;Tasaki et al., 1991), which is commonly used as a model of Parkinson's disease (Arai et al., 1990). When the pole test is performed, the mouse is placed head-upward on the top of a rough-surfaced vertical pole and the time until it descends to the floor is recorded to assess the mouse locomotor activity. The purpose of this study was to investigate the ability of the pole test to predict the extent of the lesion in 6-OHDA-lesioned mice for the purpose of selecting a mouse in which the receptive fields of the dopaminergic cells are denervated.

[1] 56w The correlation coefficient between T LA(1-3D) and striatal amine concentrations and turnover are shown in Table 1. DA, DOPAC and HVA had a significant negative correlation with T LA(1-3D) , and HVA/DA and (DOPAC+ HVA)/DA had a significant positive correlation with T LA(1-3D) , though 5-HIAA, 5-HT and DO-PAC/DA had no correlation with T LA(1-3D) .

[2] 129w The correlation between DA and T LA(1-3D) is shown in Fig. 1. The mice were divided into two groups: those whose T LA(1-3D) was more than median (T LA(1-3D) = 111-360, n =17) (poor group) and the rest (T LA(1-3D) = 18-97, n= 17) (fair group). DA, DOPAC, and HVA in poor group were significantly lower (PB 0.0001) than those in fair group. (DOPAC+ HVA)/DA in poor group was significantly higher (PB 0.005) than that in fair group (Table 2). The difference in T LA between each group over 7 days is indicated in Fig. 2. T LA increased significantly 1-3 days after 6-OHDA injection and decreased to the control level after 7 days in poor group. On the other hand, T LA was not changed in fair group.

[1] 197w The major conclusion to be drawn from this study is that DA, DOPAC and HVA in the ST 7 days after the injection of 6-OHDA were significantly correlated with T LA(1-3D) . Although T LA(1-3D) may vary according to the experimental condition, for example surface roughness, diameter and height of the pole, this result suggests that T LA(1-3D) is useful for predicting the extent of DA depletion in the ST. Since tissue DA levels are known to be a good index of the degree of striatal DA denervation after the administration of 6-OHDA (Altar et al., 1989), T LA(1-3D) can be used to choose a mouse in which receptive fields of the dopaminergic cells are denervated, similar to the amphetamine or apomorphine rotation test in rats with 6-OHDA-induced le-sions (Hudson et al., 1993). The large dispersion of DA (SD= 24.3 ng/mg protein) may be due to a technical problem, though we used India ink to check the injection technique, or to individual variability in the response to 6-OHDA. The latter seems most likely because unilateral 6-OHDA injection into the nigrostriatal pathway of rats also induces scattered DA depletion in ST (Altar et al., 1989;Hudson et al., 1993).

[2] 175w The pole test has been used to assess the effect on mice injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Ogawa et al., 1985(Ogawa et al., , 1987;;Tasaki et al., 1991), which is commonly used as a model of Parkinson's disease (Arai et al., 1990). Ohno et al. described that the pole test is of value not only in a model of MPTP-induced parkinsonism but also in evaluating the extrapyramidal side-effect associated with neuroleptics (Ohno et al., 1994). These previous reports did not address the time course of T LA . As shown in Fig. 2, T LA increased significantly 1-3 days after 6-OHDA injection and decreased to the control level after 7 days in poor group, which means behavioral recovery is possible in mice as well as in rats (Altar et al., 1989). In poor group, DA, DOPAC, and HVA were significantly lower (PB 0.0001) and (DOPAC+HVA)/ DA was significantly higher (PB 0.005) than in fair group. The increase of (DOPAC+ HVA)/DA indicates an increased dopamine turnover within the surviving terminals, which may contribute to the behavioral recovery.

[3] 25w Three days after 6-OHDA injection, the striatal DA of the mouse was lower and (DOPAC+HVA)/DA was higher than 7 days after injection (data not shown).

[4] 62w These results suggest that the compensatory changes, including accelerated dopamine metabolism and possibly activation of dopamine synthesis and release, occur rapidly (within 3 days) in the dopaminergic terminals. These observations suggest that the presynaptic neuroadaptations (Marshall, 1979;Neve et al., 1982;Robinson and Whishaw, 1988;Altar et al., 1989;Robinson et al., 1994a,b) partially contribute to the recovery and sparing of function in this animal model.

[5] 128w It has been suggested that presynaptic adaptations may not be sufficient to account for recovery of function in rats with 6-OHDA-induced lesions, because compensatory increases in DA biosynthesis, metabolism, and release are maximal within 3 days after the lesion, before behavioral recovery is complete (Marshall, 1979;Robinson and Whishaw, 1988;Altar et al., 1989). It is said that postsynaptic adaptations including proliferation of postsynaptic dopamine receptor also contribute to behavioral recovery (Neve et al., 1982;Qin et al., 1994). Robinson et al. reported that extracellular DA was significantly higher 3 -4 weeks following a 6-OHDA-induced lesion than 4 days following the lesion in rats, suggesting that the normalization of extracellular DA may be a slower process, and therefore may explain the long time needed for behavioral recovery (Robinson et al., 1994a).

[6] 40w In conclusion, the pole test is useful in studies of movement disorders of mice with 6-OHDA-induced lesions. This test can be used to study the pathophysiology of parkinsonism, develop new anti-parkinsonian agents, and evaluate the extrapyramidal side-effects associated with neuroleptics.

[1] 217w Male ICR mice weighing 30 -35 g (Charles River Japan, Kanagawa, Japan) at the beginning of the experiments were used (n=47). The animals were housed in a temperature-controlled room with a 12-h light/dark cycle and were given free access to food and water. Intracerebroventricular injection of 2 vl of 6-OHDA solution [Funakoshi, Japan; 40 mg/ml, in physiological saline containing 0.1% ascorbic acid (SA)] (n = 34) or SA (n=13) were performed under light ether anesthesia (Ogawa et al., 1994). All mice received desipramine (Sigma; 25 mg/kg i.p.) 30 min prior to 6-OHDA injection to prevent the uptake of 6-OHDA by noradrenergic neurons. Seven days after 6-OHDA injection, all mice were sacrificed by microwave irradiation (3 kW, 0.2 s). The brain was removed and the striatum (ST) was immediately dissected out. The concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), and serotonin (5-HT) were determined by high-performance liquid chromatography (HPLC) with electrochemical detection, as previously described (Ogawa et al., 1994). Tissues were homogenized in 0.2 M ice-cold perchloric acid. After centrifugation (3000×g for 10 min at 4°C), supernatants were filtered (Millipore membrane type HC, 0.45 vm pores) and then injected directly into the HPLC system. Protein contents were determined by the method of Lowry with minor modifications using bovine serum albumin as standard.

[2] 195w The pole test was performed as previously described (Ogawa et al., 1985) with minor modifications. The mouse was placed head-upward on the top of a vertical rough-surfaced pole (diameter 8 mm; height 55 cm) and the time until it descended to the floor (locomotor activity time: T LA ) was recorded with a maximum duration of 120 s. Even if the mouse descended part way and fell the rest of the way, the behavior was scored until it reached to the floor. When the mouse was not able to turn downward and instead dropped from the pole, T LA was taken as 120 s (default value) because of the maximal severity. In this experiment, two mice were not able to turn and did fall in 6-OHDA injection group. T LA was measured each day for 7 days after the lesion. T LA(1-3D) is the amount of T LA 1, 2 and 3 days after 6-OHDA injection. The reason for choosing the first 3 days after the lesion is that the effect of the lesion on TLA was maximum and plateau during these days. The rater was blind as to the experimental condition of the animal.

[3] 44w Data are presented as means 9S.E.M. Significant differences between the groups were evaluated using one-way or two-way ANOVA followed by posthoc Scheffe's test and the Mann-Whitney U-test. Correlation coefficients were obtained by linear regression analysis. The null hypothesis is rejected at the 0.05 level.