112 F
cleared from the striatum [11]. The time-course of the 1.5 ml of ice-cold buffer 50 mM Tris, 5 mM EDTA, 320
recovery process of striatal DA innervation following an mM sucrose; pH 7.5 and centrifuged at 10003g for 10
intermediate dose of MPTP was evaluated sacrificing mice min at 48C. The pellet was discarded and the supernatant
from each group at four distinct time intervals. In par- centrifuged at maximum speed in a microfuge for 30 min
ticular, the original four groups of mice were further at 48C. The pellet was re-suspended in 1.2 ml of binding
divided into four subgroups each composed of 10 animals buffer 50 mM Tris, 300 mM NaCl, 5 mM, 0.1 ascorbic
3
which were sacrificed at 5 days, and 1, 2, and 3 months, acid; pH 7.9 by sonication. After addition of [ H]GBR
respectively, after MPTP administration. 12935 DuPont NEN; final concentration 3 nM, 40–60
During chronic
L
-DOPA administration mice were Ci mmol, membrane preparations were incubated for 45
weighted once a week in order to maintain a constant min at 258C. Non-specific binding was determined in the
amount of
L
-DOPA body weight. Injections were carried presence of DA 200 mM. Incubation was stopped by
out intraperitoneally i.p., using a constant volume 10 centrifugation for 30 min. The pellet was washed twice
ml kg. Mice were sacrificed at 40 h after the last
L
- with 1 ml of binding buffer and radioactivity was mea-
DOPA saline injection and their brains were immediately sured by scintillation counting.
removed. The left striatum and the olfactory bulb were dissected
2.5. Data analysis and processed for measuring monoamine levels, whereas
the right striatum from the same animals was dissected and For monoamine assay, a standard curve was prepared
processed for measuring presynaptic striatal DA uptake using known amounts of DA, NE, 5-HT and metabolites
sites. Sigma, dissolved in 0.1 M perchloric acid containing a
constant amount 10 ng ml of the internal standard 2.2.2. Dose–response study
DBA, as used for tissue samples. The standard curve for Since in the time-course study see Section 3 we
each compound DA, NE, 5-HT or metabolites was observed a slight decrease of striatal DA levels at early
calculated using regression analysis of the peak areas for time intervals 5 days and 1 month after
L
-DOPA 50 known concentrations of each compound. For binding
mg kg administration to intact mice, we explored this studies data are expressed as percentage of specific bind-
effect in detail by increasing the dosage of
L
-DOPA. In this ing; the latter was calculated from the ratio between the
additional experimental step, 100 C57 Black mice were cpm mg protein of specific binding, and the cpm mg
divided into five groups each composed of 20 mice. Group protein of total binding.
A received saline. Group B was administered with
L
-DOPA For NE, DA, 3,4-dihydroxyphenylacetic acid DOPAC,
at 50 mg kg. Group C received
L
-DOPA at 100 mg kg. homovanillic
acid HVA,
5-HT and
5-hydroxy-in- Group D was administered with
L
-DOPA at 200 mg kg. doleacetic acid 5-HIAA levels, results are expressed as
Group E was injected with
L
-DOPA at 400 mg kg. From the mean6S.E.M. of eight to 10 animals per group. For
each group, 10 animals were sacrificed at 5 days after a binding studies, results are expressed as the mean6S.E.M.
daily saline
L
-DOPA administration, whereas 10 mice of eight to 10 animals per group. Effects of MPTP and or
were sacrificed after 1 month of daily treatment. In these
L
-DOPA on monoamine levels in the striatum and olfactory mice we measured striatal monoamine levels.
bulb as well as on striatal GBR 12935 binding sites, were evaluated using analysis of variance with Sheffe’s post-hoc
2.3. Assay of monoamines analysis.
The striatum and the olfactory bulb were sonicated in 0.6 ml of ice-cold 0.1 M perchloric acid containing 10
3. Results
ng ml of 3,4-dihydroxybenzylamine DBA, Sigma, San Louis, MO as the internal standard. An aliquot of the
3.1. Effects of MPTP administration on the time-course homogenate 50 ml was assayed for proteins [20]. After
of striatal DA and the DA transporter centrifugation at 80003g for 10 min, 20 ml of the clear
supernatant were injected into an HPLC system where DA, As shown in Fig. 1A, 5 days after administration of the
norepinephrine NE, serotonin 5-HT and metabolites neurotoxin MPTP 30 mg kg we measured an inter-
were analysed as previously described [12]. mediate degree of striatal DA loss at 5 days compared with
control values. This effect became less evident at pro- 2.4. Measurement of striatal DA uptake sites
longed time intervals. In particular, at 1 month there was an increase in DA levels in MPTP-treated mice, which
For radioligand binding measurement of the plasma became closer to control values. At 3 months, there were
membrane DA transporter DAT the striatum was quickly no longer significant differences between MPTP-treated
removed, frozen in liquid nitrogen and kept at 2808C until mice and controls. A similar trend was observed for the
assayed. For each assay one striatum was homogenised in DA metabolites DOPAC Fig. 1B and HVA Fig. 1C,
F . Fornai et al. Brain Research 887 2000 110 –117
113 Table 1
Monoamine and metabolite levels values are given in ng mg protein in
a
the olfactory bulb at different time intervals 5 days
30 days 60 days
90 days DA
Saline 4.2360.69
3.4460.49 4.2060.45
4.0160.28 MPTP
3.9260.50 4.0460.37
4.7460.71 3.9060.57
L
-DOPA 4.3660.64
3.8060.39 4.4060.37
4.0260.29
L
-DOPA1MPTP 3.8560.70
4.2360.48 3.9460.57
4.3960.62 DOPAC
Saline 1.0460.08
0.9060.14 1.2060.20
0.9060.07 MPTP
0.926 0.10 0.8760.09
0.9260.11 1.0360.06
L
-DOPA 0.9960.07
1.1060.06 1.0260.09
0.9160.14
L
-DOPA1MPTP 1.1160.14
1.0060.12 0.8760.15
0.9060.06 HVA
Saline 1.4660.17
1.8960.20 1.3660.10
1.1160.08 MPTP
1.3960.14 1.7060.18
1.5360.08 1.6160.13
L
-DOPA 1.2960.20
1.3360.11 1.6060.14
1.7360.21
L
-DOPA1MPTP 1.2260.28
1.2160.19 1.5460.23
1.6060.30 NE
Saline 5.6460.21
6.0860.45 4.3360.30
5.2960.44 MPTP
4.2760.23 4.8660.47
5.0760.41 6.2360.58
L
-DOPA 5.1360.37
4.6160.40 5.2560.63
5.3160.48
L
-DPA1MPTP 4.9060.29
5.6860.43 5.8260.41
6.0660.51 5HT
Saline 3.856042
4.0160.50 4.3260.50
3.6860.41 MPTP
4.1160.36 3.6760.28
4.1260.24 4.2060.24
L
-DOPA 3.9960.43
3.6060.28 4.5160.63
4.1160.30
L
-DOPA1MPTP 3.6560.22
3.8560.52 3.7860.26
3.8060.48
a
Male C57 Black mice have been sacrificed at different time intervals after a single MPTP 30 mg kg administration following a chronic daily
dosage of either
L
-DOPA 50 mg kg or an equivalent volume of saline. The olfactory bulb have been processed and assayed for monoamine
Fig. 1. Effects of daily
L
-DOPA administration at different time intervals leves. Data are given as the mean6S.E.M of eight to 10 animals per
after MPTP injection. Male C57 Black mice were sacrificed at different group. No differences in monoamine and metabolite levels have been
time intervals 5, 30, 60, 90 days after an i.p. injection of MPTP 30 `
measured by using the ANOVA with Sheffe’s post-hoc analysis. mg kg and a daily administration of
L
-DOPA 50 mg kg i.p.. Mice were sacrificed, the left striata were dissected and processed for HPLC
analysis. The levels of dopamine DA A, dihydroxyphenylacetic acid DOPAC B, and homovanillic acid HVA C were evaluated. For
there was an intermediate decrease in DA binding sites
each time interval results were obtained from eight to 10 animals per group and are given as the mean6S.E.M. At each time interval
compared with controls saline, 18.3962.59; MPTP,
differences between groups were evaluated using ANOVA with Sheffe’s
8.2562.46, whereas at 2 and 3 months striatal DAT
post-hoc analysis. P,0.05 compared with controls.
levels in MPTP-treated mice were close to controls. although the depleting effect of MPTP for DA metabolites
3.2. Effects of
L
-DOPA per se was less pronounced. Levels of 5-HT and its metabolite
5-HIAA within the striatum were not affected by MPTP As shown in Fig. 1A, chronic administration of
L
-DOPA administration at any time interval data not shown. No
did not modify striatal DA levels. At 5 days and 1 month significant difference was measured in saline-injected
after daily
L
-DOPA administration there was a slight controls during the 3 months observation time Fig. 1. As
decrease in striatal DA levels; however, in the following shown in Table 1, no significant effect was produced in the
time intervals, this effect was no longer present Fig. 1A. olfactory bulb by MPTP administration at any time interval
Similarly, striatal DA uptake sites were not affected by compared with controls. Similarly, in the olfactory bulb
chronic
L
-DOPA administration Fig. 2. Neither striatal MPTP administration did not modify NE levels Table 1.
5-HT levels data not shown nor levels of monoamines in As shown in Fig. 2, measurement of striatal plasma
the olfactory bulb Table 1 were modified during the membrane DAT matched the data obtained by assaying
time-course of
L
-DOPA administration compared with DA levels Fig. 1A. Five days after MPTP administration
controls.
114 F
3.3. Effects of higher doses of
L
-DOPA Since at 5 days and 1 month there was a slight decrease
in DA levels of
L
-DOPA-treated mice, we increased the daily dose of
L
-DOPA up to 8-fold. As shown in Fig. 3, no effects were observed either at 5 days Fig. 3A and 1
month Fig. 3B after a daily administration of
L
-DOPA up to 400 mg kg.
3.4. Effects of a daily administration of
L
-DOPA on the recovery following MPTP-induced neurotoxicity
Animals injected with MPTP and then receiving a daily
Fig. 2. Effects of daily
L
-DOPA on striatal dopamine uptake sites at
L
-DOPA administration had the same striatal DA levels as
different time intervals after MPTP administration. Male C57 Black mice
MPTP-treated animals receiving a chronic administration
were sacrificed at three different time intervals 5, 60, 90 days after an i.p. injection of MPTP 30 mg kg and a daily administration of
L
-DOPA
of saline Fig. 1. This was confirmed by assaying DAT
50 mg kg i.p.. Animals from each group were sacrificed, their right
binding sites Fig. 2. Therefore,
L
-DOPA administration
striata were dissected and processed for the measurement of specific
did not modify the spontaneous recovery process occurring
binding of the dopamine transporter DAT. For each time interval results
in partially lesioned nigrostriatal DA terminals Figs. 1 and
were obtained from eight to 10 animals per group and are expressed as
2. In this case, we did not observe the slight decrease in
the mean6S.E.M. At each time interval the differences between groups were evaluated by using ANOVA with Sheffe’s post-hoc analysis. P,
DA levels which was detected at early time intervals in
0.05 compared with controls.
intact mice injected with
L
-DOPA. Striatal 5-HT and 5- HIAA levels were not affected at any time interval.
Similarly, no effect was detected on monoamine levels in the olfactory bulb Table 1.
4. Discussion
