Brain Research, 501 (1989) 1-10 1 Elsevier BRES 14906 Research Reports MPTP treatment combined with ethanol or acetaldehyde selectively destroys dopaminergic neurons in mouse substantia nigra Alessandro Zuddas 1, Giovanni U. Corsini 1, Sergio Schinelli 1, Jan N. Johannessen 3, Umberto di Porzio 2'4 and Irwin J. Kopin 1 1Clinical Neuroscience Branch and 2Laboratory of Neurophysiology, NINDS, 3Laboratory of Clinical Sciences, NIMH, National Institutes of Health, Bethesda, MD (U.S.A.) and 4Istituto lnternazionale di Genetica e Biofisica, CNR, Naples (Italy) (Accepted 28 March 1989) Key words: 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Ethanol; Acetaldehyde; Neurotoxicity; Dopamine; Substantia nigra We have previously reported that ethanol and acetaldehyde potentiate 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, enhancing dopamine (DA) depletion in the striatum. The present study was designed to determine whether such enhancement of neurotoxicity was specific for the nigro-striatal DA pathway. In 5-week*old mice acetaldehyde treatment did not enhance DA depletion seen 7 days after MPTP treatment. In 8-week-old animals, however, acetaldehyde or ethanol given with MPTP decreased striatal DA content to about 10% of controls, whereas the depletion was to 43% of controls when MPTP was given alone. In acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and frontal cortex were similar to that in controls. Contents of norepinephrine and serotonin in striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde and MPTP-treated mice. Moreover, both tyrosine hydroxylase (TH) immunocytochemistry and Cresyl violet staining showed an extensive and selective cell loss in the pars compacta of the substantia nigra (SNc) of the mice treated with acetaldehyde or ethanol and MPTP, whereas MPTP alone caused only a limited cell degeneration. INTRODUCTION 1 - Methyl- 4- phenyl- 1, 2, 3, 6 - tetrahydropyridine (MPTP) induces in humans 13'2s and in monkeys 6'29 a neurological syndrome presenting all the features of idiopathic parkinsonism. Striatal dopamine (DA) and DA metabolites are depleted and DA neurons in pars compacta of the substantia nigra (SNc) are destroyed to a greater extent than aminergic neurons in any other brain area. The MPTP-induced symptoms of Parkinson's dis- ease have not been observed in rodents 9, but in mice, the systemic administration of high doses of MPTP causes a rapid and long-lasting reduction of DA and its metabolites, such as dihydroxyphenyl- acetic acid (DOPAC) and homovanillic acid (HVA) in striatum 2L25. These biochemical changes seem to reflect nerve terminal damage rather than nigrostri- atal DA neuronal death 21. Only a limited loss of DA neurons has been observed in mouse SNc 21'25'4° and a substantial, although still incomplete recovery of striatal DA depletion occurs 2-3 months after MPTP administration 8. This suggests that in mice the injured DA neurons regenerate or sprout new terminals. For such reasons, the MPTP-treated mouse has been questioned as a valid model of Parkinson's disease 4°. Nevertheless, MPTP experi- ments in mice have provided a useful model for the study of MPTP toxicity. The molecular mechanism of MPTP toxicity is not completely understood. MPTP decreases electrical activity of DA neurons 22 and inhibits monoamine oxidase (MAO) activity a'26. Drugs which inhibit MAO type B as well as, at least in mice, those that Correspondence: A. Zuddas, NIH, Bldg 10, room 5N214, Bethesda, MD 20892, U.S.A.
Transcript
Brain Research, 501 (1989) 1-10 1 Elsevier
BRES 14906 Research Reports
MPTP treatment combined with ethanol or acetaldehyde selectively destroys dopaminergic neurons in
mouse substantia nigra
Alessandro Zuddas 1, Giovanni U. Corsini 1, Sergio Schinelli 1, Jan N. Johannessen 3, Umber to di Porzio 2'4 and Irwin J. Kopin 1
1Clinical Neuroscience Branch and 2Laboratory of Neurophysiology, NINDS, 3Laboratory of Clinical Sciences, NIMH, National Institutes of Health, Bethesda, MD (U.S.A.) and 4Istituto lnternazionale di Genetica e Biofisica, CNR, Naples (Italy)
(Accepted 28 March 1989)
Key words: 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine; Ethanol; Acetaldehyde; Neurotoxicity; Dopamine; Substantia nigra
We have previously reported that ethanol and acetaldehyde potentiate 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice, enhancing dopamine (DA) depletion in the striatum. The present study was designed to determine whether such enhancement of neurotoxicity was specific for the nigro-striatal DA pathway. In 5-week*old mice acetaldehyde treatment did not enhance DA depletion seen 7 days after MPTP treatment. In 8-week-old animals, however, acetaldehyde or ethanol given with MPTP decreased striatal DA content to about 10% of controls, whereas the depletion was to 43% of controls when MPTP was given alone. In acetaldehyde or ethanol and MPTP-treated mice, changes in DA levels were observed only in the striatum. DA contents in the hypothalamus, olfactory bulb and frontal cortex were similar to that in controls. Contents of norepinephrine and serotonin in striatum, hypothalamus, olfactory bulb and cerebral cortex were not affected by any of the treatments. Three months after MPTP alone, striatal DA recovered to 74% of controls in 8-week-old mice, whereas no recovery occurred in acetaldehyde and MPTP-treated mice. Moreover, both tyrosine hydroxylase (TH) immunocytochemistry and Cresyl violet staining showed an extensive and selective cell loss in the pars compacta of the substantia nigra (SNc) of the mice treated with acetaldehyde or ethanol and MPTP, whereas MPTP alone caused only a limited cell degeneration.
INTRODUCTION
1 - Me thy l - 4- pheny l - 1, 2, 3, 6 - t e t rahydropyr id ine (MPTP) induces in humans 13'2s and in monkeys 6'29 a
neurological syndrome present ing all the features of
id iopathic parkinsonism. Striatal dopamine ( D A )
and D A metabol i tes are dep le ted and D A neurons
in pars compac ta of the substant ia nigra (SNc) are
des t royed to a grea ter extent than aminergic neurons
in any o ther brain area.
The MPTP- induced symptoms of Parkinson 's dis-
ease have not been observed in rodents 9, but in
mice, the systemic adminis t ra t ion of high doses of
MPTP causes a rap id and long-lasting reduct ion of
D A and its metabol i tes , such as d ihydroxyphenyl-
acetic acid ( D O P A C ) and homovani l l ic acid ( H V A ) in s t r ia tum 2L25. These biochemical changes seem to
reflect nerve te rminal damage ra ther than nigrostri-
atal D A neuronal dea th 21. Only a l imited loss of D A neurons has been observed in mouse SNc 21'25'4° and
a substantial , a l though still incomple te recovery of
striatal D A deple t ion occurs 2 -3 months after MPTP
adminis t ra t ion 8. This suggests that in mice the
in jured D A neurons regenera te or sprout new
terminals . For such reasons, the MPTP- t r ea t ed
mouse has been ques t ioned as a valid model of
Parkinson 's disease 4°. Never theless , MPTP experi-
ments in mice have prov ided a useful mode l for the
study of MPTP toxicity.
The molecular mechanism of MPTP toxici ty is not comple te ly unders tood. MPTP decreases electrical
activity of D A neurons 22 and inhibits monoamine
oxidase ( M A O ) activity a'26. Drugs which inhibit
M A O type B as well as, at least in mice, those that
block DA uptake, prevent MPTP toxicity 26"3°'34'37.
Conversion of MPTP into the toxic metabolite 1-methyl-4-phenylpyridinium ion (MPP+) 4'32 and its subsequent intraneuronal accumulation appear to be essential for neurotoxicity.
We have previously reported that diethyldithio- carbamate (DDC), a copper chelating agent, en- hances MPTP toxicity in mice 11. DDC is a potent inhibitor of various enzymes, including brain super- oxide dismutase (SOD) 24, but DDC also appears to retard disappearance of MPP ÷ from the brain 27.
Inhibition of SOD was suggested as responsible for this effect on MPTP toxicity 11. More recently, we
showed that similar enhancement of MPTP neuro- toxicity was obtained with ethanol or acetaldehyde in mice 12. Depletion of striatal DA and DA metab- olites after pretreatment with ethanol or acet- aldehyde was also enhanced. We now report further biochemical and histological evidence for a relatively selective degeneration of DA nigral cells induced by MPTP in ethanol- or acetaldehyde-treated mice. These effects are age-dependent and there is no evidence of significant recovery for several months after the treatment. Some of these results have appeared elsewhere in abstract form 4s.
M A T E R I A L S A N D M E T H O D S
Animals
Two groups of male C57BL/6N mice (Charles River, Wilmington, MA), 4-5 or 8-9 weeks old and weighing 14-17 or 22-25 g respectively, were housed 6 per cage, at 24 °C with 12 h light/dark cycles. Water and commercial food pellets were available ad libitum. Food was withdrawn 12 h prior to each combined treatment.
Drug" treatment
The mice were treated intraperitoneally according to one of the following schedules:
(1) Ethanol (Midwest Solvent Company of Illi- nois, Pekin, IL) in 0.2 ml 0.9% NaCI, was admin- istered at a dose of 1 g/kg, 10 min before MPTP hydrochloride (36 mg/kg) (RBI, Natick, MA) dis- solved in 0.2 ml of 0.9% NaC1. This combined treatment was repeated 16 h later.
(2) Acetaldehyde (Baker Chemical Co., Phillips- burg, N J) at a dose of 250 mg/kg, chilled to 4 °C and
diluted to 0.2 ml in 0.9% NaCi, was administered 3 times: 10 min before and 10 and 30 min after MPTP hydrochloride (36 mg/kg).
Controls were animals of the same age and weight as the corresponding experimental subjects receiving either ethanol or acetaldehyde in saline, MPTP in saline, or saline alone.
Brain dissection
Ethanol-MPTP-treated mice were sacrificed 7 days after treatment; acetaldehyde-MPTP-treated animals were divided into 3 groups and sacrificed 1, 4 or 12 weeks after the last injection. Mice were killed by cervical dislocation, brains were removed, quickly chilled in ice-cold 0.9% NaC1 and dissected. Dissection was performed as described by Glowinski and Iversen 19 with minor modification.
Briefly: first the bulbi olfactorii were separated from the rest of the brain by a transverse section on the fissura rhinalis. A second transverse section was made midway between the optic chiasma and the frontal pole of the brain to separate the frontal cortex. The rhomboencephalon was then separated and another transverse section was made at the level of the optic chiasma: this section divided this portion of the brain into frontal (A) and caudal (B) portions.
r ~
z
ae
2 0 0 -
1 0 0 '
m..
[] Controls [] n c e ~ y ~
• AcetaI.lVIPTP
5 WEEKS 8 WEEKS
AGE OF MICE
Fig. 1. Effect of age on vulnerability to MPTP toxicity. Two groups of C57BL mice 5 and 8 weeks old respectively, were administered acetaldehyde (250 mg/kg, i.p.) 10 min before and 10 and 30 min after MPTP (30 mg/kg, i.p.). Mice were killed 7 days after the last injection. Data are mean values (+ S .E.M.) for a group of 10 animals, and are expressed as ng/mg protein. The Scheffe F-test was used to compare data. *P < 0.001 compared to control mice. **P < 0.001 compared to animals treated with MPTP.
TABLE I
Ethanol and acetaldehyde increase MPTP toxicity: effects on brain dopamine
C57BL mice, 8 weeks, were administered ethanol (1 g/kg i.p.) 10 rain before MPTP (30 mg/kg) twice with a 16 h interval between doses, or acetaldehyde (250 mg/kg, i.p.) was administered 10 min before, and 10 and 30 min after MPTP (30 mg/kg, i.p.). Mice were killed 7 days after the last injection. Data are mean values (+ S.E.M.) for a group of 12 animals, and are expressed as ng/mg protein.
The brain was removed and stored in perfusion fluid at 4 °C for at least 72 h. After fixation, the brain was dehydrated with 15% sucrose in PBS for
24 h, frozen in 2-methylbutane chilled by dry ice,
sectioned on a cryostat (16/zm) and the sections mounted serially onto gelatin-coated slides. The
slides were washed in PBS, incubated at room
temperature for 30 min in blocking solution contain-
ing 3% normal goat serum in PBS. The sections were
then reacted for 48 h at 4 °C with rabbit antiserum against tyrosine hydroxylase (TH) (Eugene Tech Inc., Allandale, N J) diluted 1/1000 in PBS with 0.1%
Triton X-100. Sections were again washed in PBS (3 times), reacted with goat anti-rabbit IgG serum
diluted 1/50, washed 3 times, and incubated with rabbit peroxidase-antiperoxidase diluted 1/80 in
PBS containing 2% normal goat serum. The perox- idase was localized by reaction with 0.04% 3,
3-diaminobenzidine (DAB) and 0.01% H20 2. TH-immunoreact ive cell bodies were counted at
the level of the intrapeduncular nucleus (coronal level 50 in the atlas by Sidman et a1.42).
Alternate sections of the same brain were stained with Cresyl violet, to demonstrate that the loss of TH immunoreactivity corresponded to an actual loss of neuronal perikarya.
RESULTS
Brain catecholamine content Effect of age on vulnerability to MPTP (Fig. 1). As
previously reported 12, in adult mice (8 weeks old), 7
days after two doses of MPTP (30 mg base/kg)
administered 16 h apart, striatal D A , D O P A C and
HVA levels are greatly reduced and in this study were 43, 48 and 51% of control (162.7 + 12.2, 8.6
200
c~
z .~ 100 eL
~I+MPTP
I WEEK 5 WEEKS 12 WEEKS
12
+
.
0 1 WEEK 5 WEEKS 12 WEEKS
20 '
k kil 10'
i '~ 0 1 WEEK 5 WEEKS 12 WEEKS
T I M E A F T E R T R E A T M E N T
Fig. 2. Absence of recovery from combined acetaldehyde and MPTP treatment. C57BL mice, 8 weeks old, received acet- aldehyde (250 mg/kg, i.p.) 10 min before, and 10 and 30 min after MPTP (30 mg/kg, i.p.). Mice were killed 7, 35 or 84 days after the last injection. Bars represent the striatal content of dopamine or its metabolites in controls (hatched), MPTP- alone (solid), and acetaldehyde and MPTP (stippled) treated mice. Data are mean values (+ S.E.M.) for a group of 10 animals, and are expressed as ng/mg protein. The Scheffc F-test was used to compare data. *P < 0.001 compared to control mice; **P < 0.001 compared to animals treated with MPTP alone; ++P < 0.001 compared to MPTP-treated animals one week after injection; + no difference and "P < 0.01 in comparison to acetaldehyde-MPTP-treated animals one week after injection.
+ 1.5 and 16.9 + 2.4 ng/mg protein, respectively); in
the mice treated with ethanol before each dose of MPTP, the levels are much lower and in this study
were 9, 24 and 26% of controls. Ethanol alone did not modify the content of dopamine or its metabo-
lites. Acetaldehyde, given 10 min before, and 10 and 30
min after MPTP, also appeared to potentiate the
effects of the toxin (one dose of MPTP 30 mg base/kg). Seven days after this combined treatment striatal DA, DOPAC and HVA levels were 7, 20 and
26% of controls, respectively, similar to those seen
after two doses of ethanol and MPTP. In the younger mice (4-5 weeks old), however,
acetaldehyde did not enhance striatal DA depletion caused by MPTP. Seven days after this combined
treatment the striatal DA was 82.5 + 6.3 ng/mg
protein, and in MPTP-alone treated mice was 110.5
+ 10.7 ng/mg protein, respectively 50 and 57% of controls (165.7 + 13.4 ng/mg protein).
For this reason all the subsequent experiments
reported in this paper were carried out in 8 to 9 week
old mice. Specificity of MPTP for the nigrostriatal DA
pathway. MPTP combined with either ethanol or
acetaldehyde, as well as MPTP alone, appeared to selectively deplete striatal dopamine. Seven days
after either treatment, no changes were observed in the
dopamine content of the olfactory bulb, hypothalamus
or frontal cortex (Table I). None of these treatments
modified the levels of serotonin or norepinephrine in
any of the studied areas (Table II).
....... i~,ii,~,~ .......... ~ i ¸̧ ̧ ....... ~ .
Fig. 3. Tyrosine hydroxylase immunocytochemistry. Sections show mesencephalic DA neurons in controls (A), after MPTP alone (B), or after ethanol and MPTP (C) treatments in mice (8 weeks old). In ethanol-MPTP the number of DA cell bodies in SNc (A9 cell group) is markedly reduced. In some (3 out of 15), cell loss in the SNc was less extensive after combined treatments, but the number of cells was still markedly reduced (D). SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area. Bar: 200 pm.
A B r •
Fig. 4. Tyrosine hydroxylase immunocytochemistry and Cresyl violet staining. Alternate sections of ventral mesencephalon of MPTP alone (A,C) or acetaldehyde and MPTP (B,D) treated mice (8 weeks old). Cresyl violet staining (C,D) shows extensive loss of large basophil cell bodies (arrowheads) in the SNc of the acetaldehyde- and MPTP-treated mice (D) indicating that the loss of TH immunoreactivity (B) reflects actual cell death. Bars: 200/~m.
Recovery of DA after combined ACE-MPTP treatment. Five weeks after injection of MPTP alone,
significant recovery in the striatal D A content was observed (65 versus 50% of controls one week after treatment). Twelve weeks after MPTP alone, the striatal D A content was 123.4 + 12.1 ng/mg protein, corresponding to 74% of controls (164.6 + 13.0 ng/mg protein). There was also partial recovery in striatal D O P A C content and by 12 weeks after injections, the striatal HVA content in these animals
was similar to controls. In the acetaldehyde- and MPTP-treated mice, 5
and 12 weeks after injections the striatal D A content
was 16.8 + 4.7 and 17.7 + 5.0 ng/mg protein (10%
of controls), respectively (Fig. 2). Furthermore,
striatal D O P A C content remained low in these mice, although there was a small but significant increase in striatal HVA content (31 and 43% of controls, 5 and 12 weeks respectively).
TH immunocytochemistry Seven days after treatment with either saline-
acetaldehyde or saline-ethanol, no changes in TH- immunoreactive cells were observed in substantia nigra or in ventral tegmental area, in comparison to
control animals.
In saline-MPTP treated mice (Figs. 3B and 4A), there was only a limited decrease in the number of TH-positive cells in comparison to control animals (177.2 + 10.3 in MPTP-alone treated mice versus 206.2 + 13.2 in controls) (Fig. 3A), whereas very few TH-immunoreactive cells were counted in the SNc of ethanol-MPTP (Fig. 3C) and acetaldehyde- MPTP (Fig. 4B) treated mice. The cell loss was almost complete in the medial and central parts of the SNc, whereas few TH-positive neurons were still present in the more lateral part of SNc. In these animals there was minimal cell loss in the ventral tegmental area (A10 cell group) and no changes were observed in the retrorubral nucleus or in the locus coeruleus (not shown). In some acetaldehyde- MPTP or ethanol-MPTP treated mice (1 out of 5), the cell loss in the SNc was less extensive (Fig. 3D), but the decrease in the number of the cells was still marked (94.7 + 4.0 TH-positive cells) and the pattern of the lesion remained the same.
Cresyl violet staining clearly showed loss of large basophil cell bodies in the SNc of these animals (Fig. 4D) but not in the SNc of mice treated with MPTP alone (Fig. 4C), acetaldehyde or ethanol alone, nor untreated controls (not shown). These results indi- cated that the loss of TH immunoreactivity was not due to a decrease in the content of TH in surviving nigral DA neurons, but reflected an actual cell loss. Cresyl violet-stained sections indicated minimal in- volvement of the VTA neurons and normal mor- phology of the other surrounding cells (Fig. 4D). This analysis was restricted to the mesencephalon since TH-positive cells were spared in the other catecholaminergic regions studied.
No recovery in number of TH-positive cells was observed in the SNc of animals receiving combined treatments and sacrificed 12 weeks after MPTP injection.
DISCUSSION
Our data show that both ethanol and acet- aldehyde potentiate MPTP toxicity in mice, enhan- cing depletion of DA and DA metabolites in the striatum and markedly increasing MPTP-induced death of DA neurons in the substantia nigra. This enhancement of MPTP toxicity is age related and specific for the nigrostriatal DA pathway; recovery
does not appear to occur, at least not in the 3 months following the treatment studied.
Acetaldehyde, the major ethanol metabolite, ap- pears to be more potent than ethanol in potentiating MPTP neurotoxicity. A single MPTP treatment combined with acetaldehyde markedly enhanced depletion of striatal DA and induced loss of TH- immunoreactive cells similar to that obtained with two MPTP treatments combined with ethanol. These data suggest that the effects of ethanol might be related to acetaldehyde formation.
In mice very high doses of MPTP have been reported to deplete the striatal DA content to less than 10% of controls and cause a marked decrease in the number of the DA neurons in the SNc 25'36'43. After such doses, DA depletion was evident also in other brain areas and decreases in NE and serotonin were found in several discrete brain regions 43. However, others, using similar doses of MPTP, observed a small depletion in catecholamines in the SNc and no evidence of neuronal destruction 21'22'44.
Recent experiments with [3H]mazindol autoradio- graphy 15 and silver staining 4° have further confirmed that SNc DA neurons are largely preserved after high doses of MPTP, although striatal DA terminals appear to be destroyed.
Reports differ in the degree to which nigral DA cell degeneration occurs in mice, probably due to variability in sensitivity of different strains of mice 5' 43, different litters of the same strain 23'36, route and
frequency of MPTP administration and, perhaps more importantly, age 2°'39. Older mice do not appear to show recovery of striatal DA content 38'41. The discrepancies regarding involvement of other catecholaminergic nuclei within the brain may also be related to different ages of the animals. In both monkeys 18'33 and mice 2°, VTA and locus coeruleus have been shown damaged by MPTP in old but not in relatively young animals (21- versus 8-week-old mice).
In the present study, a dose of MPTP which selectivity decreased the striatal DA content to about 50% of controls was used. This dose caused little if any decrease in the number of the TH- immunoreactive cells of the SNc. When combined with acetaldehyde or ethanol, however, MPTP pro- duced a dramatic loss of TH-immunoreactive cells in the SNc. The decrease in the number of TH-
immunoreac t ive neurons was more extensive in the
medial and central parts of SNc than in the lateral
part : this pa t te rn is similar to that observed by
Elsworth et al. 16 in MPTP- t rea ted monkeys.
The loss of the large basophil cell bodies seen with
Cresyl violet staining and the absence of any recov-
ery in the striatal D A content 3 months after the
combined t rea tment , clearly indicate that the loss of
TH immunoreact iv i ty in the SNc is not re la ted to a
selective decrease of this enzyme, but is due to
selective and extensive destruct ion of D A neurons in
the SNc. This is fur ther confirmed by behavioral
symptoms (evoked by adminis trat ion of D A recep-
tor agonists and antagonists) that accompany the
lesion from the beginning up to several months of
t rea tment (manuscript in preparat ion) .
The enhancement of MPTP toxicity by acet-
a ldehyde does not appear to be an 'aging process '
caused by aceta ldehyde, but a selective effect on the
nigrostr iatal D A neurons. In fact, enhancement of
MPTP neurotoxici ty appeared to be selective for the
dopaminergic nigrostr iatal pathway: no changes
were observed in D A content of the other areas
studied nor in the levels of norepinephr ine or
serotonin. Moreove r histological lesions were con-
fined to the A9 area, with minimal, if any, involve-
ment of the VTA, and complete sparing of o ther
catecholaminergic nuclei, such as the re t rorubral
nucleus (A8) or the locus coeruleus.
On the o ther hand, enhancement of MPTP
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It has been suggested that unders tanding the
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In this and in previous papers 11"12 we showed how
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ethanol , are in wide use.
In the accompanying pape r we show the effects of
ethanol and ace ta ldehyde on the MPTP metabol i sm
which might explain the mechanism of their en-
hancement of MPTP neurotoxici ty.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. Jeffery L. Barker
for helpful suggestions and for comments on the
manuscript .
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