Reduction of Zinc-Positive TerminalFields in Striatum of Mouse after

1-Methyl-4-Phenylpyridinium NeurotoxicityPatricia Rojas 1,3,*, Javier E. Franco-Perez 1, Carolina Rojas 2, Julio Rojas-Castaneda 4,

Manuchair Ebadi 3, Francisca Fernandez-Valverde 1, Norma Serrano-Garcıa 1

1Laboratory of Neurotoxicology, Instituto Nacional de Neurologıa y Neurocirugıa, Manuel Velasco Suarez, SS, Av.

Insurgentes Sur No. 3877, C.P. 14269 Mexico D.F., Mexico2 Instituto de Investigaciones Biomedicas, Department of Cellular Biology and Physiology, Universidad Nacional

Autonoma de Mexico Apartado Postal 70228, C.P. 04510 Mexico D.F., Mexico3Department of Pharmacology, University of North Dakota, School of Medicine and Health Sciences, 501 North

Columbia Road, Grand Forks, ND 58203, USA4Laboratory of Histomorphology, Instituto Nacional de Pediatrıa, Av. Insurgentes Sur No.3700-C, Col. Insurgentes

Cuicuilco, C.P. 04530 Mexico D.F., Mexico

Received 4 October 2004; accepted 4 April 2005

Available online 9 June 2005

Abstract

Zinc is an essential trace element in the central nervous system and is located in three distinct pools: free zinc,

vesicular zinc and protein-bound zinc. Zinc may serve as an endogenous neuromodulator and has been associated with

neuropathologies. This study was undertaken to determine whether levels of vesicular zinc in neuronal terminals would

decrease in response to the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium ion (MPP+). Adult male C-57 black

mice were injected with MPP+ (0.72 mg/kg) into their right lateral ventricle. All animals were killed at 1, 2, 24 h and 7

days after MPP+ or saline administration. The brains were stained for zinc sulfides and the density of zinc-positive

terminal fields was evaluated after MPP+ administration. The relative optical density analysis of zinc-positive terminal

fields showed significant decreases in the striatum at 1, 2 and 24 h (24, 18 and 14%, respectively, versus control) and

ventricular epithelium (1, 2, 24 h and 7 days). The hippocampus showed increase in the stratum oriens and stratum

radiatum at different times. MPP+ administration reduced dopamine levels at 24 h and 7 days (36 and 40%, respectively,

versus control) as a result of the neurotoxic action of MPP+. The decrease of zinc-positive neuronal terminal fields in the

striatum after MPP+ administration is most likely due to a neuronal release of vesicular zinc in response to its

dopaminergic neurotoxicity.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Zinc; 1-Methyl-4-phenylpyridinium; Striatum; Histochemistry; Timm’s method

NeuroToxicology 26 (2005) 959–968

INTRODUCTION

Parkinson’s disease (PD) is a neurological disorder

characterized by degeneration and death of the dopa-

minergic neurons of the nigrostriatal pathway in the

* Corresponding author. Tel.: +52 55 5606 4040;

fax: +52 55 5424 0808.

E-mail address: prcastane@hotmail.com (P. Rojas).

0161-813X/$ – see front matter # 2005 Elsevier Inc. All rights reserv

doi:10.1016/j.neuro.2005.04.007

brain (Jenner, 1989). Death of these neurons produces a

decrease in striatal dopamine (DA) content. Although

several molecular mechanisms have been proposed for

the pathogenesis of this disorder, its etiology remains

unclear. However, there is increasing evidence suggest-

ing that oxidative stress may be involved in the neu-

ronal death (Owen et al., 1996).

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MP-

TP) is regarded as the best available experimental model

ed.

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968960

for the neurochemical sequelae of PD (Gerlach et al.,

1991). MPTP is a compound, which selectively affects

the dopaminergic (DAergic) neurons of the nigrostriatal

pathway. The neurotoxic mechanism of the drug is not

well understood, but its toxicity has been related to

oxidative stress via the production of reactive oxygen

species (Adams and Odunze, 1991). MPTP toxicity

depends on its conversion to the toxic metabolite 1-

methyl-4-phenylpyridinium (MPP+) by monoamine

oxidase (MAO) type B (Chiba et al., 1984). The high

concentration of MPP+ inside mitochondria blocks com-

plex I activity in the respiratory chain. MPP+ induces

free radical production in mitochondria, a process that

can further inhibit the function of complex I in mito-

chondria (Adams et al., 1993).

On the other hand, manganese and copper constitute

part of the active sites of the superoxide dismutase, an

enzyme that catalyzes the destruction of superoxide

free radicals. We found depletion of manganese and

copper following MPTP administration (Rıos et al.,

1995). Pretreatment with these metals protected

against MPTP/MPP+ neurotoxicity (Rojas and Rıos,

1995; Alcaraz-Zubeldia et al., 2001).

Assuming the great ability of MPTP to produce free

radicals by a metal-catalyzed reaction, the effects of

metals on this process would seem particularly useful

for a better understanding of both the mechanisms

underlying the neurotoxicity of MPTP/MPP+ and the

suggested involvement of this neurotoxin in the patho-

genesis of PD (Kienzl et al., 1995).

In particular, with the exception of calcium and

magnesium, zinc is the most abundant cation in the

brain. There are three main sources of zinc in the brain,

namely: (a) a vesicular pool localized in the synaptic

vesicles of nerve terminals; (b) a membrane-bound,

metalloprotein, or protein–metal complex pool

involved in both metabolic reactions and non-meta-

bolic reactions, such as structural support for biomem-

branes and protein folds; (c) an ionic pool of free or

loosely bound ions in the cytoplasm (Frederickson and

Danscher, 1990).

In the brain, in addition to its general functions as a

cofactor of several enzymes (Vallee and Falchuk,

1993), zinc is present in nerve terminals throughout

the mammalian central nervous system, where it plays

important roles as an endogenous signaling substance

(Walsh et al., 1994). They are more prominent parti-

cularly in the neocortex, hippocampal formation, piri-

form cortex, amygdaloid complex, striatum and

septum (Perez-Clausell and Danscher, 1985). Zinc is

actively taken up by central neurons, stored in their

synaptic vesicles (Palmiter et al., 1996) and released

into the brain interstitial space after neuronal depolar-

ization (Frederickson and Danscher, 1990).

Zinc can affect numerous membrane-spanning pro-

teins besides the glutamate receptors, including the

GABA receptors, the DA pump and some types of Na+

and K+ channels (for a review, see Frederickson and

Mocrieff, 1994).

Zinc is a non-transition metal ion, which has often

been associated with the pathogenesis of PD (Forsleff

et al., 1999). Zinc plays a protective role in physiolo-

gical conditions and its antioxidant properties in rela-

tion to brain oxidative stress have been reported (Ebadi

et al., 1996; Tate et al., 1999).

This study was undertaken to determine whether

levels of vesicular zinc in striatum would decrease in

response to MPP+ administration using Timm’s method.

MATERIALS AND METHODS

Animals

All experiments were conducted on male C-57 black

mice (25–30 g) aged 11–13 weeks and 6–8 animals for

each experimental group, obtained from Harlan (Mex-

ico City, Mexico). Animals were maintained in stan-

dard conditions (12-h light:12-h dark cycle, 21 � 2 8C,

relative humidity 40%) and allowed access to food and

water ad libitum. All animals were treated humanely to

minimize discomfort in accordance with the ethical

principles and regulations specified by Animal Care

and Use Committee of our Institution and the standards

of the National Institutes of Health of Mexico.

Fifty-four male C-57 black mice were allocated in

one of the eight groups: group I (n = 6), saline solution

(intracerebroventricular, i.c.v.) for 1 h treatment; group

II (n = 6), MPP+ (i.c.v.) group for 1 h treatment; group

III (n = 6), saline solution (i.c.v.) for 2 h treatment;

group IV (n = 6), MPP+ (i.c.v.) group for 2 h treatment;

group V (n = 8), saline solution (i.c.v.) for 24 h treat-

ment; group VI (n = 6), MPP+ (i.c.v.) group for 24 h

treatment; group VII (n = 8), saline solution (i.c.v.) for

7 days treatment; group VIII (n = 8), MPP+ (i.c.v.)

group for 7 days treatment.

Chemicals

MPP+ iodine was obtained from Research Biochem-

icals Incorporated (Wayland, MA), sodium sulfide

from Fisher Scientific (Pittsburgh, PA) and parafor-

maldehyde, glutaraldehyde and other reagents from

Sigma Chemical Co. (St. Louis, MO).

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968 961

MPP+ Administration and Tissue Preparation

MPP+ at a dose of 0.72 mg/kg was injected into the

right lateral ventricle of mice as we previously

described (Rojas and Rıos, 1993). This MPP+ dose

has been shown to produce significant damage to

dopaminergic neurons (Mihatsch et al., 1988). Animals

similarly injected with saline solution, served as con-

trols. At 1, 2, 24 h and 7 days mice from both groups

were anesthetized with ether and perfused transcar-

dially with 0.1 M Na2S in 0.2 M phosphate buffer

followed by 1% paraformaldehyde and 1.25% glutar-

aldehyde in 0.1 M phosphate buffer at pH 7.4 (West

and Hodges-Savola, 1983; West and Dewey, 1984).

Sulfide perfusion produces precipitation of endogenous

zinc, which is silver-amplified later on tissue sections.

After perfusion, the brains were removed and post-

fixed in the same fixative to which has been added 30%

sucrose. They remained in this solution until they sank

to the bottom of the container. Brains were sectioned in

the coronal and frontal planes on a cryostat (Jung

Frigocut 2800 N, Leica). At every 240 mm interval,

three consecutive sections were cut at a thickness of

20 mm. The series were collected on gelatin-coated

slides and processed using a modification of Timm’s

sulfide–silver technique (Cintra et al., 1997). Devel-

opment was performed in the dark at 26 8C for 60 min.

Two series were counterstained with cresyl violet or

Toluidine Blue.

Relative Optical Density

Densitometric analysis of zinc sulfide-positive

Timm-stained terminals was done using a Metamorph

Imaging System (Version 4.5, Metamorph Imaging

Corporations, PA, USA). The pictures were converted

to a standard video signal using a black and white CCD

video camera (Sony CCD-Iris), connected to a Nikon

Eclipse E600 microscope. The video signal was digi-

tized using an eight bit flash converter with an imagen

size of 512 pixels � 480 pixels frames, providing grey

scale values between 0 and 255. The density of Timm

stain was examined in striatum, hippocampus, hypotha-

lamus and ventricles at 10� magnification. The optical

density was measured by placing 50 circular cursors (5

for each section), each 40 mm2 in area, along 10 digi-

tized images from each animal. For each group, tissue

equivalent sections were identified, corresponding

approximately to anteroposterior �0.62 mm from

bregma for striatum, �0.46 mm for choroids plexus

and ventricular epithelium, �1.46 mm for arcuate

hypothalamic nucleus and �1.82 for hippocampus.

Circles in central region of striatum were placed with

distance of 300 mm each one. Circles in choroids plexus

and ventricular epithelium were placed at random, in

arcuate hypothalamic nucleus circles were placed two to

each side of the third ventricle and other below the third

ventricle. Circles in hippocampus were placed along of

each region.

Electron Microscopy

Five control and five experimental male mice at 24 h

of treatment were used to confirm the ultrastructural

localization of zinc in hippocampus following the

procedure described by Danscher (1981) with modifi-

cations. Animals were anesthetized with ether and

perfused transcardially as described above. After per-

fusion, the brains were removed and placed in the same

fixative for 1 h. The left striatal and hippocampal

formation were isolated and stained using a modifica-

tion of Timm’s sulfide–silver technique as described

above. The tissue was subsequently dehydrated and

finally embedded in Epon. The blocks were polymer-

ized for 16–18 h at a temperature of 60 8C. From each

block, 1-mm semi-thin sections were cut and stained

with Toluidine Blue. This procedure allowed unambig-

uous identification of the area of interest prior to

ultrathin sectioning, which was made from striatum

and stratum radiatum of hippocampal field CA2. From

each block 11–13 serial ultrathin sections (60–80 nm)

were cut with a diamond knife. The sections were

mounted on single-slot grids, which had been coated

with formvar film and contrasted with uranyl acetate

and lead citrate.

Determination of Dopamine

Additional 54 mice were allocated in one of the

eight groups as previously described. Animals were

treated with MPP+ or saline solution and included to

analyze DA content in striatum. Mice were sacrificed

by cervical dislocation at 1, 2, 24 h and 7 days after

MPP+ administration. Mice brains were immediately

removed and corpora striata were dissected out as

described above. DA concentrations were analyzed

according to the method described previously (Rojas

and Rıos, 1993). An aliquot (500 ml) of perchloric

acid–sodium metabisulfite solution (0.1%, w/v) was

added to the weighed tissue and sonicated with a Lab-

line ultratip labsonic system (Lab-line instruments,

Melrose Park, IL). Samples were then centrifuged at

4,000 g for 10 min, and the supernatants were kept at

�70 8C until analyzed.

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968962

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adjusted to 0.8 V versus Ag/AgCl reference electrode.

The mobile phase consisted of aqueous phosphate buffer

(pH 3.1), which contained 0.2 mM sodium octyl sulfate,

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were constructed for DA and concentration was

obtained by interpolation of the respective standard

curve. The results were expressed as micrograms of

compound per gram of tissue.

Statistical Analysis

Results were analyzed using two-way analysis of

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RESULTS

Zinc-rich terminal fields were observed in various

telencephalic areas: striatum, hippocampus, hypotha-

lamus and ventricles (Fig. 1; Table 1). The highest

zinc-rich terminal field in control animals was recorded

in hippocampus (black > dark brown), such as dentate

gyrus > CA3–field pyramidale > CA2–field pyrami-

dale > CA1–field pyramidale. The lowest zinc-rich

terminal field in control animals was seen in some

layers of hippocampus (light brown), such as CA2–

field moleculare > CA1–field moleculare and stria-

tum.

Examination at high magnification in the light

microscope showed that the precipitate was virtually

exclusively in the neuropil (presumably in axonal

boutons). Electron microscopic examination showed

that silver grains were almost always in boutons as seen

in control and experimental group (Fig. 1E and F), as

has been reported before (Friedman and Price, 1984).

Striatum

Statistical two-way ANOVA test revealed signifi-

cant effects for the treatment in the striatum

(F = 99.32, P < 0.01). The measured mean density

of zinc sulfide–silver precipitate in the Timm staining

was significantly lower in the striatum after MPP+

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968 963

Fig. 1. Representative microscopic images of striatum (A and B) and hippocampus (C and D) after MPP+ neurotoxicity showing loss or enhancement of zinc-

positive Timm-stained terminal fields, magnification 4�: (A) frontal section of the striatum demonstrating intense Timm’s stain for vesicular zinc in the control

group and loss in MPP+ group at 2 h (B) compared to that in control mice; (C) frontal section of the hippocampus demonstrating intense Timm’s stain for

vesicular zinc in the control group and MPP+ at 7 days (D). MPP+ administration showed increased of zinc staining in stratum oriens and stratum radiatum of

CA1 and CA2. The reaction was heavier in stratum radiatum of CA3 after MPP+ neurotoxicity. Sections were counterstained with cresyl violet or Toluidine Blue

and the color of the images correspond to the intensity of zinc staining. The lowest zinc-rich terminal field is shown by light brown color and highest zinc-rich

terminal field is represented by black > dark brown color. Results were repeated with five independent experiments. Electron micrographs demonstrating

axonal boutons of the hippocampus at 24 h of control group (E) and hippocampus at 24 h after MPP+ administration (F) of neurons containing zinc precipitated

as zinc sulfide in the Timm staining. Compare the heavier distribution of silver grain in MPP+ group. Magnification (E and F) 21,600�. MPP+, 1-methyl-4-

phenylpyridinium ion; CA1, CA1 field of the hippocampus; CA2, CA2 field of the hippocampus; CA3, CA3 field of the hippocampus; DG, dentate gyrus; Rad,

stratum radiatum; Or, stratum oriens; Py, stratum pyramidale; Mol, stratum moleculare. Ax, axonal boutons.

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968964

Fig. 2. Decrease on relative optical density of zinc-positive Timm-stained

terminal fields in striatum (A) and ventricular epithelium (B). Results are

expressed as mean � S.E.M. of n = 6–8 independent experiments: (A)

**statistically significant effect of treatment (F = 99.32, P < 0.01), two-

way ANOVA followed by Tukey’s test; (B) **statistically significant effect

of treatment (F = 147.74, P < 0.01), two-way ANOVA followed by Tukey’s

test. MPP+, 1-methyl-4-phenylpyridinium ion.

administration at 1 h (Fig. 2A). This decrease was

significant in zinc-positive terminal staining continued

through all times examined up to 24 h (Figs. 1A, 1B

and 2A) after MPP+ administration. The largest

decrease was at 2 h (24%), followed by 1 h (18%)

and 24 h (14%) after MPP+ neurotoxicity when com-

pared with control groups.

Ventricles

The two-way ANOVA test showed an important

significant effect for the treatment in ventricular

epithelium (F = 147.74, P < 0.01), as well as the

interaction between treatment and time (F = 8.61,

P < 0.01). Staining in ventricular epithelium shows

a drastic decrease at 2 h (44%) after MPP+ admin-

istration (Fig. 2B). At 1 h, 24 h and 7 days the

decrease was 21, 29 and 15%, respectively, after

MPP+ administration compared to respective control

groups.

In the choroid plexus, statistical two-way ANOVA

test revealed no changes in staining after MPP+ neu-

rotoxicity versus control group (Table 1).

Hypothalamus

In the hypothalamus, no changes were observed in

arcuate nucleus after MPP+ neurotoxicity versus con-

trol group (Table 1; two-way ANOVA test).

Hippocampus

Density changes were found after Timm staining in

the hippocampus in two strata (oriens and radiatum) of

the hippocampus (CA1, CA2 and CA3; Figs. 1C, 1D

and 3).

The two-way ANOVA statistical analysis revealed a

significant effect for the treatment in CA1 stratum

oriens (F = 12.48, P < 0.01) and the interaction

between treatment and time (F = 7.22, P < 0.01). Zinc

staining in CA1 stratum oriens after MPP+ adminis-

tration enhanced at 24 h (50%) and 7 days (41%) when

compared to control group (Fig. 3A).

The two-way ANOVA statistical analysis revealed a

significant effect for the treatment in CA1 stratum

radiatum (F = 11.72, P < 0.01), as well as for the

interaction between treatment and time (F = 5.77,

P < 0.01). The reaction in CA1 stratum radiatum after

MPP+ administration shows an enhancement at 24 h

(39%) and 7 days (47%) as compared to control values

(Fig. 3B).

Statistical two-way ANOVA test shows effect for the

treatment in CA2 stratum oriens (F = 8.14, P < 0.01),

as well as for the interaction treatment and time

(F = 3.98, P < 0.01). In CA2, the stratum oriens shows

an increase at 7 days (25%) after MPP+ administration

versus control group (Fig. 3C).

Statistical analysis showed a clear effect for the

treatment in CA2 stratum radiatum (F = 12.03,

P < 0.01) as well as the interaction treatment and time

(F = 4.55, P < 0.01). The stratum radiatum in CA2

presented higher staining at 24 h (41%) and 7 days

(42%) as compared to control group (Fig. 3D).

In the stratum oriens lighter staining was observed in

CA3 but not significant changes versus control group

(Fig. 3E).

The two-way ANOVA showed an important effect in

the treatment in CA3 stratum radiatum (F = 5.80,

P < 0.01) and the interaction between treatment and

time (F = 4.11, P < 0.01). In the stratum radiatum, the

reaction was heavier in CA3 at 7 days (39%) after

MPP+ neurotoxicity versus control group (Fig. 3F).

Administration of MPP+ did not produce significative

changes in dentate gyrus, CA1 (strata pyramidale and

moleculare), CA2 (strata pyramidale and moleculare)

and CA3 (strata pyramidale and moleculare).

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968 965

Fig. 3. Relative optical density of zinc sulfide–silver precipitate increase in stratum oriens and stratum radiatum of hippocampus fields (A and B) CA1 (C and D)

CA2 and (E and F) CA3. Results are expressed as mean � S.E.M. of n = 6–8 independent experiments: (A) **statistically significant effect of treatment

(F = 12.48, P < 0.01), two-way ANOVA followed by Tukey’s test; (B) **statistically significant effect of treatment (F = 11.72, P < 0.01), two-way ANOVA

followed by Tukey’s test; (C) **statistically significant effect of treatment (F = 8.14, P < 0.01), two-way ANOVA followed by Tukey’s test; (D) **statistically

significant effect of treatment (F = 12.03, P < 0.01), two-way ANOVA followed by Tukey’s test. (F) **statistically significant effect of treatment (F = 5.80,

P < 0.01), two-way ANOVA followed by Tukey’s test. MPP+, 1-methyl-4-pheylpyridinium ion.

Fig. 4. Striatal dopamine content 1, 2, 24 h and 7 days after a single

intracerebroventricular administration of MPP+ (0.72 mg/kg). Results are

expressed as mean � S.E.M. of 6–8 animals per group; **statistically

significant effect of the treatment (F = 11.72, P < 0.01), two-way ANOVA

followed by Tukey’s test. MPP+, 1-methyl-4-phenylpyridinium ion.

Striatal Dopamine Content

Statistical two-way ANOVA test revealed signifi-

cant effects for the treatment (F = 25.31, P < 0.01) and

the interaction between treatment and time (F = 11,

P < 0.01). Striatal DA content after MPP+ administra-

tion is shown in Fig. 4. Saline administration to mice

did not produce significant alteration in DA content at

all times tested. Mice in the MPP+ group at 24 h and 7

days presented markedly reduced DA levels (36 and

40%, respectively, versus control) as a result of the

neurotoxic action of MPP+. No alterations at 1 and 2 h

were found after MPP+ administration. These signifi-

cant decreases in DA concentration in striatum at 24 h

and 7 days but not at 1 h and 2 h, suggest that decreases

in vesicular zinc preceded the DA depletory effect of

MPP+.

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968966

DISCUSSION

Approximately 90% of the brain’s total zinc is in

metalloproteins. The rest is in the presynaptic vesicles

and histochemically reactive as revealed by Timm’s

sulfide–silver staining method (Frederickson, 1989).

Vesicular zinc, probably ionic zinc, may play a role in

synaptic neurotransmission in the mammalian brain

including the DA pump (for a review, see Frederickson

and Mocrieff, 1994). Zinc is transported into the brain

via not only the blood brain barrier but also the blood–

CSF barrier.

Zinc is taken up by neurons, which may have two

zinc uptake sites, i.e. the soma and the neuron terminal

and also by glia cells, and it is then incorporated into

zinc-binding proteins.

We undertook the present study to examine whether

the levels of Timm stainable vesicular zinc in neuronal

terminals would decrease in response to MPP+ neuro-

toxicity. The intensity of the staining reflects the

number of zinc-rich boutons in a terminal field or

the intensity of labeling enclosed in these boutons as

reported previously (Martınez-Guijarro et al., 1987).

Our results show reduction of zinc-positive terminals in

striatum following MPP+ administration. This occurs

within 1 h after MPP+ administration and continues up

to 24 h after MPP+ administration. Our electron micro-

scopy studies show that the reaction for zinc is confined

within synaptic boutons as reported previously (Fried-

man and Price, 1984). Therefore, the terminal field

staining observed in our results was considered to be

located in synaptic boutons.

Our results showed a loss of zinc-positive terminal

fields in striatum and decreases in DA levels after

MPP+ administration. This suggests that loss of zinc

is associated with the occurrence of DA decrease in

striatum. The decrease in the density of Timm stainable

terminal zinc found in the striatum, most probably

represents a release of vesicular zinc from the synaptic

terminals by MPP+ neurotoxicity. This free zinc

induced by MPP+ could avoid DA reuptake and might

produce reduction in DA release in synaptic cleft and

its production. These results suggest that this alteration

in zinc could be related to the DAergic neurotoxicity of

MPP+. Our results showed a loss of zinc-positive

terminal fields in striatum at 1–24 h after MPP+ admin-

istration and also showed decreases in DA levels but

starting at 24 h. This difference could be because MPP+

produces its action showing reduction in zinc-rich

boutons in striatum starting at 1 h and this effect is

not affecting the synaptic neurotransmission and the

DA production at this time analyzed. The reduction of

zinc and DA content occur at 24 h and 7 days when the

DAergic neurotoxin produces cell death and /or reduc-

tion in DA production after MPP+ injection (for a

review, see Gerlach et al., 1991).

It has been shown that zinc and MPP+ inhibits

uptake of DA (Richfield, 1993; Scholze et al.,

2002), an effect that is accounted for by direct binding

of zinc to human DA transporter (Norregaard et al.,

1998). Thus, there is some regulation of DA transport

by zinc. It has been suggested that the release of

glutamate depolarizes the dendrites, resulting in trans-

port reversal and subsequent release of DA non-exo-

cytotically from the dendritic region (Falkenburger

et al., 2001). Thus, when zinc is co-released with

glutamate, it may greatly augment the efflux of DA.

The enhancement of zinc-positive terminal fields

found in oriens and radiatum strata of hippocampus, is

thought to be due to uptake of zinc released by

surrounding zinc-positive terminals. This neuronal

uptake of zinc in hippocampus could represent an

attempt to buffer the concentration increase in extra-

cellular zinc that presumably takes place in relation to

the MPP+ neurotoxicity in the striatum. Another alter-

native explanation is that the neuronal zinc represents a

pathologic influx of zinc into the hippocampus, as

neuronal accumulation of zinc has been shown to

precede neurodegeneration (Koh et al., 1996).

Staining reduction was observed in ventricular

epithelium. This decrease in staining may reflect the

reduction of the passage of zinc through the blood-

brain barrier, the main route of entry for zinc into the

brain (Takeda, 2000). The mechanism of zinc secretion

from the brain capillary endothelial cells into the brain

extracellular fluid and the CSF, respectively, is

unknown.

Zinc is an important metal, because it is necessary

for the formation of a vast associational network of

neurons, while a portion of zinc is stored in the synaptic

vesicles in zinc-containing neuron terminals in the

telencephalon (Frederickson and Mocrieff, 1994).

Zinc is known to be accumulated dramatically and

selectively in the region of substantia nigra of the brain,

which has often been related to the pathogenesis of PD

(Forsleff et al., 1999). Regarding zinc, antioxidant

properties in relation to brain oxidative stress have

been reported (Ebadi et al., 1996; Tate et al., 1999). It

has been suggested that zinc does not promote the

formation of free radicals since zinc is not redox active

(Berg and Shi, 1996). Although it has been suggested

that these antioxidant properties may be related to the

capacity shown by zinc to induce the synthesis of

metallothionein, a stable endogenous cysteine-rich

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968 967

protein that may participate in scavenging free radicals

(Ebadi et al., 1996), the precise mechanism of this

effect is still unclear. Also, the mobilization of zinc

from metallothionein under conditions of oxidative

stress (Maret, 1995) can also happen in MPP+ neuro-

toxicity (Rojas et al., 1996). However, it must be kept

in mind that synaptic zinc is just a small fraction of the

total zinc pool in the brain (for a review, see Ebadi

et al., 1996).

Our findings show that the decrease of zinc-positive

terminal fields in the striatum after MPP+ administra-

tion is most likely due to a neuronal release of vesicular

zinc in response to MPP+ neurotoxicity.

ACKNOWLEDGEMENTS

The authors thank Dr. Sultan Habeebu for his com-

ments. Partially supported by the National Council of

Science and Technology of Mexico (CONACyT)

28605-M Grant.

REFERENCES

Adams JD Jr, Odunze IN. Biochemical mechanisms of 1-methyl-4-

phenyl-1,2,3,6-biochemical mechanisms of 1-methyl-4-phenyl-

1,2,3,6-tetrahydropyridine toxicity. Could oxidative stress be

involved in the brain?. Biochem Pharmacol 1991;41:1099–105.

Adams JD, Klaidman LK, Leung A. MPP+ and MPDP+ induced

oxygen radical formation with mitochondrial enzymes. Free

Radic Biol Med 1993;15:181–6.

Alcaraz-Zubeldia M, Rojas P, Boll C, Rıos C. Neuroprotective

effect of acute and chronic administration of copper(II) sulfate

against MPP+ neurotoxicity in mice. Neurochem Res

2001;26:59–64.

Berg JM, Shi Y. The galvanization of biology: a growing apprecia-

tion for the role of zinc. Science 1996;271:1081–5.

Chiba K, Trevor A, Castagnoli N. Metabolism of the neurotoxic

tertiary amine MPTP, by brain monoamine oxidase. Biochem

Biophys Res Commun 1984;120:574–8.

Cintra L, Granados L, Aguilar A, Kemper T, DeBassio W, Galler J,

et al. Effects of prenatal protein malnutrition on mossy fibers of

the hippocampal formation in rats of tour age groups. Hippo-

campus 1997;7:184–91.

Danscher G. Histochemical demonstration of heavy metals. His-

tochemistry 1981;71:1–16.

Ebadi M, Leuschen MP, El Refaey H, Hamada FM, Rojas P. The

antioxidant properties of zinc and metallothionein. Neurochem

Int 1996;29:159–66.

Falkenburger BH, Barstow KL, Mintz IM. Dendrodendritic inhibi-

tion through reversal of dopamine transport. Science 2001;293:

2465–70.

Forsleff L, Schauss AG, Bier ID, Stuart S. Evidence of functional

zinc deficiency in Parkinson’s disease. J Altern Complem Med

1999;5:57–64.

Frederickson CJ. Neurobiology of zinc and zinc-containing neu-

rons. Int Rev Neurobiol 1989;31:145–238.

Frederickson CJ, Danscher G. Zinc-containing neurons in hippo-

campus and related CNS structures. Prog Brain Res

1990;83:71–84.

Frederickson CJ, Mocrieff DW. Zinc-containing neurons. Biol

Signals 1994;3:127–39.

Friedman B, Price JL. Fiber systems in the olfactory bulb and

cortex: a study in adult and developing rats, using Timm method

with the light and electron microscope. J Comp Neurol

1984;223:88–109.

Gerlach M, Riederer P, Przuntek H, Youdim MBH. MPTP mechan-

isms of neurotoxicity and their implications for Parkinson’s

disease. Eur J Pharmacol Mol Pharmacol 1991;208:273–86.

Jenner P. Clues to the mechanism underlying dopamine cell death

in Parkinson’s disease. J Neurol Neurosurg Psychiatry 1989;22–

8 [Special Suppl.].

Kienzl E, Puchinger L, Jellinger K, Linert W, Stachelberger H,

Jameson RF. The role of transition metals in the pathogenesis of

Parkinson’s disease. J Neurol Sci 1995;134:69–78.

Koh JY, Suh SW, Gwag BJ, He YY, Hsu CY, Choi DW. The role of

zinc in selective neuronal death after transient global cerebral

ischemia. Science 1996;272:1013–6.

Maret W. Metallothionein/disulfide interactions, oxidative stress,

and the mobilization of cellular zinc. Neurochem Int

1995;27:111–7.

Martınez-Guijarro FJ, Molowny A, Lopez Garcıa C. Timm-stain-

ing intensity is correlated with the density of Timm-positive

presynaptic structures in the cerebral cortex of lizards. Histo-

chemistry 1987;86:315–9.

Mihatsch W, Russ H, Przuntek H. Intracerebroventricular adminis-

tration of MPP+ in mice: effects of simultaneously administered

nomifensine, deprenyl, and 1-butyl-4, 4-diphenylpiperidine. J

Neural Transm 1988;71:177–88.

Norregaard L, Frederiksen D, Nielsen EO, Gether U. Delineation of

an endogenous zinc-binding site in the human dopamine trans-

porter. EMBO J 1998;17:4266–73.

Owen AD, Schapira AHV. Jenner P, Marsden CD. Oxidative stress

and Parkinson’s disease. Ann NY Acad Sci 1996;786:217–23.

Palmiter RD, Cole TB, Quaife CJ, Findley SD. ZnT-3, a putative

transporter of zinc into vesicles. Proc Natl Acad Sci USA

1996;93(25):1434–9.

Perez-Clausell J, Danscher G. Intravesicular localization of zinc in

rat telencephalic boutons: a histochemical study. Brain Res

1985;337:91–8.

Richfield EK. Zinc modulation of drug binding, cocaine affinity

states, and dopamine uptake on the dopamine uptake complex.

Mol Pharmacol 1993;43:100–8.

Rıos C, Alvarez-Vega R, Rojas P. Depletion of copper and man-

ganese in brain after MPTP treatment of mice. Pharmacol

Toxicol 1995;76:348–52.

Rojas P, Rıos C. Increased striatal lipid peroxidation after intracer-

ebroventricular MPP+ administration to mice. Pharmacol Tox-

icol 1993;72:364–8.

Rojas P, Rıos C. Short-term manganese pretreatment partially

protects against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine

neurotoxicity. Neurochem Res 1995;20(10):1217–23.

Rojas P, Cerutis DR, Happe HK, Hao R, Murrin LC, Pfeiffer RF, et

al. 6-Hydroxydopamine mediated induction of rat brain

metallothionein I mRNA. Neurotoxicology 1996;17(2):

323–34.

P. Rojas et al. / NeuroToxicology 26 (2005) 959–968968

Scholze P, Nørregaard L, Singer E, Freissmuth M, Gether U, Sittle

HH. The role of zinc ions in reverse transport mediated by

monoamine transporters. J Biol Chem 2002;277(24):21505–13.

Takeda A. Movement of zinc and its functional significance in the

brain. Brain Res Rev 2000;34:137–48.

Tate DJ, Miceli M, Newsome DA. Zinc protects against oxidative

damage in cultured human retinal pigment epithelial cells. Free

Radic Biol Med 1999;26:704–13.

Vallee VL, Falchuk KH. The biochemical basis of zinc physiology.

Physiol Rev 1993;73:79–117.

Walsh CT, Sandstead HH, Prasad AS, Newberne PM, Fraker PJ.

Zinc: health effects and research priorities. Environ Health

Perspect 1994;102(Suppl. 2):5–46.

West JR, Hodges-Savola CA. Permanent hippocampal mossy fiber

hyperdevelopment following prenatal ethanol exposure. Neu-

robehav Toxicol Teratol 1983;5:139–50.

West JR, Dewey SL. Mossy fiber sprouting in the fascia dentate

after unilateral entorhinal lesions: quantitative analysis using

computer-assisted image processing. Neuroscience 1984;13:

337–84.