Neurochemistry International 52 (2008) 1305–1309

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Zinc transporter 3 immunohistochemical tracing of sprouting mossy fibres

Zhi-Hong Chi a, Xin Wang a, Ji-Qun Cai a, Meredin Stoltenberg b, Gorm Danscher b, Zhan-You Wang a,*
a
Department of Histology and Embryology, China Medical University, 92 Bei-Er Road, Heping District, Shenyang 110001, PR China
b
Department of Neurobiology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark

A R T I C L E I N F O A B S T R A C T

Article history: Zinc transporter 3 (ZNT3) has been shown to transport zinc ions from the cytosol into presynaptic vesicles
Received 27 February 2008 in the mammalian brain. Several studies have stated that the zinc ion containing synaptic vesicles of zinc-
Accepted 28 February 2008 enriched neurons (ZEN) are loaded with ZNT3 proteins in their membranes. This fact makes it possible to
Available online 6 March 2008
trace sprouting mossy fibres in the temporal lobe epileptic hippocampus. In the present study, we
examined the expression and distribution patterns of ZNT3 protein and chelatable zinc ions in the mouse
Keywords:
hippocampus after pilocarpine treatment. Our results demonstrate that both ZNT3 immunostaining and
Zinc
Zinc transporter 3
autometallography reveal identical patterns of sprouting mossy fibres in the inner molecular layer in the
Mossy fiber sprouting mouse hippocampus. Using ZNT3 immuno-electron microscopic analysis we confirmed the presence of
Autometallography ectopic mossy fibre terminals in the inner molecular layer and found additionally by immuno-blotting a
Immunohistochemistry significant increase of ZNT3 in the pilocarpine-treated mouse hippocampi compared to age-matched
Mouse controls. The increase of ZNT3 after pilocarpine treatment was time-dependent. The results support the
notion that ZNT3 immunohistochemistry provides an excellent tool for tracing sprouting of ZEN
terminals. The progressive increase of ZNT3 immunostaining in the temporal lobe epileptic hippocampus
may relate to the increased levels of vesicular zinc ions during seizure.
ß 2008 Elsevier Ltd. All rights reserved.

1. Introduction 2006). The NeoTimm AMG technique selectively labels synaptic

Temporal lobe epilepsy (TLE) is a common type of epilepsy in
adult humans, characterized by a permanent change in brain
circuitry that leads to increased seizure susceptibility (Ben-Ari,
1985; Babb et al., 1991; Dudek and Spitz, 1997). Pathologically TLE
is characterized by several histological aberrations in the
hippocampus, such as axon sprouting, synaptic reorganization,
and loss of neurons.
Mossy fibres are boutons en passage plastered axons of the
dentate granule cells, filling the dentate hilus and stratum lucidum
with giant boutons that connect synaptically with hilar and CA3
neurons (Henze et al., 2000). In the TLE hippocampus, however, the
mossy fibre axons sprout collaterally and project through the
granule cell layer into the molecular layer of fascia dentata,
forming an abnormal termination field in the inner molecular layer
(IML), in which the sprouted mossy fibres make synaptic contacts
with granule cell dendrites, i.e. the same cells from which they
originate. This abnormality causes the creation of excitatory loops
(Laurberg and Zimmer, 1981; Buckmaster et al., 2002).
The most commonly used methods for MFS detection are the
autometallographic (AMG) techniques (Danscher and Stoltenberg,
* Corresponding author. Tel.: +86 24 23256666 5305;
fax: +86 24 23256666 5305.
E-mail address: wangzy@mail.cmu.edu.cn (Z.-Y. Wang).
vesicles in the mossy fibres boutons (MFBs) by accumulating zinc
ions in the vesicles (Zn2+) in zinc–sulphur nanocrystals that are
subsequently silver enhanced (Danscher, 1981). However, this
approach is gradually replaced by the selenium AMG technique
which, based as it is on in vivo binding of zinc ions in zinc–
selenium nanoparticles, is unsurpassed in specificity (Danscher
and Stoltenberg, 2006). Apart from the endogenous zinc methods,
biocytin and other neurotracer labelings can also be used to trace
MFBs (Sutula et al., 1998; Wenzel et al., 2000).
ZNT3, a synaptic-vesicle-specific zinc transporter, is essential
for the accumulation of zinc ions within synaptic vesicles (Palmiter
et al., 1996) and appears to be limited to the zinc enriched (ZEN)
terminals (Jo et al., 2000; Wang et al., 2002a). In the adult mouse
hippocampus, a correspondence between ZNT3 expression and
synaptic zinc ion staining has been demonstrated (Palmiter et al.,
1996; Wenzel et al., 1997). ZNT3 immunostaining has even been
proposed as an alternative to AMG tracing of zinc ions in the
central nervous system (CNS) (Cole et al., 1999).
A previous study indicates that tissue from pilocarpine-treated
animals displays a dense band of ZNT3 labeling within the IML of
fascia dentata. This strong immunostaining is believed to arise
from invading mossy fibres as no ZNT3 staining was seen in the IML
of control animals (Pierce et al., 2005). Using the AMG techniques,
IML normally contains tiny ZEN terminals that give rise to a
homogenous brown colour while in the pilocarpine-treated mice
the staining becomes black and uneven. This underlines the fact

0197-0186/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuint.2008.02.008
1306 Z.-H. Chi et al. / Neurochemistry International 52 (2008) 1305–1309

that the AMG zinc–selenium technique is manifold more sensible
than what can be obtained by immunohistochemistry. However,
the only way of proving that a MFS takes place in pilocarpine-
treated animals is to analyze tissues from the two approaches in
the electron microscope. We therefore looked into the distribution
of ZNT3 and Zn2+ in fascia dentata at both light microscopical and
ultrastructural levels and additionally measured the level of ZNT3
protein using western blot analysis.
We found that MFBs are present in the IML of the fascia dentata
of pilocarpine-treated mice and that they can be detected with
ZNT3 immunohistochemistry as precisely as with AMG.
2. Materials and methods
2.1. Experimental animals
Eighty adult male CD-1 mice (8–10 weeks old, 30–35 g, provided by the
experimental animal center of China Medical University) were used in the present
study. They were housed under standard laboratory conditions and had access to
tap water and diet ad libitum.
For light microscopy, samples were cut into 10 mm coronal sections in a cryostat.
The sections were rinsed twice in Tris-buffered saline (TBS, pH 7.4) and treated with
3% hydrogen peroxide (H2O2) in PB for 10 min to reduce endogenous peroxidase
activity. After rinsing with TBS all tissue sections were preincubated for 1 h with 5%
bovine serum albumin (BSA) and 3% goat serum in TBS to reduce nonspecific
staining. The sections were incubated overnight at 4 8C with anti-ZNT3 serum
(kindly provided by Dr. R.D. Palmiter) (Wenzel et al., 1997) diluted 1:100 in TBS
containing 3% goat serum, 1% BSA and 0.3% Triton-X 100. After extensive washing in
TBS, sections were incubated with a 1:200 diluted biotinylated anti-rabbit IgG for
1 h at room temperature (RT). The ABC Kit was then applied for 1 h at RT in order to
visualize the reaction sites. The ABC solution was diluted 1:100 in TBS. A brown
colour appeared in the sections after incubation of the sections in 0.025% 3,30 -
diaminobenzidine with 0.0033% H2O2 for 10 min at RT. The sections were
dehydrated in graded alcohols and coverslipped with neutral balsam for light
microscopy.
For electron microscopy, the immunolabeling procedures were performed in
accordance with the standard ABC method as above. Sections were eventually
osmicated for 30 min in 1% osmium tetroxide (OsO4) made in 0.1 M PB at RT. After
alcohol dehydration and propylene substitution, sections were flat-embedded in
Epon 812. Ultrathin sections were cut, stained with uranyl acetate for 10 min, and
examined in an electron microscope (JEM-1200EX).

2.4. Autometallography (AMG)

2.2. Induction of epilepsy in mice
The pilocarpine model of epilepsy was used to induce spontaneous recurrent
seizures (SRS) after status epilepticus (SE) by well-established procedures (Shibley
and Smith, 2002). Briefly, mice were administered an intraperitoneal (i.p.) injection
of methylscopolamine at a dose of 1 mg/kg body weight 15–30 min prior to
injection of pilocarpine in order to suppress the peripheral cholinergic effects.
Experimental animals were then injected i.p. with a single dose of 280–290 mg/kg
of pilocarpine in a 0.1 ml sterile saline vehicle (0.9% NaCl). Control mice were age-
matched with pilocarpine-treated mice and either administered a comparable
volume of vehicle or not injected after the initial methylscopolamine treatment.
Onset of SE was determined by the presence of continuous class 4–5 level seizures
as assessed using the Racine scale (Racine, 1972). Following pilocarpine treatment,
the mice were monitored for 2 h/day, 3–5 days/week (minimum 6 h/week) for the
occurrence of spontaneous category 3–5 seizures.
2.3. Immunohistochemistry
After a survival time of 30 or 60 days, the mice were deeply anesthetized with
sodium pentobarbital (50 mg/kg, i.p.), and perfused transcardially with isotonic
saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4) for
light microscopic immunohistochemistry, or 4% paraformaldehyde and 0.025%
glutaraldehyde in 0.1 M PB for immuno-electron microscopy. The brains were
carefully removed and further post-fixed by immersion into the corresponding
fixatives for 3 h at 4 8C.
At 30 or 60 days after pilocarpine treatment, the mice were anesthetized with
sodium pentobarbital (50 mg/kg, i.p.) and injected intraperitoneally with sodium
selenite (20 mg/kg). After 1.5 h survival time, the mice, still under deep anesthesia,
were perfused transcardially with 2.5% glutaraldehyde in 0.1 M PB. For light
microscopy, samples were cut into 10 mm coronal sections in a cryostat. For
electron microscopy, samples were cut into 50 mm thick slices in a vibratome.
Samples were then incubated in the AMG developer in a water bath for 1 h at 26 8C.
Subsequently, sections were immersed in a 5% thiosulphate solution to stop the
AMG reaction. The sections were then dehydrated and coverslipped with neutral
balsam for light microscopy. For electron microscopy, ultrathin sections were
processed as above.
2.5. Western blot analysis
After a survival time of 15, 30, or 60 days the mice were killed by i.p. injection of
an overdose of sodium pentobarbital and decapitated. The brains were removed
immediately, and the hippocampi dissected and homogenized 1:5 (w:v) in ice-cold
lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, 0.25%
sodium deoxycolate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/
ml leupeptin, 1 mM Na3VO4 and 1 mM NaF). The resulting homogenate was
centrifuged at 12,000  g for 30 min at 4 8C. The supernatant was collected and total
protein levels were measured by a BCA protein assay kit (Pierce Biotechnology).
Sample protein (20 mg) was separated on 10% SDS-polyacrylamide gels, and the
proteins were transferred onto PVDF membranes (Millpore, CA) in an electronasfer

Fig. 1. Comparison of the spatial distribution of mossy fibre collaterals in the IML (arrows) of AMG-stained (a–f) and ZNT3 immuno-stained (g–l) sections of mouse
hippocampi. The lack of AMG staining seen in the hilus of c and d is a well-known phenomenon of the selenium AMG technique and has no relevance for the study. In the
control group, no mossy fibre outgrowth could be detected in the IML, neither in ZNT3 immunohistochemistry nor in AMG staining sections (a, b, g and h). In groups of 30 and
60 days after pilocarpine treatment, both ZNT3 and AMG stained sections displayed a dense band in the IML consistent with aberrant mossy fibre sprouting, and there was a
progressively increasing pattern of staining intensity of ZNT3 and AMG staining in the IML and granular layer from 30 to 60 days after pilocarpine treatment (c, d, e, f, i, j, k and
l). Same magnification in a, c, e, g, i and k. Scale bar = 500 mm. Same magnification in b, d, f, h, j and l. Scale bar = 100 mm.
 Z.-H. Chi et al. / Neurochemistry International 52 (2008) 1305–1309 1307

device (45 V, 15 h). Then the membranes were blocked in 5% nonfat milk in TBS
containing 0.05% Tween 20 (TTBS) for 3 h and incubated in primary antibody
overnight at 4 8C. The dilutions of primary antibodies were 1:500 for ZNT3 and
1:12,000 for GAPDH. Membranes were washed with TTBS and followed by
incubation with horseradish peroxidase-conjugated secondary antibody for 2 h at
RT with constant agitation. The membranes were washed and reacted with an
enhanced chemiluminescence (ECL) kit (Pierce, CA), and visualized by exposure to
Kodak-XAR film.
2.6. Statistical analysis
ZNT3 protein expression level was determined by densitometry of the bands
using Image-pro Plus 6.0 analysis software. Mean protein levels of ZNT3 were
compared between pilocarpine-treated mice and age-matched controls using a
Student’s t-test. P < 0.05 was considered significant.
3. Results
3.1. Mossy fibre sprouting revealed by the ZNT3 and AMG techniques
sprouted mossy fibres had grown even more pronounced
(Fig. 1e, f, k and l).
At electron microscopic level, ectopic terminals stained by AMG
and ZNT3 were found in the IML of fascia dentata after 30 days and
60 days of pilocarpine treatment (Fig. 2). Additional small ZEN
terminals were recorded in the zone, corresponding to the rather
homogen AMG staining. It was apparent that not all synaptic
vesicles of the MFBs contained AMG grains, i.e. silver enhanced in
vivo and in situ created zinc–selenium nanocrystals (Fig. 2a and c),
whereas almost all membranes of the synaptic vesicles were ZNT3
positive (Fig. 2b and d). In the sprouted mossy fibre boutons of the
IML of fascia dentata the amount of AMG stained synaptic vesicles
was much higher than in the normal hilus and CA3 MFBs. In
general, there was an increasing amount of ectopic MFBs at day
30–60 following the pilocarpine treatment. For control mice, no
ectopic terminals were recorded with either of the two techniques.

As anticipated, ZNT3 antigens and AMG detectable zinc ions
stained identically and massively in hilus fasciae dentatae and
the mossy fibre field. In experimental animals that had survived
for 15 days MFBs were additionally recorded in the inner zone
of the molecular layer of fascia dentata. In the control group,
this zone was void of MFBs with both techniques (Fig. 1a, b, g, h).
However, 30 days after pilocarpine treatment the hilus
and mossy fibres stained strongly compared to the controls,
and both ZNT3 and AMG stained sections displayed a dense
band of MFBs in the IML of fascia dentata. This pattern is
consistent with aberrant mossy fibre sprouting (Fig. 1c, d, i and
j). Two months after pilocarpine treatment, the pattern of
3.2. Increased expression of ZNT3 in the hippocampus of pilocarpine-
treated mice
The representative western blots of ZNT3 expression in
mouse hippocampi after 15, 30, and 60 days of pilocarpine
treatment and age-matched controls are shown in Fig. 3a. The
semiquantitative analysis of immuno-blots showed statistically
significant elevations of ZNT3 in mouse hippocampi 15 days after
seizures as compared to age-matched control mice (P < 0.05). At
30 and 60 days after exposure the significance had raised to
P < 0.01 (Fig. 3b) revealing a clear time dependent increase of
elevation in ZNT3 from day 15 to 60 (Fig. 3b).

Fig. 2. ZNT3 immunocytochemical and AMG-stained electron micrographs from the IML of mouse hippocampi after pilocarpine treatment. AMG granules, i.e. zinc ions, could
be found in the synaptic vesicles of the sprouted MFBs (a and c). The sprouted MFBs also showed ZNT3 immunoreactivity (b and d). There was a progressively increasing
tendency for the quantity or intensity of zinc ions and ZNT3 immunoreactivity in ectopic terminals in IML from 30 days to 60 days after pilocarpine treatment. Arrows indicate
the synapses with ZNT3-positive or zinc-positive presynaptic elements. Same magnification in a–d. Scale bar = 200 nm.
1308 Z.-H. Chi et al. / Neurochemistry International 52 (2008) 1305–1309
Fig. 3. A representative western blot of the ZNT3 proteins in age-matched controls
and pilocarpine-treated mouse hippocampi, 15, 30 and 60 days after SE (a). The
semiquantitative analysis was performed on the immuno-blots for ZNT3 in
pilocarpine-treated and age-matched control mouse hippocampi (b). Data are given
as means W S.E.M. (n = 5). Single asterisk indicates statistical significance at
P < 0.05, double asterisk indicates significance at P < 0.01.
4. Discussion
The present study reports a correspondence between ZNT3
immunoreactivity and AMG zinc staining in the hippocampus of
temporal lobe epileptic CD-1 mice at defined time points after
pilocarpine treatment. Our ultrastructural results confirm the
presence of zinc ions and ZNT3 proteins in the synaptic vesicles of
ectopic mossy fibre terminals in the IML of fascia dentata of
pilocarpine-treated mice. Both techniques showed a delicately
stained inner zone of fascia dentata reflecting the presence of tiny
ZEN terminals of the perforant pathway connecting the entorhinal
cortex with hippocampus. Based on these findings we suggest
ZNT3 immunohistochemistry to be an obvious alternative to the
AMG zinc tracing techniques for tracing sprouting ZEN terminals.
Double- or multilabeling with other antibodies is easy due to its
common means of fixation and staining, and if the experiment
includes chelation of zinc ions it is the only approach available. The
intimate connection between ZNT3 immunohistochemistry and
AMG has been revealed in a number of papers (Wang et al.,
2002a,b; Danscher et al., 2003). ZNT3 knock-out mice contain no
zinc ions in the synaptic vesicles of ZEN terminals (Palmiter et al.,
1996; Cole et al., 1999). In a recent study the spatial distribution of
zinc has been mapped in the hippocampus with microprobe
synchrotron X-ray fluorescence. The results prove that the ZNT3
protein controls the amount of zinc ions in the synaptic vesicles of
mossy fibres (Linkous et al., 2007).
The functional significance of vesicular zinc ions in epilepsy is
not very well understood. Alteration of zinc homeostasis in the
brain may be associated with manifestation of epileptic seizures
(Takeda et al., 2003b). It has been found that the concentration of
zinc in the hippocampus of epileptic mice was significantly lower
than that of control mice (Fukahori et al., 1988). The massive
release of glutamate during seizures was accompanied by an
equally massive release of Zn2+ from the presynaptic boutons
(Frederickson et al., 1988; Suh et al., 2001). Moreover, the
excessive release of zinc and glutamate from the ZEN terminals
under seizures was accompanied by a loss of zinc from the brain
(Takeda et al., 2003b). The susceptibility to kaninate-induced
seizures in dietary zinc deficient rats was enhanced and cell death
increased (Takeda et al., 2003a, 2005). Znt3/ knock-out mice
were more susceptible than normal Znt3+/+ mice to kainic acid-
induced seizures and the cell death was also more violent (Cole
et al., 2000). In hippocampal slices from Znt3/ mice there was a
greater attenuation of GABAA-mediated inhibitory postsynaptic
potentials during titanic stimulation (Lopantsev et al., 2003). The
increase in glutamate concentration in the entorhinal cortex
during perfusion of the CA1 with glutamate was inhibited by the
addition of zinc in CA1 (Takeda et al., 2003c). These data suggested
that the overall effect of zinc was to dampen excitability in the
hippocampus (Cole et al., 2000).
Our morphological results suggested a progressive increase in
the amount of zinc ions and ZNT3 proteins in ectopic terminals of
the inner zone of the molecular layer of fascia dentata. Western
blotting results indicated an increased tendency for ZNT3 content
in the hippocampus after pilocarpine treatment. Therefore, it is
reasonable to speculate that increased ZNT3 expression may affect
zinc homeostasis in the TLE hippocampus. Sprouting ZEN
terminals have also been found in rats following denervation of
temporal structures in a rat model of mnemonic dysfunction
(Myhrer et al., 2003), and changes in the amount of zinc ion loaded
synaptic vesicles in ZEN terminals resulting from traumatic brain
injury have been observed at both light and electron microscopic
levels (Doering et al., 2007). The functional significance of the
abundant vesicular zinc in ectopic MFBs or surviving ZEN terminals
adjacent to traumatised neocortex is unknown at present, but it
definitely calls for further investigation.
Acknowledgements
For linguistic assistance we thank Ms. K. Wiedemann.
Supported by Natural Science Foundation of China (30770680,
30670722, 30370452), Specialized Research Fund for the Doctoral
Program of Higher Education (SRFDP-20060159001), Program for
New Century Excellent Talents in University (NCET-04-0288), and
China Postdoctoral Science Foundation (2005037008). The Augus-
tinus Foundation, the Aarhus University Research Foundation, Aase
& Ejnar Danielsens Fond, the Danish Medical Association Research
Fund (‘‘Boet efter Johanne Dorthe Due’’), and the Lundbeck
Foundation of Denmark.
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