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First published online 4 July 2007
doi: 10.1242/dev.002329
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,
1 Department of Medical Biochemistry and Biophysics, Karolinska Institute,
Center of Excellence in Developmental Biology, Stockholm, Sweden.
2 Department of Cell and Molecular Biology, Medical Nobel Institute, Center of
Excellence in Developmental Biology, Karolinska Institute, Stockholm,
Sweden.
Author for correspondence (e-mail:
Andras.Simon{at}ki.se)
Accepted 4 June 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
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Key words: Neurogenesis, Newt, Regeneration, 6-hydroxydopamine, Behaviour, Dopamine, Parkinson's disease
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INTRODUCTION |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
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;
Bjorklund et al., 2002;
Kim et al., 2002). Human
foetal mesencephalic tissue has been tested in transplantation trials
(Winkler et al., 2005), but
methodological difficulties and ethical considerations have prevented the
clinical development of these techniques
(Bjorklund et al., 2003;
Freed et al., 2003).
Alternative approaches could involve adult somatic stem cell transplantation,
or stimulation of endogenous neurogenesis in situ
(Lindvall et al., 2004).
To address whether regeneration of DA neurons is feasible in the adult
vertebrate brain, we studied an aquatic salamander, the newt, in which the
mechanisms of cellular plasticity and functional tissue repair could be
analysed. Following injury or tissue removal, adult newts regenerate large
body structures, such as entire limbs and tails
(Tanaka, 2003). Regeneration
in salamanders could involve, depending on the site of the injury, both the
activation of stem cells and/or reprogramming of differentiated cells
(Brockes and Kumar, 2005;
Grogg et al., 2005;
Morrison et al., 2006). Tail
regeneration, for example, involves lineage-shifting events in which radial
glia-like cells (so called ependymoglial cells) give rise not only to neurons
and glia, but also integrate into newly formed muscle and cartilage
(Echeverri and Tanaka,
2002).
Little is known about how newts respond to brain injury, although available
reports indicate increased proliferation and possibly neurogenesis after
mechanical lesions (Minelli et al.,
1990; Minelli et al.,
1987). A toxin model of DA cell loss in amphibia has been
presented previously, but the issue of neurogenesis or behavioural recovery
was not addressed (Barbeau et al.,
1985).
We endeavoured to examine the regenerative/neurogenic capacity of the
salamander brain following cell type-specific chemical lesioning with
6-hydroxydopamine (6-OHDA), a neurotoxin selective for catecholamine neurons
and which is widely used to create parkinsonian animal models
(Deumens et al., 2002).
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
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Immunohistochemistry
Animals were anesthetised by immersion in 0.1% MS-222 (Sigma) and perfused
with 4% paraformaldehyde in PBS. Brains were removed, post-fixed for 1 hour
and left at 4°C overnight in 20% sucrose in PBS. Lesion size was assessed
as the total number of tyrosine hydroxylase (TH)-positive neurons in a
lesioned animal relative to sham-lesioned newts. A 1:5 series was cut on the
coronal plane through the brain with a section thickness of 18 µm. The
diencephalic-mesencephalic DA population extended through 
10-12 sections
per brain (1000 µm in length on a rostrocaudal axis). This DA
population commenced immediately caudal to the preoptic area and ended at the
rostral tegmental area of the midbrain (TM), and comprised 780 TH+
neurons in total. For an illustration of the DA midbrain region of the newt
brain, as previously mapped by others
(Gonzalez and Smeets, 1994;
Marin et al., 1997), see Fig.
S1 in the supplementary material. These cells project to the striatum, which
was confirmed in our studies by the loss of striatal fibres corresponding to
loss of DA cell bodies (see Fig. S1 in the supplementary material). The TM
contained 1700 cells in total, of which 1200 were NeuN+ and 300
TH+. The TM extended through three sections per brain. Counts were
made by overlaying a grid on the TM, aligning the midline and ventral pial
surface of the brain in each section. The same grid area was counted for each
animal. The grid overlay corresponded with the TH+ population
within the TM. Repeatability and low standard deviation of counts within
groups confirmed the consistency of the counting method.
Total engrailed 1 and Nurr1 counts were made in adjacent series of sections. All engrailed 1-labelled and Nurr1-labelled cells were counted within the sections of the TM for each animal.
For immunohistochemistry, sections were incubated with one of the following primary antibodies: rabbit anti-TH (1:250, Chemicon); mouse anti-TH (1:250, Chemicon); mouse anti-NeuN (1:100, Ab Cam); mouse anti-GFAP (1:500, Sigma); rat anti-BrdU (1:250, Accurate Chemical and Scientific Corporation); rabbit anti-Nurr1 (1:250, Santa Cruz); mouse anti-engrailed 1 [1:10, Developmental Studies Hybridoma Bank (DSHB)]; mouse anti-Msx1/2 (1:3, DSHB); Sox2 (1:100, a kind gift from T. Edlund, Umeå University, Sweden); Foxa2 (1:10, DSHB). The following day, sections were incubated for 2 hours with the appropriate secondary antibodies (Alexa 546 and Alexa 448 IgG, 1:1000 dilution; Molecular Probes). TUNEL-staining was performed in accordance to the manufacturer's protocol for cryosections (Fluorescein-FragEL, Oncogene Research Products, MA). Cells were observed using a Zeiss upright microscope, and pictures were captured by a colour CCD camera. A confocal laser-scanning microscope (Zeiss) was used to detect and quantify BrdU+ TH+ and BrdU+ GFAP+ cells. Cell counts are presented as the number of cells per animal unless indicated otherwise in the figure legend.
6-OHDA lesions
Newts were anaesthetised by immersion in 0.1% MS-222 and the head secured
in a neonatal stereotaxic head frame. A 6 µg/µl solution of 6-OHDA was
prepared with ascorbic acid (0.2 mg/ml) and kept on ice until the time of
injection. A glass micropipette was coupled with tubing to a 2 µl Hamilton
syringe (with a 26-gauge needle) and mounted in the stereotaxic frame. The
micropipette containing the 6-OHDA was inserted into the brain through a small
(0.5 mm) hole drilled at the junction of the parietal and frontal bones in the
cranial midline. The micropipette was inserted to a level 0.8 mm below the
surface of the brain and the toxin (200 nl) was injected into the third
ventricle, at the level of diencephalic/mesencephalic DA neurons. On
completion of the injection, the needle was left in situ for 1 minute and then
slowly withdrawn. Sham-lesioned animals were injected with 200 nl of 0.9%
saline. Following surgery, the bone hole was sealed with dental cement and the
animals left to recover overnight in 0.5% sulfamerazine (Sigma). Accuracy of
injection was previously confirmed using injections of indian ink. TH counts
revealed that we were able to obtain consistent, dose-dependent bilateral
lesions of the newt DA cell groups with lesion sizes ranging from 5-97% of
total TH+ cells.
Behavioural assessment
Behaviour was assessed between 13.00 and 16.00 hours CET with temperature
and lighting similar to the newt's normal housing conditions. The observation
plates were clear plastic 15 cm-diameter dishes with a 1.5 cm-high wall and a
3 cm-square grid on the base. The plates were filled with 50 ml tap water. On
the day of testing, newts were injected intraperitoneally with amphetamine (25
mg/kg) and placed into the observation plate. Animal behaviour was captured on
a digital video camera (Sony). Each animal was observed for 1 minute every 10
minutes, commencing 5 minutes after injection, for a 50-minute period. For
regeneration/neurogenesis studies, only animals with lesions greater than 70%,
based upon their behaviour, were used. Sham and control animals showed similar
results at each of the analysed time points and data for sham groups were set
as 100% for each data point.
BrdU pulse-labelling
BrdU (Sigma, 300 mg/kg) was injected intraperitoneally at day 1, 3, 5, 7,
10 and 19. Twenty-four hours after the single BrdU pulse, animals were
anaesthetised, perfused and the brains processed for immunohistochemistry. For
BrdU TH double-labelling experiments, newts were pulsed twice daily with BrdU
(20 mg/kg) for 10 days and then daily from day 11-30. After 30 days, newts
were sacrificed. In additional experiments, lesioned newts were pulsed daily
on days 0-3 (during the period of DA cell death) or on days 4-23 (after cell
death and during the neurogenic period) and analysed at 23 days for
TH+ BrdU+ cells. Brains were sectioned and
immunohistochemistry, using antibodies against TH and BrdU, was performed on
all sections. Using confocal microscopy, the total number of TH+
BrdU+ cells was counted.
AraC treatment
Animals were lesioned and behaviour tests were performed at 3 days. AraC
(500 mg/kg, Sigma) was administered by intraperitoneal injection three times
daily (day 4-8) or twice daily (days 4-14) into lesion and control animals.
Animals received two pulses of BrdU (150 mg/kg) either on day 7 or 13 and
behavioural analysis was performed at day 8 or 14. All brains were sectioned
and stained for BrdU and TH.
RESULTS
To identify DA neurons, we used immunohistochemical analyses with
antibodies raised against tyrosine hydroxylase (TH, the rate-limiting enzyme
in dopamine biosynthesis), Nurr1 and engrailed 1
(Arenas, 2005). We focused our
study on 780 ventral diencephalic/mesencephalic TH+ cells (see Fig.
S1 in the supplementary material), which were consistently affected by toxin
administration into the third ventricle (see below). Whereas DA neurons in
fish have only been observed in the diencephalon, the medial part of the
diencephalic posterior tuberculum (TPm) in newts extends and forms a continuum
with the mesencepahlic DA cells of the rostral tegmental area (TM) (see Fig.
S1G,H in the supplementary material). The catecholaminergic system in newts
has been extensively studied (Gonzalez and
Smeets, 1994; Marin et al.,
1997), and these studies suggested that the continuous
diencephalic and mesencephalic DA population is functionally homologous to the
mammalian ventral tegmentum and substantia nigra.
To lesion the DA system, 6-OHDA was stereotaxically injected into the third ventricle, in the vicinity of diencephalic/mesencephalic DA neurons (Fig. 1L). Consistent bilateral lesions were seen in the DA groups shown in the supplementary material (see Fig S1E'-H' in the supplementary material). Three days after 6-OHDA injection, we observed the loss of TH+ cells and fibres in the diencephalic/mesencephalic regions, as compared with the uninjected controls (Fig. 1A-I). To confirm cell death, and not just a loss of phenotype, as a result of the neurotoxin, we performed double-immunostaining for fragmented DNA (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, TUNEL) and TH. The presence of abundant TUNEL+ TH+ cells indicated that DA neurons died as a result of the 6-OHDA injection (Fig. 1J,K). Moreover, we found a significant increase in the number of pyknotic cells at 12 hours after lesioning, which further increased at 24 hours (Fig. 1M). Cell death was also evident from the loss of nuclei in the neuronal layers in the TM of the mesencephalon (Fig. 1N). In total, 1700 cells compose the entire TM as revealed by DAPI staining. Of these 1700 cells, 1200 are NeuN+ and 300 express TH. In addition to the loss of TH+ cells, we detected a significant reduction in the number of cells expressing the DA markers Nurr1 and engrailed 1, as compared with the sham injections (Fig. 1O). The number of TH-expressing cells was unchanged in control and sham-injected animals (Fig. 1I). Similarly sham injection did not alter the number of Nurr1- or engrailed 1-expressing cells (data not shown). These data indicate that 6-OHDA injection led to the death of DA neurons, not just to a loss of phenotype.
To assess the functional significance of the loss of DA cells, we devised a
quantifiable, pharmacological locomotion assay. Animals received a single
intraperitoneal injection of amphetamine, which stimulates DA release
(Ungerstedt, 1971), and were
placed in separate grid chambers. We quantified movement by counting the
number of occasions a newt's tail crossed a line in a grid while swimming, as
the typical movement of a newt can be described as a sine wave that starts at
the head region and propagates to the tail
(Davis et al., 1990). In
addition, tail movement should not be affected by our regimen of 6-OHDA
administration because the cell groups we lesion do not project to the spinal
cord in salamanders (Sanchez-Camacho et
al., 2001). Movies 1 and 2 (see Movies 1 and 2 in the
supplementary material) show sham- and 6-OHDA-injected newts with or without
an amphetamine challenge. Amphetamine induced a significant increase in
locomotion only in uninjected and sham-injected animals, in which the DA
system was intact, but not in 6-OHDA-injected animals
(Fig. 2A). It was also evident
that 6-OHDA-injected animals moved less than the shams, indicating that the
damage was so extensive that it reduced baseline movements. Importantly,
behavioural responses were in strong correlation with the number of
TH+ cells (Fig. 2B);
thus, in subsequent experiments, the degree of lesion could be estimated by
this behavioural test.
We quantified the amphetamine response and the number of TH+ cells over time in those animals that, 3 days after 6-OHDA injection, showed a greater than 70% reduction in amphetamine-induced locomotion, as compared with shams. As shown in Fig. 2C, we observed substantial improvements in behavioural responses, with animals reaching normal motor scores by 30 days. The kinetics of behavioural improvement followed the timing of the reappearance of TH+ cells (Fig. 2D). After 30 days, full recovery of the number and distribution of TH+ cell bodies along the entire anterior-posterior axis of the diencephalon and mesencephalon was detected (Fig. 2E-G, and see Fig. S2 in the supplementary material). We did not observe any difference in the recovery of the different TH+ subpopulations within the diencephalon/mesencephalon (as depicted in Fig. S1 in the supplementary material). Thus, newts were able to regenerate the DA system as assessed by cellular and functional analyses.
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During tail regeneration, ependymoglia cells of the spinal cord can act as
neural stem cells (Echeverri and Tanaka,
2002). Similar to the central canal of the spinal cord, we found
that glial fibrillary acidic protein (GFAP)-positive, radial glia-like
ependymoglia cells line the third ventricle of the brain in the vicinity of
neurons (Fig. 3B'). Cells
lining the ventricle also expressed Sox2
(Fig. 3B''), which has
been shown to be expressed in neural stem and progenitor cells and to repress
proneural genes (Bylund et al.,
2003). Animals received a single pulse of BrdU immediately upon
completion of the lesion. Six hours after the single BrdU pulse,
BrdU+ cells were seen most prominently in the GFAP+ cell
layer (Fig.
3C',C''). Thirteen days after a single BrdU pulse in
lesioned animals, significantly more BrdU+ cells were observed in
deeper cell layers (Fig.
3C',C'''), where NeuN+ cells are found
(Fig. 3B').
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To further examine whether neurogenesis was involved in the regenerative process, we examined the total number of cells and neurons in the TM after lesion. Whereas at 3 days the total cell number and the number of neurons were reduced, at 30 days both populations were restored, showing that cells were indeed eliminated and new cells appeared (Fig. 3E).
To determine the extent to which cellular proliferation was responsible for
behavioural and cellular recovery, newts were treated with cytosine
ß-D-arabinofuroside (AraC), an antimitotic compound used to
block neurogenesis in rodent models
(Doetsch et al., 1999). We did
not detect any proliferating cells after AraC treatment
(Fig. 3F'''). AraC
treatment had no effect on the behaviour of control animals
(Fig. 3F'), indicating
the lack of toxicity in our assay. However, AraC treatment from day 4-8 or day
4-14 blocked the behavioural recovery and significantly reduced cellular
recovery as reflected in the number of TH+ neurons
(Fig. 3F''). Thus, our
results indicate that the behavioural recovery in the newt is indeed
contributed by adult DA neurogenesis.
As neurogenesis was a response to the loss of TH+ cells evoked
by 6-OHDA lesion, we examined whether the mechanisms of tissue repair in the
parkinsonian salamander were similar, to some extent, to the process of
epimorphic regeneration following limb or tail amputation. The transcriptional
repressors Msx1/2 have been found to be regulated by and functionally linked
to the process of regeneration in several models, including the limb of
salamanders, the tail of frogs and the digit tip of mice
(Carlson et al., 1998;
Odelberg, 2004).
Interestingly, the ablation of Msx1 in mice results in loss of Wnt1
(Bach et al., 2003), a factor
that plays a crucial role in the generation of midbrain DA neurons
(Prakash et al., 2006).
Furthermore, Msx1 was recently found to be required for the development of DA
neurons in the ventral mesencephalon
(Andersson et al., 2006). We
noted that Msx1/2 expression was not restricted to the neuroepithelium, as in
the developing mammalian midbrain
(Andersson et al., 2006), but
was also expressed in neuronal layers. We observed that the number of
Msx1/2-expressing cells transiently decreased in the lesioned area at 24 hours
(Fig. 4A,B''') and,
subsequently, significantly increased above control levels at 7 days
(Fig. 4A,B''''). No
change was observed after sham injections
(Fig. 4A,B'') and no
increase in the number of Msx1/2-expressing cells was detected dorsally (see
Fig. S4 in the supplementary material), arguing for the specificity of the
regulation. We next investigated whether the expression of proteins found in
mature DA neurons and DA precursors, Nurr1 and engrailed 1
(Arenas, 2005), changed during
DA regeneration. We found that the number of Nurr1+ cells gradually
increased in the ventral midbrain after the loss of DA neurons, and that Nurr1
expression preceded the expression of TH
(Fig. 4C), implying that Nurr1
is also a marker for DA precursors in newts. A similar temporal change was
observed for engrailed 1 (see Fig. S3 in the supplementary material).
Together, these results indicate that adult DA regeneration in the newt
recapitulates some of the key molecular events that take place during
embryonic DA neurogenesis in mammals.
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DISCUSSION |
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INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
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), Msx1
has been implicated in skeletal muscle dedifferentiation
(Kumar et al., 2004). At
present we cannot exclude the possibility that dedifferentiation also plays a
role in DA regeneration in the salamander brain. Dedifferentiation could in
principle generate a proliferating DA progenitor that subsequently
differentiates into DA neurons, as illustrated here. Future studies will aim
at addressing this possibility. However, the observation that ependymoglia
cells are first to incorporate BrdU, and that the label appears in deeper
(neural) layers after a chase experiment, suggest that activation of
GFAP/Sox2-expressing ependymoglia is one important component of the process.
Interestingly, two recent protocols have reported that radial glia-like cells
with neurogenic capacity can be derived from mammalian embryonic stem cells
(Bibel et al., 2004;
Conti et al., 2005). It
remains to be determined whether endogenous or stem cell-derived mammalian
cells can be differentiated into ependymoglial-like cells capable of
undergoing DA neurogenesis and whether these cells might contribute to
functional recovery.
We show that injury evokes increased cellular proliferation and midbrain DA
neurogenesis. The existence of adult mammalian neurogenesis has been described
and characterised previously in the subventricular zone-olfactory bulb pathway
and in the dentate gyrus (Alvarez-Buylla
and Lim, 2004; Kempermann et
al., 2004). It has also been suggested that neurons could be
generated in other parts of the brain, such as DA neurons in the adult
midbrain (Frielingsdorf et al.,
2004; Lie et al.,
2002; Zhao et al.,
2003). However, this is a controversial issue because adult DA
neurogenesis in mammals is either undetectable or a rare and inefficient
process that is difficult to study. By contrast, the salamander 6-OHDA model
revealed robust DA regeneration/neurogenesis. A comparable process has not
been observed in other adult vertebrate models of Parkinson's disease, making
the salamander model a useful complement to other currently available models.
We see that the adult newt brain can reactivate quiescent cells upon injury,
and contains sufficient extracellular cues to direct activated neural
progenitors towards a specific neural subtype within an existing brain
structure. Hence, the 6-OHDA-induced regeneration model in salamander provides
a basis for identifying and understanding the cues required for adult DA
neurogenesis/regeneration. In the future, it will be possible to test whether
activation or manipulation of these cellular and molecular programs could
contribute to regeneration in mammalian models.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2881/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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These authors contributed equally to this work
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
DISCUSSION
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