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Keywords:

  • dopaminergic neuron;
  • dopamine transporter;
  • zebrafish

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We have generated a line of transgenic zebrafish, Tg(dat:EGFP), in which the green fluorescent protein (GFP) is expressed under the control of cis-regulatory elements of the dopamine transporter (dat) gene. In Tg(dat:EGFP) fish, dopamine (DA) neurons are labeled with GFP, including those in ventral diencephalon (vDC) clusters, amacrine cells in the retina, in the olfactory bulb, in the pretectum, and in the caudal hypothalamus. In the vDC, DA neurons of groups 2–6 are correctly labeled with GFP, based on colocalization analyses. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) treatments induced a modest but significant loss of DA neurons in groups 2–6 of the vDC. This transgenic line will be useful for the study of DA neuron development and in models of DA neuron loss. Developmental Dynamics 240:2539–2547, 2011. © 2011 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

In the central nervous system (CNS), the neurotransmitter dopamine plays important roles in a variety of physiological and behavioral processes such as voluntary movement, cognition, memory and reward (Bjorklund and Dunnett, 2007). Dopaminergic (DA) neurons synthesize dopamine from tyrosine through the action of tyrosine hydroxylase (TH), the first and rate-limiting enzyme, and DOPA decarboxylase. After release of dopamine by DA neurons, the excess dopamine in the synaptic cleft can be re-uptaken back into DA neurons by means of the action of the dopamine transporter (DAT), which is an intra-membrane protein expressed in DA neurons (Ciliax et al., 1995, 1999).

Deficiency of dopamine neurotransmission is implicated in several neurological diseases including Parkinson's disease (PD). The main pathological hallmark of PD is the progressive loss of DA neurons in the substantia nigra pars compacta (SNc) of the midbrain, which results in a deficiency in released dopamine in the striatum and causes movement disorders. Although several genes have been found to be associated with familial inheritable PD, the detailed etiology of PD is still not well understood (Cookson, 2005). A majority of PD cases are sporadic, which may result from environmental factors. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin known to induce PD-like symptoms in human (Langston and Ballard, 1983), has been used to induce DA neuron loss in various animal models.

The zebrafish has proven to be an excellent vertebrate model for developmental biology and the study of human diseases (Driever et al., 1994, 1996; Zon, 1999). Transgenesis is widely used in zebrafish due to its transparent and externally developing embryos (Lin, 2000). Despite notable differences, the overall organization of the zebrafish brain shows similarities to that of the human brain. In zebrafish, the development of DA neurons has been examined by morphological and genetic approaches (Ma, 1994, 2003; Rink and Wullimann, 2002). Although no DA neurons have been found in the midbrain of zebrafish, several groups of DA neurons in the posterior tuberculum of ventral diencephalon have been suggested as homologues of DA neurons in SNc of human as they send ascending projections to the subpallium, similar to SNc DA projections to the striatum (Rink and Wullimann, 2001). However, a recent analysis of the catecholaminergic projectome in zebrafish has shown that this homology may need to be reconsidered and that the main dopamine source in the zebrafish telencephalon may be derived locally (Tay et al., 2011). Several genes have been suggested to be involved in DA neuron development (Guo et al., 1999; Ryu et al., 2007).

In this study, we took advantage of the fact that DAT expression distinguishes DA neurons from other catecholaminergic (CA) neurons in the developing embryo (Holzschuh et al., 2001) and produced a line of transgenic fish in which the green fluorescent protein (GFP) is expressed under the control of regulatory elements of the zebrafish dat gene. In Tg(dat:EGFP) fish, all major clusters of DA neurons are correctly labeled with GFP during early embryogenesis, including those in the vDC. MPTP treatment caused the death of a significant albeit modest number of GFP-positive DA neurons in the vDC of these fish. This transgenic line will be very useful to study DA neuron development and in models of Parkinson's disease pathogenesis.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Generation of Tg(dat:EGFP) Lines

To screen for clones containing the dopamine transporter (dat) gene from a zebrafish genomic PAC library (P1 artificial chromosome; RZPD, Berlin, Germany), a partial sequence of exon 1 of zebrafish dat gene was amplified and radioactively labeled by polymerase chain reaction (PCR), and later used as a probe for DNA hybridization. A PAC clone (BUSMP706K0187Q9, RZPD) was identified and confirmed to contain the whole genomic sequence of dat except for its last coding exon and the 3′-flanking region. The enhanced green fluorescent protein (EGFP) was inserted in frame into dat exon 1 by homologous recombination in bacteria (Fig. 1A, Liu et al., 2003). A 27-kb fragment (including approximately 13 kb of dat 5′-flanking region) was then cloned into a pGEM-Tol2 vector in another round of homologous recombination. More than 90% of embryos injected with this construct in the presence of tol2 transposase mRNA showed GFP expression in areas located roughly between the eyes at 2 days post-fertilization (dpf). Among a total of 42 founder fish, 6 fish were identified to have germ-line transmission and 4 of them gave similar GFP expression patterns in transgenic F1 fish.

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Figure 1. The Tg(dat:EGFP) line. A: A schematic map of the DNA fragment used in Tol2-based dat transgenesis. The total size of the inserted DNA is 27 kb, containing the whole dat genomic sequence except for the last coding exon and 3′-flanking region. EGFP (in green) was inserted in frame at the beginning of exon 1. A stop codon and a polyadenylation signal sequence (pA) at the end of green fluorescent protein (GFP) are indicated. B: Reporter gene expression in Tg(dat:EGFP) larvae. A composite of different fluorescent focal planes was generated using the Image Pro software and merged with bright field image. MHB, midbrain–hindbrain boundary; Ob, olfactory bulb; Pr, pretectum; Tel, telencephalon; vDc, ventral diencephalon. The arrow indicates a group of cells in the hindbrain that express GFP and may correspond to some of the dat-expressing cells reported by Holzschuh et al. (2001). C: Double fluorescent in situ hybridization on 3 days post-fertilization (dpf) larvae with GFP (red) and dat (green) cRNA probes. The arrow indicates a group of cells in the preoptic area that express the transgene and th (not shown) but not dat. Scale bars = 100 μm.

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To determine GFP expression in Tg(dat:EGFP) fish, we crossed transgenic F1 fish with wild-type fish and examined their progeny under a fluorescence microscope at different time points (Fig. 1B and data not shown). GFP-positive neurons become visible at approximately 20 hours post-fertilization (hpf) in the telencephalon (data not shown). By 2–3 dpf, several groups of GFP-positive cells are found specifically in the telencephalon, diencephalon, midbrain, in the eye, and in the pharyngeal arch area. This GFP expression pattern persists in 15 dpf juveniles and in adults (Fig. 1B and data not shown). Double fluorescent in situ hybridization with GFP and dat cRNA probes indicates that the transgene is expressed as predicted in dat-expressing cells, notably in the ventral diencephalon (Fig. 1C), although there are also a few cells that express GFP mRNA but not dat (Fig. 1C, arrow). Additional sites of ectopic GFP expression included the jaw and groups of cells at or near the midbrain–hindbrain boundary. These were seen in at least three independent transgenic lines are thus unlikely due to transgene integration effects.

Comparative Expression of tyrosine hydroxylase (th) and dopamine transporter (dat)

The tyrosine hydroxylase (th) and dopamine transporter (dat) genes are commonly used as markers for mature DA neurons. We examined their expression in zebrafish by whole-mount in situ hybridization (Fig. 2). During embryogenesis, both th and dat are expressed in DA neurons, based on their position in the vDC.

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Figure 2. Whole-mount in situ hybridizations of tyrosine hydroxylase (th) and dopamine transporter (dat) in zebrafish embryos and larvae. Whole-mount in situ hybridization with a tyrosine hydroxylase (th) (A–F) or a dopamine transporter (dat) cRNA probe (G–L). The dopaminergic (DA) neurons in the ventral diencephalon are shown by arrowheads. A,C,E,G,I,K: Lateral views with anterior to the left. B,D,F,H,J,L: Ventral views. Scale bar = 100 μm.

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To better compare Tg(dat:EGFP) transgene expression with the position of DA neurons, we performed whole-mount in situ hybridization with a th1 probe which, in our hand gives us more robust signals than the dat probe, although it has the disadvantage of not marking DA neurons exclusively (Fig. 3). At 3 dpf, similar to th1 expression, GFP is expressed mainly in the olfactory bulb (Ob), pretectum (Pr), and vDC. GFP expression was also seen in the caudal hypothalamus (Hc), although this did not coincide with th1 expression (Fig. 3; see the Discussion section).

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Figure 3. Comparison of th and green fluorescent protein (GFP) expression patterns in Tg(dat:EGFP) larvae. A,B: Whole-mount in situ hybridization showing tyrosine hydroxylase (th) expression in 3 days post-fertilization (dpf) larvae. C,D: Live images showing GFP expression in 3 dpf Tg(dat:EGFP) larvae. Panels A, C are lateral views with anterior to the left; panels B, D are ventral views. The following abbreviations are used: olfactory bulb (Ob), pretectum (Pr), ventral diencephalon (vDC), amacrine cells (Ac) and caudal hypothalamus (Hc). Scale bars = 100 μm.

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The Majority of DA Neurons Are Correctly Labeled With GFP

To further determine whether the various clusters of DA neurons in the zebrafish brain are labeled by GFP, double immunofluorescence staining for TH and GFP was performed in Tg(dat:EGFP) embryos. At 3 dpf, DA neurons are mainly found in the vDC, Ob, Pr, Hc, and Ac in the retina based on the immunostaining with TH antibodies. In the vDC, DA neurons are normally divided into six different groups based on their location, morphology and function (Rink and Wullimann, 2002). In the vDC of 3 dpf Tg(dat:EGFP) larvae, the TH-positive DA neurons of groups 2, 3, 4/5, and most of those in group 6 are labeled with GFP, although those of group 1 are not (Fig. 4). Furthermore, the GFP-positive neurons of groups 2, 3, 4/5, and most of those in group 6 are TH-positive (Fig. 4A–B′″). This was also confirmed by confocal microscopy analysis of whole-mount larvae immunostained for both GFP and TH at 3 dpf (Fig. 5). Double immunostaining on transverse cryosections of 3dpf Tg(dat:EGFP) larvae also showed colocalization of GFP and TH in DA neuron groups of 2, 3, 4/5, and 6, but not in group 1 (Fig. 6). This colocalization pattern was also observed at 5 dpf (Fig. 7).

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Figure 4. Double immunostaining for green fluorescent protein (GFP) and tyrosine hydroxylase (TH) in 3 days post-fertilization (dpf) Tg(dat:EGFP) larvae. Horizontal cryosections of 3 dpf Tg(dat:EGFP) larvae were stained with anti-GFP and anti-TH antibodies. GFP-positive cells, TH-positive cells and GFP/TH-positive cells are shown in green, red and yellow, respectively. The following abbreviations are used: olfactory bulb (Ob), pretectum (Pr), ventral diencephalon (vDC), amacrine cells (Ac) and caudal hypothalamus (Hc). Numbers in A′″ and B′″ indicate different groups (1–6) of DA neurons in the vDC. Arrows show GFP/TH-positive cells in the retina. Arrowheads show GFP/TH-positive cells in the Ob, Pr and Hc. Section (A–A′″) and section (B–B′″) are the same section, focusing on different cell groups when imaging. All sections are shown as anterior to the left. Scale bars = 100 μm in A–A″,B–B″,C–C″,D–D″,E–E″; 25 μm in A′″,B′″,C′″,D′″,E′″.

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Figure 5. Whole-mount immunostaining for green fluorescent protein (GFP) and tyrosine hydroxylase (TH) in 3 days postfertilization (dpf) Tg(dat:EGFP) larvae. The 3 dpf Tg(dat:EGFP) larvae were stained with anti-GFP and anti-TH antibodies, followed by confocal microscopy. GFP-positive cells and TH-positive cells are shown in green and red respectively. A–H: A series of confocal images focusing at different levels in the ventral diencephalon (vDC). I: Projection of confocal images A–H. Numbers in I indicate different DA neuron groups (1–6) in vDC. The animals are shown as dorsal views with anterior to the left. Scale bars = 25 μm.

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Figure 6. Double immunostaining for green fluorescent protein (GFP) and tyrosine hydroxylase (TH) on transverse cryosections of Tg(dat:EGFP) larvae at 3 days postfertilization (dpf). The 3 dpf Tg(dat:EGFP) embryos were transversely cryosectioned and stained with anti-GFP and anti-TH antibodies. GFP-positive cells and TH-positive cells are shown in green and red respectively. GFP expression in the optic nerve (A″) may relate to the previously reported DAT expression in astrocytes of the optic nerve (Holzschuh et al., 2001). Numbers (1–6) indicate different groups of DA neurons in the ventral diencephalon. Pr: pretectum. All sections are shown as dorsal to the top. Scale bar = 100 μm.

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Figure 7. Double immunostaining for green fluorescent protein (GFP) and tyrosine hydroxylase (TH) on horizontal cryosections of Tg(dat:EGFP) larvae at 5 days post-fertilization (dpf). The 5 dpf Tg(dat:EGFP) embryos were horizontally cryosectioned and stained with anti-GFP and anti-TH antibodies. GFP-positive cells and TH-positive cells are shown in green and red respectively. Numbers (1–6) indicate different groups of DA neurons in the ventral diencephalon. All sections are shown as anterior to the top. Scale bar = 100 μm.

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Co-labeling indicates that all TH-positive DA neurons in the Ac, Ob, and Pr are also GFP-positive (Fig. 4B–D′″). In the Hc, all TH-immunoreactive DA neurons express GFP, but not all GFP-expressing neurons are TH-positive (Fig. 4E–E′″). One possible explanation for this discrepancy might be that the anti-TH antibody used here can only recognize a fraction of TH-positive neurons in the Hc (Chen et al., 2009; Fillipi et al., 2010; Yamamoto et al., 2010, 2011, more about this in the Discussion section).

MPTP Affects the Survival of DA Neurons in the Ventral Diencephalon

We have used the DA neurons in the vDC of Tg(dat:EGFP) fish as a live model to examine the effects of MPTP on DA neuron survival. MPTP is a neurotoxin that causes PD-like symptoms in humans. It is suggested that MPTP can interrupt the electron transfer chain in mitochondria at complex I, and this eventually causes cell death (Nicklas et al., 1987). Conflicting results were obtained from previous studies with respect to the effects of MPTP on DA neuron survival in zebrafish (Bretaud et al., 2004; Lam et al., 2005; McKinley et al., 2005; Wen et al., 2008; Sallinen et al., 2009). Here, we re-examined this issue using the Tg(dat:EGFP) fish.

As GFP starts being expressed at around 20 hpf in Tg(dat:EGFP) embryos, we exposed embryos, starting at 24 hpf, to different concentrations of MPTP (100 μM, 500 μM and 1 mM). A concentration of 1 mM was found to cause the most severe reductions in DA neuron numbers in the vDC (data not shown). After 2 days of 1 mM MPTP treatment, the number of GFP-positive neurons in the vDC (groups 2–6) is significantly reduced from 49.8 ± 3.9 (n = 19) to 38.9 ± 6.1 (n = 18, Student's t-test, P < 0.01; Fig. 8A,B,E). After 4 days of MPTP treatment, the number of GFP-positive neurons (groups 2–6) is also significantly reduced from 74.0 ± 2.4 (n = 14) to 59.8 ± 4.8 (n = 16, Student's t-test, P < 0.001; Fig. 8C–E). The DA neurons of groups 4/5 are the first to die after MPTP treatment, and seem more sensitive to MPTP than those of other groups (Fig. 8A–D). This was also confirmed by double immunostaining for GFP and TH on cryosections of control and MPTP-treated Tg(dat:EGFP) embryos (Fig. 8F–G′″).

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Figure 8. Effects of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) on green fluorescent protein (GFP) -positive neurons in the ventral diencephalon. Tg(dat:EGFP) embryos were untreated (Ctrl) or treated with MPTP at 1 mM from 24 hours post-fertilization (hpf), and examined under fluorescence microscope at 3 days post-fertilization (dpf) (A,B) and 5 dpf (C,D). The numbers of GFP-positive neurons in the ventral diencephalon (dopaminergic [DA] neuron groups 2–6) were counted and statistically analyzed (E; **P < 0.01, ***P < 0.001). F–G′″: Double immunostaining for GFP and tyrosine hydroxylase (TH) on Ctrl or MPTP-treated embryos at 3 dpf. A–D: Anterior is to the bottom; F–G′″: Anterior is to the left. More than 30 larvae were examined in each group. Scale bars = 25 μm.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Transgene Expression in Zebrafish DA Neurons

Several attempts have been made to produce transgenic zebrafish lines that express reporter genes in DA neurons but none of them successfully labeled all six groups of DA neurons in the vDC (Gao et al., 2005; Meng et al., 2008; Wen et al., 2008; Bai and Burton, 2009; Fujimoto et al., 2011). A 4.5-kb rat th promoter fragment could only drive GFP expression in retina cells (Gao et al., 2005). A 12 kb zebrafish th promoter could not drive GFP expression in DA neurons (Meng et al., 2008). Constructs containing either a 7-kb or a 11-kb promoter fragment from the dat gene were shown to drive GFP expression in DA neurons but only in the pretectal region (Bai et al., 2009). A new enhancer from the otpb gene drove GFP expression in diencephalic DA neurons but only in groups 4/6 (Fujimoto et al., 2011). An enhancer trap transgenic line, ETvmat2:GFP, can label most monoaminergic neurons with GFP but was not specific for DA neurons (Wen et al., 2008).

Here, we introduced EGFP in frame within the first exon of dat in a PAC that contains almost the entire dat transcription unit. Using this reporter gene, we generated a transgenic zebrafish line Tg(dat:EGFP) that specifically recapitulates most of the endogenous DA neuron patterning. This included the DA neurons in the vDC (groups 2–6). Consistent with a previous study (Wen et al., 2008), the DA neurons of group 1 and some neurons in group 6 are not labeled with GFP.

It is suggested that there are two tyrosine hydroxylase genes in zebrafish (th1 and th2) due to the teleost-specific whole genome duplication (Candy and Collet, 2005). Most of th2-positive neurons are located in the caudal hypothalamus (Hc) during brain development (Chen et al., 2009; Filippi et al., 2010; Yamamoto et al., 2010). We showed that GFP expression is detected in cells of the Hc in Tg(dat:EGFP) fish. These GFP-positive neurons are probably th2-positive neurons. However, not all GFP-positive neurons are TH-positive possibly due to the fact that the anti-Th antibody used in our study has a relatively lower affinity for the TH2 protein (Yamamoto et al., 2010).

MPTP Toxicity on Zebrafish DA Neurons

Previous studies showed conflicting results after MPTP treatment on zebrafish larvae. It was shown that almost all the DA neurons in the vDC seemed to be lost after MPTP treatment (Bretaud et al., 2004; Lam et al., 2005). However, some studies showed only a partial loss of DA neurons in the vDC after MPTP (McKinley et al., 2005; Wen et al., 2008; Sallinen et al., 2009). In all these studies, DA neurons were identified based on the TH/th-immunoreactivity and on their locations. The expression of TH/th is more widespread than that of Dat/dat (Holzschuh et al., 2001), which is specifically found in DA neurons. Furthermore, MPTP is metabolized into the toxic MPP+ which, in turn, is taken into DA neurons through Dat. Here, we re-examined this issue by observing MPTP-treated Tg(dat:EGFP) embryos alive under a fluorescence microscope. We tried different concentrations of MPTP on embryos and chose a 1 mM MPTP concentration, the highest concentration used in past studies. DA neurons of different groups in the vDC were previously shown to have different sensitivities to MPTP (Wen et al., 2008; Sallinen et al., 2009). In the current study, DA neurons of groups 4/5 seem to be more sensitive to MPTP than those of other groups (Fig. 8A–D). However, we did not observe a severe loss of DA neurons in the vDC (approximately 20%), which is consistent with some previous observations (Sallinen et al., 2009).

Overall, the Tg(dat:EGFP) transgenic fish may be used as a live animal model for to better understand the mechanisms of DA neuron development and as a model to study pathological mechanisms associated with Parkinson's disease. It will facilitate live observation of DA neurons under various conditions such as: (1) over-expression of genes by mRNA injection or (2) transgenesis of dominantly inherited PD-linked genes, (3) morpholino-knockdown of gene function or (4) mutant forms of recessively inherited PD-linked genes, and (5) screening for chemical compounds with therapeutic potential under the above conditions.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

Animal Maintenance

Zebrafish and embryos were maintained according to methods described by Nüsslein-Volhard and Dahm (2002). Embryos were staged as hours (hpf) or days (dpf) post-fertilization according to specific morphological features outlined by Kimmel et al. (1995). All experiments were performed according to the guidelines of the Canadian Council on Animal Care and were approved by the University of Ottawa animal care committee.

Construct for Transgenesis

A partial sequence of exon 1 of the zebrafish dat gene was amplified by PCR with oligonucleotides 5′-ATGCTGAGAGGCAGACCGG-3′ and 5′-GTAGCACAGGTATGGGAACC-3′, and radioactively labeled. This probe was used to screen a zebrafish genomic PAC library (RZPD, Berlin, Germany) according to the protocol recommended by the manufacturer. A PAC clone (BUSMP706K0187Q9) was identified to contain the dat gene. The EGFP coding sequence was cloned in frame into dat exon 1 using an E. coli strain (DY380) with temperature inducible RecET homologous recombination system according to procedures described by Liu et al. (2003). A fragment of 27 kb from this PAC-EGFP construct, containing the dat genomic sequences, including approximately 13 kb of its 5′-flanking region, but excluding the last coding exon and 3′-flanking region, was cloned into a modified pGEM vector with Tol2 arms at both ends by homologous recombination. This Tol2-PAC-EGFP construct was used for producing transgenic zebrafish lines.

Zebrafish Transgenesis

The Tol2-PAC-EGFP plasmid DNA (54 ng/μl) and 35 ng/μl transposase mRNA were co-injected into one-cell stage zebrafish embryos according to established procedures (Xi et al., 2010). Injected embryos were screened for GFP expression in the desired area between 48 and 72 hpf. GFP-positive individuals were raised to sexual maturity and outcrossed with wild type adult fish to identify transgenic carriers.

Whole-Mount In Situ Hybridization and Immunostaining

Embryos for whole mount in situ hybridization and immunostaining were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), then dehydrated in 100% methanol, and stored at −20°C in 100% methanol until use. Whole-mount in situ hybridization, immunostaining, and confocal microscopy observation were performed as described by Xi et al. (2010). A th antisense probe was used as previously described (Xi et al., 2010). The dat antisense probes were synthesized from a sub-clone of a dat cDNA. This sequence corresponds to a fragment of approximately 790 bp spanning from positions 890 to 1681 in NCBI sequence NM131755.1.

Double fluorescent in situ hybridization was performed as described in MacDonald et al. (2010). The dat probe was made using digoxigenin and revealed with tyr-fluorescein. The GFP probe was labeled with DNP and revealed with tyr-Cy3. Images were taken with a Zeiss LSM 510 Meta confocal microscope.

The following primary antibodies were used: Mouse anti-GFP (1:300, Invitrogen, A-11120, Burlington, ON, Canada); Rabbit anti-TH (1:100; Chemicon, AB152, Ottawa, ON, Canada). The following secondary antibodies were used: Goat anti-mouse AlexaFluor 488 (1:200, Invitrogen, A-10667, Burlington, ON, Canada); Goat anti-rabbit AlexaFluor 594 (1:200, Invitrogen, A-11012, Burlington, ON, Canada).

Double Immunohistochemistry on Forebrain Cryosections

Embryos and larvae for double immunohistochemistry were fixed in 4% PFA/1× PBS overnight at 4°C, rinsed in 1× PBS and placed in 30% sucrose/1× PBS to equilibrate. Embryos were then embedded in Shandon cryomatrix (ThermoFisher Scientific, 2860015, Ottawa, ON, Canada), sectioned on a Leica CM1850 (Leica Microsystems, Weltzar, Germany) cryostat at a thickness of 10–20 μm and stored at −20°C until use. After 3× washes in 1×PBS for 10 min, the sections were preincubated with a blocking solution containing 10% new calf serum and 0.1% Tween 20 for 2 hr at room temperature, and then incubated with the same blocking solution containing the primary antibodies (see below) overnight in a humid chamber at 4°C. After removal of the primary antibodies with 3× PBS washes, the appropriate secondary antibodies were incubated on the slides for 2 hr at room temperature. After the final washes, the slides were mounted using Vectashield mounting medium (Vector labs, H-1000, Burlington, ON, Canada). Signals were visualized on a Nikon Eclipse E3600 stereomicroscope with filters for both fluorescent stains. The following primary and secondary antibodies were used: Mouse anti-GFP (1:1,000, Invitrogen, A-11120); Rabbit anti-TH (1:500; Chemicon, AB152); Goat anti-mouse AlexaFluor 488 (1:300, Invitrogen, A-10667); Goat anti-rabbit AlexaFluor 594 (1:300, Invitrogen, A-11012).

MPTP Treatment

MPTP (Sigma-Aldrich, Oakville, ON, Canada) was dissolved in distilled water to 10 mM as a stock solution. Embryos were obtained from an outcross between the dat:EGFP transgenic fish and wild-type individuals. At 24 hpf, GFP-positive embryos were collected, manually dechorionated, and transferred into a six-well plate containing 4 ml of E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) with 0.003% phenylthiourea (PTU, Sigma-Aldrich, Oakville, ON, Canada) and MPTP at the following concentrations (100 μm, 500 μm and 1 mM). The MPTP-containing buffer was changed daily. The embryos were examined under a fluorescence microscope, and then fixed in 4% PFA (paraformaldehyde) in PBS for immunostaining. Control groups received no MPTP. MPTP exposures were performed and toxins were disposed according to appropriate safety protocols (Przedborski et al., 2001).

Statistical Analysis

All data quantification and statistical analysis were performed with Microsoft Excel 2003. Student's t-tests were used when applicable. Data were expressed as mean ± SD. P values of < 0.05 (two-sided) were considered as statistically significant. All experiments were independently repeated at least three times.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES

We thank Dr. Mélanie Debiais-Thibaud for her assistance with the double-fluorescent in situ hybridization and Jing Zhang and Dr. Marie-Andrée Akimenko for comments and technical support. We also thank Joel Ryan and Vishal Saxena for maintaining the Tg(dat:EGFP) fish line. This work was supported by a grant from the Canadian Institutes of Health Research to M.E. Y.X. was supported by a University of Ottawa Excellence Scholarship and an Ontario Graduate Scholarship (OGS).

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. EXPERIMENTAL PROCEDURES
  7. Acknowledgements
  8. REFERENCES