Targeting retinal dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein transgenic zebrafish

Transgenic zebrafish

To generate the Tg(−12th:MmGFP) transgenic zebrafish, we isolated a genomic P1-derived artificial chromosome (PAC) clone, BUSMP706E03252Q3, containing the zebrafish tyrosine hydroxylase 1 (th1) gene promoter region by screening the zebrafish PAC library (BUSMP706) with a probe containing a part of th1 cDNA sequence. To identify genomic fragments containing the th1 promoter region, we digested the PAC clone with several restriction enzymes and performed duplicate Southern hybridization using two probes. The first probe was a genomic PCR product of the 5′UTR region of the th1 locus. Following identification of the th1 5′UTR by RACE, we designed two primers as shown in Table 1 for the genomic PCR. The second probe was generated from a previously published partial th1 cDNA clone [17] by PvuII-BglII digest and contained approximately 100 bp of carboxyterminal portion of the coding region of the th1 gene. To identify a genomic fragment, which contains mostly the promoter region and not the coding region, we sought to isolate PAC restriction fragments, based on Southern analysis, that are positive for the 5′UTR probe but are negative for the carboxyterminal probe. A XbaI-XhoI fragment was identified, which fulfilled this criterion. The XbaI-XhoI fragment was further digested with EcoRI (EcoRI restriction site is found immediately downstream of the Th1 start ATG) and XhoI, and was cloned into a pBluescript II vector. The end sequencing of this fragment using T7 primer revealed that the fragment contains the th1 genomic region of chromosome 25 starting at position 20376290 (Ensembl Zv7 assembly). Since the th1 transcript begins at the position 20364304, and since th1 is oriented in reverse direction on this chromosome, the EcoRI-XhoI fragment encompasses 11986 bp of th1 genomic promoter region; therefore, we refer to this fragment as th1 12 kb promoter fragment. The MmGFP chromophore coding region and SV40 polyA sequence was PCR-amplified from pG1 vector (gift of Chi-Bin Chien and Darren Gilmour, University of Utah, Salt Lake City, UT and EMBL) and cloned downstream of the th1 12 kb promoter fragment in the pBluescript II vector. To increase the translational efficiency, we adjusted the translational initiation context to better match the consensus Kozak sequence (GCCATGG). Transgenic zebrafish were generated by microinjection of 1–2 nl of 50 ng/μl linearized DNA into 1- to 2-cell stage embryos (University of Freiburg, Freiburg, Germany). The founders were crossed with wild-type fish and their progeny were screened to establish transgenic lines. The transgene integration used for this study has the allele designation Tg(−12th:MmGFP)^m899.

Screen of transgenic offspring

Tg(−12th:MmGFP) transgenic zebrafish hemizygous for the Tg(−12th:MmGFP) transgene were crossed with wild-type AB* fish to produce the hemizygous Tg(−12th:MmGFP) fish used in this study. Eggs were collected in the morning, 1 h after mating, and treated with 0.003% (w/v) phenyl-2-thiourea (PTU; Sigma, St. Louis, MO) at 24 hpf to prevent the development of melanin pigment. The transgenic offspring were screened by visualizing GFP expression at 24–30 hpf with a fluorescent microscope (Leica MZFLIII, Leica Microsystems, Bannockburn, IL). The fish were treated in accordance with National Institutes of Health and Vanderbilt University Division of Animal Care guidelines.

Imaging

Tg(−12th:MmGFP) expression in the zebrafish embryos was visualized using a fluorescent dissecting microscope (Leica MZFLIII) connected to a QImaging Retiga 1300 CCD digital camera. The bright-field or fluorescent images were captured using QCapture v 1.2.0 software. All other images, including whole mount retina and immunostained vertical sections of adult zebrafish retina, were visualized with a laser scanning confocal microscope (Zeiss LSM5 Pascal, Carl Zeiss, Jena, Germany) at excitation wavelengths of 488 nm or 543 nm. The z-stack images were scanned at an optical slice of 4 μm for whole-retina expression in embryos 3 dpf and 1 μm for individual cells in living retina. For cell counting on complete retinas, tile-scans of 16 images, measuring 512×512 µm², were collected from double immunostained whole mount retinas.

Immunocytochemistry

Transgenic fish (approximately 1–3 months old) were kept in darkness overnight and then euthanized on ice. Both eyes were removed from the fish and then hemisected to remove the cornea and lens. The eyecups were fixed for 2 h and then rinsed three times for 5 min each in phosphate buffered saline (PBS; 137 mM NaCl, 2.6 mM KCl, 5.4 mM Na₂HPO₄, 1.8 mM KH₂PO₄), pH 7.4, cryoprotected in 30% sucrose overnight, and cryosectioned at 15 µm. Cryostat sections were then blocked with 1% bovine serum albumin (BSA) and 0.3% Triton X-100 in 0.1 M PBS for 2 h, and stained with 1:20,000 rabbit anti-GFP (Molecular Probes, Carlsbad, CA), and 1:2,000 sheep anti-TH (Chemicon, Temecula, CA) primary antibodies overnight at room temperature. After rinsing, a secondary incubation was performed for 2 h in a mixture containing 1:500 cy3-conjugated donkey antirabbit IgG(H⁺L) and 1:500 cy5-conjugated donkey antisheep IgG(H⁺L) antibodies (Molecular Probes). Whole mount retina immunocytochemistry was performed similarly, except that isolated retinas, instead of whole eyecups, were processed for staining, and the retinas were incubated in primary antibodies for 48 h.

Single-cell RT–PCR

To make dispersed cell cultures, we isolated retinas in Leibovitz’s L-15 medium (Gibco, Invitrogen, Carlsbad, CA). These were digested with 20 units/ml papain for 20 min and gently triturated in L-15 medium. The cell suspensions were distributed into 35 mm culture dishes, which were kept at room temperature for 30 min. For collection, cells were observed with an inverted microscope (Nikon, Lewisville, TX), and perfusion system was used to avoid contamination [24]. The inlet tube perfused the target cell with an extracellular solution for zebrafish that contained 137 mM NaCl, 2.5 mM KCl, 2.5 mM MgCl₂, 2.5 mM CaCl₂, 10 mM HEPES, and 10 mM glucose (pH 7.4). Individual cells were aspirated into the pipette and after washing with extracellular solution, the tip of the pipette harboring the target cell was broken into a 0.5 ml tube with 14 μl of Master Mix 1 and maintained on dry ice. Reverse transcription (RT) was performed using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations. For seminested polymerase chain reaction (PCR), we used the primers shown in Table 1. Seminested PCR was performed using an Eppendorf Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany). The first round reaction was incubated at 94 °C for 5 min, then at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s for 15 cycles, followed by 72 °C for 10 min. Nested-PCR reactions were performed using 1 μl of the first round PCR product in a 100 μl sample. The second round reaction was performed at 94 °C for 5 min, then at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s for 30 cycles, followed by 72 °C for 10 min.

Electrophysiological recordings

Tg(−12th:MmGFP) zebrafish were dark-adapted overnight before the day of recording. The separation of retina was performed in oxygenated extracellular solution under dim red light. The extracellular solution contained the following: 116 mM NaCl, 2.4 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 1 mM NaH₂PO₄, 28 mM NaHCO₃, 10 mM glucose. This solution was bubbled with a gaseous mixture of 95% O₂ and 5% CO₂. The retina was placed photoreceptor-side down in the recording chamber and maintained in the dark for at least 1 h before recording. The chamber was then mounted on the stage of an upright conventional fluorescent microscope (Leica DM LFSA; Leica Microsystems, Wetzlar, Germany). Oxygenated medium was continuously perfused into the recording chamber at a rate of roughly 2–3 ml/min, and the recording was performed at approximately 21 °C.

During recording, GFP+ cells were identified by fluorescence microscopy using a brief illumination of fluorescent excitation light. GFP-labeled cells and glass electrodes were visualized using infrared differential interference contrast (DIC) optics for recording. The glass electrodes were prepared using DMZ universal puller (Zeitz-Instrumente, Munich, Germany) and filled with a solution containing 150 mM NaCl and 10 mM HEPES (pH 7.5). The resistance of pipettes filled with this solution was 9–10 MΩ. The recording was made from the soma of GFP-labeled cells using a pipette with a holding potential of 0 mV. Experiments were executed and data were recorded using Clampex 8.0 software connected to an AxoClamp-2B amplifier (Molecular Devices, Palo Alto, CA), via a digitizer 1322A (Molecular Devices). Data were analyzed with Clampfit 9.2 (Molecular Devices). Action potential frequency was calculated from 40 s recordings from each cell. Values are mean ± standard deviation.

Generation of Tg(−12th:MmGFP) transgenic fish

An approximately 12 kb genomic fragment containing the zebrafish tyrosine hydroxylase gene th1 promoter was isolated and ligated with green fluorescence protein (MmGFP) followed by a SV40 polyadenylation tail (pA; Figure 1A). The recombinant DNA was injected into 1–2 cell zebrafish embryos that were allowed to grow to adulthood. The founder fish were then crossed with wild-type fish and screened for germ-line transmission of the transgene. Larval fish with GFP expression were raised as transgenic F1 fish.

th-driven GFP expression during development

To determine the GFP expression pattern in these Tg(−12th:MmGFP) fish, we crossed hemizygous transgenic F1 fish with wild-type fish and examined GFP expression in their progeny using fluorescence microscopy. The transgenic embryos showed robust GFP expression in the embryonic brain region, including the midbrain and hindbrain boundary, as well as the hindbrain and spinal cord at 24 hpf (Figure 1C). At 72 hpf, individual neurons exhibiting GFP fluorescence were identified within the retina (Figure 1E). Imaging of nontransgenic wild-type fish at 72 hpf revealed a lack of GFP fluorescence (Figure 2).

Colocalization of GFP with TH

To determine the overlap of -12th:MmGFP with native TH expression, we performed double-labeled immunocytochemistry using antibodies for TH and GFP on vertical sections and whole mount adult retinas. Figure 3A shows a vertical section in which GFP was expressed within two cells in the inner nuclear layer (INL). The cell on the right was co-labeled with TH antibody (arrow), while the one on the left was not (arrowhead). GFP+ cells were uniform in their morphological characteristics with cell bodies located in the proximal cellular row of the inner nuclear layer and branched processes extending to an extensive fiber network in the inner plexiform layer (Figure 4). Application of GFP antibody to nontransgenic wild-type zebrafish, or application of secondary antibodies alone without primary TH or GFP antibodies, resulted in a lack of staining. Immunocytochemistry of whole mount retinas was performed to examine the colocalization of GFP-immunoreactivity (IR) and TH-IR (Figure 3B). Arrows indicate cells that express both GFP and TH. Note that while the majority of TH-IR cells also expressed GFP, there were cells single-labeled by TH antibody (arrowhead). To quantify the colocalization rate of TH and GFP in the whole retina, we processed four whole-mount retinas from 4 fish for immunocytochemistry, and we analyzed the images of each entire retina. On average, 29.2 ± 1.7% (mean ± SD, n=4) of GFP-expressing neurons also expressed TH, and GFP-expressing neurons exhibited a density of 286 ± 43 cells/mm². Meanwhile, TH-expressing cells had a density of 129 ± 21 cells/mm² throughout the retina and 65.0 ± 3.4% of TH-expressing cells were labeled by GFP. Images of entire retinas that had been double-labeled for GRP-IR and TH-IR showed that both GFP-IR neurons and TH-IR neurons had a relatively even distribution throughout the retinas (data not shown).

We noted a marked variation in GFP intensity across labeled neurons and that there was a trend toward a greater incidence of colocalization with TH for dimmer GFP+ cells, whereas the neighboring bright GFP-stained cells were often single labeled. Average gray value analysis using MetaMorph on confocal images of GFP-immunostained whole-mount retinas indicated a twofold difference in fluorescent intensity between bright GFP- and dim GFP-expressing cells (Figure 5). While these two subpopulations were distinguishable in fluorescence intensity (Figure 6A), they exhibited similar cell size and shape.

Expression of th and dopamine transporter mRNA in GFP-labeled cells

To further assess what proportion of GFP-expressing cells are dopaminergic, and to test the possibility that molecular components of GFP cells could be analyzed in vitro, we sought to detect th and dat expression in isolated living cells. We acutely dissociated the retinas and plated retinal cells into culture dishes, and performed single-cell RT–PCR, using primers specific for th and dat, markers for DA neurons. To increase reaction specificity, seminested PCR primers spanning different exons of the genes were used (Figure 6B). Bright or dim GFP-expressing cells were collected and analyzed by reverse transcription followed by seminested PCR. Amplicons from the second round of PCR were visualized on agarose gels stained with ethidium bromide (Figure 6C). Among the seven dim GFP-expressing cells collected, five cells expressed both th and dat, while only one out of eight bright GFP-expressing cells did (Figure 6D). These results showed that most dim GFP-expressing cells coexpressed both dopamine-cell marker genes.

Spontaneous spike activity of GFP-expressing neurons in situ

Mammalian dopaminergic amacrine cells generate spontaneous action potentials in dissociated culture or in whole mount mouse retinas [15,25,26]. To examine the neuronal activity of dopaminergic cells in the Tg(−12th:MmGFP) zebrafish retina, we performed extracellular loose-patch recordings on GFP-expressing cells. Based on the initial observation that most dim-GFP-expressing cells are dopaminergic neurons, for recording we prioritized cells with relatively low GFP expression (see Figure 7A for overlaid fluorescent and DIC images of an example cell). Among the 20 GFP+ cells, 11 exhibited spontaneous spikes. Figure 7B shows a typical recording from a GFP+ cell. The cell fired in single-spike pattern with a spiking rate of 0.75 Hz. For the 11 cells exhibiting spontaneous spikes, the firing rate ranged from 0.47 - 4.70 Hz with an average of 1.48±1.17 Hz (mean ± SD).