ARTICLE |
Correspondence to: Takeshi Kaneko, Dept. of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. E-mail: kaneko@mbs.med.kyoto-u.ac.jp
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Summary |
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A new recombinant virus which labeled the infected neurons in a Golgi stain-like fashion was developed. The virus was based on a replication-defective Sindbis virus and was designed to express green fluorescent protein with a palmitoylation signal (palGFP). When the virus was injected into the ventrobasal thalamic nuclei, many neurons were visualized with the fluorescence of palGFP in the injection site. The labeling was enhanced by immunocytochemical staining with an antibody to green fluorescent protein to show the entire configuration of the dendrites. Thalamocortical axons of the infected neurons were also intensely immunostained in the somatosensory cortex. In contrast to palGFP, when DsRed with the same palmitoylation signal (palDsRed) was introduced into neurons with the Sindbis virus, palDsRed neither visualized the infected neurons in a Golgi stain-like manner nor stained projecting axons in the cerebral cortex. The palDsRed appeared to be aggregated or accumulated in some organelles in the infected neurons. Anterograde labeling with palGFP Sindbis virus was very intense, not only in thalamocortical neurons but also in callosal, striatonigral, and nigrostriatal neurons. Occasionally there were retrogradely labeled neurons that showed Golgi stain-like images. These results indicate that palGFP Sindbis virus can be used as an excellent anterograde tracer in the central nervous system.
(J Histochem Cytochem 49:14971507, 2001)
Key Words: Sindbis virus, green fluorescent protein, DsRed, palmitoylation, Golgi stain-like labeling, neuronal tracer, rat, brain
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Introduction |
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SINDBIS VIRUS, which is an enveloped virus with a positive-strand RNA genome and which belongs to the alphavirus genus of the Togavirus family, causes acute encephalomyelitis in mice (for review cf.
The introduction of palmitoylation signals into a reporter protein was demonstrated to be effective in targeting the protein to the cell membrane of cultured cells using adenovirus vectors (
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Materials and Methods |
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These experiments were approved by the Committee for Animal Care and Use of the Graduate School of Medicine at Kyoto University and that for Recombinant DNA Study in Kyoto University.
Construction of Recombinant Sindbis Viruses
The reporter proteins were designed as the fusion protein of N-terminal icosapeptide (palmitoylation signal) of growth-associated protein-43 (GAP43) and enhanced GFP (Clontech; Palo Alto, CA) or DsRed (Clontech). The cDNA fragment encoding GFP with the palmitoylation signal (palGFP) was obtained from plasmid pGG16 (expression vector of palGFP; a generous gift from Dr. T. Kagawa, National Institute for Physiology, Okazaki, Japan;
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pGG16 or p-palDsRed1-N1 was double digested with XhoI and NotI, and a DNA construct containing the sequence for palGFP or palDsRed was blunt-ended. The construct was inserted into the PmaCI site of pSinRep5 (Invitrogen; Carlsbad, CA). The production of Sindbis virus was performed according to the instructions with the Sindbis Expression System (Invitrogen), as follows. The capped transcript of recombinant RNA was synthesized from the pSinRep5 containing the construct (pSinRep5-palGFP or pSinRep5-palDsRed). Sindbis viral particles were obtained by co-transfecting baby hamster kidney (BHK) cells electrophoretically with the recombinant RNA transcript and DH (26S) 5'SIN helper RNA encoding the structural protein. The viral particles in the culture supernatant were concentrated to a 2055% sucrose interface by ultracentrifugation (160,000 x g, 90 min). The titer was adjusted to 2 x 1010 infective U/ml and the virus was stored in aliquots at -80C until used for delivery to brain tissue. The resulting Sindbis virus was replication-deficient and had the least chance of production of parent viral particles in the infected cells (
Cell Culture and Transfection of Expression Vectors
Chinese hamster ovary (CHO) cells (American Type Culture Collection CCL-61) were grown in Dulbecco's modified Eagle's medium (Gibco, Gaithersburg, MD, and Invitrogen; Rockville, MD) supplemented with 10% (v/v) fetal bovine serum. One microgram of expression vector with cytomegalovirus (CMV) promoter, pEGFP-N3, pGG16, pDsRed1-N1, or p-palDsRed1-N1, was mixed with 3.6 µl of FuGENE 6 reagent (Roche; Basel, Switzerland) in 100 µl of serum-free medium and then incubated with CHO cells at 80% confluency. The cells were fixed with 4% formaldehyde in 0.1 M sodium phosphate (pH 7.0) 36 hr after the transfection. The nuclei of some fixed cells were stained with 1 µg/ml of 4',6-diamidino-2-phenylindole-2HCl (DAPI) in 5 mM phosphate-buffered 0.9% (w/v) saline, pH 7.4 (PBS). The cells were coverslipped and observed under an Axiophot fluorescence microscope (Zeiss; Oberkochen, Germany) with an appropriate filter set for GFP (excitation 450490 nm, emission 515565 nm), DsRed (excitation 540552, emission 575 nm), or DAPI (excitation 359371, emission 397490 nm).
Injection of Viruses, Fixation, and Immunohistochemistry
Thirty-six Wistar rats (200300 g) were deeply anesthetized with chloral hydrate (350 mg/kg body weight). The virus (12 µl of 2 x 1010 infectious U/ml) was injected through glass micropipettes equipped with Picospritzer II (General Valve Corporation; East Hanover, NJ) into the ventrobasal nuclei of the thalamus, somatosensory cortex, neostriatum, and substantia nigra. The rats were allowed to survive for 4.5 hr to 28 days after the injection.
The rats were anesthetized again with chloral hydrate (350 mg/kg body weight) and perfused transcardially with 200 ml of PBS, followed by 300 ml of 4% (w/v) formaldehyde in 0.1 M sodium phosphate, pH 7.0. The brains were removed, cut into several blocks, and placed for 2 hr at 4C in the same fixative. After cryoprotection with 30% (w/w) sucrose in PBS, the blocks were cut into 40-µm-thick frontal sections on a freezing microtome. Some sections were mounted on a glass slide, coverslipped with 50% (v/v) glycerol and 2.5% (w/v) triethylene diamine (anti-fading reagent) in PBS, and observed with the epifluorescence microscope as described above.
The other sections were immunostained with an affinity-purified rabbit antibody to GFP (-carrageenan, and 0.5% (v/v) donkey serum (PBS-XCD). After a rinse with PBS containing 0.3% (v/v) Triton X-100 (PBS-X), they were incubated for 1 hr with 10 µg/ml of biotinylated anti-rabbit IgG donkey antibody (Chemicon; Temecula, CA) in PBS-XCD and then for 1 hr with avidinbiotinylated peroxidase complex (ABC Elite; Vector, Burlingame, CA) in PBS-X. Finally, the sections were reacted for 1030 min with 0.2% (w/v) diaminobenzidine-4HCl and 0.003% (v/v) H2O2 in 50 mM Tris-HCl, pH 7.6. The sections were mounted on a glass slide, dehydrated with an ethanol series, cleared in xylene, and coverslipped with organic mounting medium MX (Matsunami; Kishiwada, Japan). Adjacent sections were stained with Cresyl violet for cytoarchitecture.
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Results |
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The differences in intracellular localization between GFP, palGFP, DsRed and palDsRed were examined with CHO cultured cells and expression vectors pEGFP-N3, pGG16, pDsRed1-N1, and p-palDsRed1-N1, all of which used a CMV promoter for protein synthesis in mammalian cells (Fig 2). palGFP appeared to be more associated with cell membranes (arrowheads in Fig 2a) than GFP (Fig 2b), while palDsRed showed aggregated fluorescence in the cytoplasm (Fig 2c). Under a Nomarski differential interference contrast microscope, the form of palDsRed-expressing cells was spherical rather than spindle-shaped (arrowheads in Fig 2c). Double fluorescence observation showed that both palGFP and palDsRed were not located in DAPI-positive cell nuclei (not shown). In contrast, both GFP- and DsRed-expressing CHO cells were spindle-shaped and displayed diffuse fluorescence throughout the cytoplasm and cell nuclei (Fig 2b and Fig 2d). This indicates that the addition of exactly the same palmitoylation signal had clearly different effects on the respective soluble molecules, GFP and DsRed.
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It was surprising to us that CHO cultured cells could not be infected with the Sindbis virus vectors (not shown). This might be caused by the very low susceptibility of CHO cells to Sindbis virus, although Sindbis virus was reported to have a broad host range in animal cells (
Because the gene expression by Sindbis virus system was reported to reach peak between 24 and 48 hr after infection in the brain (
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To determine the best survival time of rats for more precise neuronal labeling, we analyzed the change of GFP immunoreactivity at the injection sites of the thalamus from 4.5 hr to 28 days after inoculation of palGFP Sindbis virus (Fig 4). Many small immunoreactive cells (arrows in Fig 4a) and amorphous or bushy deposits showing intense immunoreactivity (arrowheads) were found in the injection site 4.5 hr after the injection. Nine hours after the injection, larger immunoreactive cells with long processes, probably neurons, appeared among the amorphous immunopositive staining (Fig 4b). From 18 to 72 hr after the injection, immunoreactivity was very intense and was mostly confined to neuronal structures such as cell bodies, dendrites, and axons (Fig 4c4e). However, at 7 days of survival, neurons showed degenerative changes, such as beaded dendrites (arrowheads in Fig 4f) and shrinkage of the cell bodies (arrow). Immunoreactive neurons had almost completely disappeared by 28 days after the injection, except for remnant punctate immunoreactivity (arrowheads in Fig 4h). Therefore, survival times of 18 hr to 3 days (72 hr) were suitable for neuronal labeling in the injection sites.
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The labeling of palDsRed was compared with that of palGFP by co-injection of the same titers of palGFP and palDsRed Sindbis viruses (Fig 5a5d). Thirty-six hours later at the injection site in the ventrobasal thalamic nuclei, DsRed immunoreactivity was localized to the cell bodies and appeared to be aggregated or accumulated in some cytoplasmic structures (Fig 5c). Although no DsRed immunoreactivity was observed in the cerebral cortex, many GFP-immunoreactive axon fibers were observed in the somatosensory areas (Fig 5b5d). When the sections were observed without immunostaining under the fluorescence microscope, a few neurons were found to express both palGFP and palDsRed at the injection site, although their fluorescence was weaker than that of neurons expressing either palGFP or palDsRed alone (not shown). The latter finding might suggest a competition between palGFP and palDsRed protein synthesis. In those co-expressing neurons, palDsRed was localized to cell bodies, whereas palGFP was distributed in fine dendrites as well as in cell bodies, as shown in Fig 3b.
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When the palDsRed virus was injected into the cerebral cortex (Fig 5f), intense DsRed immunoreactivity was mostly found in the cell bodies and much weaker immunoreactivity was found along the apical dendrites (arrows). In contrast, when the palGFP virus was injected into the cerebral cortex (Fig 5e), many intensely immunoreactive pyramidal cells showed Golgi stain-like images with heavily spiny dendrites (arrow) and many axon collaterals (arrowheads) around the injection site. These results suggest that palDsRed is not as efficiently targeted to cell membranes as palGFP but is mostly aggregated or accumulated in some cytoplasmic structures.
The characteristics of palGFP Sindbis virus as an anterograde tracer were examined in several brain regions (Fig 6). When the virus was injected into the motor cortex, many projecting axon fibers were labeled in the corpus callosum (arrows in Fig 6a) and the terminals were in the contralateral motor cortex (Fig 6b). After injection of the virus into the neostriatum (Fig 6c) or substantia nigra (Fig 6f), intensely GFP-immunoreactive axon fibers and terminals were found in the reticular part of the substantia nigra (Fig 6d and Fig 6e) or in the neostriatum (Fig 6h), respectively. In the case of nigrostriatal projection, patchy distribution of labeled axon fibers in the neostriatum was observed as reported previously (
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In addition to intense anterograde labeling with palGFP Sindbis virus, retrogradely labeled neurons were occasionally seen (Fig 7). Five to seven pyramidal neurons in Layer VI (Fig 7a) or four to six medium-sized spiny neurons in the caudate putamen (Fig 7b) were labeled retrogradely after injection of 2 µl of 2 x 1010 infectious U/ml palGFP virus solution into the ventrobasal thalamic nuclei or substantia nigra, respectively. In both cases, the labeled neurons showed Golgi stain-like images with richly spiny dendrites (arrows in Fig 7c and Fig 7d) and axon collaterals surrounding the cell bodies.
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Discussion |
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The present study showed the advantages of palGFP Sindbis virus as in vivo anterograde and retrograde neuronal tracers with Golgi stain-like labeling of neurons. In contrast, palDsRed Sindbis virus was not suitable as an anterograde or retrograde tracer. DsRed immunoreactivity was mostly localized or aggregated inside of the cell bodies of neurons that were infected with palDsRed Sindbis virus. Very recently, DsRed was shown by analytical centrifugation to form a tetramer in living cells (
At a very early stage of palGFP Sindbis virus infection, GFP immunoreactivity was located in small cells and amorphous bushy structures at the injection site (Fig 3a). The amorphous or bushy immunoreactivity was similar to that reported in the previous study using a recombinant adenovirus encoding palGFP (Fig 1C and Fig 2A in
The palGFP Sindbis virus was proved to be an excellent tracer for anterograde axonal labeling (Fig 5). It could be applied to thalamocortical and corticocortical glutamatergic neurons, striatonigral GABAergic neurons, and nigrostriatal dopaminergic neurons in the present experiments, indicating that the Sindbis virus does not choose a target neuron. One of the possible advantages of viral vectors is that the virus can label axons sparsely but intensely; in the best case, intensely stained axons from a single neuron can be traced. On the other hand, in the case of the previous axonal tracers, the smaller the number of labeled neurons, the weaker the intensity of axonal labeling. This is not the case for viruses because of their proliferative property. The recombinant Sindbis virus produces reporter proteins massively, although it can not produce mature viral particles. In addition, using palGFP Sindbis virus, we can determine, by their Golgi stain-like images, the morphological type of the neurons that send axon fibers to the target. This has been impossible with ordinary anterograde tracers, such as Phaseolus vulgaris leukoagglutinin. We hope that the palGFP Sindbis virus will be helpful in analyzing neuronal connections of the central nervous system by its outstanding properties.
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Acknowledgments |
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Supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (12308039, 12680731, 13035020, 13035026, 13041024, 13041025, and 13210071).
We are grateful for the photographic help from Mr Akira Uesugi and Ms Keiko Okamoto.
Received for publication April 23, 2001; accepted August 15, 2001.
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