Chem. Senses 27: 215-223,
2002
© Oxford University Press 2002
Adenovirus-mediated WGA Gene Delivery for Transsynaptic Labeling of Mouse Olfactory Pathways
1 Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan 2 Department of Biochemistry, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
Correspondence to be sent to: Yoshihiro Yoshihara, Laboratory for Neurobiology of Synapse, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. e-mail: yoshihara{at}brain.riken.go.jp
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Abstract |
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Detailed knowledge of neuronal connectivity patterns is indispensable for studies of various aspects of brain functions. We previously established a genetic strategy for visualization of multisynaptic neural pathways by expressing wheat germ agglutinin (WGA) transgene under the control of neuron type-specific promoter elements in transgenic mice and Drosophila. In this paper, we have developed a WGA-expressing recombinant adenoviral vector system and applied it for analysis of the olfactory system. When the WGA-expressing adenovirus was infused into a mouse nostril, various types of cells throughout the olfactory epithelium were infected and expressed WGA protein robustly. WGA transgene products in the olfactory sensory neurons were anterogradely transported along their axons to the olfactory bulb and transsynaptically transferred in glomeruli to dendrites of the second-order neurons, mitral and tufted cells. WGA protein was further conveyed via the lateral olfactory tract to the olfactory cortical areas including the anterior olfactory nucleus, olfactory tubercle, piriform cortex and lateral entorhinal cortex. In addition, transsynaptic retrograde labeling was observed in cholinergic neurons in the horizontal limb of diagonal band, serotonergic neurons in the median raphe nucleus, and noradrenergic neurons in the locus coeruleus, all of which project centrifugal fibers to the olfactory bulb. Thus, the WGA-expressing adenovirus is a useful and powerful tool for tracing neural pathways and could be used in animals that are not amenable to the transgenic technology.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Information transfer between neurons takes place at the synapse. The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. Accordingly, detailed knowledge on neuronal connectivity patterns is essential for understanding the wide range of brain functions.
Plant lectins have been used as highly sensitive tracers in neuroanatomical
studies for mapping central neural pathways. They are efficiently taken up by
neurons and transported in axons and dendrites in both anterograde and
retrograde directions. In some cases, injection of lectins in well-mapped
neural pathways results in labeling of both first- and second-order neurons
and their processes, suggesting that the lectins undergo an interneuronal
transfer. Among various lectins, wheat germ agglutinin (WGA) has been studied
extensively and proved to be transferred most efficiently between neurons
(Broadwell and Balin, 1985
;
Fabian and Coulter, 1985). In
the visual system, for example, the intraocular injection of WGA protein in
monkeys results in labeling of ocular dominance columns in the visual cortex
(Itaya and Van Hoesen, 1982;
Ruda and Coulter, 1982;
Trojanowski, 1983). In the
rodent olfactory system, the intranasal administration of WGA protein leads to
visualization of the primary and secondary olfactory pathways, from the
olfactory epithelium to the bulb and then to the cortex
(Shipley, 1985;
Baker and Spencer, 1986;
Itaya, 1987).
We have recently developed a novel genetic strategy for visualization of
selective neural pathways across synapses by combining a neuroanatomical
tracing method with a transgenic technology
(Yoshihara et al.,
1999; Tabuchi et al.,
2000). By introducing cDNA encoding WGA, as a transgene under the
control of specific promoter elements, selective and functional transsynaptic
neural pathways could be visualized. For example, when WGA transgene was
expressed specifically in Purkinje cells in the mouse cerebellum under the
control of L7 promoter, WGA protein produced by the Purkinje cells
(first-order neurons) was transported through their axons to nerve terminals
and transferred across a synapse to the second-order neurons in the deep
cerebellar nuclei. Furthermore, WGA was conveyed to the third-order neurons in
the midbrain red nucleus and the thalamic ventrolateral nucleus, permitting us
to track the cerebellar efferent pathways. Similarly, we have succeeded in
visualization of mouse olfactory pathways and Drosophila visual
pathways. Thus, the WGA transgene technology provides an extremely valuable
tool for the studies of formation, maintenance and remodeling of neural
networks in the brain.
Viral vectors are becoming increasingly important tools for foreign gene expression in various studies of neuroscience. In contrast to the transgenic mouse system, viral vectors can be applied to transfer genes in time- and place-specific manners in fully developed animals. In this paper, the WGA transgene technique was modified, combining it with the recombinant adenoviral vector system, which was successfully applied to visualizing the mouse olfactory system. This new technique makes the process more convenient and potentially opens it up to wider applications.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Cells
HEK293 human embryonic kidney cells and N2a mouse neuroblastoma cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum.
Viral preparation
Recombinant adenovirus for expression of WGA was prepared according to the
method using a cassette cosmid pAxCAwt
(Miyake et al.,
1996). Briefly, truncated WGA cDNA which encodes WGA protein
without carboxyterminal propeptide
(Yoshihara et al.,
1999) was bluntended and inserted into pAxCAwt at the SwaI site.
In this way, the inserted cDNA is transcribed under the control of a powerful
and ubiquitous CAG promoter which harbors the cytomegalovirus immediate early
enhancer and chicken ß-actin promoter
(Niwa et al., 1991).
The cosmid DNA was cotransfected with the EcoT22I-digested DNA-terminal
protein complex of Ad5-D1X into HEK293 cells to generate the recombinant virus
by homologous recombination. The recombinant virus, designated AxCAWGA, was
propagated in HEK 293 cells. After the fourth propagation, virions were
extracted, purified by double CsC1 step-gradient centrifugation
(Kanegae et al.,
1994), dialyzed against a vehicle solution containing 10% glycerol
in PBS, pH 7.4, and stored at -80°C. The titer of recombinant virus was
determined by the modified end-point cytopathic effect assay on HEK293 cells
(Kanegae et al.,
1994) and expressed in plaque-forming units (pfu) per ml. Positive
expression of the inserted gene product was confirmed by Western blot analysis
and immunocytochemical detection using mouse neuroblastoma N2a cells.
Experiments using recombinant adenovirus were approved by the Recombinant DNA
Committee of RIKEN and performed according to institutional guidelines.
Viral infection
For cultured cell infection, AxCAWGA (3 x 108 pfu/ml, 2µ1) was added to N2a cells plated onto either six-well plates (5 x 105 cells/well) or glass chamber slides (1 x 105 cells/well) (Becton Dickinson, Franklin Lakes, NJ). After 48 h, the cells were analyzed for WGA protein expression by Western blotting and immunocytochemistry using anti-WAGA antibody (Sigma, St Louis, MO).
For in vivo infection, 4- to 12-week-old male ddY mice (Nihon SLC,
Hamamatsu, Japan) were used. Animals were anesthetized with pentobarbital (2.5
mg/mouse, i.p.). A drop (
0.5 µ1) of PBS solution containing the virus
AxCAWGA at a titer of 2-5 x 109 pfu/ml was put at the
entrance of right nasal cavity. After all the solution entered into the cavity
by animal's spontaneous respiration, another drop was applied. This process
was repeated >100 times over a period of 60 min, until the total
volume of applied virus solution reached 50-70 µ1. This simple method
enabled us to infect the virus throughout the nasal epithelium almost evenly.
After 7, 12, 20, 30 and 50 days of survival, the mice were sacrificed for
analysis of WGA expression.
WGA immunohistochemistry
Paraformaldehyde-perfused mouse nasal epithelia or brains were cut with a cryostat or a sliding microtome to obtain 20 or 50 µm sections, respectively. The sections were pretreated for 30 min with 0.3% H2O2 in PBS to inactivate endogenous peroxidase activity and incubated for 30 min with PBS containing 0.2% Triton X-100 and 5% normal goat serum (PBST/NGS) for permeabilization of cells and blocking of nonspecific protein-binding sites. The sections were then incubated for 2 h with anti-WGA polyclonal antibody (3 µg/ml, Sigma) in PBST/NGS that had been pre-absorbed with 1% acetone powder of mouse brains. This pre-absorption procedure of anti-WGA antibody was necessary for accurate detection of the WGA transgene product, because the non-absorbed antibody cross-reacted with unknown endogenous molecule(s) in the mouse brain. The sections were then incubated with biotin-labeled anti-rabbit IgG (Jackson, West Grove, PA) followed by Vectastain ABC elite kit (Vector, Burlingame, CA). Signals were visualized with Ni2+-intensified diaminobenzidine/peroxide reaction. Specimens were observed by using a light microscope equipped with differential interference contrast optics (Olympus, Tokyo, Japan: Provis), a cooled CCD camera (Sony, Tokyo, Japan), and an image analysis system (Adobe Photoshop, San Jose, CA). For immunofluorescence labeling, Cy3-conjugated anti-rabbit IgG (Jackson) was used as the second antibody. The labeled sections were analyzed with a confocal laser scanning microscopy system (Leica TCS SP, Heidelberg, Germany).
Evaluation of WGA immunoreactivity levels in various regions of interest was performed as a blind experiment by comparing the stained sections of all mice perfused at different time points in accordance with following criteria: very high (+++), high (++), weak but significant (+), faint (±), or no expression (-) of the WGA transgene product.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Adenovirus-mediated WGA expression in cultured neurons
A recombinant adenoviral vector for WGA expression under the control of
strong and ubiquitous CAG promoter elements was constructed, purified and
concentrated. We first examined whether this virus can efficiently mediate a
high level of WGA expression in cultured cells. When the recombinant virus
AxCAWGA was infected into mouse neuroblastoma N2a cells, an intense
immunoreactive band was detected by Western blot analysis, whose molecular
size (18 kDa) corresponded to that of authentic WGA protein
(Figure 1A). Immunofluorescence
labeling revealed that WGA protein was associated strongly with the
intracellular granule-like structures of N2a cells
(Figure 1B), which is
reminiscent of the electron microscopical localization of exogenously
administered WGA in Golgi-derived vesicles, dense core granules, endosomes and
synaptic vesicles in neurons (Broadwell and
Balin, 1985). Thus, it was confirmed that adenovirus-mediated WGA
expression occurs efficiently in cultured neurons.
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Adenovirus-mediated WGA expression in the olfactory epithelium in vivo
In this study, mice were followed for up to 50 days post-infection (Table 1). A relatively large volume of virus solution (50-70 µl) was required for uniform infection throughout the nasal cavity. There was no evidence of cell loss or tissue damage due to the viral infection. In addition, all of the experimental animals that recovered after the infection remained healthy until being sacrificed, without exhibiting any behavioral abnormalities.
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Figure 2A shows transverse cryosections of the olfactory epithelium at 7 days post-infection, immunofluorescently stained with anti-WGA antibody. Abundant WGA expression was detected throughout the olfactory epithelium including nasal septum (S), ventral ectoturbinate (VE), second endoturbinate (II), and third endoturbinate (III). In addition to cell bodies lining along the olfactory epithelium, numerous WGA-containing axonal bundles in the lamina propria were clearly observed.
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At higher magnification (Figure 2B), WGA expression was observed in all three types of cells in the olfactory epithelium (olfactory sensory neurons, sustentacular cells and basal cells), as judged from their characteristic shapes and their typical layered positions. Among them, the olfactory sensory neurons in the middle layer of the epithelium exhibited the most predominant WGA immunoreactivity with intracellular granule-like profiles.
Anterograde transsynaptic transfer of WGA to the olfactory centers
Axons leaving from the olfactory epithelium project into spherical neuropil structures, glomeruli, in the main olfactory bulb, where they make synaptic connections with dendrites of the second-order neurons, mitral and tufted cells. In the main olfactory bulb, intense WGA immunoreactivity was detected not only in the olfactory nerve layer and the glomerular layer, but also in the external and internal plexiform layers and the mitral cell layer (Figure 3A). A higher magnification revealed that perikarya of the mitral and tufted cells were strongly WGA-positive (Figure 3B), indicating that WGA protein underwent the anterograde transsynaptic transfer from the sensory axon terminals to the bulbar relay neurons. The presence of WGA protein in the mitral cells was observed at all time points examined, with the strongest signals at 20 days post-infection (Table 1).
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The mitral and tufted cells in the main olfactory bulb project their axons to four major terminal fields in the olfactory cortex. The WGA immunoreactivity was observed in all these fields: the anterior olfactory nucleus (Figure 4A,E), the olfactory tubercle (Figure 4B,F), the piriform cortex (Figure 4C,G), and the lateral entorhinal cortex (Figure 4D,H). The most intense signals were detected at 20 and 30 days post-infection (Table 1). Many neurons in these areas of the olfactory cortex contained WGA protein (Figure 4E-H), indicating the transsynaptic labeling of the third-order neurons. These results suggest that WGA protein produced in the olfactory sensory neurons by infection of AxCAWGA was transferred across two synapses from the olfactory epithelium to the bulb, and further to the cortex.
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Retrograde labeling of centrifugal modulatory inputs to the olfactory bulb
After 30 days of AxCAWGA infection to the olfactory epithelium, WGA immunoreactivity was detected in neurons of several brainstem nuclei that are known to send a large number of centrifugal modulatory projections to the olfactory bulb. These include cholinergic neurons in the horizontal limb of diagonal band (Figure 5A,D), serotonergic neurons in the midbrain raphe nucleus (Figure 5B,E), and noradrenergic neurons in the locus coeruleus (Figure 5C,F). The retrograde labeling was very intense in the horizontal limb of diagonal band and in the raphe nucleus, while only weak immunoreactivity was detected in the locus coeruleus. This result indicates that WGA protein produced in the olfactory epithelium and transported to the main olfactory bulb was taken up into nerve terminals of efferent axons, retrogradely conveyed, and reached to neuronal somata in these distant nuclei.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Adenovirus-mediated gene transfer in olfactory sensory neurons
Previously, Zhao et al. and Holtmaat et al. reported the
usefulness of replication-deficient recombinant adenoviral vector system for
studies of the rodent olfactory system
(Holtmaat et al.,
1996; Zhao et al.,
1996). In both papers, the adenoviral vector carrying LacZ
(ß-galactosidase) gene under the control of a strong transcriptional
activator (cytomegalovirus promoter/enhancer) was infused into the nasal
cavity and efficiently directed transgene expression to the cells in the
olfactory epithelium. X-gal staining showed predominant ß-galactosidase
activity in the olfactory sensory neurons compared with other types of cells
such as sustentacular cells and basal cells
(Holtmaat et al.,
1996; Zhao et al.,
1996). A similar preference of the olfactory sensory neurons in
adenovirus-mediated transgene expression was observed also in another report
by Zhao et al. (Zhao et
al., 1998) and in this study. This may be due to the
difference in effectiveness of adenoviral infection that critically depends on
the expression of its cellular receptor CAR
(Bergelson et al.,
1997; Bergelson,
1999; Asaoka et al.,
2000), although the expression pattern of CAR in the rodent
olfactory epithelium has not been investigated.
In the two previous papers, the authors used different methods of virus
application onto the olfactory epithelium. Zhao et al. injected the
virus solution (10 µ1) with a micro-syringe inserted through a small hole
in the bone/cartilage at the top of nasal cavity
(Zhao et al., 1996).
Holtmaat et al. performed slow infusion (2.5 µ1/min) of virus
solution (50 µ1) into the nasal cavity using a microinfusion pump
(Holtmaat et al.,
1996). However, both of these procedures did not result in uniform
infection throughout the olfactory epithelium. In contrast, we infected the
olfactory sensory neurons by putting a drop of virus solution one by one at
the entrance of the nasal cavity and letting the mice inhale it spontaneously
over 60 min to a total volume of 50-70 µl. With this simple method, we
succeeded in relatively uniform infection of the adenoviral vector to the
olfactory sensory neurons throughout the epithelium
(Figure 2A), which is also
evident from thorough labeling of all the glomeruli in the olfactory bulb
(Figure 3A).
Olfactory pathways visualized with WGA-expressing adenoviral vector
In this paper, we have demonstrated the feasibility of transsynaptic
labeling by using the WGA-expressing adenoviral vector in the mouse olfactory
system. By simply infusing the virus solution into mouse nostril, the
olfactory neural pathways were clearly visualized with great accuracy and high
reproducibility from the olfactory epithelium to the olfactory bulb, and
further to the olfactory cortex. In the second-order neurons (mitral cells) in
the olfactory bulb, the transgene product, WGA protein, was detected from 7 to
50 days post-infection with a maximal expression at 20-30 days post-infection.
In the case of ß-galactosidase, it was reported that the transgene
expression in the olfactory sensory neurons persisted for 8-12 days and
decreased at 25 days (Holtmaat et
al., 1996). These results indicate that the WGA protein was
so stable to be sufficiently accumulated in relay neurons, resulting in clear
visualization of the olfactory neural pathways.
Granule cells in the olfactory bulb are GABAergic interneurons that play
important roles in lateral inhibition between neighboring mitral cells. The
granule cells and the mitral cells make dendrodendritic reciprocal synapses.
Transsynaptic labeling of the granule cells in the adenovirus-mediated
WGA-expressing mice in this paper (Figure
3A) was significantly weaker than that in the OMP-WGA transgenic
mice in our previous study (Yoshihara
et al., 1999). One possible explanation is that WGA
transfer across the dendrodendritic synapse may be not so efficient that
chronic, long-lasting expression would be required for definite labeling of
the granule cells.
The mammalian olfactory bulb receives profuse innervation of centrifugal
fibers from the nonolfactory subcortical modulatory systems. In addition to
the anterograde transfer of WGA from the olfactory epithelium to the bulb and
further to the cortex, we observed the retrograde transsynaptic labeling from
the olfactory bulb to these brainstem nuclei
(Figure 6). WGA
immunoreactivity was very intense in cholinergic neurons in the horizontal
limb of diagonal band and serotonergic neurons in the midbrain raphe nucleus,
but faint in noradrenergic neurons in the locus coeruleus. This difference in
WGA appearance among three types of neurons may be attributable to different
termination fields in the olfactory bulb. The cholinergic and serotonergic
terminals are distributed densely in the glomerular layer
(McLean and Shipley, 1987;
Shipley et al., 1995)
where a large amount of WGA is transported directly from the olfactory
epithelium. In contrast, the noradrenergic terminals are found mostly in the
granule cell layers (Halasz and Shepherd,
1983; McLean et al.,
1989) where WGA is scarcely present.
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Compared to the conventional method in which WGA protein was infused into
the nostril (Shipley, 1985;
Itaya, 1987), we could detect
more strongly and reliably the transsynaptically transferred WGA protein in
the olfactory cortical neurons and the median raphe nucleus. This may be due
to the efficient infection of adenovirus to the olfactory sensory neurons and
to the extremely strong promoter elements (CAG promoter) we used for robust
expression of WGA.
Further applications of the WGA-expressing adenoviral vector system
Detailed knowledge of the neural connectivity patterns is indispensable for
understanding a wide range of brain functions, especially higher cognitive
functions that involve combinations of intricate neural networks widely
distributed in the brain. The technique described in this paper enabled us to
deliver plant lectin WGA to olfactory sensory neurons with the aid of the
adenoviral vector system and to visualize the olfactory neural pathways. This
method will be useful not only in the olfactory system, but also in various
neural systems focusing on the development, anatomy and functions of the
brain. Because of the wide host range of adenovirus, furthermore, this
technique will be most useful for the application to other mammalian species
such as monkeys, cats and ferrets that are not amenable to the transgenic
technology, but are useful in neuroscience research
(Bohn et al., 1999;
Lakatos et al., 2000;
Lawrence et al.,
1999; Liu et al.,
2000).
In this paper, we utilized a CAG promoter for the expression of WGA. Under
the control of the CAG promoter, a robust expression of the downstream
transgene is driven in any type of mammalian cells, including neurons
(Miyake et al.,
1996). In contrast, cell type-specific expression of the WGA
transgene will be induced by choosing appropriate promoter elements. In a
preliminary study, we have succeeded in adenovirus-mediated WGA expression in
a subpopulation of the olfactory sensory neurons by using a promoter element
of olfactory zone-specific cell adhesion molecule, OCAM
(Yoshihara et al.,
1997; N.K., T.M. and Y.Y., unpublished result). Thus, the
adenoviral vectors containing WGA cDNA downstream of neuron type-specific
promoter elements will be used for the restricted expression of WGA in time-,
place- and cell type-specific ways.
A further refinement of the technique would be to introduce a bicistronic
transgene consisting of WGA and another reporter molecule with internal
ribosome entry sequence (IRES) in-between. For example, the WGA-IRES-GFP
transgene could be used conveniently for discrimination of the first-order
neurons (WGA- and GFP-producing neurons) from the second- and third-order
neurons (transferred WGA-containing neurons). Furthermore, if a retrograde
direction-specific transneuronal tracer such as tetanus toxin C fragment (TTC)
can be used as a transgene (Coen et
al., 1997), a simultaneous expression of three molecules
(WGA, TTC and GFP) will provide us with more detailed information on neuronal
connectivity patterns originating from the infected neurons. In conclusion,
the use of a WGA-expressing adenoviral vector system should greatly facilitate
studies on the anatomical and functional organization of the developing and
mature nervous system.
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Acknowledgments |
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This work was supported in part by the Special Coordination Funds for Promoting Science and Technology from the Japan Science and Technology Corporation (JST) and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture. We thank Dr Izumi Saito (University of Tokyo) for recombinant adenovirus production system, Dr Junichi Miyazaki (Osaka University) for CAG promoter, and Drs Kensaku Mori (University of Tokyo) and Yasuyoshi Watanabe (Osaka Bioscience Institute) for consistent support.
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Accepted November 8, 2001
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