1 Department of Neuropathology, University of Bonn Medical Center,
Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
2 Institute of Reconstructive Neurobiology, University of Bonn Medical Center,
Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
3 Department of Neurosurgery, University of Bonn Medical Center,
Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
4 Department of Epileptology, University of Bonn Medical Center,
Sigmund-Freud-Strasse 25, D-53105 Bonn, Germany
Author for correspondence (e-mail:
brustle{at}uni-bonn.de)
Accepted 9 July 2003
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SUMMARY |
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Key words: ES cells, Glia, Electrophysiology, Gap junction, Hippocampus, Slice culture
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Introduction |
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In vivo, astrocytes are connected via gap junctions and form extensive
networks (Giaume and McCarthy,
1996). Incorporation of transplanted ES cell-derived astrocytes
into such a network structure could provide new perspectives for both
cell-mediated delivery of small molecules and modulation of neuronal function.
Despite these attractive prospects, little is known about the integration of
glia into the host CNS. In this study, we have explored the potential of
engrafted ESGPs to (1) undergo functional maturation, and (2) incorporate into
the host glial network. To make these functional properties accessible to
experimentation, we took advantage of an organotypic slice-culture paradigm
that permits the study of donor cells under controlled conditions.
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Materials and methods |
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Slice culture and in vitro transplantation
Using a vibroslicer (VSLM1; Campden Instruments, Sileby, UK),
400-µm-thick slices encompassing the dentate gyrus, hippocampus and
entorhinal/temporal cortex (Fig.
1A) were prepared from 9-day-old Wistar rats (Charles River,
Sulzfeld, Germany). Slices were propagated as interface cultures
(Stoppini et al., 1991) on
clear polyester membranes (Transwell-Clear; Corning, Bodenheim, Germany).
Media were changed on day one in culture and then on every other day. Although
cultures were started in a horse-serumcontaining medium, this was replaced
gradually and at day 5 slices were cultured in a serum-free, defined solution
based on DMEM/F12, and including N2 and B27 supplements (Cytogen, Sinn,
Germany). The histoarchitecture of the majority of the slices (>75%) was
remarkably well preserved for up to 5 weeks
(Fig. 1B). Field excitatory
postsynaptic potentials, recorded according to Newman et al.,
(Newman et al., 1995
),
revealed synaptic connectivity between perforant path and dentate gyrus as
well as between Schaffer collaterals and CA1 pyramidal neurons for up to 33
days in culture (n=9; Fig.
1B inset). Anterograde tracing with rhodamine-conjugated dextran
(Micro-Ruby®; Molecular Probes, Eugene, OR), performed as described by
Kluge et al. (Kluge et al.,
1998
), confirmed the integrity of the perforant path
(n=4; Fig. 1C) and
TIMM staining showed an appropriate organization of the mossy fiber system
(n=4; Fig. 1D) up to
the end of the culture period.
|
Electrophysiology
Patch-clamp analysis of ESGPs on coverslips and in slice cultures was
performed under continuous oxygenation in a bath solution containing (in mM):
150 NaCl, 5 KCl, 2 MgSO4, 2 CaCl2, 10 HEPES and 10
glucose, or DMEM/F12, pH 7.4 (with NaOH) as described
(Kressin et al., 1995;
Steinhäuser et al.,
1994
). The standard pipette solution contained (in mM): 130 KCl, 2
MgCl2, 0.5 CaCl2, 5 BAPTA, 10 HEPES, 3
Na2ATP, and 0.1% Lucifer yellow (LY; Sigma), pH 7.25. Current
signals were filtered at 3 or 10 kHz, and sampled at 5 or 30 kHz. Capacitance
and series resistance compensation (up to 60%) were used to improve
voltage-clamp control. Cells engrafted into slice cultures were identified by
virtue of their green fluorescent protein (GFP) expression and chosen at
random for functional analysis. During recording, donor cells were filled with
LY by dialyzing the cytoplasm with the patch pipette solution. Following
fixation, recorded cells were found at variable depths (20-110 µm) in the
slice preparation. Double-immunolabeling with antibodies to glial fibrillary
acidic protein (GFAP) and S100ß was used to confirm the astroglial
phenotype of the recorded cells (Fig. S1 and Table S1 at
http://dev.biologists.org/supplmental).
Dye coupling
Slices were transferred to a submerged recording chamber
(Luigs&Neumann; Neuss, Germany) and maintained under continuous
oxygenation (95% O2, 5% CO2, 35°C). Using sharp
microelectrodes (resistance 10-25 M), 0.1% LY (Molecular Probes) was
injected iontophoretically over a 30-minute period
(Konietzko and Müller,
1994
). Dye coupling was documented at 1 second, then at 1, 5, 7,
10, 15, 20, 25 and 30 minutes. Evaluation of dye spread was performed in fixed
slices using confocal microscopy (Leica; Pulheim, Germany) and digital 3D
reconstruction. Double immunolabeling with antibodies to M2 and GFAP or M2 and
S100ß confirmed the astroglial identity of both the injected donor cell
and the coupled host-cell cluster.
Immunocytochemistry
For morphological analysis, the tissue was fixed in 4% paraformaldehyde,
15% picric acid and 0.1% glutaraldehyde (GA) for 15 minutes and postfixed
without GA overnight at 4°C. Slices were then washed in PBS (Seromed;
Berlin, Germany) and soaked in a phosphate-buffered 30% sucrose solution at
4°C overnight. Unless stated, series of 10 µm horizontal cryostat
sections were prepared from each hippocampal slice specimen, mounted on either
gelatin or polylysine-coated slides, air-dried and stored at 4°C until
further use. ESGP invasion of host tissue was determined by evaluating the
presence of GFP+ cell bodies in serial sections (n=27, see Results)
and in cross sections of fixed slice cultures (n=4) derived from
three independent experiments. Data are expressed as mean±s.d.
Statistical analysis was performed using the two-tailed Student's
t-test. P values of <0.05 were considered
significant.
For immunocytochemistry, a basic buffer was used that contained PBS (Seromed) and 10% fetal calf serum (Invitrogen). 0.1% Triton X-100 (Sigma) was added for the labeling of intracellular antigens. After preincubation in 5% normal goat serum (1-2 hours), primary antibodies to the following antigens were applied overnight at room temperature: BrdU (1:100, monoclonal; BD Biosciences, Heidelberg, Germany); CNP (1:100, monoclonal; Sigma); Connexin43 (1:300, polyclonal; Zymed, Berlin, Germany), GFAP (either 1:100, monoclonal; ICN, Costa Mesa, CA, or 1:400, polyclonal; DAKO, Hamburg, Germany); M2 (1:10, monoclonal; gift from C. Lagenaur); MBP (1:500, monoclonal; Chemicon, Temecula, CA); nestin (1:5, monoclonal; Rat-401, developed by Sue Hockfield and obtained from the Developmental Studies Hybridoma Bank, University of Iowa); NG2 (1:500, polyclonal; Chemicon); and S100ß (1:5000, polyclonal; Swant, Bellinzona, Switzerland). After thorough washing, antigens were visualized by appropriate TRITC-, Cy3-, and Cy5-conjugated secondary antibodies (Vector, Burlingame, CA and Dianova, Hamburg, Germany) applied for 45 minutes at room temperature. Following another washing step, sections were mounted and analyzed using confocal microscopy and appropriate software for 3D reconstruction and image documentation (Leica). To determinate cell-type-specific antigen expression, 19 hippocampal slice cultures from days 11±4 after in vitro transplantation (from 4 independent experiments) were evaluated. For each antigen, immunofluorescence analysis of GFP+ cells was performed in 7-9 randomly assorted cryostat sections. Data are expressed as mean±s.d.
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Results |
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Independent of their location, the donor cells exhibited moderate mitotic activity during the first days after transplantation. Between days 2 and 4 post engraftment, 7.2±6.4% of the GFP+ cells incorporated BrdU (n=177). By contrast, no BrdU uptake was observed in 144 GFP+ cells evaluated between days 10 and 12 after engraftment. Thus, donor-cell migration and proliferation ceased in the second week after transplantation.
During the first week after deposition, most GFP+ cells had bipolar,
migratory phenotypes. In the second week, they developed increasingly
multipolar, astrocytic and oligodendroglial morphologies. Immunofluorescence
analyses at 11±4 days after deposition revealed that 9.4±5.6%
(n=212) of GFP-labeled cells expressed NG2, a marker of glial
precursors (Fig. 2D)
(Diers-Fenger et al., 2001).
Nestin immunoreactivity was retained in 30.4±9.3% of the cells
(n=280). Astrocytes expressed GFAP (27.5±6.2%, n=251)
and/or S100ß (40.5±7.7%, n=227) and had a nonpolarized
morphology with round-oval cell bodies from which emanated numerous prominent
processes (Fig. 2F). Many
process-bearing cells with astrocytic morphology expressed nestin
(Fig. 2E). Typically,
donor-derived CNP+ oligodendrocytes (30.3±9.8%, n=238) had
small, round cell bodies with delicate branched processes
(Fig. 2G). No correlation was
noted between donor-cell differentiation and association with specific
anatomical compartments. However, some GFP+ oligodendrocytes bearing
characteristic tubular processes were found either in clusters or individually
within fiber bundles of the recipient tissue (e.g. in the CA3-CA1 molecular
layer, the alveus and the subgranular layer of the DG). The longer the time
after deposition, the more these cells expressed myelin basic protein (MBP;
Fig. 2H). Whereas the `tubular'
appearance of the cell processes and the expression of CNP and MBP indicated
that some of the donor cells had differentiated into myelinating
oligodendrocytes, ultrastructural studies are required to confirm this
assumption.
In summary, the morphological and immunohistochemical data indicate that most ESGPs undergo terminal differentiation into astroglia and oligodendroglia by two weeks after implantation.
Changing functional properties of engrafted ESGPs
Patch-clamp analysis was used to study the functional characteristics of
ESGPs incorporated into the hippocampus and to compare them with ESGPs that
proliferate and differentiate in monolayer cultures in the absence of host
tissue. Whole-cell currents were elicited by stepping the membrane to
increasing depolarizing and hyperpolarizing potentials between 160 and
+20 mV (50 ms, holding potential 70 mV, see
Fig. 3A,B insets). Delayed
rectifier outward K+ currents [IK(D)] were isolated by
depolarizing the membrane to 40 mV for 300 ms. This pre-pulse was
followed by a 3 ms interval at 70 mV, and then the membrane was stepped
up to +70 mV (Fig. 3C, middle).
Transient outward K+ currents [IK(A)] were separated by
subtracting the outward currents evoked after the 40 mV pre-pulses from
those activated after a 110 mV pre-pulse
(Fig. 3C, right). In the
presence of FGF2 and EGF, ESGPs proliferated
(Brüstle et al., 1999) and
displayed the properties of immature glia, with all cells (n=9)
expressing IK(D) (Fig.
3A). IK(A) was present in 3/9 cells. Inwardly
rectifying K+ (IKir) and background K+
currents [IK(P)], which are characteristic of mature glia
(Bordey et al., 2001
;
Verkhratsky and Steinhäuser,
2000
), could not be demonstrated.
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|
Tail current analysis of IKir and IK(P) revealed reversal potentials of 61.3±13 mV (n=3) and 68.4±8.9 mV (n=9), which indicates that these currents were mainly carried by K+ ions (data not shown). Similar to astrocyte development in vivo, the resting potential of the grafted cells decreased in response to an increasing contribution of IKir and IK(P) (Table 1).
ES cell-derived astrocytes establish gap junctions with recipient
glia
A hallmark of astrocyte development in vivo is the formation of gap
junctions. Individual astrocytes are coupled to dozens of neighboring cells to
form an extensive syncytial network of interconnected glia
(Giaume and McCarthy, 1996).
We were interested in whether transplanted ES cell-derived astrocytes are
inserted into such a network structure. To assess gap junction coupling,
incorporated GFP+ astrocytes were filled with LY
(Fig. 4, Fig. 5A). Cells were chosen
randomly, taking care that no other GFP+ donor cells were present in the
vicinity of the injected cell. Typically, LY spread from the incorporated ES
cell-derived astrocyte to one or two neighboring host glia cells
(Fig. 4A). From there, dye
diffusion proceeded in all three dimensions, decorating additional astrocytes
of the host glial network. The resulting LY-filled cell clusters were
reminiscent of the astroglial syncytium in native rodent hippocampus
(Konietzko and Müller,
1994
; Theis et al.,
2003
). After fixation, dye coupling was quantified using digital
3D-reconstructions of LY-filled cell clusters
(Fig. 4B). Three weeks after
deposition, individual donor cells filled with LY for 30 minutes were coupled
with up to 50 endogenous astrocytes (34±8). Subsequent serial
sectioning of the hippocampal tissue and immunofluorescence analysis using the
mouse-specific antibody M2 (Lagenaur and
Schachner, 1981
) confirmed the presence of a single, donor-derived
astrocyte in each cluster of LY-filled cells
(Fig. 4C,D). The remainder of
cells represented endogenous astrocytes that expressed S100ß and/or GFAP.
No LY-filled neurons were observed in the coupled cell clusters (data not
shown).
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Dye coupling of donor cells appeared to be independent of their location in
the slice preparation. All donor cells (n=6) studied at 22-25 days
after engraftment revealed extensive dye coupling to adjacent host glia
(Fig. 5A-D). Out of four donor
cells injected 12 days after engraftment, only three exhibited dye coupling
(Fig. 5A,E). The spread of LY
from these cells was limited to three (TC region), 21 (EC region), and 24 (DG
region) host cells, respectively. In addition, one donor cell in the TC region
did not appear to be connected to the endogenous glial network. Although more
detailed analyses are required to interpret these observations, they could
indicate regional differences in the efficiency of glial gap junction
formation. However, the differences observed might simply reflect the dynamic
nature of gap junction coupling in astrocytes
(Giaume and McCarthy,
1996).
We next studied the time course of endogenous glial cell coupling and
whether coupling between host cells is affected by incorporated ESGPs. Dye
coupling was performed at days 0-2, 9-12 and 31-34 in culture, using
hippocampal slices not subjected to in vitro transplantation (nine cells in
nine individual slices at each time point;
Fig. 5A). In accordance with in
vivo studies in rodent CNS (Binmöller
and Müller, 1992; Kressin
et al., 1995
), the complexity of gap junction coupling between
host astrocytes increased with time in culture and maturation of the tissue
(Fig. 5E). Implanting ESGPs
appeared to have no effect on the development of gap junctions between host
cells. No morphological differences were observed between LY-labeled
donor-host and host-host cell clusters, and junctional coupling between donor
and host cells also increased with time in culture.
In all, nine out of 10 injected donor-derived astrocytes were coupled with host cells, demonstrating that integration of grafted ESGPs into the glial network is a robust phenomenon. The increase in coupling efficiency with the time in culture indicates that the recruitment of ES cell-derived glial precursors into the endogenous glial network depends on the developmental stage of the host tissue.
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Discussion |
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An in vitro model for neural transplantation
Organotypic hippocampal slice cultures represent a 3D model system of the
CNS that is used increasingly to study a variety of developmental and
disease-related processes (Gähwiler
et al., 1997; McKinney et al.,
1997
; Ullrich et al.,
2001
). In the past, the introduction of donor cells into CNS slice
cultures has been employed successfully for the analysis of donor cell
attachment (Förster et al.,
1998
), neurite outgrowth
(Shetty and Turner, 1999
), and
microglia and glioma cell invasion (Hailer
et al., 1997
; Ohnishi et al.,
1998
).
Refining the methodology, we have established conditions that permit the propagation of vital, functionally active hippocampal slice preparations for up to 5 weeks in serum-free media. We observed a remarkable preservation of the neuronal subpopulations (Fig. 1B,D) and functional maintenance of the fiber tracts (Fig. 1B inset; Fig. 1C,D). Used as `recipient' tissue, this culture system provides an opportunity to study the incorporation, differentiation and function of neural precursors in unprecedented detail. ESGPs deposited on the surface of the slice rapidly invaded the preparation and continued to migrate within the tissue (Fig. 2A,B). After a 2-week period, migration ceased and postmitotic donor cells with complex 3D morphologies were found at various depths of the slice preparation (Fig. 2B inset). Immunohistochemical analyses revealed that the incorporated bipotential ESGPs differentiate into astroglial and oligodendroglial phenotypes (Fig. 2C-G). Thus, this model system provides optimal prerequisites for a functional analysis of engrafted precursor cells.
Engrafted ES cell-derived glial cells undergo functional
maturation
Unlimited self-renewal, pluripotency and amenability to genetic
manipulation make ES cells a highly attractive donor source for cell
replacement. We have shown previously that bipotential glial precursors can be
efficiently derived from mouse ES cells. These cells proliferate in the
presence of FGF2 and EGF and, upon growth factor withdrawal, differentiate
into astrocytes and oligodendrocytes. Although ES cell-derived
oligodendrocytes repair myelin in dysmyelinating mutants
(Brüstle et al., 1999),
the functional properties of integrated ESGPs remain unclear.
During normal development, differentiating astrocytes undergo a
characteristic change of ion channel expression
(Kressin et al., 1995).
Whereas predominating voltage-activated K+ currents are
characteristic of immature astrocytes, IKir and IK(P)
are expressed in more mature glia (Bordey
et al., 2001
; Verkhratsky and
Steinhäuser, 2000
). We found that ESGPs proliferating in
monolayer cultures express only voltage-gated K+ currents
(Fig. 3A). Interestingly,
differentiation induced by growth factor withdrawal did not elicit
IKir and IK(P) (Fig.
3B,C). It was only after incorporation into hippocampal slice
cultures that most GFP-labeled donor-derived astrocytes expressed prominent
IKir or IK(P) (Fig.
3E,F). These data provide the first evidence for functional
maturation of transplanted glial precursors. Moreover, they indicate that
these cells might adopt the distinct astroglial phenotypes that exist in vivo
(Matthias et al., 2003
). The
dynamic changes in ion currents observed following engraftment of ESGPs into
hippocampal slice cultures appear to recapitulate developmental patterns in
vivo. The absence of IKir and IK(P) during propagation
in vitro indicates that full, functional maturation of ESGPs depends on
additional environmental cues in the recipient brain tissue.
It is difficult to address whether the electrophysiological properties of
the engrafted donor-derived astrocytes represent a normal or a `reactive'
state. Functional data from acute brain slices indicate that, in lesioned or
sclerotic CNS, there is a dramatic reduction in the number of astrocytes with
a `passive' current phenotype and a downregulation of astrocytic
IKir in the remaining astrocytes
(Bordey et al., 2001;
Hinterkeuser et al., 2000
;
Schröder et al., 1999
).
Certainly, the organotypic cell culture imposes environmental conditions on
the cells distinct from in vivo conditions. Hence, quantitative comparison of
biophysical parameters (e.g. IKir density) between cells in culture
and in situ does not seem sensible. However, the qualitative changes we
observed during development after engraftment (i.e. upregulation of
IKir density and the appearance of astrocytes with a passive
current phenotype) resemble aspects of astroglial maturation in vivo, and are
opposite to the alterations that astrocytes undergo in response to brain
damage and disease. Thus, we conclude that donor cells expressing significant
IKir densities or a dominant passive current phenotype represent
`normal' astrocytes whereas ES cell-derived astrocytes that lack
IKir might represent a `reactive' state.
Glial network integration of grafted ES cell-derived astrocytes
Gap junction coupling between hippocampal astrocytes is established
gradually during early postnatal development
(Binmöller and Müller,
1992; Konietzko and
Müller, 1994
). This phenomenon is preserved in hippocampal
slice cultures where endogenous astrocytes show an increased amount of dye
coupling with time in culture (Fig.
5E). Surprisingly, ESGPs introduced into different areas of
hippocampal tissue possess a remarkable capacity to integrate into this
network structure (Fig. 5A-D).
Functional dye coupling between donor and host cells was verified by injecting
LY, a low-molecular-weight fluorescent dye, into individual donor-derived
astrocytes (Fig. 4A,B). Following injection of an individual donor cell, LY spread typically into one
or two neighboring host cells before distributing further into the adjacent
glia. This indicates that coupling to one or two host cells is sufficient to
efficiently integrate the donor cells into the established host glial network.
This is supported by the observation that coupling ratios between donor and
host cells were comparable to those observed among the endogenous cells
(Fig. 5E).
Gap junctions are composed of connexin molecules that form pores between
neighboring astrocytes (Giaume and
McCarthy, 1996). Permeable to ions and small signaling molecules,
gap junctions are thought to play an important role in spatial ion buffering
and functional synchronization of the glial network. Connexin43, the key
component of astrocyteastrocyte gap junctions
(Rash et al., 2001
;
Theis et al., 2003
), was
detected consistently at contact zones between incorporated donor cells and
adjacent processes of host cells (Fig.
4C-F).
We found no evidence of gap junction coupling between engrafted ES
cell-derived astrocytes and host neurons, such as described between endogenous
cells in some CNS regions (Alvarez-Maubecin
et al., 2000; Rash et al.,
2001
). However, mechanisms of glio-neuronal interactions become
increasingly well defined (e.g. Aguado et
al., 2002
; Haydon,
2001
; Kojima et al.,
1999
; Verkhratsky and
Steinhäuser, 2000
), and it is tempting to speculate that the
transplanted cells might communicate with neurons via pathways other than
junctional coupling. Considering that ESGPs appear to integrate into any given
glial network structure and that neuronal cells can be easily included in the
grafted population, the slice culture paradigm might be particularly useful
for addressing these issues.
Currently, it is unclear whether and to what extent the integrated ES cell-derived astrocytes elicit functional changes in endogenous glial cells and whether they affect functioning of host neurons. However, on a translational level, gap junction coupling between donor and host glia might offer previously unrecognized opportunities for transplant-based treatment strategies. The introduction of genetically modified astrocytes into the host network could permit transcellular delivery of small foreign molecules to large areas of the CNS. Considering the amenability of ES cells to genetic modification, such a strategy might be used for compound delivery as well as for transglial modification of neuronal function.
Potential and caveats of the slice culture paradigm
Used as an `in vitro transplantation' model, the slice culture system
offers a number of advantages. In contrast to in vivo grafts, it permits
direct experimental access to donor cell migration and integration. Direct
visualization allows precise placement of very small cell numbers. The ex vivo
approach circumvents immunological problems and facilitates the analysis of
allogeneic or xenogeneic donor cells. Last, the model can accommodate donor
cells and host tissues from different genetic backgrounds, which enables a
broad range of transgenic studies.
Devised as a reductionist model, the `graft-in-a-slice' paradigm also has
several limitations. Deposition of the donor cells on the slice surface
restricts the analysis to the cells that invade the slice tissue within 48
hours. Thus, more differentiated cells with reduced migratory potential might
escape analysis. In addition, differences are expected with respect to the
efficiency with which individual donor cell types can be studied in different
slice tissues. Although we have focused primarily on the properties of ES
cell-derived astrocytes, the morphological data and the expression of myelin
proteins by incorporated donor cells indicate that the slice culture paradigm
could, in principle, be well suited for studying oligodendrocyte-axon
interactions as well as functional implications of donor-derived myelin
formation. However, because the postnatal hippocampus contains only few
myelinated areas (although preserved in the interphase culture conditions
applied here) (Berger and Frotscher,
1994), slice cultures from other brain regions such as the
cerebellum might be more suitable for addressing topics associated with myelin
formation (Dusart et al.,
1997
; Seil,
1989
).
Despite the organotypic and functional preservation of the slice tissue,
great care should be taken when extrapolating results to in vivo conditions.
Factors produced by reactive or proliferative astroglia and microglia as well
as reorganizing neuronal subpopulations are known to complicate the
interpretation of slice culture data (Caesar and Aertsen, 1991;
del Rio et al., 1991;
Derouiche et al., 1993
;
Hailer et al., 1996
). Thus,
although it improves accessibility and provides a highly controlled
experimental setting, the slice culture system is suited primarily to
providing proof-of-principle data that can be validated in an in vivo
scenario.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Neuroscience, Brain Institute, University of
Florida, PO Box 100244, Gainesville, FL 32610-0244, USA
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