1Department of Anatomy and Cell Biology and 2Program in Neuroscience, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814
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ABSTRACT |
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Rahimi, Omid and
Sharon L. Juliano.
Transplants of NGF-Secreting Fibroblasts Restore Stimulus-Evoked
Activity in Barrel Cortex of Basal-Forebrain-Lesioned Rats.
J. Neurophysiol. 86: 2081-2096, 2001.
Cholinergic nuclei in the basal forebrain supply the cerebral cortex
with acetylcholine (ACh). Depletion of cholinergic fibers following
basal forebrain lesion results in reduced stimulus-evoked functional
activity in rat barrel cortex in response to whisker stimulation. We
showed previously that exogenous delivery of nerve growth factor (NGF)
to the lateral ventricle restores reduced functional activity toward
normal despite persistent reductions in cortical cholinergic activity.
Gene transfer of therapeutic peptides using genetically engineered
cells allows for localized and biological delivery of compounds to the
CNS, circumventing systemic administration or repetitive invasive
surgery. In this study, we grafted genetically engineered fibroblasts
that secrete NGF (NGF+) into three CNS loci of rats with unilateral
basal forebrain lesions, along with control fibroblasts (NGF) that
did not secrete NGF. Only NGF+ fibroblasts grafted into ACh-depleted
somatosensory cortex resulted in improvement of functional activity
following cholinergic depletion. NGF+ fibroblast transplants into the
lateral ventricle or basal forebrain did not improve functional
activity nor did NGF
fibroblasts in any site. Similar to our previous experiments using intraventricular NGF injections, despite improvements in functional activity, the affected barrel cortex remained depleted of
acetylcholinesterase-stained fibers following insertion of NGF+
fibroblasts. These data support the idea that NGF can act directly on
the cerebral cortex following reductions in cholinergic innervation.
The mechanism of NGF action is illusive, however, since the presence of
its high-affinity receptor, trkA, in the cerebral cortex is controversial.
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INTRODUCTION |
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Nerve growth factor (NGF) and
the family of neurotrophins play a role in protection and regeneration
of CNS neurons (Levi-Montalcini 1987). NGF is implicated
specifically in the protection and maintenance of cholinergic neurons
in the basal forebrain. It has long been known that NGF improves
neuronal survival and increases sprouting of cholinergic neurons in
vivo and in vitro (Dekker et al. 1991
; Hefti et
al. 1985
). Moreover, NGF protects cholinergic neurons against
toxicity and damage (Hefti 1986
; Hefti et al.
1989
; Montero and Hefti 1989
;
Tuszynski and Gage 1990
; Wilcox et al.
1995
). These and many other observations suggest a potential
therapeutic role for NGF and other neurotrophins in disease states that
involve cholinergic neuronal atrophy, such as Alzheimer's and other
dementia. Although examination of NGF as a wonder drug in clinical
trials of patients with Alzheimer's disease has yielded mixed results (Eriksdotter Jonhagen et al. 1998
; Seiger et al.
1993
), many studies show that neurotrophins play a much wider
role in physiological functions. For example, in addition to
neuroprotective roles, NGF influences cells throughout the endocrine
and immune systems (Levi-Montalcini et al. 1996
).
Neurotrophins may also be involved in higher processes such as
information processing and cortical plasticity (Berardi and
Maffei 1999
). Despite the large number of studies fueled by the
therapeutic potential of NGF, we still lack a clear understanding of
the mechanisms, sites of action, and the role of NGF and neurotrophins
in various CNS systems.
Because of the well-known role of NGF in protecting cholinergic basal
forebrain cells from destruction, we began a series of experiments that
delivered NGF to rats concurrent with ipsilateral lesions of the basal
forebrain expecting to improve the survival of these cells. We knew
from previous studies that unilateral lesions of the basal forebrain
lead to decreased functional activity evoked in the somatosensory
barrel cortex in response to whisker stimulation (Jacobs et al.
1991; Ma et al. 1989
). In an attempt to
"rescue" basal forebrain cholinergic neurons and concomitantly improve functional activity in the lesioned hemisphere, we delivered intraventricular injections of NGF to rats. Despite dramatic
improvement of functional responses after NGF treatment, there was no
increase in cholinergic innervation of cerebral cortex, which remained more than 50% depleted of ACh after the basal forebrain lesion (Rahimi et al. 1999
).
The data from Rahimi et al. (1999) suggest that NGF may
act directly on cortical neurons to restore functional activity toward normal, but the precise site and mechanism of action is unclear. Treatment using intraventricular injections of neurotrophins is not
ideal because acute delivery of large doses of NGF limits the duration
and the amount of NGF reaching target sites throughout the CNS, while
increasing the possibility of deleterious side effects
(Taglialatela et al. 1997
). In addition, NGF may
diffusely act on multiple locations within the CNS following
intraventricular injections. In the study reported here, we used a
different form of delivery by transplanting fibroblasts genetically
engineered to secrete NGF into basal-forebrain-lesioned rats. To
precisely define the site of NGF action and deliver more physiologic
amounts of neurotrophin, we inserted NGF+ fibroblasts in various loci most likely to be affected by NGF, including the acetylcholine (ACh)-depleted neocortex, the lateral ventricle, and the basal forebrain. Our experiments revealed that NGF+ fibroblasts grafted into
the neocortex ipsilateral to the basal-forebrain-lesioned hemisphere
restore stimulus-evoked functional responses within the barrel cortex
of rats. Transplants into the lateral ventricle or the basal forebrain
do not result in improved functional responses in the barrel cortex.
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METHODS |
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Female Fisher rats (180-200 g) were divided into three groups.
All animals received unilateral basal forebrain lesions using 192-IgG-saporin. Each treatment group included rats that received grafts of NGF+ fibroblasts and control rats that received NGF fibroblasts. The transplants were delivered immediately following the
lesion and were all in the ipsilateral hemisphere. Thus each animal
presented with one experimental/treated hemisphere (control or
NGF-secreting fibroblasts) and one untreated, otherwise normal, control, hemisphere. The first group received grafts into the cerebral
cortex just medial to the barrel representation. The second group
received transplants into the lesioned basal forebrain, while the third
group received transplants into the lateral ventricle. Animals survived
for 1, 2, or 4 wk after transplantation. Functional activity was
assessed using
[14C]2-deoxy-D-glucose (2DG, ARC,
St. Louis, MO) uptake in barrel cortex during whisker stimulation.
Brain tissue was processed for cytochrome oxidase (CO) and
acetylcholinesterase (AChE) activity, immunohistochemistry to identify
the transplant, as well as 2DG autoradiography. A fourth group of
animals was used for electrophysiological experiments after we
determined the efficacy of the fibroblast transplants on 2DG uptake.
This group included rats that received a basal forebrain lesion and
NGF+ fibroblast transplant, followed by electrophysiological recording
4 wk later. Control rats included animals with basal forebrain lesion
only and no transplant.
Basal forebrain lesion
Each rat received a unilateral basal forebrain lesion as
previously described (Jacobs et al. 1991; Rahimi
et al. 1999
). Briefly, rats were anesthetized using ketamine
(60 mg/kg im) and rompun (4 mg/kg im) and placed in a stereotaxic
apparatus. A small hole was drilled 4 mm anterior and 2.3 mm lateral to
the bregma. 192-IgG-saporin (0.087 µg/µl, Chemicon, Temecula, CA)
was delivered using a Hamilton syringe according to previously
determined coordinates: 35° from vertical and 12° from lateral
(Jacobs et al. 1991
). The syringe penetrates the brain
medially and moves laterally in a rostral to caudal direction,
approaching the basal forebrain over the olfactory bulb while
preserving the parietal cortex. These coordinates target the nucleus
basalis, the substantia innominata, and the ventromedial globus
pallidus. Immunotoxin (1.0 µl) is injected at two sites in 0.5 µl
volumes, the first at 8.0 mm below dura and the second at 7.0 mm below
dura. This procedure results in consistent depletion of the cholinergic
cells of the basal forebrain, while leaving other structures such as
cerebral cortex and thalamus intact.
Bioassay
We performed a bioassay to examine the biological viability of the NGF secreted from genetically modified fibroblasts. Sympathetic ganglia were extracted from chick embryos at embryonic day 8. Neuronal suspensions were prepared and cultured into four groups. Each group received one of the following media conditions and subsequently incubated for 48 h at 37°C and 10% CO2. Dulbecco's modified Eagle's medium (DMEM, Irvine Scientific, Santa Ana, CA) was conditioned as follows: cultured with normal fibroblasts (control, from rat skin, not genetically altered), cultured with NGF+ fibroblasts, fresh media (DMEM) without any additives, and fresh media with synthetic NGF additive. Following a 48-h incubation with conditioned media, neuronal cultures were assessed for survival.
Culture and transplant of fibroblasts
Following the lesion procedure, cell suspensions of genetically
engineered and control fibroblasts were grafted at either of three loci
ipsilateral to the lesion. The genetically modified fibroblast cell
line (Fisher rat skin) was generously donated to us by Dr. F. Gage (The
Salk Institute, La Jolla, CA). The fibroblasts were grown in vitro
using standard tissue culture procedures (Kawaja et al.
1992). Briefly, fibroblasts were cultured using DMEM (Irvine Scientific) with the following additives: 10% fetal bovine serum, 2.5 µg/ml fungizone, 2 mM glutamine, and 50 µg/ml gentamycin. G418 (400 µg/ml, Geneticin, Life Technologies, Grand Island, NY) was used as an
additive only for the NGF+ fibroblasts that were also genetically
altered to be resistant to this antibiotic, ensuring the purity of the
NGF+ fibroblast population. Cultures were maintained using standard
culture conditions at 37°C in 10% CO2.
Fibroblasts were fed once every 3-4 days with fresh conditioned media.
Once confluent (approximately once per week), the cells were passaged using trypsin-EDTA solution (ATV solution, Irvine Scientific). NGF+ and
control fibroblasts were used at six to seven passages or less for CNS
grafting. The cells harvested for grafting were at resting state
(postconfluence). Fibroblasts were dislodged from culture plates using
trypsin-EDTA solution and collected using grafting media (grafting PBS
and 2% rat serum). Grafting PBS consisted of sterile PBS supplemented
with 1 µg/ml MgCl2 and CaCl2 and 0.1% glucose. After centrifugation at
70 g for 5 min, the cells were washed with 10 ml of fresh
grafting PBS, counted, and examined for viability using an acridine
orange-ethidium bromide solution (0.3% solution). Only fibroblast cell
suspensions with a viability of 90% or better were used for
transplantation. The suspension was subsequently centrifuged at 200 g and transferred to an eppendorf tube to yield a final
concentration of 1.0-1.5 × 105 cells/µl.
Higher concentrations of cells may be transplanted with no deleterious
effect on the brain (personal communication, Dr. F. Gage). Using a 10 µl Hamilton syringe, 2.5 or 5.0 µl of the cell suspension (in
grafting PBS solution) was inserted at the desired site within the CNS.
An injection rate of 1 µl/min was used. The surgical procedure and
transplant locations were similar to those described previously
(Jacobs et al. 1994
; Rahimi et al. 1999
).
Coordinates for cortical transplants included three sites along an axis
1.1 mm lateral to midline at 0.1, 1.1, and 2.1 mm posterior to bregma,
and 1.1 mm below dura (7.5 µl). Intraventricular grafts were made at
coordinates 0.5 mm posterior to bregma, 1.1 mm lateral to midline, and
3.4 mm below dura (about 5 µl of cell suspension). Fibroblast grafts
into the lesioned basal forebrain were performed in the same manner as
the lesion procedure described in the preceding text (about 6 µl of
cell suspension).
2DG experiments
Functional responses were assessed in all rats 1, 2, or 4 wk
after the grafting procedure. A 2DG experiment was performed on each
rat, and the area of stimulus-evoked metabolic uptake in barrel cortex
was measured as previously described (Rahimi et al.
1999). Briefly, each rat was lightly anesthetized using isoflurane. All whiskers were removed except three or four pairs of
matched vibrissae on the rat's face. Nonadjacent whiskers used for
stimulation included whiskers from B or D rows. Each vibrissa was
fitted with a piece of mu-metal, an alloy of iron and
75-80% nickel, 3 mm long and 350 µm in diameter, using glue (Type
201 Aron Alpha, Borden, Columbus, OH). The mu-metal was
placed at equal distances, approximately 1.0-1.2 cm away from the face
on each remaining whisker. Following recovery from anesthesia (1-1.5 h), each rat was placed in a cylindrical cage, 16 cm in diameter, surrounded by an electromagnetic coil. Our whisker stimulator system is
similar to the "Lausanne whisker stimulator" developed by Melzer
and colleagues (Melzer et al. 1985
). A Grass stimulator attached to the coil generates magnetic field bursts every 50 ms. The
magnetic field mechanically moves the whiskers with attached mu-metals at 20 Hz while the rat is moving freely in the
cylinder. Stimulation began 5 min prior to injection of
[14C]2-deoxy-D-glucose (20 µCi/100 g ip) and continued for 1 h following injection. Each
rat was then injected with an overdose of pentobarbital sodium (50 mg/kg ip) and perfused intracardially with saline followed by 0.1 M
phosphate buffered paraformaldehyde with sucrose (4%). Each brain was
then quickly removed and frozen in isopentane at
35°C and stored at
80°C until processed.
Tissue processing
Brains were cut into 30-µm-thick coronal sections using a
cryostat at 19°C; alternate sections were saved for 2DG
autoradiography, CO, AChE histochemistry, and immunohistochemistry. The
procedures for tissue processing were previously described
(Jacobs et al. 1991
, 1994
). Sections saved for 2DG
autoradiography were collected using 2% gelatin coated slides and
placed on a hot plate (60°C) for immediate dehydration. These slides
were exposed to X-ray film (Kodak MR-1; Sigma, St. Louis, MO) with
14C methylacrylate standards (2 nCi; Amersham,
Piscataway, NJ) for 3-5 days and subsequently developed using standard
procedures. These sections were later used for staining to identify
Nissl substance. AChE and CO histochemistry were performed according to
previously described protocols (Jacobowitz and Creed
1983
; Rahimi et al. 1999
; Wong-Riley
1979
).
Localization of transplants
The fibroblast transplants were localized during processing of
the tissue using several different methods. The NGF+ transplants were
identified using antibodies directed against NGF or fibronectin to
localize the fibroblasts. The control NGF transplants were visualized
using immunoreactivity against fibronectin or by labeling with a
fluorescent dye. Appropriate sections were sectioned at 30 µm and
collected on gelatin slides. Immunohistochemistry for NGF was performed
according to standard procedures using anti NGF primary antibody
(Polyclonal anti-NGF-beta, Chemicon) at 1.0 to 2.0 µg/ml. NGF
labeling was visualized using biotinylated secondary antibody kit (goat
anti-rabbit, and avidin/biotin peroxidase, Vector Labs, Burlingame, CA)
followed by a 5- to 10-min incubation in a Tris-buffered saline
solution containing 0.05% diaminobenzidine (DAB) and 0.06%
H2O2. Fibronectin
immunostaining was performed similarly, using a monoclonal antibody
against fibronectin (Boehringer Mannheim, Indianapolis, IN) at 10 µg/ml, followed by a biotinylated secondary antibody kit (horse
anti-mouse, and avidin/biotin peroxidase, Vector Labs) for
visualization with DAB.
Carboxyflurescein diacetate, succinimidyl ester (CFDA SE) cell tracer (Molecular Probes, Eugene, OR) was used to fluorescently label control fibroblasts, prior to CNS grafting. Fibroblast cell suspensions were labeled with a 10 µM solution of CFDA SE and DMSO. The cell suspension was incubated with prewarmed (37°C) PBS containing the dye for 15 min at 37°C. The PBS with dye was removed and cells resuspended in fresh PBS, and incubated for 30 min. Cell suspensions were washed with grafting PBS and used for transplantation.
Measurement of AChE distribution
Cholinergic depletion following basal forebrain lesion was
measured using AChE histochemistry. Staining for AChE, the catabolic enzyme for acetylcholine, is not a direct measure of acetylcholine but
is useful as a tool to assess cholinergic depletion (Hohmann and
Coyle 1988; Jacobs et al. 1994
; Ma et al.
1989
). Optical-density measurements were used to determine AChE
reactive fiber density as previously described (Jacobs and
Juliano 1995
; Jacobs et al. 1994
). The density
of AChE-positive fibers was assessed and quantified in both hemispheres
of all rats using the sections adjacent to 2DG-labeled autoradiographs
that contained activated barrels. Optical-density levels were measured
within a rectangular region of 1.0 × 0.5 mm, which extended from layer
II-V. An average value of optical density was derived across sections
assessed from each hemisphere. This allowed us to quantify and compare
AChE staining in the treated barrel cortex to that of the control,
untreated cortex. Optical-density values in the lesioned hemisphere
were calculated and represented as a percentage of the density in the untreated, normal cortex, set at 100%.
Barrel-associated metabolic uptake
A video-based image-processing system was used to visualize and
quantify metabolic uptake in the activated barrels revealed on 2DG
autoradiographs. Each autoradiograph was digitized using a previously
described protocol (Jacobs and Juliano 1995).
Radioactive standards were used to convert optical density values to
color or gray scales. Variability of the 2DG label was quantified in each section by measuring optical-density values for specific regions
and expressed as a percent above background, where background is set to
optical density of white matter. Using a digitizing tablet, layer IV of
the frontoparietal cortex within each 2DG autoradiograph was flattened
and represented in two dimensions using software that partitions and
designates regions of cortex into vertical and tangential arrays of
high resolution (Tommerdahl et al. 1985
). Bins (50 µm)
spanning layers II-V are generated and collapsed to a single point
containing the density values. Files from all sections are aligned and
displayed as unfolded maps that display evoked-metabolic uptake in
barrels as specific foci of high activity. Values 1.5 SD above the
average density of the entire map were set to black in the unfolded
map. Areal measurements were obtained from the barrel-associated foci
and comparisons made between the treated, experimental cortex and the
untreated, normal cortex.
Electrophysiological experiments
Neuronal response characteristics following whisker stimulation were measured using extracellular recordings of single units within layer IV of ACh-depleted barrel cortex. Recordings were obtained from neurons located in matched barrels from both hemispheres. Both spontaneous activity and response magnitude of single units were assessed. Recording experiments were done in a "blind" fashion, that is, the experimenter did not know the nature of the animal treatment.
Rats were anesthetized with a ketamine (60 mg/kg) and rompun (4 mg/kg) mixture (injected intramuscularly) and maintained on this mixture for the duration of the recording experiment. Each rat was monitored regularly during the procedure and supplemental doses of anesthesia were administered to ensure a uniform state. Each rat was placed in a stereotaxic apparatus, and an opening was made in the skull overlying the posteromedial barrel subfield. The dura was removed and the brain covered with mineral oil for protection. An opening was also made in the cisterna magna to drain cerebrospinal fluid and reduce pulsations and swelling of the brain.
Response characteristics were assessed in layer IV of the
cortical representation for each designated whisker. Cortical single units corresponding to at least two different matched whisker barrels
were located (e.g., B2-3, D2-3, C2-3 whiskers), using tungsten-in-glass recording electrodes (0.5-1.0 M). The electrode was lowered to the surface of the cortex at the specific stereotaxic coordinates of a designated whisker. The cortical representation of
each whisker was confirmed electrophysiologically by locating the
cortical site from which single units responded most vigorously to
displacement of the designated vibrissae. The electrode was lowered
through the cortex as single or multiple units were encountered in
response to stimulation of the designated or surrounding whiskers. Recorded signals were subsequently amplified, filtered, and displayed while simultaneously sent to a personal computer equipped with the
BrainWave data-acquisition and -analysis software (DataWave Technologies, Long Mont, CO). The location and depth of each recording site was noted, and response activity was recorded in multiple units
and subsequently sorted into single units off-line. Neuronal activity
was recorded during 30 sweeps of 1-s epochs. The first 500 ms of each
sweep provided spontaneous activity of the unit recorded. A uniform
stimulus was applied to the appropriate whisker using a switching
device (whisker displacement apparatus) fitted with a glass pipette.
The switching device delivered a stimulus that consisted of a motion
that deflects the whisker by 1 mm (1.14°) in the upward direction.
The whisker was trimmed to 15-mm length and fitted inside the glass
pipette attached to the switching device, approximately 10 mm away from
the skin. Cortical units displayed optimal response to this excursion
as reported by Simons and colleagues (Simons 1978
). The
response properties and spontaneous activity of each unit was recorded
and averaged over 30 stimulus sweeps. The electrode was then advanced
to other sites of responsive neuronal units, and the recording
procedure repeated. At the end of each penetration site, we made a
small electrolytic lesion by a passing current through the electrode (2 mA, anodal current; 5-8 s) to identify the electrode track.
Single-unit recordings in the same layer were assessed in matched
barrels of the opposite, otherwise normal hemisphere. At the end of the
recording session, each animal was deeply anesthetized and perfused
intracardially with paraformaldehyde (4%) and sucrose (4%). Brains
were removed and immediately frozen using isopentane at
35°C. All
brains were stored at
80°C until processed. Brains were cut on a
cryostat at 30 µm, and three series of sections were collected for
Nissl substance staining, CO activity, and AChE histochemistry. The laminar location of each recording site was verified using depth of the
recording electrode and the site of the electrolytic lesion identified
in Nissl-stained sections.
Multiple-unit recordings were analyzed off-line and isolated into single units of activity using the BrainWave program. Single units were recognized by their waveform characteristics and isolated. Each unit was distinguished using conservative and consistent criteria and represented by peri-stimulus time histograms (PSTHs). The spontaneous firing rate for each single unit was also determined and represented using PSTHs. Spontaneous activity of an individual neuron was the firing rate from a 500-ms prestimulus epoch of each stimulus trial, averaged over 30 trial sweeps. The response magnitude of individual neurons was represented on PSTHs as the number of spikes/stimulus for the 50-ms period following a whisker stimulus. Statistical analysis was used to compare evoked activity in the treated hemisphere to that of the untreated, otherwise normal hemisphere.
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RESULTS |
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Immunotoxin lesions of cholinergic basal forebrain deplete the cortical distribution of AChE
Similar to our previous experiments, lesions of the basal forebrain produced persistent depletion of the cortical cholinergic input from basal forebrain nuclei. Correct placement of the lesion was verified using CO histochemistry and AChE reactivity. Immunotoxin lesions using 192-IgG-saporin specifically target and destroy cholinergic neurons that express the low affinity NGF receptor, p75NTF. In these experiments, injections of immunotoxin destroyed cholinergic neurons within the basal forebrain confined to the nucleus basalis magnocellularis, substantia innominata, and ventromedial globus pallidus, while sparing other systems (Fig. 1).
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We compared the density of AChE staining in sections adjacent to stimulus-activated barrels in normal and treated hemispheres. The number of animals in all the experimental groups can be seen in Table 1. All rats receiving a unilateral basal forebrain lesion displayed reduced levels of AChE fiber staining irrespective of graft placement. The AChE depletion was homogenous throughout the cortical layers, ranging from 85 to 95% below the AChE fiber staining within the normal cortex (Fig. 2). That is, despite whether the NGF producing fibroblasts were placed in the cortex, the lateral ventricle, or basal forebrain ipsilateral to the lesion, AChE reactivity remained significantly reduced in comparison to the control, otherwise normal, hemisphere (Fig. 3). Although we generally found few differences related to the survival time after the transplant, in animals with cortical grafts of NGF+ fibroblasts that survived for 4 wk following treatment, slight increases in AChE-positive fibers were present surrounding the area of the graft. This increase in AChE fiber staining, however, was limited to the site of the graft and did not extend significantly into surrounding cortical regions. In addition, the optical density measurements did not show significant overall increases in the density of AChE fiber staining in the treated hemispheres in these animals compared with the animals with transplants of shorter duration. Other animals with similar treatment and shorter survival periods, i.e., 1 or 2 wk survival, following cortical transplantation did not show increases in AChE fiber staining.
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Secretion of biologically active NGF from genetically engineered fibroblasts
To ensure that the genetically altered fibroblasts produced and
secreted biologically active NGF in our hands, prior to grafting into
CNS loci, we used conditioned media obtained from the fibroblast cultures in a bioassay. Neuronal cultures were prepared from embryonic sympathetic neurons, which are dependent on NGF for survival. The
cultured sympathetic neurons were then incubated for 48 h with
conditioned media obtained from fibroblast cultures. The neurons were
grown either with conditioned media from NGF+ fibroblast cultures or
from NGF fibroblast cultures. The conditioned media was extracted
from cultured fibroblasts after 5 days of active fibroblast growth.
Positive and negative controls included sympathetic neurons cultured
with control media containing synthetic NGF additives or no additives.
Our results show that the sympathetic cultured neurons survived when
incubated with media from NGF+ fibroblasts or media with NGF additives
but not when cultured with control media or conditioned media from the
control fibroblasts. Figure 4
demonstrates typical healthy sympathetic neurons after conditioned media culture obtained from NGF+ fibroblasts and nonneuronal cells (i.e., no surviving neurons) seen after culture with conditioned media
obtained from the control fibroblasts.
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Localization of transplants
The fibroblast transplants remained near the injection site and were viable for up to 4 wk post transplantation. The immunoreactivity for NGF peptide or fibronectin revealed labeled cells at the site of the transplant and at distances up to 500 µm away. Positive NGF immunoreactivity was seen in animals that survived up to 4 wk following treatment (Fig. 5). Fibronectin immunoreactivity in a control transplant was observed at the site of transplant. The final method for identifying the fibroblasts, using fluorescent dye, CFDA SE cell tracer, can be seen in Fig. 6, which shows labeled cells in a transplant placed in the basal forebrain.
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2DG uptake
The metabolic uptake, in response to whisker stimulation,
occurred in column-like patches that corresponded to locus of the individual barrels, as we reported previously (e.g., Ma et al. 1989). The 2DG patches were compared with adjacent sections
that were reacted for CO activity and observed to coincide with the individual barrels (Fig. 7A).
We also found that the dimension of the barrels, measured by CO, did
not change after basal forebrain lesion as we have reported previously
(Jacobs and Juliano 1995
; Jacobs et al.
1991
; Ma et al. 1989
).
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The fibroblast transplants were placed in three different locations, for both the control and the NGF+ cells. In the animals that received control fibroblasts, 2DG uptake following whisker stimulation remained diminished on the side of the lesion, regardless of transplant placement, including cerebral cortex (e.g., Fig. 8A). Basal-forebrain-lesioned animals with NGF+ fibroblast transplants placed either in the basal forebrain (Fig. 8C) or the lateral ventricle (Fig. 8D), also demonstrate reduced activity in response to whisker stimulation. When an NGF+ transplant was placed in the cerebral cortex of a basal-forebrain-lesioned animal, the 2DG uptake elicited in response to whisker stimulation was similar to that in the normal hemisphere (Fig. 8B).
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When the activity was viewed as two-dimensional digitized maps, 2DG uptake appears as foci of stimulus-evoked activity that correspond to activated barrels. An example of the relation between the foci of 2DG uptake as represented in the two-dimensional maps and the CO staining of the barrels is indicated in Fig. 7B. The maps in this figure were prepared by indicating the boundaries of the cytochrome oxidase-stained barrels, using the sections adjacent to the 2DG autoradiographs, and making a separate map of the CO barrel boundaries. The map of the CO boundaries was then superimposed on the 2DG map, indicating the correspondence between the 2DG foci and the CO-stained barrels.
Illustrated in the two-dimensional maps of 2DG activity are examples of
several experimental conditions (Figs. 9
and 10). The only condition that led
to improved functional responses was the transplantation of NGF+
fibroblasts into the cerebral cortex. In this condition, the regions of
2DG uptake produced in the transplanted hemispheres are similar in
dimension to those in the untreated, normal hemisphere (Fig. 9). In all
other experimental conditions, whether receiving control NGF
transplants, or NGF+ grafts into the basal forebrain or lateral
ventricle, the barrel-associated 2DG uptake remained diminished,
similar to that observed in rats with basal-forebrain-lesioned
hemisphere alone (Fig. 10C). Shown in Fig. 10 are examples
of maps generated from control NGF
fibroblast transplants (Fig.
10A) and from animals receiving NGF+ fibroblast transplants
ipsilateral to the lesioned hemisphere, placed either into the basal
forebrain or the lateral ventricle (Fig. 10, B and D). In these conditions, foci of evoked activity are smaller
in dimension to that evoked in the normal hemisphere.
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To quantify these observations across all animals, the area of each
region of barrel-associated activity was measured and compared between
the treated and the untreated, otherwise normal hemisphere and
displayed as a ratio. Figure 11
demonstrates that the only experimental condition resulting in improved
functional activity was that of NGF+ fibroblasts placed in the cerebral
cortex. Other placements of NGF+ fibroblasts and control NGF
transplants led to 2DG uptake that was significantly reduced from that
evoked in the normal hemisphere.
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Timing and distance
We examined the effects of the length of NGF treatment in
ACh-depleted rats. The results presented in the preceding text included all survival times. Rats with unilateral basal forebrain lesions survived for 1, 2, or 4 wk following transplantation of either control
NGF or NGF+ fibroblasts at various loci. Deficits in functional
activity persisted in rats that received control or NGF+ grafts in
either the lateral ventricle or basal forebrain, despite the duration
that the fibroblasts remained in situ. Since restoration of
stimulus-evoked activity was restricted to rats with NGF+ grafts placed
in the ACh-depleted cortex, we compared short- and long-term effects of
NGF treatment in this group (Fig. 12).
Shown in Fig. 12 are the combined data for animals with NGF+ fibroblasts in the cerebral cortex for 1, 2, or 4 wk. The mean ratio of
the response for the treated: untreated hemisphere was plotted for each
survival time. Although the magnitude of the activated regions was
slightly larger after 1 wk of survival, after 4 wk of survival, the
magnitude of uptake in the treated hemisphere was still similar to that
in the normal hemisphere (i.e., a ratio slightly more than 1.0). In
addition, the dimensions were not significantly different from one
another (Mann-Whitney test), suggesting that cortical grafts of NGF+
fibroblasts were effective in restoring functional activity as long as
4 wk following transplantation.
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We also assessed whether the distance of the activated barrel from the transplant influenced the dimension of the 2DG uptake in the animals receiving NGF+ fibroblasts into the cerebral cortex. This information is presented in Fig. 13, which plots the size of the barrel-associated spots of label as a function of the distance from the transplant. Although there is a trend for the dimension of the uptake to reduce with increasing distance from the transplant, at the greatest distance examined, the ratio of the dimension of uptake for the treated: untreated hemisphere remains close to 1.0. In addition, statistical analysis reveals that there is no correlation between the distance from the transplant and the size of evoked activity (R2 = 0.16). It is likely, however, that if the activated regions were of greater distance from the transplant, we would have observed a decrease in the area of the activated region with increasing distance from the transplant.
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Electrophysiology
We examined neuronal responses in both hemispheres of two groups
of rats (3 rats per group). The first group received unilateral basal
forebrain lesions and no further treatment. The second group received
unilateral basal forebrain lesions and subsequent transplants of NGF+
fibroblasts into the cerebral cortex of the same hemisphere. Matched
cortical whisker representations were selected for analysis. An
electrode advanced through the barrel representation recorded the
number of spikes before and after a whisker deflection stimulus. For
each single unit, 30 stimulus trials were recorded and analyzed off-line. Following the isolation of single waveforms, PSTHs were generated for each unit activity recorded, accumulated over 30 stimulus
trials (Fig. 14). In animals that
received basal forebrain lesions only, single-unit recordings of layer
IV cells revealed reductions in the responses of neurons in
ACh-depleted somatosensory cortex compared with activity in the normal
cortex. Single units in ACh-depleted barrel cortex displayed a reduced
response in comparison to matched single units recorded from the normal
barrel cortex. Neurons in the control hemisphere revealed response
magnitudes similar to results previously reported (Simons and
Carvell 1989). Both the number of action potentials and the
average response magnitude of neurons in the depleted cortex were
significantly reduced in comparison to the response magnitude of
neurons in normal cortex (Mann-Whitney test, t-test,
P = 0.003; Fig. 15). The number of single units recorded for each group is indicated in Fig.
15. Spontaneous activity did not change significantly when comparing
the treated versus untreated hemispheres (Mann-Whitney test; Fig. 15).
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Recordings were also made from matched whisker representations in the barrel cortex of rats that received unilateral basal forebrain lesions and ipsilateral NGF+ fibroblast grafts placed in neocortex. In these animals, there were no significant differences in the number of action potentials evoked from the treated versus normal hemisphere as shown in Fig. 14B. The spontaneous activity also did not differ between hemispheres. The mean evoked response determined in the NGF treated hemisphere also did not differ significantly from that in the untreated, normal hemisphere (Mann-Whitney test; Fig. 15).
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DISCUSSION |
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Our results show that NGF delivered to the ACh-depleted barrel cortex using modified fibroblasts improves functional responses in rats with unilateral basal forebrain lesions. The restoration of activity was dependent on the placement of the fibroblast grafts. Animals with cortical transplants of NGF+ fibroblasts showed significant improvements in evoked activity, while those animals receiving NGF+ transplants in the basal forebrain or lateral ventricle or control transplants in any site did not show improvements. Restoration of functional activity was not dependent on the duration of survival following transplantation of NGF+ fibroblasts.
Rationale for study of NGF
Many studies emphasize the influence of ACh on neuronal response
properties in sensory cortex. Emerging hypotheses suggest that the
cholinergic neurons, in combination with the inhibitory input from the
basal forebrain, exert a tuning mechanism to enhance and strengthen
relevant sensory stimuli at the level of the neocortex (Dykes
1997). Numerous studies show that alteration in cholinergic input to the sensory cortices changes the physiological and behavioral processing of sensory information. Previous reports from this laboratory indicate that unilateral basal forebrain lesions, which produce significant reductions in neocortical cholinergic innervation, result in reductions of stimulus-evoked activity (Jacobs and
Juliano 1995
; Jacobs et al. 1991
). Similarly,
other studies show that manipulation of cortical cholinergic
innervation influences sensory processing in cerebral cortex. For
example, Kilgard and Merzenich recently found that the frequency
distributions in auditory cortex were significantly altered after
pairing a sound stimulus with basal forebrain stimulation
(Kilgard and Merzenich 1998
). In a different study,
Dykes and colleagues demonstrated that increased cortical responses due
to a whisker-pairing paradigm do not occur during blockade of
muscarinic receptors (Maalouf et al. 1998
). If ACh is
applied directly to sensory regions of cortex, increases in neuronal
response magnitude or increases in the size of a given receptive field
occur (Lamour et al. 1988
; Metherate
1998
). These and many other studies emphasize the importance of
the cholinergic influence originating from the basal forebrain on
cerebral cortex function.
Because of the importance of cholinergic innervation to sensory
processing, we aimed to ameliorate the effect of basal forebrain lesion
and subsequent cholinergic depletion in our model. Initially our
experiments delivered NGF to basal-forebrain-lesioned rats in an
attempt to ameliorate the destruction of the cholinergic neurons in the
lesioned site. The protective role of NGF for the cholinergic neurons
of the basal forebrain and its ability to guard the cholinergic (and
noncholinergic) neurons of the basal forebrain from damage and to
rescue them from lesion-induced atrophy is well documented
(Hefti 1986; Kromer 1987
; Williams
et al. 1986
). In addition, NGF offsets cognitive and behavioral
deficits attributed to cholinergic neuronal atrophy (Fischer et
al. 1987
, 1991
). Findings such as these encouraged us to use
intraventricular injections of NGF in an attempt to rescue basal
forebrain cholinergic neurons from lesion, restore neocortical
cholinergic innervation, and improve functional responses that were
diminished in our earlier studies. Although we previously observed
dramatic increases in functional responses after NGF treatment, to our
surprise, no improvement in cholinergic innervation of neocortex
occurred. This suggested that NGF might directly influence the cerebral cortex independent of cholinergic input (Rahimi et al.
1999
). Because the NGF was delivered intraventricularly in the
previous study and we could not determine the precise site of action,
the current experiment aimed to determine the site of action by
insertion of NGF+ fibroblasts in CNS regions likely to mediate a
positive effect. The fibroblasts in this study also delivered more
physiologic amounts of NGF compared with the relatively large
injections into the lateral ventricle.
Localized delivery of NGF via genetically engineered fibroblast transplants
Ex vivo gene manipulations of biological cell lines such as
fibroblasts allow for a continuous, biologically active delivery of
neurotrophins (Gage et al. 1990; Kawaja et al.
1991
, 1992
). Fibroblasts survive following CNS grafting and can
continue peptide secretion for periods up to 6 mo (Tuszynski et
al. 1994
). Rossner and colleagues showed 30- to 100-fold
increases of NGF in cerebrospinal fluid following grafts of NGF+ 3T3
mouse fibroblasts (Rossner et al. 1996
). In vitro
analysis of the NGF+ fibroblasts used in the present study, as reported
by Gage and colleagues, produce an average of 160 pg of NGF · h
1 · 105 cells
(Kawaja et al. 1992
). On the other hand, although
several studies describe relatively long-term survival and activity of NGF+ fibroblasts, others indicate a reduction of NGF peptide production after periods of transplantation. For example, Frim and colleagues examined the role of NGF+ fibroblasts in an excitotoxic model of
Huntington's disease. They showed that preimplantation of NGF+ fibroblasts protected striatal neurons against excitotoxic lesions by
80%. Despite a robust biological effect, after 7 or 18 days following
implantation, few cells within the transplant site stained positively
for NGF peptide or for mRNA of the transfected NGF gene (Frim et
al. 1993
).
In our study, we found that the NGF+ fibroblasts were effective in restoring functional responses when transplanted into the cerebral cortex. In addition, the bioassay demonstrated that only the NGF+ fibroblasts supported sympathetic neuronal cultures. We also found that the NGF+ fibroblasts continued to secret immunologically detectable NGF for periods up to 4 wk following transplantation into CNS. This was observed in the finding of positive NGF immunoreactivity in the fibroblasts after 4 wk in situ (e.g., Fig. 5) and secondarily in the increases of 2DG uptake after a 4-wk survival. The transplants also displayed immunoreactivity directed against fibronectin in the case of the control fibroblasts after 4 wk in vivo.
Our results suggest direct action of NGF on the cerebral cortex since
placement of the fibroblast transplants into the lesioned basal
forebrain or lateral ventricle were not effective in resorting functional activity, while direct placement into the cerebral cortex
resulted in increased functional responses. Other studies show that the
biological effects of NGF secretions from grafted fibroblasts are
dependent on the proximity of the NGF source and target cells
(Frim et al. 1993). Our previous study found that direct
injections of NGF into the lateral ventricle generated improved
stimulus-evoked activity, but the transplants of NGF+ fibroblasts used
in this study were not effective when placed in the lateral ventricle.
This may be due to the difference in dose delivered by several bolus
injections into the lateral ventricle versus the more physiologic NGF
levels delivered by the fibroblasts. The same may be the case with the
fibroblast transplants into the basal forebrain. The combination of the
amount of NGF delivered by the fibroblasts and the amount of damage as
a result of the immunotoxin may be too high to expect restoration of
the damaged cells. The NGF delivered by the fibroblasts may only be
effective locally by direct action on cortical cells.
On the other hand, we did not see significant differences in the
magnitude of effect related to the distance of an activated barrel from
the fibroblast neocortical transplant. In general, the barrel
associated 2DG uptake closer to the edge of an NGF+ transplant was only
slightly larger than regions of activity located farther away. For the
most part, however, the active barrels were relatively close to the
transplants (i.e., within 1.8 mm), which may be within a reasonable
range to expect an intracortical transplant to be effective
(Jacobs et al. 1994).
Does NGF act directly on neocortical cells?
Our data suggest that NGF acts at the level of neocortical
neurons to alter cortical function because only transplants directly into the neocortex led to positive results and improved functional responses. In addition, cholinergic innervation of the lesioned hemisphere was not improved, implying a noncholinergic mechanism of
action on the part of the NGF+ cells. Neurotrophins act using specific
high affinity tyrosine kinase receptors (trk A, B, C) and the
low-affinity receptor p75NTF. NGF is particularly
associated with actions on the trkA and the
p75NTF receptor. The idea that NGF acts directly
on cortical neurons is somewhat problematic because it is generally
believed that adult cortex lacks the high affinity receptors specific
for NGF (Chao 1994; Holtzman et al. 1995
;
Sobreviela et al. 1994
). Several studies have recently
reported the presence of these receptors across cortical fields,
however, suggesting that neocortex may be able to respond directly to
NGF through high-affinity trkA receptors (Cellerino and Maffei
1996
; Prakash et al. 1996
; Valenzuela et
al. 1993
). Although these receptors are present in neocortex, they may not occur at high levels (Cellerino and Maffei
1996
). On the other hand, prolonged NGF treatment may induce
trkA mRNA expression in cerebral cortex. Long-term exposure
to NGF induces trkA receptor immnoreactivity in PC12 cells and basal
forebrain cholinergic neurons (Holtzman et al. 1995
).
The low-affinity p75NTF receptor is present in
neocortex, but because this receptor may promote processes involved in
cell death, it is unlikely to mediate the increased responsiveness
observed after transplants of NGF+ fibroblasts, although the
p75NTF receptor may modulate NGF actions exerted
through trkA receptors. Other lines of evidence support a possible
direct action for NGF on cortical neurons of the neocortex, despite the
lack of high densities of trkA receptors. McAllister and colleagues
showed that NGF and other members of the neurotrophic family modulate dendritic growth in developing pyramidal neurons in specific cortical layers (McAllister et al. 1995
). NGF enhanced the growth
of dendritic arbors in layers IV and V of pyramidal neurons, while
partially reducing dendritic growth of neurons in layer I. In a
different set of experiments using adult neocortex, Kolb and colleagues showed that exogenous delivery of NGF influences dendritic branching and spine density (Kolb et al. 1997
). Such growth of
dendrites might be a mechanism for mediating the increases in cortical
responses seen in this study.
Specific neurotrophic influences on cortical function have also been
demonstrated. During a critical period of postnatal development, exogenous delivery of NGF to the neocortex can prevent cortical alterations due to monocular deprivation in visual cortex, thus not
allowing plastic rearrangements to occur (Berardi et al.
1993; Maffei et al. 1992
). In dark-reared rats,
NGF administration to visual cortex also prevents changes in cortical
responses due to deprivation (Pizzorusso et al. 1997
).
Other studies show that blockade of NGF function prevents normal
postnatal development of the visual cortex. Berardi and colleagues
blocked endogenous NGF activity by implanting hybridoma cells that
produced antibodies directed against NGF in the lateral ventricle. They
found that visual acuity and binocularity of cortical neurons in visual
cortex were significantly altered (Berardi et al. 1994
).
Inhibition of endogenous NGF also prolongs the period of sensitivity of
visual cortex to monocular deprivation (Domenici et al.
1993
).
The ability of NGF to acutely affect cortical function in barrel cortex
was demonstrated by Prakash and colleagues (Prakash et al.
1996). These researchers found that topical applications of NGF
revealed rapid but transient increases in the dimension of a functional
activity associated with a barrel, while brain-derived neurotrophic
factor (BDNF) produced an opposite effect, reducing barrel-associated
area of activity for longer periods. Although the precise site of
action for NGF cannot be determined from the preceding studies, taken
together, these data suggest a direct modulatory role for NGF at the
level of the neocortex in activities that involve active sensory input
and stimulation.
Do NGF+ transplants act on remaining cholinergic fibers?
In the normal situation, cortical NGF is likely to act on
cholinergic fibers originating from the basal forebrain. Although most
of the cholinergic afferent fibers are destroyed in our study, the
possibility exists that NGF may act on the remaining basal forebrain
afferent fibers to increase cortical cholinergic activity through
action on basal forebrain neurons. We cannot conclusively rule out this
possibility, but the lack of effect after transplants in the basal
forebrain, and the strength of the response after intracortical
transplants suggests that NGF action was directly on cortical cells. In
addition, the immunolesions in this study reduced the density of
cortical cholinergic fibers by 85-95%. This extent of reduction may
diminish the ability of NGF to act on the remaining afferent
cholinergic fibers in the neocortex. Winkler and colleagues recently
showed that administration of NGF could not restore cholinergic
activity in animals with an 81% reduction in cholinergic innervation
following 192-IgG-saporin lesions, while it was capable of restoring
cholinergic activity in animals with less severe lesions
(Winkler et al. 2000). The ability of NGF to act on
cortical cholinergic fields may therefore depend on levels of
cholinergic damage, dose and duration of NGF action, and method of NGF
delivery to cholinergic neurons of the basal forebrain.
Possible mechanisms of NGF action
Several studies indicate that, NGF can have direct effects on
neurons. NGF can directly increase the release of glutamate in
hippocampus and cerebral cortex (Knipper et al. 1994;
Sala et al. 1998
). Other studies show that NGF alters
calcium storage by enhancing calcium entry through voltage dependent
channels (Levine et al. 1995b
). Although the mechanism
for alterations in calcium levels is not fully understood, it
implicates NGF in playing a role in plastic and morphologic
modifications in CNS neurons.
Other studies show that neurotrophins influence synaptic transmission
and synaptic efficacy by increasing postsynaptic responsiveness. BDNF,
another member of the neurotrophin family, significantly and rapidly
increases spontaneous firing rate, as well as the frequency and
excitatory postsynaptic currents in cultured hippocampal neurons
(Levine et al. 1995a). In vitro BDNF also regulates
cortical excitability by regulating GABA-mediated inhibition
(Rutherford et al. 1997
). Other members of the
neurotrophin family, including NGF, may affect neuronal excitability in
a similar manner although there is limited direct evidence to support
this idea.
Novel methods of neurotrophin function may also operate. Studies in
developing chick brain suggest that neurotrophins travel anterogradely
from cell body to axon, are subsequently released, and taken up by a
second order neuron (von Bartheld et al. 1996). Neurotrophins have also been suggested to function in a paracrine or
autocrine fashion (Acheson et al. 1995
; Altar et
al. 1997
). In our study, exogenous NGF may augment these
processes in neocortex and enhance endogenous neurotrophin action. In
addition, NGF released from the graft site may act in a paracrine
manner on cortical neurons of barrel cortex. Neurotrophins may also
control their own release via regulated feed back loops (Canossa
et al. 1997
; Kruttgen et al. 1998
). Other
activity-dependent regulated release mechanisms may exist for NGF and
BDNF, which are induced by stimulation of glutamate and muscarinic
receptors and inhibited following GABAergic activation (da Penha
Berzaghi et al. 1993
; Zafra et al. 1990
, 1991
).
More than likely, NGF action and expression on cortical cells involves
complex interactions that are activity dependent and regulated by feed
back loop.
Further studies are necessary to determine the precise mechanism of NGF
action on cortical neurons. A better understanding of the site of
production and release as well as the uptake mechanisms and
physiological responses of neurons to NGF exposure in both short and
long term will clarify the role of NGF in cortical function and
processing of information. Recent studies indicate that trkA blockage
affects the size and number of early postnatal basal forebrain neurons
(Cattaneo et al. 1999). Functional blockade of trkA
receptors using anti-receptor monoclonal antibodies may help decipher
the role and mechanism of NGF action across cortical surfaces.
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ACKNOWLEDGMENTS |
---|
Lausanne whisker stimulator and whisker displacement apparatus were skillfully developed and fabricated by N. Fine, whose time and effort is greatly appreciated. We thank Dr. J. Coulombe for assistance with the bioassay experiments. We also thank G. F. Hill II, D. Tatham, S. Kim, and N. Prokhorenko for help with the experiments and data analysis.
This study was funded by Alzheimer's Association Grant IIRG-95-113, USUHS Grant RO7064, and Defense and Veterans Head Injury Program Grant 0996-92P6285 (all to S. L. Juliano).
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FOOTNOTES |
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Address for reprint requests: S. L. Juliano, Dept. of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20814 (E-mail: sjuliano{at}usuhs.mil).
Received 6 October 2000; accepted in final form 15 May 2001.
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REFERENCES |
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