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Volume 17, Number 19,
Issue of October 1, 1997
pp. 7288-7296
Copyright ©1997 Society for Neuroscience
Disruption of a Single Allele of the Nerve Growth Factor Gene
Results in Atrophy of Basal Forebrain Cholinergic Neurons and Memory
Deficits
Karen S. Chen1,
Merry
C. Nishimura1,
Mark P. Armanini1,
Craig Crowley2,
Susan D. Spencer3, and
Heidi S. Phillips1
Departments of 1 Neuroscience, 2 Molecular
Biology, and 3 Molecular Oncology, Genentech, Inc., South
San Francisco, California 94080
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Administration of nerve growth factor (NGF) to aged or lesioned
animals has been shown to reverse the atrophy of basal forebrain cholinergic neurons and ameliorate behavioral deficits. To examine the
importance of endogenous NGF in the survival of basal forebrain cholinergic cells and in spatial memory, mice bearing a disruption mutation in one allele of the NGF gene were studied. Heterozygous mutant mice (ngf+/
) have reduced levels of NGF mRNA
and protein within the hippocampus and were found to display
significant deficits in memory acquisition and retention in the Morris
water maze. The behavioral deficits observed in NGF-deficient mice were
accompanied by both shrinkage and loss of septal cells expressing
cholinergic markers and by a decrease in cholinergic innervation of the
hippocampus. Infusions of NGF into the lateral ventricle of adult
ngf+/ mice abolished the deficits on the water maze
task. Prolonged exposure to NGF may be required to induce cognitive
effects, because reversal of the acquisition deficit was seen after
long (5 weeks) but not short (3 d) infusion. Although NGF
administration did not result in any improvement in the number of
septal cells labeled for choline acetyltransferase, this treatment did
effectively correct the deficits in both size of cholinergic neurons
and density of cholinergic innervation of the hippocampus. These
findings demonstrate the importance of endogenous NGF for survival and
function of basal forebrain cholinergic neurons and reveal that partial
depletion of this trophic factor is associated with measurable deficits in learning and memory.
Key words:
NGF;
neurotrophin;
cholinergic;
learning;
memory;
cell
death;
gene deletion
INTRODUCTION
The basal forebrain cholinergic
system has been shown to play a critical role in learning and memory
function. Lesions of basal forebrain cholinergic neurons or their
projections produce severe memory deficits (for review, see Olton and
Wenk, 1987
). Nerve growth factor (NGF) is known to promote the
survival, sprouting, and phenotypic expression of responsive neuronal
populations to various degrees in developing and mature organisms
(Mobley et al., 1986; Levi-Montalcini, 1987; Thoenen et al., 1987;
Whittemore and Seiger, 1987). The profound response of basal forebrain
cholinergic cells to NGF in various experimental paradigms has
suggested that NGF may serve as a target-derived trophic factor to
regulate survival or function of these neurons (Hefti et al., 1989).
Intraventricular infusions of NGF can rescue axotomized cholinergic
cells and reverse associated memory deficits (Will and Hefti, 1985;
Hefti, 1986; Williams et al., 1986; Kromer, 1987; Gage et al., 1988;
Hagg et al., 1988; Koliatsis et al., 1990; Dekker et al., 1992). NGF
infusions can ameliorate age-related memory deficits and reverse
degeneration of basal forebrain cholinergic cells (Fischer et al.,
1987, 1991). These findings on the actions of exogenous NGF have given
rise to the hypothesis that NGF serves as a target-derived survival factor for developing basal forebrain cholinergic neurons and suggest a
role for NGF in the function of these neurons in the adult brain.
Studies using immunoneutralization and gene deletion have unambiguously
demonstrated the importance of NGF for survival of sympathetic and
small sensory neurons (Levi-Montalcini and Booker, 1960; Gorin and
Johnson, 1979; Johnson et al., 1980; Aloe et al., 1981; Ruit et al.,
1992; Crowley et al., 1994). Although numerous studies have indicated
that NGF is capable of exerting potent influences on basal forebrain
cholinergic neurons, the actions of endogenous NGF on these neurons has
not been rigorously examined. Studies using immunoneutralization have
been hampered by the limited diffusion of antibodies in the CNS, the
limited developmental time window and doses examined, and the potential
cross-reactivity of NGF antibodies with other members of the
neurotrophin family (Vantini et al., 1989; Nabeshima et al., 1991; Li
et al., 1995; Van der Zee et al., 1995). Although the gene deletion
approach circumvents these problems, the poor viability of animals
homozygous for NGF gene disruption precludes analysis of the role of
NGF in the survival or function of cholinergic neurons past the
neonatal period (Crowley et al., 1994).
In the present study, heterozygous NGF-deficient mutant mice
(ngf+/) were examined as a model of partial NGF
deprivation. The mice were examined for brain content of NGF mRNA and
protein, tested for spatial learning and memory function, and examined for morphological alterations in cholinergic neurons projecting from
the septum to the hippocampus. To distinguish aspects of the phenotype
of adult ngf+/ mice arising from acute insufficiency of
NGF in the adult brain from those that may result from NGF deprivation
during a critical developmental time window, the ability of NGF
treatment in adult life to reverse various phenotypic characteristics of ngf+/ mice was examined.
MATERIALS AND METHODS
Mice. All mice used for study were F1 hybrids
obtained by mating wild-type C57BL/6 females and chimeric males
carrying an ngf+/ allele on a 129 background. Thus, the
genetic background of all mice studied was identical, with the
exception of the presence or absence of one disrupted ngf
allele. All behavioral testing and data collection were conducted by an
investigator blinded to the genotype of the mice.
NGF two-site ELISA. A subgroup of animals (eight
ngf+/ and eight ngf+/+ mice) was decapitated,
and the brains were removed. The hippocampus and entorhinal cortex were
dissected and homogenized in a sample/conjugate buffer containing
Tris-HCL, NaCl, CaCl2, Triton X-100, and BSA at
4°C. An NGF two-site ELISA was performed in 96-well plates according
to a modification of the procedure described by Korsching and Thoenen
(1983), using human recombinant NGF as a standard.
Ribonuclease protection assay. The hippocampi from another
subgroup of animals (nine ngf+/ and nine ngf+/+
mice) were dissected out, and the RNAzol B method was used to extract
total RNA (Tel-Test, Inc.). Tissue was homogenized in 2.7× vol of
RNAzol B using a Polytron 3000 homogenizer and PT-DA 3007/2 generator.
Total RNA was extracted from each sample, and NGF mRNA levels were
determined using the Ambion RPA II Ribonuclease Protection Assay Kit.
33P-UTP-labeled RNA probes for mouse NGF and mouse
cyclophilin were generated using the Promega Riboprobe Gemini System.
The NGF probe was made to the region of the mouse NGF gene that spans
nucleotides 230-454. This probe spans the region of the NGF gene
deleted in the targeting construct, allowing the probe to specifically
recognize only wild-type NGF mRNA in the RNase Protection assay.
Quantitation of NGF mRNA was determined from standard curves generated
by synthetic sense strand NGF mRNA. The cyclophilin probe was made to
span the region of this gene from 36 to 166 bases.
Choline acetyltransferase (ChAT) radioenzyme assay. The
hippocampus and entorhinal cortex were dissected out from another subgroup of animals (12 ngf+/ and 14 ngf+/+
mice) and homogenized in a sodium phosphate buffer, pH 7.4, containing
Triton X-100. ChAT enzyme levels were then determined using the method
described by Fonnum (1974).
Learning and memory testing. Two studies were conducted. In
the first, ngf+/ (n = 22) and
ngf+/+ (n = 34) male and female mice were
tested in a Morris water maze. In the second study, ngf+/
(n = 25) and ngf+/+ (n = 25)
male and female mice were tested. All mice were F1 hybrids of 129 × C57BL/6 matings and were between 5 and 6 months of age at the start
of behavioral testing. The experimenter was blinded to the genotype of
the mice during all testing and data collection. The water maze
consisted of a circular pool 48 inches in diameter that was painted
black and filled with room temperature water. The mice were given four consecutive trials per day starting from four different
pseudo-randomized start locations, with a 10 sec intertrial interval
and a 90 sec maximum swim latency per trial. Data collection was
automated by a computerized video tracking system (San Diego
Instruments). The total swim distance, the percentage of swim distance
spent in the platform quadrant, and the latency to find the platform were analyzed. The correlation between each of these variables was
>90%, so only the swim latency results have been presented. Mice in
the first study were tested on the hidden platform task for 8 d
(days 1-8). After a 2 week period, all the mice were tested for
retention of location of the hidden platform (day 24). The location of
the platform was held constant throughout these days of testing. The
hidden platform was then moved to a new location on days 30 and 31. After a 6 week delay period, all the mice were tested for retention of
this new platform location (day 72).
In the second study, which assessed the effects of exogenous NGF
administration, mice were tested on the water maze task using a
procedure that was identical to that described above except that the
delay interval for retention of the second ("new") platform location was 4 weeks instead of 6 weeks, and mice were tested for a
period of 5 d instead of 8 d in the acquisition of the
initial platform location. Additionally, testing on the visible
platform task was performed before testing on the hidden platform task. After this pretesting, the ngf+/ mice were equally divided
into a group that received chronic intracerebroventricular infusions of
NGF via osmotic minipumps or a group that received infusions of vehicle
alone. The ngf+/+ mice were divided into a group that received infusions of vehicle or a group that received no infusion. Three days after implantation of the pumps, all the mice were trained
on a third hidden platform location (different from the two locations
used on testing before the infusions) to assess the effects of the
infusions on both acquisition and retention of a new platform location.
After a 4 week period, the mice were tested for retention of this
hidden platform location. Immediately after that retention testing, all
the mice were tested for the acquisition of another new platform
location. All animals included in both studies had to swim quickly and
accurately to a visible platform to screen out animals that had visual,
attentional, motoric, or other noncognitive impairments. All the mice
in both studies were able to perform the visible platform task, and
thus no mice were excluded from either study.
Pump implantation and NGF infusion. The mice were deeply
anesthetized with a combination of acepromazine, rompum, and ketamine, and a 28 gauge stainless steel cannula (Alzet brain infusion kit; Alza
Corp., Palo Alto, CA), embedded in a dental acrylic stabilization platform, was lowered into the right lateral ventricle (coordinates: anteroposterior = 0.2 mm, mediolateral = 1.2 mm, and
dorsoventral = 2.3 mm relative to Bregma). The stabilization
platform was secured to the skull by cyanoacrylate. An Alzet Model
1007D osmotic minipump (Alza), half of which was coated in wax to
reduce the flow rate to 0.25 µl/hr, was connected to the cannula with
flexible vinyl tubing. The minipump was placed subcutaneously in the
neck/shoulder area of the animal and changed after 2 weeks. The scalp
was then closed, and the mouse was returned to its home cage. During
the surgery to change the minipumps, the animals were anesthetized, and
an incision was made in the neck/shoulder area adjacent to the
minipump. The minipump was then removed and replaced with a new
minipump. The infusion vehicle was a phosphate-buffered artificial
cerebrospinal fluid containing 150 mM NaCl, 1.8 mM CaCl2, 1.2 mM
MgSO4, 2.0 mM
K2HPO4, 10.0 mM glucose, and
0.1% autologous mouse serum, pH 7.4. The concentration of human
recombinant NGF used was 50 µg/ml. NGF-infused mice received ~300
ng/d for a total of 8.4 µg of NGF over the 4 week infusion
period.
Activity level testing. After testing on the water maze, a
subgroup of mice from the first study (n = 12 ngf+/ and n = 12 ngf+/+ mice)
was tested on a habituation test. In this task, mice were placed
individually into a chamber that automatically measured their locomotor
activity (San Diego Instruments) for a period of 20 min, and then they
were returned to their home cages.
Hot plate and tail flick testing. All mice included in the
first study were tested on the hot plate and the tail flick tasks. Hot
plate testing consisted of placing the mouse on a 48°C hot plate and
measuring latency to paw lick or shake. For tail flick testing, mice
were gently manually restrained, the distal centimeter of their tail
was immersed in a beaker of 50°C water, and the latency to tail flick
was recorded.
Histology. A subgroup of animals from each behavioral study
was perfused with 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4. The brains were removed, post-fixed overnight in 4%
paraformaldehyde, and cryoprotected in 30% phosphate-buffered sucrose
for 3 d at 4°C. Forty-micrometer-thick coronal sections were cut
on a freezing microtome. A series of sections was processed
histochemically for acetylcholinesterase (AChE) (Hedreen et al., 1985)
to visualize the density of cholinergic fiber innervation in the
hippocampus. A second series of sections was stained with a polyclonal
antibody (made in goat) specific for ChAT (Chemicon International,
Temecula, CA) [for a more complete description of the
immunohistochemical staining procedure, see Chen and Gage (1995)]. A
series of sections from animals in the first study was also stained
with an antibody specific for the p75 receptor (generous gift from
Louis Reichardt, University of California, San Francisco).
Morphometric analysis. Cells in the medial septum labeled by
antibody to ChAT were counted and sized on a computerized image analysis system, the MCID Image Analysis System (Imaging Research), using a magnification of 33× (10× objective with a 3.3× photo objective). For each animal, a series of three sections, spaced at
intervals of 240 µm from each other, was analyzed. Sections from each
animal were matched for their anteroposterior level using the
decussation of the corpus callosum and the crossing of the anterior
commissure as landmarks. The ventral border of the medial septum was
defined in each of these sections by the anterior commissure. Cells
were defined as stained objects between 70 and 300 µm2 in area. The number of cells in each of the
three sections was then summed to obtain a total number of cells for
each animal. These numbers were then corrected using the Abercrombie
method (Abercrombie, 1946). The mean cell size was also obtained by
measuring the cross-sectional area of all the cells that were
counted.
Cell counts for p75-labeled cells were performed by microscopic
observation by an investigator blinded to the genotype of the
specimens. Cells were counted as defined by labeled profiles with an
identifiable nucleus.
The extent of fiber innervation into the hippocampus was determined on
the AChE-stained sections. The MCID Image Analysis System (Imaging
Research), using a magnification of 33× (10× objective with a 3.3×
photo objective), was used to quantify fiber density. Fiber density was
determined in three AChE-stained sections, spaced at 240 µm
intervals, starting with the first section with the entire dorsal and
ventral blades of the dentate gyrus visible. To reduce the variability
of the AChE staining, all of the AChE-stained sections were stained in
two batches. Variability of the AChE staining was assessed by examining
the level of background staining within the corpus callosum and the
intensity of staining within the thalamus. On the basis of visual
inspection, a small number of sections (<5%) were excluded from
analysis because of abnormally light staining. For both the CA1
(stratum lacunosum moleculare) and alveus, 20 fields were imaged from
both left and right hippocampi of each section. For the dentate gyrus,
30 fields were sampled in the ventral and dorsal blades. An optical
density measurement (percentage of total area occupied by stained
fibers) was obtained for each field. A mean value for all fields that
were quantitated per region per animal was then calculated.
RESULTS
Mice heterozygous for NGF gene deletion (ngf+/) did
not exhibit any gross morphological or behavioral abnormalities and
could not be distinguished from wild-type (ngf+/+)
littermates by visual inspection. There was a significant reduction in
NGF protein levels in the ngf+/ animals as measured by a
two-site ELISA (Table 1). NGF levels in
the hippocampus of the ngf+/ mice constitute ~25% of
the levels in wild-type mice. Ribonuclease protection assay revealed
that NGF mRNA levels were also reduced in the hippocampus of the
ngf+/ mice (Table 1). No significant differences were observed in hippocampal wet weight, total RNA levels, or cyclophilin mRNA between ngf+/+ and ngf+/ mice.
Table 1.
NGF protein and mRNA levels in hippocampus
| Groups |
NGF protein
(ng/gm wet wt) |
NGF mRNA (fg NGF mRNA/µg cyclophilin mRNA)
|
|
| ngf+/ |
0.085
± 0.023* (n = 8) |
7.60
± 0.86* (n = 9) |
| ngf+/+ |
0.321
± 0.024 (n = 8) |
11.25
± 1.23 (n = 9) |
|
|
*
Significant differences between groups
(p < 0.05).
|
|
Two separate behavioral and morphological studies were performed. In
the first study, a group of ngf+/ mice (n = 24) and their ngf+/+ littermates (n = 34)
were tested on the water maze task (Fig.
1) beginning at 5 months of age. Results
obtained using measures of swim distance were similar to those obtained
using latency, thus only the latency data has been presented. Analysis of the results obtained from male and female mice showed no significant differences and were consequently pooled for analysis. Importantly, all
animals were able to learn a cued or visible platform task, and there
were no significant differences in swim speed (not shown) or latency to
find the visible platform between the NGF-deficient and wild-type
groups of mice (Fig. 1A).
Fig. 1.
A, Mean latency to find the hidden
platform on the water maze task on each day of testing. Each
point represents the mean performance of a group of mice
on the four trials per day. Days 1-8 = performance
during the acquisition of the initial location of the hidden platform;
day 24 = performance on testing for retention of
the initial platform location after a 16 d delay; days
30 and 31 = performance during the
acquisition of a new platform location; day
72 = performance on testing for retention of the
new platform location after a 6 week delay. The point
labeled Visible represents performance on the visible
platform task. B, Mean latency to find the hidden
platform on each of the individual four trials on Day 1,
and (C) on Day 30.
D, Mean latency to find the hidden platform on the first
trial of Day 72 (*p < 0.05, **
p < 0.01, *** p < 0.0005;
one-factor ANOVA with post hoc Fisher PLSD).
[View Larger Version of this Image (36K GIF file)]
In the hidden-platform task of the water maze paradigm, both
ngf+/+ and ngf+/ groups exhibited identical
latencies to locate the platform on the first trial of testing (Fig.
1B) (F(1, 54) = 0.037;
p > 0.50). On the second and third trials, as the mice began to learn the location of the hidden platform, there was a trend
toward better performance in the ngf+/+ mice compared with
the ngf+/ mice. This difference, however, did not achieve statistical significance. By the fourth trial, both groups of mice were
not significantly different from each other. After 8 d of testing,
the ngf+/+ and ngf+/ mice had both learned to
find the location of the platform quickly and accurately, and the
performance of the two groups of mice did not differ (Fig.
1A, day 8). After a 2 week retention
period, all animals were retested on this same hidden platform
location, revealing no significant difference between the performance
of the two groups (Fig. 1A, day
24).
After this initial testing, animals were then analyzed in a more
demanding acquisition and retention paradigm. Acquisition was made more
difficult by requiring the mice to learn a novel platform location
(reversal task), whereas retention testing differed from the initial
paradigm by both decreasing the number of days of acquisition testing
(from 8 to 2 d) and increasing the retention interval (from 2 to 6 weeks). On day 30 of the experiment, all mice were tested for
acquisition of the second platform location (Fig. 1C).
Consistent with the lack of a memory component on the first trial of
this task, the NGF-deficient and wild-type mice performed identically;
however, the wild-type group acquired the new platform location more
quickly than the ngf+/ group, performing significantly
better on the second (F(1, 54) = 14.77;
p < 0.0005) and third trials (F(1,
54) = 5.65; p < 0.05). By the fourth trial, both groups had learned the new platform location and were not significantly different from each other (p > 0.10). When the performance of each group was averaged across all four
trials on the first day of the acquisition reversal task (day 30), the
ngf+/ mice were significantly impaired compared with
ngf+/+ littermates (F(1, 54) = 6.87;
p < 0.01). The two groups did not differ on the next
day of testing, and consequently testing on the second platform
location was stopped to prevent overtraining. After a retention
interval of 6 weeks, the mice were retested on day 72 for retention of the new platform location, and the ngf+/ mice were found
to be significantly impaired compared with the ngf +/+ mice
(Fig. 1A,D). By the second day of retention testing,
day 73, the two groups were no longer significantly different (not
shown).
The ngf+/ mice and their ngf+/+ littermates
were also tested for locomotor behavior in activity chambers (Table
2). There is no difference between the
activity levels of the two groups of mice, indicating that the
ngf+/ mice were neither hyperactive nor hypoactive. Both
groups of mice were tested on the tail flick and hot plate test, tasks
that assess responsiveness to thermal pain (Table 2). Although a trend
toward reduced responsivity of the ngf+/ mice was
observed, there was no significant difference between the
ngf+/+ and the ngf+/ mice on either task
(p > 0.10). These results support the
conclusion that the deficits exhibited by the ngf+/ mice
on the water maze task are not attributable to general sensory, motor,
or arousal deficits.
Table 2.
Open field activity and nociceptive function
| Groups |
Latency on the tail flick
test (sec) |
Latency on the hot plate test (sec) |
Activity chamber
(counts) |
|
| ngf+/ |
3.1 ± 0.6 |
31.8
± 2.4 |
423 ± 47 |
| ngf+/+ |
2.8
± 0.7 |
28.8 ± 1.5 |
389 ± 41 |
|
|
|
In the second study, which assessed the effects of chronic exogenous
NGF administration, another group of adult ngf+/ mice (n = 24) and their ngf+/+ littermates
(n = 30) was pretested on the water maze task in a
manner that was similar to that described above (see Materials and
Methods) (Fig. 2A). The
results obtained during this pretesting confirmed those observed in the
first study: the ngf+/ mice were significantly impaired on
both acquisition and retention of the reversal task (not shown). After
pretesting, ngf+/ mice were implanted with mini-osmotic
pumps that delivered NGF or vehicle into the lateral ventricle. Control
ngf+/+ mice were either untreated or infused with vehicle.
Because the performance of the ngf+/+ mice receiving vehicle
did not differ from unoperated ngf+/+ mice, results are
shown only for the vehicle-infused group. Three days after the start of
infusion (day 68), both groups of ngf+/ mice, NGF-infused
and vehicle-infused, were still significantly impaired compared with
wild-type mice on the acquisition of a new platform location (Fig.
2B) (p < 0.05). Four weeks
later, ngf+/ mice that received infusions of NGF were
significantly improved on the retention of this platform as compared
with vehicle-infused ngf+/ mice (F(1,
25) = 7.41; p < 0.05) (Fig. 2C). In
fact, the performance of the NGF-infused ngf+/ was not
significantly different from the performance of the wild-type mice on
this retention task.
Fig. 2.
A, Testing paradigm of the second
study, which assessed the effects of exogenous administration of NGF.
B, Mean latency to find the hidden platform on the water
maze task on the first day of acquisition after intraventricular
infusions (Day 68). * denotes a significant difference
between the two ngf+/ groups and ngf+/+ group; p < 0.05. C, Mean latency to
find the hidden platform on the first trial of the first day of
retention of this new platform location (Day 100).
D, Mean latency to find the hidden platform during an
acquisition task conducted on Day 103, 5 weeks after the
onset of NGF infusion. * denotes a difference between NGF-infused and
vehicle-infused ngf+/ mice (one-factor ANOVA with
post hoc Fisher PLSD).
[View Larger Version of this Image (34K GIF file)]
Because the mice had received NGF infusions for a period of only 3 d before acquisition testing, the lack of effect observed on
acquisition may be attributable to the short duration of NGF treatment
rather than to the inability of NGF to ameliorate the acquisition
deficit observed in the ngf+/ mice. Therefore, all of the
mice were tested for acquisition of another novel platform location. On
trials 2 and 3 of the acquisition testing (day 103), conducted 5 weeks
after the onset of NGF infusion, the ngf+/ mice that
received NGF performed significantly better than ngf+/ mice that received vehicle alone (trial 2: F(1,
25) = 6.53, p < 0.05; trial 3:
F(1, 25) = 5.44, p < 0.05) and
in fact were no longer impaired as compared with wild-type controls
(Fig. 2D).
One-half of the mice were perfused with fixative after behavioral
testing, and their brains were processed for analysis of basal
forebrain cholinergic neurons. A series of sections was stained for
ChAT immunocytochemistry, and ChAT-immunoreactive neurons within the
medial septal nucleus were sized and counted. ngf+/ mice
had significantly fewer ChAT-positive medial septal neurons than their
ngf+/+ littermates (Figs. 3,
4). Remaining ChAT-positive cells in the
ngf+/ animals were significantly shrunken compared with
ngf+/+ controls (Figs. 3, 4). A second series of sections
from the same animals was stained with an antibody raised against the
p75 receptor, the low-affinity NGF receptor. The cell counts obtained
in the p75-stained sections confirmed the loss of cholinergic cells
that was observed in the ChAT-stained sections (ngf+/ = 152 ± 14 vs ngf+/+ = 228 ± 13;
F(1, 24) = 15.63; p < 0.01). A
series of sections through the hippocampal formation was stained for
AChE histochemistry to assess the density of cholinergic projections to
this region. There was a significant reduction in AChE-positive fibers
in CA1, CA3, and dentate gyrus regions of the hippocampus in the
ngf+/ mice compared with ngf+/+ littermates (Figs. 5,
6, 7).
Fig. 3.
Photomicrograph of the medial septal region
of (A) a ngf+/ mouse that had
received vehicle, (B) a ngf+/
mouse that had received NGF, and (C) a
ngf+/+ mouse that had received vehicle stained with
antibodies to ChAT; insets show a higher magnification
of stained cells. Scale bar, 100 µm.
[View Larger Version of this Image (134K GIF file)]
Fig. 4.
Number (A) and size
(B) of ChAT-positive cells in the medial septal
region [* denotes a significant difference from either untreated or
vehicle-treated wild-type mice (p < 0.05;
one-factor ANOVA with post hoc Fisher PLSD)].
[View Larger Version of this Image (36K GIF file)]
Fig. 5.
Photomicrograph of the CA1 region of the
hippocampus of (A) a ngf+/ mouse
that had received vehicle, (B) a
ngf+/ mouse that had received NGF, and
(C) a ngf+/+ mouse that had
received vehicle stained with AChE. Scale bar, 100 µm.
[View Larger Version of this Image (108K GIF file)]
Fig. 6.
Photomicrograph of the hippocampus of
(A) a ngf+/ mouse that had
received vehicle, (B) a ngf+/
mouse that had received NGF, and (C) a
ngf+/+ mouse that had received vehicle stained with AChE. Scale bar, 100 µm.
[View Larger Version of this Image (110K GIF file)]
Fig. 7.
Density of AChE-positive fibers in the alveus,
CA1, and dentate gyrus regions in the hippocampus [* denotes a
significant difference between vehicle-treated ngf+/
and either ngf+/+ or NGF-treated ngf+/
mice (p < 0.05; one-factor ANOVA with
post hoc Fisher PLSD].
[View Larger Version of this Image (72K GIF file)]
Measurement of ChAT activity revealed a significant reduction in ChAT
levels in the hippocampus of the ngf+/ mice compared with
ngf+/+ littermates (Table 3).
ChAT levels in the entorhinal cortex were not significantly reduced in
the ngf+/ mice compared with their littermates. The
smaller reduction in ChAT levels in the cortex may reflect the presence
of intrinsic ChAT-positive cells in the cortex that are not affected by
decreased NGF levels.
Chronic intraventricular NGF infusions were able to reverse the
shrinkage of cholinergic neurons that is observed in the heterozygotes, but they did not reverse the observed cell loss (Figs. 3, 4). NGF
administration was also able to increase AChE-positive fiber innervation of the hippocampus in the ngf+/ mice (Figs.
5, 6, 7).
DISCUSSION
In the present study, mice that are heterozygous for NGF gene
disruption were found to have decreased levels of NGF mRNA and protein
within the hippocampus, a prominent target of basal forebrain cholinergic neurons. ngf+/ mice exhibited significant
acquisition and retention deficits in the water maze and displayed
morphological deficits in septal cholinergic neurons. Morphological
deficits included a loss of approximately one-third of septal cells
labeled by ChAT or p75, a reduction in the size of remaining septal
cholinergic cell bodies, and loss of both ChAT activity and
AChE-positive fiber density within the hippocampal formation. Direct
administration of NGF to the brain of adult ngf+/ mice was
able to ameliorate the retention and acquisition deficits as well as
increase the size of basal forebrain cholinergic neurons and
cholinergic fiber density within the hippocampus. This treatment,
however, did not produce any increase in the number of ChAT-positive
septal neurons, suggesting that the loss of ChAT-positive cells in the
ngf+/ mice reflects cell death caused by NGF deprivation
during a critical developmental time window.
Previous observations in mice homozygous for NGF gene disruption
indicate that in the absence of NGF, septal cholinergic neurons differentiate and express their normal complement of phenotypic markers
(Crowley et al., 1994). Because of the poor survival of ngf/ mice, however, it could not be determined whether
septal cholinergic neurons acquire dependence on NGF for survival after these cells have established their mature pattern of connections with
the target area. The present findings provide evidence that basal
forebrain cholinergic neurons do exhibit a dependence on endogenous NGF
for survival. Here, we observe that adult mice with a partial
deficiency in NGF exhibit evidence for loss of septal neurons labeling
with either ChAT or p75. The failure to regain any ChAT-positive cells
after an NGF treatment regimen that restores the size (and projections)
of remaining cells points strongly to cell loss rather than
downregulation of phenotypic markers. Thus, the availability of
endogenous NGF during development appears to be limiting for survival
of septal cholinergic neurons. Whether the reported lack of cell loss
in ngf/ mice reflects the immature developmental stage
at which these mice were studied or whether it indicates that exposure
to NGF is necessary to induce dependence of cholinergic neurons for
survival remains to be established.
In addition to the reduction in number of septal cholinergic neurons,
adult ngf+/ mice exhibited significant shrinkage of remaining ChAT-positive cells and a loss in density of
cholinesterase-positive fibers within the hippocampus. The presence of
morphological deficits in the cholinergic neurons remaining in
ngf+/ mice, and the ability of NGF infusion into adult
brain to reverse them, suggests that in addition to its limiting role
in development, NGF plays a critical role in the adult brain to
maintain proper function of basal forebrain cholinergic neurons. These
observations are consistent with previous findings indicating that NGF
administration to aged rats is capable of reversing both memory
deficits and shrinkage of cholinergic cells (Fischer et al., 1987), and
with observations that NGF can induce sprouting of mature cholinergic
neurons (Kawaja and Gage, 1991; Van der Zee et al., 1992). Thus,
although the dependence of cholinergic neurons on NGF for cell survival
may be restricted to a critical developmental time window, cholinergic
cells also appear to require optimal levels of NGF during adult life
for maintenance of an optimally functional phenotype.
In addition to the morphological effects observed on basal forebrain
cholinergic neurons, ngf+/ mice exhibited significant deficits in performance in the water maze task. Our results confirm and
extend previous findings using immunodeprivation (Nabeshima et al.,
1991; Van der Zee et al., 1995) by indicating that even a partial
reduction in NGF availability leads to measurable effects on spatial
learning and memory. Lack of a more robust behavioral deficit may be
the result of compensatory changes induced by decreased levels of NGF,
and presumably reduced numbers and sizes of basal forebrain cholinergic
cells, throughout development. The memory systems in these
ngf+/ mice may have compensated for this chronic hypofunction of the cholinergic system, and thus the resulting deficit
may not be as great as the deficit that would result from a comparable
decrease of NGF protein occurring in adulthood. Although observations
of behavioral deficits in genetically manipulated mice are frequently
confounded by the genetic background of the mice investigated (Gerlai,
1996), all of the mice in the current study were F1 hybrids between two
inbred strains and consequently are genetically identical, with the
exception of the presence or absence of one mutated allele for NGF. In
contrast to the CNS abnormalities reported in the 129 strain (Lipp et
al., 1995), F1 hybrid mice of C57 and 129 strains perform well in the
Morris water maze and exhibit robust long-term potentiation. Thus, the behavioral deficits reported here are attributable to the disruption of
a single allele of the NGF gene and not to abnormalities arising from
the genetic background of the mice.
Our observations indicate that partial depletion of NGF is associated
with deficits in both acquisition and retention of a spatial learning
task. The behavioral effects of NGF depletion appear to be specific to
learning and memory function, because the performance of
ngf+/ mice on the visible platform task did not differ
from that of wild-type littermates. This observation argues strongly
that the deficits in performance reflect poor acquisition and retention
of spatial memory rather than alterations in sensory, motor, or
motivational status of the ngf+/ mice. Consistent with
this view, the performance of ngf+/ mice was identical to
that of wild-type mice in hidden platform trials that did not involve a
memory component, i.e., the first trial of a given platform location.
Furthermore, swim speeds of ngf+/ mice were
indistinguishable from those of wild-type mice, and in an open field
test no differences in locomotor activity were observed. Additional
evidence that the poor performance in the water maze task reflects a
CNS-mediated deficit comes from the ability of direct infusions of NGF
into the brain of ngf+/ mice to reverse the deficit.
The ability of NGF infusions to reverse the deficits in performance on
the water maze point to a deficiency of NGF within the adult brain as
the source of the behavioral abnormality. The deficit seen in the
ngf+/ mice on acquisition in the water maze task, however,
does not appear to result from acute deficiency in NGF at the actual
time of cognitive testing, because it can be reversed only by chronic
(5 week) and not acute (3 day) infusion of NGF. This finding suggests
that the behavioral improvements seen may be mediated via morphological
alterations in basal forebrain cholinergic neurons rather than via
acute actions of NGF on neuronal activity. As has been seen in aged
rodents (Fischer et al., 1992; Chen and Gage, 1995), NGF administration
to ngf+/ mice effectively ameliorates learning and memory
deficits despite an irreversible loss of cholinergic neurons. These
behavioral effects are likely to be mediated via the hypertrophy and
sprouting of remaining cholinergic cells and may involve changes in
synaptic density similar to those reported with NGF treatment in aged
rats (Chen et al., 1995).
Our results indicate that inactivation of a single allele of the NGF
gene results in the loss of approximately one-third of septal
cholinergic cells, with an equivalent reduction in hippocampal ChAT
activity. These changes in the cholinergic system were accompanied by
subtle but significant and reproducible deficits in performance on a
task of learning and memory. Although the extent of cell loss (35%)
matches the relative reduction in hippocampal content of NGF mRNA quite
well (33%), it is less than might be predicted from the large
reduction in NGF protein content within the hippocampus (75%). The
differences between the ratios of mRNA to protein seen in the
ngf+/ versus ngf+/+ mice may reflect
differences in the efficiency of translation, secretion, transport, or
utilization of NGF protein. One caveat in the interpretation of the
gene dosage effect is that NGF mRNA and protein determinations were
performed on samples from adult animals, and as yet the time period of
dependence of cholinergic neurons on NGF for survival is unknown. Thus,
at present, the quantitative relationship between NGF availability and
cholinergic cell survival and function cannot be deduced. Further
studies in which both NGF mRNA and protein levels are determined at
various points in development in both the basal forebrain and in target
areas are needed.
The present findings provide direct evidence for the role of NGF as a
survival factor for basal forebrain cholinergic neurons during
development and further suggest that NGF plays a role in the mature
nervous system to regulate the function of these cells. These
observations underscore the importance of endogenous NGF to CNS
function by indicating that even partial reduction in levels of this
protein results in cell loss and measurable effects on learning and
memory.
FOOTNOTES
Received February 3, 1997; revised June 11, 1997; accepted July
18, 1997.
We thank Lanway Ling for assistance on the hot plate and tail flick
tests, David Merrill for assistance on the water maze task, David
Shelton for instruction in the ChAT assay, and Teddi Colbert and Hank
La for animal care.
Correspondence should be addressed to Dr. Heidi S. Phillips, Genentech,
Inc., 460 Point San Bruno Boulevard, South San Francisco, CA
94080.
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