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The Journal of Neuroscience, September 1, 2000, 20(17):6587-6593
Circuit-Specific Alterations in Hippocampal Synaptophysin
Immunoreactivity Predict Spatial Learning Impairment in Aged Rats
Thressa D.
Smith1,
Michelle M.
Adams1, 2,
Michela
Gallagher4,
John H.
Morrison1, 2, 3, and
Peter R.
Rapp1, 2, 3
1 Kastor Neurobiology of Aging Laboratories,
2 Fishberg Research Center for Neurobiology, and
3 Department of Geriatrics and Adult Development, Mount
Sinai School of Medicine, New York, New York 10029-6574, and
4 Department of Psychology, Johns Hopkins University,
Baltimore, Maryland 21218-2686
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ABSTRACT |
The present study examined the long-standing concept that changes
in hippocampal circuitry contribute to age-related learning impairment.
Individual differences in spatial learning were documented in young and
aged Long-Evans rats by using a hippocampal-dependent version of the
Morris water maze. Postmortem analysis used a confocal laser-scanning
microscopy method to quantify changes in immunofluorescence staining
for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the
principal relays of hippocampal circuitry. Comparisons based on
chronological age alone failed to reveal a reliable difference in the
intensity of SYN staining in any region that was examined. In contrast,
aged subjects with spatial learning deficits displayed significant
reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM)
relative to either young controls or age-matched rats with preserved
learning. SYN intensity values for the latter groups were
indistinguishable. In addition, individual differences in spatial
learning capacity among the aged rats correlated with levels of SYN
staining selectively in three regions: outer and middle portions of the
dentate gyrus molecular layer and CA3-LM. The cross-sectional area of
SYN labeling, by comparison, was not reliably affected in relation
cognitive status. These findings are the first to demonstrate that a
circuit-specific pattern of variability in the connectional
organization of the hippocampus is coupled to individual differences in
the cognitive outcome of normal aging. The regional specificity of
these effects suggests that a decline in the fidelity of input to the
hippocampus from the entorhinal cortex may play a critical role.
Key words:
circuit organization; synaptophysin; hippocampus; entorhinal cortex; aging; spatial learning; Morris water maze
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INTRODUCTION |
A substantial proportion of aged
individuals exhibit learning and memory deficits that qualitatively
resemble the effects of direct hippocampal damage (for review, see
Gallagher and Rapp, 1997 ). Related alterations are observed in
hippocampal neuronal activity in aged rats with spatial learning
deficits, including a decline in the scope of information controlling
location-specific firing, and modified place field stability (Barnes et
al., 1997; Shen et al., 1997; Tanila et al., 1997a,b). Against this
background recent stereological investigations indicate that the total
number of dentate gyrus granule cells and pyramidal neurons in fields CA3/2 and CA1 remains stable in aged mice (Calhoun et al., 1998), rats
(Rapp and Gallagher, 1996; Rasmussen et al., 1996), monkeys (Rapp,
1995; Peters et al., 1996), and humans (West, 1993). In the subset of
these studies incorporating behavioral assessment, neuron number
also was preserved among aged subjects that displayed robust
deficits on tests of learning and memory that depend on the hippocampal
formation (Rosene, 1993; Rapp, 1995; Rapp and Gallagher, 1996;
Rasmussen et al., 1996). Together, these findings have prompted a
consensus that hippocampal information processing can deteriorate
during normal aging in the absence of significant neuronal loss
(Gallagher et al., 1996; Morrison and Hof, 1997).
Studies of synaptic density suggest that hippocampal connectivity
is more susceptible to aging (for review, see Coleman and Flood, 1987;
deToledo-Morrell et al., 1988a; Geinisman et al., 1995). Of particular
note, Geinisman et al. (1986) documented a decline in a morphologically
distinct population of synapses in the dentate gyrus molecular layer
that was coupled to the magnitude of age-related learning impairment.
Other studies have reported changes in the relative volume of defined
components of hippocampal circuitry (Coleman et al., 1987; Rapp et al.,
1999), and, in one case, these effects were related to the severity of
age-related learning impairment (Rapp et al., 1999).
Commenting on available evidence, Geinisman (1999) and others
(Barnes, 1999; Nicolle et al., 1999a; Smith et al., 1999) have noted
the lack of high-resolution anatomically comprehensive analyses documenting the status of hippocampal circuitry in relation to the
cognitive outcome of aging. In addition, previous studies have focused
primarily on synapse number and density, leaving open the potential
contribution of alterations in other parameters affecting the integrity
of hippocampal connectivity. The present investigation addressed these
issues by using confocal laser-scanning microscopy (CLSM) to quantify
immunohistochemical labeling for the presynaptic vesicle marker
synaptophysin (SYN-IR). This approach allows for extensive regional
sampling in large numbers of subjects and yields multiple measures of
circuit integrity (Cabalka et al., 1992; Good et al., 1992; Masliah et
al., 1993, 1994, 1995; Chen et al., 1995; Gazzaley et al., 1996). A
central element of the experimental design was the use of a well
characterized behavioral assessment that is sensitive to hippocampal
disruption (Morris et al., 1982) and that reveals significant learning
impairment in a substantial proportion of aged subjects (Gallagher and
Rapp, 1997). By this strategy our aim was to conduct an anatomically comprehensive analysis of hippocampal circuitry in relation to individual variability in normal cognitive aging.
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MATERIALS AND METHODS |
Subjects. Twenty-seven young (6 months;
n = 10) and aged (24-28 months; n = 17) male Long-Evans hooded rats (Charles River Laboratories, Raleigh,
NC) served as subjects. Animals were housed singly in standard cages in
a climate controlled vivarium (ambient temperature, 25°C) maintained
on a 12 hr light/dark cycle. Food and water were available ad
libitum. Sentinel screening for a panel of viral antibodies proved
negative, confirming the pathogen-free status of aged animals from this
colony. Husbandry and experimental procedures followed the National
Institutes of Health Guide for the Care and Use of Laboratory Animals
and were approved by institutional Animal Care and Use Committees.
Behavioral testing procedures. The apparatus and protocol
for evaluating spatial learning in the Morris water maze were the same
as in numerous earlier experiments (Gallagher et al., 1993; Rapp and
Gallagher, 1996; Rapp et al., 1999). Briefly, rats were tested on a
standardized "place" version of the water maze task for a total of
8 d consecutively. Three trials were provided per day with
60 sec intertrial intervals. The location of the hidden escape platform
remained constant across trials relative to the distribution of spatial
cues surrounding the apparatus. Animals entered the maze at one of four
points around the perimeter of the apparatus, according to a
predetermined sequence. Rats that failed to escape within 90 sec were
guided to the platform where they remained for 30 sec. The last trial
on every other day was a probe test in which the escape platform was
unavailable for escape for the first 30 sec. Throughout testing the
search paths were monitored by a video tracking system (HVS Image
Analyzing VP-112) and analyzed with custom-designed software (developed by Richard Baker at HVS Imaging, Hampton, UK). The distance between the
rat and the escape platform was sampled 10 times/sec and averaged in 1 sec bins; then two standardized behavioral measures were derived
(Gallagher et al., 1993). To evaluate performance during training
trials, we calculated cumulative search error as the summed 1 sec averages of the proximity measure. At the outset of
training this measure has a high value but approaches zero as the rats
learn to swim directly to the platform from any start location. A
learning index score of spatial bias also was calculated on the basis
of data that were collected during probe testing; lower index scores
reflect searching focused on the target location and indicate better learning.
Animals subsequently were tested for one session of six trials (30 sec
intertrial interval) on a nonspatial hippocampal-independent version of
the water maze. For this "cued" task the platform was visible and
varied in location across trials. Previous studies indicate that
performance in aged rats from the present study population is
unimpaired on this nonspatial version of testing (for review, see
Gallagher and Rapp, 1997).
Histological and immunohistochemical procedures. Rats were
shipped to the Mount Sinai School of Medicine vivarium 7-10 d after behavioral testing. After 1-2 weeks of acclimation they were
anesthetized deeply with chloral hydrate (30%, i.p.) and were perfused
transcardially with ice-cold 1% paraformaldehyde in 0.1 M
PBS for 1 min, followed by 9-14 min of ice-cold 4%
paraformaldehyde in PBS with 0.125% glutaraldehyde at a flow rate of
65 ml/min. Animals with grossly apparent pituitary hypertrophy were
excluded. Brains were blocked in the coronal plane and post-fixed for 6 hr. Tissue blocks were stored in PBS with 0.1% sodium azide at 4°C.
Histological sections through the rostrocaudal extent of the
hippocampus were cut on vibratome at a nominal thickness of 50 µm.
Serial sections were collected in PBS with 0.1% sodium azide and
stored at 4°C until immunohistochemical processing (generally within
1 week).
A one-in-ten series of histological sections (500 µm spacing) was
labeled with a monoclonal antibody against SYN (0.10 µg/ml final
antibody dilution; Boehringer Mannheim, Indianapolis, IN). According to
this design an average of eight sections per brain (total 206) was
included in the main analysis. An alternate series of more widely
spaced sections (1 mm spacing) from the same brains was processed for
the visualization of microtubule-associated protein 2 (MAP2), using a
commercially available monoclonal antibody (20 µg/ml final antibody
dilution; Sigma, St. Louis, MO). The characteristics and specificity of
immunostaining have been documented for both antibodies (Bernhardt and
Matus, 1984; Wiedenmann and Franke, 1985; Gazzaley et al., 1996).
Material was coprocessed in batches of approximately three young
and six aged brains (representing a wide range of spatial learning
capacities) with the same reagents. This design eliminated the
possibility that differences in SYN-IR as a function of age or
cognitive status could be attributable to interexperimental variability
in immunohistochemical processing. Other aspects of immunohistochemical
staining followed previous descriptions (Gazzaley et al., 1996).
Briefly, free-floating sections were washed in PBS at room temperature
(3 × 10 min) and incubated for 48 hr at 4°C with antibodies to
either SYN or MAP2 diluted in PBS. After being rinsed (3 × 10 min
in PBS), the sections were incubated for 1.5 hr with a
fluorescein-conjugated anti-mouse IgG heavy and light chain secondary
antibody (Vector Laboratories, Burlingame, CA). After a final rinse
(3 × 10 min in PBS) the sections were mounted onto gelatin-subbed
slides and left to dry overnight. Slides were coverslipped with
Vectashield (Vector Laboratories) to reduce fluorescence quenching and
were stored at 4°C until analysis.
Quantitative CLSM. Blind-coded immunolabeled sections
through the hippocampus were analyzed with a Zeiss LSM 410 inverted confocal microscope (Oberkochen, Germany) with a Plan-Apochromat 63×/1.25 numerical aperture oil immersion objective. Fluorescein was
excited by an argon/krypton laser at 488 nm, attenuated by a neutral
density filter to 3.3% (attenuation setting of 10), and reflected to
the tissue with an FT488/568 dichroic mirror. Settings for gain,
aperture, contrast, and brightness were optimized initially and held
constant throughout the study so that all sections were digitized under
the same conditions of illumination. Given that images from different
young and aged cohorts were captured over a period of several months,
it was important to evaluate whether system sensitivity varied as a
function of waning laser strength or other factors. For this purpose,
calibration curves were calculated on days 1, 30, and 60 of image
capture by using fluorescein-containing polystyrene microspheres (2.5 µm; InSpeck Microscope Image Intensity Calibration Kit, Molecular
Probes, Eugene, OR). These commercially available microspheres are
manufactured to exhibit seven known decreasing levels of fluorescence
intensity, expressed as a relative percentage of maximum intensity.
Standard curves derived by this approach are shown in Figure
1. The curves were similar in form and
displayed comparable maxima, confirming that system sensitivity was
stable over the course of the study. Figure 1 also illustrates that the
function relating relative fluorescence intensity to measured values
was curvilinear under the imaging conditions that were used here.
Average fluorescence intensity for SYN-IR in both young and aged brains
fell within the rising portion of the curve (see Results), indicating
that the CLSM settings were appropriate for detecting potential
differences in staining intensity between brains.
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Figure 1.
Curves relating measured pixel intensity to
relative fluorescence intensity of calibrated microspheres. Images were
captured on days 1, 30, and 60 of data collection and used the same
CLSM parameters as in the SYN-IR analysis.
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Digitized images of SYN-IR and MAP2-IR fluorescence staining were
captured from the inner (IML), middle (MML), and outer (OML) portions
of the dentate gyrus molecular layer, stratum lucidum (SL), and stratum
lacunosum-moleculare (LM) of CA3, and stratum radiatum (SR) and LM of
CA1. Using material from a previous study stained by the Timm
histochemical method (Rapp et al., 1999), we determined that, in young
and aged Long-Evans rats, the IML, MML, and OML comprise ~25, 40, and 35% of the total molecular layer width, respectively. Guided by
these observations, we determined laminar boundaries in the present
investigation by calculating the total width of the dentate gyrus
molecular layer for each of the captured images (i.e., from the granule
cell/molecular layer border to the hippocampal fissure or ventricular
border) and by applying the corresponding percentages for the IML, MML, and OML. In CA3, SL was distinguished readily as a relatively wide
layer of large, bright, punctate immunostaining superficial to the
pyramidal cells. This layer tapered to a point at the CA2 border. SR of
CA1 was distinguished as the secondary and tertiary dendritic branch
points that started ~150 µm from the cell body layer and were
outlined clearly by the SYN-IR puncta. Images for LM of both CA3 and
CA1 were captured within the zone of the fine most-distal
dendrites, ~50 µm deep to the hippocampal fissure (Fig.
2).
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Figure 2.
Digital confocal images of SYN-IR puncta in each
of the relays of hippocampal circuitry that were analyzed. The
appearance of dendritic MAP2 staining in CA3-LM is illustrated also
(bottom left panels). Images represent sampling fields
included in the formal quantitative analysis and were selected from a
young subject that exhibited averaged SYN-IR intensity values near the
mean of its age group. Panels to the
right of the raw images (i.e., 2nd and
4th columns) display the same sampling
fields with a photometric threshold applied. Pixels with gray scale
values above the blue-coded threshold were considered to represent
specific immunoreactivity, and measurements of immunofluorescence
intensity and area were restricted to these pixels (see Materials and
Methods for details). Each panel represents an area of 1651 µm2.
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The overall sampling design was guided by stereological principles,
adapted to accommodate the characteristics of the immunohistochemical staining. For each lamina of interest within the dentate gyrus and CA1,
one field was sampled every 400 µm throughout the transverse axis
with a 63×/1.25 numerical aperture objective. A systematic random
selection of sites was ensured by positioning the initial sampling
field randomly within the first 400 µm interval. The same approach
was used in CA3-LM and SL, except that the within-section sampling
interval was 100 µm. Following this design, we sampled an average of
~200 sites in each hippocampus for a total of >5000 samples across
the entire study. "Zoomed" images were captured from the center
portion of each of the 63× images, decreasing experimenter bias and
potential measurement variability across the display. Each of these
individual fields of view, consisting of 512 × 512 × eight-bit pixel arrays (400 × 400 µm in the x-y plane), was scanned and digitized, using an electronic zoom factor of
5.0 (increasing the resolution to 0.793 µm/pixel and the
magnification to 315× while decreasing the area to 1651 µm2) at a predetermined
z-axis depth (i.e., distance into the tissue) that remained
constant for a given brain. The latter parameter ranged from 3 to 7 µm across brains and was selected on the basis of the depth at which
staining intensity appeared consistent while avoiding artifacts at the
cut surface of the sections. Pixel values, corresponding to the
intensity of immunofluorescence staining, were represented on a gray
scale of 0 to 255. Following a procedure outlined by Gazzaley et al.
(1996), we applied a photometric offset or "threshold" to each
image that distinguished the relatively dim background levels of
fluorescence from the more intense punctate staining characteristic of
specific SYN-IR (Fig. 2). This approach excludes from analysis the
contribution of pixels with gray scale values falling below threshold.
The principal measures of interest derived from the captured
thresholded images were the average pixel intensity and the
cross-sectional area of immunoreactive staining. As discussed later,
comparing results across these parameters provided a basis for
dissociating two potential effects of aging: changes in SYN protein
levels versus alterations in the density of presynaptic boutons.
Similar procedures were applied to material that was labeled with
antibodies against MAP2, providing a window on the overall structural
integrity of dendrites in the aged hippocampus.
The blind code was broken after the data collection. Statistical
analyses were performed with StatView 5.0 (Abacus Concepts, Berkeley,
CA) for the Macintosh platform (Apple Computer, Cupertino, CA).
Potential group differences with respect to chronological age and
cognitive status were tested by factorial ANOVA and follow-up pair-wise
contrasts (Bonferroni test). To examine potential regional selectivity
in the effects of aging, we conducted separate tests for each principal
hippocampal relay. Adopting a strategy validated in earlier studies
that used the same rat model (Baxter and Gallagher, 1996), we used a
linear correlation approach (Pearson's r) to explore the
relationship between individual differences in spatial learning and
hippocampal SYN-IR. A relatively small number of correlation
coefficients was calculated in this analysis (i.e., 19), and, because
fewer than one of these would be expected to emerge as significant by
chance alone, no correction for multiple comparisons was applied.
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RESULTS |
The outcome of behavioral testing was consistent with
earlier studies that used identical procedures (Gallagher et al., 1993; Rapp and Gallagher, 1996; Rapp et al., 1999). Briefly, Figure 3A shows the average search
error for the young and aged groups during training trials in the
hippocampal-dependent place version of the water maze. Performance did
not differ as a function of age on the first training trial, but
subsequent acquisition was impaired in the aged group. The latter
effect was confirmed in a statistical analysis that revealed a main
effect of age during training (F1,25 = 5.18; p < 0.05). Learning index scores computed from
the interpolated probe trials are shown for each rat in Figure 3B. Lower values of the learning index indicate a more
accurate search in the escape platform location. Young rats displayed
better learning than the aged subjects
(F1,25 = 11.25; p < 0.005). Note, however, that the range of scores for the aged rats
overlapped and exceeded values for the young group; some aged rats
performed comparably to young subjects, whereas others scored outside
this normative range. Rather than following a strict bimodal
distribution, performance in the aged group was distributed
continuously across a broader range than for young rats. The results
from cue training trials, when rats were allowed to escape to a visible
platform, did not differ as a function of age for either escape latency or path length (data not shown).
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Figure 3.
Performance of young (n = 10)
and aged (n = 17) rats in the hippocampal-dependent
spatial version of the Morris water maze task. A,
Average cumulative search error ± SEM, reflecting the distance of
animals from the escape platform throughout their search, over
five-trial blocks of training. Performance was nearly identical in
young and aged animals on the first training trial (TT) but was
impaired in the aged group during subsequent training.
B, Learning index scores of spatial bias for individual
animals derived from three interpolated probe tests in which the
platform was unavailable for escape. Because this measure represents
average proximity from the platform training location, lower scores are
indicative of more accurate searching. Note that learning scores for
the aged rats were distributed continuously across a broader range than
for young animals.
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The overall pattern of SYN-IR in the rat hippocampus, independent
of age, was similar to previous descriptions (Fykse et al., 1993; Chen
et al., 1995). Bright puncta of SYN-IR were visible in all of the
subfields that were examined (see Fig. 2). Staining intensity was
higher in CA1-LM than in CA1-SR (F1,26 = 20.70; p < 0.0001) and greater in CA3-SL than in
CA3-LM (F1,26 = 22.39; p < 0.0001). Consistent with previous observations
(Cabalka et al., 1992; Chen et al., 1995), SYN-IR varied across the
radial extent of the dentate gyrus molecular layer
(F2,52 = 106.3; p < 0.0001) and was less intense in the MML than in the IML
(p < 0.0001) or OML (p < 0.0001; Fig. 4). Labeling in the
latter two regions was comparable. These results confirm that the
methods used in the present study were sufficiently sensitive to detect subjectively apparent regional differences in the intensity of SYN-IR.
By comparison, quantitative ultrastructural data indicate that synaptic
density is homogeneous along the distal dendrites of the granule cells
and greater than in the IML (Curcio and Hinds, 1983; Desmond and Levy,
1986). Accordingly, available findings are consistent with the idea
that SYN-IR can reveal elements of presynaptic circuit organization
distinct from synaptic density, presumably related to the size and SYN
protein content of immunoreactive boutons (Calhoun et al., 1996)
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Figure 4.
Mean hippocampal SYN-IR intensity ± SEM for
the young (open bars) and aged groups
(filled bars), independent of spatial learning
ability. The numerically lower values observed in aged rats were not
statistically different from young controls in any region.
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There were no gross, subjectively apparent differences in the
overall quality or general characteristics of SYN labeling in the
material from young and aged animals. Statistical comparisons, independent of spatial learning ability, confirmed these observations. Specifically, there was no significant difference in the intensity or
cross-sectional area of SYN-IR across the age groups, regardless of
whether values were averaged for the entire hippocampus or analyzed
separately for each hippocampal subfield (all ANOVA p values > 0.10). In a majority of the examined regions, however, average SYN-IR intensity values for the aged group were numerically lower than in the younger cohort (Fig. 4).
Subsequent analyses focused on evaluating the SYN results in
relation to variability in the cognitive outcome of aging. On the basis
of their performance in the hippocampal-dependent spatial version of
the water maze, and adopting a criterion established across many
earlier investigations in the same animal model, operationally we
defined aged rats with learning index scores <240 (i.e., within the
range of young controls) as learning-unimpaired. Aged animals with
scores outside this normative range were classified as impaired. Previous studies confirm the validity and sensitivity of this strategy
for detecting neurobiological alterations that may not be apparent when
young and aged subjects are compared on the basis of chronological age
alone (for discussion, see Gallagher et al., 1993, 1995; Gallagher and
Rapp, 1997). In the present experiments, classifying the aged rats by
cognitive status revealed that the numerical decrease in SYN-IR,
observed for the aged rats as a group (Fig. 4), was attributable
almost entirely to a decline in the subset of aged animals that
displayed robust spatial learning deficits (Fig.
5). By comparison, average SYN-IR
intensity values for the young and aged-unimpaired groups were
virtually identical in the majority of examined regions. This pattern
of results was most compelling in CA3-LM, where the SYN staining
intensity was reduced by ~28% in the aged-impaired group relative to
either young controls or age-matched animals with preserved spatial
learning. Statistical analysis confirmed the presence of a significant
group effect for this hippocampal region
(F2,24 = 6.30; p < 0.01). Subsequent between-group comparisons demonstrated that, whereas
SYN-IR intensity values were lower in aged-impaired rats relative to
either young (p < 0.01) or aged-unimpaired
subjects (p < 0.01), results for the latter
groups were statistically indistinguishable (p > 0.9). Although the cross-sectional area of SYN-IR in CA3-LM also
differed across groups (F2,24 = 4.21;
p < 0.05), this effect was less robust and not
statistically reliable in between-group contrasts with appropriate
Bonferroni adjustments. Parallel analyses failed to reveal significant
group differences in SYN staining intensity or area for any of the
remaining hippocampal subfields. Nonetheless, a pattern of results
qualitatively similar to CA3-LM was observed in a majority of the
examined regions, including the OML and MML (Fig. 5).
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Figure 5.
Mean hippocampal SYN-IR intensity ± SEM in
young rats (open bars) and aged animals with
(filled bars) or without (shaded
bars) spatial learning impairment. Aged animals were
classified as impaired (n = 9) or unimpaired
(n = 8) on the basis of their performance in the
place version of the water maze (see Fig. 2B and
Results for details). Note that average hippocampal SYN-IR intensity
values were statistically indistinguishable between aged-unimpaired and
young rats in all of the regions that were examined. The subset of aged
rats with robust spatial learning deficits, however, displayed
significantly lower SYN-IR intensity values in CA3-LM relative to
either young animals or age-matched rats with preserved learning.
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The intensity of SYN staining among the aged rats, like their
spatial learning scores, was distributed continuously across a broader
range than among young subjects. These results raised the possibility
that individual differences in spatial learning during aging might be
coupled to variability in SYN-IR. This proposal was tested by a linear
correlation approach (Pearson's r). Data from the young and
aged groups initially were considered separately, eliminating the risk
of detecting associations that simply reflect the shared influence of
chronological age on SYN-IR and behavior rather than a meaningful
relationship between the latter measures. Lower learning index scores
among the aged rats (i.e., better performance) correlated with higher
intensity values for SYN-IR selectively in three regions: the dentate
gyrus OML (r =  0.56; p = 0.02), MML
(r = 0.57; p = 0.02), and CA3-LM
(r = 0.63; p = 0.006) (Fig.
6). These results indicate that ~30 to
40% of the variance in spatial learning among the aged rats was
accounted for by individual differences in SYN-IR in the OML, MML, and
CA3-LM. Confirming the regional selectivity of this effect, the
correlation was weaker (r = 0.43) and failed to reach
standard levels of statistical significance (p = 0.08) when SYN-IR values averaged across the entire hippocampus were
considered in the analysis.
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Figure 6.
Scatter plots relating individual spatial learning
index scores to levels of hippocampal SYN-IR in young and aged rats.
Lower learning index scores, indicative of better learning, correlated
with higher SYN-IR intensity values selectively in the dentate gyrus
OML, MML, and CA3-LM. Regression lines
and correlation coefficients refer to the relationship
between individual differences in spatial learning and SYN-IR for the
aged subjects alone.
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Parallel analysis of the data from young subjects revealed no
statistically reliable correlation between the intensity of SYN
labeling and spatial learning, either for the hippocampus as a whole or
for any individual subfield (all p values > 0.1). When
results for the young and aged rats were pooled, however, the
regionally selective inverse correlations that were observed for the
aged subjects alone remained significant (OML: r = 0.46, p = 0.02; MML: r = 0.46,
p = 0.01; CA3-LM: r = 0.54,
p = 0.003). This pattern of results suggests that
SYN-IR in specific components of hippocampal circuitry may be coupled
to spatial learning across the full range of performance capacities
observed in young and aged rats. By this account the failure to detect
a corresponding association for the young group alone might reflect the
limited statistical power provided by considering only 10 subjects in the analysis. Alternatively, hippocampal SYN-IR may be coupled less
tightly to learning in young animals, emerging selectively when the
effects of aging on relevant circuitry exceed some critical threshold.
In contrast to the intensity of SYN-IR, the cross-sectional area of
immunostaining failed to correlate with the learning index scores in
any hippocampal regions for either the young or aged animals (no
r values better than 0.30; all p values > 0.1). Thus, individual variability in the numerical density of
presynaptic boutons, which would influence the cross-sectional area of
SYN staining directly, is unlikely to account for the observed coupling between spatial learning ability and the intensity of immunoreactivity.
Finally, alternate histological sections stained with antibodies
against the cytoskeletal protein MAP2 were examined to evaluate the
possibility that the observed changes in SYN-IR were associated with generalized dendritic deterioration in the aged hippocampus. There were no significant age-related differences in either the intensity or area of MAP2-IR in any brain region that was examined (all
p values > 0.1). In addition, MAP2 staining was
comparable among the young, aged-unimpaired, and impaired groups (all
p values > 0.1). Negative results also were obtained
in analyses testing for potential correlations between the spatial
learning scores and the MAP2 measures (no r values better
than 0.30 or 0.30; all p values > 0.1).
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DISCUSSION |
The present investigation substantially advances current evidence,
providing the first demonstration that variability in the cognitive
outcome of normal aging is coupled to circuit-specific alterations in
the organization of hippocampal connectivity. Comparisons on the basis
of chronological age alone failed to reveal any reliable difference in
SYN-IR, either for the hippocampus as a whole or in individual relays
of hippocampal circuitry. In contrast, aged rats with robust spatial
learning deficits displayed significant reductions in the intensity of
SYN-IR in CA3-LM relative to both younger animals and age-matched
subjects with preserved learning. Individual variability in hippocampal
learning among the aged rats correlated with SYN immunolabeling
selectively in three areas: the OML, MML, and CA3-LM. This regional
specificity, together with the observation that SYN-IR was entirely
normal in aged rats without spatial impairment, argues against a
general, nonspecific change in the immunohistochemical staining
characteristics of the aged brain. Given that all of the affected
regions receive a prominent projection from layer II of the entorhinal
cortex, the results instead suggest that cognitive aging is linked to a
decline in the integrity of multimodal associational input that these
cells relay to the hippocampus. The absence of a corresponding alteration in the cross-sectional area of SYN staining is also informative, implying that presynaptic alterations coupled to age-related learning deficits include a decrease in hippocampal SYN
protein levels that is unaccounted for by changes in terminal density.
The findings reported here help to reconcile available data on the
status of connectivity in the aged hippocampus. In a recent experiment
that used the same animal model, Nicolle et al. (1999b) found no
differences related to age or cognitive status in the amount of three
presynaptic proteins, including SYN, measured by Western blotting in
whole homogenized hippocampi. Our results are compatible with those
findings in that the overall intensity of SYN-IR, averaged across the
principal relays of hippocampal circuitry, failed to differ as a
function of chronological age or cognitive status. Studies
incorporating greater anatomical resolution, however, suggest that
there is substantial regional selectivity in the effects of aging on
hippocampal circuitry, particularly in relation to cognitive decline
(Geinisman et al., 1986; deToledo-Morrell et al., 1988a,b; Rapp et al.,
1999). Consistent with this conclusion, age-related alterations in
SYN-IR in the present experiment were apparent only among aged rats
with pronounced spatial learning impairment. In addition, correlations
between SYN intensity and behavior were selective for subfields of the hippocampus that receive direct cortical input (see below). In the
absence of discrete regional analysis and assessment of capacities supported by the hippocampus, this pattern of results would be obscured.
Although quantitative electron microscopy remains the only
definitive means of determining synapse number, SYN-IR provides a
sensitive marker of the synapse loss that accompanies neurodegenerative disease (Goto and Hirano, 1990; Cabalka et al., 1992; Masliah et al.,
1993, 1994, 1995; Heinonen et al., 1995). In the present experiments
the overall density of presynaptic boutons, measured as the
cross-sectional area of SYN-IR, was not reliably different as a
function of chronological age or cognitive status. Chen et al. (1995)
reported essentially the same result, suggesting that any effect of
normal aging on presynaptic terminal number in the hippocampus is
sufficiently subtle to elude detection by the approach used in these
studies. Our additional findings, however, demonstrate marked effects
on the intensity of labeling for this presynaptic protein. The
interpretation of these results is informed by the observation
that age-related alterations in SYN-IR were apparent selectively among
aged rats with documented deficits in spatial learning and only in
certain relays of hippocampal circuitry. In addition, no change with
chronological age or cognitive status was detected in a parallel
analysis of labeling for the cytoskeletal protein MAP2. A distinctly
different pattern of results also was obtained when immunoreactivity
for the NR1 subunit of the NMDA receptor was quantified in alternate
histological sections from the same animals (M. Adams, T. Smith, M. Gallagher, P. Rapp, and J. Morrison, unpublished data). Differences in
antibody penetration or other nonspecific alterations in the aged brain
cannot account for this regional, behavioral, and protein specificity.
Taken together, the results instead suggest that cognitive aging is linked to circuit-specific alterations in presynaptic elements of
hippocampal connectivity (e.g., vesicle number, transmitter packaging,
etc.), independent of effects on the overall density of presynaptic boutons.
Synaptophysin and other synaptic vesicle proteins have been implicated
in mechanisms of cellular plasticity that are thought to underlie
learning (Lynch et al., 1994; Mullany and Lynch, 1998; Janz et al.,
1999). Viewed in the context of the overall circuit organization of the
hippocampal formation (for review, see Amaral and Witter, 1995), this
evidence provides a valuable framework for evaluating the functional
implications of the present results. Inputs from widespread regions of
the neocortex converge in the entorhinal cortex and are relayed to the
distal dendrites of granule cells in middle and outer portions of the
dentate gyrus molecular layer. Output from the granule cells is
directed to CA3 pyramidal neurons that, in turn, project to the CA1
field. Superimposed on this classic trisynaptic circuitry, the
entorhinal cortex also projects directly to hippocampal pyramidal
cells. These pathways follow a laminar organization such that input to
CA3-LM arises principally from the same layer II neurons that innervate
the dentate gyrus. In contrast, entorhinal cortex projections to CA1-LM originate exclusively in layer III. Thus, the present results indicate
that individual differences in the cognitive outcome of aging are
coupled to SYN-IR selectively in regions of the hippocampus that
receive direct input from layer II of the entorhinal cortex (i.e., the
OML, MML, and CA3-LM). Terminal fields targeted by intrinsic
projections (e.g., the IML, CA3-SL, and CA1-SR) or layer III neurons of
the entorhinal cortex (CA1-LM) are relatively unaffected. This pattern
of results highlights that the morphometric effects of aging are
remarkably selective, preferentially affecting circuits that convey
cortically derived information that is critical for hippocampal learning.
Recent findings substantially constrain the basis of age-related change
in hippocampal circuitry. Although it might be supposed that the
regional selectivity demonstrated in the current investigation results
from a degeneration of layer II neurons in the entorhinal cortex,
recent stereological studies indicate that neuron number is preserved
throughout many fields of hippocampal formation during normal aging
(West, 1993; Rapp, 1995; Peters et al., 1996; Rapp and Gallagher, 1996;
Rasmussen et al., 1996; Calhoun et al., 1998). In addition, sparing has
been noted in several reports that specifically examined neuron number
in layer II of entorhinal cortex (Rapp, 1995; Gomez-Isla et al., 1996;
Gazzaley et al., 1997; Merrill et al., 2000). Thus, although a
definitive answer awaits parallel analysis in the rat model used here,
there is little empirical support for the idea that alterations in the
circuit organization of the aged hippocampus are a secondary
consequence of neuron loss in the entorhinal cortex. The present
results also appear unrelated to gross structural deterioration in the
dendrites of hippocampal cells that receive afferents from the
entorhinal cortex. In addition to evidence for preserved or increased
dendritic elaboration until very late in life (for review, see Coleman
and Flood, 1987; Turner et al., 1998), our results failed to reveal any
change with age in the area or intensity of staining for the
cytoskeletal protein MAP2. Taken together, the findings point to
changes in protein conformation, trafficking, or other biochemical
alterations as more plausible candidates underlying the observed
effects of aging on SYN-IR.
The circuit-specific effects documented in this study are
positioned to influence the computational function of the hippocampus at multiple levels of processing. Specifically, our results imply that
the fidelity of entorhinal input to the dentate gyrus declines in a
subpopulation of aged rats. The outcome of processing this degraded
information is relayed to CA3 where, modulated by disrupted direct
inputs from layer II of the entorhinal cortex, additional computational
errors may accrue. Recent electrophysiological studies confirm that the
encoding properties of hippocampal pyramidal neurons are altered
substantially in aged rats with spatial learning deficits (Barnes et
al., 1997; Shen et al., 1997; Tanila et al., 1997a,b). Of particular
interest, location-specific firing of hippocampal neurons in these
animals appears to be controlled by a more limited scope of available
environmental cues than in young rats or age-matched subjects with
preserved spatial learning. Taken together with the present findings,
it is tempting to speculate that this impoverished encoding results
from age-related deterioration in the integrity of multimodal sensory
input to the hippocampus from the entorhinal cortex. Computational
modeling of hippocampal function, mimicking the modest regionally
selective alterations reported here, could provide a useful strategy
for testing this account.
| |
FOOTNOTES |
Received April 6, 2000; revised June 5, 2000; accepted June 6, 2000.
This work was supported by National Institutes of Health Grants AG09973
(P.R.R. and M.G.) and AG06647 (J.H.M.). We thank Robert McMahan for
expert technical assistance.
Correspondence should be addressed to Dr. Peter R. Rapp, Kastor
Neurobiology of Aging Laboratories, Mount Sinai School of Medicine, Box
1639, One Gustave L. Levy Place, New York, NY 10029-6574. E-mail:
rapp{at}neuro.mssm.edu.
| |
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[Abstract]
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R A Sperling, J F Bates, E F Chua, A J Cocchiarella, D M Rentz, B R Rosen, D L Schacter, and M S Albert
fMRI studies of associative encoding in young and elderly controls and mild Alzheimer's disease
J. Neurol. Neurosurg. Psychiatry,
January 1, 2003;
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[Abstract]
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M. Montag-Sallaz, M. Schachner, and D. Montag
Misguided Axonal Projections, Neural Cell Adhesion Molecule 180 mRNA Upregulation, and Altered Behavior in Mice Deficient for the Close Homolog of L1
Mol. Cell. Biol.,
November 15, 2002;
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[Abstract]
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G. C. Tombaugh, W. B. Rowe, A. R. Chow, T. H. Michael, and G. M. Rose
Theta-Frequency Synaptic Potentiation in CA1 In Vitro Distinguishes Cognitively Impaired from Unimpaired Aged Fischer 344 Rats
J. Neurosci.,
November 15, 2002;
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9932 - 9940.
[Abstract]
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P. R. Rapp, P. S. Deroche, Y. Mao, and R. D. Burwell
Neuron Number in the Parahippocampal Region is Preserved in Aged Rats with Spatial Learning Deficits
Cereb Cortex,
November 1, 2002;
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[Abstract]
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W. G. Honer, P. Falkai, T. A. Bayer, J. Xie, L. Hu, H.-Y. Li, V. Arango, J. J. Mann, A. J. Dwork, and W. S. Trimble
Abnormalities of SNARE Mechanism Proteins in Anterior Frontal Cortex in Severe Mental Illness
Cereb Cortex,
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[Abstract]
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M. D. McEchron, A. P. Weible, and J. F. Disterhoft
Aging and Learning-Specific Changes in Single-Neuron Activity in CA1 Hippocampus During Rabbit Trace Eyeblink Conditioning
J Neurophysiol,
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TOP
ABSTRACT
INTRODUCTION
