Cerebral Cortex February 2011;21:392--400
Advance Access publication June 10, 2010
Cognitive Decline Is Associated with Reduced Reelin Expression in the Entorhinal Cortex
of Aged Rats
Alexis M. Stranahan, Rebecca P. Haberman and Michela Gallagher
Department of Psychological and Brain Sciences, Johns Hopkins University, Baltimore, MD 21218, USA
Address correspondence to Michela Gallagher, PhD, Department of Psychological and Brain Sciences, Johns Hopkins University, 3400 North Charles
Street, Baltimore, MD 21218, USA. Email: email@example.com.
Brain regions and neural circuits differ in their vulnerability to
changes that occur during aging and in age-related neurodegener-
ative diseases. Among the areas that comprise the medial temporal
lobe memory system, the layer II neurons of the entorhinal cortex,
which form the perforant path input to the hippocampal formation,
exhibit early alterations over the course of aging Reelin,
a glycoprotein implicated in synaptic plasticity, is expressed by
entorhinal cortical layer II neurons. Here, we report that an age-
related reduction in reelin expression in the entorhinal cortex is
associated with cognitive decline. Using immunohistochemistry and
in situ hybridization, we observed decreases in the number of
Reelin-immunoreactive cells and reelin messenger RNA expression
in the lateral entorhinal cortex of aged rats that are cognitively
impaired relative to young adults and aged rats with preserved
cognitive abilities. The lateral entorhinal cortex of aged rats with
cognitive impairment also exhibited changes in other molecular
markers, including increased accumulation of phosphorylated tau
and decreased synaptophysin immunoreactivity. Taken together,
these findings suggest that reduced reelin expression, emanating
from layer II entorhinal neurons, may contribute to network
dysfunction that occurs during memory loss in aging.
Keywords: aging, Alzheimer’s disease, lateral entorhinal cortex, learning,
mild cognitive impairment
Cognitive deficits that commonly occur during aging are
associated with the disruption of specific neural networks.
The layer II neurons in the entorhinal cortex, which form a key
circuit in the medial temporal lobe memory system, have
a particular vulnerability that extends across a spectrum of age-
related memory loss, including amnestic mild cognitive
impairment (MCI) and Alzheimer’s disease (AD). These
neurons are functionally positioned at the intersection of
information processing in cortical networks and the hippo-
campal formation, and they provide the majority of input to the
dentate gyrus and CA3 areas of the hippocampus (van Strien
et al. 2009). The reasons for their selective vulnerability to
aging and AD, however, remain unclear.
In rodent models of aging, entorhinal cortical neurons are
not lost (Merrill et al. 2001; Rapp et al. 2002), but synaptic
connections are reduced in the terminal zones that are densely
innervated by layer II afferents (Geinisman et al. 1978; Smith
et al. 2000). Loss of innervation occurs only in aged rats with
hippocampal-dependent memory impairment, whereas rats
that age without cognitive decline have preserved entorhinal
connections (Smith et al. 2000). A similar topographical
vulnerability occurs in humans, such that entorhinal innerva-
tion of the dentate gyrus declines as a function of worsening
memory performance in normal aging, MCI and AD (Scheff
et al. 2006). Frank loss of entorhinal layer II neurons is apparent
in mild AD, further disconnecting cortical processing from the
hippocampal formation (Go ´ mez-Isla et al. 1996). Because the
cellular basis for entorhinal layer II vulnerability remains
undetermined in these conditions, it is of interest to identify
specific molecular features of those neurons in cognitive aging,
which may give clues about the earliest changes associated
with hippocampal network dysfunction.
Reelin is a large glycoprotein with widespread expression in
interneurons throughout the brain. Certain populations of
excitatory neurons also express reelin, and entorhinal layer II
neurons are among this group (Ramos-Moreno et al. 2006).
Reelin signaling negatively regulates tau phosphorylation
(Hiesberger et al. 1999); conversely, reelin depletion creates
a permissive environment for cellular events that are tied to the
pathophysiology of AD (Hiesberger et al. 1999; Hoe et al. 2009).
Reductions in reelin expression have been reported in the
entorhinal cortex of mouse models of AD and in the human AD
brain (Chin et al. 2007), but it remains to be determined
whether this is a condition specific to AD pathology. No studies
have addressed the possibility that changes in entorhinal
cortical reelin levels might be related to cognitive function
over the course of normal aging.
Individual differences in cognitive aging exist in humans and
in rodent populations. We used a well-characterized model in
which aged rats are classified based on the presence or absence
of memory impairments, with associated functional alterations
in the medial temporal lobe (Gallagher and Rapp 1997; Wilson
et al. 2006). Here, we report that reelin expression in the lateral
entorhinal cortex is reduced in aged rats with memory
impairment. This was observed in a stereological analysis and
in an assessment of reelin messenger RNA (mRNA) by in situ
hybridization. In both studies, changes in aged rats with
memory impairment clearly affected the lateral but not the
medial entorhinal cortex. Reelin depletion occurred in
conjunction with significant increases in tau phosphorylation
affecting the lateral entorhinal region, possibly recapitulating
the localization of early tangle formation in human aging and
AD (Braak H and Braak E 1995). The lateral entorhinal cortex
also exhibits a loss of synaptic marker expression with age-
related cognitive impairment. These signatures in rats with
memory impairment represent a change in aging that could
contribute to the selective vulnerability of entorhinal cortical
neurons and to reduced integrity of the perforant path circuit
innervating the hippocampal formation.
Materials and Methods
Animals and Behavioral Characterization
Male Long-Evans rats, obtained from Charles River Laboratories, were 6
(adult) or 24 (aged) months of age at the time of the studies. All rats
were individually housed for 2 months to acclimate to the vivarium
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before behavioral testing, which took place as described (Boric et al.
2008). Training occurred over 8 days in sessions of 3 trials per day with
a 60-s intertrial interval. During the trials, rats were placed in the water
at the perimeter of the pool, with starting locations varied across trials.
Each trial lasted for 90 s or until the rat successfully located the
platform. Every sixth trial was a probe trial to assess the rat’s spatial bias
during its search. Rats were permitted to escape on probe trials when
a retracted platform was made available after 30 s for completion of
those trials. An index score, derived from the proximity of the rat to the
escape platform location during the 30-s free swim on probe trials, was
used to characterize performance of the rats in the maze for the
purpose of neurobiological analyses. This index is the sum of the
weighted proximity scores measured during the probe trials, with
lower scores reflecting better spatial memory as indicated by shorter
average distances from the platform location (Gallagher et al. 1993). For
immunohistochemistry experiments, we analyzed tissue from (n = 9)
young, (n = 9) aged-impaired, and (n = 9) aged-unimpaired rats. For in
situ hybridization, we used tissue from (n = 5) young, (n = 6) aged-
impaired, and (n = 6) aged-unimpaired rats.
Euthanasia and Tissue Preparation
For immunohistochemistry, rats were anesthetized with isoflurane and
perfused transcardially with sterile saline, followed by 4% paraformalde-
hyde in phosphate buffer. After 24 h postfixation, brains were moved into
progressively increasing concentrations of glycerol in phosphate buffer.
Brains were then sectioned on the coronal plane in a 1 in 10 series at 50
lm thickness. Sections were stored in cryoprotectant at –80 ?C.
For in situ hybridization, rats were anesthetized, perfused, and brains
were postfixed as described above. After postfixation, brains were
moved into 4% paraformaldehyde in phosphate buffer containing 20%
sucrose for dehydration and cryoprotection. Brains were then frozen
on dry ice and stored at –80 ?C prior to sectioning. A 1 in 12 series of
coronal sections (30 lm) were cut using a freezing microtome and
stored in 4% paraformaldehyde at 4 ?C.
Immunohistochemistry and Immunofluorescence
For reelin peroxidase labeling, free-floating sections were incubated in
3.0% H2O2in phosphate-buffered saline (PBS) to quench endogenous
peroxidases. Sections were rinsed, then reacted in 0.1 M citric acid for
10 min. After additional rinses, the tissue was moved into primary
antibody solution containing mouse anti-reelin (1:1000, Chemicon)
with 0.25% Tween-20. The tissue was reacted in primary antibody
solution at 4 ?C for 48 h with shaking. After primary antibody
incubation, tissue sections were rinsed in PBS, then blocked in 5%
horse serum in PBS containing 0.25% Tween-20. Primary antibodies
were then detected with biotin-labeled secondary horse anti-mouse
and amplified with avidin--biotin complex (Vector Labs). Diaminoben-
zadine was used as a chromogen.
For reelin/synaptophysin double labeling, tissue was treated with
1.5% sodium borohydride in tris-buffered saline (TBS) for 30 min to
reduce autofluorescence. Sections were then reacted with primary
antibodies mouse anti-reelin (1:500, Chemicon) and rabbit anti-
synaptophysin (1:100, Santa Cruz) in TBS with 0.3% Triton-X 100.
The tissue was reacted for 48 h at 4 ?C with shaking. Following primary
antibody incubation, the tissue was blocked in 5% goat serum in TBS
with 0.3% Triton-X 100. Primary antibodies were then visualized with
fluorophore-conjugated secondary antibodies against the appropriate
species (Molecular Probes). Nuclei were counterstained with Hoechst.
For double-labeling of total tau and tau phosphorylated at serine 202,
tissue was treated with sodium borohydride as described above,
followed by blocking in 5% milk in TBS for 1 h. Sections were then
reacted overnight in primary antibodies diluted in 5% milk in TBS; total
tau antibodies were used at 1:100 (Dako Cytomation), while the
phospho-tau antibody was used at 1:500 (antibody kindly provided by
Peter Davies). The following day, sections were washed in TBS
containing 0.05% Triton-X 100 and reacted with fluorophore-conju-
gated secondary antibodies as described above.
Standard immunohistochemical controls included omission of primary
antibodies. The specificity of the reelin antibody used in these studies
has been determined previously (de Bergeyck et al. 1998). To produce
the CP13 antibody against tau phosphorylated at serine 202, mice were
immunized with paired helical filament tau purified from AD brain
tissue as described by Jicha et al. (1997). The CP13 antibody specifically
recognizes the phosphopeptide sequence GYSSPG(phospho-serine
202)PGTPGSRS and does not react with any other phosphoserine site
on the tau protein (Jicha et al. 1997). The total tau antibody was raised
against the C-terminal part (amino acids 243--441) containing the 4
repeated sequences involved in microtubule binding. The anatomical
pattern of staining for total tau was identical to that shown using this
antibody in previous studies (Planel et al. 2007), and this antibody has
previously been shown to label tau protein independently of its
phosphorylation state (Biernat et al. 2002). The synaptophysin antibody
detects an epitope corresponding to amino acids 221--313 mapping at
the C-terminus of synaptophysin of human origin, with crossreactivity
for the synaptophysin protein in rats. The spatial pattern of
synaptophysin immunoreactivity was identical to that observed in
previous studies (Stranahan et al. 2008), and we tested antibody
specificity in tissue from synaptophysin knockout mice (kindly
provided by Dr Rudolf Leube). No staining was observed in tissue
from synaptophysin knockout mice.
Unbiased Stereology and Anatomical Criteria
For stereological quantification, the optical fractionator method was
implemented using the StereoInvestigator morphometry system
(MicroBrightField), as described (Rapp et al. 2002). Slides were coded
prior to analysis and the code was not broken until the analysis was
finished. Neuron counts were derived from bilateral sections through
the entire rostrocaudal extent of the entorhinal cortex. The anatomical
borders of the lateral and medial entorhinal areas were traced under
low-power magnification, and subsequent cell counting was performed
within these borders as shown schematically in Figure 2A and as
described previously for the entorhinal cortex in this study population
(Rapp et al. 2002). In detail, the lateral entorhinal cortex was localized
anatomically by its position inferior to the rhinal sulcus and by its
rostrolateral position relative to the medial entorhinal cortex, as
described (Insausti et al. 1997). The lateral entorhinal cortex is
bordered rostrally by the perirhinal cortex. Reelin labeling in layer II
was restricted to the lateral entorhinal cortex and was not observed in
the superficial layers of the perirhinal cortex (Fig. 2B,C). In this regard,
the presence of reelin labeling in layer II is a chemoarchitectonic
marker that delineates the lateral entorhinal cortex.
The medial entorhinal cortex abuts the caudal and medial edge of the
lateral entorhinal cortex (Fig. 2A). The transition from lateral to medial
entorhinal cortex was readily apparent based on greater width of the
lamina dessicans (layer IV) in the medial relative to the lateral
entorhinal cortex. Layer II of the lateral entorhinal cortex exhibits
continuous labeling for reelin, relative to the medial entorhinal cortex,
consistent with the cellular architectonics in these regions on Nissl-
stained preparations (Rapp et al. 2002). Thus, we defined the border
between the lateral and medial entorhinal cortices using anatomical
criteria with reference to Paxinos and Watson (1998) and cytoarchi-
tectural criteria with reference to the continuity (lateral) or clustering
(medial) of layer II neurons. The medial entorhinal cortex is bordered
by the parasubiculm on its medial surface, and the transition between
the medial entorhinal cortex and the parasubiculum is demarcated by
a dramatic increase in the thickness of layer II (Rapp et al. 2002). Cells
were viewed through a 403 objective and counted as they first came
into focus within each optical disector. Counts derived from these
samples were converted to estimates of total labeled cell numbers for
Confocal Microscopy and Typhoon Imaging
Analysis of synaptophysin labeling in the entorhinal cortex was carried
out as described in Stranahan et al. (2008). Briefly, images of
synaptophysin labeling were captured on a Zeiss LSM 510 Meta
confocal microscope. The frame size was 71.43 3 71.43 lm with 512 3
512 pixel density. Ten frames per animal were collected from
superficial layers of the lateral and medial entorhinal areas, with
sampling spaced evenly throughout the rostrocaudal extent of these
regions. Quantitative analysis was carried out using LSM 510 software.
Cerebral Cortex February 2011, V 21 N 2 393
To acquire images of tau and phospho-tau labeling that could be
sampled across larger regions of the entorhinal cortex, we used
a typhoon fluorescent imaging system (GE Healthcare). For this
analysis, the 488 excitation channel was captured at the 520 emission
setting with bandpass 40 filtering. The 532 excitation channel was
captured at 610 emission with bandpass 30 filtering. For both channels,
the photomultiplier tube was set to 500 V. This scanning method has
a 10 lm minimum resolution and allowed us to measure immunore-
activity for tau and phospho-tau in the same tissue sections using
ImageJ. Grayscale optical density values for phosphorylated tau and
total tau were averaged across anatomically matched tissue sections to
give a single score for each brain region in each animal.
In Situ Probe Synthesis
Probe templates were synthesized as described in Haberman et al.
(2008). Initial primer sequences were as follows: for reelin, left,
agtactcagacgtgcagtgg, right, ctcatgaagcaaagtccaa; for DAB1, left, gaa-
caagccgtgtaccagac, right, agagccaaacacatctgcac. Polymerase chain re-
action (PCR) products were verified by restriction endonuclease
digestion. Initial PCR products were amplified further with the same
PCR primers that had been modified by the addition of T7 or SP6 RNA
polymerase-binding sites. PCR products containing T7 and SP6
extensions were purified by SVgel and a PCR cleanup kit (Promega).
35S-uridine triphosphate (UTP)--labeled riboprobe was then generated
using the Maxiscript kit (Ambion). The probe was then phenol/
choloroform extracted and precipitated in ethanol at –80 ?C. The final
probe was resuspended in RNase-free water and the specific activity
was determined by scintillation counter.
In Situ Hybridization
In situ hybridization was carried out as described by Haberman et al.
(2008). All tissue for each individual probe was processed during
a single run. Free-floating tissue sections were washed in 0.75% glycine
in 0.1 M phosphate buffer 2 times, followed by a single wash in
phosphate buffer. After that, sections were reacted in proteinase K
buffer containing 1.0 lg/mL proteinase K for 30 min at 37 ?C. Sections
were then treated with acetic anhydride solution (11.3% triethanol-
amine, 0.25% acetic anhydride, 0.04 M acetic acid) for 10 min at room
temperature. This was followed by two 15-min washes in 23 sodium
chloride/citrate buffer (SSC buffer; 203 concentration, 3 M NaCl, 0.3 M
sodium citrate). Next, sections were transferred to hybridization buffer
containing 20% formamide, 0.43 Denhardt’s solution, 4% dextran
sulfate, and 1.63 SSC) supplemented with 0.25 mg/mL transfer RNA,
0.33 mg/mL sheared salmon sperm DNA, 100 mM dithiothreitol (DTT),
and 1 3 107cpm/mL 35S-UTP--labeled probe for overnight reaction at
60 ?C. The following day, sections were washed at 60 ?C in 43 SSC/0.01
M DTT and 23 SSC/50% formamide. They were then incubated with
RNase (20 lg/mL) at 37 ?C for 30 min. Sections were washed with
progressively decreasing concentrations of SSC before mounting on
Densitometry of In Situ Hybridization mRNA Labeling
Slides processed for in situ hybridization were exposed to a phos-
phoimager screen and quantified by using ImageQuant (GE Health-
care). Digital images were acquired for entorhinal cortical sections
from the same levels for all animals. We analyzed (n = 5) sections per
animal from lateral entorhinal cortex, with sampling extending from
bregma --4.80 to bregma --8.72 (Paxinos and Watson 1998). In the
medial entorhinal cortex, we sampled (n = 3) sections per animal from
bregma --6.30 to bregma --9.16, as shown in Figure 2A. The subregion of
interest was outlined by hand by a researcher who was blind to group
conditions. Sections were averaged to obtain a single score for each
Behavioral data were compared across young and aged rats using
repeated-measures analysis of variance (ANOVA) with trial block as
a repeated measure and age as a fixed factor, followed by Bonferroni
post hoc analysis. Learning indices and physiological data were
compared across young, aged-unimpaired, and aged-impaired rats using
ANOVA with a planned post hoc comparison. The planned comparison
was designed to characterize differences between behaviorally
impaired aged rats (AI) and young and aged behaviorally intact rats
(Y+AU). Correlations between physiological measures and learning
indices among the rats in the aged cohort were made using Pearson’s
correlation analysis. Our rationale for including both aged-impaired and
aged-unimpaired in the correlation analyses is described in detail by
Baxter and Gallagher (1996). For all analyses, statistical significance was
set at P < 0.05.
Aged Rats Exhibit a Range of Cognitive Abilities on a Task
that Depends on Medial Temporal Lobe Function
Twenty-four-month-old male Long-Evans rats navigate less
effectively to the hidden platform in the Morris water maze,
relative to young rats, during training trials (Fig. 1A; F1,42=
12.64, P < 0.0001). The difference in performance on the first
block of training trials represents greater improvement across
those initial trials in young rats, as the aged rats performed on
par with the young rats during the first trial of maze training
(t42= 0.25, P = 0.81). As reported elsewhere, aged rats in this
study population exhibit individual differences as assessed
using an index score derived from the mean distance from the
platform location during probe trials (Fig. 1B; Gallagher et al.
1993). While a considerable number of the aged rats were
unimpaired with low index scores similar to young adults,
others in the aged cohort fell outside the range of young
performance. Note that the scores for the young rats, index
<240, in this study were in the normative range previously
Figure 1. Behavioral characterization on a task that recruits temporal lobe structures
identifies a subpopulation of aged rats that are impaired relative to young adults. (A)
Aged male Long-Evans rats follow a more circuitous path to the hidden platform in
the water maze over the course of training. Differences in performance on the first
block of trials are not detectable during the first trial (T1). Data points represent the
mean of blocks of 5 trials, and error bars depict the standard error of the mean. (B),
The learning index score, derived from the average distance from the platform
location during interpolated probe trials (described in Materials and Methods and in
Gallagher et al. 1993), distinguishes between aged-unimpaired (AU) and aged-
impaired (AI) rats.
Decreased Reelin Expression in the Entorhinal Cortex with Cognitive Aging
Stranahan et al.
observed with this protocol (Gallagher et al. 1993). Using this
index, we characterized the aged rats as either impaired
or unimpaired relative to young for immunohistochemical
Aged-Impaired Rats Have Fewer Reelin Immunoreactive
Cells in the Lateral Entorhinal Cortex
The lateral and medial subdivisions of the entorhinal cortex are
readily apparent on coronal sections (shown schematically in
Fig. 2A). Immunoreactivity for reelin delineates the layer II
neurons of the entorhinal cortex (Figs 2B--D and 3A,B).
Stereological analysis revealed that aged cognitively impaired
rats have fewer reelin-positive cells in the lateral entorhinal
cortex, relative to young rats and aged rats with preserved
cognition (Fig. 3B,C; F2,26= 4.51, P = 0.022). There was no
significant difference in reelin-positive cell numbers in the
medial entorhinal cortex (F2,26= 0.001, P = 0.99).
Changes in the number of reelin-immunoreactive cells are
unlikely to be attributable to loss of neurons, as previous work
in this model (Rapp et al. 2002) and in Fischer rats (Merrill et al.
2001) has shown no change in total neuron number in the
lateral entorhinal cortex with age-related cognitive impair-
ment. Taken together with the current report, these observa-
tions would indicate a loss of reelin expression in neurons in
the lateral entorhinal cortex of aged rats that are cognitively
impaired. We also looked at the relationship between reelin-
immunoreactive cell counts and behavioral performance
among rats in the aged cohort. While spatial memory
impairment, indicated by higher index scores, was generally
related to reduced reelin immunoreactive neuron numbers in
the lateral entorhinal cortex, this trend was not statistically
significant (Pearson’s r = –0.43, P = 0.075).
Reelin mRNA Expression Is Reduced in the Lateral
Entorhinal Cortex of Aged-Impaired Rats
In agreement with the observed decrease in reelin-positive cells
in lateral entorhinal cortex, a corresponding reduction in reelin
mRNA expression was observed in a separate set of aged rats.
between aged rats that are cognitively impaired or unimpaired,
relative to young rats (Fig. 4A). Aged rats that are cognitively
impaired exhibit reduced reelin mRNA expression in the lateral
entorhinal cortex (Fig. 4B,C; F2,16= 19.94, P = 0.001) with no
change in reelin mRNA expression in the medial entorhinal
cortex (F2,12= 1.76, P = 0.21). Reelin mRNA expression again
followed an anatomical gradient, such that greater signal was
detected in the lateral entorhinal cortex, relative to the medial
entorhinal cortex (Fig. 4C). There was no effect of aging or
cognitive status on mRNA for reelin’s intracellular signaling
target, ‘‘disabled’’-1 (Supplementary Fig. 1). No significant
Figure 2. Reelin is a marker expressed by neurons in the superficial layers of the
lateral entorhinal cortex. (A) Sagittal view of the rat brain, showing the anterior and
inferior anatomical position of the lateral entorhinal cortex (LEC) and the posterior
situation of the medial entorhinal cortex (MEC). The LEC is visible on coronal sections
containing the dorsal hippocampus. As sections become more caudal, the LEC
occupies more of the temporal cortex. On caudal sections that do not contain
hippocampus, the MEC is visible at the medial edge of the cortex. Eventually, the
MEC ascends to encompass the temporal cortical field. Image in (A) adapted from
Rapp et al. (2002). (B) Low-magnification image showing reelin immunoreactivity in
the superficial layers of the LEC. The diagram superimposed on the image is from
Paxinos and Watson (1998). (C) Reelin staining in layer II is confined to the LEC,
located inferior to the rhinal sulcus on coronal sections. No reelin staining is detected
in layer II of perirhinal cortex (PER) surrounding the superficial portion of the rhinal
sulcus. (D) Immunoreactivity for reelin among neurons in layer II of the lateral
Figure 3. Aged rats that are cognitively impaired have fewer reelin-immunoreactive cells in the lateral entorhinal cortex. (A) Representative photomicrograph showing reelin
labeling in the entorhinal cortex of a young rat. (B) Higher magnification image showing reelin immunoreactivity in layer II of the entorhinal cortex in a representative young rat (Y),
a representative aged-unimpaired rat (AU), and a representative aged-impaired rat (AI). (C) Aged-impaired rats have significantly fewer reelin-positive cells in the lateral entorhinal
cortex (LEC), relative to young rats. No changes were apparent in the medial entorhinal cortex (MEC). Asterisk indicates significance at P\0.05 following one-way ANOVA with
planned post hoc comparison between behaviorally impaired rats (AI) and behaviorally intact rats (AUþY). Error bars represent the standard error of the mean.
Cerebral Cortex February 2011, V 21 N 2 395
correlations were found between reelin or DAB1 mRNA
expression and the learning index among rats in this cohort of
Alterations in Tau Phosphorylation with Age-Related
Tangles composed of hyperphosphorylated tau form earlier in
the lateral entorhinal cortex, relative to the medial entorhinal
cortex, during aging and neurodegenerative disease in humans
(Arriagada et al. 1992; Braak H and Braak E 1995). While reelin
negatively regulates tau phosphorylation (Hiesberger et al.
1999), there are multiple tau kinases and several modulatory
factors that could underlie the regional susceptibility of the
lateral entorhinal cortex during aging and AD. To evaluate
another cellular event known to be a characteristic of the aging
lateral entorhinal cortex, we measured phosphorylation of tau
at serine 202 using immunohistochemistry.
Aged-impaired rats exhibit increased tau phosphorylation in
the lateral entorhinal cortex, relative to young rats (Fig. 5A,B;
F2,26= 11.03, P = 0.004). Accumulation of phosphorylated tau
was also correlated with behavioral performance among aged
rats, such that a greater increase in tau phosphorylation was
associated with worse behavioral performance (Fig. 5C;
Pearson’s r = 0.64, P = 0.004). By contrast, there was no effect
of aging or cognitive status on total tau immunoreactivity in the
lateral entorhinal cortex (F2,26= 0.74, P = 0.49; Supplementary
Fig. 2). Insofar, as total tau expression reflects neuronal
integrity, the absence of change in total tau immunoreactivity
is consistent with other evidence that total neuron number is
unchanged in the lateral entorhinal cortex of aged-impaired
rats (Merrill et al. 2001; Rapp et al. 2002).
Figure 4. Age-related cognitive impairment is associated with reduced reelin mRNA
expression in the lateral entorhinal cortex. (A) A subset of aged rats exhibits impaired
learning in the water maze. Rats were classified based on a proximity metric, with
higher scores indicating poor memory for the platform location (Gallagher et al. 1993).
(B) Representative images of entorhinal cortical sections visualizing reelin mRNA
expression in young (Y), aged-unimpaired (AU), and aged-impaired (AI) rats. (C) Aged
cognitively impaired rats exhibit reduced reelin mRNA expression in the lateral
entorhinal cortex (LEC), with no change in reelin expression in the medial entorhinal
cortex (MEC). Asterisk indicates significance at P\0.05 following one-way ANOVA
with planned post hoc comparison between behaviorally impaired rats (AI) and
behaviorally intact rats (AUþY). Error bars represent the standard error of the mean.
Figure 5. Increased phosphorylated tau immunoreactivity in the lateral entorhinal
cortex accompanies reelin depletion in aged-impaired rats. (A) Confocal micrograph
showing somatodendritic inclusions of phosphorylated tau in the lateral entorhinal
cortex of an aged-impaired rat. (B) Optical densities for the phosphorylated tau signal
on typhoon scanning images. Aged-impaired rats exhibit increased phospho-tau signal
in the lateral entorhinal cortex (LEC), with no significant change detected in the
medial entorhinal cortex (MEC). Asterisk indicates significance at P\0.05 following
one-way ANOVA with planned post hoc comparison between behaviorally impaired
rats (AI) and behaviorally intact rats (AUþY). Error bars represent the standard error
of the mean. (C), Among the aged rats, levels of phospho-tau immunoreactivity in the
lateral entorhinal cortex are correlated with cognitive performance. No such
correlation was found in the medial entorhinal cortex. Correlations were determined
using Pearson’s correlation with P \ 0.05 as described in Materials and Methods.
Decreased Reelin Expression in the Entorhinal Cortex with Cognitive Aging
Stranahan et al.
We observed no significant change in total tau expression
(F2,26 = 1.73, P = 0.20; Supplementary Fig. 2) in the medial
entorhinal cortex. There was a strong nonsignificant trend for
the main effect of aging, independent of behavioral performance
and cognitive status, on tau phosphorylation in medial entorhinal
cortex (Fig. 5B; F2,26= 3.29, P = 0.055). As expected based on
that trend, tau phosphorylation in the medial entorhinal cortex
did not correlate with behavioral alterations in the water maze
among the aged rats (Pearson’s r = 0.26, P = 0.31).
Loss of Reelin Immunoreactivity in the Lateral Entorhinal
Cortex Is Accompanied by Local Reductions in Synaptic
While neuron numbers are largely preserved throughout the
hippocampal formation and medial temporal cortex in aged
rats with cognitive impairment (Rapp and Gallagher 1996; Rapp
et al. 2002), a circuit-specific loss of synapses has been found in
aged rats with cognitive impairment involving the innervation
to the hippocampus originating in layer II entorhinal cortex
(Smith et al. 2000). Local intrinsic connections involving layer
II collaterals also exist within the entorhinal cortex itself,
through a network of ascending fibers traversing superficial
layers. (Supplementary Fig. 3, and van Strien et al. 2009). We
used synaptophysin immunoreactivity, which labels presynap-
tic terminals, to provide a preliminary estimate for loss of
synaptic integrity that might be associated with intrinsic
connections of layer II neurons.
The expression of reelin in neurons in the superficial layers
of the entorhinal cortex using immunofluorescence is shown in
Figure 6A. Fluorescence labeling for synaptophysin is also
evident in layer II of the entorhinal cortex, surrounding the
dendritic fields of reelin-labeled cells (Fig. 6A,B). Analysis of this
labeling for synaptophysin showed a significant reduction in
the lateral entorhinal cortex of aged-impaired rats (Fig. 6C;
F2,26= 4.59, P = 0.02). In contrast, there was no statistically
significant effect of aging or cognitive status on synaptophysin
immunoreactivity in the medial subregion (F2,26= 1.30, P =
0.29). These data suggest that inputs to the superficial layers
of the lateral entorhinal cortex are diminished, although the
source of those inputs, either extrinsic or intrinsic, remains to
be determined. However, we did observe that phospho-tau
accumulation was inversely correlated with synaptophysin
immunoreactivity (Pearson’s r = –0.60, P = 0.008; Fig. 6D). This
is notable because tau phosphorylation has been linked to
synaptic loss (Spires-Jones et al. 2009).
We have identified a number of changes associated with
individual differences in neurocognitive aging in the lateral
Figure 6. Reductions in synaptophysin labeling occur in concert with loss of reelin expression in the lateral entorhinal cortex of aged-impaired rats. (A) Immunoreactivity for the
synaptic marker synaptophysin is detectable in the dendritic fields of reelin-immunoreactive cells in the entorhinal cortex. (B) Confocal micrograph showing synaptophysin-
immunoreactive puncta in the superficial layers of the lateral entorhinal cortex in a young rat. (C) Labeling for the synaptic marker synaptophysin is reduced in the lateral entorhinal
cortex (LEC) of aged-impaired rats, with no significant alterations detected in the medial entorhinal cortex (MEC). Asterisk indicates significance at P\0.05 following one-way
ANOVA with planned post hoc comparison between behaviorally impaired rats (AI) and behaviorally intact rats (AUþY). Error bars represent the standard error of the mean. (D)
Synaptophysin optical intensity was inversely correlated with tau phosphorylation in aged rats. Correlations were determined using Pearson’s correlation with P \ 0.05 as
described in Materials and Methods.
Cerebral Cortex February 2011, V 21 N 2 397
entorhinal cortex. Reelin expression is reduced at both the
protein and mRNA level in aged rats that are cognitively
impaired. Changes in reelin expression occurred in the context
of increased tau phosphorylation and reduced synaptic marker
immunoreactivity. These changes followed a consistent ana-
tomical pattern, such that the lateral entorhinal cortex
emerged as a focal region in aged rats with impaired cognitive
Alterations in reelin expression, synaptophysin immunoreac-
tivity, and tau phosphorylation occurred in the lateral entorhinal
cortex of aged rats with spatial memory impairment. By contrast,
aged rats with preserved behavioral performance exhibited few
alterations compared with young adults. However, individual
differences in this model of cognitive aging are not confined to
spatial tasks. Aged rats that are behaviorally impaired in the
water maze also exhibit reduced odor recognition memory
(Robitsek et al. 2008), a function that involves the lateral
entorhinal cortex (Young et al. 1997). In this regard, neurobi-
ological alterations observed in the lateral entorhinal cortex in
the current experiments could directly contribute to olfactory
recognition deficits reported in aged rodents (LaSarge et al.
2007; Robitsek et al. 2008) and in humans with MCI and AD
(Djordjevic et al. 2008). These changes might additionally exert
downstream effects on hippocampal networks by disrupting
entorhinal connectivity, which would alter the fidelity of
information transmitted to the hippocampus.
The functional consequences of reduced reelin expression
with age-related cognitive decline have yet to be elucidated,
but the findings in the current study point to the lateral
entorhinal cortex as a relevant network for such studies. Reelin
is transported along perforant path axons and released within
the terminal zones of entorhinal layer II afferents (Martı´nez-
Cerden ˜ o et al. 2003). During aging, synaptic plasticity at lateral
perforant path synapses in the dentate gyrus is reduced (Froc
et al. 2003), but the question of whether changes in reelin
expression contribute to synaptic plasticity deficits has not
been addressed. Moreover, although there is significant support
for a reelinergic contribution to long-term potentiation at
Schaffer collateral synapses in CA1 (for review, see Herz and
Chen 2006), the association between reelin expression and
synaptic plasticity at lateral perforant path synapses has not
Reelin regulates neuronal migration during development and
synaptic number and function during adulthood (Herz and
Chen 2006). Transgenic overexpression of reelin in neurons
during adulthood increases the number of synapses in regions
of the hippocampus that receive input from layer II neurons
(Pujadas et al. 2010). In contrast, heterozygous reeler mice,
which exhibit a nearly 50% reduction in reelin levels, have
fewer hippocampal synapses (Niu et al. 2008). Functionally,
reelin enhances long-term potentiation and protects against
deficits induced by application of b-amyloid (Durakoglugil et al.
2009). This opens the possibility that reelin produced by the
entorhinal cortex could confer neuroprotection on target cells
in the hippocampus, which would be compromised with loss of
reelin expression during age-related cognitive decline.
The observation that AD mice have fewer reelin immunore-
active principal neurons in the entorhinal cortex (Chin et al.
2007) without overt loss of entorhinal neurons (Irizarry et al.
1997) suggests that reduced reelin expression may contribute to
early synaptic alterations in AD. While previous studies
examining differences in reelin expression in AD mouse models
did not differentiate between the medial and lateral entorhinal
cortices, the topography of changes in the terminal zones
innervated by entorhinal subdivisions suggests particular lateral
entorhinal sensitivity. The outer molecular layer, which receives
input from the lateral entorhinal cortex, shows the earliest
synaptic atrophy (Dong et al. 2007) and accumulation of amyloid
deposits (Reilly et al. 2003). This synaptic zone is similarly
subject to loss of connectivity in the same population of aged
rats used in the current study (Smith et al. 2000). Here, we show
that reduced reelin expression is also seen in the context of
changes in synaptic marker expression in the lateral entorhinal
cortex during normal age-associated cognitive decline.
Reduced reelin expression occurred in concert with in-
creased tau phosphorylation and reduced immunoreactivity for
the synaptic marker synaptophysin. The lateral entorhinal
cortex exhibited specific vulnerability to these molecular
alterations during cognitive impairment in aging. A lateral to
medial entorhinal topography has long been recognized in
human aging, as tangles initially appear in the transentorhinal
cortex with progressive involvement of entorhinal layer II
neurons along a lateral to medial gradient (Braak stages 1--2)
(Braak H and Braak E 1995). Although neurofibrillary tangles do
not occur in aged rats, increased phosphorylation of tau
emerges in the lateral entorhinal region in older animals
exhibiting cognitive decline. The current study is, to the best of
our knowledge, the first report of increased tau phosphoryla-
tion in the aged rat. A prior report described a redistribution of
phospho-tau from neuronal processes to the soma in aged rats
(Niewiadomska et al. 2005) but did not quantify phosphory-
lated tau immunoreactivity in relation to cognitive status. Mice,
in contrast with rats, do not display phosphorylated tau
accumulation with aging unless expression of wild-type human
tau is present under a transgene (Kimura et al. 2007). This
distinction may be attributable to differential expression of tau
isoforms between the rat and mouse (Takuma et al. 2003), with
the rat more closely resembling human tau isoforms.
The current findings have revealed a particular susceptibility
of the lateral entorhinal cortex in age-related cognitive
impairment. While these observations are best viewed at
present as correlates of behavior rather than mechanisms, we
have begun to establish a framework for understanding lateral
entorhinal vulnerability in neurocognitive aging. In our model,
cognitive impairment with aging is associated with reduced
reelin expression, increased tau phosphorylation, and synaptic
loss in the lateral entorhinal cortex. Future studies will address
the mechanistic interrelationships among these changes and
their impact on cognitive function.
material canbe foundat:http://www.cercor
National Institute on Aging at the National Institutes of Health
(T32 AG027668-02 to A.M.S.); Ford Foundation/National Re-
search Council Fellowship to A.M.S.; National Institute on Aging
at the National Institutes of Health (5P01AG009973-17 to M.G.)
We are grateful to Dr Rebecca Burwell for kindly letting us use her
StereoInvestigator system, to Dr Peter Davies of Yeshiva University for
Decreased Reelin Expression in the Entorhinal Cortex with Cognitive Aging
Stranahan et al.
the phosphorylated tau antibody, and to Dr Rudolf Leube of University
of Mainz for the synaptophysin knockout mouse tissue. Conflict of
Interest: M.G. is the founder of AgeneBio Incorporated, a biotechnology
company that is dedicated to commercializing therapies to treat
cognitive impairment in aging and she has a financial interest in the
company. Two of the authors (R.P.H. and M.G.) are inventors on Johns
Hopkins University intellectual property with patents pending under
option to license to AgeneBio. Dr Gallagher serves as a member of the
Board of Scientific Counselors to the National Institute on Aging and is
also a member of the Scientific Advisory Board of the Stanley Center at
the Broad Institute.
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