Alzheimer’s disease often results in impaired olfactory perceptual acuity—a potential biomarker of the disorder. However, the useful-
deficits that mimic those observed clinically—some evident at 3 months of age. Also, at 3 months of age, we observed nonfibrillar A?
deposition within the olfactory bulb—earlier than deposition within any other brain region. There was also a correlation between
olfactory deficits and the spatial-temporal pattern of A? deposition. Therefore, nonfibrillar, versus fibrillar, A?-related mecha-
nisms likely contribute to early olfactory perceptual loss in Alzheimer’s disease. Furthermore, these results present the odor
cross-habituation test as a powerful behavioral assay, which reflects A? deposition and thus may serve to monitor the efficacy of
therapies aimed at reducing A?.
ease wherein patients suffer from sensory, motor, and cognitive
loss. Currently, much interest exists in establishing methods to
diagnose AD before the irreversible deterioration of the brain
characteristic of the disease. One potential avenue in the early
diagnosis of AD is the use of olfactory sensory dysfunction in
combination with neuropsychological measures (Murphy, 1999;
Albers et al., 2006). Persons displaying AD often have a reduced
ability to detect, discriminate, and identify odors (for review, see
Mesholam et al., 1998; Murphy, 1999). However, the usefulness
of olfactory screens to serve as an informative biomarker for AD
is precluded by a lack of knowledge regarding why AD impacts
olfaction. Understanding the relationship between AD neuro-
perception will be an important step forward in this quest.
AD is pathologically defined by the presence of amyloid-?
(A?) plaques and neurofibrillary tangles (NFTs) within the
brain. Plaques are mostly composed of A?, which derives from
amyloid-? precursor protein (APP). NFTs are made of hyper-
Together, the presence of these markers represents the classic
diagnostic hallmarks of AD (Alzheimer et al., 1995). Models of
AD-related tauopathy and amyloidosis are well established as
contributing to our understanding of the physiological and cog-
nitive aspects of the disease (Lee et al., 2001; Hardy and Selkoe,
2002). Currently, however, neither model has been directly
tested in relation to understanding the cause of olfactory dys-
function in AD.
The mechanisms of AD-related olfactory sensory loss can be
uniquely explored through the use of APP transgenic mouse
models (Van Dijck et al., 2008; Zhuo et al., 2008; Gue ´rin et al.,
2009; Montgomery et al., 2009; Young et al., 2009). The neural
mechanisms of odor processing in rodents are relatively under-
stood. Olfaction involves processing stages spanning from sen-
sory neuron input to the olfactory bulb, decoding and plasticity
hippocampus (Wachowiak and Shipley, 2006; Wilson and
Stevenson, 2006). Although studies exist showing olfactory-
behavior deficits in APP transgenic mice (odor reversal learning,
odor memory, etc.) (Van Dijck et al., 2008; Zhuo et al., 2008;
Gue ´rin et al., 2009; Montgomery et al., 2009; Young et al., 2009),
no studies are available directly relating A? in the olfactory sys-
tem with olfactory perceptual loss. The extent of A? pathology
across this system and the involvement of these regions in AD-
see Price et al., 1991; Kova ´cs, 2004). Furthermore, despite the
2008), whether A? plays a role in contributing to AD-related
atypical olfactory behaviors is unknown.
Here, we addressed the relationship between A? deposition
within olfactory processing networks and odor perception in the
ments, Monika Pawlik for assistance with mice breeding and genotyping, and Dun-Sheng Yang for thoughtful
Correspondence should be addressed to Daniel W. Wesson, Nathan S. Kline Institute for Psychiatric Research,
TheJournalofNeuroscience,January13,2010 • 30(2):505–514 • 505
Tg2576 AD mouse model (Hsiao et al., 1996) at sequential life
may stem from deposition of nonfibrillar A? within the first
synaptic processing stage of the olfactory system.
Mice. Mice bred and maintained within the Nathan S. Kline Institute for
Psychiatric Research animal facility were used. Tg2576 mice were gener-
ated previously by overexpressing the 695 aa isoform of human APP
containing the KM670/671NL mutation, as described previously (Hsiao
et al., 1996). Age-matched nontransgenic [wild-type (WT)] mice on
B6SJLF1/J background were used as controls. To examine the possible
contributions of accumulating A? pathology throughout life on olfac-
tion, we used four separate age groups: 3- to 4-month-old (Tg, n ? 5, 5
3 females, 2 males; and WT, n ? 4, 2 females, 2 males), 16-month-old
(Tg, n ? 6, 6 males; and WT, n ? 9, 9 males), and 21- to 29-month-old
mice (mean ? SEM, 23.5 ? 0.6; Tg, n ? 6, 2 females, 4 males; and WT,
n ? 8, 4 females, 4 males). Mice were genotyped by PCR analysis of tail
dance with the guidelines of the National Institutes of Health and were
approved by the Institutional Animal Care and Use Committee of the
Nathan S. Kline Institute.
Olfactory behavior test. Mice were screened for olfactory deficits using
and Linster, 2008). Odors (n ? 7; limonene, ethyl valerate, isoamyl ace-
tate, pentanol, heptanone, propyl butyrate, and nonane; Sigma-Aldrich)
were diluted 1 ? 10?3in mineral oil and applied to a cotton applicator
stick, which was then enclosed in a piece of odorless plastic tubing to
still allow volatile odor delivery. Notably, such an odor presentation
method controls for the influence of visual and/or somatosensory influ-
ences on odor investigation. Odors were delivered for four successive
trials (one block), 20 s each, separated by 30 s intertrial intervals, by
inserting the odor stick into a port on the side of the animal’s home cage
(see Fig. 1a). Home cage testing was chosen over testing in a separate
of the new environment/context) on the behavioral measures. Testing
took place during the light phase of the animals’ 12 h dark/light cycle,
over two daily sessions (three to four odors/session) separated by 24–48
h. The duration of time spent investigating, defined as snout-oriented
trials by a single observer blind to genotypes (D. W. Wesson). Home
cages were cleaned with fresh corn cob bedding 24–48 h before behav-
ioral testing to reduce unnecessary background odors, yet still allow for
adaptation to the new bedding. The stainless-steel food bin and water
bottle were removed from cages immediately before testing.
Histology. Tissue collection was performed after either urethane anes-
4-, 6- to 7-, and 21- to 29-month-old mice) or CO2overexposure (16-
month-old mice; experimental constraints prevented the use of a single
killing agent for all groups). Mice were decapitated, and brains were
rapidly removed over ice. Hemibrains from 3- to 4-, 6- to 7-, and 21- to
29-month-old mice were placed in 10% formalin for fixation. Hemi-
brains from each animal were coronally sectioned (40 ?m) on a mic-
rotome. A subset of these sections were slide-mounted and immersed in
(three times) for 1 min in ddH2O, and subsequently coverslipped with
Vectashield hardmount with 4?,6-diamidino-2-phenylindole (DAPI)
(Vector Laboratories) for nuclear counterstain. The remaining sections
nohistochemistry (Mi et al., 2007). Sections were washed (three times at
5 min) in TBS after which they were treated with 85% formic acid for 5
buffer (0.05 M Tris-HCl, pH 7.6, 0.9% NaCl, 0.25% Triton X-100, 20%
normal goat serum, and 0.2% bovine serum albumin) three times (10
min each) before incubating for 12 h in 4G8 primary antibody at 4°C
(Signet Laboratories; 1:200 in blocking buffer). Sections were rinsed
(three times at 5 min) in blocking buffer before incubating for 2 h at
room temperature in Alexa 488 secondary antibody (Invitrogen). Fi-
and coverslipped with Vectashield hardmount with DAPI (Vector).
old Tg mouse) was placed on all slides of young (3- to 4- and 6- to
7 d after staining by use of a Zeiss Axioscope microscope (model 200M)
and a Zeiss digital camera (Carl Zeiss). Sections were excited at 488 nm,
and, because of the similar, yet still quantifiably different emission spec-
tra of Thioflavin-S/Alexa 488 and DAPI, emission from all fluorophores
was captured with a FITC filter.
Data analysis. For analysis of olfactory behavior data, all raw investi-
gatory values (in seconds) were pooled within animals and organized
according to odor presentation (trial) number. Because separate groups
of mice were used within each age group, two-way ANOVAs versus
repeated-measured ANOVAs were used to examine the influence of age
of all trial 1 odor investigations were pooled within each genotype and
(eight) followed by post hoc group comparisons using Fisher’s PLSD.
Second, as a measure of odor habituation, the raw investigatory values
were normalized to the maximum investigatory duration per animal for
was assigned a value of 1, and the lesser investigation times a proportion
of 1. These normalized investigation probabilities of subjects for each
odor were analyzed using two-way ANOVAs for independent groups
(eight) and repeated measures (four trials) within each group. Normal-
ized odor investigation durations within individual trials were collapsed
across all odors within groups across and analyzed using one-way
ANOVAs for independent groups (eight). Post hoc group comparisons
after a significant ANOVA result were made using Fisher’s PLSD tests.
Finally, normalized investigation data were again used to determine ef-
fects of genotype on odor cross-habituation (discrimination). To calcu-
late this “cross-habituation index” (Rankin et al., 2009), the normalized
investigatory values from all fourth trial odor presentations were sub-
tracted from the following first trial odor presentations. These cross-
habituation values were then analyzed using one-way ANOVAs for
independent groups (four) followed by post hoc group comparisons us-
ing Fisher’s PLSD.
Histological analysis of A? levels was performed in NIH ImageJ
(http://rsbweb.nih.gov/ij). Six individual brain areas, the olfactory bulb
(OB), orbitofrontal cortex (OFC), anterior and posterior piriform corti-
ces (aPCX and pPCX), dorsal hippocampus (Hipp), and lateral entorhi-
nal cortex (Ent) were analyzed for levels of Thioflavin-S and anti-A?
staining to quantify A? deposition (percentage area) across olfactory
structures. Fluorescence levels of anti-A? 4G8 and Thioflavin-S were
thresholded, and regions of interest (ROIs) were determined with the
(Paxinos and Franklin, 2000).
A? deposition area (percentage) was quantified within each ROI sep-
arately. For measurements of A? deposition within the OB, individual
layers were manually outlined using DAPI-labeled anatomical layers as
guides (see Fig. 2a). The aPCX and pPCX were separated by the disap-
pearance of the lateral olfactory tract (LOT) (see Fig. 3a, dashed rectan-
gle). Individual cell layers within the PCX were manually outlined using
DAPI labeling as a guide. To quantify A? deposition in the OFC, we
selected a rectangular area within the medial OFC (see Fig. 4a, dashed
region). We selected a diagonal rectangular area within the Ent to quan-
tify A? deposition (see Fig. 4b, dashed region). To quantify A? deposi-
tion in the Hipp, we manually outlined all Hipp regions (dentate, CA1,
CA2, and CA3) (see Fig. 4c, dashed region).
A? deposition (percentage area) (Mi et al., 2007) was defined as the
cumulative area of fluorescent pixels above threshold within each ROI.
At least three coronal brain sections (range, three to four) containing
each ROI per mouse were used for analysis. Percentage area values were
in some cases analyzed using one-way ANOVAs for independent groups
506 • J.Neurosci.,January13,2010 • 30(2):505–514Wessonetal.•OlfactoryDysfunctionandAmyloid-?
measures for analysis.
All statistical analyses were performed in StatVIEW (SAS Institute) or
in MatLab (Mathworks). All values are reported as mean ? SEM unless
Our primary goal was to establish whether there is a relationship
between A? neuropathology and olfactory perception in the
Tg2576 APP transgenic mouse model
(Hsiao et al., 1996). We began to address
this by examining odor-guided behavior
in an odor cross-habituation task, which
vestigation, odor learning and memory
(habituation), and odor discrimination
(i.e., cross-habituation)—all within a sin-
gle behavioral test. In this test, mice were
applicator sticks laced with odor (see Ma-
terials and Methods) (Fig. 1a). Each odor
was delivered across four successive trials
the duration of time spent investigating the
odor was recorded across trials allowing
quantification of novel odor investiga-
tion, odor habituation, and odor cross-
Habituation (and the generalization
thereof, tested as cross-habituation) is a
form of implicit or nondeclarative mem-
ory and is not hippocampus dependent.
One of the advantages of using odor ha-
bituation and cross-habituation as an as-
say in the present studies is that the
uation (as tested here) are known. Short-
term odor habituation is attributable to a
metabotropic glutamate receptor-mediated,
homosynaptic depression of afferent syn-
apses from the olfactory bulb in piriform
cortex (Wilson and Linster, 2008). The
odor specificity of this cortical adaptation
is likely attributable to plasticity of intra-
cortical association fiber synaptic plastic-
ity within piriform cortex (Linster et al.,
2009). Thus, although not a long-term
memory task, odor habituation and its
forms of synaptic plasticity and local cir-
cuit function that may be affected by AD-
We began our investigation into the pos-
sible olfactory deficits expressed by AD
model mice by examining odor investi-
gation behaviors in each age group of
Tg2576 mice. The time spent investigat-
ing novel odors can be an indicator of
arousal/motivation (Baum and Keverne,
2008). We pooled all novel (trial 1) odor
investigation durations across all odors (n ? 7) and animals
within each age group and genotype. As shown in Figure 1b, we
found that 16 month and older (21–29 months) APP transgenic
(Tg) mice investigated trial 1 odors longer than age-matched
controls (WT). ANOVA on all investigation times revealed a sig-
nificant effect of genotype (F(1,272)? 66.4; p ? 0.0001) and age
(F(3,270)? 10.5; p ? 0.0001). In particular, both 16- (F(1,87)?
36.7; p ? 0.0001) and 21- to 29-month-old Tg mice (F(1,95)?
tions. *p ? 0.0001, Tg versus WT. c, Odor habituation (normalized) (see Materials and Methods) across four successive odor
Progressive and age-dependent emergence of AD-like atypical olfactory behavior in APP transgenic mice. Results
J.Neurosci.,January13,2010 • 30(2):505–514 • 507
88.4; p ? 0.0001) investigated trial 1 odors significantly longer
compared with WT mice (Fig. 1b). To assess whether this ten-
dency for aged Tg mice to investigate trial 1 odors for prolonged
periods of time reflected simple hyperactivity and thus enhanced
numbers of approaches to the odor port, in addition to total
investigation duration, we also analyzed the number of sniff
bouts. We found that 16- and 21- to 29-month-old Tg mice dis-
played statistically similar numbers of sniff bouts in response to
trial 1 odors [mean ? SEM, 16 months: (Tg) 1.6 ? 0.2, (WT)
1.6 ? 0.1; 21–29 months: (Tg) 1.8 ? 0.2, (WT) 1.5 ? 0.2; two-
way ANOVA, p ? 0.05, Tg vs WT within each age], suggesting
21- to 29-month-old APP Tg mice reflects, at least within the
context of this task, deficient short-term odor habituation.
Previous behavioral and physiological work has demonstrated
that habituation to repeated odor exposure on a short timescale
(?30 s) is a process mediated by group III metabotropic gluta-
mate receptors (mGluRIII) (Best and Wilson, 2004) located on
presynaptic nerve terminals in the olfactory piriform cortex
(PCX) (Wada et al., 1998). Indeed, blocking mGluRIII receptors
in vivo at the level of the PCX prevents cortical odor adaptation
(Best and Wilson, 2004) and results in a failure to habituate nor-
mally to odor exposure (Yadon and Wilson, 2005). Since, here,
we found that AD model mice possess different capacities to ha-
bituate to novel (trial 1) odors within a single 20 s exposure (Fig.
1b), we next assessed odor habituation over repeated odor expo-
sures in each of four age groups of mice. To do this, odor inves-
tigation durations across all trials (four total; including the data
from trial 1) (Fig. 1b) of the odor cross-habituation task were
analyzed for differences between groups. Because of the signifi-
cant differences shown in Figure 1b in the investigation levels of
the groups within trial 1, all data were normalized before statis-
each individual odor presentation block (trials 1–4) (see Materi-
als and Methods).
tions compared with age-matched WT controls (Fig. 1c). Across
all age groups, there was a significant overall genotype effect
(F(1,266)? 98.2; p ? 0.0001), an age effect (F(3,266)? 4.6; p ?
Furthermore, within each age group, there was a significant
repeated-measures effect ( p ? 0.0001). The significant effect of
repeated measures indicated that all groups were eventually able
to habituate to odors by the fourth trial, however, with slight
differences among the groups (Fig. 1c). WT mice in the 6- to 7-,
16-, and 21- to 29-month-old age groups habituated more rap-
idly to odors compared with their age-matched Tg counter-
parts—achieving statistical significance by the second trial [6–7
months (F(1,37)? 22.4; p ? 0.0001); 16 months (F(1,88)? 16.2;
p ? 0.0001); 21–29 months (F(1,94)? 73.7, p ? 0.0001)]. We
Materials and Methods) across all four trials by subtracting the
normalized investigation duration during trial 4 from the maxi-
mum normalized investigation duration within that block (Fig.
1c). Indeed, even at our earliest time point, 3–4 months, the
habituation index was 0.08 (i.e., 8% difference between WT and
Tg mice in habituation abilities). This habituation deficit in Tg
mice increased in 6- to 7-month-old mice (31% difference with
WT) and was maintained at both the 16- and 21- to 29-month-
old age points (Fig. 1c)—demonstrating that atypical habitua-
tion to repeated odor exposure begins early in life in the Tg2576
mouse model and is maintained throughout life.
As a final behavioral measure, we assessed odor discrimination
response difference between a habituated odor and a newly
presented odor—can be used as a test of spontaneous odor dis-
crimination (Linster et al., 2002; Wesson et al., 2008), which
eliminates the confounds associated with traditional operant
odor discrimination tests (e.g., nutritional deprivation, sensori-
motor control). A strong investigation of a novel odor after
the presentation of a habituated odor (i.e., minimal cross-
or a weak investigation (i.e., strong cross-habituation) reflects
generalization or a failure to discriminate (Wilson and Linster,
2008; Rankin et al., 2009). To our knowledge, no data are cur-
Therefore, to characterize odor discrimination abilities in
Tg2576 mice, we analyzed the same odor cross-habituation data
used for Figure 1, b and c, but this time directly for odor cross-
habituation. In particular, we compared across all trials, the dif-
ference in duration of investigating all novel odors (trial 1) from
the previous trial 4. Because of differences in odor investigation
in trial 1, normalized odor investigation data (as in Fig. 1c) were
used. The investigation value for the preceding trial 4 was sub-
tracted from the novel odor trial (trial 1) to calculate a “cross-
habituation index.” Similar measures of cross-habituation as an
index for stimulus discrimination are commonly used across a
variety of sensory paradigms (Rankin et al., 2009).
We found that Tg2576 mice showed reduced odor discrimi-
nation abilities (enhanced generalization) when compared with
age-matched WT controls (Fig. 1d). Furthermore, similar to
measures of odor habituation, deficits in odor discrimination
were evident by 6–7 months of age. ANOVA on all cross-
habituation values revealed a significant effect of genotype
(F(1,223)? 74.6; p ? 0.0001) on odor cross-habituation. There
was a significant reduction in odor cross-habituation in 6- to 7
(F(1,29)? 8.96; p ? 0.0056)-, 16 (F(1,71)? 14.77; p ? 0.0003)-,
and 21- to 29 (F(1,81)? 222.0; p ? 0.0001)-month-old Tg mice
compared with controls (Fig. 1d). These results suggest that fine
aspects of odor information processing in Tg2576 mice may be
altered in a manner sufficient to yield deficiencies in odor dis-
crimination and odor learning.
cessing stream in AD model mice, paired with perceptual data,
would be valuable in understanding the pathological determi-
nants of olfactory dysfunction in AD. For instance, are A? levels
related to olfactory dysfunction in a dose-dependent manner?
Furthermore, given the differential expression of atypical olfac-
increases in A? within certain olfactory regions temporally cor-
respond with the emergence of these behaviors? Therefore, we
characterized A? levels throughout the olfactory system of mice
previously tested in odor cross-habituation with two separate
methods (see Materials and Methods)—Thioflavin-S labeling of
?-pleated sheet confirmation of proteins, including fibrillar A?
508 • J.Neurosci.,January13,2010 • 30(2):505–514Wessonetal.•OlfactoryDysfunctionandAmyloid-?
deposits, and immunostaining with anti-A? antibody (4G8)
against all A? forms including fibrillar and nonfibrillar deposits
and also other metabolites of APP and APP itself.
The first step in odor information processing occurs within
ular layer (Gl). As shown in Figure 2a, beneath the Gl there are
four layers, including the external plexiform layer (Epl), the mi-
tral/tufted cell layer (MT), the inner plexiform layer (Ipl), and
finally within the center of the OB is the granule cell layer (Grl).
All layers of the OB are essential in odor information processing
(Wachowiak and Shipley, 2006). Using 4G8 (anti-A?) and
increased across the other OB cell layers (Fig. 2b,c). In contrast,
in a given brain section) Thio-S-positive amyloid deposits in the
Grl (Fig. 2b). Notably, A? deposition at this age in the OB pre-
cedes previous reports of plaque deposition in the hippocampus
and neocortex (Hsiao et al., 1996; Kawarabayashi et al., 2001)
(also see Fig. 4b). Within both the 16- and 21- to 29-month-old
Tg mice, the Grl was strongly burdened with Thio-S-positive A?
plaques (Fig. 2b,c). Interestingly, we found that Thio-S-positive
amyloid plaques were localized almost exclusively within the Grl
of Tg2576 mice. Only rarely were any Thio-S-positive A? depos-
its observed within the Gl, Epl, MT, or Ipl (Fig. 2d). These data
demonstrate a unique property for the OB Grl to host Thio-S-
positive deposition across other layers of the OB. Furthermore,
these data suggest a broad range of potential contributors to the
olfactory behavioral phenotype of the Tg2576 mouse, including
Thio-S-positive A? plaque deposition in the Grl or potentially
anti-A?-positive deposition across all cell layers.
projects into the PCX. LOT axons terminate within superficial
layer I where they synapse onto pyramidal cells with soma in
layers II and III. Layer II/III pyramidal cells extend basal den-
drites into layer III where feedback and association fiber connec-
tions further process olfactory information (Haberly, 2001).
in odor identification and for pPCX (Fig. 3a, bottom) in odor
quality encoding (Gottfried et al., 2006; Kadohisa and Wilson,
2006b). Furthermore, disruption of PCX activity impairs odor
discrimination and learning (i.e., habituation) (Wilson et al.,
2006). Whereas we found that the LOT was spared of A? depo-
sition (Thio-S and anti-A?) (data not shown), we observed A?
deposition, at differing levels, throughout all layers of the aPCX
and pPCX. In general, layer III of both the aPCX and pPCX
developed the greatest density of A? burden of all three layers
(Fig. 3b,c). In contrast, layer II of aPCX had the least A? burden.
Furthermore, A? deposition within the PCX did not become
apparent until 16 months of age. Across all layers, anti-A? dep-
osition was greater than Thio-S-positive A? deposition alone
(Fig. 3c). These data, showing that A? does not escalate in the
genic model, that the PCX is impacted by A? neuropathology
later in life than the OB. Furthermore, PCX A? levels do not
J.Neurosci.,January13,2010 • 30(2):505–514 • 509
predict the occurrence of atypical odor habituation nor discrim-
ination, as these behaviors emerged at 6–7 months of age. How-
emergence of atypical odor investigation behavior (PCX-
dependent short-term habituation) (Wilson and Linster, 2008)
We also extended our analysis of A? deposition into three
“higher-order” olfactory regions. The OFC is located within the
frontal cortex (Fig. 4a) and has reciprocal connections with the
within the OFC starting with the 16 month age group. Further-
more, these A? levels increased into the 21–29 month age group,
with the greatest escalation in anti-A?-positive deposition (Fig.
4a). A? deposits within the OFC were similar in density as in
neighboring frontal cortex regions—suggesting that the OFC is
not particularly affected by A? pathology as was the case for the
OB Grl and layer III of the PCX. The Ent is located posterior to
the pPCX (Fig. 4b) and receives small but direct MT cell input
from the OB, as well as input from PCX (Schwob and Price,
1978). Furthermore, the Ent is involved in olfactory memory
(Eichenbaum et al., 1994). We found relatively dense A? deposi-
tion (both Thio-S- and anti-A?-positive) within the Ent of
itatively similar levels of A? deposition. Finally, the Hipp is also
involved in olfactory memory and receives olfactory-related in-
put from the Ent (Sta ¨ubli et al., 1984) (Fig. 4c). Similar to previ-
by A? deposition (Fig. 4c). A? deposition was evident starting in
the 16 month age group and progressed in levels into the 21–29
month group. Together, these results suggest that A? (deposi-
tion) pathology has the potential to impact olfactory processing
across multiple brain areas.
To provide contrast to A? deposition levels within brain re-
gions unique to olfactory processing, we also measured A? dep-
osition within the primary somatosensory and motor cortices
(cumulative percentage area in S1 and M1) (see Materials and
A? deposition did not become evident in the S1/M1 until 16
months of age (percentage area anti-A? at 3–4 months: mean ?
and 21–29 months: 5.5 ? 0.6; percentage area Thio-S at 3–4
months: 0 ? 0; 6–7 months: 0 ? 0; 16 months: 0.7 ? 0.1; and
21–29 months: 4.8 ? 0.5). Within animals, A? deposition was
notably greater within principal olfactory areas (OB and PCX)
than in S1/M1 (see Figs. 2, 3).
We observed A? deposition across the majority of olfactory
structures examined (Figs. 2–4). Therefore, we next asked
age-dependent olfactory behavioral deficits found in Tg2576
flavin S- and anti-A?-positive separately, as shown in Figs. 2–4)
from the OB, PCX, OFC, Ent, and Hipp within individual ani-
510 • J.Neurosci.,January13,2010 • 30(2):505–514Wessonetal.•OlfactoryDysfunctionandAmyloid-?
mals (all age groups) previously tested on olfactory cross-
(investigation, habituation, and discrimination) significantly
abnormal odor investigation behaviors highly correlated with
Thio-S-positive deposits in the Ent (r ? 0.75) and with anti-A?
deposits in the OB (r ? 0.72). Odor discrimination abilities were
significantly correlated with anti-A? deposits in the PCX (r ?
Hipp (r ? 0.44), but with neither markers in the OB (r ? 0.25).
Odor habituation abilities were significantly correlated with
Thio-S-positive deposition in the Hipp (r ? 0.46) and Ent (r ?
of odor processing (Gue ´rin et al., 2009), our data revealing as-
sociations between A? deposition and olfactory dysfunction
support a potential link between A? within specific areas of
the primary olfactory network and some aspects of olfactory
behavior (Fig. 6).
The present results demonstrate that Tg2576 mice present with
progressive olfactory impairments compared with age-matched
WT controls. The Tg2576 mice behavioral deficits included (1)
perseveration of initial odor investigation, (2) impaired short-
term odor habituation, and (3) impaired odor discrimination.
Furthermore, detailed analysis of fibrillar (Thio-S positive) and
fibrillar and/or nonfibrillar A? burden (anti-A? positive)
throughout major olfactory processing centers revealed that the
emergence of specific behavioral impairments corresponds with
a progressive A? burden in specific olfactory regions. For exam-
ple, although deficits in initial odor investigation strongly corre-
lated with nonfibrillar A? deposits within the OB, odor
discrimination deficits were correlated with nonfibrillar A? de-
posits within PCX, but not OB. Finally, the earliest detectable
olfactory deficits emerged as early as 3 months of age, the same
age as emergence of initial nonfibrillar A? deposits within the
first synaptic layer of the OB. Together, these findings provide
both a spatial and temporal correlation
between specific olfactory deficits and lo-
calize A? neuropathology within the ol-
factory system (Fig. 6).
A? in the neocortex at 7 months of age
playing fibrillar A? deposits in the hip-
pocampus and neocortex ?10 months of
age (Hsiao et al., 1996; Kawarabayashi et
al., 2001). A? plaques have been reported
in the OBs of aged Tg2576 mice (Lehman
et al., 2003) and humans with AD (Kova ´cs,
tion of A? deposition within the OB nor
throughout the olfactory system has been
mice.Our analysis of A? throughout the
differences both within and between ol-
factory structures in A? deposition. Al-
though several studies to date have
demonstrated that APP and/or A? serve
to modulate synaptic processing of neural activity (Kamenetz
et al., 2003; Puzzo et al., 2008), whether A? or the overproduc-
tion of other APP-processing fragments in these mice (e.g.,
C-terminal fragments) (Gao and Pimplikar, 2001) perturb net-
work processing of information in the brain is currently not well
understood. Indeed, measuring factors other than deposited A?
deposition (percentage area) within the OB, PCX, OFC, Ent, and Hipp, and group-averaged olfactory scores (investigation time,
difference between investigation duration in the first odor trial compared with the fourth. OB data, Average of plaque area in
J.Neurosci.,January13,2010 • 30(2):505–514 • 511
pathology and the observed olfactory impairments. For exam-
ple, soluble oligomeric forms of A? are important in mediat-
ing AD-like neurodegeneration (Varvel et al., 2008) and in
modulating synaptic activity (Haass and Selkoe, 2007). Thus,
although the present findings show strong relationships be-
tween both fibrillar and nonfibrillar A? deposits and atypical
olfactory behaviors, they suggest that nonfibrillar forms of A?
provide unique insights into the basis of olfactory dysfunction
in AD. Furthermore, future studies into the potential contri-
butions of other AD pathologies (e.g., NFTs) in combination
with A? deposition to the emergence of AD-related sensory
loss may provide important insights into disease pathogenesis
and allow additional understandings as to the cause of olfac-
tory sensory loss in AD.
The neural mechanisms that govern odor information pro-
cessing in mammals are fairly well understood (Wilson and
Stevenson, 2006). Such mechanisms are essential in informing
we used the odor cross-habituation task, which takes advantage
of the natural tendencies of mice to investigate novel odors
(Sundberg et al., 1982). This test is less labor-intensive than the
than fear-learning paradigms. The design of the cross-habituation
task allowed us to assay three separate aspects of olfaction in the
test will be highly advantageous in testing the efficacy of treat-
On the detection of a novel odor in their environment, rats
and mice reliably display an arousal-related orienting response
odor. Such orienting responses are mostly used for the acquisi-
tion of odor information (Wachowiak, 2009). Indeed, a single
and discrimination (Wesson et al., 2008)—arguing that pro-
not necessary to discriminate odors. Instead, the duration of
this odor investigation behavior is dependent on arousal, mo-
tivation, and past experience (Wachowiak, 2009). Short-term
habituation (decrease over repeated presentations) (Rankin et
al., 2009) to odors is associated with receptor neuron adapta-
tion (Reisert and Matthews, 2001; Verhagen et al., 2007), OB
adaptation (Wilson, 1998; Kadohisa and Wilson, 2006a). De-
spite these widespread factors, the primary contributor to
short-term habituation is PCX adaptation, mediated by de-
PCX (Best and Wilson, 2004).
In particular, it is worth noting that PCX layer I, especially
within the pPCX, had a high level of A? deposition. Given the
importance of layer I synapses in short-term odor habituation, it
is possible that the deficits in short-term odor habituation ob-
directly within layer I sensory processing. This hypothesis is
supported by the fact that A? deposition in the PCX was ap-
parent in 16-month-old mice, but not in 6- to 7-month-old
odor habituation (trial 1 odor investigation). Furthermore,
anti-A? staining was prevalent within the OB Gl layer starting at
3 months of age, which corresponds with the emergence of
atypical odor habituation. The Gl is a crucial site for the initial
processing of odor input from receptor neurons in the nasal
cavity (Wachowiak and Shipley, 2006). Thus, it is possible that
nonfibrillar A? acts directly on sensory neuron terminals
within the Gl to modulate synaptic processing of odor infor-
mation at the earliest level. Future studies will be important to
examine these hypotheses.
tion (Wachowiak and Shipley, 2006), local circuit function
within the olfactory bulb (Beshel et al., 2007; Doucette et al.,
2007), and pattern recognition and template formation of previ-
local circuit function, including granule cell-mediated feedback
and lateral inhibition, is believed to contribute to contrast en-
hancement between similar odors (Wachowiak and Shipley,
2006; Wilson and Mainen, 2006). Notably, previous work has
shown that the cross-habituation assay is sensitive to even slight
differences in odor structure (Fletcher and Wilson, 2002). Asso-
ciation fiber plasticity within PCX layers I and III are believed to
be critical for recognition and discrimination of those odor-
evoked spatiotemporal patterns of OB activity (Haberly, 2001).
observed odor discrimination deficits. As atypical odor discrim-
ination emerged before A? deposition in the PCX, it is possible
that the sparse A? deposition within the OB Gl (as in Fig. 2) is
responsible for the deficits in odor discrimination. In support of
had A? deposits in the OB had more predominant olfactory
dysfunction than the mice that did not (as reflected through
the correlations) (Fig. 5). Alternatively, maladaptive olfactory
processing may arise within regions/layers neighboring these
important layers (Liu et al., 2008). It will be important in
future studies to determine the contributions of A? within
each of these structures, independently, to the cellular pro-
cessing of odor information.
In summary, our findings provide initial insights into the
ticular, our data revealed a correlation between the magnitude
and occurrence olfactory deficits and the spatial-temporal
pattern of A? deposition. Also, at 3 months of age, we ob-
served nonfibrillar A? deposition within the olfactory bulb—
earlier than deposition within any other brain region.
Therefore, these data suggest that nonfibrillar, versus fibrillar,
OB A? escalation. Data (depicted in approximate “levels”) are based on the results dis-
512 • J.Neurosci.,January13,2010 • 30(2):505–514 Wessonetal.•OlfactoryDysfunctionandAmyloid-?
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