Occlusal disharmony attenuates glucocorticoid negative feedback in aged SAMP8 mice.

Abstract

To evaluate the mechanism underlying impaired cognitive function due to occlusal disharmony, we examined the effect of the bite-raised condition on spatial performance and hippocampal expression of glucocorticoid receptors (GR) and glucocorticoid receptor messenger ribonucleic acid (GRmRNA) using behavioral, immunohistochemical, and in situ hybridization techniques. Learning ability in the water maze test was significantly impaired in aged bite-raised mice compared with age-matched control mice. There was no difference between control and bite-raised young and middle-aged mice. Also, immunohistochemical and in situ hybridization analysis showed that the bite-raised condition enhanced the age-related decrease in GR and GRmRNA expression in the hippocampus. In particular, GR and GRmRNA expressions were significantly decreased in aged bite-raised mice compared to age-matched control mice. These findings suggest that the bite-raised condition in aged SAMP8 mice decreases GR and GRmRNA, which impairs the hypothalamic-pituitary-adrenal feedback inhibition, thereby leading to memory deficits.

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Available online at www.sciencedirect.com
Neuroscience Letters 427 (2007) 71–76
Occlusal disharmony attenuates glucocorticoid negative
feedback in aged SAMP8 mice
Yukiko Ichihashia, Yoko Arakawaa, Mitsuo Iinumaa, Yasuo Tamuraa, Kin-ya Kubob,∗,
Fumihiko Iwakub, Yuichi Satoc, Minoru Onozukad,e
aDepartment of Pediatric Dentistry, Asahi University School of Dentistry, Mizuho, Japan
bDepartment of Oral Anatomy, Division of Oral Structure, Function and Development, Asahi University School of Dentistry,
1851 Hozumi, Mizuho, Gifu 501-0296, Japan
cDepartment of Molecular Diagnostics, Kitasato University School of Allied Health Science, Sagamihara, Japan
dDepartment of Physiology and Neuroscience, Kanagawa Dental College, Yokosuka, Japan
eResearch Center of Brain and Oral Science, Kanagawa Dental College, Yokosuka, Japan
Received 17 May 2007; received in revised form 4 September 2007; accepted 12 September 2007
Abstract
Toevaluatethemechanismunderlyingimpairedcognitivefunctionduetoocclusaldisharmony,weexaminedtheeffectofthebite-raisedcondition
on spatial performance and hippocampal expression of glucocorticoid receptors (GR) and glucocorticoid receptor messenger ribonucleic acid
(GRmRNA) using behavioral, immunohistochemical, and in situ hybridization techniques. Learning ability in the water maze test was significantly
impaired in aged bite-raised mice compared with age-matched control mice. There was no difference between control and bite-raised young and
middle-aged mice. Also, immunohistochemical and in situ hybridization analysis showed that the bite-raised condition enhanced the age-related
decrease in GR and GRmRNA expression in the hippocampus. In particular, GR and GRmRNA expressions were significantly decreased in aged
bite-raised mice compared to age-matched control mice. These findings suggest that the bite-raised condition in aged SAMP8 mice decreases GR
and GRmRNA, which impairs the hypothalamic-pituitary-adrenal feedback inhibition, thereby leading to memory deficits.
© 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: Occlusal disharmony; Hippocampus; Stress; Glucocorticoid receptor; Glucocorticoid receptor mRNA; SAMP8 mice
Occlusaldisharmony,suchaslossofteethand/orincreasesinthe
vertical dimension of crowns, bridges, or dentures, causes brux-
ism or pain in the masticatory muscles and temporomandibular
joint [2], and general malaise [22]. The application of occlusal
splints elevates urinary cortisol excretion rates in monkeys [1],
andtheplacementofacryliccapsonthelowerincisorsincreases
plasma corticosterone levels in rats [25]. In recent studies using
accelerated senescence-prone mice (SAMP8), the bite-raised
condition in aged mice increased plasma corticosterone lev-
els and neuronal death in the hippocampal CA3 region, and
impaired spatial learning in the Morris water maze test [9].
These findings suggest that stress is involved in the occlusal
disharmony-induced hippocampal-dependent changes.
Glucocorticoid(GC)secretionisnegativelyregulatedbyGCs
at the level of the anterior pituitary gland [14], suprahypotha-
∗Corresponding author. Tel.: +81 58 329 1404; fax: +81 58 329 1404.
E-mail address: kubo@dent.asahi-u.ac.jp (K.-y. Kubo).
lamic limbic structures (e.g., hypothalamus, hippocampus, and
amygdale), and the mesoprefrontal system [3,11]. In particu-
lar, hippocampal corticosteroid receptors appear to be sensitive
to elevated GC levels [17] and these receptors or their mRNA
are downregulated by chronic stress [5,18]. These findings sug-
gestthatthebite-raisedcondition-inducedimpairmentofspatial
memory in the aged mice might be due to reduced negative
feedback response to chronic stress.
In this study, using immunohistochemical and in situ
hybridization techniques, we examined the effect of the bite-
raised condition on the expression of GR and GRmRNA,
together with changes in learning ability in the Morris water
maze.
MaleSAMP8mice(3-,5-,and9-mo-old;n=12foreachage)
were used in the study. The animals were bred and maintained
underconventionalconditions;housedingroupsoffiveinplastic
cages under temperature (23±1◦C), humidity (55±2%), and
light (12h; light period, 6:00–18:00; dark period, 18:00–6:00)
controlled conditions; and allowed free access to food and
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2007.09.020

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Y. Ichihashi et al. / Neuroscience Letters 427 (2007) 71–76
water. The mice were treated in accordance with the principles
approved by the Council of the Japanese Neuroscience Society.
Thebite-raisingprocedurewasperformedundersodiumpen-
tobarbital anesthesia (35mg/kg). The vertical dimension in the
bite was raised as previously described [9]. The vertical dimen-
sion in the bite was raised approximately 0.1mm by placing
ultraviolet-ray polymerization resin (UniFil®LoFlo, GC Corpo-
ration, Tokyo, Japan) on the upper molars after treatment with
a Single Bond Dental Adhesive System (3M Dental Product,
USA). Control animals underwent the same anesthetic proce-
dure except that no resin was applied. After the procedure, the
mice were allowed free access to pelleted chow and water.
The Morris water maze test is a sensitive behavioral assay
for brain abnormalities in the hippocampus [20]. Eight days
after the bite-raising procedure, Morris water maze training was
performed as described previously [8,9,24]. Briefly, a stainless
steel tank (90cm in diameter, 30cm deep) was filled with water
(approximately28◦C)toaheightof22cmandthewatersurface
was covered with floating polystyrene foam granules (approxi-
mately 2mm in diameter). A platform (12-cm in diameter) was
submerged 1cm under the water surface and located at a con-
stant position near the center of one of the four quadrants of the
pool. Seven days after the bite-raising procedure, the mice were
placed into the water from one of four points at the perimeter of
the tank with an intertrial interval of approximately 5min and
given 28 trials over 7 consecutive days (4 trials per day). The
sequenceofthestartingpositionswasrandomizeddaily.ACCD
videocameralinkedtoacomputersystem(Move-tr/2D,Library
Co.,Ltd.,Tokyo,Japan)wasusedtomeasurethelatencyofeach
mousetoreachtheplatform.Inourpreviousstudy[7],therewas
no significant difference in the probe trial performance between
the control and bite-raised condition in 9-mo-old SAMP8
mice.
For immunohistochemical analysis of GR induction in the
hippocampal formation, on the final training day (day 7), 5
miceofeachgroupwereanesthetizedwithpentobarbitalsodium
(40mg/kg) and perfused transcardially with 30ml of saline at
37◦C, followed by 100ml of 4% paraformaldehyde in 0.1M
phosphate buffer (PB), pH 7.4. The brain was then removed
and placed in 2% paraformaldehyde fixative overnight at 4◦C.
The brains were divided into two hemispheres, one used for
immunohistochemistry and the other for in situ hybridization
mRNA assay. Tissue sections (40-?m thick) were prepared on a
microslicer (DTK-1000W, Dosaka, Kyoto, Japan), and floating
sectionswereprocessedthroughastandardimmunohistochemi-
calprocedureusingtheABCmethod.Thesliceswerefirstrinsed
with phosphate-buffered saline (PBS), then incubated with 1%
H2O2for 10min at room temperature, rinsed with PBS, and
incubated for 60min at room temperature with 5% normal goat
serum.AfterrinsingagainwithPBS,thesectionswereincubated
with rabbit polyclonal anti-GR antiserum (Santa Cruz Biotech-
nology Inc., Santa Cruz, CA) diluted 1:1600 in PBS containing
0.3% Triton X-100 (PBS-T) for 48h at 4◦C, rinsed with PBS,
incubated with biotinylated goat ant-rabbit IgG (Dako Cytoma-
tion, Glostrup, Denmark) diluted 1:500 in PBS for 2h at room
temperature, rinsed with PBS and 0.05M Tris–HCl buffer, pH
7.6 (TBS), and incubated with peroxidase-conjugated streptoa-
vidin (Dako Cytomation) diluted 1:500 with TBS for 1h at
room temperature. Bound complex was visualized using 3,3?-
diaminobenzidine (0.5mg/ml) and hydrogen peroxide (0.01%)
in TBS. Control sections were treated with non-immune rabbit
immunoglobulin instead of primary antibody.
Theinsituhybridizationmethodusedinthisstudywasprevi-
ously reported [21]. Briefly, deparaffinized 3?m-thick sections
were placed first in xylene and then in a descending ethanol
series, and then treated with 2?g/ml proteinase K (Roche Diag-
nostics) for 15min at 37◦C. The sections were postfixed in 4%
paraformaldehyde, treated with 0.2N HCl, and acetylated with
0.25%aceticanhydridein0.1mol/ltriethanolamine(pH8.0)for
10mineach.Aftertreatmentwith3%hydrogenperoxidefor1h,
the sections were dehydrated and air dried. The Hybridization
mixture (50?l; mRNA In situ Hybridization Solution; Dako)
with 50ng cRNA probes [5] was loaded onto each section and
hybridizedfor16–18hat50◦C.Afterhybridization,thesections
wereimmersedbrieflyin5×SSC(1×SSC:0.15mol/lNaCland
0.015mol/lsodiumcitrate),thenwashedin50%formamide/2×
SSC for 30min at 55◦C. After rinsing in TNE (10nmol/l
Tris–HCl, pH 7.6; 1nmol/l EDTA, 0.5M NaCl) for 10min at
37◦C, the sections were treated with 10?g/ml RNase A (Roche
Diagnostics)for30minat37◦C.AfterrinsinginTNEfor10min
at37◦C,thesectionswerewashedsequentiallyin2×SSC,0.2×
SSC, and 0.1× SSC for 20min each at 55◦C. After rinsing in
TBS(2)-T(0.01mol/lTris–HCl,pH7.5;300mmol/lNaCl,0.5%
Tween-20) three times for 5min each, and in 0.5% casein/TBS
(0.01mol/l Tris–HCl pH 7.5, 150mmol/l NaCl) for 10min,
the sections were reacted with a 1:400 diluted horseradish
peroxidase-conjugated rabbit anti-DIG F(ab?) fragment anti-
body (Dako), 0.07?mol/l biotinylated tyramide solution, and
Fig. 1. Spatial learning in the water maze test. The results are expressed as
the mean score (mean±S.E., n=6 for each group) of four trials per day. Note
that 9-mo-old bite-raised mice required a significantly longer time to reach the
platform than age-matched controls.

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Fig. 2. Photomicrographs showing GR immunoreactivity in the hippocampal formation (A) and CA1, CA3, and DG subfields (B). Note that there were no GR-
immunoreactive cells (arrow in A) and there were fewer GR-immunoreactive cells within the CA1 and DG regions of 9-mo-old bite-raised mice, but there were no
differences in CA1 and DG regions of 3- and 5-mo-old bite-raised mice (B). Bars: 200?m (A) and 50?m (B).
Table 1
Results of correlation coefficient test analyses between the time to reach the platform at 7 days, the number of GR positive cells in hippocampal CA1 and DG
subfields, and the number of GRmRNA signals in hippocampal CA1, CA3, and DG subfields
Time at 7 daysGRCA1GRDGGRmRNACA1GRmRNACA3GRmRNADG
GRCA1
GRDG
GRmRNACA1
GRmRNACA3
GRmRNADG
−0.835**
−0.816**
−0.855**
−0.819**
−0.788**
0.89**
0.775**
0.778**
0.718**
0.712**
0.77**
0.735**
0 862**
0.822** 0.892**
Time at 7 days, the time to reach the platform at 7 days; GRCA1 and GRDG, the number of GR in hippocampal CA1 and DG subfields, respectively; GRmRNACA1,
GRmRNACA3, and GRmRNADG, the number of GRmRNA signals in hippocampal CA1, CA3, and DG subfields, respectively. **P<0.01, note there were strong
correlations between all factors.

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Y. Ichihashi et al. / Neuroscience Letters 427 (2007) 71–76
1:500 diluted horseradish peroxidase-conjugated streptoavidin
(Dako), for 15min each at room temperature. Finally, color was
developed using the DAB Liquid System (Dako) and counter-
stained with Mayer’s hematoxylin.
Hybridization with a ?-2-microglobulin antisense strand
probewasusedasaninternalcontroltoconfirmthepreservation
of the mRNA. Hybridization with GR sense strand probes was
used as a negative control.
Quantitative analysis of GR-immunoreactive cells was per-
formed as described previously [24]. Round, clear, medium,
and large cells were counted in the left hippocampus (Bregma:
−2.46mm using the atlas of Franklin and Paxinos [4]) on each
section per animal using a 20× microscope objective. For each
section, all cells meeting the criteria described above were
counted in the hippocampal pyramidal layer along a 280-?m
long line (the diagonal of a 200?m×200?m square) in four
distinctzonesoftheCA1region,threedistinctzonesoftheCA3
region,andtwodistinctzonesofthegranularlayerofthedentate
gyrus. The data from each zone were pooled and the mean value
for each region estimated and expressed in terms of density of
cells per square millimeter.
GRmRNA signals were counted within high power micro-
scopic fields over the hippocampus (8 fields/subregion,
6sections/animal) as described previously [19]. Results were
calculated as mean amount of signal/high power field for each
region after subtracting the background signal.
Two-wayrepeatedorfactorialANOVAfollowedbyScheffe’s
or Tukey multiple comparison was used to evaluate the statis-
tical significance of the behavioral or morphologic differences
betweenthecontrolandbite-raisedgroups.Inaddition,therela-
tionbetweenthetimetoreachtheplatformat7days,thenumber
of GR-positive cells in the hippocampal CA1 and DG subfields,
and the number of GRmRNA signals in the hippocampal CA1,
CA3, and DG subfields was investigated using the correlation
coefficient test.
First, we examined the effect of raising the bite on spatial
cognitive performance in the Morris water maze. In agreement
with our previous observation in molarless mice [9], an age-
dependentincreaseinescapelatency(F(2,30)=17.8,P<0.001)
wasseen.Therewasastatisticallysignificantincreaseonlyinthe
9-mo-old mice (P<0.05 by multiple comparison; Fig. 1). There
was an interaction between the day and group on the spatial
cognitive performance (F(12, 180)=2.6, P<0.02).
We next examined the effect of the bite-raised condition on
GR-immunohistochemistry in the hippocampus. Light micro-
scopic analysis revealed that GR-immunoreactive cells in the
CA1 and DG subfields, but not in CA3 (Fig. 2 Panel A).
Because this finding was identical to that of other reports [14],
the GR immunoreactivity in hippocampal CA1 and DG sub-
fields was assessed. The results are shown in Fig. 3. There
was an interaction between the age and group on the num-
ber of GR-immunoreactive cells in the CA1 (F(2, 24)=10.5,
P<0.0005) and no interaction on the number of DG subfields
(P>0.1). While no difference between the control and bite-
raised groups in 3 and 5 month existed (P>0.1), the number
of GR-immunoreactive cells of the control was higher than that
of the bite-raised group (P<0.05 by multiple comparison).
Fig.3. Effectofthebite-raisedconditiononthenumberofGR-immunoreactive
cells in the hippocampal CA1 and DG regions. The results are expressed as the
mean number of neurons/mm2(mean±S.E., n=5 for each group). **P<0.01.
NotethegreaterreductioninthenumberofGR-immunoreactivecellsintheCA1
and DG subfields of the 9-mo-old bite-raised mice, but there is little reduction
in the number of GR-immunoreactive cells in the CA1 and DG subfields of the
3- and 5-mo-old bite-raised mice.
There was an age-dependent decrease in the number of GR-
immunoreactivecells(F(2,24)=27.0,P<0.001)anddifference
between the two groups (F (1, 24)=29.5, P<0.001) in the DG
subfields.
Finally, we examined the effect of bite-raising on GRm-
RNAexpressioninthehippocampus.GRmRNAwasdetectedin
hippocampal CA1, CA3, and DG subfields by in situ hybridiza-
tion (Fig. 4). There was a difference of the expression in CA3
between the control and bite-raised (F(1, 24)=10.5, P<0.008).
The 9-mo-old mice had a significant lower number of positive
cells than the age-matched control mice (CA1 and DG: P=0.05
by multiple comparison).
Table 1 shows the correlation coefficient test results. There
was a strong correlation between the time to reach the platform
at 7 days, the number of GR-positive cells in the hippocampal
CA1 and DG subfields, and the number of GRmRNA signals in
the hippocampal CA1, CA3, and DG subfields.
In this study, GR immunoreactivity was not observed,
although GRmRNA was detected in the hippocampal CA3
region, which is consistent with other reports [15]. This dif-
ference is thought to depend on differences in the synthesis or
metabolic rates of GR and GRmRNA in the hippocampal CA3
region [15].
The present study, using behavioral and morphologic tech-
niques, also demonstrated that raising the bite induces an
age-related deficit in spatial learning with a concomitant
decreaseinGRimmunoreactivityinCA1andDG,anddecreased
expressionofGRmRNA-positivecellsinthehippocampalCA1,
CA3 and DG subfields. These findings indicate that in aged
SAMP8 mice, the bite-raised condition downregulates GR and
GRmRNAinthehippocampus,whichisverysimilartoprevious
reports on chronic stress [6,18].
GCs are believed to be key mediators of the perception and
management of stressful stimuli. It is generally accepted that

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Fig. 4. Photomicrographs showing GRmRNA of hippocampal CA1, CA3 and DG subfields of 9-mo-old mice (A). Note that there were fewer positive GRmRNA
signals in the CA1, CA3, and DG subfields of the 9-mo-old bite-raised mice. Bar: 50?m. (B) Number of GRmRNA signals/high power field in hippocampal CA1,
CA3, and DG subfields in 3-, 5-, and 9-mo-old mice. **P<0.01, note that there were significantly fewer GRmRNA signals in the CA1, CA3, and DG subfields of
9-mo-old bite-raised mice.
neuroendocrineactivationofthehypothalamic-pituitary-adrenal
(HPA) axis is counteracted by GR-mediated negative feedback,
which results in the termination of the stress response. In addi-
tion, GR are distributed throughout a wide variety of brain
regions (in particular, the hippocampus) [15], where GR appear
to be sensitive to elevated GC levels [17]. The hippocampus is a
region of negative feedback of GC on the HPA axis [18]. Taken
together, these findings suggest that the downregulation of GR
and GRmRNA expression induced by the bite-raised condition
reduces inhibition of the negative feedback response, ultimately
resulting in increased plasma corticosterone levels.
Our previous study showed that the bite-raised condition in
agedSAMP8micenotonlyincreasesplasmacorticosteronelev-
els, but also impairs spatial memory and a decreased number
of neurons in the hippocampal CA3 region [9]. Also, chronic
stress or glucocorticoid (GC) exposure impairs maze learning
performance [10,23] and hippocampal neuronal degeneration
[10]. Furthermore, in rats, hippocampal neuronal degeneration
is blocked when a GC synthesis inhibitor is administrated,
even in conditions of chronic stress [13]. It is very possi-
ble that, under chronic stress, the hippocampal degeneration
and impaired spatial performance are caused by increased GC

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Y. Ichihashi et al. / Neuroscience Letters 427 (2007) 71–76
through impairment of the HPA axis due to the downregulation
of GR and GRmRNA.
Corticosteroids regulate the activity of the HPA axis
and modulate learning and memory processes through two
corticosteroid receptor types in brain, i.e., GR and mineralo-
corticoid receptors (MR). MRs are close to being saturated
at basal concentrations of corticosterone, while high corti-
costerone concentrations during stress occupy mainly GR.
Oitzl et al. [16] reported that unlike the effect of overex-
posure to corticosteroids, the continuous blockade of brain
GR facilitates learning and memory processes, suggesting
that improvement versus impairment of cognitive function is
critically dependent on the context and duration of GR activa-
tion.
There are reports, however that chronic stress facilitates the
ACTH response to novel acute stressors, which is uncovered
after corticosterone removal in rats [12], and that the amount
of negative feed back from the hippocampus through GR to the
HPA axis is relatively small.
In conclusion, in aged SAMP8 mice, raising the bite induced
areductionofhippocampalGRandGRmRNAexpression,lead-
ing to HPA axis impairment.
Acknowledgement
This work was supported in part by a Grant-in Aid for Sci-
entific Research from the Ministry of Education, Science, and
Culture of Japan (18592153, 18209058) and a Miyata Research
Grant (A) for 2006.
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